Phenotypic, genotypic and proteomic variations between poor and robust colonizing Campylobacter jejuni strains.

Campylobacter jejuni is one of the major causes of bacterial gastrointestinal disease in humans worldwide. This foodborne pathogen colonizes the intestinal tracts of chickens, and consumption of chicken and poultry products is identified as a common route of transmission. We analyzed two C. jejuni strains after oral challenge with 105 CFU/ml of C. jejuni per chick; one strain was a robust colonizer (A74/C) and the other a poor colonizer (A74/O). We also found extensive phenotypic differences in growth rate, biofilm production, and in vitro adherence, invasion, intracellular survival, and transcytosis. Strains A74/C and A74/O were genotypically similar with respect to their whole genome alignment, core genome, and ribosomal MLST, MLST, flaA, porA, and PFGE typing. The global proteomes of the two congenic strains were quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and 618 and 453 proteins were identified from A74/C and A74/O isolates, respectively. Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that carbon metabolism and motility proteins were distinctively overexpressed in strain A74/C. The robust colonizer also exhibited a unique proteome profile characterized by significantly increased expression of proteins linked to adhesion, invasion, chemotaxis, energy, protein synthesis, heat shock proteins, iron regulation, two-component regulatory systems, and multidrug efflux pump. Our study underlines phenotypic, genotypic, and proteomic variations of the poor and robust colonizing C. jejuni strains, suggesting that several factors may contribute to mediating the different colonization potentials of the isogenic isolates.

Draft Genome Sequences of Two Campylobacter jejuni Strains That Show Significantly Different Colonization Potentials in Chickens.

Here, we report the draft genome sequences of robust (A74/C_24-3) and poor (A74/O_2-2) chicken-colonizing Campylobacter jejuni isolates. Whole-genome sequence analyses of these isolates will be helpful in facilitating further studies to identify genetic factors used in chicken colonization.

Molecular characterization of podoviral bacteriophages virulent for Clostridium perfringens and their comparison with members of the Picovirinae.

Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ΦCP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage Φ29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae.

Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase and novel internal amplification controls for rapid and specific detection.

Although Campylobacter is an important food-borne human pathogen, there remains a lack of molecular diagnostic assays that are simple to use, cost-effective, and provide rapid results in research, clinical, or regulatory laboratories. Of the numerous Campylobacter assays that do exist, to our knowledge none has been empirically tested for specificity using high-throughput sequencing. Here we demonstrate the power of next-generation sequencing to determine the specificity of a widely cited Campylobacter-specific polymerase chain reaction (PCR) assay and describe a rapid method for direct cell suspension PCR to quickly and easily screen samples for Campylobacter. We present a specific protocol which eliminates the need for time-consuming and expensive genomic DNA extractions and, using a high-processivity polymerase, demonstrate conclusive screening of samples in <1 h. Pyrosequencing results show the assay to be extremely (>99%) sensitive, and spike-back experiments demonstrated a detection threshold of <10(2) CFU mL(-1). Additionally, we present 2 newly designed broad-range bacterial primer sets targeting the 23S rRNA gene that have wide applicability as internal amplification controls. Empirical testing of putative taxon-specific assays using high-throughput sequencing is an important validation step that is now financially feasible for research, regulatory, or clinical applications.

Application of high-throughput sequencing to measure the performance of commonly used selective cultivation methods for the foodborne pathogen Campylobacter.

Campylobacter is an important foodborne human pathogen, which has traditionally been studied using a variety of selective cultivation methods. Here we use next-generation sequencing to ask the following: (i) how selective are commonly used Campylobacter cultivation methods relative to the initial sample and (ii) how do the specificity and sensitivity of these methods compare with one another? To answer these questions, we used 16S rRNA tagged-pyrosequencing to sequence directly from a pooled fecal sample representing a c. 16,000 bird poultry flock and compared these data to exhaustive sequencing of colonies formed after plating. We compared five commonly used media [Cefex, Cape Town, modified cefoperazone charcoal deoxycholate agar (mCCDA), Campy-Line agar (CLA), and Campy-CVA agar (CVA)], two incubation atmospheres (10% CO(2), 5% O(2), 85% N(2) and 10% CO(2), 10% H(2), 80% N(2)), and two incubation temperatures (37 and 42 °C). Analysis of 404,104 total sequence reads, including 19 472 total fecal reads, revealed Campylobacter represented only a small proportion (< 0.04%) of sequences present in the feces, but 88-97% of sequences from each media type. Incubation atmosphere had little effect on recovery, but a significant difference in media specificity (more non-Campylobacter OTUs; P = 0.028) was found at 42 vs. 37 °C. The most common non-Campylobacter sequence type was Proteus, which ranged from 0.04% of sequences (mCCDA) to 10.8% (Cape Town). High-throughput sequencing provides a novel and powerful approach to measure the performance of selective media, which remain widely used for research and regulatory purposes.

Enumeration of Campylobacter spp., Escherichia coli, and Salmonella in broiler carcass rinses before and after simulated transport in artificial ice for 24 hours.

The maintenance and survival of target pathogens during transport from the field collection site to the analytical laboratory is essential for obtaining accurate and reliable data. This study was conducted to compare the efficacy of sterile tap water (SW), buffered peptone water (BPW), and universal preenrichment broth (UP) for maintaining populations of Campylobacter spp., Salmonella, and Escherichia coli for 24 h under simulated transport conditions. Freshly processed broiler carcasses (n = 100) were rinsed in SW. The rinses were divided, and components were added to create equal volumes of rinse samples consisting of SW, BPW, and UP. The rinses were analyzed for the target organisms immediately and again after 24 h of simulated chilled transport conditions. The only meaningful difference between the different transport media was found for UP, which recovered fewer E. coli than did either SW or BPW. These findings support the conclusion that either SW or BPW should be used as a broiler carcass rinse and/or transport medium to accurately depict the levels or presence of these three target bacteria as a whole. Because potable water differs in pH and hardness across the United States, a follow-up study was conducted to investigate whether water hardness or pH within the ranges normally found across the United States would affect Campylobacter recovery from carcass rinses. No significant differences were detected.

