- Volume 58, Issue 6, 2009

Although mycoplasmal pneumonia has been generally considered to be a disease with good prognosis, a pathogenic host immune response has been associated with its occurrence. In the present study, the pathogenic significance of the immune response was examined using germ-free mice either infected intranasally with Mycoplasma pneumoniae or inoculated with M. pneumoniae antigens (soluble antigen and partially purified antigen). In gnotobiotic mice monoassociated with M. pneumoniae, 104 c.f.u. M. pneumoniae per lung were isolated 2–28 days after infection. Inflammatory changes with infiltration of lymphocytes were histopathologically detected in the perivascular area at 2 and 7 days after infection. In the mice intranasally inoculated with soluble antigen or partially purified antigens (F6 and F10 antigens), infiltration of neutrophils and lymphocytes was histopathologically detected at 2 days after inoculation. Severe pneumonia with tissue destruction was observed in the mice inoculated with F6 antigen. A gamma interferon (IFN-γ) dominant response in endogenous cytokine expression was observed in all the treated mice. These results indicate that inflammatory changes in the lung tissue were prolonged in gnotobiotic mice monoassociated with M. pneumoniae compared with mice inoculated with M. pneumoniae antigen. In addition, it was shown that IFN-γ plays an important role in the pathogenesis of pneumonia in mice either infected with M. pneumoniae or inoculated with its antigen. In particular, the F6 antigen has been considered to be an important virulence factor in terms of induction of tissue injury causing infiltration of lymphocytes and neutrophils in the lung, suggesting a close interaction between the immune response and the occurrence of M. pneumoniae pneumonia.

Paracoccidioides brasiliensis yeast cells can enter mammalian cells and may manipulate the host cell environment to favour their own growth and survival. Moreover, fibronectin and several other host extracellular matrix proteins are recognized by various components of the yeast cell extracts. The present study was designed to isolate and characterize a fibronectin-binding protein from P. brasiliensis. We also compared P. brasiliensis strain 18, tested before (Pb18a) and after (Pb18b) animal passage, in relation to its adhesion and invasion processes. Extracts from both samples, when cultured on blood agar solid medium, showed higher levels of protein expression than when the same samples were cultured on Fava-Netto solid medium, as demonstrated by two-dimensional electrophoresis and SDS-PAGE. Also, both Pb18a and Pb18b exhibited stronger adhesion to A549 epithelial cells when cultured on blood agar medium than when cultured on Fava-Netto medium. Ligand affinity binding assays revealed a protein of 54 kDa and pI 5.6 in P. brasiliensis cell-free extracts with the properties of a fibronectin-binding adhesin, which was characterized by tryptic digestion and mass spectroscopy as a homologue of enolase from P. brasiliensis. Antibody raised against this 54 kDa protein abolished 80 % of P. brasiliensis adhesion to A549 epithelial cells. Our results demonstrate that P. brasiliensis produces a fibronectin-binding adhesin, irrespective of the culture medium, and that this activity can be inhibited by a specific antibody and is involved in the adhesion of the fungus to pulmonary epithelial cells.

Aspergillus fumigatus, an opportunistic fungal pathogen, infects the human host via inhalation of airborne conidia. Adhesion of fungal conidia, to host cells and extracellular matrix (ECM) components associated with host tissue surfaces, is thought to be the primary step in the pathogenesis and dissemination of infection. To identify novel adhesion proteins (adhesins) of A. fumigatus, we screened its proteome in silico using spaan (software program for prediction of adhesins and adhesin-like proteins using neural networks). One of the predicted adhesin-encoding genes with a P ad (probability of being adhesin) value >0.9, the gene encoding extracellular thaumatin domain protein (AfCalA), was cloned and expressed in Escherichia coli. Recombinant AfCalAp showed significant binding with laminin and murine lung cells. Anti-AfCalAp antibodies inhibited the binding of AfCalAp to laminin in a dose-dependent manner. Significant binding of anti-AfCalAp antibodies to 2 h swollen conidia suggests the presence of AfCalAp on the conidial surface. AfCalA transcript was not detectable in resting conidia but was detected in conidia incubated with RPMI 1640 medium in the presence and absence of lung epithelial cell line (A539)-derived ECM. Elevated levels of IgE antibodies specific to AfCalAp were observed in the sera of two out of seven patients with allergic bronchopulmonary aspergillosis. The study confirms the relevance of the bioinformatic approach for predicting fungal adhesins and establishes AfCalAp as a novel laminin-binding protein of A. fumigatus.

