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Article

The Basis for Variations in the Biofilm Formation by Different Salmonella Species and Subspecies: An In Vitro and In Silico Scoping Study

by
Amreeta Sarjit
1,
Yi Cheah
2 and
Gary A. Dykes
3,*
1
Institute of Innovation, Science and Sustainability, Federation University Australia, Gippsland, VIC 3842, Australia
2
School of Science, Monash University Malaysia, Bandar Sunway 47500, Malaysia
3
School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD 4072, Australia
*
Author to whom correspondence should be addressed.
Appl. Microbiol. 2023, 3(3), 841-855; https://doi.org/10.3390/applmicrobiol3030058
Submission received: 29 June 2023 / Revised: 31 July 2023 / Accepted: 1 August 2023 / Published: 3 August 2023
(This article belongs to the Special Issue Exclusive Papers Collection of Editorial Board Members and Invited Scholars in Applied Microbiology)
Browse Figure
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Abstract

:
This study examined whether the presence/absence of biofilm-associated genes may indicate the potential for differences in the biofilm formation among the Salmonella species/subspecies. We conducted an in vitro study on the biofilm formation by eighteen Salmonella strains of different species/subspecies. Strains belonging to subspecies enterica were generally poorer biofilm formers than strains belonging to species bongori and subspecies arizonae, diarizonae, and indica. A broader in silico study was subsequently conducted. The presence/absence of 57 biofilm-associated genes was further investigated among 323 Salmonella whole genomes of various species/subspecies. The lpfE gene was present in in 88.2% of subspecies enterica but was absent in ~90.2–100% of other subspecies. The sirA gene was present in 11.8% of subspecies enterica and 2.9% of S. diarizonae genomes while absent in other species/subspecies. The lpfe gene and sirA gene in subspecies enterica negatively correlated with environmental biofilm formation. The csrB gene was present in 71.4% of the S. arizonae and 94.3% of S. diarizonae genomes but absent in other species/subspecies. The absence of csrB in subspecies enterica positively correlated with weaker environmental biofilm formation. This may contribute to subspecies arizonae and diarizonae being better biofilm formers.

1. Introduction

Salmonella is one of the leading causes of bacterial foodborne disease worldwide and also a common zoonotic pathogen. S. enterica and S. bongori are the two species of Salmonella. Most pathogenic Salmonella belong to the species S. enterica, which is traditionally further divided into subspecies enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb), houtenae (IV), and indica (VI) [1]. The subspecies enterica (I) has more than 2600 serovars [2]. More recently four more subspecies were identified, unnamed subsp VII and three novel subspecies A, B, and C [3]. The most common subspecies is S. enterica subspecies enterica, and this is generally isolated from humans and warm-blooded animals. Other subspecies and S. bongori are generally isolated from cold-blooded animals [4,5]. Salmonella has also been reported in environmental reservoirs, which have been seeded through contamination by animals and humans [6,7]. In most environments, it is difficult for planktonic Salmonella to survive due to a wide range of environmental stressors, which include desiccation, the scarcity of nutrients, and toxic compounds [8]. Biofilms represent one way in which Salmonella can ameliorate these stressors.
Biofilms are defined as a self-contained community of bacteria belonging to the same or different species that have attached to a biotic or abiotic surface and are enveloped by a layer of extracellular polymeric substances (EPS), which is composed of polysaccharides, proteins, lipids, and even DNA [9]. The benefit of a biofilm, especially in harsh conditions, is that the EPS layer, which is produced by the bacteria within the biofilm, confers protection against most of the external threats mentioned previously whilst still allowing essential nutrients to enter and wastes to leave. Another benefit of a biofilm is that the cells within a biofilm, especially when there is a mixture of strains and species, are able to produce compounds, such as nutrients and enzymes that break down toxins, that help one another survive better [10]. It has also been observed that within the close confines of a biofilm, bacteria are able to transfer genes horizontally primarily through plasmids. These genes can contain anything from virulence factors, including biofilm-associated genes [11]. Virulence genes occur in Salmonella Pathogenicity Islands (SPI) located on chromosomes, plasmids, or bacteriophages [12]. SPIs are acquired by Salmonella during horizontal genetic transfer events, in which a large amount of DNA is transferred leading to the quick adaptation of Salmonella to stronger virulence. There are currently 24 SPIs identified, with most SPIs associated with the enterica species [12,13]. S. bongori has been reported to have a lower G + C content as compared to the enterica species and has three SPIs associated with it [5]. Subspecies enterica was separated from the other subspecies with the identification of SPI6. Little work has been conducted on SPIs with regards to the differences between the enterica and non-enterica subspecies [5]. Previous studies have also reported that plasmid types were associated with Salmonella serovars and the source of the isolates, but little is known with the non-enterica subspecies [14]. The ability of Salmonella to form biofilms on food processing surfaces, equipment, pipes, vegetables, and on food matrices can be of great concern, especially when the antimicrobials used are ineffective [8,15,16].
A large number of studies have been conducted on S. enterica subspecies enterica biofilm formation with a focus on S. Typhimurium and S. Enteritidis, as these two serovars are the primary causes of infections in humans [5,17,18]. A better understanding of biofilm formation by the entire Salmonella genus is important because there could be some exchange of genetic material among various species/subspecies impacting the biofilm formation. There has been very little work conducted on the relationship between the biofilm formation and the carriage of the relevant genes in S. arizonae, S. diarizonae, S. houtenae, S. indica, S. salamae, and S. bongori and their relationship to biofilm formation [19,20,21]. We conducted an in vitro study on biofilm formation by eighteen Salmonella strains of different species/subspecies. The subspecies enterica strains were generally poorer biofilm formers than species bongori and subspecies arizonae, diarizonae, and indica. A broader in silico study was subsequently conducted using 323 whole genomes. The presence/absence of 57 biofilm-associated genes was investigated to determine which species/subspecies had greater genetic potential to form biofilms.

