Bulgarian Society for Microbiology Union of Scientists in Bulgaria Acta Microbiologica Bulgarica

Volume 32, Issue 1, March 2016 ISSN 0204-8809

ACTA MICROBIOLOGICA BULGARICA Bulgarian Society for Microbiology Union of Scientists in Bulgaria Acta Microbiologica Bulgarica

The journal publishes editorials, original research works, research reports, reviews, short communications, letters to the editor, historical notes, etc from all areas of microbiology

An Official Publication of the Bulgarian Society for Microbiology (Union of Scientists in Bulgaria)

Volume 32 / 1 (2016)

Editor-in-Chief Angel S. Galabov

Press Product Line Sofia Editor-in-Chief Angel S. Galabov

Editors Maria Angelova Hristo Najdenski

Editorial Board I. Abrashev, Sofia S. Aydemir, Izmir, Turkey L. Boyanova, Sofia E. Carniel, Paris, France M. Da Costa, Coimbra, Portugal E. DeClercq, Leuven, Belgium S. Denev, Stara Zagora D. Fuchs, Innsbruck, Austria S. Groudev, Sofia I. Iliev, Plovdiv A. Ionescu, Bucharest, Romania L. Ivanova, Varna V. Ivanova, Plovdiv I. Mitov, Sofia I. Mokrousov, Saint-Petersburg, Russia P. Moncheva, Sofia M. Murdjeva, Plovdiv R. Peshev, Sofia M. Petrovska, Skopje, FYROM J. C. Piffaretti, Massagno, Switzerland S. Radulovic, Belgrade, Serbia P. Raspor, Ljubljana, Slovenia B. Riteau, Marseille, France J. Rommelaere, Heidelberg, Germany G. Satchanska, Sofia E. Savov, Sofia A. Stoev, Kostinbrod S. Stoitsova, Sofia T. Tcherveniakova, Sofia E. Tramontano, Cagliari, Italy A. Tsakris, Athens, Greece F. Wild, Lyon, France ACTA MICROBIOLOGICA BULGARICA

Review

A Focus on Carbapenemase-Producing Pathogens Detection by Phenotypic Tests Evangelia Voulgari, Aggeliki Poulou, Georgia Vrioni, Athanassios Tsakris* Department of Microbiology, Medical School, University of Athens, Athens, Greece Abstract Over the past decade the medical community has witnessed the severe compromise of last resort antibiotics such as carbapenems, widely used in the treatment of severe infections, due to multidrug-resistant Enterobacteriaceae. A variety of carbapenem-hydrolyzing enzymes, located on diverse mobile genetic elements, attainable via horizontal transfer, have been associated with the resistant profiles conferred. As the therapeutic pallet diminishes prompt identification forms the cornerstone for effective containment and epidemiological surveillance, in an attempt to hinder dissemination in the hospital and community setting. Nevertheless, issues regarding detection and accurate characterization of the enzyme at hand persist due not only to the variety of proposed methodologies, but also because of the necessity for each laboratory to tailor its workflow to its individual needs and resources. We aim to describe the recent status in phenotypic detection and characterization of carbapenem-hydrolyzing enzymes, with a focus on inhibitor-based tests, which can offer a reliable, cost-effective, easily applicable alternative to other more expensive, in need of specialized personnel and equipment methods. Keywords: carbapenemases, carbapenemase-producing Enterobacteriaceae, phenotypic tests, KPC, MBL, OXA-48

Резюме През последното десетилетие медицинската общност е свидетел на падането и на последните „крепости” на антибиотиците, каквато е групата на карбапенемите, широко прилагани за лечение на остри инфекции, причинени от мултирезистентни Enterobacteriaceae. Профилите на резистентността са свързани с многообразни карбапенем-хидролизиращи ензими, гените за които са локализирани върху мобилни елементи, придобивани чрез хоризонтален трансфер. С намаляването на терапевтичните възможности, тяхното точно идентифициране се очертава като крайъгълен камък за ефективното потискане и епидемиологичния контрол за спиране на разпространението на инфекциите в болниците и сред населението. Въпреки разнообразните методологии и необходимостта за съобразяване на изследванията с възможностите на отделните лаборатории, разпознаването и прецизното охарактеризиране на ензимите си остава актуално. Целта на настоящата работа е да опишем съвременния статус на фенотипната детекция и характеризиране на карбапенем- хидролизиращите ензими. Ударението е върху тестовете за инхибиране, които биха предложили надеждни, икономически изгодни и лесно приложими алтернативи, които да заместят по-скъпите методи, изискващи специализиран персонал и оборудване.

Introduction Over the past decade the medical communi- tidrug-resistant Enterobacteriaceae (Nordmann et ty has witnessed the severe compromise of last real., 2012a; Tängdén and Giske, 2015). Carbapenem sort antibiotics such as carbapenems, widely used resistance does not encompass a uniform entity of in the treatment of severe infections, due to mul- potential mechanisms but may be caused by a variety of factors and their combinations. Acquisition *Corresponding author: Tel: +30 210 7462011; Fax: +30 210 7462210; via horizontal transfer of diverse mobile genetic E-mail: [email protected]

3 elements bearing genes responsible for the pro- hospital or community setting (Naas et al., 2008; duction of carbapenem-hydrolyzing enzymes and Poirel et al., 2012; Tängdén and Giske, 2015). This decreased antibiotic uptake due to outer membrane review aims to present in a comprehensive manner permeability issues, either in the form of efflux the current status in regards to the available phe- pump up-regulation coupled with complete loss of notypic tools for detecting and characterizing car- porin-expressing genes or debilitating mutations, bapenemase-producing Enterobactericaeae (CPE), in association with AmpC β-lactamase hyperpro- which can be applied in the clinical laboratory set- duction or ESBL expression, constitute the main ting and give insight into those limitations associat- mechanisms leading up to a carbapenem resistant ed with sensitivity and cost. profile (Cornaglia and Rossolini, 2010; Nordmann et al., 2012a). As the available therapeutic pallet Detection of carbapenemase-producing diminishes and given the complexity and diversity Enterobacteriaceae (CPE) of the underlying resistance mechanisms, the need Screening tests for timely, accurate and cost effective detection/ The lingering controversy behind which characterization of the molecular background at isolates could be characterized as carbapenem re- hand becomes even more pressing, in an attempt to sistant was addressed with the implementation of tailor the administered individual patient regimens the novel 2010 CLSI breakpoints and guidelines, (Nordmann, 2014; Jean et al., 2015; Tängdén and followed however by a debate on the necessity of Giske, 2015). further confirmatory and phenotypic testing (CLSI, Therefore, focusing on the acquired carbap- 2010). Although currently further testing is not enem-hydrolyzing enzyme aspect of a carbapenem routinely recommended neither by the CLSI nor resistant phenotype, it is essential to bear in mind EUCAST accurate characterization of carbapenem the diversity of the enzymes at hand as previous- resistance mechanisms in the clinical laboratory rely grouped, based on sequence and structural ho- mains crucial not only for the implementation of mology, in the Ambler classification(Ambler, 1980; infection control measures but also for epidemio- Queenan and Bush, 2007). To date, in regards to logical surveillance, especially in areas with preex- the Enterobactericeae species, carbepenem resis- isting endemicity where various genes are likely to tance has been linked to class A enzymes of the have simultaneously disseminated. KPC, SME, IMI, NMC, GES types, class B car- The clinical laboratory is called upon to an- bapenemases of the IMP, VIM, NDM, GIM, SPM, swer two vital questions. The former addresses the SIM category and class D oxacillinases such as the presence of a carbapenem-hydrolyzing enzyme in OXA-48 and its variants, with the KPC, VIM, IMP, a carbapenem resistant Enterobacteriaceae (CRE) NDM and OXA-48 types constituting the most and the latter the nature of the enzyme detected. effective carbapenemases in terms of hydrolytic Currently in regards to the first question three potential, dissemination and geographical spread main techniques for detecting carbapenemase ac- (Tängdén and Giske, 2015). tivity are receiving considerable attention, the mod- The difficulties noted in carbapenemase de- ified Hodge test (MHT), the Carba NP test and the tection and characterization, arise from the fact that carbapenem inactivation method (CIM) all based due to the diversity observed in the active sites of on indirectly witnessing carbapenem hydrolysis. these enzymes and the coexistence or not of addi- The MHT, based on the inactivation of a car- tional resistance mechanisms, the hydrolytic pro- bapenem by a carbapenemase-producing strain, file conferred is variable not only in regards to the therefore enabling a susceptible indicator strain to specific carbapenem substrate affected but also the extend growth towards the carbapenem containing minimum inhibitory concentration (MIC) conferred disk, is currently recommended by the CLSI as (Queenan and Bush, 2007; Poirel et al., 2012). a confirmatory test for the detection of diffusible Furthermore, as these resistance genes are mostly carbapenemases (Lee et al., 2001). However, in its harbored on transposon-and/or integron-encoded original form, this test although simple and easily determinants, prone to inter/intra-species dissemi- applicable in the laboratory setting, is fraught not nation, further changes in host range and epidemi- only by false positive results in the presence of ological distribution can be anticipated, thus ren- other β-lactamase genes such as AmpCs and ES- dering prompt identification the cornerstone for BLs when presented with coexisting permeabil- effective containment, in an attempt to hinder fur- ity defects but also a low sensitivity of 50% for ther dissemination following an introduction in the NDM-producers (Pasteran et al., 2009; Carvalhaes

4 et al., 2010; Girlich et al., 2012). In an attempt to carbapem hydrolysis, have been put forward, such address issues regarding NDM-producers improved as the commercial Rapid CARB Screen Kit (Rosco- protocols have been presented with the addition of Diagnostica A/S Taastrup, Denmark) and the more supplements such as ZnSO(4) to the culture media cost effective Blue-Carba test, yet both have with- or of a nonionic surfactant (Triton X), capable of standing issues to address regarding either lower releasing the membrane bound NDM carbapene- sensitivity in comparison to the Carba NP as the mases (Girlich et al., 2012; Pasteran et al., 2016). former or need for further evaluation studies as the Detection of class A carbapenemases with a reduc- latter (Pires et al., 2013; Yusuf et al., 2014; Paster- tion in false positives is also feasible with the ad- an et al., 2015). dition of inhibitors such as boronic acid (BA) and Finally, the CIM test has recently been pro- oxacillin (OXA) (Pasteran et al., 2010). posed as an effective yet cheaper alternative to the Biochemical tests such as the Carba NP, have Carba NP test (van der Zwaluw et al., 2015). The also been introduced and in current CLSI guidelines concept behind the test relies on the inactivation of are recommended for confirmatory testing (CLSI, the carbapenem substrate harbored in a single 10μg 2015) . They have been designed to identify the dis- meropenem disk following its inoculation in a bac- ruption in the β-lactam ring of the carbapenem sub- terial suspension and subsequent incubation for a strate (imipenem), based on the produced variation minimum of two hours. In the event that the isolate in color, due to the acidic pH alteration from the being screened does produce a carbapenemase the enzymatic hydrolysis, of a phenol indicator, which substrate is hydrolyzed and therefore the applica- is rendered either yellow or orange from an initial tion of the disk on a Mueler-Hinton (MH) agar plate red (Nordmann et al., 2012b; Poirel and Nordmann, streaked with a susceptible reference strain fails to 2015). Results are readily available within 2 hours produce an inhibition zone. Despite the fact that in following sample preparation (Nordmann et al., its initial description the method yields, at a frac- 2012b). The second version of the test, Carba NP II, tion of the cost of the Carba NP test, a similar high can differentiate class A and B and indirectly predict performance (96,6% concordance), further valida- class D enzymes due to the addition of EDTA and tion is warranted (van der Zwaluw et al., 2015). tazobactam, with EDTA and tazobactam inhibiting MBLs and class A carbapenemases respectively Inhibitor-based tests for carbapenemase (Dortet et al., 2012). Another interesting aspect of characterization the assay is that in biological samples which tend to The principle applied in these tests takes ad- yield single bacterial isolates such as blood cultures vantage of the properties of specific β-lactamase the test can be applied directly using the liquid cul- inhibitors, which are class dependent and exhib- ture media, thus further reducing the time to detec- it specificity in rendering their hydrolytic effect, tion and effective antibiotic treatment (Dortet et al., therefore enabling the characterization of the car- 2014). The introduced commercial version of the bapenemase class of the enzyme under investiga- Carba NP test, Rapidec Carba NP (BioMérieux, La tion. Balme-les-Grottes, France) comes to report a specificity and sensitivity of 96% while considerably re- Single inhibitor-based tests ducing the time to detetion to <15 min for class A EDTA-based tests inhibit β-lactam hydrolysis and B producers and <30min for class C producers, by exploiting the dependence of metallo-enzymes while suggesting an additional simple step prior to on zinc ions and discriminate MBL-producing En- testing for isolates which present a mucoid pheno- terobacteriaceae from CRE due to ESBL and/or type (Poirel and Nordmann, 2015). Other tests also AmpC hyper-production in light of permeability based on the concept of biochemical detection of defects (Yong et al., 2002). Others inhibitors for Table 1. Class A, B and D carbapenemases and their respective inhibitors Inhibitor Inhibitor Inhibitor β-lactamase Class Genes Clavulanate EDTA/DPA PBA/CLO* A KPC - -/- +/- IMP, VIM, Carbapenemase Β NDM - +/+ -/- D OXA-48-like - -/- -/- *Cloxacillin

5 class B type enzymes include EDTA plus 1,10-phe- In this direction Tsakris et al. impregnated nanthroline, thiol compounds and dipicolinic acid 292 μg EDTA and 400 μg PBA separately and in (DPA) (Arakawa et al., 2000; Lee et al., 2001; Ki- combination onto 10 μg meropenem disks and ap- mura et al., 2005) (Table 1). plied a minimum 5 mm increase in inhibition zone Furthermore, in attempting to optimize the diameter as an indicator of a positive result (Tsakris sensitivity and specificity of the methods used, a et al., 2010). This protocol yielded a sensitivity and variety of potentiation tests, based either on disk specificity of 96.8% and 100%, respectively, in de- diffusion or broth dilution, have been proposed tecting simultaneous production of KPC and VIM questioning the optimal concentration of inhibitor carbapenemases, with the 100% sensitivity and applied, the distance between disks at which the ef- specificity ratio for isolates harboring either VIM or fect is potentiated and the best β-lactam substrate KPC enzymes, deterred only in regards to the spec- to be utilized (Yong et al., 2002; Migliavacca et al., ificity for KPC detection (98.8%), compromised by 2002; Pitout et al., 2005; Franklin et al., 2006). In two ESBL and AmpC producers with positive tests retrospect for the detection of MBLs in Enterobac- to meropenem/ PBA and meropenem/ PBA/EDTA teriaceae two tests are noted to perform better, the (Tsakris et al., 2010). combined-disk test using either imipenem/imipen- Giske et al. validated a carbapenemase de- em with 0.5 M EDTA or ceftazidime/ceftazidime tection test based on a five disk principal with the with 0.2 M EDTA, while applying a 7 mm differ- use of meropenem alone and supplemented with ence in inhibition zone cut-off and the double-disk 730 μg EDTA, 1000 μg DPA, 600μg APBA and synergy test where an imipenem disk is placed at a 750 μg cloxacillin (Giske et al., 2011). In contrast 10 mm distance from a 0.5M EDTA supplemented to EDTA, DPA and cloxacillin for which a 5 mm disk (Galani et al., 2008). cut-off was applied, the accurate detection of all Attempts to phenotypically detect class A KPC producing isolates mandated that for APBA carbapenemases were initially based on the use of a 4 mm increase in zone inhibition diameter should clavulanate, which inhibits weakly resistance to er- be considered as indicative of KPC production. tapenem and imipenem (Yigit et al., 2001) . Phen- AmpC hyper-producers with porin loss were pos- ylboronic acid (PBA) was latter directly associated itive in the APBA test but, unlike KPC producers, with the inhibition of class A type carbapenemas- showed cloxacillin synergy. Furthermore DPA per- es ,although the mechanism of action still remains formed better than EDTA as far as specificity was unclear (Tsakris et al., 2008) (Table 1). Following concerned in MBL-producing isolates (Giske et al., this observation different boronic acid compounds 2011). have been used as inhibitors at various concentra- In a significantly more complicated and tech- tions (Pasteran et al., 2009; Tsakris et al., 2009, nically demanding approach Birgy et al. evaluated 2011). Comparative evaluation of different com- the simultaneous preparation and use of a combina- binations of aminophenylboronic acid (APBA) (at tion of inhibitor-supplemented (EDTA, PBA, clox- 300 μg or 600 μg respectively) and PBA at 400 μg, acillin) MH agar disks in an attempt to also charac- for KPC-producing Enterobacteriaceae, highlight terize carbapenemase and ESBL producers (Birgy PBA at a concentration of 400 μg as the most effec- et al., 2012). tive KPC carbapenemase inhibitor using meropen- Accurate detection of OXA-48-like-pos- em as a substrate and evaluating an increase in zone sessing Enterobacteriaceae isolates remains prob- diameter inhibition equal or greater than 5 mm be- lematic in the absence of a reliable inhibitor and tween meropenem alone and meropenem plus PBA therefore the optimal screening agents and methods (Tsakris et al., 2011). are yet to be defined (Poirelet al., 2012). However, class D carbapenemase detection has been recently Combined inhibitor-based tests been addressed by a novel single phenotypic test, The challenge however, remains the exact the OXA-48 disk test (Tsakris et al., 2015). The identification with an easy to apply method, of these test is based on the use of EDTA to permeabilize resistance traits, in isolates likely to be simultane- the bacterial cell and inhibit MBL carbapenemase ously harboring multiple carbapenemases as well production while a solution of PBA inhibits KPC as other β-lactamase genes. Integrating compounds production. A 10μg imipenem is placed on a MH with different inhibitor properties into a single test agar plate, inoculated with a lawn of a susceptible has led to the proposal and evaluation of a variety strain, adjacent to two blank disks supplemented of phenotypic tests. with 292 μg EDTA and 292 μg EDTA/600 μg PBA,

6 respectively. The isolate under investigation is then text inhibitor based tests offer the advantage of by- applied onto the imipenem disk and following incu- passing in most cases the need for more expensive bation resulting distortions were assessed and inter- tests and can be implemented successfully in every- preted (Tsakris et al., 2015). Pending further valida- day laboratory practice. tion the sensitivity of the method was estimated at 96.3% (Tsakris et al., 2015). Also, temocillin MIC References testing on Mueller-Hinton agar supplemented with Ambler, R. P. (1980). The structure of beta-lactamases. Phi- cloxacillin was found specific for detecting OXA- los. Trans. R. Soc. Lond. B. Biol. Sci. 289: 321-331. 48 production (Maurer et al., 2015). Arakawa, Y., N. Shibata, K. Shibayama, H. Kurokawa, T. Yagi, H. Fujiwara, M. Goto (2000). Convenient test for screening metallo-beta-lactamase-producing gram-nega- Conclusion tive bacteria by using thiol compounds. J. Clin. Microbiol. 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9 ACTA MICROBIOLOGICA BULGARICA

Review

Review-Development of Models of Ethanol Synthesis and Production by Using Principles of the Theory of System Analysis. Personal Experience. Alexander Dimitrov Kroumov 1*, Aparecido Nivaldo Módenes 2 1 Institute of Microbiology „Stephan Angeloff“- Bulgarian Academy of Sciences, Department of Applied Microbiology, Acad. G. Bonchev str., Bl.26, Sofia 1113, Bulgaria 2 Department of Chemical Engineering Postgraduate Program, West Parana State University - UNIOESTE, Rua da Faculdade 645, Jd. Santa Maria, 85903-000, Toledo, PR, Brazil. Abstract The objective of this review article was to highlight the authors’ more than 30 years’ experience in the field of model development procedure of ethanol biosynthesis. During all these years a powerful system analysis theory and its decomposition principles were applied not only for studying and description of microbial kinetics but for the needs of bioprocesses and bioreactors/photobioreactors optimization and scale-up. Special attention was given to the description of phenomena and techniques involved in ethanol synthesis on single and two substrates utilization processes by wild and genetically modified strains. The method of simultaneous saccharification and fermentation of starch to ethanol (SSFSE) by genetically modified microbial Saccharomyces cerevisiae YPB-G strain is presented to fully illustrate the strength of the applied theory. Another modern method for extractive fermentation and ethanol production is presented as well. Diauxic growth phenomena in ethanol synthesis is the example showing a modeling approach by connecting knowledge from molecular and population hierarchic levels. This modeling work helped tre- mendously to find new insights on the control of internal metabolic mechanisms of the cells and how the processes can be guided to an optimal trajectory. A parameter identification procedure was performed by using the latest achievements in global search methods. The applied strategy of model development was extremely successful in obtaining new knowledge about the microbial systems’ behavior as well as for fast and robust bioprocesses development. The review article can be very useful for modelers and young scientists working in the fields of biotechnology, bioengineering, metabolic and chemical engineering. Key words-modeling, system analysis, ethanol, kinetics, parameters identification, genetic algorithm.

Резюме Целта на тази обзорна статия е да се представи тридесетгодишния научен опит на авторите в областта на моделиране на кинетиката на етанолния биосинтез. През всичките тези години се е прилагала теорията на системния анализ и нейните принципи на декомпозиция като мощен инструмент не само за изучаване и описание на микробната кинетика, но така също и за нуждите на оптимизация и мащабиране на биопроцеси и биореактори/фотобиореактори. Специално внимание бе отделено на описание на механизмите и техниките при синтеза на етанол на базата на усвояване на един или два субстрата от диви и генетично модифицирани щамове. Методът на едновременно озахаряване и ферментация на нишесте до етанол от генетично модифициран микробен шам Sac- charomyces cerevisiae YPB-G е представен за да илюстрира изцяло стабилността на приложената теория. Друг съвременен метод за производство на етанол като екстракционна ферментация също е представен. Статията за «Diauxic» растеж в синтеза на етанол е пример, показващ моделиране, чрез свързване на знанията от молекулно и популационно йерархични нива. Тази работа по моделиране помогна изключително много, за да се намерят нови идеи за контрол на вътрешните метаболитни механизми на клетките и как тези процеси могат да бъдат оптимално провеждани. Процедурата на параметрична идентификация се извършва чрез използване на най-новите достижения в областта на методите за търсене на глобален екстремум. Приложената стратегия за разработване на кинетични

10 модели беше изключително успешна за получаване на нови знания за поведението на микробните системи, както и за бързо и стабилно разработване на биопроцесите. Статията може да бъде много полезна за моделатори и млади учени, работещи в областта на биотехнологиите, биоинженерство, метаболитната и инженерната химия.

Introduction The quantitative description of bioreaction al., 2015; Crofcheck et al., 2000a; Crofcheck et al., processes must first pass through their modeling 2009b; Crofcheck et al., 2010). and simulation. By applying mass and energy bal- The review paper was intended to cast light ances, the engineer is able to evaluate almost any on ethanol production processes and their mathe- bioreactor system. Having the knowledge about matical descriptions (Kroumov, 1999; Kroumov, microbial kinetics and microbial physiology, the 2002e; Kroumov et al., 2002d; Kroumov et al., process engineer can build a complex model of the 2006; Tait et al., 2006; Tait et al., 2005a; Tait et bioprocess and bioreactor system and to solve op- al., 2005b; Tait et al., Tait et al., 2005c; Tait et al., timization and scale-up tasks. During the modeling 2005d; Tait et al., 2004a; Tait et al., 2004b; Krou- procedure scientists and engineers make an attempt mov et al., 2005a). The presented examples and all to study and analyze a real, most often complex, cited works of the kinetic models will convince the situation, where many parallel processes take place reader that the application of the theory of system in the bioreactor vessels. This usually requires sim- analysis may save time and money during the over- plification of the situation in an understandable all bioprocess development procedure. physical, chemical, and biochemical analog. In order to successfully solve the task, the mathematical Materials and Methods description must have physical sense, formulated Model development procedure based on the system in complex mathematical models. The contribution analysis theory made to biotechnology by a biochemical engineer’s System analysis theory and its principles modeling approach is an undeniable fact. Howev- The main stages of the system analysis theory er, to build a robust and working model it is not (Kaffarov, 1985; Kaffarov et al., 1979; Kaffarov et enough to use only basic fundamental principles, al., 1985) applied to the development of a biopro- such as material balances. cess kinetic model can be presented as follows: (1) There is more than that. The system analy- Determination of the task of the modeling, choice sis theory and its principles provide the strategy of the criterion of optimality; (2) Analysis of the for building a working model of any chemical or bioprocess – qualitative analysis of the structure of biological sophisticated system (Kaffarov, 1985; the biosystem, decomposition of the biosystem in Kafaroff et al., 1979; Kafaroff et al., 1985). The sub-systems; building of the models of the sub-sys- authors have used the knowledge of this powerful tems; parametric identification of the models of the tool for more than 30 years in Russia, Bulgaria, elements of the sub-systems; (3) Synthesis of the Brazil and the USA in order to describe the dynam- system – development of the complex models of ic behavior of many biotechnological (Kroumova the sub-systems and their relationships; simplifica- and Wagner, 1995; Kroumov et al., 2005c; Wenzel tion and combination of all the models into a com- et al., 2006a; Wenzel et al., 2006b], wastewater plex bioprocess kinetic model; search for optimal treatment processes (Fiorentin et al., 2015; Bor- working conditions of the bioprocess based on the ba et al., 2014; Módenes et al., 2015; Kroumov model simulations; experimental verification of the et al., 2005b; Trigueros et al., 2010a; Trigueros et developed simple and complex models; (4) Solving al., 2010b; Trigueros et al., 2007; Fagundes-Klen the modeling task – determination of the optimal et al., 2007) containig heavy metals (Borba et al., structure of the kinetic model on the basis of the 2008), bioreactors (Kroumov et al., 1986; Krou- chosen criterion of effectiveness; solving the bio- mov et al., 1987; Kroumov et al., 1990; Kroumov process scale-up problems on the basis of the devel- et al., 1991; Kroumov and Gimenes, 2002a; Krou- oped complex kinetic model. mov and Gimenes, 2002b; Kroumov and Gimenes, 2002c), photobioreactors (Kroumov, 2013a; Kro- Examples umov, 2013b; Kroumov, 2014; Kroumov, 2015; Simultaneous Saccharification and Fermentation Kroumov et al., 2015a; Kroumov et al., 2015b), and of Starch to Ethanol (SSFSE) - applications of the algae kinetics (Kroumov et. al., 2013; Kroumov et. principles of system analysis theory to SSFSE sys-

11 tem processes. The dynamics of enzyme excretion and A non-structural model describing a SSF- plasmid stability are not taken into consideration. SE process by a recombinant strain of S. cerevisiae YPB-G (Altintas et al., 2002) was developed, First hierarchic level published (Kroumov et al., 2006; Kroumov et al., The first hierarchic level (see Table1) consid- 2004) and presented elsewhere (Maicon et al., ers enzymatic hydrolysis of starch by glucoamylase 2005). The scheme of the model development algo- (this enzyme in our case is secreted by the recombi- rithm is shown below. In Figure 1 the mechanisms nant strain). A mathematical description of the hy- of the SSFSE processes were formalized in two drolysis process of starch as two substrate fractions hierarchic levels of knowledge. The first one con- was developed and published in details by Polak- siders processes of enzymatic hydrolysis of starch, ovic & Bryjak (2004). This knowledge was adapted and the second one combines microbial physiology and applied to starch hydrolysis by the recombinant

Fig. 1. A scheme of model development algorithm. strain of S. cerevisiae YPB-G. ethanol accumulation in the medium. More details Second hierarchic level about the identification procedure, the applied hy- The second hierarchic level (Table. 1, see brid genetic algorithm (GA) and the new decompo- Eq. 4-7) involves knowledge about the microbial sition approach can be found elsewhere (Maicon et kinetics of the population. Several physiological al, 2005, Kroumov et al., 2006]. The values of ki- phenomena of the recombinant strain S. cerevisiae netic and stoichiometric parameters were estimated YPB-G have been described with simple unstruc- using the experimental data of Altintas et al., 2002. tured mathematical formulas taken from our kinetic Several sets of batch data of recombinant strain cul- models catalogue database (Birol et al., 1998). The tivation on starch were examined. Here only a set microbial kinetic model is flexible, and equation (4) called SG30 was used with the above initial con- (glucose balance) is a key equation, which unites ditions. It must be noted that the identification of the first and second hierarchic levels of knowledge. the 14 model parameters is not an easy task. The The enzyme synthesis rate (see eq.7) is represented straightforward use of the search method is always as a function of the specific growth rate and total going to fail. We have developed and applied a new starch concentration. The model of SSFSE is com- decomposition approach of the objective function pleted and it describes enzyme hydrolysis of starch in order robustly to find its global extreme. The de- by glucoamylase secreted by the recombinant strain tails can be found elsewhere (Maicon et al., 2005, S. cerevisiae YPB-G. It also describes microbi- Kroumov et al., 2006). Here the experimental data al growth, glucose synthesis and utilization, and of Altintas et al., 2002 are presented.

12 Table 1. SSFSE system - two hierarchic levels of the model

The analysis of the results presented in Fig. as well as by the measurement errors of the analyt- 2 evidence the predictive power of the new devel- ical procedure used. oped kinetic model of SSFSE and the new decomposition method applied for the parameter iden- Simultaneous fermentation and ethanol extraction tification. As can be seen in Fig 2, the simulated (SFEE) dynamic profiles of starch decomposition and uti- This example shows the application of a new lization (Fig 2a) show how the resistant and sus- method of fermentation and ethanol synthesis when ceptible starch fractions were degraded through the ethanol is a strong inhibitor of its own production. SSFSE process. The enzyme profile shown offered The objective of this work was to model the con- new knowledge about the overall SSFE process tinuous mode of simultaneous fermentation and ex- when limitation, inhibition and effects take place traction of ethanol (SFEE) in order to obtain max- on two hierarchic levels of the system. The experi- imum productivity. The model considers a solvent mental data and model simulation of the total starch distribution coefficient value into the confidence degradation (Fig 2b) and glucose formation and uti- range. Different kinetic models were applied. Two lization (Fig2c) are almost perfectly predicted by kinetic models representing the significant effect of the model for the given initial conditions. This very the substrate and product inhibition were chosen. well corresponds with the profiles given in Fig. 2a. The response surface analysis (RSA) methodology Understandably, the experimental dynamic profiles was applied to study the influences of the control of biomass (Fig. 2d) and ethanol (Fig. 2e) showed variables on the SFEE system. The simulation re- significant deviations from the simulated ones. In sults have shown that the solvent phase dilution biotechnology this is a well-known fact and can be rate is influenced mainly by the product inhibition, explained by the nature of the population growth, while glucose inhibition limits its own feeding con-

13 Fig. 2. Search for minimum of least square objective function by using hybrid GA and a new developed decomposition approach: a) Simulation-Dynamic profiles of the state parameters of the new developed SSFSE kinetic model (Kroumov et al., 2006); b) Starch; c) Glucose; d) Biomass; e) Ethanol. centration. The main control variable of the sys- promising molecular modeling methodology. How- tem was the watery phase dilution rate. A hybrid ever, if satisfactory solvent characteristics cannot genetic algorithm (GA) with particular codification be reached during the procedure of solvent identifi- of the crossovers, mutation and elitism technique cation, genetic engineering can be used as a tool for was used for the optimization of ethanol produc- development of solvent resistant modified strains. tivity. The optimized control variable values were The mathematical modeling of the batch process obtained for watery and solvent phase dilution rates was considered by Honda et al. (1987) and was suc- and for feeding glucose concentration, respectively. cessfully applied for a mixture of solvents by Shi et The value of the overall productivity of 21.84 kg/ al. (1990). m³ is in accordance with the optimization results published in the literature. The SFEE model Note: In SFEE, the appropriate solvent iden- A general mathematical model for SFEE tification can be considered to be a crucial step. based on the system analysis approach (see Fig.3) Kollerup and Daugulis (1985, 1986) presented was developed. studies with excellent final results of solvent iden- The model considers continuous operation in tification. Recently, Pistikopoulos and Stefanis a well-mixed reactor. The balance equations of the (1998) have analyzed identification methods and a model are presented in Table.2.

14 Fig.3. Scheme of the algorithm of SFEE model development.

Table.2 SFEE system -Two hierarchic levels of the model

15 The factor η is defined as the ratio between generation is preserved in the next one. This is in the solvent and water phase volumes. The partition case the GA does not find a better solution during (distribution) coefficient (m) is defined as thera- the search. A higher value of this elitism operator tio between the ethanol concentration in the broth typically leads to premature convergence identified phase and the ethanol in the solvent phase. For by the GA (Katare et al., 2004). Re-initialization of convenience, some scientists fixed the value of (m) chromosomes corresponds to 20% of a new genera- (Honda et al., 1987). tion. More detail about the hybrid GA can be found Different kinetic hypotheses can be applied in Singh et al., 2005; Katare et al., 2004). If during for the model development of SFEE process. In this the search GA reaches the ith lower or upper param- work, Ghose and Thyagi’s model was analyzed and eter bound, a penalty on objective function is ap- applied (see Table.2, Eq. 14-16). The model was plied. A population size of 40 has been determined verified on the basis of experimental data presented and the GA was run for 300 generations. by Dourado et al., 1987. Results and Discussion Optimization procedure The Ghose and Thyagi’s model considers An analysis of sensitivity of the optimization a hypothesis that specific utilization and ethanol variables can be performed initially using the RSA formation rate can be modeled as a function of a method. The method shows the objective function specific growth rate. This assumption has the con- (productivity) in relation to the control variables of venience and advantage to show that in microbial optimization. When analyzing the results, some of physiology the specific growth rate of microbial the variables during the optimization process can population has key importance. Taking into account be discarded. The RSA analysis (Silva et al., 1999) this fact, the system can be formalized by building also assists in the identification of the superior and it from three dependent sub-systems, and the bio- inferior limits of the search space of each control mass balance can be considered a key factor, where variable, minimizing the computational work with the specific growth rate controls the other cell func- genetic algorithm (GA). tions. Substrate limitation and inhibition effects are The three control variables of optimization presented in the Andrews type model and ethanol are as follows: water phase dilution rate, solvent inhibition is considered a function of the critical phase dilution rate and glucose concentration in inhibitory concentration. The system can be provi- the feeding flow. For analysis purposes, a three-di- sionally divided into two hierarchic levels. The first mensional representation of the relation between one includes the knowledge about microbial phys- productivity and two control variables was chosen. iology and overproduction of ethanol by fermenta- The third variable was fixed in quasi-optimal value. tion. On this level, the system behavior is studied The system response as a function of the control for the two chosen kinetic models. The second hier- variables is shown in Figures 4-6. The overall etha- archic level contains knowledge about the physical nol productivity was considered zero near the criti- process of ethanol extraction by organic solvent. cal conditions of wash-out point and can be written The thermodynamics of liquid-liquid extraction as follows: can be represented in the simplest possible way by The range of control variables was determined two coefficients. It is assumed that the solvent does not inhibit microbial growth and ethanol produc- (17) tion, i.e. the solvent is biocompatible. For preliminary studies, the relation between the volumes of the solvent and broth phases was on the bases of their microbiological and physical fixed η=0.25, and the distribution coefficient has meaning. To perform the optimization search, a hy- a value of m=2. The value of these control vari- brid genetic algorithm (GA) was used as a global ables depends on the balance between the phases optimizer. The search for a global solution by GA and limits of solubility of the solvent in the water was carried out applying a selection method of par- phase. The RSA of the Ghose and Thyagi’s kinetic ents (tournament method). We have used the perfor- model has shown the following system responses mance of hybrid GA with the following parameter (see Figures 4-6). values: crossover=0.6 and mutations=0.2. As a rule of thumb, the crossover probability is generally in the range of 0.5-0.85. The best solution from every

16 Analyzing the graphs of Figures 4, a „wash- out“ phenomenon can be observed, which supposes a penalty on the objective function when the search procedure reaches the region near this point. On the other hand, the productivity is zero on the right- hand side of the ”wash-out” point and its maximum

is near the left-hand side of this point. The Dw variable has exerted bigger influence on the system in terms of “wash-out” phenomenon. Figure 4 shows

that the Dw-Glu gives crucial information about the maximum ethanol productivity. Based on this information, different operational conditions can be selected to perform an optimal ethanol production process. Figure 5 indicates that the search for an

optimal Dw value is a crucial step of ethanol productivity maximization. For the chosen kinetic Fig. 4. RSA of Dw versus Glu0 using Ghose and models, the D -D correlation is insignificant. The Thiagy’s kinetic model–Ds=3(h-1). s w Ds influence on the system can be neglected since the ethanol inhibition effect on cells is very low. In Figure 6, the analyzed „wash-out“ phenomenon shown in Figure 4 is not observed. The maximum productivity is reached with the increase of

both control variables, Ds and Glu0 (Dw is fixed). The system behavior was analyzed in detail by using Ghose and Thyagi’s kinetics. Response surface

analysis (RSA) showed the key role of Dw control

during the optimization procedure. The Dw values near 0.37 should be taken with caution. The applied hybrid GA performed excellently for the chosen system and optimal solutions were found within a short computational time. The main difficulty of the optimization procedure was to find the optimal value of GA parameters. The RSA can be very helpful Fig. 5. Surface analysis of Dw versus Ds for Ghose for localization of space search for system optimal and Thiagy’s model–Glu0=200 kg/m³. solutions, minimizing the computational time. The optimal control variables and productivity values for Ghose and Thyagi’s model are presented in Ta- ble 3. The productivity presented in Table 3 cor-

responds to the optimal values of Glu0, Dw and

Ds. The results obtained during the search by GA have shown process productivity of 23 kg/(m³.h). The productivity simulated with the chosen kinetic models and solvent extraction is about three times higher than the productivity obtained by the conventional ethanol production process.

