CRISPR Diversity and Microevolution in Clostridium difficile.

Virulent strains of Clostridium difficile have become a global health problem associated with morbidity and mortality. Traditional typing methods do not provide ideal resolution to track outbreak strains, ascertain genetic diversity between isolates, or monitor the phylogeny of this species on a global basis. Here, we investigate the occurrence and diversity of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) in C. difficile to assess the potential of CRISPR-based phylogeny and high-resolution genotyping. A single Type-IB CRISPR-Cas system was identified in 217 analyzed genomes with cas gene clusters present at conserved chromosomal locations, suggesting vertical evolution of the system, assessing a total of 1,865 CRISPR arrays. The CRISPR arrays, markedly enriched (8.5 arrays/genome) compared with other species, occur both at conserved and variable locations across strains, and thus provide a basis for typing based on locus occurrence and spacer polymorphism. Clustering of strains by array composition correlated with sequence type (ST) analysis. Spacer content and polymorphism within conserved CRISPR arrays revealed phylogenetic relationship across clades and within ST. Spacer polymorphisms of conserved arrays were instrumental for differentiating closely related strains, e.g., ST1/RT027/B1 strains and pathogenicity locus encoding ST3/RT001 strains. CRISPR spacers showed sequence similarity to phage sequences, which is consistent with the native role of CRISPR-Cas as adaptive immune systems in bacteria. Overall, CRISPR-Cas sequences constitute a valuable basis for genotyping of C. difficile isolates, provide insights into the micro-evolutionary events that occur between closely related strains, and reflect the evolutionary trajectory of these genomes.

Enteroaggregative Escherichia coli in Daycare-A 1-Year Dynamic Cohort Study.

Enteroaggregative Escherichia coli (EAEC) has been associated with persistent diarrhea, reduced growth acceleration, and failure to thrive in children living in developing countries and with childhood diarrhea in general in industrialized countries. The clinical implications of an EAEC carrier-status in children in industrialized countries warrants clarification. To investigate the pathological significance of an EAEC carrier-state in the industrialized countries, we designed a 1-year dynamic cohort study and performed follow-up every second month, where the study participants submitted a stool sample and answered a questionnaire regarding gastrointestinal symptoms and exposures. Exposures included foreign travel, consumption of antibiotics, and contact with a diseased animal. In the capital area of Denmark, a total of 179 children aged 0-6 years were followed in a cohort study, in the period between 2009 and 2013. This is the first investigation of the incidence and pathological significance of EAEC in Danish children attending daycare facilities. Conventional microbiological detection of enteric pathogens was performed at Statens Serum Institute, Copenhagen, Denmark, and at Hvidovre Hospital, Copenhagen, Denmark. Parents completed questionnaires regarding gastrointestinal symptoms. The EAEC strains were further characterized by serotyping, phylogenetic analysis, and susceptibility testing. EAEC was detected in 25 (14%) of the children during the observational period of 1 year. One or more gastrointestinal symptoms were reported from 56% of the EAEC-positive children. Diarrhea was reported in six (24%) of the EAEC positive children, but no cases of weight loss, and general failure to thrive were observed. The EAEC strains detected comprised a large number of different serotypes, confirming the genetic heterogeneity of this pathotype. EAEC was highly prevalent (n = 25, 14%) in Danish children in daycare centers and was accompanied by gastrointestinal symptoms in 56% of the infected children. No serotype or phylogenetic group was specifically linked to children with disease.

Childhood diarrhoea in Danish day care centres could be associated with infant colic, low birthweight and antibiotics.

AIM: Diarrhoea is very common in children attending day care centres. The aim of this study was to examine certain predisposing risk factors for an association with diarrhoea, including foreign travel, treatment with antibiotics, having household pets, infant colic, bottle feeding, using a pacifier and low birthweight. METHODS: A dynamic one-year follow-up cohort study comprising 179 children from 36 day care centres was conducted from September 2009 to July 2013 in Copenhagen, Denmark. Questionnaires were sent to the children's parents or legal guardians every two months for a year, requesting information on gastrointestinal symptoms and exposure. A logistic regression was performed to identify the odds ratios of different risk factors for diarrhoea. RESULTS: The odds ratios for diarrhoea were 1.97 (0.93-4.20) for children with a history of infant colic, 1.91 (0.90-4.04) for low birthweight children and 1.45 (0.74-2.82) for children who had used antibiotics. Having a pet in the household had a possible protective effect towards diarrhoeal events, with an odds ratio of 0.47 (0.20-1.09). CONCLUSION: A history of infant colic, low birthweight, and to a lesser extent antibiotic use, possibly increased the risk of diarrhoea in Danish children in day care centres.

