New insights for rapid evaluation of bactericidal activity: a semi-automated bioluminescent ATP assay

Aims: A new assay, much more rapid and efficient than the existing standardized tests, is introduced for the evaluation of bactericidal activity of chemical disinfectants and antiseptics under simulated practical conditions of use. Methods and Results: The bactericidal activity of biocides was quantified using a novel semi‐automated assay based on the European Norm (EN) standard suspension tests but determining bacterial cell viability by intracellular adenosine tri‐phosphate (ATP) content quantification instead of traditional culture‐based microbiological techniques. The new test was validated by comparison to the standard suspension tests EN 1276 and EN 13727. During the validation, the linearity of the ATP detection system, limit of detection, specificity, sensitivity, relative accuracy and precision (repeatability and reproducibility) were determined. Conclusions: The validation study showed that the new assay evaluates the activity of biocides as well as the EN standard suspension tests, but it allows a large number of test conditions to be efficiently analysed. Significance and Impact of the Study: The new test can therefore be applied to accurately establish the lowest active concentration (MBCs) of disinfectants or antiseptics under simulated practical conditions of use and to compare the susceptibility of a large number of strains and conditions via inactivation curves. This is not possible in any reasonably practicable way with the EN standards considering the time and cost required for each determination.     

Antibacterial activity of soluble pyridinium-type polymers

Cross-linked poly(N-benzyl-4-vinylpyridinium halide) (designated insoluble BVP) was previously reported to capture bacterial cells alive by contact with them. The corresponding linear polymer poly(N-benzyl-4-vinylpyridinium salt) (designated soluble BVP) was found to exhibit antibacterial activity. This soluble pyridinium-type polymer showed strong antibacterial activity against gram-positive bacteria, whereas it was less active against gram-negative bacteria. The antibacterial activity of this cationic, polymeric disinfectant was considerably greater than that of the corresponding monomeric compound and was approximately equal to that of conventional disinfectants such as benzalkonium chloride and chlorohexidine.     

Antibacterial activity of soluble pyridinium-type polymers.

Cross-linked poly(N-benzyl-4-vinylpyridinium halide) (designated insoluble BVP) was previously reported to capture bacterial cells alive by contact with them. The corresponding linear polymer poly(N-benzyl-4-vinylpyridinium salt) (designated soluble BVP) was found to exhibit antibacterial activity. This soluble pyridinium-type polymer showed strong antibacterial activity against gram-positive bacteria, whereas it was less active against gram-negative bacteria. The antibacterial activity of this cationic, polymeric disinfectant was considerably greater than that of the corresponding monomeric compound and was approximately equal to that of conventional disinfectants such as benzalkonium chloride and chlorohexidine.     

Biocide resistance mechanisms

Information about the mechanisms of action and resistance of biocides used as preservatives, sanitizers, and disinfectants continues to accumulate. While biocides are generally not as well-studied as antibiotics, it is becoming clear that bacteria employ the same major resistance strategies against both groups of antibacterial chemicals. The resistance strategies—inhibitor inactivation, target site alteration, and target site exclusion—are described in the context of the presumed mechanisms of action of the biocides using examples drawn from the literature.     

Influence of disinfectants on domestic wastewater treatment plant performance

The inhibition effect of the disinfectants was investigated under laboratory conditions. COD removal, nitrification process and oxygen uptake rate were the observed processes. Disinfectants can be divided into a few groups depending on the present biocides. The results of the experiments showed a significant influence of the disinfectants containing sodium hypochlorite on the activated sludge. Domestos and Savo caused the highest inhibition on the respiration, 99% and 100%, respectively; while Asanox and Clorox had the highest effect on COD removal, 97% and 100%, respectively. Bref duo active, which also contains sodium hypochlorite, caused the lowest inhibition for all observed processes. Disinfectants based on other biocides did not cause significant inhibitions.     

