THE GLOBAL SPREAD AND EPIDEMIOLOGICAL CHARACTERISTICS OF SALMONELLA SPP., ESCHERICHIA COLI AND METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA): RELATED RESEARACH STUDIES IN SERBIA

It has been established that some clones of pathogenic bacteria such as Salmonella spp., Escherichia coli ST131, and methicillin-resistant Staphylococcus aureus (MRSA) tend to spread worldwide. Th erefore, epidemiological surveys have been conducted to identify the source of infection and to break the chain of infection. In this article, it was pointed out that common international clones of Salmonella are represented with the serotypes Typhimurium, Kentucky, Infantis and Enteritidis. Serovars Typhimurium and Kentucky display multidrug-resistant phenotypes more frequently. Several sequence types of E. coli and the international clone ST131 are described, including clades C1 and C2 with the extended-spectrum cephalosporin-resistance genes ( bla CTX-M-15 or bla CTX-M-27). Th ese pathogens are oft en found in both humans and animals. It is noted that Staphylococcus aureus became resistant to methicillin almost instantly aft er its introduction into clinical practice. Soon aft erwards, MRSA found its way to farm animals and wildlife. Th e cycles of infection are bidirectional: humans can disseminate MRSA in the environment but animals may also be sources of infection for humans. Comprehensive work has been done by epidemiologists to introduce all necessary measures to eliminate MRSA from hospitals. Also, threat of new resistance and virulence mechanisms, which bacteria use to resist the hostile environment and enhance their survival in their natural habitats including humans and animals. Th erefore, the capacity of certain bacteria to spread due to their virulence mechanisms and resistance phenotypes is presented, and a brief description of the research conducted in Serbia is included.


INTRODUCTION
Coping with multidrug-resistant bacteria has been among the ultimate goals of medicine. Apart from having intrinsic resistance, bacteria can also acquire antibiotic resistance genes and mobile genetic elements and induce the exchange of genetic material within their counterparts. Th e long-lasting bacterial evolution prompts the question whether it is possible to create effi cient therapy for humans and animals in the future (Dandekar et al., 2015). Bacteria use specifi c metabolic pathways to increase their fi tness, colonize the gut and organize themselves in biofi lm communities. For example, even highly pathogenic bacteria with prominent virulence and resistance mechanisms, which may present a burden to themselves, tend to become well-established in natural environments (Beceiro et al., 2013). All these features together result in the endless dissemination of resistant and virulent clones worldwide (Pitout and Finn, 2020). Th erefore, it is very important to detect and prevent the dissemination of pathogenic and multidrug-resistant bacteria in hospital settings and food production chains. Control strategies aiming to deal with bacterial infections are more comprehensive in more economically developed countries as compared to developing countries. However, mass primary production and utilization of antibiotic feed additives as growth promoters has led to an increase in resistance to antibiotics, even in developed countries (Silbergeld et al., 2008). Th ose were the reasons why as early as since 1970 some antibiotics have been sequentially banned as feed additives in the EU and from 1 January 2006 antibiotics may no longer be used as growth promoters (Castanon, 2007). Consequently, we have witnessed a decrease in antimicrobial resistance in pathogenic and commensal bacteria which originate from the environment and food-producing animals in some European countries (Aarestrup et al., 2001). However, the antibiotic withdrawal from food-producing animals has not signifi cantly changed the resistance levels in human pathogens (Pradella et al., 2006). Th e fear of continuous development of new mechanisms of resistance in bacteria is still an important global issue, while trade and travel continuously increase the risk from dissemination of multidrug-resistant and virulent bacteria with limited fi tness cost. In this brief review, some of the prominent examples of the worldwide spread of bacterial pathogens are discussed and the brief presentation of related research in Serbia is included as well. It is important to bring up the fact that serovar Enteritidis can also be vertically transmitted, which renders them even more dangerous for public health due to the increased potential for dissemination across the food chain and subsequent increase of their spread in communities. Th erefore, one of the important goals of contemporary agricultural production is to eliminate dangerous Salmonella serovars from poultry and other food-producing animals around the globe. Subsequently, the implementation of enforced biosecurity measures in the entire food chain including pre-harvest has become important both in developed and developing countries (EFSA, 2019).

