Sequencing and multiple antimicrobial resistance of Pseudomonas fluorescens isolated from Nile tilapia fish in Egypt

Abstract Pseudomonas fluorescens is one of the main causes of septicemic diseases among freshwater fish, causing severe economic losses and decreasing farm efficiency. Thus, this research was aimed to investigate the occurrence of P. fluorescens in Nile Tilapia (O. niloticus) fish in Egypt, gene sequencing of 16SrDNA gene, and antimicrobial susceptibility. P. fluorescens strains were detected in 32% (128/400) of apparently healthy (9%; 36/400) and diseased (23%; 92/400) Nile tilapia fish. The highest prevalence was observed in gills of fish, 31.3% followed by intestine 26.9%, liver 24.2%, and kidneys 17.6%. The PCR results for the 16SrDNA gene of P. fluorescens showed 16SrDNA gene in 30% of examined isolates. Moreover, Homogeny and a strong relationship between strains of P. fluorescens was confirmed using 16SrDNA sequences. Beside the responsibility of 16SrDNA gene on the virulence of P. fluorescens. The results of antimicrobial susceptibility tests revealed that all strains were resistant to piperacillin (100%), followed by ceftazidime (29.7%), and cefepime (25.8%). The strains of P. fluorescence were highly sensitive to cefotaxime (74.2%), followed by ceftriaxone and levofloxacin (70.3% each). Interestingly, 29.7% of strains of P. fluorescens were multiple antimicrobial-resistant (MAR).

Resumo Pseudomonas fluorescens é uma das principais causas de doenças septicêmicas em peixes de água doce, causando graves perdas econômicas e diminuindo a eficiência da fazenda. Assim, esta pesquisa teve como objetivo investigar a ocorrência de P. fluorescens em peixes de tilápia-do-nilo (O. niloticus) no Egito, sequenciamento do gene 16S rDNA e suscetibilidade antimicrobiana. Cepas de P. fluorescens foram detectadas em 32% (128/400) de peixes tilápia-do-nilo aparentemente saudáveis (9%; 36/400) e doentes (23%; 92/400). A maior prevalência foi observada nas brânquias dos peixes, 31,3%, seguida pelo intestino 26,9%, fígado 24,2% e rins 17,6%. Os resultados da PCR para o gene 16SrDNA de P. fluorescens mostraram o gene 16SrDNA em 30% dos isolados examinados. Além disso, a homogeneidade e uma forte relação entre cepas de P. fluorescens foi confirmada usando sequências de 16SrDNA. Além da responsabilidade do gene 16SrDNA na virulência de P. fluorescens. Os resultados dos testes de suscetibilidade antimicrobiana revelaram que todas as cepas foram resistentes à piperacilina (100%), seguida pela ceftazidima (29,7%) e cefepima (25,8%). As cepas de P. fluorescens foram altamente sensíveis à cefotaxima (74,2%), seguida pela ceftriaxona e levofloxacina (70,3% cada). Curiosamente, 29,7% das cepas de P. fluorescens eram multirresistentes a antimicrobianos (MAR).

Sequencing and multiple antimicrobial resistance of Pseudomonas fluorescens isolated from Nile tilapia fish in Egypt / Sequenciamento e resistência antimicrobiana múltipla de Pseudomonas fluorescens isoladas de tilápia-do-nilo no Egito

Pseudomonas fluorescens is one of the main causes of septicemic diseases among freshwater fish, causing severe economic losses and decreasing farm efficiency. Thus, this research was aimed to investigate the occurrence of P. fluorescens in Nile Tilapia (O. niloticus) fish in Egypt, gene sequencing of 16SrDNA gene, and antimicrobial susceptibility. P. fluorescens strains were detected in 32% (128/400) of apparently healthy (9%; 36/400) and diseased (23%; 92/400) Nile tilapia fish. The highest prevalence was observed in gills of fish, 31.3% followed by intestine 26.9%, liver 24.2%, and kidneys 17.6%. The PCR results for the 16SrDNA gene of P. fluorescens showed 16SrDNA gene in 30% of examined isolates. Moreover, Homogeny and a strong relationship between strains of P. fluorescens was confirmed using 16SrDNA sequences. Beside the responsibility of 16SrDNA gene on the virulence of P. fluorescens. The results of antimicrobial susceptibility tests revealed that all strains were resistant to piperacillin (100%), followed by ceftazidime (29.7%), and cefepime (25.8%). The strains of P. fluorescence were highly sensitive to cefotaxime (74.2%), followed by ceftriaxone and levofloxacin (70.3% each). Interestingly, 29.7% of strains of P. fluorescens were multiple antimicrobial-resistant (MAR).

