Restriction endonuclease fingerprinting of genomic DNA of Staphylococcus species of bovine origin.

Fifty-one staphylococcal isolates from mammary secretions of cows with subclinical mastitis were examined by antibiograms and DNA restriction endonuclease fingerprinting (REF). DNA REF differentiated closely related strains of each species isolated from mammary secretions of different mammary glands of the same cow and from the same mammary gland at different periods of the lactation cycle. In addition, REF analysis provided evidence concerning persistence of infection in the same or different mammary gland over different periods of the lactation cycle, and occurrence of infection with similar and dissimilar strains of each Staphylococcus species. Antibiograms were of limited value in differentiating closely related strains. The ease by which REF analysis can be performed together with the reproducibility and clarity of REF patterns suggest that this technique is useful for differentiating closely related and unrelated strains of Staphylococcus species isolated from bovine mammary secretions.     

PCR方法快速检测食品中的金黄色葡萄球菌

目的建立聚合酶链反应(PCR)方法快速检测牛奶、冰淇淋及肉中的金黄色葡萄球菌(金葡菌)。方法针对金葡菌耐热核酸酶基因(nuc)、纤维蛋白原结合蛋白基因(ClfA)设计并合成2对引物进行PCR扩增,特异的检测金葡菌,并对52株金葡菌和31株非金葡菌进行扩增,验证方法的特异性。在牛奶、冰淇淋及肉中人工污染不同菌量,增菌培养,在不同时间段提取DNA进行PCR扩增,确定该方法在食品中的检出限。结果nuc、ClfA两对引物对金葡菌的检出均有很好的特异性,在3种食品中的检出限均为10cfu/g(ml),全部检测可在24h完成。结论建立了适用于牛奶、冰淇淋及肉中的金葡菌检测的快速、灵敏、特异的PCR方法。     

Collection of genomic DNA by buccal swabs for polymerase chain reaction-based biomarker assays.

Studies in molecular and genetic epidemiology require a high-throughput, low cost, and reliable means of genomic DNA collection. Buccal (cheek) swabs have been proposed as a means of achieving these goals, but there is little information about the practical application of this approach. From January 1995 to December 1997, we processed 995 buccal swabs for use in polymerase chain reaction (PCR)-based genotype assays in the context of ongoing molecular epidemiologic studies. Six hundred forty-seven of these swabs were processed immediately after collection and 348 were received by mail. We were able to obtain at least one genotype from 99.7% (645 of 647) of fresh-processed and 97.4% (330 of 339) of mailed biosamples. A PCR success rate of 90.3% (2,546 genotypes from 2,819 assays) was achieved. Genotypes were obtained from 96.1% (1, 865 genotypes from 1,941 assays) of fresh-processed biosamples and 77.6% (681 genotypes from 878 assays) of mailed biosamples. PCR success rates at any single locus ranged from 92.6 to 98.8% (fresh-processed) and 75.5 to 79.6% (mailed). The PCR success rate among fresh-processed biosamples was significantly higher than among mailed biosamples (Fisher's exact test p < 0.0001), and more attempts were required to obtain a successful PCR result for mailed biosamples as compared to fresh-processed biosamples. For one locus (CYP3A4), a subset of mailed biosamples was purified if two or more PCR failures occurred. Additional genotypes were obtained in 58.3% of these previously failed biosamples. Time from biosample receipt to DNA extraction had no effect on PCR success. After storage of processed biosamples for as long as 3 years, there was no appreciable decrease in the rate of PCR success. These results suggest that adequate DNA for PCR-based applications can be obtained from buccal swabs, but sampling or processing considerations may be important in obtaining optimal results.     

