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The doctoral thesis “Prevalence and reduction possibilities of resistant Escherichia coli in pigs” by Daiga Gāliņa was carried out during the period from 2014 to 2022 at the Institute of Food and Environmental Hygiene of the Latvia University of Life Sciences and Technologies (LLU) and at the Division of Molecular Biology and Microbiology of the Research Laboratory of Biotechnology of LLU. The study was carried out into three activities – one of them was dedicated to find out the prevalence of resistant E. coli in pigs in Latvia, while the other two were to reduction possibilities of resistance. In the first activity, 615 samples of pig feces were collected from seven pig breeding farms in the Republic of Latvia. Subsequently, a bacteriological examination of the samples was carried out – 615 commensal Escherichia coli were isolated and their antibiotic resistance was determined. All of the obtained faecal samples of pigs were tested for the presence of extended spectrum beta-lactamase (ESBL) producing E. coli. Presumptive ESBL-producing E. coli were phenotypically and genotypically confirmed subsequently determining their resistance to antibiotics. In the second activity, in one of the pig farms (No. L3), we randomly selected 4 litters containing sows with 14-day-old Landrace-Duroc crossbred piglets (n=44). The piglets of each litter were assigned to their own treatment groups: control group (C), probiotics group (P), probiotics and buckwheat bran group (PB) and herbal group (H). During the study, control group piglets received basal diet, P and PB groups received probiotics, that were added to the drinking water; but buckwheat bran and herbal supplement were added to the basal diet for the groups PB and H, respectively. The research period was 6 weeks. During this activity, the potential of probiotics and feed components of plant origin as an alternative solution to antimicrobial agents was evaluated, determining the effect of them on the general health of piglets, intestinal health, and the prevalence of resistant E. coli. In the third activity, the in vitro antibacterial activity of the commercial and Latvian (the author of this doctoral thesis have obtained from medicinal plants during the hydrodistillation process) essential oils was compared against phenotypical and genotypic confirmed fecal ESBL producing E. coli isolated from pigs in the first activity of this study. The hypothesis put forward in the thesis: bacteria Escherichia coli resistance to antibiotics is highly widespread in pig farming farms in Latvia and the use of probiotics, as well as herbal products, could limit the spread of E. coli and ESBL-producing E. coli. The purpose of the thesis: to study the prevalence of resistant Escherichia coli in pigs in Latvia, to evaluate the possibilities of its reduction by using feed components and means of natural origin. Tasks of the doctoral thesis: 1. find out the prevalence of the resistant to antibiotics Escherichia coli in pig farms in Latvia. To identify extended-spectrum beta-lactamase (ESBL)-producing E. coli occurrence in pigs; 2. evaluate the effect of supplementary feed containing probiotics and feed components of plant origin on the health of piglets and their intestines – prevalence of resistant E. coli, intestinal microbiot, histomorphological and immunological condition, meat quality; 3. to compare the antibacterial activity of Latvian and commercial essential oils in vitro against porcine faecal extended-spectrum beta-lactamase (ESBL)-producing E. coli, as well as to evaluate the use of essential oils enriched with caprylic acid and sodium chloride in the control of this resistant E. coli. The personal contributions: 1. bacteriological examination of faecal samples (commensal indicator bacteria E. coli isolation and biochemical confirmation; testing samples for the presence of ESBL-producing E. coli, their phenotypic confirmation; E. coli and ESBL-producing E. coli determination of antimicrobial sensitivity (antibiotic resistance) against 12 and 18 antibiotics, respectively, preparation of isolated cultures for long-term storage); 2. collection of medicinal plants, preparation of a herbal supplement containing them for the implementation of the piglet feeding study; 3. bacteriological examination of the faeces and intestinal contents of obtained piglets (Enterobacteriaceae, E. coli and Lactobacillus spp. determination of the number, isolation of E. coli and determination of its antimicrobial sensitivity (antibiotic resistance); 4. wild and cultivated medicinal plants in Latvia (T. vulgaris, T. serpyllum and S. montana) collection, extraction of essential oils using the hydrodistillation method; 5. examination of the antibacterial activity of Latvian and commercial essential oils, as well as commercial enriched (with caprylic acid and NaCl ) essential oils in vitro. To determine the prevalence of resistant E. coli, including the occurrence of extended-spectrum beta-lactamase producing E. coli, we collected faecal samples of pigs of different ages (4, 6, 8 and 12 weeks old) and sows from seven pig breeding farms in three regions of Latvia – Vidzeme, Pieriga and Zemgale; four of the farms were considered relatively large (number of sows 700-2100) and three were considered small (number of sows 15-40). We isolated commensal indicator bacteria E. coli from the faecal samples bacteriologically and tested their susceptibility to 12 antibiotics. We tested each collected sample for the presence of ESBL-producing E. coli, isolated the presumed colonies and then confirmed them phenotypically and genotypically by determining the presence of the beta-lactamase genes such as blaCTX-M, blaTEM and blaSHV. In confirmed ESBL-producing E. coli, susceptibility to 18 antibiotics was determined. We evaluated the differences of the prevalence of ESBL-producing E. coli between large and small farms, and among pigs of different ages, the more frequently observed resistance phenotypes and ESBL-encoding genes were characterized. To evaluate the effect of probiotics and feed components of plant origin on the intestinal microbiota, including the spread of resistant E. coli, the