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Objective:To analysis the macrolide resistance, molecular characteristics and plused-field gel electrophoresis(PFGE) type of Bordetella pertussis ( Bp), explore the possible resistance mechanism and the relationship between PFGE types and macrolide resistance profiles. Methods:Erythromycin, azithromycin and clarithromycin susceptibility of clinical isolates during 2016 to 2018 was determined by E-test. PCR was used to detect the drug-resistant genes and mutation sites. PFGE were employed to do molecular typing for the strains.Results:Thirty-five strains were isolated, of which 27 strains were resistant to all three antibiotics, two strains were resistant to erythromycin and azithromycin, and six strains were sensitive to all three antibiotics. Partial macrolide resistant strains carried the methylase gene ermA (27.6%, 8/29) and ermB (31.0%, 9/29); A2047G site mutation was detected in macrolide-resistant strains, while no drug-resistant genes or mutation sites were found in sensitive strains. Resistant strains were classified into BPSR23 and BPFINR9 types, while sensitive strains were other profiles. Conclusions:The clinical isolated Bp were seriously resistant to erythromy and showed signs of resistance to other macrolides. The acquisition of methylase gene and mutation of A2047G site might be the main mechanism of resistance. The macrolide resistance might have has a certain correlation with PFGE profile.
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The objectives of this study were to isolate Klebsiella pneumoniae from different sources in three dairy cattle herds, to use the pulsed-field gel electrophoresis (PFGE) to measure genotypic similarities between isolates within a dairy herd, to verify the production of extended-spectrum β-lactamases (ESBLs) by the double-disk synergy test (DDST), and to use the PCR to detect the main ESBLs subgroups genes. Three dairy farms were selected based on previous mastitis outbreaks caused by K. pneumoniae. Milk samples were collected from lactating cows and from the bulk tank. Swabs were performed in different locations, including milking parlors, waiting room, soil, animal's hind limbs and rectum. K. pneumoniae was isolated from 27 cases of intramammary infections (IMI) and from 41 swabs. For farm A isolates from IMI and bulk tank were considered of the same PGFE subtype. One isolate from a bulk tank, three from IMI cases and four from environmental samples were positive in the DDST test. All eight DDST positive isolates harbored the bla shv gene, one harbored the bla tem gene, and three harbored the bla ctx-m gene, including the bulk tank isolate. Our study confirms that ESBL producing bacteria is present in different locations in dairy farms, and may be responsible for IMI. The detection of ESBLs on dairy herds could be a major concern for both public and animal health.
Os objetivos deste estudo foram isolar Klebsiella pneumoniae de diferentes localidades em três propriedades leiteiras, utilizar a eletroforese em campo pulsátil para averiguar similaridades genotípicas entre os isolados de uma mesma propriedade, verificar a produção de beta-lactamases de espectro estendido (ESBLs) pela prova da disco-difusão dupla associada (DDST) e utilizar a PCR para detecção dos principais subgrupos genéticos de ESBLs. Três propriedades leiteiras foram selecionadas baseando-se em surtos prévios de mastites causadas por K. pneumoniae. Amostras de leite foram coletadas de vacas em lactação e do tanque de expansão. Swabs foram realizados em diferentes localidades, incluindo salas de lactação, salas de espera, solo, reto e membros posteriores de animais. K. pneumoniae foi isolada de 27 casos de infecções intramamária (IMI) e de 41 swabs. Para a propriedade A os isolados de IMI e do tanque de expansão foram considerados do mesmo subtipo molecular. Um isolado do tanque de expansão, três de casos de IMI e quatro de amostras ambientais foram considerados positivos no teste da DDST. Todos os oito isolados DDST positivos portavam o gene bla shv, um portava o gene bla tem, e três portavam o gene bla ctx-m, incluindo um isolado de tanque de expansão. Nosso estudo confirma que bactérias produtoras de ESBLs estão presentes em diferentes localidades em propriedades leiteiras, e podem ser responsáveis por quadros de IMI. A detecção de ESBLs em propriedades leiteiras pode representar uma grande preocupação para saúde pública e para a saúde animal.
