Macrorestriction Analysis Of DNA Research Articles

The Emergence and Importation of Diverse Genotypes of Methicillin-Resistant Staphylococcus aureus (MRSA) Harboring the Panton-Valentine Leukocidin Gene ( pvl ) Reveal that pvl Is a Poor Marker for Community-Acquired MRSA Strains in Ireland

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) carrying pvl is an emerging problem worldwide. CA-MRSA tends to harbor staphylococcal cassette chromosome mec type IV (SCCmec IV), to be non-multiantibiotic resistant, and to have different genotypes from the local hospital-acquired MRSA (HA-MRSA). However, in Ireland, 80% of HA-MRSA isolates have the non-multiantibiotic-resistant genotype ST22-MRSA-IV. This study investigated MRSA isolates from Ireland (CA-MRSA, health care-associated MRSA, and HA-MRSA) for the carriage of pvl and determined the genotypic characteristics of all pvl-positive isolates identified. All 1,389 MRSA isolates were investigated by antibiogram-resistogram typing and SmaI DNA macrorestriction analysis. pvl-positive isolates were further characterized by multilocus sequence typing and SCCmec, agr, and toxin gene typing. Twenty-five (1.8%) MRSA isolates belonging to six genotypes (ST30, ST8, ST22, ST80, ST5, and ST154) harbored pvl. Nineteen of these (76%) were CA-MRSA isolates, but a prospective study of MRSA isolates from 401 patients showed that only 6.7% (2/30) of patients with CA-MRSA yielded pvl-positive isolates. Thus, pvl cannot be used as a sole marker for CA-MRSA. Fifty-two percent of pvl-positive MRSA isolates were recovered from patients with skin and soft tissue infections; thirty-six percent were from patients of non-Irish ethnic origin, reflecting the increasing heterogeneity of the Irish population due to immigration. All 25 pvl-positive isolates carried SCCmec IV; 14 (56%) harbored SCCmec IV.1 or IV.3, and the remaining 11 isolates could not be subtyped. This study demonstrates that pvl is not a reliable marker for CA-MRSA in Ireland and reveals the emergence and importation of diverse genotypes of pvl-positive MRSA in Ireland.

Relatedness of Streptococcus equi subsp. zooepidemicus strains isolated from harbour seals ( Phoca vitulina) and grey seals ( Halichoerus grypus) of various origins of the North Sea during 1988–2005

The present study was designed to identify 15 beta-hemolytic streptococci isolated during a period between 1988 and 2005 from nine harbour seals and six grey seals from various origins of the North Sea. All isolates were identified as Streptococcus equi subsp. zooepidemicus. The bacteria were additionally investigated for relatedness by restriction fragment length polymorphism analysis of PCR amplified 16S-23S rDNA intergenic spacer region and gene szp and by macrorestriction analysis of chromosomal DNA of the strains by pulsed field gel electrophoresis. The molecular analysis yielded identical or closely related patterns within the strains of the present study and with the S. equi subsp. zooepidemicus strains isolated from harbour seals of German North Sea which were investigated previously [Akineden, Ö., Hassan, A.A., Alber, J., El-Sayed, A., Estoepangestie, A.T.S., Lämmler, C., Weiss, R., Siebert, U., 2005. Phenotypic and genotypic properties of S. equi subsp. zooepidemicus isolated from harbor seals ( Phoca vitulina) from the German North Sea during the phocine distemper outbreak in 2002. Vet. Microbiol. 110, 147–152]. This indicates that this single or closely related bacterial clone existed during both phocine distemper virus epidemics in 1988 and 2002 and that a direct transmission of the strains has occurred between two seal species and between seal populations of far distant regions possibly with grey seals as a vector.

