Carbapenem Resistance in Acinetobacter calcoaceticus-baumannii Complex Isolates From Kathmandu Model Hospital, Nepal, Is Attributed to the Presence of blaOXA-23-like and blaNDM-1 Genes

The Acinetobacter calcoaceticus-baumannii (ACB) complex, also known as ACB complex, consists of four bacterial species that can cause opportunistic infections in humans, especially in hospital settings. Conventional therapies for susceptible strains of the ACB complex include broad-spectrum cephalosporins, β-lactam/β-lactamase inhibitors, and carbapenems. Unfortunately, the effectiveness of these antibiotics has declined due to increasing rates of resistance. The predominant resistance mechanisms identified in the ACB complex involve carbapenem-resistant (CR) oxacillinases and metallo-β-lactamases (MBLs). This research, conducted at Kathmandu Model Hospital in Nepal, sought to identify genes associated with CR, specifically blaNDM-1, blaOXA-23-like, and blaOXA-24-like genes in carbapenem-resistant Acinetobacter calcoaceticus-baumannii (CR-ACB) complex. Additionally, the study is aimed at identifying the ACB complex through the sequencing of the 16s rRNA gene. Among the 992 samples collected from hospitalized patients, 43 (approximately 4.334%) tested positive for the ACB complex. These positive samples were mainly obtained from different hospital units, including intensive care units (ICUs); cabins; and neonatal, general, and maternity wards. The prevalence of infection was higher among males (58.14%) than females (41.86%), with the 40–50 age group showing the highest infection rate. In susceptibility testing, colistin and polymyxin B exhibited a susceptibility rate of 100%, whereas all samples showed resistance to third-generation cephalosporins. After polymyxins, gentamicin (30.23%) and amikacin (34.88%) demonstrated the highest susceptibility. A substantial majority (81.45%) of ACB complex isolates displayed resistance to carbapenems, with respiratory and pus specimens being the primary sources. Polymerase chain reaction (PCR) revealed that the primary CR gene within the ACB complex at this hospital was blaOXA-23-like, followed by blaNDM-1. To ensure the accuracy of the phenotypic assessment, 12 samples were chosen for 16s rRNA sequencing using Illumina MiSeq™ to confirm that they are Acinetobacter species. QIIME 2.0 analysis confirmed all 12 isolates to be Acinetobacter species. In the hospital setting, a substantial portion of the ACB complex carries CR genes, rendering carbapenem ineffective for treatment.


