Showing papers in "Journal of Medical Microbiology in 2007"

Effects of oregano, carvacrol and thymol on Staphylococcus aureus and Staphylococcus epidermidis biofilms.

Abstract: The aim of this study was to evaluate the effect of oregano essential oil, carvacrol and thymol on biofilm-grown Staphylococcus aureus and Staphylococcus epidermidis strains, as well as the effects of the oils on biofilm formation. For most of the S. aureus (n=6) and S. epidermidis (n=6) strains tested, the biofilm inhibitory concentration (0.125–0.500 %, v/v, for oregano, and 0.031–0.125 %, v/v, for carvacrol and thymol) and biofilm eradication concentration (0.25–1.0 %, v/v, for oregano and 0.125–0.500 %, v/v, for carvacrol and thymol) values were twofold or fourfold greater than the concentration required to inhibit planktonic growth. Subinhibitory concentrations of the oils attenuated biofilm formation of S. aureus and S. epidermidis strains on polystyrene microtitre plates.

Imbalance in the composition of the duodenal microbiota of children with coeliac disease

Abstract: Coeliac disease (CD) is the most common immune-mediated enteropathy characterized by chronic inflammation of the small intestinal mucosa. The ingestion of gluten is responsible for the symptoms of CD, but other environmental factors are also thought to play a role in this disorder. In this study, the composition of the duodenal microbiota of coeliac children with active disease, symptom-free CD patients on a gluten-free diet and control children was determined. Bacteriological analyses of duodenal biopsy specimens were carried out by fluorescent in situ hybridization coupled with flow cytometry. The proportions of total bacteria and Gram-negative bacteria were significantly higher in CD patients with active disease than in symptom-free CD patients and controls. Bacteroides and Escherichia coli groups were significantly more abundant in CD patients with active disease than in controls, whilst these bacterial deviations were normalized in symptom-free CD patients. The ratio of Lactobacillus–Bifidobacterium to Bacteroides–E. coli was significantly reduced in coeliac patients with either active or inactive disease compared with controls. The differences in Atopobium, Eubacterium rectale–Clostridium coccoides, Clostridium histolyticum, Clostridium lituseburense, sulphate-reducing bacteria and Faecalibacterium prausnitzii populations among the three groups of children were less relevant. Overall, the higher incidence of Gram-negative and potentially pro-inflammatory bacteria in the duodenal microbiota of coeliac children was linked to the symptomatic presentation of the disease and could favour the pathological process of the disorder.

Biofilm formation by enterococci.

Abstract: Enterococci are an important global cause of nosocomial infections, being increasingly associated with urinary tract infections, endocarditis, intra-abdominal and pelvic infections, catheter-related infections, surgical wound infections, and central nervous system infections. The two most common enterococci species are Enterococcus faecalis and Enterococcus faecium. Both are capable of producing biofilms, which consist of a population of cells attached irreversibly on various biotic and abiotic surfaces, encased in a hydrated matrix of exopolymeric substances. Many environmental and genetic factors are associated or have been proposed to be associated with the production of biofilm. This review discusses recent advances in knowledge about the biology and genetics of biofilm formation and the role of biofilms in enterococci pathogenesis.

Development of a simple model for studying the effects of antifungal agents on multicellular communities of Aspergillus fumigatus

Abstract: Aspergillus fumigatus is an increasingly prevalent opportunistic fungal pathogen of various immunocompromised individuals. It has the ability to form filaments within the lungs, producing dense intertwined mycelial balls, which are difficult to treat. The aim of this study was to develop a suitable model of A. fumigatus to examine the effects of antifungal challenge on these intertwined filamentous communities. A. fumigatus NCPF 7367 growth conditions were optimized on both Thermanox coverslips and on flat-bottomed microtitre plates to establish optimal conidial seeding densities. Isolates were treated with itraconazole, voriconazole, amphotericin B and caspofungin and their overall killing efficiency was measured using an XTT formazan metabolic dye assay. This was compared with the CLSI (formerly NCCLS) methodology of broth microdilution of moulds (standard M38-A). It was shown that 1x10(5) conidia ml(-1) in RPMI 1640 was the optimum concentration of spores for biofilm formation. Filamentous growth characteristics were not observed until 10 h incubation, followed by an exponential increase in the biofilm biomass (hyphae and extracellular material) and cellular activity (metabolism). When susceptibility testing of biofilms was compared with that of planktonic cells by CLSI broth microdilution testing, all antifungal drugs were at least 1000 times less effective at reducing the overall metabolic activity of 90 % of the cells. Overall, this study showed that A. fumigatus has the ability to form coherent multicellular biofilm structures that are resistant to the effects of antifungal drugs.

Artemisinin triggers induction of cell-cycle arrest and apoptosis in Leishmania donovani promastigotes.

Abstract: A major impediment to effective anti-leishmanial chemotherapy is the emergence of drug resistance, especially to sodium antimony gluconate, the first-line treatment for leishmaniasis. Artemisinin, a sesquiterpene lactone isolated from Artemisia annua, is an established anti-malarial compound that showed anti-leishmanial activity in both promastigotes and amastigotes, with IC(50) values of 160 and 22 microM, respectively, and, importantly, was accompanied by a high safety index (>22-fold). The leishmanicidal activity of artemisinin was mediated via apoptosis as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, in situ labelling of DNA fragments by terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) and cell-cycle arrest at the sub-G(0)/G(1) phase. Taken together, these data indicate that artemisinin has promising anti-leishmanial activity that is mediated by programmed cell death and, accordingly, merits consideration and further investigation as a therapeutic option for the treatment of leishmaniasis.

