24 and 48 h after inoculation, bacterial cells were collected and thoroughly resuspended by vortexing in phosphate-buffered saline (PBS). Thereafter, Lactobacillus and coliform concentrations in the co-cultures and in the controls was determined on MRS agar plates additioned with vancomycin (0.2% w/v) and MacConkey agar plates,

which are selective for Lactobacillus spp. and coliforms, respectively. Antimicrobial activity was calculated by comparing the coliform growth in the co-culture and control [8]. Results were expressed as log10 CFU/ml. The experiment was performed in triplicate. Statistical Analyses Sample size was calculated based on a difference between groups of 1.5 MK-4827 log10 CFU/g faeces. Using α = 0.05, β = 0.20 and an estimated standard deviation within groups of 2 log10 CFU/g faeces, 30 patients were needed in each group. Counts (log10 CFU/g) of the total amount of coliform bacteria were calculated for each stool sample. Data are summarized by counts

and median and range for categorical and continuous variables respectively. Differences between groups were evaluated with Mann-Whitney’s U-test for continuous variables, whereas associations between categorical variables were evaluated with Fisher’s exact test. Differences between colicky infants and controls in total amount of each species detected were evaluated with Mann-Whitney’s this website test with Bonferroni correction. Statistical significance was set at a p-value < 0.05. All statistical calculations were performed with commercially available software

(SPSS for Windows release 15Æ0 SPSS Inc., Chicago, IL, USA). Results Isolation and identification of coliforms from colicky infants Thalidomide Coliform colonies were obtained on MacConkey agar plates from faeces of all the 45 colicky infants and 42 controls. The average count of total coliforms in the 45 faecal samples of colicky infants was 5.98 (2.00-8.76) log10 CFU/g of faeces, whereas total coliforms in the control group were 3.90 (2.50-7.10) log10 CFU/g of faeces. The difference between the two groups was statistically significant (p = 0.015). A total of 145 colonies was randomly picked up from the higher dilutions agar plates (10-6-10-8) and, only from colicky infants after sub-culturing in LB agar, each purified strain was examined for gas production and characterized at species level by DNA sequencing and carbohydrate fermentation profiling. All isolated strains were found to produce gas from lactose according to the method described above and the BBL™ Enterotube™ II system. They were ascribed to six different species (Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Enterococcus faecalis, Enterobacter aerogenes, and Enterobacter cloacae), as described in Table 3. The percentage of detection of each species in the faecal samples examined was reported in descending order (Table 3). The same taxonomic identification was obtained with the two methods employed.

Increases in permeability were not the result of epithelial cell death, since cells were still present in monolayers after 16 h of infection (Figure selleck chemicals llc 2B). Figure 2 Epithelial tight junctions are disrupted by AIEC infection. MDCK-I monolayers were grown to confluence on 6.5 mm diameter Transwells and then either left uninfected (sham control; Panel A) or infected with AIEC, strain LF82 (Panel B) at a MOI of 100:1 for 16 h. Monolayers were then

washed with PBS and fixed, blocked and incubated with primary rabbit anti-ZO-1 and the appropriate secondary antibody and DAPI. Panel A: Sham control cells showed a normal distribution of ZO-1, outlining the intercellular tight junctions. Panel B: AIEC infection resulted in disruption of ZO-1 localization with large gaps between cells (arrows). Approximate original magnifications: × 630. AIEC infection alters the distribution of ZO-1 Sham control MDCK-I cells (Figure 2A) demonstrated a normal distribution of ZO-1, delineating intact apical cellular junction complexes [27]. Consistent with effects on permeability, see more 16 h infection of MDCK-I monolayers with AIEC, strain LF82 (Figure 2B) led to profound disruption of ZO-1 with large gaps between cells with punctate and interrupted distribution of ZO-1, indicating disruption of this integral tight junction

protein [28]. Nevertheless, cells in the monolayer remained viable, as demonstrated by the presence of nuclei and maintenance of normal cells shape and morphology. Disruption of MDCK-I monolayers is accompanied by AIEC invasion and bacterial replication Transmission electron microscopy of infected MDCK-I monolayers was used to define the effect of AIEC infection of polarized monolayers. In contrast to sham control epithelial monolayers, which demonstrated tightly placed cells without expanded intercellular spaces (Figure 3A), AIEC-infected MDCK-I monolayers were disordered after 4 h of incubation, with spaces evident between adjacent cells and disruption of intercellular spaces. Loss of cellular click here polarity was also observed,

