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An investigation into the relative resistances of common bacterial pathogens to quaternary ammonium cation disinfectants

An investigation into the relative resistances of common bacterial pathogens to quaternary... BioscienceHorizons Volume 10 2017 10.1093/biohorizons/hzx008 ............................................................................................ ..................................................................... Research article An investigation into the relative resistances of common bacterial pathogens to quaternary ammonium cation disinfectants Gregory Wickham Department of Biological Science, University of Chester, Parkgate Road, Chester CH1 4BJ, United Kingdom *Corresponding author: Department of Biological Science, University of Chester, Parkgate Road, Chester CH1 4BJ, United Kingdom. Email: GregoryWickham@hotmail.co.uk Supervisor: Philip Wood and Chris Davis, Department of Biological Science, University of Chester, Parkgate Road, Chester CH1 4BJ, United Kingdom, p.wood@chester.ac.uk (P.W.)/cj.davis@chester.ac.uk (C.D.) ............................................................................................ ..................................................................... Benzalkonium chloride is a common quaternary ammonium cation-based disinfectant used as an industrial-grade biocide, but little independent work has been undertaken quantifying the concentrations required for sterilization. This study investi- gated relative differences in resistance between common Gram-negative and Gram-positive bacterial pathogens and deter- mined the complete sterilization concentrations for each. A membrane filtration methodology was used to quantify an enriched isolate of deionized water, which was subjected to various concentrations of disinfectant incubated on MacConkey agar. The colony forming units at each concentration were compared to an untreated control. Three main trends, defined as ‘phases of inhibition’, were observed across all isolates studied. Phase I occurred from 0 to 1 mL disinfectant/L water and dis- played a moderate, consistent rate of inhibition. Phase II occurred from 0.1% to 0.4% biocide in solution and was character- ized by a dramatic increase in inhibition and a divergence of inhibition rates for each organism. Phase III occurred from 0.4% biocide in solution onward and was characterized by the gradual decline in rate of inhibition until each organism reached total inhibition. It was found that the Gram-negative group, comprising Escherichia coli and Pseudomonas aeruginosa, was generally more resistant than the Gram-positive group, comprising Enterococcus faecalis and Staphylococcus aureus, p < 0.001, with the individual Gram-negative organisms, having the highest complete sterilization concentrations. It was also observed that a variation in resistance existed between organisms of the same Gram stain group. This resulted in some organisms exhibiting resistances comparable to that of organisms of the opposite group, namely between the E. faecalis and P. aeruginosa, which exhibited no significance difference, p = 0.080. Therefore, a model is proposed in which the Gram stain groups can be generalized as being distinct in terms of intrinsic resistance, but also that the range of resistance exists as a spectrum within each group which can cause a similarity between individual organisms of different groups. Key words: Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, membrane filtration, benzalkonium chloride Submitted on 15 October 2016; editorial decision on 3 July 2017 ............................................................................................ ..................................................................... ............................................................................................... .................................................................. © The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. bacterial resistance to QACs and appear to be remaining Introduction prevalent in current studies in the field. Quaternary ammonium cations (QACs) are an extremely The primary mechanism by which resistance in bacteria potent, versatile family of disinfectants commonly used as develops to QACs is prolonged exposure to sub-inhibitory industrial grade biocides. Biocleanse is one such established concentrations due to intensified selective pressure, which is brand of QAC-based disinfectants and utilizes benzalkonium especially significant throughout exposure during the cellular chloride (BAC) as its active antimicrobial agent. BAC pos- exponential phase (Ibusquiza et al., 2012). This has also been sesses strong biocidal activity against most major forms of shown to contribute to biofilm formation (Ortiz, López, and pathogenic microbes including fungi, encapsulated viruses Martínez-Suárez, 2014), which is noteworthy as bacteria and bacteria. QACs exhibit antimicrobial properties due to a grown in a biofilm can be up to 1000 times more resistant to membrane-active interaction which initiates autolysis and biocides than cultured bacteria (Bastian, Alabouvette, and results in the leakage of intracellular constituents (Ioannou, Siaz-Jimenez, 2009). Hanlon, and Denyer, 2007). The first materials to evacuate Gram-negative bacteria have been shown to possess the cell are usually low molecular weight cytoplasmic bodies higher complete sterilization concentrations than Gram- and later, degradation of heavier components occurs, such as positive bacteria when exposed to BAC, therefore there is a proteins and nucleic acids. This activates autolytic enzymes greater probability of the organism being exposed to a sub- that disintegrate the cytoplasmic membrane causing lysis lethal concentration. Considering this, it is not surprising (McDonnell and Russel, 1999). that Pseudomonas spp. have been shown to quickly develop Traditionally, there was a long-held belief that development resistance to QAC-based compounds in hand sanitizers of bacterial resistance to QACs was extremely unlikely due to (Mousavi et al., 2016). Furthermore, the rate at which this its non-specific target of action (Gerba, 2014). The rationale for resistance develops in P. aeruginosa has been observed to this being that any attempts by bacteria to evolve a structural slow considerably with inhibition of multidrug resistance or biochemical component to subvert the disinfection mechan- efflux pumps (Lomovskaya et al., 2001). ism would often be responded to by a different but equally as The extent of the differences in resistance to BAC between effective membrane bound reaction on account of the amphi- Gram-positive and Gram-negative bacteria remains unquanti- philic properties of the QAC molecule (Ahlström and Edebo, fied and there is no information regarding the complete steriliza- 1998). Contrary to this, clinical reports of increasingly QAC tion concentrations stated in the product testing investigations resistant strains of methicillin-resistant Staphylococcus aureus of Biocleanse . Consequently, this knowledge would better have been on the rise in recent years, particularly in nosocomial guide the decision of determining the most appropriate dilution environments (Minbiole et al., 2016). factor to counteract the development of resistance due to the Bacterial resistance to disinfectants involves not just inacti- use of sub-inhibitory concentrations. Simultaneously, it may vating antimicrobials, or inhibiting their penetration, but also also reduce toxicity risk to individuals and the environment due removing non-endogenous molecules from the cell as quickly to the use of excessively high concentrations. as possible (Heinzel, 1998). In regards to this, the contribu- The objective of this study was to determine the minimum tion of plasmid and chromosome encoded efflux pumps has concentrations of BAC required for complete sterilization of a been increasingly stated in recent years (Blair et al., 2015). number of common bacterial pathogens in vitro, and investigate Complex efflux systems are particularly widespread in Gram- the extent and variation in resistance between organisms of dif- negative bacteria. Most have no known QAC detoxifying ferent Gram stain categories. Escherichia coli and P. aeruginosa properties, but isoforms of the resistance-nodulation-cell div- constituted the Gram-negative members, and Enterococcus fae- ision (RND) superfamily, outer membrane factor lipoprotein calis and S. aureus were used as their Gram-positive counter- family and membrane fusion protein family are transporters parts. The role of these organisms as opportunistic human known to be involved in the detoxification process of a num- pathogens is becoming increasingly relevant as they emerge as ber of compounds, including ionically charged disinfectants members of the next generation of ‘superbugs’ (Skalweit, (Piddock and Webber, 2003). These effluxes are particularly 2008). Therefore, establishing the intrinsic resistance patterns abundant in Escherichia coli, Pseudomonas aeruginosa and and subsequently, quantifying how to ensure sterilization of Salmonella enterica serovar typhi, of which there remain a them, is an important prerequisite to limiting development of number of homologues whose specificities are yet to be estab- resistance. This is particularly the case in nosocomial environ- lished (Poole, 2001). ments, where BAC-based agents are most frequently employed, Other potential explanations for the development of QAC andalsowhereasignificant reservoir of infection for many of resistance in bacteria have been proposed, including surface these pathogens exists. charge alterations and hydrophobicity changes in the local cel- The alternative hypothesis developed for this study is that lular environment, which have been examined in S. enterica Gram-negative bacteria will exhibit greater resistance to disin- serovars enteritidis, typhimurium and virchow (Hilton and fection attempts than Gram-positive bacteria and will demon- Braoudaki, 2005). Despite this, efflux pump overexpression strate a greater complete sterilization concentration. studies have been predominant in more thoroughly explaining ............................................................................................... .................................................................. 