Recipient characteristics in the transduction of methicillin resistance in Staphylococcus epidermidis.

Methicillin resistance (Mecr) was transduced into a methicillin-susceptible variant of the Mecr donor Staphylococcus epidermidis BS10. UV irradiation of phage stimulated Mecr transduction frequency. If loss of Cd and Hg ion resistance occurred in this recipient, or if the three markers Mecr, Cdr, and Hgr were co-eliminated from the donor, neither strain acted as a recipient for Mecr.     

Screening tests for the detection of methicillin resistance in Staphylococcus epidermidis.

Methods to detect resistance to methicillin in Staphylococcus epidermidis were studied in order to find a rapid screening test suitable for routine use. One hundred and forty-nine clinical isolates, 16 isolates from skin of healthy people and two reference strains were studied. Hypersecretion of beta-lactamase as a cause of methicillin resistance was eliminated in the strains studied. Tube and microtitre breakpoint, agar breakpoint and disc diffusion methods were compared. The breakpoint for methicillin resistance used was 16 mg/L in broth and 10 mg/L in agar. The discs used contained 1 and 5 micrograms oxacillin and 5 and 10 micrograms methicillin. Turbidimetric measurements in broth during incubation were carried out using the Bioscreen analysing system. The skin strains were founf to be susceptible in all tests. Using an inoculum of 10(7) cfu/mL 111/149 clinical isolates were classified as resistant after incubation for 24 h at 35 degrees C using the tube and microtitre breakpoint tests, incubation for 72 h did not increase this rate. When an inoculum of 10(5) cfu/mL was used 73% of these strains were identified within 24 h and all within 72 h with the tube breakpoint test. Using the microtitre breakpoint test, with an inoculum of 10(5) cfu/mL or lower, all resistant strains were not detected within 48 h. All agar breakpoint tests required 48 h incubation for reliable results. Only the 1 microgram oxacillin disc always separated strains found to be resistant or susceptible in the tube breakpoint test. The zone of inhibition was clearly readable after 16 h of incubation at 35 degrees C.     

Detection of biofilm-producing and methicillin resistance genes in Staphylococcus epidermidis isolated from healthy humans and in blood culture tests

The detection of biofilm-producing (ica AB) and methicillin resistance genes (mec A) was investigated in 70 blood culture isolates of Staphylococcus epidermidis and in 66 and 51 isolates from human hands and the vestibules of the nose, respectively, of 77 healthy subjects who gave consent. Of the 70 strains isolated from blood culture testing, both ica AB and mec A were detected in 36 (51.4%), and neither was detected in 4 (5.7%). The mec A gene only was detected in 30 (42.9%), but no isolate from blood culture testing possessed the ica AB gene alone. In contrast, of the 66 isolates from healthy hands, only one isolate (1.5%) possessed both genes, whereas neither was detected in 56 (84.8%), but the mec A gene was detected in 9 (13.6%). Of the 51 isolates from the nasal vestibules, both genes were detected in 12 (23.5%), and neither in 15 (29.4%). Moreover, the mec A gene was detected in 17 (33.3%). Thus, S. epidermidis strains that normally inhabit the nasal vestibule were found to carry the ica AB and mec A genes more frequently than those that inhabit the fingers. The ica AB and mec A genes were detected in S. epidermidis isolated in blood culture tests from patients diagnosed with sepsis associated with catheter-related bloodstream infection (CR-BSI). Both genes were detected in 7 (70.0%) of 10 isolates, and the mec A gene alone was detected in 3 (30.0%). In fact, we could not detect any strain carrying only the ica AB gene from S. epidermidis, an infecting organism of CR-BSI. This suggests that surviving strains carrying the mec A gene cause severe infection on empirical administration of an antibacterial drug, although biofilm formation by the ica AB gene is also important for CR-BSI. Based on these findings, most strains of S. epidermidis causing CR-BSI are biofilm-producing β-lactam-resistant (methicillin-resistant) bacteria. When S. epidermidis is isolated from blood culture testing, the identification of both ica AB and mec A genes may be significant with regard to judging whether the detected strain is the etiologic agent of CR-BSI.     