Background
The genus Acinetobacter encompasses a diverse and heterogeneous collection of bacteria, many of which have the potential to induce different forms of opportunistic infections in humans [1].Nevertheless, the prevalent Acinetobacter species found in hospital environments belong to the Acinetobacter calcoaceticus-baumannii (ACB) complex [2].The ACB complex poses a concerning nosocomial threat, primarily linked to epidemic outbreaks of infections that are seldom encountered outside clinical environments.This group includes clinically derived species such as Acinetobacter baumannii, Acinetobacter nosocomialis (previously genospecies 13TU), and Acinetobacter pittii (previously genospecies 3), along with environmentally sourced Acinetobacter calcoaceticus within the genus [3].They are commonly known as the ACB complex, given their close genetic ties, which pose challenges for differentiation using conventional clinical microbiology laboratory methods relying on observable traits [4].Among these species, Acinetobacter baumannii stands out as the most concerning pathogen for healthcare institutions and has emerged as its most notable representative [5].
The recommended treatment guidelines for antibioticsusceptible ACB complex include broad-spectrum cephalosporin (such as ceftazidime or cefepime), β-lactam/β-lactamase inhibitors (including sulbactam), and carbapenem (such as imipenem, meropenem, or doripenem) [6].To mitigate the risk of resistance development, these antibiotics are occasionally combined with an aminoglycoside or an antipseudomonal fluoroquinolone.In cases of multidrugresistant (MDR) and carbapenem-resistant Acinetobacter calcoaceticus-baumannii (CR-ACB) complex, polymyxins (polymyxin B and colistin) and tetracyclines (minocycline and tigecycline) are the preferred drugs [7].Carbapenem, belonging to the broadest spectrum β-lactam class of antibiotics, exhibits bactericidal activity against a wide range of gram-positive and gram-negative aerobes, as well as anaerobes.Carbapenems have been the primary antimicrobial agent against Acinetobacter baumannii infections since 1990 [8].However, due to increasing rates of CR, their efficacy has significantly declined [5].
Historically, Ambler's molecular structure classification [9] and Bush-Jacoby-Medeiros' functional classification [10,11] have served as frameworks for categorizing β-lactamases.Ambler's classification divides these enzymes into four groups, A, B, C, and D, based on distinctive motifs formed by the fundamental sequences constituting the protein molecules.Class B enzymes require zinc as a metal cofactor for their activity, whereas class A, C, and D enzymes utilize a serine residue as their active core [12].In the ACB complex, all four classes of β-lactamases are present, with carbapenemases belonging to classes A, B, and C, while cephalosporinases are categorized as class C enzymes.In the ACB complex, the primary mechanism of CR involves enzymatic breakdown, leading to the inactivation of carbapenems.This process is chiefly orchestrated by carbapenemase enzymes [13].
The exploration of antimicrobial resistance (AMR) genes at the genomic level is crucial for understanding resistance mechanisms.However, there has been a lack of molecular investigations into AMR in the ACB complex in Nepal [19,20].Up to the time of this research, only three molecular studies had been undertaken to identify the presence of AMR genes in the ACB complex in Nepal [21][22][23].Previous research in Kathmandu Model Hospital focused mainly on phenotypic assessments of AMR [17,[24][25][26][27][28].Specifically, within the timeframe of this study at Kathmandu Model Hospital, there had been no instances of previous molecular investigations to comprehend the presence of AMR genes within the ACB complex [29,30].Since knowledge about AMR genes within the ACB complex primarily relied on just a few studies [21][22][23], this research is aimed at determining whether the AMR genes, as identified in earlier studies, were apparent in ACB isolates obtained from Kathmandu Model Hospital, Nepal.
Based on previous investigations in another hospital [21] and a tertiary care hospital [23] of Nepal, we hypothesized that the bla NDM-1 and bla OXA-23-like carbapenemase genes are among the most widespread carbapenemase genes circulating within the ACB complex in Nepal.Additionally, the bla OXA-24-like gene, belonging to the category of commonly encountered carbapenemase genes, had not been identified in any research conducted in Nepal at the time of this study.However, given its previous isolation in neighboring countries like India and China, there was conjecture regarding its potential presence [31,32].Therefore, this study specifically focuses on the prevalence of bla NDM-1 , bla OXA-24-like , and bla OXA-23-like carbapenemase genes in CR-ACB complex isolates obtained from inpatients at Kathmandu Model Hospital.

Study Design and Selection of Participants.
A prospective cross-sectional study was conducted to assess the prevalence of CR-ACB complex among inpatients (hospital-admitted individuals) and to identify the presence of bla NDM-1 , bla OXA-23-like , and bla OXA-24-like genes associated with CR.Isolates exhibiting an imipenem zone of inhibition ≤ 18 mm and a meropenem zone of inhibition ≤ 14 mm were classified as CR-ACB complex [34].The assessment of carbapenemase activity at the phenotypic level was carried out using the modified Hodge test (MHT) with E. coli ATCC 25922 and a meropenem disc.A positive control for the test involved bla NDM-1 gene PCR-positive Klebsiella pneumoniae [36].

