Plasmid-Mediated Colistin Resistance in Salmonella enterica: A Review

Colistin is widely used in food-animal production. Salmonella enterica is a zoonotic pathogen, which can pass from animal to human microbiota through the consumption of contaminated food, and cause disease, often severe, especially in young children, elderly and immunocompromised individuals. Recently, plasmid-mediated colistin resistance was recognised; mcr-like genes are being identified worldwide. Colistin is not an antibiotic used to treat Salmonella infections, but has been increasingly used as one of the last treatment options for carbapenem resistant Enterobacteria in human infections. The finding of mobilizable mcr-like genes became a global concern due to the possibility of horizontal transfer of the plasmid that often carry resistance determinants to beta-lactams and/or quinolones. An understanding of the origin and dissemination of mcr-like genes in zoonotic pathogens such as S. enterica will facilitate the management of colistin use and target interventions to prevent further spread. The main objective of this review was to collect epidemiological data about mobilized colistin resistance in S. enterica, describing the mcr variants, identified serovars, origin of the isolate, country and other resistance genes located in the same genetic platform.


Introduction
The overuse and inappropriate use of antibiotics in diverse settings, such as human and veterinary therapeutics, animal production and agriculture, is widely accepted as one of the major causes of the emergence of antimicrobial resistance worldwide [1,2]. During the past decades, we have witnessed the evolution of bacteria by the selective pressure of antibiotics, with new resistance mechanisms and their spread across bacteria populations from various ecological niches. The antimicrobial resistance was responsible for about 700,000 deaths in 2016 and this number is estimated to increase to 10 million annual deaths by 2050 [2].
In human medicine, the treatment of infections due to multidrug resistant bacteria is a real challenge, like those caused by Pseudomonas aeruginosa, Acinetobacter baumannii and carbapenem-resistant Enterobacteria. The void of effective antibiotics led to the recent use of an old antibiotic, colistin, as one of the last-resort therapeutic options. The World Health Organization reclassified colistin as an antibiotic of critical importance in human clinical settings [3].
However, colistin has been widely used in animal production in several countries for therapeutic, prophylactic and growth promotion purposes [4,5]. The use of low-dose and prolonged course of antibiotics in livestock is clearly associate with selection of zoonotic resistant strains that can be spread by direct contact of animal-to-human or indirectly, like by the food chain [6,7]. The dissemination of resistance determinants is fueled by lateral gene transfer mechanisms, such as conjugation [8]. Animal other Gram-negative bacteria. Currently, eight types of mcr genes (mcr-1 to -8) have been described and deposited into GenBank. The first reported variants were isolated from animals in Europe and China. The mcr-2 gene was found for the first time in E. coli from pigs and calves in Belgium [26], mcr-3 in E. coli from pigs in China [13], mcr-4 in a strain of the monophasic variant S. enterica serovar Typhimurium from pigs in Italy [14], mcr-5 in S. Paratyphi B dTa+ from poultry in Germany [15], mcr-6 (previously named mcr-2.2) in Moraxella spp. isolated from pigs in Great Britain [27], mcr-7 in three isolates of Klebsiella pneumoniae from chickens in China [28] and finally mcr-8 in NDM-producing K. pneumoniae isolates from both pigs and humans in China [25].
All these findings suggest that animals are the reservoir of the mcr genes with emphasis on the pigs, mostly due to the heavy usage of polymyxins in food animal production for therapy, prophylaxis and metaphylaxis purposes, which contributes for selection of mcr producers. Furthermore, the reports of identification of mcr genes have been mostly from animal isolates when compared with human isolates, sustaining animals as the main reservoir. Moreover, some genetic elements, like other resistance genes, insertions sequences and plasmids that are more prevalent and widespread in bacteria of animal origin, are found closely associated with the mcr-like genes [29].

