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.

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.

Sensibilidad de Escherichia coli frente a fosfomicina, en infecciones urinarias provenientes de personas con discapacidad que acudieron a la Secretaría Nacional por los Derechos Humanos de las Personas con Discapacidad-SENADIS / Susceptibility of Escherichia coli against fosfomycin in urinary tract infections from people with disabilities who attended SENADIS

INTRODUCCIÓN: Fosfomicina es un antimicrobiano de amplio espectro utilizado para el tratamiento de las infecciones urinarias bajas; tiene actividad sobre bacilos gramnegativos y cocos grampositivos, así también sobre microorganismos multirresistentes, además de ofrecer una alternativa terapéutica de administración vía oral en dosis única, alcanzando una efectividad de 90%. OBJETIVO: Conocer la sensibilidad in vitro de Escherichia coli frente a fosfomicina, en infecciones urinarias provenientes de personas con discapacidad. MATERIAL Y MÉTODO: Estudio observacional, descriptivo, prospectivo, en el que se incluyó un total de 273 muestras de urocultivo, de pacientes de ambos sexos que acudieron a SENADIS, y que en el momento de la consulta presentaban síntomas de infección del tracto urinario, por lo que se les solicitó el análisis de orina simple y cultivo. De las muestras procesadas en el laboratorio de microbiología, que fueron positivas con crecimiento bacteriano significativo, se procedió a la identificación bacteriana y a la realización del antibiograma según las recomendaciones de CLSI. RESULTADOS: De estas 273 muestras, 91 fueron positivas para diferentes uropatógenos, 62/91 (68%) resultaron ser E. coli. De estas cepas de E. coli, 59/62 (95%) mostraron sensibilidad in vitro a fosfomicina. Comentario: Aunque el número de muestra obtenido es pequeño y no extrapolable ampliamente, pretendemos extender el trabajo por un tiempo más para compararlo más adelante. CONCLUSIONES: Se observa que fosfomicina presenta buena actividad in vitro frente a cepas de E. coli aisladas de urocultivo, pudiendo representar una buena alternativa terapéutica a ser utilizada en la población en estudio.

BACKGROUND: Fosfomycin is a broad-spectrum antibiotic used for the treatment of lower urinary tract infections, it is active against gramnegative bacilli and grampositive cocci, as well as against multi-resistant microorganism, in addition to offering a therapeutic alternative for oral administration in a single dose, reaching an effectiveness of 90%. AIM: To study the susceptibility of Escherichia coli to fosfomycin in urinary tract infections, of isolated strains obtained from patients with disabilities. METHODS: It is an observational, descriptive, prospective study in which a total of 273 urine culture samples of patients of both sexes who attended the SENADIS were included, and who at the time of the consultation presented symptoms of urinary tract infection. The urine positive cultures with significant bacterial growth were performed to determine its bacterial identification and the antibiogram according to CLSI recommendations. RESULTS: Of these 273 samples, 91 samples were positive for different uropathogens, with 62/91 (68%) being positive for E. coli. Of these E. coli strains, 59/62 (95%) showed in vitro susceptibility to fosfomycin. Comment: Although the number of samples obtained is small and it cannot be extrapolated, we pretend to extend the work for a while longer to be able to compare it later. CONCLUSION: Fosfomycin has good activity in vitro against E. coli isolated from urine culture in our institution, representing a good alternative to be used in our study population

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.

Clinical and etiological characteristics of infectious vulvovaginitis in children in Zhejiang province from 2009 to 2019 / 中华儿科杂志

