Showing papers in "Journal of Medical Microbiology in 2010"

Candida tropicalis: its prevalence, pathogenicity and increasing resistance to fluconazole

Abstract: Candida tropicalis has been identified as the most prevalent pathogenic yeast species of the Candida-non-albicans group. Historically, Candida albicans has been the major species responsible for causing candidiasis in immunocompromised and immunocompetent patients. However, infections (candidiasis) due to C. tropicalis have increased dramatically on a global scale thus proclaiming this organism to be an emerging pathogenic yeast. The reasons for this organism's dominance and its resistance to fluconazole have been difficult to elucidate. In addition, the mechanism of this organism's pathogenicity and the consequent immune response remain to be clarified. This paper describes certain predisposing factors potentially responsible for these characteristics and presents a 'root cause analysis' to explain the increasing prevalence of C. tropicalis in developed and undeveloped countries, as well as the organism's acquired drug resistance. Control measures against fluconazole resistance in clinical management have also been discussed.

PCR characterization and typing of Klebsiella pneumoniae using capsular type-specific, variable number tandem repeat and virulence gene targets

Abstract: A multiplex PCR is described which detects capsular types K1, K2, K5, K54 and K57, which are those most associated with invasive disease or pathogenicity, a further capsular type (K20), two putative virulence factors (rmpA and wcaG) and the 16S–23S internal transcribed spacer unit of Klebsiella pneumoniae, facilitating identification of this organism. wcaG encodes capsular fucose production and was associated with capsular types K1 and K54, but was also found in strains of other capsular types; 18 of the 543 isolates screened were PCR-positive for this gene. An eight-locus variable number tandem repeat (VNTR) scheme was designed, which provided discrimination at a level similar to that afforded by PFGE among a panel of 36 isolates representing 29 PFGE types. All isolates tested of the virulent K1 clone of CC23, associated with pyogenic liver abscesses, shared the same VNTR profile, which may be helpful in identifying this clone; such isolates were also PCR-positive for allS. These methods provide a rapid means of characterizing and typing isolates of this important agent of community-acquired and nosocomial infection.

Commensal Escherichia coli of healthy humans: a reservoir for antibiotic-resistance determinants.

Abstract: This study examined in detail the population structure of Escherichia coli from healthy adults with respect to the prevalence of antibiotic resistance and specific resistance determinants. E. coli isolated from the faeces of 20 healthy adults not recently exposed to antibiotics was tested for resistance to ten antibiotics and for carriage of integrons and resistance determinants using PCR. Strain diversity was assessed using biochemical and molecular criteria. E. coli was present in 19 subjects at levels ranging from 2.0×104 to 1.7×108 c.f.u. (g faeces)−1. Strains resistant to one to six antibiotics were found at high levels (>30 %) in only ten individuals, but at significant levels (>0.5 %) in 14. Resistant isolates with the same phenotype from the same individual were indistinguishable, but more than one susceptible strain was sometimes found. Overall, individuals harboured one to four E. coli strains, although in 17 samples one strain was dominant (>70 % of isolates). Eighteen strains resistant to ampicillin, sulfamethoxazole, tetracycline and trimethoprim in 15 different combinations were observed. One resistant strain was carried by two unrelated individuals and a susceptible strain was shared by two cohabiting subjects. Two minority strains were derivatives of a more abundant resistant strain in the same sample, showing that continuous evolution is occurring in vivo. The trimethoprim-resistance genes dfrA1, dfrA5, dfrA7, dfrA12 or dfrA17 were in cassettes in a class 1 or class 2 integron. Ampicillin resistance was conferred by the bla TEM gene, sulfamethoxazole resistance by sul1, sul2 or sul3 and tetracycline resistance by tetA(A) or tetA(B). Chloramphenicol resistance (cmlA1 gene) was detected only once. Phylogenetic groups A and B2 were more common than B1 and D. Commensal E. coli of healthy humans represent an important reservoir for numerous antibiotic-resistance genes in many combinations. However, measuring the true extent of resistance carriage in commensal E. coli requires in-depth analysis.

Butyric acid-producing anaerobic bacteria as a novel probiotic treatment approach for inflammatory bowel disease.

Abstract: Inflammatory bowel disease (IBD), of which Crohn’s disease (CD) and ulcerative colitis (UC) are the most common manifestations, is characterized by chronic inflammation of the lining of the gastrointestinal tract, which causes severe watery and bloody diarrhoea, and abdominal pain. IBD is often debilitating and is characterized by onset at a young age and extra-intestinal manifestations. Whereas CD can affect any part of the gastrointestinal tract, UC is usually confined to the colon and rectum. IBD is an emerging disease and the incidence amounts to 20/100 000 in Europe and North America. There is a link with the social and economic development of the countries: the increase in IBD was first seen in northern Europe and North America, followed by the rest of Europe, Japan, South America and certain parts of Asia (Cohen, 2000; Ouyang et al., 2005). Although the exact aetiopathogenesis of IBD are not clear, it is widely accepted that the disease derives from an inappropriate immune response in genetically susceptible individuals as the result of a complex interaction between environmental factors, the microbiota and the intestinal immune system (Danese & Fiocchi, 2006).

