Main Virulence Genes Research Articles

Inactivation of Aeromonas hydrophila in water by dielectric barrier discharge plasma: Efficiency, mechanism, and application safety

Aeromonas hydrophila (A. hydrophila) is a typical zoonotic pathogen that commonly found in water environments, capable of infecting various aquatic animals and causing different diseases in both animals and humans. In this study, the dielectric barrier discharge plasma (DBDP) is used to inactivate the A. hydrophila in water. The efficiency and mechanism of A. hydrophila inactivation by DBDP were investigated, and the application safety of DBDP also were examined. The results demonstrate that DBDP treatment for 8 min can reduce the concentration of A. hydrophila from 108 CFU/mL to 0 CFU/mL under optimal conditions. Higher discharge voltage and lower concentration is beneficial for the inactivation of A. hydrophila, and O2 as carrier gas and alkaline environment can improve the inactivation efficiency. Reactive oxygen species (ROS) produced in the DBDP system play a significant role in A. hydrophila inactivation. However, molecules like O3 and H2O2 long-lived are likely the main forces responsible for the initial destruction of cell membranes. Subsequently, ROS penetrates the cell membrane, and result in the destruction of main virulence genes of A. hydrophila and the loss of bacterial infectivity. The silver carp challenge experiment indicates that mortality rates and septicemia symptoms in silver carp are not significantly different from the control group when treated with DBDP. Moreover, the total bacterial count in the main organs can be reduced to a safe level. These findings suggest that DBDP is a safe and reliable method for inactivating A. hydrophila in water bodies.

Clinical and molecular characteristics of Klebsiella pneumoniae infection in a tertiary general hospital of Wuhan, China

ObjectivesThe aim of this study was to investigate the clinical and molecular characteristics of Klebsiella pneumoniae infection from a tertiary general hospital in Wuhan, China.MethodsFrom December 2019 to August 2022, 311 non-duplicate isolates of K. pneumoniae were collected from a tertiary hospital in Wuhan. These comprised 140 carbapenem-resistant K. pneumoniae (CRKP) isolates and 171 carbapenem-susceptible K. pneumoniae (CSKP) isolates. The clinical characteristics of patients with K. pneumoniae infection were retrospectively collected. Polymerase chain reaction (PCR) assays were used to identify the main carbapenem resistance genes, virulence genes and multi-locus sequence typing (MLST) profiles of the isolates, and the Galleria mellonella infection model was used to determine their virulence phenotypes.ResultsIndependent risk factors for CRKP infection were hypertension, neurological disorders, being admitted to the intensive care unit (ICU) and prior use of antibiotics. Patient with CRKP infection had higher mortality than those with CSKP infection (23.6% vs 14.0%, P < 0.05). One hundred and two sequence types (STs) were identified among the K. pneumoniae isolates, and the most prevalent ST type was ST11 (112/311, 36.0%). All of the ST11 isolates were CRKP. Among the 112 ST11 isolates, 105 (93.8%) harboured the carbapenem resistance gene blaKPC-2 (ST11-KPC-2), and of these isolates, 78 (74.3%, 78/105) contained all of the four virulence genes, namely rmpA, rmpA2, iroN and iucA, suggesting that these genes were widespread among the isolates responsible for K. pneumoniae infections.ConclusionIn this study, ST11-KPC-2 was responsible for most of the K. pneumoniae infection cases. Carbapenem resistance rather than the co-occurrence of the virulence genes rmpA, rmpA2, iroN and iucA was associated with K. pneumoniae infection-related mortality during hospitalisation. Furthermore, a high proportion of ST11-KPC-2 isolates carried all of the four virulence genes.