Objective: To explore the clinical characteristics, common pathogens in children with vulvovaginitis. Methods: This was a retrospective cases study. A total of 3 268 children with vulvovaginitis were enrolled, who visited the Department of Pediatric and Adolescent Gynecology, Children's Hospital, Zhejiang University School of Medicine from January 2009 to December 2019. Patients were divided into 3 groups according to the age of <7, 7-<10 and 10-18 years. Patients were also divided in to 4 groups according to the season of first visit. The pathogen distribution characteristics of infective vulvovaginitis were compared between the groups. Their clinical data were collected and then analyzed by χ2 test. Results: The were 3 268 girls aged (6.2±2.5) years. There were 1 728 cases (52.9%) aged <7 years, 875 cases (26.8%) aged 7-<10 years, and 665 cases (20.3%) aged 10-18 years. Of these cases, 2 253 cases (68.9%) were bacterial vulvovaginitis, 715 cases (21.9%) were fungal vulvovaginitis and 300 cases (9.2%) were vulvovaginitis infected with other pathogens. Bacterial culture of vaginal secretions was performed in 2 287 cases, and 2 287 strains (70.0%) of pathogens were detected, of which the top 5 pathogens were Streptococcus pyogenes (745 strains, 32.6%), Haemophilus influenzae (717 strains, 31.4%), Escherichia coli (292 strains, 12.8%), Staphylococcus aureus (222 strains, 9.7%) and Klebsiella pneumoniae (67 strains, 2.9%). Regarding different age groups, H.influenzae was the most common in children under 7 years of age (40.3%, 509/1 263), S.pyogenes (41.9%, 356/849) was predominantly in children aged 7 to 10 years, and E.coli was predominant in children aged 10 to 18 years (26.3%, 46/175). Susceptibility results showed that S.pyogenes was susceptible to penicillin G (610/610, 100.0%), ceftriaxone (525/525, 100.0%), and vancomycin (610/610, 100.0%); the resistance rates to erythromycin and clindamycin were 91.9% (501/545)and 90.7% (495/546), respectively. For H.influenzae, 32.5% (161/496) produced β-elactamase, and all strains were sensitive to meropenem (489/489, 100.0%) and levofloxacin (388/388, 100.0%), while 40.5% (202/499) were resistant to ampicillin. Among E.coli, all strains were sensitive to imipenem(100%, 175/175). The resistance rates of E.coli to levofloxacin and ceftriaxone were 29.1% (43/148) and 35.1% (59/168), respectively. A total of 48 strains of methicillin-resistant Staphylococcus aureus (MRSA) were isolated with a proportion of 28.3% (45/159) in 3 268 patients. The results of drug susceptibility test showed that all MRSA strains were sensitive to linezolid 100.0% (40/40), vancomycin (45/45, 100.0%), and tigecycline (36/36, 100.0%); the resistance rates of MRSA to penicillin G, erythromycin and clindamycin were 100% (45/45), 95.6% (43/45) and 88.9% (40/45), respectively. All methicillin-sensitive Staphylococcus aureus (MSSA) strains were sensitive to oxacillin (114/114, 100.0%), linezolid (94/94, 100.0%), vancomycin (114/114, 100.0%), and tigecycline (84/84, 100.0%); it's resistance rates to penicillin G, erythromycin and clindamycin were 78.1% (89/114), 59.7% (68/114) and 46.5% (53/114), respectively. The drug resistance rate of MSSA to penicillin G, erythromycin and clindamycin were lower than those of MRSA (χ²=11.71,19.74,23.95, respectively, all P<0.001). Conclusions: The age of consultation for pediatric infectious vulvovaginitis is mainly around 6 years. The most common pathogens are S.pyogenes, H.influenzae and Escherichia coli. Third generation cephalosporins can be used as the first choice of empirical anti-infection drugs. However, the results of drug susceptibility should be considered for targeted treatment.

Characterization of a D-mannitol oxidase from Paenibacillus sp. and its application in the preparation of D-mannose / 生物工程学报

