Home About us Contact
|
Home About us Contact | ||
|
|
||
PFGE
Terms modified by PFGE Selected AbstractsInvestigating DNA migration in pulsed fields using a miniaturized FIGE systemELECTROPHORESIS, Issue 23 2008Xiaojia Chen Abstract PFGE is a well-established technique for fractionation of DNA fragments ranging from kilobases to megabases in length. But many of these separations require an undesirable combination of long experiment times (often approaching tens of hours) and application of high voltages (often approaching tens of kV). Here, we present a simple miniaturized FIGE apparatus capable of separating DNA fragments up to 32.5,kb in length within 3,h using a modest applied potential of 20,V. The device is small enough to be imaged under a fluorescence microscope, permitting the migrating DNA bands to be observed during the course of the separation run. We use this capability to investigate how separation performance is affected by parameters including the ratio of forward and backward voltage, pulse time, and temperature. We also characterize the dependence of DNA mobility on fragment size N, and observe a scaling in the vicinity of N,0.5 over the size range investigated. The high speed, low power consumption, and simple design of this system may help enable future studies of DNA migration in PFGE to be performed quickly and inexpensively. [source] Rapid determination of genomic DNA length for new bacteriophagesELECTROPHORESIS, Issue 12 2007Philip Serwer Professor Abstract dsDNA viruses with long genomes (>200,kb) are expected to be a major source of novel genes. To rapidly characterize the genomes of newly isolated dsDNA bacteriophages, we develop here a procedure for the PFGE of intact long DNA genomes from bacteriophage particles in unfractionated, infected cell lysates of either liquid or gelled cultures. The DNA used for PFGE is suitable for sequencing after extraction with phenol. The PFGE is tuned to the range of expected DNA lengths. This procedure bypasses the isolation of bacteriophage particles and is useful for PFGE analysis of DNA from dissected zones of bacteriophage plaques. [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] Features of Saccharomyces cerevisiae as a culture starter for the production of the distilled sugar cane beverage, cachaça in BrazilJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2010C.R. Campos Abstract Aims:, To evaluate the dominance and persistence of strains of Saccharomyces cerevisiae during the process of sugar cane fermentation for the production of cachaça and to analyse the microbial compounds produced in each fermentative process. Methods and Results:, Three S. cerevisiae strains were evaluated during seven consecutive 24-h fermentation batches using recycled inocula. The UFLA CA 116 strain had the largest population of viable organisms, and the maximum population was achieved in the fourth batch after 96 h of fermentation. The UFLA CA 1162 and UFLA CA 1183 strains grew more slowly, and the maximum population was reached in the seventh batch. Molecular characterization of isolated yeast cells using PFGE (pulse field gel electrophoresis) revealed that more than 86% of the isolates corresponded to the initially inoculated yeast strain. The concentration of aldehydes, esters, methanol, alcohol and volatile acids in the final-aged beverages were within the legal limits. Conclusions:, Cachaça produced by select yeast strains exhibits analytical differences. UFLA CA 1162 and UFLA CA 116 S. cerevisiae isolates can be considered the ideal strains for the artisanal production of cachaça in Brazil. Significance and Impact of the Study:, The use of select yeast strains can improve the quality and productivity of cachaça production. Our findings are important for the appropriate monitoring of yeast during sugar cane fermentation. In addition, we demonstrate that UFLA CA 116 and UFLA CA 1162, the ideal yeast strains for cachaça production, are maintained at a high population density. The persistence of these yeast strains in the fermentation of sugar cane juice promotes environmental conditions that prevent or decrease bacterial contamination. Thus, the use of select yeast strains for the production of cachaça is a viable economic alternative to standardize the production of this beverage. [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] 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] Comparison of phenotypic and genotypic markers for characterization of an outbreak of Salmonella serotype Havana in captive raptorsJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2003M.P. Reche Abstract Aims: To establish a typing method for tracing the epidemic relationship of 16 strains of Salmonella serotype Havana isolated from captive raptors showing no symptomatology and residing in a wildlife hospital in Spain. Methods and Results: Antimicrobial susceptibility testing, ribotyping, pulsed field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) methodology were applied. Ten unrelated strains of serotype Havana were included as a control group to provide a basis of for the efficiency of the different markers used. All outbreak-related strains were resistant to nalidixic acid and streptomycin and showed the same ripotype, pulsotype and AFLP pattern. Conclusions: This is the first time that AFLP analysis has been tested with serotype Havana isolates and it has demonstrated to be the most useful