- Volume 59, Issue 5, 2010

The role of Staphylococcus aureus small-colony variants (SCVs) in the pathogenesis of biofilm-associated infections remains unclear. This study investigated the mechanism behind increased biofilm-forming potential of a menadione-auxotrophic Staphylococcus aureus SCV compared with the wild-type parental strain, as recently reported by our laboratory. SCVs displayed an autoaggregative phenotype, with a greater amount of polysaccharide intercellular adhesin (PIA), significantly reduced tricarboxylic acid cycle activity and a decreased susceptibility to aminoglycosides and cell-wall inhibitors compared with wild-type. The biofilms formed by the SCV were highly structured, consisting of large microcolonies separated by channels, and contained more biomass as well as significantly more PIA than wild-type biofilms. The surface hydrophobicity of the two phenotypes was similar. Thus, the autoaggregation and increased biofilm-forming capacity of menadione-auxotrophic Staphylococcus aureus SCVs in this study was related to the enhanced production of PIA in these variants.

Globally, specific genotypes of Mycobacterium tuberculosis have been shown to dominate in patients, suggesting that these are more successful pathogens. One such genotype, the F15/LAM4/KZN (KZN) family of M. tuberculosis, has predominated in KwaZulu-Natal, South Africa, since the early 1990s. This strain recently evolved from multidrug-resistant to extensively drug-resistant (XDR). The ability of M. tuberculosis strains belonging to the Beijing family, the KZN family, strains with unique DNA fingerprint patterns and laboratory strains (H37Rv and H37Ra) to adhere to and invade a human alveolar (A549) and a human bronchial (BBM) epithelial cell line was investigated. All strains displayed greater adhesion to and invasion of A549 cells as compared to BBM cells. The Beijing and KZN strains combined showed greater adhesion (28 %) than the unique strains (5 %) (P <0.05). The XDR variant of KZN invaded A549 cells more effectively than the other isolates. These results suggest that the successful spread of the Beijing and KZN strains might be related to their interaction with alveolar epithelium.

A multiplex PCR is described which detects capsular types K1, K2, K5, K54 and K57, which are those most associated with invasive disease or pathogenicity, a further capsular type (K20), two putative virulence factors (rmpA and wcaG) and the 16S–23S internal transcribed spacer unit of Klebsiella pneumoniae, facilitating identification of this organism. wcaG encodes capsular fucose production and was associated with capsular types K1 and K54, but was also found in strains of other capsular types; 18 of the 543 isolates screened were PCR-positive for this gene. An eight-locus variable number tandem repeat (VNTR) scheme was designed, which provided discrimination at a level similar to that afforded by PFGE among a panel of 36 isolates representing 29 PFGE types. All isolates tested of the virulent K1 clone of CC23, associated with pyogenic liver abscesses, shared the same VNTR profile, which may be helpful in identifying this clone; such isolates were also PCR-positive for allS. These methods provide a rapid means of characterizing and typing isolates of this important agent of community-acquired and nosocomial infection.

The objective of this study was to examine DNA sequence polymorphisms in the 16S–23S rRNA gene intergenic spacer (ITS) regions of five emerging pathogenic Nocardia species: Nocardia beijingensis, Nocardia blacklockiae, Nocardia thailandica, Nocardia elegans and Nocardia vinacea. A set of six isolates belonging to the species of interest and 135 isolates belonging to other Nocardia species was studied. A PCR-based reverse line blot (RLB) hybridization assay incorporating species- or intraspecies ITS rRNA gene operon-specific probes was then developed for species identification. Substantial intraspecies sequence variation among different ITS operons was identified. Four sequence types of N. thailandica, eight sequence types of N. beijingensis (four types for each of two strains) and five sequence types of N. blacklockiae, N. elegans and N. vinacea were found. The results represent the first evidence of ITS sequence heterogeneity in emerging species of Nocardia. By incorporating species/operon-specific probes into a RLB assay, unique RLB patterns were identified for each of the species and every sequence type. The PCR/RLB assay demonstrated high specificity and showed promise in both the identification and genotyping of Nocardia species. More detailed studies of the polymorphism within the ITS locus may further advance our capacity to reliably identify and subtype medically important Nocardia species.

