Thermophilic Campylobacter Isolates Research Articles

Genotypic characterization, antimicrobial susceptibility and virulence determinants of Campylobacter jejuni and Campylobacter coli isolated from pastured poultry farms.

Campylobacter is the leading bacterial pathogen that causes foodborne illnesses worldwide. Pasture farming is regarded as an important source of agricultural production for small farming communities. Consumer preference for pasture-raised animal products has increased; however, there is a paucity of information on the microbiological quality of pasture-raised poultry products. The purpose of this study was to explore genetic relatedness of thermophilic Campylobacter isolates, to assess antibiotic resistance phenotypically and genotypically, and to screen the presence of virulence determinants of Campylobacter isolates from pasture-raised poultry farms from southeastern United States. Ninety-seven Campylobacter isolates previously identified by Q7 BAX® System Real-Time PCR were genotyped by multilocus sequence typing (MLST). Campylobacter isolates were then evaluated for their phenotypic antimicrobial susceptibility against nine antimicrobial agents using Sensititre plates. Additionally, Campylobacter isolates were tested for the presence of antimicrobial resistance-associated elements. Furthermore, Campylobacter isolates were screened for the presence of 13 genes encoding putative virulence factors by PCR. These included genes involved in motility (flaA and flhA), adhesion and colonization (cadF, docC, racR, and virB11), toxin production (cdtA, cdtB, cdtC, wlaN, and ceuE) and invasion (ciaB and iamA). Among 97 Campylobacter isolates, Campylobacter jejuni (n = 79) and Campylobacter coli (n = 18) were identified. By MLST, C. jejuni isolates were assigned to seven clonal complexes. Among them, ST-353, ST-607 and ST-21 were the most common STs recognized. All C. coli (n = 18) isolates were included in CC-828. Interestingly, eight STs identified were not belonging any previous identified clonal complex. Campylobacter isolates displayed a high resistance rate against tetracycline (81.4%), while a low rate of resistance was observed against macrolides (azithromycin and erythromycin), quinolones and fluoroquinolones (nalidixic acid and ciprofloxacin), aminoglycosides (gentamicin), ketolide (telithromycin), amphenicol (florfenicol) and lincomycin (clindamycin). Thirteen isolates (13.54%) were pan-susceptible to all tested antibiotics, while nine isolates were multi-antimicrobial resistant (MAR; resist to three or more antimicrobial classes). Interestingly, there were no isolates resistant to all antimicrobial classes. Thr86Ile mutation was identified in all quinolones resistant strains. Erythromycin encoding gene (ermB) was identified in 75% of erythromycin resistant isolates. The A2075 mutation was detected in one erythromycin resistant strain, while A2074 could not be identified. The tetO gene was identified in 93.7% of tetracycline resistant isolates and six tetracycline susceptible isolates. In conclusion, the results of this study revealed that Campylobacter isolates from pasture-raised poultry farms showed the ST relatedness to Campylobacter isolates commonly associated with humans, indicating pasture-raised broiler flocks, similar to conventionally-reared broiler flocks, as a potential vector for antibiotic-resistant and pathogenic strains of thermophilic Campylobacter to humans.

Antimicrobial Resistance Determinants Circulating among Thermophilic Campylobacter Isolates Recovered from Broilers in Ireland Over a One-Year Period.

Campylobacteriosis is the leading cause of human bacterial gastroenteritis, very often associated with poultry consumption. Thermophilic Campylobacter (Campylobacter jejuni and Campylobacter coli) isolates (n = 158) recovered from broiler neck skin and caecal contents in Ireland over a one-year period, resistant to at least one of three clinically relevant antimicrobial classes, were screened for resistance determinants. All ciprofloxacin-resistant isolates (n = 99) harboured the C257T nucleotide mutation (conferring the Thr-86-Ile substitution) in conjunction with other synonymous and nonsynonymous mutations, which may have epidemiological value. The A2075G nucleotide mutation and amino acid substitutions in L4 and L22 were detected in all erythromycin-resistant isolates (n = 5). The tetO gene was detected in 100% (n = 119) of tetracycline-resistant isolates and three of which were found to harbour the mosaic tetracycline resistance gene tetO/32/O. Two streptomycin-resistant C. jejuni isolates (isolated from the same flock) harboured ant(6)-Ib, located in a multidrug resistance genomic island, containing aminoglycoside, streptothricin (satA) and tetracycline resistance genes (truncated tetO and mosaic tetO/32/O). The ant(6)-Ie gene was identified in two streptomycin-resistant C. coli isolates. This study highlights the widespread acquisition of antimicrobial resistance determinants among chicken-associated Campylobacter isolates, through horizontal gene transfer or clonal expansion of resistant lineages. The stability of such resistance determinants is compounded by the fluidity of mobile genetic element.