A genetic and virulence characterization of Brazilian strains of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae (M. hyopneumoniae) is considered the primary causative agent of porcine enzootic pneumonia (EP), a chronic contagious respiratory disease that causes economic losses. Obtaining new pathogenic isolates and studying the genome and virulence factors are necessary. This study performed a complete sequencing analysis of two Brazilian strains, UFV01 and UFV02, aiming to characterize the isolates in terms of the virulence factors and sequence type. The complete genome analysis revealed the main virulence genes (mhp385, mhp271, MHP_RS03455, p102, p97, p216, MHP_RS00555, mhp107) and ST-123, the presence of three toxin-related genes (tlyC, PLDc_2 and hcnC), and some genetic groups specific to these two isolates. Subsequently, the pathogenicity of the isolates was evaluated via an experimental infection conducted in a swine model. The study was divided into three groups, namely a negative control group (n = 4) and two test groups (n = 8), totaling 20 animals. They were challenged at 35 days of age with 107 CCU (Color Changing Units) M. hyopneumoniae via the intratracheal route. The UFV01 group showed earlier and higher seroconversion (IgG) (100%), while only 50% of the UFV02 group seroconverted. The same trend was observed when analyzing the presence of IgA in the bronchoalveolar lavage fluid (BALF) at 35 days post-infection (dpi). The UFV01 group had a mean macroscopic lesion score of 11.75% at 35 dpi, while UFV02 had 3.125%. Microscopic lesions were more severe in the UFV01 group. Based on laryngeal swab samples evaluated by qPCR, and the detection began at 14 days. The UFV01 group showed 75% positivity at 14 dpi. The UFV02 group also started excreting at 14 dpi, with a positivity rate of 37.5%. The results indicate that the UFV01 isolate exhibits higher virulence than UFV02. These findings may aid in developing new vaccines and diagnostic kits and establishing experimental models for testing.

Bacterial Pathogens in the Food Industry: Antibiotic Resistance and Virulence Factors of Salmonella enterica Strains Isolated from Food Chain Links.

Salmonella is one of the most important foodborne pathogens. Fifty-three strains of Salmonella deposited in the Culture Collection of Industrial Microorganisms-Microbiological Resources Center (IAFB) were identified using molecular and proteomic analyses. Moreover, the genetic similarity of the tested strains was determined using the PFGE method. Main virulence genes were identified, and phenotypical antibiotic susceptibility profiles and prevalence of resistance genes were analyzed. Subsequently, the occurrence of the main mechanisms of β-lactam resistance was determined. Virulence genes, invA, fimA, and stn were identified in all tested strains. Phenotypic tests, including 28 antibiotics, showed that 50.9% of the strains were MDR. The tet genes associated with tetracyclines resistance were the most frequently identified genes. Concerning the genes associated with ESBL-producing Salmonella, no resistance to the TEM and CTX-M type was identified, and only two strains (KKP 1597 and KKP 1610) showed resistance to SHV. No strains exhibited AmpC-type resistance but for six Salmonella strains, the efflux-related resistance of PSE-1 was presented. The high number of resistant strains in combination with multiple ARGs in Salmonella indicates the possible overuse of antibiotics. Our results showed that it is necessary to monitor antimicrobial resistance profiles in all food chain links constantly and to implement a policy of proper antibiotic stewardship to contain or at least significantly limit the further acquisition of antibiotic resistance among Salmonella strains.

Comparative Analysis of Expression of the Main Virulence Genes in Various &lt;i&gt;Vibrio cholerae&lt;/i&gt; О1 Strains

The aim of the work was a comparative study of the expression of the main virulence genes in Vibrio cholerae strains of the classical biovar, typical and genetically modified strains of V. cholerae, El Tor biovar.Materials and methods. Natural toxigenic strains of V. cholerae O1, classical biovar (J89, Pakistan, 1969), typical (M-887, Astrakhan, 1970) and genetically modified (301, Taganrog, 2011) strains of the El Tor biovar were used as model ones. The strains were grown under optimum conditions for the production of cholera toxin and toxin-coregulated pili. The assessment of strain growth was carried out in LB broth at room temperature with determination of the cells number on a Biowave DNA spectrophotometer (Biochrome Ltd., UK). Determination of gene expression was performed using real-time PCR with reverse transcription.Results and discussion. The expression of structural (ctxA, tcpA) and regulatory (toxR, toxT, tcpP, tcpH) virulence genes has been investigated in V. cholerae strains of the classical biovar, typical and genetically modified strains of the El Tor biovar. Significant differences have been revealed in terms of time and level of maximum expression of these genes in strains of classical and El Tor biovars. It was found that ctxA and toxR genes expression in the genovariant strain reached its maximum 1–3 h earlier than in the other strains. At the same time, the level of ctxA gene expression corresponded to the level of the classical strain. The maximum expression of the toxR gene in the genovariant strain was higher than in typical El Tor and classical strains, and also had a clear inverse correlation with ctxA gene expression. Expression of the tcpA, toxT, and tcpH genes in the classical biovar strain reached its maximum 1–2 h earlier than in the El Tor biovar strains. These differences should be taken into account when conducting research work related to the study of the expression of the main virulence genes.