Subject(s)
Animals , Female , Cattle , beta-Lactamases , Klebsiella pneumoniae/growth & development , Klebsiella pneumoniae/virology , Milk , Polymerase Chain Reaction , Polymerase Chain Reaction/veterinary , Electrophoresis, Gel, Pulsed-Field , Electrophoresis, Gel, Pulsed-Field/veterinary , Mastitis, Bovine/virologyABSTRACT
BACKGROUND: We evaluated the ability of infrequent restriction site-polymerase chain reaction (IRS-PCR) to perform molecular epidemiologic analysis of Community-Onset Extended Spectrum Beta-Lactamase (ESBL) producing Escherichia coli, and also assessed the use of PFGE as an alternative method. MATERIALS AND METHODS: IRS-PCR assay was performed using combinations of adaptors for XbaI and HhaI restriction sites on clinical isolates of E. coli (n=51). We compared the discriminatory power, quality and efficiency of IRS-PCR to PFGE. RESULTS: In E. coli, PFGE discriminated 39 (76.4%) and IRS-PCR discerned 41 (80.3%) of the total 51 strains. It took much less time to complete IRS-PCR (one day) than PFGE (at least 4 days). CONCLUSIONS: IRS-PCR is a more sensitive and rapid alternative to PFGE for molecular epidemiologic analysis of E. coli.
Subject(s)
beta-Lactamases , Electrophoresis, Gel, Pulsed-Field , Escherichia , Escherichia coli , Polymerase Chain ReactionABSTRACT
Objective To investigate the genotypic characteristics and persistence of Legionella pulsed-field gel electrophoresis (PFGE) patterns in 16 air-conditioner cooling towers in six different public sites of Shanghai. Methods From May to October, continuous sampling was operated once per month in 2007. Legionella strains isolated from the 16 cooling towers were confirmed by serological and latex agglutination. PFGE was applied for the fingerprinting of the isolates, while the culster results of PFGE were analyzed by BioNumerics software. Results 131 strains of Legionella were isolated, including L. pneumophila, L. bozemanae, L. micdadei and L anisa.52 distinguishable PFGE patterns were differentiated among the 16 cooling towers, with 37 patterns were owned by just one cooling tower, which was not shared with other cooling towers, while 15 patterns were shared by more than 2 cooling towers. All the cooling towers had ≥2 PFGE patterns,while in 13 cooling towers the same PFGE patterns were recovered during the six months. From June to October of 2007, 18 strains of Legionella belonging to the PFGE pattern of LPAs. SH0078 were isolated continuously from 6 cooling towers. Conclusion This study demonstrated great genotypic diversity and complexity of Legionella in cooling towers. Persistence of the PFGE patterns was observed in 81.25% of the cooling towers. The PFGE pattern of LPAs. SH0078 was distributed widely,suggesting it might be the dominate strain in Shanghai.
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BACKGROUND: The significance of Staphylococcus epidermidis positive blood cultures is difficult to determine, but repeated isolation of the same organism with the same genotype is suggestive of true bacteremia. MATERIALS AND METHODS: Two sequential isolates of S. epidermidis from blood cultures of the same twelve patients were genotyped by PFGE. The results were compared with those of antibiotyping and isolation time intervals between the two strains. RESULTS: The two sequential strains from each patient had identical PFGE patterns in 66.6% (8 of 12) of the patients and two different types in 33.3% (4 of 12) of the patients. Antibiotypes of the two isolates from the same patient were different in all 4 patients whose isolates had different PFGE patterns, and they were the same in 7 of 8 patients whose isolates had identical PFGE patterns:the PFGE results were in agreement with the antibiotyping for 91.7% (11/12) of patients. The isolation time interval between the two strains was or =5 days. CONCLUSION: These data showed that two consecutive isolates of S. epidermidis from blood cultures had different PFGE patterns in 33% of patients, suggesting a high prevalence of contamination. In the absence of genotyping measures, both antibiotype and isolation time interval can be alternative and useful tools for determining strain relatedness of sequential isolates of S. epidermidis from blood cultures.
