Comparative genomic analysis of <Emphasis Type="Italic">Clostridium difficile</Emphasis> ribotype 027 strains including the newly sequenced strain NCKUH-21 isolated from a patient in Taiwan

Background

Clostridium difficile is a Gram-positive anaerobe and the leading cause of antibiotic-associated diarrhea worldwide. The emergence of ribotype 027 (RT027) strains is associated with increased incidence of infection and mortality. To further understand the relationship between C. difficile NCKUH-21, a RT027 strain isolated from a patient in Taiwan, and other RT027 strains, we performed whole-genome shotgun sequencing on NCKUH-21 and comparative genomic analyses.

Results

The genome size, G+C content, and gene number for the NCKUH-21 strain were determined to be similar to those for other C. difficile strains. The core genome phylogeny indicated that the five RT027 strains R20291, CD196, NCKUH-21, BI1, and 2007855 formed a clade. A pathogenicity locus, tcdR-tcdB-tcdE-orf-tcdA-tcdC, was conserved in the genome. A genomic region highly similar to the Clostridium phage \(\upvarphi\)CD38-2 was present in the NCKUH-21 strain but absent in the other RT027 strains and designated as the prophage \(\upvarphi\)NCKUH-21. The prophage \(\upvarphi\)NCKUH-21 genes were significantly higher in G+C content than the other genes in the NCKUH-21 genome, indicating that the prophage does not match the base composition of the host genome.

Conclusions

This is the first whole-genome analysis of a RT027 C. difficile strain isolated from Taiwan. Due to the high identity with \(\upvarphi\)CD38-2, the prophage identified in the NCKUH-21 genome has the potential to regulate toxin production. These results provide important information for understanding the pathogenicity of RT027 C. difficile in Taiwan.

     

Translocation of Clostridium difficile toxin B across polarized Caco-2 cell monolayers is enhanced by toxin A

Clostridium difficile is the etiological agent of antibiotic-associated diarrhea; the most common form of nosocomial infectious diarrhea. The basis for the shock-like systemic symptoms observed in severe cases of this infection are not known. It is hypothesized that the invasion of C difficile toxins A and/or B from the gut mucosa may contribute to these symptoms.A polarized tissue culture model employing Caco-2 cells grown on transwell inserts was established to study the translocation of purified C difficile toxins A and B. C difficile toxins were ¹²⁵I labelled and inoculated onto confluent polarized Caco-2 cell monolayers to study translocation dynamics. Electrical resistance measurements were used to monitor monolayer confluence and tight junction integrity. Samples were taken from the apical and basal sides of the insert, as well as the insert itself, and tested using the human foreskin fibroblasts cell cytotoxicity assay to monitor partitioning of the radiolabelled toxins. Toxin A produced a 50% reduction in electrical resistance in 3 h whereas the same concentration of toxin B required at least 7 h to achieve the same effect. Both toxins A and B were able to translocate across confluent monolayers of Caco-2 cells. The combination of toxin A and B together was synergistic with respect to promoting the translocation of toxin B. Although the addition of toxin A resulted in a 100% increase in the amount of toxin B able to translocate, no increases in toxin A translocation were observed. These findings suggest a model of pathogenesis in which C difficile toxin A facilitates the translocation of toxin B from the gut into submucosal areas where it may play a role in inflammatory damage.Key Words: Clostridium difficile, Polarized-monolayers, Toxin A, Toxin B, TranslocationClostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea and colitis. It is reported to be responsible for 10% to 25% of all cases of antibiotic-associated diarrhea, for 60% of antibiotic-associated colitis and for almost all cases of pseudomembranous colitis (1,2). C difficile is able to overgrow in the intestine of patients whose normal microflora has been disrupted by antimicrobial or antineoplastic drugs (3,4). Despite the proliferation in the bowel, studies have shown that the organism is noninvasive, and systemic spread of the organism has not been reported. In severe cases of infection, patients with pseudomembranous colitis may experience systemic complications resulting in death (5). Presently, the basis for these systemic symptoms are not known. Studies have shown that only strains capable of producing both toxins A and B have been associated with clinical disease (6). Toxin A is a 308 kDa enterotoxin responsible for promoting intestinal fluid secretion and inflammation. It is cytotoxic and can cause rounding of many mammalian cells (7-10). Toxin B is a 270 kDa protein. Although not enterotoxic, it has a 1000-fold greater cytotoxicity than toxin A (6,8,9,11). Despite extensive research on the two toxins using animal and biological models, the model of pathogenesis is still incomplete. Both toxins A and B are required for intestinal damage in animal studies, however, the exact role of toxin B in the disease progression is unclear. Some animal studies have shown that systemic injection of minute amounts of toxin B are fatal (12-14).Despite having large molecular sizes, both toxins are internalized and act intracellularly (15,16). Once internalized, the toxins glycosylate small GTP-binding glycosylate proteins of the Rho subfamily at residue threonine 37 (17). The glycosylate process leads to an inactivation of the signalling molecules and results in a breakdown of the actin filament network, an integral network for maintaining the cytoskeletal structure of cells. The resultant depolymerization of F actin to G actin also leads to a compromise in the gastrointestinal tight junctions (18,19). Because gastrointestinal tight junctions play key roles in regulating cellular trafficking and maintenance of bowel integrity, disruption of their function could potentially lead to unregulated passage of foreign molecules into the submucosa from the gut lumen. To our knowledge, there are no published data to demonstrate whether toxins A and/or B can cross the intestinal epithelium to gain access to the submucosa. It is plausible that systemic symptoms seen in severe cases of disease may be caused by the translocation of C difficile toxins from the gut into the submucosa. Once access is made into the submcosa, the toxins would be free to induce inflammatory mediated submucosal damage and/or to act systemically.The aim of the present study was to use a polarized tissue culture model employing Caco-2 cells grown on transwell inserts to determine if either toxin A or B of C difficile can alone or in combination translocate across an intact monolayer.     

