Cluster analysis of AP-PCR generated DNA fingerprints of Vibrio vulnificus isolates from patients fatally infected after consumption of raw oysters

Arbitrarily-primed-polymerase chain reaction (AP-PCR) DNA fingerprints were generated for 10 Vibrio vulnificus strains isolated from patients who became infected and died between 1993 and 1996 as a result of consuming raw oysters. Analysis of the DNA fingerprints with gel imaging and cluster analysis software revealed significant genetic heterogeneity among these strains, suggesting that V. vulnificus has a high degree of variation in its genomic organization, and that multiple pathogenic strains with greatly diverse genomic arrangements, rather than a single type of infective strain or serogroup, caused these infections.     

Physiological and Biochemical Characteristics of Iranian Strains of Xanthomonas axonopodis pv. citri, the Causal Agent of Citrus bacterial Canker Disease

Twenty-four strains of Xanthomonas axonopodis pv. citri ( Xac ), the causal agent of bacterial canker of citrus, isolated from Mexican lime ( Citrus aurantifolia ) and lemon ( Citrus limon ) in southern Iran, were characterized phenotypically. Strains were all pathogenic on C. aurantifolia . Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed slight differences in soluble protein profiles among the strains. Based on host range specificity and phenotypic characteristics, representative strains were differentiated into two groups of Asiatic (A) and atypical Asiatic (aA) forms. DNA fingerprinting analysis using Eco RI as the restriction endonuclease showed a negligible difference in restriction pattern between the two groups. On the basis of isozymic analysis, the two groups were distinct with respect to superoxide dismutase (SOD) and esterase (EST) banding patterns. Plasmid DNA profile analysis showed that the bacterial strains were different from each other in terms of plasmid number and molecular weight. Phage typing study revealed that most of group A strains were susceptible to Cp1 and/or Cp2 and some were resistant to both phage types including the strain in aA group. Bacteriocin production test indicated that there was a variation among Xac strains using different indicators for each bacteriocin producer. It is concluded that the Iranian strains of Xac are heterogeneous and constitute a subgroup(s) within the pathotype A.     

Diversity among Streptomyces Strains Causing Potato Scab

Eighty Streptomyces isolates, including 35 potato scab-inducing strains and 12 reference strains of Streptomyces scabies, were physiologically characterized by a total of 329 miniaturized tests. Overall similarities of all strains were determined by numerical taxonomy, with the unweighted average linkage (UPGMA) algorithm and simple matching (S(sm)) and Jaccard (S(j)) coefficients used as measures for similarity. Three cluster groups (A to C) were defined at a similarity level of 80.1% (S(sm)); these groups contained 14 clusters and 24 unclustered strains defined at a similarity level of 86.5% (S(sm)). Cluster group A contained strains phenotypically related to S. griseus or S. exfoliatus, whereas cluster group B contained strains which were phenotypically related to S. violaceus or S. rochei. The majority of the pathogenic isolates and reference strains were assigned to S. violaceus (57%) and S. griseus (22%). A DNA probe derived from the rRNA operon of S. coelicolor IMET 40271 was used to detect restriction fragment length polymorphisms (RELPs) among 40 pathogenic and nonpathogenic Streptomyces isolates. Southern blots revealed a high degree of diversity among the pathogenic strains tested. No significant correlation between numerical classification and RFLP grouping of Streptomyces strains could be revealed. The results obtained suggest that RFLP data are of minor importance in classification of Streptomyces species and that genes for pathogenicity determinants are spread among different Streptomyces species by mobilizable elements.     

