Mastitis is one of the most prevalent diseases in dairy cows, producing well-recognized detrimental effects on animal health and profitability of dairy farms (Ruegg, 2017; Abd El-Aziz et al., 2021a). Treatment of mastitis is often applied without prior knowledge of cause-related agents and mostly with broad-spectrum antimicrobials, which are known to enhance resistance evolution to a greater extent. This increases the selective pressure on potentially present pathogens and is considered a possible human health hazard (Neyra et al., 2014; Abd El-Aziz et al., 2021b).

Raw or processed milk is a good environment that promotes the existence of many pathogens, which could be either directly obtained via dairy cows, or indirectly from the environment of the farm (Oliver et al., 2005). Escherichia coli (E. coli) O157:H7 outbreaks associated with the consumption of raw milk have been described (Disassa et al., 2017; Ababu et al., 2020). The indiscriminate use of antimicrobials has led to the development of multidrug-resistant (MDR) Gram-negative bacteria in raw milk, in particular E. coli O157:H7, Klebsiella pneumoniae (K. pneumoniae), Aeromonas hydrophilia (A. hydrophilia), and Proteus mirabilis (P. mirabilis) (Zelalem and Bernard, 2006; Oliver et al., 2009; Garedew et al., 2012).

Resistance to most antimicrobial classes and the lack of new antimicrobial agents against Gram-negative bacteria potentiates the re-use of old antibiotics, particularly polymyxins such as colistin (Biswas et al., 2012). Colistin is currently known as the last-resort antimicrobial agent for the treatment of infections caused by MDR Gram-negative bacteria (Poirel et al., 2017). It binds to the negatively charged lipopolysaccharide (LPS) of the outer membrane of Gram-negative bacteria, leading to disruption of the membrane, induces the cytoplasmic material to fade away, and eventually cell death (Yahav et al., 2012).

Colistin has been greatly utilized in the agricultural system and veterinary medicine for decades, as a growth promoter and for the treatment of enterobacterial infections (Catry et al., 2015). Extensive colistin usages in livestock have facilitated the rapid spread of the mcr resistance genes (Shen et al., 2016). The mcr-1 plasmid-mediated colistin resistance gene has been first reported in Enterobacterales isolated from animals, food, and humans in China (Liu et al., 2016). Numerous studies have documented plasmid-borne mcr variants (mcr-1 to mcr-9) from this time onward (Xavier et al., 2016; Yin et al., 2017; Borowiak et al., 2019; Carroll et al., 2019; Gelbíčová et al., 2019; Wang et al., 2019). However, studies focused on mcr-resistant isolates from mastitis and raw unpasteurized milk have received little attention. Here, the genome sequences and genetic relatedness between MDR and extensive drug-resistant [XDR; resistance to at least one antibiotic in all tested antimicrobial groups except one or two as demonstrated in the worksheet of Magiorakos et al. (2012)]. Gram-negative bacteria isolated from bovine milk samples have been studied for the first time. This study aimed to: (1) investigate the occurrence and resistance phenotypes of the Gram-negative bacteria in mastitic cow’s milk as well as raw unpasteurized milk that consumed by the public; (2) identify the multilocus sequence types (MLST), resistance genes, and plasmid replicon types of MDR and XDR Gram-negative isolates; (3) determine the genetic environment of mcr-10 gene and epidemiological relatedness between the study isolate and the publicly available genomes of K. pneumoniae isolates harboring mcr genes.

The development of resistance to numerous antimicrobial agents in pathogenic microbes has become a critical threat, as there are fewer or indeed no effective antimicrobial agents beneficial for treating these organisms. The recent global dissemination of MDR and XDR Gram-negative bacteria has raised the alarm of antimicrobial resistance as a serious and an urgent threat to public health. Consequently, studies have been conducted to explore the distribution of several resistance genes in different reservoirs (Tartor and El-Naenaeey, 2016; Elmowalid et al., 2018; Abd El-Aziz et al., 2021c).

