Campy-Cefex Agar Research Articles

Prevalence and Antimicrobial Resistance of Campylobacter in Conventional and No Antibiotics Ever Broiler Farms

Campylobacter is a leading cause of enteric disease worldwide. No antibiotics ever (NAE) poultry has become increasingly popular, yet little is known about the incidence and antimicrobial resistance (AMR) of Campylobacter in this production system. This study was conducted to determine the prevalence, concentration, and AMR of Campylobacter in conventional (CV) and NAE-raised broilers. Two CV and two NAE commercial broiler flocks were included in this study. Cecum (n = 420) and ileum (n = 420) of chickens were collected at different stages during the broiler grow-out phase and following transportation to the processing plant. Samples of litter (n = 24), feed (n = 24), and water (n = 24) were also collected. Screening for Campylobacter was conducted using real-time PCR assay, and enumeration was performed by direct plating on Campy Cefex agar. Campylobacter isolates were confirmed by real-time PCR, and antimicrobial susceptibility was evaluated following the National Antimicrobial Resistance Monitoring System (NARMS) methods. Whole Genome Sequencing (WGS) was used to identify AMR genes carried by the resistant isolates. Campylobacter prevalence reached 100% within the first 3 weeks of summer production under both NAE and CV rearing. A lower Campylobacter prevalence was detected in conventionally raised broilers during fall (P ≤ 0.05), yet no change in prevalence was observed in NAE birds (P > 0.05). Populations were high in the cecum, carrying an average of 6.6 Log10 CFU/g after transportation, and antimicrobial-resistant Campylobacter was isolated from CV broilers during the fall. Three isolates (1.2%), identified as C. coli, carrying the gyrA and tet(O) genes, exhibited simultaneous resistance to ciprofloxacin, tetracycline, and nalidixic acid. Results from this study can help identify important shifts in gut microbial community dynamics and Campylobacter prevalence associated with antibiotic administration within commercial poultry operations.

On-site generated peroxy acetic acid (PAA) technology reduces Salmonella and Campylobacter on chicken wings

Peroxyacetic acid (PAA) is a widely used antimicrobial during poultry processing that requires to be shipped in a concentrated solution, stored under hazardous conditions and diluted for use. On-site PAA generation using nonhazardous ingredients can help eliminate transportation and storage issues at the processing plant and reduce the risk of occupational hazards. The objective of the proposed research was to 1) evaluate the efficacy of on-site generated PAA in reducing Salmonella and Campylobacter populations compared to the commercially available PAA stock solutions and 2) to perform color measurements to evaluate any deviations between treatments. PAA solutions at 50 and 100 ppm were used for treating the chicken wings. Fresh chicken wings (0.45 kg) were inoculated with a cocktail of nalidixic acid resistant Salmonella Typhimurium (STNR) and gentamicin resistant Campylobacter coli (CCGR) and immersed in PAA solutions (50 and 100 ppm) adjusted to pH 8.5 and 10.0 or 10.5, for either 10 s or 60 min. Treated chicken wings were rinsed for 1 min in chilled BPW (100 mL), serially diluted and plated on APC Petrifilm for Salmonella, spread plated on Campy-cefex agar supplemented with gentamicin (200 ppm) for Campylobacter enumeration. Immersion of chicken wings in 100 ppm PAA for 60 min irrespective of pH levels and PAA solutions resulted in greater microbial reductions (P < 0.05) of Salmonella by 1.68 and 1.42 log CFU/mL for SaniDate, 1.82 and 1.83 log CFU/mL for OxyFusion (on-site generated). For the same treatments, Campylobacter reductions of 1.59 and 1.36 log CFU/mL for SaniDate, 1.63 and 1.71 log CFU/mL for OxyFusion were achieved. The antimicrobial efficiency of PAA was not affected by pH and type of PAA solution. No significant differences (P > 0.05) in color were observed between treatments and controls. On-site generated PAA provides poultry processors an effective, safer, and less hazardous alternative to commercially available PAA solutions, ensuring poultry workers’ health and safety.

