Genome-Wide Identi cation of Carbapenem-Resistant Gram- Negative Bacterial (CR-GNB) Isolates Retrieved From Hospitalized Patients in Bihar, India

Namrata Kumari (  nrainrai@gmail.com ) Indira Gandhi Institute of Medical Sciences Mukesh Kumar All India Institute of Medical Sciences Amit Katiyar All India Institute of Medical Sciences Abhay Kumar Indira Gandhi Institute of Medical Sciences Pallavi Priya Mahavir Cancer Sansthan Bablu Kumar Indira Gandhi Institute of Medical Sciences Nihar Ranjan Biswas Indira Gandhi Institute of Medical Sciences Punit Kaur All India Institute of Medical Sciences


Introduction
Antimicrobial-resistance is an public health issues worldwide due to the rough use of antibiotics (Katiyar et al., 2020). It has been investigated as a foremost medical and community health issues since the inadequate treatment option to cure contagions triggered by antimicrobial-resistant bacteria. The growing microbial resistance rates to most available antibiotics, including penicillin, cephalosporins and carbapenem made a severe risk (Nordmann & Poirel, 2019). The evaluation of antibiotic resistance throughout the world increases and has become very hard to control due to the growth rates of multidrug-resistance and the lack of consistent observation methods (Manandhar et al., 2020). The WHO recently enumerated β-lactams carbapenemase-producing Gram-negative bacteria (GNB) of serious importance (Wyres et al., 2020). Gram-negative bacteria (GNB), especially Escherichia coli, and Klebsiella pneumoniae have proven resistance to a wide-ranging variety of antimicrobials accountable for noteworthy mortality all over the world (Zavascki et al., 2013). The development of carbapenem-resistance in GNB is a foremost medical problem, predominantly for immunocompromised patients with serious infections (Nair et al., 2021).
Carbapenem are the most effective drug of choice against pathogenic bacteria offering a wide range of antibacterial activity (Zagui et al., 2020). This antimicrobials are painstaking as one of the last option antibiotics against drug-resistant GNB (Elbadawi et al., 2021). The pathogen which are resistant to carbapenem habitually display high intensities of resistance to commonly used antibiotics. This is not only major cause of high death rates, but also creates di cult situations for the patients who spend prolonged time in the hospital and having high medical expenditures gather, employing an sensitive, monetary liability on families, particularly in inadequate sources countries. Hence, precise identi cation of AMR in GNB is an indispensable for the appropriate administration of apposite antimicrobials. To nd AMR in GNB, in vitro cultures were used to monitor the development of bacteria for various concentrations of drugs and may need at least 72 hours to acquire precise antibiotic susceptibility results. Advancement in whole-genome sequencing (WGS) reinforced the evaluation of the complete DNA sequence of bacteria. WGS delivers vital description for genotype of an individual organism. WGS data can give mechanistic insight of the antibiotic-resistance for drugs not being tested routinely (Sawa et al., 2020).
India is a prime location for AMR pathogens because of overuse of antibiotics. However, less data on carbapenemase genes from Bihar region are available to correlate genotypes with the phenotypes. Hence, the main aim of this study was to determine the resistance patterns in carbapenemase-producing clinical isolates from in-patients at I.G.I.M.S., Patna, India.

Study design
A cross sectional investigation was conducted on CR-GNB strains isolated from routine clinical samples of hospital admitted patients coming to

Bacterial isolates
Gram-negative bacteria (GNB) including E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa were isolated and identi ed by standard manual conventional method from culture of the routine clinical samples like blood, vascular catheter tip, urine, bile, ascitic uid, pus, sputum, endotracheal tube-aspirate and broncho-alveolar lavage uid (BAL).
Detection of carbapenemase production Carbapenem-resistance screening of GNB isolates was done using meropenem (10ug) disc. The Modi ed Hodge Test (MHT) and meropenem Double Disc Synergy Test (DDST, employing EDTA disc) were used to validate phenotypic identi cation of carbapenemase production in these resistant isolates. In MHT, a susceptible lawn of E. coli was spread across the plate with a meropenem disk. The expected zone of inhibition was streaked with positive controls, negative controls, and test strains. Diffusible carbapenemase, which appears as an indentation in the zone of inhibition, protects otherwise vulnerable E. coli from being killed. In DDST, enhancement of the inhibition zone (by > 5 mm in the area between meropenem disc and the EDTA disc in comparison with the zone of inhibition on the far side of the drugs) was considered to be a positive result. All of the isolates were maintained at 80°C in nutritional broth containing 7.5 percent (v/v) glycerol. These stocked carbapenem-resistant isolates were sub-cultured on blood agar prior to undergo whole-genome sequencing.

