Emergence of IS Aba1 -linked oxacillinase genes among 2 carbapenem resistant Acinetobacter baumannii isolates 3 in a tertiary cardiac center, Nepal

Abstract


Introduction
Acinetobacter baumannii has become an obstinate nosocomial bacteria [1], in virtue of its multi-resistance phenotype [2], especially among the immune-compromised patients and mostly in intensive care units (ICUs) [3].Due to their high genetic plasticity, they are able to acquire different resistance determinants through horizontal gene transfer by mobile genetic elements (MGEs) [4].Despite the fact that carbapenems are the preferred medication to treat infections caused by multidrug resistant (MDR) Acinetobacter baumannii, resistance against this antibiotic is escalating at an alarming rate throughout the world [5].The World health organization (WHO) has also listed carbapenem resistant A. baumannii to be one of the 12 critical priority pathogens for research of new antibiotics [6].The underlying resistance mechanisms includes overexpression of efflux pumps, modifications to outer membrane proteins and carbapenemases encoded even on plasmids, integrons and transposons [7,8].
The carbapenem resistance phenotype in Acinetobacter baumannii is chiefly due to the acquisition of oxacillinase genes flanked by insertion sequence (IS) elements [9].
Insertion sequence employs a crucial part in the propagation and expression of antibiotic resistance genes in A. baumannii [10].They provide promoter for overexpression of various downstream antibiotic resistance genes [11,12].Despite the significant prevalence of carbapenem resistance in A. baumannii isolates [13,14], there is paucity of studies focused to unravel the prevailing carbapenem resistance mechanism in Nepal.
Therefore, this study was carried out in order to investigate carbapenem resistance profile, occurence of OXA carbapenemase genes (bla OXA-51-like , bla OXA-23-like , bla OXA-58 ), insertion sequence (ISAba1), and ISAba1-linked oxacillinase genes and their relationship to carbapenem susceptibility among A. baumannii isolates from cardiac patients in Nepal.

Clinical settings and study design
This hospital based cross-sectional study was conducted from April 2021 to March 2022 at Shahid Gangalal National Heart Center (SGNHC), a tertiary referral cardiac center in, Kathmandu, Nepal.The study population included patients visiting to SGNHC irrespective of any age and gender within the study period.In the study, all clinical samples that were received for routine culture and testing for antibiotic susceptibility were included.A total of 1,291 clinical specimens were processed and analyzed consistent with standard microbiological practices [15] during the study period.
Initially, isolates were classified as Acinetobacter calcoaceticus -Acinetobacter baumannii (ACB) complex utilizing cultural, biochemical and microscopic methods like growth on MacConkey agar, gram staining, oxidase, motility, oxidation-fermentation (OF), citrate utilization, triple sugar iron agar (TSIA) and growth at 42°C [16].After that, they were delivered to Tribhuvan University's Central Department of Microbiology where they were kept at -20°C in tryptic soy broth that had been added with 20% glycerol (v/v) until a subsequent downstream investigation.ACB complex were further characterized as Acinetobacter baumannii by observation of intrinsic bla OXA-51-like gene [17].As the reference strain, A. baumannii ATCC 19606 was employed.
Multi Drug Resistant (MDR) is a designation for isolates that are resistant to at least one agent in three or more antimicrobial categories, while Extensively Drug Resistant (XDR) is used to describe Acinetobacter baumannii isolates that are only susceptible to one or two categories [22].
Agar dilution technique was used to evaluate the MIC of meropenem (Sigma Aldrich, USA) against all isolates of Acinetobacter baumannii.The findings were interpreted in accordance with the clinical breakpoint of (Susceptible ≤ 2 and Resistant ≥ 8 µg/ml) as recommended by CLSI [18].

