Molecular Epidemiology of Extensively-Drug Resistant Acinetobacter baumannii Sequence Type 2 Co-Harboring bla_NDM and bla_OXA From Clinical Origin

Introduction

The development and spread of carbapenem-resistant Acinetobacter baumannii (CR-AB) represents a serious global public health dilemma.¹ Extensively drug resistant A. baumannii produce resistance to all or almost all medically approved antibiotics.² A. baumannii can cause severe infections, such as septicemia, urinary tract infections, wound infections, and ventilator-associated pneumonia, and A. baumannii has been associated with nosocomial infections.³ A. baumannii has been designated as a global priority pathogen by the World Health Organization (WHO)⁴ and is listed as an ESKAPE pathogen, which effectively resist to various antibacterial drugs, and restricting therapeutic options.⁵ Every year, over 0.7 million people die from infections due to antimicrobial-resistant (AMR) bacteria globally. By 2050, an individual is estimated to die from infection with an AMR pathogen every 3 seconds, and the global costs associated with AMR infections are expected to reach $100 trillion.⁶ The Centers for Disease Control and Prevention has reported that CR-AB is responsible for 700 mortality, and $281 million are spent on the therapeutic management of CR-AB infections annually.⁷ CR-AB develop resistance through numerous mechanisms, such as the production of β-lactam enzymes, porin proteins loss, and efflux pump overexpression.⁸ Infections caused by CR-AB pathogens are difficult to treat due to the acquisition of various resistant genes such as class D oxacillinase (OXA) and metallo-β-lactamase (MBL).⁹ The New Delhi metallo-β-lactamases (NDM) belong to a superfamily of class B MBL-producing bacteria,¹⁰ which was first identified in a Swedish patient who sought medical attention in New Delhi, India, in 2009.¹¹ NDM pathogens are resistant to β-lactams and other antibiotic classes, including aminoglycosides and fluoroquinolones, and are only susceptible to the potentially toxic colistin and polymyxin B antibiotics.¹² However, resistance to colistin has also been reported at the global level.¹³ The chimeric gene bla_NDM-1 is derived from the fusion of aminoglycoside phosphotransferase (aphA6) and an existing bla_MBL gene,¹⁴ and the promoter for ISAba125, which is encoded upstream, drives the expression of bla_NDM-1. The genes bla_OXA-23, bla_OXA-40, and bla_OXA-58 encode the most frequently identified OXAs, whereas, bla_OXA-51 is intrinsic to resistant A. baumannii and can be used to identify the Acinetobacter spp.¹⁵ These pathogens continue to represent critical issues in many healthcare facilities worldwide, despite the implementation of infection control practices.¹⁶ Currently, little evidence has supported the presence of CR-AB in Pakistan. However, a recent study documented the prevalence of NDM-1-producing A. baumannii at a tertiary care hospital in Lahore.¹¹ The aim of the present study was to determine the antimicrobial susceptibility testing, identify the most prevalent sequence types (STs), and investigation of resistant mechanism of CR-AB clinical isolates in Pakistan.

Methodology

Ethical Considerations

Before initiating this research study, ethical approval in accordance with the Declaration of Helsinki was obtained from the Ethical Review Committee, Government College University, Faisalabad. Additionally, prior to collecting the clinical samples, informed consent was obtained from each study participant. Informed consent was read to the person in the language they understand and signed appropriately. They were willing to provide a sample and utilize the isolates for the study. They were assured that the samples would be used solely for research purposes and that personal information would be kept confidential.

Clinical Isolates

A convenience sample consisting of 174 non-duplicate A. baumannii clinical isolates were collected from a tertiary care hospital in Lahore between September 2020 to December 2020. Only a single strain was collected from each case irrespective of the infection site and preserved at −80°C in Luria Bertani broth containing 16% glycerol. A. baumannii strains were primarily acquired from sputum (n=81), pus (n=42), blood (n=19), urine (n=18), throat swabs (n=7), cerebrospinal fluid (CSF) (n=3), nasal swab (n=2), seminal fluid (n=1), and high vaginal swab (n=1). The collection and transport of the clinical strains was done aseptically.

Isolate Identification by VITEK 2 System

Isolates were sub-cultured on blood and MacConkey agar (Oxoid, UK), and the plates were incubated at 37°C overnight under aerobic conditions. Isolates were identified by Gram staining and culture morphology and were confirmed using Gram-negative identification (GN) cards in the VITEK 2 compact system (bioMérieux, France).