Molecular mechanisms of antimony resistance in Leishmania.

Abstract: Leishmaniasis causes significant morbidity and mortality worldwide. The disease is endemic in developing countries of tropical regions, and in recent years economic globalization and increased travel have extended its reach to people in developed countries. In the absence of effective vaccines and vector-control measures, the main line of defence against the disease is chemotherapy. Organic pentavalent antimonials [Sb(V)] have been the first-line drugs for the treatment of leishmaniasis for the last six decades, and clinical resistance to these drugs has emerged as a primary obstacle to successful treatment and control. A multiplicity of resistance mechanisms have been described in resistant Leishmania mutants developed in vitro by stepwise increases of the concentration of either antimony [Sb(III)] or the related metal arsenic [As(III)], the most prevalent mechanism being upregulated Sb(III) detoxification and sequestration. With the availability of resistant field isolates, it has now become possible to elucidate mechanisms of clinical resistance. The present review describes the mechanisms of antimony resistance in Leishmania and highlights the links between previous hypotheses and current developments in field studies. Unravelling the molecular mechanisms of clinical resistance could allow the prevention and circumvention of resistance, as well as rational drug design for the treatment of drug-resistant Leishmania.

Antimicrobial activity of lacticin 3147 against clinical Clostridium difficile strains

Abstract: Clostridium difficile-associated diarrhoea (CDAD) is the most common hospital-acquired diarrhoea, and is a major type of gastroenteritis infection in nursing homes and facilities for the elderly. In this study the antimicrobial activity of the two-component lantibiotic, lacticin 3147, against a range of genetically distinct C. difficile isolates was studied. The bacteriocin exhibited an MIC50 of 3.6 μg ml−1 for 10 genetically distinct C. difficile strains isolated from healthy subjects, inflammatory bowel disease patients and culture collection strains. In time-kill studies, 106 c.f.u. ml−1 C. difficile ATCC 42593 and CDAD isolate DPC 6220 were killed within 120 or 20 min incubation, respectively, at a concentration of 6 μg lacticin ml−1. Interestingly, addition of lacticin 3147 to exponentially growing cells of C. difficile ATCC 43593 caused rapid lysis of the cells after an initial lag phase, as measured by the concomitant release of the intracellular enzyme, acetate kinase. The addition of a food-grade, milk-based lacticin containing powder to faecal fermentation demonstrated that lacticin is effective in completely eliminating 106 c.f.u. C. difficile ml−1 from a model faecal environment within 30 min when present at concentrations as low as 18 μg ml−1. While other culturable microflora such as total anaerobes, bacteroides, total non-spore-forming anaerobes and total Gram-negative anaerobes were unaffected, populations of lactobacilli and bifidobacteria were reduced by 3 log cycles at bacteriocin levels sufficient to eliminate over 106 C. difficile. In light of these findings, the potential of lacticin 3147 for treatment of CDAD is discussed.

One year prospective survey of Candida bloodstream infections in Scotland

Abstract: A 12 month survey of candidaemia in Scotland, UK, in which every Scottish hospital laboratory submitted all blood isolates of yeasts for identification, strain typing and susceptibility testing, provided 300 isolates from 242 patients, generating incidence data of 4.8 cases per 100000 population per year and 5.9 cases per 100000 acute occupied bed days; 27.9% of cases occurred in intensive care units. More than half the patients with candidaemia had an underlying disease involving the abdomen, 78% had an indwelling intravenous catheter, 62% had suffered a bacterial infection within the 2 weeks prior to candidaemia and 37% had undergone a laparotomy. Candida albicans was the infecting species in 50% of cases, followed by Candida glabrata (21%) and Candida parapsilosis (12%). Seven cases of candidaemia were caused by Candida dubliniensis, which was more prevalent even than Candida lusitaniae and Candida tropicalis (six cases each). Among C. glabrata isolates, 55% showed reduced susceptibility to fluconazole, but azole resistance among other species was extremely low. Multilocus sequence typing showed isolates with high similarity came from different hospitals across the country, and many different types came from the hospitals that submitted the most isolates, indicating no tendency towards hospital-specific endemic strains. Multiple isolates of C. albicans and C. glabrata from individual patients were of the same strain type with single exceptions for each species. The high prevalence of candidaemia in Scotland, relative to other population-based European studies, and the high level of reduced fluconazole susceptibility of Scottish C. glabrata isolates warrant continued future surveillance of invasive Candida infections.

Detection and prevalence of inducible clindamycin resistance in staphylococci.