Killing of adherent oral microbes by a non-thermal atmospheric plasma jet.

Abstract: Atmospheric plasma jets are being intensively studied with respect to potential applications in medicine. The aim of this in vitro study was to test a microwave-powered non-thermal atmospheric plasma jet for its antimicrobial efficacy against adherent oral micro-organisms. Agar plates and dentin slices were inoculated with 6 log(10) c.f.u. cm(-2) of Lactobacillus casei, Streptococcus mutans and Candida albicans, with Escherichia coli as a control. Areas of 1 cm(2) on the agar plates or the complete dentin slices were irradiated with a helium plasma jet for 0.3, 0.6 or 0.9 s mm(-2), respectively. The agar plates were incubated at 37 degrees C, and dentin slices were vortexed in liquid media and suspensions were placed on agar plates. The killing efficacy of the plasma jet was assessed by counting the number of c.f.u. on the irradiated areas of the agar plates, as well as by determination of the number of c.f.u. recovered from dentin slices. A microbe-killing effect was found on the irradiated parts of the agar plates for L. casei, S. mutans, C. albicans and E. coli. The plasma-jet treatment reduced the c.f.u. by 3-4 log(10) intervals on the dentin slices in comparison to recovery rates from untreated controls. The microbe-killing effect was correlated with increasing irradiation times. Thus, non-thermal atmospheric plasma jets could be used for the disinfection of dental surfaces.

Visualization of adherent micro-organisms using different techniques.

Abstract: The visualization and quantification of adherent bacteria is still one of the most relevant topics in microbiology. Besides electron microscopic techniques such as transmission electron microscopy, scanning electron microscopy and environmental scanning electron microscopy, modern fluorescence microscopic approaches based on fluorogenic dyes offer detailed insight into bacterial biofilms. The aim of the present review was to provide an overview of the advantages and disadvantages of different methods for visualization of adherent bacteria with a special focus on the experiences gained in dental research.

Gene expression of Pseudomonas aeruginosa in a mucin-containing synthetic growth medium mimicking cystic fibrosis lung sputum.

Abstract: Pseudomonas aeruginosa airway infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. Various in vitro models have been developed to study P. aeruginosa pathobiology in the CF lung. In this study we produced a modified artificial-sputum medium (ASMDM) more closely resembling CF sputum than previous models, and extended previous work by using strain PAO1 arrays to examine the global transcription profiles of P. aeruginosa strain UCBPP-PA14 under early exponential-phase and stationary-phase growth. In early exponential phase, 38/39 nutrition-related genes were upregulated in line with data from previous in vitro models using UCBPP-PA14. Additionally, 23 type III secretion system (T3SS) genes, several anaerobic respiration genes and 24 quorum-sensing (QS)-related genes were upregulated in ASMDM, suggesting enhanced virulence factor expression and priming for anaerobic growth and biofilm formation. Under stationary phase growth in ASMDM, macroscopic clumps resembling microcolonies were evident in UCBPP-PA14 and CF strains, and over 40 potentially important genes were differentially expressed relative to stationary-phase growth in Luria broth. Most notably, QS-related and T3SS genes were downregulated in ASMDM, and iron-acquisition and assimilatory nitrate reductase genes were upregulated, simulating the iron-depleted, microaerophilic/anaerobic environment of CF sputum. ASMDM thus appears to be highly suitable for gene expression studies of P. aeruginosa in CF.

Transcriptional activity of the dominant gut mucosal microbiota in chronic inflammatory bowel disease patients.

Abstract: Dysbiosis of the gut mucosa-associated microbiota (MAM) plays a pivotal role in the pathogenesis of chronic inflammatory bowel diseases (IBD). To date, dysbiosis only describes the altered composition of the different bacterial populations, but little is known about transcriptional activity, metabolism and the ‘live’ status of the MAM. In this study we investigated the transcriptional activity of the dominant intestinal bacterial populations in patients with IBD. Colonic mucosal biopsies from patients with active Crohn's disease (CD; n=10), active ulcerative colitis (UC; n=10) and healthy individuals (HI; n=10) were compared by 16S rRNA gene and rRNA profiles using clone libraries with more than 1700 sequenced clones. Bacterial richness was significantly lower in clone libraries based on rRNA compared to those based on the rRNA genes in the CD group (3.01 vs 3.91) and the UC group (3.61 vs 4.15), but showed no difference in HI (3.81 vs 3.85). The qualitative composition of rRNA and rRNA gene clone libraries was significantly different, with the phylum Bacteroidetes being the most active (P<0.01) compared to other populations in all clinical groups. In contrast, Actinobacteria and Firmicutes were inactive in the CD group, while Escherichia sp. were both abundant and active in the CD and UC groups. Most of the phylotypes showing the highest activity index ratios represented less than 1 % of the microbiota. Our findings indicate that specific bacterial populations are activated in IBD patients, while other groups are in an inactive or ‘dormant’ state. The transcriptional activity points to a more functional role of the intestinal mucosal microbiota and may lead to the identification of therapeutic targets in the active modulation of microbial factors.

Drug sensitivity and clinical impact of members of the genus Kocuria.

