Two clones of methicillin-resistant Staphylococcus aureus in Poland

Objective: To evaluate relatedness among methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in Poland.
Method: Ninety-three MRSA hospital isolates were collected from different regions in Poland from 1990 to 1992. Strains were analyzed with respect to heterogeneity of methicillin resistance, phage types, resistance patterns, crystal violet staining, chromosomal DNA Sma I restriction patterns by PFGE, ERIC1 and ERIC2 AP-PCR types and DNA repeat polymorphism within the protein A gene. Resistance to methicillin was confirmed by the detection of the mecA gene by PCR.
Results: The combined results of typing methods demonstrate that all MRSA strains analyzed could be easily divided into two distinct clones (clonally related strains). The first consisted of strains with clear heterogeneous expression of resistance to methicillin (34 isolates) and the second showed more homogeneous resistance (59 isolates). In this study the best method for epidemiologic analysis of MRSA was found to be PFGE. A good correlation between the epidemic behavior of MRSA and a high number of repetitive DNA units within the protein A gene was observed.
Conclusions: Results show that in Poland two distinct clones of epidemic MRSA have circulated in the past, easily discriminated by pheno and genotyping methods, and both could be found together in a single hospital.     

Failure of bacteriophage typing to detect an inter-hospital outbreak of methicillin-resistant Staphylococcus aureus (MRSA) in Zagreb subsequently identified by random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE)

Objective: To establish the extent of inter-hospital spread of methicillin-resistant Staphylococcus aureus (MRSA) in Zagreb and to determine the most suitable method for typing local strains.
Methods: We analyzed a collection of 33 MRSA isolates from three Zagreb hospitals together with five unrelated British MRSA isolates by antibiogram typing, bacteriophage typing, randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) after digestion with Smal restriction endonuclease. Bacteriophage typing was done with the international set of S. aureus typing phages. RAPD and PFGE profiles were analyzed visually and by using the 'GelCompar' computer program.
Results: Antibiogram typing provided eight profiles. Thirty (91%) of the 33 Croatian strains of MRSA were non-typable by phage typing. Visual analysis of RAPD products identified six, and visual analysis of PFGE fragments nine, distinct profiles. Computer analysis of RAPD data separated British isolates from the Croatian ones, but did not cluster the visually determined RAPD types. PFGE computer analysis separated British isolates and clustered isolates in concordance with visual interpretation. Thirty-one of the 38 isolates (82%) were visually grouped in the same clusters by both molecular methods. The dominant strain was present in each of the three hospitals.
Conclusions: Bacteriophage typing was unhelpful for the analysis of Croatian MRSA, since most strains were untypable with the international set of bacteriophages. RAPD and PFGE were more successful in typing the organisms and showed evidence of inter-hospital spread of one predominant MRSA strain in all three Zagreb hospitals. Thus RAPD and PFGE proved to be a useful aid in elucidating the epidemiology of MRSA infection in Zagreb hospitals and should be established in Croatia for typing MRSA.     

Characterization of isolates of methicillin-resistant Staphylococcus aureus from Hong Kong by phage typing, pulsed-field gel electrophoresis, and fluorescent amplified-fragment length polymorphism analysis

