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Sodium Hypochlorite
Abstract
Sodium hypochlorite has bactericidal (5000 mg/l, 30 min) and yeasticidal (1000 mg/l, 5 min) activity. Tuberculocidal activity was reported at 1000 mg/l available chlorine in 10 min. The antimicrobial effect of sodium hypochlorite is explained by damage to cellular components and disruption of cell membranes. Cell defence occurs through energy conservation, biofilm formation and the VBNC state. Sodium hypochlorite at 1–5 mg/l mostly increases biofilm mass, whereas 100–5050 mg/l sodium hypochlorite decreases biofilm formation in most species. Moderate removal of biofilm occurs in young biofilms (mostly 21%–87%), less removal is found in mature biofilms. Elevated MIC values indicating tolerance to sodium hypochlorite have been reported in some clinical isolates, but their relevance is uncertain. In bacterial species, there is little or no increase in MIC (≤4-fold) after low-level exposure. Sodium hypochlorite-resistant isolates have been reported for Methylobacterium spp. and R. erythropolis, with insufficient bactericidal activity in suspension tests compared to culture collection strains. No cross-resistance to antibiotics has been reported for S. aureus, but in selected strains of Salmonella spp. Cross-tolerance may be seen to benzalkonium chloride, another quaternary ammonium compound, and alkylamine (L. monocytogenes) or sodium nitrite and hydrogen peroxide (E. coli). The overall probability of resistance to sodium hypochlorite of practical relevance is very low in the absence of biofilm.
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The high pathogenicity of Pseudomonas aeruginosa is attributed to the production of many virulence factors and its resistance to several antimicrobials. Among them, sodium hypochlorite (NaOCl) is a widely used disinfectant due to its strong antimicrobial effect. However, bacteria develop many mechanisms to survive the damage caused by this agent. Therefore, this study aimed to identify novel mechanisms employed by P. aeruginosa to resist oxidative stress induced by the strong oxidizing agent NaOCl. We analyzed the growth of the P. aeruginosa mutants ΔkatA, ΔkatE, ΔahpC, ΔahpF, ΔmsrA at 1 μg/mL NaOCl, and showed that these known H2O2 resistance mechanisms are also important for the survival of P. aeruginosa under NaOCl stress. We then conducted a screening of the P. aeruginosa PA14 transposon insertion mutant library and identified 48 mutants with increased susceptibility toward NaOCl. Among them were 10 mutants with a disrupted nrdJa, bvlR, hcnA, orn, sucC, cysZ, nuoJ, PA4166, opmQ, or thiC gene, which also exhibited a significant growth defect in the presence of NaOCl. We focussed our follow-up experiments (i.e., growth analyzes and kill-kinetics) on mutants with defect in the synthesis of the secondary metabolite hydrogen cyanide (HCN). We showed that HCN produced by P. aeruginosa contributes to its resistance toward NaOCl as it acts as a scavenger molecule, quenching the toxic effects of NaOCl.
The emergence of disinfectant-resistant microorganisms poses a significant threat to public health. These resilient pathogens can survive and thrive in hospital settings despite routine disinfection practices, leading to persistent infections and the potential for outbreaks. In this study, we investigated the impact of 11 different commercial sanitizers at various concentrations and exposure times on biofilms consisting of clinical and nosocomial environmental isolates of Candida parapsilosis and Staphylococcus aureus. Among the sanitizers tested, 0.5% and 2.0% chlorhexidine (CLX), 10% polyvinyl pyrrolidone (PVP-I), a disinfectant based on quaternary ammonium compound (QAC), 2% glutaraldehyde, and 0.55% orthophthalaldehyde (OPA) demonstrated efficacy against both C. parapsilosis and S. aureus in monospecies and mixed biofilms. Analysis showed that 0.5% CLX and 10% PVP-I had fungicidal and bactericidal activity against all biofilms. However, the sanitizer based on QAC and 0.55% OPA proved to be bacteriostatic and fungicidal against both monospecies and mixed biofilms. In mixed biofilms, despite the last four sanitizers exerting fungicidal action, the reduction of fungal cells was approximately 4 log10 CFU/mL compared to monospecies biofilms, showing that the interaction provided more resistance of the yeast to the sanitizer. Formation of mixed biofilms in hospital settings can create an ecological niche that enhances the survival of pathogens against routine sanitization procedures. Therefore, effective sanitization practices, including regular cleaning with effective sanitizers, should be implemented to prevent C. parapsilosis/S. aureus biofilm formation in healthcare settings.
Phenotypic susceptibility testing of Escherichia (E.) coli is an essential tool to gain a better understanding of the potential impact of biocide selection pressure on antimicrobial resistance. We, therefore, determined the biocide and antimicrobial susceptibility of 216 extended-spectrum β-lactamase-producing (ESBL) and 177 non-ESBL E. coli isolated from swine feces, pork meat, voluntary donors and inpatients and evaluated associations between their susceptibilities. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of benzalkonium chloride, chlorhexidine digluconate (CHG), chlorocresol (PCMC), glutaraldehyde (GDA), isopropanol (IPA), octenidine dihydrochloride and sodium hypochlorite (NaOCl) showed unimodal distributions, indicating the absence of bacterial adaptation to biocides due to the acquisition of resistance mechanisms. Although MIC95 and MBC95 did not vary more than one doubling dilution step between isolates of porcine and human origin, significant differences in MIC and/or MBC distributions were identified for GDA, CHG, IPA, PCMC and NaOCl. Comparing non-ESBL and ESBL E. coli, significantly different MIC and/or MBC distributions were found for PCMC, CHG and GDA. Antimicrobial susceptibility testing revealed the highest frequency of resistant E. coli in the subpopulation isolated from inpatients. We observed significant but weakly positive correlations between biocide MICs and/or MBCs and antimicrobial MICs. In summary, our data indicate a rather moderate effect of biocide use on the susceptibility of E. coli to biocides and antimicrobials.
This study aimed to investigate the biofilm-production ability of carbapenem-resistant Acinetobacter baumannii (CRAB), the biofilm-eradication potential of 70% ethanol and 0.5% sodium hypochlorite, the effects of selenium nanoparticles (SeNPs) against planktonic and biofilm-embedded CRAB, and the relationship between biofilm production and bacterial genotypes. A total of 111 CRAB isolates were tested for antimicrobial susceptibility, biofilm formation, presence of the genes encoding carbapenemases, and biofilm-associated virulence factors. The antibiofilm effects of disinfectants and SeNPs against CRAB isolates were also tested. The vast majority of the tested isolates were biofilm producers (91.9%). The bap, ompA, and csuE genes were found in 57%, 70%, and 76% of the CRAB isolates, with the csuE being significantly more common among biofilm producers (78.6%) compared to non-biofilm-producing CRAB (25%). The tested disinfectants showed a better antibiofilm effect on moderate and strong biofilm producers than on weak producers (p < 0.01). The SeNPs showed an inhibitory effect against all tested planktonic (MIC range: 0.00015 to >1.25 mg/mL) and biofilm-embedded CRAB, with a minimum biofilm inhibitory concentration of less than 0.15 mg/mL for 90% of biofilm producers. In conclusion, SeNPs might be used as promising therapeutic and medical device coating agents, thus serving as an alternative approach for the prevention of biofilm-related infections.
Plasmids are relevant reservoirs of antimicrobial resistance genes (ARGs) which confer adaptive advantages to their host and can be horizontally transferred. The aims of this study were to develop a conjugation procedure to monitor the horizontal transfer of a 193 kb plasmid containing the extended-spectrum β-lactamase production gene blaCTX-M-14 between two Escherichia coli strains under a range of food chain-related scenarios, including temperature (20–37 °C), pH (5.0–9.0) or the presence of some biocidal agents (benzalkonium chloride, sodium hypochlorite or peracetic acid). The average conjugation rate in LB broth after 18 h at 37 °C was 2.09e-04 and similar rates were observed in a food matrix (cow's milk). The conjugation was reduced at temperatures below 37 °C, at alkaline pH (especially at pH 9.0) or in the presence of benzalkonium chloride. Peracetic acid and sodium hypochlorite slightly increased conjugation rates, which reached 5.59e-04 and 6.77e-03, respectively. The conjugation procedure described can be used to identify risk scenarios leading to an enhanced ARGs transmission via plasmid conjugation, as well as to identify novel intervention strategies impairing plasmid conjugation and tackling antimicrobial resistance.
