- Volume 59, Issue 10, 2010

The principal virulence factor of Streptococcus pneumoniae is capsular polysaccharide, and encapsulated pneumococci are more common causes of disease than unencapsulated strains. This study analysed the presence of capsular genes in 59 pneumococcal isolates using two PCR methods targeted at the cpsA and cpsB genes of the capsular biosynthesis locus. The PCR method targeted at the cpsB gene, reported to be essential for encapsulation, was developed in this study. Of 59 pneumococcal isolates, 49 (83 %) were obtained from the sputum samples of elderly patients (≥65 years) with community-acquired pneumonia (CAP) and 10 (17 %) were from those with other acute lower respiratory tract infections (ARIs). Forty (82 %) of the CAP isolates and two (20 %) of the ARI isolates were encapsulated, as assessed by conventional immunochemical methods. Forty-one (98 %) of the 42 encapsulated strains had the cpsB gene present, and in 38 strains the cpsA gene was also detected. One of the unencapsulated isolates gave a positive result for the cpsB gene, and neither of the capsular locus genes were present in all the other unencapsulated strains. The distribution of encapsulated and unencapsulated isolates differed significantly between the two patient groups regardless of whether the presence of capsule was determined immunochemically (P<0.001) or by cpsB PCR (P=0.002). The cpsB PCR developed here was found to be a rapid and reliable method to detect the pneumococcal capsule locus and may have potential in sputum diagnostics when investigating the pneumococcal aetiology of CAP.

Streptococcus pneumoniae, the aetiological agent of pneumonia and non-gonococcal urethritis, shares a high degree of DNA sequence identity with the viridans group of streptococci, particularly Streptococcus mitis and Streptococcus oralis. Although their clinical and pathological manifestations are different, discrimination between S. pneumoniae and its close viridans cocci relatives is still quite difficult. Suppression subtractive hybridization was performed to identify the genomic differences between S. pneumoniae and S. mitis. Thirty-four resulting S. pneumoniae-specific clones were examined by sequence determination and comparative DNA sequence analysis using blast. S. pneumoniae-specific primers were subsequently designed from one of the clonal DNA sequences containing the cps gene (coding for capsular polysaccharide biosynthesis). The primer specificities were evaluated using 49 viridans streptococci including 26 S. pneumoniae, 54 other streptococci, 14 Lactococcus species, 14 Enterococcus species and three Vagococcus species, and compared with the specificities of previously described autolysin (lytA), pneumolysin (ply), Spn9802 and Spn9828 primers. The newly designed cpsA-specific primer set was highly specific to S. pneumoniae and was even better than the existing primers. These findings may help improve the rapid identification and differentiation of S. pneumoniae from closely related members of the viridans group streptococci.

The UK National External Quality Assessment Service (UK NEQAS) distributes clinically relevant and educational specimens for external quality assessment (EQA). The aim of this report was to assess the suitability of using a freeze-dried specimen format for the EQA of conventional and rapid methods and to review the methods used by participants to screen for meticillin-resistant Staphylococcus aureus (MRSA). Of the 714 laboratories that returned a result, 678 reported the presence of MRSA, and results showed a mean of 73 c.f.u. per 25 μl and a median of 50 c.f.u. per 25 μl confirming that the specimen was homogeneous. Four different approaches to MRSA screening were used routinely, including: (i) liquid culture; (ii) direct plating onto conventional media; (iii) direct plating onto chromogenic media; and (iv) rapid methods. A wide variety of methods were used within each of these four categories to screen for MRSA, and many laboratories reported using more than one method. Attempts should be made to determine the most appropriate approach to MRSA screening and to standardize the protocols across the UK.

A total of 17 typical and atypical enteropathogenic Escherichia coli (EPEC) were isolated from 396 children with and without diarrhoea. Out of 12 EPEC isolates from patients with diarrhoea, 3 (25 %) were atypical EPEC while 9 (75 %) were typical EPEC. It was observed that atypical EPEC strains had colonized the intestines of healthy children and its isolation rates were higher in healthy children than in children with diarrhoea. Interestingly all of the atypical EPEC isolates carried a megaplasmid, mostly comparable with the size of EPEC adherence factor (EAF) encoding gene but no virulence gene was detected in this megaplasmid. Studies also indicated that multidrug resistance EPEC are emerging and all the atypical EPEC strains showed significantly less resistance to all antimicrobial agents used in this study than typical EPEC. This study also supports the opinion that Shiga toxin-producing E. coli does not pose a major threat to human health in India. Subtyping analysis reveals that eae-α1, eae-β2 and eae-λ could be common EPEC subtypes prevalent in children with diarrhoea in Delhi. The present study is believed to be the first report of the detection of atypical EPEC from children without diarrhoea and records of isolation of eae-γ1, eae-γ2 and the rare eae-λ subtype in India. The data also indicated that typical EPEC are a common cause of diarrhoea and atypical EPEC are emerging as colonizers of the intestine of children with and without diarrhoea in Delhi and the National Capital Region, India.

