Usha Srinivasan

PicU, a second serine protease autotransporter of uropathogenic Escherichia coli

Escherichia coli is the major aetiological agent of urinary tract infections (UTI). Like diarrhoeagenic strains of E. coli, uropathogenic isolates possess virulence determinants that distinguish them from commensal strains and allow them to produce the clinical manifestations associated with UTI. Several autotransporter proteins have been associated with the ability of E. coli, and other Gram-negative bacteria, to cause disease. Recently, we described the existence within uropathogenic E. coli (UPEC) strains of Sat, a toxin of the serine protease autotransporter of Enterobacteriaceae (SPATE) subfamily. Using features common to proteins secreted via the autotransporter pathway we have identified nine additional autotransporter proteins from the genomic sequence data of UPEC CFT073. Surprisingly, two additional members of the SPATE subfamily were identified. One protein, designated PicU, was homologous to the Pic protein identified in Shigella flexneri and enteroaggregative E. coli. The PicU protein was expressed and investigated for functional activity.

Library on a slide for bacterial comparative genomics.

BackgroundWe describe a novel application of microarray technology for comparative genomics of bacteria in which libraries of entire genomes rather than the sequence of a single genome or sets of genes are arrayed on the slide and then probed for the presence or absence of specific genes and/or gene alleles.ResultsWe first adopted a 96-well high throughput working protocol to efficiently isolate high quality genomic DNA. We then optimized conditions to print genomic DNA onto a glass slide with high density (up to 15000 spots) and to sensitively detect gene targets in each genome spot using fluorescently labeled DNA probe. Finally, we created an E. coli reference collection array and probed it for the presence or absence of the hemolysin (hly) gene using a dual channel non-competing hybridization strategy. Results from the array hybridization matched perfectly with previous tests.ConclusionsThis new form of microarray technology, Library on a Slide, is an efficient way for sharing and utilizing large strain collections in comparative genomic analyses.

Vaginal and oral microbes, host genotype and preterm birth

Preterm birth (PTB) is a leading cause of infant mortality and morbidity in the US and across the globe. Infection and associated inflammation are important initiators for PTB pathways; an estimated 40% of PTBs are attributed to amniochorionic-decidual or systemic inflammation. Historically, intrauterine infections have been implicated in PTB; recent evidence suggests that infections remote from the fetal site may also be causative. There is strong epidemiological evidence that bacterial vaginosis and periodontitis--two syndromes characterized by perturbations in the normal vaginal and oral bacterial microflora, respectively--are linked to infection-associated PTB. Oral and vaginal environments are similar in their bacterial microbiology; identical bacterial species have been independently isolated in periodontitis and bacterial vaginosis. Periodontitis and bacterial vaginosis also share many behavioral and sociodemographic risk factors suggesting a possible common pathophysiology. Genetic polymorphisms in host inflammatory responses to infection are shared between bacterial vaginosis, periodontitis and PTB, suggesting common mechanisms through which host genotype modify the effect of abnormal bacterial colonization on preterm birth. We review the state of knowledge regarding the risk of PTB attributable to perturbations in bacterial flora in oral and vaginal sites and the role of host genetics in modifying the risk of infection-related PTB. We posit that bacterial species that are common in perturbed vaginal and oral sites are associated with PTB through their interaction with the host immune system.

Identification of a Gene Encoding Heat-Resistant Agglutinin in Escherichia coli as a Putative Virulence Factor in Urinary Tract Infection

ABSTRACT Escherichia coli causes the vast majority of urinary tract infections (UTI) in both ambulatory and hospital patients. Several uropathogenic virulence factors have been identified, but half of all E. coli isolates that cause UTI have none or only one of the known virulence factors. Thus, it is reasonable to presume that other bacterial factors may be important in UTI pathogenesis. In order to find additional uropathogenic E. coli genes, we used genomic subtraction to identify DNA regions present in a uropathogenic strain of E. coli (1128-11). Genomic subtraction yielded 40 tester-specific fragments, including a novel heat-resistant agglutinin (hra) gene fragment. hra occurred in 55% of 486 UTI strains compared to 28% of 165 rectal strains (P = 0.001). The hra gene in 1128-11 was cloned, sequenced, and found to have 91% homology to the hra gene from E. coli meningitis strain RS218. The genetic organization of genes flanking hra in 1128-11 is distinct from the hra found in E. coli strains J96 and RS218. In our UTI and rectal specimen collections, hra was positively associated with a number of known virulence genes, including pathogenicity island genes hly and cnf, which are absent in 1128-11. The presence of hra in 1128-11 independent of hly/cnf suggests multiple mechanisms by which hra can be acquired by pathogenic E. coli strains. The flanking genes suggest that in 1128-11, hra may be part of a novel variant of a pathogenicity island V.

