DNA polymorphisms in strains of Legionella pneumophila serogroups 3 and 4 detected by macrorestriction analysis and their use for epidemiological investigation of nosocomial legionellosis.

Genomic DNAs of clinical and environmental isolates of Legionella pneumophila belonging to serogroups 3 and 4 were analyzed by macrorestriction analysis by pulsed-field gel electrophoresis. The restriction enzymes SfiI and NotI allowed easy visual separation of epidemiologically unrelated serogroup 3 strains. Three unrelated serogroup 3 strains that were isolated from different locations were identical by this genome mapping technique. Five unrelated serogroup 4 strains were separable by this technique. The electrophoretic patterns obtained after SfiI or NotI cleavage of the DNA of strains isolated from four patients with hospital-acquired legionellosis were identical to the patterns of strains isolated from the hot water supply systems of the buildings in which the patients were hospitalized. In conclusion, macrorestriction analysis is a valuable tool for epidemiological studies of infections caused by L. pneumophila serogroups 3 and 4.     

Molecular fingerprinting of Salmonella serotype Glostrup

Thirteen isolates of Salmonella serotype Glostrup (antigenic formula, 6.8:z10:e,n,z15) from various sources and countries were analysed by ribotyping and IS200 fingerprinting. Both methods provided a high index of strain discrimination by allowing detection of three ribotypes and eight IS200 fingerprints which, though generally related, were readily distinguishable. The findings of this analysis confirm the usefulness of ribotyping and IS200 fingerprinting for studying the epidemiology of rarely isolated salmonellae of serogroup C.     

Distribution of legionellae in a hospital water system: prevalence of immunologically and genetically related Legionella pneumophila serogroup 6 isolates

A hospital warm water system was monitored for the presence and distribution of legionellae. Subtyping of ten selected Legionella pneumophila isolates, originating from four different sites in the system by using serogroup specific antisera in an indirect immunofluorescence test, revealed that nine of the ten isolates belong to serogroup 6, while the remaining one was serogroup 10. Two monoclonal antibodies (mAbs) specific for a subgroup of serogroup 6 strains were further used for characterization. None of the strains reacted with these mAbs. Genome analysis by elaborating NotI profiles using the pulsed field gel electrophoresis (PFGE) technique revealed that nearly all serogroup 6 isolates derived from different sites, including a new building connected by a ring pipe, were identical according to restriction fragment patterns. The patterns were distinguishable from those of the two L. pneumophila serogroup 6 reference strains, and from that of the L. pneumophila serogroup 10 isolate. These data argue for a relatively homogeneous L. pneumophila serogroup 6 population in the entire water system.     

Application of PCR ribotyping and tDNA-PCR for Klebsiella pneumoniae identification

PCR analysis of 16S-23S internal transcribed spacer (PCR ribotyping) and tRNA intergenic spacer (tDNA-PCR) were evaluated for their effectiveness in identification of clinical strains of Klebsiella pneumoniae and differentiation with related species. For this purpose both methods were applied to forty-three clinical isolates biochemically identified as K. pneumoniae subsp. pneumoniae isolated from patients clinical specimens attended at five hospitals in three Brazilian cities. References strains of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, K. oxytoca, K. planticola and Enterobacter aerogenes were also analyzed. Both PCR methods showed specific patterns for each species. A conserved PCR ribotype pattern was observed for all clinical K. pneumoniae isolates, while differing from other related analyzed species. tDNA-PCR revealed five distinct patterns among the K. pneumoniae clinical isolates studied, demonstrating a predominant group with 90.6% of isolates presenting the same pattern of K. pneumoniae type strain. Both PCR-based methods were not able to differentiate K. pneumoniae subspecies. On the basis of the results obtained, both methods were efficient to differentiate the Klebsiella species analyzed, as well as E. aerogenes. Meanwhile tDNA-PCR revealed different tRNA arrangements in K. pneumoniae, suggesting intra-species heterogeneity of their genome organization, the polymorphism of the intergenic spacers between 16S and 23S rRNA genes appears to be highly conserved whithin K. pneumoniae clinical isolates, showing that PCR ribotyping can be an useful tool for identification of K. pneumoniae isolates.     