Detection of oxacillinases and ISAba1 genes
Total genomic DNA was extracted by heat shock method as stated by Hartas [26].DNA extracted, including its quantity and quality was evaluated by spectrophotometric analysis using (Nanodrop 1000, Thermofischer Scientific, USA).
The Uniplex PCR assays were conducted to assess the presence of genes encoding oxacillinases, including bla OXA-51-like , bla OXA-23-like , bla OXA-58 as well as ISAba1 genes using specific primers as described previously [27][28][29][30].All primers were purchased from Macrogen Inc. (South Korea) and the expected amplicon sizes and primer sequences are listed in (Table 1).PCR reactions were conducted in 25 µL reaction volumes with 12.5 µL of 2x master mix (Biolabs, USA), 1 µL of each forward and reverse primer, 3 µL of template DNA and 7.5 µL of nuclease free water.The gradient thermal cycler (Applied Biosystems, USA) was used for the amplification, and the target genes' specific thermal cycling settings are specified in (Table S1).Nuclease-free water served as the negative control in each PCR cycle.Acinetobacter baumannii isolates previously confirmed to have bla OXA-23-like , bla OXA-58 and ISAba1 genes [10] were kindly provided by Professor Dr. Balaji Veeraraghavan, Department of Microbiology, Christian Medical College, Vellore, India and were used as positive amplification controls.The PCR products were separated by electrophoresis on a 1.5% (w/v) agarose gel prepared in tris acetic acid -EDTA (TAE) buffer with 0.5 µg/ml ethidium bromide and observed in gel documentation system (Azure Biosystems, USA).The amplicon sizes were determined by comparison with 100 bp DNA molecular size marker (GenScript, Singapore).

Detection of ISAba1 associated with OXA-51 and OXA-23
The position of the ISAba1 sequence in relation to the bla OXA genes was investigated with the polymerase chain reaction as described previously [27].Upstream association of insertion sequence, ISAba1 to bla OXA-51-like and bla OXA-23-like gene was evaluated through PCR mapping approach using the primer pairs ISAba1F/OXA-51R and ISAba1F/OXA-23R respectively.

Data analysis
All the data obtained were entered into the Excel 2013 (Microsoft Corp., Redmond, USA).Statistical Package for the Social Sciences software version 23 (SPSS, Inc. Chicago, USA) was employed for descriptive statistical assessment.Chi-square analysis was used to examine categorical data, with a p-value of less than 0.05 indicating statistical significance.

β-lactamase profile of Acinetobacter baumannii
On the phenotypic β-lactamase production test by combined disc method, 30% of the Acinetobacter baumannii isolates were identified to be MBL producer.Two of the MBL producer also co-produced ESBL and KPC each, whereas 10% of the isolates only produced ESBL.Similarly, only one isolate (2.5%) produced AmpC β-lactamase enzyme.
Moreover, there was significant association between β-lactamase profile and resistance phenotypes of Acinetobacter baumannii (p = 0.04).Every ESBL producing A. baumannii isolates were exclusively MDR, while more number of only MBL generating A. baumannii isolates was XDR (Table 2).ISAba1 was solely found upstream of bla OXA-51-like and bla OXA-23-like genes in each 10 of the isolates, whereas ISAba1 lay upstream of both bla OXA-51-like and bla OXA-23-like genes in 20 (50%) of the A. baumannii isolates (Table 3).

Relevance of meropenem resistance level to distribution of ISAba1linked oxacillinase genes
In this study, 20 Acinetobacter baumannii isolates in which ISAba1 had upstream association to both bla OXA-51like and bla OXA-23-like genes had comparatively higher MIC 90 value of 243.20 µg/ml, than that each of the 10 isolates in which ISAba1 had upstream association only to bla OXA-23-like gene (121.60µg/ml) and bla OXA-51-like gene (64 µg/ml) (Table 3).This was further illustrated in (Fig. 1), in which isolates positive for both ISAba1F/OXA-51R and ISAba1F/OXA-23R had comparatively higher MIC values for meropenem.Additionally, the highest MIC value of 256 µg/ml was only present in isolates with ISAba1 linked upstream of both bla OXA-51-like and bla OXA-23-like genes (Fig. 1).

