The purpose of this work was to set a benchmark for a monitoring and surveillance programme on the volumes of the eighteen classes of antimicrobials available and consumed by animals for the benefit of animal health in South Africa. In setting up such a programme, risk assessment and possible management and communication strategies of the potential health risks emanating from antimicrobial resistance in bacteria from animals and man were considered and the survey was compared with other overseas surveillance programmes established in Sweden, Denmark, the United Kingdom and Australia. The aim of the study was to contribute to the establishment of future surveillance programmes that will provide direction for the prudent and rational use of antimicrobials, involving all the relevant stake-holders, in order to preserve the future efficacy of those antimicrobials that are currently available. Such programmes will harmonize with international initiatives and contribute to the provision of databases for policy recommendations in South Africa. There are several benefits to the implementation of such surveys and addressing topical and relevant issues of antimicrobial use in the domain of animal health and its possible impact on human health. Furthermore, policy decisions to address concerns regarding antimicrobial resistance may be reached in a more informed and judicious manner, with the aim that the efficacy of available antimicrobials may be preserved for use in future generations of humans and animals. The authorized antimicrobials available in South Africa were firstly reviewed and compared with the volumes of antimicrobials supplied by the veterinary pharmaceutical companies. The majority of antimicrobials were registered under the Stock Remedies Act 36 of 1947. It was found that the class of antimicrobial representing the most registered products was the tetracyclines, followed by the penicillins, the sulphonamides, macrolides, lincosamides and pleuromutilins. This correlated well with the volumes of antimicrobials supplied, as these classes of antimicrobials also represented the top four groups of antimicrobials consumed. Eight of the pool of twenty-five veterinary pharmaceutical companies approached provided more detailed information on the volumes of antimicrobials consumed for the years under review, namely 2002 to 2004. The potency of antimicrobials was also requested, in order to establish trends of increasing or decreasing potency of the active ingredients, versus the volumes of antimicrobials supplied. It was established within the scope of this study, that the majority of consumed antimicrobials was from the classes of macrolides, lincosamides, and pleuromutilins, followed by the tetracycline class, the sulphonamide group and fourthly the penicillins. The potency of the active ingredients supplied by the companies did not change and therefore had no impact on the interpretation of antimicrobials consumed. These results give cause for concern in terms of the possibility of cross-resistance between antimicrobials used in the domain of animal health, and those used in the human medical field. There is also another concern, namely the exposure of humans to veterinary drug residues in food, causing modifications in the bacterial ecology of the human gut, thereby leading to a possible perturbation in the protective human gut barrier with overgrowth and invasion of pathogenic bacteria. Although much has been written about the possibility of anaphylactic reactions occurring in sensitized human individuals from â-lactams and macrolides administered in food animals, this issue has been reviewed extensively and it has been concluded that allergies from antimicrobial residues in the diet are extremely rare. The macrolide tylosin was the most extensively sold antimicrobial of all. Tylosin is one of four antimicrobials that was banned by the EU in 1999 because of its structural relatedness to therapeutic antimicrobials used for the treatment of disease in human medicine. The other three classes mentioned above, the penicillins, tetracyclines and sulphonamides are also relevant because of well-documented evidence of the ability to select for resistance or because of their structural relatedness to human therapeutic antimicrobials and their use in humans. The value of sales of antimicrobials were provided by SAAHA (South Africa Animal Health Association) and also scrutinized within the context of this study in order to obtain meaningful data on the national consumption of antimicrobials. However, as discussed in Chapter 5, the data were not of any value within the context of this study because the sales data were provided in monitory terms only. Volumes of sales of feed were also obtained and companies that mix feed approached to ascertain the percentages of antimicrobial medicated feeds consumed. It was ascertained in this survey that 68,5% of the grand total of antimicrobials surveyed during 2002 to 2004, were administered as in-feed medications. Tylosin was the most extensively used in-feed antimicrobial, followed by oxytetracycline, zinc bacitracin and olaquindox. This result emphasizes the importance of establishing a monitoring programme for the volumes of antimicrobials used, as many growth promoters used in-feed are administered at sub-therapeutic levels over long periods of time that tend to select for antimicrobial resistance. Whereas 17,5% of the total volume of