- Volume 63, Issue 4, 2014

Streptococcal toxic shock syndrome (STSS) is a re-emerging infectious disease in many developed countries. Recent studies have suggested that mutations in CovRS, a two-component regulatory system in Streptococcus pyogenes, play important roles in the pathogenesis of STSS. However, in vivo evidence of the significance of CovRS in human infections has not been fully demonstrated. We investigated five S. pyogenes strains isolated simultaneously from the pharynx, sputum, knee joint, cerebrospinal fluid and blood of a single STSS patient. All were emm89-type strains, and multilocus sequence typing (MLST) analysis revealed that the strains of pharynx and blood were isogenic. The growth rates of the strains from pharynx and sputum were faster than those of the other strains. Protein profiles of the culture supernatants of strains from the pharynx and sputum were also different from those of the other strains. Sequence analyses revealed that strains from the knee joint, cerebrospinal fluid and blood contained a single nucleotide difference in the covS coding region, resulting in one amino acid change, compared with the other strains. Introduction of a plasmid containing the covS gene from the pharynx strain to the blood strain increased the production of SpeB protein. This suggests that the one amino acid alteration in CovS was relevant to pathogenesis. This report supports the idea that mutated CovS plays important roles in vivo in the dissemination of S. pyogenes from the upper respiratory tract of human to aseptic tissues such as blood and cerebrospinal fluid.

Mucocutaneous and cutaneous candidiasis, though common in children, is often under-reported. The prevalence of Candida dubliniensis in causing these infections in this age group is also largely unknown. A prospective epidemiological cross-sectional study for candidiasis was performed in paediatric patients clinically suspected of candidiasis with oropharyngeal lesions (75 patients), cutaneous lesions (18 patients) and lesions at both sites (2 patients). Candida species were identified by conventional tests. For C. dubliniensis, chlamydospore production, growth on tobacco agar and growth at 45 °C were performed. Nine isolates were confirmed at a reference centre. The rates of candidiasis were 77.3 % (58 out of 75 patients clinically suspected of candidiasis) and 83.3 % (15/18) in oropharyngeal and cutaneous lesions respectively, and 1 of the 2 children with lesions at both sites was diagnosed as having chronic mucocutaneous candidiasis due to C. dubliniensis. The commonest species isolated was Candida albicans, in 41 (70.7 %) patients with oropharyngeal candidiasis and 11 (73.3 %) with cutaneous lesions; C. dubliniensis was isolated from 11 and 3 children respectively. In the paediatric population, C. albicans predominates in mucocutaneous and cutaneous candidiasis, with C. dubliniensis also contributing substantially.

Conventional culture and drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and time consuming. For this reason alternative rapid culture and DST techniques are urgently needed to shorten the time for drug-resistance detection. A total of 222 smear-positive sputum samples were evaluated by the direct nitrate reductase assay (D-NRA) on Lowenstein–Jensen medium, for the rapid and simultaneous detection of resistance to isoniazid, rifampicin, kanamycin and ofloxacin. p-Nitrobenzoic acid was also included for identification of the M. tuberculosis complex. Results were compared with the BACTEC MGIT 960 as gold standard. The general performance of the D-NRA was very good, reaching a global value of 97 %. D-NRA had a turn-around time of 16.9 days to obtain results while that of the indirect MGIT 960 system was 29 days. D-NRA is a low-cost technology, easy to set up in clinical laboratories and suitable to be used for DST of M. tuberculosis in all smear-positive samples.

