Identifying and elucidating the resistance of Staphylococcus aureus isolated from hospital environment to conventional disinfectants

Abstract

Introduction
Staphylococcus aureus is a nosocomial pathogen, detection and elucidation of its resistance mechanisms to conventional disinfectants may aid in limiting its spread on environmental surfaces in healthcare settings. In the current study, disinfectant susceptibility of S. aureus strains isolated from the hospital environment as well as possible associations between the presence of disinfectant-resistance genes and reduced susceptibility to disinfectants was investigated.

Methods
A total of 245 samples were collected from the hospital environmental surfaces. The minimum inhibitory (MIC) and bactericidal concentrations (MBC) of disinfectants against S. aureus isolates were determined using the micro-broth dilution method. The qac genes (qacA, qacE, and qacΔE1) were detected by PCR and confirmed by sanger sequencing.

Results
A total of 47 S. aureus strains were isolated, with more than 85% of them showing methicillin resistance. The qacA, qacE, and qac∆E1 genes were found in 23.4%, 29.7%, and 4.2% isolates respectively. All the isolates with qac genes had higher MIC and MBC values to selected disinfectants.

Conclusion
Significant methicillin resistance S. aureus (MRSA) contamination in the hospital environment was detected. Furthermore, higher qac gene frequencies were found in MRSA isolates that also correlated with higher MIC/MBC values to different disinfectants. The study proposes that hospitals should develop policies to determine disinfectant MICs against the common environmental isolates to contain the spread of resistant strains.

Full text

Major Article
Identifying and elucidating the resistance of Staphylococcus aureus
isolated from hospital environment to conventional disinfectants
Samreen Sarwar M.Phil
a,
*, Sidrah Saleem PhD
a
, Faheem Shahzad PhD
b
, Shah Jahan PhD
b
a
Department of Microbiology, University of Health Sciences, Lahore, Punjab, Pakistan
b
Department of Immunology, University of Health Sciences, Lahore, Punjab, Pakistan
ABSTRACT
Background: Staphylococcus aureus is a nosocomial pathogen, detection and elucidation of its resistance
mechanisms to conventional disinfectants may aid in limiting its spread on environmental surfaces in health
care settings. In the current study, disinfectant susceptibility of S. aureus strains isolated from the hospital
environment as well as possible associations between the presence of disinfectant-resistance genes and
reduced susceptibility to disinfectants was investigated.
Methods: A total of 245 samples were collected from the hospital environmental surfaces. The minimum
inhibitory (MIC) and bactericidal concentrations (MBC) of disinfectants against S. aureus isolates were deter-
mined using the micro-broth dilution method. The qac genes (qacA,qacE, and qacDE1) were detected by PCR
and confirmed by sanger sequencing.
Results: A total of 47 S. aureus strains were isolated, with more than 85% of them showing methicillin resis-
tance. The qacA, qacE, and qac
Δ
E1 genes were found in 23.4%, 29.7%, and 4.2% isolates respectively. All the iso-
lates with qac genes had higher MIC and MBC values to selected disinfectants.
Conclusions: Significant methicillin resistant S. aureus (MRSA) contamination in the hospital environment
was detected. Furthermore, higher qac gene frequencies were found in MRSA isolates that also correlated
with higher MIC/MBC values to different disinfectants. The study proposes that hospitals should develop pol-
icies to determine disinfectant MICs against the common environmental isolates to contain the spread of
resistant strains.
© 2022 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All
rights reserved.
Key Words:
Susceptibility
Qac genes
Chlorhexidine
Quaternary ammonium compounds
INTRODUCTION
Hospital-acquired infections (HAIs) have been progressively rising
over the last few decades, owing in part to the emergence of drug and
disinfectant resistant bacterial strains, which are thought to have
resulted from the misuse of antibiotics and disinfectants,
respectively.
1,2
Contaminated environmental surfaces in hospitals
are known to play a significant role in the transmission of HAIs.
