Molecular mechanisms of colistin- and multidrug-resistance in bacteria among patients with hospital-acquired infections

Aim: The increasing burden of resistance in Gram-negative bacteria (GNB) is becoming a major issue for hospital-acquired infections. Therefore, understanding the molecular mechanisms is important. Methodology: Resistance genes of phenotypically colistin-resistant GNB (n = 60) were determined using whole genome sequencing. Antimicrobial susceptibility patterns were detected by Vitek®2 & broth microdilution. Results: Of these phenotypically colistin-resistant isolates, 78% were also genetically resistant to colistin. Activation of efflux pumps, and point-mutations in pmrB, and MgrB genes conferred colistin resistance among GNB. Eight different strains of K. pneumoniae were identified and ST43 was the most prominent strain with capsular type-specific (cps) gene KL30. Discussion: These results, in combination with rapid diagnostic methods, will help us better advice appropriate antimicrobial regimens.

The increasing antimicrobial resistance among bacteria causing hospital-associated infections (HAIs) is a threat to global health that increases morbidity and mortality among hospitalized patients.Gram-negative bacteria (GNB) are the most prominent causative agents of bacterial HAIs in such patients [1].In the past few decades, bacteria acquired various resistance mechanisms to almost all available antibiotics including beta-lactams, fluoroquinolones, tetracycline and aminoglycosides [2].Therefore, colistin and tigecycline remain effective antibiotics and have become the last resort of treatment for multidrug-resistant (MDR) GNB.In many countries, tigecycline has not been registered; leaving colistin as the only treatment option against multi-drug-resistant organisms.MDR organisms including, but not limited to, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae are developing a low level of resistance against colistin.Thus, GNB co-resistant to carbapenems, aminoglycosides, polymyxins and tigecycline (CAPT-resistance) are causing havoc [3][4][5].Little is known about the development of a mechanism of resistance against colistin.Some bacteria, including A. baumannii, P. aeruginosa, and Enterobacteriaceae members like Klebsiella spp., Enterobacter spp.and Escherichia coli have acquired resistance against colistin.However, other bacteria, including Serratia spp., Proteus spp.and Burkholderia spp.are naturally resistant to this antibiotic.There are very limited data on acquired resistance to colistin.In gram-negative organisms such as Salmonella enteric and P. aeruginosa, modification of lipid A, a component of LPS, is considered to be the mechanism of colistin resistance.The addition of 4-amino-4-deoxy-l-arabinose (Ara4N) or/and phosphoethanolamine to lipid A removes the negative charge of lipid, lowering the affinity of positively charged colistin and thus contributing to the development of the colistin resistance [6].Insertion of ISAba 11 into lipid biosynthesis genes like lpxA or lpxC resulted in the complete loss of LPS production and a high level of colistin resistance in A. baumannii [7].Also, our finding suggest that, a mutation in the PmrAB two-component system like at least one amino acid change in PmrB or up-regulation of pmrA and pmrB resulted in colistin resistance.Modification of lipid A with the addition of phosphoethanolamine to lipid A mediated by the pmrAB two-component regulatory system also contributed to the colistin resistance [8].In Enterobacteriaceae, changes in the regulatory loci pmrA and phoP are responsible for colistin resistance [9].The pmrA locus encodes a two-component system, PmrA-PmrB, and a putative membrane protein PmrC [10,11].The phoP locus encodes a distinct two-component system, PhoP-PhoQ, that governs resistance to several amphipathic antimicrobial peptides and was recently shown to also be required for resistance to colistin [12,13].In KPC-producing K. pneumoniae (KPC-KP), alteration of the mgrB gene resulted in colistin resistance [14].
Although a major route of adaptive polymyxin resistance involves loss of lipopolysaccharide and the multicomponent regulatory genes PmrAB, PhoPQ, ParRS and CprRS, the precise molecular details of these resistance mechanisms have also remained unclear.
Since colistin is the last resort of treatment for multidrug-resistant organisms, colistin resistance is a serious problem, especially in otherwise healthy trauma patients.Thus, we conducted the present study to identify the molecular mechanisms of colistin-and multidrug-resistance in GNB among trauma patients by whole genome sequencing.