Conclusions The process of extractive fermentation for Fig. 6. RSA of Ds versus Glu0 using Ghose and ethanol production was modeled applying two dif- -1 Thiagy’s model–Dw=0.2 (h ). ferent kinetics hypotheses. The model considers the thermodynamics of liquid–liquid extraction as sim- ply as possible, utilizing the distribution coefficient

17 Table 3. Optimal parameter values estimated by GA for Ghose and Thyagi’s model

Optimal parameter values estimated by GA for Ghose and Thyagi’s model Productivity Variable Glu D D 0 w s kg/(m³.h) Model 337 0.169 1.11 21.84 as a constant. The microbial kinetics of the Ghose growth and product formation in batch processes and Thyagi’s model can be considered practically were investigated. The developed model was a use- useful. The RSA of the system showed that the cho- ful tool for microbial kinetic experimental design sen control variables Glu0, Dw and Ds had a signif- and biochemical study of key enzymes involved in icant effect on the SFEE process. The correlation the metabolic regulation of Ruminucoccus albus between the control variables was analyzed in ac- B199. An understanding of the internal regulato- cordance with the microbial physiology theory and ry processes is important for the ruminant animal liquid-liquid extraction thermodynamics. The RSA sciences (Russell et al., 1979; Strobel, 1993; Thurs- methodology is a very helpful tool for localization ton et al., 1993, 1994), fermentation using cellulose of the space of optimal solutions. The adapted hy- and hemicellulose hydrolysates. brid GA for the optimization of the SFEE process Many theoretical models have been developed performed excellently. An optimal productivity val- to describe microbial growth on multiple substrates. ue of 21.84 kg/m³ was obtained and compared to The simple unstructured models developed by Lee the results of others. The SFEE process optimiza- et al. (1974), Tsao & Hanson (1975) and Yoon et tion results showed a three times higher productiv- al. (1977) are inadequate to explain the complex ity than that obtained by the conventional ethanol cellular regulatory process. Further contributions production process. Finally, the SFEE process is in the modeling of this system are the structural very challenging for industrial applications because models of Domach et al. (1984), Joshi & Palsson of the high reductions of the production cost. (1988), Nikolajsen et al. (1991) and Palsson and Joshi, (1987). Such models incorporate too many Modeling of Ruminucoccus albus bacterial growth parameters to be used for experimental design and on cellobiose–xylose mixture optimization of fermentation processes. The most A model was developed for mixed substrates widely accepted models of Bajpai & Ghose (1978), (cellobiose and xylose) utilization by Ruminococ- Imanaka & Aiba (1977) and Van Dedem & Moo- cus albus B199. Young (1977) were based on the lac operon theory of Jacob and Monod (1961) and included detailed Provisionally, the system was divided into mechanisms of repression and induction. two sub-systems (see Fig. 7), where phenomena taking place on molecular and population levels were analyzed and modeled. On molecular level, the model describes regulatory mechanisms of induction – repression processes, taking into consideration the available knowledge of catabolic pathways and changes of the key enzyme activity, which is considered to be a rate-controlling step for the utilization of the non-preferable substrate (xylose). On population level, the specific growth rate on mixed substrates was established as an additive function of growth rates on single substrates. Fig. 7. A scheme of the system of mixed substrate The product formation rate of Ruminococcus albus utilization. B199 could be described as partially linked with the specific growth rate or, more precisely, linked with the substrate utilization rate. By using material balances, the kinetics of mixed substrates utilization,

18 Recently, a cybernetic approach (Baloo & albus B199, the relationship between the inactive Ramkrishna, 1991a; Baloo & Ramkrishna, 1991b; repressor molecule (R), xylose and the operator can Kompala, 1982; Kompala et al., 1984; Kompala et be written as follows: al., 1986; Ramkrishna, 1982; Ramkrishna & Ram- k1 R + nXyl → R.nXyl (18) krishna, 1996; Straight & Ramkrishna, 1994; Yoon ← k et al., 1977) was applied for modeling microbial 2 (19) O+R.nXyl → O.R.nXyl growth on multiple substrates. On the other hand, ← to explain complex regulatory processes, modeling where R- is the cytoplasmic repressor; O- is should be based on the knowledge of the biochem- the operator; Xyl- is inducer; n-is the number of li- ical pathways of a particular microorganism. The gand binding sites for inducer on the repressor mol- lac operon theory of Jacob and Monod (1961) was ecule R. adapted in order to develop the induction-repres- The equilibrium constants K1 and K2 are de- sion model of cellobiose-xylose utilization by Ru- fined by: minococcus albus B199. Briefly about the theory, [R.nXyl] = (20) the “xylose” operon is assumed to consist of four K1 [R].[Xyl] [O.R.nXyl] major regions. The “R” gene codes for an inactive = (21) K2 repressor molecule. This molecule in the absence of [O].[R.nXyl] an inducer, such as xylose, is able to bind to the op- The material balances for the operator and to- erator region “O” of the operon. This binding of the tal repressor concentrations are: active repressor to the operator blocks the transcrip- [O]= [O]+ [O.R.nXyl] (22) tion process by mRNA polymerase, which begins at by assuming [R] >> [ O ] then the promoter region “P” of the operon. In this re- (23) pressed situation, the “S” gene (DNA encoding the [R] = [R]+ [R.nXyl] xylose key enzyme) is not transcribed and the re- where R.n.Xyl- is the repressor-inducer com- quired key enzyme is not formed. When there is an plex, which prevents R from binding to an operator inducer present to bind with the inactive repressor, gene where it would block the transcription of the then operator region is free and mRNA polymer- structural gene; ase travels from the promoter region throughout O.R- is the operator-repressor complex whose the operator and transcribes the “S” gene to form formation blocks mRNA polymerase formation. therequired key enzyme. The mathematical descrip- Using the low of mass action the fraction of tion of this phenomenon is based on the assumption free operator genes can be written: that a chemical equilibrium exists between the re- (24)

Inducer absent Promoter sequence Operator sequence Regulator gene Structural gene The fraction of the gene promoter occupied R P O S DNA by the catabolic repressor is expressed Imanaka & RNA polymerase Repressor Aiba (1977).

Inducer present (25)

R P O S DNA

RNA Transcription Repressor polymerase The rate of enzyme synthesis can be assumed mRNA to be proportional to the mRNA polymerase con-

Translation Inducer Inactive repressor centration in the cell, which itself is proportional to Enzyme the fraction of free operator genes. Hence,

(26) pressor molecule, inducer and the operator region. An induction-repression mechanism was successfully used for description of yeast growth on glucose-cellobiose mixture by Bajpai & Ghose (1978). where E- is the induced key enzyme (xylose isomer- Applying this to cellobiose-xylose utilization by R. ase) concentration in repressed state; E0- is the key

19 enzyme concentration under completely de-re- Microbial growth on multiple substrates can pressed state; E/E0- depends upon the fraction of be represented by the following equation: free operons made available by the inductive action of xylose and upon the extent of catabolic repres- (29) sion of cellobiose; α- is the key enzyme synthesis th rate constant; β- is the enzyme decay rate constant The utilization of the i substrate (Si) by the Thus, the induction-repression model on the biomass (B) is assumed to be catalyzed by a key molecular level is completed and is based on the enzyme (Ei) representing the whole set of enzymes following assumption: -the interaction between the catalyzing the metabolic pathways of growth on inducer and the cytoplasmic repressor can be treat- (Si). The key enzyme required for utilization of a ed as a chemical equilibrium; -the equilibrium is given substrate (cellobiose or xylose) must be syn- established instantaneously; -the number of cyto- thesized before growth can occur on the substrate. plasmic repressor molecules is large compared to Hence, growth of R. albus B199 on cellobiose-xy- the amount of operators and it is small compared to lose can be considered to be made up of contribu- the inducer molecules. tions from growth on cellobiose and xylose involv-

The E/E0 ratio represents the degree of cata- ing catabolic repression mechanism. Therefore, bolic repression occurring in the cell on the basis of O/O , R/R interaction. When E/E is equal to 1, t t 0 (30) the key enzyme is in fully de-repressed state. For values of E/E0 less than 1, the system is proportion- ally repressed. It is assumed that there is only one enzyme that is induced, or if there is more than one, only one is produced at a slow rate (limiting step) as a result of induction and synthesis of the same (31) enzyme is catabolically repressed. Hence, the E/E0 ratio can be considered to be a function of two control parameters (xylose and cellobiose concentra- Modeling of batch growth tions). For development of a growth kinetics mod- Using the above kinetic models and the mass el, it is assumed that the rate of xylose utilization is balances, mathematical expressions describing directly proportional to the key enzyme concentra- batch process of biomass accumulation and sub- tion (xylose isomerase). strate utilization can be written as follows: It is important to note that measurements of Biomass absolute key enzyme activity (concentration) is not required, which is very important for practical (32) applications. The model requires only the initial values of the relative enzyme level (relative to the Cellobiose maximum one during balanced exponential growth (33) on xylose). Such initial value can be used to char- acterize the state of the inoculum and can be determined from a single substrate batch experiment. Xylose The specific growth rate ofR. albus B199 on xylose can be written as follows: (34)

(27) VFA - volatile fatty acids (35) Therefore, E/E0 could be rewritten as the ra- µ µo µ tio of ( xylm/ xylm), where xylm is the maximum growth rate on xylose in the presence of cellobiose Ethanol o and µ xylm is the maximum growth rate on pure xylose. (36)

(28) The process of mixed substrate utilization is fully described by equations 26, 30-36. The most

20 important information of this study is the informa- Cellobiose-Xylose ratio (1-1) tion about bacterial diauxic growth as a result of 1 two substrate utilization processes controlled by a X,[g/l] 0.9 S cell,[g/l] catabolic repression mechanism. For this reason, 0.8 S xyl,[g/l] E/E0,[ - ] the information from equations 35 and 36, describ- 0.7 ing product formation, is not under consideration in 0.6 0.5 the present work. 0.4 Materials and methods are published else- 0.3 where. More details about biochemical pathways 0.2 0.1 and assays may be found elsewhere (Russell et al., 0 1979; Thurston et al., 1993; Thurston et al., 1994). 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Time, [h] Results and Discussion Series of batch experiments with different cellobiose-xylose ratio were performed to evalu- Fig. 9. Batch growth of Ruminuccocus albus B199 ate the catabolic repression hypothesis describing on CB: Xylose ratio (1:1). Ruminococcus albus B199 growth. Experimental design details can be found elsewhere (Kroumov, Parametrical identification and simulations 1999). Experimental results show that the maxi- Experiments in tubes with different cello- mum specific growth rate of R. albus on cellobiose biose-xylose ratio were performed. In all cases, is greater than on xylose substrate. In mixed sub- 3 µ the total substrate concentration of 1 kg/m and strates experiments, the decrease in xyl depends on xylose inoculum was chosen. Figure 8 shows the the decrease in E/E0. On the other hand, the relative growth curves of 9 tubes processes and substrate enzyme activity of the key enzyme (E/E0) is a func- ratio changing from pure xylose via 15:1, 10:1, 5:1, tion of the repressor-inducer ratio, and for the R. 1:1, 1:5, 1:10, 1:15 to pure cellobiose. Each curve albus B199 strain the most dramatic repression ef- represents the average value of three parallel tests. fect is observed in the cellobiose-xylose ratio (1:5; R. albus B199 utilized cellobiose first and when its 1:10) Fig. 10 and Fig. 11. The detailed description concentration fell to the low level, the utilization of of the identification procedure can be found else- xylose took place. A maximum of the growth rate where (Kroumov, 1999). The final values of con- was observed on pure cellobiose. Diauxic growth stants used in the simulations: Kinetic parame- appeared in ratios “D” and “E” where the concen- ters-µxylm=0.358 1/h; µCBm=0.18 1/h; KCB=0.07 kg/ 3 3 tration of cellobiose was lower than that of xylose. m ; Kxyl=0.2 kg/m ; YCB=0.28 kg/kg; Yxyl=0.386 kg/ In “B” and “C” ratios there was co-utilization of kg; Induction repression parameters- K1=438 [-]; xylose. By changing the ratio the diauxic lag be- K2=14723 [-]; m=1; n=2.3; k1Rt1=1.5 [-]; k2Rt2= 2.3 came progressively suppressed. These observations [-]; α=1 [-]; β=1 [-]. were in agreement with the results of others (Bajpai The initial conditions for the experiments and & Ghose, 1978; Kompala et al., 1986) on multiple integration of the model equations were determined 3 substrates. as follows: X0=0.05 kg/m ; E/E0=1. The initial concentrations of cellobiose and xylose were deter- A,B.C,G,D,E,F- RATIO, XYL,CELL mined by the substrate ratio for the total amount of 3 2.5 carbohydrates equal to 1 kg/m .

2 A- 15:1 Figure 9 shows the simulation and experi- B-10:1 C-5:1 mental results of diauxic consumption of cellobiose 1.5 G-1:1 D-1:5 and xylose for the substrate ratio 1:1 (or concen- OD E-1:10 3 1 F-1:15 tration of 0.5 kg/m for each sugar). Cellobiose is Xylose Cellobiose consumed first and because of the strong catabolic 0.5 repression, the relative enzyme activity reaches the 0 minimum. It starts increasing when cellobiose in 0 2 4 6 8 10 12 14 Time, [hours] the reactor is exhausted and then reaches the maximum, which is about 90% of its initial value. Xy- Fig. 8. Growth of R. albus B199 on cellobiose- lose consumption rate increases as the key enzyme xylose mixture. concentration builds up. The enzyme activity falls

21 again as xylose concentration drops, which results ulation results (dash lines) and experimental data ( in a reduced consumption rate. In the growth curve, dotted line) for the three substrate ratios presented there is a clear indication of a second lag phase for in Fig. 9, 10, and 11, respectively, are a very good about 3 hours, where the overall specific growth manifestation of the chosen parametrical identifi- rate is close to zero. For R. albus B199, the duration cation procedure. The error between the model and of this lag phase is determined from the time taken experimental data was in the range of 3% - 12%. for complete de-repression of operators by xylose, Finally, all biochemical and molecular level i. e. from the values of the constants in equation 26. studies were performed on the basis of simulation Figure 10 presents the results from batch ex- results of the developed model. The analysis of sen- periments for the substrate ratio of 1:5 (cellobiose sitivity applied to equation 26 showed that the pow- concentration – 0.167 kg/m3; xylose concentration er prediction of the model was high for the different –0.833 kg/m3). In the growth curve, the duration of operational conditions. the second lag phase is about 3 – 4 hours. The mini- Cellobiose-Xylose ratio (1-10) mum of the relative enzyme activity (E/E0) is slight- X,[g/l] 1 ly higher than in the substrate ratio 1:1. After the S cell,[g/l] cellobiose consumption, the relative enzyme activ- 0.9 S xyl,[g/l] 0.8 E/E0,[ - ] ity reaches the maximum of about 98% of the fully 0.7 de-repressed state. This fact is obvious because xy- 0.6 lose concentration is higher compared to that in the 0.5 substrate ratio 1:1. The appearance of a second lag 0.4 0.3 phase in all three chosen ratios is a manifestation of 0.2 repression of xylose-consuming key enzyme syn- 0.1 thesis. The evidence that the key enzyme is induced 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 by the presence of xylose is indicated by very long Time, [h] lag periods (5-7 hours, even more!) observed when actively growing cells of inoculum are cultivated on the cellobiose medium. Fig. 11. Batch growth of R. albus B199 on Figure 11 presents data about R. albus B199 CB:Xylose ratio (1:10). growth based on the cellobiose-xylose substrate ratio 1:10 (cellobiose concentration–0.09 kg/m3, and Conclusions xylose concentration- 0.9 kg/m3). In this experi- In this work, it was shown that anaerobic ment, the catabolic repression effect is similar to the growth of ruminal bacterium Ruminococcus albus one observed at 1:5 ratio. The growth curve shows B199 on cellobiose-xylose mixture can be described a very long second lag phase. The recovery of the successfully by an induction-repression model. The relative enzyme activity is about 98.6% of the ful- developed model performs well with different cel- ly de-repressed state of the key enzyme. The sim- lobiose-xylose ratios and it is a powerful tool for different practical applications. Based on the model Cellobiose-Xylose ratio (1-5) simulations, the experimental design for biochem- 1 X,[g/l] ical and strain modification studies was performed 0.9 S cell,[g/l] with unexpected great success. 0.8 S xyl,[g/l] E/E0,[ - ] 0.7 0.6 Acknowledgements 0.5 The authors are very grateful to Dr. Maya Za- 0.4 harieva, Ph.D. for her valuable suggestions, proof- 0.3

0.2 reading and formatting of the manuscript.

0.1

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Fagundes-Klen, M., L. Vaz, M. Veit, C. Borba, E. Silva, A. Kroumov, A. (2013a). A Global Analysis of CO2 Fixation Kroumov (2007). Biosorption of the copper and cadmium System on Waste Gases Example Complex Rate Based th ions–a study through adsorption isotherms analysis. Int. J. Modeling of CO2 and SO2 speciation in water, 8 Balkan Bioautomation. 7: 23–33. Congress of Microbiology, Veliko Tarnovo 2013. Fiorentin, L., A. Módenes, F. Espinoza-Quiñones, D. Triguer- Kroumov, A. (2013b). Integral Approach For Energy Produc- os, A. Kroumov, D. Manenti, C. Borba (2015). Biosorp- tion In Connection With Global Warming Problems. Crit- tion of the reactive blue 5G dye in a fixed bed column ical Review, Third National Conference with international packed with orange bagasse: experimental and mathemat- participation, “Ecological Engineering and Environmental ical modeling. Sep. Sci. Technol. 50: 2267–2275. Protection” (EEEP 2013)

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23 garia with International Participation Tryavna, October 7th diction algorithms in fluidized bed bioreactor. In: Tenth – 10th, 2014. Congress of the Bulgarian Microbiologists with Interna- Kroumov, A. (2015). A global analysis and modeling of a sys- tional Participation-Program and Abstracts. Plovdiv. 93-

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CO2 Fixation from Flue Gas, Acta Microbiol. Bulg. 31: process optimization using hybrid genetic algorithm. VIII 61-70. Seminario de Hidrólise Enzimática de Biomassas. Marin- Kroumov, A., A. Modenes, M. Tait (2006). Development of gá. new unstructured model for simultaneous saccharifica- Kroumov, A., M. Tait, A. Módenes (2005b). Analysis of an- tion and fermentation of starch to ethanol by recombinant aerobic treatment of effluents using unstructured model- strain. Biochem. Eng. J. 28: 243-255. ing approach. In: VIII VIII Seminario de Hidrólise En- Kroumov, A., A. Vinarov (1986). Numerical algorithm for the zimática de Biomassas, 2005, Maringá. solution of the cell model equations with return stream of Kroumov, A., R. Campus, R. Schreiner (2002d). A dynamic the aerobic biosynthesis in loop bioreactor. Transact. Mos- and static analysis of ethanol production industrial pro- cow Inst. Chem. Technol. 140: 134-140. (in Russian). cess. In: Tenth Congress of the Bulgarian Microbiologists Kroumov, A., B. Wenzel, A. Módenes (2005c). Development with International Participation, 2002, Plovdiv. 80-80. of an enzymatic kinetics model of vegetable crude and Kroumova, A., G. Wagner (1995). Methods for separation used oils transesterification and esterification for biodiesel of free, short, medium, and long chain fatty acids and for production. In: VIII Seminario de Hidrólise Enzimática de their decarboxylation. Analyt. Biochem. 225: 270–276. Biomassas, 2005, Maringá. Lee, I., A. Fredrickson, H. Tsuchya (1974). Diauxic growth of Kroumov, A., D. Dimitrov, L. Gordeev (1991). Prediction of Propionibacterium shermanii. Appl. Microbiol. 28: 831- the functioning of a cell bioreactor model for purification 835. of industrial effluents. Int. J. Bioamtomation.9: 11-18. (in Módenes, A., F. Scheufele, F. Espinoza-Quiñones, P. de Russian) Souza, C. Cripa, J. dos Santos, V. Steffen, A. Kroumov. Kroumov, A., G. Gacheva, P. Pilarski (2014). Mathematical (2015). Adsorption of direct of yellow ARLE dye by ac- analysis and experimental study of photobioreactor design tivated carbon of shell of coconut palm: Kinetics, equi- th system for CO2 mitigation from biogas, 13 Congress of librium and mechanism study. Int. J. Bioautomation. 19: Microbiologists in Bulgaria with International Participa- 187-206. tion Tryavna, October 7th – 10th, 2014. Nikolajsen, K., J. Nielsen J. Villadsen (1991). Structured Kroumov, A., G. Gacheva, P. Pilarski, V. Beshkov (2015a). modeling of a microbial system: III. Growth on mixed

Photobioreactor System for CO2 sequestration from bio- substrates. Biotechnol. Bioeng. 38: 24-29. gas -Modeling and experimental verification. 9th Balkan Palsson, B., A. Joshi (1987). On the dynamic order of struc- Congress of Microbiology - October 22th – 24th, 2015, tured Escherichia coli growth models. Biotechnol. Bio- Thessaloniki, Greece. eng. 29: 789-792. Kroumov, A., G. Gacheva, V. Beshkov, P. Pilarski (2015b). Pistikopoulos, E., S. Stefanis (1998). Optimal solvent design Modeling of microalgal kinetics in column tubular pho- for environmental impact minimization. Comput. Chem.

tobioreactor system when used high CO2 content in waste Eng. 22: 717-733. gases, 9th Balkan Congress of Microbiology - October 22th Polakovič, M., J. Bryjak (2004). Modelling of potato starch – 24th, Thessaloniki, Greece. saccharification by an Aspergillus niger glucoamylase. Kroumov, A., I. Iliev, P. Pilarski, G. Petkov (2013). Analysis Biochem. Eng. J. 18: 57-63.

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24 eling, simulation and optimization of an extractive con- simultânea do amido em etanol, utilizando microorganis- tinuous alcoholic fermentation process, J. Chem. Technol. mos modificados geneticamente. Ciência & Engenharia. Biotechnol. 74: 176-182. 15(2): 7-12. Singh, M., T. Banerjee, A. Khanna (2005). Genetic algorithm Tait, M., A. Módenes, A. Kroumov (2006). Mathematical to estimate interaction parameters of multi-component Analysis of the Direct Bioconversion of Starch to Ethanol systems for liquid–liquid equilibria, Comput. Chem. Eng. by Recombinant Strain, 19th International Symposium-Bi- 29: 1712–1719. oprocess Systems 2006-Proceedings-BioPS’06 Sofia, Straight, J., D. Ramkrishna (1994). Cybernetic modeling and Bulgaria, 24-25 of October 2006. I.47-I.55. regulation of metabolic pathways. growth on complemen- Tait, M., E. Silva, A. Módenes, A. Kroumov (2005a). Parame- tary nutrients. Biotechnol. Prog. 10: 574-587. ters identification of biotechnological process using hybrid Strobel, H. (1993). Evidence for catabolite inhibition in reg- genetic algorithm. In: Proceedings of VIII Symposium on ulation of pentose utilization and transport in the ruminal Enzymatic Hydrolysis of Biomass, Maringa 2005. bacterium Selenomonas ruminantium. Appl. Eviron. Mi- Thurston, B., K. Dawson, H. Strobel (1993). Cellobiose ver- crobiol. 59: 40-46. sus glucose utilization by the ruminal bacterium Rumino- Tait, M., A. Módenes, A. Kroumov (2004a). Aplicação da coccus albus. Appl. Eviron. Microbiol. 59: 2631-2637. análise sistemática para a modelagem do processo de sa- Thurston, B., K. Dawson, H. Strobel (1994). Pentose utiliza- carificação e fermentação simultânea do amido em etanol, tion by the ruminal bacterium Ruminococcus albus. Appl. utilizando microorganismos modificados genéticamente. Eviron. Microbiol. 60: 1087-1092. In: I Encontro Paranaense de Engenharia e Ciência, 2004, Trigueros, D. A. Módenes, F. Espinoza-Quiñones, A. Krou- Toledo-Pr. Anais do I Encontro Paranaense de Engenharia mov (2010a). The evaluation of benzene and phenol bio- e Ciência, 2004. degradation kinetics by applying non-structured models. Tait, M., A. Módenes, A. Kroumov (2004b) Modelagem Water Sci. Technol. 61: 1289–1298. matemática do bioprocesso simultâneo de sacarificação do Trigueros, D., A. Módenes, A. Kroumov (2007). Modeling amido e fermentação para produção de etanol utilizando Biodegradation Kinetics on Benzene and Toluene and Saccharomyces cerevisiae cepa YPG-AB geneticamente Their Mixture. Int. J. Bioautomation. 7: 9-22. modoficada. In: XIII Encontro Anual de Iniciação Cientí- Tsao, G., T. Hanson (1975). Monod equation for batch cul- fica, 2004, Londrina-Pr. Anais do XIII Encontro Anual de tures with multiple exponential phases. Biotechnol. Bio- Iniciação Científica, 2004. eng. 17: 1591-1598. Tait, M., A. Módenes, A. Kroumov (2005b). Identificação dos Van Dedem, G., M. Moo-Young (1977). A model for diauxic parâmetros dos modelos cinéticos em processos biotec- growth. Biotechnol. Bioeng. 17: 1301-1312. nológicos utilizando métodos estocásticos de otimização Wang, Y., L. Achenie (2002). Computer aided solvent de- e decomposição de sistemas complexos. In: XX CRICTE sign - ethanol extractive fermentation case study. Fluid - Congresso regional de iniciação científica e tecnológica Phase Equilib. 201: 1-18. em engenharia, 2005, Foz do Iguaçu - Pr. Anais do XX Wenzel, B., M. Tait, A. Módenes, A. Kroumov (2006a). Mod- CRICTE. 1: 291-291. eling chemical kinetics of soybean oil transesterification Tait, M., A. Módenes, A. Kroumov (2005c). Development process for biodiesel production: An analysis of molar ra- of unstructured model for direct bioconversion of starch tio between alcohol and soybean oil temperature changes to ethanol by recombinant strain. Proceedings of VIII on the process conversion rate. Int. J. Bioautomation. 5: Symposium on Enzymatic Hydrolysis of Biomass, Mar- 13–22. ingá-Pr, 2005. Wenzel, B., M., M. Tait, A. Módenes, A. Kroumov (2006b). Tait, M., A. Módenes, A. Kroumov (2005d). Produção de eta- Development of a novel enzymatic kinetic model of oils nol por fermentação extrativa: modelagem matemática, transesterification and esterification for biodiesel produc- análise de superfície e otimização com algoritimo genéti- tion, 19th International Symposium-Bioprocess Systems co. In: XX CRICTE - Congresso regional de iniciação 2006-Proceedings-BioPS’06 Sofia, Bulgaria, 24-25 of científica e tecnológica em engenharia, 2005, Foz do Ig- October. I.55-I.62. uaçu. Anais do XX CRICTE. 1: 289-289. Yoon, H., G. Klinzing, H. Blanch (1977). Competition for Tait, M., A. Módenes, A. Kroumov (2005e). Análise sistemá- mixed substrates by microbial populations. Biotechnol. tica aplicada a processos de sacarificação e fermentação Bioeng. 19: 1193-1210.

25 ACTA MICROBIOLOGICA BULGARICA

Review

Paratuberculosis in Animals and Humans – an Actual Health and Economic Problem Magdalena Bonovska1, Tanya Savova2, Violeta Valcheva1, Hristo Najdenski1 1The Stephan Angeloff Institute of microbiology, Department of Infectious microbiology, Bulgarian Academy of Sciences 2 National Diagnostic and Research Veterinary Medical Institute, Sofia, Bulgaria Abstract Mycobacterium avium subspecies paratuberculosis is the etiological agent of paratuberculosis in animals (Johne’s disease) and in humans (Crohn’s disease). The review summarized the etiology, epidemiology, pathogenesis and diagnosis of the disease in domestic and wild animals, and humans as well as future challenges to diagnostics, control prebiotics promoting the growth of probiotic compounds public health concern. Special attention is paid to the enormous economic losses worldwide of affected farms associated with decreased production of milk, meat and their products, increased incidence of mastitis and infertility in animals, and higher costs of in-patient care, ambulatory care and drugs for humans. The necessity to develop a National program for rapid diagnosis, monitoring and control of paratuberculosis in Bulgaria, similar to such programs in other affected countries, is explained. In addition, this program will enable the conducting epidemiological analyses and assessment of the risk to animal and human health. Keywords: paratuberculosis, animals, humans, etiology, pathogenesis, diagnosis, control

Mycobacterium avium subspecies paratuberculosis е етиологичният причинител на пара туберкулозата при животните (болест на Джон) и хората (болест на Крон). Този обзор разглежда етиологията, епидемиологията патогенезата и диагнозата на заболяването при домашните и дивите животни и хората, както и новите предизвикателства пред диагностиката, контрола и общественото здравеопазване. Обръща се внимание на големите икономически загуби в световен мащаб, които паратуберкулозата причинява на засегнатите ферми, поради намаленото производство на мляко, месо и продукти от тях, увеличаването на маститите и безплодието при животните и разходите за лече ние на заболелите хора – медикаменти и амбулаторно обслужване. Обосновава се и необходимостта от разработване на Национална програма за бърза диагностика, мониторинг и контрол на паратуберкулозата в България, подобно на прилаганите такива в други засегнати от заболяването страни. Тази програма ще позволи провеждане на епидемиологични анализи и съвременна оценка на риска за здравето на животните и хората.

Paratuberculosis in domestic animals History and world prevalence of paratuberculosis was proved by Bang in 1906. Later, in 1907, Mc- Paratuberculosis is one of the oldest diseases Fadyean described the disease in England, and in in cattle described in 1829 in England by Johne, 1908 Vucovic reported paratuberculosis in sheep in and Frothingham in 1895. For the first time they Bosnia (Chiodini et al., 1984). Twort (1910) and discovered an acidalcohol-resistant or the first time Twort and Inggram (1913) obtained a pure bacte- they discovered an acidalcohol-resistantbacillus in rial culture and gave a general description of the the intestine of a bovine animal affected with chron- disease named “Johne’s disease” (Huhn, 1965). ic diarrhea (Johne and Frothingham, 1895). In the The bacterium was named Mycobacterium avium USA the disease was discovered in cattle in the ear- subsp. paratuberculosis (Map) at the suggestion of ly 1900s, and in New Zealand was first diagnosed Thorel et al. (1990). in 1912 (Kopecky, 1961). Infectivity of the disease Paratuberculosis is a chronic, progressively

26 developing intestinal infection mainly affecting do- developed. In Sweden, the National Program was mestic large and small ruminants (cattle, buffalo, built on the principle of “stamping out”, and all im- sheep, goats, deer, camels, lamas, mouflons), some ported animals are tested for paratuberculosis. non-ruminant animals (horses, pigs, rabbits, mar- In 2014, in Northeastern Bulgaria sever- tens, foxes, weasels, monkeys, chimpanzees) and al cases of paratuberculosis in a dairy farm were humans (Pacetti et al., 1994; Chamberlin et al., registered (Koev et al., 2015). The diseased cows 2000; Behr and Collins, 2010; Savova et al., 2013; demonstrated diarrheal syndrome, birth of non-vi- Pradenas et al., 2014; Koev et al., 2015) able calves or presence of postnatal complications, Paratuberculosis is a widespread chronic bac- severe dehydration and death. In recent years, the terial disease of ruminants in agriculturally develop- importation of cattle from other countries has in- ing and developed countries. Most affected are the creased significantly and therefore early and reli- countries with bigger production of milk and dairy able diagnosis and the epidemiological status con- products, in which 20% -80% of herds are infected cerning this zoonotic disease in our country is im- (USA, Canada, Japan, Australia, Norway, Iceland, perative. the Netherlands, Belgium, Denmark, Chile) (Her- man et al., 2005; Masalski, 2008; Salgado et al., Paratuberculosis in wild animals 2009, 2011a, 2011b; Pradenas et al., 2014). Due Paratuberculosis is a serious problem in wild to the prevalence in the world for 2004 (7-55% in and captive animals too. Jarmi first reported cases Europe, 40% US, 9-22% Australia), the disease is of paratuberculosis in antelope living in a zoo in categorized by the World Organisation for Animal 1922. In 1949, Dorofeev and Kalacheva described Health (OIE) in sheet B as a disease with serious the disease in deer (by Belchev, 1977). Before economic and health significance (OIE, 2014). OIE 1970, isolated cases of paratuberculosis were de- records show that 44% of member countries report- scribed in deer, but later many reports of the dis- ed paratuberculosis during the past decade (Ryan ease appeared. In 1973, Strogov described cases of and Campbell, 2006). paratuberculosis in reindeer in northern Russia, and In Bulgaria, Kuyumdjiev first reported the in 1979 Riemann and co-authors observed it in Cal- presence of paratuberculosis in cattle in 1950, and ifornia in sika deer (Strogov, 1973; Riemann et al., seven years later, Ivanov described it in sheep. The 1979). Temple et al. (1979) described the disease in disease was studied in more details by Belchev, be- deer, and Jessup et al. (1981) found Map in elk and ing determined as an autohtonous infection in some white-tailed deer in the US. Pacetti et al. (1994) re- areas of the country (Belchev, 1977). Nowadays, ported paratuberculosis in wild and zoo animals. In more than three decades later, there is no accurate Spain, Marco et al. (2002) found paratuberculosis data on the prevalence of paratuberculosis in ani- in fallow deer. In the Czech Republic the disease mals in the official veterinary medical statistics in is common for fallow deer, roe deer and red deer Bulgaria. The Veterinary Service in the frame of (Pavlik et al., 2000; Machackova et al., 2004). In- Bulgarian Food Safety Agency does not register the fection with Map has been described in wild and disease because of its rare manifestation and other captive deer populations in many other countries different reasons. (de Lisle et al., 1993; Power et al., 1993; Fawcett Indirect evidence of the disease may be ob- et al., 1995; Manning et al., 1998; Godfroid et al., tained by the presence of nonspecific reactions to 2000; Paolicchi et al., 2001; Alvarez et al., 2005; PPD-tuberculin during mandatory allergy tests Machackova-Kopecna et al., 2005; van Kooten et for tuberculosis in cattle and buffaloes. These real., 2006; Mackintosh et al., 2007). Cases of para- actions are often due to infection of animals with tuberculosis in red deer in the region of the Italian other non-tuberculous mycobacteria (Bachvarova Alps and Austria have been described too (Glaw- et al., 1999). The subclinical cases of the diseas- ischnig et al., 2006). Tryland and collaborators es are not tested and monitored. The purposive and (2004) carried out serological studies in Norway systematic research of large and small ruminants and showed evidence of the disease in mice, red in the country is not regulated due to absence of deer, roe deer and reindeer. a National Surveillance Program. In many Europe- Since 1980, paratuberculosis has been an an countries (Sweden, Denmark, Holland, Germa- emerging problem in deer farming with important ny, France, Poland, Hungary, Slovenia) and in the underestimated losses, mainly due to outbreaks of USA and Australia as well, National Programs for the clinical disease (Mackintosh et al., 2002). In the the control of animals and their products have been southern regions of Chile, paratuberculosis is im-

27 ported from Argentina, New Zealand and Europe, affected dairy cows a 16% decrease was observed and is widespread among deer used for hunting and in milk production between the current lactation meat production (Mereb et al., 1994; Lyle et al., and the lactation two seasons previously. In 1987, 2003; Pradenas et al., 2013). Paratuberculosis has the net cost to EU farmers with subclinical Маp in- been reported to occur in wild ruminant species, fection in their animals was estimated at £209 per including deer (Chiodini and Kruiningen, 1983), infected dairy cow (Benedictus et al., 1987). Other bison (Buergelt and Ginn, 2000) and elk (Manning economic losses come from the cost of programs et al, 2003), as well as nonruminants, such as wild undertaken by government agencies to reduce rabbits (Greig et al., 1997), their predators, includ- the value of the loss of breeding animals (Pavlik, ing foxes and stoats, and primates, such as mandrills 2006). In the USA, the economic losses due to this and macaques, indicating a wide host range (Beard infection in livestock reach $ 1.5 billion annually. et al., 2001). Due to the close relation between wild The cost of reduced productivity reaches $ 200-250 and domestic animals in some areas, it is suggest- million per year (Harris et al., 1998). Losses from ed that other wild and captive animal populations dairy farms are about $ 100 per cow in moderately could be infected, too (Salgado et al., 2011a). In- infected herds and increase to over $ 200 per cow in fections in free-ranging and captive wildlife are heavily infected herds, mainly due to the decrease also of great concern. Up to one-third of zoos ac- in milk production and increased cow replacement credited by the American Zoo and Aquarium Asso- costs (Ott et al., 1999). ciation have reported at least one culture-confirmed The control of paratuberculosis can improve case of paratuberculosis since 1995 (Manning and animal health and welfare, increasing productivi- Ziccardi, 2000). ty, reducing potential problems in the market and In Bulgaria рaratuberculosis in wild animals improving the overall business profitability. The is not well investigated. In the period 2011-2013, 50 benefits that can be derived from the control of samples of shot and dead deer were studied, hinds paratuberculosis should be communicated to all and mouflons, grown freely in a hunting area in stakeholders in the industry to encourage the devel- Bulgaria. Histological and microbiological findings opment and implementation of control programs. typical of paratuberculosis were established in 7 of them and were confirmed by a molecular genetic Etiology and pathogenesis analysis (Savova et al., 2013). These results show The causative agent of paratuberculosis, My- the need of a systematic study on the prevalence of cobacterium avium ssp. paratuberculosis (Map), is this disease in wild animals in their natural habi- one of the four members of the Mycobacterium avi- tat. The increase of paratuberculosis in wild animal um complex (MAC), known also as M. avium intraspecies is assumed to be caused by the purchase of cellulare complex (MAIC) - M. avium ssp. avium; animals, a strong increase in suckler cow farming M. avium ssp. silvaticum, M. avium ssp. paratuber- (cow-calf herds) with a concentration of pathogens culosis and M. avium ssp. hominissuis (Inderlied, in the environment. 1993). On the basis of growth characteristics and different molecular typing, Map strains are classi- Economic losses and control of paratuberculosis fied into three types (I, II and III). Strains of type Paratuberculosis is an important disease with I and III share similar phenotypic traits and have considerable economic consequences. The disease been isolated mainly from sheep and goats, and causes significant financial losses through decreased rarely from wildlife (S’ type). Type II strains were milk production in dairy cattle and meat yield from primarily isolated from cattle but also from small slaughtered animals (around 59-60 kg less), re- ruminants, different wildlife species (predominant- duced calf production from both dairy and beef cat- ly in wild cervids) and humans (C’ type). They are tle, death of the deseased cows and increased veter- the most common in Europe. inary and related medical costs. During the course The bacteria are acid-fast, aerobic, immobile, of the disease, the absorption of nutrients in normal non-spore or capsule forming bacilli (1-2 µm by 0,5 food intake decreases and animals progressively µm). In Ziel-Neelsen (ZN) stained smears short, lose weight (Pavlik, 2006). Besides losses from ex- pink or red rods in clumps or single are observed haustion and death, other reasons for financial costs (Fig. 1a). Map is a very slow-growing organism, are a significant increase in mastitis, infertility, sus- demanding the presence of iron (mycobactin) in ceptibility to other infections and disposal of the culture media for in vitro growth and cultivation infected animals (Merkel et al., 1975). In clinically (Collins, 1996). The appearance of colonies (Fig.

