Zagazig Veterinary Journal, ©Faculty of Veterinary Medicine, Zagazig University, 44511, Egypt. Volume 48, Number 4, p. 414-432, December 2020 DOI: 10.21608/zvjz.2020.40250.1118

REVIEW ARTICLE Foodborne species: , Isolation, Virulence Attributes and Antimicrobial Resistance El-sayed Y. El-Naenaeey, Marwa I. Abd El-Hamid and Eman K. Khalifa* Department of Microbiology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, 44511, Egypt.

Article History: Received: 16/09/2020 Received in revised form: 31/10/2020 Accepted: 30/11/2020 Abstract Campylobacter is widely regarded as the main cause of foodborne diarrheal diseases worldwide. It is a curved Gram-negative rod displaying corkscrew motility via a polar non-sheathed flagellum. Campylobacter grows microaerophilically at a broad range of temperature (30–45°C), and it is considered biochemically inert. Campylobacter did not use carbohydrates to obtain energy because of lacking the 6-phosphofructokinase enzyme. Campylobacter has a positive reaction for oxidase test and a negative reaction for indole test. Laboratory isolation and detection of Campylobacter species is tricky as they are fastidious and necessitate special atmospheric requirements to grow. Relatively little information is known about the virulence attributes in or how a seemingly fragile can survive with increased pathogenicity. Moreover, the growing antimicrobial resistance of campylobacters to the clinically crucial antibiotics becomes insurmountable. Thereby, this review elucidates and discusses the taxonomy, isolation, identification, virulence attributes and the antimicrobials resistance of this particular bacterium. Keywords: Campylobacter spp., Isolation, Virulence factors, Antimicrobial susceptibility. Introduction immunosuppression and young age with Thermophilic Campylobacter species (spp.) severe infections may need is one of the highly significant four antimicrobial medications [5]. Erythromycin is microorganisms causing diarrheal diseases generally the first drug of choice, where universally. It is regarded as the most frequent fluoroquinolones and on a smaller scale cause of human gastroenteritis globally tetracycline constitute other options [6]. This causing a disease called campylobacteriosis. review centered on the family’s taxonomic Most infections occurred in humans are history and their general characteristics. attributed to C. jejuni (80-85%), while the Moreover, it discusses the isolation, residual cases are credited with C. coli identification and confirmation of [1]. This bacterium is transmitted to people campylobacters, their virulence attributes in through contaminated undercooked foods, addition to their mechanisms of resistance to especially undercooked poultry meat and various antimicrobial classes. unpasteurized dairy products [2]. Despite, Historical standpoint of Campylobacter comparatively few reports about species Campylobacter spp. virulence; these Historically in 1886, the first written report microorganisms have various virulence factors regarding campylobacter is supposed to be in regard to motility, adhesion, invasion and introduced by Theodore Escherich, he noticed toxin-activity, among others [3]. and prescribed a non-cultivable bacterium with Campylobacteriosis symptoms ranged from a spiral shape [7]. After that, in 1906, mild diarrhea to sever serious complications campylobacter was firstly detected by 2 British [4]. Human campylobacteriosis are mostly veterinarians; Stewart Stockman and John self-limiting. Despite that, patients with McFadyean, who declared a huge number of peculiar organisms that were present in the *Corresponding author e-mail: ([email protected]), Department of Microbiology, Faculty of 414 Veterinary Medicine, Zagazig University.