Antimicrobial interventions to reduce Salmonella and Campylobacter populations and improve shelf life of quail carcasses

Quail (Coturnix japonica) is processed and marketed as fresh meat, with limited shelf life. The objective of this study was to evaluate the efficacy of antimicrobial interventions during slaughter on reducing Salmonella and Campylobacter contamination and to determine the microbiological shelf life of quail during refrigerated (4°C) storage. Three antimicrobials, peracetic acid (400 ppm; PAA), Citrilow (pH 1.2), and Cecure (cetylpyridinium chloride [CPC], 450 ppm), along with a water and no-treatment control were evaluated. Quail carcasses (n = 75) were inoculated with a cocktail of nalidixic acid–resistant Salmonella Typhimurium and gentamicin-resistant Campylobacter coli. After 30 min of attachment time, quail carcasses were submerged in each antimicrobial solution for 20 s with air agitation. Noninoculated quail carcasses (n = 25) were similarly treated, packaged, and stored under refrigeration (4°C). Aerobic plate counts (APC), psychrotroph counts (PC), Enterobacteriaceae counts (ENT), total coliform counts (TCC), and Escherichia coli counts on quail carcasses were determined on 1, 4, 7, and 10 d. Salmonella and Campylobacter populations were determined by plating on Petrifilm APC supplemented with 200-ppm nalidixic acid and Campy Cefex agar supplemented with 200-ppm gentamycin, respectively. No significant reductions in (P > 0.01 log cfu/mL) in APC, PC, ENT, TCC, and E. coli counts were observed on carcasses submerged in water. However, treatments with PAA, Citrilow, and CPC significantly reduced (P ≤ 0.05) Salmonella and Campylobacter coli contamination. Citrilow showed greater (P ≤ 0.05) reduction in Salmonella and Campylobacter population (1.90 and 3.82 log cfu/mL reduction, respectively) to PAA and CPC. Greater (P ≤ 0.05) reductions in APC, PC, ENT, TCC, and E. coli counts (2.22, 1.26, 1.47, 1.52, and 1.59 log cfu/mL, respectively) were obtained with the application of CPC. Application of antimicrobial interventions resulted in a reduction in Campylobacter and Salmonella, APC, PC, and ENT populations after treatments (day 0) and throughout the storage period (day 10). Use of antimicrobial interventions after slaughter can improve the microbiological safety and shelf life of quail.

REDUCTION OF CAMPYLOBACTER IN CHICKEN LIVERS USING A LOW ACID PROCESSING AID

Liver has become a prime source for Campylobacter outbreaks and products are needed to allow processors a more efficient way of controlling foodborne pathogens. Campylobacter reductions in livers treated with a low pH processing aid (CMS PoultrypHresh), with and without a surfactant (PoultrypHresh Plus) were studied. Chicken livers (n=13/treatment group) were individually inoculated with a C. coli marker strain (107) and each dipped into sterile cups containing 100 mL of water, PoultrypHresh or PoultrypHresh Plus for 15 s, removed and allowed 5 s to drain. Each liver was placed into 50 mL buffered peptone water and hand shaken for 60 s; controls (n=10) same procedure, no treatment. Rinsates were serially diluted and plated onto Campy Cefex agar with 200 ppm gentamicin. Plates were incubated for 48 h at 42°C microaerobically, colonies counted and log transformed. Procedures were replicated 3 times. Significant reductions in treated compared to untreated for PoultrypHresh and PoultrypHresh Plus was 98.1% and 99.4%, respectively and with no change in appearance. Treating with this product may allow processors to meet rising performance standards on poultry livers.