Mastitis is considered one of the most frequent diseases in dairy cows in Egypt. Here, we studied MDR and XDR Gram-negative bacteria isolated from mastitic and raw unpasteurized milk. Many studies have confirmed the presence of Gram-negative bacteria in mastitic milk (Hogan and Larry Smith, 2003; Malinowski et al., 2006; Nam et al., 2009). Similarly, we identified a variety of Gram-negative bacteria from milk samples. The most frequent was E. coli (48.91%), followed by P. mirabilis (17.9%), while the least isolated one was C. freundii (2.1%). Comparably, Nam et al. (2009) declared that E. coli predominated the Gram-negative pathogens from milk samples, while C. freundii was the least frequent one. Our prevalence of Gram-negative bacteria in subclinical mastitis was lower than those reported earlier (Mahlangu et al., 2018). These variations in the prevalence could be attributed to the changes in the host, the management factors, and the environmental differences that impact the infection (Amin et al., 2013).

Interestingly, we reported 24.46% (45/184) of the isolated bacteria from raw unpasteurized milk, which directs our attention to the human being, especially when talking later about MDR and XDR isolates as well as colistin resistance. A recent study revealed the presence of Gram-negative bacteria in raw milk and other dairy products in Egypt and imputed this contamination to the evidence that most of Gram-negative bacteria constitute a part of the flora of the mouth and intestinal tract of humans and animals as well as their presence in soil, water, and sewage (Sobeih et al., 2020).

Multidrug-resistant isolates were known to be common in mastitis milk samples. Herein, A. hydrophila, K. pneumoniae, E. cloacae (100% each), E. coli (87.78%), and P. mirabilis (69.7%) were MDR. All MDR A. hydrophila isolates were isolated from raw milk. Previous reports declared that MDR A. hydrophila isolates were commonly isolated from water and fish samples (Igbinosa et al., 2013; Tartor et al., 2021a), which affirm the role of human beings and environment in transferring MDR A. hydrophila to the raw milk.

Similarly, although we identified all E. cloacae as MDR isolates, they were previously reported with a low frequency (7.1%) in milk samples from Egypt in 2011 (Ahmed and Shimamoto, 2011). However, they were recently detected in human samples (Huang et al., 2021), which in turn emphasizes again the concept of transferring between animal/animal products including milk from/to human and animal during these 10 years. Alternatively, MDR K. pneumoniae isolates were widely detected in milk samples (Yang et al., 2020) and this is concordant with what was reported in our study.

It is known that E. coli is the major pathogen causing environmental mastitis and the spreading of MDR E. coli is considered a key challenge in mastitis treatment, especially, when combined with an XDR phenotype (Basak et al., 2016). We identified 7.07% XDR E. coli and P. mirabilis isolates. XDR is considered a pop-up threat; the emergence of commonly XDR, carbapenem-resistant, and ESBL producing E. coli reviewing the abundant usage of the broad-spectrum antimicrobials. A former report on MDR and XDR P. mirabilis infections has revealed that aminoglycosides, penicillins, cephalosporins, carbapenems, and fluoroquinolones have become ineffective as first-line agents (Magiorakos et al., 2012). Serotyping of E. coli isolates showed 10 different serotypes. As expected from the previous reports, O111:H4 was the predominant type in clinical and subclinical mastitis, whereas O157:H7 was prevalent in raw milk (Denny et al., 2008; Sayed, 2014; Ababu et al., 2020).

Since the first colistin-resistant mutant that was reported in 1981 (Vaara et al., 1981), colistin resistance has been evolving in several microorganisms like A. baumannii (Potron et al., 2019), K. pneumoniae (Shankar et al., 2019; Tartor et al., 2021b), and E. cloacae (Huang et al., 2019). A recent study has reported mcr-harboring isolates from animals and humans (Luo et al., 2020) and another group has isolated mcr-positive E. coli and K. pneumoniae from cancerous patients in Egypt (Zafer et al., 2019). Concordant with previous studies, we reported mcr-10 harboring K. pneumoniae.

