Analysis of borderline-resistant strains of methicillin-resistant Staphylococcus aureus using polymerase chain reaction.

Identification of methicillin-resistant Staphylococcus aureus by drug-susceptibility tests alone poses a serious problem, because a considerable number of clinical S. aureus isolates are borderline resistant to methicillin. To circumvent this problem, we have developed a quick and sensitive method of PCR amplification for the detection of mecA gene, which, coding for PBP2', is the specific genetic element of methicillin-resistant Staphylococcus aureus. This method made it possible to identify MRSA strains in a short time using as few as 30 cells as a starting material for template DNA. Using this method, we found that the strains of borderline methicillin-resistance could be accurately identified. We also found one S. aureus clinical strain, T3, which lacked mecA gene in spite of its resistance to methicillin.     

Comparison of methods for the detection of methicillin resistance in Staphylococcus aureus isolates from food products

AIMS: To compare several methods for detection of methicillin resistance in Staphylococcus aureus isolates from food. METHODS AND RESULTS: Two hundred S. aureus isolates from food of animal origin were screened for methicillin resistance by a PCR assay specific for the mecA gene, an oxacillin agar screen test and a cefoxitin disk diffusion test. Six out of 200 strains (3%) were found to be methicillin-resistant Staphylococcus aureus (MRSA) by PCR. The oxacillin agar screen test detected only one of the MRSA isolates (sensitivity of 16.7%) and mischaracterized three additional strains as MRSA (specificity of 98.45%). None of the MRSA strains was detected by the cefoxitin test (sensitivity of 0%), while 15 methicillin-susceptible S. aureus (MSSA) strains were misclassified as resistant (specificity of 92.3%). Fifteen MSSA strains displayed a beta-lactamase hyperproducer-like phenotype. The six MRSA (mecA-positive) strains resembled the characteristics of heteroresistant strains. CONCLUSIONS: As MRSA of animal origin may display atypical phenotypes, PCR appears to be more reliable for detection of methicillin resistance in animal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The study stresses the need for implementing the methods of screening S. aureus from food of animal origin for methicillin resistance.     

Evaluation of susceptibility to antibiotics of Staphylococcus aureus strains resistant to methicillin]

Methicillin-resistant strains of Staphylococcus aureus (MRSA) constitute a serious diagnostic and therapeutic problem. Over 500 strains of Staphylococcus aureus were tested for susceptibility to methicillin. By application of a screening method, 13.7% of these strains were classified as methicillin-resistant. Over 95% of these strains were isolated from hospital infections. Applying criteria of belonging of these strains to methicillin-resistance classes it was found that 49.3% belonged to class II, 31.2% to class III and 19.5% to class IV. Analysis of susceptibility to antibiotics of MRSA strains demonstrated significant differences between class II and between class III and IV in resistance to imipenem, gentamycin, erythromycin and tetracycline. All tested strains were susceptible to ciprofloxacin, ofloxacin, vancomycin and teicoplanin. The screening method (25 mg methicillin/l of TSA medium) results in obtaining of univocal results of determination of methicillin-resistance in S. aureus.     

Incidence, antibiotic resistance and clonal relations of MRSA strains isolated from a Romanian university hospital

Our aim was to estimate the frequency and characteristics ofmethicillin-resistant Staphylococcus aureus (MRSA) strains occurring in a Romanian teaching hospital. We retrospectively studied isolates from infected or colonized patients treated at the intensive care and surgical units during January 2004-December 2005. The antibiotic susceptibility of MRSA strains and the presence of mecA gene were determined. Consecutively occurring strains isolated through a three-month period were typed using pulsed field gel electrophoresis. A total of 423 S. aureus strains were identified, methicillin-resistance was detected in 211 (49.9%) strains. Most of them were multiresistant. One of the MRSA genotypes identified by PFGE was commonly recovered from patients treated in the intensive care unit. According to our results, MRSA strains were frequently isolated pathogens in our hospital and there is an urgent need to enhance infection control efforts.     

Staphylococci in orthopaedic surgical wounds.

