Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Multilocus evolutionarily stable strategy models: additive effects

Forty illness associated phage-type (PT) 4 and PT 8 strains of Salmonella enteritidis were analyzed by the pulsed-field technique of clamped homogeneous electric fields (CHEF) electrophoresis. Using NotI and XbaI, the 40 strains were subdivided by each enzyme into seven restriction endonuclease digestion profiles (REDP). The 35 PT 4 isolates from Austria were subdivided into six NotI and five XbaI REDP, while the five PT 8 isolates from the United States displayed a single NotI and two XbaI REDP. When highly-concentrated, uncleaved genomic DNA was subjected to CHEF electrophoresis, plasmid DNA in the size range of 350 kb relative to a linear DNA standard was discernible in 38 of the 40 strains. Subsequent isolation and restriction analyses of plasmid DNA from one strain (E40) revealed a single plasmid (pE40; ca. 54 kb) with one XbaI and two NotI cleavage sites that was similar in size to the S. enteritidis virulence plasmid pRQ29. Hybridization of the PE40 probe with S. enteritidis genomic DNAs identified a 54 kb fragment within the XbaI REDP and two fragments, 20 and 34 kb, in NotI REDP of plasmid-positive strains. It was not possible to identify plasmid-specific bands in NotI REDP without hybridization due to comigrating chromosomal and plasmid DNA fragments. Regardless of PT, all 40 S. enteritidis strains showed highly related REDP. The similarity between PT 4 and PT 8 strains as further revealed by Dice similarity coefficients was 90% to 95% for NotI REDP and 79% to 93% for XbaI REDP. These results support the hypothesis that the pandemic observed today is the result of the efficient spread of a single clone, or clusters of closely related clones, of S. enteritidis.     

Nasal carriage of staphylococcus aureus and epidemiology of surgical-site infections in a Sudanese university hospital

Surgical site infections (SSI) due to Staphylococcus aureus among 256 male and 158 female patients (mean age, 28 years) undergoing elective surgery at the Soba University Hospital (Khartoum, Sudan) were studied. During an 11-month study period all patients were analyzed for nasal carriage of S. aureus at the time of admission. Follow-up of the development of SSI proceeded until 4 weeks after the operations. In addition, nasal swabs were obtained periodically during the same period from 82 members of the staff. In order to discriminate autoinfection from cross infection, bacterial isolates were typed by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments, and restriction fragment length polymorphism analysis of the protein A and coagulase genes. Preoperative cultures revealed the presence of S. aureus in the noses of 98 patients (24%). The overall number of postsurgical wound infections in the entire group was 57 (14%), 24 of which were due to S. aureus. Only 6 of the 98 nasal S. aureus carriers suffered from wound infections by the same species. In these six cases the infecting strain could not be genetically discriminated from the nasal inhabitant, substantiating autoinfection. However, nasal carriage of S. aureus is not a significant risk factor for the development of SSI in this setting (6 of 98 patients with autoinfection versus 18 of 316 patients [414 - 98 patients] with cross infection; P = 0.81), most probably due to the fact that noncarriers are at a significant and relatively large risk for acquiring an independent S. aureus SSI. The other S. aureus strains causing SSI showed a high degree of genetic heterogeneity, demonstrating that it is not an epidemic strain that is causing the SSI. Among the staff personnel screened, 47.4% did not carry S. aureus in the nose at any time during the study period, whereas 13. 2% persistently carried a single strain in the nose. Another 39.5% could be classified as intermittent carriers. When strains derived from staff personnel were genetically typed, it was demonstrated that most of the strains represented genetic variants clearly differing from the isolates causing SSI. On the other hand, possible cross colonization among staff personnel and even cross infection from staff personnel to patients or from patient to patient were demonstrated in some cases, but epidemic spread of a single strain or a few clonally related strains of S. aureus could be excluded.     

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.     

Intercontinental spread of a multidrug-resistant methicillin-resistant Staphylococcus aureus clone

Two hundred ten methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered between 1990 and 1997 from three Portuguese hospitals located in Lisbon and Oporto were analyzed by molecular fingerprinting techniques. The hybridization of ClaI restriction digests with the mecA- and Tn554-specific DNA probes combined with pulsed-field gel electrophoresis documented the abrupt appearance and extensive intrahospital spread of the Brazilian epidemic MRSA clone in the 1995 samples of each one of the three hospitals analyzed-suggesting the intercontinental transfer of this strain from Brazil to Portugal. The appearance of this clone may challenge the dominance of another highly epidemic imported clone-the Iberian MRSA, currently the most widely spread MRSA clone in Portuguese hospitals.     

