Low-level antibacterial resistance: a gateway to clinical resistance.

The huge amount of antibiotic substances released in the human environment has probably resulted in an acceleration in the rate of bacterial evolution. It is to note that most interactions between chemotherapeutic agents and microbial populations occur at very low antibiotic concentrations. Thus, natural selection is expected to act on very small increases in the bacterial ability to resist to antibiotic inhibitory effects. On the other hand, there is a wealth of mechanisms to resist to these low antibiotic concentrations. The progressive enrichment in low-level resistant populations favours secondary selections for more specific and effective mechanisms of resistance, particularly in treated patients. These adaptations may have a biological cost in the absence of antibiotics, but frequently compensatory mutations occur, minimizing such genetic burden. In this way, a phenomenon of directional selection takes place, with low possibilities of return to susceptibility. Moreover, low antibiotic concentrations are not only able to select low-level antibiotic resistant variants, but may produce a substantial stress in bacterial populations, that eventually influences the rate of genetic variation and the diversity of adaptive responses. More attention should be devoted to the mechanisms of low-level resistance in microorganisms, as they can serve as stepping stones to develop high level, clinically relevant resistance. These mechanisms should be identified early in the development of drugs in order to adapt the therapeutic strategies (for instance dosage) to minimize the selection of low-level resistant variants, as frequently they emerge by means of concentration-specific selection. At the same time, conventional susceptibility testing should probably be able to detect low-level resistance, and not only clinically-relevant resistance. We should be vigilant of the evolutionary trends of microorganisms; for that a purpose, knowledge of the biology and epidemiology of low-level resistance is becoming a real need.     

Reducing the development of antibiotic resistance in critical care units

Bacteria becoming resistant to an increasing number of antibiotic classes are a major problem at hospitals including critical care units worldwide. Awareness of this problem and the need to prevent the development of antibiotic resistance are very important, especially since very few new antibiotics will become available in the near future. This article gives an overview of the mechanisms of antibacterial resistance and actual resistance data worldwide of the most prevalent Gram positive (MRSA, VISA/VRSE and VRE) and Gram negative bacteria (Pseudomonas aeruginosa, Acinetobacter spp., ESBL producing Enterobacteriaceae and Stenotrophomonas maltophilia). Furthermore, strategies to reduce antibiotic resistance are reviewed. Most important is institution of infection control policies including guidelines on surveillance, isolation of colonized patients and contact precautions, hand hygiene, decolonization measures and environmental decontamination. Antimicrobial stewardship, or striking the balance between an optimal antibiotic treatment for a patient and a minimal risk of development of antibiotic resistance, is another important strategy. Finally, optimizing of antibiotic dosage regimens and thus avoiding underdosage is essential to avoid selection of the most resistant subpopulation of bacteria during antibiotic treatment. Intensive care units with knowledge of local epidemiology of resistance, an effective infection control program and antimicrobial stewardship policy tailored to their specific needs, and using optimal antibiotic dosing regimens have both locally decreased the risk of an outbreak with multi-resistant bacteria, and maybe even more important help to reduce the development of antibiotic resistance.     

Transferable antibiotic resistance in Escherichia coli isolated from healthy Nigerian school children

Three hundred and ninety-six E. coli isolates obtained from apparently healthy school children in Ile-Ife, Nigeria, were tested for their susceptibility to 11 different antibiotics. Of these, only gentamicin, cefotaxime and nalidixic acid were found to have significant in vitro activity against most of the isolates. The incidence of antibiotic resistances encountered varied between 24% for trimethoprim and 55.5% for the sulphonamide. It was further observed that 47.5% of the isolates were identified as being multiply resistant, since they were simultaneously resistant to at least three different antibiotics. The 86 trimethoprim-resistant isolates tested were found to be able to transfer this resistance trait together with resistance genes of to other antibiotics, into a plamidless strain of E. coli by conjugation. Seventy-seven of the trimethoprim-resistant isolates were also found to be classifiable into the types of dihydrofolate reductases responsible for the observed resistance on the basis of hybridization experiments. The results of this study indicate that there is a large reservoir of antibiotic resistances within the community, and that the resistance genes were easily transferable to other strains even without direct exposure to antibiotics.     

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.     

Mutation and evolution of antibiotic resistance: antibiotics as promoters of antibiotic resistance?