Phenotypic and genotypic characteristics of methicillin/oxacillin-resistant Staphylococcus intermedius isolated from clinical specimens during routine veterinary microbiological examinations

Methicillin/oxacillin resistance of 10 S. intermedius strains was investigated by conventional and molecular methods. The strains tested had been isolated in Germany during routine veterinary microbiological examinations of specimens from a small animal clinic between May and September 2005. Epidemiological relationships of the strains were studied by macrorestriction analysis of their chromosomal DNA using pulsed field gel electrophoresis (PFGE). Species identity of the 10 S. intermedius strains was confirmed by conventional methods and by PCR mediated amplification of S. intermedius specific segments of thermonuclease encoding gene nuc. As controls, four methicillin/oxacillin resistant S. intermedius (MRSI) strains obtained from specimens sent by four veterinarians and three selected methicillin/oxacillin sensitive S. intermedius (MSSI), also obtained from the small animal clinic, were tested. The 10 strains, representing approximately 6% of all S . intermedius isolated from the clinic throughout the time period mentioned above, and the four MRSI obtained from veterinarians, were methicillin/oxacillin and penicillin resistant using disk diffusion tests and could be cultivated on oxacillin resistant screening agar base (ORSAB). Both resistances could be confirmed by multiplex PCR detecting the resistance genes mecA and blaZ. The three MSSI were methicillin/oxacillin sensitive in all tests. Epidemiological investigation by macrorestriction analysis of the chromosomal DNA of the strains by pulsed field gel electrophoresis revealed that all 10 MRSI strains obtained from the clinic and the four MRSI strains obtained from veterinarians, in contrast to the three MSSI strains, represent identical or closely related bacterial clones possibly indicating a cross-infection of the animals in the clinic and the distribution of a single MRSI clone in the pet population.

SOURCES AND PATHWAYS OF SPREAD OF VANCOMYCIN-RESISTANT ENTEROCOCCI IN HEMATO-ONCOLOGICAL PATIENTS

The presented study aims at analyzing an increasing prevalence of vancomycin-resistant enterococci (VRE) isolated from various kinds of clinical material obtained from patients in the Department of Hemato-oncology (DHO), University Hospital in Olomouc, Czech Republic. Between January 1 and March 31, 2005, enterococci were isolated by standard microbiological procedures using both clinical material obtained from hospitalized patients and samples from the department environment. Resistance to vancomycin and teicoplanin was determined by a standardized microdilution method. Phenotype determination of resistance to vancomycin was verified by PCR detection of vanA and vanB genes. In VanA Enterococcus faecium, macrorestriction analysis was performed by pulsed-field gel electrophoresis. During the monitored period, a total of 128 Enterococcus sp. strains were isolated, of which 38 (30 %) isolates from 22 different patients were determined as VRE. Dominating were Enterococcus faecium VanA (63 %) and Enterococcus casseliflavus VanC (16 %) strains. At the same time, one Enterococcus faecium VanA strain was acquired from a bed-side table used by a patient in whom a similar strain had been isolated repeatedly from various clinical materials including a rectal swab taken in 2004. Based on the macrorestriction analysis of genome DNA in 24 vancomycin-resistant Enterococcus faecium VanA strains isolated from the patients' clinical material, one strain from the bed-side table surface and one strain isolated from stools in 2004, 8 unique restriction profiles with similarity ranging from 90 % to 100 % were identified, which could be classified into 3 clonal types. Thus, we can assume not only the endogenous origin of the VRE in hemato-oncological patients and their potential selection caused by therapy with broad-spectrum antibiotics but also the ability of the strains to survive in a hospital setting and, subsequently, to be spread clonally by various vectors.

Differentiation of human and animal strains of Streptococcus dysgalactiae by pulsed-field gel electrophoresis.

The genetic diversity among 54 human isolates and 33 animal isolates belonging to the species Streptococcus dysgalactiae (20 alpha-haemolytic Streptococcus dysgalactiae, 23 Streptococcus equisimilis, 43 group G streptococci and one group L streptococcus) was evaluated by macrorestriction analysis of chromosomal DNA with SmaI and resolution by pulsed-field gel electrophoresis. This technique revealed a high degree of intraspecies polymorphism, leading to the differentiation of 80 distinct banding patterns, and identified the presence of two major clusters, one containing isolates of human origin and the other isolates of animal origin. These results suggest than human and animal isolates of S.dysgalactiae are genetically distinct, and support the recent proposal of the subspecies S. dysgalactiae subsp. equisimilis for human isolates. The heterogeneity revealed within isolates from the same host type indicates that pulsed-field gel electrophoresis is a powerful epidemiological tool for studying S. dysgalactiae infections.