Acute opioid dependence in the cardiovascular system of the spinal rat

A cluster of infections caused by Enterobacter cloacae was observed among preterm neonates in a neonatal intensive care unit (NICU) of a pediatric hospital in Osnabrück, Germany. The presence of similar antimicrobial susceptibility patterns among the bacterial isolates prompted an investigation to determine whether a limited spread of a single strain existed. All 12 E. cloacae isolates from the NICU and 50 nonrelated strains were fingerprinted by small-fragment restriction endonuclease analysis (SF-REA) of EcoRI DNA digests. Selected isolates were further characterized by pulsed-field gel electrophoresis (PFGE) of NotI- or XbaI-generated genomic restriction fragments. Epidemiologically unrelated strains were clearly discriminated by both methods. Results achieved by SF-REA and PFGE revealed that of the 12 isolates from the NICU, 11 belonged to the same genotypic cluster. Since all reagents and equipment for both techniques are commercially available, DNA fingerprinting by SF-REA or PFGE is proposed as a useful tool in the microbiology laboratory for investigating the epidemiological relatedness of E. cloacae strains of clinical and environmental origin.     

Two native plasmids of Pseudomonas syringae pathovar tomato strain PT23 share a large amount of repeated DNA, including replication sequences

Strain PT23 of Pseudomonas syringae pv. tomato contains four native plasmids, designated A, B, C, and D. By DNA hybridization of genomic and plasmid DNA digests from the wild type and a plasmid-cured strain, we determined that c. 61 kb (c. 74%) of pPT23B is repeated in pPT23 A and only c. 17 kb (c. 21%) is in single copy in strain PT23. pPT23B also contains DNA repeated in the chromosome that occurs in three DNA fragments of 0.6, 4.6, and 9.6 kb that might be transposable elements. Additionally, the 9.6 kb fragment also shares sequences with the three other plasmids of strain PT23. By DNA hybridization with the origin of replication from a native plasmid of P. syringae pv. syringae and in vivo replication tests, we identified the origins of replication of plasmids A, B, and D and showed that they cross-hybridize. The putative par region from pPT23 A has also been identified and is not conserved in the other three native plasmids from strain PT23. By using the defined minimal origin of replication from pPT23 A as a probe, we showed that it is highly conserved in 14 strains belonging to nine different pathovars of P. syringae and that as many as five different native plasmids with closely related origins of replication coexist in the same cell. The duplication and reorganization of plasmids might therefore occur at high frequency and could be responsible for the existence of large numbers of native plasmids in P. syringae strains.     

Clonal heterogeneity, distribution, and pathogenicity of methicillin-resistant Staphylococcus aureus

Four thousand eighty-eight Staphylococcus aureus isolates obtained from patients hospitalised in a university clinic and four community hospitals over a period of one year were screened for methicillin resistance. A resistance rate of 5% was detected among initial isolates. Distribution of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus showed an increased prevalence of MRSA in clinically significant specimens such as blood, central venous catheter tips, bronchial secretions, and wound secretions. Typing of 110 MRSA strains (initial isolates) by macrorestriction analysis of chromosomal DNA revealed 26 different genotypes that could be divided into five epidemic and 21 sporadic strains. More than 50% of all isolates belonged to one type that was confirmed to be closely related to the "southern-German" epidemic strain. Production of virulence factors such as enterotoxin A-D and toxic shock syndrome-toxin 1 among MRSA strains (initial isolates) occurred in ten of 26 different MRSA types. A strong correlation between genotype and toxin production was demonstrated.     

Molecular epidemiology of Staphylococcus aureus and Enterococcus faecalis in endophthalmitis

Genomic DNA fingerprint analysis was performed on 39 Staphylococcus aureus and 28 Enterococcus faecalis endophthalmitis isolates collected from multiple clinical centers. Among 21 S. aureus genomic DNA fingerprint patterns identified, five clonotypes were recovered from multiple unrelated patients and accounted for 58.9% (23 of 39) of the isolates analyzed. Compared with strains having unique genomic DNA fingerprint patterns, the S. aureus clonotypes occurring more than once were more likely to result in visual acuities of 20/200 or worse (P = 0.036 [chi2 test]). In contrast to the S. aureus isolates, the E. faecalis endophthalmitis isolates were a clonally diverse population, enriched for the expression of a known toxin, cytolysin, which is plasmid encoded.     

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.     