2 Bioscience Horizons � Volume 10 2017 Research article ............................................................................................... .................................................................. Disinfectant treatment Methods The Biocleanse concentrate was obtained from Metlab Bacterial strains Supplies (Deeside, UK). The disinfectant concentrations inves- The organisms, E. coli (ATCC 11775), P. aeruginosa (ATCC tigated in this study were produced through sequential serial 9027), E. faecalis (ATCC 19433) and S. aureus (NCTC 6571), dilution of the Biocleanse concentrate, altering the disinfect- were propagated within a maximum recovery diluent (MRD) ant to sterile water ratio in order to obtain the following con- broth at 22°C(±1°C). Where available, all strains were known centrations; 1.2%, 1.1%, 1%, 0.75%, 0.6%, 0.5%, 0.4%, disease-causing human pathogens of a biosafety level of two, 0.25% and 0.1% biocide in solution of concentrate. signifying a potential mild disease causing effect, but difficult to The dilutions were prepared in sterile 15 mL culture tubes contract via aerosolization, thus minimizing risk to the oper- using a calibrated 1000 μL autopipette and disposable 10 mL ator (Emmert, 2013). In regards to the E. coli strain, a patho- pipettes. Once completed, the culture tubes were sealed with genic strain was not obtainable. As a consequence of this, the sterile microbiological lids and stored at 4°C(±3°C) in a cali- type strain ATCC 11775 was chosen instead. brated, thermally monitored refrigerator when not in use. The organisms were supplied as Vitroid discs from the The various disinfectant dilutions (1 mL) were pipetted onto Sigma-Aldrich Corporation (Gillingham, UK). Vitroids are the membrane using a calibrated 1000 μL autopipette. One certified reference material in the form of a water soluble millilitre was chosen due to it being the minimum volume tested matrix, inoculated with a defined quantity of viable microor- that fully covered the membrane without oversaturating the ganisms. Once the discs were fully dissolved within the MRD, plate. This adequately simulated a chemical persistence of the they were agitated at 1500 revolutions per minute using a vor- disinfectant that would be experienced by organisms in an tex mixer for approximately 15 s to prevent bacterial adhesion uncontrolled environmental setting. Drying was encouraged by to tube surfaces. Four standard solutions were prepared from rotating and inclining the plate about a central axis in order to the 100 colony forming unit (CFU) Vitroids , each containing ensure an even distribution of disinfectant throughout the mem- an individual isolated species. This was performed by decant- brane, normalizing disinfectant volume per unit area. Once the ing the enriched MRD into 1 L of sterile deionized water. plates were dry, they were then incubated at 37°C(±1°C) in a Isolates were homogenized vigorously prior to any inocula- cyclic incubator for 24 h (±1h). tion, ensuring an even distribution of organisms throughout the vessel. Observation of colonial growth Upon completion of the incubation cycle, the plates were Membrane filtration observed for colonial growth. E. coli exhibited easily visible, A prerequisite of using Vitroid discs required inoculum red, non-mucoid colonies. P. aeruginosa demonstrated very volumes greater than 10 mL. Using volumes below this minute brown coloured colonies that were easily identified threshold was found to produce inconsistent and unreliable under ultraviolet light due to their fluorescent properties. results. Directly pipetting this amount on an agar plate would E. faecalis appeared similar to E. coli, sharing the same red saturate absorption of the liquid into the agar, and using a colouring, however were observed to be smaller in size. S. aur- volume tolerated for absorption into the agar would not be eus appeared as opaque pale pink colonies. Seven repeats sufficient to fulfil the aforementioned volumetric requirement. were performed and recorded as CFU/10 mL. In order to be A membrane filtration technique was adopted as it allowed a able to make comparisons between the responses of the differ- large inoculum to be used through fixing the organisms onto ent organisms, a percentage CFU was calculated, with 100% a filter which was then placed onto the agar for incubation. being the CFU observed with 0% biocide in solution for each organism. An enriched isolate (10 mL) was filtered through a nitrocel- lulose membrane with a 0.45 nm pore diameter at a pressure of 60 kPa. It was ensured that only one valve was opened at a Statistical analysis time to maintain constant pressure, and closed immediately A Two-Way Analysis of Variance (ANOVA) was used to after the water was fully filtered. determine the extent of significance difference between the The membranes were aseptically removed from the filtra- Gram stain groups and between individual organisms across tion bases and applied to MacConkey agar plates using a gen- the full cohort of concentrations. Significance was defined tle rolling technique. A number of different media able to with a 95% confidence interval (p < 0.05). culture all target organisms at identical incubation parameters were trialled, and MacConkey agar was the most selective Results media trialled that exhibited a complete sterilization profile within 95% confidence intervals to that of a non-selective Gram-positive and Gram-negative cohort nutrient agar control. An uninoculated plate filtered only with 100 mL of sterile water was produced at the beginning and The Gram-negative bacteria were found to be more resistant end of each filtration period as negative controls. to disinfection compared with the Gram-positive bacteria. ............................................................................................... .................................................................. 3 Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. Fig. 1 shows that 0.7% biocide in solution was the lowest with the four organism trendlines appearing generally to concentration investigated that exhibited total inhibition of diverge from one another as concentration progressively Gram-positive bacteria. In comparison, the Gram-negative increased. The third and final region, established at the con- bacteria exhibited total inhibition was observed at 1.1% bio- centrations greater than 0.4% biocide in solution, displayed a cide in solution due to singular colonies being observed on a net increase reduction with increasing disinfectant concentra- minority of plates at 1% biocide in solution. tions, until all organisms reached maximum inhibition. Between 0% biocide in solution and 0.4% biocide in solu- Individual organism cohort tion the most significant concentrations for increasing the observed bacterial inhibition were established, as over 90% Fig. 2 shows that E. coli was more resistant than the P. aerugi- of the total bacterial inhibition was observed for the Gram- nosa up to 1% biocide in solution, although after this P. aeru- positive bacteria and over 60% of the Gram-negative. After ginosa became more resistant, albeit at 1% colony formation, 0.4% biocide in solution, the level of inhibition slowed pro- whereas E. coli was fully inhibited. P. aeruginosa was found gressively until total inhibition was observed for both sets of to be the only organism to exhibit growth at the concentra- organisms. tions greater than 0.75% biocide in solution. Three main phases of inhibition were identified and Enterococcus faecalis was found to be the more resistant appeared ubiquitously across all isolates studied. The first out of the two gram-positive organisms, but still less resistant phase, appearing between 0% biocide in solution and 0.1% than the two Gram-negative organisms. Staphylococcus aur- biocide in solution, appeared as a gradual increasing inhib- eus was least resistant to disinfection out of the entire cohort. ition, with all organism cohorts displaying extremely similar Table 1 shows all statistical comparisons of disinfectant treat- levels of inhibition. The second region, observed from the ment between organisms. concentrations 0.1% biocide in solution to 0.4% biocide in Fig. 3 displays the minimum tested disinfectant concentra- solution, exhibited a trend in which all four organisms exhib- tion in which total inhibition of bacterial pathogens was ited a phase of a pronounced increasing rate of inhibition, Figure 1. Mean percentage maximum colony formation of Gram-positive and Gram-negative bacterial pathogens observed at increasing concentrations of disinfectant. Obtained from data produced by subjecting 100 CFU of filtered isolate to 1 mL of diluted disinfectant and calculating as a percentage of the mean colony formation of a 0% control, ± standard error, n = 14. ............................................................................................... .................................................................. 