Antimicrobial resistance in nosocomial isolates of Staphylococcus haemolyticus.

Staphylococcus haemolyticus is frequently cultured from hospitalized patients and is characterized by resistance to multiple antimicrobial agents. We found that S. haemolyticus represented 70 of 524 (13%) coagulase-negative staphylococcal isolates identified by the clinical microbiology laboratories of two hospitals over 2 months. S. haemolyticus isolates were recovered from wounds (44%), urine (26%), blood (10%), and other sources (20%). All S. haemolyticus isolates were tested for susceptibility to six antimicrobial agents; 77% were resistant to three or more agents, and 41% were resistant to five or six agents. In addition, among 47 multiply resistant isolates, high MICs (greater than or equal to 6.25 micrograms/ml) of vancomycin (62% of isolates) and teicoplanin (91% of isolates) were found. DNA probes which were derived from S. epidermidis or S. aureus and which contained sequences associated with resistance to antimicrobial agents were used to detect specific genes in the total cellular and plasmid DNAs of 10 resistant S. haemolyticus isolates. Resistance gene probes and the numbers of resistant isolates hybridizing were as follows: methicillin, 10 of 10; gentamicin, 9 of 10; erythromycin, 7 of 10; and trimethoprim, 0 of 10. Genes for resistance to methicillin were found only in chromosomal locations, genes for resistance to gentamicin were found in both chromosomal and plasmid locations, and genes for resistance to erythromycin were found in plasmid locations only. With the exception of trimethoprim resistance determinants, similar genes were found among concurrently isolated multiply resistant S. epidermidis isolates from our hospitals. S. haemolyticus is a potentially important nosocomial species which readily acquires antimicrobial resistance genes and which shares, to some extent, in a common gene pool with S. epidermidis.     

Conjugative transfer of high-level mupirocin resistance from Staphylococcus haemolyticus to other staphylococci.

A conjugative plasmid, pXU10, encoding high-level mupirocin resistance was transferred from a Staphylococcus haemolyticus isolate, CN216, to other coagulase-negative staphylococci and a restriction deficient Staphylococcus aureus strain, XU21, but not to clinical isolates or a restriction-proficient laboratory strain (strain WBG541) of S. aureus. However, from XU21 it was cotransferred with a 3.5-kb chloramphenicol resistance plasmid to WBG541. The results demonstrated the ability of pXU10 to mobilize nonconjugative plasmids.     

溶血葡萄球菌的临床分布特点及耐药性分析

目的：了解溶血葡萄球菌的耐药性,为临床合理用药作出指导。方法从患者标本中分离出59株溶血葡萄球菌,并对其进行临床分布及耐药性分析。结果59株溶血葡萄球菌主要集中于儿科与 ICU 等科室,标本主要来源于痰液和咽拭子,患者以新生儿为主;溶血葡萄球菌以耐甲氧西林溶血葡萄球菌（MRSH）为主,共53株,占89．8％;耐甲氧西林溶血葡萄球菌（MR‐SH）对多种抗菌药物呈高度多重耐药,而对万古霉素、替考拉宁、利奈唑胺敏感率高,为100．0％。结论溶血葡萄球菌是临床感染的重要的凝固酶阴性葡萄球菌之一,其以 MRSH 为主,且呈高度多重耐药,临床上应根据其药敏试验结果合理选用抗菌药物。     

