Estimation, Evaluation and Characterization of Carbapenem Resistance Burden from a Tertiary Care Hospital, Pakistan

Carbapenem resistance has become major concern in healthcare settings globally; therefore, its monitoring is crucial for intervention efforts to halt resistance spread. During May 2019–April 2022, 2170 clinical strains were characterized for antimicrobial susceptibility, resistance genes, replicon and sequence types. Overall, 42.1% isolates were carbapenem-resistant, and significantly associated with Klebsiella pneumoniae (K. pneumoniae) (p = 0.008) and Proteus species (p = 0.043). Carbapenemases were detected in 82.2% of isolates, with blaNDM-1 (41.1%) associated with the ICU (p < 0.001), cardiology (p = 0.042), pediatric medicine (p = 0.013) and wound samples (p = 0.041); blaOXA-48 (32.6%) was associated with the ICU (p < 0.001), cardiology (p = 0.008), pediatric medicine (p < 0.001), general surgery (p = 0.001), general medicine (p = 0.005) and nephrology (p = 0.020); blaKPC-2 (5.5%) was associated with general surgery (p = 0.029); blaNDM-1/blaOXA-48 (11.4%) was associated with general surgery (p < 0.001), and wound (p = 0.002), urine (p = 0.003) and blood (p = 0.012) samples; blaOXA-48/blaVIM (3.1%) was associated with nephrology (p < 0.001) and urine samples (p < 0.001). Other detected carbapenemases were blaVIM (3.0%), blaIMP (2.7%), blaOXA-48/blaIMP (0.1%) and blaVIM/blaIMP (0.3%). Sequence type (ST)147 (39.7%) represented the most common sequence type identified among K. pneumoniae, along with ST11 (23.0%), ST14 (15.4%), ST258 (10.9%) and ST340 (9.6%) while ST405 comprised 34.5% of Escherichia coli (E. coli) isolates followed by ST131 (21.2%), ST101 (19.7%), ST10 (16.0%) and ST69 (7.4%). Plasmid replicon types IncFII, IncA/C, IncN, IncL/M, IncFIIA and IncFIIK were observed. This is first report describing the carbapenem-resistance burden and emergence of blaKPC-2-ST147, blaNDM-1-ST340 and blaNDM-1-ST14 in K. pneumoniae isolates and blaNDM-1-ST69 and blaNDM-1/blaOXA-48-ST69 in E. coli isolates coharboring extended-spectrum beta-lactamases (ESBLs) from Pakistan.


Introduction
Since the glorious discovery of first antibiotic, revolutionary changes occurred in the health care settings that helped in reducing the suffering of mankind by preventing the onset of infectious diseases [1]. However, due to misuse of antibiotics, the mounting rise in antimicrobial resistance (AMR) has posed greater clinical challenges and public health threats with every passing day as accepted by health regulatory systems across continents [2]. In healthcare settings, factors contributing in AMR [3] include easy access and unreasonable consumption of broad-spectrum antibiotics, inadequate guidelines for antibiotics utilization guidelines, lack of audit policies for antimicrobials, transmission of resistant strains from patient to patient and through health care providers, absence of isolation of patients colonized with resistant microbes and sub-optimal infection control measures [4]. At the moment, AMR is considered to be accountable for more than

Discussion
Carbapenem resistance is considered as one of the critical threats associated with hospital-acquired infections, especially in developing countries. Therefore, timely surveillance efforts are required to reduce the spread of CRE [42]. The current study was designed to characterize the key determinants for resistance spread in a tertiary care hospital.
The National AMR action Plan for Pakistan 2017-2018 suggested a rate of 30% CR in K. pneumoniae, while much lower CR rates were reported in P. aeruginosa isolates (6.5%) [8]. However, another study showed higher CR rates among Pseudomonas spp. (34.0%) but lower rates among E. coli (7.0%) and Klebsiella spp. (8.0%) [52]. These reports together with our data suggested that the CR trend among Pseudomonas spp. is changing with time and a notable CR increase was observed from Pakistan, as can be seen in the studies showing 24.2% in 2012 [59], 49.5% imipenem resistance in 2015 [60], 81.6% in 2019 [61], 43.2% in 2020 [62] and 66.4% meropenem resistance in 2022 [63]. In contrast to our results of Acinetobacter spp. (7.3%), data from Pakistan suggested a sharp increase of CR among Acinetobacter spp. from 50% in 2011 to 95.5% in 2015 [64][65][66], 61.89% imipenem resistant Acinetobacter spp. in 2018 and 84.0% in 2022 [56,67]. High CR rates among Acinetobacter spp. and Pseudomonas spp. are alarming in Pakistan as these species exhibit intrinsic resistance to many antibiotics, leaving few therapeutic choices available. Our results strengthen the WHO recommendations for both species as critical pathogens [30,68].
The main reason for the emergence of different STs globally is the ability of strains to disseminate carbapenemases through plasmids and their successful adaption to different healthcare environments. Our data revealed that successful high-risk clones of K.  [15][16][17]97]. Furthermore, we observed the emergence of bla KPC-2 -ST147, bla NDM-1 -ST340 and bla NDM-1 -ST14 in K. pneumoniae and bla NDM-1 -ST69 and bla NDM-1 /bla OXA-48 -ST69 in E. coli.

