Expression of an iron-regulated hemolysin by Edwardsiella tarda

Abstract The ability of Edwardsiella tarda to hemolyse red blood cells was investigated. Most E. tarda strains (> 80%) produced a hemolysin when assayed by either an agar overlay or contact-dependent hemolysis technique. This activity was cell-associated (CAH) and not released into the culture supernatant under routine conditions. When quantified, E. tarda strains significantly produced 30–40-fold higher levels of hemolytic activity against guinea pig, sheep, or rabbit erythrocytes than either E. hoshinae or E. ictaluri . When grown under iron restricted-conditions in the presence of ethylenediamine di( o -hydroxyphenylacetic acid), hemoglubin, hematin and hemin were found to stimulate growth in both liquid and agar bioassays. Hemolysin activity could be released from selected E. tarda strains when grown in L broth supplemented with EDDA; hemolytic activity was 3- to > 40-fold under these conditions when compared to L broth alone. Preliminary characterization of the hemolysin of strain ET-13 indicates that it is a heat-labile protein with active sulphydryl and thiol groups. These results indicate that, in addition to its invasive capabilities, E. tarda produces a hemolysin which is at least partially regulated by the relative availability of iron and may play a role in human disease.     

Increased Bacterial Hemolytic Activity is Conferred by Expression of TlyA Methyltransferase but not by its 2′-O-methylation of the Ribosome

Bacterial 2′-O-methyltransferase TlyA methylates either both nucleotide C1409 of 16S rRNA and C1920 of 23S rRNA or only the C1920. Both ribosomal methylations increase bacterial susceptibility to ribosome-targeting antibiotics capreomycin and viomycin. However, TlyA has been suggested to also function as a hemolysin. Here, heterologous expression of TlyA from six diverse bacteria (including Mycobacterium tuberculosis and M. smegmatis) was found to increase hemolytic ability in the Escherichia coli host. Characterizing E. coli strains expressing mycobacterial TlyA with mutated rRNA recognition domain and impaired rRNA methylations showed that the abolished C1409 methylation altogether with significantly reduced C1920 methylation did not affect E. coli hemolytic activity. Thus, the increased bacterial hemolytic function is not likely a consequence of TlyA-mediated methylations of the ribosome. Purified water-soluble TlyA showed a weak concentration-dependent hemolysis in vitro. Therefore, the TlyA isoform alone is not a potent hemolysin. The results suggested that the bacterial hemolytic function might relate to the over-expression of TlyA and its interaction to other non-ribosomal target that is associated with the hemolytic ability.     

Production of enterotoxin,verotoxin, hemolysin and cytotoxic necrotizing factor byEscherichia coli of intestinal and extraintestinal origin

Seventy-sixEscherichia coli strains were examined for heat-labile enterotoxin (LT), verotoxin (VT), hemolysin (HLy) and cytotoxic necrotizing factor (CNF). Thirty-six strains were isolated from patients suffering from diarrhea and forty from different extraintestinal infections. The number of LT-producing strains was low (2.6%) (one of intestinal and one of extraintestinal origin). Verotoxin was produced only by one extraintestinal strain. Four intestinal strains were hemolytic (11.2%) and also positive for CNF. From 24 hemolytic strains of extraintestinal origin (60%), 17 produced also CNF. Most of the hemolytic (30%) as well as CNF-producing strains (22.5%) were isolated from urine. Our results are similar to those of other studies confirming the close association between hemolysin and CNF production as well as a possible role of these toxic factors in pathogenesis of extraintestinal, infections caused byE. coli.     

Cytotoxic Effect of Hemolytic Culture Supernatant from Enterococcus faecalis on Mouse Polymorphonuclear Neutrophils and Macrophages

We reconfirmed that the LD₅₀S of hemolytic Enterococcus faecalis strains were significantly less than those of nonhemolytic E. faecalis strains in normal mice. Hemolysin produced by E. faecalis lysed human, horse, rabbit, and mouse erythrocytes, but not cow and sheep erythrocytes. Sphingomyelin comprises a part of the lipid composition of the erythrocyte membrane of all mammalian species tested. But phosphatidylcholine exists only in human, horse, rabbit, and mouse. These two lipids inhibited lysis of horse erythrocytes by hemolytic E. faecalis. Phosphatidylcholine is probably the binding component on the membrane of erythrocytes for E. faecalis hemolysin. The hemolytic culture supernatant lysed not only erythrocytes but also mouse polymorphonuclear neutrophils (PMNs) and macrophages.     

