Infrequent-restriction-site PCR Research Articles

Genomic Changes and Genetic Divergence of Vibrio alginolyticus Under Phage Infection Stress Revealed by Whole-Genome Sequencing and Resequencing.

Bacteriophages (phages) and their bacterial hosts were the most abundant and genetically highly diverse organisms on the earth. In this study, a series of phage-resistant mutant (PRM) strains derived from Vibrio alginolyticus were isolated and Infrequent-restriction-site PCR (IRS-PCR) was used to investigate the genetic diversity of the PRM strains. Phenotypic variations of eight PRM strains were analyzed using profiles of utilizing carbon sources and chemical sensitivity. Genetic variations of eight PRM strains and coevolved V. alginolyticus populations with phages were analyzed by whole-genome sequencing and resequencing, respectively. The results indicated that eight genetically discrepant PRM stains exhibited abundant and abundant phenotypic variations. Eight PRM strains and coevolved V. alginolyticus populations (VE1, VE2, and VE3) contained numerous single nucleotide variations (SNVs) and insertions/indels (InDels) and exhibited obvious genetic divergence. Most of the SNVs and InDels in coding genes were related to the synthesis of flagellar, extracellular polysaccharide (EPS), which often served as the receptors of phage invasion. The PRM strains and the coevolved cell populations also contained frequent mutations in tRNA and rRNA genes. Two out of three coevolved populations (VE1 and VE2) contained a large mutation segment severely deconstructing gene nrdA, which was predictably responsible for the booming of mutation rate in the genome. In summary, numerous mutations and genetic divergence were detected in the genomes of V. alginolyticus PRM strains and in coevolved cell populations of V. alginolyticus under phage infection stress. The phage infection stress may provide an important force driving genomic evolution of V. alginolyticus.

Validation of IRS PCR, a molecular typing method, for the study of the diversity and population dynamics of Legionella in industrial cooling circuits

Legionella bacteria are ubiquitous in aquatic environments. Members of the species Legionella pneumophila are responsible for more than 98% of cases of Legionnaires' disease in France. Our objective was to validate a molecular typing method called infrequent restriction site PCR (IRS PCR), applied to the study of the ecology of Legionella and to compare this method with reference typing methods, pulsed-field gel electrophoresis (PFGE) and sequence-based Typing (SBT). PFGE and SBT are considered as gold methods for the epidemiological typing of Leg. pneumophila strains. However, these methods are not suitable to an ecological monitoring of Legionella in natural environments where a large number of strains has to be typed. Validation of IRS PCR method was performed by the identification of 45 Leg. pneumophila isolates from cooling circuits of thermal power plants by IRS PCR, PFGE and SBT. The parameters of each method were measured and compared to evaluate the effectiveness of IRS PCR. The results of this study showed that IRS PCR has a discriminating power similar or better than that of the reference methods and thus that, by its speed and low cost represents an appropriate tool for the study of the ecology of Legionella in cooling circuits.

Enterobacter nimipressuralis as a cause of pseudobacteremia

BackgroundThe clinical significance of the Enterobacter nimipressuralis as human pathogens remains unclear.Case presentationsThe microbiologic culture monitoring system of sterile body fluids revealed on an episode of Enterobacter cloacae and Enterobacter amnigenus in blood culture results on the same day; the antibiotic sensitivity and MIC were nearly the same for both species. First patient was a healthy woman with postmenopausal syndrome, while second patient with herpes zoster. Both patients had febrile sensations without signs of bacteremia. E. amnigenus was also cultured from the unused package of salined cotton in the container through epidemiologic investigation. The cultured Enterobacter species were all identified as E. nimipressuralis through hsp60 gene sequencing and infrequent-restriction-site PCR (IRS-PCR).ConclusionWhen an unusual microorganisms such as E. nimipressuralis is isolated from blood of a patient with no clinical signs of sepsis, a pseudobacteremia should be suspected. When the antibiogram and MIC test results of bacterial cultures from two or more patients are nearly the same, although the species involved may appear different, it may be necessary to prove that they are the same species through molecular methods. The microbiologic cultures monitoring system will probably help to detect pseudobacteremia and other pseudo infections through reliable and fast identification.