Antimicrobial activity of N-alkoxycarbonylmethyl-N-alkyl-piperidinium chlorides

The aim of the study was to assay antibacterial and antifungal activity of newly synthesised N-alkoxycarbonylmethyl-N-alkyl-piperidinium chlorides. The compounds tested were found to inhibit the growth of some Gram-negative bacteria, Gram-positive strains and some representatives of yeast-type Candida. From microbiological experiments two of the compounds tested, N-dodecyloxycarbonylmethyl-N-methyl-piperidinium chloride (3) and N-dodecyl-N-ethoxycarbonylmethyl-piperidinium chloride (6), emerged as more active than the other compounds. Since the resistance of biofilms to biocides should be noted during the design and testing of new antimicrobial agents therefore, we have analysed antibacterial properties of the most active compounds towards biofilms. Our study focused on strains of Pseudomonas aeruginosa and Staphylococcus aureus that served as main model organisms for the biofilm studies.     

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.     

From Broad-Spectrum Biocides to Quorum Sensing Disruptors and Mussel Repellents: Antifouling Profile of Alkyl Triphenylphosphonium Salts

‘Onium’ compounds, including ammonium and phosphonium salts, have been employed as antiseptics and disinfectants. These cationic biocides have been incorporated into multiple materials, principally to avoid bacterial attachment. In this work, we selected 20 alkyl-triphenylphosphonium salts, differing mainly in the length and functionalization of their alkyl chains, in fulfilment of two main objectives: 1) to provide a comprehensive evaluation of the antifouling profile of these molecules with relevant marine fouling organisms; and 2) to shed new light on their potential applications, beyond their classic use as broad-spectrum biocides. In this regard, we demonstrate for the first time that these compounds are also able to act as non-toxic quorum sensing disruptors in two different bacterial models (Chromobacterium violaceum and Vibrio harveyi) as well as repellents in the mussel Mytilus galloprovincialis. In addition, their inhibitory activity on a fouling-relevant enzymatic model (tyrosinase) is characterized. An analysis of the structure-activity relationships of these compounds for antifouling purposes is provided, which may result useful in the design of targeted antifouling solutions with these molecules. Altogether, the findings reported herein provide a different perspective on the biological activities of phosphonium compounds that is particularly focused on, but, as the reader will realize, is not limited to their use as antifouling agents.     

Interaction of a polymeric biguanide biocide with phospholipid membranes

Differential scanning calorimetry (DSC) and fluorescence polarization methods have been used to study the interactions between phospholipid membranes and a polymeric biocide, poly(hexamethylene biguanide hydrochloride) (PHMB). It was found that PHMB had very little effect on neutral lipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), whereas it greatly reduced the phase transition temperature of phosphatidylglycerol (PG), an acidic lipid found in bacteria. Although the corresponding monomeric biocide had a similar effect on the PG bilayer, the behaviour towards mixed lipid bilayers of PC and PG has been shown to be completely different for the polymeric and monomeric biocides: viz. the former can induce isothermal phase separation into a PHMB-PG complex domain and a PC-enriched domain, whilst the latter cannot. This may account for the great difference in bactericidal activity between them. It is suggested that PHMB interacts primarily with negatively charged species in the membranes, inducing aggregation of acidic lipids in the vicinity of the adsorption site, where higher fluidity and higher permeability are expected. The results have shown that two factors might be crucial in the cidal activity of such types of cationic disinfectants as biguanides: phase separation and interaction with the hydrocarbon interior of the membranes. Polymeric biocides could be particularly effective by virtue of their ability to combine hydrophobic character and multiple charges within a single molecule.     