Subject(s)
Humans , Bacteremia , Electrophoresis, Gel, Pulsed-Field , Genotype , Prevalence , Staphylococcus epidermidis , StaphylococcusABSTRACT
BACKGROUND: The significance of Staphylococcus epidermidis positive blood cultures is difficult to determine, but repeated isolation of the same organism with the same genotype is suggestive of true bacteremia. MATERIALS AND METHODS: Two sequential isolates of S. epidermidis from blood cultures of the same twelve patients were genotyped by PFGE. The results were compared with those of antibiotyping and isolation time intervals between the two strains. RESULTS: The two sequential strains from each patient had identical PFGE patterns in 66.6% (8 of 12) of the patients and two different types in 33.3% (4 of 12) of the patients. Antibiotypes of the two isolates from the same patient were different in all 4 patients whose isolates had different PFGE patterns, and they were the same in 7 of 8 patients whose isolates had identical PFGE patterns:the PFGE results were in agreement with the antibiotyping for 91.7% (11/12) of patients. The isolation time interval between the two strains was or =5 days. CONCLUSION: These data showed that two consecutive isolates of S. epidermidis from blood cultures had different PFGE patterns in 33% of patients, suggesting a high prevalence of contamination. In the absence of genotyping measures, both antibiotype and isolation time interval can be alternative and useful tools for determining strain relatedness of sequential isolates of S. epidermidis from blood cultures.
Subject(s)
Humans , Bacteremia , Electrophoresis, Gel, Pulsed-Field , Genotype , Prevalence , Staphylococcus epidermidis , StaphylococcusABSTRACT
OBJECTIVE To identify the antibiotic resistance,homology and the carbapenemases determinants of imipenem-resistant strains of Acinetobacter baumannii isolated from elderly in the Zhejiang Hospital.METHODS All 142 strains of A.baumannii were isolated from Zhejiang Hospital through Jan 2005 to Jan 2007.K-B method was used to screen imipenem-resistant strains.The MICs of imipenem-resistant strains to 14 antimicrobial agents were determined by agar dilution method.The homology of these isolates was analyzed by pulse-field gel electrophoresis(PFGE).The coding genes of carbapenamases and the gene environments were investigated by PCR,clone,and sequencing.RESULTS Ninety-seven strains of imipenem-resistant A.baumannii were isolated from 142 strains.All of the strains of carbapenem resistant A.baumannii belonged to 4 epidemic PFGE-clones.Ninety carbapenem resistant strains contained OXA-23-like carbapenemase gene and 91 isolates were positive for OXA-51-like gene. OXA-23-like gene of 86 strains was just on the down-stream of insert sequence ISAba1.OXA-51-like gene of 6 strains had an ISAba1 sequence just on the up-stream.CONCLUSIONS All imipenem-resistant strains of A.baumannii are pan-resistant isolates.Clone dissemination is the most important style of strains spread.No OXA-24-like,OXA-58like,IMP-like,and VIM-like gene are detected.OXA-23-like and OXA-51-like gene are the most popular carbapenemases coding genes of these strains in the Zhejiang Hospital.ISAba1 has close relationship with OXA type carbapenemases genes in Zhejiang Hospital.
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A study of genotyping (pulsotyping) of Salmonella typhi (S. typhi) isolates using pulse-field gel electrophoresis (PFGE) methods was performed to examine their genetic diversity, and relationship between genetic characteristics and clinical outcomes. Sixty-six S. typhi isolates obtained from sporadic hospitalized typhoid fever cases were used in this study. Four isolates were found identical and the dendogram constructed showed 33 pulsotypes in which 13 of them can be divided into 30 subtypes. Diversity among them were high as shown by the Dice coefficients that ranged from 0.486 to 1.000. Cluster analysis showed 2 main clusters with 65% degree of similarity, suggested that they were not originated from one clone. Further, at 90% degree of similarity, 9 clusters containing at least 3 isolates were determined to explore any possible existence of relationship between genetic profile and particular clinical outcomes. Clinical manifestations ranged from mild to severe were in fact distributed diversely among these clusters. Although the clinical data obtained were incomplete, 2 out of 4 patients infected by the S. typhi belonged to cluster 1 showed an elevation of total bilirubin, whereas it was not found in 19 other patients distributed in other 8 clusters. Even though specific clinical manifestations were apparently not found to relate with particular clusters of genotypes, S. typhi isolates grouped in cluster 1 seemed to show trophism to hepatobiliary system.