antimicrobials utilized were parenteral antimicrobials, antimicrobials for water medication constituted 12% of the total. “Other” dosage forms such as the topical, aural, ophthalmic, and aerosol antimicrobials and intra-uterine pessaries and tablets constituted 1,5% of the total. Intramammary antimicrobials represented 0,04% of the total. In Chapters 1, 5&6 the surveillance systems for veterinary antimicrobials used by other countries are discussed and compared. It was concluded that a combination of the surveillance systems applied by Australia and the United Kingdom is the best model (with modifications) to apply to the animal health situation in South Africa. Such a surveillance system of the volumes of veterinary antimicrobials consumed should ideally be implemented in conjunction with a veterinary antimicrobial resistance surveillance and monitoring programme to generate meaningful data that will contribute to the rational use of antimicrobials in order to preserve the efficacy of the existing antimicrobials in South Africa. Possible trends of antimicrobial usage in animals may be uncovered over time from implementing a programme for the volumes of antimicrobials used and thereby lead to proactive application of rational policies for the veterinary use of antimicrobials. This information can also be compared with international data, in order to harmonize as much as possible with global monitoring programmes of veterinary antimicrobial usage. Copyright / Dissertation (MMedVet)--University of Pretoria, 2008. / Paraclinical Sciences / unrestricted

The number of foodborne disease outbreaks related to fresh produce has increased in recent years. This has coincided with a growing public demand for minimally processed fruits and vegetables. Effective produce sanitizers are therefore needed that are at least as effective as chlorine, currently the most commonly used sanitizer. Natural antimicrobials from plant extracts and essential oils are a possible alternative. These are highly effective and may also be used in situations in which chlorine is not advantageous; for instance, in situations in which chlorine has limited efficacy or because of concerns over the production of harmful by-products resulting from chlorine use. Plant derived essential oils have been shown to be antibacterial, antiviral, and antifungal. In this study we examined the use of natural antimicrobials from plant extracts and essential oils as possible alternative sanitizers. We examined these antimicrobials for their efficacy against Escherichia coli. In addition, since many of these natural compounds are believed to be membrane active, silver ions were added to some of the tests to assess the potential for synergy between the antimicrobials. Silver ions, although slow-acting on their own, often exhibit a synergistic antimicrobial effect when combined with other membrane active antimicrobials such as oxidizing agents. These studies reveal that plant derived antimicrobials are effective sanitizers with the potential to replace commonly used chlorine

Campylobacter jejuni is one of the most common causes of foodborne diarrheal illness in the U.S. and worldwide. (1-2). C. jejuni infection in humans is most often attributed to undercooked poultry (3-6). However, since 2001, the Centers for Disease Control (CDC) has confirmed 9 outbreaks of campylobacteriosis linked to consumption of beef and beef products, resulting in 297 illnesses and 10 hospitalizations, and cattle isolates have been linked to other human infections (7-10). Because Campylobacter infection is generally sporadic, and not all cases are linked to poultry, other animal reservoirs such as beef likely exist. Because beef is not commonly considered a significant source of Campylobacter, interventions regarding beef cattle are generally geared toward other pathogens, such as E. coli O157:H7. Interventions to prevent Campylobacter spread in poultry houses include reducing flock colonization and bacterial loads, (11), as well as interventions directly targeting consumer behavior. Despite these efforts, many countries have not been able to reduce the prevalence of Campylobacter in poultry. The goals of this research were to 1) determine Campylobacter loads in broilers at poultry farms and processing houses through the 3-tube MPN method, and determine baseline data for poultry production systems, 2) describe temporal relationships and prevalence of Campylobacter strains in a potentially underrepresented host/environment (cattle feedlot environment), and 3) test the efficacy of natural, plant derived compounds against C. jejuni on meat. Our results show that there is a significant positive association between pre-harvest and post-harvest Campylobacter loads in poultry, with Campylobacter levels during the final step of processing remaining at infectious levels. Beef cattle represent another potential and not well-described source of campylobacteriosis, as beef cattle and their environment become rapidly contaminated with Campylobacter from weaning through processing, and cross-contamination of carcasses is possible. This research also determined that natural plant extracts of cinnamon and oregano essential oils, when added to edible films, reduced surface contamination of retail poultry meat with C. jejuni, and thus may be a useful post-harvest intervention for future use in packaging of retail meat with a high risk of Campylobacter contamination.