Type of study
This prospective observational study was conducted from January 2020 to March 2021.

Sample collection
The clinical samples of trauma patients sent to the microbiology laboratory during the study duration who were resistant to colistin were included in the study.
The patients admitted in trauma centre are mainly accidental cases.Our microbiology laboratory receives different samples viz blood, urine, sputum, BAL samples, etc. for post-trauma microbiological investigations to advise antimicrobial regime & patient treatment.As a part of routine diagnostic tests these samples were processed as per standard microbiological procedures [18].Blood samples were collected in BacT/ALERT aerobic blood culture bottles (bioMérieux, India), incubated, and monitored regularly using the BacT/ALERT system (bioMérieux, India).All bottles which signaled positive were removed from the instrument, Gram-stained and sub-cultured on blood agar and MacConkey agar.Urine samples were cultured on Cystine Lactose Electrolyte Deficient (CLED) agar.Phenotypic identification was confirmed with a Vitek R 2 ID-GNB card (bioMérieux, India).

Whole-genome sequencing
Genomic DNA was isolated from multiple sources including tissue, blood, respiratory samples, urine and water (environmental), and was quantified using a Qubit fluorometer (Life Technologies, USA).The quality and quantity of DNA were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, MA, USA) and PFGE gel electrophoresis, respectively.The qualified DNA was cut into fragments by restriction enzymes.DNA fragments were end-repaired using deoxyadenosine bases before ligation with Illumina indexed adapters,  amplified for 10 cycles of PCR, and sequenced employing v2 and v3 chemistry with paired-end 2 × 151 bp reads on NovaSeq 6000 (Illumina, USA).Output data files were de-multiplexed and transformed with Casava v.1.8.2.into FASTQ files (Illumina, Inc, USA).

Identification of resistance genes
The resistance genes in the assembled genomes were identified using the databases of antimicrobial resistance genes including cards Resistance Gene Identifier (RGI) software (RGI 4.2.0 and CARD 2.0.3 [24,25], NCBI AMR Finder Plus [26,27] and PathogenWatch v3.12.7 [28] AMRFinderPlus utilized NCBI Bacterial Antimicrobial Resistance Reference Gene Database (BioProject PRJNA313047) to find AMR-specific genes and proteins.RGI server was used to predict resistome(s) from nucleotide data based on homology and SNP models, where the 'perfect and strict hits only' criteria were chosen for the prediction.The ResFinder (webserver 3.0) was used to predict acquired genes and chromosomal point mutations mediating antimicrobial resistance in the DNA sequence of bacteria [29,30].
The WGS analyses revealed 8 different strains of K. pneumoniae; ST43 was the most prominent strain with capsular type-specific (cps) gene KL30.The frequency of different strains and capsular genes were shown in Table 3.
WGS analyses revealed that 47/60 isolates were genotypically colistin-resistant; K. pneumoniae (n = 33/33), A. baumannii (n = 6/17), E. cloacae (n = 3/3) and P. aeruginosa (n = 5/6).Table 4 details the sensitivity and specificity of genotype versus phenotype resistance pattern among K. pneumoniae.We also evaluated genotypic and phenotypic correlation and found that most of the isolates showed resistance both genetically and phenotypically except for Amikacin and Trimethoprim/sulfamethoxazole.encoding resistance.We evaluated for correlation and did not find any correlation between the resistance gene and capsule type (r = 0.056, p = 0.203); capsule and colistin (r = 0.75, p = 0.13).

Carbapenem-resistance genes
Twelve isolates of K. pneumoniae harbored the carbapenem-resistant gene OXA-181.Seven isolates harbored the OXA-232 carbapenem-resistant gene.Twelve isolates had the combination of NDM and OXA in different allelic forms.The single isolate was resistant due to the presence of the NDM-1 gene alone.

