Impact of Hydrogen Peroxide Tolerance on Carbapenem Susceptibility in Bacteria Isolated from Poultry Environments

1. Introduction

The emergence of antimicrobial-resistant bacteria in poultry environments has become a pressing global issue. Poultry farms are now recognized as epicenters for multidrug-resistant bacteria, including those resistant to critical antibiotics such as carbapenems [1]. These last-resort antibiotics are vital in clinical settings for treating severe infections caused by multidrug-resistant pathogens [2]; [3].

Antibiotic resistance remains a great threat to public health, ranking as the third most important public health threat of the 21^st century [4];[5]. The general notion concerning the resistance of microbes to antibiotics is inappropriate usage by humans as well as in agricultural feedstuffs and veterinary practice. This situation is worsened by the progressive dissemination of resistant strains of pathogens coupled with the scarcity of a robust antibiotic pipeline [6].

Biocides are used to ensure environmental or food safety by controlling microbial cross-contamination andensuring general hygiene at many stages. Active agents utilizedin biocidal products embrace  various assortment of chemicals that will exert a biocidal or biostatic impact through a spread of various mechanisms targeting a broad spectrum of microorganisms [7];[8]. While hydrogen peroxide is extensively utilized as a disinfectant in poultry processing to combat microbial contamination, its effectiveness may be compromised by bacterial tolerance mechanisms. Grasping these dynamics is crucial for maintaining food safety and safeguarding public health.

In Gram-negative bacteria, several biocide resistance genes belonging to the quaternary ammonium compounds (QACs) group have been described. Among them, qacE11 is the most widespread, as it is found in various groups of Gram-negative bacteria.Research have shown that biocides and antibiotics are similar in their activities, mechanism of action, and resistance development [9]. This creates an impression that there is a likely relationship between the resistance or tolerance of biocide being a contributing factor to antibiotic resistance[10].

Bacterial exposure to the oxidizing agent hydrogen peroxide elicits a complex array of adaptive responses aimed at mitigating oxidative damage. Many bacteria utilize transcriptional regulators like OxyR and PerR to detect hydrogen peroxide, subsequently inducing the expression of antioxidant enzymes and repair systems[11]. Simultaneously, oxidative stress triggers biochemical modifications, including reduced intracellular iron concentrations and activation of RNA quality control pathways, to protect against reactive oxygen species[12]. Recent investigations have also assessed hydrogen peroxide tolerance in bacteria isolated from poultry environments, with evaluations of hydrogen peroxide-based nanoparticle composites showing promising antibacterial activity against pathogenic Escherichia coli [13]. These findings collectively illustrate the intricate defensive strategies employed by bacteria to withstand oxidative stress in poultry settings.

Recent years have seen extensive research on carbapenem susceptibility in bacteria derived from poultry. Animal-associated Enterobacteriaceae, such as Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae, are frequently isolated from broiler farms and often display multidrug resistance profiles, including resistance to carbapenems [2];[14]. Molecular investigations have uncovered diverse mechanisms mediating carbapenem resistance, including the production of various β-lactamases (e.g., NDM, KPC), overexpression of efflux pumps, and alterations in membrane permeability [15]; [16]. Comparative analyses across different poultry farms reveal varying prevalence rates of carbapenem-resistant bacteria, emphasizing the need for ongoing surveillance and improved management practices throughout the food supply chain.

Emerging evidence points to a potential connection between bacterial tolerance to hydrogen peroxide and carbapenem susceptibility. Researchers have hypothesized that exposure to oxidizing agents like hydrogen peroxide may select for bacterial populations with enhanced defensive capabilities, potentially co-selecting for traits that reduce antibiotic susceptibility [17]. Studies suggest that cross-protection or co-selection phenomena may occur, whereby adaptations that counteract oxidative stress indirectly contribute to decreased permeability or altered expression of efflux pumps, ultimately impacting carbapenem efficacy. These interrelationships underscore the complexity of resistance development in poultry environments and highlight the necessity for integrated infection control strategies, as well as further research to elucidate the underlying molecular connections [18]. This research assessed the survivability of Escherichia coli strains and Pseudomonas species of poultry origin exposed to hydrogen peroxide and the consequential effect of the exposure on the antibiotic susceptibility pattern of the bacteria.

