Modified Charcoal Cefoperazone Deoxycholate Agar Research Articles

Virulence and resistance genes in <i>Campylobacter spp</i> isolated from asymptomatic children at Kibera slum in Nairobi, Kenya

BACKGROUND Campylobacter spp. are among the global causes of food poisoning and harbour virulence and resistance genes which play a role in invasion and antibiotic resistance. This study aimed to detect selected resistance and virulence genes in archived Campylobacter spp. isolated from children in Kibera, Nairobi, Kenya. MATERIALS AND METHODS This was a retrospective laboratory-based cross-sectional study on archived samples of Campylobacter spp. at the Department of Public Health, Pharmacology and Toxicology, University of Nairobi. Campylobacter isolates were cultured on modified charcoal cefoperazone deoxycholate agar (mCCDA) media. The presence of selected virulence genes and resistance genes was detected by multiplex polymerase chain reaction (mPCR). STATA13 was used in data analysis. Binary logistic regression was used to ascertain the association between the genes and the Campylobacter spp. RESULTS The Campylobacter spp. under this study were Campylobacter jejuni (C. jejuni) (50%), Campylobacter coli (C. coli) (38%) and Campylobacter lari (C. lari) (12%). The virulence genes detected were cgtB 37 (25%), pldA (20%), cdtA (17%), cdtB (16%), cdtC (6%), wlaN (5%), iam (4%), ciaB (4%) and VirB11 (3%). The virulence gene waaC was not detected in any of the isolates. The resistance genes detected were TetA (42%), Tet B (26%), GyrB (18%) and GyrA (14%). CONCLUSION The study revealed the presence of various virulence and resistance genes in Campylobacter spp. isolated from children. This information could help guide the development and enhancement of guidelines for the management of Campylobacter infections.

Campylobacter jejuni and Campylobacter coli infection, determinants and antimicrobial resistance patterns among under-five children with diarrhea in Amhara National Regional State, Northwest Ethiopia.

Children with under-five year age disproportionally affected with foodborne illness. Campylobacteriosis is the most common foodborne disease next to Norovirus infection. Macrolides are commonly prescribed as the first line of treatment for Campylobacter gastroenteritis, with fluoroquinolone and tetracycline as secondary options. However, resistance to these alternatives has been reported in various regions worldwide. To determine the prevalence, associated risk-factors and antimicrobial resistance of Campylobacter jejuni and C. coli among under-five children with diarrhea. Institution-based cross-sectional study was conducted from November, 2022 to April 2023. The study sites were selected using a random sampling technique, while the study subjects were included using a convenient sampling technique. The data were collected using a structured questionnaire. Stool samples were inoculated onto modified charcoal cefoperazone deoxycholate agar and incubated for 48 hours. The suspected colonies were analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry to confirm the species. Antimicrobial susceptibility testing was performed using a disc diffusion technique. All potential covariates (independent variables) were analyzed one by one using bivariate logistic regression model to identify candidate variables with P value < 0.25. Multivariable logistic analysis was used to identify potential associated factors using the candidate variables. A p value ≤ 0.05 at a 95% confidence interval was statistically significant. Among the 428 samples, 7.0% (CI: 4.5-9.3) were confirmed Campylobacter species. The prevalence of C. jejuni and C. coli among under-five children was 5.1% (CI: 3.0-7.0) and 1.9% (CI: 0.7-3.3), respectively. C. jejuni (73.3%) was dominant over C. coli (26.7%). The resident, contact with domestic animals, and parents/guardians education level were significantly associated with campylobacteriosis among under-five children. One-third of the Campylobacter isolates (33.3%, 10/30) were resistant to ciprofloxacin and tetracycline whereas 10.0% (3/30) were resistant to erythromycin. Furthermore, 3.3% (1/30) of the Campylobacter were found to be multidrug-resistant. The prevalence of Campylobacter species was 7.0%. The resistance rate of Campylobacter species of ciprofloxacin and tetracycline-resistance strains was 33.3%. Peri-urban residence, contact with domestic animals, and low parental educational statuses were significantly associated factors with increased risk of Campylobacter infection. Continuous surveillance on antimicrobial resistance and health education of personal and environmental hygiene should be implemented in the community.

Campylobacter occurrence and antimicrobial resistance profile in under five-year-old diarrheal children, backyard farm animals, and companion pets.

