Listeria monocytogenes Contamination Leads to Survival and Growth During Enoki Mushroom Cultivation.

Two recent outbreaks of listeriosis have been linked to the consumption of enoki mushrooms. After the first outbreak, import sampling by the U.S. FDA identified that 43% of the samples evaluated were positive for Listeria monocytogenes (Lm). These observations raised questions about the potential sources of Lm contamination of enoki mushrooms. One potential source of contamination is during enoki mushroom cultivation, as growing conditions are comparatively cool and moist to induce mushroom germination, to which Lm is well adapted. Two varieties of enoki mushrooms were evaluated to determine the potential for Lm to contaminate enoki cultures when introduced at various points during cultivation (inoculation, scraping, pinning, and collaring). The results of two trials showed that Lm established contamination and grew to similar levels in the substrate regardless of when Lm was introduced and, with one exception, did not alter the rate of mushroom generation to below the control. Enumeration of Lm in enoki mushroom cultures at harvest found an average contamination of 103 cfu/g, though the results were variable. Refrigerated storage for six weeks was found to result in an increase in Lm. Additionally, no statistically significant difference in the levels of Lm was observed based on proximity to the substrate, though levels of Lm in the different enoki samples correlated with levels of Lm in the substrate at harvest, but not at scraping. The ability of Lm to grow independently in the media used to culture enoki was assessed, and Lm was found to be unable to grow but could sporadically survive in Masters Mix. No growth of Lm was observed in potato dextrose broth, though growth could occur on the agar. Overall, the data indicate a high potential for the establishment of Lm contamination at any point during enoki cultivation to result in Lm-contaminated mushrooms. These data indicate a need for active control mechanisms to prevent the introduction of Lm to enoki cultures.

A longitudinal study to examine the influence of farming practices and environmental factors on pathogen prevalence using structural equation modeling.

The contamination of fresh produce with foodborne pathogens has been an on-going concern with outbreaks linked to these commodities. Evaluation of farm practices, such as use of manure, irrigation water source, and other factors that could influence pathogen prevalence in the farming environment could lead to improved mitigation strategies to reduce the potential for contamination events. Soil, water, manure, and compost were sampled from farms in Ohio and Georgia to identify the prevalence of Salmonella, Listeria monocytogenes (Lm), Campylobacter, and Shiga-toxin-producing Escherichia coli (STEC), as well as Arcobacter, an emerging human pathogen. This study investigated agricultural practices to determine which influenced pathogen prevalence, i.e., the percent positive samples. These efforts identified a low prevalence of Salmonella, STEC, and Campylobacter in soil and water (< 10%), preventing statistical modeling of these pathogens. However, Lm and Arcobacter were found in soil (13 and 7%, respectively), manure (49 and 32%, respectively), and water samples (18 and 39%, respectively) at a comparatively higher prevalence, suggesting different dynamics are involved in their survival in the farm environment. Lm and Arcobacter prevalence data, soil chemical characteristics, as well as farm practices and weather, were analyzed using structural equation modeling to identify which factors play a role, directly or indirectly, on the prevalence of these pathogens. These analyses identified an association between pathogen prevalence and weather, as well as biological soil amendments of animal origin. Increasing air temperature increased Arcobacter and decreased Lm. Lm prevalence was found to be inversely correlated with the use of surface water for irrigation, despite a high Lm prevalence in surface water suggesting other factors may play a role. Furthermore, Lm prevalence increased when the microbiome's Simpson's Diversity Index decreased, which occurred as soil fertility increased, leading to an indirect positive effect for soil fertility on Lm prevalence. These results suggest that pathogen, environment, and farm management practices, in addition to produce commodities, all need to be considered when developing mitigation strategies. The prevalence of Arcobacter and Lm versus the other pathogens suggests that multiple mitigation strategies may need to be employed to control these pathogens.

Development and Validation of a Quantitative PCR Method for Species Verification and Serogroup Determination of Listeria monocytogenes Isolates.

