The discovery of antibiotics has been considered as one of the most remarkable scientific accomplishments of the last century. However, the extensive usage of antibiotics has led to the rapid emergence of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB), which have been recognized as one of the biggest threats to human and environmental health. While ARGs and ARB are ubiquitous in diverse environments, aquatic environments play a particularly crucial role in their prevalence and dissemination. Furthermore, the microbial complexity and various pollutants persisting in aquatic environments significantly contribute to the evolution and spread of ARGs and ARB. However, the knowledge regarding the distribution pattern of ARGs on a large scale, as well as the interaction between microbial community, specific pollutants, and ARGs and ARB, is currently limited. In this study, I conducted systematic work at multiple scales, to fill crucial knowledge gaps that could support the future management of the spread of antimicrobial resistance. In particular, on the ecosystems level I depict the biogeographical patterns of ARGs in freshwater reservoirs, on the community level I explored the selection patterns of combinations of antibiotics on multidrug resistant strains in complex community context, and finally, on the population level I investigated the impact of cigarette smoke, and waste products on the dissemination of ARGs. In my first study, samples were collected from 24 freshwater reservoirs across southeast China and the biogeographical patterns of bacterial communities and ARG profile were characterized using 16S rRNA gene high-throughput sequencing and high-throughput-quantitative PCR. A distance-decay pattern for both, microbial communities and ARG profiles, were observed. However, larger differences between reservoir ARG profiles than microbial community compositions were detected. Further, I found that the biogeographical patterns of bacterial communities were simultaneously driven by stochastic and deterministic processes, while ARG profiles were not explained by stochastic processes, and the relationship between bacterial communities and ARG profiles was weak. In summary, this study indicated a decoupling of bacterial community composition and ARG profiles in inland waters under relatively low-human-impact at a large scale. In a second study, the selection dynamics for multidrug resistance between isogenic pairs of E. coli strains under exposure to multiple selective agents in the absence and presence of the microbial community were investigated using microcosm experiments. The presence of the community significantly decreased the selection for multidrug resistant strain under exposure to a single antibiotic. While pressure through the second antibiotic significantly decreased the activity and diversity of the community, its ability to reduce selection was consistently maintained at levels comparable to those recorded in the absence of the second antibiotic. This indicates that the observed effects of community context on selection dynamics are rather based on competitive or protective effects between the focal strains and a small proportion of bacteria within the community, than on general competition for nutrients. Last but not least, the effect of cigarette-derived pollutants on the proliferation of ARGs was explored using multifaced approaches. Cigarette smoke condensate in an artificial lung sputum medium significantly elevated the transfer rates of a multi-drug-resistance encoding plasmid between Pseudomonas strains. The overproduction of reactive oxygen species (ROS) as part of the bacterial stress response was directly connected to the increasing transfer rates. Similarly, cigarette ash leachate in an environmental medium significantly increased the plasmid transfer rates, and overproduction of ROS was equally detected. Furthermore, used cigarette filters with entrapped toxicants were submerged in a wastewater stream and colonized by distinct microbial communities compared to those colonizing unused control filters. The microbial communities colonizing used cigarette filters were significantly enriched in AMR, potential pathogenic bacteria and mobile genetic elements. Overall, the insights gained within this thesis into the spread of AMR at multiple scales constitute a valuable contribution to support future management and monitoring of ARGs in diverse ecosystems.

