Detection of methicillin and mupirocin resistance in staphylococcal hospital isolates with a touchdown multiplex polymerase chain reaction

Staphylococcal hospital isolates (n = 166) were tested in a touchdown multiplex-polymerase chain reaction assay for the identification of methicillin and mupirocin resistance and discrimination of S. aureus (femA gene) from coagulase negative staphylococci and other bacteria. All isolates harbored the 16SrDNA (Staphylococcus genus specific internal control) gene, and 130 (78 %) the mecA (methicillin resistance) gene. Fifty-seven (44 %) of these were determined as methicillin-resistant S. aureus, while the remaining 73 (56 %) were methicillin-resistant coagulase-negative staphylococci. Seventy-five (45 %) isolates harbored the ileS-2 (high-level mupirocin resistance) gene and were determined as mupirocin-resistant. This assay represents a simple, rapid, reliable approach for the detection and discrimination of methicillin-and mupirocin-resistant staphylococci.     

Latex agglutination and hemagglutination tests for the rapid identification of methicillin sensitive and methicillin resistant Staphylococcus aureus

Ten latex agglutination (LA) and hemagglutination (HA) kits for the identification of Staphylococcus aureus were compared with reference methods for their reliability and performance. The ten commercial kits consisted of Accu-Staph, Bacto-Staph, Hemastaph, Staphaurex, Staph-Latex, Staphylochrome, Staphyloslide, Staph-Rapid, Sero-Stat and Veri-Staph. The conventional methods included slide coagulase test, tube coagulase test (4 hr, 24 hr), thermonuclease and growth on mannitol salt agar (MSA). A total of 583 clinical isolates of staphylococci were used and all the kits correlated well with the conventional methods (93.1-99.4% sensitivity) in their ability to identify both methicillin sensitive (MSSA) and methicillin resistant S. aureus (MRSA). Although all were rapid, easy to perform and simple to interpret, Staphaurex and Staphyloslide gave the best sensitivities and specificities.     

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.     

Characterisation of Staphylococcus aureus nasal and skin carriage among patients undergoing haemodialysis treatment

The aim of the study was to investigate the rate of Staphylococcus aureus nasal and skin carriage in patients undergoing haemodialysis. The cultured staphylococcal isolates were subsequently characterized by molecular methods. The study group comprised 43 haemodialysed patients from whom nasal and skin swabs from the vascular access sites were collected. The identification of staphylococcal isolates and antibiotic susceptibility testing were performed on the basis of conventional diagnostic procedures. The staphylococci were further characterized using Pulsed-Field Gel Electrophoresis (PFGE). S. aureus was cultured from 12 (27.9%) patients. Only one (8.3%) patient was colonized with the microorganism both in the anterior nares and the vascular access site representing a single strain, as evidenced by PFGE analysis. Antibiotic susceptibility testing identified one (7.6%) methicillin-resistant S. aureus (MRSA) strain. PFGE typing identified several S. aureus genotypes with the lack of one specific strain responsible for colonization. However, it should be noted that among two (A and D) PFGE patterns genetically indistinguishable and closely related isolates (two isolates for each pattern) were identified. The obtained results revealed a relatively low rate of S. aureus carriage accompanied by low methicillin resistance rate and a significant genetic diversity of cultured isolates with the lack of one predominant strain responsible for colonization.     

Nasal carriage of methicillin resistant Staphylococcus aureus and their antibiotic susceptibility patterns in children attending day-care centers

Nasal colonization with community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) is being increasingly reported, especially in places where people are in close contact and in reduced hygiene, such as day-care centers. In this study we investigated the frequency of MRSA colonization and their antibiotic susceptibility patterns in 1-6 years old children of day-care centers in Hamadan, West of Iran.Five hundred nasal swabs were collected from children of 27 day-care centers that had no risk factors for colonization by S. aureus. The specimens were cultured for isolation of S. aureus by standard methods. Antimicrobial susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. For evaluation of the frequency of erythromycin induced clindamycin resistance, disk approximation test (D-test) was applied.Totally, 148 (29.6%) children were colonized by S. aureus. Out of 260 male, 94 (36.2%) and of 240 female, 54 (22.5%) cases were nasal carriers of S. aureus (P value = 0.001). Six (4.1%) of the 148 S. aureus isolated from children were MRSA strains. None of MRSA and methicillin susceptible S. aureus (MSSA) was resistant to vancomycin and clindamycin. Three of the 6 strains of MRSA and 7 (4.9%) of the 142 MSSA strains were resistant to erythromycin, and D-test was positive in all of them.We conclude that the rate of colonization by S. aureus is high in children attending day-care centers but colonization with MRSA is not common in our areas. Clindamycin or trimethoprim-sulfamethoxazol could be used in mild to moderataly severe diseases caused by CA-MRSA. However, if the CA-MRSA isolates are erythromycin resistant, D-test should be carried out for detection of inducible clindamycin resistance.     