CLONAL SPREAD OF SALMONELLA SPP.
Epidemiologists who are focused to trace back the source of Salmonella spp. outbreaks, to minimize the likelihood of infection in humans, are trying to help to break the chain of infection at the very bottom of the production pyramid (Pijnacker et al., 2019). Indeed, a comprehensive genome analysis of Salmonella originating from poultry has revealed that S. Enteritidis from breeder fl ocks has been spreading through global trade for decades (Li et al., 2021). Th e exact reasons for persistent infection with serovars Enteritidis and Typhimurium can be revealed using experimental animals. It was shown that invasive serovars have more virulence factors, invade internal organs of the host more effi ciently and possess mechanisms prompting their survival inside the macrophages of the host (Velhner et al., 2018). For instance, the global spread of Salmonella Enteritidis phage type 4 (PT4) was attributed to successful infection of the hens' reproductive tract most likely by inheriting additional virulence factors which facilitate internal contamination of eggs (Velge et al., 2005). Th erefore, the enhanced management practice, vaccination and utilization of prebiotics and probiotics have been enforced in many countries of the world, which has led to a much better epidemiological situation regarding Salmonella spp. infection of food-producing animals. Many of these strategies have helped to eliminate some other serovars besides Enteritidis and Typhimurium (two serovars that are primarily targeted by vaccines) and thus reduce the burden of infection at the farms worldwide.
Th e clonal spread of Salmonella Infantis in Serbia was in part attributed to strains which become resistant to ciprofl oxacin (Velhner et al., 2014). However, in 2018, it was established that strain Infantis continues to be resistant to nalidixic acid but not longer to ciprofl oxacin, which is possibly due to restrictions on the use of enrofl oxacin for the therapy of day-old chickens. It is interesting to note that brief monitoring of antimicrobial resistance in Salmonella isolates in Serbia reveals that Salmonella Typhimurium is frequently susceptible to antibiotics unlike the isolates reported in other European countries (Jovčić et al., 2020). In Serbia, Salmonella Enteritidis (SE) was not examined by any of the available genetic methods including phage typing and the whole-genome sequencing approach. Th us, factual information about the possible clonal spread of SE from humans and animals is still lacking.

GENETIC BASIS OF THE PROGRESSION OF THE E. COLI WITH THE DISTINCTIVE SEQUENCE TYPES AND PHENOTYPE
E. coli sequence type-ST131 is primarily a human pathogen oft en causing urinary infection (Giedraitiene et al., 2016). However, due to the high dissemination rate, it can infect all animal species and contaminate the environment. Th e exact reasons for the successful global spread of the clone ST131 have not been entirely elucidated. Namely, the virulence factors, including their resistance determinants, could be assumed as moderate, while the transmission rates could be minimized with proper hygiene and disinfection in hospital facilities. Th erefore, the combination of all virulence properties perhaps contributes to the good fi tness and widespread nature of the ST131 clone but none of these factors alone is signifi cantly pronounced to cause such persistent infection and signifi cant contamination rates (Pitout and Finn, 2020). Th ere are several well-established E. coli ST131 clades. Th e clades A and B comprise strains susceptible to antibiotics but possess diff erent fi mH genes, which are a part of a fi m operon. Th e fi m operon encodes fi mbria which determines the success of bacterial attachment to the host tissue receptors (Pitout and Finn, 2020). Clade C is represented by three major groups: C0, C1 and C2. Th e major diff erences between these are the resistance to fl uoroquinolones, which is present in isolates belonging to clades C1 and C2 but absent in those from clade C0. Fluoroquinolone resistance has been developed due to the multiple mutations in topoisomerase genes (the gyrA and the parC gene). During genomic evolution of the E. coli ST131 isolates from the clades C1 and C2, the acquisition of the extended-spectrum beta-lactamase resistance genes took place, also. All of these genetic changes are attributed to the new plasmid or the presence of insertion elements required for the successful integration of other genes in already existing plasmids. However, even if these properties have helped scientists with the phylogenetic classifi cation of the ST131 clones, none of them is the key reason for the successful spread of these bacteria. Th e following additional factors are further infl uencing the abundant pathways of the E. coli ST131 strains: the level of compensatory mutations required to obtain plasmid stability and decreased fi tness cost, the horizontal integration of genomic islands and prophages including a variety of virulence genes carried on by chromosomes and plasmids (Beceiro et  Th e recent increase in infections caused by the extra intestinal pathogenic E. coli in Japan, in human patients, was attributed to the worldwide clone ST131, resistant to extended-spectrum cephalosporins due to the inheritance of the blaCTX-M-27 gene (Matsumura et al., 2016). Based on a core genome sequence analysis this clone is genetically distinctive and forms the C1-M27 clade. Th e pangenome analysis identifi ed a specifi c region in the clade C1-M27 that closely resembles a prophage-like genomic island originally identifi ed in an E. coli isolate from a pig. Th is region was termed M27PP1. Upstream the M27PP1, in the two C1-M27 isolates, an additional insertion region was identifi ed showing homology to a prophage sequence found in a chromosome of the g-proteobacterium HdN1 (Matsumura et al., 2016). Th e reservoirs of the CTX-M27 are found worldwide in non-human and environmental specimens and thus present an important clade among the ST131 variants with the capacity of further global spread.
Unlike Salmonella, which tends to disseminate as a common clone, E. coli is genetically quite diverse. Th e exceptions are the above-mentioned sequence types, which persist in various niches and disseminate thanks to their enhanced fi tness. During the investigation of the diverse, multidrug-resistant commensal E. coli from a poultry farm practicing frequent use of antibiotics in Serbia, three independent clones with the common virulence type and resistotype were determined by pulse-fi eld gel electrophoresis (Velhner et al., 2018). Th e multilocus sequence type of only a small number of E. coli isolates from gulls was done as well. It was discovered that isolates resistant to extended-spectrum cephalosporins were ST38, ST2307, ST224 or ST162 while ST131 was not detected (Velhner et al., 2021). However, more comprehensive multilocus sequence typing of commensal and pathogenic E. coli isolates from animals was not performed, and therefore, the data on the existence of particular ST is not available.

