- Volume 62, Issue 9, 2013
Volume 62, Issue 9, 2013
- Pathogenicity and virulence
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New serovars of Leptospira isolated from patients in Costa Rica: implications for public health
Leptospira strains JICH 05 and INCIENSA 04 were isolated from hospitalized leptospirosis patients in the province of Puntarenas, Costa Rica. The isolates produced agglutination titres notably against members of serogroups Pyrogenes and Tarassovi, respectively, but appeared serologically unique in the cross agglutinin absorption test (CAAT). Therefore, JICH 05 and INCIENSA 04 were considered to represent two new serovars, designated Corredores and Costa Rica of the serogroups Pyrogenes and Tarassovi, respectively. Multilocus sequence genotyping revealed that both strain INCIENSA 04 and strain JICH 05 belong to Leptospira santarosai. These two new serovars are in addition to various other recently identified highly virulent serovars, including the new L. santarosai, serovar Arenal. Considering the fact that isolation and typing of leptospires from patients has only recently been introduced in Costa Rica, these findings suggest that various known and unknown virulent serovars of Leptospira are circulating in this country and probably beyond, thus posing a severe threat to public and probably veterinary health in the region.
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Immune complex binding Streptococcus pyogenes type M12/emm12 in experimental glomerulonephritis
In a rabbit model, we have previously reported evidence for a pathogenic role of streptococcal IgG Fc-binding proteins (IgGFcBP) in poststreptococcal glomerulonephritis (PSGN). These proteins, of the M protein family, were shown to trigger anti-IgG production and enhance renal deposition of IgG and/or immune complexes (ICs), with resulting activation of complement and cytokine cascades. In the present study, type M12/emm12, group A streptococci (GAS) were found often to bind artificial ICs, viz. peroxidase–anti-peroxidase rabbit IgG (PAP) or tetanus toxoid–anti-tetanus human IgG (TAT), rather than monomeric IgG. Animals injected with each of four IC binding clinical isolates (from patients with scarlet fever or PSGN) showed pronounced inflammatory and degenerative glomerular changes, morphologically similar to human PSGN, with membrane thickening and IgG and complement C3 deposition, as well as secretion of IL-6 and TNF-α by mesangial and endothelial cells. In contrast, non-binding strains (two from asymptomatic carriers and one from a PSGN case) failed to trigger any renal changes. Only the IC binding strains induced elevated titres of anti-IgG. Though the streptococcal binding component(s) has not been demonstrated, the selective binding of ICs by type M12/emm12 strains appears important for the well-known, marked nephritogenic potential of this GAS type.
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- Host response
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Modelling anti-pertussis toxin IgG antibody decay following primary and preschool vaccination with an acellular pertussis vaccine in UK subjects using a modified oral fluid assay
Recent vaccination with pertussis vaccine can confound serological and oral fluid (OF) assays targeting anti-pertussis toxin (anti-PT) IgG antibodies as a marker of recent infection. This study sought to establish the minimum potentially confounding time period based on experimental data to assist interpretation from such samples submitted from UK subjects for pertussis diagnosis. Anti-PT IgG antibody response and decay were measured post-vaccination using a modified OF IgG antibody-capture ELISA (GACELISA). Data were obtained from 72 infants after the third acellular pertussis vaccine dose in the primary schedule (4 months of age) and from 119 children after the single dose at preschool age (3 years 4 months to 5 years 8 months of age). Specimens were taken at approximately 1 month intervals for 9 months post-primary immunization (third dose) and 13 months post-preschool booster (PSB). The modified GACELISA demonstrated a sensitivity of 52/56 (92.9 %: 95 % CI 82.7–98.0) and a specificity of 120/128 (93.8 %: 95 % CI 88.0–97.3) and showed good agreement with the National Reference Laboratory standard anti-PT IgG serum ELISA (rank correlation = 0.80) and the original OF assay (rank correlation = 0.79). Modelling of the decline in antibody titres showed a reduction of 54 % and 34 % for each doubling of time after day 14 for the post-third primary dose and post-PSB subjects, respectively. These data suggest that the minimum confounding time period is approximately 300 days for samples obtained post-primary immunization and at least 3 years for samples submitted from UK children following immunization with the PSB. These data will greatly assist the interpretation of single high diagnostic anti-PT IgG titres by allowing an estimate of the positive predictive value, when the number of days post-immunization and prevalence are known or assumed.
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- Diagnostics, typing and identification
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Genotype analysis of Porphyromonas gingivalis fimA in Korean adults using new primers
More LessStrains of Porphyromonas gingivalis, a periodontopathic bacterium, are classified into six genotypic variants based on nucleotide sequence differences in the fimA gene encoding FimA. A PCR assay using primer sets specific for each genotype has demonstrated that the most predominant fimA genotype in periodontitis patients is type II, which is now commonly referred to as the periodontitis-associated fimA genotype of P. gingivalis. However, the potential for false type II fimA positives caused by cross-hybridization of type II fimA-specific primers with type Ib fimA has complicated the genotyping. A previous study developed new primers that specifically amplified only the DNA fragment of type II fimA. The aim of the present study was to assess the prevalence of P. gingivalis fimA genotypes in Korean adults and to reconfirm the relationship between type II fimA and periodontitis using the new primers. Among 412 Korean adults, P. gingivalis was detected in 97.5 % of patients and 57.8 % of healthy subjects. Type II fimA was the most widely distributed type among healthy and periodontitis subjects. Organisms with types II, Ib and IV fimA had a significant frequency of occurrence in periodontitis subjects. Statistical analysis, however, revealed that a more significant correlation was found between periodontitis and the occurrence of type Ib fimA.
