Molecular characterization of extended-spectrum beta lactamase producing Klebsiella pneumoniae from the Stavanger region between 2003 and 2012

Master's thesis in Biological chemistryKlebsiella pneumoniae is a Gram-negative rod in the Enterobacteriaceae family, known to cause both community- and hospital-acquired infections, especially in immunocompromised patients. In the hospital setting, K. pneumoniae strains resistant to antibiotics are increasingly reported to cause infections and outbreaks. K. pneumoniae is commonly found to produce extended-spectrum β-lactamases (ESBLs), which inactivate the most important antibiotics: penicillins, third generation cephalosporins and monobactams. The most widespread ESBL enzyme among Enterobacteriaceae today is CTX-M-15, which is encoded and spread between strains by plasmids. In this project, all (n = 49) ESBLA- producing K. pneumoniae isolates isolated from clinical samples at Stavanger University Hospital between 2003 and 2012 were characterized at a molecular level. Species identification and antibiotic resistance profile was confirmed by mass spectrometry and the Vitek 2 system, respectively. ESBLA-encoding genes were identified by PCR and gene sequencing, and clonal relatedness was investigated by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmids were identified and characterized by S1-PFGE, PCR-based replicon typing and Southern blot hybridization with blaCTX-M, blaSHV and IncFIIK probes. All isolates were confirmed ESBLA - producing K. pneumoniae. Identified ESBLA encoding genes were typed as blaCTX-M-15 (n = 20), blaCTX-M-3 (n = 1), blaSHV-2 (n = 1), blaSHV-2A (n = 3), blaSHV-5 (n = 13), and blaSHV-12 (n = 5). The blaCTX-M-15 positive isolates were mainly clonally unrelated, and a common plasmid encoding blaCTX-M-15 could not be identified among these isolates. Two blaCTX-M-15 positive isolates were found to be clonally related to a strain which caused a neonatal intensive care unit outbreak at Stavanger University Hospital in 2008 – 2009. Twelve blaSHV-5 positive isolates were found to be closely related by PFGE and were typed as sequence type (ST) 29 by MLST. A ~230 kbp plasmid was found to bear blaSHV-5 in all blaSHV-5 positive ST29 isolates. The most prevalent ESBLA-encoding genes among clinical K. pneumoniae isolates from the Stavanger region were blaCTX-M-15 and blaSHV-5. blaSHV-5 positive ST29 isolates have caused sporadic infections between 2003 and 2012, and may represent an endemic clone in the Stavanger region and hospital setting. In contrast, the blaCTX-M positive isolates were associated with diverse clones and plasmids

Quinolone resistant Escherichia coli in Norwegian livestock : a comparative genomics study

The occurrence of antimicrobial resistance in Norwegian livestock is generally low. This is a consequence of good animal health and welfare, which reduces the need for antimicrobial use. Quinolones are important antimicrobial compounds both for human and animal health. An increased occurrence of resistance towards these compounds in livestock have been observed in many European countries. In Norway, the use of quinolones in livestock has been restricted. Prophylactic use of any antimicrobial is prohibited. The Norwegian monitoring programme for antimicrobial resistance in feed, food and animals (NORM‑VET) performs surveillance of antimicrobial resistance among commensal E. coli. The programme identified a low occurrence of quinolone resistance among several tested animal species. However, quinolone resistant E. coli (QREC) has been detected at low levels in a high proportion of samples from both pigs and broilers. Due to the low quinolone usage, this finding was somewhat surprising. Thus, further investigation of the origin of these bacteria was warranted. The aim of this study was to compile existing data on quinolone resistance occurrence. Furthermore, quinolone resistance mechanism characterization was performed. Relationships between the isolates were investigated by using a high resolution phylogenetic approach. The results showed a low occurrence of QREC among the included animal species. A significantly higher occurrence was observed in broilers. In silico characterization of quinolone resistance mechanisms identified chromosomal mutations as the major resistance determinant. Phylogenetic analysis of QREC provided evidence for dissemination in the broiler and pig production chains. Possible persistence of QREC was detected in the broiler production environment. Major QREC sequence types were detected among the samples from broilers. Some of these sequence types had previously been reported in other Nordic countries. Furthermore, phylogenetic analysis indicate that commensal E. coli rarely develop quinolone resistance in the broiler production environment. These results provide evidence for introduction of QREC to the Norwegian broiler production via imported breeding birds. The results highlight the importance of biosecurity measures at the top of the pyramid, to prevent dissemination of QREC.Norge har en av de laveste forekomstene av antibiotikaresistens i verden grunnet god dyrehelse og –velferd. Forbruket av kinoloner, som er svært viktige antibiotika for både dyr og mennesker, er svært lavt i Norge, og profylaktisk bruk av antibiotika er forbudt i husdyrproduksjonen. I mange andre europeiske land er det oppdaget en økende forekomst av kinolonresistente E. coli (QREC), som sannsynligvis er koblet til et økt forbruk av dette antibiotikumet. I Norge derimot viser data fra overvåkningsprogrammet for antibiotikaresistens i mikrober fra fôr, dyr og næringsmidler (NORM‑VET) en lav totalforekomst av QREC blant norske husdyr. QREC har likevel blitt detektert fra en stor andel prøver fra gris og slaktekylling, men mengden QREC i hver prøve ser ut til å være lav. Disse funnene førte til spørsmål rundt deres opphav. Målet med dette prosjektet var å sammenfatte eksisterende data på forekomst av QREC i ulike dyrearter i Norge, karakterisere kinolonresistensmekanismer i QREC stammer, samt beskrive forholdet mellom stammene ved bruk av dype fylogenetiske metoder. Resultatene viser en overordnet lav forekomst av QREC, men en signifikant høyere relativ forekomst ble observert i slaktekylling. Kromosomale mutasjoner ble identifisert som hovedmekanisme for den observerte kinolonresistensen. Fylogenetiske analyser av sekvensdataene viste en klonal spredning av QREC i både slaktekyllingproduksjonen og slaktegrisproduksjonen, og mulig persistens av QREC i miljøet der slaktekyllingene oppholder seg. Videre fylogenetisk analyse av både villtype E. coli og QREC viste at villtype E. coli i liten grad utvikler kinolonresistens i slaktekyllingproduksjonen. Resultatene viser at introduksjon av QREC fra importerte foreldredyr er hovedårsaken til den observerte forekomsten av QREC i den norske slaktekyllingproduksjonen. Disse resultatene belyser viktigheten av biosikkerhetstiltak høyere i slaktekyllingpyramiden for å hindre spredning av QREC nedover i produksjonen

