Biofilms in vitro and in vivo: do singular mechanisms imply cross-resistance?

Microbial biofilm has become inexorably linked with man's failure to control them by antibiotic and biocide regimes that are effective against suspended bacteria. This failure relates to a localized concentration of biofilm bacteria, and their extracellular products (exopolymers and extracellular enzymes), that moderates the access of the treatment agent and starves the more deeply placed cells. Biofilms, therefore, typically present gradients of physiology and concentration for the imposed treatment agent, which enables the less susceptible clones to survive. Such clones might include efflux mutants in addition to genotypes with modifications in single gene products. Clonal expansion following subeffective treatment would, in the case of many antibiotics, lead to the emergence of a resistant population. This tends not to occur for biocidal treatments where the active agent exhibits multiple pharmacological activity towards a number of specific cellular targets. Whilst resistance development towards biocidal agents is highly unlikely, subeffective exposure will lead to the selection of less susceptible clones, modified either in efflux or in their most susceptible target. The latter might also confer resistance to antibiotics where the target is shared. Thus, recent reports have demonstrated that sublethal concentrations of the antibacterial and antifungal agent triclosan can select for resistant mutants in Escherichia coli and that this agent specifically targets the enzyme enoyl reductase that is involved in lipid biosynthesis. Triclosan may, therefore, select for mutants in a target that is shared with the anti-E. coli diazaborine compounds and the antituberculosis drug isoniazid. Although triclosan may be a uniquely specific biocide, sublethal concentrations of less specific antimicrobial agents may also select for mutations within their most sensitive targets, some of which might be common to therapeutic agents. Sublethal treatment with chemical antimicrobial agents has also been demonstrated to induce the expression of multidrug efflux pumps and efflux mutants. Whilst efflux does not confer protection against use concentrations of biocidal products it is sufficient to confer protection against therapeutic doses of many antibiotics. It has, therefore, been widely speculated that biocide misuse may have an insidious effect, contributing to the evolution and persistence of drug resistance within microbial communities. Whilst such notions are supported by laboratory studies that utilize pure cultures, recent evidence has strongly refuted such linkage within the general environment where complex, multispecies biofilms predominate and where biocidal products are routinely deployed. In such situations the competition, for nutrients and space, between community members of disparate sensitivities far outweighs any potential benefits bestowed by the changes in an individual's antimicrobial susceptibility.     

Mechanisms of bacterial biocide and antibiotic resistance

Resistance to antibiotics is increasingly commonplace amongst important human pathogens. Although the mechanism(s) of resistance vary from agent to agent they typically involve one or more of: alteration of the drug target in the bacterial cell, enzymatic modification or destruction of the drug itself, or limitation of drug accumulation as a result of drug exclusion or active drug efflux. While most of these are agent specific, providing resistance to a single antimicrobial or class of antimicrobial, there are currently numerous examples of efflux systems that accommodate and, thus, provide resistance to a broad range of structurally unrelated antimicrobials--so-called multidrug efflux systems. Resistance to biocides is less common and likely reflects the multiplicity of targets within the cell as well as the general lack of known detoxifying enzymes. Resistance typically results from cellular changes that impact on biocide accumulation, including cell envelope changes that limit uptake, or expression of efflux mechanisms. Still, target site mutations leading to biocide resistance, though rare, are known. Intriguingly, many multidrug efflux systems also accommodate biocides (e.g. triclosan) such that strains expressing these are both antibiotic- and biocide-resistant. Indeed, concern has been expressed regarding the potential for agents such as triclosan to select for strains resistant to multiple clinically-relevant antibiotics. Some of the better characterized examples of such multidrug efflux systems can be found in the opportunistic pathogen Pseudomonas aeruginosa where they play an important role in the noted intrinsic and acquired resistance of this organism to antibiotics and triclosan. These tripartite pumps include an integral inner membrane drug-proton antiporter, an outer membrane- and periplasm-spanning channel-forming protein and a periplasmic link protein that joins these two. Expression of efflux genes is governed minimally by the product of a linked regulatory gene that is in most cases the target for mutation in multidrug resistant strains hyperexpressing these efflux systems. Issues for consideration include the natural function of these efflux systems and the therapeutic potential of targeting these systems in combating acquired multidrug resistance.     