Molecular organization of the alcohol dehydrogenase loci of Drosophila grimshawi and Drosophila hawaiiensis

A total of 71 Staphylococcus aureus strains isolated from bovine mammary glands were identified and subtyped. The methods used to differentiate between the S. aureus isolates were the DNA polymorphism pattern after amplification with a Polymerase Chain Reaction using several primer combinations and phage typing. The DNA fingerprinting technique using RAPD, ERIC1R and ERIC primers proved to be useful in differentiating isolates of S. aureus. Differentiation of isolates using phage typing gave no additional information compared to the DNA technique. The outbreak of S. aureus in the herd studied was mainly caused by one S. aureus strain. Other strains were only found on three occasions, twice in subclinical infections and once from a case of clinical mastitis. In the latter case the dominant strain was isolated from a different quarter of the same cow. Four of the ten cows studied suffered from clinical mastitis. From those four cows, three remained infected with the same S. aureus strain despite antibiotic treatment.     

Revised estimates of enterococcal chromosomal sizes

We previously reported that the chromosomal sizes of four strains of enterococci ranged from 2,045 to 2,761 kb. Extensive analysis and mapping subsequently confirmed the size of Enterococcus faecalis strain OG1 as 2,825 kb (prior size estimate range, 2,750-2,761 kb) (Murray et al., J. Bacteriol. 175, 5216, 1993). However, using variable conditions of electrophoresis and additional digestions, revised size estimates for the other strains are 2,852-3,093 kb for E. faecalis strain JH2-2 (prior range, 2,008-2,135 kb), 2,910-3,065 kb for E. faecalis strain HH67 (prior range, 2,170-2,288 kb), and 2,334-2,558 for E. faecium strain GE-1 (prior range, 2,045-2,155 kb). The earlier underestimations of the chromosomal sizes were due to the inconsistent presence of a large fragment, likely caused by shearing of the DNA during handling, causing it to be considered a partial digestion product, and failure to resolve multiple fragments of the same approximate size.     

Rapid identification and typing of Staphylococcus aureus by PCR-restriction fragment length polymorphism analysis of the aroA gene

The Staphylococcus aureus aroA gene, which encodes 5-enolpyruvylshikimate-3-phosphate synthase, was used as a target for the amplification of a 1,153-bp DNA fragment by PCR with a pair of primers of 24 and 19 nucleotides. The PCR products, which were detected by agarose gel electrophoresis, were amplified from all S. aureus strains so far analyzed (reference strains and isolates from cows and sheep with mastitis, as well as 59 isolates from humans involved in four confirmed outbreaks). Hybridization with an internal 536-bp DNA fragment probe was positive for all PCR-positive samples. No PCR products were amplified when other Staphylococcus spp. or genera were analyzed by using the same pair of primers. The detection limit for S. aureus cells was 20 CFU when the cells were suspended in saline; however, the sensitivity of the PCR was lower (5 x 10(2) CFU) when S. aureus cells were suspended in sterilized whole milk. TaqI digestion of the PCR-generated products rendered two different restriction fragment length polymorphism patterns with the cow and sheep strains tested, and these patterns corresponded to the two different patterns obtained by antibiotic susceptibility tests. Analysis of the 59 human isolates by our easy and rapid protocol rendered results similar to those of other assays.     

Lead intoxication in children with pervasive developmental disorders

Ten clinical isolated strains of Human cytomegalovirus (HCMV) were obtained from 73 urine specimens of different people. Isolations from urine were carried out in human embryolung fibroblasts. Viral isolates were passaged four times. HCMV DNAs of laboratory strain AD169 and 10 clinical isolated strains were extracted with Hirt method, digested with each of the restriction enzymes EcoRI, Hind II. Comparison of restriction fragment length polymorphism (RFLP) of AD169 and isolated strains were made by hybridizing digested DNA with 32P labeled with HCMV Hind II cloned subgenomic fragments as the probe (pCM1035, pCM1015). pCM1035 is located in the joining region between the long(L) and short (S) unique sequences of the virus (L-S junction) pCM1015 is located in the terminal sequences of the virus. The results showed the genomic high degree of homology existed among all strains and the variable restriction site was in the L-S junction and terminal portion. The RFLP patterns of the clinical isolates which did not have relation in epidemiology were different, but the patterns of clinical isolates related in epidemiology were quite similar. Polymorphism frequently occurred in this case of EcoRI digested fragment hybridized with the probe of pCM1035. Southern hybridization of HCMV isolations is useful to researches into the molecular epidemiology and pathogenesis of HCMV infection.     

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.     

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.     

