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Pulsed-field Gel Electrophoresis (pulsed-field + gel_electrophoresis)
Selected AbstractsCoinfection with Campylobacter species: an epidemiological problem?JOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2001J.F. Richardson Aims: To determine the frequency of coinfection with multiple strains in sporadic cases of human Campylobacter infection. Method and Results: During 1999 10 single colonies of Campylobacter were cultured from each of 53 positive faecal samples. Five isolates were taken from nonselective agar after passive filtration of faecal suspensions and five isolates were taken from selective agar plates. All isolates were sero- and phage typed and their antibiotic resistance determined. Pulsed-field gel electrophoresis and flagellin gene typing were performed on selected isolates. One patient was infected with Camp. coli, the remainder with strains of Camp. jejuni. The majority of patients was infected with a single strain of Campylobacter, but from each of four samples, 7·5%, two strains of Camp. jejuni, confirmed by molecular typing, were identified. Conclusions: Coinfection occurs in sporadic cases of campylobacteriosis. Significance and Impact of the Study: This study has implications in outbreak investigation when distinct strains have been isolated from epidemiologically related patients and/or the suspected source or vehicle. [source] TRACING THE ORIGIN OF MULTI-DRUG RESISTANT (MDR) ESCHERICHIA COLI INFECTIONS FROM URINARY CATHETERS IN ICU CANINE PATIENTSJOURNAL OF VETERINARY EMERGENCY AND CRITICAL CARE, Issue S1 2004J Ogeer-Gyles Introduction: Urinary tract infections (UTIs) in dogs with urinary catheters in intensive care units (ICUs) are frequent. Historically, multi-drug resistant (MDR) Escherichia coli account for about 10% of the UTIs. The objectives of this study were to determine the frequency of E. coli infections and of MDR E. coli in dogs with UTIs in our ICU, and to assess whether the MDR E. coli were community-acquired or nosocomial in origin. Methods: Over a 1-year period, rectal swabs were taken from all dogs in the ICU on the day of admission (D0) and on days 3 (D3), 6 (D6), 9 (D9) and 12 (D12). Urine was collected on these days from dogs with an indwelling urinary catheter (n=190). Rectal swabs and urine were routinely cultured. E. coli isolates were identified by biochemical tests. Using NCCLS guidelines, antibiotic susceptibility testing was done by disk diffusion method on fecal and urinary E. coli isolates. Twelve antimicrobial agents were used: nalidixic acid, enrofloxacin, cephalothin, cefoxitin, cefotaxime, ceftiofur, trimethoprim-sulfa, chloramphenicol, gentamicin, tetracycline, ampicillin, and amoxicillin/clavulanate. Pulsed-field gel electrophoresis (PFGE) was used to compare MDR E. coli UTI strains with fecal E. coli strains from the same patient and with MDR fecal E. coli from patients that were adjacent to, or housed in the same cages. Results: E. coli was cultured from 12 (48%) of 25 UTIs. Two of the E. coli were MDR. For one dog, PFGE showed no similarities among fecal E. coli and the urinary MDR E. coli isolates from the patient or between these isolates and fecal E. coli from a dog housed in the same kennel on the previous day. The MDR E. coli UTI was likely acquired prior to admission to the ICU, as it was present on D0. For the other dog, PFGE showed genetic similarity but not complete identity between the D3 MDR E. coli urinary isolate and the D3, D6, D9 fecal MDR isolates. This suggests that the UTI originated with the fecal E. coli. Using selective plates, fecal MDR E. coli were not found on D0. Selection of the MDR strain in the intestine by the use of antibiotics occurred while the dog was in the ICU and possibly led to the UTI. Conclusions: Multi-drug resistant E. coli accounted for 2 of 12 E. coli UTIs in dogs in the ICU over a 1-year period. Genotyping showed that one of the two MDR E. coli infections could possibly be of nosocomial origin. [source] Prevalence of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus Carriage in Three PopulationsJOURNAL OF VETERINARY INTERNAL MEDICINE, Issue 1 2010S. Kottler Background: A higher prevalence of methicillin-resistant Staphylococcus aureus (MRSA) colonization is reported in healthcare workers compared with nonhealthcare workers. Hypothesis: The prevalence of MRSA colonization differed in people and pets in households with healthcare workers as compared with households without healthcare workers. Subjects: A person and 1 dog or cat from 586 households defined as either a nonhealthcare (n = 213), veterinary healthcare (n = 211), or human healthcare (n = 162) worker household. Methods: Prospective cross-sectional study. Samples from humans and pets were cultured in vitro. Staphylococcus aureus was identified as methicillin sensitive (MSSA) or MRSA with mecA polymerase chain reaction. Pulsed-field gel electrophoresis and spa -typing were used to characterize relatedness of S. aureus and MRSA and assign USA types. Results: The prevalence of MSSA and MRSA in humans was 21.5% (126/586) and 5.63% (33/586), respectively, and 7.85% (46/586) and 3.41% (20/586), respectively, in pets. There were no differences in prevalences of either MSSA or MRSA between household types. The proportion of MRSA among all S. aureus isolates in humans and pets was 20.8% (33/159) and 30.3% (20/66), respectively. In <1.0% (4/586) of households, the same strain of MRSA was found in both a person and a pet. Conclusions and Clinical Importance: There were no differences in the prevalences of MSSA or MRSA between healthcare worker and nonhealthcare worker households. Pets and people colonized with S. aureus were as likely to be colonized with MRSA. Colonization of a person and their pet with the same strain of MRSA was rare. [source] Virulence, phenotype and genotype characteristics of endodontic Enterococcus spp.MOLECULAR ORAL MICROBIOLOGY, Issue 1 2005C. M. Sedgley Background/aims:, Enterococci have been implicated in persistent root canal infections but their role in the infection process remains unclear. This study investigated the virulence, phenotype and genotype of 33 endodontic enterococcal isolates. Methods:, Phenotypic tests were conducted for antibiotic resistance, clumping response to pheromone, and production of gelatinase, hemolysin and bacteriocin. Genotype analysis involved polymerase