Campylobacteriosis disproportionately affects children under five in low-income countries. However, epidemiological and antimicrobial resistance (AMR) information at the children-animal interface is lacking. We hypothesized that Campylobacter is a major cause of enteritis in children in Ethiopia, and contact with animals is a potential source of transmission. The objective of the study was to determine Campylobacter occurrence and its AMR in children under five with diarrhea, backyard farm animals, and companion pets. Stool from 303 children and feces from 711 animals were sampled. Campylobacter was isolated through membrane filtration on modified charcoal cefoperazone deoxycholate agar plates under microaerobic incubation, and the technique showed to be feasible for use in regions lacking organized laboratories. Typical isolates were characterized with MALDI-TOF MS and multiplex PCR. Of 303 children, 20% (n = 59) were infected, with a higher proportion in the 6 to 11-month age group. Campylobacter occurred in 64% (n = 14) of dogs and 44% (n = 112) of poultry. Campylobacter jejuni was present in both a child and animal species in 15% (n = 23) of 149 households positive for Campylobacter. MICs using the gradient strip diffusion test of 128 isolates displayed resistance rates of 20% to ciprofloxacin and 11% to doxycycline. MICs of ciprofloxacin and doxycycline varied between C. coli and C. jejuni, with higher resistance in C. coli and poultry isolates. Campylobacter infection in children and its prevalent excretion from backyard poultry and dogs is a understudied concern. The co-occurrence of C. jejuni in animals and children suggest household-level transmission As resistance to ciprofloxacin and doxycycline was observed, therapy of severe campylobacteriosis should consider susceptibility testing. Findings from this study can support evidence-based diagnosis, antimicrobial treatment, and further investigations on the spread of AMR mechanisms for informed One Health intervention.

Multidrug resistance and virulence genes in Campylobacter isolates from poultry waste in Ibadan, Nigeria

Objective: This study aims to determine antibiotic susceptibility profile and detect virulence genes in Campylobacter spp. isolated from poultry waste in Ibadan, Nigeria. Methods: Poultry droppings were collected from ten poultry farms, and Total Bacteria Count (TBC) and Total Anaerobic Count (TAC) of the samples were determined. Campylobacter species were isolated using modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA). The isolates were identified using conventional biochemical techniques, and antibiotics susceptibility testing was performed using Kirby Bauer disk diffusion assay on Mueller Hinton agar. Detection of virulence genes in selected multidrug resistant (MDR) isolates was carried out using polymerase chain reaction. Results: Campylobacter species (240) were isolated from Moniya (44.6%) and Igbo-Oloyin (55.4%) farms. Campylobacter (C.) lari was the most prevalent isolate (32.5%) while C. fetus was the least common. Resistance to streptomycin was observed in 76.7% of the isolates, while 3.3% exhibited resistance to ertapenem. Specifically, C. jejuni isolates from Igbo-Oloyin farms showed resistance rates of 86% to cephalothin and 71% to cefuroxime. In comparison, C. jejuni isolates from Moniya farms showed even higher resistance rates of 95% to cephalothin and 81% to cefuroxime. The antibiotypes of the selected isolates showed that four Campylobacter spp. were resistant to a combination of eight antibiotics. Furthermore, the detection of virulence genes from the selected MDR isolates showed that cadF and hipO genes were detected in 70.0% and 50.0% of cases, respectively. Conclusions: MDR strains with virulent genes were found in Campylobacter species isolated from poultry wastes, posing a significant health challenge. Therefore, prudent use of antibiotics in animal husbandry should be ensured in addition to preventing indiscriminate discharge of untreated poultry waste into the environment.

Antimicrobial resistance patterns and genes of Campylobacter jejuni isolated from chickens in Pasuruan, Indonesia.

Poultry is one of the most prominent sources of Campylobacter jejuni, which is also a major means of transmission to people. Campylobacter jejuni contamination in chicken meat comes from chicken feces because it naturally exists in the intestines of chickens. The purpose of this study is to identify the antibiotic resistance patterns and genes of C. jejuni, which was found in chickens in Pasuruan, Indonesia. The samples used in this study were 200 contents of the small intestine of broiler chickens from 40 farms in Pasuruan Regency. The enriched sample was streaked on the selective media of modified charcoal cefoperazone deoxycholate agar containing the CCDA selective supplement. Antimicrobial susceptibility test utilizing the Kirby-Bauer diffusion test method in accordance with Clinical and Laboratory Standards Institute standards. The polymerase chain reaction (PCR) method was used to detect the (hipO), which encodes the C. jejuni strain, fluoroquinolone resistance (gyrA), beta-lactam resistance (blaOXA-61), and tetracycline resistance (tetO) genes. The findings revealed a 14% (28/200) prevalence of C. jejuni in the small intestine of broiler chickens. These isolates showed high resistance to enrofloxacin (92.9%). All isolates (100%) were susceptible to amoxicillin-clavulanate. The PCR results showed all C. jejuni isolates (100%) detected the gyrA gene, 96.4% detected the blaOXA-61 gene, and 50% detected the tetO gene. The findings of antimicrobial resistance at a high level from the small intestine of broiler chickens illustrate the potential threat to human health. To lessen the effects now and in the future, coordinated and suitable action is needed, as well as steps to guarantee the poultry industry's economic survival and public health insurance.