Serotype-specific quantification data are essential for elucidating the complex epidemiology of Streptococcus pneumoniae and evaluating pneumococcal vaccine efficacy. Various PCR-based assays have been developed to circumvent the drawback of labour-intensive and time-consuming culture-based procedures for serotype determination and quantification of pneumococcus. Here, we applied a nanofluidic real-time PCR system to establish a novel assay. Twenty-nine primer pairs, 13 of which were newly designed, were selected for the assay to cover 50 serotypes including all currently available conjugate and polysaccharide vaccine serotypes. All primer pairs were evaluated for their sensitivity, specificity, efficiency, repeatability, accuracy and reproducibility on the Fluidigm Biomark HD System, a nanofluidic real-time PCR system, by drawing standard curves with a serial dilution of purified DNA. We applied the assay to 52 nasopharyngeal swab samples from patients with pneumonia confirmed by chest X-ray to validate its accuracy. Minimum detection levels of this novel assay using the nanofluidic real-time PCR system were comparable to the conventional PCR-based assays (between 30 and 300 copies per reaction). They were specific to their targets with good repeatability (sd of copy number of 0.1), accuracy (within ±0.1 fold difference in log10 copy number) and reproducibility (sd of copy number of 0.1). When artificially mixed DNA samples consisting of multiple serotypes in various ratios were tested, all the serotypes were detected proportionally, including a minor serotype of one in 1000 copies. In the nasopharyngeal samples, the PCR system detected all the culture-positive samples and 22 out of 23 serotypes identified by the conventional method were matched with PCR results. We conclude that this novel assay, which is able to differentially quantify 29 pneumococcus groups for 45 test samples in a single run, is applicable to the large-scale epidemiological study of pneumococcus. We believe that this assay will facilitate our understanding of the roles of serotype-specific bacterial loads and implications of multiple serotype detections in pneumococcal diseases.

We investigated the performance of a real-time PCR for the detection of extended spectrum β-lactamase genes in Enterobacteriaceae (Check-MDR ESBL PCR). Results from micro-arrays were considered as the gold standard. An analysis on 489 isolates resulted in a sensitivity of 98.9 % and a specificity of 100 % for the PCR.

A subset of Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae and Proteus mirabilis isolates collected for the Study for Monitoring Antimicrobial Resistance Trends that were positive for the Clinical and Laboratory Standards Institute (CLSI) extended-spectrum β-lactamase (ESBL) phenotypic confirmatory test (n = 3245) or had an ertapenem MIC of ≥0.5 µg ml−1 (n = 293), or both (n = 467), were analysed for ESBL genes. Most ESBL phenotype E. coli or K. pneumoniae possessed an ESBL gene (95.8 and 88.4 %, respectively), and this was 93.1 % if carbapenem-non-susceptible K. pneumoniae were removed. This rate was lower for P. mirabilis (73.4 %) and K. oxytoca (62.5 %). Virtually all ESBL-positive isolates (99.5 %) were cefotaxime non-susceptible [CLSI or European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints)]. Fewer isolates (82 %) were ceftazidime non-susceptible (CLSI breakpoints). In addition, 21.1 % of E. coli, 25 % of K. oxytoca and 78.7 % of P. mirabilis isolates were ceftazidime susceptible but ESBL positive. This suggests that CLSI breakpoints for ceftazidime are too high to detect ESBLs. The lower EUCAST breakpoints detected ESBLs in E. coli and K. oxytoca better, but 59.6 % of ESBL-positive isolates of P. mirabilis were ceftazidime susceptible. For isolates with ertapenem MICs ≥0.5 µg ml−1, more accurate ESBL phenotype analysis was observed for E. coli and K. pneumoniae (sensitivity >95 % for both, specificity 94.4 and 54.1 %, respectively). If carbapenemase-positive K. pneumoniae were excluded, the specificity increased to 78 %. The positive predictive values for the ESBL phenotypic test with E. coli and K. pneumoniae were 97.6 and 81.8 %, respectively, and negative predictive values were 75.9 and 95.2 %, respectively. We therefore suggest that it would be prudent to confirm phenotypic ESBL-positive P. mirabilis, K. pneumoniae and K. oxytoca with molecular analysis.

We conducted detailed genetic analyses of the haemagglutinin–neuraminidase (HN) gene in 272 human parainfluenza virus type 3 (HPIV3) isolates from children with acute respiratory illness during the period 2002–2009 in Yamagata prefecture, Japan. A phylogenetic tree reconstructed by the Bayesian Markov chain Monte Carlo method showed that the strains diversified at around 1946 and that the rate of molecular evolution was 1.10×10−3 substitutions per site per year. Identity was high among the present strains (<90 %) and the pairwise-distances were short. Furthermore, we found four positive selection sites and some key amino acid substitutions in active/catalytic sites of the HN protein. The results suggest that the HN gene of HPIV3 in the present strains evolved rapidly, similarly to other virus genes such as the G gene of respiratory syncytial virus. However, the biological functions and detailed structures of the HN glycoprotein in some of these strains may have been altered.