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Molecular genotyping of Acinetobacter spp. isolated in Arizona, USA, using multilocus PCR and mass spectrometry
Acinetobacter spp. are a diverse group of Gram-negative bacteria frequently implicated in nosocomial infections. Genotypic methods have been instrumental in studying Acinetobacter, but few offer high resolution, rapid turnaround time, technical ease and high inter-laboratory reproducibility, which has hampered understanding of disease incidence, transmission patterns and diversity within this genus. Here, we further evaluated multilocus PCR electrospray ionization/mass spectrometry (PCR/ESI-MS), a method that is simple and robust, and provides both species characterization and strain-level resolution of Acinetobacter spp. on a single platform. We examined 125 Acinetobacter isolates from 21 hospitals, laboratories and medical centres spanning four counties in Arizona, USA, using PCR/ESI-MS. We compared PCR/ESI-MS with an in-house amplified fragment length polymorphism (AFLP) genotyping scheme. PCR/ESI-MS demonstrated that Acinetobacter spp. from Arizonan hospitals had similar species and strain distributions to other US civilian hospitals. Furthermore, we showed that the PCR/ESI-MS and AFLP genotypes were highly congruent, with the former having the advantages of robust inter-laboratory reproducibility, rapid turnaround time and simple experimental set-up and data analysis. PCR/ESI-MS is an effective and high-throughput platform for strain typing of Acinetobacter baumannii and for identification of other Acinetobacter spp., including the emerging nosocomial pathogens Acinetobacter pittii and Acinetobacter nosocomialis.
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Performance of the Vitek MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry system for identification of Gram-positive cocci routinely isolated in clinical microbiology laboratories
More LessWe evaluated the performance of the Vitek MS for identification of Gram-positive cocci routinely isolated in clinical microbiology laboratories. With a total of 424 well-characterized isolates, the results of the Vitek MS were compared to those of conventional methods and 16S rRNA gene sequencing. The Vitek MS correctly identified 97.9 % of the isolates tested to species level. The Vitek MS correctly identified the species of 97.2 % of the staphylococci (95.9 % of coagulase-negative staphylococci), 97.8 % of the streptococci, and 100 % of the enterococci. For the identification of Gram-positive cocci isolates, the overall concordance rate between conventional identification and the Vitek MS was 94.5 %. The Vitek MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) system can be a reliable and rapid method for the identification of most relevant Gram-positive cocci. In addition, expanding the database of the Vitek MS, especially for coagulase-negative staphylococci, is needed to enhance the performance of the Vitek MS.
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- Antimicrobial agents and chemotherapy
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Antibacterial and antibiofilm effects of iron chelators against Prevotella intermedia
More LessPrevotella intermedia, a major periodontopathogen, has been shown to be resistant to many antibiotics. In the present study, we examined the effect of the FDA-approved iron chelators deferoxamine (DFO) and deferasirox (DFRA) against planktonic and biofilm cells of P. intermedia in order to evaluate the possibility of using these iron chelators as alternative control agents against P. intermedia. DFRA showed strong antimicrobial activity (MIC and MBC values of 0.16 mg ml−1) against planktonic P. intermedia. At subMICs, DFRA partially inhibited the bacterial growth and considerably prolonged the bacterial doubling time. DFO was unable to completely inhibit the bacterial growth in the concentration range tested and was not bactericidal. Crystal violet binding assay for the assessment of biofilm formation by P. intermedia showed that DFRA significantly decreased the biofilm-forming activity as well as the biofilm formation, while DFO was less effective. DFRA was chosen for further study. In the ATP-bioluminescent assay, which reflects viable cell counts, subMICs of DFRA significantly decreased the bioactivity of biofilms in a concentration-dependent manner. Under the scanning electron microscope, P. intermedia cells in DFRA-treated biofilm were significantly elongated compared to those in untreated biofilm. Further experiments are necessary to show that iron chelators may be used as a therapeutic agent for periodontal disease.
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Carbapenem-resistance mechanisms of multidrug-resistant Pseudomonas aeruginosa
More LessClonal dissemination of multidrug-resistant Pseudomonas aeruginosa (MDRPA) is a major concern worldwide. The aim of this study was to explore the mechanisms leading to the carbapenem resistance of an MDRPA clone. Isolates were obtained from a surgical wound, sputum, urine and a blood culture. Pulsed-field gel electrophoresis (PFGE) showed high genomic homogeneity of these isolates and confirmed the circulation of an endemic clone belonging to serotype O4. Outer membrane protein (OMP) bands were visualized by SDS-PAGE, meropenem accumulation was measured in a bioassay and integrons were detected by PCR. Efflux pumps were studied for several antimicrobial agents and synergic combinations thereof in the presence or absence of both carbonyl cyanide m-chlorophenylhydrazone (CCCP) and Phe-Arg-β-naphthylamide (PAβN) at final concentrations of 10 and 40 mg l−1, respectively. On OMP electrophoretic profiles, MDRPA showed a reduction of outer membrane porin D (OprD) and PCR demonstrated the presence of a class 1 integron with a cassette encoding aminoglycoside adenyltransferase B (aadB). Meropenem accumulation was slightly higher in bacilli than in the filamentous cells that formed in the presence of antibiotics. Overexpression of the efflux pump MexAB-OprM and a functional MexXY-OprM were detected in all isolates.