The purpose of this dissertation was to broadly investigate the fate of antibiotic resistance genes (ARGs) and engineered nanomaterials (ENMs) as representative contaminants of emerging concern in wastewater treatment plants (WWTPs). WWTPs may have their performance impacted by ENMs and may also serve as a reservoir and point of release for both ENMs and ARGs into the environment. Of interest were potential adverse effects of ENMs, such as stimulation of antibiotic resistance in the WWTP, toxicity to microbial communities critical for WWTP performance, and toxicity to humans who may be exposed to effluents or aerosols containing ENMs and their transformation products. Response of nine representative ARGs encoding resistance to sulfonamide, erythromycin and tetracycline to various lab-scale sludge digestion processes were examined, and factors that drove the response of ARGs were discussed. Mesophilic anaerobic digestion significantly reduced sulI, sulII, tet(C), tet(G), and tet(X) with longer solids retention time (SRT) exhibiting a greater extent of removal. Thermophilic anaerobic digesters performed similarly to each other and provided more effective reduction of erm(B), erm(F), tet(O), and tet(W) compared to mesophilic digestion. Thermal hydrolysis pretreatment drastically reduced all ARGs, but they generally rebounded during subsequent anaerobic and aerobic digestion treatments. Bacterial community composition of the sludge digestion process, as controlled by the physical operating characteristics, was indicated to drive the distribution of ARGs present in the produced biosolids, more so than the influent ARG composition. Effects of silver (nanoAg), zero-valent iron (NZVI), titanium dioxide (nanoTiO2) and cerium dioxide (nanoCeO2) nanomaterials on nitrification function and microbial communities were examined in duplicate lab-scale nitrifying sequencing batch reactors (SBRs), relative to control SBRs received no materials or ionic/bulk analogs. Nitrification function was only inhibited by high load of 20 mg/L Ag+, but not by other nanomaterials or analogs. However, decrease of nitrifier gene abundances and distinct microbial communities were observed in SBRs receiving nanoAg, Ag+, nanoCeO2, and bulkCeO2. There was no apparent effect of nanoTiO2 or NZVI on nitrification, nitrifier gene abundances, or microbial community structure. A large portion of nanoAg remained dispersed in activated sludge and formed Ag-S complexes, while NZVI, nanoTiO2 and nanoCeO2 were mostly aggregated and chemically unmodified. Thus, the nanomaterials appeared to be generally stable in the activated sludge, which may limit their effect on nitrification function or microbial community structure. Considering an aerosol exposure scenario, cytotoxicity and genotoxicity of aqueous effluent and biosolids from SBRs dosed with nanoAg, NZVI, nanoTiO2 and nanoCeO2 to A549 human lung epithelial cells were examined, and the effects were compared relative to outputs from SBRs dosed with ionic/bulk analogs and undosed SBRs, as well as pristine ENMs. Although the pristine nanomaterials showed varying extents of cytotoxicity to A549 cells, and gentoxicity was observed for nanoAg, no significant cytotoxic or genotoxic effects of the SBR effluents or biosolids containing nanomaterials were observed. Studies presented in this dissertation provided new insights in the fate of ARGs in various sludge digestion processes and ENMs in nitrifying activated sludge system in lab-scale reactors. The study also yielded toxicity data of ENMs to biological wastewater treatment microbial communities and human lung cells indicated by a variety of toxicity markers. The results will aid in identifying appropriate management technologies for sludge containing ARGs and will inform microbial and human toxicity assessments of ENMs entering WWTPs. / Ph. D.

As the population in water stressed areas increases, it is critical that wastewater treatment plants (WWTPs) continue to replenish depleted water supplies, and serve as an alternative water source. WWTPs depend on microorganisms in activated sludge to remove pollutants from wastewater and therefore an understanding of how these microorganisms are affected by various conditions and pollutants is needed. Also, as consumer products and industrial processes evolve, so do the pollutants they discharge to wastewater. In order to keep pace with these changes, understanding the effects of emerging contaminants to WWTP processes is essential. The research herein assesses microbial community dynamics of the response of nitrifying microorganisms in activated sludge to variation in ammonia concentration and evaluates the impact of engineered nanoparticles on activated sludge microbial communities and other emerging pollutants, such as antibiotic resistance genes and disinfection by-products. In order to assess microbial community dynamics of the response of nitrifying microorganisms to removal of ammonia in the feed, nitrifying activated sludge reactors were operated at various relevant temperatures and the nitrifying microbial community was characterized using activity assays and bio-molecular techniques. We found that Nitrospira spp. were the dominant nitrifying microorganisms, exhibiting stable relative abundance across multiple trials and over a range of temperatures. These results indicate the possibility of comammox bacteria in the system and highlight the complexity of nitrifying microbial communities in activated sludge relative to past understanding. Both microbial and chemical impacts of engineered nanoparticles on WWTP processes were also investigated. Metagenomic analysis of DNA extracted from activated sludge sequencing batch reactors dosed with gold nanoparticles with varied surface coating and morphology indicated that nanoparticle morphology impacted the microbial community and antibiotic resistance gene content more than surface coating. However, nanoparticle fate was controlled by surface coating more than morphology. Disinfection by-product formation in the presence of nanoparticles during WWTP disinfection was assessed using silver, titanium dioxide, ceria, and zero valent iron nanoparticles. Silver nanoparticles were found to enhance trihalomethane formation, which was attributed to the citrate coating of the nanoparticles. These studies both raise concern over the relationship between engineered nanoparticles and other emerging concerns in WWTPs, and take a step towards informing nanoparticle design in a manner that limits their associated environmental impact. / Ph. D.

