Methicillin (Oxacillin)-Resistant Staphylococcus aureus Strains Isolated from Major Food Animals and Their Potential Transmission to Humans

From May 2001 to April 2003, various types of specimens from cattle, pigs, and chickens were collected and examined for the presence of methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA). S. aureus was isolated and positively identified by using Gram staining, colony morphology, tests for coagulase and urease activities, and an API Staph Ident system. Among 1,913 specimens collected from the animals, 421 contained S. aureus; of these, 28 contained S. aureus resistant to concentrations of oxacillin higher than 2 μg/ml. Isolates from 15 of the 28 specimens were positive by PCR for the mecA gene. Of the 15 mecA-positive MRSA isolates, 12 were from dairy cows and 3 were from chickens. Antimicrobial susceptibility tests of mecA-positive MRSA strains were performed by the disk diffusion method. All isolates were resistant to members of the penicillin family, such as ampicillin, oxacillin, and penicillin. All isolates were also susceptible to amikacin, vancomycin, and trimethoprim-sulfamethoxazole. To determine molecular epidemiological relatedness of these 15 animal MRSA isolates to isolates from humans, random amplified polymorphic DNA (RAPD) patterns were generated by arbitrarily primed PCR. The RAPD patterns of six of the isolates from animals were identical to the patterns of certain isolates from humans. The antibiotypes of the six animal isolates revealed types similar to those of the human isolates. These data suggested that the genomes of the six animal MRSA isolates were very closely related to those of some human MRSA isolates and were a possible source of human infections caused by consuming contaminated food products made from these animals.     

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.     

Isolation and characterization of methicillin-resistantStaphylococcus aureus from livestock and poultry meat

Meat samples from sheep, bovine, camel and poultry were collected from Amman area and were processed and tested for the presence of methicillin (oxacillin) resistantStaphylococcus aureus (MRSA). Identity ofS. aureus was ensured by Gram-staining and a battery of biochemical tests. From 1260 meat samples, 157S. aureus positive isolates were identified. Of the 157 isolates, 30 were resistant to methicillin levels greater than 2 μg/ml and only 15 weremecA-positive MRSA originating mainly from sheep and chicken. Subjecting themecA-positive MRSA to antibiotic susceptibility testing revealed that all isolates were resistant to β-lactam antibiotics (ampicillin, penicillin, and oxacillin) and were sensitive to vancomycin, trimethoprim, chloramphenicol and cephalothin. Randomamplified polymorphic DNA (RAPD) analysis ofmecA-positive animal isolates generated six different patterns. Comparing these results with results of isolates of human origin of our laboratory there is some molecular epidemiological relatedness between both and could be a possible source of infections through consuming contaminated meat products, direct contact or meat processing.     

Incidence of Staphylococcus aureus isolated from patients treated at the Clinical Center of Skopje, Macedonia, with special attention to MRSA

The distribution of 3497 Staphylococcus aureus strains according to methicillin resistance, specimens, departmental profession and antibiotic resistance patterns was analysed. The strains were cultured from the patients of the Clinical Center of Skopje, Macedonia, between 1 January 2002 and 31 December 2004. The majority of the isolates was obtained from suppurated wounds (28.5%), nares (21%), intratracheal tubes (13%) and blood cultures (11.8%). Overall 1100 (31.4%) of the isolates was methicillin-resistant with 1 microg oxacillin disc. Of these 35.5%, 30.5% and 10.4% were cultured from wounds, intratracheal tubes and blood samples, respectively. The prevalence of MRSA strains was 78.6%, 75%, 44.2% and 37.3% in specimens of ICU, Coma Center, General Surgery and Haematology patients. There were extremely big differences in the frequency of MRSA between departments with particular specialisation. The 2397 MSSA isolates belonged to practically one antibiotic resistance pattern characterised with penicillin resistance and susceptibility to other antistaphylococcal drugs. The 1100 MRSA isolates distributed to four antibiotic resistance patterns on the basis of their resistance to oxacillin, penicillin, amoxicillin+clavulanic acid, azithromycin, clindamycin, amikacin, gentamicin, ciprofloxacin, trimethoprim+sulphamethoxasole, vancomycin and teicoplanin. All the MRSA isolates were multidrug resistant but sensitive to glycopeptides.     

Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolates from clinical specimens of patients with nosocomial infection: are there unnoticed silent outbreaks?