ABSTRACT: Listeria monocytogenes (Lm) is one of the leading causes of death because of foodborne illness, affecting the elderly, pregnant women, neonates, and people who are immunocompromised. Serologically, Lm can be classified into 13 serotypes, although only 4 are typically linked with food contamination and illness. Since 2000, a shift in serotypes involved in listeriosis outbreaks has been observed, suggesting that tracking of serotypes could help identify emerging trends. A PCR method developed in 2004 allowed detection of the four major serotypes as molecular serogroups, corresponding to broad phylogenetic groups. In this study, a novel quantitative PCR (qPCR) method was developed that uses two multiplex qPCRs, one to confirm the Listeria genus and Lm species and the second for Lm molecular serogrouping. This method was compared with the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) method for Lm and the seroagglutination method, using a 208-strain panel. Comparison of the genus and species qPCR assay with the BAM methods found an equal or slightly higher accuracy for the qPCR method (>98%), compared with the BAM protocol (>96%), when evaluated against independent characterization data. Molecular serogrouping using the qPCR method (96.6%) was more accurate than the seroagglutination assay (75.6%). The qPCR method identified Lm 4bV strains, which could not be resolved using seroagglutination. The qPCR could not identify lineage III and IV serotype 4b strains but did correctly identify 16 of 18 lineage III and IV strains. The qPCR method performed genus identification for the Listeria species Lm, L. innocua, L. welshimeri, L. ivanovii, and L. seeligeri. In addition, the method performed species identification for Lm and classified Lm into six molecular serogroups: 2A, 2B, 2C, 4B, NT, and 4bV. This method provided a rapid and accurate confirmation of Lm and serogroup determinations; furthermore, it could help identify otherwise unlinked strains by enabling whole genome sequencing analysis based on broad phylogeny, independent of other information.

Serotype to genotype: The changing landscape of listeriosis outbreak investigations.

The classical definition of a disease outbreak is the occurrence of cases of disease in excess of what would normally be expected in a community, geographical area or time period. The establishment of an outbreak then starts with the identification of an incidence of cases above the normally expected threshold during a given time period. Subsequently, the cases are examined using a variety of subtyping methods to identify potential linkages. As listeriosis disease has a long incubation period, relating a single source or multiple sources of contaminated food to clinical disease is challenging and time consuming. The vast majority of human listeriosis cases are caused by three serotypes, 1/2a, 1/2b, and 4b. Thus serotyping of isolates from suspected foods and clinical samples, although useful for eliminating some food sources, has a very limited discriminatory power. The advent of faster and more affordable sequencing technology, coupled with increased computational power, has permitted comparisons of whole Listeria genome sequences from isolates recovered from clinical, food, and environmental sources. These analyses made it possible to identify outbreaks and the source much more accurately and faster, thus leading to a reduction in number of illnesses as well as a reduction in economic losses. Initial DNA sequence information also facilitated the development of a simple molecular serotype protocol which allowed for the identification of major disease causing serotypes of L. monocytogenes, including a clade of 4b variant (4bV) strains of L. monocytogenes involved in at least 3 more recent listeriosis outbreaks in the US. Furthermore, data generated using whole genome sequence (WGS) analyses was successfully utilized to develop a pan-genomic DNA microarray as well as a single nucleotide polymorphism (SNP) based analysis. Herein, we present and compare, the two recently developed sub-typing technologies and discuss how these methods are not only important in outbreak investigations, but could also shed light on possible adaptations to different foods and environments.

Genomic and phenotypic diversity of Listeria monocytogenes clonal complexes associated with human listeriosis.

Listeria monocytogenes is a pathogen of significant concern in many ready to eat foods due to its ability to survive and multiply even under significant environmental stresses. Listeriosis in humans is a concern, especially to high-risk populations such as those who are immunocompromised or pregnant, due to the high rates of morbidity and mortality. Whole genome sequencing has become a routine part of assessing L. monocytogenes isolated from patients, and the frequency of different genetic subtypes associated with listeriosis is now being reported. The recent abundance of genome sequences for L. monocytogenes has provided a wealth of information regarding the variation in core and accessory genomic elements. Newly described accessory genomic regions have been linked to greater virulence capabilities as well as greater resistance to environmental stressors such as sanitizers commonly used in food processing facilities. This review will provide a summary of our current understanding of stress response and virulence phenotypes of L. monocytogenes, within the context of the genetic diversity of the pathogen.