Understanding the Molecular Epidemiology of the Footrot Pathogen Dichelobacter nodosus To Support Control and Eradication Programs

The Gram-negative anaerobe Dichelobacter nodosus is the primary etiologic agent of ovine footrot. Few studies of the genetic diversity and epidemiology of D. nodosus have been done, despite the economic cost and welfare implications of the disease. This study examined a large collection of Australian isolates; 735 isolates from footrot-infected sheep from 247 farms in Western Australia (WA) were tested by pulsed-field gel electrophoresis (PFGE), and a subset of 616 isolates was tested by infrequent restriction site PCR (IRS-PCR). The genetic diversity of WA isolates was compared to that of 61 isolates from three other Australian states. WA isolates were genetically diverse, with 181 molecular types resolved by PFGE, resulting in a simple diversity ratio (SDR) of 1:4 and a Simpson's index of discrimination value (D) of 0.98. IRS-PCR resolved 77 molecular types (SDR = 1:8 and D = 0.95). The isolates were grouped into 67 clonal groups by PFGE (SDR = 1:11, D = 0.90) and 36 clonal groups by IRS-PCR (SDR = 1:17, D = 0.87). Despite the high genetic diversity, three common clonal groups predominated in WA and were found in other Australian states. On some farms, molecular type was stable over a number of years, whereas on other farms genetically diverse isolates occurred within a flock of sheep or within a hoof. This study provides a large database from which to appropriately interpret molecular types found in epidemiological investigations and to identify common and unknown types that may compromise footrot eradication or control programs.

Genotyping of Vibrio alginolyticus isolates from Daya Bay by infrequent-restriction-site PCR and pulsed-field gel electrophoresis

Vibrio alginolyticus is a serious bacterial pathogen that hampered the whole industry of fish farming in Guangdong, China. In order to facilitate epidemiologic studies and improve the control of disease, we developed a highly efficient method of genotyping for V. alginolyticus using infrequent-restriction-site PCR (IRS-PCR) technology. With the enzyme combination of NotI–HhaI, optimized, unique, and easy-to-interpret patterns were generated. Forty-five V. alginolyticus isolates from aquatic animals and the marine environment in Daya Bay in Guangdong were fingerprinted by IRS-PCR and pulsed-field gel electrophoresis (PFGE). The reproducibility of the IRS-PCR method was high (100%) in conformity with PFGE. When primer PN-T or PN-G was used, IRS-PCR obtained the identical clustering as PFGE, producing 24 different patterns, respectively. Moreover, IRS-PCR presented a high discriminatory power ( D = 0.953) identical to that of PFGE. In conclusion, the IRS-PCR fingerprinting method using NotI–HhaI can provide a potentially useful tool for epidemiologic investigations of V. alginolyticus isolates.

Identification of novel DNA fragments and partial sequence of a genomic island specific of Brucella pinnipedialis

Since the 1990s, Brucella strains have been isolated from a wide variety of marine mammals and were recently recognized as two different species, i.e. Brucella pinnipedialis for pinniped isolates and Brucella ceti for cetacean isolates. The aim of this study was to identify specific DNA fragments of marine mammal Brucella strains using a previously described infrequent restriction site-PCR (IRS-PCR) method but with three new couples of restriction enzymes applied on a larger panel of marine mammal Brucella isolates ( n = 74) and one human isolate from New Zealand likely from marine mammal origin. This study revealed five DNA fragments specific of Brucella strains isolated from marine mammals. Among them two new DNA fragments were specific of B. pinnipedialis but were not detected in hooded seal isolates. DNA fragment I identified in the previous IRS-PCR study and fragment VI of this study were located on a cloned and sequenced 6 kb SacI fragment. Its nucleotide sequence revealed that it is likely part of a putative genomic island. Sequence analysis showed that it carries four ORFs coding for putative metabolic functions. Although hooded seal isolates are classified within B. pinnipedialis it was shown in this study that they do not carry this genomic island and this raises the question about their evolutionary history within B. pinnipedialis.

