Drinking water quality of various sources in Peshawar, Mardan, Kohat and Swat districts of Khyber Pakhtunkhwa province, Pakistan / Qualidade de água potável de váris fontes dos distritos de Peshawar, Mardan, Kohat e Swat da província de Khyber Pakhtunkhwa, Paquistão

Abstract The present study involves the chemical and bacteriological analysis of water from different sources i.e., bore, wells, bottle, and tap, from Peshawar, Mardan, Swat and Kohat districts of Khyber Pakhtunkhwa (KP) province, Pakistan. From each district, 50 water samples (10 samples from each source), regardless of urban and rural status, were collected from these sources and analysed for sulphates, nitrates, nitrites, chlorides, total soluble solids and coliforms (E. coli). Results indicated that majority of the water sources had unacceptable E. coli count i.e.> 34 CFU/100mL. E. coli positive samples were high in Mardan District, followed by Kohat, Swat and Peshawar district. Besides this, the some water sources were also chemically contaminated by different inorganic fertilizers (nitrates/nitrites of sodium, potassium) but under safe levels whereas agricultural and industrial wastes (chloride and sulphate compounds) were in unsafe range. Among all districts, the water quality was found comparatively more deteriorated in Kohat and Mardan districts than Peshawar and Swat districts. Such chemically and bacteriologically unfit water sources for drinking and can cause human health problems.

Resumo O presente estudo envolve a análise química e bacteriológica de água de diferentes fontes, ou seja, furo, poços, garrafa e torneira, dos distritos de Peshawar, Mardan, Swat e Kohat da província de Khyber Pakhtunkhwa (KP), Paquistão. De cada distrito, 50 amostras de água (10 amostras de cada fonte), independentemente do status urbano e rural, foram coletadas dessas fontes e analisadas para sulfatos, nitratos, nitritos, cloretos, sólidos solúveis totais e coliformes (E. coli). Os resultados indicaram que a maioria das fontes de água tinha uma contagem inaceitável de E. coli, ou seja, > 34 UFC / 100 mL. As amostras positivas para E. coli foram elevadas no distrito de Mardan, seguido por Kohat, Swat e distrito de Peshawar. Além disso, algumas fontes de água também foram contaminadas quimicamente por diferentes fertilizantes inorgânicos (nitratos/nitritos de sódio, potássio), mas em níveis seguros, enquanto os resíduos agrícolas e industriais (compostos de cloreto e sulfato) estavam em níveis inseguros. Entre todos os distritos, a qualidade da água foi considerada comparativamente mais deteriorada nos distritos de Kohat e Mardan do que nos distritos de Peshawar e Swat. Essas fontes de água química e bacteriologicamente impróprias para beber podem causar problemas à saúde humana.

Screening, biochemical characterization and antibiotics resistance/susceptibility of bacteria isolated from native soil and water samples / Triagem, caracterização bioquímica e resistência/suscetibilidade a antibióticos de bactérias isoladas de amostras nativas de solo e água

The present study was conducted to isolate and characterize bacteria from water and soil sample taken from the Lahore Canal at different sites i.e. Mall Road, Mohlanwal and Khera site. Isolated bacterial strains were identified on the basis of morphological and biochemical tests. Identification was confirmed by culturing bacteria on selective media. Antibiotic resistance test was also performed to observe the resistance of bacteria against different antibiotics. Blood agar test was performed for identification of different pathogenic bacteria. The result revealed that water and soil samples of Lahore Canal Lahore from different sites were contaminated with Escherichia coli, Salmonella sp., Vibrio sp., Bacillus spp., Enterococcus sp. and Staphylococcus spp. Due to presence of these pathogens, this water is not suitable for any domestic and irrigation use. Study also revealed that water of the Lahore Canal is harmful for human health as it is contaminated with bacteria that can cause severe disease e.g., Escherichia coli can cause gastroenteritis, Bacillus spp. can cause nausea and vomiting, Enterococcus may infect urinary tract, Salmonella sp. is responsible for Bacteremia, Staphylococcus spp. can cause mild fever and Vibrio sp. can be the reason of cholera. Thus it is rendered unfit for any kind of human use even other than drinking like swimming, bathing, washing etc., until and unless some remedial measures are employed to eradicate pathogenic microorganisms by WASA and LWMS according to standards of WHO. Similarly, it is quite harmful, when and where ever it is used for irrigation without proper treatment.

O presente estudo foi realizado para isolar e caracterizar bactérias de amostras de água e solo retiradas do Canal Lahore, em Lahore, em diferentes locais, ou seja, Mall Road, Mohlanwal e Khera. As cepas bacterianas isoladas foram identificadas com base em testes morfológicos e bioquímicos. A identificação foi confirmada por cultura de bactérias em testes de meios seletivos. O teste de resistência aos antibióticos também foi realizado para observar a resistência das bactérias a diferentes antibióticos. Foi realizado o teste de ágar sangue para identificar diferentes bactérias patogênicas. O resultado revelou que amostras de água e solo do Canal Lahore, Lahore, de diferentes localidades estavam contaminadas com Escherichia coli, Salmonella sp., Vibrio sp., Bacillus spp., Enterococcus sp. e Staphylococcus spp. Por causa da presença desses patógenos, essa água não é adequada para qualquer uso doméstico e de irrigação. O estudo revelou que a água do Canal Lahore é prejudicial à saúde humana, pois está contaminada com bactérias que podem causar doenças graves, por exemplo: Escherichia coli pode ocasionar gastroenterite; Bacillus spp. pode causar náuseas e vômitos; Enterococcus sp. pode infectar o trato urinário; Salmonella sp. é responsável pela bacteremia; Staphylococcus spp. pode causar febre leve; e Vibrio sp. pode ser a razão da cólera. Assim, torna-se imprópria para uso humano, como natação, banho, lavagem etc., até que algumas medidas corretivas sejam empregadas para erradicar microrganismos patogênicos por WASA e LWMS de acordo com os padrões da OMS. Da mesma forma, é bastante prejudicial, quando usada para irrigação sem tratamento adequado.