溶血葡萄球菌庆大霉素耐药表型与基因型分析

目的研究溶血葡萄球菌庆大霉素耐药表型与基因型。方法用琼脂稀释法测定63株溶血葡萄球菌对苯唑西林、庆大霉素和其他5种抗生素的最低抑菌浓度（MIC）,用聚合酶链反应（PCR）检测mecA和aac（6’）．aph（2”）耐药基因。结果63株溶血葡萄球菌对庆大霉素的耐药率为74．6％,MIC50、MIC90分别为64和128μg／mL.庆大霉素不敏感的甲氧西林耐药溶血葡萄球菌（MRSH）对环丙沙星和红霉素的耐药率（85．7％和87．8％）远高于庆大霉素敏感的MRSH（0．0％和46．2％,P〈0．005）。13株庆大霉素敏感的溶血葡萄球菌扩增aac（6’）＋aph（2”）基因均阴性,50株庆大霉素不敏感的溶血葡萄球菌（MIC为8～≥128μg／mL）均携带aac（6’）-aph（2”）基因,其中49株同时携带mecA基因。结论aac（6’）-aph（2”）基因是溶血葡萄球菌对庆大霉素产生耐药的主要机制。     

Plasmid-mediated resistance to antibiotic synergism in enterococci.

Mating experiments have shown that high-level resistance (minimal inhibitory concentration greater than 2,000 microgram/ml) to streptomycin and kanamycin, and resistance to penicillin-streptomycin and penicillin-kanamycin synergism are transferable by conjugation from resistant clinical isolates of enterococci to a sensitive recipient strain. Cesium chloride-ethidium bromide ultracentrifugation revealed a satellite (plasmid) band in resistant clinical isolates and the transconjugant strains but not in the sensitive recipient. Examination of these satellite bands by agarose gel electrophoresis and electron microscopy demonstrated a common plasmid with a weight of 45 megadaltons. Novobiocin treatment of a resistant clinical isolate produced simultaneous loss of high-level resistance to streptomycin and kanamycin, and of resistance to penicillin-aminoglycoside synergism. These results suggest that (a) high-level resistance to streptomycin and kanamycin among some clinical isolates of enterococci is associated with a 45 megadalton plasmid, and (b) the same plasmid is also responsible for the resistance to penicillin-aminoglycoside synergism observed in these strains.     

Plasmid-mediated high-level resistance to erythromycin in Escherichia coli.

Escherichia coli BM2195 was found to be resistant to high levels of erythromycin. This new resistance phenotype was due to the constitutive synthesis of an erythromycin esterase which inactivates the antibiotic. The gene conferring resistance to erythromycin in this strain is carried on a 61-kilobase self-transferable plasmid, pIP1100, belonging to incompatibility group X.     

Homology of mecA gene in methicillin-resistant Staphylococcus haemolyticus and Staphylococcus simulans to that of Staphylococcus aureus.

A penicillin-binding protein of molecular weight 76,000 inducible by beta-lactams was detected in methicillin-resistant Staphylococcus haemolyticus and Staphylococcus simulans. DNA from these strains hybridized to the mecA gene from Staphylococcus aureus; however, the chromosomal HindIII fragments containing the mecA genes were 3.4 kilobases in S. haemolyticus and 4.3 kilobases in S. simulans.     

Effect of lincomycin on lipase formation by Staphylococcus aureus.

The production of Staphylococcus aureus lipase could be inhibited by addition of 0.1 mug/ml lincomycin to the media without affecting growth. Addition of the same amount of drug at various stages of growth inhibited further enzyme production. The enzymatic activity of the lipase could not be inhibited at a concentration of 2.5 mug/ml lincomycin.     

Plasmid-mediated sulfonamide resistance in Haemophilus ducreyi.

Clinical isolates of Haemophilus ducreyi from patients with chancroid were shown to have one or more 4.9- to 7.0-megadalton non-self-transferable plasmids and to have in vitro resistance to sulfonamides. Transformation of Escherichia coli to sulfonamide resistance was associated with the acquisition of a 4.9-megadalton plasmid, which did not confer linked resistance to streptomycin. The guanine-plus-cytosine content of this plasmid was found to be 57%. Filter-blot hybridization and restriction endonuclease digestion studies suggested a relationship of this plasmid to RSF1010. Electron microscope heteroduplex analysis confirmed this relationship. The identification in H. ducreyi of a plasmid closely related to plasmids found in enteric species, rather than transposition of a resistance determinant to an indigenous plasmid, suggests that further dissemination of the enteric plasmid pool to this genus is possible since plasmid transfer between certain Haemophilus species is readily demonstrated.     

