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Volume 56,
Issue 6,
2007
Volume 56, Issue 6, 2007
- Pathogenicity And Virulence
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CodY-affected transcriptional gene expression of Streptococcus pyogenes during growth in human blood
More LessIn an attempt to expand the available knowledge of pathogen–host interactions during ex vivo growth of Streptococcus pyogenes (GAS) in nonimmune whole human blood, the extents to which the expression of 51 genes including regulators with known targets, established virulence factors, physiologically important transporters and metabolic enzyme genes was differentially affected in the presence or absence of a functional codY gene were determined. The results obtained by quantitative real-time PCR using the M49 strain NZ131 showed that CodY influenced GAS gene activity in a dynamic fashion, with differential responses detected for 26 genes and occasionally characterized by discordance in the blood environment compared to laboratory medium. Degenerate derivatives of the recently discovered CodY box potentially serving as a cis-regulatory element for CodY action were identified in the upstream regions of 15 genes of the NZ131 genome, and these genes featured sequence motifs identical to the NZ131 CodY box in all completely sequenced S. pyogenes genomes. As none of these genes represented a genuine virulence factor, it seems likely, therefore, that the observed differential transcription of the majority of virulence genes was caused by indirect actions of CodY as part of a regulatory network.
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Molecular characterization and analysis of a gene encoding the acidic repeat protein (Arp) of Treponema pallidum
More LessThe acidic repeat protein (arp) genes from three subspecies of the treponeme Treponema pallidum (T. pallidum subsp. pallidum, Nichols strain; T. pallidum subsp. pertenue, CDC-1 and CDC-2 strains; and T. pallidum subsp. endemicum, Bosnia A strain) were cloned and sequenced. The predicted protein sequence contained a high percentage of glutamic acid, hence the name acidic repeat protein, or Arp. The protein had a potential membrane-spanning domain and a signal peptidase I site. The gene from the Nichols strain of T. pallidum subsp. pallidum contained a set of 14 nearly identical repeats of a 60 bp sequence, which occupied ∼51 % of the length of the gene. Analyses of arp from laboratory strains showed that the 5′ and 3′ ends of the genes were conserved, but there was considerable heterogeneity in the number of repeats of this 60 bp sequence. Based on amino acid variations, the 14 sequence repeats could be classified into three types, which were named type I, type II and type III repeats. The type II repeat was the most common in the strains examined. The arp gene of the Nichols strain was subsequently cloned into the expression vector pBAD/TOPO ThioFusion. The expressed protein was detected in a Western blot assay using rabbit immune sera produced against T. pallidum, or synthetic peptides derived from the repeat sequences. Using an ELISA, rapid plasma reagin (RPR) test-positive sera reacted with synthetic peptides derived from the repeat region but not with peptides derived from N and C termini of the Arp protein. These results show that the Arp protein is immunogenic and could prove to be a useful target for serological diagnosis of T. pallidum infection.
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Comparison of virulence-associated in vitro properties of typed strains of Campylobacter jejuni from different sources
Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. This blinded study was undertaken with 40 C. jejuni isolates from different sources to determine their haemolytic, cytotoxic and adhesion and invasion activities towards mammalian cells. The results were correlated with source of isolation and genetic makeup by amplified fragment length polymorphism (AFLP) typing. The isolates had variable degrees of haemolytic activity against rabbit erythrocytes and cytotoxicity towards CaCo-2, HeLa and Vero cells. The data indicated that the haemolytic and cytotoxic activities were due to separate factors. A range of cytotoxicity was exhibited, whereby some strains had no activity against the target cells and others had activity against all three cell lines. Certain strains had activity against CaCo-2 cells but little or no activity against the other cells, while others exhibited the opposite phenotype. The data suggested that the cytotoxicity assay with the different cell lines may have detected more than one cytotoxin. A wide variation between isolates was observed for both adherence and invasion with all three cell lines, yet, overall, the strains showed a significantly greater invasion capacity for CaCo-2. There was no clear relationship between source of isolation or disease manifestation and possession of statistically significantly higher levels of particular virulence-associated factors although, in some cases, a correlation between cytotoxicity and cell invasion was evident. Five AFLP clusters, each representing two to eleven isolates with similar profiles, were observed at the 90 % similarity level. Some AFLP groups contained isolates with a common serotype, but each group had C. jejuni isolates from more than one source with the exception of group IV, which contained only human isolates. Isolates with high cytotoxic activity against CaCo-2 cells were confined to groups I, III and IV and a group of unrelated strains (U). Group II isolates had uniformly low cytotoxicity. Isolates in groups I, V and U were more invasive for CaCo-2 cells than isolates in groups II, III and IV. The strain differences in cytotoxicity or invasion did not correlate with source of isolation.
