鲍曼不动杆菌的基因分型及耐药性分析

目的分析上海某综合性医院不同科室来源的鲍曼不动杆菌菌株的同源性及耐药状况,了解鲍曼不动杆菌院内感染流行情况。方法采用重复序列PCR技术（REP-PCR）,对51株临床分离的鲍曼不动杆菌菌株进行基因分型,并用纸片扩散法进行药敏试验。结果51株鲍曼不动杆菌分为13个基因型,其中A型16株,为主要流行型别;C型、D型各6株;M型有5株;E型、F型和G型各3株;B型、H型和K型各2株;I型、J型、L型各1株。药敏试验结果显示分离出的菌株对常用抗菌药呈现出多重耐药的现象。其中对阿米卡星和亚胺培南的耐药性最低,均为33.3%;对头孢唑啉耐药性最高,为100%。结论鲍曼不动杆菌基因的同源性分析表明,该院存在着以A型鲍曼不动杆菌为主的感染流行,估计该型菌株可能以克隆株的形式播散。鲍曼不动杆菌的耐药性很强,应加强其耐药性监测,合理使用抗菌药物。     

Outbreaks of imipenem-resistant Acinetobacter baumannii producing carbapenemases in Korea

Among 53 Acinetobacter baumannii isolates collected in 2004, nine imipenem-resistant isolates were obtained from clinical specimens taken from patients hospitalized in Busan, Korea. Nine carbapenemase-producing isolates were further investigated in order to determine the mechanisms underlying resistance. These isolates were then analyzed via antibiotic susceptibility testing, microbiological tests of carbapenemase activity, pI determination, transconjugation test, enterobacterial repetitive consensus (ERIC)-PCR, and DNA sequencing. One outbreak involved seven cases of infection by A. baumannii producing OXA-23 beta-lactamase, and was found to have been caused by a single ERIC-PCR clone. During the study period, the other outbreak involved two cases of infection by A. baumannii producing IMP-1 beta-lactamase. The two clones, one from each of the outbreaks, were characterized via a modified cloverleaf synergy test and an EDTA-disk synergy test. The isoelectric focusing of the crude bacterial extracts detected nitrocefin-positive bands with pI values of 6.65 (OXA-23) and 9.0 (IMP-1). The PCR amplification and characterization of the amplicons via direct sequencing showed that the clonal isolates harbored blaIMP-1 or blaOXA-23 determinants. The two clones were characterized by a multidrug resistance phenotype that remained unaltered throughout the outbreak. This resistance encompassed penicillins, extended-spectrum cephalosporins, carbapenems, monobactams, and aminoglycosides. These results appear to show that the imipenem resistance observed among nine Korean A. baumannii isolates could be attributed to the spread of an IMP-1- or OXA-23-producing clone. Our microbiological test of carbapenemase activity is a simple method for the screening of clinical isolates producing class D carbapenemase and/or class B metallo-beta-lactamase, in order both to determine their clinical impact and to prevent further spread.     

Susceptibility, phenotypes of resistance, and extended-spectrum β-lactamases in Acinetobacter baumannii strains

Acinetobacter baumannii plays an increasing role in the pathogenesis of infections in humans. The bacilli are frequently isolated from patients treated in intensive care units. A growing resistance to antibiotics is leading to the emergence of strains that are multidrug-resistant and resistant to all available agents. The objective of this study was to assess susceptibility to antibiotics and to determine the presence and current level of the extended-spectrum β-lactamases (ESBLs) and attempt to isolate the Acinetobacter baumannii strain carrying the blaPER gene. A total of 51 strains of A. baumannii identified by phenotypic features were examined. That the strains belonged to the species was confirmed by the presence of the blaOXA-51-like; gene. A broth microdilution method was used for antibacterial susceptibility testing. The occurrence of ESBLs was determined using phenotypic double-disk synergy tests. The PCR technique was used to confirm the presence of the blaPER-1; gene encoding ESBL. The most active antibiotics were meropenem, cefepime and ampicillin/sulbactam, with susceptibility shown by 76.5%, 60.8% and 56.9% of the strains, respectively. The strains exhibited the highest resistance (> 75%) to piperacillin, tetracycline, ciprofloxacin and cefotaxime. Phenotypic tests revealed ESBL mechanism of resistance in approximately 20% of Acinetobacter baumannii isolates. However, the PCR technique did not confirm the presence of the blaPER-1; gene in any of the Acinetobacter baumannii strains examined in our hospital. Acinetobacter baumannii strains demonstrate considerable resistance to many groups of antibiotics. Our findings indicate the involvement of enzymes belonging to families other than PER β-lactamase in resistance to β-lactams in A. baumannii.     