Molecular Confirmation of A. baumannii Strains

A. baumannii were further confirmed by the detection of the intrinsic bla_OXA-51 and recA genes using specific primers, as described previously. The amplicons were subjected to gel electrophoresis, followed by examination under ultraviolet (UV) light using a gel documentation instrument (Bio-Rad, UK).

Table 1 Clinical Information for the Sources of A. baumannii Isolates (n = 174)

Minimum Inhibitory Concentration (MIC)

An antibiogram was generated using various classes of antibiotics, such as mezlocillin, piperacillin, piperacillin/tazobactam, ampicillin/sulbactam, cefepime, cefotaxime, ceftazidime, imipenem, meropenem, gentamicin, amikacin, ciprofloxacin levofloxacin, tetracycline, tobramycin, trimethoprim/sulfamethoxazole, and colistin, in the VITEK 2 compact system. The MIC for colistin was determined using the microbroth dilution method. Interpretation of the antimicrobial susceptibility testing was carried out as per CLSI 2019 guidelines.¹⁷

Detection of Carbapenemase and MBL Phenotypes

The modified Hodge test (MHT) was performed to detect carbapenemase, as described previously.¹⁸ A 0.5 McFarland Escherichia coli ATCC 25922 turbidity standard was prepared and diluted 1:10 with sterile normal saline. The bacterial suspension was lawned on Mueller-Hinton agar (MHA) plates, and a meropenem (10 µg) disk was placed in the center. Isolates were streaked from the periphery of the disk to the edge of the plates. The MHT was considered positive if the isolates displayed clover leaf-like indentation.

MBL detection was performed using the double-disk synergy test (DDST), as described previously.¹⁹ Briefly, a 0.5 McFarland bacterial suspension was lawned on MHA plates, and 2 meropenem and 2 ertapenem disks were placed at a distance of 25 mm, and 10 µL of 5 M ethylenediaminetetraacetic acid (EDTA) solution was added to one of each meropenem (10 µg) and ertapenem (10 µg) disk. MBL production was considered positive if the EDTA disks showed a zone of inhibition 5 mm larger than those for the non-EDTA disks.

Detection of the bla_NDM and bla_OXA Genotypes

DNA from the clinical isolates was extracted using a commercially available bacterial genomic DNA kit (Thermo Scientific, UK). DNA purity was checked at 260 nm and 280 nm (NanoDrop Spectrophotometer, Thermo Scientific, UK). The DNA integrity was determined by electrophoresis (Bio-Rad, UK). These were following primers; blaNDM-F-5`ATGGAATTGCCCAATATTATGCAC-3` blaNDM-R-5` TCAGCGCAGCTTGTCGGC-3`, NDMV-F- 5` TGGCTTTTGAAACTGTCGCACC-3` NDMV-R-5` CTGTCACATCGAAATCGCGCGA-3`, blaOXA-23-like-F 5`GATCGGATTGGAGAACCAGA-3′ blaOXA-23-like-R-5`ATTTCTGACCGCATTTCCAT-3′, blaOXA-24- like-F-5`GGTTAGTTGGCCCCCTTAAA-3′ blaOXA-24-like-R −5`AGTTGAGCGAAAAGGGGATT-3′, blaOXA-58-like-F-5`AAGTATTGGGGCTTGTGCTG-3′, blaOXA-58-like-R-5`CCCCTCTGCGCTCTACATAC-3′, blaOXA-51-like-F-5` TAATGCTTTGATCGGCCTTG-3′ blaOXA-51-like-R-5`TGGATTGCACTTCATCTTGG-3′, blaOXA-143-F-5′ TGGCACTTTCAGCAGTTCCT-3′ blaOXA-143-R-5′-TAATCTTGAGGGGGCCAACC-3′, and recA-F-5′ACAATGACATTGCAAGCAATTG-3′ recA-R-5′CCAATTTTCATACGAATCTGG-3′. The presence of bla_NDM was detected under the following conditions: initial denaturation at 95°C for 1 min, secondary denaturation at 95°C for 45 sec, annealing at 58°C for 45 sec, primary extension at 72°C for 1 min, and a final extension at 72°C for 5 min. To determine the specific genotypes, bla_NDM was re-amplified using another set of primers for sequencing. Multiplex PCR was performed to sequence bla_OXA-51, bla_OXA-23, bla_OXA-24, and bla_OXA-143 under the following conditions: initial denaturation at 94°C for 5 min, secondary denaturation at 95°C for 30 sec, annealing at 52°C for 40 sec, primary extension at 72°C for 50 sec, and a final extension at 72°C for 6 min.