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Use of a colorimetric assay to measure differences in cytotoxicity of Mycobacterium tuberculosis strains
Several techniques have been used to quantify the cytotoxicity produced by Mycobacterium tuberculosis bacilli on cell monolayers; however, they are semi-quantitative or time consuming. Herein, a method based on crystal violet (CV) uptake by THP-1 cell monolayers is described. This colorimetric method quantifies the cytotoxic effect as a function of the number of remaining cells after the infection with M. tuberculosis. Since this micro-organism is not stained by the dye, it does not produce a background that affects absorbance readings. As determined by CV assay (CVA), M. tuberculosis strain H37Rv destroyed 10.5 % of THP-1 cell monolayers at 24 h and 50.52 % at 72 h, while M. tuberculosis strains lacking the complete phospholipase C locus produced a reduced cytotoxic effect. The damage estimated by microscopy corresponded to the effect quantified by CVA. The results show that the use of CVA is a rapid, sensitive and reliable quantitative assay to measure the cytotoxicity of different M. tuberculosis strains.
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Diversity of biofilms produced by quorum-sensing-deficient clinical isolates of Pseudomonas aeruginosa
The quorum-sensing (QS) systems control several virulence attributes of Pseudomonas aeruginosa. Five QS-deficient P. aeruginosa clinical isolates (CI) that were obtained from wound (CI-1), tracheal (CI-2, CI-3, CI-4) and urinary tract (CI-5) infections had previously been characterized. In this study, a flow-through continuous-culture system was utilized to examine in detail the biofilms formed by these isolates in comparison with the P. aeruginosa prototrophic strain PAO1. Analysis of the biofilms by confocal laser scanning microscopy and COMSTAT image analysis at 1 and 7 days post-inoculation showed that the isolates produced diverse biofilms. In comparison with PAO1, the CI produced biofilms that scarcely or partially covered the surface at day 1, although CI-1 produced larger microcolonies. At day 7, CI-2 and CI-4 produced mature biofilms denser than that produced by PAO1, while the biofilm formed by CI-1 changed very little from day 1. CI-1 was defective in both swarming and twitching motilities, and immunoblotting analysis confirmed that it produced a reduced level of PilA protein. The twitching-motility defect of CI-1 was not complemented by a plasmid carrying intact pilA. In the 48 h colony biofilm assay, the CI varied in susceptibility to imipenem, gentamicin and piperacillin/tazobactam. These results suggest that: (1) the isolates produced biofilms with different structures and densities from that of PAO1; (2) biofilm formation by the isolates was not influenced by either the isolation site or the QS deficiencies of the isolates; (3) the behaviour of CI-1 in the different biofilm systems may be due to its lack of swarming motility and type IV pilus-related twitching motility.
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- Diagnostics, Typing And Identification
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Identification of a group of shigella-like isolates as Shigella boydii 20
Infections by Shigella species are an important cause of diarrhoeal disease worldwide. Of 4198 Shigella isolates received by the French National Reference Centre for Escherichia coli and Shigella, 180 from patients with diarrhoea and dysentery in 2000–2004 did not react with any available polyclonal rabbit antisera used to identify the established Shigella serogroups. This study describes the molecular and phenotypic characteristics of these isolates in seroagglutination tests, molecular serotyping (rfb-RFLP and fliC-RFLP), ribotyping, detection of invasivity and enterotoxins genes, and antibiotic sensitivity. All isolates gave biochemical reactions typical of Shigella boydii, were mannitol-positive and indole-negative. They all carried invasion-associated genes, enterotoxin 2 [ShET-2] and an IS630 sequence. They had a unique ribotype that was distinct from all other Shigella and E. coli patterns. Further characterization by rfb-RFLP clearly distinguished this serogroup from all other Shigella or E. coli O-groups. The fliC-RFLP pattern corresponded to P4, an F-pattern which is associated with 10 different serogroups of S. boydii. A new antiserum prepared against strain 00-977 agglutinated all 180 isolates and cross-agglutination and absorption studies with anti-00-977 serum and anti-CDC 99-4528 (reference for the newly described S. boydii serogroup 20) serum showed identical antigenic structure. Furthermore, strains 00-977 and CDC 99-4528 had the same molecular serotype, ribotype and virulence genes.
