Pulsed-field Gel Electrophoresis Method Research Articles

The Bryopsis hypnoides Plastid Genome: Multimeric Forms and Complete Nucleotide Sequence

Background Bryopsis hypnoides Lamouroux is a siphonous green alga, and its extruded protoplasm can aggregate spontaneously in seawater and develop into mature individuals. The chloroplast of B. hypnoides is the biggest organelle in the cell and shows strong autonomy. To better understand this organelle, we sequenced and analyzed the chloroplast genome of this green alga.Principal FindingsA total of 111 functional genes, including 69 potential protein-coding genes, 5 ribosomal RNA genes, and 37 tRNA genes were identified. The genome size (153,429 bp), arrangement, and inverted-repeat (IR)-lacking structure of the B. hypnoides chloroplast DNA (cpDNA) closely resembles that of Chlorella vulgaris. Furthermore, our cytogenomic investigations using pulsed-field gel electrophoresis (PFGE) and southern blotting methods showed that the B. hypnoides cpDNA had multimeric forms, including monomer, dimer, trimer, tetramer, and even higher multimers, which is similar to the higher order organization observed previously for higher plant cpDNA. The relative amounts of the four multimeric cpDNA forms were estimated to be about 1, 1/2, 1/4, and 1/8 based on molecular hybridization analysis. Phylogenetic analyses based on a concatenated alignment of chloroplast protein sequences suggested that B. hypnoides is sister to all Chlorophyceae and this placement received moderate support.ConclusionAll of the results suggest that the autonomy of the chloroplasts of B. hypnoides has little to do with the size and gene content of the cpDNA, and the IR-lacking structure of the chloroplasts indirectly demonstrated that the multimeric molecules might result from the random cleavage and fusion of replication intermediates instead of recombinational events.

Comparison of Acinetobacter baumannii multidrugs resistant Isolates obtained from French and Tunisian hospitals

Objectives: The aim of this study was to assess whether there are differences between the clustering of isolates collected between 2003 and 2005 from two different hospitals in Tunisia and France. Methods: A selection of 62 isolates of A. baumannii was studied; 31 from the French Hospital (Poitiers) and 31 from Tunisian Hospital (Rabta). Antibiograms were done using the disc diffusion method. The presence of integrons class 1 and 2 was studied by PCR. Molecular relationship was studied by Random Amplified Polymorphic DNA (RAPD) and Pulsed Field Gel Electrophoresis (PFGE). The sequence typing of adeB gene was determined to identify intraspecific groups. Results: The present study successfully focuses to compare epidemiologic status between two hospitals study. PFGE and RAPD methods were useful to distinguish epidemic and endemic clones in the two collections study. Sequence analysis of an 850-bp internal fragment drug efflux gene adeB revealed 9 novel sequence types (STs). Conclusion: We, statistically, found no significant difference related to the epidemiological situation. This study showed that different genetic types of A.baumannii were found in the two collection strains. Epidemics ones were essentially confined in the ICU and were persisting during the two years study. However, more control procedures had to be used on these clones for the Tunisian hospital.

Distribution and genetic diversity of lactic acid bacteria from traditional fermented sausage

This study was conducted to investigate the distribution and genetic diversity of the native lactic acid bacteria (LAB) population in nem chua, a popular traditional Vietnamese uncooked fermented sausage. A total of 74 LAB isolates were identified and their molecular fingerprints were obtained using repetitive-PCR (rep-PCR) and pulse-field gel electrophoresis (PFGE). The results revealed that the majority of LAB isolates were Lactobacillus plantarum (67.6%), followed by Pediococcus pentosaceus (21.6%). A minority of LAB (9.5%) were Lactobacillus brevis and Lactobacillus farciminis (1.4%). A large genetic plasticity within the same species was also observed. Both rep-PCR and PFGE methods were found to be acceptable regarding reproducibility and reliability. However, this study demonstrated the higher discriminatory power of PFGE compared to rep-PCR, as observed by the higher number of clusters generated (17 and 12 clusters of L. plantarum respectively, seven and six clusters of P. pentosaceus respectively and four and two clusters of L. brevis respectively), and lower percentage of similarity among clusters in PFGE data analysis. These results also revealed that there was not a single LAB strain that was found to predominate in the product. To our knowledge, this is the first detailed analysis of the native LAB population in nem chua. As this traditional sausage is fermented naturally or only with back-slop, knowledge about the native LAB population in the product is very important to understand the microflora that has been active in fermenting the nem chua and to ensure the safety of this uncooked sausage.