Molecular Detection of Genes.
Bacterial DNA extraction was carried out using the cetyltrimethylammonium bromide (CTAB)/NaCl method [37].In this process, approximately 1-3 mL of overnight Luria Bertani broth containing a few colonies of isolates underwent centrifugation at 12,000 rpm for 10 min.After discarding the supernatant, the pellet was suspended in 567 μL of TE buffer (Tris EDTA, pH 7.4) and subjected to extraction using the CTAB/NaCl method.Subsequently, the DNA was resuspended in 50 μL of TE buffer and stored at −20 °C until further use [38].PCR was performed for the detection of bla NDM-1 , bla OXA-23-like , bla OXA-24-like , and 16s rRNA genes.Amplification of carbapenemase genes was carried out using the primer set from Eurofins Genomics, India (Table S1) [3,39] and 16s rRNA primer (Macrogen, Korea) (Table S1) [40].The reaction mixture for CR genes, with a total volume of 10 μL, was prepared using 1 μL of template DNA, 1 μL each of forward and reverse primers (10 pmol), 2 μL nuclease-free water (Thermo Fisher Scientific, India), and 5 μL of 2× PCR master mix (QIAGEN, Germany).Amplification was conducted in a Biorad T100 thermal cycler, following the PCR conditions mentioned in Table S2.The resulting substance was observed in a 2% agarose gel, which underwent electrophoresis at 100 volts for 45 min.
2.6.16s rRNA Amplification.For the amplification of the 16s rRNA gene, a reaction volume of 15 μL was employed, comprising 1.5 μL of DNA template, 3 μL each of forward and reverse primers (10 pmol), and 7.5 μL of 2× KAPA HiFi Hot-Start ReadyMix (KAPA Biosystem, USA).The amplification conditions are provided in Table S2.The resulting product was visualized in a 2% (w/v) agarose gel, which underwent electrophoresis at 90 volts for 90 min.
A total of 12 isolates, which exhibited a positive result for the bla NDM-1 gene, underwent 16s rRNA sequencing on the Illumina MiSeq™ system.The amplification of the 16s rRNA gene was carried out using primers targeting the V3 and V4 regions of the prokaryotic 16s ribosomal RNA gene [40].The quantification of PCR products was performed using the Qubit™ 3 Fluorometer, followed by multiplexing at an even concentration.Subsequently, the samples were subjected to sequencing with a 300 bp (2 × 150 bp) paired-end configuration using the Illumina MiSeq platform (Illumina, Inc., USA) [41].

Library Preparation and Sequencing.
For each sample, 1 ng of the amplified PCR products underwent processing with the Illumina MiSeq Nextera XT DNA Library Preparation Kit (Illumina, Inc., USA).The construction of a pairedend library was carried out with an insert size of 500 bp for the 12 isolates positive for bla NDM-1 .Following the purification of PCR products using AMPure XP beads (Agencourt, USA), they underwent tagmentation and indexing using the Nextera XT Index V2 Kit (Illumina, Inc., USA).The cleaned products were quantified and evaluated once more, employing the Qubit Fluorometer (Invitrogen, USA) and the Agilent bioanalyzer with the DNA 1000 kit (Agilent Technologies, UK).Ultimately, all samples were pooled at a concentration of 4 nm and underwent paired-end sequencing with a 300 bp configuration (2 × 151 bp) on the MiSeq platform (Illumina, USA).
2.8.Data Analysis.The results obtained were analyzed using the Statistical Package for Social Sciences (SPSS) software BioMed Research International (Version 23.0), with the chi-square test employed to determine the p value.Gephi 0.9 was utilized as the tool for network analysis.
2.9.16s rRNA Bacterial Taxonomic Profiling.Data were analyzed using the QIIME Version 2.0 pipeline [42].Raw sequences were demultiplexed and then quality-filtered using demux in QIIME.The Silva_132_release database was used to assign taxonomy [43].

Results
This research involved analyzing 992 clinical inpatient samples sent to the Department of Microbiology at Kathmandu Model Hospital, Nepal, for aerobic bacterial culture and AST.Among these samples, 43 (4.334%) tested positive for the ACB complex.These positive samples were obtained from hospitalized patients in different hospital wards, such as intensive care units (ICUs), cabin wards, neonatal wards, general wards, and maternity wards.
The research indicated a greater proportion of ACB complex isolates in males (60.47%) compared to females (39.53%) (Table 1).ACB complex isolates retrieved from respiratory samples, as well as from blood and tissue, were more prevalent in males when compared to females (Table 1).In a similar vein, patients in the age bracket of 40-50 years exhibited a higher infection rate than individuals in other age groups, while the age groups < 10 and 10-20 had the lowest infection rates.