3
The
elimination of bacteria from the hospital environment is especially
important for Staphylococcus aureus because these bacteria are easily
disseminated through contact with surfaces on which they have been
proven to persist for lengthy periods (up to 7 months).
3
The public
now uses hospital-acquired Methicillin-resistant Staphylococcus
aureus (MRSA) infection as a measure of hospital hygiene.
4
S. aureus,
as a nosocomial pathogen, can cause serious infections such as pneu-
monia, primary bacteremia, and infective endocarditis, particularly in
immunocompromized patients.
5
Cleaning and disinfection of environmental surfaces are critical ele-
ments of infection control and prevention programs in hospitals.
6
Dis-
infectants are widely used in hospitals and are found in a wide range
of products. Despite their growing use, bacteria are still sensitive to
disinfectants when administered correctly. The widespread and indis-
criminate usage of disinfectants in a variety of contexts, however, has
prompted worries regarding the development of disinfectant-resistant
bacteria.
7
If disinfection failures occur due to disinfectant resistance
and are not recognized by the relevant stakeholders, this can lead to
serious repercussions such as outbreaks.
8
Therefore, whether current
disinfection regimens can achieve appropriate disinfection and elimi-
nate nosocomial pathogens including the particular S. aureus strains
* Address correspondence to Samreen Sarwar, Department of Microbiology, Univer-
sity of Health Sciences, Khayaban-e-Jamia Punjab, Lahore-54600, Pakistan.
E-mail addresses: sarwarsamreen3@gmail.com,samreen@healthsecuritypartners.
org (S. Sarwar).
Conflict of interest: None to report.
https://doi.org/10.1016/j.ajic.2022.05.018
0196-6553/© 2022 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.
American Journal of Infection Control 51 (2023) 178−183
Contents lists available at ScienceDirect
American Journal of Infection Control
journal homepage: www.ajicjournal.org

found in the hospital environment is a pressing issue that must be
addressed. In S. aureus, several plasmid-borne genes encoding efflux
pumps confer reduced susceptibility to routinely used disinfectants in
S. aureus, which warrants attention and investigation.
2
In the present study, S. aureus strains were isolated from the envi-
ronment of long-term care facilities of a tertiary care hospital in
Lahore, Pakistan. The findings of this study will guide hospitals in
terms of selecting disinfectants, preventing ineffective disinfection,
containing the spread of disinfectant-resistant strains, and reducing
the occurrence of HAIs.
METHODS
Specimen collection, isolation and identification of bacteria
It was a cross-sectional study that was approved by the Advanced
Studies and Research Board (letter no. UHS/Education/126-20/898)
and Ethical Review Committee (letter no. UHS/REG-20/ER/110) of the
university, and Institutional Review Board (IRB) of the tertiary care
hospital (letter no. SZMC/IRB/External/0028/2020). The sample size
was calculated using data from a study by Cheatham et al.
9
A total of
245 samples were collected between October 2021 and January
2022, from the environmental surfaces of medical wards (n = 4), gen-
eral surgery and surgical oncology wards (n = 4), urology wards
(n = 5), and orthopaedic wards (n = 2) at a tertiary care hospital in
Lahore, Pakistan. These wards were chosen based on the type and
epidemiology of nosocomial infections, hospital occupancy, and
duration of stay.
10
The sampling sites were categorized as follows: (i) Frequently
touched surfaces (FTSs) (n = 64); these surfaces included IV pole grab
area (n = 14), bedside table handles (n = 21), room light switches
(n = 11), tray tables (n = 11), and room door knobs (n = 7). (ii) Medical
equipment (ME) (n = 59); these included blood pressure apparatus
(n = 12), cardiac monitor (n = 10), nebulizer (n = 1), oxygen supply
unit (n = 29), surgical aspirator (n = 3), syringe pump machine (n = 1),
weight machine (n = 1), and X-ray illuminator (n = 2). (iii) Patient’s
immediate surroundings (PISs) (n = 60); these surfaces included bed
rails (n = 30), bed linen (n = 20), and patient chair (n = 10). (iv) Ward
sinks (n = 62).
