- Volume 53, Issue 11, 2004

Two hundred and fifty nine isolates of Yersinia enterocolitica and related species were examined for the production of heat-stable enterotoxin (Yersinia stable toxin; YST) as well as for the prevalence of enterotoxin genes, viz. ystA, ystB and ystC. Under the conventional conditions used for the production of Y. enterocolitica enterotoxin, i.e. in tryptic soy broth (TSB) supplemented with yeast extract at 28 °C for 48 h, 77.7 % of clinical isolates and 62.3 % of swine isolates showed enterotoxigenicity in infant mice. All isolates that produced enterotoxin at 28 °C also showed enterotoxic activity at 37 °C after 48 h incubation under an alkaline pH of 7.5, the pH present in the ileum. All Yersinia intermedia and Yersinia frederiksenii isolates were negative for enterotoxin production. All clinical isolates and 96.3 % of Y. enterocolitica isolates from swine hybridized with a probe for ystB, which indicated that the ystB gene was most prevalent in Y. enterocolitica biotype 1A strains. None of the Y. enterocolitica isolates showed hybridization with oligonucleotide probes for ystA or ystC. The study indicated that YST-b was the major contributor to diarrhoea produced by biotype 1A strains of Y. enterocolitica.

Epidemiological studies have reinforced the importance of Enterococcus faecalis in causing serious infections, and to date, our understanding of how certain virulence factors are involved in the pathogenesis of enterococcal infections is still limited. The aim of the present study was to examine the occurrence of known virulence determinants in a group of E. faecalis strains isolated from different clinical sources in Brazil. A total of 95 E. faecalis strains were investigated for the presence of nine virulence genes including aggA, cylA, cylB, cylM, eep, efaA, enlA, esp and gelE by using PCR. The data showed a relatively wide distribution of the virulence genes among the investigated strains. The clinical strains carried at least one and concomitantly up to as many as eight virulence markers, with two or three being the most common pattern. Most of the strains carried efaA (58.9 %), eep (58.9 %) and esp (57.9 %) genes, whereas the remaining virulence markers were detected in variable percentages ranging from 9.5 to 45 %. Simultaneous presence of virulence markers was observed among clinical strains regardless of their sources. In this study, the efaA + esp + gelE + profile was the virulence genotype most frequently detected among E. faecalis strains. Finally, there was no significant association between virulence markers and clinical sources.

In this study, the suitability of two repetitive-element-based PCR (rep-PCR) assays, enterobacterial repetitive intergenic consensus (ERIC)-PCR and BOX-PCR, to rapidly characterize Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis (CF) was examined. ERIC-PCR utilizes paired sequence-specific primers and BOX-PCR a single primer that target highly conserved repetitive elements in the P. aeruginosa genome. Using these rep-PCR assays, 163 P. aeruginosa isolates cultured from sputa collected from 50 patients attending an adult CF clinic and 50 children attending a paediatric CF clinic were typed. The results of the rep-PCR assays were compared to the results of PFGE. All three assays revealed the presence of six major clonal groups shared by multiple patients attending either of the CF clinics, with the dominant clonal group infecting 38 % of all patients. This dominant clonal group was not related to the dominant clonal group detected in Sydney or Melbourne (pulsotype 1), nor was it related to the dominant groups detected in the UK. In all, PFGE and rep-PCR identified 58 distinct clonal groups, with only three of these shared between the two clinics. The results of this study showed that both ERIC-PCR and BOX-PCR are rapid, highly discriminatory and reproducible assays that proved to be powerful surveillance screening tools for the typing of clinical P. aeruginosa isolates recovered from patients with CF.

Multilocus sequence typing of Streptococcus pneumoniae associated with two case clusters of disease is reported here for the first time. Isolates from the first cluster were serotype 19F, resistant to penicillin and erythromycin, and were characterized as ST 320. Isolates from the second cluster were serogroup 4, resistant to ciprofloxacin, and were characterized as ST 206. Therefore, the isolates from these clusters were antibiotic-resistant, of serotypes infrequently isolated, and of uncommon sequence types.