A Comprehensive Evaluation of the Genetic Relatedness of Listeria monocytogenes Serotype 4b Variant Strains.

Recently, we have identified a link between four listeriosis incidents/outbreaks to a variant of Listeria monocytogenes (Lm) serotype 4b strains, 4bV. Although 4bV strains have been reported from clinical specimens as well as from foods, listeriosis outbreaks occurring in 2014-2016 were the first reported outbreaks involving 4bV in the USA. Since traditional typing methods do not detect members of this group, we undertook a systematic and retrospective analysis of all Lm in the NCBI WGS Sequence Read Archive database to investigate the burden of 4bV strains among all listeriosis cases. This analysis identified the presence of isolates causing sporadic cases as well as those associated with the aforementioned outbreaks, as determined by WGS and traditional epidemiology. In total, approximately 350 Lm 4bV strains were identified from multiple parts of the USA as well as from Australia and Chile, dating back to 2001. The genomic relatedness of these strains was compared using the CFSAN SNP Pipeline and multi-virulence-locus sequence typing (MVLST). Using the CFSAN Pipeline tool, the 4bV strains were found to group into seven clusters that were separate from 4b strains. All seven clades appeared to contain isolates from both clinical and non-clinical sources. Conversely, the MVLST analysis revealed that practically all of the strains belonged to a single clade, suggesting that 4bV strains from disparate geographic regions and sources are under varied selective pressure, restricting diversity across these six virulence loci while allowing more variability across the genome as a whole. Further evaluation of these 4bV strains identified genes potentially acquired from a lineage II source external to the lmo0733-lmo0739 region, as well as highly conserved SNPs unique to the 4bV strains when compared to those from other lineages. Taken together, these data suggest that 4bV strains have undergone adaptive responses to selective pressures that may enhance survival in the environment while maintaining the pathogenic potential of serotype 4b strains.

A clade of Listeria monocytogenes serotype 4b variant strains linked to recent listeriosis outbreaks associated with produce from a defined geographic region in the US.

Four listeriosis incidences/outbreaks, spanning 19 months, have been linked to Listeria monocytogenes serotype 4b variant (4bV) strains. Three of these incidents can be linked to a defined geographical region, while the fourth is likely to be linked. In this study, whole genome sequencing (WGS) of strains from these incidents was used for genomic comparisons using two approached. The first was JSpecies tetramer, which analyzed tetranucleotide frequency to assess relatedness. The second, the CFSAN SNP Pipeline, was used to perform WGS SNP analyses against three different reference genomes to evaluate relatedness by SNP distances. In each case, unrelated strains were included as controls. The analyses showed that strains from these incidents form a highly related clade with SNP differences of ≤101 within the clade and >9000 against other strains. Multi-Virulence-Locus Sequence Typing, a third standardized approach for evaluation relatedness, was used to assess the genetic drift in six conserved, known virulence loci and showed a different clustering pattern indicating possible differences in selection pressure experienced by these genes. These data suggest a high degree of relatedness among these 4bV strains linked to a defined geographic region and also highlight the possibility of alterations related to adaptation and virulence.

Infectious Dose of Listeria monocytogenes in Outbreak Linked to Ice Cream, United States, 2015.

The relationship between the number of ingested Listeria monocytogenes cells in food and the likelihood of developing listeriosis is not well understood. Data from an outbreak of listeriosis linked to milkshakes made from ice cream produced in 1 factory showed that contaminated products were distributed widely to the public without any reported cases, except for 4 cases of severe illness in persons who were highly susceptible. The ingestion of high doses of L. monocytogenes by these patients infected through milkshakes was unlikely if possible additional contamination associated with the preparation of the milkshake is ruled out. This outbreak illustrated that the vast majority of the population did not become ill after ingesting a low level of L. monocytogenes but raises the question of listeriosis cases in highly susceptible persons after distribution of low-level contaminated products that did not support the growth of this pathogen.