Synergistic effect of Myrtus communis L. essential oils and conventional antibiotics against multi-drug resistant Acinetobacter baumannii wound isolates

Acinetobacter baumannii is a rapidly emerging, highly resistant clinical pathogen with increasing prevalence. In recent years, the limited number of antimicrobial agents available for treatment of infections with multi-drug resistant (MDR) strains reinforced tendency for discovery of novel antimicrobial agents or treatment strategies. The aim of the study was to determine antimicrobial effectiveness of three Myrtus communis L. essential oils, both alone and in combination with conventional antibiotics, against MDR A. baumannii wound isolates. The results obtained highlighted the occurrence of good antibacterial effect of myrtle oils when administered alone. Using checkerboard method, the combinations of subinhibitory concentrations of myrtle essential oils and conventional antibiotics, i.e. polymixin B and ciprofloxacine were examined. The results proved synergism among M. communis L. essential oils and both antibiotics against MDR A. baumannii wound isolates, with a FIC index under or equal 0.50. Combination of subinhibitory concentrations of essential oils and ciprofloxacin most frequently reduced bacterial growth in synergistic manner. The similar has been shown for combination with polymyxin B; furthermore, the myrtle essential oil resulted in re-sensitization of the MDR wound isolates, i.e. MICs used in combination were below the cut off for the sensitivity to the antibiotic. Time-kill curve method confirmed efficacy of myrtle essential oil and polymyxin B combination, with complete reduction of bacterial count after 6 h. The detected synergy offers an opportunity for future development of treatment strategies for potentially lethal wound infections caused by MDR A. baumannii.     

Genetic basis of antibiotic resistance in pathogenic Acinetobacter species

Antibiotic resistance in Acinetobacter spp., particularly Acinetobacter baumannii, is increasing rapidly. A. baumannii possesses two intrinsic β-lactamase genes, in addition to weak permeability and efflux systems, that together confer a natural reduced susceptibility to antibiotics. In addition, numerous acquired mechanisms of resistance have been identified in A. baumannii. The very high genetic plasticity of A. baumannii allows an accumulation of resistance determinants that give rise to multidrug resistance at an alarming rate. The role of novel genetic elements, such as resistance islands, in concentrating antibiotic resistance genes in A. baumannii requires detailed investigation in the near future.     

耐亚胺培南鲍曼不动杆菌医院内感染流行的分子机制研究

目的研究耐亚胺培南鲍曼不动杆菌的耐药谱特征及其医院内感染流行和耐药性产生的分子机制,为临床防治提供依据.方法4株耐亚胺培南鲍曼不动杆菌分离自2002年10月至2003年1月外科重症监护病房的感染患者,采用纸片扩散法及E-test进行药物敏感性检测及MIC值测定,肠杆菌科基因组内重复一致序列聚集合酶链反应(ERIC-PCR)进行克隆株的DNA分型,耐药质粒转移及消除试验、等电聚焦电泳、PCR扩增β-内酰胺酶基因及其克隆测序以识别其耐药基因和进行质粒定位.结果4株菌除对头孢哌酮/舒巴坦复合制剂的MIC值较低外,对头孢菌素类、氨基糖甙类和氟喹喏酮类等抗生素均显示出了较高水平的多重耐药性;DNA分型证实为同一克隆株;产OXA-23型碳青霉烯酶和PER-1型超广谱β-内酰胺酶(ESBLs);OXA-23定位在质粒上,PER-1定位在染色体上.结论本组耐亚胺培南鲍曼不动杆菌为多重耐药株,同一克隆株在不同感染个体间的相互传播导致了本次医院内感染的流行,产OXA-23和PER-1型β-内酰胺酶是其耐药性产生的重要原因.     

