Pulsed-field Gel Electrophoresis Banding Patterns Research Articles

Long-term Persistence of Genetic Types of Mastitis-causing Staphylococcus aureus on Three Dairy Herds

Staphylococcus aureus remains a significant cause of mastitis in the dairy industry. It has been recommended that control of S. aureus mastitis be focused on the specific genetic types of S. aureus that most commonly cause the mastitis. Genetic types can be defined using molecular techniques, including pulsed field gel electrophoresis (PFGE). There have been comparatively few studies looking at within-herd persistence over time of S. aureus genetic types as causes of mastitis. Most have dealt with very few isolates or comparatively short periods of time. The objective of this investigation was to examine PFGE band patterns (EPs) from bovine mastitis-causing S. aureus isolates from three related herds over the course of 15 years to determine if any patterns persisted long-term.

Epidemiological and microbiological characterization of infections caused by Staphylococcus aureus with reduced susceptibility to vancomycin, United States, 1997-2001.

Infections caused by Staphylococcus aureus with reduced vancomycin susceptibility (SA-RVS; minimum inhibitory concentration [MIC], >or=4 microg/mL), including vancomycin-intermediate S. aureus (VISA; MIC, 8 microg/mL), are a new clinical and public health dilemma. Prospective surveillance and a nested case-control study of patients in the United States infected with SA-RVS was conduced from March 1999 through December 2000. Control patients were persons infected with oxacillin-resistant S. aureus (MIC of vancomycin, <or=2 microg/mL). Among 19 case patients, 4 infections were due to VISA and 15 were due to non-VISA SA-RVS. Case patients with and those without VISA infection had similar clinical presentations and outcomes; the overall attributable mortality rate was 63%. Isolates recovered from case patients had heterogeneous pulsed-field gel electrophoresis banding patterns, regardless of the MIC of vancomycin. Neither dialysis nor chronic renal failure were predictive of case status compared with control status. Independent risk factors for being a case patient included antecedent vancomycin use and prior oxacillin-resistant S. aureus infection 2 or 3 months before the current infection.

Epidemic spread of a single clone of methicillin-resistant Staphylococcus aureus among injection drug users in Zurich, Switzerland.

We describe an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) among injection drug users (IDUs). From August 1994 through December 1999, we registered 31 IDUs with MRSA infections (12 with soft-tissue infection, 7 with pneumonia [fatal in 1], 7 with endocarditis [fatal in 1], 2 with osteomyelitis, 2 with septic arthritis, and 1 with ulcerative tonsillitis), with a marked increase in the number of IDUs registered during 1998 and 1999. Of 31 patients, 15 (48%) were infected with human immunodeficiency virus. A point-prevalence study among IDUs who frequented outpatient facilities in Zurich revealed an MRSA carriage rate of 10.3% (range, 0%-28.6%) in various facilities. In all but 1 case, pulsed-field gel electrophoresis banding patterns of isolates obtained from these patients were indistinguishable from isolates of the initial 31 IDUs registered. Risk factors for MRSA carriage were disability and prior hospitalization in a hospice. In summary, MRSA became endemic in IDUs in Zurich as a result of the spread of a single clone. This clone caused major morbidity and was responsible for a lethal outcome in 2 cases.

Recurrent Staphylococcus aureus bacteremia: pulsed-field gel electrophoresis findings in 29 patients.

To identify risk factors for relapse among 309 prospectively identified cases of Staphylococcus aureus bacteremia, patients with recurrent S. aureus bacteremia were identified, and pulsed-field gel electrophoresis (PFGE) was performed on isolates from both episodes. PFGE banding patterns from both isolates were identical in 23 patients, consistent with relapsed infection. Patients with PFGE-confirmed relapse were more likely by both univariate and multivariate analyses to have an indwelling foreign body (odds ratio [OR]=18.2, 95% confidence interval [CI]=7. 6-43.6; P<.001), to have received vancomycin therapy (OR=4.1, 95% CI=1.5-11.6; P=.008), or be hemodialysis-dependent (OR=4.1, 95% CI=1. 8-9.3; P=.002) than patients who did not develop recurrent bacteremia. These results suggest that recurrent episodes of S. aureus bacteremia are primarily relapses and are associated with an indwelling foreign body, receiving vancomycin therapy, and hemodialysis dependence.

Molecular typing of methicillin-resistant Staphylococcus aureus in a university teaching hospital.

Plasmid analysis and pulsed-field gel electrophoresis (PFGE) were used to study the epidemiologic relationship among methicillin-resistant Staphylococcus aureus (MRSA) strains isolated at Tokyo Medical and Dental University Hospital. We found that 263 of 276 MRSA isolates had plasmids, which could be classified into 30 different patterns according to the number and plasmid molecular weight. Strains which harboured a single plasmid of approximately 13.4 Mds in molecular weight were the most numerous (55.7% of the isolates). These strains were isolated from 14 of 17 hospital wards. The largest number of strains with this plasmid pattern (33 strains) were isolated from a single ward. PFGE typing was then performed to further confirm the relationships among these 33 strains. The PFGE banding patterns of these strains were highly similar. The antibiogram profiles of these strains were also correlated with the PFGE pattern. Thus, the results suggest that these strains are epidemiologically related and spread throughout the ward. Combined plasmid analysis and PFGE were effective for discriminating the various MRSA isolates.