随机引物聚合酶链反应对耐甲氧西林金黄色葡萄球菌基因分型的研究

目的建立耐甲氧西林金黄色葡萄球菌（ＭＲＳＡ）随机引物聚合酶链反应（ＡＰ－ＰＣＲ）基因分型的方法用于；临床流行病学调查。方法优化ＡＰ－ＰＣＲ方法并与抗生素敏感谱分型法比较，评价ＡＰ－ＦＣＲ基因分型法的可靠性与分辨力；并对４９株ＭＲＳＡ进行ＡＰ－ＲＣＲ基因分型。结果４９株医院感染的ＭＲＳＡ可分为１０个ＡＰ－ＰＣＲ谱型，主要谱型为 Ａ３型，分型率 １００％；其中 ４１株 ＭＲＳＡ为流行病学相关株。结论 ＡＰ－ＰＣＲ基因分型是一种经济、快捷、可靠的分型方法；是在分子水平上对微生物感染进行病原学、发病机理及流行病学研究较理想的工具。     

Evaluation of real-time polymerase chain reaction for the detection of methicillin-resistant Staphylococcus aureus on environmental surfaces.

We compared real-time polymerase chain reaction (RT-PCR) with in vitro culture for detecting methicillin-resistant Staphylococcus aureus in samples from environmental surfaces. The sensitivity of RT-PCR, compared with culture, was 92.5%, and the specificity was 51.4%. Because of poor specificity, the RT-PCR kit tested is not suitable for the detection of MRSA on hospital surfaces.     

Real-time polymerase chain reaction detection of methicillin-resistant Staphylococcus aureus: impact on nosocomial transmission and costs.

OBJECTIVES: To assess the impact of real-time polymerase chain reaction (PCR) detection of methicillin-resistant Staphylococcus aureus (MRSA) on nosocomial transmission and costs. DESIGN: Monthly MRSA detection rates were measured from April 1, 2000, through December 31, 2005. Time series analysis was used to identify changes in MRSA detection rates, and decision analysis was used to compare the costs of detection by PCR and by culture.Setting. A 1,200-bed, tertiary care hospital in Canada. PATIENTS: Admitted patients at high risk for MRSA colonization. MRSA detection using culture-based screening was compared with a commercial PCR assay. RESULTS: The mean monthly incidence of nosocomial MRSA colonization or infection was 0.37 cases per 1,000 patient-days. The time-series model indicated an insignificant decrease of 0.14 cases per 1,000 patient-days per month (95% confidence interval, -0.18 to 0.46) after the introduction of PCR detection (P=.39). The mean interval from a reported positive result until contact precautions were initiated decreased from 3.8 to 1.6 days (P<.001). However, the cost of MRSA control increased from Can$605,034 to Can$771,609. Of 290 PCR-positive patients, 120 (41.4%) were placed under contact precautions unnecessarily because of low specificity of the PCR assay used in the study; these patients contributed 37% of the increased cost. The modeling study predicted that the cost per patient would be higher with detection by PCR (Can$96) than by culture (Can$67). CONCLUSION: Detection of MRSA by the PCR assay evaluated in this study was more costly than detection by culture for reducing MRSA transmission in our hospital. The cost benefit of screening by PCR varies according to incidences of MRSA colonization and infection, the predictive values of the assay used, and rates of compliance with infection control measures.     

Genomic diversity of mec regulator genes in methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis.

Low-affinity penicillin-binding protein PBP-2a encoded by mecA is closely related to methicillin resistance in staphylococci, and expression of PBP-2a is controlled by regulator elements encoded by mecR1 and mecI which are located adjacent to mecA on the chromosome. Deletion or mutation which occurred in mec regulator gene is considered to be associated with constitutive production of PBP-2a. The distribution of the mec regulator genes in 176 strains of Staphylococcus aureus and 33 strains of S. epidermidis isolated from a single hospital was studied by polymerase chain reaction amplification. Most clinical isolates of methicillin-resistant S. aureus (MRSA) (94.3%) and S. epidermidis (MRSE) (83.9%) possessed both mecI and mecR1 genes (type I), whereas no mec regulator genes were detected in mecA-negative isolates. In contrast, 7 MRSA and 5 MRSE isolates were found to have incomplete regulator genes, and they were classified into three groups; strains which lacked only mecI gene (type II), strains which lacked mecI and 3''-end of mecR1 gene (type III), and strains which lacked both regulator genes (type IV). Analysis of mecI gene from all the strains having mecI by restriction fragment length polymorphism after Mse I digestion indicated that three MRSA strains possessed one of the known point mutations identified previously. These findings indicated the predominance of a single type of MRSA possessing both mecI and mecR1 in the study period and also suggested a high genomic diversity in mec regulator region of staphylococci.     

