- Volume 55, Issue 1, 2006

This study utilized suppressive subtractive hybridization between the clinical isolate Burkholderia cenocepacia J2315 and the closely related environmental isolate Burkholderia cepacia ATCC 25416T to isolate DNA fragments specific to B. cenocepacia J2315. Analysis of the resulting pools of B. cenocepacia-specific DNAs identified several fragments that may be part of putative virulence factors. Further in silico analysis of a single fragment indicated that it was internal to a gene of which the predicted product had characteristics of repeat in toxin (RTX)-like proteins and high similarity to proteins in other human or plant pathogens. In conjunction with this finding, phenotypic traits associated with known RTX proteins were assessed. A haemagglutinating activity of B. cenocepacia J2315 was identified that was absent in B. cepacia ATCC 25416T. The expression of this activity appeared to be growth phase-dependent. Analysis of the gene presence and haemagglutinating activity across the species of the B. cepacia complex showed that both were common to the ET12 lineage of B. cenocepacia, but were absent in the other species examined. Haemagglutinating activity was limited to isolates with the RTX-like gene. Expression studies utilizing quantitative PCR demonstrated an association between onset of haemagglutinating activity and increased expression of the gene, which suggests that the putative RTX determinant encodes a haemagglutinating activity.

Both O157 : H7 and non-O157 : H7 Escherichia coli strains are reported to cause haemolytic–uraemic syndrome (HUS). This study was carried out to explore the pathogenicity of O157 : H7 and non-O157 : H7 E. coli strains in experimentally inoculated dogs. Twenty 40-day-old dogs were randomly divided into four groups, and the groups (n=5) were administrated orally with E. coli O157 : H7 strains HJ2001-1 (from a patient with serious haemorrhagic diarrhoea) and HZ2001-4 (from a domestic sheep kept in the house of a patient who died from diarrhoea and subsequent acute renal failure), HZ2001-9 (a non-O157 : H7 strain, from a 6-month-old child who died from diarrhoea and subsequent acute renal failure) or a control strain, EC8099. HJ2001-1 and HZ2001-4 caused slight diarrhoea, and the dogs recovered without any complications. However, HZ2001-9 resulted in watery diarrhoea accompanied with slightly bloody stools, followed by death on the fifth or sixth day. In the fatally infected experimental animals, necrotic lesions in the liver and bacterial embolism in the kidney were observed. The primary cause of death was microvascular thrombosis caused by the bacteria, leading to renal and multiple organ failure. Therefore, the non-O157 : H7 E. coli strain HZ2001-9 causes clinical signs and pathological lesions in dogs that are consistent with those in acute renal failure or HUS in humans.

The host range of well-characterized mycobacteriophages, such as D29 and TM4, has been determined, together with that of more recently isolated mycobacteriophages, in Mycobacterium ulcerans, Mycobacterium tuberculosis, Mycobacterium bovis BCG, Mycobacterium avium, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium fortuitum and Mycobacterium chelonae. Here, a set of virulent phages for M. ulcerans, a pathogen with a dramatic increase of incidence over the last decade, is demonstrated. In this work, a mycobacteriophage replication assay was adapted for the identification and rifampicin-susceptibility testing of M. ulcerans. Mycobacteriophages have generated a number of useful tools and enabled insights into mycobacterial genetics. With regard to the neglected pathogen M. ulcerans, the findings presented in this work allow the application of a large range of phage-based vectors and markers. The potential of phage therapy can now be evaluated for this extracellular pathogen.

The aim of this study was to identify a set of genetic polymorphisms that efficiently divides methicillin-resistant Staphylococcus aureus (MRSA) strains into groups consistent with the population structure. The rationale was that such polymorphisms could underpin rapid real-time PCR or low-density array-based methods for monitoring MRSA dissemination in a cost-effective manner. Previously, the authors devised a computerized method for identifying sets of single nucleotide polymorphisms (SNPs) with high resolving power that are defined by multilocus sequence typing (MLST) databases, and also developed a real-time PCR method for interrogating a seven-member SNP set for genotyping S. aureus. Here, it is shown that these seven SNPs efficiently resolve the major MRSA lineages and define 27 genotypes. The SNP-based genotypes are consistent with the MRSA population structure as defined by eburst analysis. The capacity of binary markers to improve resolution was tested using 107 diverse MRSA isolates of Australian origin that encompass nine SNP-based genotypes. The addition of the virulence-associated genes cna, pvl and bbp/sdrE, and the integrated plasmids pT181, pI258 and pUB110, resolved the nine SNP-based genotypes into 21 combinatorial genotypes. Subtyping of the SCCmec locus revealed new SCCmec types and increased the number of combinatorial genotypes to 24. It was concluded that these polymorphisms provide a facile means of assigning MRSA isolates into well-recognized lineages.