Bacteriological and epidemiological studies were carried out on 90 isolates of methicillin-resistant Staphylococcus aureus (MRSA) at Turgut Ozal Medical Center of In?nü University, (Malatya/Turkey). MRSA isolates were obtained from patients with nosocomial infections. Staphylococcus aureus clinical isolates were collected between May 2004-May 2005. Isolates were tested for resistance to methicillin. Antimicrobial susceptibility testing and slime production evaluation was performed. Genotype studies were carried out by arbitrarily primed polymerase chain reaction (AP-PCR) and consequent cluster analysis. All of the isolates were mecA-positive in a PCR-based assay; all exhibited resistance to oxacillin, by agar dilution (MICs > or = 4 mg/L) and disc diffusion methods, and multiple antibiotics. Most MRSA isolates were collected in intensive care units. Of 90 samples, 53 were found to be unrelated to the others while the remaining 37 strains were either identical or closely related. Dendrogram analysis identified nine major clusters. These data support the opinion that MRSA are significant nosocomial pathogens in intensive care units and that resistant clones may be transmitted between patients. Molecular epidemiological tools are helpful for understanding transmission patterns and sources of infection, and are useful for measuring outcomes of intervention strategies implemented to reduce nosocomial MRSA.     

长沙地区临床分离金黄色葡萄球菌的耐药监测

目的了解长沙地区临床分离金黄色葡萄球菌（以下简称金葡菌）对常用抗菌药物的耐药现状,探讨金黄色葡萄球菌对甲氧西林的耐药水平。方法收集长沙地区11家医院2009年11月至2010年11月临床分离的非重复金葡菌279株,应用Vitek-2全自动微生物分析系统进行鉴定,K-B法检测金葡菌对24种药物的敏感性,产色头孢菌素试验检测β-内酰胺酶以及D试验检测诱导型克林霉素耐药。应用头孢西丁和苯唑西林纸片扩散法筛查耐甲氧西林的金葡菌（MRSA）,琼脂稀释法检测头孢西丁和苯唑西林的最低抑菌浓度（MIC）。结果在被检测的24种药物中,敏感率〉50%的药物为9种,未发现对万古霉素、替考拉宁和利奈唑胺耐药菌株;耐药率〉50%的抗菌药物有11种,其中以青霉素和氨苄西林的耐药率最高（均为97.1%）。MRSA的分离率达54.5%,且对常用的16种抗菌药物的耐药率均显著高于甲氧西林敏感金黄色葡萄球菌（MSSA）。279株金葡菌中,β-内酰胺酶阳性250株（89.6%）;红霉素耐药而克林霉素敏感或中介的30株中,D试验阳性22株（73.3%）。苯唑西林（OXA）和头孢西丁（FOX）MIC范围分别为0.125～〉256μg/mL和2～〉256μg/mL,苯唑西林的MIC50和MIC90分别为128μg/mL和256μg/mL,头孢西丁的MIC50和MIC90分别为64μg/mL和256μg/mL。结论长沙地区临床分离金葡菌对常用抗菌药物呈多重耐药;MRSA不仅分离率高,而且对甲氧西林呈高水平耐药。     

Staphylococcus aureus nosocomial infections: the role of a rapid and low-cost characterization for the establishment of a surveillance system

Continuous surveillance on resistance patterns and characterization of Staphylococcus aureus represent simple and low-cost techniques to understand and evaluate the effectiveness of infection control and antimicrobial prescribing measures. In this study we analyzed the antibiotic susceptibility and trends for S. aureus strains collected from bacteraemia cases in a five year period. Between 2004 and 2008 we noted a progressive decrease in the number of S. aureus isolates compared to all pathogens from clinical specimens and S. aureus bloodstream infections (BSI) reflected a similar trend. In particular we analyzed 185 isolates from blood cultures: 89 isolates were MSSA and 96 isolates were MRSA. Molecular SCCmec typing of these strains showed an absolute prevalence of types I and II, whereas five spa types from 96 isolates were obtained. Resistance pattern analysis allowed us to place MRSA strains into 12 antibiotypes and the major antibiotype was resistant to penicillin, gentamicin, erythromycin, clindamycin and ciprofloxacin. The predominant antibiotype among the MSSA isolates was resistant only to penicillin. In addition, 19.1% of MSSA are susceptible to all antibiotics tested. We also found a close association between antibiotyping 1 and genotyping t002/SCCmecI of MRSA strains, suggesting a nosocomial scenario dominated by a few particular clones.     