Pseudomonas fluorescens é uma das principais causas de doenças septicêmicas em peixes de água doce, causando graves perdas econômicas e diminuindo a eficiência da fazenda. Assim, esta pesquisa teve como objetivo investigar a ocorrência de P. fluorescens em peixes de tilápia-do-nilo (O. niloticus) no Egito, sequenciamento do gene 16S rDNA e suscetibilidade antimicrobiana. Cepas de P. fluorescens foram detectadas em 32% (128/400) de peixes tilápia-do-nilo aparentemente saudáveis ​​(9%; 36/400) e doentes (23%; 92/400). A maior prevalência foi observada nas brânquias dos peixes, 31,3%, seguida pelo intestino 26,9%, fígado 24,2% e rins 17,6%. Os resultados da PCR para o gene 16SrDNA de P. fluorescens mostraram o gene 16SrDNA em 30% dos isolados examinados. Além disso, a homogeneidade e uma forte relação entre cepas de P. fluorescens foi confirmada usando sequências de 16SrDNA. Além da responsabilidade do gene 16SrDNA na virulência de P. fluorescens. Os resultados dos testes de suscetibilidade antimicrobiana revelaram que todas as cepas foram resistentes à piperacilina (100%), seguida pela ceftazidima (29,7%) e cefepima (25,8%). As cepas de P. fluorescens foram altamente sensíveis à cefotaxima (74,2%), seguida pela ceftriaxona e levofloxacina (70,3% cada). Curiosamente, 29,7% das cepas de P. fluorescens eram multirresistentes a antimicrobianos (MAR).

Characteristics of faecal bacterial flora and volatile fatty acids in Min pig, Landrace pig, and Yorkshire pig

BACKGROUND In order to study the influence of long-term growth process and evolution environment on intestinal bacteria of different breeds, the intestinal bacteria and volatile fatty acids among the faeces of Min, Landrace and Yorkshire pigs were analysed by Illumina high-throughput sequencing of the 16S-rDNA and gas chromatography. RESULTS The shared core microbiota of Landrace, Yorkshire and Min pig were 1273, accounting for 69.56% of total abundance of organisms. The proportion of Firmicutes in Min pig faeces (57.89%) was significantly higher than that in Landrace and Yorkshire pig faeces (47.01% and 46.40%, respectively) (P < 0.05), but that of Bacteroidetes was exactly opposite. Moreover, Min pig presented more highly efficient membrane transport, environmental adaptation, carbohydrate transport, and metabolism than Yorkshire pig (P < 0.05). The acetic acid/total volatile fatty acid ratio in Min pig was significantly higher than that in Landrace pig (P < 0.05), and the isobutyric acid/ total volatile fatty acid ratio in Min pig was significantly larger than that in Yorkshire pig (P < 0.05). Furthermore, the content of branched chain volatile fatty acids in Min pig was significantly higher than that in Yorkshire pig (P < 0.05). CONCLUSION This study demonstrated that Min pig, as an excellent breed in the cold area of China, possessed special intestinal floral structure compared to the imported pigs in order to satisfy their phys iological and metabolic demands, which may influence their characteristics such as resistance to cold, diseases, and crude feeding, and the ability to deposit intramuscular fat.