The genetic relatedness of 127 methicillin-resistant Staphylococcus aureus (MRSA) isolates, belonging to five major types as identified by pulsed-field gel electrophoresis (PFGE) and antibiotic resistance profiles, was examined further using phage typing and fluorescent amplified fragment length polymorphism (FAFLP). The MRSA isolates were recovered from patients at the Prince of Wales Hospital (PWH), Hong Kong, over a 13-year period, 1988 to 2000. These strains were also compared with representatives of the well-described MRSA international clones and with epidemic MRSA strains (eMRSA) 1 to 16 from the United Kingdom. Phage typing distinguished two major "clones" at this hospital: all of the phage type 1 (PT1) isolates belonged to PFGE types A, C, D, and E, while most of the PT2 isolates were associated with PFGE type B, which exhibited a unique antibiotic resistance profile. MRSA isolates belonging to PFGE subtype A2 were indistinguishable from the British eMRSA-1, while isolates of PFGE type B were closely related to eMRSA-9 by PFGE. Based on FAFLP, all five predominant PFGE types at the PWH belonged to one group and fell into the same cluster as eMRSA-1, -4, -7, -9, and -11 isolates. Multilocus sequence typing and staphylococcal cassette chromosome mec typing classified representatives of our MRSA isolates as members of the same clone (ST239-MRSA-III). Thus, the predominant MRSA isolates frin the PWH in the last decade are closely related to early United Kingdom eMRSA clones 1, 4, and 11 and are members of a lineage that includes the Brazilian MRSA clone.     

Comparison of protein A gene sequencing with pulsed-field gel electrophoresis and epidemiologic data for molecular typing of methicillin-resistant Staphylococcus aureus

The epidemiologic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) isolates is currently determined by analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis (PFGE). We have evaluated an alternative typing system (MicroSeq StaphTrack Kit; Perkin-Elmer Biosystems) based on the sequence analysis of the chromosomally encoded polymorphic repeat X region of the S. aureus protein A (spa) gene. A total of 69 clinical MRSA isolates were divided into 18 groups according to the number and nucleotide sequences of the spa repeats. Molecular typing results obtained both by spa sequencing and from the PFGE patterns were concordant except for one group, which contained 20 isolates recovered over a 2-year period from hospitalized patients at the Mayo Clinic. Although the spa typing patterns were indistinguishable for those isolates, PFGE analysis yielded seven related but distinguishable patterns. Further coagulase gene sequence analysis subtyped those 20 strains into four groups which followed distinct temporal and geographic distributions. During a 2-year epidemic period there were up to 7 fragment changes in PFGE patterns among epidemiologically related isolates, suggesting that PFGE may be unsuitable for long-term typing of strains involved in epidemics. Although more limited than PFGE in discriminatory power, spa sequencing analysis could be used as a screening method for typing of MRSA strains because of the shorter turnaround time, ease of use, and the inherent advantages of sequence analysis, storage, and sharing of information.     

Analysis of an outbreak of non-phage-typeable methicillin-resistant Staphylococcus aureus by using a randomly amplified polymorphic DNA assay.

A cluster of methicillin-resistant Staphylococcus aureus (MRSA) infections among patients on an intensive care unit (ICU) was detected by routine infection control surveillance. In the period from 5 January to 22 June 1995, 10 patients on the ICU and a further 6 patients (5 on one ward that had received colonized patients transferred from the ICU) were affected by MRSA strains with the same antibiotic susceptibility patterns. Seven (44%) of these 16 colonized patients developed MRSA bacteremia. MRSA isolates with the same characteristics were also found on the hands of one member of the ICU staff. The isolates were untypeable by phage typing, but 15 of 17 outbreak strains analyzed genetically had identical randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) profiles. A single strain of MRSA that was nontypeable by phage typing and that was isolated on the ICU on 1 January and six nontypeable and epidemiologically unrelated MRSA isolates all had RAPD profiles distinct from that of the outbreak strain. Implementation of strict infection control measures stopped the further spread of MRSA on the ICU, the affected general ward, and seven other wards that received MRSA carriers from the ICU. Although nontypeable by phage typing and not previously recognized as an epidemic strain, this strain of MRSA was readily transmissible and highly virulent. RAPD typing was found to be a simple, rapid, and effective method for the epidemiological investigation of this outbreak, and performance of typing by this method was simpler and less time-consuming than that of typing by PFGE. RAPD typing may have more general application for the study of S. aureus infections in hospitals.     