Plasmid-mediated sulfonamide resistance in Neisseria meningitidis.

An 8.5-megadalton plasmid coding for sulfonamide resistance was found in a clinical isolate of Neisseria meningitidis, as demonstrated by plasmid elimination and transformation experiments. The plasmid complemented a mutation which determines the production of a thermosensitive dihydropteroate synthetase in Escherichia coli, thus suggesting that the mechanism of resistance involved a plasmid-encoded dihydropteroate synthetase.     

Plasmid-mediated tetracycline resistance in Haemophilus ducreyi.

Clinical isolates of Haemophilus ducreyi were shown to be resistant to tetracycline. Resistance was associated in some strains with a 30-megadalton plasmid capable of transferring resistance in conjugative matings with other strains of H. ducreyi and other species of Haemophilus. Restriction endonuclease digestion patterns suggest a relationship between H. ducreyi plasmids and other tetracycline resistance plasmids in Haemophilus. The presence of plasmid-mediated resistance to the tetracyclines limits the use of these agents for the treatment of chancroid.     

tcaA inactivation increases glycopeptide resistance in Staphylococcus aureus

The experimental deletion of the tcaRAB region has been shown to increase teicoplanin resistance in Staphylococcus aureus. By sequential genetic complementation of a tcaRAB mutant, we identified tcaA as the key gene within tcaRAB that is responsible for changes in glycopeptide resistance levels. Northern blot analysis of the tcaRAB region showed that the tcaA gene is expressed only weakly over the growth cycle and is strongly inducible by teicoplanin. Among some clinical isolates tested, glycopeptide-intermediate-resistant (GISA) strains Michigan and SA137/93G were found to have truncated tcaA genes. While the former carries a nucleotide insertion that creates a premature stop codon, the latter was found to harbor an IS256 insertion. Complementation of these two GISA strains with a functional tcaA allele reduced their levels of teicoplanin and vancomycin resistance five- to eightfold and twofold, respectively. The data presented here indicate that inactivation of tcaA contributes to and plays a relevant role in glycopeptide resistance in S. aureus clinical isolates.     

溶血葡萄球菌对抗菌药物的敏感性和mecA基因的检测

目的　了解溶血葡萄球菌的耐药情况 ,以指导临床合理用药。方法　对 133株临床分离的溶血葡萄球菌采用微量肉汤稀释法测定 16种抗菌药物的最低抑菌浓度 (MIC) ,多聚酶链反应 (PCR)法检测mecA基因 ,并与普通药敏试验比较 ,对不相符的菌株进行抑制、诱导试验。结果　 133株溶血葡萄球菌对利奈唑胺、万古霉素、去甲万古霉素无耐药 ,对替考拉宁的耐药率为 6 .0 % ;对阿米卡星、异帕米星、米诺环素、利福平、氟氧头孢的耐药性较低 ,耐药率 <2 0 % ;对青霉素、苯唑西林的耐药率 >90 %。除米诺环素、青霉素外 ,住院患者分离菌株的耐药率高于门诊患者分离的菌株。mecA基因阳性率为 90 .2 3% ,且PCR产物经克隆测序证实为mecA特异性产物。对 2株mecA基因阳性而苯唑西林MIC≤ 0 .2 5 μg/ml的菌株进行诱导试验后其MIC≥ 0 .5 μg/ml;对 3株mecA基因阴性而MIC≥ 0 .5 μg/ml的菌株进行抑制试验后其MIC值下降 8倍以上。 结论　溶血葡萄球菌对大多数抗菌药物均有较高的耐药性 ,对糖肽类抗生素的敏感性也在下降 ,需引起临床重视。     

