Journal of Medical Microbiology Publish Ahead of Print

Maternal and Neonatal Sepsis Caused by Haemophilus influenzae type d [CASE REPORT]

Warren, S. J.C., Tristram, S. G., Bradbury, R. S. — Thu, 19 Nov 2009 07:31:09 PST

A 29 year old pregnant woman was admitted to hospital with signs of sepsis and threatened pre-term labour. The premature neonate also showed signs of sepsis. Haemophilus influenzae biotype III was cultured from a mid-stream urine taken from the mother, maternal placental swabs and neonatal blood cultures. The placental and neonatal isolates were both found to be serotype d by PCR, and were indistinguishable by PFGE.

Epidemiological typing of methicillin resistant Staphylococcus aureus (MRSA) isolates from Pakistan and India [EPIDEMIOLOGY]

Thu, 19 Nov 2009 07:31:10 PST

The rates of MRSA in Pakistan and India are known to be high, but few studies have described the epidemiology of the different MRSA clones present. In order to gain an understanding of the epidemiology of MRSA within this region, sixty MRSA isolates from Pakistan (49) and India (11) were genotyped. All isolates were typed using pulsed field gel electrophoresis (PFGE), staphylococcal interspersed repeat units (SIRU), restriction modification (RM) method and SCCmec typing. A subset of isolates that were distinct by PFGE and SIRU were typed using Multi locus sequence typing (MLST).CC8 was the dominant clonal complex (57/60) and was present in both Pakistan and India. Within CC8, there were 10 SIRU profiles and 24 PFGE profiles. Two SIRU profiles were present in isolates from both India and Pakistan; seven were distinct to Pakistan and one to India. All PFGE profiles were distinct to each of the two countries. Thirty four of the 57 isolates carried SCCmec type III/IIIa and the remainder carried the type IV SCCmec. MLST analysis of 14 CC8 isolates with diverse SIRU and PFGE profiles showed all were single locus variants, with nine belonging to ST239, three to ST8 and two to ST113. From a single hospital in Pakistan three isolates belonged to CC30 and all were indistinguishable by PFGE and SIRU and carried the PVL gene. Epidemiological typing of strains from three distinct locations in India and Pakistan reveals the predominance of one clonal complex and highly related STs. The ability of SIRU and PFGE to differentiate within ST239 demonstrates their utility in defining local epidemiology in these countries.

Rapid identification of Legionella species by mass spectrometry [DIAGNOSTICS, TYPING AND IDENTIFICATION]

Thu, 19 Nov 2009 07:31:10 PST

Legionella species are facultative intracellular bacteria infecting macrophages and protozoa, these latter acting as transmission vectors to humans. These fastidious bacteria mostly cause pulmonary tract infections and are routinely identified by various molecular methods, mainly PCR targeting the mip gene and sequencing, which are time- and money-consuming. Recently, Matrix Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF-MS) has emerged as a rapid and inexpensive identification method of bacterial species. We evaluated the use of MALDI-TOF-MS for rapid species and serogroup identification for 21 Legionella species recognized as human pathogens. To this end, we developed a reference MS database including 59 Legionella type strains, and performed a blind-test using 237 strains from various species. Two hundred and twenty-three of the 237 strains (94.1 %) were correctly identified at the species level, although 10 (4.2%) were identified with a score lower than 2.0. Fourteen strains (5.9%) from 8 species were misidentified at the species level, including 7 (2.9%) with a significant score, suggesting an intraspecific variability of proteic profiles within some species. MALDI-TOF-MS was reproducible but could not identify Legionella strains at the serogroup level. When compared to mip gene sequencing, MALDI-TOF-MS exhibited a sensitivity of 99.1% and 89.9% for the identification of Legionella strains at the genus and species level, respectively. Our study demonstrates that MALDI-TOF-MS is a reliable tool for the rapid identification of Legionella strains at the species level.

Vaginal colonization and activity of Lactobacillus fermentum probiotic in a murine model of vaginal tract infection [HUMAN AND ANIMAL MICROBIAL ECOLOGY]

Pascual, L., Ruiz, F., Giordano, W., Barberis, I. L. — Thu, 19 Nov 2009 07:31:09 PST

One strain of Lactobacillus, identified as Lactobacillus fermentum L23, was selected from among 100 strains isolated from vaginal swabs of healthy, nonpregnant, premenopausal women. L. fermentum L23 was chosen on the basis of its bacteriocinogenic ability and its properties relevant to colonization, i.e., self-aggregation, adherence to vaginal epithelial cells, and coaggregation with bacterial pathogens. The antimicrobial preventive and curative effects produced by the probiotic L. fermentum L23 administrated locally against E. coli in a murine vaginal tract infection model were studied. One dose of this human strain L23 containing 108 CFU/ml colonized and persisted in the vaginal tract of the female BALB/c mice for 5 days. Infection with the pathogen at 106 CFU/ml in the vaginal tract was maintained for more than 7 days. A single administration of L23 inhibited the E. coli growth on the third day showing the preventive effect displayed by this Lactobacillus. The treatment with L. fermentum L23 during the post-infection period showed complete inhibition of the pathogen growth as of the fifth day. This in vivo study indicates that the probiotic bacteria L fermentum L23 produced both preventive and curative effects on E. coli growth. The beneficial properties and the production of antimicrobial metabolites would act in situ to inhibit a pathogenic microorganism within the vaginal environmental. L23 strain could be a good natural alternative to other therapies used for genital infections.

First detection and molecular identification of Borrelia garinii isolated from human skin in Taiwan [CORRESPONDENCE]

Chao, L.-L., Chen, Y.-J., Shih, C.-M. — Thu, 12 Nov 2009 08:01:06 PST

Borrelia garinii, a causative agent for Lyme disease, was detected and identified from human skin for the first time in Taiwan. Lyme disease infection was confirmed by Western immunoblot tests and isolation of Borrelia spirochetes from skin biopsy specimens. The genetic identity of this detected spirochete was determined by analyzing the gene sequence amplified by a genospecies-specific polymerase chain reaction (PCR) assay based on the 5S (rrf)-23S (rrl) intergenic spacer amplicon gene of B. burgdorferi sensu lato. Phylogenetic analysis reveals that the sequence similarity of this detected spirochete is highly homogeneous (97.7%-98.5%) within the genospecies of B. garinii and can be distinguished clearly from other genospecies of Borrelia spirochetes. These results provide the first convincing evidence of B. garinii spirochete detected from human skin in Taiwan and also highlight the increasing demand for investigating the existence of Lyme disease infection among patients with unusual skin lesions in the Taiwan area.

Killing of adherent microbes by a non-thermal atmospheric plasma jet [ANTIMICROBIAL AGENTS AND CHEMOTHERAPY]

Thu, 12 Nov 2009 08:01:06 PST

Objectives: 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 microorganisms. Methods: Agar plates and dentin slices were inoculated with 6 log10 colony forming units (CFUs) / cm<2> of Lactobacillus casei, Streptococcus mutans and Candida albicans, including 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 oC, dentin slices were vortexed in liquid media, and suspensions were placed on agar plates. The killing efficacy of the plasma jet was assessed by CFU-counting on the irradiated areas of agar plates, as well as by determination of the number of recovered CFUs from dentin slices. Results: A microbe-killing effect was found on irradiated parts of agar plates for L. casei, Strep. mutans, C. albicans and E. coli. The plasma jet treatment reduced the CFUs by 3 – 4 log10 intervals on the dentin slices in comparison to recovery rates from untreated controls. The microbe-killing effect was correlated to increasing irradiation times.Conclusion: Non-thermal atmospheric plasma jets can be used for the disinfection of dental surfaces.