REDUCTION OF CAMPYLOBACTER ON POULTRY THIGHS USING SEQUENTIAL TREATMENTS OF ANTIMICROBIALS

Campylobacter is a major concern for poultry processors, as USDA performance standards have become stricter. This study evaluated CMS PoultrypHresh™, a low pH processing aid, and peracetic acid using consecutive and sequential dip treatments to reduce Campylobacter in thighs. Thighs (n=3/treatment group) were inoculated with a C. coli marker strain (108) and each dipped into bags containing 1 L of treatment 1 for 6 s. Thighs were allowed 5 s to drip, placed onto foil for 60 s, and dipped into treatment 2 for 6 s. After 5 s drip time, each was placed in a bag with 150 mL buffered peptone water and hand shaken for 60 s; controls same procedure, no treatment. Rinsates were serially diluted, plated onto Campy Cefex agar with 200 ppm gentamicin and incubated microaerobically for 48 h at 42°C. Procedures were replicated 5 times. Significant reductions compared to untreated using consecutive dips of PoultrypHresh™ and PAA were 98.2% and 99.3%, respectively. Treatments of PoultrypHresh™ then peracetic acid reduced Campylobacter 99.2% from untreated thighs. Peracetic acid then PoultrypHresh™ showed significant reductions compared to all other treatments (99.9% from untreated). Treating with this sequence may allow processors to meet the strict performance standards on Campylobacter in broiler parts.

Efficacy of peroxy acetic acid in reducing Salmonella and Campylobacter spp. populations on chicken breast fillets

Poultry processors use antimicrobials to reduce the risk of pathogens on poultry and poultry products. The efficacy of selective and nonselective plating media to enumerate injured Salmonella (selective media—brilliant green sulfa agar and Petrifilm Enterobacteriaceae Plate Count; nonselective media—tryptic soy agar and Petrifilm Aerobic Plate Count) and Campylobacter (selective medium–Campy cefex agar and nonselective medium—Brucella agar) populations and the efficacy of peroxy acetic acid (PAA) to reduce Salmonella and Campylobacter populations on chicken breast fillets were evaluated. All plating media for Salmonella and Campylobacter contained nalidixic acid (200 ppm) or gentamycin (200 ppm), respectively. Breast fillets were sprayed or immersed in PAA (500 ppm) for 10 min for evaluation of the plating media. Breast fillets inoculated with a mixed Salmonella and Campylobacter cocktail were sprayed (5 or 10 s) or immersed (4–30 s) in PAA (100, 400, 500, or 1,000 ppm) for evaluation of PAA efficacy. Salmonella populations were higher (P ≤ 0.05) when plated on nonselective media compared with the selective media for the non-PAA treated fillets, although the differences in populations were low (<0.32 log CFU/mL). For both the microorganisms, populations on PAA treated (immersion or spray) fillets were similar when enumerated on nonselective or selective media within each treatment (PAA immersion or spray). Both immersion and spray applications reduced (P ≤ 0.05) the Salmonella and Campylobacter populations compared with the control. Increasing the PAA concentration to 250, 500, and 1,000 ppm resulted in greater reductions (P ≤ 0.05) in Salmonella and Campylobacter populations. Immersion of the inoculated breast fillets in 1,000 ppm PAA solution for 30 s resulted in Salmonella and Campylobacter population reductions of 1.92 and 1.87 log CFU/mL, respectively. Method of antimicrobial application (immersion and spray) did not affect the reductions in Salmonella and Campylobacter populations. Either immersion or spray application can be used to improve microbial safety of chicken breast fillets in a poultry processing plant.

Validation of the TadpoleTM Campylobacter jejuni Real-Time PCR Identification Kit.

Background: The gene-based real-time PCR method for identification of Campylobacter jejuni is more simple, rapid and accurate than the traditional biochemical method. Objective: A performance validation of the TadpoleTMCampylobacter jejuni Real-Time PCR Identification Kit was performed. Method: The assay uses TaqMan Real-time PCR technology to amplify target genes from isolated colonies. Bacterial deoxyribonucleic acid (DNA) from inclusivity and exclusivity organisms cultured on Columbia Blood Agar, Campy-Cefex agar and modified Charcoal Cefoperazone Deoxycholate was extracted and analyzed on three instruments: Applied Biosystems (ABI) 7500 Fast, ABI StepOne Plus and Bio-Rad CFX96. Results: When 57 distinct strains of C. jejuni were tested for inclusivity, all 57 strains produced positive results on the three instruments. In exclusivity testing, all 35 strains of related organisms, including 7 non-target Campylobacter strains and other common species, produced negative results on the three instruments. The Independent Laboratory validation consisting of an inclusivity and exclusivity evaluation for 10 C. jejuni isolates and 10 nontarget Campylobacter isolates also showed 100% expected results on the three instruments. In addition, in robustness testing, small, deliberate changes to the assay parameters, including cell suspension turbidity, heat lysis time, and DNA template volume in the PCR reaction, did not affect the kit performance. Finally, the combined lot-to-lot and stability study on both the ABI 7500 Fast and the ABI StepOne Plus showed that the 11 C. jejuni strains and 5 nontarget Campylobacter strains can be correctly identified by the three independently manufactured, lots and it supported a shelf life of 9 months when stored at -20°C. Conclusions: The Tadpole method offers a rapid, accurate, and robust alternative for C. jejuni identification. Highlights: Rapid and accurate method to identify C. jejuni, which has a good robustness and high stability. It is flexible and offers the advantages of reduced labor and time saving.