Abstract: Organisms in the genus Kocuria are Gram-positive, coagulase-negative, coccoid actinobacteria belonging to the family Micrococcaceae, suborder Micrococcineae, order Actinomycetales. Sporadic reports in the literature have dealt with infections by Kocuria species, mostly in compromised hosts with serious underlying conditions. Nonetheless, the number of infectious processes caused by such bacteria may be higher than currently believed, given that misidentification by phenotypic assays has presumably affected estimates of the prevalence over the years. As a further cause for concern, guidelines for therapy of illnesses involving Kocuria species are lacking, mostly due to the absence of established criteria for evaluating Kocuria replication or growth inhibition in the presence of antibiotics. Therefore, breakpoints for staphylococci have been widely used throughout the literature to try to understand this pathogen's behaviour under drug exposure; unfortunately, this has sometimes created confusion, thus higlighting the urgent need for specific interpretive criteria, along with a deeper investigation into the resistance determinants within this genus. We therefore review the published data on cultural, genotypic and clinical aspects of the genus Kocuria, aiming to shed some light on these emerging nosocomial pathogens.

Mycobacterium tuberculosis complex CRISPR genotyping: improving efficiency, throughput and discriminative power of 'spoligotyping' with new spacers and a microbead-based hybridization assay.

Abstract: The aims of the present study were to implement a microbead-based ‘spoligotyping’ technique and to evaluate improvements by the addition of a panel of 25 extra spacers that we expected to provide an increased resolution on principal genetic group 1 (PGG 1) strains. We confirmed the high sensitivity and reproducibility of the classical technique using the 43 spacer panel and we obtained perfect agreement between the membrane-based and the microbead-based techniques. We further demonstrated an increase in the discriminative power of an extended 68 spacer format for differentiation of PGG 1 clinical isolates, in particular for the East African–Indian clade. Finally, we define a limited yet highly informative reduced 10 spacer panel set which could offer a more cost-effective option for implementation in resource-limited countries and that could decrease the need for additional VNTR (variable number of tandem repeats) genotyping work in molecular epidemiological studies. We also present an economic analysis comparing membrane-based and microbead-based techniques.

Aspergillus niger: an unusual cause of invasive pulmonary aspergillosis

Abstract: Infections due to Aspergillus species cause significant morbidity and mortality. Most are attributed to Aspergillus fumigatus, followed by Aspergillus flavus and Aspergillus terreus. Aspergillus niger is a mould that is rarely reported as a cause of pneumonia. A 72-year-old female with chronic obstructive pulmonary disease and temporal arteritis being treated with steroids long term presented with haemoptysis and pleuritic chest pain. Chest radiography revealed areas of heterogeneous consolidation with cavitation in the right upper lobe of the lung. Induced bacterial sputum cultures, and acid-fast smears and cultures were negative. Fungal sputum cultures grew A. niger. The patient clinically improved on a combination therapy of empiric antibacterials and voriconazole, followed by voriconazole monotherapy. After 4 weeks of voriconazole therapy, however, repeat chest computed tomography scanning showed a significant progression of the infection and near-complete necrosis of the right upper lobe of the lung. Serum voriconazole levels were low–normal (1.0 μg ml−1, normal range for the assay 0.5–6.0 μg ml−1). A. niger was again recovered from bronchoalveolar lavage specimens. A right upper lobectomy was performed, and lung tissue cultures grew A. niger. Furthermore, the lung histopathology showed acute and organizing pneumonia, fungal hyphae and oxalate crystallosis, confirming the diagnosis of invasive A. niger infection. A. niger, unlike A. fumigatus and A. flavus, is less commonly considered a cause of invasive aspergillosis (IA). The finding of calcium oxalate crystals in histopathology specimens is classic for A. niger infection and can be helpful in making a diagnosis even in the absence of conidia. Therapeutic drug monitoring may be useful in optimizing the treatment of IA given the wide variations in the oral bioavailability of voriconazole.

Probiotic Escherichia coli strain Nissle 1917 outcompetes intestinal pathogens during biofilm formation.

Abstract: Many bacterial infections are associated with biofilm formation. Bacterial biofilms can develop on essentially all kinds of surfaces, producing chronic and often intractable infections. Escherichia coli is an important pathogen causing a wide range of gastrointestinal infections. E. coli strain Nissle 1917 has been used for many decades as a probiotic against a variety of intestinal disorders and is probably the best field-tested E. coli strain in the world. Here we have investigated the biofilm-forming capacity of Nissle 1917. We found that the strain was a good biofilm former. Not only was it significantly better at biofilm formation than enteropathogenic, enterotoxigenic and enterohaemorrhagic E. coli strains, it was also able to outcompete such strains during biofilm formation. The results support the notion of bacterial prophylaxis employing Nissle 1917 and may partially explain why the strain has a beneficial effect on many intestinal disorders.