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Rapid characterization of the normal and disturbed vaginal microbiota by application of 16S rRNA gene terminal RFLP fingerprinting
More LessBacterial vaginosis (BV) is a prevalent infection in women of reproductive age associated with numerous sequelae, including preterm delivery, amniotic fluid infections and an increased risk of acquiring sexually transmitted diseases. The vaginal microbiota in BV patients is characterized by a shift from lactobacilli to a diverse spectrum of mostly anaerobic bacteria. In this study, terminal restriction fragment length polymorphism (T-RFLP) was used to characterize the vaginal bacterial communities from 50 women with BV and 20 healthy subjects. In the BV samples, 23 species or phylotypes from 17 genera could be identified, including Atopobium vaginae, Megasphaera sp., Lactobacillus iners, Gardnerella vaginalis and three recently described phylotypes from the order Clostridiales. The number of detected species or phylotypes was on average 6.3 per sample (range 2–14). In contrast, in normal samples, only Lactobacillus species could be identified. In conclusion, T-RFLP provides a rapid and reliable technique to investigate the diversity of the predominant vaginal microbiota and allows differentiation of the flora of BV and healthy women. As such, T-RFLP may be helpful both in the diagnosis of BV from vaginal fluids and in a better understanding of the bacterial succession involved in the aetiology of BV.
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Evaluation of Albicans ID2 and Biggy agar for the isolation and direct identification of vaginal yeast isolates
More LessIn this study, 250 vaginal samples from patients with vulvovaginal candidosis were inoculated onto two chromogenic media, Albicans ID2 and Biggy agar, as well as onto Sabouraud chloramphenicol agar, yielding a total of 63 yeast (25.2 %) on all three media. These strains were identified as Candida glabrata in 20 (31.8 %) samples, Candida albicans in 15 samples (23.8 %), Candida tropicalis in 10 samples (15.9 %), Candida krusei in five samples (7.9 %), Candida kefyr in five samples (7.9 %), Candida dubliniensis in four samples (6.3 %), Candida parapsilosis in two samples (3.2 %) and Candida guilliermondii in two samples (3.2 %). Mixed fungal cultures and bacterial growth or filamentous fungi were not detected on any of the selected media. The sensitivity and specificity of the Albicans ID2 and Biggy agar with regard to the identification of C. albicans were 80.0 and 64.6 %, and 86.7 and 56.3 %, respectively. This study showed these two chromogenic media to be as effective as Sabouraud chloramphenicol agar with respect to fungal detection. However, neither Albicans ID2 nor Biggy agar was sufficient for reliable differentiation of yeasts to the species level.
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Rapid quantification of hepatitis B virus DNA by direct real-time PCR from serum without DNA extraction
More LessThe purpose of this study was to quantify hepatitis B virus DNA by direct real-time PCR from serum without the need for DNA extraction. Crossing point (Cp) values were determined automatically using the second derivative maximum mode. Since serum samples from patients are inevitably haemolysed, lipaemic or icteric, the interference of endogenous substances from the serum in real-time PCR was evaluated. The result showed that, although serum protein quenched the intensity of fluorescence, the Cp value adopted to calculate the quantity of DNA copies remained unchanged. Importantly, real-time PCR from serum with or without DNA extraction reached a high level of concordance. This direct serum PCR method without the DNA extraction and gel electrophoresis allows for substantial labour and cost savings. In addition, it is also suitable for rapid DNA quantification during clinical diagnosis.