Adaptation of a Commercial Qualitative BAX® Real-Time PCR Assay to Quantify Campylobacter spp. in Whole Bird Carcass Rinses.

Poultry is the primary reservoir of Campylobacter, a leading cause of gastroenteritis in the United States. Currently, the selective plating methodology using selective agars, Campy Cefex and Modified Charcoal Cefoperazone Deoxycholate agar, is preferentially used for the quantification of Campylobacter spp. among poultry products. Due to the specific nature of Campylobacter, this methodology is not sensitive, which can lead to skewed detection and quantification results. Therefore, Campylobacter detection and quantification methods are urgently needed. The objective was to develop a shortened enrichment-based quantification method for Campylobacter (CampyQuant™) in post-chill poultry rinsates using the BAX® System Real-Time PCR assay for Campylobacter. The specificity and sensitivity for the detection of C. jejuni, C. coli, and C. lari in pure culture were determined. The BAX® System Real-Time PCR assay consistently detected and identified each species 100% of the time with an enumeration range of 4.00 to 9.00 Log10 CFU/mL. Enrichment time parameters for low-level concentrations (0.00, 1.00, and 2.00 Log10 CFU/mL) of Campylobacter using the BAX® System Real-Time PCR assay were elucidated. It was determined that an enrichment time of 20 h was needed to detect at least 1.00 Log10 CFU/mL of Campylobacter spp. Using the BAX® System Real-Time PCR assay for Campylobacter. As a result, time of detection, detection limits, and enrichment parameters were used to develop the CampyQuant™ linear standard curve using the detected samples from the BAX® System Real-Time PCR assay to quantify the levels in post-chill poultry rinsates. A linear fit equation was generated for each Campylobacter species using the cycle threshold from the BAX® System Real-Time PCR assay to estimate a pre-enrichment of 1.00 to 4.00 Log10 CFU/mL of rinsates detected. The statistical analyses of each equation yielded an R2 of 0.93, 0.76, and 0.94 with a Log10 RMSE of 0.64, 1.09, and 0.81 from C. jejuni, C. coli, and C. lari, respectively. The study suggests that the BAX® System Real-Time PCR assay for Campylobacter is a more rapid, accurate, and efficient alternative method for Campylobacter enumeration.

Comparison of Two Culture-Based Detection Systems for the Isolation of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii in Raw Ground Poultry

Arcobacters are emerging pathogens that have been underestimated due to a lack of a standardized isolation method. The aim of this research was to evaluate the ability to isolate Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii using two Arcobacter-specific culture detection systems: (i) the Houf broth and modified charcoal cefoperazone deoxycholate agar supplemented with cefoperazone, amphotericin B, and teicoplanin (HB/mCCDA+CAT), and (ii) the Nguyen-Restaino-Juárez Arcobacter enrichment broth and chromogenic agar (NRJ-B/M). Both detection systems were evaluated for productivity ratio, sensitivity, and specificity. As a result, the productivity ratio for both plating agars were >90%, which indicates that the selective agents used in the two plating agars did not inhibit Arcobacter growth. Moreover, sensitivity evaluations using artificially inoculated retail ground poultry (n = 780) determined that both detection systems were able to isolate A. butlzeri with >95% sensitivity at the 0.1 and 1.0–2.0 CFU/g detection level. The sensitivity in A. cryaerophilus isolation was higher for NRJ-B/M (78.0% at 0.1 CFU/g; 95.1% at 1.0–2.0 CFU/g) when compared with HB/mCCDA+CAT (34.1% at 0.1 CFU/g; 51.2% at 1.0–2.0 CFU/g). Both detection systems resulted in <50% sensitivity when isolating A. skirrowii at 0.1 and 1.0–2.0 CFU/g; however, the sensitivity for NRJ-B/M was significantly higher than HB/mCCDA+CAT. At the detection level of 5.0 CFU/g, both detection systems were able to isolate A. skirrowii with 100% sensitivity. Specificity comparisons using uninoculated ground poultry samples (n = 40) indicated the growth of background microbiota were significantly inhibited or could be easily differentiated on NRJ-B/M (90.0%, specificity) when compared with HB/mCCDA+CAT (30.0%, specificity). Overall, these results show that the NRJ-B/M detection system is a more sensitive and specific detection system when isolating Arcobacter spp. from ground chicken.

