Sequencing and Functional Annotation of Avian Pathogenic Escherichia coli Serogroup O78 Strains Reveal the Evolution of E. coli Lineages Pathogenic for Poultry via Distinct Mechanisms

Avian pathogenic Escherichia coli (APEC) causes respiratory and systemic disease in poultry. Sequencing of a multilocus sequence type 95 (ST95) serogroup O1 strain previously indicated that APEC resembles E. coli causing extraintestinal human diseases. We sequenced the genomes of two strains of another dominant APEC lineage (ST23 serogroup O78 strains χ7122 and IMT2125) and compared them to each other and to the reannotated APEC O1 sequence. For comparison, we also sequenced a human enterotoxigenic E. coli (ETEC) strain of the same ST23 serogroup O78 lineage. Phylogenetic analysis indicated that the APEC O78 strains were more closely related to human ST23 ETEC than to APEC O1, indicating that separation of pathotypes on the basis of their extraintestinal or diarrheagenic nature is not supported by their phylogeny. The accessory genome of APEC ST23 strains exhibited limited conservation of APEC O1 genomic islands and a distinct repertoire of virulence-associated loci. In light of this diversity, we surveyed the phenotype of 2,185 signature-tagged transposon mutants of χ7122 following intra-air sac inoculation of turkeys. This procedure identified novel APEC ST23 genes that play strain- and tissue-specific roles during infection. For example, genes mediating group 4 capsule synthesis were required for the virulence of χ7122 and were conserved in IMT2125 but absent from APEC O1. Our data reveal the genetic diversity of E. coli strains adapted to cause the same avian disease and indicate that the core genome of the ST23 lineage serves as a chassis for the evolution of E. coli strains adapted to cause avian or human disease via acquisition of distinct virulence genes.     

Molecular epidemiology over an 11-year period (2000 to 2010) of extended-spectrum β-lactamase-producing Escherichia coli causing bacteremia in a centralized Canadian region

A study was designed to assess the importance of sequence types among extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates causing bacteremia over an 11-year period (2000 to 2010) in a centralized Canadian region. A total of 197 patients with incident infections were identified; the majority presented with community-onset urosepsis, with a significant increase in the prevalence of ESBL-producing E. coli during the later part of the study. The majority of E. coli isolates produced either CTX-M-15 or CTX-M-14. We identified 7 different major sequence types among 91% of isolates (i.e., the ST10 clonal complex, ST38, ST131, ST315, ST393, ST405, and ST648) and provided insight into their clinical and molecular characteristics. ST38 was the most antimicrobial-susceptible sequence type and predominated during 2000 to 2004 but disappeared after 2008. ST131 was the most antimicrobial-resistant sequence type, and the influx of a single pulsotype of this sequence type was responsible for the significant increase of ESBL-producing E. coli strains since 2007. During 2010, 49/63 (78%) of the ESBL-producing E. coli isolates belonged to ST131, and this sequence type had established itself as a major drug-resistant pathogen in Calgary, Alberta, Canada, posing an important new public health threat within our region. We urgently need well-designed epidemiological and molecular studies to understand the dynamics of transmission, risk factors, and reservoirs for E. coli ST131. This will provide insight into the emergence and spread of this multiresistant sequence type.     

Absence of CTX-M enzymes but high prevalence of clones, including clone ST131, among fecal Escherichia coli isolates from healthy subjects living in the area of Paris, France

