Prevalence and Diversity of Arcobacter spp. in the Czech Republic

The aim of this study was to examine 634 samples of chicken, lamb, pork, beef, fish, samples from the intensive animal industry and from poultry for slaughter, as well as from the domestic breeding of poultry, horses, pigs, and lambs, from surface water, and from clinical samples for the presence of Arcobacter. All the samples were examined with a cultivation method, followed by confirmation by multiplex PCR. The method of multiplex PCR applied directly to a liquid medium after enrichment was applied only to the samples with the highest probability of the presence of arcobacters. Arcobacter spp. were detected in 11.8% of the samples, of which A. butzleri, A. cryaerophilus, and A. skirrowii were found in 6.6, 5.1, and 0.2% of the samples, respectively. The sources of the arcobacters were chicken meat from the retail market, intensive animal production facilities, domestic chicken breeding facilities, lamb raising environments, surface water and wastewater, and beef swabs taken in a meat processing factory. No occurrence of arcobacters was identified in the swabs from slaughter turkeys, ducks, and wild poultry. No arcobacters were found in horse and pig breeding environments, on pork, or on the swabs of fish. Forty-two rectal swabs taken from humans were also free of Arcobacter. Seventeen isolates of Arcobacter were further identified by sequencing the 16S rRNA gene. Varied genotypes were observed among A. butzleri from chicken meat and chicken breeds, and A. cryaerophilus from wastewater and chicken breeds. They were similar to the genotypes present in wastewater, porcine feces, human stool, and human blood obtained from databases. Our results revealed that the chicken meat from the retail market is an important source of arcobacters. Cross-contamination during handling of chicken carcass practices could play a key role in the spread of Arcobacter.

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Prevalence and Antibiogram of Vibrio parahaemolyticus and Aeromonas hydrophila in the Flesh of Nile Tilapia, with Special Reference to Their Virulence Genes Detected Using Multiplex PCR Technique

Antibiotics ◽

10.3390/antibiotics10060654 ◽

2021 ◽

Vol 10 (6) ◽

pp. 654

Author(s):

Hanan A. Zaher ◽

Mohamad I. Nofal ◽

Basma M. Hendam ◽

Moustafa M. Elshaer ◽

Abdulaziz S. Alothaim ◽

...

Keyword(s):

Antibiotic Resistance ◽

Multiplex Pcr ◽

Aeromonas Hydrophila ◽

Vibrio Parahaemolyticus ◽

Nile Tilapia ◽

Virulence Genes ◽

Resistant Bacteria ◽

Rrna Gene ◽

Bacterial Disease ◽

Fish Samples

Vibrio parahaemolyticus and Aeromonas hydrophila are major public health problems and the main cause of bacterial disease in Nile tilapia (Oreochromis niloticus). This study was conducted to determine the prevalence, antibiotic resistance and some virulence genes of both V. parahaemolyticus and A. hydrophila isolates from Nile tilapia. From Manzala Farm at Dakahlia governorate, 250 freshwater fish samples were collected. The confirmed bacterial isolates from the examined Nile tilapia samples in the study were 24.8% (62/250) for V. parahaemolyticus and 19.2% (48/250) for A. hydrophila. multiplex PCR, revealing that the tlh gene was found in 46.7% (29/62) of V. parahaemolyticus isolates, while the tdh and trh virulence genes were found in 17.2% (5/29). Meanwhile, 39.5% (19/48) of A. hydrophila isolates had the 16s rRNA gene and 10.5% (2/19) had the aerA and ahh1 virulence genes. The Multiple Antibiotic Resistance indices of V. parahaemolyticus and A. hydrophila were 0.587 and 0.586, respectively. In conclusion, alternative non-antibiotic control strategies for bacterial infections in farmed fish should be promoted to avoid multidrug-resistant bacteria. Therefore, it is suggested that farmers should be skilled in basic fish health control and that molecular detection methods are more rapid and cost-effective than bacteriological methods.

