Better together–Salmonella biofilm-associated antibiotic resistance

ABSTRACT Salmonella poses a serious threat to public health and socioeconomic development worldwide because of its foodborne pathogenicity and antimicrobial resistance. This biofilm-planktonic lifestyle enables Salmonella to interfere with the host and become resistant to drugs, conferring inherent tolerance to antibiotics. The complex biofilm structure makes bacteria tolerant to harsh conditions due to the diversity of physiological, biochemical, environmental, and molecular factors constituting resistance mechanisms. Here, we provide an overview of the mechanisms of Salmonella biofilm formation and antibiotic resistance, with an emphasis on less-studied molecular factors and in-depth analysis of the latest knowledge about upregulated drug-resistance-associated genes in bacterial aggregates. We classified and extensively discussed each group of these genes encoding transporters, outer membrane proteins, enzymes, multiple resistance, metabolism, and stress response-associated proteins. Finally, we highlighted the missing information and studies that need to be undertaken to understand biofilm features and contribute to eliminating antibiotic-resistant and health-threatening biofilms.


Introduction
An estimated 1 in 10 people in the world fall ill every year from consuming contaminated food, with diarrhea being the most common form of these diseases. Salmonellosis is a major intestinal foodborne disease affecting approximately 200 million people globally, resulting in more than 200 000 fatal cases per year. Therefore, it is considered the cause of significant socio-economic losses and a real danger to human health, livelihood, and health care systems. 1 According to the United States Department of Agriculture Economic Research Service (USDA ERS; The Economics of Food, Farming, Natural Resources, and Rural America), the economic impact of Salmonella exceeds 3 billion euros annually in the European Union.
The genus Salmonella consists of two species, Salmonella bongori, and Salmonella enterica, the latter of which is further divided into six subspecies. S. enterica subsp. enterica, comprising more than 1,500 different serovars, is mainly responsible for 99% of salmonellosis cases in humans and warm-blooded animals. 2 Interestingly, only a few of these are clinically relevant. Each serovar is characterized by varying degrees of host adaptations and can be divided into one of the following groups: host-adapted specialists, for example, Salmonella Choleraesuis, which causes swine infections; host-restricted specialists, for example S. Typhi and S. Paratyphi, which are human pathogens; and host-unrestricted generalists such as S. Enteritidis and S. Typhimurium. 3 The molecular basis of the infection process differs between generalists and specialists, and is determined by the bacterial genomic landscape. Generalists appear to rapidly induce disease symptoms and trigger the host immune system. Subsequently, they were eliminated from the body within a few weeks. 4 In contrast, specialists may persist in the host body for decades. 5 Most serovars are host-unrestricted pathogens and can infect different hosts based on the clinical patterns of salmonellosis. Two types of salmonellosis have been identified: typhoidal, caused by specialists S. Typhi and S. Paratyphi, and non-typhoidal (NTS), caused by other serotypes. 6 The predominant NTS serovars in developing countries are S. Typhimurium and S. Enteritidis, which are increasingly resistant to many antimicrobial agents. 7 Despite these variations, many Salmonella species share the capacity to induce latent, acute, or chronic illness, existing not only as planktonic cells but also as sessile, multicellular forms -biofilms attached to surfaces.
Costerton introduced "biofilm" as a biological term in 1978. 8 He described it as an interesting community formed in response to negative environmental stimuli such as temperature changes, oxygen, osmolarity, oxidative stress (bile), nutrient availability, or pH shifts. Biofilms may be formed on many biotic or abiotic surfaces, including the environment (lakes, rivers, soil, rock cover, aquatic plants, and sediments), food, water pipelines, medical devices, food processing equipment, animals, and humans 9 ( Figure 1). Biofilm development can be divided into five stages: initial reversible attachment (1), irreversible attachment (2), maturation (3), and dispersion (4). The process begins with the initial contact between the planktonic bacteria and the surface, which is still reversible. The bacteria then form a monolayer and produce a protective extracellular matrix, consisting of extracellular polysaccharides, structural proteins, cell debris, and nucleic acids, collectively referred to as extracellular polymeric substances (EPS). The initial stages of matrix formation are dominated by extracellular DNA (eDNA), following by polysaccharides and structural proteins. The biofilm grows in a three-dimensional manner, and the attachment becomes irreversible. In the final stage, some cells in the mature biofilm detach and disperse into the environment as planktonic cells, potentially initiating a new cycle of biofilm formation 10 (Figure 2).
The structure of these microbial communities ranges from homogenous, consisting of a single species of bacteria, to heterogeneous, comprising multiple species of microorganisms. 11 Importantly, both gram-positive and gram-negative bacteria are capable of forming biofilms. Regarding Enterobacteriaceae, mechanism of biofilm formation is universal between species, and both Salmonella and E. coli utilize similar pathways to form this structure: biosynthesis of type 1 fimbriae, flagella, lipopolysaccharide, as well as production of curli fimbriae, transmembrane transport and a variety of transcriptional regulators. 12 Although there are many similarities between both bacteria, a few differences were also identified. For example, the divergent utilization of tomB which deletion aids biofilm of Salmonella, but impairs its formation in E. coli. 12 One of the most essential properties of bacteria that are an integral part of biofilms is increased resistance to environmental stress, including the presence of xenobiotics. 13 In comparison to the planktonic state, biofilms are even 1000 times more resistant to antibiotics. 14 In a biofilm, horizontal gene transfer (HGT) often takes place, which results in bacteria acquiring resistance to several groups of antibiotics. 15 Consequently, changes in gene expression profiles are believed to closely resemble those of planktonic cells in the stationary growth phase. 16 Moreover, if there is a subinhibitory concentration of antibiotics in the external environment surrounding the biofilm, the bacteria may also become resistant to the substances in this case. 17 All this can lead to the development of multidrug resistant (MDR) strains that exhibit increased resistance to nearly all classes of antibiotics. 9,15,18 Additionally, the extracellular polymeric substances (EPS) produced by bacteria limit the penetration of antibiotics into the structure of the biofilm. 11 This phenomenon is caused mainly due to the cellulose and curli fimbriae present in it, which play a role not only in adhesion to the surfaces but also in imparting resistance to the biofilm by enabling tight packing of cells covered with a hydrophobic net. 18 The race between Salmonella multidrug resistance and antibiotic (and other antibacterial substances) use and development is still ongoing. Infections and risk of loss in industries such as medicine, water, food, and energy, where these bacteria can cause significant damage, are still significant worldwide problems. 19 Therefore, further studies are needed for a more effective Salmonella biofilm control.

