Phenotypic and Genotypic Investigation of Carbapenem-Resistant Acinetobacter baumannii in Maharaj Nakhon Si Thammarat Hospital, Thailand

(1) Background: Acinetobacter baumannii is well known as a causative agent of severe hospital-acquired infections, especially in intensive care units. The present study characterised the genetic traits of biofilm-forming carbapenem-resistant A. baumannii (CRAB) clinical isolates. Additionally, this study determined the prevalence of biofilm-producing A. baumannii isolates from a tertiary care hospital and investigated the association of biofilms with the distribution of biofilm-related and antibiotic resistance-associated genotypes. (2) Methods: The 995 non-duplicate A. baumannii isolates were identified, and their susceptibilities to different antibiotics were determined using the disk diffusion method. Using the modified microtiter plate assay, the CRAB isolates were investigated for their biofilm formation ability. Hemolysin and protease activities were determined. CRABs were subjected to polymerase chain reaction (PCR) assays targeting blaVIM, blaNDM, blaIMP, blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, csuE and pgaB genes. Individual CRAB isolates were identified for their DNA fingerprint by repetitive element sequence-based (REP)-PCR. (3) Results: Among all A. baumannii isolates, 172 CRABs were identified. The major antibiotic resistance gene among the CRAB isolates was blaOXA-51-like (100%). Ninety-nine isolates (57.56%) were biofilm producers. The most prevalent biofilm gene was pgaB (79.65%), followed by csuE (76.74%). Evidence of virulence phenotypes revealed that all CRAB exhibited proteolytic activity; however, only four isolates (2.33%) were positive for the hemolytic-producing phenotype. REP-PCR showed that 172 CRAB isolates can be divided into 36-DNA fingerprint patterns. (4) Conclusions: The predominance of biofilm-producing CRAB isolates identified in this study is concerning. The characterisation of risk factors could aid in controlling the continual selection and spreading of the A. baumannii phenotype in hospitals, thereby improving patient care quality.


Introduction
Acinetobacter baumannii is a gram-negative coccobacillus responsible for hospitalacquired infections, such as ventilator-related pneumonia, secondary meningitis, skin and soft tissue infections, bacterial septicaemia, urinary tract infections and burn wound infections, particularly in intensive care units (ICUs) [1][2][3]. These infections are often related

Hemolysis and Protease Activities
In this study, only 4/172 (2.33%) CRAB isolates were positive for hemolysis activity, and all of them have a beta-hemolysis phenotype.
The primary proteolytic screening results showed that all isolates were positive for protease production based on the formation of the halos zone of hydrolysis around the reaction colonies.

Biofilm Formation Phenotypes of CRAB
Among all CRAB isolates examined for biofilm formation, 99 (57.56%) were biofilm producers and 73 (42.44%) were non-biofilm producers. The CRAB biofilm-producing Antibiotics 2023, 12, 580 4 of 12 strains were divided into three categories: 40 (23.26%) were weak biofilm producers, 39 (22.67%) were moderate biofilm producers and 20 (11.63%) were strong biofilm producers. Heatmap analysis demonstrated differences in the antibiotic resistance profiles of CRAB clinical isolates, which varied in biofilm formation capacity. In Carbapenem-resistant groups (IMP, MEM and DOR), there are a high number of isolates showing strong biofilm formation [ Figure 2]. and all of them have a beta-hemolysis phenotype.
The primary proteolytic screening results showed that all isolates were positive for protease production based on the formation of the halos zone of hydrolysis around the reaction colonies.

Biofilm Formation Phenotypes of CRAB
Among all CRAB isolates examined for biofilm formation, 99 (57.56%) were biofilm producers and 73 (42.44%) were non-biofilm producers. The CRAB biofilm-producing strains were divided into three categories: 40 (23.26%) were weak biofilm producers, 39 (22.67%) were moderate biofilm producers and 20 (11.63%) were strong biofilm producers. Heatmap analysis demonstrated differences in the antibiotic resistance profiles of CRAB clinical isolates, which varied in biofilm formation capacity. In Carbapenem-resistant groups (IMP, MEM and DOR), there are a high number of isolates showing strong biofilm formation [ Figure 2].

