Identification Of Campylobacter Research Articles

Prevalence of Thermophilic Campylobacter Isolated from Water Used in Slaughter House of Kathmandu and Ruphendehi District, Nepal

Campylobacter is a fastidious organism that is capable of surviving in wide range of environments and potentially can cause serious infection in human and animal which can commonly available in the different sources of water used for daily purpose. This research was conducted to figure out the prevalence of the thermophilic Campylobacter in the water used in various slaughter houses of Kathmandu and Ruphendehi district of Nepal. So a cross sectional study was conducted and 200 water samples (100 for each district) was collected aseptically and submitted to Bacteriological Unit for the confirmation. Isolation and identification of Campylobacter was being done as described by OIE Terrestrial Manual, 2004, Chapter 2.8.10. Laboratory finding was done to confirm the positive cases. The study revealed the prevalence status of Campylobacter in water used in the slaughter house of Ruphendehi district is 12% and Kathmandu Valley is 0.00%. Thus, Ruphendehi has comparatively more prevalence of Campylobacter than Kathmandu. The water samples tested were all from actual or potential water sources for the use in slaughter house, so there are clear implications for the transmission of Campylobacter spp. to human and animal suggesting further expanded research is required in this area.
 Int. J. Appl. Sci. Biotechnol. Vol 7(1): 75-80

Hyperspectral microscope imaging methods for multiplex detection of Campylobacter

Campylobacter is an emerging zoonotic bacterial threat in the poultry industry. The current methods for the isolation and detection of Campylobacter are culture-based techniques with several selective agars designed to isolate Campylobacter colonies, which is time-consuming, labour intensive and has low sensitivity. Several immunological and molecular techniques such as enzyme-linked immunosorbent assay (ELISA) and Latex agglutination are commercially available for the detection and identification of Campylobacter. However, these methods demand more advanced instruments as well as specially trained experts. A hyperspectral microscope imaging (HMI) technique with the fluorescence in situ hybridisation (FISH) technique has the potential for multiplex foodborne pathogen detection. Using Alexa488 and Cy3 fluorophores, the HMI (450–800 nm) technique was able to identify Campylobacter jejuni stains with high sensitivity and specificity. In addition, HMI was able to classify six bacteria using scattering intensity from their spectra without a FISH fluorophore. Overall classification accuracy of quadratic discriminant analysis (QDA) method for six bacteria including Bifidobacter longum, Campylobacter jejuni, Clostridium perfringens, Enterobacter cloacae, Lactobacillus salivarius and Shigella flexneri using the HMI technique without fluorescent markers was approximately 88.6 % with pixel-wise classification.

Genetic diversity of Campylobacter jejuni and Campylobacter coli isolated from poultry meat products sold on the retail market in Southern Brazil.

Campylobacter is regarded as the most common bacterial cause of gastroenteritis throughout the world and most cases of human campylobacteriosis can be traced back to the consumption of poultry meat. In Brazil, few studies evaluated the genetic relatedness among Campylobacter isolates. The aim of this research was to evaluate the genetic diversity of Campylobacter spp. isolated from poultry meat products sold on the retail market in Southern Brazil. The presumptive identification of Campylobacter was performed using traditional microbiological analysis, followed by molecular confirmation by PCR. The genetic diversity of isolates was analyzed by pulsed-field gel electrophoresis (PFGE). Campylobacter spp. was isolated from 91.7% (33/36) of the samples, totaling 48 isolates. Campylobacter jejuni was the most prevalent species isolated (90.8%). PFGE data revealed 26 pulsotypes and 18 PFGE patterns composed of only 1 isolate. Campylobacter isolates exhibited high genetic diversity; however, some clones were recurrent in the poultry meat products sold on the retail market. As the south region of Brazil is an important producer and exporter of chicken meat, our results highlight the need to control this pathogen in the food chain in this area of the world to reduce the risks of exposing consumers to campylobacteriosis.