Abstract: Staphylococcus aureus and coagulase-negative staphylococci (CNS) are recognized as causing nosocomial and community-acquired infections in every region of the world. The resistance to antimicrobial agents among staphylococci is an increasing problem. Clindamycin (CL) is considered to be one of the alternative agents in these infections. This study demonstrates a simple, reliable method (double-disc diffusion test) for detecting inducible resistance to CL in erythromycin-resistance (ER-R) isolates of S. aureus and CNS. A total of 883 (52.3%) isolates of S. aureus and 804 (47.7%) isolates of CNS were selected from recent (2003-2005) clinical isolates recovered in the laboratory of the authors; duplicate isolates were not included. A total of 214 (12.6%) S. aureus and 308 (18.3%) CNS isolates were selected based on ER-R and CL sensitivity using standard National Committee for Clinical Laboratory Standards disc diffusion testing. A total of 1687 staphylococcal isolates were included, consisting of 27.5% meticillin-resistant S. aureus, 24.8% meticillin-sensitive S. aureus, 36.1% meticillin-resistant CNS and 11.6% meticillin-sensitive CNS isolates: 30.9% of staphylococcal isolates (214 S. aureus and 308 CNS) that were erythromycin resistant and CL sensitive were tested for inducible resistance using the D-test. A D-shaped zone around the CL was observed for 70.9% of staphylococcal isolates (81.8% of S. aureus isolates and 63.3% of CNS isolates) with an ER-R and a clindamycin-sensitive (CL-S) phenotype. The organism was positive for inducible clindamycin resistance (CL-R). There was a 21.9% level of inducible macrolide-lincosamide-streptogramin B resistance phenotype among all the staphylococcal isolates. When the S. aureus and CNS strains among all the staphylococcal isolates were compared statistically, inducible CL-R in CNS strains was determined to be 23% more positive (P=0.028, odds ratio 0.77, 95% confidence interval 0.61-0.98). When a statistical comparison was performed among ER-R but CL-S staphylococcal isolates inducible CL-R in S. aureus strains was determined to be 2.6 times more positive (P=0.000, odds ratio 2.6, 95% confidence interval 1.68-4.04). A simple, reliable method of detecting inducible resistance to CL in ER-R isolates of S. aureus and CNS is described. Clinical microbiology laboratories should use the double-disc diffusion test as standard practice with all ER-R staphylococci. CL should not be used in patients with infections caused by inducibly resistant staphylococcal isolates. Therapeutic failures may thus be avoided.

The in vivo dynamics of Streptococcus spp., Actinomyces naeslundii, Fusobacterium nucleatum and Veillonella spp. in dental plaque biofilm as analysed by five-colour multiplex fluorescence in situ hybridization.

Abstract: The formation and composition of dental plaque biofilm in vivo are important factors which influence the development of gingivitis, caries and periodontitis. Studying dental plaque biofilm in in vitro models can cause an oversimplification of the real conditions in the oral cavity. In this study, bovine enamel slabs were fixed in an individual acrylic appliance in situ to quantify dental plaque formation and composition using multiplex fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy. Each of the five oligonucleotide probes used for FISH was specific for either eubacteria or one of four frequently isolated bacterial constituents belonging to early and late colonizers of tooth surfaces. The thickness of formed biofilm increased from 14.9±5.0 mm after 1 day to 49.3±11.6 mm after 7 days. Streptococcus spp. were predominant in 1-day-old dental plaque and decreased significantly after 7 days (P50.0061). Compared to the first day, Fusobacterium nucleatum decreased after 2 days and increased significantly after 7 days (P50.0006). The decreases of Actinomyces naeslundii content on day 2 and day 7 were significant (P50.0028). Changes in Veillonella spp. were not significant during the study period (P .0.05). The results showed that an in vivo observation period of 7 days was required to detect significant changes in Streptococcus spp. and F. nucleatum. The multiplex FISH used is suitable for analysing the dynamics of four important bacterial constituents in the oral biofilm in epidemiological studies.

Species interactions in mixed-community crystalline biofilms on urinary catheters.

Abstract: Previous experimental investigations of the crystalline biofilms that colonize and block urinary catheters have focussed on their formation by pure cultures of Proteus mirabilis. In the urine of patients undergoing long-term catheterization, P. mirabilis is commonly found in mixed communities with other urinary tract pathogens. Little is known about the effect that the other species have on the rate at which P. mirabilis encrusts catheters. In the present study, a set of data on the nature of the bacterial communities on 106 catheter biofilms has been analysed and it was found that while species such as Providencia stuartii and Klebsiella pneumoniae were commonly associated with P. mirabilis, when Escherichia coli, Morganella morganii or Enterobacter cloacae were present, P. mirabilis was rarely or never found. The hypothesis that the absence of P. mirabilis from some biofilm communities could be due to its active exclusion by other species has also been examined. Experiments in laboratory models showed that co-infection of P. mirabilis with M. morganii, K. pneumoniae or E. coli had no effect on the ability of P. mirabilis to encrust and block catheters. Co-infection with Ent. cloacae or Pseudomonas aeruginosa, however, significantly increased the time that catheters took to block (P <0.05). The growth of Ent. cloacae, M. morganii, K. pneumoniae or E. coli in the model for 72 h prior to superinfection with P. mirabilis significantly delayed catheter blockage. In the case of Ent. cloacae, for example, the mean time to blockage was extended from 28.7 h to 60.7 h (P ≤0.01). In all cases, however, P. mirabilis was able to generate alkaline urine, colonize the biofilms, induce crystal formation and block the catheters. The results suggest that although there is a degree of antagonism between P. mirabilis and some of the other urinary tract organisms, the effects are temporary and whatever the pre-existing urinary microbiota, infection with P. mirabilis is thus likely to lead to catheter encrustation and blockage.