From 50 infected surgical wounds of orthopaedic patients, 43 (86%) staphylococcal strains were isolated. 34 of all these staphylococci belonged to Staphylococcus aureus species (i.e. 68 % of all isolates from surgical wounds; 79% of staphylococcal isolates); 9 were coagulase-negative staphylococci (i.e. 21% of all isolates from surgical wounds; 18% of staphylococcal isolates). Among microorganisms isolated from the wounds we also found 2 (4%) of the Enterobacteriaceae family; 2 (4%) of the Pseudomonas genus; 3 (6%) of the Streptococcus genus. Thus, orthopaedic surgical wounds were infected by staphylococci (mainly S. aureus) more frequently than by other micro-organisms. All the staphylococcal strains were screened for methicillin resistance by agar disk diffusion testing and for the presence of mecA gene responsible for methicillin resistance by PCR. 32% of the S. aureus and 33% of the S. epidermidis strains resulted methicillin resistant and mecA-positive. The data confirm the diffusion of methicillin resistant S. aureus in surgical site infections and shows that the so-called "new pathogens", i.e. S. epidermidis and other coagulase-negative staphylococci, also exhibit a frequent and hazardous methicillin-resisting ability.     

Characteristics of defective strains of methicillin resistance Staphylococcus aureus that do not produce coagulase or CF

The aim of the study was to estimate frequency of coagulase-negative and CF-negative strains among methicillin-resistant Staphylococcus aureus (MRSA) and to assess their homogeneicity in respect of genotype, phagotype and drug resistance pattern. A total of 186 MRSA strains collected from different hospitals in Gdańsk region were studied. Gens: nuc, mecA, and coa were identified by PCR method. The coagulase tube test for staphylocoagulase and the slide test for clumping factor were used. Coagulase-negative and CF-negative MRSA strains were confirmed by PCR-RFLP method of coa gene; phage typing and drug resistance pattern were evaluated by disc diffusion test. The results of the study showed low frequency of both coagulase-negative and CF-negative MRSA strains (7.25% and 3.76% respectively). Among MRSA population tested the simultaneous occurrence of the strains lacking coagulase and clumping factor was not observed. All coagulase negative MRSA had coagulase gene (coa) and differed from CF-negative strains in respect of coa gene.     

Circulation of methicillin-resistant staphylococci in hospitals with different profiles

Analysis of prevalence of methicillin-resistant staphylococci in hospitals with different specializations was performed. 2584 strains were isolated. Methicillin-resistant (MR) strains were present in different profile hospitals and their total prevalence between isolated strains was 12.3% while significantly varied in different hospitals (from 8% to 37%). Along with MRSA (methicillin-resistant Staphylococcus aureus), MRSE (methicillin-resistant Staphylococcus epidermidis) and MRSS (methicillin-resistant Staphylococcus saprophyticus) were presented in all studied hospitals. The prevalence of MR strains was highest among strains isolated from flame burn wounds (37%), while in samples from newborns in maternity hospital resistant strains represented 12% of isolates, and in general clinical hospital--not more than 9% of isolates. Relationship between rates of isolation of methicillin-resistant staphylococci and specialization of hospital unit was noted. For example, prevalence of MR staphylococci in isolates from newborns in ICU (47.5%) differed from the same one in maternity hospital (11.6%).     

Methicillin-resistant coagulase-negative staphylococci on pig farms as a reservoir of heterogeneous staphylococcal cassette chromosome mec elements

Methicillin-resistant Staphylococcus aureus (MRSA) likely originated by acquisition of the staphylococcal cassette chromosome mec (SCCmec) from coagulase-negative staphylococci (CNS). However, it is unknown whether the same SCCmec types are present in MRSA and CNS that reside in the same niche. Here we describe a study to determine the presence of a potential mecA reservoir among CNS recovered from 10 pig farms. The 44 strains belonged to 10 different Staphylococcus species. All S. aureus strains belonged to sequence type 398 (ST398), with SCCmec types V and IVa. Type IVc, as well as types III and VI, novel subtypes of type IV, and not-typeable types, were found in CNS. S. aureus, S. epidermidis, and S. haemolyticus shared SCCmec type V. The presence of SCCmec type IVc in several staphylococcal species isolated from one pig farm is noteworthy, suggesting exchange of this SCCmec type in CNS, but the general distribution of this SCCmec type still has to be established. In conclusion, this study shows that SCCmec types among staphylococcal species on pig farms are heterogeneous. On two farms, more than one recovered staphylococcal species harbored the same SCCmec type. We conclude that staphylococci on pig farms act as a reservoir of heterogeneous SCCmec elements. These staphylococci may act as a source for transfer of SCCmec to S. aureus.     