Addition of a surfactant to tryptic soy broth allows growth of a lactic acid bacteria food antimicrobial, Escherichia coli O157:H7, and Salmonella enterica

Aims: This study aimed to determine the survival and growth of Escherichia coli O157:H7 and Salmonella enterica subsp. enterica in a medium supporting the growth of a Lactic Acid Bacteria (LAB) food antimicrobial culture. Methods and Results: Foodborne pathogens and LAB were cultured individually in tryptic soy broth (TSB), tryptic soy broth supplemented with one g l^?1 Tween 80^® (TSB‐T80), and de Man, Rogosa and Sharpe (MRS) broth. Growth of E. coli O157:H7 and Salmonella was similar in TSB and TSB‐T80 but was significantly less in MRS. Conversely, LAB growth was similar in MRS and TSB‐T80 but was significantly less in TSB. Conclusions: Supplementation of TSB with Tween 80^® allows growth of LAB to levels similar to that observed with MRS but does not inhibit the growth of E. coli O157:H7 and Salmonella. We present the formulation of a medium useful in studies useful for evaluating competitive inhibition of foodborne pathogens by LAB in vitro. Significance and Impact of the Study: This study reports the utility of TSB‐T80 for the completion of in vitro competitive inhibition assays incorporating a Lactic Acid Bacteria food safety culture.     

Enteroaggregative and cell-detaching<Emphasis Type="Italic">Escherichia coli</Emphasis> strains among polish children with and without diarrhea

To determine the association of enteroaggregative (EAEC) and cell-detaching (CDEC)Escherichia coli with diarrhea of unknown origin among children from Wroc?aw (Poland),E. coli strains isolated from stool specimens of children with diarrhea were examined for mannose-resistant adherence to HEp-2 cells. EAEC were isolated from 10 of 39 (26%) children examined with diarrhea and 4 of 20 (20%) age-matched controls. CDEC were present in 14 (36%) cases of diarrhea and 7 (35%) healthy subjects. Cell-detaching activity was distinctly associated with hemolysin production. Among hemolytic CDEC strains cytotoxic necrotizing factor 1 (CNF1) synthesis prevailed among isolates obtained from cases of diarrhea (57%) in comparison with isolates obtained from healthy controls (14.3%). Although neither EAEC nor CDECE. coli strains were associated with diarrhea of children in this setting, there were differences among EAEC and CDEC strains isolated from children with and without diarrhea.     

Characterization of Campylobacter concisus hemolysins

Campylobacter concisus is an opportunistic pathogen commonly found in the human oral cavity. It has also been isolated from clinical sources including gastroenteritis cases. Both secreted and cell-associated hemolytic activities were detected in C. concisus strains isolated from children with gastroenteritis. The secreted hemolytic activity of C. concisus strains was labile and was detected in variable levels from fresh-culture filtrates only. In addition, another secreted hemolysin/cytotoxin with a molecular weight < 10 kDa was detected in a single C. concisus strain (RCH 12). A C. concisus genomic library, constructed from strain RCH 3 in Escherichia coli XL1-Blue, was screened for hemolytic clones. Subcloning and sequence analysis of selected hemolytic clones identified ORFs for genes that enhance hemolytic activity but do not appear to be related to any known hemolysin genes found in Gram-negative bacteria. In a previous study, a stable cell-associated hemolysin was identified as an outer-membrane phospholipase A (OMPLA) encoded by the pldA gene. In this study, we report cloning of the pldA gene of the clinical strain C. concisus RCH 3 and the complementation of phospholipase A activity in an E. coli pldA mutant.     

Effect of carrier molecules on production and properties of extracellular hemolysin produced byStreptococcus agalactiae

Streptococcus agalactiae type la strain 090 produced a cell-associated hemolysin during exponential growth in medium lacking proteins. Growth of the organism in medium containing proteins or medium supplemented with Tween 40 resulted in the appearance of extracellular hemolytic activity that was filterable. Maximum extracellular hemolytic activity was obtained in the late exponential phase of growth corresponding to the maximum number of cells. Extracellular hemolysin released in medium containing proteins could be precipitated by ammonium sulfate. Cell-associated hemolysin could be extracted in the cold by purified lipoteichoic acid from the organism. Purification and characterization of the extracellular hemolysin by column chromatography showed that the hemolysin was associated with molecules eliciting its release. Hemolysin associated with lipoteichoic acid or Tween 40 had an apparent molecular weight of 1,800,000 or 60,000 daltons, respectively.     