Difficulties in using 1,3-β-D-glucan as the screening test for the early diagnosis of invasive fungal infections in patients with haematological malignancies - high frequency of false-positive results and their analysis

Abstract: We have evaluated the contribution of 1,3-s-D-glucan (BG) assay for screening of invasive fungal diseases (IFI) in patients with hematological malignancies. Serum samples from patients at risk of IFI were collected twice a week and retrospectively tested using the BG assay. BG screening was performed on 1143 samples from 91 patients during 104 anticancer treatment cycles. Proven and probable cases of IFI occurred in 9 (8.7%) treatment cycles. Depending on the criterion of positivity used (1> 60 or 1> 80 pg/ml and 2> 60 or 2> 80 pg/ml) the sensitivity was 89%, 89%, 67% and 44%. Although the test was marked as positive in 82%, 68%, 54% and 45% of all the treatment cycles, in the majority of cases, these positivities were probably false. The major limit of the BG test was extremely low PPV (10% to 12%). We have analyzed mucositis, candida colonization, bacteremia, using antimicrobials, erythrocyte and thrombocyte filtered blood products, collecting tubes or sampling via venous catheters. Even though no factor is a major source of BG, it could at least partially influence BG assay performance. Thus, the BG detection has a limited usefulness as a screening method for invasive fungal infections in patients with hematological malignancies.

Species-specific identification and differentiation of Arcobacter, Helicobacter and Campylobacter by full-spectral matrix-associated laser desorption/ionization time of flight mass spectrometry analysis

Abstract: Rapid and reliable identification of Arcobacter and Helicobacter species, and their distinction from phenotypically similar Campylobacter species, has become increasingly important, since many of them are now recognized as human and/or animal pathogens. Matrix-associated laser desorption/ionization–time of flight (MALDI-TOF) MS has been shown to be a rapid and sensitive method for characterization of micro-organisms. In this study, we therefore established a reference database of selected Arcobacter, Helicobacter and Campylobacter species for MALDI-TOF MS identification. Besides the species with significance as food-borne pathogens – Arcobacter butzleri, Helicobacter pullorum, Campylobacter jejuni and Campylobacter coli – several other members of these genera were included in the reference library to determine the species specificity of the designed MALDI Biotyper reference database library. Strains that made up the reference database library were grown on Columbia agar, and yielded reproducible and unique mass spectra profiles, which were compared with the Bruker Biotyper database, version 2. The database was used to identify 144 clinical isolates using whole spectral profiles. Furthermore, reproducibility of MALDI-TOF MS results was evaluated with respect to age and/or storage of bacteria and different growth media. It was found that correct identification could be obtained even if the bacteria were stored at room temperature or at 4 °C up to 9 days before being tested. In addition, bacteria were correctly identified when grown on Campylosel agar; however, they were not when grown on modified charcoal cefoperazone deoxycholate agar. These results indicate that MALDI-TOF MS fingerprinting is a fast and reliable method for the identification of Arcobacter and Helicobacter species, and their distinction from phenotypically similar Campylobacter species, with applications in clinical diagnostics.

In vitro synergy of eugenol and methyleugenol with fluconazole against clinical Candida isolates

Abstract: The species Candida is a group of opportunistic pathogenic commensals in immune-compromised patients. Treatment of Candida infections is becoming increasingly difficult due to antifungal drug resistance, especially with fluconazole (FLC), which is a commonly used azole. In the present study the in vitro antifungal activity of eugenol (EUG) and methyleugenol (MEUG) alone and in combination against 64 FLC-sensitive and 34 FLC-resistant clinical Candida isolates is highlighted. All the strains were susceptible to both the naturally occurring phenyl propanoids. The nature of the interaction was studied from fractional inhibitory concentration indices (FICIs) for both EUG plus FLC, and MEUG plus FLC combinations calculated from chequerboard microdilution assays. FICI values depicted a high synergism of FLC with both compounds, which was greatest with MEUG. FLC-resistant Candida isolates showed high sensitivity to both compounds. No antagonistic activity was seen in the strains tested in the present study. From these results we suggest that EUG and MEUG have great potential as antifungals, and that FLC can be supplemented with EUG and MEUG to treat FLC-resistant Candida infections.

DnaK and GroEL are induced in response to antibiotic and heat shock in Acinetobacter baumannii

Abstract: We studied the expression of DnaK and GroEL in Acinetobacter baumannii cells (strains ATCC 19606 and RS4) under stress caused by heat shock or antibiotics. A Western blot assay showed that DnaK and GroEL levels increased transiently more than 2-fold after exposure of bacterial cells to heat shock for 20 min at 50 degrees C. Heat induction of DnaK and GroEL was blocked completely when an inhibitor of transcription, rifampicin, was added 1 min before a temperature upshift to 50 degrees C, suggesting that the induction of these chaperones depends on transcription. A. baumannii cells pretreated at 45 degrees C for 30 min were better able to survive at 50 degrees C for 60 min than cells pretreated at 37 degrees C, indicating that A. baumannii is able to acquire thermotolerance. DnaK and GroEL were successfully induced in cells pre-incubated with a subinhibitory concentration of streptomycin. Moreover, bacterial cells pretreated for 30 min at 45 degrees C were better able to survive streptomycin exposure than cells pretreated at physiological temperatures. DnaK expression was upregulated in a multidrug-resistant strain of A. baumannii (RS4) in the presence of different antimicrobials (ampicillin+sulbactam, cefepime, meropenem and sulphamethoxazole+trimethoprim). This study is to the best of our knowledge the first to show that A. baumannii DnaK and GroEL could play an important role in the stress response induced by antibiotics.

