Plasmids and bacterial resistance to biocides

Plasmid-encoded fu1 bacterial resistance to antibiotics and to anions and cations (including important mercurial and silver compounds) has been widely studied. Plasmid-mediated resistance to organic cationic agents which are important biocides has been described for chlorhexidine and quaternary ammonium compounds (and also for the less important acridines, diamidines and ethidium bromide) in antibiotic-resistant Staphylococcus aureus and Staph. epidermidis strains. Plasmids may also encode reduced biocide susceptibility of Gram-negative bacteria, but intrinsic resistance is likely to be of greater significance. Antibiotic resistance and biocide resistance may be linked but this is not always found clinically.     

Active export proteins mediating drug resistance in staphylococci

Drug resistance mediated by integral membrane transporters is an important mode of cellular resistance to cytotoxic agents across all classes of living organisms. Gram-positive bacteria, such as staphylococcal species, are not encapsulated by a selective outer membrane permeability barrier. Therefore, these organisms often employ integral membrane drug transport systems to maintain cellular concentrations of antimicrobials at subtoxic levels. Staphylococcal species, including the opportunistic human pathogen Staphylococcus aureus, encode a multitude of drug exporters, encompassing transporters from each of the five currently recognized families of bacterial drug resistance transporters. A number of these transporters are chromosomally encoded and allow the host cell to realize clinically significant levels of drug resistance after minor mutations to regulatory regions. Others are plasmid-encoded and can be easily passed between staphylococcal strains and species, or acquired from other Gram-positive genera. In combination, staphylococcal drug transporters potentiate resistance to a vast array of antimicrobial compounds, including macrolide, quinolone, tetracycline and streptogramin antibiotics, as well as a broad range of biocides, such as quaternary ammonium compounds, biguanidines and diamidines. An understanding of the genetic and molecular properties of drug transporters will lead to effective treatments of staphylococcal infections. Here we provide a detailed review of the active drug transporters of the staphylococci.     

Susceptibility of antibiotic-resistant cocci to biocides

Biocide resistance has hitherto been a poorly studied subject, possibly due to the belief that such resistance was rare and clinically insignificant. Various recent findings, however, have underlined the importance of biocide resistance as a clinically relevant phenomenon. Outbreaks of biocide-resistant organisms in hospitals have been described and the genetic mechanism for resistance to quaternary ammonium compounds (QACs) in Staphylococcus aureus has now been elucidated. Mycobacteria resistant to commonly used endoscope disinfectants are now commonly reported and have caused numerous adverse clinical events. Cross-resistance between triclosan and antituberculous drugs has been demonstrated in other strains of mycobacteria. This is related to a common mechanism of action. The work presented here describes studies into the biocide resistance of antibiotic-resistant cocci and attempts to create biocide-resistant strains in vitro. Strains of staphylococci (including methicillin-resistant Staph. aureus (MRSA)) and enterococci (including vancomycin-resistant enterococci (VRE)) had their susceptibility to biocides assayed using broth macro dilution methods and resistant strains were selected by serial subculture on biocide-containing media. Mutants were created with relative ease; for instance, triclosan minimal bactericidal concentrations (MBCs) increased from 0.002 to 3.12 mg l(-1). Some strains of MRSA which have intermediate resistance to glycopeptides were demonstrated to have decreased susceptibility to some biocides. Biocide resistance amongst enterococci was demonstrated although there was no clear correlation between biocide and antibiotic resistance. The exact mechanisms of resistance in these strains are still being studied but it is clear that biocide resistance is an important clinical phenomenon.     

