Stressresponser hos Listeria monocytogenes ovenfor antimikrobielle substanser fra melkesyrebakterier : et eksplorativt studium

Listeria monocytogenes is a gram-positive bacterium that causes severe and life threatening disease both in humans and animals. Due to the severity of the disease and the fact that the bacterium is responsible for considerable economical loss, L. monocytogenes is of a great concern particularly to the food industry. L. monocytogenes is a common food contaminant and nearly all human L. monocytogenes infections are due to ingestion of contaminated food. Antimicrobial components from lactic acid bacteria may serve as safe and natural food preservatives targeting unwanted microorganisms, including L. monocytogenes. Elucidations of how L. monocytogenes respond to the antimicrobial products are crucial steps to devise a knowledge-based strategy to control this deadly bacterium in food and food-related environments using food grade bacteria. In the present study the responses of L. monocytogenes to the class IIa bacteriocin sakacin P and to acids (acetic, lactic and hydrochloric acid) were explored. Exposure to sakacin P gave spontaneous mutant strains with reduced susceptibility to the bacteriocin. Analysis of large number of the mutant strains using different approaches revealed substantial difference among the strains. The mutant strains displayed difference in (i) the level of resistance to sakacin P, (ii) the stability of the sakacin P resistance phenotype, (iii) growth fitness in various conditions, (iv) biofilm formation ability, (v) virulence potential, (vi) Fourier transform infrared spectroscopy profile, (vii) regulation of the bacteriocin receptor gene, and (viii) global transcriptome profile. Overall, this indicates that the incidence(s) giving rise to the sakacin P resistance involves a complex regulatory gene network possibly mediated by the bacteriocin receptor and have pleiotropic effects on the physiology of the resistant strains. The growth of L. monocytogenes was reduced but not completely inhibited at pH 5 when the growth medium was acidified with hydrochloric acid (HCl), 10 mM acetic acid or 20 mM lactic acid. Acetic acid had the highest antilisterial activity followed by lactic acid and HCl. Stress due to the presence of the acids induced a large number of genes associated with acid defense, virulence, and cross-protection to other types of stresses. Acidulant type dependent responses were also observed. The explorative transcriptome studies confirmed numerous results of previous studies on the response of L. monocytogenes to the class IIa bacteriocins and acid stresses. In addition, it identified a number of putative genes with possible role in the responses under investigation. Altogether, the results presented in this thesis revealed insights contributing to understand the responses of L. monocytogenes to the antimicrobial substances that may be encountered by this bacterium in fermented food and thereby opening new avenues for further studiesListeria monocytogenes er en gram-positiv bakterie som kan forårsake alvorlig og livstruende sykdom blant både dyr og mennesker. Alvorligshetsgraden av sykdommen og det faktum at bakterien er årsak til stort økonomisk tap, gjør at L. monocytogenes forårsaker stor bekymring, spesielt hos næringsmiddelindustrien. L. monocytogenes forekommer i mange typer mat, og nesten alle humane infeksjoner med L. monocytogenes skyldes inntak av forurenset mat. Antimikrobielle forbindelser fra melkesyrebakterier, som bakteriocin og syrer, kan være trygge og naturlige konserveringsmidler mot uønskete mikroorganismer, inkludert L. monocytogenes. Å avdekke hvordan L. monocytogenes responderer på disse antimikrobielle forbindelsene er viktig for å utvikle en kunnskapsbasert strategi for kontroll av denne bakterien i mat og i matrelaterte omgivelser ved bruk av “food grade” bakterier. I denne studien ble responsen til L. monocytogenes mot klasse IIa bakteriocinet sakacin P, mot lav pH og syrer (eddik- og melkesyre) undersøkt. Eksponering for sakacin P ga spontane mutanter med redusert følsomhet for bakteriocinet. Bred analyse av et stort antall mutanter viste at det var vesentlig forskjell mellom stammene. De muterte stammene hadde forskjeller i (i) resistensnivå mot sakacin P, (ii) stabilitet av resistens fenotype (iii) vekst ved ulike forhold, (iv) evne til biofilmdannelse, (v) virulenspotensial, (vi) Fourier transform infrarød spektroskopiprofil, (vii) regulering av bakteriocinreseptorgenet og (viii) global transkripsjonsprofil. Til sammen indikerer dette at hendelser som medfører økt sakacin P resistens involverer et komplekst genreguleringsnettverk, muligens styrt via bacteriocin reseptoren, som har en mangfoldig påvirkning på fysiologien til de resistente stammene. Vekst av L. monocytogenes ble redusert, men ikke fullstendig hemmet ved pH 5, når vekstmediet var surgjort med saltsyre (HCl), 10 mM eddiksyre eller 20 mM melkesyre. Eddiksyre hadde den største antilisteriaeffekten, etterfulgt av melkesyre og HCl. Stress som følge av nærvær av syrer, induserte et stort antall gener assosiert med syreforsvar, virulens og kryssbeskyttelse mot andre typer stress. Det ble også observert spesifikke responser for de ulike syrene. Transkripsjonsstudier bekreftet flere resultater fra tidligere studier på respons av L. monocytogenes til klasse IIa bakteriociner og syrestress. I tillegg ble det identifisert gener som muligens er involvert i responsene som ble studert. Til sammen gir resultatene presentert i denne avhandlingen innsikt som bidrar ytterligere til å forstå responser til L. monocytogenes mot antimikrobielle forbindelser som denne bakterien kan utsettes for, for eksempel i fermentert mat, noe som kan være interessant å undersøke videre i fremtidige studier.Norges Forskningsrå