Fluctuations in phenotypes and genotypes within populations of Pseudomonas aeruginosa in the cystic fibrosis lung during pulmonary exacerbations.

Abstract: Chronic respiratory infection by Pseudomonas aeruginosa contributes significantly to the morbidity and mortality associated with cystic fibrosis (CF). Using a series of phenotypic and genotypic tests on collections of 40 isolates per sputum sample, we analysed fluctuations within sputum populations of the P. aeruginosa Liverpool epidemic strain (LES) during pulmonary exacerbations. For each of three patients, three sequential sputum samples were analysed: (1) on presentation with exacerbation at the Regional Adult Cystic Fibrosis Unit, Liverpool; (2) a few days into intravenous antibiotic treatment; (3) when the patient had recovered. Fluctuations were observed in morphotype distribution, the production of virulence-associated quorum-sensing-dependent exoproducts (the phenazine compound pyocyanin and the elastase LasA), antibiotic susceptibility profiles and levels of auxotrophy. PCR assays were used to screen isolates for the presence of novel regions of the LES genome (islands and prophages) and to detect free phages. In one patient there was an increase in the prevalence of the LESGI-5 genomic island during the sampling period from 10 to 97.5 % carriage. LES phages 2-4 were detected in either the majority or all sputum samples tested, indicating widespread phage activity during the sampling period. The results of this study are indicative that significant fluctuations occur within P. aeruginosa populations during short periods of pulmonary exacerbation and intravenous antibiotic therapy.

Virulence gene distribution in clinical, nosocomial and environmental isolates of Pseudomonas aeruginosa

Abstract: The virulence factor genotypes of a large cohort of clinical, nosocomial environment and community environment isolates (184 in total) of Pseudomonas aeruginosa from Tasmania, Australia, were determined by PCR. The virulence factor genotype of the majority of isolates was highly conserved, with the exception of the virulence gene exoU, which demonstrated low prevalence (33 isolates; 18 %) in the population tested. Isolates collected from the environment of intensive therapy wards (intensive care unit and neurosurgical units) of the major tertiary referral hospital in Tasmania were found to be more likely (P<0.001 and P<0.05, respectively) to possess the virulence factor gene exoU than all other isolates. Adult cystic fibrosis isolates showed a decreased prevalence of the exoU gene (P<0.01) when compared to other clinical isolates (P<0.01), which may indicate decreased virulence. No specific virulence factor genotype was associated with the cystic fibrosis epidemic strains tested.

A simplified multiplex PCR assay for fast and easy discrimination of globally distributed staphylococcal cassette chromosome mec types in meticillin-resistant Staphylococcus aureus.

Abstract: A multiplex PCR assay was developed for the identification of major types and subtypes of staphylococcal cassette chromosome mec (SCCmec) in meticillin-resistant Staphylococcus aureus (MRSA) strains. The method uses a novel 9 valent multiplex PCR plus two primer pairs for S. aureus identification and detection of meticillin resistance. All 389 clinical MRSA isolates from Malaysia and 18 European isolates from the Harmony collection harbouring different SCCmec types that we tested were correctly characterized by our PCR assay. SCCmec type III and V were by far the most common types among both hospital- and community-acquired Malaysian MRSA isolates, with an apparent emergence of MRSA harbouring the IVh type.

Swarming motility, secretion of type 3 effectors and biofilm formation phenotypes exhibited within a large cohort of Pseudomonas aeruginosa clinical isolates

Abstract: Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen capable of acutely infecting or persistently colonizing susceptible hosts. P. aeruginosa colonizes surfaces in vitro by either biofilm formation or swarming motility. The choice of behaviour is influenced by the physical properties of the surface and specific nutrient availability, and subject to regulatory networks that also govern type 2 and type 3 protein secretion. Biofilm formation by clinical isolates has been well-studied. However, the swarming behaviour of human isolates has not been extensively analysed. We collected isolates from 237 hospitalized patients without cystic fibrosis and analysed motility and secretion phenotypes of each isolate. We found biofilm formation and swarming to be negatively associated, while swarming was positively associated with the secretion of both proteases and type 3 exoenzymes. Most isolates were capable of type 3 secretion and biofilm formation, even though these traits are considered to favour distinct modes of pathogenesis. Our data demonstrate that while clinical isolates display diverse motility, biofilm and secretion phenotypes, many of the predicted relationships between swarming motility and other phenotypes observed in laboratory strains also hold true for bacteria isolated from human patients.

Identification of molecularly defined Staphylococcus aureus strains using matrix-assisted laser desorption/ionization time of flight mass spectrometry and the Biotyper 2.0 database

Abstract: Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced for bacterial identification. To our knowledge, this is the first study where the Biotyper 2.0 database (Bruker Daltonics) has been applied for bacterial identification in a local strain collection of molecularly defined Staphylococcus aureus. We showed that the accuracy of the Biotyper 2.0-based identification for 602 molecularly defined strains of S. aureus, irrespective of meticillin resistance, was equivalent to that of the molecularly defined reference even at a score cut-off value of 2. Also, 412 isolates of 20 different species of non-S. aureus staphylococci were all correctly identified to species level compared to the molecularly defined reference. Moreover, the MALDI-TOF MS-based S. aureus identification approach was clearly faster than more time-consuming methods such as a molecular identification approach.

