LISTERIA MONOCYTOGENESIN RAW MILK AND DAIRY PRODUCTS; OCCURRENCE AND TRIALS FOR THEREDUCTION OF THEIR RESISTANCE TO ANTIBIOTICS

Fatma I. El-hofy 1 , Ashraf A. Abd El-tawab 2 , Elham A. Mobarez 3 , Mohamed F. Abo El-Maati, Nancy Y. Tawkol 4 and Ahmed A. Hefny 4 . 1. Bacteriology, Immunology and Mycology Department, Faculty of Veterinary Medicine, Benha University, 13736, Egypt. 2. Pharmacology Department, Animal Health Research Institute, Dokki, Giza, 12618, Egypt. 3. Biochemistry Department, Faculty of Agriculture, Zagazig University, 44511, Egypt. 4. Veterinary Hospital, Faculty of Veterinary Medicine, Zagazig University, 44511, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 01 October 2018 Final Accepted: 03 November 2018 Published: December 2018

Listeria monocytogenes is a facultative intracellular pathogen responsible for severe infection in animals and humans. This study aimed to determine the occurrence of Listeria monocytogenes in 200 samples of raw milk and dairy products marketed in Egypt, with detection the antimicrobial resistance profiles of the isolates for different antimicrobial agents. The synergistic effects of three plant extracts in combination with the used antimicrobials to control resistant L. monocytogenes isolates were assessed, with evaluation of relationship between the antibacterial activity and composition of plant extracts. A total of 5 (2.5%) L. monocytogenes isolates were identified (3 isolates from raw milk and two isolates from kariesh cheese and ice cream, one each). The isolates were sensitive to ciprofloxacin and sulphadimidine, while for amoxicillin and oxytetracycline, were resistant. The higher antimicrobial effects of basil and cinnamon extracts were detected, in addition, low concentrations of them were enough to show synergistic effect with the amoxicillin and oxytetracycline to control resistant isolates, these might be attributable to their chemical compositions. In conclusion, there is need for effectively pasteurized raw milk consumption or usage in the production of dairy products. In addition, the chemical composition of these plant extracts might help in its use to control resistant isolates with appropriate antibiotics without the need for increasing the used dosage doses.
Listeriosis is a serious infectious disease of humans and animals, caused mainly by Listeria monocytogenes. The large outbreaks in humans were caused by consumption of raw orpost-pasteurization contaminated milk and mainly characterized by low incidence but high case fatality rate (Kasalica et al., 2011). L. monocytogenesis susceptible to most antibiotics, except for cephalosporins and fosfomycin. However, major concerns have been raised due to reports of Listeria spp. that are resistant to a wide range of antimicrobial agents that are commonly used for the treatment of listeriosis. Thus, as an alternative, combination therapy has been found more effectivebecause synergistic interactions can be faster in therapy, prevent emergence of resistance and increase efficacy of the used combinations (Marr et al., 2004). In addition, it can reduce toxicity, and provide broader spectrum of activity.
The presence of different antimicrobial compounds in plants help scientists to use these plants in treatment of some infectious diseases. However, combination therapy of various plant extracts with antibiotics exhibiting bactericidal synergism is a method leading tonew ways for treatment of infectious diseases (Chanda and Rakholiya, 2011).
The current study aimed to determine the prevalence of L. monocytogenes in raw milk and dairy products; soft cheese, kariesh cheese and ice cream at Qalyubiyah and Giza governorates, Egypt. The antimicrobial resistance profile of the isolates was determined against four different classes of antimicrobial agents. Moreover, the total phenolic compounds, total flavonoids compounds and antioxidant activity of three plant extracts were evaluated. Also, the synergistic effects of the three extracts in combination with the used antimicrobials to control resistant L. monocytogenes isolates were assessed.

