Application of ribosomal RNA gene restriction patterns analysis and pulsed-field gel electrophoresis in distinguishing Salmonella weltevreden isolates in Malaysia.

A representative sample of 20 isolates of Salmonella weltevreden strains from stool cultures of patients admitted at the University Hospital, Kuala Lumpur, Malaysia were analyzed. All the strains were susceptible to ampicillin, ceftriaxone, ciprofloxacin, chloramphenicol, tetracycline, trimethoprim, gentamicin and co-trimoxazole. Ribosomal RNA gene restriction pattern analysis of PstI-digested DNA gave three ribotypes while pulsed-field gel electrophoresis (PFGE) analysis of XbaI-digested DNA gave ten distinct profiles. PFGE was more discriminative than ribotyping in distinguishing the strains. The majority of the strains analyzed were very closely related with similarity coefficient values ranging from 0.8 to 1.0. Both PFGE and ribotyping could distinguish one of the strains which was obtained from a patient following a bone marrow transplant for beta-thalassemia major, indicating that this particular strain was unrelated to the rest of the strains from patients with acute gastroenteritis.     

Clonal heterogeneity of clinical isolates of vancomycin-resistant Enterococcus faecium with unique vanS

Objective To examine the clonal diversity of vancomycin‐resistant enterococci (VRE). Methods A total of 900 clinical isolates of enterococci were obtained, and VRE isolates were subjected to antimicrobial susceptibility tests, biochemical fingerprinting with the PhPlate system (PhP), ribotyping and pulsed‐field gel electrophoresis (PFGE) typing. Results Forty‐nine of all enterococcal isolates were resistant to high levels of vancomycin (MIC ≥ 128) and identified as Enterococcus faecium. Biochemical fingerprinting with PhP showed that the VRE isolates were highly diverse (diversity index, D_i = 0.93) and belonged to 24 PhP‐types. The VRE could be separated into 34 and 27 types with PFGE and ribotyping, giving diversity indices of 0.98 and 0.97, respectively. The PFGE method was more discriminatory than ribotyping and PhP system for E. faecium isolates. A combination of either of the two typing methods resulted in at least 44 types. Furthermore, sequencing analysis of vanS of Tn1546 showed one nucleotide mutation (C→A) at position 5727 in comparison with the prototype BM4147, which was found to be unique in all Iranian VRE isolates. Conclusion The isolated clinical VRE strains were highly diverse in Tehran.     

Phylogeny of Brucella abortus strains isolated in the Russian Federation

Objective: To study Brucella(B.) abortus strains isolated in the Russian Federation, in order to identify their detailed position in the phylogenetic structure of the species global population as well as to determine genetic relationships for isolates from different geographical areas.Methods: Based on Bayesian method, the whole genome singlenucleotide polymorphism(SNP) analysis of 258 B. abortus strains from different geographical areas of the world including 20 B. abortus strains isolated in Russia was carried out. Results: The core genome SNP analysis of the B. abortus isolates allowed describing the main genetic lineages. The Russian strains entered two separate clades, including the basal branch and the C1 branch that is widely spread in Eurasia. The data on the isolation time was used for the dating of phylogenetic tree, and also the estimated time frame for the B. abortus genotype diversification was determined. There were sets of specific SNPs identified, which defined each of the genotypes and sub-genotypes.Conclusions: A significant genetic diversity of the brucellosis pathogen strains from Russia has been proven. The sets of unique specific SNPs described in our study may become one of the elements within a bio-informational analysis algorithm to be used for epidemiological study of brucellosis outbreaks, including those caused by new(atypical) genetic variants of B. abortus.     

广西犬种布鲁氏菌病流行病学调查研究

1985年在广西自治区的38个市县进行了犬种布鲁氏菌病的调查,用犬种抗原试管凝集试验对2278只家犬做了血清学检测,查出33个市县为犬种布病疫区,感染率13.7％。从19个市县分离出犬种布鲁氏菌64株,以城市郊区和平原地区的病原检出率较高,山地和丘陵地区次之。     

