Antibacterial and bacteriostatic potential of coelomic fluid and body paste of Pheretima posthuma (Vaillant, 1868) (Clitellata, Megascolecidae) against ampicillin resistant clinical bacterial isolates / Potencial antibacteriano e bacteriostático do fluido celômico e pasta corporal de Pheretima posthuma (Vaillant, 1868) (Clitellata, Megascolecidae) contra isolados bacterianos clínicos resistentes à ampicilina

Abstract Pheretima posthuma (Vaillant, 1868), a native earthworm of Pakistan and Southeast Asia, has wide utilization in vermicomposting and bioremediation process. In this study, P. posthuma coelomic fluid (PCF) and body paste (PBP) was evaluated as antibacterial agent against ampicillin (AMP) resistant five Gram positive and four Gram negative clinical isolates. The antibacterial effect of different doses (i.e. 25-100 µg/ml) of PCF and PBP along with AMP and azithromycin (AZM) (negative and positive controls, respectively) were observed through disc diffusion and micro-dilution methods. All nine clinical isolates were noticed as AMP resistant and AZM sensitive. Antibacterial effects of PCF and PBP were dose dependent and zone of inhibitions (ZI) against all clinical isolates were between 23.4 ± 0.92 to 0 ± 00 mm. The sensitivity profile of PCF and PBP against clinical isolates was noticed as 44.44 and 55.56%, respectively. Both PCF and PBP showed bacteriostatic (BTS) action against S. aureus, S. pyogenes, K. pneumonia, N. gonorrhoeae. Moreover, the cumulative BTS potential of PCF and PBP against all isolates was 66.67 and 55.56%, respectively. The MICs of PCF and PBP were ranged from 50-200 µg/ml against selected isolates. The bacterial growth curves indicated that PCF and PBP inhibited the growth of all isolates at their specific MIC concentrations. However, PBP has better antibacterial potential compared to PCF against selected isolates. Therefore, it is concluded that both PCF and PBP of P. posthuma possess antibacterial and BTS potential against ampicillin resistant clinical isolates. This organism might be considered as a second choice of antibacterial agents and can further be utilized in pharmaceutical industries for novel drug manufacturing by prospecting bioactive potential agents.

Resumo Pheretima posthuma (Vaillant, 1868), uma minhoca nativa do Paquistão e sudeste da Ásia, tem ampla utilização em processos de vermicompostagem e biorremediação. Neste estudo, o fluido celômico de P. posthuma (PCF) e a pasta corporal (PBP) foram avaliados como agente antibacteriano contra cinco isolados clínicos Gram-positivos e quatro Gram-negativos resistentes à ampicilina (AMP). O efeito antibacteriano de diferentes doses (ou seja, 25-100 µg / ml) de PCF e PBP juntamente com AMP e azitromicina (AZM) (controles negativo e positivo, respectivamente) foi observado por meio de métodos de difusão em disco e microdiluição. Todos os nove isolados clínicos foram notados como resistentes a AMP e sensíveis a AZM. Os efeitos antibacterianos de PCF e PBP foram dependentes da dose e a zona de inibição (ZI) contra todos os isolados clínicos foi entre 23,4 ± 0,92 a 0 ± 00 mm. O perfil de sensibilidade do PCF e PBP contra isolados clínicos foi observado como 44,44% e 55,56%, respectivamente. Tanto o PCF quanto o PBP mostraram ação bacteriostática (BTS) contra S. aureus, S. pyogenes, K. pneumonia, N. gonorrhoeae. Além disso, o potencial BTS cumulativo de PCF e PBP contra todos os isolados foi de 66,67% e 55,56%, respectivamente. Os MICs de PCF e PBP variaram de 50-200 µg / ml contra isolados selecionados. As curvas de crescimento bacteriano indicaram que o PCF e o PBP inibiram o crescimento de todos os isolados em suas concentrações específicas de MIC. No entanto, PBP tem melhor potencial antibacteriano em comparação com PCF contra isolados selecionados. Portanto, conclui-se que tanto o PCF quanto o PBP de P. posthuma possuem potencial antibacteriano e BTS contra isolados clínicos resistentes à ampicilina. Esse organismo pode ser considerado como uma segunda escolha de agentes antibacterianos e pode ainda ser utilizado nas indústrias farmacêuticas para a fabricação de novos medicamentos por meio da prospecção de agentes com potencial bioativo.