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Extended-spectrum β-lactamases in Enterobacteriaceae isolated in Brazil carry distinct types of plasmid-mediated quinolone resistance genes
One hundred and six nalidixic acid-resistant Enterobacteriaceae isolates from two Brazilian hospitals isolated from June to October 2010 were evaluated to characterize the co-existence of plasmid-mediated quinolone resistant (PMQR) and extended-spectrum β-lactamase (ESBL) determinants. The qnr genetic environment was determined by PCR and sequencing. Conjugation and hybridization experiments determined whether qnr-carrying plasmids were self-transferable. The aac(6′)-Ib-cr and qepA genes were also screened. Thirteen qnr-like genes (12.3 %) were identified, with qnrB1 the most common, followed by qnrS1, qnrB2 and qnrB19. No qnrA, qnrC, qnrD or qepA determinant was detected. All qnr-positive strains possessed chromosomal substitutions in gyrase- and topoisomerase-encoding genes and four harboured a aac(6′)-Ib-cr gene. The co-production of bla CTX-M was observed in ten qnr-positive strains. These results indicate the dissemination of PMQR genes shown in clinical isolates from Brazil, and their co-existence with ESBL genes emphasizes the complexity of plasmid-mediated resistance determinants among Enterobacteriaceae.
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Genetic characteristics of bla NDM-1-positive plasmid in Citrobacter freundii isolate separated from a clinical infectious patient
More LessThis study reports an infectious case involving an NDM-1-producing Citrobacter freundii and further explored the potential threat of the bla NDM-1 gene by analysing the characteristics of the NDM-1-encoding plasmid sequence. A bla NDM-1-positive C. freundii with high resistance to carbapenems was separated from a clinical patient suffering from a urinary tract infection. S1 nuclease-based plasmid analysis followed by Southern blot hybridization, a conjugation experiment and electrotransformation confirmed that the bla NDM-1 gene was located on a plasmid. High-throughput sequencing of the bla NDM-1-postive plasmid (pCFNDM-CN) showed that it was a 54 kb IncX-type plasmid and contained a backbone region and a variable region with two β-lactamase genes (bla NDM-1 and bla SHV-12). The NDM-1 composite transposon in the variable region was surrounded by IS26 and IS5-truncated ISAba125, and shared a high sequence similarity to the bla NDM-1 surrounding structure in Acinetobacter spp. Our research suggested that the NDM-1 composite transposon might play an essential role in mobilization of the bla NDM-1 gene from Acinetobacter spp. to Enterobacteriaceae.
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Detection of clonal KPC-2-producing Klebsiella pneumoniae ST258 in Korea during nationwide surveillance in 2011
More LessThis study analysed the characteristics and genetic similarity of recent Klebsiella pneumoniae carbapenemase (KPC-2)-producing Klebsiella pneumoniae isolates from Korea. Recent laboratory surveillance detected an increase in carbapenemase-producing Enterobacteriaceae in Korea. A total of 6 KPC-2-producing K. pneumoniae were identified from 277 Enterobacteriaceae clinical isolates. All were sequence type (ST) 258 and they had the same pulsotype. They had high MICs for carbapenems and multi-drug resistance. TEM-1, SHV-11 and OXA type β-lactamases were detected in all isolates, whereas CTX-M type β-lactamases and plasmid-mediated AmpC β-lactamase (PABL) were not present. A conjugation experiment failed, but bla KPC-2-harbouring plasmids from the six isolates were used to transform Escherichia coli DH5-α by electroporation. Each of the transformants harboured a bla KPC-2-positive approximately 95 kb plasmid, which was typed in the IncFII incompatibility group and co-harboured TEM-1 and OXA-9 β-lactamases. They shared the same restriction profile. This study confirms the emergence of clonal ST258 KPC-2-producing K. pneumoniae in some regions of Korea.
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A 10 year surveillance for antimicrobial susceptibility of Escherichia coli and Klebsiella pneumoniae in community- and hospital-associated intra-abdominal infections in China
Qiwen Yang, Hui Zhang, Yao Wang, Yingchun Xu, Minjun Chen, Robert E. Badal, Hui Wang, Yuxing Ni, Yunsong Yu, Bijie Hu, Ziyong Sun, Wenxiang Huang, Yong Wang, Anhua Wu, Xianju Feng, Kang Liao, Dingxia Shen, Zhidong Hu, Yunzhuo Chu, Juan Lu, Bin Cao, Jianrong Su, Bingdong Gui, Qiong Duan, Shufang Zhang, Haifeng Shao, Haishen Kong, Yunjian Hu and Huifen YeThe objective of this study was to investigate the susceptibility of hospital-associated (HA) and community-associated (CA) Escherichia coli and Klebsiella pneumoniae isolated from patients with intra-abdominal infections (IAIs) in China. From 2002 to 2011, the minimum inhibitory concentrations (MICs) of 12 antibiotics against 3074 E. coli and 1025 K. pneumoniae from 23 centres located in 16 cities were determined by the broth microdilution method. During the 10 year study period, ertapenem, imipenem, amikacin and piperacillin-tazobactam retained high and stable activity against E. coli and K. pneumoniae isolates regardless of whether their source was HA or CA and regardless of their extended-spectrum beta-lactamase (ESBL) production. However, the susceptibility of E. coli to cephalosporins and ampicillin-sulbactam decreased dramatically during the 10 years, especially for the CA isolates. Fluoroquinolones showed low activity against E. coli. During the whole study period, the ESBL rates for E. coli isolates from IAIs increased from 36.1 % in 2002–2003 to 68.1 % in 2010–2011 (P<0.001). Correspondingly, the ESBL rates in HA isolates increased from 52.2 % in 2002–2003 to 70.0 % in 2010–2011 (P = 0.001), and in CA isolates from 19.1 % in 2002–2003 to 61.6 % in 2010–2011 (P<0.001). The ESBL-positive rate in K. pneumoniae remained between 30.1 and 39.3 % of the total isolates with no significant change during the 10 years. In conclusion, carbapenems retained the highest susceptibility rates against HA and CA E. coli and K. pneumoniae. High prevalence of ESBL in HA E. coli and fast-growing resistance in CA E. coli severely limit the empirical use of the third- and fourth-generation cephalosporins in the therapy of IAIs.
