Defining genomic islands and uropathogen-specific genes in uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) strains are responsible for the majority of uncomplicated urinary tract infections, which can present clinically as cystitis or pyelonephritis. UPEC strain CFT073, isolated from the blood of a patient with acute pyelonephritis, was most cytotoxic and most virulent in mice among our strain collection. Based on the genome sequence of CFT073, microarrays were utilized in comparative genomic hybridization (CGH) analysis of a panel of uropathogenic and fecal/commensal E. coli isolates. Genomic DNA from seven UPEC (three pyelonephritis and four cystitis) isolates and three fecal/commensal strains, including K-12 MG1655, was hybridized to the CFT073 microarray. The CFT073 genome contains 5,379 genes; CGH analysis revealed that 2,820 (52.4%) of these genes were common to all 11 E. coli strains, yet only 173 UPEC-specific genes were found by CGH to be present in all UPEC strains but in none of the fecal/commensal strains. When the sequences of three additional sequenced UPEC strains (UTI89, 536, and F11) and a commensal strain (HS) were added to the analysis, 131 genes present in all UPEC strains but in no fecal/commensal strains were identified. Seven previously unrecognized genomic islands (>30 kb) were delineated by CGH in addition to the three known pathogenicity islands. These genomic islands comprise 672 kb of the 5,231-kb (12.8%) genome, demonstrating the importance of horizontal transfer for UPEC and the mosaic structure of the genome. UPEC strains contain a greater number of iron acquisition systems than do fecal/commensal strains, which is reflective of the adaptation to the iron-limiting urinary tract environment. Each strain displayed distinct differences in the number and type of known virulence factors. The large number of hypothetical genes in the CFT073 genome, especially those shown to be UPEC specific, strongly suggests that many urovirulence factors remain uncharacterized.     

Distribution of pathogenicity island markers and virulence factors in new phylogenetic groups of uropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates

The present study was aimed at investigating the relationship between the new Clermont’s phylogenetic groups, virulence factors, and pathogenicity island markers (PAIs) among uropathogenic Escherichia coli (UPEC) in Iran. This cross-sectional study was carried out on 140 UPEC isolates collected from patients with urinary tract infections in Bushehr, Iran. All isolates were subjected to phylogenetic typing using a new quadruplex-PCR method. The presence of PAI markers and virulence factors in UPEC strains was evaluated by multiplex PCR. The most predominant virulence gene was fimH (85%), followed by iucC (61.4%), papC (38.6%), hlyA (22.1%), cnf-1 (18.6%), afa (10.7%), papG and neuC (each 9.3%), ibeA (3.6%), and sfa/foc (0.7%). The most common phylogenetic group was related to B2 (39.3%), and the least common to A (0.7%). The most prevalent PAI marker was PAI IV536 (77.14%), while markers for PAI III536 (13.57%), PAI IIJ96 (12.86%), and PAI II536 (12.14%) were the least frequent among the UPEC strains. Meanwhile, the PAI IJ96 marker was not detected. There was a significant association between the phylogenetic group B2 and all the studied virulence genes and PAI markers. To our knowledge, this is the first study to compare the relationship between new phylogenetic groups, virulence genes and PAI markers in UPEC strains in Iran. The phylogenetic group B2 was predominantly represented among the studied virulence genes and PAI markers, indicating the preference of particular strains to carry virulence genes.     

Complete Genome Sequence of the Wild-Type Commensal Escherichia coli Strain SE15, Belonging to Phylogenetic Group B2

