Inhibition of efflux pumps in methicillin-resistant Staphylococcus aureus and Enterococcus faecalis resistant strains by triterpenoids from Momordica balsamina

Six cucurbitane-type triterpenoids (1-6) isolated from the aerial parts of Momordica balsamina were evaluated for their ability to inhibit the activity of bacterial efflux pumps of methicillin-resistant Staphylococcus aureus (MRSA) COL(OXA), Enterococcus faecalis ATCC 29212, Salmonella enterica subsp. I serovar Typhimurium 5408 and S. Typhimurium 5408CIP strains. The latter strain overproduces the AcrB transporter of the AcrAB-TolC efflux pump six-fold compared with its parent. Compounds 4-6 were also tested for similar activity against Escherichia coli AG100 wild-type strain and E. coli AG100TET8 that overproduces the AcrAB-TolC efflux pump. Evaluation of efflux activity was performed using a semi-automated method that measures accumulation of the universal efflux pump substrate ethidium bromide (EtBr). Some of the compounds significantly inhibited efflux of EtBr by MRSA COL(OXA) and E. faecalis ATCC 29212. A correlation between activity and the topological polar surface area of the compounds was found for MRSA COL(OXA).     

Synergistic effect of tetrandrine and ethidium bromide against methicillin-resistant Staphylococcus aureus (MRSA)

Methicillin-resistant Staphylococcus aureus (MRSA) along with other resistant bacteria have become a significant social and clinical problem. Therefore, there is an urgent need to develop bioactive compounds from natural products as alternatives to the very few antibiotics that remain effective. Recently, the efflux mechanism has been identified as the main contributor to antibiotic resistance in bacteria. This study therefore aimed to evaluate tetrandrine (TET), an efflux pump inhibitor (EPI), as a potential antibiotic against MRSA. We investigated the antimicrobial activity of TET against 17 MRSA strains, of which 3 selected strains were studied in further detail using a time-kill assay. When these bacterial strains (1 × 10(6) colony-forming units (cfu)/ml) were incubated with TET in a time-kill assay, log-scale bactericidal activity was observed, which lasted for 24 hr. In addition, TET exhibits a synergistic effect when combined with the multi-drug resistance (MDR)-efflux pump substrate ethidium bromide (EtBr). Structure-function studies of the antibiotic activity of TET in combination with EtBr may lead to the discovery of more effective efflux pump inhibitors.     

An instrument-free method for the demonstration of efflux pump activity of bacteria

The aim of the study was to develop a simple, inexpensive, reproducible ethidium bromide (EB)-agar based method that is independent of any specialized instrumentation, for the demonstration of efflux pump activity, which is responsible for antibiotic resistance of bacteria. MATERIALS AND METHODS: A series of agar plates containing varying concentrations of EB were swabbed with strains of Escherichia coli or Staphylococcus aureus, which differed with respect to efflux pump activity. The plates were incubated at different temperatures and time periods and the measurements of fluorescence were used to evaluate the efflux activity of each culture. RESULTS: This simple assay allowed us to identify the efflux of EB in different bacteria following an overnight incubation. The minimal concentration of EB that produced fluorescence was significantly greater at 37 degrees C than at 4 degrees C, suggesting the presence of an energy-dependent pump. The method was shown to simultaneously identify strains of a mixed culture that differed from each other with respect to the activity of their efflux pumps. CONCLUSION: The method, in conjunction with the use of antibiotic-containing disks, provides an additional advantage for the easy identification and selection of colonies that differ with respect to antibiotic susceptibility and degree of efflux pump activity. Because the method is very reproducible it may form the basis for interlaboratory standardization of efflux pump activity of multi-drug resistant (MDR) clinical isolates.     

泵抑制剂对嗜麦芽寡养单胞菌临床分离株氟喹诺酮类敏感性的影响

目的 了解嗜麦芽寡养单胞菌临床分离株外排泵的分布以及不同泵抑制剂对该菌氟喹诺酮类敏感性的影响。方法 用琼脂二倍稀释法检测泵抑制剂存在时，嗜麦芽寡养单胞菌对环丙沙星、氧氟沙星和诺氟沙星的敏感性变化。结果 羰基氰氯苯腙(CCCP)和利舍平降低部分嗜麦芽寡养单胞菌株对氟喹诺酮的耐药性。维拉帕米无明显泵抑制作用。泵阳性株存在于耐药和非耐药菌株中，集中于耐药性较高的菌株。泵抑制剂对该菌外排氧氟沙星的抑制大于对诺氟沙星和环丙沙星的作用。结论 多重外排泵是嗜麦芽寡养单胞菌对氟喹诺酮类耐药的原因之一。泵抑制剂可部分逆转这种耐药性。     

