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Inflammatory bowel disease (IBD) is associated with an altered gut microbiota composition, including expansion of Proteobacteria . Many species in this phylum are thought to contribute to disease under certain conditions, including adherent-invasive Escherichia coli (AIEC) strains, which are enriched in some patients.

The common marmoset (Callithrix jacchus) is an omnivorous New World primate whose diet in the wild includes large amounts of fruit, seeds, flowers, and a variety of lizards and invertebrates. Marmosets also feed heavily on tree gums and exudates, and they have evolved unique morphological and anatomical characteristics to facilitate gum feeding (gummivory). In this study, we characterized the fecal microbiomes of adult and infant animals from a captive population of common marmosets at the Callitrichid Research Center at the University of Nebraska at Omaha under their normal dietary and environmental conditions. The microbiomes of adult animals were dominated by species of Bifidobacterium, Bacteroides, Prevotella, Phascolarctobacterium, Megamonas, and Megasphaera. Culturing and genomic analysis of the Bifidobacterium populations from adult animals identified four known marmoset-associated species (B. reuteri, B. aesculapii, B. myosotis, and B. hapali) and three unclassified taxa of Bifidobacterium that are phylogenetically distinct. Species-specific quantitative PCR (qPCR) confirmed that these same species of Bifidobacterium are abundant members of the microbiome throughout the lives of the animals. Genomic loci in each Bifidobacterium species encode enzymes to support growth and major marmoset milk oligosaccharides during breastfeeding; however, metabolic islands that can support growth on complex polysaccharide substrates in the diets of captive adults (pectin, xyloglucan, and xylan), including loci in B. aesculapii that can support its unique ability to grow on arabinogalactan-rich tree gums, were species-specific. IMPORTANCE Bifidobacterium species are recognized as important, beneficial microbes in the human gut microbiome, and their ability colonize individuals at different stages of life is influenced by host, dietary, environmental, and ecological factors, which is poorly understood. The common marmoset is an emerging nonhuman primate model with a short maturation period, making this model amenable to study the microbiome throughout a life history. Features of the microbiome in captive marmosets are also shared with human gut microbiomes, including abundant populations of Bifidobacterium species. Our studies show that several species of Bifidobacterium are dominant members of the captive marmoset microbiome throughout their life history. Metabolic capacities in genomes of the marmoset Bifidobacterium species suggest species-specific adaptations to different components of the captive marmoset diet, including the unique capacity in B. aesculapii for degradation of gum arabic, suggesting that regular dietary exposure in captivity may be important for preserving gum-degrading species in the microbiome.

This study addressed a knowledge gap about longitudinal changes in the gut microbiome metabolites during animal pairing. This research in the laboratory common marmoset can control for the confounding factors such as diet and other environmental conditions. Phascolarctobacterium showed the highest contribution to the sex-biased transmission of the female to the male after pairing. Here, we observed the sex difference in the increase in short-chain fatty acid concentration in the feces of newly paired marmosets, which may be caused by the sex-biased gut microbiome transmission after pairing., The role by which the gut microbiome influences host health (e.g., energy equilibrium and immune system) may be partly mediated by short-chain fatty acids, which are bacterial fermentation products from the dietary fibers. However, little is known about longitudinal changes in gut microbiome metabolites during cohabitation alongside social contact. In common marmosets (Callithrix jacchus), the gut microbiome community is influenced by social contact, as newly paired males and females develop convergent microbial profiles. Here, we monitored the dynamics of short-chain fatty acid concentrations in common marmoset feces from the prepairing (PRE) to postpairing (POST) stages. In males, we observed that the concentrations of acetate, propionate, isobutyrate, and isovalerate significantly increased in the POST stage compared to the PRE stage. However, no significant changes were found in females. We further found that the propionate concentration was significantly positively correlated with the abundance of Phascolarctobacterium in the male feces. Thus, the sex difference in the changes in the concentrations of short-chain fatty acids might be related to sex-biased gut microbiome transmission after pairing. We suggest that the significant changes in the gut microbiomes and some short-chain fatty acids of the common marmoset during cohabitation may contribute to physiological homeostasis during pairing. IMPORTANCE This study addressed a knowledge gap about longitudinal changes in the gut microbiome metabolites during animal pairing. This research in the laboratory common marmoset can control for the confounding factors such as diet and other environmental conditions. Phascolarctobacterium showed the highest contribution to the sex-biased transmission of the female to the male after pairing. Here, we observed the sex difference in the increase in short-chain fatty acid concentration in the feces of newly paired marmosets, which may be caused by the sex-biased gut microbiome transmission after pairing.