Rapid detection of micro-organisms from blood is one of the most critical functions of a diagnostic microbiology laboratory. Automated blood-culture systems reduce the time needed to detect positive cultures, and reduce specimen handling. The false-positive rate of such systems is 1–10 %. In this study, the presence of pathogens in ‘false-positive’ bottles obtained from BACTEC 9050 (Becton Dickinson) and BacT/Alert (Biomérieux) systems was investigated by eubacterial and fungal PCR. A total of 169 subculture-negative aerobic blood-culture bottles (104 BacT/Alert and 65 BACTEC) were evaluated. Both fungal and eubacterial PCRs were negative for all BACTEC bottles. Fungal PCR was also negative for the BacT/Alert system, but 10 bottles (9·6 %) gave positive results by eubacterial PCR. Sequence analysis of the positive PCR amplicons indicated the presence of the following bacteria (number of isolates in parentheses): Pasteurella multocida (1), Staphylococcus epidermidis (2), Staphylococcus hominis (1), Micrococcus sp. (1), Streptococcus pneumoniae (1), Corynebacterium spp. (2), Brachibacterium sp. (1) and Arthrobacter/Rothia sp. (1). Antibiotic usage by the patients may be responsible for the inability of the laboratory to grow these bacteria on subcultures. For patients with more than one false-positive bottle, molecular methods can be used to evaluate the microbial DNA in these bottles. False positives from the BACTEC system may be due to elevated patient leukocyte counts or the high sensitivity of the system to background increases in CO2 concentration.

The aim of this study was to evaluate the primary, combined and post-treatment antibacterial resistance rates in 1205 Helicobacter pylori strains from non-treated (786 adults, 282 children) and treated (109 adults, 28 children) patients in Bulgaria. Susceptibility was tested by the limited agar dilution method. Overall primary resistance rates to metronidazole, clarithromycin, amoxicillin, tetracycline and both metronidazole and clarithromycin were respectively 15·0, 12·5, 1·5, 3·4 and 4·7 % in children and 25·6, 12·6, 0·8, 5·2 and 4·9 % in adults. Primary metronidazole resistance in adults was more common than in children, but the differences for other agents tested were not significant. Primary resistance rates were in the range of those reported worldwide. There was no significant increase in primary resistance rates from 1996/1999 to 2003/2004; however, clarithromycin resistance rates exhibited a slight tendency to increase. Post-treatment resistance to amoxicillin was detected in 1·6 % of 63 strains. Post-treatment resistance to metronidazole was common (81·6 %) and that to clarithromycin was considerable (36 %). Alarming emergence of strains with triple resistance to amoxicillin, metronidazole and clarithromycin was found in two non-treated and three treated patients. The results motivate a larger and continuing surveillance of H. pylori resistance in Bulgaria and worldwide.

Chronic heart failure (CHF) involves interactions between the cardiovascular, neuroendocrine and immune systems. This study investigated the seropositivity rate for anti-Toxoplasma IgG and IgM antibodies by ELISA in patients with CHF. Ninety-seven patients with CHF and 50 healthy volunteers were selected for this investigation. The seropositivity rate for anti-Toxoplasma IgG antibodies among CHF patients (68 %) was significantly higher than in healthy volunteers (36 %). Thus, parasitological screening of this group of patients should be periodically performed to prevent the possible dissemination of toxoplasmosis.

This study was carried out in order to compare two PCR-based methods in the detection of Streptococcus mutans. The first PCR method was based on primers for the 16S rRNA gene and the second method was based on specific primers that targeted the glucosyltransferase gene (gtfB). Each PCR was performed with eight different streptococci from the viridans group, five other streptococci and 17 different non-streptococcal bacterial strains. Direct use of the S. mutans 16S rRNA gene-specific primers revealed that Streptococcus gordonii and Streptococcus infantis were also detected. After amplifying the 16S rRNA gene with universal primers and subsequently performing nested PCR, the S. mutans-specific nested primers based on the 16S rRNA gene detected all tested streptococci. There was no cross-reaction of the gtfB primers after direct PCR. Our results indicate that direct PCR and nested PCR based on 16S rRNA genes can reveal false-positive results for oral streptococci and lead to an overestimation of the prevalence of S. mutans with regards to its role as the most prevalent causative agent of dental caries.

A non-tuberculous mycobacterium was isolated, following a vertebral needle aspiration, from the blood of a patient with severe spondylodiscitis. The strain turned out to be different from any known mycobacterial species and was quite drug-susceptible in vitro. The patient improved markedly following treatment with meropenem, clarithromycin and amikacin.

Pyomyositis is a disease of abscess formation deep within large striated muscles. Outside of the tropics it is a rare disease which occurs mainly in certain patient populations such as the immunosuppressed or intravenous drug users (IDUs). A case of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) pyomyositis in an IDU is described. The incidence of both CA-MRSA and pyomyositis is currently increasing. To the authors' knowledge this is the first reported case of CA-MRSA pyomyositis in the UK. Cases of CA-MRSA pyomyositis are likely to increase and it may be necessary to empirically treat certain patients with glycopeptides.

Three mycobacterial isolates, one from the blood of an HIV-infected patient and two consecutive isolates from a woman with unknown HIV status, had been identified as belonging to the Mycobacterium avium complex by conventional procedures. In both patients, using genetic analysis procedures such as PCR–restriction enzyme analysis (PRA) of the hsp65 gene, a commercially available reverse hybridization-based assay (INNO-LiPA mycobacteria) and/or sequencing analysis of the 16S–23S internal transcribed spacer (ITS), the presence of Mycobacterium lentiflavum was also demonstrated. At the time of detection, both cases were also infected with M. avium, suggesting an underestimation of infection with M. lentiflavum and co-infection with different Mycobacterium species.