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Original Article
Antibacterial effect of acetic acid during an outbreak of carbapenem-
resistant Acinetobacter baumannii in an ICU (II)
Carolina Garciglia-Mercado1,2, Ramon Gaxiola-Robles1,2, Felipe Ascencio1, Concepción Grajales-Muñiz3,
Maria Luisa Soriano Rodríguez4, Jesus Silva-Sánchez5, Maria Teresa Estrada-García6, Gracia A
Gómez-Anduro1
1 Centro de Investigaciones Biológicas del Noroeste S.C., La Paz, Baja California Sur, México
2 Hospital General de Zona No. 1, Instituto Mexicano del Seguro Social, La Paz, Baja California Sur, México
3 Coordinación de Vigilancia Epidemiológica en la División de Vigilancia Epidemiológica de Enfermedades
Transmisibles, Instituto Mexicano del Seguro Social, CDMX, México
4 Unidad Médica de Atención Ambulatoria (UMAA) con medicina familiar, 180 Delegación México oriente, Instituto
Mexicano del Seguro Social, CDMX, México
5 Instituto Nacional de Salud Pública, Centro de Investigación Sobre Enfermedades Infecciosas (CISEI),
Departamento de Diagnóstico Epidemiológico, Cuernavaca, Morelos, México
6 CINVESTAV-IPN, Departamento de Biomedicina Molecular, CDMX, México
Abstract
Introduction: Acetic acid (AA) has been commonly used in medicine as an antiseptic agent for the past 6000 years. This study evaluated the
antibacterial effect of AA during an outbreak in an intensive care unit (ICU) facility in Baja California Sur, México.
Methodology: Thirty-five environmental samples were collected, subsequently, disinfection with AA (4%) was performed, and two days
later the same areas were sampled inside the ICU facility. Carbapenem-resistant A. baumannii (CRAB) was detected with loop-mediated
isothermal amplification assay (Garciglia-Mercado et al. companion paper), targeting blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, blaOXA-58-like,
blaIMP and blaVIM genes. CRAB isolates before and after disinfection were compared by PFGE.
Results: Eighteen (54.5%) and five (14.3%) of thirty-five environmental samples were identified as Acinetobacter baumannii before and after
disinfection, respectively, showing a significant decrease of 85.7% (p < 0.05) both by Loop-mediated isothermal amplification (LAMP) and
polymerase chain reaction (PCR). Furthermore, the presence of blaOXA-23-like and blaOXA-58-like genes significantly decreased (p < 0.05) both by
LAMP and PCR methods. PFGE genotype showed high similarity among CRAB isolates before and after disinfection, suggesting wide
clonal dissemination in the ICU facility.
Conclusions: This study demonstrated the novel application of AA with the LAMP assays developed for detecting CRAB. AA promises to be
a cheap and efficacious disinfectant alternative to both developed and especially developing countries, preventing the spread of this organism
in the environment and to other susceptible patients in health care settings.
Key words: Acetic acid; Carbapenem-resistance; Acinetobacter baumannii; loop-mediated isothermal amplification.
J Infect Dev Ctries 0000; 0(00):000-000. doi:10.3855/jidc.11693
(Received 23 May 2019 – Accepted 13 April 2020)
Copyright © 2021 Garciglia-Mercado et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Introduction
Acinetobacter baumannii represents a serious
public health concern due to its ability to cause
multifacility nosocomial outbreaks [1,2]. It has shown
to survive in hospital environments for extended
periods as a result of its capacity to form sessile
biofilms found in various sites, including furniture,
patient care items and hospital equipment [3,4]. Since
carbapenems are the common choice to treat infections
caused by this pathogen, the emergence of
Carbapenem-resistant A. baumannii (CRAB) is
leaving few remaining therapeutic options [5]. The
production of class B metallo-β-lactamases (MBLs)
and class D carbapenem-hydrolyzing β-lactamases
(carbapenemases) have been recognized as the main
mechanism of carbapenem resistance [6,7]. MBLs of
particular clinical importance in A. baumannii are the
Imipenemase (IMP) and Verona Imipenemase (VIM)
[8]. Four main class D OXA carbapenemases
(oxacillinases) subgroups of A. baumannii have been
identified as OXA-23-like; OXA-24-like; OXA-51-
like; and OXA-58-like [9]. Due to the reducing
efficacy of antibiotics against CRAB, high doses of
biocides are required to treat these organisms growing
Garciglia-Mercado et al. – Antibacterial effect of AA on CRAB in an ICU J Infect Dev Ctries 0000; 0(0):000-000.
