Clonal identification of Aeromonas hydrophila strains using randomly amplified polymorphic DNA analysis

The suitability of arbitrary primer polymerase chain reaction (RAPD) as a typing technique was evaluated by comparing it with pulsed-field gel electrophoresis (PFGE) to characterize Aeromonas hydrophila strains isolated from a cluster of hospital-acquired infections. Five isolates from patients and 10 isolates from the water supply were compared to 10 epidemiologically unrelated strains isolated from patients and rivers. Two methods were used to prepare DNA and two primers (AP3 and AP5) were selected. The discriminatory power was better with the extractive DNA preparation than the boiling method. The discrimination of closely related from less related strains by PCR using AP3 was consistent with that by PFGE: water supply of Cholet hospital contaminated with Aeromonas species was not the source of the cluster of hospital infections and only two patients were infected with clonally-related strains. RAPD using primer AP3 was simpler, cheaper, and quicker to perform than pulsed-field gel electrophoresis and is well suited for the epidemiological study of A. hydrophila isolates.     

Pulsed-Field Gel Electrophoresis, Conventional, and Molecular Serotyping of Listeria monocytogenes from Food Proficiency Testing Trials Toward an Harmonization of Subtyping at European Level

Abstract The European Union Reference Laboratory for Listeria monocytogenes (EURL for L. monocytogenes) coordinates a European network of 29 National Reference Laboratories (NRLs). Depending on a national decision, NRLs undertake food, environmental, and veterinary L. monocytogenes strain surveillance in their respective countries. In the framework of the PulseNet Europe network, two pulsed-field gel electrophoresis (PFGE) subtyping proficiency testing (PT) trials were carried out in 2003 and 2006. The obtained data showed that PFGE profiles can be compared and exchanged between laboratories. However, no further PT trial had been performed since 2006. In this context, two PT trials were organized by the EURL to evaluate the ability of NRLs to perform conventional serotyping, molecular serotyping and PFGE subtyping. Eleven well-characterized isolates of L. monocytogenes were used: six and nine isolates were tested in 2009 and 2010, respectively. Three isolates were repeated between the two studies. In the 2010 panel, a strain was tested in duplicate, and two strains were related to the same epidemiological group. The strains were analyzed blind in different laboratories (17 in 2009 and 25 in 2010) using (1) their own in-house method for serotyping methods and (2) standardized protocols based on the PulseNet protocol for PFGE. For conventional serotyping, 86.0% in 2009 and 91.0% in 2010 of the serotypes obtained were in agreement with the EURL data. For molecular serotyping, 93.5% of the results in 2009 and 95.2% in 2010 matched the EURL data. For PFGE, 68.9% in 2009 and 81.7% of the combined AscI/ApaI profiles were indistinguishable from the EURL reference profiles. The variations observed could be attributed to slight standardization defaults or, in a few cases, to a failure in DNA extraction. These PT trials provided a valuable opportunity to improve the subtyping ability of NRLs and facilitate exchanges of subtyping data in the future.     

Real-time application of automated ribotyping and DNA macrorestriction analysis in the setting of a listeriosis outbreak

A cluster of three cases of listeriosis cases occurred against a background of endemic listeriosis in Western Australia. Human and environmental isolates of Listeria monocytogenes obtained during the outbreak investigation were rapidly subtyped by automated ribotyping using an EcoRI protocol and a RiboPrinter. DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) was used to confirm the relatedness of isolates. Serogroup 1/2 predominated among the food samples and the four clinical isolates from the outbreak cluster were also of this serogroup. All isolates from chicken material were serogroup 1/2 and indistinguishable by ribotype pattern. PFGE subdivided strains of this ribotype into four subtypes. The preliminary analysis had an immediate impact on hypothesis generation, environmental health investigations, environmental specimen collection and initial control measures. Sufficient typing data to guide environmental health and disease control initiatives was generated in less than one week by combining automated ribotyping with PCR-based detection of L. monocytogenes in suspect foodstuffs and an L. monocytogenes DNA probe. There were no further cases of bacteriologically confirmed listeriosis in Western Australia for six months after completion of the investigation.     

