- Volume 58, Issue 7, 2009

Staphylococcus epidermidis biofilm causes device-related meningitis in neurosurgical patients. This study assessed the contribution of polysaccharide and protein to the development of a strong biofilm-positive phenotype in four S. epidermidis isolates associated with probable device-related meningitis, under varying environmental conditions. RT-PCR analysis of the intercellular adhesion operon (icaADBC) and assessment of polysaccharide intercellular adhesin (PIA) production indicated a correlation between increased icaA transcription and PIA production in ica + isolates grown in medium with 4 % ethanol and 4 % NaCl. Treatment of biofilm with sodium metaperiodate caused dispersion of adhered cells (P <0.0001), indicating involvement of PIA. Transcriptional levels of protein factors revealed that atlE transcription levels were similar in all isolates, whilst aap levels were variable, with induction being seen in two isolates following growth in the presence of alcohol or salt. Transcription of agr did not influence protein expression and RNAIII transcription varied among the strains. Although aap transcription was induced, the treatment of biofilm with proteinase K did not always disperse the biofilm. Our data suggest that, among the three ica + S. epidermidis isolates clinically associated with meningitis that were studied, PIA contributed to the strong biofilm-positive phenotype, whereas protein factors appeared to have a secondary role.

To improve the control of Chlamydia trachomatis infection in India, a rapid, specific and cost-effective method is much needed. We developed an in-house PCR assay by targeting a unique genomic sequence encoding a protein from the C. trachomatis phospholipase D endonuclease superfamily that produces an amplified fragment of 368 bp. The specificity of the primers was confirmed using genomic DNA from other sexually transmitted disease-causing and related micro-organisms and from humans. The assay was highly sensitive and could detect as low as 10 fg C. trachomatis DNA. Clinical evaluation of the in-house-developed PCR was carried out using 450 endocervical specimens that were divided in two groups. In group I (n=274), in-house PCR was evaluated against the direct fluorescence assay. The resolved sensitivity of the in-house PCR method was 97.22 % compared with 88 % for the direct fluorescent antibody assay. In group II (n=176), the in-house PCR was compared with the commercial Roche AMPLICOR MWP CT detection kit. The resolved sensitivity of the in-house PCR assay reported here was 93.1 % and the specificity was 97.46 %, making it a cost-effective alternative for routine diagnosis of genital infection by C. trachomatis. The method should facilitate early detection leading to better prevention and treatment of genital infection in India.

Two new Clostridium difficile-selective agars, from Oxoid (according to Brazier) and from BD, were compared with cycloserine-cefoxitin-fructose agar (Oxoid) for their sensitivity of recovery of toxigenic C. difficile from stool samples. For the culture-positive samples, the sensitivities were 84.0, 42.6 and 90.4 %, respectively. In addition, a C. difficile-specific fluorescence in situ hybridization assay was developed, facilitating rapid and reliable identification of cultured isolates.

Standardization of human cytomegalovirus (CMV) PCR is highly recommended. As primer design is essential for PCR sensitivity, this study evaluated all published CMV primer pairs to identify the most sensitive for single-round real-time PCR. PubMed (1993–2004) was searched for original papers aimed at CMV PCR. Fifty-seven papers were identified revealing 82 different primer pairs. Of these, 17 primer sets were selected for empirical study, as they were either used in real-time PCR or were evaluated comparatively by conventional PCR. After optimizing the PCR conditions, these primer sets were evaluated by real-time PCR using a SYBR Green format. Analytical sensitivities were assessed by testing the reference standard CMV strain AD169. A blast search was performed to identify mismatches with published sequences. Additionally, 60 clinical samples were tested with the three primer sets showing highest analytical sensitivity and the best match to all CMV strains. Three primer sets located in the glycoprotein B (UL55) gene region were found to be the most sensitive using strain AD169. However, two of these showed a considerable number of mismatches with clinical isolates in a blast search. Instead, two other pairs from the lower matrix phosphoprotein (UL83) gene and DNA polymerase (UL54) gene showed reasonable sensitivity and no mismatches with clinical isolates. These three pairs were further tested with clinical samples, which indicated that the two primer sets from UL55 and UL54 were the most sensitive. Interestingly, the analytical sensitivity of the PCR was inversely correlated with the size of the PCR product. In conclusion, these two primer pairs are recommended for a standardized, highly sensitive, real-time PCR.