Materials and Methods:-
Bacterial isolates:-Isolation and identification Two hundred random samples from fresh milk (n=80) and dairy products(soft cheese, kariesh cheeseand ice cream, 40 each) were obtained from different outlets at Qalyubiyah and Giza Governorates, Egypt, during the period of October 2016 to January 2017. The collected samples were aseptically transported in ice box as soon as possible to the laboratory for isolation and identification of L. monocytogenes according to ISO (2004).
For isolation of L. monocytogenes, 25 gramsof collected dairy products were pre-enriched in 225 ml half strength Fraser broth (Oxoid -CM0895) for 24 h at 30°C, then followed by second enrichment of 0.1 ml of full strength Fraser broth for 48 h at 37°C. Similarly, 25 ml of collected fresh milk samples were passed the two step method for enrichment after PH adjusted to neutral. After the enrichment procedure, 0.1 ml of the broth was streaked onto PALCAM agar (Oxoid -CM0877) and incubated for 48 h at 37°C. Listeria monocytogenes were preliminary identified by their colonial morphology on PALCAM agar. The suspected colonies were sub-cultured on Tryptone Soya Yeast Extract Agar (TSYEA; Sigma-Aldrish) and subjected to Gram's staining, microscopic examination, catalase test, and oxidase test. The isolates were also sub-culturedon semisolid medium (Nutrient broth supplemented with 0.5% agar, Oxoid) at 25°C for 12-18 h for the detection the typical tumbling motility (ISO, 2004). For further identification, Microgen TM Listeria-ID System (LAB; MID-67) was used according to the manufacturers' guidelines. The phenotypically isolates were confirmed using PCR targeting 16s rRNA (Kumar et al., 2015).
Antimicrobial susceptibility testing:-Two-fold broth microdilution method was used to determine the minimum inhibitory concentrations (MIC) of theidentified strains against the antimicrobial agents amoxicillin, oxytetracycline, ciprofloxacin and sulphadimidine (Sigma-Aldrich), by cultivation in Mueller Hinton broth according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2012). There are no specific international interpretationcriteria for susceptibility testing of L. monocytogenesfor the examined agents. Therefore, the Staphylococcus spp. resistance breakpoints were used. The general resistance breakpoints of CLSI (2002) were used in case of amoxicillin, while, CLSI (2016) were used for tetracycline, ciprofloxacin and sulphadimidine. Resazurin dye as a redox indicator was used to detect the MIC values to give a high level of accuracy (Elshikh et al., 2016). Each isolate was examined in triplicate. The reference strain; L. monocytogenes (ATCC ® 35152) was used as a quality control. Results:-Bacterial isolates:-Five isolates (2.5%) from raw milk (n=3), kariesh cheese (n=1) and ice cream (n=1) of L. monocytogenes were preliminary identified by their colonial morphology. On PALCAM agar, the colonies were small 2-3 mm gray-green surrounded by diffuse black zone (esculin hydrolysis), and on TSYEA agar; pinpoint colonies were observed. The isolates were further confirmed by microscopical and biochemical examination. Molecular confirmation of the five isolates by the amplification of 1200 bp of the 16s rRNA gene; was also carried out.
The antimicrobial resistance profile determined by broth micro dilution method revealed that all L. monocytogenes isolates were resistant to amoxicillin and oxytetracycline, while, all the isolates were sensitive to ciprofloxacin and sulphadimidine.

Plant extracts:-
The best yield of extracts was obtained from (24.5%) dry basil leave. The total phenolic compounds, total flavonoid compounds and antioxidant activity of all plant extracts were detected. The results revealed that TPC, TFC, and antioxidant activity was higher in basil leave and cinnamon inner bark extracts than coriander leave extract (Table 1). Regarding antimicrobial activity of plant extracts, the MICs against clinical isolates for dry basil leave, cinnamon inner bark and coriander leave extractsare reported in Table 2.  Table 2.