Listeria monocytogenes in Stone Fruits Linked to a Multistate Outbreak: Enumeration of Cells and Whole-Genome Sequencing.

In 2014, the identification of stone fruits contaminated with Listeria monocytogenes led to the subsequent identification of a multistate outbreak. Simultaneous detection and enumeration of L. monocytogenes were performed on 105 fruits, each weighing 127 to 145 g, collected from 7 contaminated lots. The results showed that 53.3% of the fruits yielded L. monocytogenes (lower limit of detection, 5 CFU/fruit), and the levels ranged from 5 to 2,850 CFU/fruit, with a geometric mean of 11.3 CFU/fruit (0.1 CFU/g of fruit). Two serotypes, IVb-v1 and 1/2b, were identified by a combination of PCR- and antiserum-based serotyping among isolates from fruits and their packing environment; certain fruits contained a mixture of both serotypes. Single nucleotide polymorphism (SNP)-based whole-genome sequencing (WGS) analysis clustered isolates from two case-patients with the serotype IVb-v1 isolates and distinguished outbreak-associated isolates from pulsed-field gel electrophoresis (PFGE)-matched, but epidemiologically unrelated, clinical isolates. The outbreak-associated isolates differed by up to 42 SNPs. All but one serotype 1/2b isolate formed another WGS cluster and differed by up to 17 SNPs. Fully closed genomes of isolates from the stone fruits were used as references to maximize the resolution and to increase our confidence in prophage analysis. Putative prophages were conserved among isolates of each WGS cluster. All serotype IVb-v1 isolates belonged to singleton sequence type 382 (ST382); all but one serotype 1/2b isolate belonged to clonal complex 5. IMPORTANCE: WGS proved to be an excellent tool to assist in the epidemiologic investigation of listeriosis outbreaks. The comparison at the genome level contributed to our understanding of the genetic diversity and variations among isolates involved in an outbreak or isolates associated with food and environmental samples from one facility. Fully closed genomes increased our confidence in the identification and comparison of accessory genomes. The diversity among the outbreak-associated isolates and the inclusion of PFGE-matched, but epidemiologically unrelated, isolates demonstrate the high resolution of WGS. The prevalence and enumeration data could contribute to our further understanding of the risk associated with Listeria monocytogenes contamination, especially among high-risk populations.

Whole Genome Sequence Analysis Using JSpecies Tool Establishes Clonal Relationships between Listeria monocytogenes Strains from Epidemiologically Unrelated Listeriosis Outbreaks.

In an effort to build a comprehensive genomic approach to food safety challenges, the FDA has implemented a whole genome sequencing effort, GenomeTrakr, which involves the sequencing and analysis of genomes of foodborne pathogens. As a part of this effort, we routinely sequence whole genomes of Listeria monocytogenes (Lm) isolates associated with human listeriosis outbreaks, as well as those isolated through other sources. To rapidly establish genetic relatedness of these genomes, we evaluated tetranucleotide frequency analysis via the JSpecies program to provide a cursory analysis of strain relatedness. The JSpecies tetranucleotide (tetra) analysis plots standardized (z-score) tetramer word frequencies of two strains against each other and uses linear regression analysis to determine similarity (r2). This tool was able to validate the close relationships between outbreak related strains from four different outbreaks. Included in this study was the analysis of Lm strains isolated during the recent caramel apple outbreak and stone fruit incident in 2014. We identified that many of the isolates from these two outbreaks shared a common 4b variant (4bV) serotype, also designated as IVb-v1, using a qPCR protocol developed in our laboratory. The 4bV serotype is characterized by the presence of a 6.3 Kb DNA segment normally found in serotype 1/2a, 3a, 1/2c and 3c strains but not in serotype 4b or 1/2b strains. We decided to compare these strains at a genomic level using the JSpecies Tetra tool. Specifically, we compared several 4bV and 4b isolates and identified a high level of similarity between the stone fruit and apple 4bV strains, but not the 4b strains co-identified in the caramel apple outbreak or other 4b or 4bV strains in our collection. This finding was further substantiated by a SNP-based analysis. Additionally, we were able to identify close relatedness between isolates from clinical cases from 1993-1994 and a single case from 2011 as well as links between two isolates from over 30 years ago. The identification of these potential links shows that JSpecies Tetra analysis can be a useful tool in rapidly assessing genetic relatedness of Lm isolates during outbreak investigations and for comparing historical isolates. Our analyses led to the identification of a highly related clonal group involved in two separate outbreaks, stone fruit and caramel apple, and suggests the possibility of a new genotype that may be better adapted for certain foods and/or environment.

