Clostridium difficile-associated clinical burden from lack of diagnostic testing in a Chinese tertiary hospital.

Despite Clostridium difficile infection (CDI) being a common cause of diarrhoea in hospitals worldwide, diagnostic testing or management guidelines are not available in most hospitals in China. In this prospective two-year study, the incidence of CDI among 276 patients with watery diarrhoea was 23.1%. Lack of diagnostic testing for CDI was associated with improper management in 26.4% of patients, risk of nosocomial transmission from lack of isolation precautions, and risk of community transmission from discharging symptomatic toxigenic C. difficile carriers. Updating practice guidelines in line with the current evidence and implementing diagnostic testing for CDI are recommended in hospitals in China.

Self-medication practices with antibiotics among Chinese university students.

OBJECTIVES: Self-medication with antibiotics (SMA) is a serious global health problem. We sought to investigate SMA behaviors and risk factors among Chinese university students, and further explore the association between SMA practices and adverse drug events (ADEs). STUDY DESIGN: Cross-sectional study. METHODS: An online survey was conducted at Jiangsu University (JSU) in eastern China in July 2011 using a pretested questionnaire. RESULTS: Out of 2608 website visitors, 1086 participated in the survey (response rate: 41.6%), 426 respondents were excluded for not being a JSU student or repeat participation, 660 (2.2% of JSU students) were included in analysis, and 316 students (47.9%) had a lifetime history of SMA. Among self-treated students, 43.5% believed that antibiotic was suitable for viral infections, 65.9% had more than one SMA episode in the previous year, 73.5% self-medicated with at least two different antibiotics, 57.1% and 64.4% changed antibiotic dosage and antibiotics during the course, respectively. Female gender, older age, and prior knowledge of antibiotics (PKA) were identified as independent risk factors of SMA. There was no difference between students with and without PKA regarding SMA frequency, use of polyantibiotics, and switching antibiotic dosage or antibiotics. ADEs happened to 13.3% of self-medicated students. Frequent change of dosage and simultaneous use of the same antibiotic with different names were independent risk practices associated with an ADE. CONCLUSIONS: Our findings substantiate high SMA prevalence among Chinese university students. Older age and PKA are independent SMA risk factors common to Chinese university students and female gender is exclusive SMA risk factor for JSU students. Poor SMA practices are associated with ADEs. Strict regulations on antibiotic sales and public education reinforced by further health care reform are recommended.

Identification and prevalence of an enterotoxin-related gene, se-int, in Staphylococcus intermedius isolates from dogs and pigeons.

AIMS: To determine the prevalence of enterotoxin-producing Staphylococcus intermedius in dogs and pigeons. METHODS AND RESULTS: A total of 106 S. intermedius isolates from 44 dogs and 62 pigeons were tested for the production of enterotoxins A, B, C and D by reverse passive latex agglutination (RPLA) and for sec-canine by PCR. Only one isolate from dog was positive for SEC and sec-canine. Screening of sec-canine-negative strains by nested PCR led to the identification of a novel enterotoxin-related gene, se-int. SE-int showed a significant homology (59-61% identity) with SEC and (56.6% identity) SEB. All 44 isolates from dogs and five isolates (8.1%) from pigeons were se-int positive. CONCLUSIONS: While S. intermedius was isolated more frequently from pigeons than from dogs, se-int was more prevalent among the S. intermedius isolates from dogs, compared with the pigeon isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Further characterization of the se-int-positive S. intermedius strains should clarify their pathogenic potential including enterotoxigenicity and zoonotic transmissibility to human beings.

Chimaeras reveal the role of the catalytic core in the activation of the plasma membrane Ca2+ pump.

Isoform 2b of the plasma membrane calcium pump differs from the ubiquitous isoform 4b in the following: (a) higher basal activity in the absence of calmodulin; (b) higher affinity for calmodulin; and (c) higher affinity for Ca(2+) in the presence of calmodulin [Elwess, Filoteo, Enyedi and Penniston (1997) J. Biol. Chem. 272, 17981-17986]. To investigate which parts of the molecule determine these kinetic differences, we made four chimaeric constructs in which portions of isoform 2b were grafted into isoform 4b: chimaera I contains only the C-terminal regulatory region of isoform 2b; chimaera II contains the N-terminal moiety of isoform 2b, including both cytoplasmic loops; chimaera III contains the sequence of isoform 2b starting from the N-terminus to after the end of the first (small) cytoplasmic loop; and chimaera IV contains only the second (large) cytoplasmic loop. Surprisingly, chimaera I showed low basal activity in the absence of calmodulin and low affinity for calmodulin, unlike isoform 2b. In contrast, the chimaera containing both loops showed high basal activity, and Ca(2+) activation curves (both in the absence and in the presence of calmodulin) similar to those of isoform 2b. The rates of activation by calmodulin and of inactivation by calmodulin removal were measured, and the apparent K(d) for calmodulin was calculated from the ratio between these rate constants. The order of affinity was: 2b=II>4b=IV>III=I. From these results it is clear that the construct that most closely resembles isoform 2b is chimaera II. This shows that, in order to obtain an enzyme with properties similar to those of isoform 2b, both cytoplasmic loops are needed.

Genomic diversity in the pfoA region of the theta-toxin-deficient strains of Clostridium perfringens.