In diabetic foot ulcers (DFUs), biofilm formation is a major challenge that promotes wound chronicity and delays healing. Antiseptics have been proposed to combat biofilms in the management of DFUs. However, there is limited evidence on the activity of these agents against biofilms, and there are questions as to which agents have the best efficiency. Here, we evaluated the antibiofilm activity of sodium hypochlorite, polyvinylpyrrolidoneIodine (PVPI), polyhexamethylenebiguanide (PHMB) and octenidine against Pseudomonas aeruginosa strains using static and dynamic systems in a chronic-wound-like medium (CWM) that mimics the chronic wound environment. Using Antibiofilmogram®, a technology assessing the ability of antiseptics to reduce the initial phase of biofilm formation, we observed the significant activity of antiseptics against biofilm formation by P. aeruginosa (at 1:40 to 1:8 dilutions). Moreover, 1:100 to 1:3 dilutions of the different antiseptics reduced mature biofilms formed after 72 h by 10-log, although higher concentrations were needed in CWM (1:40 to 1:2). Finally, in the BioFlux200TM model, after biofilm debridement, sodium hypochlorite and PHMB were the most effective antiseptics. In conclusion, our study showed that among the four antiseptics tested, sodium hypochlorite demonstrated the best antibiofilm activity against P. aeruginosa biofilms and represents an alternative in the management of DFUs.
Salmonella Enteritidis (S. Enteritidis) can adapt to sublethal sodium hypochlorite conditions, which subsequently triggers stress resistance mechanisms in this pathogen. Hence, the current work aimed to reveal the underlying stress adaptation mechanisms in S. Enteritidis by phenotypic, proteomic, and physiological analyses. It was found that 130 ppm sodium hypochlorite resulted in a moderate inhibitory effect on bacterial growth and an increased accumulation of intracellular reactive oxygen species. In response to this sublethal treatment, a total of 492 proteins in S. Enteritidis showed significant differential abundance (p < 0.05; fold change >2.0 or <0.5), including 225 more abundant proteins and 267 less abundant proteins, as revealed by the tandem-mass-tags-based quantitative proteomics technology. Functional characterization further revealed that proteins related to flagellar assembly, two-component system, and phosphotransferase system were in less abundance, while those associated with ABC transporters were generally in more abundance. Specifically, the repression of flagellar-assembly-related proteins led to diminished swimming motility, which served as a potential energy conservation strategy. Moreover, altered abundance of lipid-metabolism-related proteins resulted in reduced cell membrane fluidity, which provided a survival advantage to S. Enteritidis. Taken together, these results indicate that S. Enteritidis employs multiple adaptation pathways to cope with sodium hypochlorite stress.
Foodborne pathogenic bacteria in multi-species biofilms in food manufacturing facilities have been suspected to be the cause of cross-contamination leading to foodborne illness. We studied if cafeteria kitchen-associated bacterial isolates can have any protective effect on E. coli O157:H7 in biofilm against extracellular polymeric substances (EPS)-degrading enzymes and sodium hypochlorite. We investigated multi-species biofilm-forming ability and the efficacy of EPS-degrading enzymes using crystal violet assay. The susceptibility of E. coli O157:H7 to sodium hypochlorite (NaClO) was evaluated using propidium monoazide combined with quantitative PCR (PMA-qPCR). Then, a combined treatment with enzymes followed by NaClO was also tested. Most cafeteria kitchen isolates of Acinetobacter and Bacillus were able to form biofilms. Several of them showed a protective effect on E. coli O157:H7 against NaClO after forming multi-species biofilms, particularly in Acinetobacter. This protective effect on E. coli O157:H7 was also noticed after the enzyme or the combined treatment with NaClO. Our results give us an insight into the protective role of food-associated environmental bacteria for E. coli O157:H7 in biofilms against common sanitizers and warrant further study to develop effective control methods. Our study also highlights the importance of preventing contamination or biofilm formation by environmental microorganisms, eventually reducing foodborne illness.
The purpose of this study was to determine the effect of sanitizer use conditions on the susceptibility, biofilm forming ability and pathogenicity of Listeria monocytogenes. Two different strains of L. monocytogenes and a non-pathogenic L. innocua were exposed to sodium hypochlorite, benzalkonium chloride and peroxyacetic acid at different concentrations (4 to 512 ppm) and treatment times (30 s to 5 min), respectively. Under the tested conditions, no significant difference (p > 0.05) in reduction was observed among the three tested sanitizers. A reduction of 1 to 8 log CFU/mL was observed depending upon the sanitizer concentration and treatment times. The survived cells at the highest sublethal concentration and treatment time of a particular sanitizer upon re-exposure to the same or different sanitizer showed either no change or increased susceptibility when compared to parent strains. Upon repeated exposure to sanitizers at progressively increasing concentrations from 1 to 128 ppm, L. innocua was able to survive concentrations of up to 32 ppm benzalkonium chloride and 64 ppm peroxyacetic acid treatments, respectively. At the tested sub-lethal concentrations, no significant difference (p > 0.05) in biofilm formation was observed among the tested strains. Caco-2 interaction with L. innocua showed a reduction in invasion ability with sublethal concentrations of sanitizers.
Background and Aim: Microsporum gallinae is the major dermatophyte species that causes avian dermatophytosis. Disinfection plays an important role in controlling and preventing dermatophytosis; however, information about the effect of common disinfection processes on M. gallinae is limited. This study aimed to investigate the disinfection efficacy of ultraviolet (UV) irradiation, heat treatment, detergents, and germicides against infective spores (arthroconidia) and vegetative mycelia of M. gallinae.
Materials and Methods: The minimum inhibitory and minimum fungicidal concentrations of benzalkonium chloride, chlorhexidine, ethanol, formaldehyde, glutaraldehyde, hydrogen peroxide, phenol, povidone-iodine, and sodium hypochlorite germicides against arthroconidia and mycelia of M. gallinae American type culture collection (ATCC) 90749
were determined by broth microdilution. Time-kill assays were used to determine the fungicidal efficacy of moist heat treatment, UV irradiation, commercially available detergents, and germicides.
Results: There were no significant differences between the arthroconidia and mycelia growth stages of M. gallinae ATCC 90749 in the magnitude of the log10 cell reductions in the number of viable fungal cells induced by the disinfection treatments (all p > 0.05). Moist heat treatment at 40°C did not reduce the number of viable fungal cells at any time (1–60 min); however, treatment at 50°C for 25 min and either 60°C or 80°C for 5 min eliminated > 99.999% of viable fungal cells. Irradiation of fungal cultures with UVC and UVB at doses higher than or equal to 0.4 and 0.8 J/cm2, respectively, resulted in a 5-log10
reduction in the number of viable fungal cells, whereas UVA only reduced the number of viable fungal cells by < 2-log10 up to a dose of 1.6 J/cm2. All the tested detergents demonstrated minimal fungicidal effects with < 1-log10 reductions in the number
of viable fungal cells at concentrations up to 8% w/v. All of the tested germicides eradicated the fungus after treatment for 1 min at 1–1000× minimum inhibitory concentration (MIC), except for hydrogen peroxide, which was not fungicidal after
treatment for 20 min at 100× MIC.
Conclusion: Moist heat treatment at temperatures greater than or equal to 50°C, UVC and UVB irradiation at doses higher than or equal to 0.4 and 0.8 J/cm2, respectively, and treatment with all tested germicides except hydrogen peroxide can be considered effective processes for disinfecting the fungus M. gallinae from the equipment employed in poultry farming. In contrast, commercially available detergents are not suitable for use as M. gallinae disinfectants.
Background
Pseudomonas aeruginosa is a common pathogen in Hospitalized patients, and its various resistance mechanisms contribute to patient morbidity and mortality. The main aims of the present study were to assess the susceptibility of biofilm-producing and non-producing P. aeruginosa isolates to the five commonly used Hospital disinfectants, to evaluate the synergistic effect of selected disinfectants and Ethylene-diamine-tetra acetic acid (EDTA), and the effect of exposure to sub-inhibitory concentrations of Sodium hypochlorite on antimicrobial susceptibility test.
Results
The results showed that sodium hypochlorite 5% and Ethanol 70% were the most and least effective disinfectants against P. aeruginosa , respectively. The addition of EDTA significantly increased the effectiveness of the selected disinfectants. The changes in the antibiotic-resistance profiles after exposure to sub-inhibitory concentrations of disinfectants were observed for different classes of antibiotics (Carbapenems, Aminoglycosides, Cephalosporins, Fluoroquinolones). As well as near the all isolates harbored efflux pump genes and 117 (97.5%) of isolates produced biofilm.
Conclusion
In the current study, the mixture of disinfectant and EDTA were the most suitable selection to disinfect Hospital surfaces and instruments. Also, it was clear that exposure to sub-inhibitory concentrations of Sodium hypochlorite results in resistance to some antibiotics in P. aeruginosa species. Strong and intermediate biofilm formers belonged to MDR/XDR strains. Future studies should include more complex microbial communities residing in the Hospitals, and more disinfectants use in Hospitals.