Subject(s)
Typhoid Fever , SalmonellaABSTRACT
BACKGROUND: Salmonella infections continue to cause gastrointestinal and systemic diseases throughout the world. S. Enteritidis and S. Typhimurium were traditionally known as typical food poisoning Salmonella agents, the isolation rate of which has been increased recently in Korea. S. Typhimurium phage type DT104 has become an important emerging pathogen. Isolates of this phage type often possess resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT resistance). The mechanism by which DT104 has accumulated resistance genes is of interest, since these genes interfere with treatment of DT104 infections and might be horizontally transferred to other bacteria, even to unrelated organisms. METHODS: All the isolates included in this study were identified as S. enterica serovar Typhimurium according to the Kauffmann-White serotyping scheme and were definitive phage type DT104 according to the phage typing scheme described by Anderson, et al. A total of 63 isolates of S. enterica serovar Typhimurium DT104 were characterized by antimicrobial resistance analysis, pulsed-field gel electrophoresis (PFGE) with the restriction enzyme XbaI. RESULTS: Four hundred ninety six S. Typhimurium isolates were divided into 28 different phage types and DT104 was the second most common phage type in Korea. A total of 63 S. Typhimurium DT104 isolates were grouped into 7 resistance phenotypes. Fourty one (65.1%) isolates were resistant to the ACSSuTTic core alone or to additional drugs as well except twenty two (33.9%) isolates were resistant to the ASSuTeTic. Four PFGE subtypes A1, A2, B1, and B2 were observed among DT104 isolates and type A1 was prevalent. CONCLUSIONS: We concluded two distinct clones were present among Korea multidrug resistant S. enterica serotype Typhimurium DT104 and multidrug resistant S. Typhimurium phage type DT104 has been an important emerging pathogen in Korea.
Subject(s)
Ampicillin , Bacteria , Bacteriophage Typing , Bacteriophages , Chloramphenicol , Clone Cells , Electrophoresis, Gel, Pulsed-Field , Foodborne Diseases , Korea , Phenotype , Salmonella enterica , Salmonella Infections , Salmonella , Serotyping , Streptomycin , Sulfonamides , TetracyclineABSTRACT
BACKGROUND: Despite the recognized increase of frequency of candiduria due to Candida tropicalis, little was known of its molecular epidemiology. We applied PFGE and RAPD assay for urinary C. tropicalis isolates and evaluated the utilities of PFGE and RAPD for the epidemiological typing of C. tropicalis isolates. METHODS: A total of urinary 57 isolates of C. tropicalis from 40 patients at two hospitals was analyzed. PFGE analysis were performed by electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA (REAG) using two restriction enzymes (BssHII and SfiI). For RAPD, a total of 31 primers (30 random 10-mer primers and M13 primer) were used. RESULTS: EK and RAPD analysis showed the same or similar patterns among the isolates. REAG with BssHII separated 57 isolates into 28 distinct types. Six patterns were generated by REAG with using SfiI. By combining the two REAG, a total of 31 different DNA types were identified among 57 isolates from 40 patients. Three strain types were common to 23 isolates from 12 patients of a University Hospital, which suggested possible nosocomial transmission. In 19 patients with serial urinary isolates, the sequential strains from each patient exhibited the same REAG pattern. CONCLUSION: These suggest that REAG with BssHII and SfiI is useful for the investigation of molecular epidemiology of C. tropicalis isolates. In addition, some clusters of C. tropicalis isolates with the same DNA type suggest that nosocomial transmission may occur.