Gut dysbiosis, inflammation and type 2 diabetes in mice using synthetic gut microbiota from diabetic humans / Disbiose intestinal, inflamação e diabetes tipo 2 em camundongos usando microbiota intestinal sintética de humanos diabéticos

Abstract The study was aimed to assess impact of high fat diet (HFD) and synthetic human gut microbiota (GM) combined with HFD and chow diet (CD) in inducing type-2 diabetes (T2D) using mice model. To our knowledge, this is the first study using selected human GM transplantation via culture based method coupled dietary modulation in mice for in vivo establishment of inflammation leading to T2D and gut dysbiosis. Twenty bacteria (T2D1-T2D20) from stool samples of confirmed T2D subjects were found to be morphologically different and subjected to purification on different media both aerobically and anerobically, which revealed seven bacteria more common among 20 isolates on the basis of biochemical characterization. On the basis of 16S rRNA gene sequencing, these seven isolates were identified as Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenes (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). The seven isolates were subsequently used as synthetic gut microbiome (GM) for their role in inducing T2D in mice. Inbred strains of albino mice were divided into four groups and were fed with CD, HFD, GM+HFD and GM+CD. Mice receiving HFD and GM+modified diet (CD/HFD) showed highly significant (P<0.05) increase in weight and blood glucose concentration as well as elevated level of inflammatory cytokines (TNF-α, IL-6, and MCP-1) compared to mice receiving CD only. The 16S rRNA gene sequencing of 11 fecal bacteria obtained from three randomly selected animals from each group revealed gut dysbiosis in animals receiving GM. Bacterial strains including Bacteroides gallinarum (MT152630), Ruminococcus bromii (MT152631), Lactobacillus acidophilus (MT152632), Parabacteroides gordonii (MT152633), Prevotella copri (MT152634) and Lactobacillus gasseri (MT152635) were isolated from mice treated with GM+modified diet (HFD/CD) compared to strains Akkermansia muciniphila (MT152625), Bacteriodes sp. (MT152626), Bacteroides faecis (MT152627), Bacteroides vulgatus (MT152628), Lactobacillus plantarum (MT152629) which were isolated from mice receiving CD/HFD. In conclusion, these findings suggest that constitution of GM and diet plays significant role in inflammation leading to onset or/and possibly progression of T2D. .

Resumo O estudo teve como objetivo avaliar o impacto da dieta rica em gordura (HFD) e da microbiota intestinal humana sintética (GM) combinada com HFD e dieta alimentar (CD) na indução de diabetes tipo 2 (T2D) usando modelo de camundongos. Para nosso conhecimento, este é o primeiro estudo usando transplante de GM humano selecionado através do método baseado em cultura acoplada à modulação dietética em camundongos para o estabelecimento in vivo de inflamação que leva a T2D e disbiose intestinal. Vinte bactérias (T2D1-T2D20) de amostras de fezes de indivíduos T2D confirmados verificaram ser morfologicamente diferentes e foram submetidas à purificação em meios diferentes aerobicamente e anaerobicamente, o que revelou sete bactérias mais comuns entre 20 isolados com base na caracterização bioquímica. Com base no sequenciamento do gene 16S rRNA, esses sete isolados foram identificados como Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenides (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). Esses sete isolados foram, posteriormente, usados ​​como microbioma intestinal sintético (GM) por seu papel na indução de T2D em camundongos. Linhagens consanguíneas de camundongos albinos foram divididas em quatro grupos e foram alimentadas com CD, HFD, GM + HFD e GM + CD. Camundongos que receberam a dieta modificada com HFD e GM + (CD / HFD) mostraram um aumento altamente significativo (P < 0,05) no peso e na concentração de glicose no sangue, bem como um nível elevado de citocinas inflamatórias (TNF-α, IL-6 e MCP-1) em comparação com os ratos que receberam apenas CD. O sequenciamento do gene 16S rRNA de 11 bactérias fecais obtidas de três animais selecionados aleatoriamente de cada grupo revelou disbiose intestinal em animais que receberam GM. Cepas bacterianas, incluindo Bacteroides gallinarum (MT152630), Ruminococcus bromii (MT152631), Lactobacillus acidophilus (MT152632), Parabacteroides gordonii (MT152633), Prevotella copri (MT152634) e Lactobacillus Gasseri (MT152635D), foram tratadas com dieta modificada / CD) em comparação com as linhagens Akkermansia muciniphila (MT152625), Bacteriodes sp. (MT152626), Bacteroides faecis (MT152627), Bacteroides vulgatus (MT152628), Lactobacillus plantarum (MT152629), que foram isoladas de camundongos recebendo CD / HFD. Em conclusão, esses resultados sugerem que a constituição de GM e dieta desempenham papel significativo na inflamação levando ao início ou/e possivelmente à progressão de T2D.