Conclusions
In this study, we reported the detailed analysis of carbapenem resistance burden and the emergence of bla KPC-2 -ST147, bla NDM-1 -ST340 and bla NDM-1 -ST14 in K. pneumoniae isolates, and bla NDM-1 -ST69 and bla NDM-1 /bla OXA-48 -ST69 in E. coli isolates coharboring ESBLs from Pakistan. Moreover, we described bla NDM-1 (n = 1) and bla OXA-48 (n = 2) in Burkholderia spp. and the coexistence of bla NDM-1 /bla OXA-48 (n = 2) in Providencia spp. for first time in the study population. Our data indicated that the lack of antimicrobial stewardship and misuse augmented by diagnostic difficulties in developing countries are accelerating the evolution and spread of high-risk STs and hyper-efficient plasmids. This situation is miserable, especially in healthcare settings with immense antimicrobial selection pressure, thereby highlighting the expansion of high-risk clones as a resistance reservoir.

Methodology
The clinical strains were collected between May 2019 and April 2022 from the routine diagnostic laboratory, Mayo hospital, Lahore, Pakistan. Mayo hospital is one of the largest hospitals in South East Asia with a 3000 bed capacity. The clinical isolates were processed as given in Figure 1. Clinical specimens were phenotypically characterized by analyzing colony morphology and Grams staining by culturing on MacConkey agar and cysteine lactose electrolyte-deficient media (Oxoid Ltd., Basingstoke, UK) for urine samples. Biochemical characterization was performed by API-20E and API-20NE (BioMerieux, Marcy-IEtoile, France).

Antimicrobial Resistance Gene Analysis
The heat lysis method was used for genomic DNA extraction [100]. In short, 2 to 3 bacterial colonies were mixed with 500 µL sterile dH2O in 1.5 mL microcentrifuge tube. The sample was incubated at 98 • C for 10 min/300 rpm in thermomixer (FischerScientific, Waltham, MA, USA). Sample was centrifuged at 1000 rpm for 10 min and supernatant containing DNA was collected in a new tube. DNA was stored at −80 • C until further processing. Carbapenemase resistance genes (bla KPC-2 , bla NDM-1 , bla VIM , bla IMP , bla OXA-48 ) and selected ESBLs (bla SHV , bla TEM and bla CTX-M ) were detected by standard PCR. The PCR reaction mixture contained 25 µL of 2 × PCR Master Mix (catalogue # K0171, Thermoscien-tific, Waltham, MA, USA), 10 µM of each primer, 0.5 ng of DNA and dH2O up to 50 µL in a thermal cycler (Proflex, ABI, Haines City, FL, USA). Amplicons were resolved by agarose gel electrophoresis (1-1.5%). The primer sequences and PCR cycling conditions are given in Table S1.

Allele Identification by Sequencing
Sanger's sequencing method was used for the bla NDM and bla KPC allele identification. BigDye terminator v3.1 kit was used for cycle sequencing as per kit instructions. Briefly, 10 µL PCR reaction mixture contained BigDye terminator 3.1 Ready Reaction Mix 4 µL, forward primer (3.2 pmol) 0.5 µL, purified DNA template (5-20 ng) 2 µL and dH2O 3.5 µL. PCR cycling conditions were 96 • C 1 min, 96 • C 10 s, 50 • C 5 s, 60 • C 2 min (35 cycles). PCR product was purified by using BigDye XTerminator purification kit as per kit instructions and capillary electrophoresis was performed by Genetic Analyzer (ABI-3500, Thermo Fischer, Waltham, MA, USA). Sequencing analysis software v6.1 and basic local alignment tool (BLAST, NCBI) were used for data analysis and interpretation. CLC Sequence Viewer 7 was used for sequence alignment and mutation analysis.

Statistical Analysis
All statistical analyses were performed by using Statistical Package for Social Sciences software (SPSS 26). Categorical data are presented as frequency and percentage. The chi-square test was used to compare the categorical data among groups. p-value ≤ 0.05 was considered as significant.

Informed Consent Statement:
The study was approved by institutional review board of the King Edward Medical University, Lahore, Pakistan. Informed consent was obtained from the study participants.

Data Availability Statement:
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest:
The authors declare no conflict of interest.