2
in fomites, but they do not prevent bacterial
colonization and biofilm formation. Therefore, interest
has grown in reevaluating the novel applications of
‘household remedies’ as salvage procedures. Acetic
acid (AA) or vinegar has been commonly used in
medicine as an antiseptic agent for the past 6000 years
[10], showing excellent bactericidal effect in the
treatment of the plague, war wounds, and ear, chest
and urinary tract infections [11,12]. It has also been
used in eliminating bacteria in beef carcass [13] and
fresh products [14]. Although clinical studies and
observations provide evidence in support of the
clinical utility of AA as an antiseptic [15], recent
literature is scarce on its use as a general disinfectant.
Considering this potential application, the objective of
this study was to evaluate the antibacterial effect of
AA during an outbreak in an intensive care unit (ICU)
facility; CRAB was detected in environmental samples
with the developed loop-mediated isothermal
amplification (LAMP) assays (Garciglia-Mercado et
al. companion paper), targeting blaOXA-23-like, blaOXA-24-
like, blaOXA-51-like, blaOXA-58-like, blaIMP and blaVIM genes.
Methodology
Bacterial Strains and DNA extraction
Reference strains used as positive controls are
addressed in our companion paper (Garciglia-Mercado
et al). CRAB DNA extraction was performed by the
phenol-chloroform method [16].
Sample collection and disinfection
First sample collection
Prior to disinfection, thirty-five environmental
samples were collected from an outbreak in an ICU at
Hospital General de Zona No. 1 Instituto Mexicano
del Seguro Social (IMSS) in La Paz, Baja California
Sur, México. The project was approved by the
Comisión Nacional de Investigación Científica del
Instituto Mexicano del Seguro Social (R-2016-785-
047). The surfaces of 3-bed spaces and a general area
were sampled. Patients 1 and 2 were females with
diagnosed pneumonia and patient 3 was a male with
bacteremia in the isolation room (Table 1). Samples
were obtained with sterile cotton swabs, and
approximately 15 cm2 of each surface was swabbed
(see Garciglia-Mercado et al. companion paper for
additional details of the sampling efforts). After
sampling, the swabs were immediately placed in
Luria-Bertani (LB) media in 2-mL tubes and incubated
at 35 °C for 24 h. After incubation in the enrichment
broth, the same swab was inoculated onto selective
MacConkey agar plates and rotated completely. Plates
were streaked for isolation and incubated at 37°C for
24 h. Light-pink or purplish colonies were selected for
DNA extraction.
Disinfection
After the first sample collection, hospital staff
performed the disinfection in the ICU using 4% (v/v)
AA with clean wipes; the contact time was 10 min. All
the areas were disinfected with the exception of the
mechanical ventilators touch screens (Table 1).
Second sample collection
Two days after disinfection, the same areas were
sampled inside the ICU facility (Table 1), and the
swabs were processed as described previously.
Loop-mediated isothermal amplification reaction
conditions
The LAMP assays were performed in a total of 25
µl reaction as previously described (Garciglia-
Mercado et al. companion paper). For the detection of
blaOXA-23-like, blaOXA-24-like and blaOXA-51-like the tubes
were incubated at 64 °C for 60 min and blaOXA-58-like,
blaIMP and blaVIM at 65 °C for 60 min. One positive
(reference strains) and one negative (ddH2O) controls
were included in each LAMP run, and the experiments
were performed in duplicate to ensure reproducibility
using a T100™ Thermal Cycler (Biorad Hercules, CA,
USA).
Garciglia-Mercado et al. – Antibacterial effect of AA on CRAB in an ICU J Infect Dev Ctries 0000; 0(0):000-000.
3
Detection of Loop-mediated isothermal amplification
products
The LAMP products were detected by color
change by staining with 10000X SYBR Green I
(Sigma, Poole, Dorset, UK) as previously described
[17]; Garciglia-Mercado et al. companion paper].
Positive amplification results were considered when a
change from orange to yellowish green was observed.
For further confirmation, the presence of the
characteristic pattern of LAMP amplification in the
positive samples was analyzed by electrophoresis in
2% agarose gels and visualized by ultra-violet (UV)
transillumination.
Polymerase chain reaction assay
The conventional PCR primers described
previously by Brown et al. (2005) [18] were used for
the intrinsic blaOXA-51-like of A. baumannii. The PCR
amplification of CRAB was performed by using F3/B3
for each gene; see Garciglia-Mercado et al. companion
paper for additional details of amplification and
primers used in the assays.
