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. (C) 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

BackgroundStreptococcus pneumoniae is one of the most common pathogens to cause mucosal and invasive infection in humans. Most of the infection could be prevented through immunization by vaccines containing capsular polysaccharides but some infection may be caused by unencapsulated strains.MethodsClinical isolates of S. pneumoniae from January 2012 to December 2015 at Chang Gung Memorial Hospital, Taiwan. Serotyping by PCR method was performed. Clinical and laboratory information of patients infected by non-typeable pneumococci (NTP) were collected and analyzed.ResultsDuring the study period, 39 NTP isolates were identified. Most (21 of 39, 53.9%) were collected from purulent upper respiratory tract secretion. Others were from corneal abscess, sputum, and one from blood of a newborn. We recorded a 3.6-fold increase in the rate of isolation from 1.4% in 2012 to 5.0% in 2015 (p = 0.063). Co-infection was found in 24 cases; the major co-infecting pathogens included non-typeable Hemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus. Most (39 of 40, 97.5%) of the isolates were susceptible to both penicillin and ceftriaxone. The dominant sequence type ST1106 and an emerging sequence type ST7502 were recognized.ConclusionsA gradual increase of NTP infection was found in northern Taiwan in the pneumococcal conjugate vaccine era. Non-typeable pneumococci can cause respiratory and ophthalmological mucosal infection. Invasive infection can occur in newborns or young infants. Most of the isolates remained susceptible to penicillin and ceftriaxone.

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.

The characteristics of patients with recurrent bacteraemia caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli or Klebsiella pneumoniae (EK) are rarely described. Flomoxef belongs to the cephamycins group and demonstrates in vitro activity against ESBL-producing organisms. Whether flomoxef may be used for the treatment of such infections remains controversial. This retrospective case-control study enrolled adult patients who had bacteraemia caused by ESBL-EK during 2005-2011. Case patients were those who had more than one episode of ESBL-EK bacteraemia. Controls were those who were matched for age and interval time of blood sampling and had only one episode of ESBL-EK bacteraemia with subsequent bacteraemia episodes caused by other non-ESBL-EK bacteria. Pulsed-field gel electrophoresis and microbiologic profiles of the initial and subsequent ESBL-EK isolates were analysed. During the study period, 424 patients were found to have at least one positive blood culture after the first ESBL-EK bacteraemia episode, and 67 (15.8%) had a second episode of ESBL-EK bacteraemia. Bacteraemia resulting from vascular catheter-related infection (odds ratio, 3.24; 95% confidence interval, 1.31-8.05), and definitive therapy with flomoxef (odds ratio, 2.99; 95% confidence interval, 1.10-8.15) were both independent risk factors for the recurrence. Among the 56 patients with available ESBL-EK isolates for analysis, 38 (67.8%) were infected by genetically similar strains. In three of these 38 recurrent ESBL-EK bacteraemia cases caused by an identical strain, the minimum inhibitory concentrations of carbapenem for the subsequent K. pneumoniae isolates were fourfold or higher than the initial isolates. Recurrent bacteraemia was not uncommon in our patients with ESBL-EK bacteraemia, and most of the episodes were caused by identical strains.

