Correlation between ability of biofilm formation with their responsible genes and MDR patterns in clinical and environmental Acinetobacter baumannii isolates

Accepted Manuscript Correlation between ability of biofilm formation with their responsible genes and MDR patterns in clinical and environmental Acinetobacter baumannii isolates Ali Mohammadi Bardbari, Mohammad Reza Arabestani, Manoochehr Karamie, Fariba Keramat, Kamran Pooshang Bagheri, Mohammad Yousef Alikhani PII:

S0882-4010(17)30175-4

DOI:

10.1016/j.micpath.2017.04.039

Reference:

YMPAT 2243

To appear in:

Microbial Pathogenesis

Received Date: 20 February 2017 Revised Date:

26 April 2017

Accepted Date: 26 April 2017

Please cite this article as: Bardbari AM, Arabestani MR, Karamie M, Keramat F, Bagheri KP, Alikhani MY, Correlation between ability of biofilm formation with their responsible genes and MDR patterns in clinical and environmental Acinetobacter baumannii isolates, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.04.039. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Correlation between ability of biofilm formation with their responsible genes and MDR patterns in clinical and environmental Acinetobacter baumannii isolates Ali Mohammadi Bardbaria, Mohammad Reza Arabestania, Manoochehr Karamieb, Fariba Keramatd,e, Kamran Pooshang Bagheri d*, Mohammad Yousef Alikhani a,e,*

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a Department of Microbiology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

b Department of Epidemiology, School of Public Health, Hamadan University of Medical Sciences, Hamadan, Iran c Department of infectious diseases, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, IR Iran

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d Venom and Biotherapeutics Molecules Lab., Biotechnology Dept., Biotechnology Research Center, Pasteur Institute of Iran. Tehran, Iran. e Brucellosis Research Center, Hamadan University of Medical Sciences, Hamadan, Iran

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Ali Mohammadi Bardbari Department of Microbiology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran. Telafax: (+98) 81- 38381795, E-mail: [email protected] Mohammad Reza Arabestani Department of Microbiology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran. Telafax: (+98) 81- 38381795, E-mail: [email protected] Manoochehr Karamie Department of Epidemiology, School of Public Health, Hamadan University of Medical Sciences, Hamadan, Iran. Telefax: (+98) 81-38380762, E-mail: [email protected] Fariba Keramat Department of infectious diseases, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran. Telafax: (+98) 81- 38381795, E-mail: [email protected]. Kamran Pooshang Bagheri Venom and Biotherapeutics Molecules Lab., Medical Biotechnology Dept., Biotechnology Research Center, Pasteur Institute of Iran. Tehran, Iran. Telefax: (+98) 21-66480780, E-mail: [email protected] Mohammad Yousef Alikhani Department of Microbiology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran. Telafax: (+98) 81- 38380755, E-mail: [email protected] Corresponding authors Mohammad Yousef Alikhani Department of Microbiology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran. Telafax: (+98) 81- 38380755, E-mail: [email protected], [email protected] Kamran Pooshang Bagheri Venom and Biotherapeutics Molecules Lab., Medical Biotechnology Dept., Biotechnology Research Center, Pasteur Institute of Iran. Tehran-Iran. Telefax: (+98) 21-66480780, E-mail: [email protected] 1

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Running title Biofilm formation and MDR patterns in Acinetobacter baumannii Abstract

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Acinetobacter baumannii potential to form biofilm and exhibit multiple antibiotic resistances may be responsible in its survival in hospital environment. Accordingly, our study was aimed to determine the correlation between ability of biofilm formation and the frequency of biofilm related genes with antibiotic resistance phenotypes, and also the categorization of their patterns in clinical and environmental isolates.

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A total of 75 clinical and 32 environmental strains of the A. baumannii were collected and identified via API 20NE. Antibiotic susceptibility was evaluated by disk diffusion and microdilution broth methods.

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Biofilm formation assay was performed by microtiter plate method. OXA types and biofilm related genes including BlaOXA-51, BlaOXA-23, BlaOXA-24, BlaOXA-58, bap, blaPER-1, and ompA were amplified by PCR.

The rate of MDR A. baumannii in clinical isolates (100%) was higher than environmental (81.2%) isolates (p<0.05). Among 10 antibiotypes, the predominant resistance pattern in clinical and environmental isolates was antibiotypes I (85.3 and 78.1%, respectively). Analysis of the frequency of

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blaOXA-23 gene revealed a statistically significant difference between clinical (85.3%) and environmental (68.7%) isolates (p < 0.05). The prevalence of strong biofilm producers in clinical and environmental isolates were 31.2% to 58.7%, respectively. In the clinical and environmental isolates, the frequencies of ompA, blaRER-1 and bap genes were 100%, 53.3%, 82.7% and 100%, 37.5%, 84.4% respectively.