Genetic Signature and Serocompatibility Evidence for Drug Resistant Campylobacter jejuni.

Campylobacteriosis, a foodborne illness, is one of the world′s leading causes of gastrointestinal illness. This study investigates the link between human campylobacteriosis and the consumption of potentially contaminated food with Campylobacter jejuni. Three hundred sixty samples were collected from humans, chicken cloaca, raw chicken meat, unpasteurized milk, and vegetables. The chickens were obtained from licensed and non-licensed slaughterhouses, and only the necks and wings were studied. Samples were enriched under microaerobic conditions then cultured on the modified charcoal cefoperazone deoxycholate agar. Bacteria was identified by staining, biochemical testing, and molecular identification by the polymerase chain reaction for the virulence genes; hipO, asp, dnaJ, cadF, cdtA, cdtB, and cdtC. The genomic homogeneity of C. jejuni between human and chicken isolates was assessed by the serological Penner test and the pulse field gel electrophoresis (PFGE). Campylobacter was not detected in the vegetables and pasteurized milk, though, only twenty isolates from chickens and clinical samples were presumed to be Campylobacter based on their morphology. The biochemical tests confirmed that five isolates were C. coli, and fifteen isolates were C. jejuni including two isolates from humans, and the remaining were from chickens. The colonization of C. jejuni in chickens was significantly lower in necks (6.66%) obtained from licensed slaughterhouses compared to those obtained from non-licensed slaughterhouses (33.3%). The antimicrobial susceptibility test showed that all identified C. jejuni isolates were resistant to antibiotics, and the majority of isolates (53.5%) showed resistance against six antibiotics, though, all isolates were resistant to ciprofloxacin, tetracycline, and aztreonam. The Penner test showed P:21 as the dominant serotype in isolates from humans, necks, and cloaca. The serohomology of C. jejuni from human isolates and chicken necks, wings, and cloaca was 71%, 36%, 78%, respectively. The PFGE analysis of the pattern for DNA fragmentation by the restriction enzyme Smal showed a complete genotypic homology of C. jejuni human isolates and chicken necks compared to partial homology with cloacal isolates. The study brings attention to the need for effective interventions to ensure best practices for safe poultry production for commercial food chain supply to limit infection with foodborne pathogens, including Campylobacter.

Prevalence and risk factors of Campylobacter infection in goats in southern provinces of Thailand.

Campylobacter is a foodborne pathogen that could be isolated from various types of livestock, including small ruminant. The objectives of the current study were to investigate the prevalence of Campylobacter spp. in goats reared in the south of Thailand and identify the risk factors associated with Campylobacter infection of goat in Thailand. A total of 600 rectal swabs of goats from 45 farms in 6 provinces of Southern Thailand were collected during 2016-2020. To analyse the risk factors associated with Campylobacter infection in goat, data collection was conducted via structured questionnaires. Campylobacter isolation was performed by direct plating on modified charcoal cefoperazone deoxycholate agar (mCCDA). Polymerase chain reaction (PCR) was used to identify genus and species of the isolates. In total, 68 out of 600 rectal swab samples (11.33%; 95% CI 8.96 to 14.21%) collected from goats were positive for Campylobacter. The prevalence of Campylobacter spp. in the province level ranged from 0 to 31.48%. Interestingly, Campylobacter lanienae was the dominant species, followed by Campylobacter coli and Campylobacter jejuni. To investigate the risk factors associated with Campylobacter infection in goats, farm management factors, i.e. deworming, fencing around the house, housing design, number of animals on farm, use of antibiotics and vaccination, were included in logistic regression analysis. Statistical analysis revealed that the use of antibiotics and deworming were associated with Campylobacter infections in goats. The current study emphasises that the prudent use of antimicrobials and good agricultural practices should be applied in goat farming.