2. Materials and Methods

2.1. Sample collection andbacterial isolation and identification

Dry and fresh poultry droppings were scooped randomly from poultry farms within the Ilorin metropolis, Nigeria, using a sterile hand trowel. The samples were placed in sterile Ziploc bags and conveyed to the laboratory in an ice chest. One gram of the fecal sample was weighed into tubes holding 9 ml sterile tryptone soy broth (TSB) (Oxoid Ltd., Reading, UK) and vortexed for 30 seconds. Then, the tubes were incubated at 37^oC for 24 h. Following the incubation, aliquots were picked from the broth culture using inoculating loop and streaked(and spot-inoculated) on solidified eosin methylene blue (EMB) agar (Oxoid Ltd., Reading, UK) and Pseudomonas agar base augmented with cephalothin-fucidin-cetrimide selective supplement (SR0103; Oxoid Ltd., Reading, UK) (Pseudomonas–CFC agar) plates. The plates were then incubated at 37^oC for 24 h. The plates were observed for greenish metallic sheen colonies on EMB agar for Escherichia coli and colorless mucoid colonies on Pseudomonas–CFC agar for Pseudomonas species after the incubation. The isolates were further identified using 16s RNA sequencing.

2.2. Biocide preparation

The proprietary disinfectant brand (Huwa-San^©)was provided by the University of Ilorin water treatment plant. It is a new-generation biodegradable biocide based on ionic silver-activated and -stabilized hydrogen peroxide.Huwa-San^© fulfills the requirements of a water disinfectant and is thought to be superior to many conventional hypochlorite-based disinfectants. Research has demonstrated its strongantimicrobial effecton a wide range of microorganisms, including bacteria, fungus, viruses, molds, spores, and amoebae.[19]. It has beenfound to be effective against the notorious Legionellabacteria and can removebiofilm[20].Huwa-San^© disinfectant has been proven safe for water treatments in livestock agriculture (fisheries and poultry), the manufacturing industry (food, beverages, cosmetics, and pharmaceutics),medical and hospitality settings(hospitals, hotels,swimming pools), and for domesticpurposes.It has also been  found effective  in sewage treatment[21]. Its uses in the treatment of drinking water and disinfection of surfaces and equipment in poultry farms has greatly increased. Manufacturer’s recommendation of a high level or “shock” disinfection of a water system using Huwa-San is at 1000 ppm peroxide or 2000 ppm Huwa-San product with a contact time of one hour. The low-level disinfection is at 100 ppm peroxide or 200 ppm Huwa-San product for 12 hours where the system is offline or 24 hours where the system is in use.

2.3. Antibiotics susceptibility test (AST)          

The susceptibility of the isolates to the following antibiotics prior to their exposure to silver-stabilized hydrogen peroxideas as well as after their recovery from the disinfection stress was determined using the Clinical Laboratory Standard Institute protocol [22]: imipenem (10 μg), meropenem (10 μg), ampicillin (10 μg), gentamicin (10 μg), ciprofloxacin (5 μg), ofloxacin (5 μg), imipenem (10 μg) augmentin (30 μg), ceftazidime (30 μg), nitrofurantoin (300 μg) and cefuroxime (30 μg). The breakpoint values were interpreted as sensitive or resistant using the [22] interpretative guideline.These antibiotics were selected because of their use as therapeutic agents in the treatment of infections with the organisms chosen for this study.

2.4. Phenotypic detection of carbapenemaseproduction

Toinvestigate carbapenemase production in the carbapenem-resistant isolates, the modified carbapenem inactivation method described by [23] was adopted. Meropenem disks were immersed in bacterial suspensions composed of 400 µl of sterile distilled water inoculated with bacterial colonies. The tubes were then incubated at 37°C for 2 h. The disk was removed from the suspension after 2 h and placed onto Mueller-Hinton agar (MHA) plates seeded with a suspension of a carbapenem-susceptible indicator organism (E. coli 25922). Following overnight incubation, the zone of inhibition was measured to determine whether the meropenem has been hydrolysed (growth of the indicator organism close to the disk) or still active (a large zone of inhibition around the disk).