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Statistical analysis revealed a significant correlation between the frequency of MDR isolates and biofilm formation ability (p = 0.008).

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The high frequency of antibiotype I would be indicated that an outbreak has been happened earlier and an endemic strain is currently being settled in the hospital environment. It would be suggested that if there was no difference in the frequency of pattern I and biofilm formation ability between clinical and environmental isolates, it is a critical point representing the higher risk of bacterial transmission from environment to the patients. The resulting data would be assisted in the improvement of disinfection strategies to better control of nosocomial infections. One dominant resistance pattern has shown among clinical and environmental isolates. The frequency of blaOXA-23 had significant difference between clinical and environmental isolates. The presence of bap gene in the A. baumannii isolates was 2

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associated with biofilm formation. There was a significant correlation between multiple drug resistance and biofilm formation. The clinical isolates had a higher ability to form strong biofilms compared to the environmental samples.

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Keywords: A. baumannii, biofilm formation, biofilm-related genes, MDR, OXA type genes

1. Introduction

A. baumannii is an aerobic, pleomorphic and non-motile gram-negative coccobacillus that has a high incidence rate among hospitalized patients (1). It causes a wide spectrum of infections including

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pneumonia, urinary tract infection, bacteremia, wound infection, meningitis and Ventilator-Associated Pneumonia (VAP). Multidrug-resistant A. baumannii is associated with high mortality in hospitalized

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patients (2).

In the last two decades, due to the widespread use of antibiotics, multi drug resistant and extensively drug resistant A. baumannii (MDR-AB, and XDR-AB) have emerged as a critical problem worldwide and have been increasingly reported (3, 4). The use of broad-spectrum antibiotics and transmission of strains among patients are considerd as selective pressures led to the emerging of MDR-AB (5). A. baumannii exhibits various mechanisms to resist multiple classes of antibiotics including the

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production of antibiotic-degradation/modification enzymes, active drug efflux pumps, decreased permeability, biofilm formation, and modification in drug targets (6). The main common mechanism responsible for carbapenem resistance in A. baumannii is production of carbapenemases, including class B metallo-β-lactamases (MBLs) and class D β-lactamases (oxacillinases) that they have been reported

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globally as the main mechanism responsible for carbapenems resistance (7, 8). Environmental contamination serves as a major reservoir for nosocomial outbreaks of this organism,

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especially in intensive care units (ICUs) (9). Because of the ability of A. baumannii to survive in the harsh hospital environment for the extended periods of time, the control of its infections is largely difficult. The potential of A. baumannii to form biofilm and exhibit multiple antibiotic resistance may be involved in its environmental survival (10). Biofilm formation on biotic and abiotic surfaces is an effective strategy to enhance the bacterial survival and its persistence under stressed conditions following antibiotic treatment or environmental conditions (6, 10). The development of antibiotic resistance and increased synthesis of exopolysaccharides are sometimes associated with biofilm production (11). 3

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Biofilm formation is a complex process employing many factors such as biofilm-associated protein (Bap), outer membrane poteinA (OmpA), beta-lactamase PER1, CsuA/BABCDE chaperone-usher pili assembly system, and the iron uptake mechanism (12). Some surface proteins including Bap, ompA, and blaPER-1 are involved not only in biofilm formation but

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also in bacterial attachment to the human epithelial cells and abiotic surfaces (13-15).

Providing a new insight into the better understanding of nosocomial A. baumannii infections through finding the correlations between survival parameters in clinical and environmental isolates and their

nosocomial A. baumannii infections in the hospital wards.

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potential transmission from environment to the patient body could be helpful for controling the

Accordingly, our study was aimed to determine the correlation between ability of biofilm formation with

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distribution of biofilm related genes and antibiotic resistance phenotypes, and also the categorization of their patterns in clinical and environmental isolates.

2. Materials and methods

2.1. Specimen collection from patients and hospital environment

During a period between November 2015 and August 2016, 428 specimens were collected from

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respiratory tracts reterived from sputum, bronchoalveolar lavage, and endotracheal aspirates of the patients hospitalized at ICU wards in three university hospitals in Hamadan, Iran. The specimens were obtained from individual patients who had been hospitalized for at least 2 days. Simultaneously, a total of 137 environmental samples were randomly collected from ventilators, sink

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area, floor, hand staff, trolleys and bedside table, pillow and linens, and other fomites. Sampling was performed according to Healthcare Infection Control Practices Advisory Committee (HICPAC)