重症监护病房呼吸机相关性肺炎中鲍曼不动杆菌耐药性的动态分析

目的探讨重症监护病房呼吸机相关性肺炎(VAP)中鲍曼不动杆菌(Acinetobacter baumannii,Ab)的分布特点和耐药性,为临床防治鲍曼不动杆菌感染提供依据。方法对机械通气>48 h的79例患者的气管抽吸物(ETA)进行细菌定量培养和药敏测定。结果回顾性分析79例VAP患者ETA培养出病原菌214株,其中鲍曼不动杆菌79株(36.9%)。鲍曼不动杆菌对常见抗菌药物的耐药率呈上升趋势,出现较多多重耐药和泛耐药菌株。头孢哌酮/舒巴坦和亚胺培南的耐药率分别是55.6%和68.4%,均呈上升趋势。结论鲍曼不动杆菌是VAP常见病原菌之一,鲍曼不动杆菌的耐药性呈上升趋势,特别是多重耐药和泛耐药菌株,应引起高度重视。     

Molecular characterization of intrinsic and acquired antibiotic resistance in lactic acid bacteria and bifidobacteria

The minimum inhibitory concentrations (MICs) of 6 different antibiotics (chloramphenicol, clindamycin, erythromycin, streptomycin, tetracycline and vancomycin) were determined for 143 strains of lactic acid bacteria and bifidobacteria using the Etest. Different MICs were found for different species and strains. Based on the distribution of these MIC values, most of the strains were either susceptible or intrinsically resistant to these antibiotics. However, the MIC range of some of these antibiotics showed a bimodal distribution, which suggested that some of the tested strains possess acquired antibiotic resistance. Screening for resistance genes was performed by PCR using specific primers, or using a DNA microarray with around 300 nucleotide probes representing 7 classes of antibiotic resistance genes. The genes identified encoded resistance to tetracycline [tet(M), tet(W), tet(O) and tet(O/W)], erythromycin and clindamycin [erm(B)] and streptomycin [aph(E) and sat(3)]. Internal portions of some of these determinants were sequenced and found to be identical to genes described in other bacteria. All resistance determinants were located on the bacterial chromosome, except for tet(M), which was identified on plasmids in Lactococcus lactis. The contribution of intrinsic multidrug transporters to the antibiotic resistance was investigated by cloning and measuring the expression of Bifidobacterium breve genes in L. lactis.     

Annual trends in antibiotic resistance of nosocomial Acinetobacter baumannii strains and the effect of synergistic antibiotic combinations

Acinetobacter baumannii is becoming increasingly resistant to antibiotics often requiring combination therapy. Annual changes of resistance to selected antimicrobials of 150 A. baumannii strains, isolated as nosocomial pathogens between 1994 and 2000 were investigated. The synergistic effects of antimicrobials were studied using a microdilution checkerboard technique in eight selected isolates resistant to third-generation cephalosporins and beta-lactam/beta-lactamase inhibitor combinations and to at least one aminoglycoside. Rates of resistance of cefepime, ceftazidime, ampicillin/sulbactam, amikacin and ciprofloxacin (before 1996 and between 1996-2000) were 29.7% - 72.6, 37.8% - 81.4%, 35.1% - 72.6%, 8.1% - 56.6%, 5.4% - 46.0% respectively (p < 0.001 for each one). Synergy was observed in at least one of the combinations of antibiotics from seven of eight isolates (87%), no antagonism was detected with any combination. Ceftazidime-amikacin (50%) and ampicillin/sulbactam-tobramycin (50%) were the most effective combinations. Due to the effectiveness of sulbactams to Acinetobacter, ampicillin/sulbactam-tobramycin combination is recommended as the first line of choice.     

Genetical conditioning of fluoroquinolone resistance mechanisms of clinical Enterococcus faecalis strains. I. Characterization of tested strains

The first part of the study presents the resistance profiles of 14 selected antibiotic agents for 180 clinical E. faecalis strains. Distribution of virulence factors for tested strains were characterized using PCR method. The results proved that clinical fluoroquinolone resistant E. faecalis strains possess MDR (multidrug-resistant) phenotype and presence of 7-8 tested virulence determinants.     

Erythromycin-inducible resistance in Staphylococcus aureus: survey of antibiotic classes involved

Certain erythromycin-resistant strains of Staphylococcus aureus remain sensitive to other macrolide antibiotics. If these strains are exposed to low levels of erythromycin, resistance to other antibiotics is induced. The antibiotics to which resistance is induced by erythromycin include: other macrolides as well as lincosaminide, streptogramin (group B) antibiotics but not chloramphenicol, amicetin, streptogramin (group A) antibiotics, tetracyclines, and aminoglycosides. Hence erythromycin induces resistance exclusively towards inhibitors of 50S ribosomal subunit function and, thus far, only with respect to three of six known classes of inhibitors which act on this subunit. In the four strains tested, erythromycin did not induce resistance to pactamycin or bottromycin, to fusidic acid (which inhibits a function involving both subunits), or to other antibiotics which do not inhibit ribosomal function. Thus, by inducing resistance erythromycin could antagonize the action of other antibiotics, and a consistent pattern of antagonism was observed to each antibiotic class in all of the strains in which this could be tested, as well as to other antibiotic members of the same chemical class in each bacterial strain.     