Mechanisms responsible for the antibiotic resistance of Salmonella in biofilms
The use of antimicrobials to fight against biofilms can reduce the number of bacterial cells in the structure but does not lead to complete eradication, contributing to the development of chronic and recurrent infections. The main resistance mechanisms of Salmonella biofilms are regulated by (1) physiological, (2) biochemical, (3) environmental, and (4) molecular factors 20 ( Figure 3).

Physiological factors
Pathogenic bacteria are exposed to unfavorable conditions in the host organism. This stress often triggers the bacterial adaptive response, which may alter gene expression, induce mutations, or promote a resistant lifestyle such as biofilm formation. 21 A biofilm is a physical barrier protecting bacteria from antimicrobial agents, as the first stage of spreading of the particles is diffusion in a dense environment. 22 Therefore, the biofilm matrix is not homogeneous and has a gradient of oxygen, pH, and nutrients ( Figure 3). A gradient of oxygen is formed, with the highest concentration in the outer layers of the biofilm, steadily decreasing deeper into the biofilm matrix. A lack of oxygen may be one of the causes of persister cell formation. As a consequence of uneven oxygen distribution, a pH gradient occurs inside the biofilm matrix. The lower pH present in the inner layers of the biofilm is a result of lack of oxygen, which induces anaerobic respiration, as well as difficulties with the removal of metabolic products, such as CO 2 . Differences in pH may also interfere with the penetration of some antibiotics depending on their type. Lower pH was also found to promote the expression of genes associated with resistance mechanisms, such as multidrug resistance pumps, in some Enterobacteriaceae. 21 It is still unclear whether the fluctuation of pH is a direct cause of some of the resistance mechanisms or the aftermath of other mechanisms. 23 Along with oxygen transportation inside the biofilm matrix, it is also difficult for nutrients to penetrate deeper layers. Therefore, a microgradient of metabolic substrates forms. This slows down the growth of bacteria embedded in biofilms, making them more resistant to antibiotics as they often target the metabolic cycle of cells. 24

Biochemical factors
Similar to that of biofilms of other species, Salmonella's biofilm contains several types of EPS, such as polysaccharides, proteins, nucleic acids, and matrix. 25 All these components play a significant role in biofilm formation and antimicrobial resistance. Although the exact contribution of each EPS type is yet to be determined, several attempts have been made to investigate it. It was determined that curli fimbriae, cellulose, O-antigen capsule, and colanic acid are involved in biofilm formation, as the deletion of these factors impairs biofilm formation. 26 Curli fimbriae had the greatest effect on biofilm formation. This is probably because the adhesive properties of these fibers, along with cellulose, have been shown to play crucial roles in cell aggregation, adhesion to surfaces, and biofilm formation. 25,27,28 Another study showed that the O antigen capsule, colanic acid, and Vi-antigen affect biofilm-mediated responses to oxidative stress. 27 Moreover, both colonic acid and Vi-antigen present in the matrix were able to protect deletion mutants and wild-type bacteria from H 2 O 2 in cooperation with catalases. Interestingly, not all polysaccharides displayed community functions in co-cultures with different mutants. O-antigen-deficient mutants were not compensated by co-culture with wild-type bacteria, and co-culture of wild-type and curli fimbriae mutants, both resistant to H 2 O 2 , resulted in the sensitization of both strains. 29 O-antigen capsules were found to play a role in protecting bacteria from desiccation stress, as it was shown that deletion of the agfD gene responsible for formulation of capsules resulted in significant loss of viability in freeze-dried samples of S. Typhimurium (31% for wild type and 1.3% for agfD mutant). 30 Quorum sensing is a method of communication between cells according to the density of the bacterial population using so-called autoinducers. 31 It is driven by the production of particular molecules and sending them out of cells by either passive or active transport. Bacteria then recognize the particles via dedicated receptors and change their gene expression. Among the autoinducers produced by Salmonella, few have been linked to biofilm formation and antimicrobial resistance induction. 32,33 Quorum sensing is also one of the factors that induces HGT in some bacteria. N-acyl homoserine lactone was found to play a role in biofilm formation by S. Typhimurium. 32 Quorum sensing (luxS) has also been linked to the activation of gene expression on Salmonella pathogenicity island-1, which is responsible for motility, virulence, and biofilm formation. 33 Salmonella, a gram-negative bacteria, is less susceptible to antibiotic treatment than gram-positive bacteria owing to its double membrane and efflux systems. 34 Although the outer barrier slows down the antibiotic particles, the pumps can transport the antimicrobial agents out of the cell and send quorum sensing signals. 35 In S. enterica, several efflux pumps are encoded in its genome as well as in plasmids carried by the bacteria. 36 In S. Typhimurium, at least ten pumps have been experimentally identified. 36,37 The aforementioned pumps are divided into five superfamilies of transporters: 1) the ATP-binding cassette superfamily (ABC transporters), 2) the major facilitator superfamily (MFS), 3) the drug/metabolite transporter (DMT), 4) the multidrug and toxic compound extraction (MATE), and 5) the resistance nodulation-cell division (RND) superfamily. 38