Bacterial target sites for biocide action

Although biocides have been used for a century, the number of products containing biocides has recently increased dramatically with public awareness of hygiene issues. The antimicrobial efficacy of biocides is now well documented; however, there is still a lack of understanding of their antimicrobial mechanisms of action. There is a wide range of biocides showing different levels of antimicrobial activity. It is generally accepted that, in contrast to chemotherapeutic agents, biocides have multiple target sites within the microbial cell and the overall damage to these target sites results in the bactericidal effect. Information about the antimicrobial efficacy of a biocide (i.e. the eta-value) might give some useful indications about the overall mode of action of a biocide. Bacteriostatic effects, usually achieved by a lower concentration of a biocide, might correspond to a reversible activity on the cytoplasmic membrane and/or the impairment of enzymatic activity. The bacteriostatic mechanism(s) of action of a biocide is less documented and a primary (unique?) target site within the cell might be involved. Understanding the mechanism(s) of action of a biocide has become an important issue with the emergence of bacterial resistance to biocides and the suggestion that biocide and antibiotic resistance in bacteria might be linked. There is still a lack of understanding of the mode of action of biocides, especially when used at low concentrations (i.e. minimal inhibitory concentration (MIC) or sublethal). Although this information might not be required for highly reactive biocides (e.g. alkylating and oxidizing agents) and biocides used at high concentrations, the use of biocides as preservatives or in products at sublethal concentrations, in which a bacteriostatic rather than a bactericidal activity is achieved, is driving the need to better understand microbial target sites. Understanding the mechanisms of action of biocides serves several purposes: (i) it will help to design antimicrobial formulations with an improved antimicrobial efficacy and (ii) it will ensure the prevention of the emergence of microbial resistance.     

Cellular impermeability and uptake of biocides and antibiotics in gram-negative bacteria

The principal targets for antibacterial agents reside at the cytoplasm and cytoplasmic membrane, damage to other structures often arising from initial events at these loci. The gram-negative bacteria offer a complex barrier system to biocides and antibiotics, regulating, and sometimes preventing, their passage to target regions. Routes of entry differ between hydrophobic and hydrophilic agents, often with a structure dependency; specialized uptake mechanisms are exploited and portage transport can occur for pro-drug antibacterials. Uptake isotherms offer insight into the sorption process and can sometimes shed light on biocide mechanisms of action. The multi-component barrier system of gram-negative bacteria offers opportunities for phenotypic resistance development where partitioning or exclusion minimizes the delivery of an antibacterial agent to the target site. Active efflux processes are recognized as increasingly relevant mechanisms for resistance, potentially offering routes to biocide:antibiotic cross-resistance. These mechanisms may be targeted directly in an attempt to compromise their role in microbial survival.     

Biofilms and Biocides: Are there Implications for Antibiotic Resistance?

Considerable controversy surrounds the use of biocides in an ever increasing range of consumer products and the possibility that their indiscriminate use might reduce biocide effectiveness and alter susceptibilities towards antibiotics. These concerns have been based largely on the isolation of resistant mutants from in vitro monoculture experiments. To date, however the emergence of biocide-resistant strains in-vivo has not been reported and a number of environmental survey studies have failed to associate biocide use with antibiotic resistance. This article gives an overview of the issues as they currently stand and reviews data generated in our laboratory over the last five years where we have used laboratory microcosms of the environment and oral cavity to better understand the possible effects of real-life biocide exposure of these high risk ecosystems. In general, whilst biocide susceptibility changes can be demonstrated in pure culture, especially for E. coli towards triclosan, it has not been possible to reproduce these effects during chronic, sublethal dosing of complex communities. We conclude from this review that whilst the incorporation of antibacterial agents into a widening sphere of personal products may not overtly impact on the patterns of microbial susceptibility observed in the environment, the precautionary principle suggests that the use of biocides should be limited to applications where clear hygienic benefits can be demonstrated.     