ALPPACA - A tooL for Prokaryotic Phylogeny And Clustering Analysis

A tooL for Prokaryotic Phylogeny And Clustering Analysis (ALPPACA) is a pipeline that allows both de-novo and reference-based phylogenetic analysis of prokaryotic genomes. The pipeline provides a suite of analyses tailored for different scenarios, designed to allow analysis of datasets represented by three different genetic diversity levels, all in one package. These levels of similarity influence what assumptions are used to consider sequences as orthologous when reconstructing the multiple alignment required for phylogenetic inference. By selecting an appropriate track for the data at hand, the user can be confident that these assumptions are taken care of within the framework of ALPPACA

Changes in General Practitioners’ consultation frequency over time for patients with hypertension or anxiety/depression symptoms: a 10-year follow-up of the Norwegian HUNT study

Objective To investigate trends in GP consultation patterns for adults according to baseline hypertension and anxiety/depression symptoms and attribution of the GP to trend differences. Methods Prospective cohort study, linking survey data and clinical measurements from the Norwegian HUNT3 study (2006–08) with national administrative data on GP list assignment and consultations with GP services. We grouped participants aged 40–59 years according to sex and their baseline status regarding hypertension and anxiety/depression symptoms. We registered GP consultations in 2007–16 and used general estimation equation models to estimate the level of GP consultations per month per year during follow-up. We used multilevel models with participants nested in their assigned regular GP to calculate GP-level intra-class correlation coefficients, reflecting to what extent patients’ consultation patterns could be attributed to the individual GP. Results In total, 47 550 HUNT3 participants were registered with 102 different GPs in Nord-Trøndelag County, Norway, in 2007. Adjusted for age, we observed an overall increase in GP consultations in 2007–16, particularly in those with a better health status at baseline. About 2% of the variance of patient consultations could be attributed to differences between GPs and 10% to the use of lengthy consultations. Out-of-hours consultations did not change much in the study period 2007–16. Conclusion Increased use of GP consultations, mainly among the healthiest participants, encourage further research into whether these patients displace patients with heavier and more complex needs.publishedVersionPaid Open Acces

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

publishedVersio

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

Increasing workload in Norwegian general practice – a qualitative study

Background General practitioners (GPs) play a key role in securing and coordinating appropriate use of healthcare services, by providing primary and preventive healthcare and by acting as gatekeepers for secondary healthcare services. Historically, European GPs have reported high job satisfaction, attributed to high autonomy and good compatibility with family life. However, a trend of increasing workload in general practice has been seen in several European countries, including Norway, leading to recruitment problems and concerns about the well-being of both GPs and patients. This qualitative interview study with GPs and their co-workers aims to explore how they perceive and tackle their workload, and their experiences and reflections regarding explanations for and consequences of increased workload in Norwegian general practice. Methods We conducted seven focus groups and four individual interviews with GPs and their co-workers in seven GPs’ offices in Mid-Norway: three in rural locations and four in urban locations. Our study population consisted of 21 female and 12 male participants; 23 were GPs and 10 were co-workers. The interviews were analysed using systematic text condensation. Results The analysis identified three main themes: (1) Heavy and increasing workload – more trend than fluctuation?; (2) Explanations for high workload; (3) Consequences of high workload. Our findings show that both GPs and their co-workers experience heavy and increasing workload. The suggested explanations varied considerably among the GPs, but the most commonly cited reasons were legislative changes, increased bureaucracy related to documentation and management of a practice, and changes in patients’ expectations and help-seeking behaviour. Potential consequences were also perceived as varying, especially regarding consequences for patients and the healthcare system. The participants expressed concerns for the future, particularly in regards to GPs’ health and motivation, as well as the recruitment of new GPs. Conclusions This study found heavy and increasing workload in general practice in Norway. The explanations appear to be multi-faceted and many are difficult to reverse. The GPs expressed worries that they will not be able to provide the population with the expected care and services in the future.publishedVersio

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.publishedVersio

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.publishedVersio

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