The effect of antimicrobial resistance plasmids carrying BlaCMY-2 on biofilm formation by escherichia coli from the broiler production chain

Extended-spectrum cephalosporin-resistant Escherichia coli (ESCR E. coli) with plasmids carrying the blaCMY-2 resistance gene have been isolated from the Norwegian broiler production chain through the Norwegian monitoring program for antimicrobial resistance in animals, food and feed, NORM-VET. The aim of the present study was to investigate the biofilm forming abilities of these strains, and in particular to see whether these might be influenced by the carriage of blaCMY-2 plasmids. The ESCR E. coli from the broiler production chain displayed relatively low biofilm forming abilities in the crystal violet biofilm assay as compared to quinolone-resistant E. coli (QREC) from the same population (mean ± SD = 0.686 ± 0.686 vs. 1.439 ± 0.933, respectively). Acquisition of two different blaCMY-2 plasmids by QREC strains reduced their biofilm production in microtiter plates, but not their biofilm production on Congo Red agar plates. Furthermore, motility was reduced, but not planktonic growth. We hypothesize that genes carried by these plasmids may have caused the observed reduction in biofilm formation, possibly mediated through changes in flagellar expression or function. Furthermore, this may help explain the different biofilm forming abilities observed between ESCR E. coli and QREC. The results also indicate that the risk of biofilm reservoirs of antimicrobial resistant E. coli on in the broiler production is lower for ESCR E. coli than for QREC.publishedVersio

Karakterisering av biofilmdannende egenskaper hos Listeria monocytogenes og evaluering av TTC-metoden som et verktøy for å måle effekten av desinfeksjonsmidler på bakterier i biofilm

Food-borne diseases are a problem throughout the world, and knowledge about when and where the food is contaminated in the processing line is important to reduce the risk of illnesses. By the use of molecular typing methods as pulsed-field gel electrophoresis (PFGE), it is possible to find relations between isolates from humans and isolates from food. Listeria monocytogenes (L. monocytogenes) is a pathogenic bacterium that is frequently isolated from fish processing facilities. It tolerates a wide range of growth conditions, which makes it a potential problem in refrigerated ready-to-eat (RTE) foods. Bacteria can persist in food environments in the shape of biofilm, and be a source of contamination. A total of 84 isolates of L. monocytogenes sampled from seven different fish processing facilities, were characterized with regard to biofilm forming ability and PFGE type. The biofilm forming ability was investigated by growing biofilm in microtiter plates, using two different growth media (LB and LB without NaCl), and incubating at 37 °C, 20 °C and 12 °C. Biofilm bacteria was quantified by using crystal violet-staining (CV assay). To investigate the susceptibility of biofilms to disinfectants, the TTC assay was evaluated as a method to investigate eradication of biofilm bacteria after treatment with disinfectants. Minimal inhibitory concentrations of six disinfectants were determined using the TTC assay and compared to the colony forming unit (CFU) count and the CV assay. The results showed that biofilm formation occurred only in LB-medium. The biofilm forming ability varied between isolates, even with the same PFGE type. There was a significant difference between the biofilm forming ability of serogroup IIa and IVb at all temperatures. The TTC assay indicated eradication of biofilm bacteria by all disinfectants, but these results were not supported by the CFU count and the CV assay. The CV assay indicated that biofilm was not removed by the disinfectants. The CFU counts showed that all disinfectants reduced the number of bacteria in the biofilms, but only two disinfectants, Aco hygiene des QA and Novadan disinfect Maxi, killed all cells after treatment with the concentration and time recommended by the supplier. The results from this study indicate that biofilm forming ability varies between isolates with similar and different PFGE-types. The combined results from the validation of the TTC assay indicates that it overestimates the efficiency of disinfectants on biofilm bacteria.Næringsmiddelbåren sykdom er et vidt utbredt problem, og kunnskap om hvordan og hvor næringsmidler kontamineres i produksjonskjeden er viktig med hensyn til å redusere risiko for sykdom. Ved bruk av molekylære typings-metoder slik som pulsfelt-gelelektroforese (PFGE) kan det knyttes sammenhenger mellom bakterier isolert fra mennesker og næringsmidler. Listeria monocytogenes (L. monocytogenes) er en patogen bakterie som ofte blir isolert fra fiskeforedlingsanlegg. Den tolererer et vidt spekter av vekstbetingelser, noe som gjør den til et potensielt problem i kjølelagret, spiseklar mat. Ved å etablere seg i produksjonslokaler i form av biofilm, kan den være en potensiell smittekilde. I denne studien ble 84 isolat av L. monocytogenes samlet fra syv ulike fiskeforedlingsanlegg, karakterisert med hensyn til biofilmdannende egenskaper og PFGE-type. De biofilmdannende egenskapene ble undersøkt ved å dyrke biofilm i mikrotiter-plater ved bruk av to ulike vekstmedium (LB og LB uten NaCl), og inkubering ved 37 °C, 20 °C og 12 °C. Bakterier i biofilm ble kvantifisert ved krystallfiolett-farging. For å undersøke toleransen biofilm har til desinfeksjonsmidler ble TTC-metoden benyttet. Det ble evaluert hvor god metoden var til å måle reduksjon av bakterier i biofilm, etter behandling med desinfeksjonsmidler. Minste hemmende konsentrasjon ble bestemt for seks ulike desinfeksjonsmidler ved bruk av TTC-metoden. Resultatene ble sammenlignet med resultater fra telling av kolonidannende enheter (CFU), og kvantifisering av bakterier i biofilm. Biofilmdannelse fant bare sted i vekstmediumet LB. Biofilmdannelse varierte mellom isolatene, både for like og ulike PFGE-typer. Det var en signifikant forskjell mellom biofilmdannende egenskaper for serogruppe IIa og IVb ved alle temperaturer. TTC-metoden indikerte at alle desinfeksjonsmidlene drepte bakteriene, men disse resultatene samsvarte ikke med resultatene fra telling av CFU og kvantifisering av biofilm. Fra kvantifiseringen av biofilm ble det indikert at desinfeksjonsmidlene ikke fjerner biofilmen. Resultater fra CFU-telling viste at alle midler reduserte bakterietallet, men bare to midler, Aco hygiene des QA og Novadan disinfect Maxi drepte alle celler etter behandling med desinfeksjonsmidler brukt etter anbefalt konsentrasjon og tid fra leverandør. Resultatene fra studien indikerer at biofilmdannende egenskaper er varierende mellom stammer av like og ulike PFGE-typer. Fra valideringen av TTC-metoden, viser de samlede resultatene at metoden overestimerer effekten desinfeksjonsmidler har på bakterier i biofilm.submittedVersionM-MA

The effect of antimicrobial resistance plasmids carrying BlaCMY-2 on biofilm formation by escherichia coli from the broiler production chain

Extended-spectrum cephalosporin-resistant Escherichia coli (ESCR E. coli) with plasmids carrying the blaCMY-2 resistance gene have been isolated from the Norwegian broiler production chain through the Norwegian monitoring program for antimicrobial resistance in animals, food and feed, NORM-VET. The aim of the present study was to investigate the biofilm forming abilities of these strains, and in particular to see whether these might be influenced by the carriage of blaCMY-2 plasmids. The ESCR E. coli from the broiler production chain displayed relatively low biofilm forming abilities in the crystal violet biofilm assay as compared to quinolone-resistant E. coli (QREC) from the same population (mean ± SD = 0.686 ± 0.686 vs. 1.439 ± 0.933, respectively). Acquisition of two different blaCMY-2 plasmids by QREC strains reduced their biofilm production in microtiter plates, but not their biofilm production on Congo Red agar plates. Furthermore, motility was reduced, but not planktonic growth. We hypothesize that genes carried by these plasmids may have caused the observed reduction in biofilm formation, possibly mediated through changes in flagellar expression or function. Furthermore, this may help explain the different biofilm forming abilities observed between ESCR E. coli and QREC. The results also indicate that the risk of biofilm reservoirs of antimicrobial resistant E. coli on in the broiler production is lower for ESCR E. coli than for QREC