The role of toxin A and toxin B in Clostridium difficile-associated disease: Past and present perspectives

Recently, we constructed and characterized isogenic tcdA and tcdB mutants of a virulent Clostridium difficile strain and used a hamster model of disease to demonstrate that toxin B, not toxin A, is essential for virulence of this emerging pathogen. Earlier studies had shown that purified toxin A alone was able to induce C. difficile disease pathology and that purified toxin B was not effective unless it was co-administered with toxin A, suggesting that the toxins act synergistically. In this addendum we discuss this paradigm-shifting conclusion in the context of current strain epidemiology, particularly with respect to naturally occurring toxin A-negative, toxin B-positive isolates and the NAP1/027 epidemic isolates. The role of toxin receptors and how variant toxins might exert their effects is also discussed in relation to the published data. We conclude that it is critical to use the natural infection process to dissect the role of toxins in disease, and that future studies are contingent on such work. The impact and importance of animal models of C. difficile virulence are therefore considered within this frame of reference.Key words: Clostridium difficile, infection, toxin, nosocomial, colitis, antibiotic     

Combination of culture,antigen and toxin detection,and cytotoxin neutralization assay for optimal Clostridium difficile diagnostic testing

BACKGROUND:

There has been a growing interest in developing an appropriate laboratory diagnostic algorithm for Clostridium difficile, mainly as a result of increases in both the number and severity of cases of C difficile infection in the past decade. A C difficile diagnostic algorithm is necessary because diagnostic kits, mostly for the detection of toxins A and B or glutamate dehydrogenase (GDH) antigen, are not sufficient as stand-alone assays for optimal diagnosis of C difficile infection. In addition, conventional reference methods for C difficile detection (eg, toxigenic culture and cytotoxin neutralization [CTN] assays) are not routinely practiced in diagnostic laboratory settings.

OBJECTIVE:

To review the four-step algorithm used at Diagnostic Services of Manitoba sites for the laboratory diagnosis of toxigenic C difficile.