The sampling sites were selected based on existing data indicating
that these sites pose the greatest risk of nosocomial infections. Simi-
larly, the wards'handwashing sinks were sampled because these sur-
faces are known to harbor bacterial biofilms due to the moist and
conducive environment.
11,12
To collect samples, 30 cm
2
levelled envi-
ronmental surfaces were swabbed for 1 minute with sterile swabs
moistened with sterile 0.85% NaCl solution. Swabs were moved
across the sampling area in a systematic manner that is, back and
forth in 1 direction, then 90° degrees to the first, then diagonally. For
unlevelled and protruding surfaces (eg, tubing and bed rails), as large
an area as possible was swabbed.
13
Upon completing the sampling, the swabs were placed in amies
transport medium (Thermo Fisher Scientific, TS0001A) and were
maintained at 2 - 8°C temperature during shipment to the lab. All
specimens were inoculated on blood agar for 24 hours at 35°C. The S.
aureus strains were identified using gram stain followed by catalase,
slide coagulase, and DNase test.
14
Antimicrobial susceptibility testing
Antibiotic Susceptibility testing was carried out by the Kirby-
Bauer disk diffusion method according to the CLSI 2021 guidelines
15
for linezolid (30mg), penicillin (10 units), cefoxitin (30mg), doxycy-
cline (30mg), trimethoprim-sulfamethoxazole (1.25/23.75mg), cipro-
floxacin (5mg), clindamycin (2mg), gentamicin (10mg), erythromycin
(15mg), and azithromycin (15mg). The susceptibility to vancomycin
and teicoplanin was tested using the standard micro-broth dilution
method.
16
For susceptibility testing, ATCC strains 25922 and 25923
were used as controls.
Determination of MICs and MBCs of disinfectants
The disinfectants used in this study were benzalkonium chlo-
ride (BAC) (Sigma, B6295), »and 99% cetrimonium bromide
(CTAB) (Sigma, H6269), and ≥99.5% chlorhexidine (CHX) (Sigma,
282227), because these are some of the most frequently used dis-
infectants in health care settings.
17
For all S. aureus isolates, the
MIC of each disinfectant was determined.
18
In the absence of any
standard breakpoints for disinfectants against S. aureus,concen-
trations modified from the data, reported by Vijayakumar and
Sandle were used.
2
For MIC testing, the concentration ranges
were 2-512 mg/ml for CHX, 1-256 mg/ml for BAC, and 4-1024 mg/
ml for CTAB. The stock solution of BAC (1mg/ml) and CTAB (2mg/
ml) was prepared in warmed distilled water. The stock solution
of CHX (1mg/ml) was prepared in DMSO.
96-well microtiter plates were set up with doubling dilutions of
the disinfectant with starting concentrations in Mueller Hinton (MH)
broth. The bacterial suspension was prepared in 5ml sterile saline
and diluted 1:100 times after being adjusted to a turbidity equivalent
to that of a 0.5 McFarland standard. A fixed volume of the bacterial
suspension was added to all wells with doubling dilutions of the dis-
infectants and positive control growth wells with no disinfectant.
Two wells, 1with distilled water and one with MH broth alone, were
used as negative controls in each test. The final concentration of the
inoculum was »1£10
6
CFU/ml in each well. After 24 hours of incu-
bation at 35°C, the MIC values were recorded as the lowest concen-
trations of disinfectant that inhibited visible growth. The MBC of
disinfectants was determined by inoculating 10ml of the liquid from
the wells with no growth onto tryptone soya agar (TSA) and incubat-
ingitfor24hoursat37°C.
18
MBC was defined as the lowest concen-
tration of disinfectants resulting in the death of 99.9% or more of the
initial inoculum.
Detection of qac genes in S. aureus isolates and sequencing
Genomic DNA from the S. aureus isolates was extracted using the
boiling method.