Gut dysbiosis, inflammation and type 2 diabetes in mice using synthetic gut microbiota from diabetic humans / Disbiose intestinal, inflamação e diabetes tipo 2 em camundongos usando microbiota intestinal sintética de humanos diabéticos

The study was aimed to assess impact of high fat diet (HFD) and synthetic human gut microbiota (GM) combined with HFD and chow diet (CD) in inducing type-2 diabetes (T2D) using mice model. To our knowledge, this is the first study using selected human GM transplantation via culture based method coupled dietary modulation in mice for in vivo establishment of inflammation leading to T2D and gut dysbiosis. Twenty bacteria (T2D1-T2D20) from stool samples of confirmed T2D subjects were found to be morphologically different and subjected to purification on different media both aerobically and anerobically, which revealed seven bacteria more common among 20 isolates on the basis of biochemical characterization. On the basis of 16S rRNA gene sequencing, these seven isolates were identified as Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenes (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). The seven isolates were subsequently used as synthetic gut microbiome (GM) for their role in inducing T2D in mice. Inbred strains of albino mice were divided into four groups and were fed with CD, HFD, GM+HFD and GM+CD. Mice receiving HFD and GM+modified diet (CD/HFD) showed highly significant (P<0.05) increase in weight and blood glucose concentration as well as elevated level of inflammatory cytokines (TNF-α, IL-6, and MCP-1) compared to mice receiving CD only. The 16S rRNA gene sequencing of 11 fecal bacteria obtained from three randomly selected animals from each group revealed gut dysbiosis in animals receiving GM. Bacterial strains including Bacteroides gallinarum (MT152630), Ruminococcus bromii (MT152631), Lactobacillus acidophilus (MT152632), Parabacteroides gordonii (MT152633), Prevotella copri (MT152634) and Lactobacillus gasseri (MT152635) were isolated from mice [...].

O estudo teve como objetivo avaliar o impacto da dieta rica em gordura (HFD) e da microbiota intestinal humana sintética (GM) combinada com HFD e dieta alimentar (CD) na indução de diabetes tipo 2 (T2D) usando modelo de camundongos. Para nosso conhecimento, este é o primeiro estudo usando transplante de GM humano selecionado através do método baseado em cultura acoplada à modulação dietética em camundongos para o estabelecimento in vivo de inflamação que leva a T2D e disbiose intestinal. Vinte bactérias (T2D1-T2D20) de amostras de fezes de indivíduos T2D confirmados verificaram ser morfologicamente diferentes e foram submetidas à purificação em meios diferentes aerobicamente e anaerobicamente, o que revelou sete bactérias mais comuns entre 20 isolados com base na caracterização bioquímica. Com base no sequenciamento do gene 16S rRNA, esses sete isolados foram identificados como Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenides (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). Esses sete isolados foram, posteriormente, usados como microbioma intestinal sintético (GM) por seu papel na indução de T2D em camundongos. Linhagens consanguíneas de camundongos albinos foram divididas em quatro grupos e foram alimentadas com CD, HFD, GM + HFD e GM + CD. Camundongos que receberam a dieta modificada com HFD e GM + (CD / HFD) mostraram um aumento altamente significativo (P < 0,05) no peso e na concentração de glicose no sangue, bem como um nível elevado de citocinas inflamatórias (TNF-α, IL-6 e MCP-1) em comparação com os ratos que receberam apenas CD. O sequenciamento do gene 16S rRNA de 11 bactérias fecais obtidas de três animais selecionados aleatoriamente de cada grupo revelou disbiose intestinal em animais que receberam GM. Cepas bacterianas, incluindo Bacteroides gallinarum (MT152630), Ruminococcus [...].

Antibacterial and bacteriostatic potential of coelomic fluid and body paste of Pheretima posthuma (Vaillant, 1868) (Clitellata, Megascolecidae) against ampicillin resistant clinical bacterial isolates / Potencial antibacteriano e bacteriostático do fluido celômico e pasta corporal de Pheretima posthuma (Vaillant, 1868) (Clitellata, Megascolecidae) contra isolados bacterianos clínicos resistentes à ampicilina

Pheretima posthuma (Vaillant, 1868), a native earthworm of Pakistan and Southeast Asia, has wide utilization in vermicomposting and bioremediation process. In this study, P. posthuma coelomic fluid (PCF) and body paste (PBP) was evaluated as antibacterial agent against ampicillin (AMP) resistant five Gram positive and four Gram negative clinical isolates. The antibacterial effect of different doses (i.e. 25-100 µg/ml) of PCF and PBP along with AMP and azithromycin (AZM) (negative and positive controls, respectively) were observed through disc diffusion and micro-dilution methods. All nine clinical isolates were noticed as AMP resistant and AZM sensitive. Antibacterial effects of PCF and PBP were dose dependent and zone of inhibitions (ZI) against all clinical isolates were between 23.4 ± 0.92 to 0 ± 00 mm. The sensitivity profile of PCF and PBP against clinical isolates was noticed as 44.44 and 55.56%, respectively. Both PCF and PBP showed bacteriostatic (BTS) action against S. aureus, S. pyogenes, K. pneumonia, N. gonorrhoeae. Moreover, the cumulative BTS potential of PCF and PBP against all isolates was 66.67 and 55.56%, respectively. The MICs of PCF and PBP were ranged from 50-200 µg/ml against selected isolates. The bacterial growth curves indicated that PCF and PBP inhibited the growth of all isolates at their specific MIC concentrations. However, PBP has better antibacterial potential compared to PCF against selected isolates. Therefore, it is concluded that both PCF and PBP of P. posthuma possess antibacterial and BTS potential against ampicillin resistant clinical isolates. This organism might be considered as a second choice of antibacterial agents and can further be utilized in pharmaceutical industries for novel drug manufacturing by prospecting bioactive potential agents.

