Genome analyses of colistin-resistant high-risk blaNDM-5 producing Klebsiella pneumoniae ST147 and Pseudomonas aeruginosa ST235 and ST357 in clinical settings.

BACKGROUND: Colistin is a last-resort antibiotic used in extreme cases of multi-drug resistant (MDR) Gram-negative bacterial infections. Colistin resistance has increased in recent years and often goes undetected due to the inefficiency of predominantly used standard antibiotic susceptibility tests (AST). To address this challenge, we aimed to detect the prevalence of colistin resistance strains through both Vitek®2 and broth micro-dilution. We investigated 1748 blood, tracheal aspirate, and pleural fluid samples from the Intensive Care Unit (ICU), Neonatal Intensive Care Unit (NICU), and Tuberculosis and Respiratory Disease centre (TBRD) in an India hospital. Whole-genome sequencing (WGS) of extremely drug-resitant (XDR) and pan-drug resistant (PDR) strains revealed the resistance mechanisms through the Resistance Gene Identifier (RGI.v6.0.0) and Snippy.v4.6.0. Abricate.v1.0.1, PlasmidFinder.v2.1, MobileElementFinder.v1.0.3 etc. detected virulence factors, and mobile genetic elements associated to uncover the pathogenecity and the role of horizontal gene transfer (HGT). RESULTS: This study reveals compelling insights into colistin resistance among global high-risk clinical isolates: Klebsiella pneumoniae ST147 (16/20), Pseudomonas aeruginosa ST235 (3/20), and ST357 (1/20). Vitek®2 found 6 colistin-resistant strains (minimum inhibitory concentrations, MIC = 4 µg/mL), while broth microdilution identified 48 (MIC = 32-128 µg/mL), adhering to CLSI guidelines. Despite the absence of mobile colistin resistance (mcr) genes, mechanisms underlying colistin resistance included mgrB deletion, phosphoethanolamine transferases arnT, eptB, ompA, and mutations in pmrB (T246A, R256G) and eptA (V50L, A135P, I138V, C27F) in K. pneumoniae. P. aeruginosa harbored phosphoethanolamine transferases basS/pmrb, basR, arnA, cprR, cprS, alongside pmrB (G362S), and parS (H398R) mutations. Both strains carried diverse clinically relevant antimicrobial resistance genes (ARGs), including plasmid-mediated blaNDM-5 (K. pneumoniae ST147) and chromosomally mediated blaNDM-1 (P. aeruginosa ST357). CONCLUSION: The global surge in MDR, XDR and PDR bacteria necessitates last-resort antibiotics such as colistin. However, escalating resistance, particularly to colistin, presents a critical challenge. Inefficient colistin resistance detection methods, including Vitek2, alongside limited surveillance resources, accentuate the need for improved strategies. Whole-genome sequencing revealed alarming colistin resistance among K. pneumoniae and P. aeruginosa in an Indian hospital. The identification of XDR and PDR strains underscores urgency for enhanced surveillance and infection control. SNP analysis elucidated resistance mechanisms, highlighting the complexity of combatting resistance.

Farm to table: colistin resistance hitchhiking through food.

Colistin is a high priority, last-resort antibiotic recklessly used in livestock and poultry farms. It is used as an antibiotic for treating multi-drug resistant Gram-negative bacterial infections as well as a growth promoter in poultry and animal farms. The sub-therapeutic doses of colistin exert a selection pressure on bacteria leading to the emergence of colistin resistance in the environment. Colistin resistance gene, mcr are mostly plasmid-mediated, amplifying the horizontal gene transfer. Food products such as chicken, meat, pork etc. disseminate colistin resistance to humans through zoonotic transfer. The antimicrobial residues used in livestock and poultry often leaches to soil and water through faeces. This review highlights the recent status of colistin use in food-producing animals, its association with colistin resistance adversely affecting public health. The underlying mechanism of colistin resistance has been explored. The prohibition of over-the-counter colistin sales and as growth promoters for animals and broilers has exhibited effective stewardship of colistin resistance in several countries.

Metagenomic Insight into Microbiome and Antibiotic Resistance Genes of High Clinical Concern in Urban and Rural Hospital Wastewater of Northern India Origin: a Major Reservoir of Antimicrobial Resistance.