溶血葡萄球菌对抗菌药物的敏感性和mecA基因的检测

目的了解溶血葡萄球菌的耐药情况,以指导临床合理用药.方法对133株临床分离的溶血葡萄球菌采用微量肉汤稀释法测定16种抗菌药物的最低抑菌浓度(MIC),多聚酶链反应(PCR)法检测mecA基因,并与普通药敏试验比较,对不相符的菌株进行抑制、诱导试验.结果 133株溶血葡萄球菌对利奈唑胺、万古霉素、去甲万古霉素无耐药,对替考拉宁的耐药率为6.0%;对阿米卡星、异帕米星、米诺环素、利福平、氟氧头孢的耐药性较低,耐药率<20%;对青霉素、苯唑西林的耐药率>90%.除米诺环素、青霉素外,住院患者分离菌株的耐药率高于门诊患者分离的菌株.mecA基因阳性率为90.23%,且PCR产物经克隆测序证实为mecA特异性产物.对2株mecA基因阳性而苯唑西林MIC≤0.25 μg/ml的菌株进行诱导试验后其MIC≥0.5 μg/ml;对3株mecA基因阴性而MIC≥0.5 μg/ml的菌株进行抑制试验后其MIC值下降8倍以上.结论溶血葡萄球菌对大多数抗菌药物均有较高的耐药性,对糖肽类抗生素的敏感性也在下降,需引起临床重视.     

Teicoplanin resistance in Staphylococcus haemolyticus is associated with mutations in histidine kinases VraS and WalK

We investigated the genetic basis of glycopeptide resistance in laboratory-derived strains of S. haemolyticus with emphasis on differences between vancomycin and teicoplanin. The genomes of two stable teicoplanin-resistant laboratory mutants selected on vancomycin or teicoplanin were sequenced and compared to parental S. haemolyticus strain W2/124. Only the two non-synonymous mutations, VraS Q289K and WalK V550 L were identified. No other mutations or genome rearrangements were detected. Increased cell wall thickness, resistance to lysostaphin-induced lysis and adaptation of cell growth rates specifically to teicoplanin were phenotypes observed in a sequenced strain with the VraS Q289K mutation. Neither of the VraS Q289K and WalK V550 L mutations was present in the genomes of 121 S. haemolyticus clinical isolates. However, all but two of the teicoplanin resistant strains carried non-synonymous SNPs in vraSRTU and walKR-YycHIJ operons pointing to their importance for the glycopeptide resistance.     

溶血葡萄球菌vga（A）LC基因与克林霉素耐药分析

目的调查溶血葡萄球菌vga（A）LC基因与克林霉素耐药特点。方法用琼脂稀释法测定红霉素和克林霉素对63株溶血葡萄球菌的MIC，PCR技术检测vga（A）LC及其他克林霉素耐药相关基因。结果23株受试菌对克林霉素耐药，其中2株对红霉素敏感（MIC均为0．25mg／L），对克林霉素耐药（MIC均为8mg／L），vga（A）LC基因检测均阳性；6株结构型大环内酯类-林可酰胺类-链阳菌素B类（MLSB）耐药，15株诱导型MLSB耐药，均未检出vga（A）LC基因；1株结构型MLSB耐药菌携带ermB基因，5株结构型MLSB和15株诱导型MLSB耐药菌均携带ermC基因；10株克林霉素耐药菌携带linA／linA’基因。结论我院发现携带vga（A）LC基因的溶血葡萄球菌。     

大肠埃希菌对氟喹诺酮类的质粒介导耐药机制研究

目的了解近年来发现的质粒介导耐药机制在临床分离大肠埃希菌中对氟喹诺酮类耐药所起的作用。方法用MIC琼脂稀释法筛选耐左氧氟沙星大肠埃希菌;采用聚合酶链反应对qnrA、qnrS、qnrB、aac(6′)-Ib基因进行扩增,并进行PCR扩增产物直接双向测序。用接合实验了解是否存在水平传播的氟喹诺酮耐药性传播机制。结果 90株耐氟喹诺酮类大肠埃希菌中7株检出aac(6′)-Ib-cr基因,未检出qnrA、qnrS、qnrB基因。78株符合供体菌标准的大肠埃希菌中有2株接合成功,接合率2.6%。结论该地区存在质粒介导的氟喹诺酮类耐药性的水平传播。     

Genetic study of plasmid-associated zonal resistance to lincomycin in Streptococcus pyogenes.