Extreme weather events and environmental contamination are associated with case-clusters of melioidosis in the Northern Territory of Australia

Melioidosis, the infection due to the environmental organism Burkholderia pseudomallei, is endemic to northern Australia and South East Asia. It is associated with exposure to mud and pooled surface water, but environmental determinants of this disease are poorly understood. We defined case-clusters in northern Australia, determined their contribution to the observed rate of melioidosis, and explored clinical features and associated environmental factors. Using geographical information systems data, we examined clustering of melioidosis cases in time and geographical space in the Top End of the Northern Territory of Australia between 1990 and 2002 using a scan statistic. DNA macrorestriction analysis, resolved by pulsed field gel electrophoresis, was performed on isolates from patients. We defined five case-clusters involving 27 patients that occurred within 7-28 days and/or a radius of 100-300 km. Clustered cases were associated with extreme weather events or environmental contamination; no difference in the clinical pattern of disease was noted from other patients not involved in clusters. Isolates from patients linked to environmental contamination were caused by isolates with similar DNA macrorestriction patterns, but isolates from patients linked to severe weather events had more diverse DNA macrorestriction patterns. Case-clusters of melioidosis where isolates exhibit diverse DNA macrorestriction patterns in our region are linked to extreme weather events and outbreaks where isolates are predominantly of the same DNA macrorestriction pattern are linked with contamination of an environmental source.

Phenotypic and genotypic properties of Streptococcus equi subsp. zooepidemicus isolated from harbor seals (Phoca vitulina) from the German North Sea during the phocine distemper outbreak in 2002

The present study was designed to identify and compare 32 beta-hemolytic streptococci isolated from 28 different harbor seals of the German North Sea during the phocine distemper outbreak in 2002. The bacteria were identified as Streptococcus equi subsp. zooepidemicus based on cultural, biochemical, serological and molecular studies. Epidemiological investigations by PCR restriction fragment length polymorphism analysis of the 16S-23S rDNA intergenic spacer region and gene szp and by macrorestriction analysis of the chromosomal DNA of the strains by pulsed field gel electrophoresis revealed that all 32 strains appeared to be identical. These results indicate that a single bacterial clone seemed to be distributed among the harbor seal population of the German North Sea during this outbreak.

Countrywide Spread of Community- and Hospital-Acquired Extended-Spectrum β-Lactamase (CTX-M-15)-Producing Enterobacteriaceae in Lebanon

A prospective study was carried out to assess the extent of carriage of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae at both hospital and community levels in Lebanon. A total of 1,442 fecal samples were collected from hospital-based patients and 58 from health care workers of six Lebanese tertiary care general hospitals located in different areas of Lebanon between January and March 2003. A total of 382 fecal samples were also collected from healthy subjects between April and June 2003. The samples analysis led to the identification of 118 strains as ESBL producers based on the synergistic effects between clavulanate and selected beta-lactams (ceftazidime and cefotaxime). These strains were isolated from 72 subjects: 61 patients, 2 health care workers, and 9 healthy subjects. One representative strain per subject was selected, and a total of 72 nonduplicate ESBL producers, including a high majority of Escherichia coli (n = 56), Klebsiella pneumoniae (n = 9), Enterobacter cloacae (n = 6), and Citrobacter freundii (n = 1), were characterized. The molecular analysis revealed that the majority of the strains (83%) express CTX-M-15 ESBL (pI 8.6). SHV-5a ESBL (pI 8.2) was produced by 18% of the strains. DNA macrorestriction analysis of ESBL-producing E. coli presented 38 different genotypes, revealing the absence of clonal link among these strains. In addition to the fact that the present study highlights the emergence and the countrywide dissemination of CTX-M-15-producing E. coli in Lebanon, it represents the first report of an SHV-5a-producing C. freundii.