Prevalencia de sepsis neonatal confirmada microbiológicamente en una maternidad de la Ciudad Autó noma de Buenos Aires / Prevalence of microbiologically confirmed neonatal sepsis at a maternity center in the City of Buenos Aires

Introducción. En neonatos internados es frecuente sospechar sepsis neonatal, pero solo en el 25 % al 30 % se confirma con cultivos positivos. La selección del esquema antibiótico basándose en la epidemiología local favorece el uso racional y minimiza sus efectos colaterales. Objetivo primario. Describir la prevalencia de sepsis precoz y tardía con rescate microbiológico y sus características clínicas. Población y método. Estudio transversal retrospectivo, realizado del 1 de enero de 2013 al 31 de diciembre de 2017, en una maternidad pública de Argentina, que incluyó todos los recién nacidos internados en la unidad con diagnóstico de sepsis precoz y tardía con rescate microbiológico, y aquellos reingresados dentro del mes de vida. Resultados. Ingresaron 3322 recién nacidos, 1296 evaluados por sospecha de sepsis precoz, cultivos positivos en 25 (1,9 %; tasa: 0,86 ‰). El 52 % eran menores de 33 semanas de edad gestacional. Microorganismos: Escherichia coli 5, Listeria monocytogenes 4, Streptococcus agalactiae (SGB) 3, Streptococcus pneumoniae 3. Sepsis tardía (tasa 8,73 ‰), el 68 % ocurridas en menores de 33 semanas. Microorganismos intrahospitalarios: Staphylococcus coagulasa negativos 115, Staphylococcus aureus 47, Escherichia coli 30, Cándida spp. 16, Enterococcus faecalis 13, Klebsiella pneumoniae 11 y Streptococcus agalactiae 10. En los reingresos: E. coli 11, S. aureus 12, SGB 3 y Haemophilus influenzae 3. Conclusiones. Se observa en el período estudiado una frecuencia de sepsis precoz similar a los reportes internacionales, con predominio de E. coli y L. monocytogenes. La tasa de sepsis tardía presentó una tendencia descendente en los años analizados, con predominio de los cocos grampositivos

Introduction. Neonatal sepsis is often suspected in hospitalized newborn infants, but only in 25­30% of cases it is confirmed via a positive culture. Selecting the antibiotics based on local epidemiology favors their rational use and minimizes their side effects. Primary objective. To describe the prevalence of early- and late-onset sepsis with microorganism isolation and their clinical characteristics. Population and method. Retrospective, cross-sectional study conducted between 01-01-2013 and 12-31-2017 in a public maternity center of Argentina in all hospitalized newborn infants with a diagnosis of early- and late-onset sepsis with microorganism isolation, and those re-admitted in their first month of life. Results. A total of 3322 newborn infants were admitted; 1296 were assessed for suspected early- onset sepsis; 25 had a positive culture (1.9%; rate: 0.86‰). Of these, 52% were born before 33 weeks of gestation. Microorganisms: Escherichia coli 5, Listeria monocytogenes 4, Streptococcus agalactiae (SGB) 3, Streptococcus pneumoniae 3. Also, 68% of late-onset sepsis cases (rate: 8.73‰) occurred in infants born before 33 weeks of gestation. Hospital-acquired microorganisms: coagulase-negative Staphylococcus 115, Staphylococcus aureus 47, Escherichia coli 30, Candida spp. 16, Enterococcus faecalis 13, Klebsiella pneumoniae 11, and Streptococcus agalactiae 10. In re-admissions: E. coli 11, S. aureus 12, SGB 3, and Haemophilus influenzae 3. Conclusions. During the study period, the frequency of early-onset sepsis was similar to international reports, with a predominance of E. coli and L. monocytogenes. The rate of late-onset sepsis showed a downward trend in the analyzed years, with a predominance of Gram-positive cocci.

Características clínicas y microbiológicas de la infección de vías urinarias bajas en población ambulatoria / Clinical and Microbiological Characteristics of Lower Urinary Tract Infections in an Outpatient Population

Introducción: El tratamiento de las infecciones del tracto urinario es casi siempre empírico, lo que genera una serie de problemas en la consulta diaria. Objetivo: Caracterizar clínica y microbiológicamente las infecciones de vías urinarias bajas no complicadas en pacientes de una clínica de primer nivel. Métodos: Se realizó un estudio transversal descriptivo. La identificación de las bacterias del cultivo de orina se efectuó por métodos establecidos. La prueba de susceptibilidad a los antimicrobianos se realizó por la técnica Kirby-Bauer. Se utilizó el programa estadístico SPSS versión 26, con la prueba de ji al cuadrado y un análisis multivariado discriminante. Se calculó también razón de momios con el programa Epi-Info. Resultados: Se incluyeron 270 pacientes, con frecuencia de 39,3 por ciento de cultivos positivos, y Escherichia coli como la especie predominante. Se identificaron, además, 31,3 por ciento de bacterias Gram positivas. Se presentó significancia estadística entre la infección urinaria y factores como el sexo, y la infección del tracto urinario previa en las mujeres. Se obtuvo 100 por ciento de cepas resistentes a ampicilina. En general, se obtuvieron porcentajes de resistencia altos en los antimicrobianos probados. Conclusiones: Escherichia coli fue la especie más frecuentemente aislada, sin embargo, existe una serie de microorganismos implicados en enfermedades del tracto genital como Gardnerella vaginalis, que parecen estar involucrados en la etiología de las infecciones del tracto urinario. Se identificaron factores de riesgo como el sexo biológico y las infecciones previas en mujeres. Se obtuvieron porcentajes de resistencia altos en los antimicrobianos probados(AU)