Analysis of intestinal patients' flora changes with severe pneumonia based on 16SrDNA sequencing technology / 中华危重病急救医学

Objective To investigate the characteristics of gut microbiota dysbosis in patients with severe pneumonia using 16SrDNA sequencing. Methods A prospective observational research was conducted. The stool samples retained by natural defecation or enema within 2 days after hospital were collected from 16 patients with severe pneumonia admitted to department of intensive care unit (ICU) of General Hospital of Ningxia Medical University from June to December in 2018 and 10 persons for physical exam were enrolled as the healthy control group. The 16SrDNA sequencing technology was used to detect fecal flora and analyze biological information. Results ① 1015475 effective sequences were obtained from the stool samples from the severe pneumonia group and the healthy control group. Using 16SrDNA method, it was found that the average effective length of the sample sequence was 458.35 bp and the average sequence number of the total samples was 39056.73. ② Analysis of α diversity of gut microbiota showed that, compared with the healthy control group, the Ace index, Chao index and the Shannon index of gut microbiota diversity in the severe pneumonia group were significantly decreased [Ace index: 167.23 (143.14, 211.26) vs. 227.71 (214.53, 247.05), Chao index: 152.38 (138.09, 182.54) vs. 228.25 (215.49, 248.95), Shannon index:2.37 (1.68, 2.89) vs. 3.39 (3.03, 3.63), all P < 0.01], and the Simpson index was significantly increased [0.21 (0.11, 0.33) vs. 0.07 (0.06, 0.12), P < 0.01], which indicated the gut microbiota diversity of the severe pneumonia group was decreased. ③ Analysis of β diversity of gut microbiota, principal coordinate analysis (PCoA) showed that gut microbiota structural with the healthy control group was similar, while that in the severe pneumonia group was different. Adonis analysis showed that the structural of the gut microflora revealing significant differences between the severe pneumonia group and the healthy control group (R2 = 0.061, P = 0.05). ④ Analysis of phylum difference gut microflora showed that, compared with the healthy control group, the proportion of Firmicutes in severe pneumonia group was decreased [27.36 (18.12, 39.28)％ vs. 52.25 (38.36, 63.82)％, P = 0.02], the proportions of Actinobacterias, Synergistetes and Fusobacterias were increased [2.30 (0.30, 4.80)％ vs. 0.02 (0.00, 0.06)％, 0.36 (< 0.01, 0.57)％ vs. < 0.01 (< 0.01, < 0.01)％, 0.01 (< 0.01, 0.08)％ vs. < 0.01 (< 0.01, < 0.01)％, all P < 0.05]. ⑤ Analysis of genus difference gut microflora showed that, the proportions of Bifidobacterium, Ruminococcus, Pseudobutyrivibrio, Coprococcus, Lachnospira and Prevotella in the severe pneumonia group were significantly lower than those in healthy control group [0.18 (0.01, 0.25)％ vs. 3.40 (0.46, 5.78)％, 0.01 (< 0.01, 0.29)％ vs. 2.26 (0.84, 4.86)％, 0.01 (< 0.01, 0.02)％ vs. 2.73 (1.87, 5.74)％, 0.02 (< 0.01, 0.07)％vs. 0.80 (0.50, 2.32)％, < 0.01 (< 0.01, < 0.01)％ vs. 0.88 (0.33, 2.08)％, 0.02 (< 0.01, 0.31)％ vs. 7.74 (0.07, 36.27)％, all P < 0.05]; the proportions of Escherichia and Enterococcus in the severe pneumonia group were higher than those in healthy control group, but there was no difference between the two groups [2.00 (0.57, 10.23)％ vs. 1.16 (0.23, 2.68)％, 0.02 (< 0.01, 0.42)％ vs. < 0.01 (< 0.01, 0.04)％, both P > 0.05]; the proportions of Fusobacterium and Staphylococcus in severe pneumonia group were significantly higher than those in healthy control group [0.01 (< 0.01, 0.08)％ vs. < 0.01 (< 0.01, < 0.01)％, 0.01 (< 0.01, 0.02)％ vs. < 0.01 (< 0.01, < 0.01)％, both P < 0.05]. Conclusion Gut microbiota dysbiosis in patients with severe pneumonia shows that the abundance and diversity decrease, structure of intestinal flora changes, and beneficial symbiotic bacteria decrease and pathogenic bacteria increase, which may be associated with the occurrence and development of severe pneumonia.

