Caracterização molecular e fenotípica de Klebsiella spp. produtoras de NDM / Molecular and phenotypic characterization of Klebsiella spp. NDM producers

O perfil de resistência, que algumas das espécies do complexo Klebsiella pneumoniae podem expressar, representa uma grande ameaça à saúde humana, particularmente quando resistentes aos carbapenêmicos, que são amplamente utilizados no tratamento de infecções graves em pacientes hospitalizados. O principal mecanismo de resistência aos carbapenêmicos é a produção de carbapenemases, particularmente dos tipos KPC e NDM. Um dos compostos desenvolvidos para o tratamento de infecções causadas por cepas produtoras de KPC é a combinação ceftazidimaavibactam (CAZ-AVI), mas que não tem atividade inibitória sobre metalo-betalactamases, a exemplo das NDMs. Os objetivos deste trabalho foram determinar a frequência das espécies do complexo K. pneumoniae e da coprodução de KPC, avaliar a clonalidade dos isolados, a sensibilidade ao aztreonam-avibactam (ATM-AVI), o desempenho do disco de meropenem (MEM) com inibidores para detecção de coprodução de NDM e KPC e desenvolver um teste de triagem para prever a sensibilidade ao ATM-AVI. Um total de 113 isolados do complexo K. pneumoniae produtoras de NDM ou coprodutoras de NDM e KPC, provenientes da coleção de bactérias do Grupo Fleury, coletadas períodos pré e pós início do uso de CAZ-AVI no Brasil, foram utilizadas neste estudo. A identificação da espécie e a presença dos genes blaNDM e blaKPC foi confirmada por PCR multiplex. A clonalidade dos isolados foi avaliada por eletroforese em campos pulsados (PFGE) após clivagem com XbaI. A produção de carbapenemases foi confirmada utilizando-se o teste Blue Carba. O desempenho dos discos de meropenem e CAZ-AVI contendo um ou mais inibidores de carbapenemases foi comparado com o teste molecular. A pré-difusão combinada foi realizada pré-incubando-se o ágar não inoculado com disco de CAZ-AVI, e a seguir aplicando-se o inóculo bacteriano e um disco de ATM após remover o disco de CAZ-AVI. Após incubação, os halos foram aferidos e correlacionados com a concentração inibitória mínima para ATM-AVI. As CIMs para ATM e ATM-AVI foram determinadas segundo o EUCAST. A identificação das espécies por PCR evidenciou as seguintes frequências: K. pneumoniae 75,2% (n=85); K. quasipneumoniae 16,8% (n=19), e K. variicola 8% (n=9). Uma fração de 12,4% (n=14) dos isolados apresentaram os genes blaNDM e blaKPC e 87,6% (n=99) apenas blaNDM. A análise dos perfis de PFGE de K. pneumoniae evidenciou a presença de cinco grupos clonais predominantes. Isolados do principal grupo clonal Ap (n=15) foram detectados nas cidades de São Paulo e Porto Alegre durante todo o período analisado. O grupo clonal Lp foi detectado nas cidades de São Paulo e Recife em 2019. Os dois principais grupos clonais no período pré-CAZ-AVI continham maior número de isolados do que aqueles no período de uso do CAZ-AVI. Os perfis de PFGE de K. quasipneumoniae evidenciaram quatro grupos clonais predominantes, e presentes apenas no estado de São Paulo, com persistência do grupo clonal Aq desde 2017. Quanto à K. variicola, foram observados dois grupos clonais predominantes Av e Bv, o primeiro presente apenas em São Paulo desde 2018 e o segundo em Porto Alegre apenas em 2019. Calculando-se a diferença entre os diâmetros de halo do disco MEM contendo EDTA e ácido fenilborônico (AFB) e o maior dos halos obtidos para MEM com EDTA ou AFB, observou-se que todos os isolados com coexpressão de KPC e NDM apresentaram diferença ≥ 5 mm. Uma fração de 42,3% dos isolados positivos apenas para blaNDM apresentaram sensibilidade para ATM (CIM ≤ 4 mg/L). Todos os isolados testados apresentaram CIM para ATM-AVI ≤ 1/4 mg/L, sendo a CIM90 0,125/4 mg/l. No teste de pré-difusão combinada, o menor diâmetro de halo obtido foi de 23 mm. A espécie predominante na amostragem foi K. pneumoniae. A disseminação clonal, observada neste estudo, contrasta com a diversidade clonal descrita em outros locais do mundo para produtores de NDM, exceto Grécia e China. Considerando os pontos de corte atuais para ATM, é provável que haja resposta clínica adequada no uso de ATM-AVI no tratamento de infecções causadas por isolados produtores de NDM e coprodutores de KPC e NDM. Utilizando-se o valor de corte de ≤ 5 mm para a diferença entre halos de inibição, de MEM com AFB e EDTA e o segundo maior halo com inibidor, a sensibilidade foi de 100% e a especificidade foi de 96,1,0%. O método de pré-difusão com CAZ-AVI e ATM é um método simples e o diâmetro ≥ 23 mm tem excelente correlação com a CIM para ATM-AVI ≤ 1/4 mg/L

