Virulence Gene Expression Research Articles

Global transcriptomic response of the AI-3 isomers 3,5-DPO and 3,6-DPO in Salmonella Typhimurium.

Bacterial intercellular signaling mediated by small molecules, also called autoinducers (AIs), enables synchronized behavior in response to environmental conditions, and in many bacterial pathogens, intercellular signaling controls virulence gene expression. However, in the intestinal pathogen Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium), although three signals, named AI-1, AI-2 and AI-3, have been described, their roles in virulence remain elusive. AI-3 is the 3,6- isomer of a previously described Vibrio cholerae signaling molecule; 3,5-dimethylpyrazin-2-ol (3,5-DPO). To elucidate the role of AI-3/DPO in S. Typhimurium, we have mapped the global transcriptomic responses to 3,5- and 3,6-DPO isomers in S. Typhimurium. Our studies showed that DPO affects expression of almost 8% of all genes. Specifically, expression of several genes involved in gut-colonization respond to DPO. Interestingly, most of the affected genes are similarly regulated by 3,5-DPO and 3,6-DPO, respectively, indicating that the two isomers have overlapping roles in S. Typhimurium.

A Novel CovS Variant Harbored by a Colonization Strain Reduces Streptococcus pyogenes Virulence.

Streptococcus pyogenes, also known as group A Streptococcus, causes a wide variety of diseases ranging from mild noninvasive to severe invasive infections. To identify possible causes of colonization-to-invasive switches, we determined the genomic sequences of 10 isolates from five pairs each composed of an invasive strain and a carriage strain originating from five infectious clusters. Among them, one pair displayed a single-nucleotide difference in covS, encoding the sensor histidine kinase of the two-component CovRS system that controls the expression of 15% of the genome. In contrast to previously described cases where the invasive strains harbor nonfunctional CovS proteins, the carriage strain possessed the mutation covST115C, leading to the replacement of the tyrosine at position 39 by a histidine. The CovSY39H mutation affected the expression of the genes from the CovR regulon in a unique fashion. Genes usually overexpressed in covS mutant strains were underexpressed and vice versa. Furthermore, the covS mutant strain barely responded to the addition of the CovS-signaling compounds Mg2+ and LL-37. The variations in the accumulation of two virulence factors paralleled the transcription modifications. In addition, the covST115C mutant strain showed less survival than its wild-type counterpart in murine macrophages. Finally, in two murine models of infection, the covS mutant strain was less virulent than the wild-type strain. Our study suggests that the CovSY39H protein compromises CovS phosphatase activity and that this yields a noninvasive strain. IMPORTANCE Streptococcus pyogenes, also known as group A Streptococcus, causes a wide variety of diseases, leading to 517,000 deaths yearly. The two-component CovRS system, which responds to MgCl2 and the antimicrobial peptide LL-37, controls the expression of 15% of the genome. Invasive strains may harbor nonfunctional CovS sensor proteins that lead to the derepression of most virulence genes. We isolated a colonization strain that harbors a novel covS mutation. This mutant strain harbored a transcriptome profile opposite that of other covS mutant strains, barely responded to environmental signals, and was less virulent than the wild-type strain. This supports the importance of the derepression of the expression of most virulence genes, via mutations that impact the phosphorylation of the regulator CovR, for favoring S. pyogenes invasive infections.

CRISPRi in Xanthomonas demonstrates functional convergence of transcription activator-like effectors in two divergent pathogens.

Functional analysis of large gene families in plant pathogens can be cumbersome using classical insertional mutagenesis. Additionally, Cas9 toxicity has limited the application of CRISPR-Cas9 for directed mutagenesis in bacteria. Here, we successfully applied a CRISPR interference strategy to investigate the cryptic role of the transcription activator-like effector (tale) multigene family in several plant-pathogenic Xanthomonas bacterial species, owing to their contribution to pathogen virulence. Single guide RNAs (sgRNAs) designed against Xanthomonas phaseoli pv manihotis tale conserved gene sequences efficiently silenced expression of all tales, with concomitant decrease in virulence and TALE-induced host gene expression. The system is readily translatable to other Xanthomonas species infecting rice, citrus, Brassica, and cassava, silencing up to 16 tales in a given strain using a single sgRNA. Complementation with plasmid-borne designer tales lacking the sgRNA-targeted sequence restored molecular and virulence phenotypes in all pathosystems. Our results evidenced that X. campestris pv campestris CN08 tales are relevant for symptom development in cauliflower. They also show that the MeSWEET10a sugar transporter is surprisingly targeted by the nonvascular cassava pathogen X. cassavae, highlighting a new example of TALE functional convergence between phylogenetically distant Xanthomonas. Overall, this novel technology provides a platform for discovery and rapid functional understanding of highly conserved gene families.