2.5. Phenotypic detection of extended-spectrum beta-lactamase production

The method adopted for this assay was the double disc synergy test [24]. Standardized organisms (ca. 1.2 × 10⁸ CFU/ml) were streaked on solidified MHA plates and amoxicillin/clavulanic was placed in the middle of the already seeded plate; ceftazidime and ceftriaxone were placed on each side of the beta-lactam at 15 mm distance to each other. The plates were then incubated at 37^oC for 18 h, after which the plates were carefully observed for the formation of a champagne bottle shape. A distortion of more the 5 mm was taken as ESBL production.

2.6. Inducing silver-stabilized H₂O₂tolerance in isolates

Hydrogen peroxide tolerance was induced in the bacterial isolates by repeated passage on Huwa-San^©-amended media. To obtain the H₂O₂-modified isolates, the wild-type organismswere exposed to Huwa-San^© at the “shock dose” of 2000 ppm (0.2%v/v) for 1 h. The modified isolates were then processed by successive passages of the cells on MHA spiked with 0.1%, 1%, 5%, and 10% of the stock biocide, progressively (i.e., isolates recovered from 0.1% were regrown on 1% until 10%). After spiking the MHA with the corresponding concentration of the filter-sterilizeddisinfectant, the solidified plates were seeded with bacterial suspension adjusted to 0.5 McFarland. The plates were then incubated at 37^oC for 24 h. The recovered isolates from the different passages were tagged as follows:

H0– wild-type isolate (no biocide passage); H1– isolates passed on from 0.1% to 1% v/v biocide formulation; H10 – isolates recovered from 1% and regrown on undiluted stock of 10% v/v biocide formulation.

2.7. Survivability of bacteria on exposure to silver-stabilized H₂O₂

This experiment was performed to assess the growth rate and survivability of the modified and wild-type isolates on exposure to silver-stabilized H₂O₂ using theBritish standardprotocol [25]. Inoculum from overnight cultures of the isolates was introduced into 10 ml sterile TSB and then incubated at 37°C for 48 h. After 48 h of incubation, 5 ml of broth culture was measured and dispensed into test tubes and centrifuged at 3500 rpm for 15 min. The procedure was repeated twice until a clear supernatant was formed over the cell pellets. Phosphate buffer solution (PBS) was then re-suspended into the tube and the centrifugation process was repeated. After pure cell pellets have been recovered, they were resuspended in 5 ml of PBS, and the turbidity of the organisms was adjusted to 1 × 10⁸ CFU/ml using a spectrophotometer. Nine milliliters(9 ml) of each concentration of the filter-sterilized disinfectant diluted in sterile deionised water (100%, 50%, 25%, 12.5%) were inoculated with 1 ml of the standardized isolates inside test tubes. An aliquot of 1 ml was picked from the mixture and carefully dispensed into a 1 ml neutralizer. After 5 min, 1 ml aliquot waspour plated on sterile EMB and Pseudomonas–CFC agar. The plates were swirled gently, allowed to solidify, and incubated at 37°C for 24 h. Samples for the assessment of the survival rates were taken from the microcosm at 0, 6, and 24 h. After incubation, enumeration of culturable viablecolonies was done.

2.8. Minimum inhibitory/bactericidal concentration

The protocol of the British Standard Institute [25] with slight modifications was adopted. Varying concentrations of the biocide were prepared by double-fold serial dilution in Mueller-Hinton broth (MHB). Each of the concentrations was then inoculated with 100 µl of the standardized overnight broth culture of the test organisms. The positive control was setup as broth with only the disinfectant while the negative control contained broth and organism. The inoculated and control tubes were incubated at 37^oC for 24 h after which they were observed for turbidity. The lowest concentration that showed no turbidity was taken as the minimum inhibitory concentration (MIC) while the lowest concentration of the disinfectant which showed no growth on plates after 24 h of incubation indicates bactericidal effect and was taken as the minimum bactericidal concentration (MBC).