Molecular typing
The CRAB isolates from the environmental
samples before and after disinfection, were also
analyzed by Pulsed-field gel electrophoresis (PFGE)
performed according to Kaufmann’s method [22].
DNA was digested with ApaI enzyme, separated in 1%
agarose gel and stained with ethidium bromide. The
macrorestriction patterns were analyzed using the
Tenover criteria [23] and compared by the Dice
similarity index, with band tolerance and optimization
set at 1.5%, using BioNumerics software (Applied
Maths, Belgium). Isolates exhibiting at least 80%
similarity were clustered into a pulsotype.
Statistical analysis
Diagnosis performance of the LAMP assays were
compared with conventional PCR by estimating
sensitivity, specificity, positive predictive value
Table 2. Carbapenem-resistance Acinetobacter baumannii detection by PCR and LAMP methods before and after disinfection with acetic
acid (4%).
Sample
no.
Identificationa
PCRb
LAMPc
blaOXA-
23-like
blaOXA-24-
like
blaOXA-51-
like
blaOXA-58-
like
blaVIM
blaIMP
blaOXA-23-
like
blaOXA-24-
like
blaOXA-51-
like
blaOXA-58-
like
blaVIM
blaIMP
Before disinfection
1
A. baumannii
+
-
+
-
-
-
+
+
+
-
-
-
2
A. baumannii
-
-
+
-
-
-
-
-
+
-
-
-
4
A. baumannii
+
-
+
+
-
-
+
-
+
+
-
-
5
A. baumannii
+
-
+
-
-
-
+
-
+
-
-
-
6
A. baumannii
+
-
+
-
-
-
+
-
+
-
-
-
7
A. baumannii
+
-
+
-
-
-
+
-
+
-
-
-
9
A. baumannii
+
-
+
-
-
-
+
-
+
-
-
-
10
A. baumannii
+
-
+
-
-
-
+
-
+
-
-
-
13
A. baumannii
+
-
+
+
-
-
+
+
+
+
-
-
14
A. baumannii
+
-
+
-
-
-
+
-
+
-
-
-
15
A. baumannii
+
-
+
-
-
-
+
+
+
-
-
-
23
A. baumannii
+
+
+
+
-
-
+
+
+
+
-
-
25
A. baumannii
+
+
+
-
-
-
+
+
+
-
-
-
26
A. baumannii
+
-
+
+
-
-
+
-
+
+
-
-
27
A. baumannii
+
-
+
-
-
-
+
-
+
-
-
-
32
A. baumannii
+
-
+
-
-
-
+
+
+
+
-
-
34
A. baumannii
-
+
+
+
-
-
-
+
+
+
-
-
35
A. baumannii
+
-
+
-
-
-
+
-
+
-
-
-
After disinfection
4
A. baumannii
-
-
+
-
+
-
+
+
+
-
+
-
11
A. baumannii
+
-
+
-
+
-
+
+
+
-
+
+
23
A. baumannii
-
-
+
-
-
-
-
-
+
-
-
-
26
A. baumannii
-
+
+
-
-
-
-
+
+
-
-
-
32
A. baumannii
+
-
+
-
-
-
+
-
+
-
+
-
a Identification by amplification and sequencing of blaOXA-51-like; b + amplification occurred; -: amplification did not occur; c + amplification was observed after
60-min incubation; - amplification was not observed after 60-min incubation.
Garciglia-Mercado et al. – Antibacterial effect of AA on CRAB in an ICU J Infect Dev Ctries 0000; 0(0):000-000.
4
(PPV), and negative predictive value (NPV) in a 2×2
contingency table with 95% Confidence Intervals (CI).
The difference between the first sample collection
(before disinfection) and the second sample collection
(after disinfection) were determined with Chi-square
test with a significance set at p < 0.05, using Minitab®
statistical software (Minitab 18, Minitab Inc. State
College, PA, USA).
Results
Identification of Carbapenem-resistant Acinetobacter
baumannii
Before disinfection with Acetic Acid
Eighteen of thirty-five (54.5%) environmental
samples were found to contain A. baumannii by
amplification and sequencing of blaOXA-51-like. These
samples were analyzed for CRAB by LAMP and PCR
assays. Sixteen (45.71%), seven (21.2%) and six
(17.1%) samples were detected to be positive by
LAMP, where sixteen (45.71%), three (8.6%) and five
(14.3%) samples were detected by PCR, for blaOXA-
23-like, blaOXA-24-like and blaOXA-58-like
respectively (Table 2). All the amplicons matched the
target gene segment spanned by F3/B3 primers and
shared 99-100% of identity. None of the samples
tested positively for blaIMP and blaVIM by LAMP and
PCR. Calculation of the clinical sensitivity, specificity,
positive predictive value (PPV) and negative
predictive value (NPV) of LAMP assays yielded 100%
for blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, blaOXA-58-like
(Table 3), while sensitivity and NPV of PCR were
42.86% (CI95%: 9.90-81.59%) and 87.50% (CI95%:
78.66-93.01%) for blaOXA-24-like, 83.33% (CI95%:
35.88-99.58%) and 96.67% (CI95%: 82.89-99.43%)
for blaOXA-58-like, respectively (Table 3).