Synergistic interaction of phenylpropanoids with antibiotics against bacteria.

Abstract: Phenylpropanoids constitute a large part of our daily diet and there is a possibility that they might interact with synthetic drugs. The present work was aimed at studying the interaction of seven phenylpropanoids (cinnamic, p-coumaric, caffeic, chlorogenic, ferulic, 3,4-dimethoxycinnamic and 2,4,5-trimethoxycinnamic acid) with five antibiotics (amikacin, ampicillin, ciprofloxacin, erythromycin and vancomycin) against Gram-negative (Escherichia coli, Enterobacter aerogenes and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria. The interaction studies were performed by chequerboard and time-kill curve assays. Both assays revealed that cinnamic, p-coumaric and ferulic acids were the most active. They combined synergistically with the majority of the antibiotics and exhibited enhanced activity against all the micro-organisms. The time-kill curve parameters were better (P<0.05) for the combinations of amikacin with ferulic, cinnamic or p-coumaric acid than for the individual treatments. Amikacin was the most favourable antibiotic and S. aureus was the most sensitive microbe to most of the combinations. These phenylpropanoids damaged the bacterial membrane as assessed by the LIVE/DEAD BacLight kit, and structure-activity relationship studies indicated that hydrophilic groups enhanced this activity.

Silicone colonization by non-Candida albicans Candida species in the presence of urine.

Abstract: Urinary tract infections (UTIs) are the most common nosocomial infections and 80 % are related to the use of urinary catheters Furthermore, Candida species are responsible for around 15 % of UTIs and an increasing involvement of non-Candida albicans Candida (NCAC) species (eg Candida glabrata, Candida tropicalis and Candida parapsilosis) has been recognized Given the fact that silicone is frequently used in the manufacture of urinary catheters, the aim of this work was to compare both the adhesion and biofilm formation on silicone of different urinary clinical isolates of NCAC species (ie C glabrata, C tropicalis and C parapsilosis) in the presence of urine Several clinical isolates of NCAC species recovered from patients with UTIs, together with reference strains of each species, were examined Adhesion and biofilm formation were performed in artificial urine and the biofilm biomass was assessed by crystal violet staining Hydrophobicity and surface charge of cells was determined by measuring contact angles and zeta potential, respectively The number of viable cells in biofilms was determined by enumeration of cfu after appropriate culture The biofilm structure was also examined by confocal laser scanning microscopy (CLSM) The results showed that all isolates adhered to silicone in a species- and strain-dependent manner with C parapsilosis showing the lowest and C glabrata the highest levels of adhesion However, these differences in adhesion abilities cannot be correlated with surface properties since all strains examined were hydrophilic and exhibited a similar zeta potential Despite a higher number of cultivable cells being recovered after 72 h of incubation, stronger biofilm formation was not observed and CLSM showed an absence of extracellular polymeric material for all isolates examined In summary, this work demonstrated that all tested NCAC species were able to adhere to and survive on silicone in the presence of urine Furthermore, C glabrata strains presented higher colonization abilities than C tropicalis and C parapsilosis strains, a fact that might explain the larger role of C glabrata colonization and disseminated infections in hospitalized and catheterized patients

A novel IS26 structure surrounds blaCTX-M genes in different plasmids from German clinical Escherichia coli isolates.

Abstract: This report focuses on the molecular characterization of 22 extended-spectrum β-lactamase-producing Escherichia coli isolates collected in a German university hospital during a period of 9 months in 2006. Relationship analysis of clinical isolates was done via PFGE, multilocus sequence typing, plasmid profiling and additionally PCR for bla ESBL detection and determination of phylogroups. After conjugal transfer, plasmid isolation and subsequent PCR for bla ESBL detection and determination of incompatibility groups were performed. Using one-primer walking, up to 3600 bp upstream and downstream of different bla CTX-M genes could be sequenced. β-Lactamases found were TEM-1 (n=14), SHV-5 (n=1) and a wide variety of CTX-M types (n=21), i.e. CTX-M-15 (n=12), CTX-M-1 (n=4), CTX-M-14 (n=2), CTX-M-9 (n=1), CTX-M-3 (n=1) and one new type, CTX-M-65 (n=1). In 18 isolates, bla ESBL genes were located on conjugative plasmids of sizes between 40 and 180 kbp belonging to incompatibility groups FII (n=9), N (n=5) and I1 (n=4). bla CTX-M was found to be associated with the common elements ISEcp1, IS26 and IS903-D, but with unusual spacer sequences for ISEcp1 in two isolates. These insertion sequences, connected to bla CTX-M as well as other genes, were located between two IS26 elements in a configuration that has not yet been described. The results reveal the emergence of bla ESBL, predominantly bla CTX-M, located on different plasmids harboured by genotypically different E. coli strains. The identical gene arrangement in the bla CTX-M neighbourhood in plasmids of different incompatibility groups indicates a main role of IS26 in distribution of mobile resistance elements between different plasmids.

