Antimicrobial Resistance and Its Mechanisms among Campylobacter coli and Campylobacter upsaliensis with a Special Focus on Streptomycin

Campylobacteriosis is the most common cause of human bacterial gastroenteritis in the developed world. The most often isolated causative agent from diseased humans is C. jejuni, but also C. coli and C. upsaliensis, common colonizers of pigs and dogs, respectively, are known to cause disease. Campylobacteriosis is usually self-limiting but antimicrobial treatment is warranted in severe cases, with macrolides and fluoroquinolones being the first and second options, respectively. Intravenous aminoglycosides are indicated in Campylobacter bacteraemia. However, high rates of fluoroquinolone-resistant Campylobacter spp. have emerged in many parts of the world. Also, in several studies, high proportions of streptomycin-resistant C. coli or C. upsaliensis, have been found. Yet, the mechanisms of STR resistance have been only partially characterized in C. jejuni and C. coli and completely ignored in C. upsaliensis. The primary aim of this thesis was to investigate the molecular mechanisms of STR resistance in porcine C. coli and canine C. upsaliensis isolates. We were able to associate high level of STR resistance in porcine C. coli to mutations in the rpsL gene. In C. upsaliensis, a mutation in rpsL was also noted in all the low- and high-level STR-resistant isolates. All highly STR-resistant C. upsaliensis isolates had, in addition to the rpsL mutation, significant truncation of rsmG, encoding a conserved methyltransferase responsible for methylation of the ribosomal STR binding site. Even though STR resistance conferring mutations in rpsL and rsmG have been well documented in other bacterial species, they were first time described in Campylobacter spp. in the present study. Further, using genomics and insertional mutagenesis, a novel STR resistance-conferring gene was identified in the intermediately STR-resistant C. coli isolates. This gene is homologous, albeit at a low level, to other previously described aminoglycoside 6-adenylyltransferase encoding genes, and does not appear to originate from Gram-positive bacterial species. Based on our findings, we hypothesize that this gene could have evolved from a proto-resistance element in Campylobacter spp. Altogether these results provide a significant advance in understanding the mechanisms of STR resistance in Campylobacter spp. and will aid in predicting the phenotypic resistance from genome data. Fluoroquinolone resistance-associated mutations in the DNA gyrase-encoding gene gyrA were characterized in porcine C. coli treated with danofloxacin as well as among canine C. upsaliensis. The commonly described C257T mutation was found in both species. In C. coli this caused the amino acid change T86I in DNA gyrase and high levels of ciprofloxacin resistance, while in C. upsaliensis the predicted amino acid change was T86M causing only minor increase in CIP MIC but a high level of nalidixic acid resistance. Therefore, danofloxacin does not seem to induce novel mutations in C. coli in vivo but the same mutation appears not to be sufficient to cause a high level of fluoroquinolone resistance in C. upsaliensis.Kampylobakteerit ovat yleisimpiä bakteeriperäisiä ihmisten suolistotulehdusten aiheuttajia. Kampylobakterioosi on zoonoosi, eli se tarttuu eläinten ja ihmisten välillä. Ihmisillä yleisin taudinaiheuttaja on C. jejuni mutta myös sioilla yleisesti esiintyvä C. coli ja koirilla yleinen C. upsaliensis aiheuttavat sairastumisia. Kampylobakterioosia ei yleensä hoideta antibiooteilla mutta vakavissa infektioissa antibioottihoito on tarpeen, ja tällöin käytetään yleensä makrolidi- ja fluorokinoloniryhmien antibiootteja. Verenmyrkytystapauksissa hoito aminoglykosidiryhmän antibiooteilla on indikoitua. Viime vuosina C. jejuni ja C. coli bakteerien vastustuskyky eli resistenssi fluorokinoloneille on kuitenkin yleistynyt monissa maissa. Useissa tutkimuksissa on lisäksi havaittu että resistenssi aminoglykosideihin kuuluvalle streptomysiinille (STR) on varsin tavallista C. coli- ja C. upsaliensis-bakteereilla. Aminoglykosidiresistenssimekanismeja on karakterisoitu kuitenkin vain osittain C. jejuni ja C. coli-bakteereilla eikä lainkaan C. upsaliensis-isolaateilla. Väitöstutkimukseni ensisijainen tavoite oli tutkia sioista eristettyjen C. coli ja koirien C. upsaliensis-bakteerien streptomysiiniresistenssin geneettisiä mekanismeja. Tutkimuksessamme löytyi yhteys C. coli-bakteerin korkean tason STR-resistenssin ja kromosomaalisen rpsL-geenin mutaatioiden välillä. C. upsaliensis-bakteerilla mutaatioita rpsL-geenissä löytyi sekä matalalla että korkealla tasolla streptomysiinille resistenteiltä isolaateilta, ja lisäksi kaikilta korkeasti streptomysiinille resistenteiltä C. upsaliensis-bakteereilta löytyi mutaatioita rsmG-geenissä. Vaikka rpsL- ja rsmG-geenien mutaatioiden yhteys STR resistenssiin tunnetaan useilla muilla bakteerilajeilla, kuvasimme ne ensimmäistä kertaa kampylobakteereilla. Lisäksi löysimme uuden STR resistenssiä aiheuttavan geenin sikojen C. coli isolaateilta. Kyseinen geeni muistuttaa etäisesti aiemmin löydettyjä STR-resistenssigeenejä, mutta, toisin kuin ne, se ei näytä olevan peräisin grampositiivisista bakteerilajeista. Löydösten perusteella oletamme kyseisen geenin kehittyneen resistenssigeeniksi kampylobakteereissa. Tulokset syventävät huomattavasti ymmärrystä kampylobakteerien STR resistenssimekanismeista ja auttavat ennustamaan bakteerien fenotyyppistä resistenssiä genomisekvensointidataan perustuen. Tutkimme myös fluorokinoloniresistenssimekanismeja danofloksasiinilla hoidettujen sikojen C. coli sekä koirien C. upsaliensis bakteereilla. Molemmilla lajeilla löytyi sama mutaatio DNA gyraasia koodaavasta geenistä gyrA, mutta johtuen geenisekvenssien eroista, kyseinen mutaatio johti C. coli ja C. upsaliensis bakteereilla eri aminohappomuutoksiin. C. coli bakteereilla todettiin korkean tason resistenssi fluorokinoloneille kun taas C. upsaliensis bakteereilla resistenssi oli vain matalalla tasolla. Kuvattu mutaatio on yleisin fluorokinoloniresistenssiin johtava mutaatio C. coli ja C. jejuni bakteereilla kun taas C. upsaliensis isolaateilla mekanismeja ei ole aiemmin juuri tutkittu. Tutkimuksen perusteella danofloksasiini ei aiheuta uusia mutaatioita gyrA geeniin C. coli bakteereilla, kun taas sama mutaatio ei riitä korkean tason fluorokinoloniresistenssiin C. upsaliensis bakteereilla