Escherichia coli SE15 (O150:H5) is a human commensal bacterium recently isolated from feces of a healthy adult and classified into E. coli phylogenetic group B2, which includes the majority of extraintestinal pathogenic E. coli. Here, we report the finished and annotated genome sequence of this organism.The complete genome sequence of Escherichia coli SE15 was determined using a combination of 2-kb and 40-kb Sanger libraries and 454 pyrosequencing. We generated 57,600 sequences (ABI 3730xl sequencers) and three sequencing runs (GS20 sequencers). The 454 pyrosequencing reads were first assembled using the Newbler assembler software (4). A hybrid assembly of 454 and Sanger reads was performed using the Phred-Phrap-Consed program (1). Remaining gaps between contigs were closed by direct sequencing of clones. Prediction and annotation of protein-coding genes were performed as described previously (6).The genome of E. coli SE15 consists of a circular 4,717,338-bp chromosome containing 4,338 predicted protein-coding genes and a 122-kb plasmid (pSE15) encoding 150 protein-coding genes. From the multilocus sequence typing analysis based on the nucleotide sequences of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA), SE15 was found to belong to E. coli reference collection group B2. In the chromosome, two prophage regions and seven integrative elements are found. Of the predicted protein-coding genes, we could assign 2,883 (64%) to known functions, 1,528 (34%) as conserved hypothetical genes and 77 (2%) as novel hypothetical genes. Of the predicted protein-coding genes on the chromosome, 3,735 (86%) are common to three uropathogenic E. coli (UPEC) genomes (CFT073, UTI89, and 536) and 263 (6%) are not identified in any of the three UPEC genomes. The 263 genes include 7 genes for the phosphoenolpyruvate:sugar phosphotransferase system involved in the uptake of carbohydrates, reflecting the adaptation of SE15 to a commensal lifestyle in the intestinal tract. pSE15 shares 121 genes (81%) with a 114-kb plasmid (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP000244","term_id":"91075696","term_text":"CP000244"}}CP000244) of UPEC UTI89, indicating that both plasmids are derived from the same origin.The chromosome contains six large segments (LSs; >30 kb) designated LSs I to VI, three of which overlap one prophage region and two integrative elements. Each of the six LSs is located at the same locus as at least one of the pathogenicity islands (PAIs) or other insertion regions in the three UPEC genomes. LS II (ECSF_1824 to ECSF_1835) and three PAIs (PAI IV_UTI89, PAI IV₅₃₆, and HPI_CFT073) are located at the same loci in each chromosome and share the ybt operon encoding the yersiniabactin iron acquisition system, indicating that the ancestral E. coli of group B2 strains may have acquired the ybt genes. LS III (ECSF_1852 to ECSF_1897), PAI VI_UTI89, PAI VI₅₃₆, and PAI_CFT073-asnW are located at the same loci in each chromosome. The three PAIs contain the pks island encoding multiple nonribosomal peptide synthases and polyketide synthases, whereas LS III in SE15 completely lacks the pks island. The commensal E. coli strain ED1a also lacks the pks island (8), but the commensal E. coli strain Nissle 1917 has the pks island (5). These data suggest that the presence of the pks island may not be common among intestinal commensal strains in group B2. LS V (ECSF_2770 to ECSF_2794) is almost identical to PAI V_UTI89, which contains the genes cluster for a type II secretion system (gsp), group II capsule synthesis (kps), and polysialic acid synthesis (neu). The neu operon between the kpsFEDUCS and kpsMT genes in PAI V_UTI89 is responsible for K1 capsule biosynthesis, and this region between the kpsFEDUCS and kpsMT genes is highly variable in E. coli (9). The corresponding region (ECSF_2777 to ECSF_2781) in LS V encodes genes different from those in the neu operon in PAI V_UTI89; differs from the corresponding regions of the CFT073 (K2 serotype), 536 (K15 serotype), and APEC O1 (K1 serotype) strains; and shows no homology with any sequence in public databases.SE15 lacks many virulence-related genes, whereas UPEC encodes virulence-related factors, including fimbrial adhesins, toxins, capsule, and serum resistance and iron uptake systems. The three UPEC strains have the genes encoding P fimbriae (pap), S fimbriae (sfa/foc), Auf fimbriae (auf), and type 1 fimbriae (fim), whereas SE15 contains only the fim genes and lacked the pap, sfa/foc, and auf genes. Amino acid replacements in FimH located at the tip of type 1 fimbriae produce a shift from a commensal-associated trimannose binding phenotype to a urinary tract infection-associated monomannose binding phenotype (7). The other sequenced B2 strains (three UPEC strains, APEC O1, LF82, and ED1a) have Ser-70 and Asn-78 residues in FimH, whereas SE15 has Asn-70 and Ser-78 residues that are conserved in intestinal E. coli strains. Of the seven chaperon-usher fimbrial operons in SE15, six (fim, yad, yde, yeh, yfc, and yqi) are conserved in the three UPEC genomes. The one remaining fimbrial operon (ECSF_0163 to ECSF_0166) is specific to SE15. The GC content (42%) of this 5-kb fimbrial region is lower than the average GC content (51%) of the chromosome. UPEC strains contain a greater number of iron acquisition systems than do commensal strains, which may be a consequence of their adaptation to the iron-limiting urinary tract environment (3). SE15 also contains iron uptake system genes encoding siderophore enterobactin, siderophore yersiniabactin, iron transporter (sit), and heme (chu) systems but lacks genes for siderophore salmochelin, siderophore aerobactin, and novel siderophore (ireA), which are encoded by PAIs of UPEC strains. Furthermore, SE15 lacks genes encoding alpha-hemolysin and cytotoxic necrotizing factor, which are known toxins encoded by PAIs of UPEC strains.It has been pointed out that extraintestinal pathogenic E. coli (ExPEC) virulence factors identified in commensal strains of group B2 may facilitate colonization of the human gut and thus act as fitness factors for commensal E. coli stains (2). SE15 contains fewer known ExPEC virulence-associated genes than other known commensal strains (ED1a and Nissle 1917) in group B2, suggesting that ExPEC virulence-related genes in the SE15 genome may be necessary for this commensal microorganism to colonize the human gut.     