Metabolism of azo dyes by human skin microbiota

Abstract: Reduction of Methyl Red (MR) and Orange II (Or II) by 26 human skin bacterial species was monitored by a rapid spectrophotometric assay. The analysis indicated that skin bacteria, representing the genera Staphylococcus, Corynebacterium, Micrococcus, Dermacoccus and Kocuria, were able to reduce MR by 74-100 % in 24 h, with only three species unable to reduce completely the dye in that time. Among the species tested, only Corynebacterium xerosis was unable to reduce Or II to any degree by 24 h, and only Staphylococcus delphini, Staphylococcus sciuri subsp. sciuri and Pseudomonas aeruginosa were able to reduce completely this dye within 24 h. MR reduction started with early-exponential growth in Staphylococcus aureus and Staphylococcus epidermidis, and around late-exponential/early-stationary growth in P. aeruginosa. Reduction of Or II, Ponceau S and Ponceau BS started during late-exponential/early-stationary growth for all three species. Using liquid chromatography/electrospray ionization mass spectrometry analyses, MR metabolites produced by Staph. aureus, Staph. epidermidis and P. aeruginosa were identified as N,N-dimethyl-p-phenylenediamine and 2-aminobenzoic acid. Searches of available genomic and proteomic data revealed that at least four of the staphylococci in this study, Staphylococcus haemolyticus, Staph. epidermidis, Staphylococcus cohnii and Staphylococcus saprophyticus, have hypothetical genes with 77, 76, 75 and 74 % sequence identity to azo1 encoding an azoreductase from Staph. aureus and hypothetical proteins with 82, 80, 72 and 74 % identity to Azo1, respectively. In addition, Staphylococcus capitis has a protein with 79 % identity to Azo1. Western analysis detected proteins similar to Azo1 in all the staphylococci tested, except Staph. delphini, Staph. sciuri subsp. sciuri and Staphylococcus auricularis. The data presented in this report will be useful in the risk assessment process for evaluation of public exposure to products containing these dyes.

Predominance of an ST11 extended-spectrum β-lactamase-producing Klebsiella pneumoniae clone causing bacteraemia and urinary tract infections in Korea

Abstract: To investigate the antimicrobial resistance, extended-spectrum β-lactamases (ESBLs) and clones of Klebsiella pneumoniae isolates causing bacteraemia or urinary tract infection (UTI) in Korea, a total of 406 K. pneumoniae isolates from patients with bacteraemia (221 isolates) and UTI (185 isolates) were collected from 10 tertiary-care Korean hospitals from July 2006 to October 2007. In vitro antimicrobial susceptibility testing was performed for all isolates and ESBL production was tested. Multilocus sequence typing (MLST) analyses were performed to characterize genotypes of ESBL-producing K. pneumoniae isolates. PFGE was performed for sequence type 11 (ST11) isolates. Forty-seven UTI isolates (25.4 %) produced ESBLs, while 30 bacteraemia isolates (13.6 %) produced ESBLs (P=0.002). Among 77 ESBL-producing isolates, thirty-two (41.6 %) produced SHV-type ESBLs. bla CTX-M genes such as bla CTX-M-14 and bla CTX-M-15 were detected in 36.4 %. MLST and PFGE analyses showed that ST11 was dominant in ESBL-producing K. pneumoniae isolates causing UTI (57.4 %) and in those causing bacteraemia (70.0 %) and has been prevalent in Korean hospitals. ST11 isolates harbour a combination of different ESBL genes. The ST11 clone of ESBL-producing K. pneumoniae isolates prevails in Korea, but most isolates might acquire ESBL genes independently or several different clones might be distributed in Korea.

Purified galactooligosaccharide, derived from a mixture produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium adhesion and invasion in vitro and in vivo.

Abstract: The prebiotic Bimuno(®) is a mixture containing galactooligosaccharides (GOSs), produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 41171 using lactose as the substrate. Previous in vivo and in vitro studies demonstrating the efficacy of Bimuno(®) in reducing Salmonella enterica serovar Typhimurium (S. Typhimurium) colonization did not ascertain whether or not the protective effects could be attributed to the prebiotic component GOS. Here we wished to test the hypothesis that GOS, derived from Bimuno(®), may confer the direct anti-invasive and protective effects of Bimuno(®). In this study the efficacy of Bimuno(®), a basal solution of Bimuno(®) without GOS [which contained glucose, galactose, lactose, maltodextrin and gum arabic in the same relative proportions (w/w) as they are found in Bimuno(®)] and purified GOS to reduce S. Typhimurium adhesion and invasion was assessed using a series of in vitro and in vivo models. The novel use of three dimensionally cultured HT-29-16E cells to study prebiotics in vitro demonstrated that the presence of ∼ 5 mg Bimuno(®) ml(-1) or ∼ 2.5 mg GOS ml(-1) significantly reduced the invasion of S. Typhimurium (SL1344nal(r)) (P<0.0001). Furthermore, ∼ 2.5 mg GOS ml(-1) significantly reduced the adherence of S. Typhimurium (SL1344nal(r)) (P<0.0001). It was demonstrated that cells produced using this system formed multi-layered aggregates of cells that displayed excellent formation of brush borders and tight junctions. In the murine ligated ileal gut loops, the presence of Bimuno(®) or GOS prevented the adherence or invasion of S. Typhimurium to enterocytes, and thus reduced its associated pathology. This protection appeared to correlate with significant reductions in the neutral and acidic mucins detected in goblet cells, possibly as a consequence of stimulating the cells to secrete the mucin into the lumen. In all assays, Bimuno(®) without GOS conferred no such protection, indicating that the basal solution confers no protective effects against S. Typhimurium. Collectively, the studies presented here clearly indicate that the protective effects conferred by Bimuno(®) can be attributed to GOS.

