Live/Dead Staining for Quantifying Viable but Not Culturable Cells in Manuka Honey-Treated Wound-Causing Bacteria

Summary

The presented protocol describes a procedure to quantify Viable But Not Culturable Cells (VBNC) in Manuka Honey-treated bacterial cultures.

Abstract

Antibiotic resistance and tolerance among bacteria pose a significant threat to global health. Mechanisms contributing to antibiotic resistance and tolerance include genetic mutations and, acquisition of resistance genes, and transition to Viable But Not Culturable (VBNC) and other dormancy states, respectively. Although genetically identical to their non-antibiotic-tolerant counterparts, VBNC cells evade antibiotic effects by remaining metabolically inactive. Antibiotics are effective only when their target processes, such as DNA replication or transcription, are active. Since environmental stressors, particularly antibiotics, can drive bacteria into dormancy, alternative antimicrobials are needed to minimize or prevent this response. The antimicrobial Manuka Honey (MH) is effective against many bacteria, with rare development of resistance. Its multifaceted antimicrobial mechanisms make it a valuable agent for treating bacterial infections. This research investigated MH recalcitrance to antibiotic resistance development by testing the hypothesis that MH induces fewer VBNC cells than conventional antibiotics. To investigate this, a protocol was developed to treat the wound-causing bacteria Staphylococcus aureus and Pseudomonas aeruginosa with minimum inhibitory concentrations of MH or the conventional antibiotics tobramycin or meropenem, that then used the viable plate count to identify metabolically active culturable cells and live/dead staining to identify all viable cells. The number of VBNC cells equaled the viable cell number minus the culturable cell number. In some experiments, the culturable cell number was higher than the viable cell number, giving a negative number of VBNC cells; thus, VBNC cell numbers were not directly compared. Instead, culturable and viable cell numbers were compared for each treatment. Only P. aeruginosa treated with tobramycin had significantly fewer culturable cells than viable cells, indicating a higher number of VBNC cells. This protocol is quick and easy and can be used to evaluate MH induction of VBNC cells in other pathogenic bacteria.

Introduction

Bacteria in the Viable But Not Culturable (VBNC) dormancy state survive antibiotic treatment and cause recurring and sometimes deadly infectious diseases^1,2,3. More familiar to us is antibiotic resistance, which occurs via a heritable genetic change⁴. The genetic change provides resistance to a specific antibiotic by several mechanisms, including limiting the uptake of an antibiotic, increasing the efflux of the antibiotic, modifying the antibiotic target, or inactivating the antibiotic⁴. By contrast, bacteria in dorman....

Protocol

Since P. aeruginosa (ATCC9721) and S. aureus (ATCC29213) are classified as Biosafety Level 2 agents, the rooms where the work is conducted must have limited access and be equipped with a biosafety cabinet for manipulations that could create splashes or aerosols, such as agitating and centrifuging cultures. In all steps of this protocol, wear personal protective equipment (PPE), including a lab coat, protective glasses, and gloves. All reagents and equipment used in the experiments are listed in the Table of Materials.

1. Preparation of sterile broth and agar culture media

1. Prepa....

Results

Viable but not culturable (VBNC) bacterial life forms are induced by stressors, including antibiotic treatment, and cause recurring infections due to their antibiotic tolerance. Because MH is a broad-spectrum antimicrobial to which resistance has rarely been detected, it was hypothesized that MH induced fewer VBNCs than conventional antibiotics. The method described here was used to quantify VBNCs formed by two wound-causing bacteria, S. aureus and P. aeruginosa. The number of VBNCs equals the number of.......

Discussion

The protocol described here allows for the detection of VBNC cell populations in wound-causing bacteria treated with minimum inhibitory concentrations (MICs) of MH and conventional antibiotics. Critical steps in the protocol included preparing the MH dilutions on the day of the experiment, preventing sample loss during live/dead staining, and calculating the analyzed volume correctly. For this and other studies, the MH MICs for S. aureus and P. aeruginosa were experimentally determined^.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Mueller Hinton broth	Fisher Bioreagents	B12322
Luria Bertani (LB) broth	Fisher Bioreagents	BP142602
BD Difco Agar	Fisher Scientific	DF0812-07-1
sodium chloride	Sigma	S3014-500g
meropenem	TargetMol	T0224
tobramycin	Selleck Chemicals	S2514
Staphylococcus aureus	ATCC	ATCC29213
Pseudomonas aeruginosa	ATCC	ATCC9721
shaking incubator	Eppendorf	M13520000
incubator	Benchmark Scientific	H2200-H
Manukaguard Manuka Honey	Amazon	NA
DMIL inverted fluorescent microscope	Leica Microsystems	11521265
Digital camera	Leica Microsystems	12730522
15 mL falcon tubes	Genesee Scientific	28-103
serological pipets	Diagnocine	DP-LB0100005, DP-LB010010
Pipet Aid Pipettor	Drummond Scientific Company	4-000-101
Bunsen burner	Fisher Scientific	S48108
Rainin pipets	Pipette.com	L-20, L-200, L-1000
balance	Mettler Toledo	XS603S
96-well plate	Falcon	351172
inoculating loop	Fisher Scientific	13-104-5
isopropanol	Fisher Scientific	BP2618-1
glass spreader	homemade	NA
Live dead staining kit for bacteria	Invitrogen	L7012
microfuge tubes	Biologix Research Company	SKU 80-1500/P80-1500
tin foil	Costco	NA
Figure making software	GraphPad	NA	GraphPad Prism was used for making figures and conducting statistical analyses.