4 Bioscience Horizons � Volume 10 2017 Research article ............................................................................................... .................................................................. Figure 2. The mean percentage maximum colony formation for each individual organism observed at increasing concentrations of disinfectant. Obtained from data produced by subjecting 100 CFU of filtered isolate to 1 mL and calculating as a percentage of the mean colony formation of a 0% control, ± standard error, n = 7. Table 1. Correlations between disinfectant concentration and organisms using a two-way ANOVA Parameters tested Subjects tested Mean Standard Significance (p) deviation Concentration Gram stain Gram-negative Gram-positive 28.88 39.34 <0.001 Organism Escherichia coli Pseudomonas aeruginosa 32.98 39.52 0.811 Escherichia coli Enterococcus faecalis 30.60 39.59 0.005 Escherichia coli Staphylococcus aureus 28.77 39.36 <0.001 Pseudomonas aeruginosa Enterococcus faecalis 28.99 39.46 0.080 Pseudomonas aeruginosa Staphylococcus aureus 27.15 39.16 <0.001 Enterococcus faecalis Staphylococcus aureus 24.78 38.87 0.037 observed across all repeats. It can be inferred from the table the complete sterilization concentrations of the most resistant that the range between the inhibitory concentrations of the organism, P. aeruginosa, and least resistant organism, S. aur- two Gram-negative organisms was lower than that of the eus, was determined to be 0.6% biocide in solution. It should Gram-positive bacteria. In addition, the total range between be noted however, that trendline interpolation from Figs 1 ............................................................................................... .................................................................. 5 Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. as a result any inhibitory affect may be accountable to a bac- teriostatic effect. This could explain why the inhibition rates at these concentrations were very similar, regardless of organism or Gram group. The magnitude of inhibition increases at Phase II was antici- pated due to the exponential nature of reaction with increasing numbers of molecules per unit area, following Fick’s first law of diffusion (Nikaido, 2000). A wealth of evidence suggests that there are operational limits to the molecular functions of micro- bial detoxification, which may be the cause of the vastly increas- ing rate of inhibition between these concentrations. This has been demonstrated in the E. coli strain HN1157, where the AcrB pump demonstrated an efflux rate that plateaued at −1 −1 approximate 0.02 nmol mg s , indicating that a saturation threshold was met (Nagano and Nikaido, 2008). As a conse- quence of this, efflux binding will become less effective as the active sites become less available to remove the non-endogenous molecules from the cell. At this point only minimal supplemen- tation of an increase in concentration is required to cause cata- strophic detoxification failure and lysis. The gradual decrease of level of inhibition during Phase III indicated that some members of the bacterial population were particularly resilient at resisting detoxification. Bacterial recovery within the MRD broth likely produced a population of organisms at different magnitudes of reactivation, and thus resilience to disinfection. This adequately simulates the vari- ation in wellbeing of wild populations of bacteria. Figure 3. The complete sterilization concentrations of Biocleanse® disinfectant in which total inhibition of bacterial pathogens was observed across all repeats. Obtained from data produced by Differences in resistance by Gram subjecting 100 CFU of filtered isolate to 1 mL of variously diluted stain group disinfectant concentrate, n = 7. Gram-negative bacteria exhibited greater resistance to BAC dis- infection attempts than Gram-positive organisms. The Gram- and 2 indicates that the true minimum complete sterilization positive group experienced a greater, statistically significant, concentration may be somewhat lower than stated in Fig. 3. level of inhibition than the Gram-negative group indicating a difference in physicochemical interaction with the disinfectant. Discussion The Gram-positive bacterial cell wall is composed of mainly peptidoglycan which is easily traversed by the BAC molecule, Phases of inhibition therefore the organism can mount little defence to the invasion It is not possible from this study to offer a definitive explan- of the disinfectant molecules, which have unparalleled access to ation for the results obtained, but several well documented the cell, resulting in disruption and cellular death (Russell et al., characteristics relating to the organisms studied may have 1998). Gram-negative cell walls are comprised of two mem- some involvement in the developments of resistance observed. branes reinforced by the expression of LPS on the cellular sur- Therefore, the following discussion offers a speculative face providing an additional protective property. account of the cellular and molecular characteristics that The twin-membrane structure of Gram-negative bacteria could have a contribution to such observations. enable efflux pumps to effectively clear lipophilic agents migrat- During Phase I, it can be inferred that at extremely low sub- ing across the periplasm through the use of accessory proteins inhibitory concentrations, there are simply not enough BAC transecting the periplasmic space (Franklin and Snow, 2013). molecules present in solution to cause significant bacterial These protein complexes are known as tripartite pumps and inhibition. At these concentrations, the LPS layer or any other are responsible for an increased efflux capacity in Gram- membranous characteristics of the Gram-negative bacteria con- negatives. Conversely, in Gram-positive organisms, the pump fers no additional resistance to the organism than protective terminals are located between the external environment and measures exhibited by Gram-positive organisms. At lower con- internal cell, therefore the migratory capture of exogenous centrations, depolarization of the cell cannot yet be achieved, molecules does not occur, and the pump mechanism can only ............................................................................................... .................................................................. 6 Bioscience Horizons � Volume 10 2017 Research article ............................................................................................... .................................................................. be activated once the disinfectant molecule has penetrated the observed that the lipid II moiety, a precursor molecule cell. This results in fewer molecules being excreted due to a low- involved in peptidoglycan synthesis, can undergo structural er surface area-to-volume ratio within the cell. modifications conferring changes in the cell envelope charge (Münch and Sahl, 2015). This has been observed to result in Differences in resistance by organism alterations in the magnitude of interaction with antimicrobial peptides, which share identical mechanistic and charge char- The high complete sterilization concentration observed in acteristics with BAC (Zasloff, 2002). This may explain the P. aeruginosa may have been a result of less-permeable mem- fundamental difference in resistance observed between the branous characteristics possessed by the organism. It is under- two Gram-positive organisms, especially as peptidoglycan stood that the threshold at which BAC becomes bactericidal plays such a large role in the intrinsic protection of Gram- over bacteriostatic is greater in Pseudomonads, which may positive organisms to exogenous compounds. Additionally, it account for the poorer sterilization response until higher con- may also explain why this difference in resistance was not centrations were met. Such properties contributing to this observed between the Gram-negative organisms, as the pep- includes membrane porins which only facilitate the entry of tidoglycan expression in the twin-membrane system makes up low molecular weight compounds, inhibiting entry of QACs a relatively low proportion of the total cell wall mass. which are typically relatively heavy due to a long alkyl chain (McDonnell and Russel, 1999). BAC has been shown to E. faecalis was unexpectedly found to have no statistical exclude divalent cations from the outer membrane, which P. difference with the P. aeruginosa. More research should be 2+ aeruginosa has in abundance in the form of Mg ions (Tabata undertaken into understanding similarities of interaction with 2+ et al.,2003). This attenuates the protective effect of the Mg biocides between the E. faecalis and P. aeruginosa, since as of ions on forming strong lipopolysaccharide linkages (Russell, yet, a plausible explanation for the similarities of interaction 2001). This may explain why throughout the second half of the has not been widely adopted. Furthermore, this outcome dataset, up to 1% biocide in solution, that P. aeruginosa had resembles the findings of another study, in which resistance consistently lower growth than the E. coli.Itis atthis point against several β-lactamase inhibitors were examined on E. where mechanical factors are believed to have the greatest con- faecalis and P. aeruginosa, which were both found to have tribution and efflux saturation will have reached a maximum. similar magnitudes of susceptibility (Klepser et al., 1997). No significant difference was detected between P. aerugi- Limitations and directions for future study nosa and E. coli cohorts. Studies have shown that many gram-negative organisms contain homologues of the same It is necessary to consider the feasibility of the 37°C incuba- forms of efflux pumps and actively exchange them via hori- tion temperature causing interactions of a magnitude non- zontal gene transfer. In addition, the functionality of foreign comparable with that when employed in clinical or domestic effluxes across different genera have been confirmed, as the settings, which have temperatures typically considered to be archetypical MDR MexA-MexB-OprM and MexC-MexD- around 21°C. Despite this, data obtained from a pilot study OprJ efflux systems in P. aeruginosa are functional and retain indicated that E. faecalis cultured on Slanetz and Bartley their substrate specificity in E. coli (Srikumar et al., 1998). (S&B) agar displayed an identical complete sterilization con- This indicates