Latest Articles Include:
- Genetic stability of vaccine strain Salmonella Typhi Ty21a over 25 years
- Int J Med Microbiol 299(4):233-246 (2009)
The attenuated live bacterial vaccine strain Salmonella enterica Serovar Typhi Ty21a is the main constituent of Vivotif, the only licensed oral vaccine against typhoid fever. The strain was developed in the 1970s by chemical mutagenesis. In the course of this mutagenesis, a number of mutations were introduced into the vaccine strain. Characterisation of the vaccine strain during development as well as release of master- and working seed lots (MSL and WSL) and commercial batches is based on phenotypic assays assessing microbiological and biochemical characteristics of Ty21a. In the current study, we have analysed by DNA sequencing the specific mutations originally correlated with the attenuation of strain Ty21a. These data demonstrate the stability of these mutations for MSLs and WSLs of Ty21a produced between 1980 and 2005. Finally, we have confirmed the correlation of these genetic mutations with the expected phenotypic attenuations for the seed lots used in vaccine m! anufacture over 25 years.
- Serine protease espP subtype α, but not β or γ, of Shiga toxin-producing Escherichia coli is associated with highly pathogenic serogroups
- Int J Med Microbiol 299(4):247-254 (2009)
Besides Shiga toxins (Stx), Stx-producing Escherichia coli (STEC) harbour several other putative virulence factors, including the serine protease EspP. We have investigated 214 STEC strains from Austria belonging to 61 different serotypes from humans, animals, and food for the presence of this serine protease gene and have determined the espP subtypes and their association with clinical outcome. espP was detected in 121 (57%) out of 214 strains. Sixty-five of 68 strains (96%) of non-sorbitol-fermenting (NSF) O157:H7/NM (NM, non-motile) were positive for espP, while none of 8 SF E. coli O157:NM isolates contained this gene. All 9 strains of serotype O145:NM and 17 of 21 strains (81%) of serotype O26:H11/NM were positive for espP. Nineteen STEC serogroups including O103 and O111 serogroups – considered to be highly pathogenic – were completely negative for espP. Only 5 of 12 strains isolated from patients suffering from haemolytic uraemic syndrome (HUS) were espP-positive (all serogroup NSF O157) as well as 28 of 39 strains from patients with bloody diarrhoea, 40 of 63 strains from patients with non-bloody diarrhoea, and 15 of 19 strains from asymptomatic patients. In O157:H7/NM, O26:H11/NM, and O145:NM only espP subtype α was found, whereas in most of the other non-O157 serogroups, subtypes β and γ were found. Subtype δ was not detected in our strain collection. Regarding the espP subtypes, only subtype α, but not β and γ, were found in HUS patients. Moreover, we could demonstrate that espP, and in particular subtype α, is associated with highly pathogenic serogroups.
- Mutational analyses of the BbCRASP-1 protein of Borrelia burgdorferi identify residues relevant for the architecture and binding of host complement regulators FHL-1 and factor H
- Int J Med Microbiol 299(4):255-268 (2009)
Borrelia burgdorferi exploits multiple strategies to evade host immune responses. One central immune escape mechanism is the inactivation of the host complement attack by acquisition host complement regulators FHL-1 and factor H via complement regulator-acquiring surface proteins (BbCRASPs). The BbCRASP-1 protein is the first bacterial factor H/FHL-1-binding protein for which the atomic structure has been solved. Previously, 3 regions including the C terminus were identified as putative contact sites for the two complement regulators by the pepspot analysis. Based on the crystallographic structure an in vitro mutagenesis approach was conducted to identify amino acid residues which are relevant for FHL-1 and factor H binding by exchanging single or multiple residues in region 1 and the C-terminally located region 3. Single changes at 4 positions in region 1 either reduced (Lys136, Lys141, Glu147) or completely eliminated (Leu146) binding of both complement regulators. S! ubstitutions clustered within the C-terminal region decreased (Glu234, Lys238, Tyr239, Lys241, Asp244, Thr245) or abolished binding (Lys240, Asp242, Leu246) of both complement regulators. Mapping the mutations onto the atomic structure of BbCRASP-1 reveals that, in contrast to earlier assumption, the C-terminal mutations act indirectly on FHL-1 and factor H binding, whilst the region 1 mutations map the site of direct complement regulator interaction. The elucidation of BbCRASP-1 structure – function may allow development of novel therapeutic strategies against Lyme disease.
- emb nucleotide polymorphisms and the role of embB306 mutations in Mycobacterium tuberculosis resistance to ethambutol
- Int J Med Microbiol 299(4):269-280 (2009)
The emb locus has been considered a target for ethambutol (EMB). Substitutions of codon 306 in Mycobacterium tuberculosis embB have been shown to be the most frequent and predictive mutations for EMB resistance; however, recent reports question the biological role of this mutation. We sequenced embB, embC and embR of 44 EMB-resistant M. tuberculosis strains and found that 30/44 (68.1%) strains had a resistance-associated mutation in one of the three genes sequenced. The majority of these mutations resulted in amino acid replacements at codon 306, 368, 378, and 406 of EmbB. The most common mutation reported in EmbC was at codon 270, followed by mutation at codon 297. Novel mutations were also reported in EmbR. Mutations in embC and embR were usually present together with mutations in embB. We found 41/44 EMB-resistant isolates to be resistant to other antituberculosis drugs as well. Our data confirm that mutation at emb306 does not confer resistance to EMB but is a rath! er common polymorphism in clinical strains of M. tuberculosis predisposing them to the development of any type of drug resistance.