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recommendations for environmental surface sampling (16). Breifly, each of the environmental swabs was immersed in 5mL of Brain Heart Infusion (BHI) broth (Merck Co., USA) and incubated overnight at 35°C. Thereafter, incubated broth were subcultured in the MacConkey agar plates (Merck Co., USA). The individual colonies were identified as A. baumannii according to the analytical profile index (API) 20NE (BioMérieux Co, France) protocol and the presence of blaOXA-51 gene (17). A. baumannii ATTC 19606 was used as a reference strain (18). 2.2. Antimicrobial susceptibility tests 2.2.1. Disk difusin 4

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Susceptibility testing was performed by the Kirby-Bauer disk diffusion method according to Clinical and Laboratory Standard Institute guidelines (CLSI) (19). The most commonly prescribed drugs for A baumannii infection were used for antibiotic susceptibility assay. The following discs (Mast Group Co, UK) including ampicillin/sulbactam (10µg /10µg), imipenem (10µg), meropenem (10µg), amikacin

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(30µg), cefepime (30µg), ciprofloxacin (5µg), colistin (10µg) and tigecycline (15µg) were used. Susceptibility to tigecycline was classified based on EUCAST criteria (zone around disk ≥17 mm) (20). Pseudomonas aeruginosa ATCC 27853 was used as quality control strain. The isolates that were resistant at least to three classes of antibiotics were considered as MDR A. baumannii as previously

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described (7). 2.2.2. Minimal inhibitory concentration

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Minimum inhibitory concentrations (MICs) of imipenem and colistin (Sigma-Aldrich, St Louis, MO, USA) were determined by broth micro dilution method in 96-well plates containing cation-adjusted Mueller– Hinton agar according to CLSI recommendations (19). Serial concentrations of colistin and imipenem were used (from 256 to 0.25µg/ml). MIC was considered as the last well in which no turbidity was observed. P. aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were used as quality control strains.

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2.3. Detection of blaOXA genes

The bacterial genomic DNA was extracted from overnight cultures of A. baumannii isolates using a DNA purification kit (Qiagen, Hilden, Germany) according to manufactureʼs protocol. All isolates were screened for the presence of the carbapenem resistance genes, including blaOXA-23,

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blaOXA-51, blaOXA-58, blaOXA-24 using a multiplex PCR technique previously described by Woodford et al (21). The list of primers used in the present study has been shown in table 1.

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The PCR mix contained 1µL (10pmol) of each primer, 2µL DNA, 25µL PCR Master Mix in a final 50µL reaction volume. DNA amplification was conducted in a thermal cycler (S1000™ Thermal Cycler, Bio-Rad, Hercules, CA, USA), under the following conditions: initial denaturation at 94°C for 5min, followed by 35 cycles of denaturation at 94°C for 45 second, an annealing temperature for each gene according to Table 1 for 1min, an extension at 72°C for 1 min, followed by a final extension at 72°C for 6 min. Electrophoresis of the amplified DNA fragments, along with a 100bp DNA ladder, was carried out using 2% agarose gel (Merck, Darmstadt,- Germany) in Tris- Borate-EDTA (TBE) buffer (pH 8.2). 2.4. Detection of biofilm related genes (ompA, bap, and blaPER-1) 5

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PCR assays for detection of ompA, bap, and blaPER-1 genes were performed by a set of primers as shown in Table 1. PCRs were carried out in 25µL reaction volume consisted of 2µL of extracted DNA, 12.5µL PCR Master Mix, 1µL (10pmol) of each primer. Conditions for the PCR were initial denaturation at 94°C for 5min, followed by 35 cycles of denaturation at 94°C for 60 second, an annealing temperature

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for each gene (according to Table 1) for 1min, an extension at 72°C for 45 second, followed by a final extension at 72°C for 5min. 2.5. Biofilm production assay

Biofilm production abilities of isolated strains were quantified by microtiter plate method using 1%

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crystal violet as previously described (22). The absorbance was measured at 560nm. Each assay was performed in triplicate and the results were reported as mean ± SD. TSB without bacteria was used as

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negative control. Biofilm production was interpreted according to the criteria of Stepannovic et al.(23). The optical density cut-off value (ODc) was established as three standard deviations (SD) above the mean of the optical density (OD) of the negative control as showen in the following formula: ODc = average OD of negative control + (3 × SD of negative control)

The results were divided into the four following categories according to their optical densities as (1) strong biofilm producer (4 x ODc < OD); (2) medium biofilm producer (2 x ODc < OD ≤ 4 x ODc); (3)

2.6. Statistical analysis

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weak biofilm producer (ODc
Statistical analysis was performed using SPSS 23.0 (SPSS, Chicago, IL, USA). The frequency of some parameters including A. baumannii positive cultures, susceptibility, OXA types genes, and also biofilm

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related genes were determined in both clinical and environmental samples. To compare categorical variables, chi-square or Fisher’s exact test were performed. The total frequencies of blaPER-1 and bap

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double positive strains were determined in all isolates and their correlation with biofilm formation was analyzed using binary logistic regression test. The association between biofilm formation ability and also the frequency of biofilm related genes with antibiotic resistance phenotypes of A. baumannii was evaluated in both groups or between groups by Fisher’s exact test. Any correlations between the origin of clinical or environmental A. baumannii isolates and particular characteristics (e.g. resistance patterns) of the bacteria were evaluated using Fisher’s exact test. All of the analyses were performed with a confidence level of 95%. P values <0.05 were considered statistically significant.