Molecular characterization of Coxiella burnetii isolates by infrequent restriction site-PCR and MLVA typing

BackgroundCoxiella burnetii, the causative agent of Q fever, has a wide host range. Few epidemiological tools are available, and they are often expensive or not easily standardized across laboratories. In this work, C. burnetii isolates from livestock and ticks were typed using infrequent restriction site-PCR (IRS-PCR) and multiple loci variable number of tandem repeats (VNTR) analysis (MLVA).ResultsBy applying IRS-PCR, 14 C. burnetii isolates could be divided into six groups containing up to five different isolates. Clustering as deduced from MLVA typing with 17 markers provided an increased resolution with an excellent agreement to IRS-PCR, and with the plasmid type of each strain. MLVA was then applied to 28 additional C. burnetii isolates of different origin and 36 different genotypes were identified among the 42 isolates investigated. The clustering obtained is in agreement with published Multiple Locus Sequence Typing (MLST) data. Two panels of markers are proposed, panel 1 which can be confidently typed on agarose gel at a lower cost and in any laboratory setting (10 minisatellite markers with a repeat unit larger than 9 bp), and panel 2 which comprises 7 microsatellites and provides a higher discriminatory power.ConclusionOur analyses demonstrate that MLVA is a powerful and promising molecular typing tool with a high resolution and of low costs. The consistency of the results with independent methods suggests that MLVA can be applied for epidemiological studies. The resulting data can be queried on a dedicated MLVA genotyping Web service.

Population genetic analysis of Bartonella bacilliformis isolates from areas of peru where Carrion's disease is endemic and epidemic.

Carrion's disease is caused by infection with the alpha-proteobacterium Bartonella bacilliformis. Distribution of the disease is considered coincident with the distribution of its known vector, the sand fly Lutzomyia verrucarum. Recent epidemics of B. bacilliformis infections associated with atypical symptomatology in nonendemic regions have raised questions regarding the historic and present distribution of this bacterium and the scope of disease that infection causes. Phylogenetic relationships and genomic diversity of 18 B. bacilliformis isolates (10 isolates from a region where Carrion's disease is epidemic, Cuzco, Peru, and 8 isolates from a region where Carrion's disease is endemic, Caraz, Peru) were assessed using genomic data generated by infrequent restriction site PCR and gene sequence analysis of the flagellin gltA and ialB genes. A population genetic analysis of the genomic diversity suggests that what was once considered an epidemic region of Peru did not result from the recent introduction of B. bacilliformis.

Classification of Brucella strains isolated from marine mammals by infrequent restriction site-PCR and development of specific PCR identification tests

Brucella strains have been isolated since the 1990s from a wide variety of marine mammals and represent potential zoonotic pathogens. They have distinctive phenotypic and molecular characteristics from the terrestrial mammal Brucella species, and two new species names have been previously proposed based on DNA polymorphism at the omp2 locus and their preferential host, i.e. Brucella cetaceae for cetacean isolates and Brucella pinnipediae for pinniped isolates. The results presented in this study on characterization of these strains by infrequent restriction site-PCR (IRS-PCR), taking into account the higher number of IS 711 elements in their genome compared to terrestrial mammal Brucella species, supports this classification. The nucleotide sequences of specific DNA fragments detected by IRS-PCR were determined and used to develop PCR identification tests for either B. cetaceae or B. pinnipediae.

Phase variation in Bartonella henselae.

