Metagenome Analysis as a Tool to Study Bacterial Infection Associated with Acute Surgical Abdomen

Background: The purpose of this study was to profile the bacterium in the ascites and blood of patients with acute surgical abdomen by metagenome analysis. Methods: A total of 97 patients with acute surgical abdomen were included in this study. Accompanied with the standard culture procedures, ascites and blood samples were collected for metagenome analysis to measure the relative abundance of bacteria among groups of patients and between blood and ascites. Results: Metagenomic analysis identified 107 bacterial taxa from the ascites of patients. A principal component analysis (PCA) could separate the bacteria of ascites into roughly three groups: peptic ulcer, perforated or non-perforated appendicitis, and a group which included cholecystitis, small bowel lesion, and colon perforation. Significant correlation between the bacteria of blood and ascites was found in nine bacterial taxa both in blood and ascites with more than 500 sequence reads. However, the PCA failed to separate the variation in the bacteria of blood into different groups of patients, and the bacteria of metagenomic analysis is only partly in accordance with those isolated from a conventional culture method. Conclusion: This study indicated that the metagenome analysis can provide limited information regarding the bacteria in the ascites and blood of patients with acute surgical abdomen.

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Actinobacillus (Haemophilus) Pleuropneumoniae: Use of Coagglutination and Complement Fixation to Determine the Relationship between Presence of Organism and Antibody Titer in Slaughterhouse Pigs

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063878900100105 ◽

1989 ◽

Vol 1 (1) ◽

pp. 12-15 ◽

Cited By ~ 5

Author(s):

Lorraine J. Hoffman

Keyword(s):

Respiratory Disease ◽

Complement Fixation ◽

Culture Method ◽

Respiratory Problems ◽

Conventional Culture ◽

Significant Difference ◽

Positive Threshold ◽

Standard Culture ◽

History Of ◽

The Difference

The conventional culture method was compared to coagglutination for detection of Actinobacillus (Haemophilus) pleuropneumoniae in 425 sets of pig lungs. Sera from the same animals were evaluated for antibodies to A. pleuropneumoniae by the complement fixation (CF) test. All samples were collected at 2 packing plants in Iowa. In 2 nonvaccinated herds with no history of respiratory disease, the difference between standard culture results and coagglutination was highly significant ( P < 0.001). None of the 57 pigs in this group were positive for A. pleuropneumoniae by conventional culture, but 7 were positive by the coagglutination test. There were 15 animals with CF titers between 1:8 and 1:32. Animals from 6 herds vaccinated for A. pleuropneumoniae and without recent respiratory problems were evaluated. One out of 118 animals tested was positive for A. pleuropneumoniae by standard culture as compared to 9 positive by coagglutination. The difference in positive results between culture and coagglutination was highly significant ( P < 0.001). Twenty-eight animals had CF titers to A. pleuropneumoniae (1:4 to ≥ 1: 128). Two hundred fifty lungs and sera samples were collected from 7 herds which had recently experienced varying degrees of respiratory disease. Thirty-nine lungs were positive for A. pleuropneumoniae by culture and 182 were positive by coagglutination. The number of positives detected by coagglutination was significantly different ( P < 0.001) from the number positive by culture. There were 172 animals with antibody titers ranging from suspect to ≥ 1:128. There were significantly fewer positive animals detected by standard culture than with the CF test ( P < 0.001). There was no significant difference between coagglutination results and CF titers when a titer of 1:4 was used as the positive threshold.

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Comparison of Salmonella Bio-EnzaBead Immunoassay Method and Conventional Culture Procedure for Detection of Salmonella in Foods

Journal of Food Protection ◽

10.4315/0362-028x-50.5.379 ◽

1987 ◽

Vol 50 (5) ◽

pp. 379-385 ◽

Cited By ~ 18

Author(s):

KARL F. ECKNER ◽

RUSSELL S. FLOWERS ◽

BARBARA J. ROBISON ◽

JEROME A. MATTINGLY ◽

DAMIEN A. GABIS ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Enzyme Immunoassay ◽

