Antibiotic Resistance Gone Wild

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1617

Antibiotic resistance gone wild

A One Health perspective on carriage, selection and transmission of Extended-Spectrum Cephalosporinase- and Carbapenemase-producing Enterobacteriaceae

CLARA ATTERBY

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-513-0817-3 UPPSALA urn:nbn:se:uu:diva-397218 2019 Dissertation presented at Uppsala University to be publicly examined in Tripple room, Navet ground floor, BMC, Husargatan 3, Uppsala, Friday, 24 January 2020 at 09:00 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Professor Nicola Williams (Institute of Infection and Global Health in Liverpool, United Kingdom).

Abstract Atterby, C. 2019. Antibiotic resistance gone wild. A One Health perspective on carriage, selection and transmission of Extended-Spectrum Cephalosporinase- and Carbapenemase- producing Enterobacteriaceae. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1617. 79 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0817-3.

Antibiotics have saved millions of lives since they came into clinical use during the Second World War in the 1940s. Today, our effective use of antibiotics is under great threat due to emerging antibiotic resistance in bacteria. This thesis addresses the problems of antibiotic resistance from a ”One Health” perspective. The focus is on antibiotic resistant Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) in the environment and wildlife, and also considering the situation in healthy humans and livestock. In Paper I-III, high occurrence of Extended-Spectrum Beta-Lactamase (ESBL) -producing E. coli and/or K. pneumoniae was detected in fecal samples from wild birds, and the bacteria had genetic similarities to bacteria that cause disease in humans. Proximity to humans was associated with higher occurrence of cephalosporinase (ESBL and pAmpC)-producing E. coli in wild gulls. In Paper IV, ciprofloxacin resistant E. coli was enriched in the gut of mallards exposed to low concentrations of ciprofloxacin, and plasmid conjugation between E. coli bacteria readily took place. In Paper V, carbapenem resistant and blaOXA-48 harbouring- E. coli/K. pneumoniae was rare, but present in healthy humans in rural Cambodia, while cephalosporinase-producing E. coli/K. pneumoniae was common in both humans and livestock. The same ESBL/pAmpC genes were detected in humans and livestock, and exposure to animal manure and slaughter products were risk factors for fecal carriage in humans. In conclusion, wild birds can function as potential resistance reservoirs and sentinels for antibiotic resistant E. coli. Environmental pollution from humans is the primary source for antibiotic resistant Enterobacteriaceae found in wildlife, but selection for antibiotic resistant bacteria may also occur in wild birds. The results indicate that transmission of cephalosporinase- producing E. coli/K. pneumoniae occur between wildlife, humans and livestock, but more in-depth molecular work is needed to determine the mechanisms of dissemination. The high community carriage of multidrug-resistant bacteria in rural Cambodia is worrying and highlights Southeast Asia as a hotspot for antibiotic resistance. Antibiotic resistance surveillance is biased towards high-income countries and research should be focused more on low- and middle-income countries, and also include the important “One Health” perspective.

Keywords: Antibiotic resistance, E. coli, K. pneumoniae, ESBL, AmpC, Carbapenemase, ciprofloxacin resistance, colistin resistance, rural, wildlife, birds, sub-MIC, MSC, Cambodia, environment, epidemiology

Clara Atterby, Department of Medical Sciences, Akademiska sjukhuset, Uppsala University, SE-75185 Uppsala, Sweden.

ISSN 1651-6206 ISBN 978-91-513-0817-3 urn:nbn:se:uu:diva-397218 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-397218)

One World – One Health

Main supervisor:

Associate professor Josef Järhult, Department of Medical Sciences, Uppsala University, Sweden

Co-supervisors:

Doctor Stefan Börjesson, Department of Animal Health and Antimicrobial Strategies, National Veterinary Institute, Uppsala and Department of Clinical and Experimental Medicine, Linköping University, Sweden

Professor Björn Olsen, Department of Medical Sciences, Uppsala University, Sweden

Opponent:

Professor Nicola Williams, Institute of Infection and Global Health, University of Liverpool, United Kingdom

Examining committee:

Associate professor Marie Sjölund, Department of Animal Health and Antimicrobial Strategies, National Veterinary Institute, Sweden

Professor Diarmaid Hughes, Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden

Professor Anita Hällgren, Department of Clinical and Experimental Medicine, Linköping University, Sweden

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Atterby, C., Börjesson, S., Ny, S., Järhult, J. D., Byfors, S., & Bon- nedahl, J. (2017). ESBL-producing Escherichia coli in Swedish gulls-A case of environmental pollution from humans? PloS One, 12(12).

II Hessman, J., Atterby, C., Olsen, B., Järhult, J. (2018). High Preva- lence and Temporal Variation of Extended Spectrum β-Lactamase- Producing Bacteria in Urban Swedish Mallards. Microbial Drug Re- sistance, 24:6, 822-829.

III Atterby, C., Ramey, A. M., Hall, G. G., Järhult, J., Börjesson, S., & Bonnedahl, J. (2016). Increased prevalence of antibiotic-resistant E. coli in gulls sampled in Southcentral Alaska is associated with urban environments. Infection Ecology & Epidemiology, 6, 32334.

IV Atterby, C., Nykvist. M., Lustig, U., Andersson, D., Järhult, J., Sandegren, L. Spread of resistance plasmids and selection of resistant bacteria among Mallards exposed to sub-inhibitory concentrations of antibiotics in their water environment. Manuscript.

V Atterby, C., Osbjer, K., Tepper, V, Rajala, E., Hernandez, J., Seng, S., Holl, D., Bonnedahl, J., Börjesson, S., Magnusson, U., Järhult, J. (2019). Carriage of carbapenemase‐ and extended‐spectrum cephalosporinase‐producing Escherichia coli and Klebsiella pneumoniae in humans and livestock in rural Cambodia; gender and age dif-

ferences and detection of blaOXA‐48 in humans. Zoonoses and Public Health, 66: 603– 617.

Reprints were made with permission from the respective publishers.

Contents

Introduction ...... 11 Bacteria and antibiotic resistance ...... 11 Escherichia coli and Klebsiella pneumoniae ...... 11 Factors that drive the emergence and transmission of resistance ...... 12 Plasmids ...... 13 β-lactams: mode of action and mechanisms of resistance ...... 14 Colistin: mode of action and resistance mechanisms ...... 16 Fluoroquinolones: mode of action and resistance mechanisms ...... 17 One Health ...... 17 Antibiotic resistance from a One Health perspective ...... 18 Extended spectrum cephalosporinase- and carbapenemase producing Enterobacteriaceae ...... 18 In humans ...... 19 In livestock ...... 20 In wildlife ...... 21 Antibiotic residue and antibiotic resistant bacteria in nature ...... 22 Selection for antibiotic resistance in nature ...... 23 Antibiotic resistance and zoonotic disease in Southeast Asia ...... 23 The situation in Cambodia ...... 24 Aims ...... 26 Methods ...... 27 Study design, sampling methods and ethical permits ...... 27 Questionnaires (Paper V) ...... 31 Multiple regression model (Paper V) ...... 32 Method of ESCE/K and CPE/K screening (Paper I-III and V) ...... 32 Sample collection and storage ...... 32 Bacterial culturing and species identification ...... 32 Phenotypic and genotyping characterization ...... 34 Mallard experiment (Paper IV) ...... 34 Construction of experimental bacterial strains ...... 34 Experimental setup ...... 35 Transconjugant screening ...... 37 Whole genome sequencing ...... 38

Results and discussion ...... 39 Resistance gone wild ...... 39 Wild birds as carriers of ESCE/K (Papers I-III) ...... 39 Wild birds and transmission of antibiotic resistant bacteria (Paper I-III) ...... 40 Wild birds and selection of antibiotic resistant bacteria (Paper IV) .... 43 Conjugation of plasmids in mallards (Paper IV) ...... 46 Climate and antibiotic resistance (Paper II) ...... 48 Antibiotic resistance in rural communities (Paper V) ...... 50 Carriage of ESCE/K and CPE/K in Cambodia ...... 50 Dissemination of ESCE/K, CPE/K and colistin resistance genes ...... 53 Main conclusions ...... 58 Future perspectives ...... 59 Emergence and transmission in the environment ...... 59 Zoonotic transmission ...... 59 Environmental contamination ...... 60 AMR in low and middle income countries ...... 60 Populärvetenskaplig sammanfattning ...... 61 Acknowledgments ...... 63 References ...... 65

Abbreviations

AMR Antimicrobial Resistance Cephalosporinase ESBL and AmpC CPE Carbapenemase-producing E. coli CPK Carbapenemase-producing K. pneumoniae ESBL Extended-Spectrum β-Lactamase ESCE Extended-Spectrum Cephalosporinase- producing E. coli ESCK Extended-Spectrum Cephalosporinase- producing K. pneumoniae MLST Multi Locus Sequence Types MIC Minimal Inhibitory Concentration pAmpC Plasmid mediated AmpC WGS Whole Genome Sequencing

Introduction

Bacteria and antibiotic resistance Ever since antibiotics came into clinical use in the 1940s, these drugs have been our most effective therapeutic option against bacterial diseases. Despite the invaluable importance of antibiotics, our future use of antibiotics is under threat. An unfailing observation is that resistance to all antibiotics emerges sooner or later, and that the discovery of new antibiotic classes has been proven very difficult (1). The increasing threat of antibiotic resistant bacteria cannot be exaggerated, as millions of lives are at stake every year if we are unable to control and prevent future development and spread of antibiotic resistance (2).

Many antibiotic resistant bacteria and antibiotic resistance genes have emerged in nature and occur naturally in the environment (1). In addition, large amounts of antibiotic resistant bacteria and antibiotic resistance genes are being released in the environment through contamination of human and animal faeces (3). It has been proposed that antibiotic resistant bacteria are capable of persisting, spreading and evolving in the environment, and that antibiotic resistance genes can be passed on to other bacterial populations present in the environment (3). Wildlife can be colonized by antibiotic resistant bacteria and serve as potential infectious sources and reservoirs for such bacteria (4). It is therefore believed that antibiotic resistant bacteria and antibiotic resistance genes that occur in the environment pose a serious risk for human health.

Antibiotic resistance is a multi-factorial problem that needs to be addressed from many different angles. This thesis will approach the AMR field from a “One Health” perspective. The focus will be on antibiotic resistant Esche- richia coli and Klebsiella pneumoniae in the environment and wildlife, and also considering the situation of antibiotic resistant Escherichia coli and Klebsiella pneumoniae in humans and livestock.

Escherichia coli and Klebsiella pneumoniae Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are Gram-negative, rod-shaped, facultative anaerobic bacterial species belonging to the family Enterobacteriaceae. Facultative anaerobes constitute approxi-

11 mately 0.1 % of the human gut microbiota and both E. coli and K. pneumoniae are members of the normal commensal flora in the gut of humans and animals, and can cause opportunistic infections (5, 6). Klebsiella spp. are ubiquitous in nature and can be found in environmental sources such as soil, surface water and plants (7). E. coli can be divided in three pathotypes; commensals that lack virulence factors and are apathogenic, extra-intestinal E. coli that can cause invasive infections like urinary tract infections and bloodstream infections, and pathogenic intestinal E. coli e.g. EHEC and Shi- gella that cause diarrheal disease (8). E. coli is the most common Gram- negative rod isolated from patients with sepsis, and responsible for more than 80 % of all community-acquired UTIs, as well as many hospital- acquired infections (6). K. pneumoniae is associated with hospital-acquired lobar pneumoniae, and can also cause wound, soft tissue and urinary tract infections (6). K. pneumoniae mainly cause disease in immunocompromised patients, and because of the ability of K. pneumoniae to spread rapidly in a hospital environment, these bacteria tend to cause nosocomial outbreaks (9).

Factors that drive the emergence and transmission of resistance Antibiotic resistance is defined as the ability of a bacterium to resist an antibiotic that would normally kill them or stop their growth. Resistance to antibiotics is achieved through four main strategies: i) reduction of membrane permeability to antibiotics; ii) inactivation of antibiotics; iii) active efflux of the antibiotic; and iv) mutation of cellular targets (10). Most antibiotics are a product of nature, and antibiotic resistance is a naturally occurring phenom- enon in microorganisms (11), meaning that bacteria have had time to evolve and fine-tune resistance mechanisms for millions of years. Mutations and horizontal gene transfer effectively give rise to new bacterial genotypes, which can spread in the larger population through adaptive evolution or random genetic drift (12). Bacteria have a short generation time, large population size and a high rate of spontaneous mutations, which enables fast and effective adaptive evolution (13). The effectiveness of which a wild-type E. coli can evolve from a susceptible strain to a highly resistant strain has been demonstrated in a simple but remarkable experiment by scientists from the Kishony Lab at Harvard Medical School in Boston (14). A very large agar plate with increasing concentration of the antibiotic trimethoprim towards the middle of the plate was constructed and inoculated with a wild-type E. coli, Figure 1. In only 11 days, the susceptible wild-type E. coli strain evolved to express phenotypic resistance to trimethoprim at a concentration 3,000-fold higher than the original minimal inhibitory concentration (MIC) (14).

Figure 1. Different E. coli linages (each represented by a separate colour), which all originates from a wild-type E. coli (blue colour) and grows on a “Mega-Plate” Petri Dish with increasing concentration of trimethoprim towards the middle. Right to reprint Elsevier (14).

The evolutionary trajectories of antibiotic resistance are complex and dependent on several factors including the mutation rate of bacteria, the level of resistance conferred by the resistance mechanism, the fitness of the resistant mutant and the strength of the selective pressure (15). The strong selective pressure exerted by centuries of misused and overused commercial antibiotics in humans, animals and agriculture have accelerated the development of antibiotic resistance in pathogenic bacteria (16). Horizontal gene transfer (HGT) is the movement of genetic information between organisms, and is crucial for the spread of antibiotic resistance (17). Through the mechanism of HGT, antibiotic resistance genes can be disseminated throughout an entire population of bacterial pathogens and spread to new bacterial species (13). HGT occurs by three genetic mechanisms; the up-take of free DNA (natural transformation), the transfer of bacterial DNA between a bacteriophage-infected bacterium and a bacteriophage-susceptible bacterium (trans- duction) and transfer of mobile genetic elements by pili structures assembled by two adjacently located bacteria (conjugation) (12). In Enterobacteriaceae, the conjugation of plasmids plays a major role for the spread of antibiotic resistance genes (18).

Plasmids Plasmids are circular extra chromosomal double- stranded DNA elements that replicate autonomously in the cytoplasm of the bacterium. Plasmids are inherited vertically (mother to daughter cell), but many plasmids can also be transferred horizontally through conjugation. They often contain non- essential genes that provide an advantage for the bacterium, for example

13 genes that confer antibiotic resistance (18). The antibiotic resistance genes are often enclosed by transposable elements that enable incorporation, rear- rangement and mobility of the resistance genes. Conjugation of plasmids is likely the main mechanism for acquired antibiotic resistance in E. coli and K. pneumoniae (13). Plasmids can be grouped based on their replicon type which separates them into different incompatibility groups (19).