Zag Vet J, Volume 48, Number 4, p. 414-432, December 2020 El-Naenaeey et al., (2020) uterus of a pregnant ewe [8]. The described troglodytes [30], C. corcagensis [31] and C. organism was probably to be Campylobacter iquaniorum [32]. Currently, the genus fetus [9]. Later, in 1913, MacFadyean and Campylobacter contains 31 species and 10 Stockman recorded a vibrio-like organism subspecies from fetuses of aborted ewes and termed it (http://www.bacterio.net/campylobacter.html, Vibrio fetus, which was recovered from bovine accessed 18.03.2018). C. ornithocola [33], C. aborted fetus in 1919 [10]. In 1927, a bunch of pinnipediorum subsp. caledonicus, C. bacteria like vibrio was discovered in feces of pinnipediorum subsp. pinnipediorum [34], C. cattle suffering from diarrhea. They were geochelonis [35] and C. hepaticus [36] are the detected by Marion Orcutt and Theobold latest identified species. Smith [11]. In 1931, a vibrio organism from General characteristics of Campylobacter calves and cows having winter dysentery was species isolated by Jones et al., Later; the organism In spite of variations in the host and source was termed as Vibrio jejuni [12]. In 1944, association, members of genus Campylobacter another vibrio was isolated by Doyle from have multiple basic similar features. They are pigs' feces suffering from diarrhea and termed Gram-negative, curved bacteria. When two it as Vibrio coli [13, 14]. In 1957, King stated campylobacters converge together, they create that Vibrio fetus was implicated in humans the “S” shape as the wing of the gull. Cells are with bloodstream infections [15]. about 0.2–0.8 μm in width and 0.5–5 μm in Campylobacters were firstly classified as length [37]. Under the microscope, cells of Vibrio spp. on grounds of their spiral shape. In certain species are straighter rods (i.e., C. 1963, Sebald and Veron have created the showae) and in C. jejuni strains, the cells are Campylobacter genus to distinguish them from usually straight bacilli [38]. When cultures members of Vibrio genus relying on the become old or undergo environmental stress requirements for the microaerobic growth conditions such as low nutrient, atmospheric conditions, the low G and C base of their oxygen and high temperature or freezing, cells DNA, non-fermentative metabolism and can turn to coccal or spherical forms [20]. elevated growth temperature [16]. During Coccoid forms are usually referred as viable, 1973, Chatelain and Veron incorporated 4 but non cultivable (VBNC) cells [4]. different species in Campylobacter genus: C. Campylobacter spp. are motile by a polar sputorum, C. fetus, C. coli and C. jejuni [17]. non-sheathed flagellum. The campylobacter Taxonomy of Campylobacter species helical shape together with their flagella Campylobacter spp. belongs to the epsilon generates a characteristic darting corkscrew- subdivision of [18] and the like motion under the phase-contrast order [19]. The family microscopy [7]. There are two exclusions for Campylobacteraceae is a various group of campylobacter motility; C. showae has a tuft environmental, commensal and pathogenic of flagella at one pole and C. gracilis and C. Gram-negative bacteria. At the present time, it hominis have no flagella [37]. has 3 genera: Sulfurospirillum (7 taxa), Campylobacter spp. proliferates at a Arcobacter (17 taxa) and Campylobacter (30 temperature range of 30–45°C [39]. taxa) [20]. From 2000 to 2009, many novel Thermophilic campylobacters (e.g. C. species were discovered and added to upsaliensis, C. coli, C. jejuni, and C. lari) have campylobacter taxa. C. lanienae [21], C. a better growth at 42°C and 37°C, but they hominis [22], C. hyolei was changed to C. coli can't grow at 25°C. However, other species [23], C. insulaenigrae [24], C. canadensis have an optimum growth at 37°C [37]. [25], C. avium [26], C. cuniculorum [27] and Multiplication of Campylobacter spp. can be C. peloridis [28]. Recently, from 2010 to inhibited at temperatures less than 30°C. 2015, six species were added; B. ureolyticus Therefore, the number of campylobacters in was changed to C. ureolyticus [7], C. foods doesn't increase at room temperature subantarcticus [29], C. volucris [29], C. (20–25°C). Despite the inability of 415 Zag Vet J, Volume 48, Number 4, p. 414-432, December 2020 El-Naenaeey et al., (2020) Campylobacters spp. to grow at temperatures Global epidemiology of campylobacter less than 30°C, it can survive at 4°C within infection humid conditions [40]. Despite good surviving According to the Centers for Disease in low temperatures, Campylobacter spp. are Control and Prevention (CDC) report, heat sensitive and are inactivated by household infection cases caused by campylobacters are cooking or pasteurization. Campylobacter spp. 14 per year per 100,000 of the population in are destroyed immediately by heating at 55– United Sates of America [49]. The problem of 60°C for few minutes [41]. infections has increased 30 times more Campylobacter spp. growth can be reported by the data on an outbreak [50]. restricted by storage at -15°C or by NaCl of a Reports from South America also highlighted concentration more than 2% w/v. its increased prevalence, i.e. 4.6 to 30.1% in C. Nevertheless, freezing cannot eliminate jejuni infections, while three studies from campylobacters. Furthermore, they are Argentina showed 0 to 1.4% prevalence rates incapable of surviving at pH below 4.9 and of C. coli. The range between 4.4 to 10.5% of above 9.0 [42]. Campylobacter sensitivity to C. jejuni infections was reported from Bolivia. ionizing radiation is greater than Salmonella The campylobacter infection ranges were 0- and Listeria species [43]. 