Human Norovirus Surrogates Persist in Nontraditional Sources of Irrigation Water in Excess of 100 Days.

Human norovirus (HuNoV) has been implicated as the leading cause of foodborne illness worldwide. The ability of HuNoV to persist in water can significantly impact food safety as agriculture and processing water could serve as vehicles of virus transmission. This study focused on the persistence and infectivity of the HuNoV surrogate viruses, murine norovirus (MNV), and Tulane virus (TV), after prolonged storage in diverse environmental water types currently used for agricultural irrigation. In this study, vegetable processing water (VW), brackish tidal surface water (SW), municipal reclaimed water (RW), and pond water (PW) were inoculated with each virus in a 1:10 v/v ratio containing virus at 3.5-4.5 logPFU/mL and stored at 16°C for 100 days. This time and temperature combination was chosen to mimic growing and harvest conditions in the mid-Atlantic area of the United States. Samples were then assayed for the presence of viral RNA using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) approximately weekly throughout the study. Persistence of MNV and TV was not significantly different (p > 0.05) from one another in any water sample (n = 7) or the control (HBSS). However, there was variability observed in viral persistence across water samples with significant differences observed between several water samples. The presence of intact viral capsids enclosing the genomes of MNV and TV were evaluated by an RNase assay coupled with RT-qPCR on specific timepoints and determined to be intact up to and at 100 days after inoculation. TV was also shown to remain infectious in a cell culture assay (TCID50) up to 100 days of incubation. These findings are significant in that the potential for not only detection of enteric viruses can occur long after a contamination event occurs but these viruses may also remain infectious.

Effect of sodium bisulfate amendments on bacterial populations in broiler litter.

The accumulation of ammonia in poultry houses is of concern to bird and human health. Acidification of the litter by application of acidifying amendments such as sodium bisulfate (SBS) retains ammonia generated by microbial degradation of uric acid as harmless ammonium in the litter. Although some studies on the effects of litter amendments on specific bacteria and groups of bacteria have been carried out previously, wide gaps in knowledge remain. In the present study, 2 types of samples were prepared and either left unamended or amended with 2.5 or 10% SBS. One set of samples consisted of a 1:1 mixture of built-up litter and fresh poultry manure (L/M); the other of fresh wood shavings and fresh poultry manure (S/M). The samples were kept in the laboratory at room temperature for 35 d. The pH of unamended mixtures increased to 7.3 and 6.9 for L/M and S/M, respectively. A pH of 6.7 and 3.9 on day 35 was observed for L/M and SM with 2.5% SBS, respectively. The corresponding values for LM and SM amended with 10% SBS were 3.5 and 2.5, respectively. Plating data indicated that coliforms became less numerous in the unamended samples than the SBS-amended samples. This difference was also seen in data obtained by high-throughput sequencing of 16S rDNA. The sequencing data also indicated that sequences from the genus Oceanisphaera accounted for as much as 80% of the sequences from L/M and about 40% of those from S/M samples early on. Sequences from members of the order Clostridiales were enriched in L/M and S/M amended with 10% SBS as were sequences from the genus Turicibacter. Weisella species sequences were more prevalent in SBS-amended samples than in unamended ones. Sequences from the genus Corynebacterium, Brachybacterium, and Arthrobacter were more common in L/M samples than in S/M samples regardless of the SBS content. The data indicate that litter amendments affect some bacteria populations and not others. Further studies are required to determine if the observed population changes such as increased survival of coliforms warrant actions to improve the microbial quality of litter to be reused.

Salmonella enterica's "Choice": Itaconic Acid Degradation or Bacteriocin Immunity Genes.

Itaconic acid is an immunoregulatory metabolite produced by macrophages in response to pathogen invasion. It also exhibits antibacterial activity because it is an uncompetitive inhibitor of isocitrate lyase, whose activity is required for the glyoxylate shunt to be operational. Some bacteria, such as Yersinia pestis, encode enzymes that can degrade itaconic acid and therefore eliminate this metabolic inhibitor. Studies, primarily with Salmonella enterica subspecies enterica serovar Typhimurium, have demonstrated the presence of similar genes in this pathogen and the importance of these genes for the persistence of the pathogen in murine hosts. This minireview demonstrates that, based on Blast searches of 1063 complete Salmonella genome sequences, not all Salmonella serovars possess these genes. It is also shown that the growth of Salmonella isolates that do not possess these genes is sensitive to the acid under glucose-limiting conditions. Interestingly, most of the serovars without the three genes, including serovar Typhi, harbor DNA at the corresponding genomic location that encodes two open reading frames that are similar to bacteriocin immunity genes. It is hypothesized that these genes could be important for Salmonella that finds itself in strong competition with other Enterobacteriacea in the intestinal tract-for example, during inflammation.

