Species from Pavonia genus (Malvaceae) are traditionally used as antioxidant, n anti inflammatory and antimicrobial remedies. Previous studies have revealed their diversity of specialized metabolites widely used in the development of new medicines. This is the first study on Pavonia glazioviana Gurke, a Brazilian species known as “Malva da Chapada”. The research led to identify fifteen compounds: 132 (S) hydroxy pheophytin a; and 132(S) hydroxy 173 ethoxyphaeophorbide, n decanol, cycloart 23Z ene 3β,25 diol, cycloart 24S 25 ene 3β,24 diol, sitosterol 3 O β D glucopyranoside, stigmasterol 3 O β D glucopyranoside, quercetin, acacetin, kaempferol, tiliroside, 5 hydroxy 3,7,8,4’ tetramethoxyflavone, 5,7 dihydroxy 3,8,4’ trimethoxyflavone, 5,7 dihydroxy 4’ methoxyflavone, 5,7,4’ trihydroxy 3,8 dimethoxyflavone and 5,7,4’ trihydroxy 3 methoxyflavone. The major flavonoids were tested for their antimicrobial activity, and the compound 5,7 dihydroxy 3,8,4’ trimethoxyflavone showed strong activity against Escherichia coli (MIC = 512 μg mL1), Pseudomonas aeruginosa (MIC = 512 μg mL1), Candida albicans (MIC = 512 μg mL1), Candida tropicalis (MIC = 512 μg mL1), Candida parapsilosis (MIC = 512 μg mL1), Aspergillus flavus (MIC = 512 μg mL1) and A. fumigantus (MIC = 512 μg mL1). The total phenolic content, flavonoid content, and radical scavenging potential were determined showing its great production of phenolics and flavonoids, along with interesting radical scavenging potential (DPPH EC50 = 6.36 ± 0.029 mg mL 1). Our findings contributed to characterize P. glazioviana as a source of methoxylated flavonoids with antimicrobial potential. Malvaceae (Malvaceae antioxidant remedies medicines Gurke Malva Chapada. Chapada . Chapada” compounds 13 S (S 132S 132(S 17 ethoxyphaeophorbide decanol Z 3β25 3β 3β,2 diol 2 3β24 24 glucopyranoside quercetin acacetin kaempferol tiliroside 3,7,8,4 3784 7 8 4 tetramethoxyflavone 57 5, 3,8,4 384 methoxyflavone 5,7,4 574 38 3, MIC 51 mL1, mL1 , A mL1. content DPPH EC EC5 636 6 36 6.3 0029 0 029 0.02 1. 1 1) P 3β2 3β, 3,7,8, 378 3,8, 5,7, 63 6. 002 02 0.0 3,7,8 37 00 0. 3,7, 3,7

Abstract Osteomyelitis pubis is a rare condition that can occur after vaginal delivery. There are few cases reported, mostly associated with normal vaginal deliveries1-11. Most common pathogen is Staphylococcus Aureus, but P. aeruginosa and polymicrobial infections evolving faecal flora are also common3,12. Although pelvic trauma has been associated with this rare condition, we present a case of osteomyelitis pubis related to uncomplicated vaginal delivery.

Resumo A osteomielite da sínfise púbica é uma condição rara que pode ocorrer após partos vaginais. Existem alguns casos descritos, maioritariamente associados a partos eutócicos1-11. O microrganismo mais frequentemente associado a esta patologia é o Staphylococcus Aureus, mas P. aeruginosa e infeções polimicrobianas que incluam flora fecal também são causas comuns3,12. Apesar de o trauma pélvico ser um fator de risco importante, neste trabalho apresentamos um caso de osteomielite puerperal após um parto eutócico sem complicações.