28 lins, 2001). Live bacteria could still be isolated after pasteurization of milk (Herman et al., 2005; Mun- do et al., 2013). Experiments with Map contaminated milk products (yoghurt and fermented milk products containing probiotic cultures) stored for 6 weeks at 6°C showed that Map counts in yoghurt did not change substantially, while in fermented milk products containing probiotic cultures, Map numbers decreased but live cultures were still isolated (Van Brandt et al., 2011). After ingestion, Map enters the intestinal tis- Fig. 1a. Light microscopy of ZN-stained sue through the M-cells in the Peyers patches of the smear (1000X) small intestine. Regional enterocytes may also be involved in the uptake of the bacterium. The bacterial cells are then phagocytosed by resident sub- epithelial macrophages or dendritic cells, in which they multiply. Map is able to survive within the macrophages by inhibiting phagosome acidifica- tion and phagolysosomal fusion. This is a crucial process in the establishment and evolution of the disease. With disease progression, infected macrophages migrate into local lymphatics, resulting in bacterial spread to mesenterial lymph nodes. The infection may be disseminated, with mycobacteria detectable in extra-intestinal lymph nodes (mammary, pulmonary, hepatic and head) and associated Fig.1b. Bacterial growth of M. paratuberculosis organs. on solid Herold‘s medium with Mycobactin J Paratuberculosis refers to “open” infections with large excretion of the causative agent 1b) usually occurs 12–16 to 24 weeks when Map is in the environment (Kruze et al., 2006). The ani- isolated from cattle, and up to 9 months when iso- mals secrete mycobacteria with faeces, semen and lated from sheep (Belchev, 1977; Masalski, 2008). breast milk without showing clinical symptoms The slow growth rate may be due to the presence (Sweeney, 1996). The bacteria can also enter the of a thick, waxy and complex cell wall, primarily bloodflow after intestinal disorders (Harris, 1998). composed of mycolic acids and arabinose deriva- Drinking water, where the paratuberculous bacteria tives. can survive for 7-9 months, has an important role Like other mycobacteria, Map possesses a in the transmission of the infection. Healthy herds lipid-rich cell wall, thus resulting in acid fastness, are infected by sharing pastures and water sources hydrophobicity and increased resistance to chem- with sick animals (Belchev, 1977). The disease has icals and chlorine. Paratuberculous mycobacteria a zonal character. Predisposing factors are acidic are also resistant to drying, low temperature and soils and soils deprived of micronutrients and ma- decay. In water they survive 3-7 months, and in cronutrients, which provide a suitable environment the soil from 3 months to 5 years (Watando, 2001). for paratuberculous mycobacteria. Bacteria are isolated from infected intestine, stored Subsequent to infection, Map is initially con- for 18 months in a refrigerator at 4°C and after one trolled by a T-helper (Thl) response with IFN-y year storage at a freezing temperature (-14ºC). UV production. The progression of the disease from a rays kill bacteria for 100 hours, but in boiling water subclinical to a clinical state is associated with a they die after 2 min (Belchev, 1977; Hruska and switch from a Thl to a Th2 immune response, lead- Kaevska, 2012). In naturally contaminated with ing to antibody production that is less effective in Map bovine faeces exposed to a variety of natural controlling infection. conditions as freezing, drying, sunlight, high and Young animals (cattle, buffaloes, sheep and low temperatures and rain, bacteria have been iso- goats) are thought to be most susceptible to natu- lated between 152 and 246 days (Manning and Col- ral infection with Map by close contact with con-

29 taminated faeces, feed, water, colostrum and milk have proved the transmission of the infectious agent or indirect contact with infected animals (Pavlik et from deer to cattle. In wildlife, paratuberculosis is al., 2010). Carnivores may be infected by eating an running subclinically being characterized by low infected prey species. Contaminated colostrum and mortality, due to the relatively low virulence of my- milk produced by infected dams is a major route cobacteria (Jessup and Williams, 1999). of transmuission for neonates. Transmission occurs Infection is not always accompanied by a dis- mainly by the horizontal faecal-oral route, or ver- ease. The organism of the infected animals may de- tically through the placenta to the fetus or through velop cell mediated immunity and remain healthy. infected semen (Larson and Kopecky, 1970; Chiod- The animal can get rid of the infection or remain ini et al., 1984; OIE, 2014). The intrauterine trans- a carrier for long period of time. This means that mission, although less prevalent, is more common the greater the amount of mycobacteria , the more in farmer deer than in cattle or sheep. likely it is for infection to occur. Judge et al. (2006) investigated routes of intra-species transmission of Map in rabbits through Clinical symptoms and phatomorphological random selection of the rabbit population. Four chances hundred and eighty-seven rabbits were sampled Johne’s disease is a chronic infectious inflam- from two sites in Scotland, where Map had previ- mation of the intestinal tract of ruminants (paratu- ously been isolated from the livestock and rabbit berculous enteritis). The subclinical form of infec- populations (Greig et al., 1997; Greig et al., 1999). tion is most commonly found in the affected popu- No pathology was noted in any animal, but Map lations. The disease remains hidden, but animals are was isolated from the testes, uterus, placenta, fetus- carriers and emitters of the infectious agent. This es, and milk in rabbits at both sites. The obtained makes them a potentially infectious threat to the results provide evidence for the potential transmis- rest of the herd. In cows with subclinical mastitis, a sion of Map in rabbit populations via vertical (trans- reduction in mammary secretion and infertility can placental), pseudovertical (ingestion of contaminat- be observed (Iliev, 1972; Belchev, 1977; Jungersen, ed milk or feces) and horizontal routes. Vertical and 2002). These animals show reduced effectiveness pseudo-vertical transmission occurred in 14% of in the utilization of the feed. offspring entering the population at 1 month of age The typical clinical manifestations in sick an- (Judge et al., 2006). imals are progressive afebrile weight loss and re- The presence of these routes of transmission duced food intake with preserved appetite, chronic within natural rabbit populations will contribute persistent greenish diarrhea, diffuse intermandibular to the maintenance of Map infections within such oedemas and eventual death (Machackova-Kopec- populations and, therefore, the environment. These na et al., 2005). Death occurs as a result of cachexia observations support the assumption that rabbits and dehydration of the organism (Suárez, 2006). In are a significant true reservoir and source of the contrast to cattle, diarrhoea is not a feature in small Map livestock. Therefore, it is essential that rabbits ruminants. This is probably due to their greater abil- should be included in the management and control ity to reabsorb water in the large intestine, though strategies of paratuberculosis in farmed ruminants. in advanced cases the faeces may become soft and In the epidemiology of Map infection, re- unformed (Machackova-Kopecna et al., 2005). cently the possible role of synanthropic rodents has Chiodini et al., (1984) reported that animals with been reported. Ruminants could be infected by sick clinical form of infection excreted up to 1010 CFU/g rodents through contaminated faeces, food or water in the faeces. Actually, animals contribute infec- (Beard et al., 2001). M. paratuberculosis has been tious material to a common environment and this isolated from internal organs and feces of asympto- environment serves as an indirect source of trans- matic rodents, e.g. from the brown rat and common mission The dominant transmission strategy for vole (Durnez et al., 2008; Kopecna et al., 2008) Map is not well known, and the distinction between Studies of authors reporting the relationship the two transmission pathways, direct or indirect, of the disease between wild and domestic animals remains poorly elucidated (Heuer et al., 2012). This have aroused the interest of veterinarians, foresters, year, Slater et al. used the term “super-shedders” ecologists, etc. In their view, wild animals can be- for infectious individuals that contribute a dispro- come infected from domestic ruminants in jointly portionate amount of infectious pathogen load to inhabited regions (Riemann et al., 1979; Marco et the environment. A super-shedder host may pro- al., 2002). Chiodini and Van Kruiningen (1983) duce up to 10 000 times more pathogens than other

30 infectious hosts. Moreover, the authors showed that bowel is folded like cerebral cortex (Figure 4). the effect of super-shedders for Map is limited and In sick animals enlarged mesenteric lymph that the effect of the individual bacterial load is lim- nodes and dilation of mesenteric lymphatic vessels ited and the relationship between bacterial load and were observed (Iliev, 1977; Savova et al., 2013; the infectiousness is highly concave. A 1000-fold Pradenas et al., 2014; Koev et al., 2015). In the mu- increase in the bacterial contribution is equivalent cosa of the small intestine, including the intestinal to up to a 2–3 fold increase in infectiousness (Slater villi and propria, a proliferative inflammation is es- et al., 2016). Paratuberculosis is characterized by tablished by histological examination. Granulomas an extended incubation period. The disease symp- without caseosation and presence of macrophages toms do not occur in animals before 3-5 years, and with foamy cytoplasm and large number of epithe- even at a more mature age (Stabel, 2004). lioid cells were observed. The giant cells of Lang- The main pathological changes affect the in- hans-type are аbsent or single cells could be found testine, lymph and lymphoid tissues. In advanced (Pradenas et al., 2014). clinical cases a gelatinous atrophy of fat stores and serous effusions in body cavities are observed. In Diagnosis of paratuberculosis sheep and goats with paratuberculosis major chang- Diagnosis of paratuberculosis, especially es are often lacking or difficult to detect (Clarke, subclinical cases, continues to be a challenge in 1997). In cows, postmortem, mostly affected are animals and humans. The diagnosis in sick rumi- the small intestine and mainly the ileum (Figure nants is based on Map detection in faeces and milk 3), but changes may be detected in the jejunum and as well as patho-anatomical and histopathological colon. They are thickened, soft to the touch, hyper- examination of tissues originating from the lower emic, and in advanced disease the surface of the part of the jejunum, ileum, ileocecal valve and the associated lymph nodes (Clarke, 1997; Kruze et al., 2013). Diagnosis of the disease is a difficult, long and labour-consuming process that depends on the stage of development of the disease and the extent of its spread in the herd. It is hampered by the lack of single diagnostic methods that can be used to detect both forms of the disease - subclinical and clinical. The histopathological observation of lesions is insufficient to confirm the disease, especially due to the similarity of lesions caused by Map and M. avium (Maa). Co-infection with Map and Maa, which causes clinical disease and possibly death, compli- cates bacteriological diagnosis of mycobacterial Fig. 3. Highly pleated small intestine of mouflon infections (Godfroid et al., 2005). Furthermore, lesions detected during the slaughter of animals subclinically infected with paratuberculosis, may lead to problems because their lesions were similar to those of tuberculous animals (Campbell, 1995). The degree of reliability of the methods used largely depends on the stage of infection in infected herds. Diagnostic techniques of paratuberculosis aimed, on the one hand, to carry out screening and monitoring of herds for the presence of disease and the degree of infection, and on the other, to determine the individual status of the infected animals. The diagnosis of paratuberculosis in the wild is hampered by the low virulence of Map, chronic or subclinical course of the disease and the lack Fig.4. Intestinal tract of deer pleated as „cerebral of effective diagnostic tests (Jessup and Williams, cortex“ 1999). Therefore, the authors focus on prevention of the disease, the aim being to reduce animal den-

31 sity and to limit their transportation (Marco et al, Dickinson) and Bact (Organon Technique). The in- 2002). For the diagnosis of Map infection in live troduction of these rapid techniques for isolation of animals two approaches are used: Map (radiometric and fluorescence based methods) - direct isolation of the bacterial causative from fecal samples, create serious problems due to agent from fecal and milk samples cultivated on a overgrowth of other bacteria (spore forms and fun- differentiating nutrient media, and in recent years gi) and are currently unenforceable (Collins et al., accomplished by PCR; 1990) - indirect diagnosis by determining the im- Molecular techniques have been very valu- munological status of the animals through tubercu- able for diagnosis and epidemiological investiga- linization and serological testing with ELISA and tions and have overturned many epidemiological gamma interferon test. assumptions. The technique of the polymerase chain reaction (PCR) may be used to identify spe- Detection of the causative agent cific mycobacteria in faeces or tissues and also to Direct microscopy of ZN-stained smears from confirm identification of colonies. The PCR test faeces, intestinal mucosa or lymph nodes is a rapid, offers an attractive alternative for the tradition- economical way for obtaining a diagnosis. The di- al culture methods. It has higher sensitivity and agnosis depends on the presence of short, red-stain- yields faster results from the culture. PCR is not ing rods clustered in clumps bacteria, showing in- only a more rapid test (1-2 days as opposed to six tracellular growth, which is typical of Map. The months), but it can also offer high levels of specific- presence of predominantly single acid-fast bacilli is ity. Today, various Map-specific insertion sequenc- unconvincing as they can be environmental myco- es (IS900, IS901, IS1245, f57, ISMap2, ISMap04) bacteria. The test has a low sensitivity in the early linked to avian virulence and applicable for detec- stages of the disease, but when the animal reaches tion and identification of Map by PCR have been the clinical stage, the test reaches virtually 100% revealed. Of these, IS900 is widely used as target sensitivity. for PCR detection of Map. This insertion sequence The isolation of a Map culture from faeces is specific for the bacterium and is present in 14 to during the progress of disease is the most sensitive 18 copies in the genome. The presence of IS900- test with 100% specificity and is therefore regarded like sequences in non-Map mycobacteria shows as the “gold standard”. However, the test is very the need to verify the result of DNA sequencing. In labour-intensive, time consuming and expensive. It order to improve the specificity and to make differ- is not standardized and varies considerably in dif- entiation between single and mixed mycobacterial ferent qualified laboratories. The decontamination infections, a successful multiplex PCR has been procedure for the tested samples, especially faeces, developed (Moss et al., 1991; Ritacco et al., 1998; is an essential step in all used methods. Cultiva- Stabel et al., 2004). tion of bacteria may take several months. In order to complete successfully the elimination of the fast Detection of an immune response to M. growing bacteria, a crucial step is the effectiveness paratuberculosis of decontamination. At present there are two basic The diagnosis of subclinical paratuberculosis methods in use for culturing Map from clinical spec- presents an important point in the control of this dis- imens: the method using oxalic acid and NaOH for ease. Immunologic assays such as complement-fix- decontamination and Löwenstein-Jenssen medium ation test (CFT), enzyme-linked immunosorbent for growth, and the method using hexadecylpyrid- assay (EL1SA) and agar gel immunodiffusion as- inium (HPC) for decontamination in combination say (AGID) are used to diagnose paratuberculosis with Herrold’s Egg Yolk Medium (HEYM). Both in a herd. All these methods rely on the presence media contain mycobactin. Colonies can be ob- of antibodies to M. paratuberculosis in the blood served after 4 to 24 weeks or more. serum. Because subclinical infected animals usual- A faster method for isolation of Map is by ly do not produce measurable antibody titers to the using the radiometric system BACTEC 460. The final stages of the disease, these tests are ineffective growth of mycobacteria is measured by the release in detecting subclinical infection in a herd. 14 of CO2 from palmitate in bacterial metabolism The complement fixation test (CFT) is the (Collins et al., 1990). The authors also introduced most widely used standard test for paratuberculosis three other rapid methods based on fluorescence - in cattle. The test has sufficiently high sensitivity BACTEC 9000, and both systems MGIT (Becton for detecting the advanced clinical cases, but is not

32 a completely reliable tool for the identification of in different animals (cattle, sheep, goats and deer). less infected animals. Despite the fact that the out- However, the used antigens are not specific forMap come of the test gives no guarantee about the status (Shermann et al., 1984). The relatively low cost of of the individual animal, the CFT test is often the the AGID make this test very attractive for use in only valuable test requested for import and export small ruminants (sheep and goats). Its sensitivity is of cattle within the EU (Ridge et al., 1991; Sockett lower than the ELISA test, being also closely re- et al., 1992; Yokomizo et al., 1991). lated to the immunopathological forms of the in- ELISA is the preferred test for herd analysis fection. Sheep and goats with focal and tuberculoid because of the convenience of sample collection, forms (non-clinical infections) are usually negative rapid laboratory turnaround time and low cost of the (Garcia Marin et al., 1993). Clinical cases of sheep test. The serum antibodies against Map are detect- and goats with numerous bacterial forms are posi- ed using different crude fractions of the bacterium; tive for AGID as it must be borne in mind that those lipoarabinomannan (LAM) or PPD’s. False positive which show lymphocyte forms are typically nega- results can occur as a result of cross reactions with tive (Pérez et al., 1997). related bacteria as Corynebacterium, Mycobacteri- In general, the sensitivity of the AGID test is um and Nocardia, or in countries where bovine tu- variable, depending on the group of investigated an- berculosis is endemic. The occurrence of false pos- imals (Nyange, 1991). Like the ELISA, the AGID itives is therefore also geographically dependant. A is based on the detection of antibodies against Map. problem in accounting of the ELISA test may occur Therefore, it has a relatively low sensitivity in the in the use of vaccination against paratuberculosis early stages of the disease, but becomes more use- in cattle. The specificity of the test is increased in ful in later stages. the so-called absorbed ELISA by absorption with sonicates from environmental mycobacteria, like The optimal diagnosis of paratuberculosis re- M. phlei or M. vaccae. Since the commercial ab- quired a combination of T-cells (for early or sub- sorbed ELISA kits became available, it has become clinical cases of Map infection) and antibody tests the most widely used serological test for diagno- (for chronic clinical cases) in order to identify both sis of paratuberculosis in cattle. To avoid receiving early and late cases. Cellular immunity tests can be ELISA false positive results, it Map specific recom- performed in vivo or in vitro, using the intradermal binant antigens can be used. test with PPD or johnin (the paratuberculosis equiv- Reports on the sensitivity of the absorbed alent) or the gamma-interferon assay, respectively. ELISA are contradictory (Collins et al., 1991; Ridge The cellular response is less pronounced than in the et al., 1991; Sweeney et al., 1995). More reliable is case of bovine tuberculosis, where the intradermal the sensitivity of the test when ELISA is used in test is still the most reliable test. In the intradermal a herd or group of animals, where the disease has test, skin thickness is measured before and 72 hours existed for a longer period of time because the se- after injection with PPD. An increase in thickness rum antibodies are increased in the later stages of of more than 2 mm is regarded as a positive reac- the disease. The test showed very low sensitivity tion. in young, recently infected animals and therefore The gamma-interferon assay is performed ELISA can be used to detect specific antibodies by stimulating aliquots of heparinised blood with in farms where the disease has started spreading PPD as the subsequent release of gama-interferon recently. Therefore, the test will be useful in pro- is measured by ELISA (Wood et al., 1991). Sensi- grams for certification of flocks where a sufficient tisation to avian-PPD or johnin is very common in number or only older animals are tested. animals because they contain many cross-reacting Diagnosis of paratuberculosis is usually antigens. This interferes with the specificity of both based on the combined results of faecal culture and tests and makes interpretation of the results more ELISA (Sweeney et al., 1995). Both tests have their difficult (Billman et al., 1992). Moreover, there is drawbacks: bacterial culturing is a slow, laborious a correlation between the positivity to cellular tests and expensive process, and the sensitivity of ELI- and animals showing focal-tuberculoid pathologi- SA is not sufficiently clarified, particularly for the cal forms. These are non-clinical and non-shedding final stage of eradication. animals (Perez et al., 1994). This results in a de- The agar gel immuno-diffusion test (AGID) creased sensitivity of the tests and reduces their val- can be used for the detection of paratuberculosis ue compared to the much cheaper ELISA.

33 Treatment and vaccination against Paratuberculosis in humans paratuberculosis History Drug treatment of paratuberculosis in animals Crohn’s disease (CD) is an inflammation of is not recommended. Vaccination in cattle was first the gastrointestinal tract (from the mouth to the introduced in 1926 by Vallee and Rinjard. The vac- anus) in humans, causing a wide range of symptoms cination was performed by subcutaneous injection such as general malaise, abdominal pain, diarrhea, of living, unattenuated paratuberculosis bacilli and vomiting, chronic weight loss, skin rashes (Chiod- was administered at 1 to 30 days of age, (subcuta- ini, 1989). OIE and WHO reported the prevalence neously) in the brisket, where the vaccinal nodule is of the disease not only among domestic and wild less obtrusive. The vaccines were used in other spe- animals in various countries of the world including cies, mainly sheep involving subcutaneous admin- Europe but also increasing cases of human disease istration in both young (15 days to several months (Suárez, 2006; OIE, 2014). old) and adult animals (Sigurdsson, 1960; Benedic- A possible relationship between Map and tus et al., 1987; Perez et al., 1995; Garcia Marin et Crohn’s disease was reported in 1913 by Dalziel al., 1997). Revaccination is not recommended. in Glasgow who first noticed similarities between According to the guidelines of the specifica- general pathology and symptoms of John’s disease in cattle and Crohn’s disease in humans. In 1904 tions of the OIE, currently the World Organisation the disease was described by the Polish surgeon for Animal Health (WOAH), vaccines may contain Antoni Leśniowski. Kron, Gainsbourg and Oppen- live attenuated or killed bacteria incorporated with heimer described in 1932 in Mount Sinai Hospital an adjuvant or freeze dried and adjuvanted on re- in New York eight cases of regional ileitis, and gave constitution bacteria (OIE Paris, 1996). The use the name Crohn’s disease (Crohn et al., 1932). Lat- of vaccination is still under debate. Following the er on, the name of regional ileitis was replaced by opinion of some authors, vaccination is the best regional enteritis when Lockhart-Mummery and way to control the infection, especially in sheep Morson, in 1960, described the bowel disease and and goats, though others conclude that vaccination accepted that the disease is not restricted to the il- alone is not sufficient for control of paratubercu- eum. Subsequently, that entity was called by the losis (Sigurdsson, 1960; Marly et al., 1988; OIE USA clinician granulomatous colitis. Together with

Paris, 1996; Garcia Marin et al., 1997). The sensi- ulcerative colitis and unclassified chronic colitis, tisation caused by vaccination not only affects the Crohn’s disease belongs to a spectrum of diseases diagnosis of paratuberculosis, but vaccinated ani- more generally designated as “Chronic inflamma- mals become also sensitised to bovine PPD used in tory bowel diseases”. the intradermal test for control and eradication of bovine tuberculosis (Stuart, 1965) Theories of Crohn’s disease After isolation of Map from blood culture Prevention and control of paratuberculosis of a patient, it was assumed that Map (the causa- The control of the disease is related to diag- tive agent of paratuberculosis in animals) is also nostic tests, combined with sanitation programs, one of the etiological factors in the human disease and elimination of infected animals. The treatment (Crohn’s disease) (Golan et al., 2009). Although of animals is unprofitable and is not recommend- epidemiological studies are absent, the data of sev- ed. In Bulgaria and in other countries, the main ap- eral authors show the relationship between these proach is the eradication of the disease, reducing two diseases (Thompson, 1994; Suárez, 2006; Behr Map positive herds and preventing the spread of and Collins, 2010; Hermon-Taylor, 2009). This de- infection to healthy flocks. termines the zoonotic nature of the disease (Sha- The best prevention is achieved by reducing fran, 2008; Golan et al., 2009). the overpopulation of animals, providing good ven- The cause of Crohn’s disease remains still tilation in barns, regular disinfection of premises, unknown, although some authors claim that in hu- proper nutrition providing the necessary minerals mans it is an autoimmune disease (Loftus, 2002; and vitamins, and timely elimination and eradica- Segal, 2006). Now it is generally believed that there tion of infected animals from the herd (Belchev, is a complex multifactorial etiology, including ge- 1977; Suárez, 2007). netic predisposition, environmental factors (infectious agent, diet or smoking), and an abnormal inflammatory response.

34 Irrespective of the etiologic agent, several initially unclassified Mycobacterium sp., identified theories of disease mechanisms in Crohn’s disease later as Map, by culturing specimens from resected are currently under consideration: 1) a persistent ileum of three young patients with Crohn’s disease infection, possibly involving mycobacteria (spe- (McFadden et al., 1987). The obtained three strains cifically Map), the measles virus, Listeria spp., Es- were cultured 18 months for primary isolation and cherichia coli; 2) a defective mucosal barrier (leaky identified as Linda, Ben and Dominic. Later the au- gut) which allows uptake of bacterial, dietary and thors reported that all the three strains were isolated other immunogenic macromolecules; 3) dysregula- initially as nonacid-fast coccobacillary forms that tion of the host immune response with loss of toler- had ultrastructual appearance of spheroplast (cell ance, aggressive cellular activations and disorders wall deficient forms), which, after several months of apoptosis; 4) genetic susceptibility factors; and of incubation, were transformed into characteristic 5) a combination of some or all of these reasons Map-like organisms (Chiodini et al., 1986). This (Sartor, 1997, 2003; Shanahan, 2002). would explain why acid-fast cells were not readily observed in sections of Crohn’s tissue by histolog- Epidemiology of Crohn’s disease ical examination. Since the reported initial isola- The disease is more prevalent in Western pop- tions by Chiodini et al. (1986), Map has been suc- ulations with northern European and Anglo-Saxon cessfully cultured from resected tissue of Crohn’s ethnic derivation than in populations of Southern diseased patients in various parts of the world Europe, Asia and Africa (Jayanthi et al., 1992). (USA, UK, The Netherlands, Australia, France, the The incidence of Crohn’s disease is around Czech Republic, etc.), and also from breast milk of 13 people per 100,000 of the population in the UK. two lactating Crohn’s patients in Florida (Naser et Similar incidence rates are reported for other coun- al., 2000). With the MGIT culture system (Becton tries in Europe (Shivananda et al., 1996). All this Dickinson) in the late 1990s, isolation rates of Map evidence suggests that the incidence of CD is in- from resected tissue or biopsies from Crohn’s pa- creasing worldwide. Every year around 3,000 new tients have improved significantly: 86% of resected cases of Crohn’s disease are diagnosed in the UK. tissues and between 20 and 42% of biopsies from Crohn’s disease typically affects young people be- Crohn’s patients, compared with 5-6 and 9% of bi- tween the ages of 16 and 25, although it can also opsies from control patients positive for Map, but occur in early childhood or later. Most patients with tested by culture (Schwartz et al., 2000; Bull et al., Crohn’s disease have a chronic, intermittent disease 2003). Naser et al. (2004) cultured for the first time course and treatment is multifaceted, including a viable Mар from peripheral blood in 14 out of 28 anti-diarrhoeals and anti-inflammatory agents to (50%) patients with Crohn’s disease, in 2 out of 9 treat symptoms, immunosuppressive drugs aimed patients (22%) with ulcerative colitis, and in 9 out at disease remission, and surgery to remove dis- of 15 individuals without inflammatory bowel diseased bowel or alleviate complications such as ob- ease. In a previous study the same research group structions, stricture or fistula formation (Podolsky, reported the first isolation of viableMар from suck- 2002). ling mother (Nasser et al., 2000). The World Congress of Gastroenterology in Based on these data, some researchers con- Vienna in 1998 categorized patients with Crohn’s clude that Mар may be the causative agent of disease into 24 possible subgroups according to the Crohn’s disease. Various professional groups, such disease location in the gastrointestinal tract, behav- as farmers, veterinarians and slaughterhouse work- iour of disease and age at diagnosis (Gasche et al., ers may be affected, although high incidence of 2000). Patients with Crohn’s disease are a heteroge- Crohn’s disease was not reported among these pro- neous group and the aetiology may not be the same fessional groups. The question of whether Maр is for all patients. important for public health as a cause of disease in humans has been reviewed by a number of expert Aetiology of disease groups in recent years, regardless of detected cases Crohn’s disease is most commonly seen in of Crohn’s disease. The opinion of many еxperts is the distal ileum, but can affect any part of the gas- that conclusive data proving a causal relationship trointestinal tract. Map has been sporadically iso- between Maр and Crohn’s disease are not available lated from humans with Crohn’s disease of resected at present. tissue specimens after incubation for very long pe- In case that Maр takes part in the pathogen- riods (>1 year). Chiodini et al. (1984) first isolated esis of Crohn’s disease, it could be assumed that

35 the infection is related with consumption of food viable Map spiked into water (Whan et al., 2001). or water. The most likely means of transmission of Map from cattle to humans are milk (and poten- Pathology tially other dairy products), beef and water. Map The earliest macroscopic lesions of Crohn’s is cultivated from the milk of cows with clinical disease appear to be tiny focal “aphthoid” ulcera- and subclinical John’s disease (Taylor et al., 1981; tions of the mucosa, usually with underlying nod- Sweeney et al., 1992; Streeter et al., 1995). It can ules of lymphoid tissue. Sometimes these lesions also occur in the milk of other ruminants such as regress; in other cases, the inflammatory process sheep and goats affected by John’s disease. Most progresses and involves all layers of the intestinal people consume raw cow’s milk from a young age wall, which becomes greatly thickened. Histologi- and in this way cow’s milk has been recognized as cally, changes are most marked in the submucosa, a means of transmission of Map from cattle to hu- with lymphoedema and lymphocytic infiltration oc- mans. As most cow’s milk is pasteurized prior to curring first, and extensive fibrosis later. Patchy ul- consumption, there is considerable interest in de- cerations develop on the mucosa. The combination termining the effectiveness of milk pasteurization of longitudinal and transverse ulcers with interven- with regard to Map. Studies report that Map is more ing mucosal oedema frequently creates a character- heat-resistant than other mycobacteria (including istic “cobblestone” appearance. M. bovis) and that occasionally low numbers of via- Segments of the diseased bowel are charac- ble Map may survive after milk pasteurization. The teristically sharply demarcated from adjacent nor- ability of Map to survive pasteurization, alows the mal bowel – hence the name “regional” enteritis. microorganism to be viable in certain dairy prod- Segmental lesions could be separated by normal ucts. For example, many cheeses are made from areas (skip lesions). With reference to disease dis- raw or minimally heat-treated cows’, sheep’s and tribution in the gut, a recent European collaborative goats’ milk. A recent study found that the number study on 125 cases has shown that the ileum alone of Map cells increased 10-fold during production of is involved in about 10% of cases (ileitis); both the cheddar cheese and Map remained culturable for 27 ileum and colon are affected in about 60% (ileocol- weeks of the period of cheese ripening (Donaghy et itis); and the colon alone is involved in about 30% al., 2004). (granulomatous colitis). Occasionally, the entire Johne’s disease affects dairy and beef cattle, small bowel (jejunoileitis) is involved, and rarely and theoretically, meat could also be a potential also the stomach, duodenum, or oesophagus. food transmitting Map to humans. It is supposed While granulomas when present are helpful that meat from old dairy cows, which are used for in distinguishing Crohn’s disease from other forms ground meat or beef for human consumption, may of inflammatory bowel diseases, it is the chronic represent a source of Map infection for consumers inflammation involving all layers of the intestinal (Manning and Collins, 2001). It is hypothesized that wall that is most characteristic of Crohn’s disease. when ground beef is obtained from animals with Both lymphocytes and macrophages are present in localized infections, for example in a lymph node, the granulomas and the cellular infiltrate. Maр can spread over the entire batch of minced The attached mesentery is thickened and beef. Water may also represent a potential means lymphoedematous; mesenteric fat typically extends for transmitting Map to humans. Everyone drinks onto the serosal surface of the bowel. Mesenteric water in its natural state or as an ingredient in other lymph nodes are often enlarged. The transmural drinks. Water is used in the production and process- inflammation, deep ulceration, oedema, and fibro- ing of food, so it can reach consumers in this way. sis are responsible for obstruction, and deep sinus Surface water contaminated with effluent from the tracts and fistulas, as well as mesenteric abscesses pasture grazed by animals with Johne’s disease, can are the major local complications. theoretically enter water supplies that are used for the supply of drinking water. The efficiency with Control and public health concern which water treatments, such as sand filtration and Althogh not currently classified as zoonot- chlorination, remove or inactivate Map present in ic agent, Map is suspected to be involved in the water destined for human consumption has not been pathogenesis of Crohn’s disease in humans. This thoroughly investigated. The levels of chlorination recognizes that CD involves the interaction of at that is typically used for water treatment only re- least 3 components: a) genetic predisposition, b) an sults in around a two log10 reduction in numbers of enviromental trigger, and c) unregulated immune

36 response. The involvment in Map is supported by Health and economic losses several factors. They include: 1) CD and Map share According to the latest data, Crohn’s disease clinical and histopathological similarities; 2) via- affects between 400,000 and 600,000 people in ble Map has been cultured from intestinal tissues, North America. In Northern Europe, there are be- blood and milk of patients with CD; and 3) antibod- tween 27 and 48 diseased in every 100,000 peo- ies to Map antigen have been identified in the blood ple (Shivananda et al., 1996; Loftus et al., 2002). of Crohn’s patients. A major multicentre European study has calculat- Recent meta-analysis studies indicate that ed an incidence rate for Crohn’s disease of 5,6 per there is an association between the presence of Map 100,000 per year. The disease is life-long, though and CD, but whether this association is causal or co- considerable periods of remission may occur. It is incidental remains a controversial issue. Arguments estimated that over 200,000 people are affected by against the role of Map in CD are TNF-gamma in- this disease in the European Union. Data of the cost hibitors, which would not be effective if Map were of Crohn’s disease to society in Europe are not avail- the cause of CD. They are effective in the treatment able. However, a Swedish study has shown that in of CD, whereas anti-mycobacterial treatment has 1994 Sweden spent over 40 million SK on patients limited efficacy. In some countries, precaution- with this condition, 29% of which were direct costs, ary control measures are taken in food sectors, as i.e. costs for in-patient care, ambulatory care and there is a probability that dairy and meat products drugs. Indirect costs, i.e. sickness leave and early are contaminated by mycobacteria. The major con- retirement constituted 71% (Ekbom, 1997). cerns are studies demonstrating that Map has been Crohn’s disease results in substantial mor- cultured from pasteurized retail milk and may be bidity and high use of health services. Mortality is present in drinking water. not usually a feature of the disease though patients, particularly the young, have double risk mainly be- Diagnosis and treatment of Crohn’s disease cause of complications associated with the disease The diagnostic techniques that can be used (Prior et al., 1981). An US study found that for a to prove the disease are influenced by the clinical representative patient, projected lifetime costs were stage as it has been developed in the affected host $125,404 using mean charges (Silverstein et al., and the prevalence in the human society. Detection 1999). Total annual medical costs in the US were of the infection of the host during this sub-clinical estimated at $1-1.2 billion for 1992 (Hay J.W. and stage is complicated by the fact that the bacterium Hay A.R., 1992). If disability payments and the does not multiply rapidly and is therefore difficult cost of early retirement are taken into account, the to detect in faeces (Ridge et al., 1991; Sockett et al., total cost of this disease is much higher. 1992; Sweeney et al., 1995). Traditionally, the treat- In conclusion, paratubrculosis remains a sub- ment of Crohn’s disease has involved suppressing ject of public concern in many countries world- the host’s immune response with corticosteroids, wide, because diagnosis and control in animals and surgical resection of severely diseased bowel, nu- humans are difficult and expensive, as a result of tritional supplementation, or administration of anti- possible zoonotic links with Crohn’s disease. biotics. The efficacy of these therapies is evaluated by monitoring disease signs, symptoms and quality References of life indicators, so called Crohn’s disease activity Alvarez, J., L. De Juan, V. Briones, B. Romero, A. Aranaz, index (CDAI). Early efforts at antibiotic therapy for J. F. Fernandez-Garayzabal, A. Mateos (2005). Mycobac- the treatment of Crohn’s disease involve standard terium avium subspecies paratuberculosis in fallow deer and wild boar in Spain. Vet. Rec. 156: 212-213. drugs used in the treatment of tuberculosis (isoni- Bachvarova, Y., G. Kostov, N. Lilkova, S. Savova, I. Bard- azid, ethambutol and pyrazinamide) (Hermon-Tay- arov, Zh. Baitchev (1999). Occurrence of the non-specific lor, 1998). This antibiotic therapy gives different tuberculin reactions in cattle and nature of the accompany- results. Unlike the microorganism that causes tu- ing patho-morfological and bacterial findings. Bul. J. 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Evaluation of four serological tests for bovine Van Brandt, L., K. Coudijzer, L. Herman, C. Michiels, M. paratuberculosis. J. Clin. Microbiol. 30(5): 1134-1139. Hendrickx, G. Vlaemynck (2011). Survival of Myco- Stabel, J. R., T. L. Bosworth, T. A. Kirkbride, R. L. Forde, R. bacterium avium ssp. paratuberculosis in yoghurt and in H. Whitlock (2004). A simple, rapid, and effective meth- commercial fermented milk products containing probiotic od for the extraction of Mycobacterium рaratuberculosis cultures. J. Appl. Microbiol. 110:1252-261. DNA from fecal samples for PCR. J. Vet. Diagn. Invest. Van Kooten, H. C., C.G. Mackintosh, A. P. Koets (2006). In- 16: 22-30. tra-uterine transmission of paratuberculosis (Johne’s dis- Streeter, R. N., G. F. Hoffsis, S. Bech-Nielsen, W. P. Shulaw, ease) in farmed red deer. New. Zeal. Vet. J. 54: 16-20. M. Rings (1995). Isolation of Mycobacterium paratuber- Whan, L., I. R. Grant, H. J. Ball, R. Scott, M. T. Rowe (2001). culosis from colostrum and milk of subclinically infected Bactericidal effect of chlorine on Mycobacterium para- cows. Am. J. Vet. Res. 56:1322-1324. tuberculosis in drinking water. Lett. Appl. Microbiol. 33: Strogov, A. K. (1973). Paratuberculosis rangiferi tarandi. Lit- 227-231. tle known contagious diseases animals, F. M. Orlov (ed.). Wood, P. R., L. A. Corner, J. S. Rothel (1991). Field compar- Izdatelstvo Kolos, pp. 231- 236. ison of the interferon-gamma assay and the intradermal Stuart P. (1965). Vaccination against Johne’s disease in cattle tuberculin test for the diagnosis of bovine tuberculosis. exposed to experimental infection. Brit. Vet. J. 121: 289- Ausr. Vet. J. 68: 286-290. 318.

41 ACTA MICROBIOLOGICA BULGARICA

Immunohistochemistry on Cryostat Tissue Sections Using Synthetic Peptide Produced Monospeciffic Anti-Sheep Pox Virus Antibody Velizar Bumbarov1, Alexander Tzymanis2, Martin Hristov3, Raiko Peshev3, Rustem Eligulashvili1 1Kimron Veterinary Institute, Department of Virology, Beit Dagan 50250, Israel 2Kaplan Medical Center, Rehovot, Israel 3National Diagnostic and Research Veterinary Medicine Institute - “Prof. Georgi Pavlov”, Blvd. Pencho Slaveykov №15, Sofia, Bulgaria

Abstract An immunohistochemistry method on frozen tissue cryostat sections based on new polyclonal monospecific affinity-purified rabbit antibodies (PMAP) against 19-residue synthetic peptides (154-171), originated from the group-specific peptide P-32 for sheep poxvirus (SPV) was developed. PMAP were used as primary antibodies in an immunohistochemistry test on frozen ovine tissue for rapid laboratory diagnosis of SPV infection. This method is highly specific, reproducible, simple and inexpensive. The total time of the procedure was within 2 hours of receiving the samples in the laboratory. Key words: cryosections, immunohistochemistry, polyclonal monospecific affinity purified antibodies, sheep poxvirus.