Quinolone-resistant and CTX-M-15-producing Escherichia coli isolates belonging to clone ST131 have been reported in the community. This study was designed to identify these E. coli isolates in the stools of 332 independent healthy subjects living in the area of Paris, France. Stools were plated on media without antibiotics, in order to obtain the dominant (Dm) fecal E. coli strain, and with nalidixic acid (NAL) and cefotaxime. Quinolone susceptibility, phylogenetic groups, and molecular profiles, including multilocus sequence types (ST), were determined for all NAL-resistant (NAL-R) isolates. Groups were also determined for the Dm strains from participants with NAL-R isolates and from a subgroup without NAL-R isolates. All B2 isolates were typed; pulsed-field gel electrophoresis was performed for the ST131 isolates, and the results were compared with those for intercontinental clone ST131. Two participants (0.6%) had extended-spectrum β-lactamase-producing (SHV-2, TEM-52) fecal E. coli isolates, and 51 (15%) had NAL-R isolates; 51% of NAL-R isolates belonged to phylogenetic group A, 31% to group D, 16% to group B2, and 2% to group B1. The Dm strain was NAL-R in 3.3% of the 332 subjects. Forty-nine percent of the NAL-R isolates belonged to clones: ST10 and ST606 for group A isolates, ST117 and ST393 for group D isolates. Of all B2 isolates studied from 100 subjects (8 NAL-R strains; 19 NAL-susceptible dominant strains), 52% belonged to three clones: ST131 (n = 7), ST95 (n = 4), and ST141 (n = 3). This is the first study to show the presence of fecal E. coli isolates of clone ST131 in 7% of independent healthy subjects not colonized by CTX-M-15-producing isolates.     

Sharing of Escherichia coli Sequence Type ST131 and Other Multidrug-Resistant and Urovirulent E. coli Strains among Dogs and Cats within a Household

A multidrug-resistant (MDR) Escherichia coli strain from a human-associated pulsotype within sequence type ST131 (O25:H4) colonized three of five dogs and cats within a household. Of the four other colonizing strains identified, two were MDR and two colonized multiple hosts. The ST131 strain uniquely exhibited high resistance and virulence scores.Within-household sharing of Escherichia coli strains among humans and pets has been documented in multiple studies (8, 12, 13, 18, 24). This phenomenon, which likely reflects host-to-host transmission, may facilitate the dissemination of virulent and antimicrobial-resistant E. coli within the community.An extensively antimicrobial-resistant E. coli clonal group, sequence type ST131 (O25:H4), has been recognized as an important human pathogen only within the last several years, suggesting recent clonal dissemination and expansion (4, 7, 25). Currently best known for its association with extended-spectrum cephalosporin resistance, ST131 has contributed importantly to the global emergence of the CTX-M-15 extended-spectrum beta-lactamase (and, perhaps, vice versa) (4, 7, 25). E. coli ST131 also commonly occurs as a fluoroquinolone-resistant (FQ-R) but cephalosporin-susceptible pathogen (1, 15, 20).Although ST131 has been isolated from a companion animal (a dog) (27), this report did not address other hosts from the same household or the isolate''s relationship to human ST131 isolates. The recent finding by the Escherichia coli Reference Center (the Pennsylvania State University) of serotype O25:H4 for sequential FQ-R E. coli urine isolates from a dog with recurrent/persisting bacteriuria (see below) suggested E. coli ST131. The source household included multiple pets, providing an opportunity to investigate within-household sharing of the index FQ-R urine strain. We assessed whether (i) the dog''s urine strain represented ST131, (ii) this strain was shared with other pets in the household, (iii) the strain resembled known human ST131 isolates, and (iv) additional sharing of antimicrobial-resistant and/or virulent E. coli clones could be found among the household pets.     

Experimental enterotoxin-induced Escherichia coli diarrhea and protection induced by previous infection with bacteria of the same adhesin or enterotoxin type.

The diarrheal response to an initial and a second infection with Escherichia coli expressing various enterotoxins (the heat-stable toxin [ST] alone or in combination with the heat-labile toxin [LT]) and colonization factor antigens (CFA/I, CFA/II, or E8775-type) was studied in the reversible tie adult rabbit diarrhea model. An initial infection with high doses (1 X 10(10) to 5 X 10(11) bacteria) of the various strains regularly induced diarrhea which was usually self-limiting (only 7 of 85 animals died). The diarrheal response to equally effective doses of different strains producing both ST and LT (ST/LT) did not differ significantly with serotype or colonization factor antigen. ST/LT-producing strains appeared to induce severe disease more regularly than ST-producing strains carrying the same adhesin. Previous infection with CFA/I-carrying, ST/LT-producing E. coli protected all animals reinfected with an otherwise highly diarrheogenic dose of the same strain as well as against challenge with a CFA/I-carrying, ST/LT-producing strain with different O-, K-, and H-antigens. Fecal excretion of bacteria was also significantly reduced in the protected animals, although not completely eliminated. When only one of the two antigens, CFA/I and LT, was shared by the immunizing and rechallenge strains, partial protection was evident consistent with independent antibacterial (anti-CFA) and antitoxic (anti-LT) immune mechanisms. Oral immunization with purified CFA/I significantly reduced fluid secretion in intestinal loops infected with CFA/I-carrying enterotoxigenic bacteria.     