Resumo Os sabonetes têm um papel importante para a nossa higiene e saúde, pois eles além de ter efeito bactericida, também removem as sujeiras presentes no corpo humano. Avaliar a eficácia de sabonetes com atividade antimicrobiana de diferentes marcas comerciais vendidas no Brasil. Foram realizados ensaios da atividade antimicrobiana dos diferentes sabonetes através do método difusão em ágar frente aos microrganismos Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Acinetobacter baumannii, Proteus mirabilis e Candida albicans. Todos os sabonetes comerciais testados apresentaram formação de halo de inibição antimicrobiana frente às bactérias S. aureus e P. aeruginosa. Apenas dois sabonetes comerciais apresentaram inibição das espécies A. baumannii e C. albicans. Nenhum dos sete produtos estudados apresentou inibição das bactérias E. cloacae, P. mirabilis e E. coli. Na comparação das informações contidas nas embalagens dos produtos com os resultados obtidos durante a pesquisa realizada foram observadas divergências. Os sabonetes que apresentaram maior eficiência contra os microrganismos testados foram aqueles das apresentações 1 e 2, que se mostraram eficazes contra as espécies de bactérias S. aureus, P. aeruginosa e A. baumannii e contra a espécie de fungo C. albicans. As marcas 3, 4, 5, 6 e 7 apresentaram o mesmo resultado de sensibilidade frente as bactérias das espécies S. aureus e P. aeruginosa, com variação quantitativa apenas do halo de inibição. Houve divergência entre as informações contidas nas embalagens dos sete produtos em estudo e os resultados dos testes experimentais.

Abstract Soaps play an important role in our hygiene and health, as they not only have a bactericidal effect but also remove dirt from the human body. To evaluate the effectiveness of soaps with antimicrobial activity from different commercial brands sold in Brazil. Tests of the antimicrobial activity of different soaps were carried out through diffusion in agar against the microorganisms Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Acinetobacter baumannii, Proteus mirabilis, and Candida albicans. All commercial soaps tested transfer antimicrobial inhibition halo formation against S. aureus and P. aeruginosa bacteria. Only two commercial soaps inhibit the species A. baumannii and C. albicans. None of the seven products studied showed inhibition of E. cloacae, P. mirabilis, and E. coli bacteria. When comparing the information contained in the packaging of the products with the results obtained during a survey, divergences were observed. The soaps that provide greater efficiency against the tested microorganisms were presented in presentations 1 and 2, which become useful against the bacteria species S. aureus, P. aeruginosa, and A. baumannii and a fungus species C. albicans. Marks 3, 4, 5, 6, and 7 parallel the same sensitivity result opposite as bacteria of the species S. aureus and P. aeruginosa, with quantitative variation only of the inhibition halo. There was a divergence between the information contained in the packaging of the seven products under study and the results of the experimental tests.

Resumo O fruto-oligossacarídeo (FOS) é amplamente utilizado em muitos alimentos e indústrias farmacêuticas, e é produzido por meio de diferentes maneiras, como extraí-lo de plantas ou produzi-lo usando enzimas de plantas e microrganismos. Em um estudo anterior, extraímos a enzima frutosiltransferase (Ftase) do resíduo de abacaxi e produzimos FOS. Neste estudo, medimos a atividade antagônica de dois simbióticos: o primeiro simbiótico contendo Lactobacillus acidophilus e o FOS produzido, e o segundo simbiótico contendo Lactobacillus acidophilus e o FOS padrão, contra bactérias patogênicas (P. aeruginosa, E. coli, S. aureus e B. cereus). Os resultados mostraram que a atividade antagônica de ambos os tipos simbióticos foi muito próxima, pois não houve diferenças significativas entre eles, exceto na atividade antagônica contra S. aureus, em que houve uma diferença significativa entre o simbiótico contendo o FOS padrão, que foi o mais alto em sua atividade antagônica, em comparação com o simbiótico contendo o FOS produzido neste estudo. A atividade do fruto-oligossacarídeo (FOS) extraído do resíduo de abacaxi ficou evidente no aumento da atividade da bactéria probiótica (L. acidophilus), que teve papel importante na produção de ácidos e compostos inibidores das bactérias patogênicas. Os diâmetros das áreas de inibição no estudo atual variaram entre 19,33 e 28 mm, e E. coli foi mais suscetível à inibição, seguida por S. aureus, P. aeruginosa e B. cereus, respectivamente.

Abstract Fructooligosaccharide is used widely in many foods and pharmaceutical industries and produced by using different ways such as extracting it from plants or producing it by using plants and microorganisms’ enzymes. In a previous study, we extracted Fructosyltransferase (Ftase) enzyme from pineapple residue and produced FOS. In this study, we measured the antagonistic activity of two synbiotics, the first synbiotic containing Lactobacillus acidophilus and the produced FOS, the second synbiotic containing Lactobacillus acidophilus and standard FOS, against pathogenic bacteria (P. aeruginosa, E. coli, S. aureus and B cereus). The results showed that the antagonistic activity of both synbiotic types was very close, as there were no significant differences between them except in the antagonistic activity against S. aureus, there was a significant difference between the synbiotic containing the standard FOS, which was the highest in its antagonistic activity compared to the synbiotic containing the produced FOS in this study. The activity of the fructooligosaccharide (FOS) extracted from pineapple residue was evident in enhancing the activity of the probiotic bacteria (L. acidophilus), which had a major role in the production of acids and compounds that inhibited the pathogenic bacteria. The diameters of inhibition areas in the current study ranged between 19.33-28 mm, and E. coli was more susceptible to inhibition, followed by S. aureus, P. aeruginosa, and B. cereus, respectively.