Genomic diversity and antimicrobial resistance of Campylobacter spp. from humans and livestock in Nigeria

BackgroundCampylobacter spp. are zoonotic pathogens, ubiquitous and are found naturally as commensals in livestock from where they can be transmitted to humans directly or through animal products. The genomic diversity and antimicrobial resistance profile of Campylobacter was investigated with a focus on C. jejuni and C. coli in humans and livestock (poultry and cattle) from Nigeria.Methods586 human stool samples and 472 faecal samples from livestock were cultured for thermophilic Campylobacter species on modified charcoal cefoperazone deoxycholate agar (mCCDA). Culture in combination with whole genome sequencing identified and confirmed the presence of Campylobacter in humans and animals from the study area. Further analysis of the sequences was performed to determine multilocus sequence types and genetic determinants of antimicrobial resistance to fluoroquinolone, betalactam, tetracycline and macrolide classes of antimicrobials.ResultsFrom the human stool samples tested, 50 (9%) were positive of which 33 (66%) were C. jejuni, 14 (28%) were C. coli while 3 (6%) were C. hyointestinalis. In livestock, 132 (28%) were positive. Thirty one (7%) were C. jejuni while 101 (21%) were C. coli. Whole genome sequencing and MLST of the isolates revealed a total of 32 sequence types (STs) identified from 47 human isolates while 48 STs were identified in 124 isolates from livestock indicating a population which was overall, genetically diverse with a few more dominant strains. The antimicrobial resistance profiles of the isolates indicated a higher prevalence of resistance in Campylobacter isolated from livestock than in humans. Generally, resistance was greatest for betalactams (42%) closely followed by fluoroquinolones (41%), tetracyclines (15%) and lastly macrolides (2%). Multidrug resistance to three or more antimicrobials was observed in 24 (13%) isolates from humans (n = 1, 4%) and chicken (n = 23, 96%).ConclusionsThis study has further contributed information about the epidemiology, genetic diversity and antimicrobial resistance profile of thermophilic Campylobacter in Nigeria.

ISOLATION METHODS FOR MOLECULAR DETECTION AND ANTIBIOTIC RESISTANCE PATTERN OF CAMPYLOBACTER SPP IN LAYER CHICKENS

This study was conducted to compare two culture methods for the isolation of Campylobacter spp from commercial layer chickens and subsequently confirmed by Polymerase Chain Reaction assays (PCR). Furthermore, the antimicrobial resistance profiles of PCR positive Campylobacter isolates were determined.Cloacal swab samples (550) from chickens randomly selected from five poultry farms in the four geographical zones in Ogun State were cultured for Campylobacter using modified charcoal Cefoperazone deoxycholate agar (MCCDA) and an improved culture method involving Preston broth pre-enrichment and subsequent subculture on Mueller Hinton agar with Campylobacter growth supplements. Putative isolates were later confirmed by PCR assay and sequencing analysis.Other isolates that grew on MCCDA and confirmed by sequencing analysis are Enterococcus faecalis, Escherichis coli, Comamonas kerstli and Pseudomonas aeroginusa . The antibiotic resistant profile of all the isolates were evaluated genotypically for resistance genes to tetracyclines (tetO), multiclasses (cmeB), aminoglycosides (aphA-3-1) and β-lactams (Blaoxa-61) using multiplex PCR (mPCR), and phenotypically for chlortetracycline, tylosin, streptomycin, ciprofloxacin and erythromycin resistance by microbroth dilution method which correspond to the antibiotic resistance genes. The apparent prevalence of Campylobacter was 16.8% by MCCDA while none of the isolates was positive to PCR. Meanwhile, prevalence rate of 26% was obtained using Preston broth pre-enrichment and Mueller Hinton agar with Campylobacter growth supplements, of which 11/50 (22%) of the isolates was confirmed positive by PCR. Genotypic characterization of PCR positive isolates showed 10/11(90%) were C. coli, 1/11(10%) other Campylobacter species and 0% C. jejuni. All the isolates carried both tetO and cmeB resistant genes. The results of minimum inhibitory concentration presented all PCR positive isolates had resistance of 10/10(100%), 9/10(90%), 6/10(60%), 9/10(90%), and 8/10(80%) to tetracycline, ciprofloxacin, erythromycin, spectinomycin and tylosin respectively. In addition, all isolates carried multiple resistance to most antibiotics tested which are commonly used in poultry practice in Nigeria. Campylobacter spp in the study areas showed diverse genotypic characteristics, and gene mediated multidrug resistance.&#x0D; &#x0D;

Improvement of the detection efficiency of 3M™ molecular detection system for Campylobacter in poultry using nitrogen-doped carbon nanodots