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- Clinical microbiology and virology
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Rapid detection of Clostridium difficile toxins from stool samples using real-time multiplex PCR
More LessIn this study, a total of 650 stool samples were tested to show that our method is capable of detecting four Clostridium difficile genes; tcdA, tcdB, encoding toxin A (TcdA) and toxin B (TcdB), and the binary toxin C. difficile transferase genes (cdtA and/or cdtB) encoding CDT toxin. Besides detecting the targeted C. difficile genes, our method can be used to detect the presence of any inhibitory components in the PCR. This assay, combined with a selective culture medium, such as the chromID™ C. difficile, can be applied directly for screening C. difficile-associated disease. The PCR-based assay developed here is rapid (4 h per 21 stool samples) and accurate in diagnosing C. difficile infection, 100 % assay sensitivity and negative predictive value (NPV) were obtained. However, the assay specificity of 99.1 % and positive predictive value (PPV) of 94.9 % were slightly lower than the optimal value of 100 %. The assay protocol outlined here can be used as a rapid screening tool to assist infection control units and in managing infected patients by reducing the number of patients requiring isolation and extended hospitalization. Rapid detection can prevent unnecessary antibiotic therapy and potentially reduce the spread of infection by emerging hypervirulent C. difficile strains.
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- Case reports
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Non-toxigenic Vibrio cholerae bacteraemia: case report and review of the literature
Vibrio cholerae is a serious public health problem worldwide, but in the UK, V. cholerae infections are rare. Here, we report a case of V. cholerae bacteraemia in an elderly patient. To our knowledge, this is the first non-travel-related V cholerae bacteraemia in the UK.
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Salmonella enterica serovar Minnesota urosepsis in a patient with Crohn’s disease in the absence of recent or current gastrointestinal symptoms
Salmonella enterica serovar Minnesota is a rarely isolated organism in clinical samples mainly grown from stool cultures. Sepsis due to Salmonella is known in severely immunocompromised patients, but so far urosepsis due to S. enterica serovar Minnesota has not been described. We report a case of a 31-year-old patient suffering from Crohn’s disease treated with infliximab and azathioprine, in whom was implanted a double-J ureteric catheter for urolithiasis. The patient presented with urinary tract infection and severe sepsis. S. enterica serovar Minnesota was grown from urine and blood cultures. After empiric antimicrobial treatment with meropenem and vancomycin, treatment was changed to ceftriaxone. Antimicrobial treatment was continued for a total of 3 weeks without evidence of Salmonella recurrence on follow-up visits. Salmonella spp. rarely cause urinary tract infection and sepsis. However, in immunocompromised patients, non-typhoidal salmonellosis merits a thorough clinical and microbiological evaluation.
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- Correspondence
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Identification of Staphylococcus aureus from enriched nasal swabs within 24 h is improved with use of multiple culture media
More LessNasal carriage of Staphylococcus aureus is commonly evaluated via culture-based methods. We found that parallel use of two media, Baird-Parker and CHROMagar™ Staph aureus, increased detection of S. aureus from a healthy population by 29 %. We suggest use of both media for optimal identification of S. aureus from healthy cohorts.
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- Special Collection: Clostridium Difficile
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- Editorial
- Review
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Gut solutions to a gut problem: bacteriocins, probiotics and bacteriophage for control of Clostridium difficile infection
More LessClostridium difficile infection (CDI) is a major cause of morbidity and mortality among hospitalized patients and imposes a considerable financial burden on health service providers in both Europe and the USA. The incidence of CDI has dramatically increased in recent years, partly due to the emergence of a number of hypervirulent strains. The most commonly documented risk factors associated with CDIs are antibiotic usage leading to alterations of the gut microbiota, age >65 years and long-term hospital stay. Since standard therapies for antibiotic-associated diarrhoea and CDI have limited efficacy, there is now an urgent need for alternative therapeutics. In this review, we outline the current state of play with regard to the potential of gut-derived bacteriocins, probiotics and phage to act as antimicrobial agents against CDI in the human gut.
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- Papers
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Expression and display of Clostridium difficile protein FliD on the surface of Bacillus subtilis spores
More LessThe endospores of Bacillus subtilis can serve as a tool for surface presentation of heterologous proteins. The unique properties of the spore protective layers make them perfect vehicles for orally administered vaccines. In this study, we successfully displayed a fragment of Clostridium difficile FliD protein on the surface of B. subtilis spores using the CotB, CotC, CotG and CotZ spore coat proteins. The presence of the fusion proteins in the spore coat was verified by Western blotting and immunofluorescence microscopy. The amount of recombinant proteins was assessed by a dot-blot technique. C. difficile is one of the most common infectious agents in nosocomial infections and is especially associated with antibiotic therapies. FliD is a flagellar cap protein of C. difficile and is known to be one of the immunogenic surface antigens of this bacterium. Therefore, its use in vaccine formulations gives a good perspective for successful immunization with a FliD-based vaccine. The recombinant spores presented here may be good candidates for C. difficile oral vaccines.