14.1% in Chile , 0-14.4% in Colombia, 0- Most Campylobacter spp. grows 23.0% in Ecuador, 0.6-18.4% in Paraguay, 0- microaerophilically. However, some species 23.0% in Peru, 0-14.3% in Uruguay and 0- can proliferate aerobically otherwise 13.0% in Venezuela [51]. anaerobically. The dominant species, C. coli A report evaluation in the European Union and C. jejuni need the microaerobic showed high prevalence rates of atmosphere (3–5% CO2, 5–10% O2 and 85% campylobacter infections in Bulgaria; i.e. N2) to grow [44]. Campylobacter jejuni can 13,500 cases per 100,000 population, while adapt the aerobic atmosphere as it can make less incidence rates in Finland and Sweden biofilms. This improves its ability for were reported [52]. In the United Kingdom, surviving and spreading in food processing the rate of campylobacter infection was 9.3% environments e.g. poultry processing [45]. per 1000 cases per year [53]. In Germany, Campylobacter spp. is somewhat inactive campylobacteriosis incidence was 53.4-81.4% in the biochemical reactions. They did not use per 100,000 of the population [54]. The reports carbohydrates to obtain energy, but they have from China showed 4.84% prevalence rate of a respiratory metabolism using intermediates C. jejuni infections with 14.9% of patients of the tricarboxylic acid cycle in addition to suffering from gastroenteritis in Beijing [19, amino acids. This is because of lacking the 6- 20]. In Japan, campylobacteriosis was found to phosphofructokinase enzyme, which is be 100 cases per 100,000 of population each contributed in the energy metabolism [46]. year [55]. In India, patients having Traditional biochemical reactions of gastroenteritis were found to be culture Campylobacter spp. involve the fumarate positive for campylobacter; C. jejuni was reduction to succinate, the positive reaction for found to be isolated from 70% of the reported oxidase test (except C. gracilis) and the cases, while another study accounted 16.2% of negative reaction for indole test. cases for various Campylobacter species [56, Campylobacters can reduce nitrate with the 57]. exception of C. jejuni subsp. doylei. Prevalence of Campylobacter species in Lecithinase and lipase enzymes are absent and Egypt this genus can't hydrolyze starch, gelatin and In 1995, Campylobacter spp. was isolated casein [47]. Moreover, they are positive for from 880 (16.8%) children with diarrhea and test except C. upsaliensis [48]. from 1079 (6.4%) healthy children. The isolation of Campylobacter species was more frequent than Salmonella, Shigella and other bacterial enteric pathogenic species [58]. From 416 Zag Vet J, Volume 48, Number 4, p. 414-432, December 2020 El-Naenaeey et al., (2020) 1986-1993 in Abbassia Fever Hospital, Cairo, considered sick because of diarrhea, the Egypt, examination of 6,278 patients with frequency of Campylobacter spp. was not acute enteric infections revealed the isolation significantly different. Beef and dairy cattle of 92 strains (63%) of C. jejuni and 54 (37%) can have significant levels of campylobacters, of C. coli [59]. In 2005, military personnel with prevalence rates of 2.5%–60%. with diarrhea were participated in a military In weaning aged pigs, campylobacters can exercise in the northwestern Egyptian desert contribute to colitis. Swine commonly carry C. and the pathogens causing diarrhea were coli and C. jejuni as intestinal commensals. identified in 53.6% of 129 enrolled cases [60]. Studies in the USA, Netherlands, Great A similar study was conducted in 2011 on 72 Britain, and Germany showed that more than personnel with traveler's diarrhea in half of the commercially raised pigs excreted Multinational Force and Observers in the the organisms. C. coli strains comprise the Southern Sinai, Egypt and the bacteriological most prevalent isolates from pigs causing first examination of their stool revealed the watery and then inflammatory diarrheal isolation of C. jejuni from 7 (10%) of the diseases. Pigs have anorexia, fever, and examined samples [61]. Later on, diarrhea for 1–5 days, followed by remission Campylobacter spp. were identified from of clinical signs, but they continue to shed C. patients with gastroenteritis in Cairo, Egypt jejuni in their feces. C. hyointestinalis using conventional and polymerase chain subsp hyointestinalis and C. mucosalis are also reaction (PCR) methods in 6.6% of human implicated as causes of enteritis in pigs. In pet stool samples [62]. animals, C. jejuni causes diarrhea in dogs and Importance of Campylobacter species in cats, which is considered a significant source veterinary fields of the bacterium for the human population. Campylobacter species are almost Diarrhea lasting 5–15 days is the most ubiquitously