Molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) in a university hospital in Italy

The molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in a university hospital in Italy was studied in a five-month period in 1996, during which all S. aureus isolated were collected. All MRSA isolates (95) and a sample of methicillin-susceptible S. aureus (20) were typed with a variety of phenotypic and genotypic methods. Clonal identities were determined by pulsed-field gel electrophoresis (PFGE) of chromosomal SmaI digests and, for MRSA isolates, by probing ClaI digests with a mecA probe and a Tn554 probe. Overall, MRSA represented 32.3% of all isolates, with very high percentages from the intensive care units (adult and neonatal). PFGE after restriction with SmaI resolved genomic DNA of 95 MRSA strains into 26 major PFGE patterns. The use of southern blot hybridization of ClaI genomic digests with mecA and Tn554 allowed us a significant increase in discrimination, differentiating at least 32 different clones. Two major clones, however, each sharing common ClaI-mecA and Tn554 type and PFGE pattern as well as a common resistance phenotype, represented more than 50% of all MRSA isolates. The recovery of these two clones in the majority of the isolates of adult and neonatal intensive care units, respectively, is indicative of typical nosocomial outbreaks and clonal spread. It is concluded that intensive care units are major areas requiring preventative interventions.     

Comparison of traditional and molecular methods for typing nontoxigenic strains of Corynebacterium diphtheriae

Thirty-eight nontoxigenic strains of Corynebacterium diphtheriae isolated between 1987 and 1992 from clinical specimens of French patients were typed by biotyping, antibiograms, bacteriophage typing, ribotyping, and restriction analysis by pulsed-field gel electrophoresis (PFGE). Excellent correlation occurred between the genotypes defined by PFGE SfiI profiles or by ribotype BstEII profiles. Genotyping revealed seven genotype patterns among the 26 biotype mitis isolates, five among the nine biotype gravis isolates, and three among the three biotype belfanti isolates. Phage typing was nonreactive for nine of the 38 isolates. A combination of all the typing methods led to the identification of 19 different types of Corynebacterium diphtheriae.     

Insertion possibility of 16S-23S space amplification and random amplified polymorphic DNA analysis for typing of methicillin-resistant Staphylococcus aureus strains in the context of nosocomial infections

Within the scope of the present study n = 183 MRSA isolates from the extended area of Düsseldorf and n = 93 international MRSA strains from seven different countries were typed by pulsed-field gel electrophoresis and two PCR methods (RAPD and 16S-23S-spacer amplification). The isolates could be subdivided into 30 different types by PFGE, into 21 by means of RAPD and 18 by 16S-23S-spacer amplification. PFGE had the highest discriminatory potential, however, a combined use of the three typing methods allows a more detailed differentiation even of those isolates with identical PFGE pattern. Both amplification procedures were rapid, easy in handling with reproductable results. For a temporary epidemiological analysis within 24 hours, both amplification methods could be combined. In case the investigated isolates were still suspected of showing a "clonal identity", they should be analysed by additional PFGE (lasting about four days). Although the international isolates were chosen by random selection, several MRSA strains with identical pattern could be found in different countries of the world. Some RAPD-, spacer- and PFGE pattern were constant over many years. This reflects a high genetic stability of single strains.     

Molecular epidemiology of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a district hospital in Taiwan

Thirty-one of 104 clinical isolates of Klebsiella pneumoniae collected over a period of 8 months were found to be putative extended-spectrum beta-lactamase (ESBL) producers. Isoelectric focusing and an iodine overlay agar method were used for preliminary identification of the ESBLs. They were further identified by DNA sequencing. Seventy-one percent of the isolates were found to produce SHV-5. The variation in the ESBL patterns of these isolates was slight, with only five patterns being identified. The strains were typed by pulsed-field gel electrophoresis (PFGE), and 16 different genotypes were identified. When the PFGE patterns were analyzed by the algorithmic clustering method called the unweighted-pair group method using arithmetic averages, five clusters were found. However, significant genetic variations were found among 11 isolates and between each cluster. A plasmid of 36 kb was found in all clinical isolates and in the transconjugants. Our results indicate that the increase in the number of ESBL-producing K. pneumoniae isolates in this hospital is due mainly to the dissemination of a resistance plasmid rather than to the clonal spread of a few epidemic strains.     