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Multiplex PCR assay for identification of Corynebacterium pseudotuberculosis from pure cultures and for rapid detection of this pathogen in clinical samples

Journal of Medical Microbiology ◽

10.1099/jmm.0.46997-0 ◽

2007 ◽

Vol 56 (4) ◽

pp. 480-486 ◽

Cited By ~ 75

Author(s):

Luis G. C. Pacheco ◽

Roberta R. Pena ◽

Thiago L. P. Castro ◽

Fernanda A. Dorella ◽

Robson C. Bahia ◽

...

Keyword(s):

Multiplex Pcr ◽

Corynebacterium Pseudotuberculosis ◽

Clinical Samples ◽

Rrna Gene ◽

Sheep And Goats ◽

Multiplex Pcr Assay ◽

Pure Cultures ◽

Biochemical Identification ◽

Biochemical Testing ◽

Mpcr Assay

Corynebacterium pseudotuberculosis is the aetiological agent of caseous lymphadenitis (CLA), a debilitating disease of sheep and goats. Accurate diagnosis of CLA primarily relies on microbiological examination, followed by biochemical identification of isolates. In an effort to facilitate C. pseudotuberculosis detection, a multiplex PCR (mPCR) assay was developed targeting three genes of this bacterium: the 16S rRNA gene, rpoB and pld. This method allowed efficient identification of 40 isolates of this bacterium that had been identified previously by biochemical testing. Analysis of taxonomically related species did not generate the C. pseudotuberculosis mPCR amplification profile, thereby demonstrating the assay's specificity. As little as 1 pg of C. pseudotuberculosis genomic DNA was detected by this mPCR assay, demonstrating the sensitivity of the method. The detection limit in clinical samples was estimated to be 103 c.f.u. C. pseudotuberculosis could be detected directly in pus samples from infected sheep and goats (n=56) with a high diagnostic sensitivity (94.6 %). The developed assay significantly improves rapid C. pseudotuberculosis detection and could supersede bacteriological culture for microbiological and epidemiological diagnosis of CLA.

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Simultaneous Detection of Bacteroides forsythus and Prevotella intermedia by 16S rRNA Gene-Directed Multiplex PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.37.5.1621-1624.1999 ◽

1999 ◽

Vol 37 (5) ◽

pp. 1621-1624 ◽

Cited By ~ 27

Author(s):

Georg Conrads ◽

Thomas F. Flemmig ◽

Ilse Seyfarth ◽

Friedrich Lampert ◽

Rudolf Lütticken

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Multiplex Pcr ◽

Simultaneous Detection ◽

Clinical Samples ◽

Rrna Gene ◽

Subgingival Plaque ◽

Prevotella Intermedia ◽

Forward Primer

In a 16S rRNA gene-directed multiplex PCR, Prevotella intermedia- and Bacteroides forsythus-specific reverse primers were combined with a single conserved forward primer. A 660-bp fragment and an 840-bp fragment that were specific for both species could be amplified simultaneously. A total of 152 clinical samples, subgingival plaque and swabs of three different oral mucosae, from 38 periodontitis patients were used for the evaluation.

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16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles

Microbiome ◽

10.1186/2049-2618-2-31 ◽

2014 ◽

Vol 2 (1) ◽

pp. 31 ◽

Cited By ~ 35

Author(s):

Jun Hang ◽

Valmik Desai ◽

Nela Zavaljevski ◽

Yu Yang ◽

Xiaoxu Lin ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Temperature Stability ◽

Clinical Samples ◽

Rrna Gene ◽

16S Rrna Gene Pyrosequencing

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Multiplex PCR with 16S rRNA Gene-Targeted Primers of Bifidobacterium spp. To Identify Sources of Fecal Pollution

Applied and Environmental Microbiology ◽

10.1128/aem.70.5.3171-3175.2004 ◽

2004 ◽

Vol 70 (5) ◽

pp. 3171-3175 ◽

Cited By ~ 56

Author(s):

X. Bonjoch ◽

E. Ballesté ◽

A. R. Blanch

Keyword(s):

16S Rrna ◽

Multiplex Pcr ◽

Fecal Pollution ◽

Sensitivity Threshold ◽

Rrna Gene ◽

Specific Primers ◽

Animal Wastewater ◽

Wastewater Samples ◽

Species Specific ◽

Different Origins

ABSTRACT Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.