Molecular cloning and characterization of mouse St7r (St7-like,St7l)

Human ST7R (ST7-like, ST7L) is a paralog of tumor suppressor gene ST7 in the human genome. ST7R gene is clustered with WNT2B gene in human chromosome 1p13, while ST7 gene is clustered with WNT2 gene in human chromosome 7q31. Here, we have cloned mouse ortholog of human ST7R using bioinformatics and cDNA-PCR. Mouse St7r was found to encode 559 amino-acid polypeptide. Mouse St7r showed 90.7% total-amino-acid identity with human ST7R isoform 4 (ST7R4), and 71.6% total-amino-acid identity with human ST7 and mouse St7. Three tyrosine-phosphorylation sites as well as ST7R homologous domains (S7H1, S7H2, and S7H3) were conserved among mouse St7r, human ST7R, mouse St7, and human ST7. Leucine zipper domain of mouse St7r was conserved in human ST7R, but not in mouse St7 and human ST7. Mouse St7r was expressed almost ubiquitously in adult mouse tissues and also in mouse embryos. Although mouse genome draft sequence including mouse St7r gene has not yet been identified, rat genome draft sequence AC106372.1 was found to include rat St7r gene as well as rat Wnt2b gene. These results indicate evolutional conservation of ST7R-WNT2B gene cluster. This is the first report on molecular cloning and initial characterization of mouse St7r.     

Molecular characterization of vancomycin-resistant enterococcus faecium isolates from mainland China

Little is known about vancomycin-resistant enterococci in China. Thirteen pulsed-field gel electrophoresis-confirmed heterogeneous VanA-type vancomycin-resistant Enterococcus faecium (VRE) isolates were obtained from five Chinese hospitals from 2001 to 2005. The isolates were typed by multilocus sequence typing into nine different sequence types (STs), including five new STs (ST18, ST25, ST78, ST203, ST320, ST321, ST322, ST323, and ST335). Vancomycin resistance in each isolate was encoded on conjugative plasmids; two of the plasmids, pZB18 (67 kbp) and pZB22 (200 kbp), were highly conjugative and were able to transfer at high frequencies of around 10(-4) and 10(-7) per donor cell in broth mating, respectively. None of the plasmids identified in these isolates carried traA, which is usually conserved in the pMG1-like highly conjugative plasmid for E. faecium, implying that pZB18 and pZB22 were novel types of a highly conjugative plasmid in enterococci. Thirteen Tn1546-like elements encoding VanA-type VRE on the conjugative plasmids were classified into six types (types I to VI), and most of them contained both IS1216V and IS1542 insertions. The isolates carrying the type II element were predominant. The six type elements were different from that of a VanA-type Enterococcus faecalis strain isolated from Chinese chicken meat. The results suggested that the disseminations of VRE in these areas were by Tn1546-like elements being acquired by the conjugative plasmids and transferred among E. faecium strains.     