Prevalence and characterization ofSalmonella Gallinarum in the chicken in Korea during 2000 to 2008

Salmonella enterica serovar Gallinarum (Salmonella Gallinarum) is the causative agent of fowl typhoid (FT), a severe systemic disease of chickens that results in high mortality. Since 1992, FT has become one of the most serious bacterial diseases in poultry in Korea. The purpose of this study was to investigate the prevalence of FT during 2000 to 2008 and characterize the phenotype and genetic diversity of Salmonella Gallinarum isolates before and after adopting national-wide vaccination with a live attenuated strain (SG 9R) in 2001. For 9 yr, a total of 983 farms were affected with FT outbreaks, and after reaching a peak in 2002 (206 farms affected), the prevalence of the disease gradually declined, whereas magnitude sales of the vaccine increased every year. According to the analysis based on the chicken breeds (n=521 farms), the incidence of FT in commercial broilers, Baeksemi (a mixed breed of male meat-type breeder and female commercial layer), commercial layers, native chickens, and broiler breeders was 47.7, 28.4, 17.2, 5.1, and 1.3%, respectively. Of the affected broilers, over 90% birds were under 2 wk of age, indicating it was possible that they were infected with Salmonella Gallinarum via vertical transmission. In the phenotypic analysis, Salmonella Gallinarum strains (n=142) isolated during 2001 to 2007 showed the same pattern in the majority of the biochemical tests such as carbohydrate fermentation and amino acid decarboxylation. Interestingly, all of the strains could not ferment rhamnose, but SG 9R could, making rhamnose a potential biomarker to distinguish the vaccine strain. In analyzing the genetic diversity by the pulsed field gel electrophoresis method with XbaI digestion, we examined a total 68 isolates of Salmonella Gallinarum obtained in 5 provinces in Korea (2001 to 2007). The pulsed field gel electrophoresis profiling produced 7 different patterns, but isolates within the same group did not show clear geographic or breed relationships. In conclusion, there were close epidemiological linkages between prevalence of FT and the sales volume of the live FT vaccine in Korea. In addition, a low phenotypic and genetic heterogeneity was observed among Korean Salmonella Gallinarum isolates and the live vaccine strain (SG 9R).

Molecular analyses of Salmonella serotype Typhi and Salmonella serotype Paratyphi B strains isolated in Turkey

To investigate the characteristics of Salmonella serotype Typhi and Salmonella serotype Paratyphi B, which still remain significant in Turkey. Materials and methods: Eighteen S. Typhi and 42 S. Paratyphi B strains isolated from clinical samples were investigated by plasmid profile analysis, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), and pulsed field gel electrophoresis (PFGE) methods. Results: Plasmid profile analysis showed that 10 out of 18 (56%) S. Typhi isolates harbored 1 to 3 plasmids, and 11 out of 42 (26.2%) S. Paratyphi B isolates harbored 1 to 4 plasmids. PFGE performed with restriction enzyme AvrII grouped both S. Typhi and S. Paratyphi B isolates into 14 clusters; with restriction enzyme XbaI, S. Typhi and S. Paratyphi B isolates were grouped into 13 and 12 clusters, respectively. Conclusion: These results showed that plasmid profile analysis and PFGE are reliable and discriminative methods complementing the phenotypic characters of S. Typhi and S. Paratyphi B isolates, and may contribute to the epidemiologic investigation of typhoid or paratyphoid diseases. The study presented here is the first report on S. Typhi and S. Paratyphi B isolates from Turkey investigated by plasmid profile analysis, ERIC-PCR, and PFGE methods.

Isolation of Salmonella enterica in laying-hen flocks and assessment of eggshell contamination in France.