3. Results

3.1. Description of isolates

Based on morphological features of the isolates on the media, four representative isolates – two each of Escherichia and Pseudomonas were presumptively identified. ThetaxonomyandphylogenyoftheisolatesS4 and Ec56 belong to the genus Escherichiaandare designated as Escherichia coli BW2952 andEscherichia coli EGE,respectively. Isolates S3 and CHWbelong to the genus Pseudomonas and are designated as Pseudomonas mucidolensand Pseudomonas aeruginosa PAO1,respectively.Escherichia coli EGE is an extended spectrum beta-lactamase (ESBL) and carbapenemase producer. Other isolates were neither ESBLnorcarbapenemase producers.

3.2. Antibiogram of isolates before chemical exposure

Notably, all the isolates exhibited reduced antibiotic susceptibility resulting in multidrug resistance. The E. coli strains showed a similar resistance pattern to cephalosporins, penicillin, and carbapenems. However, both E. coli strains showed sensitivity to ofloxacin and ciprofloxacin. Contrastingly, the Pseudomonas species showed varying susceptibility properties.P.mucidolensexhibited pandrug resistance to all the antibiotics, while P. aeruginosa was largely sensitive to the tested antibiotics except for ampicillin and amoxicillin/clavulanate

CAZ: ceftazidime; CRO: ceftriaxone; CPR: cefuroxime; AUG: augmentin; OFL: ofloxacin; MEM: meropenem; GEN: gentamicin; AMP: ampicillin; NIT: nitrofurantoin

3.3. Survival kinetics of the isolates exposed to biocide

The survival rates of the tested isolates to different H₂O₂ concentrations at progressive contact times (0,6, 24 h) are presented in Figures 1 and 2.

Generally, for the E. coli strains, viability is reduced directly with the increase in biocide concentration. Also, bacterial growth was completely inhibited by the higher concentrations (50% and 100%) from 0 to 24 h. There were, however, varying reactions to the lower concentrations by the isolates.

E. coli BW2952 (H0): On exposure to 12.5% biocide concentration, culturability declined drastically between 0 and 6 h with a slight recovery after 24 h. Within the first 0–30 min of biocide exposure, there was a 3.8 log reduction in the bacterial load while no visible colonial growth was recorded after 6 h of exposure. After 24 h, the cell recovery observed was equivalent to a 90% reduction in the bacterial cell counts at 0 h. In the 25% concentration, cell viability and culturability were limited to <6 h of exposure with an approximate 3.1 log reduction (about 99.9%) from the initial cell load (10⁸ CFU/ml).

E. coli BW2952 (H100): The modified strain persisted in the least concentration (12.5%) over the 24 h exposure time, with a consistent decline in the number of culturable surviving cells. The resultant bacterial count was a mean 1.75 log reduction after 24 h exposure. Similar to the H0strain, cell viability completely declined in <6h on treatment with 25% of biocide.

E. coli BW2952 (H10): In the 12.5% concentration treatment, cell culturability declined by 4.1 log reduction at 0–30 min of exposure. However, culturability increased slightly at 6 h and then reduced by 4 logs at 24 h. Despite the fluctuations in culturability, the strainmaintained viability for 24 h on exposure to the biocide. On exposure to 25%, culturability declined completely at 6 h, recovering at 24 h with a considerably sharp reduction (99.999%) from initial cell counts.

E. coli EGE (H0): On treatment with 12.5% concentration, cell culturability fluctuated per contact time. At 0–30 min, a 6-log reduction in cell number was observed followed by a loss of culturability at 6 h of treatment time. After 24 h, cells recovered to about 1.7 logs from counts at 0 h. Disinfection at 25% concentration notably influenced cell culturability until 24 h when cell recovery was equivalent to a 4.9-log decrease in the initial cell population.