The effects of amoxicillin treatment of newborn piglets on the prevalence of hernias and abscesses, growth and ampicillin resistance of intestinal coliform bacteria in weaned pigs

This study investigated the effects of a single amoxicillin treatment of newborn piglets on the prevalence of hernias and abscesses until the age of nine weeks. We also studied whether the treatment was associated with growth and mortality, the need for treatment of other diseases, the proportions of ampicillin resistant coliforms and antimicrobial resistance patterns of intestinal Escherichia coli (E. coli). A total of 7156 piglets, from approximately 480 litters, were divided into two treatment groups: ANT (N = 3661) and CON (N = 3495), where piglets were treated with or without a single intramuscular injection of 75 mg amoxicillin one day after birth, respectively. The umbilical and inguinal areas of weaned pigs were palpated at four and nine weeks of age. At the same time, altogether 124 pigs with hernias or abscesses and 820 non -defective pigs from three pens per batch were weighed individually. Mortality and the need to treat piglets for other diseases were recorded. Piglet faecal samples were collected from three areas of the floors of each pen at four weeks of age. The prevalence of umbilical hernias or abscesses did not differ between the groups at four weeks of age, but it was higher in the CON group than in the ANT group at nine weeks of age (2.3% vs. 0.7%, PPeer reviewe

Rifampin Reduces the Plasma Concentrations of Oral and Intravenous Hydromorphone in Healthy Volunteers

Peer reviewe

Low-level colonization of methicillin-resistant Staphylococcus aureus in pigs is maintained by slowly evolving, closely related strains in Finnish pig farms