Introduction: The management of urinary tract infections is almost always empirical, which generates a series of problems in the daily consultation. Objective: To characterize, clinically and microbiologically, uncomplicated lower urinary tract infections in patients of a primary level clinic. Methods: A descriptive and cross-sectional study was carried out. Bacterial identification in urine culture was performed by established methods. Antimicrobial susceptibility testing was performed using the Kirby-Bauer technique. The statistical software SPSS (version 26) was used, with the chi squared test and multivariate discriminant analysis. Odds ratios were also calculated with the Epi-Info program. Results: A total of 270 patients were included, with a 39.3percent frequency of positive cultures and Escherichia coli as the predominant species. In addition, 31.3percent of Gram-positive bacteria were identified. There was statistical significance between urinary tract infection and factors such as sex or previous urinary tract infection in women. One result was 100percent of ampicillin-resistant strains. In general, high percentages of resistance were obtained for the tested antimicrobials. Conclusions: Escherichia coli was the most frequently isolated species; however, there is a number of microorganisms implicated in genital tract diseases, such as Gardnerella vaginalis, which appear to be involved in the etiology of urinary tract infections. Risk factors such as biological sex and previous infections in women were identified. High percentages of resistance were obtained for the tested antimicrobials(AU)

Atividade in vitro de extratos e frações de Copernicia prunifera (Arecaceae) contra bactérias de importância clínica em saúde pública / In vitro activity of extracts and fractions of Copernicia prunifera (Arecaceae) against bacteria of clinical importance in public health / Actividad in vitro de extractos y fracciones de Copernicia prunifera (Arecaceae) frente a bacterias de importancia clínica en salud pública

Introdução: O aumento contínuo da resistência bacteriana aos antibióticos convencionais é um problema de importância global. Encontrar produtos como alternativas terapêuticas naturais é essencial. As plantas medicinais possuem uma composição química muito rica, que podem ser estruturalmente otimizadas e processadas em novos antimicrobianos. Objetivo: Avaliar o potencial antibacteriano frente a microrganismos humanos potencialmente patogênicos do extrato etanólico e frações de Copernicia prunifera. Metodologia: A triagem fitoquímica de plantas foi realizada usando métodos de precipitação e coloração e a atividade antibacteriana utilizando o método de difusão em disco e microdiluição em caldo contra cepas padronizadas de Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa e Staphylococcus aureus. Resultados: A triagem fitoquímica revela a presença de taninos, flavonoides, esteroides, triterpernóides, saponinas e alcaloides. Os extratos etanólico e frações da casca do caule e folhas tiveram atividade inibitória contra S. aureus e K. pneumonie com zona de inibição que variou de 7,0±1,73 a 9,33±0,58 mm pelo método de difusão em disco. Pelo método de microdiluição em caldo os extratos foram satisfatórios somente contra K. pneumoniae (CIM = 125 a 1000 µg/mL) S. aureus, P. aeruginosa e E. coli se mostraram resistentes aos testes (CIM > 1000 µg/mL). Conclusão: Esses resultados fornecem uma base para futuras investigações em modelos in vivo, para que os compostos de C. prunifera possam ser aplicados no desenvolvimento de novos agentes antimicrobianos contra K. pneumoniae.

Introduction: The continuous increase in bacterial resistance to conventional antibiotics is a problem of global importance. Finding products as natural therapeutic alternatives is essential. Medicinal plants have a very rich chemical composition, which can be structurally optimized and processed into novel antimicrobials. Objective: To evaluate the antibacterial potential against potentially pathogenic human microorganisms of the ethanolic extract and fractions of Copernicia prunifera. Methodology: Phytochemical screening of plants was performed using precipitation and staining methods and antibacterial activity using the disk diffusion and broth microdilution method against standardized strains of Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Results: Phytochemical screening reveals the presence of tannins, flavonoids, steroids, triterpernoids, saponins and alkaloids. The ethanolic extracts and fractions of stem bark and leaves had inhibitory activity against S. aureus and K. pneumonie with zone of inhibition ranging from 7.0±1.73 to 9.33±0.58 mm by disc diffusion method. By broth microdilution method the extracts were satisfactory only against K. pneumoniae (MIC = 125 to 1000 µg/mL) S. aureus, P. aeruginosa and E. coli were resistant to the tests (MIC > 1000 µg/mL). Conclusion: These results provide a basis for further investigation in in vivo models, so that compounds from C. prunifera can be applied in the development of new antimicrobial agents against K. pneumoniae.

Introducción: El continuo aumento de la resistencia bacteriana a los antibióticos convencionales es un problema de importancia mundial. Es esencial encontrar productos como alternativas terapéuticas naturales. Las plantas medicinales tienen una composición química muy rica, que puede optimizarse estructuralmente y transformarse en nuevos antimicrobianos. Objetivo: Evaluar el potencial antibacteriano frente a microorganismos humanos potencialmente patógenos del extracto etanólico y fracciones de Copernicia prunifera. Metodología: Se realizó el cribado fitoquímico de las plantas mediante los métodos de precipitación y tinción y la actividad antibacteriana mediante el método de difusión en disco y microdilución en caldo frente a cepas estandarizadas de Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa y Staphylococcus aureus. Resultados: El cribado fitoquímico revela la presencia de taninos, flavonoides, esteroides, triterpernoides, saponinas y alcaloides. Los extractos etanólicos y las fracciones de la corteza del tallo y las hojas presentaron actividad inhibitoria contra S. aureus y K. pneumonie con una zona de inhibición que osciló entre 7,0±1,73 y 9,33±0,58 mm por el método de difusión en disco. Por el método de microdilución en caldo, los extractos sólo fueron satisfactorios frente a K. pneumoniae (CMI = 125 a 1000 µg/mL). S. aureus, P. aeruginosa y E. coli fueron resistentes a las pruebas (CMI > 1000 µg/mL). Conclusiones: Estos resultados proporcionan una base para futuras investigaciones en modelos in vivo, de modo que los compuestos de C. prunifera puedan aplicarse en el desarrollo de nuevos agentes antimicrobianos contra K. pneumoniae.