Temporal Data on the Occurrence of the CR-ACB
Complex in Various Specimen Types.The predominant source of CR-ACB complex isolates was respiratory specimens, such as sputum, tracheal aspirate, and ET tube tips (Table S3).Respiratory samples accounted for about 54.29% of the identified CR isolates.After respiratory tract samples, pus specimens showed the highest prevalence of CR-ACB complex.No instances of CR-ACB complex were identified in urine samples.A limited number of CR-ACB complex isolates were found in fluid samples, including blood, bile, and CSF (Figure 2).Examining the temporal data (Figure 3), a notable increase in the occurrence of the ACB complex in pus, tissue, and samples from the respiratory system was observed after August.Conversely, the isolation of the ACB complex from wound swabs and blood samples decreased after August.However, before August, the ACB complex was detected in a wider range of specimen types than after that time.

Comparison of AMR Pattern of CR and Carbapenem-
Sensitive ACB Complex.In both CR-ACB complex (n = 35) and carbapenem-susceptible ACB complex (CS-ACB complex) (n = 8), colistin and polymyxin B displayed full susceptibility at 100%, while ceftriaxone and cefotaxime exhibited complete resistance at 100%.Among the CR isolates, the majority demonstrated resistance to cotrimoxazole, piperacillin/tazobactam, ciprofloxacin, and cefepime, constituting 97.1% of the cases.In contrast, the smallest percentage of isolates showed resistance to tigecycline (Table 3).
Among isolates susceptible to carbapenem, most antibiotics displayed complete susceptibility.Out of the 14 antibiotics assessed (excluding meropenem and imipenem), 8 antibiotics exhibited a 100% susceptibility rate, while the remaining 6 demonstrated varying levels of resistance.Among these six antibiotics, ceftriaxone and cefotaxime had the highest resistance rate at 100% (n = 8).Following this, doxycycline showed a resistance rate of 37.5% (n = 3), while the lowest number of isolates displayed resistance to 4 BioMed Research International cefepime, ciprofloxacin, and cefoperazone/sulbactam at 12.5% (n = 1), as depicted in (Table 3).It is noteworthy that there was a significant association between susceptibility to carbapenems and susceptibility to other antibiotics (p > 0 05), determined through chi-square testing.
3.5.Carbapenemase Production, Presence of CR Genes, and 16s rRNA Sequencing Result.The MHT was employed on CR isolates to assess carbapenemase production.Of the 35 CR-ACB complex isolates, 33 (94.28%) showed a positive result in the MHT, characterized by a cloverleaf-shaped indentation.Two samples did not demonstrate clover-leaf indentation which was regarded as MHT negative.Among the 33 carbapenemase-positive CR-ACB complex isolates, 12 (36.36%)were found to carry the bla NDM-1 gene through PCR analysis (Table 4).The 16s rRNA gene of the 12 isolates carrying the bla NDM-1 gene was sequenced using Illumina MiSeq.Analysis via QIIME 2.0 verified that all 12 isolates were Acinetobacter species.
Among the 33 carbapenemase-producing CR-ACB complex, 29 (87.87%)tested positive for the bla OXA-23-like gene via PCR analysis (Table 5).The two isolates negative in the MHT also lacked the bla OXA-23-like e gene.However, neither carbapenemase-producing nor nonproducing isolates showed a positive presence of the bla OXA-24-like gene.

Sample-Wise Distribution of bla OXA-23-like and bla NDM-1
Genes.The examination revealed that bla OXA-23-like or bla NDM-1 genes were predominantly detected in sputum, tracheal aspirate, and pus samples (Figure 4).When visualizing through network analysis, which visualizes relationships between variables, it showed that among CR samples, only 5 BioMed Research International one from a suction tip culture lacked any CR genes.Notably, a significant number of samples testing negative for the presence of the ACB complex were obtained from pus, blood, and urine samples (Figure 5).

