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Objective In this report, we aim to describe the epidemiology of extended-spectrum cephalosporin-resistant (ESC-R) and carbapenem-resistant (CR) Enterobacteriaceae infections in children. Methods ESC-R and CR Enterobacteriaceae isolates from normally sterile sites of patients aged

Background Limited studies have investigated the microbial diversity of wild marine mammals. Objectives This study characterized Escherichia coli isolates collected from fresh faecal samples of endangered southern resident killer whales (Orcinus orca) located by detection dogs. Methods WGS of each strain was done to determine ST (using MLST), clonotype (C:H), antimicrobial resistance and virulence profile. Conjugation experiments were done to determine the mobility of the tet(B) tetracycline resistance gene. Results All isolates belonged to extraintestinal pathogenic E. coli (ExPEC) clonal lineages ST73 (8/9) and ST127 (1/9), often associated with human community-acquired urinary tract disease. Clonotyping using fumC and fimH alleles showed divergence in clonal lineages, with ST73 isolates belonging to the C24:H10 clade and the ST127 isolate belonging to C14:H2. The eight ST73 isolates carried multiple acquired antibiotic resistance genes, including aadA1, sul1 and tet(B), encoding aminoglycoside, sulphonamide and tetracycline resistance, respectively. Conjugative transfer of the resistance gene tet(B) was observed for three of the eight isolates. ST127 did not carry any of these acquired resistance genes. Virulence-associated genes identified included those encoding adhesins (iha, papC, sfaS), toxins (sat, vat, pic, hlyA, cnf1), siderophores (iutA, fyuA, iroN, ireA), serum survival/protectins (iss, ompT), capsule (kpsM) and pathogenicity island marker (malX). Conclusions Orca whales can carry antibiotic-resistant potentially pathogenic strains of E. coli. Possible sources include contamination of the whale’s environment and/or food. It is unknown whether these isolates cause disease in southern resident killer whales, which could contribute to the ongoing decline of this critically endangered population.

Multi-locus sequence typing (MLST) is a useful system for phylogenetic and epidemiological studies of multidrug-resistant Escherichiacoli. Most studies utilize a seven-locus MLST, but an alternate two-locus typing method (fumC and fimH; CH typing) has been proposed that may offer a similar degree of discrimination at lower cost. Herein, we compare CH typing to the standard seven-locus method for typing commensal E. coli isolates from dairy cattle. In addition, we evaluated alternative combinations of eight loci to identify combinations that maximize discrimination and congruence with standard seven-locus MLST among commensal E. coli while minimizing the cost. We also compared both methods when used for typing uropathogenic E. coli (UPEC). CH typing was less discriminatory for commensal E. coli than the standard seven-locus method (Simpson's Index of Diversity=0.933 [0.902-0.964] and 0.97 [0.96-0.979], respectively). Combining fimH with housekeeping gene loci improved discriminatory power for commensal E. coli from cattle but resulted in poor congruence with MLST. We found that a four-locus typing method including the housekeeping genes adk, purA, gyrB and recA could be used to minimize cost without sacrificing discriminatory power or congruence with Achtman seven-locus MLST when typing commensal E. coli.

The first Oregon case of New Delhi metallo-β-lactamase-1 (NDM-1)-producing Escherichia coli was reported during November 2013. Epidemiologic investigation revealed only local outpatient medical care and no travel outside Oregon for both the patient and his household contact. Environmental sampling discovered a matching isolate from the patient’s household vacuum cleaner, suggesting environmental persistence.

We describe the rapid and ongoing emergence across multiple US cities of a new multidrug-resistant Escherichia coli clone-sequence type (ST) 1193-resistant to fluoroquinolones (100%), trimethoprim-sulfamethoxazole (55%), and tetracycline (53%). ST1193 is associated with younger adults (age

Objectives In vitro trends of cefazolin and ceftriaxone susceptibilities from pediatric clinical isolates of methicillin-susceptible Staphylococcus aureus (MSSA) between 2011 and 2016 were analyzed for surveillance. Methods Our laboratory continues to use agar disk diffusion for staphylococcal susceptibilities applying Clinical Laboratory Standard Institute’s 2012 breakpoints. Results A total of 3992 MSSA clinical isolates in the last 6 years were analyzed for their in vitro cefazolin and ceftriaxone susceptibilities. While all MSSA isolates exhibited cefazolin susceptibilities within the “susceptible” zone range, there have been a proportion of isolates with ceftriaxone susceptibilities falling in “intermediate” zones, ranging from 2.6% in 2011 to 8.3% in 2016. Conclusions Cefazolin continues to be the recommended agent for MSSA treatment at our institution, reflected by the finding that only 2% (6/321) of patients who received ceftriaxone as definitive therapy for MSSA bacteremia during the study period. We have confirmed the cefoxitin-predicted MSSA susceptibility to cefazolin, but have found concerning drifts in ceftriaxone susceptibilities by continued in vitro monitoring over the last 6 years. Electronic supplementary material The online version of this article (10.1186/s12941-018-0257-x) contains supplementary material, which is available to authorized users.