Recovery of thermophilic Campylobacter by three sampling methods from river sites in Northeast Georgia, USA, and their antimicrobial resistance genes.

Sixteen sites in the watershed of the South Fork of the Broad River (SFBR) in Northeastern Georgia, USA, were sampled in two seasons to detect Campylobacter. Sites were classified as mostly influenced by forest, pasture, wastewater pollution control plants (WPC) or mixed use. Sampling was repeated in the late spring and late fall for 2years for a total of 126 samples. Free-catch water and sediment grab samples were taken at each site; Moore's swabs were placed for up to 3days at most sites. A total of 56 isolates of thermophilic Campylobacter were recovered. Thirteen samplings were positive by two or three methods, and 26 samplings were positive by only one method; once by Moore's swab only and 25 times by free-catch water only. Campylobacter was detected at 58% of cattle pasture sites, 30% of forested sites and 81% of WPC sites. Twenty-one of the isolates carried antimicrobial resistance genes, mostly blaOXA-61. Free-catch water samples were more efficient than Moore's swabs or sediment samples for recovery of Campylobacter, which was more likely to be detected in streams near cattle pastures and human communities than in forested land. SIGNIFICANCE AND IMPACT OF THE STUDY: The role of environmental water in transmitting Campylobacter was investigated, and methods for recovery of the organism were compared. The sequence types of recovered Campylobacter correlated with adjacent land use without regard to the method used to isolate the organisms. Sequence types and antimicrobial resistance genes associated with cattle were most prevalent near pastures. Even though types were recurrent at a given site, types appeared to be lost or replaced as the water flowed downstream.

Prevalence of Thermophilic Campylobacter in Cattle Production at Slaughterhouse Level in France and Link Between C. jejuni Bovine Strains and Campylobacteriosis.

Campylobacter is the leading cause of bacterial gastroenteritis in industrialized countries, with poultry reservoir as the main source of infection. Nevertheless, a recent study on source attribution showed that cattle could be a source of human contamination in France (Thépault et al., 2017). However, few data are available on thermophilic Campylobacter epidemiology in cattle in France. The aim of this study is to collect new data of thermophilic Campylobacter prevalence in these animals and to subtype C. jejuni isolates to assess the potential implication of cattle in campylobacteriosis. A 6-month survey was carried out in one of the largest European slaughterhouse of cattle. Based on a statistical representative sampling plan, 959 intestinal content samples (483 adult cattle and 476 calves) were collected. An adapted version of the ISO 10272 standard and Maldi-Tof were used for detection and speciation of thermophilic Campylobacter isolates. Within more than 2000 thermophilic Campylobacter isolates collected, a selection of 649 C. jejuni isolates was typed with Comparative Genomic Fingerprinting (CGF40) and a subset of 77 isolates was typed using Multilocus Sequence Typing (MLST). Simultaneously, clinical isolates occurred in France were genotyped. Prevalence of thermophilic Campylobacter in the global cattle population was 69.1% (CI95% = 66.1, 72.1) at slaughterhouse level. In adult cattle, the prevalence was 39.3%, while 99.4% of calves were contaminated, and C. jejuni was the most prevalent species with prevalence of 37.3 and 98.5%, respectively and a higher genetic diversity in adult cattle. The prevalence of C. coli was lower with 3% in adult cattle and 12.5% in calves. MLST and CGF40 genotyping did not showed a high number of clusters within cattle isolates but the predominance of few clusters accounted for a large part of the population (CC-21, CC-61, CC-48, and CC-257). By comparison with clinical genotypes, genetic diversity was significantly lower in cattle. Moreover, significant overlap was observed between genotypes from both origins, with 3 of the 4 main cattle clusters present in human isolates. This study provides new insights on the epidemiology of thermophilic Campylobacter and C. jejuni in cattle production in France and their potential implication in human infection.

