Escherichia coli é um bacilo Gram-negativo, anaeróbico facultativo e de distribuição cosmopolita. E. coli coloniza o intestino de humanos e outros animais endotérmicos logo após o nascimento, estabelecendo-se como um importante membro da microbiota intestinal. Algumas cepas de E. coli podem adquirir fatores de virulência, assumindo assim, uma natureza patogênica, como é o caso das E. coli patogênicas extraintestinais (ExPEC). As cepas ExPEC apresentam a capacidade de colonizar e se disseminar em diversos nichos no hospedeiro, e são divididas em UPEC (E. coli uropatogênica), NMEC (E. coli causadora de meningite neonatal) e APEC (E. coli patogênica para aves). UPEC, NMEC e APEC compartilham fatores associados à virulência. Para serem aptas a causar doença, cepas ExPEC devem apresentar pelo menos um fator associado à adesão, um fator para captação de ferro (sideróforo) e um fator de resistência ao soro, podendo, também, apresentar genes que codificam toxinas e invasinas. Embora sejam conhecidos muitos fatores de virulência associados à patogenicidade de cepas ExPEC, a regulação da expressão de tais fatores ainda não foi elucidada. Fumarato-nitrato-redutase (FNR) é uma proteína que atua como regulador global, agindo como um sensor da presença de oxigênio em bactérias gramnegativas. Já foi demonstrado que FNR está relacionada à regulação da virulência de bactérias patogênicas como Shigella flexneri e Salmonella enterica serovar Typhimurium. Este trabalho teve como objetivo a análise epidemiológica e caracterização de cepas APEC, bem como a investigação do controle da expressão de fatores associados à virulência de ExPEC pelo regulador global FNR. Os resultados da análise epidemiológica das cepas APEC mostram o perfil de resistência aos agentes antimicrobianos, a prevalência dos fatores de virulência e dos grupos filogenéticos (de acordo com a classificação EcoR) e a relação filogenética dos isolados, fornecendo um panorama da caracterização de E. coli patogênicas aviárias de lesões severas de celulite e de infecção sistêmica oriundas da região sul do Brasil. Em relação ao FNR, este estudo mostrou a influência deste regulador sobre importantes fatores associados à virulência, estando envolvido no controle de várias etapas do estabelecimento da infecção por cepas ExPEC. A deleção de fnr na cepa UPEC CFT 073 reduziu a motilidade, a expressão das fimbrias tipo I e tipo P, reduziu a expressão da hemolisina e controlou a expressão da ilha de patogenicidade do α- cetoglutarato. Além disso, a deleção de fnr fez tornou as bactérias incapazes de invadir células dos rins e da bexiga e de causar doença in vivo em camundongos de 6 semanas. FNR também foi capaz de controlar as etapas da infecção por NMEC 56. Uma vez deletado, as bactérias perderam a capacidade de causar bacteremia, de crescer no fluido cerebrospinal e de causar doença in vivo em ratos de 5 dias de idade. A deleção de fnr em APEC O1 resultou na diminuição da expressão da proteína OmpT plasmidial, da fímbria do tipo I e do auto-transportador AatA. A principal contribuição deste trabalho foi demonstrar que FNR atua na regulação da expressão de importantes fatores associados à virulência de cepas ExPEC (UPEC, NMEC e APEC), sendo importante para o estabelecimento da infecção por essas cepas. Neste trabalho, verificamos que, além da função já conhecida de regular os genes envolvidos na manutenção de um meio anaeróbico, FNR também atua no controle de genes associados à virulência de cepas ExPEC, refletindo na capacidade de causar doença que tais cepas apresentam. / Escherichia coli is a Gram-negative bacillus, facultative anaerobic and has cosmopolitan distribution. E. coli colonizes the intestine of humans and other endothermic animals immediately after birth, establishing as an important member of the intestinal microbiota. Some strains of E. coli can acquire virulence factors thereby assuming a pathogenic nature, as in the case of extraintestinal pathogenic E. coli (ExPEC). ExPEC strains have the ability to colonize and spread out in