chain reaction amplification of virulence determinants encoding aggregation substances asa and asa373, cytolysin activator cylA, gelatinase gelE, gelatinase-negative phenotype ef1841/fsrC, adherence factors esp and ace, and endocarditis antigen efaA. Physical DNA characterization involved pulsed-field gel electrophoresis of genomic DNA, and plasmid analysis. Results:, Potential virulence traits expressed included production of gelatinase by Enterococcus faecalis (n = 23), and response to pheromones in E. faecalis culture filtrate (n = 16). Fourteen strains produced bacteriocin. Five strains were resistant to tetracycline and one to gentamicin, whereas all were susceptible to ampicillin, benzylpenicillin, chloramphenicol, erythromycin, fusidic acid, kanamycin, rifampin, streptomycin and vancomycin. Polymerase chain reaction products encoding efaA, ace, and asa were detected in all isolates; esp was detected in 20 isolates, cylA in six isolates, but asa373 was never detected. The gelatinase gene (gelE) was detected in all isolates of E. faecalis (n = 31) but not in Enterococcus faecium (n = 2); a 23.9 kb deletion sequence corresponding to the gelatinase-negative phenotype was detected in six of the eight E. faecalis isolates that did not produce gelatinase. Pulsed-field gel electrophoresis and plasmid analyses revealed genetic polymorphism with clonal types evident. Plasmid DNA was detected in 25 strains, with up to four plasmids per strain and a similar (5.1 kb) plasmid occurring in 16 isolates. Conclusions:, Phenotypic and genotypic evidence of potential virulence factors were identified in endodontic Enterococcus spp., specifically production of gelatinase and response to pheromones. [source] Strain relatedness of meticillin-resistant Staphylococcus aureus isolates recovered from patients with repeated bacteraemiaCLINICAL MICROBIOLOGY AND INFECTION, Issue 5 2010C-H. Liao Clin Microbiol Infect 2010; 16: 463,469 Abstract Information on the relatedness of isolates causing repeated meticillin-resistant Staphylococcus aureus (MRSA) bacteraemia is limited. An observational study of 177 patients with MRSA bacteraemia, admitted to the emergency department of National Taiwan University Hospital, was conducted from January 2001 to June 2006. Among these patients, 28 had a previous episode of MRSA bacteraemia and 59 died during the index episode of bacteraemia. Until December 2007, among the 118 patients who survived the index episode (101 without previous bacteraemia and 17 with previous bacteraemia), 24 (20.3%) had repeated MRSA bacteraemia. The duration from discontinuation of antimicrobial therapy to repeat episodes was in the range 35,854 days (median 86 days). Eight patients (33.3%) died as a result of the second bacteraemic episode. Clinical characteristics associated with repeated bacteraemia included the diagnosis of infective endocarditis and active malignancy. Pulsed-field gel electrophoresis and multilocus sequence typing analysis were performed for 32 pairs of available isolates recovered from patients with repeated bacteraemia and revealed that 29 of them (90.6%) were genetically closely-related strains. The majority of patients with repeated MRSA bacteraemia had recurrent infections and a high mortality rate. [source] Salmonella enterica serotype Virchow: epidemiology, resistance patterns and molecular characterisation of an invasive Salmonella serotype in IsraelCLINICAL MICROBIOLOGY AND INFECTION, Issue 10 2006M. Weinberger Abstract This study outlines the unique epidemiology of Salmonella enterica serotype Virchow in Israel. Between 1997 and 2002, the overall incidence of non-typhoid Salmonella enterica (NTS) decreased from 69.3 to 53.3 infections/100 000 population, but the incidence of S. Virchow increased (from 7.2 to 9.1 infections/100 000). Since 2000, S. Virchow has become the second-ranking NTS isolate, accounting for 17% and 27% of all stool and blood NTS isolates, respectively. Infants aged <,1 year had the highest incidence of isolation from stools (92.8/100 000). The incidence of isolation from blood was highest for infants aged <1 year (4.4/100 000). Only 6% of isolates were susceptible to all ten antibiotic agents tested; 34% were resistant to one agent, 54% to one to three agents, and 40% to four to six agents. A high proportion of the tested isolates were resistant to nalidixic acid (89%), streptomycin (56%), tetracycline (43%), trimethoprim,sulphamethoxazole (38%) and chloramphenicol (28%), but none to ciprofloxacin or ceftriaxone. Pulsed-field gel electrophoresis revealed two closely related clusters, each containing a predominant pulsotype. Coupled with its invasive propensity, the increasing incidence of highly resistant S. Virchow in Israel is of real concern. Future research should focus on the sources of S. Virchow in the food chain in order to institute effective control measures. [source] Staphylococcus lugdunensis: clinical spectrum, antibiotic susceptibility, and phenotypic and genotypic patterns of 39 isolatesCLINICAL MICROBIOLOGY AND INFECTION, Issue 1 2006C. Hellbacher Abstract Staphylococcus lugdunensis is a member of the coagulase-negative staphylococci with the potential to cause clinically significant infections. The spectrum of infections was investigated in 39 isolates of S. lugdunensis from 38 patients. Most (73%) infections were located below the waist, while those above the waist were mainly (5/7) breast abscesses. Most isolates were susceptible to the antibiotics tested, although 15.4% were ,-lactamase-positive and could be identified by the disk-diffusion method for penicillin G. There was very good concordance between the disk-diffusion method and the Etest method for oxacillin resistance. Pulsed-field gel electrophoresis (PFGE) showed that 56% of the isolates belonged to one SmaI pulsotype, while phenotypic analysis by the Phene Plate system identified three main phenotypic groups. Although the S. lugdunensis isolates analysed were obtained from different patients, treated in different wards and hospitals during a 4-year period, there was a low degree of diversity, both genotypically and phenotypically. For this reason, PFGE is not suitable for the analysis of an outbreak situation, and the homogeneity observed may indicate that S. lugdunensis is a genetically conserved species of coagulase-negative Staphylococcus. [source] Molecular diversity of Proteus mirabilis isolates producing