Novel multiplex format of an extended multilocus variable-number-tandem-repeat analysis of Clostridium difficile correlates with tandem repeat sequence typing.

Subtyping of Clostridium difficile is crucial for outbreak investigations. An extended multilocus variable-number tandem-repeat analysis (eMLVA) of 14 variable number tandem repeat (VNTR) loci was validated in multiplex format compatible with a routine typing laboratory and showed excellent concordance with tandem repeat sequence typing (TRST) and high discriminatory power.

Identification of clinical aeromonas species by rpoB and gyrB sequencing and development of a multiplex PCR method for detection of Aeromonas hydrophila, A. caviae, A. veronii, and A. media.

Conventional identification of Aeromonas species based on biochemical methods is challenged by the heterogeneous nature of the species. Here, we present a new multiplex PCR method directed toward the gyrB and rpoB genes that identifies four Aeromonas species, A. hydrophila, A. media, A. veronii, and A. caviae, and we describe the application of this method on a Danish strain collection.

Epidemiology and clinical manifestations of enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) represents a heterogeneous group of E. coli strains. The pathogenicity and clinical relevance of these bacteria are still controversial. In this review, we describe the clinical significance of EAEC regarding patterns of infection in humans, transmission, reservoirs, and symptoms. Manifestations associated with EAEC infection include watery diarrhea, mucoid diarrhea, low-grade fever, nausea, tenesmus, and borborygmi. In early studies, EAEC was considered to be an opportunistic pathogen associated with diarrhea in HIV patients and in malnourished children in developing countries. In recent studies, associations with traveler's diarrhea, the occurrence of diarrhea cases in industrialized countries, and outbreaks of diarrhea in Europe and Asia have been reported. In the spring of 2011, a large outbreak of hemolytic-uremic syndrome (HUS) and hemorrhagic colitis occurred in Germany due to an EAEC O104:H4 strain, causing 54 deaths and 855 cases of HUS. This strain produces the potent Shiga toxin along with the aggregative fimbriae. An outbreak of urinary tract infection associated with EAEC in Copenhagen, Denmark, occurred in 1991; this involved extensive production of biofilm, an important characteristic of the pathogenicity of EAEC. However, the heterogeneity of EAEC continues to complicate diagnostics and also our understanding of pathogenicity.

Metronidazole therapy for treating dientamoebiasis in children is not associated with better clinical outcomes: a randomized, double-blinded and placebo-controlled clinical trial.

BACKGROUND: There is a paucity of evidence documenting the pathogenicity of Dientamoeba fragilis, an intestinal protozoan common in children. As case reports on successful treatment are numerous, many authors advocate treatment, despite no placebo-controlled trials being available. Metronidazole is often used for treatment, though eradication rates are relatively low (60%-80%). In the present study we determined the clinical and microbiological efficacy of metronidazole in Danish children. METHODS: In this parallel placebo-controlled double-blinded trial, children aged 3-12 years with >4 weeks of gastrointestinal symptoms were allocated using block randomization in a 1:1 ratio to a 10-day course of oral metronidazole or placebo. Primary outcome was change in level of gastrointestinal symptoms, measured on a visual-analog-scale (VAS), and secondary outcome was eradication of D. fragilis infection. Participants, caregivers, investigators, and sponsor were all blinded to group assignment. The trial was registered with clinicaltrials.gov (NCT01314976) prior to start. RESULTS: Of 96 participants, 48 were allocated to the metronidazole and placebo group each. Mean VAS change from pre- to post-treatment did not differ significantly (P = .8) between the metronidazole (-1.8 CI, [-2.5, -1.1]) and the placebo group (-1.6 CI, [-2.3, -.9]). Eradication of D. fragilis was significantly greater in the metronidazole group, although it declined rapidly from 62.5% 2 weeks after end of treatment to 24.9% 8 weeks after end of treatment. CONCLUSIONS: These findings do not provide evidence to support routine metronidazole treatment of D. fragilis positive children with chronic gastrointestinal symptoms. Study funded by Statens Serum Institut. CLINICAL TRIALS REGISTRATION: Trial was registered with clinicaltrials.gov (NCT01314976).

Poultry as a possible source of non-typhoidal Salmonella enterica serovars in humans in Bangladesh.