Characterization of rabbit Pasteurella multocida isolates by use of whole-cell, outer-membrane, and polymerase chain reaction typing.

PURPOSE: To characterize Pasteurella multocida isolates from laboratory rabbits using serotyping, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins (WCPs) and outer-membrane proteins (OMPs), and polymerase chain reaction (PCR) fingerprinting. METHODS: Fifty isolates were obtained from five sources: ATCC (1), Oklahoma (4), Michigan (9), Minnesota (7), and Texas (29). The PCR fingerprinting was conducted using two minisatellite probes for M13 and a modified M13 core sequence and two microsatellite probes--(GTG)5 and (GACA)4. RESULTS: Forty-five isolates were serogroup A, and five were serogroup D. Ten WCP patterns (W1-W10) with one variation (W1a) and 10 OMP (OM1-OM10) patterns were found. Primers M13 phage, modified M13 phage, (GTG)5, and (GACA)4 generated 7, 9, 5, and 9 fingerprint types, respectively. Combination of WCP, OMP, and PCR fingerprint results yielded 39 groups with a discrimination index of 0.98. The PCR fingerprint results generally indicated clonal association among isolates within geographic locations except for the isolates from Texas, which varied markedly in PCR fingerprint types. CONCLUSION: Single primer PCR fingerprinting provided a simple and rapid means of typing P. multocida isolates from laboratory rabbits. Combinations of conventional and molecular typing enhanced differentiation among P. multocida isolated from rabbits with pasteurellosis.     

Genetic relatedness of Legionella longbeachae isolates from human and environmental sources in Australia.

The genetic relatedness of Legionella longbeachae isolated in Australia since 1987 was investigated by restriction fragment length polymorphism (RFLP) analysis and allozyme electrophoresis. Three radiolabeled probes were used in Southern hybridizations for the RFLP studies. They were Escherichia coli 16S and 23S rRNA and cloned fragments of L. longbeachae selected empirically from genomal banks in lambda and a cosmid. The legionellae included in the study comprised 11 Legionella longbeachae serogroup 1 organisms isolated from humans, 28 L. longbeachae serogroup 1 isolates from environmental sources, and 3 L. longbeachae serogroup 2 environmental isolates. These were compared with the American Type Culture Collection reference strains of both serogroups and some other related Legionella species. Results of allozyme and RFLP analysis showed that all the isolates from humans and all but three of the environmental L. longbeachae serogroup 1 isolates were closely related. They were also closely related to L. longbeachae serogroup 1 ATCC 33462. There was wider variation among the three L. longbeachae serogroup 2 environmental isolates. One of these was closely related to L. longbeachae serogroup 2 ATCC 33484. RFLP studies with the rRNA probe provided the most discrimination among isolates but did not distinguish between the two serogroups.     

Evaluation of genetic diversity among Pseudomonas citronellolis strains isolated from oily sludge-contaminated sites

The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing. Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of P. citronellolis were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of P. citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P. citronellolis.     

Differentiation of Vibrio alginolyticus strains isolated from Sardinian waters by ribotyping and a new rapid PCR fingerprinting method

We investigated the usefulness of a novel PCR fingerprinting technique, based on the specific amplification of genomic regions, to differentiate 30 Vibrio alginolyticus strains isolated in Sardinian waters. The different profiles obtained were scanned and analyzed by a computer program in order to determine genetic relationships. The results were then compared with the patterns obtained by ribotyping with HindIII, KpnI, and XbaI restriction enzymes. PCR fingerprinting could differentiate the strains analyzed into 12 different patterns, whereas ribotyping with XbaI, which produced the highest number of patterns, generated only 7 different profiles. This study revealed the superior discriminative power of the proposed technique for the differentiation of related V. alginolyticus strains and the potential use of PCR fingerprinting in epidemiological studies.     

