Prevalence of Carbapenemases in Carbapenem-Resistant Acinetobacter baumannii Isolates from the Kingdom of Bahrain

Background: Acinetobacter baumannii is regarded as a significant cause of death in hospitals. The WHO recently added carbapenem-resistant Acinetobacter baumannii (CRAB) to its global pathogen priority list. There is a dearth of information on CRAB from our region. Methods: Fifty CRAB isolates were collected from four main hospitals in Bahrain for this study. Bacterial identification and antibiotic susceptibility tests were carried out using the BD PhoenixTM and VITEK-2 compact, respectively. Using conventional PCR, these isolates were further screened for carbapenem resistance markers (blaOXA-51, blaOXA-23, blaOXA-24, blaOXA-40, blaIMP, blaNDM, blaVIM, and blaKPC). Results: All of the isolates were resistant to imipenem (100%), meropenem (98%), and cephalosporins (96–98%), followed by other commonly used antibiotics. All these isolates were least resistant to gentamicin (64%). The detection of resistance determinants showed that the majority harbored blaOXA-51 (100%) and blaIMP (94%), followed by blaOXA-23 (82%), blaOXA-24 (46%), blaOXA-40 (14%), blaNDM (6%), blaVIM (2%), and blaKPC (2%). Conclusion: The study isolates showed a high level of antibiotic resistance. Class D carbapenemases were more prevalent in our CRAB isolate collection. The resistance genes were found in various combinations. This study emphasizes the importance of strengthening surveillance and stringent infection control measures in clinical settings to prevent the emergence and further spread of such isolates.


Introduction
Acinetobacter baumannii (A.baumannii) is emerging as a significant multidrug-resistant (MDR) pathogen in hospitals, particularly in intensive care units (ICUs), and it is considered a major nosocomial pathogen causing high mortality [1,2]. The reported mortality rate is around 7.8% to 23% in hospitals and around 10% to 43% in ICUs [3]. Although there has not been any clear consensus on the associations between carbapenem-resistant Acinetobacter baumannii (CRAB) infections and an elevated risk of mortality [4], CRAB infections have shown a significant correlation with the length of ICU stays, elevated patient costs, and antibiotic use [4]. Moreover, it is also considered a significant pathogen causing hospitalacquired infections (HAIs) that increase the risk of the emergence of pan-drug resistance and outbreaks [5]. It usually infects human skin and wounds, especially the respiratory, gastrointestinal, and circulatory systems, causing serious infection [6]. Examples of HAIs are bacteremia, septicemia, wounds, meningitis, ventilator-associated pneumonia, and urinary tract infections [6]. Countries in the Mediterranean area have some of the highest resistance rates to carbapenems on A. baumannii, reaching 90%, including the Middle East,
Among the tested Class B carbapenemases, bla IMP was detected in 94% of the isolates (n = 47), and bla NDM was detected in 6% (n = 3) of the isolates. bla VIM and bla KPC were detected in one isolate each. Detailed results are presented in Figure 1. Various combinations of genes were noticed in our collection of CRAB isolates, ranging from as few as two genes to as many as six genes in the respective isolates ( Figure 1). The majority (44%) of the isolates had a combination of three genes (bla OXA-51 , bla OXA-23, bla IMP ), followed by a combination of four genes (bla OXA-51 , bla OXA-23, bla OXA-24 , and bla IMP ) in 12 isolates (24%). The detailed results are presented in Table 2. A representative PCR gel demonstrating the respective amplicons is shown in Figure 2.

Discussion
A. baumannii is a pathogen of concern worldwide in the context of nosocomial infections owing to its multidrug resistance, often including drugs of last resort such as carbapenems [4]. This is a plausible reason why the WHO has included CRAB in its global priority list. Of the various mechanisms of carbapenem resistance in CRAB, the production of carbapenem-hydrolyzing enzymes is one of the main mechanisms of resistance [4]. These enzymes are mainly produced by the genes encoding carbapenem-hydrolyzing en-