E. coli EGE (H100): Similar to the H100of E. coli BW2952, cell viability was maintained for 24 h in the 12.5% biocide concentration exposure although with a progressive decrease in cell counts. However, after 24 h of exposure, cell counts were reduced by 99.99%. Same indications in cell culturability and viability were observed from 0 to 6 h and until 24 h when a recovery equivalent to 5.1 log reduction from initial cell counts (10⁸ CFU/ml) was observed on exposure to 25% concentration.

E. coli EGE (H10): Cell viability was limited to 12.5% biocide treatment. An initial non-culturability was observed, followed by a recovery of cells at 6 h and 24 h. Cell counts however reduced by 3.6 logs from initial bacterial cell counts.

Significantly, the Pseudomonas strains persisted longer with a mean ≤4 log reduction at 24 h even for the higher concentrations of 50% and 100% undiluted stock.

Survivability of tested bacteria exposed to concentrations of H₂O₂ for 24 h. S4– Escherichia coliBW2952; Ec56 – Escherichia coliEGE; H0– wild-type isolate (no biocide passage); H10– isolates passed on from 0.1% to 10% biocide formulation; H100 – isolates recovered from 10% and regrown on undiluted stock of 100% v/v biocide formulation.

P. mucidolens (H0): In contrast to the directly proportional decline in cell culturability with increased exposure time observed largely with the E. coli strains, cell culturability increased with exposure time for this isolate and its modified strains. On exposure to 12.5% concentration, cell counts ranged from 2.20–5.52 × 10⁴ CFU/ml from 0 to 24 h with a final 3.3 log reduction from initial cell load (10⁸ CFU/ml). In 25% concentration, cell counts ranged from 1.94–3.20 × 10⁴ CFU/ml with a 3.5-log reduction after 24 h which is similar to the effects of 50% treatment. Despite the persistence of the cells in the 12.5%–50% concentrations, cell viability decreased progressively with an increase in concentration.

P. mucidolens (H100): On exposure to 12.5% and 25% concentrations, about 3.7 log (about 99.99%) reduction from initial cell load (10⁸ CFU/ml) occurred with cell counts ranging from 1.6–2.58 × 10⁴ CFU/ml and 4 × 10³–2.02 × 10⁴ CFU/ml, respectively. Contrastingly, loss of cell culturability was observed from 0 to 6 h until 24 h at 50% concentration treatment. The cell recovery at 24h was however equivalent to a 4.5 log reduction in cell counts from the initial.

P. mucidolens (H10): A 3-log reduction in cell counts was observed after 24h of biocide exposure in 12.5% and 25% concentrations, although a loss of cell culturability occurred at 0–30 min on exposure to 25% biocide. Cell viability was inhibited at 50% and 100%.

P. aeruginosa PAO1 (H10): Of all the tested isolates, this isolate and its modified strains displayed the most ability to survive in (high numbers) and maintain culturability in all concentrations over the 24h exposure time. Contrary to the observations for P. mucidolens, there was a consistent decrease in culturable cells of P. aeruginosa as exposure time increased. In 12.5%–50% concentrations, cell counts declined by about 3.2 logs from the initial 10⁸ CFU/ml after 24 h while a 3.5-log reduction was observed in 100% concentration.

P. aeruginosa PAO1 (H100): Considerably, this strain seemingly survived better in the different concentrations compared to other strains with bacterial counts range of 5.88 × 10⁴–1.66 × 10⁵cfu/ml. Bacterial counts progressively declined with increased concentration and exposure time. After 24h, bacterial counts were reduced by 3.0–3.2 log across all concentrations.

P. aeruginosa PAO1 (H10): On exposure to 12.5% and 25% biocide concentration, bacterial counts were reduced by 3.7 and 3.9-log, respectively. An increased loss in viability of 4 and 4.6 log was however observed on exposure to 50% and 100%, respectively.

Survivability of tested bacteria exposed to concentrations of H₂O₂ for 24 h. S3 – Pseudomonas mucidolens;CHW – Pseudomonas aeruginosa PAO1; H0– wild-type isolate (no biocide passage); H10–isolates passed on from 0.1% to 10% biocide formulation; H100–isolates recovered from 10% and regrown on undiluted stock of 100% v/v biocide formulation.

