Plasmid prole of extended-spectrum β-lactamase and quaternary ammonium compound E delta 1 gene held multi-resistant Shigella species isolated from raw cow milk and milk products in Egypt

Background Multi-resistant Shigella species recovered from raw cow milk and milk products has predominated all over the world especially extended spectrum β-lactamases (ESBLs) and Quaternary ammonium compound E delta 1 (qacE ∆ 1) genes. This study was conducted to investigate the prevalence, antibiotic and disinfectant resistance phenotypes and genotypes as well as plasmid proles of Shigella species isolated from raw cow milk and milk products in Egypt. The genotypic analysis was determined for the presence of β-lactamase encoding genes (blaTEM, blaCTX-M, blaOXA-1 and blaSHV), tetA(A) and qacE ∆ . Results Twenty-one (7%) of Shigella isolates (S. dysenteriae, S. exneri and S. sonnei) were recovered with S. dysenteriae as the most predominant types. Antibiotic sensitivity tests showed 71.4% of multidrug-resistant Shigella isolates. High resistance rates were observed to tetracyclines (100%), ampicillin, amoxicillin-clavulanate (90.5%, each) and cefaclor (66.7%), whilst no resistance was detected against imipenem, sulfamethoxazole/trimethoprim, and azithromycin. Disinfectant susceptibility test of Shigella isolates revealed resistance to phenolic compound (vanillic), while 85.7% of Shigella isolates were benzalkonium chloride resistant. Uniplex PCR analysis exhibit presence of β-lactamase encoding genes (blaTEM in all isolates and blaCTX-M in 28.6% of isolates), tetA(A) in all isolates and 85.7% isolates positive for qacE ∆ 1, while all isolates were negative for blaOXA-1 and blaSHV. All Shigella extended spectrum β-lactamase (ESBL) producers (6, 100%) were positive for blaTEM, blaCTX-M, and qacE ∆ 1 genes. Furthermore, plasmid proling revealed seven distinct plasmid patterns (P1-P7) ranging from 1.26 to 33.61 kb among all Shigella strains; S. dysenteriae displayed the greatest variance. The co-transfer of β-lactamase genes (blaTEM and blaCTX-M) and qacE ∆ 1 genes was observed by conjugation. Conclusions S. dysenteriae was the most common identied types in the examined sources. Also, the ndings imply the emergence of multi-resistant Shigella species either multi-resistant to antibiotics (particularly ESBL producer strains) or disinfectants in Egypt. Thus, the resistance of Shigella species should regularly be monitored and appropriate measures should be taken to manage this problem.


Background
Infections caused by Shigella species are the main causes of bacillary dysentery, which is associated with high morbidity and mortality, especially in developing countries, such as Egypt [1]. Additionally, Shigella spp. infect and cause corresponding clinical symptoms in cows, chickens, pigs, monkeys and other animals which ignored in the previous studies [2]. Members of the Shigella genus are classi ed into four species: S. dysenteriae, S. exneri, S. boydii, and S. sonnei. Endemic shigellosis in developing countries is caused mainly by S. exneri, but S. sonnei is commonly detected in industrialized countries [3]. Dairy products especially raw milk and unpasteurized cheese remain important vehicles for the transmission of Shigella to humans [4,5]. Analyzing the epidemiological characteristics and resistance patterns of Shigella is essential for animal and human health [6].
There are many ESBL variants known in a variety of pathogens (blaCTX-M,blaTEM,SHV and blaOXA-1) that have been proved to be the most successful in terms of antibiotic resistance and for epidemiological niches.
Antibiotic therapy for Shigella infection can decrease the extent and severity of the disease [7] . As a consequence of the misuse of antibiotic administration, multi-resistant (MR) Shigella species de ned as resistance to multiple antimicrobial drugs are predominant worldwide, particularly those with extended spectrum β-lactamases (ESBLs), which is one of the strongest extended resistance mechanisms [8,9]. Many ESBL variants are known to be present in a variety of pathogens, including blaCTX-M, blaTEM, SHV and blaOXA-1, which have been proven to be the most successful in terms of antibiotic resistance and epidemiological niches [9]. Among blaTEM ESBL types, blaTEM-1 is the most commonly plasmid-mediated β-lactamase of ampicillin (AM) resistant gram-negative bacteria [10]. The selection of antibiotic treatment for shigellosis is made more di cult by the generation of resistance in Shigella spp., especially the resistance to third-generation cephalosporins, which is a common public health problem, mainly in developing countries [11,12]. The evolution of multidrug-resistant (MDR) strains, termed as resistance to at least one antibiotic belonging to at least three antibiotic classes, is common due to the existence of mobile genetic elements such as; plasmids, integrons, and transposons that assist Shigella spp. in the acquisition and transfer of exogenous genes [13] . Antibiotic resistance plasmids often include genes conveying resistance to numerous different antibiotics [14].
Disinfectants, including phenolic compounds (vanillic acid) and quaternary ammonium compounds (QACs), such as benzalkonium chloride (BKC), are commonly used in dairy cattle farms. The capability of phenolic compounds to inhibit the growth of any microorganism depends on their interaction with proteins and/or on their ability to impair membrane permeability [15]. A particular concern is that repeated use of disinfectants may cause the persistence of bacteria with reduced susceptibility not only to antiseptics but also, possibly, to antibiotics [16]. One of the major mechanisms underlying such resistance is the acquisition of the resistance genes qacE and qacEΔ1, which confer resistance to QACs [17]. qacEΔ1, a mutant version of qacE, is widely distributed throughout gramnegative bacteria, mainly in Enterobacteriaceae [18].
Multiple global studies have reported the molecular basis of antibiotic resistance in clinical Shigella isolates of human origin [19,20]but, inadequate data are available on the genotypic and phenotypic characteristics of Shigella spp. isolated from raw cow milk and milk products in Egypt. Therefore, this study was planned to isolate and characterize Shigella spp. from raw cow milk and milk products in Egypt. This work was also proposed to examine the prevalence of MR Shigella particularly ESBL producing strains, screen the isolates for the presence of β-lactamase, tetA(A) and quaternary ammonium compound E delta 1 (qacE∆1) genes, and examine their plasmid pro les.

