Clostridium innocuum is a vancomycin-resistant pathogen that may cause antibiotic-associated diarrhoea.

OBJECTIVES: Clostridium innocuum can cause extraintestinal infection in patients with underlying diseases. The role of C. innocuum in antibiotic-associated diarrhoea (AAD) remains unknown. METHODS: Clinical information of 103 patients from whom C. innocuum was isolated was reviewed. We carried out cellular and animal experiments to examine the pathogenic potential of C. innocuum in AAD. RESULTS: Eighty-eight per cent (91/103) of the 103 patients received antibiotics within 2 weeks of diarrhoea onset. Patients were further classified into two groups, severe colitis and diarrhoea, according to clinical severity level. The mortality rate was 13.6% (14/103) among the patients from whom C. innocuum was isolated. The lowest concentrations at which 90% of the isolates were inhibited for metronidazole and vancomycin were 0.5 and 16 mg/L, respectively. All isolates tested were susceptible to metronidazole but resistant to vancomycin. Nineteen randomly selected isolates (ten from severe colitis group, nine from diarrhoea group) were subjected to further in vitro cellular examinations. The level of cytotoxicity to Vero cells was significantly higher in isolates from the severe colitis group at both 24 and 48 hours after inoculation (24 and 48 hours, p 0.042 and 0.033, respectively). We observed apoptotic changes that subsequently led to cell death in C. innocuum-infected Vero cells. Tissue damages, necrotic changes and oedema were observed in the mouse ileal loop infected by C. innocuum. CONCLUSIONS: Vancomycin-resistant C. innocuum may play a potential role as a causative agent of AAD. The clinical manifestations of AAD caused by C. innocuum were diarrhoea or severe colitis, including pseudomembranous colitis.

Clostridium innocuum is a significant vancomycin-resistant pathogen for extraintestinal clostridial infection.

OBJECTIVES: Extra-intestinal clostridial infection (EICI) is rare but can be fatal. Traditional phenotypic methods can only assign many of the Clostridium species to the genus level. METHODS: A total of 376 non-repetitive Clostridium isolates from sterile sites were collected and subjected to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) Biotyper analysis and 16S rRNA sequencing. Antimicrobial susceptibility was determined, and clinical characteristics of the patients were assessed. Clostridium innocuum isolates were characterized by genome sequencing and genotyping. We used molecular and cellular methods to explore the virulence and resistance mechanisms of C.innocuum. RESULTS: Clostridium innocuum was the second most common species to cause EICI, only next to Clostridium perfringens. All Clostridium isolates showed susceptibility to clindamycin, metronidazole, penicillin, piperacillin and ampicillin-sulbatam, while C. innocuum isolates were invariably resistant to vancomycin. Among 24 patients with EICI caused by C. innocuum, two (8.3%) had diarrhoea, three (12.5%) had soft-tissue infection, six (25%) had appendicitis and four (16.7%) each had shock and gastrointestinal perforation. The 30-day mortality was 16.7%. The C. innocuum isolated from different sites could not be separated from one another by genotyping. No known toxin genes were identified in the genome of C. innocuum but the species expressed cytotoxicity to epithelial cells. d-Alanine-d-alanine ligase, alanine racemase and d-alanyl-d-alanine carboxypeptidase are three main genes responsible for vancomycin resistance in C. innocuum. CONCLUSIONS: Vancomycin-resistant C. innocuum is a previously unrecognized, yet prominent, cause for EICI. Genome analysis showed that the species could carry a lipopolysaccharide-like structure that is associated with cytotoxicity to cells in vitro.

Identification of a hidden outbreak due to the spread of a VIM-3-producing, extensive drug-resistant Pseudomonas aeruginosa (XDRPA) clone at a regional hospital in Taiwan.

A review of the annual prevalence of Pseudomonas aeruginosa at a regional hospital in Taiwan revealed a significant increase in the incidence of extensive drug-resistant P. aeruginosa (XDRPA) from 2∙1% in 2003 to 5∙8% in 2007. The first XDRPA isolate was recovered in 2001 from the emergency ward. The widespread dissemination of XDRPA isolates to more than 10 other wards was discovered the following year. Six pulsotypes of 67 XDRPA isolates from 2006 onwards were identified and 91% were a single strain, suggesting the existence of a hidden outbreak. Prior to the recognition of the outbreak, the majority of cases were not considered to be healthcare-associated infections until molecular evidence was provided. A cohort measure was launched by the infection control practitioners that effectively controlled the outbreak. Patients with XDRPA were mostly referred from neighbouring long-term care facilities, which may have been the reservoir of the XDRPA clone.