Whole-genome sequencing
Genomic DNA of pathogens were isolated from a total of 17 freshly sub-cultured colonies of carbapenem-resistant bacteria using DNeasy Blood and Tissue Kit (Qiagen, Catalogue # 69505), in accordance with the manufacturer's recommendations. DNA concentration of the puri ed DNA was calculated on Qubit (Thermo Fischer Scienti c), using Qubit™ 1X dsDNA HS Assay Kit (Invitrogen, Catalogue # Q33231). The genomic DNA was stored at -20°C. The Illumina MiSeq (Illumina, San Diego, CA, 107 USA) technology was used to sequence these DNA isolates. Using the Nextera DNA Flex Library Prep Kit, a MiSeq shortread sequencing library was created with 1ng pure DNA (Illumina, Cat. No. 20018704). Bead-linked transposomes (BLTs) were employed in this kit to facilitate simultaneous DNA fragmentation and Illumina sequencing primer tagging (Tagmentation). These sequencing ready DNA fragments were ampli ed by PCR and indexes (for sample identi cation) and adapters were further added to them (using Nextera DNA CD Indexes, Cat. No.20018707). For each library, the normalization was performed to bring them to a standard concentration of 4nM. Sequencing ready fragments were further washed and pooled. The average size of the libraries was found to be around 550bp that was calculated with the help of Agilent Bioanalyzer 2100 using High Sensitivity DNA Assay Kit (5067-4626). The libraries were next loaded on to the sequencer Illumina MiSeq 500 platform for sequencing by synthesis/bridge ampli cation. Casava (v.1.8.2) was used to de-multiplex the output data les and convert them to FASTQ les (Illumina, Inc, USA).

Detection of resistance genes
The RGI-CARD (Comprehensive Antibiotic Resistance Database) and Pathogenwatch were used to predict the resistance genes in the assembled Gramnegative bacteria genomes (Center for Genomic Pathogen Surveillance databases). We utilized 50 percent sequence identity and 70 percent query coverage as cut-off criteria. The acquired antimicrobial resistance genes and genes associated with chromosomal point mutations were identi ed using the ResFinder webserver 3.0 (https://cge.cbs.dtu.dk/services/ResFinder/).

Demographic distribution
The isolates found were from different age group patients ranging from 6 years to 76 year old. Isolates from males 76.5% (13/17) were more in number as compared to isolates from female 23.5% (4/17) inpatients. Maximum numbers of these carbapenemase producing isolates were found from Medicine ICU 47% (8/17) followed by surgery ward 35.3% (6/17) admitted patients. Isolates generating carbapenemase were found in the following clinical specimens: carbapenem-resistance rate of isolated GNB is given in Table 1.

Co-resistance genes
It has been observed that the majority of GNB isolates had more than one β-lactams carbapenem-resistance gene, where co-resistance genes were mostly found in E. coli and K. pneumoniae isolates. Among, co-resistance of two genes namely "bla CTX-M + bla NDM " (1/17), and "bla CTX-M + bla SHV " (1/17), was observed in K. pneumoniae and E. coli, respectively, whereas co-resistance of three genes "bla CTX-M + bla NDM + bla OXA " was commonly observed in both K.
pneumoniae and E. coli. Likewise, "bla CTX-M + bla TEM + bla OXA " and "bla CTX-M + bla NDM + bla SHV " pattern of three genes was found in K. pneumonia, whereas "bla CTX-M + bla NDM + bla TEM" co-resistance genes pattern was found exclusively in E. coli. Co-resistance of four genes namely "bla CTX-M + bla NDM + bla SHV + bla TEM " was detected in 3 species (K. pneumoniae, E. coli and A. baumannii), whereas "bla CTX-M + bla NDM + bla OXA + bla TEM " was observed in E. coli only. Interestingly, co-resistance of ve genes "bla CTX-M + bla NDM + bla SHV + bla OXA + bla TEM " was found in 3 isolates of E. coli. A high co-resistance rate in GNB may provide further insight into the epidemiology of resistance acquisition. Table 4 shows the distribution of co-resistance genes among different Gramnegative bacteria. Analysis revealed β-lactams co-resistance genes with bla NDM in 12 (70.6%) of carbapenemase-producing isolates.