The phenomenon of zonal resistance to lincomycin, which is characteristic of most clinical isolates with lincomycin resistance in Streptococcus pyogenes, has been studied. These strains grow within a defined concentration range of lincomycin (approximately 60 to 200 microgram/ml), or at lincomycin concentrations below the minimal inhibitory concentration for susceptible strains. It is shown that the zonal growth phenomenon is a stable phenotype and results from induction of resistance only within the zonal concentration range of lincomycin. These strains also possess inducible resistance to erythromycin which is nonzonal in character. One-step mutations to constitutive resistance have been isolated which are of two types: constitutive for lincomycin or for erythromycin, but not for both. Those strains with constitutive erythromycin resistance retain their zonal resistance for lincomycin. Mutants doubly constitutive for both lincomycin and erythromycin can be obtained by a second mutational step from either of the singly constitutive mutants. Satellite deoxyribonucleic acid has been shown to be present in the zonal resistant strains. A plasmid, pSM10419, of 14.9 megadaltons, has been isolated from one of the doubly constitutive mutants and used to jointly transform Streptococcus sanguis strain Challis to constitutive resistance to both lincomycin and erythromycin. From this, a multicopy plasmid of reduced size, pSM10 (5.4 megadaltons), which retains its resistance phenotype, has been isolated and mapped with restriction endonucleases HindIII (three sites), EcoRI (one site), KpnI (one site), and HpaI (one site). The staphylococcal plasmid pC221 (2.9 megadaltons; chloramphenicol resistant) has been fused to pSM10 at the EcoRI site resulting in a chimeric plasmid, pSM10221 (8.3 megadaltons), which retains resistance to chloramphenicol, erythromycin, and lincomycin. pSM10 is therefore suggestive as an effective cloning vehicle for the genus Streptococcus.     

Plasmid-mediated florfenicol resistance in Pasteurella trehalosi

OBJECTIVES: A florfenicol-resistant Pasteurella trehalosi isolate from a calf was investigated for the presence and the location of the gene floR. METHODS: The P. trehalosi isolate 13698 was investigated for its in vitro susceptibility to antimicrobial agents and its plasmid content. A 14.9 kb plasmid, designated pCCK13698, was identified by transformation into Pasteurella multocida to mediate resistance to florfenicol, chloramphenicol and sulphonamides. The plasmid was sequenced completely and analysed for its structure and organization. RESULTS: Plasmid pCCK13698 exhibited extended similarity to plasmid pHS-Rec from Haemophilus parasuis including the region carrying the parA, repB, rec and int genes. Moreover, it revealed similarities to plasmid RSF1010 in the parts covering the mobC and repA-repC genes and to plasmid pMVSCS1 in the parts covering the sul2-catA3-strA gene cluster. Moreover, the floR gene area corresponded to that of transposon TnfloR. In addition, two complete insertion sequences were detected that were highly similar to IS1593 from Mannheimia haemolytica and IS26 from Enterobacteriaceae. Several potential recombination sites were identified that might explain the development of plasmid pCCK13698 by recombination events. CONCLUSIONS: The results of this study showed that in the bovine pathogen P. trehalosi, floR-mediated resistance to chloramphenicol and florfenicol was associated with a plasmid, which also carried functionally active genes for resistance to sulphonamides (sul2) and chloramphenicol (catA3). This is to the best of our knowledge the first report of resistance genes in P. trehalosi and only the second report of the presence of a florfenicol-resistance gene in target bacteria of the family Pasteurellaceae.