A typing system for Clostridium difficile using sequencing of the surface-layer protein A encoding gene (slpA) was evaluated and used to analyse clinical isolates in Japan. A total of 160 stool specimens from symptomatic patients in Japan was examined and 87 C. difficile isolates were recovered. slpA sequence typing was found to have reliable typability and discriminatory power in comparison with PCR ribotyping, and the typing results were highly reproducible and comparable. slpA sequence typing was used to type C. difficile in DNA extracted directly from stool specimens. Among the 90 stool specimens in which direct typing results were obtained, 77 specimens were positive for C. difficile culture, and typing results from isolated strains agreed with those from direct typing in all 77 specimens. The slpA sequence type smz was dominant at all four hospitals examined, and this endemic type was detected by culture and/or direct typing in 61 (62 %) of 99 stool specimens positive for toxic culture and/or direct slpA sequence typing. Comparison of epidemic strains reported throughout the world revealed one isolate identified as slpA sequence type gc8, which was found to correspond to PCR ribotype 027 (BI/NAP1/027), whereas no isolates were found with the slpA gene identical to that of PCR ribotype 078 strain. slpA sequence typing is valuable for comparison of C. difficile strains epidemic in diverse areas because the typing results are reproducible and can easily be shared. In addition, slpA sequence typing could be applied to direct typing without culture.

Molecular diagnostic methods are of potential value in identifying tuberculosis (TB) and drug resistance where tissue specimens have been submitted for histology but not for microbiological culture. All paraffin-embedded tissue (PET) specimens (n=60) referred to a single national centre over a 42 month study period were analysed using the INNO-LiPA Rif.TB assay; 29/60 patients had been reported to the UK Enhanced Tuberculosis Surveillance database with a diagnosis of TB based on clinical, radiological and histological evidence. Mycobacterium tuberculosis (MTB) DNA was detectable in 5 out of 29 reported TB cases (17.2 %); 12 out of 29 of the reported TB cases had a positive MTB culture from a secondary clinical specimen and MTB DNA was detectable in 2 of the 12 (16.7 %) laboratory-confirmed TB cases. Referring clinicians should be aware of the limitations of this assay on PETs, and should request molecular testing only in patients with a high clinical probability of TB and when acid-fast bacilli are seen in tissue specimens.

In this study, we investigated the anti-inflammatory and reinforcing barrier effects of Lactobacillus casei subsp. rhamnosus (Lcr35) on Caco-2 intestinal epithelial cells already exposed to Salmonella LPS. Using the Transwell co-culture model, Salmonella LPS was apically added to polarized Caco-2 cells co-cultured with peripheral blood mononuclear cells (PBMCs) in the basolateral compartment. LPS-stimulated Caco-2 cells were incubated with Lcr35 for 1, 6, 24 or 48 h. Apical inoculation of Lcr35 after 48 h significantly inhibited the basolateral secretion of interleukin-8 (IL-8) in the Caco-2/PBMC co-culture. The PCR analysis showed that Lcr35 significantly downregulated mRNA expression of monocyte chemoattractant protein 1 (MCP-1) (P<0.05) and had a trend of decreasing mRNA expression of IL-8 (P=0.05), but did not alter mRNA expression of transforming growth factor-β1 in LPS-stimulated Caco-2 cells at 48 h after addition of Lcr35. Compared to non-LPS-pretreated controls, transepithelial electrical resistance (TEER) of the polarized Caco-2 cell monolayers pretreated with LPS for 48 h was decreased by 9.9 % (P<0.05). Additionally, compared to those cells only treated with LPS, apical co-incubation with Lcr35 showed biphasic TEER levels increased by 12.1 % (P<0.001), 5.7 % (P<0.05) and 86.8 % (P<0.001) in the Caco-2 cell monolayers compared to those without Lcr35 treatment after 1, 6 and 48 h, respectively. In conclusion, Lcr35 can exert anti-inflammatory effects and ameliorate barrier dysfunction in the Salmonella LPS-pretreated inflamed intestinal epithelium in vitro.