Culture Method ◽

Food Samples ◽

Selective Enrichment ◽

Conventional Culture ◽

Significant Difference ◽

Standard Culture ◽

Raw Shrimp ◽

Culture Procedure

A rapid enzyme immunoassay screening procedure (EIA) utilizing two monoclonal antibodies specific for salmonellac was compared to the standard culture method (BAM/AOAC) on 1,289 samples representing 26 food types. The samples consisted of 760 artificially inoculated, 150 naturally contaminated, and 379 uninoculated food samples. There were 594 samples positive by the EIA (optical densities greater than 0.2 at 405 nm), of which 568 were confirmed culturally from M-broth. A total of 570 samples was positive by the BAM/AOAC procedure. Of the foods tested, there was no significant difference between the two methods, with the exception of cake mix and raw shrimp. The EIA was significantly better for detecting Salmonella in cake mix, while the culture procedure was more productive for shrimp. The method employed a 24 ± 2-h preen-richment, an 18-h selective enrichment, and a 6-h M-broth post-enrichment. The EIA assay required an additional 2 h for a total of 48 h, compared to a minimum of 4 d by BAM/AOAC.

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Evaluating Metagenomic Analysis for Pathogen Transmission in Healthcare Settings

Infection Control and Hospital Epidemiology ◽

10.1017/ice.2020.768 ◽

2020 ◽

Vol 41 (S1) ◽

pp. s224-s224

Author(s):

Curt Hewitt ◽

Katharina Weber ◽

Danielle LeSassier ◽

Anthony Kappell ◽

Kathleen Schulte ◽

...

Keyword(s):

Antibiotic Resistance ◽

Dna Analysis ◽

Pathogen Transmission ◽

Species Level ◽

Metagenomic Analysis ◽

Skin Microbiome ◽

Limited Information ◽

Culture Methods ◽

Data Set ◽

Skin Contact

Background: The prevalence of healthcare-acquired infections (HAIs) and rising levels of antimicrobial resistance place a significant burden on modern healthcare systems. Cultures are typically used to track HAIs; however, culture methods provide limited information and are not applicable to all pathogens. Next-generation sequencing (NGS) can detect and characterize pathogens present within a sample, but few research studies have explored how NGS could be used to detect pathogen transmission events under HAI-relevant scenarios. The objective of this CDC-funded project was to evaluate and correlate sequencing approaches for pathogen transmission with standard culture-based analysis. Methods: We modeled pathogen transfer via hand contact using synthetic skin. These skin coupons were seeded with a community of commensal organisms to mimic the human skin microbiome. Pathogens were added at physiologically relevant high or low levels prior to skin-to-skin contact. The ESKAPE pathogens: E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter spp plus C. difficile were employed because they are the most common antibiotic resistant HAIs. Pathogen transfer between skin coupons was measured following direct skin contact and fomite surface transmission. The effects of handwashing or fomite decontamination were also evaluated. Transferred pathogens were enumerated via culture to establish a robust data set against which DNA and RNA sequence analyses of the same samples could be compared. These data also provide a quantitative assessment of individual ESKAPE+C pathogen transfer rates in skin contact scenarios. Results: Metagenomic and metatranscriptomic analysis using custom analysis pipelines and reference databases successfully identified the commensal and pathogenic organisms present in each sample at the species level. This analysis also identified antibiotic resistance genes and plasmids. Metatranscriptomic analysis permitted not only gene identification but also confirmation of gene expression, a critical factor in the evaluation of antibiotic resistance. DNA analysis does not require cell viability, a key differentiator between sequencing and culturing reflected in simulated handwashing data. Sensitivity remains a key limitation of metagenomic analysis, as shown by the poor species identification and gene content characterization of pathogens present at low abundance within the simulated microbial community. Species level identification typically failed as ratios fell below 1:1,000 pathogen CFU:total community CFU. Conclusions: These findings demonstrate the strengths and weaknesses of NGS for molecular epidemiology. The data sets produced for this study are publicly available so they can be employed for future metagenomic benchmarking studies.Funding: NoneDisclosures: None

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Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

Journal of Food Protection ◽

10.4315/0362-028x.jfp-11-297 ◽

2012 ◽

Vol 75 (4) ◽

pp. 743-747 ◽

Cited By ~ 23

Author(s):