A 7-year surveillance for ESBL-producing Escherichia coli and Klebsiella pneumoniae at a university hospital in Taiwan: the increase of CTX-M-15 in the ICU.

To monitor the changing trend of extended-spectrum beta-lactamase (ESBL)-producing bacteria, a 7-year continuous study was launched in 2001 at the largest tertiary hospital in Taiwan. A significant increase over the study period was evident for ESBL-producing isolates of Escherichia coli (4.8-10.0%) and Klebsiella pneumoniae (15.0-23.4%). Molecular investigation conducted in three separate periods revealed the prevalent ESBL types and their genetic relatedness. CTX-M-producing isolates (73.8%) were more prevalent than SHV-type ESBLs (37.0%), the most frequent being CTX-M-14 (34.3%), CTX-M-3 (25.9%), and SHV-12 (25.7%). However, a marked increase of CTX-M-15-producing isolates from 2.1% in 2002 to 29.6% in 2007 was also noted. The increase of ESBL-producing isolates in both species may be mainly due to the horizontal transmission of resistance plasmids, while clonal expansion of some epidemic strains further added to the dispersion of ESBL-producing K. pneumoniae.

Emergence of carbapenem-resistant Escherichia coli in Taiwan: resistance due to combined CMY-2 production and porin deficiency.

Eight pairs of Escherichia coli isolates with various carbapenem susceptibilities from 8 patients were prospectively collected to study the development of resistance. All carbapenem-resistant E. coli isolates were resistant to all tested ss-lactams antibiotics except tigecycline. Identical pulsed-field gel electrophoresis (PFGE) patterns were found in carbapenem-susceptible and -resistant isolates but different PFGE patterns occurred among patients. A CMY-2 ss-lactamase was found in all E. coli isolates. No previously reported carbapenemase genes were detected. Examination of outer membrane protein (OMP) profiles revealed that OmpA was not found in all isolates, while OmpC and OmpF were lost in carbapenem-resistant isolates. Loss of both OmpC and OmpF represents the major mechanism of the development of carbapenem resistance in those patients with CMY-2-producing E. coli infections.

Isolation of Salmonella enterica serotype choleraesuis resistant to ceftriaxone and ciprofloxacin.

Salmonella enterica serotype choleraesuis (S choleraesuis) usually causes systemic infections in man that need antimicrobial treatment. We isolated a strain of S choleraesuis that was resistant to ceftriaxone and ciprofloxacin from a patient with sepsis. Ciprofloxacin resistance was associated with mutations in gyrA and parC, whereas the ampC gene (bla(CMY-2)), responsible for ceftriaxone resistance, was carried by a transposon-like mobile element. This element was found inserted into finQ of a potentially transmissible 140 kb plasmid, with an 8 bp direct repeat flanking the junction regions. The appearance of this resistant S choleraesuis is a serious threat to public health, and thus constant surveillance is warranted.

Molecular epidemiology of nalidixic acid-resistant campylobacter isolates from humans and poultry by pulsed-field gel electrophoresis and flagellin gene analysis.

To investigate the potential of poultry products as the source of human infections associated with quinolone-resistant campylobacters, 140 human and 75 poultry isolates of nalidixic acid-resistant campylobacters were collected between 1996 and 1998, and analysed by two molecular typing methods. By the analysis of restriction fragment length polymorphism of the flagellin gene, 33 distinct patterns were obtained, with 18 of which shared by both human (89%) and poultry (93%) isolates. By the pulsed-field gel electrophoresis of SmaI-restricted macrofragments, 105 different profiles were obtained, and 11 were found in both human (40%) and poultry (23%) isolates. When the two typing methods were combined, 112 unique genotypes were obtained, 11 of which were shared by both populations, including 53 (38%) human isolates and 14 (19%) poultry isolates. Although domestic poultry products are still important sources of the quinolone-resistant campylobacter infections in humans, there are other factors that might contribute to these increasing infections simultaneously. A more stringent policy in the use of antimicrobial agents in food animals can no longer be ignored.

