The Efficiency of Dettol as Detergent against Microbial Biofilm formation isolated from UTI infections

Current Research in Microbiology and Biotechnology

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The Efficiency of Dettol as Detergent against

Microbial Biofilm formation isolated from UTI

infections

Shaymaa Suhail Najim*

Baghdad University, College of Science, Biology Department, Baghdad, Iraq.

* Corresponding author:Shaymaa Suhail Najim; e-mail: ghada90m@gmail.com

ABSTRACT

In response to the widespread of Dettol using for hand cleaning in our population, this study was designed to investigate the efficiency of Dettol on the growth of biofilm producing pathogenic bacteria that is associated with urinary tract infections (UTI). Urine samples (50) were collected to identify the microorganism isolates using traditional laboratory methods along with antibiotic sensitivity test. Biofilm production against detergent was performed for most resistant bacterial isolates. Results showed growth of 38 (76%) different kinds of pathogenic bacterial isolates. Most of them showed multi-drug resistance (MDR). In this study, the results revealed that E. coli isolates are the most common pathogen followed by K. pneumonia and Enterobacter than other bacterial pathogens. The dilution 1:10 of detergent observed the MIC for the growth of bacterial isolates. Meanwhile, the highest dilution of detergent more than 1:10 lost ability to eradicate biofilm growth. Moreover, the increasing of dilution showed enhancement of biofilm production in Staph. epidermidis isolate as compared to positive control. In conclusion, the commercial Dettol soap has weak ability to eradicate biofilm growth from the surface of plate assay.

Keywords:

1. INTRODUCTION

Detergents are urgent needs in everyday life because they represent assistant chemicals for cleaning and removing dirt and germs that may attach contaminated surfaces. In response to the increasing concern about health hygiene by hand washing due to cross contaminating of pathogenic bacteria, thus the use of detergent is necessary for cleaning [1]. The goal of detergent is to totally eradicate microorganisms on body surfaces such as hands. Adhesions of a microorganism to equipment surface enable to direct or indirectly transmit to other body parts and environment [2].

Dettol is a chemical liquid with a very distinctive smell that we can recognize easily. It is an aromatic compound derived from phenol, which contains a significant chlorine atom 4-chloro-3,5-dimethylphenol

which is the molecule dettol. Dettol is usually used in domestic and hospital sites as a safe and effective way to disinfect different surfaces. It has wide range of effect on different kindes of microorganisms especially "superbug" MRSA [3]. Chloroxylenol is the key halophenol which is used in antiseptic or disinfectant formulations, it is bactericidal but P. aeruginosa and many fungi are resistant. Surprisingly, its mechanism of action has been poorly studied despite its much extended use over many years. Because of its phenolic nature, it would be expected to have an effect on microbial membranes [4].

Biofilms are described as a microbial population adhered to a substrate and surrounded by an extracellular matrix which is usually self-derived. This mode of life presents important clinical repercussions

since it is estimated that over half of all hospital infections are originated from these microbial communities [5], they represent the growth of bacteria on the biotic and abiotic surfaces in which the bacteria embed themselves in a self-produced extracellular matrix of exopolysaccharide (EPS), proteins and some micro molecules such as DNA [6]. Biofilms play an important role in nature, in which under non-optimal circumstances they can survive and in medicine, medical devices used inside and outside the body are usually coated with biofilm producing bacterium which can promote and sustain infection, they are not easily eradicated by antibiotics [7]. Several studies have shown that biofilms produced by bacteria are responsible for persistent infections in humans such as dental caries periodontitis, and many others [8,9].

Because widespread of Dettol using for hand cleaning in our population, this study designed to investigate the efficiency of Dettol on the growth biofilm producing pathogenic bacteria that is associated with UTIs.

