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Year : 2021  |  Volume : 14  |  Issue : 1  |  Page : 45-50  

Potential public health impact of the development of antimicrobial resistance in clinical isolates of Pseudomonas aeruginosa on repeated exposure to biocides In vitro

Departments of Microbiology, Armed Forces Medical College, Pune, Maharashtra, India

Date of Submission02-Jul-2020
Date of Decision21-Sep-2020
Date of Acceptance28-Sep-2020
Date of Web Publication22-Jan-2021

Correspondence Address:
Mahima Lall
Department Microbiology, Armed Forces Medical College, Pune, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/mjdrdypu.mjdrdypu_353_20

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Background: Antimicrobial resistance (AMR) is a real threat having the potential of impacting public health immensely. Biocides may contribute to AMR as they are routinely used in household formulations, often in suboptimal concentrations. Gram-negative bacteria like Pseudomonas aeruginosa (P. aeruginosa) can adapt to biocides such as chlorhexidine and cetrimide (CHX + CET) on repeated exposure and develop cross-resistance to antibiotics. Aim and Objective: The aim was to test CHX + CET laboratory-adapted clinical isolates of P. aeruginosa for the development of cross-resistance to antipseudomonal antibiotics. Materials and Methods: Ten clinical isolates of P. aeruginosa were included in the study. The initial antibiotic susceptibility pattern was noted before they were exposed to increasing concentrations of CHX + CET over several days. Briefly, 10 μl of bacterial suspension was inoculated into 10 ml of nutrient broth with the biocide and incubated at 37°C for 48 h. New series of tubes with increasing concentration of biocide were inoculated with growth from the previous tube every 48 h. Till, no further growth was obtained. Antibiotic susceptibility testing for antipseudomonal antibiotics by the disc diffusion as well as the minimum inhibitory concentration (MIC) by VITEK 2 bacterial identification system was performed and repeated before and after exposure to the biocide. The difference in the zone diameter and MIC was noted. Results: Significant difference (P < 0.05) in the mean of the zone size before and after exposure to CHX + CET was noted. Furthermore, there was an increase in MIC postexposure to the biocide. Conclusions: P. aeruginosa on exposure to biocides developed antibiotic resistance.

Keywords: Antimicrobial resistance, biocides, chlorhexidine, Pseudomonas aeruginosa

How to cite this article:
Vaz S, Lall M. Potential public health impact of the development of antimicrobial resistance in clinical isolates of Pseudomonas aeruginosa on repeated exposure to biocides In vitro. Med J DY Patil Vidyapeeth 2021;14:45-50

How to cite this URL:
Vaz S, Lall M. Potential public health impact of the development of antimicrobial resistance in clinical isolates of Pseudomonas aeruginosa on repeated exposure to biocides In vitro. Med J DY Patil Vidyapeeth [serial online] 2021 [cited 2021 Mar 9];14:45-50. Available from: https://www.mjdrdypv.org/text.asp?2021/14/1/45/307682

  Introduction Top

Antibiotic resistance or antimicrobial resistance (AMR) is a threat and a cause of concern world over.[1],[2] Biocides, which include antiseptics, disinfectants, and preservatives, are used in hospitals as well as in the community for decontamination, personal hygiene, and wound care.[3] However, increased use of biocides, probably in suboptimal concentrations may contribute to the emergence of cross-resistance among pathogens making them less susceptible not only to biocides but also to antibiotics, thus compounding the problem of AMR.[4] In recent times, biocides are being explored as one of the contributors for the development of cross-resistance to antibiotics.[5] Unlike antibiotics, the use of biocides is not monitored, especially when used in household formulations, and guidelines regarding the use of biocides are not strictly enforced.[6] The unregulated, sub-optimal concentrations of the biocide used in household products can lead to selective pressure and gradual development of AMR.[7] Internationally, there are only a few guidelines for biocide use in household formulations, no recognized or standardized methods of biocide susceptibility testing and those available are yet to be accepted and put into regulation.[8] We investigated the effects of using suboptimal concentrations of chlorhexidine (CHX) and cetrimide (CET) on clinical isolates of Pseudomonas aeruginosa (P. aeruginosa).