Isolation and Molecular Characterization of Thermophilic Campylobacter spp. in Dogs and Cats

Isolation and Molecular Characterization of Thermophilic Campylobacter spp. in Dogs and Cats

Ocurrence of Campylobacter spp. in chilled chicken carcasses slaughtered in the west region of Santa Catarina, Brazil

Background: The thermophilic bacteria of the genus Campylobacter are important agents of alimentary gastroenteritis, called campylobacteriosis. These microorganisms multiply in temperatures ranging from 25ºC to 46ºC, however, low temperatures are incompatible with their multiplication. For this reason, the seasons of the year may interfere with the level of contamination by Campylobacter sp. The main sources of transmission are contaminated meat and giblets from poultry during poorly conducted slaughter operations. The disease may present itself with a different range of forms of disease: from mild signs of gastrointestinal infection to more severe cases, such as the Guillain-Barré syndrome.Material, Methods & Results: Due to the great importance of western Santa Catarina to the poultry industry, it was necessary to verify the occurrence of the pathogen in cold carcasses of broilers slaughtered in this region, and its variation through the seasons of the year. From January 2013 to February 2015 broiler carcasses were collected weekly, after the water cooling process, in slaughterhouses under Federal Inspection of the three largest microregions of western Santa Catarina in terms of number of broilers slaughtered, totaling 808 samples. The assessment of thermophilic Campylobacter was performed according to the methodology recommended by ISO 10272-1: 2006. Of the 808 samples analyzed, the frequency of isolation of thermophilic Campylobacter was 1.82% (8/440) in microregion 1, 4.95% (10/202) in microregion 2 and 13.86% (23/166) in the microregion 3, totaling 5.07% of positive samples (41/808). Comparing the microregions, it was verified that there was no significant difference (P > 0.05) between the isolation frequencies of microregions 1 and 2. However, there was a significant difference (P < 0.05) between the isolation rates of Microregion 3 in relation to microregions 1 and 2. Microregion 1 presented the lowest percentage of Campylobacter thermophilic isolates.Discussion: Although the rates found are lower than expected when compared to those already published, consumption of these products may provide risks to consumers, mainly through cross-contamination of foods that will be consumed raw, requiring greater controls in the entire poultry industry in order to ensure a safe product for the consumer. Analyzing each season of the year individually (winter and summer), 5.88% (14/238) of the samples processed during summer (October to March) were positive for the assessed microorganism and in 4,74% (27/570) of the samples analyzed in the winter (April to September), the presence of thermophilic Campylobacter was detected. Nevertheless, there was no significant difference (P > 0.05) between the seasons. However, it is evident the need for a longer study to evaluate the actual influence of the seasons (winter and summer) on the isolation rate of thermophilic Campylobacter in broiler carcasses shortly after the cooling process. Even though the study demonstrated low levels of thermophilic Campylobacter isolation rates in the evaluated regions, just the fact of isolating the agent already shows that there is an imminent risk to consumers, mainly due to cross-contamination of foods that will be consumed raw; therefore, higher standards of microbiological controls should be practiced in the production chain and slaughter of poultry, respecting good manufacturing practices, hygiene and handling practices within the industry.

Heterogeneity of cytolethal distending toxin sequence types of Campylobacter jejuni and correlation to invasion/cytotoxicity potential: The first molecular survey from Iran

The aim of this study was to evaluate the cytotoxicity and attachment/invasion potential of thermophilic Campylobacter isolates regarding their cdtABC sequence types and virulence-associated gene content.A total of 33 Campylobacter spp. were identified from 750 stool samples isolated from patients characterized with diarrhea. The prevalence rates of flaA, ciaB, and pldA genes among the isolates were 97, 100, and 15%, respectively. The iam gene was found in 100% of the C. coli isolates while it was not detected in C. jejuni isolates. Four PCR primer pairs jointly amplifying the entire cdtABC genes array and sequence analysis revealed variations dispersed along the sequence array. The isolates attachment to HeLa cells ranged from 89 ± 2–100%, and the range of invasions was also from 0 to 11 ± 0.04%. The cytotoxicity value was between 2 and 32 in cdt+ isolates with no significant correlation to any of the cdtABC sequence types. Moreover, the cdtABC encoding strains had increased invasion to HeLa cells, and all of the related patients presented much higher white and red blood cell shedding in stool specimens (P-value≤ 0.001). No significant difference was observed between cdt+ and cdt− isolates in their attachment rate to HeLa cells.About 48% of all the Iranian Campylobacter population lacked a complete set of cdtABC genes array, suggesting low invasion and cytotoxicity potential of the isolates which are heterogeneous in their cdt genes and virulence.