The genomic structure of the pfoA-colA region in six theta-toxin-deficient strains of Clostridium perfringens was examined by Southern hybridization using the pfoR, pfoA, pbg, arcABDC and colA genes, encoding regulator for pfoA, theta-toxin, beta-galactosidase, arginine metabolism enzymes and kappa-toxin, respectively, as gene probes. It is suggested that the productivity of theta-toxin in these strains is diverse because of the multiple genetic backgrounds including single deletion of pfoA, large deletion of the pfoA-colA region and the putative point mutations.

The virR/virS locus regulates the transcription of genes encoding extracellular toxin production in Clostridium perfringens.

Extracellular toxin production in Clostridium perfringens is positively regulated by the two-component regulatory genes virR and virS. Northern (RNA) blots carried out with RNA preparations from the wild-type strain 13 and the isogenic virR and virS mutants TS133 and JIR4000 showed that the virR and virS genes composed an operon and were transcribed as a single 2.1-kb mRNA molecule. Primer extension analysis led to the identification of two promoters upstream of virR. Hybridization analysis of the mutants and their complemented derivatives showed that the virR/virS system positively regulated the production of alpha-toxin (or phospholipase C, theta-toxin (perfringolysin O), and kappa-toxin (collagenase) at the transcriptional level. However, the modes of regulation of these genes were shown to differ. The theta-toxin structural gene, pfoA, had both a major and a very minor promoter, with the major promoter being virR/virS dependent. The colA gene, which encodes the kappa-toxin, had two major promoters, only one of which was virR/virS-dependent. In contrast, the alpha-toxin structural gene, p1c, had only one promoter, which was shown to be partially regulated by the virR and virS genes. Comparative analysis of the virR/virS-dependent promoters did not reveal any common sequence motifs that could represent VirR-binding sites. It was concluded that either the virR/virS system modulates its effects via secondary regulatory genes that are specific for each toxin structural gene or the VirR protein does not have a single consensus binding sequence.

Characterization of a toxin-deficient Clostridium perfringens strain, KZ1340.

Clostridium perfringens KZ1340, previously classified as Clostridium plagarum, is an isolate from Antarctic soil, and was identified as an alpha-, theta-, and kappa-toxin non-producing variant. On Southern hybridization, the variant was found to be defective in the pfoA (theta-toxin) gene, but the plc (alpha-toxin) and colA (kappa-toxin) genes were present on the same EcoRI fragment as in the standard strain, NCTC8237. Northern analysis revealed that mature plc mRNA was transcribed in KZ1340 though less efficiently than in NCTC8237, while no mature colA mRNA was present in KZ1340. After transformation of the pfoA and plc genes into the KZ1340 via shuttle vector, pJIR418, the pfoA gene was successfully expressed but the plc gene was not efficiently expressed, suggesting that in KZ1340 there is negative regulation of plc gene expression. Toxin-deficient C. perfringens KZ1340 might be a suitable host for expression analysis of the pfoA gene and other clostridial virulence genes, if expressed efficiently, because it produces a small amount of extracellular toxins.

Sequence analysis of flanking regions of the pfoA gene of Clostridium perfringens: beta-galactosidase gene (pbg) is located in the 3'-flanking region.

The 3'-flanking region of the perfringolysin O (theta-toxin) gene (pfoA) of Clostridium perfringens was analyzed by chromosome walking. A total of 5,363 bp of the downstream region of the pfoA gene was sequenced and four open reading frames were found. ORF54 and ORF80 were found to be homologous to genes coding for membrane-bound transporter proteins of other bacteria and the beta-galactosidase gene (bgaB) of Bacillus stearothermophilus, respectively. ORF80 was named the pbg gene. Clones which showed beta-galactosidase activities were selected from a lambda FIXII genomic library of C. perfringens by blue plaque screening using X-Gal as a substrate. Four clones whose plaques showed blue appearances were obtained. Two of the four clones hybridized with the pbg probe but the others did not, indicating that there are two distinct beta-galactosidase genes in C. perfringens. The pbg gene was subcloned into pBR322 and was successfully expressed in Escherichia coli, suggesting that the pbg gene codes for a beta-galactosidase of C. perfringens.

The virR gene, a member of a class of two-component response regulators, regulates the production of perfringolysin O, collagenase, and hemagglutinin in Clostridium perfringens.

The perfringolysin O (theta-toxin) gene (pfoA) of Clostridium perfringens was cloned into an Escherichia coli-C. perfringens shuttle vector, and the pfoA gene was expressed in mutants of C. perfringens 13 which lacked the production of perfringolysin O. One group (SI117) could express the pfoA gene, and the other (SI112) could not. A mutation in the regulatory system for pfoA gene expression was suspected in SI112. A chromosomal DNA library constructed from strain 13 was transformed into strain SI112 to identify the regulatory gene(s) for the pfoA gene. Five strains of 10,000 transformants restored perfringolysin O production. All contained a 2.5-kb DNA fragment. This fragment activated the transcription of the pfoA gene and also restored the production of collagenase (kappa-toxin) and hemagglutinin in strain SI112. Deletion analysis showed that a 1.25-kb region was sufficient for the trans activity, and sequence analysis disclosed that open reading frame 2 (ORF2) was located in this region. A homology search for the deduced amino acid sequence revealed that ORF2 was homologous to a response regulator in a two-component signal transduction system. ORF2 was designated virR, and it is suggested that the virR gene plays an important role in the pathogenicity of C. perfringens.