Prevalence of Colistin Resistance among MDR Gram Negative Bacteria from Tertiary Care Hospitals

Abstract

Antibiotic resistance among Gram-negative bacterial pathogen is a global menace and is considered, a silent pandemic associated with higher morbidity and mortality worldwide. With an increase in antimicrobial resistance (AMR), and few new available drug, older toxic drug “colistin” is consider as last-resort for the treatment of these resistant pathogens. Gram-negatives, overcome the lethal effects of this drug via modification of outer membrane (OM). The mechanism responsible for colistin resistance has not been deciphered completely yet. However, resistance is thought to be associated with the plasmid-mediated carriage of mobile colistin-resistant genes (mcr), or chromosomal alterations in lipopolysaccharide (LPS) modification genes, or efflux pump. These modifications affect colistin binding to its target site or extrusion of drug. The emergence of colistin resistance and its gradual increase in clinical Gram-negatives, compelled research community to comprehend and explore other mechanisms responsible for resistance. Increase in colistin resistant (CR) Gram-negatives have been reported from multiple countries and recent studies from Pakistan highlighted the emerging resistance to colistin in Gram-negatives with detection of mcr gene. Based on previous reports from Pakistan, where low mcr gene prevalence with much higher phenotypic resistance indicated that there are other possible mechanisms for resistance in MDR Gram-negatives which need to be elucidate for better understanding of this emerging threat. Hence, there is a need for a genome-based study to gain insight of underlying colistin resistance mechanism among Gram-negatives in Pakistan. This study aims to investigate the prevalence of colistin resistance among Gram-negative bacteria and to decipher molecular mechanism of resistance in mcr negative CR MDR Gram negative bacteria. A total 566 clinical specimens were collected from patients visiting 4 major tertiary care hospitals which were, Military Hospital (MH), Rawalpindi, Pakistan Institute of Medical Science Hospital Islamabad (PIMS), Federal Government Polyclinic Hospital Islamabad (PGMI), and Capital Development Authority (CDA) Capital Hospital from Islamabad. Isolation and identification of Gram-negative bacteria was carried out using standard cultural, biochemical techniques and API10S kit. Phenotypic colistin resistance was evaluated using modified broth microdilution method and Minimum inhibitory concentration (MIC) were interpreted as per “European Committee on Antimicrobial xviii Susceptibility Testing” (EUCAST) guidelines. Antimicrobial susceptibility profiling of CR Gram-negatives against a panel of 10 commonly used antibiotics was done by using Kirby Bauer disc diffusion assay. The inhibition zone breakpoints were interpreted as per “Clinical Laboratory and Standard Institute” (CLSI) guidelines. For detection of plasmid mediated mcr genes, polymerase chain reaction (PCR) approach was used. For the understanding of other potential molecular mechanisms of resistance, whole genome sequencing (WGS) of selected mcr negative Gram-negative isolates was carried out. Whole genome data analysis was done using various in silico tools and online databases which includes Bacterial and Viral Bioinformatics Resource Center (BV-BRC), Comprehensive Antimicrobial Resistance Database (CARD), Pathogenwatch, Pasteur and ResFinder Database. Phage red recombinase system was used to knock out transporter gene (YjiJ) using E. Coli UTI89 to assess this novel approach to mitigate resistance gene. A total 528 Gram-negatives were identified from 566 collected clinical specimens, out of which 70% were Enterobacteriaceae. The most predominant member of Enterobacteriaceae was E. coli (46.6%), followed by K. pneumoniae (43%). In case of non-Enterobacteriaceae, A. baumannii (50.9%), and P aeruginosa (37.8%) were dominant. Higher number of both Enterobacteriaceae and non-Enterobacteriaceae isolates were recovered from male patients, and from age group III. Majority of the Enterobacteriaceae members were isolated from urine specimens while in case of non-Enterobacteriaceae, pus and blood were the dominant specimen type. Prevalence of phenotypic colistin resistance among Gram negative was 31% (161/525). Highest colistin resistance was observed in K. pneumoniae (88/161), followed by P. aeruginosa (26/161) and