19
Multiskan Sky (Fisher Scientific Inc.) was used to
quantify and assess the quality of extracted DNA samples. All S.
aureus isolates were screened for the presence of the quaternary
ammonium compound (qac) resistance genes qacA, qacE, and qacDE1
using PCR. Each PCR reaction consisted of 10 mL of DreamTaq Master
Mix (2X) (Thermo Fisher Scientific, Waltham, MA USA), 7 mL sterile
distilled water, 0.5 mL each of the forward and reverse primers (at
20 nM each), and 2 mL DNA to make up a total volume of 20 mL. The
primer sequences for qacA, qacE, and qac
Δ
E1 genes were F5’-
GCTGCATTTATGACAATGTTTG-3’and R5’-AATCCCACCTACTAAAG-
CAG-3’,F5’-GCCCTACACAAATTGGGAGA-3’and R5’-TTAGTGGG-
CACTTGCTTTGG-3’, and F5’-AATCCATCCCTGTCGGTGTT-3’and R5’-
CGCAGCGACTTCCACGATGGGGAT-3’respectively, as described by
Hassan and Al-Khafaji.
20
Thermal cycling was performed on an iQ5
iCycler (Bio−Rad Laboratories, Inc., Hercules, California, USA) with
annealing temperatures of 54.8°C, 57.2°C and 56°C for qacA, qacE, and
qac
Δ
E1 genes respectively. The PCR-amplified products were
detected by electrophoresis on a 2% agarose gel and viewed with a
Bio-Rad Gel Doc XR+ Gel Documentation System (Bio−Rad Laborato-
ries, Inc.). The amplicons obtained were purified and sequenced using
the conventional Sanger dideoxy sequencing method at a commercial
DNA sequencing service provider (Lab Genetix) for validation. DNA
sequence data were analyzed by using NCBI-BLAST (https://blast.
ncbi.nlm.nih.gov/Blast.cgi).
S. Sarwar et al. / American Journal of Infection Control 51 (2023) 178
−
183 179

Statistical analysis
Data were analyzed by SPSS v26. Frequency and percentages
were calculated for drug resistance and qac genes. Chi-square
was applied to determine the association between the carriage of
disinfectant resistance genes and observable reduced antiseptic
susceptibility. The MBC and MIC data were compared using the
Mann-Whitney U-test and median test. P-value≤.05 was consid-
ered statistically significant.
RESULTS
Distribution of S. aureus isolates in the hospital environment
A total of 47 (19.1%) S. aureus strains were isolated from 245 hos-
pital environmental samples. The highest number of S. aureus isolates
(25%) were obtained from orthopedic wards followed by urology
wards (20.8%), medical wards (17.2%), and general surgery and surgi-
cal oncology wards (5.9%). Bedside table handles (38.1%) were found
to be the most contaminated with S. aureus among the environmen-
tal surfaces, followed by bed rails (33.3%), oxygen supply unit (31%),
and bed linen (25%). Table 1 shows the distribution of S. aureus at var-
ious sampling sites.
Antimicrobial susceptibility testing
Out of 47 isolates, 85% (n = 40) of the S. aureus isolates were resis-
tant to cefoxitin and were identified as Methicillin-resistant Staphylo-
coccus aureus (MRSA). Only 4 and 3 MSSA S. aureus isolates were
found in the orthopedic and medical wards, respectively. Drug resis-
tance manifested by S. aureus towards penicillin and erythromycin
was 85.1%, followed by azithromycin (78.7%), ciprofloxacin (59.6%),
clindamycin (34%), gentamicin (34%), trimethoprim/sulfamethoxa-
zole (27.7%), doxycycline (14.9%), and linezolid (4.26%). Doxycycline,
ciprofloxacin, trimethoprim/sulfamethoxazole, and teicoplanin sus-
ceptibility were intermediate in 10.6%, 4.3%, 2.1%, and 2.1% of S.
aureus isolates, respectively. 79% of the isolates (n = 37) were multi-
drug-resistant (MDR), that is, they were resistant to 3 or more than 3
classes of antimicrobials. None of the multidrug-resistant isolates
was susceptible to methicillin (cefoxitin). All S. aureus isolates were
sensitive to vancomycin.