A comparison of Listeria monocytogenes serovar 4b isolates of clinical and food origin in Austria by automated ribotyping and pulsed-field gel electrophoresis

In this study, two typing methods, automated ribotyping and pulsed-field gel electrophoresis (PFGE), were evaluated for the subtyping of Listeria monocytogenes serotype 4b. The strains originated from patients and food samples collected in Austria during 2001-2005 and from Europe and North America in the World Health Organization collaborative study on the subtyping of this species. The largest group of Austrian clinical isolates was of the same PFGE subtype as those isolated from foodborne outbreaks in Switzerland and in the United States. Another subtype of clinical isolates from Austria was indistinguishable to that obtained from isolates responsible for a foodborne outbreak in the United States in 1985. Although the discriminatory power of PFGE was higher than that of automated ribotyping, some PFGE types were differentiated by ribotyping. Thus, combining data obtained by both automated ribotyping and PFGE increases the strain discrimination. Still, many of the Austrian strains remain indistinguishable from strains of foodborne outbreaks in other countries although there is no known epidemiological relation. This complies to previous studies which show the highly clonal nature of L. monocytogenes 4b strains which are responsible for both large outbreaks and sporadic cases.     

Molecular characterization of Salmonella isolates by REP-PCR and RAPD analysis

Eighteen Salmonella isolates from both human and food (non-human) sources (fish, meat, and poultry) were characterized using conventional culture methods, biochemical, serological, and molecular analyses. REP-PCR and RAPD produced DNA profiles for differentiation purposes. Enterobacterial repetitive intergenic consensus (ERIC), repetitive extragenic palindronic (REP) and BOXAIR primers were selected for REP-PCR and two arbitrary primers, namely OPP-16 and OPS-11 were used for RAPD to generate DNA fingerprints from the Salmonella isolates. REP-PCR method showed greater discriminatory power in differentiating closely related strains of the related strains of Salmonella and produced more complex banding patterns as compared with RAPD. A dendogram was constructed with both sets of profiles using SPSS Version 13.0 computer software and showed that most human isolates were separately clustered from the non-human isolates. Two of the human isolates were closely related to some of the non-human isolates. A good correlation was also observed between the serogrouping of the O antigen and the molecular profiles obtained from REP-PCR and RAPD data of the Salmonella isolates. The results of a principal coordinate analysis (PCA) corresponded to the clustering in the dendrogram.     

102株单核细胞增生李斯特菌随机扩增多态性DNA基因分型研究

目的:对单核细胞增生李斯特菌进行随机扩增多态性DNA(RAPD)基因分型研究。方法:以优化的随机引物和反应体系对生肉、熟肉、生奶、乳制品、水产品、冷冻食品、生食蔬菜共七大类食品中分离的102株单核细胞增生李斯特菌进行RAPD法基因分型,并按DNA条带数及片段大小绘制指纹图谱。结果:102株单核细胞增生李斯特菌共得26种RAPD指纹图谱,以第12、18、23和10型为主要型别,分别为15、14、8和6株。结论:RAPD技术可将单核细胞增生李斯特菌分型26种,我省食品中分离的单核细胞增生李斯特菌主要基因型为第12、18、23和10型。     

Molecular characterization of Streptococcus pyogenes isolates to investigate an outbreak of puerperal sepsis.

OBJECTIVE: To describe microbiological characteristics and epidemiologic features of an outbreak of postpartum endometritis. METHODS: Various markers were investigated in five patients and three throat carriage isolates of Streptococcus pyogenes obtained during an outbreak of endometritis occurring in a 13-week period. Molecular characterization included biotyping, T-serotyping, emm gene sequence and restriction, pulsed-field gel electrophoresis (PFGE), and random amplified polymorphic DNA (RAPD) analysis. RESULTS: Biotype, T-serotype, and genotypic data (emm analysis, PFGE, and RAPD analysis) revealed a close relationship among the isolates from three patients, suggesting that cross-contamination had occurred. These isolates were biotype 1, T type 28, and emm type 28. The isolates from one patient and one carrier differed from those of the index patient by minor variations of the emm amplicon restriction pattern, PFGE pattern, or RAPD pattern. The remaining isolates were phenotypically and genetically different. CONCLUSION: Identification of different isolates demonstrated that different strains may circulate simultaneously during a true outbreak and that the predominant strain might persist for several months.     