We investigated Salmonella enterica isolates from human clinical cases of gastroenteritis to determine the distribution of non-typhoidal Salmonella serovars in the human population, and compared them to isolates originating from poultry by serotyping, phage typing, plasmid profiling, pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) to evaluate the potential role of poultry in human non-typhoidal salmonellosis in Bangladesh. Nine different serovars were identified among the human isolates of which Salmonella Paratyphi B var Java (S. Java), S. Kentucky, S. Enteritidis, S. Virchow and S. Weltevreden also were commonly isolated from poultry. The poultry isolates belonging to S. Java, S. Kentucky and S. Enteritidis were indistinguishable from human isolates or genetically closely related, based on PFGE profiles and MLST. S. Kentucky clone ST198 and S. Java clone ST43 both well-known cause of human infections were also isolated from poultry.

Molecular characterization of motile serovars of Salmonella enterica from breeder and commercial broiler poultry farms in Bangladesh.

Contaminated poultry and poultry products are a major source of motile Salmonellae for human salmonellosis worldwide. Local circulation of any motile Salmonella serovar in poultry has a wider public health impact beyond its source of origin for being dispersed elsewhere through poultry trades or human travels. To investigate the status of motile Salmonella serovars in breeder farms in Bangladesh, multiple flocks of two breeder farms were observed for a period of six months. In addition, a cross-sectional survey was carried out to determine the prevalence and serovar distribution of motile Salmonella by randomly selecting 100 commercial broiler poultry farms. Five pooled faecal samples representing an entire housed flock of breeders or broilers were screened for presence of motile Salmonella following conventional bacteriological procedures. The Salmonella isolates obtained were subsequently serotyped, and characterized by plasmid profiling and pulsed-field gel electrophoresis (PFGE). The results revealed that both the breeder farms were positive with three Salmonella serovars: S. Virchow, S. Paratyphi B var Java (S. Java) and S. Enteritidis. Eleven of the 100 broiler farms investigated were positive for motile Salmonella, giving a farm-level prevalence of 11% (95% confidence interval 5-17%). S. Virchow and S. Kentucky were the two predominant serovars isolated from the broiler farms. The PFGE genotyping demonstrated that the isolates belonging to the same serovars were closely related due to variation in only 1-4 bands. All the S. Virchow and S. Java isolates, irrespective of breeder or broiler farm origin, were plasmid-free, except for one S. Virchow isolate from a broiler farm that harboured a 9.7 kb-sized plasmid. The S. Kentucky isolates belonged to three plasmid profiles having plasmids of four different sizes, ranging from 2.7 to 109 kb. This is the first report of any motile Salmonella serovars from breeder and commercial broiler poultry farms in Bangladesh.

Real-time TaqMan polymerase chain reaction-based genus-identification and pyrosequencing-based species identification of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus directly on stool samples.

A new method was developed for Campylobacter identification and applied directly on 599 stool samples from diarrhoeagenic patients. Here, the gyrase B gene of Campylobacter was targeted in a 2-step process: first, TaqMan polymerase chain reaction (PCR)-based identification of C. jejuni, C. coli, C. upsaliensis, C. lari, and C. fetus at the genus level, and, second, pyrosequencing-based identification at the species level. The TaqMan PCR method was compared to culturing and identified 87 Campylobacter-positive samples of which 64 were culture positive. Among the discrepant 23 samples, 18 were confirmed positive by conventional PCR, underlining a significant increase in diagnostic yield by use of this molecular and culture-independent method. For species identification, the pyrosequencing method was compared to conventional PCR and among the 87 TaqMan PCR-positive samples, 74 Campylobacter species were identified by both methods, 10 samples gave discrepant results, and 3 samples were negative by both methods.

Prevalence and characterization of motile Salmonella in commercial layer poultry farms in Bangladesh.