耐甲氧西林葡萄球菌和肠球菌blaTEM及tetM基因检测

目的了解临床分离的耐甲氧西林葡萄球菌(MRS)和肠球菌中blaTEM及tetM基因存在状况。方法分离50株耐甲氧西林金黄色葡萄球菌(MRSA),7株耐甲氧西林表皮葡萄球菌(MRSE)、5株耐甲氧西林溶血葡萄球菌(MRSH)、15株粪肠球菌和9株屎肠球菌,采用PCR技术检测耐药基因。结果MRSA、MRSE、MRSH、粪肠球菌和屎肠球菌中blaTEM基因阳性率分别为40.0%、57.1%、60.0%、6.7%和88.9%,tetM基因阳性率分别为100%、0%、0%、66.7%、0%。结论blaTEM基因阳性率在MRS中较高,在屎肠球菌中则很高;携带tetM基因是MRSA和粪肠球菌对四环素耐药的主要原因。     

Comparison of disk-diffusion method and PCR for detection of methicillin resistance in Staphylococcus aureus strains

The aim of the study was to compare the disk-diffusion (oxacillin 1 microg, cefoxitin 30 microg) method and PCR for detection of methicillin-resistance in S. aureus. The investigation were carried out on 120 S. aureus strains isolated from clinical materials of patients hospitalized in the University Hospital at the L. Rydygier Collegium Medicum in Bydgoszcz, University of Nicolaus Copernicus in Toruń. Of the 120 S. aureus strains tested, 60 (50%) were mecA-positive by PCR. Consistency of results between oxacillin disk-difussion method and PCR amounted 92.5% and cefoxitin disk-diffusion method and PCR--98.3%. The oxacillin disk-difussion method falsely identified 3 (2.5%) strains as MSSA (sensitivity 95.0%) and 4 strains as MRSA (specificity 93.3%) in comparison with PCR. The cefoxitin disk-diffusion method falsely identified 2 (1.6%) strains as MSSA (sensitivity 96.7%) and there were no false resistant results (specificity 100%). Our results showed that in disk-diffusion tests, cefoxitin is a better than oxacillin for the identification of MRSA.     

Immunological detection of penicillin-binding protein 2'' in clinical isolates of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis

The additional penicillin-binding protein (PBP 2') that is important in determining intrinsic resistance in methicillin-resistant strains of Staphylococcus aureus (MRSA) has been detected immunologically in strains from a variety of world-wide locations. This additional protein has also been definitively identified both immunologically and as a PBP in methicillin-resistant strains of S. epidermidis (MRSE). The assay described is rapid, specific and sensitive and has been used to detect PBP 2' in S. haemolyticus but not in beta-lactam resistant Streptococci.     

Cytidine deamination assay to differentiate Staphylococcus aureus from other staphylococci

AIMS: Adoption of the property of cytidine (cytosine-beta-d-riboside) deamination in staphylococci to distinguish Staphylococcus aureus from other staphylococci. METHODS AND RESULTS: A total of 560 staphylococcal strains were examined. The test demonstrated a sensitivity of 97.1% and a specificity of 98.8%. Of the 249 S. aureus strains (115 oxacillin-resistant) 58 strains were coagulase-negative S. aureus and another 16 strains were clumping factor-negative S. aureus. The 74 deficient S. aureus strains were identified by 16S rRNA gene sequencing and further investigated by spa typing and 13 spa types were found. CONCLUSIONS: The cytidine deaminase test (CDT) is useful especially for distinguishing coagulase- and clumping factor-negative S. aureus from other staphylococci and the results correlated well with 16S rRNA sequencing and the polymerase chain reaction (PCR) amplification of the nuc gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Cytidine deamination assay differentiates S. aureus from other staphylococci. This method is fast (6 h) and reliable in distinguishing between non-S. aureus and the defective (coagulase-negative, clumping factor-negative) S. aureus isolates which could have major consequences for therapy.     

