J Clin Med Res

Journal of Clinical Medicine Research, ISSN 1918-3003 print, 1918-3011 online, Open Access

Article copyright, the authors; Journal compilation copyright, J Clin Med Res and Elmer Press Inc

Journal website https://jocmr.elmerjournals.com

Original Article

Volume 17, Number 8, August 2025, pages 445-459

The Elimination Effect of Medical-Grade Honey on Pseudomonas aeruginosa Biofilms: A Systematic Review and Meta-Analysis

Figure 2. Funnel plot of included studies assessing the effect of medical-grade honey on P. aeruginosa biofilm.

Figure 3. Forest plot illustrating the effect of medical-grade honey on biofilm formation. Forest plots represent static biofilm models only.

Figure 4. Forest plot illustrating the effect of medical-grade honey on established biofilm. Forest plots represent static biofilm models only.

Figure 5. Forest plot illustrating the effect of medical-grade honey subgroup division based on bacterial strains used in the study. Forest plots represent static biofilm models only.

Figure 6. Forest plot illustrating the effect of medical-grade honey on subgroup division based on control used in the study. Forest plots represent static biofilm models only. ABC: active biofilm control; Int.: intervention; SC: sterility control.

**Table 1.** Descriptive Summary of Included Studies
No.	Study	P. aeruginosa strain	Medical-grade honey	Honey concentrations tested	CV assay	Outcomes measured	Key findings
ATCC: American Type Culture Collection; CV: crystal violet; MBEC: minimum biofilm eradication concentration; MBIC: minimal biofilm inhibitory concentration; MIC: minimum inhibitory concentration; NCTC: National Collection of Type Cultures; PBS: phosphat-buffered saline; UCBPP: University of California Berkeley Plant Pathology.
1	Camplin and Maddocks, 2014 [10]	ATCC 9027, clinical isolate 867	Medihoney	0-80% w/v	Stained with 0.25% w/v CV for 10 min. Two washes with PBS. Destaining with 7% acetic acid.	MIC, MBEC, resistance development	MIC: 15.3% to 25.6%. MBEC: 43.3% and 48.3%
2	Cooper et al, 2014 [27]	Clinical isolate	Medihoney	5-50% w/v	Stained with 0.25% w/v CV for 15 min. Three washes with PBS. Destaining with 7% acetic acid.	MIC50, MIC90, MBEC, biofilm structure	MIC50: 16.8%, MBEC: 35.3%; ≥ 40% honey significantly alter biofilm structure and viability
3	Halstead et al, 2016 [28]	ATCC 15692, NCTC 6749, clinical isolate	Surgihoney RO, Medihoney, Manuka honey	1:3 to 1:6,144 dilution	Stained with 1% w/v CV for 10 min. Two washes with PBS. Destaining with 70% ethanol.	MBIC, biofilm biomass	Honey dilution of 1:3 to 1:12 showed good results against P. aeruginosa biofilm (P < 0.05)
4	Lu et al, 2019 [13]	ATCC 15692, UCBPP-PA14	Manuka, Medihoney, Clover honey	1-80% w/v	Stained with 0.2% w/v CV for 60 min. Three washes with PBS. Destaining with 33% w/v acetic acid.	MIC, MBIC, biofilm biomass, viability	MBIC 16-32%; UCBPP-PA14 more susceptible than ATCC 15692; 64-80% honey eradicated biofilms
5	Maddocks et al, 2013 [29]	ATCC 9027, clinical isolate 867	Medihoney	0-60% w/v	Stained with 0.25% w/v CV for 10 min. Two washes with PBS. Destaining with 7% acetic acid.	Biofilm biomass, adhesion, invasion	Honey can reduce the amount of biofilm biomass to 33% (867) and 43% (ATCC 9027)
6	Morroni et al, 2018 [30]	Clinical isolate	Manuka honey, Kenya honey, Cuba honey	2-22% v/v	Stained with 0.25% w/v CV for 10 min. Two washes with PBS. Destaining with 70% ethanol.	Antimicrobial activity, biofilm biomass,	Manuka honey minimal dilution for antimicrobial: 14%. Significant biomass reduction at ≥ 8%

Table 1. Descriptive Summary of Included Studies

No.

Study

P. aeruginosa strain

Medical-grade honey

Honey concentrations tested

CV assay

Outcomes measured

Key findings

ATCC: American Type Culture Collection; CV: crystal violet; MBEC: minimum biofilm eradication concentration; MBIC: minimal biofilm inhibitory concentration; MIC: minimum inhibitory concentration; NCTC: National Collection of Type Cultures; PBS: phosphat-buffered saline; UCBPP: University of California Berkeley Plant Pathology.

Camplin and Maddocks, 2014 [10]

ATCC 9027, clinical isolate 867

Medihoney

0-80% w/v

Stained with 0.25% w/v CV for 10 min. Two washes with PBS. Destaining with 7% acetic acid.

MIC, MBEC, resistance development

MIC: 15.3% to 25.6%. MBEC: 43.3% and 48.3%

Cooper et al, 2014 [27]

Clinical isolate

Medihoney

5-50% w/v

Stained with 0.25% w/v CV for 15 min. Three washes with PBS. Destaining with 7% acetic acid.