Резюме Разработен е имунохистохимичен метод върху замразени криостатни срези на базата на нови поликлонални моно-специфични афинно пречистени заешки антитела (PMAP) срещу 19 синтетич- ни пептиди (154-171), произхождащи от групата на специфичния пептид P-32 на вируса на шарката по овцете (SPV). PMAP се използват като първични антитела в имунохистохимичния тест на замра- зени тъкани от овце за бърза лабораторна диагностика на SPV инфекция. Този метод е много специ- фичен, възпроизводим, прост и не-скъп. Общото време на процедурата е до 2 часа от получаването на пробите в лабораторията. Introduction Sheep pox (SP) is an endemic infection in Af- The basic diagnostic methods are histopathology rica, the Middle East, and Central Asia, as well as and virus isolation on cell culture. For confirma- in Russia, China, Israel (Yeruham et al., 2006) and tion of the infection, the virus neutralization test India (OIE, 2000). The disease is highly contagious or immuno-fluorescence by using hyper immune and causes mortality in young animals leading to anti-capripox serum are used (Rao and Bandyopa- significant economic losses (Kitching and Ham- dhyay, 2000). Due to the slow growth of SPV in cell moud, 1991; OIE, 2000).The sheep poxvirus (SPV) culture (it takes more than two passages of visible belongs to the family Poxviridae, genus Capripox- cytopathic effect), the isolation and identification of viruses (OIE, 2000). the virus is a long procedure (Carn, 1995; Manga- The diagnosis of SPV is still a problematic na-Vougiouka et al., 1999; Gulbahar et al., 2000). issue. The currently used diagnostic methods are Transmission electron microscopy for identification with low sensitivity (Agar Gel Precipitation Test) of typical SPV particles is used as a rapid diagnos- and the differentiation between the poxviruses is tic method, but it is not applicable for the ordinarily difficult (Rao and Negi, 1997; Mangana-Vougiou- laboratory practice (Gulbahar et al., 2000; Rao and ka et al., 1999; Mangana-Vougiouka et al., 2000). Bandyopadhyay, 2000). Recently, different techniques of PCR and ELISA have been developed in *Corresponding author: V. Bumbarov, tel.: + 972 3 9688949 Fax: + 972 3 968 17 88; e-mail: [email protected] many laboratories (Carn, 1995; Tiwari et al., 1996;

42 Mighell et al., 1998; Mangana-Vougiouka et al., USA). Forty days after the last inoculation the rab- 1999; Mangana-Vougiouka et al., 2000; Markoula- bits were bled. Specific antibodies for synthesized tos et al., 2000; OIE, 2000). peptide were separated by affinity chromatography. The aim of the study is to develop a highly Specificity and determination of optimal working specific, reproducible, inexpensive and rapid meth- conditions of purified Abs were determined by ti- od for SPV diagnosis. tration in ELISA (dilution from 1:10 to 1:1000) and IHC reaction (dilution from 1:50 to 1:500). Primary Materials and methods PMAP anti-SPV rabbit Abs, diluted 1:100 in PBS Indirect immunohistochemistry methods were added (50 μL) to the skin sections or tissue (IHC) for detection of SP viral antigen in Vero cells culture slides and were incubated for 30 min in a or frozen tissue cryostat sections by using the Avi- humid chamber at RT. After rinsing in PBS (three din-Biotin (AB) technique (Gulbahar et al., 2000; times x 2 min), they were treated for 10 min with Eligilashvili et al., 2002) and polyclonal mono- secondary Goat-Antirabbit Biotinylated Abs, at RT specific affinity-purified (PMAP) rabbit antibodies according to the manufacture’s protocol (Zymed, (Abs) against 19-residue synthetic peptides (154- USA). After incubation with secondary antibodies 171) (Vyberg and Nielsen, 1998; Eligilashvili et the slides were washed with PBS and treated for 10 al., 2002; Petrosyan et al., 2002), originated from min at RT with enzyme conjugate (Streptavidin – the group-specific peptide P-32 for sheep poxvirus Horse Radish Peroxidase). After that the substrate (SPV) were developed. 3-amino 9-aethyl carbasol (AEC) was applied for Preparation of cell culture and tissue samples 8-10 min at RT. The specimens were counterstained Vero cells (ATCC CCL-81) were infected with Mayer’s Hematoxylin 0.1% solution for 3 min with the RM 65 vaccine strain of SPV. After obser- and rinsed in tap water for 10 min. The slides were vation of visible cytopathic effect (CPE) the cells mounted with GVA-Mount with cover glass and were fixed and tested by IHC reaction at 24, 48, 72 were monitored under a light microscope Olympus h and after 5 days. CK-40 with a magnification of 100x or 200x (Elig- Small particles (8x5 mm) of skin originating ilashvili et al., 2002). from animals with clinical and pathological changes Controls of SPV infection were frozen by using 2-Methylb- Uninfected Vero cells, skin from healthy utane in liquid nitrogen. The tissue samples were sheep or sheep with other diseases peste de petit sectioned by Leica CM 1800 cryostat microtome (6 ruminants (PPR), border disease virus (BDV), en- µm thick section at T -25o C) and were mounted on terotoxaemia, rabbit PMAP anti - Rift Valey Fever poly-L-lysine-coated glass microscope slides. After (RVF), west Nile fever (WNF) Abs and rabbit iso- air-drying at room temperature (RT) for 20 min, the type control were used as negative controls. sections were fixed in cold acetone for 10 min. The endogenous peroxidase activity on frozen tissue Results section was blocked with peroxo-block (Zymed, After investigation to determine the optimal USA) for 45 s at RT following the manufacture’s working dilution and conditions of incubation, rich instructions. After that the slices were washed and dense coloring of the cytoplasm of the inoculat- three times x 2 min with phosphate buffered saline ed cell cultures at Abs dilution 1:50 was observed. (PBS). Nonspecific background of Vero cells was At 1:200 dilution of PMAP Abs the coloring was blocked with 10% Normal Goat Serum for 10 min with less density in comparison with the 1:100 di- at RT without washing. lution. The number of colored virus conglomerates Preparation of polyclonal monospecific affinity-pu- was smaller than that at the 1:100 dilution. No dif- rified rabbit antibodies (PMAP) ference in the number of colored virus conglom- The specificity of the used 19-residue synthet- erates at dilutions 1:50 and 1:100 was found. The ic peptides (154-171 ETFHNSNSRILFNQENNN) optimal dilution of PMAP was determined to be from the group-specific for SPV peptide P-32 (Vy- 1:100. berg and Nielsen, 1998; Eligilashvili et al., 2002; In IHC reaction on Vero cells infected with Petrosyan et al., 2002; Tulman et al., 2002) was the SPV strain RM-65, coloring of the cells cyto- verified by Gene Runner, ClasteW multiply align- plasm without any background was observed. IHC ment and Blast software. Rabbits were immunized reaction was performed on infected cells at 24, 48, twice with a 22-day interval with the specific syn- 72 and 120 h after infection. The number of colored thetic peptide attached to a carrier peptide (Zymed, cells containing SPV antigen was growing propor-

43 Fig. 1. Positive immunostaining in Vero cells inoculated with vaccine SPV RM-65 after infection at 24 (A), 48 (B), 72 (C) and 120 h (D). Uninfected cell culture (E). Counterstaining with Mayer’s haematoxylin. Magnification x100. tionally to the time after infection (Fig. 1 A, B, C sues. Synthetic peptides also have potential disad- and D). Background or cells with coloring of the vantages. The created synthetic peptides can have cytoplasm were not observed in uninfected cell cul- an altered three-dimensional structure of the native tures or in cell cultures infected with viruses differ- protein. Proteins similar to the desired peptides can ent than SPV (PPR, BDV, RVF, WNF and isotype exist in the investigated tissue. Both of these factors specific rabbit antibodies) (Fig. 1 E). can lead to false negative results and unsuccessful The primary PMAP Abs used in IHC reaction detection of antigen (Mandel et al., 1992; 1994). for cell cultures were applied in the same concen- For SPV detection we developed and used a tration and by the same scheme onto organs or tis- peptide-based IHC method. The specificity of the sue cryosections from diseased animals or animals IHC reaction depends on the specificity of the used which died of SPV. components. The obtaining of new classes of poly- All frozen skin sections from SPV-infected clonal Abs based on studying the peptide structure sheep were positive in the cryo-IHC test using rab- of viral strains allows the creation of highly specific bit anti-SP PMAP Abs. In the cytoplasm of epider- Abs (polyclonal monospecific) which can be con- mal and dermal cells specific coloring without any centrated and purified by affinity chromatography background staining was observed. The staining of with the column containing gel with included syn- the cell cytoplasm was clear in both parallel and thetic peptide (Vyberg and Nielsen, 1998; Eligilas- vertical sections of the skin surface (Fig.2 A and hvili et al., 2002; Petrosyan et al., 2002). B). Background or staining in the cytoplasm were Polyclonal Abs are produced by immuniza- not found in the organs or tissue section from alive tion of experimental animals with purified antigens. or infected with different than SPV viruses (PPR, The immunized animals respond by a rise of Abs BDV, RVF and WNF) animals (Fig. 2 C). which specifically recognized and reacted with the antigens used for inoculation. Polyclonal Abs have Discussion high affinity and broad reactivity but lower speci- The origin and procedures for obtaining im- ficity compared to monoclonal Abs (Hayat, 2002). munogen are very important for the preparation of Polyclonal Abs contain different Abs to the target high quality reagents for the IHC reaction. An ad- antigen and nonspecific Abs with high concentra- vantage of synthetic peptides is the knowledge of tion (up to 10 mg/mL) if they are not affinity puri- amino acid sequences. That information can be use- fied (Mighell et al., 1998; Elias, 2003). ful for the interpretation of the IHC reaction results The used nucleotide sequences of SPV p32 according to the isoforms of the desired peptides peptide (ETFHNSNSRILFNQENNN) were specif- and cross reactivity with similar peptide sequences ic for the Capripoxvirus group. A confirmation of in other proteins contained in the investigated tis- this fact is the absence of background in the IHC re-

44 2000; Kitching and Hammoud, 1991).

References Carn, V. M. (1995). An antigen trapping ELISA for detection of capripoxvirus in tissue culture supernatant and biopsy samples. J. Virol. Methods 51: 95-102. Elias, J.M. (2003). Immunohistochemical methods, in: Elias J.M. (Ed.), Immunohistopathology. A Practical Approach to Diagnosis, 2nd ed., ASCP Press, Chicago, IL, pp. 1–110. Eligilashvili, R., V. Bumbarov, Y. Hagai (2002). comparison of the avidin-biotin and new polymer detection systems for rapid recognition of PPR virus antigen in situ. Bulga- rian J. Vet. Med. 5: 229 - 232. Gulbahar, M.Y., M. Cabalar, Y. Gul, , H. Icen (2000). Immu- nohistochemical detection of antigen in lamb tissues naturally infected with sheep pox virus. J. Vet. Med. B Infect. Dis. 47: 173-181. Hayat, M.A. (2002). Fixation and embedding, in: Hayat M.A. (Ed.), Microscopy, Immunohistochemistry and Antigen Retrieval Methods for Light and Electron Microscopy. Kluwer Academic, New York, NY, pp. 71-93. Kitching, R.P., J.M. Hammoud (1991). Poxvirus infection and immunity, in: Roith, I.M., P.J. Delucs (Eds.), Enciclopedia of Immunology. Academic press, London, 3: pp. 1261- 1264. Mandel, U., B. Gaggero, J. Reibel, M.H. Therkildsen, E. Da- belsteen, H. Clausen (1994). Oncofetal fibronectins in oral carcinomas: correlation of two different types. Acta Pathol. Microbiol. Immunol. Scand. 102: 695-702. Mandel U., M.H. Therkildsen, J. Reibel, B. Sweeney, H. Mat- suura, S.I. Hakomori, E. Dabelsteen, H. Clausen (1992). Cancer-associated changes in glycosilation of fibronectin: immunohistological localisation of oncofetal fibronectin defined by monoclonal antibodies.APMIS 100: 817-826. Mangana-Vougiouka, O., P. Markoulatos, C. Koptopoulas, K. Nomikou, N. Bakandritsos, O. Papadopoulos (2000). Sheep poxvirus identification from clinical specimens by PCR, cell culture, immunofluorescence and agar gel im- munoprecipitation assay. Mol. Cell. Probes 14: 305-310. Fig. 2. Positive immunostaining in parallel (A) and Mangana-Vougiouka, O, P. Markoulatos, C. Koptopoulas, vertical (B) cryo section of sheep skin epidermis K. Nomikou, N. Bakandritsos, O. Papadopoulos (1999). Sheep poxvirus identification by PCR in cell cultures. J. with and without SPV infection (C). Counterstaining Virol. Methods 77: 75-79. with Mayer’s haematoxylin. Magnification x100. Markoulatos, P., O. Mangana-Vougiouka, G. Koptopoulas, K. Nomikou, O. Papadopoulos (2000). Detection of sheep action. In the other used methods background was poxvirus in skin biopsy samples by a multiplex polymerase chain reaction. J. Virol. Methods 84: 161-167. observed due to endogen biotin or avidin (Vyberg Mighell, A.J., W.J. Hume, P.A. Robinson (1998). An over- and Nielsen, 1998; Sabattini et al., 1998; Shi et al., view of the complexities and subtleties of immunohisto- 1999; Petrosyan et al., 2002; Taylor et al., 2002). chemistry. Oral Dis. 4: 217-223. The evidence of IHC specificity and sensitivity is OIE (2000). Manual of standards for diagnostic test and vac- the observed virus antigen in infected Vero cells cines, 4th edition, part 2, section 2.1, chapter 2.1.10. within the first 24 h after their infection. Petrosyan, K., R. Tamayo, D. Joseph (2002). Sensitivity of a novel biotin-free detection reagent (Powervision+ТМ) for It can be concluded that the developed cryo- immunohistochemistry. J. Histotechnol. 25: 247-250. IHC reaction is a suitable method for rapid recog- Rao, T.V, B.S. Negi (1997). Evaluation of different serologi- nition of SPV antigen in skin biopsies taken from cal tests for the diagnosis of goat pox using soluble anti- live animals. This method can be used for rapid gens. Trop. Anim. Health Prod. 29: 235-239. laboratory diagnosis of SPV in the early stages of Rao, T.V, S. K. Bandyopadhyay (2000). A comprehensive re- the disease and could be a useful tool for studying view of goat pox and sheep pox and their diagnosis. Anim. Health Res. Rev. 1: 127-136. the pathogenesis of SP infections (Gulbahar et al.,

45 Sabattini, E., K. Bisgaard, S. Ascani, S. Poggi, M. Piccioli, 3-43. C. Ceccarelli, F. Pieri, G. Fraternali-Orcioni, S.A. Pileri Tiwari, A.K., T.V. Rao, B.S. Negi (1996). Spot agglutination (1998). The EnVision++ system: a new immunohisto- test for the rapid diagnosis of goat pox. Trop. Anim. Health chemical method for diagnostics and research. Critical Prod. 28: 213-215. comparison with the APAAP, ChemMate CSA, LABC, Tulman, E.R., C.L. Afonso, Z. Lu, L. Zsak, J.H. Sur, N.T. and SABC techniques. J. Clin. Pathol. 51; 506-511. Sandybaev, U.Z. Kerembekova, V.L. Zaitsev, G.F. Kutish, Shi, S.R., J. Guo, R.J. Cote, L.L. Young, D. Hawes, Y. Shi, D.L. Rock (2002). The genomes of Sheep pox and Goat S. Thu, C.R. Taylor (1999). Sensitivity and detection ef- pox viruses. J. Virol. 76: 6054-6061. ficiency of a novel two-step detection system (PowerVi- Vyberg, M., S. Nielsen (1998). Dextran polymer conjugate sion) for immunohistochemistry. Appl. Immunohistochem. two step visualization system for immunohistochemistry. Mol. Morphol. 7: 201-208. Appl. Immunohistochem. 6: 3-10. Taylor, C.R., S.R. Shi, N.J. Barr, N. Wu (2002). Techniques Yeruham, I, H. Yadin, M. Vam Ham, V. Bumbarov, A. Soham, of immunohistochemistry: principles, pitfalls, and stand- S. Perl (2007). Economic and epidemiological espects of ardization, in: Dabbs, D.J. (Ed.), Diagnostic Immunohis- an outbreak of sheeppox in dairy sheep flock. The Vet. tochemistry. Churchill Livingstone, New York, NY pp. Rec. 160: 236-237.

46 ACTA MICROBIOLOGICA BULGARICA

Application of the Agar-Diffusion Plaque-Inhibition Method for Testing Combination Antiviral Effects: PTU-23 and Guanidine Hydrochloride Interactions and Combined Effects on the Replication of Poliovirus 1

Angel S. Galabov The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences

Abstract Original modifications of the agar-diffusion plaque inhibition method were used for testing the combination effect of anti-enteroviral substances. It was established that the anti-picornavirus compounds PTU- 23 and guanidine.HCl are antagonists when applied in combination against the replication of poliovirus 1 in cell cultures. Some significant differences in their activity were found: (i) guanidine antagonists did not influence the anti-poliovirus effect of PTU-23; (ii) Cu++ and Zn++ ions significantly increased the activity of PTU-23 towards poliovirus 1, whereas Zn++ ions had no effect on the antiviral activity of guanidine.HCl, and Cu++ ions manifested a marked antagonistic effect on the guanidine activity. Evidently, the anti-enterovirus activity of these compounds is directed to different targets in the virus replication cycle. Key words: agar-diffusion test, combination effects, poliovirus 1, PTU-23, guanidine.HCl

Резюме Оригинални модификации на агар-дифузионния плако-инхибиращ метод са приложени за изпитване на комбинирания ефект на анти-ентеровирусни вещества. Установено е, че анти-пикор- навирусните съединения PTU-23 и гванидин.HCl, приложени в комбинация спрямо репликацията на полиовирус 1 в клетъчни култури показват отчетлив антагонизъм. Установени са някои съществени различия в тяхната активност: (i) гванидиновите антагонисти не влияят върху анти-полиовирусния ефект на PTU-23; (ii) Cu++ и Zn++ йони усилват значително активността на PTU-23 спрямо поли- овирус 1, докато Zn++ йони са без ефект върху антивирусния ефект на гванидин.HCl, а Cu++ йони проявяват отчетлив антагонистичен ефект спрямо активността на гванидина. Очевидно, анти-ен- теровирусната активност на тези съединения е насочена към различни мишени в ентеровирусния репликативен цикъл.

Introduction Enterovirus infections are among the principal Shipman (1990) stands out among these methods. indications for application of chemotherapy, but the In the field of antivirals it definitely replaced the lack of clinically effective agents is due mainly to routine isobologram method, largely used in the the development of drug resistance. This phenom- experimental pharmacology (Fucquier and Guedj, enon is related to the unusually high mutation rate 2015; Tallarida, 2011). and the consequent composition of the enterovirus The present work describes a completely dif- progeny of billions оf quasispecies. The combined ferent approach for testing the combination effects administration of enterovirus replication inhibitors of inhibitors of enterovirus replication. It is a vari- with different mechanisms of action is considered ant of the “classical” agar-diffusion plaque-inhibi- to be a basic approach which could counteract the tion method of Rada and Zavada (1962). Its basic drug resistance (Galabov et al., 2012). In this con- advantage is the very convincing direct visualiza- nection, several various methods for studying the tion of the combination effects of the antivirals, combination effect of virus inhibitors were intro- unattainable by other methods for testing combina- duced in the experimental chemotherapy of viral tions effects. infections. The 3D spaced method of Prichard and As a model enterovirus, poliovirus 1 (Ma-

47 honey) we used, replicated in monolayer cultures (iv) pronounced inhibition of infectious virus proof FL or MK cells. The work with this polio 1 strain duction; (v) marked inhibition of viral 37S RNA was done before its stock was destroyed in the late synthesis; (vi) strong inhibition of CPE develop- 1990s. In the meantime, The Third Meeting of the ment. However, several significant differences were Advisory Committee on Poliovirus Eradication, registered: (i) PTU-23 significantly decreases the held in October 2006, proposed the establishment activity of the virus-specific RNA-dependent RNA of a “poliovirus antiviral initiative” and the appro- polymerase in a cell-free system, guanidine.HCl priate and possibly essential development of at least being without effect; (ii) PTU-23 markedly inhibits two anti-polio drugs for control in the post-eradica- the synthesis of the double-stranded RNA, where- tion era. This will be of great benefit for post-expo- as guanidine.HCl has no effect on this species of sure prophylaxis and outbreak control (Workshop virus-specific RNA; (iii) PTU-23 does not inhibit Report, Committee on Development of a Polio An- the synthesis of RNA-dependent RNA polymerase, tiviral and its Potential Role in Global Poliomyeli- strongly suppressed by guanidine.HCl; (iv) there tis Eradication, 2006; Chumakov et al., 2007). are some reasons to consider that under the effects The combination effect of two selective in- of PTU-23 and guanidine some partial degradation hibitors of enterovirus replication was investigat- of viral RNA occurs, but for PTU-23 this phenome- ed, namely guanidine.HCl and PTU-23. PTU-23 non supposedly occurs only at the end of the latent (N-phenyl-N’-3-hydroxyphenyl thiourea) was se- period; (v) PTU-23 prevents virus-induced inhi- lected and characterized in a series of publications bition of the host-cell protein and RNA syntheses within the period 1972 – 1999. PTU-23 has been independently of the multiplicity of infection; gua- characterized as a strong and specific inhibitor of nidine.HCl is without effect at m.o.i. exceeding 10; picornavirus replication with a very broad antiviral (vi) PTU-23 and guanidine.HCl do not markedly spectrum, embracing polioviruses 1-3, CVB 1-6, inhibit the poliovirus protein synthesis in contrast CVA 7/IV, echoviruses 1, 3, 6-9, 11-14, 17, 19, 20, to the pronounced inhibition of the virus-specific enterovirus 71, ECSO II-IV, EMCV, rhinoviruses RNA synthesis, quantitatively the guanidine effect 1B-532 and H-17, FMDV type C (Galabov, 1979). being better expressed; (vii) guanidine.HCl effect is The comparison of the anti-picornaviral ef- directed to the process of the posttranslational mor- fects of PTU-23 and guanidine.HCl marked several phogenesis (cleavage) of the structural proteins and substantial similarities: (i) the highest susceptibility to the virions morphogenesis; guanidine.HCl dam- of viruses belonging to Enterovirus genus; (ii) the ages the functional configuration of the structural arrangement of polioviruses by their susceptibili- proteins’ precursor NCVP1; the data on PTU-23 ty: polio 2 > polio 3 > polio1; (iii) the analogous effect on EMC virus protein synthesis evidenced a susceptibility of type C FMD virus. Nevertheless, lack of influence on the protein cleavage process; some differences were also found: (i) PTU-23 in- (viii) a single passage of poliovirus in the presence hibits EMC virus replication, insensitive to guan- of guanidine.HCl is sufficient for the resistant mu- idine.HCl; (ii) PTU-23 inhibits rhinovirus 1B-632 tant selection; in the case of PTU-23, 6-7 passag- replication, also insensitive to guanidine.HCl; (iii) es are needed for the development of drug-resist- PTU-23 possesses a marked protective effect in ex- ant progeny (Caliguiri and Tamm, 1973; Galabov, perimental picornavirus infections in vivo (in lab- 1981; Galabov and Dmitrieva, 1983; Galabov and oratory animals) - against Coxsackieviruses A6, Svitkin, 1983; Saunders et al., 1985). A7, B1, B3 and FMD virus in newborn mice and It was established that the target of guanidine against Coxsackievirus effect on picornavirus replication is the non-struc- A7-IV in cotton rats, whereas guanidine.HCl tural protein 2C (Pincus et al., 1986; Barton and does not show in vivo activity (Caliguiri and Tamm, Flanegan, 1997). According to Barton and Flanegan 1973; Galabov and Velichkova, 1974; Galabov, (1997), guanidine inhibits the 2C function that is re- 1979; Herrmann et al., 1982; Klein et al., 2000). quired for the initiation of RNA(-), but not RNA(+) It is of interest to compare of the mechanisms synthesis or RNA elongation. In addition to this tar- of anti-picornavirus action of the two compounds. get, it was found that guanidine prevents the asso- PTU-23 and guanidine.HCl are similar in the fol- ciation of 2C/2BC with a host membrane structure lowing properties: (i) lack of virucidal effect ; (ii) without affecting their association with the viral they do not influence viral adsorption and penetra- RNA (Bientz et al., 1990). Studies on the mode of tion; (iii) the drug-susceptible period ranges from anti-picornavirus effect of PTU-23 characterized 1st to 5th hour in the picornavirus replication cycle; this compound as a selective inhibitor of specific

48 viral 37S ssRNA synthesis as a result of suppres- was recorded on the basis of the size (diameter, Ø, sion of the synthesis of a viral protein with regu- in mm) of the plaque inhibition and the zone of cy- latory functions in the replication cycle (Галабов, totoxicity. 1981; Galabov and Dmitrieva, 1983; Galabov and Svitkin, 1983). Results and Discussion The comparative analysis of the data on the anti-picornaviral effects of PTU-23 and guanidine. Resultant effect of the combined application of HCl outlines the necessity to study the interaction PTU-23 and guanidine.HCl of these two specific inhibitors of picornavirus rep- The agar-diffusion plaque-inhibition test lication: (i) the resultant effect of the simultaneous of Rada and Zavada (1962) was applied in sev- application of the two compounds on poliovirus 1 eral experimental designs in FL cells monolayers replication; (ii) action of guanidine.HCl antagonists in Petri dishes. This method gives considerable on the antiviral effect of PTU-23; (iii) influence of conveniences for testing the combination effects, heavy metals (Cu++, Zn++) on the effects of PTU-23 i.e. the interaction of two or more substances ap- and guanidine.HCl on poliovirus replication. plied in combination. One of the substances could be included in the agar overlay (its concentration Materials and Methods could be varied). Depending on the size of the Petri Compounds dishes used one or more cylinders could be fixed N-phnyl-N’-3-hydroxyphenylthiourea (PTU- in the agar overlay. These cylinders may be filled 23), synthesized by G. N. Vassilev (Institute of with substances whose combination effect with a Plant Physiology, Bulgarian Academy of Sciences), substance included in the agar overly is tested. It is dissolved ex tempore in ethanol was used. also possible for two substances to be added in one Guanidine hydrochloride was supplied by and a same cylinder and their combination effect to Eastman Organic Chemicals, New York, USA, dis- be tested against a third substance. By changing the solved in physiological saline. distance between the cylinders in this method, the Cells and media combination effects manifest themselves within a Monolayer FL and MK cells were used. The part of the overlapping diffusion zones of the two growth medium consisted of a mixture of equal substances. It is also possible, to record the effect parts of medium 199 (Difco) and Hanks’ saline, as a function of the time of application of the sub- supplemented with 10% heated calf serum and an- stances in the cylinders, etc. Thus, the labour-con- tibiotics (penicillin 100 IU/ml) and streptomycin suming preliminary procedures are avoided in the 100 µg/ml). course of the cell cultures work with liquid medium Virus including determination of the substances’ individ- Poliovirus type 1 (Mahoney) was grown in ual toxicity and their combined toxic effects. In the Eagle’s Minimal Essential Medium (MEM, Flow) latter case, a combination of two methods has to with 5% heated calf serum and antibiotics. The be used in order to obtain statistically significant stock virus titer was 2-5 x 109 PFU/ml. results in the determination of the minimal subtox-

Agar-diffusion plaque-inhibition method ic or CC50 (50% cytotoxic concentration). Accord- Monolayer FL cells or MK cell cultures in ing to the described method, a preliminary deter- Anumbra (Czeck Republic) Petri dishes (Ø = 90 mination of the virus inoculation dose and of the mm) were inoculated (adsorption: 60 min at 20oC) non-toxic concentration of the substance included with 1.88 ml of a virus dose, giving semiconfluent in the agar overlay are only required. plaques after 24-48 h of incubation at 37oC. A 12.5- In the initial experimental setup, PTU-23 ap- ml agar overlay (1% agar in MM Eagle Difco con- plied at various concentrations (graded from 20 to taining 10% heated calf serum, 1.65 mg/ml sodium 60 µg/ml) was included in the agar overlay in Petri bicarbonate, 100 IU/ml penicillin and 100 µg/ml dishes (with 90 mm diameter), and guanidine.HCl streptomycin) was placed on the dish. One or more in a 0.5% solution (5 mg/ml) was added in a cylin- glass cylinders (Ø = 6 mm) were fixed in the agar der placed in the agar overlay, simultaneously with overlay, in which 0.1 ml of each substance tested the virus inoculation . In order to increase the diffu- (usually in 0.5-4% solution) was added. A second sion time of the substances in the agar overlay, FL overlay was added following incubation, contain- cells cultures were initially stored for 20 hours at 4o ing 1.5% agar and o.o2% neutral red in physiologi- C, and then incubated for 24 hours at 37 o C. cal saline. The antiviral effect of a given compound As seen in Table 1, a marked antagonistic ef-

49 Table 1. Effect of PTU-23 and guanidine.HCl on poliovirus 1 (Mahoney) growth in FL cells by the agar- diffusion plaque-inhibition test of Rada and Zavada (1962)

An analogous experiment with some modifications is shown in Figures 1A and 1B.

fect of PTU-23 towards the antiviral effect of gua- virus replication) plus 6 hours at 37 oC. nidine.HCl was registered. It was expressed by di- When the substances diffused in the agar minishment of the diameter of the inhibition zone overlay containing PTU-23 30 µg/ml (Figure 1B), around the cylinder with guanidine, increasing with the following was observed: (i) an additive increase the raising of the PTU-23 concentration (up to 60 in the PTU-23’ inhibition zone (cylinder No 2); (ii) µg/ml). At higher PTU-23 concentrations a com- a markedly weakly expressed at the combination plete inhibition of plaque formation was present. PTU-23 + guanidine.HCl in one cylinder (cylinder As seen in Figure 1A, the inhibition zones No 3); (iii) a decrease in the guanidine effect - a de- of PTU-23 40 mg/ml and of guanidine.HCl 1 mg/ crease in the inhibition zone diameter by 25% when ml were of approximately equal size in “pure” agar the compound was applied simultaneously with the overlay. The guanidine.HCl effect on such agar infection (not illustrated) and a complete disappear- overlay was one and the same irrespective of the ance of the guanidine inhibition zone when applied compound addition simultaneously with or 6 hours 6 hours post incubation at 37oC (cylinder 1). post the infection onset (inhibition zones with di- It was of interest to check the opposite, i.e. ameters 19.7 and 20.9 mm, respectively). how guanidine.HCl influences the antiviral activity In Figure 1B the agar overlay contained 30 of PTU-23 against poliovirus. An identical experi- µg/ml PTU-23, a concentration selected in order mental setup was used (Table 2): the agar overlay to reach a moderate reduction of the plaque num- contained guanidine.HCl of various concentrations ber and size, aiming a more clear marking of the (5 – 40 µg/ml), and cylinders with PTU-23 (40 mg/ inhibition zones of the substances applied in the kg) and with guanidine.HCl, respectively, were cylinders. Guanidine.HCl at concentration 1 mg/ placed simultaneously with the infection.. It was ml was added in a cylinder (No 1) simultaneous- observed that guanidine, even at a lower degree, ly with virus infection or 6 hours post incubation antagonized the effect of at 37oC of the infected cultures (immediately after PTU-23 on poliovirus replication, i.e. a two- infection the Petri dishes were stored for 20 hours way antagonism was registered. It was also seen at 4 oC). In the same Petri dish, control cylinders that guanidine.HCl slightly stimulated the toxic ef- with PTU-23 (40 mg/ml) (No 2) and with a mix- fect of PTU-23. ture of 1/1 v/v guanidine.HCl 1 mg/ml + PTU-23 These data convincingly prove the antago- 40 mg/ml (No. 3) were placed. In all cases PTU-23 nistic interaction between the effect of PTU-23 and was dropped simultaneously with the infection, i.e. guanidine.HCl. towards poliovirus replication in in one of the test groups the two compounds were FL cells. PTU-23 attained a decidedly higher con- applied simultaneously, and in the other test group centration in the poliovirus infected cells and bond- guanidine was applied 26 hours post PTU-23: 20 ed with the virus-specific target before the entry of hours at 4 oC (PTU-23 diffusion in the absence of guanidine.HCl, and vice versa. 50 a c

b d

Fig. 1A. Combination effect of PTU-23 and guanidine. Fig. 1B. Combination effect of PTU-23 and gua HCl on replication of poliovirus 1 (Mahoney) in FL nidine.HCl on replication of poliovirus 1 (Mahoney) cells by the agar-diffusion plaque-inhibition test of in FL cells by the agar-diffusion plaque-inhibition Rada and Zavada (1962) on ”pure” agar overlay: test of Rada and Zavada (1962) on agar overlay a. virus control; b. effect of guanidine.HCl (1 mg/ containing PTU-23: c. effect of PTU-23 (30 µg/ml) ml) in cylinder 1; PTU-23 (40 mg/ml) in cylinder included in the agar overlay; d. effect of guanidine. 2; PTU-23 + guanidine 1:1 v/v in cylinder 3. HCl (1 mg/ml) in cylinder.1; PTU-23 (40 mg/ml) in cylinder 2; PTU-23 + guanidine.HCl 1:1 v/v in cylinder 3.

51 Table 2. Effect of guanidine.HCl on PTU-23 effect towards poliovirus 1 (Mahoney) by the agar-diffusion plaque-inhibition test (1962)

Influence of guanidine.HCl antagonists on the PTU-23 against poliovirus 1. Among them l-me- PTU-23 effect thionine (135 mM), l-isoleucine (1150 mM) and It is known that there are substances which choline-chloride (85 mM) markedly antagonized diminish or completely cancel the antiviral effect of the guanidine effect. guanidine.HCl, the so-called guanidine antagonists In the experimental setup presented in Table (Lwoff, Lwoff, 1964; Loddo et al., 1966; Philipson 4, a “pure” agar overlay was placed on FL cells et al., 1966). monolayers in a Petri dish with a diameter of 150 The experimenmtal setup of the agar-diffusion mm. Guanidine.HCl (at a relatively high guanidine plaque-inhibition test of Rada and Zavada (1962) concentration of 20 mg/ml) was applied in a cyl- was used to test the influence of some guanidfine. inder placed in the Petri dish center, and cylinders HCl antagonists on the inhibitory effect of PTU-23. with PTU-23 (40 µg/ml) and a series of guanidine The following substances were applied: (i) from the antagonists in suitable concentrations were placed first class antagonists (amino acids) - 1-methionine, around in a 30 mm-radius circle. A marked narrow- l-valine, l-leucine, l-isoleucine and l-phenylalanine; ing of the guanidine inhibition zone was manifested and (ii) from the second class (choline-like com- by PTU-23 (whose antagonistic effect towards the pounds) - choline chloride. In parallel, the effect of guanidine activity was described above), along un- some other substances was checked-up, namely of der the effects of l-isoleucine and choline chloride, methanol (possessing also an anti-guanidine effect and at a lower extent by l-leucine and l-valine. Evi- - Loddo et al., 1966), amino acids l-glycine and dently, only substances antagonizing the guanidine l-glutamine, and uridine (RNA precursor, in view effect more strongly were selected at this experi- of the pronounced inhibitory effect of PTU-23 to- mental setup. wards virus-specific RNA synthesis). Walking all over the concept of Moser et al. For this study, a non-toxic for FL cells PTU- (1971) antagonists shift the guanidine.HCl from its 23 concentration of 60 µg/ml included in the target, by binding to a receptor locus, adjacent to agar-overlay was previously selected. It exerted the locus of guanidine. The lack of effect by gua- complete inhibition of poliovirus 1 in FL cells (in nidine antagonists on the antiviral effect of PTU- Petri dish with Ø = 90 mm) during a 24-h incuba- 23 shows that PTU-23 and guanidine.HCl bind to tion. Such properties PTU-23 demonstrated with- different receptor loci, i.e. the two enterovirus in- in the concentration range of 55-70 µg/ml. For the hibitors have different targets, although different guanidine.HCl the minimal 100% concentration receptor loci could be situated on one and the same was ≥ 30 µg/ml. target. The presumable methylating effect of antag- It was establishes (Table 3) that none of the onists, according to another concept on their mech- tested substances influenced the antiviral effect of anism of action (Lwoff, 1965; Ménard et al., 1966) 52 Table 3. Influence of guanidine antagonists on the effect of PTU-23 against poliovirus 1

Table 4. Effect of PTU-23 and guanidine antagonists on the antiviral effect of guanidine.HCl towards poliovirus 1(Mahoney) in the agar-diffusion plaque-inhibition test of Rada and Zavada (1962) in FL cells monolayers in Petri dishes

is excluded in the case of PTU-23. the probable role of chelate bonds to their virus-spe- The RNA precursor uridine, taking down ef- cific target (Galabov et al., 1980). Their structure fects of a serious of metabolites, does not influence suggests they could be considered as two-centric the PTU-23 activity. ligands. In order to verify this probable property the influence of Cu++ and Zn++ ions on the PTU-23 Influence of heavy metals (Cu++, Zn++) on effect towards poliovirus 1 growth in MK cells was the effects of PTU-23 and guanidine.HCl on studied. The modification of the plaque-inhibition poliovirus 1 replication agar-diffusion method describrd above was used: at The analysis of structure-activity relationship a constant PTU-23 concentration (40 mg/ml) in a of diphenylthioureas proposed the hypothesis about cylinder placed in the center of the Petri dish, and

53 CuSO4 and ZnCl2 were included in the agar over- in the virus RNA replicative complex; (ii) the re- lay at concentration gradients. At that, ZnCl2 was ceptors for both compounds preferably coordinate applied at concentrations below the one inhibiting Cu++; (iii) Cu++ ions suppress guanidine activity vs poliovirus plaque formation. poliovirus 1 replication, and increase the antipolio- It was established (Figure 2) that the combina- virus effect of PTU-23. Evidently, guanidine bind- tion of PTU-23 with Cu++ (5x10-7M) or Zn++ (10-5M) ing to its receptors most probably occurs through ions led to a marked increase in PTU-23 antiviral weaker and labile bonds, therefore the reversibility activity. Moreover, Cu++ effect was approximately of the compound effect is considerable in speed and 20 times stronger than the effect of Zn++. Besides, size. it was found that PTU-23 toxicity zone remains un- changed in the presence of Cu++ or Zn++ ions. These Conclusion data showed that (i) PTU-23 functions as a ligand, The studies carried out convincingly mani- coordinating heavy metals (Cu++ and Zn++); (ii) the fested the antagonistic character of the combination complex PTU-23-Cu++ is 20-fold more active than effects of guanidine.HCl and PTU-23 on the rep- the complex PTU-23-Zn++; (iii) the target of the lication of poliovirus 1, based on the concurrence complex PTU-23-Me is virus-specific (the combi- of these two picornavirus replication inhibitors for nation does not change the PTU-23 toxicity). Evi- binding to their specific targets in the viral replica- dently, the receptor of PTU-23 preferably coordi- tion cycle. nates the Cu++ ions. Further investigation of the guanidine.HCl In parallel, the combination effect of Cu++ and PTU-23 interactions needs to study the effect and Zn++ ions with guanidine.HCl (1 mg/ml in the of PTU-23 on the guanidine mutants of poliovirus cylinder placed in the Petri dish center) was tested. 1, and vice versa, the guanidine effect on poliovirus As seen in Figure 2, Zn++ ions do not influence the strains in the course of selection of PTU-23-resist- antiviral activity of guanidine.HCl, whereas Cu++ ance. ions (>5x10-7M) manifested a marked antagonistic effect on the guanidine activity, i.e. most probably, Acknowledgements Cu++ ions form a coordination complex with guani- I would like to express my great thankfulness dine receptors, interfering with the binding of gua- to Mrs Lilly Goranova for her excellent technical nidine with its virus-specific target. assistance. These results support the proposed hypothesis on the mechanism of the observed antagonism References between PTU-23 and guanidine.HCl in their effects Barton, D. J., J. B. Flanegan (1997). Synchronous replication on poliovirus type 1 replication: (i) attack on dif- of poliovirus RNA: Initiation of negative-strand RNA ferent non-structural protein targets, most probably synthesis requires the guanidine-inhibited activity of protein 2C. J. Virol. 71: 8482-8489. Bienz, K., D. Eggers, M. Troxler, L. Pasamontes (1990). Structural organization of poliovirus RNA replication in kmediated by viral proteins of the P2 genomic region. J. ++ Guanidine+Cu Virol. 64: 1156-1163. Guanidine+Zn++ 70 Caliguiri, L. A., I. Tamm (1973). Guanidine and 2-(α-hydrox- PTU-23+Zn++ ++ ybenzyl)-benzimidazole (HBB): selective inhibitors of 60 PTU-23+Cu picornavirus multiplication, in: Carter, W. A.(Ed.), Selec- 50 tive Inhibitors of Viral Functions, CRC Press, Cleveland, mm φ 40 pp. 257- 293. Chumakov, K., E. Ehrenfeld, E. Wimmer, V. Agol (2007). Vc- 30 cination against polio should not be stopped. Nature Rev.