Microbial community adaptation to quaternary ammonium biocides as revealed by metagenomics

Quaternary ammonium compounds (QACs) represent widely used cationic biocides that persist in natural environments. Although microbial degradation, sensitivity and resistance to QACs have been extensively documented, a quantitative understanding of how whole communities adapt to QAC exposure remain elusive. To gain insights into these issues, we exposed a microbial community from a contaminated river sediment to varied levels of benzalkonium chlorides (BACs, a family of QACs) for 3 years. Comparative metagenomic analysis showed that the BAC‐fed communities were dramatically decreased in phylogenetic diversity compared with the control (no BAC exposure), resulting presumably from BAC toxicity, and dominated by Pseudomonas species (> 50% of the total). Time‐course metagenomics revealed that community adaptation occurred primarily via selective enrichment of BAC‐degrading Pseudomonas populations, particularly P. nitroreducens, and secondarily via amino acid substitutions and horizontal transfer of a few selected genes in the Pseudomonas populations, including a gene encoding a PAS/PAC sensor protein and ring‐hydroxylating dioxygenase genes. P. nitroreducens isolates were reproducibly recoverable from communities after prolonged periods of no‐BAC exposure, suggesting that they are robust BAC‐degraders. Our study provides new insights into the mechanisms and tempo of microbial community adaptation to QAC exposure and has implications for treating QACs in biological engineered systems.     

Two different mechanisms are involved in the extremely high-level benzalkonium chloride resistance of a Pseudomonas fluorescens strain

A Pseudomonas fluorescens strain, PFRB, which we previously isolated as a contaminant in a batch of benzalkonium chloride (BAC) stock solution, exhibits high-level resistance, not only to BAC, but also to other cationic surfactants belonging to disinfectants classified as quaternary ammonium compounds (QACs). In this study, we analyzed the resistance mechanism of the strain to BAC and other disinfectants. We obtained results suggesting that two different mechanisms, reduced adsorption of BAC to the cell surface and an energy-dependent mechanism which is most probably an efflux system, were implicated in the high-level resistance to BAC. Reduced adsorption of BAC is likely due to the decreased negative cell surface charge of the strain. The putative efflux system seems to be unique in that it excretes only a certain range of cationic membrane-acting disinfectants belonging to QACs.     

Effect of biocides on Pseudomonas aeruginosa phage F116

Pseudomonas aeruginosa phage F116 is of interest in understanding the virucidal mechanisms of disinfectant action. Phage F116 has been used to test several disinfectants. The bacteriophage was relatively resistant to several biocides commonly used in disinfection processes. Only 0.05% cetylpyridinium chloride, 0.1% peracetic acid and 2% phenol were highly active against the phage.     

细菌的消毒剂耐药性

细菌的消毒剂耐药性指细菌与消毒剂多次接触后,使该类消毒剂的最小抑菌浓度或最小杀菌浓度升高的现象。细菌的消毒剂耐药性普遍存在,多种细菌可对一种消毒剂耐药,一种细菌可对多种消毒剂耐药。消毒剂选择件压力是耐药性产生的外在原因,产生的机制包括生化结构、遗传学途径和酶学途径。消毒剂耐药与抗生素耐药之间存在一定的关系。应加强研究,制定统一标准,加强监测,并制定合理应用消毒荆规范,以减少消毒剂耐药性的发生。     

Comparative sensitivity to antibiotics and biocides of methicillin-resistant Staphylococcus aureus strains isolated from Saudi Arabia and Great Britain

Methicillin-resistant Staphylococcus aureus (MRSA) isolated in Saudi Arabia and Great Britain were examined for susceptibility to antibiotics and biocides. The strains differed in their sensitivity patterns. None of the Saudi strains showed resistance to propamidine isethionate, but most of the British gentamicin methicillin-resistant Staph. aureus (GMRSA) strains were highly resistant to this compound and to some other nucleic acid-binding (NAB) compounds. Both groups showed a low level of resistance towards quaternary ammonium compounds (QACs), but resistance to these compounds was not associated with resistance to gentamicin in the Saudi strains. The aminoglycoside-resistant determinants were non-conjugative in these strains. Natural MRSA strains were good recipients for pWG613, but transferred this plasmid in reciprocal crosses at significantly lower rates.     