extended-spectrum ,-lactamases in a French university hospitalCLINICAL MICROBIOLOGY AND INFECTION, Issue 5 2005M. Biendo Abstract Between February 1997 and December 2002, 3340 hospitalised patients yielded samples positive for Proteus mirabilis, of whom 45 (1.3%) were colonised/infected by P. mirabilis producing extended-spectrum ,-lactamases (ESBLs). The gross incidence of patients colonised/infected by ESBL-producing P. mirabilis was 1.61/105 days of hospitalisation, with 20% of isolates being collected from patients in urology wards, most frequently (53.3%) from urine samples. Seventeen (37.7%) of the 43 isolates were obtained from samples collected within 48 h of hospitalisation, indicating that they were community-acquired. Isoelectric focusing assays and sequencing identified the TEM-24, TEM-92 and TEM-52 ESBLs. Pulsed-field gel electrophoresis revealed eight pulsotypes (I,VIII), with the two most common pulsotypes, IV and VI, comprising ten (23.3%) and 12 (26.6%) isolates, respectively. These pulsotypes were considered to represent epidemic strains and spread in various wards of the hospital. [source] A statistical model for unwarping of 1-D electrophoresis gelsELECTROPHORESIS, Issue 22 2005Chris Glasbey Professor Abstract A statistical model is proposed which relates density profiles in 1-D electrophoresis gels, such as those produced by pulsed-field gel electrophoresis (PFGE), to databases of profiles of known genotypes. The warp in each gel lane is described by a trend that is linear in its parameters plus a first-order autoregressive process, and density differences are modelled by a mixture of two normal distributions. Maximum likelihood estimates are computed efficiently by a recursive algorithm that alternates between dynamic time warping to align individual lanes and generalised-least-squares regression to ensure that the warp is smooth between lanes. The method, illustrated using PFGE of Escherichia coli O157 strains, automatically unwarps and classifies gel lanes, and facilitates manual identification of new genotypes. [source] Genotypic variability and persistence of Legionella pneumophila PFGE patterns in 34 cooling towers from two different areasENVIRONMENTAL MICROBIOLOGY, Issue 2 2008Inma Sanchez Summary Genotypic variability and clonal persistence are important concepts in molecular epidemiology as they facilitate the search for the source of sporadic cases or outbreaks of legionellosis. We studied the genotypic variability and persistence of Legionella pulsed-field gel electrophoresis (PFGE) patterns over time (period > 6 months) in 34 positive cooling towers from two different areas. In area A, radius of 70 km, 52 indistinguishable PFGE patterns were differentiated among the 27 cooling towers. In 13 cooling towers we observed , 2 PFGE patterns. Each cooling tower had its own indistinguishable Legionella PFGE pattern which was not shared with any other cooling tower. In area B, radius of 1 km, 10 indistinguishable PFGE patterns were obtained from the seven cooling towers. In four, we observed , 2 PFGE patterns. Three of these 10 indistinguishable PFGE patterns were shared by more than one cooling tower. In 27 of 34 cooling towers the same PFGE pattern was recovered after 6 months to up to 5 years of follow-up. The large genotypic diversity of Legionella observed in the cooling towers aids in the investigation of community outbreaks of Legionnaires' disease. However, shared patterns in small areas may confound the epidemiological investigation. The persistence of some PFGE patterns in cooling towers makes the recovery of the Legionella isolate causing the outbreak possible over time. [source] High phenotypic diversity in infecting but not in colonizing Staphylococcus aureus populationsENVIRONMENTAL MICROBIOLOGY, Issue 12 2007Christiane Goerke Summary In hostile environments diversity within a bacterial population may be beneficial for the fitness of the microbial community as a whole. Here we analysed the population diversity of Staphylococcus aureus in infecting and colonizing situations. In the study, performed independently in two German centres, the heterogeneity of the S. aureus population was determined by quantifying the occurrence of phenotypic variants (differences in haemolysis, pigmentation, colony morphology) in primary cultures from nose, oropharyngeal and sputum specimens from cystic fibrosis (CF) patients and in nose swabs from healthy S. aureus carriers. The proportion of heterogeneous samples, the number of clearly distinguishable isolates per sample and the qualitative differences between phenotypes was significantly higher in CF sputum specimens than in the other samples. The heterogeneity of the S. aureus population could be correlated with high bacterial densities in the sputum samples. In patients co-infected with Pseudomonas aeruginosa lower S. aureus bacterial loads and less heterogeneity in the S. aureus population were observed. Typing of all S. aureus isolates from heterogeneous samples by pulsed-field gel electrophoresis or spa typing revealed that the bacteria were polyclonal in 30%, monoclonal with minor genetic alterations in 25% or not distinguishable in 69% of the specimens. Some specimens harboured monoclonal and polyclonal variants simultaneously. Importantly, differences in antibiotic susceptibility were detected in phenotypic S. aureus variants within a single specimen. Diversification of a S. aureus population is highly favoured during chronic CF lung infection, supporting the general hypothesis that maintenance of intrahost diversity can be of adaptive value, increasing the fitness of the bacterial community. [source] Worldwide distribution of Pseudomonas aeruginosa clone C strains in the aquatic environment and cystic fibrosis patientsENVIRONMENTAL MICROBIOLOGY, Issue 7 2005Ute Römling Summary Highly successful bacterial clones have the ability to effectively colonize environmental niches and patients. However, the factors which determine the complex interplay between the colonization of environmental niches and patients are mainly unknown. In this study we show that Pseudomonas aeruginosa clone C strains are distributed worldwide and highly prone to infect cystic fibrosis (CF) patients in Canada, England, France and Germany. In Hanover, Germany and Vancouver, Canada, clone C strains are highly prevalent in the CF patient community, although the mechanisms of acquisition