Identification of Streptococcus canis Isolated from Milk of Dairy Cows with Subclinical Mastitis

Streptococcus canis was isolated from 31 milk samples from 11 cows in a dairy herd (with 49 lactating cows) affected by subclinical mastitis in north Rhine-Westphalia, Germany. Thirty-one isolates from the infected udder quarters were further characterized for their phenotypic and molecular properties. Most isolates (83.9%) produced alpha-galactosidase, and all were negative for beta-d-glucuronidase. Amplification of the 16S rRNA gene by the PCR method and digestion with the restriction enzymes RsaI, MspI, and AvaII yielded species-specific patterns. Additional identification by species-specific amplification of the 16S rRNA gene, the 16S-23S rRNA gene intergenic spacer region, the CAMP factor-encoding gene cfg, and the internal fragments of the sodA gene was consistent with S. canis. Macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis showed that the S. canis isolates originated from a single clone or were very closely related.

DNA macrorestriction analysis of nontypeable group B streptococcal isolates: clonal evolution of nontypeable and type V isolates.

Group B streptococci (GBS) are serotyped according to capsular polysaccharide (CPS) type (Ia to VIII); an isolate is classified as nontypeable (NT) if no detectable CPS is found. Surface-localized protein antigens (alpha, beta, R1, and R4) serve as additional markers to classify GBS isolates, which is particularly useful since NT isolates often express one or more of these proteins. To compare genetic resemblance among isolates with similar protein profiles, we studied 58 NT isolates digested with the SmaI macrorestriction enzyme prior to pulsed-field gel electrophoresis (PFGE). Of these 58, 15.5% expressed alpha only, 20.7% expressed alpha+beta, 15.5% expressed R4, and 25.8% expressed R1,R4, while 22.4% of the isolates expressed no detectable proteins. The largest PFGE profile group, with 48% of the isolates, was group 4, composed primarily of isolates that expressed R1,R4 or no proteins. The second most common profiles were 3 and 32, each with 13.8% of the isolates. Since NT isolates in profile group 4 were highly related to type V isolates, as demonstrated by PFGE profiles, we investigated 45 type V isolates. Two-thirds of the type V isolates within profile group 4 were classified into subgroup 4a, compared to 28.2% of 39 NT isolates. Only 11% of the V/R1,R4 isolates were identical to the prototype group 4 profile, in contrast to 75% of the NT/R1,R4 isolates. A shift of type V isolates into profile 4 subgroups may be indicative of a genetic change over time. PFGE is a valuable approach for comparison of GBS isolate relatedness and for monitoring of NT and typeable GBS isolates for potential clonal divergence.

Clonal Relatedness and Conserved Integron Structures in Epidemiologically Unrelated Pseudomonas aeruginosa Strains Producing the VIM-1 Metallo-β-Lactamase from Different Italian Hospitals

Three epidemiologically independent Pseudomonas aeruginosa isolates, representative of the first VIM-1 metallo-beta-lactamase producers detected at three different hospitals in northern Italy, were investigated to determine their genomic relatedness and to compare the structures of the genetic supports for the VIM-1 determinants. The three isolates, all of serotype O11, appeared to be clonally related according to the results of genotyping by macrorestriction analysis of genomic DNA by pulsed-field gel electrophoresis and random amplification of polymorphic DNA. Investigation of the genetic support for the bla(VIM-1) determinant revealed that it was carried on identical or almost identical integrons (named In70.2 and In70.3) located within a conserved genomic context. The integrons were structurally related to In70 and In110, two plasmid-borne bla(VIM-1)-containing integrons from Achromobacter xylosoxidans and Pseudomonas putida isolates, respectively, from the same geographic area (northern Italy) and were found to be inserted close to the res site of a Tn5051-like transposon, different from any of those described previously, that was apparently carried on the bacterial chromosome. The present findings suggest that the three VIM-1-producing isolates are members of the same clonal complex which have been spreading in hospitals in northern Italy since the late 1990s and point to a common ancestry of their bla(VIM-1)-containing integrons.

Occurrence and clonal relatedness of sec/tst-gene positive Staphylococcus aureus isolates of quartermilk samples of cows suffering from mastitis.