Sixty-seven serotype 14 pneumococci, isolated from invasive disease in Scotland during the first 6 months of 2003, were characterized. Serotype 14 pneumococci accounted for 18.2 % of the total number of cases. Serotyping, multilocus sequence typing and antibiotic susceptibility testing revealed 10 different sequence types (STs), predominantly ST 9 and ST 124; most ST 9 pneumococci were erythromycin-resistant whilst those of ST 124 were not.

The transferase gene wbeT of six clinical isolates of Vibrio cholerae O1 biotype El Tor was analysed. Two unique mutations were identified in the wbeT gene of three Inaba isolates. Due to their random nature, mutations in wbeT can be used to determine the clonal origin of clinical Inaba isolates.

The aim of this work was to determine whether diazepam could induce the multiple antibiotic resistance (Mar) phenotype in Klebsiella pneumoniae and Escherichia coli strains. The Mar phenotype is characterized by decreased susceptibility to multiple antibiotics due to the loss of porins and/or increased expression of active efflux systems. The effect of subinhibitory concentrations of diazepam on the susceptibility of different antimicrobial agents, outer-membrane protein expression and norfloxacin intracellular accumulation was studied. The results revealed that diazepam concentrations equal or twice adult dosage induced the same Mar phenotype as two well known E. coli marRAB inducers, sodium salicylate and sodium benzoate. Susceptibility to norfloxacin in a K. pneumoniae clinical isolate and E. coli strain Ag100 decreased due to enhanced active efflux and loss of porin expression. A decreased susceptibility to chloramphenicol, tetracycline, nalidixic acid and β-lactam antibiotics was also observed. In conclusion, like sodium salicylate or sodium benzoate, diazepam may induce the Mar phenotype.

Almost 50 % of all Helicobacter pylori isolates are resistant to metronidazole, which reduces the efficacy of metronidazole-containing regimens, but does not make them completely ineffective. This discrepancy between in vitro metronidazole resistance and treatment outcome may partially be explained by changes in oxygen pressure in the gastric environment, as metronidazole-resistant (MtzR) H. pylori isolates become metronidazole-susceptible (MtzS) under low oxygen conditions in vitro. In H. pylori the rdxA and frxA genes encode reductases which are required for the activation of metronidazole, and inactivation of these genes results in metronidazole resistance. Here the role of inactivating mutations in these genes on the reversibility of metronidazole resistance under low oxygen conditions is established. Clinical H. pylori isolates containing mutations resulting in a truncated RdxA and/or FrxA protein were selected and incubated under anaerobic conditions, and the effect of these conditions on the MICs of metronidazole, amoxycillin, clarithromycin and tetracycline, and cell viability were determined. While anaerobiosis had no effect on amoxycillin, clarithromycin and tetracycline resistance, all isolates lost their metronidazole resistance when cultured under anaerobic conditions. This loss of metronidazole resistance also occurred in the presence of the protein synthesis inhibitor chloramphenicol. Thus, factor(s) that activate metronidazole under low oxygen tension are not specifically induced by low oxygen conditions, but are already present under microaerophilic conditions. As there were no significant differences in cell viability between the clinical isolates, it is likely that neither the rdxA nor the frxA gene participates in the reversibility of metronidazole resistance.