Management of obstructive renal failure caused by bilateral renal aspergilloma in an immunocompetent newborn [CASE REPORT]

Thu, 12 Nov 2009 08:01:05 PST

Kidneys fungal infection is a rare condition which has been reported in premature babies and in diabetic or immunocompromised adult patients. Candida spp is the most frequent microorganism involved. This paper reports a case of an immunocompetent newborn with a bladder exstrophy who suffered from an acute renal failure caused by bilateral renal aspergilloma (Aspergillus flavus). The newborn was treated with amphotericin B urinary tract irrigation through bilateral nephrostomy catheters combined with liposomal amphotericin B and voriconazole therapy, which improved his renal function. However, due to persistent fungal colonization, long antifungal treatment and permanent ureterostomies were necessary to deal with new episodes of ureterorenal obstruction. At the moment, despite of renal injuries, renal function is conserved. The management of mechanical obstruction and choice of antifungal drugs are discussed in this peculiar case.

Traumatic endophthalmitis caused by Staphylococcus gallinarum [CASE REPORT]

Tibra, N. K., Jalali, S., Reddy, A. K., Narayanan, R., Agarwal, R. — Thu, 12 Nov 2009 08:01:05 PST

Herein, we describe what is believed to be the first case of traumatic endophthalmitis caused by Staphylococcus gallinarum following injury with an iron nail. The patient was successfully treated with vitrectomy and intravitreal injection of cefazolin and vancomycin.

Apoptosis in Candida biofilms exposed to amphotericin B [PATHOGENICITY AND VIRULENCE]

Al-Dhaheri, R. S., Douglas, L. J. — Thu, 05 Nov 2009 08:01:28 PST

Candida biofilms are resistant to a range of antifungal agents in current clinical use. The basis of this drug resistance is not clear but in some cases it could be due to the presence of a small number of drug-tolerant or persister cells. In this study, specific staining methods were used to investigate the existence of persisters and apoptosis in Candida biofilms subjected to different concentrations of amphotericin B. Fluorescein diacetate staining revealed the presence of persisters in biofilms of one of two strains of C. albicans tested, and in biofilms of C. krusei and C. parapsilosis. Caspase activity, indicative of apoptosis, was detected with SR-FLICA and D2R fluorochrome-based staining reagents in all of these biofilms. The general inhibitor of mammalian caspases, Z-VAD-FMK, when used at a low concentration (2.5 µM), increased the viability of drug-treated biofilms up to 11.5-fold (P<0.001%). Seven specific caspase inhibitors had different effects on C. albicans biofilm viability, but inhibitors of caspases-1, -9, -5, -3, and -2 all significantly increased cell survival (40-fold, 8-fold, 3.5-fold, 1.9-fold and 1.7-fold, respectively). On the other hand, histone deacetylase (HDA) inhibitors enhanced the activity of amphotericin B for biofilms of all three Candida species. Sodium butyrate and sodium valproate, for example, when added concurrently with amphotericin B, completely eliminated biofilm populations of C. albicans. Overall, our results demonstrate an apoptotic process in amphotericin-treated biofilms of three Candida species. They also indicate that HDA inhibitors can enhance the action of the drug and in some cases even eradicate persister subpopulations, suggesting that histone acetylation might activate apoptosis in these cells.

Evaluation of PCR MP method for intra-species differentiation of Trichophyton rubrum and Trichophyton interdigitale [DIAGNOSTICS, TYPING AND IDENTIFICATION]

Leibner-Ciszak, J., Dobrowolska, A., Krawczyk, B., Kaszuba, A., Staczek, P. — Thu, 05 Nov 2009 08:01:27 PST

In order to identify the sources of infection caused by dermatophytes as well as the pathogen transmission pathway there is the need to determine the methods that allow for the deep genetic differentiation of the strains within the genus. In this work for the first time PCR MP technique based on the ligation of adaptors and the difference in the melting temperatures of DNA restriction fragments was used for intraspecies genotyping of dermatophytes. Clinical isolates and reference strains of dermatophytes isolated from skin, toe and finger nails were used for this study. PCR MP (PCR Melting Profiles) and RAPD method has been applied for typing 11 isolates of T. rubrum, 40 isolates of T. interdigitale and 14 isolates of M. canis. We distinguished 5 types (containing 1 subtype) characteristic for T. rubrum, and 7 types characteristic for T. interdigitale with the PCR MP technique. Analysis conducted with RAPD method revealed 5 types for T. rubrum and 4 types for T. interdigitale isolates. No differentiation was observed in the case of M. canis isolates. These results demonstrate that PCR MP is a reliable method for the differentiation of T. rubrum and T. interdigitale strain and yields at least similar discriminatory power as the RAPD method.

In vitro interactions between primycin and different statins in their effects against some clinically important fungi [ANTIMICROBIAL AGENTS AND CHEMOTHERAPY]

Nyilasi, I., Kocsube, S., Pesti, M., Lukacs, G., Papp, T., Vagvolgyi, C. — Thu, 29 Oct 2009 08:31:20 PDT

The in vitro antifungal activities of primycin and various statins against some opportunistic pathogenic fungi were investigated. Primycin completely inhibited the growth of Candida albicans (MIC: 64 µg ml^-1) and Candida glabrata (MIC: 32 µg ml^-1), and was very effective against Paecilomyces variotii (MIC: 2 µg ml^-1), but it had little effect on Aspergillus fumigatus, Aspergillus flavus or Rhizopus oryzae (MIC: >64 µg ml^-1). The fungi exhibited different degrees of sensitivity to the statins; fluvastatin and simvastatin exerted potent antifungal activities against a wide variety of clinically important fungal pathogens. Atorvastatin, rosuvastatin and lovastatin were slightly effective against all fungal isolates tested, whereas pravastatin was completely ineffective. The in vitro interactions between primycin and the different statins were investigated by using the standard checkerboard titration method. When primycin was combined with fluvastatin, lovastatin or simvastatin, both synergistic and additive effects were observed. The extents of inhibition were higher when these compounds were applied together, and the concentrations of primycin and the given statin needed to block fungal growth completely could be decreased by several dilution steps. Similar interactions were observed when the variability of the within-species sensitivities was investigated.

Role and clinical course of verotoxigenic Escherichia coli infections in childhood acute diarrhoea, Argentina [CLINICAL MICROBIOLOGY AND VIROLOGY]

Rivero, M. A., Passucci, J. A., Rodriguez, E. M., Parma, A. E. — Thu, 22 Oct 2009 08:01:14 PDT

The aim of the study was to investigate the role and clinical course of verotoxigenic Escherichia coli (VTEC) infections in children with acute diarrhoea from Argentina, the country with the highest worldwide incidence of haemolytic uraemic syndrome (HUS). To accomplish our objective, 437 samples from children up to 6 years old with acute diarrhoea were collected and processed. More than 60% of the children studied presented watery or mucous diarrhoea without blood, and in 25.2% of the cases the samples were with blood. In a first screening, a multiplex PCR was performed to detect the presence of vt1, vt2, eae, ehxA, saa virulence genes Then, the strains were isolated and analyzed in order to characterize their serotypes, virulence genes, antibiotic susceptibility profiles and verotoxin (VT) production. Forty four of the 437 samples (10.06%) were positive to VTEC virulence genes. VTEC-infected patients presented different types of diarrhoea (27.27% belonged to non-bloody type). Several serotypes and virulence genotypes were found. Isolates belonged to the serotypes O157:H7, O145:H-, O26:H11, O121:H19, O111:H2, and O118:H2. HUS developed in 16 (36.4%) patients corresponding to positive samples. All the VTEC isolates produced a cytopathic effect on Vero cells monolayer, confirming the ability for VT expression. Despite most strains were sensitive to all antimicrobials studied a positive association between clinical progression to HUS and antibiotic therapy, both for the total of patients studied, as well as for the VTEC-positive group was observed. In conclusion, the data obtained in this study increase the knowledge on the role and clinical course of VTEC infection in childhood acute diarrhoea beyond bloody diarrhea, and might be considered for the prevention, diagnosis and management of this disease. It is possible that the optimal approach for VTEC diagnosis could be through searching by multiplex PCR for the presence of vt1, vt2, eae and ehxA genes