Phenothiazines alter resistance of methicillin-resistant strains of Staphylococcus aureus (MRSA) to oxacillin in vitro

Mechanisms of antibiotic resistance of bacteria include efflux pumps which extrude the antibiotic prior to reaching its target. Phenothiazines inhibit the activity of some efflux pumps thereby altering the susceptibility of bacteria. This study demonstrated that chlorpromazine and thioridazine reduce the susceptibility of methicillin-resistant strains (MRSA) but not that of methicillin-susceptible Staphylococcus aureus (MSSA) strains to oxacillin (MIC of oxacillin reduced from >500 to 10 mg/l). Reserpine, an inhibitor of antibiotic efflux pumps also reduced the resistance of MRSA strains to oxacillin suggesting the presence of an efflux pump that contributes to antibiotic resistance of MRSA strains.     

耐甲氧西林葡萄球菌对17种抗生素的敏感性

目的　研究耐甲氧西林葡萄球菌 (MRS)的耐药状况。方法　用琼脂筛选法、酸测定法、琼脂稀释法 ,分别检出 MRS、测定 MRS的 β-内酰胺酶及对 17种抗生素的最低抑菌浓度 (MIC)。结果　 46 6株菌中 ,金黄色葡萄球菌 (SA ) 12 3株 ,其中 5 3株为耐甲氧西林金黄色葡萄球菌 (MRSA) ,占 43.1% (5 3/12 3) ;血浆凝固酶阴性葡萄球菌 (CNS) 343株 ,其中 188株为耐甲氧西林凝固酶阴性葡萄球菌 (MRCNS) ,占 5 4.8% (188/343)。β-内酰胺酶产生率 MRS为 77.4% (4 1/5 3) ,MRCNS为 6 5 .4% (12 3/188)。 MRS对青霉素、氨苄青霉素、苯唑青霉素、四环素、红霉素严重耐药 ,耐药率为 87.8%到 10 0 % ,对氯霉素、林可霉素、妥布霉素、氟哌酸、庆大霉素高度耐药 ,耐药率超过 6 0 % ,对其他抗生素有不同程度的耐药 ,对磷霉素、利福利敏感性好 (耐药率低于 30 % )。结论　在本研究资料中 ,MRS对抗生素的耐药性非常严重 ,磷霉素、利福利可用于治疗由 MRS所致的感染。     

Identification of the plasmid-encoded qacA efflux pump gene in meticillin-resistant Staphylococcus aureus (MRSA) strain HPV107, a representative of the MRSA Iberian clone

Meticillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial bacterium for which prevention and control measures consist mainly of the application of biocides with antiseptic and disinfectant activity. In this study, we demonstrated the presence of the plasmid-located efflux pump gene qacA in MRSA strain HPV107, a clinical isolate representative of the MRSA Iberian clone. The existence of efflux activity in strain HPV107 due to the QacA pump was also established and this QacA efflux activity was linked with a phenotype of reduced susceptibility towards several biocide compounds. No association could be made with antibiotic resistance. This work emphasises the potential of QacA pump activity in the maintenance and dissemination of important MRSA strains in the hospital setting and, increasingly, in the community.     

Antimicrobial resistance in foodborne pathogens--a cause for concern?

The widespread use of antibiotics in food animal production systems has resulted in the emergence of antibiotic resistant zoonotic bacteria that can be transmitted to humans through the food chain. Infection with antibiotic resistant bacteria negatively impacts on public health, due to an increased incidence of treatment failure and severity of disease. Development of resistant bacteria in food animals can result from chromosomal mutations but is more commonly associated with the horizontal transfer of resistance determinants borne on mobile genetic elements. Food may represent a dynamic environment for the continuing transfer of antibiotic resistance determinants between bacteria. Current food preservation systems that use a combination of environmental stresses to reduce growth of bacteria, may serve to escalate development and dissemination of antibiotic resistance among food related pathogens. The increasing reliance on biocides for pathogen control in food production and processing, heightens the risk of selection of biocide-resistant strains. Of particular concern is the potential for sublethal exposure to biocides to select for bacteria with enhanced multi-drug efflux pump activity capable of providing both resistance to biocides and cross-resistance to multiple antibiotics. Although present evidence suggests that biocide resistance is associated with a physiological cost, the possibility of the development of adaptive mutations conferring increased fitness cannot be ruled-out. Strategies aimed at inhibiting efflux pumps and eliminating plasmids could help to restore therapeutic efficacy to antibiotics and reduce the spread of antibiotic resistant foodborne pathogens through the food chain.     