Bartonella henselae is a fastidious, Gram-negative bacterial pathogen of cats and humans. Previous workers have shown that serial passage in vitro leads to attenuation of virulence-associated attributes such as expression of pili, invasion of human epithelial cell lines and the stimulation of endothelial cell proliferation. In contrast to the published data, it was found that pilin expression is frequently preserved in organisms which have undergone phase variation in vitro. Transition from a slow-growing, dry agar-pitting (DAP) to a faster-growing, smooth non-agar-pitting (SNP) form appears to occur predictably and may reflect competition between two populations growing at different rates. Better survival of the slower-growing (DAP) form may explain its relatively easy retrieval from piliated SNP populations allowed to age on solid media. Pilin expression is associated with auto-agglutination in liquid suspension or broth cultures, and appears to be necessary but not sufficient for expression of the agar-pitting phenotype and for the formation of biofilms. Outer-membrane protein variation is seen in association with phase variation, but lipopolysaccharide expression is preserved in piliated as well as extensively passaged non-piliated isolates. The EagI/HhaI infrequent restriction site-PCR fingerprint, which has been previously used to discriminate between serotypes Marseille and Houston, is shown to alter with phase variation in vitro, and there is evidence that genetic change accompanies these events. The extent of genetic and phenotypic variability of phase-variant B. henselae has previously been underestimated. It may lead to new insights into the pathogenicity of this organism, and must be considered when interpreting data arising from such studies.

Limited diversity among human isolates of Bartonella henselae.

A study of 59 isolates of Bartonella henselae reveals relatively limited diversity among those of human origin (n = 28). Either of two distinct alleles of both gltA and 16S ribosomal DNA (rDNA) was found in all isolates, with a high level of congruity between 16S and gltA inheritance among proven human pathogens. Human isolates from all over Eastern Australia were most commonly 16S rDNA (Bergmans) type I, with the same gltA allele as the type strain (Houston-1). Comparable feline isolates were more commonly 16S type II, with less congruity of inheritance between 16S and gltA alleles. Previously described arbitrarily primed PCR and EagI-HhaI infrequent restriction site PCR fingerprinting techniques separated Bartonella species effectively but lacked discriminating power within B. henselae. Examination of the 16-23S intergenic spacer region revealed for several strains several point mutations as well as a repeat sequence of unknown significance which is readily detected by HaeIII restriction fragment length polymorphism analysis. The bacteriophage-associated papA gene was present in all isolates. Enterobacterial repetitive intergenic consensus PCR proved to be a useful and robust typing tool and clearly separated human isolates (including imported strains) from the majority of feline isolates. Our data are consistent with published evidence and with previous suggestions of intragenomic rearrangements in the type strain and suggest that human isolates come from a limited subset of B. henselae strains. They strengthen arguments for careful exploration of genotype-phenotype relationships and for the development of a multilocus enzyme electrophoresis and multilocus sequence typing-based approach to the phylogeny of B. henselae.

Molecular epidemiology of nosocomial infection associated with multi-resistant Acinetobacter baumannii by infrequent-restriction-site PCR

Acinetobacter baumannii was considered endemic in a university-affiliated tertiary hospital. A significant increase was noted in the proportion of nosocomial infections associated with this micro-organism from 1996 to 1999, although no apparent clusters could be found. Between July 1998 and February 2000, 58 nosocomial isolates of A. baumannii were collected and characterized by antibiotyping and a genotyping method, infrequent-restriction-site PCR (IRS-PCR). High resistance to the 14 antimicrobial agents examined was observed among the isolates. Of the 13 antibiograms detected, eight were multi-resistant to gentamicin and almost all of the traditional and extended-spectrum β-lactams. These multi-resistant strains consisted of 41 isolates (71%), distributed amongst different wards and intensive care units (ICUs). By IRS-PCR, 23 types were obtained, with one major type found among 28 (48%) isolates. All of these 28 isolates were collected from surgical ICUs. It appears that a single strain of multi-resistant A. baumannii was responsible for the prevalence of nosocomial infection amongst surgical patients, clearly differentiating this outbreak from the previous endemic situation. An efficient molecular typing method played a vital role in making this discrimination.

Characterization of Listeria monocytogenes strains involved in invasive and noninvasive listeriosis outbreaks by PCR-based fingerprinting techniques.