Stress Hormone Adrenaline Boosts the Antibacterial Properties of L. plantarum and E. hallii

Amid intense exercise, the human body’s quick adaptability to stressful conditions is vital to cellular function, initiating what we recognize as an “adrenaline rush”. Despite findings on the in vitro interaction of adrenaline with the gut microbiome to regulate its virulence gene expression through AI-3 quorum sensing, studies conducted to examine the influence of the stress hormone adrenaline released through exercise on the function of the microbiome in our gut are yet insufficient. The human gut consists of a variety of microbes such as L. plantarum and E. hallii that hold vital roles in maintaining metabolic balance in the gut microbiome. Through our research, we envisioned investigating how adrenaline treatment at different time intervals or concentrations mirroring the various circumstances of exercise may affect bacterial growth, antibacterial substance production, and their antibacterial properties. While both bacteria were not affected in its growth for 48 hours, in L. plantarum, demonstrating decreased growth in the first 8 hours, its main functions — lactic acid production — were furthered with the hormone. In E. hallii, butyric acid production, with the hormone, significantly increased in its supernatant, and the intermediate treatment of adrenaline for 8 hours served to catalyze the rate at which the bacteria reached its stationary phase of growth. The study holds great promise for future investigations on how such a mechanism can be applied to therapies boosting our existing abilities of protein disposal directly correlated to the prevention of diseases. What remains is, nevertheless, the discussion regarding the true control center of these bacteria that interact with adrenaline to cause changes in the antibiotic properties, function, and growth. In other words, future studies can be oriented towards understanding the changes in the antibacterial properties of given bacteria not indirectly through chemicals with antibacterial traits but rather through the quantitative measurement of growth inhibition with other bacteria or organisms.

Deletion of pbpC Enhances Bacterial Pathogenicity on Tomato by Affecting Biofilm Formation, Exopolysaccharides Production, and Exoenzyme Activities in Clavibacter michiganensis.

Penicillin-binding proteins (PBPs) are considered essential for bacterial peptidoglycan biosynthesis and cell wall assembly. Clavibacter michiganensis is a representative Gram-positive bacterial species that causes bacterial canker in tomato. pbpC plays a significant role in maintaining cell morphological characteristics and stress responses in C. michiganensis. The current study demonstrated that the deletion of pbpC commonly enhances bacterial pathogenicity in C. michiganensis and revealed the mechanisms through which this occurs. The expression of interrelated virulence genes, including celA, xysA, xysB, and pelA, were significantly upregulated in △pbpC mutants. Compared with those in wild-type strains, exoenzyme activities, the formation of biofilm, and the production of exopolysaccharides (EPS) were significantly increased in △pbpC mutants. It is noteworthy that EPS were responsible for the enhancement in bacterial pathogenicity, with the degree of necrotic tomato stem cankers intensifying with the injection of a gradient of EPS from C. michiganensis. These findings highlight new insights into the role of pbpC affecting bacterial pathogenicity, with an emphasis on EPS, advancing the current understanding of phytopathogenic infection strategies for Gram-positive bacteria.

Genotypic and Phenotypic Characterization of Some psms Hypervirulent Clinical Isolates of Staphylococcus aureus in a Tertiary Hospital in Hefei, Anhui.