3. Result 3.1. The frequency distribution of A. baumannii in clinical and environmental samples 6

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During the 10-months study period, 428 clinical specimens were collected. Among 249 positive cultures, 75 (30.1%) isolates were A. baumannii. The age of the patients ranged from 19 to 90 years (mean age, 65.31 ± 1.9); 38 (50.7%) patients were male and 37 (49.3%) were female. At the same period a total of 137 hospital environmental samples were obtained. Among 110 positive cultures 32 (29%)

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isolates were identified as A. baumannii (table2). 3.2. Antimicrobial susceptibility testing

A. baumannii from environment demonstrated an antibiotic resistance rate of greater than 78% to majority of the examined antibiotics (6 out of 8). Antibiotic resistance was severe among the clinical

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isolates. All of clinical and environmental isolates were susceptible to colistin and tigecycline (table 3). One hundred and one A. baumannii isolates (94.4%) were resistant to three or more antibiotic classes

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and considered as MDR. The rate of MDR A. baumannii in clinical isolates (100%) was much higher than environmental isolates (81.2%). Ten different patterns of susceptibility were detected among isolates. The susceptibility patterns and the incidence of A. baumannii antibiotypes are presented in table 4 according to the source of the samples. Among 10 antibiotypes, the predominant resistance pattern in clinical and environmental isolates was antibiotypes I (85.3 and 78.12% respectively). 3.3. Minimal inhibitory concentration

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The MIC of clinical (n=75) and environmental (n=32) A. baumannii isolates are shown in table 3. The range of MIC for colistin in clinical and environmental isolates were ranged from 0.25 to 2 µg/mL and all of them were susceptible to colistin. Colistin in concentration of 1 µg/mL was able to inhibit the growth of the most of clinical and environmental isolates. The range of MIC for imipenem in clinical

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and environmental isolates were ranged from 2 to 256 µg/mL. According to results, 94.7% and 81.2% of clinical and environmental isolates were resistant to imipenem recpectively. The majority of imipenem-

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resistant A. baumannii from clinical and environmental isolates exhibited a MIC ≥ 256 µg/mL. 3.4. Biofilm production assay

The majority of both environmental and clinical isolates were able to form varying degrees of biofilm. The mean optical densities for clinical and environmental isolates were 0.687 ± 0.280 (ranged from 0.142 to 1.381) and 0.543 ± 0.275 (ranged from 0.122 to 1.224) respectively (table 5). Based on the results, biofilm production capabilities of the isolates were classified as strong, moderate, weak, and non-biofilm producer. The prevalence of strong biofilm producers in clinical and environmental isolates were 31.2% to 58.7% respectively. 3.5. The frequency of blaOXA genes 7

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Both clinical and environmental isolates were analyzed for the presence of carbapenem resistance gene. The results for the frequency of OXA genes among the isolates are presented in table 5. All of isolates encoded blaOXA-51 which intrinsically found in all of A. baumannii. blaOXA-58 gene was not detected in all difference between clinical and environmental isolates (p < 0.05).

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of clinical and environmental isolates. Analysis of the blaOXA-23 revealed a statistically significant

3.6. The frequency of biofilm-related genes and their correlation with biofilm formation

The frequency of biofilm-related genes was shown in table 5. The mean for biofilm biomass in bap and blaPER-1 positive clinical and environmental isolates were 0.649 ± 0.277 and 0.558 ± 0.252; and 0.625

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± 0.286 and 0.589 ± 0.333 respectively.

Comparing the frequency of ompA and blaPER-1 genes revealed that no significant difference between the

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biofilm and non-biofilm producer in the both clinical and environmental isolates. The results showed that 83.2% (89 cases) of all A. baumannii isolated in the current study encoded bap gene and the presence of this gene is associated with biofilm formation (p= 0.004). This issue was shown in both clinical and environmental isolates. There was no remarkable correlation between the frequency of blaPER-1 positive strains and biofilm producer ones in all clinical and environmental isolates (p> 0.05). Total frequencies of blaPER-1 and bap double positive isolates were shown in table 3. No correlation was

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seen between the frequency of blaPER-1-bap double positive isolates and biofilm formation in both clinical and environmental isolates (p > 0.05).