Low-level methicillin resistance in strains of Staphylococcus aureus.

Two strains of Staphylococcus aureus expressing borderline or low-level methicillin resistance by one or more in vitro test methods were examined for resistance in vivo and for biochemical and genetic markers of methicillin resistance. In vivo, nafcillin was equally effective against experimental aortic valve endocarditis in rabbits, regardless of whether they were infected by a fully susceptible or a low-level-resistant strain. Resistance did not emerge during therapy. For the more resistant of the two low-level-resistant strains, methicillin was as effective as nafcillin. Nafcillin was ineffective against endocarditis caused by a truly methicillin-resistant strain, and resistance emerged on therapy. The low-level-resistant strains did not produce the low-affinity penicillin-binding protein 2a that is associated with methicillin resistance and did not contain DNA that hybridized with probes that recognized the methicillin resistance determinant. Low-level resistance in S. aureus is a phenomenon that is biochemically and genetically distinct from true methicillin resistance. These strains actually are susceptible to beta-lactam antibiotics. The clinical problem posed by these strains is not a therapeutic one but, instead, one of how to differentiate them from those that are truly methicillin resistant.     

Penicillin and tetracycline resistance plasmids in Staphylococcus epidermidis

The genetic nature of penicillin (Pc) and tetracycline (Tc) resistance plasmids in Staphylococcus epidermidis were studied and compared with those in S. aureus. Of 10 S. epidermidis strains transduced for penicillin resistance, we could isolate Pc plasmids from only 3. One of these plasmids also encoded for cadmium resistance and another encoded for resistance to ethidium bromide, traits also associated with S. aureus Pc plasmids. Endonuclease fingerprinting of the Pc plasmids from the two species revealed extensive heterogeneity. Two S. epidermidis strains were also transduced for tetracycline resistance. Both harbored plasmids indistinguishable from S. aureus Tc plasmids as judged by endonuclease fingerprinting. These data suggest that genetic exchange between S. aureus and S. epidermidis occurs in vivo.     

临床分离的表皮葡萄球菌耐药性分析

目的了解重庆地区表皮葡萄球菌所致医院感染的耐药情况。方法对临床收集的42株表皮葡萄球菌进行药物敏感试验并分析结果。结果表皮葡萄球菌对米诺环素、利福平、阿米卡星、替考拉林的敏感性较高,敏感率均达到80%以上;对青霉素G,苯唑西林,红霉素的耐药性最严重,均在80%以上;无万古霉素耐药的表皮葡萄球菌;耐甲氧西林的表皮葡萄球菌(MRSE)检出率高达85.7%。结论表皮葡萄球菌的耐药情况严重,MRSE菌株的检出率高,应引起高度重视,临床治疗必须根据药敏试验结果选择合适的抗生素。     

表皮葡萄球菌诱导克林霉素耐药及常见抗生素耐药性分析

目的 分离临床标本中表皮葡萄球菌,检测其对常用抗生素的耐药性,并检测红霉素对克林霉素诱导性耐药的发生率.方法 分离培养临床标本中的表皮葡萄球菌,用k-b法检测其对红霉素及克林霉素等抗生素的耐药性,并做纸片法D试验检测红霉素诱导克林霉素耐药发生率.用头孢西丁法检测MRSE.结果 86株表皮葡萄球菌中MRSE 72株,占83.72%,MSSE 14株 占16.28%.总D试验阳性34株,占39.53%,D试验阴性52珠,占60.47%.所有试验菌株对青霉素耐药率100%,红霉素耐药率88.37%,克林霉素24.42%,利奈唑烷100%敏感,万古霉素100%敏感.结论 临床分离表皮葡萄球菌对青霉素,红霉素和克林霉素等药物耐药率较高,常规D试验检测有助于临床合理使用大环内酯类及林可霉素类抗菌药物.     

Conjugational transfer of gentamicin resistance plasmids intra- and interspecifically in Staphylococcus aureus and Staphylococcus epidermidis.