RESULT:

One year of retrospective C difficile data using the proposed algorithm was reported. Of 5695 stool samples tested, 9.1% (n=517) had toxigenic C difficile. Sixty per cent (310 of 517) of toxigenic C difficile stools were detected following the first two steps of the algorithm. CTN confirmation of GDH-positive, toxin A- and B-negative assays resulted in detection of an additional 37.7% (198 of 517) of toxigenic C difficile. Culture of the third specimen, from patients who had two previous negative specimens, detected an additional 2.32% (12 of 517) of toxigenic C difficile samples.

DISCUSSION:

Using GDH antigen as the screening and toxin A and B as confirmatory test for C difficile, 85% of specimens were reported negative or positive within 4 h. Without CTN confirmation for GDH antigen and toxin A and B discordant results, 37% (195 of 517) of toxigenic C difficile stools would have been missed. Following the algorithm, culture was needed for only 2.72% of all specimens submitted for C difficile testing.

CONCLUSION:

The overview of the data illustrated the significance of each stage of this four-step C difficile algorithm and emphasized the value of using CTN assay and culture as parts of an algorithm that ensures accurate diagnosis of toxigenic C difficile.     

Algorithm proposal based on the C. Diff Quik Chek Complete ICT device for detecting Clostridium difficile infection

Objective

To assess a new immunochromatography (ICT) test that detects glutamate dehydrogenase (GDH) antigen and Clostridium difficile toxin A/B simultaneously, and to propose an algorithm for the diagnosis of C. difficile infection (CDI) based on this test.

Methods

We analysed 970 stool samples. Discrepant results between GDH and toxin A/B were resolved using toxigenic culture as the reference.

Results

This test enabled us to obtain a conclusive result in <30 min in 93.8% of the samples. Among the discrepant results (GDH (+)/Toxin A/B (−)), 41.7% (25/60) were found to be toxigenic C. difficile by toxigenic culture.

Conclusion

This test has a high sensitivity and specificity for the diagnosis of CDI.     

Clostridium difficile toxin CDT hijacks microtubule organization and reroutes vesicle traffic to increase pathogen adherence

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by the actions of Rho-glucosylating toxins A and B. Recently identified hypervirulent strains, which are associated with increased morbidity and mortality, additionally produce the actin-ADP–ribosylating toxin C. difficile transferase (CDT). CDT depolymerizes actin, causes formation of microtubule-based protrusions, and increases pathogen adherence. Here we show that CDT-induced protrusions allow vesicle traffic and contain endoplasmic reticulum tubules, connected to microtubules via the calcium sensor Stim1. The toxin reroutes Rab11-positive vesicles containing fibronectin, which is involved in bacterial adherence, from basolateral to the apical membrane sides in a microtubule- and Stim1-dependent manner. The data yield a model of C. difficile adherence regulated by actin depolymerization, microtubule restructuring, subsequent Stim1-dependent Ca²⁺ signaling, vesicle rerouting, and secretion of ECM proteins to increase bacterial adherence.The anaerobe and spore-forming bacterium Clostridium difficile is the causative pathogen of pseudomembranous colitis and antibiotic-associated diarrhea. During recent years, morbidity and mortality of C. difficile infections (CDIs) increased and emerged as a major health threat in developed countries (1).Main pathogenic factors of C. difficile are the Rho-inactivating toxins A and B (2, 3). Recently, so-called hypervirulent strains (e.g., NAP1/027) have been associated with severe courses of CDIs (4). These strains have deletions in a regulatory gene of the pathogenicity locus, are resistant towards fluoroquinolons, and produce the binary toxin C. difficile transferase (CDT). CDT ADP ribosylates actin at arginine-177, thereby inhibiting actin polymerization (5–7).CDT-induced depolymerization of F-actin induces formation of long microtubule-based protrusions, which form a meshwork on the surface of intestinal host cells (8). Clostridia are embedded in this microtubule-based meshwork, resulting in increased host cell adherence.Here, we report that toxin-induced protrusions contain vesicles and tubular ER structures, which are functionally linked to microtubules and to the cell membrane via Stim1. The data indicate that CDT alters the secretory machinery of host cells and reroutes ECM proteins like fibronectin from basolateral to the apical surface of host cells. This redistribution increases adherence and colonization of clostridia in a microtubule-dependent manner.     