ccrABEnt serine recombinase genes are widely distributed in the Enterococcus faecium and Enterococcus casseliflavus species groups and are expressed in E. faecium

The presence, distribution and expression of cassette chromosome recombinase (ccr) genes, which are homologous to the staphylococcal ccrAB genes and are designated ccrABEnt genes, were examined in enterococcal isolates (n=421) representing 13 different species. A total of 118 (28 %) isolates were positive for ccrABEnt genes by PCR, and a number of these were confirmed by Southern hybridization with a ccrAEnt probe (n=76) and partial DNA sequencing of ccrAEnt and ccrBEnt genes (n=38). ccrABEnt genes were present in Enterococcus faecium (58/216, 27 %), Enterococcus durans (31/38, 82 %), Enterococcus hirae (27/52, 50 %), Enterococcus casseliflavus (1/4, 25 %) and Enterococcus gallinarum (1/2, 50 %). In the eight other species tested, including Enterococcus faecalis (n=94), ccrABEnt genes were not found. Thirty-eight sequenced ccrABEnt genes from five different enterococcal species showed 94–100 % nucleotide sequence identity and linkage PCRs showed heterogeneity in the ccrABEnt flanking chromosomal genes. Expression analysis of ccrABEnt genes from the E. faecium DO strain showed constitutive expression as a bicistronic mRNA. The ccrABEnt mRNA levels were lower during log phase than stationary phase in relation to total mRNA. Multilocus sequence typing was performed on 39 isolates. ccrABEnt genes were detected in both hospital-related (10/29, 34 %) and non-hospital (4/10, 40 %) strains of E. faecium. Various sequence types were represented by both ccrABEnt positive and negative isolates, suggesting acquisition or loss of ccrABEnt in E. faecium. In summary, ccrABEnt genes, potentially involved in genome plasticity, are expressed in E. faecium and are widely distributed in the E. faecium and E. casseliflavus species groups

Global Transcriptional Analysis of Spontaneous Sakacin P-Resistant Mutant Strains of Listeria monocytogenes during Growth on Different Sugars

Subclass IIa bacteriocins have strong antilisterial activity and can control the growth of Listeria monocytogenes in food. However, L. monocytogenes may develop resistance towards such bacteriocins. In this follow-up study, the transcriptomes of a high level (L502-1) and a low level (L502-6) spontaneous sakacin P-resistant mutant strain of L. monocytogenes were compared to the wild-type (L502). The growth of the resistant strains was reduced on mannose but not affected on cellobiose and the transcriptomics was performed during growth on these sugars. The mannose phosphotransferase system (PTS) encoded by the mptACD operon (mpt) is known for transporting mannose and also act as a receptor to class IIa bacteriocins. The mpt was repressed in L502-1 and this is in accordance with abolition of the bacteriocin receptor with resistance to class IIa bacteriocins. In contrast, the mpt was induced in L502-6. Despite the induction of the mpt, L502-6 showed 1,000 times more resistance phenotype and reduced growth on mannose suggesting the mannose-PTS may not be functional in L502-6. The microarray data suggests the presence of other transcriptional responses that may be linked to the sakacin P resistance phenotype particularly in L502-6. Most of commonly regulated genes encode proteins involved in transport and energy metabolism. The resistant strains displayed shift in general carbon catabolite control possibly mediated by the mpt. Our data suggest that the resistant strains may have a reduced virulence potential. Growth sugar- and mutant-specific responses were also revealed. The two resistant strains also displayed difference in stability of the sakacin P resistance phenotype, growth in the presence of both the lytic bacteriophage P100 and activated charcoal. Taken together, the present study showed that a single time exposure to the class IIa bacteriocin sakacin P may elicit contrasting phenotypic and transcriptome responses in L. monocytogenes possibly through regulation of the mpt

Effect of high pressure processing in combination with Weissella viridescens as a protective culture against Listeria monocytogenes in ready-to-eat salads of different pH