India is one of the largest consumers and producers of antibiotics and a hot spot for the emergence and proliferation of antimicrobial resistance genes (ARGs). Indian hospital wastewater (HWW) accumulates ARGs from source hospitals and often merges with urban wastewater, with the potential for environmental and human contamination. Despite its putative clinical importance, there is a lack of high-resolution resistome profiling of Indian hospital wastewater, with most studies either relying on conventional PCR-biased techniques or being limited to one city. In this study, we comprehensively analyzed antibiotic resistomes of wastewater from six Indian hospitals distributed in rural and urban areas of northern India through shotgun metagenomics. Our study revealed the predominance of ARGs against aminoglycoside, macrolide, carbapenem, trimethoprim, and sulfonamide antibiotics in all the samples through both read-based analysis and assembly-based analysis. We detected the mobile colistin resistance gene mcr-5.1 for the first time in Indian hospital sewage. blaNDM-1 was present in 4 out of 6 samples and was carried by Pseudomonas aeruginosa in HWW-2, Klebsiella pneumoniae in HWW-4 and HWW-6, and Acinetobacter baumanii in HWW-5. Most ARGs were plasmid-mediated and hosted by Proteobacteria. We identified virulence factors and transposable elements flanking the ARGs, highlighting the role of horizontal gene transmission of ARGs. IMPORTANCE There is a paucity of research on detailed antibiotic resistome and microbiome diversity of Indian hospital wastewater. This study reports the predominance of clinically concerning ARGs such as the beta-lactamases blaNDM and blaOXA and the colistin resistance gene mcr and their association with the microbiome in six different Indian hospital wastewaters of both urban and rural origin. The abundance of plasmid-mediated ARGs and virulence factors calls for urgent AMR crisis management. The lack of proper wastewater management strategies meeting international standards and open drainage systems further complicates the problem of containing the ARGs at these hospitals. This metagenomic study presents the current AMR profile propagating in hospital settings in India and can be used as a reference for future surveillance and risk management of ARGs in Indian hospitals.

Artificial intelligence as a smart approach to develop antimicrobial drug molecules: A paradigm to combat drug-resistant infections.

Antimicrobial resistance (AMR) is a silent pandemic with the third highest global mortality. The antibiotic development pipeline is scarce even though AMR has escalated uncontrollably. Artificial intelligence (AI) is a revolutionary approach, accelerating drug discovery because of its fast pace, cost efficiency, lower labor requirements, and fewer chances of failure. AI has been used to discover several beta-lactamase inhibitors and antibiotic alternatives from antimicrobial peptides (AMPs), nonribosomal peptides, bacteriocins, and marine natural products. The significant recent increase in the use of AI platforms by pharmaceutical companies could result in the discovery of efficient antibiotic alternatives with lower chances of resistance generation.

Functional Amyloids in Pseudomonas aeruginosa Are Essential for the Proteome Modulation That Leads to Pathoadaptation in Pulmonary Niches.

Persistence and survival of Pseudomonas aeruginosa in chronic lung infections is closely linked to the biofilm lifestyle. One biofilm component, functional amyloid of P. aeruginosa (Fap), imparts structural adaptations for biofilms; however, the role of Fap in pathogenesis is still unclear. Conservation of the fap operon encoding Fap and P. aeruginosa being an opportunistic pathogen of lung infections prompted us to explore its role in lung infection. We found that Fap is essential for establishment of lung infection in rats, as its genetic exclusion led to mild focal infection with quick resolution. Moreover, without an underlying cystic fibrosis (CF) genetic disorder, overexpression of Fap reproduced the CF pathotype. The molecular basis of Fap-mediated pulmonary adaptation was explored through surface-associated proteomics in vitro. Differential proteomics positively associated Fap expression with activation of known proteins related to pulmonary pathoadaptation, attachment, and biofilm fitness. The aggregative bacterial phenotype in the pulmonary niche correlated with Fap-influenced activation of biofilm sustainability regulators and stress response regulators that favored persistence-mediated establishment of pulmonary infection. Fap overexpression upregulated proteins that are abundant in the proteome of P. aeruginosa in colonizing CF lungs. Planktonic lifestyle, defects in anaerobic pathway, and neutrophilic evasion were key factors in the absence of Fap that impaired establishment of infection. We concluded that Fap is essential for cellular equilibration to establish pulmonary infection. Amyloid-induced bacterial aggregation subverted the immune response, leading to chronic infection by collaterally damaging tissue and reinforcing bacterial persistence. IMPORTANCE Pseudomonas aeruginosa is inextricably linked with chronic lung infections. In this study, the well-conserved Fap operon was found to be essential for pathoadaptation in pulmonary infection in a rat lung model. Moreover, the presence of Fap increased pathogenesis and biofilm sustainability by modulating bacterial physiology. Hence, a pathoadaptive role of Fap in pulmonary infections can be exploited for clinical application by targeting amyloids. Furthermore, genetic conservation and extracellular exposure of Fap make it a commendable target for such interventions.