Pheretima posthuma (Vaillant, 1868), uma minhoca nativa do Paquistão e sudeste da Ásia, tem ampla utilização em processos de vermicompostagem e biorremediação. Neste estudo, o fluido celômico de P. posthuma (PCF) e a pasta corporal (PBP) foram avaliados como agente antibacteriano contra cinco isolados clínicos Gram-positivos e quatro Gram-negativos resistentes à ampicilina (AMP). O efeito antibacteriano de diferentes doses (ou seja, 25-100 µg / ml) de PCF e PBP juntamente com AMP e azitromicina (AZM) (controles negativo e positivo, respectivamente) foi observado por meio de métodos de difusão em disco e microdiluição. Todos os nove isolados clínicos foram notados como resistentes a AMP e sensíveis a AZM. Os efeitos antibacterianos de PCF e PBP foram dependentes da dose e a zona de inibição (ZI) contra todos os isolados clínicos foi entre 23,4 ± 0,92 a 0 ± 00 mm. O perfil de sensibilidade do PCF e PBP contra isolados clínicos foi observado como 44,44% e 55,56%, respectivamente. Tanto o PCF quanto o PBP mostraram ação bacteriostática (BTS) contra S. aureus, S. pyogenes, K. pneumonia, N. gonorrhoeae. Além disso, o potencial BTS cumulativo de PCF e PBP contra todos os isolados foi de 66,67% e 55,56%, respectivamente. Os MICs de PCF e PBP variaram de 50-200 µg / ml contra isolados selecionados. As curvas de crescimento bacteriano indicaram que o PCF e o PBP inibiram o crescimento de todos os isolados em suas concentrações específicas de MIC. No entanto, PBP tem melhor potencial antibacteriano em comparação com PCF contra isolados selecionados. Portanto, conclui-se que tanto o PCF quanto o PBP de P. posthuma possuem potencial antibacteriano e BTS contra isolados clínicos resistentes à ampicilina. Esse organismo pode ser considerado como uma segunda escolha de agentes antibacterianos e pode ainda ser utilizado nas indústrias farmacêuticas para a fabricação de novos medicamentos por meio da prospecção de agentes com potencial bioativo.

Gut dysbiosis, inflammation and type 2 diabetes in mice using synthetic gut microbiota from diabetic humans

Abstract The study was aimed to assess impact of high fat diet (HFD) and synthetic human gut microbiota (GM) combined with HFD and chow diet (CD) in inducing type-2 diabetes (T2D) using mice model. To our knowledge, this is the first study using selected human GM transplantation via culture based method coupled dietary modulation in mice for in vivo establishment of inflammation leading to T2D and gut dysbiosis. Twenty bacteria (T2D1-T2D20) from stool samples of confirmed T2D subjects were found to be morphologically different and subjected to purification on different media both aerobically and anerobically, which revealed seven bacteria more common among 20 isolates on the basis of biochemical characterization. On the basis of 16S rRNA gene sequencing, these seven isolates were identified as Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenes (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). The seven isolates were subsequently used as synthetic gut microbiome (GM) for their role in inducing T2D in mice. Inbred strains of albino mice were divided into four groups and were fed with CD, HFD, GM+HFD and GM+CD. Mice receiving HFD and GM+modified diet (CD/HFD) showed highly significant (P 0.05) increase in weight and blood glucose concentration as well as elevated level of inflammatory cytokines (TNF-, IL-6, and MCP-1) compared to mice receiving CD only. The 16S rRNA gene sequencing of 11 fecal bacteria obtained from three randomly selected animals from each group revealed gut dysbiosis in animals receiving GM. Bacterial strains including Bacteroides gallinarum (MT152630), Ruminococcus bromii (MT152631), Lactobacillus acidophilus (MT152632), Parabacteroides gordonii (MT152633), Prevotella copri (MT152634) and Lactobacillus gasseri (MT152635) were isolated from mice treated with GM+modified diet (HFD/CD) compared to strains Akkermansia muciniphila (MT152625), Bacteriodes sp. (MT152626), Bacteroides faecis (MT152627), Bacteroides vulgatus (MT152628), Lactobacillus plantarum (MT152629) which were isolated from mice receiving CD/HFD. In conclusion, these findings suggest that constitution of GM and diet plays significant role in inflammation leading to onset or/and possibly progression of T2D. .