Antibiotic resistance appearance and spread have been classically considered the result of a process of natural selection, directed by the use of antibiotics. Bacteria, that have to face the antibiotic challenge, evolve to acquire resistance and, under this strong selective pressure, only the fittest survive, leading to the spread of resistance mechanisms and resistant clones. Horizontal transference of resistance mechanisms seems to be the main way of antibiotic resistance acquisition. Nevertheless, recent findings on hypermutability and antibiotic-induced hypermutation in bacteria have modified the landscape. Here, we present a review of the last data on molecular mechanisms of hypermutability in bacteria and their relationship with the acquisition of antibiotic resistance. Finally, we discuss the possibility that antibiotics may act not only as selectors for antibiotic resistant bacteria but also as resistance promoters.     

淋球菌对阿奇霉素耐药性与耐药机制研究进展

阿奇霉素是15元环大环内酯类抗生素,其结构与红霉素相似,但在内酯环的9位上杂入了一个甲氨基。阿奇霉素的抗菌谱广,不仅对革兰阳性球菌、厌氧菌、支原体、衣原体有作用,对一些革兰阴性菌,包括流感嗜血菌、淋球菌等也有较好的作用,且对淋球菌的作用比红霉素强4倍。淋球菌为严重的人体寄生菌,常存在于急性尿道炎与阴道炎的脓性分泌物白细胞中,是引发淋病的病原体。如今,淋球菌正在对越来越多种类的抗生素产生耐药性,阿奇霉素便是其中一种。然而,淋球菌对阿奇霉素产生耐药性的机制尚不明确,有研究表明其机制可能与mtr系统有关。本文主要对一些国家出现的淋球菌对阿奇霉素的耐药性及其机制进行了概述。     

Prevalence and determinants of antibiotic resistance in faecal Escherichia coli among unselected patients attending general practitioners in Southwest Germany

PURPOSE: Widespread use of antibiotics is thought to be the main reason for the world-wide increase in antibiotic resistance. Although a great majority of antibiotics are prescribed outside hospitals, little is known about the prevalence and determinants of antibiotic resistance in the general population. METHODS: Escherichia coli (E. coli) was cultured from and minimal inhibitory concentrations against six commonly prescribed antibiotic substances were tested in 750 stool samples of 484 unselected, consecutive outpatients aged 40-74 years attending general practitioners. Odds ratios (OR) and their 95% confidence intervals (CI) for the association between potential risk factors and the prevalence of antibiotic resistance were estimated using generalised estimating equations. RESULTS: Prevalence of E. coli resistance against ampicillin, doxycycline, cotrimoxazole or quinolones was 24%. Current antibiotic use was strongly associated with antibiotic resistance, adjusted OR: 11.1, 95% CI: 2.3-53, but antibiotic resistance was unaffected by antibiotic use stopped weeks before. Recent hospitalisations were the only other significant predictor of an increased prevalence of resistance. CONCLUSIONS: The strong association between current use of antibiotics and colonisation with antibiotic resistant E. coli suggests a major role for selection of resistant strains while using antibiotics that seem to be quickly reversible, though.     

Antibiotic resistance of Staphylococcus aureus isolates from community acquired soft tissue infections in Bulawayo

Fifty cases of community acquired strains of Staphylococcus aureus causing superficial abscesses were tested for their sensitivity to commonly used antibiotics. Ninety-two percent of isolates were resistant to benzyl-penicillin and ampicillin. Possible causes for this very high level of resistance and the implications for antibiotic usage are discussed.     

Inhibition of efflux transporter ABCG2/BCRP does not restore mitoxantrone sensitivity in irinotecan-selected human leukemia CPT-K5 cells: evidence for multifactorial multidrug resistance.

It has been shown that the human acute lymphoblastic leukemia (ALL) T cell line (RPMI 8402) selected with irinotecan (CPT-11) is transformed to a multidrug resistant (MDR) phenotype (CPT-K5) with cross-resistance to mitoxantrone (MX). Since MX is a well-documented substrate for the efflux transporter breast cancer resistant protein (BCRP/ABCG2), we assessed the contribution of drug efflux to MX resistance in CPT-K5 cells. Our results demonstrate that CPT-K5 cells had markedly higher expression levels of BCRP, negligible expression of MRP2 and P-gp, and lower intracellular retention of MX as compared to RPMI 8402 cells. Surprisingly, MX resistance in CPT-K5 cells was not reversed by the BCRP chemical inhibitor, novobiocin (NOV), or gene-specific siRNA, although intracellular MX concentrations were significantly increased when BCRP was functionally knocked down. These results suggest that up-regulation of BCRP plays a minimal role in conferring MX resistance to CPT-K5 cells, highlighting the existence of multiple, redundant mechanisms of drug resistance. The current results support the concept of "multifactorial multidrug resistance", a recently-described phenomenon that ascribes multidrug resistance to many possible cellular mechanisms, not only by efflux drug transporters.     