A Novel Two-Component Signaling System Facilitates Uropathogenic Escherichia coli's Ability to Exploit Abundant Host Metabolites

Two-component signaling systems (TCSs) are major mechanisms by which bacteria adapt to environmental conditions. It follows then that TCSs would play important roles in the adaptation of pathogenic bacteria to host environments. However, no pathogen-associated TCS has been identified in uropathogenic Escherichia coli (UPEC). Here, we identified a novel TCS, which we termed KguS/KguR (KguS: α-ketoglutarate utilization sensor; KguR: α-ketoglutarate utilization regulator) in UPEC CFT073, a strain isolated from human pyelonephritis. kguS/kguR was strongly associated with UPEC but was found only rarely among other E. coli including commensal and intestinal pathogenic strains. An in vivo competition assay in a mouse UTI model showed that deletion of kguS/kguR in UPEC CFT073 resulted in a significant reduction in its colonization of the bladders and kidneys of mice, suggesting that KguS/KguR contributed to UPEC fitness in vivo. Comparative proteomics identified the target gene products of KguS/KguR, and sequence analysis showed that TCS KguS/KguR and its targeted-genes, c5032 to c5039, are encoded on a genomic island, which is not present in intestinal pathogenic E. coli. Expression of the target genes was induced by α-ketoglutarate (α-KG). These genes were further shown to be involved in utilization of α-KG as a sole carbon source under anaerobic conditions. KguS/KguR contributed to the regulation of the target genes with the direct regulation by KguR verified using an electrophoretic mobility shift assay. In addition, oxygen deficiency positively modulated expression of kguS/kguR and its target genes. Taken altogether, this study describes the first UPEC-associated TCS that functions in controlling the utilization of α-ketoglutarate in vivo thereby facilitating UPEC adaptation to life inside the urinary tract.     

Phylogenetic Distribution and Prevalence of Genes Encoding Class I Integrons and CTX-M-15 Extended-Spectrum β-Lactamases in Escherichia coli Isolates from Healthy Humans in Chandigarh,India

Escherichia coli is generally considered as a commensal inhabitant of gastrointestinal tract of humans and animals. The aim of this study was to gain insight on the distribution of phylotypes and presence of genes encoding integrons, extended β-lactamases and resistance to other antimicrobials in the commensal E. coli isolates from healthy adults in Chandigarh, India. PCR and DNA sequencing were used for phylogenetic classification, detections of integrase genes, gene cassettes within the integron and extended β-lactamases. The genetic structure of E. coli revealed a non-uniform distribution of isolates among the seven phylogenetic groups with significant representation of group A. Integron-encoded integrases were detected in 25 isolates with class 1 integron-encoded intI1 integrase being in the majority (22 isolates). The gene cassettes identified were those for trimethoprim, streptomycin, spectinomycin and streptothricin. The dfrA12-orfF-aadA2 was the most commonly found gene cassette in intI1 positive isolates. Phenotypic assay for screening the potential ESBL producers suggested 16 isolates to be ESBL producers. PCR detection using gene-specific primers showed that 15 out of these 16 ESBL-producing E. coli harboured the bla _CTX-M-15 gene. Furthermore, molecular studies helped in characterizing the genes responsible for tetracycline, chloramphenicol and sulphonamides resistance. Collectively, our study outlines the intra-species phylogenetic structure and highlights the prevalence of class 1 integron and bla _CTX-M-15 in commensal E. coli isolates of healthy adults in Chandigarh, India. Our findings further reinforce the relevance of commensal E. coli strains on the growing burden of antimicrobial resistance.     

Impact of Host Age and Parity on Susceptibility to Severe Urinary Tract Infection in a Murine Model

The epidemiology and bacteriology of urinary tract infection (UTI) varies across the human lifespan, but the reasons for these differences are poorly understood. Using established monomicrobial and polymicrobial murine UTI models caused by uropathogenic Escherichia coli (UPEC) and/or Group B Streptococcus (GBS), we demonstrate age and parity as inter-related factors contributing to UTI susceptibility. Young nulliparous animals exhibited 10–100-fold higher bacterial titers compared to older animals. In contrast, multiparity was associated with more severe acute cystitis in older animals compared to age-matched nulliparous controls, particularly in the context of polymicrobial infection where UPEC titers were ∼1000-fold higher in the multiparous compared to the nulliparous host. Multiparity was also associated with significantly increased risk of chronic high titer UPEC cystitis and ascending pyelonephritis. Further evidence is provided that the increased UPEC load in multiparous animals required TLR4-signaling. Together, these data strongly suggest that the experience of childbearing fundamentally and permanently changes the urinary tract and its response to pathogens in a manner that increases susceptibility to severe UTI. Moreover, this murine model provides a system for dissecting these and other lifespan-associated risk factors contributing to severe UTI in at-risk groups.     