Background Escherichia coli sequence type (ST) 131-H30 is a globally important pathogen implicated in rising rates of multidrug resistance among E. coli causing extraintestinal infections. Previous studies have focused on adults, leaving the epidemiology of H30 among children undefined. Methods We used clinical data and isolates from a case-control study of extended-spectrum cephalosporin-resistant E. coli conducted at 4 US children's hospitals to estimate the burden and identify host correlates of infection with H30. H30 isolates were identified using 2-locus genotyping; host correlates were examined using log-binomial regression models stratified by extended-spectrum cephalosporin resistance status. Results A total of 339 extended-spectrum cephalosporin-resistant and 1008 extended-spectrum cephalosporin-susceptible E. coli isolates were available for analyses. The estimated period prevalence of H30 was 5.3% among all extraintestinal E. coli isolates (95% confidence interval [CI], 4.6%-7.1%); H30 made up 43.3% (81/187) of extended-spectrum β-lactamase (ESBL)-producing isolates in this study. Host correlates of infection with H30 differed by extended-spectrum cephalosporin resistance status: Among resistant isolates, age ≤5 years was positively associated with H30 infection (relative risk [RR], 1.83 [95% CI, 1.19-2.83]); among susceptible isolates, age ≤5 years was negatively associated with H30 (RR, 0.48 [95% CI, .27-.87]), while presence of an underlying medical condition was positively associated (RR, 4.49 [95% CI, 2.43-8.31]). Conclusions ST131-H30 is less common among extraintestinal E. coli collected from children compared to reported estimates among adults, possibly reflecting infrequent fluoroquinolone use in pediatrics; however, it is similarly dominant among ESBL-producing isolates. The H30 subclone appears to disproportionately affect young children relative to other extended-spectrum cephalosporin-resistant E. coli.

BackgroundE. coli ST131-H30 is a globally important pathogen implicated in rising rates of multidrug resistance among E. coli causing extraintestinal infections. Previous studies have focused on adults, leaving the epidemiology of H30 among children undefined.MethodsWe used clinical data and isolates from a case-control study of extended-spectrum cephalosporin-resistant E. coli conducted at four US children’s hospitals to estimate the burden and identify host correlates of infection with H30. H30 isolates were identified using two-locus genotyping; host correlates were examined using log-binomial regression models stratified by extended-spectrum cephalosporin resistance status.ResultsA total of 339 extended-spectrum cephalosporin-resistant and 1008 extended-spectrum cephalosporin-susceptible E. coli isolates were available for analyses. The estimated period prevalence of H30 was 5.3% among all extraintestinal E. coli isolates (95% confidence interval [CI] 4.6%-7.1%); H30 made up 43.3% (81/187) of ESBL-producing isolates in this study. Host correlates of infection with H30 differed by extended-spectrum cephalosporin resistance status: among resistant isolates, age ≤5 years was positively associated with H30 infection (relative risk [RR] 1.83, 95% CI 1.19-2.83); among susceptible isolates, age ≤5 years was negatively associated with H30 (RR 0.48, 95% CI 0.27-0.87), while presence of an underlying medical condition was positively associated (RR 4.49, 95% CI 2.43-8.31).ConclusionsST131-H30 is less common among extraintestinal E. coli collected from children compared to reported estimates among adults, possibly reflecting infrequent fluoroquinolone use in pediatrics; however, it is similarly dominant among ESBL-producing isolates. The H30 subclone appears to disproportionately affect young children relative to other extendedspectrum cephalosporin-resistant E. coli.SummaryST131-H30 was responsible for 5.3% of all extraintestinal E. coli infections and 43.3% of ESBL-producing extraintestinal E. coli infections among US children. The clinical and demographic correlates of infection with ST131-H30 differed between extended-spectrum cephalosporin-resistant and -sensitive isolates.

The objective of this study was to assess the association between previous antibiotic use, particularly long-term prophylaxis, and the occurrence of subsequent resistant infections in children with index infections due to extended-spectrum-cephalosporin-resistant Enterobacteriaceae . We also investigated the concordance of the index and subsequent isolates. Extended-spectrum-cephalosporin-resistant Escherichia coli and Klebsiella spp. isolated from normally sterile sites of patients aged fumC-fimH ( E. coli ) or tonB ( Klebsiella pneumoniae ) type were identical to those of the index isolate. In total, 323 patients had 396 resistant isolates; 45 (14%) patients had ≥1 subsequent resistant infection, totaling 73 subsequent resistant isolates. The median time between the index and first subsequent infections was 123 (interquartile range, 43 to 225) days. In multivariable Cox proportional hazards analyses, patients were 2.07 times as likely to have a subsequent resistant infection (95% confidence interval, 1.11 to 3.87) if they received prophylaxis in the 30 days prior to the index infection. In 26 (58%) patients, all subsequent isolates were concordant with their index isolate, and 7 (16%) additional patients had at least 1 concordant subsequent isolate. In 12 of 17 (71%) patients with E. coli sequence type 131 (ST131)-associated type 40-30, all subsequent isolates were concordant. Subsequent extended-spectrum-cephalosporin-resistant infections are relatively frequent and are most commonly due to bacterial strains concordant with the index isolate. Further study is needed to assess the role prophylaxis plays in these resistant infections.