Subject(s)
Humans , Candida tropicalis , Candida , DNA Restriction Enzymes , DNA , Electrophoresis, Gel, Pulsed-Field , Karyotyping , Molecular Epidemiology , Molecular TypingABSTRACT
BACKGROUND: Vancomycin-resistant enterococci (VRE) infection is an emerging nosocomial problem. In Chonnam National University Hospital, 64 vanA VRE isolates were recovered from clinical specimens of 21 patients from January 1995 through December 1999. It is important to understand the epidemiology of VRE within a hospital for implementing appropriate infection control measures. METHODS: Twenty-one vanA VRE isolates obtained from 21 patients during over a 5-year period were analyzed by pulsed-field gel electroporesis (PFGE) fingerprinting. Medical records of the patients with VRE isolates were reviewed. RESULTS: By PFGE analysis for 21 VRE isolates, eleven genotypes were determined; 4 genotypes were shared by more than 2 patients. This four small clusters of VRE involved patients from hemato-oncologic units and ICU (intensive care unit) over 3 days to 1 year period. CONCLUSION: Although the VRE epidemic at our hospital is polyclonal, some spread of VRE from a clonal origin were found in this hospital. The implementation of careful isolation and infection control measures, prudent use of antibiotics, especially vancomycin, and periodic screening of patients populations, are required.
Subject(s)
Humans , Anti-Bacterial Agents , Dermatoglyphics , Epidemiologic Studies , Epidemiology , Genotype , Infection Control , Mass Screening , Medical Records , VancomycinABSTRACT
PURPOSE: In this study we tried to look at the spreading, duration of colonization, and acquisition of new streptococci which were obtained in one geographical area, as well as the bacteriologic and molecular epidemiology of normal school children carrying group A streptococci and their clonal relationship through the combined application of the serotype of T antigen and Pulsed Field Gel Electrophoresis(PFGE). METHODS: A total of 88 strains of group A streptococci were isolated from 396 normal school children. All isolates were classified in groups by Streptex and serotyped by T. agglutination. Restriction enzyme digestion of DNA was taken using Sma I. DNA fragments were separated by PFGE. RESULTS: A total of 33 strains were allocated their epidemiologic characteristics. Four out of 33 strains were not restricted by enzyme(Sma I). Twenty nine strains out of 33 strains showed 12 subtypes with 8-12 fragments between 40kbp and 500kbp of DNA fragments on PFGE. Eight strains of NT and T6 war same fragment patterns on PFGE, respectively. Three strains out of 4 strains of T8/25 were not restricted and the other one showed different, unique patterns. One strain out of 8 stains of T12 was not restricted, and the others were classified as 5 different subtypes. Two strains of Tl were different patterns from each other, and 2 strains of T4 showed the samefragment pattern CONCLUSION: T serotypes with PFGE will be useful as a screening and molecular epidemiologic method in a country where anti-M antisera is not available, after recognizing the advantages and disadvantages of M and T serotyping.
Subject(s)
Child , Humans , Agglutination , Antigens, Viral, Tumor , Colon , Coloring Agents , Digestion , DNA , Epidemiologic Methods , Epidemiologic Studies , Immune Sera , Mass Screening , Molecular Epidemiology , Serotyping , Streptococcus pyogenes , StreptococcusABSTRACT
BACKGROUND: Clostridium difficile is a major cause of nosocomial infectious diarrhea. Nosocomial clusters of C. difficile disease have been ascribed to the transfer of the organism form patient to patient. The aim of this study was to survey the nosocomial acquisition of C. difficile infection and to evaluate the efficacy and efficiency of epidemiologic typing systems by molecular analysis of the isolates. METHODS: A surveillance study for C. difficile acquisition was performed in patients admitted to neurology ward (NW) and medical intensive care unit (MICU) in an 800-bed tertiary-care hospital from August 1998 to October 1998. Stool specimens were taken weekly for culture of C. difficile. All isolates were examined for toxin B gene by PCR assay. Three molecular typing methods, including pulsed-field gel electrophoresis (PFGE), ribotyping, and arbitrarily primed polymerase chain reaction (AP-PCR) were used to differentiate individual strains of C. difficile isolates. Their performance characteristics were compared according to the consensus guidelines by the European Society for Clinical Microbiology and Infectious Disease. RESULTS: A total of 38 C. difficile strains were isolated from 308 stool cultures. The period prevalence was 7.4/1000 patient-days and 21.2/1000 patient-days in the NW and MICU, respectively (P=0.034). The acquisition incidence of C. difficile infection was 1.85/ 1,000 patient-days and 5.33/1,000 patient-days in NW and MICU, respectively. The toxin B gene was detected in 38% (8/21) of C. difficile isolates; 62.5% from diarrheal patients and 23% from asymptomatic patients. In a comparison of the three typing systems, the typeability was 0.444 by PFGE, 0.972 by AP-PCR and 1 by ribotyping, and the discrimination index was 0.975 by PFGE, 0.810 by AP-PCR and 0.777 by ribotyping. All three typing systems were highly reproducible. AP-PCR was the least costly and most rapid method. CONCLUSION: The relatively high prevalence of C. difficile infection in the hospital might indicate a potential nosocomial spread, even though the acquisition incidence was low. AP-PCR appears to be an efficacious and efficient method for the epidemiologic study of C. difficile infection, and its suboptimal discriminative power may be enhanced by complementary PFGE.