Sugar sulfates are not hydrolyzed by the acid-inducible sulfatase AslA from Salmonella enterica Enteritidis NalR and Kentucky 3795 at pH 5.5.

The open reading frames SEN0085 and SeKA_A4361, from Salmonella enterica serovar Enteritidis NalR and serovar Kentucky 3795, respectively, corresponding to the acid-inducible sulfatase gene aslA from Salmonella enterica serovar Typhimurium, were previously suggested by microarray analysis to be differentially expressed under acid conditions. However, growth and enzyme activity tests in the present study demonstrated that both wild-type strains exhibited sulfatase activity with 4-nitrophenyl sulfate and 5-bromo-4-chloro-3 indolyl sulfate at pH 5.5. The acid sulfatase does not appear to be involved in sugar sulfate, tyrosine sulfate, 4-hydroxy-3-methoxyphenylglycol sulfate, heparin sulfate, or chondroitin sulfate hydrolysis at pH 5.5. Adhesion and invasion assays did not reveal differences between the serotypes and their corresponding aslA deletion mutants. Thus, the role and substrate(s) of AslA, a protein unique to salmonella and encoded in all sequenced Salmonella strains, remain elusive.

Current Status of the Preharvest Application of Pro- and Prebiotics to Farm Animals to Enhance the Microbial Safety of Animal Products.

The selection of microorganisms that act as probiotics and feed additives that act as prebiotics is an ongoing research effort, but a sizable range of commercial pro-, pre- and synbiotic (combining pro- and prebiotics) products are already available and being used on farms. A survey of the composition of commercial products available in the United States revealed that Lactobacillus acidophilus, Enterococcus faecium, and Bacillus subtilis were the three most common species in probiotic products. Of the nearly 130 probiotic products (also called direct-fed microbials) for which information was available, about 50 also contained yeasts or molds. The focus on these particular bacteria and eukaryotes is due to long-standing ideas about the benefits of such strains, research data on effectiveness primarily in laboratory or research farm settings, and regulations that dictate which microorganisms or feed additives can be administered to farm animals. Of the direct-fed microbials, only six made a claim relating to food safety or competitive exclusion of pathogens. None of the approximately 50 prebiotic products mentioned food safety in their descriptions. The remainder emphasized enhancement of animal performance such as weight gain or overall animal health. The reason why so few products carry food safety-related claims is the difficulties in establishing unambiguous cause and effect relationships between the application of such products in varied and constantly changing farm environments and improved food safety of the end product.

Contribution of the hdeB-like gene (SEN1493) to survival of Salmonella enterica enteritidis Nal(R) at pH 2.

Periplasmic proteins are particularly vulnerable to denaturation upon entry into a highly acid environment. In Escherichia coli, a level of protection of these proteins is afforded by acid-inducible chaperonins encoded by hdeAB. In contrast, Salmonella enterica only harbors an hdeB-like gene and it is currently not known what function it plays in this genus. In the present study, the hdeB-like gene was deleted in Salmonella enterica Enteritidis Nal(R) and Salmonella enterica Kentucky 3795. When grown overnight in tryptic soy broth (TSB) medium buffered at pH 5.5 and then exposed to TSB pH 2 for 20 min, Enteritidis wild-type strain experienced a 0.5-log10 reduction in colony-forming units, whereas the deletion strain's surviving cells were reduced by 1.6 log10. No difference in survival was observed in the corresponding Salmonella enterica Kentucky 3795 strains treated the same way. Exposure of the strains to pH 2.5 or 3 resulted in the same log reduction regardless of the presence of the hdeB-like gene. When wild-type and deletion strains of both serovars were grown in medium buffered at pH 7 prior to exposure to the acidic pHs, no difference in survival with respect to serovar or presence/absence of the hdeB-like gene was found. Salmonella enterica Enteritidis Nal(R) carrying its own or the intragenic region upstream of the hdeB-like from Salmonella enterica Kentucky 3795 cloned in front of the gfp gene from pFPV25 showed maximum fluorescence when grown at pH 5.5, whereas the corresponding plasmid-carrying Salmonella enterica Kentucky strains did not exhibit fluorescence regardless of the pH of the growth medium. Therefore, the hdeB-like gene in Salmonella enterica Enteritidis, but not in Salmonella enterica Kentucky 3795, contributed to survival at pH 2 and its expression is responsive to the pH of the medium.