different niches of the host, and are divided into UPEC (uropathogenic E. coli), NMEC (newborn meningitis E. coli) and APEC (avian pathogenic E. coli). UPEC, NMEC and APEC share virulence factors. To be able to cause disease, ExPEC strains must produce virulence factors required for adherence, for iron uptake (siderophore) and for resistance to serum and may also contain genes encoding toxins and invasins. Although many virulence factors associated with the pathogenicity of ExPEC strains are known, the regulation of the expression of these factors has not yet been fully elucidated. Fumarate nitrate reductase (FNR) is a global regulatory protein, acting as a sensor of oxygen in Gram- negative bacteria. It has been shown that FNR relates virulence of pathogenic bacteria such as Shigella flexneri and Salmonella enterica serovar Typhimurium. The aim of this study was to do an epidemiological analysis and characterization of APEC strains as well as the investigation of regulation of ExPEC’s virulence factors by the global FNR regulator. The results of epidemiological analysis of APEC strains showed the profile of antimicrobial resistance , the prevalence of virulence factors and phylogenetic groups (according to the EcoR group) and the phylogenetic relationship of the isolates, providing an overview of the characterization of avian pathogenic E. coli causing severe cellulitis lesions and systemic infection originating from southern Brazil. In relation to FNR, this study showed the influence of this important regulator of virulence factors that is involved in controlling various stages of establishment of infection by ExPEC strains. Deletion of fnr in UPEC strain CFT 073 reduced motility and expression of type I and type P fimbriae, reduced the expression of hemolysin and control the expression of the pathogenicity island of α -ketoglutarate. Furthermore, fnr mutant strains were unable to invade cells of kidney and bladder, and to colonize the urinary tract of 6 weeks-old mice. FNR was also able to control the stages of infection of NMEC 56. The fnr mutant lost its ability to cause bacteremia, grow in cerebrospinal fluid, cause disease in 5 days old rats. Deletion of fnr in APEC O1 resulted in decreased expression of genes corresponding to the plasmid encoded OmpT protein, type I fimbriae and autotransporter AatA. The main contribution of this work was to demonstrate that FNR regulates expression of important virulence factors of ExPEC strains (UPEC, NMEC and APEC), which is important for the establishment of infection by these strains. In this work, we found that, besides the already known function in regulating genes involved in maintaining an anaerobic environment, FNR also acts in the control of virulenceassociated genes of ExPEC strains, reflecting the ability of these strains to cause disease.

Escherichia coli patogênica aviária (APEC) é a causa de doenças extra-intestinais em aves, que se manifestam na forma de doenças localizadas ou infecções sistêmicas, gerando grandes perdas econômicas para a indústria aviária. O objetivo desse trabalho foi estudar isolados de APEC do sul do Brasil em relação à resistência a agentes antimicrobianos; a prevalência de fatores associados à virulência; a tipagem filogenética e o perfil filogenético. Na avicultura, os agentes antimicrobianos são muito utilizados na prevenção de infecções e na forma de promotores de crescimento. Foram utilizadas 41 isolados de E. coli obtidos de infecções sistêmicas e 144 isolados de celulite, utilizando o método de difusão com discos. Isolados APEC apresentaram baixos níveis de resistência, com excessão da tetraciclina e das sulfonamidas; e para isolados de colissepticemia, também foi observado uma resistência aos antimicrobianos que atuam na parede celular (ampicilina, cefalotina, bacitracina e ceftiofur). Vários fatores de virulência têm sido investigados em cepas APEC, os que têm sido mais frequentemente associados com a patogenicidade são as fímbrias de aderência F1 e Tsh (hemaglutinina sensível à temperatura); o