Impact of preoperative screening for meticillin-resistant Staphylococcus aureus by real-time polymerase chain reaction in patients undergoing cardiac surgery

We report a significant reduction in the number of surgical site infections (SSIs) due to meticillin-resistant Staphylococcus aureus (MRSA) in patients undergoing cardiac surgery after the introduction of preoperative screening using a same-day polymerase chain reaction (PCR) test. This was an observational cohort study set in a cardiac surgery unit based in southwest England. We studied 1462 patients admitted for cardiac surgery between October 2004 and September 2006. The IDI MRSA PCR test was used preoperatively to screen 765 patients between October 2005 and September 2006. Patients identified as carriers were treated with nasal mupirocin ointment and topical triclosan for five days, with single-dose teicoplanin instead of flucloxacillin as perioperative antibiotic prophylaxis. The rate of SSI following cardiac surgery in this group was compared to 697 patients who underwent surgery without screening between October 2004 and September 2005. After introduction of PCR screening, the overall rate of SSI fell from 3.30% to 2.22% with a significant reduction in the rate of MRSA infections (relative risk reduction: 0.77; 95% confidence interval: 0.056-0.95). PCR screening combined with suppression of MRSA at the time of cardiac surgery is feasible in routine clinical practice and is associated with a significant reduction in subsequent MRSA SSIs.     

Identification of human and bovine rotavirus serotypes by polymerase chain reaction.

The use of the polymerase chain reaction (PCR) for identifying serotypes of human and bovine rotaviruses was examined. In the identification of 115 human rotavirus samples in stools, results with PCR showed excellent agreement with results of serotyping by an enzyme-linked immunosorbent assay (ELISA) using serotype-specific monoclonal antibodies. Furthermore, the PCR showed a much higher sensitivity (93%) than the ELISA test (82.6%). The PCR method could also be applied for identifying the serotype of bovine rotaviruses.     

Statistical methods for determining the accuracy of quantitative polymerase chain reaction-based tests

Quantitative polymerase chain reaction (PCR)-based tests are used in several different scientific fields to determine levels of a target DNA sequence of interest (the target molecule). The accuracy of quantitative PCR-based tests can be assessed by using the assay to determine the number of copies of the target molecule in a sample with a known concentration of the target molecule. For example, a sample with a known concentration of a target DNA sequence is serially diluted into replicate aliquots and these are tested to determine if the observed quantity of the target is close to the expected quantity (given the concentration in the original sample and the dilution). Statistical methods that are conventionally used to assess the accuracy of these assays do not take into account the variability in the number of target molecules in each aliquot from the original sample. We develop methods that take into account this extra variation and which determine the accuracy of quantitative PCR-based tests in estimating the number of target molecules based on a set of assays of serial dilutions from an original sample with a known concentration of target molecules. These methods are applied to data from an experiment to test the accuracy of a real-time PCR assay at low HIV-1 DNA copy levels.     

金黄色葡萄球菌随机扩增DNA多态性分型研究

目的建立金黄色葡萄球菌随机扩增ＤＮＡ多态性（ＲＡＰＤ）分型技术，以应用于临床菌种鉴定及流行病学调查。方法对临床分离的１５株金黄色葡萄球菌，酚氯仿法提取其ＤＮＡ后行ＲＡＰＤ分型，同时进行噬菌体分型。结果１５株临床分离株大多数产生独特而稳定的ＲＡＰＤ带型，可分成５个噬菌体型和１４个ＲＡＰＤ谱型。采用同一反应体系，产肠毒素金黄色葡萄球菌未出现ＲＡＰＤ谱型。结论ＲＡＰＤ基因分型技术不需已知核酸序列，分型率高，分辨力强，简便快捷，可为金黄色葡萄球菌临床分离株提供分型标志，是分子流行病学研究的有效方法。     