epidemiological tool for discriminating between unrelated and outbreak-related strains of this serotype. The results obtained suggest that all the Salmonella serotype Havana isolates represented a common outbreak strain whose origin of contamination could not be established although it is thought that it was the poultry meat used for raptors'diet. Significance and Impact of the Study: Our study suggests the importance of microbiological analysis of these products in order to prevent contamination and dissemination of Salmonellae in this kind of Hospital. [source] Investigation of the genetic diversity among isolates of Salmonella enterica serovar Dublin from animals and humans from England, Wales and IrelandJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2002E. Liebana Aims: To assess the degree of genetic diversity among animal Salmonella Dublin UK isolates, and to compare it with the genetic diversity found among human isolates from the same time period. Methods and Results: One hundred isolates (50 human and 50 animal) were typed using plasmid profiling, XbaI-pulsed field gel electrophoresis (PFGE) and PstI- SphI ribotyping. Antimicrobial resistance data to 16 antibiotics was presented, and the presence of class-I integrons was investigated by real-time PCR. Seven different plasmid profiles, 19 ribotypes and 21 PFGE types were detected. A combination of the three methods allowed clear differentiation of 43 clones or strains. Eighteen isolates were resistant to at least one antimicrobial; five of them were multi-resistant and of these, only three presented class I integrons. Conclusions: Ribotyping data suggest the existence of at least three very different clonal lines; the same distribution in well-defined groups was not evident from the PFGE data. The existence of a variety of clones in both animals and humans has been demonstrated. A few prevalent clones seem to be widely disseminated among different animal species and show a diverse geographical and temporal distribution. The same clones were found in animals and humans, which may infer that both farm and pet animals may act as potential vehicles of infection for humans. Some other clones seem to be less widely distributed. Clustering analysis of genomic fingerprints of Salmonella Dublin and Salm. Enteritidis isolates confirms the existence of a close phylogenetic relationship between both serotypes. Significance and Impact of the Study: This paper describes the utility of a multiple genetic typing approach for Salm. Dublin. It gives useful information on clonal diversity among human and animal isolates. [source] Genotypic and phenotypic heterogeneity among lactococci isolated from traditional Pecorino Sardo cheeseJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2000L. Mannu Twenty-nine Lactococcus lactis isolates from one traditional 24 h-old Pecorino Sardo cheese were characterized phenotypically, technologically and genotypically in order to assess the biodiversity within this wild microbial population. Two DNA-based techniques, plasmid profiling and PFGE, were used for the genetic typing of the isolates. All 29 isolates were characterized at strain level and eight different genotypes were recognized. In addition, by combining the results from plasmid profile analysis and PFGE, it was possible to identify closely related isolates probably belonging to the same clonal lineage. The dominant biotype was identified in the 24 h-old cheese, as were the strains believed to act as starters for the curd. Atypical lactococci, able to grow in 6·5% NaCl, were isolated. The results suggest that wild bacterial populations should be preserved in order to protect the traditional raw milk cheeses, and to select new starter strains for the dairy industry. [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] 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] Persistence of Shiga toxin-producing Escherichia coli O26 in cow slurryLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2007B. Fremaux Abstract Aims: The main objective of this study was to evaluate the growth and survival of Shiga toxin-producing Escherichia coli (STEC) O26 in cow slurry; this serogroup is regarded as an important cause of STEC-associated diseases. Methods and Results: Four STEC were examined by polymerase chain reaction (PCR) to determine whether they harbour key virulence determinants and also by pulsed-field gel electrophoresis (PFGE) to obtain overview fingerprints of their genomes. They were transformed with the pGFPuv plasmid and were separately inoculated at a level of 106 CFU ml,1 in 15 l of cow slurry. All STEC O26 strains could be detected for at least 3 months in cow slurry without any genetic changes. The moisture content of the slurry decreased over time to reach a final value of 75% while the pH increased from 8·5 to 9·5 units during the last 50 days. Conclusion: STEC O26 strains were able to survive in cow slurry for an extended period. Significance and Impact of the Study: Long-term storage of waste slurry should be required to reduce the pathogen load and to limit environmental contamination by STEC O26. [source] DNA-based subtyping of verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2 strains from human and raw meat sourcesLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2003G. Domingue Abstract Aims:, To investigate subtyping methods for verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2. Methods