Effect of some antibiotics and biocides on plasmid transfer in Staphylococcus aureus

The effects of some antibiotics and biocides on the conjugative transfer of the Staphylococcus aureus gentamicin resistance plasmid pWG613 were investigated. Gentamicin and vancomycin were found to stimulate plasmid transfer frequency by 10- to 20-fold whereas methicillin and three inhibitors of protein synthesis each reduced it by various degrees. Most significantly, mupirocin inhibited plasmid transfer frequency by more than 1000-fold. All the biocides tested (cationic agents, sodium dodecyl sulphate and an organomercurial) reduced plasmid transfer.     

Effects of quaternary-ammonium-based formulations on bacterial community dynamics and antimicrobial susceptibility

Quaternary ammonium compounds (QACs) are widely used as adjuncts to hygiene in domestic cleaning products. Current concern that the increased use of such biocides in consumer products might contribute to the emergence of antibiotic resistance has led us to examine the effects of a QAC-containing domestic cleaning fluid on the population dynamics and antimicrobial susceptibility of domestic sink drain biofilm communities. QAC susceptibilities of numerically dominant, culturable drain bacteria (15 genera, 17 species) were determined in vitro before and after repeated QAC exposure (14 passages). A fully characterized drain microcosm was then exposed to short-term (12 days) and long-term (3 months) dosing with a QAC-containing domestic detergent (QD). QAC exposure of isolated cultures caused both increases (three species) and circa twofold decreases (six species) in QAC susceptibility. The susceptibility of Ralstonia sp. was considerably decreased following 14 consecutive QAC passages. Control drain microcosm biofilms maintained dynamic stability, as evidenced by culture and denaturing gradient gel electrophoresis (DGGE) analysis. Bacterial population densities were largely unaffected during short-term exposure to use levels of QD, although 50% QD caused circa 10-fold viability reductions. DGGE analysis supported these observations; identified the major microcosm genera as Pseudomonas, Pseudoalteromonas, Erwinia, and Enterobacter, and showed that aeromonads increased in abundance under 10 to 50% QD. Long-term exposure of the microcosms to QD did not significantly alter the pattern of antimicrobial susceptibility. These data demonstrate the recalcitrance of domestic drain biofilms toward QAC and that although repeated QAC exposure of drain isolates in pure culture results in susceptibility change in some test bacteria, such changes do not necessarily occur within complex communities.     

Specific variations of fatty acid composition of Pseudomonas aeruginosa ATCC 15442 induced by quaternary ammonium compounds and relation with resistance to bactericidal activity

The role of membrane fatty acid composition in the resistance of Pseudomonas aeruginosa ATCC 15442 to the bactericidal activity of Quaternary Ammonium Compounds (QACs) was investigated. The strain was grown in a medium with increasing concentrations of a QAC, benzyldimethyltetradecylammonium chloride (C14) and two non-QACs, sodium dichloroisocyanurate and tri-sodium phosphate. In the presence of C14 only, the strain was able to grow in concentrations higher than the minimal inhibitory concentration. As the strain adapted to C14, resistance to bactericidal activity of the same biocide increased. For the non-QACs, no change was noted when cells were grown in the presence of biocides. The C14-adapted cells showed variations in membrane fatty acid composition. A hierarchical clustering analysis was used to compare all fatty acid compositions of cultures in the presence, or not, of the three biocides used here and another QAC studied previously. The clusters obtained underlined specific variations of membrane fatty acids in response to the presence of QACs. Furthermore, with a simple linear regression analysis, a relationship was shown between the membrane fatty acids and the resistance developed by the strain against the bactericidal activity of C14.     