Results
Occurrence of Shigella species in raw cow milk and milk products A total of twenty-one (7%) organisms were isolated and identi ed as Shigella spp. S. dysentriae was the most frequently identi ed, comprising 12 isolates (57.1%), followed by S. exneri with 6 isolates (28.6%) and S. sonnei with 3 isolates (14.3%). The occurrence of the isolates in the samples is presented in Table 1. The occurrence of Shigella spp. was higher in Kareish cheese (13%) obtained from markets than in raw cow milk (8%) obtained from dairy farms. In addition, the absence of Shigella spp. was observed in all examined yoghurt samples. These are indicative of high contamination level at market point sampled kareish cheese.
With regard to disinfectant resistance (Table 2), phenolic compound (vanillic acid) showed no efect on the growth of the Shigella isolates, no inhibition zone around the discs, as well as the effect of saline used as control. Although, it had larger zone of inhibition (19mm) against Escherichia coli ATCC 25922 which was used as quality control strain. Approximately 18 (85.7%) of the Shigella isolates exhibited BKC tolerance (no inhibition zone around the discs).

Characterization of antibiotic and disinfectant resistance genes
Uniplex PCR assay results revealed that all Shigella spp. isolates were positive for blaTEM, and only 6 (28.6%) isolates were positive for blaCTX-M, while all the Shigella isolates were negative for blaOXA-1 and blaSHV (Table  4) (Figures S1, S2, S3, S4). Out of the 21 Shigella isolates, 6 (28.6%, 3 S. exneri and 3 S. sonnei) were considered ESBL producers based on CTX and CAZ resistance phenotype, alone and in combination with clavulanate, by the double-disc synergy test. The genotype analysis demonstrated the existence of ESBL-encoding genes that were responsible for ESBL production in Shigella isolates. All Shigella ESBL producers (6, 100%) were positive for blaTEM and blaCTX-M genes, while none of the isolates harboured the blaOXA-1 and blaSHV genes. The TE resistance gene, tetA(A), was identi ed in all Shigella isolates. Furthermore, 18 (85.7%) of the Shigella isolates possessed a QAC resistance gene (qacE∆1) ( Figure S5). The association of qacE∆1 with antibiotic resistance is presented in Table 4, which shows that all MR strains harbour the qacE∆1 gene. In addition, all qacE∆1 genepositive strains were blaTEM and tetA(A) genes positive.

Plasmid pro ling and conjugative transfer
Plasmid pro ling (PP) revealed seven distinct plasmid patterns (P1-P7) ranging from 1.26 to 33.61 kb among the Shigella strains; S. dysenteriae yielded the greatest variance (Table 4) ( Figure S6). All plasmid patterns (P1-P7) were distributed in a similar percentage ( According to spatial information, there were spatial relationships observed among S. dysenteriae strains (P1, P3) isolated from raw milk samples collected from Gamsa and Belqas districts, as showed in Table 4.
The co-transfer of β-lactamase genes (blaTEM and blaCTX-M) and qacE∆1 genes was observed by conjugation. After the conjugative test using the plating mating method, the co-transfer of β-lactamase genes (blaTEM and blaCTX-M) and qacE∆1 genes was observed by conjugation from all ESBL producing strains (n=6) as donor strains to the azide-resistant E. coli J53 as the recipient strain (Figures. S1, S2, S5). All obtained transconjugants successfully acquired these resistance (blaTEM, blaCTX-M and qacE∆1) genes from donor strains.

Discussion
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. exneri as the predominant species. Tambekar and Bhutda [22] detected 8.7% S. exneri 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 e cient 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 con rm 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 rst-generation cephalosporins, was identi ed in all isolates. Additionally, the ESBL-encoding gene blaCTX-M was identi ed in 28.6% of the isolates. The high incidence of βlactamase-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 identi ed 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 ndings 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-en coded resistance to disinfectants and antibiotics, such as ESBLs, which could quickly be transmitted to several other strains, has been demon strated [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 e ciency. 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 nding 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 nding 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 rst 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.