Immunological Interrelationships Between Cholera Toxin and the Heat-Labile and Heat-Stable Enterotoxins of Coliform Bacteria

Cholera toxin (CT) and the heat-labile (LT) toxin of Escherichia coli are known to share antigenic properties. The present study examined the immunological relationship of CT and the LT and heat-stable (ST) toxins of E. coli, Klebsiella pneumoniae, and Enterobacter cloacae. The neutralizing capacity of equine CT antiserum and of antiserum raised in rabbits to the LT toxin of the three species of coliform bacteria was evaluated by determining their capacity to inhibit the action of purified CT and semipurified ultrafiltration preparations of the coliform LT and ST toxins in inducing water secretion as assayed by the in vivo marker perfusion technique in the rat jejunum. One milliliter of antiserum to CT and to E. coli and Klebsiella LT completely neutralized the secretory action of each of these three toxins; effective serial dilutions of CT antiserum extended to 1 to 4, whereas those of the antisera to LT were limited to 1 to 2 in most instances. One milliliter of antiserum to E. cloacae LT partially neutralized each of the three coliform LT toxins; serial dilutions were inactive. Antiserum to E. cloacae LT did not neutralize CT. Antiserum to CT and to each of the three coliform LT toxins also had a weak neutralizing effect on the ST toxins of E. coli and Klebsiella, but they did not affect E. cloacae ST. Adsorption of the antiserum to CT and to each of the three LT toxins by incubation with a heat-inactivated preparation of either the homologous or a heterologous LT toxin completely abolished the neutralizing capacity of the antisera towards both LT and ST. These observations indicate that the immunological interrelationship of CT and E. coli LT extends to the LT toxins of Klebsiella and E. cloacae and, further, that these immunological properties are shared to a lesser extent by the ST toxins of E. coli and Klebsiella.     

A hypothesis on the pathogenesis of rheumatoid and other non-specific synovitides. IV A. The possible intermediate role of local hypoxia and metabolic alterations

According to the original hypothesis, synovial tissue (ST) oedema and synovial fluid (SF) volume increase contribute to local hypoxia and metabolic alterations and to inflammation (A 1). Studies on biochemical mechanisms (A 2) in synovitides show that the SF volume correlates to SF hypoxia that correlates to lactate increase, acidosis and to some decrease in glucose. Normal ST produces lactate by glycolytic and pentose phosphate pathway activity, both, as well as the normally low oxygen utilization, being increased in synovitides . In ST very little carbohydrate seems to pass directly into the citric acid cycle and oxidation of fat may be involved, but it is not known if the fat is carried to ST by the blood or if it is synthetized locally (B). ST oedema and effusions may be most important as causes of local hypoxia (C). Acidosis alters physico-chemical properties of proteins, possibly changing their chemotactic and antigenic qualities, etc. Hypoxia and fuel supply might be related to fibroblast, macrophage and neutrophil reactions, and, in view of the high metabolic demands of villi, they may contribute to, e.g., ST necroses, and to erosions (D). I shall summarize some essentials of my present views on the pathogenesis (E 1 a) and causes in single cases (E 3 b) of synovitis, and comment on two other new hypotheses on rheumatoid arthritis (E 2) and on therapeutic (E 4) and other implications of this concept (E).     

Genetic diversity and antimicrobial resistance profile of Enterococcus faecalis isolated from broilers with vertebral osteomyelitis in Southeast Brazil

Vertebral osteomyelitis (VO) is a worldwide emerging disease that affects broilers. Recently, the isolation of Enterococcus faecalis in cases of the disease has been described. This study aimed at determining the genetic diversity and antimicrobial resistance profile of 12 E. faecalis strains isolated from broilers with VO. Strains were isolated from nine flocks from six farms in a high-density poultry production area in Southeast Brazil and were evaluated using multilocus sequence typing and phylogenetic analysis. Antimicrobial susceptibility tests and PCR were performed to detect antimicrobial resistance genes. E. faecalis isolates belonged to different sequence types (ST), six of which (ST49, ST100, ST116, ST202, ST249, and ST300) have been previously described. Strains ST708 and ST709 were newly identified in this study. Strain ST49 was most frequently isolated (50% of the flocks) from the analysed VO cases. No phylogenetic or phylogeographic relationship was found among the strains. The VO isolated E. faecalis strains showed highest resistance to aminoglycosides, mainly gentamicin (40%), but were highly susceptible to vancomycin (10%). Aminoglycoside resistance genes were detected in seven E. faecalis strains, and AAC6′-APH2″ genes were most frequently detected. The results showed that E. faecalis strains isolated from recently reported VO cases were highly diverse genetically. The diversity of genotypes in circulation in the analysed flocks, without apparent relationship among them, raises questions on aetiopathogenesis of the disease in broilers and evolutionary aspects of E. faecalis.     