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Prevalence and diversity of Arcobacter spp. in retail chicken meat in Turkey

Microbiology Research ◽

10.4081/mr.2016.6578 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 1

Author(s):

Celenk Molva ◽

Halil Ibrahim Atabay

Keyword(s):

Polymerase Chain Reaction ◽

Multiplex Pcr ◽

Chicken Meat ◽

Species Level ◽

Waterborne Pathogens ◽

Chain Reaction ◽

Retail Markets ◽

Specific Polymerase Chain Reaction ◽

Polymerase Chain ◽

Retail Chicken Meat

Arcobacters are food and waterborne pathogens associated with human and animal infections. The objective of the present study was to investigate the prevalence and diversity of Arcobacter spp. in commercially sold chicken meat in İzmir region of Turkey. For this purpose, 100 samples including legs (n=40), 17 chicken quarters (n=17), drumstickers (n=16), breasts (n=11), wings (n=10), and carcasses (n=6) were collected from different retail markets. A total of 65 isolates were confirmed as Arcobacter spp. from 55 samples by genus-specific polymerase chain reaction (PCR). The prevalence of Arcobacter spp. was 32.5, 81.3, 64.7, 72.7, 83.3, and 50% for legs, drumstickers, chicken quarters, breasts, carcasses and wings, respectively. Based on the multiplex-PCR, most of the isolates were identified as A. butzleri (n=45, 80%), followed by A. cryaerophilus (n=2, 3.6%), A. skirrowii (n=1, 1.8%) and 17 isolates (30.9%) could not be identified at the species level.

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Simplified Fluorescent Multiplex PCR Method for Evaluation of the T-Cell Receptor Vβ-Chain Repertoire

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.12.4.477-483.2005 ◽

2005 ◽

Vol 12 (4) ◽

pp. 477-483 ◽

Cited By ~ 2

Author(s):

Sanjit Fernandes ◽

Surendra Chavan ◽

Vivek Chitnis ◽

Nina Kohn ◽

Savita Pahwa

Keyword(s):

T Cell ◽

Multiplex Pcr ◽

T Cell Receptor ◽

Cell Receptor ◽

T Cell Subsets ◽

Clinical Samples ◽

Cdr3 Length ◽

Pcr Products ◽

Pcr Method ◽

T Cell Receptor Vβ

ABSTRACTRationale: evaluation of the T-cell receptor (TCR) Vβ-chain repertoire by PCR-based CDR3 length analysis allows fine resolution of the usage of the TCR Vβ repertoire and is a sensitive tool to monitor changes in the T-cell compartment. A multiplex PCR method employing 24 labeled upstream Vβ primers instead of the conventionally labeled downstream Cβ primer is described. Method: RNA was isolated from purified CD4 and CD8 T-cell subsets from umbilical cord blood and clinical samples using TRI reagent followed by reverse transcription using a Cβ primer and an Omniscript RT kit. The 24 Vβ primers were multiplexed based on compatibility and product sizes into seven reactions. cDNA was amplified using 24 Vβ primers (labeled with tetrachloro-6-cardoxyfluorescein, 6-carboxyfluorescein, and hexachloro-6-carboxyfluorescein), an unlabeled Cβ primer, and Taqgold polymerase. The fluorescent PCR products were resolved on an automated DNA sequencer and analyzed using the Genotyper 2.1 software. Results: Vβ spectratypes of excellent resolution were obtained with RNA amounts of 250 ng using the labeled Vβ primers. The resolution was superior to that obtained with the labeled Cβ primer assay. Also the numbers of PCRs were reduced to 7 from the 12 required in the Cβ labeling method, and the sample processing time was reduced by half. Conclusion: The method described for T-cell receptor Vβ-chain repertoire analysis eliminates tedious dilutions and results in superior resolution with small amounts of RNA. The fast throughput makes this method suitable for automation and offers the feasibility to perform TCR Vβ repertoire analyses in clinical trials.