<p>Chlamydia trachomatis, a pathogen responsible for major diseases of significant clinical and public health importance, remains poorly characterized because of its intractability to molecular genetic manipulation. The development of a system(s) for genetic analysis would significantly accelerate our ability to identify genes that enable Chlamydia to establish infection, survive within its host, and cause disease. This thesis describes two methods used to assess gene function in Chlamydia and to provide insights into its biology and pathogenesis. The first method described is based on specific inhibitors and is used to probe the role of lipooligosaccharide (LOS), a main lipid components of bacterial outer membranes. Using this approach, we show that small molecule inhibitors of LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase], the enzyme that catalyzes the first committed step in the biosynthesis of lipid A, blocks the synthesis of LOS in C. trachomatis. In the absence of LOS, Chlamydia remains viable and establishes a pathogenic vacuole ("inclusion") that supports robust bacterial replication. However, bacteria grown under these conditions were no longer infectious. In the presence of LpxC inhibitors, replicative reticulate bodies accumulated in enlarged inclusions but failed to express selected late-stage proteins and transition to elementary bodies, a Chlamydia developmental form that is required for invasion of mammalian cells. These findings suggest the presence of an outer membrane quality control system that regulates Chlamydia developmental transition to infectious elementary bodies and highlights the potential application of LpxC inhibitors as unique class of anti-chlamydial agents.</p><p> The second part of this thesis describes the development of a system with which to perform forward genetics in C. trachomatis. Forward genetics approaches set out to identify the gene or set of genes that contributes to a specific biological process and usually entails generating random mutations in a large number of organisms, isolating mutants with an aberrant phenotype, and identifying the alleles associated with the mutant phenotype. In this approach, chemical mutagenesis is coupled with whole genome sequencing (WGS) and a system for DNA exchange within infected cells to generate Chlamydia mutants with distinct phenotypes, map the underlying genetic lesions, and generate isogenic strains. We identified mutants with altered glycogen metabolism, including an attenuated strain defective for Type II secretion. The coupling of chemically induced gene variations and WGS to establish genotype-phenotype associations should be broadly applicable to the growing list of microorganisms intractable to traditional genetic mutational analysis.</p> / Dissertation

O Brasil é hoje um dos maiores produtores e exportadores de alimento no mundo, destacando-se no cenário de produção de proteína animal como o 1º país exportador em volume de carne de frango, dentre tantas conquistas recentes do agronegócio brasileiro. Essas conquistas foram alcançadas pelo país graças à sua inegável vocação agropecuária, resultado de uma série de fatores, como extensão territorial e áreas agricultáveis, clima, disponibilidade de água e ambiente regulatório favorável à adoção de tecnologias. Dentre as tecnologias existentes e disponíveis que tornam possível um país de vocação agrícola se tornar um dos maiores produtores mundiais de alimentos estão os aditivos zootécnicos melhoradores do desempenho e, dentre eles, os antimicrobianos. Na posição de um dos atores principais no cenário internacional de produção de alimentos, o Brasil vem-se deparando com questões relacionadas à preocupação global com relação à segurança do alimento e aos escândalos de contaminação de alimentos ocorridos em diversos países, inclusive na Europa. A preocupação com a segurança do alimento tem levado os diversos organismos internacionais, dentre eles, a Organização Mundial da Saúde e a Organização Mundial de Saúde Animal a estabelecerem critérios de análise de risco com relação aos principais patógenos envolvidos em surtos de toxinfecções alimentares e ao risco de transmissão de resistência bacteriana através dos alimentos. Sendo assim, o presente estudo foi desenvolvido com base na lista de antimicrobianos criticamente importantes, elaborada em conjunto pela OMS e OIE, na determinação do perfil de resistência destes frente a Salmonella isolada de aves e materiais avícolas em diferentes períodos. Foram utilizadas 100 amostras de Salmonella spp isoladas em granjas no Brasil, sendo 68 amostras colhidas durante o período atual (de 2008 a 2010) e 32 amostras colhidas durante a década de 90 (de 1989 a 1999), as quais passaram por testes de difusão em disco, determinação da concentração inibitória mínima, sorotipificação e determinação clonal por eletroforese em campo pulsado. Com relação às amostras colhidas no período atual (2008 a 2010), evidenciou-se uma redução no perfil de sensibilidade para diferentes antimicrobianos, marcadamente para a classe das cefalosporinas, com 19 (22%) das amostras resistentes e 38 (56%) das amostras apresentando resistência intermediária. Várias das amostras desse período apresentaram multirresistência. Para 32 das amostras isoladas na década de 90, não se evidenciou resistência a nenhum dos antimicrobianos selecionados, embora se tenham encontrado 13 amostras (41%) com perfil de resistência intermediário para o ceftiofur. Ainda, detectaram-se 10 diferentes sorovares de Salmonella com somente um perfil clonal para as amostras antigas e perfil variado para as amostras atuais. Os resultados comprovaram o aumento de resistência aos antimicrobianos criticamente importes em amostras recentes isoladas de aves e material avícola. Tais dados comprovam que cada vez mais a atenção deve ser focada no uso prudente dos antimicrobianos na produção animal. / Brazil is currently one of the largest producers and exporters of food in the world, appearing in the animal protein production ranking as the 1st exporter of poultry, in terms of volume, among many recent achievements of the brazilian agribusiness. These achievements have been made possible due to the country natural agriculture vocation, which is the result of a number of factors, such as land extension and available land for agriculture, climate, water availability and regulatory environment open to technology adoption. Among the existing technologies available that enable a country to become one of the largest producers of food are the feed additives and performance enhancers, including the antibiotics. In the position of one of the key actors in the international food production environment, Brazil has been facing issues related to the global concerns on food safety and the food contamination scandals occurred in several countries, including in Europe. These concerns has led several international organizations, including the World Health Organization and the International Organization of Animal Health to establish criteria for risk analysis in relation to the main pathogens involved in foodborne disease outbreaks and the risk of transmission of resistant bacteria from food. Therefore, this study was developed based on the list of the critically important antimicrobials, developed jointly by WHO and the OIE, in determining the resistance profile of them against Salmonella isolated from poultry and poultry materials in different periods. It have been used 100 samples of Salmonella isolated from poultry farms in Brazil, 68 samples taken during the current period (2008 to 2010) and 32 samples taken during the 90s (1989-1999), which have been tested for antibiotic disk diffusion, minimum inhibitory concentration, serotyping and clone determination by pulsed field gel electrophoresis. In the samples collected in the current period (2008-2010), there was a reduction in susceptibility to different antimicrobials, notably for the class of cephalosporins with 19 (22%) of isolates resistant and 38 (56%) of samples presenting intermediate resistance. Several samples from this period showed multidrug resistance. For 32 of the isolates in the 90s, there was no evidence of resistance to any antimicrobial selected, although it was found 13 samples (41%) with intermediate resistance profile for ceftiofur. It have been identified 10 different Salmonella serovars, among them only one clone profile for the old samples and different clone profiles for the current samples. Results showed increased antimicrobial resistance to the critically important antimicrobials for the more recent isolates from poultry and poultry material. These data demonstrate that more attention should be focused on the prudent use of antimicrobials in animal production.

The fruit fly Drosophila melanogaster is recognised as the most widely established genetic model of immunity of the contemporary scientific era, exhibiting a high degree of conservation between Drosophila and mammalian innate immunity genes. However, whilst the majority of Drosophila immunity studies have previously been performed in adults and larvae, the embryo has recently emerged as a potentially viable model system; aiding in vivo studies and providing a more amendable system to undertake live imaging, hence evading many of the caveats associated with current immunity models. This project aimed to further develop the Drosophila embryo as a more potent and insightful immunity model, focusing on the immune response to bacterial infection. Initial results demonstrated that the Stage 15 Drosophila embryo was able to mount a relatively robust immune response to bacterial infection. This included induction of antimicrobial peptide (AMP) genes upon a range of bacterial stimuli; a response which was able to effectively discriminate between differential types of bacterial infection via the characterised Drosophila systemic immunity pathways. Live-imaging studies also showed that the cellular immune response was functional within the Stage 15 embryo. Subsequently, immune competence was shown to arise at approximately mid-embryogenesis, under the control of 20-hydroxyecdysone (20-HE) signalling, as demonstrated by the partial rescue of AMP expression and bacterial clearance in early stage embryos upon 20-HE co-administration with infective agents. Further analysis of the global transcriptional response of the Drosophila embryo to infection and damage via microarray studies confirmed the immune potential of this system, but also permitted the identification of genes upregulated uniquely upon Gram-positive or Gram-negativ infection. Moreover, wounding via sterile laser ablation induced significant upregulation of a subset of AMP genes an a network of cuticular genes, providing an insight into the embryonic damage response. Parallel analysis of the hemocyte transcriptional profile upon infection and damage elucidated that these immune cells may play a role in the regulation of the immune response via 20-HE signalling and production of ROS, although this remains subject to further validation. As such, transcriptional profile analysis of the embryo has been successful in identifying candidate genes for further validation and study.

O Brasil é hoje um dos maiores produtores e exportadores de alimento no mundo, destacando-se no cenário de produção de proteína animal como o 1º país exportador em volume de carne de frango, dentre tantas conquistas recentes do agronegócio brasileiro. Essas conquistas foram alcançadas pelo país graças à sua inegável vocação agropecuária, resultado de uma série de fatores, como extensão territorial e áreas agricultáveis, clima, disponibilidade de água e ambiente regulatório favorável à adoção de tecnologias. Dentre as tecnologias existentes e disponíveis que tornam possível um país de vocação agrícola se tornar um dos maiores produtores mundiais de alimentos estão os aditivos zootécnicos melhoradores do desempenho e, dentre eles, os antimicrobianos. Na posição de um dos atores principais no cenário internacional de produção de alimentos, o Brasil vem-se deparando com questões relacionadas à preocupação global com relação à segurança do alimento e aos escândalos de contaminação de alimentos ocorridos em diversos países, inclusive na Europa. A preocupação com a segurança do alimento tem levado os diversos organismos internacionais, dentre eles, a Organização Mundial da Saúde e a Organização Mundial de Saúde Animal a estabelecerem critérios de análise de risco com relação aos principais patógenos envolvidos em surtos de toxinfecções alimentares e ao risco de transmissão de resistência bacteriana através dos alimentos. Sendo assim, o presente estudo foi desenvolvido com base na lista de antimicrobianos criticamente importantes, elaborada em conjunto pela OMS e OIE, na determinação do perfil de resistência destes frente a Salmonella isolada de aves e materiais avícolas em diferentes períodos. Foram utilizadas 100 amostras de Salmonella spp isoladas em granjas no Brasil, sendo 68 amostras colhidas durante o período atual (de 2008 a 2010) e 32 amostras colhidas durante a década de 90 (de 1989 a 1999), as quais passaram por testes de difusão em disco, determinação da concentração inibitória mínima, sorotipificação e determinação clonal por eletroforese em campo pulsado. Com relação às amostras colhidas no período atual (2008 a 2010), evidenciou-se uma redução no perfil de sensibilidade para diferentes antimicrobianos, marcadamente para a classe das cefalosporinas, com 19 (22%) das amostras resistentes e 38 (56%) das amostras apresentando resistência intermediária. Várias das amostras desse período apresentaram multirresistência. Para 32 das amostras isoladas na década de 90, não se evidenciou resistência a nenhum dos antimicrobianos selecionados, embora se tenham encontrado 13 amostras (41%) com perfil de resistência intermediário para o ceftiofur. Ainda, detectaram-se 10 diferentes sorovares de Salmonella com somente um perfil clonal para as amostras antigas e perfil variado para as amostras atuais. Os resultados comprovaram o aumento de resistência aos antimicrobianos criticamente importes em amostras recentes isoladas de aves e material avícola. Tais dados comprovam que cada vez mais a atenção deve ser focada no uso prudente dos antimicrobianos na produção animal. / Brazil is currently one of the largest producers and exporters of food in the world, appearing in the animal protein production ranking as the 1st exporter of poultry, in terms of volume, among many recent achievements of the brazilian agribusiness. These achievements have been made possible due to the country natural agriculture vocation, which is the result of a number of factors, such as land extension and available land for agriculture, climate, water availability and regulatory environment open to technology adoption. Among the existing technologies available that enable a country to become one of the largest producers of food are the feed additives and performance enhancers, including the antibiotics. In the position of one of the key actors in the international food production environment, Brazil has been facing issues related to the global concerns on food safety and the food contamination scandals occurred in several countries, including in Europe. These concerns has led several international organizations, including the World Health Organization and the International Organization of Animal Health to establish criteria for risk analysis in relation to the main pathogens involved in foodborne disease outbreaks and the risk of transmission of resistant bacteria from food. Therefore, this study was developed based on the list of the critically important antimicrobials, developed jointly by WHO and the OIE, in determining the resistance profile of them against Salmonella isolated from poultry and poultry materials in different periods. It have been used 100 samples of Salmonella isolated from poultry farms in Brazil, 68 samples taken during the current period (2008 to 2010) and 32 samples taken during the 90s (1989-1999), which have been tested for antibiotic disk diffusion, minimum inhibitory concentration, serotyping and clone determination by pulsed field gel electrophoresis. In the samples collected in the current period (2008-2010), there was a reduction in susceptibility to different antimicrobials, notably for the class of cephalosporins with 19 (22%) of isolates resistant and 38 (56%) of samples presenting intermediate resistance. Several samples from this period showed multidrug resistance. For 32 of the isolates in the 90s, there was no evidence of resistance to any antimicrobial selected, although it was found 13 samples (41%) with intermediate resistance profile for ceftiofur. It have been identified 10 different Salmonella serovars, among them only one clone profile for the old samples and different clone profiles for the current samples. Results showed increased antimicrobial resistance to the critically important antimicrobials for the more recent isolates from poultry and poultry material. These data demonstrate that more attention should be focused on the prudent use of antimicrobials in animal production.

>Magister Scientiae - MSc / Antimicrobials are chemical compounds that destroy or inhibit the growth of microorganisms. The majority of these antimicrobials are actually natural products or natural product derived with key examples being the pioneer antibiotics penicillin and cephalosporin. Antimicrobials are an extremely important class of therapeutic agents; however, the development of drug resistance and slow pace of new antibiotic discovery is one of the major health issues facing the world today. There is therefore a crucial need to discover and develop new antibacterial agents. In this study, the potential of marine algae as a source of new antibiotics was explored. Crude organic extracts and chromatographic fractions obtained from small-scale extraction of 17 different marine algae were used to prepare a pre-fractionated library that would be tested against several disease causing microorganisms. The activity of the pre-fractionated library and purified compounds was determined against a panel of drug resistant microorganisms namely Acinetobacter baumannii ATCCBAA®-1605™, Enterococcus faecalis ATCC® 51299™, Escherichia coli ATCC® 25922™, Staphylococcus aureus subsp. aureus ATCC® 33591™ and Candida albicans ATCC® 24433™. Finally, cytotoxicity tests of 50 selected library extracts and isolated compounds were done against two cell lines namely MCF-7 (breast cancer) and HEK-293 (kidney embryonic). Based on their antimicrobial activity and interesting chemical profiles, the seaweeds Plocamium sp. and Stypopodium multipartitum were selected for further study. Three new and unusual halogenated monoterpenes (4.16, 4.17 and 4.18) were isolated from Plocamium sp., and an unusual meroditerpenoid (5.8) was isolated from Stypopodium multipartitum. The metabolites were purified using preparative (silica gel) chromatography as well as semipreparative normal phase HPLC. The structures of purified compounds were determined from spectroscopic data, including nuclear magnetic resonance (NMR) spectroscopy. A small library of 153 fractions was generated from collections of South African marine algae. Pre-fractionated crude extracts showed excellent antimicrobial activity against all microbes but particularly against Staphylococcus aureus. The compounds were generally active against the Gram positive bacteria and the yeast. In conclusion, three antimicrobial halogenated monoterpenes and an unusual monoterpene were isolated from a Plocamium sp. and Stypopodium multipartitum respectively. Antimicrobial activity of crude fractions was excellent but that of isolated compounds was not as great as anticipated.

Bacterial isolates from animals, foods and clinical samples with resistances to one or more antibiotics are being found frequently each year. Selective pressure exerted by antibiotic growth promoters in food animals has been considered a main cause for the development of antibiotic resistance and antibiotic use has been strongly criticized as a serious public health threat. The gastrointestinal tract of animals not only serves as a reservoir of zoonotic agents but also as a spot for exchange of genetic information between pathogenic and commensal bacteria. Humans get infections from resistant bacteria either through the food chain, contaminated water or by direct contact with animals. In this situation much of the concern has been directed against the use of antibiotic growth promoters in animals. The removal of synthetic antibiotics from animal diets created other problems such as a decline in animal welfare and an increase in the use of therapeutic antibiotics. So there is a need for new alternatives to antimicrobial drugs to overcome resistance development and related problems. Plants and plant-derived compounds have long been considered to posess antimicrobial activity since they were frequently used in ancient medicine as natural remedies to treat human infections. Identifying new sources of natural antimicrobials and inhibitors of resistance development will yield novel therapeutic drugs and extend the useful life of existing antibiotics. In the present work, individual and combined effects of five essential oils (eugenol, thymol, carvacrol, cinnamaldehyde, allyl isothiocyanate (AIT)) and a formaldehyde-based feed additive, Termin 8, with antibiotics against 4 antibiotic resistant bacteria with known determinants for resistance were tested using broth microdilution and the checker board assay. The bacteria showed considerable susceptibility towards these antimicrobials and a significant reduction in the minimum inhibitory concentrations (MICs) of antibiotics was noted when paired combinations of antibiotic and antimicrobial were used. The synergistic interaction was further confirmed by the extent of decrease in logarithmic count or viable population (Log DP). Although most of the combinations were synergistic by fractional inhibitory concentration (FIC) values, fewer combinations showed synergistic interaction when Log DP was considered. Gram-positive bacteria were more sensitive to the antimicrobials than Gram-negative bacteria. In combination studies, carvacrol was more effective and showed synergistic interaction with at least three antibiotics. When used alone, AIT was more effective and the concentration needed to exhibit antimicrobial action was much lower when compared to other compounds. An in vitro study was conducted to assess the antibacterial effects of Termin 8 and thymol in chicken caecal digesta and poultry feed samples by using a thin agar layer (TAL) method. Concentrations greater than the MIC of both the compounds was required to exert antimicrobial activity in the feed and digesta samples. The natural antimicrobials and Termin 8 had significant inhibitory effects on the drug resistant bacteria and synergistically enhanced the efficacy of antibiotics when used in combination. Further studies are needed to test their effectiveness in animal models.

Bacterial isolates from animals, foods and clinical samples with resistances to one or more antibiotics are being found frequently each year. Selective pressure exerted by antibiotic growth promoters in food animals has been considered a main cause for the development of antibiotic resistance and antibiotic use has been strongly criticized as a serious public health threat. The gastrointestinal tract of animals not only serves as a reservoir of zoonotic agents but also as a spot for exchange of genetic information between pathogenic and commensal bacteria. Humans get infections from resistant bacteria either through the food chain, contaminated water or by direct contact with animals. In this situation much of the concern has been directed against the use of antibiotic growth promoters in animals. The removal of synthetic antibiotics from animal diets created other problems such as a decline in animal welfare and an increase in the use of therapeutic antibiotics. So there is a need for new alternatives to antimicrobial drugs to overcome resistance development and related problems. Plants and plant-derived compounds have long been considered to posess antimicrobial activity since they were frequently used in ancient medicine as natural remedies to treat human infections. Identifying new sources of natural antimicrobials and inhibitors of resistance development will yield novel therapeutic drugs and extend the useful life of existing antibiotics. In the present work, individual and combined effects of five essential oils (eugenol, thymol, carvacrol, cinnamaldehyde, allyl isothiocyanate (AIT)) and a formaldehyde-based feed additive, Termin 8, with antibiotics against 4 antibiotic resistant bacteria with known determinants for resistance were tested using broth microdilution and the checker board assay. The bacteria showed considerable susceptibility towards these antimicrobials and a significant reduction in the minimum inhibitory concentrations (MICs) of antibiotics was noted when paired combinations of antibiotic and antimicrobial were used. The synergistic interaction was further confirmed by the extent of decrease in logarithmic count or viable population (Log DP). Although most of the combinations were synergistic by fractional inhibitory concentration (FIC) values, fewer combinations showed synergistic interaction when Log DP was considered. Gram-positive bacteria were more sensitive to the antimicrobials than Gram-negative bacteria. In combination studies, carvacrol was more effective and showed synergistic interaction with at least three antibiotics. When used alone, AIT was more effective and the concentration needed to exhibit antimicrobial action was much lower when compared to other compounds. An in vitro study was conducted to assess the antibacterial effects of Termin 8 and thymol in chicken caecal digesta and poultry feed samples by using a thin agar layer (TAL) method. Concentrations greater than the MIC of both the compounds was required to exert antimicrobial activity in the feed and digesta samples. The natural antimicrobials and Termin 8 had significant inhibitory effects on the drug resistant bacteria and synergistically enhanced the efficacy of antibiotics when used in combination. Further studies are needed to test their effectiveness in animal models.