Although milk and milk products represent as important vehicles for foodborne disease transmission to humans, in developing countries, limited publications have documented shigellosis outbreaks related to the consumption of milk and milk products. The present study showed a high prevalence of Shigella spp. (7%) with the predominance of S. dysenteriae, in comparison to the results of Ahmed and Shimamoto [21], who reported that Shigella spp. were detected in 0.5% of raw cow,s milk samples and 0.9% of Kareish cheese samples with S. flexneri as the predominant species. Tambekar and Bhutda [22] detected 8.7% S. flexneri in milk product (pedha) samples in India. In the current study, the level of contamination with Shigella spp. was higher in kareish cheese obtained from markets. The high rate of Shigella prevalence in this study might indicate poor hygienic measures used during milking, processing, preparation, handling, and storage of milk and milk products. Therefore, basic hygienic measures must be enforced in animal farms to reduce the risk of spread of Shigella to other animals and human.
The risk of Shigella might be higher in the raw cows milk and cheese, as compared to yoghurt products because yoghurt has very effectively inhibitory effect on the growth of the most common enteric pathogens such as Shigella [23,24]. In addition, the production process of yoghurt is entirely industrial type, while in the case of karish cheese, this process is completely hand made. Therefore, results revealed whatever the type of the sample, the presence of Shigella, but the risk appears to decrease as we move from products obtained in informal to those are industrially manufactured [25]. Overall, it is important to observe all hygienic measures while dealing with milk and milk products.
The antibiotic resistance of Shigella spp. isolated from raw cow milk and milk product samples in this study was compared with previous reports from Egypt to observe the trend in antibiotic resistance taking into account the sample collection method for each study. Overall, this study revealed the presence of harmful level of Shigella spp. resistant to the prevalently used antibiotics (TE, AM, AMC, CEC) among human and livestocks [26,27], though the presence of policies in Egypt regarding the use of antibiotics in both livestock and humans according to the World Health Organization. This resistance might be due to the frequent and improper use of such antibiotics either in animal therapy or as a growth promoter in the veterinary context in Egypt. Additionally, the current study showed reduced susceptibility to CTX, CAZ (third-generation cephalosporins) and CIP, which are considered preferable drugs for shigellosis treatment [12]. Thus, the appearance of such resistance would pose a great challenge for the efficient treatment of shigellosis.
In this study, approximately 71.4% of Shigella isolates were resistant to at least three of the antimicrobial classes with a MAR index of 0.2-0.5. In addition, many isolates of Shigella spp. were shown to have multi-resistance phenotypes against TE, AM, AMC, and CEC. Similarly, a high prevalence of MR Shigella isolates in dairy products (90.9%) was reported by Ahmed and Shimamoto [21] in Egypt. Therefore, some measures must be considered to confirm that the currently available antibiotics remain effective. These measures may include increasing the awareness among the public, healthcare professionals and the food-agriculture sector regarding the importance of the proper use of these medicines.
The presence of Shigella in the the analysed samples was an indicator of poor hygiene and sanitation during milking, post milking and during milk processing. The effectiveness of disinfection depended on the use of a suitable disinfectant, which is considered the most critical aspect of hygienic measures used in dairy cattle farms. Phenolic compounds and BKC are widely used as farm disinfectants due to their antimicrobial activity [28]. BKC is a cationic, surface-active QAC commonly used as a farm disinfectant for cleaning and sanitizing livestock buildings, equipment, milk utensils, and vehicles. The present study demonstrated that all the tested strains were resistant to phenolic compounds, while 85.7% of the isolates were resistant to BKC. Similarly, Bouzada et al. [29] found that gram-negative rods of Enterobacteriaceae exhibited low susceptibility to BKC. Moreover, this work demonstrated that most of the Shigella isolates (85.7%) harboured the qacE∆1 gene.