in the environment and they are common clinical sign in dogs <6 months old. found in the intestinal tract of many wild and Occasionally, the diarrhea becomes chronic domestic warm-blooded animals, usually and may be accompanied by fever and without showing clinical symptoms [63]. increased the white blood cells` counts [68]. Birds are naturally infected via the Zoonotic importance of Campylobacter fecal-oral route and ingestion of species campylobacters numbers as few as 35 CFU Human infection with campylobacter, can be sufficient for successful colonization leading to campylobacteriosis, is caused by the [64]. The organisms colonize primarily the thermophilic members of the genus ceca and the colon and to a lesser extent the Campylobacter; C. jejuni, C. coli, C. lari and small intestine [65]. Young birds (less than 2– C. upsaliensis. Interestingly, C. jejuni and C. 3 weeks) are rarely infected with coli are the most frequently identified species campylobacter due to the maternal antibodies associated with food borne illness. They [66]. The colonization of the intestine has been together account for approximately 95% of associated with jejunal histomorphological human campylobacteriosis cases worldwide changes, higher intestinal permeability, altered [69]. An infective dose as low as 500-800 live intestinal electrolyte transport with a decrease campylobacter cells may be sufficient to cause in the intestinal nutrient absorption and an illness in humans [70]. intracellular Ca2+signaling interference [67]. Generally, the human campylobacter In cattle and sheep, Campylobacter spp. infections are considered foodborne. It occurs cause enteritis and abortion. They include C. via consumption of undercooked meat and jejuni, C. fetus subsp fetus, C. meat products, mainly poultry as well as raw hyointestinalis subsp hyointestinalis, and C. or contaminated milk and contaminated sputorum, which causes abortions in sheep. untreated water. Contact with pet animals has However, in studies that compared C. also been proposed as a mode of transmission jejuni prevalence in healthy cattle and in cattle of campylobacteriosis in humans [71]. 417 Zag Vet J, Volume 48, Number 4, p. 414-432, December 2020 El-Naenaeey et al., (2020) Campylobacter is also recovered from Multiple enrichment broths such as Bolton seawater and bovine manure compost [37]. broth, Preston broth and Campylobacter Campylobacteriosis is an acute self-limiting enrichment broth have been tested for their foodborne enteric illness. The incubation effectiveness [81]. Solid media such as period ranges from 2-5 days, but it has been Preston, charchoal cefoperazone deoxycholate reported to be up to 10 days. In most patients, (CCDA) and Butzler agars have been proved symptoms include diarrhea, abdominal to possess the same effectiveness [82]. The cramps, myalgia, fever and occasionally latest standard method [9] for isolation and vomiting [72]. Additionally, serious post- detection of Campylobacter spp. uses CCDA infectious complications such as Guillain- as the selective medium, while Bolton broth is Barré syndrome and reactive arthritis may be utilized for the enrichment step. Moreover, the occurred in 0.1% and 1% of the population, microaerobic incubation at 42°C is typically respectively [73]. the preferred method as it enables more Isolation and identification of campylobacter strains to proliferate [83]. Campylobacter species Alternative combinations of enrichment broth Isolation of Campylobacter species and plating were used to detect and enumerate Laboratory isolation and identification of campylobacters in meat of chicken [84] and Campylobacter species is laborious as they are they look like to give significantly better fastidious and necessitate special atmospheric results. requirements to grow. The isolation step is Identification of Campylobacter species further compounded by the existence of Among the Campylobacter spp. growing at commensal bacteria that grow rapidly and 42°C, C. coli and C. jejuni are the most vying with campylobacter on the medium [74]. common emerged species from samples; All Campylobacter spp. selective media however, low frequencies of other species have scavengers of oxygen such as charcoal, were also reported. Speciation is difficult due blood, and ferrous iron as campylobacter is to the consequence of the complicated and the sensible to oxygen. The selective agents are swiftly changing taxonomy in addition to the particularly a mixture of antibiotics to which inertness of Campylobacter spp. to campylobacter are resistant e.g vancomycin, biochemical tests. These problems have colistin, cycloheximide and nystatin. These generated phenotypic and genotypic methods antibiotics suppress the proliferation of several to distinguish the members of that group [60]. microbial flora exist in samples and therefore Campylobacters are biochemically inactive allowing the Campylobacter spp. to proliferate compared with other bacteria. As a [75, 76]. consequence, the phenotypic tests used to Methods of campylobacter isolation mostly differentiate them to the species level are few. necessitate a pre-enrichment in broth before In general, C. jejuni