Gene expression analysis of Salmonella enterica Enteritidis Nal(R) and Salmonella enterica Kentucky 3795 exposed to HCl and acetic acid in rich medium.

In the United States, serovar Kentucky has become one of the most frequently isolated Salmonella enterica serovars from chickens. The reasons for this prevalence are not well understood. Phenotypic comparisons of poultry Salmonella isolates belonging to various serovars demonstrated that serovar Kentucky isolates differed from those of most other serovars in their response to acid. Microarray and qPCR analyses were performed with aerated exponentially growing poultry isolates, Salmonella enterica serovar Kentucky 3795 and Enteritidis Nal(R), exposed for 10 min to tryptic soy broth (TSB) adjusted to pH 4.5 with HCl and to pH 5.5 with HCl or acetic acid. Data obtained by microarray analysis indicated that more genes were up- or down-regulated in strain Kentucky 3795 than in Enteritidis Nal(R) under acidic conditions. Acid exposure in general caused up-regulation of energy metabolism genes and down-regulation of protein synthesis genes, particularly of ribosomal protein genes. Both strains appear to similarly utilize the lysine-based pH homeostasis system, as up-regulation of cadB was observed under the acidic conditions. Expression of regulatory genes (rpoS, fur, phoPQ) known to be involved in the acid response showed similar trends in both isolates. Differences between Kentucky 3795 and Enteritidis Nal(R) were observed with respect to the expression of the hdeB-like locus SEN1493 (potentially encoding a chaperone important to acid response), and some differences in the expression of other genes such as those involved in citrate utilization and motility were noted. It appears that the early stages of the transcriptional response to acid by isolates Kentucky 3795 and Enteritidis Nal(R) are similar, but differences exist in the scope and in some facets of the response. Possibly, the quantitative differences observed might lead to differences in protein levels that could explain the observed differences in the acid phenotype of serovar Kentucky and other Salmonella serovars.

Gene expression profiling of a pressure-tolerant Listeria monocytogenes Scott A ctsR deletion mutant.

Listeria monocytogenes is a food-borne pathogen of significant threat to public health. High hydrostatic pressure (HHP) treatment can be used to control Listeria monocytogenes in food. The CtsR (class three stress gene repressor) protein negatively regulates the expression of class III heat shock genes. A spontaneous pressure-tolerant ctsR mutant 2-1 that was able to survive under HHP treatment has been identified previously. So far, there is only limited information about the mechanisms of survival and adaptation of this mutant to high pressure. Microarray technology was used to monitor the gene expression profiles of the ctsR mutant 2-1 under HHP treatment. Compared to pressure-treated L. monocytogenes Scott A wild type, 17 genes were up-regulated (>2-fold increase) in the ctsR mutant 2-1, whereas 58 genes were down-regulated (<-2-fold decrease). The entire clpC operon was up-regulated in the ctsR mutant 2-1, indicating that the mutant CtsR protein was not a functional repressor. The increased levels of expression of stress-related genes in ctsR mutant 2-1 may contribute to its survival under high pressure. The reduced expression levels of the genes related to virulence, flagella synthesis, and cell division in the ctsR mutant 2-1 correlate with its characteristics (elongated cells, reduced virulence, and absence of flagella). The gene expression changes determined by microarray assays were confirmed by real-time reverse transcriptase PCR analyses. This study enhances our understanding of how Listeria monocytogenes survives under HHP and may contribute to the design of effective and economically feasible HHP treatment in food processing.

Presence of arsenic resistance in Salmonella enterica serovar Kentucky and other serovars isolated from poultry.

