Antimicrobial Activity of Psidium guajava Aqueous Extract against Sensitive and Resistant Bacterial Strains.

The inappropriate use of antimicrobials, along with environmental conditions, can lead to the emergence of resistant microorganisms. The use of phytopharmaceuticals and herbal medicines has a positive impact and represents a promising alternative. Psidium guajava extracts have been widely reported to have antimicrobial potential; however, studies reporting their activity against resistant bacterial strains are scarce. Because of the emerging resistance, the aim of this study was to analyze the antimicrobial capacity of the aqueous extract of guava leaves against wild-type and resistant bacterial strains. The aqueous extract obtained from the leaves of P. guajava was evaluated by HPLC for the content of total phenolics and tannins, antioxidant activity, and chemical composition. The antimicrobial activity of the extracts was analyzed by the disk diffusion and broth microdilution methods. The results of the chemical analysis of the extracts showed total phenolics content of 17.02 ± 6.87 mg/g of dry extract, total tannin content of 14.09 ± 1.20 mg of tannic acid equivalents/g of dry extract, and moderate antioxidant capacity with an EC50 value of 140 µg/mL. Flavonoids are the major compounds (rutin, hesperidin, and quercetin), followed by phenolic acids. Disk diffusion test results showed the presence of inhibition halos for Gram-positive bacteria (Staphylococcus aureus, sensitive and resistant; Staphylococcus pseudintermedius, sensitive and resistant; and Streptococcus spp., beta-hemolytic), while for Gram-negative bacteria (Escherichia coli, sensitive and resistant), there was no inhibition in the tested concentration range. The Minimal Inhibitory Concentration was 6.8 mg/mL for all Gram-positive strains evaluated. The present study demonstrated the antimicrobial activity of the aqueous extract of P. guajava against sensitive and resistant Gram-positive bacteria. The better antimicrobial activity found in the present study compared with previously reported activity should be highlighted and may be related to the higher concentration of total phenolics present in the tested extract. Moreover, the content of tannins found suggests a species with high quality that produces tannins. These new findings suggest an innovative profile regarding therapeutic resources that can be adopted to combat resistant microbial strains.

Production of extended-spectrum beta-lactamases in Escherichia coli isolated from poultry in Rio de Janeiro, Brazil.

The overuse of antimicrobials in poultry has led to the development and dissemination of multidrug-resistant bacteria in the poultry industry. One of the most effective mechanisms of resistance found in Escherichia coli is the production of extended-spectrum ß-lactamases (ESBL); there are several ESBLs, including the TEM, SHV, and CTX-M families. This resistance mechanism and the risks associated with transmitting these resistant microorganisms between animals, the environment, and humans can occur through direct contact and consumption of infected animals. This study aimed to determine the prevalence of E. coli in samples isolated from three broiler farms in Rio de Janeiro, Brazil, and screen the isolates for ESBL genes. The findings of this study demonstrated the presence of ESBL-producing E. coli in all farms studied. The findings of this study highlight the urgency for a program to monitor the poultry industry value chains at the regional level to control the spread of antimicrobial resistance. Therefore, we recommend that the enzyme subtypes produced by bacterial isolates should be determined to effectively characterize the distribution of genes related to antimicrobial resistance.

O uso excessivo de antimicrobianos em frangos de corte tem contribuído para o desenvolvimento e disseminação de bactérias multirresistentes, e um dos mais relevantes mecanismos de resistência encontrados em Escherichia coli é a produção de enzimas denominadas ß-lactamases de espectro estendido (ESBL). CTX-M, SHV e TEM são as ß-lactamases mais comumente encontradas nesta espécie e as ESBL mais prevalentes globalmente. Esse mecanismo de resistência e o risco associado à transmissão desses microrganismos resistentes entre animais, meio ambiente e seres humanos se devem principalmente ao contato direto e ao consumo de origem animal. Este trabalho buscou elucidar a prevalência de E. coli em amostras de três granjas de frangos de corte localizadas no Rio de Janeiro, Brasil, e caracterizá-las de acordo com seu genótipo. O estudo demonstrou uma presença consistente de E. coli produtora de ESBL com presença abundante do gene bla SHV nos isolados de todas as fazendas estudadas. Deste modo, este estudo teve como objetivo contribuir com dados epidemiológicos relativos à distribuição de genes relacionados às ß-lactamases na produção animal, conscientizando sobre a transmissão desses microrganismos resistentes entre animais, meio ambiente e seres humanos contribuindo com dados epidemiológicos e de sua importância em uma perspectiva de saúde única.