The present investigation was conducted in conjunction with the European Union baseline study for the estimation of Salmonella prevalence in laying-hen flocks. It aimed at evaluating eggshell contamination in farms positive for Salmonella, characterizing the genetic patterns of Salmonella strains and identifying the factors associated with Salmonella contamination of eggshells. For this purpose, a total of 4,200 eggs were collected from 28 positive flocks and analyzed according to draft Annex D of International Organization for Standardization Method 6579. Molecular characterization of the Salmonella strains was obtained by the pulsed-field gel electrophoresis method with two restriction enzymes, XbaI and BlnI. The relationship between the presence of Salmonella on eggshells and rearing practices was studied by using multiple correspondence analysis. Results showed that 39.3% of the positive flocks had at least one positive eggshell, with a total of 1.05% of eggshells testing positive for Salmonella. We detected the same serovars on samples taken from the farm and from eggshells within a given flock, with isolates sharing the same genetic pattern in 7 of 11 flocks. Eggshells tested positive for Salmonella in flocks (i) located where delivery trucks pass near air entrances of the poultry house, (ii) with high holding capacity (>30,000 laying hens), and (iii) with more than five positive samples coming from the farm environment, as well as in cases of flocks with a maximum egg-laying rate of >96% and in cases where farmers worked in other animal production. This study provided valuable information that could be used for risk management and risk assessment studies.

Molecular Investigation of <i>Salmonella</i> Choleraesuis and <i>Salmonella</i> Hadar Strains Isolated from Humans in Turkey

Eleven Salmonella Choleraesuis and seven Salmonella Hadar strains isolated from various clinical humand samples were investigated by plasmid profile analysis, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and pulsed-field gel electrophoresis (PFGE) in order to obtain information at a molecular level on the epidemiology of S. Choleraesuis and S. Hadar, which are significantly present in Turkey. Plasmid profile analysis showed that 10 (90.9%) of 11 S. Choleraesuis isolates harbored one to two plasmids with sizes of 2.0, 5.0 or 6.5 kb; and 5 (71.4%) of 7 S. Hadar isolates harbored one to three plasmids ranging from 2.5 to 70 kb. ERIC-PCR was performed using ERIC-2 primers; since isolates within each serotype showed similar band models, we concluded that ERIC-PCR is not suitable for differentiating isolates within the same serotype and for grouping into clusters. In PFGE using the AvrII enzyme, S. Choleraesuis isolates formed three clusters, and S. Hadar isolates formed three clusters; using the XbaI enzyme, S. Choleraesuis isolates formed two clusters, and S. Hadar isolates formed four clusters. These results showed that plasmid profile analysis and PFGE are reliable and discriminative methods that would complement antibiograms, and could contribute to the investigation of outbreak epidemiology. This is the first report on S. Choleraesuis and S. Hadar isolates from Turkey investigated by plasmid profile analysis, ERIC-PCR and PFGE methods.

Differentiation of strains identified asBifidobacterium animalissubsp.lactis

Six strains (Bi11, Bi30, Bi36, Bi50, Bi52 and Bi55) isolated from bio-yoghurts and two strains (KD10 and KD11) derived from human faeces were identified by genus- and species specific polymerase chain reaction (PCR) with reference to the type strains of B. animalis subsp. lactis DSM 10140 and B. animalis subsp. animalis DSM 20104. The isolates were differentiated by using Bcu I ( Spe I), Xba I and Dra I endonucleases for subsequent pulsed field gel electrophoresis (PFGE) technique and by API 50 CHL tests.All the isolates tested were classified to B. animalis subsp. lactis species. The reliable identification as B. animalis subsp. lactis (by PCR with Bflact2/Bflact5 primers), however, required confirmation by a negative result of B. animalis subsp. animalis -specific PCR.Differentiation of the B. animalis subsp. lactis isolates with PFGE method enabled to distinguish KD11 strain with all the restriction enzymes applied, and Bi11 and Bi30 — exclusively with Dra I and Spe I enzymes, respectively. The biochem...