BACKGROUND:Outbreaks of urinary tract infections (UTIs) due to contaminated ureteroscopes have been rarely reported. AIM:To report such an outbreak at a regional teaching hospital in southern Taiwan. METHODS:From October to December 2010, ertapenem-resistant Enterobacter cloacae were identified from urine cultures of 15 patients who had undergone ureteroscopy prior to the infection. Three batches of surveillance cultures were obtained from the environmental objects and healthcare workers related to the procedures. Pulsed-field gel electrophoresis (PFGE) was used for bacterial typing. Antimicrobial susceptibility was assessed by disc diffusion and E-test methods. Polymerase chain reaction and sequencing were used to analyse β-lactamase genes. FINDINGS:A total of 70 specimens were obtained during the first surveillance operation. One ertapenem-resistant E. cloacae was isolated from a ureteroscope. Although the disinfection protocols for ureteroscopes were revised and implemented, seven additional UTI cases were identified thereafter. The pathogen was identified from two subsequent surveillance cultures and was not eliminated until ethylene oxide sterilization was added to the disinfection protocol. PFGE revealed that all 15 isolates from the patients and the three isolates from the ureteroscope shared a common pattern with minor variance. Most isolates were resistant to gentamicin, levofloxacin, ceftriaxone, ceftazidime, and ertapenem. All isolates were susceptible to amikacin, imipenem, and meropenem. SHV-12 and IMP-8 genes were simultaneously identified in 16 of the 18 isolates. CONCLUSION:The outbreak of ertapenem-resistant E. cloacae was caused by a contaminated ureteroscope and was terminated by the implementation of a revised disinfection protocol for ureteroscopes.

SUMMARYA 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.

Multidrug-resistant Salmonella infection is a global problem, and carbapenems may represent the last therapeutic choice. We report a case of infection caused by ceftriaxone-resistant and ciprofloxacin-resistant Salmonella enterica serotype Typhimurium. A bla(CMY-2) -containing Tn6092, located on a self-transferable IncI1 plasmid, was found in all isolates derived from the patient. During ertapenem treatment, the strain developed carbapenem resistance. Apart from the OmpD deficiency found in all isolates, the strain further developed OmpC deficiency through a single gene mutation, and became carbapenem-resistant. Salmonella appears to be very plastic in developing antimicrobial resistance. Care must be taken by physicians when treating multidrug-resistant Salmonella infection.

SUMMARY To monitor the changing trend of extended-spectrum β-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 .

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 beta-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 beta-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.

Pandrug-resistant Acinetobacter baumannii (PDRAB) emerged in Taiwan in the early 2000s but was not identified in the Children's Hospital (Hospital A) until March 2005 when a patient was transferred from a respiratory care hospital (Hospital B). PDRAB was recovered from an eye swab taken on admission; once aware of the culture result, in addition to implementing infection control precautions, an epidemiological investigation was conducted in both hospitals. A total of 212 specimens were taken from 30 hospital inpatients (seven in Hospital A, 23 in Hospital B), clinical equipment and ward environment. Thirteen (15.5%) of 84 specimens obtained from Hospital A and 23 (18%) of 128 specimens obtained from Hospital B were positive for A. baumannii; of these, six isolates from two patients and clinical equipment in Hospital A and five from three patients in Hospital B were PDRAB. One patient stayed in both hospitals, and had one PDRAB isolate detected at each. Of the 36 A. baumannii isolates, there were nine IRS–PCR (infrequent restriction site–polymerase chain reaction) patterns, 12 PFGE (pulsed-field gel electrophoresis) patterns and six antibiogram patterns identified. Twenty-five isolates belonged to a major IRS–PCR type (four PFGE patterns) and presented with either pandrug resistance (all 11 PDRAB isolates clustered in this type) or multidrug resistance (only susceptible to imipenem). A. baumannii is an ubiquitous organism that can be isolated from patients and their equipment. A clone of A. baumannii with multi- or pandrug resistance was circulating in both hospitals.