β-lactams: mode of action and mechanisms of resistance β-lactam antibiotics (penicillins, cephalosporins, monobactams and carbapenems) are very important drugs due to their effective and broad mode of action on different bacterial pathogens combined with their low toxicity in humans and animals (20). WHO has classified the cephalosporins (3rd and higher generations) as critically important antimicrobials for human medicine (21). β-lactam antibiotics acts by binding to penicillin binding proteins (PBP), which inhibits the bacterial cell wall synthesis (20). The most important resistance mechanism in bacteria is the production of β-lactamases that hydrolyses β-lactamase antibiotics (22). β-Lactamases are ancient enzymes, whose origin can be traced back millions, or sometimes billions of years (23). The group β-lactamases consists of approximately 2800 unique proteins, and were evolved in microorganisms to protect them from naturally occurring β-lactams in the environment (23). β-lactamases are classified biochemically into two groups; serine- β-lactamases and metallo- β- lactamases, according to the mechanism by which they hydrolyse β-lactam antibiotics. The molecular classes A, B, C and D were later established as sequence analyses revealed four major molecular differentiations for key β- lactamases, and serine- β-lactamases were subdivided as A, C or D while metallo- β-lactamases were classified as B. The four molecular classes were further subdivided based on functional capability related to substrate and inhibitor profiles into 17 functional groups (23). The classic Extended- Spectrum β-lactamases, or ESBLs, are found in the functional group 2be, denoting ability to degrade penicillin, extended-spectrum cephalosporins and inhibition by clavulanic acid (24). Some ESBL-genes originated through different point mutations that alter the amino acid configuration around the active sites in naturally occurring β-lactamase genes e.g. TEM-1, OXA-1 and SHV-1, resulting in the emergence of numerous extended-spectrum β- lactamase genes e.g. TEM-10, OXA-12 and SHV-28 in potential pathogenic bacteria like E. coli and K. pneumoniae. Other ESBL-genes emerged in non- pathogenic bacteria, and were horizontally transferred to pathogenic bacteria, the most important example being the CTX-M ESBLs that are believed to originate from environmental bacteria belonging to the genus Kluyvera (23). CTX-M-type ESBLs have spread into a global pandemic and now rep- resent the major ESBL-genes found in the clinic, the community and the environment (25).

14 AmpC β-lactamases are found in the molecular class C and functional group 1, and they are characterized by their ability to hydrolyse most penicillins, extended-spectrum cephalosporins and showing resistance to clavulanic acid (23). AmpC β-lactamases are encoded on the chromosome of many bacteria belonging to the family Enterobacteriaceae, and chromosomal AmpC was actually the first β-lactamase reported in literature in 1940 (26). They are inducible in many bacteria and mutation can lead to overexpression, which leads to resistance to broad-spectrum cephalosporins like cefotaxime and ceftazidime (27). AmpC genes have been transferred to transmissible plasmids and can thus appear in bacteria lacking or poorly expressing chromosomal AmpC, such as E. coli and K. pneumoniae (27). Plasmid-mediated AmpC (pAmpC) enzymes are generally harder to detect, broader in spectrum and less common compared to plasmid-mediated ESBL enzymes, but certain pAmpC enzymes are prevalent in livestock and humans in some regions. Differences in amino acid sequenced have given rise to different pAmpC families, e.g FOX, MOX, DHA, MIR, ACT, LAT, ACC, and, the most commonly encountered pAmpC family called CMY (27). In this thesis, ESBLs and pAmpCs will be collectively referred to as extended-spectrum cephalosporinases (ESC).

Carbapenemases belonging to three molecular classes have become clinically significant: class A (KPC), class B (metallo-β-lactamases such as IMP, VIM, NDM), and class D (OXA enzymes) (28-30). KPC, IMP, VIM, NDM and OXA-48, make up the so called ‘big five’ carbapenemase families that cause the greatest clinical concern due to their ability to hydrolyse carbapenems (31). Carbapenems are beta-lactam antibiotics with the broadest activity and they are used for critically ill patients as last-line antibiotics (31). KPC, IMP, VIM and NDM enzymes are able to hydrolyse penicillins, broad-spectrum cephalosporins and carbapenems. While KPCs are often inhibited by clavulanic acid, IMPs, VIMs and NDMs are not (32). NDM-1 genes are often located on plasmids that harbour multiple other resistance genes, making most NDM-1 producers only susceptible to a handful of antibiotics like tigecycline, colistin and fosfomycin (32). The resistance profile of OXA-48 producers is peculiar as they only weakly hydrolyse some broad- spectrum cephalosporins and carbapenems, making them more difficult to detect (32). Antibiotic resistance in OXA-48 producers increases when additional resistance mechanisms like ESBLs or permeability defects are present in the bacteria (32).

Multiple bacteria in the family of Enterobacteriaceae can produce ESC/carbapenemases but the most commonly isolated producers worldwide are E. coli and K. pneumoniae (33). ESC-producing E. coli (ESCE) and ESC-producing K. pneumoniae (ESCK) are at the top-four list of causative agents in infections with antibiotic resistant bacteria in Europe, both in terms of number of cases and number of deaths (2). Carbapenemase-producing E.

15 coli (CPE) and carbapenemase producing K. pneumoniae (CPK) is a growing concern, and CPK have reached endemic levels in some countries, Greece being one example. The European Antimicrobial Resistance Surveil- lance Network reported in 2016 that more than 50 % of K. pneumoniae isolated from bloodstream infections and meningitis in Greece are non- susceptible to carbapenems (34). It has been proposed that the successful emergence of ESC and carbapenemase genes in Gram-negative bacteria is due to the genes’ association to transferrable genetic elements like plasmids, transposons and integrons (35). It is also important to acknowledge that the dominance of some ESC genes (e.g. blaCTX-M-15) is greatly influenced by a few highly successful clones of bacteria (e.g. ST131 blaCTX-M-15 harbouring E. coli clones) that have spread globally like a pandemic (36). The ESC and carbapenemase genes travel together with resistance factors for other antibiotic classes, and sometimes resistance to non-antibiotic molecules like heavy metals, leading to the enrichment of multidrug resistant bacteria in response to a variety of different selective pressures (10). ESCs are commonly associated to plasmids belonging to the incompatibility groups IncF, IncI1, IncK and IncN, and carbapenemases are often associated to IncA/C, IncL/M, IncF, IncI1 and IncN (37).

Colistin: mode of action and resistance mechanisms Colistin is a polypeptide antibiotic belonging to the group polymyxins, con- sisting of 5 chemically different compounds (polymyxin A-E), out of which only polymyxin B and polymyxin E (colistin) have been used in clinical practice (38). The outer cell membrane in Gram-negative bacteria is the site of action for Polymyxin B and colistin (39). These drugs bind to the lipid A component of the lipopolysaccharide (LPS) and act to displace divalent cati- ons (Calcium and Magnesium), thereby disrupting the outer membrane and triggering the release of phospholipids, which results in cell wall leakage and cell death (39). Polymyxins were discovered many decades ago (39). They are effective against Enterobacteriaceae, and therefore particularly interesting due to the limited number of effective antibiotics towards such bacteria, but has been used very limited in human medicine due to kidney toxicity (39). Patients with cystic fibrosis that suffer from recurrent, severe lower respiratory tract infections are often treated with colistin through inhalation (40), and polymyxin B have been extensively used as treatment for ear infections through topical administration (41). Colistin can also be administrated intravenously. In later years, colistin has been considered a last line drug to treat multidrug-resistant infections caused by Gram-negative bacteria such as K. pneumoniae and Pseudomonas aeruginosa, and should be preserved for such purposes (39). However, since the 1960s and still today, colistin is used for prevention, treatment and growth promotion in animals (42). A variety of gene mutations can cause resistance to colistin through different modifica- tions of the LPS in the outer cell membrane (43). The recent emergence of

16 transferrable plasmid-mediated colistin resistance (mcr-1-9 genes) is very worrying, especially since colistin resistance genes and other antibiotic resistance genes sometimes co-exist on the same plasmid (42-44). Livestock has been proposed to be the main reservoir for colistin resistance (45), and thus, the One Health aspect is particularly important.

Fluoroquinolones: mode of action and resistance mechanisms The mode of action of fluoroquinolones is by inhibition of DNA synthesis in bacteria. Fluoroquinolones bind to DNA gyrase and topoisomerase IV, which are enzymes responsible for supercoiling and relaxation of bacterial DNA during DNA replication (46). The use of fluoroquinolones in the clinic is extensive and their effectiveness are very important as they serve as the first line drug for several infections, e.g. febrile urinary tract infections. Re- sistance to fluoroquinolones is mediated through chromosomal mutations in genes encoding the subunits of the DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE), as well as mutations in genes that affect the expression of diffusion channels and efflux pumps (46). DNA gyrase is the primary fluoroquinolone target in Gram-negative bacteria. High-level fluoroquinolone resistance in E. coli commonly arise due to stepwise accumulation of spontaneous mutations in gyrA and subsequent amino acid substitutions such as the change of serine at position 83 to tryptophan (gyrA S83L genotype) or aspartic acid at position 87 to tyrosine (gyrA D87Y genotype) (47). Plasmid-mediated fluoroquinolone resistance was discovered in the late 1990s, and since then, several plasmid associated qnr genes have been detected (48). The mechanism of resistance is mediated through i) protection of the cellular targets DNA gyrase and topoisomerase IV through amino acid substitutions, ii) acetylation of quinolones and iii) enhanced efflux pumps (48). Plasmid-mediated fluoroquinolone resistance genes confer only low- level resistance but makes infections harder to treat and also allow for selection of higher-level resistance (48).

One Health “One Health” is a concept that acknowledges that humans, animals and the environment are inextricably linked. One application of “One Health” is intersectional work in infectious medicine between veterinarians, physicians, molecular biologists, ecologists and environmental chemists. Historically, the collaborations between veterinarians and physicians when it comes to infectious disease and public health have been very important. For example, the term “vaccine” is derived from the Latin word “vacca”, meaning cow, and relates back to the first vaccine developed by physician Jenner in the 18th century (49). Observations had been made that milkmaids who had fallen ill with cowpox would never suffer from smallpox thereafter, and this led to the

17 development of the first vaccine from dried pus extracted from cowpox vesi- cles (49). Dr Theobald Smith and Dr Kilbourne are another great example of collaborative work between a physician and veterinarian that led to the discovery that insects could transmit infectious disease (50). Unfortunately, as medicine became increasingly specialized, the important collaborations between physicians and veterinarians have been largely lost (51).

Antibiotic resistance from a One Health perspective Resistant bacteria arising in humans, animals or the environment do not rec- ognize geographic or species borders and may spread between different environments and between countries (52). Antimicrobials used to treat various infectious diseases in animals or used as growth promoters in livestock are often identical to those used for humans. The World Health Organization acknowledged the need for a One Health approach when dealing with the problem of antimicrobial resistance, as stated in the Global Action Plan on AMR (53). In response, the Global Antimicrobial Resistance system (GLASS) was developed (54). However, the One Health aspect is largely missing as the global surveillance is biased towards samples from humans, while animal surveillance (in many regions) and environmental surveillance (in most regions) is lacking. Studies on carriage, selection and transmission of antibiotic resistant bacteria need to have a “One Health” approach in order to prevent further spread and development of important pathogens and antibiotic resistance genes.

Extended spectrum cephalosporinase- and carbapenemase producing Enterobacteriaceae Antimicrobial resistance in commensal flora is a serious threat because a very highly populated ecosystem, such as the gut, may function as a reservoir for AMR. The antibiotic resistant bacteria may cause invasive infections, spread to other hosts, or transfer mobile genetic elements to other bacteria in the gut (55). In fact, positive faecal culture for ESBL-producing En- terobacteriaceae is a risk factor for subsequent invasive infections caused by such bacteria (56, 57) Thus, community carriage can function as a reservoir for antibiotic resistant bacteria, although far from all carriers develop infection. Further, the presence of antibiotic resistant bacteria in the gut is an environmental threat, as it has been shown that faecal pollution is a major con- tributor to the abundance of antibiotic resistance genes in anthropogenically impacted environments (58)

18 In humans In human medicine, the first clinical isolates of ESC-producing Enterobacte- riaceae appeared in the 1980s (59) and blaSHV and blaTEM were the first rec- ognized ESBL-genes (60). Initial outbreaks were restricted to European hospital environments such as intensive care units (25), but in the 1990s, ESC- associated community acquired urinary tract infections appeared (61). In the 2000s, it was discovered that healthy humans could carry community acquired ESBL-producing bacteria in the gut, and at the same time, the blaCTX- M genes became increasingly common. Since then, the spread of blaCTX-M- genes have evolved into a global pandemic, causing challenges to patients and clinicians worldwide (25). The level of faecal community carriage of ESC-producing Enterobacteriaceae varies dramatically globally, reports ranging from 5 % in Sweden and Norway (62, 63) to 68 % in a community in Thailand (64).

Since the first carbapenemase producer (CP) in Enterobacteriaceae (NmcA producer) was identified in 1993 (65), a large variety of CPs have been detected (66). The first KPC producer was identified in the United States in 1996 (67), and within a few years, KPC had spread globally and outbreaks from South America, Asia and Europe were described (32). KPC production has mainly been reported from K. pneumoniae isolates and especially one K. pneumoniae clone (ST-258) has been highly successful and spread globally (68). Although KPC producers often differ from each other by MLST, plasmid type and number, and additional beta-lactamase mechanisms, the blaKPC genes are always associated with a single genetic element (transposon Tn4401) (68). Community-acquired KPC producers have been rarely reported (32). Since the first class B metallo-beta-lactamase (MBL) IMP-1 was reported from Japan in 1991 (69), infections caused by MBLs have been described worldwide, often caused by hospital acquired K. pneumoniae (32). In some countries like Greece, Taiwan and Japan, VIM- and IMP-type enzymes have reached endemic levels (32). The important class B MBL-gene blaNDM-1 was first detected in 2008 in Sweden (70). The patient had been previously hospitalized in India and had contracted a K. pneumoniae infection, and in addition, the patient had an NDM-1 positive E. coli in his gut flora (70). Following this finding, an active search for NDM-1 positive En- terobacteriaceae was initiated, and unfortunately, the blaNDM-1 gene proved to be common in Enterobacteriaceae in hospitals in India and Pakistan (71). This exemplifies how novel antibiotic resistance can emerge and spread in silence, and highlights the importance of continuous active infection surveillance. NDM-1 genes are of particular public health concern due to their ability to disseminate to many bacterial species and the environment, and NDM- 1 has been identified in the pandemic E. coli ST131 clone as a source of community acquired infection (32). The most common class D carbapenemase is the OXA-48 enzyme, and several OXA-48 clones have been identified world-wide (32). The low level of resistance conveyed by OXA-

19 48 producers likely has resulted in an underestimation of their prevalence, as they are often not detected through regular screening of CPs (32).