2. MATERIALS AND METHODS

Urine samples (50) were collected from UTIs patients attending different hospitals in Baghdad; they were all inoculated on MacConkey agar and blood agar, incubated at 37C° for 24 hr. The colonies were purified by sub-culturing on nutrient agar. The isolates were identified according to the morphological characteristics on culture medium, microscopically on the slide and biochemically with the use of API system kit [10].

2.2 Antibiotic sensitivity test: - (Disk diffusion method)

Antibiogram of bacterial isolates was tested for selected antibiotics representing various classes of antimicrobial agents, as shown in Table 1. The kirby-baure method was used according to [11, 12] to carry out antimicrobial susceptibility. The isolates were interpreted as susceptible, intermediate or resistant to antibiotics used in study by comparison with standard inhibition zones as mentioned in Table (2).

Table 1: Antimicrobial discs used in this study.

Code Disc potency

g/disc)µ( Antibiotics (Antimicrobial discs) No. AK 10 Amikacin 1. C 10 Chloramphenicol 2. IC 10 Imipenem Cilastin 3. TE 10 Tetracycline 4. CIP 10 Ciprofloxacin 5.

Table 2: Inhibition zones of antibiotic discs according to CLSI (2013).

Diameter of inhibition zones (mm) Code Disc potency (µg/disc) Antibiotic No. Resistant Susceptible Interpreted ≤ 14 ≥ 17

15 - 16 AK 10 Amikacin 1. ≤ 19 20–22 ≥ 23 IC 10 Imipenem 2. ≤ 11 12–14 ≥ 15 TE 10 Tetracycline 3. ≤ 12 ≥ 18

13 - 17 C 10 Chloramphenicd 4. ≤ 15 ≥ 21

16 - 20 CIP

10 Ciprofloxacin

5.

2.3 Preparation of Inoculum

Bacterial strains were prepared in inoculum normal saline to adjust its turbidity by taking 3-4 colonies from an 18 hr. fresh culture by sterilized loop and suspended in 5 ml normal saline to get a culture with 1.5×108

CFU/ml and comparing with turbidity standard of McFarland No. 0.5.

2.3.1 Inoculation and Applying antibiotic discs

By a sterile cotton swab, a portion of bacterial culture (swab dipped into inoculum tube) was transferred and carefully evenly spread on Muller -Hinton agar medium and left for 10 min. Subsequently, the anti-microbial discs were placed on the agar medium using a forceps (sterilized with alcohol and flam) and pressed on the agar to ensure contact with the surface. The plates were incubated at 37 C° for 18 hr.

2.3.2 Reading the inhibition zone

Inhibition zone for each antibiotic was measured by a metric ruler in millimeters (mm) according to the clinical laboratories standards institute (CLSI) [13]. There are many of factors effect diameter of inhibition zone that should be considered such as the thickness of medium (not more than 4-6 mm as it reduces the size of the zone), and too thin medium may exaggerate zone appear, also humidity and the age of medium may also effect zone size [14].

2.4 Detection of Biofilm production with Crystal Violet staining

Overnight cultures of trypticase soy broth (TSB) supplemented with (1% glucose) were diluted until 108

CFU/ml in TSB. Individual wells of polystyrene, flat-bottomed 96 well plates were filled with 200 microliter aliquots of the cultures which were further incubated for 24 hr. at 37C°. Next, the wells were washed 3 times with 200 microliters of sterile phosphate buffer saline (pH: 7.2) and biofilm was fixed with heating at 60 C° for 15 min. 200 microliter of crystal violet solution (0.1% wt. /vol.) was added to all wells and left for (15 min).

Excess crystal violet was rinsed with distilled water and air dried overnight. Bounded crystal violet was released by adding 200 microliters of 96% ethanol and absorbance was measured spectrophotometrically at 490 nm (A490) and was proportional to biofilm biomass.

The test was performed in triplicates and negative control wells contained TBS, performed in triplicates. The results were calculated according to [16] as in Table (3).

Table 3: Values of biofilm formation by bacterial isolates.