that some homogeneity between the efflux centration with those incubated on MacConkey. The incuba- mechanisms of P. aeruginosa and E. coli, and possibly similar tion parameters of S&B were far lengthier and warmer than magnitudes of operation. MacConkey, indicating that if temperatures around this range did influence biological activity of the disinfectant, it was neg- The data indicate that there is resistance variation between ligible. The same was observed with E. coli on membrane lac- organisms of the same Gram stain category, as displayed by tose glucuronide agar, which has a shorter, cooler incubation the significant difference between the S. aureus and E. faeca- period than MacConkey agar. lis. Enterococcus faecalis is known to contain an additional component in its peptidoglycan structure in the form of One must consider the possibility that the nutritional environ- lysine-alanine side chains (de Pedro, Blanot, and Vollmer, ment provided by MacConkey agar may select against growth 2008). These repeated sequences of dipeptides have been of certain organisms investigated in this study. Regarding this, observed to modify the surface charge density and structural trial data indicated that the concentration of complete steriliza- organization of the peptidoglycan caused a decrease in sensi- tion of MacConkey agar remained within 95% confidence inter- tivity to cationic compounds (Grare et al., 2010). This may vals, therefore if there was any inhibition of cellular growth, it explain why E. faecalis was observed to have a greater resist- did not significantly affect colony formation. ance than S. aureus, despite both organisms being Gram- It has been recognized in this study that there is a significant positive, as BAC is also cationic and therefore subject to the difference between the average Gram-negative and Gram- same charge-related interactivity predispositions. positive organism, and also that there is a degree of variation Another plausible reason for the high variation of resist- and overlap within and between these groups. However, only ance in the Gram-positive bacteria may be due to differences two organisms of each category were examined in this study, in the membrane structure imparted during peptidoglycan therefore the extent to which this variation is present has not formation in different Gram-positive species, as it has been yet been established. ............................................................................................... .................................................................. 7 Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. In order to confirm if the double-membraned structure, and for their greatly appreciated feedback and supervision during other properties unique to Gram-negative bacteria, confers a the production of this report. significantly greater intrinsic resistance, it is necessary to repeat and increase the scope of this study. Additional organisms that Funding are similarly relevant in human bacterial infection should be investigated in order to better understand the magnitude of Funding for this study was derived from the Faculty of variation that is present between these organisms. Biological Science, University of Chester. Furthermore, it is believed that BAC has a bacteriostatic effect at some lower concentrations. An additional useful met- References ric for determining intrinsic resistance would be to establish the endpoints of the bacteriostatic effect at the point where it Ahlström, B. and Edebo, L. (1998) Hydrolysis of the soft amphiphilic becomes bactericidal for each organism investigated. antimicrobial agent tetradecyl betainate is retarded after binding to In this study, efflux pumps were postulated to be a sig- and killing Salmonella typhimurium, Microbiology (Reading, England), nificant contributor to the intrinsic differences in resistance 144, 2497–2504. observed between organisms. In order to support these Bastian, F., Alabouvette, C. and Siaz-Jimenez, C. (2009) Bacteria and results, future studies should implement nucleic acid-probe free-living amoeba in the Lascaux Cave, Research in Microbiology, techniques for the detection of the genes that encode these 160, 38–40. pumps. Furthermore, inactivation of these genes through site-directed mutagenesis and subsequent evaluation on its Blair, J. M., Webber, M. A., Baylay, A. J. et al. (2015) Molecular mechan- effect on sterilization concentration would provide add- isms of antibiotic resistance, Nature Reviews Biotechnology, 13 (1), itional support to the conjectures made. 42–51. de Pedro, M. A., Blanot, M. A. and Vollmer, W. (2008) Peptidoglycan Conclusions structure and architecture, FEMS Microbiology Reviews, 32 (2), 149–167. In conclusion, the results demonstrate the well-established phe- Emmert, E. A. B. (2013) Biosafety guidelines for handling microorgan- nomenon of increased Gram-negative resistance to biocides, isms in the teaching laboratory: development and rationale, and consequently highlight the increased risk of development of Journal of Microbiology & Biology Education, 14 (1), 78–83. resistance. However, the results also indicate the presence of a spectrum of resistances existing in which Gram-positive bac- Franklin, T. J. and Snow, G. A. (2013) Biochemistry and molecular biol- teria are believed to have an increased range of variants. It is ogy of antimicrobial drug action, in G. A. Snow, ed., Biochemistry feasible that the relative resistances between groups can be gen- and Molecular Biology of Antimicrobial Drug Action, Springer Science eralized as being distinct, but more importantly, that being cate- +Business Media, Heidelberg, p.112. gorized as Gram-positive or Gram-negative may not be the absolute determination of magnitude of resistance as previously Gerba, C. P. (2014) Quaternary ammonium biocides: efficacy in applica- thought. In order to solidify these findings and identify the tion, Applied and Environmental Microbiology, 81 (2), 464–469. extent to which this spectrum exists, study of additional organ- Grare, M., Dibama, H. M., Lafosse, S. et al. (2010) Cationic compounds isms is necessary. with activity against multidrug-resistant bacteria: interest of a new compound compared with two older antiseptics, hexamidine Author biography and chlorhexidine, Clinical Microbiology and Infection, 16 (5), 432–438. Gregory Wickham is a first class honours biology graduate Heinzel, M. (1998) Phenomena of biocide resistance in microorganisms, from the University of Chester. He has a particular interest in International Biodeterioration & Biodegradation, 41, 225–234. microbiology and molecular biotechnology and, since 2015, has worked as a microbiologist at ALS Environmental, the Hilton, A. C. and Braoudaki, M. (2005) Mechanisms of resistance in United Kingdom’s largest independent provider of environ- Salmonella enterica adapted to erythromycin, benzalkonium chlor- mental analysis. He is currently studying for an MSc in med- ide and triclosan, International Journal of Antimicrobial Agents, ical microbiology at the University of Manchester and intends 25 (1), 31–37. to undertake a PhD in molecular microbiology upon the com- Ibusquiza, P. S., Herrera, J. J., Vázquez-Sánchez, D. et al. (2012) A new pletion of his Master’s degree. and efficient method to obtain benzalkonium chloride adapted cells of Listeria monocytogenes, Journal of Microbiological Methods, Acknowledgements 91 (1), 57–61. The author would like to express his sincere gratitude to Ioannou, C. J., Hanlon, G. W. and Denyer, S. P. (2007) Action of disinfect- ALcontrol Laboratories for generously allowing the under- ant quaternary ammonium compounds against Staphylococcus aur- taking of this project, and offer thanks C. Davis and P. Wood eus, Antimicrobial Agents and Chemotherapy, 51 (1), 296–306. ............................................................................................... .................................................................. 8 Bioscience Horizons � Volume 10 2017 Research article ............................................................................................... .................................................................. Klepser, M. E., Marangos, M. N., Zhu, Z. et al. (1997) Comparison of the bac- Ortiz, S., López, V. and Martínez-Suárez, J. V. (2014) The influence of tericidal activities of piperacillin-tazobactam, ticarcillin-clavulanate, subminimal inhibitory concentrations of benzalkonium chloride on and ampicillin-sulbactam against clinical isolates of Bacteroides fragilis, biofilm formation by Listeria monocytogenes, International Journal Enterococcus faecalis, Escherichia coli,and Pseudomonas aeruginosa, of Food Microbiology, 189, 106–112. Antimicrobial Agents and Chemotherapy, 41 (2), 435–439. Piddock, L. J. V. and Webber, M. A. (2003) The importance of efflux Lomovskaya, O., Warren, M. S., Lee, A. et al. (2001) Identification and pumps in bacterial antibiotic resistance, Journal of Antimicrobial characterization of inhibitors of multidrug resistance efflux pumps Chemotherapy, 51 (1), 9–11. in Pseudomonas aeruginosa: novel agents for combination therapy, Poole, K. (2001) Multidrug resistance in Gram-negative bacteria, Antimicrobial Agents & Chemotherapy, 45 (1), 105–116. Current Opinion in Microbiology, 4 (5), 500–508. McDonnell, G. and Russel, D. A. (1999) Antiseptics and disinfectants: activ- Russell, A. D. (2001) Mechanism of bacterial insusceptibility to biocides, ity, action, and resistance, Clinical Microbiology Reviews,12(1),147–179. American Journal of Infection Control, 29 (4), 259–261. Minbiole, K. P. C., Jennings, M. C., Ator, L. E. et al. (2016) From antimicro- Russell, A. D., Tattawasart, U., Maillard, J.