Resumo O presente trabalho de pesquisa apresenta a importância antiviral e antibacteriana da gordura corporal de Saara hardwickii, comumente chamado de lagarto de cauda espinhosa. O óleo foi extraído de gorduras corporais localizadas na região ventral desse animal usando hidrocarbonetos, por exemplo, n-hexano, metanol, butanol e acetato de etila, como solvente. A atividade antibacteriana do óleo do lagarto foi testada em linhagens padrão e multirresistentes de Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa e Proteus vulgaris, de forma isolada e com antibiótico ampicilina. Para o potencial antibacteriano, acetato de etila e extrato de butanol apresentaram melhor zona de inibição (7 mm) com P. aeruginosa e S. aureus, respectivamente. Para o potencial antiviral, o extrato de butanol e o extrato de metanol apresentaram melhor título de hemaglutinação de 4 com as cepas virais NDV e IBV, respectivamente. Conclui-se que o óleo do lagarto possui potencial antimicrobiano contra diferentes cepas de patógenos (vírus e bactérias).

Abstract Present research work represents antiviral and antibacterial value of body fat of Saara hardwickii commonly called as spiny tailed lizard. Oil was extracted from body fats located in the ventral region of this animal using hydrocarbons e.g., n-hexane, methanol, butanol and ethyl acetate as a solvent. The antibacterial activity of lizard oil was tested against standard as well as multi-resistant lines ofEscherichia coli, Styphalococcus aureus, Pseudomonas aeruginosa and Proteus vulgaris alone and with antibiotic ampicillin. For antibacterial potential, Ethyl acetate and Butanol solvent extract showed best zone of inhibition (7mm) with P. aeruginosa and S. aureus respectively. For antiviral potential, Butanol and Methanol extract showed best HA (Hemagglutination) titer of 04 with NDV and IBV viral strain respectively. It is concluded that lizard oil has antimicrobial potential against different pathogens strains (virus, bacteria).

Resumo Nanopartículas de óxido de zinco foram sintetizadas a partir do extrato foliar de Brassica oleracea L., grupo Acephala (couve), seguidas de sua caracterização em Microscópio Eletrônico de Varredura (MEV) e Raio X por Energia Dispersiva (EDX). As propriedades antibacterianas das nanopartículas de zinco foram testadas em bactérias Gram-negativas, Pseudomonas aeruginosa (ATCC® 9027™), Escherichia coli (ATCC® 8739™) e Klebsiella pneumoniae (ATCC® BAA-1705™), e bactérias Gram-positivas, Staphylococcus aureus (ATCC® 6538™) e Listeria monocytogenes (ATCC® 13932™), em quatro concentrações diferentes (50,00 µg / ml; 100,00 µg / ml; 500,00 µg / ml; e 1 mg / ml) de suspensão de nanopartículas de óxido de zinco. Os resultados revelaram que as nanopartículas sintetizadas exibem fortes efeitos antibacterianos contra P. aeruginosa, L. monocytogenes, K. pneumonia, S. aureus e E. coli em concentrações de 500,00 µg / ml-1 mg / ml. Um aumento na eficácia das nanopartículas com a diminuição de seu tamanho também foi evidente. Este é o primeiro relatório sobre nanopartículas à base de B. oleracea L. que demonstra que 500,00 µg-1 mg / ml de concentração de nanopartículas de óxido de zinco têm atividade antibacteriana contra bactérias Gram-negativas e Gram-positivas e que essas nanopartículas têm potencial para ser consideradas um agente antibacteriano no futuro.