as demonstrated by presence of microvilli on the lateral aspect of infected cells. Furthermore, consistent with previous reports [29], multiple bacteria were seen within cells 4 h after infection with effective replication, indicating that these organisms survive within the cytoplasm of epithelial cells (Figure 3B). Extension of bacterial infection to 48 h resulted in profound disruption of the monolayer, with complete separation between cells and terminal changes in cells, including loss of membrane integrity, chromatin condensation and ballooning of mitochondria (Figure 3C). This effect may be the result of bacterial overgrowth after 48 h of infection. Figure 3 AIEC disrupts MDCK-I monolayers and replicates in the cell cytoplasm.

As such, the design of this study should allow for the results to be more generalizable to the habitual consumption of bottled water than would results from a laboratory controlled study. Influence on Acid-Base Balance When compared with the consumption of the placebo bottled water,

habitual consumption of AK water in the present study was associated with GSK690693 clinical trial an increase in both urine (Table 7) and blood (Figure 3) pH while measures of both daily PA (Table 4) and dietary composition remained stabile. Previous research by Welch et al. [11] demonstrated that urinary pH from 24-hour collection samples could function as an effective surrogate marker for changes in acid-base balance when evaluating differences in dietary intake. König et al [10] used this information as a premise for determining that consumption of a mineral-rich supplement significantly increased both urine (5.94 to 6.57) and blood pH (7.40 to PF-6463922 mw 7.41). Similarly, Berardi et al. [9] showed that urinary pH increased from 6.07 to 6.21 and 6.27

following one and two weeks of ingestion, respectively, of a plant-based supplement. The observations from these studies [9, 10] are consistent with the changes in urine (6.23 to 7.07) and blood pH (7.52 to 7.69) observed by the present study for the Experimental group. Thus, the habitual consumption of AK water under free-living conditions had a similar influence on urinary and blood pH as has been shown to occur with nutrition supplements specifically designed to impact the body’s acid-base balance. The above observations, however, are not without limitations as the onset and magnitude of the urine alkalization within the Experimental group was influenced by daily PA, SRWC, and computed dietary PRAL (Table 9). Specifically, urine pH tended to increase sooner within the treatment period and to a higher pH level for those who habitually engaged in more physical activity, self-reported drinking more AK water, IMP dehydrogenase as well as those who regularly reported higher nutritionally-induced acid loads (Table 9). Thus, the actual impact of consuming the AK water’s mineral-based alkalizing

agents on urine pH may be dose dependent. This observation would certainly explain the differences in urinary pH between “”low”" and “”high”" levels of AK water consumption and daily PA, but a study that precisely controls AK water intake is needed to support the speculation of a dose-response relationship. It is interesting to note that the blood pH values reported for this study are somewhat higher than the 7.35-7.45 range typically ascribed as the ideal range for blood pH. It is likely that the measurement procedures used (i.e., fingertip samples collected in heparinized capillary tubes and refrigerator stored for 6-10 hrs) allowed the samples to slightly increase pH prior to the actual measurement of pH.

These results do not entirely fit the expectation of the consensus [12], which predicts an optimal adsorption rate that maximizes the Crenigacestat order plaque size (Figure 1B). One possible explanation for the discrepancy is because our phage collection has a narrower range of adsorption rates than those used in the models. Consequently, the observed diminishing negative relationship could simply be a reflection of the fact that all our phages have medium to high adsorption rates when compared to the model simulations. Though whether this is the case remains to be seen, it should be pointed out that it makes an intuitive

sense that a lower adsorption rate, at some point, should result in a smaller plaque size. After all, for a phage with a very low adsorption rate, it would spend proportionally more time in the extracellular phase diffusing before it initiates an actual infection. By the time the phage clears enough host cells to reveal a visible plaque, the host physiology may have already switched to the unproductive phase. That is, for selleck products a phage with a very low adsorption rate, the plaque would be small, and possibly blurry, due

to host over-growth (Abedon, per. comm.; [19] for smaller plaques due to lowered adsorption rate via withholding cofactor; [34] and [35] for low adsorption rate and turbid plaques in ht mutants). Because the ratio tests of each model showed that none of these models could consistently reproduce the observed ratios of plaque radius and plaque productivity (Figure 4), it suggests that other factors may Glutamate dehydrogenase also be important in the formation of a plaque. For example, for a high-adsorption phage, the time spent in the extracellular phase would be shorter when compared to a low-adsorption one. That is, there would be less time for a high-adsorption

phage to diffuse too far away from where it was released before it encounters another host cell. Consequently, on average, a higher proportion of the released progeny would be adsorbed onto the cells that are in their immediate vicinities. There are several consequences from such a scenario: (i) One likely consequence of the high adsorption rate in a spatially restricted environment is that many of the host cells nearby would be multiply infected. Multiple infection would potentially shorten the lysis time (the latent period) by producing more holin proteins inside the cell [36]. On the other hand, it may also increase the burst size per infected cell because more genomes would contribute to the synthesis of virion components. For example, infection of phage λ to E coli strains expressing λ’s morphogenetic genes B, D, or W would increase 20 to 40% of the normal burst size (Shao & Wang, unpublished data). But the progeny produced per infected phage would likely be lower than when the host is singly infected (for phage ϕ6, P. Turner, per. comm.).

Purification of FabF1 and FabZ Plasmid pHW76 and pHW74m were introduced into strain BL21 (DE3), respectively, and the proteins were overexpressed and purified as described previously[20]. The enzymes were homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The E. coli FabD, FabH, FabG, FabA, FabZ, FabB,

FabI and Vibrio harveyi AasS proteins were purified by their hexahisitidine tags described previously [18, 20]. Assay of FabF1 and FabZ activity in vitro Fatty acid synthesis selleck was reconstituted in vitro to assay FabF1 and FabZ activity using the purified enzymes that catalyze the fatty acid biosynthesis essentially. The assay utilized the AasS acyl-ACP synthetase from Vibrio harveyi [18] to generate 3-hydroxydecanoyl-ACP. The reaction mixtures to synthesize 3-hydroxydecanoyl-ACP contained 20 μM ACP, 10 mM this website ATP, 10 mM MgSO4, 5 mM DTT, 0.1 M sodium phosphate buffer (pH 7.0), 100 μM 3-hydroxydecanoic acid

(Sigma) and AasS (0.2 μg) in a final volume of 50 μl. The mixtures were incubated at 37°C for 1 h. To assay C. acetobutylicium FabF1, the following incubation 1 μg each of E. coli FabD, FabG and FabA, 100 μM NADPH, 100 μM NADH, 100 μM malonyl-CoA, and 1 μg of either E. coli FabB or C. acetobutylicium FabF1 was added. To assay C. acetobutylicium FabZ, the following incubation contained 1 μg each of E. coli FabD, FabG and FabB, 100 μM NADPH, 100 μM NADH, 100 μM malonyl-CoA, and 1 μg of E. coli FabA or C. acetobutylicium FabZ was added. The resulting mixture was incubated for an additional 1 h and the reaction products were analyzed by conformationally sensitive gel electrophoresis on 20% polyacrylamide gels containing 2.5 M urea [20, 24]. The gel was stained with Coomassie Brilliant Blue R250. Acknowledgements This work was supported by the President Foundation of South China Agricultural University and NIH Pembrolizumab manufacturer grant AI15650. We are grateful to Professor Hiroshi Kobayashi (Graduate School of Pharmaceutical Sciences, Chiba University Japan) for critical reading. Electronic supplementary material Additional file 1: Bacterial strains, plasmids and oligonucleotides used in this work. The data provided bacteria strains,

plasmids and oligonucleotides used in this work. (PDF 104 KB) References 1. Cronan JE: Bacterial membrane lipids: where do we stand? Annu Rev Microbiol 2003, 57:203–224.CrossRefPubMed 2. Mansilla MC, de Mendoza D: The Bacillus subtilis desaturase: a model to understand phospholipid modification and temperature sensing. Archives of microbiology 2005,183(4):229–235.CrossRefPubMed 3. Bloch K, Baronowsky P, Goldfine H, Lennarz WJ, Light R, Norris AT, Scheuerbrandt G: Biosynthesis and metabolism of unsaturated fatty acids. Fed Proc 1961, 20:921–927.PubMed 4. Scheuerbrandt G, Goldfine H, Baronowsky PE, Bloch K: A novel mechanism for the biosynthesis of unsaturated fatty acids. J Biol Chem 1961, 236:PC70-PC71.PubMed 5. Bloch K: Beta-Hydroxythioester dehydrase.

PLoS Biol 2011, 9:e1000622.PubMedCrossRef 29. Dutech C, Enjalbert J, Fournier E, Delmotte F, Barrès B, Carlier J, Tharreau D, Giraud T: Challenges of microsatellite isolation in fungi. Fungal Genet Biol 2007, 44:933–949.PubMedCrossRef

Competing interest No conflicts of interest. The authors have no financial relationship with the organizations that sponsored the research. Authors’ contributions RA carried out the experimental studies. RA, AA, and LG conceived the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.”
“Background Bacteriophage therapy is one of the emerging methods used to overcome bacterial infections [1–3]. Bacteriophages are viruses that infect and kill bacteria. Theoretically, phages have several advantages over antibiotics. They are highly specific and very effectively lyse AG-881 mouse targeted pathogenic bacteria. They are safe because they have no activity against animal or plant cells. Phages are ubiquitous, so isolation

of new phages is a relatively rapid process that can frequently be accomplished in days or weeks. The use Histone Methyltransferase inhibitor of phages as therapeutic agents was initiated in 1919, 3 years after their discovery, for the treatment of bacillary dysentery and continued until the 1940s. Over this time period, phages were used to treat a variety of infectious diseases [4]. However, with the advent of antibiotics, commercial production of therapeutic Amisulpride phages ceased in most of the Western world [5]. Currently, there is renewed interest in phage research and the applications of bacteriophages as potentially powerful antibacterial agents due to the emergence of drug-resistant pathogens and the dearth of new antibiotics. Several studies have shown that bacteriophages can

be used successfully for therapeutic purposes, both in humans and animals [6–9]. However, more research is required before clinical use can be re-initiated. Before using a phage for therapeutic purposes, the isolation of lytic phages and characterization of the phage are essential. In this study, clinical isolates of Acinetobacter baumannii were collected and used as indicator hosts to screen phages from water samples. A. baumannii mostly infects debilitated patients in intensive care units and is associated with high mortality rates [10, 11]. Since its discovery, A. baumannii has become resistant to many common antibiotics [12]. The increasing prevalence of multidrug- and pandrug-resistant A. baumannii strains in clinics has rendered it one of the most important nosocomial pathogens [12–15]. Fortunately, lytic phages specific to A. baumannii might provide an alternative to antibiotics for the prevention and treatment of infections caused by this bacterium. However, to the best of our knowledge, very few detailed characterizations of A. baumannii phages have been published [16, 17].

After a mean follow-up of 41.4 months, there have been 2 cases of ASBO recurrence in the icodextrin group and 10 cases in the control group (p < 0.05). Only one patient in the first group was submitted to surgery showing an Adhesion Severity Score = 2,

whereas three patients in the latter ATM Kinase Inhibitor group were operated, and the ASS was respectively 3,2 and 3. In accordance with this data, the use of icodextrin 4% solution seems to be safe and effective to prevent intra-abdominal adhesion formation and the risk of re-obstruction [100]. Intergel solution (Lifecore Biomedical, Inc, Chaska, MN), which contains .5% ferric hyaluronate, is another product used for adhesion prevention. In preliminary studies it has been shown to reduce the number, severity, and extent of adhesions

in peritoneal surgery [101]. However, the use of Intergel in abdominal surgery in which the gastrointestinal tract was opened still led to an unacceptably high rate of postoperative complications [102]. An interesting experimental finding is the reduction of both number and type of adhesions after postoperative stimulation of gastrointestinal motility by a prokinetic agent [103]. Finally merits mention that peritoneal infusion A-1210477 in vivo with cold saline has shown to decrease the degree of postoperative intra-abdominal adhesion formation in an animal model [104]. Adhesions quantification Among the different adhesions scoring

systems which have been proposed mainly by gynecologists, the more complete and easy to use one is the PAI score proposed by Coccolini et al. [105]. In fact, specific attention should be paid to uniformity of measurement. We therefore Selleck Verteporfin suggest a regimented classification system for adhesions in an effort to standardize their definition and subsequent analysis. In this way, different surgeons in different treatment centers can more effectively evaluate patients and compare their conditions to past evaluations using a universal classification system (Figure 3). This classification is based on the macroscopic appearance of adhesions and their extent to the different regions of the abdomen. Using specific scoring criteria, clinicians can assign a peritoneal adhesion index (PAI) ranging from 0 to 30, thereby giving a precise description of the intra-abdominal condition [105]. Figure 3 Peritoneal adhesion index: by ascribing to each abdomen area an adhesion related score as indicated, the sum of the scores will result in the PAI. Conclusions ASBO is a common disease. Non operative management should be attempted in absence of signs of peritonitis or strangulation.