Prevalence of Yersinia enterocolitica in pigs slaughtered in Chinese abattoirs

The distribution of Yersinia enterocolitica in slaughtered pigs in China was studied. A total of 8,773 samples were collected and examined from different pig abattoirs in 11 provinces from 2009 to 2011. Of these, 4,495 were oral-pharyngeal swab (tonsils) samples from pigs, 1,239 were from intestinal contents, and 3,039 were feces samples from abattoirs or local pigpens. The data showed that 1,132 strains were obtained, from which the isolation rate for Yersinia enterocolitica was 19.53% (878/4,495) from the tonsil samples, 7.51% (93/1,239) from intestinal contents, and 5.30% (161/3,039) from feces. Of the 850 pathogenic Yersinia strains, except for three of bioserotype 2/O:9 and three of bioserotype 4/O:3, most (844/850) were of bioserotype 3/O:3. Interestingly, pathogenic Y. enterocolitica accounted for the majority of the isolated strains from most provinces (85.17% to 100%), whereas from Heilongjiang, 96.52% (111/115) were classified as nonpathogenic biotype 1A with various serotypes, and only 3.48% of the strains (4/115) were pathogenic 3/O:3. All of the pathogenic strains were analyzed using pulsed-field gel electrophoresis (PFGE), and 49 patterns were obtained for the O:3 pathogenic strains; most of them were K6GN11C30021 (53.13%: 450/847) and K6GN11C30012 (21.37%: 181/847). Several strains from diarrhea patient samples revealed PFGE patterns identical to that from samples of local pigs, suggesting a possible link between porcine isolates and human infection. The results above suggested that Yersinia enterocolitica in slaughtered pigs from Chinese abattoirs was characterized by region-specific PFGE patterns and confirmed that strains isolated from pigs are closely related to those from human infections.     

G1 antigen: a cell-surface immunoprotective 96 kDa glycoprotein from the virulent fish pathogen Enterococcus seriolicida, its purification and characterization

Strains of the fish pathogen Enterococcus seriolicida were identified as agglutinating and non-agglutinating, according to their reaction with anti-serum raised against type strain YT-3 (ATCC49156). The non-agglutinating strains are highly pathogenic in contrast to agglutinating strains. A 96 kDa immunoprotective glycoprotein G1 antigen from non-agglutinating Ent. seriolicida strain SS91-014 (N) was purified and characterized. The purification procedure entailed extraction of antigen by glass bead agitation, 80% (NH4)(2)SO4 precipitation, gel filtration and electroelution. An immunofluorescence microscopy study using monoclonal antibody M3A5 raised against G1 antigen revealed that G1 antigen is present only on the cell surface of non-agglutinating strains. Therefore, the G1 antigen of virulent Ent. seriolicida could be a potential candidate for protective vaccine against enterococcosis in fish.     

Characterization of pathogenic and nonpathogenic strains of Xanthomonas axonopodis pv. manihotis by PCR-based DNA fingerprinting techniques

Strains of Xanthomonas axonopodis pv. manihotis (Xam) were characterized for pathogenicity and for DNA polymorphism using different PCR-based techniques. Using amplified restriction fragment length polymorphism (AFLP), strains were distinguished from each other and also from other Xanthomonas strains. Cluster analysis showed a high correlation between DNA polymorphism and pathogenicity. Four Xam strains were further analyzed using three PCR-based techniques, AFLP, AFLP-pthB and RAPD-pthB. Various primer combinations were used including primers specific to a Xam pathogenicity gene (pthB) along with RAPD or AFLP primers. The AFLP primer combinations EcoRI+T/MseI+A and EcoRI+T/MseI+T were the most efficient to discriminate among pathogenic and nonpathogenic Xam strains. Polymorphic bands were excised from the gel, amplified and cloned. Sequences analysis showed significant homology with bacterial pathogenicity island, genes involved in pathogenic fitness and regulators of virulence. Three cloned AFLP fragments were used as probes in DNA blot experiments and two of them showed significant polymorphism.     