Inhibition zone, 20 Microbiology 5: 952-958. 10 Foucquier, J., M. Guedj (2015). Analysis of drug combinations: current methodological landscape. Pharmacol. Res. 0 -7 -6 -5 -4 & Perspectives 3 (3) (/doi/10.1111/prp2.2015.3.issue-3/ 10 10 10 10 ++ ++ Concentration, M [Cu , Zn ] issuetoc) June 2015. Galabov, A. S., E. H. Velichkova (1974). Antiviral activity of N-phenyl-N’-aryl- or alkylthiourea derivatives in Fig. 2. Effects of PTU-23 and guanidine.HCl Coxsackie virus infections in mice. Antimicrobial Agents in combination with Cu++ and Zn++ ions on the Chemother. 5: 1-18. replication of poliovirus 1 (Mahoney) in MK cells Galabov, A. S. (1979). Thiourea derivatives as specific inhibitors of picorna viruses. Arzneim.-Forsch./Drug Res.

54 29(II): 1863-1868. Loddo, B., G. L. Gessa, M. L. Schivo, A. Spanedda, G. Brot- Galabov, A. S., B. S. Galabov, N. A. Neykova (1980). Struc- zu, W. Ferrari (1966). Antagonism of the guanidine inter- ture-activity relationship of dophenylthiourea antivirals. ference with poliovirus replication by simple methylated J. Medic. Chem. 23: 1048-1051. and ethylated compounds. Virology 28: 707-712. Galabov, А. S. (1981). The study of inhibitory effect of N-phe- Lwoff, A. (1965). The specific effectors of viral development. nyl-N’-3-hydroxy-phenyl thioura (PTU-23) on poliovirus Biochem. J. 96: 289-901. 1 reproduction, in: Votyakov, V. I. (Ed.), Nucleosides, Lwoff, A., M. Lwoff (1964). Neutralization par divers me- Bicycloheptane and Adamantane Derivatives, Other An- tabolites de l’effet inhibiteur de la guanidine sur le devel- tiviral Substances (Materials of the Session of All-Union opppement du poliovirus. C. R. Acad. Sci. (Paris) 259: Problem Commission “Chemotherapy and Chemoprophy- 949-952. laxis of Viral Infections” and United Research Session, Menard, D., M. Lwoff, A. Lwoff (1966). Role de la methi- 1-2.12.1981 Минск (in Russian). Bielorussian Research onine dans las resistance du poliovirus a guanidine. C. R. Institute of Epidemiology and Microbiolkogy, Minsk, pp. Acad. Sci. (Paris) D262: 1163. 125-139. Mosser, A. G., L. A. Caliguiri, I. Tamm (1971). Blocking ac- Galabov, A. S., T. M. Dmitrieva (1983). Inhibitory effect of tion of guanidine action of poliovirus multiplication. Vi- N-phenyl-N’-3-hydroxyphenylthiourea (PTU-23) on the rology 45: 653-663. reproduction of encephalomyocarditis virus in Krebs-II Phillipson, L., S. Bengtsson, Z. Dinter (1966). The reversion cells. Zbl. Bakt. Hyg., I Abt. Orig. A254: 291-305. of guanidine inhibition of polioviruws synthesis. Virology Galabov, A. S., V. A. Ginevskaya, Yu. V. Svitkin (1983). Ef- 29: 317-329. fect of N-phenyl-N’-3-hydroxyphenylthiourea (PTU-23) Pincus, S. E., D. C. Diamond, E. A. Emini, , E. Wimmer on the protein synthesis in Krebs-II cells infected with (1986). Guanidine-selected mutants of poliovirus: map- encephalomyocarditis virus. Zbl. Bakt. Hyg., I Abt. Orig. ping of point mutations to polypeptide 2C. J. Virol. 57: A254: 306-317. 638-646. Galabov, A. S., L. Nikolaeva-Glomb, I. Nikolova, R. Vassi- Prichard, M. N., C. Jr. Shipman (1990): A three-dimension- leva-Pencheva (2012). Perspectives for effective chemo- al model to analyze drug-drug interactions. Antiviral Res. therapy of enterovirus infections, in: Najdenski, H., M. 14; 181-206. Angelova, S. Stoitsova (Eds.),. New Trends of Microbiol- Rada, B., J. Zavada (1962). Screening test for cytostatic and ogy (65th Anniversary of the Stephan Angeloff Institute of virostatic substances. Neoplasma 9: 57-65. Microbiology). The Stephan Angeloff Institute of Micro- Saunders, K., A. M. King, D. McCahon, J. W. Newman, W. biology, Bulgarian Academy of Sciences, Sofia, pp. 47-81. R. Slade, S. Forss (1985). Recombination and oligonucle- Herrmann, E. C. Jr, J. A. Herrmann, D. C. Delong (1982). otide analysis of guanidine-resistant foot-and-mouth dis- Prevention of death in mice infected with coxsackievirus ease virus mutants. J. Virol. 56: 921-929. A16 using guanidine HCl mixed with substituted benzim- Tallarida, R. J. (2011). Quantitative methods for assessing idazoles. Antiviral Res. 2: 339-346. drug synergism. Genes Cancer. 2: 1003-1008. Klein, M., D. Hadaschik, H. Zimmermann, H. J. Eggers, B. Workshop Report, Committee on Development of a Polio An- Nelsen-Salz (2000). The picornavirus replication inhibi- tiviral and its Potential Role in Global Poliomyelitis Erad- tors HBB and guanidine in the echovirus-9 system: The ication (ed. Press, T. N. A.) (National Research Council of significance of viral protein 2C. J. Gen. Virol. 81: 895- the National Academy, Washington, DC, 2006). 901.

55 ACTA MICROBIOLOGICA BULGARICA

Screening for Antimicrobial Activity of Bacillus subtilis and Paenibacillus Alvei Isolated From Rotten Apples Compost Natalija Atanasova-Pancevska1*, Ilina Popovska1, Katarina Davalieva2, Dzoko Kungulovski1 1Department of Microbiology and Microbial Biotechnology, Institute of Biology, Faculty of Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, Skopje, Macedonia 2Research Centre for Genetic Engineering and Biotechnology “Georgi D Efremov”, Macedonian Academy of Sciences and Arts, Skopje, Republic of Macedonia Abstract New antimicrobial compounds are continuously required to combat antibiotic-resistant bacteria and pathogenic yeast as such resistance increasingly limits the effectiveness of current antimicrobial drugs. New screening approaches, including the search for novel targets and exploration of non-conventional places as sources of producer microorganisms, are needed. Rotten apples compost samples from the composting plant in Resen, Macedonia, were screened for microbial colonies which form a clear zone of inhibition. The isolated bacterial strains were further screened for their antimicrobial activity against different bacteria and fungi by diffusion agar method. The isolates were later identified as Paenibacillus alvei and Bacillus subtilis by morphological studies and sequencing of the 16S rRNA bacterial gene. The maximum growth of P. alvei and B. subtilis for different cultivation media, pH, temperature, time of incubation and glucose concentration were determined. Maximum growth of P. alvei and B. subtilis was observed in MHB, at pH 6 after 24 hours of incubation at 30 °C for P. alvei, and at pH 7 after 48 hours at 40 °C for B. subtilis. In conclusion, the identified strains with antimicrobial activity in this study are a useful addition to the worldwide effort for new antibiotic discovery. Key words: Paenibacillus alvei; Bacillus subtilis; antimicrobial activity; optimization; 16S rRNA squen cing.

Резюме В борбата с резистентните бактерии и патогенни дрожди непрекъснато се изискват нови антимикробни съединения, тъй като тяхната резистентност ограничава ефективността на настоящите антимикробни лекарства. За това са необходими нови подходи за скрининг, включително и за търсенето на нови целеви места и неконвенционални източници на микроорганизми-продуценти. Проби от гниещи ябълки, събрани от завода за компостиране в Ресен, Македония, са скринирани за колонии от микроорганизми, образуващи зона на инхибиране. Изолираните бактериалните щамове са изследвани допълнително за тяхната антимикробна активност срещу различни бактерии и гъби по метода за дифузия в агар. Изолатите са идентифицирани като Paenibacillus alvei и Bacillus subtilis чрез морфологични проучвания и секвениране на 16S рРНК. Установен е максималня растеж на P. alvei и B. subtilis върху различни среди за култивиране, рН, температура, време на инкубиране и концентрация на глюкоза. Максимален растеж на P. alvei се наблюдава в среда МБ и рН 6, след 24 часа инкубиране при 30°С и за B. subtilis ва същата среда с рН 7, след 48 часа инкубиране при 40°С. В заключение, изолираните и идентифицирани щамове с антимикробиална активност в настоящето изследване са полезно допълнение към световните усилия за откриване на нови антибиотици. Introduction Due to the increasing numbers of resistant isting antibiotics, new antimicrobial compounds pathogenic bacteria and side effects caused by ex- with effective properties are needed (Devasahayam et al., 2010). *Corresponding author: [email protected]

56 Antimicrobial peptides are attracting increas- Screening for antimicrobial activity ing interest as a potential substitute and the best al- The isolates were initially screened for their ternatives for biological control (Papagianni, 2003; antimicrobial activity by a diffusion agar method Lee et al., 2008). Antimicrobial peptides are sec- (Hasegawa et al., 1990). They were inoculated into ondary metabolites produced by microorganisms Muller Hinton broth medium and incubated for via enzymatic catalysis involving a series of bio- 48 hours at 27 °C. As test microorganisms were synthetic pathways (Demain et al., 1983). These used freshly prepared (i.e, 24-hour-old) cultures peptides occur in living organisms and are proof Gram-positive bacteria (Bacillus subtilis ATCC duced as defense molecules against pathogens such 6633, Staphylococcus aureus ATCC 6538, Micro- as bacteria. Therefore, antimicrobial peptides are coccus luteus), Gram-negative bacteria (Escheri- considered the first line of defense in invaded eu- chia coli ATCC 8739 and Pseudomonas aerugino- karyotic and prokaryotic cells (Papagianni, 2003). sa ATCC 9027 inoculated in MHB medium), yeasts It was found that the effect of different factors (Candida albicans ATCC 10231, Saccharomyces affecting growth such as incubation period, temper- cerevisiae ATCC 9763) and molds (Aspergillus ni- ature, pH, aeration, addition of surfactants and the ger ATCC 16404, cucumber mold, tangerine mold constituents of the production medium used, espe- and Penicillium sp. inoculated in Sabouraud dex- cially carbon sources, nitrogen sources and miner- trose broth medium). One hundred microliters of als, depends on the microbial strain and its growth each test microorganism were transferred into 15 rate (Vincent and Priestly, 1975; Kaur et al., 2001). ml of appropriate molten medium at 45 °C and This study aimed to screen for antimicrobi- poured into a separate empty sterile petri plate. The al activity of isolates from rotten apples compost agar was allowed to solidify for about 30 minutes against some microorganisms, to identify the strains and 10 µl of the isolates which showed antimicro- using a molecular identification method based on bial activity were transferred on 5 mm sterile filter 16S rRNA sequencing and to suggest the optimum paper disks, previously placed on properly marked conditions for maximum antimicrobial activity of places on the surface of agar plates inoculated with the selected strains. test microorganisms. Sterile distilled water (10 µl) was used as a negative control and gentamicin (or Materials and methods cycloheximide) antimicrobial susceptibility test Collection of compost samples disks (10 µg) were used as a positive control. The Rotten apples compost samples were collect- petri plates were kept in a refrigerator for 1 hour ed from the composting plant in Resen, Macedonia. before incubation to permit the diffusion of antimi- The collected samples were stored into sterile glass crobial substances (Rizk et al., 2007). The plates screw cap bottles. The collected compost sample were then incubated for 5 days at 25 °C for yeast was kept for screening and isolation of different and molds, and 72 hours at 37 °C for bacteria. Af- microorganisms with antimicrobial potential. ter incubation, the diameters of the inhibition zones Isolation of microorganisms with antimicrobial were measured. potential Identification of the microorganisms by 16S rRNA Fifty grams of compost were taken and add- sequencing technique ed to 250 ml sterile distilled water in a 500 ml Er- The phenotypic properties of the selected lenmeyer flask. The flask was shaken on an orbital strains were determined using the methods de- shaker for 30 min at 27 °C and serial dilutions from scribed in Bergey’s Manual of Determinative Bac- 10-1 to 10-6 were performed. From each dilution, teriology (Holt et al., 1994). The selected antimi- about 0.5 ml of sample was taken and placed on crobial strains were identified by sequencing of the Muller Hinton agar (MHA) medium along with an- 16S rRNA gene. First, DNA from each strain was timycotic cycloheximide (5 g mL-1) using pour plate isolated. Each pure colony was grown overnight in technique and incubated at 27 °C for 1 week. After the appropriate medium, cells were harvested by the incubation period, the plates were observed for centrifugation (14000 rpm, 10 min), washed twice microbial colonies which had formed a clear zone with 1xPBS buffer (140 mM NaCl, 2.7 mM KCl, of inhibition. They were selected and picked up by 100 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) and a sterilized wire loop and sub-cultured on MHA to kept at -20oC until further processing. DNA extrac- obtain pure bacterial colonies. The pure cultures tion was done using PrepManUltra reagent (Ap- were preserved on agar slants of Muller Hinton me- plied Biosystems), following the protocol for cul- dium for further studies. ture broth samples. The concentration of DNA was

57 A.

Fig. 1. Identification of bacterial strains with highest antimicrobial activity. Phylogenetics trees were obtained by BLAST pairwise alignments of NCBI bacterial 16S ribosomal RNA sequences against 16S rRNA sequences of AA11 and AA3 strains. The strains were identified as strains of Bacillus subtilis (A) and Paenibacillus alvei (B). The magnification of the microscopic images shown is 100×. determined spectrophotometrically. DNA working was performed with forward and reverse primers solution of 2.7 – 3.1 ng/µl was prepared by dilut- for each amplified product according to the instruc- ing the stock DNA. The sequence of the 16S ribo- tions provided by the kit with one exception: the somal RNA gene (rDNA) of bacterial strains was final volume of the sequencing reactions was 10 µl. determined using MicroSeq Full Gene Kit (Applied After cycle sequencing, excess dye terminators and Biosystems), composed of two parts: MicroSeq® primers were removed from the cycle sequencing Full Gene 16S rDNA Bacterial Identification PCR reactions by precipitation in separate tubes with 2 Kit and MicroSeq® Full Gene 16S rDNA Bacterial µl 5M Na-acetate and 50 µl ethanol. After incuba- Identification Sequencing Kit. Amplification of the tion at room temperature for 30 min, the tubes were three fragments of the 16S ribosomal RNA gene centrifuged at 14000 rpm for 30 min, the superna- was done using 7.5 µl DNA working solution in a tant was discarded, the precipitate was dried for 5 reaction volume of 15 µl on 2720 Thermal Cycler min at room temperature and re-suspended in 20 (Applied Biosystems). Purification of the ampli- µl of Hi-Di™ Formamide. Sequence analyses were fied products was done using ExoSAP-IT® reagent performed on a 3500 Genetic Analyzer (Applied (USB) according to the manufacturer’s instruc- Biosystems). tions prior to sequencing. The cycle sequencing

58 Fig. 2. Determination of optimal cultivation medium for growth of P. alvei and B. subtilis in a stationary series (first row) and series on a rotary shaker (second row).

Optimization of various parameters for maximum activity Various parameters such as cultivation medium, pH, temperature, time of incubation and glucose concentration were tested to determine the optimal conditions for microbial growth. In order to determine the most effective medium for mass cell production of isolates, four different media were tested: Muller Hinton broth, Nutri- ent broth, NYD medium (Li et al., 2009) and Man- nitol broth. Fifty mililiters of each medium were aliquoted in four separate 100 ml Erlenmeyer flasks and autoclaved. Each flask was inoculated with 1.5 ml of 24-hour-old cultures from each isolate. Two Erlenmeyer flasks of each medium were incubated stationary at 25°C and the remaining two on a rotary shaker at 120 rpm, at ambient room tempera- Fig. 3. Effect of various pH levels on the ture (22-28°C). Bacterial growth was monitored by growth of P. alvei and B. subtilis in a stationary measuring the turbidity of a culture at the beginning series. and every 24 hours untill 144 hours using a spectro-

Table 1. Changes in the pH of the media after 72 hours of incubation

Before pH 5 pH 6 pH 7 pH 8 incubation P. alvei After pH 6.77 pH 7.00 pH 7.04 pH 7.35 incubation Before pH 5 pH 6 pH 7 pH 8 incubation B. subtilis After pH 5.14 pH 5.53 pH 6.72 pH 7.15 incubation

59 Fig. 4. Effect of temperature and time of incubation on the growth of P. alvei and B. subtilis.

A. B.

Fig. 5. Diffusion agar method for determining A) the antifungal activity of P. alvei against Penicillium sp. B) the antibacterial activity of B. subtilis against S. aureus ATCC 6538. C) P. alvei and B. subtilis show no antibacterial activity against P. aeruginosa ATCC 9027.

Table 2. Antibacterial activity of P. alvei and B. subtilis against different bacterial species

Diameter of zone of inhibition (mm) Control Control Test organism P. alvei B. subtilis (10 µg gentamicin) (sd H2O) 13 bc 0 E. coli ATCC 8739 15 bs1 25 bc2 27 bs B. subtilis ATCC 14 bc 8 bc 28 bc 0 6633 P. aeruginosa ATCC 0 0 17 bc 0 9027 S. aureus ATCC 6538 15 bc 40 bc 26 bc 0 M. luteus 20 bc 0 26 bc 0 1- bactericidal effect; 2- bacteriostatic effect

60 Table 3. Antifungal activity of P. alvei and B. subtilis against different fungal species

Diameter of zone of inhibition (mm) Control Control Test organism P. alvei B. subtilis (10 µg (sd H O) cycloheximide) 2 C. albicans ATCC 20 fc1 0 0 0 10231 S. cerevisiae ATCC 15 fc 0 46 fc 0 9763 A. niger ATCC 18 fc 0 12 fs2 0 16404 Cucumber mold 27 fc 0 0 0 Tangerine mold 17 fc 0 11 fs 0 Penicillium sp. 24 fc 0 14 fs 0 1- fungicidal effect; 2- fungistatic effect photometer (Lovibond, Photometer MaxiDirect) to ator at +4°C and on glass beads at -20°C. Further determine transmission or absorbance. screening was done to determine the bioactivity of The influence of pH on bacterial growth was the isolates. tested using the determined optimal medium at pH ranging from 5-8. pH was adjusted before autoclav- Identification of microorganisms ing. For each pH, two Erlenmeyer flasks with 50 ml Morphological studies showed that the iso- medium were prepared, autoclaved, then inoculat- lates are Gram-positive, sporulating, rod shaped ed with 1.5 ml of inoculum of 24-hour-old cultures bacteria (Fig. 1). Alignment of the 16S rRNA se- and incubated stationary at 25 °C. The turbidity of a quences of the 2 bacterial species revealed identi- culture was measured before autoclaving and every ty of 99% to the genus Bacillus. Isolates AA3 and 24 hours untill 72 hours, after which the pH of the AA11 were identified as Paenibacillus alvei and medium was again registered in order to observe Bacillus subtilis, respectively. Inoculated on MHA, media pH changes. P. alvei produced large, circular, rough, white-yel- The effects of temperature and incubation pe- lowish colonies with irregular margins and B. riod on the bacterial growth were studied at 25, 30, subtilis produced very small, circular, convex and 35 and 40°C under different incubation periods (0, smooth, white in color colonies. The spores of B. 24, 48, 72 and 96 hours) using determined optimal subtilis and P. alvei are smooth, spherical and green medium and determined optimal pH. For each cul- in color using the Schaeffer and Fulton staining ture, eight tubes with 7 ml medium were prepared, method. inoculated with 70 µl 24-hour-old culture isolates and incubated at four different temperatures. The Optimization of various parameters for bacterial turbidity of a culture was measured before auto- growth claving and every 24 hours untill 96 hours. Maximum growth of P. alvei in a stationary and rotary shaker series was observed in MHB after Results 24 and 96 hours of incubation, respectivеly (Fig. 2). Isolation of microorganisms with antimicrobial Maximum growth of B. subtilis in a stationary and potential rotary shaker series was observed in MHB after 48 In the course of screening for novel substanc- and 24 hours of incubation, respectivеly (Fig. 2). es with antimicrobial potential from rotten apples Regarding the optimal pH of the growth me- compost samples collected from the composting dium according to the results of the previous exper- plant in Resen, Macedonia, two bacterial colonies iment, maximum growth for P. alvei was observed (AA3 and AA11) were isolated and screened using at pH 6, and for B. subtilis at pH 7 after 72 hours MHA medium. The isolated cultures were sub-cul- of incubation (Fig. 3). After 72 hours of incubation, tured on MHA slants and preserved in a refriger- changes in the pH of the media inoculated with P.

61 alvei and B. subtilis were observed (Table 1). crobial activity. The effects of temperature and incubation The identified B. subtilis shows potent anti- period on the bacterial growth of P. alvei and B. bacterial activity. The most sensitive strain to its subtilis were studied in MHB at pH 6 and pH 7, antibacterial activity was S. aureus ATCC 6538, respectively. The results showed that the optimal against whom B. subtilis forms a 40 mm inhibition temperature for growth of P. alvei is 30 °C and the zone. Awais et al. (2007) studied the inhibitory ef- optimal incubation period is 24 hours. The optimal fects of a Bacillus sp. isolate against two pathogen- temperature for growth of B. subtilis is 35 °C and ic strains of Micrococcus luteus ATCC 10240 and the optimal incubation period is 48 hours (Fig. 4). Staphylococcus aureus ATCC 6538 and determined an 18 mm inhibition zone for these two indicator Preliminary screening for antimicrobial activity strains. In our study, B. subtilis showed no antifun- The isolated bacterial strains from rotten gal activity, but antimicrobial studies of Moshafi apples compost were screened for secondary meet al. 2015 determined its ability to inhibit C. albi- tabolites with antimicrobial activity by diffusion cans, A. flavus and A.niger as well. agar method under conditions which showen as The other identified strain with antibacte- optimal in previous experiments. Paenibacillus rial activity was P. alvei. P. alvei are Gram-posi- alvei showed potential antifungal activity against tive, rod-shaped, motile, spore-forming and cat- all tested yeasts and molds, and antibacterial activ- alase-positive bacteria (Najafi et al., 2011). The ity. Fig. 5A shows the potential antifungal activity first report of antimicrobial peptide production by of P. alvei against Penicillium sp. Bacillus subti- these bacteria was by Anandaraj et al. 2009, who lis showed potential antibacterial activity against isolated a strain from fermented tomato fruit and some Gram-positive and Gram-negative bacteria, detected two antimicrobial peptides, Paenibacillin but no antifungal activity. Fig. 5B shows antibac- P and Paenibacillin N. P. alvei has potent antibacte- terial activity of B. subtilis against S. aureus ATCC rial and antifungal activity and showed the highest 6538. Fig. 5C shows that P. alvei and B. subtilis zones of inhibition against M. luteus (20 mm) and have no antibacterial activity against P. aeruginosa cucumber mold (27 mm). The antibacterial activity ATCC 9027. P. alvei and B. subtilis also exhibited of P. alvei AN5 against different bacterial strains a potent antimicrobial activity against a wide range was confirmed by Alkotaini et al., (2013) and Al- of both Gram-negative and Gram-positive bacteria Obaidy (2010). These studies showed that P. alvei, and fungi (Table 2 and Table 3). isolated from rhizosphere soil of garden flowers, had a wide range of antifungal activities toward Discussion different kinds of pathogenic fungi. Soil is a prosperous source of microorganism In order to achieve rapid and unambiguous that produces a wide range of antibiotics includ- identification of the strains that showed the highest ing peptide antibiotics (Awais et al., 2008; Janabi, antimicrobial activity, molecular methods of identi- 2006). The development of antibiotics resistance fication were preferred over culture-based methods. and lesser safety margins provoked scientists to Determinative bacteriology based on culture-based search for antimicrobial agents with modified prop- methods involves time-consuming isolation, cul- erties and maximum activity. Bacillus species have tivation and characterization of phenotypic traits, been considered as extremely useful microorgan- which is often not discriminatory and can take days isms for producing antimicrobial agents (Amin et to weeks for unambiguous identification. The 16S al., 2012). Bacillus species are aerobic or faculta- rRNA gene, universally present in all bacteria, has tive anaerobic, sporulating, rod-shaped, Gram-pos- both highly conserved and more variable domains, itive bacteria (Ali Janabi et al., 2006; Graumann, which makes it an ideal target for studying phy- 2007). Some species may turn Gram-negative with logenetic relationships and obtaining precise and age (Baron, 1996). The Bacillus species are known reliable identification. for the synthesis of secondary metabolites with In this study different experiments were per- remarkable diversity both in structure and func- formed to determine the most favorable conditions tion (SiloSuh et al., 1994). The Bacillus species for growth of B. subtilis and P. alvei. The highest are most popular for producing peptide antibiotic turbidity of P. alvei and B. subtilis in a stationary compounds such as polymyxin, colistin and circu- series and on a rotary shaker incubation was ob- lin (Katz and Demain, 1997). In this study we have served in MHB. The observed increasing turbidity also identified a Bacillus strain with strong antimi- in the NYD medium as a function of the incuba-

62 tion period may be due to the high concentration Awais, M., A. Pervez, S. Qayyum, M. Saleem (2008). Effects of sugars in the medium (10 gL-1 glucose). Glucose of glucose, incubation period and pH on the production of initially inhibited the growth of the cells and then as peptide antibiotics by Bacillus pumilus. Afr. J. Microbiol. Res. 2: 114-119. the cells adapted and began to exploit sugars, their Baron, S. (1996). Medical Microbiology. 4th ed. Texas, TX, concentration decreased and subsequently, the bac- USA: University of Texas Medical Branch. terial cells growth increased. The optimal pH for Demain, A. L., Y. Aharonowitz, J. F. Martin (1983). Metab- P. alvei and B. subtilis was determined to be pH 6 olite control of secondary biosynthetic pathways. in: L. and pH 7, respectively, after 72 hours of incubation. C. Vining (Ed.), Biochemistry and Genetic Regulation of Amin et al. (2012) studied the effect of pH on zones Commercially Important Antibiotics. 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64 ACTA MICROBIOLOGICA BULGARICA

In Vitro Antimicrobial and Antioxidant Activity of Extracts from Wild Small Fruits Spread in Bulgaria Hristo M. Najdenski1*, Violeta S. Kondakova2, Ekaterina T. Krumova1, Maya M. Zaharieva1, Lyuba J. Doumanova1, Maria B. Angelova1 1 The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str. 26, Sofia 1113, Bulgaria 2 Agrobioinstitute, Dragan Tzankov bul, 8, Sofia, 1164, Bulgaria

Abstract The antimicrobial and antifungal activities of the methanol extracts from 5 different wild small berries fruits originating from Bulgaria and collected in 2012 were evaluated against selected Gram-positive and Gram-negative bacteria as well as the fungus Candida albicans by agar diffusion method. Microbial growth was measured by the microdilution test. The antioxidant potential of the same extracts was determined by two complementary methods - DPPH-radical scavenging test and superoxide-anion scavenging assay. Different bacterial species exhibit varying sensitivities towards total extracts, but all extracts exhibited a strong antioxidant capacity. No antifungal activity was found in all examined berry extracts. In general, the wild Bulgarian berry fruits are a good source of antioxidant compounds with defined antimicrobial properties and can be used as a potential value-added ingredient in the food, cosmetic and pharmaceutical industry. Ключови думи: диви дребни горски плодове, антимикробна активност, антиоксидантна активност

Резюме Антимикробната и антимикотична активности на метанолни екстракти от 5 различни вида диворастящи дребни горски плодове, събрани през 2012 г. на територията на България, бяха изследвани върху избрани Грам положителни и Грам отрицателни бактерии и фунги от рода Candida albicans с помощта на агар-дифузионен метод. Микробният растеж беше измерен с микротитърен тест. Антиоксидантния потенциал беше определен по два комплементарни метода - DPPH-радикал и супероксид-анйон улавяща активност. Различните бактериални видове показаха различна чувствителност спрямо тоталните екстракти, всички от които имаха силна антиоксидантна активност. Не беше установена антимикотична активност при нито един от изследваните екстракти. В заключение, диворастящите български горски плодове са добър източник на антиоксиданти с дефинирани антимикробни свойства и могат да бъдат използвани като потенциални продукти с висока добавена стойност във фармацевтичната индустрия. Introduction Berries are traditional part of the European stances like vitamins (ascorbic acid, vitamins A diet and represent good sources of beneficial bio- and E), phenolic compounds (flavonoids, phenolic logically active compounds, which may play an im- acids, lignans, and polymeric tannins), titratable portant role in the maintenance of the human health acids, sugars, anthocyanins, essential oils, carote- (Howell, 2002; Puupponen-Pimia et al., 2001; Rau- noids, minerals, fibres etc. (Lee et al., 2012; Nile ha et al., 2000). Wild berries are distinguished by and Park, 2014; Nohynek et al., 2006; Paredes-Lo- a wide spectrum of pharmacologically active sub- pez and Valverde, 2012). The beneficial preventive and therapeutic effects of these compounds for hu- th *Corresponding author: Prof. H. Najdenski, PhD, DSc man health have been known from at least the 16 Address: The Stephan Angeloff Institute of Microbiology, century (Cisowska et al., 2011). Nowadays, their Bulgarian Academy of Sciences, Acad. G. Bonchev Str. 26, Sofia 1113, Bulgaria; Tel.: +3592 979 3161; Fax: +3592 987 01 09; antimicrobial, antitumor, antiulcer, antioxidant and E-mail address: [email protected] anti-inflammatory activities are being intensively 65 investigated and documented (Ayachi et al., 2009; et al., 2014). Kähkönen et al., 2001; Nakajima et al., 2004; Pan- Phytochemicals such as flavonoids and other ico et al., 2009; Ribera et al., 2010; Rufino et al., phenolics may have antioxidant activity that helps 2010; Vasco et al., 2008) due to their high potential protect cells against the oxidative damage caused for use in the food, cosmetic and pharmaceutical by free radicals (Nile and Park, 2014). Due to a high industry. Recently, there is an increasing interest to content and wide diversity of phenolic compounds, measure the antimicrobial and antioxidant capacity berries exhibit high biological activity against free of different berries (Ayachi et al., 2009; Panico et radicals, including superoxide radicals, hydrogen al., 2009; Vasco et al., 2008). peroxide, hydroxyl radicals, and singlet oxygen Among the berries, lingonberries, blackcur- (Panico et al., 2009) reducing the oxidative damage rant, cloudberries, red raspberries, strawberries, of human cells (Bazzano et al., 2003). The major blackberry, and bilberries possess clear antimicro- contribution to the antioxidant capacity of berries bial effects against various human pathogens. Berry of the genus Rubus and Fragaria are the ellagitan- ellagitannins (ETs) are strong antimicrobial agents nins, which represent between 51% and 88% of acting as possible anti-adherence compounds in all phenolic compounds depending on the growth preventing the colonization and infection by many and stress conditions (Jimenez-Garcia et al., 2012). pathogens (Nile and Park, 2014). Several studies Blackberry and strawberry phenolic compounds have shown the capacity of berry fruits’ extracts are known for their protective effects on age-re- to inhibit the growth of food-borne, urinary tract lated neurodegenerative diseases and bone loss in and skin pathogens such as Salmonella, Enterococ- vivo and can inhibit low-density lipoprotein and li- cus, Staphylococcus and Streptococcus sp., E. coli, posomal oxidation in vitro (Giampieri et al., 2012; Mycobacterium phlei and Neisseria meningitidis, Kaume et al., 2012). Strawberry fruits also demon- as well as pathogenic fungi such as Candida albi- strate scavenging effect against radicals thus pre- cans (Cavanagh et al., 2003; Toivanen et al., 2011). venting lipid peroxidation in in vitro experiments Lingonberries (Vaccinium vitis-idaea) products are (Giampieri et al., 2012; Ozsahin et al., 2012). most commonly applied for prevention and treat- The antimicrobial and antioxidant properties ment of urinary tract infections caused by uropath- of cultivated plants are usually well documented ogenic E. coli being included in the composition in difference to the wild plants. However, the lit- of multiple dietary supplements (Česonienė et al., erature data indicate that wild berry fruits possess 2009; Wojnicz et al., 2012). They possessed antimi- higher concentration of sugar, organic and phenolic crobial activity also on a wide range of other human compounds than the cultivated and domesticated pathogenic bacteria, both Gram-negative (Salmo- cultivars of the same species or genus (Bunea et al., nella typhimurium) and Gram-positive (Enterococ- 2011; Mikulic-Petkovsek et al., 2012). Differenc- cus faecalis, Listeria monocytogenes, Staphylococ- es between investigated cultivars and wild plants cus aureus, and Bacillus subtilis) (Česonienė et al., are usually based on geographic region, genotype, 2009). In addition, purified flavonol glycosides and growing season, maturity, period of harvesting, procyanidins isolated from V. vitis-idaea inhibited etc. Giovanelli and Buratti (2009) reported that the biofilm formation of Streptococcus mutans, a hu- antioxidant activity of wild Italian blueberries is man oral pathogen routinely associated with the much higher as compared to cultivated ones. Un- formation of human dental caries (Riihinen et al., fortunately, the beneficial biological properties of 2014). Berry juices from Ribes species demonstrat- wild small fruits are poorly investigated by now ed antifungal properties against human pathogenic and there are only few scientific reports available Candida species (Krisch et al., 2009). Crude ex- about their antimicrobial and antioxidant activities. tracts from Ribes nigrum fruits displayed antiviral Further detailed studies are required in order to elu- activity against herpes and influenza viruses (Krisch cidate their pharmacological potential. et al., 2009; Roschek et al., 2009). Rubus idaeus The consumption of small wild berries has L. extracts are traditionally used in eastern parts of been a long time tradition in Bulgaria, especially Europe as herbal remedy in common cold, fever in the mountain areas where they have been used and flu-like infections. Recent studies revealed that in folk medicine as water extracts and dried fruits young shoots of R. idaeus stand out as a valuable (Dimkov, 2001). It should be noted that the infor- source of sanguiin H-6 and ellagic acid, thus pos- mation of the antimicrobial and antioxidant prop- sessing antioxidative, antimicrobial and cytotoxic erties of the Bulgarian berries is scare in general. properties (Denev et al., 2014; Krauze-Baranowska Therefore, the principal objective of this study was

66 to provide data about the in vitro antimicrobial layer. Briefly, 0.2 ml of bacterial/fungal suspen- and antioxidant activity of the total extracts from sion (~1.0 × 105 CFU ml-1) was plated on the agar 5 different wild berries fruits (blackberries, straw- layer into Petri dishes (10 cm d). After preparing 6 berries, raspberries, lingonberries, and bilberries) wells per dish with a diameter of 9 mm, 0.1 ml of naturally growing in Bulgaria. each sample was dropped into each well. The Petri dishes were placed for pre-diffusion at 4oC for 2 h Material and Methods for improvement the sensitivity of the test system. Plant Collection Antimicrobial activity was measured by the diam- Fruits of blackberries (Rubus ursinus), straw- eter of inhibitory zones in the agar layer after 48 h berries (Fragaria vesca), raspberries (Rubus idae- incubation at 37 °C. Extracts giving an inhibitory us L.), lingonberries (Vaccinium vitis – idaea) and zone with a diameter (d) less than 13 mm indicat- bilberries (Vaccinium myrtillus) were collected in ed a lack of activity. Experiments with methanol 2012, in west Rhodope mountain (nearby Bozyo- as a respective solvent were carried out as nega- va GPS: 41°55‘51.73“N; 23°46‘14.98“E; altitude: tive control. Ceftazidime (Biochemie, Austria) and 1600 m). The samples were freeze-dried, ground Nistatin (Actavis, Bulgaria) were used as reference and stored at –80◦C prior to extraction. substances (positive control). The assays were per- Chemicals formed in triplicates for each berry extract. DPPH, nitro blue tetrazolium (NBT), me- Minimal inhibitory concentration (MIC) thionine, riboflavin, NaCN, superoxide dismutase The minimal inhibitory concentration (MIC) (SOD) from bovine erytrocytes and caffeic acid of all samples was determined by the microdilution were provided by Sigma-Aldrich Chemie GmbH, method described by Andrews (Andrews, 2001) by Deisenhofen, Germany. using 96-well standard microtiter plates. Briefly, 50 Extraction Procedure µl of twofold serial dilutions of examined samples Frozen wild berry fruits were used for prepa- were added to 50 µl of microbial suspension ad- ration of total methanol extracts. The samples of justed to yield approximately 1.0 × 105 CFU ml-1. wild berry fruits were placed in 1 L round bottom MIC was encountered as the lowest concentration flasks and lyophilized. Determined standard quan- of examined sample that inhibits the visible micro- tity of lyophilized powder of each sample was bial growth after 24 h incubation at 37 °C. Negative moistened with methanol in ratio 1:20, stirred and controls were included too. kept in the dark at a room temperature 24 h. Then DPPH radical scavenging assay a second extraction under the same conditions was The scavenging of DPPH radicals was de- carried out. termined following the procedure described by Individual extracts of wild berry fruits were Kovatcheva-Apostolova et al. (Kovatcheva-Ap- filtered through a filter paper and both filtrates of ostolova et al., 2008). Two milliliters of 100 μM each sample were mixed and concentrated under DPPH solution were mixed with 0.1 mL solution, vacuum at 40°C to a defined volume. The concen- containing enhanced concentration of total extract tration of each of filtrates used in the next biologi- of above mentioned small fruits (from 0 to 120 μg/ cal tests was determined and calculated. mL). The inhibition of DPPH radicals (in %) was Bacterial strains and growth conditions determined after 5 min by reading the absorbance at The test bacteria Staphylococcus aureus 209, 516 nm (Shimadzu 1240 UV/vis spectrophotome- Streptococcus pyogenes 10535, (Gram-positive) ter). Inhibition of free radical DPPH in percent was and Еscherichia coli WF+ and Salmonella typhi- calculated according the formula: murium 1923, (Gram-negative) were obtained from Inhibition %= (Ablank – Asample/Ablank) x 100, the Bulgarian Type Culture Collection. All bacteria where Ablank is the absorbance of the control were cultivated on Tryptic Soy Agar (Difco, USA). reaction (containing all reagents except the test

Candida albicans 562 grown on Sabouraud agar compound) and Asample is the absorbance of the test

(Difco, USA) was used for screening antifungal ac- compound. Further the IC50 values, which represent tivity. the necessary concentration of sample for 50% re- Antibacterial and antifungal activity duction of the absorbance of DPPH radicals, were Antibacterial and antifungal activity was calculated. Tests were carried out in triplicate. The checked by the agar diffusion method of Spooner synthetic antioxidant buthylated hydroxy toluene and Sykes (Spooner and Sykes, 1972) encountering (BHT) was included in the experiments as positive the diameter of the inhibitory zone in a soft agar control.