A total of 32 Listeria monocytogenes strains (16 from a recent outbreak of invasive listeriosis and 16 from two outbreaks of noninvasive listeriosis, all three occurring in Italy) were characterized by PCR-ribotyping, arbitrarily primed PCR (AP-PCR), and the recently developed infrequent-restriction-site PCR (IRS-PCR). The discriminatory ability of the techniques, first evaluated on 29 unrelated L. monocytogenes food isolates using Simpson's index of diversity, was 0.714 for PCR-ribotyping, 0.690 for AP-PCR, and 0.919 for IRS-PCR. IRS-PCR was also more capable of distinguishing among strains from the invasive listeriosis outbreak: three different clusters were identified by IRS-PCR compared to two clusters identified by both PCR-ribotyping and AP-PCR. Within each of the two outbreaks of noninvasive listeriosis, the patterns were practically identical, as demonstrated by all three techniques. Only IRS-PCR succeeded in clearly discriminating the strains related to noninvasive listeriosis from all of the other strains included in this study, including those from the outbreak of invasive listeriosis. This finding may suggest the presence of unique differences in their DNA sequences.

Suitability of PCR fingerprinting, infrequent-restriction-site PCR, and pulsed-field gel electrophoresis, combined with computerized gel analysis, in library typing of Salmonella enterica serovar enteritidis.

Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting interassay and intercenter reproducibility was low and was only increased when DNA samples were extracted at the same time and amplified with the same reaction mixtures. Reproducibility of IRS-PCR technique reached 100%, but discrimination was low (D = 0.52). The PFGE procedure showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI, BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.0 software. Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance of Salmonella serovar Enteritidis, specially if the prevalence of genetic events that could be responsible for changes in PFGE profiles in this serovar was low.

Molecular investigation of two clusters ofhospital-acquired bacteraemia caused by multi-resistant Klebsiella pneumoniae using pulsed-field gel electrophoresis andinfrequent restriction site PCR

Two molecular typing methods, DNA macrorestriction analysis with XbaI resolved by pulsed-field gel electrophoresis (PFGE) and infrequent restriction site PCR (IRS-PCR) assay with adapters designed for XbaI andHhaI restriction sites, were used to investigate two clusters of hospital-acquired bacteraemia associated with multi-resistant Klebsiella pneumoniae which occurred in a paediatric intensive care unit (PICU). A total of 56 K. pneumoniae isolates were analysed. These included 10 bacteraemic isolates from eight patients, 26 isolates obtained during an epidemiological survey, and 20 epidemiologically non-related isolates incorporated as controls. One major pattern was demonstrated in 22 of the 56 isolates analysed. These included nine of the 10 bacteraemic isolates, a single rectal isolate, two hand culture isolates and 10 sink isolates. All of these 22 isolates illustrated identical antibiograms, whilst the other 34 isolates shared six antibiograms and 31 unique patterns by either PFGE or IRS-PCR assay. The two clusters of bacteraemia appeared to be outbreaks induced by the same strain of K. pneumoniae which may have utilized sinks as reservoirs and been transmitted through the hands of medical personnel to patients. IRS-PCR demonstrates concordant results with PFGE analysis in studying the genetic relationships among K. pneumoniae isolates, and serves as an excellent epidemiological tool for this bacterium.

Differentiation of pathogenic Bartonella species by infrequent restriction site PCR.