Development of a multiplex PCR assay for identification of Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis.

Abstract: A multiplex PCR assay has been developed for the identification of the six common Campylobacter taxa associated with human gastroenteritis and/or septicaemia, namely Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. The assay was developed using a combination of newly designed and published primers. It provided a specific PCR product for each of the five Campylobacter species and the one subspecies, and each of the PCR products was sufficiently distinguished by a difference in size by agarose gel electrophoresis. On evaluation of efficacy with 142 Campylobacter strains, the assay correctly identified all strains as 1 of the 6 Campylobacter taxa. This multiplex PCR assay is a rapid, simple and practical tool for identification of the six Campylobacter taxa commonly associated with gastroenteritis and/or septicaemia in humans, and offers an effective alternative to conventional biochemical-based assays.

Enteroaggregative Escherichia coli: epidemiology, virulence and detection.

Abstract: Enteroaggregative Escherichia coli (EAEC) is a subgroup of diarrhoeagenic E coli (DEC) that during the past decade has received increasing attention as a cause of watery diarrhoea, which is often persistent EAEC have been isolated from children and adults worldwide As well as sporadic cases, outbreaks of EAEC-caused diarrhoea have been described The definition of EAEC is the ability of the micro-organism to adhere to epithelial cells such as HEp-2 in a very characteristic ‘stacked-brick’ pattern Although many studies searching for specific virulence factor(s) unique for this category of DEC have been published it is still unknown why the EAEC cause persistent diarrhoea In addition, the aggregative property of EAEC causes a lot of problems in serotyping due to the cells auto-agglutinating The gold standard for identification of EAEC includes isolation of the agent and an adherence assay using tissue culture, viz HEp-2 cells This assay is in most cases reliable; however, emergence of ‘atypical’ EAEC has been described in several publications In addition, the HEp-2 assay is time consuming, demands a tissue culture lab and trained staff Several molecular biological assays have been described, however, none show 100 % specificity

Real-time TaqMan PCR for rapid detection and typing of genes encoding CTX-M extended-spectrum β-lactamases

Abstract: The prevalence of CTX-M-producing members of the Enterobacteriaceae is increasing worldwide. A novel, multiplex, real-time TaqMan PCR assay to detect and type bla(CTX-M) genes is described which is an improvement on previously described techniques with respect to reduced assay time, elimination of the need for protracted post-PCR processing and the convenience of a single reaction vessel. Based on beta-lactam antibiogram and MIC data, 478 of 1279 Enterobacteriaceae isolates from clinical blood and urine culture specimens were selected and tested for extended-spectrum beta-lactamase (ESBL) production using phenotypic methods. The new TaqMan assay detected and typed bla(CTX-M) genes in 21 of 28 ESBL-producing isolates.

Phospholipase, proteinase and haemolytic activities of Candida albicans isolated from oral cavities of patients with type 2 diabetes mellitus

Abstract: The aim of this study was to biotype and characterize phospholipase, proteinase and haemolytic activities of oral Candida albicans isolates from 210 Chinese patients with type 2 diabetes mellitus (DM) and 210 age- and sex-matched healthy controls. Seventy-six and 50 C. albicans isolates were obtained from type 2 DM patients and controls, respectively, using the oral rinse technique. The isolates were characterized with a biotyping system based on enzyme profiles, carbohydrate assimilation patterns and boric acid resistance of the yeasts, and the isolates were further tested for in vitro phospholipase, proteinase and haemolytic activities. The major biotypes of C. albicans isolates from the type 2 DM and control groups were A1R (42.1 %) and J1R (36.0 %), respectively. Significantly higher proteinase and haemolytic activities were found in the isolates from the type 2 DM group (P or =10 years of DM history than those with <10 years (P<0.05). Haemolytic activity was significantly higher in isolates from female DM patients than in those from male counterparts (P<0.05). These data provide evidence of increased extracellular enzyme activity in Candida isolates taken from DM patients.

Clinical and microbiological features of nocardiosis 1997-2003.

Abstract: Nocardiosis has been believed to be caused by the members of the Nocardia asteroides complex and the Nocardia brasiliensis species. However, recent advances in genotypic identification have shown that the genus exhibits considerable taxonomic complexity and the phenotypic markers used in the past for its identification can be ambiguous. The aim of this study was to assess the species distribution of Nocardia isolates and to determine whether there are differences in pathogenicity or antimicrobial susceptibility between the different species identified. Nocardia isolates obtained over a 7 year period were retrospectively reviewed. The isolates were identified genotypically, their antibiotic susceptibility was tested and the clinical data of the 27 patients were retrieved. Eight different Nocardia species were identified: Nocardia farcinica (n=9), Nocardia abscessus (n=6), Nocardia cyriacigeorgica (n=6), Nocardia otitidiscaviarum (n=2), Nocardia nova (n=1), N. nova complex (n=1), Nocardia carnea (n=1) and Nocardia transvalensis complex (n=1). All species were susceptible to co-trimoxazole but different patterns of susceptibility to other agents were observed. All patients had active comorbidities at the time of infection. A total of 19 patients were immunosuppressed, due to human immunodeficiency virus infection, chronic corticosteroid therapy, immunosuppressive therapy or haematological malignancies. Six patients displayed a Charlson comorbidity index score above 4. Global mortality was 50 % while attributable mortality was 34.6 %. Patients infected with N. farcinica--the most resistant species--had the highest Charlson index score and the highest mortality rate. Accurate identification of the species and susceptibility testing of Nocardia isolates may play an important role in diagnosis and treatment.