Prevalence and Level of Listeria monocytogenes in Ice Cream Linked to a Listeriosis Outbreak in the United States.

A most-probable-number (MPN) method was used to enumerate Listeria monocytogenes in 2,320 commercial ice cream scoops manufactured on a production line that was implicated in a 2015 listeriosis outbreak in the United States. The analyzed samples were collected from seven lots produced in November 2014, December 2014, January 2015, and March 2015. L. monocytogenes was detected in 99% (2,307 of 2,320) of the tested samples (lower limit of detection, 0.03 MPN/g), 92% of which were contaminated at <20 MPN/g. The levels of L. monocytogenes in these samples had a geometric mean per lot of 0.15 to 7.1 MPN/g. The prevalence and enumeration data from an unprecedented large number of naturally contaminated ice cream products linked to a listeriosis outbreak provided a unique data set for further understanding the risk associated with L. monocytogenes contamination for highly susceptible populations.

A novel gene, lstC, of Listeria monocytogenes is implicated in high salt tolerance.

Listeria monocytogenes, causative agent of human listeriosis, has been isolated from a wide variety of foods including deli meats, soft cheeses, cantaloupes, sprouts and canned mushrooms. Standard control measures for restricting microbial growth such as refrigeration and high salt are often inadequate as L. monocytogenes grows quite well in these environments. In an effort to better understand the genetic and physiological basis by which L. monocytogenes circumvents these controls, a transposon library of L. monocytogenes was screened for changes in their ability to grow in 7% NaCl and/ or at 5 °C. This work identified a transposon insertion upstream of an operon, here named lstABC, that led to a reduction in growth in 7% NaCl. In-frame deletion studies identified lstC which codes for a GNAT-acetyltransferase being responsible for the phenotype. Transcriptomic and RT-PCR analyses identified nine genes that were upregulated in the presence of high salt in the ΔlstC mutant. Further analysis of lstC and the genes affected by ΔlstC is needed to understand LstC's role in salt tolerance.

Whole-Genome Sequencing Identifies an Atypical Listeria monocytogenes Strain Isolated from Pet Foods.

Four Listeria isolates, including an atypical strain, were isolated from various pet foods and sequenced. We report here the draft genome sequences of these isolates and a comparative genomic analysis with a closely related human clinical isolate. An analysis of the atypical strain identified a frameshift mutation in the prfA gene.

Genomic characterization of novel Listeria monocytogenes serotype 4b variant strains.

Over 90% of the human listeriosis cases are caused by Listeria monocytogenes serotypes 1/2a, 1/2b and 4b strains. As an alternative to antigen-antibody based serotyping, a PCR-based method for serogrouping has been developed and validated. In this communication, we report an in-depth analysis of five 4b variant strains, four clinical isolates from Australia and one environmental isolate from USA. Although these five strains were serotype 4b by classical serotyping method, the serogrouping PCR profiles of these strains show the presence of a 1/2a-3a specific amplicon in addition to the standard 4b-4d-4e specific amplicons. These strains were further analyzed by pulsed field gel electrophoresis, binary gene typing, multi-locus variable-number-tandem-repeat analysis and a high density pan-genomic Listeria microarray. Using these sub-typing results, the clinical isolates were grouped into two distinct genomic groups- one of which could be part of an unidentified outbreak. The microarray results when compared with our database of other 4b outbreak isolates indicated that the serotype 4b variant strains represent very different genotypic profiles than the known reported 4b outbreak strains representing major epidemic clones. The acquisition of serotype 1/2a gene clusters by the 4b variant strains appears to be independent in origin, spanning large areas of geographical and temporal space and may indicate predisposition of some 4b strains towards accepting DNA from related organisms.