Over the past two decades, livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has become widely prevalent in pig production in Europe. The carriage status of LA-MRSA is known to vary among individual pigs, but bacterial load in pigs has rarely been studied. We assessed the quantity of LA-MRSA in nasal and skin samples of pigs and investigated the genetic diversity of the strains together with sequenced strains from national surveillance and pathology samples from the Finnish Food Authority. On two farms with assumed MRSA-positive status, farm 1 and farm 2, 10 healthy pigs were sampled three times during 2 weeks from the nares and skin (study A). On farm 1, 54 additional pigs were sampled and from confirmed MRSA-positive animals, 10 were randomly selected and transported to a clean, controlled environment for further sampling (study B). From the samples taken on farms 1 and 2 and in the controlled environment, MRSA was isolated both by direct plating and enrichment on selective media. spa types, multilocus sequence types, staphylococcal cassette chromosome mec types, resistance and virulence genes were determined. Core genome multilocus sequence typing (cgMLST) analysis was performed, including the sequences deriving from the surveillance/pathology samples from the Finnish Food Authority.Peer reviewe

Population genetics and antimicrobial susceptibility of canine Campylobacter isolates collected before and after a raw feeding experiment

In recent years, increasing numbers of consumers have become interested in feeding raw food for their pet dogs as opposed to commercial dry food, in the belief of health advantages. However, raw meat and internal organs, possibly contaminated by pathogens such as Campylobacter spp., may pose a risk of transmission of zoonoses to the pet owners. Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans but C. upsaliensis has also been associated with human disease. In this study we investigated the effect of different feeding strategies on the prevalence of Campylobacter spp. in Finnish dogs. We further characterized the isolates using multilocus sequence typing (MLST), whole-genome (wg) MLST and antimicrobial susceptibility testing. Dogs were sampled before and after a feeding period consisting of commercial raw feed or dry pellet feed. Altogether 56% (20/36) of the dogs yielded at least one Campylobacter-positive fecal sample. C. upsaliensis was the major species detected from 39% of the dogs before and 30% after the feeding period. Two C. jejuni isolates were recovered, both from raw-fed dogs after the dietary regimen. The isolates represented the same genotype (ST-1326), suggesting a common infection source. However, no statistically significant correlation was found between the feeding strategies and Campylobacter spp. carriage. The global genealogy of MLST types of dog and human C. upsaliensis isolates revealed weakly clonal population structure as most STs were widely dispersed. Major antimicrobial resistance among C. upsaliensis isolates was against streptomycin (STR MIC > 4mg/l). Apart from that, all isolates were highly susceptible against the antimicrobials tested. Mutations were found in the genes rpsL or rpsL and rsmG in streptomycin resistant isolates. In conclusion, increasing trend to feed dogs with raw meat warrants more studies to evaluate the risk associated with raw feeding of pets in transmission of zoonoses to humans.Peer reviewe

Antimicrobial use, biosecurity, herd characteristics, and antimicrobial resistance in indicator Escherichia coli in ten Finnish pig farms

We investigated connections between antimicrobial use (AMU), biosecurity, and the numbers of pigs and staff in ten Finnish farrow-to-finish herds. Data on AMU in each herd were collected for 12 months. AMU was quantified as treatment incidences per 1000 days at risk (TI) using the consensus defined daily dose calculation. Biosecurity was scored using the Biocheck.UGent T system. We also examined antimicrobial resistance patterns of indicator E. coli isolated from faeces of selected pigs. In each herd, two groups of five pigs were formed: 1) antimicrobial treatment group (ANT: at least one pig in the litter was identified as sick and treated with antimicrobials) and 2) non-antimicrobial treatment group (NON: the litter was not medicated). Faecal samples were taken from these pigs at 5 and 22 weeks of age, cultured, and indicator E. coli isolates were tested for antimicrobial susceptibilities. The AMU varied considerably between the herds. Altogether, most of the antimicrobial treatment courses were assigned to weaned piglets. When AMU was quantified as TIs, suckling piglets had the highest TI (mean 46.6), which was significantly higher (P 0.05). We found few connections: enhanced external biosecurity levels found in the large herds co-occurred with lower use of antimicrobials and herds with low biosecurity scores - especially in the internal subcategories - appeared to have higher proportions of resistant isolates. Conclusively, we suggest that enhancing internal biosecurity might contribute to a reduction in the spreading of antimicrobial resistance in pig herds.Peer reviewe

