Prevalence and Antimicrobial Resistance of Virulent Listeria monocytogenes and Cronobacter sakazakii in Dairy Cattle, the Environment, and Dried Milk with the In Vitro Application of Natural Alternative Control

Abstract

This study aims to detect the prevalence and antimicrobial resistance of Listeria monocytogenes and Cronobacter sakazakii in three dairy households and dried milk from different suppliers, and evaluate the antimicrobial effect of rose water, rose, and orange essential oils. In total, 360 samples were collected from cattle, the environment, and dried milk (n = 30). Antimicrobial activity was evaluated with twofold microtube dilution and the time-kill method. L. monocytogenes was identified in all households (13.3%) with a prevalence in the range of 5.8-17.5%, while C. sakazakii was identified in one household (5.3%). The former and latter pathogens were highly isolated from the feces at 20% and 2.5% and bedding at 12.5% and 1.6%, respectively. L. monocytogenes was isolated only from milk at 7.5%, but C. sakazakii was not detected in either milk or dried milk. L. monocytogenes strains were screened for virulence genes (iap, hylA, and actA). All strains were positive for the iap gene, while for hlyA and actA, the percentages were (35.4% 16.6%, respectively). L. monocytogenes strains showed high resistance against sulfamethoxazole-trimethoprim (100%), followed by gentamicin, penicillin, and imipenem (95.8%, 95.8%, and 91.6%, respectively). All C. sakazakii strains were susceptible to all tested antibiotics. The bactericidal activity of orange oil was the strongest, appeared after 1 h for both pathogens, followed by rose oil and then rose water.

Keywords: C. sakazakii; L. monocytogenes; alternative; antimicrobial resistance; essential oils.

Figure 1

Agarose gel electrophoresis of PCR of a specific oligonucleotide sequence as part of cgcA (genus-specific marker sequences) at 492 bp for detection of C. sakazakii. Lane M: 100 bp ladder as molecular size DNA marker. Lane C+: Control positive C. sakazakii. Lane C–: Control negative. Lanes 1 to 5: positive C. sakazakii strains.

Figure 2

Agarose gel electrophoresis of multiplex PCR of iap (131 bp), hylA (456 bp) and actA (839 bp) virulence genes for the characterization of L. monocytogenes. Lane M 100 bp ladder as molecular size DNA marker. Lane C+: control positive L. monocytogenes for iap, hylA and actA genes. Lane C–: control negative. Lanes 1 and 4: positive L. monocytogenes strains for iap and actA genes. Lanes 2, 3, and 5: positive L. monocytogenes strains for iap and hylA genes. Lane 2: positive L. monocytogenes strain for iap, hylA, and actA genes.

Figure 3

Inhibitory effect of (A) rose water, (B) rose oil, and (C) orange oil at different concentrations; (b) MIC (dotted line) and (c) 2 × MIC (dot-dashed line). (a) Control (solid line) on the viable counts of L. monocytogenes inoculated into MHB and incubated at different time points of 0, 1, 3, 6, and 24 h. Values are the mean ± standard deviation of three independent trials. ND means not detected.

Figure 3

Inhibitory effect of (A) rose water, (B) rose oil, and (C) orange oil at different concentrations; (b) MIC (dotted line) and (c) 2 × MIC (dot-dashed line). (a) Control (solid line) on the viable counts of L. monocytogenes inoculated into MHB and incubated at different time points of 0, 1, 3, 6, and 24 h. Values are the mean ± standard deviation of three independent trials. ND means not detected.

Figure 4

Inhibitory effect of (A) rose water, (B) rose oil, and (C) orange oil at different concentrations; (b) MIC (dotted line) and 2 × MIC (dot-dashed line). (a) Control (solid line) on the viable counts of C. sakazakii inoculated into MHB and incubated at different points of time (0, 1, 3, 6 and 24 h). Values are the mean ± standard deviation of three independent trials. ND, not detected.