Development of a selective broth medium for the detection of injured Campylobacter jejuni by capacitance monitoring.

The purpose of these studies was to develop a conductimetric method for the rapid detection of Campylobacter jejuni. Numerous basal medium components were analyzed to develop a growth-enhancing broth medium for detection of freeze-injured Campylobacter cells using a conductimetric system. The final medium was composed of a modified Campy-Line agar from which the agar and triphenyltetrazolium chloride were removed and the amino acid, L-arginine was added. Pure isolates of C. jejuni. (frozen and thawed to produce stressed cells) were utilized to test the detection methodology. Monitoring of significant changes in the capacitance signal was found suitable for detection of Campylobacter proliferation. Using stressed pure cultures, Campylobacter growth was repeatedly detected at very low inoculum levels (about one cell per well). There was a direct linear relationship between detection times (DTs) and the initial inoculum level. For example, using a single strain, the mean DT (n = 20) at the 10 CFU/ml inoculum level was 28.6 h, with 100% of the inoculated wells detecting. The mean DTs at the 100, 1,000, and 10,000 CFU/ml inoculum levels were 24.9, 21.4, and 17.0 h, respectively. This study demonstrates that conductimetric methods can be utilized for the rapid detection of C. jejuni.

Comparison of Three Methods for Recovery of Campylobacter spp. from Broiler Carcasses.

We compared three enrichment methods, with different sampling times (mid-and endpoint incubation) and various dilutions of enrichment culture for productivity in the isolation of Campylobacter spp. from 50 retail-level chicken carcasses. We subcultured the enrichment cultures onto three Campylobacter spp.-selective media (Campy-BAP, CCDA, Campy-Cefex) to compare yields of the organism. The highest yield (43/50) of Campylobacter spp. from these carcasses was derived by using the 24-h enrichment culture of Hunt & Radle diluted 1:100 before plating onto any of the three selective plating media. When all carcass analyses were combined, Campylobacter spp. was recovered from 49 of 50 broilers. This study indicates the optimum approaches for the recovery of Campylobacter spp., as well as the high incidence of the organism among the broiler carcasses tested.

Lethality of Heat to Escherichia coli 0157:H7: D-Value and Z-Value Determinations in Ground Beef.

D-values and z-values were determined for lean (2.0% fat) and fatty (30.5% fat) ground beef inoculated with approximately 107 Escherichia coli 0157:H7 cells per g. Inoculated ground meat was sealed in glass thermal death time tubes which were completely immersed in a circulating water bath and held at prescribed temperatures for predetermined lengths of time. Survival was determined by enumeration on plate count agar (PCA) containing 1% sodium pyruvate and by the 2-h indole test of Lee and McClain (7). D-values for fatty ground beef exceeded those for lean ground beef at the temperatures tested. D-values for lean and fatty ground beef at 125°F were 78.2 and 115.5 min, respectively, as enumerated on PCA plus pyruvate. D-values at 135°F were 4.1 and 5.3 min for lean and fatty beef. At 145°F D-values were determined to be 0.3 and 0.5 min. D-values calculated from 2-h indole test data for lean and fatty ground beef at 125°F were 80.1 and 121.0 min, respectively. D-values at 135°F were 4.0 and 7.4 min for lean and fatty beef and at 145°F a D-value of 0.2 min was calculated for lean beef only, due to insufficient survival of E. coli 0157:H7 in fatty beef at this temperature. The z-values determined for lean beef and fatty beef using PCA were 8.3 and 8.4°F respectively. The z-value for lean beef using the 2-h indole data was 7.8°F. No z-value for fatty beef using 2-h indole data could be determined.

Lethality of Heat to Listeria monocytogenes Scott A: D-Value and Z-Value Determinations in Ground Beef and Turkey.

D-Values and z-values for Listeria monocytogenes strain Scott A were determined in lean (2.0% fat) and fatty (30.5%) ground beef inoculated with approximately 107cells/g. Inoculated ground meat was sealed in glass thermal death time tubes which were completely immersed in a circulating water bath and held at prescribed temperatures for predetermined lengths of time. Survival was determined by enumeration on Columbia CNA agar base containing 1% sodium pyruvate with a CNA + 4% horse blood overlay (CBNA) and on Listeria Plating Medium (LPM). D-values for L. monocytogenes in lean and fatty ground beef at 125° were 81.3 and 71.1 min, respectively, as enumerated on CBNA plus pyruvate. D-values at 135°F were 2.6 and 5.8 min in lean and fatty beef. At 145°F, D-values were determined to be 0.6 and 1.2 min. D-values calculated from LPM recovery data from fatty ground beef at 125°F were 56.1 and 34.5 min, respectively. D-values at 135°F were 2.4 and 4.6 min in lean and fatty beef. At 145°F a D-value of 0.5 min was calculated in lean beef and a D-value of 1.1 min was determined in fatty beef. The z-values determined in lean beef and fatty beef using CBNA recovery data were 9.3 and 11.4°F, respectively. The z-value in lean beef using LPM recovery data was 9.8°F. The z-value in fatty beef using LPM recovery data was 13.2°F. A D-value for ground turkey meat at 160°F could not be determined under the conditions of this study. Problems encountered are discussed.