A collection of 125 Salmonella enterica poultry isolates (71 serovar Kentucky isolates, and the remainder belonging to serovars Alachua, Enteritidis, Hadar, Heidelberg, Montevideo, Mbandaka, Senftenberg, Typhimurium, and Worthington) were tested for the ability to grow on tryptic soy agar containing sodium arsenite [As(III)] or arsenate [As(V)]. All serovar Kentucky isolates and 18 of the non-Kentucky isolates were able to grow in the presence of 0.1 mM As(III), and 69 grew in the presence of 1 mM As(V). Thirty of the non-Kentucky isolates did not grow at these As(III) and As(V) concentrations, but seven grew at 1 mM As(III) and 10 mM As(V). PCR-based analysis demonstrated the presence of arsB and arsD sequences in all Kentucky isolates, whereas one or both of these sequences were present in only 30 of the other isolates. It remains to be determined if these arsenic-resistance determinants benefit Salmonella exposed to man-made arsenic-containing compounds in poultry environments.

Comparison of genetic and physiological properties of Salmonella enterica isolates from chickens reveals one major difference between serovar Kentucky and other serovars: response to acid.

For unknown reasons, Salmonella enterica Kentucky has become the serovar most frequently isolated from chickens and chicken carcasses in the United States. In an attempt to identify traits that may underlie this phenomenon, genetic and physiological features of 30 serovar Kentucky chicken isolates were compared with those of chicken isolates belonging to a range of other S. enterica serovars. Most of the well-known Salmonella virulence genes were detected in the serovar Kentucky isolates by PCR, but the cdtB, spvB, spvC, and pefA genes were not found. The serovar Kentucky isolates were as invasive as the non-Kentucky isolates in in vitro assays involving chicken embryo hepatocytes, but were less invasive than the Enteritidis, Mbandaki, and Typhimurium isolates when incubated with human HCT-8 cells. Statistical comparison of growth, biofilm formation, and stress survival data from the serovar Kentucky and those from the serovar Enteritidis, Hadar, Mbandaka, Senftenberg, Typhimurium, and Worthington isolates demonstrated either no differences or differences with only a few of the serovars; however, three data sets were different. These data sets included the OD(600) values of cultures grown in tryptic soy broth (TSB) adjusted to pH 5.5 with acetic acid and survival counts of cells grown in either TSB pH 7 or TSB adjusted to pH 5.5 with acetic acid and then transferred into TSB adjusted to pH 2.5 with HCl. Most notable was the log(10) reduction for acetic acid pre-exposed Kentucky isolates of 3.1 versus <1 log(10) for the other isolates upon transfer to pH 2.5. The connection, if any, between this acid response phenotype and the prevalence of the serovar Kentucky in poultry remains to be elucidated, but it is possible that slightly better growth in the presence of acetic acid in conjunction with not mounting a strong, energy-consuming acetic acid-induced adaptive acid response provides a small competitive advantage to this serovar in low acid environments such as the cecum where the pH is around 5.5.

Differences in pressure tolerance of Listeria monocytogenes strains are not correlated with other stress tolerances and are not based on differences in CtsR.

Thirty strains of Listeria monocytogenes were screened for their pressure tolerance phenotype at 400 MPa for 2 min at 21 degrees C. The strains exhibited reductions ranging from 1.9 to 7.1 log(10)CFU/ml in tryptic soy broth with 6% yeast extract (TSBYE). The 3 most and the 3 least pressure-tolerant strains were further tested for their thermal resistance (based on their ability to survive at 55 degrees C), acid tolerance (based on their ability to survive in acidified TSBYE; pH 2.0) and for their nisin sensitivity. No correlation between pressure tolerance and heat, acid or nisin resistances was found. Nucleotide sequence analysis of the ctsR region in these 6 strains demonstrated that this gene codes for a CtsR protein with identical predicted amino acid sequences. The sequences of the 200-bp region located immediately upstream of the ctsR start codon of the different strains were virtually identical and it is therefore likely that differences in pressure tolerance are based on factors other than the stress gene regulator CtsR. The pressure sensitivity of a cocktail of the 2 most pressure-resistant strains and a cocktail of the 2 most-sensitive strains was investigated when the cocktails were inoculated into a real food system consisting of ground chicken meat. We demonstrated that the nature of the suspending substrate or the temperature did not change the expected pressure tolerance of the cocktails.

Use of nisin-coated plastic films to control Listeria monocytogenes on vacuum-packaged cold-smoked salmon.

