Upregulation of abeM, amvA, and qacEΔ1 efflux pump genes associated with resistance of Acinetobacter baumannii strains to disinfectants

Abstract Background and aims Acinetobacter baumannii is among the most concerning cause of nosocomial infections due to its high level of antibiotic resistance and high mortality. The aim of this study was to determine the role of efflux pumps in resistance of A. baumannii strains to three disinfectants, including MICROZED ID‐MAX, NANOSIL D2, and OPIDEX OPA. Methods Twenty‐eight environmental and clinical isolates of A. baumannii were collected from selected hospitals of central Iran. The minimum inhibitory concentrations of the disinfectants were determined and real time reverse transcriptase‐PCR was performed to investigate the expression level of qacEΔ1, amvA, abeM, and adeB efflux pump genes. Results Considering both clinical and environmental isolates, there was a significant difference in the mean expression level of qacEΔ1 gene between susceptible and resistant strains to MICROZED ID‐MAX disinfectant, of amvA and abeM genes between susceptible and resistant strains to NANOSIL D2 disinfectant and of abeM gene in susceptible and resistant strains to OPIDEX OPA disinfectant (all P ˂ .05). The expression levels of abeM and amvA genes were higher in the environmental isolates that were resistant to NANOSIL D2 disinfectant compared to those that were susceptible (P ˂ .05). Conclusions This study provided evidence for the role of abeM and amvA genes in the resistance of environmental isolates to disinfectants, particularly hydrogen peroxide derivatives.

A. baumannii are nonfermentative gram-negative coccobacilli that colonize various organs of hospitalized patients and could survive for long time on both humid and dry environmental surfaces. 3,4 There are increasing reports of carbapenem-resistant and multidrug resistant A. baumannii and several evidence show dissemination of common clones of drug resistant A. baumannii strains among patients within or between hospitals. A. baumannii can tolerate desiccation and survive in the inanimate healthcare environment including surfaces and equipment for long period of time. A combination of these properties and the enhanced resistance of A. baumannii to antibiotic and biocide, make it a great challenge in healthcare settings. A. baumannii causes pneumonia, bacteremia, secondary meningitis, burn and wound infection, urinary tract infection, and soft tissue infection. [5][6][7] Disinfectants are chemicals that inactivate pathogenic microorganisms on contaminated equipment and surfaces by different mechanisms. Various commercially available disinfectants are currently used to reduce or completely eliminate the microbial burden of health care facilities 8 and their activity depends on several factors such as temperature, concentration, chemical nature, and pH. 5 One of the key mechanisms of low susceptibility/resistance of bacteria to biocides is the function of efflux transport systems. Efflux pumps are proteins which are localized in plasma membrane of bacteria 9 and efflux function allows the microorganisms to regulate their internal environment by removing toxic substances, including antimicrobial agents, metabolites, and biocides. According to their substrates, composition, number of membrane spanning segments, and energy sources, bacterial efflux pumps are classified into seven families including the resistancenodulation-cell division superfamily (RND), the small multidrug resistance family (SMR), the major facilitator superfamily (MFS), the ATP-binding cassette superfamily (ABC), the multidrug and toxic compound extrusion protein family (MATE), the p-aminobezoyl-glutamate transporter, and the proteobacterial antimicrobial compound efflux. [10][11][12] A. baumannii have been isolated from surfaces, equipment, solutions, monitors, and beds in hospitals and most of the A. baumannii infections are directly associated with the length of hospital stay, especially in the intensive care units. To control the emergence and outbreaks of multiple drug resistance (MDR) A. baumannii in health care settings, there is a need to focus simultaneously on environmental isolates in order to disrupt transmission of A. baumannii clones between environment and hospitalized patients and/or healthcare-workers. 13 In this study, we have analyzed the transcription levels of four efflux pumps genes from four different families including: qacEΔ1 (SMR), adeB (RND), amvA (MFS), and abeM (MATE) in the environmental and clinical isolates of A. baumannii.
The association of activity of efflux pumps with susceptibility/resistance to common commercially available disinfectants was also evaluated.

| Disinfectants
Three commonly known disinfectants, including MICROZED ID-MAX (Atrineh Saziba, Iran), NANOSIL D2 (Kimiafaam Pharmaceutical, Iran), and OPIDEX OPA (Sung Kwang Pharm, Korea), which are routinely used in hospitals of Iran to disinfect surfaces, equipment, and medical devices, were selected. MICROZED ID-MAX is a concentrated disinfectant solution classified as quaternary ammonium compounds. NANOSIL D2 is a ready to use disinfectant for surfaces and might be used in a wide range of ambient temperatures (0 C-95 C) composed of hydrogen peroxide and silver ions. OPIDEX OPA is a ready to use solution composed of 0.55% ortho-phthalaldehyde (Table 1).