Prolonged exposure of methicillin-resistant Staphylococcus aureus (MRSA) COL strain to increasing concentrations of oxacillin results in a multidrug-resistant phenotype

Our previous studies demonstrated that exposure of a bacterium to increasing concentrations of an antibiotic would increase resistance to that antibiotic as a consequence of activating efflux pumps. This study utilises the same approach; however, it employs the methicillin-resistant Staphylococcus aureus (MRSA) COL strain, which is highly resistant to oxacillin (OXA). MRSA COL was adapted to 3200 mg/L of OXA. Changes in resistance to other antibiotics were evaluated and efflux pump activity during the adaptation process was determined. MRSA COL was exposed to stepwise two-fold increases of OXA. At the end of each step, minimum inhibitory concentration determination for erythromycin (ERY) and other antibiotics was conducted. Reserpine (RES) was employed to evaluate whether resistance to ERY was dependent on efflux pump activity. Efflux pump activity was also evaluated using the ethidium bromide (EB) assay. DNA typing of the products of each culture step was conducted to assess purity. Serial exposure of MRSA COL to increasing concentrations of OXA resulted in increased resistance to ERY, which could be eliminated with RES. Evaluation of efflux pump activity by the EB method indicated increased efflux activity. Resistance to ERY was accompanied by resistance to kanamycin, amikacin, ofloxacin, norfloxacin, ciprofloxacin and rifampicin. This is the first time that a multidrug-resistant phenotype has been experimentally produced as a consequence of exposure of the organism to an antibiotic to which it is initially highly resistant.     

肺炎克雷伯茵耐氟喹诺酮机理研究

目的：探讨肺炎克雷伯菌耐氟喹诺酮类药物的机理。方法：采用NCCLS推荐的K—B法测定50株肺炎克雷伯菌对氟喹诺酮类药物的耐药性并初步筛选出耐环丙沙星菌株。试管稀释法测定耐环丙沙星菌株对环丙沙星和左氧氟沙星的最低抑菌浓度（MIC）。对此23株菌GyrA的基因（gyrA）进行PCR扩增和基因序列的分析比较。结果：23株耐环丙沙星肺炎克雷伯菌中,20株存在gyrA变异：Ser83（TCC）→Phe（TTC）、Tyr（TAC）,Asp87（GAC）→Asn（AAC）、Ala（GCC）。结论：肺炎克雷伯菌对氟喹诺酮类药物耐药机理主要与药物作用靶位gyrA基因的突变有关,并且gyrA基因突变位点越多其耐药程度越高。     

亚最低杀菌浓度环丙沙星体外诱导铜绿假单胞菌标准质控菌株耐药研究

目的:研究亚最低杀菌浓度(minimum bactericidal concentration,Sub-MBC)环丙沙星体外诱导对铜绿假单胞菌MIC值影响,为防止临床不合理使用抗菌药物导致细菌耐药性,提供新思路。方法:以铜绿假单胞菌质控菌株ATCC27853为研究对象,以环丙沙星为诱导耐药抗菌药物,用微量稀释法首先测定其MBC值,然后用1/2MBC值环丙沙星浓度进行体外诱导培养,每天观察其MBC值变化,及时调整诱导浓度为变化后MBC值的1/2,直到诱导菌株MBC值升高到原始MBC值的64倍时,停止诱导,并记录诱导天数;诱导出的耐药菌株传代3 d,再采用微量稀释法对其MBC进行测定,并通过全自动微生物药敏仪对其进行耐药性鉴定。结果:铜绿假单胞菌标准质控菌株的环丙沙星MBC值为0.5μg·ml^-1;采用0.25μg·ml^-1环丙沙星浓度体外诱导7 dMBC值明显升高,诱导30 d升高至原始菌株MBC的64倍;诱导出的铜绿假单胞菌针对环丙沙星的耐药菌株,经3 d传代后,微量稀释法测定其MBC仍为原始菌株的64倍,全自动药敏分析仪鉴定为环丙沙星耐药菌。结论:亚最低杀菌浓度环丙沙星可体外诱导铜绿假单胞菌标准质控菌株出现耐药,且耐药性随亚最低杀菌浓度诱导时间延长而增加,诱导出的铜绿假单胞菌耐药菌株的耐药性能稳定传代,提示低剂量使用抗菌药物可导致细菌产生耐药性。     

Tackling antimicrobial resistance

Bacteria resistant to antibiotic therapy are becoming much more common and this has led to mounting concern in the UK and worldwide. Many pathogens are now 'multiresistant', that is, they are resistant to several classes of antimicrobial drug. Infection with such organisms may be particularly difficult to treat. In this article, we briefly discuss how resistance and multiresistance occur. We consider some of the important pathogens involved and the problems they pose in hospitals and the community. We discuss strategies for slowing the accumulation of antibiotic resistance and the implications for doctors treating patients with common infections.     