Discussion
Acinetobacter baumannii being notorious pathogen can cause infection in any parts of human body.Acinetobacter baumannii isolates were recovered from diverse clinical specimens including sputum, blood, endotracheal secretion, urine and wound swab even in this study.Most of the A. baumannii (77.5%) was isolated from samples of the respiratory tract (sputum and endotracheal secretion).This was in line with earlier investigations [13,14,27] which indicated that the respiratory system was the most often infected area by A. baumannii.Contamination of respiratory equipment is often responsible as a source of nosocomial outbreak [31].
All Acinetobacter baumannii isolates in this investigation were from inpatient source.
This accentuates that A. baumannii has established its niche in this cardiac center.Their persistence in clinical environment is facilitated by their minimal nutrient requirements, ability to survive for long period in desiccants and resistance to key antimicrobial drugs and disinfectants [1].Moreover, inpatients in this hospital are mostly cardiac patients which account as a high risk group for A. baumannii infection, as they are immunecompromised or have undergone some invasive procedures [32].Acinetobacter baumannii has emerged to be the most prevalent nosocomial pathogen, especially in ICUs worldwide, and confers 10 -43% mortality rate in patients admitted to ICUs [33].

Numerous studies have documented high proportion and elevated antibiotic resistance of
Acinetobacter baumannii in ICUs [13,27,34].Accordingly, majority of the A. baumannii isolates (60%) were recovered from ICUs in this study.Environmental contamination, particularly invasive mechanical ventilation as well as other invasive procedures frequently performed in ICUs is typically linked to nosocomial A. baumannii infection in ICUs [27].
Acinetobacter baumannii is increasingly being resistant to almost commonly prescribed drugs causing therapeutic failure [35].All isolates in this research were also resistant to ciprofloxacin, piperacillin/tazobactam and extended spectrum cephalosporins.Similarly, resistance rate to gentamicin, meropenem and imipenem were also greater than 90%.
These data suggested that these antibiotics are ineffective for empirical therapy against A.
baumannii infection.High resistance rate was also observed against most other tested antibiotics except polymyxins and tigecycline, implying their potent usage in the treatment of A. baumannii infection in healthcare settings in Nepal.Due to their severe adverse effects and low plasma levels, these medications are typically used sparingly and only in life-threatening situations, which may explain their high susceptibility rate [36,37].
The prevalence of MDR and XDR Acinetobacter baumannii has risen sharply worldwide in recent years, which raises a serious concern [38].Various studies from China, India and Iran have reported almost 100% incidence of MDR A. baumannii [2,39,40].Similar to these studies, every single isolate of A. baumannii in this investigation were MDR and around one-third of isolates were XDR.Acinetobacter baumannii have high genetic plasticity with outstanding capacity to obtain various resistance determinants following lateral gene transfer, which when coupled with up-regulated innate resistance mechanism, it leads to multidrug resistance phenotype [41].This process is even aided by the selective pressure caused by extensive misuse of antibiotics.
are widely and indiscriminately prescribed in Nepal, this might elaborate the increased resistance rate to these antibiotics [48].Resistance to carbapenems has also been associated with production of β-lactamases, modification in exterior membrane and penicillin binding proteins and overexpression of AdeABC efflux system [8].
Oxacillinase type (OXA) carbapenemases are the primary factors responsible for carbapenem resistance in Acinetobacter baumannii globally [49].As phenotypic methods for determining the presence of OXA carbapenemases in A. baumannii have not yet been documented [45], PCR was used to identify different OXA carbapenemases, including bla OXA-51-like , bla OXA-23-like and bla OXA-58 .OXA-23-like enzymes are the most common oxacillinases and the primary reason behind carbapenem resistance in A. baumannii [4,30].Thereby, the 100% resistance rate to carbapenem in this research might presumably be due to the notion that all isolates had harbored bla OXA-23-like gene.There are numerous reports with 100% incidence of bla OXA-23-like gene in A. baumannii [5,13,14].This clearly depicts inclining widespread dispersion of bla OXA-23-like gene in A.