Isolation and Identification of Campylobacter spp. from Poultry and Poultry By-Products in Tunisia by Conventional Culture Method and Multiplex Real-Time PCR

Thermophilic Campylobacter spp. are one of the primary causes of bacterial human diarrhea. The consumption of poultry meats, by-products, or both is suspected to be a major cause of human campylobacteriosis. The aims of this study were to determine the prevalence of thermophilic Campylobacter spp. in fresh poultry meat and poultry by-products by conventional culture methods and to confirm Campylobacter jejuni and Campylobacter coli isolates by using the multiplex PCR assay. Two hundred fifty fresh poultry samples were collected from a variety of supermarkets and slaughterhouses located in Sfax, Tunisia, including chicken (n =149) and turkey (n =101). The samples were analyzed using conventional microbiological examinations according to the 2006 International Organization for Standardization method (ISO 10272-1) for Campylobacter spp. Concurrently, a real-time PCR was used for identification of C. jejuni and C. coli . Of the 250 samples of poultry meat and poultry by-products, 25.6% (n = 64) were contaminated with Campylobacter spp. The highest prevalence of Campylobacter spp. was found in chicken meat (26.8%) followed by turkey meat (23.7%). Among the different products, poultry breasts showed the highest contamination (36.6%) followed by poultry by-products (30%), poultry wings (28%) and poultry legs (26%) showed the lowest contamination, and no contamination was found on neck skin. Of the 64 thermophilic Campylobacter isolates, C. jejuni (59.7%) was the most frequently isolated species and 10.9% of the isolates were identified as C. coli . All of the 64 Campylobacter isolates identified by the conventional culture methods were further confirmed by PCR. The seasonal peak of Campylobacter spp. contamination was in the warm seasons (spring and summer). The study concluded that high proportions of poultry meat and poultry by-products marketed in Tunisia are contaminated by Campylobacter spp. Furthermore, to ensure food safety, poultry meats must be properly cooked before consuming.

Epidemiology and RAPD-PCR typing of thermophilic campylobacters from children under five years and chickens in Morogoro Municipality, Tanzania.

BackgroundCampylobacter species are gram negative and flagellated bacteria under the genus Campylobacter, family Campylobacteriaceae. These pathogens cause zoonotic infections among human and animal populations. This study was undertaken between December 2006 and May 2007 to determine prevalence, risk factors and genetic diversity of thermophilic Campylobacter isolates from children less than 5 years and chickens in Morogoro Municipality, Tanzania.MethodsThe Skirrow’s protocol was used for isolation and identification of Campylobacter from 268 human stool specimens and 419 chicken cloacal swabs. Patient biodata and risk factors associated with human infection were also collected. Genetic diversity of Campylobacter isolates was determined by a RAPD-PCR technique using OPA 11 primer (5′-CAA TCG CCG T-3′). Phylogenetic analysis and band pattern comparison were done by Bionumerics software and visual inspection.ResultsStool samples from 268 children and 419 cloacal swabs from chickens were analyzed. Prevalence of thermophilic Campylobacters in children was 19% with higher isolation frequency (p = 0.046) in males (23.5%) than females (13.8%). Campylobacter jejuni (78.4%) was more isolated (p = 0.000) than C. coli (19.6%) and 2% were unidentified isolates. In chickens, the prevalence was 42.5% with higher isolation rate (p = 0.000) of C. jejuni (87%) than C. coli (13%). Campylobacters were more frequently recovered (p = 0.000) from indigenous/ local chickens (75.0%) followed by cockerels (52.2%), broilers (50.0%) and lowest in layers (22.7%). Keeping chickens without other domestic animals concurrently (p = 0.000), chicken types (p = 0.000) and flock size (p = 0.007) were risk factors for infection in chickens. One hundred and fifty two (152) thermophillic Campylobacter isolates were genotyped by RAPD-PCR of which 114 were C. jejuni (74 from chickens and 40 humans) and 38 C. coli (28 from chickens and 10 humans). Comparison of Campylobacter isolates from children and chickens revealed high diversity with only 6.1% of C. jejuni and 5.3% of C. coli being 100% genetically similar.ConclusionsThis study has recorded high prevalence of thermophilic Campylobacter in children less than 5 years and chickens in Morogoro municipality. The observed genetic similarity among few C. jejuni and C. coli isolates from children and chicken suggests existence of cross transmission of these pathogens between children under 5 years and chickens.