Abstract Zinc oxide nanoparticles were synthesized from the leaf extract of Brassica oleracea L. Acephala group (collard green) followed by their characterization using Scanning Electron Microscope (SEM), and Energy Dispersive X-ray (EDX). The antibacterial properties of zinc nanoparticles were tested against Gram-negative bacteria, Pseudomonas aeruginosa (ATCC ® 9027™), Escherichia coli (ATCC ® 8739™), Klebsiella pneumoniae (ATCC® BAA-1705™) and Gram-positive bacteria, Staphylococcus aureus (ATCC ® 6538™) and Listeria monocytogenes (ATCC ® 13932™), at four different concentrations (50.00 µg/ml, 100.00 µg/ml, 500.00 µg/ml and 1 mg/ml) of zinc oxide nanoparticles suspension. Results revealed that the synthesized nanoparticles exhibit strong antibacterial effects against Pseudomonas aeruginosa, Listeria monocytogenes, Klebsiella pneumonia, Staphylococcus aureus and Escherichia coli at 500.00 µg/ml-1 mg/ml concentrations. An increase in efficacy of nanoparticles with the decrease of their size was also evident. This is a first ever report on Brassica oleracea, L. based nanoparticles which demonstrates that 500.00 µg-1 mg/ml conc. of zinc oxide nanoparticles have antibacterial activity against both Gram -ve and Gram +ve bacteria and have the potential to be considered as an antibacterial agent in future.

SESQUITERPENES ALKALOIDS FROM Maytenus distichophylla (CELASTRACEAE). The phytochemical study of the roots of Maytenus distichophylla Mart. ex Reissek (Celastraceae), resulted in the isolation of the seven sesquiterpenes pyridine alkaloids euojaponine F (1), euojaponine K (2), ebinifoline W-I (3), wilforine (4), maiteine (5), euojaponine A (6) and ebinifoline E-II (7). The structures of compounds were established using spectroscopic techniques such as 1D and 2D NMR. All compounds were subjected to an in silico study through of a predictive model and then submitted to in vitro testing against S. epidermidis, P. aeruginosa, C. parapsilosis and C. albicans for evaluation of their antimicrobial activity. The compounds tested showed bacterial and fungal growth inhibition with MIC values 1024 and 256 µg mL-1, respectively. CELASTRACEAE. CELASTRACEAE . (CELASTRACEAE) Mart Celastraceae, Celastraceae , (Celastraceae) 1, 1 (1) 2, 2 (2) WI W I 3, 3 (3) 4, 4 (4) 5, 5 (5) 6 (6 EII E II 7. 7 (7) D NMR S epidermidis P aeruginosa C activity 102 25 mL1, mL1 mL mL-1 respectively (CELASTRACEAE (Celastraceae (1 (2 (3 (4 (5 ( (7 10 mL-

Abstract Biofilm degradation with amylase is one of the effective ways for controlling bacterial biofilm. Although amylase can be obtained from several sources, microbial amylase is preferred. Information of the new source of amylase and its activity is therefore fundamental for new applications and enzyme technology advancement. In this study, amylase was extracted from bacteria isolated from soil in Nakhon Ratchasima, Thailand. Two different soil isolates AMPB10 and AMPB31 were selected for the purification of amylase; they were identified as Bacillus tequilensis and Bacillus subtilis, respectively. The efficiencies of purified amylase in degradation of biofilm of Staphylococcus aureus TISTR 1466, Staphylococcus epidermidis TISTR 518, and Pseudomonas aeruginosa TISTRA 781 biofilms were measured. The amylase from AMPB10 and AMPB31 degraded 70.9% and 66.1% of S. aureus biofilm, 59.6% and 64.1% of S. epidermidis biofilm, and 57.8% and 60.1% of P. aeruginosa biofilm, respectively. Amylase produced from AMPB10 had greater biofilm degrading activity on S. aureus than AMPB31, while amylase from AMPB31 was more effective against P. aeruginosa and S. epidermidis at high concentration. However, AMPB10 amylase showed stronger degrading activity on P. aeruginosa at intermediate concentration. To the best of our knowledge, this is the first report demonstrating a successful use of B. tequilensis amylase to degrade the bacterial biofilm. sources preferred advancement study Ratchasima Thailand AMPB AMPB1 AMPB3 subtilis respectively 1466 518 78 measured 709 70 9 70.9 661 66 1 66.1 S 596 59 6 59.6 641 64 64.1 578 57 8 57.8 601 60 60.1 P concentration However knowledge B 146 51 7 70. 66. 5 59. 64. 57. 60. 14

