Resistência "in vitro" aos antimicrobianos e microbiota bucal de cães diagnosticada por microbioma e espectrometria de massas
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Data
2020-04-24
Autores
Portilho, Fábio Vinícius Ramos
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Universidade Estadual Paulista (Unesp)
Resumo
O estreitamento da relação entre tutores e animais de companhia, nas últimas décadas, aumentou consideravelmente o risco de transmissão de patógenos dos animais para os humanos. A microbiota da cavidade oral de animais de companhia é polimicrobiana e estes agentes podem potencialmente infectar humanos pelas mordeduras ou contato direto com mucosas ou feridas de pele. No entanto, são escassas as informações sobre a identificação destes micro-organismos por técnicas moleculares (microbioma, proteômica). Ainda, o perfil de sensibilidade microbiana in vitro da microbiota bacteriana bucal de cães e a etiologia dos agentes envolvidos em mordeduras em humanos não são completamente elucidados, posto que muitos cães são errantes e evadem após a agressão. Com efeito, o presente estudo investigou a presença de agentes de origem bacteriana e fúngica na cavidade oral de 100 cães hígidos por técnicas de cultivo microbiano convencional, sequenciamento genético em larga escala (microbioma) e espectrometria de massas (MALDI-TOF MS), bem como investigou o perfil de sensibilidade/resistência in vitro dos isolados. Foram identificados 213 micro-organismos de origem bacteriana e 20 de origem fúngica. Os agentes bacterianos mais prevalentes no diagnóstico microbiológico e espectrometria de massas foram Staphylococcus pseudintermedius (40/100=40%), Streptococcus α-hemolítico (37/100=37%) e Pasteurella stomatis (22/100=22%). O gênero de fungo mais prevalente foi Aspergillus (10/100=10%). Imipenem (207/213=97,2%), ceftiofur (196/213=92%) e amoxicilina/ácido clavulânico (194/213=91,1%) foram os antimicrobianos mais eficazes, enquanto a maior resistência dos isolados foi observada com o uso de sulfametoxazol/trimetoprim (77/213=36,1%), penicilina (75/213=35,2%) e azitromicina (48/213=22,5%). Multirresistência bacteriana foi identificada em 18,8% dos isolados (40/213), além da detecção do gene de resistência mecA em três isolados (3/49=6,1%) de estafilococos coagulase-positivos, sendo um S. aureus. e dois S. pseudintermedius. No sequenciamento em larga escala foram identificados predominantemente os gêneros Porphyromonas (32,5%), Moraxella (16,3%), Fusobacterium (12,8%) e Conchiformibius (9,5%), seguidos em menor frequência por Bergeyella (5%), Campylobacter (3,8%) e Capnocytophaga (3,4%). Alta complexidade de micro-organismos foi identificada na microbiota oral de cães aparentemente hígidos, incluindo agentes de potencial zoonótico. Ainda, os isolados revelaram alta multirresistência aos antimicrobianos convencionais, sendo um problema emergente em saúde pública. No geral, a natureza polimicrobiana da microbiota oral de cães e a multirresistência dos patógenos aos antimicrobianos convencionais reforçam o risco potencial de lesões contaminadas causadas por mordeduras de cães em humanos e a necessidade de tratamento dessas lesões com base na identificação etiológica e perfil de sensibilidade antimicrobiana dos isolados.