may have been different. All clone C strains showed highly related macrorestriction fragment pattern of the whole genome as visualized by pulsed-field gel electrophoresis and harboured the 102 kbp plasmid pKLC102. Comparison of three prevalent P. aeruginosa clones with different distribution between the environment and patients revealed that neither enhanced biofilm formation nor antibiotic resistance was responsible for the spread of clone C. Clone M, which was highly prevalent in the clinical environment such as sanitary facilities, lacked motility, which could explain its relatively low prevalence in CF patients. Elucidation of the mechanisms which lead to the prevalence of clone C strain in patients and the environment requires the investigation of additional phenotypes. [source] Ecotype diversity in the marine picoeukaryote Ostreococcus (Chlorophyta, Prasinophyceae)ENVIRONMENTAL MICROBIOLOGY, Issue 6 2005Francisco Rodríguez Summary The importance of the cyanobacteria Prochlorococcus and Synechococcus in marine ecosystems in terms of abundance and primary production can be partially explained by ecotypic differentiation. Despite the dominance of eukaryotes within photosynthetic picoplankton in many areas a similar differentiation has never been evidenced for these organisms. Here we report distinct genetic [rDNA 18S and internal transcribed spacer (ITS) sequencing], karyotypic (pulsed-field gel electrophoresis), phenotypic (pigment composition) and physiological (light-limited growth rates) traits in 12 Ostreococcus strains (Prasinophyceae) isolated from various marine environments and depths, which suggest that the concept of ecotype could also be valid for eukaryotes. Internal transcribed spacer phylogeny grouped together four deep strains isolated between 90 m and 120 m depth from different geographical origins. Three deep strains displayed larger chromosomal bands, different chromosome hybridization patterns, and an additional chlorophyll (chl) c -like pigment. Furthermore, growth rates of deep strains show severe photo-inhibition at high light intensities, while surface strains do not grow at the lowest light intensities. These features strongly suggest distinct adaptation to environmental conditions encountered at surface and the bottom of the oceanic euphotic zone, reminiscent of that described in prokaryotes. [source] Molecular heterogeneity in Yersinia enterocolitica and ,Y. enterocolitica -like' species , Implications for epidemiology, typing and taxonomyFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2005Jugsharan S. Virdi Abstract Yersinia enterocolitica is an extremely heterogeneous species. Serotyping and biotyping have been used extensively, in the past, to study its heterogeneity and epidemiology. Application of methods like ribotyping, pulsed-field gel electrophoresis and a host of other genomic techniques have further revealed molecular heterogeneity in this species. Furthermore, these methods may be used effectively to supplement serotyping and biotyping schema for studying epidemiology of Y. enterocolitica. This is evident from the ability of some of these methods to subtype strains belonging to serogroups O:3, O:9 and O:8 , which are most commonly encountered in human Yersiniosis. Multilocus enzyme electrophoresis and nucleotide sequencing have reiterated the taxonomic relationships of this organism. However there is paucity of information about the molecular heterogeneity of ,Y. enterocolitica -like' species, which need to be addressed in the future. Also, newer techniques such as amplified fragment length polymorphism, VNTR-based typing and multilocus sequence typing should be applied to further understand epidemiology, population structure and evolutionary genetics of Y. enterocolitica and ,Y. enterocolitica -like' species. [source] Genotypes of multidrug-resistant Salmonella enterica serotype typhimurium from two regions of KenyaFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2000Samuel Kariuki Abstract A combination of phage typing and pulsed-field gel electrophoresis of XbaI-digested chromosomal DNA has been used to study the epidemiological relationships of multidrug-resistant Salmonella enterica serotype typhimurium from Nairobi (64 isolates) and Kilifi (40 isolates) collected over the period 1994,1997. Isolates from Nairobi belonged to 11 definitive phage types (DTs) encompassing eight different PFGE patterns. In contrast, isolates from Kilifi were mainly DT 56 (60%) and all fell into a single PFGE pattern. The remaining isolates did not conform to a recognisable phage type. We conclude that multidrug-resistant S. typhimurium infections from Nairobi were caused by multiple strains while those from Kilifi were likely to be from a microepidemic caused by a single clone. [source] Mechanisms of antimicrobial resistance and genetic relatedness among enterococci isolated from dogs and cats in the United StatesJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2010C.R. Jackson Abstract Aims:, In this study, mechanisms of antimicrobial resistance and genetic relatedness among resistant enterococci from dogs and cats in the United States were determined. Methods and Results:, Enterococci resistant to chloramphenicol, ciprofloxacin, erythromycin, gentamicin, kanamycin, streptomycin, lincomycin, quinupristin/dalfopristin and tetracycline were screened for the presence of 15 antimicrobial resistance genes. Five tetracycline resistance genes [tet(M), tet(O), tet(L), tet(S) and tet(U)] were detected with tet(M) accounting for approx. 