Porphyromonas gingivalis infection causes umbilical vein endothelial barrier dysfunction in vitro by down-regulating ZO-1, occludin and VE-cadherin expression / 南方医科大学学报

OBJECTIVE@#To explore the molecular mechanisms of Porphyromonas gingivalis infection-induced umbilical vein endothelial barrier dysfunction in vitro.@*METHODS@#Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and after the formation of the endothelial barrier, the cells were infected with P. gingivals at a multiplicity of infection (MOI). The transepithelial electrical resistance (TEER) of the cell barrier was measured, and FITC-dextran trans-endothelial permeability assay and bacterial translocation assay were performed to assess the endothelial barrier function. The expression levels of cell junction proteins including ZO-1, occludin and VE-cadherin in the cells were examined by qRT-PCR and Western blotting.@*RESULTS@#In freshly seeded HUVECs, TEER increased until reaching the maximum on Day 5 (94 Ωcm2), suggesting the formation of the endothelial barrier. P. gingivals infection caused an increase of the permeability of the endothelial barrier as early as 0.5 h after bacterial inoculation, and the barrier function further exacerbated with time, as shown by significantly lowered TEER, increased permeability of FITC-dextran (40 000/70 000), and increased translocation of SYTO9-E. coli cross the barrier. MTT assay suggested that P. gingivals infection did not significantly affect the proliferation of HUVECs (P>0.05), but in P. gingivalsinfected cells, the expressions of ZO-1, occludin and VE-cadherin increased significantly at 24 and 48 h after bacterial inoculation (P < 0.05).@*CONCLUSION@#P. gingivals may disrupt the endothelial barrier function by down-regulating the expressions of the cell junction proteins (ZO-1, occludin, VE-cadherin) and increasing the permeability of the endothelial barrier.

Clinical Outcome and Risk Factors of Treatment Failure of Peritoneal Dialysis Associated Peritonitis Caused by Klebsiella Pneumoniae:A Multicenter Study / 中国医学科学院学报

Objective To investigate the treatment outcomes,prognosis,and risk factors of treatment failure of peritoneal dialysis associated peritonitis (PDAP) caused by Klebsiella pneumoniae,and thus provide clinical evidence for the prevention and treatment of this disease. Methods The clinical data of PDAP patients at four peritoneal dialysis centers from January 1,2014 to December 31,2019 were collected retrospectively.The treatment outcomes and prognosis were compared between the patients with PDAP caused by Klebsiella.pneumoniae and that caused by Escherichia coli.Kaplan-Meier method was employed to establish the survival curve of technical failure,and multivariate Logistic regression to analyze the risk factors of the treatment failure of PADP caused by Klebsiella pneumoniae. Results In the 4 peritoneal dialysis centers,1034 cases of PDAP occurred in 586 patients from 2014 to 2019,including 21 cases caused by Klebsiella pneumoniae and 98 cases caused by Escherichia coli.The incidence of Klebsiella pneumoniae caused PDAP was 0.0048 times per patient per year on average,ranging from 0.0024 to 0.0124 times per patient per year during 2014-2019.According to the Kaplan-Meier survival curve,the technical failure rate of Klebsiella pneumoniae caused PDAP was higher than that of Escherichia coli caused PDAP (P=0.022).The multivariate Logistic regression model showed that long-term dialysis was an independent risk factor for the treatment failure of Klebsiella pneumoniae caused PDAP (OR=1.082,95%CI=1.011-1.158,P=0.023).Klebsiella pneumoniae was highly sensitive to amikacin,meropenem,imipenem,piperacillin,and cefotetan,and it was highly resistant to ampicillin (81.82%),cefazolin (53.33%),tetracycline (50.00%),cefotaxime (43.75%),and chloramphenicol (42.86%). Conclusion The PDAP caused by Klebsiella pneumoniae had worse prognosis than that caused by Escherichia coli,and long-term dialysis was an independent risk factor for the treatment failure of Klebsiella pneumoniae caused PDAP.

Prokaryotic expression, polyclonal antibody preparation, spatio-temporal expression profile and functional analysis of c-Myc of Helicoverpa armigera (Lepidoptera: Noctuidae) / 生物工程学报

c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni2+-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells. The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.

Development of a whole-cell biosensor for detecting organophosphorus pesticide methyl parathion in the farmland soil / 生物工程学报

The evaluation of the bioavailability of pollutants in soil is crucial to accurately assess the pollution risk, and whole-cell biosensor is one of the important tools for such evaluation. This study aimed to develop a novel whole-cell biosensor for the detection of methyl parathion in soil using. First, a whole-cell biosensor was constructed by the screened methyl parathion hydrolase mpd gene, the existing specific induction element pobR, and the pUC19 plasmid skeleton. Then, the detection method of methyl parathion in soil extracts was established using 96-well microtiter plate as carrier and five whole-cell biosensors as indicator. The method was applied in the detection of methyl parathion in tested and field soil extracts. Taking E. coli DH5α/pMP-AmilCP with the best detection performance as an example, this biosensor had a detection limit of 6.21-6.66 µg/L and a linear range of 10-10 000 µg/L for methyl parathion in four soil extracts. E. coli DH5α/pMP-RFP and E. coli DH5α/pMP-AmilCP methods have good detection performance for the analysis of methyl parathion in soil extract samples. This biosensor method can help to quickly assess the bioavailability of methyl parathion in soil, and thus help to understand the risk of soil pollution caused by organophosphorus pesticide methyl parathion.