The aim of the present case control study was to investigate the prevalence of atypical enteropathogenic Escherichia coli (EPEC) and its possible role in causing diarrhoea among children < 5 years of age in Norway. Stool specimens received in the laboratory from children with suspected gastroenteritis (n = 251) were, in addition to routine testing, analysed for the presence of EPEC by PCR of the eae, bfpA and stx genes. Specimens from healthy children (n = 210) recruited from Maternal and Child Health Centres were analysed for EPEC only. EPEC isolates (eae +, stx −) were classified as typical (bfpA +) or atypical (bfpA −), and were tested for O : K serogroup. Information on duration of diarrhoea was recorded in a questionnaire and from referral forms. Atypical EPEC was diagnosed in 37 patients (14.7 %) compared to 21 (10.0 %) of the healthy controls [Odds ratio (OR) = 1.4, P = 0.3]. Only three isolates, all from patients, belonged to EPEC serogroups. One patient had typical EPEC. Twenty (22.5 %) of 89 patients with diarrhoea lasting ⩾14 days had atypical EPEC. The association between atypical EPEC and prolonged diarrhoea (OR = 2.1, P = 0.04) was caused by a high prevalence among female patients (40.6 %). In conclusion, atypical EPEC was found to be slightly more prevalent in patients than controls, without any overall significant association with diarrhoea. However, a significant association was observed with diarrhoea lasting 14 days or more, a finding that may indicate a role for atypical EPEC in prolonged disease.

Enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli are important diarrhoeagenic pathogens; infection is dependent on translocation of a number of type III effector proteins. Until recently all the known effectors were encoded on the LEE pathogenicity island, which also encodes the adhesin intimin and the type III secretion apparatus. Recently, a novel non-LEE effector protein, EspI/NleA, which is required for full virulence in vivo and is encoded on a prophage, was identified. The aim of this study was to determine the distribution of espI among clinical EHEC and EPEC isolates. espI was detected in 86 % and 53 % of LEE+ EHEC and EPEC strains, respectively. Moreover, the espI gene was more commonly found in patients suffering from a more severe disease.

Despite frequent use of immunosuppressive drugs in patients with inflammatory bowel disease (IBD) and reports of cytomegalovirus (CMV) infection following post-transplant immunosuppression, data on the frequency and clinical significance of CMV in patients with IBD are scant. Sixty-three patients with IBD (61 ulcerative colitis and two Crohn's disease) were evaluated for CMV using serology (IgM antibody, μ-capture ELISA), PCR for CMV DNA in colonic biopsy and histological assessment of haematoxylin and eosin-stained colonic biopsy. Positive result in any test was considered as CMV infection. Various parameters associated with CMV infection were analysed using univariate and multivariate analysis. Ten of 63 (15.8 %) patients (age 36.0 ± 11.2 years, 31 female) were infected with CMV (DNA alone in four, IgM antibody alone in two and both in four, inclusion body in one). Patients with CMV infection were more often female (8/10 vs 23/53, P < 0.05), had pancolitis (10/10 vs 33/53, P < 0.05), histological activity (9/10 vs 17/53, P < 0.005) and used azathioprine (5/10 vs 7/53, P = 0.04; Fisher exact test for all). On multivariate analysis, female gender, pancolitis and histological activity were the independent factors associated with infection. Patients with CMV infection more often required surgical treatment for IBD (4/10 vs 4/53, P = 0.01) and had fatal outcome (3/10 vs 0/53, P = 0.003). CMV infection in patients with IBD may be common and is associated with poor outcome. PCR of rectal biopsy was the most sensitive method of detection followed by IgM antibody for diagnosis.

Surveillance performed after the introduction of general Haemophilus influenzae serotype b (Hib) vaccination in Denmark identified 13 cases of invasive bacteraemic H. influenzae serotype f (Hif) disease in adults over a period of 7 years. Bacteraemic respiratory tract infections accounted for 61 % of cases, but meningitis, epiglottitis and osteoarthritis were also seen. Recent Danish isolates were compared to recent American isolates, historical Hif strains and non-Hif invasive strains. Results of conventional serotyping were confirmed by PCR detection of the serotype-f-specific cap and bexA gene sequences. Multilocus enzyme electrophoresis typing revealed that recent Danish and American isolates belonged to a single Hif clone, which may be undergoing expansion. The need for accurate serotyping of H. influenzae to enable reliable monitoring for Hib replacement by other capsular types is emphasized.