Biofilm production and beta-lactamic resistance in Brazilian Staphylococcus aureus isolates from bovine mastitis

Abstract Staphylococcus spp. play an important role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most relevant species due to the production of virulence factors such as slime, which is required for biofilm formation. This study aimed to evaluate biofilm production and its possible relation to beta-lactamic resistance in 20 S. aureus isolates from bovine mastitic milk. The isolates were characterized by pheno-genotypic and MALDI TOF-MS assays and tested for genes such as icaA, icaD, bap, agr RNAIII, agr I, agr II, agr III, and agr IV, which are related to slime production and its regulation. Biofilm production in microplates was evaluated considering the intervals determined along the bacterial growth curve. In addition, to determine the most suitable time interval for biofilm analysis, scanning electron microscopy was performed. Furthermore, genes such as mecA and blaZ that are related to beta-lactamic resistance and oxacillin susceptibility were tested. All the studied isolates were biofilm producers and mostly presented icaA and icaD. The Agr type II genes were significantly prevalent. According to the SEM, gradual changes in the bacterial arrangement were observed during biofilm formation along the growth curve phases, and the peak was reached at the stationary phase. In this study, the penicillin resistance was related to the production of beta-lactamase, and the high minimal bactericidal concentration for cefoxitin was possibly associated with biofilm protection. Therefore, further studies are warranted to better understand biofilm formation, possibly contributing to our knowledge about bacterial resistance in vivo.

The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) identification versus biochemical tests: a study with enterobacteria from a dairy cattle environment

Abstract Mastitis adversely affects milk production and in general cows do not regain their full production levels post recovery, leading to considerable economic losses. Moreover the percentage decrease in milk production depends on the specific pathogen that caused the infection and enterobacteria are responsible for this greater reduction. Phenotypic tests are among the currently available methods used worldwide to identify enterobacteria; however they tend to misdiagnose the species despite the multiple tests carried out. On the other hand The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique has been attracting attention for its precise identification of several microorganisms at species level. In the current study, 183 enterobacteria were detected in milk (n = 47) and fecal samples (n = 94) from cows, and samples from water (n = 23) and milk lines (n = 19). All these samples were collected from a farm in Rio de Janeiro with the specific purpose of presenting the MALDI-TOF MS technique as an efficient methodology to identify Enterobacteriaceae from bovine environments. The MALDI-TOF MS technique results matched the biochemical test results in 92.9% (170/183) of the enterobacteria species and the gyrB sequencing confirmed 100% of the proteomic technique results. The amino acid decarboxylation test made the most misidentifications and Enterobacter spp. was the most misidentified genus (76.9%, 10/13). These results aim to clarify the current biochemical errors in enterobacteria identification, considering isolates from a bovine environment, and show the importance for more careful readings of phenotypic tests which are often used in veterinary microbiology laboratories.

Biofilm production and beta-lactamic resistance in Brazilian Staphylococcus aureus isolates from bovine mastitis.

Staphylococcus spp. play an important role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most relevant species due to the production of virulence factors such as slime, which is required for biofilm formation. This study aimed to evaluate biofilm production and its possible relation to beta-lactamic resistance in 20 S. aureus isolates from bovine mastitic milk. The isolates were characterized by pheno-genotypic and MALDI TOF-MS assays and tested for genes such as icaA, icaD, bap, agr RNAIII, agr I, agr II, agr III, and agr IV, which are related to slime production and its regulation. Biofilm production in microplates was evaluated considering the intervals determined along the bacterial growth curve. In addition, to determine the most suitable time interval for biofilm analysis, scanning electron microscopy was performed. Furthermore, genes such as mecA and blaZ that are related to beta-lactamic resistance and oxacillin susceptibility were tested. All the studied isolates were biofilm producers and mostly presented icaA and icaD. The Agr type II genes were significantly prevalent. According to the SEM, gradual changes in the bacterial arrangement were observed during biofilm formation along the growth curve phases, and the peak was reached at the stationary phase. In this study, the penicillin resistance was related to the production of beta-lactamase, and the high minimal bactericidal concentration for cefoxitin was possibly associated with biofilm protection. Therefore, further studies are warranted to better understand biofilm formation, possibly contributing to our knowledge about bacterial resistance in vivo.

The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) identification versus biochemical tests: a study with enterobacteria from a dairy cattle environment.