Discussion
A lack of knowledge exists regarding the prevalence of genes associated with CR and the current state of the CR-ACB complex in Nepali hospitals [17,21,24,[26][27][28].The country needs to establish an annual monitoring system to trace the presence of CR-ACB complex genes and their distribution within various healthcare settings [44].In recognition of this requirement, a research project was carried out at Kathmandu Model Hospital.The aim was to identify three genes (bla NDM-1 , bla OXA-23- like , and bla OXA-24-like ) from the numerous genes associated with CR within the ACB complex.This investigation represented the inaugural instance of such a study within the ACB complex framework at Kathmandu Model Hospital [29,30].
The ACB complex stands out as a significant nosocomial pathogen, leading to a variety of diseases.Effectively handling infections caused by the ACB complex poses a challenge due to the tendency of isolated ACB complex strains to demonstrate resistance to multiple drugs [2,6].In the absence of proper antimicrobial prescription, these infections can prove to be highly fatal.Carbapenems were traditionally the preferred drugs for treating MDR-ACB complex infections, but recent occurrences of resistance or decreased susceptibility to carbapenems are emerging as a serious clinical issue [8].In this study, the presence of the ACB complex among hospitalized patients was found to be 4.334%, a rate exceeding the results of prior research   6 BioMed Research International conducted in Nepal [25,45].Nevertheless, it is lower than the prevalence documented in another study in Nepal [46][47][48].The unique pattern observed in our study is likely due to our exclusion criteria (i.e., outpatients).Some studies have included both inpatients and outpatients in their analysis [27,49].In this investigation, a higher percentage of isolates were obtained from males (58.14%) compared to females (41.86%), aligning with trends found in previous studies in Nepal (Table 1) [27] and Northern Vietnam [50,51].
The ACB complex possesses the capability to infect various organs and systems in the human body, allowing its detection in a diverse range of clinical samples [52].In this study, the ACB complex was identified in various sample types, including bile, CSF, pus, wound swabs, blood, tissue, sputum, tracheal aspirate, urine, bed sores, ET tips, and CT tips (Figures 2 and 3 and Table S3).Despite being present in various samples, a significant majority of ACB complex isolates were found in respiratory samples, such as tracheal aspirate, sputum, and ET tips, constituting 48.83% of the cases.These results are consistent with previously published findings from Nepal [23,45,47,53] and other countries [54,55].Respiratory infections are the most common form of Acinetobacter infections, occurring predominantly in critically ill patients, especially those admitted to ICUs with mechanical ventilation [56].Examining the temporal data (Figure 3), it is evident that there was an increase in the incidence of infections from respiratory, pus, and tissue samples starting in August and continuing through November (late summer to early winter months).In another study, a similar trend of elevated respiratory-related infections was noted [57].Additionally, a study conducted in Western Nepal documented a general increase in Acinetobacter baumannii infections during the same month [48].
Regarding susceptibility testing to antimicrobial agents, all isolates of the ACB complex were resistant to thirdgeneration cephalosporins, such as ceftriaxone and cefotaxime (Table 2), which aligns with previous findings [58].Resistance to fourth-generation cephalosporin (i.e., cefepime) was found to be 83.72%;however, it is noteworthy that a level of resistance (i.e., 65.4% [27], 74.4% [49], 88.6% [59], and 89.79% [48]) has  7 BioMed Research International been reported in other studies.The combination of cefoperazone and sulbactam exhibited an higher susceptibility rate (18.6%) compared to cefepime (Table 2).Sulbactam, serving as a β-lactamase inhibitor, along with other inhibitors like clavulanate and tazobactam, possesses intrinsic antibacterial activity against Acinetobacter species [60].Consequently, the cefoperazone/sulbactam combination demonstrated greater antimicrobial activity than other cephalosporins, despite cefoperazone being a third-generation cephalosporin.A similar rationale may be applied to the increased susceptibility of piperacillin/tazobactam.In this study, 65.11% of isolates exhibited resistance to amikacin, and 69.77% were resistant to gentamicin (Table 2).Comparable resistance rates for amikacin (67.24%) and gentamicin (70.68%) were previously documented in Nepal by Bhandari et al. [61].