In livestock In livestock, carriage of ESC-Enterobacteriaceae has been a concern since it was first detected in the 2000s (72). ESCE/K have been detected in several farm animals like cattle, pigs and poultry (72-74). In several studies, poultry stands out as being most frequently colonized with ESCE and prevalence rates of 40-100 % have been reported in Europe (75-77). Also in Swedish poultry, prevalence rates were high (34 %) when selective screening was first implemented in the surveillance program in 2010 (78). Detection of CPE/K from livestock such as pigs, dairy cows and poultry has been reported in EU although the prevalence appears to be low. A mandatory monitoring for ESCE and CPE was initiated by EFSA (European Food Safety Agen- cy) and performed in 23 EU member states in 2015. The ceacal content of 6,167 pigs, 2,347 calves and 10,679 meat samples resulted in high prevalence of ESBL producers (31.9 % and 36.8 % in pigs and calves, respectively), and lower for pAmpC producers (9.75 % and 4.8 % in pigs and calves, respectively) (31). Only two CPEs were identified, one from a German pig and one from pig meat in Belgium (31). Prevalence studies of CPE/K in some non-EU countries have revealed more worrying results. In Egypt, a study on broiler chickens reared in different farms found CPK from 15 % of broilers and 6 % of water samples (79). In China, approximately 50 % of randomly picked E. coli (using non-selective plates) from faecal samples of diseased chicken carried colistin resistance gene mcr-1 and/or carbapenemase gene blaNDM, and all investigated E. coli (n=78) were highly resistant to third generation cephalosporins (80). Selective pressure by antibiotics used in livestock, as well as transmission of CP from human sources, are the likely explanations for the occurrence of CPs in livestock (31). Food- producing animals, especially chicken, have been proposed as resistance gene reservoirs (80), and there is a concern that meat and milk could become a source of CPs for the consumers, especially in countries were infections with CP are more prevalent (31).

There is a conflict between medicine/public health and agriculture when it comes to the use of antimicrobials in livestock. Medical and public health leaders argue that the use of nontherapeutic antibiotic in livestock is a public health risk, but agricultural leaders insists that the benefits outweigh the risks as successful intensive agriculture is dependent on antibiotics (51). Sweden is a country with a relatively prudent antibiotic use and has historically been a leading country in acknowledging antibiotic resistance and promote pre- ventions. Swedish veterinarians were very early to voice concerns about the growing use of antibiotics in agriculture and already in 1986, Sweden became the first country to stop the use of antibiotic as growth promoters in animal feed (81). Swedish veterinarians believed that antibiotic supplements

20 was used to compensate for a lacking animal welfare such as appropriate housing, handling and care for food producing animals. Also, consumers in Sweden were strongly opposed the use of antibiotics as growth promoters (51). The focus on disease prevention and correct use of antimicrobials led to a 50 % reduction in antibacterial drug use in animals. This showed that it is possible to successfully rear poultry, pigs and calved without antibiotics as growth promoters (81). In 2006, an EU-wide ban on the use of antibiotics as growth promoters entered into effect (82). Still today, livestock worldwide are exposed to large quantities of antibiotics, and outside the EU, antibiotics are being used as growth promoters (51). In fact, 50-70 % of all antibiotics sold in most countries of the world are used for livestock (83).

In wildlife In the last decade, antibiotic resistant bacteria in natural reservoirs and wildlife have received increasing interest from the scientific community. The potential risk posed by antibiotic resistant bacterial colonization of wildlife and the ensuing contamination of the environment has been acknowledged (84). It is believed that the majority of emerging infectious diseases in humans have a wildlife reservoir (85) and the potential transfer of antibiotic resistant bacteria from wildlife/environment to crops, humans and domestic animals should not be neglected (84).

Since ESBL-producing E. coli was first detected in wild birds in Portugal in 2006 (86), ESC- producing Enterobacteriaceae have been isolated from a wide range of bird species across the world (4, 87). The highest prevalence of antibiotic resistant bacteria has been recorded in birds of prey and in aquatic-associated species (4, 87). As of today, carbapenem resistance in wild animals is still rare, but the emergence of NDM-1 and IMP carbapenemases in wild birds is of great concern (88, 89). Wild birds have been proposed as sentinels, reservoirs, and spreaders of antibiotic resistance and they appear to be one of the main wildlife hosts for antibiotic resistant En- terobacteriaceae (87). Many birds are migratory, and flocks of birds meet during breeding and over-wintering, which can allow for the dissemination of antibiotic resistant bacteria between birds from different regions. Several gull species are opportunistic feeders and are able to forage on anthropogenic food resources, such as domestic refuse or landfills. High availability to anthropogenic food resources has been associated to larger gull populations, probably due to decreased winter mortality and increased reproductive suc- cess (90). It has been postulated that ESC-producing bacteria in nature is mostly due to a spillover effect from human waste, human faeces and farm manure (4, 58, 91, 92). Anthropogenic inputs into the local environment, or a relative lack thereof, influence the prevalence of antibiotic resistant bacteria among wildlife inhabiting that area (93-99). Nevertheless, birds in areas with limited human presence have also been found to host antibiotic resistance (100, 101). Animal experiments performed by our group showed that ESBL-

21 producing E. coli can be maintained in the intestine of mallards for at least a month in absence of antibiotic selective pressure, and be transmitted to other mallards, which would allow long distance migration and possible spread to new areas and other birds (102). The carriage and transmission of antibiotic resistant bacteria in wild birds is evident, but the magnitude of resistance development and selection that take place in nature remains poorly defined.

Antibiotic residue and antibiotic resistant bacteria in nature Antibiotics and antibiotic resistant bacteria, originating from wastewater treatment plants, hospital and pharmaceutical industry effluents, surface water effluents, aquaculture facilities and animal husbandry facilities are released in large amounts in natural ecosystems (3, 103). Some environments are more contaminated than others, such as sewage waters, soils treated with manure and water contaminated with pharmaceutical effluents (104, 105). Varying levels of antibiotics have been reported in aqueous environmental matrices worldwide (106, 107). Just as the amounts of antibiotics released in the environment differs markedly from site to site, the degradation rates of antibiotics are also different depending on temperature, mois- ture, chemical composition of the environment (i.e. pH), and the microbiota that contribute to biodegration (105). Some antibiotics, such as fluoroquinolones are chemically very stable and can as a result accumulate and persist in the environment (108). As an example, ciprofloxacin concentrations are generally detected at lower levels between 0-100ng/ml in Europe, but higher concentrations can also be found (106). In two European rivers, fluoroquinolones concentrations were measured at 19ng/ml in Switzerland (109) and ranging between 20-2745ng/ml in Poland (110). India has been highlighted as a country with unusually high degree of antibiotic pollution after Larsson and colleagues (2007) published an alarming report that revealed that ciprofloxacin levels up to 31,000 ug/L had been measured in water effluent from pharmaceutical companies, exceeding the MIC of many bacteria by over 1000-fold (111). In more pristine Indian waters, levels of ciprofloxacin ranging between 12-2500ng/ml were measured in rivers and 2800ng/ml was measured in a lake (112).

ESC-producing bacteria have been repeatedly detected in the environmental waters. Fluoroquinolone-resistant-ST131 E. coli harbouring CTX-M-14 was detected in a river in the UK (113) and ESBL-producing K. pneumoniae and E. coli were detected in municipal wastewater treatment plant effluents in the Czech Republic (114). CMY harbouring E. coli were detected on recreational beaches and in private drinking water in Canada (115), as well as in the Han River in Korea (116). CP-bacteria have also been reported from environmental samples. NDM-1 harbouring bacteria was detected in drinking water in New Delhi, India, which raised immense concerns (117). Other reports include CPK in samples from rivers in Switzerland (118) and Tunisia

22 (119), and IMP-harbouring Enterobacter cloacae and Enterobacter asburiae from US rivers (120).

Selection for antibiotic resistance in nature It has been speculated that antibiotic resistant bacteria could be enriched in the outer environment due to contaminating antibiotics (105). The hypothesis is that antibiotic pressure on the environmental microbiota results in selection for antibiotic-resistant mutants and favours the acquisition of antibiotic resistance determinants by gene-transfer elements such as conjugation of plasmids (105). A recent publication suggest that environmental selection of antibiotic resistant bacteria is only relevant in environments polluted by very high levels of antibiotics (58). However, previous in vitro studies showed that sub-MIC levels of antibiotics can enrich for pre-existing resistant strains (121-124) as well as select high-level resistant mutants de novo from a susceptible strain (121-124). Studies of sub-MIC exposure to antibiotics to determine the MSC in vivo are lacking. The extent of AMR selection that takes place in nature is unclear, and the importance of environmental antibiotic contamination when it comes to resistance development and maintenance in wildlife needs further research.

Antibiotic resistance and zoonotic disease in Southeast Asia Southeast Asia has been suggested as the region in the world with the highest risk of emergence and spread of antibiotic resistance (125). The following factors related to the high emergence, selection and transmission in Southeast Asia were identified by Chereau and colleagues; indiscriminate use of antibiotics in humans and animals, the release of antibiotics and antibiotic resistant bacteria in the environment, poor hospital hygiene, poor food hygiene and inadequate sanitation systems, Figure 2 (125). The context is crucial to the problem of emergence and spread of AMR (126), and only by understanding the context and local challenges can we address the causes and solutions regarding AMR. Looking at the food hygiene aspect, an estimated 35 % of the population in Southeast Asia are exposed to faeces contaminated drinking water, with higher contamination in rural areas (125). Another aspect is the limited access to effective antibiotics in poor communities, which can be a much bigger public health threat than excessive and unnecessary use of antibiotics (127).

Figure 2. The emergence and spread of antibiotic resistant bacteria in Southeast Asia (125). The right to reprint was obtained from the corresponding author.

The situation in Cambodia Cambodia is a tropical country in Southeast Asia that is bordered by Thai- land, Lao PDR, Vietnam and the Gulf of Thailand. Cambodia is the home of 16 million people, out of which 77 % live in rural areas (2018) (128). Fol-

24 lowing more than two decades of strong economic growth, Cambodia has attained the lower middle-income status (129). Poverty continues to fall in Cambodia, but the vast majority that has now escaped poverty did so by a small margin (129). Health and education remain important challenges for Cambodia. Malnutrition in children is a major public health concern and 32 % children are estimated to be stunted (below the median height-for-age) (130). The life expectancy for females is 67.9 years and 62.7 years for males, resulting in place 182 out of 223 at country comparison (highest lowest number of years) (128). In rural areas, 31 % of the population are exposed to unsafe drinking water and 70 % of the population has unsafe sanitation access (e.g. toilet not piped to a sewer system or no toilets) (128). The risk of acquiring a major infectious disease such as bacterial diarrhoea, hepatitis A, malaria or dengue fever is considered to be very high (2016)(128). The la- bour force is overwhelmingly agricultural-oriented and keeping livestock is deeply embedded in society and customs in Cambodia (131). Most rural households keep livestock, mainly pigs, cattle, chicken, ducks, cattle and water buffalo in traditional backyard farming (131). The average farmer is a smallholder and keeps enough livestock to meet household consumption needs and minor cash expenses, although market-oriented intensive, large- scale commercial production also exists (131).

Non-prescription antibiotic use is a common practice in the Cambodian community (132). Unregulated sale of antibiotics and the absence of national documentation of antibiotic sales make the situation regarding antibiotic use in Cambodia difficult to overview. However, studies have shown that Cam- bodian physicians are lacking knowledge of local antibiotic resistance patterns and also use old treatment regimens for common disease such as respiratory infections, urinary tract infections and diarrhoea (133). Physicians were also concerned regarding the sale of substandard and falsified drugs (133), which are common problems in countries that lack proper legislation on manufacturing and distribution of pharmaceutical products. Interviews with Cambodian patients have revealed that pervasive antibiotic misuse was driven by a habitual supplier-seeking behaviour and that self-medication with a drug-cocktail was widespread and included broad-spectrum antibiotics for mild illness (134).

There are limited reports regarding the prevalence of ESCE/K and CPE/K in Cambodia, but results from recently published studies reveal that Enterobac- teriaceae resistant to third-generation cephalosporins appear common both in patients and asymptomatic community carriers, while carbapenem resistant isolates are rare (135, 136).

25 Aims

The overall aim of the thesis was to investigate carriage, transmission and selection of extended spectrum cephalosporinase- and carbapenemase- producing E. coli and K. pneumoniae (ESCE/K and CPE/K) from a One Health perspective, focusing on the environment, wild birds, domestic animals and humans.

Specific aims were to:

• Determine the occurrence and characterization of ESCE/K in gulls and mallards

• Investigate if carriage of antibiotic resistant E. coli in gulls is associated to urban environments

• Compare the occurrence and characteristics of ESCE/K isolates in wild gulls to isolates from other sectors of society in Sweden

• Determine if there is a temporal variation in the occurrence of ESCE/K in urban mallards

• Determine the minimal selective concentration (MSC) of ciprofloxacin for a ciprofloxacin resistant E. coli strain in vivo, using a mallard model

• Investigate the characteristics and frequency of plasmid conjugation events in vivo, using a mallard model

• Determine the occurrence and characteristics of ESCE/K and CPE/K in humans and animals in rural Cambodia

• Identify risk factors associated with faecal carriage of ESCE/K in rural Cambodia

26 Methods

Study design, sampling methods and ethical permits Paper I: This was a cross-sectional study of ESCE in gulls in urban Sweden. The timing and location of sample collection was coordinated with another Swedish project (“ESBL-bildande E. coli i vår omgivning – livsmedel som spridningsväg till människa”) by three governmental institutions; Swedish Veterinary Institute, the Swedish Food Health Agency and the Public Health Agency in Sweden. The studies aimed to determine the occurrence and over- laps of ESBL/pAmpC-producing E. coli in Sweden, Figure 3.

Figure 3. Samples from different sectors in Sweden were collected for isolation and characterization of ESCE (137). The original picture has been edited and the right to reprint was obtained from the authors.

27 The prevalence and characteristics of ESCE was investigated in different sectors in Sweden; i) Swedish livestock; ii) imported and domestic meat; iii) intestinal colonization of Swedish healthy community carriers; iv) bloodstream isolates from Swedish patients; v) environment (urban gulls, paper I) and vi) sewage water.