Multilocus Sequence Subtypes of Campylobacter Detected on the Surface and from Internal Tissues of Retail Chicken Livers

Foodborne campylobacteriosis has been traced to undercooked chicken liver. The objectives of this study were to determine the prevalence of Campylobacter associated with chicken livers at retail and to determine which subtypes are detected on the surface and in the internal tissues of the livers. Fifteen packages of fresh chicken livers, each representing a unique combination of processing plant and sell-by date, were collected at each of three retail grocery stores in Georgia. Three intact, undamaged livers per container ( n = 45) were selected and sampled using each three methods: outside swab, inside swab accessed by pressing through a heat-sterilized outer surface, and whole liver blended in enrichment broth. Each liver sample with 0.1 mL of exudate from packages was cultured for Campylobacter by plating on Campy-Cefex agar. The most prevalent Campylobacter colony type from each positive sample was subjected to whole genome sequencing and multilocus sequence typing. Campylobacter was detected in at least one sample from every package. Surface swabs were positive for 29 of 45 livers, but significantly fewer swabs of internal tissue were positive, 14 of 45 ( P < 0.01). Campylobacter was detected in 30 of 45 blended whole liver samples. Multiple subtypes were detected from eight livers. In four livers, a different subtype was dominant on the surface than was dominant internally. In one liver, three subtypes were detected. Various subtypes of Campylobacter can be readily isolated from fresh retail chicken livers; therefore, undercooked chicken livers pose a food safety risk.

Detecting Campylobacter coli in young chicks using two different cloacal swab techniques

SUMMARY It is important to determine whether valuable broiler-breeder chicks are contaminated with Campylobacter. It is important to have a non-destructive method to determine whether microorganisms such as Campylobacter are present without sacrificing the animal. The objective of this study was to evaluate the reliability of cloacal swabs to detect Campylobacter in young chicks. Day-old chicks (n = 25) were gavaged with 101–3 or 106Campylobacter coli (C. coli) gentamicin-resistant marker strain. Batches of chicks were placed in separate isolation units, and 7, 10, 14, or 21 d post challenge, 10 birds per group were cloacally swabbed shallow (9 mm) and deep (24 mm). Swabs were placed into 5 mL of Tecra® broth, vortexed, and streaked for isolation onto Campy Cefex agar plus 200 ppm of gentamicin. After swabbing, birds were sacrificed and one cecum was quantitatively analyzed for C. coli from the control group; both ceca from all challenged birds were analyzed for C. coli. At 14 d post challenge, 95% of the shallow and 90% of the deep swabs were positive. Even with a low inoculum of 103, C. coli achieved a high degree of cecal colonization, and the cloacal swab (either shallow or deep) proved reliable for detecting C. coli. Birds challenged with >102, after 7 and 14 d were colonized with >106 cells. After 7 d, all shallow and deep swabs were positive for C. coli, regardless of challenge dose. Since it might not be practical in industry to process the swabs the d of collection, we looked at the reliability of cloacal swabs after freezing for up to 21 days. When the level in the ceca was high, recovery of C. coli was excellent, but when the level was low (± 102 inoculum level), recovery was very unreliable. If the levels of Campylobacter are relatively high (log ≥ 6.0) in the ceca, both the shallow and deep swabs, unfrozen or frozen, are reliable, nondestructive methods to detect this microorganism.