D-mannose has many functional activities and is widely used in food, medicine, agriculture and other industries. D-mannitol oxidase that can efficiently convert D-mannitol into D-mannose has potential application in the enzymatic preparation of D-mannose. A D-mannitol oxidase (PsOX) was found from Paenibacillus sp. HGF5. The similarity between PsOX and the D-mannitol oxidase (AldO) from Streptomyces coelicolor was 50.94%. The molecular weight of PsOX was about 47.4 kDa. A recombinant expression plasmid pET-28a-PsOX was constructed and expressed in Escherichia coli BL21(DE3). The Km and kcat/Km values of PsOX for D-mannitol were 5.6 mmol/L and 0.68 L/(s·mmol). Further characterization of PsOX showed its optimal pH and temperature were 7.0 and 35 ℃, respectively, while its enzyme activity could be stably remained below 60 ℃. The molar conversion rate of 400 mmol/L D-mannitol by PsOX was 95.2%. The whole cells of PsOX and AldO were used to catalyze 73 g/L D-mannitol respectively. The reaction catalyzed by PsOX completed in 9 h and 70 g/L D-mannose was produced. PsOX showed a higher catalytic efficiency compared to that of AldO. PsOX may facilitate the enzymatic preparation of D-mannose as a novel D-mannose oxidase.

Multicellular coupling fermentation for 3'-sialyllactose conversion using N-acetyl-glucosamine and lactose / 生物工程学报

Sialyllactose is one of the most abundant sialylated oligosaccharides in human milk oligosaccharides (HMOs), which plays an important role in the healthy development of infants and young children. However, its efficient and cheap production technology is still lacking presently. This study developed a two-step process employing multiple-strains for the production of sialyllactose. In the first step, two engineered strains, E. coli JM109(DE3)/ pET28a-BT0453 and JM109(DE3)/pET28a-nanA, were constructed to synthesize the intermediate N-acetylneuraminic acid. When the ratio of the biomass of the two engineered strains was 1:1 and the reaction time was 32 hours, the maximum yield of N-acetylneuraminic acid was 20.4 g/L. In the second step, E. coli JM109(DE3)/ pET28a-neuA, JM109(DE3)/ pET28a-nst and Baker's yeast were added to the above fermentation broth to synthesize 3'-sialyllactose (3'-SL). Using optimal conditions including 200 mmol/L N-acetyl-glucosamine and lactose, 150 g/L Baker's yeast, 20 mmol/L Mg2+, the maximum yield of 3'-SL in the fermentation broth reached 55.04 g/L after 24 hours of fermentation and the conversion rate of the substrate N-acetyl-glucosamine was 43.47%. This research provides an alternative technical route for economical production of 3'-SL.

Expression of BmSPI38 tandem multimers in Escherichia coli and its antifungal activity / 生物工程学报

The aim of this study was to prepare tandem multimeric proteins of BmSPI38, a silkworm protease inhibitor, with better structural homogeneity, higher activity and stronger antifungal ability by protein engineering. The tandem multimeric proteins of BmSPI38 were prepared by prokaryotic expression technology. The effects of tandem multimerization on the structural homogeneity, inhibitory activity and antifungal ability of BmSPI38 were explored by in-gel activity staining of protease inhibitor, protease inhibition assays and fungal growth inhibition experiments. Activity staining showed that the tandem expression based on the peptide flexible linker greatly improved the structural homogeneity of BmSPI38 protein. Protease inhibition experiments showed that the tandem trimerization and tetramerization based on the linker improved the inhibitory ability of BmSPI38 to microbial proteases. Conidial germination assays showed that His6-SPI38L-tetramer had stronger inhibition on conidial germination of Beauveria bassiana than that of His6-SPI38-monomer. Fungal growth inhibition assay showed that the inhibitory ability of BmSPI38 against Saccharomyces cerevisiae and Candida albicans could be enhanced by tandem multimerization. The present study successfully achieved the heterologous active expression of the silkworm protease inhibitor BmSPI38 in Escherichia coli, and confirmed that the structural homogeneity and antifungal ability of BmSPI38 could be enhanced by tandem multimerization. This study provides important theoretical basis and new strategies for cultivating antifungal transgenic silkworm. Moreover, it may promote the exogenous production of BmSPI38 and its application in the medical field.