Analysis of intestinal patients' flora changes with severe pneumonia based on 16SrDNA sequencing technology / 中华危重病急救医学

Objective@#To investigate the characteristics of gut microbiota dysbosis in patients with severe pneumonia using 16SrDNA sequencing.@*Methods@#A prospective observational research was conducted. The stool samples retained by natural defecation or enema within 2 days after hospital were collected from 16 patients with severe pneumonia admitted to department of intensive care unit (ICU) of General Hospital of Ningxia Medical University from June to December in 2018 and 10 persons for physical exam were enrolled as the healthy control group. The 16SrDNA sequencing technology was used to detect fecal flora and analyze biological information.@*Results@#① 1 015 475 effective sequences were obtained from the stool samples from the severe pneumonia group and the healthy control group. Using 16SrDNA method, it was found that the average effective length of the sample sequence was 458.35 bp and the average sequence number of the total samples was 39 056.73. ② Analysis of α diversity of gut microbiota showed that, compared with the healthy control group, the Ace index, Chao index and the Shannon index of gut microbiota diversity in the severe pneumonia group were significantly decreased [Ace index: 167.23 (143.14, 211.26) vs. 227.71 (214.53, 247.05), Chao index: 152.38 (138.09, 182.54) vs. 228.25 (215.49, 248.95), Shannon index: 2.37 (1.68, 2.89) vs. 3.39 (3.03, 3.63), all P < 0.01], and the Simpson index was significantly increased [0.21 (0.11, 0.33) vs. 0.07 (0.06, 0.12), P < 0.01], which indicated the gut microbiota diversity of the severe pneumonia group was decreased. ③ Analysis of β diversity of gut microbiota, principal coordinate analysis (PCoA) showed that gut microbiota structural with the healthy control group was similar, while that in the severe pneumonia group was different. Adonis analysis showed that the structural of the gut microflora revealing significant differences between the severe pneumonia group and the healthy control group (R2 = 0.061, P = 0.05). ④ Analysis of phylum difference gut microflora showed that, compared with the healthy control group, the proportion of Firmicutes in severe pneumonia group was decreased [27.36 (18.12, 39.28)% vs. 52.25 (38.36, 63.82)%, P = 0.02], the proportions of Actinobacterias, Synergistetes and Fusobacterias were increased [2.30 (0.30, 4.80)% vs. 0.02 (0.00, 0.06)%, 0.36 (< 0.01, 0.57)% vs. < 0.01 (< 0.01, < 0.01)%, 0.01 (< 0.01, 0.08)% vs. < 0.01 (< 0.01, < 0.01)%, all P < 0.05]. ⑤ Analysis of genus difference gut microflora showed that, the proportions of Bifidobacterium, Ruminococcus, Pseudobutyrivibrio, Coprococcus, Lachnospira and Prevotella in the severe pneumonia group were significantly lower than those in healthy control group [0.18 (0.01, 0.25)% vs. 3.40 (0.46, 5.78)%, 0.01 (< 0.01, 0.29)% vs. 2.26 (0.84, 4.86)%, 0.01 (< 0.01, 0.02)% vs. 2.73 (1.87, 5.74)%, 0.02 (< 0.01, 0.07)% vs. 0.80 (0.50, 2.32)%, < 0.01 (< 0.01, < 0.01)% vs. 0.88 (0.33, 2.08)%, 0.02 (< 0.01, 0.31)% vs. 7.74 (0.07, 36.27)%, all P < 0.05]; the proportions of Escherichia and Enterococcus in the severe pneumonia group were higher than those in healthy control group, but there was no difference between the two groups [2.00 (0.57, 10.23)% vs. 1.16 (0.23, 2.68)%, 0.02 (< 0.01, 0.42)% vs. < 0.01 (< 0.01, 0.04)%, both P > 0.05]; the proportions of Fusobacterium and Staphylococcus in severe pneumonia group were significantly higher than those in healthy control group [0.01 (< 0.01, 0.08)% vs. < 0.01 (< 0.01, < 0.01)%, 0.01 (< 0.01, 0.02)% vs. < 0.01 (< 0.01, < 0.01)%, both P < 0.05].@*Conclusion@#Gut microbiota dysbiosis in patients with severe pneumonia shows that the abundance and diversity decrease, structure of intestinal flora changes, and beneficial symbiotic bacteria decrease and pathogenic bacteria increase, which may be associated with the occurrence and development of severe pneumonia.

Changes of Intestinal Microecology in Patients with Primary Sjogren's Syndrome after Therapy of Yangyin Yiqi Huoxue Recipe () / 中国结合医学杂志

OBJECTIVE@#To explore the change of intestinal microecology in patients with primary Sjogren's syndrome (pSS) and correlation with disease activity, and also discuss the therapy effect of Yangyin Yiqi Huoxue Recipe (, YYHD).@*METHODS@#Sixteen pSS patients were enrolled in the present study, who received 3-month treatment of YYHR, 200 mL orally twice daily. Their pre-and post-test ESSDAI scores, erythrocyte sedimentation rate (ESR) and serum immunoglobulin G (IgG) levels were measured respectively. The 16SrDNA metagenomic sequencing was used to detect and analyze the abundance and diversity of intestinal bacteria flora and the proportion of bacteria at the levels of phylum, family, and genus, in comparision with those of 6 healthy subjects in the control group.@*RESULTS@#The abundance and diversity of intestinal bacteria flora in pSS patients were lower than those of healthy subjects (P0.05).@*CONCLUSIONS@#There exists an imbalance of intestinal microecology in pSS patients, which can be improved through the treatment with YYHD. Besides, such treatment can also improve the disease activity and adjust the diversity of intestinal bacteria flora, the composition and the abundance of intestinal flora.

Study on ecologic microorganisms in lesions of acute guttate psoriasis induced by upper respiratory tract infection / 天津医药

Objective To investigate the characteristics of lesions of acute guttate psoriasis induced by upper respiratory tract infection, and to compare the difference in the different species between patients and healthy controls. Methods A total of 11 cases of acute guttate psoriasis induced by upper respiratory tract infection and 11 cases of healthy control without skin lesions of any dermatosis were included in this study. The 16SrDNA sequencing technology was used for analyzing data. The aseptic cotton swabs were used for sampling. DNA extraction and quality inspection were then performed. PCR amplification, library construction, microbial gene extraction, purification and recovery process were also performed. Then the gene samples were sent to be sequenced and to annotate the species. Finally, the data were analyzed by α and β diversity analysis to find the differences in microbial species and the diversity of microbial community. LEfSe analysis was used to find the species with significant difference, and the results were verified by the rank test. Results There was no significant difference in α diversity analysis between the two groups. There was a trend of difference in β diversity analysis between the two groups. However, LEfSe analysis (LED Score was 4) and rank test (P＜0.05) found that acinetobacter was a statistically significant different species and played a major role in the lesions of acute guttate psoriasis. Conclusion The skin lesions of microbiota is developing from one steady state to another one in the primary or recurrent acute guttate psoriasis patients with the history of upper respiratory infection. The different species of acinetobacter may play a key role in this change. However there is no significant difference in the overall microbial community between two groups.

The identification of the 16SrDNA of the diatoms in rabbits' internal organs in the summer and winter in Yongjiang River of Ningbo / 中国法医学杂志

Objective To evaluate the application value of identification on drown by detection 16SrDNA of the diatoms in rabbits' internal organs in summer month of July and winter month of December in YongJiang River of Ningbo. Methods 60 Rabbits were randomly and medially divided into three groups in summer and winter: drowning group, postmortem immersion group and using only lethal aeroembolism as control group. Specimen including heart, liver, lung and kidney from each rabbit were tested with diatom 16SrDNA PCR method. Results Compared with postmortem immersion group, detection rate of diatom 16SrDNA of heart, liver, lung, renal tissue in drowning group was significantly higher than that in summer month of July (P<0.05), In December, the 16SrDNA of the drowning group was detected in heart and lung tissues, There was no significant difference compared with postmortem immersion group (P>0.05) In summer month of July, detection rate of 16SrDNA of heart, liver, lung, renal tissues in drowning group was significantly higher than that in winter month of December (P<0.05). Diatom 16SrDNA of heart, liver, lung, kidney tissues in air embolism group were not detected In summer month of July and winter month of December. Conclusion With the higher detection rate of diatom 16SrDNA in drowning rabbit in summer, the diatom 16SrDNA PCR method can be used for the diagnosis of drowning in Yongjiang River of Ningbo; while in winter , it should be carefully apllied with the lower detection rate of diatom 16SrDNA.