Passage of Campylobacter jejuni and Campylobacter coli Subtypes through 0.45- and 0.65-Micrometer-Pore-Size Nitrocellulose Filters

Campylobacter can be difficult to recover from complex samples due to overgrowth by background bacteria. A 0.45- or 0.65-μm-pore-size filter overlaid on agar plates can be used as a means to separate Campylobacter from confounding non-Campylobacter cells, facilitating detection on solid plating media. It is unclear what percentage of cells in a Campylobacter suspension passes through a filter and results in visible colonies. The objective of this study was to compare the number of Campylobacter cells detected by the filter method with those detected by direct plating and determine if the filter method can be used to estimate cellular density of an unknown Campylobacter in suspension. Overnight liquid cultures of six subtypes of Campylobacter jejuni and six of Campylobacter coli, all originally detected in chicken samples, were used for this study. Motility of isolates was tested by using a stab into soft agar, incubating plates, and measuring colony size. Each subtype was applied to Campy-Cefex agar directly and through a 0.45- or 0.65-μm-pore-size filter. Filters were removed, plates were incubated, and colonies were counted; three replications were conducted. Mean recovery by direct plating was 8.3 log CFU/mL. Regardless of pore size, the overall mean number of Campylobacter detected by using the filter method was significantly less than that using direct plating (P < 0.05). The mean difference between direct plating and plating though a 0.65-μm-pore-size filter for motile Campylobacter was log 2.4 CFU/mL, with a 95% confidence interval of ±0.2 log CFU/mL.

Efficacy of Syringe Filtration for the Selective Isolation of Campylobacter from Chicken Carcass Rinse

We investigated the efficacy of syringe filtration for selective isolation of Campylobacter from chicken carcass rinse by combining syringe filtration with the conventional culture method. Whole chicken carcass rinses were incubated in Bolton enrichment broth, set aside or subjected to syringe filtration, and streaked on Campy-Cefex agar with or without cefoperazone antibiotic supplement. Compared with the conventional method without filtration, 0.65-μm-pore-size syringe filtration resulted in a significantly higher number of Campylobacter-positive samples (23.8 to 37.5% versus 70.0 to 72.5%; P < 0.05), a lower number of plates contaminated with non-Campylobacter (93.8% versus 6.3 to 26.3%), and a lower growth index (1 = growth of a few colonies; 2 = growth of colonies on about half of the plate; and 3 = growth on most of the plate) for competing microbiota (2.9 to 3.0 versus 1.2 to 1.4). When syringe filtration was applied, agar plates containing the antibiotic had significantly less contamination (6.3% versus 26.3%; P < 0.05) and a lower growth index (1.2 versus 1.4) compared with plates without the antibiotic, although the Campylobacter isolation rate was similar (P > 0.05). Syringe filtration combined with conventional enrichment improved the rate and selectivity of Campylobacter isolation from chicken carcasses.

Treatment with a low pH processing aid to reduce Campylobacter counts on broiler parts

New regulations and performance standards for Campylobacter have been implemented by the USDA - Food Safety and Inspection Service (FSIS). The objective of this study was to evaluate treatment with a low pH processing aid (CMS1 PoultrypHresh), a formulated low pH processing aid, to reduce numbers of Campylobacter which could help companies meet regulatory requirements. Two experiments (3 replicates each) were conducted. Experiment 1, in each of 3 replicates, skin-on split chicken breasts (n = 15) were obtained from a local grocery and divided into groups of 5. The skin of each part was inoculated with approximately 107 cells of a gentamicin resistant C. coli (CCGR) marker strain in an area of approximately 6.5 cm2. CCGR cells were allowed to attach for 5 min prior to treatment. Ten inoculated breasts were individually placed into separate 6 L plastic storage boxes containing either 3.5 L deionized water or PoultrypHresh solution at a pH of 1.4. Parts were subjected to agitation (bubbled air) for 25 s. After treatment, each part was removed, allowed to drain for 5 s, and placed into a plastic bag prior to mechanical rinsing with 150 mL of buffered peptone water for 60 s. Five inoculated breasts served as controls, were untreated with a dip or agitation and sampled as above. Experiment 2 procedures were repeated using skin-on thighs under the same conditions. Rinsates were collected from each chicken part, serially diluted, and plated onto Campy Cefex agar with 200 ppm gentamicin (CCGen). All plates were incubated microaerobically (5% O2, 10% CO2, 85% N2) for 48 h at 42°C, colonies were counted and the cfu/mL was log transformed. The use of PoultrypHresh on split breast produced a 99.6% reduction compared to untreated controls, while thighs showed a 99.4% reduction. This study demonstrated an approximate 3 log reduction (P < 0.05) using a 25 s air agitation treatment in PoultrypHresh at pH 1.4 with no observable damage, which will help processors meet FSIS regulations.