MIC50, MIC90, MBEC, biofilm structure

MIC50: 16.8%, MBEC: 35.3%; ≥ 40% honey significantly alter biofilm structure and viability

Halstead et al, 2016 [28]

ATCC 15692, NCTC 6749, clinical isolate

Surgihoney RO, Medihoney, Manuka honey

1:3 to 1:6,144 dilution

Stained with 1% w/v CV for 10 min. Two washes with PBS. Destaining with 70% ethanol.

MBIC, biofilm biomass

Honey dilution of 1:3 to 1:12 showed good results against P. aeruginosa biofilm (P < 0.05)

Lu et al, 2019 [13]

ATCC 15692, UCBPP-PA14

Manuka, Medihoney, Clover honey

1-80% w/v

Stained with 0.2% w/v CV for 60 min. Three washes with PBS. Destaining with 33% w/v acetic acid.

MIC, MBIC, biofilm biomass, viability

MBIC 16-32%; UCBPP-PA14 more susceptible than ATCC 15692; 64-80% honey eradicated biofilms

Maddocks et al, 2013 [29]

ATCC 9027, clinical isolate 867

Medihoney

0-60% w/v

Stained with 0.25% w/v CV for 10 min. Two washes with PBS. Destaining with 7% acetic acid.

Biofilm biomass, adhesion, invasion

Honey can reduce the amount of biofilm biomass to 33% (867) and 43% (ATCC 9027)

Morroni et al, 2018 [30]

Clinical isolate

Manuka honey, Kenya honey, Cuba honey

2-22% v/v

Stained with 0.25% w/v CV for 10 min. Two washes with PBS. Destaining with 70% ethanol.

Antimicrobial activity, biofilm biomass,

Manuka honey minimal dilution for antimicrobial: 14%. Significant biomass reduction at ≥ 8%

**Table 2.** Quality Assessment for Included Studies Using QUIN Tool
No.	Criteria	Microbiological relevance	Camplin and Maddock, 2014 [10]	Cooper et al, 2014 [23]	Halstead et al, 2016 [24]	Lu et al, 2019 [27]	Maddocks et al, 2013 [25]	Morroni et al, 2018. [26]
ANOVA: analysis of variance; OD: optical density; QUIN: Quality Assessment Tool for In Vitro Studies.
1	Clearly stated aims/objectives	The objectives are clearly stated. The specific strain of P. aeruginosa and type of medical-grade honey is defined.	2	2	2	2	2	2
2	Detailed explanation of sample size calculation	A sufficient number of replications had been performed for valid results.	1	1	1	1	1	1
3	Detailed explanation of sampling technique	The studies describe how the biofilm was harvested, how it was diluted, how the samples were processed to accurately measure the outcome.	1	1	1	1	1	1
4	Details of comparison group	The studies had defined the negative control and any positive controls.	2	2	2	2	2	2
5	Detailed explanation of methodology	The methodology of studies includes specific microbiological details: bacterial strain, culture conditions, the method used to grow the biofilm, the concentration and form of honey used, the exposure time.	2	2	2	2	2	2
6	Operator details	Operator details must meet standardized protocols. However, it is less critical for automated OD measurements.	0	0	0	0	0	0
7	Randomization	The studies did not provide details regarding sequence generation and allocation concealment.	0	0	0	0	0	0
8	Method of measurement of outcome	The method to measure outcome of biofilm biomass is crystal violet assay for optical density measurement.	2	2	2	2	2	2
9	Outcome assessor details	The studies did not state number of outcome assessors and details regarding training and calibration of assessor(s).	0	0	0	0	0	0
10	Blinding	No details regarding blinding of operator(s), outcome assessor(s), and statistician.	0	0	0	0	0	0
11	Statistical analysis	The studies use valid statistical tests (e.g., t-test, ANOVA).	2	0	1	2	1	2
12	Presentation of results	The studies provide summary statistics and visual representations.	2	2	2	2	2	2

Table 2. Quality Assessment for Included Studies Using QUIN Tool

No.

Criteria

Microbiological relevance

Camplin and Maddock, 2014 [10]

Cooper et al, 2014 [23]

Halstead et al, 2016 [24]

Lu et al, 2019 [27]

Maddocks et al, 2013 [25]

Morroni et al, 2018. [26]

ANOVA: analysis of variance; OD: optical density; QUIN: Quality Assessment Tool for In Vitro Studies.

Clearly stated aims/objectives

The objectives are clearly stated. The specific strain of P. aeruginosa and type of medical-grade honey is defined.

Detailed explanation of sample size calculation

A sufficient number of replications had been performed for valid results.

Detailed explanation of sampling technique

The studies describe how the biofilm was harvested, how it was diluted, how the samples were processed to accurately measure the outcome.

Details of comparison group

The studies had defined the negative control and any positive controls.

Detailed explanation of methodology

The methodology of studies includes specific microbiological details: bacterial strain, culture conditions, the method used to grow the biofilm, the concentration and form of honey used, the exposure time.

Operator details

Operator details must meet standardized protocols. However, it is less critical for automated OD measurements.

Randomization

The studies did not provide details regarding sequence generation and allocation concealment.

Method of measurement of outcome

The method to measure outcome of biofilm biomass is crystal violet assay for optical density measurement.

Outcome assessor details

The studies did not state number of outcome assessors and details regarding training and calibration of assessor(s).

Blinding

No details regarding blinding of operator(s), outcome assessor(s), and statistician.

Statistical analysis

The studies use valid statistical tests (e.g., t-test, ANOVA).

Presentation of results

The studies provide summary statistics and visual representations.