sistema sideróforo aerobactina, a proteína iss (increased serum survival), a cápsula K e a produção de colicina V. Foram realizadas reações da polimerase em cadeia multiplex (PCR), testando um total de 33 fatores nos isolados APEC. Os fatores relacionados a adesão, sideróforos e resistência ao soro foram presentes em todas as amostras. Os fatores que apresentaram maior prevalência nas amostras foram, fimC, ompA, crlA e traT. A tipagem filogenética foi realizada através do método descrito por Clermont et al., (2000), que permite separar os isolados em 4 grupos filogenéticos principais (A, B1, B2 e D). Observamos a maior presença de isolados APEC pertencentes ao grupo D, tanto para celulite quanto para colissepticemia. A análise do perfil filogenético foi realizada pelo método ARDRA, que é baseado na variação da região do espaçamento intergênico (ISR) na região 16S-23S do DNA ribossômico. Os resultados foram analisados formando um dendrograma que mostra a proximidade filogenética dos isolados, indicando que não foi possível separar os clones de celulite dos de colissepticemia e não existem clones endêmicos nas regiões analisadas. Os resultados obtidos no presente estudo, fornecem um panorama geral da resistência aos agentes antimicrobianos, prevalência dos fatores de virulência, tipagem filogenética e perfil filogenético encontrados nos isolados de E. coli aviária do sul do Brasil de infecções de colissepticemia e celulite. / extra-intestinal infections in poultry, called colibacilose, and cause great economic losses to the poultry industry. The aim of this work was to examine APEC strains, isolated from avian cellulitis and colisepticemic chickens from South Brazil, for antibiotic resistance, presence of virulence-associated genes (VAGs), phylogenetic typing and phylogenetic analyses. In poultry, antibiotics are routinely used to prevent infection and promote growth. We analyzed the susceptibility to 15 antibiotics in 144 E. coli isolates collected from cellulitis lesions and in 41 isolates from septicemic chickens. APEC isolates have shown low levels of resistance excepting tetracycline and sulphonamides, and colisepticemic isolates were resistant to antibiotics that act in the cell wall, such as ampicilin, cephalotin, ceftiofur and bacitracin. The virulence factors most frequently associated with APEC pathogenicity are the adhesins F1 and Tsh (Temperature sensitive hemagglutinin), the iron acquisition system aerobactin, the protein Iss (increased serum survival), K1 capsule and production of colicin V. To investigate the presence of VAGs in the isolates, we performed multiplex polymerase chain reactions to test 33 virulence factors. Virulence factors related to adhesion, iron acquisition and serum resistance were present in almost all strains. The factors fimC, ompA, crlA and traT were the most frequent in APEC isolates. The phylogenetic typing were done using the Clermont et al. (2000) method, which classifies E. coli strains into four main phylogenetic groups (A, B1, B2, and D). Our phylogenetic typing has shown that cellulitis and colisseptisemic isolates were more related to group D. Amplified ribossomal restriction analysis (ARDRA) was performed in colissepticemic and celullitis isolates from broiler chickens from Southern Brasil. The similarity among isolates were observed with a dendrogram based on the band pattern. Our results have shown that clones of celullitis and colissepticemic isolates could not be distinguished and there is no endemic clones in regions observed analised. The results of the present work give us a panorama of the susceptibility to antibiotics, virulence factors, phylogenetic typing and phylogenetic analyses currently found in APEC isolates from severe lesions of cellulites and colibacillosis in south Brazil. Besides that, these analyses could be helpful for monitoring and preventing outbreakes of colibacilosis. Controling the first stages of the disease and detecting more prevalent clones in this region may reduce the incidence of the disease.