Detection of E. coli IncX1 Plasmid-Mediated mcr-5.1 Gene in an Indian Hospital Sewage Water Using Shotgun Metagenomic Sequencing: A First Report.

Colistin is used against a multitude of multidrug-resistant and extremely drug-resistant Gram-negative bacterial infections. The emergence of colistin resistance is highly concerning as it may lead to the failure of this last-resort antibiotic. Since the identification of first mobile colistin resistance (mcr) genes, several variants of mcr genes have been reported, but still there are limited studies detecting mcr genes in hospital sewage water. The prevalence of mcr in the hospital environment is extremely hazardous putting health care workers, patients, and visitors at a higher risk of exposure. It may lead to a multidrug-resistant bacterial infection outbreak. In this study, we report mcr-5.1 gene in an Indian hospital sewage water using shotgun metagenomics, as a first report. The mcr-5.1 gene in the metagenome has been explored using RGI, ABRicate, NCBI database, CARD, and Resfinder. This mcr-5.1 gene harbored by Escherichia coli is a plasmid-mediated gene carried by an IncX1 plasmid pSGMCR103. The bioinformatics analysis revealed the genetic environment of mcr-5.1 gene, which consisted of mobile element protein, ChrB domain protein, putative major facilitator superfamily type transporter, and a hypothetical protein.

Vaccines against antimicrobial resistance: a promising escape route for multidrug resistance.

Antibiotic resistance has become a global health problem requiring urgent intervention. The pace of development and frequency of transmission of antimicrobial resistance have tremendously surpassed the number of antibiotics developed in the past few decades. Emergence and transmission of multidrug-resistant genes, for example, mcr-1 and mcr-5.3, against the last resort of antibiotics has challenged the treatment options. Vaccination is a promising approach with no instance of antimicrobial resistance generation or transmission reported so far. The time required for developing a vaccine, extensive pre- and post-licensure studies and the financial constraints for the R&D has hampered vaccine development over the past few decades. Vaccine can prove to be an effective future strategy for combating antimicrobial resistance.

Patents in chemotherapy: nanoparticles as drug-delivery vehicles.

The high statistics of cancer cases and mortalities throughout the world signify the urgent need for an advanced technology to target tumor cells. Nanotechnology has emerged as one such revolutionary platform to specifically target cancerous tissues and to enhance the efficacy for various anticancer drugs. This report is a snapshot of the patents in chemotherapy from January 2010 to May 2020 involving nanoparticles, novel methods developed for their synthesis and their impact as efficient drug-delivery vehicles.

Whole-genome sequence analysis of multidrug-resistant Staphylococcus epidermidis ST35 strain isolated from human ear infection of an Iraqi patient.

OBJECTIVES: Staphylococcus epidermidis is the most common coagulase-negative Staphylococcus colonising the human skin and mucous membranes and is a major cause of nosocomial infections. Antimicrobial resistance (AMR) in S. epidermidis has increased significantly in the last few decades, threatening human health globally. METHODS: This study explored the AMR status in an S. epidermidis isolate (AK-612) from an ear infection of an Iraqi student who had undergone treatment for the same. Whole-genome sequence analysis was also performed. RESULTS: Strain AK-612 is a methicillin-resistant S. epidermidis (MRSE) isolate possessing an SCCmec type V element. Strain AK-612 belongs to sequence type 35 (ST35), clonal complex 2 (CC2), which to the best of our knowledge has not been reported previously in Iraq, having been previously reported only in Portugal and Germany. In Portugal, S. epidermidis ST35 was reported to colonise the nasal area of a bird of prey (Buteo buteo) that undergoes a migratory period extending its range from Europe to Asia. The plasmid of S. epidermidis AK-612 showed greatest identity with plasmid 1 of S. epidermidis strain PM221, which was isolated in Finland from an intramammary bovine infection. This ST35 strain may have crossed continental boundaries and expanded its occurrence in animals and humans. CONCLUSION: This is a matter of serious concern as the dissemination of multidrug-resistant S. epidermidis in human infections is a major hindrance for the treatment of these infections. Transmission of this isolate across continental boundaries will make infection control a difficult task.