Detection of<Emphasis Type="Italic">pap</Emphasis>-,<Emphasis Type="Italic">sfa</Emphasis>- and<Emphasis Type="Italic">afa</Emphasis>-specific DNA sequences in<Emphasis Type="Italic">Escherichia coli</Emphasis> strains isolated from extraintestinal material

P-fimbriae, S-fimbriae and AFA-adhesins are virulence factors responsible for adherence ofEscherichia coli strains to extraintestinal host-cell surface. Detection ofpap-,sfa- andafa-specific sequences performed by PCR revealed 74%pap ⁺, 65%sfa ⁺, and 8.3%afa ⁺ strains in a group of 84 extraintestialE. coli isolates. Detection in a group of fecal strains showed 29%pap ⁺, 21%sfa ⁺ and 4%afa ⁺ strains.pap together withsfa were found as the most frequent combination (56%) among extraintestinal isolates probably due to localization ofpap-andsfa-operons on a common pathogenicity island. The occurrence ofafa-specific sequence among 56 urine strains was 11%, although noafa ⁺ strain was detected among 28 gynecological isolates. No strains with detected adhesin operons were found among twenty (24%) extraintestinalE. coli strains.     

Signature-Tagged Mutagenesis in a Chicken Infection Model Leads to the Identification of a Novel Avian Pathogenic Escherichia coli Fimbrial Adhesin

The extraintestinal pathogen, avian pathogenic E. coli (APEC), known to cause systemic infections in chickens, is responsible for large economic losses in the poultry industry worldwide. In order to identify genes involved in the early essential stages of pathogenesis, namely adhesion and colonization, Signature-tagged mutagenesis (STM) was applied to a previously established lung colonization model of infection by generating and screening a total of 1,800 mutants of an APEC strain IMT5155 (O2:K1:H5; Sequence type complex 95). The study led to the identification of new genes of interest, including two adhesins, one of which coded for a novel APEC fimbrial adhesin (Yqi) not described for its role in APEC pathogenesis to date. Its gene product has been temporarily designated ExPEC Adhesin I (EA/I) until the adhesin-specific receptor is identified. Deletion of the ExPEC adhesin I gene resulted in reduced colonization ability by APEC strain IMT5155 both in vitro and in vivo. Furthermore, complementation of the adhesin gene restored its ability to colonize epithelial cells in vitro. The ExPEC adhesin I protein was successfully expressed in vitro. Electron microscopy of an afimbriate strain E. coli AAEC189 over-expressed with the putative EA/I gene cluster revealed short fimbrial-like appendages protruding out of the bacterial outer membrane. We observed that this adhesin coding gene yqi is prevalent among extraintestinal pathogenic E. coli (ExPEC) isolates, including APEC (54.4%), uropathogenic E. coli (UPEC) (65.9%) and newborn meningitic E. coli (NMEC) (60.0%), and absent in all of the 153 intestinal pathogenic E. coli strains tested, thereby validating the designation of the adhesin as ExPEC Adhesin I. In addition, prevalence of EA/I was most frequently associated with the B2 group of the EcoR classification and ST95 complex of the multi locus sequence typing (MLST) scheme, with evidence of a positive selection within this highly pathogenic complex. This is the first report of the newly identified and functionally characterized ExPEC adhesin I and its significant role during APEC infection in chickens.     

Urinary Tract Infections in Kidney Transplant Patients Due to Escherichia coli and Klebsiella pneumoniae-Producing Extended-Spectrum β-Lactamases: Risk Factors and Molecular Epidemiology