Escherichia coli é um bacilo Gram-negativo, anaeróbico facultativo e de distribuição cosmopolita. E. coli coloniza o intestino de humanos e outros animais endotérmicos logo após o nascimento, estabelecendo-se como um importante membro da microbiota intestinal. Algumas cepas de E. coli podem adquirir fatores de virulência, assumindo assim, uma natureza patogênica, como é o caso das E. coli patogênicas extraintestinais (ExPEC). As cepas ExPEC apresentam a capacidade de colonizar e se disseminar em diversos nichos no hospedeiro, e são divididas em UPEC (E. coli uropatogênica), NMEC (E. coli causadora de meningite neonatal) e APEC (E. coli patogênica para aves). UPEC, NMEC e APEC compartilham fatores associados à virulência. Para serem aptas a causar doença, cepas ExPEC devem apresentar pelo menos um fator associado à adesão, um fator para captação de ferro (sideróforo) e um fator de resistência ao soro, podendo, também, apresentar genes que codificam toxinas e invasinas. Embora sejam conhecidos muitos fatores de virulência associados à patogenicidade de cepas ExPEC, a regulação da expressão de tais fatores ainda não foi elucidada. Fumarato-nitrato-redutase (FNR) é uma proteína que atua como regulador global, agindo como um sensor da presença de oxigênio em bactérias gramnegativas. Já foi demonstrado que FNR está relacionada à regulação da virulência de bactérias patogênicas como Shigella flexneri e Salmonella enterica serovar Typhimurium. Este trabalho teve como objetivo a análise epidemiológica e caracterização de cepas APEC, bem como a investigação do controle da expressão de fatores associados à virulência de ExPEC pelo regulador global FNR. Os resultados da análise epidemiológica das cepas APEC mostram o perfil de resistência aos agentes antimicrobianos, a prevalência dos fatores de virulência e dos grupos filogenéticos (de acordo com a classificação EcoR) e a relação filogenética dos isolados, fornecendo um panorama da caracterização de E. coli patogênicas aviárias de lesões severas de celulite e de infecção sistêmica oriundas da região sul do Brasil. Em relação ao FNR, este estudo mostrou a influência deste regulador sobre importantes fatores associados à virulência, estando envolvido no controle de várias etapas do estabelecimento da infecção por cepas ExPEC. A deleção de fnr na cepa UPEC CFT 073 reduziu a motilidade, a expressão das fimbrias tipo I e tipo P, reduziu a expressão da hemolisina e controlou a expressão da ilha de patogenicidade do α- cetoglutarato. Além disso, a deleção de fnr fez tornou as bactérias incapazes de invadir células dos rins e da bexiga e de causar doença in vivo em camundongos de 6 semanas. FNR também foi capaz de controlar as etapas da infecção por NMEC 56. Uma vez deletado, as bactérias perderam a capacidade de causar bacteremia, de crescer no fluido cerebrospinal e de causar doença in vivo em ratos de 5 dias de idade. A deleção de fnr em APEC O1 resultou na diminuição da expressão da proteína OmpT plasmidial, da fímbria do tipo I e do auto-transportador AatA. A principal contribuição deste trabalho foi demonstrar que FNR atua na regulação da expressão de importantes fatores associados à virulência de cepas ExPEC (UPEC, NMEC e APEC), sendo importante para o estabelecimento da infecção por essas cepas. Neste trabalho, verificamos que, além da função já conhecida de regular os genes envolvidos na manutenção de um meio anaeróbico, FNR também atua no controle de genes associados à virulência de cepas ExPEC, refletindo na capacidade de causar doença que tais cepas apresentam. / Escherichia coli is a Gram-negative bacillus, facultative anaerobic and has cosmopolitan distribution. E. coli colonizes the intestine of humans and other endothermic animals immediately after birth, establishing as an important member of the intestinal microbiota. Some strains of E. coli can acquire virulence factors thereby assuming a pathogenic nature, as in the case of extraintestinal pathogenic E. coli (ExPEC). ExPEC strains have the ability to colonize and spread out in different niches of the host, and are divided into UPEC (uropathogenic E. coli), NMEC (newborn meningitis E. coli) and APEC (avian pathogenic E. coli). UPEC, NMEC and APEC share virulence factors. To be able to cause disease, ExPEC strains must produce virulence factors required for adherence, for iron uptake (siderophore) and for resistance to serum and may also contain genes encoding toxins and invasins. Although many virulence factors associated with the pathogenicity of ExPEC strains are known, the regulation of the expression of these factors has not yet been fully elucidated. Fumarate nitrate