Development of highly efficient electrocompetent cells for electroporation of Geobacillus thermoglucosidasius NCIMB 11955 / 生物工程学报

Geobacillus thermoglucosidasius is a kind of Gram-positive facultative anaerobic bacteria. The fast growth rate under high temperature and less susceptibility to microbial contamination enable G. thermoglucosidasius to be a desirable producer of biofuels and high-value-added chemicals for the next-generation industrial biotechnology. However, compared with the classical model strain Escherichia coli, the applications of G. thermoglucosidasius are hampered by its low transformation efficiency. This study aimed at obtaining competent cells with high transformation efficiency through inactivating restriction enzymes, adding cell membrane inhibitors and cell wall weakening agents. The results showed that the electro-transformation efficiency achieved 1.2×104 CFU/(μg DNA) by knocking out four genes encoding restriction enzymes. Adding a certain amount of tween 80, dl-threonine and glycine further increased the competent efficiency about 22.5, 44, and 334 times, respectively. The electro-transformation efficiency was enhanced to 4.6×106 CFU/(μg DNA) under the optimized conditions, laying a foundation for genetic manipulation and metabolic engineering of G. thermoglucosidasius.

Secretory expression and fermentation optimization for extracellular production of pullulanase in Vibrio natriegens / 生物工程学报

Pullulanase is a starch debranching enzyme, which is difficult in secretory expression due to its large molecular weight. Vibrio natriegens is a novel expression host with excellent efficiency in protein synthesis. In this study, we achieved secretory expression of the full-length pullulanase PulA and its truncated mutant PulN2 using V. natriegens VnDX strain. Subsequently, we investigated the effects of signal peptide, fermentation temperature, inducer concentration, glycine concentration and fermentation time on the secretory expression. Moreover, the extracellular enzyme activities of the two pullulanases produced in V. natriegens VnDX and E. coli BL21(DE3) were compared. The highest extracellular enzyme activity of PulA and PulN2 in V. natriegens VnDX were 61.6 U/mL and 64.3 U/mL, which were 110% and 62% that of those in E. coli BL21(DE3), respectively. The results indicated that V. natriegens VnDX can be used for secretory expression of the full-length PulA with large molecular weight, which may provide a reference for the secretory expression of other large molecular weight proteins in V. natriegens VnDX.

Synthesis of cello-oligosaccharides which promotes the growth of intestinal probiotics by multi-enzyme cascade reaction / 生物工程学报

Soluble cello-oligosaccharide with 2-6 oligosaccharide units is a kind of oligosaccharide with various biological functions, which can promote the proliferation of intestinal probiotics such as Bifidobacteria and Lactobacillus paracei. Therefore, it has a regulatory effect on human intestinal microbiota. In this study, a Cc 01 strain was constructed by expressing cellodextrin phosphorylase (CDP) in Escherichia coli. By combining with a previously constructed COS 01 strain, a three-enzyme cascade reaction system based on strains COS 01 and Cc 01 was developed, which can convert glucose and sucrose into cello-oligosaccharide. After optimization, the final titer of soluble cello-oligosaccharides with 2-6 oligosaccharide units reached 97 g/L, with a purity of about 97%. It contained cellobiose (16.8 wt%), cellotriose (49.8 wt%), cellotetrose (16.4 wt%), cellopentaose (11.5 wt%) and cellohexose (5.5 wt%). When using inulin, xylo-oligosaccharide and fructooligosaccharide as the control substrate, the biomass (OD600) of Lactobacillus casei (WSH 004), Lactobacillus paracei (WSH 005) and Lactobacillus acidophilus (WSH 006) on cello-oligosaccharides was about 2 folds higher than that of the control. This study demonstrated the efficient synthesis of cello-oligosaccharides by a three-enzyme cascade reaction and demonstrated that the synthesized cello-oligosaccharides was capable of promoting intestinal microbial proliferation.