DNA sequence analysis of cagA 3' motifs of Helicobacter pylori strains from patients with peptic ulcer diseases [PATHOGENICITY AND VIRULENCE]

Salih, B. A, Bolek, B. K., Arikan, S. — Thu, 22 Oct 2009 08:01:15 PDT

The H. pylori cagA gene is a major virulence factor that plays an important role in gastric pathologies. DNA sequence data of the cagA 3' region of Western isolates differs markedly in their EPIYA motifs from those of East Asian isolates. An increase in the number of these motifs is known to be associated with gastric cancer. Whether such association is also the case for peptic ulceration was investigated in this study. Gastric biopsies were collected from 96 patients with duodenal ulcer (DU), gastric ulcer (GU) and gastritis. The type of the EPIYA motifs detected by PCR among 28 DU strains were 13 ABC, 8 ABCC, 6 ABCCC, and in one patient with mixed infection of ABC and ABCCCCC; among the 9 GU strains were 2 ABC, 5 ABCC, and 2 ABCCC; and in 40 gastritis strains were 35 ABC, and 5 ABCC. DNA sequencing was done to confirm the detection of the EPIYA motifs type and to analyze their peptide sequences. A significant association was found between the number of the EPIYA-C motifs (>/2) and peptic ulceration (P = 0.00001) as compared to gastritis. In conclusion, this study shows that our patients harbor cagA positive H. pylori strains with EPIYA motifs of the Western type and the increase in the number of EPIYA-C motifs was significantly associated with DU and GU than with gastritis indicating predictive association with the severity of the disease.

Clostridium sordellii lethal toxin gene is not detectable by PCR in the intestinal flora of Sudden Infant Death Syndrome cases or infants who died of other causes [CORRESPONDENCE]

Highet, A. R, Gibson, C. S, Goldwater, P. N — Thu, 22 Oct 2009 08:01:14 PDT

Infection caused by Clostridium sordellii translocated from the gastrointestinal tract has been reported to cause septic shock, often resulting in fatality. The organism's major virulence factor, lethal toxin (LT), is responsible for fatal outcome after C. sordellii infection. We designed an experiment to explore the possibility of C. sordellii colonising the intestinal tract contributing to Sudden Infant Death Syndrome, possibly via a fatal toxaemia. The feasibility of the methodology was demonstrated using a spiked culture of intestinal contents. Cultures grown from intestinal contents of fifty infants meeting the 1991 definition of SIDS, and thirteen cases of non-SIDS death were tested for the LT gene by PCR. None of the SIDS or non-SIDS infant samples tested positive for LT. The results of this investigation suggest that intestinal colonization by LT toxigenic C. sordellii is unlikely to contribute to SIDS. However, based on these results alone we cannot completely exclude the role of C. sordellii bacteraemia or toxaemia in SIDS.

Moraxella catarrhalis bacteraemia associated with prosthetic vascular graft infection [CASE REPORT]

Thu, 22 Oct 2009 08:01:13 PDT

Moraxella catarrhalis, formerly called Branhamella catarrhalis, Neisseria catarrhalis or Micrococcus catarrhalis, is a gram-negative, aerobic diplococcus frequently found as a colonizer of the upper respiratory tract. Over the last 20–30 years, this bacterium has emerged as a genuine pathogen and is now considered an important cause of otitis media in children and an etiologic agent in pneumonia in adults with chronic obstructive pulmonary disease. However, bacteraemia due to M. catarrhalis has rarely been reported. Presented below, is a case of M. catarrhalis bacteraemia associated with prosthetic vascular graft infection along with a review of the relevant literature.

Fatal spontaneous bacterial peritonitis and necrotizing fasciitis with bacteremia caused by Bacillus cereus in a patient with cirrhosis [CASE REPORT]

Lee, Y.-L., Shih, S.-D., Weng, Y.-J., Chen, C., Liu, C.-E. — Thu, 22 Oct 2009 08:01:13 PDT

We report a case of spontaneous bacterial peritonitis and necrotizing fasciitis caused by Bacillus cereus in a cirrhotic patient without preceding disruption of skin or symptoms of gastroenteritis. This rapidly fatal infection due to B. cereus adds to the long list of etiologies of infectious complications among patients with cirrhosis of the liver.

Disease burden due to Streptococcus dysgalactiae subsp. equisimilis (group G and C streptococci; GGS/GCS) is higher than due to S. pyogenes among Mumbai school children [EPIDEMIOLOGY]

Thu, 15 Oct 2009 07:31:04 PDT

SummaryStreptococcus pyogenes (group A Streptococcus, GAS), a human pathogen and S. dysgalactiae subsp. equisimilis (human groups G and C streptococci; GGS/GCS) are evolutionarily related, share the same tissue niche in humans, exchange genetic material, share up to half of virulence-associated genes and cause similar spectrum of diseases. Yet, GGS/GCS is often considered as commensal bacterium and its role in streptococcal disease burden is under-recognized. While reports of GGS/GCS recovery from normally sterile sites are increasing, studies describing GGS/GCS throat colonization rates relative to GAS in the same population are very few. This study was carried out in India where burden of streptococcal diseases, including rheumatic fever (RF) and rheumatic heart disease (RHD), is high. As part of surveillance study, throat swabs were taken from 1504 children attending seven municipal schools in Mumbai, India during 2006-08. GAS and GGS/GCS were identified on the basis of beta-haemolytic activity, group carbohydrate and PYR-test, and subsequently typed. The GGS/GCS carriage rate (166/1504, 10%) was eight-fold higher than the GAS carriage (22/1504, 1.4%) rate in this population. The 166 GGS/GCS isolates collected represented 21 different emm-types (molecular types), and the 22 GAS isolates represented 15 different emm-types. Although the rate of pharyngitis associated with GGS/GCS is marginally lower than with GAS, high rates of throat colonization by GGS/GCS underscores its importance in the pathogenesis of pharyngitis.

The Changing Aetiology of Paediatric Bacteraemia in England and Wales, 1998-2007 [EPIDEMIOLOGY]

Thu, 15 Oct 2009 07:31:05 PDT

Bacteraemia in children is a potentially life threatening condition. The objective of this study was to determine trends in the aetiology of bacteraemia in children aged 1 month - 15 years in England and Wales by collecting data voluntarily reported by National Health Service hospital microbiology laboratories. Over the ten-year period, 1998-2007, a total of 51,788 bacteraemia cases involving 105 genera/species of bacteria were reported. Total annual reports of bacteraemia increased from 4,125 to 6,916, with an average increase of 6.5% per year (95% CI: 1.3%-12.1%). In 2007, just over half the cases were accounted for by four groups of organisms: coagulase-negative staphylococci (28%), Staphylococcus aureus (10%), non-pyogenic streptococci (9%), and Streptococcus pneumoniae (7%). These organisms along with a further 13 species/genera accounted for 90% of the cases. The commonest Gram-negative organisms were Neisseria meningitidis and Escherichia coli which each accounted for 5% of total bacteraemia reports in 2007. There was a significant decrease in reports of bacteraemia due to the three vaccine preventable pathogens, Haemophilus influenzae, N. meningitidis and S. pneumoniae, following the introduction of each vaccine programme or catch-up campaign. This study identifies the commonest causes of bacteraemia in children in England and Wales, and highlights the shifts in trends observed over time.