Pulsed field gel electrophoresis as a new epidemiological tool for monitoring methicillin-resistant Staphylococcus aureus in an intensive care unit.

Pulsed field gel electrophoresis (PFGE) of bacterial DNA was used in a 1-month epidemiological study of methicillin-resistant Staphylococcus aureus (MRSA) in a 15-bed Intensive Care Unit (ICU). Patient and hospital staff carriage as well as distribution of MRSA in the ICU environment were investigated, and a total of 3802 samples produced 175 isolates. The stability and the reproducibility of the PFGE method were satisfactory. Moreover, the plasmid content of the strains so far examined had no influence on the PFGE profiles of the MRSA strains. The polymorphic profiles observed also account for the use of this method as an epidemiological tool for investigating MRSA. Among 30 patients who stayed more than 4 days in the unit, PFGE analysis showed 11 episodes of colonization in nine patients, whereas lysotyping and plasmid DNA analysis demonstrated only eight and seven such episodes in the same patients, respectively. The combination of PFGE with lysotyping and plasmid analysis may provide a greater discriminatory capacity between MRSA isolates.     

广东省食品中单核细胞增多性李斯特菌脉冲场凝胶电泳技术分子分型的研究

目的应用脉冲场凝胶电泳技术（PFGE）对广东省食品中分离的单核细胞增多性李斯特菌（Lm）进行分子分型,并建立PFGE数据库。方法参照美国PulseNet的Lm PFGE分子分型标准方法进行检测。应用BioNumerics软件对不同食物种类、时间和地点的分离株进行比对,分析菌株之间的相关性。结果107株Lm分成41个PFGE型,型别较为分散;从鸡肉中分离的菌株数最多（37株）,PFGE分型也最多（19个）;冷藏和冷冻食品的分离率较高,其中有26株菌仅存在1-2个电泳条带的差异,相似度极高;韶关和惠州地区分离的Lm存在着地区特异性;随时间变迁,部分菌株仍存在不同程度的相关性。结论广东省食品中分离的Lm在整体上表现出较大的遗传多样性,但部分菌株有不同程度的相关性。     

High Level of Heterogeneity Among Listeria monocytogenes Isolates from Clinical and Food Origin Specimens in Greece

Abstract In order to examine the genetic variation of clinical and food isolates of Listeria monocytogenes in Greece, a total of 61 L. monocytogenes non-duplicate isolates, recovered from clinical specimens (n=19) and food (n=42), were serotyped and genotyped using two different Random Amplification of Polymorphic DNA (RAPD) protocols and Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). Serotype group 4b, 4d, 4e prevailed (39.4%), among both clinical and food isolates, followed by serotype group 1/2a, 3a (23.0%), which nevertheless was detected only among food isolates. The most discriminatory typing protocol was MLVA, which grouped four isolates into two pairs, while the remaining isolates produced unique fingerprints. Similar results were obtained when taking into account the combination of the two RAPD protocols (Simpson index 0.999); six isolates were grouped into three pairs, two of which were the pairs that were identified also by MLVA. Single use of each RAPD protocol resulted in inferior discrimination (Simpson index 0.978 and 0.997, respectively). In conclusion, the two molecular procedures, MLVA, and the combined RAPD protocols, produced similar results, showing that L. monocytogenes isolates from clinical and food specimens were highly heterogenous and that clustering was very uncommon.     

Molecular typing of Mannheimia (Pasteurella) haemolytica serotype A1 isolates from cattle in Japan

Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) methods were applied for molecular typing of 130 Mannheimia (Pasteurella) haemolytica serotype A1 isolates obtained from 13 prefectures in Japan. These isolates were divided into 15 ApaI PFGE profiles that formed six distinct clusters (clusters A-F). Fifty-three (40.7%) isolates were classified in cluster B, and 20.0, 13.8, 12.3, 6.9 and 6.1% of isolates were in clusters E, A, F, D and C, respectively. The isolates of cluster B were differentiated into seven subtypes (B1-B7) and subtype B5 contained 63% (34/53) of isolates. RAPD revealed four banding patterns (types I-IV), and among 130 isolates 60.7% (79/130) of isolates were RAPD type I. All of the RAPD type I isolates were grouped into clusters A-C by PFGE. There was no relationship between molecular typing and geographic origin of these isolates. These results indicate that isolates of M. haemolytica A1 strain with various molecular profiles have already spread in Japan and may have caused sporadic infections.     