reductase (FNR) is a global regulatory protein, acting as a sensor of oxygen in Gram- negative bacteria. It has been shown that FNR relates virulence of pathogenic bacteria such as Shigella flexneri and Salmonella enterica serovar Typhimurium. The aim of this study was to do an epidemiological analysis and characterization of APEC strains as well as the investigation of regulation of ExPEC’s virulence factors by the global FNR regulator. The results of epidemiological analysis of APEC strains showed the profile of antimicrobial resistance , the prevalence of virulence factors and phylogenetic groups (according to the EcoR group) and the phylogenetic relationship of the isolates, providing an overview of the characterization of avian pathogenic E. coli causing severe cellulitis lesions and systemic infection originating from southern Brazil. In relation to FNR, this study showed the influence of this important regulator of virulence factors that is involved in controlling various stages of establishment of infection by ExPEC strains. Deletion of fnr in UPEC strain CFT 073 reduced motility and expression of type I and type P fimbriae, reduced the expression of hemolysin and control the expression of the pathogenicity island of α -ketoglutarate. Furthermore, fnr mutant strains were unable to invade cells of kidney and bladder, and to colonize the urinary tract of 6 weeks-old mice. FNR was also able to control the stages of infection of NMEC 56. The fnr mutant lost its ability to cause bacteremia, grow in cerebrospinal fluid, cause disease in 5 days old rats. Deletion of fnr in APEC O1 resulted in decreased expression of genes corresponding to the plasmid encoded OmpT protein, type I fimbriae and autotransporter AatA. The main contribution of this work was to demonstrate that FNR regulates expression of important virulence factors of ExPEC strains (UPEC, NMEC and APEC), which is important for the establishment of infection by these strains. In this work, we found that, besides the already known function in regulating genes involved in maintaining an anaerobic environment, FNR also acts in the control of virulenceassociated genes of ExPEC strains, reflecting the ability of these strains to cause disease.

BACKGROUND AND OBJECTIVES: Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal infections and deaths in human. It can also cause infections in pregnant women and non-pregnant adults. Penicillin and ampicillin are antibiotics of choice for the treatment of GBS infections. Erythromycin and clindamycin are used as alternative therapy in penicillin allergic patients, however resistance to these agents has been increasingly observed. This present study was undertaken to determine the colonization rate of GBS, susceptibility profile and the mechanism of antibiotic resistance in pregnant women and their babies at Dr. George Mukhari Academic Hospital in Pretoria. METHODS: Rectal and vaginal swabs were collected from pregnant women; ear and umbilical swabs from newborns over an 11 month period. Samples were cultured on selective media (CNA agar and Todd-Hewitt broth) and GBS positively identified using morphological and biochemical tests including Gram staining, hemolytic activity, catalase test, bile esculin, CAMP test and Latex agglutination test. The susceptibility testing was done using the Kirby-Bauer and E-test methods. The D-test method was used to determine the inducible clindamycin resistance. Multiplex PCR with were used to detect different genes coding for resistance. RESULTS: Out of the 413 patients evaluated, 128 (30.9%) were positive with GBS. All isolates were sensitive to penicillin and ampicillin. Erythromycin and clindamycin resistance was 21.1% and 17.2% respectively; of which 69% harbouring constitutive MLBB, 17.4% inducible MLSB. The alteration of ribosomal target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was detected in one of isolates. Conclusion: This study reaffirms the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it raises the challenges of resistance to the macrolides and lincosamides. More GBS treatment options for penicillin allergic patients need to be researched. / Health Studies / M.Sc. (Life Sciences (Microbiology))