Bacteria of the Burkholderia cepacia complex (Bcc) are opportunistic human pathogens that can cause serious infections in the lungs of cystic fibrosis patients. The Bcc is a complex taxonomic group and comprises 17 closely related species of both biotechnological and clinical importance that have been discriminated by a polyphasic taxonomic approach. In this study we focused on the hisA gene, which encodes a 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino) methylideneamino] imidazole-4-carboxamide isomerase involved in histidine biosynthesis, as a new target gene to discriminate among the Bcc species. PCR primers were designed to amplify a hisA DNA fragment of 442 bp from 78 strains representative of all the 17 Bcc species known at the time of writing. The nucleotide sequences of the amplicons were determined and aligned with the 54 Bcc sequences available in databases. Then a phylogenetic tree was constructed on the basis of this alignment and this revealed that this hisA region allows discrimination of all Bcc species, suggesting that this gene fragment can be used for the identification of Bcc strains. In addition, an 11 nucleotide letter code for the rapid discrimination of Bcc species was identified.

To obtain the genotype and antimicrobial susceptibility profiles of Campylobacter jejuni isolates from north China, 93 C. jejuni isolates (56 isolates from patients with diarrhoea, 7 isolates from Guillain–Barré syndrome patients and 30 isolates from chicken stools) were selected for multilocus sequence typing (MLST), PFGE and drug resistance testing. A total of 49 sequence types (STs) were identified from the entire panel of 93 C. jejuni isolates. Fifty-six isolates belonged to 14 clonal complexes, while 37 isolates could not be assigned to any known clonal complex. The most frequently observed clonal complexes were ST-21 (11 isolates), ST-353 (10 isolates) and ST-443 (6 isolates). Fifty-three PFGE SmaI patterns were identified among 93 isolates. No erythromycin-, gentamicin- or streptomycin-resistant isolates were found among the 44 strains isolated in 2008. Resistance to nalidixic acid, levofloxacin and ciprofloxacin was observed in 100 % (44/44) of the tested isolates. This study has shown the genetic characteristics of C. jejuni isolates in north China. In addition, overlapping clonal groups were defined by both MLST and PFGE for C. jejuni human and chicken isolates.

To understand the domestic population structure of Mycobacterium tuberculosis clinical isolates in the Republic of Korea, we genotypically analysed 80 isolates obtained from various geographical origins in the country. Of these, 64 (80.0 %) isolates were identified as Beijing family strains. It is particularly interesting that their phylogenetic classification, based on the ancient/modern separation and the presence/absence of the genomic region RD181, revealed a majority of the ancient (RD181+) subfamily in the population. The 15 loci of variable number of tandem repeat(s) of mycobacterial interspersed repetitive units (15-MIRU-VNTR) were also analysed. Combination with the previous VNTR data reported from surrounding countries revealed that the topology of the minimum spanning tree was linked tightly not to the geographical origins of the patients but to the phylogenetic characteristics of the isolates. These results show that the phylogeographical distribution of the M. tuberculosis Beijing family around far-eastern Asia could be estimated using international accumulation and comparison of VNTR genotyping data.

Bronchoscopes and the filters used for washing them were found to yield high numbers of Mycobacterium avium isolates sharing the same repetitive sequence-based PCR (rep-PCR) fingerprint pattern as M. avium isolates recovered from patient samples collected by bronchoscopy. Water and biofilm samples collected from the bronchoscopy preparation laboratory yielded M. avium, Mycobacterium intracellulare, Mycobacterium malmoense and Mycobacterium gordonae. Several M. avium and M. intracellulare isolates from water samples in the bronchoscopy laboratory had rep-PCR patterns matching those of patient bronchoscopy isolates. Five of the 22 (23 %) M. avium patient bronchoscopy isolates and 42 of the 56 (75 %) M. intracellulare patient bronchoscopy isolates could have been due to contamination from the water supply.