3.7. Association of biofilm formation and antibiotic resistance The mean for biofilm biomass in clinical and environmental MDR isolates were 0.687±0.280 and

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0.540±0.265, respectively. The mean for biofilm biomass in total MDR and non-MDR isolates were 0.649±0.289 and 0.556±0.346 respectively. Statistical analysis revealed a significant correlation

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between MDR phenotypes and biofilm formation ability (p=0.008).

4. Discussion

A. baumannii nosocomial infections are generally transmitted directly from environmental surfaces or via health-care workers to patients because of the ability of this organism to be survive in the hospital environment for a long period of time (24). Colonization of A. baumannii have been frequently reported from surgical and ICU wards where the most of nosocomial infections are occurred (25). To better management of infection control in a hospital, particularly in intensive care units, important parameters should be evaluated to provide an applicable approach that they could be considered as a 8

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practical guideline for infection control committees. Moreover, the physicians should also use these data to exploit effective treatment strategies in a hospital to avoid prolonged hospitalization of the patients, death, wasting budget, clinical complications, antibiotic resistance due to overload pressure of antibiotic consumption, and transmission of resistant bacteria as well. To achieve to this aim, the current study was

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designed to evaluate different parameters (i.e. biofilm formation ability, the frequency of biofilm related genes, oxa type genes, and etc.,) and their correlation with antibiotic resistance phenotypes.

Among 249 positive culture collected from patients’ respiratory tract samples, 75 (30.1%) A. baumannii

result is in accordance with previous studies (26, 27).

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were isolated. A. baumannii is the most common organism was isolated from respiratory tract and our

In our study, the incidence of A. baumannii isolated from environmental samples was greater than the

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earlier studies in which the frequency of environmental isolates in intensive care units were 4.017% (25), 7.3% (28), 13.1% (29) and 9.8% (9). However in the study of Obeidat et al., the incidence of A. baumannii in the environmental samples was 49.7% (30). Environmental isolates were mostly recovered from ventilator (37.5%) followed by pillow and bed linens (25%). This issue is raised a public health caution for hospitalized patients especially those who are admitted to surgical and ICU wards of the hospitals. The high prevalence of A. baumannii in hospital environment may imply that there is an

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improper infection control strategy in the hospitals.

Consistent with the previous studies, our disk diffusion results showed a higher rate of resistance in clinical isolates compared to environmental ones (29, 31). Colistin and tigecycline are currently the only effective antibiotics and have become the last-line therapy for antibiotic-resistant A. baumannii

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infection. While colistin-resistant A. baumannii isolates have been reported in some studies but our study was revealed that all of the clinical and environmental isolates were susceptible to colistin and

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tigecycline that was consist with the earlier studies (29, 30). Based on Fishers Exact Test, this study revealed that environmental MDR isolates have less frequent compared with those isolates colonizing the respiratory tract of hospitalized patients over the same period (p=0.001).

There are increasing reports on occurrence of MDR and extensive-drug resistant (XDR) clinical isolates of A. baumannii worldwide. Likewise, our results are in agreement with the occurrence of MDR A. baumannii described from different regions of the world (ranged from 67-100%) (10, 25, 30, and 32). Inversely, Greene et al. were showed that environmental isolates were almost more likely to be MDR than clinical isolates (10). 9

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Results of this study have shown one dominant resistance pattern among clinical and environmental A. baumannii isolates (i.e. SAMR, AKR, CIPR, IPMR, MEMR, CPMR, COLS, and TGCS). There was no significant difference in the frequency distribution of antibiotype I obtained from the clinical and

endemic strain is currently settled in hospital environment.

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environmental isolates (p >0.05). This issue indicated that an outbreak has been happened earlier and an

Our findings showed 100% frequency of blaOXA-51-like gene in clinical and environmental isolates that is in agreement with earlier reports (3, 7). Among carbapenem hydrolyzing class D β-lactamase genes (CHDL), blaOXA−23 was the most prevalent gene that has been reported in A. baumannii isolates

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worldwide and is mediated both by chromosomal integration and plasmids (4). Our study indicated that blaOXA-23 was the most prevalent gene in the clinical (85.3%) and environmental 68.7% isolates. Our

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analysis also showed that the frequency of blaOXA-23 had significant difference between clinical and environmental isolates (p < 0.05). This issue may indicate high horizontal transmission rate of this gene between isolates. As aforementioned, the presence of oxacillinase genes, mostly in the form of plasmidmediated blaOXA-23 enzymes could be correlated with high incidence rate of MDR A. baumannii (3, 33). blaOXA-24 gene was found in 30.7% and 31.2% cases of clinical and environmental isolates, respectively. Based on the previous reports, the blaOXA−24 gene could also be confined to bacterial chromosome or

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plasmids, however it appears to be less widespread than the blaOXA−23 gene (4). Similar to our findings, many recent studies reported similar prevalence of the blaOXA-23 and blaOXA-24 resistance genes in A. baumannii clinical and environmental isolates (4, 7, 30). According to current and the previous reports, high incidence of these genes may explain why the MDR clinical and environmental A. baumannii

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isolates are widespread (10, 30, 33). The frequency of double positive blaOXA-23 and blaOXA-24 clinical and environmental isolates were 30.7 and 31.2% respectively that interestingly all of them were categorized

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in pattern I.