Clonal structure of the methicillin-resistant Staphylococcus aureus (MRSA) population in Poland: revision and update

The clonal structure of the methicilin-resistant Staphylococcus aureus (MRSA) population in Poland has been analyzed in several reports since the mid-1990s. The present study was performed on 253 MRSA isolates (146 archival and 107 new isolates) recovered in 26 hospitals between 1990 and 2001. Whereas all isolates were typed by pulsed-field gel electrophoresis (PFGE) and the analysis of the ClaI::mecA and ClaI::Tn554 RFLP polymorphism, selected isolates were also subjected to multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) comparisons. Based on the PFGE data, 15 MRSA clones were discerned, seven of which were observed in multiple hospitals. Five of these were related to the pandemic Hungarian (MLST clonal complex, CC8), Iberian (CC8), Pediatric (CC5), Mexican (CC30), and Brazilian clones (CC8). MLST confirmed the earlier reports on the similarity of the Hungarian and Brazilian clones, and it revealed that one of the two remaining epidemic clones was related to the Hungarian/Brazilian, and the other--to the Berlin clones. A local strain from the Northeastern part of the country was found to be similar to a minor Greek clone. The MRSA clonal structure and the increasing complexity of the relationships between the genetic and phenotypic traits of this micro-organism in Poland has now been firmly established.     

Evolutionary relationships between sporadic and epidemic strains of healthcare-associated methicillin-resistant Staphylococcus aureus

National surveillance of healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) isolates by pulsed-field gel electrophoresis (PFGE) typing allowed identification of rarely occurring 'sporadic' isolates with patterns significantly distinct from those of major epidemic clones of methicillin-resistant S. aureus (MRSA) circulating in Belgian hospitals. The aim of the present study was to compare the genetic background, antibiotic susceptibility profile and in vitro growth rates of 36 MRSA isolates with either 'epidemic' or 'sporadic' PFGE profiles to identify factors that could be involved in the epidemic behaviour of S. aureus . Sequence analysis of seven housekeeping genes (multilocus sequence typing) and seven surface-associated genes, combined with staphylococcal cassette chromosome mec (SCC mec ) typing and spa typing results, segregated sporadic isolates into four groups: (1) isolates phylogenetically distant from epidemic HA-MRSA clones that possessed several properties of community-acquired MRSA strains; (2) isolates derived from the same methicillin-susceptible S. aureus ancestor as epidemic isolates but possessing a distinct type of SCC mec ; and (3) and (4) isolates that were closely related to epidemic strains, either as recent descendants of these or as intermediate evolutionary steps between epidemic HA-MRSA strains and their putative ancestors. Sporadic isolates did not show slower growth in vitro than epidemic isolates. These findings suggest that the SCC mec type and insertion/deletion of other mobile genetic elements may be involved in modulating the epidemic behaviour of MRSA strains of similar genetic background, independently of fitness cost.     

In vivo stability and discriminatory power of methicillin-resistant Staphylococcus aureus typing by restriction endonuclease analysis of plasmid DNA compared with those of other molecular methods.

We evaluated test discriminatory power and DNA type alterations among methicillin-resistant Staphylococcus aureus strains by testing 199 sequential isolates from 39 patients collected over 30 to 228 days. Isolates were typed by one or three different methods (restriction endonuclease analysis of plasmid DNA [REAP] with or without pulsed-field gel electrophoresis of genomic DNA [PFGE] and immunoblotting [IB]). REAP was highly discriminatory compared with PFGE and IB. However, the initial isolates from 4 of the 39 patients lacked detectable plasmid DNA and could not be typed by REAP. Typing of individual patient isolates showed that a different REAP type was identified only once every 138 days. Among 25 comparisons, seven sequential isolate pairs demonstrating REAP differences were also different by PFGE and IB. This likely represented the presence of more than one strain. Eighteen other pairs with REAP differences were identical or related to one another by PFGE and IB typing, and 17 of these differences were likely caused by a single genetic alteration within the same strain or clone. The rate of PFGE differences explicable by single genetic alterations among sequential isolates identical by REAP was similar to the overall rate for REAP differences in the whole collection. We conclude that REAP and PFGE typing differences explicable by single genetic alterations are relatively infrequent but not rare. These isolates should be examined by alternative typing systems to further support or refute clonality.     