Evaluation of Genetic Diversity among Pseudomonas citronellolis Strains Isolated from Oily Sludge-Contaminated Sites

The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing. Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of P. citronellolis were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of P. citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P. citronellolis.     

Legionella pneumophila serogroup 1 isolates from cooling towers in Japan form a distinct genetic cluster

Thirty-one epidemiologically unrelated Legionella pneumophila serogroup 1 isolates (10 from cooling towers, 10 from public spas and/or hot spring baths, and 11 from patients) were analyzed by pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) using 6 loci, flaA, pilE, asd, mip, mompS, and proA. The results of PFGE and SBT analysis indicated that all 10 isolates from cooling towers clustered into a unique type, which was distinct from strains of other environmental sources.     

Comparison of methods for discrimination between strains of Listeria monocytogenes from epidemiological surveys.

Total cellular DNA from 28 strains of Listeria monocytogenes isolated from food implicated in food-borne illness and from patients with listeriosis was digested with the restriction endonucleases HindIII, HaeIII, and EcoRI. Following agarose gel electrophoresis, the fragments were subjected to Southern blot hybridization with a digoxigenin-labeled cDNA probe transcribed from Escherichia coli 16S and 23S rRNA. The patterns of bands from genomic (DNA fingerprints) and rDNA fingerprints (ribotypes) were used for classifying L. monocytogenes strains, and the resulting subtypes were compared with serotyping and multilocus enzyme electrophoresis classification schemes. A total of 15 distinct and identical groups were obtained when genomic DNA was digested with either HindIII or HaeIII. The most discriminating enzyme for ribotyping of strains was EcoRI, which divided the 28 strains of L. monocytogenes into 6 ribotype groups. DNA fingerprinting and ribotyping differentiated L. monocytogenes from other Listeria spp., including L. ivanovii, L. welshimeri, and L. innocua as well as the lactic acid bacteria Lactococcus lactis subsp. lactis and subsp. cremoris. L. monocytogenes strains isolated from four independent food-borne illness incidents were analyzed by all typing methods. Patient and product isolates were not distinguishable by serotyping, ribotyping, or multilocus enzyme electrophoresis. DNA fingerprinting was the only method capable of differentiating these strains, or conversely, of proving relatedness of patient-product pairs of isolates. This method was a relatively simple, sensitive, reproducible, and highly discriminating method for epidemiological tracking of L. monocytogenes implicated in food-borne illness.     

Diversity of Burkholderia isolates from woodland rhizosphere environments

AIMS: Determination of genetic diversity among UK Burkholderia cepacia isolates from various environmental niches, principally woodland tree rhizospheres and onions. METHODS AND RESULTS: Genus determination was made using polymerase chain reaction (PCR) amplification and fatty acid methyl ester profiling. Genetic diversity was investigated by repetitive sequence genetic PCR fingerprinting. Several onion isolates were similar to clinical isolates but others were diverse. Some environmental isolates were possibly synonymous with B. cepacia and B. gladioli but most from woodland rhizospheres were distinct and clustered together. The 16S rRNA genes of representatives from these clusters were PCR amplified, sequenced and phylogenetically compared with all known Burkholderia and related species. This revealed that the rhizospheric isolates had closest affinity with Burkholderia spp. with known bioremediative and biocontrol capabilities and were unrelated to taxa comprising plant or human pathogenic strains. CONCLUSIONS: All of the analyses investigated revealed that environmental and onion isolates of B. cepacia complex bacteria are genetically diverse but that woodland rhizospheric isolates are related to each other and unrelated to plant or human pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Woodland rhizospheric isolates of B. cepacia are potentially good candidates for use in bioremediation and biocontrol, as they appear distinct from plant or human pathogenic strains.     

