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Ascertaining mycobacterial inducible resistance to peptidoglycan synthesis inhibitors
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A tuberculose (TB), doenƧa infeciosa causada pelo agente patogĆ©nico Mycobacterium tuberculosis (Mtb), Ć© um grave problema de saĆŗde pĆŗblica e uma significante causa de morte. A situaĆ§Ć£o epidemiolĆ³gica Ć© agravada pela emergĆŖncia e propagaĆ§Ć£o de estirpes resistentes, nomeadamente TB multirresistente (MDR-TB) e TB extensivamente resistente (XDR-TB), que reduzem as opƧƵes terapĆŖuticas e levam a um maior risco de insucesso do tratamento. Esta situaĆ§Ć£o levou Ć necessidade de reavaliar opƧƵes alternativas ao tratamento padrĆ£o, incluindo a potencial adiĆ§Ć£o de Ī²-lactĆ¢micos aos regimes terapĆŖuticos.
Os Ī²-lactĆ¢micos sĆ£o uma eficaz classe de antibiĆ³ticos, que tĆŖm como alvo as transpeptidases envolvidas na sĆntese do peptidoglicano (PG), um componente da parede celular comum a todas as bactĆ©rias. Anteriormente, os Ī²-lactĆ¢micos tinham sido excluĆdos do tratamento de TB devido Ć resistĆŖncia intrĆnseca de Mtb. Esta Ć© conferida pela presenƧa de uma eficaz Ī²-lactamase, BlaC, e pela peculiar parede celular micobacteriana, com um cross-linking do PG atĆpico, sintetizado por L,D-transpeptidases, enzimas resistentes Ć inibiĆ§Ć£o pela maioria dos Ī²-lactĆ¢micos. No entanto, estudos recentes demonstraram que a combinaĆ§Ć£o de um Ī²-lactĆ¢mico com um inibidor da Ī²-lactamase, adicionado a um regime de tratamento da TB tem resultados promissores contra estirpes resistentes de Mtb. De modo a otimizar a aplicaĆ§Ć£o dos Ī²-lactĆ¢micos, Ć© importante caracterizar o seu potencial de induĆ§Ć£o de resistĆŖncia em micobactĆ©rias. Assim, o objetivo desta tese foi explorar o potencial de induĆ§Ć£o de resistĆŖncia de inibidores da sĆntese do PG, nomeadamente Ī²-lactĆ¢micos, em micobactĆ©rias, apĆ³s tratamento com estes fĆ”rmacos, e caracterizar os isolados obtidos.
Para este fim, isolados resistentes da estirpe nĆ£o-patogĆ©nica M. smegmatis (Msm) foram selecionados apĆ³s tratamento com elevadas concentraƧƵes de vĆ”rios antibiĆ³ticos. Obtiveram-se trinta e nove isolados: vinte e quatro derivados da estirpe Msm mc2155 (WT), selecionados com amoxicilina, meropenem (com e sem adiĆ§Ć£o de clavulanato), vancomicina, rifampicina, isoniazida e etambutol; trĆŖs derivados da estirpe Msm PM965, selecionados com meropenem; e doze isolados submetidos a dois tratamentos de seleĆ§Ć£o, o primeiro com isoniazida ou etambutol, e o segundo com amoxicilina ou meropenem. Para cada tratamento, a frequĆŖncia de resistĆŖncia foi determinada, com valores entre 1x10-6 e 1x10-10, sendo os mais elevados observados para a isoniazida, a amoxicilina e o meropenem. A determinaĆ§Ć£o das concentraƧƵes mĆnimas inibitĆ³rias (CMIs) confirmou o fenĆ³tipo de resistĆŖncia para a maioria dos isolados; e evidĆŖncia de resistĆŖncia cruzada foi observada para os dois Ī²-lactĆ¢micos, meropenem e amoxicilina, e ainda para outros trĆŖs antibiĆ³ticos pertencentes a classes distintas, amoxicilina, vancomicina e isoniazida.
Os isolados selecionados com um Ī²-lactĆ¢mico foram ainda caracterizados atravĆ©s da realizaĆ§Ć£o de ensaios de hidrĆ³lise da nitrocefina e de efluxo de brometo de etĆdio. DiferenƧas entre os isolados selecionados com os dois Ī²-lactĆ¢micos (meropenem e amoxicilina), observadas neste ensaio, sugerem a aĆ§Ć£o de diferentes mecanismos de resistĆŖncia, embora, em ambos os casos, ter sido registado um aumento da CMI da amoxicilina. Poucas diferenƧas entre isolados e estirpes parentais foram observadas nos ensaios de efluxo, com a exceĆ§Ć£o de dois isolados selecionados com amoxicilina e um selecionado com amoxicilina e clavulanato que sofreram uma inesperada diminuiĆ§Ć£o da capacidade de efluxo.
