All vertebrates produce gastric acid. Its main function is inactivation of ingested microorganisms. The majority of microbiological pathogens ingested never reaches the intestine because of the gastric barrier. Although gastric hypochlorhydria is fairly common due to atrophic gastritis, gastric surgery or use of inhibitors of gastric acid secretion, the resulting susceptibility to infection has not been studied extensively. Drug-induced blockade of acid secretion leads to gastrointestinal bacterial overgrowth; the clinical significance of this is still controversial. Gastric acidity is known to protect against non-typhoid salmonellosis and cholera and it is suspected that it protects against several parasitic diseases as giardiasis and strongyloides. There is a lack of studies focusing on the impact of the gastric acidic barrier on viral infections. Concerning prion infections only a single study has been performed, demonstrating a possible role of gastric acidity in the protection against foodborne prion disease in mice. The combination of malnutrition and hypochlorhydria may contribute to the high prevalence of gastrointestinal infections in developing countries. Further studies are needed to evaluate the clinical consequences of impaired gastric acidity with respect to susceptibility to infections.

The role of atypical enteropathogenic Escherichia coli (EPEC) in childhood diarrhea is controversial. The aim of the present study was to search for genes linked with diarrhea in atypical EPEC strains from a case-control study among Norwegian children. Using DNA microarray analysis, genomic DNAs from strains isolated from children with (n ‫؍‬ 37) and without (n ‫؍‬ 20) diarrhea were hybridized against 242 different oligonucleotide probes specific for 182 virulence genes or markers from all known E. coli pathotypes. PCR was performed to test the strains for seven putative virulence genes not included in the microarray panel. The OI-122 gene efa1/lifA was the gene with the strongest statistical association with diarrhea (P ‫؍‬ 0.0008). Other OI-122 genes (set/ent, nleB, and nleE) and genes with other locations (lpfA, paa, ehxA, and ureD) were also associated with diarrheal disease. The phylogenetic marker gene yjaA was negatively associated with diarrhea (P ‫؍‬ 0.0004). Atypical EPEC strains could be classified in two main virulence groups based on their content of OI-122, lpfA, and yjaA genes. Among children with diarrhea, atypical EPEC isolates belonging to virulence group I (OI-122 and lpfA positive, yjaA negative) were the most common, while the majority of isolates from healthy children were classified as virulence group II strains (OI-122 negative, lpfA and yjaA positive; P < 0.001). In conclusion, using DNA microarray analysis to determine the virulence gene profile of atypical EPEC isolates, several genes were found to be significantly associated with diarrhea. Based on their composition of virulence genes, the majority of strains could be classified in two virulence groups, of which one was seen mainly in children with diarrhea.

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Background and purpose Low-virulence implant infections are characterized by bacterial colonization of the implant with subsequent biofilm formation. In these cases, soft tissue biopsies often prove to be culture negative. Consequently, detachment of the causative adherent bacteria is crucial for correct microbiological diagnosis. Using an in vitro model, we compared 4 methods of biofilm sampling from metal surfaces.Methods Discs of titanium and steel were incubated in the presence of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Propionibacterium acnes in Mueller Hinton broth. Non-adherent bacteria were removed by repeated rinsing of the discs. 10 parallels of each disc were subjected to 1 of 4 methods for bacterial recovery: (A) sonication of the discs, (B) scraping of the discs using surgical blades followed by streaking of the blades onto agar plates, (C) scraping of the discs followed by vortex mixing of the surgical blades, and (D) scraping of the discs followed by sonication of the surgical blades. Quantitative bacterial cultures were performed for each sampling method.Results With the exception of S. epidermidis on steel, sonication efficiently and reliably dislodged biofilm bacteria. The scraping methods employed did not detach bacteria embedded in biofilm.Interpretation Scraping of metal surfaces is not an adequate method for sampling of biofilm bacteria in vitro.

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 ¼ 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 ¼ 210) recruited from Maternal and Child Health Centres were analysed for EPEC only. EPEC isolates (eae þ , stx À ) were classified as typical (bfpA þ ) or atypical (bfpA À ), 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) ¼ 1 . 4, P ¼ 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 ¼ 2 . 1, P ¼ 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.