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Surface-layer (S-layer) of human and animal Clostridium difficile strains and their behaviour in adherence to epithelial cells and intestinal colonization
Clostridium difficile is a frequent cause of severe, recurrent post-antibiotic diarrhoea and pseudomembranous colitis. The surface layer (S-layer) is the predominant outer surface component of C. difficile which is involved in pathogen–host interactions critical to pathogenesis. In this study, we characterized the S-layer protein A (SlpA) of animal and human strains belonging to different PCR-ribotypes (PR) and compared the in vitro adherence and in vivo colonization properties of strains showing different SlpA variants. Since each SlpA variant has been recently associated with an S-layer cassette, we were able to deduce the cassette for each of our strains. In this study, an identity of 99–100 % was found among the SlpA of isolates belonging to PR 012, 014/020, 045 and 078. One exception was the SlpA of a poultry isolate, PR 014/020, which showed 99 % identity with that of strain 0160, another PR 014/020 which contains an S-layer cassette 6. Interestingly, this cassette has also been found in a PR 018 strain, an emerging virulent type currently predominant in Italy. Five other SlpA variants (v014/020a–e) were identified in strains PR 014/020. In vitro adherence assays and in vivo colonization experiments were performed on five PR 014/020 strains: human 1064 (v014/020e), human 4684/08 (v014/020b), human IT1106 (v078a), poultry P30 (v014/020d) and poultry PB90 (v014/020b) strains. Adhesion assays indicate that C. difficile strains vary in their capacity to adhere to cells in culture and that adhesion seems to be independent of the SlpA variant. Colonization properties were assessed in vivo using a dixenic mouse model of colonization. The kinetics of faecal shedding and caecal colonization were similar when human 4684/08 (v014/020b) strain was compared with human 1064 (v014/020e) and poultry PB90 (v014/020b) strain. In contrast, poultry P30 (v014/020d) strain outcompeted both human 4684/08 (v014/020b) and IT1106 (v078a) strains and its adherence to caeca at day 7 was significantly higher. The peculiar characteristics of C. difficile P30 seem to advantage it in colonizing the intestinal mice niche, increasing its ability to compete and adapt. The results obtained underline the need of an increased attention to the genetic evolution of C. difficile to prevent and limit the consequences of the emergence of increasingly virulent strains.
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Systemic antibody responses induced by a two-component Clostridium difficile toxoid vaccine protect against C. difficile-associated disease in hamsters
Clostridium difficile infection (CDI) has been identified as the leading cause of nosocomial diarrhoea and pseudomembranous colitis associated with antibiotic therapy. Recent epidemiological changes as well as increases in the number of outbreaks of strains associated with increased virulence and higher mortality rates underscore the importance of identifying alternatives to antibiotics to manage this important disease. Animal studies have clearly demonstrated the roles that toxins A and B play in gut inflammation as well as diarrhoea; therefore it is not surprising that serum anti-toxin A and B IgG are associated with protection against recurrent CDI. In humans, strong humoral toxin-specific immune responses elicited by natural C. difficile infection is associated with recovery and lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. The first generation of C. difficile vaccine that contained inactivated toxin A and B was found to be completely protective against death and diarrhoea in the hamster C. difficile challenge model. When tested in young healthy volunteers in Phase I clinical trials, this investigational vaccine was shown to be safe and immunogenic. Moreover, in a separate study this vaccine was able to prevent further relapses in three out of three patients who had previously suffered from chronic relapsing C. difficile-associated diarrhoea. Herein we examined the immunogenicity and protective activity of a next-generation Sanofi Pasteur two-component highly purified toxoid vaccine in a C. difficile hamster model. This model is widely recognized as a stringent and relevant choice for the evaluation of novel treatment strategies against C. difficile and was used in preclinical testing of the first-generation vaccine candidate. Intramuscular (i.m.) immunizations with increasing doses of this adjuvanted toxoid vaccine protected hamsters from mortality and disease symptoms in a dose-dependent manner. ELISA measurements of pre-challenge sera showed that the median anti-toxin A and anti-toxin B IgG titres in the group of surviving animals were significantly higher than the median values in the group of animals that did not survive challenge. Assessment of the neutralizing activity of these sera revealed a statistically significant difference between the levels of both toxin A and toxin B neutralizing titres in protected versus unprotected animals as the median anti-toxin A and anti-toxin B neutralizing titres from surviving animals were higher than the median values from animals that succumbed to challenge. Statistically significant correlations between the toxin-specific binding titres and toxin neutralizing titres were seen for both toxin A and toxin B responses. The role of circulating anti-toxin antibodies in immunity against disease was evaluated by passive transfer of immune sera against C. difficile toxoids to naïve hamsters. Passively immunized animals were protected against morbidity and mortality associated with C. difficile challenge. Taken together, these results indicate the ability of i.m. immunization with inactivated toxins A and B to induce robust dose-dependent anti-toxin A and anti-toxin B IgG responses, the principal role of circulating anti-toxin antibody in immunity against disease and that antibody toxin binding and neutralization titres can serve as correlates of protection in the hamster challenge model of C. difficile.