Faecal samples were collected from gulls inhabiting the two Swedish cities Malmö and Gothenburg. Samples were collected from freshly deposited faeces from a variety of gulls (Larus marinus, Larus argentatus, Larus canus and Croicocephalus ridibundus). Gulls were considered an appropriate indicator for ESBL-producing E. coli in wildlife due to previous screening results, and their frequent contact with anthropogenic impacted environments such as landfills and urban environments. The target number of samples collected in paper I (n=97 in Malmö and n=74 in Gothenburg) was based on previous experience and the estimated ESCE prevalence of 10-30 %, but no sample size calculation was made. The study did not require ethical permits.

Paper II: This was an observational study of ESCE/K in mallards in urban Uppsala. The longitudinal study design was chosen to see the potential seasonal variation in ESCE/K carriage. Mallard were chosen due to their all- year presence in Svandammen, and because we assumed that it would be possible to trap them. We aimed to trap and tag mallards to enable individual follow-up. But trapping mallards proved surprisingly difficult and had to be disregarded early in the project. The population in Svandammen is probably not fixed throughout the year, but more likely consists of three populations; i) an overwintering population (November-March); ii) a small non-breeding population during summer (July-June), and iii) an autumn population con- sisting of young mallards and moulting adult mallards (July-October). The longitudinal aspect could be applied within each of those three time-periods, but not for the whole year. The sample collection site was chosen due to its urban location and proximity to the University Hospital in Uppsala, and the presence of mallards throughout the year. The target number of samples (n=20-100/sampling occasion, and total n=813) was based on an estimated ESCE prevalence of 10-30 %. The variation per sampling occasion was due to problems with catching birds, and at some events limited by numbers of mallards present at the site. Ethical approval for trapping birds was obtained from the Uppsala Animal Ethical Committee (Reference Number C228/12).

Paper III: This was a cross-sectional study of antibiotic resistant E. coli in gulls from one remote and one urban site in Alaska. Alaska is an interesting area to study as large parts are unaffected by mankind and agriculture. Large groups of migratory birds from North America, Southeast Asia, South Amer- ica and Australia gather in Alaska to overwinter, and thus, Alaska could serve as a centre for AMR dissemination through migratory birds. The two

28 collection sites were chosen due to the remote location of Middleton Island and the urban location of the Kenai Peninsula, and the presence of gulls at both sites. The hypothesis was that gulls in remote Middleton Island would be far less colonized by antibiotic resistant E. coli, as compared to gulls in urban Kenai Peninsula. The target number of samples (n=80 from Middleton Island and n=80 from Kenai Peninsula) was estimated to give enough power to detect the potential difference in occurrence of antibiotic resistant E. coli between the two sites, but no sample size calculation was made. The study did not require ethical permits.

Paper IV: This was an experimental study of selection and plasmid conjugation of fluoroquinolone resistant E. coli in an in vivo model. The mallard model was previously used by our group to study Influenza and Campylo- bacter, and set up at a BSL 2 facility at the Animal House at the National Veterinary Institute in Uppsala. Mallards were chosen as a wild bird representative that reside in aquatic environments and can carry multidrug resistant bacteria in a wildlife setting. The number of mallards used in the experiment (n=5/group in gyrA(D87Y)-experiment and n=6/group in gy- rA(S83L)-experiment) was limited by ethical and practical considerations, and was not based on a power calculation. The animals were kept at the laboratory in accordance with ethical guidelines and approvals from Uppsala Ethical Committee on Animal Experiments (C201/11, C125/12 and C63/13)

Paper V: This was a cross-sectional study of ESCE/K and CPE/K in humans and livestock in rural Cambodia. The sample collection had been previously performed for a project on Influenza and Campylobacter in Cambo- dia by collaborators at the Swedish Agricultural University. Ten villages were selected for inclusion with the specific selection criteria: the village had to be situated within 5 km from a main road; it had to have various species of livestock; and there had to be interactions between humans, domestic animals and wildlife. Within each village, purposive samples were selected from the 10 households keeping as many different livestock species as possible. Simple random sampling of villages and households would have been optimal from a statistical perspective, but was impractical given the logisti- cal arrangements for sample collection and transport. The target number of samples collected was based on sample size calculation for expected influenza and Campylobacter prevalence in humans and livestock. The study population consisted of smallholder livestock producers with small-scale backyard/garden production. Pigs were generally kept in confinement areas, while poultry and ruminants were allowed to roam free in the village. More details regarding living conditions, socio-economic status and zoonosis awareness amongst the study population in Kampong Cham, Cambodia can be read elsewhere (131). Ethical approval (43 NECHR, 8th April 2011) was obtained prior to the survey from the National Ethics Committee for Health Research, Ministry of Health, Cambodia, and an advisory ethical statement

29 (Dnr 2011/63) was obtained from the Regional Board for Research Ethics in Uppsala, Sweden. All procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as re- vised in 2008.

Figure 4. Sampling collection from water buffalo in Cambodia. Dr. Kristina Osbjer provided the photo.

Figure 5. Free-range poultry in Cambodia. Dr. Kristina Osbjer provided the photo.

Figure 6. Pigs kept in confinements. Dr. Kristina Osbjer provided the photo.

30 Questionnaires (Paper V) The female head of the household was interviewed using a questionnaire. In practice, however, it was difficult to exclude men during the interviews, as illustrated in figure 7. The questions that were relevant to this study were related to household practices (Table 1), meat consumption and zoonosis awareness. The interviews were carried out in Khmer language, and subsequently translated by two independent translators and compared for con- sistency. Measures were taken to reduce bias in the interviews, such as questionnaire pre-testing, validation questions and double translation. Around half of the households allowed livestock access to sleeping and food preparation areas, facilitated by open housing construction among the poorer households. Home-slaughter was reported in 76 % of households. The threat of zoonosis was not a concern for the majority of households, and few respond- ents considered disease transmission between livestock, humans and wildlife to be likely in their village.

Table 1. Self-reported household practices in the 100 Cambodian households interviewed Which of these practices do you employ in this household? Percentage Slaughter domestic animals 76 Allow animals in sleeping and food preparation areas 57 Consume unsafe water (untreated well or pond water) 36 Wash hands with soap after handling live animals 29 Bury or burn meat waste products 21 Collect manure indoors and outdoors daily 20 Wash hands with soap before and after cooking 15 Consume undercooked meat 7

Figure 7. Interviewing one of the households in Cambodia. Dr. Kristina Osbjer provided the photo.

31 Multiple regression model (Paper V) Statistical analysis was performed in SAS for Windows 9.3 (SAS Institute Inc.). The eight potential risk factors (Table 1) were screened using univari- able logistic regression and selected for multivariable logistic regression if p < 0.2. A multivariable logistic regression model was used to investigate the association between faecal carriage of ESCE/K and potential risk or pro- tective factors at individual level. Manual backward elimination was used until all remaining variables showed a p ≤ 0.05. The model was investigated for interactions between all included variables in the final model. The statistical models had three levels of nested factors in the hierarchy, where each person sampled was clustered within households that were clustered within villages. All variables in the model were categorical except for the continuous variable meat consumption.

Method of ESCE/K and CPE/K screening (Paper I-III and V) Sample collection and storage Faecal samples were collected from animals/humans in all papers, and placed in vials with bacterial freeze medium. In paper II, a sub-set of samples (n=173) were collected and analysed in duplicates, which let to an increase of ESCE/K positive samples by a factor of 1.3 (17 % to 22 %). Sam- ples from paper I-III were frozen once before culturing. At the January sampling in paper II, an additional sample replicate was collected for culturing without previous freezing (n=20). The non-frozen samples generated six ESBL-producing isolates, while the corresponding previously frozen replicates only yielded two ESBL-producing isolates. In paper V, all livestock samples were frozen once before culturing, while 60 % of human samples had been previously frozen-thawn-frozen before inclusion in this project. The previous thawing of samples probably influenced the detection rate of ESCE/K and CPE/K as bacteria often undergo lysis when they are thawed. All frozen samples were stored in -80 degrees Celsius. In paper III and V, shipment of samples to Sweden was performed on dry ice by a couri- er company. Samples in paper I, II and III were stored for maximum six months prior to culturing, while samples in paper V were stored for four years prior to culturing. Prolonged storage time may have influenced the detection rate of ESCE/K and CPE/K in paper V.

Bacterial culturing and species identification Different techniques for bacterial isolation and identification were used in the projects.

32 In paper I and II, faecal samples were enriched in Brain Heart infusion (BHI) broth with 16 mg/L Vancomycin and subsequently cultured on chromID ESBL bacterial plates (BioMérieux). In paper III, faecal samples were enriched in two Luria-Bertani (LB) broth containing cefpodoxime (vial 1) and ertapenem (vial 2) and subsequently cultured on MacConkey agar plates with cefpodoxime and ertapenem discs. In paper V, faecal samples were enriched in buffered peptone water and subsequently cultured on three agar plates: chromID OXA-48, chromID CARBA and CHROMagar C3G. Enrichment of faecal samples after storage in -80 degrees is necessary to enable representative bacterial recovery. BHI-broth is a highly nutritious broth, and Vancomycin was added to inhibit over-growth of Gram-positive bacteria. LB broth is another nutritious broth, commonly used in over-night culturing of bacteria. Buffered peptone water is recommended as enrichment broth by EFSA (European food safety agency) for the screening of ESCE/K and CPE/K in food. Buffered peptone water entails the necessary nutrients for bacterial growth but is less nutritious compared to BHI and LB. The more highly nutritious broths will increase the clonal expansion of bacteria and lead to higher sensitivity, but there is a concern that this might lead to plasmid conjugation in vitro during enrichment and lead to false positive results, according to EFSA scientific report from 2011 (138). The bacterial agar plates chromID ESBL, chromID OXA-48, chromID CARBA and CHROMagar C3G are all supplemented with selective chromogenic agar that allow for relatively easy phenotypic detection of different members of the Enterobacteriaceae family. For example, E. coli that produces beta- glucoronidase appears pink on the chromogenic plates, figure 8.

Figure 8. The typical pink appearance of an pAmpC-producing E. coli on the CHROMagar C3G plate. Photo by Clara Atterby.

The bacterial plate chromID ESBL (BioMérieux) is selective for ESBL- producing Enterobacteriaceae. Unpublished results have shown that chromID ESBL plates are sub-optimal for the screening of AmpC-producing Enterobacteriaceae, which probably is due to an AmpC-inhibitor incorporated in the plate but the manufacturer does not make the list of contents available. The CHROMagar C3G is more suitable for screening of both ESBL- and AmpC-producing Enterobacteriaceae, and therefore part of our

33 current ESCE/K screening process. ChromID OXA-48 was used due to its selective screening for OXA-48-producers, which might go undetected if only chromID CARBA plates are in use. Species identity of presumptive E. coli and K. pneumoniae was assessed using matrix-assisted laser desorption ionization-time of flight mass spectrometer (MALDI-TOF) in paper I, III and V. MALDI-TOF is a very fast and user-friendly technique, as compared to the more time-consuming traditional typing method through biochemistry tests used in paper II.

Phenotypic and genotyping characterization In paper I-III, the ESBL phenotype was confirmed by preparing the isolates on bacterial plates to asses the inhibition of bacterial growth around specific antibiotic discs. Phenotypic confirmation prior to molecular testing is the traditional approach in ESBL-screening. In paper V, the isolates were subjected to molecular characterization without prior phenotypic confirmation. The vast majority of E. coli/K.pneumoniae isolates that grew on CHRO- Magar C3G were found to harbour ESBL/pAmpC-genes. Confirmation of the ESBL phenotype was only performed in isolates that were negative in all multiplex-PCRs targeting ESBL and pAmpC genes. If the isolates expressed AmpC phenotype, the likely resistance mechanism was considered to be an over-expression of chromosomal resistance mechanisms, and those isolates were not further included. Our approach in paper V saved some time and did not cause a lot of unnecessary molecular work. Susceptibility to antibiotics was assessed through different techniques; disk diffusion, microdilution and E-tests. Genotypic characterization was performed using specific sin- gleplex- and multiplex- PCRs and subsequent Sanger sequencing to determine the MLST (paper I) and ESBL/pAmpC/Carbapenemase resistance genes (paper I-III and V). PCR screening for colistin resistance mcr genes was performed in paper II and V. In paper I, plasmid transfer was assessed in a subset of randomly selected isolates by electroporation. All trans- formants positive for an ESBL gene were tested for plasmid replicon type, and IncI1 plasmids were subjected to plasmid MLST.

Mallard experiment (Paper IV) Construction of experimental bacterial strains Extended Spectrum Beta-Lactamase (ESBL)-producing E. coli used in the experiment (SP132 and SP163) were originally isolated from Yellow-legged gulls (Larus michaelis) in Spain 2009 (139). The strains were selected based on their wild bird origin and different phenotypic traits that would make them easily distinguishable from each other and the normal gut flora of the mallards. SP132 is cefotaxime resistant due to ESBL-gene blaCTX-M-1 and

34 SP163 is tetracycline resistant due to plasmid bound tetA. SP132 is susceptible to streptomycin and ciprofloxacin in its wildtype form. Isogenic pairs of streptomycin and streptomycin + ciprofloxacin resistant mutants from SP132 were selected by picking spontaneous streptomycin resistant mutants on LB- agar plates supplemented with streptomycin and LA-agar plates supplemented with ciprofloxacin. Mutants were screened with PCR targeting the rpsL and gyrA genes. This procedure was performed twice, resulting in two isogenic pairs with different gyrA mutations. The pairs were named DA26187 (rpsL K43T) and DA26192 (rpsL K43T/gyrAD87Y) (referred to as Gy- rAwt/GyrAD87Y) and DA28261 (rpsL K43R) and DA28400 (rpsL K43R/gyrAS83L) (referred to as GyrAwt/GyrAS83L). To determine the fitness cost of the gyrA mutations the strains were competed without antibiotics, and for measurement of minimum selective concentration (MSC) the strains were competed in the presence of different sub-MIC concentrations of ciprofloxacin. In the absence of ciprofloxacin, the GyrAD87Y mutant was outcompeted by GyrAwt due to the cost of the gyrA mutation that was determined to 0,6 % per generation, but at 1 ng/ml and above it was enriched and the MSC was estimated to 0,6 ng/ml. The gyrAS83L mutation barely had any cost with only 0,06 % mean fitness cost per generation in the absence of antibiotic, in accordance with previously published data (122). The low cost of the gyrA S83L mutation makes it hard to differ from neutrality with the method used here, and the reliability of the MSC become low. However, previous studies using more sensitive methods to measure fitness cost (122) has determined the MSC for this particular mutation to 0,1 ng/ml in accordance with our positive selection of the mutant at concentrations of 0,25 ng/ml ciprofloxacin and above. For plasmid conjugation studies between bacteria in the gut of the mallards, strain SP163 containing a tetA gene on a conjugative plasmid was included and named DA26200.