Background Antibiotic-resistant E. coli (EC) infections represent a major cause of morbidity and mortality, and pose a challenge to antibiotic stewardship. At present, clinicians in outpatient facilities may not have access to local antibiogram data to guide stewardship. Additionally, antibiotic resistance may vary between types of outpatient practices. Methods Using the database of a major clinical reference lab, this study analyzed several years of antibiotic susceptibility results for outpatient urinary EC isolates from Washington State. We compared rates of resistance to antibiotics between different types of outpatient practices, categorized using a modification of published ambulatory practice categories. Logistic regression was used to examine the association of outpatient practice type with antibiotic resistance, controlling year, sex, and age. Results After adjusting for year, sex, and age, logistic regression found significantly higher odds of resistance in urology compared with the reference groups of general family practice for ampicillin (OR 1.35), ciprofloxacin (OR 2.27), trimethoprim-sulfa (OR 1.51) and gentamicin (OR 1.73). We also saw increased odds of resistance to ciprofloxacin in patients from an oncology clinic (OR 1.56) as well as patients from “All other specialties” (OR 1.37). A lower odds of resistance was found in OBGYN clinics for ampicillin (OR 0.86), trimethoprim-sulfa (0.81) while a greater odds or resistance in OBGYN clinics was found for nitrofurantoin (OR 1.36). Conclusion Antibiotic resistance in EC urinary isolates can vary across types of outpatient practices according to clinical practice type. This may reflect differences in patient morbidity and/or differences in antibiotic stewardship practices and deserves further investigation. Patients with recurrent cases of resistant UTIs are generally referred to a urologist, and this was reflected in our data as there a higher odds of resistance was found in urology clinics. Similarly, we found higher odds of resistance into nitrofurantoin, a commonly prescribed antibiotic for UTIs in pregnant women, in OBGYN clinics that may reflect prescribing practices. Use of clinical data to create facility and specialty-specific antibiograms in outpatient settings may enable improved and “precise” antibiotic stewardship. Disclosures All authors: No reported disclosures.

The ability to identify bacterial pathogens at the subspecies level in clinical diagnostics is currently limited. We investigated whether splitting Escherichia coli species into clonal groups (clonotypes) predicts antimicrobial susceptibility or clinical outcome. A total of 1,679 extraintestinal E. coli isolates (collected from 2010 to 2012) were collected from one German and 5 U.S. clinical microbiology laboratories. Clonotype identity was determined by fumC and fimH (CH) sequencing. The associations of clonotype with antimicrobial susceptibility and clinical variables were evaluated. CH typing divided the isolates into >200 CH clonotypes, with 93% of the isolates belonging to clonotypes with ≥2 isolates. Antimicrobial susceptibility varied substantially among clonotypes but was consistent across different locations. Clonotype-guided antimicrobial selection significantly reduced “drug-bug” mismatch compared to that which occurs with the use of conventional empirical therapy. With trimethoprim-sulfamethoxazole and fluoroquinolones, the drug-bug mismatch was predicted to decrease 62% and 78%, respectively. Recurrent or persistent urinary tract infection and clinical sepsis were significantly correlated with specific clonotypes, especially with CH40-30 (also known as H30), a recently described clonotype within sequence type 131 (ST131). We were able to clonotype directly from patient urine samples within 1 to 3 h of obtaining the specimen. In E. coli , subspecies-level identification by clonotyping can be used to significantly improve empirical predictions of antimicrobial susceptibility and clinical outcomes in a timely manner.

We describe a 16-year-old neutropenic patient from the Middle East with bloodstream infection caused by two carbapenemase-producing Escherichia coli isolates that we characterized by whole-genome sequencing. While one displayed meropenem resistance and was bla NDM positive, the other demonstrated meropenem susceptibility yet harbored bla OXA181 (which encodes a bla OXA48 -like enzyme). This report highlights the challenge of laboratory detection of bla OXA48 -like enzymes and the clinical implications of genotypic resistance detection in carbapenemase-producing Enterobacteriaceae .

FimH, the adhesive subunit of type 1 fimbriae expressed by many enterobacteria, mediates mannose-sensitive binding to target host cells. At the same time, fine receptor-structural specificities of FimH from different species can be substantially different, affecting bacterial tissue tropism and, as a result, the role of the particular fimbriae in pathogenesis. In this study, we compared functional properties of the FimH proteins from Escherichia coli and Klebsiella pneumoniae , which are both 279 amino acids in length but differ by some ∼15% of residues. We show that K. pneumoniae FimH is unable to mediate adhesion in a monomannose-specific manner via terminally exposed Manα(1-2) residues in N-linked oligosaccharides, which are the structural basis of the tropism of E. coli FimH for uroepithelial cells. However, K. pneumoniae FimH can bind to the terminally exposed Manα(1-3)Manβ(1-4)GlcNAcβ1 trisaccharide, though only in a shear-dependent manner, wherein the binding is marginal at low shear force but enhanced sevenfold under increased shear. A single mutation in the K. pneumoniae FimH, S62A, converts the mode of binding from shear dependent to shear independent. This mutation has occurred naturally in the course of endemic circulation of a nosocomial uropathogenic clone and is identical to a pathogenicity-adaptive mutation found in highly virulent uropathogenic strains of E. coli , in which it also eliminates the dependence of E. coli binding on shear. The shear-dependent binding properties of the K. pneumoniae and E. coli FimH proteins are mediated via an allosteric catch bond mechanism. Thus, despite differences in FimH structure and fine receptor specificity, the shear-dependent nature of FimH-mediated adhesion is highly conserved between bacterial species, supporting its remarkable physiological significance.

