Occurrence and molecular characterization of metallo- β-lactamases (MBLs) among Acinetobacter baumannii isolates from cancer patients

Background:During the last decade, the prevalence of carbapenemresistant infection associated with multidrug resistant (MDR) Acinetobacter baumanniiin patients has been continuously increasing. This prospective study aimed to determine the occurrence and molecular characterization of metallo-β-lactamases (MBLs) and carbapenem hydrolyzing oxacillinases among A. baumannii isolates from cancer patients over a period of 6-month. Methods:Antimicrobial susceptibility profile of 70 randomly collected A. baumannii isolates was first determined using disc diffusion test, and second, the MICs of 45 representative multidrug resistant (MDR) isolates were tested to most clinically used drugs in treatment of their infections using E-test. PCR assays were used to detect the common four types of class D carbapenem hydrolyzing oxacillinases, two types of class A carbapenemases, four types of class B metallo-β –lactamases, and prevalence of Class 1 Integron among MDR isolates. Results:All 70 isolates were MDR, including 100% resistance to meropenem, aztreonam, piperacillin/tazobactum and 99% to carbapenem. All isolates carried blaOXA-23 and blaOXA-51, but none carried a blaOXA-24 like or blaOXA-58. The isolates were also positive for NDM-1, VIM, GES, KPC and SPM at the rates of 29%, 20%, 29%, 19%, 7% and 2%, respectively. Class 1 Integron was positive in 82% of A. baumannii isolates. The clonal relationship of 42 MDR A. baumannii isolates using ERICPCR and constructed dendrogram showed 3 major genotype clusters Occurrence and molecular characterization of metalloβ-lactamases (MBLs) among Acinetobacter baumannii isolates from cancer patients Husam F. qouzah1, Feras Hawari2, Luay F. Abu-Qatouseh3, Asem A. Shehabi1 1 Department of Pathology-Microbiology, School of Medicine, The Jordan University, Amman, Jordan 2 King Husain Cancer Center (KHCC), Amman, Jordan 3 Faculty of Pharmacy, Department of Biomedical Sciences, University of Petra, Amman, Jordan. Contact information:


Introduction
In recent years worldwide including Jordan, most clinical A. baumannii isolates have developed resistant to most clinically used drugs, and caused increased morbidity and mortality among hospitalized patients in intensive care units (ICUs) [1][2][3][4].
A. baumannii acts first often as colonizer rather than a pathogen because of its wide distribution and colonizing capability [1,7], especially in immune-compromised and among patients with prolonged hospital stay [8]. The spectrum of infections caused by this pathogen include ventilator-associated pneumonia (VAP) which is developed after prolonged hospitalization or mechanical ventilation and prior use of antibiotics [4,[9][10].
Many factors contribute to the ability of A. baumannii to cause infections in hospitalized patients. First, A. baumannii has the capacity to survive for long periods in various body parts of patients and their environment. Secondly, the genomic makeup of this organism allows it to acquire resistance to many antibiotics within a short period [11]. However, the rates of antibiotic resistance vary from region to region and among hospitals [12]. Almost all studies indicate that MDR A. baumannii infections are difficult to treat, spread quickly among hospitalized patients and can be associated with high mortality due to blood sepsis or ventilator associated pneumonia [2,4,[12][13][14].
This study describes the occurrence and the mechanism of antibiotic resistance of A. baumannii isolated from cancer patients.

Collection of A. baumannii isolates
This prospective study included a total of 70 A. baumannii isolates, were recovered from clinical specimens of patients who were investigated for presence of A. baumannii during hospitalization or investigation as outpatients at a Cancer Center in Amman, Jordan, over a period of 6-months (2016) as shown in the Table 2. Approval was obtained from the Institution Ethical Review Board (IERB) at the King Hussein Cancer Center (KHCC), Amman, Jordan, for conducting and publishing this study.

Identification of A. baumannii isolates
All isolateswere first identified using BioMerieux VI-TEK 2 Automated Microbiology System (France). Later all collected isolates were sub-cultured on Blood and MacConkey agar plates and incubated at 37°C for 24 hr. Pure growth was again confirmed as A. baumannii according to the following characteristics; negative oxidase test, negative lactose and glucose fermentation in Kligler iron tubes, and later by presence of OXA-51 gene using PCR. Five fresh colonies were inoculated in brain-heart infusion agar plus 15% glycerol and kept frozen at -70 ºC until used for further investigation.