Another possibility is that there are other, as yet

unannotated proteins that play a role in a putative flagellar system in C. pneumoniae. For example, along with the FliH/FliI complex that is formed in other bacteria, another protein, FliJ, which is a general chaperone, is believed to be involved in this complex [39, 44]. FliJ has not been identified in C. pneumoniae. In the absence of a genetic manipulation system for the chlamydiae, direct evidence for the role of these flagellar proteins remains elusive. The fact that FliI is enzymatically active and forms complexes in vitro with other flagellar proteins, all of which are present in all other chlamydiae sps. studied to date, suggests that these proteins play an important role in chlamydial replication or survival. Further see more studies using heterologous systems and genetic complementation could help to decipher the exact role of these flagellar proteins in chlamydiae. Methods Expression Plasmids C. pneumoniae CWL029 (VR1310:ATCC) (GenBank accession # AE001363) was the strain used to isolate genomic DNA for cloning and protein expression. Full length fliI, Cpn0859, cdsL, copN, Cpn0322, and fragments of flhA, fliF, and fliI were amplified from CWL029 using AttB-containing primers (Gateway; Invitrogen).

The amplified products were cloned into pDONR201 (Gateway; Invitrogen) to generate pENT vectors. The pENT vectors were then Ro 61-8048 research buy used in LR reactions (Gateway; Invitrogen) to produce pEX vectors containing the genes of interest. We used either pEX17 (N terminal His tag) or pEX15 (N terminal GST tag) vectors for our protein expression. All constructs were confirmed by sequencing at the Molecular Biology Facility at McMaster University. To identify protein interactions we utilized the bacterial-2-hybrid Exoribonuclease system [39]. Genes of interest were

cloned into either pT18 or pT25 plasmids, each of which expresses a different fragment of adenylate cyclase. When these two plasmids are co-transformed, expressing the protein of interest fused to the adenylate cyclase fragment, any interaction between the two proteins results in production of cAMP. Increases in cAMP results in an increase in the β-galactosidase gene that can be monitored using β-galactosidase activity assays. pT18 and pT25 were digested with KpnI (New England Biolabs) as well as genes amplified from CWL029 (fliI, flhA, fliF, cdsL, Cpn0322, copN) that had a KpnI site designed into the primers. Ligation was performed overnight at 16°C using T4 Ligase (Invitrogen) and the resulting mixture was used to transform E. coli XL-1 cells and transformants were selected on 100 μg/μL ampicillin and 34 μg/μL chloramphenicol (Luria Bertani) LB plates. Plasmids were prepared using the GenElute Plasmid Miniprep Kit (Sigma). Protein Expression All constructs were expressed in E. coli Rosetta pLysS. Expression plasmids were used to transform E.

Results No

significant changes were measured for body mass (BM) or lean body mass (LBM) in either group. A group x time effect for total body fat percent (P=0.01; www.selleckchem.com/products/mek162.html mean ± SE; PL baseline, 42.3 ± 0.2% to post, 42.6 ± 0.2%,+ 0.71 %; MIDS baseline, 44.5 ± 0.2% to post, 43.8 ± 0.2%,-2.24%) and android fat percent (P= 0.03; PL baseline, 49.1 ± 0.2% to post, 49.3 ± 0.2%,+ 0.4%; MIDS baseline, 51.8 ± 0.3 % to post, 50.9 ± 0.3%, – 0.9%) was observed. There was a main time effect where satiety increased (P= 0.004) and desire to eat decreased (P=0.007). No other changes were reported. The side effects reported with MIDS were headache (n=1), anxiety (n=1), and jitteriness (n=1) and for PL were headache (n=1), bloated feelings (n=1), and improved bowel movements (n=1). Conclusion Eight weeks of MIDS supplementation significantly decreased total and android fat percent. A main time effect was observed for