A correlation between δ over baseline (DOB) values of the [13C]urea breath test (UBT) and Helicobacter pylori clarithromycin resistance has been reported, suggesting a possible predictive role of UBT in therapeutic outcome. However, available data are limited and conflicting. This study aimed to clarify this issue, assessing the possible relationship between H. pylori resistance towards different antibiotics (clarithromycin, metronidazole and levofloxacin) and UBT values. The data showed similar DOB values between susceptible and resistant strains for clarithromycin (46.9±32.3 vs 45.7±30.6; P=0.8), metronidazole (46.4±29.6 vs 47.4±37.9; P=0.8), and levofloxacin (45.0±30.2 vs 54.2±38.4; P=0.08). Likewise, comparable DOB values were observed between susceptible and multidrug-resistant strains (45.4±29.6 vs 54.8±44.8; P=0.1). In conclusion, our data failed to find a significant correlation between UBT values and H. pylori antibiotic resistance.

Delayed and reversible hearing loss occurred in a 55-year-old male patient with murine typhus infection. The patient had the initial symptoms of headache, fever and chills, followed by the occurrence of bilateral hearing loss on day 9 from fever onset. Murine typhus was diagnosed with a high IgM titre by indirect immunofluorescence assay. After treatment with azithromycin and prednisolone, the fever and other symptoms subsided gradually and bilateral hearing loss improved 3 weeks later. Though an adverse reaction to azithromycin could not be ruled out, delayed onset of hearing loss was more likely a complication of murine typhus, mainly because the hearing loss did not occur during the azithromycin usage period. Although hearing loss due to murine typhus is rare, clinicians should be alert to the existence of such a delayed complication.

Lactobacilli are Gram-positive rod-shaped bacteria that inhabit the oral cavity, gastrointestinal tract, vagina and nasal cavity. In this report, a rare case of Lactobacillus jensenii endocarditis in a 47-year-old immunocompetent patient is described. Blood cultures and a replaced mitral valve were positive for L. jensenii as assessed by 16S rRNA gene sequencing. Based on susceptibility tests the patient was successfully treated with a mixture of teicoplanin and meropenem antimicrobial therapy.

Disseminated histoplasmosis and nocardiosis typically affect immunocompromised hosts. We report a case of gastrointestinal and adrenal histoplasmosis, presenting as protein-losing enteropathy and hypogammaglobulinaemia, coincident with Nocardia infection, in a HIV-negative patient in whom a specific immunological defect could not be identified. Clinicians in areas of non-endemicity should be vigilant for rare manifestations of histoplasmosis.

Chronic pulmonary infection with Pseudomonas aeruginosa occurs in up to 85 % of individuals with cystic fibrosis (CF) by the time they reach adulthood, and is the major cause of morbidity and mortality: nearly all patients die from progressive respiratory failure due to repeated pulmonary exacerbations. However, despite the predilection of this organism for the lungs of CF people, infection of the pleura is much less common and is not well described in the CF population. We describe what is believed to be the first case of pleural empyema due to a particularly pathogenic transmissible strain of P. aeruginosa (the Liverpool epidemic strain) in an adult CF patient.

Mycobacteria other than tuberculosis infections in patients taking various tumour necrosis factor (TNF)-α inhibitors have been reported in the literature. We describe sporotrichoid spread of Mycobacterium marinum in a man with Crohn's disease treated with infliximab. After starting ethambutol and rifampicin and discontinuing infliximab, a worsening appeared. M. marinum infection may have a potential local spread and systemic dissemination in patients treated with TNF-α inhibitors.