Microbial biocenosis of the nasal mucosa in healthy persons and in Staphylococcus carriers

The normal microflora of the nasal mucosa in man is differentiated into main, complementary and casual microflora, taking into account its isolation rate and characteristics of microbial contamination. The main microflora of adults, in contrast to children, is represented, in addition to coagulaso-negative (CN) staphylococci, by bacteria of the genus Corynebacterium. In staphylococcal carrier state a decrease in the isolation rate and the amount of bacteria belonging to the main and complementary microflora (CN staphylococci, Micrococcus, Streptococcus, Bacillus) was observed. Moreover, in carriers the weakening of the degree of association between CN staphylococci, Micrococcus, Corynebacterium, Streptococcus in symbiotic pairs was noted. In the absence of S. aureus carrier state the main and complementary microflora was characterized by the combination of the factors of antagonism and persistence, while in carrier state the complex of colonization factors was defective in the main microflora and well pronounced in coagulase-positive staphylococcal strains and enterobacteria.     

Simultaneous PCR detection of ica cluster and methicillin and mupirocin resistance genes in catheter-isolated Staphylococcus.

Recent data show that more than 50% of catheter-associated bloodstream infections are caused by staphylococci. Staphylococcal infections produced by intercellular-adhesion cluster (ica) carriers can be even more problematic due to the presence of methicillin and mupirocin resistance genes. In the present study, a multiplex PCR protocol that allows the simultaneous identification of staphylococci and detection of both the ica and methicillin and/or mupirocin resistance genes was developed. Furthermore, the method allows differential detection of the ica locus from Staphylococcus aureus and Staphylococcus epidermidis.     

Investigation of aminoglycoside modifying enzyme genes in methicillin-resistant staphylococci

Methicillin-resistant staphylococci may also be resistant to some other antibiotics as well as beta-lactams. In this study, co-existence of resistance to methicillin and aminoglycosides was genetically investigated in staphylococci. A total of 50 staphylococci from in-patients, 17 Staphylococcus aureus and 33 coagulase negative staphylococci (CNS) that contained mecA (gene encoding PBP 2a, an altered penicillin-binding protein) determined by polymerase chain reaction (PCR) were included in the study. Aminoglycoside modifying enzyme (AME) genes were investigated using multiplex-PCR. Aminocyclitol-6'-acetyltransferase-aminocyclitol-2'-phosphotransferase [aac(6')/aph(2')] gene (encoding bifunctional acetyltransferases/phosphotransferases) was determined in 66% of the isolates, aminocyclitol-4'-adenylytransferase (ant(4')-Ia) gene (encoding phosphotransferases) in 24%, and aminocyclitol-3'-phosphotransferase (aph(3')-IIIa) gene (encoding nucleotidyltransferases) in 8%. Two isolates contained all these three genes. Thirty-six (72%) isolates had at least one of these genes. Three CNS and one S. aureus isolates sensitive to oxacillin had the mecA gene. In conclusion, a high rate of aminoglycoside resistance was determined in methicillin-resistant staphylococci. The aac(6')/aph(2') was the most frequently detected.     

Evaluation of methicillin-resistance in Staphylococcus aureus by the agar disk diffusion method and PCR

Staphylococcus aureus is a widespread human pathogen. One the most striking characteritics of this bacterium is resistance to methicillin and all beta-lactam antibiotics. The agar disk diffusion method is the most widely used in vitro susceptibility test, but recently molecular methods, e.g. Polymerase Chain Reaction, have been also introduced. We compared the detection of methicillin resistant coagulase positive Staphylococcus aureus isolated from clinical materials in Silesian microbiological laboratories by diffusion method and PCR through the detection of nuc and mec A genes. Our results show that PCR used for the detection of mec A gene increases the detection of methicillin-resistant Staphylococcus aureus strains by 10% as compared to the agar disk diffusion method. Among Staphylococcus aureus strains, detected as methicillin-resistant, 17% of organisms showed no presence of mec A gene.     