The resistance rates to imipenem and meropenem were lower at 81.4% in this study, contrary to findings in most other studies conducted within Kathmandu Valley [39,47,62] which reported higher resistance rates.However, some studies, like those conducted in Lalitpur District [27], Eastern Nepal [63], and Western Nepal [48], reported lower percentages of resistance.In Nepal, the percentage of CR has significantly increased, with reported resistance rates ranging from 17.24% [61] to 97.7% [23] indicating a critical state of CR.Concerning carbapenemase genes, the most frequently identified one is bla OXA-23-like [3,21].However, in recent times, bla NDM-1 has become widespread across various bacteria, primarily carried by Acinetobacter spp.and Enterobacteriaceae [39,[64][65][66][67][68].With this in mind, we conducted PCR testing to identify the presence of bla NDM-1 , bla OXA-24-like , and bla OXA-23-like genes in the CR-ACB complex.Among 35 CR isolates, 82.5% (29/35) tested positive for the bla OXA-23-like gene, and 34.28% (12/35) were positive for the bla NDM-1 gene (Tables 4 and 5 and Figures 4 and 5).Among these isolates, 31.42%(11/35) were positive for both bla OXA-23-like and bla NDM-1 genes.The sole isolate contained only the bla NDM-1 gene, and none of the isolates carried the bla OXA-24-like gene.In total, 30 isolates (85.71%) contained the tested genes.Notably, five of the isolates did not possess any of the tested genes, which is a study limitation since we did not examine these isolates for other CR genes like bla OXA-58-like , bla IMP , bla SIM , bla VIM , bla KPC , bla SME [3,16,69,70] or nonenzymatic CR mechanisms such as efflux pumps, loss of porin proteins, and altered penicillin-binding proteins [5].CR in these cases could potentially be attributed to the mentioned mechanisms.Additionally, two of the isolates tested negative for the MHT, indicating that nonenzymatic methods might be responsible for their CR [71].
In previous molecular investigations carried out in Nepal, a higher incidence of the bla OXA-23-like gene was observed compared to the findings of our current research.Two studies demonstrated elevated incidence rates of the bla OXA-23-like gene, with 95.08% and 100% [21,23].Additional studies conducted between 2012 and 2019 found a gene incidence rate of 36% [68], 63.2% [62], and 69.59% [47].The prevalence of the bla NDM-1 gene stood notably higher (34.285%) compared to earlier research findings in Nepal.Three studies reported a prevalence of the bla NDM-1 gene at 13.6% in Kathmandu, 16.7% in Eastern Nepal [49], and 20.7% in Lalitpur [68], which is significantly lower than the prevalence discovered in our study.This indicates a rising trend and increased dissemination of the bla NDM-1 gene in Nepal.This trend may be attributed to Acinetobacter baumannii's capacity to transfer this gene to other recipients through conjugation, with Tn125 being the primary mechanism for its widespread distribution [72].MBLs, such as bla NDM-1 , bla IMP , and bla SIM , provide a significantly higher level of resistance to carbapenems, approximately 100-1000 times more resistance than OXA-type carbapenemases [5,73].Furthermore, bla NDM-1 imparts resistance not only to carbapenems but also to penicillins and cephalosporins [74].Similarly, bla OXA-23-like confers resistance to carbapenems, oxyiminocephalosporins, piperacillin, aminopenicillins, oxa-cillin, and aztreonam [5,9,16,75].This may provide a plausible explanation for the observed antibiotic resistance patterns, wherein cefoperazone/sulbactam, piperacillin/tazobactam, and cefepime exhibit almost 100% susceptibility in CS-ACB complex while being 100% resistant in CR isolates (Table 3) [69].
The presence of the bla OXA-24-like gene was not detected in any of the isolates in this study.Although a recent study [76] reported the cooccurrence of bla OXA-23-like and bla OXA-24-like genes in a single isolate, most other studies did not observe such cooccurrence [21,23,66,77].The coexistence of bla OXA-23-like and bla NDM-1 gene in our study might be the cause of the absence of the bla OXA-24-like gene.Another possible explanation for the lack of the bla OXA-24-like gene may be related to variations in sample size and the duration of the study.The earlier study that identified the presence of the bla OXA-24-like gene covered a sampling period from 2012 to 2018, involving 382 Acinetobacter spp.[78].In contrast, our study spanned only 6 months and included only 43 ACB complex isolates.
CR isolates presented a limited array of treatment options, with polymyxins (colistin and polymyxin B) being the sole available choices, despite concerns about their previously documented side effects [79].In our study, both colistin and polymyxin B exhibited 100% susceptibility (Table 2), while tigecycline exhibited the next highest level of susceptibility at 60.47%.Like some of the past research [21,23,78], this investigation was unable to investigate clonal types through multilocus sequence typing (MLST) or whole genome sequencing because of insufficient funds, which is a major limitation of this study.However, three samples with intriguing results (AST and genes) underwent whole genome sequencing, and the results will be published separately as a case study.Additionally, it is important to note that this research did not provide details about patient outcomes following infection, primarily because most cases were successfully treated using third-line antibiotics.Lastly, due to inadequate funding, minimum inhibitory concentration (MIC) testing of carbapenem or other antibiotics could not be conducted.
This research emphasized the growing challenge posed by the CR-ACB complex in clinical environments.The CR-ACB complex, when isolated, demonstrated resistance to broad categories of antibiotics, indicating a significant issue [13].Precise microbial diagnostic techniques are essential for the accurate identification of this organism to facilitate appropriate treatments [80].Managing and minimizing infections caused by this organism can be effectively achieved through regular fumigation of ICUs and hospital wards, routine monitoring of organisms in these areas, and a judicious restriction on the use of antibiotics [81].