Resumo O estudo teve como objetivo avaliar o impacto da dieta rica em gordura (HFD) e da microbiota intestinal humana sintética (GM) combinada com HFD e dieta alimentar (CD) na indução de diabetes tipo 2 (T2D) usando modelo de camundongos. Para nosso conhecimento, este é o primeiro estudo usando transplante de GM humano selecionado através do método baseado em cultura acoplada à modulação dietética em camundongos para o estabelecimento in vivo de inflamação que leva a T2D e disbiose intestinal. Vinte bactérias (T2D1-T2D20) de amostras de fezes de indivíduos T2D confirmados verificaram ser morfologicamente diferentes e foram submetidas à purificação em meios diferentes aerobicamente e anaerobicamente, o que revelou sete bactérias mais comuns entre 20 isolados com base na caracterização bioquímica. Com base no sequenciamento do gene 16S rRNA, esses sete isolados foram identificados como Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenides (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). Esses sete isolados foram, posteriormente, usados como microbioma intestinal sintético (GM) por seu papel na indução de T2D em camundongos. Linhagens consanguíneas de camundongos albinos foram divididas em quatro grupos e foram alimentadas com CD, HFD, GM + HFD e GM + CD. Camundongos que receberam a dieta modificada com HFD e GM + (CD / HFD) mostraram um aumento altamente significativo (P 0,05) no peso e na concentração de glicose no sangue, bem como um nível elevado de citocinas inflamatórias (TNF-, IL-6 e MCP-1) em comparação com os ratos que receberam apenas CD. O sequenciamento do gene 16S rRNA de 11 bactérias fecais obtidas de três animais selecionados aleatoriamente de cada grupo revelou disbiose intestinal em animais que receberam GM. Cepas bacterianas, incluindo Bacteroides gallinarum (MT152630), Ruminococcus bromii (MT152631), Lactobacillus acidophilus (MT152632), Parabacteroides gordonii (MT152633), Prevotella copri (MT152634) e Lactobacillus Gasseri (MT152635D), foram tratadas com dieta modificada / CD) em comparação com as linhagens Akkermansia muciniphila (MT152625), Bacteriodes sp. (MT152626), Bacteroides faecis (MT152627), Bacteroides vulgatus (MT152628), Lactobacillus plantarum (MT152629), que foram isoladas de camundongos recebendo CD / HFD. Em conclusão, esses resultados sugerem que a constituição de GM e dieta desempenham papel significativo na inflamação levando ao início ou/e possivelmente à progressão de T2D.

Antibacterial and bacteriostatic potential of coelomic fluid and body paste of Pheretima posthuma (Vaillant, 1868) (Clitellata, Megascolecidae) against ampicillin resistant clinical bacterial isolates

Abstract Pheretima posthuma (Vaillant, 1868), a native earthworm of Pakistan and Southeast Asia, has wide utilization in vermicomposting and bioremediation process. In this study, P. posthuma coelomic fluid (PCF) and body paste (PBP) was evaluated as antibacterial agent against ampicillin (AMP) resistant five Gram positive and four Gram negative clinical isolates. The antibacterial effect of different doses (i.e. 25-100 µg/ml) of PCF and PBP along with AMP and azithromycin (AZM) (negative and positive controls, respectively) were observed through disc diffusion and micro-dilution methods. All nine clinical isolates were noticed as AMP resistant and AZM sensitive. Antibacterial effects of PCF and PBP were dose dependent and zone of inhibitions (ZI) against all clinical isolates were between 23.4 ± 0.92 to 0 ± 00 mm. The sensitivity profile of PCF and PBP against clinical isolates was noticed as 44.44 and 55.56%, respectively. Both PCF and PBP showed bacteriostatic (BTS) action against S. aureus, S. pyogenes, K. pneumonia, N. gonorrhoeae. Moreover, the cumulative BTS potential of PCF and PBP against all isolates was 66.67 and 55.56%, respectively. The MICs of PCF and PBP were ranged from 50-200 µg/ml against selected isolates. The bacterial growth curves indicated that PCF and PBP inhibited the growth of all isolates at their specific MIC concentrations. However, PBP has better antibacterial potential compared to PCF against selected isolates. Therefore, it is concluded that both PCF and PBP of P. posthuma possess antibacterial and BTS potential against ampicillin resistant clinical isolates. This organism might be considered as a second choice of antibacterial agents and can further be utilized in pharmaceutical industries for novel drug manufacturing by prospecting bioactive potential agents.