This study was performed to examine whether the use of nitrogen-doped carbon nanodots (N-CNDs) can improve the detection sensitivity of the 3 M™ molecular detection system (MDS) for Campylobacter. N-CNDs were added to a Campylobacter enrichment broth (CEB) at concentrations of 5 and 10 mg/mL (NCEB-5 and NCEB-10, respectively). Campylobacter coli, C. jejuni, and C. lari were inoculated into the broths. The broth cultures were then irradiated with light-emitting diode (LED) at 425 nm for 1 h and incubated at 42 °C for 6 h, and then grown on modified charcoal cefoperazone deoxycholate agar (mCCDA). The detection rates of the MDS and a conventional method (plating an enriched sample on mCCDA and analyzing a colony on mCCDA with PCR) for Campylobacter in chicken and duck carcasses were compared. The detection rates from the MDS were compared after enrichment in CEB and NCEB-5 at 3, 5, 6, 7, 9, 12, and 24 h. When 5 mg/mL of N-CNDs was added to the CEB followed by irradiation at 425 nm, growth of the Campylobacter was accelerated. In addition, the qualitative test was more sensitive in the MDS than in the conventional method, and the detection time was shortened in CEB enriched with N-CNDs. These results indicate that adding N-CNDs to CEB can improve the detection efficiency of MDS.

Method-Dependent Implications in Foodborne Pathogen Quantification: The Case of Campylobacter coli Survival on Meat as Comparatively Assessed by Colony Count and Viability PCR.

The aim of the present study was to address method-dependent implications during the quantification of viable Campylobacter coli cells on meat over time. Traditional colony counting on selective and non-selective culture media along with an optimized viability real-time PCR utilizing propidium monoazide-quantitative PCR (PMA-qPCR), spheroplast formation and an internal sample process control (ISPC), were comparatively evaluated for monitoring the survival of C. coli on fresh lamb meat during refrigeration storage under normal atmospheric conditions. On day zero of three independent experiments, lamb meat pieces were artificially inoculated with C. coli and then stored under refrigeration for up to 8 days. Three meat samples were tested on different days and the mean counts were determined per quantification method. An overall reduction of the viable C. coli on lamb meat was observed regardless of the applied quantification scheme, but the rate of reduction followed a method-dependent pattern, the highest being observed for colony counting on modified charcoal cefoperazone deoxycholate agar (mCCDA). Univariate ANOVA indicated that the mean counts of viable C. coli using PMA-qPCR were significantly higher compared to Columbia blood agar (CBA) plating (0.32 log10 cell equivalents, p = 0.015) and significantly lower when mCCDA was compared to CBA plating (0.88 log10 CFU, p < 0.001), indicating that selective culture on mCCDA largely underestimated the number of culturable cells during the course of meat storage. PMA-qPCR outperformed the classical colony counting in terms of quantifying both the culturable and viable but non-culturable (VBNC) C. coli cells, which were generated over time on meat and are potentially infectious and equally important from a public health perspective as their culturable counterparts.

Campylobacteriosis Agents in Meat Carcasses Collected from Two District Municipalities in the Eastern Cape Province, South Africa.

Raw meats are sometimes contaminated with Campylobacter species from animal faeces, and meats have repeatedly been implicated in foodborne infections. This study evaluated the prevalence, virulence genes, antimicrobial susceptibility patterns, and resistance gene determinants in Campylobacter species isolated from retailed meat carcasses. A total of 248 raw meat samples were collected from butcheries, supermarkets, and open markets; processed for enrichment in Bolton broth; and incubated at 42 °C for 48 h in 10% CO2. Thereafter, the broths were streaked on modified charcoal cefoperazone deoxycholate agar (mCCDA) plates and incubated at the same conditions and for the same amount of time. After incubation, colonies were isolated and confirmed by Polymerase chain reaction using specific oligonucleotide sequences used for the identification of the genus Campylobacter, species, and their virulence markers. The patterns of antimicrobial resistance profiles of the identified isolates were studied by disk diffusion method against 12 antibiotics, and relevant resistance genes were assessed by PCR. From culture, 845 presumptive Campylobacter isolates were obtained, of which 240 (28.4%) were identified as genus Campylobacter. These were then characterised into four species, of which C. coli had the highest prevalence rate (22.08%), followed by C. jejuni (16.66%) and C. fetus (3.73%). The virulence genes detected included iam (43.14%), cadF (37.25%), cdtB (23.53%), flgR (18.63%), and flaA (1.96%), and some of the isolates co-harboured two to four virulence genes. Of the 12 antibiotics tested, the highest phenotypic resistance displayed by Campylobacter isolates was against clindamycin (100%), and the lowest level of resistance was observed against imipenem (23.33%). The frequency of resistance genes detected included catll (91.78%), tetA (68.82%), gyra (61.76%), ampC (55%), aac(3)-IIa (aacC2)a (40.98%), tetM (38.71%), ermB (18.29%), tetB (12.90%), and tetK (2.15%). There is a high incidence of Campylobacter species in meat carcasses, suggesting these to be a reservoir of campylobacteriosis agents in this community, and as such, consumption of undercooked meats in this community is a potential health risk to consumers.