Genotypes, Antibiogram, and Pulsed-Field Gel Electrophoresis Profiles of Escherichia Coli Strains from Piglets in Korea

Adherence factors and enterotoxins are major virulence factors of enterotoxigenic Escherichia coli (ETEC). Antibiotics have been used frequently for the treatment and prevention of ETEC infection in piggeries worldwide, including Korea. Therefore, data on both virulence profiles and antibiotic resistance patterns are useful in the epidemiological study of ETEC. A total number of 198 E. coli field isolates were examined. The most prevalent pathotype was F1, followed by a combination of F1 and EAST1. All of the 71 isolates were resistant to more than 2 antibiotics used in a disk diffusion test, and 87.94% of the isolates were found to be resistant to more than 4 antibiotics. Investigations were also conducted to correlate the virulence gene profiles with antibiogram and pulsed-field gel electrophoresis (PFGE). Although a high degree of polymorphism was noted among strains having the same virulence patterns, the highest similarity pattern was observed carrying the same virulence profiles and similar antibiogram. Thus, investigation of both virulence profiles and antibiogram is essential to the epidemiological study of ETEC. Moreover, the PFGE method might be applicable as a tool to reveal genetic relatedness among E. coli strains from piggeries in Korea.

Genetic characterization of Streptococcus phocae strains isolated from Atlantic salmon, Salmo salar L., in Chile

Streptococcus phocae is a beta-haemolytic bacterium frequently involved in disease outbreaks in seals causing pneumonia or respiratory infection. Since 1999, this pathogen has been isolated from diseased Atlantic salmon, Salmo salar, causing serious economic losses in the salmon industry in Chile. In this study, we used different molecular typing methods, such as pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), repetitive extragenic palindromic PCR (REP-PCR) and restriction of 16S-23S rDNA intergenic spacer regions to evaluate the genetic diversity in S. phocae. Thirty-four strains isolated in different years were analysed. The S. phocae type strain ATCC 51973(T) was included for comparative purposes. The results demonstrated genetic homogeneity within the S. phocae strains isolated in Chile over several years, suggesting the existence of clonal relationships among S. phocae isolated from Atlantic salmon. The type strain ATCC 51973(T) presented a different genetic pattern with the PFGE, RAPD, ERIC-PCR and REP-PCR methods. However, the fingerprint patterns of two seal isolates were distinct from those of the type strain.

Application of pulsed-field gel electrophoresis for the discrimination of leptospiral isolates in Brazil

Leptospirosis is a public health problem worldwide. Traditionally, microscopic agglutination test (MAT) and cross-agglutinin absorption test (CAAT) are used to identify leptospires. However, these techniques are laborious and time-consuming, requiring the maintenance of a collection of more than 200 reference strains and correspondent rabbit antisera. The purpose of this study was to evaluate the pulsed-field gel electrophoresis (PFGE) method for discrimination of Leptospira serovars. Fourteen clinical isolates of Leptospira spp. were analysed by MAT before being characterized by PFGE. The isolates were compared with a library of 206 different reference Leptospira serovars. All the isolates gave clear profiles with high resolution. PFGE and MAT results were in agreement for all clinical isolates evaluated. Twelve isolates were classified as serovar Icterohaemorrhagiae/Copenhageni by PFGE. By MAT, these isolates were classified as serogroup Icterohaemorrhagiae with titres ranging from 3200 to 25 600. Two isolates were classified as serovar Canicola by PFGE, and as serogroup Canicola by MAT with titres higher than 3200. PFGE offers the advantages of simple, reliable and reproducible results. PFGE provides a convenient tool for the identification of clinical isolates.

Association of the shuffling of Streptococcus pyogenes clones and the fluctuation of scarlet fever cases between 2000 and 2006 in central Taiwan

BackgroundThe number of scarlet fever occurrences reported between 2000 and 2006 fluctuated considerably in central Taiwan and throughout the nation. Isolates of Streptococcus pyogenes were collected from scarlet fever patients in central Taiwan and were characterized by emm sequencing and a standardized pulsed-field gel electrophoresis (PFGE) method. National weekly report data were collected for investigating epidemiological trends.ResultsA total of 23 emm types were identified in 1,218 S. pyogenes isolates. The five most prevalent emm types were emm12 (50.4%), emm4 (23.2%), emm1 (16.4%), emm6 (3.8%) and emm22 (3.0%). PFGE analysis with SmaI suggested that, with a few exceptions, strains with a common emm type belonged to the same clone. There were two large emm12 clones, one with DNA resistant to cleavage by SmaI. Each prevalent emm clone had major PFGE strain(s) and many minor strains. Most of the minor strains emerged in the population and disappeared soon after. Even some major strains remained prevalent for only 2–3 years before declining. The large fluctuation of scarlet fever cases between 2000 and 2006 was associated with the shuffling of six prevalent emm clones. In 2003, the dramatic drop in scarlet fever cases in central Taiwan and throughout the whole country was associated with the occurrence of a severe acute respiratory syndrome (SARS) outbreak that occurred between late-February and mid-June in Taiwan.ConclusionThe occurrences of scarlet fever in central Taiwan in 2000–2006 were primarily caused by five emm types, which accounted for 96.8% of the isolates collected. Most of the S. pyogenes strains (as defined by PFGE genotypes) emerged and lasted for only a few years. The fluctuation in the number of scarlet fever cases during the seven years can be primarily attributed to the shuffling of six prevalent emm clones and to the SARS outbreak in 2003.