We have previously reported the transfer of gentamicin resistance (Gmr) plasmids in a mixed culture inter- and intraspecifically between strains of Staphylococcus aureus and Staphylococcus epidermidis isolated at Michael Reese Hospital (Jaffe et al., Antimicrob. Agents Chemother. 21:773-779, 1982). We have now shown that representatives of these plasmids were transferred between apparently nonlysogenic strains of S. aureus either in mixed culture in broth or by filter-mating on agar medium. The mechanism of transfer appeared to be conjugation. A transferable Gmr plasmid (pSH8) mobilized or cotransferred a tetracycline R-plasmid and a chloramphenicol R-plasmid that were not independently transferable. The transfer of Gmr plasmids was accompanied by a high incidence of deletion mutations with varied loss of plasmid resistance determinants and, with some mutants, loss of the ability to effect self-transfer. Restriction endonuclease digestion of pSH8 and its deletion mutants made it possible to assign the property of self-transfer to a specific segment of the pSH8 genome and provided the basis for a physical and genetic map of that plasmid. Similar Gmr plasmids from S. aureus strains isolated in locations remote from Michael Reese Hospital had resistance determinants and transfer properties comparable to those of pSH8. Our observations provide evidence for the conjugal transfer of some staphylococcal plasmids, apparently independent of the presence of phage. This mechanism may be of significance in the intra- and interspecific dissemination of resistance to aminoglycosides and other antibiotics in Staphylococcus spp.     

Novel mechanism for plasmid-mediated erythromycin resistance by pNE24 from Staphylococcus epidermidis.

We describe an unusual type of erythromycin resistance (Emr) mediated by a plasmid designated pNE24 from Staphylococcus epidermidis. This 26.5-kilobase plasmid encodes resistance strictly to 14-membered macrolide antibiotics, erythromycin, and oleandomycin. Resistance to other macrolide-lincosamide-streptogramin B (MLS) antibiotics was not observed even after a prior induction stimulus with various MLS antibiotics. Plasmid pNE24 was found to express resistance constitutively and manifested a low to intermediate MIC (62.5 micrograms/ml) for erythromycin. The resistance gene, designated erpA, appears to mediate resistance by altering the permeability of the host cell for erythromycin, because the measured uptake of 14C-labeled erythromycin by strain 958-2 (containing pNE24) was lower than for the erythromycin-susceptible, isogenic strain 958-1. No inactivation of erythromycin in overnight broth culture supernatants could be detected. In addition, no significant loss in binding affinity between [14C]erythromycin and ribosome could be detected for ribosomes isolated from strain 958-2 relative to 958-1, indicating that pNE24 probably does not produce a modification of the bacterial ribosome. No other selectable marker was found associated with pNE24; however, a 60,000-dalton protein was present only in the membrane fractions of cells (958-2) containing pNE24 and may play a role in mediating resistance to erythromycin.     

Cloning and expression of Staphylococcus aureus plasmid-mediated quaternary ammonium resistance in Escherichia coli.

The Staphylococcus aureus plasmid pSK1 carries Tn4001, a 4.7-kilobase (kb) transposon which specifies resistance to gentamicin, tobramycin, and kanamycin. In addition, pSK1 mediates resistance to trimethoprim and linked resistance to ethidium bromide (Ebr) and to quaternary ammonium compounds (Qar). Restriction endonuclease analysis of pSK1 and a deleted derivative of pSK1 revealed that the gene(s) responsible for Ebr Qar lies within a 5.2-kb HindIII fragment. This fragment has been cloned into the Escherichia coli plasmid vector pBR322, and transformants of an E. coli K-12 strain exhibited Ebr Qar. Subcloning of the 5.2-kb insert, combined with data from electron microscopic analysis of deleted derivatives of pSK1, located the Ebr Qar determinant(s) on a 2.3-kb segment of pSK1 DNA.     

Cloning and expression of the norA gene for fluoroquinolone resistance in Staphylococcus aureus.