-Y. et al. (1998) Possible link bial activity to mechanism of resistance: the multifaceted role of between bacterial resistance and use of antibiotics and biocides, simple quaternary ammonium compounds in bacterial eradication, Antimicrobial Agents and Chemotherapy, 42 (8), 2151. Tetrahedron, 72 (25), 3559–3566. Skalweit,H.M. (2008) β-lactams against emerging ‘superbugs’:pro- Mousavi, Z. E., Condell, O., Fanning, S. et al. (2016) Quaternary ammonium gress and pitfalls, Expert Reviews in Clinical Pharmacology, 1 (4), compounds (QACs) induced inactivation of Pseudomonas spp.: effect 559–571. of material surface, Food and Bioproducts Processing, 98, 71–78. Srikumar, R., Kon, T., Gotoh, N. et al. (1998) Expression of Pseudomonas aer- Münch, D. and Sahl, H.-G. (2015) Structural variations of the cell wall uginosa multidrug efflux pumps MexA-MexB-OprM and MexC-MexD- precursor lipid II in Gram-positive bacteria – impact on binding and OprJ in a multidrug-sensitive Escherichia coli strain, Antimicrobial efficacy of antimicrobial peptides, Biochimica et Biophysica Acta, Agents and Chemotherapy,42(1),65–71. 1848 (11), 3062–3071. Tabata, A., Nagamune, H., Maeda, T. et al. (2003) Correlation between Nagano, K. and Nikaido, H. (2008) Kinetic behavior of the major multi- resistance of Pseudomonas aeruginosa to quaternary ammonium drug efflux pump AcrB of Escherichia coli, Proceedings of the compounds and expression of outer membrane protein OprR, National Academy of Sciences of the United States of America, Antimicrobial Agents and Chemotherapy, 47 (7), 2093–2099. 106 (14), 5854–5858. Zasloff, M. (2002) Antimicrobial peptides of multicellular organisms, Nikaido, H. (2000) Crossing the envelope: how cephalosporins reach Nature, 415, 389–395. their targets, Clinical Microbiology and Infection, 6 (3), 22–26. ............................................................................................... .................................................................. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

An investigation into the relative resistances of common bacterial pathogens to quaternary ammonium cation disinfectants

Bioscience Horizons , Volume 10 – Jul 28, 2017

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BioscienceHorizons Volume 10 2017 10.1093/biohorizons/hzx008 ............................................................................................ ..................................................................... Research article An investigation into the relative resistances of common bacterial pathogens to quaternary ammonium cation disinfectants Gregory Wickham Department of Biological Science, University of Chester, Parkgate Road, Chester CH1 4BJ, United Kingdom *Corresponding author: Department of Biological Science, University of Chester, Parkgate Road, Chester CH1 4BJ, United Kingdom. Email: GregoryWickham@hotmail.co.uk Supervisor: Philip Wood and Chris Davis, Department of Biological Science, University of Chester, Parkgate Road, Chester CH1 4BJ, United Kingdom, p.wood@chester.ac.uk (P.W.)/cj.davis@chester.ac.uk (C.D.) ............................................................................................ ..................................................................... Benzalkonium chloride is a common quaternary ammonium cation-based disinfectant used as an industrial-grade biocide, but little independent work has been undertaken quantifying the concentrations required for sterilization. This study investi- gated relative differences in resistance between common Gram-negative and Gram-positive bacterial pathogens and deter- mined the complete sterilization concentrations for each. A membrane filtration methodology was used to quantify an enriched isolate of deionized water, which was subjected to various concentrations of disinfectant incubated on MacConkey agar. The colony forming units at each concentration were compared to an untreated control. Three main trends, defined as ‘phases of inhibition’, were observed across all isolates studied. Phase I occurred from 0 to 1 mL disinfectant/L water and dis- played a moderate, consistent rate of inhibition. Phase II occurred from 0.1% to 0.4% biocide in solution and was character- ized by a dramatic increase in inhibition and a divergence of inhibition rates for each organism. Phase III occurred from 0.4% biocide in solution onward and was characterized by the gradual decline in rate of inhibition until each organism reached total inhibition. It was found that the Gram-negative group, comprising Escherichia coli and Pseudomonas aeruginosa, was generally more resistant than the Gram-positive group, comprising Enterococcus faecalis and Staphylococcus aureus, p < 0.001, with the individual Gram-negative organisms, having the highest complete sterilization concentrations. It was also observed that a variation in resistance existed between organisms of the same Gram stain group. This resulted in some organisms exhibiting resistances comparable to that of organisms of the opposite group, namely between the E. faecalis and P. aeruginosa, which exhibited no significance difference, p = 0.080. Therefore, a model is proposed in which the Gram stain groups can be generalized as being distinct in terms of intrinsic resistance, but also that the range of resistance exists as a spectrum within each group which can cause a similarity between individual organisms of different groups. Key words: Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, membrane filtration, benzalkonium chloride Submitted on 15 October 2016; editorial decision on 3 July 2017 ............................................................................................ ..................................................................... ............................................................................................... .................................................................. © The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. bacterial resistance to QACs and appear to be remaining Introduction prevalent in current studies in the field. Quaternary ammonium cations (QACs) are an extremely The primary mechanism by which resistance in bacteria potent, versatile family of disinfectants commonly used as develops to QACs is prolonged exposure to sub-inhibitory industrial grade biocides. Biocleanse is one such established concentrations due to intensified selective pressure, which is brand of QAC-based disinfectants and utilizes benzalkonium especially significant throughout exposure during the cellular chloride (BAC) as its active antimicrobial agent. BAC pos- exponential phase (Ibusquiza et al., 2012). This has also been sesses strong biocidal activity against most major forms of shown to contribute to biofilm formation (Ortiz, López, and pathogenic microbes including fungi, encapsulated viruses Martínez-Suárez, 2014), which is noteworthy as bacteria and bacteria. QACs exhibit antimicrobial properties due to a grown in a biofilm can be up to 1000 times more resistant to membrane-active interaction which initiates autolysis and biocides than cultured bacteria (Bastian, Alabouvette, and results in the leakage of intracellular constituents (Ioannou, Siaz-Jimenez, 2009). Hanlon, and Denyer, 2007). The first materials to evacuate Gram-negative bacteria have been shown to possess the cell are usually low molecular weight cytoplasmic bodies higher complete sterilization concentrations than Gram- and later, degradation of heavier components occurs, such as positive bacteria when exposed to BAC, therefore there is a proteins and nucleic acids. This activates autolytic enzymes greater probability of the organism being exposed to a sub- that disintegrate the cytoplasmic membrane causing lysis lethal concentration. Considering this, it is not surprising (McDonnell and Russel, 1999). that Pseudomonas spp. have been shown to quickly develop Traditionally, there was a long-held belief that development resistance to QAC-based compounds in hand sanitizers of bacterial resistance to QACs was extremely unlikely due to (Mousavi et al., 2016). Furthermore, the rate at which this its non-specific target of action (Gerba, 2014). The rationale for resistance develops in P. aeruginosa has been observed to this being that any attempts by bacteria to evolve a structural slow considerably with inhibition of multidrug resistance or biochemical component to subvert the disinfection mechan- efflux pumps (Lomovskaya et al., 2001). ism would often be responded to by a different but equally as The extent of the differences in resistance to BAC between effective membrane bound reaction on account of the amphi- Gram-positive and Gram-negative bacteria remains unquanti- philic properties of the QAC molecule (Ahlström and Edebo, fied and there is no information regarding the complete steriliza- 1998). Contrary to this, clinical reports of increasingly QAC tion concentrations stated in the product testing investigations resistant strains of methicillin-resistant Staphylococcus aureus of Biocleanse . Consequently, this knowledge would better have been on the rise in recent years, particularly in nosocomial guide the decision of determining the most appropriate dilution environments (Minbiole et al., 2016). factor to counteract the development of resistance due to the Bacterial resistance to disinfectants involves not just inacti- use of sub-inhibitory concentrations. Simultaneously, it may vating antimicrobials, or inhibiting their penetration, but also also reduce toxicity risk to individuals and the environment due removing non-endogenous molecules from the cell as quickly to the use of excessively high concentrations. as possible (Heinzel, 1998). In regards to this, the contribu- The objective of this study was to determine the minimum tion of plasmid and chromosome encoded efflux pumps has concentrations of BAC required for complete sterilization of a been increasingly stated in recent years (Blair et al., 2015). number of common bacterial pathogens in vitro, and investigate Complex efflux systems are particularly widespread in Gram- the extent and variation in resistance between organisms of dif- negative bacteria. Most have no known QAC