Relationship among enterotoxigenic phenotypes, serotypes, and sources of strains in enterotoxigenic Escherichia coli.

The relationship among O groups, O:H serotypes and enterotoxigenic phenotypes was examined in 76 Escherichia coli strains isolated in Brazil from different sources. Of the 17 heat-labile and -stable enterotoxin (LT/ST)-producing strains whose O antigens were identified, 15 belonged to serotypes O6:H16 (7 strains), O63:H- (5 strains), and O139:H28 (3 strains). All 11 ST strains were in group OO128PAC, which was represented by four O:H serotypes. The 23 LT strains with the O antigen identified were distributed among serotypes of 14 O groups. Colonization factor CFA/I was not found in any of the LT strains, but it was found in six LT/ST and three ST strains. On the whole, each E. coli O:H serotype had a particular fermentation pattern. LT/ST as well as ST strains were all isolated from patients with diarrhea, whereas LT strains were isolated from patients with diarrhea, normal children, food, and river water.     

Effects of choleragenoid and glucose on the response of dog intestine to escherichia coli enterotoxins.

Culture superantes of Escherichia coli from diarrhoea patients were tested in dog jejunal loops for thermostable (ST) and thermolabile (LT) enterotoxins. Pre-treatment with choleragenoid blocked LT but not ST. Glucose was found to reduce fluid accumulation induced by both types of enterotoxin. The findings confirm and extend previous evidence of similarities between cholera toxin and E. coli LT and differences between it and E. coli ST.     

Interleukin-8 response in an intestinal HCT-8 cell line infected with enteroaggregative and enterotoxigenic Escherichia coli

This study examined the interleukin-8 (IL-8) response of the intestinal adenocarcinoma HCT-8 cell line to infection with enteroaggregative and enterotoxigenic Escherichia coli pathotypes isolated from patients with travelers' diarrhea. Individual diarrheagenic E. coli strains (enteroaggregative E. coli [EAEC]; n = 30), heat-stable enterotoxin (ST)-producing enterotoxigenic E. coli (ETEC ST; n = 11), heat-labile enterotoxin (LT)-producing enterotoxigenic E. coli (ETEC LT; n = 10), and ST- and LT-producing enterotoxigenic E. coli (ETEC ST:LT; n = 8) were coincubated with HCT-8 cells for 3 h. Tissue culture supernatants were assayed for IL-8 content by enzyme-linked immunosorbent assay. Fifty percent of EAEC (72% of those EAEC carrying the virulence factors aggR, aggA, and aspU and 40% of those EAEC not carrying virulence factors) and 64% of ETEC ST elicited IL-8 production. In contrast, 10% of ETEC LT elicited the production of IL-8 above baseline. These results suggest that (i) the HCT-8 cell line infection model can be used as a tool to differentiate proinflammatory E. coli from noninflammatory isolates; (ii) EAEC has a heterogeneous ability to induce the production of IL-8, and this may be associated with the presence of virulence factors; and (iii) ETEC ST can elicit an inflammatory response and helps explain our earlier findings of increased fecal IL-8 in patients with ETEC diarrhea.     

Enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxin.