Core genes comprising the ubiquitous backbone of bacterial genomes are not subject to frequent horizontal transfer and generally are not thought to contribute to the adaptive evolution of bacterial pathogens. We determined, however, that at least one-third and possibly more than one-half of the core genes in Escherichia coli genomes are targeted by repeated replacement substitutions in the same amino acid positions—hotspot mutations. Occurrence of hotspot mutations is driven by positive selection, as their rate is significantly higher than expected by random chance alone, and neither intragenic recombination nor increased mutability can explain the observed patterns. Also, commensal E. coli strains have a significantly lower frequency of mutated genes and mutations per genome than pathogenic strains. E. coli strains causing extra-intestinal infections accumulate hotspot mutations at the highest rate, whereas the highest total number of mutated genes has been found among Shigella isolates, suggesting the pathoadaptive nature of such mutations. The vast majority of hotspot mutations are of recent evolutionary origin, implying short-term positive selection, where adaptive mutations emerge repeatedly but are not sustained in natural circulation for long. Such pattern of dynamics is consistent with source-sink model of virulence evolution.

FimH is an adhesive subunit of type 1 fimbriae expressed by different enterobacterial species. The enteric bacterium Klebsiella pneumoniae is an environmental organism that is also a frequent cause of sepsis, urinary tract infection (UTI), and liver abscess. Type 1 fimbriae have been shown to be critical for the ability of K. pneumoniae to cause UTI in a murine model. We show here that the K. pneumoniae fimH gene is found in 90% of strains from various environmental and clinical sources. The fimH alleles exhibit relatively low nucleotide and structural diversity but are prone to frequent horizontal-transfer events between different bacterial clones. Addition of the fimH locus to multiple-locus sequence typing significantly improved the resolution of the clonal structure of pathogenic strains, including the K1 encapsulated liver isolates. In addition, the K. pneumoniae FimH protein is targeted by adaptive point mutations, though not to the same extent as FimH from uropathogenic Escherichia coli or TonB from the same K. pneumoniae strains. Such adaptive mutations include a single amino acid deletion from the signal peptide that might affect the length of the fimbrial rod by affecting FimH translocation into the periplasm. Another FimH mutation (S62A) occurred in the course of endemic circulation of a nosocomial uropathogenic clone of K. pneumoniae. This mutation is identical to one found in a highly virulent uropathogenic strain of E. coli , suggesting that the FimH mutations are pathoadaptive in nature. Considering the abundance of type 1 fimbriae in Enterobacteriaceae , our present finding that fimH genes are subject to adaptive microevolution substantiates the importance of type 1 fimbria-mediated adhesion in K. pneumoniae .

Enterotoxigenic Escherichia coli (ETEC) strains produce a type IV pilus named Longus. We identified a 16-gene cluster involved in the biosynthesis of Longus that has 57 to 95% identity at the protein level to CFA/III, another type IV pilus of ETEC. Alleles of the Longus structural subunit gene lngA demonstrate a diversity of 12 to 19% at the protein level with strong positive selection for point replacements and horizontal transfer.

OBJECTIVES High levels of β-lactamase production can impact treatment with a β-lactam/β-lactamase inhibitor combination. Goals of this study were to: (i) compare the mRNA and protein levels of CTX-M-15- and CTX-M-14-producing Escherichia coli from 18 different STs and 10 different phylotypes; (ii) evaluate the mRNA half-lives and establish a role for chromosomal- and/or plasmid-encoded factors; and (iii) evaluate the zones of inhibition for piperacillin/tazobactam and ceftolozane/tazobactam. METHODS Disc diffusion was used to establish zone size. RNA analysis was accomplished using real-time RT-PCR and CTX-M protein levels were evaluated by immunoblotting. Clinical isolates, transformants and transconjugants were used to evaluate mRNA half-lives. RESULTS mRNA levels of CTX-M-15 were up to 165-fold higher compared with CTX-M-14. CTX-M-15 protein levels were 2-48-fold less than their respective transcript levels, while CTX-M-14 protein production was comparable to the observed transcript levels. Nineteen of 25 E. coli (76%) had extended CTX-M-15 mRNA half-lives of 5-15 min and 16 (100%) CTX-M-14 isolates had mRNA half-lives of

BACKGROUNDWe identified an outbreak of AmpC–producingEscherichia coliinfections resistant to third-generation cephalosporins and carbapenems (CR) among 7 patients who had undergone endoscopic retrograde cholangiopancreatography at hospital A during November 2012–August 2013. Gene sequencing revealed a shared novel mutation in ablaCMYgene and a distinctivefumC/ fimHtyping profile.OBJECTIVETo determine the extent and epidemiologic characteristics of the outbreak, identify potential sources of transmission, design and implement infection control measures, and determine the association between the CRE. coliand AmpCE. colicirculating at hospital A.METHODSWe reviewed laboratory, medical, and endoscopy reports, and endoscope reprocessing procedures. We obtained cultures from endoscopes after reprocessing as well as environmental samples and conducted pulsed-field gel electrophoresis and gene sequencing on phenotypic AmpC isolates from patients and endoscopes. Cases were those infected with phenotypic AmpC isolates (both carbapenem-susceptible and CR) and identicalblaCMY-2,fumC, andfimHalleles or related pulsed-field gel electrophoresis patterns.RESULTSThirty-five of 49 AmpCE. colitested met the case definition, including all CR isolates. All cases had complicated biliary disease and had undergone at least 1 endoscopic retrograde cholangiopancreatography at hospital A. Mortality at 30 days was 16% for all patients and 56% for CR patients. Two of 8 reprocessed endoscopic retrograde cholangiopancreatography scopes harbored AmpC that matched case isolates by pulsed-field gel electrophoresis. Environmental cultures were negative. No breaches in infection control were identified. Endoscopic reprocessing exceeded manufacturer’s recommended cleaning guidelines.CONCLUSIONRecommended reprocessing guidelines are not sufficient.Infect Control Hosp Epidemiol2015;00(0): 1–9

