Clinical and Genomic Evolution of Carbapenem-Resistant Klebsiella pneumoniae Bloodstream Infections over Two Time Periods at a Tertiary Care Hospital in South India: A Prospective Cohort Study.

INTRODUCTION: The objective of this study was to examine the evolution of carbapenem-resistant Klebsiella pneumoniae (CRKp) infections and their impact at a tertiary care hospital in South India. METHODS: A comparative analysis of clinical data from two prospective cohorts of patients with CRKp bacteremia (C1, 2014-2015; C2, 2021-2022) was carried out. Antimicrobial susceptibilities and whole genome sequencing (WGS) data of selected isolates were also analyzed. RESULTS: A total of 181 patients were enrolled in the study, 56 from C1 and 125 from C2. CRKp bacteremia shifted from critically ill patients with neutropenia to others (ICU stay: C1, 73%; C2, 54%; p = 0.02). The overall mortality rate was 50% and the introduction of ceftazidime-avibactam did not change mortality significantly (54% versus 48%; p = 0.49). Oxacillinases (OXA) 232 and 181 were the most common mechanisms of resistance. WGS showed the introduction of New Delhi metallo-ß-lactamase-5 (NDM-5), higher genetic diversity, accessory genome content, and plasmid burden, as well as increased convergence of hypervirulence and carbapenem resistance in C2. CONCLUSIONS: CRKp continues to pose a significant clinical threat, despite the introduction of new antibiotics. The study highlights the evolution of resistance and virulence in this pathogen and the impact on patient outcomes in South India, providing valuable information for clinicians and researchers.

India-discovered levonadifloxacin & alalevonadifloxacin: A review on susceptibility testing methods, CLSI quality control and breakpoints along with a brief account of their emerging therapeutic profile as a novel standard-of-care.

BACKGROUND: Levonadifloxacin (intravenous) and alalevonadifloxacin (oral prodrug) are novel antibiotics based on benzoquinolizine subclass of fluoroquinolone, licensed for clinical use in India in 2019. The active moiety, levonadifloxacin, is a broad-spectrum antibiotic with a high potency against methicillin-resistant Staphylococcus. aureus, multi-drug resistant pneumococci and anaerobes. OBJECTIVE: This review, for the first time, critically analyses the antimicrobial susceptibility testing methods, Clinical Laboratory & Standards Institute (CLSI)-quality control of susceptibility testing and breakpoints of levonadifloxacin. Further, the genesis, discovery and developmental aspects as well as therapeutic profile of levonadifloxacin and alalevonadifloxacin are briefly described. CONTENTS: In order to aid the scientific and clinician communities with a single comprehensive overview on all the key aspects of levonadifloxacin and alalevonadifloxacin, the present article covers the reference MIC and disk diffusion methods for levonadifloxacin susceptibility testing that were approved by CLSI and the reference ranges for quality control strains published in the CLSI M100 document. The breakpoints of levonadifloxacin were derived in concordance to US FDA, European Committee on Antibiotic Susceptibility Testing (EUCAST) and CLSI approaches. Further, the article provides a brief account of challenges encountered during the discovery stages of levonadifloxacin and alalevonadifloxacin, activity spectrum and safety benefits accruing from structural novelty-linked mechanism of action. Further, the review also covers in vitro and in vivo activities, registrational clinical studies and patient-friendly features of levonadifloxacin/alalevonadifloxacin. Cumulatively, levonadifloxacin has a potential to offer a long awaited new standard-of-care treatment for the resistant Gram-positive bacterial infections.

Genome sequencing and molecular characterisation of XDR Acinetobacter baumannii reveal complexities in resistance: Novel combination of sulbactam-durlobactam holds promise for therapeutic intervention.