Acinetobacter baumannii hospital infections are difficult to treat due to the rapid emergence of multidrug-resistant (MDR) strains. In addition, A. baumannii can survive in numerous adverse environments, including in the presence of common hospital antiseptics. We hypothesized that in addition to accumulating drug resistance determinants, MDR A. baumannii strains also accumulate mutations that allow for greater microbicide tolerance when compared to pan-susceptible (PS) strains. To test this hypothesis, we compared the survival of five MDR and five PS patient isolates when exposed to bleach, ethanol, quaternary ammonium compounds, chlorhexidine gluconate, and povidone. We evaluated bacteria in a free-living planktonic state and under biofilm conditions. Each disinfectant eliminated 99.9% of planktonic bacteria, but this was not the case for bacterial biofilms. Next, we characterized strains for the presence of the known microbicide-resistance genes cepA, qacEΔ1, qacE, and qacA. MDR strains did not survive more than PS strains in the presence of microbicides, but microbicide-resistant strains had higher survival rates under some conditions. Interestingly, the PS strains were more likely to possess microbicide-resistance genes. Microbicide resistance remains an important topic in healthcare and may be independent of antimicrobial resistance. Hospitals should consider stricter isolation precautions that take pan-susceptible strains into account.
Background
: Biofilm formation is a major factor in the resistance mechanism of Klebsiella pneumoniae. This study aimed to evaluate the effects of sodium hypochlorite on the biofilm of K. pneumoniae with different drug resistance.
Methods
: We collected three different types of K. pneumoniae respectively. The growth trend of biofilms of different drug-resistant K. pneumoniae was quantified by measuring the OD590 for seven consecutive days using crystal violet staining. Scanning confocal fluorescence microscopy was used to observe biofilm morphology.
Results
: After adding sodium hypochlorite, there were significant differences between the OD590 value of the 200, 500 and 1000 µg/ml groups and the positive control group (all P < 0.05) on the fifth day. Concentrations of 2000 and 5000 µg/ml sodium hypochlorite were added after the biofilm had matured. In the 5000 µg/ml sodium hypochlorite group, the OD590 of K. pneumoniae biofilm in the three groups decreased significantly compared with the blank control group (all P < 0.05).
Conclusion
: Sodium hypochlorite inhibited and cleared the biofilm of K. pneumoniae with different drug resistance, and the effect was enhanced with the increase of concentration in the range of bacteriostatic and bactericidal concentration.
When selecting effective doses of antimicrobials, be they biocides or antibiotics, it is essential to know the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of these substances. The present research determined the MICs and MBCs for three biocides, sodium hypochlorite (SH), benzalkonium chloride (BC), and peracetic acid (PAA), and nine antibiotics in eight strains of Listeria monocytogenes of varying serotypes. Marked intra-species differences were observed in the resistance of L. monocytogenes to the biocides and antibiotics. The MICs (ppm) for the biocides ranged between 1750 and 4500 for SH, 0.25 and 20.00 for BC, and 1050 and 1700 for PAA. Their MBCs (ppm) ranged from 2250 to 4500 for SH, 0.50 to 20.00 for BC, and 1150 to 1800 for PAA. The MICs (ppm) for antibiotics lay between 1 and 15 for ampicillin, 8 and 150 for cephalothin, 20 and 170 for cefoxitin, 0.05 and 0.20 for erythromycin, 4 and 50 for chloramphenicol, 3 and 100 for gentamicin, 2 and 15 for tetracycline, 2 and 80 for vancomycin, and 160 and 430 for fosfomycin. The corresponding MBCs (ppm) were from 5 to 20 for ampicillin, 9 to 160 for cephalothin, 70 to 200 for cefoxitin, 4 to 5 for erythromycin, 9 to 70 for chloramphenicol, 5 to 100 for gentamicin, 3 to 30 for tetracycline, 3 to 90 for vancomycin, and 160 to 450 for fosfomycin. Notably, erythromycin showed considerable efficacy, demonstrated by the low values for both MIC and MBC. Based on EUCAST and the CLSI criteria, all strains were susceptible to erythromycin. All strains were resistant to cephalothin, cefoxitin, gentamicin, and fosfomycin. Further values for resistance were 87.50% for ampicillin and vancomycin, 75.00% for tetracycline, and 62.50% for chloramphenicol. The high prevalence of antibiotic resistance is a matter for concern. A positive correlation was found between MIC and MBC values for most of the biocides and antibiotics. The higher the hydrophobicity of the cell surface, the higher the susceptibility to biocides, suggesting that surface characteristics of bacterial cells influence resistance to these compounds.
A global upsurge in antibiotic resistant Acinetobacter baumannii requires supervised selection of biocides and disinfectants to avert nosocomial infections by reducing its spread. On the other hand, inadequate and improper biocides have been reported as a contributing aetiology of antimicrobial resistance (AMR). Regardless of the manner of administration, a biocidal concentration that does not kill the target bacteria creates a stress response, propagating the resistance mechanisms. This is an essential aspect of the disinfection program and the overall bio-contamination management plan. Knowing the mechanisms of action of biocides and resistance modalities may open new avenues to discover novel agents. Here, we describe the mechanisms of action of some biocides, resistance mechanisms, and approaches to study susceptibility/resistance to these agents.
A novel 4% hydrogen peroxide disinfectant was effective against methicillin-resistant Staphylococcus aureus (MRSA), Clostridioides difficile spores, carbapenem-resistant Escherichia coli, and 2 strains of Candida auris. In laboratory testing, a sodium hypochlorite disinfectant caused fading and loss of pliability of a hospital mattress, but the hydrogen peroxide disinfectant did not. These findings suggest that the hydrogen peroxide-based disinfectant may be a useful addition to the sporicidal disinfectant products available for use in healthcare settings.
The presence of virulence genes, phylogenetic relationships, biofilm formation index (BFI), and ultrastructure in S. Minnesota at different temperatures (4, 25, and 36 °C) were analyzed. In addition, the ability of biocidal agents (chlorhexidine1%, sodium hypochlorite 1%, and peracetic acid 0.8%) to inhibit biofilms formed by 20 strains isolated from broiler slaughter plants from two Brazilian companies in 2009, 2010, and 2014 was determined. The presence of specific genes was evaluated by PCR and phylogeny between strains by pulsed-field gel electrophoresis. The BFI was determined using tryptone soy broth with 5% of chicken juice, and its structure was observed by scanning electron microscopy. The presence of specific genes indicated that S. Minnesota has the potential to cause disease in humans, adapting to adverse conditions. Temperatures of 25 and 36 °C favored biofilm formation, although at 4 °C, there was still biomass that could contaminate the final product. Tolerance to all biocides was identified in 12/20 (60%), representing a real risk of adaptation mechanisms development, especially regarding to resistance to sodium hypochlorite. Phylogenetic analysis indicated cross-contamination and spread among companies, which was probably related to biofilms formation. Results show the necessity of attention to this serovar considering its resistance to sodium hypochlorite, including the need for rigorous control, adopting low temperatures to prevent biofilms formation in the poultry industry.
Biocide use is essential and ubiquitous, exposing microbes to sub-inhibitory concentrations of antiseptics, disinfectants, and preservatives. This can lead to the emergence of biocide resistance, and more importantly, potential cross-resistance to antibiotics, although the degree, frequency, and mechanisms that give rise to this phenomenon are still unclear. Here, we systematically performed adaptive laboratory evolution of the gut bacteria Escherichia coli in the presence of sub-inhibitory, constant concentrations of ten widespread biocides. Our results show that 17 out of 40 evolved strains (43%) also decreased the susceptibility to medically relevant antibiotics. Through whole-genome sequencing, we identified mutations related to multidrug efflux proteins ( mdfA and acrR ), porins ( envZ and ompR ), and RNA polymerase ( rpoA and rpoBC ), as mechanisms behind the resulting (cross)resistance. We also report an association of several genes ( yeaW , pyrE , yqhC , aes , pgpA , and yeeP - isrC ) and specific mutations that induce cross-resistance, verified through mutation repairs. A greater capacity for biofilm formation with respect to the parent strain was also a common feature in 11 out of 17 (65%) cross-resistant strains. Evolution in the biocides chlorophene, benzalkonium chloride, glutaraldehyde, and chlorhexidine had the most impact in antibiotic susceptibility, while hydrogen peroxide and povidone-iodine the least. No cross-resistance to antibiotics was observed for isopropanol, ethanol, sodium hypochlorite, and peracetic acid. This work reinforces the link between exposure to biocides and the potential for cross-resistance to antibiotics, presents evidence on the underlying mechanisms of action, and provides a prioritized list of biocides that are of greater concern for public safety from the perspective of antibiotic resistance.