Development and Evaluation of Internal Amplification Controls (IACs) for use in a real time duplex PCR assay for detection of Campylobacter coli and C. jejuni [DIAGNOSTICS, TYPING AND IDENTIFICATION]

Randall, L., Leema, F., Rodgers, J., Vidal, A., Clifton-Hadley, F. — Thu, 15 Oct 2009 07:31:06 PDT

A common problem of both conventional and real time PCR assays is failure of DNA amplification due to the presence of inhibitory substances in samples. In view of this, our aim was to develop and evaluate Internal Amplification Controls (IACs) for use with an existing duplex real time PCR assay for Campylobacter coli and C. jejuni. Both competitive and non-competitive IACs were developed and evaluated. The competitive approach involved a DNA fragment of the coding region of the viral fish haemorrhagic septicaemia virus, flanked by the mapA PCR primers, whilst the non-competitive approach utilised an extra set of universal 16S primers. Both IAC PCR assay types were evaluated using cultures of Campylobacter and chicken caecal content samples. Both IACs were sensitive to caecal inhibitors, making them suitable to detect inhibition which could lead to false negatives. Results showed that both IACs at optimum concentrations worked well without reducing the overall sensitivity of the PCR. Compared to culture, the optimized competitive IAC-PCR assay detected 45/47 positives (sensitivity 93.6%, specificity 80.1%), however it had the advantage over culture in that it could detect mixed infections of C. coli and C. jejuni and was capable of giving a result for a sample within a day.

Rapid detection of Streptococcus agalactiae from swabs by Peptide Nucleic Acid Fluorescence in situ hybridization (PNA FISH) [DIAGNOSTICS, TYPING AND IDENTIFICATION]

Thu, 15 Oct 2009 07:31:03 PDT

The applicability of the PNA FISH (peptide nucleic acid fluorescence in situ hybridization) method for detection of Streptococcus agalactiae (Group B streptococci - GBS) from swab samples was evaluated. Three swab sample processing protocols with different time-to-result (TTR) were compared, including (i) direct smearing of fresh swabs onto microscope slides (n = 153; TTR 2.5 h), (ii) further extraction and concentration of cells from these same swabs (n = 153; TTR 2.7 h), and (iii) short term LIM broth enrichment culture incubation (7 h, 37 °C) of fresh swabs (n = 120; TTR 9.5 h). Sensitivity, specificity, positive predictive value, and negative predictive value for GBS PNA FISH for sample processing procedures with TTR of 2.5 h, 2.7 h, and 9.5 h were 68%, 100%, 100%, 95%; 91% 100%, 100%, 98%; and 100%, 100%, 100%, 100% respectively. Improved test results were achieved by subjecting swabs to an extraction procedure or abbreviated LIM broth enrichment culture incubation prior to performing GBS PNA FISH.

Genetic Analysis of High-Level Mupirocin Resistance in ST80 clone of Community-Associated Methicillin-Resistant Staphylococcus aureus [ANTIMICROBIAL AGENTS AND CHEMOTHERAPY]

Udo, E. E, Sarkhoo, E. — Thu, 15 Oct 2009 07:31:02 PDT

Four Community- associated MRSA isolates expressing high-level mupirocin resistance (MIC: >1024mg/L) were isolated from four sites of a diabetic patient and characterized for the genetic location of their resistance determinants and typed using pulsed-field gel electrophoresis (PFGE), SCCmec coagulase gene and multilocus sequence typing to ascertain their relatedness. The presence of genes for resistance to high-level mupirocin (mupA), tetracycline (tetK) and fusidic acid (far 1), Panton-Valentine leukocidin (PVL), accessory gene regulators (agr) and capsular polysaccharide (cap) were detected in PCR assays. They were resistant to kanamycin, streptomycin, tetracycline, fusidic acid and cadmium acetate. They harboured mupA, tetK, far1, PVL, agr 3 and cap8. They had identical PFGE pattern, coagulase gene type, possessed type IV SCCmec element and belonged to sequence type 80 (ST80). In contrast, they had three different plasmid profiles consisting of (1) 28.0 and 26.0 kb, (2) 28.0, 21.0 and 4.0 kb, (3). 41.0 and 4.0 kb. Genetic studies located resistance to tetracycline, fusidic acid and cadmium acetate on the 28 kb plasmids and mupA on related non conjugative 26 kb and 21 kb plasmids. One of the 21-kb mupirocin resistance plasmids was derived from the 41 kb plasmid during transfer experiments. The emergence of high-level mupirocin resistance in ST80-SCCmec-IV MRSA clone demonstrates the increasing capacity of CA-MRSA clones to acquire resistance to multiple antibacterial agents. The presence of different plasmid profiles in genetically identical isolates created difficulty in interpretation of typing results and highlighted the weakness of using plasmid analysis as a sole method for strain typing.

Phylogenetic studies of genus Nocardia species based on gyrB gene analyses [DIAGNOSTICS, TYPING AND IDENTIFICATION]

Takeda, K., Kang, Y., Yazawa, K., Gonoi, T., Mikami, Y. — Thu, 15 Oct 2009 07:31:01 PDT

Phylogenetic analyses of 56 type species of Nocardia were conducted using the partial nucleotide sequences of gyrase B (gyrB) gene. The interspecies similarities of gyrB gene for 56 type species of Nocardia are 82.4-99.9%, which correspond to 270-2 nucleotide differences in the partial gene sequences around 1,200 bp. In comparison to phylogenetic relations, gyrB gene sequence information is generally consistent with that of 16S rRNA gene sequences with minor exceptions. However, the degree of divergence of gyrB gene sequences is approximately 3.6 times greater than that of 16S rRNA gene sequences, suggesting a higher discriminative power of gyrB sequence information than that of 16S rRNA within the species of Nocardia. Nocardia type species are clustered based on gyrB sequence similarity values of 93.5% and above. Among the 56 type species, 37 species are distributed into 13 clusters, each comprising 2-7 species. The remaining 19 species are classified into an independent cluster, in which the similarities between each species and other 55 Nocardia species are lower than 93.5%. Among the eight mycolic-acid-containing actinomycete genera in the suborder Corynebacterineae, Nocardia was clearly differentiated from other actinomycete genera such as Rhodococcus by gyrB gene analyses (similarity values of gyrB sequences of Nocardia and Rhodococcus are 75-85%), indicating that gyrB gene is a useful alternative to 16S rRNA gene for determination of phylogenetic relations between the genus Nocardia and seven other actinomycete genera.

Visualization of adherent micro organisms - different techniques [REVIEW]

Hannig, C., Follo, M., Hellwig, E., Al-Ahmad, A. — Thu, 08 Oct 2009 08:31:04 PDT

Visualization and quantification of adherent bacteria still is one of the most important topics in microbiology. The aim of the present short review was to give an insight into traditional and current methods for the visualization and quantification of adherent bacteria or biofilms with a special focus on the experiences gained in dental research.