Resumo Pheretima posthuma (Vaillant, 1868), uma minhoca nativa do Paquistão e sudeste da Ásia, tem ampla utilização em processos de vermicompostagem e biorremediação. Neste estudo, o fluido celômico de P. posthuma (PCF) e a pasta corporal (PBP) foram avaliados como agente antibacteriano contra cinco isolados clínicos Gram-positivos e quatro Gram-negativos resistentes à ampicilina (AMP). O efeito antibacteriano de diferentes doses (ou seja, 25-100 µg / ml) de PCF e PBP juntamente com AMP e azitromicina (AZM) (controles negativo e positivo, respectivamente) foi observado por meio de métodos de difusão em disco e microdiluição. Todos os nove isolados clínicos foram notados como resistentes a AMP e sensíveis a AZM. Os efeitos antibacterianos de PCF e PBP foram dependentes da dose e a zona de inibição (ZI) contra todos os isolados clínicos foi entre 23,4 ± 0,92 a 0 ± 00 mm. O perfil de sensibilidade do PCF e PBP contra isolados clínicos foi observado como 44,44% e 55,56%, respectivamente. Tanto o PCF quanto o PBP mostraram ação bacteriostática (BTS) contra S. aureus, S. pyogenes, K. pneumonia, N. gonorrhoeae. Além disso, o potencial BTS cumulativo de PCF e PBP contra todos os isolados foi de 66,67% e 55,56%, respectivamente. Os MICs de PCF e PBP variaram de 50-200 µg / ml contra isolados selecionados. As curvas de crescimento bacteriano indicaram que o PCF e o PBP inibiram o crescimento de todos os isolados em suas concentrações específicas de MIC. No entanto, PBP tem melhor potencial antibacteriano em comparação com PCF contra isolados selecionados. Portanto, conclui-se que tanto o PCF quanto o PBP de P. posthuma possuem potencial antibacteriano e BTS contra isolados clínicos resistentes à ampicilina. Esse organismo pode ser considerado como uma segunda escolha de agentes antibacterianos e pode ainda ser utilizado nas indústrias farmacêuticas para a fabricação de novos medicamentos por meio da prospecção de agentes com potencial bioativo.

Antibacterial and bacteriostatic potential of coelomic fluid and body paste of Pheretima posthuma (Vaillant, 1868) (Clitellata, Megascolecidae) against ampicillin resistant clinical bacterial isolates.

Pheretima posthuma (Vaillant, 1868), a native earthworm of Pakistan and Southeast Asia, has wide utilization in vermicomposting and bioremediation process. In this study, P. posthuma coelomic fluid (PCF) and body paste (PBP) was evaluated as antibacterial agent against ampicillin (AMP) resistant five Gram positive and four Gram negative clinical isolates. The antibacterial effect of different doses (i.e. 25-100 µg/ml) of PCF and PBP along with AMP and azithromycin (AZM) (negative and positive controls, respectively) were observed through disc diffusion and micro-dilution methods. All nine clinical isolates were noticed as AMP resistant and AZM sensitive. Antibacterial effects of PCF and PBP were dose dependent and zone of inhibitions (ZI) against all clinical isolates were between 23.4 ± 0.92 to 0 ± 00 mm. The sensitivity profile of PCF and PBP against clinical isolates was noticed as 44.44 and 55.56%, respectively. Both PCF and PBP showed bacteriostatic (BTS) action against S. aureus, S. pyogenes, K. pneumonia, N. gonorrhoeae. Moreover, the cumulative BTS potential of PCF and PBP against all isolates was 66.67 and 55.56%, respectively. The MICs of PCF and PBP were ranged from 50-200 µg/ml against selected isolates. The bacterial growth curves indicated that PCF and PBP inhibited the growth of all isolates at their specific MIC concentrations. However, PBP has better antibacterial potential compared to PCF against selected isolates. Therefore, it is concluded that both PCF and PBP of P. posthuma possess antibacterial and BTS potential against ampicillin resistant clinical isolates. This organism might be considered as a second choice of antibacterial agents and can further be utilized in pharmaceutical industries for novel drug manufacturing by prospecting bioactive potential agents.

Gut dysbiosis, inflammation and type 2 diabetes in mice using synthetic gut microbiota from diabetic humans.