Metabolic engineering of Escherichia coli for adipic acid production / 生物工程学报

Adipic acid is a high-value-added dicarboxylic acid which is primarily used in the production of nylon-66 for manufacturing polyurethane foam and polyester resins. At present, the biosynthesis of adipic acid is hampered by its low production efficiency. By introducing the key enzymes of adipic acid reverse degradation pathway into a succinic acid overproducing strain Escherichia coli FMME N-2, an engineered E. coli JL00 capable of producing 0.34 g/L adipic acid was constructed. Subsequently, the expression level of the rate-limiting enzyme was optimized and the adipic acid titer in shake-flask fermentation increased to 0.87 g/L. Moreover, the supply of precursors was balanced by a combinatorial strategy consisting of deletion of sucD, over-expression of acs, and mutation of lpd, and the adipic acid titer of the resulting E. coli JL12 increased to 1.51 g/L. Finally, the fermentation process was optimized in a 5 L fermenter. After 72 h fed-batch fermentation, adipic acid titer reached 22.3 g/L with a yield of 0.25 g/g and a productivity of 0.31 g/(L·h). This work may serve as a technical reference for the biosynthesis of various dicarboxylic acids.

Modular engineering of Escherichia coli for high-level production of l-tryptophan / 生物工程学报

As an essential amino acid, l-tryptophan is widely used in food, feed and medicine sectors. Nowadays, microbial l-tryptophan production suffers from low productivity and yield. Here we construct a chassis E. coli TRP3 producing 11.80 g/L l-tryptophan, which was generated by knocking out the l-tryptophan operon repressor protein (trpR) and the l-tryptophan attenuator (trpL), and introducing the feedback-resistant mutant aroGfbr. On this basis, the l-tryptophan biosynthesis pathway was divided into three modules, including the central metabolic pathway module, the shikimic acid pathway to chorismate module and the chorismate to tryptophan module. Then we used promoter engineering approach to balance the three modules and obtained an engineered E. coli TRP9. After fed-batch cultures in a 5 L fermentor, tryptophan titer reached to 36.08 g/L, with a yield of 18.55%, which reached 81.7% of the maximum theoretical yield. The tryptophan producing strain with high yield laid a good foundation for large-scale production of tryptophan.

A new biosynthesis route for production of 5-aminovalanoic acid, a biobased plastic monomer / 生物工程学报

5-aminovalanoic acid (5AVA) can be used as the precursor of new plastics nylon 5 and nylon 56, and is a promising platform compound for the synthesis of polyimides. At present, the biosynthesis of 5-aminovalanoic acid generally is of low yield, complex synthesis process and high cost, which hampers large-scale industrial production. In order to achieve efficient biosynthesis of 5AVA, we developed a new pathway mediated by 2-keto-6-aminohexanoate. By combinatory expression of L-lysine α-oxidase from Scomber japonicus, α-ketoacid decarcarboxylase from Lactococcus lactis and aldehyde dehydrogenase from Escherichia coli, the synthesis of 5AVA from L-lysine in Escherichia coli was achieved. Under the initial conditions of glucose concentration of 55 g/L and lysine hydrochloride of 40 g/L, the final consumption of 158 g/L glucose and 144 g/L lysine hydrochloride, feeding batch fermentation to produce 57.52 g/L of 5AVA, and the molar yield is 0.62 mol/mol. The new 5AVA biosynthetic pathway does not require ethanol and H2O2, and achieved a higher production efficiency as compared to the previously reported Bio-Chem hybrid pathway mediated by 2-keto-6-aminohexanoate.

Synergistic effect of β-thujaplicin and tigecycline against tet(X4)-positive Escherichia coli in vitro / 生物工程学报

The widespread of tigecycline resistance gene tet(X4) has a serious impact on the clinical efficacy of tigecycline. The development of effective antibiotic adjuvants to combat the looming tigecycline resistance is needed. The synergistic activity between the natural compound β-thujaplicin and tigecycline in vitro was determined by the checkerboard broth microdilution assay and time-dependent killing curve. The mechanism underlining the synergistic effect between β-thujaplicin and tigecycline against tet(X4)-positive Escherichia coli was investigated by determining cell membrane permeability, bacterial intracellular reactive oxygen species (ROS) content, iron content, and tigecycline content. β-thujaplicin exhibited potentiation effect on tigecycline against tet(X4)-positive E. coli in vitro, and presented no significant hemolysis and cytotoxicity within the range of antibacterial concentrations. Mechanistic studies demonstrated that β-thujaplicin significantly increased the permeability of bacterial cell membranes, chelated bacterial intracellular iron, disrupted the iron homeostasis and significantly increased intracellular ROS level. The synergistic effect of β-thujaplicin and tigecycline was identified to be related to interfere with bacterial iron metabolism and facilitate bacterial cell membrane permeability. Our studies provided theoretical and practical data for the application of combined β-thujaplicin with tigecycline in the treatment of tet(X4)-positive E. coli infection.

