- Volume 59, Issue 3, 2010

Helicobacter pylori is classified as a class I carcinogenic factor and its persistent colonization in the stomach induces gastric cancer. Cancerous Inhibitor of PP2A (CIP2A) is a newly identified oncoprotein overexpressed in gastric cancer. Serving as a key oncoprotein, CIP2A also participates in regulation of senescence and proliferation of gastric cells. The combination of aberrant CIP2A expression inducing unlimited cell proliferation, and H. pylori infection eliciting aberrant expression of some key proteins, results in the onset of gastric tumorigenesis. However, the relationship between H. pylori infection and CIP2A expression still remains undefined. The aim of our study was to verify the effect of H. pylori infection on CIP2A expression levels and identify H. pylori signalling molecules and corresponding pathways influencing CIP2A expression. Following plasmid-mediated expression of CagA in human gastric cell lines, the cells were infected with H. pylori and CIP2A expression levels were examined by immunoblotting. Signal inhibitors were used to verify which signal pathways were involved. We also performed CIP2A depletion and H. pylori infection after depletion in AGS cells. H. pylori infection-induced CIP2A expression was dependent on cagA gene expression and CagA phosphorylation. Bacterial oncoprotein CagA upregulated CIP2A expression and this upregulation effect was dependent on Src and Ras/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways. H. pylori infection-induced Myc stabilization was partially attenuated by CIP2A depletion. The results of our study provide further information for understanding the mechanism of H. pylori carcinogenesis.

Legionella species are facultative, intracellular bacteria that infect macrophages and protozoa, with the latter acting as transmission vectors to humans. These fastidious bacteria mostly cause pulmonary tract infections and are routinely identified by various molecular methods, mainly PCR targeting the mip gene and sequencing, which are expensive and time-consuming. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has emerged as a rapid and inexpensive method for identification of bacterial species. This study evaluated the use of MALDI-TOF-MS for rapid species and serogroup identification of 21 Legionella species recognized as human pathogens. To this end, a reference MS database was developed including 59 Legionella type strains, and a blind test was performed using 237 strains from various species. Two hundred and twenty-three of the 237 strains (94.1 %) were correctly identified at the species level, although ten (4.2 %) were identified with a score lower than 2.0. Fourteen strains (5.9 %) from eight species were misidentified at the species level, including seven (3.0 %) with a significant score, suggesting an intraspecific variability of protein profiles within some species. MALDI-TOF-MS was reproducible but could not identify Legionella strains at the serogroup level. When compared with mip gene sequencing, MALDI-TOF-MS exhibited a sensitivity of 99.2 and 89.9 % for the identification of Legionella strains at the genus and species level, respectively. This study demonstrated that MALDI-TOF-MS is a reliable tool for the rapid identification of Legionella strains at the species level.

A total of 456 non-repetitive Escherichia coli isolates from human clinical specimens (urinary, n=134; cervix, vagina and prostate, n=52; blood, pus and wounds, n=45), healthy animals (cattle, n=45; poultry, n=20) and diseased animals (cattle, n=53; swine, n=64; poultry, n=43) obtained in Lithuania during the period 2005–2008 were studied for trimethoprim (TMP) resistance and the prevalence of dfr genes. A TMP resistance rate in the range of 18–26 % respective to the origin was found in clinical isolates, 23–40 % in isolates from diseased animals and 9–20 % in isolates from healthy animals. Of 112 TMP-resistant isolates, 103 carried at least one of the six dfrA genes (dfrA1, dfrA5, dfrA8, dfrA12, dfrA14 and dfrA17) as determined by multiplex PCR and RFLP. The dfrA1 and dfrA17 genes were found most frequently in clinical isolates (17 and 19 isolates, respectively), whilst dfrA1 and dfrA14 genes dominated in isolates of animal origin (25 and 13 isolates, respectively). The dfrA5, dfrA12 and dfrA8 genes were detected at lower frequencies. The association with class 1/class 2 integrons was confirmed for 73–100 % of dfr genes found in most groups of isolates, except for the isolates from diseased swine. In this group, the majority of dfr-positive isolates (67 %, 8/12) carried dfrA8 (6/12) or dfrA14 genes (2/12) that were not associated with integrons. Non-integron location was also confirmed for the remaining dfrA8 genes (six clinical isolates and one isolate from diseased cattle) and for dfrA14 genes (two isolates from diseased cattle and swine each). All cassette-independent dfrA14 genes were found to be located within the strA gene. This study on the prevalence and distribution of TMP resistance genes among E. coli isolates of human and animal origin in Lithuania demonstrates that dfr genes are carried most frequently as gene cassettes within class 1 and/or class 2 integrons. However, TMP resistance in some of the isolates was found to be mediated by non-integron-associated dfrA8 and dfrA14 genes, indicating the existence of alternative sources for the spread of resistance.

The levels of meticillin-resistant Staphylococcus aureus (MRSA) in Pakistan and India are known to be high, but few studies have described the epidemiology of the different MRSA clones present. In order to gain an understanding of the epidemiology of MRSA within this region, 60 MRSA isolates from Pakistan (49) and India (11) were genotyped. All isolates were typed using PFGE, staphylococcal interspersed repeat units (SIRUs), a restriction–modification method and staphylococcal cassette chromosome mec (SCCmec) typing. A subset of isolates that were distinct by PFGE and SIRUs were typed using multilocus sequence typing (MLST). Clonal complex (CC) 8 was the dominant clonal complex (57/60) and was present in both Pakistan and India. Within CC8, there were 10 SIRU profiles and 24 PFGE profiles. Two SIRU profiles were present in isolates from both India and Pakistan, whilst seven were distinct for Pakistan and one for India. All PFGE profiles were distinct for each of the two countries. Thirty-four of the 57 isolates carried SCCmec type III/IIIa and the remainder carried type IV SCCmec. MLST analysis of 14 CC8 isolates with diverse SIRU and PFGE profiles showed that all were single-locus variants, with nine belonging to sequence type (ST) 239, three to ST8 and two to ST113. From a single hospital in Pakistan, three isolates belonged to CC30 and all were indistinguishable by PFGE and SIRUs and carried the Panton–Valentine leukocidin gene. Thus, epidemiological typing of strains from three distinct locations in India and Pakistan revealed the predominance of one clonal complex and highly related STs. The ability of SIRUs and PFGE to differentiate within ST239 demonstrates their utility in defining local epidemiology in these countries.