Susceptibility of Candida spp. clinical isolates to antimycotics and disinfectants

The incidence of candidiasis among immunocompromised patients and emergence of antimycotics resistant strains has increased significantly. The aims of this study were: to examine the in vitro activity of antimycotics and biocides against Candida clinical isolates; to detect cross-resistance of fungi to these preparations and to estimate whether disinfectants applied in hospital areas are active against clinical Candida isolates. In vitro susceptibility of 102 Candida isolates to eight antimycotics was examined by Etest and ATB Fungus. Sensitivity of these strains to four disinfectants and an antiseptic agent was tested according to EN 1275:2005. Amphotericin B, caspofungin and 5-fluorocytosine were the most effective antimycotics against all Candida isolates. Resistance to itraconazole and fluconazole was observed among C. krusei and C. glabrata. The MICs (Minimal Inhibitory Concentrations) for ketoconazole, voriconazole and posaconazole against Candida albicans ranged: 0.003 - >32 μg/ml and one strain was resistant to three agents tested. All analysed Candida strains were sensitive to biocides containing either chlorine, aldehyde, alcohol mixtures, glucoprotamin or chlorhexidine gluconate with isopropanol. Sensitivity to these agents was observed at concentrations lower than those concentrations recommended by manufacturers to achieve proper biocidal activity to those preparations. Our data suggest that these disinfectants can be effectively applied in clinical wards to prevent nosocomial Candida infections.     

Structure-function analyses involving palindromic analogs of tritrypticin suggest autonomy of anti-endotoxin and antibacterial activities

Neutralization of invading pathogens by gene-encoded peptide antibiotics has been suggested to manifest in a variety of different modes. Some of these modes require internalization of the peptide through a pathway that involves LPS-mediated uptake of the peptide antibiotics. Many proline/tryptophan-rich cationic peptides for which this mode has been invoked do, indeed, show LPS (endotoxin) binding. If the mechanism of antibiotic action involves the LPS-mediated pathway, a positive correlation ought to manifest between the binding to LPS, its neutralization, and the bacterial killing. No such correlation was evident based on our studies involving minimal active analogs of tritrypticin. The anti-endotoxin activities of these analogs appear not to relate directly to their antibiotic potential. The two palindromic analogs of tritrypticin, NT7 (RRFPWWW) and CT7 (WWWPFRR), showed comparable antibacterial activities. However, while NT7 exhibited anti-endotoxin activity, CT7 did not. The LPS binding of two tritrypticin analogs correlated with their corresponding structures, but the antibacterial activities did not. Further structure-function analysis indicated specific structural implications of the antibacterial activity at the molecular level. Studies involving designed analogs of NT7 incorporating either rigid or flexible linkers between the specifically distanced hydrophobic and cationic clusters modulate the LPS binding. On the other hand, not knowing the target receptor for antibacterial activity is a drawback since the precise epitope for antibacterial activity is not definable. It is apparent that the anti-endotoxin and antibacterial activities represent two independent functions of tritrypticin, consistent with the emerging multifunctionality in the nature of cathelicidins.     

A review on biocide reduced susceptibility due to plasmid-borne antiseptic-resistant genes—special notes on pharmaceutical environmental isolates

Biocides (antiseptics and disinfectants) are widely used in hospitals and pharmaceutical industries for contamination control. The emergence of reduced susceptibility to biocides is the major concern and this is caused by various factors, among which plasmid-mediated resistance is common. Many publications describe the antibiotic resistance and mechanisms in a clinical setting. However, there are only limited studies available worldwide addressing the molecular mechanisms of biocide resistance in the pharmaceutical sector. In addition, there is a considerable lack of scientific reports regarding minimum inhibitory concentration (MIC) values of typical biocides against pharmaceutical cleanroom environmental isolates. This review analyses the plasmid-mediated resistance in typical pharmaceutical micro-organisms and prevalence of biocide-resistant genes among common clinical and pharmaceutical isolates. This review discusses the MIC values of biocides in pharmaceutical environmental isolates, indicating the importance of the correlation between the presence or absence of biocide-resistant genes and reduced susceptibility of MIC values. This review recommends that pharmaceutical organizations adopt policies and test methodologies to examine the MICs of common cleanroom biocides against the most common types of cleanroom environmental isolates.     

Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm

A new, quick method, using the resazurin dye test as a bacterial respiration indicator, has been developed to assay the antibacterial activity of various substances used as disinfectants against bacterial biofilm growth on clinical devices. Resazurin was used to measure the presence of active biofilm bacteria, after adding disinfectant, in relation to a standard curve generated from inocula in suspension of the same organism used to grow the biofilm. The biofilm was quantified indirectly by measuring the fluorescent, water-soluble resorufin product produced when resazurin is reduced by reactions associated with respiration. Four products used as disinfectants and the biofilm growth of five bacterial species on carriers made of materials commonly found in clinical devices were studied. Under test conditions, chlorhexidine, NaOCl, ethanol, and Perasafe at concentrations of 0.2, 0.01, 350, and 0.16 mg/ml, respectively, all produced 5-log reductions in biofilm cell numbers on the three different carriers. The redox-driven test depends on bacterial catabolism, for which reason resazurin reduction produces an analytic signal of the bacterial activity in whole cells, and therefore could be used for determining disinfectant efficacy in an assay based on the metabolic activity of microorganisms grown as biofilm or in suspension.     

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%).     

Effect of permeabilizing agents on antibacterial activity against a simple Pseudomonas aeruginosa biofilm

A simple Pseudomonas aeruginosa G48 biofilm on stainless steel discs provided a useful primary screen of the potentiating effects of various permeabilizing agents on antibacterial agents. Experiments with Ps. aeruginosa suspensions could not be used to predict the effects of biocides and permeabilizers on biofilms. Although antibacterial activity against biofilms was less than demonstrated in suspension tests, potentiation by some permeabilizers was still observed.     

Synthesis and biological evaluation of novel 1,3,5-triazine derivatives as antimicrobial agents

Numerous studies have contributed to the development of natural and synthetic antimicrobial peptides as a prospective source of antibiotic agents. Based on the concept that cationic charge, bulk, and lipophilicity are major factors determining antibacterial activity in these peptides, we designed and screened several combinatorial libraries based on 1,3,5-triazine as a template. A set of compounds were identified to show potent antimicrobial activity together with low hemolytic activity.     

Cationic polypeptides are required for antibacterial activity of human airway fluid

In a search for direct evidence leading to the biological relevance of airway secretions in innate host defense, we characterized the antibacterial function of cationic polypeptides within minimally manipulated nasal fluid. In this study, we show that cationic antimicrobial polypeptides are responsible for most of the bactericidal activity of whole nasal fluid. The removal of cationic polypeptides using a cation-exchange resin ablated the activity of nasal fluid against Escherichia coli, Listeria monocytogenes, and Pseudomonas aeruginosa. By using a novel proteomic approach, we identified a dozen cationic peptides and proteins within nasal fluid, all of which either are known antimicrobial polypeptides or have other proposed roles in host defense. Of the three most abundant cationic polypeptides in nasal fluid, lysozyme was more effective than either lactoferrin or secretory leukoprotease inhibitor in restoring the antibacterial activity of the cationic polypeptide-depleted fluid against a mucoid cystic fibrosis isolate of P. aeruginosa.     

Phospholipases and cationic peptides inhibit Cryptosporidium parvum sporozoite infectivity by parasiticidal and non-parasiticidal mechanisms

