Impact of Sublethal Disinfectant Exposure on Antibiotic Resistance Patterns of Pseudomonasaeruginosa

Disinfectant-induced changes [1] in gram-negative bacilli have resulted in decreased permeability to some antibiotics [2‒4] and increased susceptibility to dyes and detergents [5, 6]. Contact with disinfectants in sublethal concentrations allows survival and multiplication of bacteria. Serial passage of bacteria through diluted disinfectants may increase antibiotic minimal inhibitory concentrations, MICs [7] and lead to phenotypic changes in antibiograms [8]. It was suggested that the disinfectants probably altered sites in the bacterial ribosomes making it selectively less susceptible to certain antibiotics. Others suggested that enzymes involved in peptidoglycan synthesis might be destroyed causing resistance to penicillin’s and cephalosporins. Destruction of periplasmic enzymes by groups of disinfectants is also another contributing factors [9]. The most commonly used disinfectant in microbiology laboratory and hospitals are ethanol, Dettol, chlorohexidine, and soap [10]. Ethanol, as a dehydrating agent causes cell membrane damage, denaturalization of protein and cell lyses [11]. Dettol affect bacterial cells by denaturation of protein and also act on the cytoplasmic membrane of microorganisms. Bleach with a main constituent of sodium hypochlorite effect can be achieved by oxidizing of the cell of microorganism of attaching essential cell component including protein, lipid and DNA, while Hibitane (chlorohexidine) act by disruption of membranes, precipitation of proteins, and inactivation of enzymes [12]. The present study was performed to determine the effect of disinfectants exposure on the antibiotics sensitivity and resistance pattern of Pseudomonas aeruginosa.

Fifty isolates of P. aeruginosa obtained from the Children’s Hospital in Mosul, Iraq were studied. These strains were isolated from rectal swabs from infants with diarrhea. Swabs were transferred to the laboratory in sterile nutrient broth. After incubation at 37°C for 18 h, a loopful was subcultured on Pyocyanogel agar (bio-Merieux) and incubated at 37°C for 18 h. Colonies were identified using standard methods [6, 13].

Four less toxic, less irritant, and commonly used disinfectants in hospitals were selected as follows: chlorhexidine gluconate (Hibitane 5% w/v, ICI); cetrimide (Cetavlon 40% w/v, ICI); chloroxylenol (Dettol 4.8% w/v, Reckitt and Colman); and chlorhexidine 1.5%/cetrimide 15% mixture (Savlon, ICI).

Suitable working solutions of disinfectants were prepared as a doubling dilution (Table 1) in distilled water and sterilized at 115°C for 15 min. Disinfectants were incorporated at the desired concentrations into molten Mueller-Hinton (MH) agar (Oxoid) cooled to about 50°C. Plates were poured and when set dried for 1 h at room temperature. A loopful of 3-h subculture in nutrient broth (10⁵–10⁶ cfu mL⁻¹) was inoculated onto the agar surface and the sublethal disinfectant concentrations for each strain were determined after incubation at 37°C for 18 h. The sublethal disinfectant concentration was defined as the highest disinfectant concentration that allowed bacterial growth. The disinfectant-exposed variants, i.e., isolates grown in higher concentrations of the disinfectants than the majority of the cells in the inoculum, were passed from the various disinfectants into all four disinfectants in the following order: chlorhexidine, cetrimide, chloroxylenol, and finally to the cetrimide/chlorhexidine mixture. Plates containing no disinfectant were included in each series, as were unexposed strains. The identity of each strain was checked at every passage by gram strain, pyocyanin production, and oxidase test. After passages through the four disinfectants, all strains were grown in disinfectant-free nutrient broth for 3 h at 37°C and antibiotic susceptibility tests were performed.

Antibiotic resistance patterns before and after disinfectant exposure were tested by the disc diffusion method on MH agar. The following antibiotic-impregnated paper discs (Oxoid) were used (µg/disc): tetracycline (30); carbenicillin (100); co-trimoxazole (100); amikacin (10); gentamicin (10); cephaloridine (30); clindamycin (2); neomycin (30); kanamycin (30); and bacitracin (10 i.u.). A 4 mL quantity of 3-h subculture in nutrient broth of each strain was poured onto the dried agar plates. After 10 min, the inoculum was removed and the plates left at room temperature for 1 h to dry. The antibiotic-impregnated discs were then placed onto the surface of the seeded MH agar plates and incubated at 37°C for 18 h. The antibiotic resistance pattern of each strain was then determined. Strains were collectively considered as sensitive when there was a zone of inhibition of more than 2 mm around the edge of the disc, and as resistant when there was a zone of inhibition of less than 2 mm [14].