A molecular survey on virulence associated genotypes of non-O1 non-O139 Vibrio cholerae in aquatic environment of Tehran, Iran

Thirty-seven Vibrio cholerae strains were isolated from surface water sources at 5 different locations in Tehran, Iran during 2006 and were identified as non-O1 and non-O139 isolates. PCR for SXT element and class 1 integron was positive for 19% and 5.4% of isolates, respectively. PCR for virulence associated-genes within the vibrio pathogenicity island (VPI) gene cluster showed the presence of LJ, int and RJ in 8, 59 and 30% of the isolates, respectively. None of the V. cholerae isolates contained the toxin encoding genes ( ace, zot, ctx) in the CTX genetic element. Biochemical fingerprinting using PhPlate system (PhP-RV) was able to type all strains and resulted in 8 common types (containing 78% of the isolates) and 8 single types (22%). Out of 37 isolates, only 26 isolates were typeable with pulsed-field gel electrophoresis (PFGE) producing banding patterns. The results presented in this study showed no genotyping correlation between the V. cholerae isolated from surface water and the clinical setting which had been reported previously by this laboratory. Furthermore, combination of PFGE and PhP-RV methods was proved beneficial for non-typeable V. cholerae isolates.

Comparison ofStreptococcus mutansstrains from children with caries-active, caries-free and gingivitis clinical diagnosis by pulsed-field gel electrophoresis

A study was conducted to compare the DNA structure of Streptococcus mutans strains in children with caries-active, caries-free, and gingivitis clinical diagnosis. Twenty-eight Streptococcus mutans strains from 100 children's plaques were examined by pulsed-field gel electrophoresis (PFGE) method. The classified strains were closely related to one another, though the strains originated from different disease groups. Three identical pairs were found, but the pairs in two cases belonged to different disease groups. The results of the PFGE experiments suggest that there is no correlation between the different DNA patterns ofS. mutans strains and their cariogenecity. So the different DNA strains ofS. mutans are not the only determining factor in the development of dental caries.

USE OF PHAGE TYPING COMBINED WITH PULSED-FIELD GEL ELECTROPHORESIS AND POLYERASE CHAIN REACTION IN CHARACTERIZATION OF HUMAN SALMONELLA ENTERITIDIS ISOLATES

Phage typing (PT) combined with pulsed-field gel electrophoresis (PFGE) and Polymerase chain reaction (PCR) analysis methods, using 3 kinds of primers ( ERIC, SERE, RAPD) was used in order to characterize human isolates of Salmonella Enteritidis strains. Thirty-six epidemiologically unrelated isolates from outbreaks of foodborne gastroenteritis were studied. Analysis with Xba I, Bln I and Spe I enzymes showed that Xba I gave the highest index of discrimination among strains. Molecular typing by PCR methods was not able to differentiate the isolates belonging to different phage types. We identified eight phage types, with a predominance of PT 8 (42% of isolates). Three PFGE pattern types were obtained with Xba I restriction endonuclease enzyme among the unrelated isolates. The establishment of a highly reliable and discriminatory method for epidemiologic analysis is necessary in order to trace the sources of specific pathogens and, consequently, to control and prevent the spread of epidemic Salmonella Enteritidis isolates to humans. It is concluded that PFGE, in combination with phage typing, represents a suitable tool for the epidemiological typing of Salmonella Enteritidis strains which could be used for investigations or surveillance of the international spread of these clones

Usefulness of mec-associated direct repeat unit (dru) typing in the epidemiological analysis of highly clonal methicillin-resistant Staphylococcus aureus in Scotland