Many antibiotic resistance genes (ARGs) conferring resistance to a broad range of antibiotics have often been detected in aquatic environments such as untreated and treated wastewater, river and surface water. ARG proliferation in the aquatic environment could depend upon various factors such as geospatial variations, the type of aquatic body, and the type of wastewater (untreated or treated) discharged into these aquatic environments. Likewise, the strong interconnectivity of aquatic systems may accelerate the spread of ARGs through them. Hence a comparative and a holistic study of different aquatic environments is required to appropriately comprehend the problem of antibiotic resistance. Many studies approach this issue using molecular techniques such as metagenomic sequencing and metagenomic data analysis. Such analyses compare the broad spectrum of ARGs in water and wastewater samples, but these studies use comparisons which are limited to similarity/dissimilarity analyses. However, in such analyses, the discriminatory ARGs (associated ARGs driving such similarity/ dissimilarity measures) may not be identified. Consequentially, the reason which drives the dissimilarities among the samples would not be identified and the reason for antibiotic resistance proliferation may not be clearly understood. In this study, an effective methodology, using Extremely Randomized Trees (ET) Algorithm, was formulated and demonstrated to capture such ARG variations and identify discriminatory ARGs among environmentally derived metagenomes. In this study, data were grouped by: geographic location (to understand the spread of ARGs globally), untreated vs. treated wastewater (to see the effectiveness of WWTPs in removing ARGs), and different aquatic habitats (to understand the impact and spread within aquatic habitats). It was observed that there were certain ARGs which were specific to wastewater samples from certain locations suggesting that site-specific factors can have a certain effect in shaping ARG profiles. Comparing untreated and treated wastewater samples from different WWTPs revealed that biological treatments have a definite impact on shaping the ARG profile. While there were several ARGs which got removed after the treatment, there were some ARGs which showed an increase in relative abundance irrespective of location and treatment plant specific variables. On comparing different aquatic environments, the algorithm identified ARGs which were specific to certain environments. The algorithm captured certain ARGs which were specific to hospital discharges when compared with other aquatic environments. It was determined that the proposed method was efficient in identifying the discriminatory ARGs which could classify the samples according to their groups. Further, it was also effective in capturing low-level variations which generally get over-shadowed in the analysis due to highly abundant genes. The results of this study suggest that the proposed method is an effective method for comprehensive analyses and can provide valuable information to better understand antibiotic resistance. / MS

The microbial source tracking (MST) methods of antibiotic resistance analysis (ARA) and fluorometry (to detect optical brighteners in detergents) were used in the summers of 2004 and 2005 to determine the origins of fecal pollution at beaches with a past history of, or the potential for, high enterococci counts and posted advisories. At Hilton and Anderson beaches, ARA and fluorometry in the summer of 2004 detected substantial human-origin pollution in locations producing consistently high counts of Enterococcus spp. Investigations by municipal officials led to the fluorometric detection and subsequent repair of sewage infrastructure problems at both beaches. The success of these mitigation efforts was confirmed during the summer of 2005 using ARA and fluorometry, with the results cross-validated by pulsed-field gel electrophoresis (PFGE). Results at other beaches indicated that birds and/or wildlife were largely responsible for elevated enterococci levels during 2004 and 2005. The application of fluorometry proved difficult in opens waters due to high levels of dilution, but showed potential for use in storm drains. An additional study developed and tested a new library-based MST approach based on the pattern of DNA band lengths produced by the amplification of the 16S-23S rDNA intergenic spacer region, and subsequent digestion using the restriction endonuclease MboI. Initial results from small known-source libraries yielded high average rates of correct classification (ARCC). However, an increase in the library size was accompanied by a reduction in the ARCC of the library and the method was deemed unsuccessful, and unsuitable for field application. A final study focused on the potential for classification bias with disproportionate source category sizes using discriminant analysis (DA), logistic regression (LR), and k-nearest neighbor (K-NN) statistical classification algorithms. Findings indicated that DA was the most robust algorithm for use with source category imbalance when measuring both correct and incorrect classification rates. Conversely k-NN was identified as the most sensitive algorithm to imbalances with the greatest levels of distortion obtained from the highest k values. Conclusions of this project include: 1) application of a validation set, as well as a minimum detectable percentage to known-source libraries aids in accurately assessing the classification power of the library and reducing the false positive identification of contributing fecal sources; 2) the validation of MST results using multiple methods is recommended for field applications; 3) fluorometry displayed potential for detecting optical brighteners as indicators of sewage leaks in storm drains; 4) the digestion of the 16S-23S rDNA intergenic spacer region of Enterococcus spp. using MboI does not provided suitable discriminatory power for use as an MST method; and 5) DA was the least, and k-NN the most, sensitive algorithm to imbalances in the size of source categories in a known-source library. / Ph. D.