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Germination efficiency of clinical Clostridium difficile spores and correlation with ribotype, disease severity and therapy failure
P. Moore, L. Kyne, A. Martin and K. SolomonSpore germination is an important part of the pathogenesis of Clostridium difficile infection (CDI). Spores are resistant to antibiotics, including those therapeutically administered for CDI and strains with a high germination rate are significantly more likely to be implicated in recurrent CDI. The role of germination efficiency in cases of refractory CDI where first-line therapy fails remains unclear. We investigated spore germination efficiencies of clinical C. difficile isolates by measuring drop in OD600 and colony forming efficiency. Ribotype 027 isolates exhibited significantly higher germination efficiencies in the presence of 0.1 % (w/v) sodium taurocholate (51.66±8.75 %; 95 % confidence interval (CI) 47.37–55.95 %) than ribotype 106 (41.91±8.35 %; 95 % CI 37.82–46 %) (P<0.05) and ribotype 078 (42.07±8.57 %, 95 % CI 37.22–46.92 %) (P<0.05). Spore outgrowth rates were comparable between the ribotype groups but the exponential phase occurred approximately 4 h later in the absence of sodium taurocholate. Spore germination efficiencies for isolates implicated in severe CDI were significantly higher (49.68±10.00 %, 95 % CI 47.06–52.30 %) than non-severe CDI (40.92±9.29 %, 95 % CI 37.48–44.36 %); P<0.01. Germination efficiencies were also significantly higher in recurrent CDI or when metronidazole therapy failed than when therapy was successful [(49.00±10.49 %, 95 % CI 46.25–51.75 %) versus (41.42±9.43 %, 95 % CI 37.93–44.91 %); P<0.01]. This study suggests an important link between C. difficile spore germination, CDI pathogenesis and response to treatment; however, further work is warranted before the complex interplay between germination dynamics and CDI outcome can be fully understood.
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Cellular uptake of Clostridium difficile TcdA and truncated TcdA lacking the receptor binding domain
More LessThe combined repetitive oligopeptides (CROPs) of Clostridium difficile toxins A (TcdA) and B (TcdB) induce clathrin-mediated endocytosis of the toxins. Inconsistently, CROP-truncated TcdA1–1874 is also capable of entering host cells and displaying full cytotoxic properties although with less potency. Pre-incubation of cells with isolated CROPs, however, reconstitutes the reduced uptake of TcdA1–1874 to the level of the full-length toxin. We believe that TcdA exhibits an additional binding motif beyond the C-terminally located CROP domain, which might interact with cellular receptor structures that are associated with alternative internalization pathways. This study therefore evaluated endocytosis routes of CROP-dependent cellular uptake for TcdA and CROP-independent cellular uptake for TcdA1–1874. Clathrin knockdown or inhibition with chlorpromazine affected subsequent internalization of TcdA and TcdA1–1874, although only to some extent, arguing for alternative, clathrin-independent endocytosis routes. Inhibition of dynamin, a GTPase essentially involved in clathrin-mediated endocytosis as well as in various clathrin-independent uptake mechanisms, affected uptake of TcdA to the same extent as clathrin inhibition. In contrast, uptake of TcdA1–1874 was almost completely eliminated in dynamin-inhibited cells. Thus, clathrin-independent uptake of TcdA1–1874 presumably depends on dynamin. These findings demonstrate that the toxins are endocytosed via complex pathways involving clathrin and dynamin, putatively enabling them to adapt to mechanisms of various cell types. With regard to the emergence of C. difficile strains producing C-terminally truncated toxins, this study emphasizes the relevance of elucidating toxin uptake as a prerequisite for the development of toxin intervention strategies.
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Isolation of Clostridium difficile from faecal specimens – a comparison of chromID C. difficile agar and cycloserine-cefoxitin-fructose agar
More LessThe culture of toxigenic Clostridium difficile from stool specimens is still seen as the gold standard for the laboratory diagnosis of C. difficile infection (CDI). bioMérieux have released ChromID Cdiff chromogenic agar (CDIF) for the isolation and identification of C. difficile in 24 h. In this study, we compared CDIF to pre-reduced cycloserine-cefoxitin-fructose agar with sodium taurocholate (TCCFA) in the examination of glutamate dehydrogenase-positive faecal specimens that were either GeneOhm positive or negative, using direct culture or culture following alcohol shock. Direct culture on CDIF had a sensitivity of 100 % and recovery of 94 % while for TCCFA these were 87 % and 82 %, respectively. For GeneOhm-positive alcohol-shocked faecal samples, sensitivity and recovery on CDIF was similar to direct culture while on TCCFA they were about 10 % higher. For direct culture, there was a significant difference between growth on CDIF at 24 h and TCCFA at 48 h (P = 0.001) and between the two media at 48 h (P<0.001). A total of 142 strains of C. difficile were recovered in pure culture from all GeneOhm-positive samples used in this study and 11 (7.7 %) of these were A−B−CDT− and may represent mixed infections of toxigenic and non-toxigenic C. difficile. The most dominant ribotype was UK 014 (14.7 %) followed by 002 (11.9 %) and 020 (11.9 %), and 36 % of toxigenic isolates, including an A−B+CDT− strain, could not be assigned a UK ribotype. CDIF outperformed pre-reduced TCCFA by negating the need for alcohol shock treatment and by giving a time saving of 24 h in the isolation of C. difficile. CDIF plates were also more selective than TCCFA and C. difficile colonies were easy to identify and subculture prior to strain typing.