Experimental setup Our aim was to study if exposure to sub-MIC levels of ciprofloxacin enrich- es for ciprofloxacin resistant E. coli and affects the plasmid conjugation in vivo. We wanted to mimic a wildlife situation where wild mallards are carrying ciprofloxacin resistant E. coli in their intestines, and at the same time are being exposed to low concentrations of ciprofloxacin present in the environment. Male mallards (Anas platyrhynchos) of minimum 10 weeks and maximum 6 months were included in the experiment. They were purchased from a commercial breeder one day post-hatched and bred in closed facilities at the Animal House at the National Veterinary Institute in Uppsala. As they were introduced in the experiments, they were separated from their flock and moved to a smaller experimental room where they could roam freely and have ad libitum access to water and feed. Their faeces were screened for the presence of E. coli resistant to cefotaxime, tetracycline and ciprofloxacin prior to inclusion in the experiment. Their sole water source was a 170 L

35 pool used for drinking and swimming. Faeces were excreted in the pool during swimming. Different concentrations of ciprofloxacin were added to the water, which was changed daily. Ciprofloxacin was our drug of choice because of its importance and vast clinical use, as well as its chemical stability that enables ciprofloxacin to be transmitted to, and remain in the environment in its active form. Paper V consists of two experiments with two different setups and different bacterial strains (Figure 9).

gyrAwt/gyrAD87Y 0 13710 Days post infection Inoculation

WATER + CIPROFLOXACIN Inoculated birds † Transmission birds

Fecal sample obtained DA 26187 and DA 26192

gyrAwt/gyrAS83L 0 4h 4h 1 1.5 2 2.5 3 3.5 5 7 10

WATER + WATER + WATER + CIPROFLOXACIN CIPROFLOXACIN CIPROFLOXACIN †

DA 28261 and + sacrifies DA 26200 DA 28400 index birds

Figure 9. Experimental setup in paper V. Isogenic E. coli strains (ciprofloxacin resistant and ciprofloxacin sensitive) were inoculated in mallards exposed to different concentrations of ciprofloxacin in two experiments, gyrAwt/gyrAD87Y and gy- rAwt/gyrAS83L. Faecal samples were obtained and cultured, and the ratio of the ciprofloxacin resistant:ciprofloxacin sensitive strains was calculated. In the gy- rAwt/gyrAS83L experiment, a third genetically distinct E. coli strain was introduced after 4 hours to study conjugation of plasmids.

The GyrAwt/GyrAD87Y experiment comprised four exposures with ciprofloxacin, each with five different mallards and a concentration of ciprofloxacin in the water of 0 ng/ml, 10 ng/ml, 20 ng/ml and 32 ng/ml, respectively. This experiment studied selective pressure of ciprofloxacin. Each mallard was inoculated intra-esophageally with 1 mL 1:1mix of DA26187 and DA26192 containing approximately 1010 colony forming units (CFU) of each strain. Faecal samples were obtained at 1, 3, 7 and 10 dpi. The experiments were interrupted on day 10.

The GyrAwt/GyrAS83L experiment comprised four exposures with ciprofloxacin, each with six different mallards and ciprofloxacin concentrations of 0 ng/ml, 1 ng/ml, 5 ng/ml and 20 ng/ml respectively. This experiment studied selective pressure of ciprofloxacin and conjugation of plasmids. Three of the birds (index birds) were inoculated with 1 mL 1:1mix of DA28261 and

36 DA28400 containing approximately 1010 colony forming units (CFU) of each strain, and placed in the experimental room together with three other birds (transmission birds). After four hours, the index birds were inoculated by a third, genetically different strain DA26200 (cefotaximeS, ciproflox- acinR, tetracyclineR). The index birds were euthanized 24 hours after the inoculation and only the three transmission birds remained in the experiment. Faecal samples were obtained twice daily the first four days, and once daily at 5, 7 and 10 dpi. The experiments were interrupted on day 10. The setup with index and transmission birds was previously used to study transmission of ESBL-producing E. coli (102).

For analysis, faecal samples were plated on selective Eosin Methylene-Blue Lactose (EMBL) plates supplemented with 500μg/ml streptomycin. 50 colonies from each streptomycin plate were patched to a 0.04μg/ml ciprofloxacin EMBL plate and the cipR and cipS colonies were counted. A linear regression analysis was made for the average log ratio of CipR:CipS strains in each group and plotted as a function of the number of days of growth at each concentration of antibiotic. The slope of each linear curve determined the selection coefficient of each ciprofloxacin concentration.

The number of birds that could be included in the experiments was restricted due to ethical considerations and practical limitations. The birds in each ciprofloxacin concentration group roamed freely in the same room and shared the same drinking/swimming pool, and thus, shared bacteria amongst each other. As we have to assume that selection for the resistant mutant is the result of the enrichment in the group as a whole, each group must be considered as only one biological replicate. This led to decreased power, and more uncertainty in the measured selection coefficients. Future experiments should consider using several separate groups of birds.

Transconjugant screening In the GyrAwt/GyrAS83L experiment, faecal samples were plated on an additional EMBL agar plate to detect putative transconjugants. The SP132 deri- vates were resistant to cefotaxime and susceptible to tetracycline, while the DA26200 strain was susceptible to cefotaxime and resistant to tetracycline. By plating faecal samples on EMBL-plates containing cefotaxime (10 ug/ml) and tetracycline (10 μg/ml), putative transconjugants could be identified. Before the GyrAwt/GyrAS83L experiment started, some of the birds excreted tetracycline resistant E. coli in their feaces. These were susceptible to ciprofloxacin, cefotaxime and streptomycin and could be differentiated from the experimental strains through these phenotypic traits. Putative transconjugants were categorized based on what group (0, 1, 5 or 20 ng/ml ciprofloxacin exposure) they were detected in, resistance profiles, PCR targeting regions on the SP163/SP132 plasmids and the tetA gene from the SP163 plas-

37 mid. One isolate from each category was whole genome sequenced and analysed regarding gMLSR, resistance genes and plasmid replicons.

Whole genome sequencing Total bacterial DNA was prepared with the Epicentre MasterPure DNA Puri- fication Kit (Illumina) according to the manufacturer’s instructions. Whole genome sequencing was performed on an Illumina MiSeq using the Nextera XT Library Prep Kit (Illumina) and sequenced with paired-end read length of 100bp. Sequences were analysed with CLC Genomics workbench v12 (Qiagen) by de novo assembly and contigs were analysed for resistance genes, plasmid replicons and virulence genes by the ResFinder (140), Plasmid- Finder (141) and VirulenceFinder (142) applications.

38 Results and discussion

Resistance gone wild Wild birds as carriers of ESCE/K (Papers I-III) Main conclusions - Wild birds are frequent carriers of ESCE/K - Wild birds can function as a resistance reservoir for ESCE/K - Wild birds can function as sentinels for antibiotic resistance present in the community

Main results The presence and characterization of ESCE in wild birds were studied in paper I, II and III. In paper II, ESCK was also included. In paper I (n=170), we found a 17 % occurrence of ESBL-producing E. coli in gulls inhabiting the two Swedish cities Malmö and Gothenburg in 2013. In paper II (n=813), the occurrence of ESBL-producing E. coli and ESBL-producing K. pneumoniae in mallards residing in an urban pond in Uppsala was 47 % and 3 % respectively. In paper III (n=160), the occurrence of ESCE in gulls residing in an urban site Kenai Peninsula in Alaska was 16 %, while no ESCE was detected in gulls residing in the remote site Middleton Island, Alaska. No carbapenem-resistant isolates were detected in paper I-III.

Discussion The ESBL-producing E. coli occurrence of 17 % in Swedish urban gulls (paper I) is slightly higher than the reported occurrence in gulls sampled in Stockholm in 2010, 9 % (143), and similar to the level reported from gulls in the Swedish city Hudiksvall in 2009, 21 % (139). These results indicate that the colonization level of ESBL-producing E. coli in the Swedish gull population is at relatively stable levels during the last few years, although direct comparisons must be made with caution due to small sample sizes (n=200- 300), and variation in sampling location and to some extent species of gulls. The ESBL-producing E. coli occurrence of 47 % in Swedish urban mallards (paper II) is considerably higher compared to the previously reported presence in Swedish wild birds (139, 144, 145), and from ducks in Europe (146- 148). Interestingly, the faecal carriage occurrence of ESBL-producing E. coli is about three times higher in Swedish gulls (17 %), and 10 times higher in Swedish mallards (47 %), compared to the latest report on community car-

39 riage in Swedish healthy humans (5%) (62). The relatively high colonization in Swedish wild birds suggests that wildlife could serve as a resistance reservoir. ESCE/K in wild birds was first detected in Alaska in 2010, when high occurrence (37 %, n=150) was detected in faecal samples from glaucous winged gulls in Barrow, Alaska (149). E. coli isolates mainly carried blaCTX- M-14 or blaTEM-19, whereas K. pneumoniae isolates mainly carried blaCTX-M- 15, blaSHV-12, or blaSHV-102. This was surprising, as a previous study from 2005 had revealed no ESCE in gulls from the same area (150). Our results from paper III showed a 17 % occurrence of ESCE in gulls from urban Kenai Peninsula, while no ESCE was detected in the remote area Middleton Island. The occurrence from previously mentioned studies cannot be directly compared to our work as there are differences in sampling locations and screening methods, but our results emphasizes the introduction and maintenance of ESCE in wild birds in Alaska. No CPE/K were detected in Barrow or our paper III study from Kenai Peninsula, Alaska. However, a recent study found seven E. coli isolates positive for the carbapenemase genes blaKPC-2 or blaOXA-48 from 1000 faecal samples from gulls in Alaska (151). Although the prevalence of carbapenem-resistance in gulls was low in the previously mentioned study, the result was still surprising since only four imported cases of carbapenem resistance in human infections has been reported in Alaska since the surveillance started in 2013 (152). Similar to the situation in Alaska, another study found that 40 % of gulls (n=120) sampled in a single nesting colony in Australia carried IMP-4-group metallo-beta-lactamase producing Enterobacteriaceae, meanwhile, the prevalence of CP bacteria in human clinical samples was below 1 % (89). The source of resistant isolates was most likely human refuse from a nearby landfill, which was used as food supply by the gulls during breeding season (89). These studies show that wild birds, especially those living in urban environments, could function as sentinels for antibiotic resistance in the community.

Wild birds and transmission of antibiotic resistant bacteria (Paper I-III) Main conclusions - Environmental pollution from humans is the main source for antibiotic resistant Enterobacteriaceae found in urban gulls and mallards

Main results Results from paper I and II showed that the genetic characteristics of the ESBL-producing E. coli population in Swedish wild birds and Swedish humans are similar. Paper I was conducted in conjunction with a large study on ESBL-producing E. coli in Sweden (153), which allowed direct comparison between our gull isolates with isolates collected at the same time period 2010-2013 from bloodstream infections, healthy community carriers, food producing animals and surface water. The same type of ESBL-plasmids and

40 ESBL-genes were found in gulls, bloodstream infections and healthy carriers, Figure 10. The majority of isolates from gulls were of the same ST-type as isolates from healthy human carriers and bloodstream infections, Figure 11.

Figure 10. Frequency of overlapping ESBL genes (A) and plasmid replicon types (B) in Swedish gulls (paper I), community carriers, bloodstream infections, poultry, pigs/calves ((153)) and surface waters (154)

Figure 11. Frequency of multi-locus sequence types (MLST) in ESBL producing E. coli isolates from Swedish gulls (current study), community carriers, bloodstream infections, poultry, pigs/calves (153) and surface waters (154)

ESBL-plasmids of IncF- and IncI1-type and ESBL-genes blaCTX-M-15 and blaCTX-M-14 were the most common in isolates from gulls, bloodstream infections and healthy human carriers. On the contrary, isolates from gulls dif- fered to isolates from food-producing poultry in terms of ESBL-genes and ST-types. In poultry, blaCTX-M-1 gene was dominating. There was no evidence of clonal overlap, i.e. no isolate from gull had the same combination of ST- type, plasmid-type and ESBL-gene as any isolate from human. In paper II, the ESBL-gene of ESBL-producing E. coli and K. pneumoniae isolates in mallards was determined, but not the ST-type or plasmid-type. Identical to isolates from gulls, bloodstream infections and healthy human carriers, the

41 most common ESBL-gene in Swedish urban mallards were blaCTX-M-15 and blaCTX-M-14.

Faecal samples in paper III were collected from two sites, urban Kenai Pen- insula and remote Middleton Island. The Kenai Peninsula is a landmass ex- tending south of Anchorage, Alaska, the most populous city in the state. Middleton Island is located 115 km offshore from the mainland and is unin- habited by humans. Of the E. coli isolates recovered from gulls habituating in the urban site, 55 % were resistant to at least one of the tested antibiotics, as compared to 8 % of the isolates recovered from gulls in the remote site, and 22 % of E. coli isolates from the urban site were classified as multi-drug resistant (resistant to three or more antibiotic classes), as opposed to 2 % from the remote site. From the urban site, eight ESBL- or pAmpC-producing E. coli were detected in gull faeces, and found to harbour blaCTX-M-15 or blaC- MY-2 genes, as compared to zero ESBL/pAmpC-producing E. coli isolates from the remote site.

Discussion The similar genetic characteristics indicate dissemination between the human community and wildlife, and the introduction of ESBL-producing E. coli in Swedish gulls and mallards are likely due to environmental pollution from humans. Although the characteristics of ESBL-producing E. coli was similar between gulls and humans, the lack of clonal overlap between the sectors in paper I indicates that the spread of resistance elements between wildlife and human is mainly through horizontal gene transfer rather than clonal spread. The very high occurrence of ESBL-producing E. coli and K. pneumoniae in Swedish urban mallards (paper II) is most likely due to the habitat of the mallards. The pond “Svandammen” where the faecal samples were obtained is located in urban Uppsala, within 200 meters from the Upp- sala University Hospital and close to Uppsala wastewater treatment plant. The pond is frequently inhabited by other bird species such as gulls. It allows frequent interactions between humans and mallards, as it is common that people visit the pond to feed the ducks. On the same note, our results from paper III show that increased prevalence of antibiotic-resistant E. coli in gulls in Alaska was associated with urban environments. In concordance with our conclusion, the increase in antibiotic resistance as a direct effect of anthropogenic activities were shown in other studies on Arctic birds (155), Baboons (97) and a variety of wild and domestic birds from urban and rural areas (156). Wildlife exposure to human waste on landfills has been suggested as a major anthropogenic-induced driver for antibiotic resistant bacteria in wild birds (99, 152). Another potential source of antibiotic resistance is wildlife exposure to urban wastewater treatment plants (WWTPs) (157). In paper I, we found that isolates from Swedish gulls harbour similar genetic characteristics to isolates from Swedish surface water, suggesting WWTPs as one possible transmission route.