Campylobacter in broiler slaughter samples assessed by direct count on mCCDA and Campy-Cefex agar.

Campylobacter spp. cause foodborne illnesses in humans primarily through the consumption of contaminated chicken. The aim of this study was to evaluate the United States Department of Agriculture's (USDA) recommended methodology, protocol MLG 41.02, for the isolation, identification and direct plate counting of Campylobacter jejuni and C. coli samples from the broiler slaughtering process. A plating method using both mCCDA and Campy-Cefex agars is recommended to recover Campylobacter cells. It is also possible to use this method in different matrices (cloacal swabs and water samples). Cloacal swabs, samples from pre-chiller and post-chiller carcasses and samples of pre-chiller, chiller and direct supply water were collected each week for four weeks from the same flock at a slaughterhouse located in an abattoir in southern Brazil. Samples were analyzed to directly count Campylobacter spp., and the results showed a high frequency of Campylobacter spp. on Campy-Cefex agar. For the isolated species, 72% were identified as Campylobacter jejuni and 38% as Campylobacter coli. It was possible to count Campylobacter jejuni and Campylobacter coli from different samples, including the water supply samples, using the two-agar method. These results suggest that slaughterhouses can use direct counting methods with both agars and different matrices as a monitoring tool to assess the presence of Campylobacter bacteria in their products.

Method-Dependent Variability in Determination of Prevalence of Campylobacter jejuni and Campylobacter coli in Canadian Retail Poultry

Campylobacter is the most frequent cause of bacterial gastroenteritis in Canada, and the illness is commonly associated with poultry consumption. Whereas Canadian retail poultry is often contaminated with campylobacters, studies on the prevalence of this organism are inconsistent due to variability in sampling and microbiological methodology. To determine the current microbiological status of Canadian poultry, and to evaluate two commonly used microbiological methods, 348 raw poultry samples were collected at retail across Canada over a period of 3 years (2007 to 2010) and were analyzed for the presence of thermophilic Campylobacter species. The overall prevalence of Campylobacter spp. was found to be 42.8% by a combination of the two testing methods, with 33.9% of the samples positive for C. jejuni, 3.7% of the samples positive for C. coli, and 5.2% of the samples positive for both. Variability in Campylobacter spp. prevalence was observed in samples obtained from different regions across Canada and from poultry with or without skin, but this was not statistically significant. In co-contaminated samples, C. jejuni was preferentially recovered from Preston agar compared with mCCDA and Campy-Cefex agar, with an increase in recovery of C. coli on all selective media after 48 h of enrichment. A subset of 214 of the poultry rinses were analyzed by both Health Canada's standard method, MFLP-46 (enrichment in Park and Sanders broth), and a second method requiring enrichment in Bolton broth. Significantly more positive samples were obtained with the MFLP-46 method (40.6%) than with the alternate method (35.0%). This improved recovery with MFLP-46 may be due to the omission of cycloheximide from this method. These results demonstrate that determination of prevalence of Campylobacter spp. on poultry products may be significantly impacted by the choice of microbiological methods used. Canadian poultry continues to be a source of exposure to Campylobacter spp.