Silkworm excrement bacterial communities diversity in different instars based on 16S rDNA sequence analysis / 中国中药杂志

This paper investigated the diversity of the silkworm excrement bacterial communities in different ages before and after drying, aiming to clarify the differences of bacterial communities in composition and bacterial abundance and the influences of drying treatment, and provide scientific basis for the efficacy of scientific connotation and utilization of silkworm excrement. High-throughput sequencing technique was used to measure the sequence of 16S rDNA-V4 variable region of bacteria in silkworm excrement. QIIME, Mothur and PICRUSt software programs were employed to sort and calculate the number of sequences and operational taxonomic units (OTUs) for each sample. Thereafter, the abundance, distribution, alpha diversity index of species, beta diversity and bacterial communities diversity among different sample groups and predicted the bacterial gene functions were analyzed. In this study, the numbers of effective sequences for six samples were 259 250; the rarefaction curves showed a sufficient sequencing depth, and the number of OTUs was close to saturation. The bacteria in silkworm excrement belonged to the following five phylums: Proteobacteria (89.3%), Actinobacteria (5.0%), Firmicutes (4.4%), Bacteroidetes (1.1%) and Cyanobacteria (0.2%). The dominant specie was Cyanobacteria of the total bacteria identified, respectively. The abundances and diversities of the silkworm excrement bacterial communities have been reduced after drying treatment, especially the silkworm excrement of the fifth instar. PICRUSt analysis was performed to show that abundance of the functional genes such as membrane transport, carbohydrate metabolism, amino acid metabolism, lipid metabolism, nucleotide metabolism, cellular processes and signaling were relatively high. The result showed that the drying treatment could decreased the species and numbers of pathogenic bacteria in silkworm excrement obviously and improve the quality of medicinal materials. Compared with the lower ages, silkworm excrement of fifth instar seems like to be more suitable for use in medicine. Illumina MiSeq high-throughput sequencing system provides a more accurate and scientific data resource for the study of bacteria in silkworm excrement.

Application of DetectingVibrio Cholerae Combined with Serum Agglutination and Gene Sequencing / 现代检验医学杂志

Objective To avoid false positive detection ofVibrio cholerae and improve the detection correct rate.Methods 1~7 months of 2013 were randomly selected,the national various provinces and cities CDC to China cholera CDC positive screening 14 strains.LB nutrient agar 12 hours,take single colony to Vibrio cholera serum agglutination,extraction of strain DNA at the same time boiled template method.For Vibrio 16SrDNA sequence and design primers for PCR detection of Vib-rio,16SrDNA,electrophoresis were used to observe the 16SrDNA products,16SrDNA positive products sent to sequencing company sequencing,sequencing results were Blast comparison on the NCBI website for the analysis and comparison of ser-um agglutination and Blast alignment.Results 12 strains was positive for agglutination and 2 strains of non agglutination in 14 strains.The Vibrio 16SrDNA amplification,electrophoresis were used to observe the 14 isolates that were amplified frag-ment corresponding,that the selected strains were vibrio.The 16SrDNA positive products were 14 strains,and the sequen-cing the Blast results:2 strains of bacteria were Vibrio harveyi for non agglutination,in 12 positive strains of serum aggluti-nation;1 strains was Vibrio natriegen and 11 strains wereVibrio cholerae .Conclusion Detection ofVibrio cholerae cholerae combined with serum agglutination and gene sequencing can avoid false positive result ofVibrio cholerae ,and improve cor-rect rate of the detection.

The Isolation and Phylogenetic Analysis of Facultative Anaerobic Cellulase-producing Strain / 微生物学通报

A bacillus strain was isolated from the dejecta of the Giant Panda " Xiangxiang" returned to wild, afforded by China Giant Panda Protection and Research Center, Wolong Sichuan. By primary research, the strain was identified as Serratia and called as Serratia JF-1116. The 16S rDNA gene of the bacteria strain, was amplified , cloned and sequenced. BLAST of the sequence in GenBank indicated that the species with close similarity to JF-1116 were from genus Enterobacter only. The phylogenetic tree was producted from 16S rDNA of JF-1116 and other 18 Enterobacter species with the high similarity. JF-1116 was clustered with 3 strains of Serratia.