Intracellular polyamine pools, oligopeptide-binding protein A expression, and resistance to aminoglycosides in Escherichia coli

The role of intracellular free polyamine (putrescine and spermidine) pools in multiple resistance to aminoglycoside antibiotics was investigated among in vitro selected kanamycin-resistant Escherichia coli J53 mutants expressing diminished oligopeptide-binding protein (OppA) levels and/or defective ornithine decarboxylase (ODC) activity. The results suggest that diminished OppA content, but not defective ODC activity expression, increased the relative concentration of free spermidine as compared to the wild type strain. Moreover, by adding exogenous polyamines or polyamine synthesis inhibitors to cultures with different mutant strains, a direct relationship between the intracellular OppA levels and resistance to kanamycin was revealed. Collectively these results further suggest a complex relation among OppA expression, aminoglycoside resistance and polyamine metabolism.

Epithelial cell signaling responses to enterohemorrhagic Escherichia coli infection

Enterohemorrhagic Escherichia coli, including the serotype O157:H7 that is most commonly identified with human disease, cause both sporadic cases and outbreaks of non-bloody diarrhea and hemorrhagic colitis. In about 10% of infected subjects, the hemolytic uremic syndrome (hemolytic anemic, thrombocytopenia, and acute renal failure) develops, likely as a consequence of systemic spread of bacterial-derived toxins variously referred to as Shiga-like toxin, Shiga toxin, and Verotoxin. Increasing evidence points to a complex interplay between bacterial products - for example, adhesins and toxins - and host signal transduction pathways in mediating responses to infection. Identification of critical signaling pathways could result in the development of novel strategies for intervention to both prevent and treat this microbial infection in humans.

Genetic variability of hepatitis A virus strain HAF-203 isolated in Brazil and expression of the VP1 gene in Escherichia coli

The hepatitis A virus (HAV) HAF-203 strain was isolated from an acute case of HAV infection. The primary isolation of HAF-203 in Brazil and its adaptation to the FRhK-4 cell lineage allowed the production of large amounts of viral particles enabling molecular characterization of the first HAV isolate in Brazil. The aim of our study was to determine the nucleotide sequence of the HAF-203 strain genome, compare it to other HAV genomes and highlight its genetic variability. The complete nucleotide sequence of the HAF-203 strain (7472 nucleotides) was compared to those obtained earlier by others for other HAV isolates. These analyses revealed 19 HAF-specific nucleotide sequence differences with 10 amino acid substitutions. Most of the non-conservative changes were located at VP1, 2C, and 3D genes, but the 3B region was the most variable. The availability of HAF-203 complementary DNA was useful for the production of the recombinant VP1 protein, which is a major determinant of viral infectivity. This recombinant protein was shown by enzyme-linked immunoassay and blotting, to be immunogenic and resemble the native protein, therefore suggesting its value as a reagent for incorporation into diagnostic tests.

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

Escherichia coli (E.coli) Bacteraemia Surveillance

Charts showing E.coli and ESBL Bacteraemias in Northern Ireland, by quarter, from 2005 to 2014.

Genetic relationship of diarrheagenic Escherichia coli pathotypes among the enteropathogenic Escherichia coli O serogroup

The genetic relationship among the Escherichia coli pathotypes was investigated. We used random amplified polymorphic DNA (RAPD) data for constructing a dendrogram of 73 strains of diarrheagenic E. coli. A phylogenetic tree encompassing 15 serotypes from different pathotypes was constructed using multilocus sequence typing data. Phylogram clusters were used for validating RAPD data on the clonality of enteropathogenic E. coli (EPEC) O serogroup strains. Both analyses showed very similar topologies, characterized by the presence of two major groups: group A includes EPEC H6 and H34 strains and group B contains the other EPEC strains plus all serotypes belonging to atypical EPEC, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). These results confirm the existence of two evolutionary divergent groups in EPEC: one is genetically and serologically very homogeneous whereas the other harbors EPEC and non-EPEC serotypes. The same situation was found for EAEC and EHEC.

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.

In vitro activity of fosfomycin against extended-spectrum-beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae: comparison of susceptibility testing procedures

The agar dilution, broth microdilution, and disk diffusion methods were compared to determine the in vitro susceptibility of 428 extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae to fosfomycin. Fosfomycin showed very high activity against all ESBL-producing strains. Excellent agreement between the three susceptibility methods was found for E. coli, whereas marked discrepancies were observed for K. pneumoniae.