Comparison of nasopharyngeal nylon flocked swabs with universal transport medium and rayon bud swabs with a sponge reservoir of viral transport medium in the diagnosis of paediatric influenza [CLINICAL MICROBIOLOGY AND VIROLOGY]

Thu, 08 Oct 2009 08:31:05 PDT

This study compared a kit containing a nasopharyngeal nylon flocked swab and a tube with a liquid universal transport medium (UTM) with a kit containing a plastic shafted rayon budded swab with a sponge reservoir of viral transport medium for the molecular detection of influenza viruses in children. Respiratory samples were collected from 314 children aged <5 yrs with influenza-like illness (186 males; mean age, 2.32 ± 2.27 yrs) using both swabs in a randomised sequence for each patient. The flocked swabs permitted to detect 28 influenza A (8.9%) and 45 influenza B cases (14.3%), and the rayon bud swabs 26 influenza A (8.2%) and 43 influenza B cases (13.7%), with detection rates of respectively 23.2% and 21.9%, and similar cycle threshold values. Pediatricians and laboratory staff were significantly more satisfied with both the simplicity (p<0.0001) and rapidity (p<0.0001) of the nasopharyngeal flocked swabs with UTM. These findings show that the flocked swabs with UTM and the rayon bud swabs with a sponge transport medium are similarly efficient in preserving influenza viruses nucleic acid, but the kit containing a flocked swab with a UTM allows the easier and more rapid collection and processing of specimens.

Diversity in cag pathogenicity island (cag PAI) of Helicobacter pylori isolates from North and South Indian populations [PATHOGENICITY AND VIRULENCE]

Kumar, S., Kumar, A., Dixit, V. K — Thu, 08 Oct 2009 08:31:02 PDT

The cag pathogenicity island (cagPAI) has been reported to be the major virulence determinant of Helicobacter pylori-related diseases. In the present study diversity in cagA gene and integrity of cagPAI in one hundred fifty eight H. pylori strains of Varanasi (North India) and Hyderabad (South India) were studied by amplifying cagA gene (ca 3.5 kb) followed by PCR-RFLP analysis. Results revealed significant differences in cagA gene and the integrity of cagPAI between North and South Indian isolates. Out of 158 isolates, 40 (34.8%) from Varanasi and 20 (46.5%) of Hyderabad were found to carry intact cagPAI. Partially deleted cagPAI was present in 75 (65.2%) isolates of Varanasi and 23 (53.5%) Hyderabad. None of the isolates showed complete deletion of cagPAI. Differences in cagA 5' and cagA 3' region were also noted and eleven isolates (8 from Varanasi and 3 from Hyderabad) which were found cagA negative with primers for 5' region turned out to be cagA positive with primers of 3' variable region. It is tentatively concluded that the 3' variable region may be a better marker for cagA typing. Results of PCR-RFLP of cagA gene (3.5 kb) showed 29 distinguishable digestion patterns and cluster analysis of RFLP types placed all the 32 isolates into five groups. Our results demonstrate that significant differences in cagPAI occur among isolates of North and South India and RFLP of cagA could be employed for elucidating genetic variations among various isolates of H. pylori.

Vibrio cholerae O1 Ogawa detoxified lipopolysaccharide structures as inducers of cytokines and oxidative species in macrophages [HOST RESPONSE]

Paulovicova, E., Kovacova, E., Bystricky, S. — Thu, 08 Oct 2009 08:31:02 PDT

Multi-drug resistance to several strains of Vibrio cholerae supported the anti-cholera vaccine developmental attempts via various sub-cellular moieties. In order to examine immunological efficacy of dLPS derived saccharidic immunogens, ex vivo activation of mouse peritoneal macrophage was investigated. Immunomodulatory effect was evaluated via induction of pro-inflammatory cytokines TNF-, IL-1 , IL-6 and acceleration of nitric oxide and reactive oxygen species (NOS/ROS). Immunologically active structures triggered mouse peritoneal macrophages to cytokine secretion and NOS/ROS release, even at very low concentrations of 12.5µg/mL. It could be assumed that O-specific polysaccharidic moiety is more immunologically efficient than glycolipidic one, probably due to KDO position. Present study revealed effective structure-immunomodulating relationships of detoxified LPS–derived moieties, desirable in sub-cellular anti-cholera vaccine design.

Mycoplasma salivarium detected in a microbial community with Candida glabrata in the biofilm of an occluded biliary stent [CASE REPORT]

Thu, 08 Oct 2009 08:31:01 PDT

Mycoplasma salivarium, preferentially an inhabitant of the human oral cavity, has rarely been found in other locations associated with disease. We describe here for the first time, the detection of M. salivarium, together with Candida glabrata, in an occluded biliary stent of an icteric, cholestatic patient.

Accelerated Identification of Staphylococcus aureus from Blood Cultures by a Modified Fluorescence in situ Hybridisation (FISH) procedure [DIAGNOSTICS, TYPING AND IDENTIFICATION]

Poppert, S., Riecker, M., Wellinghausen, N., Frickmann, H., Essig, A. — Thu, 01 Oct 2009 07:31:22 PDT

We evaluated fluorescence in situ hybridisation (FISH) for rapid identification of Staphylococcus aureus and coagulase-negative staphylococci directly from blood cultures. Initially 360 blood cultures containing Gram-positive cocci were investigated by a previously described microwave-FISH procedure. 44 of 49 (89.7%) S. aureus and 298 of 299 (99.7%) coagulase-negative staphylococci were correctly identified. Because FISH proved useful and reliable, but handling was found inconvenient, the method was modified by employing a recently developed slide chamber. The required time was thereby reduced from one hour to half an hour. The simplified execution allowed integration of the method into the workflow of a routine laboratory without difficulty. By identifying 37 of 37 (100%) S. aureus and 169 of 172 (98.2%) coagulase- negative staphylococci directly from blood cultures the modified method proved highly reliable.

Potential use of a liposome-encapsulated mixture of lipopolysaccharide core types (R1, R2, R3 and R4) of Escherichia coli in controlling colisepticaemia in chickens [VETERINARY MICROBIOLOGY]

Dissanayake, D. R. A., Wijewardana, T. G, Gunawardena, G. A, Poxton, I. R — Thu, 01 Oct 2009 07:31:21 PDT

Infections caused by Escherichia coli make an economically significant impact in the poultry industry and a non-serotype-specific vaccine appears to be the most logical method of controlling it. The core oligosaccharide-lipid A region of bacterial lipopolysaccharide (LPS) is well conserved and highly immunogenic but toxic. This study determined the possible use of a liposome encapsulated mixture of rough LPSs of core types R1, R2, R3 and R4 in controlling infections caused by E. coli in chickens. The liposome which encapsulated LPS consisted of egg phosphatidylcholine, bovine brain phosphatidylserine and cholesterol. As determined by Limulus amoebocyte lysate assay, the endotoxicity of liposome incorporated LPS was at least 700 times lower than free LPS. Induction of nitric oxide production and expression of inflammatory genes by free LPS and liposome-incorporated LPS, when tested on chicken macrophage cell line (HD11), showed that liposome incorporated LPS produced a significantly lower amount of nitric oxide (>5 µM) than free LPS (22 µM). Transcription of the genes for interleukin-1 beta (IL-1β) and inducible nitric oxide synthase (iNOS) were lower in cells treated with liposome incorporated LPS than in cells treated with free LPS. Chickens when immunized with 0.2µg, 1 µg and 5 µg of liposome encapsulated mixture of LPS core types showed that the antibody response increased with increasing dose and the birds which received 5µg of liposome encapsulated LPS had significantly higher (p<0.001) anti-LPS core antibody titres than the chickens in all other groups and this also protected the birds against lethal challenge with E. coli O78.