The mechanism of resistance to biocides is both due to horizontal gene transfer and to the selective pressure exerted by the biocide, leading to the development of efflux pumps and cellular impermeability.[9] Since similar mechanisms are at play in antibiotic resistance, concern has been raised that the use of antiseptics may contribute to the development of antibiotic resistance among microorganisms.[10],[11] This selection pressure may eventually contribute to the emergence of multidrug-resistant (MDR) bacteria both in the hospital as well as the community with potential public health impact.

P. aeruginosa is a Gram-negative bacteria (GNB) which is a cause of hospital-acquired infections, particularly in immunocompromised, burns patients and in neonatal intensive care unit (ICUs). A species of considerable medical importance, P. aeruginosa is a MDR bacterium recognized for its ubiquity, its intrinsic antibiotic resistance, and its pathogenicity.[12] Infections caused by this bacterium are difficult to treat due to its intrinsic and acquired antibiotic resistance mechanisms. They also have the ability to remain viable or grow at in-use dilutions of disinfectants. This survival advantage for Pseudomonas results from their nutritional versatility, their unique outer membrane that constitutes an effective barrier to the passage of biocides and efflux pumps.

CHX is a biguanide antiseptic, preservative, and a disinfectant which is effective against bacteria, viruses, and fungi.[13] It causes leakage of bacterial intracellular contents, inhibits bacterial respiratory enzymes and causes coagulation of the cytoplasmic contents.[14] It is used in the home environment include hand washes, mouthwashes, and creams and in the hospital set up as a solution for cleaning instruments. Due to its easy availability and low cost, it is commonly used in the health-care setting often as a combination with CET in a concentration of 0.12%–4%. However, concerns are being raised on the development of cross-resistance in bacteria due to unregulated and increased use.[15]

Aim and Objectives

To develop CHX + CET adapted strains of P. aeruginosa in vitro. To observe antibiotic sensitivity patterns and minimum inhibitory concentrations (MICs) of the adapted strains to antipseudomonal antibiotics before and after exposure to CHX + CET.

  Materials and Methods Top

The institutional ethical committee gave permission to conduct the study (IEC/2019/341 dated 19 Oct 2019). Study design: Laboratory experiment, Study site: Bacteriology laboratory of a tertiary care center. Duration of the study: 6 months (April to October 2019). Inclusion criteria: Ten clinical isolates from ICU yielding positive P. aeruginosa growth on culture. Exclusion criteria: Mixed growth samples. Sample collection: Samples routinely received from the ICU at the laboratory. Processing of samples: Growth, on culture was confirmed as a GNB on Gram stain, further confirmed as Pseudomonas by the oxidase test and conventional biochemical tests, later VITEK 2 bacterial identification system was used to confirm the identity of the isolates grown. Strains of P. aeruginosa were grown on nutrient agar. Isolated colonies were picked for preparing the bacterial suspension of 0.5 McFarland turbidity standard for antibiotic sensitivity testing by the Kirby-Bauer disc diffusion method. This was performed on cation adjusted Mueller–Hinton agar. Each study isolate was tested for antibiotics of the following group; cephems, βlactams, carbapenems, and aminoglycosides using the following antibiotic discs from the Hi-Media laboratories; ceftazidime (CAZ) (30 μg), piperacillin (PIP) (100 μg) (PIP + TAZOBACTAM [TZP] disc was not available), imipenem (IPM) + ethylenediaminetetraacetate (10/750 μg) ([IPM] disc was not available), Meropenem (MEM) (10 μg) and amikacin (AMK) (30 μg).

The MIC for each isolate was tested initially before it was adapted to the biocide using VITEK 2 Gram-negative antibiotic sensitivity testing card (GN AST card). The MIC values of antipseudomonal antibiotics as per M100, Clinical Laboratory Standards Institute (CLSI) guidelines 2020 were tabulated. The MIC values of Ticarcillin/Clavulanic acid (TIM), PIP/TZP, CAZ, IPM, MEM, AMK, Gentamicin (GEN), ciprofloxacin (CIP), and Levofloxacin were included in the study. These were noted for each isolate again after biocide exposure.