Emerging nosocomial strains of Acinetobacter baumannii are of recent concern as they are expressing extensive drug resistance (XDR). Using whole-genome sequencing and molecular characterisation analysis, the current study reveals the presence of carbapenemase genes in 92.86% of studied Indian isolates. These included blaOXA-51 , blaOXA-23 , blaOXA-58 , and blaNDM genes, with over a third expressing dual carbapenemase genes. As per the MLST scheme, IC2Oxf /CC2Pas was the predominant clone, with 57.14% isolates belonging to this lineage. The presence of these carbapenemase genes resulted in sulbactam (SUL) resistance (MIC: 16-256 µg/ml) in all of the studied isolates. The efficacy of durlobactam (DUR), a novel ß-lactamase inhibitor that also inhibits PBP2 was assessed through in silico intermolecular interaction analysis. Several nonsynonymous single nucleotide polymorphisms were identified in PBP2 (G264S, I108V, S259T) and PBP3 (A515V, T526S) sequences. Minimal variations were recorded in the protein backbone dynamics in active-site motifs of wild-type and mutants, which correlated with negligible binding energy fluctuations for the PBP3-SUL (-5.85 ± 0.04 kcal/mol) and PBP2-DUR (-5.16 ± 0.66 kcal/mol) complexes. Furthermore, higher binding affinities and low inhibition constants were noted in OXA23-DUR (-7.36 kcal/mol; 4.01 µM), OXA58-DUR (-6.44 kcal/mol; 19.07 µM), and NDM-DUR (-6.82 kcal/mol; 10.01 µM) complexes when compared with the conventional drugs avibactam and aztreonam. Stable interaction profiles of DUR with carbapenemases can possibly restore SUL activity against both PBP3WT and PBP3MTs . The study establishes the efficacy of the novel SUL-DUR combination as a successful treatment strategy in combating emerging XDR strains of A. baumannii.

Aerobactin Seems To Be a Promising Marker Compared With Unstable RmpA2 for the Identification of Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae: In Silico and In Vitro Evidence.

Background: The incidence of hypervirulent (hv) carbapenem-resistant (CR) Klebsiella pneumoniae (Kp) is increasing globally among various clones and is also responsible for nosocomial infections. The CR-hvKp is formed by the uptake of a virulence plasmid by endemic high-risk clones or by the uptake of plasmids carrying antimicrobial resistance genes by the virulent clones. Here, we describe CR-hvKp from India belonging to high-risk clones that have acquired a virulence plasmid and are phenotypically unidentified due to lack of hypermucoviscosity. Methods: Twenty-seven CRKp isolates were identified to possess rmpA2 by whole-genome sequencing; and resistance and virulence determinants were characterized. By in silico protein modeling (and validation), protein backbone stability analysis, and coarse dynamics study, the fitness of RmpA, RmpA2, and aerobactin-associated proteins-IucA and IutA, were determined to establish a reliable marker for clinical identification of CR-hvKp. Results: The CR-hvKp belonged to multidrug-resistant (MDR) high-risk clones such as CG11, CG43, ST15, and ST231 and carried OXA-232 as the predominant carbapenemase followed by NDM. The virulence plasmid belonged to IncHI1B replicon type and carried frameshifted and truncated rmpA and rmpA2. This resulted in a lack of hypermucoviscous phenotype. However, functional aerobactin was expressed in all high-risk clones. In silico analysis portrayed that IucA and IutA were more stable than classical RmpA. Furthermore, IucA and IutA had lower conformational fluctuations in the functional domains than the non-functional RmpA2, which increases the fitness cost of the latter for its maintenance and expression among CR-hvKp. Hence, RmpA and RmpA2 are likely to be lost among CR-hvKp owing to the increased fitness cost while coding for essential antimicrobial resistance and virulence factors. Conclusion: Increasing incidence of convergence of AMR and virulence is observed among K. pneumoniae globally, which warrants the need for reliable markers for identifying CR-hvKp. The presence of non-functional RmpA2 among high-risk clones highlights the significance of molecular identification of CR-hvKp. The negative string test due to non-functional RmpA2 among CR-hvKp isolates challenges phenotypic screening and faster identification of this pathotype. This can potentially be counteracted by projecting aerobactin as a stable, constitutively expressed, and functional marker for rapidly evolving CR-hvKp.

Molecular characterization of colistin-resistant Klebsiella pneumoniae & its clonal relationship among Indian isolates.