Resumo O objetivo do presente estudo foi avaliar a capacidade da Pseudomonas aeruginosa, microencapsulada em goma de gelana, para descontaminar águas residuais de laticínios e explorar o potencial reuso das microcápsulas. A P. aeruginosa foi microencapsulada usando a técnica interna de gelificação iônica, empregando goma de gelana de baixo acil como material de parede. As formas livres e microencapsuladas de P. aeruginosa foram inoculadas em 150 mL de água residual estéril e incubadas em um frasco agitado (150 rpm) a 30°C. Posteriormente, o modelo de Baranyi foi utilizado para calcular os parâmetros de crescimento da P. aeruginosa. Ao mesmo tempo, foram determinadas a Demanda Bioquímica de Oxigênio (DBO) e a Demanda Química de Oxigênio (DQO). Os resultados obtidos indicaram que o processo de microencapsulação reduziu a taxa de crescimento do microorganismo encapsulado. No entanto, as bactérias microencapsuladas alcançaram porcentagens de redução de DQO e DBO de 61,54% e 64,05%, respectivamente. Da mesma forma, ao reutilizar as microcápsulas, foram alcançadas porcentagens de remoção superiores a 57,00%. Essas descobertas podem ter implicações significativas para a indústria em termos de redução da contaminação do efluente causada por quantidades substanciais de poluentes. microcápsulas P iônica parede 15 (15 rpm 30C C 30 30°C Posteriormente tempo (DBO DQO. . (DQO) encapsulado entanto 6154 61 54 61,54 6405 64 05 64,05% respectivamente forma 5700 57 00 57,00% poluentes 1 (1 3 (DQO 615 6 5 61,5 640 0 64,05 570 57,00 ( 61, 64,0 57,0 64, 57,

Abstract This study assessed the ability of Pseudomonas aeruginosa, microencapsulated in gellan gum, to decontaminate dairy wastewater and explored the potential reuse of microcapsules. P. aeruginosa was microencapsulated using the internal ionic gelation technique, employing low-acyl gellan gum as the wall material. The free and microencapsulated P. aeruginosa were inoculated into 150 mL of sterile wastewater and incubated in a shaking flask (150 rpm) at 30°C. Subsequently, the Baranyi Model was employed to calculate the growth parameters of P. aeruginosa. Concurrently, Biological Oxygen Demand (BOD) and Chemical Oxygen Demand (COD) were determined. The obtained results indicated that the microencapsulation process reduced the growth rate of the encapsulated microorganism. However, the microencapsulated bacteria achieved COD and BOD reduction percentages of 61.54% and 64.05%, respectively. Similarly, when reusing the microcapsules, removal percentages exceeding 57.00% were achieved. These findings could have significant implications for the industry in terms of reducing effluent contamination caused by substantial amounts of pollutants. microcapsules P technique lowacyl low acyl material 15 (15 rpm 30C C 30 30°C Subsequently Concurrently (BOD (COD determined microorganism However 6154 61 54 61.54 6405 64 05 64.05% respectively Similarly 5700 57 00 57.00 pollutants 1 (1 3 615 6 5 61.5 640 0 64.05 570 57.0 ( 61. 64.0 57. 64.

Abstract I. paraguariensis St. Hil. is a south American species of agronomic interest with studies supporting its medicinal properties. As the investigation of active ingredients with antimicrobial effect from medicinal plants is a suitable approach to the current antibacterial resistance problem, the aim of the present study was to determine the antibacterial activity of yerba mate ethanolic extracts against carbapenemase-producing gram-negative bacteria (reference strains and clinical isolates). Extracts showed antibacterial activity against Klebsiella pneumoniae ATCC® BAA-2342™ (KPC producing), Providencia rettgeri (NDM producing), Pseudomonas aeruginosa (MBL producing) and P. aeruginosa (VIM producing) at the concentrations tested. The Minimal-Inhibitory-Concentration and Minimal-Bactericidal-Concentration values ranged between 1 and 32 mg.ml-1 for the reference strains, and between 0.125 and 1 mg.ml-1 for the clinical isolates. The MBC/MIC index characterized the extracts as bactericidal. The combinations of commercial antibiotics and extracts showed a synergistic action on the reference strains studied. The lethal concentration 50 obtained using the Artemia salina toxicity assay were higher than 1 mg.ml-1 for all the extracts, indicating a low toxicity. The in vitro activity and low toxicity suggest that ethanolic I. paraguariensis leaf extracts constitute an outstanding source for new antibacterial compounds, and further studies should be carried out to understand their mechanism of action. I St Hil properties problem carbapenemaseproducing carbapenemase producing gramnegative gram negative isolates . isolates) ATCC BAA2342™ BAA2342 BAA 2342™ 2342 BAA-2342 KPC producing, , NDM MBL P VIM tested MinimalInhibitoryConcentration Minimal Inhibitory Concentration MinimalBactericidalConcentration Bactericidal 3 mg.ml1 mgml1 mgml mg.ml mg ml mg.ml- 0125 0 125 0.12 MBCMIC MBC MIC bactericidal studied 5 compounds BAA234 234 BAA-234 ml1 012 12 0.1 BAA23 23 BAA-23 01 0. BAA2 2 BAA-2 BAA-