satiety and desire to eat. Health indices of blood pressure, heart rate and blood lipids did not differ between groups. Acknowledgements This study was supported by an independent research grant from the International Society of Sports Nutrition to MJO.”
“Background An intact composition of extracellular matrix (ECM) collagens, proteoglycans and elastic fibres are responsible for the constitutional strength of tendons and ligaments [1, 2]. It is known that pathophysiological changes in the ECM could lead to reduced extension properties and diminished capacity of energy absorption of ligaments and tendons and could Selleck SB202190 promote diseases like patellar tip syndrome, tendinopathy and rupture [3, 4]. In a clinical study it could be demonstrated that the oral ingestion of specific collagen peptides improved extension properties of the finger joints L-gulonolactone oxidase [5]. Aim of the present study was to investigate the impact of a specific

collagen peptide composition (FORTIGEL®) on the extracellular matrix of ligaments and Achilles tendons. Previous experimental studies confirmed the stimulatory impact of these bioactive collagen peptides on the ECM biosynthesis of joint cartilage tissue [6–8]. Methods Primary fibroblasts derived from human ligaments and tendons were isolated by enzymatic digestion and seeded in monolayer cultures in a humidified incubator in 5 % CO2 atmosphere at 37° C. After 80 % cell confluence regular culture medium was supplemented with 0.5 mg/ml of a specific collagen hydrolysate (FORTIGEL®, GELITA AG, Germany). The RNA expression of matrix molecules and degenerative metalloproteinases was determined via real-time PCR after a stimulation time period of 24 h. Moreover, the collagen, proteoglycan and elastin biosynthesis of tendon and ligament derived fibroblasts was determined using validated methods like western blotting, alcian blue staining or 14[C]-incorporation assay. Results The biosynthesis of ligament and tendon matrix molecules was clearly stimulated by FORTIGEL®.

Inactivation of the antibiotic resistance gene (bla CTX-M-14) on pCT also had no effect on the plasmid or bacterial host biology in the absence of selective antibiotic pressure [18]. Therefore, we proposed that alternative plasmid encoded factors were responsible for the successful persistence and global distribution of pCT. In order to test this hypothesis, we used an inactivation technique adapted from a novel gene inactivation method previously used on multi-copy plasmids [18, 19] to systematically inactivate candidate genes and operons previously associated with ‘plasmid success’. Using a functional genomic

approach analogous to that which has been broadly AP26113 datasheet employed in studying chromosomal genes of various eukaryotic and prokaryotic organisms, we examined selleck the impact of plasmid genes on pCT persistence and conjugation and upon the bacterial host. Results and discussion Inactivation of six selected genes Based upon our previous work [15, 18], six loci on pCT were identified as candidates predicted to encode fundamental factors contributing to the success of this plasmid. Comparative genomics with other characterised Incompatibility group I plasmids (including IncI, IncB, IncK and IncZ) identified: a region of pCT encoding a toxin-antitoxin

addiction system, pndACB (pCT_065) which we hypothesised to be involved in stable inheritance of the plasmid into daughter cells [20]; operons involved in plasmid conjugation, the tra and pil loci (pCT_068 and pCT_103) [21] including a gene likely to determine mating pair recipient specificity, shufflon recombinase gene rci (pCT_093) [22]; an unusual putative sigma 70 factor (pCT_066) and a putative parB gene involved in

plasmid segregation (pCT_057) [15]. Therefore, the effects of inactivating the pndACB operon, rci, pCT_066 and key structural pilus protein genes traY (tra locus), pilS (pil locus) and the putative parB Rebamipide gene were investigated to establish the role of each element in plasmid ‘success’ (Figure 1). Figure 1 Plasmid map of pCT showing the relative positions of each target genes. Each gene was inactivated by homologous recombination using hybrid amplimers encoding an aph cassette encoding kanamycin resistance, flanked by regions homologous to the target. Mutants were created within an intermediate Lambda Red recombinase encoding E. coli SW102 host [23] and confirmed by sequencing across the mutated region to ensure the aph cassette has been inserted to inactivate the target gene. All six recombinant plasmids were then transformed into E. coli DH5α, and transferred to S. Typhimurium SL1344 to prevent further recombination events and for further analysis.