OD values Biofilm Formation

<ODc None

ODc<ODt ≤ 2*ODc Weak 2*ODc<ODt ≤ 4*ODc Moderate

4*ODc<ODt High

2.5 Investigation of Detergent effect on Biofilm production

2.5.1 Minimum inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) determination

The MICs and MBCs tests were performed in 96-well microtiter plates. Twenty four bacterial isolates were determined for MIC using McFarland standard (5×106_{CFU/ml) by diluting 1: 1000 using (TSB) Tryptic}

soy broth. Antibiotics were diluted 1: 10 in TSB containing 5×103 _{CFU/ml inoculums. positive control}

containing (only medium and inoculum), and negative control containing (only medium). After 24 h incubation at 37 C, the MIC was measured as the lowest concentration of antimicrobial agent resulting in complete inhibition of visible growth [17]. To determine MBCs, wells with no visible growth were used. The medium (100 µl) of each well was removed and was placed onto agar plates spread by a glass spreader and incubated at 37 C for 24 hr. The positive controls and growth-negative controls were included in the MBC test. MBC was defined as the lowest concentration of antimicrobial agent at which bacterial isolates in the culture were killed.

2.5.2 Dettol eradication for Bacterial Biofilm Growth

To measure eradication of the detergents, adhering cells in the 96-well microtiter plates were mixed with 100 µl of bacterial culture containing 5×103 _CFU/ml.

The wells in rows one to six of the microtiter plate included bacterial cultures (100 µl) with MIC detergent (100 µl) . Positive control (medium and inoculum), negative control (only medium). Then incubated at 37 °C for 24 h. In order to determine the detergent effect on biofilms production, the optical density (OD490) measured and compared to the control reading.

2.6 Statistical analysis

Statistical analysis was performed with SPSS 21.0 statistical package (SPSS Inc., Chicago, Illinois, USA). The frequencies for different groups were compared

between them using chi-square test. The values for P < 0.05 were considered statistically significant.

3. RESULTS AND DISCUSSION

3.1 Bacterial Isolation and Identification-:

Clinical specimens (50) collected from urine and showed growth of 38 (76%) different kinds of pathogenic bacterial isolates, they were tested for their susceptibility to five groups of antibiotics as summarized in Table (4). Most of them showed multi-drug resistant (MDR). Results revealed that E. coli

isolates were the most common pathogen followed by

K. pneumonia and Enterobacter than other bacterial pathogens in this study. The significant appearance of specific bacterial isolates compared with them that attribute in the current study (x2_{=22.28, P<0.05).}

More than 70% of bacterial isolates appeared resistant against tetracycline, chloramphenicol, and amikacin antibiotic discs. Whereas, the resistance against imipenem cilastin and ciprofloxacin were showed 57.89% and 47.37%, respectively. Generally, most of the isolates considered resistance to antimicrobial agents in this study.

3.2 Detection of Biofilm production by some pathogenic bacterial isolates against Dettol soap

In this study, biofilm assay pointed out that most of the isolates have the ability to produce biofilm, thus the selection of the isolates along depending on multi-drug resistance results to perform the effect of Dettol inhibition for biofilm growth. Meanwhile, the dilution 1:10 of detergent observed the MIC for the growth of bacterial isolates. Biofilm formation is the most important factor in the development of chronic infection and allows for immune evasion as well as resistance to antimicrobial agents [18] so that the only method of successful treatment is its removal from the injured tissues or devices coated with such biofilms [19].

Regarding the effect of Dettol for biofilm inhibition of all tested isolates, the biofilm inhibition appeared at dilution 1:10 (MIC) of detergent for most bacterial isolates when compared with positive control, exception the Acinetobacter isolate exhibited high producer biofilm along with detergent, Table (5).

Meanwhile, the highest dilution of detergent more than 1:10 lost ability to eradicate biofilm growth. Moreover, the increasing of dilution showed enhancement of biofilm production in Staph. epidermidis isolate as compared to positive control.