Catheter-tract infections in rhesus macaques (Macaca mulatta) with indwelling intravenous catheters

Development of catheter-tract infections in experimental animals can have devastating consequences on animal health and the functional lifespan of surgical implants. To measure the incidence of catheter-tract infections in animals with exteriorized intravenous catheters in this facility and assess the effects of these infections on mean catheter lifespan, health records of 31 Macaca mulatta with catheters were reviewed. Records spanned the interval of January 1, 1996 through October 1, 1997. Catheter-tract infections in 16 of 53 (30.2%) monkeys with catheters were diagnosed based on a combination of clinical signs of infection and results of bacterial culture. Segmental catheter-tract infections reduced mean catheter lifespan to 147 days, compared with 354 days for uninfected catheters. Exit-wound, local tunnel, and surgical-site infections did not significantly reduce catheter lifespan. Bacterial culture reports documented 31 isolates; 41.9% (13 of 31) were coagulase-negative staphylococci, and 22.6% (7 of 31) were Staphylococcus aureus. Of 20 isolates tested, 15 (75%) were resistant to methicillin/oxacillin in vitro. Antimicrobial susceptibility testing of methicillin-resistant and methicillin-susceptible isolates indicated that, compared with methicillin-sensitive isolates, methicillin-resistant isolates had a pattern of multiple antibiotic resistance. Catheter-tract infections were common in this colony of rhesus macaques, and clinically severe infections caused a drastic reduction in catheter lifespan. Approximately half (48%) the bacterial isolates were methicillin-resistant gram-positive bacteria.     

Bacterial interference with skin colonization in germfree hairless mice

Bacterial colonization of the digestive tract and the skin was studied over a 3-week period in a group of 10 germfree HRS mice using Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa. Sequential utilization of two strains allowed us to carry out six assays and to show the presence of interference phenomena during colonization of the skin. When P. aeruginosa was given after challenge with S. aureus or S. epidermidis, it did not colonize the skin. If the first challenge was done with P. aeruginosa, this bacteria was eliminated within 10 days by S. aureus and S. epidermidis on the skin, but it succeeded in colonizing the digestive tract. When the first challenge was done with S. aureus, colonization of the skin and the digestive tract with S. epidermidis was prevented, whereas these two species were found in association when S. aureus was given in second place. None of the in vitro assays (mixed culture, bacteriocin production, adherence inhibition, antimicrobial activity) could explain the in vivo observations.     

Staphylococci Isolated from Carriage Sites and Infected Sites of Dogs as a Reservoir of Multidrug Resistance and Methicillin Resistance

The aim of this study was to compare the spread of multidrug-resistant (MDR) and methicillin-resistant (MR) staphylococci in healthy dogs and in dogs with evident symptoms of infection. The samples from 172 healthy and 197 infected dogs were examined. The staphylococci were identified with conventional methods and by means of the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method (MboI). Susceptibility to 15 antibiotics from 10 different antimicrobial classes was tested. Resistance to methicillin was confirmed by the presence of Staphylococcus aureus mecA and S. sciuri mecA genes. Multidrug resistance was defined as resistance to three or more antimicrobial classes. The oral mucosa to be the most frequent site of staphylococcal colonization (55.8 %), followed by nasal cavity (44.2 %), and anus (32.6 %). The prevalence of MDR staphylococci in infected dogs was significantly higher than in the healthy animals (74/137 vs. 34/95, P = 0.006). The MR strains of S. pseudintermedius (2.9 %) originated solely from infected dogs. In contrast, the MR coagulase-negative strains (7.4 %) were isolated solely from healthy dogs. S. aureus strains originated from nasal swabs, MRSA strains were not isolated. MDR staphylococci and MR S. pseudintermedius are more common among infected dogs, but coagulase-negative staphylococci (mostly S. sciuri) seem to be a reservoir of methicillin resistance in healthy dogs.     