Changing Molecular Epidemiology of Group B Streptococcus in Korea

The prevalence of group B streptococcus (GBS) among pregnant women and disease burdens in neonates and adults are increasing in Korea. Colonizing isolates, collected by screening pregnant women (n=196), and clinical isolates collected from clinical patients throughout Korea (n=234), were serotyped and screened for antibiotic resistance. Serotype III (29.8%) and V (27.7%) predominated, followed by Ia (17.0%). Antibiotic resistance was higher among clinical than colonizing isolates for erythromycin (35.1% and 26.9%; P=0.10) and for clindamycin (49.4% and 42.1%; P=0.17). erm(B) occurred in 91.9% of erythromycin resistant isolates, and 84.0% of isolates resistant to clindamycin. Only five isolates (4.2%) resistant to erythromycin were susceptible to clindamycin; by contrast, and unique to Korea, 34% of isolates resistant to clindamycin were erythromycin susceptible. Among these 60 erythromycin-susceptible & clindamycin-resistant isolates, 88% was serotype III, and lnu(B) was found in 89% of strains. Four fifths of the serotype V isolates were resistant to both erythromycin and clindamycin. Further characterization of the genetic assembly of these resistance conferring genes, erm(B) and lnu(B), will be useful to establish the clonal lineages of multiple resistance genes carrying strains.

Selected Vaginal Bacteria and Risk of Preterm Birth: An Ecological Perspective

We examined the community ecology of vaginal microbial samples taken from pregnant women with previous preterm birth experience to investigate whether targeted pathogenic and commensal bacteria are related to risk of preterm birth in the current pregnancy. We found a significant correlation between the community structure of selected bacteria and birth outcome, but the correlation differed among self-reported racial/ethnic groups. Using a community ordination analysis, we observed infrequent co-occurrence of Mycoplasma and bacteria vaginosis associated bacteria 3 (BVAB3) among black and Hispanic participants. In addition, we found that the vaginal bacteria responded differently in different racial/ethnic groups to modifications of maternal behavioral (ie, douching and smoking) and biological traits (ie, body mass index [BMI]). Even after accounting for these maternal behaviors and traits, the selected vaginal bacteria was significantly associated with preterm birth among black and Hispanic participants. By contrast, white participants did not exhibit significant correlation between microbial community and birth outcome. Findings from this study affirm the necessity of considering womens race/ethnicity when evaluating the correlation between vaginal bacteria and preterm birth. The study also illustrates the importance of studying the vaginal microbiota from an ecological perspective, and demonstrates the power of ecological community analysis to improve understanding of infectious disease.

Comparative whole-genome analysis of Streptococcus mutans isolates within and among individuals of different caries status.

INTRODUCTION Genotypic analyses of Streptococcus mutans using fingerprinting methods depend on a few genetic loci being different but do not reveal the underlying genome-wide differences between strains. METHODS We used comparative genomic hybridization (CGH) with 70-mer oligonucleotide microarrays containing open reading frames (ORFs) from S. mutans strain UA159 to examine the genetic diversity of 44 isolates from nine children selected from a local study population in Eastern Iowa. RESULTS Unique strains (clones) within each child initially identified by arbitrary-priming polymerase chain reaction were confirmed by CGH. There was a wide range of variation in the hybridization patterns of the 1948 ORFs among the test isolates examined. Between 87 and 237 ORFs failed to give a positive signal among individual isolates. A total of 323 of the UA159 ORFs were absent from one or more of the test strains. These 323 variable genes seemed to be distributed across the entire UA159 genome and across all the predicted functional categories. CONCLUSION This set of very close geographically and temporally collected S. mutans isolates had a degree of gene content variation as high as a previously examined global set of strains. Comparing the frequency of these variable genes, the majority of which have unknown function, among strains of different origins (i.e. different caries status) could help to determine their relevance in S. mutans cariogenicity.