脉冲场凝胶电泳用于副溶血性弧菌的分子流行病学研究

目的运用脉冲场凝胶电泳技术和荧光PCR技术对成都市2008-2009年分离的副溶血性弧菌进行分子分型和毒素基因检测,分析感染来源。方法利用PCR检测副溶血性弧菌分离株耐热直接溶血素(tdh)基因和耐热相关溶血素(trh)基因。用脉冲场凝胶电泳(PFGE)对菌株进行分型,所得结果用BioNumerics V 4.0软件UPGMA方法进行聚类分析。结果所试38株分离菌tdh基因阳性有31株,2株为trh阳性。以SfiⅠ酶切后PFGE分型,38株菌被分成了24个带型,大多数暴发有各自优势型别,其中有两起暴发优势型别一致;环境分离株菌型分散。结论多起暴发事件分离株之间PFGE型别差异大,成都市副溶血性弧菌暴发的感染来源复杂。     

Molecular tracking of the lineage of strains of Vibrio cholerae O1 biotype El Tor associated with a cholera outbreak in Andaman and Nicobar Islands, India

A large outbreak of acute watery diarrhoea involving all age groups of mongoloid tribal aborigines occurred during October-November, 2002 in the Nancowry group of Andaman and Nicobar Islands in the Indian Ocean. Twenty-one of the 67 stool samples from 67 patients were positive for toxigenic Vibrio cholerae O1, serotype Ogawa biotype El Tor, which showed striking similarity in its antibiogram with some of the strains of V. cholerae O1 Serotype Ogawa biotype El Tor isolated in Kolkata. The Nancowry and Kolkata isolates were compared with molecular tools involving random amplified polymorphic DNA (RAPD) fingerprinting, ribotyping and pulsed-field gel electrophoresis (PFGE). RAPD fingerprinting and ribotyping techniques revealed that all the V. cholerae strains associated with the outbreak in these islands were clonal in nature and identical to a population of isolates obtained from Kolkata since 1993. PFGE could discriminate within these Kolkata isolates further and established that a particular subtype of this population reached the remote Nancowry islands and was responsible for the outbreak.     

Molecular characterization of enterohemorrhagic Escherichia coli O157:H7 isolates dispersed across Japan by pulsed-field gel electrophoresis and multiple-locus variable-number tandem repeat analysis

We identified seven distinct subtypes of enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were derived from sporadic cases and outbreaks from multiple prefectures in Japan in 2005. A surveillance system utilizing pulsed-field gel electrophoresis (PFGE), PulseNet Japan, was used. Some strains showed indistinguishable PFGE patterns using another restriction enzyme (BlnI or SpeI) in each subtype of EHEC O157:H7 isolates that were routinely subtyped by the XbaI PFGE pattern. In order to examine the genotypic relatedness of these strains, we carried out a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA). By using the MLVA system, we found that three of seven subtypes of EHEC O157:H7 strains that were isolated from sporadic cases dispersed across multiple prefectures within a few months showed indistinguishable PFGE patterns and identical MLVA types. Strains belonging to the other four subtypes of EHEC O157:H7 in the PFGE analysis were further classified into different clusters of EHEC O157:H7. Therefore, compared to PFGE, MLVA showed greater discriminatory power with respect to analysis of the isolates in this study.     

Brucella canis. A cause of undulant fever.

Brucella canis, recognized in 1967 as a cause of canine abortion, is an uncommonly recognized human pathogen. Our patient, a 48 year old man, probably acquired his infection by contact with an infected dog. His clinical course was characterized by intermittent fever and bacteremia over a 4 month interval. His course, which expands the clinical spectrum of human infections due to Br. canis, is discussed in the context of the other reported instances of community and laboratory acquired cases.     

Molecular epidemiological analysis of Salmonella enterica serotype Derby infections in Hong Kong

OBJECTIVES: We aimed to study the antimicrobial susceptibilities and molecular epidemiology of Salmonella enterica serotype Derby, a unique and common salmonella serotype in Hong Kong. METHODS: Salmonella Derby strains isolated from stools of patients in a large general hospital in Hong Kong from 1989 to 1994 and from food samples isolated in the Public Health Laboratory were randomly selected and investigated for the antimicrobial susceptibilities by determining the minimal inhibitory concentrations of 19 antimicrobial agents and their relatedness using plasmid analysis, ribotyping, pulsed-field gel electrophoresis (PFGE) and total DNA fingerprinting. RESULTS: About 50% of the 127 isolates studied were susceptible to all the 19 antibiotics tested, although resistance to tetracycline (49%) and sulfamethoxazole (38%) was high. Only 12% did not harbour any detectable plasmids, while the rest contained plasmids in 51 profiles. There were two predominant clones, one comprising of 35% of isolates that could not be pulsotyped because discrete bands were not discernible after PFGE and another comprising 34% of isolates that could be pulsotyped. The remaining 31% belonged to a variety of types. CONCLUSIONS: Approximately 70% of S. Derby belonging to two clones were endemic in the community, while the remaining isolates belonged to a variety of types which were probably a result of sporadic infection. The sources of human infections were foods, since most isolates from foods also belonged to the two endemic clones. Typing of S. Derby isolates from other sources such as animals or the environment would help elucidate how foods were contaminated. PFGE might not be universally applicable to all salmonella strains.     