In Recurrent C. difficile, the CRP Response to the Primary C. difficile Infection Predicts Whether the Same Strain or a Different Strain will Cause a Second Infection

Background

Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and is associated with an increase in morbidity and mortality. There is a wide variance in disease severity with some patients suffering a single, self-limiting episode of diarrhoea while others suffer more intractable problems with recurrent attacks or toxic dilatation. Numerous different C. difficile ribotypes exist, some of which are considered hypervirulent. The magnitude of toxin production alone is not sufficient to explain the varying virulence of these ribotypes, suggesting the involvement of other mechanisms.

Methods

To test the same patient’s response to infection with different C. difficile ribotypes, we reviewed 45 patients who suffered two episodes of C. difficile infection and determined by ribotyping and MLVA whether the second episode was due to the same strain or a different strain.

Results

Patients harbouring a different strain had significantly higher C-reactive protein (CRP) responses on the first assessed infection (143 mg/L ± 20 vs. 55 ± 9.63, p = 0.0001) and a significantly lower CRP on reinfection (p = 0.048). Same strain patients had a non-significant increase in CRP response on second infection.

Conclusions

This suggests that the inflammatory response to C. difficile is determined by an interaction between host immunobiology, previous exposure and C. difficile strain.     

Clinical and economic consequences of vancomycin and fidaxomicin for the treatment of Clostridium difficile infection in Canada

BACKGROUND:

Clostridium difficile infection (CDI) represents a public health problem with increasing incidence and severity.

OBJECTIVE:

To evaluate the clinical and economic consequences of vancomycin compared with fidaxomicin in the treatment of CDI from the Canadian health care system perspective.

METHODS:

A decision-tree model was developed to compare vancomycin and fidaxomicin for the treatment of severe CDI. The model assumed identical initial cure rates and included first recurrent episodes of CDI (base case). Treatment of patients presenting with recurrent CDI was examined as an alternative analysis. Costs included were for study medication, physician services and hospitalization. Cost effectiveness was measured as incremental cost per recurrence avoided. Sensitivity analyses of key input parameters were performed.

RESULTS:

In a cohort of 1000 patients with an initial episode of severe CDI, treatment with fidaxomicin led to 137 fewer recurrences at an incremental cost of $1.81 million, resulting in an incremental cost of $13,202 per recurrence avoided. Among 1000 patients with recurrent CDI, 113 second recurrences were avoided at an incremental cost of $18,190 per second recurrence avoided. Incremental costs per recurrence avoided increased with increasing proportion of cases caused by the NAP1/B1/027 strain. Results were sensitive to variations in recurrence rates and treatment duration but were robust to variations in other parameters.

CONCLUSIONS:

The use of fidaxomicin is associated with a cost increase for the Canadian health care system. Clinical benefits of fidaxomicin compared with vancomycin depend on the proportion of cases caused by the NAP1/B1/027 strain in patients with severe CDI.     

Comparison of MALDI-TOF MS,gene sequencing and the Vitek 2 for identification of seventy-three clinical isolates of enteropathogens

Objective

This study was performed to evaluate the analytical and practical performance of the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) compared to the sequencing method and the Vitek 2 system for identification of enteropathogens in the clinical microbiology laboratory.

Methods

Ten type strains and 73 clinical isolates of enteropathogens representing eight genera were analyzed by MALDI-TOF MS. All isolates were also characterized by gene sequencing allowing interpretation of the results from MALDI-TOF MS. In addition, MALDI-TOF MS was compared with the Vitek 2 system for the identification of ten isolates of Aeromonas and six of Salmonella.

Results

As previously known, identification between Shigella and Escherichia coli is not possible to distinguish. MALDI-TOF MS produced the correct identifications for all other type strains and clinical isolates to the genus level. Fifteen Campylobacter jejuni, six Campylobacter coli, three Plesiomonas shigelloides, three Yersinia enterocolitica, two Clostridium difficile, one Vibrio parahaemolyticus, one Vibrio fluvialis, and one Vibrio cholera were all correctly identified to the species level. Genus and species identifications of ten Aeromonas and six Salmonella isolates by MALDI-TOF MS were consistent with those by the Vitek 2, but with much less cost and about ten times faster.