2. Materials and Methods

2.1. In Vitro Study of Biofilm Formation by Selected Salmonella Strains

2.1.1. Bacterial Culture

Eighteen Salmonella strains isolated from previous studies [22,23] were used. These consisted of 10 isolates obtained from lizard stools, 7 isolates obtained from supermarket vegetables, and 1 isolate obtained from a bovine stool sample in Peninsular Malaysia. S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076, obtained from the American Type Culture Collection (Manassas, VA, USA), were also used in this study. All strains were grown on xylose lysine deoxycholate (XLD; Oxoid, Basingstoke, UK) or in Tryptone Soy Broth (TSB; Oxoid, Basingstoke, UK) at 37 °C for 24 h under aerobic conditions. The subspecies of the Salmonella isolates were determined using a multiplex polymerase chain reaction (PCR) procedure as described by Lee et al. [24]

2.1.2. Biofilm Formation Assay

The biofilm assay was carried out as described previously by Mireles et al. [25] with modifications. The crystal violet staining method is a common method used to quantify the Salmonella biofilm formed [26,27]. It is based on the fact that crystal violet binds to the surface molecules of the bacteria and extracellular biofilm matrix. Bacterial cultures were diluted in TSB to obtain an optical density of ~108 cfu/mL. Briefly, 200 µL of the bacterial culture was inoculated into a flat bottomed 96-well polystyrene microtiter plate (TPP®, Trasadingen, Switzerland). Uninoculated TSB was used as blanks. The microtiter plates were incubated under aerobic conditions for 18 h at 37 °C statically. We were interested in examining environmental biofilm formation, which is why the biofilms were grown under aerobic conditions, as described previously [28]. Biofilm formation under anaerobic, microaerophilic, or other conditions may be different. After incubation, broth cultures were poured out of the titer plates and the wells were gently rinsed twice with 200 µL of distilled water to remove the loosely attached bacteria. The plates were air dried for 20 min, and the wells were then stained using 200 µL of crystal violet for 15 min. The crystal violet was then removed, and the wells were rinsed twice again with 200 µL of distilled water and left to air dry for 20 min. The wells were then destained using 200 µL of 80:20 alcohol:acetone solution. The absorbance values of the plates were immediately read at 550 nm using Tecan Infinite® M200 (Tecan, Männedorf, Switzerland). The Pseudomonas aeruginosa strain MR2 isolated from the sewage treatment near Monash University Sunway Campus was used a control.

2.1.3. Screening of the Biofilm-Associated Genes

The experiment was repeated using PCR as described by Lee et al. [24] with slight modifications. Due to the significantly different annealing temperatures of the different primers used for the screening of biofilm-associated genes, monoplex PCRs were performed. The PCR reaction was carried out using a 25 µL reaction mixture which was composed of 14.4 µL of sterile distilled water, 5 µL 5× Green GoTaq Flexi buffer, 1.5 µL 25 mM MgCl2, 0.5 µL of 10 mM dNTP mixture, 1.25 µL of 0.1 mM forward and reverse primers, respectively, 0.125 µL of GoTaq® Flexi DNA polymerase, and 1.0 µL template genomic DNA. All the PCR procedures started with an initial denaturation step at 95 °C for 10 min, followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at the temperatures shown in Table 1 for 1 min, and extension at 72 °C for 6 min. There was a final extension at 72 °C for 10 min before a final hold at 4 °C. The positive controls used in this study were S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076 for all genes except tcfA, since tcfA has been known to be S. Typhi specific. The amplicons were visualized on 2% agarose gel.

2.1.4. Expression of the Curli and Cellulose Assay

Colonies of all the bacterial isolates were analyzed on Luria Agar (LA; BD Difco, Sparks, MD, USA) without salt supplemented with Congo Red 40 µg/mL (Sigma Aldrich, St. Louis, MA, USA) as described by Malcova et al. [34] with some minor modifications. Briefly, each suspension was inoculated with each Salmonella isolate at ~108 cfu/mL. Serial decimal dilutions were conducted in phosphate-buffered saline (PBS; 2.7 mM KCl, 10 mM Na2HPO4, 17 mM KH2PO4, and 137 mM NaCl at pH 7.4; 1st Base, Singapore) and were inoculated onto the media. The inoculated media were then incubated for 24 h at 37 °C, followed by another 48 h at 24 °C. The different colony morphologies were recorded, and the respective expressions of fimbriae and cellulose genes were recorded as described by Bokranz et al. [35]. Briefly, a smooth and white (saw) morphology represented that neither curli fimbriae nor cellulose were expressed; a pink, dry, and red (pdar) morphology represented that only cellulose was expressed; brown, dry, and red (bdar) morphology represented that only curli fimbriae was expressed; and a red, dry, and rough (rdar) morphology represented both curli fimbriae and cellulose being expressed.