Reliability Evaluation of MALDI-TOF MS Associated with SARAMIS Software in Rapid Identification of Thermophilic Campylobacter Isolated from Food

Thermophilic Campylobacter species represent the most frequent cause of food-borne disease worldwide. Biochemical and serological tests, according to ISO 10272:2017, are routinely used in microbiology laboratories for their detection/identification. However, these procedures require a lot of time and can incur in diagnostic mistakes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is considered a promising identification method for bacteria and fungi from clinical isolates. The aim of this study was to evaluate the reliability of MALDI-TOF MS associated with SARAMIS software, in rapid identification of food-related Campylobacter spp., considering that this method was tested only on human and animal clinical strains. A total of 276 putative thermophilic Campylobacter strains, isolated from different foodstuffs according to ISO 10272-1:2006, were identified comparing the results of MALDI-TOF MS/SARAMIS with Multiplex PCR assay. MALDI-TOF MS/SARAMIS correctly identified 96% (n = 267) of strains at the species level, while only 2.5% (n = 7) of strains were not identified. Misidentification occurred in 0.7% (n = 2) of samples. The performance of this method demonstrated an overall sensitivity (Se) of 97.45% and specificity (Sp) of 100%. Thus, this method can be used for the rapid identification of Campylobacter strains isolated from food samples according to ISO 10272:2017.

Isolation and Molecular Identification of Campylobacter spp. from Vaginal Swab Sample Obtained from Sheep Herds with Abort History

Isolation and Molecular Identification of Campylobacter spp. from Vaginal Swab Sample Obtained from Sheep Herds with Abort History

Comparison of the Filtration Culture and Multiple Real-Time PCR Examination for Campylobacter spp. From Stool Specimens in Diarrheal Patients.

Campylobacter is one of the most common pathogens leading to the bacterial diarrheal illness. In order to set up one effective culture independent assay for the screen of the Campylobacter infection in the diarrheal patients, the quadruple real-time PCR method comparing to the culture based on the enriched filtration method which was recognized as the most effective isolation method was assessed for 190 stool samples from the diarrheal patients collected during the Foodborne Diseases Active Surveillance Network in Beijing. This multiple real-time PCR was designed to identify the Campylobacter genus, C. jejuni, C. coli, and C. lari simultaneously. With the enrichment culture method, 23 (12.1%, 23/190) Campylobacter isolates were obtained (20 C. jejuni and 3 C. coli), however, 31 samples (16.3%, 31/190) were detected positively with the real-time PCR (21 C. jejuni, 8 C. coli, and 2 Campylobacter genus only). With the comparison, the real-time-PCR method is more sensitive than the enrichment filtration method (16.3 vs. 12.1%, p = 0.021). Among the culture-positive samples, 95.7% (22/23) were detected positively by PCR which indicate the specificity of this method was higher. These two methods were consistent well (Kappa = 0.785, p < 0.05). Comparing to the culture methods, the result of the multiple real-time PCR method is sensitive, reliable and rapid. The present study indicated this multiple real-time PCR can be used both for the surveillance network and the preceding screen for bacteria isolation. This is first comparative study between the culture and multiple real-time PCR method for Campylobacter identification in stool specimens from the diarrheal patients.

Application of MALDI-TOF MS Systems in the Rapid Identification of Campylobacter spp. of Public Health Importance

Campylobacteriosis is an infectious gastrointestinal disease caused by Campylobacter spp. In most cases, it is either underdiagnosed or underreported due to poor diagnostics and limited databases. Several DNA-based molecular diagnostic techniques, including 16S ribosomal RNA (rRNA) sequence typing, have been widely used in the species identification of Campylobacter. Nevertheless, these assays are time-consuming and require a high quality of bacterial DNA. Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS is an emerging diagnostic technology that can provide the rapid identification of microorganisms by using their intact cells without extraction or purification. In this study, we analyzed 24 American Type Culture Collection reference isolates of 16 Campylobacter spp. and five unknown clinical bacterial isolates for rapid identification utilizing two commercially available MADI-TOF MS platforms, namely the bioMérieux VITEK® MS and Bruker Biotyper systems. In addition, 16S rRNA sequencing was performed to confirm the species-level identification of the unknown clinical isolates. Both MALDI-TOF MS systems identified the isolates of C. jejuni, C. coli, C. lari, and C. fetus. The results of this study suggest that the MALDI-TOF MS technique can be used in the identification of Campylobacter spp. of public health importance.