High throughput multiple locus variable number of tandem repeat analysis (MLVA) of Staphylococcus aureus from human, animal and food sources

Staphylococcus aureus is a major human pathogen, a relevant pathogen in veterinary medicine, and a major cause of food poisoning. Epidemiological investigation tools are needed to establish surveillance of S. aureus strains in humans, animals and food. In this study, we investigated 145 S. aureus isolates recovered from various animal species, disease conditions, food products and food poisoning events. Multiple Locus Variable Number of Tandem Repeat (VNTR) analysis (MLVA), known to be highly efficient for the genotyping of human S. aureus isolates, was used and shown to be equally well suited for the typing of animal S. aureus isolates. MLVA was improved by using sixteen VNTR loci amplified in two multiplex PCRs and analyzed by capillary electrophoresis ensuring a high throughput and high discriminatory power. The isolates were assigned to twelve known clonal complexes (CCs) and--a few singletons. Half of the test collection belonged to four CCs (CC9, CC97, CC133, CC398) previously described as mostly associated with animals. The remaining eight CCs (CC1, CC5, CC8, CC15, CC25, CC30, CC45, CC51), representing 46% of the animal isolates, are common in humans. Interestingly, isolates responsible for food poisoning show a CC distribution signature typical of human isolates and strikingly different from animal isolates, suggesting a predominantly human origin.     

Polymerase chain reaction fingerprints of methicillin-resistant Staphylococcus aureus clinical isolates in Greece are related to certain antibiotypes

Characterization of clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and representatives of three MRSA clones from other hospitals was performed by arbitrarily primed polymerase chain reaction (AP-PCR) and antibiotic susceptibility patterns. All isolates were mecA-positive and two main antibiotypes have been characterized. Two major clones were identified with AP-PCR related to the aforementioned antibiotypes. The combination of antibiotypes with AP-PCR patterns successfully identified the two major clones in our hospital, which are identical with the MRSA clones previously characterized in Athens and in Central and North Greece.     

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.     

Molecular characterization and antimicrobial susceptibility of nasal Staphylococcus aureus isolates from a Chinese medical college campus

Staphylococcus aureus colonization and infection occur more commonly among persons living or working in crowded conditions, but characterization of S. aureus colonization within medical communities in China is lacking. A total of 144 (15.4%, 144/935) S. aureus isolates, including 28 (3.0%, 28/935) MRSA isolates, were recovered from the nares of 935 healthy human volunteers residing on a Chinese medical college campus. All S. aureus isolates were susceptible to vancomycin, quinupristin/dalfopristin and linezolid but the majority were resistant to penicillin (96.5%), ampicillin/sulbactam (83.3%) and trimethoprim/sulfamethoxazole (93.1%). 82%, (23/28) of the MRSA isolates and 66% (77/116) of the MSSA isolates were resistant to multiple antibiotics, and 3 MRSA isolates were resistant to mupirocin--an agent commonly used for nasal decolonization. 16 different sequence types (STs), as well as SCCmec genes II, III, IVd, and V, were represented among MRSA isolates. We also identified, for the first time, two novel STs (ST1778 and ST1779) and 5 novel spa types for MRSA. MRSA isolates were distributed in different sporadic clones, and ST59-MRSA-VId- t437 was found within 3 MRSA isolates. Moreover, one isolate with multidrug resistance belonging to ST398-MRSA-V- t571 associated with animal infections was identified, and 3 isolates distributed in three different clones harbored PVL genes. Collectively, these data indicate a high prevalence of nasal MRSA carriage and molecular heterogeneity of S. aureus isolates among persons residing on a Chinese medical college campus. Identification of epidemic MRSA clones associated with community infection supports the need for more effective infection control measures to reduce nasal carriage and prevent dissemination of MRSA to hospitalized patients and health care workers in this community.     