In Vivo Selection of Fluoroquinolone-Resistant Escherichia coli Isolates Expressing Plasmid-Mediated Quinolone Resistance and Expanded-Spectrum β-Lactamase

A ciprofloxacin-resistant Escherichia coli isolate, isolate 1B, was obtained from a urinary specimen of a Canadian patient treated with norfloxacin for infection due to a ciprofloxacin-susceptible isolate, isolate 1A. Both isolates harbored a plasmid-encoded sul1-type integron with qnrA1 and blaVEB-1 genes. Isolate 1B had amino acid substitutions in gyrase and topoisomerase.

Identification of uropathogenic Escherichia coli clonal group A (CgA) in hospitalised patients

This study provides the first description of healthcare-associated infections with Escherichia coli clonal group A (CgA) isolates in Latin America. Isolates were typed by enterobacterial repetitive intergenic consensus-PCR, pulsed-field gel electrophoresis, E. coli phylogenetic grouping, multilocus sequence typing and fimH single nucleotide polymorphism analysis. Out of 42 E. coli hospital isolates studied, three belonged to E. coli phylogenetic group D and ST69 and had fimH sequences identical to that of the CgA reference strain ATCC BAA-457. E. coli CgA is another potential source of resistant infections in hospitals.

Bacteremia due to extended-spectrum beta-lactamase-producing Escherichia coli in the CTX-M era: a new clinical challenge.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli, particularly those producing CTX-M types of ESBL, are emerging pathogens. Bacteremia caused by these organisms represents a clinical challenge, because the organisms are frequently resistant to the antimicrobials recommended for treatment of patients with suspected E. coli sepsis. METHODS:A cohort study was performed that included all episodes of bloodstream infection due to ESBL-producing E. coli during the period from January 2001 through March 2005. Data on predisposing factors, clinical presentation, and outcome were collected. ESBLs were characterized using isoelectric focusing, polymerase chain reaction, and sequencing. RESULTS: Forty-three episodes (8.8% of cases of bacteremia due to E. coli) were included; 70% of the isolates produced a CTX-M type of ESBL. The most frequent origins of infection were the urinary (46%) and biliary tracts (21%). Acquisition was nosocomial in 21 cases (49%), health care associated in 14 cases (32%), and strictly community acquired in 8 cases (19%). Thirty-eight percent and 25% of patients had obstructive diseases of the urinary and biliary tracts, respectively, and 38% had recently received antimicrobials. Nine patients (21%) died. Compared with beta-lactam/beta-lactamase-inhibitor and carbapenem-based regimens, empirical therapy with cephalosporins or fluoroquinolones was associated with a higher mortality rate (9% vs. 35%; P=.05) and needed to be changed more frequently (24% vs. 78%; P=.001). CONCLUSIONS: ESBL-producing E. coli is a significant cause of bloodstream infection in hospitalized and nonhospitalized patients in the context of the emergence of CTX-M enzymes. Empirical treatment of sepsis potentially caused by E. coli may need to be reconsidered in areas where such ESBL-producing isolates are present.

Clinical and molecular epidemiology of extended-spectrum beta-lactamase-producing Escherichia coli as a cause of nosocomial infection or colonization: implications for control.