42 Overall, there is strong evidence that support human-to-wildlife directed transmission of antibiotic resistant bacteria/genetic elements, but can birds transmit antibiotic resistance to the human population? Wild birds are very mobile and excrete their faeces freely in the environment, and can contami- nate animal feed, pastures, crops, urban environments and reservoir of drinking or recreational water (35). Wild birds were found to transmit Campylo- bacter to crops of peas that led to an outbreak of Campylobacteriosis in humans in Alaska (158). An outbreak of bovine salmonellosis in Japan was associated with Salmonella enterica in wild sparrow habitats near the farm (159). Mallards and gulls are waterfowls that feed and defecate in water, and faeces contaminated surface-water could serve as a dissemination route of ESBL-producing E. coli from wild birds back to humans. The sampling site of the urban gulls in Alaska (paper III) is located adjacent to the Kenai Riv- er, a popular place for salmon fishing, and bird faeces-contamination of the river is a potential public health risk. Other settings such as faeces- contaminated agricultural land fields and playgrounds for children could also serve as transmission routes from wild birds to humans.

Wild birds and selection of antibiotic resistant bacteria (Paper IV) Main conclusions: - Selection for antibiotic resistant E. coli can occur in wild birds exposed to sub-MIC concentrations of ciprofloxacin in their water environment - Plasmid transfer between pathogenic strains and the normal flora readily takes place in the gut of mallards

Main results In paper IV, we performed two animal experiments (gyrAwt/gyrAD87Y and gyrAwt/gyrAS83L) where we orally infected mallards with competing ciprofloxacin susceptible and ciprofloxacin resistant E. coli, and exposed the mallards to low concentrations of ciprofloxacin in their water environment. A linear regression analysis was made for the average log ratio of CipR:CipS strains in each group and plotted as a function of the number of days of growth at each concentration of antibiotic, Figure 12. The slope of each linear curve determined the selection coefficient of each ciprofloxacin concentration. Standard errors of the linear slopes were incorporated as error bars to illustrate the margin of error of the selection coefficient, Figure 12 (B and D). We found that selection for ciprofloxacin resistant E. coli occurs in vivo at sub-MIC concentrations of ciprofloxacin, Figure 12.

Figure 12. In vivo competition of isogenic strains in Mallards. Linear regression of the ratios GyrAwt/GyrAD87Y (A) and GyrAwt/GyrAS83L (C) at different concentrations of ciprofloxacin. B and D. Plots of the selection coefficient per day at each specific antibiotic concentration in GyrAwt/GyrAD87Y (B) and GyrAwt/GyrAS83L (D) with inserted trendlines and standard errors of the slopes (calculated from the linear slopes in A and C).

The lowest ciprofloxacin concentration that conferred enrichment for the resistant mutant was 1 ng/ml in the gyrAwt/gyrAS83L experiment and 20 ng/ml in the gyrAwt/gyrAD87Y experiment, corresponding to 12-fold below the MIC and 1.3-fold above the MIC of the sensitive strain, respectively. Previous in vitro studies have shown that selection of the resistant gyrA S83L mutant takes place at ciprofloxacin concentrations 1/230 of the MIC, corresponding to approximately 0.1ng/ml (122). However, in the present study the MSC is based on the water concentration and the concentration of ciprofloxacin in the bird intestine is likely much lower than in the drinking water. Pharmaco- kinetic data for humans indicate that 20-35% of an oral dose can be recovered from faeces within 5 days (160). Assuming similar pharmacokinetics in birds, ciprofloxacin concentrations of 1ng/ml (gyrAwt/gyrAS83L) and 20ng/ml (gyrAwt/gyrAD87Y) in drinking water would result in approximately 0.33ng/ml and 6.6ng/ml liquid in bird intestines (36- and 2.4-fold below the MIC, respectively). The dilutive effect by the forage of the gut content must also be considered, as the birds were eating straw and pellets. In addition, it is well known that fluoroquinolones bind strongly to particular matter (e.g. faecal matter), resulting in a further reduction in free (i.e. selective concentrations) of the drug (161).

44 Discussion From my work presented in this thesis and the work of several other researchers, we know that the spread and accumulation of antibiotic resistant bacteria in the environment and wild birds take place. But the magnitude and mechanisms of resistance development and selection that takes place in nature remains poorly defined. Although the majority of multidrug resistant bacteria detected in the environment are probably due to contamination by human faeces and waste (58), our results imply that selection for antibiotic resistant bacteria may also occur in wildlife. Fluoroquinolones are highly stable molecules and end up in the environment from patient and animal treatment courses and the manufacturing industry and can be found in soil and water sources (162, 163). Fluoroquinolones have been repeatedly detected in water environments such as rivers, waste water treatment plants, water effluent from drug manufacturers and surface water at concentrations ranging from 0 ng/ml to several ug/ml (109-112). Our finding that concentrations as low as 1 ng/ml of ciprofloxacin can selectively enrich for resistant bacteria merits strong reductions in the contaminating concentrations in water environments.

Four other selection experiments where an animal was experimentally inoculated with a test bacterium, and then exposed to antibiotics have been reported. In two studies, the urinary tract of mice were infected with isogenic E. coli strains carrying different qnr genes, conferring low-level resistance to ciprofloxacin, and then exposed the mice to therapeutic levels of ciprofloxacin (164, 165). Although the ciprofloxacin concentration in urine by far ex- ceeded the MICs for the qnr harbouring strains (by >1000 fold), the effect of ciprofloxacin was significantly lower in qnr strains compared to the wild type. The third study experimentally infected zebrafish with isogenic Staphy- lococcus aureus or Pseudomonas aeruginosa strains, and found that exposure to sub-curative concentrations of tetracycline, oxacillin or erythromycin selected for the resistant mutant (166). The fourth study infected mice with a CTX-M-15 harbouring E. coli strain, and while exposure to therapeutic concentrations of cefotaxime, dicloxacillin and clindamycin promoted over- growth of the experimental strain, ciprofloxacin surprisingly did not (167). Another interesting study did not use experimental strains but instead investigated how drug residues in milk affect the normal intestinal flora of calves. Resistant E. coli isolates in the normal flora were enriched when calves were fed milk containing sub-MIC concentrations of tetracycline, ceftiofur and ampicillin (168). Unfortunately, no estimation of the MSC was considered in any of these studies, making it hard to set breakpoints regarding which concentrations could be deemed safe from sub-MIC selection of resistance.

45 Conjugation of plasmids in mallards (Paper IV)

Main conclusions: - Plasmid transfer between pathogenic strains and the normal flora readily takes place in the gut of mallards - Conjugation frequency was not affected by ciprofloxacin exposure

Main results In the GyrAwt /GyrAS83L experiment, we included a third strain of a different genetic origin to test if sub-MIC concentrations of ciprofloxacin could increase the conjugational frequency of a plasmid in the gut of the mallards. The isogenic strains, derived from SP132, contained an IncI1 plasmid harbouring the cefotaxime resistance gene blaCTX-M-1 while the third strain, DA26200 (cefotaximeS, ciprofloxacinR, tetracyclineR), derived from SP163, contained an IncF plasmid harbouring the tetracycline resistance gene tetA. Some mallards excreted tetracycline resistant E. coli in faeces prior to inclusion in the experiment, here named endogenous tetR E. coli. These were however sensitive to ciprofloxacin, cefotaxime and streptomycin. Due to these varying phenotypic traits, all strains could be differentiated from each other, and the emergence of new combinations of resistance could be identified. Totally 88 putative transconjugants were found in nine birds from all four experiments. A subset of 52 isolates were further categorised into possible independent transfer events by group (0, 1, 5 or 20 ng/ml ciprofloxacin), resistance profiles, PCR targeting regions on the SP163/SP132 plasmids and the tetA gene from the SP163 plasmid. One isolate from each of the resulting groups was whole genome sequenced and analysed regarding gMLST, resistance genes and plasmid replicons, Table 2.

In a total of eight different transfer events, the SP132 derivate had functioned as a plasmid recipient in one event and a donor in seven events. The SP163 derivate had functioned as a plasmid recipient in two events. The tetracycline resistance in endogenous tetR E. coli were due to the presence of a tetA or tetB gene. Endogenous tetR E. coli belonging to three different ST- types (SP48, SP165 and novel ST) had functioned as plasmid recipients in five events, and endogenous tetR E. coli belonging to ST-type 409 had functioned as a plasmid donor in one transfer event. Most transconjugants were detected in samples from the index birds obtained within the first 48 hours after inoculation. No association between exposure to ciprofloxacin and the number of recovered transconjugants was observed.

Table 2. Characteristics of the experimental strains and distribution of eight possible different transfer events that had occurred in 52 transconjugants. The 52 transconju- cants are grouped based on experiment (0, 1, 5 and 20 ng/ml ciprofloxacin (Cip) exposure), MLST-type, resistance genes on the SP132/SP163 plasmids and plasmid replicon type. TG = number of transconjugants, End tetR = endogenous tetracycline resistant E. coli. The table was simplified to fit the format and the full table is available in paper IV.

Resistance Plasmid Strain TG MLST genes replicons ctx-m-1, IncI1 SP132 - ST409 mdf(A) tet(A) IncFIB, IncFIB, SP163 - ST359 mdf(A) IncFIC Cip Transfer event

tet(A), ctx-m- IncI1, IncFIB, SP163 recipient 4 ST359 1, mdf(A) IncFIB, IncFIC SP132 donor 0 end tetR recipient 5 Novel tet(A), ctx-m-1 IncI1 SP132 donor

end tetR recipient 5 Novel tet(A), ctx-m-1 IncI1 SP132 donor

tet(A), ctx-m- end tetR recipient 1 1, mdf(A), IncI1, IncFIB, SP132 donor 17 ST48 aadA5, sul2, IncFIB, IncFII dfrA17 SP163 recipient 12 ST359 tet(A), ctx-m-1 IncI1 SP132 donor

end tetR recipient tet(A), ctx-m- 5 ST165 IncI1 SP132 donor 1, mdf(A)

5 SP132 recipient tet(B), ctx-m- IncI1, IncHI2, 2 ST409 end tetR donor 1, mdf(A) IncHI2A

end tetR recipient tet(A), ctx-m- 20 2 ST165 IncI1 SP132 donor 1, mdf(A)

Discussion Our results in paper V show that while conjugation of plasmids readily took place in the gut of mallards. The frequent plasmid transfer between the ex-

47 perimental ESBL-producing E. coli strains and the normal E. coli flora in the mallards highlights the risk of dissemination of resistance genes in wildlife exposed to bacterial pollution. In paper V, there was no detectable association between plasmid conjugation and ciprofloxacin exposure. Sub-MIC levels of antibiotics have been reported to increase the rate of plasmid conjugation in certain settings (169). However, some scientists suggest that the effects of antibiotic exposure on plasmid conjugation frequency have been overestimated (170). One study showed an increase in blaCTX-M-borne plasmid dissemination in the intestines of piglets exposed to sub-therapeutic ceftiofur or enrofloxacin (171). Furthermore, exposure to sub-MIC tetracycline concentrations increased the conjugation frequency of a multidrug resistance plasmid pB10 from E. coli to enteric bacteria present in sludge from wastewater treatment plants (172). Others have shown that exposure to tetracycline and erythromycin (above MIC concentrations) enhances the oppor- tunities for conjugative transfer in gnotobiotic rats (173, 174). In contrast, another study found no association between ceftiofur exposure and plasmid conjugation frequency in Salmonella (175).

Climate and antibiotic resistance (Paper II) Main conclusion: - The occurrence of ESBL-producing E. coli and K. pneumoniae in wild urban Swedish mallards correlates with higher temperatures

Main results In paper II, 813 faecal samples from urban Swedish mallards were collected at the urban pond “Svandammen” in Uppsala at 11 different occasions, once each month from January to November. The occurrence varied dramatically between months, ranging from 4.2 % in May to 84 % in July, Figure 13. There was a significantly higher occurrence of ESBL-producing E. coli and K. pneumoniae during the warmer 6 months of the year (May to October), compared to the colder 5 months (November and January to April). No sampling was performed in December.

48 90 80 70 60 50 40 30 Occurrence, % 20 10 0

Figure 13. Monthly occurrence of ESBL-producing E. coli and K. pneumoniae in faecal samples from urban Swedish mallards.

Discussion The potential association between climate and the emergence and spread of antibiotic resistant bacteria is largely unclear, but definitely worth looking deeper into. Emergence and spread of infectious disease is believed to be affected by climate change through different factors; higher proliferation and reproduction rates at higher temperature, extended transmission seasons, changes in ecological balances, and climate-related migration of vectors, reservoir hosts, or human populations (176). The temporal variation in the occurrence of ESBL-producing E. coli and K. pneumoniae in wild birds that we observed is important as most other prevalence reports in wild birds (as well as prevalence reports in domestic animals and humans) is the result of one single sampling. In concordance with our results, other researchers have found that the prevalence of antibiotic resistant bacteria in wild rodents ex- hibited a seasonal cycle with peaks in early- to mid-summer in mice, and late summer and autumn in voles (177). Looking at clinical isolates in humans, a study from Canada found an association between increasing local temperature and increasing antibiotic resistance (percent resistant) in E. coli, K. pneumoniae and S. aureus (178). MacFadden and colleagues propose several factors that could explain the observed associations between prevalence of antibiotic resistant bacteria and temperature: i) temperature is one of the most potent modifiers of bacterial growth and could affect the carriage and transmission of antibiotic resistant bacteria both in human and animal hosts ii) higher temperature could enhance the survival of antibiotic resistant bacteria in the environment and iii) higher temperature might facilitate horizontal gene transfer (178). Other studies found summer peaks in the incidence of

49 infections caused by Gram-negative bacteria E. coli, A. baumanni, P.aureginosa and E. cloacae (179), and seasonality of MRSA infections with higher incidence during summer and autumn (180). Another bacterial infection that has been repeatedly found to exhibit distinct seasonality is human campylobacteriosis (181, 182). A recent publication concludes that the increased campylobacteriosis cases in warmer months are probably an effect of changes in human behaviour during summer time that lead to increased risk exposure (182). In Europe, southern countries like Italy and Greece have a higher incidence of infections due to ESC- and CP- producing Enterobacteriaceae (183), and many recent emergences of highly mobile genetic elements of resistance originates from southern countries like China and India (117, 184). Results from a recent metagenomic study on antibiotic resistance gene abundance in urban sewage water from 69 countries revealed high AMR gene abundance in countries near the equator (185). The high incidence of antibiotic resistant bacteria in some countries is generally ex- plained by overuse/misuse of antibiotics, inadequate infection control in hospitals, inadequate sanitation systems and poor public health (178, 185). In our study (paper II), the high occurrence of ESC- and CP- producing bacteria during the warmer months is likely due to enhanced bacterial growth and improved survival of bacterial cells in the environment, facilitating transmission of bacteria between birds. Thus, the temperature should be regarded as a factor in the emergence and spread of antibiotic resistant bacteria.