Expression, purification, and characterization of the histidine kinase CarS from Fusobacterium nucleatum / 生物工程学报

Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity. In this paper, we focused on the CarRS two-component system of F. nucleatum, and the histidine kinase protein CarS was recombinantly expressed and characterized. Several online software such as SMART, CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein. The results showed that CarS is a membrane protein with two transmembrane helices and contains 9 α-helices and 12 β-folds. CarS protein is composed of two domains, one is the N-terminal transmembrane domain (amino acids 1-170), the other is the C-terminal intracellular domain. The latter is composed of a signal receiving domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP), a phosphate receptor domain (histidine kinase domain, HisKA), and a histidine kinase catalytic domain (histidine kinase-like ATPase catalytic domain, HATPase_c). Since the full-length CarS protein could not be expressed in host cells, a fusion expression vector pET-28a(+)-MBP-TEV-CarScyto was constructed based on the characteristics of secondary and tertiary structures, and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL. CarScyto-MBP protein was purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography with a final concentration of 20 mg/ml. CarScyto-MBP protein showed both protein kinase and phosphotransferase activities, and the MBP tag had no effect on the function of CarScyto protein. The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F. nucleatum.

Genotyping Characteristics of Human Fecal Escherichia coli and Their Association with Multidrug Resistance in Miyun District, Beijing / 生物医学与环境科学（英文）

OBJECTIVE@#To explore the genotyping characteristics of human fecal Escherichia coli( E. coli) and the relationships between antibiotic resistance genes (ARGs) and multidrug resistance (MDR) of E. coli in Miyun District, Beijing, an area with high incidence of infectious diarrheal cases but no related data.@*METHODS@#Over a period of 3 years, 94 E. coli strains were isolated from fecal samples collected from Miyun District Hospital, a surveillance hospital of the National Pathogen Identification Network. The antibiotic susceptibility of the isolates was determined by the broth microdilution method. ARGs, multilocus sequence typing (MLST), and polymorphism trees were analyzed using whole-genome sequencing data (WGS).@*RESULTS@#This study revealed that 68.09% of the isolates had MDR, prevalent and distributed in different clades, with a relatively high rate and low pathogenicity. There was no difference in MDR between the diarrheal (49/70) and healthy groups (15/24).@*CONCLUSION@#We developed a random forest (RF) prediction model of TEM.1 + baeR + mphA + mphB + QnrS1 + AAC.3-IId to identify MDR status, highlighting its potential for early resistance identification. The causes of MDR are likely mobile units transmitting the ARGs. In the future, we will continue to strengthen the monitoring of ARGs and MDR, and increase the number of strains to further verify the accuracy of the MDR markers.

Temporal association between antibiotic use and resistance in Gram-negative bacteria / Associação temporal entre o uso de antibióticos e resistência em bactérias gram-negativas

Abstract The β-lactam/lactamase inhibitors (BLBLIs) combination drugs are considered an effective alternative to carbapenems. However, there is a growing concern that the increased use of BLBLIs may lead to increased resistance. This study determined the temporal association between the consumption of BLBLI and the antimicrobial resistance in Gram-negative bacteria. In this retrospective study, electronic data on the Gram-negative bacterial isolates, including A. baumannii, P. aeruginosa, E. coli, and K. pneumoniae from in-patients and susceptibility testing results were retrieved from the medical records of the clinical laboratory. A linear regression and cross-correlation analysis were performed on the acquired data. Increasing trends (p<0.05) in the consumption of BIBLI and carbapenem with a median use of 27.68 and 34.46 DDD/1000 PD per quarter were observed, respectively. A decreased trend (p=0.023) in the consumption of fluoroquinolones with a median use of 29.13 DDD/1000 PD per quarter was observed. The resistance rate of K. pneumoniae was synchronized with the BIBLI and carbapenem consumptions with a correlation coefficient of 0.893 (p=0.012) and 0.951 (p=0.016), respectively. The cross-correlation analysis against the consumption of BIBLI and meropenem resistant K. pneumoniae was peaked at 0-quarter lag (r=951, p=0.016). There was an increasing trend in the consumption of BLBLI and carbapenems. The increasing trend in the rates of resistance to piperacillin/tazobactam, in line with the increasing consumption of BLBLI, suggests that BLBLI has to be used with caution and cannot be directly considered as a long-term alternative to carbapenems.

Resumo Os medicamentos combinados de β-lactâmicos / inibidores da lactamase (BLBLIs) são considerados uma alternativa eficaz aos carbapenêmicos. No entanto, existe uma preocupação crescente de que o aumento do uso de BLBLIs pode levar ao aumento da resistência. Este estudo determinou a associação temporal entre o consumo de BLBLI e a resistência antimicrobiana em bactérias gram-negativas. Neste estudo retrospectivo, os dados eletrônicos sobre as bactérias gram-negativas isoladas, incluindo A. baumannii, P. aeruginosa, E. coli e K. pneumoniae de pacientes internados e os resultados dos testes de suscetibilidade foram recuperados dos registros médicos do laboratório clínico. Uma regressão linear e análise de correlação cruzada foram realizadas nos dados adquiridos. Foram observadas tendências crescentes (p < 0,05) no consumo de BIBLI e carbapenem com uma mediana de uso de 27,68 e 34,46 DDD/1000 PD por trimestre, respectivamente. Foi observada uma tendência de diminuição (p = 0,023) no consumo de fluoroquinolonas com uma mediana de uso de 29,13 DDD/1000 PD por trimestre. A taxa de resistência de K. pneumoniae foi sincronizada com os consumos de BIBLI e carbapenem com coeficiente de correlação de 0,893 (p = 0,012) e 0,951 (p = 0,016), respectivamente. A análise de correlação cruzada contra o consumo de BIBLI e K. pneumoniae resistente ao meropenem atingiu o pico no intervalo de 0 quarto (r = 951, p = 0,016). Houve uma tendência de aumento no consumo de BLBLI e carbapenêmicos. A tendência crescente nas taxas de resistência a piperacilina/tazobactam, em linha com o consumo crescente de BLBLI, sugere que BLBLI deve ser usado com cautela e não pode ser considerado diretamente como alternativa de longo prazo aos carbapenêmicos.

