Diagnostics and molecular epidemiology of cpe-positive Clostridium perfringens type A

The present thesis reveals new information, showing that healthy human is a rich reservoir for Clostridium perfringens enterotoxin (CPE)- producing Clostridium perfringens type A. The results show that healthy foodhandlers are commonly asymptomatic carriers of this pathogen. The findings indicate that persons handling foods should be considered a possible source of contamination for C. perfringens type A food poisoning. This work also found that the genotypes that previously were considered as atypical causative agents in food poisonings, in fact cause a notable proportion of C. perfringens type A food poisonings. CPE- producing C. perfringens type A is considered one of the most common causes of food poisonings in the industrialized world. This pathogen is also involved in antibiotic-associated diarrhea and sporadic diarrhea in humans as well as in animals. Recent findings of the variable loci (plasmid vs. chromosome) and different genetic arrangements adjacent to enterotoxin gene (cpe) have shown that CPE- producing C. perfringens type A isolates form in different subpopulations. These subpopulations are responsible for different CPE-associated diseases, i.e. chromosomal cpe-bearing strains are involved in food poisonings, whereas strains carrying cpe in the plasmid are typical in antibiotic-associated or sporadic diarrhea cases as well as in animal diarrheas. Not much is known about reservoirs or transmission routes of cpe-positive isolates. The present thesis aimed to improve diagnostics of cpe-positive C. perfringens by developing molecular methods for detection and isolation. Other objectives were to improve identification and typing of C. perfringens. With novel assays, molecular epidemiology of cpe-positive C. perfringens type A was further investigated. When investigating the presence of cpe-positive C. perfringens in the feces of healthy food- handlers, the organism was carried by 18% of the individuals. The presence of strains representing all previously identified genotypes (plasmid-borne IS1151-cpe, plasmid-borne IS1470-like-cpe, and chromosomal IS1470-cpe) as well as strains carrying previously unrecognized genetic arrangements attached to cpe shows that various different subpopulations of cpe-positive C. perfringens type A occur in the human gastrointestinal tract and that the healthy human is a rich reservoir for cpe-positive C. perfringens type A. The common carriage of cpe-positive C. perfringens type A of healthy food-handlers and the full capacity of these strains to produce CPE indicate that humans handling food should be considered a possible source of contamination of food. By showing that 25% of investigated C. perfringens type A food poisoning outbreaks were caused by strains carrying plasmid-borne cpe, we reveal that plasmid-borne cpe carrying C. perfringens type A is a common cause of food poisonings. The new data presented in this thesis improve the understanding of reservoirs and movements of enterotoxin producing C. perfringens type A. Therefore, CPE-associated diseases may be better controlled and prevented.Väitöskirjatutkimus tuo esiin aiemmin tuntematonta tietoa, jonka mukaan ihminen on ruokamyrkytyksiä aiheuttavan Clostridium perfringens tyyppi A-bakteerin merkittävä varasto luonnossa ja toimii ruokamyrkytystapauksissa mahdollisena ruuan saastuttajana. Uutta tietoa on myös se, että bakteerin genotyyppi, jota aiemmin on pidetty epätyypillisenä ruokamyrkytysten aiheuttajana, aiheuttaa merkittävän osan ruokamyrkytystapauksista. Enterotoksiinia tuottava C. perfringens tyyppi A bakteeri kuuluu yleisimpien ruokamyrkytyksen aiheuttajien joukkoon kaikissa teollisuusmaissa. Viime vuosikymmeninä kyseinen bakteeri aiheutti 20% kaikista raportoiduista ruokamyrkytyksistä maassamme. Enterotoksiinia tuottavat C. perfringens tyyppi A bakteerikannat voidaan jakaa eri genotyyppeihin enterotoksiinigeenin sijainnin mukaan (bakteerin kromosomissa tai plasmidissa). Kromosomaaliseen genotyyppiin kuuluvat kannat tunnetaan ruokamyrkytysten aiheuttajina, kun taas plasmidillisia kantoja löydetään muista ripulitaudeista sekä ihmisiltä että eläimiltä. Väitöskirjatyön tavoitteena oli parantaa enterotoksiinia tuottavan C. perfringens tyyppi A bakteerin diagnostiikkaa kehittämällä molekyylibiologisia menetelmiä bakteerin eristämiseen, tunnistamiseen ja tyypittämiseen. Tavoitteena oli myös käyttää työssä kehitettyjä menetelmiä apuna selvitettäessä bakteerin eri genotyyppien esiintymistä terveillä ihmisillä ja osallisuutta ruokamyrkytystapauksissa. Suomalaisia elintarviketyöntekijöitä käsitellyt tutkimus toi esiin aiemmin tuntematonta tietoa, jonka mukaan 18% terveistä ihmisistä toimii ruokamyrkytyksiä aiheuttavan C. perfringens tyyppi A:n oireettomana kantajana. Tarkempi tarkastelu osoitti ihmisillä esiintyvän sekä plasmidillisia että kromosomaalisia kantoja. Lisäksi löydettiin aiemmin tuntemattomia genotyyppejä. Tutkimus osoittaa, että ihminen on enterotoksiinia tuottavan C. perfringens tyyppi A bakteerin merkittävä varasto luonnossa, ja että ruokaa käsittelevä ihminen on otettava huomioon ruuan mahdollisena saastuttajana C. perfringens tyyppi A ruokamyrkytystapauksissa. Tutkittaessa eri genotyyppien osallisuutta ruokamyrkytyksissä havaittiin, että vastoin aiemmin vallinneita käsityksiä, plasmidilliset kannat ovat yleisiä aiheuttajina ruokamyrkytyksissä. Jopa 25%:ssa tutkituista ruokamyrkytystapauksista todettiin aiheuttajaksi plasmidilliseen genotyyppiin kuuluva C. perfringens tyyppi A-kanta. Väitöskirjatutkimuksessa saatu tieto on oleellista C. perfringens tyyppi A:n aiheuttaman ruokamyrkytyksen torjuntatyössä. Tutkimustietoa voidaan hyödyntää myös muiden C. perfringens tyyppi A- ripulitautien diagnostiikassa ja syntymekanismien selvittelyssä