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.     

Associations between antibiotic use and changes in susceptibility patterns of Pseudomonas aeruginosa in a private, university-affiliated teaching hospital: an 8-year-experience: 1995-2002

Increasing resistance in Pseudomonas aeruginosa to multiple antibiotics has been observed and is posing therapeutic dilemmas. Antibiotic utilization is one factor that has been associated with the emergence of antimicrobial resistance. We examined the overall and specific antimicrobial use in relation to changes in susceptibility patterns in P. aeruginosa. Regression analysis was performed to explore the relationships between annual antibiotic use and the incidence of resistant P. aeruginosa. There were statistically significant relationships between increasing anti-pseudomonal cephalosporin and levofloxacin use and the increasing incidence of ciprofloxacin resistant P. aeruginosa. However, there was not an association between other fluoroquinolone or overall fluoroquinolone use and this change. In addition, there was no association between increasing anti-pseudomonal cephalosporin use and cefepime resistant P. aeruginosa. No statistical relationship was seen with overall antibiotic use and the development of resistance in P. aeruginosa, suggesting that the development of resistance is associated with the use of individual agents, rather than overall antibiotic consumption.     

Comparing the selective and co-selective effects of different antimicrobials in bacterial communities

Bacterial communities are exposed to a cocktail of antimicrobial agents, including antibiotics, heavy metals and biocidal antimicrobials such as quaternary ammonium compounds (QACs). The extent to which these compounds may select or co-select for antimicrobial resistance (AMR) is not fully understood. In this study, human-associated, wastewater-derived bacterial communities were exposed to either benzalkonium chloride (BAC), ciprofloxacin or trimethoprim at sub-point-of-use concentrations for one week to determine selective and co-selective potential. Metagenome analyses were performed to determine effects on bacterial community structure and prevalence of antibiotic resistance genes (ARGs) and metal or biocide resistance genes (MBRGS). Ciprofloxacin had the greatest co-selective potential, significantly enriching for resistance mechanisms to multiple antibiotic classes. Conversely, BAC exposure significantly reduced relative abundance of ARGs and MBRGS, including the well characterised qac efflux genes. However, BAC exposure significantly impacted bacterial community structure. Therefore BAC, and potentially other QACs, did not play as significant a role in co-selection for AMR as antibiotics such as ciprofloxacin at sub-point-of-use concentrations in this study. This approach can be used to identify priority compounds for further study, to better understand evolution of AMR in bacterial communities exposed to sub-point-of-use concentrations of antimicrobials.     

Antimicrobials: modes of action and mechanisms of resistance

After six decades of widespread antibiotic use, bacterial pathogens of human and animal origin are becoming increasingly resistant to many antimicrobial agents. Antimicrobial resistance develops through a limited number of mechanisms: (a). permeability changes in the bacterial cell wall/membrane, which restrict antimicrobial access to target sites; (b). active efflux of the antimicrobial from the cell; (c). mutation in the target site; (d). enzymatic modification or degradation of the antimicrobial; and (e). acquisition of alternative metabolic pathways to those inhibited by the drug. Numerous bacterial antimicrobial resistance phenotypes result from the acquisition of external genes that may provide resistance to an entire class of antimicrobials. These genes are frequently associated with large transferable extrachromosomal DNA elements called plasmids, on which may be other mobile DNA elements such as transposons and integrons. An array of different resistance genes may accumulate on a single mobile element, presenting a situation in which multiple antibiotic resistance can be acquired via a single genetic event. The versatility of bacterial populations in adapting to toxic environments, along with their facility in exchanging DNA, signifies that antibiotic resistance is an inevitable biological phenomenon that will likely continue to be a chronic medical problem. Successful management of current antimicrobials, and the continued development of new ones, is vital to protecting human and animal health against bacterial pathogens.     