Thioridazine reduces resistance of methicillin-resistant staphylococcus aureus by inhibiting a reserpine-sensitive efflux pump

Previous studies suggested that the phenothiazine chlorpromazine (CPZ) could reverse or reduce the antibiotic resistance of bacteria. In some areas of the world, the majority of Staphylococcus aureus isolates are now resistant to methicillin, prompting this study to see whether such resistance can be altered by phenothiazine thioridazine (TZ), an agent with equal antibacterial activity, which is free of the severe side-effects associated with chronic administration of CPZ. The results indicated that, whereas methicillin-sensitive strains of Staphylococcus aureus (MSSA) were not rendered more susceptible to oxacillin, resistance to oxacillin by highly-resistant strains (MRSA) could be significantly reduced by sub-inhibitory concentrations of TZ. Reserpine, an inhibitor of efflux pumps, was also shown to reduce the resistance of MRSA strains to oxacillin in a concentration-dependent manner. The phenothiazines have been shown, by others, to inhibit the efflux pumps of bacteria and the mechanism by which MRSA are rendered more susceptible to oxacillin in the presence of TZ is believed to be due to a similar efflux pump.     

Thanatin activity on multidrug resistant clinical isolates of Enterobacter aerogenes and Klebsiella pneumoniae

Efflux pumps protect bacterial cells by ejecting intracellular toxic molecules such as antibiotics, detergents and defensins that have penetrated the cell envelope. Some of these efflux pumps recognise structurally unrelated compounds (mdr pump) and account for the resistance of some organisms to two or more agents. It would be of interest to identify molecules that are able to circumvent the problems created by multidrug resistance phenotypes during antibiotic therapy. We have studied the activity of thanatin, a 21-residue cationic antimicrobial peptide produced by an insect, against three bacterial species. The antibacterial effect depended on the size of lipopolysaccharide side chains. In clinically resistant isolates of Enterobacter aerogenes and Klebsiella pneumoniae, the biological activity of thanatin is independent of the membrane permeability, possibly controlled by one or more porins, and/or the expression of drug efflux pumps, two mechanisms which confer high level antibiotic resistance. In addition, thanatin was able to improve the activity of structurally unrelated antibiotics (norfloxacin, chloramphenicol, tetracycline) on a multidrug- resistant E. aerogenes clinical isolate.     

Efflux pumps: their role in antibacterial drug discovery

The emergence of active efflux as a major causative factor in antibiotic resistance has been one of the most significant trends in antiinfective chemotherapy over the last decade. The phenomenon affects virtually all classes of antibiotics and frequently results in multi-drug resistant phenotypes. This review analyzes efflux pumps of clinical significance and examines their impact on different antibiotic classes relative to other mechanisms of resistance. Progress in strategies to combat efflux-mediated resistance by modification of existing antibiotics or identification of efflux pump inhibitors is also reviewed.     

耐甲氧西林金黄色葡萄球菌多重耐药研究

目的了解本地区耐甲氧西林金黄色葡萄球菌（MRSA）的多重耐药情况。方法收集经头孢西丁纸片扩散试验和青霉素结合蛋白2a胶乳凝集实验分离出的51株MRSA,用β-内酰胺酶实验、D试验、Kirby-Bauer法检测MRSA产β-内酰胺酶以及对红霉素、克林霉素、4种氟喹诺酮类药物（氧氟沙星、诺氟沙星、左旋氧氟沙星、加替沙星）和万古霉素耐药性。结果51株MRSA头孢西丁纸片抑菌圈直径介于6～20mm,产β-内酰胺酶率为84.3%,对红霉素、克林霉素、氟喹诺酮类、万古霉素耐药率分别为98.0%、86.3%、88.2%、0,红霉素诱导克林霉素耐药率为50.0%（3/6）。结论MRSA呈多重耐药,轻度感染可根据药敏结果选万古霉素与氟喹诺酮类？生素联合用药。     

Practical applications and feasibility of efflux pump inhibitors in the clinic--a vision for applied use