Conclusions

This study demonstrates that MALDI-TOF MS is a powerful tool for fast, accurate and low-cost identification of enteropathogens in the clinical microbiology laboratory.     

Evaluation of the clinical value of ELISA based on MPT64 antibody aptamer for serological diagnosis of pulmonary tuberculosis

Background

Antibiotic associated diarrhoea complicates 5?C39% of courses of antibiotic treatment. Major risk factors are increased age and admission to hospital. Of particular importance is C. difficile associated diarrhoea which occurs in about 4% of antibiotic courses and may result in severe illness, death and high healthcare costs. The emergence of the more virulent 027 strain of C. difficile has further heightened concerns. Probiotics may prevent antibiotic associated diarrhoea by several mechanisms including colonization resistance through maintaining a healthy gut flora.

Methods

This study aims to test the hypothesis that administration of a probiotic comprising two strains of lactobacilli and two strains of bifidobacteria alongside antibiotic treatment prevents antibiotic associated diarrhoea. We have designed a prospective, parallel group trial where people aged 65?years or more admitted to hospital and receiving one or more antibiotics are randomly allocated to receive either one capsule of the probiotic or a matching placebo daily for 21?days. The primary outcomes are the frequency of antibiotic associated and C. difficile diarrhoea during 8?C12?weeks follow-up. To directly inform routine clinical practice, we will recruit a sufficient number of patients to demonstrate a 50% reduction in the frequency of C. difficile diarrhoea with a power of 80%. To maximize the generalizability of our findings and in view of the well-established safety record of probiotics, we will recruit a broad range of medical and surgical in-patients from two different health regions within the UK.

Discussion

Antibiotic associated diarrhoea constitutes a significant health burden. In particular, current measures to prevent and control C. difficile diarrhoea are expensive and disrupt clinical care. This trial may have considerable significance for the prevention of antibiotic associated diarrhoea in hospitals.

Trial registration

International Standard Randomised Controlled Trial Number Register ISRCTN70017204.     

First case of autochthonous Clostridium difficile PCR ribotype 027 detected in Spain

Introduction

Clostridium difficile ribotype 027 (Cd027) has caused outbreaks in the United States, Canada, and Europe since 2001. In Spain, the importance of Cd027 is still unknown. In 2007, we began active surveillance of Cd027 to determine its incidence in our hospital.

Methods

From January 2007 to April 2012, isolates of C. difficile by multiplex PCR were studied to detect toxin genes. Binary toxin-positive isolates were characterized using PCR-ribotyping. Cd027 were further characterized by toxino-typing, sequencing of tcdC gene, and MLVA (multilocus-variable-number-tandem-repeat-analysis).

Results

Only 8 strains were Cd027 from 3666 isolates of C. difficile analyzed during the study period. These strains were isolated from 4 patients: a Spanish patient previously hospitalized in the UK, a pregnant laboratory technician, a British tourist, and a Spanish patient without epidemiological antecedents for acquiring Cd027. MLVA typing of Cd027 isolates revealed 4 different patterns. The first patient had 2 episodes of diarrhea caused by different Cd027. The strains from the first episode of patient 1 and the strain from patient 2 were grouped in the same clonal cluster (these cases were previously published as laboratory transmission), while strains from patients 3 and 4 were genetically unrelated to each other, and to the strains from patients 1 and 2.

Conclusion

We report the first finding of an autochthonous case of non-severe Cd027 infection. Our results indicate that Cd027 diarrhea is uncommon in our area, and it appears mainly as imported cases. MLVA typing enables us to distinguish different genotypes among our Cd027 isolates.     

Risk factors for nasopharyngeal carriage of drug-resistant Streptococcus pneumoniae: data from a nation-wide surveillance study in Greece

Background

A nation-wide surveillance study was conducted in Greece in order to provide a representative depiction of pneumococcal carriage in the pre-vaccination era and to evaluate potential risk factors for carriage of resistant strains in healthy preschool children attending daycare centers.