Significance Statement
Bacterial resistance and decreased susceptibility to antimicrobials is of utmost concern. There is evidence that improper biocide (antiseptic and disinfectant) use and discard may select for bacteria cross-resistant to antibiotics. Understanding the cross-resistance emergence and the risks associated with each of those chemicals is relevant for proper applications and recommendations. Our work establishes that not all biocides are equal when it comes to their risk of inducing antibiotic resistance; it provides evidence on the mechanisms of cross-resistance and a risk assessment of the biocides concerning antibiotic resistance under residual sub-inhibitory concentrations.
In this contribution, the antimicrobial susceptibility toward 11 antibiotics and four biocides of a panel of 205 Listeria monocytogenes (Lm) strains isolated from different ecological niches (i.e., food, animals and natural environment) was evaluated. The impact of exposure to biocides on the antibiotic susceptibilities of Lm was also investigated. Lm strains isolated from food exhibited overall a lower susceptibility (higher minimal inhibitory concentrations, MIC) for ammonium quaternary compounds (QACs) and peracetic acid (PAC) than strains isolated from animals and natural environments. Conversely, the ecological origins of Lm strains did not significantly affect their susceptibilities towards antibiotics. Interestingly, repeated exposure to QACs recurrently led to a decrease in susceptibility toward ciprofloxacin (CIP), a fluoroquinolone antibiotic, largely used in human medicine. Moreover, these lower levels of susceptibility to CIP remained stable in most Lm strains even after subcultures without biocide selection pressure, suggesting an adaptation involving modifications at the genetic level. Results underlined the ability of Lm to adapt to biocides, especially QACs, and the potential link between this adaptation and the selection of resistance toward critical antibiotics such as ciprofloxacin. These data support a potential role of the extensive use of QACs from “farm to fork” in the selection of biocide and antibiotic resistance in pathogenic bacteria such as Lm.
Background:
A catheter allowing a release of antibacterial substances such as antiseptics into the bladder could be a new way of preventing biofilm formation and subsequent catheter-associated urinary tract infections.
Methods:
Minimal inhibitory and bactericidal concentration (MIC/MBC) determinations in cation-adjusted Mueller-Hinton broth and artificial urine were performed for 4 antiseptics against 3 uropathogenic biofilm producers, Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis. Furthermore, effects of octenidine and polyhexanide against catheter biofilm formation were determined by quantification of biofilm-producing bacteria.
Results:
Sodium hypochlorite showed MIC/MBC values between 200 and 800 mg/L for all strains tested. Triclosan was efficient against E. coli and P. mirabilis (MIC ≤2.98 mg/L) but ineffective against P. aeruginosa. Octenidine and polyhexanide showed antibacterial activity against all 3 species tested (MIC 1.95-7.8 and 3.9-31.25 mg/L). Both octenidine and polyhexanide were able to prevent biofilm formation on catheter segments in a concentration dependent manner. Furthermore, adding 250 mg/L of each biocide disrupted biofilms formed by E. coli and P. mirabilis, whereas even 500 mg/L was not sufficient to completely destroy P. aeruginosa biofilms.
Conclusion:
Octenidine- and polyhexanide-containing antiseptics showed a broad effect against typical uropathogenic biofilm producers even in high dilutions. This study provides a basis for further investigation of the potential of octenidine and polyhexanide as prophylaxis or treatment of catheter biofilms.
A good disinfectant for the human body should have various properties, such as strong bactericidal activity, harmlessness to living tissues, and resistance against biological fluids (or other organic contaminants). Peroxynitric acid (PNA) showed a bactericidal effect that was several tens up to several hundred times higher per unit of molarity than that of sodium hypochlorite and peracetic acid, which are used as general disinfectants for medical equipment. Moreover, the high resistance of PNA to organic load was confirmed, indicating that PNA will inactivate bacteria effectively even on contaminated surfaces, such as used medical devices or the human body surface. Therefore, we propose that PNA can be used as a strong disinfectant for the human body.
Biofilms pose a relevant factor for wound healing impairment in chronic wounds. With 78% of all chronic wounds being affected by biofilms, research in this area is of high priority, especially since data for evidence-based selection of appropriate antimicrobials and antiseptics is scarce. Therefore, the objective of this study was to evaluate the anti-biofilm efficacy of commercially available hypochlorous wound irrigation solutions compared to established antimicrobials. Using an innovative complex in-vitro human plasma biofilm model (hpBIOM), quantitative reduction of Pseudomonas aeruginosa, Staphylococcus aureus, and Methicillin-resistant S. aureus (MRSA) biofilms by three hypochlorous irrigation solutions [two <0.08% and one 0.2% sodium hypochlorite (NaClO)] was compared to a 0.04% polyhexanide (PHMB) irrigation solution and 0.1% octenidine-dihydrochloride/phenoxyethanol (OCT/PE). Efficacy was compared to a non-challenged planktonic approach, as well as with increased substance volume over a prolonged exposure (up to 72 h). Qualitative visualization of biofilms was performed by scanning electron microscopy (SEM). Both reference agents (OCT/PE and PHMB) induced significant biofilm reductions within 72 h, whereby high volume OCT/PE even managed complete eradication of P. aeruginosa and MRSA biofilms after 72 h. The tested hypochlorous wound irrigation solutions achieved no relevant penetration and eradication of biofilms despite increased volume and exposure. Only 0.2% NaClO managed a low reduction under prolonged exposure. The results demonstrate that low-dosed hypochlorous wound irrigation solutions are significantly less effective than PHMB-based irrigation solution and OCT/PE, thus unsuitable for biofilm eradication on their own. The used complex hpBIOM thereby mimics the highly challenging clinical wound micro-environment, providing a more profound base for future clinical translation.
Sodium hypochlorite (NaOCl) and its active ingredient, hypochlorous acid (HOCl), are the most commonly used chlorine-based disinfectants. HOCl is a fast-acting and potent antimicrobial agent that interacts with several biomolecules, such as sulfur-containing amino acids, lipids, nucleic acids, and membrane components, causing severe cellular damage. It is also produced by the immune system as a first-line of defense against invading pathogens. In this review, we summarize the adaptive responses of Gram-negative bacteria to HOCl-induced stress and highlight the role of chaperone holdases (Hsp33, RidA, Cnox, and polyP) as an immediate response to HOCl stress. We also describe the three identified transcriptional regulators (HypT, RclR, and NemR) that specifically respond to HOCl. Besides the activation of chaperones and transcriptional regulators, the formation of biofilms has been described as an important adaptive response to several stressors, including HOCl. Although the knowledge on the molecular mechanisms involved in HOCl biofilm stimulation is limited, studies have shown that HOCl induces the formation of biofilms by causing conformational changes in membrane properties, overproducing the extracellular polymeric substance (EPS) matrix, and increasing the intracellular concentration of cyclic-di-GMP. In addition, acquisition and expression of antibiotic resistance genes, secretion of virulence factors and induction of the viable but nonculturable (VBNC) state has also been described as an adaptive response to HOCl. In general, the knowledge of how bacteria respond to HOCl stress has increased over time; however, the molecular mechanisms involved in this stress response is still in its infancy. A better understanding of these mechanisms could help understand host-pathogen interactions and target specific genes and molecules to control bacterial spread and colonization.
Surface disinfectants are regularly used in prophylactic and infection control measures. Concern has been raised whether residues of sub-inhibitory disinfectant concentrations may constitute a selective pressure and could contribute to the development of strains which are tolerant and/or resistant to biocides including antibiotics. The current study investigated whether Staphylococcus (S.) aureus ATCC® 29213™ and ATCC® 6538™ would change their growth characteristics and antimicrobial susceptibility profiles after prolonged treatment with sub-inhibitory concentrations of sodium hypochlorite (NaOCl). NaOCl is a fast-acting disinfectant with a broad-spectrum activity, inexpensive and widely used in healthcare and the food production industry. Minimum inhibitory concentration (MIC) for NaOCl was determined by broth macrodilution according to the guidelines for disinfectant efficacy testing provided by the German Veterinary Medical Society. Serial passages after 24 h and 72 h, respectively, in defined sub-inhibitory concentrations of NaOCl resulted in a number of phenotypic variants. Two of these variants, derived from S. aureus ATCC® 29213™, showed elevated MICs of oxacillin and were considered as in vitro-generated borderline oxacillin-resistant S. aureus (BORSA). Transmission electron microscopy revealed a significantly thickened cell wall in these isolates, a phenomenon that has also been described for Listeria monocytogenes after low-level exposure to NaOCl. Whole genome sequencing revealed an early stop codon in the gene coding for the GdpP protein and thereby abolishing the function of this gene. GdpP represents a phosphodiesterase that regulates gene expression, and loss of function of the GdpP protein has been described in association with borderline oxacillin resistance. Our findings suggest that a mutation in the GdpP protein gene and morphological changes of the cell wall were induced by repeated exposure to sub-lethal NaOCl concentrations, and most likely accounted for a BORSA phenotype in two variants derived from S. aureus ATCC® 29213™.