Mastitis adversely affects milk production and in general cows do not regain their full production levels post recovery, leading to considerable economic losses. Moreover the percentage decrease in milk production depends on the specific pathogen that caused the infection and enterobacteria are responsible for this greater reduction. Phenotypic tests are among the currently available methods used worldwide to identify enterobacteria; however they tend to misdiagnose the species despite the multiple tests carried out. On the other hand The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique has been attracting attention for its precise identification of several microorganisms at species level. In the current study, 183 enterobacteria were detected in milk (n=47) and fecal samples (n=94) from cows, and samples from water (n=23) and milk lines (n=19). All these samples were collected from a farm in Rio de Janeiro with the specific purpose of presenting the MALDI-TOF MS technique as an efficient methodology to identify Enterobacteriaceae from bovine environments. The MALDI-TOF MS technique results matched the biochemical test results in 92.9% (170/183) of the enterobacteria species and the gyrB sequencing confirmed 100% of the proteomic technique results. The amino acid decarboxylation test made the most misidentifications and Enterobacter spp. was the most misidentified genus (76.9%, 10/13). These results aim to clarify the current biochemical errors in enterobacteria identification, considering isolates from a bovine environment, and show the importance for more careful readings of phenotypic tests which are often used in veterinary microbiology laboratories.

Impairments of mecA gene detection in bovine Staphylococcus spp.

Staphylococcus aureus antimicrobial resistance, especially to beta-lactams, favors treatment failures and its persistence in herd environment. This work aimed to develop a more specific primer for mecA gene detection based on the comparison of the conserved regions from distinct host origins and also investigated the presence of homologue mecA(LGA251) in bovine strains. A total of 43 Staphylococcus spp. were included in this study, comprising 38 bovine S. aureus, two human and three equine coagulase-negative staphylococci (CNS). Phenotypical methicillin-resistance detection was performed through oxacillin agar-screening and cefoxitin disk-diffusion test. None isolate tested positive for mecA(LGA251) gene. For mecA gene PCR, new primers were designed based on the sequences of human S. aureus (HE681097) and bovine S. sciuri (AY820253) mecA. The new primers based on the S. aureus mecA sequence amplified fragments of human and equine CNS and the ones based on S. sciuri mecA sequence only yielded fragments for S. aureus bovine strains. Multiples alignments of mecA gene sequences from bovine, human and equine revealed punctual but significant differences in bovine strains that can lead to the mecA gene detection impairment. The observed divergences of mecA gene sequences are not a matter of animal or human origin, it is a specificity of bovine samples.

Impairments of mecA gene detection in bovine Staphylococcus spp.

Staphylococcus aureus antimicrobial resistance, especially to beta-lactams, favors treatment failures and its persistence in herd environment. This work aimed to develop a more specific primer for mecA gene detection based on the comparison of the conserved regions from distinct host origins and also investigated the presence of homologue mecA LGA251 in bovine strains. A total of 43 Staphylococcus spp. were included in this study, comprising 38 bovine S. aureus, two human and three equine coagulase-negative staphylococci (CNS). Phenotypical methicillin-resistance detection was performed through oxacillin agar-screening and cefoxitin disk-diffusion test. None isolate tested positive for mecA LGA251 gene. For mecA gene PCR, new primers were designed based on the sequences of human S. aureus (HE681097) and bovine S. sciuri (AY820253) mecA. The new primers based on the S. aureus mecA sequence amplified fragments of human and equine CNS and the ones based on S. sciuri mecA sequence only yielded fragments for S. aureus bovine strains. Multiples alignments of mecA gene sequences from bovine, human and equine revealed punctual but significant differences in bovine strains that can lead to the mecA gene detection impairment. The observed divergences of mecA gene sequences are not a matter of animal or human origin, it is a specificity of bovine samples.

Caracterização fenotípica da resistência a antimicrobianos e detecção do gene mecA em Staphylococcus spp. coagulase-negativos isolados de amostras animais e humanas / Phenotypic characterization of antimicrobial resistance and detection of the mecA gene in coagulase-negative Staphylococcus spp. isolates from animal and human samples

