Effect of Nk-lysin peptides on bacterial growth, MIC, antimicrobial resistance, and viral activities

Figures & data

Table 1. The amino acids sequence of the three Nk-lysin peptides.

Peptide name	Amino acid sequence	M.W (Dalton)
Chicken-Nk-lysin	PDEDAINNALNKVCSTGRRQRSICKQLLKK	3399.92
Bovine- Nk-lysin	RPSKNVIIHVTSNVCSKMGLWSILCNQMMK	3419.16
Human-Nk-lysin	PTQRSVSNAATRVCRTGRSRWRDVCRNFMR	3568.07

Table 2. The primers list for detection and characterization of beta-lactamases and quinolone resistance genes.

Gene	Forward primer (5′ to 3′)	Reverse primer (5′ to 3′)	Amplicon size (bp)	References
BLATem	ATGAGTATTCAACATTTCCG	GACAGTTACCAATGCTTAATCA	869	Hendriksen et al. [71]
CTX-M1	GACGATGTCACTGGCTGAGC	AGCCGCCGACGCTAATACA	499	Hendriksen et al. [71]
TOHO1	GCGACCTGGTTAACTACAATCC	CGGTAGTATTGCCCTTAAGCC	351	Hendriksen et al. [71]
CTXM8	CGCTTT GCCATGTGCAGCACC	GCT CAGTACGATCGAGCC	307	Hendriksen et al. [71]
CTXM9	GCTGGAGAAAAGCAGCGGAG	GTAAGCTGACGCAACGTCTG	474	Hendriksen et al. [71]
Gyrase A	AAATCTGCCCGTGTCGTTGGT	GCCATACCTACTGCGATACC	344	Harrois et al. [72]
QnrA	ATTTCTCACGCCAGGATTTG	GATCGGCAAAGGTTAGGTCA	516	Piddock [69]
QnrB	GATCGTGAAAGCCAGAAAGG	ACGATGCCTGGTAGTTGTCC	469	Piddock [69]
QnrD	CGAGATCAATTTACGGGGAATA	AACAAGCTGAAGCGCCTG	565	Kruger et al. [73]
QnrS	ACGACATTCGTCAACTGCAA	TAAATTGGCACCCTGTAGGC	417	Piddock [69]

Figure 1. Antimicrobial activity of Nk-lysin peptides of chicken, bovine and human against Salmonella typhimurium. Data presented as means (±SD) of three independent repeats in triplicate (*p˂0.03 compared to control group).

Table 3. Antimicrobial activity of Nk-lysin peptides of chicken, bovine and human against gram-positive and gram-negative bacteria.

Bacterial strain	Nk-lysin chicken		Nk-lysin bovine		Nk-lysin human		Kanamycin
	MIC (µg/ml)	MBC (µg/ml)	MIC (µg/ml)	MBC (µg/ml)	MIC (µg/ml)	MBC (µg/ml)	MIC (µg/ml)	MBC (µg/ml)
Salmonella typhimurium ATCC 14028	15 ± 0.098	30 ± 0.10	15 ± 0.10	25 ± 0.12	15 ± 0.090	70 ± 0.092	62.5 ± 0.00	62.5 ± 0.00
Klebsiella Pneumonia (isolate)	15 ± 0.030	25 ± 0.010	15 ± 0.03	25 ± 0.010	15 ± 0.040	15 ± 0.040	31.25 ± 0.00	31.25 ± 0.00
St. pyogenes ATCC 19615	15 ± 0.00	25 ± 0.010	15 ± 0.00	25 ± 0.010	15 ± 0.00	25 ± 0.010	62.5 ± 0.00	62.5 ± 0.00
Streptococcus mutans (isolate)	15 ± 0.00	15 ± 0.00	15 ± 0.0	15 ± 0.0	15 ± 0.010	25 ± 0.012	62.5 ± 0.023	62.5 ± 0.23
Escherichia coli ATCC 11775	15 ± 0.012	25 ± 0.010	15 ± 0.012	25 ± 0.010	15 ± 0.014	25 ± 0.012	15.62 ± 0.011	15.62 ± 0.00
Klebsiella oxytoca ATCC 49131	15 ± 0.015	25 ± 0.10	15 ± 0.015	25 ± 0.10	15 ± 0.012	15 ± 0.012	31.25 ± 0.00	31.25 ± 0.00
Pseudomonas aeruginosa P.aeruginosa ATCC 9027	15 ± 0.012	25 ± 0.013	15 ± 0.012	25 ± 0.013	15 ± 0.010	15 ± 0.010	62.5 ± 0.00	62.5 ± 0.00
Shigella sonnei ATCC 25931	15 ± 0.033	25 ± 0.010	15 ± 0.033	25 ± 0.010	15 ± 0.025	25 ± 0.012	7.81 ± 0.026	7.81 ± 0.026

Figure 2. Colony counting assay of Nk-lysin peptides of chicken, bovine, human against Salmonella typhimurium. Data presented as means (±SD) of three independent repeats in triplicate (*p˂0.01 compared to control group).