Methods

A study group was organized with the responsibility to collect nasopharyngeal samples from children. Questionnaires provided demographic data, data on antibiotic consumption, family and household data, and medical history data. Pneumococcal isolates were tested for their susceptibility to various antimicrobial agents and resistant strains were serotyped.

Results

Between February and May 2004, from a total population of 2536 healthy children, a yield of 746 pneumococci was isolated (carriage rate 29.41%). Resistance rates differed among geographic regions. Recent antibiotic use in the last month was strongly associated with the isolation of resistant pneumococci to a single or multiple antibiotics. Serotypes 19F, 14, 9V, 23F and 6B formed 70.6% of the total number of resistant strains serotyped.

Conclusion

Recent antibiotic use is a significant risk factor for the colonization of otherwise healthy children's nasopharynx by resistant strains of S pneumoniae. The heptavalent pneumococcal conjugate vaccine could provide coverage for a significant proportion of resistant strains in the Greek community. A combined strategy of vaccination and prudent antibiotic use could provide a means for combating pneumococcal resistance.     

Diagnostic algorithm using a sensitive broth culture method for detection of Clostridium difficile toxin from stool samples

BACKGROUND:

The two-step glutamate dehydrogenase antigencytotoxicity neutralization assay algorithm has been found to be reliable for the diagnosis of toxigenic Clostridium difficile. However, the high sensitivity of the screening method is compromised by the relative low sensitivity of the second step, the direct cytotoxin neutralization assay (DCNA) using a fecal filtrate. The objective of the present study was to compare the DCNA with an indirect cytotoxin neutralization assay (ICNA).

METHODS:

For ICNA, the cytotoxin B of C difficile was obtained from a broth culture of the stools and neutralized according to a standard cytotoxin assay using MRC-5 fibroblast cells.

RESULTS:

A total of 923 stool specimens from adults were tested during a three-month period from June to August 2008. The prevalence of toxigenic C difficile was 13.5%. The sensitivity of the two-step algorithm was 88%. With the ICNA, 12% toxigenic C difficile were detected that were missed by DCNA.

CONCLUSIONS:

The use of broth for the ICNA is convenient, and results in increased sensitivity of detection of toxigenic C difficile. It can be implemented in routine diagnosis.     

Clonal diversity among Burkholderia cepacia complex isolates from cystic fibrosis patients in a reference unit

Introduction

The epidemiology of Burkholderia cepacia complex (Bcc) in cystic fibrosis (CF) is not widely known.

Methods

All CF patients with Bcc between 2002 and 2011 were reviewed, and a molecular analysis of isolates was performed.

Results

The prevalence of Bcc infection was 7.2% (18/250). Molecular analysis of 16 Bcc isolates showed 5 species (7 B. contaminans, 6 B. cepacia, 1 B. cenocepacia, 1 B. multivorans, and 1 B. stabilis) and 13 sequence types. There were no cases of cross-transmission.

Conclusion

A high diversity of Bcc species was found in infected CF patients.     

A novel mechanism regulating insulin secretion involving Herpud1 in mice

Aims/hypothesis

Type 2 diabetes results from beta cell dysfunction after prolonged physiological stress, which causes oversecretion of insulin. We recently found that insulin hypersecretion is mediated by at least two genes. Among mouse models of type 2 diabetes, the DBA/2 mouse strain is more susceptible to diabetes than is the C57BL/6J (B6J) strain. One distinctive feature of the DBA/2 mouse is that it hypersecretes insulin, independent of changes in insulin sensitivity; we identified Nnt as a gene responsible for this trait.

Methods

To identify the other gene(s) affecting insulin hypersecretion, we tested a panel of recombinant inbred BXD strains, which have different combinations of B6 and DBA/2 alleles.