A. baumannii (15/161). Among all CR, 84% displayed MDR phenotype against panel of tested antibiotics. While 74.53% resistance was observed to last line empirical drug “carbapenem”. Most of the CR Gram-negatives were from male patients (71.4%), highest percentage were from age group II (30.4%), while most common specimen type were urine (26.7%), blood (22.4%) and pus (19.9%). Carriage of plasmid-mediated colistin resistance genes was assessed among CR-Gram negative isolates (n=161). The carriage of mcr genes was low among CR-Gram negatives (12%), as percentage positivity of mcr-1, mcr-2 and mcr-3 gene were 0.6%, 9% and 5.5% respectively. Highest mcr-2 (6%) and mcr-3 (2%) gene carriage was detected among CR K. Pneumoniae followed by P. aeruginosa, and A. baumannii. xix To assess the underlying mechanism of colistin resistance, selected mcr negative CR Gram-negatives (n=23) were characterized using WGS. In all the 23 sequenced genomes, 100 non-synonymous mutations were detected in various Lipid A modification genes, of which 65 mutations were novel. Majority of the novel variations were in CR-E. coli and CR-E. hormaechei genomes. A total of 18 variations were predicted to have deleterious impact and were annotated to be preset in various crucial protein domains. Apart from carrying multiple mutations, the acquisition of putative phosphoethanolamine (PEA) and glycosyl transferase genes via phage mediated integration was also predicted in CR-Gram negative pathogens. Microbial Genome characterization further revealed the carriage of blaNDM1, blaNDM-5 and blaNDM-7 gene along with other crucial carbapenemase, Extended Spectrum β lactamase (ESBL), and AMR determinants. The potential carriage of diverse siderophores, adhesion, invasion and biofilm associated determinants were also identified in MDR CR strains depicting a potentially convergent hypervirulent and MDR genotype emergence. The acquisition of these AMR and virulent determinants might be associated with mobile genomic elements as various (Insertional sequence) IS elements, plasmid replicons, conjugative elements and CRISPR-Cas regions were predicted in the sequenced genomes. Multi Locus Sequence Typing (MLST), predicted that most of the CR-Gram-negative isolates belonged to globally distributed high risk MDR lineages. The identified STs were, ST-11 and ST-2096 in K. pneumoniae, ST-448 and ST-617 in E. coli while ST-93 in E. hormaechei. Some potentially emerging high risk MDR clonal lineages were also observed these were ST-1503, ST-208 in CR A. baumannii. S. maltophilia was identified to carry a novel ST in this study as remained unmatched with available ST in Pasteur, Center of Genomic Epidemiology (CGE) and Pathogenwatch databases. In current study, a novel therapeutic approach was used to render drug resistant strain drug sensitive strain for enhance eradication of resistant bacteria with conventional drugs. A single colony on chloramphenicol containing agar plates was detected after knock out of yjiJ transport gene, where targeted gene was replaced with chloramphenicol (CamR) selective marker gene. However, despite phenotypic inactivation and gene inactivation, a PCR based detection yjiJ gene was seen in the transformed colony which suggested possible multiple copy nature of this yjiJ transporter gene in E. coli model genome. This xx approach had limited success, however such technique for development of anti-infective therapy. The current study reported much higher phenotypic colistin resistance compared to a low plasmid mediated mcr gene carriage among Gram negative bacterial isolates pointing toward the possible role of chromosomal mechanism in resistance. This is the first study to provide a snapshot of multiple underlying chromosomal mechanisms in CR MDR Gram negative isolates, thought to be associated with LipidA modification as were having novel non-synonymous variations, with deleterious impact on crucial protein domains of Two component system (TCS) associated proteins, and transferases, putative PEA and glycosyl transferases using WGS approach. Moreover, the detection of convergent high-risk MDR+hypervirulent clonal lineages in these CR isolates reflects the emergence of potential pan drug resistant with highly pathogenic potential in clinical setting, require immediate attention. To cope this emerging threat therapeutic potential of membrane associated transporter genes needs to be explored as a possible option against MDR bacteria.