2.2. In silico Study of Biofilm-Associated Genes Across a Range of Salmonella Genomes

The presence of 57 biofilm-associated genes across 323 Salmonella genomes consisting of S. enterica subspecies enterica (n = 51), S. enterica subspecies arizonae (n = 70), S. enterica subspecies diarizonae (n = 70), S. enterica subspecies indica (n = 21), S. enterica subspecies houtenae (n = 51), S. enterica subspecies salamae (n = 52), and S. bongori (n = 8) were identified to determine whether the presence/absence of biofilm genes was linked to Salmonella subspecies and their ability to form biofilms. The 57 biofilm-associated genes were chosen from previous studies conducted mostly on S. enterica subspecies enterica [36,37]. The number of genomes per species/subspecies was selected based on the availability of the genomes on the National Centre for Biotechnology Information (NCBI) website and the information provided such as the year, location, and source of isolation. These strains were isolated from various sources and geographical locations globally from the year 1905 to 2022. The biofilm-associated genes were generally screened using a well characterized reference genome, S. Typhimurium strain LT2. The sequence of each reference gene was BLAST-searched against the 57 genomes respectively with a maximum e-value of 10−30 [38]. The reads of all these genomes were downloaded from the sequence read archive (SRA) in the NCBI and assembled using SPADES. The Quality Assessment Tool (QUAST) was used to determine the genome qualities of the final assemblies followed by annotation, using Rapid Annotations using Subsystems Technology (RAST) [39,40,41]. All the genomes were selected based on the availability of accession numbers and genome information as shown in the Supplementary Table S1. EvalG and EvalCon as described by Parks et al. [42] were used to determine the genome quality. The genomes were also annotated using the PROKKA annotation in order to determine the pan genome using the ROARY tool [43]. The pan genome was visualized by constructing a phylogenetic tree using the core genome sequences obtained from Roary. The phylogenetic tree was viewed and annotated using FigTree version 1.4.3, UK.

2.3. Statistical Analysis

All assays were conducted in triplicate. In the case of the biofilm formation assays, and viability assays, three independently grown bacterial broths were used. The data obtained were analyzed using SPSS 20 software (IBM Inc., Armonk, NY, USA). The data collected on biofilm formation by the respective strains were analyzed using a one-way analysis of variance (ANOVA) and a pairwise comparison of means that was performed using a post hoc Tukey test at 95% confidence level.

3. Results and Discussion

3.1. In Vitro Study of Biofilm Formation by Selected Salmonella Strains

The results of the biofilm assay using the microtiter plate method are shown in Figure 1. When the strains were arranged according to subspecies, a trend was observed where subspecies enterica appeared to be a weaker biofilm former than the other subspecies. Out of the ten strains under subspecies enterica, only ATCC 13076, M11, and M16 were at least half as good at forming biofilms as the P. aeruginosa control. Out of the remaining ten strains belonging to the other subspecies and S. bongori, all of them, except U3, produced at least half as much biofilm as P. aeruginosa, which was the control strain used in the study. S. arizonae, S. diarizonae, and S. indica were evidently better biofilm formers as compared to S. bongori in this study.
Subspecies enterica tended to have more biofilm-associated genes with all ten enterica strains carrying between 57.1 and 85.7% of these. In comparison, the majority of the strains belonging to subspecies arizonae, diarizonae, and indica carried between 0 and 57.1% of the biofilm-associated genes. Subspecies enterica are better adapted at colonization and survival within the hosts as compared to the other subspecies [44]. Interestingly, the presence of several genes associated with biofilm formation in the genomes of the strains analyzed in this study may not influence the ability to form biofilms on polystyrene surfaces under conditions that simulate the environment. A limitation of our study was the number of analyzed strains of each subspecies. However, it can be observed that the higher percentage of biofilm-associated genes in subspecies enterica may be negatively correlated with biofilm formation in the environment, possibly making subspecies enterica a poorer biofilm former outside the host. Similarly, S. bongori carries a higher percentage of these genes as compared to subspecies arizonae, diarizonae, and indica, and it was a weaker biofilm former as compared to these three subspecies. Further studies can be carried out to analyze the role of these genes in the formation of biofilms by these Salmonella subspecies.
The expressions of curli and cellulose by the strains are presented in Table 2. Of the 20 strains screened, only two strains, S. enterica M11 and S. arizonae U3 expressed neither curli nor cellulose. The remaining 18 strains exhibited at least one of the two with seven of that 18 expressing both curli and cellulose. The formation of biofilms in S. enterica have been shown to rely heavily on the presence of curli fimbriae, which facilitates autoagglutination, and cellulose, which forms the protective coating of a biofilm [29,32]. Previous studies have reported that the curli and cellulose production is variable in S. enterica, while certain strains of subspecies arizonae and diarizonae were not able to produce both cellulose and curli [5]. The expressions of both curli and cellulose in this study were at odds with the screening of the biofilm-associated genes as well with the ability to form biofilms. This shows that there could be other regulatory pathways that may be involved that are not present or active in these Salmonella strains. This is in agreement with previous studies that showed rdar, pdar, bdar, and saw morphotypes did not solely correlate with biofilm formation [45,46].
S. indica M4 was a strong biofilm former despite not possessing the agfA, bcsA, and yshA biofilm-associated genes investigated in this study, which are responsible for curli adhesin, cellulose production, and cellulose secretion, respectively, [29,32,33]. These three genes play a role in biofilm formation regardless of the surface being tested. Some bacteria have been shown to form biofilms without cellulose as the main component of the biofilm [47]. Another inconsistency that arose was the fact that S. enterica ATCC 14028, S. enterica M12, and S. enterica M13, which possessed all these three biofilm-associated genes, were poor biofilm formers. A possible reason for this is that mutations in the bcs operon, of which the bcsA gene is a part of, can adversely affect biofilm formation [48].
Based on the results shown in Figure 1, whilst each subspecies group had strong and weak biofilm formers, subspecies enterica was generally a weaker biofilm former as compared to the other groups. One possible explanation for the observed differences is that subspecies arizonae, diarizonae, indica, and houtenae, are generally found in cold-blooded animals as opposed to subspecies enterica, which is commonly found in warm-blooded animal hosts. We recognize that this study was limited with respect to both the number of strains and the conditions under which biofilm formation was investigated. It did, however, provide some interesting results, and we therefore undertook an in silico study to establish whether there was a broader basis for the results and to establish whether further research in the area is warranted.

3.2. In Silico Study of Biofilm-Associated Genes Across a Range of Salmonella Genomes

Across the 323 Salmonella genomes, the pan genome analysis indicated 1774 core genes present within ≥99% of Salmonella genomes. A phylogenetic tree was generated based on the entire pan genome as shown in Supplementary Figure S1, which consisted of a total of 60,259 genes. The genomes are listed according to the pan genome in Supplementary Table S1. The source of the Salmonella strains, presence/absence of biofilm-associated genes, and gene function are shown in Supplementary Table S1 and S2. The source of isolation was further categorized into clinical, nonclinical, and unclassified. Based on these categories, there were 166 clinical sources, 136 nonclinical sources, and 21 unclassified. To determine whether there was a more distinct relationship between the presence/absence of these biofilm-associated genes in each subspecies/species, we also categorized the source of isolation into fruit, herb, mammal, marsupial, reptile, meat, seafood, spice, and unclassified. Sometimes, other subspecies apart from enterica can be found in warm- blooded animals, cold-blooded animals, and humans, as seen in the screening of the 323 genomes. The numbers of each biofilm-associated gene present across species/subspecies are shown in Table 3. Among all the species/subspecies investigated, the percentage of all biofilm-associated genes carried by S. enterica and S. arizonae was 94.7% respectively, S. diarizonae was 96.5%, S. houtenae was 92.9%, S. indica was 87.7%, S. salamae was 89.5.%, and S. bongori was 82.46%. Within the S. enterica cohort, the presence/absence of genes was consistent across most of the genomes unlike the S. arizonae genomes.
The fimbriae (peF) are encoded by genes on the Salmonella plasmids [49]. These genes include pefA and pefC, which aid in the adhesion of Salmonella to surfaces such as epithelial cells [49,50]. Both pefA and pefC genes were reported in 15.7% of the subspecies enterica genomes, 1.4% of the subspecies arizonae genomes, and 5.8% of the subspecies houtenae genomes in this study. The presence of the pefA gene has been reported to be low in previous studies, with a recent study only reporting the presence of the pefA gene in 10.3% of the subspecies enterica strains investigated in their study [51,52,53]. Previous studies have shown that S. Enteritidis and S. Typhimurium plasmids carry the pefA and pefC genes [49]. Similarly, our in silico study showed that both the pefA gene and pefC gene were present across both these serovars. Both these genes were present across 60% of S. Enteritidis genomes and 40% of S. Typhimurium genomes, but our study also reported it across 50% of S. Bovismorbificans genomes among subspecies enterica. The pefA gene was also evident in both the S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076 strains used in the in vitro study, which were weaker biofilm formers. The presence of pefA maybe negatively correlated with biofilm formation, and a possible reason for this could be that our study was investigating Salmonella biofilms in the environment rather than within a host. As such, there could be other regulatory pathways preventing it from being a strong biofilm former. The pefA gene and pefC gene were only present in 1.4% of the subspecies arizonae and 5.8% of the subspecies houtenae genomes, which could be associated with carrying the pef operon. However, with the screening of the in vitro study, both S. arizonae R32 and S. diarizonae R2, which carried the pefA gene, were strong biofilm formers. The presence of plasmids plays a role in recombination and virulence in Salmonella, and this could enhance its ability to form biofilms [54].
The lpfE gene, which codes for long polar fimbriae, aids in the colonization of the host intestinal wall [29]. Previous studies have reported that the plasmid encoded phase 2 flagellar regions carrying both lpf (long polar fimbriae) and pef (plasmid encoded fimbriae) genes, commonly associated with human salmonellosis were absent in S. Sofia [5,55]. This correlates with the results of this study, which showed that all the subspecies salamae genomes, which were characterized to S. Sofia lacked the lpfe, pefA, and pefC genes. All the S. salamae genomes irrespective of serovar and the source of isolation investigated in this study were reported to lack the pefA and pefC genes, while the lpfE gene was only evident in 1.9% of the S. salamae genomes. The lpfE gene was present in 70% of S. enterica and in 100% of S. bongori strains, while it was absent across the remaining strains in the in vitro study. This correlates with the broader in silico investigation, as 88.2% of the S. enterica genomes and 100% of the S. bongori genomes carried the lpfe gene. The lpfE gene was absent in 100% of the S. indica genomes but was present in 4.3% of the S. arizonae, 4.3% of the S. diarizonae, 9.8% of the S. houtenae, and 1.9% of the S. salamae genomes. The lpfE gene was only present in the S. salamae genome isolated from a reptile. In the case of S. arizonae, lpfE was only present in strains isolated from animal-based sources, while for S. diarizonae, the lpfE gene was only present in strains isolated from food or humans. This was not the case when compared with subspecies enterica, where 88.2% of subspecie enterica genomes carried this gene irrespective of the source. The lpfE gene was present in 88.2% of subspecies enterica but was absent in ~90.2–98.1% of the other subspecies. The relatively high presence of the lpfE gene in subspecies enterica is correlated with previous studies that showed that the lpf operon has been associated with subspecies enterica [56]. Although subspecies enterica has a high occurrence of the lpfE gene, which aids in the colonization of the host intestinal wall, it was a weaker biofilm former in our study. The presence of this gene may be negatively correlated with the biofilm formation of subspecies enterica in the environment rather than in the host.
Previous studies have determined the expression of cellulose using morphotypic screening to determine the expression of extracellular cellulose and curli fimbriae using Congo Red impregnated onto the culture media to enable the differentiation of the bacterial gene expression based on the colony morphology, which prompted us to use this methodology [34]. The rdar morphotype is caused by the co-expression of the curli fimbriae and cellulose associated genes, bdar is associated with the curli fimbriae synthesis genes, saw is associated with the cellulose synthesis genes, and pdar is associated with the presence of cellulose production but the absence of fimbriae biosynthesis genes [48,57,58,59]. As mentioned earlier, the bcsA gene is essential for cellulose secretion and was only absent in 66.7% of the S. indica strains in the in vitro study. This was not the same with the broader in silico investigation, as this gene was present across all species and subspecies. The presence/absence of this bcsA gene was only variable across the S. arizonae genomes investigated in the in silico study with regard to biofilm formation. The adrA gene is essential in enhancing the cellulose synthesis to enhance biofilm formation. In the in silico study, all species and subspecies carried this gene with the exception of 5.7% of the subspecies arizonae genomes. Bacterial cells exhibiting the rdar and pdar morphotypes are related to biofilm formation and are closely related to the adrA gene. However, in the in vitro study, only 10% of subspecies enterica, 33% of subspecies arizonae, and subspecies diarizonae, respectively, did not express the cellulose morphotype. This may suggest that most Salmonella genomes irrespective of subspecies may carry this gene with some exceptions. The other essential gene involved in exhibiting the rdar morphotype is csgD, as both adrA and csgD are involved in co-expressing cellulose and curli fimbriae [46]. There were eleven S. arizonae genomes from mostly clinical sources lacking csgD. Some of these S. arizonae genomes were also lacking the bcsC involved in cellulose production. This is consistent with our in vitro study, as two strains of S. arizonae did not express the curli fimbriae. This could be attributed to the fact that they might not be carrying csgD.
The sigma factor, rpoS, regulates the expression of ~100 genes associated with environmental stressors, while the hilA gene is a transcriptional activator involved in Salmonella invasion associated with the transcription of genes in SPI1 [36,60]. Both rpoS and hilA, which is essential in the biofilm formation of S. Typhimurium, were observed to be present across all species and subspecies with variation observed among the S. arizonae genomes [36]. The luxS gene is involved in the molecular or Salmonella communication signals associated with biofilm formation [61]. The luxS gene, mostly evident in S. enterica genomes, was also observed to be present across most species/ subspecies with the exception of some S. arizonae genomes and one S. salamae genome. The serovar of these Salmonella genomes may have contributed to the absence of the luxS in the S. salamae genome, as the other S. salamae genomes with a known serovar such as Sofia were observed to carry this gene.
The bssR gene, which is essential in cell signaling [62] was present across all subspecies investigated in this study with some variation across the S. arizonae genomes. The bssR gene involved in biofilm regulation has been previously reported in S. enterica, and its expression was downregulated in S. Typhi biofilm [63]. However, this same gene was significantly upregulated in Escherichia coli biofilms, which is very closely related to Salmonella [63]. The yciZ and ynfC genes are reported to co-occur with bssR. To our knowledge, there has been no work conducted with yciZ in relation to the biofilm formation of Salmonella. Approximately 88.6% of the S. arizonae genomes lacking this gene clustered together in the pan genome irrespective of the source of isolation. The yciZ gene was evident in ~98.6–100% of the genomes of other subspecies and species, which may help with its biofilm formation. The gatC gene, a gene regulator for biofilm formation has been reported among antimicrobial resistant S. enterica subspecies enterica isolated from poultry [64]. Among the subspecies enterica genomes investigated in this study, 66.7% of the genomes carried the gatC gene. This gene was absent in all the S. Dublin and S. Enteritidis genomes, 16.7% of the S. Bovismorbificans genomes, and 40% of the S. Saintpaul and S. Newport genomes, among the subspecies enterica. However, in the non-enterica subspecies investigated in our study, gatC was present in 57.7% of the subspecies salamae, 7.8% of the subspecies houtenae, 4.8% of the subspecies indica, 4.3% of the subspecies arizonae, and 2.8% of the subspecies diarizonae. The gatC gene has been implicated to be associated with invasive isolates in a host [65]. Thus, the presence of this gene in subspecies enterica may be negatively correlated with strong biofilm formation in the environment.
The fimF, fimG, and fimH genes are responsible for the adhesive capability and for the initial contact of biofilm formation on host cells [66,67]. The fimF and fimH associated with type 1 fimbriae in S. Typhimurium were reported across all subspecies in this study. These genes were only absent across all S. bongori genomes. Some of the genes investigated in this study could be Salmonella species/subspecies/serovar determinant genes, which could contribute towards better biofilm formation. To our knowledge, these genes have yet to be reported in S. bongori, as there is very little work on it. Previous studies have reported that certain strains of S. Typhimurium carrying fimF were good biofilm formers, while fimH is important for the initial attachment on epithelial cells [68]. The fimG gene associated with Type 1 fimbriae is essential for the initial attachment and has been reported to enhance the biofilm formation in E. coli [69]. This was shown with a mutant of fimG that decreased the biofilm formation of E. coli. This gene has rarely been reported among subspecies enterica and has not been reported in S. Typhimurium [70]. This agrees with our study, as all subspecies enterica lacked this gene. In addition, this gene was also absent across all other subspecies and species with the exception of one genome of S. diarizonae.