The world of antibiotic drug discovery and development is driven by the necessity to overcome antibiotic resistance in common Gram-positive and Gram-negative pathogens. However, the lack of Gram-negative activity among both recently approved antibiotics and compounds in the developmental pipeline is a general trend despite the fact that the plethora of covered drug targets are well-conserved across the bacterial kingdom. Such intrinsic resistance in Gram-negative bacteria is largely attributed to the activity of multidrug resistance (MDR) efflux pumps. Moreover, these pumps also play a significant role in acquired clinical resistance. Together, these considerations make efflux pumps attractive targets for inhibition in that the resultant efflux pump inhibitor (EPI)/antibiotic combination drug should exhibit increased potency, enhanced spectrum of activity and reduced propensity for acquired resistance. To date, at least one class of broad-spectrum EPI has been extensively characterized. While these efforts indicated a significant potential for developing small molecule inhibitors against efflux pumps, they did not result in a clinically useful compound. Stemming from the continued clinical pressure for novel approaches to combat drug resistant bacterial infections, second-generation programs have been initiated and show early promise to significantly improve the clinical usefulness of currently available and future antibiotics against otherwise recalcitrant Gram-negative infections. It is also apparent that some changes in regulatory decision-making regarding resistance would be very helpful in order to facilitate approval of agents aiming to reverse resistance and prevent its further development.     

Bacteriological evaluations of combination therapies with minocycline and beta-lactams for methicillin-resistant Staphylococcus aureus. I. Cefotiam plus minocycline]

Since methicillin-resistant Staphylococcus aureus (MRSA) is resistant to multiple antibiotics, only a limited number of antibacterial agents shows efficacy against this bacteria. Therefore, combination therapy is often attempted for MRSA infections. Most of the MRSA strains recently isolated, however, have been found to show very high resistance, and some of the antibiotics which had previously been effective have been failing to produce good responses in increasing numbers of patients. Thus, the drugs used for combination therapy in MRSA infections need to be reevaluated. We assessed the bacteriological efficacy of cefotiam (CTM) plus minocycline (MINO) therapy against MRSA in an in vitro system (CTM shows relatively strong antibacterial activities against MRSA with moderate resistance, and MINO shows strong antibacterial activities against highly resistant MRSA. 1. Against MINO-susceptible MRSA strains, CTM + MINO demonstrated potent antibacterial activities at MINO concentrations of MIC or sub-MIC levels, irrespective of the MIC of CTM against MRSA strains being tested. 2. Against MINO-resistant MRSA strains (strains for which MICs of MINO exceeded the upper limit of the clinically expected plasma MINO level), CTM + MINO showed no significant antibacterial activity. These results suggested that the effect of this combination was dependent on the antibacterial activity of MINO. Therefore, the usefulness of this combination in patients with MRSA infections can be predicted based on susceptibilities of involved strains to MINO. 3. The potent antibacterial effect of this combination against MINO-susceptible MRSA strains was considered to be the result of damage to the cellular membrane by MINO and the subsequent antibiotic effect of CTM.(ABSTRACT TRUNCATED AT 250 WORDS)     

临床标本中金黄色葡萄球菌的分布情况及其耐药性分析

目的 了解金黄色葡萄球菌感染及耐药情况,为合理使用抗菌药物提供科学依据.方法 对106株自临床标本分离的金黄色葡萄球菌进行分析,菌株鉴定采用法国生物梅里埃公司提供的VITEK32全自动微生物鉴定仪,药敏试验采用K-B纸片扩散法.结果 106株金黄色葡萄球菌中有60株(56.60％)来自呼吸道分泌物,25株(23.58％)来自伤口分泌物;从科室分布来看,以ICU为主,占48例(45.28％);金黄色葡萄球菌对多种抗菌药物呈普遍耐药,而对万古霉素、呋喃妥因、利奈唑胺的敏感率为100％,对复方新诺明的耐药率为10.38％(耐药菌株为11株);检出耐甲氧西林金黄色葡萄球菌(MRSA) 82株,检出率为77.36％,MRSA对抗菌药物的耐药率均高于甲氧西林敏感金黄色葡萄球菌.结论 金黄色葡萄球菌对多种抗菌药耐药率高,应规范临床用药,加强MRSA耐药性检测,隔离MRSA感染者,防止医源性传播.     