Antimicrobial Resistance of Salmonella Serovars and Campylobacter spp. Isolated from an Opportunistic Gull Species, Yellow-legged Gull ( Larus michahellis ).

Wildlife is a natural reservoir of Salmonella and Campylobacter, the most important human foodborne pathogens worldwide. Free-living birds have the potential to transport, over large distances, such zoonotic bacteria that may harbor antimicrobial resistance traits. On the northeastern Iberian coast, we assessed the role of Yellow-legged Gulls ( Larus michahellis ) as reservoirs of antimicrobial resistance in Salmonella and thermophilic Campylobacter isolates recovered from gulls at three colonies, with varying degrees of dependence on refuse dumps as food sources. Of the 39 Salmonella isolates we tested, 17 were multiresistant (resistance to three antimicrobial families), with eight being Salmonella enterica serovar Typhimurium. Other clinically relevant Salmonella serovars showing multiresistance included Hadar, Bredeney, and Virchow. Relevant Campylobacter antimicrobial resistances were detected among three Campylobacter jejuni isolates, of which all three showed resistance to nalidixic acid, two were resistant to ciprofloxacin, one was resistant to enrofloxacin, and one was resistant to tetracycline. Our results highlight the importance of free-living gulls with opportunistic feeding habits in the dissemination of enteric pathogens resistant to multiple antimicrobial agents of public health concern.

Detection of virulence, antibiotic resistance and toxin (VAT) genes in Campylobacter species using newly developed multiplex PCR assays

Campylobacter species are one of the leading causes of bacterial gastroenteritis in humans worldwide. This twofold study was sought to: i) develop and optimize four single-tube multiplex PCR (mPCR) assays for the detection of six virulence (ciaB, dnaJ, flaA, flaB, pldA and racR), three toxin (cdtA, cdtB and cdtC) and one antibiotic resistance tet(O) genes in thermophilic Campylobacter spp. and ii) apply and evaluate the developed mPCR assays by testing 470 previously identified C. jejuni, C. coli and C. lari isolates from agricultural water. In each mPCR assay, a combination of two or three sets of primer pairs for virulence, antibiotic resistance and toxin (VAT) genes was used and optimized. Assay 1 was developed for the detection of dnaJ, racR and cdtC genes with expected amplification sizes of 720, 584 and 182bp. Assay 2 generated PCR amplicons for tet(O) and cdtA genes of 559 and 370bp. Assay 3 amplified cdtB ciaB, and pldA genes with PCR amplicon sizes of 620, 527 and 385bp. Assay 4 was optimized for flaA and flaB genes that generated PCR amplicons of 855 and 260bp. The primer pairs and optimized PCR protocols did not show interference and/or cross-amplification with each other and generated the expected size of amplification products for each target VAT gene for the C. jejuni ATCC 33291 reference strain. Overall, all ten target VAT genes were detected at a variable frequency in tested isolates of thermophilic Campylobacter spp. where cdtC, flaB, ciaB, cdtB, cdtA and pldA were commonly detected compared to the flaA, racR, dnaJ and tet(O) genes which were detected with less frequency. The developed mPCR assays are simple, rapid, reliable and sensitive tools for simultaneously assessing potential pathogenicity and antibiotic resistance profiling in thermophilic Campylobacter spp. The mPCR assays will be useful in diagnostic and analytical settings for routine screening of VAT characteristics of Campylobacter spp. as well as being applicable in epidemiological studies by providing information that could be related to the risk of human infection.