An outbreak of Serratia marcescens infections related to contaminated chlorhexidine.

An outbreak of Serratia marcescens infections occurred in a university tertiary-care hospital. Alcohol-free chlorhexidine solutions were contaminated with S marcescens. The majority of patient and chlorhexidine strains had similar pulsed field-gel electrophoresis banding patterns. Chlorhexidine was recalled, and the rate of S marcescens isolation returned to baseline. Chlorhexidine without alcohol should not be used as an antiseptic.

An Outbreak of Serratia marcescens Infections Related to Contaminated Chlorhexidine

An outbreak of Serratia marcescens infections occurred in a university tertiary-care hospital. Alcohol-free chlorhexidine solutions were contaminated with S marcescens. The majority of patient and chlorhexidine strains had similar pulsed field-gel electrophoresis banding patterns. Chlorhexidine was recalled, and the rate of S marcescens isolation returned to baseline. Chlorhexidine without alcohol should not be used as an antiseptic.

Evidence of genomic instability in Campylobacter jejuni isolated from poultry.

Poultry isolates of Campylobacter jejuni derived from a survey of meat processing batches were genotyped by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA to establish the clonal relationships between single-colony isolates. In the majority of batches studied, one or two genotype patterns predominated. However, in one batch (batch A), 21 single-colony isolates gave 14 different PFGE genotypes. The banding patterns obtained with SmaI were sufficiently different to distinguish between genotypes, although the patterns also produced many common bands. The question of whether these isolates represented different clones or had a common clonal ancestry was addressed by additional genotypic and phenotypic methods. Restriction length polymorphism of PCR products obtained from the flagellin genes showed an identical flagellin genotype for all of these isolates. In contrast, unrelated control isolates resulted in different flagellin genotypes. Moreover, all 14 different PFGE genotypes of batch A had identical Penner serotypes and identical or similar biotypes and phage types. It was concluded that the isolates were of clonal origin and that the diversity in the PFGE banding patterns had most likely originated from genomic rearrangements. However, the PFGE genotypes were shown to be stable upon subculturing in vitro and after in vivo passage in chickens, and natural transformation between isogenic mutants carrying antibiotic markers did not occur in vivo in a chick colonization model. The possible mechanisms for the hypothesized genomic recombinations and the conditions that allow, induce, or select for such events are discussed.

Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin

Since the discovery of the vancomycin-resistant Staphylococcus aureus (VRSA) strain Mu50 (minimum inhibitory concentration [MIC] 8 mg/L), there has been concern about the potential spread of such strains throughout Japanese hospitals. Two important questions need to be answered: (1) what is the prevalence of VRSA, and (2) by what mechanism does vancomycin resistance occur. The vancomycin susceptibilities of three methicillin-resistant S aureus (MRSA) strains (Mu50, Mu3, and H1) and the methicillin-susceptible S aureus type strain FDA209P were compared by MIC determinations and population analysis. Mu3 (MIC 3 mg/L) was isolated from the sputum of a patient with pneumonia after surgery who had failed vancomycin therapy. H1 (MIC 2 mg/L), which is a representative vancomycin-susceptible MRSA strain, was isolated from a patient with pneumonia who responded favourably to vancomycin therapy. Subclones of Mu3 with increased resistance against vancomycin were selected with serial concentrations of vancomycin and their MICs were determined. The prevalence of VRSA and Mu3-like strains in Japanese hospitals was estimated by population analysis from 1149 clinical MRSA isolates obtained from 203 hospitals throughout Japan. The genetic traits of the Mu3 and Mu50 strains were compared with clonotypes of MRSA from around the world. Mu3 and Mu50 had an identical pulsed-field gel electrophoresis banding pattern. When grown in a drug-free medium, Mu3 produced subpopulation of cells with varying degrees of vancomycin resistance, thus demonstrating natural heterogeneity, or variability, in susceptibility to vancomycin. In the presence of vancomycin, Mu3 produced subclones with resistance roughly proportional to the concentrations of vancomycin used. Selection of Mu3 with 8 mg/L or more of vancomycin gave rise to subclones with vancomycin resistance equal to that of Mu50 (MIC 8 mg/L) at a frequency of 1/1,000,000. During screening of Japanese MRSA strains, no strain of VRSA additional to Mu50 was found. The prevalence of MRSA isolates heterogeneously resistant to vancomycin was 20% in Juntendo University Hospital, 9.3% in the other seven university hospitals, and 1.3% in non-university hospitals or clinics. Heterogeneously resistant VRSA is a preliminary stage that allows development into VRSA upon exposure to vancomycin. Heterogeneously resistant VRSA was found in hospitals throughout Japan. This finding could explain, at least partly, the frequent therapeutic failure of MRSA infection with vancomycin in Japan.