Discussion:-
L.monocytogenes is one of the important emerging bacterial zoonotic diseases worldwide. It is associated with consumption of raw milk or dairy products made from unpasteurized milk (Dewé, 2014). The current studyaimed to investigate the occurrence of L. monocytogenes in raw milk, and dairy products (soft cheese, kariesh cheese and ice cream), obtained from retail outlets at Qalyubiyah and Giza Governorates, Egypt, and to determine the antimicrobial resistance profile. Furthermore, the current study evaluated a trial to reduce the resistance of multidrug resistantisolates by using different plant extracts which have antimicrobial effect with different concentrations.
The isolation rate of L. monocytogenesin raw milk samples was 3.75%. In accordance, Meshref et al. (2015) reported that the prevalence rates of L. monocytogenes in raw milk samples was 3.92%. However, fresh cow milk samples were free from L. monocytogenes in another study (Al Kassaa et al., 2016). Infected animals, poor silage quality and insufficient hygienic conditions during milking and storage are likely the most common causes of L. monocytogenes contamination in raw milk.
The Kariesh cheese is a home-made cheese made in Egypt and Palestine. The current study showed that the prevalence of L. monocytogenes was 2.5% in Kariesh cheese samples. monocytogenes isolates from 110 soft cheese samples. Regarding, ice cream samples; the prevalence rate of L. monocytogenes was 2.5%. Similar result was reported in Egypt (Tantawy, 2011). On contrary, high prevalence rate (26%) was reported in Greece (Effimia, 2015). The prevalence of L. monocytogenes in dairy products might reveal insufficient heat treatment of contaminated milk and improper sanitation of equipment.
The increased rate of L. monocytogenes resistance to one or more clinically relevant antibiotics has been reported (Morvan et al., 2010). The current study revealed that all isolates were sensitive to both ciprofloxacin and sulphadimidine. Similar results were previously reported (Karadal and Yildirim, 2014). However, all L. monocytogenes in the current study were resistant to amoxicillin and oxytetracycline. In accordance, Peter et al. (2016) reported that 16 L. monocytogenes isolates in Nigeria were resistant to amoxicillin and tetracycline. High resistance rates to amoxicillin and oxytetracycline may be partly attributable to overuse of these antibiotics in veterinary field, which increase genesmutation rate, in addition to acquisition of mobile genetic elements.
The continuous increase in microbial resistance to different types of antibiotics increases the need to find new classes of antibiotics. Antimicrobials of plant origin may give a hope to find a solution to this problem. Phenolic compounds are mainly considered as scavengers of free radicals (Gülçin et al., 2005). The bio-analytical analysis of plant extracts used in the current study demonstrated that basil and cinnamon had higher amount of TPC than coriander. In accordance, the high amount of TPC inbasil and cinnamon were reported (Snigdha and Monika, 2014). Also, higher amounts of TFC were reported in the current study than coriander. Similar finding was reported (Jangra et al., 2018).
The ability of natural antioxidants to inhibit the growth of bacterial pathogens has been documented to have influence on infectious diseases (Roginsky and Lissi, 2005). In the current study, the highest antioxidant activity was detected in basil followed by cinnamon then coriander. The antimicrobial activities of the ethanol extract of plant extracts against bacterial isolates were determined by evaluating the presence of MIC values, and thus, their potencies were assessed quantitatively (Yildiz, 2016). In the current study, the higher antimicrobial effects of basil and cinnamon were detected. These results might be conducted to the positive relationship between antibacterial activity and composition of plant extracts from TPC and TFC, in addition to antioxidant activity of plant extracts (Yildiz, 2016). The yield of extracts recovered from the examined dried plants in the current study ranged from 8.675 to 24.5%. The variation in the extraction yields from different plants is attributed to their varied chemical composition, efficiency of extraction and the surrounding climatic conditions, in addition to the phytochemical properties and differences among bacterial species (Brant et al., 2008). Also, the variations in the ability of antibiotics and plant extracts combinations to reduce drug resistant in bacteria might be conducted to their effect on efflux pumps, which need to be evaluated (Wagner and Ulrich-Merzenich, 2009).  (2006) reported that although the antimicrobial activity of a plant extract has not been relatively high, the synergism rates with other antibiotics were relatively high. In the current study, low concentrations of amoxicillin and oxytetracycline showed synergism with all plant extracts used against resistant isolates, especially, with basil or cinnamon extracts showed relatively high synergism than coriander extract. These findings confirmed that the activity of antibacterial combinations were more effective than individual agents. The same findings were reported in other studies. These results are consistent with a previous study mentioned that the double attack of two agents on different target sites of the bacteria could theoretically lead to synergistic effect (Olajuyigbe and Afolayan, 2012). For instance, agents that inhibitprotein synthesis (tetracycline) or interfere with cell wall synthesis (amoxicillin) had strongest synergistic effect with plant extracts (Betoni et al., 2006;Marzoog, 2013). Also, the antagonistic effect in vitro between coriander in combination with amoxicillin /clavulanic acid against L. monocytogeneswas reported (Shaaban et al., 2013). Ciprofloxacin and sulphadimidine showed neither synergistic nor additive effect against L. monocytogenes in the current study with the investigated plants. These results are consistent with those obtained byBetoni et al. (2006) who reported that none or weak synergism activity between nucleic acid synthesis inhibitors and plant extracts. In contrary, Shaaban et al. (2013) reported antagonistic effect between norfloxacin (Quinolones) and coriander. The absence of effect between plant extracts and ciprofloxacin or sulphadimidine in the current study might be because the isolates were sensitive to them. In conclusion, this study indicated that raw milk and dairy productscould be considered as sources of L. monocytogenes. Therefore, raw milk must be effectively pasteurized before consumption or usage in the production of dairy products. The chemical characters of ethanol extracts of basil, cinnamon and coriander, including; TPC, TFC and anti-oxidant properties, was in parallel to their synergistic effect with antibiotics. Furthermore, their concomitant administration together could be useful in treating L. monocytogenes infectionswith minimal concentrations of antibiotics, and subsequently minimal side effects from antibiotics.

Disclosure Statement:-
No competing financial interests exist.