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Real-time PCRs for detection of Trichomonas vaginalis β-tubulin and 18S rRNA genes in female genital specimens
More LessTrichomonas vaginalis is the cause of one of the most common types of vaginitis, trichomoniasis. The incidence of trichomoniasis in developed countries has decreased substantially during the past decade, but high prevalence of this disease can still be found in rural and remote areas of Australia. Clinical manifestations of symptomatic women are generally non-specific, but include vaginal discharge, vaginitis and irritation. T. vaginalis infection has also been linked to the increased risk of human immunodeficiency virus transmission. Current diagnosis of T. vaginalis relies on the visualization of motile organisms in a wet-mount preparation. Culture is used mainly in reference laboratories. The latter two methods require viable organisms and would not be suitable for use where transportation of specimens can be delayed. Two real-time fluorescence resonance energy transfer (FRET) hybridization probe PCR assays were used in this study to test for T. vaginalis DNA, targeting the β-tubulin and 18S rRNA genes. We tested 500 randomly selected female patients, in an STD setting, for T. vaginalis DNA. The FRET PCRs targeting the β-tubulin gene and the 18S rRNA gene detected 96 % (85/89) and 100 % (89/89) , respectively, of the positive specimens (first-void urine sample or genital swabs). Wet-mount microscopy was performed on 76 of these PCR-positive specimens and showed a sensitivity of 38 % (29/76). The prevalence, by PCR, of trichomoniasis was 18 % in this study. The two real-time PCRs developed in this study, targeting different genetic regions of the organism, provide a rapid, sensitive and specific diagnosis of T. vaginalis infection.
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Species identification and strain differentiation of clinical Candida isolates using the DiversiLab system of automated repetitive sequence-based PCR
The DiversiLab system, which uses repetitive sequence-based PCR (rep-PCR) to genotype micro-organisms, was evaluated as a molecular typing tool for members of the genus Candida. Initially, 41 clinical Candida spp. (7 Candida krusei, 10 Candida parapsilosis, 7 Candida albicans, 10 Candida tropicalis and 7 Candida glabrata), previously identified at the species level by morphological and biochemical analysis, were analysed with the DiversiLab system. Species identification was confirmed by DNA sequence analysis of the contiguous internal transcribed spacer (ITS) region (ITS1–5.8S–ITS2). On the basis of an 80 % similarity threshold, rep-PCR consistently clustered like species and this set of isolates, along with five ATCC reference strains, was used to create a DNA fingerprint library with the DiversiLab software. Subsequently, an additional set of 115 clinical Candida isolates, identified biochemically as C. albicans (n=94), C. glabrata (n=8), C. parapsilosis (n=5), C. tropicalis (n=3), C. krusei (n=3) and Candida lusitaniae (n=2), isolated at a regional reference laboratory, were typed using DiversiLab. One hundred and six of these isolates clustered with members of the Candida library at >80 % similarity and thus could be assigned species identification, and initial calculations showed that identification via rep-PCR fingerprinting was 95 % concordant (101/106) with the biochemical/morphological identification. However, ITS region sequencing of the five discrepant samples, as well as the nine isolates that were <80 % similar to the database samples, showed that nine were misidentified with traditional biochemical/morphological methods. For the misidentified isolates, the sequence-based identification was in agreement with the DiversiLab clustering, yielding an actual correlation of >99 %. As traditional techniques can take several days to provide information about Candida at the genus/species level, genotyping with the DiversiLab system holds promise for more-rapid speciation of members of this genus. This system may also be useful for epidemiological studies such as source tracking that require Candida subspecies discrimination.