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal
Concentration (MBC) to disinfectants
The MIC90 ranges observed for CHX and CTAB were 2-256 mg/ml,
while for BAC MIC90 range was 1-16 mg/ml. The MBC90 ranges
observed for CHX and CTAB were 8-512 mg/ml and 2-512 mg/ml
respectively, while for BAC MBC90 range was 1 −64 mg/ml (Table 2).
Majority of the of S. aureus isolates showed higher MIC90 values that
is, ≥64 mg/mL for CHX (87.2%) as compared to BAC (0%) and CTAB
(4.2%). For BAC, the isolates mainly displayed MIC90 values of 1 mg/
mL (38.3%), 2 mg/mL (23.4%) and 4 mg/mL (19.1%). For CTAB, the iso-
lates mainly displayed MIC90 values of 1 mg/mL (38.3%), 2 mg/mL
(23.4%) and 4 mg/mL (19.1%).
Detection of qac genes in S. aureus isolates
Representative amplicons of each gene (qacA, qacE and qac
Δ
E1)
detected on gel electrophoresis are shown in Figure 1A. The qacA
gene was found in 11 isolates (23.4%). The qacE gene was detected in
14 (29.7%) isolates, while qac
Δ
E1 was found in only 2 isolates (4.2%).
MRSA isolates showed a higher frequency of qac genes (35%) as com-
pared to Methicillin-sensitive Staphylococcus aureus (MSSA) (14.3%)
isolates; however, it was statistically insignificant (P= .4) (Fig 1B).
There was also no statistically significant association between the
presence of qac genes and resistance to antibiotics tested against
these isolates. Positive isolates with both qacA and qacE genes were
most frequent (7%), followed by the qacE gene alone (4%). Isolates
positive for qac
Δ
E1 always carried the qacE gene. S. aureus isolates
from the patient's immediate surroundings had the highest number
of qac genes, followed by frequently touched surfaces.
Association between the presence of qac genes and MIC values
Isolates carrying the qacA and qacE genes had higher MIC90 values
to CHX, although it was not statistically significantly different from
qacA and qacE negative S. aureus isolates. qac
Δ
E1 positive S. aureus
isolates had higher MIC90 values to CHX, which were statistically sig-
nificantly different from qac
Δ
E1 negative S. aureus isolates (P= .02).
Isolates carrying qacA genes had statistically significantly higher
MIC values to BAC (P= .01), similarly, isolates carrying qacE and
qac
Δ
E1 genes had higher MIC90 values to BAC, however, the differ-
ence between qacE/qac
Δ
E1 positive and qacE/qac
Δ
E1 negative S.
Table 1
Distribution of S. aureus among different sampling sites
Sampling category Total number of samples
(number & percentage of
samples positive for S. aureus)
Sampling sites Total number of samples
(number & percentage of
samples positive for S. aureus)
FTSs 64 (n = 12, 25.5%) Bedside table handle 21 (8, 38.1%)
IV pole grab area 14 (2, 14.3%)
Room doorknob 7 (1, 14.3%)
Room light switch 11 (1, 9.1%)
Tray table 11 (0, 0%)
PISs 60 (n = 16, 34%) Patient chairs 10 (1, 10%)
Bed linen 20 (5, 25%)
Bed rail 30 (10, 33.3%)
ME 59 (n = 12, 25.5%) Cardiac monitor 10 (1, 10%)
Oxygen supply unit 29 (9, 31%)
Surgical aspirator 3 (1, 33.3%)
Syringe pump machine 1 (1, 100%)
Blood pressure apparatus 12 (0, 0%)
Nebulizer 1 (0, 0%)
Weight machine 1 (0, 0%)
X-ray illuminator 2 (0, 0%)
WSs 62 (n = 7, 11.3%) Ward sink 62 (7, 11.3%)
FTSs = Frequently touched surfaces; PISs = Patient’s immediate surroundings; ME = Medical equipment; WS = Ward sinks
180 S. Sarwar et al. / American Journal of Infection Control 51 (2023) 178
−
183

aureus isolates were statistically insignificant. Isolates with qacA,
qacE, and qac
Δ
E1 had higher MIC values to CTAB however, the differ-
ence was statistically insignificant (Table 2).