Discussion
A. baumannii is a pathogen of concern worldwide in the context of nosocomial infections owing to its multidrug resistance, often including drugs of last resort such as carbapenems [4]. This is a plausible reason why the WHO has included CRAB in its global priority list. Of the various mechanisms of carbapenem resistance in CRAB, the production of carbapenem-hydrolyzing enzymes is one of the main mechanisms of resistance [4]. These enzymes are mainly produced by the genes encoding carbapenem-hydrolyzing enzymes [4].
It is interesting to note that the Middle East was historically linked to A. baumannii, often known as "Iraqibacter," due to an epidemic of resistant strains among the US military during the Iraq War. Since then, hospitals around the Middle East, including those in the United Arab Emirates, Saudi Arabia, Bahrain, Palestine, and Lebanon, have isolated this bacteria [6]. A. baumannii has been subjected to numerous investigations testing its susceptibility to various antibiotic classes. According to a study from the holy cities of Saudi Arabia, the screening of carbapenem-resistant A. baumannii isolates revealed that imipenem and meropenem resistance was widespread in 81% and 84% of the strains, respectively, while the majority of the organisms were colistin-and tigecycline-susceptible [12]. Another study from Saudi Arabia concluded that eight isolates (30%) were resistant to colistin, 15 isolates (56%) were resistant to tigecycline (56%), and 24 isolates (89%) were resistant to one or more carbapenems (imipenem and meropenem] [13]. The present study showed significant resistance to imipenem, meropenem, and cephalosporins and comparatively lower resistance to minocycline, tigecycline, and colistin. Similarly, a study in China reported a high resistance rate to carbapenems and cephalosporins and a lower resistance rate to levofloxacin, minocycline, and tigecycline [14]. A two-year retrospective study from Saudi Arabia also documented that almost all isolates of A. baumannii were carbapenemresistant (98%). It was interesting to note that these isolates had higher resistance to colistin (15%) when compared to tigecycline (3%) [15]. These high levels of resistance rates were caused by the overuse of imipenem and meropenem for the treatment of A. baumannii infection in patients [16]. As a result, recommendations for the administration of infection control procedures are required to curb the spread of these isolates in hospital settings [16].
To date, we believe that no study has determined the prevalence of carbapenemase genes from Bahrain in a relatively large cohort of CRAB isolates. Earlier, in 2015, a collaborative effort was performed in a published joint research paper incorporating A. baumannii isolated from the Gulf Cooperation Council (GCC) countries. Most isolates were collected from Saudi Arabia, whereas a small proportion were collected from other GCC countries (only eight isolates were gathered from Bahrain hospitals) [17]. In that study, the researchers collected a total of 117 CRAB isolates from six countries (mainly Saudi Arabia) and reported the presence of bla OXA-51-type in all the isolates (100%; 117/117) and that of bla OXA-23-type in 91% (107/117) of the isolates [17]. Cumulatively, bla OXA-40-type was detected in 4% (5/117) of the isolates; all five isolates positive for this gene type were from Bahrain, and none of the isolates from other GCC countries demonstrated this gene type. Among the Bahraini isolates, all eight (100%) demonstrated the presence of bla OXA-51-type , followed by bla OXA-40-type (62.5%), and three isolates (38%; 3/8) showed bla OXA-23-type [17]. The authors also reported the non-detection of bla OXA-58 , bla KPC , and metallo-beta-lactamases (MBLs) like bla IMP and bla VIM [17].
In another report published based on a study in Bahrain in 2009, where eight isolates were again molecularly tested for these carbapenemase genes [18], the most prevalent reported gene was bla OXA-40-like (in five isolates), followed by bla OXA-23 (two isolates) and bla OXA-58 (one isolate) [18].
In the current study, bla OXA-51 , bla OXA-23 , bla OXA-24 , and bla IMP were the most commonly detected carbapenemase-producing genes, occurring at frequencies of 100%, 82%, 46%, and 94%, respectively. In contrast to the previous reports, our collection of CRAB isolates also showed the presence of bla VIM , bla NDM , bla KPC , and bla OXA-40 , though with lesser frequency. None of our isolates showed the presence of bla OXA-48 or bla OXA-58 . Even though it is too early to speculate on the present context due to the small number of isolates tested previously, it looks as if the molecular epidemiology in Bahrain has changed over the years, with the predominant gene now being bla OXA-23 , which was comparatively less prevalent earlier. The bla OXA-40 gene has become less prevalent, being the predominant gene reported in previous studies. It is also alarming to note the presence of a combination of carbapenem-resistance genes in some isolates at a level as high as six genes. MBLs and bla KPC were rarely reported in A. baumannii isolates, except for bla IMP . However, in our isolates, we found the presence of bla IMP in a significantly higher percentage (94%), which is also alarming. It was noticed that there was no correlation between the combinations of carbapenemase genes and the antibiotic resistance pattern. The CRAB isolates were highly resistant to all carbapenems and most cephalosporins with lower resistance to other antibiotics.
On the other hand, bla OXA-23 is a significant cause of carbapenem resistance in A. baumannii [19,21,22]. It has been reported as a prevalent gene in various studies published in several countries, including Saudi Arabia and Iran [19,21]. In contrast to this, a few other international studies have observed the presence of bla OXA-23 at a lower frequency, as reported in Bosnia, Poland, and Croatia [21]. The bla OXA-24 gene is also reported as a common gene, albeit at variable percentages [19,22,23]. On the Arabian Peninsula, another study from Egypt investigated the prevalence of carbapenemase genes in 40 CRAB isolates [26]. The bla OXA-51 gene was amplified in all isolates, whereas bla OXA-23 , bla OXA-24 , and bla OXA-58 were present in 50%, 7.5%, and 5% of the isolates. All these isolates lacked bla KPC or MBLs [26].
In a study conducted in Iran in 2015, Azizi O et al. observed that bla OXA-51 and bla OXA-23 were present in all isolates but were negative for bla OXA-58 [19]. In South Africa, Lowings M. et al. reported two genes (bla OXA-51 and bla OXA-23 ) among 100 MDR A. baumannii isolates (99 and 77%, respectively) [25]. The other genes, such as bla OXA-24 , bla OXA-58 , bla KPC , and MBLs, were negative [25]. Various other international studies outside of the GCC region also reported the presence of these genes, albeit with varying frequencies [24,27]. However, it is interesting to note that many of these studies reported the absence of genes such as bla OXA-24 , bla OXA-58 , bla IMP , bla VIM , and bla KPC [27].
Alarmingly, a significant proportion of the isolates in our collection also demonstrated concomitant resistance to fluoroquinolones and the aminoglycoside group of antibiotics. For other last-resort antibiotics such as tigecycline and colistin, even though the isolates demonstrated a lower frequency of resistance, the appearance of resistance is quite alarming.
This study has a few limitations. One is that the analysis of resistance determinants using molecular methods was limited to carbapenemases in CRAB isolates and did not include the details of ESBL and other antibiotic resistance mechanisms. In addition, sequencing was not performed to search for gene mutations. However, to the best of our knowledge, this is the first report describing the prevalence (and molecular characterization) of CRAB isolates in a relatively large cohort from the Kingdom of Bahrain.