Salmonella is a globally widespread food-borne pathogen having major impact on public health. All motile serovars of Salmonella enterica of poultry origin are zoonotic, and contaminated meat and raw eggs are an important source to human infections. Information on the prevalence of Salmonella at farm/holding level, and the zoonotic serovars circulating in layer poultry in the South and South-East Asian countries including Bangladesh, where small-scale commercial farms are predominant, is limited. To investigate the prevalence of Salmonella at layer farm level, and to identify the prevalent serovars we conducted a cross-sectional survey by randomly selecting 500 commercial layer poultry farms in Bangladesh. Faecal samples from the selected farms were collected following standard procedure, and examined for the presence of Salmonella using conventional bacteriological procedures. Thirty isolates were randomly selected, from the ninety obtained from the survey, for serotyping and characterized further by plasmid profiling and pulsed-field gel electrophoresis (PFGE). Results of the survey showed that the prevalence of motile Salmonella at layer farm level was 18% (95% confidence interval 15-21%), and Salmonella Kentucky was identified to be the only serovar circulating in the study population. Plasmid analysis of the S. Kentucky and non-serotyped isolates revealed two distinct profiles with a variation of two different sizes (2.7 and 4.8 kb). PFGE of the 30 S. Kentucky and 30 non-serotyped isolates showed that all of them were clonally related because only one genotype and three subtypes were determined based on the variation in two or three bands. This is also the first report on the presence of any specific serovar of Salmonella enterica in poultry in Bangladesh.

[Intracerebral abscess after infection with non-toxigenic Vibrio cholerae]. / Cerebral absces med Vibrio cholerae non-01 efter badning i dansk havvand.

We present the first case of intracerebral abscess after blood-borne infection with non-toxigenic Vibrio cholerae (non-01). The patient was a 66 year-old woman who was infected after swimming in Danish seawater during an unusually hot summer. She had predisposing haemochromatosis and a skin lesion on the ankle. We treated the patient with meropenem and ciprofloxacin for six weeks followed by ciprofloxacin for four weeks, and she recovered with hemiparesis and speech impairment. Marine Vibrio species may produce intracranial infection in predisposed individuals, even in temperate climate zones.

Multiplex PCR method for detection of Clostridium difficile tcdA, tcdB, cdtA, and cdtB and internal in-frame deletion of tcdC.

A multiplex PCR method was developed for the detection of Clostridium difficile toxin genes tcdA, tcdB, ctdA, and cdtB and the major in-frame deletion types (18, 39, and 54 bp) of tcdC. The method has high specificity for PCR ribotype 027 and may identify other C. difficile strains of clinical and epidemiological importance.

Binary toxin and death after Clostridium difficile infection.

We compared 30-day case-fatality rates for patients infected with Clostridium difficile possessing genes for toxins A and B without binary toxin (n = 212) with rates for patients infected with C. difficile possessing genes for A, B, and binary toxin. The latter group comprised patients infected with strains of PCR ribotype 027 (CD027, n = 193) or non-027 (CD non-027, n = 72). Patients with binary toxin had higher case-fatality rates than patients without binary toxin, in univariate analysis (relative risk [RR] 1.8, 95% confidence interval [CI] 1.2-2.7) and multivariate analysis after adjustment for age, sex, and geographic region (RR 1.6, 95% CI 1.0-2.4). Similar case-fatality rates (27.8%, 28.0%) were observed for patients infected with CD027 or CD non-027. Binary toxin either is a marker for more virulent C. difficile strains or contributes directly to strain virulence. Efforts to control C. difficile infection should target all virulent strains irrespective of PCR ribotype.

Five commercial DNA extraction systems tested and compared on a stool sample collection.

In this study, 5 different commercial DNA extraction systems were tested on a stool sample collection containing 81 clinical stool specimens that were culture-positive for diarrheagenic Escherichia coli, Campylobacter jejuni, Salmonella enterica, or Clostridium difficile. The purified DNAs were analyzed by polymerase chain reaction (PCR) directed toward the relevant organisms. The results showed that conventional PCR combined with the extraction systems BioRobot EZ1 (Qiagen, Hilden, Germany), Bugs'n Beads (Genpoint, Oslo, Norway), ChargeSwitch (Invitrogen, Paisley, UK), QIAamp Stool Mini Kit (Qiagen), and 2 protocols (generic and Specific A) for EasyMag (BioMérieux, Marcy I'Etoile, France) were able to identify 89%, 62%, 85%, 88%, 85%, and 91%, respectively, of the pathogens originally identified by conventional culture-based methods. When TaqMan PCR was combined with the EasyMag Specific A protocol, 99% of the samples were correctly identified. The results demonstrate that the extraction efficiencies can vary significantly among different extraction systems, careful optimization may have a significant positive effect, and the use of sensitive and specific detection methods like TaqMan PCR is an ideal choice for this type of analysis.

Eradication of Blastocystis carriage with antimicrobials: reality or delusion?