For CHX, isolates with qacA, qacE and qac
Δ
E1 genes showed com-
parable MBC values to qacA, qacE, and qac
Δ
E1 negative isolates. For
BAC, isolates carrying qacA genes had higher MBC values that were
statistically significantly different from qacA negative isolates
(P= .003). Similarly, MBC values were higher in isolates carrying qacE
and qacΔE1 genes to BAC, however, the difference was statistically
insignificant. All isolates with qacA, qacE, and qacΔE1 genes had
higher MBC values to CTAB, however only qacE
Δ
1positive isolates
had statistically significantly higher MBC values (P= .03) (Table 2).
DISCUSSION
In this study, 245 environmental samples were collected from a
1030-bed tertiary care hospital to determine the prevalence of S.
aureus in the hospital environment. S. aureus was found to be present
on 19.1% of the environmental surfaces. Other Pakistani studies
revealed S. aureus contamination of hospital environmental surfaces
at 28.7%,
21
32%,
22
29%,
23
and 40%.
24
However, in this study, methicil-
lin resistance was found in a higher percentage (85.1%) of isolates
than in another study from Pakistan (57.81%).
22
This high prevalence
of MRSA in the hospital setting might be due to natural selection,
especially by natural transformation, which permits methicillin resis-
tance to be transferred to methicillin-sensitive strains.
25
Further-
more, the greater frequency of qac genes in MRSA isolates in the
current study compared to MSSA isolates may also have contributed
to the selection pressure on MRSA isolates.
In this study, the highest number of S. aureus isolates were found
in orthopedic (25%) and urology (20.8%) wards, of which 84% and
100% isolates were MRSA respectively. Several studies have reported
S. aureus as one of the most frequently isolated pathogens in orthope-
dic wards and orthopedic surgical site infections.
26,27,28
This high
prevalence highlights the importance of having a basic understand-
ing of the bacterium, its symptoms, and treatment options in the
orthopedic community. A few studies have found urinary MRSA to be
a potential HAI reservoir, resulting in occasional outbreaks of HA-
MRSA in urology wards.
29,30
This necessitates vigilant surveillance
for successful outbreak prevention.
The sampling sites and frequency of exposure, particularly those
that come into close contact with human hands, affect the types and
distribution of bacteria isolated. for example, bedside table handles
were found to be the most contaminated (38.1%) surfaces among the
FTSs, possibly because of a high touch frequency from patients and
attendants, who are the most uninformed in terms of infection and
prevention and control. Similarly, among the PISs, the most contami-
nated surfaces were bed rails (33%), possibly due to the high fre-
quency with which the staff who are responsible for shifting patients
on beds and changing the bed linens come into contact with them.
Most housekeeping workers are usually from low socioeconomic
backgrounds, with limited education, awareness, and training in
infection prevention and control, putting them and others at risk of
acquiring HAIs.
31
In short, bacterial contamination was more fre-
quent on inanimate surfaces with direct hand contact by people who
were maybe the most uninformed of IPC practices. The distribution
of S. aureus isolates varied depending on the equipment used.
11
How-
ever, S. aureus was not isolated from blood pressure equipment,
which is typically handled by nurses and doctors. Ward sinks, on the
other hand, had lower levels of S. aureus contamination. This is con-
sistent with previous studies, which found that S. aureus was more
commonly isolated from PISs, MEs, and FTSs.