Table 4: Antibiotic Susceptibility of some pathogenic bacterial isolates.

Isolate (No.) Antibiotics with their results

CIP IC C AK TE

Acinetobacter (2) R (2) S (1),R(1) R (2) R (2) R (2)

Strep. Pneumonia (1) S (1) R (1) R (1) R (1) R (1)

Strep. Pyogenes (1) S (1) S (1) R (1) I(1) R (1)

Strep. Fecalis(1) R (1) S (1) R (1) S (1) R (1)

Staph. Epidermidis (3) S (1), R

(1),I(1)

S(3) I(1), R (2) R (2),S(1) R (1),S(2)

Staph. Aureus (4) R(2), I (2) R (4) I(1), R (3) R (4) S (1), R (3)

Proteus (3) S(3) S(2),R(1) R (3) I(3) R (3)

Vibrio cholera (2) S (2) S,I I(1), S (1) R (2) S (1), R (1)

E. coli (8) I (1), R (7) R(4),S(3), I(1) I(2), R (6) R (8) R (8)

Pseudomonas (3) I (I), S(2) R (1),S(2) R (3) S (1), R(1) I(1) R (3)

Enterobacter (5) S (5) R (5) I(2), R (3) R (5) R (5)

K. pneumonia (5) R (5) R (5) R (5) S (1), I (3) R(1) S (1), R (4)

Total : 38 - - - -

-S: Sensitive, I: Intermediate, R: Resistance, (no. of isolates)

mTable 5: Biofilm formation by some pathogenic bacteria in presence of Dettol soap.

Dilutions Staph1 _{Pseud Acineto Entero E.coli Strep}2 _Strep3 _K.4 _K.4 _Staph5 _Staph5 _Vibrio Dilution 1

(1:10) N N H N N N W N W W N W

Dilution 2 W W H W W W M W M M N W

Dilution 3 M M H W M M M W M M W M

Dilution 4 M M H M M M M M H M W M

Dilution 5 M H H M M M M M H M W M

Dilution 6 M H H M M M M M H M M M

- ve control 0.084 0.081 0.087 0.096 0.088 0.093 0.095 0.098 0.097 0.089 0.081 0.08 +ve control 0.135 0.281 0.332 0.254 0.329 0.269 0.304 0.286 0.305 0.325 0.249 0.291

1: Staphylococcus.epidermidis, 2: Streptococcus.pyogenes, 3: Streptococcus pneumonia, 4: K. pneumonia, 5: Staphylococcus.aureus, -ve: detergent

without bacteria, +ve: bacteria without detergent. H: high, M: moderate, W: weak, N: non-producers

The potential to produce biofilm increased the high prevalence of resistant bacteria in UTI. Also, pathogenic bacteria with MDR showed high to moderate biofilm production as similar to bacterial biofilm producers in Dettol presence.This explained the weak Dettol ability to eradicate biofilm growth from hands when it was used for hands washing and cleaning after.

Most pathogenic bacteria sustain its pathogenicity from antibiotic resistance and its related genes [20,21], the main reason for antibiotic resistance is due to the uncapability of antibiotics diffusion through the biofilm matrix and the bacterial decreased metabolic activity. Researches have studied the strategies that facilitate bacteria to produce biofilm [22], that biofilm producing bacteria secrete certain chemicals that protect it from disinfectants, antimicrobial and immunologic systems

[23]. The intercellular adhesion gene (ica) regulates extracellular polysaccharide adhesion. The accessory gene regulator (agr) quorum sensing (QS) system has been related to biofilm formation and regulates microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) binding extracellular matrix (e.g. fibronectin, fibrinogen, and collagen) [24]. PS/A mediate initial adherence to solid surfaces and PIA mediate accumulation of cells on biofilms [25].

4. CONCLUSION

© 2017; AIZEON Publishers; All Rights Reserved This is an Open Access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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