The aim of this study was to investigate the high level of pulmonary Mycobacterium abscessus infections and implement a surveillance programme among 43 ventilator-dependent patients, 15 with pulmonary M. abscessus infections, in a hospital long-term respiratory care ward (RCW) in central Taiwan. M. abscessus isolates were obtained from 35 patients in the RCW of hospital A, 6 patients in the RCWs of another three hospitals (B, C and D), and from 4 water sources in two of the hospitals (A and B). Strains were characterized by methods including hsp65 PCR–RFLP and PFGE. The patients were followed-up by chest X-ray for 1 year. All clinical isolates were type I and II, and belonged to ten distinct clusters of PFGE patterns. Five clinical strains in two hospitals belonged to a single cluster, whilst four clinical strains in the other two hospitals belonged to a single unique cluster. The strains from hospital A fell into nine clusters and were distinct from the strains isolated from the water supply. Patients infected with type I strains showed a significantly more rapid progression of disease. The number of different strains involved suggested either that there had been a polyclonal outbreak or that a high level of endemic infections was present in the RCW of hospital A. This and the lack of homology between the clinical and environmental isolates from hospital A raised the possibility that pulmonary M. abscessus infections may have been spread by the movement of patients between RCWs, a routine practice in Taiwan's integrated delivery system.

Data from 4727 invasive isolates of Streptococcus pneumoniae submitted to the Scottish Haemophilus, Legionella, Meningococcus and Pneumococcus Reference Laboratory between 1999 and 2007 were analysed to establish susceptibility profiles to penicillin, erythromycin and cefotaxime. Pneumococcal resistance to penicillin over the study period remained low, with only 0.2 % (n=7/4727) of isolates falling into this category (MIC ≥2 mg l−1). These isolates have been sporadic, and have mainly represented serogroup 14 (ST9) and 9 (ST156). In comparison, the ‘intermediate sensitivity’ group (MIC 0.12–1 mg l−1) ranged between 2 and 6 % per year, the majority from serogroup 9 (ST156). Over the study period, we found that 12 % (n=585/4727) of isolates were erythromycin-resistant (MIC >0.5 mg l−1), with the majority (n=467; 80 %) of these isolates identified as serogroup 14 (ST9). Cephalosporin resistance (cefotaxime MIC >1 mg l−1) was found in only 0.06 % (n=2/3135) of isolates. Internationally recognized clones (Pneumococcal Molecular Epidemiology Network) accounted for 35 % (n=28/81) of the penicillin non-susceptible isolates and 75 % (n=248/330) of the macrolide-resistant isolates, with ST9 and ST306 predominating. Between 1999 and 2007 we found that 11.6 % (n=18/155) of the penicillin non-susceptible isolates and 4.8 % (n=28/585) of the macrolide-resistant isolates were from serogroups not covered by the 7-valent conjugate pneumococcal vaccine in use in the UK since 2006. Susceptibility to first-line antimicrobial agents for invasive pneumococcal disease in Scotland remained high over the period 1999–2007.

Osteoarticular tuberculosis is the fourth leading type of extrapulmonary tuberculosis. The disease has a progressive course and the diagnosis is often made in the later stages of bone destruction. We describe a case of a foot ulcer caused by drug-resistant Mycobacterium tuberculosis in a patient with known diabetes where the diagnosis was not suspected initially. Although tuberculous foot ulcers are rare, they should be included in the differential diagnosis of unknown foot ulcers. A greater awareness of this rare clinical entity may help in commencing specific evidence-based therapy quickly and preventing undue morbidity and mortality.

Primary laryngeal aspergillosis is extremely rare, especially in an immunocompetent host. It is commonly found as part of a systemic infection involving the respiratory system in immunocompromised people. Two cases of laryngeal aspergillosis with no systemic extension and no generalized immune deficiency are presented here. We report what is to the best of our knowledge only the second case of Aspergillus infection in a vocal cord cyst. Aspergillus species were identified in tissue sections and confirmed by PCR studies. We present a literature review of laryngeal aspergillosis cases and discuss predisposing factors, clinical presentation, histopathology, PCR, diagnosis and treatment of Aspergillus laryngitis. The known aetiological causes of the disease are increasing and include iatrogenic factors, vocal abuse, vocal-fold cysts and occupational factors, and immunocompetent patients are susceptible to these predisposing factors.

Ureaplasma species are usually associated with infection of the urogenital tract. An unusual case of a sternal wound infection caused by Ureaplasma urealyticum in a 41-year-old male after aortic valve replacement is described.