Rapid in-house detection method of Campylobacter spp. from food by redox potential monitoring combined with real-time PCR.

The rapid detection of Campylobacter spp. is of utmost importance for the reduction of infections in humans by contaminated food products. The standard culturing method (ISO 10272-1:2006) involves a high time and labour demand. In this paper, we present a method that reduces the detection time of Campylobacter spp. to or below one third as compared to the ISO method, at a reduced cost per test. We used redox potential change of enrichment cultures (Bolton broth with Bolton selective supplement) for reliably selecting Campylobacter-contaminated raw milk and broiler meat samples. Identification of Campylobacter spp. in the contaminated samples was done by real-time PCR method. Culturing time to conclusive redox monitoring varied between 6 and 24 h for positive samples, depending on the contamination rate, in contrast to 136 h with the standard culturing process. However, now the Campylobacter-negative majority of food samples will not need to be tested by real-time PCR because redox potential monitoring can identify them in the selective enrichment phase. This method could be potentially used as a faster alternative to the current standard ISO 10272-1:2006, for nonregulatory monitoring purposes.

The Prevalence of Campylobacter spp. in Polish Poultry Meat.

The prevalence, count and molecular identification of Campylobacter spp. in Polish poultry meat were analysed. 181 samples of meat from chicken (70), turkey (47), duck (54) and goose (10) were studied. Campylobacter spp. was found in 64% of meat samples. The highest prevalence of this pathogen was detected for duck meat. On average 80% of duck samples were contaminated with Campylobacter spp. The counts of Campylobacter spp. in positive samples remained under ten colony forming units per gram of product in 59% of poultry meat. C. jejuni was more frequently detected in poultry meat than C. coli.

Campylobacter coli in Swine Slaughtering Flowchart and Research of cdt Genes

Background: Campylobacter spp. are among the microorganisms most commonly associated with foodborne disease. Campylobacter spp. isolation from pigs during the slaughter and final products have been reported in several countries, including Brazil. However, very little is known about the sources of contamination in the slaughtering flowchart and how these microorganisms are spread in processing plants. Considering the possibility of the pigs carry Campylobacter spp. since the farm or its products are contaminated in the slaughterhouse, this study had as aim to track Campylobacter spp. in pig slaughtering flowchart to understand the behavior of these pathogens in the production line.Materials, Methods &amp; Results: Forty animals of 10 lots, four from each lot, were followed during slaughter. Stool samples were collected from the floor of each enclosure where the pigs were housed on the farm and immediately after stunning on slaughterhouse. Samples from carcass surface were collected after removal of the animals from scrap machine, after evisceration and before the refrigeration chamber. It was also collected surface samples from jowls and samples from the scalding tank water before and after the passage of animals. The swabs containing samples were plated onto Columbia agar supplemented with activated charcoal, oxygen reduction solution and antibiotics supplement, and incubated at 42°C for 48 h under microaerobic conditions. The colonies which presented with a shiny and moist appearance were analyzed by Gram staining for identification of Campylobacter by morphology, and then tested for catalase and oxidase. The Campylobacter isolates were identified for species C. jejuni or C. coli by PCR. Bands profiles were determined by rep-PCR and used to compare the strains. Campylobacter was isolated from 19 (9.5%) of the 200 pig samples analyzed, seven (36.8%) of the rectum, seven (36.8%) after evisceration and five (26.3%) before the refrigeration chamber. Campylobacter was not isolated from jowls and from scalding tank water. All isolates were C. coliand cdtnegative.Persistence of strains originating from the farm and cross contaminations during the slaughtering flowchart was identified by the analysis of the bands profiles obtained by rep-PCR.Discussion: C. coli was the species of Campylobacter present in the swine intestinal tract and in the swine slaughterhouse. The animals, once contaminated, can carry the microorganism during the stages of the slaughtering flowchart. The farm where the animals came from is an important source of contamination during processing, however cross contamination also plays a relevant role. The evisceration was considered the most critical stage, due to the greater number of isolates obtained after this procedure, what emphasize the importance of the hygienic-sanitary management in this stage. Campylobacter spp. can survive, despite not being able to multiply, in foods at refrigeration temperatures (-1 to 5°C) for one to three weeks. Therefore, the high percentage of isolates obtained from the carcass before the refrigeration chamber may represent a problem, since the contamination of the carcasses that enter in this sector can be maintained until the food reaches the consumer. There was no similarity between strains isolated from different lots, indicating that there were no persistence of strains both in the farm and in the slaughterhouse.