Background & objectives: Klebsiella pneumoniae (KP), a common cause of invasive infections, is often extensively drug resistant in India. At present, studies on resistance mechanism and clonal relationship of KP from India are limited. The present study was undertaken to determine the resistance mechanism and clonal relationship of colistin-resistant isolates obtained from various specimens. Carbapenemases were also determined since the isolates were carbapenem resistant. Methods: Sixty five isolates from blood, exudates and respiratory specimens collected between 2016 and 2017 were studied. Colistin minimum inhibitory concentration (MIC) was performed by broth-micro dilution method. Multiplex PCR was carried out to determine carbapenemases. Targeted sequencing was performed to determine mutations in mgrB, phoP, phoQ and multilocus sequence typing was performed to determine the prevalent clones. Results: Colistin MIC ranged from 4 to 256 µg/ml. SHV, TEM and CTX-M were co-produced in 60 per cent and OXA48-like in 71 per cent. Thirteen isolates had mutations in mgrB. Mutations included a premature stop codon at 21st amino acid, the presence of insertion sequences such as IS903, IS Kpn 14 and ISK pn 26; and elongation of mgrB. Novel mutations were also observed among phoP and phoQ genes. Colistin resistance due to mcr genes was absent. Fifteen clonal types were seen with ST231, ST14 and ST2096 being predominant. Interpretation & conclusions: This study revealed the changing trend of carbapenem resistance mechanism predominantly to OXA48-like from NDM. Known mgrB mutations and novel mutations in phoP and phoQ were detected. There was no plasmid-mediated colistin resistance. ST14 and ST231 were international clones associated with carbapenem resistance. Colistin-resistant KP was of diverse clones with predominantly ST231, ST14 and ST2096.

Specimen Collection, Processing, Culture, and Biochemical Identification of Acinetobacter spp.

Specimen collection and processing is an important aspect of clinical microbiology laboratory. The reports are dependent on the quality of the specimen and the time between the collection and processing. Appropriate methodology needs to be followed for the collection, amount, type, labeling, transportation, and processing of the specimens especially for organism like Acinetobacter species. Various biochemical tests are used for identification of various organisms. Such identification depends on the ability of organisms to produce certain enzymes or to utilize certain compound to be identified by biochemical tests.

Is it safe to do a single-stage implant exit and primary hip replacement? clinical and microbiological profiling.

Background: A single-stage implant revision for failed fixation of proximal femoral fractures is performed only when there is no evidence of infection. Else, a two-staged revision is preferred - where the definitive revision surgery is done a few months after the implant exit. This study aims to audit the safety and incidence of culture positivity in single-stage revisions. Materials and Methods: Forty one of 284 patients that presented over the last 12 years for implant exchange of the hip, had a single stage revision surgery for failed fixation of a fracture of the hip, as there was no obvious evidence of infection at the time of implant exit. Results: Micro-organisms were grown in 51% of the 41 hips. 76% were gram positive, of which 63% were Coagulase negative staphylococci (CoNS). 50% of CoNS and 75% of S. aureus were resistant to oxacillin, but susceptible to Vancomycin. Of the gram negative organisms, 2 (Enterobacter sp) were resistant to carbapenam, while others were susceptible. Preoperative ESR and CRP, individually, had low specificity - 50% for ESR >30mm at 1 hour and 62% for CRP>10. The combined use of ESR > 30mm and CRP>10 increased the specificity to 90%. 12% of the patients had immediate postoperative complications that required a wash out in theatre. The long term clinical follow up of these patients is limited. Conclusion: This study suggests that implant exit and simultaneous arthroplasty for failed fracture fixation should be done with caution due to the high possibility of infection. It may be prudent to opt for a 2 stage revision.

First genome report on novel sequence types of Neisseria meningitidis: ST12777 and ST12778.

OBJECTIVES: Neisseria meningitidis is an important causative agent of meningitis and/or sepsis with high morbidity and mortality. Baseline genome data on N. meningitidis, especially from developing countries such as India, are lacking. This study aimed to investigate the whole genome sequences of N. meningitidis isolates from a tertiary care centre in India. METHODS: Whole-genome sequencing was performed using an Ion Torrent™ Personal Genome Machine™ (PGM) with 400-bp chemistry. Data were assembled de novo using SPAdes Genome Assembler v.5.0.0.0. Sequence annotation was performed through PATRIC, RAST and the NCBI PGAAP server. Downstream analysis of the isolates was performed using the Center for Genomic Epidemiology databases for antimicrobial resistance genes and sequence types. Virulence factors and CRISPR were analysed using the PubMLST database and CRISPRFinder, respectively. RESULTS: This study reports the whole genome shotgun sequences of eight N. meningitidis isolates from bloodstream infections. The genome data revealed two novel sequence types (ST12777 and ST12778), along with ST11, ST437 and ST6928. The virulence profile of the isolates matched their sequence types. All isolates were negative for plasmid-mediated resistance genes. CONCLUSIONS: To the best of our knowledge, this is the first report of ST11 and ST437 N. meningitidis isolates in India along with two novel sequence types (ST12777 and ST12778). These results indicate that the sequence types circulating in India are diverse and require continuous monitoring. Further studies strengthening the genome data on N. meningitidis are required to understand the prevalence, spread, exact resistance and virulence mechanisms along with serotypes.