BACKGROUND: Extended-spectrum beta-lactamase (ESBL)-producing members of the Enterobacteriaceae family are important nosocomial pathogens. Escherichia coli producing a specific family of ESBL (the CTX-M enzymes) are emerging worldwide. The epidemiology of these organisms as causes of nosocomial infection is poorly understood. The aims of this study were to investigate the clinical and molecular epidemiology of nosocomial infection or colonization due to ESBL-producing E. coli in hospitalized patients, consider the specific types of ESBLs produced, and identify the risk factors for infection and colonization with these organisms. METHODS: All patients with nosocomial colonization and/or infection due to ESBL-producing E. coli in 2 centers (a tertiary care hospital and a geriatric care center) identified between January 2001 and May 2002 were included. A double case-control study was performed. The clonal relatedness of the isolates was studied by repetitive extragenic palindromic-polymerase chain reaction and pulsed-field gel electrophoresis. ESBLs were characterized by isoelectric focusing, polymerase chain reaction, and sequencing. RESULTS: Forty-seven case patients were included. CTX-M-producing E. coli were clonally unrelated and more frequently susceptible to nonoxyimino-beta-lactams. Alternately, isolates producing SHV- and TEM-type ESBL were epidemic and multidrug resistant. Urinary catheterization was a risk factor for both CTX-M-producing and SHV-TEM-producing isolates. Previous oxyimino-beta-lactam use, diabetes, and ultimately fatal or nonfatal underlying diseases were independent risk factors for infection or colonization with CTX-M-producing isolates, whereas previous fluoroquinolone use was associated with infection or colonization with SHV-TEM-producing isolates. CONCLUSIONS: The epidemiology of ESBL-producing E. coli as a cause of nosocomial infection is complex. Sporadic CTX-M-producing isolates coexisted with epidemic multidrug-resistant SHV-TEM-producing isolates. These data should be taken into account for the design of control measures.

Biochemical properties, enterohaemolysin production and plasmid carriage of Shiga toxin-producing Escherichia coli strains

Thirty-eight strains of Shiga toxin-producing Escherichia coli (STEC) were characterised in terms of biochemical properties, enterohaemolysin production and plasmid carriage. A wide variation in the biochemical properties was observed among the STEC, with 14 distinct biotypes identified. Biotype 1 was the most common, found in 29% of the strains. Enterohaemolysin production was detected in 29% of the strains. Most of the bacterial strains (95%) carried one or more plasmids and considerable heterogeneity in size and combinations was observed. Seven distinct plasmid profiles were identified. The most common profile, characterised by the presence of a single plasmid of ~90 kb, was found in 50% of these strains. These data indicate extensive diversity among STEC strains. No correlation was found among biotype, serotype, enterohaemolysin production and plasmid profile.

Expression of bacterial virulence factors and cytokines during in vitro macrophage infection by enteroinvasive Escherichia coli and Shigella flexneri: a comparative study

Enteroinvasive Escherichia coli (EIEC) and Shigellaspp cause bacillary dysentery in humans by invading and multiplying within epithelial cells of the colonic mucosa. Although EIEC and Shigellashare many genetic and biochemical similarities, the illness caused by Shigellais more severe. Thus, genomic and structure-function molecular studies on the biological interactions of these invasive enterobacteria with eukaryotic cells have focused on Shigella rather than EIEC. Here we comparatively studied the interactions of EIEC and of Shigella flexneriwith cultured J774 macrophage-like cells. We evaluated several phenotypes: (i) bacterial escape from macrophages after phagocytosis, (ii) macrophage death induced by EIEC and S. flexneri, (iii) macrophage cytokine expression in response to infection and (iv) expression of plasmidial (pINV) virulence genes. The results showed thatS. flexneri caused macrophage killing earlier and more intensely than EIEC. Both pathogens induced significant macrophage production of TNF, IL-1 and IL-10 after 7 h of infection. Transcription levels of the gene invasion plasmid antigen-C were lower in EIEC than in S. flexneri throughout the course of the infection; this could explain the diminished virulence of EIEC compared to S. flexneri.

The ability of haemolysins expressed by atypical enteropathogenic Escherichia coli to bind to extracellular matrix components

Typical and atypical enteropathogenic Escherichia coli (EPEC) are considered important bacterial causes of diarrhoea. Considering the repertoire of virulence genes, atypical EPEC (aEPEC) is a heterogeneous group, harbouring genes that are found in other diarrheagenic E. coli pathotypes, such as those encoding haemolysins. Haemolysins are cytolytic toxins that lyse host cells disrupting the function of the plasma membrane. In addition, these cytolysins mediate a connection to vascular tissue and/or blood components, such as plasma and cellular fibronectin. Therefore, we investigated the haemolytic activity of 72 aEPEC isolates and determined the correlation of this phenotype with the presence of genes encoding enterohaemolysins (Ehly) and cytolysin A (ClyA). In addition, the correlation between the expression of haemolysins and the ability of these secreted proteins to adhere to extracellular matrix (ECM) components was also assessed in this study. Our findings demonstrate that a subset of aEPEC presents haemolytic activity due to the expression of Ehlys and/or ClyA and that this activity is closely related to the ability of these isolates to bind to ECM components.