鲍曼不动杆菌耐药程度与其主动外排泵蛋白的相关性研究

目的:探讨鲍曼不动杆菌耐药程度与其主动外排泵蛋白的相关性。方法:首先用纸片扩散法检测64株临床鲍曼不动杆菌对8种抗菌药物的敏感性;将其分为A组(0～2种抗生素耐药)、B组(对3～5种抗生素耐药)和C组(对6～8种抗生素耐药);检测64株临床鲍曼不动杆菌对罗丹明6G的外排情况,筛选出罗丹明6G外排明显增加的菌株;并用逆转录-聚合酶链反应(RT-PCR)方法检测主动外排泵基因AdeABC的表达水平。结果:64株鲍曼不动杆菌中有4株对0～2种抗生素耐药(A组),对3～5种抗生素耐药的有33株(B组),对6～8种抗生素耐药的有27株(C组);多重耐药组鲍曼不动杆菌罗丹6G外排明显增高,外排程度A组相似文献   

In vitro activity of cefamandole, cefoxitin, cefuroxime, and carbenicillin, alone and in combination with aminoglycosides against Serratia marcescens.

Synergistic antibiotic studies were undertaken to compare the effectiveness of two new beta-lactamase resistant cephalosporins, cefamandole, and carbenicillin, with four aminoglycosides against clinical strains of Serratia marcescens. The strains demonstrated various combinations of resistance and/or susceptibility to the antibiotics tested. Tobramycin was the most effective aminoglycoside when used in combination with beta-lactam antibiotics. Carbenicillin and cefamandole demonstrated similar activity with aminoglycosides in synergy experiments. Tobramycin-carbenicillin was found to be the superior pairs as indicated by the total number of strains inhibited. This combination was the only one effective against certain high drug resistant strains and the strain resistant to all four aminoglycosides. Carbenicillin or cefamandole with tobramycin exhibited comparable activity against multiple drug resistant organisms. However, mutants significantly more resistant to cefamandole developed during susceptibility testing. The findings of this study have clinical relevance for treating infections by this formidable pathogen.     

Synergy of Vancomycin with Penicillins and Cephalosporins Against Pseudomonas,Klebsiella, and Serratia

A model of antibiotic synergy based on a molecular mechanism of action which blocked sequential steps in a single metabolic pathway was tested. Twenty-five strains each of Pseudomonas, Klebsiella, and Serratia were tested in vitro against three different two drug combinations of vancomycin, carbenicillin, or cephalothin. Synergy was observed when vancomycin was combined with either carbenicillin or cephalothin against isolates of Pseudomonas or Serratia, whereas the combination of carbenicillin and cephalothin did not result in significant synergy against these isolates. The presence of synergy was not related to the sensitivity or resistance of the isolates to the drugs in the combination. Synergy was also observed with all three antibiotic combinations against Klebsiella isolates which may be related to enzyme inactivation by one of the drugs in the combination. These observations support the hypothetical model of antibiotic synergy based on sequential blocking of one biochemical pathway.     

Interaction of aminoglycosides and the other antibiotic on selected bacterial strains

The aim of this study was to analyse the activity interaction of aminoglycosides (gentamicin, kanamycin, streptomycin and dihydrostreptomycin) when combined with other antibiotics (lincomycin, benzylpenicillin, amoxicillin, cephalexin, spectinomycin and erythromycin), on selected clinical bacterial strains. The checkerboard method has been selected from the traditional assays for the measurement of antibiotic interaction. Checkerboard results for all strains demonstrated synergism for nine cases (9/112--8%). Additive effects were predominant--73/112--65.2%. In 12.5% neutral effects were shown, but in 11.6% of combinations FIC indexes were not possible to calculate, because of the resistance of clinical strains to the highest concentration of at least one antibiotic. The best results were achieved for combinations of dihydrostreptomycin with procaine penicillin because of higher number of cases synergy effect was observed. Antagonism of aminoglycosides and beta-lactams in case of gentamicin and amoxicillin for E. coli and E. cloacea strains were shown. Potential activity for combination of streptomycin and erythromycin was shown.     