Dynamic regulation using a quorum-sensing circuit enhances the production of tyrosol by Escherichia coli / 生物工程学报

Tyrosol is a natural polyphenolic product that is widely used in chemical, pharmaceutical and food industries. Currently, the de novo synthesis of tyrosol by Escherichia coli suffers from issues such as low cell density and poor yield. Therefore, the phenylpyruvate decarboxylase mutant ARO10F138L/D218G obtained in our previous study was fused with an alcohol dehydrogenase from different microorganisms for fusion expression, and the optimal ARO10F138L/D218G-L-YahK produced 1.09 g/L tyrosol in shake flasks. In order to further improve tyrosol production, feaB, a key gene in the competing pathway of 4-hydroxyphenylacetic acid, was knocked out, and the resulted strain produced 1.26 g/L tyrosol with an increase of 21.15% compared to that of the control. To overcome the low cell density in tyrosol fermentation, the quorum-sensing circuit was used to dynamically regulate the tyrosol synthesis pathway, so as to alleviate the toxic effect of tyrosol on chassis cells and relieve the growth inhibition. Using this strategy, the yield of tyrosol was increased to 1.74 g/L, a 33.82% increase. In a 2 L fermenter, the production of tyrosol in the engineered strain TRFQ5 dynamically regulated by quorum-sensing reached 4.22 g/L with an OD600 of 42.88. Compared with those in the engineered strain TRF5 statically regulated by induced expression, the yield was increased by 38.58% and the OD600 was enhanced by 43.62%. The combination of blocking the competing pathway using gene knockout technology, and reducing the inhibitory effect of tyrosol toxicity on chassis cells through quorum-sensing dynamic regulation increased the production of tyrosol. This study may facilitate the biosynthesis of other chemicals with high toxicity.

Expression, thermal stability modification and application in PHB degradation of polyhydroxyalkanoate depolymerase from Thermomonospora umbrina / 生物工程学报

Polyhydroxyalkanoate depolymerase (PHAD) can be used for the degradation and recovery of polyhydroxyalkanoate (PHA). In order to develop a PHAD with good stability under high temperature, PHAD from Thermomonospora umbrina (TumPHAD) was heterelogously expressed in Escherichia coli BL21(DE3). At the same time, a mutant A190C/V240C with enhanced stability was obtained via rational design of disulfide bonds. Characterization of enzymatic properties showed that the mutant A190C/V240C had an optimum temperature of 60 ℃, which was 20 ℃ higher than that of the wild type. The half-life at 50 ℃ was 7 hours, at 50 ℃ which was 21 times longer than that of the wild type. The mutant A190C/V240C was used for the degradation of polyhydroxybutyrate (PHB), one of the typical PHA. At 50 ℃, the degradation rate of PHB being treated for 2 hours and 12 hours was 2.1 times and 3.8 times higher than that of the wild type, respectively. The TumPHAD mutant A190C/V240C obtained in this study shows tolerance to high temperature resistance, good thermal stability and strong PHB degradation ability, which may facilitate the degradation and recovery of PHB.

Efficient synthesis of L-methionine by engineering the one carbon module of Escherichia coli / 生物工程学报

L-methionine, also known as L-aminomethane, is one of the eight essential amino acids required by the human body and has important applications in the fields of feed, medicine, and food. In this study, an L-methionine high-yielding strain was constructed using a modular metabolic engineering strategy based on the M2 strain (Escherichia coli W3110 ΔIJAHFEBC/PAM) previously constructed in our laboratory. Firstly, the production of one-carbon module methyl donors was enhanced by overexpression of methylenetetrahydrofolate reductase (methylenetetrahydrofolate reductase, MetF) and screening of hydroxymethyltransferase (GlyA) from different sources, optimizing the one-carbon module. Subsequently, cysteamine lyase (hydroxymethyltransferase, MalY) and cysteine internal transporter gene (fliY) were overexpressed to improve the supply of L-homocysteine and L-cysteine, two precursors of the one-carbon module. The production of L-methionine in shake flask fermentation was increased from 2.8 g/L to 4.05 g/L, and up to 18.26 g/L in a 5 L fermenter. The results indicate that the one carbon module has a significant impact on the biosynthesis of L-methionine, and efficient biosynthesis of L-methionine can be achieved through optimizing the one carbon module. This study may facilitate further improvement of microbial fermentation production of L-methionine.