Among several virulence factors, the ability to form biofilm is a major factor attributes to pathogenicity of A. baumannii (2). The current study proved that almost all of clinical and environmental isolates are able to form biofilm which is in line with previous studies (2, 30). No significant difference was observed in biofilm forming ability between clinical and environmental isolates. This result was similar to the results obtained by Obeidat et al. in 2014 (30) and de Campos et al. in 2016 (34). Inversely, Greene et al. showed that clinical isolates produced less biofilm than environmental isolates (10). Clinical isolates in our study were significantly shown to have a higher ability to form strong biofilms compared to those recovered from environmental samples (p=0.009). This matter also demonstrates that 10

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classification of the collected isolates based on the biofilm formation ability could guide us to get additional insight into potential pathogenicity of environmental isolates. Consequently, it could help us to improve disinfection strategies and better control of nosocomial infections. Previous studies reported that the frequency of MDR phenotype of A. baumannii was linked to biofilm

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production ability (6, 35). In line with the previous investigations, our statistical analysis revealed a significant difference between the frequency of MDR and biofilm formation (p=0.008).

The frequency of biofilm-related genes in the clinical and environmental A. baumannii isolates was similar to previous studies (32, 36, 37). Regarding to the frequency of biofilm-related genes and ability

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of biofilm formation, no significant statistical difference was observed between environmental and clinical A. baumannii strains. In current study, most of the clinical and environmental isolates of A.

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baumannii encoded bap gene. The presence of this gene was associated with biofilm formation (p=0.004). Sung et al. (22) and Azizi et al. (32) showed that biofilm formation ability in A. baumannii isolates carrying the bap gene were significantly differ from those without this gene. Our results also proved the major role of bap gene in biofilm formation. It has been already reported that there was a positive correlation between the biofilm formation and blaPER-1 gene among the A. baumannii isolates (15) but in our study, there was no remarkable correlation in both clinical and environmental isolates (p

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> 0.05). Our findings are in accordance with some previous studies (38). High frequency of antibiotype I could indicate that an outbreak has been happened earlier and subsequently an endemic strain currently settled in the hospital environment. The high incidence rate of OXA type genes may explain the wide spread of the MDR A. baumannii isolates among both the

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environment and clinical isolates. Categorization of biofilm formation ability and the frequency of dominant MDR antibiotype would be clarified the risk of infection from environment to the hospitalized

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patients. There were no significant correlation between the source of clinical (sputum, bronchoalveolar lavage and endotracheal aspirate) and environmental (ventilation, sink area, floor, handstaff, Trolleys & bedside table, Trolleys & bedside table and other fomite) isolates and particular characteristics (i.e. biofilm-related genes, OXA type, and antibiotic resistance patterns). By comparing the frequency of pattern I and biofilm formation ability between clinical and environmental isolates, we did not find a significance difference (p > 0.05). Thus, this predictive value could be considered as a cut-off point to represent the current status of environmental and clinical isolates regarding biofilm formation and concomitantly being MDR. It would be suggested that if the p 11

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value was greater than 0.05, this could be considered as a critical status for infection control committees; representing the higher risk of infection transmission from environment to the patient bodies. In the current study, some signifigant tips concerning the parametrs involved in the risk of bacterial transmission from environment to patients were succsesfully determined. Our data would be applied in

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the improvement of disinfection strategies to control nosocomial infections. Therefore, deepen knowledge of the mechanisms underlying biofilm formation and development of drug resistance will allow us to more effectively control and/or treat biofilm-related infections. Conclusion

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One dominant resistance pattern has shown among clinical and environmental isolates. The frequency of blaOXA-23 had significant difference between clinical and environmental isolates. The presence of bap

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gene in the A. baumannii isolates was associated with biofilm formation. There was a significant correlation between multiple drug resistance and biofilm formation. Clinical isolates had a higher ability to form strong biofilms compared to environmental samples.

Acknowledgments

This investigation is a part of Ph.D. thesis of Ali Mohammadi Bardbari, approved and financially

Hamedan, Iran.