From August 2004 to July 2005, 210 clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were collected prospectively from 173 children admitted to Chang Gung Children's Hospital in Taiwan. A comparative molecular analysis of the 111 community-associated (CA) isolates from 102 children and the 99 healthcare-associated (HA) isolates from 71 children was conducted. In comparison to the HA isolates (31%), the CA isolates (90%) were more likely to have been isolated from pus (p <5 x 10(-8)). For each patient with MRSA infection, only the first isolate was selected for molecular analysis. The molecular characteristics differed significantly between the CA and the HA isolates (p <5 x 10(-8)). The clone characterized as sequence type (ST)59/pulsotype D (similar to USA1000)/staphylococcal chromosomal cassette (SCC)mec V(T)/Panton-Valentine leukocidin (PVL)-positive accounted for 69% of the CA isolates, and another clone, characterized as ST239/pulsotype A (Hungary clone)/SCCmec III/PVL-negative, accounted for 45% of the 71 HA isolates. The CA clone of ST59 also accounted for 20% of the HA isolates, including 47% of the 17 community-onset isolates. It was concluded that the molecular characteristics of clinical MRSA isolates from children differed significantly between the CA and the HA isolates in northern Taiwan. However, the CA clone of ST59 was also identified in a substantial proportion of HA isolates.

SUMMARY Two hundred and four Salmonella enterica serotype Typhi (S. Typhi) isolates were collected from seven Asian countries during 2002–2004. Multidrug-resistant S. Typhi (resistant to ⩾3 antibiotics) was detected in 84 (41·2%) isolates and 142 (69·6%) showed reduced susceptibility to ciprofloxacin (minimum inhibitory concentration=0·125–1·0 mg/l). This study highlights the worsening situation of antimicrobial resistance of S. Typhi in Asia.

CTX-M-3 is the most common extended-spectrum β-lactamase produced by Enterobacteriaceae in Taiwan. The present study was conducted to characterise the genetic environment surrounding blaCTX-M-3. A total of 11 ceftriaxone-resistant isolates were studied: Escherichia coli (n = 4), Klebsiella pneumoniae (n = 5) and Salmonella enterica serotypes Anatum (SA831R) and Potsdam (SC72). Molecular methods used included polymerase chain reaction, sequencing, DNA–DNA hybridisation, conjugation, physical mapping and restriction fragment length polymorphism (RFLP) analysis. All isolates examined carried blaCTX-M-3 on large plasmids (>70 kb). The resistance plasmids of the two Salmonella and two K. pneumoniae strains (KP104 and KP116) were confirmed to be conjugative in vitro. RFLP analysis indicated that the plasmids were different. Physical mapping also revealed the difference between the two Salmonella plasmids, pSA831R (82 kb) and pSC72 (74 kb). An insertion sequence, ISEcp1, was found upstream of each blaCTX-M-3 gene. However, sequencing of downstream regions of the bla genes showed two different patterns: the presence of orf477 in pSA831R and of orf1-mucA in pSC72, pKP104 and pKP116. IncI1-type oriT and nikA sequences were present in the plasmids of all the clinical isolates tested, except S. Anatum. Different blaCTX-M-3-carrying plasmids were identified among the enterobacteria studied. The presence of ISEcp1 in all isolates may be associated with the widespread resistance among Enterobacteriaceae. Although the plasmids were not identical, they appeared to belong to the same incompatibility group (IncI1-like plasmids), suggesting that they are genetically related but may have evolved divergently over time.

Genetic determinants of antibiotic resistance (AR) have been extensively investigated. High-throughput sequencing allows for the assessment of the relationship between genotype and phenotype. A panel of 672 Pseudomonas aeruginosa strains was analysed, including representatives of globally disseminated multidrug-resistant and extensively drug-resistant clones; genomes and multiple antibiograms were available. This panel was annotated for AR gene presence and polymorphism, defining a resistome in which integrons were included. Integrons were present in >70 distinct cassettes, with In5 being the most prevalent. Some cassettes closely associated with clonal complexes, whereas others spread across the phylogenetic diversity, highlighting the importance of horizontal transfer. A resistome-wide association study (RWAS) was performed for clinically relevant antibiotics by correlating the variability in minimum inhibitory concentration (MIC) values with resistome data. Resistome annotation identified 147 loci associated with AR. These loci consisted mainly of acquired genomic elements and intrinsic genes. The RWAS allowed for correct identification of resistance mechanisms for meropenem, amikacin, levofloxacin and cefepime, and added 46 novel mutations. Among these, 29 were variants of the oprD gene associated with variation in meropenem MIC. Using genomic and MIC data, phenotypic AR was successfully correlated with molecular determinants at the whole-genome sequence level.