60% (130/216) of tetracycline resistance; erm(B) was also widely distributed among 96% (43/45) of the erythromycin-resistant enterococci. Five aminoglycoside resistance genes were also detected among the kanamycin-resistant isolates with the majority of isolates (25/36; 69%) containing aph(3,)-IIIa. The bifunctional aminoglycoside resistance gene, aac(6,)-Ie -aph(2,)-Ia, was detected in gentamicin-resistant isolates and ant(6)-Ia in streptomycin-resistant isolates. The most common gene combination among enterococci from dogs (n = 11) was erm(B), aac(6,)-Ie- aph(2,)-Ia, aph(3,)-IIIa, tet(M), while tet(O), tet(L) were most common among cats (n = 18). Using pulsed-field gel electrophoresis (PFGE), isolates clustered according to enterococcal species, source and antimicrobial gene content and indistinguishable patterns were observed for some isolates from dogs and cats. Conclusion:, Enterococci from dogs and cats may be a source of antimicrobial resistance genes. Significance and Impact of the Study:, Dogs and cats may act as reservoirs of antimicrobial resistance genes that can be transferred from pets to people. Although host-specific ecovars of enterococcal species have been described, identical PFGE patterns suggest that enterococcal strains may be exchanged between these two animal species. [source] Host range and lytic capability of four bacteriophages against bovine and clinical human isolates of Shiga toxin-producing Escherichia coli O157:H7JOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2009Y.D. Niu Abstract Aims:, To evaluate host range and lytic capability of four bacteriophages (rV5, wV7, wV8 and wV11) against Escherichia coli O157:H7 (STEC O157:H7) from cattle and humans. Methods and Results:, Four hundred and twenty-two STEC O157:H7 isolates (297 bovine; 125 human) were obtained in Alberta, Canada. The four phages were serially diluted and incubated for 5 h with overnight cultures of STEC O157:H7 to estimate their multiplicity of infection (MOI). All bovine STEC O157:H7 were subjected to pulsed-field gel electrophoresis (PFGE) and phage typing (PT). Phage wV7 lysed all human and bovine isolates irrespective of PFGE genotype or PT phenotype and exhibited the lowest MOI (0·004,0·006, P < 0·0001) of all phages. Phages rV5 and wV11 exhibited a lower MOI (0·002,0·04, P < 0·0001) than did phage wV8 (25,29) and they had a narrower host range than wV7 or wV8. Phages rV5, wV11 and wV8 lysed 342 (81·0%), 321 (76·1%) and 407 (96·4%), respectively, of the 422 isolates. Susceptibility of bovine STEC O157:H7 to rV5, w11 and wV8 was influenced by PFGE genotype and/or PT phenotype. Conclusions:, Phages exhibited activity against the majority of bovine and human STEC O157:H7 isolates. PFGE genotype and/or PT phenotype of the host-target influenced their vulnerability to phage attack. Susceptibility of bovine STEC O157:H7 to phage may also differ among farms. Both lytic capability and host range should be considered in the selection of therapeutic phage for on-farm control of STEC O157:H7. Significance and Impact of the Study:, The present work indicates that a four-phage cocktail should be equally effective at mitigating STEC O157:H7 isolates both of bovine and of human origin. Given that some STEC O157:H7 exhibited resistance to some but not all phages, a phage cocktail is the logical approach to efficacious on-farm therapy. [source] Virulence genes of bovine Staphylococcus aureus from persistent and nonpersistent intramammary infections with different clinical characteristicsJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2007M. Haveri Abstract Aims:, To screen putative virulence genes in Staphylococcus aureus causing persistent and nonpersistent bovine intramammary infections (IMI) with different clinical characteristics. To examine, whether a possible relationship exists between genetic profile and infection persistence, clinical signs of infection, clonal type determined by pulsed-field gel electrophoresis (PFGE), and antimicrobial resistance. Methods and Results:, One-hundred and sixty-one S. aureus isolates derived from bovine IMI, consisting of 17 different PFGE types, were screened by conventional and multiplex-polymerase chain reaction (PCR) for 24 virulence genes for haemolysins (hla-hlg), leukocidins (lukED, lukM), exfoliative toxins (eta, etb), enterotoxins (sea-seo, seu), toxic-shock syndrome toxin (tst), and genes encoding penicillin (blaZ) and methicillin resistance (mecA). The majority of S. aureus isolated at the onset of mastitis carried haemolysin genes (76·7,97·4%), lukED (96·6%), and at least one gene for pyrogenic toxin superantigen (PTSAg) (69·0%). Strains carrying PTSAg-encoding genes were more common among predominant PFGE types and in persistent IMI. Strains concomitantly possessing sed, sej, and blaZ, putatively plasmid-encoded, were typically found in connection with persistent IMI. Conclusions:, Our results suggest that certain genetic elements are over-representative in S. aureus isolates especially from persistent bovine mastitis. This phenomenon seems to be in connection with clonal type and is often concomitant with penicillin resistance. Significance and Impact of the Study:, This is the first study to investigate associations between a large number of bacterial factors and outcome of S. aureus mastitis. The finding that widespread clonal types of S. aureus causing bovine mastitis of low treatment response may harbour characteristic genes could be improved for strain-specific diagnostic purposes. [source] Direct comparison of four bacterial source tracking methods and use of composite data setsJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2007E.A. Casarez Abstract Aims:, Four bacterial source tracking (BST) methods, enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR), automated ribotyping using HindIII, Kirby-Bauer antibiotic resistance analysis (KB-ARA) and pulsed-field gel electrophoresis (PFGE) were directly compared using the same collection of Escherichia coli isolates. The data sets from each BST method and from composite methods were compared for library accuracy and their ability to identify water isolates. Methods and Results:, Potential sources of faecal pollution were identified by watershed sanitary surveys. Domestic sewage and faecal samples from pets, cattle, avian livestock, other nonavian livestock, avian wildlife and nonavian wildlife sources were collected for isolation of E. coli. A total of 2275 E. coli isolates from 813 source samples were screened using ERIC-PCR to exclude clones and to maximize library diversity, resulting in 883 isolates from 745 samples selected for the library. The selected isolates were further analysed using automated ribotyping with HindIII, KB-ARA and PFGE. A total of 555 E. coli isolates obtained from 412 water samples were analysed by the four BST methods. A composite data set of the four BST methods gave the highest rates of correct classification (RCCs) with the fewest unidentified isolates than any single method alone. RCCs for the four-method composite data set and a seven-way split of source classes ranged from 22% for avian livestock to 83% for domestic sewage. Two-method composite data sets were also found to be better than individual methods, having RCCs similar to the