EVOLUTION OF THE METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS
cillin-resistant Staphylococcus aureus. MRSA is characterized by the mecA gene that is incorporated in a staphylococcal cassette chromosome SCCmec, which diff ers in size and genetic composition. Th e mecA gene encodes the penicillinbinding protein PBP2a of the bacterial cell wall, which has a low affi nity for beta-lactam antibiotics (Enright et al., 2002). MRSA isolates are multidrugresistant and oft en only glycopeptide antibiotics such as vancomycin are eff ective against them. However, some strains became resistant also to these antibiotic classes (such isolates are called GISA), which renders successful treatment impossible (Enright et al., 2002). MRSA emerged primarily as a hospital pathogen, but it is now widespread in the community and can be isolated from animals and the environment. Th e evolution of MRSA is still a puzzle. Since genetic analysis has confi rmed genetic divergence between strains, it has been proposed that independent inheritance of the mecA gene contributed to the evolution of MRSA rather than the diverse inheritance of genetic material that has arisen in a single clone (Enright et al., 2002). Th e multilocus sequence typing (MLST) utilizes analysis of the seven housekeeping genes to obtain the specifi c allelic profi le. Th is method is benefi cial in the classifi cation of various MRSA lineages that have appeared in the past decades and more recently. It was elucidated that early MRSA strains evolve from the ST250 ancestor, who in turn arose from the methicillin-susceptible (MSSA) ST8 linage. Th e ST247 clone that emerged independently from ST250 is currently circulating in European hospitals. Several MRSA clones have the same allelic composition as their methicillin-susceptible (MSSA) counterparts indicating the independent horizontal acquisition of the mecA gene. Th is fact is in a way supported by the independent introduction of the four SCCmec types within MRSA strains of the same sequence type (Enright et al., 2002).
Th e community-acquired MRSA strains (CA-MRSA) possess more virulence determinants than hospital strains. Th e production of the Panton-Valentine leucocidin (PVL) exotoxin is pronounced in community-acquired strains and its detection could be used to distinguish CA-MRSA from the healthcare MRSA (HA-MRSA). PVL toxin is recognized in those strains that cause necrotizing pneumonia and invasive skin diseases although the mechanism of these virulence factors is not known. Other toxins produced primarily by the CA-MRSA are toxic shock syndrome toxin-1, staphylococcal enterotoxin B or C, α-haemolysin and phenol-soluble modulins (Hassoun et al., 2017).
Livestock-associated MRSA (LA-MRSA) are also very important pathogens (Deiters et al., 2015). Not only may farm workers or those in contact with raw meat become infected with LA-MRSA but they might also transmit this pathogen to hospitals. In the vice versa manner, humans infected with LA-MRSA can transfer the pathogens to animals. Th is type of two-way directional transmission makes it even more diffi cult to keep infections under control in both human and veterinary medicine. However, with the proper treatment and management in hospitals and on farms it is possible to eliminate and prevent the spread of MRSA. Th e principle "search and destroy" is very eff ective in healthcare settings in eliminating this pathogen. Firstly, the MRSA carriers are to be detected, and then the medical treatment is to be applied to eliminate the pathogen and interrupt its transmission pathways. A similar principle is applied on farms, but there it is also important to prevent the purchase of positive herds to keep the MRSA infection under control (Crespo-Piazuelo and Lawor, 2021).Th erefore, eff ective management programmes are required to be rewarded with a healthier environment and better treatment options for both human and animal patients.
It was shown that MRSA isolates among patients and health care workers from the Serbian University Clinical Hospital were mostly of the CC-5-SCCmec type I. Th ese isolates were predominantly found in patients from emergency departments and medical departments. In addition, various genetic lineages were found in isolates from healthcare workers in surgical departments. MRSA isolates from healthcare workers carried mostly smaller SCC elements of type IV and V. Th is was the most comprehensive MRSA detection in Serbia and, as noted, has shown the increased risk of MRSA carriage not only among patients but also among the healthcare workers (Cirkovic et al., 2015). Th e presence of MRSA in pigs was estimated on one farm from the north of Serbia, revealing that out of 84 nasal swabs, six tested positive for MRSA. Th ose isolates belonged to the ST45, spa type t015 and in addition possessed SCCmec IVa. Th e MRSA sequence type 45 is a global MRSA lineage of particular strain diversity and common nosocomial isolate in Europe (Velebit et al., 2010).

CONCLUSION
Th e important goal in human and veterinary medicine is to follow up the work of epidemiologists to break the infection cycles of all persistent and dangerous pathogens. If collective eff orts in improving management are introduced in everyday practice, it will be possible to reduce the burden of infections with pathogenic bacteria.