The Use of Machine Learning Methodologies to Analyse Antibiotic and Biocide Susceptibility in Staphylococcus aureus

Background

The rise of antibiotic resistance in pathogenic bacteria is a significant problem for the treatment of infectious diseases. Resistance is usually selected by the antibiotic itself; however, biocides might also co-select for resistance to antibiotics. Although resistance to biocides is poorly defined, different in vitro studies have shown that mutants presenting low susceptibility to biocides also have reduced susceptibility to antibiotics. However, studies with natural bacterial isolates are more limited and there are no clear conclusions as to whether the use of biocides results in the development of multidrug resistant bacteria.

Methods

The main goal is to perform an unbiased blind-based evaluation of the relationship between antibiotic and biocide reduced susceptibility in natural isolates of Staphylococcus aureus. One of the largest data sets ever studied comprising 1632 human clinical isolates of S. aureus originated worldwide was analysed. The phenotypic characterization of 13 antibiotics and 4 biocides was performed for all the strains. Complex links between reduced susceptibility to biocides and antibiotics are difficult to elucidate using the standard statistical approaches in phenotypic data. Therefore, machine learning techniques were applied to explore the data.

Results

In this pioneer study, we demonstrated that reduced susceptibility to two common biocides, chlorhexidine and benzalkonium chloride, which belong to different structural families, is associated to multidrug resistance. We have consistently found that a minimum inhibitory concentration greater than 2 mg/L for both biocides is related to antibiotic non-susceptibility in S. aureus.

Conclusions

Two important results emerged from our work, one methodological and one other with relevance in the field of antibiotic resistance. We could not conclude on whether the use of antibiotics selects for biocide resistance or vice versa. However, the observation of association between multiple resistance and two biocides commonly used may be of concern for the treatment of infectious diseases in the future.     

Theoretical aspects of enzyme induction and inhibition leading to the reversal of resistance to biocides

The well-known phenomena of enzyme induction and inhibition have been applied in the enunciation of two mechanisms which could be used in the reversal of resistance which organisms develop towards biocides (drugs and pesticides) in many cases. For those biocides active per se which are metabolized by inducible enzymes to non-toxic metabolites, resistant organisms would be those possessing high levels of drug-metabolizing enzymes (Mechanism 1). For those biocides inactive per se but requiring metabolic activation for activity, resistant organisms would be those possessing low levels of drug metabolizing enzymes (Mechanism 2). In mechanism 1, the addition of enzyme inhibitors to the biocide would be effective in reversing resistance. In mechanism 2 the addition of an enzyme inducer to the biocide would increase the susceptibility of the resistant organisms. An ectoparasite insecticide 2-chloro-1-(2,4 dichlorophenyl) vinyl diethylphosphate (chlorfenvinphos or supona) is used as an example for mechanism 1. The malarial drugs primaquine and chloroquine are used as examples of mechanism 2.     

Comparative analysis of antibiotic and antimicrobial biocide susceptibility data in clinical isolates of methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa between 1989 and 2000