Exploring the nature of interaction between shiga toxin producing Escherichia coli (STEC) and free-living amoeba - Acanthamoeba sp

Free-living amoebae (FLA) are widely distributed protozoa in nature, known to cause severe eye infections and central nervous system disorders. There is growing attention to the potential role that these protozoa could act as reservoirs of pathogenic bacteria and, consequently, to the possibility that, the persistence and spread of the latter may be facilitated, by exploiting internalization into amoebae. Shiga toxin-producing strains of Escherichia coli (STEC) are zoonotic agents capable of causing serious diseases, such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Cattle represent the main natural reservoir of STEC, which are frequently found also in other domestic and wild ruminants, often without causing any evident symptoms of disease. The aspects related to the ecology of STEC strains in animal reservoirs and the environment are poorly known, including the persistence of these microorganisms within niches unfavorable to survival, such as soils or waters. In this study we investigated the interaction between STEC strains of serotype O157: H7 with different virulence gene profiles, and a genus of a wild free-living amoeba, Acanthamoeba sp. Our results confirm the ability of STEC strains to survive up to 20 days within a wild Acanthamoeba sp., in a quiescent state persisting in a non-cultivable form, until they reactivate following some stimulus of an unknown nature. Furthermore, our findings show that during their internalization, the E. coli O157 kept the set of the main virulence genes intact, preserving their pathogenetic potential. These observations suggest that the internalization in free-living amoebae may represent a means for STEC to resist in environments with non-permissive growth conditions. Moreover, by staying within the protozoa, STEC could escape their detection in the vehicles of infections and resist to the treatments used for the disinfection of the livestock environment.

Prevalence, virulence determinants, and genetic diversity in Yersinia enterocolitica isolated from slaughtered pigs and pig carcasses

Yersinia enterocolitica is an important zoonotic foodborne pathogen that could be transferred from infected pigs to their carcasses at slaughter, with subsequent introduction of the pathogen into the food chain. The aim of the present study was to study the prevalence, virulence characteristics, and genetic diversity of Y. enterocolitica isolates present in slaughtered pig tonsils and carcasses by using the WGS approach. A total of 200 slaughtered pig tonsils from 11 pig farms were collected in 2020–2021 at six slaughterhouses located in Latvia. Out of these samples, n = 190 were obtained from slaughtered pigs raised on Latvian farms while n = 10 were of Lithuanian origin, with the number of farms sampled being 10 and 1, respectively. Additionally, 30 pig carcasses were sampled at five slaughterhouses from pigs originating from five farms in 2021. Samples were investigated microbiologically, Y. enterocolitica isolates were biotyped and serotyped. Y. enterocolitica 4/O:3 was screened for antimicrobial resistance with the EUVSEC test panels. Whole genome sequence analysis (WGS) was performed in order to detect virulence genes and to assess the genetic diversity of Y. enterocolitica isolates. A total of 139 isolates, including one to three isolates from 84 Y. enterocolitica positive slaughtered pig tonsils and 13 pig carcass samples, were subjected to WGS analysis. The prevalence of Y. enterocolitica 4/O:3 in slaughtered pig tonsils and carcasses was 35% (70/200) and 13% (4/30), respectively. Antimicrobial resistance to ampicillin and tetracycline was detected in 97% (72/74) and 1% (1/74) of Y. enterocolitica 4/O:3 isolates. Y. enterocolitica 4/O:3 was represented only by ST18, while Y. enterocolitica 1A by ST3, ST147, ST304, ST307, and ST473. The ST18 isolates harbored the same main chromosomal (ail, inv, myfA, ystA) and majority shared plasmid-borne virulence genes (virF, yadA, yop virulon). The main virulence genes were not identified within the STs of Y. enterocolitica 1A and only minor differences were found between ST3, ST147, ST304, ST307, and ST473. Among ST18 isolates, cgMLST analysis revealed 43 cgMLST genotypes while 16 cgMLST genotypes were found among Y. enterocolitica 1A STs. The present study has shown the distribution of genetically distant cgMLST genotypes in slaughtered pigs from pig farms located in different geographical regions of Latvia, with one to 11 cgMLSTs identified within each sampled farm. The presence of undistinguishable cgMLST genotypes in slaughtered pig tonsils and the respective carcasses supported the link between the slaughter of Y. enterocolitica - positive pigs and carcass contamination with Y. enterocolitica 4/O:3.