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Advances in molecular surveillance of Clostridium difficile in Bulgaria
The increasing incidence of Clostridium difficile infection (CDI) in Bulgaria has indicated the need to implement better surveillance approaches. The aim of the present work was to improve the current surveillance of CDI in Bulgaria by introducing innovative methods for identification and typing. One hundred and twenty stool samples obtained from 108 patients were studied over 4 years from which 32 C. difficile isolates were obtained. An innovative duplex EvaGreen real-time PCR assay based on simultaneous detection of the gluD and tcdB genes was developed for rapid C. difficile identification. Four toxigenic profiles were distinguished by PCR: A+B+CDT− (53.1 %, 17/32), A−B+CDT− (28.1 %, 9/32), A+B+CDT+ (9.4 %, 3/32) and A−B−CDT− (9.4 %, 3/32). PCR ribotyping and multilocus variable number of tandem repeat analysis (MLVA7) were used for molecular characterization of the isolates. In total, nine distinct ribotypes were confirmed and the most prevalent for Bulgarian hospitals was 017 followed by 014/020, together accounting for 44 % of all isolates. Eighteen per cent of the isolates (6/32) did not match any of the 25 reference ribotypes available in this study. Twenty-four MLVA7 genotypes were detected among the clinical C. difficile isolates, distributed as follows: five for 017 ribotype, two for 014/020, 001, 002, 012 and 046 each, and one each for ribotypes 023, 070 and 078. The correlation between the typing methods was significant and allowed the identification of several clonal complexes. These results suggest that most C. difficile cases in the eight Bulgarian hospitals studied were associated with isolates belonging to the outbreak ribotypes 017 and 014/20, which are widely distributed in Europe. The real-time PCR protocol for simultaneous detection of gluD and tcdB proved to be very effective and improved C. difficile identification and confirmation of clinical C. difficile isolates.
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Contamination of ready-to-eat raw vegetables with Clostridium difficile in France
More LessThe presence of Clostridium difficile in food like shellfish, vegetables and meat has been reported in several publications during the past few years. The objective of this study was to assess the prevalence of ready-to-eat raw vegetables contaminated with C. difficile in France. One hundred and four ready-to-eat salads and vegetables were studied. Toxigenic C. difficile strains were isolated in three samples (2.9 %): two ready-to-eat salads (one heart of lettuce and one lamb’s lettuce salad) and one portion of pea sprouts. The strains belonged to three different PCR ribotypes: 001, 014/020/077 and 015. The detection thresholds for vegetative cells and spores cells varied between 1 and 3 c.f.u. in 20 g salad and between 6 and 15 c.f.u. in 20 g salad, respectively, for the method employed.
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Determination of the attP and attB sites of phage ϕCD27 from Clostridium difficile NCTC 12727
More LessThe attP region of the Clostridium difficile phage ϕCD27 was identified, located immediately downstream of the putative recombinase. The phage could integrate into two specific sites (attB) in the C. difficile genome, one of which was in an open reading frame encoding a putative ATPase of an ABC transporter and the other in an open reading frame encoding a putative ATPase of the flagella protein export apparatus. The prophage was capable of excision and formation of a circular molecule and phages were spontaneously released at a low frequency during growth. Infection and lysogeny of a C. difficile strain previously shown to be sensitive to ϕCD27 were demonstrated, leading to a reduction in toxin production. Finally, a putative repressor was identified which is likely to be involved in maintaining lysogeny in these strains.
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Diversity of cwp loci in clinical isolates of Clostridium difficile
An increased incidence of Clostridium difficile infection (CDI) is associated with the emergence of epidemic strains characterized by high genetic diversity. Among the factors that may have a role in CDI is a family of 29 paralogues, the cell-wall proteins (CWPs), which compose the outer layer of the bacterial cell and are likely to be involved in colonization. Previous studies have shown that 12 of the 29 cwp genes are clustered in the same region, named after slpA (cwp1), the slpA locus, whereas the remaining 17 paralogues are distributed throughout the genome. The variability of 14 of these 17 cwp paralogues was determined in 40 C. difficile clinical isolates belonging to six of the currently prevailing PCR ribotypes. Based on sequence conservation, these cwp genes were divided into two groups, one comprising nine cwp loci having highly conserved sequences in all isolates, and the other five loci showing low genetic conservation among isolates of the same PCR ribotype, as well as between different PCR ribotypes. Three conserved CWPs, Cwp16, Cwp18 and Cwp25, and two variable ones, Cwp26 and Cwp27, were characterized further by Western blot analysis of total cell extracts or surface-layer preparations of the C. difficile clinical isolates. Expression of genetically invariable CWPs was well conserved in all isolates, whilst genetically variable CWPs were not always expressed at comparable levels, even in strains containing identical sequences but belonging to different PCR ribotypes. This is the first report on the distribution and variability of a number of genes encoding CWPs in C. difficile.
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Mortality in patients with Clostridium difficile infection correlates with host pro-inflammatory and humoral immune responses
Host anti-toxin immune responses play important roles in Clostridium difficile disease and outcome. The relationship between host immune and inflammatory responses during severe C. difficile infection (CDI) and the risk of mortality has yet to be defined. We aimed to investigate the host systemic IgG anti-toxin immune responses, the in vitro cytotoxicity of the infecting C. difficile ribotyped strain, and the host inflammatory markers and their relationship to CDI disease severity and risk of mortality. Inflammatory markers, co-morbidities and CDI outcomes were recorded in a prospective cohort of 150 CDI cases. Serum anti-cytotoxin A (TcdA) and anti-TcdB IgG titres were measured by ELISA and the infecting C. difficile isolate was ribotyped and the in vitro cytotoxin titre assessed. A low median anti-TcdA IgG titre was significantly associated with 30-day all-cause mortality (P<0.05). Ribotype 027 isolates were significantly more toxinogenic than other ribotypes (P<0.00001). High cytotoxin titres correlated with increased inflammatory markers but also higher anti-TcdA and -TcdB (P<0.05) IgG responses resulting in a lower risk of mortality. On multivariate analysis, predictors of mortality were peak white cell count >20×109 l−1 [odds ratio (OR) 11.53; 95 % confidence interval (CI) 2.38–55.92], creatinine concentration >133 µmol l−1 (OR 6.54; 95 % CI 1.47–29.07), Horn’s index >3 (OR 4.09; 95 % CI 0.76–22.18) and low anti-TcdA IgG (OR 0.97; 95 % CI 0.95–0.99), but not ribotype, cytotoxin titre or anti-TcdB IgG. Thus, host pro-inflammatory and humoral responses correlate with the cytotoxin titre of the infecting strain and effective anti-toxin immune responses reduce the risk of mortality.