The sensitivity of an enzyme-linked immunosorbent assay (ELISA) to detect pure native Escherichia coli heat-stable toxin (ST) and to identify ST-producing strains among clinical isolates was determined. Two synthetically produced ST preparations were used to raise hyperimmune antisera in rabbits and goats: ST(S), which has the same antigenicity as native ST; and ST(C), which is 15-fold more immunogenic. These antisera were used in the double-sandwich technique as either crude double-species antisera or pure single-species antibody. The sensitivity of the assay was increased by using either a purer antibody preparation or the antiserum to the more potent immunogen; the assay in which pure antibody to ST(C) was used was 2,857-fold more sensitive in detecting ST than the assay in which crude antiserum to ST(S) was used. The minimum amount of ST detectable by the ST(C) ELISA was 140 pg/ml, which was an amount 285-fold smaller than that detectable by the suckling mouse assay. Among 50 human E. coli isolates examined by both the ST(C) ELISA and an ELISA for heat-labile toxin (LT), which had a sensitivity of 290 pg/ml for LT, the respective toxins were consistently identified in broth cultures of 10 LT+ and ST-, 15 LT+ and ST+, and 10 LT- and ST+ strains, and there were no false-positive responses. The ST(C) ELISA also detected ST in all of seven ST - producing E. coli strains tested of human origin, which had been shown elsewhere by DNA hybridization probes to have ST-coding genes of either human or porcine origin, and in all of three ST-producing E. coi strains tested of porcine origin. These results indicate that the sensitivity of the ST(C) ELISA is the same as that of previously described LT ELISAs. The concomitant use of both ST and LT ELISAs provides a rapid, simple, and sensitive method for identifying among clinical isolates enterotoxigenic strains of E. coli which produce either toxin.     

Isolation and nucleotide sequence determination of a gene encoding a heat-stable enterotoxin of Escherichia coli.

A gene encoding a heat-stable enterotoxin (ST) from an Escherichia coli strain isolated from a human with diarrhea was cloned and characterized by nucleotide sequence analysis. The gene was found to be partially homologous to a previously characterized ST gene from an E. coli strain of bovine origin. Hybridization studies showed that most ST-producing strains of E. coli isolated from humans with diarrhea possess genes highly homologous to either the ST gene from the bovine strain or the ST gene characterized in the present study.     

Virulence of Escherichia coli clinical isolates in a murine sepsis model in relation to sequence type ST131 status, fluoroquinolone resistance, and virulence genotype

Escherichia coli sequence type ST131 (O25b:H4) has emerged over the past decade as a globally disseminated, multidrug-resistant pathogen. Unlike traditional antimicrobial-resistant E. coli, ST131 derives from virulence-associated phylogenetic group B2 and exhibits extraintestinal virulence factors. This, plus preliminary evidence of virulence in experimental animals, has suggested that ST131's epidemic emergence may be due to high virulence potential, compared with other E. coli types. To test this hypothesis, we compared a large number of matched ST131 and non-ST131 E. coli clinical isolates, both fluoroquinolone resistant and susceptible, plus isolates from classic extraintestinal pathogenic E. coli (ExPEC) sequence types (STs) and case report ST131 household transmission isolates, for virulence in a mouse subcutaneous sepsis model. Overall, in mice, the study isolates produced a wide range of lethality and clinical illness. However, neither ST131 status nor fluoroquinolone phenotype correlated with this diversity of illness severity, which occurred within each of the 6 study groups. In contrast, multiple known or suspected ExPEC virulence genes, including pap (P fimbriae), vat (vacuolating toxin), kpsM II (group 2 capsule), ibeA (invasion of brain endothelium), and clbB/N (colibactin synthesis), plus molecularly defined ExPEC status, were significantly associated with virulence. These findings point away from ST131 isolates as having higher virulence potential compared with other E. coli types in causing invasive extraintestinal infections and suggest instead that ST131's epidemiological success may reflect enhanced fitness for upstream steps in pathogenesis or in colonization and transmission. Additionally, the extensive within-ST virulence diversity suggests an opportunity to compare closely related strains to identify the responsible genetic determinants.     

Prevalence and characterisation of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates in healthy volunteers in Tunisia

