Quinolone resistance despite low antimicrobial usage - mechanisms and possible preventive measures

The Norwegian livestock production with low antimicrobial usage is a unique platform for building basic knowledge and gaining insight intothe development and spread of antibiotic resistance caused by mechanisms other than the use of antibiotics. Although the use of quinolones is nearly negligible in Norwegian livestock production, quinolone resistant bacteria are present at low levels. There is an interspecies variation with the highest occurrence in broilers and wild birds. The genetic mechanisms responsible for the detected quinolone resistance in E. coli from the livestock production is mainly chromosomal mutations, while plasmid encoded genes are present only to a small extent. Whole genome studies (WGS) of quinolone resistant E. coli (QREC) from broilers, swine, red fox and wild birds showed a high diversity of QREC among these animal species, though some highly similar QREC was identified from different animal species, indicating a between animal species dissemination. The isolates from broilers were more similar to each other than the isolates within the other species indicating a disseminations through the broiler production line. It is unclear how far up in the production chain these QREC have developed. However, in a follow up study comparing quinolone sensitive E. coli with QREC from the same broiler production site, only a few potential sporadic local resistance development was detected. Studies have been performed to investigate if different feed ingredients can cause or inhibit development of antimicrobial resistance in the intestinal bacteria. In vitro testing of E. coli in culturing together with ingredients from soy feed (luteolins) and under different physical factors did not result in QREC. A small pilot study found differences in QREC levels in animals with lowest levels in ruminants. One possible explanation may be that the QREC levels is related to the anatomy and physiological processes of the digestive tract. Feed ingredients will be treated differently in the digestive tract depending on the physiological processes. It may be that QREC is not developed / amplified to a large extent in adult ruminants due to an active fermentation in the rumen. Any feed ingredients contributing to development and dissemination of QREC could be broken down by the microbiota in the rumen before transported to the parts of the digestive tract where E. coli is located. In calves not yet developed into fully ruminants, QREC will then be able to develop. Experiments already established in other research projects (?Feed Mileage? and ?Foods of Norway?) were used to test whether different feed ingredients are drivers of QREC development. In two different feed experiments where rape seed and yeast cells replaced a major part of normally used soy as protein source in the concentrate to fattening pigs, QREC was not developed to the degree seen in ordinary fattening pigs. The feed types used also included the normally employed soy containing concentrate feed that resulted in 67% QREC in the pig samples in the pilot study. The feed used in the experiment for the fattening pigs that were sampled at slaughter did not contain formic acid as used in commercial feed. We decided to test the hypothesis that soy as feed ingredient with or without addition of formic acid could have an impact on the development of QREC in the intestines in a broiler model in a field farm. One trial with and without soy included in the diet, and a second trial with and without formic acid added in a diet with soy has been conducted. A third trial is planned to test parameters that varies in the two first trials. Preliminary results show differences in time for when the QREC develops after the start of feeding after hatching between the two trials. However, the analyses of the data from these trials have not been completed, and results from the third trial would be favourable before concluding. There has been several outbreaks of upper respiratory disease in pigs caused by Actinobacillus pleuropneumoniae (APP) in Norway. To prevent the risk for outbreaks and further spread of the infection it has previously been attempted to eradicate APP from a few herds using quinolones. We have sampled five pig herds treated with quinolones and five respective control herds without treatment for detection of QREC. Results show very low occurrence of QREC in both case and control herds, though some higher occurrence of QREC were seen in the case herds treated with quinolones. The project addresses scientifically complex issues that are challenging to communicate to the authorities and the industry, as well as to the public. Good risk communication practice is emphasized in the dissemination of knowledge and results to stakeholders.

Prosjektet har styrket kompetansen om forekomst av QREC, de bakenforliggende genetiske mekanismene for kinolonresistensen, samt forståelse av disse bakterienes klonalitet og mulig spredningsmønster hos forskjellige dyrearter. Resultatene fra prosjektet vil kunne benyttes som kunnskapsgrunnlag for faglige vurderinger til nytte for både husdyrnæringen og myndigheter, samt være grunnlag for videre forskning. Veterinærinstituttet har benyttet prosjektet som en mulighet for kompetanseutvikling innen helgenomsekvensering og bioinformatikk, og har i den anledning implementert flere bioinformatiske verktøy til nytte for en rekke forsknings- og utviklingsprosjekter ved instituttet, samt ved beredskapshendelser. Prosjektet har med andre ord bidratt til at Veterinærinstituttet har fått økt kompetanse og kapasitet på helgenomsekvensering og analyser av slike data. Dette er kompetanse som vil være essensiell i årene framover, både innen forskning, overvåking, og i beredskapssammenheng.

It is well known that usage of antimicrobials promotes the development and spread of resistance. In addition to resistance driven by antimicrobial usage, development and dissemination of antimicrobial resistance can occur through other mechanisms that are currently unidentified or otherwise poorly characterized or understood. The Norwegian animal production, with low antimicrobial usage, provides a unique platform for building basic knowledge and gaining insight into alternative mechanisms behind the development and spread of antimicrobial resistance. In Norwegian livestock production the use of fluoroquinolones is nearly negligible and resistance to this class of antimicrobial agents among bacteria from production animals has usually been a rare finding. However, after the implementation of a selective method in the Norwegian monitoring program for antimicrobial resistance in the veterinary sector (NORM-VET), it has been shown that quinolone resistant Escherichia coli (QREC) is present at low levels in a high proportion of the samples. There is currently a substantial knowledge gap regarding how, why, when and where this resistance has developed. Moreover, it is unclear if there is any association between the findings of QREC-isolates in the various production chains, and/or with environmental factors. It is crucial to understand these mechanisms to be able to identify, test and implement measures for prevention of resistance development and spread, which is important for maintaining our favourable national situation. Additionally, knowledge based risk management and transparent risk communication is critical to minimize loss of trust and negative economic consequences, and to ensure that relevant and adequate measures are taken. All these aspects will be addressed in the current project.