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.

Cleaning and disinfection programs against Campylobacter jejuni for broiler chickens: productive performance, microbiological assessment and characterization

Detailed cleaning and disinfection programs aims to reduce infection pressure from microorganisms from one flock to the next. However, studies evaluating the benefits to poultry performance, the sanitary status of the facilities, and the sanitary quality of the meat are rarely found. Thus, this study was designed to evaluate 2 cleaning and disinfecting programs regarding their influence on productive performance, elimination of Campylobacter, and characterization of Campylobacter jejuni strains when applied to broiler chickens’ facilities. Two subsequent flocks with 960 birds each were distributed into 32 pens containing 30 birds each. In the first, the whole flock was inoculated with a known strain of Campylobacter jejuni in order to contaminate the environment. In the second flock, performance and microbiological evaluations were done, characterizing an observational study between 2 cleaning and disinfection programs, regular and proposed. The regular program consisted of sweeping facilities, washing equipment and environment with water and neutral detergent. The proposed cleaning program consisted of dry and wet cleaning, application of 2 detergents (one acid and one basic) and 2 disinfectants (250 g/L glutaraldehyde and 185 g/L formaldehyde at 0.5% and 210 g/L para-chloro-meta-cresol at 4%). Total microorganism count in the environment and Campylobacter spp. identification were done for the microbiological assessment of the environment and carcasses. The positive samples were submitted to molecular identification of Campylobacter spp. and posterior genetic sequencing of the species identified as Campylobacter jejuni. The birds housed in the facilities and submitted to the proposed treatment had better performance when compared to the ones in the regular treatment, most likely because there was a smaller total microorganism count on the floor, walls, feeders and drinkers. The proposed program also resulted in a reduction of Campylobacter spp. on floors, drinkers and birds. Moreover, it was possible to identify 6 different Campylobacter jejuni strains in the facilities. The proposed treatment resulted in a positive influence on the birds’ performance and reduction of environment contamination for broiler chickens.

Identification and differentiation of Campylobacter species by high-resolution melting curve analysis.

Campylobacter jejuni and Campylobacter coli are the important food-born zoonotic pathogen, also are leading causes of human food borne illnesses worldwide. cadF gene is expressed in all C.jejuni and C.coli strains and mediates cell binding to the cell matrix protein, Fibronectin. High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The goal of this study was to apply HRM analysis to identification of C.jejuni and C.coli. A total of 100 samples were obtained from chicken in Kermanshah, Iran. HRM analysis based on cadF gene and Eva Green was developed to the identification of Campylobacter to the species level. Fifty-five of 100 samples were positive as campylobacter (7 C.jejuni, 29 C.coli, 16 mixes and 3 unknown). Minor variations were observed in melting point temperatures of C.coli or C.jejuni isolates and this variation can be used to the differentiation between C.coli or C.jejuni isolates. The results of this study indicated that HRM curve analysis can be a suitable technique and rapid technique for distinguishing between C.jejuni and C.coli isolates.