Molecular characterization of Listeria monocytogenes of the serotype 4b complex (4b, 4d, 4e) from two turkey processing plants

Most foodborne outbreaks of listeriosis have been found to involve a small number of closely related strains of Listeria monocytogenes serotype 4b. The ecology of these organisms and their reservoirs in nature or in the processing plant environment, however, remain poorly understood. Surveys of environmental samples from two turkey processing plants in the United States indicated presence of L. monocytogenes of the serotype 4b complex (serotype 4b and the closely related serotypes 4d and 4e). In addition, environmental and raw product samples from one plant repeatedly yielded isolates with genetic markers typical of two major serotype 4b epidemic clonal groups, ECI and ECII. The pulsed field gel electrophoresis (PFGE) profiles of these isolates, however, were clearly distinct from those of confirmed epidemic-associated strains. Furthermore, we observed minor but consistent differences in PFGE profiles of isolates that harbored ECI- or ECII-specific genetic markers, and that were obtained at different sampling times from the same plant. The findings suggest processing plant persistence (or repeated introductions) and genomic diversification of L. monocytogenes serotype 4b isolates that harbor ECI- or ECII-specific genetic markers. Such diversification would need to be taken into consideration in further efforts to elucidate the evolution and epidemiology of these organisms.     

Epidemiological study of invasive pulmonary aspergillosis in a haematology unit by molecular typing of environmental and patient isolates of Aspergillus fumigatus

In order to determine the possible relationship between environmental contamination by Aspergillus fumigatus and occurrence of invasive aspergillosis, a one-year prospective study was carried out in the haematology ward of Hautepierre Hospital, Strasbourg, France. During the study period, 21 environmental isolates and 26 clinical isolates of A. fumigatus were collected. Each was genotyped using a random amplification of polymorphic DNA (RAPD) technique. Thirty-four distinct profiles were identified by RAPD analysis, indicating the great genetic diversity of A. fumigatus isolated from infected patients and from the environment. For two patients, RAPD analysis demonstrated concurrent infection by at least two different strains. In two cases, a genetic similarity was noted between isolates obtained from a patient and from the environment.     

Irish-1 and Irish-2: UK epidemic meticillin-resistant Staphylococcus aureus strains associated with Northern Ireland

Since 1998, an increasing number of meticillin-resistant Staphylococcus aureus (MRSA) isolates with one of two characteristic phage patterns have been referred to the authors' laboratory from Northern Ireland. These strains were designated 'Irish-1' and 'Irish-2'. Analysis of 956 submitted isolates classified as Irish-1 or Irish-2 showed that 97% of the former and 95% of the latter were from Northern Ireland. Only 0.2% and 3%, respectively, were from England. Eleven Irish-2 isolates had been referred from Western Australia as representatives of an epidemic strain originally isolated there in 1994. Ninety isolates with the Irish-1 phage pattern and 91 isolates with the Irish-2 phage pattern, from numerous hospitals, were characterized by SmaI pulsed-field gel electrophoresis (PFGE), toxin gene carriage and antibiotic susceptibility. PFGE showed that, within each collection, a few isolates represented unrelated strains, but the majority were within six band differences of the most common profiles. Half of the Irish-1 isolates were homogeneous, with 22 DNA profiles among the remainder. Irish-2 isolates had two common profiles, D1 and D2, equally divided between one-third of the isolates and differing from each other by two bands; the remaining isolates shared 31 DNA profiles. Cluster analysis showed some overlap in DNA profiles between the Irish-1 and Irish-2 strains, but clear separation from other epidemic MRSA strains. There was no obvious correlation between PFGE profile and either antibiotic resistance pattern or toxin gene possession. All but three Irish-1 isolates possessed only the staphylococcal enterotoxin A (sea) gene, whereas almost all Irish-2 isolates were negative for all 12 enterotoxin genes. Sixty-nine percent of Irish-2 isolates were resistant to ciprofloxacin, erythromycin, kanamycin, neomycin and streptomycin, while 90% of Irish-1 isolates were resistant to all these plus gentamicin and mupirocin. All isolates were sensitive to quinupristin/dalfopristin, teicoplanin and vancomycin. Urease production was negative in both strains. The results suggest that Irish-1 and Irish-2 are distinct epidemic strains, identifiable by phage typing, DNA profiles, antibiotic resistance and toxin gene carriage.     

