Detection of Klebsiella pneumoniae in healthy poultry: Insights and perspectives from culturing and metagenomics.

Previously, Klebsiella pneumoniae was found to occur more frequently in healthy turkey flocks than in healthy broiler flocks in Norway. This study aimed to investigate whether this higher occurrence could be attributed to a greater abundance of K. pneumoniae in turkey flocks. We compared culturing, qPCR, and shotgun metagenomic sequencing for the detection and quantification of K. pneumoniae. Using qPCR, we found that 20.7% of broiler flock samples and 63.9% of turkey flock samples were positive for K. pneumoniae. Culturing revealed a significantly higher abundance of K. pneumoniae in turkey flocks compared to broiler flocks. However, metagenomic analysis showed no difference in the relative abundance of Klebsiella spp. between broiler and turkey flocks, and no correlation between the results of culturing and metagenomic quantification. Interestingly, the differential abundance of K. quasipneumoniae was significantly different between the two hosts. Our results indicate that Klebsiella spp. are present in both turkey and broiler flocks at relatively low levels but with a higher abundance in turkey flocks. Our findings also suggest that shotgun metagenomic studies targeting low-abundance taxa such as Klebsiella have poor sensitivity when comparing groups, indicating that reliance on results from metagenomic analysis without experimental validation should be done with caution.

Population dynamics and characteristics of Klebsiella pneumoniae from healthy poultry in Norway.

Klebsiella pneumoniae is an important opportunistic pathogen widely studied in relation to human infection and colonization. However, there is a lack of knowledge regarding other niches that K. pneumoniae may inhabit. K. pneumoniae isolated from healthy broiler and turkey flocks in Norway in 2018 have previously been described with regard to population structure, sequence types (STs), and the presence of virulence- and antimicrobial resistance (AMR) genes. In the present study we aimed to evaluate the dynamics of the K. pneumoniae population in poultry over time, with regards to AMR and virulence, and with a special focus on persistence of STs. A total of 391 flocks sampled in 2020 were included in the present study, of which 271 were from broiler flocks and 120 from turkey flocks. Similar to findings from 2018, the occurrence of K. pneumoniae was significantly higher based on culturing in turkey flocks (62.5%) compared to broiler flocks (24.0%). Major STs in 2020 included ST5827 (n = 7), ST37 (n = 7), ST370 (n = 7), ST17 (n = 5), and ST4710 (n = 5). Several STs persisted over time in both host species, including ST35, ST37, ST590, and ST17. This persistence may be due to local re-circulation or reintroduction from parent flocks. Of these five major STs, only ST590 carried AMR genes, indicating that the persistence was not associated with the presence of AMR genes. An ST4710 strain with a hypervirulence-encoding plasmid (p4710; iro5, iuc5) was recovered from turkeys in 2018. The same strain was present in turkeys in 2020, but the plasmid had lost the salmochelin locus. This loss may be attributed to reductive evolution due to the presence of several siderophores within the same isolates. In this study we also characterized a clinical ST4710 isolate from a turkey with airsacculitis. The isolate was closely related to two intestinal ST4710 isolates from healthy turkeys in 2018. These three isolates were sampled within the same location and time frame in 2018, and all carried the full p4710 virulence plasmid. These findings highlight the transmission- and infectious potential of ST4710 in turkeys.

Within-patient and global evolutionary dynamics of Klebsiella pneumoniae ST17.