Results

We found that 25% of the BXD strains hypersecreted insulin in response to glucose. Microarray profiling of islets from high- and low-secretor strains showed that at least four genes were differentially expressed. One gene was consistently underexpressed in islets from both DBA/2 and the high-secretor BXD strains. This gene (Herpud1 or Herp) encodes the 54 kDa endoplasmic reticulum stress-inducible protein (HERP) that resides in the integral endoplasmic reticulum membrane. To test directly whether Herpud1 can interact with Nnt, Herpud1 was either knocked down or overexpressed in MIN6 cells. These results showed that when Herpud1 was suppressed, Nnt expression was reduced, while overexpression of Herpud1 led to increased Nnt expression. Furthermore, Herpud1 suppression resulted in significantly decreased glucose-stimulated insulin secretion in the DBA/2 islets but not B6J islets.

Conclusions/interpretation

We conclude that Herpud1 regulates insulin secretion via control of Nnt expression.     

Recurrent outbreaks of Serratia marcescens among neonates and infants at a pediatric department: an outbreak analysis

Purpose

Serratia marcescens is a known cause of bloodstream infections (BSIs) and outbreaks in neonates receiving intensive care. Our aim was to analyze clinical and epidemiological characteristics of two outbreaks detected in our unit to prevent and control further epidemic infections.

Methods

Two episodes of BSI outbreaks in neonates have been investigated in a 20-month period at a pediatric department of a medical university in Hungary. We collected all S. marcescens strains that were isolated in the study period, and two strains that were isolated before the outbreaks. Strains were analyzed by pulsed-field gel electrophoresis (PFGE). Clinical data were collected for the BSIs during and between the outbreaks (n = 14).

Results

Out of the 28 S. marcescens isolates investigated by PFGE, 16 were blood isolates. All isolates represented four PFGE types. Pathogenic strains that caused epidemic BSIs were related to a single PFGE type (SM009). Strains with the same pulsotype could be detected before, between, and after the outbreak periods from surveillance cultures of neonates, and a water tap in the infant care unit despite intensive infection control measures. Case fatality rate of BSIs was 29 %. Rate of complications in central nervous system was high: 3/14 neonates developed meningitis.

Conclusions

Rapid spread and high mortality rate of S. marcescens infections necessitate a high suspicion when isolating this species in neonatal intensive care. Early identification of outbreaks is essential, that can be facilitated by determination of clonal relatedness using molecular methods, and with regular surveillance cultures of patients and environment.     

Low prevalence of VRE gastrointestinal colonization of hospitalized patients in Manitoba tertiary care and community hospitals

OBJECTIVE:

To determine the prevalence of vancomycin-resistant enterococci (VRE) bowel colonization in hospitalized patients in Manitoba who had stool specimens collected for Clostridium difficile toxin and/or culture testing.

DESIGN:

Two tertiary care and five community hospitals in Winnipeg and three rural Manitoba community hospitals participated in this study. From January 1 to December 31, 1997 stool specimens, one per patient, submitted to hospital microbiology laboratories for C difficile toxin and/or culture testing were screened for VRE on colistin-nalidixic acid-vancomycin (6 μg/mL) (CNAV) agar plates. The study was divided into six, eight-week intervals. Stool specimens received in the first two weeks of each eight week interval were screened for VRE.

MAIN RESULTS:

A total of 1408 stool specimens were submitted over the 48-week study period. Sixty-seven (4.8%) patients with VRE colonization of their lower gastrointestinal tract were identified. Three of the 67 (4.5%) VRE isolates were Enterococcus faecium, with the remaining 64 (95.5%) were Enterococcus gallinarum. The three vancomycin-resistant E faecium -VREF- (from two different Winnipeg hospitals) demonstrated the vanA genotype, and were resistant to vancomycin, teicoplanin and ampicillin. All three VREF isolates also demonstrated high level resistance to both gentamicin and streptomycin but were susceptible to quinuprisitin/dalfopristin and {"type":"entrez-nucleotide","attrs":{"text":"LY333328","term_id":"1257359838"}}LY333328.