New dominant spa type t2741 in livestock-associated MRSA (CC398-MRSA-V) in Finnish fattening pigs at slaughter

Background: The emergence of livestock-associated MRSA has become a growing public health concern worldwide. Studies elucidating the population structure, as well as resistance phenotypes and virulence gene profiles of livestock-associated MRSA strains are needed to improve risk assessment and to develop effective control measures. The objective of this study therefore was to determine i) clonal complexes and spa types, as well as ii) resistance phenotypes and iii) virulence and resistance gene profiles of livestock-associated MRSA isolated from Finnish fattening pigs at slaughter. Methods: Fifty MRSA isolates collected from Finnish fattening pigs at slaughter were characterized by spa typing and DNA microarray profiling. In addition, antimicrobial susceptibility testing was performed using the Kirby Bauer disk diffusion method. Results: MRSA isolates were assigned to clonal complexes CC1 (n = 4) and CC398 (n = 46). One dominant spa type (t2741) was present in 33 out of 50 investigated isolates, originating from 15 out of 18 farms. The remaining isolates were assigned to spa types t034 (n = 7), t108 (n = 5), and t011 (n = 1). Although each herd exhibited isolates assigned to one clonal complex only, five herds harbored MRSA isolates of either two or three different spa types. All tested MRSA isolates were phenotypically resistant to penicillin, oxacillin, cefoxitin, and tetracycline. With the exception of the isolates assigned to t108, all isolates exhibited resistance to clindamycin. On the genomic level, all isolates exhibited mecA, blaZ/I/R, and tetK, and were assigned to SCCmec type V. Many isolates also harbored tetM (46/50 isolates), lnuB (41/50 isolates), ermB (26/50 isolates), and one isolate was positive for aadD. DNA microarray profiling showed that all isolates of the dominant CC398/t2741 MRSA-V type belonged to agr type I, capsule type 5, and were negative for fnbB. Interestingly, one isolate of CC398/t2741 MRSA-V was agr negative and also lacked hld. Conclusions: A new dominant LA-MRSA clone (CC398/t2741, SCCmec type V) was identified among fattening pigs in Finland. This is the first study identifying t2741 as a common spa type in LA-MRSA in pigs.Peer reviewe

Genomic analysis of sewage from 101 countries reveals global landscape of antimicrobial resistance

Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, evolution, and transmission of individual antibiotic resistance genes (ARGs) is essential to develop sustainable strategies combatting this threat. Here, we use metagenomic sequencing to analyse ARGs in 757 sewage samples from 243 cities in 101 countries, collected from 2016 to 2019. We find regional patterns in resistomes, and these differ between subsets corresponding to drug classes and are partly driven by taxonomic variation. The genetic environments of 49 common ARGs are highly diverse, with most common ARGs carried by multiple distinct genomic contexts globally and sometimes on plasmids. Analysis of flanking sequence revealed ARG-specific patterns of dispersal limitation and global transmission. Our data furthermore suggest certain geographies are more prone to transmission events and should receive additional attention.Peer reviewe