The resistance profile, which some species of the Klebsiella pneumoniae complex may express, represent a great threat to human health, particularly when resistant to carbapenems, which are widely used in the treatment of severe infections in hospitalized patients. The main mechanism of resistance to carbapenems is the production of carbapenemases, particularly KPCs and NDMs. One of the compounds developed for the treatment of infections caused by KPC-producing strains is the combination ceftazidime-avibactam (CAZ-AVI), but which has no inhibitory activity on metallobetalactamases, as is the case for NDMs. The objectives of this work were to determine the frequency of K. pneumoniae complex species and KPC co-production, evaluate the clonality of isolates, the susceptibility to aztreonam-avibactam (ATM-AVI), the performance of meropenem (MEM) disks with inhibitors for detecting NDM co-production and KPC and develop a screening test to predict sensitivity to ATM-AVI. A total of 113 NDM-producing or NDM and KPC co-producing K. pneumoniae complexes, from the Fleury Group's bacteria collection, collected in the pre- and post-starting periods of CAZ-AVI use in Brazil, were used in this study. Species identification and the presence of the blaNDM and blaKPC genes were confirmed by multiplex PCR. The clonality of the isolates was evaluated by pulsed field electrophoresis (PFGE) after cleavage with XbaI. Carbapenemase production was confirmed using the Blue Carba test. The performance of MEM and CAZ-AVI disks containing one or more carbapenemase inhibitors was compared with the molecular test. Combined pre-diffusion was performed by preincubating the uninoculated agar with a CAZ-AVI disk, and then applying the bacterial inoculum and na ATM disk after removal of the CAZ-AVI disk. After incubation, halos were measured and correlated with the minimum inhibitory concentration (MIC) for ATM-AVI. ATM and ATM-AVI MICs were determined according to EUCAST. The identification of species by PCR evidenced the following frequencies: K. pneumoniae 75.2% (n=85); K. quasipneumoniae 16.8% (n=19), and K. variicola 8% (n=9). A fraction of 12.4% (n=14) of the isolates had the blaNDM and blaKPC genes and 87.6% (n=99) had only blaNDM. The analysis of the PFGE profiles of K. pneumoniae evidenced the presence of five predominant clonal groups. Isolates from the main clonal group Ap (n=16) were detected in the cities of São Paulo and Porto Alegre throughout the analyzed period. The clonal group Lp was detected in the cities of São Paulo and Recife 2019. The PFGE profiles of K. quasipneumoniae showed four predominant clonal groups, present only in the state of São Paulo, with persistence of the clonal group Aq since 2017. As for K. variicola, two predominant clonal groups Av and Bv were observed, the first present only in São Paulo since 2018 and the second in Porto Alegre only in 2019. Calculating the difference between the inhibition zone diameters of the MEM disk containing EDTA and phenylboronic acid (AFB) and the largest of the inhibition zone diameters obtained for MEM with EDTA or AFB, it was observed that all isolates with co-expression of KPC and NDM showed a difference 5 ≥mm. A fraction of 42.3% of isolates positive only for blaNDM showed sensitivity to ATM (MIC ≤ 4 mg/L). All tested isolates presented MIC for ATM-AVI ≤ 1/4 mg/L, being the MIC90 0.125/4 mg/l. In the combined pre-diffusion test, the smallest inhibition zone diameter obtained was 23 mm. The predominant species in the sample was K. pneumoniae, but a significant fraction of the other species in the complex was also observed in the sample. The clonal spread observed in this study contrasts with the clonal diversity described elsewhere in the world for NDM-producing isolates, except Greece and China. Considering the current cut-off points for ATM, it is likely that there is an adequate clinical response in the use of ATM-AVI in infections caused by NDM-producing and KPC-NDM co-producing isolates in Brazil. Using the cutoff value of 5 mm for the difference between inhibition zones, of MEM with AFB and EDTA and the second largest zone of MEM with inhibitor, the sensitivity was 100% and the specificity was 96.1%. The pre-diffusion method with CAZ-AVI and ATM is a simple method and the diameter ≥ 23 mm has excellent correlation with the MIC for ATM-AVI ≤ 1/4 mg/L