In this controlled study, we collected longitudinal fecal samples from 16 male and female marmoset monkeys for 2 weeks prior to and for 8 weeks after pairing in male-female dyads. We report for the first time that marmoset monkeys undergo significant changes to the gut microbiome following pairing and that these changes are sex-biased; i.e., females transmit more microbes to their social partners than males do. Marmosets exhibit pair bonding behavior such as spatial proximity, physical contact, and grooming, and sex biases in these behavioral patterns may contribute to the observed sex bias in social transmission of gut microbiomes., Social behavior can alter the microbiome composition via transmission among social partners, but there have been few controlled experimental studies of gut microbiome transmission among social partners in primates. We collected longitudinal fecal samples from eight unrelated male-female pairs of marmoset monkeys prior to pairing and for 8 weeks following pairing. We then sequenced 16S rRNA to characterize the changes in the gut microbiome that resulted from the pairing. Marmoset pairs had a higher similarity in gut microbiome communities after pairing than before pairing. We discovered sex differences in the degrees of change in gut microbiome communities following pairing. Specifically, the gut microbiome communities in males exhibited greater dissimilarity from the prepairing stage (baseline) than the gut microbiome communities in females. Conversely, females showed a gradual stabilization in the rate of the gut microbiome community turnover. Importantly, we found that the male fecal samples harbored more female-source gut microbes after pairing, especially early in pairing (paired test, P

The Nebraska Sandhills region contains over 1,500 geochemically diverse interdunal lakes, some of which are potassium rich, alkaline, and hypersaline. Here, we report 16S rRNA amplicon pyrosequencing data on the water and sediment microbial communities of eight alkaline lakes in the Sandhills of western Nebraska.

The LEE pathogenicity island has been acquired on multiple occasions within the different lineages of enteropathogenic and enterohemorrhagic Escherichia coli . In each lineage, LEE expression is regulated by complex networks of pathways, including core pathways shared by all lineages and lineage-specific pathways. Within the O157:H7 lineage of enterohemorrhagic E. coli , strain-to-strain variation in LEE expression has been observed, implying that expression patterns can diversify even within highly related subpopulations. Using comparative genomics of E. coli O157:H7 subpopulations, we have identified one source of strain-level variation affecting LEE expression. The variation occurs in prophage-dense regions of the genome that lie immediately adjacent to the late regions of the pch prophage carrying pchA, pchB, pchC , and a newly identified pch gene, pchX . Genomic segments extending from the holin S region to the pchA, pchB, pchC , and pchX genes of their respective prophage are highly conserved but are nonetheless embedded within adjacent genomic segments that are extraordinarily variable, termed pch adjacent genomic regions ( pch AGR). Despite the remarkable degree of variation, the pattern of variation in pch AGR is highly correlated with the distribution of phylogenetic markers on the backbone of the genome. Quantitative analysis of transcription from the LEE1 promoter further revealed that variation in the pch AGR has substantial effects on absolute levels and patterns of LEE1 transcription. Variation in the pch AGR therefore serves as a mechanism to diversify LEE expression patterns, and the lineage-specific pattern of pch AGR variation could ultimately influence ecological or virulence characteristics of subpopulations within each lineage.