The study was aimed to assess impact of high fat diet (HFD) and synthetic human gut microbiota (GM) combined with HFD and chow diet (CD) in inducing type-2 diabetes (T2D) using mice model. To our knowledge, this is the first study using selected human GM transplantation via culture based method coupled dietary modulation in mice for in vivo establishment of inflammation leading to T2D and gut dysbiosis. Twenty bacteria (T2D1-T2D20) from stool samples of confirmed T2D subjects were found to be morphologically different and subjected to purification on different media both aerobically and anerobically, which revealed seven bacteria more common among 20 isolates on the basis of biochemical characterization. On the basis of 16S rRNA gene sequencing, these seven isolates were identified as Bacteroides stercoris (MT152636), Lactobacillus acidophilus (MT152637), Lactobacillus salivarius (MT152638), Ruminococcus bromii (MT152639), Klebsiella aerogenes (MT152640), Bacteroides fragilis (MT152909), Clostridium botulinum (MT152910). The seven isolates were subsequently used as synthetic gut microbiome (GM) for their role in inducing T2D in mice. Inbred strains of albino mice were divided into four groups and were fed with CD, HFD, GM+HFD and GM+CD. Mice receiving HFD and GM+modified diet (CD/HFD) showed highly significant (P<0.05) increase in weight and blood glucose concentration as well as elevated level of inflammatory cytokines (TNF-α, IL-6, and MCP-1) compared to mice receiving CD only. The 16S rRNA gene sequencing of 11 fecal bacteria obtained from three randomly selected animals from each group revealed gut dysbiosis in animals receiving GM. Bacterial strains including Bacteroides gallinarum (MT152630), Ruminococcus bromii (MT152631), Lactobacillus acidophilus (MT152632), Parabacteroides gordonii (MT152633), Prevotella copri (MT152634) and Lactobacillus gasseri (MT152635) were isolated from mice treated with GM+modified diet (HFD/CD) compared to strains Akkermansia muciniphila (MT152625), Bacteriodes sp. (MT152626), Bacteroides faecis (MT152627), Bacteroides vulgatus (MT152628), Lactobacillus plantarum (MT152629) which were isolated from mice receiving CD/HFD. In conclusion, these findings suggest that constitution of GM and diet plays significant role in inflammation leading to onset or/and possibly progression of T2D. .

Isolated primary bone tumours of the lesser trochanter: Demographics, diagnosis and management.

Primary bone tumours arising from the lesser trochanter (LT) are rare and the literature describing them is sparse. In this paper, we describe the largest series of LT tumours describing the demographics, diagnosis and management. METHODS: A retrospective search of prospectively maintained radiology and oncology databases was performed to identify bone tumours of the LT diagnosed between 2007 and 2018. Metastatic lesions were excluded. All cases were re-reviewed by a senior Radiologist and all case of isolated tumours of the LT were included. RESULTS: 23 cases of isolated LT tumours were identified. There were 15 males and 8 females. Mean age of our cohort was 32 (14-63) years. Most (n = 19, 82.6%) cases had classic radiological (Radiographic, MR Imaging and CT) features and therefore did not undergo biopsy. 4 patients had equivocal radiological investigations that required biopsy to confirm the diagnosis. MR imaging was the most commonly used imaging modality for diagnosis (n = 17, 73.9%)There was a broad range of tumour subtypes, and osteochondroma (n = 17, 73.9%) the most frequently diagnosed. Surgical excision was performed in 4 patients (all osteochondromas) and 4 patients underwent therapeutic radiological guided hip injections for symptomatic relief. The remaining cases were managed conservatively and where they were identified incidentally, no intervention was required. CONCLUSION: We report the largest case series of isolated primary bone tumours of the LT. All isolated primary bone tumours of LT are benign. Osteochondroma is the most common. The diagnosis can be made with on radiological investigations in most patients.

Infrapopliteal angioplasty using a combined angiosomal reperfusion strategy.

INTRODUCTION: Infra-popliteal angioplasty continues to be widely performed with minimal evidence to guide practice. Endovascular device selection is contentious and there is even uncertainty over which artery to treat for optimum reperfusion. Direct reperfusion (DR) targets the artery supplying the ischaemic tissue. Indirect reperfusion (IR) targets an artery supplying collaterals to the ischaemic area. Our unit practice for the last eight years has been to attempt to open all tibial arteries at the time of angioplasty. When successful, this results in both direct and indirect; or combined reperfusion (CR). The aim was to review the outcomes of CR and compare them with DR or IR alone. METHODS: An eight year retrospective review from a single unit of all infra-popliteal angioplasties was undertaken. Wound healing, limb salvage, amputation-free and overall survival data as well as re-intervention rates were captured for all patients. Subgroup analysis for diabetics was undertaken. Kaplan Meier curves are presented for survival outcomes. All odds and hazard ratios (HR) and p values were corrected for bias from confounders using multivariate analysis. RESULTS: 250 procedures were performed: 22 (9%) were CR; 115 (46%) DR and 113 (45%) IR. Amputation-free survival (HR 0.504, p = 0.039) and re-intervention and amputation-free survival (HR 0.414, p = 0.005) were significantly improved in patients undergoing CR compared to IR. Wound healing was similarly affected by reperfusion strategy (OR = 0.35, p = 0.047). Effects of CR over IR were similar when only diabetic patients were considered. CONCLUSIONS: Combined revascularisation can only be achieved in approximately 10% of patients. However, when successful, it results in significant improvements in wound healing and amputation-free survival over simple indirect reperfusion techniques.