Difference of Phenotype and Genotype Between Human and Environmental: Isolated Vibrio cholerae in Surabaya,Indonesia

Cholera due to Vibrio cholerae has been spreading worldwide, although the reports focusing on Indonesian V. cholerae are few. In this study, in order to investigate how V. cholerae transmitted to human from environment. We extended an epidemiological report that had investigated the genotype of V. cholerae isolated from human pediatric samples and environmental samples. We examined 44 strains of V. cholerae isolated from pediatric diarrhea patients and the environment such as shrimps or oysters collected in three adjacent towns in Surabaya, Indonesia. Susceptibilities were examined for 11 antibiotics. Serotype O1 or O139 genes and pathogenic genes including cholera toxin were detected. Multi-locus sequence typing (MLST) and enterobacterial repetitive intergenic consensus (ERIC)-PCR were also performed to determine genetic diversity of those isolates. Serotype O1 was seen in 17 strains (38.6%) with all pathogenic genes among 44 isolates. Other isolates were non-O1/non-O139 V. cholerae. Regarding antibiotic susceptibilities, those isolates from environmental samples showed resistance to ampicillin (11.4%), streptomycin (9.1%) and nalidixic acid (2.3%) but those isolates from pediatric stools showed no resistance to those 3 kinds of antibiotics. MLST revealed sequence type (ST) 69 in 17 strains (38.6%), ST198 in 3 strains (6.8%) and non-types in 24 strains (54.5%). All the ST69 strains were classified to O1 type with more than 95% similarity by ERIC-PCR, including all 6 (13.6%) isolates from environmental samples with resistance to streptomycin. In conclusion, V. cholerae O1 ST69 strains has been clonally spreading in Surabaya, exhibiting pathogenic factors and antibiotic resistance to streptomycin, especially in the isolates from environment.     

Genetic relatedness amongst intestinal spirochaetes isolated from rate and brids.

Multilocus enzyme electrophoresis was used to determine genetic relationships amongst 32 intestinal sprrochaetes ( Serpulina spp.) isolated from rats (17), rheas (7), chickens (4), ducks (2), a swan (1) and a flamingo (1). The strains were divided into 20 electrophoretic types (ETs), with a mean genetic diversity per locus of 0.62. The results were compared with those previously published for procine intestinal spirochaetes. One strain from a healthy rat, and three rhea strains which were recovered from cases of necrotizing typhlitis, were grouped in the same ETs as certain procine strains of Serpulina hydysenteriae . The rhea strains could be differentiated from these by pulsed-field gel electrophoresis. Fifteen of the rat strains could be differentiated from these by pulsed-field gel electrophoresis. Fifteen of the rat strains were genetically and phenotypically closely related. In contrast the avian strains were genetically more heterogeneous, with pathogenic isolates located in three different genetic groups.     

Virulence of Streptococcus pneumoniae strains belonging to different serovars

The study of the virulence of 352 S. pneumoniae strains isolated from patients with inflammatory lung diseases revealed that their virulence depended, to a certain extent, on the state of the polysaccharide capsule of streptococci, as among 299 typed cultures 38.1% were virulent, while out of 53 nontypable strains of these bacteria only 8 strains (15.1%) proved to be highly pathogenic for white mice and all S. pneumoniae R-forms proved to be avirulent. All 11 S. pneumoniae strains under study belonging to serovars 1 and 2 and 87% of the cultures belonging to serovar 3, isolated from patients with inflammatory lung diseases in Leningrad, were highly virulent. The characteristic feature of S. pneumoniae cultures of other serotypes was their wide spectrum of pathogenicity. S. pneumoniae cultures isolated from the spinal fluid of patients with pneumococcal meningitis also differed in their pathogenicity levels but strains highly pathogenic for mice prevailed.     

Genetic relatedness of a non-motile variant O157 enteropathogenic Escherichia coli (EPEC) strain and E. coli strains belonging to pathogenic related groups

The study was undertaken to determine the clonal relationship and the genetic diversity among Escherichia coli isolates by comparing a non-motile O157 variant with three O157:H7 EHEC isolates and one O55:H7 enteropathogenic E. coli (EPEC) strain. E. coli strains were characterized by sorbitol phenotype, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, random amplification polymorphic DNA, and the presence of specific virulence genes (stx, E-hly and LEE genes). Sorbitol fermentation was observed in O157:H- (strain 116I), O55:H7 and O157:H7 (strain GC148) serotypes. stx1 or stx2 and E-hly genes were only detected among O157:H7 isolates. LEE typing revealed specific allele distribution: eaegamma, tirgamma, espAgamma, espBgamma associated with EPEC O55:H7 and EHEC O157:H7 strains (B1/1 and EDL 933), eaealpha, tiralpha, espAalpha, espBalpha related to the 116I O157:H- strain and the GC148 strain presented non-typable LEE sequences. Multilocus enzyme profiles revealed two main clusters associated with specific LEE pathotypes. E. coli strains were discriminated by random amplification of polymorphic DNA-polymerase chain reaction and pulsed-field gel electrophoresis methodologies. The molecular approaches used in this study allowed the determination of the genetic relatedness among E. coli strains as well as the detection of lineage specific group markers.     