Prevalence of Drug Resistance, Biofilm-Related Genotypes and Genetic Diversity of CRAB
In this study, CRAB isolates harboured the drug resistance-associated genes, including the blaOXA51-like gene, at a rate of 100% (172 isolates) and were the most prevalent genotype. In contrast, the blaVIM, blaIMP, blaNDM and blaOXA23-like genes were present in 137 isolates (79.65%), one isolate (0.58%), 21 isolates (12.21%) and 161 isolates (93.60%), respectively. The blaOXA24-like gene was not found in CRAB isolates.
The PCR experiments showed positive results for biofilm-related genes in only the csuE gene in 19 (11.05%) isolates, only the pgaB gene in 24 (13.395%) isolates and both the csuE and pgaB genes in 113 (65.70%) isolates. However, there were 16 (9.30%) isolates that did not carry both genes. Our findings revealed a high prevalence of biofilm-forming and biofilm-related genes (csuE and pgaB) in CRAB strains in the study region [ Table 2]. Moreover, Figure 3 depicts an unweighted pair group method with an arithmetic mean dendrogram derived from REP-PCR of 172 CRAB strains. The REP-PCR cluster for bacteria with at least 70% coefficient similarity generated 36 clonal diversities.

Prevalence of Drug Resistance, Biofilm-Related Genotypes and Genetic Diversity of CRAB
In this study, CRAB isolates harboured the drug resistance-associated genes, including the bla OXA51-like gene, at a rate of 100% (172 isolates) and were the most prevalent genotype. In contrast, the bla VIM , bla IMP , bla NDM and bla OXA23-like genes were present in 137 isolates (79.65%), one isolate (0.58%), 21 isolates (12.21%) and 161 isolates (93.60%), respectively. The bla OXA24-like gene was not found in CRAB isolates.
The PCR experiments showed positive results for biofilm-related genes in only the csuE gene in 19 (11.05%) isolates, only the pgaB gene in 24 (13.395%) isolates and both the csuE and pgaB genes in 113 (65.70%) isolates. However, there were 16 (9.30%) isolates that did not carry both genes. Our findings revealed a high prevalence of biofilm-forming and biofilm-related genes (csuE and pgaB) in CRAB strains in the study region [ Table 2]. Moreover, Figure 3 depicts an unweighted pair group method with an arithmetic mean dendrogram derived from REP-PCR of 172 CRAB strains. The REP-PCR cluster for bacteria with at least 70% coefficient similarity generated 36 clonal diversities.