Bacterial Isolates and Hospital Setting
From February 2021 to June 2022, 50 random, nonrepetitive CRAB isolates were collected from the microbiology labs of four different hospitals (Al-Salmaniya Medical Complex, Bahrain Defense Force Hospital, King Hamad University Hospital, and Bahrain Specialist Hospital) in the Kingdom of Bahrain. These isolates were cultured from specimens such as endotracheal aspirates, urine, sputum, blood cultures, wounds, pus, and rectal swabs. The isolates obtained from the lab were preserved in glycerol milk at certain volumes (3 mL and 4 mL) with 13 mL of deionized water and stored at −80 • C until further testing [28].

Bacterial Identification and Antibiotic Susceptibility Testing
The bacterial species-level identification and antibiotic susceptibility testing of the isolates were performed with automated microbiological systems (Vitek2 automated system) at the Bahrain Defense Force Hospital and Bahrain Specialist Hospital and a BD Phoenix TM automated system at the Al-Salmaniya Medical Complex and King Hamad University Hospital. Only the isolates that were identified as A. baumannii resistant to carbapenems were included for further molecular analysis. As per each hospital's antibiotic policies, the isolates were tested against certain antibiotics. The tested and non-tested antibiotics are presented in Figure 1A.

Conclusions
This study provides a clear picture of the currently prevalent bla-carbapenemases in the Kingdom of Bahrain. Oxacillinases (Class D) were the predominant carbapenemases; the most common genes detected were bla OXA-51 and bla OXA-23 . From Class B, bla IMP was also detected at a significantly higher percentage. The presence of other Class B genes (such as bla NDM and bla VIM ) and Class A genes (bla KPC ), though in smaller percentages, is quite alarming. The rate of resistance to most antibiotics is high in our region. These results emphasize the significance of rational antibiotic therapy and ongoing stringent surveillance and infection control strategies to successfully curb the spread of these clinical strains.

Data Availability Statement:
We confirm that all the data are presented in this article.