This study established an experimental model of replicative Legionella longbeachae infection in A/J mice. The animals were infected by intratracheal inoculation of 103–109 c.f.u. L. longbeachae serogroup 1 (USA clinical isolates D4968, D4969 and D4973). The inocula of 109, 108, 107 and 106 c.f.u. of all tested L. longbeachae serogroup 1 isolates were lethal for A/J mice. Inoculation of 105 c.f.u. L. longbeachae caused death in 90 % of the animals within 5 days, whilst inoculation of 104 c.f.u. caused sporadic death of mice. All animals that received 103 c.f.u. bacteria developed acute lower respiratory disease, but were able to clear Legionella from the lungs within 3 weeks. The kinetics of bacterial growth in the lungs was independent of inoculum size and reached a growth peak about 3 logarithms above the initial inoculum at 72 h after inoculation. The most prominent histological changes in the lungs were observed at 48–72 h after inoculation in the form of a focal, neutrophil-dominant, peribronchiolar infiltration. The inflammatory process did not progress towards the interstitial or alveolar spaces. Immunohistological analyses revealed L. longbeachae serogroup 1 during the early phase of infection near the bronchiolar epithelia and later co-localized with inflammatory cells. BALB/c and C57BL/6 mice strains were also susceptible to infection with all L. longbeachae serogroup 1 strains tested and very similar changes were observed in the lungs of infected animals. These results underline the infection potential of L. longbeachae serogroup 1, which is associated with high morbidity and lethality in mice.

The aim of this study was to investigate the prevalence and characteristics of ACME (arginine catabolic mobile element)-arcA-positive isolates among meticillin-resistant Staphylococcus haemolyticus (MRSH). ACME-arcA, native arcA and SCCmec elements were detected by PCR. Susceptibilities to 10 antimicrobial agents were compared between ACME-arcA-positive and -negative isolates by chi-square test. PFGE was used to investigate the clonal relatedness of ACME-arcA-positive isolates. The phylogenetic relationships of ACME-arcA and native arcA were analysed using the neighbour-joining methods of mega software. A total of 42 (47.7 %) of 88 isolates distributed in 13 PFGE types were positive for the ACME-arcA gene. There were no significant differences in antimicrobial susceptibility between ACME-arcA-positive and -negative isolates. A novel ccr allotype (ccrAB SHP) was identified in ACME-arcA-positive isolates. Among 42 ACME-arcA-positive isolates: 8 isolates harboured SCCmec V, 8 isolates harboured class C1 mec complex and ccrAB SHP; 22 isolates harbouring class C1 mec complex and 4 isolates harbouring class C2 mec complex were negative for all known ccr allotypes. The ACME-arcA-positive isolates were first found in MRSH with high prevalence and clonal diversity, which suggests a mobility of ACME within MRSH. The results from this study revealed that MRSH is likely to be one of the potential reservoirs of ACME for Staphylococcus aureus.

Anthrax is a zoonotic disease caused by Bacillus anthracis. The infection is associated with inflammation and sepsis, but little is known about the acute-phase response during disease and the nature of the bacterial factors causing it. In this study, we examined the levels of the acute-phase proteins (APPs) in comparative experiments using mice challenged with spores and a purified B. anthracis protease InhA as a possible factor mediating the response. A strong increase in the plasma levels of APPs such as haptoglobin and serum amyloid A was observed during infection. Protein and mRNA levels of plasminogen activator inhibitor (PAI)-1 in the liver were also increased concurrently with bacterial dissemination at 72 h post-infection. Similar effects were observed at 6 h post injection with InhA. Induction of hepatic transforming growth factor-β1, a PAI-1 inducer, was also found in the liver of InhA-injected mice. PAI-1 elevation by InhA resulted in an increased level of urokinase-type plasminogen activator complex with PAI-1 and a decreased level of D-dimers indicating inhibition of blood fibrinolysis. These results reveal an acute liver response to anthrax infection and provide a plausible pathophysiological link between the host inflammatory response and the pro-thrombotic haemostatic imbalance in the course of disease through PAI-1 induction in the liver.