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。     

Methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci: epidemiological and molecular aspects

Infections caused by the genus Staphylococcus are of great importance for human health. Staphylococcus species are divided into coagulase-positive staphylococci, represented by S. aureus, a pathogen that can cause infections of the skin and other organs in immunocompetent patients, and coagulase-negative staphylococci (CNS) which comprise different species normally involved in infectious processes in immunocompromised patients or patients using catheters. Oxacillin has been one of the main drugs used for the treatment of staphylococcal infections; however, a large number of S. aureus and CNS isolates of nosocomial origin are resistant to this drug. Methicillin resistance is encoded by the mecA gene which is inserted in the SCC mec cassette. This cassette is a mobile genetic element consisting of five different types and several subtypes. Oxacillin-resistant strains are detected by phenotypic and genotypic methods. Epidemiologically, methicillin-resistant S. aureus strains can be divided into five large pandemic clones, called Brazilian, Hungarian, Iberian, New York/Japan and Pediatric. The objective of the present review was to discuss aspects of resistance, epidemiology, genetics and detection of oxacillin resistance in Staphylococcus spp., since these microorganisms are increasingly more frequent in Brazil.     

Bacterial competition for human nasal cavity colonization: role of Staphylococcal agr alleles

We examined the bacterial aerobic nasal flora of 216 healthy volunteers to identify potential competitive interactions among different species, with special emphasis on the influence of staphylococcal agr alleles. The Staphylococcus aureus colonization rate correlated negatively with the rate of colonization by Corynebacterium spp. and non-aureus staphylococci, especially S. epidermidis, suggesting that both Corynebacterium spp. and S. epidermidis antagonize S. aureus colonization. Most of the S. aureus and S. epidermidis isolates were agr typed by a PCR method. Only one S. aureus agr (agr(Sa)) allele was detected in each carrier. Multiple logistic regression of the two most prevalent agr(Sa) alleles (agr-1(Sa) and agr-2(Sa)) and the three S. epidermidis agr (agr(Se)) alleles showed a specific influence of the agr system. The results of this model did not support conclusions drawn from previous in vitro agr-specific cross-inhibition experiments. Our findings suggest that the agr alleles, which are strongly linked to the bacterial genetic background, may simply be associated with common biological properties--including mediators of bacterial interference--in the strains that bear them.     

Inhibition of Fibrinogen Reaction by Polysaccharide of Encapsulated Staphylococcus aureus

Encapsulated and nonencapsulated strains of Staphylococcus aureus which lack coagulase or clumping factor (bound coagulase), or both, were examined for the antigen associated with the fibrinogen-cell clumping reaction. Extracts of the cells were tested for the ability to react with fibrinogen or to inhibit fibrinogen precipitation. Antisera prepared against encapsulated (coagulase-positive, clumping factor-negative) variants, as well as against nonencapsulated wild-type (coagulase-positive, clumping factor-positive) S. aureus strains, contained high titers of clumping-inhibiting antibody. When coagulase-negative, clumping factor-negative mutants were the immunizing agents, antisera contained no demonstrable clumping-inhibiting antibody. Phenol extracts of all coagulase-positive strains tested precipitated fibrinogen, regardless of the ability of cells to clump in the presence of fibrinogen. Polysaccharide extracts of encapsulated, clumping factor-negative strains inhibited this fibrinogen-precipitating activity, whereas similar extracts of nonencapsulated staphylococci did not inhibit the fibrinogen reaction. From these results, it appeared that the coagulase-positive, encapsulated staphylococci which do not clump in fibrinogen solution possess clumping factor, but that their capsular polysaccharide inhibits clumping activity. These findings suggested a closer association of clumping factor and coagulase than is now recognized.     

A combination of methods to evaluate biofilm production may help to determine the clinical relevance of Staphylococcus in blood cultures

Staphylococcus is the most prevalent pathogen causing bacteremia and many of its isolates possess the ability to form biofilm. In this study Staphylococcus isolates from the blood of patients with bacteremia were analyzed by two biofilm detection phenotypic methods: Congo red agar (CRA) and microtiter-plate adherence (MPA) in relation to the presence of ica genes, detected by PCR. Their oxacillin susceptibility was also evaluated. Among 127 isolates evaluated, 47 were S. aureus and 80 were coagulase negative staphylococci (CNS). Seventy-four (58.3%) isolates were mecA gene positive (27.7%S. aureus and 76.3% CNS isolates). Among the 40 S. aureus isolates which were positive for the ica genes, 25 (62.5%) were positive in MPA and 27 (67.5%) in CRA, whereas both methods combined detected 34 (85%) isolates as biofilm producers. Among 12 S. epidermidis isolates carrying ica genes, 8 were positive in MPA and 5 in CRA. The combination of CRA and MPA methods provided a better prediction of the presence of ica genes in S. aureus isolates than did either method alone.     