The serotyping of O and H antigens is an important first step in the characterization of Salmonella enterica. However, serotyping has become increasingly technically demanding and expensive to perform. We have therefore sequenced additional S. enterica O antigen gene clusters to provide information for the development of DNA-based serotyping methods. Three S. enterica isolates had O antigen gene clusters with homology to the Escherichia coli O123 O antigen region. O antigen clusters from two serogroup O58 S. enterica strains had approximately 85 % identity with the E. coli O123 O antigen region over their entire length, suggesting that these Salmonella and E. coli O antigen regions evolved from a common ancestor. The O antigen cluster of a Salmonella serogroup O41 isolate had a lower level of identity with E. coli O123 over only part of its O antigen DNA cluster sequence, suggesting a different and more complex evolution of this gene cluster than those in the O58 strains. A large part of the Salmonella O41 O antigen DNA cluster had very close identity with the O antigen cluster of an O62 strain. This region of DNA homology included the wzx and wzy genes. Therefore, molecular serotyping tests using only the O41 or O62 wzx and wzy genes would not differentiate between the two serogroups. The E. coli O123 O-antigenic polysaccharide and its repeating unit were characterized, and the chemical structure for E. coli O123 was entirely consistent with the O antigen gene cluster sequences of E. coli O123 and the Salmonella O58 isolates. An understanding of both the genetic and structural composition of Salmonella and E. coli O antigens is necessary for the development of novel molecular methods for serotyping these organisms.

Real-time PCR was employed to detect a region of the Mycoplasma genitalium mg219 gene, a gene of unknown function, in clinical samples. Amplification of DNA and signal production from 15 other species of human mycoplasmas and 14 other bacteria and viruses did not occur. Using a panel of 208 genital and rectal samples, the sensitivity when compared to the modified mgpa gene (encoding the major surface protein MgPa) real-time PCR assay was found to be 100 % and the specificity of the assay 99.5 % with a positive predictive value of 80 % and a negative predictive value of 100 %. The mg219 gene was found to be in all strains of M. genitalium and was highly conserved. M. genitalium was detected in 3.9 % (11/280, 95 % CI 2.1–6.9) of all male specimens, in 7.7 % (10/130, 95 % CI 4.1–13.7) of patients with non-gonococcal urethritis (NGU) and in 0.7 % (1/150, 95 % CI <0.01–4.1) of patients without urethritis. The presence of M. genitalium was significantly associated with NGU (P ≤0.01; 95 % Cl 0.88–0.98) and non-chlamydial-non-gonococcal urethritis (P=0.0005; 95 % Cl 0.84–0.97).

Non-tuberculous mycobacteria (NTM) causing clinical disease have become increasingly common and more diverse. A new reverse line probe assay, GenoType Mycobacterium CM/AS (Hain Lifescience), was evaluated for identification of a broad range of NTM. It was compared with phenotypic (HPLC) and molecular (DNA probes, in-house real-time multiplex species-specific PCR, 16S rRNA gene PCR and sequencing) identification techniques, which together provided the reference ‘gold standard’. A total of 131 clinical isolates belonging to 31 Mycobacterium species and 19 controls, including 5 non-Mycobacterium species, was used. Concordant results between the GenoType Mycobacterium assay and the reference identification were obtained in 119/131 clinical isolates (90.8 %). Identification of Mycobacterium abscessus and Mycobacterium lentiflavum by the assay was problematic. The GenoType Mycobacterium assay enables rapid identification of a broad range of potentially clinically significant Mycobacterium species, but some species require further testing to differentiate or confirm ambiguous results.

The isolation of Shiga toxin-producing Escherichia coli (STEC) other than serogroup O157 from clinical stool samples is problematic due to the lack of differential phenotypic characteristics from non-pathogenic E. coli. The development of molecular reagents capable of identifying both toxin and serogroup-specific genetic determinants holds promise for a more comprehensive characterization of stool samples and isolation of STEC strains. In this study, 876 stool samples from paediatric patients with gastroenteritis were screened for STEC using a cytotoxicity assay, commercial immunoassay and a conventional PCR targeting Shiga-toxin determinants. In addition, routine culture methods for isolating O157 STEC were also performed. The screening assays identified 45 stools presumptively containing STEC, and using non-differential culture techniques a total of 20 O157 and 22 non-O157 strains were isolated. These included STEC serotypes O157 : H7, O26 : H11, O121 : H19, O26 : NM, O103 : H2, O111 : NM, O115 : H18, O121 : NM, O145 : NM, O177 : NM and O5 : NM. Notably, multiple STEC serotypes were isolated from two clinical stool samples (yielding O157 : H7 and O26 : H11, or O157 : H7 and O103 : H2 isolates). These data were compared to molecular serogroup profiles determined directly from the stool enrichment cultures using a LUX real-time PCR assay targeting the O157 fimbrial gene lpfA, a microsphere suspension array targeting allelic variants of espZ and a gnd-based molecular O-antigen serogrouping method. The genetic profile of individual stool cultures indicated that the espZ microsphere array and lpfA real-time PCR assay could accurately predict the presence and provide preliminary typing for the STEC strains present in clinical samples. The gnd-based molecular serogrouping method provided additional corroborative evidence of serogroup identities. This toolbox of molecular methods provided robust detection capabilities for STEC in clinical stool samples, including co-infection of multiple serogroups.