To go deeper in the genetic and molecular characterization of MDR and XDR Gram-negative isolates, we selected the isolates with the highest MAR indices for WGS analysis. E. coli were found to have two-allele sequence types; ST2165 and ST7624. Although ST2165 was previously detected in colistin-resistant E. coli carrying mcr-1 from residents of long-term-care facilities (Giufrè et al., 2016), it was isolated elsewhere from chickens and store-bought produce (Reid et al., 2020; Foster-Nyarko et al., 2021). To our knowledge, this is the first isolation of this sequence type from milk. Similarly, E. coli ST7624 was not identified before from milk samples, although it was reported in wildlife (Arribas, 2019). In the view of plasmid replicon types, we found that the three E. coli isolates belonging to ST2165 have plasmid replicon types; IncFIA, IncFIB, IncFII, and IncQ1. Likewise, Foster-Nyarko et al. (2021) reported that the MDR isolates often co-carried large IncF plasmids like IncFIA, IncFIB, or IncFIC.

Herein, we found that E. coli isolates belonging to ST2165 are carrying several antibiotic resistance genes (Table 2), which confirms that IncF plasmids can play a potential role in disseminating antimicrobial resistance (Adenipekun et al., 2019). Comparably, the E. coli isolate belonging to ST7624 possessed multiple resistance genes to B-lactams (bla_CMY–4, bla_TEM–1B and bla_{CTX–M–14b}), aminoglycosides [aadA1, aph(3′)-Ia, aac(6′)-Iaa and aadA2b], tetracyclines [tet(A)], sulfonamides (sul3), macrolides (erm), lincosamide (lnuF), and phenicols (floR, cmlA1), that might be attributed to the presence of IncFII, IncHI2, IncHI2A, and IncI1α replicon types. In a previous study, 27 acquired antimicrobial resistance genes were located on the IncHI2-IncHI2A-type plasmid, including bla_CTX–M–65, fosA3, mphA, qepA, and rmtB (Zhang et al., 2021).

On the other hand, WGS analysis of Klebsiella species showed that K. pneumoniae had a novel ST, that was closely matched to ST1224, and ST48 alleles. In earlier studies, K. pneumoniae ST1224 was isolated from neonates (Chen et al., 2019), while K. pneumoniae ST48 was reported in chicken meat in Western Algeria, one of the Middle East region (Chaalal et al., 2021) indicating that these STs are not host specific and could be easily transmitted to humans from food animals and their products.

On contrary, although we identified ST11 in K. aerogenes, a previous study found ST11 in K. pneumoniae (Hao et al., 2019).

Interestingly, this study is the first isolation of mcr-10-carrying K. pneumoniae isolate from a raw milk sample. The mcr-10 gene flanked by xerC and IS26 in K1 isolate, xerC and IS2 in isolate Ecl_20_981 (CP048651.1) and xerC and IS21 in isolate STW0522-51 (AP022432.1). This possibly indicates the role of xerC gene, that encodes a tyrosine recombinase, in the mobilization of mcr-10 gene (Wang et al., 2020; Xu et al., 2021). Although the mcr-10 gene was detected recently in clinical strains of Enterobacter roggenkampii from different sources such as chickens (Lei et al., 2020), a patient (Wang et al., 2020), and sewage water (Xu et al., 2021), it is foremost found here in the raw milk sample that sequentially implicating the human in the transmission. Numerous isolates carrying mcr-1 gene obtained from hard cheese made from raw milk were reported in Egypt (Hammad et al., 2019; Ombarak et al., 2021), which in turn highlights the importance of raw milk quality control check in Egypt focusing on farm workers as well as the animal itself.