Changes in the clonal nature and antibiotic resistance profiles of methicillin-resistant Staphylococcus aureus isolates associated with spread of the EMRSA-15 clone in a tertiary care Portuguese hospital

Two hundred eighty methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates recovered from a tertiary care hospital in Oporto, Portugal, between 2003 and 2005 were studied by a combination of molecular typing techniques in order to investigate the genetic backgrounds associated with the changes in the resistance phenotypes observed since 2001 and compare them to those previously found in the hospital. All MRSA isolates were grouped into resistance profiles for a panel of seven antibiotics and characterized by pulsed-field gel electrophoresis (PFGE) and SCCmec (staphylococcal cassette chromosome mec) typing. Representative isolates of PFGE types were further studied by spa typing and multilocus sequence typing. Our findings clearly document that the increasing isolation of nonmultiresistant MRSA strains was associated with the decline (from 69% in 1996 to 2000 to 12% in 2003 to 2005) and massive replacement of the multiresistant Brazilian clone (ST239-IIIA) by the epidemic EMRSA-15 clone (ST22-IV), in which resistance to antibiotics other than beta-lactams is very rare, as the major clone (80% of isolates). The Iberian clone (ST247-IA), a major clone in 1992 to 1993, was represented in the present study by just one isolate. Two other pandemic MRSA clones were detected, as sporadic isolates, for the first time in our hospital: the New York/Japan (ST5-II) and the EMRSA-16 (ST36-II) clones. Furthermore, the pattern of susceptibility of MRSA isolates both to gentamicin and to trimethoprim-sulfamethoxazole was shown to be an excellent phenotypic marker for the discrimination of the EMRSA-15 clone from other nonmultiresistant MRSA clones present in our hospital.     

Wide geographic distribution of a unique methicillin-resistant Staphylococcus aureus clone in Hungarian hospitals

Objective: To determine the genetic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from six provincial hospitals in Hungary between 1993 and 1994.
Method: Molecular fingerprinting methods were used: hybridization with a mecA -specific DNA probe after Cla I restriction; hybridization with a probe for Tn 554 ; and pulsed-field gel electrophoresis after Sma I digestion of chromosomal DNA.
Results: All strains were resistant to penicillin, oxacillin, erythromycin, gentamicin, tetracycline, imipenem, and cephalosporins, and variably resistant to ofloxacin, clindamycin and tobramycin; all isolates were susceptible to vancomycin. Forty-eight of the 51 isolates carried the mecA gene as determined by Southern hybridization, using a mecA -specific DNA probe, indicating that the methodology used for initial identification may have been in error in three of the cases. Forty-seven of the 48 mecA -positive isolates showed very similar genetic backgrounds as defined by pulsed-field gel electrophoresis (PFGE) patterns after Sma I digestion of chromosomal DNAs: a unique PFGE pattern was seen in 32 isolates and minor variants of it in 15 additional isolates. All the 47 isolates carried the same mecA polymorph ( Cla I type III), as determined by DNA hybridization after Cla I digestion of chromosomal DNA. Only one of the MRSA isolates had a completely different PFGE pattern and a novel mecA polymorph.
Conclusions: The findings demonstrate the existence of a unique, epidemic MRSA clone, in both invasive and colonizing strains, which is widely dispersed in Hungarian hospitals hundreds of kilometers apart.     

Concordant clonal delineation of methicillin-resistant Staphylococcus aureus by macrorestriction analysis and polymerase chain reaction genome fingerprinting.