Conflict of interest

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supported by the vice chancler of research and technology of Hamedan university of Medical Sciensec,

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Refernces

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None of the authors have any conflicts of interest to this article.

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[20] Perez F, Hujer AM, Hujer KM, Decker BK, Rather PN, Bonomo RA. Global challenge of multidrug-resistant Acinetobacter baumannii. Antimicrobial agents and chemotherapy. 51 (2007) 3471-3484. [21] Woodford N, Ellington MJ, Coelho JM, Turton JF, Ward ME, Brown S, et al. Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. International journal of antimicrobial agents. 27 (2006) 351-353.

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[31] Gong Y, Shen X, Huang G, Zhang C, Luo X, Yin S, et al. Epidemiology and resistance features of Acinetobacter baumannii isolates from the ward environment and patients in the burn ICU of a Chinese hospital. Journal of Microbiology. 54 (2016) 51-58.

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[32] Azizi O, Shahcheraghi F, Salimizand H, Modarresi F, Shakibaie MR, Mansouri S, et al. Molecular Analysis and Expression of bap Gene in Biofilm-Forming Multi-Drug-Resistant Acinetobacter baumannii. Rep Biochem Mol Biol. 5 (2016) 62-72. [33] Chung H-S, Lee Y, Park ES, Lee DS, Ha EJ, Kim M, et al. Characterization of the Multidrug-Resistant Acinetobacter species Causing a Nosocomial Outbreak at Intensive Care Units in a Korean Teaching Hospital: Suggesting the Correlations with the Clinical and Environmental Samples, Including Respiratory Tract-rela. Annals of Clinical Microbiology. 17 (2014) 29-34. [34] de Campos PA, Royer S, da Fonseca Batistão DW, Araújo BF, Queiroz LL, de Brito CS, et al. Multidrug Resistance Related to Biofilm Formation in Acinetobacter baumannii and Klebsiella pneumoniae Clinical Strains from Different Pulsotypes. Current microbiology. 72 (2016) 617-627.

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[35] Rao RS, Karthika RU, Singh SP, Shashikala P, Kanungo R, Jayachandran S, et al. Correlation between biofilm production and multiple drug resistance in imipenem resistant clinical isolates of Acinetobacter baumannii. Indian journal of medical microbiology. 26 (2008) 333-337. [36] Liu H, Wu Y-Q, Chen L-P, Gao X, Huang H-N, Qiu F-L, et al. Biofilm-Related Genes: Analyses in Multi-Antibiotic Resistant Acinetobacter Baumannii Isolates From Mainland China. Medical Science Monitor. 22 (2016) 1801-1807.

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[39] McConnell MJ, Pérez-Ordóñez A, Pérez-Romero P, Valencia R, Lepe JA, Vázquez-Barba I, et al. Quantitative real-time PCR for detection of Acinetobacter baumannii colonization in the hospital environment. Journal of clinical microbiology. 50 (2012) 1412-1414.

Table1: The primers used in this study for detection of carbapenem resistance and biofilm related genes.

blaOXA-51 blaOXA-58

blaOXA-23 bap ompA

ATT TCT GAC CGC ATT TCC AT TGCTGACAGTGACGTAGAACCACA TGCAACTAGTGGAATAGCAGCCCA TCTTGGTGGTCACTTGAAGCACTCTTGTGGTTGTGGAGCA GCAACTGCTGCAATACTCGG ATGTGCGACCACAGTACCAG

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blaPER-1

R F R F R F R

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thblaOXA-40

F R F R F R F

➔3') Sequence primer (5'➔ TAA TGC TTT GAT CGG CCT TG TGG ATT GCA CTT CAT CTT GG AAG TAT TGG GGC TTG TGC TG CCC CTC TGC GCT CTA CAT AC GGT TAG TTG GCC CCC TTA AA AGT TGA GCG AAA AGG GGA TT GAT CGG ATT GGA GAA CCA GA

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Target gene

15

Amplication size(bp)

Annealing temperature

Ref

353

52

(21)

599

52

(21)

246

52

(21)

501

52

(21)

184

57

(13)

85

50

(39)

900

50

(3)

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Table 2. The frequency distribution of A. baumannii in clinical and environmental samples based on the location of specimen collection. Positive Culture No. (%)

A. baumnnii isolates No. (%)

Ventilator Sink area Floor Hand staff

44 17 15 13

40 (36.4) 14 (12.7) 13 (11.8) 8 (7.3)

12 (37.5) 2 (6.2) 3 (9.4) 3 (9.4)

Trolleys & bedside table Pillow& Bed linens b Other fomite Total environmental Sputum Bronchoalveolar lavage Endotracheal aspirate Total clinical

14

11 (10.0)

19 15 137 138 70 220 428

16 (14.5) 8 (7.3) 110 43 (17.3) 22 (8.8) 184 (73.9) 249

Hospital source

Hospital 1

Hospital 2

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Samples Cultured, No.