Objectives
Triclosan is usually employed as a disinfectant in a wide range of medical and consumer care products, which may have imposed a selective pressure on bacteria. This study was designed to evaluate the resistance mechanisms of triclosan and molecular epidemiology of triclosan-resistant isolates of Acinetobacter baumannii in Wenzhou, China.
Methods
A collection of 626 A. baumannii were isolated from the First Affiliated Hospital of Wenzhou Medical University during 2016-2017 and antimicrobial susceptibility testing of these isolates were performed via agar dilution method. Molecular mechanisms of triclosan resistance, including the existence of mutations in reductase (FabI) were investigated by PCR and sequencing. Furthermore, quantitative RT-PCR was conducted to evaluate the expression levels of the fabI gene and efflux pump genes (adeB, adeG, adeJ, abeM, amvA and abeS) at normal condition and sub-inhibitory concentration of triclosan, and the epidemiological characteristics were analyzed by PFGE and MLST.
Results
2.7% (17/626) of A. baumannii exhibited resistance to triclosan. The FabI mutation Gly95Ser was found in one triclosan resistant strain. The expression of fabI and adeB gene were significant difference between triclosan-resistant and susceptible strains (P < 0.05). The expression of fabI, adeG, adeJ and abeM were increased after triclosan induction. The clones of these resistant isolates were diverse and sporadic.
Conclusions
The hyper-expression of fabI was probably the main mechanism of triclosan resistance in this study, and the efflux pump AdeB, AdeG, AdeJ and AbeM might also be related to decreased triclosan susceptibility.
Chemical biocides are used for the prevention and control of infection in health care, targeted home hygiene or controlling microbial contamination for various industrial processes including but not limited to food, water and petroleum. However, their use has substantially increased since the implementation of programmes to control outbreaks of methicillin-resistant Staphylococcus aureus, Clostridioides difficile and severe acute respiratory syndrome coronavirus 2. Biocides interact with multiple targets on the bacterial cells. The number of targets affected and the severity of damage will result in an irreversible bactericidal effect or a reversible bacteriostatic one. Most biocides primarily target the cytoplasmic membrane and enzymes, although the specific bactericidal mechanisms vary among different biocide chemistries. Inappropriate usage or low concentrations of a biocide may act as a stressor while not killing bacterial pathogens, potentially leading to antimicrobial resistance. Biocides can also promote the transfer of antimicrobial resistance genes. In this Review, we explore our current understanding of the mechanisms of action of biocides, the bacterial resistance mechanisms encompassing both intrinsic and acquired resistance and the influence of bacterial biofilms on resistance. We also consider the impact of bacteria that survive biocide exposure in environmental and clinical contexts.
Background:
Dry surface biofilms (DSB) have been found abundantly across hospital surfaces within intensive care units and may explain how nosocomial pathogens can remain virulent and persist on surfaces for extended periods. Testing standards governing the performance of disinfectant products employ planktonic models under routine growth conditions, which are known to be less tolerant than their biofilm counterpart.
Aim:
To evaluate biofilm models cultured under artificial human sweat (AHS), a source of nutrient expected on touch surfaces, to assess the antimicrobial performance of common cleaning agents, including a quaternary ammonium, hydrogen peroxide and active chlorine.
Method:
5 single-species biofilms, using pathogenic bacteria such as Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis, were generated on stainless steel substrates using a sedimentation protocol under both AHS and nutrient rich conditions for a direct comparison of phenotypic tolerance. The biofilm models were grown over 5 days followed by desiccation cycles, before being submerged into the disinfectant solutions for up to 25 minutes. Epifluorescence (EF) microscopy using LIVE/DEAD™ stain was used to visualise microcolony viability.
Findings:
The results revealed biofilms cultured under AHS exhibited a greater antimicrobial tolerance and reduced speed of kill for all cleaning agents compared with the routine media; an average reduction of 72.4% vs 96.9%, respectively. EF microscopy revealed traces of viable bacteria across all coupons after disinfection indicating a potential opportunity for regrowth and recontamination.
Conclusion:
The notable difference in biocidal performance between the two growth conditions highlights potential pitfalls within current antimicrobial test standards, and the importance of accurate representation of the microbial challenge.
Objective:
Antiseptics are widely used in wound management to prevent or treat wound infections, and have been shown to have antibiofilm efficacy. The objective of this study was to assess the effectiveness of a polyhexamethylene biguanide (PHMB)-containing wound cleansing and irrigation solution on model biofilm of pathogens known to cause wound infections compared with a number of other antimicrobial wound cleansing and irrigation solutions.
Method:
Staphylococcus aureus and Pseudomonas aeruginosa single-species biofilms were cultured using microtitre plate and Centers for Disease Control and Prevention (CDC) biofilm reactor methods. Following a 24-hour incubation period, the biofilms were rinsed to remove planktonic microorganisms and then challenged with wound cleansing and irrigation solutions. Following incubation of the biofilms with a variety of concentrations of the test solutions (50%, 75% or 100%) for 20, 30, 40, 50 or 60 minutes, remaining viable organisms from the treated biofilms were quantified.
Results:
The six antimicrobial wound cleansing and irrigation solutions used were all effective in eradicating Staphylococcus aureus biofilm bacteria in both test models. However, the results were more variable for the more tolerant Pseudomonas aeruginosa biofilm. Only one of the six solutions (sea salt and oxychlorite/NaOCl-containing solution) was able to eradicate Pseudomonas aeruginosa biofilm using the microtitre plate assay. Of the six solutions, three (a solution containing PHMB and poloxamer 188 surfactant, a solution containing hypochlorous acid (HOCl) and a solution containing NaOCl/HOCl) showed increasing levels of eradication of Pseudomonas aeruginosa biofilm microorganisms with increasing concentration and exposure time. Using the CDC biofilm reactor model, all six cleansing and irrigation solutions, except for the solution containing HOCl, were able to eradicate Pseudomonas aeruginosa biofilms such that no viable microorganisms were recovered.
Conclusion:
This study demonstrated that a PHMB-containing wound cleansing and irrigation solution was as effective as other antimicrobial wound irrigation solutions for antibiofilm efficacy. Together with the low toxicity, good safety profile and absence of any reported acquisition of bacterial resistance to PHMB, the antibiofilm effectiveness data support the alignment of this cleansing and irrigation solution with antimicrobial stewardship (AMS) strategies.
We evaluated the bactericidal efficacy of two alternative hand antiseptics, based on sodium hypochlorite or sodium hypochlorite and hypochlorous acid, compared to isopropanol on hands of volunteers artificially contaminated with E. coli using EN 1500. The reference alcohol was applied according to the norm and the study formulations were used as common in practice (3 mL for 30 seconds). Both products showed mean log10-reductions of 1.63 and 1.89 which was inferior to the reference treatment (4.78). Due to the failure to achieve sufficient bactericidal efficacy within 30 seconds, sodium hypochlorite (0.05% - 0.06%) is not an option for hand disinfection.
Introduction
Acquisition of reduced susceptibility to biocides may contribute to the dissemination of high-risk (HR) clones of carbapenemase-producing Klebsiella pneumoniae (CP-Kp). The aim of this study was (a) to determinate the activity of biocides against CP-Kp, and (b) to analyse the relationship between biocide activity and the presence of efflux pumps.
Methods
The minimal inhibitory concentrations (MICs) of 6 biocides (sodium hypochlorite, chlorhexidine digluconate, benzalkonium chloride, povidone-iodine, ethanol and triclosan) were determined in triplicate at 25 °C and 37 °C in Mueller-Hinton broth (MHB) and M9 minimum medium, against 17 CP-Kp isolates representing different clones (HR and no-HR), sequence-types (STs) and carbapenemases. Efflux pumps genes were detected by whole genome sequencing (MiSeq).