Subject(s)
Humans , Clostridioides difficile , Clostridium , Communicable Diseases , Consensus , Diarrhea , Discrimination, Psychological , Electrophoresis, Gel, Pulsed-Field , Epidemiologic Studies , Incidence , Intensive Care Units , Molecular Typing , Neurology , Polymerase Chain Reaction , Prevalence , RibotypingABSTRACT
BACKGROUND: Clostridium difficile is a major cause of nosocomial infectious diarrhea. Nosocomial clusters of C. difficile disease have been ascribed to the transfer of the organism form patient to patient. The aim of this study was to survey the nosocomial acquisition of C. difficile infection and to evaluate the efficacy and efficiency of epidemiologic typing systems by molecular analysis of the isolates. METHODS: A surveillance study for C. difficile acquisition was performed in patients admitted to neurology ward (NW) and medical intensive care unit (MICU) in an 800-bed tertiary-care hospital from August 1998 to October 1998. Stool specimens were taken weekly for culture of C. difficile. All isolates were examined for toxin B gene by PCR assay. Three molecular typing methods, including pulsed-field gel electrophoresis (PFGE), ribotyping, and arbitrarily primed polymerase chain reaction (AP-PCR) were used to differentiate individual strains of C. difficile isolates. Their performance characteristics were compared according to the consensus guidelines by the European Society for Clinical Microbiology and Infectious Disease. RESULTS: A total of 38 C. difficile strains were isolated from 308 stool cultures. The period prevalence was 7.4/1000 patient-days and 21.2/1000 patient-days in the NW and MICU, respectively (P=0.034). The acquisition incidence of C. difficile infection was 1.85/ 1,000 patient-days and 5.33/1,000 patient-days in NW and MICU, respectively. The toxin B gene was detected in 38% (8/21) of C. difficile isolates; 62.5% from diarrheal patients and 23% from asymptomatic patients. In a comparison of the three typing systems, the typeability was 0.444 by PFGE, 0.972 by AP-PCR and 1 by ribotyping, and the discrimination index was 0.975 by PFGE, 0.810 by AP-PCR and 0.777 by ribotyping. All three typing systems were highly reproducible. AP-PCR was the least costly and most rapid method. CONCLUSION: The relatively high prevalence of C. difficile infection in the hospital might indicate a potential nosocomial spread, even though the acquisition incidence was low. AP-PCR appears to be an efficacious and efficient method for the epidemiologic study of C. difficile infection, and its suboptimal discriminative power may be enhanced by complementary PFGE.
Subject(s)
Humans , Clostridioides difficile , Clostridium , Communicable Diseases , Consensus , Diarrhea , Discrimination, Psychological , Electrophoresis, Gel, Pulsed-Field , Epidemiologic Studies , Incidence , Intensive Care Units , Molecular Typing , Neurology , Polymerase Chain Reaction , Prevalence , RibotypingABSTRACT
Pulsed field gel electrophoresis(PFGE)is a new type technique of gel electrophoresis which can be used to separate large DNA molecules.It has been widely applied to the karyotype analysis,identification of species groups,genetic orientation and genetic analysis for fungi.This article describes the principle,development and general manipulative procedure of PFGE,and elaborates the application in the molecular research of fungi.