Relationship between phylogenetic groups, genotypic clusters, and virulence gene profiles of Escherichia coli strains from diverse human and animal sources

Escherichia coli strains in water may originate from various sources, including humans, farm and wild animals, waterfowl, and pets. However, potential human health hazards associated with E. coli strains present in various animal hosts are not well known. In this study, E. coli strains from diverse human and animal sources in Minnesota and western Wisconsin were analyzed for the presence of genes coding for virulence factors by using multiplex PCR and biochemical reactions. Of the 1,531 isolates examined, 31 (2%) were found to be Shiga toxin-producing E. coli (STEC) strains. The majority of these strains, which were initially isolated from the ruminants sheep, goats, and deer, carried the stx(1c) and/or stx(2d), ehxA, and saa genes and belonged to E. coli phylogenetic group B1, indicating that they most likely do not cause severe human diseases. All the STEC strains, however, lacked eae. In contrast, 26 (1.7%) of the E. coli isolates examined were found to be potential enteropathogenic E. coli (EPEC) strains and consisted of several intimin subtypes that were distributed among various human and animal hosts. The EPEC strains belonged to all four phylogenetic groups examined, suggesting that EPEC strains were relatively widespread in terms of host animals and genetic background. Atypical EPEC strains, which carried an EPEC adherence factor plasmid, were identified among E. coli strains from humans and deer. DNA fingerprint analyses, done using the horizontal, fluorophore-enhanced repetitive-element, palindromic PCR technique, indicated that the STEC, potential EPEC, and non-STEC ehxA-positive E. coli strains were genotypically distinct and clustered independently. However, some of the potential EPEC isolates were genotypically indistinguishable from nonpathogenic E. coli strains. Our results revealed that potential human health hazards associated with pathogenic E. coli strains varied among the animal hosts that we examined and that some animal species may harbor a greater number of potential pathogenic strains than other animal species.     

Chromosome sizes and phylogenetic relationships between serotypes of Actinobacillus pleuropneumoniae

The genome size of Actinobacillus pleuropneumoniae was determined by pulsed field gel electrophoresis of AscI and ApaI digested chromosomal DNA. The genome size of the type strain 4074^T (serotype 1) was determined to be 2404±40 kb. The chromosome sizes for the reference strains of the other serotypes range between 2.3 and 2.4 Mb. The restriction pattern profiles of AscI, ApaI and NheI digested chromosomes showed a high degree of polymorphism among the different serotype reference strains and allowed their discrimination. The analysis of the macrorestriction pattern polymorphism revealed phylogenetic relationships between the different serotype reference strains which reflect to some extent groups of serotypes known to cross-react serologically. In addition, different pulsed fields gel electrophoresis patterns also revealed heterogeneity in the chromosomal structure among different field strains of serotypes 1, 5a, and 5b, while strains of serotype 9 originating from most distant geographical places showed homogeneous ApaI patterns in pulsed field gel electrophoresis.     

Two-dimensional analysis of exoproteins of methicillin-resistant Staphylococcus aureus (MRSA) for possible epidemiological applications