Random amplified polymorphic DNA typing versus pulsed-field gel electrophoresis for epidemiological typing of vancomycin-resistant enterococci

Sixty vancomycin-resistant vanA mutant Enterococcus faecium (VRE) isolates, collected during a 40-month period from 48 patients hospitalized in a French Cancer Referral Center, were typed by using random amplified polymorphic DNA (RAPD), and the results were compared with those previously obtained by typing with SmaI pulsed-field gel electrophoresis (PFGE), which is currently recognized as the "gold standard." The discriminating power of RAPD typing, with seven primers and 11 combinations of primers, was tested on 18 strains, and only the most discriminating combination was further tested on the whole collection. We compared the epidemiological usefulness of RAPD typing of 60 clinical VRE isolates with that of SmaI PFGE typing. With primers AP4 and ERIC1R, RAPD generated 30 patterns versus the 36 patterns generated by SmaI PFGE. However, this did not hamper the epidemiologically correct clustering of 15 related strains and the detection of multiple colonization in nine patients. We conclude that this simple RAPD technique is well suited to the epidemiological typing of VRE and the monitoring of its nosocomial spread.     

Variations in glucocorticoid levels within the physiological range affect plasma leptin levels

In March 1996, an epidemic of Shigella sonnei infection occurred in Ooamishira-sato Town, Chiba Prefecture. Colicine typing, antibiotic resistance patterns, plasmid profiles, pulsed-field gel electrophoresis (PFGE) and random amplified poly-morphic DNA (RAPD) were used for the investigation of the epidemic. Ninety-four isolates from patients exhibited three different colicine types and five different antibiotic resistance patterns. But the patterns of plasmid profile, PFGE and RAPD were uniform among the isolates with different colicine type and antibiotic resistance pattern. It is possible that these isolates belonged to a single bacterial clone and circulated through human to human.     

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.     

Comparative classification of Acinetobacter baumannii strains using seven different typing methods

A group of 49 Acinetobacter baumannii strains obtained from several hospital outbreaks and some sporadic cases were typed by biotyping, antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), plasmid typing, multilocus enzyme electrophoresis, whole-cell protein profile, and Fourier-transform infrared (FT-IR) spectroscopy. All these methods have shown a high degree of reproducibility and are capable of recognising strains from the same epidemiological event. However, their power to discriminate between epidemiologically unrelated strains varies, with PFGE being superior to the other methods investigated. FT-IR spectroscopy, which has not yet been used for typing of Acinetobacter strains, proved to be a very rapid and highly reproducible method, but was somewhat limited in its discriminating power.     

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.     

Differentiation of Yersinia enterocolitica serotype O:5,27 strains by phenotypic and molecular techniques

Restriction endonuclease analyses of virulence plasmid DNA (REAP) and chromosomal DNA and other phenotypic characteristics were used to study the differentiation of Yersinia enterocolitica serotype O:5,27 strains. There was a close correlation between REAP patterns and the geographical distribution of serotype O:5,27. Human isolates produced only one REAP pattern, which was also found with isolates from pigs and dogs.     

Impact of control measures on the course of an outbreak of methicillin-resistant Staphylococcus aureus