Fulminant necrotizing fasciitis due to Vibrio parahaemolyticus [CASE REPORT]

Tena, D., Arias, M., Alvarez, B. T., Mauleon, C., Jimenez, M. P., Bisquert, J. — Thu, 01 Oct 2009 07:31:21 PDT

Necrotizing soft-tissue infection due to V. parahaemolyticus is exceptional. We report a case of necrotizing fasciitis due to V. parahaemolyticus in a 92-year-old woman with a history of chronic renal failure, diabetes mellitus and malnutrition. Clinical evolution was fulminant and the patient died after 6 hours of admission. The review of all cases previously reported shows that the infection occur in patients with underlying diseases through ingestion of raw oysters or inoculation via traumatic injury in marine environments. Mortality rate of all reviewed cases was 42.8%. In conclusion, V. parahaemolyticus should be considered as a possible causative agent of necrotizing fasciitis, especially in patients with underlying diseases. Early diagnosis and prompt aggressive debridement associated with antibiotic therapy are essential in order to save the patient's life, because clinical evolution can be fulminant and mortality rates are high.

Allele variability of critical virulence genes (eae, bfpA and perA) in typical and atypical EPEC in Peruvian children [PATHOGENICITY AND VIRULENCE]

Thu, 01 Oct 2009 07:31:20 PDT

Enteropathogenic Escherichia coli (EPEC) are a leading cause of infantile diarrhea in developing countries. The aim of this study was to describe the allelic diversity of critical virulence EPEC genes and their association with clinical characteristics. 120 EPEC strains isolated from a cohort diarrhea study in Peruvian children were characterized for the allele type of eae (intimin), bfpA (bundlin pilin protein of bundle-forming pilus) and perA (plasmid encoded regulator) genes by PCR-restriction fragment length polymorphism. Atypical EPEC strains (eae+, bfp-) were the most common pathotype in diarrhea (54/74, 73%) and control samples from children without diarrhea (40/46, 87%). Overall there were 13 eae alleles; the most common were beta (34/120, 28%), theta (24/120, 20%), kappa (14/120, 12%) and mu (8/120, 7%). There were 5 bfpA alleles; the most common were beta1/7 (10/26), alpha3 (7/26) and beta5 (3/26). There were 3 perA alleles: beta (8/16), alpha (7/16) and gamma (1/16). The strains belonged to 36 distinct serogroups; O55 was the most frequent. The gamma-intimin allele was more frequently found in diarrhea episodes of longer duration (>7 days) than shorter (3/26, 12% vs. 0/48, 0%, p<0.05). The kappa-intimin allele had the highest clinical severity score in comparison to other alleles (p<0.05). In Peruvian children the virulence genes of EPEC strains are highly variable. Further studies are needed to evaluate additional virulence markers to determine whether relationships exist between specific variants and clinical features of disease.

Molecular identification of Mycobacterium avium subspecies paratuberculosis in an Italian patient with Hashimoto's thyroiditis and Melkersson-Rosenthal syndrome [CORRESPONDENCE]

D'Amore, M., Lisi, S., Sisto, M., Cucci, L., Dow, C. T. — Thu, 01 Oct 2009 07:31:22 PDT

Recently we described the case of a 52-year-old woman with Melkersson-Rosenthal syndrome in association with Hashimoto's thyroiditis. In view of the literature supporting the involvement of Mycobacterium paratuberculosis(MAP)in autoimmune disease and an autoimmune etiology of MRS, we performed blood tests for the presence of MAP. The PCR data show a band that co-migrates with the positive control at the predicted 298 bp amplicon size. The sequenced DNA of the representative sample band showed 100 % identity with MAP IS900.

Detection of Cardiobacterium valvarum in a patient with aortic valve infective endocarditis by broad-range PCR [CASE REPORT]

Thu, 01 Oct 2009 07:31:20 PDT

Cardiobacterium valvarum, a fastidious gram-negative bacterium, was detected in the aortic valve of a previously healthy 63-year-old man by broad-range PCR and 16S rRNA gene sequencing. In contrast to five previously published cases, our patient had neither congenital bicuspid nor prosthetic aortic valve. Here, we present a case of C. valvarum native tricuspid aortic valve infective endocarditis and a review of the literature.

Nocardia cyriacigeorgica: First case of endocarditis with disseminated soft tissue infection and a review of the literature [CASE REPORT]

Cargill, J. S, Boyd, G. J, Weightman, N. C — Thu, 01 Oct 2009 07:31:20 PDT

Nocardia cyriacigeorgica is a common environmental organism. The organism has been isolated from clinical samples in Europe, Asia and North America, predominantly from respiratory samples but from several other sites. We present a case report of an 85 year old female patient in the UK who was found to have a multi-focal soft tissue infection from which N. cyriacigeorgica was isolated. She had a background history of chronic obstructive pulmonary disease and corticosteroid use for polymyalgia rheumatica. During the course of her treatment echocardiography showed the presence of a mobile heart mass attached to a valve leaflet, a major Dukes criterion for endocarditis. We suggest that in cases of disseminated Nocardia infection, endocarditis should be excluded, particularly in cases failing to respond to treatment. We also review the previous reports of both N. cyriacigeorgica infection and of endocarditis due to Nocardia species and related genera.

Postoperative spondylodiscitis due to Kytococcus schroeteri in a diabetic woman [CASE REPORT]

Thu, 01 Oct 2009 07:31:19 PDT

Kytococcus schroeteri, a cocci gram-positive bacterium, is a usually regarded as a part of human skin flora. It has been described in prosthetic valve endocarditis but never involved in osteoarticular infections. We report the first case of a spondylodiscitis due to Kytococcus schroeteri identified by the 16S rRNA gene sequencing.

Evaluation of ITS2-RFLP-analysis for the identification of dermatophytes (Fungi) [DIAGNOSTICS, TYPING AND IDENTIFICATION]

De Baere, T., Summerbell, R., Theelen, B., Boekhout, T., Vaneechoutte, M. — Thu, 24 Sep 2009 08:01:17 PDT

A total of 95 isolates, belonging to 33 species of 5 dermatophyte genera, i.e. Arthroderma (15 species), Chrysosporium (2), Epidermophyton (1), Microsporum (3) and Trichophyton (12), were studied with ITS2-PCR-RFLP analysis (ITS2-RFLP), consisting of amplification of the ITS2 region, restriction digestion with BstUI (CG/CG) and restriction fragment length determination by capillary electrophoresis on an ABI Prism 310 (Applied BioSystems). ITS2-RFLP-analysis proved to be most useful for identification of species of the genera Arthroderma, Chrysosporium and Epidermophyton, but could not distinguish between several Trichophyton species. The identification results are in correspondence with established and recent taxonomical insights of the dermatophytes, e.g. because highly related species also had closely related and sometimes difficult-to-discriminate ITS2-RFLP patterns. In some cases, several ITS2-RFLP-groups could be distinguished within species, again mostly in correspondence with the taxonomic delineations of subspecies and/or genomovars, confirming the relevance of ITS2-RFLP-analysis as an identification technique and a useful taxonomic approach.

A quadraplexed real-time PCR assay for rapid detection and differentiation of the Clostridium botulinum toxin genes A, B, E and F [DIAGNOSTICS, TYPING AND IDENTIFICATION]

Thu, 24 Sep 2009 08:01:17 PDT

Clostridium botulinum is the etiologic agent of botulism, a disease marked by flaccid paralysis that can progress to asphyxiation and death. This species is defined by the production of one of the botulinum neurotoxins (BoNTs) which are the most potent toxins known. Because of their potency, these toxins have the potential to be used as biological weapons, and therefore, C. botulinum has been classified as a category A select agent. There are four related but antigenically distinct BoNT types that cause disease in humans, (A, B, E, and F). The mouse bioassay is the current gold standard by which BoNTs is confirmed. However, this method is expensive, slow, and very labor intensive. Although PCR-based assays have been used extensively for the detection of BoNT-producing bacteria in food, animals, and fecal samples, and recently, to help diagnose disease in humans, no real-time quantitative PCR (qPCR) assay has yet been developed that can identify and differentiate all four BoNTs that cause disease in humans. This report describes the development of a qPCR single-tube assay that uniquely identifies these four BoNTs responsible for human disease. A total of 79 C. botulinum isolates with varying toxin types were evaluated in this study, as well as numerous near-neighbors and other bacterial species. Results showed that this quadraplexed assay was capable of detecting any of the four toxin genes in a given sample at a sensitivity of about 130-840 fg of genomic DNA and could detect the presence of up to all four BoNT genes simultaneously in a given sample. The assay was also functional in the presence of extraneous organic matter commonly found in various environmental samples.