Babesia DNA detection in canine blood and Dermacentor reticulatus ticks in southwestern Siberia, Russia

Babesia infection was studied in 21 blood samples of dogs with symptoms of babesiosis and among 72 Dermacentor reticulatus and 70 Ixodes persulcatus ticks from southwestern Siberia, Russia. Babesia DNA was detected by hemi-nested PCR based on the 18S rRNA gene with subsequent direct sequencing. All of the analyzed canine blood samples and three D. reticulatus, but none from I. persulcatus ticks studied were shown to contain Babesia DNA. Nucleotide sequences of the Babesia 18S rRNA gene fragment of 354 bp long for all 24 positive samples appeared to belong to the subspecies Babesia canis canis and differed only at three positions. The Babesia nucleotide sequences from 17 canine blood samples and from one D. reticulatus tick were identical to each other and to previously known B. canis canis from canine blood in Slovenia. Four canine blood samples and the second tick sample contained a mixture of two nucleotide sequences previously found in canine blood. B. canis canis nucleotide sequence from the third tick differed in the unique nucleotide transition and could correspond to a new genetic variant. Thus, the main etiological agent of canine babesiosis in Novosibirsk region is B. canis canis, and D. reticulatus, but not I. persulcatus, ticks could serve as a vector of this infectious agent. To our knowledge, this is the first report of the B. canis canis nucleotide sequences from ticks.     

Molecular epidemiological analysis of an outbreak of Campylobacter jejuni using restriction enzyme double-digestion technique for genotyping of isolates by pulsed-field gel electrophoresis

In an outbreak of gastroenteritis in elementary school students and their families in Chiba Prefecture, Japan, Campylobacter jejuni was isolated from the stools of 14 patients who developed diarrheal illness after a one-day bus trip. C. jejuni was also isolated from the stools of 3 patients not going on the bus trip. Pulsed-field gel electrophoresis (PFGE) analysis was done on 17 isolates of C. jejuni to study genetic relationships among them. PFGE profiles of isolates treated with restriction enzymes Sma I, Ksp I and Kpn I were separated into 9, 10, and 10 types, but the relationship between PFGE profiles and epidemiological profiles was unclear. Dendrograms of PFGE of isolates double-digested with both Sma I and Ksp I were typed into D1, D2, D3 and D4, and profiles compared to profiles of serotyping and flagellin typing of isolates and epidemiological profiles to evaluate genetical and epidemiological relationships. Thirteen isolates of PFGE type D1 possessed serotype G and flagellin type Al and were isolated from patients going on the bus trip. Type D2 isolated from a student going on the bus trip and type D3 isolates from two students not going on the bus trip had serotype B and flagellin type A2. C. jejuni of PFGE type D4, serotype UT, and flagellin type A3 was also isolated from a student not going on the trip. Our results show that at least two outbreaks of C. jejuni occurred simultaneously in people related to the school. Restriction enzyme double-digestion PFGE was thus useful in the molecular epidemiological analysis of the C. jejuni outbreak.     