Isolation, identification and differentiation of Campylobacter spp. using multiplex PCR assay from goats in Khartoum State, Sudan

The aim of this study was to identify and characterize thermophilic Campylobacter species in faecal samples from goats in Khartoum State, Sudan, by application of multiplex polymerase chain reaction. Campylobacteriosis is a zoonotic disease of global concern, and the organisms can be transmitted to human via food, water and through contact with farm animals and pets. There are five clinically related Campylobacter species: Campylobacter jejuni (C. jejuni). Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis and Campylobacter fetus. Conventional cultural methods to diagnose campylobacteriosis are tedious and time consuming. Wide ranges of genes have been reported to be used for PCR-based identification of Campylobacter spp. We used a multiplex PCR assay to simultaneously detect genes from the major five clinically significant Campylobacter spp. The genes selected were hipO (hippuricase) and 23S rRNA from glyA (serine hydroxymethyl transferase) from each of C. jejuni. C. coli, C. lari, and C. upsaliensis; and sapB2 (surface layer protein) from C. fetus subsp. fetus. The assay was used to identify Campylobacter isolates recovered from 336 cultured faecal samples from goats in three localities in Khartoum State. C. coli was the most predominant isolate (234; 69.6%), followed by C. jejuni (19; 5.7%), C. upsaliensis (13; 3.9%), C. fetus subsp. fetus (7; 2.1%) and C. lari (6; 1.8%). Twenty-nine goats showed mixed infection with Campylobacter spp., 21 of which harbored two Campylobacter spp., while eight animals were infected with three species. Ten out of twelve goats that displayed diarrhea harbored C. coli only. C. coli, C. jejuni and C. upsaliensis showed significant variation with localities. The prevalence of C. coli was significantly higher (87; 25.9%) in goats from Omdurman, whereas C. jejuni and C. upsaliensis were significantly higher (11; 3.3%, 9; 2.7%) in goats from Khartoum. The multiplex PCR assay was found to be rapid and easy to perform and had a high sensitivity and specificity for characterizing the isolates, even in mixed cultures. The study demonstrated the significance of goats as reservoirs in the dissemination of Campylobacter spp. which could be considered as potential agent of caprine enteritis and abortion as well as contamination of the wider environment posing serious public health concern in Khartoum State.

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.

Identification of genus Campylobacter up to species level using internal features of 16S rRNA gene sequences

Introduction: 16S rRNA sequencing of novel isolates is one of the preliminary steps in characterization of bacteria, especially when the isolates are of medical relevance. The genus Campylobacter belongs to Class e-proteobacteria under the Phylum Proteobacteria. It represents economically important species which are gastrointestinal pathogens in humans and livestock animals. Currently, more than 400 16S rDNA sequences belonging to 28 species of this genus are present in the RDP database. But heterogeneity has led to the misplacement of many of these sequences within wrong species. Also, various sequences belonging to Campylobacter have been deposited as orphans. The current study aimed to explore the internal features of 16S rRNA gene sequences in order to develop methods for identification of Campylobacter up to species level. Methods: 428 16S rRNA sequences of 28 species of Campylobacter were analyzed. 392 sequences (>1200 nucleotides, nts) of 16 species were considered for (i) phylogenetic framework analysis and (ii) in silico restriction digestion. 28 uncharacterized sequences present in the database were also investigated in the present study. Results: Phylogenetic framework analysis allowed the identification of genetic variability within Campylobacter species and helped to segregate certain uncharacterized sequences up to species level. 12 out of the 16 species under study showed homogenous behavior, but heterogeneity was observed between C. jejuni and C. coli and C. helveticus and C. upsaliensis respectively. Unique restriction enzymes were identified for six species. Conclusions: The present approach clearly showed that internal features of 16S rRNA is a useful tool for characterization of novel isolates up to the species level. Studies have revealed that niche overlap and consequent increase in the horizontal gene transfer between C. coli and C. jejuni, due to anthropogenic factors, maybe the reason for their heterogeneous nature. This explains the difficulties faced in segregation of the members of these species. 16S rRNA gene proved to be a viable and excellent marker for characterizing the uncharacterized Campylobacter strains leading to a significant diminution in database redundancy. Further, the approaches used in the study might assist in easier identification of the various Campylobacter sequences present in the database.

