- Volume 48, Issue 4, 1999

To assess the influence of incubation conditions on the resistance of Helicobacter pylori this study compared the effect of micro-aerophilic and anaerobic incubation followed by micro-aerophilic incubation on the measurement of metronidazole resistance of 102 H. pylori isolates, by both disk diffusion and Epsilometer (E)-tests. Anaerobic incubation for 24 h before micro-aerophilic incubation for 48 h consistently increased metronidazole activity in both assay methods. Although statistically significant, this was microbiologically less significant, as only 4 of 102 isolates gave discrepant readings (all four were resistant in micro-aerophilic conditions but susceptible in anaerobic/micro-aerophilic conditions). In all four cases variation was by a few millimeters in zone size (i.e., all were close to the cut-off point). There was 100% agreement between disk diffusion and E-test results. Of 104 observations (52 duplicate assays: 13 strains, two atmospheric conditions, two methods of determining resistance) there was 100% intra-observer and inter-observer agreement with regard to susceptibility and resistance status for both E-test and disk diffusion methods. Anaerobic incubation followed by micro-aerophilic incubation had little effect on the estimation of prevalence of metronidazole resistance and seemed to add little, if any, significant advantage over micro-aerophilic incubation alone.

Antimicrobial resistance patterns of Salmonella serotype Typhimurium isolates obtained during the period 1987-1994 were examined and the molecular epidemiology and the mechanisms of resistance to ampicillin, chloramphenicol and trimethoprim were investigated in 24 strains isolated during 1994. Resistance to ampicillin increased from 18% to 78%, to chloramphenicol from 15% to 78%, to tetracycline from 53% to 89% and to co-trimoxazole from 3% to 37%, whereas resistance to norfloxacin remained at 0%. Of Salmonella serotype Typhimurium strains isolated during 1994, all ampicillin-resistant strains had an MIC > 256 mg/L, except one strain in which the MIC was 64 mg/L. Twelve strains (52%) had a TEM-type β-lactamase, nine (39%) a CARB-type β-lactamase and two strains (8%) had an OXA-type β-lactamase. Chloramphenicol acetyl-transferase activity was detected in only nine (47%) of 19 chloramphenicol resistant strains, whereas all eight trimethoprim-resistant strains produced a dihydrofolate reductase type Ia enzyme. Three different epidemiological groups were defined by either low-frequency restriction analysis of chromosomal DNA and pulsed-field gel electrophoresis or repetitive extragenic palindromic-PCR. The latter technique provided an alternative, rapid and powerful genotyping method for S. Typhimurium. Although quinolones provide a good therapeutic alternative, the multiresistance of S. Typhimurium is of public health concern and it is important to continue surveillance of resistance levels and their mechanisms.

Streptococcus oralis has emerged as one of the most important organisms of the viridans streptococcus group in terms of infections and is recognised as an agent of infective endocarditis and, in immunocompromised patients, septicaemia. The mechanisms by which this organism proliferates in vivo are unknown. However, host-derived sialic acids - including N-acetylneuraminic acid (NeuNAc) which is present in serum and cell-associated glycoproteins - are a potential source of fermentable carbohydrate for bacterial proliferation, especially for sialidase-producing bacteria, including S. oralis. To further elucidate the role of NeuNAc in supporting growth, this study determined the ability of S. oralis strain AR3 (isolated from a patient with infective endocarditis) to transport NeuNAc and characterised the transport system. The transport of [14C]-labelled NeuNAc into S. oralis was monitored and this transport system was induced by growth of the bacteria in the presence of the N-acetylated sugars NeuNAc, N-acetylglucosamine and N-acetylmannosamine. The transport system followed typical Michaelis-Menten kinetics, with a Km of 21.0 μm and a Vmax of 2.65 nmoles of NeuNAc transported/min/mg of dry cell mass. NeuNAc transport was inhibited by the presence of exogenous N-glycolylneuraminic acid, a related sialic acid. Chlorhexidine, NaF and 2,4-dinitrophenol were potent inhibitors of the transport system, suggesting that the uptake of NeuNAc occurs via a proton motive force-dependent permease system. This is the first report of the mechanism by which NeuNAc transport occurs in pathogenic streptococci. This transport process may have relevance to the acquisition of a source of fermentable carbohydrate and thus bacterial proliferation in vivo.

An earlier report described the discovery of a micro-organism in the form of a double helix in human small bowel biopsies. Mucosal biopsies of the stomach and small bowel obtained from patients with rheumatic diseases and dyspepsia by enteroscopy and gastroscopy were fixed for scanning electron microscopy to investigate the organism further. In 62% of biopsies, an organism in the form of a double helix with bifid ends, 5-30 μm long, was found lying free on the surface of the mucosa. The organism has been demonstrated in the stomach, duodenum and small bowel. Flagella were never seen to be associated with the organism. In spite of its helical form, the organism lacks many of the factors associated with spirochaete morphology. It is suggested that this, as yet unnamed organism, may be found throughout the length of the digestive tract. Its pathological significance is not known.