| Settings and sampling
Samples were collected from environmental surfaces, equipment, and hospitalized patients of intensive care unit (ICU) and neonatal intensive care unit wards of four hospitals located at central Iran including Qom and the capital Tehran provinces. For environmental sampling, the sterile swab was moistened with sterile saline and rubbed by swab over a 10 cm square surface of environment. In the case of liquids, 1 mL of the solution was used as sample for culture. 14 Clinical MDR strains were isolated from skin ulcers, trachea, and bronchoalveolar specimens of the hospitalized patients. Also, environmental strains were isolated from bed sheets, sinks, faucets, carrying tables, and medical devices such as suction tubes. The clonal distribution of the isolates was previously determined which belongs to nine different clones. 14

| Identification of A. baumannii strains
The swabs were directly cultured on blood agar medium and then were subcultured on MacConkey agar medium (Merck, Germany) and incubated at 37 C for 48 hours. The resulting single colonies were used further for culture and biochemical tests. MacConkey agar, OF (oxidative fermentative basal agar), TSI (triple sugar iron agar) (all from Merck, Germany), oxidase (Padtan Teb Co, Iran), and growth test at 44 C were used to identify A. baumannii. 14 Nonfermentative Gram-negative bacilli which identified as A. baumannii were further confirmed by PCR targeting bla OXA-51 gene. For this purpose, the strains were cultured on trypticase soy agar (TSA) medium and incubated at 37 C for 18 to 24 hours. DNAs were extracted by alkaline lysis method, 15

| MIC and MBC determination
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of disinfectants were determined by microdilution broth method.  16 Overall, the isolates that were resistant to at least one of the three disinfectants were considered resistant, and isolates that were susceptible to all three disinfectants were considered susceptible.

| Minimum bactericidal concentration
After MIC determination, the well containing MIC and the well after that were cultured on TSA medium and incubated at 37 C for 18 to 24 hours. Then, plates without colony growth with the lowest disinfectant concentration were designated as MBC. The housekeeping gene 16S rRNA was used as the internal standard for normalization. qPCR results were calculated based on Pfaffl method using C t value in 2 ÀΔΔCt formula. 17 The relative expression of each gene was calculated as the normalized ratio of gene expression of a given strain to gene expression in reference strain. A. baumannii strain ATCC19606 was used as the standard strain.

| Statistical analysis
Data were analyzed using SPSS software ver. 20.0 (SPSS Inc., Chicago, Illinois). Parameters such as mean and SD were calculated as T A B L E 2 Sequence of primers of efflux genes

| The overall frequency of susceptible/ resistant strains among clinical and environmental isolates of A. baumannii
The frequency of resistant/susceptible strains to different disinfectants among environmental and clinical strains is shown in Figure 2.
Of the total 28 strains, 10  resistant. According to these data, the frequency of resistance to all three disinfectants in environmental strains (n = 6 of 10) was significantly higher than that of clinical strains (n = 9 of 18) (P < .05) ( Figure 2). The mean expression levels of abeM and amvA genes were significantly higher in environmental isolates which were susceptible (amvA = 1.82 ± 0.37; abeM = 3.02 ± 2.14) compared to those that were resistant (amvA = 4.09 ± 2.02; abeM = 23.86 ± 31.59) to NANOSIL D2 disinfectant (P < .05). However, there was no significant difference between the mean expression of abeM, qacEΔ1, amvA, and adeB genes in resistance vs susceptible clinical isolates (Figure 3).

| The correlation between the rates of expression of the studied genes
The correlation between the expression of four studied genes was investigated, and the results showed that the relationship between gene expression of amvA and adeB was positive, strong, and significant F I G U R E 2 The frequency of susceptible and resistant strains of Acinetobacter baumannii to each disinfectant, in terms of environmental/ clinical sources. Resistance of bacterial isolates to different disinfectants was calculated based on the minimum inhibitory concentrations. Overall, 60% of environmental and 50% of clinical isolates were designated as resistant strain (P < .001), between abeM and adeB (P < .05) and between abeM and amvA (P < .05) was also significant.