A One Year Study of Newly Constructed Broiler Houses for the Prevalence of Campylobacter

3 Abstract: In 2009, the USDA Food Safety and Inspection Service announced the development of ne w pathogen reduction performance standards for Salmonella and Campylobacter both on-farm and in th e processing plant. The objective of this study was to evaluate the prevalence and distribution of Campylobacter in 3 newly constructed broiler houses for the first 4 flocks placed. Litter and fecal samples were collected from each house at 0, 28 and 48 d of production. Samples were serially diluted and spread onto Campy Cefex agar plates. Two 40 mL water samples were collected each production day and filtered through a 0.45 µm membrane before being placed onto a Campy Cefex agar plate. All plates were purged with a microaerophilic gas and incubated for 36 h at 42°C. Individual plates were screened for characteristic Campylobacter colonies and suspect colonies were confirmed using a latex agglutination kit. An additional 50 g of litter was collected from the evaporative cooling inlets, middle and tunnel ventilation fans to determine litter moisture and pH. Inside and outside temperatures were also collected. Out of 2300 litter, 900 fecal and 45 water samples, only 5, 6 and 1 of the collected samples, respectively, were confirmed Campylobacter positive. The middle of the house contained a higher litter moisture level (37%) than the evaporative cooling inlet end (33%) and tunnel ventilation fan end (34%) (p<0.05). Litter pH was not different across days, locations or flocks. Temperature averaged 26.8 C inside and 27.6 C outside. In conclusion, the newly o o

Comparison of 2 types of broths and 3 selective agars for the detection of Campylobacter species in whole-chicken carcass-rinse samples

In this study, we compared the effectiveness of 2 types of Bolton broths and 3 selective media for isolating Campylobacter spp. from naturally contaminated whole-chicken carcass-rinse samples. One hundred chickens were rinsed with buffered peptone water, and the rinses were added to 2× Bolton broth (with or without blood supplementation). The samples were incubated and then streaked onto Preston agar, modified cefoperazone charcoal deoxycholate agar (mCCDA), and Campy-Cefex agar, which was followed by incubation under microaerobic conditions. No statistical differences were observed (P > 0.05) in isolation rate and selectivity between the 2 types of Bolton broths. Among the 3 selective agars, Preston agar yielded a significantly (P < 0.05) better isolation rate and selectivity. The Campy-Cefex agar, which is recommended by many food authorities for its high quantitative detection ability, showed extensive contamination with competing microorganisms and exhibited the lowest isolation rate and selectivity.

Improvement of Modified Charcoal-Cefoperazone-Deoxycholate Agar by Supplementation with a High Concentration of Polymyxin B for Detection of Campylobacter jejuni and C. coli in Chicken Carcass Rinses

Modified charcoal-cefoperazone-deoxycholate agar (mCCDA) was improved by supplementation with a high concentration of polymyxin B. The ability of the supplemented medium to isolate Campylobacter jejuni and C. coli from chicken carcass rinses was compared to that of Campy-Cefex agar and mCCDA. Modification of mCCDA with increased polymyxin B yielded a significantly (P < 0.05) higher isolation rate and greater selectivity than those achieved using Campy-Cefex agar and mCCDA.

In vitro Selection of Enteric Microflora for Potential Use as a Competitive Exclusion Culture Against Campylobacter in Poultry

3 Abstract: The administration of nonpathogenic microflora in neonatal poultry has been employed to reduce or eliminate the colonization of enteric pathogens. This concept, also called Competitive Exclusion (CE), although effective against Salmonella, has not consistently worked against Campylobacter. Most CE cultures are developed by randomly collecting enteric bacteria without any preselection criteria for bacteria. It may be possible to enhance the efficacy of a CE against Campylobacter by preselecting enteric microflora with the ability to inhibit Campylobacter, in vitro. With this goal, an assay was developed to test individual isolates with the ability to reduce or eliminate Campylobacter growth, in vitro . Individual isolates (n = 137) were collected from ceca of both juvenile and adult poultry and tested for efficacy against Campylobacter. Isolates were serially diluted (10 or 10 CFU/well) and added to 96 well polystyrene plates containing 1 x 10 CFU 4 5 4 C. jejuni or C. coli /well. Plates were incubated at 42°C in a microaerophilic environment for 24-48 h. Following incubation, a 1 µl loop from each well was streaked onto Campy-Cefex agar plate and incubated at 42°C in a microaerophilic environment for 24-48 h. Twenty-three isolates were identified with the ability to inhibit C. jejuni or C. coli growth in vitro . This research demonstrates that it is possible to pre-scree n enteric isolates for Campylobacter inhibition for use as competitive exclusion cultures.