11
In this study, multi-
drug-resistant (MDR) MRSA with resistance to combinations of oxa-
cillin, fluoroquinolones, and macrolides was frequently isolated,
suggesting the existence of selection pressure for this bacterium.
Table 2
Comparison of MIC and MBC values to chlorhexidine (CHX), benzalkonium chloride (BAC), and cetrimonium bromide (CTAB) for S. aureus isolates with and without qac genes
Biocide MIC90 MBC90 Gene Number of isolates (n) MIC90 MBC90
Median (mg/ml) Range (mg/ml) Median (mg/ml) Range (mg/ml) Median (mg/ml) Range (mg/ml) P-value Median (mg/ml) Range (mg/ml) P-value
CHX 128 2-256 256 8-512 qacA Positive 11 128 32-128 .5 256 128-256 .5
Negative 36 96 02-256 256 08-512
qacE Positive 14 128 32-128 0.1 256 128-256 .2
Negative 33 64 2-256 256 8-512
qac
Δ
E1 Positive 2 128 64-128 .02 256 256-256 .5
Negative 45 96 2-256 256 8-512
BAC 2 1-16 8 1-64 qacA Positive 11 4 1-16 .01 16 8-32 .003
Negative 36 2 1-16 8 1-64
qacE Positive 14 4 1-16 .2 12 4-32 .1
Negative 33 2 1-16 8 1-64
qac
Δ
E1 Positive 2 2 1-1 .1 8 4-4 .1
Negative 45 1 1-16 4 1-64
CTAB 4 2-256 16 2-512 qacA Positive 11 8 2-256 .4 32 8-512 .3
Negative 36 4 2-64 16 2-128
qacE Positive 14 6 2-256 .2 48 8-512 .03
Negative 33 4 2-64 16 2-128
qac
Δ
E1 Positive 2 10 4-16 .4 48 32-64 .2
Negative 45 4 2-256 16 2-512
AP-value of .05 was considered statistically significant.
S. Sarwar et al. / American Journal of Infection Control 51 (2023) 178
−
183 181

To our knowledge, this is the first study to report biocide suscepti-
bility and the presence of the qac A, qacE, and qac
Δ
E1 genes in S.
aureus isolated from a hospital environment in Pakistan. In 2013,
there was only 1 study on S. aureus clinical isolates from 2 Peshawar
tertiary care teaching hospitals that reported biocide susceptibility.
32
The aforementioned study found lower MIC90 values to CHX
(median = 0.25 mg/ml), however, the current study found substan-
tially higher MIC90 values to CHX (median = 128 mg/ml). In the cur-
rent study, the median BAC and CTAB MIC90 values were lower than
in the above-mentioned study, but the MIC90 ranges were broader,
with upper limits of 16 mg/ml and 256 mg/ml, respectively, compared
to 3 mg/ml and 15 mg/ml.
32
This shows that, even though the majority
of the strains had lower MIC90 values, the current study found S.
aureus strains with significantly higher MIC90 values in the hospital
environment. Several studies have demonstrated decreased suscepti-
bility to chlorhexidine,
33,34,35
although the current study found the
highest MIC90 values to date. This indicates that S. aureus isolates in
the hospital environment may have been exposed to significant
amounts of biocides, and strains with lower susceptibility are spread-
ing into the hospital environment. However, the clinical significance
of this finding is debatable because the concentration of biocides
used in situ is typically much higher than the MICs, even for strains
with reduced susceptibility. This is likely to become more important
when other factors are taken into account, such as biofilm formation,
which compromises the activity of the biocides. Besides these limita-
tions, it is possible to determine proper disinfection concentrations
for a facility’s sanitization program using MIC determination and
activity spectrum classification of disinfectants. Some other studies
showed similar or lower MIC90 values to BAC and CTAB.
