Antibacterial effect of acetic acid during an outbreak of carbapenem- resistant Acinetobacter baumannii in an ICU (II)

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.


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-51like; 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 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 loopmediated isothermal amplification (LAMP) assays (Garciglia-Mercado et al. companion paper), targeting bla OXA-23-like , bla OXA-24-like , bla OXA-51-like, bla OXA-58-like , bla IMP and bla VIM genes.

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 cm 2 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 bla OXA-23like , bla OXA-24-like and bla OXA-51-like the tubes were incubated at 64 °C for 60 min and bla OXA-58-like , bla IMP and bla VIM at 65 °C for 60 min. One positive (reference strains) and one negative (ddH 2 O) 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).

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 bla OXA-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 Table 2. Carbapenem-resistance Acinetobacter baumannii detection by PCR and LAMP methods before and after disinfection with acetic acid (4%).
A. baumannii + - A. baumannii + -+ ---+ -+ -+ a Identification by amplification and sequencing of bla OXA-51-like; b + amplification occurred; -: amplification did not occur; c + amplification was observed after 60min incubation; -amplification was not observed after 60-min incubation. sensitivity, specificity, positive predictive value (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).

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 bla OXA-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 bla OXA-23-like, bla OXA-24-like , bla VIM , and one (2.9%) for bla IMP 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 bla OXA-23-like and bla OXA-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 bla OXA-23-like , bla OXA-24-like , bla OXA-51-like , bla VIM and bla IMP after disinfection. Sensitivity and NPV of PCR were 33.33% (CI95%: 0.84-90.57%) and 94.12% (CI95%: 87.79-97.27%) for bla OXA-24-like , 66.67% (CI95%: 9.43-99.16%) and 96.97% (CI95%: 86.59-99.37%) for bla OXA-23-like and bla VIM , 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%).    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 Grampositive 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, bla OXA-24-like and an elimination of bla OXA-58-like in the environmental samples. However the presence of bla VIM and bla IMP 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. 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. 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.