耐甲氧西林金黄色葡萄球菌随机引物DNA扩增多态性分型研究

目的了解某院耐甲氧西林金黄色葡萄球菌（MRSA）基因型别及流行情况,为医院感染的监测与控制提供科学的依据。方法对临床分离的金黄色葡萄球菌通过头孢西丁纸片扩散法初筛和mecA基因检测确定MRSA,对筛选出的MRSA应用随机引物DNA扩增多态性（RAPD）技术进行基因型别分析。结果MRSA的分离率为72.15%;临床分离株均产生独特而稳定的RAPD带型,图谱显示某院MRSA的基因型相似,且以A型为主。结论MRSA存在医院感染,必须采取有效的措施减少其发生;RAPD是一种快速、经济、灵敏、可靠的分型技术。     

Predictive value and cost-effectiveness analysis of a rapid polymerase chain reaction for preoperative detection of nasal carriage of Staphylococcus aureus.

OBJECTIVE: To determine the accuracy and cost-effectiveness of a polymerase chain reaction (PCR) for detecting nasal carriage of Staphylococcus aureus directly from clinical specimens. CROSS-SECTIONAL STUDY: This occurred in a tertiary-care hospital in Cleveland, Ohio, and included 239 consecutive patients who were scheduled for a cardiothoracic surgical procedure. Conventional cultures and a PCR for S. aureus from nasal swabs were used as measurements. COST-EFFECTIVENESS ANALYSIS: Data sources were market prices and Bureau of Labor Statistics. The time horizon was the maximum period for availability of culture results (3 days). Interventions included universal mupirocin therapy without testing; initial therapy, with termination if PCR negative (treat-PCR); initial therapy, with termination if culture negative (treat-culture); treat PCR-positive carriers (PCR-guided treatment); and treat culture-positive carriers (culture-guided treatment). The perspective was institutional and costs and the length of time to treatment were outcome measures. RESULTS: Sixty-seven (28%) of the 239 swabs grew S. aureus. Rapid PCR was 97.0% sensitive and 97.1% specific for the detection of S. aureus. For populations with prevalences of nasal S. aureus carriage of up to 50%, the PCR assay had negative predictive values of greater than 97%. PCR-guided treatment had the lowest incremental cost-effectiveness ratio (1.93 dollars per additional day compared with the culture strategy). Among immediate treatment strategies, treat-PCR was most cost-effective. The universal therapy strategy cost 38.19 dollars more per additional day gained with carrier identification compared with the PCR strategy. CONCLUSION: Rapid real-time PCR is an accurate, rapid, and cost-effective method for identifying S. aureus carriers for preoperative intervention.     

Antimicrobial susceptibility of Staphylococcus aureus and characterization of methicillin-resistant Staphylococcus aureus isolated from bovine mastitis in Korea