Pulsed-field gel electrophoresis of DNA macrorestriction fragments (macrorestriction analysis) allows epidemiologic typing and delineation of genetic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) by indexing variations in the global chromosome architecture. Polymerase chain reaction (PCR)-mediated genome fingerprinting can also discriminate MRSA strains by detecting locally variable DNA motifs. To assess the correlation between these methods, 48 epidemic MRSA strains collected from 20 hospitals over a 10-year period were tested in a blind comparison by (i) macrorestriction analysis with SstII or SmaI endonuclease and (ii) PCR fingerprinting with four primer sets aimed at the mecA gene, enterobacterial repetitive intergenic consensus sequences, and arbitrary sequences. Isolates were discriminated into 22 macrorestriction patterns and 15 PCR fingerprints. MRSA strains belonging to 12 distinct clones by macrorestriction analysis showed 11 distinct PCR genotypes distinguished by multiple band differences. In contrast, 34 of 37 MRSA strains found to be clonally related by macrorestriction analysis clustered in two highly related PCR genotypes that differed by a single DNA fragment (P < 0.0001). These data demonstrate concordant clonal delineation of epidemic MRSA by macrorestriction analysis and PCR fingerprinting and thereby indicate that the rapid PCR assay may be an efficient epidemiologic typing system.     

Clinical isolates of Staphylococcus aureus from the Arkhangelsk region,Russia: antimicrobial susceptibility,molecular epidemiology,and distribution of Panton‐Valentine leucocidin genes

A total of 91 consecutive clinical isolates of Staphylococcus aureus were collected at the Regional Hospital of Arkhangelsk, Russia, from May to December 2004, and examined for antimicrobial susceptibility, methicillin resistance and presence of Panton‐Valentine leucocidin (PVL) genes. Epidemiological typing was performed by pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Methicillin‐resistant S. aureus (MRSA) isolates were examined by staphylococcal cassette chromosome mec (SCCmec) typing. High‐to‐moderate rates of resistance to penicillin (β‐lactamase production; 93%), tetracycline (40%), erythromycin and clindamycin (32%) were observed. Forty out of ninety‐one (44%) isolates were positive for PVL genes. Thirty‐six (40%) PVL‐positive methicillin‐susceptible S. aureus (MSSA) strains were shown by PFGE and MLST typing (ST121, ST681, ST837) to be part of a nosocomial outbreak caused by clonal complex (CC) 121. PFGE, MLST and SCCmec typing revealed three MRSA clones. Sequence type (ST) 239‐III (n=11), ST1097‐III (n=1) and ST8‐IV (n=3) belong to CC8 of epidemic multiresistant MRSA, whereas ST426‐MRSA‐IV/CC395 (n=1) has not been reported previously. All MRSA strains were PVL negative. The overall results underline the necessity of microbiological sampling, antimicrobial susceptibility testing, and epidemiological typing as a rational basis for antimicrobial treatment of S. aureus infections, and infection control measures to limit the spread of multiresistant MRSA and epidemic MSSA clones.     

Clonal analysis and identification of epidemic strains of methicillin-resistant Staphylococcus aureus by antibiotyping and determination of protein A gene and coagulase gene polymorphisms.

Forty-three methicillin-resistant Staphylococcus aureus (MRSA) isolates with known genetic and epidemiological relatedness and different degrees of transmission were analyzed by antibiotyping, protein A gene polymorphism analysis, and coagulase gene polymorphism analysis. The three typing systems were evaluated for their performance and convenience to define clones and to discriminate between epidemic MRSA (EMRSA) and sporadic MRSA (SMRSA). Antibiotyping and AluI restriction fragment length polymorphism analysis of the coagulase gene were able to define clones in the same way as DNA macrorestriction analysis (SmaI). However, both techniques presented disadvantages, making neither of them useful as a single typing method. Protein A gene polymorphism analysis appeared to be of no value for clonal analysis. None of the three typing methods was able to differentiate between EMRSA and SMRSA.     