and Results:, Eleven human and food strains isolated over a 15-year period were examined. All were intimin (eae)-negative, but all possessed enterohaemolysin and VT1-encoding sequences which in nine strains were vtx1c variant. Ten strains had VT2 genes which were all vtx2d. Plasmid profiles and randomly amplified polymorphic DNA-PCR were not discriminatory. Long-PCR restriction fragment length polymorphism of amplicons bound by the p gene and the VT2A subunit had screening potential. Pulsed field gel electrophoresis (PFGE) using XbaI gave fine discrimination although VT2 sequences were located on a 220 kbp fragment conserved in nine strains and on a 200 kbp fragment in the 10th. Conclusions:, As a result of apparent clonality, PFGE proved essential for differentiation. Long-PCR has promise for screening but requires further evaluation of inter-strain variable sequences. Significance and Impact of the Study:, A combined phenotypic and genotypic screen, and PFGE for selected strains was effective. [source] A small outbreak of listeriosis potentially linked to the consumption of imitation crab meatLETTERS IN APPLIED MICROBIOLOGY, Issue 2 2000J.M. Farber A small outbreak of listeriosis involving two previously healthy adults occurred in Ontario. Food samples obtained from the refrigerator of the patients included imitation crab meat, canned black olives, macaroni and vegetable salad, spaghetti sauce with meatballs, mayonnaise and water. All of the samples except the water contained Listeria monocytogenes. The three most heavily contaminated samples were the imitation crab meat, the olives and the salad which contained 2·1 × 109, 1·1 × 107 and 1·3 × 106 cfu g,1, respectively. L. monocytogenes serotype 1/2b was isolated from the patients, as well as from the opened and unopened imitation crab meat. Molecular typing of the isolates by both randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) typing demonstrated the imitation crab meat and clinical strains to be indistinguishable. Challenge studies performed with a pool of L. monocytogenes strains showed that imitation crab meat, but not olives, supported growth of the organism. In this study we have shown for the first time the potential involvement of imitation crab meat in a small outbreak of listeriosis. In terms of disease prevention, temperature control is critical to prevent or reduce the growth of this foodborne pathogen. In addition, with refrigerated products having a long (> 30 d) shelf life, additional safety factors must be used to prevent the growth of foodborne pathogens such as L. monocytogenes. [source] Streptococcus suis outbreak investigation using multiple-locus variable tandem repeat number analysisMICROBIOLOGY AND IMMUNOLOGY, Issue 7 2010Wei Li ABSTRACT Two outbreaks of Streptococcus suis ST7 occurred in humans in 1998 and 2005 in China. PFGE of chromosome restriction fragments found all ST7 isolates to be indistinguishable. Due to the genetic homogeneity of ST7 isolates, development of a rapid sub-typing method with high discriminatory power for ST7 isolates is required. In this study, a novel method, MLVA, was developed to type S. suis serotype 2 strains. Further, this method was used to analyze outbreak-associated ST7 strains in China. A total of 144 ST7 S. suis isolates were sub-typed into 34 MLVA types. Among these, eight isolates from the 1998 outbreak were sub-typed into five MLVA types, of which four MLVA types were also detected in Sichuan in 2005. These data indicate that the pathogens responsible for the two outbreaks had the same origin. In addition, some observations also provided molecular evidence for the transmission route, possibly indicating that the MLVA method has usefulness in epidemiology. The developed MLVA scheme for S. suis has greater discriminative power than PFGE. The method described here may be useful for identifying the source of S. suis infection and monitoring its spread. [source] The identification of circular extrachromosomal DNA in the nuclear genome of Trypanosoma bruceiMOLECULAR MICROBIOLOGY, Issue 2 2003N. S. Alsford Summary Nuclear extrachromosomal DNA elements have been identified in several kinetoplastids such as Leishmania and Trypanosoma cruzi, but never in Trypanosoma brucei. They can occur naturally or arise spontaneously as the result of sublethal drug exposure of parasites. In most cases, they are represented as circular elements and are mitotically unstable. In this study we describe the presence of circular DNA in the nucleus of Trypanosoma brucei. This novel type of DNA was termed NR-element (NlaIII repeat element). In contrast to drug-induced episomes in other kinetoplastids, the T. brucei extrachromosomal NR-element is not generated by drug selection. Furthermore, the element is stable during mitosis over many generations. Restriction analysis of tagged NR-element DNA, unusual migration patterns during pulsed field gel electrophoresis (PFGE) and CsCl/ethidium bromide equilibrium centrifugation demonstrates that the NR-element represents circular DNA. Whereas it has been found in all field isolates of the parasites we analysed, it is not detectable in some laboratory strains notably the genome reference strain 927. The DNA sequence of this element is related to a 29 bp repeat present in the subtelomeric region of VSG-bearing chromosomes of T. brucei. It has been suggested that this subtelomeric region is part of a transition