Environmental factors
One of the ways in which bacteria resist antibiotics is by spontaneously forming persister cells mainly in the deeper layers of the biofilms. These cells are dormant and their metabolism is muted. Therefore, antimicrobial agents that interfere with metabolic reactions have little effect on the persister cells. 39 Persister cells are often associated with chronic diseases and can turn back into regular cells and their planktonic form to further infect host organisms. Biofilms are a way for bacteria to withstand higher concentrations of antimicrobial agents, and the presence of antibiotics may also induce the formation of biofilms. Some antibiotics, such as nalidixic acid, spectinomycin, tetracycline, and neomycin, were found to induce biofilm formation in S. Infantis cultures at sub-minimum inhibitory concentrations (MIC). 40 A study on S. Typhi showed a similar effect to that of cefetoxime. 41 Although some antibiotics may promote biofilm formation, others have the opposite effect, as shown in a study with S. Typhimurium. 42

Molecular factors
Gene expression in biofilms is substantially different from that in the planktonic form. Apart from changes in gene regulation, mutations can aid in the resistance of bacteria to antimicrobial agents. Gene mutations that occur in bacteria may be part of an adaptive response to stressful environments. 2 For example, a mutation in soxRS, a gene directly involved in the control of the AcrAB-TolC multidrug efflux system and indirectly with limitation of antimicrobial uptake, has been found in several serovars of Salmonella, as well as in other gramnegative bacteria. 43,44 HGT plays a major role in antibiotic resistance. Microbes can share additional genes between themselves and, therefore, help each other fight antimicrobial agents. Plasmids are shared from one cell to another by a conjugation pilus and can carry multiple antibiotic-resistance genes simultaneously. 45,46 It has been shown that antibiotic-resistance genes can be transferred between different species, from Escherichia coli to Salmonella. 47 Although there are many studies regarding the differences in gene expression and gene mutations in Salmonella, their effects on biofilm resistance and formation have not been thoroughly studied. Therefore, we attempted to gather all available data on the topic in the following chapters.
Taken together, these factors provide protection against antibiotics, osmotic shifts, metallic cations, oxidative stress, UV, desiccation, biocides, and even from washing or cleaning. In addition, biofilms enable competition for nutrients and space. 48 Biofilms provide a wide range of benefits for Salmonella and are a threat to humans. These bacterial consortia play a negative role in chronic human infections. Salmonella spp. adheres to and forms biofilms in the gastrointestinal tract. 9 Furthermore, S. Typhi can form robust biofilms on the cholesterol gallstones present in the gallbladder. 49 Consequently, the human immune system is mobilized to fight pathogens that are significantly straitened for biofilms as compared to planktonic cells. Considering this, biofilms are an exceptional adaptation mechanism that allows bacteria to survive in unfavorable conditions; therefore, it is a crucial threat to public health.

Upregulation of antibiotic resistance-associated gene expression in Salmonella biofilms
Molecular biology techniques have revolutionized the study of bacterial pathogenicity processes, including the formation of biofilms. These techniques allow researchers to investigate the genetic and molecular mechanisms underlying bacterial biofilm formation, providing insights into the factors that contribute to bacterial virulence.
Besides quantitative PCR (qPCR), microarray analysis and RNA sequencing are the most common strategies, which allow the simultaneous monitoring of thousands of genes, providing a comprehensive quantitative view of the gene expression pattern that occurs during biofilm formation. 50,51 More recently, CRISPR-Cas technology has emerged as a robust tool for investigating bacterial pathogenicity, allowing for targeted editing of bacterial genomes and enabling researchers to investigate the function of specific genes involved in virulence. 52 Although these techniques are useful for identifying mutations and genetic changes in bacterial populations, they do not provide information on the functional consequences of these changes or how they affect bacterial fitness. Therefore, recent studies have focused on another powerful strategy to characterize the genetic mechanisms of resistance: experimental evolution coupled with whole-genome sequencing (WGS). 53,54 By propagating bacterial populations in the presence of antibiotics, this strategy allows for the selection of clones capable of surviving antibiotic exposure. WGS of these populations or clones reveals the genetic causes of the resistance phenotype and sheds light on the interaction between selection, chance, and historical contingency in microbial populations. Previous studies used this approach to track the evolution of bacterial resistance to antibiotics in different contexts. This method has been used to study the evolutionary dynamics of combining antibiotics, deploying them sequentially, and adapting to changes in temporal or spatial drug concentration. 55,56 The study revealed that resistance evolution is influenced by divergent genetic changes resulting from different genetic backgrounds and drug exposure. 54 It is worth noting, that the evolution of decreased susceptibility to antibiotics in biofilm and planktonic populations is not identical, as different mechanisms and trajectories are involved. Mutations in genes that code for antibiotic targets are commonly found in planktonic populations evolved in the presence of antibiotics, while mutations in efflux and metabolism genes are often observed in evolved biofilm populations. 57 Growth in well-mixed planktonic cultures tends to select for high-level resistance under subinhibitory concentrations of antibiotics, while growth in spatially structured biofilms favors mutants with lower levels of resistance. 58 However, this is not always the case when using stepwise increasing or lethal concentrations of antibiotics during evolution. Evolved biofilm populations maintain a higher diversity than corresponding planktonic populations, which may protect against a negative selection of less fit-resistant mutants. 59 Mutations in different genes may lead to similar phenotypes, suggesting that the fundamental mechanisms behind reduced biofilm susceptibility could be similar for different classes of antibiotics and organisms.
Experimental evolution can help elucidate the interplay of resistance, tolerance, and persistence behind the reduced antimicrobial susceptibility of biofilms. However, identifying complex patterns of mutations, gene expression, and metabolism will require an interdisciplinary and holistic approach. In this study, we utilized recently published data to investigate the drugassociated gene expression profile of biofilmforming S. enterica. We focused on identifying the genes that are upregulated in multicellular aggregates compared to planktonic cells (Table 1; Figure 4).