Molecular epidemiology for local outbreaks of methicillin resistantStaphylococcus aureus (MRSA)

Subtyping isolates may be useful for epidemiological studies of methicillin-resistant-Staphylococcus aureus (MRSA) outbreaks. Among subtyping methods, DNA-based techniques have been applied very effectively for this purpose. An outbreak of MRSA infections took place in one hospital in Barcelona early during 1991. From the beginning of the outbreak to December 92, 70 MRSA isolates from different patients and sources were collected. All strains were evaluated by restriction endonuclease analysis of plasmid DNA (REAP) and macrorestriction endonuclease analysis of genomic DNA usingSma I and pulsed-field-gel-electrophoresis (PFGE). Plasmid screening and REAP usingHind III demonstrated two plasmid subtypes: subtype A showing a large plasmid, and subtype B showing the same large plasmid plus a smaller one. Subtypes A and B corresponded to the more recent and older isolates, respectively, suggesting the loss of the small plasmid during the epidemic. PFGE usingSma I displayed two closely related profiles (PFGE subtype A and A'; CS=0.90). These subtypes were different from those subtypes exhibited from 4 methicillin-susceptible-Staphylococcusaureus (MSSA) isolates from the same hospital and from 2 epidemiologically unrelated MRSA isolates. Almost all isolates showing PFGE subtype A preceded those isolates showing PFGE subtype A'. This fact and the similarity between both subtypes suggested minor chromosomal DNA rearrangement during the outbreak from a unique strain. While PFGE usingSma I is a useful tool in evaluation of clonal dissemination, our data suggest epidemic or local outbreaks may need several methods to best delineate the source and spread of MRSA strains. The reproducibility and discriminatory power of REAP makes it a useful adjunct in this context.     

Discriminating between strains of Escherichia coli using pulsed-field gel electrophoresis and BOX-PCR

In this study, we evaluated two biomolecular techniques for discriminating between strains of Escherichia coli isolated form a variety of sources. The DNA of 211 strains of E. coli collected from dairy farms, calves, feces, pigs, primates, humans, and food products was analyzed by pulsed-field gel electrophoresis (PFGE) and repetitive-element polymerase chain reaction using the BOXA1 primer (BOX-PCR). Objectives of the present study were to compare PFGE and BOX-PCR for discriminating among strains of E. coli and investigate their capability in clustering E. coli strains according to the origin of bacterial isolation. Our results showed that PFGE and BOX-PCR were both able to distinguish closely related strains of E. coli; however, PFGE was able to discriminate between isolates indistinguishable by BOX-PCR and interpretation of PFGE data was easier. BOX-PCR proved to have good discrimination power, was less expensive, and could be performed in a PCR thermocycler. Neither of the methods used were effective in clustering E. coli strains according to the source of the organism.     

Genotypic analysis of Staphylococcus aureus from milk of dairy cows with mastitis in Argentina

Staphylococcus aureus is the most prevalent pathogen causing mastitis of dairy ruminants. This study was developed to ascertain the genotypes and genealogical relationship among strains isolated from milk of bovines with mastitis in Argentina. Molecular epidemiological analysis of S. aureus was performed on 112 isolates from 21 districts. Clonality was assessed by SmaI pulsed-field gel electrophoresis (PFGE) typing, automated EcoRI ribotyping and restriction enzyme analysis of plasmid (REAP) DNA profiles. A total of 22 band patterns distributed in four clusters were found by SmaI PFGE analysis. The similarity of clusters 2, 3 and 4 with cluster 1 was 0.73, 0.69 and 0.33, respectively, and 101 of 112 isolates belonged in cluster 1. PFGE band patterns from 42 isolates within cluster I were indistinguishable from each other (type A). The second largest group of isolates with indistinguishable PFGE band patterns was subtype A11, which was composed of 19 isolates. Automated ribotyping assigned the 112 isolates into 13 ribotypes. Among these, the most prevalent ribotypes I and VI were composed of 49 and 35 isolates respectively. Although there was certain correspondence between PFGE genotypes and ribotypes, further discrimination was achieved by combining both methods. REAP DNA profile analysis was useful to provide even further discrimination between isolates with identical PFGE genotype and ribotype. The most prevalent S. aureus strains A/I and A11/VI were widely distributed in the country and were not restricted to individual nearby locations. Prevalence of these two strains varied consecutively within a period of 8 years. Whether the shift in type prevalence was due to selection of a phenotypic trait remains undisclosed.     