Ten tetracycline-resistant strains of P. aeruginosa were selected and subjected to antibiotic resistance transfer experiment as donors before and after the disinfectant passages. Escherichia coli J53.2 (F⁻ pro met rif^r) was used as a recipient strain, which was kindly provided by Dr. K.J. Towner (University of Nottingham, UK) and the procedure performed as follows: donors and recipients were grown in 10 mL nutrient broth at 37°C for 6 h. Three ml of the resistant donor broth was then mixed with 3 mL of the recipient broth and the mixtures incubated at 37°C for 18 h. Then, 10⁻¹ and 10⁻² dilutions of the mating mixtures were prepared in 1:4 Ringer’s solution, and 0.1 mL of these, together with undiluted mating mixture, were seeded onto selective plates of MH agar containing 100 µg mL⁻¹ rifampicin and 50 µg mL⁻¹ tetracycline. The plates were incubated at 37°C for 18 h for isolation of transconjugants.

A paired sample t test correlation coefficient (R) and coefficient of determination (R²) were calculated.

Sublethal inhibitory concentrations of the disinfectants for different strains did not vary markedly (Table 2). For example, cetrimide at concentrations of 1,562 mg L⁻¹ and 3,125 mg L⁻¹ inhibited 34 (68%) and 15 (30%) of strains, respectively, but the remaining strain was inhibited at higher concentration, 12,500 mg L⁻¹. The results of antibiotic susceptibility tests are shown in (Table 2). All of the unexposed strains were resistant to one or more antibiotics, i.e., multiple resistant and all were resistant to individual antibiotics, e.g., tetracycline, carbenicillin, co-trimoxazole, cephaloridine, clindamycin, or bacitracin. Passage of P. aeruginosa in subinhibitory concentrations of different disinfectants induced susceptibility to most of the antibiotics tested (Table 3 and Figure 1). For example, sensitivity to tetracycline or carbenicillin was exhibited by almost 50% of the disinfectant variants. Exposure of strains to disinfectants induced resistance to kanamycin and neomycin in 2% and 4% of strains, respectively. The statistical analyses revealed a high significant difference between disinfectants – nonexposed and disinfectants – exposed isolates to disinfectants and the p value was 0.007 using a paired sample t test. The correlation coefficient (R) between exposed and nonexposed isolated showed a high association with value equal to 0.959714. The present results showed that the calculated coefficient (R²) value was equal to 0.001 for disinfectants – exposed isolates whereas R² of disinfectants – unexposed was equal to 0.0347. Before exposure to disinfectants, current strains belonged to only three antibiotic resistance patterns, but after disinfectant passage there were nine different patterns of sensitivity. Seven isolates of P. aeruginosa showed seven different antibiotic resistance patterns; the resistance pattern (TE, PY, SXT, CR, DA, K, B) was found among 24 strains (Table 4).

The transfer experiments showed no antibiotic marker transferred from either disinfectant-exposed or disinfectant-unexposed P. aeruginosa donor strains to the recipient E. coli J53.2 strain, i.e., the two types of pseudomonads were unable to transfer any of their resistance markers to the recipient strain, E. coli.

The problem of hospital cross-infection due to contamination of disinfectants has been recognized [3]. The passage of bacteria through diluted disinfectants may not only bring about phenotypic changes in their antibiograms [15] but also changes in phage susceptibility patterns [10, 16]. Sublethal inhibitory concentrations of the disinfectants for different strains did not markedly differ between strains. Passage through disinfectants induced susceptibility to most of the antibiotics in many of the strains in the present study. It was found that, in the case of E. coli, the loss of phosphatases and heptose in the lipopolysaccharide resulted in increased susceptibility to antibiotics, such as novobiocin, spiramycin, and actinomycin D [2]. In the present study, a few disinfectants exposed strains became resistant to neomycin and kanamycin, but not to gentamicin or amikacin.

The mechanisms of antibiotic resistance in these variants are still under investigation. One hypothesis would be that the disinfectants altered the target site in the bacterial ribosome, making it less susceptible to neomycin and kanamycin but not to gentamicin or amikacin. Sivaji, Mandal and Agarwal [9] observed that disinfectant derived variants of Staphylococcus aureus became resistant to streptomycin but not to gentamicin or kanamycin. Destruction of various periplasmic enzymes by disinfectants has also been reported [16‒19]. Decreased uptake of antibiotics can also be another contributory factor to the resistance. However, microorganisms exposed to subinhibitory concentrations of antimicrobials tend to adopt an adaptative response or develop resistance mechanisms in order to overcome this selective pressure [20]. It may occur as a horizontal gene transfer, thus enabling the spread of antimicrobial resistance genes and alteration of antimicrobial susceptibility profiles [21].