Detection of microorganisms with antibacterial activities from different industrial wastes and GC-MS analysis of crude microbial extract / Detecção de microrganismos com atividades antibacterianas de diferentes resíduos industriais e análise por GC-MS do extrato microbiano bruto

Abstract Many soil microorganisms' i.e., bacteria and fungi produce secondary metabolites called antibiotics. These are used for the treatment of some of the bacterial, fungal and protozoal diseases of humans. There is a need for isolation of a broad spectrum of antibiotics from microorganisms due to the emergence of antibiotic resistance. In the present study two antibiotic producing bacteria Klebsiella pneumoniae and Bacillus cereus were isolated from pharmaceutical and poultry feed industry of Hattar, Haripur Pakistan. Total 10 waste samples were collected from different industries (Marble, Ghee, Soap, Mineral, Steel, Poultry Feed, Pharmaceutical, Qarshi, Cosmetic and Glass). Thirty-three bacterial strains were isolated from industrial wastes of these ten different industries. Fourteen out of thirty-three bacterial strains exhibited antimicrobial activities against at least one of the test microbes considered in this study including Escherchia coli, Staphylococcus aureus and Salmonella typhi. The bacteria were isolated by standard serial dilution spread plate technique. Morphological characterization of the isolates was done by Gram staining. Nine bacterial isolates out of fourteen were initially identified as B. cereus and five as K. pneumoniae through biochemical characterization. The antibacterial activities were tested by well diffusion method. Maximum number of antibiotic producing bacteria were isolated from pharmaceutical and poultry feed industry based on the results of primary screening, the most potential isolates S9, S19, S20, S22 and S23 were selected for secondary screening. The maximum activity against E. coli and S. aureus was recorded by bacterial isolate S19 i.e zones of inhibition of 6.5mm and 9mm while S20 showed 7.5mm and 6mm zones respectively. Molecular identification was carried out on the basis of 16S rRNA sequence analysis. Finally, the isolates were identified as B. cereus accession number LC538271and K. pneumoniae accession number MT078679. Analysis of bacterial extract S20 through GC-MS indicated the presence of 8 compounds of diverse nature and structure. Present study suggests that wastes of pharmaceutical and poultry feed industry may have antibiotic producing bacteria. These bacteria could be utilized for the production of antibiotics. B. cereus and K. pneumoniae isolated from wastes of poultry feed and pharmaceutical industries have the potential to produce antibiotics and could be used to control the microbial growth.

Resumo Muitos microrganismos do solo, ou seja, bactérias e fungos produzem metabólitos secundários chamados antibióticos. Eles são usados ​​para tratamento de algumas doenças bacterianas, fúngicas e protozoárias em humanos. Há necessidade de isolamento de um amplo espectro de antibióticos de microrganismos devido ao surgimento de resistência aos antibióticos. No presente estudo, duas bactérias produtoras de antibióticos, Klebsiella pneumoniae e Bacillus cereus, foram isoladas da indústria farmacêutica e de ração avícola de Hattar, Haripur, Paquistão. Um total de 10 amostras de resíduos foi coletado de diferentes indústrias (mármore, ghee, sabão, mineral, aço, ração para aves, farmacêutica, Qarshi, cosmética e vidro). Trinta e três cepas bacterianas foram isoladas de resíduos industriais dessas dez diferentes indústrias. Quatorze das 33 cepas bacterianas exibiram atividades antimicrobianas contra pelo menos um dos micróbios de teste considerados neste estudo, incluindo Escherchia coli, Staphylococcus aureus e Salmonella typhi. As bactérias foram isoladas pela técnica de placa de diluição em série padrão. A caracterização morfológica dos isolados foi feita por coloração de gram. Nove isolados bacterianos de 14 foram inicialmente identificados como B. cereus e cinco como K. pneumoniae por meio de caracterização bioquímica. As atividades antibacterianas foram testadas pelo método de difusão em poço. O número máximo de bactérias produtoras de antibióticos foi isolado da indústria farmacêutica e de ração avícola com base nos resultados da triagem primária, os isolados mais potenciais S9, S19, S20, S22 e S23 foram selecionados para a triagem secundária. A atividade máxima contra E. coli e S. aureus foi registrada pelo isolado bacteriano S19, ou seja, zonas de inibição de 6,5 mm e 9 mm, enquanto S20 mostrou zonas de 7,5 mm e 6 mm, respectivamente. A identificação molecular foi realizada com base na análise da sequência 16S rRNA. Finalmente, os isolados foram identificados como B. cereus número de acesso LC538271 e K. pneumoniae número de acesso MT078679. A análise do extrato bacteriano S20 por meio de GC-MS indicou a presença de oito compostos de natureza e estrutura diversas. O presente estudo sugere que resíduos da indústria farmacêutica e de ração para aves podem conter bactérias produtoras de antibióticos. Essas bactérias podem ser utilizadas para a produção de antibióticos B. cereus e K. pneumoniae isolados de resíduos de rações de aves e indústrias farmacêuticas têm potencial para produzir antibióticos e podem ser usados ​​para controlar o crescimento microbiano.