Analysis for the presence of determinants involved in the transport of mercury across bacterial membrane from polluted water bodies of India

Abstract Mercury, which is ubiquitous and recalcitrant to biodegradation processes, threatens human health by escaping to the environment via various natural and anthropogenic activities. Non-biodegradability of mercury pollutants has necessitated the development and implementation of economic alternatives with promising potential to remove metals from the environment. Enhancement of microbial based remediation strategies through genetic engineering approaches provides one such alternative with a promising future. In this study, bacterial isolates inhabiting polluted sites were screened for tolerance to varying concentrations of mercuric chloride. Following identification, several Pseudomonas and Klebsiella species were found to exhibit the highest tolerance to both organic and inorganic mercury. Screened bacterial isolates were examined for their genetic make-up in terms of the presence of genes (merP and merT) involved in the transport of mercury across the membrane either alone or in combination to deal with the toxic mercury. Gene sequence analysis revealed that the merP gene showed 86–99% homology, while the merT gene showed >98% homology with previously reported sequences. By exploring the genes involved in imparting metal resistance to bacteria, this study will serve to highlight the credentials that are particularly advantageous for their practical application to remediation of mercury from the environment.

The Effect of Temperature on Nitrate and Phosphate Uptake from Synthetic Wastewater by Selected Bacteria Species.

Aim: The aim of this study was to ascertain the effect of temperature on nutrient uptake ability of four bacterial species. Methodology: A total of four bacterial species (Klebsiella sp., Pseudomonas sp., Lysinibacillus sp. and Staphylococcus sp.) were used for the study. The media used for the investigation was synthetic wastewater. Four different temperatures (25ºC, 30ºC, 35ºC and 40ºC) were used for the investigation. The study was carried out under shake flasks conditions. Immediate after inoculation with the respective test bacterial species and every 24 h for a 96 h incubation time, aliquot wastewater samples were removed from the flasks for the estimation of total phosphate, nitrate, pH and growth rate, using standard procedures. Results: The results revealed phosphate and nitrate removal ranges of 10.84 % to 55.55 % and 90.67 % to 97.27 %, respectively in the presence of the Klebsiella sp. In the presence of the Pseudomonas sp, Lysinibacillus sp. and Staphylococcus sp., phosphate removals ranges of 0.36 % to 46.98 %, 11.89 % to 50.80 % and 2.74 % to 51.21 % were observed, respectively. For nitrate concentrations, removal levels that ranged from 2.19 % to 92.95 %, 0.97 % to 23.12 % and 7.56 % to 91.66 % were observed in the presence of Pseudomonas sp, Lysinibacillu ssp. and Staphylococcus sp., respectively. All the test bacterial species showed some measure of efficiency in phosphate removal. For nitrate removal, the Lysinibacillus sp. did not exhibit remarkable nitrate removal ability at any of the temperatures. In addition, the optimum temperatures for phosphate removals were observed to be 30ºC to 40ºC for the Klebsiella sp. and Pseudomonas sp; and 30ºC to 35ºC for the Lysinibacillus sp. and Staphylococcus sp. For nitrate removal, optimum temperatures for removal were observed to be 25ºC to 40ºC, for the Klebsiella sp and 25ºC to 35ºC, for the Pseudomonas sp. and Staphylococcus sp. Conclusion: The study was able to reveal the optimum temperatures for phosphate and nitrate uptake in synthetic wastewater by the test bacterial species.