The Aerogenic Response of Escherichia coli and Strains of Aerobacter in EC Broth and Selected Sugar Broths at Elevated Temperatures

Gas formation by 116 strains of Escherichia coli and 104 strains of Aerobacter was determined in a specially constructed and accurately controlled water bath employing EC, lactose, maltose, sucrose, glucose, levulose, and galactose broths at temperatures ranging from 44.5 to 46.5 C.

Greatest gas activity occurred in EC broth. In the range 44.9 to 45.5 C over 92% of the E. coli cultures formed gas, but the Aerobacter strains dropped from 68 to 2%. A natural point of separation of the two groups occurred at 45.5 C.

Inhibition of the gas-forming mechanism rather than death is the universal response of the Escherichia organisms to these temperatures. The inhibition increases with rising temperatures and is readily reversible. At 46.5 C, 64.5% of all the Escherichia cultures were inhibited and 69.1% of all the cultures were actually viable.

In EC broth it was found that as a group atypical E. coli (-+--) were the most resistant gas-positive types. Least resistant in EC broth was a group of known typical fecal isolates of E. coli (++--). Of intermediate resistance between the two groups was the large body of typical E. coli (++--) organisms.

Certain individual strains of E. coli excelled in the production of gas in the variety of sugar broths tested at elevated temperatures. The Aerobacter strains did not exhibit this property.

Finally it is suggested that elevated temperature incubation studies of this type be conducted in critically controlled water baths with an ascertained accuracy in the vicinity of 45.5 ± 0.1 C under full load.

     

An alternative approach for evaluating the phenotypic virulence factors of pathogenic Escherichia coli

Escherichia coli is a recognized zoonotic food-borne pathogen; however, the use of polymerase chain reaction (PCR) in the underdeveloped countries to differentiate pathogenic from non-pathogenic E. coli is a problematic issue. Our grail was to assess the phenotypic virulence markers motility, hemolysin, congo red agar, embryo lethality assay and serum resistance for pathogenic E. coli (PEC) correlated to PCR tests which is currently used world-wide to evaluate the PEC. The 448 strains of Escherichia coli that were isolated from different sources, were characterized for phenotypic virulence factors such as motility, hemolysin, Congo red binding, Embryo Lethality assay (ELA) and serum resistance, as well as antibiotic susceptibility using disc diffusion method to 23 antibiotics. Results exhibited 100% motility and Congo red binding, 97.1% for hemolysin production and 90.2% in the ELA. As a result, we were able to hypothetically conclude that the aforementioned virulence markers are plain, straightforward, economical, rapid, more dynamic, uncomplicated methodology, duplicatable and cost next to nothing when compared to the molecular PCR. Their implementation in a diagnostic microbiology laboratory for vetting is a rewarding task in the underdeveloped countries. It augments endeavors to minimize the use of PCR in our investigations especially during epidemiological and outbreak investigations of PEC.     

Efficacy of culture filtrates of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> against larvae of <Emphasis Type="Italic">Anopheles stephensi</Emphasis> and <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis>

Efficacy of culture filtrates of five strains of Metarhizium anisopliae isolated from insects were evaluated against Anopheles stephensi and Culex quinquefasciatus. The culture filtrates released from the strains of M. anisopliae in the YpSs and chitin broths were filtered and used for the bioassays after a growth of 7 days. Among the culture filtrates of five strains, M. anisopliae 892 was found to be more effective against both the mosquitoes. The LC(50) values of culture filtrates of M. anisopliae 892 in chitin broth was lower than the LC(50) of culture filtrates in YpSs broth against first and fourth instars of both the mosquitoes. The LC(50) values of culture filtrates were significantly different between first and fourth instars of A. stephensi (t test; P = 0.0001) and C. quinquefasciatus (t test; P = 0.02). The larvae of A. stephensi were more susceptible than C. quinquefasciatus except in two cases. This is the first report of efficacy of culture filtrates produced by M. anisopliae in chitin broth against mosquitoes and have potential as a biological control agent of mosquitoes.     

Prevalence of Hemolysin Genes and Comparison of ehxA Subtype Patterns in Shiga Toxin-Producing Escherichia coli (STEC) and Non-STEC Strains from Clinical,Food, and Animal Sources

Shiga toxin-producing Escherichia coli (STEC) belonging to certain serogroups (e.g., O157 and O26) can cause serious conditions like hemolytic-uremic syndrome (HUS), but other strains might be equally pathogenic. While virulence factors, like stx and eae, have been well studied, little is known about the prevalence of the E. coli hemolysin genes (hlyA, ehxA, e-hlyA, and sheA) in association with these factors. Hemolysins are potential virulence factors, and ehxA and hlyA have been associated with human illness, but the significance of sheA is unknown. Hence, 435 E. coli strains belonging to 62 different O serogroups were characterized to investigate gene presence and phenotypic expression of hemolysis. We further investigated ehxA subtype patterns in E. coli isolates from clinical, animal, and food sources. While sheA and ehxA were widely distributed, e-hlyA and hlyA were rarely found. Most strains (86.7%) were hemolytic, and significantly more hemolytic (95%) than nonhemolytic strains (49%) carried stx and/or eae (P < 0.0001). ehxA subtyping, as performed by using PCR in combination with restriction fragment length polymorphism analysis, resulted in six closely related subtypes (>94.2%), with subtypes A/D being eae-negative STECs and subtypes B, C, E, and F eae positive. Unexpectedly, ehxA subtype patterns differed significantly between isolates collected from different sources (P < 0.0001), suggesting that simple linear models of exposure and transmission need modification; animal isolates carried mostly subtypes A/C (39.3%/42.9%), food isolates carried mainly subtype A (81.9%), and clinical isolates carried mainly subtype C (66.4%). Certain O serogroups correlated with particular ehxA subtypes: subtype A with O104, O113, and O8; B exclusively with O157; C with O26, O111, and O121.     

Altered hemolysin production in urine-grown uroisolates ofEscherichia coli

Fifteen uroisolates ofE. coli were studies for both cell-free and cell-bound hemolysin production. Estimations were done inTrypticase soy broth (TSB, providing iron-replete medium) TSB +2,2′-bipyridine (providing experimentally created iron-depleted conditions) and pooled normal human urine (providing natural iron-depleted growth medium). In TSB 40% of strains showed no detectable cell-free hemolysin, they were able to produce it in the presence of 2,2′-bipyridine and more so when grown in urine. The cell-bound hemolysin was produced by all the strains in TSB, but in the presence of 2,2′-bipyridine and urine an insignificant increase was observed. All the strains when given 2nd and 3rd passage in urine, were found to elaborate significantly more cell-free as well as cell-bound hemolysin.     

Molecular cloning,characterization, and sequencing of the hemolysin gene from Edwardsiella tarda

Hemolysis is a major symptom of diseased eels infected by Edwardsiella tarda. The hemolysin gene of E. tarda strain ET16 was cloned into plasmid pSK and expressed in Escherichia coli. The mol. mass of the functional β-hemolysin was estimated to be approximately 34 kDa by gel filtration and by SDS-PAGE followed by in situ hemolysin activity analysis. The cloned fragment containing the β-hemolysin locus from E. tarda strain ET16 expressed in E. coli was estimated to be 5.3 kb in length; the deduced gene product was identical in mol. mass and properties to the extracellular products of E. tarda strain ET16. The presence of EcoRI and XbaI sites within the β-hemolysin gene of E. tarda was determined from the loss of hemolytic activity in subclones. Analysis of the DNA sequence of a 2,436-bp HaeIII-HindIII fragment that included EcoRI and XbaI sites revealed three ORFs organized as an operon that encoded three predicted polypeptides of 15,874, 7,055, and 34,804 Da. A 34-kDa polypeptide expressing hemolytic activity in cell lysates of the clone DH5α(pETH3E) is very likely the β-hemolysin encoded by the third ORF. The observation that hemolytic activity appeared in the culture medium of E. tarda, but not in that of E. coli strain DH5α(pETH3E) indicates the existence of a mechanism for transporting the hemolysin across the cell envelope in E. tarda that is different from that of E. coli. Received: 7 July 1995 / Accepted: 24 October 1995     

Diarrheal and Environmental Isolates of Aeromonas spp. Produce a Toxin Similar to Shiga-Like Toxin 1

Diarrheal and environmental isolates of 39 strains of Aeromonas spp. were studied for detection of virulence factors. Although these 39 strains did not produce either heat-labile or heat-stable enterotoxins, culture filtrates of 31 strains produced cytopathic effects on Vero cells. Among these, culture filtrates of three strains of Aeromonas hydrophila and one strain of Aeromonas caviae could be neutralized by Escherichia coli O157:H7 Shiga-like toxin 1 antiserum. A single band of plasmid DNA of 2.14 kbp was isolated from these strains of Aeromonas spp. and E. coli O157:H7, which could be amplified by the polymerase chain reaction (PCR), employing oligonucleotide primers from the Shiga-like toxin 1 (SLT1) gene of E. coli O157:H7. E. coli HB 101 cells when transformed with the same plasmid showed cytopathic effects on Vero cells, which indicates that the SLT 1 homolog gene(s) of Aeromonas spp. is plasmid encoded. These results suggest that Aeromonas spp. may also produce Shiga-like toxin 1, or at least a cytotoxin with some homology with the Shiga-like toxin 1 of E. coli O157:H7.     

Phylogenetic Groups and Pathogenicity Island Markers in Fecal Escherichia coli Isolates from Asymptomatic Humans in China

The study of phylogenetic groups and pathogenicity island (PAI) markers in commensal Escherichia coli strains from asymptomatic Chinese people showed that group A strains are the most common and that nearly half of all fecal strains which were randomly selected harbor PAIs.Escherichia coli is a well-diversified commensal species in the intestine of healthy humans but also includes intestinal or extraintestinal pathogens. It has been reported that pathogenic E. coli may be derived from fecal strains by acquisition of virulence determinants (11). The relationship between the E. coli genetic background and the acquisition of virulence factors is now better understood (1, 5). Extraintestinal E. coli strains may harbor several virulence factors, such as adhesins, fimbriae, and hemolysin, which can contribute to bacterial pathogenesis. These traits are usually encoded on pathogenicity islands (PAIs), which have been studied in pathogenic E. coli previously (15). The E. coli population includes 4 major phylogroups (A, B₁, B₂, and D) (2). Pathogenic strains belong mainly to groups B₂ and D, while most fecal isolates belong to groups A and B₁. Strains of groups B₂ and D often carry virulence factors that are lacking in group A and B₁ strains (3, 9, 13).In this study, we examined the distribution of phylogroups and the prevalence of PAIs in commensal E. coli strains isolated from asymptomatic persons in one region of China.     

Efficient Transfer of the Pheromone-Independent Enterococcus faecium Plasmid pMG1 (Gmr) (65.1 Kilobases) to Enterococcus Strains during Broth Mating

Plasmid pMG1 (65.1 kb) was isolated from a gentamicin-resistant Enterococcus faecium clinical isolate and was found to encode gentamicin resistance. EcoRI restriction of pMG1 produced five fragments, A through E, with molecular sizes of 50.2, 11.5, 2.0, 0.7, and 0.7 kb, respectively. The clockwise order of the fragments was ACDEB. pMG1 transferred at high frequency to Enterococcus strains in broth mating. pMG1 transferred between Enterococcus faecalis strains, between E. faecium strains, and between E. faecium and E. faecalis strains at a frequency of approximately 10⁻⁴ per donor cell after 3 h of mating. The pMG1 transfers were not induced by the exposure of the donor cell to culture filtrates of plasmid-free E. faecalis FA2-2 or an E. faecium strain. Mating aggregates were not observed by the naked eye during broth mating. Small mating aggregates of several cells in the broth matings were observed by microscopy, while no aggregates of donor cells which had been exposed to a culture filtrate of E. faecalis FA2-2 or an E. faecium strain were observed, even by microscopy. pMG1 DNA did not show any homology in Southern hybridization with that of the pheromone-responsive plasmids and broad-host-range plasmids pAMβ1 and pIP501. These results indicate that there is another efficient transfer system in the conjugative plasmids of Enterococcus and that this system is different from the pheromone-induced transfer system of E. faecalis plasmids.     

Development of antigen-delivery systems,based on the Escherichia coli hemolysin secretion pathway

We describe the development of plasmid vectors carrying the expression sites, an hlyA cassette and the secretion genes of Escherichia coli hemolysin. These allow the synthesis and secretion of heterologous microbial antigens in E. coli and attenuated Salmonella aroA strains. Genes or gene fragments encoding microbial antigens are inserted in-frame into a residual part of the hlyA gene which essentially encodes the HlyA secretion signal (HlyA₈). In general, the fused genes, carrying the hlyA_s sequence at the 3' terminus, are efficiently expressed, and the synthesized antigens are secreted into the culture supernatant of the producing strain. Attenuated Salmonella strains synthesizing either HlyA_s-fused listeriolysin or p60 of Listeria monocytogenes were constructed by this procedure and shown to provide protective immunity against L. monocytogenes in mice. The most effective protection was obtained when these microbial antigens were secreted by the attenuated Salmonella strains. We further present new approaches which may allow the application of this antigen-delivery system to any microbial antigen.     

A comparison and optimization of methods and factors affecting the transformation of Escherichia coli

DNA manipulation routinely requires competent bacteria that can be made using one of numerous methods. To determine the best methods, we compared four commonly used chemical methods (DMSO, MgCl₂–CaCl₂, CaCl₂ and Hanahan''s methods) on frequently used Escherichia coli (E. coli) strains: DH5α, XL-1 Blue, SCS110, JM109, TOP10 and BL21-(DE3)-PLysS. Hanahan''s method was found to be most effective for DH5α, XL-1 Blue and JM109 strains (P<0.05), whilst the CaCl₂ method was best for SCS110, TOP10 and BL21 strains (P<0.05). The use of SOB (super optimal broth) over LB [Luria–Bertani (broth)] growth media was found to enhance the competency of XL-1 Blue (P<0.05), dampened JM109′s competency (P<0.05), and had no effect on the other strains (P>0.05). We found no significant differences between using 45 or 90 s heat shock across all the six strains (P>0.05). Through further optimization by means of concentrating the aliquots, we were able to get further increases in transformation efficiencies. Based on the optimized parameters and methods, these common laboratory E. coli strains attained high levels of TrE (transformation efficiency), thus facilitating the production of highly efficient and cost-effective competent bacteria.     

Extraintestinal Pathogenic and Antimicrobial-Resistant Escherichia coli Contamination of 56 Public Restrooms in the Greater Minneapolis-St. Paul Metropolitan Area

How extraintestinal pathogenic Escherichia coli (ExPEC) and antimicrobial-resistant E. coli disseminate through the population is undefined. We studied public restrooms for contamination with E. coli and ExPEC in relation to source and extensively characterized the E. coli isolates. For this, we cultured 1,120 environmental samples from 56 public restrooms in 33 establishments (obtained from 10 cities in the greater Minneapolis-St. Paul, MN, metropolitan area in 2003) for E. coli and compared ecological data with culture results. Isolates underwent virulence genotyping, phylotyping, clonal typing, pulsed-field gel electrophoresis (PFGE), and disk diffusion antimicrobial susceptibility testing. Overall, 168 samples (15% from 89% of restrooms) fluoresced, indicating presumptive E. coli: 25 samples (2.2% from 32% of restrooms) yielded E. coli isolates, and 10 samples (0.9% from 16% of restrooms) contained ExPEC. Restroom category and cleanliness level significantly predicted only fluorescence, gender predicted fluorescence and E. coli, and feces-like material and toilet-associated sites predicted all three endpoints. Of the 25 E. coli isolates, 7 (28%) were from phylogenetic group B2(virulence-associated), and 8 (32%) were ExPEC. ExPEC isolates more commonly represented group B2 (50% versus 18%) and had significantly higher virulence gene scores than non-ExPEC isolates. Six isolates (24%) exhibited ≥3-class antibiotic resistance, 10 (40%) represented classic human-associated sequence types, and one closely resembled reference human clinical isolates by pulsed-field gel electrophoresis. Thus, E. coli, ExPEC, and antimicrobial-resistant E. coli sporadically contaminate public restrooms, in ways corresponding with restroom characteristics and within-restroom sites. Such restroom-source E. coli strains likely reflect human fecal contamination, may pose a health threat, and may contribute to population-wide dissemination of such strains.