A molecular analysis of the bacteria present within oral squamous cell carcinoma.

Abstract: In order to characterize the bacterial microbiota present within oral cancerous lesions, tumorous and non-tumorous mucosal tissue specimens (approx. 1 cm(3)) were harvested from ten oral squamous cell carcinoma (OSCC) patients at the time of surgery. Any microbial contamination on the surface of the specimens was eliminated by immersion in Betadine and washing with PBS. Bacteria were visualized within sections of the OSCC by performing fluorescent in situ hybridization with the universal oligonucleotide probe, EUB338. DNA was extracted from each aseptically macerated tissue specimen using a commercial kit. This was then used as template for PCR with three sets of primers, targeting the 16S rRNA genes of Spirochaetes, Bacteroidetes and the domain Bacteria. PCR products were differentiated by TA cloning and bacterial species were identified by partial sequencing of the 16S rRNA gene fragments. A total of 70 distinct taxa was detected: 52 different phylotypes isolated from the tumorous tissues, and 37 taxa from within the non-tumorous specimens. Differences between the composition of the microbiotas within the tumorous and non-tumorous mucosae were apparent, possibly indicating selective growth of bacteria within carcinoma tissue. Most taxa isolated from within the tumour tissue represented saccharolytic and aciduric species. Whether the presence of these bacteria within the mucosa has any bearing on the carcinogenic process is a concept worthy of further investigation.

Genetically similar isolates of Klebsiella pneumoniae serotype K1 causing liver abscesses in three continents

Abstract: The magA gene was sought in hypermucoviscous isolates of Klebsiella spp., the Klebsiella K serotype reference strains and in isolates of the K1 serotype of Klebsiella pneumoniae from the UK, Hong Kong, Israel, Taiwan and Australia. Only K1 isolates were PCR positive for magA; this gene was found in all such isolates tested. Hypermucoviscosity was not confined to magA positive isolates, nor was it found in all magA positive isolates. Comparison of XbaI PFGE profiles revealed that most (19/23) of the magA positive isolates clustered within 72 % similarity, with a further subcluster of isolates, from three different continents, clustering within >80 %. All of the 16 isolates tested within the main cluster had the same sequence type (ST 23) by multilocus sequence typing, with the exception of one isolate, which had a single nucleotide difference at one of the seven loci. This study indicates that a genotype strongly associated with highly invasive disease in Taiwan, where large numbers of cases have been reported, is geographically very widespread.

Multiplex PCR assay for identification of Corynebacterium pseudotuberculosis from pure cultures and for rapid detection of this pathogen in clinical samples

Abstract: Corynebacterium pseudotuberculosis is the aetiological agent of caseous lymphadenitis (CLA), a debilitating disease of sheep and goats. Accurate diagnosis of CLA primarily relies on microbiological examination, followed by biochemical identification of isolates. In an effort to facilitate C. pseudotuberculosis detection, a multiplex PCR (mPCR) assay was developed targeting three genes of this bacterium: the 16S rRNA gene, rpoB and pld. This method allowed efficient identification of 40 isolates of this bacterium that had been identified previously by biochemical testing. Analysis of taxonomically related species did not generate the C. pseudotuberculosis mPCR amplification profile, thereby demonstrating the assay's specificity. As little as 1 pg of C. pseudotuberculosis genomic DNA was detected by this mPCR assay, demonstrating the sensitivity of the method. The detection limit in clinical samples was estimated to be 10(3) c.f.u. C. pseudotuberculosis could be detected directly in pus samples from infected sheep and goats (n=56) with a high diagnostic sensitivity (94.6 %). The developed assay significantly improves rapid C. pseudotuberculosis detection and could supersede bacteriological culture for microbiological and epidemiological diagnosis of CLA.

Formation and properties of in vitro biofilms of ica-negative Staphylococcus epidermidis clinical isolates

Abstract: Coagulase-negative Staphylococcus epidermidis has become the leading cause of foreign-body infections due to its biofilm formation on all kinds of medical-device surfaces. The biofilm development of S. epidermidis includes two steps: the initial attachment phase and the accumulative phase. In the accumulative phase, the polysaccharide intercellular adhesin (PIA), encoded by the icaADBC locus, is the major component mediating intercellular adhesion. However, recent studies have revealed the emergence of biofilm-positive/ica-negative staphylococcal clinical isolates. In this report, two ica-negative S. epidermidis clinical strains, SE1 and SE4, exhibited their heterogeneity in biofilm architecture under static and flow conditions, compared with the biofilm-positive/ica-positive RP62A strain. Strains with this type of absence of PIA from biofilms also displayed intermediate resistance to vancomycin. More importantly, the cells of both SE1 and SE4 strains were more tolerant than those of RP62A to exposure to lysostaphin and vancomycin. Based on the results, it is suggested that the biofilm-positive/ica-negative strain represents a newly emergent subpopulation of S. epidermidis clinical strains, arising from selection by antibiotics in the nosocomial milieu, which displays a survival advantage in its host environment. Recent epidemiological data support this suggestion, by showing a tendency towards an increasing proportion of this subpopulation in staphylococci-associated infections.