Antibiotic resistance in rural communities (Paper V) Carriage of ESCE/K and CPE/K in Cambodia Main conclusions The presence of OXA-48 harbouring CPE/K isolates in the Cambo- dian community could be a public health threat Healthy humans and animals in rural low-income villages in Cam- bodia commonly carry multidrug-resistant E. coli and K. pneumoniae The same ESBL/pAmpC genes detected in healthy Cambodian community carriers are present in Cambodian patient isolates Exposure to animal manure and slaughter products are risk factors for carriage of ESCE/K in humans

Main results In paper V, faecal samples from humans and livestock living in 100 households in 10 rural villages in Cambodia in 2011 were analysed. We detected three blaOXA-48-harbouring meropenem-resistant E. coli/K. pneumoniae isolates and found that the occurrence rate of ESCE/K was 20 % in humans and 23 % in livestock. A multilevel regression model determined association

50 between faecal carriage of ESCE/K and CPE/K and household practices/meat consumption. We found that 96 % of the ESCE/K and CPE/K isolates were multi-drug resistant and that 11 isolates harboured colistin resistance genes mcr-1-like and mcr-3-like. Women (23 %) had higher occurrence compared to men (11 %), and small children at age-group 0-5 years (30 %) had higher occurrence compared to children at age-group 6-15 years (13 %). In livestock, pigs (46 %) and poultry (28 %) had higher occurrence compared to ruminants (7 %). All three CPE/K isolates detected in our study harboured the blaOXA-48 gene and were detected in two women. The most common ESBL-genes in E. coli from both humans and animals were of CTX-M-type group 1 and 9, and especially blaCTX-M-55 (group 1), blaCTX-M-27 (group 9), blaCTX-M-14 (group 9) and blaCTX-M-15 (group 1) were frequently detected. The most common pAmpC-gene in E. coli from both humans and animals was blaCMY-2. The ESBL/pAmpC gene distribution in K.pneumoniae was more heterogenic compared to the genes in E. coli, and the following seven genes were detected in nine K. pneumoniae isolates: blaSHV-2, blaSHV- 11, blaSHV-28, blaCTX-M-27, blaCTX-M-14, blaCTX-M-15 and blaDHA-1.

Discussion A publication by van Aartsen and colleagues was published a few months before paper V and was first to report community carriage of ESCE/K and CPE/K in healthy children in Cambodia (136). In this study, 148 faecal samples that had been obtained from children/adolescents enrolled in an intestinal parasite prevalence study were analysed for the presence of ESCE/K and CPE/K, and isolated were consecutively whole genome sequenced. Similar to our study, the only carbapenemase gene detected was blaOXA-48 in one E. coli and one K. pneumoniae from two individuals. This implies that the blaOXA-48 has been successful in colonizing the Cambodian community. Fae- cal carriage of CPE/K in asymptomatic humans and animals is a rare find, and large surveillance studies from Sweden, Germany and the Netherlands did not detect CPs amongst healthy humans (62, 186, 187). A few other reports on faecal community carriage do however exist; one study identified NDM-5 producers in healthy carriers in China (188), OXA-48 producers were found in Lebanese elderly (189) and VIM-1 producers were identified from non-hospitalized patients in Spain (190). A review by Kelly and colleagues identified 10 studies that reported community-associated or community-onset infections by carbapenem-resistant Enterobacteriaceae at a percentage ranging from 0.04 % to 29.5 % (191).

Other studies have supported our conclusion that multi-drug resistant ESCE/K are widespread in the Cambodian community. Van Aartsen and colleagues found a 55 % occurrence of ESCE/K and CPE/K in Cambodian children/adolescents (136), and Turner and colleagues reported 23 % ESCE and 33 % ESCK occurrence in hospitalized neonates in Cambodia on initial admission (192). To compare our work to other studies from the region, a

51 recent report from Vietnam found that 20 % of chicken farmers and 23 % of chickens were colonized by ESBL-producing E. coli (193), while higher occurrence rates were reported from humans in Thailand (62 %) (64) and Vietnam/Laos (41 %-70 %) (194). The lower detection rate in paper V compared to some of the previously mentioned studies could be due to the rural habitat of the study population or that previous thawing of human samples led to an underestimation of occurrence. Faecal samples were only obtained at one single occasion in paper V, which is a limiting factor. A recent study showed that the prevalence rate of ESCE in veterinary hospital staff and students increased from 6 % (point prevalence) to 26 % when a sub- population was enrolled in a longitudinal study and sampled on multiple occasions during a 6 week period (195).

In concordance with our findings in paper V, van Aartsen found that the most common ESBL-mechanism in isolates from children in Cambodia were blaCTX-M 1 and 9 sub-family variants. Examining clinical isolates, one study found that 50 % of Enterobacteriaceae from bloodstream infections in Phnom Penh (2007-2010) were resistant to cefotaxime, and that the resistance was often due to blaCTX-M-15 and blaCTX-M-14 genes (196). Resistance to extended-spectrum cephalosporins was also identified in 36-44% of urinary tract isolates in 2004-2005 and 2007-2011 (197, 198). A reference microbiology laboratory in Cambodia investigated the presence of ESBL- production in E. coli and K. pneumoniae strains from patient samples (blood, stool, lungs, pus, genito-urinary, and other body fluids) between 2012-2015 (135). In this study, phenotypic ESBL-production in E. coli increased significantly from 29 % in 2012 to 48 % in 2015, while phenotypic ESBL- production in K. pneumoniae strains was 34 % without significant increase.

With regard to the specific gene variants found in paper V, the same ESBL/pAmpC genes detected in E. coli and K.pneumoniae from humans and livestock (blaCTX-M-55, blaCTX-M-27, blaCTX-M-15, blaCMY-2 and blaDHA-1) were previously detected in E. coli and K.pneumoniae isolated from bloodstream infections in Phnom Penh, Cambodia (196). This indicates that the gut func- tions as a reservoir for extra-intestinal pathogenic bacteria, as been previously suggested by Carlet and colleagues (199). However, it is important to acknowledge that additional molecular work is needed to understand the relatedness between isolates from different sectors.

In paper V, certain household practices were associated with faecal carriage of ESCE/K in rural Cambodia. The practice of daily collection of animal manure indoor and outdoor decreased the risk of faecal carriage of ESCE/K, indicating that households that reduce environmental contamination are less likely to get exposed to antibiotic resistant bacteria present in faeces. The practice of burying or burning meat waste in the household increased the risk of faecal carriage of ESCE/K. This appears contradictory, as meat waste

52 could be contaminated with antibiotic-resistant bacteria (200), and getting rid of the meat waste would limit the environmental exposure. We believe that the association between burning/burying meat waste could be a con- founding factor to home-slaughter, as only the households’ that home- slaughter would need to bury and burn the waste. However, home-slaughter was also analysed as an explanatory factor, and was not significantly associated to faecal carriage. That slaughter could lead to dissemination of antibiotic resistant bacteria from the slaughter product to the abattoir has been previously shown (201). In contrast to previous studies that have identified consumption of undercooked meat and regular meat consumption as risk factors for community carriage of ESBL-producing Enterobacteriaceae (64, 202), we did not find any associations between meat consumption and faecal carriage.

Dissemination of ESCE/K, CPE/K and colistin resistance genes Main conclusions Transmission of ESCE/K between humans and livestock in rural Cambodia is likely Gender-related household work might affect the dissemination of ESBL-producing Enterobacteriaceae Colistin resistance genes mcr-1-like and mcr-3-like were detected in humans and livestock

Main results The majority of ESBL/pAmpC genes were detected in E. coli from both humans and livestock, Figure 14. There was a similar distribution of blaCTX- M-15, blaCTX-M-55, blaCTX-M-14, blaCTX-M-27 and blaCMY-2 in humans, ruminants, pigs and poultry. The carbapenemase-gene blaOXA-48 was only detected in three isolates from two women.

53 n=57 n=7 n=18 n=41 100% Unknown 90% 80% blaOXA-48 70% blaCMY-42 60% blaCMY-2 50% 40% blaCTX-M-27 30% blaCTX-M-14 20% 10% blaCTX-M-55 0% blaCTX-M-15 Human Ruminant Pigs Poultry

Figure 14. Distribution of ESCE/K and CPE/K genes in E. coli isolates from humans and livestock living in rural Cambodia

The combined occurrence of CPE/K and ESCE/K in women (23 %) were significantly higher compared to men (11 %) (Person´s chi-square test, p=0.03), Figure 15.

17%, 3%, 3%, Female ESCE 1%, 1% n=135n=135 ESCK 11% ESCE and K Male CPE n=54n 54 CPE and K 0 10 20 30 Figure 15. ESCE/K and CPE in women and men (<15 years) in rural Cambodia

In children, the occurrence of ESCE/K isolates was significantly higher in age-group 0-5 years (30 %), compared to age group 5-15 years (13 %) (Per- sons chi-square test, p = 0.04), Figure 16.

0-5 years 27 %, ESCE 3 % n=33 ESCK 6-15 years 12 %, 1 %

n=12 ESCE and K 0 20 40

Figure 16. ESCE/K in children in rural Cambodia

54 Colistin resistance genes mcr-1-like or mcr-like-3 were identified in ten ESCE isolates and one ESCK isolate from two humans and nine livestock

Discussion The population sampled in paper V lived in rural conditions where contact between livestock and humans were frequent, making the samples ideal to study direct zoonotic transmission of antibiotic resistant bacteria. The strik- ing similarities in ESBL/pAmpC gene distribution detected in livestock and humans imply that transmission of ESCE/K occurs between species in the study population. That resistance plasmids can spread from animals to humans has been known since 1976 when Levy and colleagues reported of an infection experiment performed on a chicken farm. They inoculated chicken with an E. coli that harboured a resistance plasmid that was later detected in the faeces of chicken farmers (203). Since then, the spread of resistant bacteria from livestock to human have been proposed for several resistant mechanisms such as vancomycin resistant enterococci (VRE) (204), MRSA (205) and colistin resistance gene mcr-1 (206). Regarding ESBL-producing bacteria, the mechanisms and impact of zoonotic dissemination are not fully un- derstood. The prevalence of ESCE in poultry is high in several European countries, leading to a public health concern that handling and ingestion of poultry products might lead to colonization and infections of ESCE in humans. Previous European studies have found that ESCE disseminates from poultry to humans, however, the methods used had low discriminatory power (e.g., ESBL genotyping and multilocus sequence typing) (187, 207-210). Subsequent WGS of some of the reported clonal ESCE isolates from poultry and humans in the Netherlands concluded that none of the isolates were clonal (211). Similarly, a Swedish study found limited evidence of overlap between the ESCE population in poultry and humans (153). A recent study by Mughini-Gras and colleagues from 2019 investigated the source attribu- tion of intestinal carriage of ESCE from individuals in the open Dutch community, to different human (e.g. human-human transmission, returning trav- ellers and patients) and non-human sources (e.g. companion animals, livestock, wild birds and food). Approximately one third of community-acquired ESCE was attributable to non-human sources (212). Most studies performed on the potential zoonotic aspects of ESCE have been performed in high- income European countries, and the findings might not be applicable to low- income countries. Zoonotic transmission is more likely in rural Cambodia, as compared to industrialized countries, where the community is agricultural oriented and there is more frequent and closer contact with the livestock. Also, the livestock manure is a potential source of ESCE/K to humans, as manure is often dumped in the environment or used (untreated) as fertilizer on crops (213). However, a recently published study from Vietnam found a limited dissemination between chicken and chicken farmers in non-intensive chicken farms (193). The molecular data that we obtained from our isolates in paper V is not detailed enough to draw further conclusions regarding the

55 possible dissemination of ESCE/K between hosts in the villages. Our group is currently performing whole genome sequencing of the ESCE/K isolates.

The difference in community carriage rate of CPE/K and ESCE/K in men compared to women observed in paper V contrasts to community carriage studies from Western Europe where no difference between genders was observed (62, 186, 214). A case-control study from Spain revealed that female sex was a risk factor for infections caused by ESCE, and the association was possibly related to women´s increased prevalence of recurrent urinary tract infections (and subsequent antibiotic treatment) (215). A study on out- patients in a multinational survey targeting 6 centres in Europe, Asia and North America found that male gender was a risk factor for infection with ESBL-producing Enterobacteriaceae (216). There are no obvious biological reasons for the observed difference in community carriage between genders in paper V, and the explanation could be gender-related behaviour leading to transmission between populations. The women in our study group are more often responsible for the care of young children (high level ESCE/K colonized group) and the less valuable livestock like poultry and pigs (high level ESCE/K colonized livestock), while men generally handle the expen- sive ruminants (low level ESCE/K colonized livestock) (131). Thus, we hy- pothesize that the higher occurrence of ESCE/K seen in women could be due to the close contact with young children, poultry and pigs. The idea that small children could be reservoirs for ESCE/K in the community is supported by another community carriage study that reported higher occurrence of ESCE/K in children 0-5 years (5.7 %) compared to children >5 years (1.7 %) (217). Also, Kaarme and colleagues reported a surprisingly high occurrence of ESBL-producing E. coli of 30 % in young children at Swedish preschools (218), which can be compared to the overall Swedish community carriage rate of 5 % (62). Young children are incontinent, have less developed hygiene and intimate contact with the environment, animals and their caregiv- er, which probably makes them more likely to participate in the dissemination of antibiotic resistant bacteria. The presence of multidrug resistant bacteria in communities poses a specific threat to the youngest children, especially in the developing world. The under five mortality rate in Cambodia is 29 per 1000 births, which is 10 times higher compared to Sweden (219). Infectious diseases like malaria, diarrheal disease, tuberculosis and pneumoniae are the leading cause of under-five mortality globally (219). The high burden of infections in children in low-income countries is multifacto- rial, and associated to increased exposure of certain infectious agents, and the immune response of the child. Malnourishment undermines the function of the innate and adaptive immune system through decreased effectiveness of lymphocytes, macrophages and granulocytes (220). Around half of the infection related deaths in children under five are associated with malnourishment (220). Further, neonates are naturally immunocompromised because of their immature immune system, which makes neonates more susceptible

56 to serious infections (221). 99 % of neonatal deaths occur in low and middle income countries, and 26 % is caused by severe infections such as sepsis, meningitis and pneumoniae (222). In India, antimicrobial resistance in bacteria causing sepsis is a growing problem and many neonates in South Asia are treated with carbapenems as a first-line therapy for sepsis (223). Among 770 neonates admitted to a neonatal unit in Tanzania, 300 (39 %) were diagnosed with sepsis, and 50 % of detected Gram-negative bloodstream isolates were resistant to third generation cephalosporins (224). Death occurred in 19 % of neonates and positive blood culture, Gram-negative sepsis and infection with ESBL-producing or MRSA- isolates were factors that predicted death (224).