In this study, the association between genotypic and selected phenotypic characteristics was examined in a collection of Canadian Escherichia coli O157:H7 strains isolated from humans and cattle in the provinces of Alberta, Ontario, Saskatchewan, and Quebec. In a subset of 69 strains selected on the basis of specific phage types (PTs), a strong correlation between the lineage-specific polymorphism assay (LSPA6) genotype and PT was observed with all strains of PTs 4, 14, 21, 31, 33, and 87 belonging to the LSPA6 lineage I (LSPA6-LI) genotype, while those of PTs 23, 45, 67, and 74 belonged to LSPA6 lineage II (LSPA6-LII) genotypes. This correlation was maintained when additional strains of each PT were tested. E. coli O157:H7 strains with the LSPA6-LI genotype were much more common in the collection than were the LSPA6-LII or lineage I/II (LSPA6-LI/II)-related genotypes (82.6, 11.2, and 5.8%, respectively). Of the strains tested, proportionately more LSPA6-LI than LSPA6-LII genotype strains were isolated from humans (52.7% versus 19.7%) than from cattle (47.8% versus 80.2%). In addition, 96.7% of the LSPA6-LII strains carried the stx 2c variant gene, while only 50.0% of LSPA6-LI/II and 2.7% of LSPA6-LI strains carried this gene. LSPA6-LII strains were also significantly more likely to possess the colicin D gene, cda (50.8% versus 23.2%), and have combined resistance to streptomycin, sulfisoxazole, and tetracycline (72.1% versus 0.9%) than were LSPA6-LI strains. The LSPA6 genotype- and PT-related characteristics identified may be important markers of specific ecotypes of E. coli O157:H7 that have unique epidemiological and virulence characteristics.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a major cause of both large-scale epidemics and sporadic cases of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in many countries around the world (13, 34, 36, 54, 56). The annual incidence of reported E. coli O157:H7 infections in Canada and the United States ranges from 1.7 to 5.3 per 100,000 persons and may be much higher in certain regions (14, 56). Within the United States alone, it has been estimated that approximately 73,000 cases of E. coli O157:H7 infection occur annually (33). The most common E. coli O157:H7 isolates are motile, non-sorbitol fermenting (SOR−), and β-glucuronidase negative (GUD−), while a nonmotile SOR+ GUD+ O157:H− clone has also been isolated in Germany (20) and a nonmotile SOR− GUD− O157:H− clone is commonly isolated in Australia (46). Population genetic analysis has shown that E. coli O157:H7 and O157:H− isolates belong to a geographically disseminated clone complex that acquired virulence genes independently from other EHEC isolates (31, 45, 61, 62). Despite the clonal nature of E. coli O157:H7 and O157:H− isolates, significant variability was observed when they were tested by high-resolution genomic typing methods, such as pulsed field gel electrophoresis and octamer-based genome scanning (OBGS) (12, 22, 23, 53), implying that subpopulations are diverging quite rapidly. OBGS is a large-scale genome comparison method based on pattern analysis of PCR amplification products generated using overrepresented octamers as primers. Recent studies using OBGS suggest that extant populations of O157:H7 isolates have diverged through two primary lineages, lineage I and lineage II, and that these lineages can be detected in geographically unlinked regions, such as the United States and Australia (22, 23). The origin of these two lineages, therefore, appears to predate the geographical spread of E. coli O157:H7 and the regional evolution of the SOR− GUD− O157:H− clone commonly isolated in Australia (23). More recently, the lineage-specific polymorphism assay (LSPA-6) was developed, based on six loci that show bias in their allelic distribution between the two lineages. The LSPA-6 is therefore a more efficient alternative for inferring lineage assignments compared to laborious OBGS typing (63). The two methods were demonstrated to generate highly concordant data (63). All lineage I isolates were LSPA-6 genotype 111111 (lineage I allele at each locus), while the majority of lineage II isolates were LSPA-6 genotypes 222222, 211111, and 212111. In the initial OBGS studies and in the LSPA-6 study, a low proportion of human strains were observed in OBGS lineage II and LSPA-6 genotype 222222, respectively (22, 63). The paucity of OBGS lineage II and LSPA-6 genotype 222222 human isolates led workers to postulate that these E. coli O157:H7 isolates may be deficient in their abilities either to be transmitted to humans or to cause clinically significant human infections (22, 63). Several other studies also suggest that there are clear differences in the expression of virulence attributes, such as Shiga toxin and the locus for enterocyte effacement (LEE) proteins, by E. coli O157:H7 isolates from humans and from cattle (27, 32, 47, 48). These latter studies, however, did not consider the population structure of E. coli O157:H7 (e.g., lineage of descent) as a variable. In this study, suppression subtractive hybridization (SSH) was used to identify genomic regions present in E. coli O157:H7 lineage I (LSPA-6 111111) strains but absent from lineage II (LSPA-6 222222) strains. We show that lineage I strains do indeed share a set of unique genes that are largely absent in lineage II strains. Several of these genes encode proteins that could contribute to virulence characteristics or which are known to regulate expression of virulence genes.