FimH, the mannose-specific, type 1 fimbrial adhesin of Escherichia coli, acquires amino acid replacements adaptive in extraintestinal niches (the genitourinary tract) but detrimental in the main habitat (the large intestine). This microevolutionary dynamics is reminiscent of an ecological "source-sink" model of continuous species spread from a stable primary habitat (source) into transient secondary niches (sink), with eventual extinction of the sink-evolved populations. Here, we have adapted two ecological analytical tools-diversity indexes DS and alpha--to compare size and frequency distributions of fimH haplotypes between evolutionarily conserved FimH variants ("source" haplotypes) and FimH variants with adaptive mutations (putative "sink" haplotypes). Both indexes show two- to threefold increased diversity of the sink fimH haplotypes relative to the source haplotypes, a pattern that ran opposite to those seen with nonstructural fimbrial genes (fimC and fimI) and housekeeping loci (adk and fumC) but similar to that seen with another fimbrial adhesin of E. coli, papG-II, also implicated in extraintestinal infections. The increased diversity of the sink pool of adhesin genes is due to the increased richness of the haplotypes (the number of unique haplotypes), rather than their evenness (the extent of similarity in relative abundances). Taken together, this pattern supports a continuous emergence and extinction of the gene alleles adaptive to virulence sink habitats of E. coli, rather than a one-time change in the habitat conditions. Thus, ecological methods of species diversity analysis can be successfully adapted to characterize the emergence of microbial virulence in bacterial pathogens subject to source-sink dynamics.

Type 1 fimbriae of Escherichia coli mediate mannose-specific adhesion to host epithelial surfaces and consist of a major, antigenically variable pilin subunit, FimA, and a minor, structurally conserved adhesive subunit, FimH, located on the fimbrial tip. We have analysed the variability of fimA and fimH in strains of vaginal and other origin that belong to one of the most prominent clonal groups of extraintestinal pathogenic E. coli, comprised of O1:K1-, O2:K1- and O18:K1-based serotypes. Multiple locus sequence typing (MLST) of this group revealed that the strains have identical (at all but one nucleotide position) eight housekeeping loci around the genome and belong to the ST95 complex defined by the publicly available E. coli MLST database. Multiple highly diverse fimA alleles have been introduced into the ST95 clonal complex via horizontal transfer, at a frequency comparable to that of genes defining the major O- and H-antigens. However, no further significant FimA diversification has occurred via point mutation after the transfers. In contrast, while fimH alleles also move horizontally (along with the fimA loci), they acquire point amino acid replacements at a higher rate than either housekeeping genes or fimA. These FimH mutations enhance binding to monomannose receptors and bacterial tropism for human vaginal epithelium. A similar pattern of rapid within-clonal structural evolution of the adhesive, but not pilin, subunit is also seen, respectively, in papG and papA alleles of the di-galactose-specific P-fimbriae. Thus, while structurally diverse pilin subunits of E. coli fimbriae are under selective pressure for frequent horizontal transfer between clones, the adhesive subunits of extraintes-tinal E. coli are under strong positive selection (Dn/ Ds > 1 for fimH and papG) for functionally adaptive amino acid replacements.

A multidrug-resistant NDM-1 carbapenamase-producing Escherichia coli sequence type 131 (ST131) organism was obtained from vacuum cleaner dust collected from the home of a case patient. Here, we report the assembly and annotation of its genome.

Escherichia coli NU14, a cystitis isolate used to study the pathogenesis of cystitis and to develop a FimH (type 1 fimbrial adhesin) vaccine, was assessed for extended virulence genotype, phylogenetic background, and FimH sequence and binding phenotype(s). NU14 exhibited the same virulence genotype and was derived from the same (meningitis- and cystitis-associated) subclone of E. coli O18:K1:H7 as the archetypal neonatal bacterial meningitis (NBM) isolate RS218. NU14 also displayed the same Ser62Ala FimH polymorphism as did NBM isolates RS218 and IHE3034-conferring both collagen binding and a distinct monomannose binding capability (which characterizes uropathogenic but not commensal E. coli and dramatically increases adherence to uroepithelial cells). These findings establish that strain NU14 exhibits numerous urovirulence-associated traits and derives from the single most prevalent clonal group in acute cystitis. They provide further evidence of clonal and pathotypic similarities between cystitis and NBM isolates of E. coli O18:K1:H7.

A case of enterococcal meningitis in a toddler is presented. The organism was highly resistant to all drugs previously used for pediatric Gram-positive meningitis. She was successfully treated with intraventricular and intravenous daptomycin and intravenous tigecycline. The organism was characterized as a member of CC17, a notorious emerging nosocomial clone of Enterococcus faecium.