Molecular epidemiology of nosocomial infection associated with multi-resistant Acinetobacter baumannii by infrequent-restriction-site PCR.

Acinetobacter baumannii was considered endemic in a university-affiliated tertiary hospital. A significant increase was noted in the proportion of nosocomial infections associated with this micro-organism from 1996 to 1999, although no apparent clusters could be found. Between July 1998 and February 2000, 58 nosocomial isolates of A. baumannii were collected and characterized by antibiotyping and a genotyping method, infrequent-restriction-site PCR (IRS-PCR). High resistance to the 14 antimicrobial agents examined was observed among the isolates. Of the 13 antibiograms detected, eight were multi-resistant to gentamicin and almost all of the traditional and extended-spectrum beta-lactams. These multi-resistant strains consisted of 41 isolates (71%), distributed amongst different wards and intensive care units (ICUs). By IRS-PCR, 23 types were obtained, with one major type found among 28 (48%) isolates. All of these 28 isolates were collected from surgical ICUs. It appears that a single strain of multi-resistant A. baumannii was responsible for the prevalence of nosocomial infection amongst surgical patients, clearly differentiating this outbreak from the previous endemic situation. An efficient molecular typing method played a vital role in making this discrimination.

Secular trends in incidence and antimicrobial resistance among clinical isolates of Salmonella at a university hospital in Taiwan, 1983-1999.

The incidence and antimicrobial resistance among clinical isolates of salmonella at a university hospital in Taiwan between 1983 and 1999 are summarized in this report. A total of 7986 isolates were analysed. Serogroup B has been the most prevalent over the years, with an apparently continuous decline after 1995. Concordant decrease was also found among S. choleraesuis and S. typhi isolates in recent years. In contrast, the proportion of serogroup D strains increased significantly after 1996. S. typhi remained relatively susceptible to most of the antimicrobial agents examined. For non-typhoidal isolates, antimicrobial resistance to ampicillin (62%), chloramphenicol (67%), and sulfamethoxazole-trimethoprim (37%) was relatively higher than that reported elsewhere. Newer generation cephalosporins and fluoroquinolones remained effective over the years, although emerging resistance to these drugs has been noticed since 1992. A more prudent selection and use of antimicrobial agents, in both humans and animals, and a continuous surveillance of resistance are essential in the future.

Outbreaks of nosocomial bloodstream infections associated with multiresistant Klebsiella pneumoniae in a pediatric intensive care unit.

BACKGROUND: Between June and October 1997, and during April 1998, a cluster of nosocomial bloodstream infections (BSIs) associated with Klebsiella pneumoniae was observed in 8 premature neonates from 1 pediatric intensive care unit (TPICU) in a 4000-bed medical center in northern Taiwan. An investigation was conducted to identify the possible reservoirs and mode of transmission. METHODS: Epidemiologic surveillance and infection control interventions were executed. The environment was checked by submitting several swab samples for microbiological studies. The antibiograms and results from 2 molecular typing methods (pulsed-field gel electrophoresis and infrequent-restriction site polymerase chain reaction) of all bacteremic and environmental isolates of K. pneumoniae were compared. RESULTS: Totally 39 K. pneumoniae isolates, including 9 from bacteremia, 26 from the environment, and 4 controls, were analyzed. One major pattern was found in 21 isolates, which included 8 bacteremic isolates with identical antibiograms, a single isolate from rectal swab screening, 2 of 8 isolates from hand cultures of medical staff, and 10 of 17 isolates from swabs of sinks in the TPICU. All 21 isolates illustrated identical antibiograms, while the other 18 isolates shared 4 antibiograms and 15 unique patterns. CONCLUSIONS: The nosocomial BSIs appeared to be an outbreak induced by 1 multiresistant K. pneumoniae strain. The sinks may have acted as reservoirs for this outbreak strain. During washing, splattered water droplets containing the bacterial particles may have contaminated the hands of medical personnel and were then further transmitted to patients.