The growth and activity of some Lactobacillus and Bifidobacterium strains are stimulated by the presence of nondigestible fructooligosaccharides (FOS), which are selectively fermented by specific intestinal bacteria. Consumption of FOS, therefore, enriches for those bacteria that possess metabolic pathways necessary for FOS metabolism. In this study, a DNA microarray consisting of 7,680 random genomic library fragments of Lactobacillus paracasei 1195 was used to examine genes involved in the utilization of FOS in this organism. Differential expression profiles between cells grown on FOS and those grown on glucose provided a basis for identifying genes specifically induced by FOS. Several of the FOS-induced genes shared sequence identity with genes encoding β-fructosidases and components of phosphoenolpyruvate-dependent phosphotransferase systems (PTS). These genes were organized in a putative operon, designated the fos operon, that may play an essential role in FOS utilization. The complete 7,631-bp nucleotide sequence of the putative fos operon was determined and consists of fosABCDXE genes, which encode a putative fructose/mannose PTS (FosABCDX) and a β-fructosidase precursor (FosE). The latter contains an N-terminal signal peptide sequence and cell wall sorting signals at the C-terminal region, suggesting its localization at the cell wall. Inactivation of the fosE gene led to impaired growth on FOS and other β-fructose-linked carbohydrates. Transcriptional analysis by reverse transcriptase PCR suggested that fosABCDXE was cotranscribed as a single mRNA during growth on FOS. Expression array analysis revealed that when glucose was added to FOS-grown cells, transcription of the FOS-induced genes was repressed, indicating that FOS metabolism is subject to catabolite regulation.

Comparative genome hybridization of the Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica populations have shown that genome content is highly conserved, with relatively few genes in the F. tularensis subsp. tularensis genome being absent in other F. tularensis subspecies. To determine if organization of the genome differs between global populations of F. tularensis subsp. tularensis and F. tularensis subsp. holarctica , we have used paired-end sequence mapping (PESM) to identify regions of the genome where synteny is broken. The PESM approach compares the physical distances between paired-end sequencing reads of a library of a wild-type reference F. tularensis subsp. holarctica strain to the predicted lengths between the reads based on map coordinates of two different F. tularensis genome sequences. A total of 17 different continuous regions were identified in the F. tularensis subsp. holarctica genome (CR holar c tica ) which are noncontiguous in the F. tularensis subsp. tularensis genome. Six of the 17 different CR holarctica are positioned as adjacent pairs in the F. tularensis subsp. tularensis genome sequence but are translocated in F. tularensis subsp. holarctica , implying that their arrangements are ancestral in F. tularensis subsp. tularensis and derived in F. tularensis subsp. holarctica . PCR analysis of the CR holarctica in 88 additional F. tularensis subsp. tularensis and F. tularensis subsp. holarctica isolates showed that the arrangements of the CR holarctica are highly conserved, particularly in F. tularensis subsp. holarctica , consistent with the hypothesis that global populations of F. tularensis subsp. holarctica have recently experienced a periodic selection event or they have emerged from a recent clonal expansion. Two unique F. tularensis subsp. tularensis -like strains were also observed which likely are derived from evolutionary intermediates and may represent a new taxonomic unit.