This study explored the effect of HPP (400 MPa/1 min) and a Weissella viridescens protective culture, alone or in conjunction, against Listeria monocytogenes in ready-to-eat (RTE) salads with different pH values (4.32 and 5.59) during storage at 4 and 12 °C. HPP was able to reduce the counts of the pathogen after treatment achieving approximately a 4.0 and 1.5 log CFU/g reduction in the low and higher pH RTE salad, respectively. However, L. monocytogenes was able to recover and grow during subsequent storage. W. viridescens grew in both RTE salads at both storage temperatures, with HPP resulting in only a small immediate reduction of W. viridescens ranging from 0.50 to 1.2 log CFU/g depending on the pH of the RTE salad. For the lower pH RTE salad, the protective culture was able to gradually reduce the L. monocytogenes counts during storage whereas for the higher pH RTE salad in some cases it delayed growth significantly or exerted a bacteriostatic effect. exerted a bacteriostatic effect. The results revealed that the increased storage temperature led to an increase in the inactivation/inhibition of L. monocytogenes in the presence of W. viridescens. The combination of HPP and W. viridescens is a promising strategy to control L. monocytogenes and can increase safety even when a break in the chill chain occurs

Modelling the growth kinetics of Listeria monocytogenes in pasta salads at different storage temperatures and packaging conditions

The aim of this study was to model Listeria monocytogenes growth kinetics in ready to eat full meal pasta salads, containing fresh and cooked ingredients. With this aim, laboratory prepared salads, representing two formulations of commercial pasta salads, were spiked with L. monocytogenes and tested under categorised packaging and storage temperature conditions. L. monocytogenes enumeration results collected in 15 different laboratory prepared salad datasets were analysed with primary and secondary models. The models showing the best fit to describe L. monocytogenes growth kinetics in the laboratory prepared salads were then validated within commercial pasta salads. Baranyi no-lag was the best primary model fitting datasets collected at 12 \ub0C, whereas the exponential model gave the best results for datasets collected at 4 \ub0C. The maximum microbial specific growth rate (\u3bcmax) mean values obtained at 4 and 12 \ub0C for salads packaged under air packaging conditions were 0.008 \ub1 0.003 and 0.036 \ub1 0.006 log10(cfu/g) h 121, respectively. At the same temperatures, the \u3bcmaxmean values obtained under modified atmosphere were 0.005 \ub1 0.005 and 0.026 \ub1 0.005 log10(cfu/g) h 121, respectively. The Gamma secondary model predicted the growth kinetics of L. monocytogenes at both temperatures and packaging conditions and the \u3bcmaxat the optimum temperature and the optimum pH for Listeria growth (\u3bcopt) estimated by the model corresponded to 0.247 \ub1 0.009 log10(cfu/g) h 121. Baranyi model without lag phase was used to generate growth kinetics under different scenarios. In the comparison of the predicted log10concentrations respect to the observed ones the residues rarely exceeded 1 Log10cfu/g. The selected models can be applied to describe the growth kinetics of L. monocytogenes in similar types of pasta salads with comparable pH, shelf life and storage conditions

Characteristics of pulmonary multidrug-resistant tuberculosis patients in Tigray Region, Ethiopia: A cross-sectional study.

BackgroundTuberculosis (TB) is among the top 10 causes of mortality and the first killer among infectious diseases worldwide. One of the factors fuelling the TB epidemic is the global rise of multidrug resistant TB (MDR-TB). The aim of this study was to determine the magnitude and factors associated with MDR-TB in the Tigray Region, Ethiopia.MethodThis study employed a facility-based cross-sectional study design, which was conducted between July 2018 and August 2019. The inclusion criteria for the study participants were GeneXpert-positive who were not under treatment for TB, PTB patients' ≥15 years of age and who provided written informed consent. A total of 300 participants were enrolled in the study, with a structured questionnaire used to collect data on clinical, sociodemographic and behavioral factors. Sputum samples were collected and processed for acid-fast bacilli staining, culture and drug susceptibility testing. Drug susceptibility testing was performed using a line probe assay. Logistic regression was used to analyze associations between outcome and predictor variables.ResultsThe overall proportion of MDR-TB was 16.7% (11.6% and 32.7% for new and previously treated patients, respectively). Of the total MDR-TB isolates, 5.3% were pre-XDR-TB. The proportion of MDR-TB/HIV co-infection was 21.1%. A previous history of TB treatment AOR 3.75; 95% CI (0.7-2.24), cigarette smoking AOR 6.09; CI (1.65-2.50) and patients who had an intermittent fever (AOR = 2.54, 95% CI = 1.21-5.4) were strongly associated with MDR-TB development.ConclusionsThe magnitude of MDR-TB observed among new and previously treated patients is very alarming, which calls for an urgent need for intervention. The high proportion of MDR-TB among newly diagnosed cases indicates ongoing transmission, which suggests the need for enhanced TB control program performance to interrupt transmission. The increased proportion of MDR-TB among previously treated cases indicates a need for better patient management to prevent the evolution of drug resistance. Assessing the TB control program performance gaps and an optimal implementation of the WHO recommended priority actions for the management of drug-resistant TB, is imperative to help reduce the current high MDR-TB burden in the study region