Discussion
Recently, the role of A. baumannii in nosocomial infections, its notable ability to develop antimicrobial resistance and its involvement in severe clinical infections have raised considerable attention [23]. Treating these bacteria, particularly MDR and broad-spectrum beta-lactamase strains, is of critical importance [24]. Carbapenems are currently the treatment of choice for MDR A. baumannii infections that are resistant to third generation cephalosporins. However, the number of carbapenem-resistant strains is rising [25]. As a result of the large prevalence rate of this infection and the varying patterns of antibiotic resistance in various geographical locations, the surveillance of the prevalence rate and antibiogram in different parts of the world is crucial. These data would help determine the distribution of resistance patterns and select the most suitable medication regimen [26].
In our investigation, 172 of the 995 A. baumannii isolates from clinical samples were carbapenem-resistant. Moreover, all CRAB isolates were MDR (100%). The resistance to the cephalosporin antibiotics, gentamicin and ciprofloxacin, was more than 80%. All isolates were resistant to imipenem, meropenem and doripenem. The antibiotic resistance in the present study was similar to the reports from Iran [27]. Almost all the isolates in our study carried the bla OXA-51-like genes. The intrinsic bla OXA-51-like gene's identification of the A. baumannii isolates provided proof of their identity. The bla OXA-51-like genes are an easy and robust tool for identifying A. baumannii [28][29][30]. These carbapenem-resistant isolates have been attributed to a strong promoter-driven upregulation of the bla OXA51-like genes when related to the ISAba1 gene 7 bp upstream, as previously reported [31]. The bla OXA-51-like gene was the most frequently detected carbapenemase gene among all clinical CRAB isolates (100%) in the current study, and it is also commonly found in health care facilities worldwide, followed by the bla OXA-23-like , bla VIM , bla NDM and bla IMP genes at 93.60%, 79.65%, 12.21% and 0.58%, respectively. Furthermore, the spread of CRAB carrying carbapenem-resistance genes was proven in many reports from other countries [26,28,[32][33][34]. Additionally, none of the isolates in this study possessed the bla OXA-24-like gene, which is consistent with others [35][36][37][38]. The finding is presumed to correlate with clonal expansion [37][38][39]. Moreover, the resistance to carbapenems in these strains may be clarified by the participation of other mechanisms of resistance to carbapenem, such as the modified permeability or additional carbapenemase enzymes not examined in this study. Examples of non-carbapenemase carbapenem-resistance mechanisms in A. baumannii include reduced membrane porin density [40], decreased drug affinity caused by PBP downregulation [41] and efflux pump (EP) mechanisms.
A. baumannii's pathogenicity and resistance to unfavourable environmental conditions correlate with numerous virulence factors, such as the capacity to generate hemolysin, lipase, lecithinase and protease, as well as the ability to form biofilms and quorum sensing [42,43]. The growth behaviour of bacteria in biofilms is altered, reducing their susceptibility to specific antimicrobial treatments [44]. Along with known traditional drug-resistant mechanisms, alternative strategies contribute to the resilience of bacteria in biofilms, such as slow or partial permeation of antimicrobial drugs into the biofilm, a unique microenvironment in the biofilm and altered growth behaviour of microbes within biofilms. Since biofilms are multicellular, these mechanisms result in bacterial resistance and unsuccessful treatment efforts [45]. Approximately 57.56% of the CRAB isolates in this study were biofilm-forming strains. According to previous studies, biofilm-forming bacteria have a substantially longer life than those that do not form biofilms (36 versus 15 days, p-value < 0.001) [46,47]. The capacity of A. baumannii to form biofilms improved colonisation and persistence, allowing for higher rates of nosocomial infections, particularly device-associated illnesses [48]. Several factors, including environmental factors and numerous cell signals, influence A. baumannii biofilm development by influencing signalling, cell-to-cell interaction and scaffolding functions [49]. Furthermore, the genes associated with biofilms offer a comprehensive perspective on surface adhesion and biofilm development. Among these genes were pgaB and csuE [50]. The Csu chaperone-usher pili assembly system is regulated by the BfmS/BfmR two-component system (pgaB), and pgaABCD is responsible for producing poly-1,6-N-acetylglucosamine [51]. The initial surface attachment phase of biofilm formation is mediated by pili, composed of Antibiotics 2023, 12, 580 7 of 12 proteins encoded by the csu operon. Previous research demonstrated that BfmR stabilised the transcription of the csu operon genes, which are vital in biofilm formation [52,53]. The presence of pgaB and csuE as biofilm encoding genes in CRAB isolates from admitted patients was studied. Our results revealed that pgaB and csuE were present in most isolates similar to those reported by Zeighami et al. [3] who reported these genes in A. baumannii recovered from ICU patients. In this study, the vital virulence activities of CRAB illustrated the evidence of biofilm production, hemolysis and protease activities. This finding is consistent with a previous study by Dahdouh and Hajjar [54], which revealed that the isolates produced biofilm, caused blood hemolysis in an agar plates and exhibited proteolytic activity.
PCR-based fingerprinting methods, such as REP-PCR, are easy, quick and low-cost, with strong discriminatory power for identifying A. baumannii. The results of REP-PCR can be achieved in a reasonably short amount of time, as demonstrated by a review by Sabat et al. This is also the reason this procedure is less expensive. REP-PCR is highly discriminating for several bacterial species [55]. Previous research found that the REP-PCR discrimination power was adequate and correlated with PFGE [56,57]. REP-PCR was helpful in the epidemiological analysis of hospital epidemics. Many investigations employed PCR-based fingerprinting to identify A. baumannii clinical isolates [3,58]. By typing A. baumannii with REP-PCR, the obtained patterns were classified as distinct REP-PCR clusters that provided evidence of possible clonal expansion among the different isolates. Despite several reports of clonality in the literature, it is possible to identify the levels of clonal diversity among the CRAB strains.
However, the underlying molecular reason for the bacterium's rising prevalence and antibiotic resistance still needs to be fully understood. Molecular detection and wholegenome sequencing should be employed to better understand drug resistance and bacterial pathogenesis mechanisms. Furthermore, integrating molecular, genomic and bioinformatics tools resulted in genomic epidemiology approaches, which gradually increased in various fields of pathogen surveillance and developed control strategies.