- Detection of non-tuberculous mycobacteria in hospital water by culture and molecular methods
- Int J Med Microbiol 299(4):281-290 (2009)
Non-tuberculous mycobacteria (NTM) are emerging pathogens in immunocompromised patients. Therefore, it is important for hospitals to consider tap water as a source for these infections. Aim of the study was to establish highly sensitive and specific techniques to detect and identify NTM in hospital drinking water. A Mycobacterium genus-specific 16S rDNA-based real-time LightCycler PCR assay with internal inhibition control and a M. xenopi-specific PCR were developed. Ninety-three water samples from 53 taps from 4 hospitals were investigated. NTM were cultured from 21 of 49 (43%) cold and 32 of 44 (73%) warm water samples. M. chelonae, M. flavescens, M. frederiksbergense, M. gordonae, M. moriokaense, M. mucogenicum, M. vaccae, and M. xenopi were identified with molecular methods. All 93 water samples were positive in the genus-specific PCR. M. xenopi DNA was detected in 40 of 44 (91%) warm and 33 of 49 (67%) cold water samples including 45 of 65 (69%) M. xenopi culture-! negative samples. In conclusion, selective culture followed by molecular identification methods enabled detection of rare species of NTM in hospital drinking water and may be used in further studies investigating the epidemiology of NTM in water samples. The real-time PCR assays have a high sensitivity and allow rapid quantification of mycobacterial DNA in water samples.
- Broad-range real-time PCR assay for the rapid identification of cell-line contaminants and clinically important mollicute species
- Int J Med Microbiol 299(4):291-300 (2009)
Polymerase chain reaction assays have become widely used methods of confirming the presence of Mollicutes species in clinical samples and cell cultures. We have developed a broad-range real-time PCR assay using the locked nucleic acid technology to detect mollicute species causing human infection and cell line contamination. Primers and probes specifically for the conserved regions of the mycoplasmal tuf gene (encoding elongation factor Tu) were designed. Cell culture supernatants, clinical specimens (vaginal swabs, sputum, cryopreserved heart valve tissues), and reference strains were tested for mollicute contamination as well as to exclude cross-reaction to human nucleic acids and other bacterial species. Nucleic acids were extracted using magnetic separation technology. The coamplification of the human β2-microglobulin DNA served as an internal control. The PCR assay was highly specific and obtained an analytical sensitivity of one copy per μl sample. The 95% dete! ction limit was calculated to 10 copies per μl sample for Mycoplasma pneumoniae and M. orale. No false-positive results were observed due to cross-reaction of walled bacterial, fungal, and human nucleic acids. To evaluate the PCR, we compared the results to two commercialized test systems. Moreover, in combination with a previously developed broad-range RT-PCR assay for the detection of bacteria in blood products, both mollicute and walled bacterial contamination can be detected simultaneously using multiplex real-time RT-PCR.
- Regulation of extracellular matrix expression and distribution in Trypanosoma cruzi-infected cardiomyocytes
- Int J Med Microbiol 299(4):301-312 (2009)
Alterations in the extracellular matrix have been observed in the cardiomyopathy of Chagas disease caused by Trypanosoma cruzi infection. However, the mechanism of extracellular matrix regulation in T. cruzi-infected cultured cardiomyocytes (CMs) is unclear. Using confocal laser microscopy, we demonstrated that treatment of these cultures with transforming growth factor beta (TGF-β) and tumor necrosis factor alpha (TNF-α) leads to an enhancement of the fibronectin matrix only in uninfected CMs, while infected myocytes displayed low fibronectin expression. Digital image analysis also revealed low superposition of the fibronectin signal with parasite nests in cytokine treated and untreated cultures. Cytochalasin D treatment resulted in microfilament disarray that induced a disturbance in the fibronectin network of CMs, suggesting that cytoskeleton disruption caused by T. cruzi infection disorganizes the fibronectin matrix. Western blot analysis revealed a 2-fold increa! se in the fibronectin expression in CM cultures after cytokine treatment, whereas T. cruzi infection significantly reduced fibronectin levels in all conditions. In contrast, no change in the laminin expression was detected after cytokine treatment. Laminin distribution was altered in T. cruzi-infected CMs, with intense laminin labeling only at the cell periphery even after cytokine treatment. Our observations indicate that TGF-β and TNF-α stimulates fibronectin expression only in uninfected cells of the T. cruzi-infected cultures, whereas the cells harboring the parasites display low or no fibronectin fibrils.