Pasteurella multocida pneumonia with molecular evidence of zoonotic transmission

We report the case of a patient with pneumonia caused by Pasteurella multocida (P. multocida). An 83-year-old woman admitted for bronchiectasis, productive cough, and a high fever was found in chest radiography on admission to have a new infiltrative shadow in the left lower lung field. Chest computed tomography indicated bilateral bronchiectasis and an infiltrative lingular shadow. P. multocida was isolated from sputum samples. Biapenem and minocycline therapy ameliorated symptoms. Pulsed-field gel electrophoresis (PFGE) showed that the clinical isolate recovered was identical to the P. multocida isolate recovered from the pharyngeal swab specimen culture obtained from the patient's dog. Several years later, P. multocida subsp. gallicida isolates were recovered from human sputum and a canine pharyngeal swab specimen. PFGE confirmed these isolates to be identical and PFGE band patterns were identical to those of previous P. multocida isolates. These findings suggest that P. multocida was transmitted from the patient's dog to the patient and underscore the need to inform susceptible hosts at risk of contracting zoonotic agents about basic hygiene rules for keeping pets. PFGE data strengthened the evidence of zoonotic transmission of P. multocida through casual contact in a patient with a respiratory infection caused by this organism. PFGE should prove useful in both epidemiological tracing and preventing pasteurellosis and clarifying its pathology. Case reports of respiratory infections including pneumonia caused by P. multocida are increasing in humans, and P. multocida respiratory infections appear to occur mostly in patients with chronic respiratory disease. A history of pet exposure, even without bites or scratches, should alert the physician to P. multocida as a potential respiratory pathogen.     

A serological diagnostic survey for Brucella canis infection in Turkish patients with Brucellosis-like symptoms

The incidence of Brucella canis infection in humans is unknown in Turkey. In this study, we investigated the prevalence of B. canis infection in human sera obtained from six regions in Turkey and comparatively evaluated the results obtained by agglutination-based techniques using standardized antigens made from B. canis. The patients (n = 1,746) presented with clinical symptoms that were similar to those of brucellosis. All patients who tested negative in the Rose Bengal test for the smooth Brucella strains (abortus, melitensis, and suis) were screened for evidence of B. canis infection using the rapid slide agglutination test (RSAT), the microagglutination test (MAT), and the 2-mercaptoethanol RSAT test (2ME-RSAT). Of the samples tested, 157 (8.9%), 68 (3.8%), and 66 (3.7%) were positive for B. canis, as determined by RSAT, MAT, and 2ME-RSAT, respectively. The diagnostic sensitivity, specificity, positive predictive value, and negative predictive value of RSAT were 100%, 94.6%, 42%, and 100%, respectively, and of MAT were 100%, 99.9%, 97%, and 100%, respectively. We recommend the routine use of MAT and 2ME-RSAT to check the sera of all patients with symptoms of brucellosis who are negative for brucellosis using a smooth Brucella antigen.     

Usefulness of pulsed-field gel electrophoresis in tracking two outbreaks of invasive meningococcal disease serogroup C in British Columbia

Two major outbreaks of invasive meningococcal disease serogroup C (IMD-C) were identified in British Columbia between 2000 and 2004. Pulsed-field gel electrophoresis (PFGE) and porA gene sequencing of all retained IMD-C isolates were used to assess correlations between genotypes and epidemiological patterns. PFGE patterns of IMD-C genotypes correlated with epidemiological patterns between 2000 and 2004 in British Columbia, and demonstrated that PFGE can identify outbreak-related cases. Both IMD-C outbreaks correlated with a respective PFGE pattern. PFGE analysis demonstrated that the 2004 British Columbia outbreak strain in men who have sex with men was closely related to the 2001 Abbotsford outbreak strain. PorA sequencing data indicated low diversity of class 1 outer membrane proteins in British Columbia, and did not correlate with epidemiological trends. There was a trend for outbreak-associated PFGE types to demonstrate higher case fatality rates.     

Clonal diversity of Salmonella enterica serotype Typhi isolated from patients with typhoid fever in Tehran

In this study, antimicrobial susceptibility test and genetic typing were used to characterize 15 Salmonella enterica serotype Typhi (S. Typhi) isolates recovered from sporadic cases of typhoid fever in Tehran, Iran during 2004. Antimicrobial susceptibility test showed that all isolates were susceptible to 20 antimicrobials examined in this study. Analysis of insertion elements showed that 2 IS200 types with 10 and 11 copies were present. 11 of the 15 isolates were found to possess 10 IS200 elements residing on fragments from 23 to 2.3 kb. Comparison of the RiboPrinter (automated ribotyping) patterns of S. Typhi showed that 60% (9/15) of the isolates belonged to a single ribotype. PCR based random amplified polymorphic DNA analysis (RAPD) and enterobacterial repetitive intergenic consensus (ERIC) and pulsed-field gel electrophresis (PFGE) were also performed. ERIC and RAPD-PCR method showed 2 and 3 genotyping patterns amongst the isolates, respectively. The PFGE typing was carried out by using XbaI restriction enzyme, and 7 restriction patterns were observed. Overall, the molecular typing methods applied in this study showed that the isolated S. Typhi populations were highly polyclonal as shown by PFGE.     