67 Superoxide anion-scavenging activity teric pathogens E. coli and S. typhimurium, and The inhibition of NBT-reduction by photo- the fungus C. albicans. The highest antibacterial • - chemically generated O2 was used to determine activity against the Gram-positive species S. au- the superoxide anion-scavenging activity of the reus and S. pyogenes demonstrated the extract test samples(Beauchamp and Fridovich, 1971). The from strawberry with inhibitory zones of 17.3±1.2, reaction mixture contained 56 mM (NBT), 0.01 M respectively 20.7±1.1 mm (Table 1). Similar high methionine, 1.17 mM riboflavin, 20 mM NaCN and activity against S. pyogenes was found by extracts 0.05 M phosphate buffer pH 7.8. Superoxide was from raspberry and lingonberry (inhibitory zones: measured by the increasing amount of the absorb- 20.3±0.6, respectively 18.7±1.2 mm). Interesting ance at 560 nm at 30° C after 6 min incubation from finding was the antibacterial activity of lingon- the beginning of illumination. The plant extracts and berry’s extract not only against the Gram-positive the reference substance caffeic acid were assayed at species S. pyogenes (inhibitory zone 18.7±1.2), but varying concentrations with three repetitions (inhi- against the Gram-negative enteropathogen S. typh-

bition between 20 and 80%). IC50 (concentrations, imurium (inhibitory zone 17.0±1.0 mm). Bacteria required to inhibit NBT reduction by 50%) values of the species E. coli were not inhibited by any of were calculated from dose-inhibition curves. The the tested extract in difference to the applied posi- influence of superoxide dismutase (SOD from bo- tive control ceftazidime (34.3±3.1). Similarly, the vine erythrocytes, 4870 U/mg) on non-enzymatic antifungal drug nistatin was applied against the reduction of NBT was determined by addition of fungus C. albicans (Table 1). No antifungal activity varying amounts of SOD to the above reaction mix- was found in all examined berry extracts. ture. Total methanol extracts of strawberry, raspberry, and lingonberry were subjected also to broth Results microdilution assay. Only the Gram-positive bac- The antimicrobial and antifungal activities teria (S. aureus and S. pyogenes) were inhibited by of five berry extracts were first measured by agar the extract of strawberry in a concentration of 15 diffusion which is a suitable for semi-quantitative mg ml-1 (Table 1). A weaker activity was found for estimation followed by MIC evaluation as a valu- the extracts of raspberry and lingonberry against S. able quantitative and reproducible assay. For agar pyogenes in concentrations of 28.1 mg ml-1, respec- diffusion and microdilution assays all total extracts tively 33.1 mg ml-1. No activity was found against from blackberry, strawberry, raspberry bilberry and the Gram-positive S. aureus and the Gram-negative lingonberry were dissolved in methanol in concen- pathogens E. coli and S. typhimurium, as well as tration varying from 225.2 to 264.8 mg/ml. Their against C. albicans. antimicrobial and antifungal activities are exam- The antioxidant potential of total extracts ined against the Gram-positive bacterial pathogens was determined by two complementary methods. S. pyogenes and S. aureus, the Gram-negative en- The free radical scavenging activity of all extracts,

Table 1. Antimicrobial activity of methanol berry extracts MIC* of methanol Wild berry / Inhibitory zone of methanol berry extracts berry extracts Test pathogen S. aureus S. pyogenes E. coli S. typhimurium C. albicans S. aureus S. pyogenes Blackberry 15,5±1 14,6±1,2 13,5±1 12,2±1,4 11,8±1,2 0.0 0.0 Strawberry 17.3±1.2* 20.7±1.1 11,6±0,6 10,8±0,8 11,2±1 15.0§ 15.0 Raspberry 10,6±0,6 20.3±0.6 12,4±0,8 11,4±1 11,0±0,8 0.0 28.1 Lingonberry 12,1±1 18.7±1.2 13,0±1,2 17.0±1.0 12,4±1 0.0 33.1 Bilberry 13,2±1,4 12,4±1 12,8±1,0 12,0±1,0 11,8±0,8 0.0 0.0 Methanol 0.0 0.0 0.0 0.0 0.0 n.t. n.t. Ceftazidime 29.3±2.5 27.0±2.0 34.3±3.1 37.0±3.6 0.0 n.t. n.t. Nistatin n.t. n.t. n.t. n.t. 38.5±5.0 n.t. n.t.

Legend: * - minimal inhibitory concentration (MIC); & - inhibitory zone (mm); § - MIC (mg ml-1); n.t. – not tested. 68 evaluated by the DPPH method, is presented in Fig. 1. All extracts tested exhibited a strong antioxidant Blachberry 0,5 Strawberry capacity. They were able to reduce the stable free Raspberry ] radical DPPH to yellow-colored diphenylpicrylhy- 0,4 Lingonberry nm Bilberry drazine. The extracts can be divided into two groups SOD 560

E Caffeic acid according to the detected inhibitory activity. Straw- [ 0,3 berry, lingonberry and bilberry, demonstrated bet- 0,2 ter radical scavenging activity than the blackberry 0,1

and raspberry. The extracts from first group showed Absorbance 100% inhibitions in variants with concentration 0,0 above 80 mg/ml. IC50 values in DPPH method were calculated: blackberry - 91 mg/ml; strawberry - 56 0 20 40 60 80 100 120 140 160 mg/ml; raspberry - 91 mg/ml; lingonberry - 57 mg/ Concentration [µg/ml] ml, and bilberry - 52 mg/ml. The most promising Fig. 2. Inhibitory effect of total extracts on the re- antioxidant in terms of DPPH radicals was E5 (bil- duction of NBT by photochemically generated su- berry). peroxide anion radicals. Samples contained 50 mg/ mL of the corresponding preparations. Control does not contain any extract. Error bars represent stan- 100 dard deviations.

blackberry 80 strawberry raspberry SOD 60 lingonberry bilberry Bilberry + SOD 40 Lingonberry + SOD

Inhibition [%] 20 Raspberry + SOD Strawberry + SOD 0 Blackberry +SOD 0 20 40 60 80 100 120 Concentration [µg/ml] Control 0,0 0,1 0,2 0,3 0,4 0,5 Fig. 1. Free radical scavenging effect of the total Absorbance (E560 nm) extracts from blackberry (¢), strawberry (), raspberry (▲), lingonberry (£), and bilberry Fig. 3. Dose-dependence of the superoxide anion ( ) (DPPH assay). Error bars represent standard scavenging effect of the total extracts from black- deviations. berry (¢), strawberry (), raspberry (▲), lingon- △ berry (£), and bilberry ( ). Caffeic acid () and SOD (Û) were used as positive controls. Com- Further, the antioxidant potential of total ex- mercial SOD from bovine△ erythrocytes with 4870 tracts was evaluated by suppression of the super- U/mg protein and 4870 U/mg solid was used; the oxide anion radicals generated in a photochemical working solution was prepared as 1 mg/mL. Error system in the presence of the test samples. The bars represent standard deviations. results are shown in Fig. 2. All of the investigat- • ed extracts in concentration 50 mg/ml showed O2ˉ For a detailed study of radical scavenging –• radical scavenging activity. The order of scaveng- capacity, we investigated the O2 scavenging ac- ing effectiveness was: bilberry (62.5% inhibition) tivities of samples at different concentrations, and ≥ strawberry (56.3%) ≥ raspberry (40%) ≥ lingon- dose-response curves were formed (Fig. 3). Caffe- berry (36.8) ≥ blackberry (25.5). These results were ic acid and commercial SOD were used as positive lower than those of positive controls, caffeic acid controls. The results indicated that the best scav- and commercial SOD, which were used in concen- enging activity among tested extracts demonstrated trations 6.5 and 3.5 mg/mL, respectively (data not E2 (strawberry) and E5 (bilberry). It is worthy to shown). note that all extracts exhibited less scavenging activity than SOD and caffeic acid.

69 • The 50% O2ˉ scavenging concentrations typhimurium. The extracts were also more active

(IC50) of the total extracts were calculated from the against S. pyogenes in comparison with S. aureus. dose-activity curves. The IC50 were found to be 85, Notably, the strawberry extract was found to be 43.9, 80.5, 63 and 41.8 mg/ml for blackberry, straw- highly active against both S. aureus and S. pyo- berry, raspberry, lingonberry, and bilberry, respec- genes. Highest activity against S. pyogenes showed tively. As positive controls, 50% inhibitory concen- the strawberry and raspberry extracts followed by trations of SOD and caffeic acid were detected as the lingonberry extract. A decrease in the antimicro- 1.04 and 4.9 mg/mL, respectively. bial activity against S. pyogenes as compared to the • Further we studied the combined O2ˉ scav- untreated control was observed in the following or- enging effect of the fruit preparations in the dose der: strawberry > raspberry > lingonberry > black- 50 mg/mL, applied together with 5.5 U/mL of com- berry > bilberry. As far as the various compounds mercial SOD (Fig. 4). The combination resulted in available in the extract should be attributed to anti- 50% and 70% decrease in NBT-reduction respec- microbial activities additional studies are required. tively, compared to the inhibition caused by SOD Studies on Nordic berries reveal that strawberry alone. The results outlined the possibilities to use extract contains ETs which may explain their mod- these alone or in combination with SOD effectively erate antimicrobial effects against Staphylococcus in cases, when the cells are exposed to oxidative and Salmonella bacteria (Puupponen-Pimia et al., stress. 2005). Ellagitannins were detected in berries from the Rosaceae family (cloudberry, raspberry, rose Bilberry hip, sea buckthorn, and strawberry), ranging from 21.7 to 83.2 mg/100 g (Giampieri et al., 2012). Oth- Lingonberry er studies discussed in the same review reported ET Raspberry content in strawberries from 25 to 59 mg/100 g in fresh samples. The representative ET in strawber- Strawberry ries and raspberries is sanguiin H-6. Other investi- gators have reported the presence of galloylbishex- Blackbarry ahydroxydiphenoyl-glucose, previously found in Control the Rubus berry (Mullen et al., 2003). These findings support the results obtained in our study for the 0,0 0,1 0,2 0,3 0,4 moderate to high activity of the strawberry extract E560 nm in S. aureus and S. pyogenes (important causative agents of skin lesions) and of the raspberry extract Fig. 4. Inhibitory effect of the total extracts against S. pyogenes. Different mechanisms of ac- on the NBT-reduction by photochemically gener- tion proposed to explain tannin antimicrobial activ- ated superoxide anions in the presence of SOD. ities including inhibition of extracellular microbi- Samples contained 5.5 U/mL SOD from bovine al enzymes, deprivation of the substrates required erythrocytes and 50 mg/mL of the corresponding for microbial growth or direct action on microbial preparation. Control does not contain any extract. metabolism through inhibition of oxidative phos- Error bars represent standard deviations. phorylation (Puupponen-Pimia et al., 2005). The same authors found that freeze-dried lingonberry Discussion showed moderate inhibitory activity against Salmo- The recent study provides data on antibacte- nella similarly to our finding with crude methanol rial and antioxidant activities of the most popular extract against S. typhimurium. The antimicrobi- wild small berries naturally growing in Bulgaria al properties of lingonberry juice are known for a like blackberries, strawberries, raspberries, ling- long time. Moderate effect of lingonberry extract onberries, and bilberries. We determined the anti- against E. coli was observed in our study too. V. vi- bacterial activity of crude methanol extracts against tis-idaea fruits‘ extracts have been found to inhibit selected bacterial pathogens including also the fun- the growth of uropathogenic Escherichia coli rods gus C. albicans. In general, all berry extracts tested by influencing the virulence factors expression and were found to inhibit the growth of Gram-positive biofilm formation (Česonienė et al., 2009; Wojnicz S. aureus and S. pyogenes but not the Gram-nega- et al., 2012). The effect should be associated with tive species, excepting the extract of lingonberries inhibition of bacterial adherence to mucosal surfac- which was active against the virulent serovar S. es (Schmidt and Sobota, 1988). Evidently anti-ad-

70 hesion might be one mechanism in action in antimi- sponsible for beneficial health effects (Moyer et al., crobial activity of berry compounds in general but 2001). the mode of action of tannins probably depends on Further investigation of the total extracts was the individual microorganism. performed in a non-enzymatic system - NBT, me- • The berry phenolics attribute to many diverse thionine and riboflavin. There O2ˉ were generated biological functions. In parallel to their antimicrobi- photochemically and SOD inhibited the reduction al and antifungal activities, they provide also ultra- of NBT. Although the superoxide anion radical is violet radiation protection, chelation of toxic heavy a weak oxidant, it gives rise to the generation of metals, and antioxidant quenching of free radicals more powerful and dangerous hydroxyl radicals as generated during photosynthesis. Anthocyanins, well as singlet oxygen, both of which contribute catechin, ellagic acid, gallic acid and quercetin are to the oxidative stress (Fridovich, 1998). Our re- • potent antioxidants with possible health effects in sults showed that the best O2ˉ scavenging activity humans due to their reported positive effect on blood was determined by the bilberry extract, followed vessels, as well as hepatoprotective, antirheumatic by the strawberry extract (Fig. 2). These prepara- and anticancer activities (Lee et al., 2012; Nile and tions inhibited the development of the color, pro- • Park, 2014). All these observations provoked us duced during the reaction of O2ˉ with NBT by 62 to investigate the capacity of those 5 total extracts and 56% respectively. On the other hand, the lowest to scavenge the reactive oxygen species (ROS). activity showed blackberry and lingonberry. More- • Generally, ROS in the body are generated by ex- over, the samples of total extracts suppressed O2ˉ ogenous agents (e.g., radiation, cigarette smoke, release in a dose-dependent manner (Fig. 3). The • atmospheric pollutants, toxic chemicals, over nutri- 50% O2ˉ scavenging concentrations were between m tion, changing food habits, etc.) and/or endogenous 85.0 and 41.8 g/ml respectively. IC50 values of sources (inflammation, mitochondria, cytochrome bilberry (41.8 mg/ml) and strawberry (43.9 mg/ml) P-450 metabolism, microsomes, and peroxisomes) demonstrated better inhibition of superoxide level (Pham-Huy et al., 2008). When ROS level reach- compared to the other extracts. These values were es above threshold, an imbalance between oxidants comparable to the total extracts from other berries and antioxidants take place in the cells. This situa- (Kim et al., 2009; Palíková et al., 2008). • tion results in oxidative stress that causes various The O2ˉ scavenging effect of the extracts, ob- degenerative diseases (Fridovich, 1998). In this tained from wild Bulgarian berries was confirmed context, antioxidants may play an important role in by measurement of inhibition of NBT-reduction in preventing and repairing ROS-induced damages by presence of commercial SOD (Fig. 4). The combi- scavenging free radicals and repairing the enzymes nation of SOD (5.5 U/mL) with the total extracts involved in the process of cellular development. (50 mg/ml) resulted in 50% and 70% decrease in Present investigations confirmed the antioxidant NBT-reduction respectively, compared to the inhi- potential of all tested extracts. The scavenging of bition by SOD alone. These results demonstrated the stable free radical 1,1-Diphenyl-2-picrylhydra- synergistic effect between the antioxidant enzyme zyl by the DPPH method gave reliable information and the tested preparations suggesting SOD-like concerning the antioxidant activity defined as the activity of the extracts from berries. mean of free radical scavenging capacity (Fig. 1). The obtained results show that the wild Bul- The highest in vitro antioxidant capacity was deter- garian berry fruits are a good source of antioxidant mined for bilberry extract. A decrease in intracellu- compounds and can be used as a potential val- lar oxidation compared to control was observed in ue-added ingredient in the food, cosmetic and in the the following order: bilberry > strawberry > ling- pharmaceutical industry. onberry > blackberry = raspberry. High scavenging capacity was also found for juices of chokeberry, Conclusion blueberry and bilberry (Slatnar et al., 2012) and The results from the present experiments extracts from strawberry, blackberry and raspberry demonstrated that the total extracts from wild Bul- leaves (Buřičová et al., 2011). In small fruits (Pan- garian small berry fruits manifested strong antiox- ico et al., 2009) the antioxidant capacity has been idant activities in model systems and moderate to correlated to a significant degree with anthocyanin high activity against the Gram-positive pathogens content. Moreover, phenolic phytochemicals with S. aureus and S. pyogenes, as well as the fungus antioxidant properties are now believed to be an C. albicans. The in vitro antioxidant properties important component in fruits and vegetables re- of these preparations might be contributing to the

71 overall antimicrobial effect on the selected bacteri- Giovanelli, G., S. Buratti (2009). Comparison of polyphe- al pathogens. The demonstrated antibacterial activ- nolic composition and antioxidant activity of wild Italian ity is a good prerequisite for further studies on skin blueberries and some cultivated varieties. Food Chem. 112: 903-908. infection model systems in vitro and in vivo. Howell, A. B. (2002). Cranberry proanthocyanidins and the maintenance of urinary tract health. Crit. Rev. Food Sci. Acknowledgments Nutr. 42: 273-278. We are acknowledged Iva Tzvetkova for the Jimenez-Garcia, S. N., R. G. Guevara-Gonzalez, R. Miran- excellent technical assistance. This study was sup- da-Lopez, A. A. Feregrino-Perez, I. Torres-Pacheco, M. ported by a research grant DO02-334/08 from the A. Vazquez-Cruz (2012). Functional properties and quality characteristics of bioactive compounds in berries: Bi- Bulgarian Scientific Fund. ochemistry, biotechnology, and genomics. Food Res. 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72 crobial properties and mechanisms of action against se- Ribera, A. E., M. Reyes-Diaz, M. Alberdi, G. E. Zuñiga, M.L. vere human pathogens. Nutr. Cancer 54: 18-32. Mora (2010). Antioxidant compounds in skin and pulp Ozsahin, A. D., Z. Gokce, O. Yilmaz, O. A. Kirecci (2012). of fruits change among genotypes and maturity stages in The fruit extract of three strawberry cultivars prevents li- highbush blueeberry (Vaccinium corymbosum L.) grown pid peroxidation and protects the unsaturated fatty acids in Southern Chile. J. Soil Sci. Plant Nutr. 10: 509-536. in the Fenton reagent environment. Int. J. Food Sci. Nutr. Riihinen, K. R., Z. M. Ou, T. Gödecke, D. C. Lankin, G. F. 63: 353-357. Pauli, C. D. Wu (2014). The antibiofilm activity of lingon- Palíková, I., J. Heinrich, P. Bednár, P. Marhol, V. Kren, L. berry flavonoids against oral pathogens is a case connect- Cvak, K. Valentová, F. Růzicka, V. Holá, M. Kolár, V. ed to residual complexity. Fitoterapia 97: 78-86. Simánek, J. Ulrichová (2008). Constituents and antimi- Roschek, B., Jr., R. C. Fink, M. D. McMichael, D. Li, R. S. crobial properties of blue honeysuckle: a novel source for Alberte (2009). Elderberry flavonoids bind to and prevent phenolic antioxidants. J. Agric. Food Chem. 56: 11883- H1N1 infection in vitro. Phytochemistry 70: 1255-61. 11889. Rufino, M. d. S. M., R. E. Alves, E. S. de Brito, J. Pérez-Jimén- Panico, A. M., F. Garufi, S. Nitto, R. Di Mauro, R. C. Longh- ez, F. Saura-Calixto, J. Mancini-Filho (2010). Bioactive itano, G. Magri, A. Catalfo, M. E. Serrentino, G. De Gui- compounds and antioxidant capacities of 18 non-tradition- di (2009). Antioxidant activity and phenolic content of al tropical fruits from Brazil. Food Chem. 121: 996-1002. strawberry genotypes from Fragaria xananassa. Pharm. Schmidt, D. R., A. E. Sobota (1988). An examination of the Biol. 47: 203-208. anti-adherence activity of cranberry juice on urinary and Paredes-Lopez, O., M. E. Valverde (2012) Berries: Bioac- nonurinary bacterial isolates. Microbios 55: 173-81. tive Constituents and Their Impact on Human Health, in: Slatnar, A., J. Jakopic, F. Stampar, R. Veberic, P. Jamnik Tuberoso, C., (Ed.), Berries: Properties, Consumtion and (2012). The effect of bioactive compounds on in vitro and Nutrition, Nova Biomedical Books, New York, 2012. in vivo antioxidant activity of different berry juices. PLoS Pham-Huy, L.A., H. He, C. Pham-Huy (2008). Free radicals, One 7: e47880. antioxidants in disease and health. Int. J. Biomed. Sci. 4: Spooner, D. F., G. Sykes (1972) Laboratory assessment of 89-96. antibacterial activity, in: Norris, J. R., D. W. Ribbons, Puupponen-Pimia, R., L. Nohynek, S. Hartmann-Schmidlin, (Eds.), Methods in Microbiology, Academic Press, Lon- M. Kähkönen, M. Heinonen, K. Määttä-Riihinen, K. M. don, 1972, pp. 216-7. Oksman-Caldentey (2005). Berry phenolics selectively Toivanen, M., S. Huttunen, S. Lapinjoki, C. Tikkanen-Kau- inhibit the growth of intestinal pathogens. J. Appl. Micro- kanen (2011). Inhibition of adhesion of Neisseria mening- biol. 98: 991-1000. itidis to human epithelial cells by berry juice polyphenolic Puupponen-Pimia, R., L. Nohynek, C. Meier, M. Kahkonen, fractions. Phytother. Res. 25: 828-832. M. Heinonen, A. Hopia, K. M. Oksman-Caldentey (2001). Vasco, C., J. Ruales, A. Kamal-Eldin (2008). Total phenol- Antimicrobial properties of phenolic compounds from ic compounds and antioxidant capacities of major fruits berries. J. Appl. Microbiol. 90: 494-507. from Ecuador. Food Chem. 111: 816-823. Rauha, J. P., S. Remes, M. Heinonen, A. Hopia, M. Kahko- Wojnicz, D., A. Z. Kucharska, A. Sokol-Letowska, M. Kicia, nen, T. Kujala, K. Pihlaja, H. Vuorela, P. Vuorela (2000). D. Tichaczek-Goska (2012). Medicinal plants extracts af- Antimicrobial effects of Finnish plant extracts containing fect virulence factors expression and biofilm formation flavonoids and other phenolic compounds. Int. J. Food by the uropathogenic E. coli Urol. Res. 40: 683-697. Microbiol. 56: 3-12.

73 ACTA MICROBIOLOGICA BULGARICA

Oligosaccharide Production from Lactose and Lactulose by β-Galactosidase from Lactobacillus plantarum S30

Plamena Zheleva, Tonka Vasileva, Veselin Bivolarski, Ilia Iliev* Department of Biochemistry & Microbiology, Plovdiv University, 24,Tzar Assen Str, 4000, Plovdiv, Bulgaria

Abstract A β-Galactosidase preparation from ten strains Lactobacillus plantarum was evaluated as a biocata- lyst for the synthesis of oligosaccharides. Among them, the enzymes from L. plantarum S26 and L. plantarum S30 were selected for further studies. The correlation between organic acid production, α-galactosidase, β-galactosidase and lactate dehydrogenase activity and the initial concentration of lactose and lactulose was studied. It could be suggested that the studied β-galactosidase catalyzes the trans-glycosylation reaction at 20% lactose and 20% lactulose concentrations. Key words: lactose, lactulose, prebiotic potential, Lactobacillus, prebiotics, β-galactosidase, α-galactosidase

Резюме β-Галактозидаза от 10 щама Lactobacillus plantarum е изследвана като биокатализатор за синтезата на олигозахариди.Сред тях ензимите от L. plantarum S26 и L. plantarum S30 са селектирани за последващи изследвания. Изследвана е корелацията между продукцията на органични киселини, α-галактозидазна, β-галактозидазна и лактат дехидрогеназна активност от една страна и началната концентрация на лактоза и лактулоза. Получените резултати показват възможностите на изследваната β-галактозидаза да катализира реакция на трансгликозилиране при концентрации на лактоза 20% и лактулоза 20%.

Introduction Several strains from the genus Lactobacillus charides (FOS), galacto–oligosaccharides (GalOS), have been considered as probiotics due to their ben- xylo-oligosaccharides (XOS), lactulose, etc. act as eficial effect on the host by improving the intestinal prebiotics. Prebiotics are galacto-oligosaccharides microflora, helping in the immune system matura- that promote the growth of probiotic compounds, tion, and presenting inhibitory activity toward the and are neither digested nor absorbed in the human growth and adhesion to epithelial cells or intestinal gut. Several studies have evaluated the fermenta- mucus of pathogenic microorganisms (Tannok et tion of prebiotic compounds by LAB strains. Ga- al., 2004; Mnnoz et al., 2012). lOS are mixtures consisting of numerous different Lactobacillus plantarum is a widespread oligosaccharides varying in their degree of polym- member of the genus Lactobacillus, commonly erization (DP), structure and glycosidic linkage found in many fermented food products, includ- (Iqbal et al., 2010a). The degree of polymerization ing sauerkraut, pickles, brined olives, Korean kim- of the chain of galacto-oligosaccharides generated chi, Nigerian Ogi, sourdough, and other fermented by an enzymatic reaction depends on the lactose plant materials, and also some cheeses, fermented concentration in the media (Rustom et al., 1998). sausages, and stockfish. Quantitatively, the amount of the different GOS It has also been shown that different non-di- products present appears to follow the order: di- > gestible di- and oligosaccharides as fructo-oligosac- tri- > tetra- > higher- saccharides, and the linkages

*Corresponding author: Ilia Iliev e-mail: [email protected] synthesized are predominantly β-(1→6); β-(1→3) and β-(1→2) (Smart, 1993). Trisaccharides, espe-

74 cially galactosyl 1→6 lactose, can be identified at control as well as lactose (Merck, Germany). Each most lactose levels. Tetra- and higher saccharides carbohydrate was sterilized on 0.2 μm sterile filter have been reported only when using much higher (Sartorius), and pH was not adjusted. All examina- starting lactose levels, although they are consid- tions were performed at least twice. ered to be formed at most lactose concentrations Fermentation but inquantities too small to be detected (Mahoney, Lactobacilli were routinely grown in MRS 1998). broth (Merck). Overnight grown cells were washed β-Galactosidase (β-D-galactoside galactohy- twice in saline (0.85% NaCl solution) and 10% of drolase, EC 3.2.1.23) is an enzyme that catalyzes the bacterial suspension (107 cfu/mL-1) was used two basic reactions, hydrolysis of lactose and struc- to inoculate mMRS broth medium (pH 6.8) from turally related galactosides, and transglycosylation 5% to 25% lactose and from 2% to 25% lactulose. reactions, resulting for example, in a mixture of ga- The anaerobic fermentations were performed for lacto-oligosaccharides when lactose is the starting lactobacilli in 50 mL PS bottles at 37ºC for 48 h material for the latter reaction (Iqbal et al., 2010b). (Mandadzhieva et al., 2011). α-Galactosidase enzymes (α-D-galactosil galacto- Microbial growth hydrolases, E.C. 3.2.1.22) hydrolise the α-1,6 link- Bacterial growth was measured by a turbi- ages that join the residue of galactose to the glu- dimetric method at 650 nm and calibrated against cose present in raffinose, producing free galactose cell dry-weight using a spectrophotometer (UV/ and sucrose. Possible sources of these two enzymes Vis Beckman Coulter DU 800, USA). For each ex- are plant, bacteria, fungi, animal organisms and periment, data was analyzed using Excel statistical moulds. Lactic acid bacteria have been studied in- package. The optical density (OD) readings and tensively with respect to their enzymes for various standard deviations were calculated from duplicate different reasons including their GRAS status. Lac- samples from two separate experiments. tobacillus plantarum is a versatile lactic acid bac- Analysis of metabolites terium, which is encountered in a range of environ- Lactic acid was determined enzymatically mental niches including dairy, meat, and vegetable with L-lactate dehydrogenase and D-lactate dehy- fermented foods (Saarela et al., 2003). drogenase (commercially available kit code 10 139 The aim of the present work was to study the 084 035, Boehringer, Mannheim, Germany,). activity α-galactosidase and β-galactosidase from Acetic acid was determined enzymatical- the studied strains Lactobacillus plantarum S26 ly with acetyl-CoA synthetase, citrate synthase, and Lactobacillus plantarum S30, cultivated in a malate dehydrogenase (commercially available kit medium with more than 15% lactose and 15% lact- code 10 148 261 035, Boehringer, Mannheim, Ger- ulose as a sole carbon substrate. many). Ethanol was determined enzymatically with Materials and methods alcohol dehydrogenase and aldehyde dehydroge- Bacterial strains and culture conditions nase (commercially available kit code 10 176 290 In this study two strains, Lactobacillus plan- 035, Boehringer Mannheim, Germany). tarum S26 and Lactobacillus plantarum S30, from Analysis of carbohydrates the collection of the Department of Microbiology, Oligosaccharides were analyzed by HPLC

Sofia University, Bulgaria, were used (Stoyanovski using a Symmetry C18 column (4.6 x 150 mm) and et al., 2009). They were identified using 16S RNA a Waters 1525 Binary HPLC Pump (Waters, Mil- techniques. The strains were cultured overnight ford, MA, USA). Oligosaccharides were detected (16-18 h) on MRS (de Mann Rogosa Sharpe broth, by using a Waters 2414 refractive index detector. Merck, Darmstadt, Germany) at 37ºC and in lim- The products were identified in the chromatograms itation of oxygen (BBL® Gas Pak anaerobic system as described by Remaud-Simeon et al. (1994). Envelopes, Becton Dickinson, Franklin Lakes, NJ Sugars (residual glucose, xylose, and xylooli- USA). gosaccharides in fermentation broth after fermenta- Carbohydrates used in this study tion) were determined by HPLC, using Zorbax car- Lactose (Merck, Germany) and lactulose bohydrate column (4.6 x 150 mm; Agilent, Santa (Lactulose Crystals EP, Viccio, Italy) contained Clara, CA, USA), analytical guard column Zorbax lactulose 97.5%, galactose 0.5%, lactose 0.5%, epi- NH2 (4.6 x 12.5 mm), and a mobile phase of 75/25 lactose 0.5%, tagatose 0.5%, fructose 0.5%. Glu- acetonitrile/water. Breeze Chromatography Man- cose (purity 99%, Merck, Germany) was used as a ager Software (Waters) was used for data treatment.

75 Enzyme activity the examined strains utilized 15% of lactose and The β-galactosidase activity assays were 5% lactulose with different capacity for changing carried out using ONPG( ortho-Nitrophenyl-β-ga- biomass concentration, pH, and enzyme activity lactopyranoside) with substrate prepared in cit- (Zheleva, et al., 2014). The next investigation was rate-phosphate buffer solution. One β-galactosi- focused on the production of organic acids during dase unit (U) was defined as the amount of enzyme the cultivation in mMRS media with 15% lactose which liberated 1 µmol of ONP(ortho-Nitrophe- and 5% lactulose (Tables 1 and 2). The fermenta- nyl) per min per mg of protein at 37°C and pH 6.0 tion pattern depends on the physiological condi- (Kneifel et al., 2000). tions of the growing cells. Homofermentative LAB The α-galactosidase activity assays were can ferment hexoses via glycolysis with 90% of the carried out using PNPG (p-Nitrophenyl α-D-ga- glucose being metabolized to lactic acid, and heter- lactopyranoside) with substrate prepared in citrate– ofermentative LAB metabolized hexoses to lactic phosphate buffer solution. One α-galactosidase unit acid, acetic acid and ethanol. It is known that L. (U) was defined as the amount of enzyme which plantarum is a facultative homofermentative LAB liberated 1 µmol of ONP per min per mg of protein species. When cultivated on 15% lactose and 5% at 37°C and pH 6.5 (Kontula et al., 2002). lactulose, the studied strains L. plantarum S26 and Proteins were assayed by the method of L. plantarum S30 produced different amounts of Bradford (1976) by using bovine serum albumin as D-lactate, L-lactate, acetic acid, and ethanol (Ta- standard (Olano et al., 2009). bles 1 and 2). When the fermentation end-products, Preparation of resting cells obtained using the high concentration of lactose Bacterial cells of L. plantarum S26 and L. and lactulose as growth substrates, were compared plantarum S30, cultivated in mMRS-10% lactose to those observed on glucose, the main effect was in the logarithmic phase of growth were harvested that the production of lactic acid was lower while by centrifugation at 6000xg for 10 min (4°C), and the production of acetic acid and ethanol increased. then washed twice with physiological saline. Both studied strains showed the same behavior. Hydrolysis of lactose and lactulose by resting cells In the current study, the activity of lactate The hydrolysis of lactose and lactulose was dehydrogenase of the two studied strains cultivat- performed with 5g-wet/l resting cells in physiolog- ed on mMRS medium with 5%, 10%, 12,5%, and ical saline at 37°C for 60 min. After boiling for 10 15% lactose were determined. The study of the en- min., the sample was analyzed by HPLC for the de- zyme profiles concluded that the activity of lactate tection of galactose, glucose, lactose, lactulose and dehydrogenase was not strongly associated with oligosaccharides as described above. cell growth (data not shown). The activity of the studied enzyme was evaluated during the growth Results of the strains (Fig. 1). The maximum lactate dehy- As a result of the performed screening pro- drogenase activity was found when the strains were cedure of lactobacillus strains, it was found that cultivated in 5% lactose on the 12th h of the process.

Table 1. Dynamics of organic acids and ethanol producing of L. plantarum S26, cultivated on mMRS with 5% lactulose

Biomass D-lactate L-lactate acetate ethanol h pH [g/L] [g/L] [g/L] [g/L] [g/L] 6 1.21±0.9 5.68 0.59±0.1 0.47±0.2 1.84±0.3 0.01±0.001 18 13.8±0.2 3.62 4.71±0.3 5.01±0.1 1.06±0.1 0.02±0.001 24 15.4±0.5 3.34 6.09±0.5 6.16±0.3 1.26±0.3 0.09±0.002 48 15.9±0.8 3.22 6.32±0.1 6.68±0.2 0.94±0.1 0.14±0.3

76 Table 2. Dynamics of organic acids and ethanol producing of L. plantarum S30, cultivated on mMRS with 5% lactulose

Biomass D-lactate L-lactate acetate ethanol h pH [g/L] [g/L] [g/L] [g/L] [g/L] 6 0.48±0.1 6.02 0.27±0.1 0.25±0.1 1.89±0.3 0.007±0.0001 18 5.40±0.2 4.27 4.37±0.25 3.99±0.21 1.11±0.2 0.008±0.0003 24 9.90±0.5 3.69 1.99±0.22 4.10±0.28 1.38±0.2 0.001±0.0001 48 12.40±0.8 3.26 6.93±0.25 6.65±0.31 1.40±0.3 0.015±0.009

A 40% (L. plantarum S26) and 80% (L. plantarum lactulose concentration on the hydrolase and trans- S30) reduction of the enzyme activity was detected ferase activity of β-galactosidase from the studied when the strains were cultivated in mMRS media strains was investigated. Lactose was converted with 15% lactose. On the other hand, the maximum by resting cells of L. plantarum S26 and S30 incu- lactate dehydrogenase activity was found during bated at 37ºC for 24 hours. The total activity was cultivation in 12.5% on the 24th h of fermentation expressed as the concentration of glucose and no for both strains. inhibitory effect of the concentration of initial lac- Further studies were performed to prove the tose for the first hour of the reaction was detected induction or inhibition of β-galactosidase activity (Fig. 2). Glucose is a non-competitive inhibitor of in the presence of lactose and lactulose in concen- β-galactosidase as confirmed by the results from trations from 1% to 25%. The effect of lactose/ 24 h of incubation.

А) Lactose

B) Lactulose

Fig. 1. Hydrolysis on different lactose (А) or lactulose (B) concentrations with β-galactosidases of resting cells of strains L. plantarum S26 and L. plantarum S30 with duration of the reaction 1h.