Sulfonamide resistance gene
Nineteen isolates harbored the sulphonamide resistance gene; all attributed to the presence of Sul gene.

Discussion
A major cause of concern is the increasing number of reports of CAPT resistance across the globe [31].Moreover, there is a scarcity of clinical data to guide the treatment of MDR-GNB, and are based exclusively on small case series and a few case reports.To aim for timely and targeted treatment of patients with infections by such MDR GNB, a better understanding of resistance mechanisms is essential.Although at present, a synergistic combination of older agents for example-fosfomycin-or polymyxin-based synergistic combinations, may be the last resort option, their use against CAPT-resistant GNB requires further investigation [32].
Our study focused on the most problematic species, namely K. pneumoniae, P. aeruginosa and A. baumannii among others.WGS analyses showed that only 78% of the phenotypically colistin-resistant isolates were genotypically colistin-resistant.Thus, the phenotypic tests probably do not reflect the actual resistance burden.Previous studies conducted at our center also highlighted the possibilities of a complex combination of mutations that led to colistin resistance [33,34].Activation of efflux pumps, and point-mutations in pmrB, and MgrB genes conferred colistin resistance among these GNB.In addition to genotypic analyses of genes conferring colistin resistance in these isolates, we studied the genes encoding the most predominant resistance enzymes among these MDR-GNB viz.β-lactams resistance genes, β-lactams + inhibitor resistance genes, ESBL resistance genes, carbapenem-resistance genes and sulfonamide resistance gene.As the previous studies reported a high proportion of ESBL production from the isolates of middle east Asia [35], this study also found a high proportion of isolates were positive for ESBL.Hence, continuous and large-scale surveillance of molecular mechanisms is required and emphasis should be laid on culture-based and synergistic antimicrobial treatment regimes.
The limitation of our study remains the limited number of isolates studied at a single centre.More such data from different geographies would help gain more insights.Until new drugs become widely available in clinical practice, more research encompassing the study of molecular epidemiological mechanisms of resistance, pharmacokinetics & pharmacodynamics, and outcome studies is crucial.This understanding becomes particularly advantageous if there is access to accurate & rapid laboratory methods that can determine the molecular mechanisms of resistance.Many such methods are available, including lower-cost phenotypical assays which are suitable for microbiology laboratories of any capacity.However, PCR tests and high-throughput diagnostic tests would not only be expensive but also require expert and trained personnel to interpret such data.These data will guide the selection of appropriate antimicrobials for the management of MDR GNB, especially the CAPT-resistant GNB.

Conclusion
This study found K. pneumoniae to be the commonest organism with ST43 being the most prominent strain encoding capsular type-specific (cps) gene KL30.All the organisms contained several antimicrobial resistant genes, particularly genes for antibiotic efflux pumps, pmrB and MgrB.It appears that these complementary mutations conferred colistin resistance in these organisms.Since colistin is the 'last resort' antimicrobial, uniform surveillance of molecular mechanisms would enable adoption of the synergistic antimicrobial treatment regimes.

Summary points
• In this study, we found that 78% of the phenotypically colistin-resistant isolates were genotypically colistin resistant.• Thus, the phenotypic tests probably do not reflect the actual resistance burden.
• Activation of efflux pumps, and point-mutations in pmrB, and MgrB genes conferred colistin resistance among these Gram-negative bacteria (GNB).• We also elucidated the genes encoding the most predominant resistance enzymes among these MDR-GNB viz.

†
Others samples included fluid, bile and central venous pressure tip.

Table 1 .
Distribution of source of samples and pathogens.

Table 2 .
Phenotypic antimicrobial resistance pattern of different pathogens.

Table 3 .
Frequency of different strains and capsular specific type genes.

Table 4 .
The sensitivity and specificity of genotype versus phenotype resistance pattern among K. pneumoniae.

Table 5 .
Details of colistin resistance isolates and genes encoding resistance.

Table 6 .
Genes encoding for resistance to different classes of antimicrobials.