Control of Salmonella Enteritidis on food contact surfaces with bacteriophage PVP-SE2

Salmonella is one of the worldwide leading foodborne pathogens responsible for illnesses and hospitalizations, and its capacity to form biofilms is one of its many virulence factors. This work evaluated (bacterio)phage control of adhered and biofilm cells of Salmonella Enteritidis on three different substrata at refrigerated and room temperatures, and also a preventive approach in poultry skin. PVP-SE2 phage was efficient in reducing both 24- and 48-h old Salmonella biofilms from polystyrene and stainless steel causing 2 to 5 log CFU cm2 reductions with a higher killing efficiency at room temperature. PVP-SE2 phage application on poultry skins reduced levels of Salmonella. Freezing phage-pretreated poultry skin samples had no influence on the viability of phage PVP-SE2 and their in vitro contamination with S. Enteritidis provided evidence that phages prevented their further growth. Although not all conditions favor phage treatment, this study endorses their use to prevent and control foodborne pathogen colonization of surfaces.Catarina Milho acknowledges the Portuguese Foundation for Science and Technology (FCT) grant [SFRH/BD/94434/ 2013]. Sanna Sillankorva is an Investigador FCT [IF/01413/ 2013]. This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 [POCI-01-0145-FEDER-006684] and BioTecNorte operation [NORTE-01-0145-FEDER-000004] funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte and the Project RECI/BBB-EBI/0179/2012 [FCOMP-01-0124-FEDER-027462].info:eu-repo/semantics/publishedVersio

In Vitro Activity of Quaternary Ammonium Surfactants against Streptococcal, Chlamydial, and Gonococcal Infective Agents

Free PMC Article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4879390/Quaternary ammonium compounds (QAC) are widely used, cheap, and chemically stable disinfectants and topical antiseptics with wide-spectrum antimicrobial activities. Within this group of compounds, we recently showed that there are significant differences between the pharmacodynamics of n-alkyl quaternary ammonium surfactants (QAS) with a short (C12) alkyl chain when in vitro toxicities toward bacterial and mammalian epithelial cells are compared. These differences result in an attractive therapeutic window that justifies studying short-chain QAS as prophylactics for sexually transmitted infections (STI) and perinatal vertically transmitted urogenital infections (UGI). We have evaluated the antimicrobial activities of short-chain (C12) n-alkyl QAS against several STI and UGI pathogens as well as against commensal Lactobacillus species. Inhibition of infection of HeLa cells by Neisseria gonorrhoeae and Chlamydia trachomatis was studied at concentrations that were not toxic to the HeLa cells. We show that the pathogenic bacteria are much more susceptible to QAS toxic effects than the commensal vaginal flora and that QAS significantly attenuate the infectivity of N. gonorrhoeae and C. trachomatis without affecting the viability of epithelial cells of the vaginal mucosa. N-Dodecylpyridinium bromide (C12PB) was found to be the most effective QAS. Our results strongly suggest that short-chain (C12) n-alkyl pyridinium bromides and structurally similar compounds are promising microbicide candidates for topical application in the prophylaxis of STI and perinatal vertical transmission of UGI.This work, including the efforts of Otilia V. Vieira, was funded by FCT (PTDC/BIA-BCM/112138/2009). This work, including the efforts of Otilia V. Vieira, was funded by FCT (HMSP-ICT/0024/2010). This work, including the efforts of Otilia V. Vieira, was funded by FCT (iNOVA4Health - UID/Multi/04462/2013).info:eu-repo/semantics/publishedVersio

Influenza seroprotection correlates with predominant circulating viruses during 2014/15 and 2015/16 seasons in Portugal