77 According to the results in this study, the formation of galacto-oligosaccharides is limited by the accumulation of glucose during incubation of resting cells for 24h (Fig. 3). When lactulose is a substrate for β-galactosidase from resting cells of L. plantarum S26 and S30, the total activity was expressed as the concentration of fructose (Fig. 2). As shown in Fig. 4. of the transferase reaction, the products synthesized with β-galactosidase from the studied strains displayed two main prod- А) 15% Lactose ucts, galacto-oligosaccharides with DP 3 and DP 4, using 20% lactose as a substrate (Figure 4). Among the GalOS, only tri- and tetrasaccharides were synthesized and oligosaccharides with longer chains than DP 5 were not found at any time during the course of the reaction. The results demonstrate that the β-galactosidase from strain S26 efficiently synthesized oligosaccharides with DP 3 overall yield of 24% using 25% initial lactose concentration at 40°C. The yield of the GalOS with DP 3 is 14% using 20% initial lactulose concentration. B/ 15% Lactulose Discussion It has been shown in this study that two LAB Fig. 2. Hydrolysis of 15% lactose (А) and 15% strains identified as L. plantarum S26 and L. plan- lactulose (B) with resting cells of strains L. tarum S30 isolated from home made sausages can plantarum S26 and L. plantarum S30 be cultivated in media with lactose in concentration more than 20%. It is well known that most lactobacilli can use 5 % lactose easily, however, this study has shown that only a few strains from L. plantarum possess the ability to grow in media with 15% lactose. In this study, it has been demonstrated that in the presence of 12.5% lactose and 5% lactulose, the studied strain L. plantarum S26 showed correlation between the highest α-galactosidase and β-galactosidase activities and lactate dehydrogenase activity during the first 12 h of fermentation. β-Galactosidase (EC.3.2.1.23) catalyzed both reactions – hydrolysis and transgalactosylation, using lactose and lactulose as substrate. In this regard, our results on the enzyme reaction demonstrated in vitro the capacity of β-galactosidase from L. plantarum S26 to synthesize galacto-oligosaccharides using lactose and lactulose in a concentration from 20 to 25%. This observation confirms the results described by Smart et al. (1993), according to whom high lactose concentrations favor the synthesis of short chain oligosaccharides. It was observed that complete inhibition of oligosaccharide synthesis Fig. 3. Dynamics of lactate dehydrogenase activity occurred when the monosaccharide content of the of strains L. plantarum S26 and L. plantarum S30, reaction medium reached the total concentration cultivated on media mMRS with 5%, 10%, 12,5% of galacto-oligosaccharides. The optimum reaction and 15% lactose 78 Fig. 4. Distribution of oligosaccharides by degree of polimerisation received through transfer reaction by β-galactosidase of strain L. plantarum S26, in the presence of different percentages of lactulose conditions for galacto-oligosaccharide synthesis Kneifel, W., A. Rajal, K. D. Kulbe (2000). In vitro growth with DP 3, substrate concentration and enzyme behaviour of probiotic bacteria in culture media with car- activity using β-galactosidase from strain L. plan- bohydrates of prebiotic importance. Microb. Ecol. Health Dis. 12: 27-34. tarum S26 were determined. Since lactulose is a Kontula, P., L. Nollet, M. Saarela, T. Vilpponen-Salmela, W. substrate for β-galactosidase, when the hydrolysis Verstraete, T. Mattila-Sandholm A. von Wright (2002). of lactulose is conducted in the presence of fruc- The effect of lactulose on the survival of Lactobacillus tose, its effect on the hydrolysis of lactulose does rhamnosus in the simulator of the human intestinal mi- not correspond to a competitive inhibitor. crobial ecosystem (SHIME) and in vivo. Microb. Ecol. Health Dis. 14: 90–96. Mahoney, R. (1998). Galactosyl-oligosaccharides formation Conclusion during lactose hydrolysis: a review. Food Chem. 63: 147- It has been shown in this study that the strain 154. of L. plantarum S26 produces α-galactosidase and Mandadzhieva, T., T. Ignatova-Ivanova, S. Kambarev, I. I. β-galactosidase during cultivation in a medium Ivanova (2011). Utilization of different prebiotics by Lac- with lactose in 15% concentration as a sole carbon tobacillus spp. and Lactococcus spp. Biotechnol. Biotech. source. It could be suggested that the studied β-ga- Eq. 25/2011/4, Suppl: 117-120. Mnnoz, M., A. Mosquera, C. J. Almeciga-Diaz, O. F. Melen- lactosidase catalyzes the trans-glycosylation reac- dez, A. P. Sanchez (2012). Fructooligasaccharides metab- tion at 20% and 25% concentrations of lactose. The olismand effect on bacteriocin production in Lactobacillus selectivity of the reaction of galacto-oligosaccha- strains isolated from ensild corn and molasses. Anaerobe, rides synthesis with β-galactosidase was dependent 18(3): 321-330. on the enzyme source. The selectivity of the select- Olano, A., N. Corzo (2009). Lactulose as a food ingredient. J. ed enzymes was influenced by the structure and DP Sci. Food Agric. 89(12): 1987-1990. Rustom, I., M. Foda, M. Lopez-Leiva (1998). Formation of of the synthesized oligosaccharides. oligosaccharides from whey UF-permeate by enzymatic hydrolysis: analysis of factors. Food Chem. 62: 141-147. Acknowledgements Saarela, M., K. Hallamaa, T. Mattila-Sandholm, J. Matto This work was supported by research grant of (2003). The effect of lactose derivatives lactulose, lactitol NSF Bulgaria BG B01/7-2012. and lactobionic acid on the functional and technological properties of potentially probiotic Lactobacillus strains. Int. Dairy J. 13: 291–300. References Iqbal, S., T-H. Nguyen, T. T. Nguyen, T. Maishberger, D. Hal- Iqbal, S., T. Nguyen, T., D. Haltrich (2010). β-Galactosidase trich (2010). β-Galactosidase from Lactobacillus planbta- from Lactobacillus plantarum WCFS1: biochemical char- rum WCFS1: biochemical characterization and formation acterization and formation of prebiotic galactooligosac- of prebiotic galacto-oligosaccharides. Carbohydr. Res. charides. Carbochydr. Res. 345: 1408-14016. 345: 1408-1416.

79 Stoyanovski, S., V. Chipeva, S. G. Dimov, S. Danova, I. Dim- bacteria and bifidobacteria on lactose and lactose-related itrova, L. Yocheva, S. Atanasova-Nikolova, I. Ivanova mono-, di-, and trisaccharides and correlation with distri- (2009). Characterization of lactic acid bacteriafrom dry bution of β-galactosidase and phospho-β-galactosidase. J. sausages. Biotechnol. Biotech. Eq. 23/2009/Se Special Diary Res. 60: 557-568. Edition/on-line, 598-601. Zheleva, Pl., T. Vasileva, T. Mandadjieva, I. Ivanova, I. Iliev Tannok, G.W. (2004). A special fondness for lactobacilli, (2014). Influence of lactose concentration on the α-ga- Appl. Environ. Microbiol. 70: 3189-3194. lactosidase and β-galactosidase activity of Lactobacillus Smart, J., C. Pillidge, J. Garman (1993). Growth of lactic acid plantarum, J. BioSci. Biotech. Se/online: 71-74.

80 ACTA MICROBIOLOGICA BULGARICA

Presence of N-Acetylneuraminate Pyruvate-Lyase Activity in Sialidase Producing Strains of Erysipelothrix rhusiopathiae and Oerskovia sp. Rumyana Eneva*, Stephan Engibarov, Ignat Abrashev Department of General microbiology, Section of Microbial biochemistry, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 “Acad G. Bonchev” Str, 1113 Sofia, Bulgaria

Abstract The enzyme N-acetylneuraminate pyruvate-lyase (sialate aldolase) was found in two sialidase producing strains of the pathogen species Erysipelothrix rhusiopathiae and the saprophyte genus Oerskovia. The enzyme is secreted in the medium in both strains and its activity reaches similar values - 12.5 and 13.3 U/(mg protein) for E. rhusiopathiae B40 and Oerskovia sp./14 respectively. Intracellular enzyme activity of 10.7 U/(mg protein) was also detected in the latter strain. The data obtained for the sialate aldolase and sialidase activities were compared to analogical data for Vibrio cholerae and Aeromonas sp. sialidase producer strains. The presence of sialate aldolase and sialidase enzymes in all these strains supports the concept that sialic acid metabolism contributes to the pathogenesis of microorganisms. Key words: sialate aldolase, sialidase, Erysipelothrix rhusiopathiae, Oerskovia sp.

Резюме Ензимът N-ацетил неураминат пируват-лиаза (сиалат алдолаза) е установен при два щама про- дуценти на сиалидаза – съответно от патогенния вид Erysipelothrix rhusiopathiae и сапрофитния род Oerskovia. И при двата щама ензимът се секретира извън клетката, като активността му има сходни нива – съответно 12.5 и 13.3 E/(мг белтък). При щам Oerskovia sp./14 беше установена и вътрекле- тъчна сиалат алдолазна активност (10.7 Е/мг белтък). Данните, получени за продукцията на алдо- лаза и сиалидаза при двата изследвани щама са сравнени с аналогични данни за други сиалидаза продуциращи щамове (Vibrio cholerae и Aeromonas sp.). Наличието на сиалат алдолаза и сиалидаза при всички тези щамове подкрепя схващането за приноса на сиалометаболизма към патогенезата на микроорганизмите.

Introduction N-acetylneuraminate lyases (4.1.3.3; N-ace in numerous cell-cell and cell-environment interac- tylneuraminate pyruvate-lyases; sialate aldolases) tions. In bacteria, sialic acid metabolism is engaged are enzymes that reversibly split N-acetylneur- in various functions, depending on microorganism’s aminic (sialic) acid into N-acetyl-D-mannosamine strategy for adaptation to certain ecological niche. (ManNAc) and piruvate (Comb and Roseman, The first step in the catabolism of these compounds 1960). Sialic acids are a family of nine-carbon keto is the cleavage of their terminal Neu5Ac residues sugar acids, derivatives of N-acetylneuraminic acid (the reaction being catalyzed by sialidases), thus re- (Neu5Ac). They are abundantly represented in the leasing free sialic acids. They are further degraded tissues of higher animals and irregularly in some by sialic acid aldolases to form piruvate that enters of their microbial commensals or pathogens. Sial- either Embden-Meyerhof-Parnas pathway (as phos- ic acid metabolism is an object of interest for sci- phoenolpyruvate) or citric acid cycle, and ManNAc entists in the areas of biochemistry, microbiology, which is converted to fructose-6-P for entrance into medicine, etc, since sialoconjugates are involved glycolysis (Vimr, 2004). Sialate aldolases also reg- ulate the intracellular sialic acid concentration and *Corresponding author: Rumyana Eneva Address: The Stephan Angeloff Institute of Microbiology, prevent its rising to toxic levels (Vimr and Troy, Bulgarian Academy of Sciences, 26 “Acad G. Bonchev” Str, 1113 Sofia, Bulgaria, 1985). [email protected]

81 According to BRENDA Enzyme Database grown on Hottinger broth (Bulbio, NCIPD, Sofia, (http://www.brenda-enzymes.org/), approximately Bulgaria), рН 7.6, in 100/20 ml Erlenmeyer flasks 100 prokaryote species are reported to harbor sion rotary shaker (50 r/min). All cultivations were alidase genes. Since this enzyme is considered as for 24 h, at 37°С. a virulence factor, it has always been an object of Enzyme assays thorough investigation. About 20 bacterial siali- Sialidase and acylneuraminate pyruvate-lyase dases, most of them from pathogens (Vibrio chol- activities were measured quantitatively by colori- erae, Clostridium perfringens, Corynebacterium metric determination of free sialic acid by the thio- diphtheriae, Salmonella enterica, Bacteroides fra- barbituric acid method, modified by Uchida et al. gilis, Arthrobacter ureafaciens, Micromonospo- (1977). One unit of sialidase or aldolase activity ra viridifaciens, etc.), are characterized in detail, was defined as the amount that releases or degrades the data including their full gene sequences, some 1 μg of N-acetylneuraminic acid (Neu5Ac) for 1 crystal structures and 3D models. There are also min at 37°C using glycomacropeptide (GMP) as a commercial products of several proteins from well- substrate. GMP is isolated by us from milk whey in known producer species. laboratory conditions. The carbohydrate moiety of Sialate aldolases are far less studied, though GMP includes galactose, N-acetylgalactosamine, the number of species that have these enzymes and terminally linked sialic acids (predominantly identified as gene sequences is even slightly higher Neu5Ac). The sialic acid content of GMP is 7–9 % – more than 110. As for biochemical studies, there (Abrashev et al., 1980; Neelima et al., 2013). are some data of only about 15 bacterial species The extracellular and the cell-bound sialidase until now (Drzeniek et al., 1972; Muller, 1973; and sialate aldolase activities were assayed as ei- Sanchez-Carron et al., 2011, etc.). ther the supernatant or the biomass was used. The Sialometabolism is related to the patholo- culture liquid was centrifuged at 3000 rpm for 20 gy of some bacterial species (Steenbergen et al., min, and enzyme activities of the cell-free superna- 2005). Therefore, we find it intriguing to determine tant were determined. The cell pellet was suspend- if sialate aldolases occur together with sialidase ed in phosphate buffer at pH 7.2 and homogenized. activity in Erysipelothrix and Oerskovia - genera, After centrifuging at 10,000 rpm for 15 min, the which aldolase activity has not been studied until supernatant from disrupted cells was collected and now. The aim of this work is to investigate the pres- subjected to further centrifugation at 14,500 rpm ence, location and levels of aldolase and sialidase for 15 min. The supernatant (cell-free extract) was activity in two bacterial strains, representatives of assayed for intracellular sialidase, sialate aldolase these genera. activities and protein content. The preparations of cell-free extracts were carried out at 0-4°C. Materials and Methods Protein concentrations were measured by the Strains, Media, and Cultivation Conditions Lowry procedure (Lowry et al., 1951), using bo- The sialidase-producing strains Oerskovia vine serum albumin as standard. Enzyme activities sp./14 (O/14, NBIMCC №8734) (www.nbimcc. are expressed as units per mg protein [U/(mg pro- org) and E. rhusiopathiae B40 (B40) were test- tein)]. ed for aldolase and sialidase activity. They were

6 secreted sialidase 4 secreted aldolase

2 intracellular sialidase

Enzyme activity, U/(mg protein) intracellular aldolase 0 E. rhusiopathiae/B 40 Oerskovia sp./ 14

Fig.1. Levels of sialidase and aldolase activities in E. rhusiopathiae B40 and Oerskovia sp./14 82 Results As steps of a common metabolite pathway, The levels of secreted and cellular sialidase sialidase and aldolase are met together in many cas- and aldolase activities of the strains are presented es (C. perfringens, C. diphtheriae, V. cholerae, P. in Fig. 1. Both strains produced these enzymes, as multocida, S. pneumoniae), but there are also spe- their activities were higher in Oerskovia. Their ex- cies that carry just one of them. For example, E. tracellular aldolase activity was almost equal – 12.5 coli and L. plantarum do not produce sialidase. As and 13.3 U/(mg protein) respectively for B40 and can be concluded by our results, E. rhusiopathiae O/14. In Oerskovia sp./14 we could also measure B40 and Oerskovia sp./14 can be added to the list the intracellular activity of the enzyme – 10.7 U/ of species that carry both enzymes. It is notewor- (mg protein). Sialidase activity was registered in thy that most of these species, including E. rhusi- the culture liquids and the cell-free extracts in B40 opathiae B40, are pathogens. Oerskovia species are and O/14. The levels of the secreted and the cellular saprophytic nocardialike bacteria that have been as- sialidase activity in each strain were close. Inter- sociated with human infections only rarely, in cases estingly, the presented activities were reached only of immunocompromised patients (Maguire et al., when cultures were grown with agitation on rotary 1996). It can be suggested that, in these cases the si- shaker, providing good aeration. When the same alic acid metabolism acts in favor of pathogenesis, cultures were cultivated statically we could detect besides nutrition. only traces of sialidase and aldolase activities (data The aldolases of Aeromonas sp.40/02, E. rhu- not shown). siopathiae B 40 and Oerskovia sp./14 are secret- The data obtained in this study are compared ed enzymes, indicating that these bacteria uptake to the analogical results, estimated earlier for two ManNAc and piruvate after their production from other strains – Vibrio cholerae non-O1/13 and Ae- sialic acid outside the cell and use them for energy romonas sp. 40/02 (Table 1.). All the strains pro- metabolism (as is the case in T. vaginalis – Vimr, duce secreted sialidase. V. cholerae non-O1/13 2004). The relation of aeration conditions to the ex- does not produce extracellular aldolase. Intracellu- pression of sialidase was observed also in V.choler- lar aldolase in B40 could not be detected so far. The ae and Aeromonas 40/02. In the former species the aeration conditions in which the production of sial- mechanism of this phenomenon was explained re- idase and aldolase is expressed to the higher extend cently by Liu et al. (2011). The authors reveal that appear to be different for each strain. the pathogenicity and virulence factors of V. cholerae are expressed at low oxygen concentrations. Discussion Since the four strains listed in the table show dif- Sialidase has been reported for E. rhusi- ferences in this characteristic, it would be interest- opathiae and Oerskovia (Cellulomonas) turbata ing to examine the possible relations between the (Abrashev and Orozova, 2006; Muller, 1995). Al- expression of the enzymes of sialic acid metabo- dolase is reported for E. rhusiopathiae only as a lism and the conditions at which pathogenic mech- sequence based annotation, while there are no data anisms are revealed. to our knowledge for occurrence of this enzyme in Oerskovia, until now.

Table 1. Comparison of sialidase and aldolase production in the strains investigated in this study to analogical data from previous studies

Enzyme activity, E. rhusiopathiae Oerskovia Aeromonas sp. V. cholerae (U/mg protein) B40 sp./ 14 40/02 non-O1/13 secreted 2.13 4.5 0.65 11.75 Neuraminidase cellular 3.2 4.4 4.9 4.2 secreted 12.4 13.3 1.9 - Aldolase cellular - 10.7 15.0 3.5 Aerobic on Aerobic on Aerobic static Aeration conditions Microaerophilic shaker shaker Engibarov et al., Eneva et al., 2011, Source This study This study 2015 2015

83 References Maguire, J. D., M. C. McCarthy, C. F. Decker (1996). Oer- Abrashev, I., P. Velcheva, P. Nikolov, J. Kourteva (1980). skovia xanthineolytica bacteremia in an immunocompro- Substrate for colorimetric determination of enzyme activ- mised host: case report and review. Clin. Inf. Dis. 22: 554- ity. Bulgarian patent N 47647/IIR. 556. Abrashev, I., P. Orozova (2006). Erysipelothrix rhusiopathi- Müller, H. E. (1973). Occurrence of neuraminidase and acyl- ae neuraminidase and its role in pathogenicity. Z. Natur- neuraminate pyruvate lyase in a strain of Vibrio falling forsch. C. 61(5-6): 434-438. into Heiberg‘s group II isolated from a patient with diar- Comb, D. G. and S. Roseman (1960). The Sialic Acids. I. The rhea. Inf. Immun. 8(3): 430-433. structure and enzymatic synthesis of n-acetylneuraminic Müller, H. E. (1995). Detection of sialidase activity in Oersk- acid. J. Biol. Chem., 235: 2529-2537. ovia (Cellulomonas) turbata. Zentralbl. Bakteriol. 282(1): Drzeniek, R., W. Scharmann, and E. Balke (1972). Neurami- 13-17. nidase and N-Acetylneuraminate Pyruvate-Lyase of Pas- Neelima, R. Sharma, Y. S. Rajput, and B. Mann (2013). teurella multocida. J. Gen. Microbiol. 72: 357-368. Chemical and functional properties of glycomacropeptide Eneva, R., S. Engibarov, T. Strateva, R. Abrashev, I. Abra- (GMP) and its role of the detection of cheese whey adul- shev. (2011). Biochemical studies on the production of teration in milk: a review. Dairy Sci. Technol, 93: 21-43. neuraminidase by environmental isolates of Vibrio choler- Sanchez-Carron, G. M. I. García-García, A.B. Lopez- ae non-O1 from Bulgaria. Can. J. Microbiol., 57(7): 606- Rodríguez, S. Jimenez-García, A. Sola-Carvajal, F. 610. doi: 10.1139/w11-042. García-Carmona, and A. Sanchez-Ferrer (2011). Molecu- Eneva, R.T., S.A. Engibarov, P. Petrova, R. Abrashev, T. Strat- lar characterization of a novel N-acetylneuraminate lyase eva, V. Kolyovska, I. Abrashev (2015). High Production of from Lactobacillus plantarum WCFS1. Appl. Environ. Neuraminidase by a Vibrio cholerae non-O1 Strain - the Microbiol. 77(7): 2471-2478. First Possible Alternative to Toxigenic Producers. Appl. Steenbergen, S. M., C. A. Lichtensteiger, R. Caughlan, J. Biochem. Biotechnol. DOI 10.1007/s12010-015-1584-4. Garfinkle, T. E. Fuller, and E. R. Vimr (2005). Sialic Engibarov, S., R. Еneva, I. Abrashev (2015). Neuraminidase acid metabolism and systemic pasteurellosis. Inf. Immun. (sialidase) from Aeromonas sp. strain 40/02 - isolation and 73(3):1284-1294. partial purification. Ann. Microbiol. doi 10.1007/s13213- Uchida, Y., Y. Tsukada, T. Sugimori (1977). Distribution of 014-0990-0. neuraminidase in Arthrobacter and its purification by af- Liu Z., M. Yang, G. L. Peterfreund, A. M. Tsou, N. Selam- finity chromatography. J. Biochem. 82: 1425-1433. oglu, F. Daldal, et al. (2011). Vibrio cholerae anaerobic Vimr E. R. and F. A. Troy (1985). Identification of an induc- induction of virulence gene expression is controlled by ible catabolic system for sialic acids (nan) in Escherichia thiol-based switches of virulence regulator AphB. PNAS coli. J. Bacteriol. 164(2): 845-853. 108(2): 810-815. Vimr, E., A. Kathryn, E.Deszo, S. Steenbergen (2004). Diver- Lowry, H., J. Rosenberg, L. Farr, J. Randall, (1951). Pro- sity of microbial sialic acid metabolism. Microbiol. Mol. tein measurement with the Folin phenol reagent. J. Biol. Biol. Rev., 68: 132-153. Chem. 193: 265-275.

84 ACTA MICROBIOLOGICA BULGARICA

Dynamics of Biodegradation Kinetics of 1,2-DCE and TCE in Bioreactor Coupled to Ultrafiltration Membrane Unit. Modeling Procedure Daniela Estelila Goes Trigueros2, Alexander D. Kroumov1* 1Institute of Microbiology „Stephan Angeloff“- Bulgarian Academy of Sciences, Department of Applied Microbiology, Acad. G. Bonchev str., Bl.26, Sofia 1113, Bulgaria. 2Department of Chemical Engineering-Postgraduate Program, West Parana State University - UNIOESTE, Rua da Faculdade 645, Jd. Santa Maria, 85903-000, Toledo, PR, Brazil

Abstract The paper presents the modeling approach to aerobic biodegradation of 1,2-dichloroethane (1,2-DCE) by the microorganism Xanthobacter autotrophicus GJ10, and of trichlorethylene (TCE) by the microorganism Pseudomonas cepacia PR131 in a bioreactor with an ultrafiltration (UF) membrane unit. The aim of the study was to estimate the kinetics of the bioprocess and to perform a parameter identification procedure. The specific growth rate was assumed to be a function of 1,2-DCE and TCE and oxygen substrate concentrations. The parameter values were estimated based on experimental data of continuous and discontinuous systems published elsewhere. For this purpose, the Particle Swarm optimization algorithm, coded in Maple 15® software, was applied. Simulations with the developed kinetic model showed new highlights and an optimal trajectory that can help to increase organochlorine wastewater treatment efficiency. Keywords: Biodegradation of organochlorine; kinetics modeling; bioreactor with recycle; ultrafiltration membrane unit.

Резюме Представен е подход за моделиране на процеса на микробиологично разграждане в аеробни условия на 1,2-дихлороетан (1,2-DCE) от Xanthobacter autotrophicus GJ10 и на трихлороетилен (ТСЕ) от Pseudomonas cepacia PR131 в биореактор снабден с ултрафилтрационна мембрана. Целта на изследването е да се определи кинетиката на биопроцеса и да се извърши параметрична идентификация на модела. Беше допуснато, че специфична скорост на растеж е функция на 1,2-DCE и TCE както и от концентрации на разтворения кислород. Стойностите на параметрите са изчислени на основата на експериментални данни от непрекъснати и прекъснати операции публикувани в литературата. За тази цел беше използван алгоритъм на рояка на частиците, кодиран в MAPLE 15® софтуер. Симулациите с разработения кинетичен модел показаха нови свойства на системата и под- чертаха една оптимална траектория, която може да се използва за увеличаване на ефективността на пречистване на отпадъчни води от органохлорни съединения.

Introduction Volatile organic compounds are produced posed of a double bond between the carbon atoms. in many manufacturing processes (Scheutz et al, The maximum allowable concentration of both 2011; Frascari et al., 2013b. ), especially in the pro- compounds in drinking water is 5 µg L-1 (US EPA, duction of pesticides, plastics and paper, and thus 2009). 1,2-DCE is widely used in the production comprise an important group in the treatment of in- of chlorinated solvents like 1,1,1-trichloroethane, dustrial effluents (Arjoonet al., 2013; Huang et al., TCE and perchloroethylene (PCE), and especially 2014). 1,2-DCE and TCE are two organochlorine in the manufacture of vinyl chloride (Gwinn et al., compounds, well-known for their chemical stabil- 2011), which is an essential substance for the pro- ity and high toxicity (Yeh and Kastenberg, 1991). duction of polyvinyl chloride (PVC) (Yuan et al., Both have similar physical and chemical properties 2015). TCE is widely used as a degreasing agent (Table 1), and their molecular structure is com- and a solvent, and has the characteristic of migrat-

85 Table 1. Physical and chemical properties of 1,2-DCE and TCE

Description Units 1,2-DCE TCE

Molecular formula - C2H2Cl2 C2HCl3 Molecular weight MW(g.mol-1) 98.97 131.39a Liquid density (25°C) Ρ(g.cm-3) 1.253 1.460a Solubility in water S(g.cm-3) 8.606x10-3 1.1x10-3a Dynamic viscosity (20°C) µ(cP) 0.84 0.55 Cinematic viscosity ν 2.41x10-3

a Vapor pressure Vp(torr) 79 73 a Fusion point Tm(°C) -35 -86 a Boiling point Tb(°C) 85 87 Coefficient of octanol-water K 1.48 1.86a partition ow Sorption coefficient (organic K 1.52 2.42a carbon) oc Henry‘s Law Constant H(atm.m3mol-1) 1.2 11.7

2 -1 -2a Diffusion coefficient and clean air Dair(m .h ) - 2.84x10 2 -1 -6a Diffusion coefficient in pure water Dwater(m .h ) - 3.75x10

a(Pant and Pant, 2010) ing through the soil making it a threat to groundwa- capable of completely mineralizing compounds at a ter (Folsom et al., 1990). With respect to damage to low cost (Kocamemi Ceçen, 2010). humans, 1,2-DCE is classified as a possible human In order to group the strengths of treatments carcinogen (group B2) by the International Agen- and to achieve simultaneous separation and biodeg- cy for Research on Cancer (IARC, 1999). TCE has radation, the aerobic or anaerobic biodegradation been classified by the Agency for Toxic Substanc- methods can be performed in membrane reactors es and Disease Registry (ATSDR, 2015) as the 16th (MBR) (Inguva et al., 1998; Xing et al., 2000; Min greatest potential threat to human health, setting up аnd Ergas, 2006; Bolzonella et al., 2007). The ma- for a probable human carcinogen group (Group B1) jor advantages of combining both treatments in- (US EPA, 2001). clude improved control of microbiological activity, The treatment of wastewaters contaminated lower operational costs. where the released efflu- with organic compounds has been accomplished ent is free of bacteria and pathogens (Cicek, 2003). using various physical, chemical and biological Furthermore, studies have shown that microbial ac- methods such as: tivity improves the flow of the solute through the Chemical oxidation (Vilve et al., 2010; Che membrane (Aziz et al., 1995; Inguva et al., 1998). and Lee, 2011); reduction employing iron (Zhang Min and Ergas (2006) conducted a study of et al., 2011; Dror et al., 2012; Fu et al., 2014); pho- biodegradation of volatile compounds: acetalde- tocatalytic degradation (Joo et al., 2013; Lin et al., hyde, butyraldehyde and vinyl acetate. The experi- 2014; Gas et al., 2015; Priya and Philip, 2015); sep- ment was designed in two CSTRs reactors in series aration membranes (Oliveira et al., 2001; Das et al., coupled to an UF membrane. The biomass retained 2006); ultrasound (Jiang et al., 2002). on the membrane module was returned to the first Aerobic and anaerobic biodegradation (Ingu- reactor through a recycle stream. The results of va and Shreve, 1999; Min and Ergas, 2006; Kocam- this approach verified the need to use a reactor that emi and Çeçen, 2009; Frascari et al., 2013a, 2013b; enabled a long solids residence time, under high Yu et al., 2013; Hasan and Jabeen, 2015) are nat- organic content. This helped to maximize the dis- ural methods of breaking down organic matter by solved oxygen concentration and to minimize vol- microorganisms. They have the advantage of being atilization of biodegradable products. Inguva et al.,

86 1998 also conducted experiments for biodegrada- an acceptable range of possible solutions. There- tion of 1,2-DCE and TCE using an UF membrane, fore, the interpretation of mass-transfer phenomena as well as a source of biomass such as Xanthobacter in the bioreactor can be performed using empirical autotrophicus GJ10 and Pseudomonas cepacia correlations related to the estimation parameters, PR131. The results showed an improvement in the such as diffusivity coefficient of the solute in the flow of 1,2-DCE and TCE through the membrane medium. compared to the system without use of microorgan- The parametric identification procedure was isms. More particularly, the authors determined the a critical phase of the development of the mathe- diffusivity of the compounds 1,2-DCE and TCE in matical model, as it can help in the search for ex- the UF membrane and the solute flows through the perimentally unknown parameters. In this relation, membrane in the presence and absence of microbial there are several methods, especially those based degradation of the solute into the permeate side. on probability techniques such as the Genetic Al- The phenomena that arise during microbi- gorithm and Particle Swarm (Kroumov et al., 2006; al activities govern the processes in reactors for Schwaab et al., 2008; Trigueros et al., 2010; Ghov- wastewater treatment and have great relevance vati et al., 2015). The Particle Swarm optimization for the treatment of natural ecosystems. Phenom- technique was developed by Kennedy and Eber- enological models are widely used in bioprocesses hart, 1995, and was successfully applied for esti- (Esser et al., 2015), being based on the formulation mation of parameters of different models describ- of hypotheses and theoretical and/or empirical cor- ing sophisticated phenomena of biotechnological relations to describe the phenomena and inter-rela- processes (Kroumov et al., 2006; Trigueros et al., tions between process variables. The pioneer work 2010; Feisther et al., 2015). of Monod (1949) on modeling of microbial growth In this context, this paper presents the devel- kinetics is still considered fundamental when a sub- opment of a mathematical model able to describe strate limitation process is described. The Monod- the biodegradation of the compounds 1,2-DCE and based expressions (see Table 2) describe the growth TCE in an aerobic bioreactor coupled to an UF rate of biomass μ(h-1) as a function of (S) the sub- module. During the model development, the Par- strate concentration, where different effects on the ticle Swarm global search method was applied to growth take place (Schimidell, 2001). estimate the kinetic, stoichiometric and diffusivity Considering the difficulty of measuring accu- parameters. rately all the variables involved in the biodegradation process, the study and application of a set of al- Materials and Methods gebraic and differential equations formulated from Laboratory scale. Bioreactor modeling physical, chemical and/or biological relationships The biodegradation process was modeled us- between variables was a useful tool to adequately ing experimental data taken from the literature (In- describe the process within the required range of guva et al., 1998). The authors used an experimental precision (Gombert and Nielsen, 2000). Thus, an apparatus, basically consisting of a glass container elaborate mathematical modeling should be able to and a polypropylene ultrafiltration membrane mod- predict the system output variable(s) from the input ule. This UF module divided the bioreactor space data correlated to the independent process varia- into two compartments: an effluent feed chamber bles. and a permeate chamber where the cellular biomass Due to the complexity of the mechanism of acted. Two strains were used by the authors: Xan- diffusion in a medium, it is reasonable to estimate thobacter autotrophicus GJ10 and Pseudomonas the diffusion coefficient of the compounds within cepacia PR131, for degradation of 1,2-DCE and

Table 2. Kinetic models of microbial growth

87 -2 -1 The mass balance for a batch system (Eq.1) is given by the diffusive flux ji (M.L .T ) of the compound i (1,2-DCE and TCE) over the UF membrane. A P ji = K 0 (Si − Si ) (1) -1 A where K0 - is the global mass-transfer coefficient (L.T ); Si -is the substrate concentration in the feed P chamber; Si -is the substrate concentration in the permeate chamber. Equation 2 gives the removal rate of the compounds in the feed chamber: A (2) dSi A = − ji dt VA

3 2 where VA -is the volume of the feed (L ); A is the contact surface area of the UF module (L ). The biodegradation rate of the permeate chamber (Eq.3) considers the solution flux through the membrane plus the biological reaction rate (Ri) for each compound i (Eq.4).

P dSi A (3) = j + Ri dt VP µ R = − i X (4) i Y x / si

3 -1 where VP -is the volume of the permeate chamber (L ), µi -is the specific growth rate of the biomass (T ) th -3 for the "i " compound; X -is the biomass concentration (M.L ); / -is the yield coefficient of conversion of "ith" compound to biomass (M.M-1). 𝑥𝑥 𝑠𝑠𝑠𝑠 The dynamics of changes in the concentration of biomass and oxygen𝑌𝑌 in the permeate chamber are given by equations 5 and 6, respectively. dX (5) = µ X dt i dC µ ox = K a (C eq − C ) − i X (6) dt L ox ox ox Y oxi

-3 th TCE, respectively. The followingwhere assumptions Cox-is the were oxygen oxygen concentration to biomass (ML (MM-1)) in the for liquid the i compound.phase; KLa-is the volumetric mass transfer -1 -3 considered for modeling the aerobiccoefficient biodegradation in the liquid phaseThe (T mass); Ceq transfer-is the equilibrium coefficients concentration of the film of the oxygen (ML ) in the A P -1 th of compounds: (i) the flow throughliquid, theand Ymembraneoxi -is the yieldon thecoefficient feed kf for and oxygen the permeate to biomass kf (MM (LT -)1) were for the i compound. A P -1 was in a pseudo-steady state; (ii) the diffusivityThe mass co transfer- calculated coefficients using of equation the film 7 (Coltonon the feed1969);kf andand K 0 the permeate kf (LT ) were -1 -1 efficient of the solute was constant;calculated and (iii) using perfect equation (LT 7 (Colton) was calculated 1969); and using K0 (LT equation) was 8 calculated (Kreulen etusing equation 8 (Kreulen et al., mixing of the liquid phase. 1993). al., 1993).