Acinetobacter baumannii detected on modified charcoal-cefoperazone-deoxycholate agar in a waste stabilization pond.

Campylobacter is a recommended reference pathogen for the verification and validation of water recycling schemes in Australia and globally. In a larger study investigating the efficacy of pathogen removal in waste stabilization ponds (WSP), we cultivated bacteria from wastewater samples on modified charcoal-cefoperazone-deoxycholate agar (mCCDA) targeting the growth of Campylobacter. A high number of colonies characteristic of Campylobacter grew on this selective medium, but this did not correlate with qPCR data. Using primers targeting the 16S rRNA gene, and additional confirmatory tests to detect VS1, ompA, blaOXA-51-like, blaOXA-23-like genes, we tested 80 random colonies from 10 WSP samples. All 80 were identified as Acinetobacter baumannii. Wastewater grab samples taken three times over 6 months throughout the WSP system showed removal of A. baumannii in the WSP at rates similar to that of Escherichia coli. Our study suggests that mCCDA agar is not a suitable medium for isolating Campylobacter from environmental samples and that A. baumannii can be used as an indicator for removal of pathogens in WSPs.

Prevalence of pathogenic Arcobacter species in South Korea: Comparison of two protocols for isolating the bacteria from foods and examination of nine putative virulence genes

Contamination of foodstuffs by potentially enteropathogenic Arcobacter spp. is becoming a concern worldwide. However, few studies have examined virulence-associated genes in isolates of Arcobacter spp. from food. Here, we investigated the prevalence of three pathogenic Arcobacter species, A. butzleri, A. cryaerophilus, and A. skirrowii, in chicken, pork, and leafy green vegetables (n = 323) in South Korea. Samples were examined using two different protocols selected from a literature review: Acrobacter selective broth (ASB) II + Arcobacter selective medium (ASM) II (protocol A), and ASB II + modified charcoal cefoperazone deoxycholate agar supplemented with CAT (protocol B). Overall, Arcobacter spp. were detected in 45.8% of food samples, and the recovery rate of protocol B (37.8%) was significantly higher than that of protocol A (30.7%) (p < 0.05). Refrigerated chicken gizzard samples showed the highest detection rate (100%), followed by refrigerated chicken wing (79.5%), intestine (77.3%), neck skin (63.3%), pork (55.6%), frozen chicken legs (5.0%), and leafy green vegetables (4.4%) (p < 0.05). All isolates from chicken and leafy green vegetables were identified as A. butzleri, whereas A. cryaerophilus and A. skirrowii were mainly detected in pork. Most samples (95.8%) harbored more than one of nine putative virulence factors (cadF, ciaB, cj1349, hecA, hecB, mviN, pldA, irgA, and tlyA), and 91.3% harbored more than two. Isolates harboring all nine putative virulence genes were obtained from 1.9% of samples: five pork and one chicken. This study provides comprehensive and de facto evidence regarding prevalence of an emerging pathogen, Arcobacter spp., in various foods, along with their virulence potential. The results justify further research with respect to their role in food safety.

Comparision of Five Different Enrichment Broth-Agar Combinations for the Isolation of Campylobacter jejuni

Campylobacter is a fastidious microaerophilic bacterium, it is a leading cause of bacterial gastroenteritis in humans globally. In the present study, the efficacy of five enrichment broths in combination with five selective agars for the isolation of Campylobacter jejuni was compared to identify a best possible combination of broth- agar for enrichment. Where, C. jejuni standard culture (ATCC®33560 TM) was enriched in five enrichment broths viz., Brucella, Preston enrichment, modified Charcoal Cefoperazone Deoxycholate broth (mCCDB), Park and Sanders, and Bolton broths, and consequently plated onto modified Charcoal Cefoperazone Deoxycholate agar (mCCDA), Karmali agar, Columbia blood agar, Campylobacter Cefex agar and Campylobacter agar base (CAB). Bolton broth and mCCD agar combination showed the maximum enrichment efficiency and the colony recovery by Bolton broth significantly differed when compared to the Park and Sanders and Brucella broths. The result of the current study shows the combination of enrichment broth and agar most appropriate for the isolation of C. jejuni.