After disinfection with Acetic Acid
After disinfection with AA five of thirty-five
(14.3%) environmental samples were identified as A.
baumannii by amplification and sequencing of blaOXA-
51-like, showing a significant decrease (P < 0.05) both
by LAMP and PCR of its presence in the ICU facility.
These samples were analyzed for CRAB by LAMP
and PCR assays. Three (8.6%) samples were detected
to be positive for blaOXA-23-like, blaOXA-24-like, blaVIM, and
one (2.9%) for blaIMP by LAMP, respectively, where
two (5.7%), one (2.9%), two (5.7%) and 0 (0%)
samples were detected by PCR (Table 2). The
presence of blaOXA-23-like and blaOXA-58-like genes
significantly decreased (p < 0.05) after disinfecting
both by LAMP and PCR methods (Table 3). Clinical
sensitivity, specificity, PPV, and NPV of LAMP
assays yielded 100% for blaOXA-23-like, blaOXA-24-like,
blaOXA-51-like, blaVIM and blaIMP after disinfection.
Sensitivity and NPV of PCR were 33.33% (CI95%:
0.84-90.57%) and 94.12% (CI95%: 87.79-97.27%) for
blaOXA-24-like, 66.67% (CI95%: 9.43-99.16%) and
96.97% (CI95%: 86.59-99.37%) for blaOXA-23-like and
blaVIM, respectively. The specificity and PPV was
detected to be 100% for both LAMP and PCR.
Overall, comparing with PCR, LAMP results
highlighted a robust performance of the assays.
Table 3. Evaluation of LAMP and PCR methods for the detection of CRAB in an ICU before and after disinfection with acetic acid (4%).
LAMP VS PCR
No. of samples
Sensitivity
(%)
CI
(%)
Specificity
(%)
CI
(%)
PPV
(%)
CI
(%)
NPV
(%)
CI
(%)
LAMP
PCR
Positive
Negative
Positive
Negative
Before disinfection
blaOXA-23-like
16
19
16
19
100/100
79.41-100/79.41-100
100/100
82.35-100/82.35-100
100/100
-/-
100/100
-/-
blaOXA-24-like
7
28
3
32
100/42.86
59.04-100/9.90-81.59
100/100
87.66-100/87.66-100
100/100
-/-
100/87.50
-/78.66-
93.01
blaOXA-51-like
18
17
18
17
100/100
81.47-100/81.47-100
100/100
80.49-100/80.49-100
100/100
-/-
100/100
-/-
blaOXA-58-like
6
29
5
30
100/83.33
54.07-100/35.88-99.58
100/100
88.06-100/88.06-100
100/100
-/-
100/96.67
-/82.89-
99.43
blaVIM
0
35
0
35
-/-
-/-
-/-
-/-
-/-
-/-
-/-
-/-
blaIMP
0
35
0
35
-/-
-/-
-/-
-/-
-/-
-/-
-/-
-/-
After disinfection
blaOXA-23-like
3a
32
2a
33
100/ 66.67
29.24-100/9.43-99.16
100/100
89.11-100/ 89.11-
100
100/100
-/-
100/ 96.97
-/ 86.59-
99.37
blaOXA-24-like
3
32
1
34
100/33.33
29.24-100/0.84-90.57
100/100
89.11-100/89.11-100
100/100
-/-
100/94.12
-/87.79-
97.27
blaOXA-51-like
5a
30
5a
30
100/100
47.82-100/47.82-100
100/100
88.43-100/88.43-100
100/100
-/-
100/100
-/-
blaOXA-58-like
0a
35
0a
35
-/-
-/-
-/-
-/-
-/-
-/-
-/-
-/-
blaVIM
3
32
2
33
100/66.67
29.24-100/9.43-99.16
100/100
89.11-100/89.11-100
100/100
-/-
100/96.97
-/86.59-
99.37
blaIMP
1
34
0
35
100/0
2.50-100/0.00-97.50
100/100
89.72-100/89.72-100
-/-
-/-
100/97.14
-/97.14-
97.14
PPV: Positive predictive value; NPV: Negative predictive value; CI: Confidence intervals (95%); -: not applicable; a: Statistical difference (P < 0.05), n = 35.