BACKGROUND AND OBJECTIVES: Streptococcus agalactiae (Group B Streptococcus, GBS) is the leading cause of neonatal infections and deaths in human. It can also cause infections in pregnant women and non-pregnant adults. Penicillin and ampicillin are antibiotics of choice for the treatment of GBS infections. Erythromycin and clindamycin are used as alternative therapy in penicillin allergic patients, however resistance to these agents has been increasingly observed. This present study was undertaken to determine the colonization rate of GBS, susceptibility profile and the mechanism of antibiotic resistance in pregnant women and their babies at Dr. George Mukhari Academic Hospital in Pretoria. METHODS: Rectal and vaginal swabs were collected from pregnant women; ear and umbilical swabs from newborns over an 11 month period. Samples were cultured on selective media (CNA agar and Todd-Hewitt broth) and GBS positively identified using morphological and biochemical tests including Gram staining, hemolytic activity, catalase test, bile esculin, CAMP test and Latex agglutination test. The susceptibility testing was done using the Kirby-Bauer and E-test methods. The D-test method was used to determine the inducible clindamycin resistance. Multiplex PCR with were used to detect different genes coding for resistance. RESULTS: Out of the 413 patients evaluated, 128 (30.9%) were positive with GBS. All isolates were sensitive to penicillin and ampicillin. Erythromycin and clindamycin resistance was 21.1% and 17.2% respectively; of which 69% harbouring constitutive MLBB, 17.4% inducible MLSB. The alteration of ribosomal target encoded by ermB genes was the commonest mechanism of resistance observed in 55% of isolates, 38% of isolates had both ermB and linB genes and efflux pump mediated by mefA genes was detected in one of isolates. Conclusion: This study reaffirms the appropriateness of penicillin as the antibiotic of choice for treating GBS infection. However it raises the challenges of resistance to the macrolides and lincosamides. More GBS treatment options for penicillin allergic patients need to be researched. / Health Studies / M. Sc. (Life Sciences (Microbiology))

Le suivi de la viabilitié, la croissance et le métabolisme cellulaire des bactéries peut contribuer de manière significative au diagnostic précoce de la maladie, mais peut aussi aider à améliorer le rendement des produits bactériens dans des expériences industrielle ou à petite echelle. Les méthodes conventionnelles utilisées pour l'étude de la sensibilité des bactéries aux antibiotiques sont basées principalement sur la culture, une technique qui prend au moins 12 heures pour rendre un résultat. Ce retard conduit au surtraitement d'un large éventail d'infections par des antibiotiques à large spectre, ce qui est coûteux et peut conduire à l'apparition de résistance à ces antibiotiques précieux, tandis que la détection rapide d'une infection virale ou l'absence de bactéries pourrait prévenir de tels traitements et, dans le cas d'une infection bactérienne, l'identification de la sensibilité aux antibiotiques pourrait permettre l'utilisation d'antibiotiques à spectre étroit. Le projet décrit dans le présent document vise à surveiller les activités biologiques des bactéries vivantes immobilisées sur les surfaces biofonctionnalisées de microstructures composées de semi-conducteurs quantiques (QS). Le procédé dépend de la sensibilité de la photoluminescence (PL) émise par des semi-conducteurs à la perturbation du champ électrique induit par la charge électrique des bactéries immobilisées sur la surface de ces structures. Dans la première phase du projet, nous avons étudié une méthode innovante impliquant la surveillance par PL de l'effet de photocorrosion dans des hétérostructures GaAs/AlGaAs. Le maintien d'un équilibre entre la sensibilité et la stabilité du biocapteur dans l'environnement aqueux nous a permis de détecter Escherichia coli K12 dans des solutions salines tamponnées au phosphate (PBS) avec une limite de détection attrayante de 103 UFC/ml en moins de 2 heures. Suite à cette recherche, nous avons émis l'hypothèse que ces hétérostructures pourraient être utilisés pour développer une méthode à faible coût et quasiment en temps reel de la croissance et de la sensibilité des bactéries aux antibiotiques. L'un des éléments clés dans le développement de cette plate-forme de biocapteurs était de démontrer que le GaAs (001), normalement utilisé pour recouvrir les hétérostructures de GaAs/AlGaAs, ne nuira pas à la croissance des bactéries. Dans la deuxième phase du projet, nous avons exploré la capture et la croissance de E. coli K12 sur des surfaces nues et biofonctionnalisées de GaAs (001). Il a été déterminé que la couverture initiale et les taux de croissance de bactéries dépendent de l'architecture de biofonctionnalisation utilisée pour capturer les bactéries: les surfaces biofonctionnalisées avec d'anticorps présentaient une efficacité de capture significativement plus élevée. En outre, on a trouvé que pour des suspensions contenant des bactéries à moins de 105 UFC/ml, la surface des plaquettes de GaAs ne supportait pas la croissance des bactéries, quel que soit le type d'architecture de biofonctionnalisation. Dans la troisième phase du projet, nous avons suivi la croissance et la sensibilité aux antibiotiques de E. coli K12 et E. coli HB101. Tandis que la présence de bactéries retardaient d’apparition du maximum de PL, la croissance des bactéries retardaient encore plus ce maximum. Par contre, en presence d’antibiotiques efficaces, la croissance des bactéries était arrêtée et le maximum de PL est arrivé plus tôt. Ainsi, nous avons pu distinguer entre des E. coli sensibles ou résistantes à la pénicilline ou à la ciprofloxacine en moins de 3h. En raison de la petite taille, du faible coût et de la réponse rapide du biocapteur, l'approche proposée a le potentiel d'être appliquée dans les laboratoires de diagnostic clinique pour le suivi rapide de la sensibilité des bactéries aux antibiotiques. / Abstract : Monitoring the viability, growth and cellular metabolism of bacteria can contribute significantly to the early diagnosis of disease, but can also help improve yield of bacterial products in industrial- or small-scale experiments. Conventional methods applied for investigation of antibiotic sensitivity of bacteria are mostly culture-based techniques that are time-consuming and take at least 12 h to reveal results. This delay leads to overtreatment of a wide range of infections with broad spectrum antibiotics which is costly and may lead to the development of resistance to these precious antibiotics, whereas rapid detection of a viral infection or absence of bacteria could prevent such treatments and, in the case of bacterial infection, identification of antibiotic susceptibility could allow use of narrow spectrum antibiotics. The project outlined in this document aims at monitoring biological activities of live bacteria immobilized on biofunctionalized surfaces of quantum semiconductor (QS) microstructures. The method takes advantage of the sensitivity of photoluminescence (PL) emitting semiconductors to the perturbation of the electric field induced by the electric charge of bacteria immobilized on the surface of these structures. Our hypothesis was that bacteria growing on the surface of biofunctionalized QS biochips would modify their PL in a different, and measurable way in comparison with inactivated bacteria. In the first phase of the project, we investigated an innovative method involving PL monitoring of the photocorrosion effect in GaAs/AlGaAs heterostructures. Maintaining the balance between device sensitivity and stability in the biosensing (aqueous) environment allowed us to detect Escherichia coli K12 in phosphate buffered saline solutions (PBS) at an attractive limit of detection of 103 CFU/mL in less than 2 hours. Following this research, we hypothesised that these heterostructures could be employed to develop a method for inexpensive and quasi-real time monitoring of the growth and antibiotic susceptibility of bacteria. One of the key elements in the development of this biosensing platform was to demonstrate that GaAs (001), normally used for capping PL emitting GaAs/AlGaAs heterostructures, would not inhibit the growth of bacteria. In the second phase of the project, we explored the capture and growth of E. coli K12 on bare and biofunctionalized surfaces of GaAs (001). It has been determined that the initial coverage, and the subsequent bacterial growth rates are dependent on the biofunctionalization architecture used to capture bacteria, with antibody biofunctionalized surfaces exhibiting significantly higher capture efficiencies. Moreover, for suspensions containing bacteria at less than 105 CFU/mL, it has been found that the surface of GaAs wafers could not support the growth of bacteria, regardless of the type of biofunctionalization architecture. In the third phase of the project, we used PL to monitor the growth and antibiotic susceptibility of E. coli K12 and E. coli HB101 bacteria. While immobilization of bacteria on the surface of GaAs/AlGaAs heterostructures retards the PL monitored photocorrosion, growth of these bacteria further amplifies this effect. By comparing the photocorrosion rate of QS wafers exposed to bacterial solutions with and without antibiotics, the sensitivity of bacteria to the specific antibiotic could be determined in less than 3 hours. Due to the small size, low cost and rapid response of the biosensor, the proposed approach has the potential of being applied in clinical diagnostic laboratories for quick monitoring of antibiotic susceptibility of different bacteria.