弗氏柠檬酸杆菌随机扩增多态性DNA法基因分型

目的建立弗氏柠檬酸杆菌(Citrobacter freundii)随机扩增多态性DNA(RAPD)法基因分型图谱. 方法以优化的反应体系及单一随机引物L8,对临床分离的116株C.freundii进行RAPD分析,并按指纹图上DNA条带数及片段大小绘制基因分型图谱. 结果 116株临床分离C.freundii共得78种RAPD型. 结论 RAPD法可将C.freundii分型78种.     

通州区6类食品单增李斯特菌污染状况及分子流行病学特征调查研究

目的:了解通州区6类食品中单增李斯特菌的污染情况,探讨所污染菌株携带的三个毒力基因(hlyA、iap、prfA)的状况和DNA随机扩增多态性PCR分型情况。方法:依据国标方法,采用全自动微生物分析仪和API Listeria试剂条鉴定系统对样本进行单增李斯特菌分离和生化鉴定;采用聚合酶链反应检测实验菌株携带毒力因子的情况,使用随机引物PCR的方法对菌株进行分子分型。结果:从通州区共采集6类食品样品121件,检出单增李斯特菌18株,检出率为14.9%;18株实验菌株均携带单核细胞增多性李斯特菌特异的毒力因子;随机引物PCR方法将实验菌株分成6个随机引物PCR型别。结论:本区食品中单增李斯特菌污染较为严重,且毒力基因prfA、hly、iap同时存在,说明有潜在的致病能力,应引起相关部门关注;通州区在不同时间、不同地点分离的单增李斯特菌其PCR型别差别较大,有多种型别的细菌存在;也可根据PCR分型看出在食品加工过程中存在着内部污染问题。但来源不同的菌株之间PCR分型无关联,无流行趋势。     

A longitudinal analysis of methicillin-resistant Staphylococcus aureus in a Hong Kong teaching hospital.

OBJECTIVES: To review the incidence and trends of MRSA during a 12-year (1989-2000) period at a university teaching hospital and the relationship between strain distribution by antibiogram and molecular typing. DESIGN: Retrospective review of laboratory-based surveillance records on MRSA isolation and characterization of strains by antimicrobial susceptibility and PFGE. A patient episode was counted at the time when MRSA was first isolated. SETTING: A 1,350-bed university teaching hospital in Hong Kong. PATIENTS: Those with clinical isolates of MRSA. RESULTS: During 1989 to 2000, the hospital recorded 1,203,175 deaths and discharges (D&D) and encountered 5,707 patient episodes of new MRSA isolation. The overall incidence of patient episodes of MRSA was 0.47/100 D&D. In 1989, the incidence was 0.81/100 D&D and fell to a low of 0.33/100 D&D in 1995, but then rose to 0.50/100 D&D in 2000. Antibiogram and DNA typing identified 5 major types. PFGE type A constituted 68% (211/312) of isolates and was present throughout the 12-year period. PFGE type B constituted 13% (40/312) of isolates and was only present from 1995 to 2000. These isolates form a distinct clone and had unique antibiotic resistance profiles. CONCLUSIONS: The study showed the establishment of a dominant MRSA clone (PFGE type A group) in the intensive care, medical, and surgical units and the appearance of a new MRSA strain in 1995 (PFGE type B), which partly explained the rise in incidence of MRSA cases and a disproportionate rise in MRSA bacteremia from 1995 to 2000.