To investigate the prevalence of sec/tst-gene positive Staphylococcus aureus in bovine mastitis and to get information about the clonal relatedness of these clinical isolates. A total of 533 Staph. aureus strains isolated from bovine mastitic quartermilk samples at 493 randomized dairy farms in Hessia, Germany, from January 1997 until June 1998 were examined for enterotoxin C (sec) gene and toxic shock syndrome toxin (tst) gene by multiplex polymerase chain reaction. Fifty-three (9.3%) of the strains were sec/tst-gene positive. Phenotypic TSST-1 production was found in all positive strains by reversed passive latex agglutination test. With DNA macrorestriction analysis, sec/tst-gene positive strains were divided into five different macrorestriction types. Type I (10 isolates) and III (40 isolates) were found to be the predominant types in terms of frequency of isolation in the investigated area. These DNA macrorestriction types differed in only two bands in the 500 and 270 bp region. Closely related Staph. aureus strains seem to be responsible for an unusual large proportion of bovine mastitis cases in geographically widely distinct locations. This is one of the first reports about the relatedness of sec/tst-gene positive Staph. aureus clinical isolates from bovine mastitis.

Extended-spectrum beta-lactamases in Klebsiella spp and Escherichia coli obtained in a Brazilian teaching hospital: detection, prevalence and molecular typing

His study was performed to compare the methods of detection and to estimate the prevalence of extendedspectrum β-lactamases (ESBL) among Klebsiella spp and E. coli in a university hospital in southern Brazil. We also used a molecular typing method to evaluate the genetic correlation between isolates of ESBL K. pneumoniae. Production of ESBL was investigated in 95 clinical isolates of Klebsiella spp and Escherichia coli from Hospital de Clinicas de Porto Alegre, using Kirby-Bauer zone diameter (KB), double-disk diffusion (DD), breakpoint for ceftazidime (MIC CAZ), increased zone diameter with clavulanate (CAZ/CAC) and ratio of ceftazidime MIC/ceftazidime-clavulanate MIC (MIC CAZ/CAC). Molecular typing was performed by DNA macrorestriction analysis followed by pulsed-field gel electrophoresis. The KB method displayed the highest rates of ESBL (up to 70% of Klebsiella and 59% of E. coli), contrasting with all the other methods (p < 0.05). The confirmatory methods (DD, MIC CAZ, CAZ/CAC and MIC CAZ/CAC) showed a range of ESBL production from 8 to 13% for E. coli and from 33 to 40% for Klebsiella species. Therefore, the KB method was useful only as a screening method as it provided several false positive results. Molecular typing of 17 ESBL K. pneumoniae indicated that the isolates had no clonal relation. We found a good correlation among the confirmatory methods for ESBL detection although the methods which evaluate inhibition of the β-lactamase by clavulanate appeared to be more specific. The high prevalence of ESBL Klebsiella in our hospital is probably due to individual selection of resistant strains rather than the transmission of a common strain.

Real-time application of automated ribotyping and DNA macrorestriction analysis in the setting of a listeriosis outbreak.

A cluster of three cases of listeriosis cases occurred against a background of endemic listeriosis in Western Australia. Human and environmental isolates of Listeria monocytogenes obtained during the outbreak investigation were rapidly subtyped by automated ribotyping using an EcoRI protocol and a RiboPrinter. DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) was used to confirm the relatedness of isolates. Serogroup 1/2 predominated among the food samples and the four clinical isolates from the outbreak cluster were also of this serogroup. All isolates from chicken material were serogroup 1/2 and indistinguishable by ribotype pattern. PFGE subdivided strains of this ribotype into four subtypes. The preliminary analysis had an immediate impact on hypothesis generation, environmental health investigations, environmental specimen collection and initial control measures. Sufficient typing data to guide environmental health and disease control initiatives was generated in less than one week by combining automated ribotyping with PCR-based detection of L. monocytogenes in suspect foodstuffs and an L. monocytogenes DNA probe. There were no further cases of bacteriologically confirmed listeriosis in Western Australia for six months after completion of the investigation.