Salmonella and Escherichia coli mannose-binding type 1 fimbriae exhibit highly similar receptor specificities, morphologies, and mechanisms of assembly but are nonorthologous in nature, i.e., not closely related evolutionarily. Their operons differ in chromosomal location, gene arrangement, and regulatory components. In the current study, we performed a comparative genetic and structural analysis of the major structural subunit, FimA, from Salmonella and E. coli and found that FimA pilins undergo diverse evolutionary adaptation in the different species. Whereas the E. coli fimA locus is characterized by high allelic diversity, frequent intragenic recombination, and horizontal movement, Salmonella fimA shows structural diversity that is more than 5-fold lower without strong evidence of gene shuffling or homologous recombination. In contrast to Salmonella FimA, the amino acid substitutions in the E. coli pilin heavily target the protein regions that are predicted to be exposed on the external surface of fimbriae. Altogether, our results suggest that E. coli, but not Salmonella, type 1 fimbriae display a high level of structural diversity consistent with a strong selection for antigenic variation under immune pressure. Thus, type 1 fimbriae in these closely related bacterial species appear to function in distinctly different physiological environments., IMPORTANCE E. coli and Salmonella are enteric bacteria that are closely related from an evolutionary perspective. They are both notorious human pathogens, though with somewhat distinct ecologies and virulence mechanisms. Type 1 fimbriae are rod-shaped surface appendages found in most E. coli and Salmonella isolates. In both species, they mediate bacterial adhesion to mannose receptors on host cells and share essentially the same morphology and assembly mechanisms. Here we show that despite the strong resemblances in function and structure, they are exposed to very different natural selection environments. Sequence analysis indicates that E. coli, but not Salmonella, fimbriae are subjected to strong immune pressure, resulting in a high level of major fimbrial protein gene shuffling and interbacterial transfer. Thus, evolutionary analysis tools can provide evidence of divergent physiological roles of functionally similar traits in different bacterial species.

Analysis of genetic differences (gene presence/absence and nucleotide polymorphisms) among strains of a bacterial species is crucial to understanding molecular mechanisms of bacterial pathogenesis and selecting targets for novel antibacterial therapeutics. However, lack of genome-wide association studies on large and epidemiologically well-defined strain collections from the same species makes it difficult to identify the genes under positive selection and define adaptive polymorphisms in those genes. To address this need and to overcome existing limitations, we propose to create a "microbial variome"--a species-specific resource database of genomic variations based on molecular evolutionary analysis. Here, we present prototype variome databases of Escherichia coli and Salmonella enterica subspecies enterica (http://depts.washington.edu/sokurel/variome, last accessed March 26, 2013). The prototypes currently include the point mutations data of core protein-coding genes from completely sequenced genomes of 22 E. coli and 17 S. enterica strains. These publicly available databases allow for single- and multiple-field sorting, filtering, and searching of the gene variability data and the potential adaptive significance. Such resource databases would immensely help experimental research, clinical diagnostics, epidemiology, and environmental control of human pathogens.

Background. Fluoroquinolone-resistant Escherichia coli are increasingly prevalent. Their clonal origins—potentially critical for control efforts—remain undefined. Methods. Antimicrobial resistance profiles and fine clonal structure were determined for 236 diverse-source historical (1967–2009) E. coli isolates representing sequence type ST131 and 853 recent (2010–2011) consecutive E. coli isolates from 5 clinical laboratories in Seattle, Washington, and Minneapolis, Minnesota. Clonal structure was resolved based on fimH sequence (fimbrial adhesin gene: H subclone assignments), multilocus sequence typing, gyrA and parC sequence (fluoroquinolone resistance-determining loci), and pulsed-field gel electrophoresis. Results. Of the recent fluoroquinolone-resistant clinical isolates, 52% represented a single ST131 subclonal lineage, H30, which expanded abruptly after 2000. This subclone had a unique and conserved gyrA/parC allele combination, supporting its tight clonality. Unlike other ST131 subclones, H30 was significantly associated with fluoroquinolone resistance and was the most prevalent subclone among current E. coli clinical isolates, overall (10.4%) and within every resistance category (11%–52%). Conclusions. Most current fluoroquinolone-resistant E. coli clinical isolates, and the largest share of multidrug-resistant isolates, represent a highly clonal subgroup that likely originated from a single rapidly expanded and disseminated ST131 strain. Focused attention to this strain will be required to control the fluoroquinolone and multidrug-resistant E. coli epidemic.

Single-nucleotide polymorphisms (SNPs) in structural genes can have a dramatic effect on the biology of whole organisms, from bacteria and viruses to mammals. Here, we underscore the importance of SNPs in bacterial genes that contribute to the ability of pathogens to cause disease. SNPs that confer an adaptive advantage for bacterial pathogens have been discovered in the genes encoding the FimH and Dr adhesins of Escherichia coli and, most recently, Salmonella enterica sv. Typhimurium FimH.