Use of RAPD to investigate the epidemiology of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> infection in Malaysian hospitals

Summary Randomly ampli.ed polymorphic DNA (RAPD) with four different decamer oligonucleotide primers was performed on 50 clinical Staphylococcus aureus isolates obtained from di.erent hospitals in Malaysia. All the four primers generated polymorphisms in all 50 isolates of S. aureus studied, revealing DNA markers with sizes ranging from 100 to 7000 bp. The dendrogram generated from the RAPD analysis revealed two major groups (Groups I-II) with three clusters each in one group. S. aureus strains isolated from the same hospital were found to be genetically closely related and most of them were placed in the same cluster. In addition RAPD di.erentiated between MRSA and non-MRSA based on the clustering, where all MRSA and non-MRSA were placed in their respective clusters. The RAPD analysis showed that there could be four to .ve clones of S. aureus spreading around Malaysia, of which two clones may be MRSA. The overall genetic distances ranged from 0.088235 to 0.954545 among the isolates. This technique was found to be a simple and e.ective method for epidemiological investigation. Because of these e.cient features, this technique may have more general application for the study of S. aureus infections in hospitals and the community.     

猪源致病性金黄色葡萄球菌的分离鉴定及其耐药性分析

目的鉴定引起猪渗出性皮炎的病原,并分析猪源致病性金黄色葡萄球菌的耐药性,为临床用药提供依据。方法采集患渗出性皮炎的仔猪标本进行细菌分离培养,联合应用形态学检查、生理生化试验和PCR方法鉴定分离菌株,并进行致病性和药物敏感性试验。结果先后从病猪标本中分离鉴定获得PSA1、PSA2、PSA3和PSA4四株金黄色葡萄球菌,其中PSA1和PSA3分离株的致病性较强。药敏试验结果显示PSA1、PSA2和PSA3分离株为MRSA菌株,PSA4分离株为MSSA菌株。MRSA菌株对14种抗菌药物均呈现不同程度的耐药,尤其是对青霉素、链霉素、四环素、强力霉素、环丙沙星和氧氟沙星等6种抗菌药物的耐药率达100%。所有分离株对万古霉素与替考拉宁均敏感。结论合肥地区猪渗出性表皮炎的病原为金黄色葡萄球菌。猪源致病性金黄色葡萄球菌合肥分离株具有多重耐药性,治疗猪渗出性皮炎应建立在体外药敏试验的基础上,有针对性选择抗菌药物。     

Lack of clonal relationship between non-typhi Salmonella strain types from humans and those isolated from animals living in close contact

Antibiogram patterns and chromosomal DNA typing were used to compare 151 non-typhoidal Salmonella spp. (NTS) isolated from patients and 78 from animals, environmental or food specimens obtained within or near the homes of patients with invasive salmonellosis. The majority of NTS from humans (137; 90.7%) were Salmonella enterica serotype Typhimurium (S. Typhimurium) and S. Enteritidis. Chicken specimens and feeds produced (24; 52.2%) S. Enteritidis, while S. Agona was the predominant (20; 77%) serovar among pigs and dairy cows. The majority (97; 64.2%) of NTS from humans were multidrug resistant, while NTS from cows, pigs, beef carcass swabs and sewers were fully susceptible to all antibiotics tested. Pulsed-field gel electrophoresis patterns of XbaI-digested genomic DNA of NTS from the humans and the chickens were different. However, S. Enteritidis from chickens, and S. Braenderup and S. Agona from cows and pigs were clustered together in one group. There was no significant relatedness between NTS isolates from humans and those from animals, food or the environment in close contact to humans.     

RAPD-PCR analysis of Staphylococcus aureus strains isolated from bovine and human hosts

Staphylococcus aureus is one of the most important pathogens in humans and animals. In this study eighty strains were analyzed by RAPD-PCR to assess the genetic relationship between S. aureus isolates from bovine and human hosts. Results were compared with those obtained by biotyping. Fifty-two percent of the S. aureus isolates belonged to a host specific biotype (human, bovine and poultry). Bovine and human ecovars were the most prevalent. Dendrogram obtained by RAPD results showed that all the isolates clustered into eleven groups (A-K) at a relative genetic similarity of less than 30% when analyzed with the three primers. Group A clustered 95% of the human host isolates and the remaining groups (B-K) clustered the bovine host isolates. Principal coordinate analysis also showed that the isolates could be arbitrarily divided into two groups, bovine and human, by the second coordinate. Only 9 isolates (11%) were not clustered into these groups. The genetic diversity among the S. aureus isolates from bovine hosts is relatively low compared to that of isolates from human hosts. There were no statistically significant differences among isolated from bovine and human hosts. This study shows that RAPD-PCR assayed with three primers can be successfully applied to assess the genetic relationship of S. aureus isolates from different hosts.     