Efflux pumps
The first group comprises genes related to membrane transport encoding MFS, multidrug resistance, and RND efflux pumps. Efflux pumps are a key element of resistance for gram-negative bacteria, especially planktonic cells, which are more exposed to antibiotics than bacterial consortia, forming a biofilm structure. Therefore, it is not surprising that there is a tendency to reduce the expression of genes responsible for the efflux of substances from cells in the biofilm structure. Biofilm, which is a mechanical barrier limiting the access of harmful substances from the external environment, reduces the need for cells to develop a system of efflux pumps. However, several genes associated with efflux pumps encoding structural or regulatory proteins are upregulated in biofilm cells. 51,60-66

MFS (major facilitator superfamily)
MFS is a family of secondary transport proteins and one of five efflux pumps described in prokaryotes. They are primarily engaged in the uptake of sugars; however, numerous MFS proteins are also implicated in drug efflux systems, and they increase antibiotic resistance in gram-negative bacteria, including Salmonella. Many microbial genomes contain MFS transporters that typically function as single-component pumps capable of moving small solutes through the inner membrane. 68 Five of the nine known MDR efflux pumps (AcrAB, MdtK, AcrEF, EmrB, and MdfA) in S. Typhimurium genome can export quinolones when overexpressed. 69,70 In clinical S. Typhimurium, an active MDR efflux pump has recently been identified as the factor for the main fluoroquinolone-resistant mechanism. It was also shown that deletion of efflux pumps such as acrD, acrEF, emrAB, macAB, mdfA, mdsABC, mdtBC, and mdtK reduced the ability of S. enterica serovars to produce biofilms. 71 Based on comparative analysis, two efflux pumpcoding genes mdtK (ydpH) mdtL and (yidY), were significantly upregulated in the biofilm cells compared to that in the planktonic cells of S. Typhimurium isolated from stainless steel surfaces. 51 MdtK is present in all Enterobacteriaceae families and shares 90% similarity with its homologs in Salmonella and E. coli, indicating its high conservation among the Enterobacteriaceae member family. 70 By inhibiting the accumulation of antimicrobial dipeptides through quorum sensing, the MdtK pump may be crucial for the efflux of dipeptides in Salmonella cells. It was demonstrated that MdtK imparted acriflavine, doxorubicin, and norfloxacin resistance. 72 Antibiotics and several antimicrobial peptide components with antibacterial action are extruded by the superfamily MdtK from the PhoP/ PhoQ system. 73 This observation has also been reported for efflux dipeptides, such as alanylglycine, in E. coli. 74 Despite antibiotic resistance, Salmonella's capacity to produce biofilms is reduced in S. Typhimurium when MdtK is deleted or its activity is chemically inhibited. 71 Another gene involved in multidrug resistance is mdtL. Studying global gene expression in E. coli, mdtL showed a high 18-fold upregulation in biofilms compared to that in the  65 planktonic form. 75 The multidrug resistance efflux protein MdtL belongs to the drug/H+ antiporter (DHA) subfamily of the MFS superfamily. Its overexpression led to 2-fold higher chloramphenicol resistance. 76 In Salmonella (except for serovar S. Typhimurium), mdtL is directly adjacent to Salmonella genomic island 1 (SGI1). 77 Considering that E. coli and Salmonella are closely related, a similar observation can be expected in Salmonella biofilms. Different gene expression patterns between planktonic cells and multicellular aggregates S. Typhimurium 14,028 biofilm was also found in the case of another representative of the MFS superfamily, the galactose permease GalP. 60 It has been shown that overproduction of inner membrane proteins, like carbohydrate permeases, can alter antibiotic resistance mediated by those pumps in E. coli and Salmonella cultures, using a non-PTS carbohydrate as the only carbon source. 78 One such permease, GalP, induces exclusion-dependent galactose permease that transports sugar through the bacterial membrane, and also transports glucose, whose expression is triggered by galactose. 79,80 When a non-PTS carbohydrate was the only carbon source, S. Typhimurium showed increased susceptibility to paraquat, indicating that the overproduction of permeases such as GalP caused by the presence of non-PTS sugars interfered with the efflux pumps linked to paraquat resistance. 78