Multilocus Sequence Typing (MLST)

Multilocus sequence typing (MLST) was performed to determine the genetic diversity of carbapenem-resistant isolates according to the Pasteur scheme. The DNA sequences of 7 housekeeping genes (cpn60, fusA, gltA, pyrG, recA, rplB, and rpoB) were amplified, followed by purification and sequencing. The sequences were analyzed using the PubMLST database https://pubmlst.org/bigsdb?db=pubmlst_abaumannii_seqdef.

Results

Clinical Information of the Isolates

Of the 174 A. baumannii clinical isolates obtained in this study, the majority were recovered from male (64.9%) patients compared to female (35.1%) patients, with an age range from 1 year to 70 years (mean age: 35.8 years). Isolates were primarily recovered from sputum (n = 81; 46.5%), followed by pus (n = 42; 24%) and blood (n = 19; 10.9%) samples. There were 3 (1.7%) strains of A. baumannii isolates isolated from cerebrospinal fluid samples. A significant proportion of the clinical samples (n = 131; 75.2%) were obtained from patients who were admitted to the intensive care unit (ICU) followed by the male surgical ward (MSW; n = 13; 7.4%) and the female medical ward (FMW; n = 11; 6.3%). The clinical information for the recovered samples is provided in Table 1.

Antimicrobial Resistance Phenotypes

The antimicrobial susceptibility testing for 113 (64.9%) CR-AB isolates displayed 100% resistance to cephalosporins [cefepime (≥32 µg/mL), ceftazidime (≥32 µg/mL), cefotaxime (≥64 µg/mL)], β-lactam inhibitors [piperacillin/tazobactam (≥128/4 µg/mL), ampicillin/sulbactam (≥32/16 µg/mL)], and carbapenems (imipenem and meropenem), with MIC breakpoints ≥8 µg/mL. In addition, 87.6% isolates were resistant to fluoroquinolones (ciprofloxacin), 75.2% to tetracycline (doxycycline), 81.4% to amikacin (aminoglycoside), and 75.2% to co-trimoxazole. However, all of the strains were susceptible to colistin (Figure 1). The WHO AWaRe categorization assigns medically relevant antibiotics into Access (first- and second-line antibiotics), Watch (critical antibiotics), and Reserve (last-resort antibiotics) groups.²⁰ Interestingly, 76 A. baumannii isolates displayed resistance against 16 different AWaRe antibiotics, 78 were resistant to 15 antibiotics, 81 to 14 antibiotics, and 113 to 9 antibiotics (Figure 2).

Figure 1 The hierarchical clustering of carbapenem-resistant Acinetobacter baumannii. The antibiotics are shown on the x-axis, aligned with the bacterial strains on the y-axis. The scale on the y-axis shows bacterial resistance, intermediate resistance, and sensitivity from 1 to 3, respectively. The dendrogram shows the relationship among the different antibacterial drugs. Abbreviations: CT, colistin; MEM, meropenem; IPM, imipenem; CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; SAM, ampicillin/sulbactam; PIP, piperacillin; MEZ, mezlocillin; TZP, piperacillin/tazobactam; TOB, tobramycin; SXT, trimethoprim/sulfamethoxazole; LEV, levofloxacin; TET, tetracycline; CIP, ciprofloxacin; CN, gentamicin; AK, amikacin.

Figure 2 Carbapenem-resistant A. baumannii shows simultaneous resistance to multiple classes of antibiotics.

Phenotypic Detection of Carbapenemase and MBL

Of the 113 CR-AB isolates identified, 89 (78.7%) were positive for both carbapenemase and MBL production (Figure 3). A significant proportion (n = 64; 71.9%) of the MBL-producing CR-AB isolates were detected in ICU patients, followed by 14 (15.7%) in the MSW, 8 (9%) in the operation theater (OT), 2 (2.2%) in the male medical ward (MMW), 6 (6.7%) in the FMW, and 2 (2.2%) in the female surgical ward (FSW).