Bakgrund: Antibiotikaresistens är ett globalt folkhälsoproblem vars ökning kan leda till att antibiotikabehandlingens verkan uteblir. Att förebygga antibiotikaresistens kan göras på många olika sätt, där utgångspunkten är att minska dagens antibiotikaanvändning för att i sin tur minska förekomsten av resistenta bakterier så framtida generationer kan behandla svåra infektioner. Sjuksköterskan är central i detta arbete till följd av omvårdnadsarbetets täta patientkontakt.  Syfte: Syftet var att beskriva sjuksköterskans kunskaper om förebyggande arbete gällande antibiotikaresistens.  Metod: Metoden var en allmän litteraturöversikt baserad på tio vetenskapliga artiklar.  Resultat: Tre teman identifierades: Sjuksköterskans kunskap om antibiotika och antibiotikaresistens, där sjuksköterskans kunskap om antibiotika generellt visades god medan kunskapen om antibiotikaresistens var något mer varierande. Sjuksköterskans förmåga och kunskap att arbeta enligt förebyggande praxis, vilket visade att sjuksköterskan till stor del hade god följsamhet att förebygga infektioner och därmed antibiotikaresistens. Desto högre utbildning och erfarenhet, desto bättre kunskap och följsamhet, vilket visade olika orsaker som ledde till bättre kunskap och följsamhet.  Slutsats: Litteraturöversikten visade att kunskap om antibiotikaresistens är nödvändigt för sjuksköterskor att ha för att de ska kunna arbeta förebyggande. Att öka kunskapen hos sjuksköterskor gällande antibiotikaresistens och hur det förebyggs i kliniskt arbete kan leda till en framtid där vi fortfarande kan behandla patienter med antibiotika. / Background: Antibiotic resistance is a global public health problem whose increase can cause antibiotic treatment to fail. Preventing antibiotic resistance can be done in many ways, where the basis is to reduce today’s antibiotic use in order to reduce the occurrence of resistant bacteria so that future generations can treat severe infections. The nurse is central in this work because of the frequent patient contact in nursing care.  Aim: The aim was to describe nurse’s knowledge of preventative work regarding antibiotic resistance.  Method: The method used was a general literature review based on ten research articles.  Results: Three themes were identified: Nurse’s knowledge regarding antibiotics and antibiotic resistance, where the nurse’s knowledge of antibiotics was generally shown to be good, while the knowledge of antibiotic resistance was somewhat more variable. Nurse’s ability and knowledge to work preventative, which showed that the nurse largely had good compliance to prevent infections and thus antibiotic resistance. The higher the education and experience, the better the knowledge and compliance, which showed different reasons that led to better knowledge and compliance.   Conclusions: This literature review showed that knowledge about antibiotic resistance is necessary for nurses to possess in order to prevent it in their profession. Increased nursing knowledge regarding antibiotic resistance and how to prevent it in clinical work results in a future where we are still able to treat patients with antibiotics.

The antibiotic resistance is a natural phenomenon that can be considered harmful for humans when antibiotic resistant bacteria transfers resistance genes to the human pathogens at uncontrollable rate. One of the potential environments for spreading of antibiotic resistance is found to be in biogas reactors due to their mesophilic conditions that are optimal for the bacterial reproduction. The sudden changes in the environment of biogas reactors could negatively affect both biogas production rate but also microbial reproduction ability. This experimental research was done to determine to what extend does changes in pH with the increased volume dosing of acetic acid affect the persistence of antibiotic resistant enterobacteria in biogas reactors. Additionally, this experimental research is aiming to determine the difference in the enterobacterial persistence between year 2023 and year 2024 for the two biogas reactors (Selena and Taylor) fed with the chemically based substrate. The results show that the general persistence of antibiotic resistant enterobacteria in both reactors increased year 2024 compared to year 2023. According to the blank samples, the average persistence of enterobacteria increased in Selena by 500% and in Taylor by 250%. However, overall increase of enterobacteria that are antibiotic resistant cannot be determined where it can only be observed that the resistance has increased against majority of tested antibiotics. The obtained results also show a trend that in most tested groups quantity of antibiotic resistant enterobacteria is gradually increasing with the higher volume of acetic acid. The pH level decreased with the increased volume of acetic acid prior the incubation process with an average of 6,4 for Taylor biogas reactor and 6,7 for Selena biogas reactor. This indicates that the composition of fatty acid affected the environment so that it became more acidic. However, after the incubation process, the increased pH was detected in all groups compared to the results prior the incubation with the pH average of 8,1 for Taylor biogas reactor and 7,9 for Selena biogas reactor. This indicates that the acetic acid was consumed by the present bacteria and a decrease of acetic acid quantity resulted in an increase of pH. With that being said, the acetic acid can be considered as the enrichment substrate for the enterobacterial colonies adequate for efficient exchange of antibiotic resistant genes hence reproduction. However, the statistical analysis (nonparametric Kruskal-Wallis) results show that only between minority of tested groups the significant difference was detected.