2,36,37
The prevalence of qac genes is frequently reported in S. aureus
clinical isolates, however, very limited data is available on the pres-
ence of qac genes in environmental isolates with no studies from
Pakistan. Disinfectants are routinely used in the hospital environ-
ment, and microorganisms come into regular contact with these sub-
stances. These genes confer reduced susceptibility to commonly used
disinfectants, such as cetrimide, benzalkonium chloride and chlor-
hexidine. qacA gene is commonly associated with reduced suscepti-
bility to disinfectants in staphylococci
35
and has been found in 0.9 to
83.3% of MRSA isolates from various countries,
35,37
however, in the
present study, the qacE gene was the most frequently isolated gene
(29.7%) followed by qacA (23.4%), which is a rare finding. To our
knowledge, only 1 study has described qacE genes in Gram-positive
bacteria, including staphylococci,
38
although this observation has not
been confirmed afterwards. However, the presence of Qac
Δ
E1 in S.
aureus has been confirmed in a few other studies.
39
Isolates carrying the qacA, qacE,andqac
Δ
E1 genes had higher
MIC90 values, however, only the qacA and qac
Δ
E1 genes were statisti-
cally significantly associated with higher MIC90 values for BAC and
CHX respectively. Only qacA and qac
Δ
E1 genes showed statistically sig-
nificant differences in MBC90 values for BAC and CTAB respectively.
Other studies have found a statistically significant association between
the qacA gene and higher BAC and CHX MIC values.
16,40,41,42
However,
there is limited data to compare the presence of qacE and qac
Δ
E1 genes
with MIC values in S. aureus isolates. Moreover, heterogenicity exists
among several studies regarding the association of disinfectant resis-
tance genes with reduced susceptibility to disinfectants. In our study,
isolates with qac genes had higher MIC values than isolates without
qac genes, but the difference was statistically insignificant. This statis-
tically insignificant difference can be clinically significant
43
and could
be due to the small sample size or other environmental factors.
44
In the current study, a few strains of S. aureus carrying qacA genes
had lower MIC values to chlorhexidine than qacA negative strains.
Measuring RNA expression of disinfectant resistance genes during
exposure to disinfectants is one way to further investigate this vari-
ability. Staphylococci can have many disinfectant efflux mediated
resistance genes, which are part of tightly regulated virulence sys-
tems. As a result, it is critical to understand how bacteria regulate
and express different genes of these virulence systems in response to
various environmental factors, as well as the broader implications of
regulatory networks that could lead to efflux-based resistance.
CONCLUSION
The current study found significant MRSA contamination in the
hospital environment. Their distribution in the hospital environment
and the isolation of multiple MRSA isolates indicate that environment
may be an important source of nosocomial pathogens. Furthermore,
higher qac gene frequencies were found in MRSA isolates that also
correlated with higher MIC/MBC values to different disinfectants in
isolates carrying qac genes. The use of disinfectants is probably the
best option for disease control in a post-antibiotic era, however, it is
critical to fully understand the mechanisms of resistance to disinfec-
tants to prevent their misuse to a point where the majority of nosoco-
mial pathogens develop resistance to disinfectants. Selective pressure
in the hospital environment allows such strains to persist and spread.
Therefore, it is also important to keep track of the acquisition of resis-
tance genetic elements. This study suggests that hospitals should
implement policies and techniques to determine MICs of disinfec-
tants against the most frequent types of environmental isolates until
Fig 1. Detection of qac genes and their distribution in Methicillin-resistant and sensitive S. aureus isolates from hospital environment (A) Detection of qacA, qacE, and qac
Δ
E1 genes
in S. aureus isolates by PCR agarose gel electrophoresis: 1-2 represent qac
Δ
E1, 3-4 represent qacE, 5-6 represent qacA and M represents DNA marker size (100bp), (B) Distribution of
qac genes in MRSA and MSSA isolates.
182 S. Sarwar et al. / American Journal of Infection Control 51 (2023) 178
−
183

scientists can fully understand these complicated disinfectant resis-
tance processes.
Acknowledgment
The authors sincerely acknowledge the University of Health Scien-
ces Lahore for supporting this study.
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