Antibiotic susceptibility tests
The susceptibility of A. baumannii isolates to antibiotics was determined using the disc diffusion method according to the guidelines of CLSI 2016 [15]. The minimum inhibitory concentrations (MICs) of isolates were determined by the E-test (ABBioMérieux, France) for imipenem, amikacin and colistin. Interpretations of the MICs in the E-test were done according to the guidelines of CLSI 2016 [15].

Genomic DNA Extraction from A. baumannii Isolates
Genomic DNA was extracted according to the manufacturer's instructions of Wizard Genomic DNA purification kit (Promega, USA).

Plasmid DNA extraction from A. baumannii isolates
The bacterial plasmid was extracted using the EZ-10 Spin Column Plasmid DNA Minipreps Bio Basic kit (Canada) according to manufactures protocol.
The following control strains were used in the experiment, which were kindly donated by Prof. PCR tests were carried out in 25 μl reaction with 2.5 μl of extracted DNA, 20 pmol of each primer (Alpha DNA, Montreal, Canada), 12.5 μl GoTaq ® Green master mix (Promega, USA). The volume was made up to 25 µl using nuclease free water. DNA concentrations of each sample was evaluated using Nanodrop 2000c (Thermo scientific, USA). The PCR amplification assays for the target genes were performed using programmable PCR-Thermocycler (Bioerxp cycler, China). Control tubes containing master mix without template DNA were included in each run as negative control. Tubes were held at 4°C when the cycles were ended. The amplified products and the PCR DNA marker were separated via electrophoresis on 2% agarose gels containing 15% Red safe™ stain (5 µl), for 40-50 min at 120 volts, and then visualized using Gel documentation system including:UV camera, monitor and printer (UVP, USA).

Enterobacterial repetitive consensus (ERIC)-PCR
This test was performed in 50 μl volumes containing 10 ng of genomic DNA from A. baumannii clinical isolates, 4 mM MgCl2, 50 pM each of primer ERIC-1 and 2 as described by Jeong et al. [22].

Statistical analysis
Statistical analysis was done using SPSS version 20, chi square test and t-test were used to analyzed categorical variablesand to measure the significance of the association between the study variables. If less than 5 a fisher exact test was used. P-value of ≤ 0.05 was considered significant \. Table 2 Shows the sources of 70 A. baumannii isolates within the period of study (March 2016 to October 2016). The majority of samples were collected from nasal swabs (30%) and perianal swabs (21%). Table 3 shows the demographic characteristics of cancer patients with positive A. baumannii isolates. The majority of patients (56%) were over 50 years old and were in-patient (90%) rather than out-patient (10%). Most of the patients (68%) have received medication prior to collection their specimens. The results of antimicrobial susceptibility using disc diffusion method are presented in Table 4 A. baumannii isolates were 100% resistant   Table 4.
The genetic relationship of 42 MDR A. baumannii isolates from hospitalized patients as shown in the dendrogram indicated 3 major genotype clusters of genetically related isolates (Figure 1). These include