Conclusion
This study demonstrated the prevalence of MR Shigella species that were either MR to antibiotics (particularly ESBL-producing strains) or disinfectants in raw cow milk and milk products in Egypt, posing a possible hazard to animal and public health and causing di culty in controlling outbreaks. Thus, the resistance of Shigella species should regularly be monitored, and appropriate measures should be taken to avoid the emergence and spread of MR strains. Additionally, strict hygienic measures must be applied throughout dairy industry chain to prevent infection through the consumption of dairy products.

Sample collection and microbial analysis
A total of 300 dairy product samples (100 raw cow milk from different dairy farms, 100 Kareish cheese, and 100 yoghurt samples from shops, and supermarkets) were collected randomly from three districts "Gamasa, Belqas and Dekernes" in Dakahlia Governorate, Egypt, throughout 2018. Raw cow milk samples were collected from the udder of cows (a composite sample from four quarters for each cow). After collection, the samples were stored at 4°C until examination (within 3-4 h). Then, homogenization of 25 ml or g of each sample was performed in 225 ml of 0.1% buffered peptone water (Oxoid, England) by shaking for 5 min in sterile Stomacher bags and incubating for 24 h at 44 °C and 42 °C for Shigella sonnei and other Shigella species, respectively, for recovery. A loop from the enriched cultures was directly inoculated into Selenite F broth and then subcultured onto Salmonella-Shigella (S-S) agar, MacConkey agar and xylose-lysine-deoxycholate (XLD) agar (Oxoid, UK), followed by incubation at 37°C for 24 h. The presumptive Shigella isolates (colourless and non-lactose fermenting on S-S agar, white and translucent on MacConkey agar, and pink to red colonies on XLD agar) were biochemically con rmed with triple sugar iron (TSI) agar, lysine iron agar (LIA), methyl red, Voges-Proskauer (VP) broth, the indole test, urea agar (UA), Simmon's citrate agar (SCA) and a motility test. Serotypes of the isolates were determined by slide agglutination assays, using a commercially available kits as described by the manufacturer (Difco Laboratories). All bacterial isolates were stored at −80 °C in tryptic soy broth (TSB) containing 25% glycerol for further analysis.
Antibiotic resistance evaluation Antibiotic susceptibility testing was performed using the disc diffusion method according to the standards and interpretative rules described by the guidelines of the . The sensitivity pattern was scored as tolerance (-) and sensitive (+).

Screening for antibiotic and disinfectant resistance genes
For ampli cation of β-lactamase-encoding genes (blaTEM, blaCTX-M, blaOXA-1, and blaSHV), TE resistance genes and QAC resistance genes (qacE∆1), uniplex PCR assays were performed with primers provided by Metabion (Germany) ( Table 5). These resistance genes were mostly encoded in gram-negative bacteria. Chromosomal DNA from the Shigella isolates was extracted by the QIAamp DNA Mini Kit (Qiagen, Germany, GmbH) with the modi cations recommended by the manufacturer. The PCR ampli cation reaction was carried out in an Applied Biosystems 2720 thermal cycler using speci c pro les ( Table 5). The PCR products were subjected to electrophoresis on a 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer using a gradient of 5 V/cm. After staining with ethidium bromide, the gel was visualized under UV light. Escherichia coli ATCC 25922 was used as a negative control in the PCR assay.

Plasmid pro le analysis and Conjugative transfer
The Plasmid Midi kit (Qiagen, Germany, GmbH) was used for plasmid DNA isolation from all clinical isolates (n=21). Gel electrophoresis of the PCR products on an 0.8% agarose gel (Applichem, Germany, GmbH) was performed. The fragment sizes were determined by a GeneRuler 1Kb Plus DNA Ladder (Fermentas, Thermo Scienti c, Germany) without using any restriction enzymes. In a trial to prove the association of these plasmids with ESBL-based antibiotic resistance, conjugation experiments were carried out using the azide-resistant E. coli J53 as the recipient and all Shigella ESBL producers (n=6) as the donors, as described previously [53]. Transconjugants were detected by plating mating mixtures on MacConkey agar supplemented with 150 mg/L sodium azide and 2 mg/L cefotaxime. Plasmid DNA was extracted from E.coli J53 transconjugants and cotransfer of resistance determinants was determined through ampli cation of the relevant genes (blaTEM, blaCTX-M and qacE∆1) in the transconjugants by PCR as previously described.

Declarations
Availability of data and materials The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
This article does not contain any animal studies performed by any of the authors.

Competing interests
The authors have declared that they have no competing interests.
Author contributions RME, RAE, MME, and MMA conceived and designed the experiments. RME, RAE, MME, and MMA performed the experiments. RME and MME analysed the data. RME, RAE, MME, and MMA contributed reagents/materials/analysis tools. RME, RAE, MME, and MMA wrote the paper. All authors reviewed the manuscript.       Figure S1. Representative gel showing ampli cation of the blaTEM gene (516 bp) from Shigella isolates.