Listeria monocytogenes strains belonging to phylogenetic lineage II (serotypes 1/2a, 1/2c, and 3a) carry a lineage-specific genome segment encoding a putative sigma subunit of RNA polymerase (lmo0423, herein referred to as sigC), a gene of unknown function (lmo0422) similar to the padR family of regulators, and a gene that is similar to the rodA-ftsW family of cell wall morphology genes (lmo0421). To understand the function of this set of genes, their expression patterns and the effects of null mutations in the lineage II L. monocytogenes strain 10403S were examined. The data are consistent with the three genes comprising an operon (the sigC operon) that is highly induced by temperature upshift. The operon is transcribed from three different promoters, the proximal of which (P1) depends upon sigC itself. Null mutations in sigC or lmo0422 increase the death rate at lethal temperatures and cause loss of thermal adaptive response, whereas the lmo0421 mutation causes only a loss of the adaptive response component. Only the sigC mutation affects transcription from the P1 promoter, whereas ectopic expression of lmo0422 from the PSPAC promoter complements the individual lmo0422 and sigC null mutations, showing that lmo0422 is the actual thermal resistance regulator or effector while sigC provides a mechanism for temperature-dependent transcription of lmo0422 from P1. Our genetic and phylogenetic analyses are consistent with lmo0422—renamed lstR (for lineage-specific thermal regulator)—and sigC comprising a system of thermal resistance that was ancestral to the genus Listeria and was subsequently lost during divergence of the lineage I L. monocytogenes population. Although only a relatively small number of cases of human listeriosis occur each year in the United States (estimated at 2,500 per year), the associated rates of morbidity and mortality are substantial, with nearly 30% mortality in some outbreaks (40, 58). Because the causative agent of the disease, Listeria monocytogenes, is ubiquitous in nature and possesses durable physiological characteristics, the organism is one of the most significant food safety problems in the food production industry.

The emergence in the United States of resistance to expanded-spectrum cephalosporin (e.g., ceftriaxone) within the salmonellae has been associated primarily with three large (>100-kb) plasmids (designated types A, B, and C) and one 10.1-kb plasmid (type D) that carry theblaCMY-2gene. In the present study, the distribution of these four knownblaCMY-2-carrying plasmids among 35 ceftriaxone-resistantSalmonellaisolates obtained from 1998 to 2001 was examined. Twenty-three of these isolates wereSalmonella entericaserotype Newport, 10 wereSalmonella entericaserotype Typhimurium, 1 wasSalmonella entericaserotype Agona, and 1 wasSalmonella entericaserotype Reading. All 23 serotype Newport isolates carried a type C plasmid, and 5, 4, and 1 serovar Typhimurium isolate carried type B, A, and C plasmids, respectively. Both the serotype Agona and serotype Reading isolates carried type A plasmids. None of the isolates carried a type D plasmid. Hybridization data suggested that plasmid types A and C were highly related replicons. DNA sequencing revealed that the region surroundingblaCMY-2was highly conserved in all three plasmid types analyzed (types B, C, and D) and was related to a region surroundingblaCMY-5from theKlebsiella oxytocaplasmid pTKH11. These findings are consistent with a model in whichblaCMY-2has been disseminated primarily through plasmid transfer, and not by mobilization of the gene itself, to multipleSalmonellachromosomal backbones.