Introduktion: Världshälsoorganisationen (WHO) har förklarat att antibiotikaresistens är en global folkhälsokris som finns i både låginkomstländer och höginkomstländer. Faktorer som sjukvård, förskrivarnas kunskap om infektionssjukdomar och deras kommunikation påverkar antibiotikaanvändningen och dess förskrivning. Därför metoder som påverkar kommunikationsstrategier för en rationell användning av antibiotika kan vara fördelaktiga i samhället då förökad kunskap kan bidra till bättre användning av antibiotika . Syfte: Syftet med denna studie var att granska informationsaktiviteter i svenska regionala läkemedelskommittéer för att förebygga antibiotikaresistens: lärdomar och områden för förbättring. Metod: 21 läkemedelskommittéer i Sverige fick 5 frågor via e-post att besvara på gällande antibiotika kommunikation i kommunen. Detta för att sedan analysera svaren med hjälp av kvalitativ tematisk analys. Resultat: 18 läkemedelskommittéer besvarade på frågorna därav tre teman och ett undertema identifierades; Nationell reglering och övervakningsverktyg är viktiga för länen, undertema, frekvens av informationsutbyte om antibiotikaförskrivning, Covid-19-pandemins påverkan på informationsaktivitet och Patienternas medvetenhet – ett område att fokusera på. Slutsats: I Sverige har länen en viktig roll att förmedla information, uppdateringar av riktlinjer och följa upp antibiotikaförskrivningar på förskrivarnivå. Covid-19 pandemin har ökat kunskapen om att fysiska möten är mer efektiva för att förmedla information. Det finns även ett behov av att utbilda allmänheten även om regionerna är fokuserade på denna aspekt, de flesta län måste fortfarande uppmärksamma detta specifika område. / Introduction: The World Health Organization (WHO) has declared that antibiotic resistance is a global public health emergency that exists in both low income countries and high income countries. Factors like healthcare, prescribers’ knowledge in infectious diseases and their communication affect antibiotic use and its prescription. Therefore methods that impact communication strategies enhance rational use of antibiotics. Aim: The purpose of this study was to explore information activities of Swedish Regional Pharmaceutical Committees for preventing antibiotic resistance. Also we tried to explore if there were certain lessons to be learned regarding effective information activities, and to identify any rooms for improvements.  Methods: 21 Pharmaceutical committees in Sweden were sent 5 questions via email to answer regarding antibiotic communication in the county. This to then analyze the responses using qualitative thematic analysis. Results: 18 counties responded to the questions whereas three themes and one sub-theme were identified; National regulation and surveillance tools are important for counties, subtheme, frequency of information sharing regarding antibiotic prescription, the COVID-19 pandemic’s impact on information activity and Patient’s awareness – an area to focus on. Conclusion: In Sweden, the counties have an important role to relay information, update on guidelines and follow up on antibiotic prescription at prescriber level. The Covid-19 pandemic has increased the knowledge that physical meetings are more effective to convey information. There is also a need to educate the general public though counties are focused on this aspect, majority of counties still need to bring their attention to this particular area.