BACKGROUND: Outbreaks of nosocomial infection by methicillin resistent Staphylococcus aureus (MRSA) are a problem in many hospitals with the control measures to be adopted being controversial. An outbreak of MRSA in a 550-bed university hospital is herein described and the impact of the adopted control measures on the evolution of the epidemic in the general hospitalization area (GHA) was analyzed. PATIENTS AND METHODS: The adopted control measures in the GHA were: microbiologic surveillance, cutaneous isolation measures, treatment of nasal carrier, and the early discharge of the cases. Hand washing was reinforced and a study of carriers was carried out on detection of sporadic cases (not related to the ICU). A molecular study of 70 strains of MRSA was performed with analysis of total plasmids, plasmid restriction pattern and chromosomic DNA analysis by pulsed field gel electrophoresis (PFGE). RESULTS: From December 1990 to December 1993, 273 cases of MRSA were reported. One hundred seventy-two cases originated in the ICU and 101 cases in the GHA (sporadic cases). The incidence of MRSA in 1991-1993 was 13.6, 14.3, and 6.6% in the ICU and 0.17, 0.36, and 0.15% in the GHA, respectively. Molecular study of MRSA isolates (1991 and 1992) demonstrated two plasmid and two chromosomic patterns. The latter had a similarity coefficient > 0.90, probably belonging to the same "clone". CONCLUSIONS: Despite the control measures adopted in the GHA the outbreak of MRSA originated in the ICU thereafter extending to the GHA. The rates of colonization detected, however, remained stable during the 3 years studied. On the other hand, the observation of a single "clone", responsible for the epidemic, suggest that most of the sporadic cases were autoctonous and due to failure in fulfillment of the established norms.     

Restriction endonuclease analysis of plasmid DNA of Yersinia pseudotuberculosis infections in Shimane Prefecture, Japan

The epidemiology of Yersinia pseudotuberculosis infections in a limited area of Shimane Prefecture, Japan, was examined by serotyping and restriction endonuclease analysis of virulence plasmid DNA of Y. pseudotuberculosis strains isolated from humans, wildlife animals and river water. Almost all isolates from three sources belonged to serotype 1b REAP pattern D and serotype 4b REAP patterns B, G and L. The identity of the distribution of serotype and REAP patterns among isolates from humans, wildlife animals and river water shows that Y. pseudotuberculosis is transmitted to humans through environmental substances contaminated by wildlife animals infected with this species.     

Genotyping of clinical isolates of MRSA by PFGE and AP-PCR

Genotyping of 67 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) separated from patients in a hospital in Mizusawa City in 1994 and 1995 was studied by pulsed field gel electrophoresis (PFGE) and arbitrarily primed-polymerase chain reaction (AP-PCR). Two main genotypes were observed by PFGE, and more than 70% of the 67 MRSA isolates produced coagulase type II. One group diverged well and gained higher tolerance in 1994, but was not isolated in 1995. The other group was continually isolated during the two-year period and showed moderate tolerance in 1994, and higher tolerance in 1995. AP-PCR was able to classify the genotypes of MRSA into 6 subgenotypes under the present conditions, which supported the results obtained by PFGE. These results suggest that AP-PCR could become a convenient and useful typing method by improving both sequence and length of a primer.     

Evaluation of pulsed-field gel electrophoresis as a typing system for Candida rugosa: comparison of karyotype and restriction fragment length polymorphisms

Nosocomial infections with Candida species have emerged as an increasingly important cause of morbidity and mortality in intensive care units. Ten Candida rugosa isolates from a previously documented cluster of C. rugosa infections in one hospital (nine burn unit isolates and one isolate from another hospital ward) and eight C. rugosa isolates recovered in a referral fungus testing laboratory (comparison isolates) from distinct geographic areas were investigated by molecular techniques. Isolates were from multiple anatomic sites. Pulsed-field gel electrophoresis (PFGE) of whole-cell DNA was performed with the 18 C. rugosa isolates as a marker of strain identity. The PFGE karyotypes of the C. rugosa isolates were demonstrated from four to seven chromosome bands. Karyotyping revealed the same PFGE pattern for the nine outbreak isolates from the burn unit, confirming clonal strain transmission. The isolate from the other hospital ward had a distinct karyotype. Distinct PFGE karyotype patterns were demonstrated for the eight comparison isolates. Restriction fragment length polymorphisms (RFLP) generated from whole-cell DNA digested with SfiI demonstrated the same RFLP pattern among outbreak isolates. Among comparison isolates, karyotyping distinguished some isolates that were indistinguishable by RFLP patterns. Karyotyping by PFGE appears to be the most useful molecular typing tool for discrimination among strains of C. rugosa and will be a useful marker for evaluating the epidemiology of future C. rugosa infections.     