Urinary tract infection (UTI) is a common complication after kidney transplantation, often associated to graft loss and increased healthcare costs. Kidney transplant patients (KTPs) are particularly susceptible to infection by Enterobacteriaceae-producing extended-spectrum β-lactamases (ESBLs). A retrospective case-control study was conducted to identify independent risk factors for ESBL-producing Escherichia coli and Klebsiella pneumoniae in non-hospitalized KTPs with UTI. Forty-nine patients suffering from UTI by ESBL-producing bacteria (ESBL-P) as case group and the same number of patients with UTI by ESBL negative (ESBL-N) as control-group were compared. Clinical data, renal function parameters during UTI episodes, UTI recurrence and relapsing rate, as well as risk factors for recurrence, molecular characterization of isolates and the respective antimicrobial susceptibility profile were evaluated. Diabetes mellitus (p <0.007), previous antibiotic prophylaxis (p=0.017) or therapy (p<0.001), previous UTI (p=0.01), relapsing infection (p=0.019) and patients with delayed graft function after transplant (p=0.001) represented risk factors for infection by ESBL positive Enterobacteriaceae in KTPs. Interestingly, the period of time between data of transplantation and data of UTI was shorter in case of ESBL-P case-group (28.8 months) compared with ESBL-N control-group (50.9 months). ESBL-producing bacteria exhibited higher resistance to fluoroquinolones (p=0.002), trimethoprim-sulfamethoxazole (p<0.001) and gentamicin (p<0.001). Molecular analysis showed that bla _CTX-M was the most common ESBL encoding gene (65.3%), although in 55.1% of the cases more than one ESBL gene was found. In 29.4% of K. pneumoniae isolates, three bla-genes (bla _CTX-M-bla _TEM-bla _SHV) were simultaneously detected. Low estimated glomerular filtration rate (p=0.009) was found to be risk factor for UTI recurrence. Over 60% of recurrent UTI episodes were caused by genetically similar strains. UTI by ESBL-producing Enterobacteriaceae in KTPs represent an important clinical challenge regarding not only hospitalized patients but also concerning outpatients.     

Comparative Analysis of Virulence Genes, Genetic Diversity, and Phylogeny of Commensal and Enterotoxigenic Escherichia coli Isolates from Weaned Pigs

If the acquisition of virulence genes (VGs) for pathogenicity were not solely acquired through horizontal gene transfers of pathogenicity islands, transposons, and phages, then clonal clusters of enterotoxigenic Escherichia coli (ETEC) would contain few or even none of the VGs found in strains responsible for extraintestinal infections. To evaluate this possibility, 47 postweaning diarrhea (PWD) ETEC strains from different geographical origins and 158 commensal E. coli isolates from the gastrointestinal tracts of eight group-housed healthy pigs were screened for 36 extraintestinal and 18 enteric VGs using multiplex PCR assays. Of 36 extraintestinal VGs, only 8 were detected (fimH, traT, fyuA, hlyA, kpsMtII, k5, iha, and ompT) in the ETEC collection. Among these, hlyA (α-hemolysin) and iha (nonhemagglutinating adhesin) occurred significantly more frequently among the ETEC isolates than in the commensal isolates. Clustering analysis based on the VG profiles separated commensal and ETEC isolates and even differentiated serogroup O141 from O149. On the other hand, pulsed-field gel electrophoresis (PFGE) successfully clustered ETEC isolates according to both serotype and geographical origin. In contrast, the commensal isolates were heterogeneous with respect to both serotype and DNA fingerprint. This study has validated the use of VG profiling to examine pathogenic relationships between porcine ETEC isolates. The clonal relationships of these isolates can be further clarified by PFGE fingerprinting. The presence of extraintestinal VGs in porcine ETEC confirmed the hypothesis that individual virulence gene acquisitions can occur concurrently against a background of horizontal gene transfers of pathogenicity islands. Over time, this could enable specific clonotypes to respond to host selection pressure and to evolve into new strains with increased virulence.     

Virulence characteristics and antimicrobial susceptibility of uropathogenic Escherichia coli strains

Eight virulence factors associated with uropathogenic Escherichia coli (UPEC) were investigated in 204 clinical isolates of E. coli recovered from urine cultures at counts ≥10(5). The bacteria were classified into two groups according to the number of leukocytes in urine samples from which they were isolated: group I ≤8 leukocytes/hpf, 104 strains; group II >8 leukocytes/hpf, 100 strains. Two multiplex PCR systems were used to detect genes encoding adhesin P (pap), adhesin S (sfa), afimbrial adhesin I (afa), siderophore aerobactin (aer), alpha-hemolysin (hly), cytotoxic necrotizing factor type 1 (cnf1), and traT associated with serum resistance. The PAI marker for the virulence island identified in strains CFT072 and CVD432, a marker of enteroaggregative E. coli, was also investigated using PCR. The susceptibility profile of E. coli strains was determined by disk diffusion method. Ninety percent UPEC showed at least one of the virulence genes, the prevalence being traT (76%), aer (41%), PAI (32%), sfa (26%), pap (25%), cnf1 (18%), afa (6%), and hly (5%). There was no significant difference in the distribution of virulence genes between groups I and II. A significantly higher degree of virulence was detected in UPEC group II. The CVD432 gene was not detected in any of the UPECs. Fifty-nine percent of the strains were resistant to at least one of the antimicrobials that we tested; the most common being resistance to ampicillin (51%) and trimethoprim-sulfamethoxazole (44%).     