detoxifying ferent Gram stain categories. Escherichia coli and P. aeruginosa properties, but isoforms of the resistance-nodulation-cell div- constituted the Gram-negative members, and Enterococcus fae- ision (RND) superfamily, outer membrane factor lipoprotein calis and S. aureus were used as their Gram-positive counter- family and membrane fusion protein family are transporters parts. The role of these organisms as opportunistic human known to be involved in the detoxification process of a num- pathogens is becoming increasingly relevant as they emerge as ber of compounds, including ionically charged disinfectants members of the next generation of ‘superbugs’ (Skalweit, (Piddock and Webber, 2003). These effluxes are particularly 2008). Therefore, establishing the intrinsic resistance patterns abundant in Escherichia coli, Pseudomonas aeruginosa and and subsequently, quantifying how to ensure sterilization of Salmonella enterica serovar typhi, of which there remain a them, is an important prerequisite to limiting development of number of homologues whose specificities are yet to be estab- resistance. This is particularly the case in nosocomial environ- lished (Poole, 2001). ments, where BAC-based agents are most frequently employed, Other potential explanations for the development of QAC andalsowhereasignificant reservoir of infection for many of resistance in bacteria have been proposed, including surface these pathogens exists. charge alterations and hydrophobicity changes in the local cel- The alternative hypothesis developed for this study is that lular environment, which have been examined in S. enterica Gram-negative bacteria will exhibit greater resistance to disin- serovars enteritidis, typhimurium and virchow (Hilton and fection attempts than Gram-positive bacteria and will demon- Braoudaki, 2005). Despite this, efflux pump overexpression strate a greater complete sterilization concentration. studies have been predominant in more thoroughly explaining ............................................................................................... .................................................................. 2 Bioscience Horizons � Volume 10 2017 Research article ............................................................................................... .................................................................. Disinfectant treatment Methods The Biocleanse concentrate was obtained from Metlab Bacterial strains Supplies (Deeside, UK). The disinfectant concentrations inves- The organisms, E. coli (ATCC 11775), P. aeruginosa (ATCC tigated in this study were produced through sequential serial 9027), E. faecalis (ATCC 19433) and S. aureus (NCTC 6571), dilution of the Biocleanse concentrate, altering the disinfect- were propagated within a maximum recovery diluent (MRD) ant to sterile water ratio in order to obtain the following con- broth at 22°C(±1°C). Where available, all strains were known centrations; 1.2%, 1.1%, 1%, 0.75%, 0.6%, 0.5%, 0.4%, disease-causing human pathogens of a biosafety level of two, 0.25% and 0.1% biocide in solution of concentrate. signifying a potential mild disease causing effect, but difficult to The dilutions were prepared in sterile 15 mL culture tubes contract via aerosolization, thus minimizing risk to the oper- using a calibrated 1000 μL autopipette and disposable 10 mL ator (Emmert, 2013). In regards to the E. coli strain, a patho- pipettes. Once completed, the culture tubes were sealed with genic strain was not obtainable. As a consequence of this, the sterile microbiological lids and stored at 4°C(±3°C) in a cali- type strain ATCC 11775 was chosen instead. brated, thermally monitored refrigerator when not in use. The organisms were supplied as Vitroid discs from the The various disinfectant dilutions (1 mL) were pipetted onto Sigma-Aldrich Corporation (Gillingham, UK). Vitroids are the membrane using a calibrated 1000 μL autopipette. One certified reference material in the form of a water soluble millilitre was chosen due to it being the minimum volume tested matrix, inoculated with a defined quantity of viable microor- that fully covered the membrane without oversaturating the ganisms. Once the discs were fully dissolved within the MRD, plate. This adequately simulated a chemical persistence of the they were agitated at 1500 revolutions per minute using a vor- disinfectant that would be experienced by organisms in an tex mixer for approximately 15 s to prevent bacterial adhesion uncontrolled environmental setting. Drying was encouraged by to tube surfaces. Four standard solutions were prepared from rotating and inclining the plate about a central axis in order to the 100 colony forming unit (CFU) Vitroids , each containing ensure an even distribution of disinfectant throughout the mem- an individual isolated species. This was performed by decant- brane, normalizing disinfectant volume per unit area. Once the ing the enriched MRD into 1 L of sterile deionized water. plates were dry, they were then incubated at 37°C(±1°C) in a Isolates were homogenized vigorously prior to any inocula- cyclic incubator for 24 h (±1h). tion, ensuring an even distribution of organisms throughout the vessel. Observation of colonial growth Upon completion of the incubation cycle, the plates were Membrane filtration observed for colonial growth. E. coli exhibited easily visible, A prerequisite of using Vitroid discs required inoculum red, non-mucoid colonies. P. aeruginosa demonstrated very volumes greater than 10 mL. Using volumes below this minute brown coloured colonies that were easily identified threshold was found to produce inconsistent and unreliable under ultraviolet light due to their fluorescent properties. results. Directly pipetting this amount on an agar plate would E. faecalis appeared similar to E. coli, sharing the same red saturate absorption of the liquid into the agar, and using a colouring, however were observed to be smaller in size. S. aur- volume tolerated for absorption into the agar would not be eus appeared as opaque pale pink colonies. Seven repeats sufficient to fulfil the aforementioned volumetric requirement. were performed and recorded as CFU/10 mL. In order to be A membrane filtration technique was adopted as it allowed a able to make comparisons between the responses of the differ- large inoculum to be used through fixing the organisms onto ent organisms, a percentage CFU was calculated, with 100% a filter which was then placed onto the agar for incubation. being the CFU observed with 0% biocide in solution for each organism. An enriched isolate (10 mL) was filtered through a nitrocel- lulose membrane with a 0.45 nm pore diameter at a pressure of 60 kPa. It was ensured that only one valve was opened at a Statistical analysis time to maintain constant pressure, and closed immediately A Two-Way Analysis of Variance (ANOVA) was used to after the water was fully filtered. determine the extent of significance difference between the The membranes were aseptically removed from the filtra- Gram stain groups and between individual organisms across tion bases and applied to MacConkey agar plates using a gen- the full cohort of concentrations. Significance was defined tle rolling technique. A number of different media able to with a 95% confidence interval (p < 0.05). culture all target organisms at identical incubation parameters were trialled, and MacConkey agar was the most selective Results media trialled that exhibited a complete sterilization profile within 95% confidence intervals to that of a non-selective Gram-positive and Gram-negative cohort nutrient agar control. An uninoculated plate filtered only with 100 mL of sterile water was produced at the beginning and The Gram-negative bacteria were found to be more resistant end of each filtration period as negative controls. to disinfection compared with the Gram-positive bacteria. ............................................................................................... .................................................................. 3 Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. Fig. 1 shows that 0.7% biocide in solution was the lowest with the four organism trendlines appearing generally to concentration investigated that exhibited total inhibition of diverge from one another as concentration progressively Gram-positive bacteria. In comparison, the Gram-negative increased. The third and final region, established at the con- bacteria exhibited total inhibition was observed at 1.1% bio- centrations greater than 0.4% biocide in solution, displayed a cide in solution due to singular colonies being observed on a net increase reduction with increasing disinfectant concentra- minority of plates at 1% biocide in solution. tions, until all organisms reached maximum inhibition. Between 0% biocide in solution and 0.4% biocide in solu- Individual organism cohort tion the most significant concentrations for increasing the observed bacterial inhibition were established, as over 90% Fig. 2 shows that E. coli was more resistant than the P. aerugi- of the total bacterial inhibition was observed for the Gram- nosa up to 1% biocide in solution, although after this P. aeru- positive bacteria and over 60% of the Gram-negative. After ginosa became more resistant, albeit at 1% colony formation, 0.4% biocide in solution, the level of inhibition slowed pro- whereas E. coli was fully inhibited. P. aeruginosa was found gressively until total inhibition was observed for both sets of to be the only organism to exhibit growth at the concentra- organisms. tions greater than 0.75% biocide in solution. Three main phases of inhibition were identified and Enterococcus faecalis was found to be the more resistant appeared ubiquitously across all isolates studied. The first out of the two gram-positive organisms, but still less resistant phase, appearing between 0% biocide in solution and 0.1% than the two Gram-negative organisms. Staphylococcus aur- biocide in solution, appeared as a gradual increasing inhib- eus was least resistant to disinfection out of the entire cohort. ition, with all organism cohorts displaying extremely similar Table 1 shows all statistical comparisons of disinfectant treat- levels of inhibition. The second region, observed from the ment between organisms. concentrations 0.1% biocide in solution to 0.4% biocide in Fig. 3 displays the minimum tested disinfectant concentra- solution, exhibited a trend in which all four organisms exhib- tion in which total inhibition of bacterial pathogens was ited a phase of a pronounced increasing rate of inhibition, Figure 1. Mean percentage maximum colony formation of Gram-positive and Gram-negative bacterial pathogens observed at increasing concentrations of disinfectant. Obtained from data produced by subjecting 100 CFU of filtered isolate to 1 mL of diluted disinfectant and calculating as a percentage of the mean colony formation of a 0% control, ± standard error, n = 14. ............................................................................................... .................................................................. 