Doctor of Philosophy / Department of Entomology / Ludek Zurek / Enterococci are a group of commensal bacteria that are important nosocomial pathogens. They are abundant in human sewage and wastewater treatment facilities (WWTF). This study focused on the role of house flies, Musca domestica, in the ecology of enterococci at WWTF in both field and laboratory experiments. The first study objective focused on sampling and characterizing enterococci from house flies and wastewater sludge from four WWTF in northeastern Kansas. Enterococci were quantified, identified, and screened for antibiotic resistance and virulence traits, and genotyped. The profiles of enterococci (spp. diversity, antibiotic resistance and virulence) from WWTF sludge and the house flies were similar, indicating that the flies successfully acquired the bacteria from the WWTF substrate. Enterococci with the greatest amount of antibiotic resistant and virulence traits originated from the WWTF that processed meat waste from a commercial sausage plant. Genotyping of E. faecalis revealed clonal matches from sludge and house flies. The second study objective involved tracking the fate of E. faecalis in the digestive tract of house flies in laboratory assays. Colony forming unit (CFU) counts were highest in the midgut at 1 h and declined during the first 24 h. In the labellum, foregut and hindgut, E. faecalis concentrations were more variable but were overall higher after 24 h. Observations from CFU counts and visualizations under a dissecting microscope revealed that E. faecalis peaked in the crop after 48 h suggesting active proliferation in this region. The third objective of the study involved tracking the emergence of calyptrate muscoid flies from stockpiled biosolid cake at one of the four WWTF. Traps were employed at the site for a total of 47 weeks, totaling 386 trap-weeks. A total of 11,349 calyptrate muscoid flies were identified with the two most common species being stable flies (Stomoxys calcitrans) (9,016, 80.2%) and house flies (2022, 18.0%). Numbers of stable flies and house flies peaked around mid-July each year and a second, smaller peak was observed for stable flies 5-8 weeks later. Estimated annual emergence of stable flies was 551,404 and for house flies 109,188.

Doctor of Philosophy / Department of Grain Science and Industry / Subramanyam Bhadriraju / Ludek Zurek / Feed samples and live stored-product insects from feed mills and swine farms were collected and cultured for Enterococcus spp. The mean concentration of enterococci in insect and feed were 2.7 ± 0.5 × 101 cfu/insect and 6.3 ± 0.7 × 103 cfu/g respectively. A total of 362 isolates of enterococci collected from 89 feed samples and 228 stored-product insects were identified to the species level using PCR. These isolates were represented by Enterococcus casseliflavus (53.0%), E. gallinarum (20.4%), E. faecium (16.2%), E. hirae (5.2%), and E. faecalis (5.0%). Enterococci were phenotypically resistant to tetracycline (48.0%), erythromycin (14.3%), streptomycin (16.8%), kanamycin (12.1%), ciprofloxacin (11.0%), ampicillin (3.3%), and chloramphenicol (1.1%). All isolates were susceptible to vancomycin and gentamicin. Tetracycline resistance was encoded by tetM (50.0%), tetO (15.1%), tetK (0.5%), tetS (0.2%) and other unknown tetracycline determinants. Enterococci carried virulence genes including gelatinase (gelE; 21.5%), an enterococcus surface protein (esp; 1.9%), and cytolysin (cylA; 2.2%). An aggregation substance (asa1) gene was detected in 61.0% of E. faecalis isolates. Fifty perncet of E. faecalis isolates were phenotipically tested positive for aggregation substances. Enterococci with cylA genes were hemolytic (52.0%) and with gelE genes were gelatinolytic (18.5%). The ermB gene, encoding erythromycin resistance was detected in 8.8% of the total isolates. The Tn916/1545 family of conjugative transposons was detected in 10.7% of the isolates. Laboratory experiments showed that adults of the red flour beetle, Tribolium castaneum (Herbst), fed on poultry and cattle feeds inoculated with E. faecalis OG1RF:pCF10, were able to successfully acquire enterococci and contaminate sterile poultry and cattle feeds. To assess the potential of horizontal gene transfer, conjugation assays were carried out with E. faecalis using a donor (wild strains) and recipient (E. faecalis OG1SSP) in ratio of 1:10. Only one isolate (1 out of 18 E. faecalis) could transfer tetM to a recipient using broth mating. However, filter mating assay, followed by PCR confirmation revealed that 89.0% (16 out of 18 E. faecalis) of isolates could transfer tetM to E. faecalis. Transfer ratios of transconjugant per recipients ranged from 2.6 × 10-4 to 1 × 10-9. In summary, feed (52.0%) and stored-product insects (41.6%) collected from feed mills and swine farms carried antibiotic-resistant and potentially virulent enterococci. Our study showed that T. castaneum, a pest commonly associated with feed, served as a potential vector for enterococci in the feed environment. Conjugation assays followed by PCR confirmed presence of the tetM gene on a mobile genetic element(s) such as Tn916 and may be horizontally transferred to other Enterococcus species and to other bacteria of clinical significance.