Cold-smoked (Salmo salar) salmon samples were surface-inoculated with a cocktail of three nisin-resistant strains of L. monocytogenes (PSU1, PSU2 and PSU21) to a level of approximately 5 x 10(2) or 5 x 10(5) CFU/cm2 of salmon surface. The inoculated smoked salmon samples were vacuum-packaged with control film (no nisin) or nisin-coated plastic films and stored at either 4 or 10 degrees C. When the inoculated smoked salmon samples were packaged with film coated with 2000 IU/cm2 of nisin, a reduction of 3.9 log CFU/cm2 (compared with control) was achieved at either temperature for samples inoculated with 5 x 10(2) CFU/cm(2 of L. monocytogenes after 56 (4 degrees C) and 49 (10 degrees C) days of storage while reductions of 2.4 and 0.7 log CFU/cm2 were achieved for samples inoculated with a high level of L. monocytogenes (5 x 10(5) CFU/cm2) after 58 (4 degrees C) and 43 (10 degrees C) days, respectively. For samples packaged in film coated with 500 IU/cm2 of nisin, reductions of 0.5 and 1.7 log CFU/cm2 were achieved for samples inoculated with a low level of L. monocytogenes (5 x 10(2) CFU/cm2) after 56 (4 degrees C) and 49 (10 degrees C) days of storage while reductions of 1.8 and 0.8 log CFU/cm2 were achieved for samples inoculated with high level of L. monocytogenes after 58(4 degrees C) and 43 (10 degrees C) days, respectively. In addition, nisin inhibited the proliferation of background microbiota on smoked salmon in a concentration-dependent manner at both storage temperatures although the bacteriostatic effect was more pronounced at refrigeration temperature. This work highlights the potential for incorporating nisin into plastic films for enhancing the microbial safety of smoked salmon as well as controlling its microbial spoilage.

Characterization of a spontaneous, pressure-tolerant Listeria monocytogenes Scott A ctsR deletion mutant.

A spontaneous, pressure-tolerant mutant of Listeria monocytogenes Scott A, designated 2-1, was isolated after several rounds of pressure treatments at 500 MPa for 10 min. Mutant 2-1 was almost 100,000-fold more resistant than the wild type to a pressure of 350 MPa, and about 100-fold more resistant to 450 MPa when pressurized in growth medium. Approximately ten times more mutant cells than wild-type cells survived a 20-min exposure to 55 degrees C, and the mutant appears also to be more resistant to 0.2% H(2)O(2), although the difference could not be confirmed statistically. About 10 times more wild-type than mutant cells survived exposure to growth medium adjusted to pH 2.5 with HCl. The mutant is about 16-fold more sensitive to nisin than the wild type. Mutant 2-1 is non-motile, produces hemolytic activity, is able to grow in fetal calf serum as well as the wild type, and exhibits a lower level of invasiveness of human ileocecal adenocarcinoma cells than the wild type. The mutation in strain 2-1 is a deletion in the ctsR gene that results in the predicted production of truncated CtsR of 20 amino acids compared to a CtsR of 152 amino acids in the wild type. With the exception of its response to pH and possibly also to H(2)O(2), mutant 2-1 shares most of the phenotypes of the previously described ctsR mutant, AK01. The isolation of another spontaneous, pressure-resistant ctsR mutant confirms the central role of this regulatory gene in pressure tolerance of L. monocytogenes. Although such mutants appear of lesser concern to human health then the wild type, current detection methods for Listeria monocytogenes are not able to distinguish between these mutants and wildtype cells.

Pressure inactivation kinetics of phage lambda cI 857.

Inactivation curves of phage lambda cI 857 inactivated by high hydrostatic pressure were obtained at three pressure levels (300, 350, and 400 MPa) in buffered media and ultrahigh-temperature 2% reduced fat milk. Pressurization of phage lambda in buffered media at 300 MPa for 300 min, 350 MPa for 36 min, and 400 MPa for 8 min reduced the titer of phage lambda by 7.5, 6.7, and 7.7 log, respectively. Pressurization of phage lambda in milk at 300 MPa for 400 min, 350 MPa for 80 min, and 400 MPa for 20 min reduced the titer of phage lambda by 5.4, 6.4, and 7.1 log, respectively. Tailing was observed in all inactivation curves, indicating that the linear model was not adequate for describing these curves. Among the three nonlinear models studied, the Weibull and log-logistic models consistently produced best fits to all inactivation curves, and the modified Gompertz model the poorest. Because there were no significant differences in the values of shape factor (n) for suspension medium buffer, we reduced the number of parameters in the Weibull model from two to one by setting n at the mean value. The simplified Weibull model produced a fit comparable to the full model. Additionally, the simplified Weibull model allowed predictions to be made at pressures different from the experimental pressures. Menstruum was found to significantly affect the pressure resistance of phage lambda. Comparison of pressure inactivation of hepatitis A virus and phage lambda indicated that phage lambda is more sensitive to pressure than hepatitis A virus in Dulbecco's modified Eagle medium with 10% fetal bovine sera.