The aim of this study was to investigate the role and clinical course of verotoxigenic Escherichia coli (VTEC) infections in children with acute diarrhoea from Argentina, the country with the highest worldwide incidence of haemolytic uraemic syndrome (HUS). To accomplish this objective, 437 samples from children up to 6 years old with acute diarrhoea were collected and processed. More than 60 % of the children studied presented watery or mucous diarrhoea without blood, and in 25.2 % of the cases the samples contained blood. In a first screening, a multiplex PCR was performed to detect the presence of the vt 1, vt 2, eae, ehxA and saa virulence genes. The strains were then isolated and analysed to characterize their serotypes, virulence genes, antibiotic susceptibility profiles and verotoxin (VT) production. Forty-four of the 437 samples (10.1 %) were positive for VTEC virulence genes. VTEC-infected patients presented different types of diarrhoea (27.3 % belonged to the non-bloody type). Several serotypes and virulence genotypes were found. Isolates belonged to the serotypes O157 : H7, O145 : H−, O26 : H11, O121 : H19, O111 : H2 and O118 : H2. HUS developed in 16 (36.4 %) patients positive for VTEC virulence genes. All of the VTEC isolates produced a cytopathic effect on Vero cell monolayers, confirming the ability to express VT. Despite most strains being sensitive to all of the antimicrobials studied, a positive association between clinical progression to HUS and antibiotic therapy was observed for the total number of patients studied, as well as for the VTEC+ group. In conclusion, the data obtained in this study increase our knowledge of the role and clinical course of VTEC infection in childhood acute diarrhoea beyond bloody diarrhoea, and might be considered for the prevention, diagnosis and management of this disease. It is possible that the optimal approach for VTEC diagnosis could be using multiplex PCR to search for the presence of the vt 1, vt 2, eae and ehxA genes.

The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of humans, and various species of animals and birds, in which it may induce a mild colitis and diarrhoea. The aim of the current study was to evaluate the use of putative oligopeptide-binding proteins of B. pilosicoli as vaccine components. A partial genome sequence of B. pilosicoli porcine strain 95/1000 was subjected to bioinformatics analysis, and six genes predicted to encode oligopeptide-binding proteins were selected. Following a PCR-based distribution study of the genes across different strains of the spirochaete, they were amplified from B. pilosicoli human strain WesB and cloned in Escherichia coli. The recombinant histidine-tagged proteins were purified and subjected to in vitro and in vivo immunogenicity analysis. Recombinant products (P-1 and P-3) from two genes that were immunogenic and recognized by sera from pigs that had recovered from B. pilosicoli infections were tested in a mouse model of intestinal spirochaetosis. For each recombinant protein, groups of 12 C3H/HeJ mice were vaccinated subcutaneously with 100 μg protein emulsified in Freund's incomplete adjuvant, twice with a 2 week interval. Two weeks later the vaccinated and non-vaccinated control animals were challenged orally with B. pilosicoli strain WesB. Both proteins induced systemic and local colonic IgG antibody responses, and, following experimental infection, the cumulative number of colonization days was significantly (P<0.001) less in both groups of vaccinated mice compared to the control mice. There were significantly (P=0.012) fewer mice colonized in the group vaccinated with P-1 than in the non-vaccinated control group. The results suggest that oligopeptide-binding proteins may have potential for use as components of vaccines for B. pilosicoli.

A strain of Lactobacillus, identified as Lactobacillus fermentum L23, was selected from among 100 strains isolated from vaginal swabs of healthy, non-pregnant, pre-menopausal women. L. fermentum L23 was chosen on the basis of its bacteriocinogenic ability and its properties relevant to colonization, i.e. self-aggregation, adherence to vaginal epithelial cells and co-aggregation with bacterial pathogens. The antimicrobial preventative and curative effects produced by the probiotic L. fermentum L23 administered locally against Escherichia coli in a murine vaginal tract infection model were studied. One dose of the human strain L23 containing 108 c.f.u. ml−1 colonized and persisted in the vaginal tract of the female BALB/c mice for 5 days. Infection with the pathogen at 106 c.f.u. ml−1 in the vaginal tract was maintained for more than 7 days. A single dose of L23 administered 24 h pre-infection inhibited E. coli growth on day 3 post-infection, showing the preventative effect displayed by this Lactobacillus strain. Treatment with L. fermentum L23 during the post-infection period showed complete inhibition of pathogen growth from day 5. Thus, this in vivo study indicated that the probiotic bacterium L. fermentum L23 produced both preventative and curative effects on E. coli growth. The beneficial properties and the production of antimicrobial metabolites may act in situ to inhibit a pathogenic micro-organism within the vaginal environment. Strain L23 could be a good natural alternative to other therapies used for genital infections.

Herein, we describe what we believe to be the first case of traumatic endophthalmitis caused by Staphylococcus gallinarum, following injury with an iron nail. The patient was successfully treated by vitrectomy and intravitreal injection of cefazolin and vancomycin.