3.4. Antibiogram profile of survivors

Figure 4 shows the AST profile of surviving isolates after exposure to the biocide. A variation in susceptibility pattern was observed between the wild-type and modified strains of the Escherichia coli strains. More pronounced sensitivity to carbapenems was observed in the recovered cells of E. coli BW2952 and E. coli EGE.

Notably, the survivors of P. aeruginosa PAO1 exhibited resistance to meropenem post-exposure contrary to the significant sensitivity observed for the unexposed/wild-type strain.

Susceptibility of surviving bacteria to meropenem post-exposure to concentrations of H₂O₂ for 24 h. S4– Escherichia coliBW2952; Ec56 – Escherichia coliEGE; H0– wild-type isolate (no biocide passage); S3 – Pseudomonas mucidolens;CHW – Pseudomonas aeruginosa PAO1; H0– wild-type isolate (no biocide passage); H10–isolates passed on from 0.1% to 10% biocide formulation; H100–isolates recovered from 10% and regrown on the undiluted stock of 100% v/v biocide formulation.

3.5. Minimum inhibitory/bactericidal concentration of surviving isolates

The MIC of the strains of E. coli and their modified types ranged from 25%–50% while the MBC ranged from 25%–100% with the highest recorded for the ESBL-producing strain (E. coli EGE). The Pseudomonas strains had higher MIC values; MIC for P. mucidolenswas 50% or 100% while P. aeruginosa was neither inhibited nor killed at the highest concentration under 24 h

S4- Escherichia coliBW2952; Ec56 – Escherichia coliEGE; CHW – Pseudomonas aeruginosa PAO1; S3: Pseudomonas mucidolens. H0– wild-type isolate (no biocide passage); H10–isolates passed on from 0.1% to 10% biocide formulation; H100–isolates recovered from 10% and regrown on undiluted stock of 100% v/v biocide formulation.

4. Discussion

In this study, carbapenem-resistant pathogens were identified in poultry droppings. The increasing prevalence of MDR bacteria in poultry and livestock environments generally calls for intensified active surveillance. The use of antibiotics in livestock production as growth promoters and prophylactics has escalated in developing countries to an alarming rate. This incessant use of antibiotics coupled with reckless waste disposal or management protocols continuously increases the risk of dissemination of MDR pathogens and consequently fatal disease outbreaks [26]; [27]. Effective antibiotics surveillance can therefore be enhanced through the adoption of a one-health initiative and molecular investigations into genetic/clonal relatedness of resistant strains and genes as well as resistance mechanisms.

This study reports the notable persistence of MDRE. coli and Pseudomonas sp. in poultry environment despite exposure to increasing concentrations of hydrogen peroxide 10 folds higher than the recommended proprietary use in disinfection. The tolerance exhibited by the bacteria especially Pseudomonas aeruginosa PAO1, through consecutive exposure to sublethal and lethal concentrations of hydrogen peroxide is suggestive of adaptive mutations. Adaptation of E. coli to physical and chemical stresses has been reported [28]; [29]. Reports have suggested that changes in the environment or a point mutation in E. coli cells influence the expression of a number of regulator proteins, resulting in physiological variabilities [30]; [31]. Similarly, Pseudomonas possesses the genetic ability to adapt and switch phenotypically in response to induced stress. This ability plays a critical role in colonization and biofilm formation [32]. These physiological variabilities may confer stress tolerance and survival on the cells, and in some cases enhanced virulence [33].  The biocide used in this study is a disinfectant designed for either shock disinfection of environmental surfaces or constant dosing of water systems; hence inappropriate use (particularly inadequate concentrations) may select for biocide tolerance in isolates and consequent risks of ineffectiveness.