Conclusion
In summary, the objective of this study was to evaluate the presence of three genes associated with CR within the ACB complex at Kathmandu Model Hospital, Nepal.The results indicated a prevalence of 4.334% for the ACB complex.Respiratory samples, particularly from patients on mechanical BioMed Research International ventilation in the ICUs, were the primary source of most ACB complex isolates.The highlighted that the bla OXA-23-like and bla NDM-1 genes are the most frequently occurring CR genes in Kathmandu Model Hospital, and there is an apparent increase in the prevalence of the bla NDM-1 gene.Furthermore, except for third-line antibiotics, many other antibiotics showed either no susceptibility or reduced susceptibility in the CR-ACB complex.This implies that the CR-ACB complex is going to be a formidable foe for clinicians.Therefore, it is recommended that all healthcare providers make a united effort through rigorous execution of infection prevention and control measures, prompt diagnosis, and responsible use of antibiotics to lessen the impact of AMR on both patients and healthcare facilities.

Figure 3 :
Figure 3: A bar graph illustrating the distribution of ACB complex in various samples throughout the study (June-November 2018).

Figure 5 :
Figure5: Using Gephi 0.9, a graphical depiction of data related to carbapenem resistance and bla OXA-23-like or bla NDM-1 genes across various samples is presented."No" indicates sensitivity to carbapenems, while "Yes" denotes resistance.The font size reflects the frequency of variables, with larger fonts indicating higher frequency.The strength of association between variables is represented by the darkness or boldness of connecting arrows.
The sample collection took place between June 2018 and November 2018 at Kathmandu Model Hospital.A total of 992 clinical samples from inpatients at Kathmandu Model Hospital during that period were chosen as the study cohort, irrespective of age, gender, or clinical symptoms.
2.4.CR and Carbapenemase Production Testing.CR was identified through susceptibility testing with imipenem and meropenem, utilizing the Kirby-Bauer disc diffusion method.

Table 1 :
Gender-wise distribution of ACB complex among positive specimens.

Table 3 :
Difference in antimicrobial susceptibility pattern of CR-ACB and CS-ACB complex as well as chi-square test p values (p > 0 05) to assess the association between antimicrobial susceptibility of other antibiotics and carbapenem resistance.

Table 4 :
Carbapenemase production in CR-ACB complex isolates and the presence of bla NDM-1 gene.

Table 5 :
Carbapenemase production in CR-ACB complex isolates and the presence of bla OXA-23-like gene.