Quantitative proteomic analysis of cell wall and plasma membrane fractions from multidrug-resistant Acinetobacter baumannii

Acinetobacter baumannii is a Gram-negative, nonmotile aerobic bacterium that has emerged as an important nosocomial pathogen. Multidrug-resistant (MDR) A. baumannii is difficult to treat with antibiotics, and treatment failure in infected patients is of great concern in clinical settings. To investigate proteome regulation in A. baumannii under antibiotic stress conditions, quantitative membrane proteomic analyses of a clinical MDR A. baumannii strain cultured in subminimal inhibitory concentrations of tetracycline and imipenem were performed using a combination of label-free (one-dimensional electrophoresis-liquid chromatography-tandem mass spectrometry) and label (isobaric tag for relative and absolute quantitation) approaches. In total, 484 proteins were identified, and 302 were classified as outer membrane, periplasmic, or plasma membrane proteins. The clinical A. baumannii strain DU202 responded specifically and induced different cell wall and membrane protein sets that provided resistance to the antibiotics. The induction of resistance-nodulation-cell division transporters and protein kinases, and the repression of outer membrane proteins were common responses in the presence of tetracycline and imipenem. Induction of a tetracycline resistant pump, ribosomal proteins, and iron-uptake transporters appeared to be dependent on tetracycline conditions, whereas β-lactamase and penicillin-binding proteins appeared to be dependent on imipenem conditions. These results suggest that combined liquid chromatography-based proteomic approaches can be used to identify cell wall and membrane proteins involved in the antibiotic resistance of A. baumannii.     

皖北地区耐碳青霉烯鲍曼不动杆菌产金属β-内酰胺酶检测

目的了解皖北地区ICU病房内耐碳青霉烯鲍曼不动杆菌产金属β-内酰胺酶（metallo-β-lactamase,MBL）情况。方法收集皖北地区3家教学医院ICU病房中不动杆菌,K-B纸片扩散法检测其对碳青霉烯类抗生素耐药情况;PCR法检测blaIMP、blaVIM等MBL基因。结果共收集35株耐亚胺培南、美罗培南非重复分离鲍曼不动杆菌菌株,其中9株（25.7%）金属酶初筛阳性;PCR检测5株（5/35,14.3%）产IMP型MBL,1株（1/35,2.9%）VIM型MBL。结论在本地区耐碳青霉烯鲍曼不动杆菌中首次发现IMP和VIM型金属酶,提示产金属酶机制参与本地区鲍曼不动杆菌对碳青霉烯耐药,值得进一步关注。     

Interaction of neomycin with other antibiotics on selected bacterial strains

Antimicrobial combinations are used most frequently to provide broad-spectrum empirical coverage in the treatment of bacterial infections. However, combination of two antibiotics may not influence their activity, may lead to synergy or antagonism in the activity. Neomycin may be combined with one of the following antibiotics: ampicillin, procaine penicillin, gramicidin, bacitracin, polymyxin B, lincomycin, oxytetracycline, and erythromycin in some human and veterinary multiantibiotic drugs distributed in Poland. The checkerboard method has been one of the traditional assays for the measurement of antibiotic interactions. The aim of this study was to analyse the activity interaction of neomycin with second antibiotic in multiantibiotic drugs distributed in Poland on standards and clinical bacterial strains. Checkerboard results for all strains demonstrated synergism for 2.5% of combinations, only for standards strains. In one case Salmonella Enteritidis, in combination of neomycin with bacitracin, inhibition effect was observed. Additive effects were predominant--49%. In 18% neutral effects were shown, but in 26% of combinations FIC indexes were not possible to calculate, because of the resistance of clinical strains to the highest concentration of at least one antibiotic. In combination of aminoglycoside (neomycin) with beta-lactams antibiotics (ampicillin, procaine penicillin) in vitro, no synergy was observed for all examined strains. The best results were achieved for combinations of neomycin with peptide antibiotics (polymyxin, gramicidin and bacitracin)--5 for all 6 synergy effect observed.     

Transferability of antibiotic resistance determinants in strains of Staphylococcus aureus belonging to different phage groups

A total of 107 donor strains of Staphylococcus aureus isolated from clinical material, with a high incidence of multiresistant strains belonging predominantly to phage group III, were tested for transmission of determinants of resistance to 6 antibiotics using mixed cultures of donor strains and the recipient Staphylococcus aureus strain 5849-fur-r, rif-r. The capability of strains to transfer resistance markers to the recipient was found to depend neither on phage group nor phage type to which the donor strain belonged, but strains possessing multiple resistance to antibiotics effectuated transfers at comparatively higher frequencies.