Antimicrobial resistance pattern of methicillin-resistant Staphylococcus aureus in the food industry

There is increasing concern about the impact on public health of methicillin-resistant Staphylococcus aureus (MRSA) associated with animal food products. MRSA remains a serious problem because of the high incidence and multidrug resistance of the strains, even for strains isolated from foods, food environments and food handlers. The objectives of this study are: (i) to evaluate the susceptibility of S. aureus strains isolated from food, food handlers and food-processing environments to 14 antibiotics currently used in veterinary and human therapy; (ii) to assess the presence of the mecA gene. A total of 1007 samples were collected from food, food handlers, and environments and were analyzed for the presence of S. aureus. S. aureus was present in 165 of the 1007 samples. A total of 157 isolates were methicillin-susceptible S. aureus (MSSA) and 8 isolates were MRSA. In particular, out of 8 MRSA strains detected, 4 strains harboured the mecA gene. All MRSA strains were resistant to at least one of the tested antibiotics and 6 strains demonstrated multi-resistance. Considering the high level of resistances in S. aureus and the isolation of MRSA strains, the surveillance of antimicrobial resistance and the spreading of this pathogen is of crucial importance in the food production chain. These data are useful in improving background data on antimicrobial resistance of S. aureus isolated from food, processing environments and food handlers, supporting the prudent use of antibiotics and the development of international control programs.     

Methicillin-resistant Staphylococcus epidermidis carrying biofilm formation genes: detection of clinical isolates by multiplex PCR

Staphylococcus epidermidis is the most prevalent coagulase-negative Staphylococcus (CNS) and is a major cause of hospital bacteremia. Based on 18 reference strains and 149 Staphylococcus clinical strains, used in a novel multiplex PCR method, the aim of this study was to identify S. epidermidis with respect to the sequence of three genes: recN, which encodes a recombination/repair protein, mecA (methicillin resistance), and icaAB, which is involved in biofilm formation. Amplicons of 219 bp (S. epidermidis-recN gene), 154 bp (mecA gene), and 546 bp (icaAB genes) were obtained. Reliable results were achieved for 100% of the evaluated strains, suggesting that this new multiplex-PCR approach could be useful for the accurate identification of methicillin-resistant S. epidermidis with the potential to produce biofilm.     

MRSA气溶胶诱导烫伤SKH-1无毛小鼠皮肤感染合并肺炎模型

目的探讨烫伤条件下,建立MRSA气溶胶致SKH-1无毛小鼠皮肤感染合并肺炎模型的方法及其评价指标。方法 48只SKH-1无毛小鼠,随机分成PBS对照组,单纯感染组和烫伤合并感染组。合并感染组小鼠皮肤烫伤后,MRSA（ST-239）气溶胶呼吸道感染,采用体重、血常规、菌血症率、皮肤和脏器荷菌量、病理组织学变化和分子生物学等指标对模型进行综合评价。结果与其他两组相比,合并感染组小鼠第3天体重下降明显;白细胞数显著增多;菌血症率达100%;皮肤和肺荷菌量较高,分别达108CFU/g和106CFU/g;镜下观察皮肤和肺炎症病变明显;免疫组化结果显示皮肤组织内CD138呈阳性表达;multi-PCR测定感染组织内nuc和mecA基因均为阳性。结论 SKH-1无毛小鼠烫伤再辅以MRSA气雾攻击的方式,可成功建立能够模拟临床上烧烫伤患者继发MRSA肺炎的动物模型,此模型对于抗MRSA药物药效评价研究具有重要临床应用价值。     

Rapid detection of methicillin-resistant Staphylococcus aureus in screening samples by relative quantification between the mecA gene and the SA442 gene

Rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) is important to identify patients colonized with this pathogen and implement infection control precautions. We aimed to improve the combined use of the mecA gene polymerase chain reaction (PCR) and the SA442 PCR to detect MRSA in clinical screening samples. In a true MRSA the mecA copy number will be equal to the SA442 copy number, whereas in samples with a methicillin-sensitive Staphylococcus aureus (MSSA) combined with a methicillin-resistant coagulase-negative Staphylococcus (MRCNS) the copy numbers will usually differ. Here we introduce a PCR system, relative quantification PCR (RQ-PCR), which takes this difference into account. RQ-PCR identifies true MRSA in clinical samples with a specificity that is comparable to the SCCmec-based PCRs.     