Klebsiella pneumoniae sequence type (ST) 17 is a global problem clone that causes multidrug-resistant (MDR) hospital infections worldwide. In 2008-2009, an outbreak of MDR ST17 occurred at a neonatal intensive care unit (NICU) in Stavanger, Norway. Fifty-seven children were colonized. We observed intestinal persistence of ST17 in all of the children for up to two years after hospital discharge. Here, we investigated the within-host evolution of ST17 in 45 of those children during long-term colonization and compared the outbreak with 254 global strains. Ninety-two outbreak-related isolates were whole-genome sequenced. They had capsule locus KL25, O locus O5 and carried yersiniabactin. During within-host colonization ST17 remained stable with few single nucleotide polymorphisms, no acquisition of antimicrobial resistance (AMR) or virulence determinants, and persistent carriage of a bla CTX-M-15-encoding IncFII(K) IncFIB(K) plasmid (pKp2177_1). The global collection included ST17 from 1993 to 2020 from 34 countries, that were from human infection (41.3%), colonization (39.3%) and respiratory specimens (7.3%), from animals (9.3%), and from the environment (2.7%). We estimate that ST17 emerged mid-to-late 19th century (1859, 95 % HPD 1763-1939) and diversified through recombinations of the K and O loci to form several sublineages, with various AMR genes, virulence loci and plasmids. There was limited evidence of persistence of AMR genes in any of these lineages. A globally disseminated sublineage with KL25/O5 accounted for 52.7 % of the genomes. It included a monophyletic subclade that emerged in the mid-1980s, which comprised the Stavanger NICU outbreak and 10 genomes from three other countries, which all carried pKp2177_1. The plasmid was also observed in a KL155/OL101 subclade from the 2000s. Three clonal expansions of ST17 were identified; all were healthcare-associated and carried either yersiniabactin and/or pKp2177_1. To conclude, ST17 is globally disseminated and associated with opportunistic hospital-acquired infections. It contributes to the burden of global MDR infections, but many diverse lineages persist without acquired AMR. We hypothesize that non-human sources and human colonization may play a crucial role for severe infections in vulnerable patients, such as preterm neonates.

A molecular epidemiological study on Escherichia coli in young chicks with colibacillosis identified two possible outbreaks across farms.

Avian pathogenic Escherichia coli (APEC) is the cause of colibacillosis outbreaks in young poultry chicks, resulting in acute to peracute death. The high morbidity and mortality caused by colibacillosis results in poor animal welfare, reduced sustainability and economical loss worldwide. To advance the understanding of the molecular epidemiology, genomic relatedness and virulence traits of APEC, we performed systematic sampling from 45 confirmed colibacillosis broiler flocks with high first week mortality (FWM) during 2018-2021. From these flocks, 219 APEC isolates were whole genome sequenced (WGS) and bioinformatic analyses were performed. The bioinformatic analyses included sequence typing (ST), serotyping, detection of virulence-associated genes (VAGs) and phylogenetic analysis. Our results showed a high prevalence of ST23, ST429 and ST95 among APEC isolates from Norwegian broiler flocks, and identified ST23, ST429, ST117 and ST371 to cause disease more often alone, compared to ST95, ST69 and ST10. Phylogenetic analyses, together with associated metadata, identified two distinct outbreaks of colibacillosis across farms caused by ST429 and ST23 and gave insight into expected SNP distances within and between flocks identified with the same ST. Further, our results highlighted the need for combining two typing methods, such as serotyping and sequence typing, to better discriminate strains of APEC. Ultimately, systematic sampling of APEC from multiple birds in a flock, together with WGS as a diagnostic tool is important to identify the disease-causing APEC within a flock and to detect outbreaks of colibacillosis across farms.

Highly conserved composite transposon harbouring aerobactin iuc3 in Klebsiella pneumoniae from pigs.