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Typing of Histoplasma capsulatum strains by fatty acid profile analysis
More LessThe performance of fatty acid profiling for strain differentiation of Histoplasma capsulatum was assessed. Total fatty acids were isolated from the yeast-phase cells of seven stock and two previously unreported clinical strains of H. capsulatum var. capsulatum, as well as from one unreported clinical strain and one stock strain of H. capsulatum var. duboisii, and one strain of each of three other dimorphic zoopathogenic fungal species, Blastomyces dermatitidis, Paracoccidioides brasiliensis and Sporothrix schenckii. Different colony morphology and pigmentation types of the H. capsulatum strains were also included. The most frequently occurring fatty acids were oleic, palmitic, stearic and linoleic acids. There were variations in the relative percentage fatty acid contents of H. capsulatum strains that could be used for strain identification and discrimination. Differentiation between H. capsulatum strains was achieved by the comparison of detected fatty acids accompanied by principal component analysis using calculated Varimax-rotated principal component loadings. Statistical analysis yielded three major principal components that explained over 94 % of total variance in the data. All the strains of H. capsulatum var. capsulatum RFLP classes II and III were grouped into two distinct clusters: the heterogenic RFLP class I formed a large, but also well-defined group, whereas the outgroup strains of H. capsulatum var. duboisii, B. dermatitidis, P. brasiliensis and S. schenckii were shifted away. These data suggest that fatty acid profiling can be used in H. capsulatum strain classification and epidemiological studies that require strain differentiation at the intraspecies level.
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- Antimicrobial Agents And Chemotherapy
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In vitro activity of piperacillin/tazobactam and ertapenem against Bacteroides fragilis and Escherichia coli in pure and mixed cultures
Ertapenem and piperacillin/tazobactam are β-lactam antibiotics with a broad spectrum of activity used for the treatment of mixed infections in which Bacteroides fragilis and Escherichia coli play an important aetiological role. In this study, the activities of piperacillin/tazobactam and ertapenem (MIC and time–kill kinetics) against these bacteria were compared. MICs were determined by the agar dilution method, and the time and slope of time–kill curves were analysed. In the in vitro pharmacodynamic assays, pure and mixed cultures of E. coli and B. fragilis were exposed to peak concentrations of ertapenem (8.0 μg ml−1) and piperacillin/tazobactam (64.0/8.0 μg ml−1) for 48 h. Treatment with ertapenem reduced the viability of E. coli and/or B. fragilis by 3 logs in all experiments, whereas piperacillin/tazobactam only affected the viability of B. fragilis. Both drugs exhibited their fastest rates of killing when bacteria were grown in mixed cultures. According to the results, ertapenem exhibited activity similar to that of piperacillin/tazobactam against B. fragilis alone or in mixed culture. However, ertapenem exhibited a markedly higher activity against E. coli alone or in combination with B. fragilis relative to piperacillin/tazobactam.
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Two diallyl sulphides derived from garlic inhibit meticillin-resistant Staphylococcus aureus infection in diabetic mice
More LessThe inhibitory effect of diallyl sulphide (DAS) and diallyl disulphide (DADS) against meticillin-resistant Staphylococcus aureus (MRSA) infection in diabetic mice was studied. The influence of these agents on the plasma levels of fibronectin, C-reactive protein (CRP), fibrinogen, interleukin (IL)-6 and tumour necrosis factor-α (TNF-α), and on the activity of plasminogen activator inhibitor-1 (PAI-1), antithrombin III (AT-III) and protein C, in MRSA-infected diabetic mice was examined. To induce diabetes, mice were treated intraperitoneally with streptozotocin for 5 consecutive days. Ten clinical MRSA isolates obtained from infected patients were used in this study. Diabetic mice were infected by injecting 200 μl MRSA/PBS suspension containing 107 c.f.u. via the tail vein. At day 4 post-infection, 200 μl DAS or DADS was administrated twice orally with an interval of 12 h. Eight hours after each administration, the blood and organs of mice were collected. Results showed that DAS and DADS significantly decreased MRSA viability in the kidney (P <0.05), with administration of each agent twice showing a greater inhibitory effect than when given once (P <0.05). MRSA infection in diabetic mice significantly elevated the plasma levels of IL-6 and TNF-α (P <0.05). DAS or DADS given once did not affect the plasma levels of IL-6 and TNF-α (P >0.05); however, DAS or DADS given twice significantly decreased the plasma levels of both IL-6 and TNF-α (P <0.05). DAS and DADS treatments also significantly reduced the plasma levels of CRP, fibronectin and fibrinogen (P <0.05). DAS or DADS treatment did not affect PAI-1 activity (P >0.05), but DAS or DADS given twice significantly increased AT-III activity (P <0.05). DADS given twice elevated protein C activity (P <0.05). MRSA infection significantly increased malondialdehyde levels in the kidney and spleen (P <0.05), and these levels were significantly decreased by treatment with DAS or DADS (P <0.05). These data suggest that DAS and DADS could provide multiple protective functions against MRSA infection in diabetic mice.