Genotype analysis of enterohemorrhagic Escherichia coli O157:H7 strains isolated in Iwate Prefecture from 1996 to February 1997

Genotypes of 38 isolates of enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolated from 11 sporadic cases and one outbreak in Iwate Prefecture from 1996 to February 1997 were studied by pulsed-field gel electrophoresis (PFGE), in comparison with a strain of EHEC O157:H7 isolated in 1992 in Ohazama-Cho, Iwate Prefecture, and two isolates of EHEC O157:NM. In order to substantiate the genotypes classified by PFGE, Southern blotting was performed to investigate integration sites of the Shiga toxin genes (stx) in the XbaI-digested genome DNA fragments. The stx1 gene existed on an approximately 130 kb fragment in all isolates except two ones. On the other hand, the stx2 gene was observed on 11 DNA fragments in different length from 600 kb to 155 kb, indicating that the stx2 gene integrates into more heterogeneous sites of genome DNA than stx1 does. From these analyses, EHEC O157:H7 isolates examined were classified into 7 genotypes. Since half of the isolates were the same genotype as that of the isolate in 1992, it is suggested that this type of EHEC O157:H7 strain is expanding from Ohazama-Cho and Morioka City in Iwate Prefecture.     

The distribution of adipose tissue and the metabolic profile in hypertensive and normotensive adults

Fifty isolates belonging to the six validly described species of the genus Thermus (T. aquaticus, T. filiformis, T. thermophilus, T. scotoductus, T. brockianus, and T. oshimai) isolated from hot springs of different geographical areas were compared using macrorestriction analysis of genomic DNA and ribotyping. With the exception of presumed clones, the macrorestriction patterns of isolates obtained with EcoRI or NdeI were distinct. However, isolates belonging to the same species exhibited similar profiles particularly when they were isolated from the same hot spring. The estimated genomic size of strains of the Thermus spp. varied between approximately 1.8 and 2.5 Mbp. Ribotyping with BamHI and HindIII produced 30 and 35 distinct ribotypes, respectively. In spite of the variability of the hybridization patterns produced, the ribotypes obtained for isolates belonging to the same species also shared, in general, several fragments of identical size, and these fragments were similar when isolates originated from the same spring.     

Widespread detection of PER-1-type extended-spectrum beta-lactamases among nosocomial Acinetobacter and Pseudomonas aeruginosa isolates in Turkey: a nationwide multicenter study

We studied the prevalence and molecular epidemiology of PER-1-type beta-lactamases among Acinetobacter, Klebsiella, and Pseudomonas aeruginosa strains isolated over a 3-month period in eight university hospitals from distinct regions of Turkey. A total of 72, 92, and 367 Acinetobacter, Klebsiella, and P. aeruginosa isolates were studied, respectively. The presence of blaPER was determined by the colony hybridization method and later confirmed by isoelectric focusing. We detected PER-1-type beta-lactamases in 46% (33/72) of Acinetobacter strains and in 11% (40/367) of P. aeruginosa strains but not in Klebsiella strains. PER-1-type enzyme producers were highly resistant to ceftazidime and gentamicin, intermediately resistant to amikacin, and susceptible or moderately susceptible to imipenem and meropenem. Among PER-1-type-beta-lactamase-positive isolates, five Acinetobacter isolates and six P. aeruginosa isolates from different hospitals were selected for ribosomal DNA fingerprinting with EcoRI and SalI. The EcoRI-digested DNAs were later hybridized with a digoxigenin-labelled PER-1 probe. The ribotypes and the lengths of blaPER-carrying fragments were identical in four Acinetobacter strains. A single isolate (Ac3) harbored a PER gene on a different fragment (approximately 4.2 kbp) than the others (approximately 3.4 kbp) and showed a clearly distinguishable ribotype. Ribotypes of P. aeruginosa strains obtained with EcoRI showed three patterns. Similarly, in Pseudomonas strains two different EcoRI fragments harbored blaPER (approximately 4.2 kbp in five isolates and 3.4 kbp in one isolate). PER-1-type beta-lactamases appear to be restricted to Turkey. However, their clonal diversity and high prevalence indicate a high spreading potential.     

Persistence of Staphylococcus aureus strains among cystic fibrosis patients over extended periods of time

Pulsed-field gel electrophoresis (PFGE) of SmaI macrorestriction fragments of chromosomal DNA was used to confirm the persistence of methicillin-sensitive Staphylococcus aureus isolates in the sputum of 25 cystic fibrosis patients in five French hospitals. Three-to-eight consecutive isolates, with the same esterase electrophoretic type isolated from each patient over a period of 12-28 months, were analysed. Consecutive isolates with indistinguishable PFGE profiles were found in 12 patients (48%) and consecutive isolates with similar PFGE profiles showing minor differences of one-to-four fragments (similarity coefficient >/=84%) were found in 11 patients. Consecutive isolates with different PFGE profiles were obtained from only two patients, but the profiles found in each patient were more closely related to each other than to other profiles. The results were in agreement with esterase electrophoretic typing for 23 patients, and we considered that those patients were infected with a single persistent strain. For any given patient, variations in antibiotypes and phage types of consecutive isolates were not associated with major genotypic variations. PFGE is useful in confirming the persistence of S. aureus strains in cystic fibrosis patients over long periods.