RND (resistance-nodulation-cell division)
AcrAB-TolC is the best-characterized RND system in Enterobacteriaceae including E. coli, Salmonella, and Klebsiella pneumoniae. The RND efflux system family has the greatest clinical significance as it is associated with multidrug resistance. 61,81 Clinically important is the induction of acrAB-tolC by bile, fatty acids, and cationic peptides. 81 This pump is composed of a TolC channel, with AcrB and AcrA proteins positioned in the inner membrane and periplasmic space. 82 It removes a wide range of substances from the bacterial cell, including antibiotics such as quinolones, aminoglycosides, chloramphenicol, and tetracycline, which provide resistance to a broad spectrum of antibiotics. 61,83 This system is present both in planktonic bacteria and biofilms, but changes in the expression of its components are visible. Such differences are related to the tolC gene, which encodes the TolC channel, with higher tolC expression observed in the planktonic form of S. Typhimurium. Significantly, changes in gene expression were observed not only between planktonic and biofilm bacteria but also between single-species biofilms and those composed of a multispecies bacterial community. Upregulation of tolC was observed in mixed biofilms composed of two S. Typhimurium strains and one E. coli strain. 61 Moreover, in the presence of eugenol, the expression of acrA and acrB in biofilm cells was higher than that in planktonic cells. 62 S. Typhi biofilm is also characterized by a higher expression of genes responsible for energy production and conversion: marA, marB, and marR. MarA is a global regulator that affects acrAB expression as well as many other genes that play a role in bacterial response to stress. 67,84 MarA acts mainly as an activator and directly or indirectly regulates many genes, including acrAB and tolC. 67,85 The MarR protein, on the other hand, is a repressor of the marAB operon. 86 MarR blocks MarA transcripts when there are no signals from the environment induced by phenolic compounds, antibiotics, or oxidative stress. Derepression of the marAB operon results in the expression of marA, which encodes the global transcriptional activator MarA, which in turn is subject to positive feedback regulation and represses MarR, allowing marA to be active. 81 Perera and Grove demonstrated that the Mar regulon is associated with antibiotic resistance and stress response. 87 Increased MarA expression was observed in the biofilm form of Salmonella in the presence of triclosan biocide. Triclosan increased the expression of marA and acrAB, which was activated by marA. In stationary cells, no significant effects on the expression of marA and acrA were observed. 66 According to Prieto et al., who demonstrated that the genes marA and marB were upregulated when exposed to bile, the Mar regulon also controls bacterial virulence factors, such as cell wall proteins and surface adhesins. 88 Therefore, it is conceivable that the Mar regulon prepares the Salmonella cells for possible antibiotic resistance or for stressors such as bile utilized to form biofilms. Another reason may be that some proteins have multiple roles. For example, the efflux pump in pathogenic organisms can pump antibiotics out of the cytoplasm of cells and into the surrounding environment, in addition to serving as a mechanism to expel toxic chemicals from inside the cells to the external environment. Therefore, the Mar regulon may perform two tasks: pumping out bile or antibiotics.

ABC (ATP-binding cassette)
One of the largest superfamily of transport system are ABC transporters which use energy from ATP hydrolysis to transport substrates across the membrane. Representative of this group is the YbhFSR transporter consisting of three proteins: YbhF, YbhR, and YbhS. YbhF is an ATP-binding component, whereas the next two are membrane components. In this case, a 2-fold upregulation of ybhF gene was observed in S. Typhimurium biofilms compared to those in the planktonic cells. 65 It has been shown that the main antibiotic substrates for YbhFSR are tetracyclines such as tetracycline, oxytetracycline, chlortetracycline, and doxycycline. 89 Additionally, deletion of ybhF resulted in increased sensitivity to cefoperazone (a third-generation cephalosporin) and a decreased growth rate compared to that of the wild type.

Outer membrane proteins
Drug transport through the outer membrane is another bacterial mechanism that plays a role in resistance to environmental stresses like antibiotics. 88 Porins are associated with antibiotic resistance by participating in the passive transport of antibiotics through the outer membrane of gram-negative bacteria. 90 Therefore, when threatened with such substances, microbial cells reduce the expression of genes that lead to the formation of porins or hinder the penetration of harmful compounds into cells. Changes in the expression of porins -reduction in porin levels and point mutations within them -favor the emergence of resistance. 91 The better-studied porins in the Enterobacteriaceae family are the highly conserved OmpF and OmpC proteins 92 whose expression is regulated by OmpR in a manner that depends on the osmolarity of the environment. 93 By considering the correlation between the expression of genes and their regulators, we suggest that overexpression of ompF and ompC could be associated with the elevated expression of ompR. It is one of the regulatory genes for the biofilm and an important virulence factor for Salmonella. Its deletion prevents biofilm formation by repressing pili and cellulose. 94 ompR is also involved in cell adhesion as one of the genes that regulate curli biosynthesis as OmpR controls the transcription of csgD whose product regulates the expression of curli subunits CsgA and CsgB. 95 Another gene involved in the regulation of OmpF is micF, a stress response gene whose protein controls the expression of the ompF gene found in E. coli, Salmonella, and related bacteria, to ensure resistance to carbapenems or βlactams. 96,97 OmpF is a nonspecific porin that is involved in the transport of antibiotics. 90 Through OmpF porins, antibiotics, such as β-lactams and fluoroquinolones, enter the cell; therefore, ompF mutations in gram-negative bacteria can cause resistance to these antibiotics. 96 The expression of the micF gene in S. Typhimurium in the presence of triclosan was higher than that in the 16sRNA controls, but was similar for both biofilm and planktonic forms. 66 However, in a study conducted by Chin et al. in which NGS was used to determine the transcriptome of S. Typhi in planktonic and biofilm forms, a higher expression of micF in the biofilm form not exposed to any chemical compound was noted. 63 OmpC is an important factor in preserving membrane integrity but also participates in the transport of antibiotics. 90 Loss of OmpC causes Salmonella resistance to cephalosporins. 98 However, its expression in biofilm is reduced compared to planktonic form. 63 Overexpression of another OMP was observed in E. coli biofilms. The OmpA protein was 4-fold overexpressed in the biofilms compared to its expression in the planktonic form in proteome studies, as confirmed by immunoassay enzyme-linked immunosorbent assay (ELISA) and western blotting. OmpA is a conserved OMP of Enterobacteriaceae with several different functions. 64 OmpA allows the passive transport of many small chemicals, including antibiotics, because it is a nonspecific porin. Deletion of OmpA has been shown to increase antibiotic susceptibility in several cases. E. coli deletion mutants showed increased susceptibility to antibiotics, such as β-lactams, glycopeptides, amphenicols, and lincosamides, whereas in S. Typhimurium, OmpA has been shown to increase bacterial survival when exposed to two β-lactam antibiotics, ceftazidime and meropenem. 99 Additionally, in the case of Acinetobacter baumannii, increased susceptibility to β-lactams was noted. 100 The protective role of OmpA against the harmful effects of β-lactam antibiotics is probably due to the maintenance of outer membrane stability. 99 Another OMP gene ompX showed an almost 5-fold upregulation in S. Typhimurium. 65 OmpX is a small porin, and similar to OmpF, it is involved in the permeability control of β-lactams and fluoroquinolone antibiotics. During exposure to drugs and environmental stress, the expression of ompX, encoding the outer membrane protein, is increased. 101 This protein was originally described in Enterobacter cloacae but is also present in various enterobacterial species, including E. coli, Enterobacter aerogenes, and S. Typhimurium. [101][102][103]