Rede Portuguesa de Laboratórios para o Diagnóstico da GripeBACKGROUND: Population immune profile for influenza is highly affected by circulating influenza viruses, thus changing the risk of infection for influenza. This study aims to assess influenza immunity in the Portuguese population by age groups, during 2014 and 2015 and establish a relationship between seroprotection and circulating influenza viruses in 2014/15 and 2015/16 seasons. METHODS: Two cross-sectional studies were developed based on a convenience serum sample collected in June 2014 (n=626) and July 2015 (n=675) in hospitals from mainland and Azores and Madeira.Serums equally represent all age groups. Antibody titers were evaluated by HI assay for strains recommended for seasonal influenza vaccine northern hemisphere,2014/15 and 2015/2016. Seroprevalences were estimated for each strain by age group and the association with seasonal cumulative influenza-like illness (ILI) rates for influenza virus during both seasons was analised. RESULTS: In June 2014 the highest seroprotection was observed for influenza A(H3) (39.0%; 95% CI: 36.2-43.8%) and A(H1)pdm09 (29.7; 95% CI: 26.3-33.4%), with higher levels in children 5-14 years old. In 2014/2015 a dominant circulation of influenza B/Yamagata was observed with high incidence rates in individuals under 65 years old, the ones that had lower seroprotection. Although before the start of the season high protection for A(H3) was observed, the circulation of the new drift A(H3) strains had gained an immunological advantage,in accordance with A(H3) elevated incidence rates observed during 2014/15. In July 2015 the highest seroprotection was observed for influenza B/ Yamagata (55.1%; 95% CI: 51.4-58.9%), 2.4 times the estimated 2014.This increase was even more pronounced in younger (≤ 4 years old), 6.3 times increase in 2015.This fact is in agreement with the predominant influenza B virus detected and the high ILI incidence rate observed in children during 2014/2015 epidemic. Seroprotection levels for influenza A in July 2015 were not significantly different from 2014.During 2015/16 season, influenza A(H1N1)pdm09 was predominant, with high incidence rate in < 65 year old. Influenza B/Victoria lineage,although detected at low levels increased in frequency, in agreement with the lowest level of seroprotection detected in the general population before the start of 2015/2016 season (21.8%; 95% CI: 18.7-24.0%). CONCLUSIONS There was a correlation between virus circulation, incidence rates for each age group and the previous seroprotection for seasonal influenza viruses.Our study highlights the value of measuring the serological profile for influenza to establishe risk groups for infection for which an increase preventive measures, including vaccination, should be fostered.info:eu-repo/semantics/publishedVersio

Identification of Novel Type III Secretion Chaperone-Substrate Complexes of <em>Chlamydia trachomatis</em>

<div><p><i>Chlamydia trachomatis</i> is an obligate intracellular bacterial pathogen of humans that uses a type III secretion (T3S) system to manipulate host cells through the delivery of effector proteins into their cytosol and membranes. The function of T3S systems depends on small bacterial cytosolic chaperone-like proteins, which bind T3S substrates and ensure their appropriate secretion. To find novel T3S chaperone-substrate complexes of <i>C. trachomatis</i> we first searched its genome for genes encoding proteins with features of T3S chaperones. We then systematically tested for interactions between candidate chaperones and chlamydial T3S substrates by bacterial two-hybrid. This revealed interactions between Slc1 (a known T3S chaperone) or CT584 and several T3S substrates. Co-immunoprecipation after protein expression in <i>Yersinia enterocolitica</i> and protein overlay binding assays indicated that Slc1 interacted with the N-terminal region of the known T3S substrates Tarp (a previously described substrate of Slc1), CT694, and CT695, and that CT584 interacted with a central region of CT082, which we identified as a <i>C. trachomatis</i> T3S substrate using <i>Y. enterocolitica</i> as a heterologous system. Further T3S assays in <i>Yersinia</i> indicated that Slc1 or CT584 increased the amount of secreted Tarp, CT694, and CT695, or CT082, respectively. Expression of CT584 increased the intra-bacterial stability of CT082, while Slc1 did not affect the stability of its substrates. Overall, this indicated that in <i>C. trachomatis</i> Slc1 is a chaperone of multiple T3S substrates and that CT584 is a chaperone of the newly identified T3S substrate CT082.</p> </div

Slc1 promotes type III secretion (T3S) of Tarp, CT694, and CT695 in <i>Y. enterocolitica</i>.

<p>(A) T3S-deficient <i>Y. enterocolitica</i> ΔHOPEMT ΔYscU and T3S-proficient ΔHOPEMT strains expressing the indicated proteins were incubated in T3S-inducing conditions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056292#pone.0056292-Sorg1" target="_blank">[42]</a>. Proteins in culture supernatants (S - secreted proteins), and in bacterial pellets (P - non-secreted proteins) from ∼5×10⁸ and ∼5×10⁷ bacteria, respectively, were analyzed by immunoblotting with the indicated antibodies. Immunodetection of TEM-1 β-lactamase (encoded by pBAD/Myc-His–derived plasmids expressing Myc-tagged proteins) ensured that presence of proteins in culture supernatants was not a result of bacterial lysis or contamination. (B) The amount of protein in bacterial pellets and in secreted fractions of assays done with ΔHOPEMT-derived strains was estimated by densitometry analyses of immunoblot images. We calculated the ratio between the amounts of each protein (Tarp-Myc, CT694-Myc, CT695-Myc or CT621-Myc) in the presence of Slc1-HA relative to when the proteins were expressed alone (+ Slc1-HA/− Slc1-HA). The dashed line indicates a ratio of 1. Data are the mean ± SEM from 3 independent experiments. (C) <i>Y. enterocolitica</i> ΔHOPEMT strains expressing the indicated proteins were grown in non-secreting conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056292#s2" target="_blank">Materials and Methods</a>). Chloramphenicol was added (time = 0 min) to stop bacterial protein synthesis. Samples were then taken at the depicted time points and analyzed by immunoblotting with the indicated antibodies.</p

Bacterial two-hybrid screen for novel type III secretion (T3S) chaperone-substrate complexes of <i>C. trachomatis</i>.

<p>(A) The open reading frames within the genome of <i>C. trachomatis</i> L2/434 strain were analyzed for putative proteins with a predicted molecular mass of <30 kDa and isolectric point (pI) of <6 or with a molecular mass of 10–20 kDa and a pI of 6–7. We then selected those showing amino acid similarity to known T3S chaperones or with an unknown function. To determine if the 21 proteins thus identified could form dimers, we analyzed if they self-interacted using the bacterial adenylate cyclase two-hybrid (BACTH) system (B and data not shown). To determine if small and acidic proteins that can self-interact bind <i>C. trachomatis</i> T3S substrates, we systematically analyzed for protein-protein interactions using the BACTH) system (C). (B) and (C) β-galactosidase (β-gal) activity in <i>E. coli</i> BTH101 (<i>cya-99</i>) harboring plasmids encoding the indicated T18 and T25 fusion proteins. P, positive control: <i>E. coli</i> BTH101 expressing fusions of a leucine zipper protein to T18 and T25. N, negative control: <i>E. coli</i> BTH101 expressing T18 and T25. Data are the mean ± SEM from 3 independent experiments. Asterisks denote statistical significant differences relative to the negative control (P<0.05; student’s <i>t</i>-test).</p

CT584 interacts with a central region of CT082.

<p>(A) <i>Y. enterocolitica</i> ΔHOPEMT strains expressing the indicated proteins were grown in non-secreting conditions. The bacterial cells were lysed and proteins in the lysate supernatants (input) were immunoprecipitated with mouse monoclonal anti-Myc or anti-HA antibodies (as depicted) bound to Protein G agarose beads (output). The input (Inp.) and output (Outp.) fractions from the immunoprecipitations (IPs) were analyzed by immunoblotting with rabbit polyclonal anti-Myc and rat monoclonal anti-HA antibodies. (B) Scheme of the truncated GST-tagged CT082 proteins analyzed to determine the binding region of CT584. (C) Protein overlay binding assays to identify the binding region of CT584 within CT082. Two identical SDS-PAGE were loaded with extracts of <i>E. coli</i> expressing the indicated GST fusion proteins. The electrophoresed proteins were transferred onto nitrocellulose membranes. One membrane was immunodetected with anti-GST antibodies while the other was probed with purified (6x)His-CT584 before being immunodetected with anti-His antibodies. Asterisks indicate the position corresponding to the predicted migration on SDS-PAGE of full-length GST fusion proteins (upper image) and the predicted position where an anti-His-dependent signal should appear if (6x)His-CT584 would bind the GST fusion proteins (lower image).</p

Effect of the expression of Slc1 or CT584 on <i>Yersinia</i> type III secretion (T3S).

<p>(A) <i>Y. enterocolitica</i> ΔHOPEMT carrying plasmids encoding Slc1-HA, CT584-HA or CT790-HA, and YopE-Myc (as indicated) were incubated in T3S-inducing conditions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056292#pone.0056292-Sorg1" target="_blank">[42]</a>. Proteins in culture supernatants (S - secreted proteins), and in bacterial pellets (P - non-secreted proteins) were analyzed by SDS-PAGE followed by Coomassie staining and immunoblotting with the indicated antibodies. Immunodetection of TEM-1 β-lactamase (encoded by the plasmid expressing YopE-Myc, a pBAD-Myc-His–derivative) ensured that presence of proteins in culture supernatants was not a result of bacterial lysis or contamination. Proteins from culture supernatants corresponding to ∼5×10⁸ or ∼2.5×10⁸ bacteria and from bacterial pellets corresponding to ∼5×10⁷ or ∼2.5×10⁷ bacteria were analyzed by Coomassie staining or by immunoblotting, respectively. (B) The amount of YopE-Myc in the bacterial pellets and in the secreted fractions was analyzed by densitometry from images of immunoblots. (C) The amount of YscP (graph on the left) and of YopB, YopD, LcrV and YopN (graph on the right) in secreted fractions was analyzed by densitometry from images of Coomassie-stained gels. YopB, YopD, LcrV, and YopN were analyzed all together because single bands corresponding to these proteins could not be easily distinguished in Coomassie-stained gels. The bands corresponding to YopB, YopD, LcrV, YopN, or YscP were deduced from the well known separation on SDS-PAGE of <i>Yersinia</i> secreted proteins after a T3S assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056292#pone.0056292-Sorg1" target="_blank">[42]</a>. In (B) and (C), we calculated the ratio between the amounts of YopE-Myc, YscP, or YopB, YopD, LcrV, and YopN when they were expressed in the presence of chlamydial proteins (Slc1-HA, CT584-HA, or CT790-HA) relative to when they were expressed alone (+ chlamydial proteins/− chlamydial proteins). All data are the mean ± SEM from 3 independent experiments.</p

CT584 promotes type III secretion (T3S) and intra-bacterial stability of CT082 in <i>Y. enterocolitica</i>.

<p>T3S-deficient <i>Y. enterocolitica</i> ΔHOPEMT ΔYscU and T3S-proficient <i>Y. enterocolitica</i> ΔHOPEMT carrying plasmids encoding Myc-tagged CT082 or CT695, and HA-tagged CT584 (as indicated) were incubated in T3S-inducing conditions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056292#pone.0056292-Sorg1" target="_blank">[42]</a>. Proteins in culture supernatants (S - secreted proteins), and in bacterial pellets (P - non-secreted proteins) from ∼5×10⁸ and ∼5×10⁷ bacteria, respectively, were analyzed by immunoblotting with anti-Myc, anti-HA, and anti-TEM or anti-SycO antibodies. Immunodetection of TEM-1 β-lactamase (encoded by pBAD/Myc-His–derived expressing Myc-tagged proteins) or the <i>Y. enterocolitica</i> T3S chaperone SycO <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056292#pone.0056292-Letzelter1" target="_blank">[4]</a> in the CT082-Myc or CT695-Myc T3S assays, respectively, ensured that presence of proteins in culture supernatants was not a result of bacterial lysis or contamination. (B) The amount of protein in bacterial pellets and in secreted fractions of assays done with ΔHOPEMT-derived strains estimated by densitometry analyses of immunoblot images. We calculated the ratio between the amounts of each protein (CT082-Myc or CT695-Myc) in the presence of CT584-HA relative to when the proteins were expressed alone (+ CT584-HA/− CT584-HA). The dashed line indicates a ratio of 1. Data are the mean ± SEM from 4 independent experiments. (C) <i>Y. enterocolitica</i> ΔHOPEMT carrying plasmids encoding Myc-tagged CT082 or CT695, and HA-tagged Slc1 (as indicated) were grown in non-secreting conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056292#s2" target="_blank">Materials and Methods</a>). Chloramphenicol was added (time = 0 min) to stop bacterial protein synthesis and samples were taken at the indicated time points. Samples were analyzed by immunoblotting using anti-Myc, anti-HA, and anti-TEM antibodies.</p