Metronidazole constitutes a mainstay in the antimicrobial therapy of intestinal protozoa, and is also traditionally considered first-line therapy in cases where there is a requirement to treat Blastocystis, a common protist of disputable clinical significance. Many compounds have been used in attempts to eradicate the parasite, and an accumulating body of data indicates that successful antimicrobial eradication of Blastocystis is far from straightforward. This review focuses on some issues that prevent us from reaching a clear understanding of how to eradicate Blastocystis based on chemotherapeutic intervention, by focusing on conflicting reports on the efficacy of metronidazole and other compounds and study design and data limitations. The review provides a comprehensive overview of antimicrobials used to target Blastocystis, and discusses issues pertaining to drug resistance, treatment failure, and reinfection. Finally, key methodological and molecular diagnostic tools that will assist in the generation of data required to improve current knowledge are identified and discussed.

Escherichia coli isolates from broiler chicken meat, broiler chickens, pork, and pigs share phylogroups and antimicrobial resistance with community-dwelling humans and patients with urinary tract infection.

Escherichia coli is the most common cause of urinary tract infection (UTI). Phylogroup B2 and D isolates are associated with UTI. It has been proposed that E. coli causing UTI could have an animal origin. The objective of this study was to investigate the phylogroups and antimicrobial resistance, and their possible associations in E. coli isolates from patients with UTI, community-dwelling humans, broiler chicken meat, broiler chickens, pork, and pigs in Denmark. A total of 964 geographically and temporally matched E. coli isolates from UTI patients (n = 102), community-dwelling humans (n = 109), Danish (n = 197) and imported broiler chicken meat (n = 86), Danish broiler chickens (n = 138), Danish (n = 177) and imported pork (n = 10), and Danish pigs (n = 145) were tested for phylogroups (A, B1, B2, D, and nontypeable [NT] isolates) and antimicrobial susceptibility. Phylogroup A, B1, B2, D, and NT isolates were detected among all groups of isolates except for imported pork isolates. Antimicrobial resistance to three (for B2 isolates) or five antimicrobial agents (for A, B1, D, and NT isolates) was shared among isolates regardless of origin. Using cluster analysis to investigate antimicrobial resistance data, we found that UTI isolates always grouped with isolates from meat and/or animals. We detected B2 and D isolates, that are associated to UTI, among isolates from broiler chicken meat, broiler chickens, pork, and pigs. Although B2 isolates were found in low prevalences in animals and meat, these sources could still pose a risk for acquiring uropathogenic E. coli. Further, E. coli from animals and meat were very similar to UTI isolates with respect to their antimicrobial resistance phenotype. Thus, our study provides support for the hypothesis that a food animal and meat reservoir might exist for UTI-causing E. coli.

[First Danish case of Clostridium difficile ribotype 027]. / Første påvisning af Clostridium difficile type 027 i Danmark.

Increasing rates of Clostridium difficile-associated diarrhoea (CDAD) with an unusual, severe course have been reported in Canada, USA and several European countries since 2003. A new virulent strain, PCR ribotype 027 (CD027), is associated with this increase. We report the first Danish case of CDAD caused by CD027. A 85-year-old woman was admitted to hospital with pneumonia. Following treatment initially with penicillin, secondly with moxifloxacin she developed bloody diarrhoea. A stool specimen showed CD027. She was treated with metronidazol for ten days and recovered completely.

Subtype-specific suppression of Shiga toxin 2 released from Escherichia coli upon exposure to protein synthesis inhibitors.

Shiga toxins (Stx) are important virulence factors in the pathogenesis of severe disease including hemolytic-uremic syndrome, caused by Stx-producing Escherichia coli (STEC). STEC strains increase the release of Stx in vitro following the addition of fluoroquinolones, whereas protein synthesis inhibitors previously have been reported to suppress the release of Stx. The amount of Stx released from wild-type STEC strains incubated with protein synthesis inhibitors was examined by a Vero cell cytotoxicity assay. The amounts released were compared to the Stx type (Stx1 or Stx2) and additionally to the individual subtypes and toxin variants of Stx2. In general, Stx2 release was suppressed significantly upon exposure to protein synthesis inhibitors at MICs, which was not observed in the case of Stx1. Also, the average amount of different Stx2 toxin variants released was suppressed to various levels ranging from 14.0% (Stx2-O157-EDL933) to 94.7% (Stx2d-O8-C466-01B). Clinical studies exploring protein synthesis inhibitors as future candidates for treatment of intestinal infections caused by Stx2-producing STEC should therefore include knowledge of the toxin variant in addition to the subtype.