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Clostridium difficile erm(B)-containing elements and the burden on the in vitro fitness
More LessIn Clostridium difficile, resistance to the macrolide-lincosamide-streptogramin B group of antibiotics generally relies on erm(B) genes. In this study, we investigated elements with a genetic organization different from Tn5398, the mobilizable non-conjugative element identified in C. difficile strain 630. Our results suggested that the elements most frequently found in strains isolated during the European surveillance study in 2005 were related to Tn6194, the conjugative transposon recently detected in different C. difficile types, including PCR-ribotype 027. We characterized a Tn6194-like and a novel element rarely found in clinical isolates. A burden on the in vitro fitness of C. difficile was observed after the acquisition of these elements as well as of Tn5398.
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Clostridium difficile infections in South East Scotland: mortality and recurrence in a region without PCR ribotype 027
More LessThree hundred and thirty-five patients with laboratory-confirmed Clostridium difficile infections (CDIs) were studied for epidemiological features, clinical presentation and laboratory markers. They were followed up for 1 year to determine recurrence and mortality. Four hundred and thirty-two episodes were recorded. One year mortality was 41.8 % of which CDI was listed on 20 % of the death certificates. One year recurrence rate was 22.9 %. PCR ribotype 001 was the commonest epidemiological type and ribotype 027 was not detected. High total leucocyte count and low albumin were significantly associated with mortality, as was the absence of a GI-invasive procedure in the 12 weeks preceding CDI diagnosis, probably due to patients being unfit for the procedure. No association with acid suppressants, deletion in the tdcC anti-sigma factor or vancomycin-resistant enterococcus/methicillin-resistant Staphylococcus aureus co-infection was detected. One year mortality was higher in patients who developed recurrent infections (P<0.001). Differences in ribotype were observed in 2.3 %, 11.11 %, 20 % and 32.4 % isolates with time intervals between sampling of 0–20, 21–40, 41–60 and >60 days, respectively, suggesting that the arbitrary cut-off of 28 days to call a repeat infection a reinfection may not be correct in some cases.
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Antimicrobial susceptibility of animal and human isolates of Clostridium difficile by broth microdilution
A total of 188 human (n = 92) and animal (n = 96) isolates of Clostridium difficile of different PCR ribotypes were screened for susceptibility to 30 antimicrobials using broth microdilution. When comparing the prevalence of antimicrobial resistance, the isolates of animal origin were significantly more often resistant to oxacillin, gentamicin and trimethoprim/sulfamethoxazole (P<0.01). The most significant difference between the animal and human populations (P = 0.0006) was found in the level of imipenem resistance, with a prevalence of 53.3 % in isolates of human origin and 28.1 % in isolates of animal origin. Overall, the results show similar MICs for the majority of tested antimicrobials for isolates from human and animal sources, which were collected from the same geographical region and in the same time interval. This supports the hypothesis that C. difficile could be transmissible between human and animal hosts. Resistant isolates have been found in all animal species tested, including food and companion animals, and also among non-toxigenic isolates. The isolates of the most prevalent PCR ribotype 014/020 had low resistance rates for moxifloxacin, erythromycin, rifampicin and daptomycin, but a high resistance rate for imipenem. Multiresistant strains were found in animals and humans, belonging to PCR ribotypes 012, 017, 027, 045, 046, 078 and 150, and also to non-toxigenic strains of PCR ribotypes 010 and SLO 080.
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- Case Report
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Two cases of fulminant colitis due to binary toxin-positive Clostridium difficile that are not PCR ribotype 027 or type 078
More LessTwo cases of fulminant colitis due to Clostridium difficile occurred within ten weeks of each other on the same ward of a hospital in Japan. The patients died 2 and 4 days after the onset of colitis. C. difficile isolates obtained from both patients were toxin A-positive, toxin B-positive and binary toxin-positive. These isolates yielded identical results by both PCR ribotyping and slpA sequence typing. However, the banding patterns and slpA sequences of the isolates differed from those of PCR ribotype 027, as well as those of PCR ribotype 078. The tcdC sequences of the isolate differed from those of C. difficile 027, but a single base-pair deletion at position 117 and an 18 bp deletion, both of which were identical to the sequence of the reference strain of 027, were found. This type may be a new hypervirulent strain, but further studies of the epidemiology and pathogenicity of the strain are needed.
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Volumes and issues
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Volume 73 (2024)
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Volume 72 (2023 - 2024)
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Volume 71 (2022)
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Volume 70 (2021)
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Volume 14 (1981)
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Volume 11 (1978)
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Volume 9 (1976)
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Volume 5 (1972)
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Volume 4 (1971)
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Volume 3 (1970)
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Volume 2 (1969)
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Volume 1 (1968)