Abstract This study presents a novel approach to obtaining nano-antimicrobial hybrid material by integrating electrospun nanofibers based on cellulosic biopolymer (BP) associated with antimicrobial agents, specifically silver nanoparticles (AgNPs) and Clavanin A (ClavA), an antimicrobial peptide obtained from the marine tunicate Styela clava. The electrospinning technique produced the blended polyvinyl alcohol:BP nanofibers. Chemical crosslinking was performed to ensure the stability of the nanofibers. The nanofibers had an average diameter of 568 nm for PVA nanofibers and 648 nm for PVA nanofibers functionalized with silver nanoparticles. The nanohybrid material demonstrates significant inhibition zones against Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa, Klebsiella pneumoniae) bacteria. P. aeruginosa exhibits a substantial inhibition zone of 15 mm. Thus, the nanohybrid material was effective against this challenging pathogen. Combining electrospun nanofibers, bacterial cellulose hydrogel, and antimicrobial agents establishes a solution that could combat microbial threats in wound care. nanoantimicrobial nano BP (BP AgNPs (AgNPs ClavA, ClavA , (ClavA) clava alcoholBP alcohol 56 64 Grampositive Gram positive Bacillus subtilis aureus Gramnegative negative Pseudomonas pneumoniae bacteria P 1 mm Thus pathogen hydrogel care (ClavA 5 6

Abstract Pseudomonas aeruginosa is an opportunistic nosocomial pathogen with a pathogenicity that is an important factor in public health and is the result of the functioning of the Quorum Sensing (QS) system. Currently, compounds of natural origin have shown promising biological effects on certain pathogens in recent years. The aim of the present study was to determine the possible inhibitory effect of five naturally occurring molecules on some genes associated with two pathways of the QS system, as well as on elastase protease and pyocyanin production in two resistant strains of P. aeruginosa. For this purpose, RT-qPCR assays were performed to know the expression of some QS-associated genes (lasR, rhlR, lasI, aprA and lasB) and spectrophotometric assays to evaluate the production of elastase, protease and pyocyanin in the presence or absence of the molecules. It was found that sesamin and ferulic acid were the molecules with the highest inhibitory effect, higher than 40 %, on most of the genes evaluated (lasR, rhlR, aprA and lasB) in the two P. aeruginosa strains BAA-47 and 27853. Likewise, ferulic acid was the most active compound on two of the three virulence factors evaluated even though in general the effect of the molecules was not greater than 50 %. Therefore, these compounds are considered to have a possible multitarget effect by inhibiting the expression of genes associated with the two main systems of P. aeruginosa. (QS system Currently years P purpose RTqPCR RT qPCR QSassociated lasR, lasR (lasR rhlR lasI lasB 4 % BAA47 BAA 47 BAA-4 27853 Likewise 5 Therefore BAA4 BAA- 2785 278 27 2

Abstract. Substrate specificity is instrumental in enzyme catalysis and a major determinant of the enzyme’s physiological role. Nevertheless, the degree of substrate specificity may vary in an ample range; some enzymes exhibit very high specificity while others show a relaxed specificity or promiscuity. The latter is used by evolution for the emergence of new enzymes able to perform novel metabolic roles. The basis of substrate specificity is substrate recognition, which is achieved in the enzyme active site by chemical and structural mechanisms. Here, we exemplify that specificity may exist within promiscuity by comparatively analyzing kinetic and structural data of the four Pseudomonas aeruginosa aminoaldehyde dehydrogenases-PA5373, PA5312, PA4189, and PA0219. Despite their apparent substrate promiscuity, we found that these enzymes show a significant degree of substrate specificity. They have evolved to preferentially oxidize different aminoaldehydes, even though each of them can use as in vitro substrates most of the aminoaldehydes preferred by the others. We focus on the role played in these enzymes by two active-site residues-one acidic and the other aromatic, both belonging to the so-called “anchor” loop-in binding the aldehyde amino group, as well as on the importance of the anchor loop conformation in defining the size and shape of the active-site cavity. Our results support the notion that natural selection has fine-tuned the active-site structural and chemical features of the P. aeruginosa AMADH enzymes to the structural and chemical features of their physiological aminoaldehydes substrates.