Study Settings and Ethical Approval
The non-duplicate 995 A. baumannii isolates were attained from February to September 2021 at the Maharaj Nakhon Si Thammarat Hospital. The study was approved by the Human Research Ethics Committee of Walailak University (protocol number: WUEC-21-027-01).

Bacterial Isolation and Identification
The stored isolates were recovered and confirmed as A. baumannii by conventional microbiological methods such as gram stain, gram-negative coccobacilli; the oxidase test, negative; triple sugar iron test, K/N; Simmons citrate agar, positive; motility-indole-lysine, negative-negative-positive; OF-maltose, non-oxidiser; OF-glucose, oxidiser and growth on MacConkey agar at 42 • C [59].

Hemolysis Assay and Protease Activity
The phenotypic hemolysin activity was determined using the streaking and spot methods on a blood agar plate assay, as described previously [62]. All plates were incubated for 24 h at 37 • C. On blood agar plates, the hemolysis was visualised. The presence of a zone that is lightly cleared in the media and around bacterial colonies when using the streaking method indicated that CRAB could haemolyse red blood cells. Based on the appearance of a greyish-greenish colony encircled by a clear zone, beta-hemolysis was detectable using the spot method. Additionally, the skim milk plate method tested all isolates for proteolytic activity. The inoculated plates were incubated for 4 h at 37 • C. A clear zone surrounding a bacterial colony indicated a positive outcome. The experiment was carried out in triplicate.

Biofilm Formation Using Microtiter Plate Assay
The microtiter plate technique assessed the biofilm-forming capacity of CRAB clinical isolates. The isolates were cultivated overnight at 37 • C in tryptic soy broth (TSB) (Oxoid, Basingstoke, UK) and adjusted to No. 0.5 McFarland standards. A 96-well flat-base plate was inoculated with 20 microliters of fresh bacterial culture and incubated for 24 h at 37 • C in 180 microliters of TSB supplemented with 0.25% (w/v) glucose. The plates were rinsed thrice with phosphate-buffered saline after incubation. Crystal violet (1%; v/v) was used to stain the attached cells for 20 min. The stained dye of the adhering cells was dissolved with absolute ethanol. The solution's optical density was measured at 580 nm [63]. This absorbance value of the solution indicated the biofilm-forming capacity of the isolate. As a negative control, sterile TSB supplemented with glucose was used. The average reading was achieved with three replicated experiments. The term 'ODc' was defined as the mean optical density (OD) of the negative control plus three standard deviations (cut-off OD). The respective biofilm formation degrees of the CRAB isolates were reported as follows: strong biofilm formation (4 × ODc < OD), moderate biofilm formation (2 × < ODc < OD 4 × ODc), weak biofilm formation (ODc < OD < 2 × ODc) and non-biofilm formation (OD < ODc) [16].