Figure 3 Carbapenem-resistant phenotypes. The left photograph shows the DDST for MBL production, and the right photograph displays the MHT for carbapenemase production. Abbreviations: ETP, ertapenem; MEM, meropenem; EDTA, ethylenediaminetetraacetic acid; MHT, modified Hodge's test; DDST, double disk susceptibility test; +ve, positive control; -ve, negative control; T-70, 71, tested organisms.

Sequence Typing of Carbapenem Resistant A. baumannii

The MLST results for the 113 CR-AB clinical isolates revealed 7 different STs across the various samples and hospital wards (Table 2). A total of 53 (46.9%) CR-AB isolates belonged to ST2, which was the most predominant ST identified in this study, corresponding to clonal complex 2 (CC2), followed by 18 (15.9%) isolates that belonged to ST1 (CC1), 14 (12.3%) to ST589 (CC1), and 11 (9.7%) to ST7 (CC1). ST2 strains were primarily isolated from sputum samples (n = 27; 50.9%) and were more prevalent in patients admitted to the ICU (n = 21), whereas 13 (24.5%) ST2 isolates were detected in pus samples. Furthermore, 13 (72.3%) ST1 isolates were identified in sputum samples, most of which were found in the ICU (n = 12) cases. In addition, 5 (35.7%) ST589 isolates were also obtained from sputum collected from ICU cases.

Table 2 Prevalence of Carbapenem-Resistant Acinetobacter baumannii Sequence Types in Different Clinical Wards

Genotypic Detection of Carbapenem-Resistant Genes

The CR-AB isolates (n = 113) were further screened for the presence of carbapenem-resistant genes. All CR-AB isolates harbored the intrinsic bla_OXA-51 gene, with a high prevalence of bla_OXA-23 (n = 56; 49.5%), followed by bla_NDM-1 (n = 28; 24.7%), bla_OXA-58 (n = 22; 19.4%), and bla_OXA-143 (n = 3; 2.6%). In addition, 28 (50%) isolates harbored bla_OXA-23, 14 (50%) harbored bla_NDM-1, 12 (54.5%) harbored bla_OXA-58, and 8 (80%) harbored bla_OXA-24, all of which belonged to ST2. Interestingly, 11 (9.7%) isolates harbored bla_OXA-51, bla_NDM-1, and bla_OXA23 together, 3 (2.6%) isolates harbored bla_OXA-51, bla_OXA-23, bla_OXA-24, and bla_NDM-1, and 7 (6.2%) harbored bla_OXA-51, bla_OXA-23, bla_OXA-58, and bla_NDM-1. In addition, 6 (5.3%) isolates harbored both bla_OXA-51 and bla_OXA-23, and 17 (15%) harbored bla_OXA-24, bla_OXA-51, bla_OXA-23, and bla_OXA-58 (Table 3).

Table 3 The Co-Detection and Distribution of Carbapenem-Resistant Genes in Different CR-AB Alleles