A anĆ”lise genĆ³mica revelou que os isolados selecionados com um fĆ”rmaco anti-TB desenvolveram mutaƧƵes nos genes que codificam o alvo destes antibiĆ³ticos, confirmando assim que o mĆ©todo de seleĆ§Ć£o utilizado neste trabalho foi bem-sucedido e as conclusƵes que permitiu obter podem ser associadas ao organismo patogĆ©nico Mtb. As mutaƧƵes identificadas nos isolados selecionados com amoxicilina ou vancomicina localizam-se em genes reguladores, que apesar de nĆ£o serem diretamente afetados pelo antibiĆ³tico podem regular a expressĆ£o de genes associados aos alvos destes fĆ”rmacos.
Estes resultados confirmam o desenvolvimento de resistĆŖncia induzida a inibidores da sĆntese do PG, assim como o envolvimento de diferentes mecanismos de resistĆŖncia. A anĆ”lise genĆ³mica de vĆ”rios isolados estĆ” ainda a decorrer e juntamente com uma anĆ”lise de RNA-sequencing, irĆ”, no futuro, fornecer mais esclarecimentos em relaĆ§Ć£o aos mecanismos envolvidos na resistĆŖncia aos antibiĆ³ticos.
Tuberculosis (TB), caused by the pathogenic organism Mycobacterium tuberculosis (Mtb), is a major public health problem and a leading cause of death. The epidemiologic situation is aggravated by the emergence and spreading of resistant strains, namely multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB), which reduce the treatment options and result in a higher risk of therapy failure. This led to the necessity of revaluating alternative options for the standard treatment of TB, including the potential addition of Ī²-lactams to therapy regimens. Ī²-lactams are a highly successful class of antibiotics which target the transpeptidases involved in the biosynthesis of the peptidoglycan (PG), a cell wall (CW) component common to all bacteria. Ī²-lactams were previously excluded from TB therapy due to the intrinsic resistance of Mtb to this antibiotic class, conferred by the presence of a highly effective Ī²-lactamase, BlaC, and the unusual mycobacterial CW, with a PG cross-linking performed by L,D-transpeptidases resistant to inhibition by most Ī²-lactams. However, recent studies showed that the combination of a Ī²-lactam with a Ī²-lactamase inhibitor added to an anti-TB treatment regimen has promising results against drug-resistant Mtb strains. To ensure the optimized application of Ī²-lactams it is important to characterize their resistance-inducing potential in mycobacteria. Thus, the aim of this thesis was to explore the induction of resistance to PG synthesis inhibitors, namely Ī²-lactams, in mycobacteria, upon exposure to these antibiotics, and to characterize the obtained isolates. To do this, resistant isolates of the non-pathogenic organism M. smegmatis (Msm) were selected by exposure to high concentrations of several antibiotics. Thirty-nine isolates were obtained: twenty-four Msm mc2155 (WT)-derivatives selected with amoxicillin and meropenem, in the presence and absence of clavulanate, vancomycin, rifampicin, isoniazid and ethambutol; three Msm PM965-derivatives, selected with meropenem; and twelve isolates subjected to two different selections, the first with isoniazid or ethambutol, and the second with amoxicillin or meropenem. Resistance frequency was determined for each treatment, with values ranging from 1x10-6 to 1x10-9, with the highest frequencies registered for isoniazid, amoxicillin and meropenem. Furthermore, minimal inhibitory concentration (MIC) assays confirmed the resistance phenotype for most of the isolates; evidence of cross-resistance was also noticeable between the two Ī²-lactams, meropenem and amoxicillin; and three antibiotics of different classes amoxicillin, vancomycin, and isoniazid. To further characterize the isolates selected with a Ī²-lactam antibiotic, nitrocefin hydrolysis and ethidium bromide efflux assays were performed. The difference in nitrocefin hydrolysis values between meropenem and amoxicillin-selected isolates suggests different mechanisms of resistance are at play despite the increase in amoxicillin MIC observed in both cases. Regarding the efflux quantification, few variations were observed between isolates and parental strains, with the exceptions of two amoxicillin and one amoxicillin/clavulanate isolates which presented an unexpected decrease in efflux capacity. Furthermore, whole genomic analysis of the isolates selected with the anti-TB antibiotics revealed mutations in the expected drug target genes, confirming that the method used in this work to obtain resistant isolates was successful and the conclusions taken from it can be associated with Mtb. Mutations in amoxicillin and vancomycin-selected isolates were identified on regulatory genes, which albeit not directly targeted by the antibiotics might regulate the expression of genes associated with the drug targets. These findings confirm the development of inducible resistance to PG synthesis inhibitors and the involvement of different resistance mechanisms; the genomic analysis of the remaining resistant isolates is still in progress and will in the future, together with an RNA-sequencing analysis, provide more insight into the pathways involved in the development of drug resistance.