Hospital 3

3 0 0 1

4 0 2 0

5 2 1 2

1 (3.1)

0

1

0

8 (25) 3 (9.4) 32 (100) 23 (30.7) 12 (16.0) 40 (53.3) 75 (100)

3 0 7 9 6 15 30

3 2 12 6 4 12 22

2 1 13 8 2 13 23

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Site of isolation

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Table 3. The antimicrobial resistant patterns in clinical and environmental A. baumannii isolates MIC for imipenem (%)

Antibiotic resistance (%) COL

IPM

TGC

CPM*

97.4

94.7

97.3

0

94.7

0

100

5.3

94.7

100

0

78.1

81.2

81.2

81.2

0

81.2

0

81.2

18.7

81.2

100

0

90.7

90.7

93.5

92.5

0

90.7

0

94.4

9.3

90.7

100

0

0.007

0.062

0.003

0.009

-

0.062

-

0.001

-

-

-

-

S (≤2)

16

S (≤4)

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MEM *

96

R (≥16)

CIP *

Clinical (n = 75) environmental (n = 32) Total (n=107) P value

R (≥16)

AK

SAM*

Source

MIC for colistin (%)

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AK, amikacin; MEM, meropenem; IPM, imipenem; CPM, cefepime; COL, colistin; TGC, tigecycline; CIP, ciprofloxacin; SAM, Ampicillin-sulbactam; S: susceptible; R: resistant; Asterisks (*) assign show statistical significance results (P < 0.05) between clinical and environmental isolates.

Table 4. Classification of clinical and environmental isolates to different resistance patterns. Environmental No (%)

Total No (%)

MDR/NonMDR

64 (85.3 )

25 (78.1)

89 (83.2)

MDR

R

1 (1.3)

-

1 (0.9)

MDR

R

3 (4.0)

-

3 (2.8)

MDR

R

1 (1.3)

-

1 (0.9)

MDR

R

1 (1.3)

1 (3.1)

2 (1.9)

MDR

R

2 (2.7)

-

2 (1.9)

MDR

SAM*

AK*

CIP*

MEM*

COL*

IPM*

TGC*

CPM*

I

R*

R

R

R

S*

R

S

R

II

R

R

R

S

S

S

S

III

R

S

R

R

S

R

S

IV

R

R

S

R

S

S

S

V

S

S

R

R

S

R

S

VI

S

R

R

R

S

R

S

VII

R

R

R

R

S

S

S

R

2 (2.7)

-

2 (1.9)

MDR

VIII

R

R

R

S

S

R

S

R

1 (1.3)

-

1 (0.9)

MDR

IX

S

R

S

R

S

R

S

S

-

1 (3.1)

1 (0.9)

MDR

X

S

S

S

S

S

S

S

S

-

5 (15.6)

5 (4.7)

Non MDR

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Pattern

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Clinical No (%)

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R, resistant; S, sensitive; SAM, Ampicillin-sulbactam; AK, amikacin; CIP, ciprofloxacin; MEM, meropenem; COL, colistin; IPM, imipenem; TGC, tigecycline; CPM, cefepime.

Table 5. The frequency distribution of biofilm formation ability, OXA type genes and biofilm-related genes in clinical and environmental isolates. Biofilm formation (%)

Biofilm-related genes (%) blaPER-1 +bap

blaPER-1

ompA

bap

OXA-23 +OXA-24

OXA-58

17

OXA-24

OXA-23 *

OXA-51

Strong * biofilm

Moderate biofilm

Weak biofilm

Non biofilm

Source

OXA type genes (%)

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1.3

2.7

37.3

58.7

100

85.3

30.7

0

30.7

82.7

100

53.3

41.3

6.9

9.4

53.1

31.2

100

68.7

31.2

0

31.2

84.4

100

37.5

34.4

Total (n=107) P value

2.8

4.7

42.1

50.5

100

80.4

30.8

0

30.8

83.2

100

48.6

39.2

0.212

0.157

0.13

0.021

-

0.048

0.952

-

0.952

0.829

-

0.134

0.537

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Clinical (n = 75) environmental (n = 32)

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Asterisks (*) assign show statistical significance (P < 0.05) between clinical and environmental isolates.

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Highlights

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There was a significant correlation between multiple drug resistance and biofilm formation. One dominant resistance pattern has shown among clinical and environmental isolates. The presence of bap gene in the A. baumannii isolates was associated with biofilm formation. The frequency of blaOXA-23 had significant difference between clinical and environmental isolates. Clinical isolates had a higher ability to form strong biofilms compared to environmental samples.

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•