The expanding global distribution of multi-resistant Klebsiella pneumoniae demands faster antimicrobial susceptibility testing (AST) to guide antibiotic treatment. Current ASTs rely on time-consuming differentiation of resistance and susceptibility after initial isolation of bacteria from a clinical specimen. Here we describe a flow cytometry workflow to determine carbapenem susceptibility from bacterial cell characteristics in an international K. pneumoniae isolate collection (n = 48), with a range of carbapenemases. Our flow cytometry-assisted susceptibility test (FAST) method combines rapid qualitative susceptible/non-susceptible classification and quantitative MIC measurement in a single process completed shortly after receipt of a primary isolate (54 and 158 minutes respectively). The qualitative FAST results and FAST-derived MIC (MICFAST) correspond closely with broth microdilution MIC (MICBMD, Matthew’s correlation coefficient 0.887), align with the international AST standard (ISO 200776-1; 2006) and could be used for rapid determination of antimicrobial susceptibility in a wider range of Gram negative and Gram positive bacteria.

Carbapenemases are increasingly important antimicrobial resistance determinants. Little is known about the carbapenem resistance mechanisms in Sri Lanka. We examined 22 carbapenemresistant Klebsiella pneumoniae from Sri Lanka to determine their b-lactam resistance mechanisms. The predominant resistance mechanisms we detected in this study were OXA-181, NDM-1 carbapenemases and extended-spectrum b-lactamase CTX-M-15. All isolates were then genotyped by pulsed-field gel electrophoresis, variable-number tandem repeat sequence analysis and multilocus sequence typing, and seven distinct genotypes were observed. Five OXA-181-positive Klebsiella pneumoniae isolates were genotypically related to an isolate of Indian origin. Multilocus sequence typing found that these related isolates belong to ST-14, which has been associated with dissemination of OXA-181 from the Indian subcontinent. Other genotypes we discovered were ST-147 and ST-340, also associated with intercontinental spread of carbapenemases of suspected subcontinental origin. The major porin genes ompK35 and ompK36 from these isolates had insertions, deletions and substitutions. Some of these were exclusive to strains within single pulsotypes. We detected one ompK36 variant, ins AA134-135GD, in six ST-14-and six ST-147, bla OXA-181 -positive isolates. This porin mutation was an independent predictor of high-level meropenem resistance in our entire Sri Lankan isolate collection (P50.0030). Analysis of the Sri Lankan ST-14 and ST-147 ins AA134-135GD-positive isolates found ST-14 was more resistant to meropenem than other isolates (mean MIC: 32±0 mg ml -1 and 20±9.47 mg ml -1 , respectively, P50.0277). The likely international transmission of these carbapenem resistance determinants highlights the need for regional collaboration and prospective surveillance of carbapenem-resistant Enterobacteriaceae.

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Peritoneal dialysis (PD) peritonitis cases require rapid clinical interventions to ensure the best possible patient outcomes. Culture-dependent microbiology tools are slow and cannot provide clinicians with evidence to guide antimicrobial prescription practices in an appropriate time frame. Genotypic methods have met with limited success for analyzing continuous ambulatory PD effluent, with most centers still relying on culture-dependent microbiology. We present a case study in which we apply flow cytometry techniques to antibiotic-compromised effluent. We demonstrate, with supporting evidence, direct enumeration of bacterial and human immune cells, with results reported within 2 hours of receiving the clinical specimen.