The incidence of the epidemic methicillin-resistant Staphylococcus aureus (EMRSA) strains EMRSA-15 and EMRSA-16 in Scotland has increased dramatically, now accounting for c. 70% and c. 20% of isolates, respectively. Epidemiological tracking of these EMRSA strains is difficult, as c. 50% of EMRSA-15 and c. 35% of EMRSA-16 isolates are indistinguishable using pulsed-field gel electrophoresis (PFGE) and other typing methods. The usefulness of mec-associated direct repeat unit (dru) sequence analysis as a more sensitive approach to tracking the persistence and spread of these 'clonal' EMRSA strains in Scotland was evaluated. Analysis of 47 EMRSA-15 and 57 EMRSA-16 isolates (including two separately cultured isolates of the Harmony collection type strain) obtained from 22 hospital laboratories over an 8-year period (1997-2005) revealed 13 and 12 different dru types, respectively. Whereas some types appeared to be endemic in multiple hospitals, subtypes that may represent specific strain movement among hospitals in a given geographical region were identified in other instances. These results suggest that mec-associated dru typing may have potential for identifying and tracking specific subtypes of otherwise indistinguishable epidemic MRSA isolates such as those in Scotland.

Comparison of pulsed-field gel electrophoresis and three rep-PCR methods for evaluating the genetic relatedness ofStenotrophomonas maltophiliaisolates

In this study, three facile repetitive-sequence PCR (rep-PCR) techniques have been compared with the pulsed-field gel electrophoresis (PFGE) method for differentiating the genetic relatedness of clinical Stenotrophomonas maltophilia isolates. The dendrograms of 20 S. maltophilia isolates were constructed based on the data obtained from PFGE and three PCR-based methods, i.e. enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), BOX-PCR and repetitive extragenic palindromic-PCR (REP-PCR). When compared with PFGE, ERIC-PCR displayed a much lower discriminatory power, whereas BOX-PCR and REP-PCR had a comparable discriminatory power for close genetic-related isolates. BOX-PCR and REP-PCR can be convenient and effective methods for evaluating the close genetic relatedness of clinical S. maltophilia isolates. A rapid method for determining S. maltophilia's close genetic relatedness provides a convenient tool for understanding the epidemiology of S. maltophilia.

Molecular typing of staphylococcal communities isolated during municipal solid waste composting process

This study investigates the molecular typing occurrence of staphylococcal communities during composting process of municipal solid waste. One hundred staphylococcal strains were isolated during the composting process and analysed either by phenotypic and genotypic methods. Amplified ribosomal DNA restriction analysis (ARDRA), internal transcripted spacer-polymerase chain reaction (ITS-PCR) and pulsed-field gel electrophoresis (PFGE) were used as molecular tools. During the digestion phase of composting process three staphylococcal species were recovered:Staphylococcus xylosus (48%),Staphylococcus lentus (40%), andStaphylococcus hominis (12%). In the maturation phase in addition to the latter species four species were also recovered at low frequencies:Staphylococcus lugdunensis, Staphylococcus haemolyticus, Staphylococcus warneri andStaphylococcus sciuri. Therefore, the composition of staphylococcal communities was temperature-, and phase dependant. The molecular methods showed that the ARDRA was not able to differentiate between strains of the same species. However, the ITS-PCR and the PFGE methods allowed interspecies and intraspecies discrimination, respectively.

Molecular Differentiation of Mycoplasma gallisepticum and Mycoplasma imitans Strains by Pulsed-Field Gel Electrophoresis and Random Amplified Polymorphic DNA

Pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis were used to compare 21 Mycoplasma gallisepticum strains and five M. imitans strains. Each strain of M. gallisepticum typed by PFGE and RAPD methods was genetically quite unique and RAPD and PFGE fingerprinting enabled strain characterization. Relationships between the M. gallisepticum and M. imitans strains were established and dendrograms were drawn from PFGE and RAPD patterns. PFGE group A and RAPD group D were significantly associated with M. imitans strains (P < 0.05). Three M. imitans strains shared the same PFGE and RAPD patterns. The two M. gallisepticum vaccine strains had singular PFGE and RAPD patterns. Thus, PFGE and RAPD can be used to investigate disease outbreaks in vaccinated flocks or for epidemiological tracking. For M. gallisepticum, the RAPD and PFGE discriminatory powers were superior to 0.95 and the in vitro, in ovo and in vivo reproducibility of RAPD and PFGE was 100%. The RAPD drawback was the inconsistent band intensity complicating the interpretation of patterns, while the PFGE limit was its low typeability (86%). Thus, these two molecular typing methods seemed complementary for M. gallisepticum epidemiological studies.