Resumen. La especificidad de sustrato es fundamental para la catálisis enzimática y un importante determinante del papel fisiológico de una enzima. Sin embargo, el grado de especificidad de las enzimas puede variar en un amplio intervalo; algunas enzimas exhiben una especificidad estricta por sus sustratos mientras que otras exhiben una especicificidad relajada o promiscuidad. Esto último es utilizado por la evolución para que emerjan nuevas enzimas capaces de llevar a cabo funciones metabólicas novedosas. La base de la especificidad es el reconocimiento del sustrato, lo que se logra en el sitio activo de la enzima mediante mecanismos químicos y estructurales. En este trabajo mostramos que la especificidad puede existir dentro de la promiscuidad, analizando resultados cinéticos y estructurales de las cuatro aminoaldehído deshidrogenasas de Pseudomonas aeruginosa-PA5373, PA5312, PA4189 y PA0219. A pesar de su aparente promiscuidad, encontramos que estas enzimas presentan un alto grado de especificidad. Han evolucionado para oxidar preferencialmente algunos aminoaldehídos, aunque cada una de ellas pueda in vitro usar como sustratos la mayoría de aminoaldehídos preferidos por las otras. Enfocamos nuestro análisis en el papel que desempeñan dos residuos del sitio activo-uno ácido y el otro aromático, ambos pertenecientes a la llamada asa “ancla”-en la unión de los grupos aminos de sus aldehídos sustrato, así como en la importancia de la conformación de esta asa para definir el tamaño y la forma de la cavidad del sitio activo. Nuestros resultados apoyan la idea de que la selección natural ha finamente ajustado las características químicas y estructurales del sito activo de las AMADHs a las características químicas y estructurales de sus sustratos aldehídos fisiológicos.

RESUMEN Las plantas purificadoras de agua que carecen de un adecuado sistema de control de calidad pueden generar problemas de salud pública. El objetivo de este estudio fue examinar la calidad microbiológica del agua proveniente de pequeñas plantas purificadoras de la ciudad de Puebla, así como, determinar la existencia de bacterias Aeromonas sp y Pseudomonas sp, y caracterizar si presentan un fenotipo patógeno oportunista. Se recolectaron 70 muestras de garrafones de agua de 25 establecimientos. La cuantificación bacteriana se realizó mediante el método de goteo en placa. Se comprobaron los géneros microbianos mediante análisis bioquímico. En las cepas que mostraron discrepancia se utilizó la identificación molecular con base a secuencias parciales del gen 16S rRNA para confirmar su especie y se les evaluaron sus características de patogenicidad: multirresistencia a antibióticos, producción de biopelícula y actividad hemolítica. El 40 % de las plantas purificadoras no cumplieron con la calidad microbiológica del agua para consumo humano. El 41.4 % de los garrafones de agua muestreados incumplió la normativa, presentando coliformes totales 35.7 %, Pseudomonas 30 %, Enterococcus faecalis 8.6 % y bacterias coliformes fecales el 5.7 %. Se obtuvieron 56 aislados, provenientes de los 29 garrafones contaminados; 10 de ellos se caracterizaron molecularmente, resultando 7 aislados relacionados con especies diferentes de P. aeruginosa y 3 con especies de Aeromonas. De los aislados de Pseudomonas, 5 presentaron resistencia a 2 familias de antibióticos y 2 mostraron multirresistencia. El 36 % de los 10 aislados produjeron hemólisis y biopelícula. Dos cepas de Aeromonas mostraron resistencia a Cefalosporina 3a generación pero no produjeron hemólisis. Los 10 aislados analizados fueron clasificados como no patógenos. Es necesario un seguimiento sanitario más estricto para lograr el cumplimiento de las normas nacionales e internacionales relacionadas con el consumo de agua purificada, para evitar dañar la salud de los consumidores.