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Karakteristik Genus Bakteri Pada Karkas Ayam Broiler Dari Swalayan di Kota Pontianak

Jurnal Protobiont ◽

10.26418/protobiont.v7i3.29066 ◽

2018 ◽

Vol 7 (3) ◽

Author(s):

Prianti Rahmawati Diah Wulandari Rousdy

Keyword(s):

Broiler Chicken ◽

Pathogenic Bacteria ◽

Chicken Meat ◽

Bacterial Isolates ◽

Bacterial Genus ◽

Chicken Carcass

The availability of nutrients in chicken carcasses can cause chicken meat to be an excellent medium for the growth of pathogenic and non-pathogenic bacteria. This study aims to determine the characteristics of the bacterial genus in broiler chicken carcasses from supermarkets in Pontianak City. Based on the results of the study found 23 bacterial isolates in broiler chicken carcass samples from supermarkets in Pontianak City, which included members of the Aeromonas, Acetobacter, Alcaligenes, Amphibacillus, Bacillus, Brevibacterium, Camphylobacter, Carnobacterium, Erwinia, Erysipelothrik, Eubacterium, Hafnia, Kluyvera, Klebsiella, Kurthia, Lactobacillus, Listeria, Proteus, Pseudomonas, Shigella, Sporolactobacillus, Serratia, and Yersinia.

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Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009779 ◽

2021 ◽

Vol 15 (10) ◽

pp. e0009779

Author(s):

Fakhriddin Sarzhanov ◽

Funda Dogruman-Al ◽

Monica Santin ◽

Jenny G. Maloney ◽

Ayse Semra Gureser ◽

...

Keyword(s):

Next Generation Sequencing ◽

Gastrointestinal Symptoms ◽

Molecular Methods ◽

Clinical Samples ◽

Rrna Gene ◽

Next Generation ◽

Dientamoeba Fragilis ◽

Significant Difference ◽

Generation Sequencing ◽

Immunocompetent Patients

Introduction The clinical significance of Blastocystis sp. and Dientamoeba fragilis in patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. subtypes using next generation sequencing. Material and methods Individual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes. Results The prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients. Conclusion and recommendation Our findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.

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Development of Novel Multiplex PCR for Diagnosis of Co-infected Hemo-parasites in Cattle

Indian Journal of Animal Research ◽

10.18805/ijar.b-4695 ◽

2021 ◽

Author(s):

Pankaj Kumar ◽

Abhay Kumar ◽

Kamal Sarma ◽

Paresh Sharma ◽

Rashmi Rekha Kumari ◽

...

Keyword(s):

Multiplex Pcr ◽

Large Scale ◽

Parasitic Infection ◽

Epidemiological Studies ◽

Kappa Coefficient ◽

Detection Methods ◽

Diagnostic Process ◽

Specific Gene ◽

Rrna Gene ◽

Babesia Bigemina

Background: A novel, rapid and specific multiplex polymerase chain reaction was developed to diagnose hemo-parasitic infection in bovine blood co-infected with three of the most common hemo-parasites. Methods: The diagnostic process relied on the detection of the three different bovine hemoparasites isolated from red blood cells (RBCs) of cattle (N=30) by conventional Giemsa stained blood smear (GSBS) and confirmed by multiplex PCR. The multiplex PCR system was used to diagnose GSBS positive blood samples (N=12) found infected or co-infected with hemoparasites. The designed multiplex primer sets was attempted to amplify 205, 313 and 422 bp fragments of apocytochrome b, sporozoite and macroschizont 2 (spm2) and 16S rRNA gene for Babesia bigemina, Theileria annulata and Anaplasma marginale, respectively. Result: This multiplex PCR was sensitive with the ability to detect the presence of 150 ng of genomic DNA. The primers used in this multiplex PCR also showed highly specific amplification of specific gene fragments of each respective parasite. Comparing the two detection methods revealed that 58.33% of specimens showed concordant diagnoses with both techniques. The specificity, positive predictive value and kappa coefficient of the agreement was highest for diagnosis of B. bigemina and lowest for A. marginale. The overall Kappa coefficient for diagnosis based on GSBS for multiple pathogens compared to multiplex PCR was 0.56, slightly behind the threshold of 0.6 of agreement. Therefore, confirmation should always be based on PCR to rule out false positives due to differences in subjective observations, stain particles and false negatives due to low parasitemia. The simplicity and rapidity of this specific multiplex PCR method make it suitable for large-scale epidemiological studies and follow-up of drug treatments.

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