4 Bioscience Horizons � Volume 10 2017 Research article ............................................................................................... .................................................................. Figure 2. The mean percentage maximum colony formation for each individual organism observed at increasing concentrations of disinfectant. Obtained from data produced by subjecting 100 CFU of filtered isolate to 1 mL and calculating as a percentage of the mean colony formation of a 0% control, ± standard error, n = 7. Table 1. Correlations between disinfectant concentration and organisms using a two-way ANOVA Parameters tested Subjects tested Mean Standard Significance (p) deviation Concentration Gram stain Gram-negative Gram-positive 28.88 39.34 <0.001 Organism Escherichia coli Pseudomonas aeruginosa 32.98 39.52 0.811 Escherichia coli Enterococcus faecalis 30.60 39.59 0.005 Escherichia coli Staphylococcus aureus 28.77 39.36 <0.001 Pseudomonas aeruginosa Enterococcus faecalis 28.99 39.46 0.080 Pseudomonas aeruginosa Staphylococcus aureus 27.15 39.16 <0.001 Enterococcus faecalis Staphylococcus aureus 24.78 38.87 0.037 observed across all repeats. It can be inferred from the table the complete sterilization concentrations of the most resistant that the range between the inhibitory concentrations of the organism, P. aeruginosa, and least resistant organism, S. aur- two Gram-negative organisms was lower than that of the eus, was determined to be 0.6% biocide in solution. It should Gram-positive bacteria. In addition, the total range between be noted however, that trendline interpolation from Figs 1 ............................................................................................... .................................................................. 5 Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. as a result any inhibitory affect may be accountable to a bac- teriostatic effect. This could explain why the inhibition rates at these concentrations were very similar, regardless of organism or Gram group. The magnitude of inhibition increases at Phase II was antici- pated due to the exponential nature of reaction with increasing numbers of molecules per unit area, following Fick’s first law of diffusion (Nikaido, 2000). A wealth of evidence suggests that there are operational limits to the molecular functions of micro- bial detoxification, which may be the cause of the vastly increas- ing rate of inhibition between these concentrations. This has been demonstrated in the E. coli strain HN1157, where the AcrB pump demonstrated an efflux rate that plateaued at −1 −1 approximate 0.02 nmol mg s , indicating that a saturation threshold was met (Nagano and Nikaido, 2008). As a conse- quence of this, efflux binding will become less effective as the active sites become less available to remove the non-endogenous molecules from the cell. At this point only minimal supplemen- tation of an increase in concentration is required to cause cata- strophic detoxification failure and lysis. The gradual decrease of level of inhibition during Phase III indicated that some members of the bacterial population were particularly resilient at resisting detoxification. Bacterial recovery within the MRD broth likely produced a population of organisms at different magnitudes of reactivation, and thus resilience to disinfection. This adequately simulates the vari- ation in wellbeing of wild populations of bacteria. Figure 3. The complete sterilization concentrations of Biocleanse® disinfectant in which total inhibition of bacterial pathogens was observed across all repeats. Obtained from data produced by Differences in resistance by Gram subjecting 100 CFU of filtered isolate to 1 mL of variously diluted stain group disinfectant concentrate, n = 7. Gram-negative bacteria exhibited greater resistance to BAC dis- infection attempts than Gram-positive organisms. The Gram- and 2 indicates that the true minimum complete sterilization positive group experienced a greater, statistically significant, concentration may be somewhat lower than stated in Fig. 3. level of inhibition than the Gram-negative group indicating a difference in physicochemical interaction with the disinfectant. Discussion The Gram-positive bacterial cell wall is composed of mainly peptidoglycan which is easily traversed by the BAC molecule, Phases of inhibition therefore the organism can mount little defence to the invasion It is not possible from this study to offer a definitive explan- of the disinfectant molecules, which have unparalleled access to ation for the results obtained, but several well documented the cell, resulting in disruption and cellular death (Russell et al., characteristics relating to the organisms studied may have 1998). Gram-negative cell walls are comprised of two mem- some involvement in the developments of resistance observed. branes reinforced by the expression of LPS on the cellular sur- Therefore, the following discussion offers a speculative face providing an additional protective property. account of the cellular and molecular characteristics that The twin-membrane structure of Gram-negative bacteria could have a contribution to such observations. enable efflux pumps to effectively clear lipophilic agents migrat- During Phase I, it can be inferred that at extremely low sub- ing across the periplasm through the use of accessory proteins inhibitory concentrations, there are simply not enough BAC transecting the periplasmic space (Franklin and Snow, 2013). molecules present in solution to cause significant bacterial These protein complexes are known as tripartite pumps and inhibition. At these concentrations, the LPS layer or any other are responsible for an increased efflux capacity in Gram- membranous characteristics of the Gram-negative bacteria con- negatives. Conversely, in Gram-positive organisms, the pump fers no additional resistance to the organism than protective terminals are located between the external environment and measures exhibited by Gram-positive organisms. At lower con- internal cell, therefore the migratory capture of exogenous centrations, depolarization of the cell cannot yet be achieved, molecules does not occur, and the pump mechanism can only ............................................................................................... .................................................................. 6 Bioscience Horizons � Volume 10 2017 Research article ............................................................................................... .................................................................. be activated once the disinfectant molecule has penetrated the observed that the lipid II moiety, a precursor molecule cell. This results in fewer molecules being excreted due to a low- involved in peptidoglycan synthesis, can undergo structural er surface area-to-volume ratio within the cell. modifications conferring changes in the cell envelope charge (Münch and Sahl, 2015). This has been observed to result in Differences in resistance by organism alterations in the magnitude of interaction with antimicrobial peptides, which share identical mechanistic and charge char- The high complete sterilization concentration observed in acteristics with BAC (Zasloff, 2002). This may explain the P. aeruginosa may have been a result of less-permeable mem- fundamental difference in resistance observed between the branous characteristics possessed by the organism. It is under- two Gram-positive organisms, especially as peptidoglycan stood that the threshold at which BAC becomes bactericidal plays such a large role in the intrinsic protection of Gram- over bacteriostatic is greater in Pseudomonads, which may positive organisms to exogenous compounds. Additionally, it account for the poorer sterilization response until higher con- may also explain why this difference in resistance was not centrations were met. Such properties contributing to this observed between the Gram-negative organisms, as the pep- includes membrane porins which only facilitate the entry of tidoglycan expression in the twin-membrane system makes up low molecular weight compounds, inhibiting entry of QACs a relatively low proportion of the total cell wall mass. which are typically relatively heavy due to a long alkyl chain (McDonnell and Russel, 1999). BAC has been shown to E. faecalis was unexpectedly found to have no statistical exclude divalent cations from the outer membrane, which P. difference with the P. aeruginosa. More research should be 2+ aeruginosa has in abundance in the form of Mg ions (Tabata undertaken into understanding similarities of interaction with 2+ et al.,2003). This attenuates the protective effect of the Mg biocides between the E. faecalis and P. aeruginosa, since as of