Garciglia-Mercado et al. – Antibacterial effect of AA on CRAB in an ICU J Infect Dev Ctries 0000; 0(0):000-000.
5
Molecular typing
PFGE fingerprint analysis was performed in the 23
CRAB environmental isolates. A major clone A with 9
subtypes (A to A9) was identified, including all the 23
isolates from before and after disinfection (Figure 1).
The subtype variants showed similar values that were
always higher than 91%.
Discussion
Earlier studies have reported the use of AA as a
topical agent for the treatment of burn infections and
skin and soft tissue infections [19]. Planktonic growth
prevention and biofilm eradication has generally been
reported in problematic Gram-negative but also in
Gram-positive bacteria, with concentrations ranging
from 0.31% to 5% [10,12]. Moreover, the authors
found excellent antibacterial effect against multi-drug
resistant organisms, such as Pseudomonas aeruginosa
and A. baumannii [10,15].
The antibacterial effect of AA has been thought to
be due to a variety of mechanisms, of which a physical
alteration of the bacterial cell wall is the most accepted
theory. Since weak acids can cross bacterial
membranes more easily than strong ones, the
internalized AA will dissociate, acidifying the
cytoplasm, which can cause acid-induced protein
unfolding, membrane, and DNA damage [12]. This
effect could explain the mayor susceptibility of Gram-
negative compared to Gram-positive bacteria, caused
by the lack of murein layer.
With the ever-limited antibiotic and disinfectant
choices for multi-drug resistant strains as CRAB, AA
is a potential alternative to traditional antimicrobials
(e.g. chlorine and cefotaxime) for preventing
colonization and biofilm formation in hospital
environments. This study found that in the disinfected
areas with AA, A. baumannii showed a decrease of
85.7% (p < 0.05).
Interestingly, PFGE genotype showed high
similarity among CRAB isolates before and after
disinfection, suggesting wide clonal dissemination in
the ICU facility (Fig. 1) where transmission through
contaminated hands of healthcare staff and equipment
is likely to be the main route for the spread of the
microorganisms.
Furthermore, the presence of the target genes for
carbapenem resistance was diminished after
disinfection, showing a decrease in blaOXA-23-like,
blaOXA-24-like and an elimination of blaOXA-58-like in the
environmental samples. However the presence of
blaVIM and blaIMP increased in the same areas that were
not disinfected (mechanical ventilators touch screens
and buttons, and the telephone dial pad and handset
from staff), confirming that these organisms acquire
carbapenem resistance under antimicrobial selective
pressure [20,21]. Finally, the validation of the LAMP
assays with the PCR method showed a higher
detection rate for CRAB samples and a higher
diagnosis-based performance with no false positive or
false negative observed, as previously described
(Garciglia-Mercado et al. companion paper).
Conclusions
This study demonstrated the potential use of AA in
critical hospital areas through the LAMP assays
developed for detecting CRAB, which promises to be
a cheap and efficacious disinfectant alternative to both
developed and especially developing countries,
preventing the spread of this organism in the
environment and to other susceptible patients in
hospital settings.
Acknowledgements
Figure 1. PFGE comparison of CRAB isolates before (B) and
after (A) disinfection with acetic acid (4%). Letter B or A was
assigned next to the corresponding sample number (Table 1).
Similarity (%) among patterns is represented by the numbers
beside the nodes.
Garciglia-Mercado et al. – Antibacterial effect of AA on CRAB in an ICU J Infect Dev Ctries 0000; 0(0):000-000.
6
The authors are grateful to CIBNOR (PAZA) for funding
this study to G.A.G-A; to the Instituto Mexicano del Seguro
Social; the CONACyT (Mexican Council for Science and
Technology) for the granted scholarship to C.G-M and the
grant No. 256927; to Q. Alejandro Sánchez Perez for his
valuable participation in the PFGE analysis; to Diana
Fischer for providing editorial help in English and proof
reading the article.
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Corresponding author
Gracia Alicia Gomez Anduro, PhD.
Molecular Biology of Plants Group, Centro de Investigaciones
Biológicas del Noroeste S.C. (CIBNOR), Av. IPN 195
23096, La Paz, B.C.S. México.
Phone: +52 612 1238484 ext 3419
E-mail: ggomez@cibnor.mx
Conflict of interests: No conflict of interests is declared.