Editorial : From genomics to antibiotic resistance in emerging pathogens

Non peer reviewe

Antibiotikaresistens ur One Health-perspektiv - hur antibiotikaanvändning hos djur inverkar på människors hälsa

English summar

Plasmids conferring resistance to extended-spectrum beta-lactamases including a rare IncN+IncR multireplicon carrying blaCTX-M-1 in Escherichia coli recovered from migrating barnacle geese (Branta leucopsis) [version 1; peer review: 2 approved]

Peer reviewe

Risk Perceptions of Antibiotic Usage and Resistance: A Cross-Sectional Survey of Poultry Farmers in Kwara State, Nigeria

Overwhelming empirical evidence has highlighted the contribution of indiscriminate antibiotic usage (ABU) in food animals to the overall burden of antibiotic resistance (ABR) in humans, thus making antibiotic use the main selective pressure driving antibiotic resistance. The social and behavioral perspective on antibiotic use and resistance in poultry is limited. Our study therefore aimed at obtaining information on antibiotic usage, awareness of ABR, and the attitude and perceptions towards prudent antibiotic usage and ABR. A cross-sectional survey using a structured questionnaire was conducted in 125 poultry farms in Kwara state in December 2019. Most farmers (69.6%, n = 87/125) were aware of ABR and had satisfactory knowledge about ABR with a mean knowledge score of 3.2 ± 1.5. Age (older farmers; OR: 1.1, 95% CI: 1.0, 1.2) and gender (male respondents, OR: 8.5, 95% CI: 3.0, 23.9; p < 0.01) were more likely to have satisfactory knowledge of ABR. Tertiary education was significantly associated with ABR awareness (OR: 4.7; 95% CI: 0.1, 0.7; p = 0.007) and the ABR knowledge level (OR: 7.8; 95% CI: 3.3, 18.7; p < 0.01). Higher flock size was significantly associated with a satisfactory knowledge of ABR (OR: 9.5; 95% CI: 3.8, 23.6; p < 0.01). Most of the poultry farmers (68%) had positive attitudes towards prudent antibiotic use with a mean score of 2.7 ± 0.9. On the contrary, only 32.8% of poultry farmers had a desirable perception of ABR with a mean perception score of 4.9 ± 1.1. The ABR knowledge level was significantly associated with the perceptions of farmers (p < 0.05) but not their attitudes toward ABU and ABR (p = 0.083). There was evidence of unprescribed use of antibiotics in poultry and a failure to observe antibiotic withdrawal periods. These constitute a risk of exposure to unacceptable levels of drug residues from poultry products and an increased risk of ABR. Improving education and communication on antibiotic stewardship programs are crucial to prevent the looming antibiotic threat

Humans as Reservoir for Enterotoxin Gene–carrying Clostridium perfringens Type A

Humans may play a role in the transmission of gastrointestinal diseases caused by C. perfringens

Direct vertical transmission of ESBL/pAmpC-producing Escherichia coli limited in poultry production pyramid

Correction: Veterinary Microbiology 239 (2019) 100-106, article 108503Extended-spectrum beta-lactamase (ESBL) and plasmidic AmpC (pAmpC) producing Escherichia coli are found in the poultry production even without antibiotic use. The spread of these bacteria has been suggested to occur via imported parent birds, enabling transmission to production level broilers vertically via eggs. We studied transmission of ESBL/pAmpC-producing E. coli and E. coli without antibiotic selection by sampling imported parent birds (n = 450), egg surfaces prior to and after the incubation period (n = 300 and n = 428, respectively) and the laying house environment (n = 20). Samples were additionally taken from embryos (n = 422). To study the prevention of transmission, a competitive exclusion (CE) solution was added onto freshly laid eggs prior to incubation period (n = 150). Results showed carriage of ESBL/pAmpC-producing E. coil in parent birds (26.7%), the environment (5%) and egg surfaces before the incubation period (1.3%), but not from egg surfaces or embryos after the incubation period. Whole genome sequencing revealed ESBL/pAmpC-producing E. coil isolates belonging to clonal lineages ST429 and ST2040. However, the finding of E. coli cultured without antibiotic selection in two (2.2%) embryos strengthens the need to study E. coil transmission in poultry production in more depth. Since ESBL/pAmpC-producing E. coli seem not to persist on egg surfaces, there is no need to use CE solution ex ovo as a prevention method. The results indicate that other routes, such as for example transmission through fomites or horizontal gene transfer by other bacterial species, could be more important than vertical transmission in the spread of resistance in broiler production.Peer reviewe