BWALYA LUNGU ◽

W. DOUGLAS WALTMAN ◽

ROY D. BERGHAUS ◽

CHARLES L. HOFACRE

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Salmonella Enteritidis ◽

Culture Method ◽

Pcr Assay ◽

Culture Methods ◽

The Real ◽

Selective Enrichment ◽

Conventional Culture ◽

Field Samples

Conventional culture methods have traditionally been considered the “gold standard” for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis–specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis–specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

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Evaluation of StrepBSelectChromogenic Medium and the Fast-Track Diagnostics Group B Streptococcus (GBS) Real-Time PCR Assay Compared to Routine Culture for Detection of GBS during Antepartum Screening

Journal of Clinical Microbiology ◽

10.1128/jcm.00043-17 ◽

2017 ◽

Vol 55 (7) ◽

pp. 2137-2142 ◽

Cited By ~ 4

Author(s):

Deirdre L. Church ◽

Heather Baxter ◽

Tracie Lloyd ◽

Oscar Larios ◽

Daniel B. Gregson

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Fast Track ◽

Group B Streptococcus ◽

Culture Method ◽

Predictive Values ◽

Content Type ◽

Standard Culture ◽

Group B ◽

The Cost

ABSTRACTLife-threatening infection in neonates due to group BStreptococcus(GBS) is preventable by screening of near-term pregnant women and treatment at delivery. A total of 295 vaginal-rectal swabs were collected from women attending antepartum clinics in Calgary, Alberta, Canada. GBS colonization was detected by the standard culture method (Strep B Carrot Broth subcultured to blood agar with a neomycin disk) and compared to recovery with Strep Group B Broth (Dalynn Biologicals) subcultured to StrepBSelectchromogenic medium (CM; Bio-Rad Laboratories) and the Fast-Track Diagnostics GBS real-time PCR (quantitative PCR [qPCR]) assay (Phoenix Airmid Biomedical Corp.) performed with broth-enriched samples and the Abbottm2000sp/m2000rt system. A total of 62/295 (21%) women were colonized with GBS; 58 (19.7%) cases were detected by standard culture, while CM and qPCR each found 61 (20.7%) cases. The qPCR and CM were similar in performance, with sensitivities, specificities, and positive and negative predictive values of 98.4 and 98.4%, 99.6 and 99.6%, 98.4 and 98.4%, and 99.6 and 99.6%, respectively, compared to routine culture. Both qPCR and CM would allow more rapid reporting of routine GBS screening results than standard culture. Although the cost per test was similar for standard culture and CM, the routine use of qPCR would cost approximately four times as much as culture-based detection. Laboratories worldwide should consider implementing one of the newer methods for primary GBS testing, depending on the cost limitations of different health care jurisdictions.

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Prevalence and Antimicrobial Susceptibility of Salmonella Isolated from a Variety of Raw Meat Sausages in Gaborone (Botswana) Retail Stores

Journal of Food Protection ◽

10.4315/0362-028x.jfp-11-438 ◽

2012 ◽

Vol 75 (4) ◽

pp. 637-642 ◽

Cited By ~ 4

Author(s):

RONALD GAELEKOLWE SAMAXA ◽

MAITSHWARELO IGNATIUS MATSHEKA ◽

SUNUNGUKO WATA MPOLOKA ◽

BERHANU ABEGAZ GASHE

Keyword(s):

Antimicrobial Susceptibility ◽

Salmonella Enterica ◽

Antimicrobial Agents ◽

Culture Method ◽

Multidrug Resistant ◽

Pcr Assay ◽

Raw Meat ◽

Conventional Culture ◽

The Difference ◽

Conventional Culture Method

The objective of the study was to provide baseline data on the prevalence and antimicrobial susceptibility of Salmonella in different types of raw meat sausages directly accessible to the consumers in Gaborone, Botswana. A total of 300 raw sausages comprising 79 beef, 78 pork, 72 chicken, and 71 mutton samples were concurrently analyzed for the presence of Salmonella using a conventional culture method and a validated PCR method. The PCR assay results were in full concordance with those of the conventional culture method for the detection of Salmonella. Sixty-five (21.7%) of 300 samples were positive for Salmonella by both the conventional culture method and PCR assay. Even though more chicken samples contained Salmonella than did any other sausage type, the difference in the presence of Salmonella among the four sausages types was not significant. Eleven serotypes were identified, and Salmonella enterica subsp. salamae II was most prevalent in all the sausage types. Beef sausages generally had higher mesophilic bacterial counts than did the other three sausage types. However, higher microbial counts were not reflective of the presence of salmonellae. Susceptibility of the Salmonella enterica serotypes to 20 antimicrobial agents was determined, and Salmonella Muenchen was resistant to the widest array of agents and was mostly isolated from chicken sausages. Regardless of the meat of origin, all 65 Salmonella isolates were resistant to at least four antimicrobial agents: amikacin, gentamicin, cefuroxime, and tombramycin. This resistance profile group was the most common in all four sausage types, comprising 90% of all Salmonella isolates from beef, 71% from pork, 63% from mutton, and 35% from chicken. These results suggest that raw sausages pose a risk of transmitting multidrug-resistant Salmonella isolates to consumers.

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A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

Journal of Food Protection ◽

10.4315/0362-028x-70.5.1080 ◽

2007 ◽

Vol 70 (5) ◽

pp. 1080-1087 ◽

Cited By ~ 63

Author(s):

V. M. BOHAYCHUK ◽

G. E. GENSLER ◽

M. E. McFALL ◽

R. K. KING ◽

D. G. RENTER

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Culture Method ◽

Pcr Assay ◽

Culture Methods ◽

The Real ◽

Conventional Culture ◽

Food Animal ◽

Highly Sensitive ◽

Field Samples

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

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Assessment of Various Laboratory Diagnostic Methods in Diagnosis of Cutaneous Tuberculosis. A Study from A Tertiary Care Hospital of North India

Journal of Pure and Applied Microbiology ◽

10.22207/jpam.15.2.06 ◽

2021 ◽

Author(s):

Shoaib Khan ◽

Nahid Nahvi ◽

Umara Amin ◽

Yousuf Ul Bashir ◽

Danish Zahoor

Keyword(s):

Tertiary Care ◽

Culture Method ◽

Diagnostic Methods ◽

North India ◽

Care Hospital ◽

Age Group ◽

Cutaneous Tuberculosis ◽

Standard Culture ◽

Lowenstein Jensen ◽

Culture Positive

Cutaneous tuberculosis (CTB) is the rarest case of extrapulmonary TB comprising 2% of total cases. It’s often a challenge both clinically and diagnostically. 1) To determine prevalence, age & gender-wise distribution of CTB. 2) To assess various diagnostic, microbiological modalities for the diagnosis of CTB. 76 skin biopsy specimens from suspected CTB lesions were analysed using following methods – Acid-fast Bacilli (AFB) staining (Ziehl-Neelsen method), growth of mycobacteria in culture (Lowenstein-Jensen media), and Gene Xpert MTB/RIF, Histopathological (H&E staining). Of the 76 specimens, 44 were males and 32 were females. The most commonly affected age group was 40–59 years. Infections were least common in 0-19 years age group. AFB was not seen in any of the primary smears. 10 were confirmed as CTB by the recovery of Mycobacterium in solid culture. Of the 10 culture positives, 9 were confirmed as MTB, and 1 was found to be NTM. Staining of 10 culture positive specimens revealed acid fast, beaded rods. Detection of MTB by Gene Xpert gave positive result in 9 cases with all RIF sensitive. All 9 PCR confirmed cases were also culture positive, all 9 were slow growers with a minimum of 5 weeks required for growth on the LJ slant. PCR is the test of choice and should be performed on all specimens of suspected CTB. However when coupled with the “gold standard” culture method, the diagnostic accuracy improves. Also, further, culture helps in identification and isolation of NTM’s.

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Physical and Chemical Properties of Soil in the Sahelian Region: Case Study in Nioro du Rip, Senegal

Journal of Geography Environment and Earth Science International ◽

10.9734/jgeesi/2020/v24i530229 ◽

2020 ◽

pp. 73-82

Author(s):

Landing Biaye ◽

Fary Diome ◽

Seybatou Diop ◽

Modou Mbaye ◽

Djibril Tine ◽

...

Keyword(s):

Agricultural Land ◽

Organic Matter Content ◽

Chemical Properties ◽

Principal Component ◽

Fertility Level ◽

Physical And Chemical Properties ◽

Limited Information ◽

Geochemical Composition ◽

Physical And Chemical ◽

And Chemical Properties

The locality of Nioro du Rip is facing intense erosion, loss of agricultural land, soil pollution and soil degradation. Today, there is limited information about the soil physical and chemical properties in the locality. In this work, we describe the main essential factors or mechanism that control the evolution of the soil in the study area. The physical and chemical properties of soils encountered along a NE-SW transect in are analyzed in this paper. The statistical analysis results revealed low structural stability of soils in general, due to their low organic matter content and exchangeable bases and their predominantly silty texture. A net trend towards acidification, which is more pronounced in the lower-bottom and terrace soils, provides information on the conditions that are increasingly unfavorable to agricultural development. The multivariate principal component analysis (PCA) identified the preponderance of two factors among the four primarily involved in soil geochemical composition. These include a mineralization process (expressed through the first principal component (PC1), which causes soils to be rich in elements (Ca2+, K+,C, N, MO) controlling their structure and fertility level; The PC2 axis expresses the spatial differentiation phenomenon of the soil granulometric composition: soils forming cluster poles according to their textural affinity in the projection of the plane formed by these two components , with on one side the sandy-dominated soils of the shallows and terraces and on the other the clay-dominated soils of the plateau and the slope. A clear reversal of textural polarity in the studied topo sequence that must be blamed on the strong water erosion in this area.

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Distinct gene expression patterns in vector-residing Leishmania infantum identify parasite stage-enriched markers

10.1101/679712 ◽

2019 ◽

Cited By ~ 1

Author(s):

Iliano V. Coutinho-Abreu ◽

Tiago D. Serafim ◽

Claudio Meneses ◽

Shaden Kamhawi ◽

Fabiano Oliveira ◽

...

Keyword(s):

Leishmania Infantum ◽

Developmental Stages ◽

Expression Patterns ◽

Principal Component ◽

Sand Fly ◽

Parasite Development ◽

Lutzomyia Longipalpis ◽

Specific Marker ◽

Limited Information ◽

Natural Sand

AbstractPromastigotes of Leishmania infantum undergo a series of extracellular developmental stages inside the natural sand fly vector Lutzomyia longipalpis to reach the infectious stage, the metacyclic promastigote. There is limited information regarding the expression profile of L. infantum developmental stages inside the sand fly vector, and molecular markers that can distinguish the different parasite stages are lacking. We performed RNAseq on unaltered midguts of the sand fly Lutzomyia longipalpis after infection with L. infantum parasites. RNAseq was carried out at various time points throughout parasite development. Principal component analysis mapped the sequences corresponding to the procyclic, nectomonad, leptomonad or metacyclic promastigote stage into distinct positions, with the procyclic stage being the most divergent population. Transcriptional levels across genes varied on average between 10- to 100-fold. Comparison between procyclic and nectomonad promastigotes resulted in 836 differentially expressed (DE) genes; between nectomonad and leptomonad promastigotes in 113 DE genes; and between leptomonad and metacyclic promastigotes in 302 DE genes. Most of the DE genes do not overlap across stages, highlighting the uniqueness of each stage. Furthermore, the different stages of Leishmania parasites exhibited specific transcriptional enrichment across chromosomes. Using the transcriptional signatures exhibited by distinct Leishmania stages during their development in the sand fly midgut, we determined the genes predominantly enriched in each stage, identifying multiple stage-specific markers for L. Infantum. Leading stage-specific marker candidates include genes encoding a zinc transporter in procyclics, a beta-fructofuranidase in nectomonads, a surface antigen-like protein in leptomonads, and an amastin-like surface protein in metacyclics. Overall, these findings demonstrate the transcriptional plasticity of the Leishmania parasite inside the sand fly vector and provide a repertoire of stage-specific markers for further development as molecular tools for epidemiological studies.

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