Multi-Drug Resistant Acinetobacter: Detection of blaNDM-1 genotype and biofilm formation at a tertiary care hospital in Eastern Nepal

Background: Acinetobacter species is an important hospital acquired pathogen. Rapid development of resistance to multiple drug and ability to form biofilm make these bacteria more adaptable to survive in health care facilities, thus posing a challenge for its effective management. This study was aimed to characterize clinical isolates of Acinetobacter spp, study their antimicrobial susceptibility pattern and ability to form biofilm. Resistant Acinetobacter was further analyzed for the detection of extended spectrum β-lactamases (ESBLs), metallo β-lactamases (MBLs) production and presence of blaNDM-1 (New Delhi metallo-beta-lactamase-1) gene. Results: A total of 324 Acinetobacter species isolated from various clinical specimens submitted to Department of Microbiology, B. P. Koirala Institute of Health Sciences, Dharan, were studied Antibiotic susceptibility testing, detection of ESBL and MBL production, and biofilm formation was performed by standard microbiological methods. PCR was done to determine the presence of blaNDM-1 gene. Predominant Acinetobacter species isolated was A calcoaceticus-baumannii Complex (Acb complex) 167 (51.5%). 13 (4.0%) were ESBL producers, 61.9% (70/113) were MBL, 10.6% (12/113) were carbapenemase producers. About 40% were Multi-drug resistant (MDR). Among MDR, producers of ESBL, MBL and carbapenemase were 1.6%, 57% and 9.6%, respectively. Thirty seven percent isolates formed biofilm. A significant proportion of biofilm producers were MDR (p<0.001). Majority was resistant to cefotaxime (73.8%) and cefepime (74.4%). blaNDM1 gene was present in 33 isolates. Conclusions: Resistant Acinetobacter formed a substantial proportion in our hospital with presence of blaNDM-1 gene in significant numbers. A matter of great concern is association of Multi-drug resistant phenotype with biofilm formation. This situation warrants a stringent surveillance and adherence to Infection prevention and control practice.


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Background Acinetobacter, a widely distributed, saprophytic bacterium in nature has established itself as the most common nosocomial pathogen [1,2]. Although different species of Acinetobacter have potential to cause infection, 80% of infections are caused by Acinetobacter baumannii. Ease of survival even in adverse environment, ability to form biofilms on the surfaces and possession of many genes for antimicrobials resistance have made this bacteria an important pathogen. The potential ability to form biofilms, indeed, could explain outstanding antibiotic resistance and survival properties in the harsh environment of hospital, particularly the intensive care setting [3,4].
Over the past few decades, its clinical importance has been increased by its ability to receive antimicrobial resistance factors [5] through the transfer of plasmid or transposons that contain antimicrobial resistant genes particularly in hospital setting where usage of antibiotics is huge leading to selective pressure. Multi-drug resistant (MDR) Acinetobacter species is defined as the isolate resistant to at least three classes of antimicrobial agents -all penicillins and cephalosporins, fluoroquinolones, and aminoglycosides [6]. These strains are implicated in variety of life threatening infections such as ventilator-associated pneumonia (VAP), urinary tract infections, blood stream infections, surgical site infections, and infections associated with medical devices occurring especially in patients in intensive care unit. Moreover, significant correlation between biofilm formation and multidrug resistance has been attributed to the threat imposed by Acinetobacter to the current antibiotic era [7].
Diagnosis of multi-drug resistant Acinetobacter infections is a great challenge owing to the distribution of various species in relation to the type of infection, their antimicrobial profile and biofilm forming phenotype. Hence, it is crucial to minimize the risk associated with Acinetobacter infection in health care facilities, from effective management and infection control perspectives. Present study was conducted to characterize the clinical Acinetobacter isolates with special reference to the detection of antimicrobial resistance, biofilm formation and presence of bla NDM−1 ( New Delhi metallo-beta-lactamase-1) gene.
Among the different specimens analyzed, frequency of yield of Acinetobacter from blood in the descending order of frequency was A. haemolyticus (37%), A. lowffi (33%), A. radioresistens (30%), Acb complex (20%), and A. junii (16.5%), as shown in Table 1. In case of pus samples, Acb complex was predominant (37%), whereas from urine sample it was A. radioresistens (33%). In this study, 26% of the samples were obtained from medical ward, 20% from ICU, 11% from surgery and pediatrics, 6% from gynecology, 12% were from OPD, 4% were from emergency, NICU and orthopedic department. Acb complex was predominant in ICU (76.7%).  Table 2. Acb complex was found to have had highest drug resistant phenotypes to analyzed antibiotics with 84.2% being resistant to imipenem. Acinetobacter isolates from ICU were comparatively resistant to the antibiotics than those from other wards (Fig. 1).   Table 4 & Table 5. The biofilm formations were consistently found in isolates from ICU ( Fig. 2) and Acb complex (Fig. 3) as shown by principle component analysis.  Table 5 Association between antibiotic resistance, ESBL production, MBL production, biofilm production and  [20].
The gold standard for the identification of carbapenemase production and detection of bla NDM−1 gene is PCR assay [30]. Moreover, other assay such as Loop mediated isothermal amplification [31] has been developed for the detection of bla NDM−1 gene. In this study, NDM producing Acinetobacter isolates was 10.2% (33/324) which was consistent with reports by Devkota et al. 2017 (12.8%) [32]. The evidence of Acinetobacter with presence of bla NDM−1 gene had already been reported worldwide [33,34]. In this study, 91% NDM producers were resistant to second and third generation cephalosporins. Moreover, 25 [6]. We found that isolates with highest biofilm producer was Acb complex (77.7%) which was marginally higher than 60% reported by Yadav et al. 2017. This disagreement may be due to the variation in the nature of clinical isolates from different geographical locations. Biofilm forming isolates found mostly samples from medical devices (37.2%) followed by pus (29.8%) which was consistent with previous studies [36,37]. In addition, the biofilm forming isolates were predominating in ICU (64.4%) followed by wards (31.1%) which was consistent with other studies [38]. Among the biofilm formers in this study, we found resistance to imipenem 62.0% (75/121) which was higher than 46.7% reported by Yadav et al 2017 [25]. Moreover, association between biofilm producer and imipenem has also shown by Chaudhary et al.
had both phenotype biofilm producers and multi-drug resistant [40]. However, a few variable results are seen in several studies due to different scenario that might be due to difference in geography, arrangement of specimens in the study groups, condition and use of antibiotic.
The data from this study demonstrates that Acinetobacter species are resistant to many available antimicrobial agents making this nosocomial pathogens one of the most significant microbial challenges to control in future.