The norA gene for fluoroquinolone resistance in Staphylococcus aureus TK2566 was cloned from chromosomal DNA into fluoroquinolone-susceptible Escherichia coli TG1. The resulting transformant, NY11, contained a recombinant plasmid, designated pTUS1, with a cloned 5.5-kilobase (kb) HindIII fragment of staphylococcal DNA. The MIC of norfloxacin for the strain increased from 0.1 to 3.13 micrograms/ml. Furthermore, when the fragment was recloned into S. aureus, the transformant NY12, containing recombinant plasmid pTUS20, had the same level of resistance to norfloxacin as did the original strain, although it was less resistant to ofloxacin and ciprofloxacin. A single KpnI-HaeIII fragment was found to be the minimum size able to express norfloxacin resistance, suggesting that the norA gene is located within the 2.6- to 3.2-kb region of the original 5.5-kb fragment. The 5.5-kb fragment hybridized to DNA from a fluoroquinolone-susceptible S. aureus strain.     

Reassessment of the number of auxiliary genes essential for expression of high-level methicillin resistance in Staphylococcus aureus.

A new transposon library constructed in the background of the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL yielded 70 independent insertional mutants with reduced levels of antibiotic resistance. Restriction analysis with HindIII, EcoRV, EcoRI, and PstI and then Southern hybridization with probes for the transposon and for the femA-femB gene demonstrated that 41 of the 70 Tn551 mutants carried distinct and novel, as yet undescribed insertion sites, all of which were outside of the mecA gene and were also outside the already-characterized auxiliary genes femA, femB, femC, and femD. All previously described Tn551 mutations of this type were in genes located either on SmaI fragment A or SmaI fragment I. In contrast, inserts of the new library were located in 7 of the 16 SmaI chromosomal fragments, fragments A, B, C, D, E, F, and I. In all of the mutants, expression of methicillin resistance became heterogeneous, and the MIC for the majority of cells was reduced (1.5 to 200 micrograms ml-1) from the homogeneous methicillin MIC (1,600 micrograms ml-1) of the parental cells. Although identification of the exact number of genes inactivated through the new set of transposon inserts will require cloning and sequencing, a rough estimate of this number from mapping data suggests a minimum of at least 10 to 12 new genetic determinants, all of which are needed together with femA, femB, femC, and femD for the optimal expression of methicillin resistance.     

Methicillin-resistant Staphylococcus epidermidis.

Staphylococcus epidermidis is frequently associated with infection of prosthetic heart valves, prosthetic orthopedic devices, and neurosurgical shunts. Penicillinase-resistant semisynthetic penicillins, such as methicillin, have been the therapeutic and prophylactic agents of choice for S epidermidis infection. However, more S epidermidis isolates are now resistant to methicillin and other penicillins. In our laboratory 41% of S epidermidis isolates were resistant to methici-lin. All of the methicillin-susceptible isolates and 82% of the methicillin-resistant isoates were susceptible to cephalothin. Cephalothin should replace methicillin as the prophylactic and therapeutic agent of choice in institutions with a high percentage of methicillin-resistant S epidermidis.     

Altered production of penicillin-binding protein 2a can affect phenotypic expression of methicillin resistance in Staphylococcus aureus.

Altered production of penicillin-binding protein 2a (PBP 2a) may affect the phenotypic expression of resistance in methicillin-resistant Staphylococcus aureus (MRSA). COL, an MRSA strain that constitutively produces PBP 2a, was transformed with a recombinant plasmid containing the two beta-lactamase regulatory genes, blaI and blaR1, with either the beta-lactamase gene, blaZ, or a truncated blaZ. Both of the transformed MRSA strains now produced an inducible PBP 2a, and the MICs of nafcillin, methicillin, and imipenem for these strains were similar to those for the parental strain. A mutation in blaR1 that resulted in the complete repression of PBP 2a production altered the phenotypic expression of methicillin resistance in that strain, as evidenced by efficiency-of-plating experiments. Rather than being homogeneously resistant like COL, the blaR1 mutant strain now appeared to have a small resistant subpopulation. Gene products that regulate PBP 2a production may contribute to the organism's expression of methicillin resistance, but additional chromosomally located factors are required.