Various β-lactamases, which hydrolyse the β-lactam ring and thereby inactivate β-lactam antibiotics, have been described, but TEM-, OXA-, SHV- and CTX-M-type β-lactamases are dominant in gram-negative bacteria [30]. Thus, in this investigation, the presence of these β-lactamase-encoding genes in isolates was recognized by molecular methods, which provided data to support the present study. The blaTEM gene, a narrow-spectrum β-lactamase gene that confers resistance to penicillins and first-generation cephalosporins, was identified in all isolates. Additionally, the ESBL-encoding gene blaCTX-M was identified in 28.6% of the isolates. The high incidence of β-lactamase-encoding genes (blaTEM-1, blaCTX-M, in 2 isolates; blaOXA, in 4 isolates) had been detected previously in Shigella strains isolated from dairy products in Egypt [21]. The blaTEM gene was the dominant β-lactamase gene in Shigella spp. in this work, while blaCTX-M was the most common type of cefotaximases identified among Shigella isolates in a previous study [11]. Alarmingly, in this research, the prevalence of ESBL-producing Shigella isolates, accounting for 28.6% of all Shigella isolates, was higher than the detection rates observed in other countries, such as England [31]. This discrepancy between these findings and previous studies might be attributed to the misuse of antibiotics during the treatment of bacterial infections. In addition, the high TE resistance in all Shigella isolates might be explained by the potential distribution of the tetA(A) resistance gene [32].
For epidemiological investigations of various enteric pathogens, PP could be an attractive tool. Shigella spp. usually harbour various plasmids, with 2 to 10 plasmids being harboured by one strain. These plasmids are required for antibiotic resistance and for bacterial invasion of intestinal epithelial cells [33]. Seven plasmid patterns, with relative plasmid sizes ranging from 1.26 to 33.61 kb, were detected in this study. It had been reported previously that Shigella spp. in Egypt harbour varying numbers of plasmids ranging in size from 1.0 to 120 MDa [34]. All MR strains, particularly ESBL producing strains, carried plasmids with pattern P7 as the predominant pattern in this study. The ESBL-encoding gene (blaCTX-M) is present on plasmids with greater frequency than genes encoding other class A β-lactamases [35]. The results of the conjugation experiment aided the determination of plasmid locations of the blaTEM, blaCTX-M, and qacE∆1 genes because transconjugants of the MR Shigella isolates were grown on MacConkey agar. Over the last half-century, the extraordinary ability of different isolates to acquire plasmid-encoded resistance to disinfectants and antibiotics, such as ESBLs, which could quickly be transmitted to several other strains, has been demonstrated [36,37].
Antibiotics and disinfectants have been commonly used in dairy farms in Egypt. The most commonly used antimicrobials are the β-lactams, tetracyclines, aminoglycosides, lincosamides, macrolides and sulfonamides [38]. Antibiotics may be used indiscriminately for the treatment of bacterial diseases or they may be used to enhance animal growth and feed efficiency. The ongoing hazard of antibiotic resistance is one of the biggest challenges to public health that is faced not only by the African people, but also by the human population worldwide [39].
However, the susceptibility of Shigella to disinfectants and its contribution to the multidrug resistance phenotype and genotype by plasmid co-selection had never been reported. Plasmid-mediated multidrug resistance should be considered when studying infectious diseases. Therefore, this work was planned to explore the link between the qacE∆1 gene, plasmids, and antibiotic resistance. The results of this study showed that all qacE∆1 gene-positive strains were MR strains and harboured plasmids. Recently, it was demonstrated that the qacC gene confers resistance to a number of β-lactam antibiotics [40]. The ability of qac genes to directly acquire resistance to antibiotics was found. This finding indicated a close relationship between resistance to antibiotics and antiseptics [41]. Plasmids frequently transfer qac genes with a number of other antibiotic resistance genes [42,43]. The plasmid analysis and conjugation experiments showed that the isolates harboured various detectable plasmids and that the antibiotic and disinfectant resistance genes could be co-transferred. This finding indicated that the resistance was plasmid-mediated, so there was a high risk for the spread of antibiotic and disinfectant resistance genes among the bacteria.
The limitations of this study should be mentioned. Although this work explored for the first time the relationship of resistance to antibiotics and disinfectants with plasmids in MR Shigella spp. in Egypt, it focused on raw cow milk and milk product samples which collected randomly from only one province of Egypt and did not elucidate such relationships in other provinces. Therefore, additional studies are warranted to explore such relationships in other provinces of Egypt.