Klebsiella pneumoniae is an important opportunistic pathogen associated with severe invasive disease in humans. Hypervirulent K. pneumoniae, which are K. pneumoniae with several acquired virulence determinants such as the siderophore aerobactin and others, are more prominent in countries in South and South-East Asia compared to European countries. This Klebsiella pathotype is capable of causing liver abscesses in immunocompetent persons in the community. K. pneumoniae has not been extensively studied in non-human niches. In the present study, K. pneumoniae isolated from caecal samples (n=299) from healthy fattening pigs in Norway were characterized with regard to population structure and virulence determinants. These data were compared to data from a previous study on K. pneumoniae from healthy pigs in Thailand. Lastly, an in-depth plasmid study on K. pneumoniae with aerobactin was performed. Culturing and whole-genome sequencing was applied to detect, confirm and characterize K. pneumoniae isolates. Phylogenetic analysis described the evolutionary relationship and diversity of the isolates, while virulence determinants and sequence types were detected with Kleborate. Long-read sequencing was applied to obtain the complete sequence of virulence plasmids harbouring aerobactin. A total of 48.8 % of the investigated Norwegian pig caecal samples (n=299) were positive for K. pneumoniae. Acquired virulence determinants were detected in 72.6 % of the isolates, the most prominent being aerobactin (69.2 %), all of which were iuc3. In contrast, only 4.6 % of the isolates from Thailand harboured aerobactin. The aerobactin operon was located on potentially conjugative IncFIBK/FIIK plasmids of varying sizes in isolates from both countries. A putative, highly conserved composite transposon with a mean length of 16.2 kb flanked by truncated IS3-family IS407-group insertion sequences was detected on these plasmids, harbouring the aerobactin operon as well as several genes that may confer increased fitness in mammalian hosts. This putative composite transposon was also detected in plasmids harboured by K. pneumoniae from several countries and sources, such as human clinical samples. The high occurrence of K. pneumoniae harbouring aerobactin in Norwegian pigs, taken together with international data, suggest that pigs are a reservoir for K. pneumoniae with iuc3. Truncation of the flanking ISKpn78-element suggest that the putative composite transposon has been permanently integrated into the plasmid, and that it is no longer mobilizable.

High sequence similarity between avian pathogenic E. coli isolates from individual birds and within broiler chicken flocks during colibacillosis outbreaks.

Avian pathogenic E. coli (APEC) cause high first week mortality (FWM) in broiler chickens worldwide. In order to investigate the epidemiologic aspects of colibacillosis in broiler flocks it is important to develop reliable and cost-effective sampling guidelines. In this context, it is particularly important to define the minimum number of samples required to reliably identify the causative APEC clone during outbreaks of colibacillosis. This study describes the diversity of E. coli isolates between and within three flocks with high FWM due to colibacillosis. Each flock was represented by five animals, showing typical lesions of colibacillosis, and spleen, liver and one other organ from each animal was sampled for APEC. A total of 47 E. coli isolates, one per organ, and approximately 15 isolates per flock were whole genome sequenced and compared by multilocus sequence typing (MLST), serotyping and phylogenetic analysis to deduce their relationship. The results revealed that within individual birds there was little or no sequence type (ST) or serotype diversity between APEC isolates from different organs. Based on phylogenetic analysis, isolates belonging to the same ST and serotype showed a low number of single nucleotide polymorphisms (SNPs) across more than 95 % of the genome. Isolates from the liver always represented the major disease-causing APEC in individual birds, even when more than one ST was detected within an individual bird and flock. This study guides us towards an economically efficient way of sampling for future epidemiological studies on colibacillosis, by determining the causative APEC-clone at flock level.

Closed Genome Sequences of Providencia alcalifaciens Isolates from Dogs.

Eight Providencia alcalifaciens isolates from eight different dogs in Norway with acute hemorrhagic diarrhea were sequenced. Based on Illumina and Oxford Nanopore Technologies sequencing, all of the genomes were complete and closed after hybrid assembly.

Exploring Klebsiella pneumoniae in Healthy Poultry Reveals High Genetic Diversity, Good Biofilm-Forming Abilities and Higher Prevalence in Turkeys Than Broilers.