Efficacy of Phenotypic, PCR and MALDI-ToF Identification Methods for Campylobacter spp.

This study compared phenotypic and genotypic identification methods of Campylobacter spp. against the polymerase chain reaction (PCR) in terms of sensitivity, specificity, positive-predictive value and negative-predictive value. Thermophilic Campylobacter isolates were identified using conventional biochemical tests, specifically hippurate hydrolysis, matrix assisted laser desorption ionization- time of flight (MALDI-ToF) mass spectrometry and PCR with primers unique to C. jejuni and C. coli. MALDI-ToF was shown to be superior to biochemical tests for identification of C. coli but equivalent to biochemical tests for C. jejuni .

Molecular identification of Campylobacter jejuni and coli from chicken, calves and dogs to determine its potential threat on human being.

Aim:Campylobacter is an emerging zoonotic pathogen and one of the leading cause of foodborne infection worldwide and it has been isolated from a variety of animal species. The aim of this study was to identify Campylobacter jejuni and Campylobacter coli from dogs, calves, and poultry using polymerase chain reaction (PCR).Methodology:A total of 104 number of samples comprising cloacal swab from poultry (38), a rectal swab from dogs (40), and calves (26) were collected for the isolation of thermophilic Campylobacters using conventional culture method. PCR was used for identification of mapA gene for C.jejuni and ceuE for C.coli.Results:The overall presence of Campylobacter was found to be 67(64.42%) from the samples, out of which 6 isolates belongs to C. jejuni species, were 5(18.51%) from chicken and 1(4.17%) from dog was recorded and about 17 isolates belongs to C. coli species were 9(33.33%), 6 (25%), and 1(9.09%) from chicken, dog and calves was recorded.Conclusion:Results suggested that Campylobacter reservoirs chicken, calves and pet dogs can play a role as the source of infection to human beings and PCR can be an ideal tool for molecular confirmation at the species level.

Genetic Characterization of Campylobacter Jejuni and C. coli Isolated From Broilers Using flaA PCR-Restriction Fragment Length Polymorphism Method in Shiraz, Southern Iran.

Background:Thermophilic campylobacters, particularly Campylobacter jejuni and C. coli are the main agents of human campylobacteriosis. Campylobacter contaminated chicken products is the most important source of foodborne gastroenteritis. Evaluation of genetic diversity among Campylobacter population is critical for understanding the epidemiology of this bacterium and developing effective control strategies against Campylobacter infections and other related disorders.Objectives:The aim of this study was to investigate the polymorphism of thermophilic Campylobacter isolated from broiler fecal samples in Shiraz, southern Iran.Materials and Methods:Ninety Campylobacter isolates were recovered from broiler feces using enrichment process followed by cultivation method. The isolates were species typing on the basis of polymerase chain reaction (PCR) detection of 16SrRNA and multiplex PCR for determining two thermophilic species. To evaluate strain diversity of thermophilic Campylobacter isolates, flaA PCR-Restriction Fragment Length Polymorphism (RFLP) was performed using DdeI restriction enzyme.Results:All 90 Campylobacter isolates confirmed by m-PCR were successfully typed using flaA-PCR-RFLP. Eleven different types were defined according to flaA-typing method and the RFLP patterns were located at three separate clusters in RFLP image analysis dendrogram.Conclusions:Campylobacter jejuni isolates significantly showed more variety than C. coli isolates. A relatively low genetic diversity existed among C. jejuni and C. coli isolated from broilers in Shiraz, southern Iran. In our knowledge, this was the first report of genetic diversity among broiler originated human pathogen thermophilic campylobacters in Shiraz, southern Iran.

Assessment of two different types of sample for the early detection and isolation of thermophilic Campylobacter in broiler farms

In order to assess the optimal method for the early detection and isolation of thermophilic Campylobacter in broilers at farm level, two types of samples were compared: caecal contents obtained by necropsy and cloacal swabs transported in charcoal Amies medium. The study was conducted in five batches of broilers from five different farms, where weekly samples (caecal contents and cloacal swabs) from 30 birds were obtained. Samples were plated onto selective agar (modified charcoal cefoperazone desoxycholate agar, mCCDA) for Campylobacter isolation. Four out of five batches were positive for Campylobacter. No marked differences in sensitivity of both sample types were observed. However, a higher percentage of positive birds were detected when cloacal swabs were used. The results show that cloacal swab samples are adequate, and in some cases even better than caecal samples for the early detection of Campylobacter in broiler flocks at farm level. Also, this sample avoids sacrificing birds to test Campylobacter, which not only allows saving time in sample collection, transportation and processing at the laboratory, but also improves bird welfare and cost of sampling.

Occurrence, species distribution and antimicrobial resistance of thermophilic Campylobacter isolates from farm and laboratory animals in Morogoro, Tanzania

Occurrence, species distribution and antimicrobial resistance of thermophilic Campylobacter isolates from farm and laboratory animals in Morogoro, Tanzania

Occurrence of virulence genes and strain diversity of thermophilic campylobacters isolated from cattle and sheep faecal samples

The objective of this study was to investigate the genotypic characteristics of Campylobacter isolates in Shiraz, Iran. A total of 40 Campylobacter isolates including 20 C. jejuni and 20 C. coli were recovered from both cattle and sheep faeces by cultivation methods. The isolates were identified on the basis of polymerase chain reaction (PCR) detection of 16SrRNA and multiplex PCR to determine two species. For confirmed isolates, PCR was carried out for the presence of virulence genes using specific primers. Other than verifying the genetic diversity of thermophilic Campylobacter isolates, flaA PCR-RFLP was performed. Results showed the high prevalence (100%) of the cadF gene and three genes associated with cytolethal distending toxin (CDT). Plasmid virB11 gene was not found in any Campylobacter isolate, and dissimilarities and discrepancies occurred in pldA, iamA, wlaN, waaC and cgtB genes. Among the 40 Campylobacter isolates studied, nine different types were defined by flaA-typing. Results indicated genetic diversity among Campylobacter isolates recovered from cattle and sheep faecal samples. Findings showed the potential ability of C. jejuni and C. coli with cattle and sheep origins to cause infection in humans.

Sağlıklı ve İshalli Pet Hayvanlarda Termofilik Campylobacter spp.’nin Üç Kültür Metodu ile İzolasyonu ve İzolatların Multipleks Polimeraz Zincir Reaksiyonu m(PZR) İle İdentifikasyonu

In this study, the determination of prevalence of thermophilic Campylobacter species in dogs and cats with and without diarrhoea using 3 different cultural methods was aimed. For this purpose, rectal swabs were collected from 120 dogs and 15 cats and 14 of them (12 dogs and 2 cats) were taken from diarrhoeic animals. The isolation of thermophilic Campylobacter spp. was conducted by direct plating onto modified charcoal cefoperazone deoxycholate agar (mCCDA) supplemented with CCDA (cefoperazone, amphotericin B) or CAT (cefoperazone, amphotericin and teicoplanin) for all samples and membrane filtration method onto Mueller-Hinton Agar supplemented with 5% defibrinated sheep blood for samples from diarrhoeic pets and identification of isolates was performed using multiplex polymerase chain reaction (mPCR). The overall prevalence of Campylobacter species was found to be 40.0% and 26.7% in dogs and cats, respectively. Campylobacter jejuni was the most frequent bacterium isolated from 36 dogs and 4 cats. C. upsaliensis, C. coli and C. lari were isolated from 10 dogs and 1 cat, 5 dogs and 2 healthy dogs respectively. For the isolation of thermophilic Campylobacter spp., whilst the method using CAT as selective supplement being more sensitive in dogs, the membrane filtration appeared as the most suitable method in diarrhoeic dogs. These results showed the occurrence of a relatively high carriage of Campylobacter spp., particularly in healthy dogs that may constitute a non negligible risk for public health.