The objective of this investigation was to analyse the carriage rate of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in faecal samples of healthy humans in Tunisia and to characterise the recovered isolates. One hundred and fifty samples were inoculated on MacConkey agar plates supplemented with cefotaxime (2 μg/ml) for ESBL-positive E. coli recovery. The characterisation of ESBL genes and their genetic environments, detection of associated resistance genes, multilocus sequence typing (MLST) and phylogroup typing were performed by polymerase chain reaction (PCR) and sequencing. The presence and characterisation of integrons and virulence factors were studied by PCR and sequencing. ESBL-positive E. coli isolates were detected in 11 of 150 faecal samples (7.3%) and one isolate/sample was further characterised. These isolates contained the blaCTX-M-1 (ten isolates) and blaTEM-52c genes (one isolate). The ISEcp1 (truncated by IS10 in four strains) and orf477 sequences were found upstream and downstream, respectively, of all bla (CTX-M-1) genes. Seven different sequence types (STs) and three phylogroups were identified among CTX-M-1-producing isolates [ST/phylogroup (number of isolates)]: ST58/B1 (3), ST57/D (2), ST165/A (1), ST155/B1 (1), ST10/A (1), ST398/A (1) and ST48/B1 (1). The TEM-52-producing isolate was typed as ST219 and phylogroup B2. Six ESBL isolates contained class 1 integrons with the gene cassettes dfrA17-aadA5 (five isolates) and dfrA1-aadA1 (one). Healthy humans in the studied country could be a reservoir of CTX-M-1-producing E. coli.     

Assessment of high-level gentamicin and glycopeptide-resistant Enterococcus faecalis and E. faecium clonal structure in a Portuguese hospital over a 3-year period

This study focussed on the clonal structure and temporal distribution of E. faecalis and E. faecium with high-level resistance to gentamicin (HLGR) and glycopeptides (GR) collected from clinical samples during 2004 to 2006 at a Portuguese Hospital. The findings were an E. faecalis-dominant and epidemic clone (PFGE-AO), the maintenance of a major epidemic E. faecium clone (PFGE-c) and a high prevalence of putative virulence genes—asa1 (aggregation substances), gelE (gelatinase), cylA (cytolysin), esp (enterococcal surface protein), and hyl (hyaluronidase)—most of them significantly associated with the major clones of both species. The E. faecalis GR isolates ST6 and the E. faecium GR isolates ST17, ST18 and ST280 belong to the clonal complexes E. faecalis-CC2 and E. faecium-CC17, which are well adapted to the nosocomial setting and are disseminated worldwide. This study highlights the need for continuous and active surveillance in this Portuguese hospital in order to follow the evolution of these epidemic and persistent clones.     

Evaluation of non-radioactive trivalent DNA probe (LT, ST1a, ST1b) for detecting enterotoxigenic Escherichia coli.

AIMS: To evaluate a digoxigenin-labelled trivalent DNA probe (LT, ST1a, ST1b) for detecting enterotoxigenic Escherichia coli (ETEC), by comparison with a cell culture assay for detecting LT, individual DNA probes for LT, ST1a and ST1b, and an enzyme immunoassay for detecting ST1. METHODS: A 1268 base pair DNA fragment, containing parts of the genes for E coli heat labile enterotoxin (LT) and heat stable enterotoxins (ST1a and ST1b), was random prime labelled with digoxigenin-dUTP. The labelled DNA was used as a probe in colony hybridisation reactions to examine 180 E coli strains of which 92 had previously been shown by a cell culture assay to produce LT. Six LT negative ST1 positive E coli, 34 Verotoxin producing E coli (VTEC), and 84 organisms from other genera were also examined. All organisms other than VTEC were isolated from travellers returning from abroad with diarrhoea. All E coli strains were retested by cell culture for LT, and were tested by enzyme immunoassay (EIA) for ST1, and by the trivalent and individual DNA probes. RESULTS: All 81 isolates, that on retesting by cell culture were positive for LT, also hybridised with the trivalent and LT probes; 27 of these were also enzyme immunoassay (EIA) positive for ST1 of which 24 hybridised with the ST1b probe and three with the ST1a probe. Of 99 isolates, that on retesting by cell culture were negative for LT, all were negative by LT probe and only three were EIA positive for ST1; these three were positive by both trivalent and ST1b probes. Four isolates were positive by the trivalent probe but negative by cell culture and EIA; all four were positive by ST1b probe. Compared with the cell culture assay for LT, the probe had a sensitivity and specificity both of 100%; compared with the EIA for ST1, the probe had a sensitivity of 100% and specificity of 88%. CONCLUSIONS: The trivalent DNA probe is a sensitive, specific, and reliable method for detecting ETEC that should be considered for use by diagnostic microbiology laboratories.