Genome Sequences of Listeria monocytogenes Serotype 4b Variant Strains Isolated from Clinical and Environmental Sources.

Listeria monocytogenes strains that show a novel PCR serotyping profile (IVb-v1) have been reported recently. Here, we announce the draft genome sequences of five L. monocytogenes IVb-v1 strains isolated from the United States and Australia that harbor a 6.3-kb DNA cassette characteristic of serotype 1/2a strains.

Genomic characterization of Listeria monocytogenes strains involved in a multistate listeriosis outbreak associated with cantaloupe in US.

A multistate listeriosis outbreak associated with cantaloupe consumption was reported in the United States in September, 2011. The outbreak investigation recorded a total of 146 invasive illnesses, 30 deaths and one miscarriage. Subtyping of the outbreak associated clinical, food and environmental isolates revealed two serotypes (1/2a and 1/2b) and four pulsed-field gel electrophoresis two-enzyme pattern combinations I, II, III, and IV, including one rarely seen before this outbreak. A DNA-microarray, Listeria GeneChip®, developed by FDA from 24 Listeria monocytogenes genome sequences, was used to further characterize a representative sample of the outbreak isolates. The microarray data (in the form of present or absent calls of specific DNA sequences) separated the isolates into two distinct groups as per their serotypes. The gene content of the outbreak-associated isolates was distinct from that of the previously-reported outbreak strains belonging to the same serotypes. Although the 1/2b outbreak associated isolates are closely related to each other, the 1/2a isolates could be further divided into two distinct genomic groups, one represented by pattern combination I strains and the other represented by highly similar pattern combinations III and IV strains. Gene content analysis of these groups revealed unique genomic sequences associated with these two 1/2a genovars. This work underscores the utility of multiple approaches, such as serotyping, PFGE and DNA microarray analysis to characterize the composition of complex polyclonal listeriosis outbreaks.

Listeria monocytogenes mutants with altered growth phenotypes at refrigeration temperature and high salt concentrations.

Listeria monocytogenes can survive and grow in refrigerated temperatures and high-salt environments. In an effort to better understand the associated mechanisms, a library of ∼ 5,200 transposon mutants of LS411, a food isolate from the Jalisco cheese outbreak, were screened for their ability to grow in brain heart infusion (BHI) broth at 5°C or in the presence of 7% NaCl and two mutants with altered growth profiles were identified. The LS522 mutant has a transposon insertion between secA2 and iap and showed a significant reduction in growth in BHI broth at 5°C and in the presence of 7% NaCl. Reverse transcriptase quantitative PCR (RT-qPCR) revealed a substantial reduction in the expression of iap. Additionally, a hypothetical gene (met), containing a putative S-adenosylmethionine-dependent methyltransferase domain, downstream of iap had downregulated expression. In-frame deletion mutants of iap and met were created in LS411. The LS560 (LS411 Δiap) mutant showed reduced growth at 5°C and in the presence of 7% salt, confirming its role in cold and salt growth attenuation. Surprisingly, the LS655 (LS411 Δmet) mutant showed slightly increased growth during refrigeration, though no alteration was seen in salt growth relative to the wild-type strain. The LS527 mutant, containing an insertion 36 bp upstream of the gbu operon, showed reduced expression of the gbu transcript by RT-qPCR and also showed growth reduction at 5°C and in the presence of 7% salt. This attenuation was severely exacerbated when the mutant was grown under the combined stresses. Analysis of the gbu operon deletion mutant showed decreased growth in 7% salt and refrigeration, supporting the previously characterized role for this gene in cold and salt adaptation. These studies indicate the potential for an intricate relationship between environmental stress regulation and virulence in L. monocytogenes.