Studies on the survival dynamics of E. coli and Pseudomonas sp. in biocides are documented, however, there is limited emphasis on the rate of switch and recovery to and from the ‘viable, non-culturable (VBNC) state. The term ‘VBNC’ here describes the observed loss of the ability of cells to grow on nutrient media due to exposure to chemical stress; followed by a recovery of the cells through a suggestive physiological adaptation. In this study, exposure of E. coli and Pseudomonas sp. to concentrations of hydrogen peroxide over time resulted in VBNC states peaking at 6 h contact time for most of the isolates. Recovery of some cells after 24 h of exposure thus suggests a possible reduction in the efficacy of the biocide as the contact time increases. A unique survival strategy in response to drastic or adverse environmental conditions exhibited by bacteria is the VBNC phase. The attainment of a VBNC state by pathogens in disinfection processes is particularly of public health concern because resuscitation and restart of metabolic processes may occur under optimum growth conditions such as the host habitat [34]. Studies have shown that indicator and pathogenic bacteria in the environment have a VBNC state [35], thus negative routine tests post disinfection may not be sufficient to pronounce the safety of an environment, especially in water purification. Better quantification of the viability and culturability of these cells would have been achieved with a combined method that is microscopy and culture-based.

Adaptation to biocide seemingly increased the survivability chances of the modified strains of the test bacteria, although the strains modified through successive passages on increasing concentrations of the biocide (H10) survived significantly lesser than the ones grown directly on absolute undiluted stock. This may be attributed to the loss of physiological integrity that may have been induced by the repeated exposure to the biocide. Also, the E. coli strains were less tolerant to hydrogen peroxide than the Pseudomonas isolates, notably Pseudomonas aeruginosa PAO1. This strain of P. aeruginosa has been described as a versatile and ubiquitous opportunistic pathogen usually used as a model for understanding the environmental and infectious versatility of P. aeruginosa [36]. Both E. coli and Pseudomonas sp. are of high clinical significance, causing hospital and community-acquired infections; therefore, their tolerance to bactericidal concentrations of commonly used disinfectants is a concern. This work demonstrated that E. coli strains EGE and BW2952, Pseudomonas aeruginosa PAO1, and Pseudomonas mucidolenshave the capacity to resist inactivation by hydrogen peroxide even at concentrations 10 folds higher than the recommended minimum bactericidal during-use concentration. Tolerance of E. coli and Pseudomonas sp. to other biocides such as triclosan, sodium hypochlorite, benzalkonium chloride, and chlorhexidine have also been documented [37]; [38]. It is however necessary to note that biocide efficacy can be altered by factors such as inappropriate storage conditions, improper dosage, and total suspended solids among others [39].

Despite the seemingly better survival kinetics of the modified strains of the E. coli isolates, a notable increase in sensitivity to carbapenem was observed in some of the recovered cells. This is contrary to most reports highlighting increased or unchanged resistance to antibiotics of cells recovered from biocide exposure. This observation may suggest that the induced chemical stress triggered certain metabolic changes in the cells (such as an increase in cell permeability) which led to increased susceptibility; however, this mechanism is not properly understood. For P. aeruginosa PAO1 however, a significant reduction in meropenem susceptibility was obtained. Studies have postulated and established the relationship between biocide tolerance and increased antibiotic resistance; for Gram-negative bacteria, a non-specific efflux pump has been suggested [40]; [29]. Carbapenems are last-resort antibiotics used in treating MDR Gram-negative bacterial infections [41]. The world is currently tackling the dissemination of carbapenem-resistant pathogens in humans, animals, and the environment. Therefore, the selection of carbapenem resistance in pathogens through hydrogen peroxide tolerance presents a critical public health risk.

The findings of this study have provided knowledge on the capacity of MDR opportunistic pathogens to persist on exposure to hydrogen peroxide by switching to VBNC states. It also highlights the need to consider the utilization of a combination of biocidal agents with different contact time potency in disinfection. More so, the issuance of recommended during-use concentrations of biocides should be duly monitored with cognizance of the recoverability of cells over time.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Authors’ Contributions

AOO& EA, designed the study, performed the statistical analysis, wrote the protocol, and wrote the first draft of the manuscript. GID managed the analyses of the study, while KTM was involved with manuscript preparation, review, and editing of the manuscript All authors read and approved the final manuscript.”

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