The up-regulated genes include 3 hypothetical proteins and 18 ribosomal subunit genes. The down-regulated genes include 9 hypothetical proteins. NCBI BLAST analysis showed that the gene STY4905 was similar to gene DUF1435, a putative membrane protein in Salmonella enterica subsp. enterica serovar Senftenberg. Gene STY1229 was found to code for a protein which was closely related to ribosomal protein L32p in Salmonella enterica subsp. enterica serovar Weltevreden. The gene STY3469 had no orthologs, which suggests that it is a unique protein associated with S. Typhi biofilm genesis.
In the down-regulated group, only 2 genes were identified; STY1854 and STY2264. NCBI BLAST showed that STY2264 was an ortholog of the gene yeeX, found in Salmonella enterica subsp. enterica serovar Typhimurium strain YU15. The gene STY1854 was identified to be a histidine kinase protein unique to Salmonella enterica subsp. enterica serovar Typhi. The other 7 genes were unknown and did not have any orthologs.
For a deeper analysis of the possible functions of the genes in their role for biofilm formation in S. Typhi, the genes were grouped according to the following categories: 1. Genes responsible for the bacteria membrane matrix, 2. Genes responsible for antibiotic resistance, 3. Genes with general metabolic functions, and 4. Genes involved in biofilm regulation.
Genes responsible for bacteria membrane matrix
The gene that was most highly up-regulated in the biofilm cells was STY1254, a multiple stress resistance protein, bhsA. It plays a role in the membrane structure of S. Typhi . According to a study by Zhang et al. , the bhsA gene increases the stickiness of the membrane protein in E. coli, allowing it to stick to surfaces during biofilm formation. The researchers showed that deletion of the bhsA gene in E. coli caused more biofilm to form as the bacteria was not able to stick to the apparatus surface, causing them to form more biofilm matrix for protection. Gene STY1255 is involved in the peptidoglycan biosynthesis pathway and is part of the cell wall biogenesis . It also plays a part in anchoring the major outer membrane Braun lipoprotein to the peptidoglycan to stabilize the cell wall, giving biofilm cells a thicker cell wall and added stability .
Based on the functions of the genes STY1254 (bhsA) and STY1255, and the results from our transcriptome study, the two genes may be responsible for the binding of S. Typhi to the surface of the polypropylene tubes, thus allowing biofilm formation and maintenance. Not only were the two genes responsible for adhesion, they also affected the properties of the surface membrane, which presumably allows the bacteria to survive the acidic environment of the biofilm culture media [16,17,18,19]. It was also found in a study by Salazar et al.  that ycfR/bhsA promotes the attachment of S. Typhimurium to the surface of glass, polystyrene, spinach leaves and tomato fruit.
The gene STY1389, which codes for cyclic-di-GMP-binding biofilm dispersal mediator protein, was also found to be highly up-regulated in mature biofilm (log2-fold change = 1.79). In a review paper by Valentinin and Filloux , it was shown that cyclic-di-GMP regulated biofilm formation in P. aeruginosa by affecting various pathways such as flagella rotation to Type IV pili retraction, exopolysaccharide production, surface adhesin expression, antimicrobial resistance, and biofilm dispersion. Our result suggests that the gene STY1389 is responsible for mediating biofilm dispersion and propagation of infection at the mature stage of the biofilm cycle.
Genes responsible for antibiotic resistance
Based on the transcriptome data, the Mar regulon genes, marA and marR, were significantly up-regulated in the biofilm cells as compared to the planktonic cells. Perera and Grove  indicated that the Mar regulon was associated with antibiotic resistance and stress responses. The Mar regulon also regulates bacterial virulence factors, such as cell wall proteins and surface adhesins, as reported by Prieto and colleagues  which showed that the genes marA and marB were up-regulated when exposed to bile. Thus, it can be hypothesized that the Mar regulon prepares the S. Typhi cells for potential antibiotic resistance, or against stress factors, such as the bile used in growing the biofilm.
Yet another reason could be that some proteins serve several functions, such as the efflux pump in pathogenic organisms which serve to pump toxic chemicals out from the inside of the cells to the external environment, but can also serve as a method to extricate antibiotics from the cell cytoplasm to the external environment [22, 23]. Thus, the Mar regulon may serve two functions, ie. extrication of bile and antibiotics.
Genes associated with general metabolic mechanisms
The cold shock-like protein family, cspE, cspC and cspB, was significantly down-regulated in biofilm cells. Also, it was shown that the DNA protection and DNA binding genes, STY4154 and hupA, respectively, were down-regulated. Many membrane transport proteins, such as tatE, were also down-regulated in the biofilm cells. These results showed that while the S. Typhi cells remained in the mature biofilm, the cells entered a state of stasis and reduce the energy requirements.
Ribosomal Modulation Factor (rmf) was the most down-regulated gene in the mature biofilm cells. Based on an article by Niven and El-Sharoud , rmf was used for stabilizing ribosomes during stress conditions; that is, it would be expected for rmf to be highly up-regulated during the early stage of biofilm formation. However, it can also be theorized that the biofilm cells collected in this study at 24 h post-incubation were already matured and have entered the stasis stage. Thus, to reduce or inhibit general protein translation in the cells, there was a need to down-regulate rmf. This hypothesis is supported by a paper published by Yamagishi et al.  that showed a reduction in rmf caused cells to lose their viability and ability to form ribosome dimers as well as when the cells transitioned from the growth to stationary state. It was also hypothesized in the paper that rmf is down-regulated under certain growth conditions.
This study found that ribosomal RNA (rRNA), such as multiple ribosomal subunit genes rplD, rplW, rpsS and rplU, were significantly up-regulated in the biofilm cells. This suggests that general protein translation continues even in the sessile biofilm tissue.
Genes involved in biofilm regulation
STY0893, the bssR gene for biofilm regulation, was significantly down-regulated in the biofilm cells. According to the paper by Domka et al., , the bssR gene, also called yliH in E. coli cells, was up-regulated during biofilm formation. However, deletion of the yliH gene in E. coli caused the biofilm mass to increase by as much as 290-fold, but did not affect growth of the cells under normal conditions. However, this transcriptome study on S. Typhi biofilm cells showed that the bssR gene (STY0893) was significantly down-regulated as compared to the planktonic cells. BLAST analysis comparison between E. coli K-12 and S. Typhi CT18 showed that while the gene bssR is present in both species, it only has a 75% nucleotide similarity. Also since the base pair length is different between the two species; it can be assumed that the two proteins are different. The difference in results between this study (on S. Typhi) and the studies on E. coli  could be due to other parameters, such as different growth conditions, media and surfaces used for biofilm culture.
YjfO is a gene that encodes for biofilm stress and motility protein A. In a study done on E. coli, yjfO was found to be associated with cell survival by formation of biofilm in response to peroxide stress . It was shown that yjfO was up-regulated in E. coli during the micro-colony formation and biofilm maturation processes. However, it was also shown to be unnecessary for planktonic cell growth or initial surface adhesion. Comparison of the results of the study by Weber et al.  with our data suggests that in S. Typhi there could possibly be a different function for the gene yjfO, as it was down-regulated in S. Typhi for biofilm formation. However, this can also be explained that since yjfO is responsible for micro-colony formation, it is also possible that the transcription rate of the gene in mature biofilm tissue was suppressed as there is no further need for the gene in the static environment of the biofilm tissue.