Miniaturised broth microdilution for simplified antibiotic susceptibility testing of Gram negative clinical isolates using microcapillary devices.

Antibiotic resistance is a major global challenge. Although microfluidic antibiotic susceptibility tests (AST) offer great potential for rapid and portable testing to inform correct antibiotic selection, the impact of miniaturisation on broth microdilution (BMD) is not fully understood. We developed a 10-plex microcapillary based broth microdilution using resazurin as a colorimetric indicator for bacterial growth. Each capillary had a 1 microlitre capillary volume, 100 times smaller than microplate broth microdilution. The microcapillary BMD was compared to an in-house standard microplate AST and commercial Vitek 2 system. When tested with 25 uropathogenic isolates (20 Escherichia coli and 5 Klebsiella pneumoniae) and 2 reference E. coli, these devices gave 96.1% (441/459 isolate/antibiotic combinations) categorical agreement, across 17 therapeutically beneficial antibiotics, compared to in-house microplate BMD with resazurin. A further 99 (50 E. coli and 49 K. pneumoniae) clinical isolates were tested against 10 antibiotics and showed 92.3% categorical agreement (914/990 isolate/antibiotic combinations) compared to the Vitek 2 measurements. These microcapillary tests showed excellent analytical agreement with existing AST methods. Furthermore, the small size and simple colour change can be recorded using a smartphone camera or it is feasible to follow growth kinetics using very simple, low-cost readers. The test strips used here are produced in large batches, allowing hundreds of multiplex tests to be made and tested rapidly. Demonstrating performance of miniaturised broth microdilution with clinical isolates paves the way for wider use of microfluidic AST.

Draft genome sequence of a carbapenemase-producing (NDM-1) and multidrug-resistant, hypervirulent Klebsiella pneumoniae ST11 isolate from Pakistan, with a non-hypermucoviscous phenotype associated with rmpA2 mutation.

OBJECTIVES: ST11 is a high-risk sequence type associated with carbapenem-resistant Klebsiella pneumoniae strains. Carbapenemase-producing hypervirulent K. pneumoniae (hvKp) are a major concern as they harbour a diverse range of pathogenicity traits. Here we describe the characteristics of K. pneumoniae strain KP75w isolated from a tertiary-care hospital in Pakistan. METHODS: Antimicrobial susceptibility testing was performed by the Kirby-Bauer disk diffusion test and broth microdilution assay. The virulence phenotype was determined by string test as well as biofilm and cell adhesion assays. Genome sequencing was performed using MiSeq and HiSeq 2500 platforms with 30 × coverage. RESULTS: Antimicrobial resistance profiling characterised strain KP75w as a multidrug-resistant carbapenemase-producing strain with a meropenem minimum inhibitory concentration (MIC) of 4 µg/mL, which is above the CLSI susceptible breakpoint (≤1 µg/mL). The annotated contigs indicated a genome size of 5 644 609 bp with 5679 coding regions. KP75w (ST11) was designated as a carbapenemase-producing hvKp strain on the basis of the presence of a carbapenemase gene (blaNDM-1) and hypervirulence genes (rmpA2, iucABCD-iutA, fyuA, irp, mrk, ybt, fep and virB2). KP75w was found to contain a 163-kb virulence region showing 58.8% identity to the large virulence plasmid pLVPK, supporting the hypervirulence of KP75w. CONCLUSION: KP75w is a novel non-hypermucoviscous carbapenemase-producing hvKp ST11 strain that appears to represent the convergence of multidrug resistance with hypervirulence traits in clinical K. pneumoniae strains from the Southeast Asian region.

Analysis of Antibiotic Resistance and Virulence Traits (Genetic and Phenotypic) in Klebsiella pneumoniae Clinical Isolates from Pakistan: Identification of Significant Levels of Carbapenem and Colistin Resistance.

BACKGROUND: The emergence of carbapenem-resistant and hypervirulent hypermucoviscous Klebsiella pneumoniae strains poses a significant public health challenge. We determined the MDR profiles, antibiotic resistance factors, virulence gene complement, and hypermucoviscous features of 200 clinical K. pneumoniae isolates from two major tertiary care hospitals in Islamabad and Rawalpindi, Pakistan. METHODS: Susceptibility profiling and phenotypic analysis were performed according to the CLSI guidelines. Genetic determinants of antibiotic resistance and virulence were detected by PCR. Biofilm formation analysis was performed by microtiter plate assay. RESULTS: The isolates displayed a high degree of antibiotic resistance: 36% MDR-CRKP; 38% carbapenem resistance; 55% gentamicin resistance; 53% ciprofloxacin resistance; and 59% aztreonam resistance. In particular, the level of resistance against fosfomycin (22%) and colistin (15%) is consistent with previous reports of increased resistance levels. Combined resistance to carbapenem and colistin was 7%. Genetic factors associated with colistin resistance (mcr-1 and mcr-2 genes) were detected in 12 and 9% of the isolates, respectively. Significant differences in resistance to gentamicin and levofloxacin were observed between the 200 isolates. Many of the isolates harbored genes specifying extended-spectrum and/or carbapenem-resistant ß-lactamases: bla CTX-M-15 (46%), bla NDM-1 (39%), and bla OXA-48 (24%). The prevalence of the hypermucoviscous phenotype was 22% and 13% of the MDR isolates carried the rmpA gene (regulator for mucoid phenotype). Key virulence factor genes detected include those encoding: porins (ompK35 and ompK36; at 56 and 55% prevalence, respectively); adhesins (fimH, mrkD, and ycfM; at 19, 18, and 22% prevalence, respectively); and the polysaccharide regulator, bss, at 16% prevalence. CONCLUSION: This report highlights carbapenem-resistant K. pneumoniae (CRKP) prevalence, emerging resistance to fosfomycin, and the presence of mcr-1 and mcr-2 in colistin-resistant isolates. Further, the detection of rmpA signifies the prevalence of the hypermucoviscous trait in CRKP clinical isolates from Pakistan.