Resistance to extended-spectrum β-lactams is increasing worldwide among Escherichia coli and has been linked to a small number of emergent clones (e.g. ST38, ST131 and ST405) recovered from extraintestinal infections in community and hospital settings. There are, however, limited data about the relative contributions of bacterial strains, plasmids and β-lactamase genes to extended-spectrum β-lactam resistance in paediatric infections. We performed an extensive molecular analysis of phylogenetic, virulence and antibiotic resistance-related properties of 49 previously reported serial E. coli isolates recovered during 1999–2007 at Seattle Children's Hospital (Seattle, WA). Class C enzyme CMY-2 and class A enzyme CTX-M-15 were the most prominent extended-spectrum β-lactam resistance enzymes in the collection, first appearing in this patient population in 2001 and 2003, respectively, and then steadily increasing in frequency over the remainder of the study period. Among 19 CMY-2-positive isolates, 16 distinct STs were detected ( D =98.25%, 95% CI 96–100.25%), indicating that CMY spread is non-clonal at the host strain level. In contrast, among ten CTX-M-15-positive isolates, three STs were detected ( D =37.78%, 95% CI 2.36–73.20%), of which eight represented the worldwide-disseminated ST131 lineage, consistent with clonal spread of CTX-M-15-associated resistance. fimH TR subtyping of ten ST131 isolates (including two CTX-M-negative isolates) revealed that, within ST131, carriage of allele fimH TR 30 correlated with CTX-M-15 positivity, whilst carriage of non- fimH TR 30 alleles correlated with carriage of non-CTX-M enzymes. Thus, spread of CMY-2 is non-clonal at the ST level, but clonal spread of CTX-M-15 may be associated with a specific fimH TR -defined sublineage of ST131.

Escherichia coli sequence type ST131 (from phylogenetic group B2), often carrying the extended-spectrum-β-lactamase (ESBL) gene bla CTX-M-15 , is an emerging globally disseminated pathogen that has received comparatively little attention in the United States. Accordingly, a convenience sample of 351 ESBL-producing E. coli isolates from 15 U.S. centers (collected in 2000 to 2009) underwent PCR-based phylotyping and detection of ST131 and bla CTX-M-15 . A total of 200 isolates, comprising 4 groups of 50 isolates each that were (i) bla CTX-M-15 negative non-ST131, (ii) bla CTX-M-15 positive non-ST131, (iii) bla CTX-M-15 negative ST131, or (iv) bla CTX-M-15 positive ST131, also underwent virulence genotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). Overall, 201 (57%) isolates exhibited bla CTX-M-15 , whereas 165 (47%) were ST131. ST131 accounted for 56% of bla CTX-M-15 -positive- versus 35% of bla CTX-M-15 -negative isolates ( P < 0.001). Whereas ST131 accounted for 94% of the 175 total group B2 isolates, non-ST131 isolates were phylogenetically distributed by bla CTX-M-15 status, with groups A ( bla CTX-M-15 -positive isolates) and D ( bla CTX-M-15 -negative isolates) predominating. Both bla CTX-M-15 and ST131 occurred at all participating centers, were recovered from children and adults, increased significantly in prevalence post-2003, and were associated with molecularly inferred virulence. Compared with non-ST131 isolates, ST131 isolates had higher virulence scores, distinctive virulence profiles, and more-homogeneous PFGE profiles. bla CTX-M-15 was associated with extensive antimicrobial resistance and ST131 with fluoroquinolone resistance. Thus, E. coli ST131 and bla CTX-M-15 are emergent, widely distributed, and predominant among ESBL-positive E. coli strains in the United States, among children and adults alike. Enhanced virulence and antimicrobial resistance have likely promoted the epidemiological success of these emerging public health threats.

Multilocus sequence typing (MLST) is usually based on the sequencing of 5 to 8 housekeeping loci in the bacterial chromosome and has provided detailed descriptions of the population structure of bacterial species important to human health. However, even strains with identical MLST profiles (known as sequence types or STs) may possess distinct genotypes, which enable different eco- or pathotypic lifestyles. Here we describe a two-locus, sequence-based typing scheme for Escherichia coli that utilizes a 489-nucleotide (nt) internal fragment of fimH (encoding the type 1 fimbrial adhesin) and the 469-nt internal fumC fragment used in standard MLST. Based on sequence typing of 191 model commensal and pathogenic isolates plus 853 freshly isolated clinical E. coli strains, this 2-locus approach—which we call CH ( fum C / fim H ) typing—consistently yielded more haplotypes than standard 7-locus MLST, splitting large STs into multiple clonal subgroups and often distinguishing different within-ST eco- and pathotypes. Furthermore, specific CH profiles corresponded to specific STs, or ST complexes, with 95% accuracy, allowing excellent prediction of MLST-based profiles. Thus, 2-locus CH typing provides a genotyping tool for molecular epidemiology analysis that is more economical than standard 7-locus MLST but has superior clonal discrimination power and, at the same time, corresponds closely to MLST-based clonal groupings.