ABSTRACT Water purification establishments that lack an adequate quality control system can cause public health problems. The objective of this study was to examine the microbiological quality of water from small purification establishments in the city of Puebla, as well as to determine the existence of Aeromonas sp and Pseudomonas sp bacteria, and to characterize whether they present an opportunistic pathogenic phenotype. 70 water jug samples were collected from 25 establishments. Bacterial quantification was performed using the drop plate method. Microbial genera were determined by biochemical analysis using the standard methodology. In the strains that showed discrepancy, molecular identification based on partial sequences of the 16S rRNA gene was used to confirm their species, and their pathogenic characteristics were evaluated: multiresistance to antibiotics, biofilm production, and hemolytic activity. The results showed that 40 % of the purification establishments did not comply with the microbiological quality of water for human consumption. Similarly, 41.4 % of the jugs of water sampled failed to comply with the regulations, presenting total coliforms 35.7 %, Pseudomonas 30 %, Enterococcus faecalis 8.6 % and fecal coliform bacteria 5.7 %. Likewise, 56 isolates were obtained from the 29 contaminated jugs, of which 10 were molecularly characterized, resulting in 4 different species for P. aeruginosa and 3 for Aeromonas. Of the 7 Pseudomonas isolates, 5 presented resistance to 2 families of antibiotics and 2 showed multiresistance. In total, 36 % of the 10 isolates produced hemolysis and biofilm. Two Aeromonas strains showed resistance to 3rd generation Cephalosporin but did not produce hemolysis. The 10 isolates analyzed were classified as non-pathogenic. A stricter sanitary monitoring is necessary to achieve compliance with national and international standards related to the consumption of purified water, to avoid harming the health of consumers.

RESUMEN: La aparición de cepas de Pseudomonas aeruginosa con resistencias a diversos antibióticos es un problema clínico de gran relevancia. Los métodos de detección de dichas resistencias son laboriosos y lentos, lo que genera una complicación al momento de tratar a los pacientes oportunamente. En este trabajo desarrollamos un método simple de detección simultánea de varios genes de resistencia a carbapenem, mediante un sistema de PCR múltiple. El ensayo de PCR desarrollado, seguido de una separación electroforética de los amplicones, permite distinguir simultáneamente la presencia de 6 genes de resistencia a antibióticos: bla-VIM (261 pb), bla-IMP (587 pb), bla-SPM (648 pb), bla-GIM-1 (753 pb), bla-NDM-1 (813 pb) y bla-KPC (882 pb). Analizamos 7 aislados clínicos obtenidos en Chile, encontrando en 5 de ellos los genes de resistencia bla-VIM, bla-IMP, bla-SPM, bla-GIM y bla-NDM. Encontramos una perfecta correlación entre la detección de diversos genes de resistencia y los resultados obtenidos mediante antibiogramas. Interesantemente, 2 de las cepas mostraron poseer simultáneamente 3 genes de resistencia distintos. Por último, en este trabajo encontramos la presencia de 3 genes nunca antes descritos en aislados clínicos de P. aeruginosa en Chile (bla-IMP, bla-SPM y bla-GIM-1). Hemos desarrollado un test rápido de PCR múltiple, para la detección simultánea de hasta 6 genes de resistencia a antibióticos de la familia.a de las metallo-b-lactamases en P. aeruginosa.

SUMMARY: The appearance of Pseudomonas aeruginosa strains with multi-resistance to antibiotics is a clinical problem of great relevance. The methods for detecting these resistances are laborious and slow, which is a complication when treating patients promptly. In this work, we developed a simple method for simultaneous detection of several carbapenem resistance genes using a multiplex PCR assay. The PCR assay developed, followed by electrophoretic separation of fragments, allows to simultaneously identify the presence of 6 antibiotic resistance genes: bla-VIM (261 bp), bla-IMP (587 bp), bla-SPM (648 bp), bla-GIM-1 (753 bp), bla-NDM-1 (813 bp) and bla-KPC (882 bp). We analyzed 7 clinical isolates of P. aeruginosa obtained in Chile, finding the resistance genes bla-VIM, bla-IMP, bla-SPM, bla-GIM, and bla-NDM in 5 of them. We found a perfect correlation between the detection of various resistance genes by PCR and the results obtained by antibiograms. Interestingly, 2 of the strains possessed 3 different resistance genes simultaneously. Finally, in this work, we found the presence of 3 genes never described before in clinical isolates of P. aeruginosa in Chile (bla-IMP, bla-SPM, and bla-GIM-1). We developed a rapid multiplex PCR test for the simultaneous detection of up to 6 antibiotic resistance genes of the metallo-β-lactamase family in P. aeruginosa.