can be distinguished from plating on the agar plates [77]. Campylobacter others relying on hippurate hydrolysis as it is isolation from faecal samples is done by the only species that have the hippuricase gene plating directly on selective media followed by giving a hippurate positive result. However, microaerobic incubation at 42°C [78] as feces variability in the hippurate reaction has been frequently contain viable campylobacters with noticed in some of C. jejuni strains resulting in high number [79]. However, environmental hippurate-negative results [86]. There are other samples and food products usually have few biochemical tests used in species identification stressed cells; thus, the initially enrichment such as catalase test, which is negative in C. step in the liquid medium is required [77]. upsaliensis and indoxyl acetate hydrolysis test, The first utilized selective medium for which is negative in C. lari [9]. campylobacter culturing was reported by In earlier times, nalidixic acid and Skirrow. After that, more than forty liquid and cephalothin susceptibility testing have been solid selective culture media have been listed relied on for the species identification [87]. and evaluated [80]. Both C. coli and C. jejuni are cephalothin 418 Zag Vet J, Volume 48, Number 4, p. 414-432, December 2020 El-Naenaeey et al., (2020) resistant [78]. In contrast, C. upsaliensis is Bacterial motility, adherence, invasion ability sensitive to cephalothin [9]. Nowadays, the and toxins production have been described as sensitivity to nalidixic acid may yield virulence factors for Campylobacter spp. [92, difficulties in the interpretation [88] due to the 93] (Figure 1) [94] as following: increasing resistance to fluoroquinolones [89]. Motility Therefore, antimicrobial susceptibility tests Motility is important to get away from the cannot be utilized for the phenotypic stressful environments [95]. Campylobacter identification of campylobacter isolates [85]. motility system requires a chemosensory As a consequence of the difficulties and the system and flagella that drives the bacterial inaccuracy of the phenotypic identification, movement in reliance of the environmental multiple molecular methods were used to conditions. The spiral shape of campylobacter supplement the biochemical tests or to replace in addition to its flagella allow it to swim them [90]. Detecting the species -specific through the thick mucus layer covering the sequences using PCR is auxiliary; specifically, intestine enabling the campylobacter to reach when the distinction between hippuricase efficiently to their favored site of colonization. negative C. jejuni strains and C. coli is This system is formed by many proteins, required and using biochemical tests alone is which have different roles e.g. the main inadequate [91]. flagellum filament proteins, FlaA (the major Campylobacter species virulence factors flagellin subunits) and FlaB (the minor Specific virulence mechanisms are not subunit), those were encoded by flaA and flaB clearly demonstrated for Campylobacter genes, respectively [3]. Other crucial protein, species up till now, probably owing to the CheY that is a response regulator demanded paucity of pathogenesis resemblance between the flagellar rotation [96]. campylobacters and the other bacteria [92].

Figure 1: Diagram demonstrates the virulence attributes of Campylobacter species taken from a previos report [94]. IL-8: interleukin-8, NF-Kb: nuclear factor kappa-light-chain-enhancer of activated B cells

419 Zag Vet J, Volume 48, Number 4, p. 414-432, December 2020 El-Naenaeey et al., (2020) The bacterial flagellum is also relevant with invasion of Campylobacter spp. [106]. bacterial adhesion and invasion into the host Indeed, inhibitor reports with cells. Campylobacter flagellum has chloramphenicol, that retards the Cia protein components analogous to the classical Type III expression, but not FlpA or CadF, propose that secretion systems (T3SS) that assist in the invasion of the host cell necessitates more of a transport of the non-flagellar proteins (FlaC, constitutive expression of the last 2 adhesins FspA, CiaB, CiaC and CiaI) [97] playing a [106]. It is noticed that there was a correlation pivotal role in campylobacter pathogenesis between the degree of adherence of [98]. campylobacters to host cells and the extreme Additional to their essential role in bacterial of the clinical manifestations in infected mobility, it can modulate the host responses peoples [92]. through Toll-like receptor 5 (TLR5). The Invasion TLR5 usually triggers a potent host response, Campylobacter invasion ability is a vital which gives critically important signals to pathogenicity factor. The clinical maintain the immune homeostasis of intestine. manifestations of campylobacteriosis are Despite that, C. jejuni can escapes from the consistent with the cellular invasion. A variety recognition of TLR5 because of the structure of proteins termed campylobacter invasion of its flagellin protein is clearly different from antigens (Cia), are considered the key for other bacterial flagellins stimulating this invasion to host cell and survival, they are system [99]. transferred