VopF, a type III effector protein from a non-O1, non-O139 V. cholerae strain demonstrates toxicity in Saccharomyces cerevisiae model [PATHOGENICITY AND VIRULENCE]

Thu, 24 Sep 2009 08:01:15 PDT

VopF, a type III effector protein has been identified as a contributory factor to the intestinal colonization of T3SS positive non O1, non-O139 V. cholerae strains. To gain more insight of the function of VopF, a yeast model was developed. We observed that ectopic expression of VopF conferred toxicity in yeast.

Subinhibitory concentrations of moxifloxacin decrease adhesion and biofilm formation of Stenotrophomonas maltophilia from cystic fibrosis [ANTIMICROBIAL AGENTS AND CHEMOTHERAPY]

Thu, 17 Sep 2009 07:31:13 PDT

Stenotrophomonas maltophilia is an emerging nosocomial bacterial pathogen which is currently isolated with increasing frequency from the airways of cystic fibrosis (CF) patients. We evaluated the effect of subinhibitory concentrations of moxifloxacin against adhesion, biofilm formation, and cell-surface hydrophobicity of two strains of Stenotrophomonas maltophilia isolated from CF patients. Adhesion and biofilm formation assays were carried out on polystyrene and quantified by colony counts. CSH was determined by adhesion to n-hexadecane test. Moxifloxacin at 0.03x and 0.06xMIC caused a significant decrease of adhesion and biofilm formation by both strains tested. A significant reduction of the cell-surface hydrophobicity following exposure to sub-MICs of moxifloxacin was observed for one strain only. Results of the present study provided an additional rationale for the use of moxifloxacin in CF patients and, more generally, in biofilm-related infections involving S. maltophilia.

Daptomycin is not active against rapidly growing Mycobacteria [CORRESPONDENCE]

Bastian, S., Brossier, F., Wichlacz, C., Jarlier, V., Veziris, N. — Thu, 10 Sep 2009 08:01:01 PDT

We evaluated in vitro the activity of daptomycin against rapidly growing mycobacteria (RGM). According to EUCAST breakpoints, the MIC measured classifies RGM as resistant to daptomycin.

Alterations on the structure of Leishmania major induced by N arylisoquinolines correlate with compound accumulation and disposition [ANTIMICROBIAL AGENTS AND CHEMOTHERAPY]

Ponte Sucre, A., Gulder, T., Gulder, T., Vollmers, G., Bringmann, G., Moll, H. — Thu, 10 Sep 2009 08:01:01 PDT

Naphthylisoquinoline alkaloids equipped with an N,C-hetero-'biaryl' axis and, in particular, simplified synthetic analogs thereof, kill intracellular Leishmania major at concentrations in the low sub-micromolar range, while being significantly less toxic to their major host cell, the macrophage, at the same concentrations. To further investigate their mechanism of action we evaluated the morphological and ultrastructural changes induced by specific arylisoquinolines in L. major and the correlation of these changes with compound accumulation and disposition by the parasite. After 24 h of treatment with the synthetic arylisoquinolinium salts 3 or 4, dramatic structural changes and cell death were observed. Furthermore, the auto-fluorescent derivative 3 accumulates continually in intracellular compartments. Our results thus suggest that the leishmanicidal effect of arylisoquinolinium salts may involve their ability to accumulate and precipitate in intracellular organelles, form a huge vacuole and eventually promote cell lysis.

Molecular emm genotyping and antibiotic susceptibility of Streptococcus dysgalactiae subsp. equisimilis isolated from invasive and noninvasive infections [EPIDEMIOLOGY]

Thu, 10 Sep 2009 08:01:03 PDT

To analyze characteristics of infections caused by Streptococcus dysgalactiae subsp. equisimilis, clinical isolates (n=145) were collected at 11 medical institutions between September 2003 and October 2005. These isolates belonged to Lancefield group A (n=5), group C (n=18), or group G (n=122). Among all isolates, 42 strains were isolated from sterile samples such as blood, synovial fluid, and tissue specimens from patients, - mostly over 50 years with invasive infections -, and included 7 cases of streptococcal toxic shock syndrome and necrotizing fasciitis. In contrast, the remaining 103 were mainly isolated from patients of all age groups with noninvasive infections such as pharyngotonsillitis. These isolates were classified into 25 types based on emm genotyping. A significant difference in emm types was observed between isolates from invasive and noninvasive infections (P <0.001); stG485, stG6792, and stG2078 predominated among isolates from invasive infections. A phylogenic tree of complete open reading frames of emm genes in this organism showed high homology with those of Streptococcus pyogenes, but not with those of other streptococci. The presence of 5 different clones was estimated based on from DNA profiles of isolates from invasive infections obtained by PFGE. Genes for resistance to macrolides [erm(A), 3 isolates; erm(B), 5; mef(A), 7] and levofloxacin (mutations in gyrA and parC, 4) were identified in this organism. These results suggest the need for further nationwide surveillance of invasive infections caused by S. dysgalactiae subsp. equisimilis.

Zoonotic transmission of Streptococcus equi subsp. zooepidemicus from a dog to a handler [CASE REPORT]

Thu, 10 Sep 2009 08:01:03 PDT

This is the first case report to describe the direct transmission of Streptococcus equi subsp. zooepidemicus from an infected dog to a handler who subsequently developed severe systemic infection. Characterisation of the haemolytic streptococci isolated from both patients, by phenotypic and molecular analysis, confirmed the canine and human isolates were identical.

Identification of virulence determinants of Mycobacterium avium which impact the ability to resist host killing mechanisms [PATHOGENICITY AND VIRULENCE]

Li, Y., Danelishvili, L., Wagner, D., Petrofsky, M., Bermudez, L. — Thu, 10 Sep 2009 08:01:02 PDT

230 Summary31 Mycobacterium avium is an opportunistic pathogen associated with pulmonary disease in non-32 AIDS patients and disseminated infection in patients with AIDS. The chief route of infection is33 by colonizing and invading mucosa of gastrointestinal tract, but infection through the respiratory34 route also occurs. After crossing the mucosa, M. avium infects and replicates within tissue35 macrophages.36 To identify M. avium genes required for survival in vivo, a library of signature-tagged37 transposon mutants was constructed and screened for clones attenuated in mice. Thirty-two38 clones were found attenuated for their virulence, from which eleven have been sequenced and39 tested further. All the mutants studied grew in vitro similarly to the wild-type MAC104. Ten40 mutants were tested individually in mice, confirming the attenuated phenotype. MAV_2450, a41 polydetide synthase homologue to Mycobacterium tuberculosis pks12, was identified. STM542 and STM10 genes (encoding for two hypothetical proteins MAV_4292 and MAV_4012) were43 associated with susceptibility to oxidative products. Mutants MAV_2450, MAV_4292,44 MAV_0385 and MAV_4264 live in macrophage vacuole with acidic pH (below 6.9). Mutants45 MAV_4292, MAV_0385 and MAV_4264 were susceptible to NO in vitro. The study of46 individual mutants can potentially lead to new knowledge about M. avium pathogenic47 mechanisms.