ions on forming strong lipopolysaccharide linkages (Russell, yet, a plausible explanation for the similarities of interaction 2001). This may explain why throughout the second half of the has not been widely adopted. Furthermore, this outcome dataset, up to 1% biocide in solution, that P. aeruginosa had resembles the findings of another study, in which resistance consistently lower growth than the E. coli.Itis atthis point against several β-lactamase inhibitors were examined on E. where mechanical factors are believed to have the greatest con- faecalis and P. aeruginosa, which were both found to have tribution and efflux saturation will have reached a maximum. similar magnitudes of susceptibility (Klepser et al., 1997). No significant difference was detected between P. aerugi- Limitations and directions for future study nosa and E. coli cohorts. Studies have shown that many gram-negative organisms contain homologues of the same It is necessary to consider the feasibility of the 37°C incuba- forms of efflux pumps and actively exchange them via hori- tion temperature causing interactions of a magnitude non- zontal gene transfer. In addition, the functionality of foreign comparable with that when employed in clinical or domestic effluxes across different genera have been confirmed, as the settings, which have temperatures typically considered to be archetypical MDR MexA-MexB-OprM and MexC-MexD- around 21°C. Despite this, data obtained from a pilot study OprJ efflux systems in P. aeruginosa are functional and retain indicated that E. faecalis cultured on Slanetz and Bartley their substrate specificity in E. coli (Srikumar et al., 1998). (S&B) agar displayed an identical complete sterilization con- This indicates that some homogeneity between the efflux centration with those incubated on MacConkey. The incuba- mechanisms of P. aeruginosa and E. coli, and possibly similar tion parameters of S&B were far lengthier and warmer than magnitudes of operation. MacConkey, indicating that if temperatures around this range did influence biological activity of the disinfectant, it was neg- The data indicate that there is resistance variation between ligible. The same was observed with E. coli on membrane lac- organisms of the same Gram stain category, as displayed by tose glucuronide agar, which has a shorter, cooler incubation the significant difference between the S. aureus and E. faeca- period than MacConkey agar. lis. Enterococcus faecalis is known to contain an additional component in its peptidoglycan structure in the form of One must consider the possibility that the nutritional environ- lysine-alanine side chains (de Pedro, Blanot, and Vollmer, ment provided by MacConkey agar may select against growth 2008). These repeated sequences of dipeptides have been of certain organisms investigated in this study. Regarding this, observed to modify the surface charge density and structural trial data indicated that the concentration of complete steriliza- organization of the peptidoglycan caused a decrease in sensi- tion of MacConkey agar remained within 95% confidence inter- tivity to cationic compounds (Grare et al., 2010). This may vals, therefore if there was any inhibition of cellular growth, it explain why E. faecalis was observed to have a greater resist- did not significantly affect colony formation. ance than S. aureus, despite both organisms being Gram- It has been recognized in this study that there is a significant positive, as BAC is also cationic and therefore subject to the difference between the average Gram-negative and Gram- same charge-related interactivity predispositions. positive organism, and also that there is a degree of variation Another plausible reason for the high variation of resist- and overlap within and between these groups. However, only ance in the Gram-positive bacteria may be due to differences two organisms of each category were examined in this study, in the membrane structure imparted during peptidoglycan therefore the extent to which this variation is present has not formation in different Gram-positive species, as it has been yet been established. ............................................................................................... .................................................................. 7 Research article Bioscience Horizons � Volume 10 2017 ............................................................................................... .................................................................. In order to confirm if the double-membraned structure, and for their greatly appreciated feedback and supervision during other properties unique to Gram-negative bacteria, confers a the production of this report. significantly greater intrinsic resistance, it is necessary to repeat and increase the scope of this study. Additional organisms that Funding are similarly relevant in human bacterial infection should be investigated in order to better understand the magnitude of Funding for this study was derived from the Faculty of variation that is present between these organisms. Biological Science, University of Chester. Furthermore, it is believed that BAC has a bacteriostatic effect at some lower concentrations. An additional useful met- References ric for determining intrinsic resistance would be to establish the endpoints of the bacteriostatic effect at the point where it Ahlström, B. and Edebo, L. (1998) Hydrolysis of the soft amphiphilic becomes bactericidal for each organism investigated. antimicrobial agent tetradecyl betainate is retarded after binding to In this study, efflux pumps were postulated to be a sig- and killing Salmonella typhimurium, Microbiology (Reading, England), nificant contributor to the intrinsic differences in resistance 144, 2497–2504. observed between organisms. In order to support these Bastian, F., Alabouvette, C. and Siaz-Jimenez, C. (2009) Bacteria and results, future studies should implement nucleic acid-probe free-living amoeba in the Lascaux Cave, Research in Microbiology, techniques for the detection of the genes that encode these 160, 38–40. pumps. Furthermore, inactivation of these genes through site-directed mutagenesis and subsequent evaluation on its Blair, J. M., Webber, M. A., Baylay, A. J. et al. 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It is ogy of antimicrobial drug action, in G. A. Snow, ed., Biochemistry feasible that the relative resistances between groups can be gen- and Molecular Biology of Antimicrobial Drug Action, Springer Science eralized as being distinct, but more importantly, that being cate- +Business Media, Heidelberg, p.112. gorized as Gram-positive or Gram-negative may not be the absolute determination of magnitude of resistance as previously Gerba, C. P. (2014) Quaternary ammonium biocides: efficacy in applica- thought. In order to solidify these findings and identify the tion, Applied and Environmental Microbiology, 81 (2), 464–469. extent to which this spectrum exists, study of additional organ- Grare, M., Dibama, H. M., Lafosse, S. et al. (2010) Cationic compounds isms is necessary. with activity against multidrug-resistant bacteria: interest of a new compound compared with two older antiseptics, hexamidine Author biography and chlorhexidine, Clinical Microbiology and Infection, 16 (5), 432–438. Gregory Wickham is a first class honours biology graduate Heinzel, M. (1998) Phenomena of biocide resistance in microorganisms, from the University of Chester. He has a particular interest in International Biodeterioration & Biodegradation, 41, 225–234. microbiology and molecular biotechnology and, since 2015, has worked as a microbiologist at ALS Environmental, the Hilton, A. C. and Braoudaki, M. (2005) Mechanisms of resistance in United Kingdom’s largest independent provider of environ- Salmonella enterica adapted to erythromycin, benzalkonium chlor- mental analysis. He is currently studying for an MSc in med- ide and triclosan, International Journal of Antimicrobial Agents, ical microbiology at the University of Manchester and intends 25 (1), 31–37. to undertake a PhD in molecular microbiology upon the com- Ibusquiza, P. S., Herrera, J. J., Vázquez-Sánchez, D. et al. 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(2016) Quaternary ammonium gress and pitfalls, Expert Reviews in Clinical Pharmacology, 1 (4), compounds (QACs) induced inactivation of Pseudomonas spp.: effect 559–571. of material surface, Food and Bioproducts Processing, 98, 71–78. Srikumar, R., Kon, T., Gotoh, N. et al. (1998) Expression of Pseudomonas aer- Münch, D. and Sahl, H.-G. (2015) Structural variations of the cell wall uginosa multidrug efflux pumps MexA-MexB-OprM and MexC-MexD- precursor lipid II in Gram-positive bacteria – impact on binding and OprJ in a multidrug-sensitive Escherichia coli strain, Antimicrobial efficacy of antimicrobial peptides, Biochimica et Biophysica Acta, Agents and Chemotherapy,42(1),65–71. 1848 (11), 3062–3071. Tabata, A., Nagamune, H., Maeda, T. et al. (2003) Correlation between Nagano, K. and Nikaido, H. (2008) Kinetic behavior of the major multi- resistance of Pseudomonas aeruginosa to quaternary ammonium drug efflux pump AcrB of Escherichia coli, Proceedings of the compounds and expression of outer membrane protein OprR, National Academy of Sciences of the United States of America, Antimicrobial Agents and Chemotherapy, 47 (7), 2093–2099. 106 (14), 5854–5858. Zasloff, M. (2002) Antimicrobial peptides of multicellular organisms, Nikaido, H. (2000) Crossing the envelope: how cephalosporins reach Nature, 415, 389–395. their targets, Clinical Microbiology and Infection, 6 (3), 22–26. ............................................................................................... ..................................................................

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Bioscience HorizonsOxford University Press

Published: Jul 28, 2017

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