| DISCUSSION
In this study, the resistance of A. baumannii isolates to disinfectants (MICROZED ID-MAX, NANOSIL D2, and OPIDEX OPA) was measured based on MIC. Then, the association between efflux gene expression rate and MIC changes was determined. The serial dilutions of the three disinfectants used in this study were prepared considering the recommended concentrations of the manufacturers. However, the recommended concentration, which is also applied in hospitals, was higher than enough to inhibit the growth and the isolates were inhibited or killed with lower dilutions. The maximum concentration of OPIDEX OPA disinfectant that inhabited the growth was a solution of 25% while the manufacturer recommended the concentration of 100%, and for NANOSIL D2 it was 0.6% while the recommended concentration was 100%. Similarly, in several other studies, the MIC of the biocides used was less than that of recommended by manufacturers. 18,19 For instance, in the study of the effect of hospital disinfectants including quaternary ammonium compounds (similar to MICROZED ID-MAX) on Staphylococcus epidermidis, it was suggested that the MIC was from 6 to 8 times lower than the concentrations recommended by the manufacturer. 20 However, there are studies that reported that all A. baumannii isolates were resistant to recommended concentrations of disinfectants. 21 The use of concentrations above the effective level may lead to the emergence and spread of more resistant strains due to selective pressure and becomes a great challenge in nosocomial infection control. 19,[21][22][23][24] It was noted that the high concentrations of biocides are toxic to humans and the environment, in addition to imposing cost to health system. 22 It seems that the difference in the effective con- attributed to the similarity of their chemical structure. 16 According to this study, the percentage of resistant strains in environmental isolates (60%) was higher than that of resistant strains in clinical isolates (50%). This may be due to the fact that environmental strains are more exposed to disinfectants than clinical strains. In contrast to the current study, a study showed that clinical strains of F I G U R E 3 (A-D). Relative expression of efflux genes in Acinetobacter baumannii strains isolated from clinical and environmental settings. Total RNA was extracted from each strain and reverse transcription to cDNA was performed using M-MLV enzyme. Real-time PCR was set on cDNA samples using SYBR Green I system and specific primer pairs. Threshold cycles (C t s) of each amplicon was used for further analysis. The relative quantities of the target genes were normalized against the 16 seconds rRNA gene. Fold-expression changes of (A) qacEΔ1, (B) adeB, (C) amvA, (D) abeM genes were calculated in each strain compared to reference strain and represented A. baumannii were more resistant to disinfectants than environmental strains. 27 The reason for the difference can be related to different testing conditions such as geographical location, type of disinfectants, and to the method of using disinfectants. The effectiveness of a disinfection method depends on the contact time, temperature, concentration of the active substance and the persistence of disinfectants, 2 and the type of growth of the isolates in the environment (biofilm or plankton). 28 In this study, the level of efflux gene expression was compared in environmental and clinical strains and showed that the mean expression of amvA and adeB genes was significantly higher in the environmental than clinical isolates of A. baumannii. There is further evidence for the role of adeB and amvA gene expression in resistance to biocides. In one study, strains of A. baumannii exposed to chlorhexidine digluconate had increased adeB gene expression. 29 Also, the expression of the adeB gene in A. baumannii, which was resistant to triclosan, was significantly higher than the susceptible strains. 30,31 In MDR A. baumannii, which were exposed to methyl viologen and ethidium bromide, amvA expression levels increased compared to the susceptible strains and inhibiting the amvA pump confirmed the role of this gene in resistance. 32 The most probable reason for the high resistance of environmental isolates is that they are more exposed to biocides than clinical isolates.
Based on the current result, the difference in the expression of qacEΔ1 gene was significant between susceptible and resistant strains to MICROZED ID-MAX disinfectant. Previously, a relationship between the presence of qac gene and increased MIC in A. baumannii isolates was reported. 33 MICs were suggested to be significantly high in strains of Salmonella spp., Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus containing the qacEΔ1 gene. 25,34 In contrast, there are other studies that do not suggest a significant difference between the presence or absence of qacE gene and susceptible/ resistant strains of A. baumannii. 18,23 This study showed that among clinical isolates, there was no significant difference in amvA gene expression between susceptible and resistant ones. There was also no significant difference in abeM gene expression between susceptible and resistant clinical isolates. In contrast, there was a significant difference in efflux gene expression between resistant and susceptible isolates of environmental source.
A study by Rajamohan et al suggested that by inactivation of these genes, susceptibility to multiple antibiotics and disinfectants was increased. 32 While we did not find a role for adeB efflux gene The results showed that the effective concentrations of disinfectants in inhibiting the growth of A. baumannii strains are less than the amounts recommended by manufacturing companies. Therefore, to prevent selective pressure and consequently the spread of biocideresistant strains, it is advisable to keep the concentration of the disinfectants according to their calculated MICs and bacterial type. EU GMP and US FDA recommend the use of disinfectants by rotations to prevent microbial resistance. 37

| CONCLUSIONS
The significant difference in the expression of efflux pumps genes studied in this study between the susceptible and resistant strains of A. baumannii shows the association of efflux function in resistance to disinfectants. Particularly abeM and amvA genes contribute to resistance of environmental isolates of A. baumannii to hydrogen peroxide derivatives. However, the contribution of efflux pumps mechanism in reducing the susceptibility of the clinical strains to disinfectants was not as evident as in the environmental isolates.
discrepancies from the study as planned (and, if relevant, registered) have been explained.

DATA AVAILABILITY STATEMENT
The authors confirm that the data supporting the findings of this study are available within the article.