Moreover, the change in susceptibility does not imply development of resistance [22, 23] because tolerance or adaptation as a phenotypic display may be considered instead [24]. Hence, cross-resistance can be proposed when the use of biocides drives selective pressure toward antimicrobial resistance regarding some microbial subpopulations [25]. The involved stress induces an adaptative response that protects pathogens, producing cellular changes that may affect the native antimicrobial susceptibility pattern [26]. This situation is particularly important in healthcare settings and infrastructures where contamination plays a significant role in healthcare-associated infections [27]. However, comprehensive efforts, including basic infection control education, improved selection, use of products and practical training are required to minimize harmful cleaning and disinfection exposures without reducing the effectiveness of infection prevention [28]. Furthermore, the evolution of antibiotic-resistant healthcare acquired microorganisms after treatment with sub-MICs of different disinfectants has been investigated elsewhere [27]. The results of previous studies suggested that exposure to subinhibitory doses of various disinfectants can induce antibiotic resistance in clinical Pseudomonas isolates through either natural selection process or enforcement of acquiring resistance mechanisms to antibiotics as an adaptation to the new environment. Moreover, in contrast the present data revealed that the exposure of antibiotic-resistant bacteria induced sensitivity to these therapeutic agents. Inducing antibiotic sensitivity might be due to genetic, anatomical, and/or physiological changes occurring in bacterial cells when they are under disinfectants exposure. It has been reported previously that the loss of phosphatases and heptose in the liposaccharide of E.coli resulted in increased susceptibility to antibiotics [17]. However, the coefficient of determination (R²) was very low (0.001) which indicated a very weak relationship between different sensitive exposed isolates tested. Thus, the use of appropriate bactericidal concentrations of various disinfectants should be emphasized by the infection prevention and control specialist as a part of infection control program standards in healthcare settings. Sodium hypochlorite and didecyldimonium chloride are still recommended for low- and intermediate-level disinfection in hospitals. However, precautions are in place regarding the use of appropriate concentrations as recommended by the manufacturers, particularly for prevention of infections caused by antibiotic-resistant bacteria such as P. aeruginosa [28, 29].

The changing susceptibility of P. aeruginosa to antibiotics as a result of exposure to disinfectants has been demonstrated, and 32% of the antibiotic-resistant strains became sensitive to all the antibiotics tested. Apart from antibiograms, other typing procedures for similarity were not performed and the stability of the antibiograms was not tested in control strains similarity cultured but unexposed to disinfectants. The growing concerns about the development of biocide resistance and cross-resistance with antibiotics among pseudomonads have been suggested. It is clear that clinical isolates particularly of P. aeruginosa should be under continuous surveillance and possible mechanisms associated with disinfectant-resistance should be further investigated particularly among hospitals where patients who are mostly immunocompromised are resident [30]. However, the present findings revealed that exposure of some hospital pseudomonads to disinfectants could change the antibiotic sensitivity pattern, and this might lead to the erroneous conclusion that strains are unrelated. In these circumstances, other typing methods are required.

The present findings revealed that exposure of some hospital pseudomonads to disinfectants unexpectedly increased the sensitivity to most of antibiotics tested, e.g., more than 40% of the resistant isolates to tetracycline, carbenicillin, and co-trimoxazole became sensitive to the three antibiotics after exposure to subinhibitory concentrations of disinfectants. The explanations of the present results would be due to genetical, anatomical, and/or physiological changes of the bacterial cells due to exposure to subinhibitory concentration of disinfectants. The resistant patterns of antibiotics for isolates were gathered into three different mode of resistance before exposure and these patterns changed to be nine after exposure to disinfectants. This might lead to the erroneous conclusion that strains are unrelated. In these circumstances, other typing methods are required as an epidemiological tool particularly in hospitals, and this situation calls for further investigations to explore more details of bacterial cellular changes that might occur when the pathogens are under impact of disinfectants exposure.

The author extends his appreciation to the Department of Scientific Research at University of Mosul for funding this work. I would like to acknowledge Prof. Dr. K.J. Towner (University of Nottingham, UK) for providing me with E. coli J53.2 (F⁻ pro met rif^r) as a recipient strain.

All the procedures involving human participation were conducted in strict accordance with the ethical standards of Institutional Research Committee, Department of Scientific Research, Mosul University, as well as the 1964 Helsinki Declaration and its subsequent amendments or equivalent ethical norms.

The author declares that he has no conflicts of interest, financial, or otherwise.

The author extends his appreciation to the Department of Scientific Research of University of Mosul, Iraq.

Mohemid Maddallah Al-Jebouri carried out study design, data collection, manuscript preparation, statistical analyses and literature search.

The data that support the findings of this study are available from the corresponding author upon reasonable request.