to the host cells cytosol by a Adhesion flagellar T3SS [107]. In that regard, flagellar The capability of campylobacter to adhere mutants have considerably reduced the to the epithelial cells of the host invasiveness ability of campylobacters [101]. gastrointestinal tract is a fundamental There are 3 Cia proteins; CiaB, which is precondition for colonization. This process is needed for target cells adherence, CiaC, which mediated by variety of adhesins existing on the is concerned with complete invasion of cells bacterial surface; in addition, it is essential to and CiaI, which is requisite for surviving generate a disease [100]. within cells. Recently, CiaD has been detected Adhesion of campylobacter to fibronectin is as an essential factor needed for the best brokered by CadF (fibronectin binding outer invasion of cell hosts [108]. membrane protein) [101], that is encoded by Moreover, the CiaB mutants have the chromosomal cadF gene [102]. Binding to decreased the campylobacter invasiveness fibronectin (glycoprotein found in GIT ability by a marked decrease in their adherence epithelial cells) stimulates signals that lead to capability. Other proteins e.g. FspA, IamA, an activation of Cdc42 and GTPases Rac1 that FlaC, VirK, CeuE and HtrA have been spur the campylobacter cell internalization. proposed to play a role in host cells invasion, Many studies with mutants exhibited that the however the right mechanisms are not absence of this protein inhibits the understood probably up till now [3]. campylobacter colonization [103]. Toxin production Furthermore, some proteins were recognized Cytolethal distending toxin (CDT) is in the colonization process e.g. CapA (auto broadly spread among Gram negative bacteria transporter), PEB1 (periplasmic binding [109]. It is regarded to be the best toxin protein) and JlpA (surface-exposed generated by Campylobacter spp. It is a lipoprotein) [3]. Initially, the FlpA was serious virulence factor that has been listed for regarded to be important for binding to this pathogen [110]. Campylobacter spp. epithelial cells of poultry intestine [104]. including C. lari, C. jejuni, C. coli, C. fetus However, other studies proposed that FlpA is subsp. fetus, C. fetus subsp. venerealis, C. capable of binding to fibronectin [105] and it hyointestinalis and C. upsaliensis can produce is proposed that CadF plus FlpA work together toxins [111]. during the adhesion and the subsequent 420 Zag Vet J, Volume 48, Number 4, p. 414-432, December 2020 El-Naenaeey et al., (2020) The CDT is comprised of 3 subunits: CdtA, trimethoprim, oxacillin, nafcillin and CdtB and CdtC. The CdtA coupled with CdtC vancomycin. However, other resistance types are accountable for the binding and the occur during the use of therapeutic internalization into host cells, while the CdtB antimicrobials in humans and animals [118]. subunit is the toxic subunit, which is driven Antimicrobial resistance mechanisms in into the cell nucleus and induces cleavage in campylobacters are well documented for many the DNA, arrest in cell cycle followed by cell antimicrobial classes as following: death [3, 92, 112]. In fact, all the three Aminoglycosides products of the cdt gene should be existed Aminoglycosides are bacterial protein together to be functionally active [110, 113]. synthesis inhibitors which execute their The CDT activity differs somewhat relying antimicrobial activities through binding to the on the affected eukaryotic cell exhibiting 3 30S ribosome. The principle mechanism of types of avenues: (1) in keratinocytes, campylobacter resistance to aminoglycoside is epithelial and endothelial cells subjected to G2 through the plasmid borne aminoglycoside- cell cycle arrest and death, (2) the fibroblasts modifying enzymes (AphD, AphA, Sat and subjected to G1 and G2 arrest, cell distension AadE). Meanwhile, the efflux contribution to and death and (3) the immune cells subjected their resistance is unclear up till now [96, 116]. to G2 arrest with subsequent apoptosis. Tetracyclines Because of these actions, CDT participates in ]Tetracyclines are antimicrobial agents that campylobacter pathogenesis through inhibiting are utilized frequently in veterinary and human both humoral and cellular immunity through medicine [119]. These agents inhibit the apoptosis of the cell’s immune response [114]. elongation process of protein synthesis Any changes occur at the level of actin and through binding to the bacterial 16S rRNA microtubules filaments could block the toxin; within the ribosome minor subunit [120]. therefore, it cannot reach the nucleus and Campylobacter resist tetracyclines by thereby it prevents the DNA damage and leads expressing one of these mechanisms: alteration to alterations in cell cycle [115]. in tetracycline ribosomal target, 16Sr RNA Antimicrobial resistance mechanisms of and efflux pumps [121]. The most common Campylobacter species distributing mechanism among Campylobacter Campylobacter is a commensal bacterium spp. is through protecting the binding site of many farm animal species so, it exposed to between the ribosome and the antimicrobial high number of veterinary antibiotics. agents. This is brokered by