Rapid Detection of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari in Fresh Chicken Meat and By-Products in Bangkok, Thailand, Using Modified Multiplex PCR

A multiplex PCR assay for simultaneous detection and differentiation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari was developed and validated to assess the occurrence of these bacteria in fresh chicken meat and by-products in Bangkok, Thailand, by using a new combination of four previously published PCR primers for C. jejuni, C. coli, C. lari, and a universal 16S rDNA gene as an internal control. The specificity was determined by using 13 strains of other bacteria. With pure culture DNA, the detection limit was 0.017 ng/PCR for C. jejuni and C. coli and was 0.016 ng/PCR for C. lari. It can detect 10 CFU of C. jejuni, C. coli, and C. lari in 2 g of chicken meat within a 16-h enrichment time. Our multiplex PCR assay was applied for identification of Campylobacter spp. in 122 supermarket samples and 108 fresh market samples. Of the 230 samples evaluated by multiplex PCR, 54.0, 3.3, and 10.7% of supermarket samples were positive for C. jejuni, C. coli, and mixed C. jejuni and C. coli, respectively, and 56.5 and 33.3% of fresh market samples were positive for C. jejuni and mixed C. jejuni and C. coli, respectively. No sample was positive for C. lari. Fresh market samples had significantly higher C. jejuni and C. coli contamination than those from supermarkets (relative risk: 1.3; P = 0.0001). Compared with the culture method (a gold standard), the sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of multiplex PCR were 97.7, 86.8, 96.1, 92.0, and 95.2%, respectively. No significant difference was observed between results from two methods (P = 0.55). Therefore, the established multiplex PCR was not only rapid and easy to perform but had a high sensitivity and specificity to distinguish between C. jejuni, C. coli, and C. lari, even in samples containing mixed contamination. Our study indicated that fresh chicken meat and by-products from fresh markets were significantly less hygienic than those from supermarkets.

Mo1952 Phenotypic and Genotypic Identification of Campylobacters in Diarrheal Patients and Poultry in and Around Chandigarh, India

Background: Campylobacter is an important cause of bacterial gastroenteritis with C. jejuni and C. coli being commonly associated with it. Campylobacteriosis is a disease of foodborne origin, with poultry as the major food vehicle. The present study was aimed to investigate the prevalence of Campylobacters in diarrheal patients and poultry from Chandigarh by phenotypic and genotypic methods. Methods: Stool samples of diarrheal patients (n=1145) and healthy subjects (n=102) from hospital and community in and around Chandigarh were investigated. Apart from this, 127 intestinal specimens from broilers of the same region were also cultured microaerophilically on Campylobacter media for 48 h. The suspected colonies were identified by Gram stain, biochemical and serologic investigations. Molecular confirmation was done using specific primers to unique regions of 16SrRNA, C. jejuni (hipO), C. coli (aspK), C. lari (glyA) and C. upsaliensis (lpxA) genes. Identification of specific genes to look for resistance to nalidixic acid, ciprofloxacin, tetracycline and streptomycin was also done. Results: Campylobacters were isolated from 30 (2.6%) diarrheal samples of which 90% were C. jejuni and 10% were C. coli. The most frequent C. jejuni serogroups were Group S:27, Group B:2, Group Z5:52 and Group V:32 in descending order. No Campylobacter was isolated from healthy subjects. Amongst poultry, Campylobacters were isolated from 57 (44.9%) samples. Genotypically 78.9% isolates were C. jejuni, 1.8% C. coli and 19.3% were co-infections of C. coli and C. jejuni.The most frequent serogroups were Group B:2, Group S:27, Group Z5:52 and Group Z7:57 in descending order. Neither C. lari nor C. upsaliensis was isolated from either human or poultry samples. Antibiotic resistance in humans and poultry isolates were 40.0% and 36.8% for nalidixic acid, 23.3% and 35.0% for ciprofloxacin, 50% and 31.5% for tetracycline and 20% and 31.5% for streptomycin respectively. Conclusion: C. jejuni with high degree of antibiotic resistance was most commonly isolated from both poultry and humans. Poultry in the environment of Chandigarh is a good reservoir of Campylobacters and may be responsible for continued transmission to humans in this region.