Results
Median MICs were slightly higher at 37 °C than at 25 °C (p ≤ 0.05), except for benzalkonium chloride, triclosan and ethanol. MIC medians were much higher in MHB than in M9, except for triclosan. No significant differences were observed in the median MICs, regarding the type of clone, ST or carbapenemase; cepA, acrAB, kpnEF and oqxAB genes were detected in all isolates, whereas qacE and qacA were not detected; smvAR, and qacΔE genes were detected in 94% and 47% of isolates, respectively.
Conclusions
Triclosan, chlorhexidine digluconate, benzalkonium chloride and ethanol were the most active biocides. The activity of some biocides is affected by temperature and growth media, suggesting that standardised procedures for biocide susceptibility testing based on MIC determination are required. This activity, in terms of MICs, are not related to the type of clone, ST, carbapenemase or the presence of the efflux pump genes.
The development, validation, and use of new quantitative methodologies for testing the effectiveness of antimicrobial products are necessary to meet the regulatory challenges associated with an ever-changing marketplace, novel product claims, new infection control practices, and the emergence of new clinical pathogens. A series of four interlaboratory studies were conducted in a standardized manner on an interim quantitative method for testing liquid treatments against bacteria to assess its statistical performance. The Quantitative Method, a derivative of ASTM E2197, is designed to enumerate the number of viable microbes remaining on a test carrier following exposure to a liquid antimicrobial treatment; a log10 reduction (LR) in viable bacteria is calculated based on the difference between the mean log10 density values of the untreated control and treated carriers. The Quantitative Method uses 1 cm diameter disks (carriers) of brushed stainless steel as the material to represent a hard, non-porous surface. The LR value is used as the measure of product effectiveness, where higher LR values are indicative of greater microbial kill. The test microbes were Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium terrae. The liquid antimicrobial treatments used in these studies were highly relevant to those in the marketplace and provided a wide range of mean LR outcomes. The focus of the statistical assessment was on the repeatability of the LRs across experiments within a lab (Sr) and the reproducibility of the LRs across labs (SR). Due to the additional sources of variability, the SR is expected to be higher than the variability within a laboratory (Sr); this was observed in the studies reported here. Across the studies, the Sr values for LR were small (i.e., less than 0.84), most notably for treatments generating high mean LRs (5 or above) where the Sr was as small as 0.12. Overall, the SR values ranged from 0.227 to 1.217. Only three of the twenty-four treatment combinations over the study period resulted in SR values above 1.0 – the associated LRs for the three treatments ranged from 2.22 to 3.26. Antimicrobial treatments with a LR of 4.5 or higher exhibited SR of 0.561 or less. The statistical attributes reported here for the draft Quantitative Method when used to test P. aeruginosa, S. aureus, and M. terrae provide information for decision makers when considering the method as a candidate regulatory procedure. The data and statistical analyses contained in this report are historical in nature and provide useful baseline information for individuals conducting additional technical review of the method. Based on the data, the Quantitative Method displays a statistical profile consistent with other standard methods approved by standard-setting organizations where method performance data are available.
Pathogens such as Salmonella can be difficult to control in low-moisture food (LMF) processing plants and because of this its presence especially in biofilm mode should be prevented in such an environment. This study evaluated the capacity of Salmonella strains isolated from the peanut supply chain (S. Muenster, S. Miami, S. Glostrup, S. Javiana, S. Oranienburg and S. Yoruba) to form biofilm as well as their resistance to sanitizers (sodium hypochlorite, peracetic acid, quaternary ammonium, alkaline chlorinated solution and biguanide). Regarding biofilm formation, there was no significant difference (p > 0.05) among the strains tested singly on AISI 304 stainless steel (SS) and polypropylene (PP) coupons at the same temperature. However, a difference (p < 0.05) between the temperatures was noted in the first hours of incubation. The sessile cells reached counts between 3 and 4 log CFU/cm² at 25 °C whereas more than 5 log CFU/cm² was observed at 37 °C after 8 h. From 24 h the counts were above 6 log CFU/cm² for both temperatures. Nevertheless, the SEM images of the 6-strain pool showed the highest density of adhered cells after 48 h at 25 °C and 24 h at 37 °C on PP, and after 48 h at both temperatures on SS. Peracetic acid (300 mg/L) had the shortest action time against 24-h biofilm on SS and PP, with sessile cell counts below the limit of detection (0.59 log CFU/cm²) after 3 min. For 48-h and 96-h biofilm, sodium hypochlorite (200 mg/L) decreased more than 4 log CFU/cm² within 5 min. Quaternary ammonium (350 mg/L) and chlorinated alkaline detergent (200 mg/L) showed intermediate performances. Only biguanide (800 mg/L) did not reduce the biofilm counts to below the limit of detection in any of the conditions evaluated. The results indicated high biofilm formation ability of the Salmonella strains isolated from the peanut supply chain. Nevertheless, in general the biofilms were sensitive to most sanitizers within 15 min of treatment. This was the first study which evaluated biofilm formation by Salmonella isolated from the peanut supply chain. Data obtained here will contribute to optimize the hygiene practices in LMF manufacturing plants.
The current study determined the antibacterial and antibiofilm activity of six organic acids including acetic acid, citric acid, formic acid, tartaric acid, malic acid and maleic acid along with different chemicals (disinfectants, surfactants and chelating agents) either alone or in a combination of organic acid with chemicals against C. sakazakii. All the organic acids and only three chemicals viz., EDTA, NaCl, NaOCl were found to possess anti-Cronobacter activity and were evaluated for antibiofilm efficacy. All the agents tested for antibiofilm activity were quite effective against formation of biofilm as well as prefomed mature biofilms. Subtractive screening based on antibiofilm activity of different combinations revealed malic acid (0.0625 mol l⁻¹) with NaOCl (0.00004 mol l⁻¹) as the most effective combination with fractional inhibitory concentration index (FICI) of 0.38. Biofilm microstructures were rarely observed after treatment as revealed by light microscopy and SEM analysis and a reduction of 7.06 ± 0.40 log CFU ml⁻¹ in viable cell counts as compared to control groups. The findings indicate that combination of malic acid with NaOCl has synergistic effect and may have implications in its usage in eradication of C. sakazakii biofilms from abiotic surfaces in production plant and clinical settings.
The effect that the hydrodynamic conditions under which biofilms are formed has on their persistence is still unknown. This study assessed the behaviour of Pseudomonas fluorescens biofilms, formed on stainless steel under different shear stress (τw) conditions (1, 2 and 4 Pa), to chemical (benzalkonium chloride – BAC, glutaraldehyde – GLUT and sodium hypochlorite – SHC) and mechanical (20 Pa) treatments (alone and combined). The biofilms formed under different τw showed different structural characteristics. Those formed under a higher τw were invariably more tolerant to chemical and mechanical stresses. SHC was the biocide which caused the highest biofilm killing and removal, followed by BAC. The sequential exposure to biocides and mechanical stress was found to be insufficient for effective biofilm control. A basal layer containing biofilm cells mostly in a viable state remained on the surface of the cylinders, particularly for the 2 and 4 Pa-generated biofilms.
The spread of OXA-48-encoding plasmids from Klebsiella pneumoniae (OXA-48-Kpn), especially successful high-risk (HR) clones, is a growing concern. Biofilm formation can contribute to the dissemination of OXA-48-Kpn. It is not known whether biocides can affect the transfer of OXA-48-Kpn in biofilm. The aim of this study was to evaluate the effect of biocides on the conjugation frequency (CF) of OXA-48-Kpn in both biofilm and planktonic cultures. For that, seven OXA-48-Kpn isolates (4 belonging to HR clones and 3 to non-HR clones) were selected as donors. Each isolate was mixed (1:1) with Escherichia coli J53 (recipient) and grown on polystyrene microplates without biocides (control) and with 0.25x MIC of triclosan (TRI), chlorhexidine digluconate (CHX), povidone-iodine (POV), sodium hypochlorite (SOD) or ethanol (ETH). The CF was calculated as the number of transconjugants/number of E. coli J53. The results showed that for isolates growing in the absence of biocide, the mean fold change in the CF in biofilm with respect to that determined in planktonic cells (CF-BF/CF-PK) was 0.2 in non-HR isolates and ranged from 2.0-14.7 in HR isolates. In HR isolates grown in the presence of biocide, especially CHX, TRI, and ETH, the fold changes in CF-BF/CF-PK decreased, whereas in non-HR isolates the fold changes were similar or increased slightly with CHX, ETH, SOD and POV. In conclusion, the CF is higher in biofilms of HR isolates than in those of non-HR isolates. Biocides can reduce the CF in biofilms of HR isolates, whereas in non-HR isolates, biocides have no significant effect, or produce only a slight increase in the CF.