Tuberculosis (TB), caused by the pathogenic organism Mycobacterium tuberculosis (Mtb), is a major public health problem and a leading cause of death. The epidemiologic situation is aggravated by the emergence and spreading of resistant strains, namely multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB), which reduce the treatment options and result in a higher risk of therapy failure. This led to the necessity of revaluating alternative options for the standard treatment of TB, including the potential addition of Ī²-lactams to therapy regimens. Ī²-lactams are a highly successful class of antibiotics which target the transpeptidases involved in the biosynthesis of the peptidoglycan (PG), a cell wall (CW) component common to all bacteria. Ī²-lactams were previously excluded from TB therapy due to the intrinsic resistance of Mtb to this antibiotic class, conferred by the presence of a highly effective Ī²-lactamase, BlaC, and the unusual mycobacterial CW, with a PG cross-linking performed by L,D-transpeptidases resistant to inhibition by most Ī²-lactams. However, recent studies showed that the combination of a Ī²-lactam with a Ī²-lactamase inhibitor added to an anti-TB treatment regimen has promising results against drug-resistant Mtb strains. To ensure the optimized application of Ī²-lactams it is important to characterize their resistance-inducing potential in mycobacteria. Thus, the aim of this thesis was to explore the induction of resistance to PG synthesis inhibitors, namely Ī²-lactams, in mycobacteria, upon exposure to these antibiotics, and to characterize the obtained isolates. To do this, resistant isolates of the non-pathogenic organism M. smegmatis (Msm) were selected by exposure to high concentrations of several antibiotics. Thirty-nine isolates were obtained: twenty-four Msm mc2155 (WT)-derivatives selected with amoxicillin and meropenem, in the presence and absence of clavulanate, vancomycin, rifampicin, isoniazid and ethambutol; three Msm PM965-derivatives, selected with meropenem; and twelve isolates subjected to two different selections, the first with isoniazid or ethambutol, and the second with amoxicillin or meropenem. Resistance frequency was determined for each treatment, with values ranging from 1x10-6 to 1x10-9, with the highest frequencies registered for isoniazid, amoxicillin and meropenem. Furthermore, minimal inhibitory concentration (MIC) assays confirmed the resistance phenotype for most of the isolates; evidence of cross-resistance was also noticeable between the two Ī²-lactams, meropenem and amoxicillin; and three antibiotics of different classes amoxicillin, vancomycin, and isoniazid. To further characterize the isolates selected with a Ī²-lactam antibiotic, nitrocefin hydrolysis and ethidium bromide efflux assays were performed. The difference in nitrocefin hydrolysis values between meropenem and amoxicillin-selected isolates suggests different mechanisms of resistance are at play despite the increase in amoxicillin MIC observed in both cases. Regarding the efflux quantification, few variations were observed between isolates and parental strains, with the exceptions of two amoxicillin and one amoxicillin/clavulanate isolates which presented an unexpected decrease in efflux capacity. Furthermore, whole genomic analysis of the isolates selected with the anti-TB antibiotics revealed mutations in the expected drug target genes, confirming that the method used in this work to obtain resistant isolates was successful and the conclusions taken from it can be associated with Mtb. Mutations in amoxicillin and vancomycin-selected isolates were identified on regulatory genes, which albeit not directly targeted by the antibiotics might regulate the expression of genes associated with the drug targets. These findings confirm the development of inducible resistance to PG synthesis inhibitors and the involvement of different resistance mechanisms; the genomic analysis of the remaining resistant isolates is still in progress and will in the future, together with an RNA-sequencing analysis, provide more insight into the pathways involved in the development of drug resistance.
DescriĆ§Ć£o
Palavras-chave
Tuberculose Mycobacterium tuberculosis Inibidores do peptidoglicano ResistĆŖncia a antibiĆ³ticos