Análise do tecido hepático de camundongos alimentados com dieta enteral contendo prebióticos e contaminadas com Klebsiella sp / Analysis of liver tissue of mice fed an enteral diet containing probiotics and infected with Klebsiella sp / Análisis del tejido del hígado de ratones alimentados con una dieta enteral que contiene probióticos e infectados con Klebsiella sp

Objetivo: Avaliou-se, neste trabalho, o efeito da administração de frutoligossacarídeo (FOS) e insulina, adicionados à dieta enteral, na translocação de Klebsiella sp. para o fígado de camundongos. Métodos: Camundongos albinos suiços, com quatro a seis semanas de vida divididos em três grupos. Os animais do grupo I (controle) receberam apenas dieta AIN93-G. No tratamento II, os animais receberam dieta enteral contendo FOS, insulina, antibiótico e corticóide. No tratamento III, os animais receberam antibiótico e corticóide, dieta enteral sem FOS e insulina. No quinto dia após o início de oferecimento das dietas, os animais dos tratamentos II e III foram alimentados com dieta enteral contaminada com, aproximadamente, 10 UFC/g de Klebisella pneumoniae. Dois animais de cada grupo foram eutanasiados 24h após o fornecimento da dieta contaminada. O fígado desses animais foi coletado assepticamente e mantido em solução fixadora para estudos histológicos. Resultados: Análises histológicas demonstraram características mais intensas de inflamação no fígado dos animais que foram imunodeprimidos com corticóide, alimentados com dieta enteral contaminada com K. pneumoniae e sem prebióticos, em relação aos animais imunodeprimidos que receberam dieta enteral com prebióticos contaminada com com K. pneumoniae. Conclusão: Dietas enterais contendo ingredientes prebióticos diminuíram o grau de lesões degenerativas no tecido hepático.

Objective: We evaluated in this study, the effect of a fructoligosaccharides (FOS) and insulin, added to the enteral diet on translocation of Klebsiella sp. to the liver of mice. Methods: Swiss albino mice, with four to six weeks of life divided into three groups. Animals in group I (control) received only diet AIN 93-G. In treatment II, animals received enteral diet containing FOS, insulin, antibiotics and steroids. In treatment III, animals given antibiotics and corticosteroids, enteral nutrition and FOS without insulin. On the fifth day after start offering the diets, the animals in treatments II and III were fed with enteral feeding contaminated with approximately 10 CFU/g Klebisella pneumoniae. Two animals from each group were euthanized 24 hours after the diet contaminated. The liver of these animals were collected aseptically and kept in fixative solution for histological studies. Results: Histological analysis showed features of more intense inflammation in the liver of animals that were immunosuppressed with corticosteroids, enteral feeding on contaminated with K. pneumoniae and without probiotics, for immunosuppressed animals that received enteral feeding with prebiotics-contaminated with K. pneumoniae. Conclusion: Enteral diets containing prebiotic ingredients decreased the degree of degenerative lesions in liver tissue.

Objetivo: Se evaluó en este estudio, el efecto de una fructoligosaccharides (FOS) y la insulina, sumado a la dieta enteral en la translocación de Klebsiella sp. para el hígado de ratones. Métodos: ratones albino suizo, con cuatro a seis semanas de vida dividido en tres grupos. Animales en el grupo I (control) recibió dieta sólo AIN 93-G. En el tratamiento II, los animales recibieron la dieta enteral que contiene FOS, insulina, antibióticos y esteroides. En el tratamiento III, los animales que recibieron antibióticos y corticosteroides, la nutrición enteral y FOS sin insulina. En el quinto día comenzará después de ofrecer las dietas, los animales en los tratamientos II y III fueron alimentados con la alimentación enteral contaminados con aproximadamente 10 UFC/g Klebisella pneumoniae. Dos animales de cada grupo se sacrificaron 24 horas después de la dieta contaminada. El hígado de estos animales fueron recogidos asépticamente y se mantienen en solución fijadora para estudios histológicos. Resultados: El análisis histológico demostró características de más intensa inflamación en el hígado de animales que fueron inmunodeprimidos con corticosteroides, la nutrición enteral en contaminadas con K. pneumoniae y sin probióticos, por inmunodeprimidos animales que recibieron alimentación enteral con prebióticos contaminados con K. pneumoniae. Conclusión: enteral con dietas que contenían ingredientes prebióticos redujo el grado de lesiones degenerativas en el tejido hepático.