the TetO protein, Moreover, the hypervariable genomic which is encoded by tet (O) gene. The TetO sequences and the natural competence of protein binds to the ribosomal A site leading to campylobacter are conferring for its separation of the attached tetracycline [117]. substantial genomic plasticity, which The tet (O) gene in most strains is encoded by responsible for these resistances [116]. plasmid. In contrast, few numbers of isolates Several mechanisms have been implicated have a chromosomal copy of this gene [122]. in the development of antimicrobial resistance β-Lactams such as (i) production of inactivating enzymes The β-lactam ring in β-lactam antibiotics is to antibiotics, (ii) protection or alternations of needed for the antimicrobial activity. β-lactam the antibiotic targets, (iii) extrusion of drugs antibiotics inhibit the cell wall biosynthesis by away from the bacterial cells via the active binding to the bacterial peptidoglycan efflux transporters and (iv) decreasing the transpeptidases resulting in lacking the permeability to antibiotics. Some of structural integrity of their walls [116]. There campylobacter resistance traits are are 2 main mechanisms responsible for endogenous, while others acquired either by campylobacter β-lactam resistance: (1) the the genetic transfer or mutations [117]. enzymatic inactivation by β-lactamases Campylobacter spp. has an intrinsic enzymes and (2) efflux pumps [116]. In resistance to sulfamethoxazole, cloxacillin, campylobacters the expression of β-lactamases 421 Zag Vet J, Volume 48, Number 4, p. 414-432, December 2020 El-Naenaeey et al., (2020) accounts for the resistance to ticarcillin, is traditionally reported in multiple European ampicillin and amoxicillin, that is may be Union members at higher levels [125]. antagonized by clavulanic acid, sulbactam and Campylobacters acquire resistance to these tazobactam. These enzymes have no effect on agents by 4 mechanisms; (i) mutations in 23S the susceptibility to carbapenems and rRNA genes, (ii) ribosomal proteins mutations, cephalosporins [116]. Lately, OXA61 β - (iii) ribosomal methylation encoded by erm lactamase has been discovered in C. jejuni (B) gene and (iv) efflux through CmeABC [123]. This enzyme is responsible for the [126]. Lately, the previously mentioned third resistance to piperacillin, oxacillin, ampicillin, way has been detected in Spain in a C. coli penicillin, carbenicillin and amoxicillin- isolate from broilers. This was the first record clavulanate [116]. Moreover, the CmeABC in Europe recording the existence of erm (B) in efflux pump has been participating in Campylobacter species [125]. Macrolides campylobacter resistance to ampicillin. work via targeting the 50S subunit of bacterial Insertional mutagenesis of cmeB gene leads to ribosome in addition to inhibiting the bacterial increasing in the ampicillin susceptibility. RNA-dependent protein synthesis. Moreover, Alterations in the porins of the outer the 23S rRNA nucleotides 2059 and 2058 membrane have a potential mechanism of serve as key binding sites for macrolide. This resistance to β-lactam antibiotics, even so the results in ribosomal conformational changes underlying mechanism is still not described followed by ending the peptide chain clearly [124]. These antibiotics are applied elongation process. In campylobacter widely in veterinary medicine; however, their chromosome, 3 copies of 23S rRNA gene were emerging resistance has compromised their found and generally in strains resistant to use. erythromycin. All copies possess macrolide Quinolones resistance-associated mutations; however, In campylobacters, quinolones inhibit the presence of wild-type alleles doesn't seem like DNA gyrase enzyme that is needed for DNA to have an impact on the level of resistance supercoiling and replication [124]. [124]. Moreover, efflux pumps are considered Campylobacter species resist quinolones via a another resistance mechanism. In single mutation in the gyrA gene and by the campylobacter, at least 8 efflux systems were CmeABC efflux pump activity [116]. Many described, but CmeABC pump is the virtually different single modifications in gyrA were relevant efflux pump, which operates in a reported in Campylobacter species such as synergistic manner with mutations in 23S Thr86Ile, Thr86Lys and Thr86Ala. However, rRNA gene attributing for the high resistance the more frequent one is the Thr86Ile toward macrolides [127]. substitution. This mutation accounts for the Multidrug efflux pump system high campylobacter resistance level to this The efflux pumps in Campylobacter spp. group [124]. In all European State members, cooperate with the other mechanisms of human campylobacter isolates showed high resistance. They are related to the intrinsic ciprofloxacin resistance levels. Consequently, resistance mechanisms toward a broad range fluoroquinolones have ceased to be the proper of antibiotics. Variety types of these pumps drug for routine treating of campylobacterosis were found in campylobacters e.g. CmeABC [125]. Many reports have demonstrated a clear (the best stated efflux pump), CmeDEF and correlation between using fluoroquinolone in CmeG [128]. CmeABC