Environmental Vibrio Cholerae O139 May Be the Progenitor of Outbreak of Cholera in Coastal Area of Orissa, Eastern India, 2000: Molecular Evidence

Cholera has been reported in the state of Orissa, India during the past decades. An outbreak of diarrheal disease occurred during November 1 to November 9, 2000 in Rusipada village near Puri, which was inhabited by a population of approximately 560. During the outbreak, Vibrio cholerae O139 strains were isolated from clinical specimens collected from patients with acute diarrhea admitted to the infectious diseases (ID) hospital in Puri and from environmental samples collected from multiple bodies of water in the village. The index case with acute diarrhea was a 60-year-old female resident of Rusipada who had not visited any known outbreak-related areas, including an outbreak 1 week prior to her symptom onset in a nearby village. All the isolated strains were positive for ctxA, tcpA, ace, and zot genes, produced cholera toxin, and exhibited a similar antibiogram pattern. Comparison of DNA fingerprinting analysis by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) method and dendrogram constructed from RAPD revealed that genetic homogeneity exist between the clinical and environmental O139 strains. Epidemic and endemic cholera is a major public health problem in many developing countries and continues to be an important cause of morbidity in many areas of Asia, Africa, and Latin America. Among more than 200 serogroups of V. cholerae so far identified, 1 only O1 and recently identified O139 serogroup 2–5 are capable of causing epidemic cholera. It is now widely accepted that O139 Bengal like O1 and non-1 and non-O139 Vibrio may survive better in aquatic environments 6–8 and environmental water is the reservoir for infectious V. cholerae. 7,9 In this report we investigated the clonal relationship between strains of V. cholerae collected both from stool specimens from persons with acute diarrhea and from environmental sources of water. During the outbreak of diarrhea, 23 non-randomly selected rectal swabs were collected from the hospitalized diarrhea patients in ID hospital, Puri in Cary-Blair Transport (CBT, DIFCO,USA) medium and transported to the Microbiology Department of Regional Medical Research Centre (RMRC), Bhubaneswar and bacteriologically analyzed following standard technique. 10 Subsequently after the isolation of V. cholerae O139 as the etiological agent of the outbreak, 20 water samples non-randomly selected were collected from water bodies situated at various distances in that village to determine the source of contamination following a previous method. 11 Presumptive identification of 10 and 6 V. cholerae were isolated from rectal swabs and water samples respectively and the strains were agglutinated with monoclonal O139 antiserum supplied by NICED, Kolkata, India and were confirmed to belong to V. cholerae serogroup O139. To investigate the similarities of clinical and environmental strains of V cholerae O139, drug susceptibility test, cholera toxin assay, detection of virulent genes by polymerase chain reaction (PCR) assay, RAPD finger printing assay, and PFGE were performed. A monosialoganglioside (GM1) enzyme-linked immunosorbent assay (ELISA) was used to examine cholera toxin production in V. cholerae O139 strains by the method Svennerholm and Holmgren. 12 Drug susceptibility test was performed following the method described elsewhere, 13 with the antibiotics (Hi-media Laboratories, Bombay, India) ampicillin (A, 10 g), chloramphenicol (C, 30 g), co-trimoxazole (Co, 25 g), ciprofloxacin (Cf, 5 g), furazolidone (fz, 100 g), gentamicin (G, 10 g), neomycin (N, 30 g), nalidixic acid (Na, 30 g), norfloxacin (Nx, 10 g), streptomycin (S, 10 g) and tetracycline (T, 30 g). Characterization of strains as susceptible or resistant was based on size of the inhibition zone around each disc according to manufacturer’s instructions, which matched interpretive criteria recommended by the WHO. 14 Strains showing an intermediate zone of inhibition were interpreted as resistant to that drug on the basis of previous MIC studies conducted with V. cholerae. 15