Antibacterial activity of rokitamycin against fresh clinical isolates]

We obtained bacterial strains which were clinically isolated and identified from outpatients with various infections in medical institutions throughout Japan. Possible antibacterial activities of rokitamycin (RKM) were examined against these isolates. Minimum inhibitory concentrations (MICs) were determined through a comparative study with reference drugs. The results of the study are summarized as follows. 1. Resistance patterns of 400 isolates which were highly resistant to macrolides (MLs) with MIC values > 100 micrograms/ml were classified into 55 patterns. Staphylococcus spp. showed cross resistance to 14-membered ring MLs with 100% cross resistance observed between erythromycin (EM) and clarithromycin (CAM), and 85.2% between EM and oleandomycin (OL). Fewer isolates showed strong resistance to 16-membered ring MLs than to 14-membered ring MLs. Cross resistances observed among the Staphylococcus isolates were 100% between acetylmidecamycin (MDM-AC) and kitasamycin (leucomycin (LM)), 93.9% between MDM-AC and josamycin (JM), and 53.3% between MDM-AC and RKM. Streptococcus spp. and Peptococcus spp. showed very similar resistance patterns to both 14- and 16-membered ring MLs, but resistance patterns to RKM were quite different. Most of anaerobic streptococci and Bacteroides fragilis group had similar resistance patterns to 14- and 16-membered ring MLs, but in some cases a pattern similar to that of Staphylococcus spp. was observed. 2. When ML-resistant bacteria isolated during 1975 to 1980 were compared to those isolated in 1986 and 1989, it was observed that resistance of Staphylococcus aureus remained almost unchanged, that of Streptococcus pyogenes was lower in the later years than during 1975 to 1980, but that of Streptococcus pneumoniae increased. 3. Most of ML-resistances of the resistant isolates were inducible, but extents of induction varied depending on drugs tested. Strong inductions were observed when 14-membered ring MLs were used, but inductions were minimal with 16-membered ring MLs. RKM appeared to induce resistance to the least extent. From these results, it appears that the RKM is quite useful clinically even in the 1990s.     

Mechanisms of bacterial resistance to antibiotics in infections of COPD patients

A key characteristic of airway inflammation in chronic obstructive pulmonary disease (COPD) is the persistent presence of bacteria in the lower airways. The most commonly isolated bacteria in the lower respiratory tract of COPD patients are nontypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae, with growing evidence of the significance of Pseudomonas aeruginosa infections in severe COPD disease. This review focuses on the antibiotic resistant mechanisms associated with the gram-negative bacteria H. influenzae and M. catarrhalis and comparison with P. aeruginosa infection because of the recent evidence of its significance in patients with severe COPD disease. These mechanisms of resistance to β-lactams in H. influenzae and M. catarrhalis are mostly associated with serine β-lactamases of class A type, whereas P. aeruginosa strains exhibit a much broader repertoire with class A-D type mechanisms. Other mechanisms of antibiotic resistance include membrane permeability, efflux pump systems and mutations in antimicrobial targets. Antimicrobial resistance within biofilm matrices appears to be different to the mechanisms observed when the bacteria are in the planktonic state. P. aeruginosa exhibits a more numerous and diverse range of antimicrobial resistance mechanisms in comparison to M. catarrhalis and H. influenzae. The recognition that P. aeruginosa is associated with exacerbations in patients with more severe COPD and that turnover in infecting strains is detected (unlike in cystic fibrosis patients), then further investigation is required to better understand the contribution of antimicrobial resistance and other virulence mechanisms to poor clinical outcomes to improve therapeutic approaches.     

单纯疱疹病毒1型对抗生素17997的耐药及交叉耐药研究

目的 体外训练单纯疱疹病毒1型（HSV－1）对抗生素17997的耐药突变及体内、外研究抗生素17997与阿昔洛韦（ACV）的交叉耐药。方法 HSV－1在抗生素17997或ACV的存在下连续传代，选择一定代数测定HSV－1野株及训练株的IC50。用HSV－1的ACV耐药株感染兔用膜造模，分别用抗生素17997或ACV局部给药治疗，以局部病损及病毒分离判断疗效。结果 HSV－1在抗生素17997存在下连传41代仍保持对抗生素17997的敏感性，未产生耐药变株；但HSV－1在ACV存在下，仅传一代，就发生耐药变株。抗生素17997与ACV无交叉耐药。抗生素17997对HSV－1 ACV耐药株实验感染角膜炎有明显治疗效果，可减轻病损，降低病毒排出量；阳性对照药ACV无任何疗效。结论 体外HSV－1对抗生素17997不易产生耐药。抗生素17997与ACV体内、外均无交叉耐药。