Molecular analysis of glycopeptide-resistant Enterococcus faecium isolates collected from Michigan hospitals over a 6-year period

The purpose of this study was to evaluate the molecular relatedness of clinical isolates of glycopeptide-resistant Enterococcus faecium isolates collected from hospitals in Michigan. A total of 379 isolates used in this study were all vancomycin-resistant E. faecium isolates collected from 28 hospitals and three extended-care facilities over a 6-year period from 1991 to 1996. For the 379 isolates, there were 73 pulsed-field gel electrophoresis (PFGE) strain types. Within strain types, there were as many as six restriction fragment differences. Most isolates (70%) belonged to six strain types, which were designated M1 (36%), M2 (3%), M3 (18%), M4 (6%), M10 (4%), and M11 (3%). PFGE strain M1 was cultured from 135 patients in 13 hospitals during the period 1993 to 1996. Strain type M2 was cultured from 11 patients in two hospitals during the period 1991 to 1992 and was not observed after 1992. Strain type M3 was cultured from 70 patients in 10 hospitals during the period of 1994 to 1996. Both M4 and M10 were cultured from 23 patients in three hospitals and from 15 patients in two hospitals, respectively, during 1995 to 1996. M11 was cultured from 13 patients in four hospitals during 1996. A total of 23 of 28 hospitals had evidence of clonal dissemination of some isolates. Plasmid content and hybridization analysis done on 103 isolates from one hospital and two affiliated extended-care facilities indicated that the strains contained from one to eight plasmids. Mating experiments indicated transfer of vancomycin resistance from 94 of these isolates into plasmid-free E. faecium GE-1 at transfer frequencies of <10(-9) to 10(-4). Gentamicin resistance and erythromycin resistance were cotransferred at various frequencies. A probe for the vanA gene hybridized to the plasmids of 23 isolates and to the chromosomes of 72 isolates. A probe for the vanB gene hybridized to the chromosomes of 8 isolates. The results of this study suggest inter- and intrahospital dissemination of vancomycin-resistant E. faecium strains over a 6-year period in southeastern Michigan. The majority of isolates studied belonged to the same few PFGE strains, indicating that clonal dissemination was responsible for most of the spread of resistance that occurred.     

Epidemiological typing of isolates from an outbreak of infection with multidrug-resistant Enterobacter cloacae by repetitive extragenic palindromic unit b1-primed PCR and pulsed-field gel electrophoresis

An outbreak of multidrug-resistant Enterobacter cloacae infection lasted for 4 months in a neonatal intensive care unit (NICU). Forty-six isolates from the NICU and 20 epidemiologically unrelated strains were characterized by pulsed-field gel electrophoresis (PFGE) and repetitive extragenic palindromic unit b1-primed PCR (REPUb1-PCR) typing. The PFGE patterns after XbaI restriction of the bacterial DNA were analyzed by computer software (Gelcompar) using the UPGMA (unweighted pair group method with arithmetic averages) clustering method and the Dice coefficient. The 46 isolates from the NICU were classified by PFGE typing into five clusters: A (further classified into 7 subtypes, A1 to A7), B, C, D, and E. This outbreak was attributed to multiple genetically related strains of cluster A which had a similarity of 85.8% +/- 4.6%. The minor band differences among strains of cluster A were probably due to minor genetic mutations. The type A1 and A3 strains were isolated from the clinical specimens of patients and hands of nurses. It was probable that these outbreak strains were transmitted among patients via the hands of personnel. REPUb1-PCR typing of the 46 isolates also demonstrated five types, in agreement with results obtained by the PFGE technique, but could not detect the minor mutations among the cluster A strains. Twenty epidemiologically unrelated strains were well distinguished by both PFGE and REPUb1-PCR typing. We conclude that PFGE is a highly discriminatory but time-consuming method for epidemiological typing of E. cloacae and that REPUb1-PCR is a more rapid method with good reproducibility and discriminatory power comparable to that of PFGE.     