Fresh pork sausage is produced without a microbial kill step and therefore chilled or frozen to control microbial growth. In this report, the microbiota in a chilled fresh pork sausage model produced with or without an antimicrobial combination of sodium lactate and sodium diacetate was studied using a combination of traditional microbiological methods and deep pyrosequencing of 16S rRNA gene amplicons. In the untreated system, microbial populations rose from 10 2 to 10 6 CFU/g within 15 days of storage at 4°C, peaking at nearly 10 8 CFU/g by day 30. Pyrosequencing revealed a complex community at day 0, with taxa belonging to the Bacilli , Gammaproteobacteria , Betaproteobacteria , Actinobacteria , Bacteroidetes , and Clostridia . During storage at 4°C, the untreated system displayed a complex succession, with species of Weissella and Leuconostoc that dominate the product at day 0 being displaced by species of Pseudomonas ( P. lini and P. psychrophila ) within 15 days. By day 30, a second wave of taxa ( Lactobacillus graminis , Carnobacterium divergens , Buttiauxella brennerae , Yersinia mollaretti , and a taxon of Serratia ) dominated the population, and this succession coincided with significant chemical changes in the matrix. Treatment with lactate-diacetate altered the dynamics dramatically, yielding a monophasic growth curve of a single species of Lactobacillus ( L. graminis ), followed by a uniform selective die-off of the majority of species in the population. Of the six species of Lactobacillus that were routinely detected, L. graminis became the dominant member in all samples, and its origins were traced to the spice blend used in the formulation.

The activity of ς B in Listeria monocytogenes is stimulated by high osmolarity and is necessary for efficient uptake of osmoprotectants. Here we demonstrate that, during cold shock, ς B contributes to adaptation in a growth phase-dependent manner and is necessary for efficient accumulation of betaine and carnitine as cryoprotectants.

The phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) utilizes high-energy phosphate present in PEP to drive the uptake of several different carbohydrates in bacteria. In order to examine the role of the PTS in the physiology of Listeria monocytogenes , we identified the ptsH and ptsI genes encoding the HPr and enzyme I proteins, respectively, of the PTS. Nucleotide sequence analysis indicated that the predicted proteins are nearly 70% similar to HPr and enzyme I proteins from other organisms. Purified L. monocytogenes HPr overexpressed in Escherichia coli was also capable of complementing an HPr defect in heterologous extracts of Staphylococcus aureus ptsH mutants. Additional studies of the transcriptional organization and control indicated that the ptsH and ptsI genes are organized into a transcription unit that is under the control of a consensus-like promoter and that expression of these genes is mediated by glucose availability and pH or by by-products of glucose metabolism.

Biosynthesis of the Caulobacter crescentus polar flagellum requires the expression of a large number of flagellar (fla) genes that are organized in a regulatory hierarchy of four classes (I to IV). The timing of fla gene expression in the cell cycle is determined by specialized forms of RNA polymerase and the appearance and/or activation of regulatory proteins. Here we report an investigation of the role of the C. crescentus transcriptional regulatory protein FlbD in the activation of sigma 54-dependent class III and class IV fla genes of the hierarchy by reconstituting transcription from these promoters in vitro. Our results demonstrate that transcription from promoters of the class III genes flbG, flgF, and flgI and the class IV gene fliK by Escherichia coli E sigma 54 is activated by FlbD or the mutant protein FlbDS140F (where S140F denotes an S-to-F mutation at position 140), which we show here has a higher potential for transcriptional activation. In vitro studies of the flbG promoter have shown previously that transcriptional activation by the FlbD protein requires ftr (ftr for flagellar transcription regulation) sequence elements. We have now identified multiple ftr sequences that are conserved in both sequence and spatial architecture in all known class III and class IV promoters. These newly identified ftr elements are positioned ca. 100 bp from the transcription start sites of each sigma 54-dependent fla gene promoter, and our studies indicate that they play an important role in controlling the levels of transcription from different class III and class IV promoters. We have also used mutational analysis to show that the ftr sequences are required for full activation by the FlbD protein both in vitro and in vivo. Thus, our results suggest that FlbD, which is encoded by the class II flbD gene, is a global regulator that activates the cell cycle-regulated transcription from all identified sigma 54-dependent promoters in the C. crescentus fla gene hierarchy.

Francisella tularensis subsp. holarctica is widely disseminated in North America and the boreal and temperate regions of the Eurasian continent. Comparative genomic analyses identified a 1.59-kb genomic deletion specific to F. tularensis subsp. holarctica isolates from Spain and France. Phylogenetic analysis of strains carrying this deletion by multiple-locus variable-number tandem repeat analysis showed that the strains comprise a highly related set of genotypes, implying that these strains were recently introduced or recently emerged by clonal expansion in France and the Iberian Peninsula.