Impact of UV and Peracetic Acid Disinfection on the Prevalence of Virulence and Antimicrobial Resistance Genes in Uropathogenic Escherichia coli in Wastewater Effluents

Wastewater discharges may increase the populations of pathogens, including Escherichia coli, and of antimicrobial-resistant strains in receiving waters. This study investigated the impact of UV and peracetic acid (PAA) disinfection on the prevalence of virulence and antimicrobial resistance genes in uropathogenic Escherichia coli (UPEC), the most abundant E. coli pathotype in municipal wastewaters. Laboratory disinfection experiments were conducted on wastewater treated by physicochemical, activated sludge, or biofiltration processes; 1,766 E. coli isolates were obtained for the evaluation. The target disinfection level was 200 CFU/100 ml, resulting in UV and PAA doses of 7 to 30 mJ/cm² and 0.9 to 2.0 mg/liter, respectively. The proportions of UPECs were reduced in all samples after disinfection, with an average reduction by UV of 55% (range, 22% to 80%) and by PAA of 52% (range, 11% to 100%). Analysis of urovirulence genes revealed that the decline in the UPEC populations was not associated with any particular virulence factor. A positive association was found between the occurrence of urovirulence and antimicrobial resistance genes (ARGs). However, the changes in the prevalence of ARGs in potential UPECs were different following disinfection, i.e., UV appears to have had no effect, while PAA significantly reduced the ARG levels. Thus, this study showed that both UV and PAA disinfections reduced the proportion of UPECs and that PAA disinfection also reduced the proportion of antimicrobial resistance gene-carrying UPEC pathotypes in municipal wastewaters.     

Escherichia coli from retail meats carry genes associated with uropathogenic Escherichia coli, but are weakly invasive in human bladder cell culture

Aims: The aim of this study was to determine the uropathogenic potential of Escherichia coli isolated from retail meats. Methods and Results: Two hundred E. coli isolates recovered from retail meats, which were previously identified molecularly as extraintestinal pathogenic E. coli, were investigated for the presence of 21 uropathogenic E. coli (UPEC) virulence‐associated genes. Twenty‐three E. coli isolates were selected based on their serogroups and the number of virulence genes they contained, and further characterized using multilocus sequence typing, and by tissue culture assays for adherence to and invasion of T‐24 human bladder cells and for their induction of interleukin (IL)‐6 secretion. All virulence genes tested, except afa/dra and hlyD, were detected among the E. coli isolates. Multilocus sequence typing analysis of 23 selected isolates revealed that 17 isolates belonged to STs associated with human UPEC. Nearly all 23 isolates exhibited lower level of adherence and invasion compared to a clinical strain, UPEC CFT073. Conclusions: These observations suggested that a small proportion of E. coli isolates from retail meats carry uropathogenic associated virulence genes and thus may serve as a reservoir of these genes to UPEC in the human intestine. Their virulence potential seemed limited as they were only weakly invasive in human bladder cell culture. Significance and Impact of the Study: These findings support the hypothesis that retail meat E. coli may play a role in relation to urinary tract infection (UTI) and may be considered in development of a UTI prevention strategy.     

Phenotypic and Molecular Characterization of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Bangladesh

Background

Resistance to cephalosporins in Enterobacteriaceae is mainly due to the production of extended-spectrum beta-lactamase (ESBL). Little is known about ESBL-producing bacteria in Bangladesh. Therefore, the study presents results of phenotypic and molecular characterization of ESBL-producing Escherichia coli from hospitals in Bangladesh.

Methods

A total of 339 E. coli isolated from patients with urinary tract and wound infections attending three different medical hospitals in urban and rural areas of Bangladesh between 2003–2007 were screened for ESBL-production by the double disk diffusion test. Isolates with ESBL-phenotype were further characterized by antibiotic susceptibility testing, PCR and sequencing of different β-lactamase and virulence genes, serotyping, and XbaI-macrorestriction followed by pulsed-field gel electrophoresis (PFGE).

Results

We identified 40 E. coli with ESBL phenotype. These isolates were resistant to ceftriaxone, ceftazidime, cefotaxime, aztreonam, cefepime, and nalidixic acid but remained susceptible to imipenem. All but one isolate were additionally resistant to ciprofloxacin, and 3 isolates were resistant to cefoxitin. ESBL genes of blaCTX-M-1-group were detected in all isolates; blaTEM-type and blaOXA-1-type genes were detected in 33 (82.5%) and 19 (47.5%) isolates, respectively. Virulence genes that are present in diarrhoeagenic E. coli were not found. Class-1 integron was present in 20 (50%) isolates. All the ESBL-producing E. coli isolates harbored plasmids ranging between 1.1 and 120 MDa. PFGE-typing revealed 26 different pulsotypes, but identical pulsotype showed 6 isolates of serotype O25:H4.

Conclusion

The prevalence of multidrug-resistant ESBL-producing E. coli isolates appears to be high and the majority of the isolates were positive for bla _CTX-M. Although there was genetic heterogeneity among isolates, presence of a cluster of isolates belonging to serotype O25:H4 indicates dissemination of the pandemic uropathogenic E. coli clone in Bangladesh.     

The CTX-M-15-Producing Escherichia coli Clone O25b: H4-ST131 Has High Intestine Colonization and Urinary Tract Infection Abilities

Increasing numbers of pyelonephritis-associated uropathogenic Escherichia coli (UPEC) are exhibiting high resistance to antibiotic therapy. They include a particular clonal group, the CTX-M-15-producing O25b:H4-ST131 clone, which has been shown to have a high dissemination potential. Here we show that a representative isolate of this E. coli clone, referred to as TN03, has enhanced metabolic capacities, acts as a potent intestine- colonizing strain, and displays the typical features of UPEC strains. In a modified streptomycin-treated mouse model of intestinal colonization where streptomycin was stopped 5 days before inoculation, we show that TN03 outcompetes the commensal E. coli strains K-12 MG1655, IAI1, and ED1a at days 1 and 7. Using an experimental model of ascending UTI in C3H/HeN mice, we then show that TN03 colonized the urinary tract. One week after the transurethral inoculation of the TN03 isolates, the bacterial loads in the bladder and kidneys were significantly greater than those of two other UPEC strains (CFT073 and HT7) belonging to the same B2 phylogenetic group. The differences in bacterial loads did not seem to be directly linked to differences in the inflammatory response, since the intrarenal expression of chemokines and cytokines and the number of polymorphonuclear neutrophils attracted to the site of inflammation was the same in kidneys colonized by TN03, CFT073, or HT7. Lastly, we show that in vitro TN03 has a high maximum growth rate in both complex (Luria-Bertani and human urine) and minimum media. In conclusion, our findings indicate that TN03 is a potent UPEC strain that colonizes the intestinal tract and may persist in the kidneys of infected hosts.     

Comparison of Virulence Gene Profiles of Escherichia coli Strains Isolated from Healthy and Diarrheic Swine

A combination of uni- and multiplex PCR assays targeting 58 virulence genes (VGs) associated with Escherichia coli strains causing intestinal and extraintestinal disease in humans and other mammals was used to analyze the VG repertoire of 23 commensal E. coli isolates from healthy pigs and 52 clinical isolates associated with porcine neonatal diarrhea (ND) and postweaning diarrhea (PWD). The relationship between the presence and absence of VGs was interrogated using three statistical methods. According to the generalized linear model, 17 of 58 VGs were found to be significant (P < 0.05) in distinguishing between commensal and clinical isolates. Nine of the 17 genes represented by iha, hlyA, aidA, east1, aah, fimH, iroN_{E. coli}, traT, and saa have not been previously identified as important VGs in clinical porcine isolates in Australia. The remaining eight VGs code for fimbriae (F4, F5, F18, and F41) and toxins (STa, STb, LT, and Stx2), normally associated with porcine enterotoxigenic E. coli. Agglomerative hierarchical algorithm analysis grouped E. coli strains into subclusters based primarily on their serogroup. Multivariate analyses of clonal relationships based on the 17 VGs were collapsed into two-dimensional space by principal coordinate analysis. PWD clones were distributed in two quadrants, separated from ND and commensal clones, which tended to cluster within one quadrant. Clonal subclusters within quadrants were highly correlated with serogroups. These methods of analysis provide different perspectives in our attempts to understand how commensal and clinical porcine enterotoxigenic E. coli strains have evolved and are engaged in the dynamic process of losing or acquiring VGs within the pig population.     

Zoonotic Potential of Escherichia coli Isolates from Retail Chicken Meat Products and Eggs

Chicken products are suspected as a source of extraintestinal pathogenic Escherichia coli (ExPEC), which causes diseases in humans. The zoonotic risk to humans from chicken-source E. coli is not fully elucidated. To clarify the zoonotic risk posed by ExPEC in chicken products and to fill existing knowledge gaps regarding ExPEC zoonosis, we evaluated the prevalence of ExPEC on shell eggs and compared virulence-associated phenotypes between ExPEC and non-ExPEC isolates from both chicken meat and eggs. The prevalence of ExPEC among egg-source isolates was low, i.e., 5/108 (4.7%). Based on combined genotypic and phenotypic screening results, multiple human and avian pathotypes were represented among the chicken-source ExPEC isolates, including avian-pathogenic E. coli (APEC), uropathogenic E. coli (UPEC), neonatal meningitis E. coli (NMEC), and sepsis-associated E. coli (SEPEC), as well as an undefined ExPEC group, which included isolates with fewer virulence factors than the APEC, UPEC, and NMEC isolates. These findings document a substantial prevalence of human-pathogenic ExPEC-associated genes and phenotypes among E. coli isolates from retail chicken products and identify key virulence traits that could be used for screening.     

Occurrence of ESBL-Producing Escherichia coli in Livestock and Farm Workers in Mecklenburg-Western Pomerania,Germany

In recent years, extended-spectrum β-lactamases (ESBL) producing bacteria have been found in livestock, mainly as asymptomatic colonizers. The zoonotic risk for people working in close contact to animal husbandry has still not been completely assessed. Therefore, we investigated the prevalence of ESBL-producing Escherichia spp. in livestock animals and workers to determine the potential risk for an animal-human cross-transmission.In Mecklenburg-Western Pomerania, northeast Germany, inguinal swabs of 73 individuals with livestock contact from 23 different farms were tested for ESBL-producing Escherichia spp. Two pooled fecal samples per farm of animal origin from 34 different farms (17 pig farms, 11 cattle farms, 6 poultry farms) as well as cloacal swabs of 10 randomly selected broilers or turkeys were taken at each poultry farm. For identification, selective chromogenic agar was used after an enrichment step. Phenotypically ESBL-producing isolates (n = 99) were tested for CTX-M, OXA, SHV and TEM using PCR, and isolates were further characterized using multilocus sequence typing (MLST). In total, 61 diverse isolates from different sources and/or different MLST/PCR results were acquired. Five farm workers (three from cattle farms and two from pig farms) harbored ESBL-producing E. coli. All human isolates harbored the CTX-M β-lactamase; TEM and OXA β-lactamases were additionally detected in two, resp. one, isolates. ESBL-producing Escherichia spp. were found in fecal samples at pig (15/17), cattle (6/11) and poultry farms (3/6). In total, 70.6% (24/36) of the tested farms were ESBL positive. Furthermore, 9 out of 60 cloacal swabs turned out to be ESBL positive. All isolated ESBL-producing bacteria from animal sources were E. coli, except for one E. hermanii isolate. CTX-M was the most prevalent β-lactamase at cattle and pig farms, while SHV predominated in poultry. One human isolate shared an identical MLST sequence type (ST) 3891 and CTX-M allele to the isolate found in the cattle fecal sample from the same farm, indicating a zoonotic transfer. Two other pairs of human-pig and human-cattle E. coli isolates encoded the same ESBL genes but did not share the same MLST ST, which may indicate horizontal resistance gene transfer. In summary, the study shows the high prevalence of ESBL-producing E.coli in livestock in Mecklenburg- Western Pomerania and provides the risk of transfer between livestock and farm workers.     

Extended-Spectrum β-Lactamase- and Carbapenemase-Producing Enterobacteriaceae Isolated from Egyptian Patients with Suspected Blood Stream Infection

ObjectivesThe aim of the study was to investigate the prevalence of extended-spectrum β-lactamase and carbapenemase production among Enterobacteriaceae isolated from Egyptian patients with suspected blood stream infection.MethodsNinety-four Enterobacteriaceae blood culture isolates from Egyptian patients with suspected blood stream infection were collected, one isolate per patient. Identification of bacterial isolates was performed with MALDI-TOF (MS-based Vitek MS system, bioMerieux). Screening for ESBLs and carbapenemases production was done with the Vitek 2 system (bioMérieux). ESBL production was confirmed using the combined disk diffusion method for cefotaxime, ceftazidime, and cefepime, all with and without clavulanic acid (Rosco). Real-time PCR and sequencing were used to characterize the resistance genes. The phylogenetic groups of E. coli were identified by a PCR-based method.ResultsOf the 94 Enterobacteriaceae isolates 46 (48.93%) showed an ESBL phenotype. One Enterobacter spp isolate was ESBL-producer and meropenem-resistant. The genetic analysis showed that CTX-M was present in 89.13% (41/46) of the ESBL-producing Enterobacteriaceae, whereas TEM and SHV were detected in 56.52% (26/46) and 21.74% (10/46) respectively (47.83%) of the ESBL-producing isolates were multidrug resistant (MDR). Eleven out of 30 ESBL-producing E-coli isolates were assigned to phylogroup B2, followed by groups B1 (8 isolates), A (6 isolates) and D (5 isolates).ConclusionsThe high ESBL-E rates (48.93%) found in this study together with the identification of one carbapenem-resistant Enterobacter spp isolate is worrisome. Our results indicate that systems for monitoring and detection of ESBL-producing bacteria in Egyptian hospitals have to be established. Also strict hospital infection control policies with the restriction of the consumption of extended-spectrum cephalosporins are necessary.