Atypical enteropathogenetic Escherichia coli (EPEC) strains are frequently detected in children with diarrhea but are also a common finding in healthy children. The aim of this study was to compare the phylogenetic ancestry and virulence characteristics of atypical (eae positive, stx and bfpA negative) EPEC strains from Norwegian children with (n ‫؍‬ 37) or without (n ‫؍‬ 19) diarrhea and to search for an association between phylogenetic ancestry and diarrhea. The strains were classified in phylogenetic groups by phylogenetic marker genes and in sequence types (STs) by multilocus sequence typing. Phylogenetic ancestry was compared to virulence characteristics based on DNA microarray analysis. Serotyping and pulsed-field gel electrophoresis (PFGE) were also performed. All four phylogenetic groups, 26 different STs, and 20 different clonal groups were represented among the 56 atypical EPEC strains. The strains were separated into three clusters by overall virulence gene profile; one large cluster with A, B1, and D strains and two clusters with group B2 strains. There was considerable heterogeneity in the PFGE profiles and serotypes, and almost half of the strains were O nontypeable. The efa1/lifA gene, previously shown to be statistically linked with diarrhea in this strain collection (J. E. Afset et al., J. Clin. Microbiol. 44:3703-3711, 2006), was present in 8 of 26 STs. The two phylogenetic groups B1 and D were weakly associated with diarrhea (P ‫؍‬ 0.06 and P ‫؍‬ 0.09, respectively). In contrast, group B2 was isolated most frequently from healthy controls (P ‫؍‬ 0.05). In conclusion, the atypical EPEC strains were heterogeneous both phylogenetically and by virulence profile. Phylogenetic ancestry was less useful as a predictor of diarrhea than were specific virulence genes.

We report an outbreak of vancomycin-variable vanA+ enterococci (VVE) able to escape phenotypic detection by current guidelines and demonstrate the molecular mechanisms for in vivo switching into vancomycin resistance and horizontal spread of the vanA cluster. Forty-eight vanA+ Enterococcus faecium isolates and one Enterococcus faecalis isolate were analyzed for clonality with pulsed-field gel electrophoresis (PFGE), and their vanA gene cluster compositions were assessed by PCR and whole-genome sequencing of six isolates. The susceptible VVE strains were cultivated in brain heart infusion broth containing vancomycin at 8 μg/ml for in vitro development of resistant VVE. The transcription profiles of susceptible VVE and their resistant revertants were assessed using quantitative reverse transcription-PCR. Plasmid content was analyzed with S1 nuclease PFGE and hybridizations. Conjugative transfer of vanA was assessed by filter mating. The only genetic difference between the vanA clusters of susceptible and resistant VVE was an ISL3-family element upstream of vanHAX, which silenced vanHAX gene transcription in susceptible VVE. Furthermore, the VVE had an insertion of IS1542 between orf2 and vanR that attenuated the expression of vanHAX. Growth of susceptible VVE occurred after 24 to 72 h of exposure to vancomycin due to excision of the ISL3-family element. The vanA gene cluster was located on a transferable broad-host-range plasmid also detected in outbreak isolates with different pulsotypes, including one E. faecalis isolate. Horizontally transferable silenced vanA able to escape detection and revert into resistance during vancomycin therapy represents a new challenge in the clinic. Genotypic testing of invasive vancomycin-susceptible enterococci by vanA-PCR is advised.

We studied the basic release patterns of antibiotics from cancellous bone in vitro. Antibiotic-impregnated bone was compressed into a wire-mesh cylinder and the release of antibiotic was assessed by two different in vitro methods: agar diffusion and broth elution. The zones of inhibition were measured on seeded agar and the amounts of antibiotics released in elution tubes were assessed by a bioassay. The study continued for 21 days with daily transfer of the cylinders. The results indicated that benzylpenicillin, dicloxacillin, cephalotin, netilmicin, clindamycin, vancomycin, ciprofloxacin and rifampicin were adsorbed to cancellous bone in vitro. Compared to broth elution, agar diffusion showed a prolonged period of release, owing to the small amounts of antibiotic leaking out of the cylinder into the agar. The betalactams had antibacterial activity in broth for a shorter time than the other antibiotics. The release patterns of the betalactams were similar, in spite of their differences in thermal stability. Only rifampicin showed a concentration higher than MIC for longer than 21 days.