Uropathogenic Escherichia coli are less likely than paired fecal E. coli to have CRISPR loci.

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are short fragments of DNA that act as an adaptive immune system protecting bacteria against invasion by phages, plasmids or other forms of foreign DNA. Bacteria without a CRISPR locus may more readily adapt to environmental changes by acquiring foreign genetic material. Uropathogenic Escherichia coli (UPEC) live in a number of environments suggesting an ability to rapidly adapt to new environments. If UPEC are more adaptive than commensal E. coli we would expect that UPEC would have fewer CRISPR loci, and--if loci are present--that they would harbor fewer spacers than CRISPR loci in fecal E. coli. We tested this in vivo by comparing the number of CRISPR loci and spacers, and sensitivity to antibiotics (resistance is often obtained via plasmids) among 81 pairs of UPEC and fecal E. coli isolated from women with urinary tract infection. Each pair included one uropathogen and one commensal (fecal) sample from the same female patient. Fecal isolates had more repeats (p=0.009) and more unique spacers (p<0.0001) at four CRISPR loci than uropathogens. By contrast, uropathogens were more likely than fecal E. coli to be resistant to ampicillin, cefazolin and trimethoprim/sulfamethoxazole. However, no consistent association between CRISPRs and antibiotic resistance was identified. To our knowledge, this is the first study to compare fecal E. coli and pathogenic E. coli from the same individuals, and to test the association of CRISPR loci with antibiotic resistance. Our results suggest that the absence of CRISPR loci may make UPEC more susceptible to infection by phages or plasmids and allow them to adapt more quickly to various environments.

Probe Hybridization Array Typing: a Binary Typing Method for Escherichia coli

ABSTRACT The ability to distinguish between Escherichia coli strains is critical for outbreak investigations. Binary typing, based on the presence or absence of genetic material, provides a high-throughput alternative to gel- and PCR-based typing techniques that generate complex banding patterns and lack uniform interpretation criteria. We developed, validated, and determined the discriminatory power of an E. coli binary typing method, probe hybridization array typing (PHAT). In PHAT, the absence or presence of genetic material is identified by using DNA hybridization to produce a reproducible and portable fingerprint for each genome. PHAT probes were generated from genome subtractive hybridization experiments. We PHAT typed the ECOR collection of strains from a variety of geographical locations, and 33 rectal E. coli strains selected from college-aged women with urinary tract infection. In the set of 33 human rectal strains, the discriminatory power of PHAT (98%) equaled that of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. However, for ECOR strains, which include nonhuman strains, the current set of PHAT probes was less discriminating than MLST, ribotyping, and enterobacterial repetitive intergenic consensus sequence PCR (80% versus 97, 92, and 97%, respectively). When we limited the analysis to ECOR strains of B2 and D lineage, which are associated with human infection, current PHAT probes were highly discriminatory (94%). PHAT can be applied in a high-throughput format (i.e., “library on a slide”), the discriminatory ability can be varied based on the probe set, and PHAT is readily adapted to other bacterial species with high variation in genetic content.

Combining microarray technology and molecular epidemiology to identify genes associated with invasive group B streptococcus.

Many bacterial species function as both commensals and pathogens; we used this dual nature to develop a high-throughput molecular epidemiological approach to identifying bacterial virulence genes. We applied our approach to Group B Streptococcus (GBS). Three representative commensal and one invasive GBS isolates were selected as tester strains from a population-based collection. We used microarray-based comparative genomic hybridization to identify open reading frames (ORFs) present in two sequenced invasive strains, but absent or divergent in tester strains. We screened 23 variable ORFs against 949 GBS isolates using a GBS Library on a Slide (LOS) microarray platform. Four ORFs occurred more frequently in invasive than commensal isolates, and one appeared more frequently in commensal isolates. Comparative hybridization using an oligonucleotide microarray, combined with epidemiologic screening using the LOS microarray platform, enabled rapid identification of bacterial genes potentially associated with pathogenicity.