This study evaluated bactericidal efficacy test of sanitisers for benzalkonium chloride, sodium hypochlorite and peracetic acid, determined minimum inhibitory concentration (MIC) of sanitisers and assessed their effect on the reduction of biofilms produced by Staphylococcus spp. strains isolated from organic and conventionally produced Minas Frescal cheese dairy products. The MIC was determined at three different contact times (5, 10, and 15 min). Biofilms formed on stainless steel and the efficiency of sanitisers in removing the biofilms were investigated at contact times of 5 and 10 min and incubated for 24 and 48 h. All strains tested were able to form biofilm within 24 and 48 h for all evaluated sanitisers, indicating the MIC used in this study was not sufficient to inactivate biofilm formation. Therefore, the MICs obtained through the analysis of the microorganism in its planktonic form were not efficient to eliminate the biofilms formed.
Foodborne pathogens, such as Salmonella are problematic in food processing environments, and understanding the means of persistence is critical in the development of effective control measures. This study determined the antimicrobial tolerance of Salmonella isolates from the processing environment and characterized their biofilm production and antibiotic resistance. Twenty-five Salmonella isolates were previously recovered from poultry processing equipment in commercial production facilities after sanitation. The minimum inhibitory concentration (MIC) of two antimicrobials; chlorine and quaternary ammonium compounds (QAC) that were frequently used for sanitation was determined for these isolates using the Clinical and Laboratory Standards Institute (CLSI) guidelines. Biofilm forming ability was assessed using the crystal violet assay and antibiotic susceptibility was also determined. These isolates were further characterized based on their genes that were responsible for biofilm formation and resistance to sanitizers and antibiotics. MIC values between 500 and 1000 ppm for chlorine, or 3 to 25 ppm for QAC were observed amongst these Salmonella isolates. These isolates possessed strong (24%), moderate (28%), and weak (48%) biofilm forming ability. All isolates were resistant to multiple antibiotics, and 64% exhibited resistance to aminoglycosides and β-lactams. Molecular characterization showed that the isolates possessed specific genes for biofilm formation, sanitizer tolerance, and antibiotic resistance. These results suggest that Salmonella isolates with low tolerance to sanitizers may remain on surfaces because of their strong biofilm forming ability.
Aims:
This work aimed to determine the occurrence, virulence, antibiogram, carbapenem resistance genes and susceptibility to disinfectants of Pseudomonas aeruginosa isolated from animals, environment and workers in intensive farms.
Methods and results:
A total of 610 samples from intensive beef cattle and sheep farms in Kafr El Sheikh Governorate, Egypt were screened for the presence of P. aeruginosa using bacteriological assays. The isolates were characterized by PCR and tested for susceptibility to antibiotics using disk diffusion method and disinfectants by quantitative suspension test. In all, 60 P. aeruginosa isolates were recovered in this study and all isolates harboured at least one of the virulence genes tested. Human P. aeruginosa isolates were highly resistant to cephalosporins, fluroquinolones, aminoglycosides, carbapenems and penicillins+β-lactamase inhibitors than non-human isolates. Colistin resistance was higher in non-human than human P. aeruginosa isolates, whereas low resistance to aztreonam was observed in non-human and human isolates. Carbapenem-resistant P. aeruginosa (CRPA) strains were recovered from workers (56.5%), sheep (8.3%) and cattle (8.3%). All CRPA harboured at least one of the carbapenem resistance genes tested and most of them showed multidrug resistance (MDR) or extensive drug resistance (XDR) phenotypes. Glutaraldehyde 1% and hydrogen peroxide 3% eliminated P. aeruginosa completely in the absence and presence of organic matter within short contact time compared with other disinfectants.
Conclusions:
This study reported the occurrence of CRPA in animals and workers in intensive farms. Glutaraldehyde and hydrogen peroxide were the most effective disinfectants against P. aeruginosa.
Significance and impact of the study:
The occurrence of CRPA in intensive livestock farms is a serious challenge that threatens animal and human health and increases the risk of P. aeruginosa infection in the community. Therefore, it is vital to control the spread of CRPA by banning or restricting the use of antibiotics and applying proper cleaning and disinfection protocols in livestock farms.
This study aimed at assessing the impact of the physiological state of fungal spores on inactivation by sodium hypochlorite, 0.1% and 0.2% active chlorine, and 3% hydrogen peroxide. In this context, two physiological states were compared for 4 fungal species (5 strains). The first physiological state corresponded to fungal spores produced at 0.99 aw and harvested using an aqueous solution (laboratory conditions), while the second one corresponded to fungal spores produced under a moderate water stress (0.95 aw) and dry-harvested (mechanical harvesting without use of any water, mimicking food plant conditions). Aspergillus flavus “food plant” conidia were more resistant to all tested fungicide molecules than the “laboratory” ones. The same phenomenon was observed for Penicillium commune UBOCC-A-116003 conidia treated with hydrogen peroxide. However, this isolate did not exhibit any inactivation difference between “laboratory” and “food plant” conidia treated with sodium hypochlorite. Similarly, the physiological state of Cladosporium cladosporioides conidia did not impact the efficacy of the tested biocides. P. commune UBOCC-A-112059 “food plant” and “laboratory” conidia were more resistant to hydrogen peroxide and sodium hypochlorite, respectively. As for Mucor circinelloides, “laboratory” spores were more resistant to all disinfectant than the “food plant” ones. Noteworthy, regardless of the physiological state, all M. circinelloides and C. cladosporioides conidia were inactivated for 5 min treatment at 0.2% active chlorine and for 2.5 min treatment at 0.1% active chlorine, while the conidia of all the other species remained viable for these treatments. The obtained data indicate that the efficacy of disinfectant molecules depends not only on the encountered fungal species and its intraspecific diversity but also on the spore physiological state.
The substantial use of disinfectants has increased antibiotic resistance, thereby mediating serious ecological safety issues worldwide. Accumulating studies have reported the role of chlorine disinfectants in promoting disinfectant resistance. The present study sought to investigate the role of chlorine disinfectants in developing multiple resistance in Pseudomonas sp. isolated from the river through antioxidant enzyme measurement, global transcriptional analyses, Gene Ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The results demonstrated that 100 mg/L sodium hypochlorite could increase disinfectant resistance and antibiotic resistance. The SOS response (a conserved response to DNA damage) triggered by oxidative stress makes bacteria resistant to chlorine. An increase in antibiotic resistance could be attributed to a decreased membrane permeability, increased expression of MuxABC-OpmB efflux pump, beta-lactamase, and antioxidant enzymes. Additionally, KEGG enrichment analysis suggested that the differentially expressed genes were highly enriched in the metabolic pathways. In summary, the study results revealed the impact of chlorine disinfectants in promoting microbial disinfectant resistance and antibiotic resistance. This study will provide insight into disinfectant resistance mechanisms.
Objective:
The abundance and prevalence of dry-surface biofilms (DSBs) in hospitals constitute an emerging problem, yet studies rarely report the cleaning and disinfection efficacy against DSBs. Here, the combined impact of treatments on viability, transferability, and recovery of bacteria from DSBs has been investigated for the first time.
Methods:
Staphylococcus aureus DSBs were produced in alternating 48-hour wet-dry cycles for 12 days on AISI 430 stainless steel discs. The efficacy of 11 commercially available disinfectants, 4 detergents, and 2 contactless interventions were tested using a modified standardized product test. Reduction in viability, direct transferability, cross transmission (via glove intermediate), and DSB recovery after treatment were measured.
Results:
Of 11 disinfectants, 9 were effective in killing and removing bacteria from S. aureus DSBs with >4 log10 reduction. Only 2 disinfectants, sodium dichloroisocyanurate 1,000 ppm and peracetic acid 3,500 ppm, were able to lower both direct and cross transmission of bacteria (<2 compression contacts positive for bacterial growth). Of 11 disinfectants, 8 could not prevent DSB recovery for >2 days. Treatments not involving mechanical action (vaporized hydrogen peroxide and cold atmospheric plasma) were ineffective, producing <1 log10 reduction in viability, DSB regrowth within 1 day, and 100% transferability of DSB after treatment.
Conclusions:
Reduction in bacterial viability alone does not determine product performance against biofilm and might give a false sense of security to consumers, manufacturers and regulators. The ability to prevent bacterial transfer and biofilm recovery after treatment requires a better understanding of the effectiveness of biocidal products.