Isolation and characterization of bacteria residing in the oral, gut, and fecal samples of different pheasant species / Isolamento e caracterização de bactérias residentes nas amostras orais, intestinais e fecais de diferentes espécies de faisão

Abstract There is a paucity of research conducted on microbial prevalence in pheasants. The microbiota of captive birds has zoonotic significance and must be characterize. Present study is therefore planned to assess the microbiota from oral, fecal and gut content of captive avian species. It will be helpful in characterization of harmful microbes. Different samples taken from oral, gut and feces of ring-necked pheasants (Phasianus colchicus), green pheasants (Phasianus versicolor), golden pheasant (Chrysolophus pictus) and silver pheasant (Lophura nycthemera). Samples were collected, diluted, and inoculated onto different agar plates (MacConkey, SS agar, MSA and nutrient agar) for cultivation of bacterial species. Colonies of E.coli, Staphylococcus spp. Brachyspira spp. and Campylobacter spp were observed based on colony morphology. Colony forming unit showed E. coli as frequently found bacteria in fecal, oral and gut contents of all the above pheasants. The overall significance difference was found among bacterial species of golden pheasants, green pheasant, ring-necked pheasant, and silver pheasants. It was concluded that E.coli is predominant isolated from heathy pheasants followed by Campylobacter, Staphylococcus and Brachyspira.

Resumo Há uma escassez de pesquisas realizadas sobre a prevalência microbiana em faisões. A microbiota de aves em cativeiro tem significado zoonótico e deve ser caracterizada. O presente estudo está, portanto, planejado para avaliar a microbiota do conteúdo oral, fecal e intestinal de espécies aviárias em cativeiro. Será útil na caracterização de micróbios nocivos. Diferentes amostras retiradas da boca, intestino e fezes de faisões de pescoço redondo (Phasianus colchicus), faisões verdes (Phasianus versicolor), faisões dourados (Chrysolophus pictus) e faisão prateado (Lophura nycthemera). As amostras foram coletadas, diluídas e inoculadas em diferentes placas de ágar (MacConkey, ágar SS, MSA e ágar nutriente) para o cultivo de espécies bacterianas. Colônias de E. coli, Staphylococcus spp., Brachyspira spp. e Campylobacter spp foram observados com base na morfologia da colônia. A unidade formadora de colônia mostrou E. coli como bactéria frequentemente encontrada no conteúdo fecal, oral e intestinal de todos os faisões acima. A diferença de significância geral foi encontrada entre as espécies bacterianas de faisões dourados, faisões verdes, faisões de pescoço anelado e faisões prateados. Verificou-se que a E.coli é predominantemente isolada de faisões saudáveis, seguida por Campylobacter, Staphylococcus e Brachyspira.

Antibacterial and antifungal activity of methanolic extracts of Salix alba L. against various disease causing pathogens / Atividade antibacteriana e antifúngica de extratos metanólicos de Salix alba L. contra vários patógenos que causam doenças

Abstract The present study was aimed to manifest the antibacterial and antifungal activity of methanolic extracts of Salix alba L. against seven Gram-positive and Gram-negative bacterial pathogens e.g. Streptococcus pyogenes, Staphylococcus aureus (1), S. aureus (2), Shigella sonnei, Escherichia coli (1), E. coli (2) and Neisseria gonorrhoeae and three fungal isolates from the air such as Aspergillus terreus, A. ornatus, and Rhizopus stolonifer. Two different serotypes of S. aureus and E. coli were used. The agar well-diffusion method results showed the dose-dependent response of plant extracts against bacterial and fungal strains while some organisms were found resistant e.g. E. coli (1), S. sonnei, A. terreus and R. stolonifer. The highest antibacterial activity was recorded at 17.000±1.732 mm from 100 mg/mL of leaves methanolic extracts against S. pyogenes while the activity of most of the pathogens decreased after 24 h of incubation. The highest antifungal activity was reported at 11.833±1.0 mm against A. ornatus at 50 mg/mL after 48 h of the incubation period. These experimental findings endorse the use of S. alba in ethnopharmacological formulations and suggest the use of methanolic extracts of the said plant to develop drugs to control the proliferation of resistant disease causing pathogenic microbes.

Resumo O presente estudo teve como objetivo manifestar a atividade antibacteriana e antifúngica de extratos metanólicos de Salix alba L. contra sete patógenos bacterianos Gram-positivos e Gram-negativos. Streptococcus pyogenes, Staphylococcus aureus (1), S. aureus (2), Shigella sonnei, Escherichia coli (1), E. coli (2) e Neisseria gonorrhoeae e três isolados de fungos do ar, como Aspergillus terreus, A. ornatus, e Rhizopus stolonifer. Dois sorotipos diferentes de S. aureus e E. coli foram usados. Os resultados do método de difusão em ágar mostraram a resposta dependente da dose de extratos de plantas contra cepas de bactérias e fungos, enquanto alguns organismos foram considerados resistentes, e.g. E. coli (1), S. sonnei, A. terreus e R. stolonifer. A maior atividade antibacteriana foi registrada em 17.000 ± 1.732 de 100 mg/mL de extratos metanólicos de folhas contra S. pyogenes, enquanto a atividade da maioria dos patógenos diminuiu após 24 h de incubação. A maior atividade antifúngica foi relatada em 11,833 ± 1,0 contra A. ornatus a 50 mg/mL após 48 h do período de incubação. Esses achados experimentais endossam o uso de S. alba em formulações etnofarmacológicas e sugerem o uso de extratos metanólicos da referida planta para o desenvolvimento de fármacos que controlem a proliferação de doenças resistentes que causam micróbios patogênicos.

Immobilization of mutated xylanase from Neocallimastix patriciarum in E. coli and application for kraft pulp biobleaching / Imobilização de xilanase mutada de Neocallimastix patriciarum em E. coli e aplicação para polpa kraft

Abstract As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.

Resumo Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.