Variation in Campylobacter Multilocus Sequence Typing Subtypes from Chickens as Detected on Three Plating Media.

The objective of this study was to compare subtypes of Campylobacter jejuni and Campylobacter coli detected on three selective Campylobacter plating media to determine whether each medium selected for different subtypes. Fifty ceca and 50 carcasses (representing 50 flocks) were collected from the evisceration line in a commercial broiler processing plant. Campylobacter was cultured and isolated from cecal contents and carcass rinses on Campy-Cefex, Campy Line, and RF Campylobacter jejuni/coli agars. When a positive result was obtained with all three media, one colony of the most prevalent morphology on each medium was selected for further analysis by full genome sequencing and multilocus sequence typing. Sequence types were assigned according to PubMLST. A total of 49 samples were positive for Campylobacter on all three media. Forty samples contained only C. jejuni , three had only C. coli , and both species were detected in six samples. From 71% of samples, Campylobacter isolates of the same sequence type were recovered on all three media. From 81.6% of samples, isolates were all from the same clonal complex. From significantly fewer samples (26%, P < 0.01), one medium recovered an isolate with a sequence type different from the type recovered on the other two media. When multiple sequence types were detected, six times the medium with the odd sequence type was Campy-Cefex, four times it was Campy-Line, and six times it was RF Campylobacter jejuni/coli . From one sample, three sequence types were detected. In most cases, all three plating media allowed detection of the same type of Campylobacter from complex naturally contaminated chicken samples.

Proposed model for the high rate of rearrangement and rapid migration observed in some IncA/C plasmid lineages.

IncA/C plasmids are a class of plasmids from the Enterobacteriaceae that are relatively large (49 to >180 kbp), that are readily transferred by conjugation, and that carry multiple antimicrobial resistance genes. Reconstruction of the phylogeny of these plasmids has been difficult because of the high rate of remodeling by recombination-mediated horizontal gene transfer (HGT). We hypothesized that evaluation of nucleotide polymorphisms relative to the rate of HGT would help to develop a clock to show whether anthropic practices have had significant influences on the lineages of the plasmid. A system was developed to rapidly sequence up to 191 known open reading frames from each of 39 recently isolated IncA/C plasmids from a diverse panel of Salmonella enterica and Escherichia coli strains. With these data plus sequences from GenBank, we were able to distinguish six distinct lineages that had extremely low numbers of polymorphisms within each lineage, especially among the largest group designated as group 1. Two regions, each about half the plasmid in size, could be distinguished with a separate lineal pattern. The distribution of group 1 showed that it has migrated extremely rapidly with fewer polymorphisms than can be expected in 2,000 years. Remodeling by frequent HGT was evident, with a pattern that appeared to have the highest rate just upstream of the putative conjugation origin of transfer (oriT). It seems likely that when an IncA/C plasmid is transferred by conjugation there is an opportunity for plasmid remodeling adjacent to the oriT, which was also adjacent to a multiple antimicrobial resistance gene cassette.

Comparison of cumulative drip sampling with whole carcass rinses for estimation of Campylobacter species and quality indicator organisms associated with processed broiler chickens.

The whole carcass rinse (WCR) procedure is routinely used as a sampling method for determining the presence and number of quality-indicator organisms or pathogens associated with broiler chicken carcasses in processing facilities. Collection of a cumulative drip sample by placing collection vessels under the processing line could potentially capture a more representative sample of bacterial populations associated with an entire flock with less labor than individual bird rinses. The purpose of this study was to evaluate a cumulative drip sampling method for recovery of Campylobacter spp. and 3 types of quality indicator organisms from broiler carcasses. Cumulative drip and WCR samples were collected on 14 d from a commercial broiler processing facility over a 3-mo period. No statistically significant difference was demonstrated between the WCR and cumulative drip sampling methods in recovery of Campylobacter spp., total aerobes, Enterobacteriaceae, or Escherichia coli associated with the postevisceration samples (P > 0.01). Analysis of the pyrosequencing census data demonstrated high interbird variability and indicates cumulative sampling may be required to obtain fully representative sampling of a flock. For most bacterial taxa, the relative abundance in individual WCR was correlated with cumulative drip samples, but some taxa were undercounted or missed entirely by individual WCR. Consequently, individual carcass rinses may not be representative of the flock microbial community. The cumulative drip sampling technique may save labor and provide a more representative summary of process control in poultry processing facilities.

Comparison of an automated most-probable-number technique with traditional plating methods for estimating populations of total aerobes, coliforms, and Escherichia coli associated with freshly processed broiler chickens.

An instrument (TEMPO) has been developed to automate the most-probable-number (MPN) technique and reduce the effort required to estimate some bacterial populations. We compared the automated MPN technique with traditional microbiological plating methods and Petrifilm methods for estimating the total viable count of aerobic microorganisms (TVC), total coliforms (CC), and Escherichia coli populations (EC) on freshly processed broiler chicken carcasses (postchill whole carcass rinse [WCR] samples) and cumulative drip-line samples from a commercial broiler processing facility. Overall, 120 broiler carcasses, 36 prechill drip-line samples, and 40 postchill drip-line samples were collected over 5 days (representing five individual flocks) and analyzed by the automated MPN and direct agar plating and Petrifilm methods. The TVC correlation coefficient between the automated MPN and traditional methods was very high (0.972) for the prechill drip samples, which had mean log-transformed values of 3.09 and 3.02, respectively. The TVC correlation coefficient was lower (0.710) for the postchill WCR samples, which had lower mean log values of 1.53 and 1.31, respectively. Correlations between the methods for the prechill CC and EC samples were 0.812 and 0.880, respectively. The estimated number of total aerobes was generally greater than the total number of coliforms or E. coli recovered for all sample types (P < 2e⁻¹6). Significantly more bacteria were recovered from the prechill samples than from the postchill WCR or cumulative drip samples (P < 9.5e⁻¹² and P < 2e⁻¹6, respectively). When samples below the limit of detection were excluded, 92.1% of the total responses were within a single log difference between the traditional plating or Petrifilm methods and the automated MPN method.