The study isolates were then adapted over days to the biocide. The biocide solution containing CHX gluconate (7.5% w/v) and CET (15% w/v) (CGS supply, mfg. by Unicure India Ltd.) was used for the study. Tubes of 10 ml nutrient broth containing 100 μl of 7.5% w/v CHX (7.5μg) + CET (15% w/v) were inoculated using 10 μl of an overnight nutrient broth culture. They were then incubated at room temperature for 48 h and examined for growth, which was observed by increased turbidity of the broth noted by the unaided eye. MIC of the biocide was the lowest concentration of the biocide at which no growth was observed. The growth was checked by subculturing it aerobically on nutrient agar each time the isolate was adapted to a higher concentration of the biocide (7.5 μg/ml per successful subculture). The growth thus obtained was then inoculated into a new series of tubes containing CHX + CET. The biocide was gradually increased as follows; 100 μl, 150 μl, 200 μl, 250 μl, till no further growth was obtained. This was performed every 48 h. The antibiotic susceptibility of the adapted isolates was repeated by the Kirby–Bauer disc diffusion method, and the difference in zone diameters for the aforementioned antibiotics after exposure to the biocide was noted and interpreted as per the CLSI 2020.[16] MIC values of the study isolates were obtained by VITEK 2 bacterial identification system after biocide exposure, these were tabulated as MIC values before biocide exposure and after to antipseudomonal antibiotics.


The mean of the difference in zone diameters before and after biocide exposure was tested for significance, and P value was determined. P < 0.05 was considered statistically significant. The MIC values for various antibiotics were tabulated and compared.

  Results Top

The resistance of P. aeruginosa to CHX + CET increased after step-wise exposure to increasing concentrations of CHX + CET, though it was slow to develop at first and growth was obtained only after 48 h. Of incubation for all the isolates except isolate number one (which failed to grow after the first subculture). The repeat tests to confirm the identity of isolate one by the VITEK bacterial identification system revealed it to be Pseudomonas putida and not P. aeruginosa. [Table 1] shows the mean of the difference in zone diameters before and after biocide exposure. These were found to be significant, and value of P < 0.05. [Table 2] shows the MIC values for antipseudomonal antibiotics before and after biocide exposure. [Table 3] shows the number of isolates with an increase in the MIC values after exposure to the biocide. Antibiotics to which cross-resistance occurred in the majority of the isolates were CAZ and PIP; however, the adapted isolates did not show the development of resistance to TZP.
Table 1: Mean zone sizes (in mm) before and after stepwise exposure of study isolates to chlorhexidine + cetrimide for the following antibiotics

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Table 2: Minimum inhibitory concentration breakpoints (μg/ml) of antipseudomonal antibiotics (GN AST Card VITEK) before and after exposure of the study isolates to biocide chlorhexidine and cetrimide

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Table 3: Number of isolates showing an increase in minimum inhibitory concentration breakpoints after exposure to the biocide

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  Discussion Top

Biocides have played a pivotal role in the disinfection of hospitals as well as in regular domestic use; they are active chemical molecules used for controlling the growth or killing of bacteria.[17] Biocides usage is largely unregulated, and antibacterial cleaning agents and hygiene products are an emerging risk for AMR in the community.[18] One of the first documented episodes which laid the foundation of legislative action against the unregulated use of biocides came in 1998, when Triclosan, a non-specific biocide being used in clinics and homes for over 30 years as a component of antibacterial soaps, body washes, and toothpastes was found to induce a selection pressure leading to the emergence of many Triclosan resistant bacteria, which showed cross-resistance to antibiotics. However, it took until 2016 for the US Food and Drug Administration to ban Triclosan in antimicrobial soaps used by the general population at home.[19] Antiseptic resistance has since been found in many different bacteria showing resistance to a wide spectrum of disinfectants and antiseptics, including iodophors, quaternary ammonium compounds, biguanides, peroxides, and glutaraldehyde.[20] Many other antibiotic-resistant strains of bacteria also show resistance to biocides.[21],[22] Extensive and unregulated use of biocides could lead to the selection of clinical isolates showing resistance to both antibiotics and biocides.[23]