Klebsiella pneumoniae is a well-studied human pathogen for which antimicrobial resistant and hypervirulent clones have emerged globally. K. pneumoniae is also present in a variety of environmental niches, but currently there is a lack of knowledge on the occurrence and characteristics of K. pneumoniae from non-human sources. Certain environmental niches, e.g., animals, may be associated with high K. pneumoniae abundance, and these can constitute a reservoir for further transmission of strains and genetic elements. The aim of this study was to explore and characterize K. pneumoniae from healthy broilers and turkeys. A total of 511 cecal samples (broiler n = 356, turkey n = 155), included in the Norwegian monitoring program for antimicrobial resistance (AMR) in the veterinary sector (NORM-VET) in 2018, were screened for K. pneumoniae by culturing on SCAI agar. K. pneumoniae was detected in 207 (40.5%) samples. Among the broiler samples, 25.8% were positive for K. pneumoniae, in contrast to turkey with 74.2% positive samples (p < 0.01). Antibiotic susceptibility testing was performed, in addition to investigating biofilm production. Whole genome sequencing was performed on 203 K. pneumoniae isolates, and analysis was performed utilizing comparative genomics tools. The genomes grouped into 66 sequence types (STs), with ST35, ST4710 and ST37 being the most prevalent at 13.8%, 7.4%, and 5.4%, respectively. The overall AMR occurrence was low, with only 11.3% of the isolates showing both pheno- and genotypic resistance. Genes encoding aerobactin, salmochelin or yersiniabactin were detected in 47 (23.2%) genomes. Fifteen hypervirulent genomes belonging to ST4710 and isolated from turkey were identified. These all encoded the siderophore virulence loci iuc5 and iro5 on an IncF plasmid. Isolates from both poultry species displayed good biofilm-forming abilities with an average of OD595 0.69 and 0.64. To conclude, the occurrence of K. pneumoniae in turkey was significantly higher than in broiler, indicating that turkey might be an important zoonotic reservoir for K. pneumoniae compared to broilers. Furthermore, our results show a highly diverse K. pneumoniae population in poultry, low levels of antimicrobial resistance, good biofilm-forming abilities and a novel hypervirulent ST4710 clone circulating in the turkey population.

Complete Genome Sequences of 12 Quinolone-Resistant Escherichia coli Strains Containing qnrS1 Based on Hybrid Assemblies.

In total, 12 quinolone-resistant Escherichia coli (QREC) strains containing qnrS1 were submitted to long-read sequencing using a FLO-MIN106 flow cell on a MinION device. The long reads were assembled with short reads (Illumina) and analyzed using the MOB-suite pipeline. Six of these QREC genome sequences were closed after hybrid assembly.

Actinobacillus pleuropneumoniae Eradication with Enrofloxacin May Lead to Dissemination and Long-Term Persistence of Quinolone Resistant Escherichia coli in Pig Herds.

Norway has a favourable situation with regard to health status and antimicrobial usage in the pig production sector. However, one of the major disease-causing agents in the commercial pig population is Actinobacillus pleuropneumoniae (APP). In some herds, APP eradication has been performed by using enrofloxacin in combination with a partial herd depopulation. The aim of this study was to investigate the long-term effects of a single treatment event with enrofloxacin on the occurrence of quinolone resistant Escherichia coli (QREC). The study was designed as a retrospective case/control study, where the herds were selected based on treatment history. Faecal samples were taken from sows, gilts, fattening pigs and weaners for all herds where available. A semi-quantitative culturing method was used to identify the relative quantity of QREC in the faecal samples. A significant difference in overall occurrence and relative quantity of QREC was identified between the case and control herds, as well as between each animal age group within the case/control groups. The results indicate that a single treatment event with enrofloxacin significantly increased the occurrence of QREC in the herd, even years after treatment and with no subsequent exposure to quinolones.

Comparative Genome Analyses of Wild Type- and Quinolone Resistant Escherichia coli Indicate Dissemination of QREC in the Norwegian Broiler Breeding Pyramid.