FINRES-Vet 2021 : Finnish Veterinary Antimicrobial Resistance Monitoring and Consumption of Antimicrobial Agents

Authors and institutions participating in making the FINRES-Vet report: Finnish Food Authority: Suvi Nykäsenoja, Satu Olkkola, Marie Verkola, Mia Biström, Thomas Grönthal, Tiina Autio, Liisa Kaartinen, Mirja Raunio-Saarnisto Finnish Medicines Agency Fimea: Katariina Kivilahti-Mäntylä, Tita-Maria Muhonen Faculty of Veterinary Medicine, University of Helsinki: Katarina EskolaSales of veterinary antibiotics turned to a 5% increase in 2021. The result, however, was the second lowest since the monitoring began. The majority of overall sales consisted of products for individual treatment and the proportion of products for group treatment was roughly over a quarter. The biggest increase in sales was noted for orally administered sulfa-trimethoprim-combination and is partly explained by the increased manufacture of medicated feed for fur animals. Increased sales were also noted for antibiotic tablets for companion animals. Injectable penicillin continued to be the most sold veterinary antibiotic. Sales of critically important antibiotics (HPCIA, WHO) for treatment of animals decreased further and remained very low. The antibiotic resistance situation in bacteria from animals and food has remained relatively good in Finland. However, in certain bacterial species resistance was detected in moderate or high levels. Therefore, the need remains to further emphasise the preventive measures and prudent use of antibiotics. It is important to follow the Finnish recommendations for the use of antimicrobials in animals. Among salmonella from food-producing animals and campylobacter from broilers, resistance levels were low. Since 2014, the proportions of fluoroquinolone and tetracycline resistant broiler campylobacter isolates have varied. Among porcine campylobacter, fluoroquinolone resistance has increased. Resistance situation among indicator E. coli from pigs has remained good. The prevalence of ESBL/AmpC-producing bacteria in slaughtered pigs increased in 2021 while no ESBL/AmpC-producing bacteria were detected in pork and beef at retail. MRSA bacteria were detected more than previously in fresh pork at retail. The resistance situation among pathogenic bacteria isolated from food-producing animals remained similar to 2020. Resistance was overall low in bovine and porcine respiratory pathogens as well as in pathogens isolated from broilers. Resistance was still detected most in enterotoxigenic E. coli from pigs. Among bacteria isolated from companion animals, the changes in resistance situation were mostly small. The proportion of canine E. coli strains resistant to third-generation cephalosporins was the lowest since the start of the monitoring

FINRES-Vet 2018 : Finnish Veterinary Antimicrobial Resistance Monitoring and Consumption of Antimicrobial Agents

Consumption of veterinary antimicrobials in food-producing animal species in Finland is low and in recent years, has decreased further. Drop is noted in sales of almost all antimicrobial classes. Particularly sales of orally administered products have decreased. Sales of tablets intended to companion animals has almost halved during this decade. Majority, two thirds, of all antimicrobial products sold in 2018 was for treatment of individual animals and the remaining third products applicable for group treatment. Narrow spectrum penicillin G was the most used antimicrobial for animals and the proportion of highest priority critically important antimicrobials (HPCIA) was very low. The antimicrobial resistance situation in bacteria from animals and food has remained relatively good in Finland. However, in certain bacteria resistance was detected in moderate or high levels. Therefore, there is a need to further emphasise the preventive measures and prudent use of antimicrobials. It is important to follow the Finnish recommendations for the use of antimicrobials in animals. Among salmonella and campylobacter isolated from Finnish food-producing animals, resistance levels were mainly low. For the first time in Finland, multidrug resistant S. Kentucky was isolated from cattle in 2018. From 2014, the occurrence of fluoroquinolone and tetracycline resistance in campylobacter from broilers have varied. The occurrence of fluoroquinolone resistance in indicator E. coli has increased although the resistance is still low. Among pathogenic bacteria isolated from food-producing animals the most notifiable change was the worsening of resistance in some bovine respiratory disease pathogens. In other pathogens from food-producing animals the resistance situation remained similar as in previous years. The proportion of resistant bacterial isolates from companion animals and horses decreased for nearly all antimicrobials. However, the proportion of resistant isolates is still high for some antimicrobials. ESBL/AmpC-producing bacteria were still encountered in broilers and broiler meat; prevalence of these bacteria in broiler meat was somewhat lower in 2018 compared to 2016