Figure 3. Growth kinetic activities of 2xMIC of chicken, bovine and human Nk-lysin peptide against Streptococcus pyogenes (ATCC 19615) (*p˂0.02 compared to control group) and Streptococcus mutans (isolate) (*p˂0.02 compared to control group), Escherichia coli (ATCC 11775) (*p˂0.03 compared to control group), Klebsiella oxytoca (ATCC 49131) (*p˂0.03 compared to control group), Pseudomonas aeruginosa (ATCC 9027) (*p˂0.04 compared to control group), Salmonella typhimurium (ATCC 14028), Klebsiella Pneumonia (isolate) (*p˂0.02 compared to control group)and Shigella sonnei (ATCC 25931) (*p˂0.03 compared to control group). Data presented as means (±SD) of three independent repeats in triplicate.

Figure 4. The bacterial lytic effect of 1× MIC bovine Nk-lysin peptide against Salmonella typhimurium (ATCC 14028) at different inoculum concentrations. Data presented as means (±SD) of three independent repeats in triplicate. (* p ˂ 0.01 compared to control group).

Figure 5. The bacterial lytic effect of 1x MIC bovine Nk-lysin peptide against Salmonella typhimurium (ATCC 14028) at different inoculum concentrations. Data presented as means (±SD) of three independent repeats in triplicate. (*p˂0.01 compared to control group).

Figure 6. DNA/RNA release of Salmonella typhimurium (ATCC 14028) bacterial cells after incubation with (Nk-lysin peptides of chicken, bovine, human) at 37 °C. Data presented as means (±SD) of three independent repeats in triplicate. (*p˂0.01 compared to control group).

Figure 7. The amplified fragment of CTX-M1 gene with 499 bp, where M (DNA ladder =100 bp), 1 (Salmonella typhimurium ATCC 14028 untreated and treated cells), 2 (Pseudomonas aeruginosa ATCC 9027 untreated and treated cells), 3 (Klebsiella oxytoca ATCC 49131 untreated and treated cells), 4 (Streptococcus pyrogens ATCC 19615 untreated and treated cells) and arrows indicate gene in untreated bacteria whereas empty well indicates treated bacteria after challenge with Nk-lysin.

Figure 8. The amplified fragment of CTX-M8 gene with 307 bp, where M (DNA ladder =100 bp), 1 (Salmonella typhimurium ATCC 14028 untreated and treated cells), 2 (Pseudomonas aeruginosa ATCC 9027 untreated and treated cells), 3 (Klebsiella oxytoca ATCC 49131 untreated and treated cells), 4 (Streptococcus pyrogens ATCC 19615 untreated and treated cells) and arrows indicate gene in untreated bacteria whereas empty well indicates treated bacteria after challenge with Nk-lysin.

Figure 9. The amplified fragment of CTX-M9 gene with 474 bp, where M (DNA ladder =100 bp), 1 (Salmonella typhimurium ATCC 14028 untreated and treated cells), 2 (Pseudomonas aeruginosa ATCC 9027 untreated and treated cells), 3 (Klebsiella oxytoca ATCC 49131 untreated and treated cells), 4 (Streptococcus pyrogens ATCC 19615 untreated and treated cells) and arrows indicate gene in untreated bacteria whereas empty well indicates treated bacteria after challenge with Nk-lysin.

Table 4. Detection of beta-lactam and quinolone resistance genes after treatment with Nk-lysin peptides.

Gene name	Untreated bacteria	treated bacteria
BLATem	(+) all	(+) all
TOHO1	(−) all	(−) all
CTX-M1	(+) all	(−) all
CTXM8	(+) all	(−) all
CTXM9	(+) all	(−) all
Gyrase A	(+) all	(+) all
QnrA	(−) all	(−) all
QnrB	(−) all	(−) all
QnrD	(−) all	(−) all
QnrS	(−) all	(−) all

(+) all, the gene is present in all bacteria.

(−) all, the gene is absent in all bacteria.

Table 5. The antiviral activity of Nk-lysin peptides of chicken, bovine and human at concentration (80 µg/ml) against Rotavirus sa-11.

Tested extract	TCID50/ml	Log10TCID50/ml	Log reduction	% Reduction
Initial titer	1.95 × 10^6	6.29	N.A	N.A
cNk-lysin	4.11 × 10^5	5.61	0.7	79%
bNk-lysin	1.95 × 10^5	5.29	1.00	90%
hNk-lysin	1.00 × 10^6	6.00	0.29	49%

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