Quinolones are important antimicrobials for both humans and animals, and resistance toward these compounds is a serious threat to public health. In Norway, quinolone resistant E. coli (QREC) have been detected at low levels in a high proportion of broiler flocks, even without the use of quinolones in rearing of broilers. Due to the pyramidal structure of broiler breeding, QREC isolates may be disseminated from grandparent animals down through the pyramid. However, quinolone resistance can also develop in wild type E. coli through specific chromosomal mutations, and by horizontal acquisition of plasmid-mediated quinolone resistance genes. The goal of this study was to determine whether QREC is disseminated through the broiler breeding pyramid or developed locally at some stage in the broiler production chain. For this purpose, we whole genome sequenced wild type- and QREC isolates from broiler and parent flocks that had been isolated in the Norwegian monitoring program for antimicrobial resistance in feed, food and animals (NORM-VET) between 2006 and 2017, from 22 different production sites. The sequencing data was used for typing of the isolates, phylogenetic analysis and identification of relevant resistance mechanisms. Highly similar QREC isolates were identified within major sequence types from multiple production sites, suggesting dissemination of QREC isolates in the broiler production chain. The occurrence of potential resistance development among the WT E. coli was low, indicating that this may be a rare phenomenon in the Norwegian broiler production. The results indicate that the majority of the observed QREC at the bottom of the broiler production pyramid originates from parent or grandparent animals. These results highlight the importance of surveillance at all levels of the broiler production pyramid and of implementation of proper biosecurity measures to control dissemination of QREC.

Dissemination of Quinolone-Resistant Escherichia coli in the Norwegian Broiler and Pig Production Chains and Possible Persistence in the Broiler Production Environment.

In Norway, the use of quinolones in livestock populations is very low, and prophylactic use is prohibited. Despite this, quinolone-resistant Escherichia coli (QREC) isolates are present at low levels in several animal species. The source of these QREC isolates is unknown. The aim of this study was to characterize and compare QREC isolates from different animal species to identify putative factors that may promote the occurrence of QREC. A total of 280 QREC isolates, from broilers, pigs, red foxes, and wild birds, were whole-genome sequenced and analyzed. Well-known chromosomal and plasmid-mediated resistance mechanisms were identified. In addition, mutations in marR, marA, and rpoB causing novel amino acid substitutions in their respective proteins were detected. Phylogenetic analyses were used to determine the relationships between the isolates. Quinolone resistance mechanism patterns appeared to follow sequence type groups. Similar QREC isolates with similar resistance mechanism patterns were detected from the samples, and further phylogenetic analysis indicated close evolutionary relationships between specific isolates from different sources. This suggests the dissemination of highly similar QREC isolates between animal species and also the persistence of QREC strains within the broiler production chain. This highlights the importance of both control measures at the top of the production chain as well as biosecurity measures to avoid the further dissemination and persistence of QREC in these environments.IMPORTANCE Since antimicrobial usage is low in Norwegian animal husbandry, Norway is an ideal country to study antimicrobial resistance in the absence of selective pressure from antimicrobial usage. In particular, the usage of quinolones is very low, which makes it possible to investigate the spread and development of quinolone resistance in natural environments. Comparison of quinolone-resistant E. coli (QREC) isolates from livestock and wild animals in light of this low quinolone usage provides new insights into the development and dissemination of QREC in both natural and production environments. With this information, preventive measures may be taken to prevent further dissemination within Norwegian livestock and between other animals, thus maintaining the favorable situation in Norway.

Occurrence of quinolone resistant E. coli originating from different animal species in Norway.

The aim of this study was to describe and compare the occurrence of quinolone resistant Escherichia coli (QREC) in various animal species in relation to human population density. Data from the Norwegian monitoring programme for antimicrobial resistance in feed, food and animals from 2006 to 2016 was compiled and analysed. In total, 4568 E. coli isolates were included in this study. The isolates originated from broilers, layers, cattle, turkeys, dogs, wild birds, red foxes, reindeer, sheep, horses and pigs. Data regarding the geographical location of sampling was obtained for 4050 of these isolates and used to categorize the isolates depending on the human population density of the area. In total, 1.4% of the isolates were categorized as quinolone resistant. Compared to most European countries, there was an overall low occurrence of QREC in various animal species in Norway, though with an interspecies variation with the highest occurrence in broilers and wild birds (p < 0.05). Human population density was not associated with the occurrence of QREC. Since fluoroquinolones are not used prophylactically and in almost negligent amounts in various species in Norway, the interspecies variation in the occurrence of QREC suggests that other factors than fluoroquinolone use may be important in the development of QREC.