The apicomplexan parasite Cryptosporidium parvum is an important cause of diarrhea in humans and cattle, and it can persistently infect immunocompromised hosts. No consistently effective parasite-specific pharmaceuticals or immunotherapies for control of cryptosporidiosis are presently available. The innate immune system represents the first line of host defense against a range of infectious agents, including parasitic protozoa. Several types of antimicrobial peptides and proteins, collectively referred to herein as biocides, constitute a major effector component of this system. In the present study, we evaluated lactoferrin, lactoferrin hydrolysate, 5 cationic peptides (lactoferricin B, cathelicidin LL37, indolicidin, β-defensin 1, β-defensin 2), lysozyme, and 2 phospholipases (phospholipase A2, and phosphatidylinositol-specific phospholipase C) for anti-cryptosporidial activity. The biocides were evaluated either alone or in combination with 3E2, a monoclonal antibody (MAb) against C. parvum that inhibits sporozoite attachment and invasion. Sporozoite viability and infectivity were used as indices of anti-cryptosporidial activity in vitro. All biocides except lactoferrin had a significant effect on sporozoite viability and infectivity. Lactoferrin hydrolysate and each of the 5 cationic peptides were highly parasiticidal and strongly reduced sporozoite infectivity. While each phospholipase also had parasiticidal activity, it was significantly less than that of lactoferrin hydrolysate and each of the cationic peptides. However, each phospholipase reduced sporozoite infectivity comparably to that observed with lactoferrin hydrolysate and the cationic peptides. Moreover, when 3 of the cationic peptides (cathelicidin LL37, β-defensin 1, and β-defensin 2) were individually combined with MAb 3E2, a significantly greater reduction of sporozoite infectivity was observed over that by 3E2 alone. In contrast, reduction of sporozoite infectivity by a combination of either phospholipase with MAb 3E2 was no greater than that by 3E2 alone. These collective observations suggest that cationic peptides and phospholipases neutralize C. parvum by mechanisms that are predominantly either parasiticidal or non-parasiticidal, respectively.     

The effects of disinfectant foam on microbial biofilms

This investigation examined the effects of common aqueous biocides and disinfectant foams derived from them on Pseudomonas aeruginosa biofilms. Biofilms were grown on stainless steel coupons under standardised conditions in a reactor supplemented with low concentrations of organic matter to simulate conditions prevalent in industrial systems. Five-day-old biofilms formed under ambient conditions with continuous agitation demonstrated a low coefficient of variation (5.809%) amongst viable biofilm bacteria from independent trials. Scanning electron microscopy revealed biofilms on coupons with viable biofilm bacteria observed by confocal microscopy. An aqueous solution of a common foaming agent amine oxide (AO) produced negligible effects on bacterial viability in biofilms (p>0.05). However, significant biofilm inactivation was noted with aqueous solutions of common biocides (peracetic acid, sodium hypochlorite, sodium ethylenediaminetetraacetic acid) with or without AO (p<0.05). Aereation of a mixture of AO with each of these common biocides resulted in significant reductions in the viability of biofilm bacteria (p<0.05). In contrast, limited effects were noted by foam devoid of biocides. A relationship between microbial inactivation and the concentration of biocide in foam (ranging from 0.1-0.5%) and exposure period were noted (p<0.05). Although, lower numbers of viable biofilm bacteria were recovered after treatment with the disinfectant foam than by the cognate aqueous biocide, significant differences between these treatments were not evident (p>0.05). In summary, the studies revealed significant biofilm inactivation by biocidal foam prepared with common biocides. Validation of foam disinfectants in controlled trials at manufacturing sites may facilitate developments for clean in place applications. Advantages of foam disinfectants include reductions in the volumes of biocides for industrial disinfection and in their disposal after use.     

A target theory description of the chemical inactivation of membrane functions

The basic ideas of target theory (currently employed in radiobiology) are applied to describe the chemical inactivation of any biological function arising from the summation of a large number of identical individual contributions. A general equation giving the fractional survival of a biological activity as a function of the mean number of hits received by each target from the inactivating molecules and on the number of individual targets forming the functional assemblies is established. It properly accommodates phenomena as diverse as the decrease by anesthetics of the compound action potential in nerve trunks, the inhibition by protein cross-linking reagents of the active transport of sodium across epithelia and the killing of bacteria by disinfectants. This approach seems able to extract molecular level information from macroscopic data.