Non-sorbitol-fermenting, β-glucuronidase-negative Escherichia coli O157:H7 strains are regarded as a clone complex, and populations from different geographical locations are believed to share a recent common ancestor. Despite their relatedness, high-resolution genotyping methods can detect significant genome variation among different populations. Phylogenetic analysis of high-resolution genotyping data from these strains has shown that subpopulations from geographically unlinked continents can be divided into two primary phylogenetic lineages, termed lineage I and lineage II, and limited studies of the distribution of these lineages suggest there could be differences in their propensity to cause disease in humans or to be transmitted to humans. Because the genotyping methods necessary to discriminate the two lineages are tedious and subjective, these methods are not particularly suited for studying the large sets of strains that are required to systematically evaluate the ecology and transmission characteristics of these lineages. To overcome this limitation, we have developed a lineage-specific polymorphism assay (LSPA) that can readily distinguish between the lineage I and lineage II subpopulations. In the studies reported here, we describe the development of a six-marker test (LSPA-6) and its validation in a side-by-side comparison with octamer-based genome scanning. Analysis of over 1,400 O157:H7 strains with the LSPA-6 demonstrated that five genotypes comprise over 91% of the strains, suggesting that these subpopulations may be widespread.

Thirteen different serotypes ofListeria monocytogenescan be distinguished on the basis of variation in somatic and flagellar antigens. Although the known virulence genes are present in all serotypes, greater than 90% of human cases of listeriosis are caused by serotypes 1/2a, 1/2b, and 4b and nearly all outbreaks of food-borne listeriosis have been caused by serotype 4b strains. Phylogenetic analysis of these three common clinical serotypes places them into two different lineages, with serotypes 1/2b and 4b belonging to lineage I and 1/2a belonging to lineage II. To begin examining evolution of the genome in these serotypes, DNA microarray analysis was used to identify lineage-specific and serotype-specific differences in genome content. A set of 44 strains representing serotypes 1/2a, 1/2b, and 4b was probed with a shotgun DNA microarray constructed from the serotype 1/2a strain 10403s. Clones spanning 47 different genes in 16 different contiguous segments relative to the lineage II 1/2a genome were found to be absent in all lineage I strains tested (serotype 4b and 1/2b) and an additional nine were altered exclusively in 4b strains. Southern hybridization confirmed that conserved alterations were, in all but two loci, due to absence of the segments from the genome. Genes within these contiguous segments comprise five functional categories, including genes involved in synthesis of cell surface molecules and regulation of virulence gene expression. Phylogenetic reconstruction and examination of compositional bias in the regions of difference are consistent with a model in which the ancestor of the two lineages had the 1/2 somatic serotype and the regions absent in the lineage I genome arose by loss of ancestral sequences.

Polyclonal antibodies to Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase were used in coagglutination tests for identification of these three enzymes in cell lysates. Enzyme capture assays were also developed for the detection of E. coli beta-galactosidase and beta-glucuronidase. The enzymes were released by using a gentle lysis procedure that did not interfere with antibody-enzyme interactions. All three enzymes were detected in 93% (51 of 55) of the E. coli strains tested by coagglutination; two of the three enzymes were identified in the remaining 7%. Of 42 non-E. coli tested by coagglutination, only four nonspecifically agglutinated either two or three of the anti-enzyme conjugates. Thirty-two (76%) non-E. coli isolates were negative by coagglutination for all three enzymes. The enzyme capture assay detected the presence of beta-galactosidase in seven of eight and beta-glucuronidase in all eight strains of E. coli tested. Some strains of beta-galactosidase-positive Citrobacter freundii and Enterobacter cloacae were also positive by the enzyme capture assay, indicating that the antibodies were not entirely specific for E. coli beta-galactosidase; however, five other gas-positive non-E. coli isolates were negative by the enzyme capture assay. The coagglutination tests and enzyme capture assays were rapid and sensitive methods for the detection of E. coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase.