Comparison of DNA fingerprinting by PFGE and PCR-RFLP of the coagulase gene to distinguish MRSA isolates

Staphylococcus aureus isolates were collected from epidemiologically unrelated clinical sources in Japan between 1991 and 1993. A total of 40 isolates, five each of eight coagulase types, were analysed by polymerase chain reaction (PCR) of the coagulase gene, PCR-restriction fragment length polymorphism (RFLP) after AluI digestion, and pulsedfield gel electrophoresis (PFGE) of chromosomal DNA after SmaI digestion. The efficiency of discrimination among the isolates increased in the order of PCR < PCR-RFLP < PFGE, yielding five, 13 and 31 different types, respectively. To assess the clinical use of these methods, 42 additional methicillin-resistant S. aureus (MRSA) isolates collected from 27 inpatients in a hospital were analysed. PFGE and PCR-RFLP were able to discriminate 11 and four types, respectively. PFGE analysis detected cross-infection between four postoperative patients in an intensive-care unit, and in six neonates in intensive care. We conclude that of the three methods tested, PFGE analysis currently allows the most effective discrimination of MRSA strains.     

应用IRS-PCR对金黄色葡萄球菌分型的研究

目的探讨低频限制性位点聚合酶链反应(infrequent-restriction-site PCR，IRS-PCR)在金黄色葡萄球菌(简称金葡菌)基因分型中的应用价值。方法建立本实验室IRS-PCR方法。同时用IRS-PCR和脉冲场凝胶电泳(PFGE)对金葡菌进行基因分型。根据49株社区感染分离菌的分型结果计算辨别力指数(ID)值估计分辨力。对其中30株社区感染菌重复实验一次估计重复性。比较两种基因分型方法的分型率、分辨力、重复性、结果的一致率及操作特点。结果建立IRS-PCR对金葡菌基因分型的方法。70株金葡菌均可被2种方法分型，分型率100％。IRS-PCR分为38个型，21株院内感染菌分为6个型，49株社区感染菌分为32个型，计算ID值为0．981。PFGE分为40个型，21株院内感染菌分为6个型，49株社区感染菌分为34个型，计算ID值为0．983。两种分型方法的重复性均为100％。对院内感染菌，两种方法分型的一致率为100％；对社区感染菌，两种方法分型的一致率为92％(45／49)。与PFGE相比，IRS-PCR更简单、省时、易于操作、不需特殊昂贵仪器。结论IRS-PCR能对金葡菌简易快速可靠分型，适合检验科对临床标本的快速有效分型，是一种有价值的分子流行病学研究工具。     

Comparison of multiple-locus variable-number tandem repeat analysis and pulsed-field gel electrophoresis in a setting of polyclonal endemicity of vancomycin-resistant Enterococcus faecium.

In order to assess whether multiple-locus-variable number tandem repeat analysis (MLVA) could replace pulsed-field gel electrophoresis (PFGE) for genotyping vancomycin-resistant isolates of Enterococcus faecium (VREF), this study compared the typeability, discriminatory power, concordance and costs of these methods for VREF isolates obtained from patients, environmental samples and the hands of healthcare workers (HCWs) in a medical intensive care unit (ICU) where VREF was endemic. Over a 58-day period, 393 VREF isolates (373 vanA, one vanA/B, 19 vanB) were cultured from patient rectal swabs (n = 76), the environment (n = 270) and the hands of HCWs (n = 47). PFGE was able to divide 358 (91.1%) isolates into 19 PFGE types (>six bands different) and 24 subtypes (one to three bands different). MLVA was able to type 391 (99.5%) isolates into 11 genotypes. The discriminatory power of PFGE subtypes was 83%, as compared to 68% for MLVA. Concordance between the two methods, based on matched or mismatched MLVA types and PFGE types or subtypes, was 67.5% and 82.8%, respectively. Using PFGE, 13 isolates could be genotyped in 3 days; MLVA genotyped 94 isolates in 2 days. For both methods, the estimated costs were Euro 7 ($10)/isolate. PFGE and MLVA produced highly concordant results when assigning genotypes to nosocomial VREF isolates. MLVA was faster, but PFGE subtyping was more discriminatory.     

Localization of yeast glucoamylase genes by PFGE and OFAGE

Summary Chromosomes of two closely related yeast strains, the amylolytic Saccharomyces diastaticus and the non-amylolytic Saccharomyces cerevisiae, were resolved by pulsed field gel electrophoresis (PFGE) and orthological field alteration gel electrophoresis (OFAGE). Electrophoretic karyotypes of these two strains are identical. Sixteen cloned Saccharomyces genes of known chromosomal location were used to identify individual chromosomes by Southern hybridization analyses. The Southern blots were reprobed with a cloned fragment of the STA2 glucoamylase gene of S. diastaticus. STA2 exhibits homology to STA1 and STA3 as well as the sporulation-specific glucoamylase (SGA) gene from both Saccharomyces strains. The three unlinked, homologous genes, STA1 (DEX2, MAL5), STA2 (DEX1) and STA3 (DEX3) encoding the extracellular glucoamylase isozymes GAI, GAII and GAIII in S. diastaticus were then assigned to chromosomes IV, II and XIV, respectively. The SGA gene, encoding an intracellular glucoamylase in both S. diastaticus and S. cerevisiae, was assigned to chromosome IX. Electrophoretic mapping of the STA and SGA genes is at present the only way to localize these genes, since glucoamylase repressor gene(s) (STA10, INH1 and/or IST2) are present in most laboratory strains of S. cerevisiae and the SGA phenotype is only detectable during sporulation.     

Prevalence of emm types 1 and 12 from invasive Streptococcus pyogenes disease in Greece—results of enhanced surveillance

Among a total of 101 isolates from the first systematic multicentre surveillance effort concerning invasive Streptococcus pyogenes disease in Greece, conducted between 2003 and 2005 and covering 38% of the population, emm types 1 and 12 were prevalent, being responsible for 27 and nine cases, respectively. The isolates from the remaining 65 cases were assigned to 26 other emm types. Erythromycin resistance (12 isolates) was primarily mef (A)-mediated, although all emm type 1 strains were susceptible. Tetracycline resistance, due mostly to tet (M), was detected in 26 isolates. Subtyping by pulsed-field gel electrophoresis yielded 50 chromosomal fingerprints, thus discriminating further among ten of the 28 observed emm types.     

A fluorescent amplified fragment length polymorphism study of Salmonella enterica serovar Sofia, the major Salmonella serovar isolated from chickens in Australia

Fluorescent amplified fragment length polymorphism (FAFLP) analysis was performed on 68 isolates of Salmonella enterica subsp. salamae serovar Sofia (S. Sofia). Fifty eight isolates were obtained over a period of approximately 15 years from a range of human, chicken industry and environmental sources throughout Australia. A further ten isolates were identified from human and poultry sources in Israel from 1972 to 1987. Analysis of FAFLP profiles for fragments between 50 to 500 base pairs in length indicated distinct clusters of isolates. All but seven isolates clustered into four groups of >90% similarity and all isolates displayed at least 70% similarity with each other. No cluster could be attributed to a particular geographical, temporal or source-of-isolation origin. It is concluded that S. Sofia is genetically variable with certain clones persisting over time but no group appears unique to Australia.     

Isolation,characterization and chromosomal mapping of an actin gene from the primitive red alga Cyanidioschyzon merolae

Based on the results of cytological studies, it has been assumed that Cyanidioschyzon merolae does not contain actin genes. However, Southern hybridization of C. merolae cell-nuclear DNA with a yeast actin-gene probe has suggested the presence of an actin gene in the C. merolae genome. In the present study, an actin gene was isolated from a C. merolae genomic library using a yeast actin-gene probe. The C. merolae actin gene has no intron. The predicted actin is composed of 377 amino acids and has an estimated molecular mass of 42003 Da. Southern hybridization indicated that the C. merolae genome contains only one actin gene. This gene is transcribed at a size of 2.4 kb. When Southern hybridization was performed with C. merolae chromosomes separated by pulsed-field gel electrophoresis, a band appeared on unseparated chromosomes XI and XII. A phylogenetic tree based on known eucaryote actin-gene sequences revealed that C. merolae diverged after the division of Protozoa, but before the division of Fungi, Animalia and Chlorophyta.     

Clonality among ampicillin-resistant Enterococcus faecium isolates in Sweden and relationship with ciprofloxacin resistance

Objective  To investigate clonal relationships in a nationwide sample of human Enterococcus faecium isolates.
Methods  Biochemical fingerprinting (PhP (PhenePlate) typing) was used to compare 180 fecal ampicillin-resistant E. faecium (ARE) isolates with 169 matched fecal ampicillin-susceptible E. faecium (ASE) isolates from patients in 23 hospitals, collected in 1998, and to study 39 fecal ARE isolates from non-hospitalized individuals collected in 1998, and five ARE and 29 ASE isolates from the early 1990s. Representative ARE and ASE isolates were subjected to pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA and sequencing of the regions encoding the fluoroquinolone targets of the enzymes GyrA and ParC.
Results  Both PhP and PFGE results showed a higher homogeneity among ARE than among ASE isolates ( P  < 0.001). One PhP type (FMSE1) comprised 73% of the hospital ARE isolates (53% of ARE isolates from non-hospitalized individuals, and four of five ARE isolates from the early 1990s), but only 1% of the ASE isolates. PFGE of the hospital E. faecium isolates revealed that 23 of the 25 ARE isolates and one of the 22 ASE isolates were of one dominating type. High-level resistance to ciprofloxacin (MIC > 16 mg/L) was present in 91% of ARE isolates, whereas only low-level resistance (MIC 4–16 mg/L; 35% of isolates) was found among ASE isolates. One mutation in parC (codon 80) and one of two mutations in gyrA (codons 83 or 87) were detected in all ARE isolates tested with high-level ciprofloxacin resistance, but were lacking in ARE and ASE isolates with low-level ciprofloxacin resistance.
Conclusion  Most ARE isolates in Sweden were clonally related. High-level ciprofloxacin resistance was found in ARE isolates of PhP type FMSE1 as well as in other PhP types, but never in ASE isolates.     

International spread of major clones of methicillin resistant staphylococcus aureus: nosocomial endemicity of multi locus sequence type 239 in Saudi Arabia and Romania

Phenotypically identified methicillin resistant Staphylococcus aureus (MRSA) strains from several hospitals in Romania and Saudi Arabia (n = 103 and 68, respectively) were confirmed to be MRSA by mecA PCR and PBP-2' based latex agglutination. Subsequently, strains were differentiated at the sub-species level using pulsed field gel electrophoresis (PFGE) of SmaI DNA macro-restriction fragments. Comparison of the PFGE fingerprints identified major clusters of strains, persistently present in the various hospitals. Endemicity of certain strains was identified, amongst others one due to a particularly methicillin resistant type in the burn wound sector of the Romanian hospital. No PFGE-based overlap was found between the Saudi and Romanian strains. However, multi locus sequence typing (MLST), performed for 20% of all strains, revealed that genuine genetic similarity was obscured by the PFGE analysis. In both the Romanian and Saudi hospitals the renowned sequence type (ST) 239 was very over-represented. This was especially apparent in Saudi Arabia, where all strains except two shared the ST 239 genotype. This clonal type has previously been identified in a variety of other countries. Despite the MLST concordance, PFGE data indicate that ST 239 diversifies while maintaining its core genome intact. ST 80, another previously but less frequently identified clone, was introduced in 2000 in the Romanian institutes and persisted over the past 3 years as a frequent cause of infections in a surgical department. The successful MRSA types can acquire prominent positions in hospitals of previously low-endemicity MRSA status.