Enhanced Antibacterial Activity of Silver Nanoparticles Combined with Hydrogen Peroxide Against Multidrug-Resistant Pathogens Isolated from Dairy Farms and Beef Slaughterhouses in Egypt

Figures & data

Table 1 Antibiotics Used to Test Antimicrobial Susceptibility of Five Types of Bacteria

Bacteria	Antimicrobials
S. typhimurium	Sulfamethoxazole-trimethoprim (25 µg), Chloramphenicol (30 µg), Ciprofloxacin (5 µg), Amoxicillin (25 µg), Norfloxacin (10 µg), Nalidixic acid (30 µg), Cefotaxime (30 µg), Cephalexin (30 µg)
Listeria monocytogenes	Rifampicin (5 µg), Oxacillin (1 µg), Tetracycline (30 µg), Amoxicillin (10 µg), Gentamycin (10 µg), Cefotaxime (30 µg), Cefepime (30 µg), Sulfamethoxazole-trimethoprim (25 µg), Carbenicillin (30 µg), Ampicillin (10 µg), Chloramphenicol (30 µg)
Klebsiella pneumoniae	Neomycin (30 µg), Kanamycin (30 µg), Tetracycline (30 µg), Nalidixic acid (30 µg), Streptomycin (10 µg), Gentamycin (10 µg), Chloramphenicol (30 µg), Ampicillin (10 µg)
Pseudomonas aeruginosa	Ciprofloxacin (5 µg), Neomycin (30 µg), Norfloxacin (10 µg), Streptomycin (10 µg), Kanamycin (30 µg), Levofloxacin (5 µg), Carbapenem (100 µg), Cephalexin (30 µg), Cefepime (30 µg)
E. coli O157:H7	Chloramphenicol (30 µg), Ampicillin (10 µg), Tetracycline (30 µg), Streptomycin (10 µg), Cefotaxime (30 µg), Carbenicillin (30 µg), Erythromycin (15 µg), Cephalexin (30 µg)

Table 2 Frequency Distribution of Bacterial Species Recovered from Dairy Farms and Slaughterhouse

Sampling Site	Sampling Source	Prevalence of Bacterial Isolates – Number (%)
		E. coli O157:H7	L. monocytogenes	S. typhimurium	P. aeruginosa	K. pneumoniae
Dairy farms	BTM	12 (30)	8 (20)	8 (20)	2 (5)	10 (25)
	MU	10 (25)	8 (20)	14 (35)	4 (10)	4 (10)
	WHSs	6 (15)	10 (25)	10 (25)	8 (20)	6 (15)
Total		23.3	21.7	26.7	11.7	16.7
Beef slaughterhouse	Carcass	6 (30)	6 (30)	6 (30)	2 (10)	0
	KSs	8 (40)	4 (20)	2 (10)	4 (20)	2 (10)
	WSs	2 (10)	8 (40)	6 (30)	4 (20)	0
	WHSs	8 (40)	2 (10)	4 (20)	4 (20)	2 (10)
Total		30	25	22.5	17.5	5

Notes: Number of samples; 120/dairy farm samples, 80/slaughterhouse samples.

Abbreviations: BTM, bulk tank milk; MU, milking utensils; KSs, knives’ swabs; WSs, wall swabs; WHSs, workers’ hands swabs.

Table 3 Multidrug Resistance Patterns (MDR) of Recovered Bacterial Species (N=40) from Dairy Farms and a Beef Slaughterhouse

Bacterial Species	% of Resistant Dairy Farm Isolates	% of Resistant Beef Abattoir Isolates	MDR Pattern*	MAR Index
E. coli O157:H7	35.7	25	E, AM, TE, S	0.5
L. monocytogenes	46.2	20	RA, CTX, TE, CN, C	0.45
S. typhimurium	25	44.4	SXT, C, NOR, AM, CIP	0.63
P. aeruginosa	28.6	85.7	CIP, NOR, S, LEV	0.44
K. pneumoniae	70	50	N, K, TE, C, NA, AM, CN	0.88

Notes: *Erythromycin (E; 15 µg), Rifampicin (RA; 5 µg), Chloramphenicol (C; 30 µg), Norfloxacin (NOR; 10 µg), Levofloxacin (LEV; 5 µg), Neomycin (N; 30 µg), Nalidixic acid (NA; 30 µg), Ciprofloxacin (CIP; 5 µg), Tetracycline (TE; 5 µg), Amoxicillin (AM; 25 µg), Cefotaxime (CXT; 30 µg), Kanamycin (K; 30 µg), Trimethoprim/sulphamethoxazole (SXT; 25 µg), Gentamycin (CN; 10 µg), Streptomycin (S; 10 µg).

Abbreviation: MAR, multiple antibiotic resistance index.

Figure 1 Characterization of AgNPs-H₂O₂ product, TEM micrographs and size distribution for silver (scale bar: 100 nm), micrographs displays AgNPs-H₂O₂ with sizes ranging from (30.17–67.92 nm) based on TEM images.

Figure 2 Zeta-potential of AgNPs-H₂O₂ product, −0.192 mV surface charge – good quality nanoparticles.

Table 4 MIC, MBC Values, and MBC/MIC Ratio of Commercial AgNPs-H₂O₂ Product Against MDR Bacterial Species

MDR Bacteria	Concentration (µg/mL)		MBC/MIC Ratio	P-value	Degree of Agreement Between MIC and MBC Tests %*
	MIC	MBC
E. coli O157:H7	6.25	12.5	2	<0.0001	82
L. monocytogenes	12.5	50	4	<0.0001	30
S. typhimurium	3.125	6.25	2	<0.0001	83
P. aeruginosa	6.25	50	8	<0.0001	30
K. pneumoniae	25	50	2	<0.0001	80

Note: *Kappa test association.

Figure 3 Time-kill assays of AgNPs-H₂O₂ against multidrug-resistant bacteria. (A) L. monocytogenes, (B) P. aeruginosa, (C) S. typhimurium, (D) E. coli O157:H7, (E) K. pneumoniae, and (F) comparison of time-kill assay of AgNPs-H₂O₂ against all tested MDR bacteria. The measurements were made at 0, 2, 4, 6, 8, 12, and 24 hours post-treatment, viability of microorganisms expressed by CFU/mL, data points represented by mean±SEM for triplicates of each experiment.