This study was undertaken to determine the antibacterial activity of eight cationic antimicrobial peptides towards strains of genomovars I–V of the Burkholderia cepacia complex (Bcc) in time–kill assays. All but one of the peptides failed to show activity against the panel of test strains. The exception was magainin II, a 23 aa peptide isolated from the epidermis of the African clawed frog, Xenopus laevis, which exhibited significant bactericidal activity for Bcc genomovars most frequently associated with lung infection of patients with cystic fibrosis. In vitro studies indicated that magainin II protected a human bronchial epithelial cell line (BEAS-2B) from killing by Bcc and suggest that this peptide may have therapeutic potential against these organisms.

The aim of this study was to evaluate the presence and prevalence of multidrug antibacterial resistance in Helicobacter pylori in Bulgaria from 2005 to 2008. The resistance in 828 untreated adults, 124 treated adults and 105 untreated children was, respectively, 26.5, 50.8 and 16.2 % for metronidazole; 18.4, 45.2 and 19 % for clarithromycin; 1, 2.4 and 0 % for amoxicillin; 4.4, 10.6 and 1.9 % for tetracycline; and 9, 14.5 and 5.8 % for ciprofloxacin. Triple resistance to the evaluated agents was uncommon and was detected in 1 % of the untreated children, 3.5 % of the untreated adults and 13.6 % of the treated adults. Five H. pylori strains were resistant to amoxicillin, metronidazole and clarithromycin, two of them exhibiting quadruple resistance. Resistance to four of the five antibacterials tested was found in 0.7 % of the untreated and 1.8 % of the treated adults. The overall level of multidrug resistance in the treated adults (15.4 %) was higher than that in the untreated adults (4.2 %, P=0.0001) and the untreated children (1 %, P=0.0001). The presence of multidrug H. pylori resistance in Bulgaria could be associated with many factors, among them the slightly increasing national use of macrolides, lincosamides and streptogramins and of quinolones since 2000, the significant increase in primary H. pylori clarithromycin resistance, the high tetracycline use between 1994 and 1999, and, in individual cases, the use of azithromycin-based regimens or reuse of nitroimidazoles. In conclusion, for the first time in a European country during the last 5 years, H. pylori strains harbouring a worrying quadruple antibacterial resistance were found in treated as well as in untreated patients. H. pylori susceptibility patterns have a tendency to become unpredictable and should be monitored constantly at both national and global levels.

Biofilms on catheters are responsible for catheter-related bloodstream infections (CRBSIs), which cause significant mortality and morbidity. Antimicrobial catheter-lock solutions may salvage precious catheters by eradicating biofilms. Staphylococcus epidermidis and Candida albicans are frequently isolated organisms in CRBSIs. We evaluated N-acetylcysteine (NAC), EDTA, ethanol and talactoferrin (TLF) individually and in combination with antibiotics against biofilms of S. epidermidis and C. albicans to identify effective catheter-lock solutions. Minimum biofilm-eradication concentrations causing 50 % inhibition (MBEC50) for EDTA, NAC, ethanol and TLF were determined against biofilms of S. epidermidis and C. albicans formed on 96-well microtitre plates. Biomass, mean thickness and viability of S. epidermidis and C. albicans biofilms were evaluated after exposure to MBEC50 concentrations of EDTA, NAC, ethanol and TLF. Antimicrobial combinations of EDTA, NAC, ethanol and TLF with nafcillin, vancomycin, fluconazole and amphotericin B were evaluated systematically for synergy using combination indices (CIs). EDTA, NAC, ethanol and TLF significantly reduced biofilm biomass and mean thickness (P<0.05, one-way ANOVA) of monomicrobial and polymicrobial biofilms as evaluated by confocal microscopy. CIs evaluated at equipotency ratios, and 50, 75 and 90 % effects, showed that EDTA, NAC, ethanol and TLF were synergistic (CI <1) with antibiotics (with few exceptions) against biofilms of S. epidermidis and C. albicans. EDTA, NAC, ethanol and TLF inhibit monomicrobial and polymicrobial biofilms of neonatal strains of S. epidermidis and C. albicans, and are synergistic with antibiotics. Catheter-lock solutions of EDTA, NAC and ethanol alone or in combination with antibiotics may be used to salvage infected catheters, which will directly impact on patient morbidity and health-care costs.

We report a case of a 60-year-old lady with a history of a heel ulcer that had not responded to antibiotic therapy. This progressed to involve the right leg, which was swollen and erythematous. Radiological imaging revealed the presence of gas within the fascial planes. Blood cultures on admission yielded Morganella morganii. Due to the extent of the gas gangrene and her co-morbidities the patient was not suitable for surgical intervention and was treated conservatively with antibiotics. She deteriorated and died within 72 h of presentation. Non-clostridial gas gangrene is relatively rare, and diagnosis is frequently delayed and often missed. Early aggressive surgical intervention combined with appropriate antibiotic therapy is essential. Bacterial species other than Clostridium should be considered in all cases of gas gangrene.

Aspergillus species are widely distributed in nature, and more than 30 species have been reported to be involved in human and animal infection. Cutaneous infections due to Aspergillus terreus are particularly rare. In this report, we describe a case of cutaneous infection caused by A. terreus in a paediatric patient who underwent surgical treatment for an open tibial fracture secondary to an agricultural accident.