The gene sirA is important in enhancing biofilm formation by increasing the expression of type 1 fimbriae and is essential in activating the csrB for biofilm formation. This gene has been reported to be associated with Salmonella host cell invasion [71]. It was apparent in our in silico study that this gene was only present in 11.8% of subspecies enterica and 2.8% of subspecies diarizonae genomes. It is interesting to note that S. Dublin and S. Newport were reported to carry the sirA gene among subspecies enterica in this study. The presence of this gene in subspecies enterica was negatively correlated with the biofilm formation of Salmonella in the environment. None of the subspecies enterica genomes carried the csrB gene. The absence of the csrB gene in subspecies enterica may contribute to this subspecies being a poorer biofilm former. The presence of the csrB gene was evident in 71.4% of subspecies arizonae and 94.3% of subspecies diarizonae genomes. All the remaining subspecies lacked csrB. The presence of the csrB gene in both subspecies arizonae and diarizonae was positively correlated with biofilm formation and may contribute to these subspecies having better potential in biofilm formation as compared to the other subspecies and species.
The ygcB gene, which is involved in the CRISPR-associated helicase/endonuclease Cas3, is associated with the regulation of S. enterica biofilm formation. This gene was absent across 60% of S. Newport and 100% of S. Bovismorbificans genomes among the subspecies enterica [72]. This included S. Newport 2393, which lacked all cas genes, and this was related to its stress response to heat in the presence of iron [73]. The presence/absence of this gene in subspecies enterica could be serovar specific [73]. This gene was also absent in 90.2% of the S. houtenae genomes clustering together in the pan genome.
An important factor that can contribute to enhance the virulence of Salmonella is the occurrence of recombination and the presence of novel genes which may impact the diversity of the lipopolysaccharide antigenic factor [54,74]. These changes can also contribute to the presence of lipopolysaccharides-associated genes such as waaL, waaJ, waaK, waaB, and waaO, which are essential in biofilm formation. The waaL, waaJ, and waaB genes are generally conserved among S. Typhimurium [75]. However, in this study, only waaJ and waaB were present across all subspecies enterica genomes irrespective of serovar, while waal was absent across all subspecies enterica genomes irrespective of serovar. The gene waaL was only evident in 1.4% of the S. diarizonae genomes investigated in this study. Previous studies reported that the deletion of the waaJ gene in subspecies enterica resulted in a lower amount of lipopolysaccharide being produced than the deletion of the waaL gene in S. enterica [76]. In addition, the deletion of the waaJ gene in subspecies enterica also exhibited a decrease in resistance to ultraviolet-, acid-, and alkaline-based treatments [76]. This shows that the presence of this gene is essential to ensure the integrity of the lipopolysaccharide layer to form better biofilms as well as to resist intervention strategies using ultraviolet-, acid-, and alkaline-based treatments. In our study, the waaJ gene was absent across all S. bongori genomes and was present across all genomes of S. diarizonae, S. houtenae, S. indica, and S. salamae. Similar to most other genes in S. arizonae, there was a variation in the presence/absence of waaJ. The pagC has been shown to influence lipopolysaccharide production in S. enterica [77]. In our study, the pagC gene was present across most genomes irrespective of subspecies. The bdca gene aids in biofilm dispersal in E. coli by interacting with the intracellular signal cyclic diguanylate (c-di GMP) when there is a reduced concentration of c-di GMP [78]. This leads to an increase in extracellular DNA production and a lower amount of extracellular polysaccharide production (EPS) and cellular aggregation, finally leading to the biofilm dispersal [31]. In this study, we noted that bdcA was only present across all the genomes of S. enterica, S. houtenae, S. indica, and S. bongori. To our knowledge, the concentration of c-di GMP has yet to be investigated in detail with regard to Salmonella, which may lead to uncertainties about whether this gene may enhance or decrease biofilm formation.
The marT gene is a regulator in S. Typhimurium biofilms [68]. The presence of this gene is essential in regulating the expression of 14 other genes including csgA and csgD to enhance biofilm formation. This gene was present in 100% of subspecies enterica, indica and S. bongori, 94.4% of subspecies diarizonae, 94.2% of subspecies salamae, 9.8% of subspecies houtenae, and 5.7% of subspecies arizonae genomes. As marT is only present in a number of S. houtenae and S. arizonae genomes, this may impact the expression of csgA and csgD to enhance biofilm formation. Although the marT gene was present across all subspecies enterica genomes, it may not be expressed or might not be regulating both the csgA and csgD genes to enhance biofilm formation, making this subspecies a poorer biofilm former.
Some of the genes screened in this study have been investigated in the human host in previous studies instead of in the environment for biofilm formation. As such, the presence of some genes may be negatively correlated with biofilm formation in some subspecies as mentioned above. All the genomes and strains investigated in our in vitro and in silico study, respectively, were characterized to the species and subspecies level. However, there were only two strains characterized to the serovar level in the in vitro study. These two strains were S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076, which were also used as positive controls for the screening of the biofilm-associated genes. With regard to the genomes investigated in the in silico study, all the subspecies enterica genomes and some of the other subspecies genomes were characterized to the serovar level. Future studies should more deeply characterize all genomes and strains to the serovar level to have a better understanding of biofilm formation.