Recent molecular studies have led to the recognition of three distinct species within the Candida parapsilosis complex, namely Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis. As currently available yeast identification systems fail to differentiate these species, there is a paucity of information on their occurrence in different geographical regions. This study describes a simple PCR-based protocol for rapid discrimination among C. parapsilosis, C. orthopsilosis and C. metapsilosis strains by using primers derived from unique sequences within the internally transcribed spacer 1 (ITS1)–5.8 rRNA–ITS2 region. Retrospective analysis of 114 C. parapsilosis-complex isolates recovered from clinical specimens in Kuwait identified 109 as C. parapsilosis, five as C. orthopsilosis and none as C. metapsilosis. The results were further validated by PCR-RFLP patterns of the secondary alcohol dehydrogenase gene fragment. DNA sequencing of the ITS region and the D1/D2 regions of the 28S rRNA gene confirmed the species-specific identification of all five C. orthopsilosis strains. The amplicon length of the intergenic spacer between the 28S and 5S rRNA genes (IGS1) was also species-specific, and PCR-RFLP analyses of the IGS1 region identified two distinct genotypes among the five C. orthopsilosis strains, which corresponded with the ITS region sequence data. The three bloodstream C. orthopsilosis strains were confined to a single genotype. Among 81 randomly selected C. parapsilosis strains, two genotypes were detected by IGS1 region analyses, indicating limited genotypic heterogeneity among C. parapsilosis sensu stricto strains. As far as is known, this is the first report on the identification of C. orthopsilosis from a bloodstream infection in the Arabian Gulf region.

Mycobacterium leprae, the causative agent of leprosy, is uncultivable in defined media. Development of new diagnostic tools which do not depend on growth of bacteria is needed for the early detection of M. leprae and for monitoring the effectiveness of chemotherapy. We used a real-time PCR-based assay to quantify the copy number of bacterial DNA and hsp18 mRNA from 47 leprosy patients using paraffin-embedded biopsy samples. The assay used was specific, sensitive and reproducible. The applicability of this approach in monitoring the chemotherapy of leprosy was examined. A reduction in DNA and mRNA during chemotherapy was observed and hsp18 mRNA could not be detected in patients who underwent 2 years of multidrug therapy (MDT). However, a considerable amount of M. leprae DNA could be detected even after 2 years of MDT. A significant amount of hsp18 mRNA was found in reactional cases as well. This raises important questions regarding the role of bacterial antigens in leprosy reactions and the rationale of omitting antibiotics in the treatment of reactional cases. Results in this study show that real-time PCR could be a better tool for the careful monitoring of bacillary DNA and mRNA in lesions, which will help to improve diagnosis, disease progression and the treatment regimen.

The distribution of different Chlamydia trachomatis serovars in Hungary has not been reported previously. The objective of this study was to determine the distribution and prevalence of C. trachomatis serovars in a high-risk population by genotyping. The endocervical specimens of 484 female sex workers (FSWs) were screened for C. trachomatis by plasmid PCR. Genotyping was performed in all C. trachomatis-positive samples by PCR-based RFLP analysis of the omp1 gene. A total of 32 specimens (6.6 %) were positive for C. trachomatis. Age was an important risk factor for C. trachomatis infection in FSWs. The highest prevalence was detected in women under the age of 20 (18.8 %). All positive specimens were successfully genotyped and seven serovars were identified. The most prevalent was serovar D (34.4 %), followed by E (21.9 %), F (18.8 %), G (9.4 %), J (9.4 %), H (3.1 %) and I (3.1 %). A heterogeneous distribution of C. trachomatis serovars was observed in the study group, where the most common serovars were D, E and F comprising 75 % of the positive samples. This PCR-based RFLP method could be used in epidemiological studies on the prevalence of C. trachomatis infection to provide more information and to compare the serovar distribution among different cohorts.