Survey of retail alfalfa sprouts and mushrooms for the presence of Escherichia coil O157:H7, Salmonella, and Listeria with BAX, and evaluation of this polymerase chain reaction-based system with experimentally contaminated samples.

BAX, a polymerase chain reaction (PCR)-based pathogen detection system, was used to survey retail sprouts and mushrooms for contamination with Escherichia coli O157:H7, Salmonella, Listeria spp., and Listeria monocytogenes. No Salmonella or E. coli O157:H7 was detected in the 202 mushroom and 206 alfalfa sprout samples screened. L. monocytogenes was detected in one sprout sample, and seven additional sprout samples tested positive for the genus Listeria. BAX also detected Listeria species in 17 of the mushroom samples. Only 6 of 850 PCR assays (0.7%) failed to amplify control DNA, and therefore reagent failures and the inhibition of PCR by plant compounds were rare. The sensitivity of the detection system was evaluated by assaying samples inoculated with 10 CFU of each of the pathogens. One hundred seventy-two alfalfa sprout samples were inoculated with E. coli O157:H7, and two sets of 130 samples were experimentally contaminated with Salmonella Enteritidis and L. monocytogenes. The frequency of detection depended on the protocols used for inoculation and culturing. Inoculation of samples with approximately 10 CFU from frozen stocks yielded detection rates of 87.5 and 94.5% for L. monocylogenes and Salmonella Enteritidis, respectively, in mushrooms. The corresponding rates for alfalfa sprouts were 94.5 and 76.3%. The E. coli O157:H7 detection rate was 100% for mushrooms but only 48.6% for sprouts when standard BAX culture protocols were used. The substitution of an overnight incubation in modified E. coli medium for the 3-h brain heart infusion incubation increased the rate of E. coli O157:H7 detection to 75% for experimentally contaminated sprouts. The detection rate was 100% when E. coli O157:H7 cells from a fresh overnight culture were used for the inoculation. Test sensitivity is therefore influenced by the type of produce involved and is probably related to the growth of pathogens in the resuscitation and enrichment media.

16S rRNA-based analysis of microbiota from the cecum of broiler chickens.

The microbiota of the intestinal tract of chickens plays an important role in inhibiting the establishment of intestinal pathogens. Earlier culturing and microscopic examinations indicated that only a fraction of the bacteria in the cecum of chickens could be grown in the laboratory. Therefore, a survey of cecal bacteria was done by retrieval of 16S rRNA gene sequences from DNA isolated from the cecal content and the cecal mucosa. The ribosomal gene sequences were amplified with universal primers and cloned or subjected to temporal temperature gradient gel electrophoresis (TTGE). Partial 16S rRNA gene sequences were determined from the clones and from the major bands in TTGE gels. A total of 1,656 partial 16S rRNA gene sequences were obtained and compared to sequences in the GenBank. The comparison indicated that 243 different sequences were present in the samples. Overall, sequences representing 50 phylogenetic groups or subgroups of bacteria were found, but approximately 89% of the sequences represented just four phylogenetic groups (Clostridium leptum, Sporomusa sp., Clostridium coccoides, and enterics). Sequences of members of the Bacteroides group, the Bifidobacterium infantis subgroup, and of Pseudomonas sp. each accounted for less than 2% of the total. Sequences related to those from the Escherichia sp. subgroup and from Lactobacillus, Pseudomonas, and Bifidobacterium spp. were generally between 98 and 100% identical to sequences already deposited in the GenBank. Sequences most closely related to those of the other bacteria were generally 97% or less identical to those in the databases and therefore might be from currently unknown species. TTGE and random cloning indicated that certain phylogenetic subgroups were common to all birds analyzed, but sequence data from random cloning also provided evidence for qualitative and quantitative differences among the cecal microbiota of individual birds reared under very similar conditions.