Conclusion
The clinical isolates of Acinetobacter in our setting were multi-drug resistant and biofilm former as revealed by the present study. In addition, the biofilm formation could be the potential marker to determine the multi-drug resistant (MDR) phenotype. These isolates have been proven to cause nosocomial infection in health care settings. Therefore and detection of growth after the recommended duration was done by standard microbiological techniques [8]. Suspected smooth, opaque colonies on blood agar corresponding non lactose fermenting colonies on MacConkey and on CLED agar plates were presumed as Acinetobacter and processed further. Species identification of the genus Acinetobacter was done by several biochemical tests [9].
Antimicrobial Susceptibility test

Detection of ESBL phenotype
According to the CLSI guidelines, the probable ESBL producing isolate had zone of inhibition for ceftazidime (30 µg) ≤ 22 mm and cefotaxime (30 µg) ≤ 27 mm [8]. In order to confirm ESBL production, ceftazidime (30 µg) and ceftazidime + clavulanate (30/10 µg) disc were placed in Acinetobacter culture. Zone of inhibition were compared with the ceftazidime and cefotaxime discs alone and compared with the combined ceftazidime + clavulanate disc. An enhanced zone of diameter of ≥ 5 mm in combination with clavulanate confirmed isolates as ESBL. Combined disc diffusion test was employed to determine the MBL producing phenotype in Acinetobacter. On MHA plate lawn culture of Acinetobacter, imipenem disc (10 µg) and imipenem disc with 10 µl of 0.5 M EDTA were applied 20 mm apart from center to center.
Isolate demonstrating zone of inhibition of more than 7 mm around the imipenem-EDTA disc than imipenem disc alone was considered as MBL producer [10].
b. Carbapenemase production test Phenotypic detection of carbapenemase producing MDR Acinetobacter was determined by Modified Hodge Test [8]. New Delhi metallo-beta-lactamase-1 (bla NDM − 1 ) is a novel MBL that confers resistance to all β-lactam antibiotics with the exception of aztreonam. In this study, the multi-drug resistant organisms were selected to detect bla NDM − 1 gene by conventional PCR [11].

DNA extraction of bacterial isolates
The overnight broth cultures were centrifuged at 3,500 rpm for 10 minutes at 4 °C. Then, the supernatant was discarded and the pellet was washed twice with 5 ml phosphate buffered saline (PBS) followed by centrifugation. The pellet was re-suspended in 1 ml PBS and centrifuged at 10,000 rpm for 10 minutes at 4 °C. Finally, the supernatant was discarded and the remaining pellet was stored at -20 °C till assayed. [12].

Detection of Biofilm formation
Detection of biofilm formation was performed by the standard laboratory methods described elsewhere [14][15][16][17]. The bacterial isolates were grown overnight at 37 °C in 5 ml of tryptic soy broth (TSB). Methicillin-sensitive Staphylococcus aureus (MSSA) ATCC-25923 and P. aeruginosa ATCC-27853 were used as negative and positive controls, respectively. Interpretation of OD measurements: The average OD values were calculated for all tested strains and negative controls. The cut-off value (ODc) was calculated as three standard deviations (SD) above the mean OD of negative control. ODc = average OD of negative control + (3 × SD of negative control). Strains were divided into four categories which are based upon OD values: (a) OD ≤ ODc = no biofilms producer, (b) ODc < OD ≤ 2 × ODc = weak biofilm production (+), (c) 2 × ODc < OD ≤ 4 × ODc = moderate biofilm production (++), and (d) 4 × ODc < OD = strong biofilm production (+++).

Analysis
Data were entered in MS Excel worksheet and statistical analysis was done by using R package [18]. The principle component analysis among the several factors was done by using "prcomp" function of R stat package and visualization of the plot was demonstrated by ggbiplot package [19].