Multidrug-resistant Iberian epidemic clone of methicillin-resistant Staphylococcus aureus endemic in a hospital in northern Portugal

Forty-two methicillin-resistant Staphylococcus aureus (MRSA) isolates collected during 1992-1995 at a hospital in the north of Portugal were characterized by a variety of genomic fingerprints. Hybridization of ClaI and SmaI restriction digests with the mecA- and Tn554-specific DNA probes was used to identify polymorphs and determine their localization in chromosomal DNA preparations, and pulsed-field gel electrophoretic analysis of SmaI digests was used to determine chromosomal backgrounds. A major clone (and its variants) carrying the mecA polymorph I, Tn554 type E in the PFGE background of pattern A, accounted for 85% of all MRSA tested in 1992-1993 and 66% in 1994-1995. This clone is closely related to the epidemic Iberian clone that was associated with outbreaks in Spain during 1989-1993 and was endemic in 1992-1993 in two hospitals in Lisbon (Portugal).     

Genetic diversification of methicillin-resistant Staphylococcus aureus as a function of prolonged geographic dissemination and as measured by binary typing and other genotyping methods

The aim of the present study was to determine the extent of genome evolution among methicillin-resistant Staghylococcus aureus (MRSA) strains. Three different collections of strains were analysed, comprising locally, nationally and internationally disseminated genotypes. Various genotyping assays displaying different levels of resolution were used. Geographically and temporally diverse MRSA strains comprised the international group. MRSA strains recovered during an outbreak in a New York City hospital and Portuguese MRSA isolates, all resembling the so-called Iberian clone, were included in the local and national collections, respectively. Genotypes were determined by genome scanning typing techniques and procedures which analyse specific DNA elements only. The outbreak strains showed subclonal variation, whereas the Portuguese isolates displayed an increased number of genotypes. Among the epidemiologically unrelated MRSA strains, the different genotyping techniques revealed a wide heterogeneity of types. Different typing techniques appeared to show different levels of resolution, which could be correlated with the extent of geographic spread; the more pronounced the spread, the higher the degree of genome evolution. Binary typing and randomly amplified polymorphic DNA analysis are the typing methods of choice for determining (non)identity among strains that have a recent common ancestor and have undergone yet limited dissemination.     

Oxacillin- and quinolone-resistant Staphylococcus aureus in Sao Paulo, Brazil: a multicenter molecular epidemiology study

OBJECTIVE: To investigate the possibility of interhospital spread of multiresistant Staphylococcus aureus in Sao Paulo, Brazil. DESIGN: We evaluated 13 nosocomial S aureus strains selected because of resistance to oxacillin and ciprofloxacin. SETTING: The strains were collected between March 1991 and September 1991 from four different hospitals in Sao Paulo. Two were teaching hospitals, and two were private hospitals. PATIENTS: Each strain was isolated from a different patient. All patients were hospitalized when the strains were isolated. INTERVENTIONS: The strains were typed by restriction endonuclease analyses of plasmid DNA (REAP) using EcoRI, HindIII, RsaI, and AluI and by extended antibiogram profile (34 drugs). RESULTS: All strains had identical plasmid and antibiogram profile. They demonstrated the same plasmid pattern as previously described in one of the hospitals studied. CONCLUSIONS: Our results suggest the dissemination of a unique oxacillin- and quinolone-resistant strain of S aureus in several hospitals of Sao Paulo, Brazil.     

Use of the health history as a psychiatric screening tool

Epidemiologic supervision of P. aeruginosa infections is a daily care for microbiologists. The aim of this study is to evaluate quantitative antibiogram, which compare strains by calculation of euclidian distances from inhibition zone diameters on agar, compared with pulse field gel electrophoresis (PFGE) results. 53 isolates of P. aeruginosa obtained from bronchial secretions of 26 patients in intensive care unit are typed by conventional disk diffusion test on agar with 11 usual antibiotics. Dendrogram of euclidian distances is performed by Taxotron computer program (P.A.D. Grimont, Institute Pasteur, Paris); results are compared with 53 PFGE patterns after restriction with Spe I (Genepath system, BIO-RAD). Quantitative antibiogram has a high discriminatory power for beta-lactamin resistant strains: 27 isolates from 11 patients clustered in 5 types where PFGE discerned 5 patterns. The discriminatory power is low for beta-lactamin sensible strains: 26 isolates from 15 patients clustered in 8 types where PFGE discerned 19 patterns, but serotyping method combined with quantitative antibiogram restore a high disriminatory power for beta-lactamin sensible strains.