Quaternary Ammonium Biocides: Efficacy in Application

Quaternary ammonium compounds (QACs) are among the most commonly used disinfectants. There has been concern that their widespread use will lead to the development of resistant organisms, and it has been suggested that limits should be place on their use. While increases in tolerance to QACs have been observed, there is no clear evidence to support the development of resistance to QACs. Since efflux pumps are believe to account for at least some of the increased tolerance found in bacteria, there has been concern that this will enhance the resistance of bacteria to certain antibiotics. QACs are membrane-active agents interacting with the cytoplasmic membrane of bacteria and lipids of viruses. The wide variety of chemical structures possible has seen an evolution in their effectiveness and expansion of applications over the last century, including non-lipid-containing viruses (i.e., noroviruses). Selection of formulations and methods of application have been shown to affect the efficacy of QACs. While numerous laboratory studies on the efficacy of QACs are available, relatively few studies have been conducted to assess their efficacy in practice. Better standardized tests for assessing and defining the differences between increases in tolerance versus resistance are needed. The ecological dynamics of microbial communities where QACs are a main line of defense against exposure to pathogens need to be better understood in terms of sublethal doses and antibiotic resistance.     

Mechanisms of bacterial resistance to biocides

Bacterial resistance to biocides is basically of two types: (i) intrinsic, a natural chromosomally-controlled property of an organism, (ii) acquired, resulting from genetic changes in a cell and arising either by mutation or by the acquisition of genetic material. Both types of resistance are discussed together with the underlying biochemical mechanisms where known. Specific examples of organisms are provided by reference to bacterial spores, mycobacteria, other Gram-positive bacteria and Gram-negative bacteria. The stability of resistance to biocides is considered, as is the possible linkage between biocide and antibiotic resistance.     

Importance of porins for biocide efficacy against Mycobacterium smegmatis

Mycobacteria are among the microorganisms least susceptible to biocides but cause devastating diseases, such as tuberculosis, and increasingly opportunistic infections. The exceptional resistance of mycobacteria to toxic solutes is due to an unusual outer membrane, which acts as an efficient permeability barrier, in synergy with other resistance mechanisms. Porins are channel-forming proteins in the outer membrane of mycobacteria. In this study we used the alamarBlue assay to show that the deletion of Msp porins in isogenic mutants increased the resistance of Mycobacterium smegmatis to isothiazolinones (methylchloroisothiazolinone [MCI]/methylisothiazolinone [MI] and octylisothiazolinone [2-n-octyl-4-isothiazolin-3-one; OIT]), formaldehyde-releasing biocides {hexahydrotriazine [1,3,5-tris (2-hydroxyethyl)-hexahydrotriazine; HHT] and methylenbisoxazolidine [N,N'-methylene-bis-5-(methyloxazolidine); MBO]}, and the lipophilic biocides polyhexamethylene biguanide and octenidine dihydrochloride 2- to 16-fold. Furthermore, the susceptibility of the porin triple mutant against a complex disinfectant was decreased 8-fold compared to wild-type (wt) M. smegmatis. Efficacy testing in the quantitative suspension test EN 14348 revealed 100-fold improved survival of the porin mutant in the presence of this biocide. These findings underline the importance of porins for the susceptibility of M. smegmatis to biocides.     

Comparative sensitivity to antibiotics and biocides of methicillin-resistant Staphylococcus aureus strains isolated from Saudi Arabia and Great Britain

S.B. AL-MASAUDI, A.D. RUSSELL AND M.J. DAY. 1991. Methicillin-resistant Staphylococcus aureus (MRSA) isolated in Saudi Arabia and Great Britain were examined for susceptibility to antibiotics and biocides. The strains differed in their sensitivity patterns. None of the Saudi strains showed resistance to propamidine isethionate, but most of the British gentamicin methicillin-resistant Staph. aureus (GMRSA) strains were highly resistant to this compound and to some other nucleic acid-binding (NAB) compounds. Both groups showed a low level of resistance towards quaternary ammonium compounds (QACs), but resistance to these compounds was not associated with resistance to gentamicin in the Saudi strains. The aminoglycoside-resistant determinants were non-conjugative in these strains. Natural MRSA strains were good recipients for pWG613, but transferred this plasmid in reciprocal crosses at significantly lower rates.     

Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei

Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.     