Toxic megacolon complicating a Clostridium difficile infection in a pregnant woman : case report [CASE REPORT]

Candiotto, A., Pascoli, I., Gritti, A., Busato, E., Dal Pozzo, G. — Thu, 10 Sep 2009 08:01:02 PDT

Clostridium difficile infection (CDI) in non hospitalized patients has been reported with increased frequency. An association between CDI and pregnancy has not been stressed. We report a case of severe CDI during pregnancy in a patient who did not have the traditional risk factors for CDI, such as antibiotic use, concurrent hospitalizations or immunodeficiency. There is recent evidence that Clostridium difficile-associated disease severity and frequency may be increasing not only in high-risk populations (hospitalized, immunocompromised or elderly patients) but also among younger and healthier patients from the community. Further research into the scope and risk factors during pregnancy for CDI is warranted

Development of ertapenem resistance in a patient with mediastinitis caused by an extended-spectrum beta-lactamase producing Klebsiella pneumoniae. [CASE REPORT]

Thu, 10 Sep 2009 08:01:03 PDT

To study the clinical and microbiological features associated with a carbapenem-resistant K. pneumoniae isolate that has been selected in vivo by an ertapenem-containing regimen in a patient with mediastinitis despite high blood and mediastinal levels of ertapenem. Carbapenem-resistance was characterized by conjugation, PCR, DNA sequencing and analysis of outer-membrane proteins. The isolates susceptible and resistant to the carbapenems were compared by ribotyping and PFGE. Resistance to all available beta-lactams was most probably due to combined production of extended spectrum beta-lactamase CTX-M-15 and loss of OmpK36 porin. The results of ribotyping and PFGE suggest that the carbapenem resistant strain was a derivative of the original mediastinal isolate rather than a superinfecting isolate. This observation stresses the risk of selection of pan-penem resistant strains of enterobacteria when ertapenem is used for the treatment of severe infections due to ESBL producing enterobacteria.

Metabolism of azo dyes by human skin microbiota [HUMAN AND ANIMAL MICROBIAL ECOLOGY]

Stingley, R., Zou, W., Heinze, T., Chen, H., Cerniglia, C. — Thu, 03 Sep 2009 07:31:16 PDT

Reduction of Methyl Red and Orange 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 Methyl Red by 74-100% in 24 h, with only three species unable to reduce completely the dye in that time. Among the species tested, only C. xerosis was unable to reduce Orange II to any degree by 24 h, and only Staphy. delphini, Staphy. sciuri sciuri, and Pseudomonas aeruginosa were able to reduce completely this dye within 24 h. Methyl Red reduction started with early exponential growth in Staphy. aureus and Staphy. epidermidis, and around late exponential/early stationary growth in P. aeruginosa. Reduction of Orange II, Ponceau S, and Ponceau BS started during late exponential/early stationary growth for all three species. Using LC/ESI-MS/MS analyses, Methyl Red metabolites produced by Staphy. aureus, Staphy. 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, Staphy. haemolyticus, Staphy. epidermidis, Staphy. cohnii, and Staphy. saprophyticus, have hypothetical genes with 77, 76, 75, and 74% sequence identity to azo1 encoding an azoreductase form Staphy. aureus and hypothetical proteins with 82, 80, 72, and 74% identity to Azo1, respectively. In addition, Staphy. capitis has a protein with 79% identity to Azo1. Western analysis detected proteins similar to Azo1 in all the staphylococci tested, except Staphy. delphini, Staphy. sciuri sciuri, and Staphy. 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.

New complex class 1 integron carrying an ISCR1 element in Escherichia coli clinical isolates harbouring the blaCMY-11 gene [CORRESPONDENCE]

Song, J. S., Jang, S. J., Bae, I. K., Lee, H. J., Jeong, B. C., Lee, S. H. — Thu, 03 Sep 2009 07:31:14 PDT

Plasmid profiles, Southern blot hybridization, and conjugation assays revealed that the blaCMY-11 gene, responsible for β-lactam resistance, was located on a noble complex class 1 integron within a conjugative plasmid. A sul1-type class 1 integron, harboring dfrA12, orfF, and aadA2a gene cassettes, was identified upstream of an ISCR1 element and ended with a truncated 3' conserved segment. The nucleotide sequence analyses of blaCMY-1, blaCMY-8, blaCMY-9, and blaCMY-11 genes indicate that there might be past transposition events by the ISCR1 element upstream of blaCMY-11. For the first time, a unique gene (yqgF-yqgE-gshB-orf97-orf105) array was detected between blaCMY-11 and a duplicate of the 3' conserved segment.

Identification of Burkholderia cepacia complex (Bcc) bacteria with a lipopolysaccharide specific monoclonal antibody [DIAGNOSTICS, TYPING AND IDENTIFICATION]

AuCoin, D. P, Crump, R., Thorkildson, P., Nuti, D., LiPuma, J., Kozel, T. — Thu, 03 Sep 2009 07:31:15 PDT

The genus Burkholderia includes many bacteria that cause serious human infections. As is the case with other Gram-negative bacteria, Burkholderia species produce lipopolysaccharides (LPS) which is an abundant component of the bacterial cell surface. Burkholderia cepacia complex (Bcc) bacteria (including at least 17 separate species) produce LPS structures that are quite different. In an attempt to determine the degree of LPS epitope variation among Bcc species, we produced a monoclonal antibody (mAb), designated 5D8, specific for the LPS of B. cepacia. Western blot analysis determined that mAb 5D8 was able to produce the classic 'ladder pattern' when used to probe B. cepacia and B. anthina lysates, although 5D8 did not produce this pattern with the other seven Bcc species tested. mAb 5D8 reacted with varying intensity to most but not all of the additional B. cepacia and B. anthina strains tested. Therefore, there seems to be significant epitope variation among Bcc LPS both between and within species. Additionally, mAb 5D8 reacted with a proteinase K-sensitive 22 kD antigen in all Bcc strains and also in a strain of Burkholderia pseudomallei.

Syphilis serology in HIV patients: a need to re-define VDRL cut-off for biological false positives [CORRESPONDENCE]

sharma, M., Wanchu, A., Biswal, M., Banga, S. s., Sethi, S. — Thu, 03 Sep 2009 07:31:14 PDT

No abstract

Helicobacter pylori oipA, vacA and dupA genetic diversity in individual hosts [CLINICAL MICROBIOLOGY AND VIROLOGY]

Thu, 30 Jul 2009 08:01:28 PDT

Helicobacter pylori putative virulence factors can undergo a continuous evolving mechanism as an approach to bacterial adaptation to host changing environment during chronic infection. oipA, vacA and dupA genetic diversity among isolates from multiple biopsies (niches) from antrum and corpus of 40 patients was investigated. A set of 229 isolates was examined. Direct DNA sequence analysis of amplified fragments was used to study oipA 'on/off' expression status as well as the presence of C or T insertion in jhp0917 that originates a continuous (jhp0917-jhp0918) dupA gene. vacA alleles were identified by PCR-multiplex. Different inter-niches oipA CT repeat patterns were observed in 9 patients; in 6 of these, 'on'and 'off' mixed patterns were found. In 3 of these 9 patients, different vacA alleles were also observed in a single host. Inter-niches dupA differences involved absence and presence of jhp0917 and/or jhp0918 or mutations in dupA, including those that may originate a non-functional gene, and they were also present in 2 patients with mixed oipA CT patterns and in other 7 patients. Evidence of mixed infection was observed in 2 patients only. In conclusion, oipA and dupA genes showed similar inter-niches variability, close to 1/4 patients. Conversely, vacA allele microevolution seems to be a less common event, close to 1/10 patients, probably due to the mechanism this gene evolves 'in vivo'.