Diphtheria in the Republic of Georgia: Use of Molecular Typing Techniques for Characterization of Corynebacterium diphtheriae Strains

Sixty-six Corynebacterium diphtheriae strains (62 of the gravis biotype and 4 of the mitis biotype) isolated during the Georgian diphtheria epidemic of 1993 to 1998 and 13 non-Georgian C. diphtheriae strains (10 Russian and 3 reference isolates) were characterized by (i) biotyping, (ii) toxigenicity testing with the Elek assay and PCR, (iii) the randomly amplified polymorphic DNA (RAPD) technique, and (iv) pulsed-field gel electrophoresis (PFGE). Fifteen selected strains were ribotyped. Six RAPD types and 15 PFGE patterns were identified among all strains examined, and 12 ribotypes were found among the 15 strains that were ribotyped. The Georgian epidemic apparently was caused by one major clonal group of C. diphtheriae (PFGE type A, ribotype R1), which was identical to the predominant epidemic strain(s) isolated during the concurrent diphtheria epidemic in Russia. A dendrogram based on the PFGE patterns revealed profound differences between the minor (nonpredominant) epidemic strains found in Georgia and Russia. The methodologies for RAPD typing, ribotyping, and PFGE typing of C. diphtheriae strains were improved to enable rapid and convenient molecular typing of the strains. The RAPD technique was adequate for biotype differentiation; however, PFGE and ribotyping were better (and equal to each other) at discriminating between epidemiologically related and unrelated isolates.     

Typing of Staphylococcus aureus and Staphylococcus epidermidis strains by PCR analysis of inter-IS256 spacer length polymorphisms.

IS256 elements are present in multiple copies in the staphylococcal genome, either flanking the transposon Tn4001 or independent of it. PCR-based analysis of inter-IS256 spacer polymorphisms was developed for typing of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis strains. Using SmaI macrorestriction analysis resolved by pulsed-field gel electrophoresis (PFGE) as the reference method for MRSA typing, excellent reproducibility (100%), discriminatory power (97%), and in vivo stability were observed. Good concordance of the results with those of other molecular typing methods was found for two MRSA collections. Inter-IS256 PCR analysis of a U.S. collection of MRSA strains (n = 36), previously characterized by 15 typing methods, showed more limited discrimination. Agreement was 78% with PFGE analysis and 83% with ribotyping (HindIII). Analysis of a second set of Belgian MRSA strains (n = 17), categorized into two widespread epidemic clones by PFGE analysis, showed 65% agreement. For typing of S. epidermidis strains (n = 26), inter-IS256 PCR showed complete typeability (100%) and good discriminatory power (85%). Inter-IS256 PCR analysis is proposed as an efficient molecular typing assay for epidemiological studies of MRSA or S. epidermidis isolates.     

Epidemiologic typing and delineation of genetic relatedness of methicillin-resistant Staphylococcus aureus by macrorestriction analysis of genomic DNA by using pulsed-field gel electrophoresis.

To evaluate the usefulness of phenotypic and genotypic analyses for the epidemiologic typing of methicillin-resistant Staphylococcus aureus (MRSA), we characterized 64 epidemic MRSA isolates and 10 sporadic methicillin-susceptible S. aureus isolates from a university hospital and 18 MRSA isolates from hospitals in different geographical areas. Chromosomal DNA macrorestriction analysis with SstII was resolved by pulsed-field gel electrophoresis and compared with antibiotype analysis, phage type analysis, and standard genomic DNA restriction analysis with BglII. Indices of the discriminatory ability of these methods were 0.982, 0.959, 0.947, and 0.959, respectively. Macrorestriction patterns of 94% of MRSA isolates from patients, personnel, and the environment associated with a nosocomial outbreak were closely related (similarity coefficient, 85 to 100%). In contrast, methicillin-susceptible S. aureus isolates showed a marked diversity of macrorestriction patterns (median similarity, 41%). MRSA isolates from other geographical areas showed diverse macrorestriction patterns, with the exception of four isolates displaying identical or closely related patterns; these isolates were associated with concurrent outbreaks in four other Belgian hospitals. A concordance of genomic DNA macrorestriction typing with phenotypic methods was observed for 60 to 65% of MRSA isolates, and a concordance with standard DNA restriction analysis was found for 79 to 98% of these isolates. In conclusion, genomic DNA macrorestriction analysis was a useful complement to phenotypic methods for delineating epidemic isolates of MRSA, for identifying their nosocomial reservoirs, and for tracing their intra- and interhospital spread. The genetic relatedness of MRSA isolates, as estimated by this technique, appeared to correlate with their space-time clustering.     