Pseudomonas, known for its resistance to antibiotics, has been documented to show resistance against many biocides, including quaternary ammonium compounds and Triclosan. Resistance against CHX has been demonstrated in Pseudomonas in both, in vitro and in clinical isolates.[24] Being a common cause of nosocomial infections, which are often fatal, this is a cause for concern. CHX, owing to its availability, accessibility, and convenience, is one of the most commonly used disinfectants in the ICU and clinical settings. However, there is a 32-fold difference between the lowest and the highest concentrations used (0.12%–4%) in clinical practice. Such variations in concentration are problematic due to concerns about the potential for under-dosing and development and the spread of resistance. The resistance of P. aeruginosa to CHX has started posing problems of clinical concern worldwide. P. aeruginosa also has been reported to contaminate disinfectants (e.g., CHX, benzalkonium and Triclosan) in the hospital or other such environments, thereby compromising the disinfectant's ability to reduce or eliminate bacterial contamination. In Trinidad, 11 of 120 disinfectant solutions were found to be contaminated with Pseudomonas spp., with resistance rates to CIP of 58.3%, to norfloxacin of 50.0%, to tobramycin of 45.8%, and to GEN with 41.7%, which were used in pediatric and surgical settings.[25] In Kolkata, strains of Pseudomonas resistant to CHX were isolated from clinical specimens, which showed resistance to PIP, MEM, IPM, and AMK. Due to these studies, there is reason to believe that the high prevalence of drug-resistant Pseudomonas in wards and ICUs can be, in part, due to the indiscriminate and incorrect use of antiseptics like CHX.[26]

Strengths of our study include the following; it is the first in vitro study from western Maharashtra reporting cross-resistance and the potential of MDR hospital pathogens (ICU clinical isolates) developing on repeated exposure to sub-inhibitory concentrations of biocides. We noted increasing MICs of such adapted strains to antibiotics indicating the development of cross-resistance. Few studies have reported similarly.[27] In our study, isolate one did not adapt to increasing concentrations of CHX + CET and failed to grow in concentrations higher than 7.5 μg/ml, on VITEK it was later identified as P. putida. Growth of P. aeruginosa in nutrient broth containing CHX + CET was noted at concentrations up to 30 μg/ml. Since mechanisms of antibiotic and biocide resistance overlap, we showed biocide adapted strains of Pseudomonas becoming resistant to antipseudomonal antibiotics.[28] Our study raises concern about the use of antiseptics contributing to the development of antibiotic cross-resistance among bacteria. We developed in the laboratory strains with reduced susceptibility to CHX + CET that demonstrated decreased susceptibility to antibiotics. Excessively diluted antiseptic and disinfectant solutions used for cleaning in the hospital and domestic environment may select resistant bacterial strains. Limitations of our study include a small sample size.

  Summary and Conclusions Top

The study hypothesis was to test CHX resistant strains of P. aeruginosa for antibiotic resistance. We exposed the clinical isolates of P. aeruginosa to varying concentrations of biocide. We carried out antibiotic susceptibility testing of the isolates to note their sensitivity pattern before and after exposure to CHX. The difference in the sensitivity patterns, pre- and post-exposure was noted. The effect of biocide on the bacterial isolate was developed through exposure to increasing concentrations of biocide through five subcultures and sensitivity of the study isolates to selected antibiotics was performed by the Kirby–Bauer disc diffusion test. MICs were determined by the VITEK 2 bacterial identification system. CHX is a frequently used antiseptic in the community and the hospital setting. Increasing prevalence of its usage may lead to the development of resistant strains of bacteria, which can cause outbreaks in a community as well as nosocomial infections in hospitals. Research in antiseptic resistance may pave the way for better guidelines on usage and significantly reduce morbidity and mortality. Suboptimal concentration exposure of biocides can select bacterial strains that confer AMR. Household use of antimicrobial chemicals and biocides is often overlooked as a factor of persistent low-level exposure contributing to the selection of antibiotic-resistant strains.[29] Our study may pave the path toward larger studies testing different classes of biocides and pathogenic Enterobacteriaceae and cross-resistance with commonly used antibiotics. Finally, our study though small has brought to the fore legislative and policy concerns, which may need to be considered by the government to control AMR.[30]


To the Head of the Department of Microbiology for the support. Senior Laboratory Technician Sub Prakash for technical help.

Financial support and sponsorship

The grant for the work was from the Indian Council Medical Research Short Term Studentship (ICMR-STS-2019).

Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Table 1], [Table 2], [Table 3]


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