Os estafilococos coagulase-negativos (ECN) fazem parte da microbiota normal da pele e, apesar de terem sido considerados saprófitas por muito tempo, o seu significado clínico como agente etiológico tem aumentado com o passar dos anos. Neste estudo, foram obtidos 72 isolados de ECN a partir de amostras do conduto auditivo de cães, de mastite bovina e de infecções humanas. Staphylococcus xylosus foi o microrganismo mais isolado, nas amostras animais, e S. cohnii subsp. cohnii em humanos. Os isolados foram avaliados de modo a traçar o perfil fenotípico de sua resistência aos antimicrobianos mais indicados no tratamento de infecções estafilocócicas. Foi detectado um elevado nível de resistência à penicilina e ampicilina. A gentamicina, a vancomicina e a associação ampicilina+sulbactam foram eficientes frente aos isolados testados. A resistência à oxacilina foi avaliada por meio dos testes de difusão em disco modificada, ágar screen, microdiluição em caldo e diluição em ágar para constatar, se à semelhança do que ocorre com os estafilococos coagulase-positivo, esta pode ser mediada pelo gene mecA e apresentada de forma heterogênea. A presença do gene mecA foi determinada pelo método da Reação em Cadeia de Polimerase (PCR), sendo 5,6 por cento dos isolados mecA positivos.

Coagulase-negative staphylococci (SCN) make part of the normal microbiota skin and although they have been considered saprophytics for years, nowadays their clinical significance as an etiologic agent has increased. In this study, 72 SCN isolates obtained from external ear canals of dogs, bovine mastitis and human nosocomial infections were evaluated. Staphylococcus xylosus was the most prevalent microorganism in animal samples and S. cohnii subsp. cohnii in human samples. SCN isolates were evaluated in order to establish a phenotypical resistance pattern towards the most indicated antibiotics for staphyloccocal infections. A high level of resistance to penicillin and ampicillin was detected. The most efficient antibiotics evaluated were gentamicin, vancomicin and the association between ampicillin and sulbactam. To certify the heterogeneous resistance pattern, oxacillin resistance was phenotypically detected by a modified-disc-diffusion test, agar screen, broth micro-dilution and agar dilution. The presence of the mecA gene was detected in 5.6 percent of the SCN isolates by Polimerase Chain Reaction (PCR).

Mapeamento do perfil de resistência e detecção do gene mecA em Staphylococcus aureus e Staphylococcus intermedius oxacilina-resistentes isolados de espécies humanas e animais / Resistance pattern and detection of mecA gene in oxacillin-resistant isolates of Staphylococcus aureus and Staphylococcus intermedius from animal and human samples

Espécies de Staphylococcus spp resistentes a antimicrobianos representam um problema cosmopolita, sendo o controle de sua disseminação um importante desafio. O perfil de resistência a antimicrobianos de isolados de Staphylococcus aureus e Staphylococcus intermedius em amostras clínicas humanas e animais foi avaliado através do método de difusão em disco, no qual foi possível detectar um elevado nível de resistência à ampicilina e à penicilina. A avaliação da resistência à oxacilina, devido à heterogeneidade de resposta do gênero estudado, foi desenvolvida também através das seguintes técnicas: difusão em ágar modificado, ágar screen e microdiluição em caldo, e posteriormente correlacionada com a detecção do gene mecA, pela técnica de PCR, nas amostras consideradas resistentes em pelo menos um dos testes utilizados. A correlação entre os resultados obtidos nos testes fenotípicos com a presença do gene de resistência, considerado um método de referência, foi utilizada para validar a sensibilidade destes. De um total de 80 amostras avaliadas, 28 apresentaram resistência à oxacilina, sendo possível detectar a presença do gene de resistência mecA em 12 destas amostras. Os testes de suscetibilidade à oxacilina apresentaram sensibilidade superior a 50,0 por cento, sendo a difusão em disco simples e o ágar screen considerados mais sensíveis, e a difusão em disco modificada, o de menor sensibilidade.

Antimicrobial resistant Staphylococcus species represent an important cosmopolitan problem, and its spreading control is a significative challenge. Resistance pattern of Staphylococcus aureus and Staphylococcus intermedius species isolated from animals and humans clinical samples to different antibiotics was evaluated through disk diffusion method, where ampicillin and penicillin presented the highest level of resistance. The evaluation of the resistance to oxacillin, due to the heterogeneity of the response of the studied genus was carried out through the following tests: modified agar diffusion, agar screen and microdilution, and further correlation with the detection of mecA gene in samples that showed resistance in at least one of the susceptibility tests used. The correlation between the results obtained from phenotypic methods and the detection of resistance gene, considered as a reference method, was used in order to validate its sensitivity. Eighty clinical staphylococcal isolates (29 human and 51 animal isolates) were evaluated, 28 were oxacillin-resistant, mecA gene being detected in 12 samples. Susceptibility assessment tests to oxacillin presented above 50 percent of specificity, disk diffusion and agar screen being the most sensitive one, while modified disk diffusion presented the lowest sensibility rate. Ampicillin and penicillin presented the highest level of resistance.