Staphylococcus aureus is a highly successful pathogen that can cause various infectious diseases, from relatively mild skin infections to life-threatening severe systemic diseases. The widespread pathogenicity of S. aureus is mainly due to its ability to produce many virulence factors that help destroy various host cells, causing disease. Our primary goal in this study was to explore the genes of highly virulent strains, to identify genes closely associated with high virulence, and to provide ideas for the treatment of infection by highly virulent clinical strains. This study collected 221 clinical strains from The First Affiliated Hospital Of The University of Science and Technology of China (USTC); their hemolytic abilities were tested. Eight isolates were selected based on their highly hemolytic ability and tested their hemolytic activity again; their phenotypes and gene sequences were also explored. Whole-genome sequencing (WGS) showed six plasmids (pN315, pNE131, pSJH901, pSJH101, SAP106B, and MSSA476), eight antibiotic resistance genes [blaR1, blaI, blaZ, mecA, erm(C), erm(T), tet(38), and fosB-Saur] and seventy-two virulence related genes. Three highly virulent strains, namely X21111206, 21092239, and 21112607, were found according the Galleria mellonella infection model. Therefore, we selected 10 representative virulence genes for qRT-PCR: psmα, psmβ, hlgA, hlgB, hlgC, hla, clfA, clfB, spa, and sak. Among them, the expression levels of psmα and psmβ, the three isolates, were significantly higher than the positive control NCTC8325. Significant differences appear in the expression of virulence genes in the highly virulent strains, particularly the psmα and psmβ, It may be that the high expression of psm gene is the cause of the high virulence of Staphylococcus aureus. We can reduce the pathogenicity of Staphylococcus aureus by inhibiting the expression of psm gene, which may provide a strong basis for psm as a new target for clinical treatment of S. aureus infection.

2nd Workshop in Microbial Molecular Biology

2nd Workshop in Microbial Molecular Biology

N-acetylcysteine (NAC) attenuates quorum sensing regulated phenotypes in Pseudomonas aeruginosa PAO1

The expression of many virulence genes in bacteria is regulated by quorum sensing (QS), and the inhibition of this mechanism has been intensely investigated. N-acetylcysteine (NAC) has good antibacterial activity and is able to interfere with biofilm-related respiratory infections, but little is known whether this compound has an effect on bacterial QS communication. This work aimed to evaluate the potential of NAC as a QS inhibitor (QSI) in Pseudomonas aeruginosa PAO1 through in silico and in vitro analyses, as well as in combination with the antibiotic tobramycin. Initially, a molecular docking analysis was performed between the QS regulatory proteins, LasR and RhlR, of P. aeruginosa with NAC, 3-oxo-C12-HSL, C4-HSL, and furanone C30. The NAC sub-inhibitory concentration was determined by growth curves. Then, we performed in vitro tests using the QS reporter strains P. aeruginosa lasB-gfp and rhlA-gfp, as well as the expression of QS-related phenotypes. Finally, the synergistic effect of NAC with the antibiotic tobramycin was calculated by fractional inhibitory concentrations index (FICi) and investigated against bacterial growth, pigment production, and biofilm formation. In the molecular docking study, NAC bound to LasR and RhlR proteins in a similar manner to the AHL cognate, suggesting that it may be able to bind to QS receptor proteins in vivo. In the biosensor assay, the GFP signal was turned down in the presence of NAC at 1000, 500, 250, and 125 μM for lasB-gfp and rhlA-gfp (p < 0.05), suggesting a QS inhibitory effect. Pyocyanin and rhamnolipids decreased (p < 0.05) up to 34 and 37%, respectively, in the presence of NAC at 125 μM. Swarming and swimming motilities were inhibited (p < 0.05) by NAC at 250 to 10000 μM. Additionally, 2500 and 10000 μM of NAC reduced biofilm formation. NAC-tobramycin combination showed synergistic effect with FICi of 0.8, and the best combination was 2500–1.07 μM, inhibiting biofilm formation up to 60%, besides reducing pyocyanin and pyoverdine production. Confocal microscopy images revealed a stronger, dense, and compact biofilm of P. aeruginosa PAO1 control, while the biofilm treated with NAC-tobramycin became thinner and more dispersed. Overall, NAC at low concentrations showed promising anti-QS properties against P. aeruginosa PAO1, adding to its already known effect as an antibacterial and antibiofilm agent.

Antimicrobial Activity of Sertraline on Listeria monocytogenes.

We explored the antimicrobial activity of sertraline on Listeria monocytogenes and further investigated the effects of sertraline on biofilm formation and the virulence gene expression of L. monocytogenes. The minimum inhibitory concentration and minimum bactericidal concentration for sertraline against L. monocytogenes were in the range of 16-32 μg/mL and 64 μg/mL, respectively. Sertraline-dependent damage of the cell membrane and a decrease in intracellular ATP and pHin in L. monocytogenes were observed. In addition, sertraline reduced the biofilm formation efficiency of the L. monocytogenes strains. Importantly, low concentrations (0.1 μg/mL and 1 μg/mL) of sertraline significantly down-regulated the expression levels of various L. monocytogens virulence genes (prfA, actA, degU, flaA, sigB, ltrC and sufS). These results collectively suggest a role of sertraline for the control of L. monocytogenes in the food industry.