Metabolism and stress response-associated proteins
In addition to genes related to biofilm development and xenobiotic transport, genes involved in stress tolerance, oxidative stress, heat shock, cell envelope stress, putative stress, osmotic stress, acid tolerance responses, and DNA replication and repair were also upregulated in biofilms. 104 It was established that S. Typhimurium could utilize two possible routes to gain nitrate from the host: (1) direct nitrate absorption from the environment via nitrate transmembrane transporters NarK and NarU, which are encoded by the narK and narU genes, respectively, and (2) synthesis of nitrate from NO and O2 by a flavohemoprotein. 105,106 NarU was shown to function as a nitrate/nitrite antiporter, or, more likely, a nitrate/H+ or nitrite/H+ channel. 107 Although there is no evidence of the contribution of this gene to the antimicrobial resistance of Salmonella, it was demonstrated that chlorhexidine resulted in significantly higher transcript levels of narU (5-fold). 108 During nutritional deprivation or extremely slow growth, NarU, which is abundant in the stationary phase, confers a selection advantage. Following chlorhexidine therapy, the increased transcription of a gene related to nitrate absorption may indicate the activation of processes resembling anaerobic respiration.
The second group consisted of genes associated with reactive oxygen stress (ROS)-inducing response: ahpC and katG which showed increased expression in multicellular aggregates relative to planktonic cells isolated from S. Typhimurium 14,028 cultures. 60 ROS is a weapon against lipids, proteins, and nucleic acids, causing several types of intracellular damage. 109 Bacterial oxidative stress responses involve the action of enzymes, small compounds, and regulatory proteins that directly detoxify or defend against ROS. AhpC scavenges endogenous H 2 0 2 at the physiological level in E. coli whereas catalase KatG provides protection at higher H 2 0 2 concentrations. 110 The substrates of S. Typhimurium AhpC are small hydroperoxides and organic hydroperoxides, including alkyl hydroperoxides, which can be intracellularly produced from unsaturated fatty acids and nucleic acids. 111,112 Mature biofilm bacteria are essentially starved for iron, as evidenced by very low catalase levels and increased Mn-containing superoxide dismutase activities. 113 Thus, the low expression of catalase in biofilms mediated by alterations in overall iron metabolism, relative to planktonic bacteria, may be one factor that allows for increased expression of AhpCF in response to H 2 O 2 . The level of OxyR-regulated AhpC remains high in mature biofilms, and deletion of the ahpC gene appears to promote early biofilm formation by inducing exopolysaccharide production. Furthermore, the addition of exogenous H 2 O 2 or antioxidants modulates biofilm formation by altering exopolysaccharide production. 114 KatG is referred to as hydroperoxidase I or catalase peroxidase. KatG has been shown to decrease susceptibility to aminoglycosides. According to these findings, kanamycin helped the peroxidase reaction by acting as an electron donor, bringing KatG's oxidized ferryl intermediates back to their resting state. 115 Increased expression levels of genes associated with metabolism and xenobiotic biodegradation, cutC and sodAC, in S. Typhimurium, isolated from stainless steel surfaces, were also demonstrated. 51 CutC, a cytosolic protein of 248 amino acids, is responsible for copper homeostasis. 116 In 2008, the United States Environmental Protection Agency (US EPA) recognized copper as the first antimicrobial metal. In in vitro assays, solid copper surfaces killed 99.9% of microorganisms within two hours of contact. 117 A mutant lacking cutC was more susceptible to copper than its parental strain, supporting the theory that cutC is a cytoplasmic copper-binding protein. 118 Interestingly, this protein contains a putative copper-binding motif and can confer copper tolerance to both cutC and cutF mutants of E. coli. 118 Cadmium exposure causes oxidative stress, and the enzymes that are able to detoxify superoxide anions contribute to cadmium resistance. The capacity of bacteria to resist cadmium toxicity is attributed to the activity of the two cytoplasmic superoxide dismutases, the manganese-containing MnSOD, encoded by sodA, and the iron-containing FeSOD, encoded by sodB. 119 The importance of periplasmic Cu-and Zn-SOD in Salmonella pathogenesis is underscored by the existence of two distinct and unlinked sodC genes. The first of the discovered genes is designated sodCI and appears to be encoded on a cryptic A-like bacteriophage. The second locus, sodCII, was more closely related to the E. coli sodC gene. Because the combination of mutations in both sodC genes provides a higher reduction in virulence in mice than single mutations in either sodC gene alone, both SodC proteins appear to be functionally significant. The biological function of sodA in antimicrobial resistance was also investigated. SodA deactivation resulted in a reduced bacterial growth rate, low SOD activity, and high sensitivity to reactive oxygen species and chicken serum. 104 Dps (DNA-binding protein from starved cells) is a stress-related protein similar to ferritin, and it showed an almost 6-fold increase in expression in S. Typhimurium biofilms. 65 Because of its DNAbinding properties, it protects DNA from oxidative damage and is also able to protect cells during periods of nutrient deficiency and other environmental stresses, including ultraviolet and gamma irradiation, metal toxicity (iron and copper), thermal stress, and acid/base shocks. 120,121 Dps is common in starved cells, and mutants lacking Dps exhibit significant changes in protein synthesis in response to starvation stress. It forms very stable complexes with DNA and the complex formation does not require sequence specificity. 121,122 When examining the response of the dps deletion mutant of S. Enteritidis, hypersensitivity to four different antibiotics was observed compared to the wild strain: two quinolones (nalidixic acid and norfloxacin), an aminoglycoside (streptomycin), and rifampicin, which is a semisynthetic drug. However, these differences were completely reduced when the cultures were supplemented with an iron chelator. 120

