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-resist- ant 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 re- al., 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 vari- ety 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 re- ly 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