Evaluation of real-time PCR and conventional diagnostic methods for the detection of Clostridium difficile-associated diarrhoea in a prospective multicentre study.

Abstract: In this prospective multicentre study, an enzyme-linked fluorescent assay (VIDAS CDA2; bioMerieux), an enzyme-linked assay [Premier Toxins A and B (PTAB); Meridian] and an in-house real-time PCR amplifying the tcdB gene were compared with the cell cytotoxicity assay used as the 'gold standard' for diagnosis of Clostridium difficile-associated diarrhoea (CDAD). Faecal samples from patients with a request for C. difficile diagnosis and samples from patients with diarrhoea hospitalized for at least 72 h were collected for 3 consecutive months from four university medical centres in The Netherlands. In total, 547 faecal samples were obtained from 450 patients. Of 540 samples available for all of the assays, 84 (15.6 %) showed a positive result in one or more assays. The cell cytotoxicity assay was positive in 31 samples (5.7 %) from 28 patients. A diagnosis of CDAD was not considered by the physician in 5 (23.8 %) of 21 patients with CDAD who were hospitalized for at least 72 h. Compared with the cell cytotoxicity assay, the sensitivity of VIDAS, PTAB and PCR was 83.9, 96.8 and 87.1 %, respectively. The specificity of VIDAS, PTAB and PCR was 97.1, 94.3 and 96.5 %, respectively. The positive and negative predictive values for VIDAS, PTAB and PCR were 63.4 and 99.0 %, 50.9 and 99.8 %, and 60.0 and 99.2 %, respectively. Of 61 samples that were positive in one, two or three of the assays, 56 were available for discordance analysis. Discordance analysis was performed by culture of toxinogenic strains. The concordance of VIDAS, PTAB and PCR with culture was 53.6 % (30/56), 55.4 % (31/56) and 71.4 % (40/56), respectively. It was concluded that real-time PCR had the highest concordance with toxinogenic culture and is therefore the preferred method for diagnosing CDAD in faecal samples. It was also concluded that diagnosis of patients with diarrhoea who have been hospitalized for more than 72 h should focus mainly on the detection of C. difficile, irrespective of the physician's request.

Multilocus sequence typing of Staphylococcus aureus isolates recovered from cows with mastitis in Brazilian dairy herds.

Abstract: Staphylococcus aureus is a major pathogen associated with bovine mastitis, one of the most important infectious diseases occurring in dairy cattle herds worldwide. In the present study, S. aureus isolates recovered from cows with mastitis in dairy herds located in the south-east of Brazil were genotyped by PFGE and multilocus sequence typing (MLST). PFGE identified 60 pulsotypes (PTs), which were found to be distributed among six clonal complexes (CCs) by MLST. All PTs with similarity percentages greater than 65 % belonged to the same CC. Most of the PTs belonged to CC126 (n=28) and CC97 (n=19), which were represented by 91 % of the isolates. These CCs have also been recovered from cows with mastitis in countries located in different continents, but they have rarely been isolated from human specimens. Few isolates were represented by PTs belonging to CCs that are frequently isolated from human specimens (CC1, CC5 and CC30). These data reinforce the hypothesis that a limited number of S. aureus CCs are responsible for most bovine mastitis cases internationally. Specific features of the specialized clones should be studied for use as future targets of mastitis control measures.

Sequential multiplex PCR for determining capsular serotypes of pneumococci recovered from Brazilian children.

Abstract: Capsular serotype surveillance of clinical isolates of Streptococcus pneumoniae is essential for evaluation of the potential impact of introducing multivalent capsular serotype-based vaccines in Latin America. Here, a previously described sequential multiplex PCR method was revised for optimal targeting of prevalent serotypes in Latin America. The revised protocol successfully serotyped 139/147 pneumococci (94.6%) from Brazilian children, demonstrating a labour-efficient, accurate method requiring only conventional PCR capability.

Species distribution and antifungal susceptibility of Candida bloodstream isolates in Kuwait: a 10-year study.