baumannii, which could be justified by its allocation in plasmids [50].Allied to this, ISAba1 insertion element is also involved in spread of bla OXA-23-like gene forming composite transposons [51].Plasmids (RepAci6 and pAZJ221) and transposons (Tn2006 and Tn2009) have mostly contributed on horizontal dissemination of bla OXA-23-like gene, promoting its global distribution [4,52].Furthermore, only 5% of isolates from this study had the bla OXA-58 gene.Previously it was reported only in one isolate in a study [14] from Nepal.Majorly, this gene was more common in European isolates of A. baumannii [53].
To the greatest extent of our understanding, this present study is the initial research from Nepal to document clinical isolates of A. baumannii co-harboring both ISAba1 and bla OXA-58 genes.The presence of such rare carbapenemases along with ISAba1 in this current study signals the possible increment of this gene in Nepal as A. baumannii have remarkable capacity to acquire new resistance traits thorough lateral gene transfer [41], which eventually may increase resistance to carbapenem.
Carbapenem resistance genes are primarily expressed and disseminated through the insertion sequence, ISAba1 in Acinetobacter baumannii [10].ISAba1 brackets carbapenemase gene forming a transposon, which actively mobilize and thereby aids in the dissemination of carbapenemase gene [54].Such transposons are even disseminated further through plasmids horizontally [55].In addition, insertion sequence elements contain promoter that contributes in the overexpression of down-stream antibiotic resistance genes [56].Acinetobacter baumannii isolates possess ISAba1 upstream of the genes bla OXA-51-like , bla OXA-23-like and bla OXA-58 , contributing to the over-production of these enzymes further facilitating resistance to carbapenem [57].Kobs et al. [27] reported that the isolates lacking ISAba1 or ISAba1 located not near to oxacillinase genes were found to be susceptible to carbapenem.In this work, ISAba1 was found in every isolate either upstream of bla OXA-23-like or bla OXA-51-like genes.This most plausibly explains none of the isolates being susceptible to carbapenem, as ISAba1 aids in dissemination and overexpression of oxacillinases in these isolates.High meropenem MICs (32-256 µg/ml) with high MIC 90 value of 243.20 µg/ml was reported among 50% of the isolates with ISAba1 linked upstream to both bla OXA-51-like and bla OXA-23-like genes.But comparatively lower MIC 90 value of 121.60 µg/ml and 64 µg/ml was documented in isolates with ISAba1 upstream only of bla OXA-23-like and bla OXA-51-like genes respectively.In this respect, it can be said that increased rates of carbapenem hydrolysis could be attributed due to overexpression of both bla OXA-23-like and bla OXA-51-like genes by ISAba1.
There were several limitations in the study.As this study was limited to single hospital, its findings cannot be generalized to the entire nation or worldwide.Confirmation of the location of insertion sequence can only be done through nucleotide sequencing, which was infeasible due to financial constraints.
In conclusion, there is agonizing state of carbapenem resistance among Acinetobacter baumannii in regard to high distribution of genes encoding oxacillinases and insertion sequence in this region.The emergence and spread of ISAba1-linked oxacillinase genes further enhanced carbapenem resistance level creating havoc.Thus, all the medical fraternity and stakeholders need to be vigilant and enable the targeted approaches.

Figure 1 :
Figure 1: Meropenem MICs in Acinetobacter baumannii with ISAba1 linked to Acinetobacter spp. on account of various biochemical tests and growth temperatures.Of the 45 ACB complex, 40 (88.9%) were identified to be A. baumannii based on the presence of an endogenous bla OXA-51-like gene.The prevalence of A. baumannii among total bacteria isolated was thus 11.8%.
All the 40 isolates of Acinetobacter baumannii were from inpatient source.Of them, predominant isolates (60%) came from ICU.The most prevalent specimen to yield A. baumannii isolates was sputum (57.5%) proceeded by endotracheal secretion (20%).Equal number of isolates (10% each) was recovered from blood and wound swab and least only 1 (2.5%) isolate was obtained from urine sample.
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made

Table 2 : Association of β-lactamase profile of A. baumannii with resistance phenotypes
plausible upstream location of ISAba1 above bla OXA-23-like gene.On the same line, ISAba1 was detected upstream of bla OXA-51-like gene in 30 (75%) of the isolates, as a band of around 359 bp was observed in a PCR employing the ISAba1F and OXA-51R primers.