four-method composite and identification of the same major sources of faecal pollution. Conclusions:, The use of BST composite data sets may be more beneficial than the use of single methods. Significance and Impact of the Study:, This is one of the first comprehensive comparisons using composite data from several BST methods. While the four-method approach provided the most desirable BST results, the use of two-method composite data sets may yield comparable BST results while providing for cost, labour and time savings. [source] Comparison of Salmonella enterica serotype Infantis isolates from a veterinary teaching hospitalJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2007M. Dunowska Abstract Aims:, To compare Salmonella enterica serotype Infantis isolates obtained from patients or the environment of a veterinary teaching hospital over a period of 9 years following a nosocomial outbreak to determine whether isolates were epidemiologically related or represented unrelated introductions into the hospital environment. Methods and Results:, Fifty-six S. Infantis isolates were compared based on their phenotypic (antimicrobial drug [AMD] susceptibility pattern) and genotypic (pulsed-field gel electrophoresis [PFGE] pattern and presence of integrons) characteristics. Epidemiologically unrelated S. Infantis isolates clustered separately from all but two of the hospital isolates, and several isolates from different years and various sources were indistinguishable from each other in cluster analysis of two-enzyme PFGE results. A high percentage of isolates (80·3%) were resistant to at least one AMD, with 67·8% showing resistance to >5 AMD. The majority (74·1%) of isolates tested contained type 1 integrons. Conclusion:, Results strongly suggest that there was nosocomial transmission of S. Infantis during the initial outbreak, and that contamination arising from this outbreak persisted across years despite rigorous hygiene and biosecurity precautions and may have led to subsequent nosocomial infections. Significance and Impact of the Study:, Evidence of persistence and transmission of Salmonella clones across years, even in the face of rigorous preventive measures, has important implications for other facilities that have experienced outbreaks of Salmonella infections. [source] Soil survival of Escherichia coli O157:H7 acquired by a child from garden soil recently fertilized with cattle manureJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2006A. Mukherjee Abstract Aims:, This investigation was conducted to determine the survival of a naturally occurring Escherichia coli O157:H7 in garden soil linked to a sporadic case of E. coli O157 infection in Minnesota. Methods and Results:, The presence and viability of E. coli O157:H7 was monitored in manure-contaminated garden soil for several weeks. Bacterial isolates were characterized using PCR and pulsed-field gel electrophoresis (PFGE). Isolates obtained from the patient and the garden plots during this investigation had indistinguishable PFGE patterns and had the same virulence factors (stx1, stx2, eaeA, ehxA). The E. coli O157:H7 levels obtained from the garden plots declined gradually for a period of 2 months, and on day 69 only one garden plot of four had detectable levels of pathogen. All plots were negative on day 92. The rate of decline in the soil samples stored at 4°C was faster compared with soil samples that remained in ambient conditions, and in refrigerated storage E. coli O157:H7 could not be detected after 10 days. Conclusions:,E. coli O157:H7 strains can survive on manure-amended soil for more than 2 months, and this survival could be reduced by low temperature. Significance and Impact of the Study:, This is one of the few reports that have investigated the survival of a proven virulent strain in naturally contaminated soil samples. This case stresses the importance of avoiding the use of raw cattle manure to amend soil for cultivation of foods, including soils in residential garden plots. [source] Genotyping of Campylobacter spp. from retail meats by pulsed-field gel electrophoresis and ribotypingJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2006B. Ge Abstract Aims:, To determine the genetic relatedness of Campylobacter spp. from retail meat products, and compare the discriminatory power of pulsed-field gel electrophoresis (PFGE) and automatic ribotyping. Methods and Results:, A total of 378 Campylobacter isolates recovered from 159 raw meats (130 chicken, 25 turkey, three pork and one beef) sampled from 50 retail grocery stores of four supermarket chains in the Maryland suburban area from August 1999 to July 2000 were analysed by PFGE with SmaI, 120 isolates of which were also characterized by ribotyping with PstI using RiboPrinter® system. A total of 148 unique PFGE patterns were identified, 91 of which were present in multiple Campylobacter isolates and 24 in multiple meat samples. Nineteen Campylobacter clones with identical PFGE patterns recurred frequently (up to nine times) throughout the sampling period. Comparing ribotyping with PFGE, we identified 44 PFGE patterns and 22 RiboGroups among the 120 isolates tested. Multiple PFGE patterns within one RiboGroup were commonly observed, as well as multiple RiboGroups within one PFGE pattern. Conclusions:, Although Campylobacter present in retail meats were genetically diverse, certain clones persisted in poultry meats. PFGE had a greater discriminatory power than ribotyping, and the two methods were complementary in genotyping Campylobacter. Significance and Impact of the Study:, Genomic DNA fingerprinting of Campylobacter confirmed diverse and recurrent Campylobacter clones in the retail meats, which provides additional data for a better understanding of the epidemiological aspect of Campylobacter infection. [source] Genetic heterogeneity and functional properties of intestinal bifidobacteriaJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2004J. Mättö Abstract Aims:, The aim of the present study was to compare several molecular methods for the identification and genotyping of bifidobacteria, and further to investigate genetic heterogeneity and functional properties of bifidobacterial isolates from intestinal samples of Finnish adult subjects. Methods and Results:, A total of 153 intestinal bifidobacterial isolates were included in initial screening and 34 isolates were further characterized. Identification