Abstract: Bloodstream infections due to Candida species are important complications in severely ill hospitalized patients. This study presents data on species distribution and antifungal susceptibility profiles of Candida bloodstream isolates obtained from Kuwait during a 10-year period. All the bloodstream isolates were identified to species level by the germ tube test and carbohydrate assimilation profile using the VITEK 2 yeast identification system. Using E-test strips for amphotericin B, fluconazole, 5-flucytosine and voriconazole, MICs were determined on RPMI agar supplemented with 2% glucose. The MIC breakpoints for resistance were based on Clinical and Laboratory Standards Institute criteria or those published by reference laboratories, and were as follows: amphotericin B, >1 m gm l ”1 ; fluconazole, ¢64 m gm l ”1 ; 5-flucytosine, ¢32 m gm l ”1 ; and voriconazole, 4 m gm l ”1 . In all, 607 bloodstream yeast isolates were obtained over the past 10 years in Kuwait. Candida albicans was the predominant species (39.5%), followed by Candida parapsilosis (30.6%), Candida tropicalis (12.4%), Candida glabrata (5.6%) and Candida krusei (1.6%). All C. albicans, C. tropicalis and C. glabrata isolates were susceptible to amphotericin B. Of 186 isolates of C. parapsilosis tested, only four (2%) exhibited an MIC for amphotericin B of >1 m gm l ”1 . Resistance to fluconazole was observed in nine (3.8%) C. albicans isolates, two (5.8%) C. glabrata isolates and four (40%) C. krusei isolates. Resistance to 5-flucytosine was observed in two (0.8%) C. albicans isolates, seven (9.3%) C. tropicalis isolates, three (1.6%) C. parapsilosis isolates and all ten (100%) C. krusei isolates. All the isolates of C. albicans, C. tropicalis, C. parapsilosis, C. glabrata and C. krusei were susceptible to voriconazole, including those resistant to fluconazole. Although amphotericin B and fluconazole are widely used in clinical practice in Kuwait, resistance to these drugs remained low.

Evaluation of susceptibility of Trichophyton mentagrophytes and Trichophyton rubrum clinical isolates to antifungal drugs using a modified CLSI microdilution method (M38-A).

Abstract: Onychomycosis is a common adult human mycosis, and dermatophytes of the Trichophyton genera are the most common causative agent. Many antimycotic agents are safe and highly effective for the treatment of dermatophytosis, and are available for clinical practice. Successful treatment depends on the ability of antifungal drugs to eradicate the fungal isolates. The aim of this work was to determine the MICs of four antifungal drugs (fluconazole, itraconazole, terbinafine and griseofulvin) recognized for ungual dermatophytosis treatment caused by Trichophyton species, especially Trichophyton mentagrophytes and Trichophyton rubrum. MICs were determined using a broth microdilution method in accordance with Clinical and Laboratory Standards Institute approved standard M38-A with some modifications, such as an incubation temperature of 28 degrees C, an incubation time of 7 days and inocula constituted of only microconidia. The results showed that the activities of terbinafine and itraconazole were significantly higher (MICs of <0.007-0.031 and 0.015-0.25 microg ml(-1), respectively) than other tested agents. All isolates had reduced susceptibility to fluconazole (1-64 microg ml(-1)). The MIC of griseofulvin varied among strains (MICs of 0.062-1 microg ml(-1)). The parameters adopted to perform susceptibility testing of T. rubrum and T. mentagrophytes to antifungal agents appeared to be suitable and reliable, and could contribute to the possible development of a standard protocol.

Acinetobacter baumannii lipopolysaccharides are potent stimulators of human monocyte activation via Toll-like receptor 4 signalling.

Abstract: Acinetobacter baumannii is a major nosocomial pathogen and frequent cause of hospital-acquired pneumonia, surgical wound infections and sepsis. As very little is known of the endotoxic potential of A. baumannii lipopolysaccharide (LPS) with respect to human cells or of its ability to stimulate inflammatory signalling via human Toll-like receptors (TLRs), the biological activity of these endotoxins was investigated in human monocytic THP-1 cells and in TLR-deficient HEK-293 cells transfected with human TLR2 and TLR4 constructs. Endotoxins derived from five clinical isolates of A. baumannii and one of Acinetobacter ‘genomospecies 9’ showed high potency, which was comparable to that of Escherichia coli strain R1 NCTC 13114 LPS, in the induction of the Limulus amoebocyte reaction and interleukin 8 and tumour necrosis factor alpha release from THP-1 cells. Whole UV-killed cells of A. baumannii and Acinetobacter ‘genomospecies 9’ stimulated both TLR2- and TLR4-dependent signalling, whereas pure endotoxins of all investigated strains induced signalling via TLR4, but not TLR2.

Helicobacter pylori heat-shock protein 60 induces interleukin-8 via a Toll-like receptor (TLR)2 and mitogen-activated protein (MAP) kinase pathway in human monocytes.

Abstract: Previous reports have indicated that Helicobacter pylori heat-shock protein 60 (H. pylori-HSP60), as an immunodominant antigen, induces interleukin (IL)-8 production in human monocytes. The exact mechanism by which H. pylori-HSP60 induces IL-8 production in monocytes has not been fully elucidated. In the present study, the downstream pathway by which H. pylori-HSP60 induces IL-8 secretion in human monocytic cell lines was investigated. Intact H. pylori, heat-killed H. pylori and H. pylori recombinant HSP60 (rHpHSP60) all induced the secretion of IL-8 and the activation of mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) and p38, but not c-Jun N-terminal kinase (JNK), up to 24 h in NOMO1 cells. The specific inhibitors PD98059 and U0126 (for ERK1/2 signalling) and SB203580 (for p38 MAPK signalling) down-regulated IL-8 secretion from rHpHSP60-treated NOMO1 cells. An anti-Toll-like receptor (TLR)2 antibody or TLR2 small interfering RNA (siRNA) partially inhibited the secretion of IL-8, and anti-TLR2 antibody also suppressed activation of ERK and p38 MAPK in rHpHSP60-treated NOMO1 cells. These reactions were associated with nuclear factor-kappaB (NF-kappaB)-mediated transcriptional activation, since U0126, SB203580 and the anti-TLR2 antibody decreased NF-kappaB activation. Taken together, the results suggest that ERK and p38 MAPK signalling linked to the TLR2 recognition receptor in human monocytes may be an important pathway in H. pylori-HSP60-induced IL-8 secretion.