Multiple resistance proteins
Upregulation of genes associated with microbial resistance, including ugtL and bhsA, was observed in biofilms compared to their expression in the planktonic form. 51 UgtL is a PhoQ accessory protein that contributes to monophosphorylated lipid A formation. 123 It was demonstrated that the Salmonella ugtL mutant strain has a deficiency in gut colonization, using a mouse model of streptomycin-induced diarrhea. Outer membrane integrity and sensitivity to magainin 2, an alpha-helical CAMP, were also diminished in the ugtL mutant strain. Additionally, it was discovered that PhoPactivated ugtL is necessary for polymyxin B resistance. 124 BhsA is a protein encoded by a porin gene that regulates membrane permeability in response to ROS generated by H 2 0 2 and HOCl. 125 It is also referred to as the STY1254 gene, which is one of the most highly upregulated genes in S. Typhi biofilms. 63 Zhang et al. indicated that the bhsA gene in E. coli increases the stickiness of the protein to the membrane, enabling it to adhere to surfaces during biofilm formation. The scientists demonstrated that the deletion of the bhsA gene in E. coli led to increased biofilm formation because bacteria were unable to adhere to the surface, leading them to produce more biofilm matrix as a form of defense. 126 Additionally, Salazar et al. discovered that bhsA contributes to S. Typhimurium adhesion to the surface of glass, polystyrene, spinach leaves, and tomato fruit. 127 The two genes, bhsA and STY1255, may be responsible for the adherence of S. Typhi to the surface of polypropylene tubes, enabling the development and maintenance of biofilms, according to their activities and the findings of transcriptome investigation. 63 In addition, the absence of bhsA increases the resistance of E. coli to carbon monoxide-releasing molecule-2 (CORM-2), which acts as an inhibitor of bacterial growth, even in MDR strains. 128

Enzymes
Another group of genes upregulated in Salmonella biofilms encodes enzymes. The gene osmC of E. coli belongs to a family of homologs distributed across a wide range of species, including Salmonella. Several studies on the regulation of these genes have demonstrated that they are induced by a variety of stressors. OsmC is also activated in Salmonella biofilms. 65 The involvement of OsmC in defense against oxidative stress was proposed in a study investigating the survival of E. coli cells in media with different NaCl concentrations, in which the hydroperoxide activity of OsmC was subsequently demonstrated. 129 However, the biochemical functions of the OsmC-like proteins remain unknown. Its homolog in Xanthomonas campestris, ohr, is involved in protection against organic hydroperoxides. 130 This was also the case for osmC in E. coli. Indeed, the osmC mutant exhibited a higher sensitivity to H 2 O 2 . 129