Modified Enrichment Strategies Coupled with Molecular and Conventional Methods to Detect and Quantify Campylobacter jejuni in Chicken Meat from the Market

AbstractCampylobacter spp. are currently the bacterial foodborne pathogen that causes the highest number of gastrointestinal diseases in developed countries, according to the World Health Organization. The aim of this study was to compare the performance of four Campylobacter enrichment broths: Bolton broth, Bolton broth with blood, Preston broth and Preston broth with blood using both culture‐dependent methods and an optimized quantitative polymerase chain reaction (qPCR) protocol. The enrichment in Bolton broth with blood allowed most positive samples for Campylobacter jejuni to be detected when 40 chicken meat samples from the market were tested. Correlation between cultural methods and molecular methods was poor. Only in a few cases could C. jejuni and Campylobacter spp. be identified by PCR. Out of 480 colonies isolated throughout the enrichment process, only seven colonies could be identified as Campylobacter spp., three of which were C. jejuni. Enrichment of chicken meat samples with Bolton broth added with blood, coupled with qPCR resulted to be the most suitable method to detect samples contaminated with C. jejuni.Practical ApplicationsDetection and identification of Campylobacter spp. in food is still a challenge and there is lack of consensus on the methodology that should be used in order to recover these fastidious microorganisms. The application of molecular methods, such as the amplification of a target gene sequence by PCR, in order to rapidly and inequivocally detect and identify foodborne pathogens in foodstuff, offers a valid alternative to traditional microbiological testing. In this study, we investigated the performances of combined enrichment strategies with a specific C. jejuni qPCR protocol. The molecular approach resulted to be superior with respect to traditional plating and was able to detect a higher number of C. jejuni positive samples. Among the enrichment broths tested, Bolton broth added with blood resulted to be the most suitable for the detection of Campylobacter spp. in poultry meat.

Multilocus sequence typing (and phylogenetic analysis) of Campylobacter jejuni and Campylobacter coli strains isolated from clinical cases in Greece

BackgroundThe molecular epidemiology of C. jejuni and C. coli clinical strains isolated from children with gastroenteritis, was investigated using the multilocus sequence typing method (MLST). This analysis establishes for the first time in Greece and constitutes an important tool for the epidemiological surveillance and control of Campylobacter infection in our country.MethodsThe MLST genotypes were compared with those gained by other typing methods (HS-typing, PFGE and FlaA typing) and were also phylogenetically analyzed, in order to uncover genetic relationships.ResultsAmong 68 C. jejuni strains, 41 different MLST-Sequence Types (MLST-STs) were found. Fifty six strains or 34 MLST-STs could be sorted into 15 different MLST-Sequence Type Complexes (MLST-STCs), while twelve strains or seven MLST-STs did not match any of the MLST-STCs of the database. Twenty C. coli strains belonged to 14 different MLST-STs. Eleven MLST-STs were classified in the same MLST-STC (828), and three were unclassifiable. There was no significant association between the MLST-STs and the results of the other typing methods.Phylogenetic analysis revealed that some strains, classified to the species of C. jejuni, formed a separate, phylogenetically distinct group. In eight strains some alleles belonging to the taxonomic cluster of C. jejuni, were also detected in C. coli and vice versa, a phenomenon caused by the genetic mosaic encountered inside the genus Campylobacter.ConclusionsThe MLST-ST determination proved to be a very useful tool for the typing as well as the identification of Campylobacter on the species level.