Salmonella enterica

Extracted DNA from the strains Escherichia coli K-12 MG1655 (700926), Bacillus cereus str. Frankland and Frankland (10876), Vibrio parahaemolyticus (17802D-5), and Clostridioides difficile (9689D-5) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The additional bacteria and their sources used in specificity testing included Bacillus megaterium, Bacillus subtilis, Bacillus thuringiensis, Citrobacter freudii, Enterobacter aerogenes, E. coli (ATCC 25922), Klebsiella oxytoca, Listeria monocytogenes, Micrococcus luteus, Salmonella enterica, Serratia macrescens, Shigella flexneri, Staphylococcus capitis, and Staphylococcus saprophyticus, as listed in Table 1. B. cereus and E. coli bacterial cultures were also obtained from the Carolina Biological Supply Company (Burlington, NC, USA). E. coli and B. cereus were inoculated in Luria–Bertani (LB) medium and grown at 37 °C. A hemocytometer and a compound light microscope were used to obtain cell counts. Human HL-60 DNA was also tested in the specificity studies and was obtained from ATCC.

Eight clinical isolates were tested in this study, among which MRSA (Malaysian Type Culture Collection MTCC 381123), Bacillus cereus (MTCC 131621) and Streptococcus pyogenes (ATCC 49399) were Gram-positive; while, Escherichia coli K1 (MTCC 710859), Pseudomonas aeruginosa (American Type Culture Collection ATCC 10145), Klebsiella pneumonia (ATCC 13883), Salmonella enterica (ATCC 14028) and Serratia marcescens (ATCC 13880) were Gram-negative. All the strains were resistant to two or more antibiotics (Table 1). A 24 h old bacterial broth culture was used for experiments as previously described (Khan et al. 2008 (link)).Table 1

Antibiotic susceptibility profile of bacteria used in this study

Bacteria/ID no	Antibiotic susceptibility profile
	amx 25 µg	amc 20/10 µg	cip 10 µg	cst 10 µg	enr 5 µg	gen 10 µg	lcn 15 µg	nxn 10 µg	tcn 30 µg	sxt 1.25 + 23.75 µg
Methicillin-resistant S. aureus MTCC 381123	R	R	R	R	R	S	S	R	S	R
E. coli K1 MTCC 710859	R	R	S	S	S	S	R	R	S	S
S. pyogenes ATCC 49399	R	R	S	R	S	S	R	S	S	I
B. cereus MTCC 131621	R	R	S	R	R	S	S	S	S	S
P. aeruginosa ATCC 10145	R	R	S	R	R	S	R	S	R	R
K. pneumonia ATCC 13883	R	S	S	S	R	S	R	S	R	S
S. enterica ATCC 14028	S	S	S	S	S	R	R	S	I	S
S. marcescens ATCC 13880	R	R	S	R	S	S	R	S	S	S

The antibacterial and antifungal activities of the extracts were determined according to methods described in detail by Pires et al. (2018) [35 (link)] and Heleno et al. (2013) [46 (link)], respectively. Five Gram-negative bacteria (Enterobacter cloacae—ATCC 49741, Escherichia coli—ATCC 25922, Pseudomonas aeruginosa—ATCC 9027, Salmonella enterica—ATCC 13076, and Yersinia enterocolitica—ATCC 8610) and three Gram-positive bacteria (Bacillus cereus—ATCC 11778, Listeria monocytogenes—ATCC 19111 and Staphylococcus aureus—ATCC 25923), besides two fungi strains (Aspergillus fumigatus—ATCC 204305 and Aspergillus brasiliensis—ATCC 16404) purchased from Frilabo (Porto, Portugal) were used. All work was performed with sterile materials handled under laminar flow. Prior to the assays, microorganisms were incubated (37 °C ± 0.5 °C for 24 h for bacteria and 25 °C ± 0.5 °C for 72 h for fungi) with appropriate media for each strain to reach a state of exponential growth. Negative controls of the extract and culture medium were prepared, and the antibiotics streptomycin, methicillin, ampicillin and antifungal ketoconazole served as positive controls. The antimicrobial potential was assessed as the minimum concentration required to inhibit the microorganism growth (MIB) and to cause bacteria or fungi death (MBC or MFC, respectively).

Ampicillin (APC) and vancomycin (VAN) were purchased from Macklin Biochemical Co., Ltd (Shanghai, China). Mueller-Hinton broth (MHB) medium was ordered from Hope Biotechnology Co., Ltd (Qingdao, China). Pepsin, papain, and trypsin were all sourced from Aladdin (Shanghai, China). Escherichia coli CMCC44102, Escherichia coli CMCC 44817, Escherichia coli ATCC 8739, Escherichia coli CMCC 44102, Escherichia coli ATCC 8739, Salmonella enterica CMCC 50335, Shigella flexneri CMCC 51571, Shigella sonnei CMCC 51592, Shigalla dysenteria CMCC 51252, Shigella flexneri CMCC 51572, Bacillus cereus CMCC 63301, Bacillus pumilus CMCC 63202, Staphylococcus aureus ATCC 43300, Staphylococcus aureus ATCC 29213, Staphylococcus aureus CMCC 26003, Staphylococcus aureus ATCC 12600, Staphylococcus aureus ATCC 6538, MRSA N315, and MRSA ATCC 43300 were provided by University of Science and Technology of China. Caco-2 cells were obtained from BeNa Biotechnology Co., Ltd (Beijing, China).

Enterobacter cloacae (ATCC BAA-2341), Listeria monocytogenes (ATCC 19111), Salmonella enterica (ATCC 13076), and Staphylococcus aureus (ATCC 25923) were obtained from LGC Standards (Teddington, GB) in the form of a spore suspension. Before their use in the experiments, the suspensions were defrosted and washed with distilled water to remove glycerol. The bacteria strains were cultured in nutrient media: tryptic soy agar (TSA) for S. aureus and S. enterica, and brain heart agar (BHA) for L. monocytogenes and E. cloacae. Before each experiment, the bacteria’s suspension in distilled buffer saline was prepared according to the appropriate density in the McFarland scale [26 ].