Population genetic structure of Legionella pneumophila inferred from RNA polymerase gene (rpoB) and DotA gene (dotA) sequences

The population structure of Legionella pneumophila was studied by using partial RNA polymerase gene (rpoB) and DotA gene (dotA) sequences. Trees inferred from rpoB sequences showed that two subspecies of L. pneumophila, Legionella pneumophila subsp. pneumophila and Legionella pneumophila subsp. fraseri, were clearly separated genetically. In both rpoB and dotA trees, 79 Korean isolates used in this study constituted six clonal populations, four of which (designated subgroups P-I to P-IV) were identified in L. pneumophila subsp. pneumophila and two of which (designated subgroups F-I and F-II) were identified in L. pneumophila subsp. fraseri. Although the relationships among subgroups were not identical, such subgrouping was congruent between the rpoB and dotA trees. Type strains of several serogroups did not belong to any subgroup, presumably because isolates similar to these strains were not present among our local sample of the population. There was evidence that horizontal gene transfer or recombination had occurred within L. pneumophila. Contrary to the phylogeny from rpoB and the taxonomic context, subgroups P-III and P-IV of L. pneumophila subsp. pneumophila proved to be closely related to those of L. pneumophila subsp. fraseri or showed a distinct clustering in the dotA tree. It can be inferred that dotA of subgroups P-III and P-IV has been transferred horizontally from other subspecies. The diverse distribution of serogroup 1 strains through the gene trees suggests that surface antigen-coding genes that determine serogroup can be exchanged. Thus, it can be inferred that genetic recombination has been important in the evolution of L. pneumophila.     

PCR-酶切技术快速鉴定军团菌

[目的]建立一种新型的军团菌鉴定方法,并探讨该法在鉴定环境水源和临床标本军团菌菌株中的应用价值.[方法]根据军团菌16S rRNA基因保守序列设计引物,以分离培养得到的可疑军团菌菌株作为模板,采用PCR法对模板扩增,并用限制性内切酶对PCR产物进行酶切分析,建立一种嗜肺军团菌及非嗜肺军团菌的鉴定方法.对16株嗜肺军团菌、22株非嗜肺军团菌及12株其他细菌标准菌株进行检测,验证该方法的可靠性,最后用该法检测广州地区分离的169株可疑军团菌菌株并进行基因测序.[结果]该PCR方法检测嗜肺军团菌及非嗜肺军团菌所有标准菌株均为阳性,非军团菌检测结果均为阴性;进一步的Hinf Ⅰ酶切分析可准确的区分嗜肺军团菌标准菌株;广州地区分离的169株可疑军团菌菌株经该法检测发现160株为军团菌,其中79株为嗜肺军团菌,与基因测序检测结果一致.[结论]PCR-酶切技术可快速、特异地检测军团菌及嗜肺军团菌,适用于环境水源和临床标本可疑军团菌菌株的检测.     

Genetic characterization of Legionella pneumophila serogroup 1 associated with respiratory disease in Australia.

Techniques were developed for genetic characterization of Legionella pneumophila serogroup 1 by using restriction fragment length polymorphism analysis. Allozyme analysis provided an index of the discrimination achieved by restriction fragment length polymorphism. Isolates from human cases of legionellosis were examined by both methods, and their profiles were compared with reference strains of L. pneumophila serogroup 1 obtained from the American Type Culture Collection. Eighteen distinct clones were evident among the isolates examined. Both methods could be used to trace the source of an outbreak of legionellosis caused by L. pneumophila serogroup 1.     

Genetic characterization of Legionella pneumophila serogroup 1 associated with respiratory disease in Australia.

Techniques were developed for genetic characterization of Legionella pneumophila serogroup 1 by using restriction fragment length polymorphism analysis. Allozyme analysis provided an index of the discrimination achieved by restriction fragment length polymorphism. Isolates from human cases of legionellosis were examined by both methods, and their profiles were compared with reference strains of L. pneumophila serogroup 1 obtained from the American Type Culture Collection. Eighteen distinct clones were evident among the isolates examined. Both methods could be used to trace the source of an outbreak of legionellosis caused by L. pneumophila serogroup 1.     