The success of Pseudomonas aeruginosa in cystic fibrosis (CF) and other chronic infections is largely attributed to its ability to grow in antibiotic-resistant biofilm communities. This study investigated the effects of limiting iron levels as a strategy for preventing/disrupting P. aeruginosa biofilms. A range of synthetic and naturally occurring iron-chelating agents were examined. Biofilm development by P. aeruginosa strain PAO1 and CF sputum isolates from chronically infected individuals was significantly decreased by iron removal under aerobic atmospheres. CF strains formed poor biofilms under anaerobic conditions. Strain PAO1 was also tested under anaerobic conditions. Biofilm formation by this model strain was almost totally prevented by several of the chelators tested. The ability of synthetic chelators to impair biofilm formation could be reversed by iron addition to cultures, providing evidence that these effective chelating compounds functioned by directly reducing availability of iron to P. aeruginosa. In contrast, the biological chelator lactoferrin demonstrated enhanced anti-biofilm effects as iron supplementation increased. Hence biofilm inhibition by lactoferrin appeared to occur through more complex mechanisms to those of the synthetic chelators. Overall, our results demonstrate the importance of iron availability to biofilms and that iron chelators have potential as adjunct therapies for preventing biofilm development, especially under low oxygen conditions such as encountered in the chronically infected CF lung.

Phenotypic identification of AmpC, KPC and extended-spectrum β-lactamases (ESBLs) among members of the Enterobacteriaceae remains challenging. This study compared the Phoenix Automated Microbiology System (BD Diagnostics) with the Clinical and Laboratory Standards Institute confirmatory method to identify ESBL production among 200 Escherichia coli and Klebsiella pneumoniae clinical isolates. The Phoenix system misclassified nearly half of the isolates as ESBL-positive, requiring manual testing for confirmation. Inclusion of aztreonam±clavulanic acid (CA) and cefpodoxime±CA in the testing algorithm increased the ESBL detection rate by 6 %. Boronic acid-based screening identified 24 isolates as AmpC+, but in a subset of genotypically characterized isolates, appeared to have a high false-positivity rate. PCR screening revealed eight KPC+ isolates, all of which tested as ESBL+ or ESBL+ AmpC+ by phenotypic methods, but half were reported as carbapenem-susceptible by the Phoenix system. Overall, these results indicate that laboratories should use the Phoenix ESBL results only as an initial screen followed by confirmation with an alternative method.

The pathogenic yeast Candida albicans can grow in multiple morphological states including budded, pseudohyphal and true hyphal forms. The ability to interconvert between budded and hyphal forms, herein termed the budded-to-hyphal transition (BHT), is important for C. albicans virulence, and is regulated by multiple environmental and cellular signals. To identify small-molecule inhibitors of known cellular processes that can also block the BHT, a microplate-based morphological assay was used to screen the BIOMOL–Institute of Chemistry and Cell Biology (ICCB) Known Bioactives collection from the ICCB-Longwood Screening Facility (Harvard Medical School, Boston, MA, USA). Of 480 molecules tested, 53 were cytotoxic to C. albicans and 16 were able to block the BHT without inhibiting budded growth. These 16 BHT inhibitors affected protein kinases, protein phosphatases, Ras signalling pathways, G protein-coupled receptors, calcium homeostasis, nitric oxide and guanylate cyclase signalling, and apoptosis in mammalian cells. Several of these molecules were also able to inhibit filamentous growth in other Candida species, as well as the pathogenic filamentous fungus Aspergillus fumigatus, suggesting a broad fungal host range for these inhibitory molecules. Results from secondary assays, including hyphal-specific transcription and septin localization analysis, were consistent with the inhibitors affecting known BHT signalling pathways in C. albicans. Therefore, these molecules will not only be invaluable in deciphering the signalling pathways regulating the BHT, but also may serve as starting points for potential new antifungal therapeutics.