Complement activation and development of murine bacteraemia and meningitis following intranasal instillation of cell-bound fimbriated or non-fimbriated organisms were compared to clarify the role of fimbriae in the pathogenesis of illnesses caused by Haemophilus influenza type b (Hib). In-vitro resistance of non-fimbriate bacteria to the bactericidal effects of normal human serum was at least 400 times greater than that of fimbriate bacteria. The amount of C3 bound to fimbriate Hib was more than that to non-fimbriate Hib. When mice were infected with fimbriate bacteria, 11.5% died. When mice were infected with non-fimbriate bacteria, the mean number of viable organisms gradually increased or was constant up to day 7; 38.5% of these mice died. These in-vivo results were coincident with the in-vitro data. However, the content of polyribosyl ribitol phosphate (PRP) in fimbriate organisms was the same as in non-fimbriate organisms. These results indicate that fimbriate Hib may be less likely to produce bacteraemia and meningitis, correlating with the greater susceptibility to complement-mediated bacteriolysis and the lower mortality seen with this type of organism, although fimbriae increase adherence to epithelial cells (mucosal surface).

Infection and associated disease caused by Helicobacter pylori are common in Poland, as in much of Eastern Europe, although the genotypes of strains have not been much studied, especially in terms of traits that might be important in disease. This study developed a sensitive and efficient polymerase chain reaction (PCR) test for the presence of H. pylori in gastric biopsy samples with ureA gene-specific primers and primers for the virulence-associated cag pathogenicity island (PAI). These tests were used with biopsy samples from 246 symptomatic children (age range 1-17 years) and 82 adults (age range 18-53 years) in Warsaw. An assessment was also made of the success of metronidazole-based therapy intended to eradicate infection. H. pylori was detected by ureA-specific PCR in 83 (76.9%) children and in 41 (87.2%) adults with histologically proven gastritis, and in 28.4% and 29.2%, respectively, of the 38 children and 7 adults with little or no evidence of gastritis. In general, H. pylori was detected more often by PCR than by culture (70.3% compared with 52.8% in children and 62.8% compared with 38.6% in adults), although in several cases a negative PCR was associated with a positive culture result. The rate of H. pylori infection increased with age from 5.4% in children up to 5 years old to 29.2% to age 10 and 65.4% to age 18. The tests detected the cagPAI in 97 (75%) and 44 (85%) of the H. pylori-infected children and adults, respectively. Some H. pylori-infected patients with a ureA+ PCR result contained the ‘empty site’ of the cagPAI and only four patients were infected with mixed cag+ cag- strains. PCR with cagPAI and ‘empty site’ of the cagPAI represents a novel tool for fast screening of mixed cag+ cag- infection. These results confirm and further illustrate that direct PCR of biopsy specimens can be useful for detection of infection and genotyping of resident strains, and that H. pylori infection is very common among children as well as adults in Poland. They also show that Polish strains vary with regard to the presence or absence of the cagPAI, and suggest that the proportion of strains that are cag+ is higher in Poland than in Western European countries, which may reflect the relatively higher risk of infection in this society.

The ubiquity of Legionella pneumophila in aquatic habitats means that epidemiological evaluation is important for the investigation and control of nosocomial outbreaks of legionellosis. Pulsed-field gel electrophoresis (PFGE) of chromosomal DNA following digestion with SfiI is considered to be one of the most discriminative methods for detecting DNA polymorphisms amongst L. pneumophila serogroup 1 (Lp1) isolates. This paper describes an arbitrarily primed PCR (AP-PCR) method with three different primers (20-mers) for detecting DNA polymorphisms of Lp1 isolates. The AP-PCR assay was compared with PFGE analysis. Both experimental methods were found to have good discriminatory power (discrimination index of 98% and 94.3%, respectively) with 27 unrelated isolates from different geographical areas collected between 1987 and 1997. Furthermore, when the AP-PCR was used in the epidemiological investigation of nosocomial cases of infection, convergent results with the three primers allowed an epidemiological link to be established between isolates from patients and their environment. The AP-PCR method, which is rapid and easy to perform, gave results at least as discriminatory as those obtained with the PFGE method and is proposed for use in the molecular typing of Lp1 outbreaks.

A murine model with immunocompetent animals was used in a comparative study of experimental pathogenicity of 13 isolates belonging to the four most frequent pathogenic species of Fusarium in man (F. solani, F. oxysporum, F. verticillioides and F. proliferatum). Inocula of 5 × 106 conidia/mouse of each isolate of Fusarium were injected into a lateral vein of the tail of the mice to produce a systemic infection. F. solani was the most virulent species; the five strains to this species assayed caused the death of all the animals tested in <19 days. The other species of Fusarium were not virulent in this model. The organs mainly affected by F. solani were the kidneys and the heart. These findings correlate with the clinical evidence and demonstrate that there is a high risk associated with infection by F. solani, especially for immunocompromised patients.