CONCLUSION:

VRE colonization in hospitalized patients in Manitoba is infrequent and most commonly due to E gallinarum. The prevalence of VREF colonization in the patients studied was 0.2% (three of 1408).Key Words: Manitoba, Prevalence, Vancomycin-resistant enterococciVancomycin-resistant Enterococcus faecium (VREF) accounts for up to 65% of E faecium isolates in hospitalized patients across the United States and is endemic in many North American tertiary care institutions (1,2). The management of these infections presents a significant clinical challenge because species of the genus Enterococcus, and in particular E faecium, are frequently resistant to several antimicrobial agents (3). High level penicillin resistance, high level aminoglycoside resistance and most recently vancomycin resistance are emerging as significant concerns in the treatment of enterococcal infections. This has prompted the development and evaluation of new antimicrobial agents such as quinupristin/dalfopristin and {"type":"entrez-nucleotide","attrs":{"text":"LY333328","term_id":"1257359838"}}LY333328, a glycopeptide, which may offer activity against enterococci resistant to conventional therapy (2).VREF is not endemic in Manitoba hospitals, and infection with VREF is extremely rare (4). However, the prevalence of VREF lower gastrointestinal tract (GIT) carriage, which frequently precedes infection (5,6), is presently unknown for patients hospitalized in Manitoba. To determine whether the lack of VREF endemnicity correlated with an absence of lower GIT colonization, we assessed lower GIT carriage of VREF for patients hospitalized in 10 Manitoba hospitals from January 1 to December 31, 1997. Our study was consistent with Centers for Disease Control and Prevention guidelines (Atlanta, Georgia) that suggest surveillance programs for vancomycin-resistant enterococci (VRE) be undertaken on an intermittent basis in areas where VRE is not known to be endemic (6). Isolates of VREF identified were phenotypically and genotypically characterized, and tested for their susceptibilities against a panel of antimicrobial agents.     

A dynamic estimation of the daily cumulative cases during infectious disease surveillance: application to dengue fever

Background

To control multidrug resistant tuberculosis (MDR-TB), the drug susceptibility profile is needed to guide therapy. Classical drug susceptibility testing (DST) may take up to 2 to 4 months. The GenoType^® MTBDR plus test is a commercially available line-probe assay that rapidly detects Mycobacterium tuberculosis (MTB) complex, as well as the most common mutations associated with rifampin and isoniazid resistance. We assessed sensitivity and specificity of the assay by using a geographically representative set of MTB isolates from the South of Vietnam.

Methods

We re-cultured 111 MTB isolates that were MDR, rifampin-resistant or pan-susceptible according to conventional DST and tested these with the GenoType^® MTBDR plus test.

Results

By conventional DST, 55 strains were classified as MDR-TB, four strains were rifampicin mono-resistant and 52 strains were susceptible to all first-line drugs. The sensitivity of the GenoType^® MTBDR plus was 93.1% for rifampicin, 92.6% for isoniazid and 88.9% for the combination of both; its specificity was 100%. The positive predictive value of the GenoType^® MTBDR plus test for MDR-TB was 100% and the negative predictive value 90.3%.

Conclusions

We found a high specificity and positive predictive value of the GenoType^® MTBDR plus test for MDR-TB which merits its use in the MDR-TB treatment program in Vietnam.     

OspA heterogeneity of <Emphasis Type="Italic">Borrelia valaisiana</Emphasis>confirmed by phenotypic and genotypic analyses

Background

Although European Borrelia burgdorferi sensu lato isolates have been divided into five genospecies, specific tools for the serotype characterization of only three genospecies are available. Monoclonals antibodies (mAbs) H3TS, D6 and I17.3 identify B. burgdorferi sensu stricto (ss.), B. garinii and B. afzelii respectively, but no mAbs are available to identify B. valaisiana. In the same way, specific primers exist to amplify the OspA gene of B. burgdorferi ss., B. garinii and B. afzelii. The aim of the study was to develop species-specific mAb and PCR primers for the phenotypic and genetic identification of B. valaisiana.

Results

This study describes a mAb that targets OspA of B. valaisiana and primers targeting the OspA gene of this species. As the monoclonal antibody A116k did not react with strains NE231, M7, M53 and Frank and no amplification was observed with strains NE231, M7 and M53, the existence of two subgroups among European B. valaisiana species was confirmed.

Conclusions

The association of both monoclonal antibody A116k and primers Bval 1F and Bval 1R allows to specific identification of the B. valaisiana isolates belonging to subgroup 1.