Salmonella enterica: Salmonellosis and Enteric Fever in Humans
S. enterica infections are an important public health concern worldwide. S. enterica serovars can be separated in two main groups: The typhoidal Salmonella that comprise S. enterica serovar Typhi (from now on designated as S. Typhi), S. Paratyphi A, S. Paratyphi B, and S. Paratyphi C, whereas all the other serovars are called as non-typhoidal Salmonella (NTS) [30].
Animals are the primary reservoir of NTS, and NTS infections, generally called salmonellosis, are a huge threat in developing countries especially in infants, young children and in HIV-carriers, while in developed countries infection is mostly acquired through the food chain by ingestion of commercially contaminated produced animal-derived food [7,31,32]. It is estimated that NTS gastroenteritis is responsible for about 93.8 million illness and 155,000 deaths each year worldwide, and of these, it is estimated that 80.3 million cases are foodborne, with very high associated costs, most of them in developing countries, which contrasts with the reality in developed countries, where this rate is lower [33].
Despite food producing animals behave as the main reservoirs of S. enterica, a small group of serovars are capable of infecting and colonizing only determined hosts. For example, typhoidal serovars are human host-restricted organisms that cause typhoidal fever and para-typhoid fever (both also known as enteric fever) [30,34].
All typhoidal Salmonella serovars are responsible for 27 million annual cases of enteric fever, which results in more than 200,000 deaths worldwide [35]. In developing countries, where sanitary conditions and clean water are a problem of public health, enteric fever is generally endemic. Fecal-oral route is the main cause for spread of typhoidal Salmonella. In some countries, especially in Southeast Asia, S. Paratyphi infections are increasing. It is estimated that this serovar is responsible for about half of all enteric fever cases [36].
Currently, colistin is not used to treat human infections caused by this bacterium, and the development of colistin resistance is clinically not relevant. However, in vivo colistin resistance has been observed in S. enterica from food-producing animals [37][38][39][40], and the resistance determinants when inserted in genetic mobile elements (e.g., mcr-like genes) can be laterally transferred to other species, commensals or pathogens of animal and human origin. Moreover, the genetic platforms carrying mcr-like genes frequently host resistance genes that hinder the efficacy of other antibiotic classes [41]. Therefore, the presence of mcr-like genes should not be neglected in this zoonotic pathogen.