Discussion

CR-AB infections are difficult to treat and represent a persistent threat to Pakistan’s fragile healthcare system.²¹ These highly resistant pathogens are associated with extended hospital stays, increased mortality and morbidity, and added financial burden.²² In this study, the intrinsic bla_OXA-51 was detected in all A. baumannii isolates, stressing the importance of using the bla_OXA-51 gene as an alternative marker for Acinetobacter spp., identification, as described previously.^23,24 A. baumannii is considered among the major nosocomial pathogens and has been identified in various areas of the hospital environment, including on medical devices and intravenous catheters.²⁵ In the present study, A. baumannii were recovered from numerous clinical specimens, primarily from sputum (46%) and pus (24%) samples, and were prevalent among patients admitted to the ICU (75%). Previously, multiple studies have indicated that the prevalence of A. baumannii in the ICU was becoming a serious nosocomial problem.^26–28 The CR-AB isolates in this study displayed resistance to all available β-lactam drugs, including carbapenems, which are considered to be the ultimate therapy for the infections produced by multidrug-resistant bacteria; although colistin remains the utmost effective drug, colistin is associated with considerable side effects. Among the identified isolates, 76 and 78 isolates showed simultaneous resistance to 16 and 15 medically relevant antibiotics, respectively, which have been categorized in the WHO AWaRe system. Previous data from Pakistan and other parts of the world have documented a similar scenario, in which the primary methods used to treat infections are no longer able to fight these pathogens.^{11,21,29–31} AMR is primarily caused by the acquisition of resistant determinants, which increase AMR phenotypes. The OXA-type-β-lactamase is the most widely distributed class D enzyme found in gram-negative bacteria.³² In this study, bla_OXA-23 was the predominant resistance determinant. OXAs are encoded by plasmid-mediated genes that can easily be transferred by conjugation. The main reservoir for the bla_OXA-23 gene exists in Asiatic countries, such as Pakistan, India, China, and Nepal. In addition, an extensive study conducted by the SENTRY Antimicrobial Surveillance Program in various Asian-Pacific counties also revealed the heavy presence of bla_OXA-23 in A. baumannii.³³ We also found bla_OXA-24, bla_OXA-58, and bla_OXA-143 genes, which had not been widely reported in Pakistan. The A. baumannii harboring bla_OXA-58 are primarily prevalent in China,³⁴ whereas A. baumannii harboring bla_OXA-143 are prevalent in Brazil³⁵ and Mexico.³⁶ The bla_NDM-1 gene encodes the most powerful class B MBL enzyme, which can hydrolyze a wide range of medically relevant antibiotics. The Indian subcontinent is considered to represent the primary reservoir for the emergence and spread of the bla_NDM-1 gene globally.^6,11,37 This study documented the prevalence of bla_NDM-1 in clinical A. baumannii isolates (24.7%) and found that this gene could be detected in isolates that simultaneously harbor the class D genes bla_OXA-23, bla_OXA-24, and bla_OXA-58. Thus far, various studies have provided evidence to support the spread of bla_NDM-1 in Enterobacteriaceae in Pakistan rather than in non-fermenting bacteria. However, recently published data have focused on the bla_NDM-1-producing A. baumannii isolates in Pakistan.^11,38 The previously published data have also described the global detection of bla_OXA-23 and bla_NDM-1 genes in isolates that also harbor the bla_OXA gene. The NDM gene can be readily transferred to A. baumannii via Tn125 by conjugation, and IS_Aba125 acts as a powerful promoter of the bla_NDM-1 gene.^15,39,40

MLST is a promising technique for studying molecular epidemiology and investigating bacterial strain outbreaks in clinical and environmental settings. In this study, the A. baumannii isolates belonged to 7 different STs: ST1, ST2, ST7, ST23, ST25, ST158, and ST589. Of these, the majority of the A. baumannii isolates (46.9%) belonged to ST2 (CC2). This clone was especially prevalent among critical care patients and was detected in isolate that also harbored the genes bla_OXA-23, bla_OXA-58, and bla_NDM-1. The international clones, CC1 and CC2, were primarily associated with the enzymes bla_OXA-23, bla_OXA-24, and bla_OXA-158. Mediterranean counties have documented the emergence of A. baumannii belonging to ST2, with fewer identified isolates belonging to ST1.^41,42 A Lebanon study reported the detection of ST2 A. baumannii harboring NDM-1.⁴³ Several studies from Pakistan have described the detection of A. baumannii ST2 clones.^29,39 ST2 (CC2) can survive in the clinical settings for longer times, allowing for the acquisition of AMR genes, which can complicate treatment. This report represents the first detection of the ST7 strain in Pakistan to date and need further comprehensive study to determine its diversification. This study was a compressive study examining the genes harbored by various CR-AB isolates that fills a gap by providing previously unavailable data regarding the coexistence of bla_NDM-1 and bla_OXA genes in A. baumannii ST1, ST23, ST25, and ST158 isolates at the national level.

Conclusion

This study highlights the significance of the intrinsic gene bla_OXA-51, which can be used to rapidly detect A. baumannii. All strains were observed to be resistant to medically relevant antibacterials, with few treatment options available for infections associated with these isolates. Therefore, active monitoring and urgent management are necessary for clinical settings. The international clone ST2 was the most widespread isolates identified in public hospitals, simultaneously harboring bla_OXA-23, bla_OXA-24, bla_OXA-58, and bla_NDM-1, indicating that the circulation of these clones occurs at the international level, and this report represents the first reported detection of ST7 in Pakistan. Due to the impending threat associated with AMR bacteria, Pakistan’s Ministry of Health has recently developed a National Action Plan (NAP) on Antimicrobial Resistance. Stakeholders must implement the NAP rapidly and effectively to halt the spread of these super-bacteria.