AIMS: To analyse population minimum inhibitory concentrations (MICs) data from clinical strains of Staphylococcus aureus and Pseudomonas aeruginosa for changes over a 10-year period and to look for correlations between the antimicrobials tested. METHODS AND RESULTS: Data from the MIC study of 256 clinical isolates of Staph. aureus [169 methicillin-sensitive Staph. aureus (MSSA), 87 methicillin-resistant Staph. aureus (MRSA)] and 111 clinical isolates of Ps. aeruginosa against eight antimicrobial biocides and several clinically relevant antibiotics was analysed using anova, Spearman-Rho correlation and principal component analysis. Comparisons suggest that alterations in the mean susceptibility of Staph. aureus to antimicrobial biocides have occurred between 1989 and 2000, but that these changes were mirrored in MSSA and MRSA suggests that methicillin resistance has little to do with these changes. Between 1989 and 2000 a sub-population of MRSA has acquired a higher resistance to biocides, but this has not altered the antibiotic susceptibility of that group. In both Staph. aureus and Ps. aeruginosa several correlations (both positive and negative) between antibiotics and antimicrobial biocides were found. CONCLUSIONS: From the analyses of these clinical isolates it is very difficult to support a hypothesis that increased biocide resistance is a cause of increased antibiotic resistance either in Staph. aureus or in Ps. aeruginosa. SIGNIFICANCE AND IMPACT OF THE STUDY: The observation of negative correlations between antibiotics and biocides may be a useful reason for the continued use of biocides promoting hygiene in the hospital environment.     

微生物消毒剂抗性机理

在物体表面和传播介质中,消毒剂能有效抑制或杀死微生物,广泛用于食品、卫生、健康、防疫等领域.在新型冠状病毒肺炎(COVID-19)疫情期间,全球消毒剂的使用量激增,对有效防控病毒传播和防止疫情扩散起到重要作用.但消毒剂的不正确使用会降低其有效性,甚至会诱导微生物产生抗性,从而增加传染性疾病的传播风险.微生物的消毒剂抗性...     

综述与专论：微生物消毒剂抗性机理

在物体表面和传播介质中,消毒剂能有效抑制或杀死微生物,广泛用于食品、卫生、健康、防疫等领域。在新型冠状病毒肺炎(COVID-19)疫情期间,全球消毒剂的使用量激增,对有效防控病毒传播和防止疫情扩散起到重要作用。但消毒剂的不正确使用会降低其有效性,甚至会诱导微生物产生抗性,从而增加传染性疾病的传播风险。微生物的消毒剂抗性基因还会通过繁殖传代增殖或在不同种属间水平转移而加剧其污染和传播风险,严重威胁到公共卫生安全。目前,抗生素抗性基因(ARG)的广泛出现引起了全球对公共卫生的关注,但对消毒剂的抗性认识非常有限。本文综述了近年来微生物对消毒剂抗性的研究,着重就微生物通过形成生物膜、降低细胞膜通透性、过量表达外排泵、产生消除或减弱消毒剂的特异性酶、改变作用靶点等方式产生抗性的机理进行综述。另外针对微生物消毒剂抗性的获得和传播,对染色体和质粒介导的抗性基因、环境中微生物消毒剂抗性与抗生素抗性的关联进行了论述。消毒剂抗性基因能通过质粒、噬菌体等可移动遗传元件,以转化、转导或接合的方式转移传播,对科学消毒提出新要求。     

Introduction of biocides into clinical practice and the impact on antibiotic-resistant bacteria

Biocides and other antimicrobial agents have been employed for centuries. Much later, iodine found use as a wound disinfectant, chlorine water in obstetrics, alcohol as a hand disinfectant and phenol as a wound dressing and in antiseptic surgery. In the early part of the twentieth century, other chlorine-releasing agents (CRAs), and acridine and other dyes were introduced, as were some quaternary ammonium compounds (QACs, although these were only used as biocides from the 1930s). Later still, various phenolics and alcohols, formaldehyde and hydrogen peroxide were introduced and subsequently (although some had actually been produced at an earlier date) biguanides, iodophors, bisphenols, aldehydes, diamidines, isocyanurates, isothiazolones and peracetic acid. Antibiotics were introduced clinically in the 1940s, although sulphonamides had been synthesized and used previously. After penicillin came streptomycin and other aminoglycosides-aminocyclitols, tetracyclines, chloramphenicol, macrolides, semi-synthetic beta-lactams, glycopeptides, lincosamides, 4-quinolones and diaminopyrimidines. Bacterial resistance to antibiotics is causing great concern. Mechanisms of such resistance include cell impermeability, target site mutation, drug inactivation and drug efflux. Bacterial resistance to biocides was described in the 1950s and 1960s and is also apparently increasing. Of the biocides listed above, cationic agents (QACs, chlorhexidine, diamidines, acridines) and triclosan have been implicated as possible causes for the selection and persistence of bacterial strains with low-level antibiotic resistance. It has been claimed that the chronological emergence of qacA and qacB determinants in clinical isolates of Staphylococcus aureus mirrors the introduction and usage of cationic biocides.     