Molecular epidemiology and antibiotic susceptibility of enterococci in Cincinnati, Ohio: a prospective citywide survey.

To determine patterns of antimicrobial susceptibility among enterococci and to assess molecular characteristics of vancomycin-resistant enterococci, 157 clinical blood isolates of enterococci from 10 hospitals in Cincinnati, Ohio, were prospectively collected during a 6-month period from February to July 1995. The isolates included 108 (69%) E. faecalis isolates, 46 (29%) E. faecium isolates, and 1 isolate each of E. avium, E. durans, and E. gallinarum. The E. faecalis and E. faecium isolates differed in their susceptibilities to ampicillin (100 versus 20%), ampicillin-sulbactam (100 versus 13%), vancomycin (100 versus 57%), imipenem (94 versus 2%), and high levels of gentamicin (59 versus 83%). Supplemental susceptibility testing of the 21 vancomycin-resistant isolates showed that 21 (100%) were susceptible to chloramphenicol and that only 7 (33%) were susceptible to doxycycline. Nineteen (90%) of the vancomycin-resistant E. faecium isolates were of the VanB phenotype, with vanB resistance genes detected by PCR and hybridization with gene-specific probes; and the E. gallinarum isolates demonstrated the VanC phenotype with the vanC1 gene. One vancomycin-resistant E. faecium isolate was highly resistant to both teicoplanin and vancomycin, corresponding to the VanA phenotype; however, it was found to have the vanB gene. Pulsed-field gel electrophoresis (PFGE) revealed that all of the 19 E. faecium isolates with the VanB phenotype had identical to closely related banding patterns. Hybridization of restriction enzyme-digested DNA separated by PFGE with a vanB gene probe demonstrated differences in the locations of vanB genes that corresponded closely to the PFGE banding patterns. Our study has documented that the emerging vancomycin resistance in our city was mainly due to the clonal dissemination of a single strain of E. faecium VanB.

A comparison of methods to determine whether clinical isolates of Staphylococcus epidermidis from the same patient are related

Staphylococcus epidermidis is a major cause of hospital-acquired infections but also part of the normal skin flora. A common clinical question is whether repeated isolation of S. epidermidis from one patient represents the same strain; because if different strains are isolated, they are often thought to be contaminants. In this study, different typing methods were compared to answer this question. Twenty isolates of S. epidermidis from five different patients were investigated. The isolates from each patient had identical or very similar antibiograms, and were recovered on different occasions. Typing was performed by antibiogram, biotype, slime production, plasmid profile, and pulsed-field gel electrophoresis (PFGE) banding pattern of SmaI digests of chromosomal DNA. In addition, the level of resistance to methicillin was determined by growth curves in broth containing methicillin for a series of different inocula for each isolate. The results showed that the isolates from each patient belonged to the same clone, but examples of instabilities in their antibiograms, plasmid profiles, as well as their PFGE banding patterns were seen. A change in the level of methicillin resistance was observed in one strain; otherwise this characteristic was found to be strain-specific and stable in vivo. It was concluded that in combination with biotyping and antibiotic resistance testing the level of resistance to methicillin could be used as an aid to distinguish between two or more clinical isolates of S. epidermidis from the same patient.

Genetic variability of Bacillus anthracis and related species

We evaluated the abilities of pulsed-field gel electrophoresis (PFGE) and sequences of intergenic spacer regions (ISRs) between two highly conserved genes, 16S-23S rDNA and gyrB-gyrA ISRs, to detect variation in strains of Bacillus anthracis as well as two closely related species, B. cereus ATCC 14579 and B. mycoides ATCC 6462. For each restriction enzyme, (NotI, SfiI, and SmaI), the PFGE banding patterns for three B. anthracis strains (Ames, Vollum, and Sterne) were identical. However, closely related species could be differentiated from B. anthracis and from each other. PCR amplification of the 16S-23S rDNA ISR yielded a 143- to 144-bp fragment, showing identical sequences for B. anthracis strains, one nucleotide deletion between B. cerus and B. anthracis, and 13 nucleotide differences between B. mycoides and B. anthracis. The gyrase ISR sequences (121 bp) in B. anthracis strains were also identical, but those in B. cereus and B. mycoides differed from that in B. anthracis by 1 and 2 nucleotides, respectively, and from each other by only 1 nucleotide. Given the diverse geographic origins of these B. anthracis strains, this species is very homogenous. We conclude that methods such as PFGE and sequences of ISRs may be useful in separating B. anthracis from closely related species, but more sensitive methods are needed for strain identification of B. anthracis.