Evaluating 3 gas-delivery systems for culturing Campylobacter jejuni in a microaerophilic environment

Campylobacter spp. require a microaerophilic environment (80% N(2), 10% CO(2), 5% H(2), and 5% O(2)) for growth. Since the late 1800s, several systems for creating and maintaining specific microbial atmospheres have been developed and applied. The objective of this study was to evaluate Campylobacter jejuni growth by means of 3 commonly used gas-delivery systems for generating a microaerophilic environment: automated, gas-generating sachet, and plastic storage bag. Pure culture C. jejuni cells were suspended in Brucella broth and spread onto campy cefex agar plates. For the automated gas-delivery system, plates were positioned in a Mart anaerobic jar and flushed with a microaerophilic gas mixture using an Anoxomat Mart II system (Mart Microbiology B. V., Netherlands). For the sachet samples, plates were placed in a Mart anaerobic jar and 3 Gaspak EZ campy sachets (Becton Dickinson and Company, Franklin Lakes, NJ) were activated to induce a microaerophilic gas environment. The plates placed in plastic storage bags were flushed with a microaerophilic gas mixture from a premixed tank. For all 3 systems, plates were placed in a low-temperature incubator at 42°C for 24 h. After 24 h, plates were removed from the incubator and colonies were counted. The entire experiment was repeated 5 times. Results indicated no significant difference in colony counts among the gas-delivery systems tested, but colonies grown under the sachet-generated environment were smaller than colonies in the other 2 methods. Smaller colonies could have resulted from the type of media used or the length of time the plates were incubated. In conclusion, all 3 gas-delivery methods were able to produce similar Campylobacter growth results. Initial and long-term costs of equipment, as well as laboratory space availability, may be influential when choosing a gas-delivery method for generating a microaerophilic environment.

Evaluation of different selective media and culturing techniques for the quantification of Campylobacter ssp. from broiler litter

Poultry is a major reservoir for Campylobacter, the leading cause of foodborne illness in the United States, but how broilers become initially colonized is still under debate. Broiler litter is a potential source, but the best technique for quantifying Campylobacter from litter is still unknown. Therefore, our objectives were to determine if certain media are more selective for quantifying Campylobacter and if enrichment allows for the detection of stressed or viable but nonculturable cells from broiler litter samples. In this trial, 5 media and 2 culturing techniques were used to enumerate Campylobacter from broiler litter. The media used were campy-Line agar (CLA), campy-cefex agar (CCA), modified CCA, Campylobacter agar plates (CAP), and modified charcoal cefoperazone deoxycholate agar. Litter samples were obtained from a commercial broiler house. Each sample was equally divided and diluted 10-fold into peptone, for direct plating, or 4-fold into Campylobacter enrichment broth. Samples diluted in peptone were direct-plated onto each media and incubated under microaerophilic conditions for 48 h at 42 degrees C. Samples diluted in enrichment broth were incubated under the same conditions for 24 h, then further diluted to 10-fold before plating. Plates from enriched samples were incubated for an additional 24 h after plating. After incubation, all plates (direct and enriched) were counted and presumptive positive colonies were confirmed using a Campylobacter latex agglutination kit. Results indicated that there was no difference in the ability of any of the selective media tested to grow Campylobacter. Direct-plated samples had a higher Campylobacter isolation rate compared with enriched samples. The CLA and CAP were able to suppress total bacterial growth better than modified charcoal cefoperazone deoxycholate, modified CCA, and CCA. The CLA and CAP were the only media able to detect total bacterial population shifts over time. In conclusion, it is important before making a final decision on a selective medium to consider the medium's ability to suppress total bacterial growth as well as isolate Campylobacter.