Molecular typing of fluoroquinolone-resistant and fluoroquinolone-susceptible Escherichia coli isolated from blood of neutropenic cancer patients in a single center

Objectives: To investigate the molecular epidemiology of fluoroquinolone-resistant (FQ-R) and fluoroquinolone-susceptible (FQ-S) bacteremic Escherichia coli isolates from neutropenic patients by pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis.
Methods: Nineteen FQ-R and 27 FQ-S isolates of E. coli , obtained from patients on a hematologic ward over a 7-year period, were genotyped by PFGE and RAPD using two different random primers (1247 and 1283).
Results: PFGE analysis was able to type all FQ-S isolates and most (17/19, 89%) FQ-R isolates of E. coli . All isolates were genotypically unrelated, with the exception of two indistinguishable FQ-R isolates from different patients in the same period. RAPD analysis typed all isolates, including those FQ-R isolates untypable by PFGE, but was unable to distinguish between some isolates that were different by PFGE. Using primer 1247, RAPD analysis identified six pairs and one triad, while primer 1283 identified seven pairs and one triad of indistinguishable isolates.
Conclusions: No spread of epidemic FQ-R or FQ-S E. coli isolates was documented among neutropenic patients. RAPD analysis is a powerful genotyping method, but appeared to be less reproducible and discriminatory than PFGE for investigating E. coli isolates.     

Subtyping of methicillin-resistant Staphylococcus aureus isolates from the North-West of England: a comparison of standardised pulsed-field gel electrophoresis with bacteriophage typing including an inter-laboratory reproducibility study.

Bacteriophage typing is currently the recognised methodology for the typing of methicillin-resistant Staphylococcus aureus (MRSA) in the UK. Bacteriophage typing is less discriminatory and does not type all isolates compared with some molecular methods for typing MRSA. Chromosomal genotyping by pulsed-field gel electrophoresis (PFGE) is increasingly recognised as an improved method for typing MRSA, providing increased discrimination and typability. In this study the results of a comparison of bacteriophage typing and PFGE typing and subtyping are presented for a large collection of isolates from the North-West of England. Isolates belonging to the most frequently isolated epidemic methicillin-resistant Staphylococcus aureus (EMRSA) bacteriophage types 15 and 16 were typed by PFGE with further discrimination of common PFGE types possible into a number of subtypes. These results for a large collection of isolates demonstrate the improved typing of MRSA with PFGE. The widespread acceptance of PFGE for typing MRSA isolates has been hampered by the lack of standardised methodologies. Recently, a standardised PFGE strain typing system, known as the GenePath system has become available. The results of an inter-laboratory comparison of PFGE typing for a collection of isolates demonstrated good reproducibility with this system.     

Comparison of Typing Results Obtained for Methicillin-Resistant Staphylococcus aureus Isolates with the DiversiLab System and Pulsed-Field Gel Electrophoresis