Development of Nalidixic Acid Amphotericin B Vancomycin (NAV) Medium for the Isolation of Campylobacter Ureolyticus from the Stools of Patients Presenting with Acute Gastroenteritis

Recently, Campylobacter ureolyticus has been detected for the first time in the faeces of patients with acute gastroenteritis using polymerase chain reaction (PCR) techniques. Cultural isolation of C. ureolyticusis is not possible using the established selective methods for the isolation of thermophilic Campylobacter spp. from faeces. The aim of the current study is to develop a new selective medium capable of isolating C. ureolyticus from faecal samples. The newly-developed medium consists of Anaerobe Basal Agar with 10 g/L additional agar, 2 g/L sodium formate and 3 g/L sodium fumarate dibasic, to which 10 mg/L nalidixic acid, 10 mg/L amphotericin B and 20 mg/L vancomycin (NAV) are added as selective agents. Validation studies have shown that this experimental selective medium completely inhibits growth of Candida spp. and of Enterococcus spp. and permits reduced growth of selected coliforms and Proteus spp. Growth of Campylobacter ureolyticus on NAV medium is optimal in anaerobic and enriched hydrogen atmospheres. Additionally, an overnight enrichment step using Bolton broth to which 2 g/L sodium formate, 3 g/L sodium fumarate dibasic and the NAV supplement are added, in place of the commercial Bolton broth supplement, allows improved recovery of C. ureolyticus from patients’ faeces.

Identification of Campylobacter spp. Isolates with Phenotypic Methods and Multiplex Polymerase Chain Reaction and Their Antibiotic Susceptibilities

The aims of this study were to detect the frequency of isolation of thermophilic Campylobacter spp. from acute gastroenteritis cases by phenotypic and molecular methods and to evaluate the antibiotic susceptibilities of the isolates. A total of 3287 stool samples obtained from diarrheal patients who were admitted to Kayseri Training and Research Hospital, Kayseri, Turkey, between March 2010 - March 2011 and sent to the microbiology laboratory, were included in the study. Cefoperazone, amphotericin B and teicoplanin (CAT) supplemented Campylobacter Blood-free Selective Medium (modified CCDA-Preston, Oxoid CM739, UK) was used for the isolation of Campylobacter spp. The media inoculated with stool samples were incubated at 42°C microaerobically for 72 to 96 hours. The genus and species level identifications of the thermophilic Campylobacter spp. isolates defined by phenotypic tests were carried out with multiplex polymerase chain reaction (mPCR). Antibiotic susceptibilities of the isolates were detected by disc diffusion method and the results were evaluated according to the CLSI guidelines. The thermophilic Campylobacter spp. were isolated from 5.4% (179/3287) of the patients' stool samples. Of Campylobacter positive cases 71% (127/179) were children and 58% (104/179) were male. The prevalence rate was estimated as 7.5% (127/1683) for children and 3.2% (52/1604) for adults. Of the isolates, 146 (82%) were identified as C.jejuni, 24 (13%) were C.coli, 6 (3%) were C.lari and 3 (2%) were C.upsaliensis with phenotypic tests. By using mPCR, 152 (85%) and 27 (15%) of 179 isolates were identified as C.jejuni and C.coli, respectively. Three of the six isolates identified as C.lari by the phenotypic methods were identified as C.jejuni and the remaining three as C.coli by mPCR. Phenotypically identified three C.upsaliensis isolates were shown to be C.jejuni (n= 2) and C.coli (n= 1). On the other hand one C.coli isolate was found to be C.jejuni by mPCR. The rates of resistance of the isolates were 92.6% for trimethoprim-sulfamethoxazole, 79.5% for nalidixic acid, 75.6% for levofloxacin, 73.9% for ciprofloxacin, 40.3% for ampicillin, 35% for cefotaxime, 33.4% for piperacillin-tazobactam, 24% for tetracycline, 14.6% for clindamycin, 11.2% for amikacin and 6.3% for erythromycin. During the 13 months study period, the highest isolation rates were detected between March-June (mean rate 66%). Our data concerning the prevalence and antibiotic resistance rates revealed the significance of campylobacters in gastroenteritis cases. Therefore, specific microbiological isolation and identification methods should be applied in routine microbiology laboratories to investigate the presence of campylobacters in gastroenteritis etiology. Besides, determination of the antibiotic susceptibilities of the isolates on routine basis should be encouraged to help to guide the antimicrobial treatment approaches in case of gastroenteritis. The results of this study also indicated that phenotypic tests were adequate for the identification of campylobacters at the genus-level, however, for accurate identification at the species level and for reliable epidemiological data molecular analysis might be added to the detailed identification procedures.