Prevalence of trimethoprim resistance genes in Escherichia coli isolates of human and animal origin in Lithuania

Abstract: A total of 456 non-repetitive Escherichia coli isolates from human clinical specimens (urinary, n=134; cervix, vagina and prostate, n=52; blood, pus and wounds, n=45), healthy animals (cattle, n=45; poultry, n=20) and diseased animals (cattle, n=53; swine, n=64; poultry, n=43) obtained in Lithuania during the period 2005-2008 were studied for trimethoprim (TMP) resistance and the prevalence of dfr genes. A TMP resistance rate in the range of 18-26 % respective to the origin was found in clinical isolates, 23-40 % in isolates from diseased animals and 9-20 % in isolates from healthy animals. Of 112 TMP-resistant isolates, 103 carried at least one of the six dfrA genes (dfrA1, dfrA5, dfrA8, dfrA12, dfrA14 and dfrA17) as determined by multiplex PCR and RFLP. The dfrA1 and dfrA17 genes were found most frequently in clinical isolates (17 and 19 isolates, respectively), whilst dfrA1 and dfrA14 genes dominated in isolates of animal origin (25 and 13 isolates, respectively). The dfrA5, dfrA12 and dfrA8 genes were detected at lower frequencies. The association with class 1/class 2 integrons was confirmed for 73-100 % of dfr genes found in most groups of isolates, except for the isolates from diseased swine. In this group, the majority of dfr-positive isolates (67 %, 8/12) carried dfrA8 (6/12) or dfrA14 genes (2/12) that were not associated with integrons. Non-integron location was also confirmed for the remaining dfrA8 genes (six clinical isolates and one isolate from diseased cattle) and for dfrA14 genes (two isolates from diseased cattle and swine each). All cassette-independent dfrA14 genes were found to be located within the strA gene. This study on the prevalence and distribution of TMP resistance genes among E. coli isolates of human and animal origin in Lithuania demonstrates that dfr genes are carried most frequently as gene cassettes within class 1 and/or class 2 integrons. However, TMP resistance in some of the isolates was found to be mediated by non-integron-associated dfrA8 and dfrA14 genes, indicating the existence of alternative sources for the spread of resistance.

Zoonotic transmission of Streptococcus equi subsp. zooepidemicus from a dog to a handler

Abstract: This is, to the best of our knowledge, the first case report to describe the apparent transmission of Streptococcus equi subsp. zooepidemicus from an infected dog to a handler who subsequently developed severe systemic infection. Characterization of the haemolytic streptococci isolated from both the patient and the dog, by phenotypic and molecular analysis, confirmed the canine and human isolates were identical.

Curcumin alone and in combination with augmentin protects against pulmonary inflammation and acute lung injury generated during Klebsiella pneumoniae B5055-induced lung infection in BALB/c mice.

Abstract: Acute lung injuries due to acute lung infections remain a major cause of mortality. Thus a combination of an antibiotic and a compound with immunomodulatory and anti-inflammatory activities can help to overcome acute lung infection-induced injuries. Curcumin derived from the rhizome of turmeric has been used for decades and it exhibits anti-inflammatory, anti-carcinogenic, immunomodulatory properties by downregulation of various inflammatory mediators. Keeping these properties in mind, we investigated the anti-inflammatory properties of curcumin in a mouse model of acute inflammation by introducing Klebsiella pneumoniae B5055 into BALB/c mice via the intranasal route. Intranasal instillation of bacteria in this mouse model of acute pneumonia-induced inflammation resulted in a significant increase in neutrophil infiltration in the lungs along with increased production of various inflammatory mediators [i.e. malondialdehyde (MDA), myeloperoxidase (MPO), nitric oxide (NO), tumour necrosis factor (TNF)-alpha] in the lung tissue. The animals that received curcumin alone orally or in combination with augmentin, 15 days prior to bacterial instillation into the lungs via the intranasal route, showed a significant (P 0.05) as compared to the control group. We therefore conclude that curcumin ameliorates lung inflammation induced by K. pneumoniae B5055 without significantly (P <0.05) decreasing the bacterial load in the lung tissue whereas augmentin takes care of bacterial proliferation. Hence, curcumin can be used as an adjunct therapy along with antibiotics as an anti-inflammatory or an immunomodulatory agent in the case of acute lung infection.