Furthermore, our mcr-10-producing K. pneumoniae isolate was also carrying fosA5 gene, which is responsible for fosfomycin resistance. Fosfomycin was known to be always effective in the treatment of MDR Enterobacterales (Falagas et al., 2010). However, such identification of the fosA5 gene in one of Enterobacterales species; K. pneumoniae (K1), isolated mainly from raw milk, is considered a terrifying situation. Recently, some studies reported the fosfomycin resistance in isolates from beef samples (Sadek et al., 2020), human sputum (Soliman et al., 2020), and chicken feces (Soliman et al., 2021) in Egypt. This might be attributed to the habitual utilization of fosfomycin in combination with aminoglycosides widely in Egypt for the treatment of numerous diseases. Due to the misuse of different antimicrobial agents in Egypt, we found K. pneumoniae isolate was carrying not only the mcr-10 and fosA5 genes, but also multiple resistance genes to ß-lactamases, aminoglycosides, quinolones, tetracyclines, macrolides, and folate pathway inhibitors; unfortunately, these antimicrobials are used at both human and veterinary levels.

To correlate the genetic character of our K1 isolate with similar K. pneumoniae isolates worldwide, we performed a phylogenetic analysis to find out their epidemiological relationship.

Probably, we found that our isolate was clustered with 25 K. pneumoniae isolates from different sources including human, environment, and animal (Figure 3) supporting the direct/indirect transmission between human and animals and this in tune with the recent global concept of dissemination of mobilized colistin resistance genes from animals to humans (Luo et al., 2020). Followingly, this result emphasizing “the One Health” concept; human health is connected to the health of animals and the environment (Luo et al., 2020). Remarkably, this result affirms that mcr genes debilitate human wellbeing and contaminate the environment due to the plausibility of their worldwide spread brought from the raising, generation, worldwide exchange, and food utilization of animals (Anyanwu et al., 2020).

Similarly, when comparing the study isolate (K1) with different mcr-10 harboring K. pneumoniae, our isolate presented in a clade consisting of isolates from numerous sources such as human and environment. Strikingly, this is the first identification of colistin mcr-10 resistance gene in K. pneumoniae in the Middle East. Entirely, all formerly identified mcr-10 carrying K. pneumoniae were detected in China, Europe, and Canada from clinical or environmental sources only. Given this close genetic distance between K1 isolate and the other mcr-10 isolates (Figure 4), we investigated the sequence type of each isolate and they showed different sequence types. This agrees with what was reported by Luo et al. (2020), who documented that the sequence type diversity of the mcr-positive E. coli isolates showed a scattered and non-clonal prevalence.

The emergence of mcr-10 and fosA5 harboring K. pneumoniae from raw milk is alarming and poses a public health threat. The existence of multiple resistance genes in MDR and XDR Gram-negative bacteria from dairy cattle provides evidence for its spread from the veterinary sector to humans and heralds the penetration of the last-resort antibiotics. Hence, prudent use of antibiotics in both humans and animals and antimicrobial surveillance plans are urgently required to fight XDR and MDR bacteria.

The data that support the findings of this study are available in the SRA database under the bioproject PRJNA713498.

Ethical review and approval was not required for the animal study because this study did not include any experimental animals. Written informed consent was obtained from the owners for the participation of their animals in this study.

YHT and NKA contributed equally in the conception and design of the study and participated with RMAG and HME in the application of classical microbiological and molecular techniques. ME participated with HR in whole-genome sequence analysis. HR performed the bioinformatics. RMAG, HME, SE, EAS, ASAA, SSA, MMB, YAE-S, and ME conceived the study and participated in the design. RMAG, HME, SE, EAS, ASAA, SSA, MMB, YAE-S, and ME participated in interpretation of data. YHT, NKA, RMAG, HME, SE, and HR wrote the initial draft of the manuscript. All authors revised the manuscript critically for important intellectual content and gave the final approval of the version to be published.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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