Role of efflux mechanisms on fluoroquinolone resistance in Streptococcus pneumoniae and Pseudomonas aeruginosa

Prokaryotic efflux mechanisms can effectively increase the intrinsic resistance of bacteria by actively transporting antibiotics out of cells, thus reducing the effective concentration of these agents. The fluoroquinolones, similar to most other antimicrobial classes, are susceptible to efflux mechanisms, particularly in Gram-negative organisms, such as Pseudomonas aeruginosa. Resistant P. aeruginosa clones isolated after fluoroquinolone therapy frequently over express at least one of the multiple efflux pump mechanisms found in this organism. Gram-positive bacteria, such as Streptococcus pneumoniae, also possess efflux mechanisms, though their effect on fluoroquinolone resistance seems to be more limited and selective. In the future, efflux pump inhibitors may offer effective adjunctive therapy to antibiotics for the treatment of difficult infections by efflux mutants. In the meantime, appropriate antibiotic selection and optimal dosing strategies should aim to eradicate the causative pathogen before a resistant efflux mutant can emerge.     

Measurement of MICs on 16 antimicrobial agents for freshly isolated methicillin-resistant Staphylococcus aureus strains

MICs of 16 antimicrobial agents including a newly synthesized aminoglycoside antibiotic, arbekacin (HBK), were determined against methicillin-resistant Staphylococcus aureus (MRSA) strains which had been isolated since 1988 from patients with refractory infections. 1. The proportion of highly methicillin-resistant strains was large, especially among those isolated from blood and respiratory tract. 2. All the MRSA strains were resistant to all the beta-lactam antibiotics tested. 3. Most of the strains were also resistant to aminoglycosides. However, HBK inhibited the growth of 95% of the strains at 3.13 micrograms/ml or less, showing potent activities even against highly resistant MRSA strains. 4. The MIC distribution of HBK against MRSA strains showed no change since 1986 when year to year data were compared. Based on our studies on the drug resistance pattern of MRSA since 1986, a large proportion of MRSA strains isolated from blood and respiratory tract appeared to be coagulase type II. 5. Netilmicin-resistant MRSAs have increased since 1986, and MRSA resistant to fosfomycin, minocycline, ofloxacin and norfloxacin have also increased in 1989 compared to 1988. 6. The drug resistance pattern of MRSA has been changing every year and they are acquiring multi-drug resistance. Thus, in the treatment of MRSA infections, it is very important to identify the drug susceptibility of MRSA quickly and accurately.     

我院临床标本中病原菌的分布特点及抗菌药物耐药分析

目的:了解我院病原菌检出分布特点及抗菌药物耐药趋势。方法:对我院2005年7月~2007年6月临床所采集标本分离出的细菌,分析各种细菌的分布特点,并对抗菌药物耐药情况进行统计。结果:以痰、血标本的分离率最高,细菌分布以G+球菌为主,多重耐药菌(耐甲氧西林金黄色葡萄球菌、耐甲氧西林凝固酶阴性葡萄球菌、产超广谱β-内酰胺酶的大肠埃希菌和肺炎克雷伯菌)检出率呈上升趋势,部分抗菌药物的耐药率较高。结论:加强病原菌分布及耐药监测,合理规范应用抗菌药物势在必行。     

In vitro additive effect of Hyptis martiusii in the resistance to aminoglycosides of methicillin-resistant Staphylococcus aureus

Context: Bacterial infectious agents represent a risk to populations, where they are responsible for the high morbidity and mortality. In combating these pathogens, our main line of defense is the use of antibiotics. However, the indiscriminate use of these drugs select resistant strains to these same drugs.

Objective: In this study the ethanol extract of Hyptis martiusii Benth. (EEHM) (Lamiaceae) was tested for its antimicrobial activity against aminoglycoside multi-resistant Staphylococcus aureus (MRSA).

Materials and methods: In this study, the ethanol extract of H. martiusii was prepared and tested with chlorpromazine for its antimicrobial activity using the microdilution method. Chlorpromazine and the ethanol extract were used alone and also in combination with aminoglycosides against a MRSA strain resistant to these antibiotics to determine the participation of efflux systems in resistance mechanisms. The FIC index was calculated and evaluated by the checkerboard method.

Results: A potentiating effect between this extract and aminoglycosides was demonstrated. Similarly, a potentiating effect of chlorpromazine with kanamycin was detected, indicating the involvement of an efflux system in the resistance to this aminoglycoside. The checkerboard method with combinations of aminoglycosides and EEHM demonstrated additive effect with kanamycin and gentamicin. It is therefore suggested that extracts from H. martiusii could be used as a source of plant-derived natural products with resistance- modifying activity.

Conclusion: This is the first report about the modifying antibiotic activity of Hyptis martiusii, constituting a new approach against bacterial resistance to antibiotics as aminoglycosides.