Evidence for transmission of pertussis in schools, Massachusetts, 1996: epidemiologic data supported by pulsed-field gel electrophoresis studies

In 1996, 18 of 20 pertussis outbreaks reported in Massachusetts occurred in schools. Pertussis surveillance data were reviewed and a retrospective cohort study was conducted in a high school that experienced an outbreak. Bordetella pertussis isolates from 9 school cases and from 58 cases statewide were examined by use of pulsed-field gel electrophoresis (PFGE). Statewide incidence rates were highest among children aged <1 year, 10-14 years, and 15-19 years (106, 117, and 104 cases per 100,000, respectively). Among 34 confirmed and 20 probable cases at the school, 61% had cough onset within 8 weeks of school opening. Five different PFGE types were identified among the 58 B. pertussis isolates from throughout the state. All 9 isolates from the affected high school were the same PFGE type. School-aged children may play an important role in pertussis epidemics. Consideration should be given to use of acellular pertussis vaccines among school-aged children.     

我国山丘疫区与平原疫区利什曼虫分离株分子核型分析

目的：分析我国山丘疫区和平原疫区利什曼原虫分离株分子核型。方法：脉冲场电泳。采用１％琼脂糖凝胶，电压６Ｖ／ｃｍ，０．５×ＴＢＥ，１４℃，脉冲时间６０ｓ，电泳１５ｈ和脉冲时间９０ｓ，电泳９ｈ。结果：各个分离株均分离出分子量范围在２００ｋｂ－２２００ｋｂ的ＥＢ染色条带为１５条左右，其中６个山丘疫区分离株之间的核型相似，且犬 株与人株的亦相似，并与Ｌ．ｉｎｆａｎｔｕｍ的核型接近；２个平原疫区分离株之间的核型相似；山丘疫区与平原疫区分离株之间的核型不同。结论：山丘疫区分离株之间和平原疫区分离株之间均各自存在着同源性，山丘疫区分离株与Ｌ．ｉｎｆａｎｔｕｍ之间存有部分同源性，但两个疫区分离株之间存在着异源性；保虫宿主——家犬，是我国山丘疫区内脏利什曼病的重要传染源。     

从北京某地分离的2株犬种布氏菌的鉴定报告

目的对从北京某地分离的2株布氏菌进行鉴定。方法布氏菌常规鉴定分型方法,布氏菌噬菌体裂解试验,同时辅以脉冲场电泳基因分型方法。结果2株待测菌株均为犬种布氏菌,2株待测菌的脉冲场电泳图谱与国际标准犬种布氏菌RM6/66株的图谱完全一致。结论采用传统布氏菌分型方法同时辅以脉冲场电泳基因分型方法对布氏菌进行分型鉴定,可以增加鉴定工作的稳定性与正确性。     

Stability of strain genotypes of Burkholderia pseudomallei from patients with single and recurrent episodes of melioidosis

The constancy of strain genotypes of multiple isolates of Burkholderia pseudomallei from 13 patients with melioidosis was examined by Bam HI ribotyping and pulsed-field gel electrophoresis (PFGE) of Xba I digests of DNA. Seven of 8 patients with single episodes of melioidosis each yielded genetically identical isolates and only one of five patients with recurrent episodes was infected with a new strain clearly distinct from the original primary strain. Variation was observed in PFGE patterns of primary and relapse isolates of another patient but this was insufficient to define genetically distinct strains. We conclude that most patients with single or multiple episodes of melioidosis retain a single strain.     

First report of an unclassified Demodex mite causing demodicosis in a Venezuelan dog

For the first time in Venezuela, a case of canine demodicosis apparently caused by a simultaneous infection with an unclassified Demodex and Demodex canis has been observed. The most severely affected area of the dog involved, a 3-month-old male Doberman Pinscher, was the head, particularly the peri-orbital skin. Skin samples of the affected areas were taken and evaluated by both light and scanning electron microscopy. The mites of the unclassified Demodex were smaller than those of D. canis and appeared relatively stout. In the skin scrapings, D. canis outnumbered the unclassified Demodex by a ratio of 30:1, making it unclear if the unclassified Demodex is pathogenic to dogs. A combination of superficial and deep skin scrapings, from a suspected case of demodicosis, may help to reveal other infestations of the unclassified Demodex that would otherwise be masked by concurrent infections with D. canis.