Audit of early syphilis: Teesside experience 2005-2007.

A retrospective audit of all cases of early syphilis seen in genitourinary (GU) medicine clinics in Teesside was undertaken between 2005 and 2007. In all 80 patients early syphilis was identified. Data on patient sexuality, treatment, and serological follow-up and partner notification were collected and analysed. There were 48 heterosexual cases. There were 21 heterosexual males, and of the 27 females, 12 (44%) were diagnosed during antenatal care. Intramuscular injections of benzathine penicillin were used to treat 75 out of the 80 patients at the clinics. The remaining five patients received oral therapy for two weeks: four had a course of doxycycline and one received amoxicillin plus probenecid. The treatment rate of the population was 100%. In total, there were 115 contactable partners, of whom 87 (75.7%) were screened and/or treated. Successful treatment was defined as a four-fold or greater titre decrease in the Venereal Disease Reference Laboratory test within 3-6 months after treatment. Twenty-one patients were excluded when assessing this due to incomplete serological follow-up. Satisfactory titre declines occurred in 56 (94.9%) of the 59 patients. Overall, the clinics were shown to have adhered well with national standards. The rise in heterosexual and antenatal incidence is of concern.

Expression of V1A and GRP receptors leads to cellular transformation and increased sensitivity to substance-P analogue-induced growth inhibition.

Small-cell lung cancer (SCLC) is a particularly aggressive cancer, which metastasises early. Despite initial sensitivity to radio- and chemo-therapy, it invariably relapses, so that the 2-year survival remains less than 5%. Neuropeptides particularly arginine vasopressin (AVP) and gastrin-releasing peptide (GRP) act as autocrine and paracrine growth factors and the expression of these and their receptors are a hallmark of the disease. Substance-P analogues including [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance-P (SP-D) and [Arg6,D-Trp7,9,NmePhe8]-substance-P (6-11) (SP-G) inhibit the growth of SCLC cells by modulating neuropeptide signalling. We show that GRP and V1A receptors expression leads to the development of a transformed phenotype. Addition of neuropeptide provides some protection from etoposide-induced cytotoxicity. Receptor expression also leads to an increased sensitivity to substance-P analogue-induced growth inhibition. We show that SP-D and SP-G act as biased agonists at GRP and V1A receptors causing blockade of Gq-mediated Ca2+ release while directing signalling to activate ERK via a pertussis toxin-sensitive pathway. This is the first description of biased agonism at V1A receptors. This unique pharmacology governs the antiproliferative properties of these agents and highlights their potential therapeutic potential for the treatment of SCLC and particularly in tumours, which have developed resistance to chemotherapy.

Increased gastrin-releasing peptide (GRP) receptor expression in tumour cells confers sensitivity to [Arg6,D-Trp7,9,NmePhe8]-substance P (6-11)-induced growth inhibition.

[Arg(6),D-Trp(7,9),N(me)Phe(8)]-substance P (6-11) (SP-G) is a novel anticancer agent that has recently completed phase I clinical trials. SP-G inhibits mitogenic neuropeptide signal transduction and small cell lung cancer (SCLC) cell growth in vitro and in vivo. Using the SCLC cell line series GLC14, 16 and 19, derived from a single patient during the clinical course of their disease and the development of chemoresistance, it is shown that there was an increase in responsiveness to neuropeptides. This was paralleled by an increased sensitivity to SP-G. In a selected panel of tumour cell lines (SCLC, non-SCLC, ovarian, colorectal and pancreatic), the expression of the mitogenic neuropeptide receptors for vasopressin, gastrin-releasing peptide (GRP), bradykinin and gastrin was examined, and their sensitivity to SP-G tested in vitro and in vivo. The tumour cell lines displayed a range of sensitivity to SP-G (IC(50) values from 10.5 to 119 microM). The expression of the GRP receptor measured by reverse transcriptase-polymerase chain reaction, correlated significantly with growth inhibition by SP-G. Moreover, introduction of the GRP receptor into rat-1A fibroblasts markedly increased their sensitivity to SP-G. The measurement of receptor expression from biopsy samples by polymerase chain reaction could provide a suitable diagnostic test to predict efficacy to SP-G clinically. This strategy would be of potential benefit in neuropeptide receptor-expressing tumours in addition to SCLC, and in tumours that are relatively resistant to conventional chemotherapy.