Molecular investigation of two clusters of hospital-acquired bacteraemia caused by multi-resistant Klebsiella pneumoniae using pulsed-field gel electrophoresis and in frequent restriction site PCR. Infection Control Group.

Two molecular typing methods, DNA macrorestriction analysis with XbaI resolved by pulsed-field gel electrophoresis (PFGE) and infrequent restriction site PCR (IRS-PCR) assay with adapters designed for XbaI and HhaI restriction sites, were used to investigate two clusters of hospital-acquired bacteraemia associated with multi-resistant Klebsiella pneumoniae which occurred in a paediatric intensive care unit (PICU). A total of 56 K. pneumoniae isolates were analysed. These included 10 bacteraemic isolates from eight patients, 26 isolates obtained during an epidemiological survey, and 20 epidemiologically non-related isolates incorporated as controls. One major pattern was demonstrated in 22 of the 56 isolates analysed. These included nine of the 10 bacteraemic isolates, a single rectal isolate, two hand culture isolates and 10 sink isolates. All of these 22 isolates illustrated identical antibiograms, whilst the other 34 isolates shared six antibiograms and 31 unique patterns by either PFGE or IRS-PCR assay. The two clusters of bacteraemia appeared to be outbreaks induced by the same strain of K. pneumoniae which may have utilized sinks as reservoirs and been transmitted through the hands of medical personnel to patients. IRS-PCR demonstrates concordant results with PFGE analysis in studying the genetic relationships among K. pneumoniae isolates, and serves as an excellent epidemiological tool for this bacterium.

DNA polymorphism of Mycobacterium abscessus analyzed by infrequent-restriction-site polymerase chain reaction.

BACKGROUND: Mycobacterium abscessus is an important pathogen that has been increasingly associated with many clinical and nosocomial infections. A reliable molecular typing scheme is essential for the epidemiological study of this rapidly growing mycobacterium. Pulsed-field gel electrophoresis (PFGE), considered to be the gold standard among molecular typing methods, has failed to provide satisfactory results in the molecular typing of this bacterium. A newly developed molecular typing method, infrequent-restriction-site polymerase chain reaction (IRS-PCR), was examined in this study to determine its suitability for fingerprinting M. abscessus isolates. METHODS: Eight clinical isolates of M. abscessus and two reference strains (M. abscessus ATCC 19977 and M. chelonae ATCC 35749) were studied by DNA macrorestriction analysis with XbaI resolved by PFGE, and IRS-PCR assay with adaptors designed for XbaI and HhaI restriction sites. RESULTS: By PFGE, different banding patterns were found in two clinical isolates of M. abscessus; the other isolates yielded only broken DNA and could not be assessed. By IRS-PCR, unique patterns were noted for the 10 isolates; the 10 appeared to be genetically different. CONCLUSION: IRS-PCR may be an efficient substitute for PFGE in analyzing the DNA polymorphism and epidemiology of M. abscessus.

Molecular biology of hepatitis D virus: research and potential for application.

Superinfection by hepatitis D virus (HDV) leads to acute hepatitis and causes progression to liver cirrhosis in a significant proportion of hepatitis B surface antigen (HBsAg) carriers. Current regimens (interferon) to treat hepatitis D patients has only transient but no lasting effects. New approaches are, therefore, warranted. Recently, several laboratory studies have discovered interesting properties of HDV that may become targets for antiviral chemicals. Viral replication requires the small hepatitis delta antigen (s-HDAg). The s-HDAg is a nuclear phosphoprotein. There is evidence indicating that phosphorylation is important for HDV replication. A second step of replication requires HDV-RNA self-cleavage and self-ligation. Interestingly, one group of antibiotics, the aminoglycosides, exerts strong suppression effects on HDV ribozyme activities. In the following stage of viral assembly, two post-translational modifications, namely isoprenylation of large HDAg and glycosylation of HBsAg are involved. Agents capable of blocking the two modifications should reduce viral production. These four possible targets are reviewed. For prevention, effective vaccines are not yet available. Two novel approaches are discussed. The first demonstrates the immunogenicity of a nucleic acid vaccine in mice. The second approach assembled an empty HDV particle in yeast. Advances on such laboratory investigations may provide new methods for the control of hepatitis D in the future.