A total of 402 Staphylococcus aureus isolates from bovine mastitis milk collected during 2003-2009 in Korea were tested for susceptibility to 20 antimicrobial agents. All S. aureus isolates were susceptible to 11 of 20 antimicrobials tested; no resistance was observed against pirlimycin, telithromycin, novobiocin, penicillin/novobiocin, quinupristin/dalfopristin, clindamycin, rifampin, ciprofloxacin, trimethprim/sulfamethoxazol, vancomycin, and linezolid. Over 66% of the S. aureus isolates were resistant to penicillin. Resistance was also seen for gentamicin (11.9%), erythromycin (7.7%), methicillin (oxacillin and cefoxitin, 6.2%), and tetracycline (4.2%). No noticeable change was observed in penicillin, gentamicin, and erythromycin resistance over the 7-year period. Tetracycline resistance appeared to decrease consistently, whereas methicillin resistance was observed from 2005. About 2.7% (11/402) were resistant to three or more antimicrobials. Genotyping of the 17 methicillin-resistant S. aureus (MRSA) isolated from each cow revealed two staphylococcal cassette chromosome mec (SCCmec) types (IV and IVa), three spa types (t286, t324, and untypable), and two sequence types (ST1 and ST72). Eleven of 17 (64.7%) MRSA strains belonged to SCCmec IVa-t324-ST72. The rest of strains belonged to SCCmec IVa-t286-ST1 (n?=?3) and SCCmec IV-untypable-ST72 (n?=?3). None of the MRSA carried the Panton-Valentine leukocidin gene. These characteristics are the same as those found in community-acquired (CA) MRSA strains prevalent in humans in Korea. Three pulsed-field gel electrophoresis types (A-C) were observed among the 17 MRSA strains examined, and 14 strains belonged to the same pulsed-field gel electrophoresis pattern regardless of their geographical origin and year of isolation. The results of this study provide evidence of CA-MRSA infection in dairy cattle for the first time in Korea.     

Emergence of methicillin-resistant Staphylococcus aureus of animal origin in humans

In 2003 in the Netherlands, a new methicillin-resistant Staphylococcus aureus (MRSA) strain emerged that could not be typed with Sma1 pulsed-field gel electrophoresis (NT-MRSA). The association of NT-MRSA in humans with a reservoir in animals was investigated. The frequency of NT-MRSA increased from 0% in 2002 to >21% after intensified surveillance was implemented in July 2006. Geographically, NT-MRSA clustered with pig farming. A case-control study showed that carriers of NT-MRSA were more often pig or cattle farmers (pig farmers odds ratio [OR] 12.2, 95% confidence interval [CI] 3.1-48.6; cattle farmers OR 19.7, 95% CI 2.3-169.5). Molecular typing showed that the NT-MRSA strains belonged to a new clonal complex, ST 398. This study shows that MRSA from an animal reservoir has recently entered the human population and is now responsible for >20% of all MRSA in the Netherlands.     

金黄色葡萄球菌基因组DNA提取方法的比较研究

目的:对金黄色葡萄球菌DNA不同提取方法进行评价.方法:采用4种不同金黄色葡萄球菌DNA提取方法,比较提取得率和质量,对提取的DNA进行PCR扩增并评价效果.结果:在DNA提取之前加入70%乙醇可有效处理细菌细胞,增加细胞对裂解液的敏感性,再用溶菌酶处理能很好地被消化,获得了一种快速、简洁和有效的提取方法.结论:这是一种适用于金黄色葡萄球菌和其它病原细菌的有效提取方法(方法4),可用于金黄色葡萄球菌和其它病原细菌的分子生物学操作和快速诊断技术.     

Prevalence of Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella spp. in seafood products using multiplex polymerase chain reaction

Although several etiological agents can be transmitted through seafood consumption, Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella spp. are considered among the most important pathogens in terms of public health and disease. In this study, multiplex polymerase chain reaction (PCR), as a rapid and cost-effective method, was used to determine the prevalence of these pathogens in 245 samples of raw/fresh, frozen, and ready-to-eat (RTE) seafood products marketed in Iran. The prevalence of L. monocytogenes in raw/fresh fish and shrimp samples was 1.4%, whereas 2.9% of the raw/fresh fish and 7.1% of the shrimp samples were contaminated with V. parahaemolyticus. No contamination with L. monocytogenes and V. parahaemolyticus was found in frozen and RTE seafood products. The prevalence of S. aureus was found to be higher than other investigated pathogens. S. aureus was detected in 5% of the raw/fresh samples of fish and shrimp, 17.5% of the frozen, and 12.3% of the RTE samples. Further, our findings indicate that 2.9% of the fish samples, 4.3% of the shrimp samples, and 1.5% of the RTE samples were contaminated with Salmonella spp. Owing to the potential hazard of these pathogenic bacteria, multiplex PCR can provide a rapid and cost-effective method for the surveillance of these pathogens in seafood products.