The high occurrence of ESCE/K in poultry and pigs could be related to inap- propriate use of antibiotics, as previous studies have shown that antibiotic use in the Cambodian poultry and pig production is widespread and uncon- trolled (225, 226). A qualitative study investigated Cambodian poultry farmers experience and knowledge of antibiotics and antibiotic resistance (225). Quotes from farmer in the previously mentioned study:“If we treat ducks for two days and they aren’t cured we change to human drugs. We cocktail 10 tablets of this, 10 tablets of that and 20 tablets of this one. Altogether 200 tablets are mixed in 100 or 200 L of water for the ducks to drink.” “No one taught me, just my experiences.” Broad- spectrum antibiotics were believed to be necessary to prevent illness: “On the first day when we bring in the chicks, we let them drink Enro [enrofloxacin] and vitamins to make them resist to the weather.” The poultry farmers were unaware of the concern of antibiotic resistance (225). Low awareness of the risks and consequences related to antimicrobial use and AMR was also found amongst Cambodian pig farmers in another study (226). Commensal E. coli from the pigs exhibit- ed high prevalence of resistance to several antimicrobials of critical importance for human medicine, including ampicillin, ciprofloxacin and colistin, but 45 % of the farmers had never heard the term ‘antimicrobial resistance’ (226).

Further, high level of ESCE colonization in poultry is common, even in countries where the general antibiotic resistant burden is low (153, 227). The high colonization of poultry in industrialized countries is mainly due to ESCE transmission from ESCE-colonized imported grandparent animals through the production pyramid via vertical transmission and environmental contamination (228). In the rural setting in paper V, livestock housing conditions could be a factor that explains high-level colonization in pigs, as pigs are often housed in crowded confinement (131), which allows for transmission of bacteria.

57 Main conclusions

• Wild birds can function as potential resistance reservoirs and sentinels for antibiotic resistant E. coli

• Environmental pollution from humans is the primary source for antibiotic resistant Enterobacteriaceae found in wildlife

• Selection for antibiotic resistant bacteria may also occur in wild birds

• The high community carriage of multidrug-resistant bacteria in rural Cambodia is worrying and highlights Southeast Asia as a hotspot for antibiotic resistance

• Transmission of cephalosporinase-producing E. coli/K. pneumoniae is likely occurring between humans and livestock in rural Cambodia, but more in-depth molecular work is needed to determine the mechanisms of dissemination

58 Future perspectives

The future perspectives for the research of One Health perspectives of carriage, transmission and selection of antibiotic resistant bacteria are certainly extensive and varying. Just like my thesis has shown, future research will need many different and complementary approaches.

Emergence and transmission in the environment The surveillance of AMR in the environment should have a global and systematic approach. Standardized surveillance methods for the detection and quantification of antimicrobials, bacteria/bacterial DNA in different environmental matrices should be implemented. A continuous surveillance of resistance in faecal bacteria in e.g. untreated sewage water could potentially serve as an indicator of emerging clinically relevant antimicrobial resistance. The surveillance of AMR in wildlife should not be limited to screening of randomly selected populations and regions, but rather harmo- nized globally by selecting appropriate wildlife species as indicators for AMR, using comparable methods and comprehensive report systems.

Zoonotic transmission From the epidemiology perspective of carriage and zoonotic transmission of ESCE/K and CPE/K, population based modelling studies are one way for- ward to understand the sources of ESCE/K and CPE/K in the community. When comparing isolates between sectors in society, it is important to use high-resolution methods to draw correct conclusions regarding bacterial/plasmid/gene dissemination. ESCE/K and CPE/K isolates collected in paper V are being analysed using whole genome sequencing by our group at the moment, and our aim is to investigate if cross-species transmission occurred in our study population. Another important question is whether the bacteria found in different species have different properties that would make them more or less likely to colonize certain species. Species adaption is a trait found in other bacterial species like Campylobacter.

59 In the field of food hygiene and AMR, seafood and vegetables deserve greater attention from the scientific community. ESCE/K of human origin could be enriched in seafood and vegetables through contaminated surface water and irrigation water.

Environmental contamination From the perspective of environmental antibiotic contamination, the impact of sub-MIC levels of antibiotics present in the environment on the emergence and transmission of resistant bacteria remains largely unknown. Thus, in vivo experiments are needed to establish whether selection and development of resistant bacteria occurs in wildlife exposed to antibiotics. Such studies could be performed in birds like we did in paper IV, or in a mamma- lian model e.g. rodents. The results could lead to guidelines regarding ac- ceptable emission levels of antimicrobials. The level and characteristics of co-selection conferred by non-antibiotic compounds, such as heavy metals and antibacterial biocides are also important future research questions.

It is important to study how waste management can become more effective in removing antibiotic contaminants and bacteria. Advanced waste water treatment solutions like ozonation, activated carbon, biofilters, wetland- treatment etc should be evaluated.

AMR in low and middle income countries The global action plan on AMR called for increased AMR surveillance in low and middle income countries, and the Fleming Fund was developed to aid countries. But AMR surveillance is presently highly biased toward high income countries. To illustrate the huge difference between GLASS surveillance reports between countries, Germany (population 81 M) has 1840 AMR registered surveillance sites in GLASS out of which 337 reported data to GLASS, while Cambodia (population 16 M) has eight registered surveillance sites but none reported any data. It is important that future surveillance studies are planned and financed with some perspective of equality. As low and middle income countries are often hotspots for the emergence of new antibiotic resistance, a lacking global surveillance system can lead to a global spread of new multidrug resistant bacteria. Further, the inadequate system of microbiology analysis in some countries pose a direct threat to the population, and will also lead to unnecessary broad antibiotic treatment. A future research question could be to study the attributable fraction of resistant infections due to inadequate AMR surveillance systems and undeveloped microbiology facilities.

60 Populärvetenskaplig sammanfattning

Antibiotika har räddat miljontals liv sedan dessa läkemedel började användas på 1940-talet under andra världskriget. Innan antibiotika fanns tillgängligt kunde friska, unga personer dö av vanliga bakteriella infektioner. Olika typer av antibiotika fungerar mot olika typer av bakterier. Tyvärr har många bakterier utvecklat resistens, det vill säga motståndskraft, mot antibiotika och spridningen av sådana bakterier går fort. Antibiotikaresistens är ett problem i hela världen, och vissa regioner i Afrika och Asien är särskilt hårt drabbade. Escherichia coli (E. coli) och Klebsiella pneumoniae (K. pneumoniae) är bakterier som bland annat kan orsaka urinvägsinfektioner, blodförgiftningar, sårinfektioner och lunginflammationer. Antibiotika av typen cefalosporiner och karbapenemer samt en annan typ av antibiotika som kallas ciprofloxacin är mycket viktiga mot dessa infektioner. Bakterier som är resistenta mot cefalosporiner och karbapenemer kallas fortsättningsvis för CF-resistenta respektive KP-resistenta. Antibiotikaresistenta bakterier förekommer bland sjuka och friska människor, husdjur, livsmedelsproducerande djur, vilda djur, livsmedel och i miljön. Uppkomsten, utvecklingen och spridningen av antibiotikaresistens sker ofta i naturen och bland människor och djur i sam- hället, men det saknas kunskap om hur detta går till. Den här avhandlingen adresserar problemet med antibiotikaresistens ur perspektivet ”En värld - En hälsa”. I fem arbeten (I-V) undersöks antibiotikaresistenta varianter av bakterierna E. coli och K. pneumoniae i miljön, vilda djur, livsmedelsproducerande djur och friska människor.

I arbete I-III hittades CF-resistenta E. coli i avföringsprover från vilda fåg- lar i Sverige och dessa bakterier har många genetiska likheter med de bakterier som man hittar hos sjuka och friska människor i Sverige. I arbete I och II var förekomsten av CF-resistenta E. coli 17 % i måsfåglar och 47 % i gräsänder. Detta är betydligt högre än förekomsten av CF-resistenta E. coli i avföringsprover från friska svenska människor under samma tid (5 %). Ar- bete II visade att måsfåglar som bor i närheten av människor har högre före- komst av antibiotikaresistenta E. coli, jämfört med måsfåglar som bor på avlägsna platser. I arbete IV infekterades gräsänder med två stammar av E. coli bakterier som var identiska bortsett från att den ena stammen var resi- stent mot ciprofloxacin. När ciprofloxacin tillfördes till gräsändernas bad- och dricksvatten växte den resistenta stammen bättre, även då nivåerna av ciprofloxacin var mycket låga. I arbete V hittades ett fåtal KP-resistenta E.

61 coli och K. pneumoniae i avföringen hos människor som bor i jordbruksbyar i Kambodja. CF-resistenta E. coli/K.pneumoniae visade sig vara vanligt i byarna, och förekom hos 20 % av människorna och 23 % av de livsmedelsproducerande djuren. Bakterierna förekom i högre utsträckning bland kvin- nor och små barn under 5 år. Samma typer av antibiotikaresistensgener hittades i både människor och djur, och exponering för slaktavfall och djur- spillning ökade risken för bärarskap av CF-resistenta bakterier hos männi- skorna.

Slutsatserna av avhandlingen är att vilda fåglar kan fungera som potentiella reservoarer och indikatorer för antibiotikaresistenta bakterier i samhället. Majoriteten av de resistenta bakterierna som hittas i avföringen från vilda fåglar beror på miljöförorening av mänsklig avföring och avfall. Uppkomst och utveckling av antibiotikaresistenta bakterier kan sannolikt även ske i tarmen på vilda fåglar, särskilt om de exponeras för antibiotika som släppts ut i miljön. Det är viktigt att undersöka förekomsten av antibiotika och antibiotikaresistenta bakterier i miljön och att förebygga vidare föroreningar genom korrekt avfallshantering. Den höga förekomsten av antibiotikaresistenta bakterier i jordbrukssamhällen i Kambodja är oroande, och understry- ker Sydostasien som en region med särskilt mycket antibiotikaresistens. Forskning och satsningar kring antibiotikaresistens behöver fördelas mer rättvist bland världens länder och mer fokus måste riktas på låg- och mede- linkomstländer. Mycket talar för att CF-resistenta E. coli/K. pneumoniae sprids mellan djur och människor, men det behövs mer ingående studier av bakterierna för att uttala sig om hur spridningen går till.

62 Acknowledgments

First of all, I would like to thank myself for being curious, ambitious and stub- born enough to produce this thesis!

As a 5th semester medical student, I was enrolled in a programme at Uppsala University called the MD/PhD-program, founded by Peter Bergsten. After graduating medical school, I was hired as a Forskar-AT at the University Hos- pital in Uppsala, which allowed me to finish my PhD. I was introduced to the One Health field of research by my enthusiastic and supportive co-supervisor Professor Björn Olsen, and thereafter guided by main-supervisor Josef Jä- rhult. Josef is encouraging, ambitious and a genuinely warm person, who always takes time for his students. It has been fun and exciting to work with you Josef, and I have grown a lot as a researcher and person under your watch. Stef- an Börjesson has done more for me than what could be expected from a co- supervisor. The million questions I have asked Stefan throughout my PhD were always answered quickly in a friendly and supportive way. I could not have done all this work without Stefans’ expertise and commitment.

My research group Zoonosis Science Center is located at BMC, Uppsala Uni- versity and led by Professor Åke Lundkvist, a wise man with whom I have shared many rewarding conversations. Many people have worked and studied at “the Zoolab” during my PhD years, and it has been great to get to know everyone. I want to send a special acknowledgement to Patrik, Tove, Anna, Frida, Olivia, Per, Michelle, Evangelos, Rachel, Jiaxin, Erik, Gabriel, Mahmoud, Jenny, John, Badrul, Janina, Erik, Helen and Tomas. A special thanks to Viktoria for excellent work in the lab and for the friendship – you are missed! To Jon for your great work on Paper II and for teaching me about birds. To Tanja for the support, love and many telephone talks. And to Mia – for being wonderful and for all the laughter, may we never forget the time we had with Mr Capoeira!

Some of my work was performed at Professor Dan Anderssons lab at IMBIM, Uppsala Univeristy. I want to thank everyone at D7:3 and especially Ulrika Lustig and Linus Sandegren for your guidance and patients. And Dan, both professor and family member with a soft shell and even softer heart – thank you for everything!

63 Thank you One Health Sweden for allowing Deryn Ramsey to use your logo as inspiration for her amazing design that resulted in the beautiful illus- tration on the cover of this thesis.

I want to thank Jonas Bonnedahl at the Linneus University in Kalmar for our fruitful collaborations in several projects. My warmest thanks to my collaborators Kristina Osbjer and Ulf Magnusson at SLU for providing me with the unique samples for the Cambodia-project, and to Elisabeth Rajala for performing the statistical analysis. Thank you Viktor Ljungström for support, input and friendly words at time of need. Thank you to researchers Annica Landén and Mattias Myrenås at SVA for your kind help.

I am blessed with many amazing friends and a large family. Mum Christina and dad Anssi brought up four daughters to become the power-women that we are today. My sisters Johanna, Carin, Saga and bonus-siblings Julia and Si- mon are my foundation. I also want to thank the inspiring leaders of the matriar- chy - “mormor emerita” Ingegärd and dear farmor Anneli. Many thanks to my wonderful brother-in-law Lutz for statistics help. A special thanks to dear Michaela and Axel who have been there since childhood. I also want to send my love to my father’s fiancée Ann, and to my extended Johansson/Jonsson family in Umeå.

I started veterinary school at 19 and found myself surrounded by strong and supportive women! I want to acknowledge my wonderful veterinary class “Corvets” with a special thanks to the fantastic Caroline, Sanna, Lisa, Cecilia, Rebecka, Emma, Karin, Hanna, Josefin, Tuva, Isabel, Kerstin, Markus and Eva.

Later in medical school, I yet again depended on fantastic women who led me all the way to graduation – Anna, Ulrika, Anna, Lovisa, Christine, Ylva, Emelie, Ingrid, Sofie and Felicia.

In recent years, I have had the fortune to spend a lot of time with like-minded and inspiring Olivia, Ebba, Janna, Katja, Annabel, Louise and Paliz. Your friendship and support made all the difference.

My fiancée Linus has been with me throughout the majority of medical school and PhD-studies as my main support. Linus always respects and encourage my goals and I could not have asked for a better and more loving partner. Your positivity and strength never ceases to amaze me! When all of this is over, we will move on to our next big adventure!

And to little Dennis – our son and sunlight – you are loved beyond words.

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79 Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1617 Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine”.)

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