Enrichment Broth for the Detection of Campylobacter jejuni and Campylobacter coli in Fresh Produce and Poultry

Although campylobacteriosis caused by Campylobacter jejuni and Campylobacter coli has been increasingly reported worldwide owing to the consumption of contaminated poultry and fresh produce, the current detection protocols are not selective enough to inhibit unspecific microbes other than these pathogens. Five antibiotics were separately added to Bolton broth, and the survival rates of 18 Campylobacter spp. and 79 non-Campylobacter spp. were evaluated. The survival rate of the non-Campylobacter spp. was the lowest in Bolton broth with rifampin (6.3%), followed by cefsulodin (12.7%), novobiocin (16.5%), and potassium tellurite and sulfamethozaxole (both 17.7%). Also the most effective concentration of rifampin was found to be 12.5 mg/L, which markedly inhibited non-Campylobacter strains while not affecting the survival of Campylobacter strains. After the Campylobacter spp. were enriched in Bolton broth supplemented with 12.5 mg/L rifampin (R-Bolton broth), CampyFood Agar (CFA) was found to be better in selectively isolating the pathogens in the enrichment broth than the International Organization for Standardization method of using modified charcoal cefoperazone deoxycholate agar (mCCDA) for this step. When applied to natural food samples-here, romaine lettuce, pepper, cherry tomato, Korean leek, and chicken-the R-Bolton broth-CFA combination decreased the number of false-positive results by 50.0, 4.2, 20.8, 50.0, and 94.4%, respectively, compared with the International Organization for Standardization method (Bolton broth-mCCDA combination). These results demonstrate that the combination of R-Bolton broth and CFA is more efficient in detecting C. jejuni and C. coli in poultry and fresh produce and thus should replace the Bolton broth-mCCDA combination.

High Rates of Contamination of Poultry Meat Products with Drug-Resistant Campylobacter in Metro Manila, Philippines.

A total of 265 chicken parts were collected from 15 wet markets and 15 supermarkets in Metro Manila, Philippines. Campylobacter spp. was isolated on modified charcoal cefoperazone deoxycholate agar plates and identified through biochemical tests and PCR amplification of genus- and species-specific genes. Antimicrobial resistance profiles were determined following the protocols of the Clinical and Laboratory Standards Institute. Two hundred and seven (78.1%) Campylobacter spp. isolates were obtained. Campylobacter jejuni and Campylobacter coli were detected in 170 (64.2%) and 32 (12.1%) of the samples, respectively. Liver and skin samples showed the greatest levels of contamination. Most of the isolates were resistant to clindamycin (98.6%), erythromycin (98.6%), nalidixic acid (98.1%), tetracycline (94.2%), gentamicin (65.2%), and chloramphenicol (52.7%). The results indicated that poultry meat sold in markets in Metro Manila is contaminated with drug-resistant Campylobacter spp.

Sensitivity of Direct Culture, Enrichment and PCR for Detection of Campylobacter jejuni and C.coli in Broiler Flocks at Slaughter.

Broiler chicken flocks are a significant source of Campylobacter jejuni and Campylobacter coli that result in the major public health problem of campylobacteriosis. Accurate estimates of the prevalence of both C.coli and C.jejuni in flocks would enhance epidemiological understanding, risk assessment and control options. This study combined results from a panel of 10 detection tests (direct culture, enrichment and PCR) on caecal samples from flocks at slaughter. A parallel interpretation approach was used to determine the presence of Campylobacter spp. and for C.jejuni and C.coli individually. The sample was considered positive if at least one method detected the target and this interpretation was taken to represent a 'proxy gold standard' for detection in the absence of a gold standard reference test. The sensitivity of each individual method to detect Campylobacter spp., C.jejuni and C.coli was then estimated relative to the proxy gold standard. Enrichment in adapted Exeter broth (deficient in polymyxin B) with a resuscitation step was 100% sensitive, whilst direct culture on modified charcoal cefoperazone deoxycholate agar (mCCDA) was highly sensitive (97.9%). Enrichment methods using Preston broth and Bolton broth were significantly less sensitive. Enrichment in Exeter broth promoted the recovery of C.jejuni, whilst enrichment in Bolton broth favoured C.coli. A RT-PCR detection test could identify 80% of flocks that were co-colonised with both species. This study found that 76.3% (n=127) of flocks were colonised with Campylobacter spp. The majority (95.9%) of Campylobacter-positive flocks were colonised with C.jejuni; however, approximately one-third of positive flocks were simultaneously colonised with both C.jejuni and C.coli. The findings highlight the impact of different detection methodologies on the accuracy of the estimated incidence of both C.jejuni and C.coli entering the abattoir within broiler flocks and the associated public health risks.