Antibiotic resistant Staphylococcus aureus: a paradigm of adaptive power

Nothing documents better the spectacular adaptive capacity of Staphylococcus aureus than the response of this important human and animal pathogen to the introduction of antimicrobial agents into the clinical environment. The effectiveness of penicillin introduced in the early 1940s was virtually annulled within a decade because of the plasmid epidemics that spread the ss-lactamase gene through the entire species of S. aureus. In 1960 within one to two years of the introduction of penicillinase resistant ss-lactams (methicillin), methicillin resistant S. aureus (MRSA) strains were identified in clinical specimens. By the 1980s, epidemic clones of MRSA acquired multidrug resistant traits and spread worldwide to become one of the most important causative agents of hospital acquired infections. In the early 2000s, MRSA strains carrying the Tn1546 transposon-based enterococcal vancomycin resistant mechanism were identified in clinical specimens, bringing the specter of a totally resistant bacterial pathogen closer to reality. Then, in the late 1990s, just as effective hygienic and antibiotic use policies managed to bring down the frequency of MRSA in hospitals of several countries, MRSA strains began to show up in the community.     

Antibiotic resistance in bacterial isolates from laboratory animal colonies naive to antibiotic treatment

Antiobiogrammes were made of a number of isolates of Staphylococcus aureus, Escherichia coli and Pasteurella pneumotropica derived from rodent, rabbit or minipig colonies never treated with antibiotics. For S. aureus no differences between rats and mice were found in the percentage of resistant isolates. Gentamicin and erythromycin were found to be the most efficient, while the highest percentages of resistance were found to be against penicillins and sulphonamides. In general, the results from antibiogrammes on E. coli were rather uniform, with only slight differences between isolates from different species, except that more vancomycin and tetracycline-resistant minipig isolates were found. In almost all isolates of E. coli, resistance was shown against penicillin, fucidin, macrolides, lincosamides and tiamulin. For a number of antibiotics, mouse isolates of P. pneumotropica were more frequently found to be sensitive than rat isolates. The resistance patterns of E. coli from the minipigs were quite similar to resistance patterns found in farm pigs, but apart from this, the resistance patterns of the bacterial species tested did not resemble human or farm animal patterns in any of the animal species, and, therefore, these studies do not support the theory that S. aureus and E. coli in laboratory animal colonies derive from the normal flora of the human caretakers. The fact that rodent species of E. coli, in contrast to human and farm animal species, are sensitive to ampicillin, tetracyclines, and the combination of sulphonamides and trimethoprim, might be due to the fact that these antibiotics are not used in rodent populations.     

Accuracy of phenotypic methicillin susceptibility methods in the detection of Staphylococcus aureus isolates carrying different SCCmec types

A total of 138 isolates, 118 methicillin-resistant Staphylococcus aureus (MRSA) isolates (staphylococcal cassette chromosome type II, 20 isolates, type III, 39 isolates and type IV, 59 isolates) and 20 methicillin-sensitive S. aureus isolates were evaluated by phenotypic methods: cefoxitin and oxacillin disk diffusion (DD), agar dilution (AD), latex agglutination (LA), oxacillin agar screening (OAS) and chromogenic agar detection. All methods showed 100% specificity, but only the DD tests presented 100% sensitivity. The sensitivity of the other tests ranged from 82.2% (OAS)-98.3% (AD). The LA test showed the second lowest sensitivity (86.4%). The DD test showed high accuracy in the detection of MRSA isolates, but there was low precision in the detection of type IV isolates by the other tests, indicating that the genotypic characteristics of the isolates should be considered.     

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。     

A CD-1 mouse model of infection with Staphylococcus aureus: influence of gender on infection with MRSA and MSSA isolates

Staphylococcus aureus is an important pathogen of humans and other animals, causing bacteremia, abscessation, toxemia, and other infectious diseases. An animal model using CD-1 mice was developed to study the pathogenesis of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA). When inoculated into the CD-1 mouse model, it was shown that both MSSA isolates, (HR 78 and CSA-1) and MRSA isolates (MRSA 456 and MRSA 457) led to chronic infection of the kidney. Female CD-1 mice inoculated with MRSA 456 proved to be more susceptible to infection and mortality than their male counterparts. Castrated mice became more susceptible to infection than intact male mice, suggesting a hormonal involvement in the infection process.