Stem-Loop Structures in Iron-Regulated mRNAs of Giardia duodenalis.

Giardia duodenalis is a significant cause of waterborne and foodborne infections, day-care center outbreaks, and traveler's diarrhea worldwide. In protozoa such as Trichomonas vaginalis and Entamoeba histolytica, iron affects the growth, pathogenicity mechanisms, and expression of virulence genes. One of the proposed iron regulatory mechanisms is at the post-transcriptional level through an IRE/IRP-like (iron responsive element/iron regulatory protein) system. Recently, the expression of many putative giardial virulence factors in the free-iron levels has been reported in subsequent RNAseq experiments; however, the iron regulatory mechanism remains unknown. Thus, this work aimed to determine the effects of iron on the growth, gene expression, and presence of IRE-like structures in G. duodenalis. First, the parasite's growth kinetics at different iron concentrations were studied, and the cell viability was determined. It was observed that the parasite can adapt to an iron range from 7.7 to 500 µM; however, in conditions without iron, it is unable to survive in the culture medium. Additionally, the iron modulation of three genes was determined by RT-PCR assays. The results suggested that Actin, glucosamine-6-phosphate deaminase, and cytochrome b5 mRNA were down-regulated by iron. To investigate the presence of IRE-like structures, in silico analyses were performed for different mRNAs from the Giardia genome database. The Zuker mfold v2.4 web server and theoretical analysis were used to predict the secondary structures of the 91 mRNAs analyzed. Interestingly, the iron-induced downregulation of the genes analyzed corresponds to the location of the stem-loop structures found in their UTR regions. In conclusion, iron modulates the growth and expression of specific genes, likely due to the presence of IRE-like structures in G. duodenalis mRNAs.

GrlR, a negative regulator in enteropathogenic E. coli, also represses the expression of LEE virulence genes independently of its interaction with its cognate partner GrlA.

Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) belong to a group of pathogens that share the ability to form "attaching and effacing" (A/E) lesions on the intestinal epithelia. A pathogenicity island known as the locus of enterocyte effacement (LEE) contains the genes required for A/E lesion formation. The specific regulation of LEE genes relies on three LEE-encoded regulators: Ler activates the expression of the LEE operons by antagonizing the silencing effect mediated by the global regulator H-NS, GrlA activates ler expression and GrlR represses the expression of the LEE by interacting with GrlA. However, despite the existing knowledge of LEE regulation, the interplay between GrlR and GrlA and their independent roles in gene regulation in A/E pathogens are still not fully understood. To further explore the role that GrlR and GrlA in the regulation of the LEE, we used different EPEC regulatory mutants and cat transcriptional fusions, and performed protein secretion and expression assays, western blotting and native polyacrylamide gel electrophoresis. We showed that the transcriptional activity of LEE operons increased under LEE-repressing growth conditions in the absence of GrlR. Interestingly, GrlR overexpression exerted a strong repression effect over LEE genes in wild-type EPEC and, unexpectedly, even in the absence of H-NS, suggesting that GrlR plays an alternative repressor role. Moreover, GrlR repressed the expression of LEE promoters in a non-EPEC background. Experiments with single and double mutants showed that GrlR and H-NS negatively regulate the expression of LEE operons at two cooperative yet independent levels. In addition to the notion that GrlR acts as a repressor by inactivating GrlA through protein-protein interactions, here we showed that a DNA-binding defective GrlA mutant that still interacts with GrlR prevented GrlR-mediated repression, suggesting that GrlA has a dual role as a positive regulator by antagonizing GrlR's alternative repressor role. In line with the importance of the GrlR-GrlA complex in modulating LEE gene expression, we showed that GrlR and GrlA are expressed and interact under both inducing and repressing conditions. Further studies will be required to determine whether the GrlR alternative repressor function depends on its interaction with DNA, RNA, or another protein. These findings provide insight into an alternative regulatory pathway that GrlR employs to function as a negative regulator of LEE genes.

Group B streptococcus virulence factors associated with different clinical syndromes: Asymptomatic carriage in pregnant women and early-onset disease in the newborn.

Group B streptococcus (GBS) harbors many virulence factors but there is limited data regarding their importance in colonization in pregnancy and early-onset disease (EOD) in the newborn. We hypothesized that colonization and EOD are associated with different distribution and expression of virulence factors. We studied 36 GBS EOD and 234 GBS isolates collected during routine screening. Virulence genes (pilus-like structures-PI-1, PI-2a, PI-2b; rib and hvgA) presence and expression were identified by PCR and qRT-PCR. Whole genome sequencing (WGS) and comparative genomic analyses were used to compare coding sequences (CDSs) of colonizing and EOD isolates. Serotype III (ST17) was significantly associated with EOD and serotype VI (ST1) with colonization. hvgA and rib genes were more prevalent among EOD isolates (58.3 and 77.8%, respectively; p < 0.01). The pilus loci PI-2b and PI-2a were more prevalent among EOD isolates (61.1%, p < 0.01), while the pilus loci PI-2a and PI-1 among colonizing isolates (89.7 and 93.1% vs. 55.6 and 69.4%, p < 0.01). qRT PCR analysis revealed that hvgA was barely expressed in colonizing isolates, even though the gene was detected. Expression of the rib gene and PI-2b was two-fold higher in EOD isolates compared to colonizing isolates. Transcription of PI-2a was three-fold higher in colonizing isolates compared to EOD isolates. ST17 isolates (associated with EOD) had a smaller genome size compared ST1 and the genome was more conserved relative to the reference strain and ST17 isolates. In a multivariate logistic regression analysis virulence factors independently associated with EOD were serotype 3, and PI-1 and PI-2a was protective. There was a significant difference in the distribution of hvg A, rib, and PI genes among EOD (serotype III/ST17) and colonizing (serotype VI/ST1) isolates suggesting an association between invasive disease and these virulence factors. Further study is needed to understand the contribution of these genes to GBS virulence.

Hesperidin methyl chalcone, a citrus flavonoid, inhibits Aeromonas hydrophila infection mediated by quorum sensing

Plant-derived phytocompounds are effective in treating a variety of ailments and disorders, the most common of which are bacterial infections in humans, which are a major public health concern. Flavonoids, one of the groups of phytocompounds, are known to have significant antimicrobial and anti-infective properties. Hence, the current study investigates the efficacy of the citrus flavonoid hesperidin methylchalcone (HMC) in addressing this major issue. The results of this study indicate that the anti-quorum sensing (anti-QS) action against Aeromonas hydrophila infections is exhibited with a decrease in biofilm development and virulence factors production through in vitro and in silico analyses. In addition, the qPCR findings indicate that HMC has antivirulence action on A. hydrophila by reducing the expression of QS-related virulence genes, including ahyR, ahyB, ahh1, aerA, and lip. Interestingly, HMC significantly rescued the A. hydrophila-infected zebrafish by reducing the internal colonization, demonstrating the in vivo anti-infective potential of HMC against A. hydrophila infection. Based on these results, this study recommends that HMC could be employed as a possible therapeutic agent to treat A. hydrophila-related infections in humans.

Glucose-Induced Enhanced Virulence in Strains of Multidrug-Resistant Pseudomonas aeruginosa Isolated from Diabetic Patients.

Pseudomonas aeruginosa is known for its metabolic versatility and uses a variety of substrates; interestingly, glucose is not the favored carbon source. Although glucose is not readily utilized by them, there is a possibility that the increased susceptibility of diabetics to infections with P. aeruginosa is related to the effect of glucose on the expression of virulence genes. The curiosity in understanding the effect of glucose on virulence gene expression in P. aeruginosa and the lacuna of studies in this field prompted us to undertake the current investigation. It included the quantification of various virulence factors and their gene expression upon supplementation with glucose in clinical MDR P. aeruginosa isolates recovered from diabetics. Interestingly, the study observed a remarkable difference in the virulence attributes in the isolates with and without glucose supplementation. External glucose was found to be modulating the QS gene expression, thus altering the elaboration of other virulence factors. Variations in the gene expressions induced by glucose partly explain the increased susceptibility of diabetic patients to P. aeruginosa infections.

Effect of acidic conditions on the growth and expression of two virulence genes of Listeria monocytogenes serotype 4b

In this work, the effect of the usual acidic conditions of dry-cured fermented foods (pH values between 4.5 and 6), on the growth and expression of the virulence genes, hly and inlA, of Listeria monocytogenes serotype 4b, was evaluated. To analyse the expression of the inlA gene, a novel real-time PCR (qPCR) method using SYBR® Green methodology was developed. L. monocytogenes levels increased as the pH did and they were kept constant throughout incubation time at pH 4.5. However, a significant increase in the relative expression of the virulence genes was detected in most of the acidic conditions in all the incubation times. The most pronounced upregulation of the relative expression of the virulence genes was found at pH 4.5. The efficient inlA-based qPCR method could be of interest to check changes in the expression of such virulence gene of this pathogenic bacterium in acidic environments.

Effect of Extracts, Fractions, and Isolated Molecules of Casearia sylvestris to Control Streptococcus mutans Cariogenic Biofilm

The effects of extracts, fractions, and molecules of Casearia sylvestris to control the cariogenic biofilm of Streptococcus mutans were evaluated. First, the antimicrobial and antibiofilm (initial and pre-formed biofilms) in prolonged exposure (24 h) models were investigated. Second, formulations (with and without fluoride) were assessed for topical effects (brief exposure) on biofilms. Third, selected treatments were evaluated via bacterium growth inhibition curves associated with gene expression and scanning electron microscopy. In initial biofilms, the ethyl acetate (AcOEt) and ethanolic (EtOH) fractions from Brasília (BRA/DF; 250 µg/mL) and Presidente Venceslau/SP (Water/EtOH 60:40 and Water/EtOH 40:60; 500 µg/mL) reduced ≥6-logs vs. vehicle. Only the molecule Caseargrewiin F (CsF; 125 µg/mL) reduced the viable cell count of pre-formed biofilms (5 logs vs. vehicle). For topical effects, no formulation affected biofilm components. For the growth inhibition assay, CsF yielded a constant recovery of surviving cells (≅3.5 logs) until 24 h (i.e., bacteriostatic), and AcOEt_BRA/DF caused progressive cell death, without cells at 24 h (i.e., bactericidal). CsF and AcOEt_BRA/DF damaged S. mutans cells and influenced the expression of virulence genes. Thus, an effect against biofilms occurred after prolonged exposure due to the bacteriostatic and/or bactericidal capacity of a fraction and a molecule from C. sylvestris.

Draft genome of clinical isolate Salmonella enterica Typhimurium ms204 from Odisha, India, reveals multi drug resistance and decreased virulent gene expression

Salmonellosis, a food-borne illnesses caused by enteropathogenic bacterium Salmonella spp., is a continuous concern in both developed and developing countries. This study was carried out to perform an in-depth examination of an MDR Salmonella strain isolated from gastroenteritis patients in Odisha, India, in order to understand the genomic architecture, distribution of pathogenic island regions, and virulence factor diversity. Fecal samples were obtained from individuals with acute gastroenteritis and further subjected to panel of biochemical tests. The IlluminaHiSeq X sequencer system was used to generate whole-genome sequencing. The draft genome was submitted to gene prediction and annotation using RAST annotation system. Pathogenicity Island database and bioinformatics pipeline were used to find Salmonella pathogenicity islands (SPI) from the built scaffold. The gene expression in SPI1 and SPI2 encoded regions was investigated using qRT-PCR. The taxonomic position of Salmonella enterica subsp. enterica serovar Typhimurium was validated by serotype analysis and 16S rRNA based phylogenetic analysis. The de-novo genome assembly showed total length of 5,034,110 bp and produced 37 contigs. There are nine prophage areas, comprising of 12 regions and scaffold 8 contained a single plasmid, IncFIB. The isolate contains six known SPI genes content which was shown to be largely conserved from SPI1 to SPI2. We identified the sit ABCD cluster regulatory cascade and acquired antibiotic resistance genes in S. enterica Typhimurium ms204. Further research may aid in the correct diagnosis and monitoring of MDR Salmonella strains with a variety of physiological activities.

Effects of BlueM® against Streptococcus mutans biofilm and its virulence gene expression

This study evaluated the antimicrobial capacity of BlueM® mouthwash against the bacterium Streptococcus mutans and its influence on gbpA gene expression as well as its cytotoxic effect on fibroblast cells. BlueM® showed antimicrobial activity, with MIC and MBC values of 0.005% and 0.01%, respectively. The MBIC was 6.25% for S. mutans. CFU count and confocal microscopy revealed significant effect of BlueM® on S. mutans biofilm pre-formed on dentin surfaces. Interestingly, the analysis of gbpA gene expression indicated a decrease in gene expression after 15 min of treatment with BlueM® at a concentration of 25%. Moreover, BlueM® exhibited low levels of cytotoxicity. In conclusion, our results showed the antimicrobial effectiveness of BlueM® against S. mutans, its ability to modulate the expression of the gbpA gene and its low cytotoxicity. This study supports the therapeutic potential of BlueM® as an alternative agent for the control of oral biofilm.

Epidemiological Features and Impact of High Glucose Level on Virulence Gene Expression and Serum Resistance of Klebsiella pneumoniae Causing Liver Abscess in Diabetic Patients.

Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative bacterium that is predominantly associated with liver abscesses in global diabetic patients. High levels of glucose in the surrounding of K. pneumonia increase its pathogenicity including capsular polysaccharide (CPS) and fimbriae. Other important virulent factors include outer membrane protein A (ompA) and regulator mucoid phenotype A (rmpA). The objective of this investigation was to elucidate the effects of high glucose on rmpA and ompA gene expression and serum resistance of K. pneumoniae causing liver abscess. The clinical history of 57 patients suffering from K. pneumoniae-causedliver abscesses (KLA) was acquired and their clinical and laboratory manifestations in the presence or absence of diabetes were analyzed. The antimicrobial susceptibility, serotypes, and virulence genes were tested. Clinical isolates of 3 serotype-K1 hypervirulent K. pneumoniae (hvKP) were used to detect the effect of exogenous high glucose on rmpA, ompA, and clbB genes expression, and bacterial serum resistance. KLA patients with diabetes showed higher C-reactive protein (CRP) compared to non-diabetic KLA patients. Furthermore, the diabetic group showed increased incidences of sepsis and invasive infections, and their length of hospital stay was also prolonged. Pre-incubation of K. pneumoniae in high glucose (0.5%) concentration up-regulated rmpA, ompA, and clbB genes expression. However, cAMP supplementation, which was inhibited by environmental glucose, reversed the increase of rmpA and ompA in a cAMP-dependent manner. Moreover, hvKP strains incubated in high glucose also exhibited enhanced protection from serum killing. High glucose levels reflected by poor glycemic control has increased gene expression of rmpA and ompA in hvKP by the cAMP signaling pathway and enhanced its resistance to serum killing, thus providing a new and reasonable explanation for the high incidences of sepsis and invasive infections in KLA patients with diabetes.

Impact of short chain fatty acids (SCFAs) on antimicrobial activity of new β-lactam/β-lactamase inhibitor combinations and on virulence of Escherichia coli isolates

In a healthy gut microbiota, short chain fatty acids (SCFAs) are produced. The antibacterial action of SCFAs against intestinal pathogens makes them useful for ensuring the safety of food and human health. In this study, we aimed to assess the in vitro inhibitory activity of SCFAs, and to report, for the first time, their impact on the activity of new β-lactam/β-lactamase inhibitor combinations. The minimum inhibitory concentrations of acetic, propionic, and butyric acids were determined against E. coli clinical isolates recovered from gastrointestinal infections. Cefoperazone/sulbactam, ceftazidime/avibactam and cefepime/enmetazobactam are new β-lactam/β-lactamase inhibitor combinations that were studied for their combined therapeutic effects. Also, the effects of pH and concentration of SCFAs were evaluated on in vitro bacterial growth and expression of genes encoding for motility, adhesion, invasion, and biofilm formation. SCFAs were tested at concentrations of 12 mM at pH 7.4 (ileum-conditions), in addition to 60 mM and 123 mM, at pH 6.5 (colon-conditions). The tested SCFAs showed the same MIC (3750 μg ml−1 ≃ 60 mM) against all isolates. Furthermore, the addition of SCFAs to the tested β-lactam/β-lactamase inhibitor combinations greatly restored the susceptibility of the isolates. SCFAs had significant effect on bacterial growth and virulence in a pH and concentration-dependent manner; low ileal concentration potentiated E. coli growth, while higher colonic concentration significantly suppressed growth and down-regulated the expression of virulence genes (fliC, ipaH, FimH, BssS). Therefore, the significant inhibitory effect of colonic SCFAs on β-lactam/β-lactamase inhibitor combinations might lead to the development of promising treatment strategies.