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Role of AmpD, OprF and penicillin-binding proteins in β-lactam resistance in clinical isolates of Pseudomonas aeruginosa
More LessIn this study, the mechanisms leading to increased chromosomal AmpC β-lactamase expression and the contributory roles of the outer-membrane protein OprF and penicillin-binding proteins were analysed in 33 characterized clinical isolates of Pseudomonas aeruginosa. The genes ampD and ampE were analysed by PCR and DNA sequencing. Expression of the gene oprF was assessed using real-time RT-PCR, and penicillin-binding proteins were analysed using a chemiluminescence assay. Several of the isolates with increased ampC expression had major deletions affecting ampD, although in some isolates the mechanism of increased ampC expression could not be ascertained. Occasional isolates had increased expression of both ampC and oprF but remained susceptible to cephalosporins, suggesting that increased β-lactamase activity could not offset increased outer-membrane permeability. There were no discernible changes in penicillin-binding proteins. Genomic deletions in ampD were observed in selected clinical isolates of P. aeruginosa with increased expression of the AmpC β-lactamase. For some isolates, cephalosporin resistance was dependent upon the interplay of ampC and oprF expression.
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Post-antifungal effects of the antifungal compound 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate on Aspergillus fumigatus
More LessThe post-antifungal effect (PAFE) of the antifungal compound 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate (DHP) upon Aspergillus fumigatus was investigated. The conidia of A. fumigatus were exposed to DHP at concentrations of 1× and 4× MIC90 for variable times at 37 °C. Amphotericin B (AmB)-treated or drug-free controls were included in the study. DHP as well as AmB exposure resulted in prolonged lag phases of the turbidimetric growth curves. Both the treatments gave rise to delayed growth, with lag phases of 11 h upon treatment with a concentration of 4× MIC90 for 4 h. Furthermore, it was observed that DHP inhibited the expression of three A. fumigatus secretory proteins of 18, 42 and 55 kDa. One protein of 42 kDa was found to be a metalloprotease, which is an important virulence factor. Analysis of time-dependent antigenic profiles showed the early expression of high-molecular-mass antigens. Expression of low-molecular-mass antigens started after 24 h culture. The antigens of A. fumigatus that are expressed during the early phase of growth were observed to be adversely affected after treatment with DHP. Although the mechanism of action of DHP to inhibit these proteins/antigens is unknown, the observations may be valuable to understand their role in the virulence of the pathogen, as well as the antigen-mediated responses caused by A. fumigatus.
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- Epidemiology
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Pneumococci responsible for invasive disease and discharging ears in children in Sydney, Australia
The serotypes and molecular clones of penicillin-nonsusceptible Streptococcus pneumoniae (PNSP) responsible for invasive pneumococcal disease (IPD) and discharging ears in metropolitan New South Wales were characterized to form a baseline prior to introduction of the heptavalent conjugate pneumococcal vaccine in Australia. Pneumococci isolated between 1 July 2000 and 30 June 2003 in Sydney from children <15 years were tested for antibiotic susceptibilities and serotyped. Penicillin-nonsusceptible pneumococci were typed by multilocus sequence typing and BOX PCR. During this period, 97 (13.9 %) of 698 pneumococci from IPD that were serotyped were penicillin-nonsusceptible. Of 607 pneumococci from discharging ears, 157 (26.1 %) were penicillin-nonsusceptible. Serotype 14 was the predominant serotype responsible for IPD and serotype 19F predominated from discharging ears. The heptavalent vaccine serotypes accounted for 613 (87.8 %) of all invasive isolates and 420 (69.8 %) of all isolates from discharging ears. Representatives of the major international clones were present among the PNSP. The majority of serotypes and clones that showed penicillin-nonsusceptibility are present within the vaccine. Serotype switching was also noted to have occurred prior to introduction of the vaccine. This study provides a characterization of the pneumococcal serotypes associated with IPD and discharging ears that will be useful for detecting potential selective effects of the vaccine. This surveillance should be continued, as it will be important to monitor the frequency and distribution of serotypes in the post-vaccine era.