Ribotyping of Clostridium perfringens from industrially produced ground meat

AIMS: Clostridium (Cl.) perfringens is a common cause of food poisoning outbreaks. Ribosomal DNA analysis (ribotyping), a method which analyses restriction fragment length polymorphisms in the chromosomal genes that encode rRNA, has been shown to be useful for microbial species identification and subtyping. METHODS AND RESULTS: The current study has used ribotyping to examine 111 Cl. perfringens isolates from industrially produced ground meat in order to collect a basis for a contamination survey. Among the 111 isolates 107 distinctly different ribopatterns were detected. In only four cases two Cl. perfringens isolates showed an identical ribopattern. The isolates gave identical ribotype patterns in three different runs, carried out 3-4 months apart from each other. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The discriminatory index for EcoRI ribotyping of the Cl. perfringens isolates was 0 x 99. Results showed that ribotyping is suitable for subtyping Cl. perfringens isolates from raw meat. Ribotyping appeared to be a useful tool for profound epidemiologic studies of Cl. perfringens-contamination in food production and processing.     

Different strategies for molecular differentiation of Mycobacterium bovis strains isolated in Sardinia, Italy

Different genetic markers were used to analyze 22 Mycobacterium bovis strains isolated from cattle in Sardinia and one human isolate. IS6110 DNA fingerprinting differentiated the strains into six patterns, whereas with enterobacterial repetitive consensus sequence primers produced seven clusters. PCR ribotyping followed by digestion with HaeIII and PvuII produced five and seven patterns, respectively. PCR with the (GTG)5 oligonucleotide primer showed the best discriminatory power, generating eight clusters among the strains analyzed.     

Differentiation of Vibrio alginolyticus Strains Isolated from Sardinian Waters by Ribotyping and a New Rapid PCR Fingerprinting Method

We investigated the usefulness of a novel PCR fingerprinting technique, based on the specific amplification of genomic regions, to differentiate 30 Vibrio alginolyticus strains isolated in Sardinian waters. The different profiles obtained were scanned and analyzed by a computer program in order to determine genetic relationships. The results were then compared with the patterns obtained by ribotyping with HindIII, KpnI, and XbaI restriction enzymes. PCR fingerprinting could differentiate the strains analyzed into 12 different patterns, whereas ribotyping with XbaI, which produced the highest number of patterns, generated only 7 different profiles. This study revealed the superior discriminative power of the proposed technique for the differentiation of related V. alginolyticus strains and the potential use of PCR fingerprinting in epidemiological studies.     

Characterization by numerical taxonomy and ribotyping of Vibrio splendidus biovar I and Vibrio scophthalmi strains associated with turbot cultures

Twelve Vibrio strains were examined phenotypically in 91 biochemical characters and genotypically by ribotyping. Ten were isolated from sea water and two from diseased turbot (Scophthalmus maximus). All isolates originated from one experimental system located in Ría de Vigo (Galicia, north-west Spain). Different type strains were used for comparative purposes. The taxonomic position was analysed with the NTSYST-pc and similarities among strains were calculated by the Simple Matching coefficient (SSM). rRNA gene restriction patterns were performed with the HindIII enzyme. The SSM coefficient separated the 12 Vibrio strains into two groups which included strains that showed a SSM coefficient quite similar to V. splendidus biovar 1 (ATCC 33125) and V. scophthalmi (CECT 4638). None of 91 phenotypical characters were specific in distinguishing both species. The ribotyping confirmed the taxonomic classification of strains. The pathogenicity of each strain was evaluated; 10 environmental strains were avirulent and two, isolated from diseased turbot, were virulent. Different biotypes and ribotypes were found among the avirulent isolates. This work showed ribotyping to be a valuable tool for identification and confirmed the necessity of extending the ribotype database within closely related Vibrio species in order to clarify the taxonomic position.