The objective of this study was to investigate the trends and patterns of resistance in β-lactamase-producing members of the family Enterobacteriaceae in a children's hospital over a 9-year period (1999 to 2007). Clinically significant isolates of the Enterobacteriaceae were screened for patterns of broad-spectrum resistance to β-lactams. The strains likely to be resistant were subsequently confirmed by an inhibitor-based disc test. The plasmid-mediated resistance determinants in these isolates were identified by PCR and by in vitro transformation, which successfully reproduced the AmpC phenotype unrestricted by the species of the host organisms. Among 8,048 Enterobacteriaceae isolates belonging to the four chromosomal ampC -negative or -nonfunctional genera, 86 (1.07%) isolates (56 Escherichia coli isolates, 22 Klebsiella species isolates, 1 Proteus mirabilis isolate, and 7 Salmonella species isolates) exhibited broad-spectrum β-lactam resistance patterns. These organisms collectively produced three classes of β-lactamases, including class A extended-spectrum β-lactamases ( n = 47), class C or AmpC β-lactamases ( n = 36, including 4 isolates that produced both class A and class C enzymes), and class A or B carbapenem-hydrolyzing β-lactamases ( n = 3). The proportion increased from 0.46% during the first 3 years to 1.84% during the last 3 years (relative risk [RR], 4.04; 95% confidence interval [CI], 2.28 to 7.42; P < 0.001). The increase was mainly due to the emergence of a plasmid-mediated bla CMY-2 β-lactamase, the incidence of which increased from 0.11% during the first 3 years to 0.96% during the last 3 years (RR, 9.11; 95% CI, 2.76 to 30.1; P = 0.001). Class A-type resistance increased slightly during the study period, from 0.35% during the first 3 years to 0.85% during the last 3 years (RR, 2.42; 95% CI, 1.15 to 5.07; P = 0.02). A Proteus mirabilis strain was documented to possess a novel bla DHA determinant. Of special concern, three carbapenemase-producing isolates were identified between 2003 and 2006. The infections caused by resistant isolates of the Enterobacteriaceae mainly affected hospitalized patients with underlying conditions; however, 19 (22%) episodes were of community onset in otherwise well children. The rate of resistance to broad-spectrum β-lactams among isolates of the Enterobacteriaceae is increasing in children in both hospital- and community-acquired settings, and the resistance is driven largely by plasmid-mediated AmpC β-lactamases. These data have important implications for empirical antimicrobial strategies targeting serious pediatric infections. Further study of this problem is warranted.

Spread of biological species from primary into novel habitats leads to within-species adaptive niche differentiation and is commonly driven by acquisition of point mutations in individual genes that increase fitness in the alternative environment. However, finding footprints of adaptive niche differentiation in specific genes remains a challenge. Here we describe a novel method to analyze the footprint of pathogenicity-adaptive, or pathoadaptive, mutations in the Escherichia coli gene encoding FimH-the major, mannose-sensitive adhesin. Analysis of distribution of mutations across the nodes and branches of the FimH phylogenetic network shows (1) zonal separation of evolutionary primary structural variants of FimH and recently derived ones, (2) dramatic differences in the ratio of synonymous and nonsynonymous changes between nodes from different zones, (3) evidence for replacement hot-spots in the FimH protein, (4) differential zonal distribution of FimH variants from commensal and uropathogenic E. coli, and (5) pathoadaptive functional changes in FimH brought by the mutations. The selective footprint in fimH indicates that the pathoadaptive niche differentiation of E. coli is either in its initial stages or undergoing an evolutionary "source/sink" dynamic.

Escherichia coli K1 is the leading cause of gram-negative bacterial meningitis in neonates. It is principally due to our limited understanding of the pathogenesis of this disease that the morbidity and mortality rates remain unacceptably high. To identify genes required for E. coli K1 penetration of the blood-brain barrier (BBB), we used the negative selection strategy of signature-tagged transposon mutagenesis (STM) to screen mutants for loss or decreased invasion of human brain microvascular endothelial cells (HBMEC) which comprise the BBB. A total of 3,360 insertion mutants of E. coli K1 were screened, and potential HBMEC invasion mutants were subjected to a secondary invasion screen. Those mutants that failed to pass the serial invasion screens were then tested individually. Seven prototrophic mutants were found to exhibit significantly decreased invasive ability in HBMEC. We identified traJ and five previously uncharacterized loci whose gene products are necessary for HBMEC invasion by E. coli K1. In addition, cnf1 , a gene previously shown to play a role in bacterial invasion, was identified. More importantly, a traJ mutant was attenuated in penetration of the BBB in the neonatal rat model of experimental hematogenous meningitis. This is the first in vivo demonstration that traJ is involved in the pathogenesis of E. coli K1 meningitis.

FimH is the tip adhesin of mannose-specific type 1 fimbriae of Escherichia coli , which are critical to the pathogenesis of urinary tract infections. Point FimH mutations increasing monomannose (1M)-specific uroepithelial adhesion are commonly found in uropathogenic strains of E. coli . Here, we demonstrate the emergence of a mixed population of clonally identical E. coli strains in the urine of a patient with acute cystitis, where half of the isolates carried a glycine-to-arginine substitution at position 66 of the mature FimH. The R66 mutation induced an unusually strong 1M-binding phenotype and a 20-fold advantage in mouse bladder colonization. However, E. coli strains carrying FimH-R66, but not the parental FimH-G66, had disappeared from the patient's rectal and urine samples collected from 29 to 44 days later, demonstrating within-host instability of the R66 mutation. No FimH variants with R66 were identified in a large (>600 strains) sequence database of fimH -positive E. coli strains. However, several strains carrying genes encoding FimH with either S66 or C66 mutations appeared to be relatively stable in the E. coli population. Relative to FimH-R66, the FimH-S66 and FimH-C66 variants mediated only moderate increases in 1M binding but preserved the ability to enhance binding under flow-induced shear conditions. In contrast, FimH-R66 completely lost shear-enhanced binding properties, with bacterial adhesion being inhibited by shear forces and lacking a rolling mode of binding. These functional trade-offs may determine the natural populational instability of this mutation or other pathoadaptive FimH mutations that confer dramatic increases in 1M binding strength.