Effect of some antibiotics and biocides on plasmid transfer in Staphylococcus aureus

S.B. AL-MASAUDI, M.J. DAY AND A.D. RUSSELL. 1991. The effects of some antibiotics and biocides on the conjugative transfer of the Staphylococcus aureus gentamicin resistance plasmid pWG613 were investigated. Gentamicin and vancomycin were found to stimulate plasmid transfer frequency by 10- to 20-fold whereas methicillin and three inhibitors of protein synthesis each reduced it by various degrees. Most significantly, mupirocin inhibited plasmid transfer frequency by more than 1000-fold. All the biocides tested (cationic agents, sodium dodecyl sulphate and an organomercurial) reduced plasmid transfer.     

Effect of Biocides on MS2 and K Coliphages

Several biocides commonly used in disinfection processes as antibacterial and antifungal agents were tested for activity against MS2 and K coliphages. MS2 was resistant to most biocides; only glutaraldehyde (0.5%) and peracetic acid (1%) achieved a 4-log₁₀ titer reduction in 20 min. In contrast, K phage was sensitive to most biocides, being resistant only to phenol (2%) and chlorhexidine (1%).     

Effects of Quaternary-Ammonium-Based Formulations on Bacterial Community Dynamics and Antimicrobial Susceptibility

Quaternary ammonium compounds (QACs) are widely used as adjuncts to hygiene in domestic cleaning products. Current concern that the increased use of such biocides in consumer products might contribute to the emergence of antibiotic resistance has led us to examine the effects of a QAC-containing domestic cleaning fluid on the population dynamics and antimicrobial susceptibility of domestic sink drain biofilm communities. QAC susceptibilities of numerically dominant, culturable drain bacteria (15 genera, 17 species) were determined in vitro before and after repeated QAC exposure (14 passages). A fully characterized drain microcosm was then exposed to short-term (12 days) and long-term (3 months) dosing with a QAC-containing domestic detergent (QD). QAC exposure of isolated cultures caused both increases (three species) and circa twofold decreases (six species) in QAC susceptibility. The susceptibility of Ralstonia sp. was considerably decreased following 14 consecutive QAC passages. Control drain microcosm biofilms maintained dynamic stability, as evidenced by culture and denaturing gradient gel electrophoresis (DGGE) analysis. Bacterial population densities were largely unaffected during short-term exposure to use levels of QD, although 50% QD caused circa 10-fold viability reductions. DGGE analysis supported these observations; identified the major microcosm genera as Pseudomonas, Pseudoalteromonas, Erwinia, and Enterobacter, and showed that aeromonads increased in abundance under 10 to 50% QD. Long-term exposure of the microcosms to QD did not significantly alter the pattern of antimicrobial susceptibility. These data demonstrate the recalcitrance of domestic drain biofilms toward QAC and that although repeated QAC exposure of drain isolates in pure culture results in susceptibility change in some test bacteria, such changes do not necessarily occur within complex communities.     

Active efflux,a common mechanism for biocide and antibiotic resistance

Energy-driven drug efflux systems are increasingly recognized as mechanisms of antibiotic resistance. Chromosomally located or acquired by bacteria, they can either be activated by environmental signals or by a mutation in a regulatory gene. Two major categories exist: those systems energized by proton motive force and those dependent on ATP. The pumps may have limited or broad substrates, the so-called multiple drug resistance pumps, which themselves form a number of related families. The multiple antibiotic resistance (mar) locus and mar regulon in Escherichia coli and other members of the Enterobacteriaceae is a paradigm for a generalized response locus leading to increased expression of efflux pumps. One such pump, the AcrAB pump extrudes biocides such as triclosan, chlorhexidine and quaternary ammonium compounds as well as multiple antibiotics. In Pseudomonas aeruginosa, a number of multidrug efflux pumps export a broad range of substrates. Since bacteria expressing these pumps thwart the efficacy of both kinds of therapeutic agents which control infectious diseases--biocides which prevent transmission of infectious disease agents and antibiotics which treat and cure infectious diseases--they are of particular concern. The prudent use of antibiotics and biocides will guard against the selection and propagation of drug-resistant mutants and preserve the efficacy of these valuable anti-infective agents.