Genotypic Characterisation of Antimicrobial Resistance and Biofilm-Associated Genes by Polymerase Chain Reaction (PCR)
The CRAB isolates were cultivated in Luria-Bertani broth at 37 • C. The DNA template for the PCR experiment was prepared using a genomic DNA extraction kit (Geneaid, Taiwan). Briefly, the bacterial pellet was collected from 1 mL of an overnight bacterial culture by centrifuging for 1 min at 14,000× g. Then, the pellet was resuspended in 180 µL of GT buffer and mixed by vortex. The suspension was added to 20 µL of Proteinase K and incubated at 60 • C for 10 min. The sample was added with 200 µL of GB Buffer, mixed and incubated at 70 • C for 10 min. Two-hundred microliters of absolute ethanol were added into the sample and mixed well. The sample solution was transferred into a GD Column, then centrifuged at 14,000× g for 2 min. The sample was washed with washing buffers. The extracted DNA was eluted from the column by elution buffer and determined the DNA quantity using a NanoDrop-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, NC, USA).
PCR was used to detect the presence of drug resistance-associated genes, including bla VIM , bla IMP , bla NDM , bla OXA-23-like , bla OXA-24-like and bla OXA-51-like genes and biofilmrelated genes, such as csuE and pgaB genes. The primer sets, annealing temperature and sizes of the expected amplicons are demonstrated in Table S1. The amplification reaction (25 µL) contains Taq DNA polymerase, 10× Taq buffer, 25 mM MgCl 2 , 10 mM dNTPs, 10 mM of each forward and reverse primer solution, 1.25 units/L (Thermo Scientific, Wilmington, NC, USA) and 100 ng/L of DNA template. The thermal cycles were carried out with the initial denaturation at 95 • C for 5 min, followed by 35 cycles of denaturing at 95 • C for 30 s, annealing at a temperature specific for each primer pair for 30 s and extension at 72 • C for 30 s, with a final extension at 72 • C for 7 min. The PCR amplicon was separated on a 1% (w/v) agarose gel, stained with the nucleic acid staining dye, SafeView TM FireRed (ABM Good, Richmond, BC, Canada), and examined under UV transillumination using the ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA). Subsequently, the purified PCR amplicons were sequenced (Macrogen, Seoul, Republic of Korea). The NCBI web-based genome analysis, including the DDBJ/EMBL/GenBank databases, was used for sequence analysis to confirm the target amplicons.

Molecular Typing and Clonal Relationship between CRAB Strains by Repetitive Element Sequence-Based PCR (REP-PCR)
To distinguish between bacterial strains, DNA fingerprinting was investigated by using REP-PCR, a molecular typing method. The REP-like elements in the genomic DNA isolated from CRAB isolates were amplified using the primer pairs REP1 (5 -IIIGCGCCGICATCAGGC-3 ) and REP2 (5 -ACGTCTTATCAGGCCTAC-3 ), as previously described [6]. Briefly, a 25 µL reaction mixture comprising 100 ng of chromosomal DNA, 2.5 µL of 10× Taq buffer, 0.5 µL of 10 mM dNTP mix, 1.5 U of Taq DNA polymerase (Thermo Fisher Scientific, Wilmington, NC, USA), 50 pmoL of each primer and 1.25 µL of dimethyl sulfoxide was added. The amplification procedure included initial denaturation at 94 • C for 10 min, followed by 30 cycles of denaturation (94 • C, 1 min), annealing (40 • C, 1 min), extension (72 • C, 2 min) and a single final extension (72 • C, 16 min). The PCR products were electrophoresed in a 1.2% (w/v) agarose gel. The lanes and bands were determined against a 100 bp plus ladder lane (Thermo Fisher Scientific, Wilmington, NC, USA).
For determining genotyping and clonal relationships between CRAB strains, the gels were stained and the DNA bands were photographed and visualised using a UV transilluminator. The images of DNA banding patterns were analysed with GelJ software version 3.0 (San Diego, CA, USA) and the dendrogram was created using the unweighted pair group method with arithmetic averages and Dice's similarity coefficient with a tolerance of 1.0% [64,65]. A similarity of 70% or greater indicated the same REP-PCR genotype, whereas a similarity of less than 70% indicated different REP-PCR genotypes.

Statistical Data Analysis
The data were analysed using GraphPad Prism 9. All analyses were performed using three separate experiments. The prevalence of CRABs, genotypic and phenotypic biofilm formation and drug resistance were demonstrated as percentages. The relationship between biofilm formation and antibiotic susceptibility was demonstrated by heatmap analysis and the chi-squared test where a p-value of <0.05 was considered to be statistically significant.

Conclusions
The high occurrence of biofilm-producing MDR clinical isolates found in this study is concerning. The rising prevalence of carbapenem resistance is alarming. Our findings highlighted the significance of csuE and/or pgaB genes, carbapenemase-encoding genes and the emergence of drug resistant strains as potential risk factors linked with biofilmproducing A. baumannii clinical isolates. These surveillance outcomes that continually monitor the epidemiological and susceptibility profiles of antimicrobials used against disseminating pathogens in healthcare environments might be one of the policies that help treat nosocomial infections caused by bacteria and limit the spread of virulent strains. It could assist health officials and policymakers in managing continuous selection and transfer of this trait within healthcare facilities, thereby improving patient care quality.