is a resistance- avian and the increasing resistance in avian nodulation cell division (RND) type of and human campylobacter isolates [117]. multidrug efflux pump. It is composed of 3 Macrolides proteins; CmeA (periplasmic fusion protein), Historically, the macrolides resistance CmeB (inner membrane drug transporter) and incidence was low, especially in C. jejuni. CmeC ( outer membrane protein) [100]. However, in last years, macrolides resistance CmeABC expression is modulated by CmeR, in campylobacters has increased. Moreover, it which inhibit cj0561c gene. The CmeABC has 422 Zag Vet J, Volume 48, Number 4, p. 414-432, December 2020 El-Naenaeey et al., (2020) a fundamental contribution in the patterns [137]. At present, whole-genome antimicrobial resistance of campylobacters sequencing (WGS) is considered as the most attributed to that the cmeB inactivation or informative and discriminative typing method using an inhibitor to efflux pump induce of bacterial pathogens, allowing for increasing in susceptibility to various comprehensive phylogenetic analyses of antibiotics even the antimicrobial agent to numerous traits associated with virulence or which the campylobacters are intrinsically antibiotic resistance [138]. resistant [129]. Conclusion Modern and new molecular techniques to Despite campylobacteriosis is a disease of detect the virulence and antibiotic zoonotic importance in Egypt, there is a gap of resistance genes knowledge about the disease’s epidemiology Variations of PCR have been developed in different localities, which hinders the and employed to detect virulence and accurate assessment of the human health resistance genes such as multiplex PCR [130] burden. There is an urgent need for and enter bacterial repetitive intergenic collaborative surveillance and intervention consensus PCR (ERIC-PCR) analyses [131]. national plans for controlling such infection. Identification of virulence factors successively From the standpoint of one health approach, a relied on biochemical approaches, or complete health surveillance program of systematic molecular screening of a panel of campylobacter infections must be done genes demonstrated to play a role in nationally to provide data about the disease pathogenesis using molecular cloning and/or occurrence and the common routes of mutagenesis [132]. In addition, several multi- transmission. Notifications of the disease in all criteria genome analysis tools enable detecting suffering regions should be happen rapidly. As virulence factors. The MvirDB combines well, it is very important to collect, analyze several databases including PRINTS and and interpret data to create relationships VFDB to identify virulence factors [133]; the between campylobacter isolates of human and Pathosystems resource integration center those of animal origins. Raising the public (PATRIC) combines the VFDB, Victors and health awareness, education and training of the PATRIC VF databases to detect virulence target populations (veterinarians, farm, abattoir factors and host–pathogen protein–protein and restaurant workers, household, nurses, interactions [134] and the PHI-base doctors in hospitals, etc…) are very crucial. (pathogen–host interaction) database can Biosecurity measures, vaccination or using identify virulence factors and host–pathogen natural competitive exclusion compounds are protein–protein interactions also enables very critical to reduce the risk of infection in detecting 2875 virulence genes and 4102 host– the farms and consequently reduce the level of pathogen interactions [135]. transmission to humans. Multilocus sequence typing (MLST), Conflict of interest based on sequence comparison of seven None of the authors have any conflict of housekeeping genes defined as sequence types interest to declare

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الملخص العربي تسميه٬ عزل٬ سمات الضراوة و المقاومة للمضادات الحيوية النواع الكامبيلوباكتر المنقولة عن طريق األغذية السيد يوسف النعناعي ٬ مروة ابراهيم عبد الحميد ٬ ايمان خالد خليفة قسم الميكروبيولوجيا – كلية الطب البيطري- جامعة الزقازيق- مصر علي نطاق واسع٬ تعتبر الكامبيلوباكتر السبب الرئيسي المراض االسهال المنقولةةة عةةي ريةةق االي يةةة اةةي جميةةع انحةةا العالم. اهي بكتريا سالبه الجرام تتحرك بواسطه سو قطبي٬ تنمو اى وجود نسبة قليلة مةةي االوكسةةجيي واةةي نطةةاق واسةةع مةةي درجه الحرارة مي 30 الي 45 درجة مئوية. اضة ع عةي فلةه اة ن هة البكتريةةا خاملةةة اةةي االختبةةارائ الكيمائيةةة وال تسةةت دم الكربوهيدرائ للحصول على الطاقة٬ حيث يرجع فله الي عدم وجةةود زنةةزيم 6 اوسةةفواركتوكيناز. هةة البكتريةةا لةةديها تفاعةة ايجابي اي اختبار االكسدة وتفاع سلبي الختبار االندول. زن عزل وتش يص أنواع الكامبيلوباكتر اةةي الم تبةةرائ أمةةر ةةعب ألنهةةا تنمةةو بصةةعوبة وتتطلةةب ئةةروا هوائيةةة خا ةةة لنموهةةا. وهنةةاك معلومةةائ قليلةةة نسةةبيا عةةي عوامةة الضةةراوة ال ا ةةة بالكامبيلوباكتر او كيةةف يمكةةي لبكتريةةا هشةةة ان تيةة علةةي قيةةد الحيةةا مةةع زيةةادة الحالةةه المر ةةية. باال ةةااة الةةي ان مقاومةةة الكامبيلوباكتر المتناميةةة للمضةةادائ الحيويةةة السةةريرية ا ةةبح مستعصةةية. لةة له هةة ا المقةةال يو ةة وينةةاقز تسةةمية وعةةزل وتصنيف وسمائ الضراو ومقاومة المضادائ الحيوية له البكتريا.

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