Phylogenetic analysis of bacterial and archaeal species in symptomatic and asymptomatic endodontic infections.

Abstract: Phylogenetic analysis of bacterial and archaeal 16S rRNA was used to examine polymicrobial communities within infected root canals of 20 symptomatic and 14 asymptomatic patients. Nucleotide sequences from ∼750 clones amplified from each patient group with universal bacterial primers were matched to the Ribosomal Database Project II database. Phylotypes from 37 genera representing Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria were identified. Results were compared to those obtained with species-specific primers designed to detect Prevotella intermedia, Porphyromonas gingivalis, Porphyromonas endodontalis, Peptostreptococcus micros, Enterococcus sp., Streptococcus sp., Fusobacterium nucleatum, Tannerella forsythensis and Treponema denticola. Since members of the domain Archaea have been implicated in the severity of periodontal disease, and a recent report confirms that archaea are present in endodontic infections, 16S archaeal primers were also used to detect which patients carried these prokaryotes, to determine if their presence correlated with severity of the clinical symptoms. A Methanobrevibacter oralis-like species was detected in one asymptomatic and one symptomatic patient. DNA from root canals of these two patients was further analysed using species-specific primers to determine bacterial cohabitants. Trep. denticola was detected in the asymptomatic but not the symptomatic patient. Conversely, Porph. endodontalis was found in the symptomatic but not the asymptomatic patient. All other species except enterococci were detected with the species-specific primers in both patients. These results confirm the presence of archaea in root canals and provide additional insights into the polymicrobial communities in endodontic infections associated with clinical symptoms.

Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification.

Abstract: A loop-mediated isothermal amplification (LAMP) assay was developed to detect Vero toxin (VT)-producing Escherichia coli rapidly (within 60 min). The 24 strains of VT-producing E. coli were successfully amplified, but 6 strains of non-VT-producing E. coli and 46 bacterial species other than E. coli were not. The sensitivity of the LAMP assay was found to be >0.7 c.f.u. per test using serogroups O157, O26 and O111 of VT-producing E. coli; this sensitivity is greater than that obtained by PCR assay. Furthermore, the LAMP assay was examined for its ability to detect VT-producing E. coli in food because of the difficulty of detection in food samples. The recovery of VT-producing E. coli by LAMP assay from beef and radish sprouts inoculated with the pathogen was high, similar to that obtained using culture methods with direct plating and/or plating after immunomagnetic separation. Although PCR assay was unable to recover VT-producing E. coli from half of the radish samples, LAMP assay was successful in most samples. In addition, VT-producing E. coli was successfully detected in cultures of the beef samples by LAMP assay, but not by the culture method. The LAMP products in naturally contaminated beef samples were analysed to confirm the specific amplification of the VT-encoding gene, and were found to show a specific ladder band pattern on agarose gel after electrophoresis. Additionally the sequences of the LAMP products coincided well with the expected sequences of the VT-encoding gene. These results indicate that the proposed LAMP assay is a rapid, specific and sensitive method of detecting the VT-producing E. coli.

Expression of biofilm-associated genes of Streptococcus mutans in response to glucose and sucrose.

Abstract: Streptococcus mutans is known as a primary pathogen of dental caries, one of the most common human infectious diseases. Exopolysaccharide synthesis, adherence to tooth surface and biofilm formation are important physiological and virulence factors of S. mutans. In vitro comparative gene expression analysis was carried out to differentiate 10 selected genes known to be mostly involved in S. mutans biofilm formation by comparing the expression under biofilm and planktonic environments. Real-time RT-PCR analyses indicated that all of the genes tested were upregulated in the biofilm compared to cells grown in planktonic conditions. The influence of simple dietary carbohydrates on gene expression in S. mutans biofilm was tested also. Among the tested genes, in the biofilm phase, the greatest induction was observed for gtf and ftf, which are genes encoding the extracellular polysaccharide-producing enzymes. Biofilm formation was accompanied by a 22-fold induction in the abundance of mRNA encoding glucosyltransferase B (GTFB) and a 14.8 -fold increase in mRNA encoding GTFC. Levels of mRNA encoding fructosyltransferase were induced approximately 11.8-fold in biofilm-derived cells. Another notable finding of this study suggests that glucose affects the expression of S. mutans GS5 biofilm genes. In spite of a significant upregulation in biofilm-associated gene expression in the presence of sucrose, the presence of glucose with sucrose reduced expression of most tested genes. Differential analysis of the transcripts from S. mutans, grown in media with various nutrient contents, revealed significant shifts in the expression of the genes involved in biofilm formation. The results presented here provide new insights at the molecular level regarding gene expression in this bacterium when grown under biofilm conditions, allowing a better understanding of the mechanism of biofilm formation by S. mutans.