Infrequent restriction site PCR (IRS-PCR) is a recently described DNA fingerprinting technique based on selective amplification of restriction endonuclease-cleaved fragments. Bartonella isolates associated with human disease and related nonhuman isolates were analyzed by IRS-PCR genomic fingerprinting. Preparation of DNA templates began with double digestion using three different restriction endonuclease combinations. Combinations included the frequently cutting endonuclease HhaI in conjunction with an infrequently cutting endonuclease, EagI, SmaI, or XbaI. Digestion was followed by ligation of oligonucleotide adapters designed with ends complementary to the restriction endonuclease sites. Amplification of fragments flanked with an EagI, SmaI, or XbaI site in combination with an HhaI site produced a series of different-sized amplicons resolvable into patterns by polyacrylamide gel electrophoresis (PAGE). The pattern complexity was varied by the addition of selective nucleotides to the 3' ends of the EagI-, SmaI-, or XbaI-specific primers. Amplicons were also generated with fluorescently labeled primers and were subsequently resolved and detected by capillary electrophoresis. Analysis by traditional slab PAGE and capillary electrophoresis provided suitable resolution of patterns produced with the enzyme combinations EagI-HhaI and SmaI-HhaI. However, the combination of XbaI-HhaI produced too many fragments for sufficient resolution by traditional PAGE, thus requiring the better resolving properties of capillary electrophoresis. Due to the flexibility in modulating the pattern complexity and electrophoresis methods, these techniques allow for a high level of experimental optimization. The results provide evidence of the discriminatory power, ease of use, and flexibility of the IRS-PCR method as it applies to the identification of human-pathogenic Bartonella species.

Intérêt et limites des techniques d'épidémiologie moléculaire dans les infections a legionella pneumophila

Molecular epidemiology methods are useful to investigate cases of legionellosis. As Legionella pneumophila is widely distributed in the aquatic environment, its isolation from water samples does not allow, by itself, to identify the contamination source. Several molecular methods can be used : plasmid analysis, ribotyping, restriction enzyme analysis, pulsed field gel electrophoresis, arbitrarily primed-PCR, amplified fragment lengh polymorphism, infrequent restriction site PCR, sequencing. These methods should always be associated with an epidemiological investigation and should be validated in a given area on a large set of strains non epidemiologicaly linked.

Application of infrequent-restriction-site PCR to clinical isolates of Acinetobacter baumannii and Serratia marcescens.

We applied infrequent-restriction-site PCR (IRS-PCR) to the investigation of an outbreak caused by 23 isolates of Acinetobacter baumannii in an intensive care unit from November 1996 to May 1997 and a pseudoepidemic caused by 16 isolates of Serratia marcescens in a delivery room from May to September 1996. In the epidemiologic investigation of the outbreak caused by A. baumannii, environmental sampling and screening of all health care workers revealed the same species from the Y piece of a mechanical ventilator and the hands of two health care personnel. IRS-PCR showed that all outbreak-related strains were genotypically identical and that three strains from surveillance cultures were also identical to the outbreak-related strains. In a pseudoepidemic caused by S. marcescens, IRS-PCR identified two different genotypes, and among them one genotype was predominant (15 of 16 [93.8%] isolates). Extensive surveillance failed to find any source of S. marcescens. Validation of the result of IRS-PCR by comparison with that of field inversion gel electrophoresis (FIGE) showed that they were completely concordant. These results suggest that IRS-PCR is comparable to FIGE for molecular epidemiologic studies. In addition, IRS-PCR was less laborious and less time-consuming than FIGE. To our knowledge, this is the first report of the application of IRS-PCR to A. baumannii and S. marcescens.

Comparative Analysis of Infrequent-Restriction-Site PCR and Pulsed-Field Gel Electrophoresis for Epidemiological Typing of Legionella pneumophila Serogroup 1 Strains

Two methods were compared for the analysis of 48 unrelated and epidemiologically related Legionella pneumophila serogroup 1 isolates. These are the infrequent-restriction-site PCR (IRS-PCR) assay with adapters designed for XbaI and PstI restriction sites and the pulsed-field gel electrophoresis (PFGE) analysis determined after DNA restriction with SfiI. Both methods demonstrated a high level of discrimination with a similar capacity for differentiating 23 of the 24 unrelated isolates. PFGE analysis and IRS-PCR assay were both able to identify epidemiologically related isolates of L. pneumophila from three outbreaks. Hence, IRS-PCR assay appears to be a reproducible (intergel reproducibility, 100%) and discriminative (discriminatory index, > or = 0.996) tool for typing of Legionella. Compared to PFGE, however, IRS-PCR presented an advantage through ease of performance and with attributes of rapidity and sensitivity of target DNA.