Genotype and phenotype correlations
The ndings of the genotypic method (WGS) were compared with phenotypic method (MHT/DDST) and observed that the concordance between genotypic and phenotypic was 100% for β-lactams carbapenem-resistance genes ( Table 5). We also observed resistance for ceftazidime, piperacillintazobactum, meropenem and imipenem antimicrobials genotypically and phenotypically for E. coli, K. pneumoniae, A. baumannii and P. aeruginosa strains. These ndings concluded a strong correlation between genotypic and phenotypic correlations among GNB isolates.

Discussion
Carbapenemase-producing Gram-negative bacterial (GNB) infection is increasing worldwide including India where it is a cause for major concern (Meletis, 2015). Carbapenems are currently the medicine of choice for treating serious hospital-acquired infections, however carbapenem-resistance has been reported very high in India and the Indian subcontinent in recent decades. The accurate identi cation of carbapenemase-producing microbes is interesting, and it necessitates phenotypic and genotypic studies to identify all genes linked to carbapenemase-production.
This study was carried out in 1060 bedded super-specialty tertiary care hospital in Bihar, India. The majority of patients were referred after using antimicrobials. In addition, 47% of the isolates in the study were from the intensive care unit, where patients are more prone to undergo invasive procedures. The abundance of bla CTX-M genes in different species suggests horizontal gene transfer is occurring now or in the past. For example, E. coli from healthy food animals can be key repositories of bla genes and may contribute to the spread and transmission of these β-lactamase genes, and lateral transfer of resistance genes between animals and humans. In contrast, bla NDM and bla OXA was observed to be highly prevalent in GNB isolates in Tamil nadu (Nachimuthu et al., 2016) as well as Mumbai (Kazi et al., 2015). Likewise, bla VIM (Okoche et al., 2015), and bla IMP (Mushi et al., 2014) was observed to be the most common gene in CR-GNB isolates. We found 3 types of carbapenemase gene namely bla NDM (4.65%), bla OXA (6.90%) and bla DIM (1.15%) in our study. Though these carbapenemase gene are not common, yet it is concerning because it can be resistant to even more number of antibiotics. Carbapenem-resistant isolates may exhibit multidrug-resistance as they possessed bla NDM-1 or bla NDM-5 , along with other antimicrobial-resistance factors. Among, subclass B1 metallo-betalactamase (bla NDM ), higher prevalence of bla NDM-5 was detected in GNB isolates which may confers higher resistance against carbapenems than bla NDM-1 as reported earlier (Hornsey et al., 2011). Varying geographic locations, different levels of healthcare institutions engaged, different levels of exposure to healthcare environments, antibiotic use, and antibiotic stewardship procedures may all contribute to these disparities.
In our research, multiple co-existence genes within the same isolate were observed, where β-lactams co-resistance genes with bla NDM was found in 70.6% of carbapenemase-producing isolates. Carbapenem co-resistance retain genes that make them resistant to other antibiotics, making them multi-drug resistant and it threatens global antibiotic chemotherapy, patients' recovery, and the economy (Kopotsa et al., 2020); (Mmatli et al., 2020). Resistance to carbapenem can be caused by the presence of bla NDM , bla CTX-M , bla TEM , bla SHV , and bla OXA gene family as well as impermeability (Rawat & Nair, 2010). This is particularly problematic in India, where β-lactams carbapenemase prevalence is quite high. This suggests that the detection of carbapenemase-encoding genes is an important index for phenotype in CR-GNB isolates. In our study, more than half of the isolates tested positive for multidrug resistance (MDR) to the most commonly used antibiotics. The acquisition and horizontal transfer of resistant genes from a variety of sources, including pathogenic bacteria, the environment, and animals, could be the main causes of resistance's uncontrolled expansion (Fair & Tor, 2014). Poor infection management in the country might be another reason for the high incidence of MDR and the acquisition of resistance genotypes, necessitating immediate action to combat the burgeoning AMR.

Conclusions
Carbapenemase-producing bacteria were detected in abundance in the Bihar region. The present study highlights the overwhelming threat of the β-lactam group to explore the mechanism of carbapenem-resistance in GNB. A high co-resistance rate in multidrug resistant GNB was observed which may provide further insight into the epidemiology of resistance acquisition. The prevalence of carbapenemase-encoding genes (bla NDM , bla OXA and bla DIM) found from this study is a rising threat in India which requires immediate attention from the healthcare perspective. Therefore, strict antibiotic policy to prevent the misuse of antibiotics should be imposed to control the drug-resistance in India.        Isolates-wise prevalence and distribution of carbapenemase genes in Gram-negative bacteria

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. FinalSupplementaryScirep91121.pdf