Relationship between nosocomial Acinetobacter species occurring in two geographical areas (Greece and the UK)

Fifty-two isolates of Acinetobacter spp. obtained from three Greek and one UK hospital, were studied using partial 16 S ribosomal DNA sequence analysis, repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR) mediated fingerprinting and DNA macro-restriction analysis. The aim was twofold: first, to discern the major differences in the population of Acinetobacter spp. between the two countries. Second, to compare a simple PCR-based typing scheme with pulsed-field gel electrophoresis (PFGE). The multi-resistant Greek isolates were within DNA groups 2 and TU13, and clustered into three types both by REP-PCR and PFGE. By contrast, the more susceptible Oxford isolates were heterogeneous on 16 S RNA sequence analysis and distinguishable on typing. The need for studies that elucidate the phylogeny of Acinetobacter spp. inside and outside hospitals are important, as this will help clarify the relationship between organisms that are increasingly recognized as causes of severe infections.

Molecular typing of Aeromonas isolates in natural mineral waters.

A total of 103 isolates of Aeromonas spp. were obtained over a 3-year period from a natural mineral water and from surface streams located within the boundaries of the watershed of the natural mineral water wells and were typed by macrorestriction analysis of genomic DNA with XbaI and by pulsed-field gel electrophoresis. All Aeromonas caviae isolates from the natural mineral water belonged to the same clone, and an analogous clonal identity was found among Aeromonas hydrophila isolates. These two clones expressed no hemolytic or cytotoxic activities. Aeromonas isolates from surface waters showed high molecular heterogeneity and were not related to the clones found in the natural mineral water. The presence of aeromonads chronically found in the natural mineral water was a likely consequence of a localized development of a biofilm, with no exogenous contamination of the aquifer. Molecular fingerprinting of drinking water isolates is a useful tool in explaining possible reasons for bacterial occurrences.

DNA macrorestriction analysis by pulsed-field gel electrophoresis of Pseudomonas aeruginosa isolates from mastitis in dairy sheep.

DNA macrorestriction analysis by pulsed-field gel electrophoresis of Pseudomonas aeruginosa isolates from mastitis in dairy sheep.

Epidemiological typing of imipenem-resistant Pseudomonas aeruginosa

The level of genetic heterogeneity of nine isolates of Pseudomonas aeruginosa resistant or intermediately susceptible to imipenem was determined by epidemiological typing using macrorestriction analysis of chromosomal DNA. The strains were isolated between December 2000 and February 2001 from a variety of specimens from nine patients hospitalized in five different departments of the University Medical Center in Ljubljana and in the nursing home. They belonged to seven genotypes or clones (A–G). Six isolates were heterogeneous, with different B–G genotypes. Three isolates had an identical A genotype but it is more likely that this genotype was more prone to develop imipenem resistance than to spread among the patients.

Prevalence of erm(A) and mef(B) erythromycin resistance determinants in isolates of Streptococcus pneumoniae from New Zealand.

One hundred and twenty-four erythromycin-resistant pneumococcal isolates were examined for the presence of macrolide resistance genes. The erm(B) gene was detected in 118 (95.2%) isolates and the mef(A) gene in 83 (66.9%) isolates. Both the mef(A) and erm(B) genes were detected in 77 (62.1%) isolates. DNA macrorestriction analysis of these isolates identified them as belonging to a single multi-resistant clone.

Comparison between human and animal isolates of Shiga toxin-producing Escherichia coli O157 from Australia.

There is very little human disease associated with enterohaemorrhagic Escherichia coli O157 in Australia even though these organisms are present in the animal population. A group of Australian isolates of E. coli O157:H7 and O157:H- from human and animal sources were tested for the presence of virulence markers and compared by XbaI DNA macrorestriction analysis using pulsed-field gel electrophoresis (PFGE). Each of 102 isolates tested contained the gene eae which encodes the E. coli attaching and effacing factor and all but one carried the enterohaemolysin gene, ehxA, found on the EHEC plasmid. The most common Shiga toxin gene carried was stx2c, either alone (16%) or in combination with stx1 (74%) or stx2 (3%). PFGE grouped the isolates based on H serotype and some clusters were source specific. Australian E. coli O157:H7 and H- isolates from human, animal and meat sources carry all the virulence markers associated with EHEC disease in humans therefore other factors must be responsible for the low rates of human infection in Australia.