Colistin Resistance in Salmonella enterica
S. enterica strains have developed resistance to a variety of antimicrobials. Chloramphenicol was the first antibiotic used in the treatment of typhoid fever, but emergence of resistance soon after its introduction lead to the replacement by trimethoprim-sulfamethoxazole and ampicillin or amoxicillin. Multidrug resistant strains emerged with the overuse of these first-line treatment drugs, and fluoroquinolones, such as ciprofloxacin, and extended-spectrum cephalosporins, such as ceftriaxone, were introduced in the treatment of Salmonella infections. However, resistance to these antimicrobials is now also frequent [7,30,42].
In S. enterica, the chromosomal colistin resistance involve activation of the PmrA/PmrB and PhoP/PhoQ two-component regulatory systems, which are responsible for the biosynthesis of L-Ara4N and PEtn. The activation of these systems is related with environmental stimuli, such as low concentration of Mg 2+ , or with specific mutations in the two-component regulatory systems-encoding genes [4,23,43]. These mutations lead to the constitutive expression of PmrA/PmrB and PhoP/PhoQ, with consequent activation of operons arnBCADTEF and pmrCAB, and permanent addition of L-Ara4N and PEtn, respectively, to lipid A [23].
Other alterations, such as deacylation of lipid A by PagL [23,44], and activation of the transcription of genes involved in adaptation and survival of the bacterial cells by RpoN [23,45], can also lead to colistin resistance in S. enterica, but are less common.
Plasmid-mediated colistin resistance conferred by mcr-1 [46], mcr-2 [47], mcr-3 [48], mcr-4 [14] and mcr-5 [15] genes have been already identified in different serovars of S. enterica. Like in other bacterial species, mcr-like genes have been detected in isolates from different origin, such as food-producing animals, food products and human samples, and are inserted in diverse genetic environments and plasmid backbones. It is of note that the presence of the mcr genes can be associated with low level of resistance to colistin [4,14,15,46,[49][50][51], allowing to persist undetectable. Table 1 summarizes the reports on mcr-like genes and their variants in this species and the key findings of each study. Briefly, S. Typhimurium is the most prevalent serotype harbouring mcr genes. This serotype is also one of the most frequent to cause human infections [52]. Monophasic variants of S. Typhimurium such as 1,4, [5],12:i:-are also widely reported. It is still worth noting that mcr positive Paratyphi B are isolated from animal samples, though this serotype usually infects humans and cause invasive disease [52]. Food-producing animals appear to be the main reservoir of mcr positive S. enterica strains. Poultry and swine animals are the most reported sources of isolates. Nonetheless, there are isolates from human clinical sources, which suggests dissemination from animals to humans along food chain [53]. In addition, China is the country where more mcr positive S. enterica strains are identified. This is consistent with the high rates of use of colistin in livestock and veterinary medicine, which leads to the emergence of resistance [10]. Nevertheless, in European countries, such as Italy and Portugal, where colistin is frequently used for therapeutic and metaphylactic purposes in animal husbandry, the reports are emerging [10,41,53]. On the other hand, European countries are more engaged in screening and surveillance activities, which justifies the high number of European reports [14,20,48,54,55]. These studies evidence the wide and ubiquitous spread of mcr genes around the world. Although the first report of mcr-1 only occurred in 2015 from an E. coli isolate [9], these genes are also carried by S. enterica at least since 2008 [56]. Finally, several mcr-carrying S. enterica isolates show multidrug resistance profiles, with several genes conferring resistance to tetracyclines, beta-lactams including cephalosporins, quinolones, sulfamethoxazole/trimethoprim and streptomycin, which limits the therapeutic options for treatment of S. enterica infections.
The existence of colistin resistance genes embedded into mobile genetic elements, such as plasmids, is a huge concern because they can be horizontally spread across different bacteria. Furthermore, mcr genes can be located in plasmids encoding other resistance genes, such as bla CTX-M , floR and/or qnr, originating strains resistant to several antibiotic classes, including polymyxins, the majority of beta-lactams, including broad-spectrum cephalosporins and monobactams [48,57,58], amphenicols [51] and quinolones [48,59], respectively. For instance, mcr-1 and bla CTX-M-1 genes embedded into plasmid IncHI2 were co-transferred from S. enterica isolated from swine retail meat by conjugation under colistin selection [41]. The co-selection of resistance might compromise treatment of complicated gastroenteritis and invasive infections caused by S. enterica.   • First report of the mcr-5 gene • The transfer of colistin-resistance-mediating phosphoethanolamine transferase genes from bacterial chromosomes to mobile genetic elements has occurred in multiple independent events raising concern regarding their variety [15] MDR, multidrug resistant

Conclusion
Here we reviewed the epidemiology of mcr-like genes identified in S. enterica serovars. It is not expected that colistin will be an antibiotic to treat human enteric fever or gastroenteritis caused by this pathogen; nonetheless, mcr-like genes are carried in conjugative plasmids that spread among bacterial populations. The zoonotic feature of S. enterica cannot be neglected and plasmid-mediated colistin resistance genes may reach human microbiota through the food chain. Genetic multidrug resistant platforms can be selected not only by colistin but also by the other antibiotics used in livestock, such as quinolones. It is of paramount importance to understand where resistant pathogens are emerging in order to implement infection control measures to prevent their spread. Emergence of mcr-like genes are not confined to Asia, as initially supposed, and are found in countries where a higher antibiotic restriction is used in animal production, even in strains isolated ten years ago, raising questions of the stability of these plasmids in bacterial populations, their impact on bacterial fitness. Further research on mcr-like genes in zoonotic pathogen populations is necessary to unveil the true impact in human health and to manage colistin use to minimize selection, proliferation and spread of drug-resistant bacteria.