Metabolic engineering of Escherichia coli for production of salicylate 2-O-β-d-glucoside / 生物工程学报

Salicylate 2-O-β-d-glucoside (SAG) is a derivative of salicylate in plants. Recent reports showed that SAG could be considered as a potential anti-inflammatory substance due to its anti-inflammatory and analgesic effects, and less irritation compared with salicylic acid and aspirin. The biological method uses renewable resources to produce salicylic acid compounds, which is more environmentally friendly than traditional industry methods. In this study, Escherichia coli Tyr002 was used as the starting strain, and a salicylic acid producing strain of E. coli was constructed by introducing the isochorismate pyruvate lyase gene pchB from Pseudomonas aeruginosa. By regulating the expression of the key genes in the downstream aromatic amino acid metabolic pathways, the titer of salicylic acid reached 1.05 g/L in shake flask fermentation. Subsequently, an exogenous salicylic acid glycosyltransferase was introduced into the salicylic acid producing strain to glycosylate the salicylic acid. The newly engineered strain produced 5.7 g/L SAG in shake flask fermentation. In the subsequent batch fed fermentation in a 5 L fermentation tank, the titer of SAG reached 36.5 g/L, which is the highest titer reported to date. This work provides a new route for biosynthesis of salicylate and its derivatives.

Regulation of intracellular level of ATP and NADH in Escherichia coli to promote succinic acid production / 生物工程学报

Succinic acid is an important C4 platform chemical that is widely used in food, chemical, medicine sectors. The bottleneck of fermentative production of succinic acid by engineered Escherichia coli is the imbalance of intracellular cofactors, which often leads to accumulation of by-products, lower yield and low productivity. Stoichiometric analysis indicated that an efficient production of succinic acid by E. coli FMME-N-26 under micro-aeration conditions might be achieved when the TCA cycle provides enough ATP and NADH for the r-TCA pathway. In order to promote succinic acid production, a serial of metabolic engineering strategies include reducing ATP consumption, strengthening ATP synthesis, blocking NADH competitive pathway and constructing NADH complementary pathway were developed. As result, an engineered E. coli FW-17 capable of producing 139.52 g/L succinic acid and 1.40 g/L acetic acid in 5 L fermenter, which were 17.81% higher and 67.59% lower than that of the control strain, was developed. Further scale-up experiments were carried out in a 1 000 L fermenter, and the titer of succinic acid and acetic acid were 140.2 g/L and 1.38 g/L, respectively.

Causative Microorganisms Isolated from Patients with Intra-Abdominal Infections and Their Drug Resistance Profiles: An 11-Year (2011-2021) Single-Center Retrospective Study / 生物医学与环境科学（英文）

OBJECTIVE@#To investigate the distribution and antimicrobial susceptibility of causative microorganisms recovered from patients with intra-abdominal infections (IAIs).@*METHODS@#A total of 2,926 bacterial and fungal strains were identified in samples collected from 1,679 patients with IAIs at the Peking Union Medical College Hospital between 2011 and 2021. Pathogenic bacteria and fungi were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing (AST) was performed using the VITEK 2 compact system and the Kirby-Bauer method. AST results were interpreted based on the M100-Ed31 clinical breakpoints of the Clinical and Laboratory Standards Institute.@*RESULTS@#Of the 2,926 strains identified, 49.2%, 40.8%, and 9.5% were gram-negative bacteria, gram-positive bacteria, and fungi, respectively. Escherichia coli was the most prevalent pathogen in intensive care unit (ICU) and non-ICU patients; however, a significant decrease was observed in the isolation of E. coli between 2011 and 2021. Specifically, significant decreases were observed between 2011 and 2021 in the levels of extended-spectrum β-lactamase (ESBL)-producing E. coli (from 76.9% to 14.3%) and Klebsiella pneumoniae (from 45.8% to 4.8%). Polymicrobial infections, particularly those involving co-infection with gram-positive and gram-negative bacteria, were commonly observed in IAI patients. Moreover, Candida albicans was more commonly isolated from hospital-associated IAI samples, while Staphylococcus epidermidis had a higher ratio in community-associated IAIs. Additionally, AST results revealed that most antimicrobial agents performed better in non-ESBL-producers than in ESBL-producers, while the overall resistance rates (56.9%-76.8%) of Acinetobacter baumanmii were higher against all antimicrobial agents than those of other common gram-negative bacteria. Indeed, Enterococcus faecium, Enterococcus faecalis, S. epidermidis, and S. aureus were consistently found to be susceptible to vancomycin, teicoplanin, and linezolid. Similarly, C. albicans exhibited high susceptibility to all the tested antifungal drugs.@*CONCLUSION@#The distribution and antimicrobial susceptibility of the causative microorganisms from patients with IAIs were altered between 2011 and 2021. This finding is valuable for the implementation of evidence-based antimicrobial therapy and provides guidance for the control of hospital infections.