We applied two-dimensional gel electrophoresis (2-DE) to the total exoproteins secreted from pathogenic MRSA strains and identified major protein spots by N-terminal amino acid sequence analysis. In approximately 300 to 500 spots visualized on each gel, various exoproteins and cell-associated proteins were identified and their sites on the gels confirmed for construction of a reference map. Major exotoxins such as enterotoxins SEA, SEB, and SEC,, toxic shock syndrome toxin-1 (TSST-1), and hemolysins were distributed in the region of pI 6.8 to 8.1 and MW 21 to 35 kDa. Although the differences between calculated and observed values of pI and MW were relatively small in each exoprotein, those of several proteins including alpha-hemolysin and SEB were considerably deviated from the positions of the expected values. Some exoproteins were detected as multiple spots. These included beta-hemolysin, enterotoxins SEA, SEB, and SEC3, glutamic acid-specific endopeptidase, glycerophosphoryl diester phosphodiesterase and triacylglycerol lipase. The multiple spots of these exoproteins may be generated by the action of own proteases. Certain similarities of 2-DE patterns among strains belonging to the same coagulase types were observed. On the basis of 2-DE image analysis, coagulase type II strains secreted somewhat larger amounts of SEB and SEC3 as well as TSST-1 than the strains belonging to other coagulase types. Taken together, 2-DE analysis of exoproteins is applicable to epidemiological studies for MRSA, as compared with pulsed field gel electrophoresis of restricted chromosomal DNA.     

Emergence of uropathogenic extended-spectrum beta lactamases-producing Escherichia coli strains in the community

The aim of this study was to determine the virulence characteristics and resistance pattern of the extended-spectrum/lactamases (ESBLs)-producing Escherichia coli strains isolated from urine of outpatients in the Zagreb region during a five-month period, and to compare them with the non ESBLs-producing E. coli strains isolated in the same period. Out of 2451 E. coli strains isolated from urine of nonhospitalized patients with significant bacteriuria, a total of 39 ESBLs-producing strains (1.59%) were detected by a double-disk diffusion technique and by the broth-dilution minimal inhibitory concentration reduction method. The 45 non ESBLs-producing strains were randomly chosen, and phenotype of the two groups of strains was characterized and compared. Serogroup O4, hemolysin production, expression of P- and type 1 fimbriae as well as resistance to gentamicin and amikacin were significantly more prevalent characteristics among the ESBLs-producing strains than among non ESBLs-producing strains (p < 0.01), while higher prevalence of trimethoprim-sulfamethoxazole resistance among ESBLs-producing strains was not statistically significant (p > 0.05). Chromosomal DNA analysis by pulsed-field gel electrophoresis exhibited a great genomic similarity among ESBLs-producing strains and revealed that those highly virulent and resistant E. coli strains isolated from urine of outpatients in the Zagreb region had a clonal propagation.     

Molecular and physiological differentiation between pathogenic and nonpathogenic Acanthamoeba

In this study, 14 isolates of Acanthamoeba from both clinical and environmental sources belonging to seven different species were assayed for tolerance of high osmotic pressure, temperature tolerance, extracellular proteases, and cytopathic effects (CPE) on immortalized rabbit corneal epithelial cells. On the basis of the results, amoeba isolates were divided into pathogenic and nonpathogenic groups. Ribosomal DNA sequencing was performed on these isolates. Phylogenetic relationships revealed that all the pathogenic strains tested clustered together as one group, while nonpathogenic strains clustered into other groups. Sequence comparisons with previously published sequences determined that among the six new pathogenic isolates used in this study, five belong to T4 genotype and one to T11. This is the first report of a T11 genotype being found in Acanthamoeba keratitis. Received: 1 November 2001 / Accepted: 31 December 2001     

Epidemiological study of Vibrio cholerae using variable number of tandem repeats

By conventional genetic methods, including pulse-field gel electrophoresis and multilocus sequence typing, most pathogenic, cholera toxin-positive O1 and O139 isolates of Vibrio cholerae cannot be distinguished. We evaluated relationships among 173 V. cholerae isolates collected between 1992 and 2007 from different geographic areas in India by analyzing five variable number of tandem repeat (VNTR) loci. Each VNTR locus was highly variable, with between 5 and 19 alleles. eburst analysis revealed four large groups of genetically related isolates. Two groups contained genotypes of isolates with the O139 serogroup (which emerged for the first time in epidemic form in 1992), with the other two groups containing O1 strains. In subsequent analysis, it was possible to track the spread of specific genotypes across time and space. Our data highlight the utility of the methodology as an epidemiologic tool for assessing spread of isolates in both epidemic and endemic settings.     