Carvacrol is an important component of essential oils and recently has attracted much attention as a result of its biological properties, such as a wide spectrum of antimicrobial activity. The aim of this study was to evaluate the effect of carvacrol in liquid and vapour phase on preformed biofilms of Staphylococcus aureus and Staphylococcus epidermidis by determining biofilm biomass and cultivable cell numbers, and by using epifluorescence and scanning electron microscopy. Carvacrol was able to reduce biofilm biomass and cell viability more effectively when used with liquid contact rather than with vapour phase. The efficacy of treatment with carvacrol vapour was found to be dependent on exposure time. The predominance of red fluorescence using a LIVE/DEAD BacLight Viability kit (Molecular Probes) and the partially destroyed biofilm architecture as determined by microscopy in treated samples provided evidence for the efficacy of carvacrol. The findings of this investigation suggest a potential application for carvacrol in the inactivation of staphylococcal biofilms.

Variable-number tandem repeat (VNTRs) occur throughout the chromosome of Mycobacterium tuberculosis. Although these polymorphic VNTRs, also known as mycobacterial interspersed repetitive units (MIRUs), have proved to be useful tools in molecular epidemiology, their biological significance is less well understood. This study investigated the polymorphism of the VNTR 3690 locus located in the intergenic region between rv3304 and rv3303c (encoding the gplD2 and lpdA genes, respectively) and its possible function in the regulation of gene expression. The copy number of VNTR 3690 was found to vary among Indian clinical isolates of M. tuberculosis (one to twelve copies), M. tuberculosis H37Rv TMC102 (four copies), M. tuberculosis H37Ra (two to four copies), Mycobacterium bovis BCG (one copy). The expression of lpdA as measured by quantitative RT-PCR was 12-fold higher in M. tuberculosis H37Rv than in M. bovis BCG. Using a GFP reporter system in which the 5′-flanking region of lpdA was fused to the gfp gene, the effect of VNTRs on gene expression was measured in an M. bovis BCG host background by real-time PCR. Compared with one VNTR repeat, a 12.5-fold upregulation of GFP expression was found with a flanking region containing four VNTR 3690 repeats, indicating that there is a good correlation between VNTR copy number and transcription of lpdA.

The envelope glycoprotein G of rabies virus in vaccines induces the production of neutralizing antibodies important in the protection against the disease. The measurement of anti-envelope glycoprotein antibodies is a good predictor of the degree of humoral immunity in people during anti-rabies treatment or after vaccination. Several assays exist for the serological determination of antibody protection against rabies virus infection. Antibody neutralization by the rapid fluorescent focus inhibition test (RFFIT) or the fluorescent antibody virus neutralization (FAVN) test is currently the gold standard. Performance of the highly complex RFFIT and FAVN tests, however, requires specialized reference laboratories with expertise with this assay. Although not widely used, ELISA test kits are available and may be an additional option for testing that is more accessible. The aim of the present study was to evaluate available ELISA assays for the determination of anti-rabies antibodies. We compared the Bio-Rad Platelia Rabies II ELISA, DRG Rabies Virus IgG Ab ELISA and Focus Diagnostics Rabies Antibody Detection by ELISA to RFFIT. Bland–Altman plots comparing the Bio-Rad Platelia assay and the Focus Diagnostics assay to RFFIT showed a low degree of variability between the ELISA assays and RFFIT results except in samples with high RFFIT values. The agreement, sensitivity and specificity of Bio-Rad Platelia Rabies II ELISA when compared to RFFIT were 95.1 %, 94.1 % and 95.8 %, respectively. The DRG Rabies assay compared to RFFIT had an agreement of 77.7 %, a sensitivity of 86.7 % and a specificity of 69.4 %. The agreement, sensitivity and specificity of Focus Diagnostics Rabies Detection by ELISA when compared to RFFIT were 82.2 %, 91.7 % and 73.0 %, respectively. Overall, the Bio-Rad Platelia assay showed higher accuracy and specificity than either the DRG or Focus assays. All of these ELISAs, however, measure all antibody types and do not discriminate the neutralizing antibodies as measured by functional assays (RFFIT and FAVN) and cannot be relied upon to predict the neutralizing activity of the sera. The results of this study offer insight into the availability of alternative, less-complex methods to monitor rabies antibody titres in at-risk individuals following vaccination.