Surveillance of bacterial resistance in children aged 0-14 years from 2018 to 2022 / 中华儿科杂志

Objective: To understand the distribution and antimicrobial resistance of common bacteria from children aged 0-14 years from China Antimicrobial Resistance Surveillance System. Methods: Bacterial resistance data of 2 575 040 strains from children aged 0-14 years were extracted from the national bacterial resistance surveillance reports from October 2018 to September 2022 and resistance changes were further analyzed by comparing with all data in each year. Results: The total number of bacteria isolated from children in 2018-2022 ranged from 415 306-588 016 strains, accounted for 15.9% (514 193/3 234 372), 16.2% (572 107/3 528 471), 12.8% (415 306/3 249 123), 13.0% (485 418/3 743 027), and 12.2% (588 016/4 828 509), respectively. The proportions of gram-positive bacteria among children were 45.4% (233 456/514 193), 44.5% (254 869/572 107), 44.7% (185 756/415 306), 42.6% (206 903/485 418), and 41.7% (245 044/588 016), respectively. The top five isolates of gram-positive bacteria were Staphylococcus aureus (36.0%-38.8%), Streptococcus pneumoniae (27.1%-31.7%), Staphylococcus epidermidis (7.3%-9.3%), Enterococcus faecium (4.0%-4.8%), and Enterococcus faecium (2.5%-3.6%), and the top five isolates of gram-negative bacteria were Escherichia coli (21.8%-26.2%), Haemophilus influenzae (14.4%-26.4%), Klebsiella pneumoniae (10.1%-14.7%), Moraxella catarrhalis (7.3%-11.9%), and Pseudomonas aeruginosa (5.5%-6.8%). The bacteria from children aged 0-14 years commonly isolated from sputum samples (48.8%-57.0%). The prevalence of methicillin-resistant S. aureus was 28.7%-30.1%. The detection rates of vancomycin-resistant E. faecalis or E. faecium were 0.1%-0.3%. The proportions of non-cerebrospinal fluid-derived penicillin-resistant S. pneumoniae were 0.7%-1.6%. The prevalence of cefotaxime and (or) ceftriaxone-resistant E. coli and K. pneumoniae decreased were 43.7%-50.0% and 31.8%-42.7%, respectively. The resistant rates of E. coli to imipenem and meropenem were 1.2%-1.9% and 1.2%-2.0%, respectively, and the resistant rates of K. pneumoniae to imipenem and meropenem were 7.3%-10.1% and 8.2%-12.2%, respectively. About 6.6%-10.2% and 5.3%-9.6% of the P. aeruginosa isolates showed resistant to imipenem and meropenem, respectively, while 17.2%-24.0% and 19.0%-29.4% of the Acinetobacter baumannii isolates were resistant to imipenem and meropenem, respectively. Conclusions: There is no significant change in the composition of common clinical pathogens in children aged 0-14 years from 2018 to 2022. The prevalence of some resistant bacteria such as methicillin-resistant S. aureus and carbapenem-resistant Enterobacterales is decreasing. However, it is necessary to pay attention to antimicrobial resistance of bacteria from children and long-term monitoring of the prevalence of resistant bacteria should be conducted.