We investigated the genetic properties of imipenem-resistant Acinetobacter baumannii collected from a regional hospital in Taiwan. Pulsed-field gel electrophoresis demonstrated that the isolates were genetically diverse. Polymerase chain reaction, DNA sequencing, and DNA-DNA hybridisation showed that the bla(IMP-1) gene resided as a cassette in a plasmid-borne class 1 integron in two isolates. The majority of the resistant isolates were plasmid-less and carried no bla(IMP), bla(VIM) or bla(CFI) genes, indicating that other uncharacterised metallo-beta-lactamases or mechanisms other than enzyme production are involved in carbapenem resistance in this group of A. baumannii. We conclude that multidrug resistance of A. baumannii was a combined effect of lateral gene transfer and clonal spread of multiple resistant clones. Strict measures should be implemented to control the further spread of resistance.

OBJECTIVES:The dissemination of cephamycin resistance in Enterobacteriaceae and its correlation with a transposon-like DNA element consisting of a specific tnpA-bla(CMY-2)-blc-sugE structure were investigated.METHODS:A total of 140 enterobacterial isolates belonging to 17 species (10 genera) of Enterobacteriaceae phenotypically characterized as putative AmpC-producers were evaluated. The isolates were examined by PCR analysis, DNA-DNA hybridization and nucleotide sequencing.RESULTS:The bla(CMY-2)-carrying element was detected in 34 isolates from 10 species (9 genera), including all 14 Salmonella and 4 Shigella isolates as well as 7 of the 10 Escherichia coli isolates tested. The remaining 9 isolates were from 112 isolates of the other 14 species tested. The genetic structure of the bla(CMY-2)-carrying element was identical in 29 isolates, while in 3 E. coli and 2 Citrobacter isolates an additional insertion sequence IS1 was found inserted at various nucleotide positions close to the 3' end, either within or downstream, of tnpA. In 12 of the 14 representative isolates examined, the bla(CMY-2)-carrying element was found inserted in the finQ gene of various-sized plasmids with highly conserved 8 bp direct repeats flanking the junction regions. Among the other 106 non-CMY-2-producing isolates, plasmid-mediated ampC genes were found only in one isolate of Enterobacter aerogenes which carried a bla(DHA-1)-like gene.CONCLUSIONS:bla(CMY-2) is the most prevalent plasmid-mediated ampC gene among Enterobacteriaceae. All the bla(CMY-2) genes identified in the present study were associated with a specific transposon-like element that may be responsible for the spread of bla(CMY-2) among Enterobacteriaceae.

Chryseobacterium meningosepticum causes severe infections in infants or adults with underlying illness. The species is highly heterogeneous, genetically composed of subgroups with different pathogenicity. Eight strains of C. meningosepticum, representing four different genomic subgroups, were evaluated for their ability to penetrate Madin-Darby Canine Kidney (MDCK) epithelial cell monolayers and serum resistance. None of the strains showed cytotoxicity or penetration to the MDCK cells. All displayed resistance to the bactericidal activity of various normal human sera. A murine pulmonary infection model was used to compare the pathogenicity between a clinical isolate and an environmental isolate. C. meningosepticum were cleared from the lung of infected mice within 7 days following the intratracheal challenge. Electron microscopy demonstrated the large membrane protrusions, indicative of ruffles, and smaller, less organized membrane structures of the respiratory epithelial cells induced by the clinical isolate. Bacteria were observed to enter the cells as single entities in spacious vacuoles. Suppressive subtraction hybridization identified in the invasive isolate 35 distinct sequences associated with systems of energy production and conversion, transport, and secretion. In most cases, the identities between the references and the amino acid sequences deduced were low, suggesting that the functions of these sequences remain unknown.