4. Conclusions

Our in vitro study on biofilm formation showed that subspecies enterica were generally poorer biofilm formers than strains belonging to the species bongori and subspecies arizonae, diarizonae, and indica. The broader in silico study investigating Salmonella whole genomes of all species and subspecies supported our in vitro study. It showed that the lpfE gene was present in 88.2% of subspecies enterica genomes but was absent in ~90.2–100% of other subspecies genomes. The sirA gene was present in 11.8% of subspecies enterica and 2.8% of subspecies arizonae genomes but absent in other species and subspecies. The presence of the lpfE gene and sirA gene in subspecies enterica may be negatively correlated with Salmonella biofilm formation in the environment. The csrB gene was present in 71.4% of the S. arizonae and 94.3% of S. diarizonae genomes but absent in other species/subspecies. The absence of csrB in subspecies enterica may be positively correlated with weaker biofilm formation in the environment. These differences among Salmonella species/subspecies contribute to the potential for differences in environmental biofilm formation, possibly making subspecies enterica a poorer biofilm former as compared to the other species and subspecies. Further studies should focus on in vitro studies of biofilm formation across all species and subspecies from various sources worldwide. Gene expression studies utilizing whole transcriptome sequencing should also be conducted to complement the in vitro studies of Salmonella biofilm formation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/applmicrobiol3030058/s1. Figure S1: Phylogenetic tree of core genome sequences of Salmonella genomes (n = 323). The maximum-likelihood method was used to generate the tree across the core genomes. Table S1: Pan genome; Table S2: Gene Function.

Author Contributions

Conceptualization, G.A.D; Formal analysis, A.S. and Y.C.; Investigation, Y.C.; Writing–original draft, A.S. and Y.C.; Writing–review & editing, G.A.D.; Supervision, G.A.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

All publicly available whole genome sequence of Salmonella strains have been deposited at the PATRIC database listed in Supplementary Table S1. Supplementary Figure S1: Phylogenetic tree of core genome sequences of Salmonella genomes (n = 323).

Conflicts of Interest

The authors declare no conflict of interest.