Chlamydia psittaci is a zoonotic pathogen with a wide range of avian hosts and may be transmitted to humans and cause severe disease. To assess the risk of psittacosis posed by pet birds, the seroprevalence of Chlamydia psittaci antibodies in 360 Eurasian siskins (Carduelis spinus), 289 oriental skylarks (Alauda arvensis) and 36 black-tailed grosbeaks (Coccothraustes migratorius) in Gansu province, north-western China was detected by an indirect haemagglutination assay. Twenty-seven out of 289 (9.34 %) Alauda arvensis, 45 out of 360 (12.50 %) Carduelis spinus and 2 out of 36 (5.56 %) Coccothraustes migratorius were positive for Chlamydia psittaci infection at a cut-off dilution of 1 : 16. The prevalence of Chlamydia psittaci was higher in Carduelis spinus (12.5 %) than in Alauda arvensis (9.34 %) and Coccothraustes migratorius (5.56 %); however, the differences were not statistically significant (P>0.05). Statistical analysis indicated that Chlamydia psittaci seroprevalence in adult pet birds (12.4 %, 67/540) was significantly higher than that in juvenile pet birds (4.83 %, 7/145) (P<0.01). There was no statistical difference in Chlamydia psittaci seroprevalence between male (12.4 %) and female (8.27 %) birds. To our knowledge, this is the first report indicating the seroprevalence of Chlamydia psittaci exposure in pet birds in China. Our results indicate that close contact with pet birds poses the risk of zoonotic transmission of Chlamydia psittaci.

Urinary tract infections (UTIs) are primarily caused by Escherichia coli with the patient’s own faecal flora acting as a reservoir for the infecting E. coli. Here we sought to characterize the E. coli faecal flora of UTI patients and healthy controls who had never had a UTI. Up to 20 E. coli colonies from each rectal swab were random amplified polymorphic DNA (RAPD) typed for clonality, dominance in the sample and correlation to the infecting UTI isolate in patients. Each distinct clone was phylotyped and tested for antimicrobial susceptibility. Eighty-seven per cent of the UTI patients carried the infecting strain in their faecal flora, and faecal clones causing UTI were more often dominant in the faecal flora. Patients had a larger diversity of E. coli in their gut flora by carrying more unique E. coli clones compared to controls, and patient faecal clones were more often associated with multidrug resistance compared to controls. We found a similar phylotype distribution of faecal clones from UTI patients and healthy controls, including a large proportion of B2 isolates in the control group. Faecal-UTI isolates from patients were more often associated with multidrug resistance compared to faecal-only clones, indicating a link between UTI virulence and antimicrobial resistance. Intake of any antibiotic less than 6 months prior to inclusion in the experiment occurred significantly more in patients with UTI than in controls. In contrast, presence of an intrauterine device was significantly more common in controls indicating a protective effect against UTI. In conclusion, healthy controls have a large proportion of potentially pathogenic E. coli phylotypes in their faecal flora without this causing infection.

Although rarely isolated from cystic fibrosis (CF) patients, Burkholderia dolosa is associated with accelerated lung function decline. During 18 years of epidemiological surveillance in the major Portuguese CF centre in Lisbon, only one patient was infected with B. dolosa. Pulmonary deterioration, associated with the evolution of forced expiratory volume in 1 s, occurred during 5.5 years of colonization with this B. dolosa clone (with the new sequence type ST-668). Transient co-colonization with Burkholderia cenocepacia and other bacterial and fungal pathogens occurred, but B. dolosa prevailed until the patient’s death. The systematic assessment of relevant phenotypes for the sequential clonal isolates examined in this retrospective study (14 of B. dolosa and four of B. cenocepacia) showed that they were variants, although in general no isolation time-dependent pattern of alteration was identified. However, the first B. dolosa isolate retrieved was more susceptible to gentamicin, imipenem and tobramycin, and exhibited a higher swarming motility compared with most of the isolates obtained during the later stages of disease progression and antimicrobial therapy.