Infection by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae has been increasing in Taiwan. Accurate identification of the ESBL genes is necessary for surveillance and for epidemiological studies of the mode of transmission in the hospital setting. We describe herein the development of a novel system, which consists of a multiplex PCR to identify bla(SHV), bla(CTX-M-3)-like, and bla(CTX-M-14)-like genes and a modified SHV melting-curve mutation detection method to rapidly distinguish six prevalent bla(SHV) genes (bla(SHV-1), bla(SHV-2), bla(SHV-2a), bla(SHV-5), bla(SHV-11), and bla(SHV-12)) in Taiwan. Sixty-five clinical isolates, which had been characterized by nucleotide sequencing of the bla(SHV) and bla(CTX-M) genes, were identified by the system. The system was then used to genotype the ESBLs from 199 clinical isolates, including 40 Enterobacter cloacae, 68 Escherichia coli, and 91 Klebsiella pneumoniae, collected between August 2002 and March 2003. SHV-12 (80 isolates) was the most prevalent type of ESBL identified, followed in order of frequency by CTX-M-3 (65 isolates) and CTX-M-14 (36 isolates). Seventeen (9%) of the 199 clinical isolates harbored both SHV- and CTX-M-type ESBLs. In contrast to Enterobacter cloacae, the majority of which produced SHV-type ESBLs, E. coli and K. pneumoniae were more likely to possess CTX-M-type ESBLs. Three rare CTX-M types were identified through sequencing of the bla(CTX-M-3)-like (CTX-M-15) and bla(CTX-M-14)-like (CTX-M-9 and CTX-M-13) genes. The system appears to provide an efficient differentiation of ESBLs among E. coli, K. pneumoniae, and Enterobacter cloacae in Taiwan. Moreover, the design of the system can be easily adapted for similar purposes in areas where different ESBLs are prevalent.

BACKGROUND:Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infection in children without health care-associated risk factors has emerged. To evaluate the clinical features and genotyping analysis of CA-MRSA in children in Taiwan, we conducted this study.METHODS:Between July 2000 and June 2001, 198 episodes of S. aureus infection were identified in 191 children hospitalized at Chang-Gung Children's Hospital. The medical records of these children were retrospectively reviewed. The MRSA clinical isolates from each episode of infection, if available, were collected for pulsed-field gel electrophoresis (PFGE) typing and staphylococcal cassette chromosome mec (SCCmec) type determination.RESULTS:Among the 198 episodes of S. aureus infection, MRSA accounted for 47% of 114 CA infections and 62% of 84 hospital-acquired (HA) infections, respectively. Among 54 CA-MRSA infections, 32 episodes (59%) occurred in children without risk factors. Similar to HA-MRSA isolates, these CA-MRSA isolates were also multiresistant, with a high rate of resistance to clindamycin (93%) and erythromycin (94%), but 91% of them were susceptible to trimethoprim/sulfamethoxazole. Superficial soft tissue infection was the most common presentation, accounting for 65% of CA-MRSA infections. All 54 children with CA-MRSA infection recovered without sequelae, though 63% of 35 children with superficial soft tissue infection were treated with in vitro nonsusceptible antibiotics. Of the 83 clinical isolates (40 CA, 22 with no identified risk) available for analysis, 6 genotypes with 30 subtypes were identified. Three major PFGE patterns were identified, accounting for 94% of the isolates (92.5% for CA isolates, 95% for HA isolates). Type IV SCCmec was carried by 25 and 40% of CA and HA isolates, respectively.CONCLUSION:In Taiwan, MRSA was prevalent among pathogens of CA infections in children, and these CA isolates were multiresistant and genetically associated with HA isolates. In an area with a high prevalence of methicillin resistance, for children with putative S. aureus infection, even community-acquired, a glycopeptide-containing regimen should be considered for initial empirical therapy in the case of serious infection.