zone on chromosomes separating the relatively stable telomeric repeats from the recombinationaly active region downstream of VSG genes. Therefore, we discuss a functional connection between the occurrence of this circular DNA and subtelomeric recombination events in T. brucei. [source] Genome size of human oral Treponema species by pulsed-field gel electrophoresisMOLECULAR ORAL MICROBIOLOGY, Issue 2 2004F. F. Correia The genome sizes of seven strains of oral treponemes were determined using pulsed-field gel electrophoresis (PFGE). These strains represent members from six of the currently known cultivable oral treponeme groups. The PFGE fragments were digitally recorded and then quantitated using GIMP v 1.2, an image manipulation program. The results show that the six oral treponeme genomes are comparable in size, ranging from approximately 2.2 to 2.5 Mbp. The genome sizes of these strains are 20,25% smaller than Treponema denticola strains, which have genome sizes of approximately 2.8,3.0 Mbp. [source] Systemic neonatal candidosis: the karyotyping of Candida albicans strains isolated from neonates and health-workersMYCOSES, Issue 1 2010J. Ben Abdeljelil Summary Candida albicans has become an important cause of nosocomial infections in neonatal intensive care units (NICUs). The aim of the present study was to compare C. albicans strains isolated from neonates (NN) suffering from systemic candidosis and from nurses in order to determine the relatedness between NN and health workers' strains. Thirty-one C. albicans strains were isolated from 18 NN admitted to the NICU of the neonatology service of Farhat Hached Hospital of Sousse, Tunisia and suffering from systemic candidosis, together with five strains recovered from nurses suffering from C. albicans onychomycosis. Two additional strains were tested, one from an adult patient who developed a systemic candidosis and the second from an adult with inguinal intertrigo. All strains were karyotyped by pulsed-field gel electrophoresis (PFGE) with a CHEF-DR II system. Analysis of PFGE patterns yielded by the 38 strains tested led to the identification of three pulsotypes that were designated I, II and III, and consisted of six chromosomal bands with a size ranging from 700 to >2500 kbp. The most widespread was the pulsotype I, which was shared by 17 NN and the five nurses' strains. The identity between NN and nurses' strains is very suggestive of a nosocomial acquisition from health-workers. [source] Karyotyping of Candida albicans and Candida glabrata from patients with Candida sepsisMYCOSES, Issue 5-6 2000Klempp-Selb The aim of this study was to determine the relatedness of Candida strains from patients suffering from Candida septicaemia by typing of Candida isolates from blood cultures and different body sites by pulsed field gel electrophoresis (PFGE using a contour-clamped homogenous electric field, CHEF). We studied 17 isolates of Candida albicans and 10 isolates of Candida glabrata from six patients. Four patients suffered from a C. albicans septicaemia, one patient from a C. glabrata septicaemia, and one patient had a mixed septicaemia with C. albicans and C. glabrata. Eight isolates from blood cultures were compared with 19 isolates of other sites (stool six, urine four, genital swab four, tip of central venous catheter three, tracheal secretion one, sputum one). PFGE typing resulted in 10 different patterns, four with C. albicans and six with C. glabrata. Five of the six patients had strains of identical PFGE patterns in the blood and at other sites. Seven isolates of a 58-year-old female with a C. glabrata septicaemia fell into five different PFGE patterns. However, they showed minor differences only, which may be due to chromosomal rearrangements within a single strain. Thus it appears, that the colonizing Candida strains were identical to the circulating strains in the bloodstream in at least five of six patients. [source] Sjögren's syndrome sufferers have increased oral yeast levels despite regular dental careORAL DISEASES, Issue 2 2008KCM Leung Aim:, To investigate the prevalence and quantity of oral yeasts and their association with oral candidiasis in Sjögren's syndrome (SS) patients receiving regular dental care. Materials and methods:, Yeasts in oral rinse and full-mouth supra-gingival plaque samples from 25 primary SS, 27 secondary SS and 29 control subjects were selectively cultured. All yeasts except single-species isolates were genotyped using pulsed field gel electrophoresis (PFGE). Results:, Ten (19%) SS sufferers had symptomless candidiasis. SS subjects had a higher prevalence (73%vs 7%) and quantity of yeasts than controls in both oral rinse and plaque samples (P < 0.05). The prevalence of yeasts in plaque was associated with candidiasis regardless of denture wearing (P , 0.04). Candida albicans was the predominant yeast isolated. PFGE showed 20 (66% of total) C. albicans isolate pairs, i.e. C. albicans species isolated from plaque and oral rinse samples of the same individual, were of closely related genetic clonal types (P < 0.01). Conclusions:, Despite effective oral hygiene, more SS subjects than controls had detectable levels of oral yeasts and their presence in supra-gingival plaque was associated with candidiasis. Candida albicans colonized supra-gingival biofilm even in well-maintained SS individuals, posing a challenge to the control of oral candidiasis. [source] | |||||||||||||