health of pigs and their intestines, as well as meat quality, we selected four litters of 14-day-old Landrace-Duroc crossbred piglets (n=44) in farm No L3. Each litter of piglets was assigned into one of 4 research groups – control group (group C), probiotic group (group P), probiotic and buckwheat bran group (PB group) and herbal group (group H). During the study, we added a probiotics to the drinking water for the groups P and PB at certain concentrations depending on the age of the piglets (1%, 0.75%, 0.45%, 0.34%, and 0.32% according to the 14th, 28th, 35th, 42nd, and 49th). In addition, piglets of group PB received a buckwheat bran supplement – 3% of the basic feed mass. Group H received a feed supplement of medicinal plants – plantain (Plantago major L.), nettle (Urtica dioica L.) and St. John’s wort (Hypericum perforatum L.) in a ratio of 1:1:1 in the amount of 1.5% of the basic feed mass. The research period was 6 weeks, i.e., from the 14th to the 56th day of piglet life. We investigated the impact of probiotics and feed components of plant origin on the haematological and serum biochemical parameters; faecal and intestinal microbiota (the number of Enterobacteriaceae, E. coli and Lactobacillus spp.) the prevalence of resistant E. coli; histomorphological measurements of the intestine; the number and density of goblet cells, including neutral and acidic mucin-producing goblet cells; epithelial cell proliferation and infiltration of lymphocytes (immunohistochemical analysis of the intestine); as well as fatty acid content in muscular tissue. In order to evaluate the potential use of commercial and Latvian Lamiaceae plant-derived products (essential oils) to limit the spread of ESBL-producing E. coli on pig farms, we performed in vitro determination of the antimicrobial activity commercial essential oils and hydrodistillation-derived essential oils from medicinal plants grown in Latvia (breckland thyme (Thymus serpyllum L.), common thyme (Thymus vulgaris L.) and montain savory (Satureja montana L.). The chemical composition of essential oils was determined, the factors affecting their antibacterial activity were analysed, and the antibacterial efficiency of pure and supplemented (with caprylic acid and NaCl) commercial essential oils against faecal ESBL-producing E. coli was compared. High level of resistance to tetracycline, ampicillin, trimethoprim, sulfamethoxazole-trimethoprim, and amoxicillin-clavulanic acid were observed in E. coli, isolated from the pig population and they were characterized by very diverse resistance phenotypes; a significantly higher prevalence of antibiotic-resistant E. coli could be observed in large farms. The carriage of resistant E. coli in pigs decreased at the end of the production cycle, that is, significantly fewer resistant E. coli isolates were observed from fattening pigs. The presence of fecal ESBL-producing E. coli was confirmed on three large farms (L1, L2, L3) and one small (S1) farm, moreover, the rates of occurrence varied significantly between farms. Very often ESBL-producing E. coli was classified as multidrug-resistant, in addition, the isolates from the large farms had demonstrated more extensive MDR phenotypes. The most frequently detected ESBL-encoding genes were blaTEM and blaCTX-M, followed by blaSHV. The gene blaSHV was dominant on the small farm and it characterized by a positive association with resistance to cefixime, ceftazidime and amoxicillin-clavulanic acid, while the gene blaCTX-M was dominant in isolates on the large farms and it was positively associated with resistance to cephalexin and several non-beta-lactam antibiotics. In the piglet feeding study, probiotics increased the number of Lactobacillus spp. in the small and large intestine and reduced the number of Enterobacteriaceae in the faeces on the 35th day of piglet life, while the herbal supplement reduced the number of Enterobacteriaceae and E. coli in the faeces on the 56th day of piglet life. Resistant E. coli (to at least one antibiotic) were detected slightly more before the study compared to the conclusion in all study groups. Furthermore, the herbals reduced the phenotypic diversity of resistant E. coli. All of the supplemented groups showed an improvement of intestinal histomorphological parameters – the villus height; the villus height to crypt depth ratio in the jejunum; the crypt depth in the colon; and the number of proliferating (Ki67 positive) epithelial cells. The inclusion of probiotics in the basal diet was increased the total number of goblet cells in crypts of intestine, furthermore, increasing acidic mucin producing goblet cells in jejunum; in contrast the herbal supplement decreased the density of goblet cells and acidic mucin producing goblet cells in jejunum villi. Probiotics increased the number of intraepithelial lymphocytes, the relative frequency of T cell (CD3 positive cells) in Peyer’s patches and lamina propria. Herbals had a dual effect – on the one hand, they reducing the number of intraepithelial lymphocytes, but increasing the relative frequency of T cell (CD3 positive cells) in the Peyer’s patches, and the lamina propria of the colon. Probiotics significantly increased α-linolenic and palmitoleic acids, but decreased stearic acid levels in muscle tissue. By comparing the antibacterial efficiency of pure commercial essential oils and the essential oils of Lamiaceae family plants grown in Latvia against faecal ESBL-producing E. coli, it was found that commercial essential oils had higher inhibitory and bactericidal activity. In addition, the antibacterial activity of commercial essential oils was significantly improved when they were enriched with sodium chloride or caprylic acid. Pure essential oils had lower activity against ESBL and multidrug-resistant (MDR) ESBL-producing E. coli, but enrichment of essential oils with caprylic acid and, especially sodium chloride, significantly improved antibacterial activity against ESBL and MDR ESBL-producing E. coli. Ten conclusions and five practical recommendations have been formulated at the end of the doctoral thesis. There are 140 pages, 23 tables, 30 figures and attached two appendices. The bibliography contains 340 references.