Antimicrobial resistance is considered by many prominent researcher and scientist as a profound global health crisis that us humans must face in the next decade. It is threatening the effectiveness of these once-reliable weapons against bacterial infections and leaving us susceptible to pathogenic agents. The indiscriminate overprescription of antibiotic in healthcare and animal husbandry, has led to an increased emergence of “super bugs”— a resistant strain of bacteria that were once susceptible to antibiotic—. The escalating creation of those resistant bacteria has been coupled with a proliferation of research papers that seek to explain the working mechanism of antibiotics and their efficacy on the bacterial pathogens, however these efforts often fall short of explaining the impact that antibiotics has on the bacterial metabolism. This project utilizes an established technology, specifically silicone nano-wire ion-selective field-effect transistor in an innovative approach to discern alteration in the metabolic pathways induced by various antibiotics. The methodology involves measuring extracellular acidity of the tested culture and converting it to an electrical signal to extract valuable information about the metabolic process of the bacteria, and how is altered in the presence of antibiotics. Empirical observations pertaining bacteriostatic antibiotics suggests comprehensive suppression of metabolic pathways, encompassing the efflux transition from acetyl-CoA to acetate, resulting an elevated pH level in cultures treated with bacteriostatic agents relative to their wild-type counterparts. Our experimental data also indicates a shift in bacterial metabolic and physiological responses to bactericidal antibiotic-induced stress which include an increased respiration rate, and a heightened activity of the TCA cycle in the test group with bactericidal antibiotics, causing acetate uptake from the medium and decelerating the acidification of the treated culture compared to the wild-type. The results clearly demonstrate a successful utilization of the chip to further study the effects that antibiotics have on bacteria and the interplay between bacterial metabolism and antibiotic efficacy.

Background Broth microdilution (BMD) is a gold-standard reference method to determine minimum inhibitory concentration (MIC) of antibiotics. For this, a standardized concentration of bacterial inoculum (2e5–8e5 colony-forming units, CFU/ml) is added to progressively higher concentrations of antibiotics. Bacteria stop growing at a particular antibiotic concentration termed MIC. Like other assays, various biological and/or technical factors can affect BMD results.   Aims To investigate the effects of inoculum concentration (5e4–5e6 CFU/ml), growth-medium concentration (cation-adjusted Mueller-Hinton Broth (CAMHB)), ranging 0.5x to 2x (1x as standard)) and age (<6-months or >1-year old) of fastidious medium on MIC results. And to compare BMD results using 5 different brands of CAMHBs and 1 cation-non-adjusted MH-broth (non-CAMHB).   Methods 12 isolates of bacteria (gram-positive (n=3), gram-negative(n=5), fastidious isolates (n=7)) and custom-made antibiotics-containing plates for gram-positive (11 antibiotics) or gram-negative bacteria (10 antibiotics) were used. Overnight-grown colonies were used to prepare BMD solutions (MH-broth + inoculum +/- fastidious) which were plated on antibiotic-plates as well as diluted prior to plating on agar-plates. Antibiotic- and agar-plates were incubated (18–20hr, 35°C) and used to determine MICs (following European Committee on Antimicrobial Susceptibility Testing instructions) and actual number of viable bacteria in BMD solutions, respectively.   Results Increasing inoculum concentration increased MICs of all antibiotics except cefoxitin. Piperacillin–tazobactam, levofloxacin, benzylpenicillin and ampicillin were especially sensitive to increase in inoculum and showed a 4-fold increase in >50% isolates. MICs for tobramycin, tigecycline and gentamicin increased by 2-fold in >50% isolates every time MH-broth concentration increased. Age of fastidious medium had no decipherable pattern of effects on MIC. All MH-broths gave similar results except when testing daptomycin which gave higher MICs with non-CAMHB compared to CAMHB.    Conclusion This research reveals some technical factors affecting MIC results. These results could help define parameters for automated BMD-performing-systems. However, this research shows only trends as more replicates are needed to determine statistically significant results.