The mass balance for a batch system (Eq.1) 1/3 0,75 D  v   ωd 2  is given by the diffusive flux j (M.L-2.T-1) of the kf = 0,0443 AB     (7) (7) i d  D   v  compound i (1,2-DCE and TCE) over the UF  AB    -21 -1 1 δ 1 The mass balance formembrane. a batch system (Eq.1) is given by the diffusive flux ji (M.L .T ) of the (8) = A + + P (8) compound i (1,2-DCE and TCE) over the UF membrane. K0 kf Dm kf A P (1) (1) ji = K 0 (Si − Si ) 2 -1 2 - where DAB is the thermalwhere diffusivity DAB iscoefficient the thermal of the diffusivity solute (L coefficient.T ); ν -is the kinematic viscosity (L .T -1 A -2 -1 2 -1 whereTheK0 mass- is the balance global for mass a -batchtransfer system coefficient (Eq.1) (L.Tis given1); S by-is thethe diffusivesubstrate fluxconcentrationof theji ( M.Lsolute.T in(L )the .Tof );feedthe ν -is the kinematic viscosity -1 where K - is the global mass-transfer); di -is the coefficient length of the impeller (L); ɷ -is the velocity of the agitation (rotation speed,T ); Dm - is the compound i (1,P 2-DCE and TCE) over0 the UF membrane. 2 -1 2 -1 -1 A coefficient molecular diffusivity(L .T ); dof -is the the solute length in of the the membrane impeller (L);(L . ɷT -is); the-is membrane thickness (L). chamber; Si -is the substrate(L.T concentration); S -is the substrateinA the permeateP concentration chamber in. the feed ji = K (S − S ) velocity of the agitation(1) (rotation speed,T-1); D - is Equation 2 gives thechamber; removali SrateP -is 0of the thei substratecompoundsi concentration in the feed chamberin the : -2 -1 m The mass balance for a batch systemi (Eq.1) is given-1 A by the diffusive fluxthe jcoefficienti (M.L .T ) molecularof the diffusivity of the solute𝛿𝛿 in where K0 - is the global masspermeate-transfer chamber. coefficient (L.T ); Si -is the substrate concentration in the feed compound i (1,2-DCE and TCE) over thedS AUF membrane.A the membrane (L2.T-1);(2 -is) membrane thickness (L). P Equationi 2 AgivesP the removal rate of the -2 -1 chamber; Si -is the substrate concentrationj = K ( =Sin− the−ji S permeate) chamber. (1) The mass balance for a batchi system0 i (Eq.1)i is given by the diffusive flux jThei (M.L molecular.T ) of diffusivity the coefficient (Dm) of compounds indt the feedV chamber:A compoundEquation i (1, 2 -givesDCE theand removalTCE) over rate the of UFthe membrane.compounds-1 inA the feed chamber: 1 where K0 - is the global mass-transfer coefficient (L.T ); S -is the substrate concentration1,2-DCE and in TCE the feedin the Metricel membrane was A P i 3 determined2 experimentally(1) by Inguva et al. (1998) P ji =AK 0 (Si − Si ) (2) chamber;where VAS-is- isthe thevolume substrate of theconcentration feed (LdSi); A inis the the Apermeatecontact surface chamber area. of the UF module (L ). -8 -8 2 - i -1 A (2) = − ji and their values were 1,692.10 and 5,076.10 m h whereTheK0 -biodegradationis the global massrate -oftransfer the permeate coefficient chamber(L.T (Eq.3)); Si -is consider the substrates the solutionconcentration1 flux through in the thefeed Equation 2 gives the removal ratedt of the compoundsVA in the feed chamber: , respectively. The values of the parameters such as membrane plusP the biological reaction rate (Ri) for each compound i (Eq.4). chamber; Si -is the substrate concentrationA in the permeate chamber. Metricel membrane thickness, diameter and contact 3 (2) dS3 Pi A 2 where VAEquation-is the volume 2 gives of thewhere the removal feed VA (L-is rate ) ;theA =of is−volume the jthe compoundscontact of the surface feed in the(L area );feed A of is chamberthe the UF module:area used (L by). Inguva et al, (1998) were as follows: dSi Ai 2 (3) contact surfacedt =areaj ofV+ AtheRi UF module (L ). -2 -4 2 The biodegradation rate of the permeateA chamber (Eq.3) considers the solution89 µm; flux 4,7.10 through m andthe 17,4.10 m , respectively. dtdS VP A (2) membrane plus the biological reactionThe biodegradationratei (Ri) for each rate compound of the i (Eq.permeate4). The recipient container used with a total volume of 3 = − ji 2 chamber (Eq.3) considersµ the solution flux through -6 3 where VA -is the volume of the feed (L P)dt; A is thei VcontactA surface area of the UF module70.10 (Lm ).was maintained(4) on a magnetic stirrer, as- Ri = − X The biodegradation therate membrane of thedS permeatei plus Athe chamber biological (Eq.3) reaction consider rate (Rs the) solution flux through the(3) -2 = jYx / s+ R i suming the length of the stir bar was 1.10 m. 3 i i 2 membranewhere VA plus-is the biologicalvolume forof reactiontheeach feed compounddt rate(L ) ;(RAVi) is iPfor (Eq.4).the eachcontact compound surface iarea(Eq. of4). the UF moduleIn (Lthis). study, several models with different 3 -1 The biodegradation rate of theP permeateµ chamber (Eq.3) considers the levelssolution of fluxsophistication through the were tested in order to rep- where VP -is the volume of the permeatedS chamberAi (L ), µi -is the specific growth(3) rate of the biomass (T(34)) th Rii = − X -3 formembrane the "i " plus compound the biological; X -is reaction the biomass rate= j (R iconcentration) +forRi each compound (M.L );i (Eq. 4). -is theresent yield the coefficienteffects of substrate of (1,2-DCE, TCE, and Yx / s / th dt VP-i1 conversion of "i " compound to biomass P(M.M ). oxygen) limitation (Monod (1949), Tessier (1942) dSi µ A 𝑌𝑌𝑥𝑥 𝑠𝑠𝑠𝑠 (3) The dynamics of changes in the concentration= j i + 3R iof biomass and oxygen(4) in theand permeate Moser (1958)) chamber and (are-41 inhibition) (Andrews (1968)) where VP -is the volume of the permeateRi =chamber− X(L ), µi -is the specific growth rate of the biomass (T ) dt VP and Wu et al. (1988)). (see Table 2). The Ks oxy- given by equationsth 5 and 6, respectively. Yx / s -3 ox for the "i " compound; X -is the biomass concentrationi (M.L ); / -is the yield coefficient of -3 th where V -is the volume-µ1 i of the permeate chamber gen saturation constant was set at 10-4 kg m , far conversion of "i " compound to biomassP RdX (M= .−M ). X (5()4) 3 i 3 𝑥𝑥 𝑠𝑠𝑠𝑠 below the critical oxygen-1 concentration. This en- where V -is the volume of (Lthe), permeate µ -is the chamber specific= µYi X (L growth), µ - israte the ofspecific the biomass𝑌𝑌 growth rate of the biomass (T ) TheP dynamics of changes ini the concentrationx / si of biomassi and oxygen in the permeate chamber are th -1 dt th -3 sured about 10-30% of the oxygen saturation con- givenfor the by "i equations" compound 5 and; 6,X(T respectively-is) forthe thebiomass . “i ” compound;concentration X -is(M .theL ); biomass / - is the yield coefficient of th dCox eq -3-1 µi centration (C ) in the cultivation medium (Schim- conversion of "i " compoundconcentration to biomass= K a (M(M.L(C.M−);).C -is) − 3the yieldX coefficient of eq (6-)1 where VP -is the volume of the permeateL ox chamberox ox (L ), µi -is the specific𝑥𝑥 𝑠𝑠𝑠𝑠 growth rate of the biomass (T ) th dt dX th Y -3 -1 idell, 2001). The equilibrium(5) oxygen concentration The dynamics of changesconversion in the of concentration “i ” compound of biomassoxtoi biomass and (M.M oxygen𝑌𝑌 ). in the permeate chamber are for the "i " compound; X -is the biomass= µi Xconcentration (M.L ); / -is the yield coefficient of th The dynamicsdt of- 1changes in the concentration (Ceq) in the medium was estimated to be about 6.5 givenconversion by equations of "i "5 compoundand 6, respectively to biomass. (M.M ). -1 -3 𝑥𝑥 𝑠𝑠𝑠𝑠 to 7.6 mg L at the given temperature (Schimidell, where Cox-is the oxygen concentrationofdC biomassox and (ML oxygeneq ) in in the the µ liquidpermeatei phase; chamber KLa𝑌𝑌- isare the volumetric mass transfer The dynamics of changes= -in1 the concentration− − of biomass and oxygen in the permeate chamber-3 (6 are) K LadXox (Cox Cox ) X 2001). This corresponded(5) to 10 up to 15% lower coefficient in the liquid phasegivendt (T by );equations Ceq -=isµ the5 and equilibrium 6, Yrespectively. concentration of the oxygen (ML ) in the given by equations 5 and 6, respectively. i X oxi th liquid, and Yoxi -is the yield coefficient fordt oxygen to biomass (MM-1) for the i compound.concentration than the saturation concentration of A P -1 The mass transfer dCcoefficients ofdX the-3 film onµ the feed kf and (5)the permeatethe oxygenkf in(LT water.) were The(5 ) value of the volumetric where C -is the oxygen concentrationox = (MLeq=−)µ in the− liquidi phase; K a-is the volumetric mass transfer(6) ox K Laox (Cox Ci Xox ) -1 X L mass transfer coefficient K a of oxygen varied from calculated using equation 7 (Coltondt -1 1969);dt and K0 (LTY ) was calculated using equation 8 (Kreulen-3 et al., L coefficient in the liquid phase (T ); Ceq -is the equilibriumoxi concentration of the oxygen (ML-1 ) in the 1993). dC µ th 50 to 150 h . The initial O2 concentration was con- liquid, and Yoxi -is the yield coefficientox = K fora o(Cxygeneq − C to biomass) − i X (MM-1) for the(6) i compound. (6) L ox -3ox 1/3ox sidered to be equal to the saturation concentration 2 0,75 A P -1 where CTheox-is mass the oxygentransfer concentration coefficientsdt of(ML the ) filmin the  onω liquid theYox feed phase;kf KandLa-is the the permeate volumetrickf mass(LT transfer) were DAB v d i (7) kf -=1 0,0443    -1 (Ceq). -3 coefficientcalculated usingin the equation liquid phase 7 (Colton (T ); 1969); Ceq - isand the K 0equilibrium(LT ) was concentrationcalculated using-3 of equationthe oxyg 8en (Kreulen (ML ) inet al.,the where C -is thed  DoxygenAB   v concentration (ML ) ox -3 th The kinetic and stoichiometric parameters of 1993).liquid,where and C Y-isox i the-is theoxygenyield concentrationcoefficient for (MLoxygen) in to thebiomass liquid (MM phase;-1) forK a the-is ithecompound. volumetric mass transfer ox in the liquid1 phase;1 δ K a-is1 the volumetricA Lmass P -1 -1 = + +L the kinetic model-3 (μmax(8, )Ks and Yx/s), and the coeffi- coefficientThe mass in the transfer liquid phasecoefficients (T ); Cofeq theA-is filmthe1/3 equilibrium onP the0,75 feed concentrationkf-1 and the ofpermeate the oxygkfen (LT(ML )) werein the transfer coefficientK0 kf  Dinm thekfω liquid2-1 phase (T ); C -is DAB v d eq thcient of diffusivity DAB(7 )and stirring speeds ɷ in the calculatedliquid, and using Y equation-is the yield 7 (Colton coefficientkf = 0 ,1969);0443 for and oxygen K0 (LT to biomass) was calculated (MM-1) forusing -3the equationi compound. 8 (Kreulen et al., oxi the equilibriumd concentration D   v  of the oxygen (ML ) feed and permeate chambers were estimated by ap- 1993).  AB    2 -1 A P -1 2 - where DABTheis massthe thermal transfer diffusivity coefficients coefficient of the offilm the on solute the (Lfeed.T kf); νand -is thethe kinematicpermeate viscositykf (LT (L) were.T in the liquid,1 and1 Yδoxi -is1 the -1 yield coefficient for 1 1/3 -1 calculated using equation 7 (Colton 1969);= + and +K (LT2 0,75) was calculated using equation 8 (Kreulen et(8 al.,) ); d -is the length of the impeller (L); ɷ -Ais the velocity0ωP  of the agitation (rotation speed,T ); Dm - is the K kfDAB Dv kf d (7) coefficient1993). molecular diffusivitykf of= 0 the,04430 solute inm the membrane (L2.T-1); -is membrane88 thickness (L). d  DAB   v  1/3 0,75 1 1 δ  1 ω 2  2 -1 2 - where DAB is the thermal diffusivity coefficient= DAB+ vof+ the soluted (L .T ); ν -is the kinematic viscosity (L .T(8()7) 1 kf = 0,0443 A   P  𝛿𝛿 -1 ); d -is the length of the impeller (L);K0 ɷkf-isd theDDm ABvelocitykf v of the agitation (rotation speed,T ); Dm - is the 2 -1 coefficient molecular diffusivity of the1 solute1 in theδ membrane1 (L .T ); -is membrane thickness (L). = + + 2 -1 2 (-8) where DAB is the thermal diffusivity coefficientK kf A Dof thekf soluteP (L .T ); ν -is the kinematic viscosity (L .T1 1 0 m -1 ); d -is the length of the impeller (L); ɷ -is the velocity of the agitation𝛿𝛿 (rotation speed,T ); Dm - is the 22 -1-1 2 - coefficientwhere DAB molecularis the thermal diffusivity diffusivity of the coefficientsolute in the of membrane the solute (L(L.T.T ));; ν --isis membranethe kinematic thickness viscosity (L) .(L .T 1 -1 ); d -is the length of the impeller (L); ɷ -is the velocity of the agitation (rotation speed,T ); Dm - is 1the coefficient molecular diffusivity of the solute in the membrane (L2.T-1)𝛿𝛿; -is membrane thickness (L).

𝛿𝛿 1

1 plying the Particle Swarm Optimization Algorithm. pounds 1,2-DCE and TCE. The values of the ki- The mass transfer coefficients in the film (kfA and netic and stoichiometric constants of this model are kfP) were calculated using the expression obtained shown in Table 4. from literature kf= f(DAB, ω, ν, d) (Eq.7). The over- The specific growth rate of the biomass was -1 all mass transfer coefficient for 1,2-DCE and TCE higher when 1,2-DCE (µmax=0,080h ) was used. A P was calculated using the following K0=f (kf , kf , The value of the saturation constant (Ks=0,723

Dm, δ) (Eq.8). ppm) showed that X. autotrophicus GJ10 had high- Parameter Identification Procedure er affinity to the 1,2-DCE substrate. On the other The Particle Swarm optimization algorithm is hand, P. cepacia PR131 showed affinity to TCE based on the simulation of the movement of groups (Ks=2,5 ppm). The TCE yield coefficient was high- of animals in search for food and/or shelter, accord- er than on DCE (Yx/s=0,52kgcell/kgTCE). The bio- ing to their individual and collective contributions. mass initial concentration was set up in the range of More details can be found elsewhere (Kennedy and 0,010-0,050 kg.m-3 (Table 4). Eberhardt, 2001). The diffusivity coefficients of 1,2-DCE and The predefined values of algorithm parame- TCE in the permeate chamber (4.88 × 10-5 and 5.65 ters were as follows: 500 particles; 30 iterations; × 10-5 m2h-1, respectively) were smaller than their -5 -5 c1=c2=1.5; ωinicial=0.9; ωfinal=0.4.The algorithm was values in the feed chamber (7.35 × 10 7.94 × 10 coded in Maple15® software, and the ordinary dif- m2h-1, respectively). These facts can be explained ferential equations were solved using the Rosen- by the presence of microorganisms in the permeate brock method. The least squares objective function chamber. The mass transfer coefficients in the film (Eq. 9) was applied to minimize the difference be- (kf) for 1,2-DCE and TCE were also lower in the tween the model and experimental profiles (Eq. 9). permeate chamber (0.010 and 0.015 m.h-1, respectively) than the estimated values for the effluent (9) feed chamber (0.068 and 0.073 m.h-1, respectively). The same interpretation can be applied to the kf where ymod stands for the simulated values value. The smaller kf value corresponded to greater th and yij is the experimental value of the i dependent resistance to the mass transfer flow. This is because variable at jth data point of the independent variable. of the presence of microorganisms in the permeate All simulations were carried out on Intel Core i7 chamber (Table 4). computer (1.8 GHz 1.8 GHz cache memory) with The profiles presented in Figure 1 show the 8 GB RAM. kinetics of biodegradation of 1,2 DCE and TCE in the feed and permeate chamber (Fig.1A and Fig.1B, Simulation Results and Discussion respectively); the kinetics of microbial growth of Batch mode Xanthobacter autotrophicus GJ10 associated with All models applied to describe the substrate the 1,2 DCE utilization and the dynamics of Pseu- limitation (Monod, 1949; Tessier, 1942; and Mos- domonas cepacia PR131I growth on TCE (Fig.1C er, 1958) were evaluated based on the experimen- and Fig.1D, respectively); DO profiles for 1,2 DCE tal data of Inguva et al., 1998. The obtained values and TCE are shown, as well (Fig.1E and Fig.1F, re- of their objective function and the correlation co- spectively). efficient were similar (see Table 3). The values of Analyzing the flow profiles across the mem- kinetic parameters of the models Andrews, 1968, brane (see Fig 1A, Fig.1B) two phases of state can and Wu et al., 1988 showed no effect of inhibition. be distinguished. Initially, the flows of 1,2 DCE and Hence, the Monod model was further used to de- TCE had maximum and minimum values, since the scribe the process of biodegradation of the com- concentration gradients among the two cameras

2 Table 3. Values of the objective function (Fobj) and correlation coefficient (R ) for all tested models used in simulations Model Monod (1949) Megee et al. (1972) Tessier (1942) Moser (1958) Substrate 1,2-DCE TCE 1,2-DCE TCE 1,2-DCE TCE 1,2- TCE DCE Fobj 3,5×10-2 6,7×10-3 3,3×10-3 6,7×10-3 3,3×10-2 6,4×10-3 3,3×10-3 5,3×10-3 R2 0.9933 0.9771 0.9928 0.9771 0.9923 0.9773 0.9928 0.9776

89 were large. Then the flow profiles followed each centration profiles are in accordance with the theory other up to the end of the process, where the con- and their minimum values (see Fig. 1E and 1F) cor- centrations of 1,2-DCE and TCE reached zero on respond to the maximum value of biomass concen- both sides of the UF membrane module. The effect trations. Further, the DO concentrations approach of substrate utilization by the cells can be indirect- the Ceq values because of the complete utilization ly seen in the dynamics of the increase in the bio- of organic compounds. It is noteworthy that dur- mass concentrations (see Fig 1C, Fig.1D). Moreo- ing the process neither DO concentration reached ver, when analyzing the biomass profiles (see Fig. the critical values that affected cellular metabolism. 1C and 1D), the strain that had higher growth was The critical values for both substrates (1,2 DCE and Pseudomonas cepacia PR131, associated with the TCE) can be obtained by using RSA methodology consumption of TCE (0.062 kg.m-3). The simula- (see Fig. 2). tion results about the dissolved oxygen (DO) con-

Table 4. Values of the estimated model parameters and experimental data of aerobic biodegradation of 1,2-DCE and TCE compounds in reactor supplied with UF membrane module. Batch mode.

Parameters Value Units Estimated Parameters 1.2-DCE TCE -1 µmax 0.080 0.035 h -3 -3 3 Ks 0.723×10 2.5×10 kg.m -1 Yx/s 0.450 0.520 kg.kg F -5 -5 2 -1 DAB 7.35×10 7.94×10 m h P -5 -5 2 -1 DAB 4.88×10 5.65×10 m h ɷF 100 100 rpm ɷP 10 10 rpm Calculated Parameters 1.2-DCE TCE kfF 0.068 0.092 m.h-1 kfP 0.010 0.012 m.h-1 K0 0.006 0.0092 m.h-1 Note: Superscript symbols-”F”-stands for feed chamber; “P”-stands for permeate chamber; rpm-stands for rotation per minute.

Figure 2 shows the response surface of the specific growth rate of biomass (μ) as a function of substrates and DO concentration. It can be seen in Figure 2A that concentrations below 1.5 ppm (20% of saturation concentration) of DO value limit the growth of X. autotrophicus GJ10. The limiting concentration of 1,2-DCE for the same strain was found to be below 20 ppm. Figure 2B shows that a concentration below 30 ppm of TCE limits the growth of P.cepacia PR131, and that the value of 1.5 ppm of DO can be considered as a limiting level Fig. 2. Response Surface Analysis of the specific growth for the system. rate as a function of substrates: (Fig.2A) 1,2-DCE and dissolved oxygen; (Fig.2B) TCE and dissolved Conclusions oxygen. The paper presents the modeling approach to the process of aerobic biodegradation of 1,2-DCE by the microorganism Xanthobacter autotrophicus GJ10 and TCE by the microorganism Pseudomonas

90 Fig. 1. Kinetics of 1,2-DCE and TCE biodegradation in reactor supplied with UF membrane module. Batch mode. (experimental data are taken from Inguva et al., 1998): (A,B) compounds removal in feed and permeate: (●) 1,2-DCE in the feed chamber; (○)1,2-DCE in the permeate chamber; (■) TCE in the feed chamber; (□) TCE in the permeate chamber; (----) Simulation results for the feed chamber; (▬)Simula- tion results for the permeate chamber; (C,D) simulation results for biomass growth on 1,2-DCE and TCE substrates, respectively; (E,F) simulation results of the DO -dissolved oxygen concentration in the culture medium in the presence of 1,2-DCE and TCE substrates, respectively. cepacia PR131 in a bioreactor coupled to an UF ed based on experimental data of batch mode. For membrane unit. The aim of the study was to esti- this purpose, the Particle Swarm optimization algo- mate the kinetics of the bioprocess and to perform rithm was used, encoded in Maple 15® software. a parameter identification procedure based on the The simulation results obtained using the developed real experimental data published in the literature. kinetic model showed new highlights and optimal The specific growth rate was assumed to be a func- trajectory that can help to increase organochlorine tion of the 1,2-DCE and TCE and oxygen substrates wastewater treatment efficiency for the bioreactor concentrations. The parameter values were estimat- system coupled to an UF membrane module.

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Bateson, K. Z. Guyton tion of water with chlorinated hydrocarbons and heavy (2011). A review of the genotoxicity of 1,2-dichloroethane metals: Challenges and current bioremediation strategies. (EDC). Mutat. Res. 727: 42-53. Int. J. Environ. Sci. Technol. 10: 395-412. Hasan, S. A., S. Jabeen (2015). Degradation kinetics and Aziz, C. E., M. W. Fitch, L.K. Linquist, J. G. Pressman (1995). pathway of phenol by Pseudomonas and Bacillus species. Methanotropic biodegradation of of trichlorodbyke i n a Biotechnol. Biotechnol. Eq. 29: 45-53. hollow fiber membrane bioreactor. Environ. Sci. Technol. Huang, B., C. Lei, C. Wei, G. Zeng (2014). Chlorinated vola- 29: 2574-2583. tile organic compounds (Cl-VOCs) in environment-sourc- Bolzonella, D., P. Battistoni, F. Fatone, E. Cola, A.L. Eusebi es, potential human health impacts, and current remedia- (2007). Micropollutants removals and operating strategies tion technologies. Environ. Int. 71: 118-138. in ultrafiltration membrane systems for municipal waste- Inguva, S., M. Boensch, G. S. Shreve (1998). Microbial En- water treatment: preliminary results. Ind. Eng. Chem. Res. hancement of TCE and 1,2-DCE Solute Flux in UF-Mem- 46: 6716-6723. brane Bioreactors. AIChE J. 44: 2112-2122. Che, H., W. Lee (2011). Selective redox degradation of chlo- Inguva, S., G. S. Shreve (1999). Biodegradation kinetics of rinated aliphatic compounds by Fenton reaction in pyrite trichloroethylene and 1,2-dichloroethane by Burkholderia suspension. Chemosphere 82: 1103-1108. (Pseudomonas cepacia PR1 and Xanthobacter autotro- Cicek, N. (2003). A review of membrane bioreactors and phicus GJ09). Int. Biodeterior. Biodegrad. 43: 57-61. their potential application in the treatment of agricultural International Agency for Research on Cancer (IARC), (1999). wastewater. Can. Biosyst. Eng./Le Genie des Biosyst. au 1,2-Dichloroethane by the IARC Monographs, vol 71. 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92 Min, K., S. J. Ergas (2006). Volatilization and biodegradation Drinking Water Contaminants-Standards and Regula- of VOCs in membrane bioreactors (MBR). Water Air Soil tions. http://www.cdph.ca.gov/certlic/drinkingwater/ Pollut. 6: 83-96. Documents/DWdocuments/EPAandCDPH-11-28-2008. Monod, J. (1949). The growth of bacterial culture. Annu. Rev. pdf (accessed 2.12.15). Microbiol. 3: 371-394. U.S. Environmental Protect Agency (US EPA), (2001). Tri- Moser, H. (1958). The Dynamics of Bacterial Populations chloroethylene Health Risk Assessment: Synthesis and Maintained in the Chemostat. Carnegie Institution of Characterization. US EPA Office of Research and Devel- Washington, pp. 136 opment. Washington, DC 20460. EPA/600/P-01/002A Oliveira, T. A. C., J. T. Scarpello, A. G. Livingston (2001). Vilve, M., S. Vilhunen, M. Vepsäläinen, T.A. Kurniawan, N. Pervaporation-biological oxidation hybrid process for re- Lehtonen, H. Isomäki, M. Sillanpää (2010). Degradation moval of volatile organic compounds from wastewaters. J. of 1,2-dichloroethane from wash water of ion-exchange Memb. Sci. 195: 75-88. resin using Fenton’s oxidation. Environ. Sci. Pollut. Res. Pant, P., S. Pant (2010). A review: Advances in microbial re- Int. 17: 875-84. mediation of trichloroethylene (TCE). J. Environ. Sci. 22: Wu, Y. C., O. J. Hao, K. C. Ou, R. J. Schoelze (1998). Treat- 116-126. ment of leachate from solid waste landfill site using a two- Priya, V. S., L. Philip (2015). Photocatalytic Degradation of stage anaerobic filter. Biotechnol. Bioeng. 31: 257-266. Aqueous VOCs Using N Doped TiO 2 : Comparison of Xing, C.-H., E. Tardieu, Y. Qian, X.-H. Wen (2000). Ultrafil- Photocatalytic Degradation under Visible and Sunlight Ir- tration membrane bioreactor for urban wastewater recla- radiation. Int. J. Environ. Sci. Dev. 6: 286-291. mation. J. Memb. Sci. 177: 73-82. Scheutz, C., N. D. Durant, M. H. Hansen, P. L. Bjerg (2011). Yeh, H. C., W.E. Kastenberg (1991). Health risk asessment of Natural and enhanced anaerobic degradation of 1,1,1-tri- biodegradable volatile organic chemicals: A case study of chloroethane and its degradation products in the subsur- PCE, TCE, DCE and VC. J. Hazard. Mater. 27: 111-126. face - A critical review. Water Res. 45: 2701-2723. Yu, R., H. S. Peethambaram, R. W. Falta, M. F. Verce, J. K. Schimidell,W., U. Lima, E. Aquarone, W. Borzani (2001). Henderson, C. E. Bagwell, R. L. Brigmon, D. L. Freed- Biotecnologia Industrial Vol II: Engenharia Bioquímica. man (2013). Kinetics of 1,2-dichloroethane and 1,2-di- Ed. Edgard Blucher Ltda, São Paulo, pp. 560. bromoethane biodegradation in anaerobic enrichment cul- Schwaab, M., E. C. Biscaia, Jr. J. L. Monteiro, J. C. Pinto tures. Appl. Environ. Microbiol. 79: 1359-1367. (2008). Nonlinear parameter estimation through particle Yuan, M.-H., C.-C. Chang, C.-Y. Chang, W.-C. Liao, W.-K. swarm optimization. Chem. Eng. Sci. 63: 1542-1552. Tu, J.-Y. Ji, D.-R. Tseng, J.-L. Shie, Y.-H. Chen (2015). Tessier, G. (1942). Croissance des populations bactériennes Ozone-catalytic oxidation for gaseous 1,2-dichloroethane et quantite â d’aliment disponible. Rev. Sci. Paris. 80-209. in air over Pt/Al2O3 catalyst. J. Taiwan Inst. Chem. Eng. Trigueros, D. E. G., A.N. Módenes, A. D. Kroumov, F. R. 53: 52-57. Espinoza-Quiñones (2010). Modeling of biodegradation Zhang, M., F. He, D. Zhao, X. Hao (2011). Degradation of process of BTEX compounds: Kinetic parameters estima- soil-sorbed trichloroethylene by stabilized zero valent iron tion by using Particle Swarm Global Optimizer. Process nanoparticles: Effects of sorption, surfactants, and natural Biochem. 45: 1355-1361. organic matter. Water Res. 45: 2401-2414. U.S. Environmental Protect Agency (US EPA), (2009).

93 ACTA MICROBIOLOGICA BULGARICA

In memoriam was discovered 103 unknown agents of diseases, from which over 60 on cultural plants. The major part of his scientific work was connected with plant virology. Prof. Kovachevsky together with Prof. Dimitar Atanassov were founders of plant virology in Bulgaria. Corr. Member Prof. Dr. Kovachevsky first marshaled method of experimental infection of test (indicator) of each virus plants. It is no over- straining to emphasize that Prof. Kovachevsky is researched viral diseases on extraordinarily large number herbaceous cultural and wild plants and with this large envelopment of plant species of different botanical families he holds leadership among the plant virologist in Bulgaria. Virus diseases on Ivan Kovachevski important for our country agricultural cultures were 1904 – 1955 investigated: maize, beans, vegetables (tomato, pepper, eggplant, cucumbers, melons, pumpkins as well as vegetables from Apiaceae and Cruciferae Prof. Dr. Ivan Christov Kovachevsky, Cor- families. Virus diseases on ornamentals, aromatic respondent Member of the Bulgarian Academy of and medicinal plants from 22 families were studied Sciences was born in 10 May 1904 in Sofia and in his publications. It could say, Prof. Kovachevsky was sprung from (of) the Bulgarian national re- is fixed the beginning of the investigations of viral vival genus. A hundred twelve years have passed diseases on aromatic and curative plants in Bulgaria. since his birth to 2016. He was died of age from 91 He was identified viruses on strategically important years (15.03.1995). Prof. Dr. Kovachevsky began agricultural cultures. Fundamental studies for Bul- as an assistant phytopathologist in Department of garian Plant Virology were Prof. Kovachevsky’s “Plant-growing production” of the Agriculture-For- studieеs for Cucumber mosaic virus, Alfalfa mosaic estry Faculty of the Sofia University “Saint Kliment virus, Potato Y virus, Cucumber green mottle mo- Ohridsky” (1927-1930). He was scholar of Hum- saic virus, Cauliflower mosaic virus, Turnip mosaic bolt’s Foundation in Botanical Garden in Darmsh- virus and etz. More important monographies and tad near Berlin to specialize plant virology. He was papers reflected his researches were: “ Virus and Chief-section of the Agricultural Trial Station in So- Virus-like Diseases of Crop Plants”, “Cucumber fia among 1931-1935. Prof. Dr. Kovachevsky was mosaic viroses in Bulgaria”, “Viroses on pepper, Head of the Institute of Plant Protection in Bulgaria “Potato Y viruses (sypanitza) on tobacco, “ Studies in the period 1941-1973. He was chosen for Cor- on tomato vein necrosis, “Necrotic bronzing on to- respondent Member of the Bulgarian Academy of mato”. As a Head of Plant Protection Institute Prof. Sciences in 1961 and also was Correspondent Mem- Dr. Kovachevsky ensured the most of new for their ber of the Academy of the Agricultural Sciences of time scientific journals and books for the library. Germany and Member of the American Phytopatho- This literature is used from the researchers and at logical Society (1928). the present time. Corr. Member Prof. Dr. Ivan Kovachevsky Corr. Member Prof. Dr. Ivan Kovachevsky began his scientific work with the studies of fungal was and remains model for the following generations diseases on agricultural cultures, also and myco- of phytopathologists and plant virologists with his plasma diseases, more specially “stolbur” on toma- scientific work and exceptionally positive qualities to and other plants from Solanaceae family (Inves- of scientific researcher. tigations of the disease “stolbur” in Bulgaria). He Bistra Dikova

94 Cronicle

DAY OF THE STEPHAN ANGELOFF INSTITUTE OF MICROBIOLOGY BULGARIAN ACADEMY OF SCIENCES

On 14 March 2014, the Stefan Angeloff Institute of Microbiology at the Bulgarian Academy of Sciences (BAS) held a formal meeting. Founded as a microbiological centre sixty-nine years ago (14 March 1947), today the Institute is a well-established leader in the Balkan region. For more than a decade it has been associated with the Pasteur Institute, Paris, and with the International Network of Pasteur Institutes, which comprises 32 research centres in 25 countries in five continents. Thus, in partnership, the Stefan Angeloff Institute has been successfully resolving scientific and practical problems, as became evident at the last meeting. Among the presidium members at the scientific forum were Academician Angel Galabov, Chairman of the Stefan Angeloff Foundation and a long-time director of the Institute of Microbiology at BAS, and the current director of the Institute of Microbiology, Hristo Naidenski (Corresponding Member). Guests attending the gala day were Prof. Athanassios Tsakris, Head of Department of Microbiology at the Faculty of Medicine at University of Athens, Prof. Todor Kantardzhiev, Director of the National Centre of Infectious and Parasitic Diseases, Prof. Encho Savov, Head of Department of Military Epidemiology and Microbiology at the Military Medical Academy (MMA), Prof. Dimitar Kadiyski, Director of the Institute of Experimental Morphology, Pathology and Anthropology with museum (BAS), Assoc. Prof. Galina Sachanska, Head of the Biolaboratory at the Department of Natural Sciences at New Bulgarian University, Assoc. Prof. Giannis Papathanasiou from the Medical University in Plovdiv, Valery Andreev MPharm, manager of Sevex Pharma. As Corresponding Member Hristo Naidenski emphasized in his welcome speech: “Our Institute is a recognized centre of the microbiological sciences in Bulgaria and South East Europe. It has gained popularity and European identity. Our Institute has learned from a multitude of microbiologists of international renown, and in turn has been attracting young scientists for training in the fields of microbiology, virology, and biotechnologies.” Prof. Naidenski went on to point out that today, despite the financial constraints, Bulgarian scholars have retained their high publication activity and fully participate in the European research collaboration by responding adequately to the new realities and challenges. The participants in the forum welcomed with great interest the guest speaker, Prof. Athanassios Tsakris, author of over 270 scientific publications and member of the Science Advisory Board of the European Union for the research on antimicrobial resistance, and member of the editorial boards of over 10 specialized journals. In his academic speech Prof. Tsakris talked about the molecular mechanisms of microbial resistance to therapeutic agents, the control of certain infections, and the antibiotic policies. He presented his own methods now applied in many countries for determining microbial resistance to antibiotics. The cost of each method is about 0.5 Euros, which means that they are far less expensive compared to other tests. Prof. Tsakris noted that because of today’s unjustified use of antibiotics there is an increase in drug-resistant pathogens, and that the problem can be controlled by modern diagnostic tests and other innovative approaches. Traditionally, on the day commemorating the establishment of the Institute, a young scientist is awarded a prize for best work in the field of microbiology. This year a jury from the Stefan Angeroff Foundation distinguished two first-prize winners: Adelina Stoyanova for her research on consistent alternative application of triple combination of viral inhibitors in enteroviral neuroinfections, and Maya Hadzhieva for her studies on the biological role of immunoglobulins with induced poly-specificity. The awards were handed out by Academician Angel Galabov, Chairman of the Foundation. Yet another award - a plaque of honour - caused further excitement at the forum as it was conferred to Prof. Maria Angelova for her contribution to the overall development of the Institute. In turn, she thanked her colleagues, underlying that such a distinction is never a single person’s merit but the work of many people. The academic atmosphere of the meeting was followed by the positive emotions of a concert and came to a solemn end with “Na Mnogaya Leta”. That was, in fact, the wish of all attendees – to see the Institute of Microbiology at BAS working, progressing and conquering new scientific heights. Na mnogaya leta (For many years to come!)! Dr. Vanya Shipochlieva, MD

95 Fourth Congress of Virology (Days of Virology in Bulgaria) with International Participation

May 18-20, 2016, Sofia, Bulgaria

Big Hall of Bulgarian Academy of Sciences 1 “15h November” Str., Sofia

www.bulvirologydays2016.com

The Organizing Committee informs that the Fourth Congress of Virology (Days of Virology in Bulgaria) with International Participation will be held on May 19-20, 2016 in the Big Hall of the Bulgarian Academy of Sciences, 1 “15h November” Str., Sofia, Bulgaria. The Congress will be attended by virologists, specialists of the medical, veterinary and plant virology from all over the country. The scientific program contains plenary lectures given by leading scientists from Europe (Balkan region included), USA and Japan, large poster session and organized discussions on present milestones in virology. The registration details will be given in the website: www.virologycongress.bg The Congress reports in English will be published in Acta Microbiologica Bulgarica, volume 32, issues 2 and 3 (2016). The reports have to be prepared according to the „Guide of Authors” of the Journal.

96 Workshop on Food-Borne Pathogens and Food Safety

May 26-27, 2016, Sofia, Bulgaria

www.microbio.bas.bg

The Organising Committee has the pleasure to invite you to attend the workshop Food-Borne Pathogens and Food Safety, which will take place during May 26-27, 2016, at The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 26, Sofia 1113, Bulgaria. It is a pre-conference meeting within the frame of the International Conference on Food Microbiology that will be held on August 08-10, 2016, in Birmingham, UK. The meeting will bring together microbiologists, immunologists and biotechnologists, experts in pathogenic microorganisms, food control, medical and veterinary microbiology from Bulgaria and other Balkan countries. During the plenary lectures, oral presentations, and poster sessions will be shared newest data and information on key points in the field of food-borne pathogens and food safety. The authors may choose to publish the presented results as full-text or short communications in Acta microbiologica bulgarica after approval by the Editorial Board.

International Conference on Food Microbiology

August 08-10, 2016, Birmingham, UK.

http://foodmicrobiology.conferenceseries.com

OMICS International welcomes you to attend the International Conference on Food Microbiology during August 08-10, 2016 at Birmingham, UK. It will be joined by participants around the globe who are interested in sharing their knowledge and research in the area of Food Microbes and Microbial Technology. The meeting includes Keynote lectures, Oral and Poster presentations. The attending delegates include Editorial Board Members of related OMICS International Journals. This is an excellent opportunity for the delegates from Universities and Institutes to interact with the world class scientists. The importance of Food Microbiology is growing rapidly, influencing the industrial sector and scientific community. The significance can be gauged by the fact that it has made huge advancements over the course of time and is continuing to influence various zones.The main theme of the conference is “Food Microbiology 2016: Meeting Needs for Sustainable and Safe Future”.

97 Acta Microbiologica Bulgarica

GUIDE FOR AUTHORS

The journal Acta Microbiologica Bulgarica is organ of the Bulgarian Society for Microbiology (Union of Scientists in Bulgaria). The journal is continuation of the edited till 1993 journal of the same name, cited in Index Medicus and Medline. The new edition is published two times per year.

Types of articles

The journal publishes editorials, original research works, research reports, reviews, short communications, letters to the editor, historical notes, etc from all areas of microbiology. The manuscripts should not represent research results which the authors have already published or submitted in other books or journals. The papers submitted for publication in Acta Microbiologica Bulgarica are peer-reviewed by two experts from the respective scientific field who remain anonymous to the authors.

Article structure

The papers are published in English, accompanied by a bilingual summary (English and Bulgarian). The papers should be typed with double-spacing (28-30 lines), on a white paper in A4 format, with margins of 3 cm. The length of the original research paper, including the annexes (tables, figures, etc.) should not exceed a signature or printer’s sheet (30,000 signs, that is, 16 pages with 30 lines each) in Times New Roman 12. The length of shorter reports should not exceed seven pages.The submitted manuscript must contain the name/s and surname/s of the author/s, the name and address of the institution and or organisation where it was prepared. The name and address of the corresponding author should be noted. The abstract should not exceed 250 words and should represent briefly the goals, methods, main results (with numerical data) and basic conclusions of the research.The most essential six key words must be added to the abstract. The manuscript contains the following sections: introduction, materials and methods, results, discussion, acknowledgements, references. The introduction must be concise with a clearly defined goal and with previous knowledge of the problem. The materials and methods ought to contain sufficient data to enable the reader to repeat the investigation without seeking additional information. The results should be presented briefly and clearly, and the discussion should explain the results.The measuring units and other technical data should be given according to the Sl-system. Illustrations (tables and figures) are submitted separately and their places in the text should be clearly indicated. Tables should be given in a separate sheet, numbered and above-entitled. The figures must be accompanied by legends in a separate sheet.

Reference style

The references used are cited in the manuscript as follows: • In the case of single author - the author’s surname and the year of publication (Petrov, 2012); • In the case of two authors - the authors surnames and the year of publication (Petrov and Vassileva, 2013); • In the case of more than two authors - the first author’s surname, at all., and the year of publication (Christova et al., 2014). The references included in the section “References” of the manuscript should be arranged first alphabetically and then further sorted chronologically if necessary. More then one reference from the same author(s) in the same year must be identified by the letters “a”, “b”, “c”, etc. place after the year of publication. 98 Examples: Reference to a journal publication: Georgiev, P., V. Simeonov, T. Ivanov (2010). Production of thermostables enzymes. Biotechnol. Biotec. Eq. 27: 231-238. Reference to a book: John, R., W. Villiam, G. Wilmington (2011). New approach for purification of enteroviral proteins, in: Peterson, K., A. Smith (Eds.), Methods in Proteinology. Elsevier, London, pp. 281-304. Journal names should be abbreviated according to the List of Title World Abbreviations: http://www.issn.org/services/online-services/access-to-the-ltwa/ Manuscripts should be submitted to the Editorial Board of the journal Acta Microbiologica Bulgarica in electronic version of the paper to the following address: [email protected] The publications in Acta Microbiologica Bulgarica are free of charge. The authors of the manuscripts need to cover the costs of printing collared illustrations. https://library.caltech.edu/reference/abbreviations/

The Edition of Acta Microbiologica Bulgarica volume 32 issue 1 is financially supported by ProViotic AD, Sofia

99 Vol. 32, Issue 1 ACTA MICROBIOLOGICA BULGARICA March 2016

CONTENTS Review Articles

A Focus on Carbapenemase-Producing Pathogens Detection by Phenotypic Tests Evangelia Voulgari, Aggeliki Poulou, Georgia Vrioni, Athanassios Tsakris 3

Review-Development of Models of Ethanol Synthesis and Production by Using Principles of the Theory of System Analysis. Personal Experience. Alexander D. Kroumov, Aparecido Nivaldo Módenes 10

Paratuberculosis in Аnimals and Humans – an Actual Health and Economic Problem Magdalena Bonovska, Tanya Savova, Violeta Valcheva, Hristo Najdenski 26 Regular Papers

Immunohistochemistry on Cryostat Tissue Sections Using Synthetic Peptide Produced Monospeciffic Anti-Sheep Pox Virus Antibody Velizar Bumbarov, Alexander Tzymanis, Martin Hristov, Raiko Peshev, Rustem Eligulashvili 42

Screening for Antimicrobial Activity of Bacillus Subtilis and Paenibacillus alvei Isolated from Rotten Apples Compost Natalija Atanasova-Pancevska, Ilina Popovska, Katarina Davalieva, Dzoko Kungulovski 56

In Vitro Antimicrobial and Antioxidant Activity of Extracts From Wild Small Fruits Spread in Bulgaria Hristo M. Najdenski, Violeta S. Kondakova, Ekaterina T. Krumova, Maya M. Zaharieva, Lyuba J. Doumanova, Maria B. Angelova 65

Oligosaccharide Production from Lactose and Lactulose by β-Galactosidase from Lactobacillus plantarum S30 Plamena Zheleva, Tonka Vasileva, Veselin Bivolarski, Ilia Iliev 74

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Workshop on Food-Borne Pathogens and Food Safety, May 26-27, 2016, Sofia 97 International Conference on Food Microbiology, August 08-10, 2016, Birmingham, UK 97