Three clinical strains of Escherichia coli (p168, p517 and p667) were collected in 2006 from three hospitals in Anhui Province (China). PCR and DNA sequencing revealed that E. coli p168 carried a novel extended-spectrum β-lactamase (ESBL), which was designated CTX-M-87. The extended-spectrum β-lactamase which was carried by E. coli p517 and E. coli p667 was previously named CTX-M-65. The deduced amino acid sequence of CTX-M-87, with pI 9.1, differed from that of CTX-M-14 by the substitutions Ala77→Val and Pro167→Leu. Like CTX-M-14, CTX-M-87 had a more potent hydrolytic activity against cefotaxime than against ceftazidime and had high affinity for cefuroxime and cefotaxime. These data show that mutations at position 167 in CTX-M do not always affect catalytic activity and substrate preference.

Inhalational anthrax is the most severe form of anthrax. It has been shown in small-animal and non-human primate models that relatively large pools of ungerminated Bacillus anthracis spores can remain within the alveolar spaces for days to weeks post-inhalation or until transported to areas more favourable for germination and bacillary outgrowth. In this study, spores of the Ames strain that were exposed to germination-inducing media prior to intranasal delivery were significantly less infectious than spores delivered in either water or germination-inhibitory medium. The effect of manipulating the germination potential of these spores within the lungs of infected mice by exogenous germination-altering media was examined. The data suggested that neither inducing germination nor inhibiting germination of spores within the lungs protected mice from the ensuing infection. Germination-altering strategies could, instead, significantly increase the severity of disease in a mouse model of inhalational anthrax when implemented in vivo. It was shown that germination-altering strategies, in this study, were not beneficial to the infected host and are impractical as in vivo countermeasures.

Clostridium perfringens type C-induced enteritis necroticans is a rare but often fatal disease in humans. A consistent histopathological finding is an acute, deep necrosis of the small intestinal mucosa associated with acute vascular necrosis and massive haemorrhage in the lamina propria and submucosa. Retrospective immunohistochemical investigations of tissues from a diabetic adult who died of enteritis necroticans revealed endothelial localization of C. perfringens β-toxin in small intestinal lesions. Our results indicate that vascular necrosis might be induced by a direct interaction between C. perfringens β-toxin and endothelial cells and that targeted disruption of endothelial cells plays a role in the pathogenesis of enteritis necroticans.

A rare case of a severe prosthetic joint infection in a 71-year-old immunocompetent woman is presented. Listeria monocytogenes was identified in two consecutive samples using broad-range PCR and sequencing, whereas cultivation remained negative for the first sample and streptococci of a non-group A streptococci, non-group B streptococci type were detected for the second one. This report demonstrates that the phenotypic approach may lead to misidentification of L. monocytogenes in a routine clinical setting. Molecular methods of pathogen detection might be useful when a rare and/or unexpected micro-organism is present or the sample is collected during antibiotic treatment.

We report an occurrence of treatment failure after oral spiramycin therapy in a man with secondary syphilis and a reported penicillin and tetracycline allergy. Molecular detection revealed treponemal DNA in the blood of the patient and sequencing of the 23S rDNA identified an A to G transition at the gene position corresponding to position 2059 in the Escherichia coli 23S rRNA gene. The occurrence of this novel 23S rDNA mutation was examined among 7 rabbit-propagated syphilitic strains of Treponema pallidum and among 22 syphilis patient isolates from the Czech Republic. The prevalence of A2058G and A2059G mutations among clinical specimens was 18.2 and 18.2 %, respectively.