results obtained with PCR,ELISA and ribotyping were well in accordance with each other, while randomly amplified polymorphic DNA (RAPD) gave tentative identification only to Bifidobacterium bifidum and to 65% of the B. longum isolates. The most commonly detected species were B. longum biotype longum followed by B. adolescentis and B. bifidum. In addition, B. animalis (lactis), B. angulatum and B. pseudocatenulatum were found. Ribotyping and pulsed-field gel electrophoresis (PFGE) proved to be discriminatory methods for bifidobacteria, but also RAPD revealed intraspecies heterogeneity. Besides two B. animalis (lactis) isolates with very close similarity to a commercially available probiotic strain, none of the intestinal isolates showed optimal survival in all tolerance (acid, bile and oxygen) or growth performance tests. Conclusions:, Several species/strains of bifidobacteria simultaneously colonize the gastrointestinal tract of healthy Finnish adults and intestinal Bifidobacterium isolates were genetically heterogeneous. Functional properties of bifidobacteria were strain-dependent. Significance and Impact of the Study:, Applicability of ribotyping with the automated RiboPrinter® System for identification and genotyping of bifidobacteria was shown in the present study. [source] The diversity of Listeria monocytogenes strains from 10 Icelandic sheep farmsJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2004K.B. Gudmundsdottir Abstract Aims:, The purpose of this study was to examine the diversity of Listeria monocytogenes strains from healthy sheep, winter feed and environment of sheep farms in Iceland. Methods and Results:, A total of 104 L. monocytogenes isolates from animals, winter feed and environment on 10 Icelandic sheep farms were compared by serotyping, ribotyping, and pulsed-field gel electrophoresis with ApaI and AscI. The isolates were divided into 24 genotypes, all identified as serovars 1/2a, 1/2b, or 4b. Nine genotypes were detected on more than one farm. On three of the farms there seemed to be a dominant strain of L. monocytogenes. Isolates from incidents of listeriosis in animals occurring on two of the farms belonged to the genotype most commonly found on the particular farm. Nine of the 24 genotypes found on the sheep farms have been associated with disease in animals and/or humans elsewhere in Iceland. Conclusions:, Certain strains of L. monocytogenes seem to be widely distributed on Icelandic sheep farms. On some farms there appears to be a dominant strain of L. monocytogenes. Incidents of listeriosis in animals may tend to be associated with strains commonly found on the farm. Significance and Impact of the Study:, This study demonstrates the diversity of L. monocytogenes present in healthy sheep and their environment. [source] Discrimination of Staphylococcus aureus biotypes by pulsed-field gel electrophoresis of DNA macro-restriction fragmentsJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2003J.A. Hennekinne Abstract Aims: To examine whether pulsed-field gel electrophoresis (PFGE) of DNA macro-restriction fragments could provide better discrimination among the different biotypes previously described within the species Staphylococcus aureus than the traditional biochemical approach. Methods and Results: Seventy three Staph. aureus strains from various sources (human, animal or food origin) and belonging to eight biotypes, including the poultry-like biotype, tentatively designated as an ,abattoir' biotype, were genotyped by PFGE after SmaI digestion of DNA. The PFGE patterns were compared using the average linkage matching method (UPGMA) with the Dice coefficient. A total of 61 PFGE patterns were observed, showing between 31 and 100% similarity. In most cases, strains with the same biotype were grouped specifically into one, two or three separate sub-clusters. Strains from the ,abattoir' biotype were clustered in one separate sub-cluster. Conclusions: The PFGE typing is useful to distinguish the traditional biotypes of Staph. aureus and has a more discriminatory power than the biochemical typing. Significance and Impact of the Study: The PFGE typing confirms the ,abattoir' biotype as a separate group on a genetic level and is well suited to investigate modes of staphylococcal contamination of food. [source] Clustering of Saccharomyces boulardii strains within the species S. cerevisiae using molecular typing techniquesJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2002G. Mitterdorfer Aims: This study was undertaken to characterize and differentiate therapeutically relevant Saccharomyces yeasts. Among the isolates were so-called Saccharomyces boulardii strains, which are considered as probiotic agents, but whose taxonomic assignment is controversial. Moreover, the discriminative power of the applied molecular typing techniques should be evaluated. Methods and Results: Genotyping was performed using species-specific polymerase chain reaction (PCR), randomly amplified polymorphic DNA-PCR, restriction fragment length polymorphism analysis of rDNA spacer regions and pulsed-field gel electrophoresis. Species-specific PCR assigned all of the product isolates to the species S. cerevisiae. By combining the other techniques, all isolates could be discriminated. Moreover, it could be demonstrated that probiotic S. boulardii strains form a separate cluster located within the species. Conclusions: With the exception of species-specific PCR, all of the applied methodologies were suitable for subspecies typing and indicated a close relationship between the probiotic strains. Significance and Impact of the Study: The methods applied in this study are considered powerful tools for quality control of therapeutically relevant yeasts. It is of crucial importance, especially regarding S. boulardii yeasts, to verify the identity of the correct strain, since the beneficial properties are considered to be strain-specific. [source] Use of specific PCR primers to identify three important industrial species of Saccharomyces genus: Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces pastorianusLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2010G.V. De Melo Pereira Abstract Aim:, To develop species-specific primers capable of distinguishing between three important yeast species in alcoholic fermentation: Saccharomyces bayanus, Saccharomyces cerevisiae and Saccharomyces pastorianus. Methods and