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Phenotypic and genotypic traits and epidemiological implication of Vibrio cholerae O1 and O139 strains in India during 2003
During 2003, Vibrio cholerae O1 Ogawa was the predominant serotype among diarrhoeal patients admitted to different hospitals in India. With the exception of 3 strains from Kolkata, none of 172 strains examined exhibited resistance to tetracycline, but 45.7 % showed reduced susceptibility to ciprofloxacin. Extensive molecular characterization using randomly amplified polymorphic DNA analysis, ribotyping and PFGE revealed that almost all the strains within a serogroup were clonally related. Along with the H pulsotype, a newly described L pulsotype of recently emerged O1 Inaba strains was detected among the O1 Ogawa strains from 2003. The striking similarity in their molecular properties and antibiograms indicated that at least certain clones of recently emerged Inaba strains from 2004 may have evolved from O1 Ogawa strains. This view was further supported by the detection of a nearly identical wbeT region among the O1 Ogawa and recently emerged Inaba strains, the latter differing only by a single point mutation. Since 2003, a hiatus in the isolation of serogroup O139 was observed and these strains share the same PFGE profiles as those isolated during 2000. Organization of tandemly arranged CTXEl, CTXCal and truncated CTXCal (devoid of ctxAB) prophages was unique among the majority of these O139 strains.
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Role of the plasmid-encoded tet(O) gene in tetracycline-resistant clinical isolates of Campylobacter jejuni and Campylobacter coli
More LessThe prevalence of tetracycline resistance, tetracycline MICs and tet(O) gene localization were investigated in 83 Campylobacter isolates from patients suffering from acute gastroenteritis in Germany. Combined biochemical and molecular markers identified 74 isolates (89 %) as Campylobacter jejuni, including seven atypical isolates that failed to hydrolyse hippurate, and nine isolates (11 %) as Campylobacter coli. Tetracycline resistance was detected in six out of nine Campylobacter coli isolates (67 %) and 13 out of 74 C. jejuni isolates (18 %). Low-level tetracycline resistance was observed for C. coli (MIC 16 μg ml−1 for all strains), whereas C. jejuni showed high-level resistance (MIC >256 μg ml−1 for all strains). Both low- and high-level tetracycline resistance was associated with the presence of the tet(O) gene. In C. jejuni, tet(O) was plasmid-encoded in 54 % of tetracycline-resistant isolates, whereas in C. coli, tet(O) appeared to be located on the chromosome.
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Diversity of antibiotic resistance integrative and conjugative elements among haemophili
More LessThe objective of this study was to investigate the sequence diversity in a single country of a family of integrative and conjugative elements (ICEs) that are vectors of antibiotic resistance in Haemophilus influenzae and Haemophilus parainfluenzae, and test the hypothesis that they emerged from a single lineage. Sixty subjects aged 9 months – 13 years were recruited and oropharyngeal samples cultured. Up to 10 morphologically distinct Pasteurellaceae spp. were purified, and then the species were determined and differentiated by partial sequence analysis of 16S rDNA and mdh, respectively. ICEs were detected by PCR directed at five genes distributed evenly across the ICE. These amplicons were sequenced and aligned by the neighbour-joining algorithm. A total of 339 distinguishable isolates were cultured. ICEs with all 5 genes present were found in 9 of 110 (8 %) H. influenzae and 21 of 211 (10 %) H. parainfluenzae, respectively. ICEs were not detected among the other Pasteurellaceae. A total of 20 of 60 (33 %) children carried at least 1 oropharyngeal isolate with an ICE possessing all 5 genes. One of the five genes, integrase, however, consisted of two lineages, one of which was highly associated with H. influenzae. The topology of neighbour-joining trees of the remaining four ICE genes was compared and showed a lack of congruence; though, the genes form a common pool among H. influenzae and H. parainfluenzae. This family of antibiotic resistance ICEs was prevalent among the children studied, was genetically diverse, formed a large gene pool, transferred between H. influenzae and H. parainfluenzae, lacked population structure and possessed features suggestive of panmixia, all indicating it has not recently emerged from a single source.
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