We compared the results of typing methicillin-resistant Staphylococcus aureus (MRSA) isolates using the DiversiLab system (DL) to the results obtained using pulsed-field gel electrophoresis (PFGE). One hundred five MRSA isolates of PFGE types USA100 to USA1100 and the Brazilian clone, from the Centers for Disease Control and Prevention (CDC) and Project ICARE strain collections, were typed using DL. In addition, four unique sets of MRSA isolates from purported MRSA outbreaks that had been previously typed by DL, each consisting of six isolates (where five isolates were classified as indistinguishable by DL and one was an unrelated DL type) were typed by PFGE. DL separated the 105 MRSA isolates of known USA types into 11 clusters and six unique banding patterns. DL grouped most of the USA100, USA200, and USA1100 isolates into unique clusters. Multilocus sequence type 8 isolates (i.e., USA300 and USA500) often clustered together at >95% similarity in DL dendrograms. Nevertheless, USA300 and USA500 DL patterns could be distinguished using the pattern overlay function of the DL software. Among the hospital outbreak clusters, PFGE and DL identified the same “unrelated” organism in three of four sets. However, PFGE showed more pattern diversity than did DL, suggesting that two of the sets were less likely to represent true outbreaks. In summary, DL is useful for screening MRSA isolates to rule out potential outbreaks of MRSA in hospitals, but PFGE provides better discrimination of potential outbreak strains and is more useful for confirming strain relatedness and specific USA types.Although pulsed-field gel electrophoresis (PFGE) is often considered the gold standard for typing methicillin-resistant Staphylococcus aureus (MRSA) isolates for epidemiologic studies (8, 12, 13), PFGE requires several days to complete and the results are often difficult for inexperienced users to interpret. On the other hand, DNA sequence-based methods, such as spa typing, which has also been shown to be useful for epidemiologic studies of MRSA (3), are not practical for many clinical laboratories in the United States, which lack access to DNA sequencing facilities. An alternative strain typing method, which is available commercially, is the DiversiLab typing system (DL) (bioMérieux, Inc., Durham, NC), which uses the presence of DNA repetitive elements present in the organism''s genome to determine the genetic relatedness of bacterial and fungal isolates (4-6, 9, 18). DL has been used successfully in several MRSA typing studies to distinguish sporadic from outbreak-related isolates and is noted to be more rapid to perform and easier to learn than PFGE (14, 15). Agreement between DL clusters of organisms and USA PFGE types, as defined by McDougal et al. (12), was reported for five well-defined U.S. outbreaks, although specific data were not shown (14). However, a recent study of representative MRSA strains from the Harmony collection in Europe concluded that while DL, PFGE, and multilocus sequence typing (MLST) provided concordant classification of strains, PFGE showed a higher level of strain discrimination than either DL or MLST (17). Thus, whether DL can differentiate accurately among USA types remains an open question.The goal of this study was to use DL to characterize a series of MRSA isolates of known PFGE types from U.S. hospitals to determine whether DL could (i) differentiate among PFGE types USA100 through USA1100, (ii) identify DL banding patterns that correlated with specific USA types, and (iii) differentiate contemporary outbreak-related MRSA isolates from sporadic isolates collected from U.S. hospitals.     

Characterization of a possible nosocomial aspergillosis outbreak

Objective   To study the epidemiologic aspects of a suspected outbreak of nosocomial invasive aspergillosis.
Methods   Sixteen Aspergillus fumigatus strains were isolated from bronchoalveolar washings or sputa of 10 patients during a 9-month period. Furthermore, two environmental samples, isolated in a microbiological screening of the hospital, were also available for analysis. Random amplified polymorphic DNA analysis (RAPD) was carried out.
Results   The analysis performed by RAPD clearly demonstrated substantial genetic variation among the isolates. Both of the two different primers selected for RAPD analysis (R-108 and AP12h) were able to demonstrate that the strains isolated from all patients infected with the same fungal species and the environmental samples were genotypically distinct. The results by RAPD typing demonstrated that this technique could detect variability among isolates of Aspergillus fumigatus from different patients and even from the same patient.
Conclusions   RAPD genotyping proved that the outbreak of invasive aspergillosis consisted of a series of events, non-related, and probably not coming from the same source within the hospital. This type of analysis is an easy, quick and highly discriminatory technique that may help in planning epidemiologic studies of aspergillosis.