Discussion
This study has demonstrates that many body sites of cancer patients are frequently colonized with A. baumannii, especially their nares andperianal site. Invasive blood infection was detected among few percentage (6%)of hospitalized cancer patients during the 6-month study period ( Table 2).The rest of the isolates can be considered colonizers and might become potential pathogens for patients under certain condition, especially in association with the use of invasive procedures [23][24]. There is no significance difference in general demographic characteristics between male and female patients or their ages, whereas treatment with antibiotics showed increased significantly the chance of (P=0.033) colonization with A. baumanni. Additionally, hospitalized patients were at increased risk for colonization with AB (90%; P=0.026) when compared to outpatients (10%) with A. baumannii (Table 3). Recent studies from Jordan and other countries reported that A. Baumannii frequently colonizes hospitalized patients, causing sporadic invasive infection and nosocomial outbreaks, especially among critically ill patients and in ICUs [2,[25][26][27].
This study demonstrates that the majority of A. baumannii species isolates were resistant in the range between 76% to 100% to commonly useful antimicrobial agents in the treatment of gram-negative infections, including infections due to A. baumannii (Table 4). Previous studies which has been performed over the last few years at the Jordan University Hospital and KHCC in Amman, have also found that the majority of A. baumannii isolates from patients and hospital environment sources was also multidrug resistant [2,3,28].
The ability to acquire resistance to a broad range of antimicrobial agents within a short period and during treatment is commonly observed in A. baumannii [4,29]. High mortality rates among critically ill patients infected with MDRor extensively drugresistant (XDR) A. baumannii strains (resistant to all antibiotics except colistin and tigecycline, have been recently described in many regions [30][31][32]. This study shows extremely high rate of carbapenems-resistance (99-100%) among A. baumannii isolates. The prevalence of imipenem-resistance among A. baumannii isolates was 99%. This rate is very high compared to resistance rates of clinical isolates reported few years ago from Jordan and neighboring countries which ranged between 90% to 64% [3,31,32,33]. A previous Jordanian study has also indicated that majority of Abaumannii isolates from environmental and clinical sources was multidrug resistant, except for colistin and tigecycline similar to the case in other countries such as Lebanon [3,32].
Carbapenems are frequently used as the drugs of choice in treatment of infections caused by MDR Gram-negative bacteria including A. baumannii. Increased resistance to carbapenems in A. baumannii has raised special concerns during the last decade, especially since it is associated mostly with the production of acquired carbapenemases belonging to either carbapenem-hydrolyzing OXA-type class D -β-lactamases or class B metallo-β-lactamases [32,[35][36]. The results of this study demonstrate that all representative examined 45 MDR A. baumannii isolates were 100% positive for genes of blaOXA-51, and blaOXA-23, but none of the isolates was positive to OXA-24 or OXA-58. A recent Jordanian study carried at the Jordan University Hospital also showed that all examined A. baumannii isolates harbored a blaOXA-51gene, 58% has a blaOXA-23gene, and 38.8% has a blaOXA-24 gene [3]. It is well known that blaOXA-51 gene, is an enzyme that naturally exists in A. baumannii and has very weak carbapenem hydrolyzing activity [37].While the presence of plasmid-borne blaOXA-23 and blaOXA-58 genes have been shown to contribute significantly to carbapenem resistance in A. baumannii worldwide [38]. However, the distribution of carbapenem-hydrolyzing oxacillinases varies among regions and hospitals [3,[32][33][34][35]38]. A study used multivariate analysis demonstrated that the risk factors for acquisition of MDR A. baumannii were not related specifically to presence of cancer, but rather due to exposure of patients to health care procedures, especially dialysis, antibiotic treatment and length of stay in intensive care unities [4,7].
The present study also demonstrates that all A. baumannii isolates were susceptible to colistin. This result is similar to most recent studies from neighboring countries which has reported high susceptibility rates of clinical A. baumannii isolates to colistin [2-3, 25, 32, 35]. However, with an increase of using colistin to treat carbapenem-resistant A. baumannii infections, colistin resistance could be later emerged [39].
This study revealed that 37 of the 45 (82%) MDR A. baumannii isolates harbored class-1 Integrons (Table 5). Previous studies from Jordan has demonstrated that class 1-integrons is frequently found in clinical and environmental isolates of Gram-negative bacteria such as A. baumannii and E. coli and are often associated with their MDR strains [28,40]. The presence of the integrase gene in the majority of our A. baumannii clinical isolates in association with other resistance markers provided a strong evidence that these isolates have the potential for acquisition more antimicrobial resistance genes in future. It has been often observed that MDR A. baumannii strains involved in hospital outbreaks have carried class 1 integrons [41][42].
The ERIC-PCR analysis as shown by dendrogram demonstrated that few A. baumannii isolates might have been circulated among cancer patients ( Figure  1). Further testing of these isolates using multilocus sequence typing (MLST) or DNA sequencing could confirm this result.
In conclusion, the results of this study demonstrate that body colonization with A. baumannii increased in association with antibiotic treatment of cancer patients. The majority of A. baumannii are carrying a wide spectrum of genetic resistance factors, especially many important types of blaOXAcarbapnemases which will limit using valuable drugs in treatment of A. baumannii invasive infection.