Results:, Two sets of primers with sequences complementary to the HO genes from Saccharomyces sensu stricto species were used. The use of the ScHO primers produced a single amplificon of c. 400 or 300 bp with species S. cerevisiae and S. pastorianus, respectively. The second pair of primers (LgHO) was also constructed, within the HO gene, composed of perfectly conserved sequences common for S. bayanus species, which generate amplicon with 700 bp. No amplification product was observed in the DNA samples from non- Saccharomyces yeasts. Saccharomyces species have also been characterized via electrophoretic karyotyping using pulsed-field gel electrophoresis to demonstrate chromosomal polymorphisms and to determine the evolutionary distances between these species. Conclusions:, We conclude that our novel species-specific primers could be used to rapidly and accurately identify of the Saccharomyces species most commonly involved in fermentation processes using a PCR-based assay. Significance and Impact of the Study:, The method may be used for routine identification of the most common Saccharomyces sensu stricto yeasts involved in industrial fermentation processes in less than 3 h. [source] Characterization of antimicrobial resistance and seasonal prevalence of Escherichia coli O157:H7 recovered from commercial feedlots in Alberta, CanadaLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2010M. Aslam Abstract Aims:, To characterize antimicrobial resistance (AMR) and determine the seasonal prevalence of Escherichia coli O157:H7 isolated from commercial feedlots. Methods and Results:,Escherichia coli O157:H7 were isolated from faecal and oral samples collected at monthly intervals from three commercial feedlots over a 12-month period. A total of 240 isolates were characterized using pulsed-field gel electrophoresis (PFGE) technique. A subset of 205 isolates was analysed for AMR using Sensititre system and AMR genes (tet, sul and str) by PCR. Seven PFGE clusters (,90% Dice similarity) were identified, and two clusters common to all three feedlots were recovered year-round. The majority of isolates (60%) were susceptible to all antimicrobials and were closely related (P < 0.001), whereas isolates with unique AMR patterns were not related. The prevalences of AMR from feedlots A, B and C were 69%, 1% and 38%, respectively. Resistance to tetracycline (69%) and sulfisoxazole (68%) was more prevalent in feedlot A than other two feedlots. The presence of strA and strB genes was linked in the majority of isolates, and tet(A) and tet(B), and sul1 and sul2 genes were present individually. Escherichia coli O157:H7 were genetically diverse during summer and fall, and strains from winter and spring months were more closely related. Conclusions:, Antimicrobial resistance was more common in E. coli O157:H7 obtained from two of the three commercial feedlots, and the phenotypic expression of resistance was correlated with the presence of resistant genes. A highly diverse E. coli O157:H7 population was found during summer and fall seasons. Significance and Impact of the Study:, Information would help understanding the dynamics of AMR in E. coli O157:H7 from commercial feedlots. [source] Clonal groups of high-level gentamicin-resistant Enterococcus faecium isolated from municipal wastewater and clinical samples in Tehran, IranLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2009M. Saifi Abstract Aims:, Clonality among high-level gentamicin-resistant Enterococcus faecium (HLGR-EF) isolates obtained from clinical and sewage treatment plants (STP) were investigated using PhePlate system (PhP), ribotyping and pulsed-field gel electrophoresis (PFGE). Methods and Results:, During 1 year study (September 2005,2006), a total of 106 HLGR-EF isolates were collected from clinical (n = 48) and STP (n = 58) samples in Tehran, Iran. Biochemical fingerprinting of these isolates using the PhP showed the presence of 21 PhP types (diversity index, Di = 0·97) among the clinical and 21 PhP types (Di = 0·91) among the STP isolates. Representative isolates of each PhP type (n = 42) were further characterized by the ribotyping method. Sixteen ribotypes were identified among the isolates with five types shared between the clinical and STP isolates. PFGE recognized 24 clonal types among these isolates with three pulsotypes shared between the clinical and STP isolates. Combination of the two techniques (PFGE and ribotyping) resulted in 24 (Di = 0·96) and 16 (Di = 0·93) types among the strains isolated from clinical and STP samples, respectively. Conclusions:, We concluded that the combination of PhP typing, ribotyping and PFGE could be extremely discriminatory when examining HLGR-EF isolates. Significance and Impact of the Study:, The emergence of highly diverse HLGR-EF population in Iran is of serious concern especially because of their multi-resistances. [source] Comparison of automated ribotyping and pulsed-field gel electrophoresis for subtyping of Vibrio choleraeLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2009H.-J. Zhou Abstract Aims:, To compare the discriminatory power of an automated ribotyping method for Vibrio cholerae subtyping with the pulsed-field gel electrophoresis (PFGE), to evaluate the possibility of automated ribotyping in use of outbreak investigations and surveillance of cholera. Methods and Results:, Eight-one epidemiologically unrelated isolates of V. cholerae, and 19 isolates from seven cholera outbreaks were used as the panels. When comparing the two methods using the epidemiologically unrelated isolates, automated ribotyping using PvuII distinguished 38 different ribotypes with a D -value of 0·8956. When combined with serotyping, the D -value is 0·9466. However, PFGE with NotI and SfiI digestions had higher D -values of 0·9951 and 0·9948, respectively. PFGE could cluster the isolates from each outbreak into the same pattern, and distinguish different patterns from different outbreaks, whereas automated ribotyping had lower discriminatory ability. Conclusions:, The automated ribotyping has lower discriminatory ability compared to PFGE, and is limited to application in V. cholerae subtyping and outbreak investigation. Significance and Impact of the Study:, The study evaluated the limitation in subtyping of automated ribotyping for V. cholerae, and raise the question of improvement for the automated ribotyping in subtyping. [source] | |||||||||||||