Evaluation of a serotyping scheme using a combination of an antibody-based serogrouping method and a multiplex PCR assay for identifying the major serotypes of Listeria monocytogenes.

To evaluate a simplified serotyping scheme, we used a combination of an antibody-based serogrouping assay that identified only type 1 and type 4 strains and a multiplex PCR-based serogrouping assay to analyze 362 L. monocytogenes isolates collected over more than 20 years. The multiplex PCR assay also incorporated a set of primers specific for L. monocytogenes hlyA gene to verify the species identification of these isolates. A subset (n = 120) of these isolates were also serotyped with the Denka Seiken serotyping scheme, which is often considered the "gold standard" for serotyping of L. monocytogenes. The results indicate that the multiplex PCR-based assay, in combination with an antibody-based serogrouping assay, correctly identified serotypes of 96% of the previously serotyped isolates. Compared with the Denka Seiken method, the combination method also performed better in identifying serotypes of 120 previously unserotyped L. monocytogenes isolates. Thus, the combination scheme appears to be a simple and rapid way to identify serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, and 4b isolates, which are the predominant L. monocytogenes serotypes found in food, environmental, and clinical samples.

The chlamydial invasin-like protein gene conundrum.

Variants of an ilp (invasin-like protein) gene have been identified previously in Chlamydia caviae and in Chlamydia suis. The C. caviae ilp gene is interrupted by two frame shift mutations while the C. suis gene is intact. Characterization of the ilp gene in C. caviae passaged minimally in vitro showed that the two frameshift mutations were present in the original isolates. The gentamicin protection assay was used to determine if E. coli bacteria expressing the intact C. suis ilp could adhere to or invade HEp-2 cells. While inv+ clones showed increased adherence and invasion, no increase in adherence or invasion was observed for ilp+ clones. However, these clones were found to produce detectable amounts of ilp transcript. In a 48 h time course of C. suis culture, ilp transcript was initially detected at 8 h, peaked at 16 h, and declined subsequently. Antibodies specifically recognizing the putative functional domain of Ilp failed to detect any ilp-specific gene product in either E. coli or C. suis cultures. These data suggest that ilp does not encode a functional protein and raise questions about how ilp was introduced and maintained in Chlamydia.

Proteus mirabilis genes that contribute to pathogenesis of urinary tract infection: identification of 25 signature-tagged mutants attenuated at least 100-fold.

Proteus mirabilis, a common cause of urinary tract infections (UTI) in individuals with functional or structural abnormalities or with long-term catheterization, forms bladder and kidney stones as a consequence of urease-mediated urea hydrolysis. Known virulence factors, besides urease, are hemolysin, fimbriae, metalloproteases, and flagella. In this study we utilized the CBA mouse model of ascending UTI to evaluate the colonization of mutants of P. mirabilis HI4320 that were generated by signature-tagged mutagenesis. By performing primary screening of 2088 P. mirabilis transposon mutants, we identified 502 mutants that ranged from slightly attenuated to unrecoverable. Secondary screening of these mutants revealed that 114 transposon mutants were reproducibly attenuated. Cochallenge of 84 of these single mutants with the parent strain in the mouse model resulted in identification of 37 consistently out-competed P. mirabilis transposon mutants, 25 of which were out-competed >100-fold for colonization of the bladder and/or kidneys by the parent strain. We determined the sequence flanking the site of transposon insertion in 29 attenuated mutants and identified genes affecting motility, iron acquisition, transcriptional regulation, phosphate transport, urease activity, cell surface structure, and key metabolic pathways as requirements for P. mirabilis infection of the urinary tract. Two mutations localized to a approximately 42-kb plasmid present in the parent strain, suggesting that the plasmid is important for colonization. Isolation of disrupted genes encoding proteins with homologies to known bacterial virulence factors, especially the urease accessory protein UreF and the disulfide formation protein DsbA, showed that the CBA mouse model and mutant pools are a reliable source of attenuated mutants with mutations in virulence genes.