The close relationship between humans and companion animals in recent decades has strongly increased the risk of transmission of pathogens from pets-to-humans. The microbiota of the oral cavity from companion animals is polymicrobial and these agents may potentially infect humans through bites or by direct contact with mucous membranes or cutaneous lesions. Nonetheless, the identification of these microorganisms by molecular techniques (microbiome, proteomics) is scarce. Besides, the in vitro microbial susceptibility pattern of oral bacterial microbiota from dogs and the etiology of agents involved in human bites are not fully understood, since many dogs are homeless and/or evade after aggression. The present study investigated the presence of bacterial and fungal agents in the oral cavity of 100 healthy dogs based on conventional microbiological culture, large-scale DNA sequencing (microbiome), and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). In vitro antimicrobial susceptibility profile of the isolates was assessed as well. A total of 213 bacterial and 20 fungal microorganisms were identified. The most prevalent bacterial agents diagnosed by microbiological culture and mass spectrometry were Staphylococcus pseudintermedius (40/100=40%), α-hemolytic Streptococcus (37/100=37%), and Pasteurella stomatis (22/100=22%), whereas the most common genus of fungi was Aspergillus (10/100=10%). Imipenem (207/213=97.2%), ceftiofur (196/213=92%), and amoxicillin/clavulanic acid (194/213=91.1%) were the most effective antimicrobials. Conversely, the highest resistance of isolates was observed to sulfamethoxazole/trimethoprim (77/213=36.1%), penicillin (75/213=35.2%), and azithromycin (48/213=22.5%). Multidrug resistance was identified in 18.8% of the bacterial isolates (40/213), in addition to the detection of the resistance gene mecA in three isolates of coagulase-positive staphylococci (3/49=6.1%) (one S. aureus and two S. pseudintermedius). In large-scale sequencing, the most predominant genera were Porphyromonas (32.5%), Moraxella (16.3%), Fusobacterium (12.8%), and Conchiformibius (9.5%). Less frequently, there were identified Bergeyella spp. (5%), Campylobacter spp. (3.8%), and Capnocytophaga spp. (3.4%). High complexity of agents was identified in oral microbiota of apparently healthy dogs, including pathogens with zoonotic potential. Also, isolates revealed a high multidrug resistance to conventional antimicrobials, an emergent public health issue. Overall, the polymicrobial nature of oral microbiota of dogs and multi-resistance of the agents to conventional drugs highlights the potential risk of contaminated lesions caused by bites from dogs-to-humans, and need for treatment of these lesions based on etiology identification with a combination of methods, and antimicrobial susceptibility pattern.
The close relationship between humans and companion animals in recent decades has strongly increased the risk of transmission of pathogens from pets-to-humans. The microbiota of the oral cavity from companion animals is polymicrobial and these agents may potentially infect humans through bites or by direct contact with mucous membranes or cutaneous lesions. Nonetheless, the identification of these microorganisms by molecular techniques (microbiome, proteomics) is scarce. Besides, the in vitro microbial susceptibility pattern of oral bacterial microbiota from dogs and the etiology of agents involved in human bites are not fully understood, since many dogs are homeless and/or evade after aggression. The present study investigated the presence of bacterial and fungal agents in the oral cavity of 100 healthy dogs based on conventional microbiological culture, large-scale DNA sequencing (microbiome), and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). In vitro antimicrobial susceptibility profile of the isolates was assessed as well. A total of 213 bacterial and 20 fungal microorganisms were identified. The most prevalent bacterial agents diagnosed by microbiological culture and mass spectrometry were Staphylococcus pseudintermedius (40/100=40%), α-hemolytic Streptococcus (37/100=37%), and Pasteurella stomatis (22/100=22%), whereas the most common genus of fungi was Aspergillus (10/100=10%). Imipenem (207/213=97.2%), ceftiofur (196/213=92%), and amoxicillin/clavulanic acid (194/213=91.1%) were the most effective antimicrobials. Conversely, the highest resistance of isolates was observed to sulfamethoxazole/trimethoprim (77/213=36.1%), penicillin (75/213=35.2%), and azithromycin (48/213=22.5%). Multidrug resistance was identified in 18.8% of the bacterial isolates (40/213), in addition to the detection of the resistance gene mecA in three isolates of coagulase-positive staphylococci (3/49=6.1%) (one S. aureus and two S. pseudintermedius). In large-scale sequencing, the most predominant genera were Porphyromonas (32.5%), Moraxella (16.3%), Fusobacterium (12.8%), and Conchiformibius (9.5%). Less frequently, there were identified Bergeyella spp. (5%), Campylobacter spp. (3.8%), and Capnocytophaga spp. (3.4%). High complexity of agents was identified in oral microbiota of apparently healthy dogs, including pathogens with zoonotic potential. Also, isolates revealed a high multidrug resistance to conventional antimicrobials, an emergent public health issue. Overall, the polymicrobial nature of oral microbiota of dogs and multi-resistance of the agents to conventional drugs highlights the potential risk of contaminated lesions caused by bites from dogs-to-humans, and need for treatment of these lesions based on etiology identification with a combination of methods, and antimicrobial susceptibility pattern.
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Palavras-chave
Cavidade oral, Mordida de cães, Sequenciamento em larga escala, MALDI-TOF, Oral cavity, Dog bites, Large scale sequencing