Several methods have been developed to generate microaerobic conditions for the growth and multiplication of Campylobacter spp. These methods are routine and are consistently used during the enrichment of food samples or during the incubation of inoculated plate media. However, little is known about the actual changes in O2 content in enrichment broth media during incubation (37°C or 42°C). Our experiments were aimed at determining the changes of O2 content in the broth and in the air of the head space of the bags used to enrich the samples for the isolation of Campylobacter from retail broiler meat. The premises of this work was that the incubation of enrichment broth may naturally create microaerobiosis conducive to the grow of Campylobacter spp. Samples were therefore divided in two subsamples which were in turn incubated under microaerobic conditions (M) or aerobic conditions (A).
We used an unpaired sample design, where the enrichment conditions differ between the reference (subsamples M) and the alternative method (subsamples A), and confirmed all presumptive positives using the same molecular protocols. Because the comparison of two qualitative methods is best accomplished near the limit of detection of these methods, we used naturally contaminated broiler meat samples, which have the lowest contamination that can be naturally found [4; 17]. The statistical analyses of data from unpaired samples are performed in the same way as for paired samples, mainly using McNemar's chi square test . The number of Campylobacter positive subsamples was statistically similar between subsamples M and A, and all isolates were clearly identified as C. jejuni or C. coli. These results demonstrate that enrichment broths incubated under normal, aerobic conditions are sufficient to detect Campylobacter spp. in retail broiler meat. There was an increase in number of total positive samples by 10% when combining the result of the two subsamples. These findings have been already reported several times for commercial broiler meat naturally contaminated with Campylobacter spp. [4; 17]. In addition, a ROC curve of the data showed a high true positive fraction, or rate, and a very low false positive fraction, which indicated a very strong correspondence in the results between the reference (subsamples M) and the alternative methods (subsamples A).
The traditional methodology of enriching 25 g of meat is the one suggested by the Bacteriological Analytical Manual of the Food and Drug Administration (FDA) , the Microbiology Laboratory Guidebook of the Food Safety and Inspection Services of the U. S. Department of Agriculture (FSIS UDSA) , the International Organization for Standardization , the Health Protection Agency of the UK , and several other countries' regulatory agencies. However, this methodology does not appear to be optimized to detect the true prevalence of Campylobacter spp. in retail broiler meat. PCR analysis of the isolates showed that C. jejuni or C. coli species are the only Campylobacter spp. found in retail broiler meat. Some samples can be contaminated with both species  but again the current methodology used in food samples is not accurate enough to reveal the extent of contamination of the same product with different Campylobacter strains. PFGE analysis further demonstrated that a single meat sample could be contaminated with two, or maybe more, isolates from the same species. For all practical purposes, C. jejuni and C. coli are the only two Campylobacter spp. found in retail poultry meat because no C. lari has been identified since the introduction of molecular techniques for routine identification of Campylobacter isolates, approximately 15 years ago . The data collected with the O2 sensors showed that the amount of O2 in the enrichment broth was stable around 5-7 ppm after 6 h of enrichment. These O2 levels can be obtained by pressing out the air before closing the sample bags, and without the need of any vacuum, as is required when removing the air from a hard container. Whirl-Pak or ziplock bags performed similarly, showing that they are impervious to changes in the air trapped inside . The fact that bags with only the enrichment broth (without meat or blood) created microaerobic conditions has encouraged us to continue this line of research, and we are currently testing other broths without blood to isolate Campylobacter spp. from retail broiler meat. Therefore, an inexpensive, simplified method can be developed for routinely use in the isolation and detection of Campylobacter spp. from food products.
Incubation of broth under normal aerobic conditions, with or without airspace, was done in the early 1980s to isolate Campylobacter spp. from fecal samples , and the use of 10% O2, 10% CO2 and 80% of N2 facilitated and sustained the growth of Campylobacter spp. . The ISO normative 10272-1:2006 requires a microaerobic environment but provides for an alternative incubation in a microaerobic atmosphere created by "screw-capped bottles or flasks filled with enrichment broth, leaving a headspace of less than 2 cm, and tightly closing the caps" . But much has been speculated about the need to have a higher surface area of the meat samples during enrichment to yield a higher number of positive samples under microaerobic conditions , or the exact depth of the airspace, the appropriate ratio of air to broth , and the correct type of incubation container to promote the growth of Campylobacter jejuni  to avoid significant difference in the results if a microaerobic atmosphere is not used . Therefore, the microaerobic conditions are routinely used to isolate Campylobacter spp. However, our results do not suggest any correlation between surface and microaerobic conditions and do not support the notion that air to broth ratio and the type of container are indispensable to isolate Campylobacter spp. Our results point to the simple fact that any closed plastic bag naturally produces microaerobic environments conducive to the growth of Campylobacter spp. without the need to add any microaerobic gas mix. In our experiments, bags were closed to leave a minimum airspace and the samples were mixed, without stomaching, for few seconds. Thus, bags with subsamples M had the same contact surface as bags with subsamples A.
The microbial population of the enriched samples in Bolton broth, as assessed by RISA and DGGE, was diverse. There are no current data on the microbial assemblage of retail broiler meat as a predictor to the presence of a bacterial pathogen, such as Campylobacter. Most of the work on the bacterial community of broiler meat was done more than 20 years ago using direct bacterial counts, and very few research studies have used culture-independent methods to study the microbial profile of these foods . It is known, however, that some cold-tolerant bacteria, such as Enterobacteriaceae, Acinetobacter and Pseudomonas, are commonly present on broiler meat . These bacteria are primarily facultative anaerobes or microaerobic organisms, and the ribosomal RNA gene sequences recovered in our samples, especially form the most prominent bands from DGGE gels, had a high similarity to these bacterial groups.
RISA and DGGE can be used to broadly characterize the total microbial population in complex samples. The results from these techniques were analyzed using the Pearson correlation, which is the standard procedure for comparison of densitometric curves [31; 32]. We analyzed the results with the Pearson correlation and also the Dice coefficient, which takes into account only the band position and not the band thickness, as it is the case in densitometric curves. Although the Dice correlation showed a higher DNA relatedness among corresponding M and A subsamples, the variability in the bacterial populations in each set of subsamples was still large and appeared to be more attributable to the original bacterial composition of the sampled meat itself than to the enrichment conditions (aerobic vs. microaerobic). A significant limitation of DGGE-derived phylogenetic data with the primers used in this study is the relatively short rDNA sequence obtained from each amplicon, thereby reducing the degree of phylogenetic inference that may be assigned to each band. Yet, both RISA and DGGE produced consistent results regarding the variability in the bacterial assemblages associated with retail broiler meat samples.