Detection of a large linear plasmid in Borrelia spielmanii isolate

Borrelia spielmanii belongs to human pathogenic species within the Borrelia burgdorferi sensu lato complex in Europe, which is a causative agent of Lyme disease. So far, the human disease caused by B. spielmanii has been associated with skin manifestations. The aim of the study was to analyze 4 human B. spielmanii isolates by pulsed-field gel electrophoresis and to localize genes of 3 important Borrelia proteins: OspA, DbpA, and VlsE. The analysis revealed variation within linear plasmid profiles among the strains; isolate PSigII contained a large plasmid of 100 kb compared with a 50 kb plasmid present in the 3 other B. spielmanii isolates, all carried the genes ospA and dbpA. Differences in the size of linear plasmids among the Borrelia strains may be a result of host-pathogen interactions, as the PSigII strain was the only strain of the 4 tested strains to be isolated from a patient with a previous history of Lyme disease, whereas 3 other patients were diagnosed with this disease for the first time.     

Comparative Genomics to Delineate Pathogenic Potential in Non-O157 Shiga Toxin-Producing Escherichia coli (STEC) from Patients with and without Haemolytic Uremic Syndrome (HUS) in Norway

Shiga toxin-producing Escherichia coli (STEC) cause infections in humans ranging from asymptomatic carriage to bloody diarrhoea and haemolytic uremic syndrome (HUS). Here we present whole genome comparison of Norwegian non-O157 STEC strains with the aim to distinguish between strains with the potential to cause HUS and less virulent strains. Whole genome sequencing and comparisons were performed across 95 non-O157 STEC strains. Twenty-three of these were classified as HUS-associated, including strains from patients with HUS (n = 19) and persons with an epidemiological link to a HUS-case (n = 4). Genomic comparison revealed considerable heterogeneity in gene content across the 95 STEC strains. A clear difference in gene profile was observed between strains with and without the Locus of Enterocyte Effacement (LEE) pathogenicity island. Phylogenetic analysis of the core genome showed high degree of diversity among the STEC strains, but all HUS-associated STEC strains were distributed in two distinct clusters within phylogroup B1. However, non-HUS strains were also found in these clusters. A number of accessory genes were found to be significantly overrepresented among HUS-associated STEC, but none of them were unique to this group of strains, suggesting that different sets of genes may contribute to the pathogenic potential in different phylogenetic STEC lineages. In this study we were not able to clearly distinguish between HUS-associated and non-HUS non-O157 STEC by extensive genome comparisons. Our results indicate that STECs from different phylogenetic backgrounds have independently acquired virulence genes that determine pathogenic potential, and that the content of such genes is overlapping between HUS-associated and non-HUS strains.     

Characterization of Listeria monocytogenes isolates from food animal clinical cases: PFGE pattern similarity to strains from human listeriosis cases

Twenty-one isolates of Listeria monocytogenes from food animal clinical cases that involved meningitis or meningoencephalitis, encephalitis, mastitis and abortion were characterized by serotyping and pulsed-field gel electrophoresis (PFGE) in order to improve our understanding of the genetic links between individual strains and strains recovered from human listeriosis cases. Results showed that five of the isolates were serotype 1/2a, six were 1/2b, nine were 4b, and one was untypeable. A caprine, two bovine and an ovine brain isolate shared identical PFGE patterns indicating that strains of L. monocytogenes are not host specific. Other isolates exhibited distinct patterns that were not shared, indicating a genetic diversity. Dendrogram analysis revealed that PFGE patterns of the isolates clustered primarily according to serotype. We compared the PFGE types obtained for these isolates with PFGE types for human clinical isolates present in the CDC national PulseNet database. Six (29%) of the twenty-one strains had patterns that were indistinguishable from pathogenic human isolates in the database. Our observations offer preliminary evidence that food animals could be significant reservoirs of L. monocytogenes that lead to human infections and support the inclusion of PFGE patterns of veterinary clinical isolates in the national PulseNet database for increased surveillance.