Droplet Digital PCR (ddPCR) Analysis for Detecting Shiga-Toxin-Producing Escherichia coli (STEC)

Verocytotoxin-producing Escherichia coli, also referred to as Shiga-toxin-producing Escherichia coli (STEC), can be transmitted to humans through person-to-person contact, consumption of contaminated food or water, or by direct contact with animals. Its clinical and economic consequences have prompted the development of alternative approaches to the official method of analysis “UNI CEN ISO/TS 13136: 2012”, which describes the identification of STEC through the detection of its main virulence genes. Recently, droplet digital PCR (ddPCR) has been proposed as a technique for the sequence-specific detection and direct quantification of nucleic acids. The present study aimed to investigate if ddPCR could be able to detect STEC in less time than that required by the official method. This study consisted of the ddPCR of slices of beef contaminated with STEC and of the sponges used for beef official control at the slaughter stage. The results showed the ability of ddPCR to detect STEC in slices of beef already after sample incubation for 7 h at 37 °C while, in the case of sponges used for official controls, 9 h at 37 °C was needed. In this way, the ddPCR could represent an efficient method for detecting STEC and providing results in less time than the official method.

Virulence genes expression profiling of different Shigella flexneri serotypes in response to sub-inhibitory concentrations of azithromycin and ciprofloxacin

BackgroundShigellosis is a self-limiting disease that antibiotic therapy could decrease its complications and duration. However, sublethal levels of antibiotics, may lead to alteration in disease state, besides its role in the emergence of resistant variants. To understand this link, we investigated diversity of Shigella serogroups in children with diarrhea, diversity of S. flexneri serotypes, cytotoxic potential, resistance patterns to antibiotics, and alteration in transcriptional expression of main virulence genes in response to sub-inhibitory concentrations of azithromycin and ciprofloxacin.ResultsThe most frequently isolated serogroups were S. sonnei (70.3%), followed by S. flexneri (29.1%) and S. boydii (0.6%). Ten serotypes were characterized among the S. flexneri isolates, including 2b, 1b, 2a, 1c, 4a, 3a, 3b, 6 and X and/or Xv. Antimicrobial susceptibility testing showed low frequency of multi-drug resistance phenotype among S. flexneri isolates with minimum inhibitory concentrations (MIC) of 0.5–64 and 0.25–8 µg/mL for azithromycin and ciprofloxacin, respectively. Gene expression analysis showed upregulation of icsA in serotype 4a after exposure with azithromycin, whereas other genes in the VirF pathway were downregulated, and downregulation of virB in serotypes 2a and 3a after exposure with ciprofloxacin, while upregulation of noted genes was detected.ConclusionsAlteration in transcription of key virulence genes of S. flexneri serotypes was shown in response to sublethal concentration of antibiotics. The detected incongruency in the extent of gene transcription proposed that diverse regulatory pathways are possibly mediating response to sub-MIC concentrations of antibiotics in S. flexneri.

Comparison of Drug Resistance of Helicobacter pylori Between Children and Adults in Jilin, China.

The drug resistance of Helicobacter pylori in children is gaining more and more attention. Polymerase chain reaction-reverse dot blot was used to analyze main virulence genes and drug resistance of H. pylori. (1) The main H. pylori vacA virulence genotypes were s1/m1 and s1/m2 in Jilin, China. There was no significant difference in H. pylori virulence genotypes between children and adults. (2) The resistance rates of H. pylori to clarithromycin, metronidazole, and levofloxacin were high, the resistance rate to tetracycline and amoxicillin were relatively low. The drug-resistance rate of clarithromycin in children was significantly higher than that in adults. The single drug-resistance rate of metronidazole in adults was significantly higher than that in children. (3) The mutation sites of clarithromycin resistance in H. pylori were mainly A2143G of 23S rRNA gene, G616A of rdxA gene in metronidazole, N81K and D91G/N/Y of gyrA gene in levofloxacin, T556S and N562D/Y of PBP1 gene in amoxicillin, AGA926-928TTC and AG926-927GT of 16S rRNA gene in tetracycline. There was significant difference in D91Y of gyrA gene in levofloxacin between children and adults. (4) In different groups, the drug-resistance rate of clarithromycin in male children was higher than that in male adults. The drug-resistance rate of clarithromycin in children with peptic ulcers was higher than that in adults. There are some differences in drug resistance of H. pylori between children and adults, which indicated us to pay attention to the infection of H. pylori in children.

Antimicrobial resistance and molecular epidemiology of virulence genes among multi-drug resistant Acinetobacter baumannii clinical isolates in Iran

IntroductionMultidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii) is considered a serious threat level bacterial pathogen. This study aims to investigate the prevalence rate of main virulence genes within clinically MDR isolates of A. baumannii and extract their antibiotic susceptibility patterns. MethodsIn the present study, various clinical specimens were collected from general hospitals of Tehran, Iran in two years. The identification of A. baumannii isolates was performed by conventional biochemical tests. The antibiotic resistance profile of A. baumannii isolates was determined using the disk diffusion method and results were interpreted according to the CLSI guidelines. The existence and frequency of main virulence genes were screened by PCR method in MDR A. baumannii isolates. ResultsA total of 198 A. baumannii were isolated from clinical specimens in selected general hospitals in Iran, out of which 90 (45.45%) isolates were recognized as MDR A. baumannii isolates. The highest frequency of MDR A. baumannii isolates was detected in blood (42.22%) and urine (24.44%) specimens, respectively. Moreover, most MDR A. baumannii isolates (43.33%) were isolated from ICU. All of MDR A. baumannii isolates were resistant to piperacillin/tazobactam and cefepime. The prevalence of surveyed virulence genes among MDR A. baumannii isolates was as follows: pld (76.67%), bap (70%), traT (66.67%) lasB (56.67%), omp33-36 (52.22%), and ompA (23.33%). ConclusionThe results of our study revealed that the prevalence of MDR A. baumannii isolates and virulence-associated genes in general hospitals was high. It is suggested that virulence genes were associated with antibiotic resistance in A. baumannii isolates.

Detection and Virulence Characterization of Listeria monocytogenes Strains in Ready-to-Eat Products

The public health risk posed by Listeria monocytogenes in ready-to-eat (RTE) foods depends on the effectiveness of its control at every stage of the production process and the strain involved. Analytical methods currently in use are limited to the identification/quantification of L. monocytogenes at the species level, without distinguishing virulent from hypovirulent strains. In these products, according to EU Regulation 2073/2005, L. monocytogenes is a mandatory criterion irrespective of strain virulence level. Indeed, this species encompasses a diversity of strains with various pathogenic potential, reflecting genetic heterogeneity of the species itself. Thus, the detection of specific L. monocytogenes virulence genes can be considered an important target in laboratory food analysis to assign different risk levels to foods contaminated by strains carrying different genes. In 2015-2016, a severe invasive listeriosis outbreak occurred in central Italy, leading to the intensification of routine surveillance and strain characterization for virulence genetic markers. A new multiplex real-time polymerase chain reaction targeting main virulence genes has been developed and validated against the enzyme-linked fluorescent assay (ELFA) culture-based method. Results of the improved surveillance program are now reported in this study.

Adaptation of Staphylococcus aureus in a Medium Mimicking a Diabetic Foot Environment.

Staphylococcus aureus is the most prevalent pathogen isolated from diabetic foot infections (DFIs). The purpose of this study was to evaluate its behavior in an in vitro model mimicking the conditions encountered in DFI. Four clinical S. aureus strains were cultivated for 16 weeks in a specific environment based on the wound-like medium biofilm model. The adaptation of isolates was evaluated as follows: by Caenorhabditis elegans model (to evaluate virulence); by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) (to evaluate expression of the main virulence genes); and by Biofilm Ring test® (to assess the biofilm formation). After 16 weeks, the four S. aureus had adapted their metabolism, with the development of small colony variants and the loss of β-hemolysin expression. The in vivo nematode model suggested a decrease of virulence, confirmed by qRT-PCRs, showing a significant decrease of expression of the main staphylococcal virulence genes tested, notably the toxin-encoding genes. An increased expression of genes involved in adhesion and biofilm was noted. Our data based on an in vitro model confirm the impact of environment on the adaptation switch of S. aureus to prolonged stress environmental conditions. These results contribute to explore and characterize the virulence of S. aureus in chronic wounds.

Core and Accessory Genome Analysis of Vibrio mimicus.

Vibrio mimicus is an emerging pathogen, mainly associated with contaminated seafood consumption. However, little is known about its evolution, biodiversity, and pathogenic potential. This study analyzes the pan-, core, and accessory genomes of nine V. mimicus strains. The core genome yielded 2424 genes in chromosome I (ChI) and 822 genes in chromosome II (ChII), with an accessory genome comprising an average of 10.9% of the whole genome for ChI and 29% for ChII. Core genome phylogenetic trees were obtained, and V. mimicus ATCC-33654 strain was the closest to the outgroup in both chromosomes. Additionally, a phylogenetic study of eight conserved genes (ftsZ, gapA, gyrB, topA, rpoA, recA, mreB, and pyrH), including Vibrio cholerae, Vibrio parilis, Vibrio metoecus, and Vibrio caribbenthicus, clearly showed clade differentiation. The main virulence genes found in ChI corresponded with type I secretion proteins, extracellular components, flagellar proteins, and potential regulators, while, in ChII, the main categories were type-I secretion proteins, chemotaxis proteins, and antibiotic resistance proteins. The accessory genome was characterized by the presence of mobile elements and toxin encoding genes in both chromosomes. Based on the genome atlas, it was possible to characterize differential regions between strains. The pan-genome of V. mimicus encompassed 3539 genes for ChI and 2355 genes for ChII. These results give us an insight into the virulence and gene content of V. mimicus, as well as constitute the first approach to its diversity.

Unraveling the anti-bacterial mechanism of Litsea cubeba essential oil against E. coli O157:H7 and its application in vegetable juices

Recently, natural essential oils have been extensively studied for anti-bacterial application in foods due to their safety and high biological activity. Herein, Litsea cubeba essential oil (LC-EO) was applied as a natural anti-bacterial agent for exploring its anti-bacterial mechanism against Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7). The LC-EO could effectively inhibit the growth of EHEC O157:H7 and the minimal inhibitory concentration (MIC) was 0.5 mg/mL. In the study of anti-bacterial mechanism, the LC-EO was proved with good membrane penetration ability, which could destroy bacterial cell structure and disorder membrane permeability, thereby causing the leakage of intracellular organic matters. Furthermore, the inhibitory effects of LC-EO on physiological metabolism of EHEC O157:H7, including respiratory metabolism, enzyme activity, the replication of nucleic acid and the transcription level of main virulence genes (stx1, stx2, ehxA, eae), were also demonstrated in this study. Specially, the possible action mechanism of different components of LC-EO on bacterial genetic material was revealed deeply on molecular level by the molecular docking technology. Finally, the results of application evaluation indicated that the addition of LC-EO at MIC in different vegetable juices could maintain anti-bacterial rate above 99.9% for 4 days without remarkable influence on foods sensory quality. The information in this study provides the necessary theoretical foundation for extending the application of LC-EO in food preservation.

Experimental infection of the white snook Centropomus viridis Lockington (1877) with Vibrio ponticus: Histopathological manifestations and screening for putative virulence genes

Juvenile C. viridis (5.26 ± 0.72 g) were intraperitoneally injected with three doses of Vibrio ponticus (Vp) CAIM 1751 from 104 to 106 CFU/g, and histological analysis, haemolytic activity assessment and putative virulence genes identification were performed. The time of experimental challenge was 168 h. The virulence of Vp was dose-dependent. The mortality after 25 h post infection (p.i.) was 100% at 3.1 × 106 CFU/g and, 83% at 60 h p.i. at 2.9 × 105 CFU/g. The LC50 was 2.9 × 105 CFU/g at 32 h p.i. Congestion and petechial haemorrhages in the ventral area were observed in the infected fish. Similar histological lesions were observed at the three doses as necrosis, congestion and bacterial masses in different organs and tissues. This is the first report where Vp caused in C. viridis pathological changes associated with an acute peritonitis that caused irreversible damage to the internal organs, except in the kidney. Greater severity was identified in the liver and pancreas of the organisms exposed to the higher dose and in the stomach, intestine and nervous system of the fish infected with the lower dose. The main virulence genes of Vp CAIM 1751 were auto inducers, toxins and iron acquisition, including three types of haemolysins and metalloproteases. Furthermore, this strain had a strong β-haemolytic reaction on erythrocytes of the Pacific white snook. The combined action of the proteases and haemolysins may be responsible for the vascular fragility allowing for the rupture of small vessels and haemorrhages in the tissues. This study contributes to our understanding of Vibrio ponticus as an emerging finfish aquaculture pathogen.

Thymol and Carvacrol Downregulate the Expression of Salmonella typhimurium Virulence Genes during an In Vitro Infection on Caco-2 Cells

Salmonella typhimurium is one of the major bacteria responsible for gastroenteritis in humans caused by foodborne pathogens. As pork is one of the main routes of transmission, bioactive compounds used as feed additives may be an important strategy to control Salmonella typhimurium. The aim of this study was to assess the antimicrobial activity of several organic acids and nature identical compounds against Salmonella typhimurium ATCC®® 6994™. Moreover, the effect of sub-lethal concentrations of thymol and carvacrol in counteracting a Salmonella typhimurium in vitro infection on Caco-2 cells was evaluated, focusing on the maintenance of the epithelial barrier and the alteration of Salmonella virulence genes. The results showed a protective effect of the compounds on the integrity of the intestinal monolayer, improving transepithelial electrical resistance and bacterial translocation compared to the non-treated cells. A real-time PCR study highlighted a significant downregulation of the main virulence genes of Salmonella (hilA, prgH, invA, sipA, sipC, sipD, sopB, sopE2). These findings indicate that thymol and carvacrol could be good candidates for the control of Salmonella typhimurium in pigs.

Association of diarrheagenic Escherichia coli with virotypes and sensitivity to antimicrobials in children of the Mexico City

Diarrheal diseases continue to be a public health problem worldwide. Escherichia coli is one of the main etiological agents of diarrhea. The aim of the present work was to identify, through the polymerase chain reaction assay, the main virulence genes that characterize the different E. coli pathotypes that cause diarrhea in children under 5 years of age. A total of 151 stool samples from children with diarrhea were analyzed, of which 284 strains of E. coli were identified and DNA extracted. The virulence genes LT, STx1, STx2, EAST1, eaeA, bfpA, ial, aat, and astA were identified. Of the 284 strains analyzed, 179 (63.1%) were positive for at least one of the genes and 105 (36.9%) were negative for the nine genes studied. Of the 63%, enterotoxigenic E. coli was identified in a total of 63%, enterohemorrhagic E. coli in 25.0%, enteroaggregative E. coli in 25%, enteroinvasive E. coli in 20.7%, and enteropathogenic E. coli in 11.4%; These data are due to a strain of E. coli sharing genes with another strain. Children under 2 years old were the most affected. The adhesion phenotype, both aggregative and diffuse, was related to the presence of the aat and astA genes. The antibiogram showed that the strains of E. coli were resistant to more than three antibiotics, mainly ampicillin, cephalothin, amoxicillin/clavulanic acid, sulfamethoxazole-trimethoprim, and sensitive to amikacin, cefotaxime, and cefoxitin

Virulence potential of commensal multidrug resistant Escherichia coli isolated from poultry in Brazil

There is an increasing number of reports worldwide about multidrug resistance (MDR) with potential of ExPEC in commensal E. coli. The present study evaluated the potential ExPEC in selected 44 MDR E.coli isolates, collected from livestock. ExPEC isolates were characterized by analysis of five main groups of virulence genes (papA and/or papC, sfa and/or foc, afa and/or dra, kpsMT II and iutA). We also determined the increased virulence potential analyzing other 29 virulence genes, the epidemiology of these isolates. Additionally, fifteen ExPEC isolates were selected to evaluate the adhesion and invasion capacity in vitro using Caco-2 cells. Based on the analysis of the five main virulence genes, 72.7% (32/44) strains were classified as ExPEC. The presence of each gene was iutA 88.6%, KpsMT II 70.4%, papC 25%, sfa/focDE 4.5%; afa/draBC genes were not found. All E. coli isolates were classified into: phylogenetic groups A (34%), B1 (10%), B2 (20%), and D (36%). MLST revealed 7 different STs among isolates, including a new ST identified (ST5687). The in vitro assay in Caco-2 cells showed that all isolates were capable to adhere or invade the epithelial cells, although this occurred at variable levels. The ExPEC isolate LO122 reached similar levels of invasion to the positive control strain Salmonella Typhimurium LT2. These results showed that the apparently commensal microbiota of poultry harbors MDR ExPEC isolates with high adhesion and invasion potential.

Dysbiotic Biofilms Deregulate the Periodontal Inflammatory Response

Periodontal diseases originate from a dysbiosis within the oral microbiota, which is associated with a deregulation of the host immune response. Although little is known about the initiation of dysbiosis, it has been shown that H2O2 production is one of the main mechanisms by which some commensal bacteria suppress the outgrowth of pathobionts. Current models emphasize the critical nature of complex microbial biofilms that form unique microbial ecologies and of their change during transition from health (homeostatic) to disease (dysbiotic). However, very little is known on how this alters their virulence and host responses. The objective of this study was to determine differences in virulence gene expression by pathobionts and the inflammatory host response in homeostatic and dysbiotic biofilms originating from the same ecology. Quantitative polymerase chain reaction was performed to quantify the pathobiont outgrowth. Expression analysis of bacterial virulence and cellular inflammatory genes together with cytokine enzyme-linked immunosorbent assays were used to detect differences in bacterial virulence and to analyze potential differences in inflammatory response. An increase in pathobionts in induced dysbiotic biofilms was observed compared to homeostatic biofilms. The main virulence genes of all pathobionts were upregulated in dysbiotic biofilms. Exposure of these dysbiotic biofilms to epithelial and fibroblast cultures increased the expression of interleukin (IL)–6, IL-1β, tumor necrosis factor–α, and matrix metalloprotease 8, but especially the chemokine CXCL8 (IL-8). Conversely, homeostatic and beneficial biofilms had a minor immune response at the messenger RNA and protein level. Overall, induced dysbiotic biofilms enriched in pathobionts and virulence factors significantly increased the inflammatory response compared to homeostatic and commensal biofilms.