Punicalagin, a pomegranate polyphenol sensitizes the activity of antibiotics against three MDR pathogens of the Enterobacteriaceae.

BACKGROUND: Multidrug resistance (MDR) in the family Enterobacteriaceae is a perniciously increasing threat to global health security. The discovery of new antimicrobials having the reversing drug resistance potential may contribute to augment and revive the antibiotic arsenal in hand. This study aimed to explore the anti-Enterobacteriaceae capability of bioactive polyphenols from Punica granatum (P. granatum) and their co-action with antibiotics against clinical isolates of Enterobacteriaceae predominantly prevalent in South Asian countries. METHODS: The Kandhari P. granatum (Pakistani origin) extracts were tested for anti-Enterobacteriaceae activity by agar well diffusion assay against MDR Salmonella enterica serovar Typhi, serovar Typhimurium and Escherichia coli. Predominant compounds of active extract were determined by mass spectrometry and screened for bioactivity by agar well diffusion and minimum inhibitory concentration (MIC) assay. The active punicalagin was further evaluated at sub-inhibitory concentrations (SICs) for coactivity with nine conventional antimicrobials using a disc diffusion assay followed by time-kill experiments that proceeded with SICs of punicalagin and antimicrobials. RESULTS: Among all P. granatum crude extracts, pomegranate peel methanol extract showed the largest inhibition zones of 25, 22 and 19 mm, and the MICs as 3.9, 7.8 and 7.8 mg/mL for S. typhi, S. typhimurium and E. coli, respectively. Punicalagin and ellagic acid were determined as predominant compounds by mass spectrometry. In plate assay, punicalagin (10 mg/mL) was active with hazy inhibition zones of 17, 14, and 13 mm against S. typhi, S. typhimurium and E. coli, respectively. However, in broth dilution assay punicalagin showed no MIC up to 10 mg/mL. The SICs 30 µg, 100 µg, and 500 µg of punicalagin combined with antimicrobials i.e., aminoglycoside, ß-lactam, and fluoroquinolone act in synergy against MDR strains with % increase in inhibition zone values varying from 3.4 ± 2.7% to 73.8 ± 8.4%. In time-kill curves, a significant decrease in cell density was observed with the SICs of antimicrobials/punicalagin (0.03-60 µg/mL/30, 100, 500 µg/mL of punicalagin) combinations. CONCLUSIONS: The P. granatum peel methanol extract exhibited antimicrobial activity against MDR Enterobacteriaceae pathogens. Punicalagin, the bacteriostatic flavonoid act as a concentration-dependent sensitizing agent for antimicrobials against Enterobacteriaceae. Our findings for the therapeutic punicalagin-antimicrobial combination prompt further evaluation of punicalagin as a potent activator for drugs, which otherwise remain less or inactive against MDR strains.

Evaluation of rapid polymyxin Nordmann Poirel test for detection of polymyxin resistance in clinical isolates of Enterobacteriaceae.

Polymyxins play a significant role against carbapenem-resistant Enterobacteriaceae (CRE). A total of 121 clinical samples yielded growth of CRE that were included in the study. Rapid Polymyxin NP test was performed on all the isolates as described by Nordmann P et al. and results were compared with broth microdilution method. Majority of the isolates were Klebsiella pneumoniae (70.2%) followed by Escherichia coli (17.4%). A total of 71 isolates were found resistant and 50 as susceptible by broth microdilution. Sensitivity and specificity of rapid polymyxin NP test were found to be 97.2% and 100%, respectively. Our study concluded that rapid polymyxin NP test is reliable and can be used as an alternative to broth microdilution in resource limited settings.