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Applmicrobiol 03 00058 g001
Figure 1. Biofilm formation on polystyrene surfaces by Salmonella strains. All results are presented as the mean ± SD where n = 3. The different letters represent significant differences in biofilm formation across the strains where p < 0.05.
Figure 1. Biofilm formation on polystyrene surfaces by Salmonella strains. All results are presented as the mean ± SD where n = 3. The different letters represent significant differences in biofilm formation across the strains where p < 0.05.
Applmicrobiol 03 00058 g001
Table 1. Base sequence, expected amplicon size, and annealing temperature of the primers used for the monoplex virulence gene PCR and the gene function.
Table 1. Base sequence, expected amplicon size, and annealing temperature of the primers used for the monoplex virulence gene PCR and the gene function.
GeneSequenceAnnealing Temperature
(°C)		Size of Amplicons (bp)EffectReference
lpfE5′-TTTGATGCCAGCGTGTTTACTG-3′
5′-AGTAGACCACCAGCAGAGGGAAAG-3′		50525Codes for long polar fimbriae that aid in the colonization of host intestinal wallBäumler et al. [29]
agfA5′-TGCAAAGCGATGCCCGTAAATC-3′
5′-TTAGCGTTCCACTGGTCGATGGTG-3′		61151Codes for curli adhesins that facilitate autoaggregation during biofilm formationBäumler et al. [29]
tcfA5′-CATTTATTCTCAGGGGGAGCG-3′
5′-CATCCTCTTTATCTGTTGCCACG-3′		511070Typhi specific fimbriae required for virulence in humansTownsend et al. [30]
bcfA5′-TCCCCCGGGGATACTACAACCGTCACTGG-3′
5′-GCGGATAAATCACCCTGGTC-3′		57698Codes for fimbriae involved in colonization of bovine gastrointestinal colonizationTownsend et al. [30]
pefA5′-GGGAATTCTTGCTTCCATTATTGCACTGGG-3′
5′-TCTGTCGACGGGGGATTATTTGTAAGCCACT-3′		58526Encodes for fimbriae necessary for adhesion to the murine gastrointestinal tractBäumler et al. [31]
bcsA5′-GTCCCACATATCGTTACCGTCCTG-3′
5′-CGCCGCATCATTTCTTCTCCC-3′		55119Plays a role in the production of extracellular cellulose required for biofilm formationBarak et al. [32]
yshA5′-CGGGATCCTTTTCTCTTGTATCGCCTTC-3’
5′-CCCAAGCTTGAAGAAATACTTCGCCCCGA-3′		571000Involved in the formation and secretion of extracellular polysaccharides required for biofilm formation. Especially under stress conditions.Villareal et al. [33]
Table 2. Phylogenetic groups of Salmonella carrying biofilm-associated genes and its phenotypic traits on Congo Red agar.
Table 2. Phylogenetic groups of Salmonella carrying biofilm-associated genes and its phenotypic traits on Congo Red agar.
Strain NameSourceSubspecies/SpeciesGenesColony MorphologyCurliCelluloseTotal Genes (%)
lpfEagfAbcsAyshAbcfApefAtcfA
ATCC14028 - enterica ++++++-rdar√√85.7
ATCC13076 - enterica ++++++-rdar√√85.7
M11 Spinach enterica +++++--saw××71.4
M12 Spinach enterica +++++--pdar×√71.4
M13 Spinach enterica +++++--pdar×√71.4
M15 Cabbage enterica +++++-+rdar√√85.7
M16 Spinach enterica -++++--pdar×√57.1
R11 Rural Lizard enterica -++++-+pdar×√71.4
U30 Urban Lizard enterica +++++--pdar×√71.4
U5 Urban Lizard enterica -++-++-rdar√√57.1
R32 Rural Lizard arizonae-++-++-pdar×√57.1
R36 Rural Lizard arizonae-++-+--rdar√√42.9
U3 Urban Lizard arizonae-++-+--saw××42.9
R1 Rural Lizard diarizonae-++-+--pdar×√42.9
R2 Rural Lizard diarizonae-++-++-bdar√-42.9
U68 Urban Lizard diarizonae-++-+--rdar√√42.9
B3 Cow S. bongori +++++--rdar√√71.4
M3 Cabbage indica -+--+--pdar×√28.6
M4 Lettuce indica -------pdar×√0.0
U56 Urban Lizard indica -++-+--pdar×√42.9
+ indicates that the Salmonella strain carries the biofilm-associated gene of concern, whilst – denotes that the Salmonella strain does not carry the biofilm gene of concern; √ exhibits the positive phenotype for a given trait, while × does not exhibit the phenotype for a given trait.
Table 3. Number of Salmonella strains carrying biofilm-associated genes.
Table 3. Number of Salmonella strains carrying biofilm-associated genes.
Subspecies/
Species		nBiofilm-Associated Gene
bssRyciZynfCbssSygjKgatCwcaMnarGfimFfimGfimHcsgAcsgBcsgEcspAbapAcsgDcspEadrAtrpEompRrpoSbcsCbcsEygcBsirAhilAhilCbarAcsrB
S. enterica51 34 426
S. arizonae705985560583535634 665962305059596066645353555357 57535850
S. diarizonae70 2 1 2 66
S. houtenae51 450 5
S. indica21 1
S. salamae52 30 24
S. bongori8
Subspecies/SpeciesnBiofilm-associated gene
yjcCfabRflgKrfbAnusBwaaGrfaBpnpwaaLwaaJwaaKwaaBwaaObcsAbcsBbcsZlpfEcsgCyshAluxScsrAsdiAbdcApefApefCmarTpagC
S. enterica51 45 88
S. arizonae706558556055566456 575646259595535551575457311467
S. diarizonae7069 1 69 369 3 66
S. houtenae51 5 335
S. indica21
S. salamae52 33 51 1515051515148 49
S. bongori8
Applmicrobiol 03 00058 i001 means that all the genomes of the particular subspecies/species carry the biofilm gene of concern. Applmicrobiol 03 00058 i002 means all genomes of the particular subspecies/species do not carry the biofilm gene of concern. The numbers corresponding to each gene represent numbers of genomes within a subspecies/species carrying the biofilm gene of concern.
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Sarjit, A.; Cheah, Y.; Dykes, G.A. The Basis for Variations in the Biofilm Formation by Different Salmonella Species and Subspecies: An In Vitro and In Silico Scoping Study. Appl. Microbiol. 2023, 3, 841-855. https://doi.org/10.3390/applmicrobiol3030058

AMA Style

Sarjit A, Cheah Y, Dykes GA. The Basis for Variations in the Biofilm Formation by Different Salmonella Species and Subspecies: An In Vitro and In Silico Scoping Study. Applied Microbiology. 2023; 3(3):841-855. https://doi.org/10.3390/applmicrobiol3030058

Chicago/Turabian Style

Sarjit, Amreeta, Yi Cheah, and Gary A. Dykes. 2023. "The Basis for Variations in the Biofilm Formation by Different Salmonella Species and Subspecies: An In Vitro and In Silico Scoping Study" Applied Microbiology 3, no. 3: 841-855. https://doi.org/10.3390/applmicrobiol3030058

APA Style

Sarjit, A., Cheah, Y., & Dykes, G. A. (2023). The Basis for Variations in the Biofilm Formation by Different Salmonella Species and Subspecies: An In Vitro and In Silico Scoping Study. Applied Microbiology, 3(3), 841-855. https://doi.org/10.3390/applmicrobiol3030058

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