Exposure of Salmonella enterica serovar Typhimurium to high level biocide challenge can select multidrug resistant mutants in a single step

Background

Biocides are crucial to the prevention of infection by bacteria, particularly with the global emergence of multiply antibiotic resistant strains of many species. Concern has been raised regarding the potential for biocide exposure to select for antibiotic resistance due to common mechanisms of resistance, notably efflux.

Methodology/Principal Findings

Salmonella enterica serovar Typhimurium was challenged with 4 biocides of differing modes of action at both low and recommended-use concentration. Flow cytometry was used to investigate the physiological state of the cells after biocide challenge. After 5 hours exposure to biocide, live cells were sorted by FACS and recovered. Cells recovered after an exposure to low concentrations of biocide had antibiotic resistance profiles similar to wild-type cells. Live cells were recovered after exposure to two of the biocides at in-use concentration for 5 hours. These cells were multi-drug resistant and accumulation assays demonstrated an efflux phenotype of these mutants. Gene expression analysis showed that the AcrEF multidrug efflux pump was de-repressed in mutants isolated from high-levels of biocide.

Conclusions/Significance

These data show that a single exposure to the working concentration of certain biocides can select for mutant Salmonella with efflux mediated multidrug resistance and that flow cytometry is a sensitive tool for identifying biocide tolerant mutants. The propensity for biocides to select for MDR mutants varies and this should be a consideration when designing new biocidal formulations.     

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.     

A functional gene array for detection of bacterial virulence elements

Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples.     

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.     

Antimicrobial susceptibility changes and T-OMP shifts in pyrithione-passaged planktonic cultures of Pseudomonas aeruginosa PAO1

AIMS: The aim of this study was to determine whether passaging Pseudomonas aeruginosa PAO1 with sub-MICs of the pyrithione biocides results in both the induction of decreased susceptibility towards these antimicrobials and associated outer membrane profile changes. METHODS AND RESULTS: Previous work by this group has shown that it is possible to induce susceptibility changes towards the isothiazolone biocides in Ps. aeruginosa PAO1 by successive passages in the presence of increasing sub-MICs of biocide. This procedure was accompanied by the loss of a 35 kDa outer membrane protein, T-OMP. In this experiment, this process was repeated with the biocides sodium pyrithione (NaPT), zinc pyrithione (ZnPT) and cetrimide. The pattern of susceptibility was similar to that observed with the isothiazolone biocides. Upon removal of biocide, the observed MIC did not return to the original pre-exposure value. The onset and development of resistance was accompanied by the loss of T-OMP from outer membrane profiles, which suggests that this is a non-specific membrane channel whose production within the cell is sensitive to biocide presence. The T-OMP reappeared when the cells were passaged in the absence of pyrithione. Cross-resistance studies indicated that induced resistance to one biocide yields partial resistance towards other members of the group and the positive control. CONCLUSIONS: These results indicate that the pyrithione biocides have similar susceptibility profiles in Ps. aeruginosa to those exhibited by the isothiazolones, but that the acquired changes in susceptibility to the pyrithiones is largely irreversible. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that acquired susceptibility changes towards sub-MICs of selected biocides are multifactorial in nature.