Biofilm-forming Salmonella: a threat to health and environment and strategies for prevention and control
The gene expression profile of bacteria in biofilms differ significantly from that of their planktonic counterparts. This is due to the complex, threedimensional structure of the biofilm and the presence of an extracellular matrix that can alter genetic patterns. One of the most significant implications of altered gene expression in biofilms is their increased antimicrobial resistance. These problem appears in healthcare settings, where biofilms form on medical devices and lead to increased risk of treatment failure. 131 Furthermore, altered genetic patterns have important consequences in the spread of infectious diseases. Biofilms can provide a protective environment for bacteria, allowing them to evade the immune system and persist in the host even in harsh conditions. 132 This persistence results in chronic infections that can be not only difficult to treat but also promote the formation of new, more virulent strains of bacteria. Moreover, biofilms facilitate the horizontal transfer of genetic material, including virulence factors and antibiotic-associated genes, which can lead to the spread of these traits to other microorganisms. 133 While Salmonella is a type of enteric pathogen, its biofilm is a significant concern in the food processing and packaging industries as it can contaminate both fresh and processed food products. 134 This corruption occurs either directly during processing or indirectly when uncompromised products come into contact with previously contaminated machinery or surfaces. It was shown that Salmonella adhere to different materials used in industrial settings, including stainless steel, glass, and polystyrene, and persist in such environment for a long time. 135 In addition to causing foodborne illness, Salmonella biofilms contaminate water systems. 136 Pathogens can survive in water for extended periods, allowing corruption of drinking water and recreational water sources such as lakes and swimming pools. What is more, Salmonella alters the physical and chemical properties of the surface, resulting in changes in nutrient cycling and water quality. 137 Although sanitation practices are implemented to disinfect possible sources of crosscontamination, Salmonella biofilms are more challenging to disinfect compared to their planktonic counterparts, making such practices less effective. It leads to the need for more aggressive cleaning agents, such as biocides which have a detrimental environmental impact and deleterious consequences for human health. 138 Additionally, the use of biocides could act as selection pressures for increased microbial resistance to antimicrobial compounds, and the sanitizers may lose effectiveness gradually. This phenomenon, known as antimicrobial cross-resistance, is considered as one of the most challenging to public health worldwide. 139 Addressing this problem requires a multifaceted approach that considers both socioeconomic and bacterial perspectives. There are two main strategies to overcome biofilms: inhibiting their formation or dispersing established biofilms. 140 Preventing biofilm development involves inhibiting bacterial adherence to surfaces and their subsequent growth. Surface modification, such as imprinting 3D patterns or targeting physicochemical properties, can be effective at preventing early attachment. 141 Another approach is preconditioning surfaces with surfactants that inhibit bacterial adherence. 142 Improving sanitation and hygiene practices in industries is therefore crucial to prevent biofilm-associated Salmonella contamination. Awareness campaigns, education, and community outreach programs can also play a vital role in educating the public about the importance of proper food handling and storage practices.
Strategies aimed at blocking bacterial establishment on surfaces often exploit stimuli to control the genes involved in biofilm formation. 143,144 Understanding the molecular mechanisms involved in this process can help identify potential targets for the development of anti-biofilm agents. Molecular biology techniques, such as genome sequencing and transcriptomics, can be used to identify genes involved in biofilm formation and develop drugs that specifically target these genes or their products, leading to more effective therapy. On the contrary, mature biofilms can be dispersed by breaking off biofilm assemblies and promoting detachment. This requires disrupting EPS polymers by enzymes or programming cells to disperse. 145 Given the challenge to conventional antimicrobial therapies due to increased resistance and genetic exchange of biofilm, a therapeutic approach that increases sensitivity to current methods or combines various modes of action with low cost is necessary.

Summary
The biofilm lifestyle of Salmonella enables its persistence in the environment and in medical, veterinary, and industrial settings; it is a natural attribute, with the bacteria being significantly more widespread in its biofilm-producing form than in the planktonic form. The main reasons for this are enhanced tolerance to nutrient deficiencies and increased resistance to aggressive environmental conditions, including antimicrobial treatments. Indeed, infections caused by biofilmforming bacteria require a substantially more intensive combination of antibiotics. Consequently, Salmonella becomes resistant to the medications being used, posing a threat to public health.
In this work, we have discussed the background of the factors regulating the main resistance mechanisms of Salmonella biofilms, with a particular emphasis on molecular agents. We have summarized the current knowledge of advancements in research dedicated to antibioticresistance-associated genes overexpressed in biofilms and described their role in Salmonella pathogenicity. Although much attention has been paid to this topic, there are still important scientific concerns that remain unanswered.
There are already known proteins directly associated with antibiotic resistance, including multiple resistance, transporters, xenobiotic degradation, and stress response proteins, while the roles of others, such as those involved in energy metabolism and outer membrane proteins or enzymes, are not primarily associated with enhanced treatment tolerance. Dozens of genes that encode these important proteins have been identified as upregulated in biofilms of Enterobacteriaceae. Although these genes or their homologs are present in Salmonella, most of the investigations presented transcriptomic data only and did not report their role in Salmonella pathogenicity. In addition, their biological functions were primarily validated in E. coli strains; therefore, we can only assume that their roles in Salmonella are analogous. We anticipate that this research will be broadened with comprehensive laboratory experiments consisting of the generation of deletion mutants and functional analysis using eukaryotic cell lines and various abiotic surfaces being utilized by Salmonella. There is no doubt that a large-scale xenobiotic resistance analysis should be performed to identify and study the antibiotic resistance profile of Salmonella. Another crucial factor that needs to be considered in future investigations is the serotype. It was previously shown that the outcomes can significantly differ due to different genetic backgrounds, such as single nucleotide polymorphisms (SNPs) in virulence factor-coding sequences. Thus, we believe that information obtained from a comparative analysis of representative strains would be beneficial.
Due to the increasing number of antibioticresistant biofilm-forming Salmonella, treatment of salmonellosis is impeded. This has led to the overuse of antibiotic-based therapies, especially in underdeveloped countries. As a result, selective pressure from antimicrobial therapy and antibiotic resistance crises have been observed worldwide. According to the developed models, antimicrobial resistance can cause 10 million deaths annually in the human population worldwide by 2050. Therefore, understanding the molecular patterns of biofilm formation, as well as defining the antibiotic resistance profile of biofilms under various environmental conditions, is required for the development of antibiofilm strategies. Considering the zoonotic potential of Salmonella, it is essential for both scientific research institutions and the food industry to develop techniques that effectively prevent biofilm formation and remove mature biofilms from the environment, food, and medical devices.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Data availability statement
Data sharing not applicable -no new data generated.