The aim of this study was to determine the prevalence of fecal ESBL-producing Escherichia coli (E. coli) in pigs on large and small farms in Latvia, to characterize beta-lactamase genes and establish an antimicrobial resistance profile. Fecal samples (n = 615) were collected from 4-week, 5-week, 6-week, 8-week, 12-week and 20-week-old piglets, pigs and sows on four large farms (L1, L2, L3, L4) and three small farms (S1, S2, S3) in Latvia. ChromArt ESBL agar and combination disc tests were used for the screening and confirmation of ESBL-producing E. coli. The antimicrobial resistance was determined by the disc diffusion method and ESBL genes were determined by polymerase chain reaction (PCR). Subsequently, ESBL-producing E. coli was confirmed on three large farms, L1 (64.3%), L2 (29.9%), L3 (10.7%) and one small farm, S1 (47.5%), n = 144 (23.4%). The prevalence of ESBL-producing E. coli differed considerably between the large and small farm groups (26.9% vs. 12.7%). Of ESBL E. coli isolates, 96% were multidrug-resistant (MDR), demonstrating there were more extensive MDR phenotypes on large farms. The distribution of ESBL genes was blaTEM (94%),&nbsp, blaCTX-M (94%) and blaSHV (48%). On the small farm, blaSHV dominated, thus demonstrating a positive association with resistance to amoxicillin-clavulanic acid, ceftazidime and cefixime, while on the large farms, blaCTX-M with a positive association to cephalexin and several non-beta lactam antibiotics dominated. The results indicated the prevalence of a broad variety of ESBL-producing E. coli among the small and large farms, putting the larger farms at a higher risk. Individual monitoring of ESBL and their antimicrobial resistance could be an important step in revealing hazardous MDR ESBL-producing E. coli strains and reviewing the management of antibiotic use.

The aim of the study was to evaluate the effect of probiotics and herbal products on the intestinal histomorphological and immunological development in piglets. Accordingly, 2-week-old piglets were allocated in 4 groups: C (basal diet), Pro (basal diet + probiotics), Pro+B (basal diet + probiotics + buckwheat bran), and H (powder of herbs). After 6 weeks of the experiment, 4 piglets from each experimental group were randomly selected and slaughtered at a slaughterhouse. Samples of tissue and digestive content from the jejunum and colon were collected for bacteriological, histological, and immunohistochemical examination. The results showed that probiotics increased the number of Lactobacillus spp. in the small p<0.05 and large intestines. The intestinal histomorphology was improved p<0.05 in all experimental groups by an increased villus height, VH : CD ration, colon crypt depth, and number of Ki-67+ epithelial cells. A higher number p<0.05 of goblet cells and their acidification were observed in group Pro, while the density of goblet cells was decreased by the herbs. Probiotics increased p<0.05 the number of intraepithelial lymphocytes (IELs), density of CD3+ cells in Peyer’s patches (PPs), and lamina propria (LP). In group H, a dual effect on the CD3+ cell distribution was observed. The herbs reduced p<0.05 the number of IELs and CD3+ in LP but increased the distribution of CD3+ cells in PPs. In the colon, herbs increased CD3+ cells in LP as well. It suggests that probiotics and herbs had influence on the intestinal histomorphology and the ability to modulate the mucosal immune system; however, the combination of probiotics and buckwheat bran was not so convincing, probably due to the inhibitory effect of the buckwheat bran on the probiotics used.

The present study was designed to evaluate antimicrobial resistance of Escherichia coli (E.coli) isolated from liquid pig manure and to determine in vitro the antibacterial activity of nine traditional Latvian medical plants against various profiles of isolated antibiotic resistant E.coli. The commensal E.coli and extended-spectrum Beta-lactamase (ESBL)-producing E.coli isolates were tested to 12 antibiotics by the disk diffusion method. The in vitro antimicrobial activities of extracts of plants were evaluated, using well diffusion technique. Results showed that overall 72% of the E.coli isolates were antibiotic resistant, being divided into 55.5% resistant to up to three different classes of antibiotics and 44.5% resistant to four or more (up to 6) different classes. In the present study, majority of tested plants (Urtica dioica, Hypericum perforatum, Hippophae rhamnoides, Viburnum L., Vaccinium macrocarpon L., Cuminum cyminum L.) have not demonstrated antimicrobial activity to E.coli isolates. However, the oil extract of leaves of Plantago major, aqueous extract of green husk of Juglans regia L. and ethanol extract of seeds of Tropaeolum majus L. demonstrated activity against test cultures.