Public transport as a reservoir of methicillin‐resistant staphylococci

Aims: The aim of this study was to explore the occurrence of methicillin‐resistant staphylococci in a large urban public transport system. Methods and Results: Samples were taken from hand rails, which passengers hold onto when they are standing. In total, 1400 swabs taken from 55 vehicles (trolleybuses, trams and buses) were examined. As many as 30·1% samples were positive for the presence of methicillin‐resistant coagulase‐negative staphylococci (MRCoNS), but none for methicillin‐resistant Staphylococcus aureus (MRSA). MRCoNS were isolated from all 55 vehicles. Nearly 50% of MRCoNS isolates displayed resistance not only to beta‐lactams, but at least to two or more other classes of antimicrobials as well. Conclusions: This study demonstrated widespread occurrence of MRCoNS on hand rails in public transport vehicles. MRSA was not detected. Significance and Impact of the Study: The recovery of methicillin‐resistant staphylococci from public transport system implies a potential risk for transmission of these bacteria in an out‐hospital environment.     

Characterization of staphylococci

A total of 158 Staphylococcus strains from various sources were characterized by biochemical, physiological, and morphological tests. Numerical taxonomy was applied by using these features. Taxonomic analysis was done with programs run under the MVS-TSO system of the IBM 370 complex and PDP-10 system of the National Institutes of Health. DNA-DNA hybridization with nitrocellulose filters was done to compare selected atypical cultures with American Type Culture Collection reference strains. We found that the use of the nomenclature of Bergey's Manual (8th edition) to identify these strains by species was not adequate. DNA homology values supported the formation of Staphylococcus hyicus subsp. hyicus separate from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. The three tests that best separated these strains into four species were (i) tube coagulase (6-h or 24-h porcine plasma or 24-h Difco rabbit plasma), (ii) production of acetoin or acid aerobically from ribose, maltose, or trehalose, and (iii) growth in the presence of novobiocin. Four strains of S. hyicus subsp. hyicus (VII76, VII113, VII131, and VA519) gave typical enterotoxigenic responses in monkey-feeding tests but were negative for enterotoxins A through E, suggesting the presence of one or more new enterotoxins. Two coagulase-negative, heat-stable DNase-positive strains (D143 and ARM) could not be classified by either DNA-DNA hybridization or numerical taxonomy, and D143 was enterotoxigenic as measured by the monkey-feeding bioassay. DNA homology showed that strain FRI-698M was more closely related to S. epidermidis than to S. aureus, yet it produced enterotoxin D. These data suggest the occurrence of coagulase-negative enterotoxigenic strains that are not S. aureus; nonetheless, a positive tube coagulase test and heat-stable DNase test should together be useful for routine screening of most potentially enterotoxigenic staphylococci in foods.     

Composition of AME encoding gene in Gram positive Cocci--study on spread of aminoglycoside resistance

Aminoglycoside modifying enzymes (AMEs) are major factors which confer aminoglycoside resistance on bacteria. Composition of six genes encoding AMEs (including lately described aph 2"-genes) was investigated by PCR for 16 clinical isolates of Enterococcus faecalis, 16 clinical isolates of coagulase-positive (S. aureus) and 13 clinical isolates of coagulase negative staphylococci (S. haemolyticus, S. epidermidis) collected in Gdańsk region (Northern Poland) in the years 1998-2001. Diversity of AME encoding gene profiles (composition) was used to analyze spread of AME encoding gene among and within studied group of cocci. According to presence of particular genes we distinguish eleven different AME encoding gene profiles: seven profiles were unique for particular species while the most common was shared among S. aureus, coagulase negative staphylococci and enterococci. Regarding profile frequency statistical analysis (Fstat, AMOVA, cluster analysis UPGMA) shows: the difference between S. aureus and enterococci and coagulase-negative staphylococci, lack of difference between enterococci and coagulase-negative staphylococci, higher variability within than between studied species and presence of multispecies cluster. On the basis of the reports about ability of staphylococci to synthesis enterococcal pheromones, this finding lets assume that spread of aminoglycoside resistance gene among gram (+) cocci is limited only by the ability of stains to synthesis or induction of synthesis conjugation protein.