Gene sequences and specific detection for Panton-Valentine leukocidin

A new category of methicillin-resistant Staphylococcus aureus (MRSA), called community-acquired MRSA (CA-MRSA), has emerged worldwide. In contrast to previous MRSA, most CA-MRSA carries the Panton-Valentine leukocidin (PVL) genes (lukPVSF) as a virulence genetic trait. Sequence analysis of the lukPVSF gene of a Japanese isolate demonstrated that the gene has more similarity to methicillin-susceptible S. aureus from France than MRSA from the United States. Based on the sequences, we developed a real-time PCR assay for the three key genes of CA-MRSA; that is, lukPVSF, mecA (for methicillin resistance), and spa (for S. aureus). Dual or triple assay for lukPVSF, mecA, and spa in one test tube became possible. The detection limit of the assay with probe and SYBR Green methods was between 2.7 and 2.7 x 10(1) CFU/ml. The assay detected PVL-positive MRSA in clinical (blood) isolates.     

医院感染葡萄球菌菌种变迁与耐药性近况

目的：了解近9年来医院感染葡萄球菌菌种的变迁与近3年来葡萄球菌药状况。方法：1993年1月至2001年12月我院传染病科等13科室住院病人的各种标本采用血琼脂培养,所分离的葡萄球菌采用美国DADE公司生产的MICROSCAN WALKAY-40全自动微生物分析仪鉴定到种及其亚种。药敏试验药物有青霉素、苯唑西林、氨苄西林、哌拉西林、阿莫西林／克拉维酸（阿莫仙）、头孢唑啉、头孢噻肟、头孢哌酮／舒巴坦（舒普深）、庆大霉素、复方新诺明、环丙沙星、氧氟沙星、利福平、万古霉素、红霉素、林可霉素、四环素、伊米配能／西司他丁（泰能）共18种。采用液体稀释法测定每株葡萄球菌对受试药物的最小抑菌浓度（MIC）,操作按说明书进行。质控菌ATCC25923。依据新近NCCLS标准判读结果。结果：1993年至1998年分离的葡萄球菌中,金黄色葡萄球菌（金葡萄）占71.43%,凝固酶阴性葡萄球菌（CNS）占28.57%,包括表皮葡萄球菌（表葡菌）、腐生葡萄球菌、里昂葡萄球菌和头状葡萄球菌4种。1999年至2001年分离的424株葡萄球菌中,金葡菌仅占29.01%,CNS增至13种,占70.995,以表葡菌、溶血葡萄球菌、松鼠葡萄球菌为主。近3年来分离的各种葡萄球菌甲氧西林耐药率在73.03%-100%之间,除对舒普深、复方新诺明、利福平和万古霉素较敏感外,对其余抗菌药物的耐药率均超过60％,以金葡菌、溶血葡萄球菌、松鼠葡萄球菌的耐药率为最高。MRS耐药率普遍高于MSS,且均呈多重耐药。5.42%(23/424)菌株万古霉素MIC＞16mg/L,除1株为MSCNS外,其余22株均为MRS。结论：3年来医院感染葡萄球菌菌种构成比发生了显著变化,以CNS为主。对抗菌药物呈多重耐药,部分菌株对万古霉素敏感性降低,应予警惕。     

mecA基因PCR扩增法检测耐甲氧西林金黄色葡萄球菌

目的 应用mecA基因PCR扩增法检测耐甲氧西林金黄色葡萄球菌（methicillin resistant staphylococcus aureus,MRSA）。方法 临床分离的70株金黄色葡萄球菌,应用mecA基因PCR扩增法鉴定MRSA,并与苯唑西林纸片扩散法进行比较。结果 70株金黄色葡萄球菌用PCR扩增法和纸片扩散法有6株鉴定有差异,4株。mecA基因阳性而纸片扩散法鉴定为敏感,1株mecA基因阳性纸片扩散法鉴定为临界耐药,1株mecA基因阴性却表现为苯唑西林耐药,2种方法符合率为91．43％。结论 mecA基因PCR扩增法可以准确、快速判定MRSA,特别是对隐匿型或低水平耐药菌株的检出有重要的价值。