In this study, we investigated the efficacy of chlorine-based disinfectants (sodium hypochlorite and chlorine dioxide) against Salmonella Enteritidis planktonic cells in the presence of different level of organic substances. Also, the reduction effect against S. Enteritidis biofilms formed on stainless steel, silicone rubber, plastic, and chicken skin were evaluated. At the same effective chlorine concentration under clean condition, chlorine dioxide (ClO2) had significantly (P < 0.05) higher planktonic cell reduction than sodium hypochlorite (NaOCl). And when chlorine-based disinfectants were treated under dirty condition, the disinfectant efficacy was significantly (P < 0.05) decreased than that was in clean condition. The disinfectant efficacy of NaOCl and ClO2 against the biofilms formed on food contact surfaces and chicken skin was also evaluated. Likewise before, ClO2 showed significantly (P < 0.05) increased biofilm removal ability than NaOCl in food contact surfaces and chicken skin at all treated times and concentrations. The reduction value of NaOCl and ClO2 at 100 μg/mL depends on the type of surface was decreased in the order of stainless steel (4.91 or 6.55 log CFU/cm²), silicone rubber (3.79 or 6.68 log CFU/cm²), plastic (4.20 or 6.60 log CFU/cm²), and chicken skin (0.58 or 1.33 log CFU/cm²). The results indicated that chlorine-based disinfectants (NaOCl and ClO2) could be used in poultry industry, especially on food contact surfaces, for control planktonic cells and biofilms of Salmonella Enteritidis to enhance food safety.
The objective of this study was to determine if the adaptation at planktonic stage to subinhibitory concentrations (SIC) of sodium hypochlorite (NaOCl) could modulate the biofilm forming ability of five Listeria monocytogenes strains V7, Scott A, FSL-N1-227, FSL F6-154 and ATCC 19116 representing serotypes 1/2a, 4b and 4c. Biofilm formation by NaOCl nonadapted and adapted L. monocytogenes planktonic cells was measured in the presence or absence of SIC of NaOCl. The biofilm formation ability of NaOCl nonadapted and adapted L. monocyotgenes planktonic cells was reduced only in the presence of NaOCl (P < 0.05). Scanning electron microscopy revealed that the continuous exposure of NaOCl induced morphological changes in the L. monocytogenes biofilm structure and reduced its attachment to polystyrene surface. The qRT-PCR results also showed that the subinhibitory NaOCl reduced biofilm formation related gene expression such as motility and quorum sensing signals (P < 0.05). These findings indicate that subinhibitory NaOCl can reduce the ability of L. monocytogenes planktonic cells to form biofilms on polystyrene surface.
Pseudomonas spp. contamination was screened from a Minas Frescal cheese processing plant. Biofilm formation of Pseudomonas fluorescens and Pseudomonas aeruginosa and the effect of three sanitisers, peracetic acid, sodium hypochlorite, and chlorhexidine gluconate, on the biofilm production was also studied. The biofilms were formed in milk according to a central composite rotatable design (CCRD) at 0, 1.2, 4, 6.8, and 8 days and 7, 13, 27, 41, and 47 °C. Pseudomonas spp. counts were observed in milking machine, tank, wall, door handle, floor, drain, and cheese. All Pseudomonas spp. isolates showed lipolytic activity and some proteolytic activity. P. fluorescens was able to form biofilms at 9.8–47 °C and 0.9–8 days, while P. aeruginosa formed biofilms at 11–47 °C and 0.7–8 days. Complete biofilm removal was not observed for all sanitisers studied, showing difficulty in sanitising the surfaces after the biofilm formation.
The presence of mycotoxin-producing fungi in food production environments is a cause of concern since they can contaminate food products, synthesizing toxic compounds in later steps. To avoid this, an effective hygiene and sanitation process of the environment and equipment should be adopted, using sanitizing agents with adequate antifungal efficacy. This work evaluated the effectiveness of different chemical sanitizers: benzalkonium chloride (0.3%, 1.2%, 2 %), biguanide (2%, 3.5%, 5%), iodine (0.2%, 0.6%, 1%), peracetic acid (0.3%, 0.6%, 1%), sodium hypochlorite (0.5%, 0.75%, 1%), besides a new non-polluting technology, the electrolyzed water, both the acid in the chlorine concentrations of 60, 85 and 121 ppm and the respective basic electrolyzed water formed against strains of toxigenic Aspergillus flavus, Aspergillus nomius, Aspergillus parasiticus, Aspergillus carbonarius, Aspergillus niger, Aspergillus ochraceus and Aspergillus westerdijkiae through the methodology recommended by the European Committee for Standardization. Benzalkonium chloride and iodine were the most effective sanitizers to eliminate Aspergillus from the Flavi and Nigri section. Peracetic acid showed the best elimination of the growth of Aspergillus from Circumdati section. Sodium hypochlorite, biguanide, and electrolyzed water agents were the least effective, reducing less than 3 logs from initial control, not being the most suitable agents for the control of toxigenic fungi in food industries.
Biofilms on the surface of food processing equipment act as potential reservoirs of microbial contamination. Bacterial interactions are believed to play key roles in both biofilm formation and antimicrobial tolerance. In this study, Aeromonas hydrophila, Chryseobacterium oncorhynchi, and Pseudomonas libanensis, which were previously isolated from Chinese raw milk samples, were selected to establish two dual-species biofilm models (P. libanensis plus A. hydrophila and P. libanensis plus C. oncorhynchi) on stainless steel at 7°C. Subsequently, three disinfectants, hydrogen peroxide (100 ppm), peracetic acid (100 ppm), and sodium hypochlorite (100 ppm), were used to treat the developed sessile communities for 10 min. Structural changes after exposure to disinfectants were analyzed with confocal laser scanning microscopy. The cell numbers of both A. hydrophila and C. oncorhynchi recovered from surfaces increased when grown as dual species biofilms with P. libanensis. Dual-species biofilms were more tolerant of disinfectants than were each single-species biofilm. Peracetic acid was the most effective disinfectant for removing biofilms, followed by hydrogen peroxide and sodium hypochlorite. The results expand the knowledge of mixed-species biofilms formed by psychrotrophic bacteria and will be helpful for developing effective strategies to eliminate bacterial mixed-species biofilms.
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Periodontitis pathogenesis involves activation of host immune responses triggered by microbial dysbiosis. Therefore, controlling periodontal pathogens in-vivo is a main goal of periodontal therapy. New antimicrobials might help to control periodontal infection and improve treatment outcomes at "the dark times" of increasing antibiotic resistance. Here, we determined the biological activity of collinin and isocollinin against 8 bacterial strains. Antimicrobial activity of collinin and isocollinin, chlorhexidine digluconate (CHX) and sodium hypochlorite (NaClO) was evaluated against clinically relevant periodontal bacteria, like Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Dialister pneumosintes strains and superinfectants like Escherichia coli, Staphylococcusaureus, and Pseudomonasaeruginosa strains. A broth microdilution test was carried out to determine the minimum inhibitory concentration of collinin and isocollinin against those strains, and bacterial viability was determined by resazurin assay at diverse concentration and exposure times. P. gingivalis was the most susceptible strain to collinin and isocollinin (MIC 2.1 μg/mL and 4.2 μg/mL respectively). Other periodontal pathogens showed MICs <17 μg/mL for collinin and MICs between 20 and 42 μg/mL for isocollinin, whereas CHX and NaClO showed MICs of 62 and 326 μg/mL, respectively. Collinin and isocollinin also exhibited antimicrobial activity against superinfectant bacteria (MIC < 21 and < 42 μg/mL, respectively). Overall, collinin and isocollinin showed a remarkable antibacterial activity against relevant periodontal and superinfective bacteria, especially against P. gingivalis (MIC 2.1 μg/mL and 4.2 μg/mL respectively) and the highly virulent P. aeruginosa (MIC 5.2 and 20.8 μg/mL, respectively).
Hypermucoviscous K. pneumoniae (HMV) are emergent zoonotic pathogens associated with increased invasiveness and pathogenicity in terrestrial and marine mammals. In this study, HMV and non-HMV isolates recovered from stranded pinnipeds were used to investigate: 1) their persistence in sea and fresh water microcosms at 10 and 20°C, 2) their capacity to form biofilms, and 3) the biocide efficacy of four disinfectants on their planktonic and biofilm phenotypes. Results indicated that although HMV isolates were significantly more mucoviscous, non-HMV isolates displayed significantly greater capacity to form biofilms (p < 0.05). Additionally, non-HMV isolates persisted in greater numbers in both sea- and freshwater, particularly at 20°C. These two phenomena could be associated with the greater growth observed for non-HMV isolates in in-vitro growth-curve assays (p < 0.05). Similar susceptibility to disinfectants was detected in HMV and non-HMV isolates when exposed for 24 h; however, the minimal biofilm disinfectant eradication concentration for HMV isolates was significantly higher than that for non-HMV when exposed to disinfectants for 0.5 h. This information should be taken into consideration when developing biosecurity protocols in facilities holding marine mammals in captivity.