The ZKIR Assay, a novel Real-Time PCR Method for the Detection of Klebsiella pneumoniae and Closely Related Species in Environmental Samples

ABSTRACTKlebsiella pneumoniae (Kp) is of growing public health concern due to the emergence of strains that are multidrug-resistant, virulent, or both. Taxonomically, Kp includes seven phylogroups, with Kp1 (K. pneumoniae sensu stricto) being medically prominent. Kp can be present in environmental sources such as soils and vegetation, which could act as reservoirs of animal and human infections. However, the current lack of screening methods to detect Kp in complex matrices limits research on Kp ecology. Here we analysed 4222 genome sequences and found that existing molecular detection targets lack specificity for Kp. A novel real-time PCR method, the ZKIR assay, was developed and used to detect Kp in 96 environmental samples. Results were compared to a culture-based method using SCAI agar medium coupled to MALDI-TOF mass spectrometry identification. Whole-genome sequencing of environmental Kp was performed. The ZKIR assay was positive for the 48 tested Kp reference strains, whereas 88 non-Kp strains were negative. The limit of detection of Kp in spiked soil microcosms was 1.5 × 10-1 CFU g-1 after enrichment for 24 h in LB supplemented with ampicillin, and 1.5 × 103 to 1.5 × 104 CFU g-1 directly after soil DNA extraction. The ZKIR assay was more sensitive than the culture method. Kp was detected in 43% of environmental samples. Genomic analysis of the isolates revealed a predominance of phylogroups Kp1 (65%) and Kp3 (32%), a high genetic diversity (23 MLST sequence types), a quasi-absence of antibiotic resistance or virulence genes, and a high frequency (50%) of O-antigen type 3. This study shows that the ZKIR assay is an accurate, specific and sensitive novel method to detect the presence of Kp in complex matrices, and indicates that Kp isolates from environmental samples differ from clinical isolates.IMPORTANCEThe Klebsiella pneumoniae species complex (Kp) includes human and animal pathogens, some of which are emerging as hypervirulent and/or antibiotic resistant strains. These pathogens are diverse and classified into seven phylogroups, which may differ in their reservoirs and epidemiology. Proper management of this public health hazard requires a better understanding of Kp ecology and routes of transmission to humans. So far, detection of these microorganisms in complex matrices such as food or the environment has been difficult due to a lack of accurate and sensitive methods. Here, we describe a novel method based on real-time PCR, which enables detection of all Kp phylogroups with high sensitivity and specificity. We used this method to detect Kp isolates from environmental samples, and show based on genomic sequencing that they differ in antimicrobial resistance and virulence gene content, from human clinical Kp isolates. The ZKIR PCR assay will enable rapid screening of multiple samples for Kp presence and will thereby facilitate tracking the dispersal patterns of these pathogenic strains across environmental, food, animal and human sources.

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The ZKIR Assay, a Real-Time PCR Method for the Detection of Klebsiella pneumoniae and Closely Related Species in Environmental Samples

Applied and Environmental Microbiology ◽

10.1128/aem.02711-19 ◽

2020 ◽

Vol 86 (7) ◽

Cited By ~ 1

Author(s):

Elodie Barbier ◽

Carla Rodrigues ◽

Geraldine Depret ◽

Virginie Passet ◽

Laurent Gal ◽

...

Keyword(s):

Public Health ◽

Klebsiella Pneumoniae ◽

Real Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Health Concern ◽

Complex Matrices ◽

Content Type ◽

Novel Method ◽

Pcr Method

ABSTRACT Klebsiella pneumoniae is of growing public health concern due to the emergence of strains that are multidrug resistant, virulent, or both. Taxonomically, the K. pneumoniae complex (“Kp”) includes seven phylogroups, with Kp1 (K. pneumoniae sensu stricto) being medically prominent. Kp can be present in environmental sources such as soils and vegetation, which could act as reservoirs of animal and human infections. However, the current lack of screening methods to detect Kp in complex matrices limits research on Kp ecology. Here, we analyzed 1,001 genome sequences and found that existing molecular detection targets lack specificity for Kp. A novel real-time PCR method, the ZKIR (zur-khe intergenic region) assay, was developed and used to detect Kp in 96 environmental samples. The results were compared to a culture-based method using Simmons citrate agar with 1% inositol medium coupled to matrix-assisted laser desorption ionization–time of flight mass spectrometry identification. Whole-genome sequencing of environmental Kp was performed. The ZKIR assay was positive for the 48 tested Kp reference strains, whereas 88 non-Kp strains were negative. The limit of detection of Kp in spiked soil microcosms was 1.5 × 10−1 CFU g−1 after enrichment for 24 h in lysogeny broth supplemented with ampicillin, and it was 1.5 × 103 to 1.5 × 104 CFU g−1 directly after soil DNA extraction. The ZKIR assay was more sensitive than the culture method. Kp was detected in 43% of environmental samples. Genomic analysis of the isolates revealed a predominance of phylogroups Kp1 (65%) and Kp3 (32%), a high genetic diversity (23 multilocus sequence types), a quasi-absence of antibiotic resistance or virulence genes, and a high frequency (50%) of O-antigen type 3. This study shows that the ZKIR assay is an accurate, specific, and sensitive novel method to detect the presence of Kp in complex matrices and indicates that Kp isolates from environmental samples differ from clinical isolates. IMPORTANCE The Klebsiella pneumoniae species complex Kp includes human and animal pathogens, some of which are emerging as hypervirulent and/or antibiotic-resistant strains. These pathogens are diverse and classified into seven phylogroups, which may differ in their reservoirs and epidemiology. Proper management of this public health hazard requires a better understanding of Kp ecology and routes of transmission to humans. So far, detection of these microorganisms in complex matrices such as food or the environment has been difficult due to a lack of accurate and sensitive methods. Here, we describe a novel method based on real-time PCR which enables detection of all Kp phylogroups with high sensitivity and specificity. We used this method to detect Kp isolates from environmental samples, and we show based on genomic sequencing that they differ in antimicrobial resistance and virulence gene content from human clinical Kp isolates. The ZKIR PCR assay will enable rapid screening of multiple samples for Kp presence and will thereby facilitate tracking the dispersal patterns of these pathogenic strains across environmental, food, animal and human sources.

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Efficient nested-PCR-based method development for detection and genotype identification of Acanthamoeba from a small volume of aquatic environmental sample

Scientific Reports ◽

10.1038/s41598-021-00968-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tsui-Kang Hsu ◽

Jung-Sheng Chen ◽

Hsin-Chi Tsai ◽

Chi-Wei Tao ◽

Yu-Yin Yang ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Nested Pcr ◽

In Silico ◽

Environmental Samples ◽

Method Development ◽

Small Volume ◽

Detection Efficiency ◽

Genotyping Method ◽

Pcr Method

AbstractAcanthamoeba spp. are opportunistic human pathogens that cause granulomatous amoebic encephalitis and keratitis, and their accurate detection and enumeration in environmental samples is a challenge. In addition, information regarding the genotyping of Acanthamoeba spp. using various PCR methods is equally critical. Therefore, considering the diverse niches of habitats, it is necessary to develop an even more efficient genotyping method for Acanthamoeba spp. detection. This study improved the sensitivity of detection to avoid underestimation of Acanthamoeba spp. occurrence in aquatic environmental samples, and to accurately define the pathogenic risk by developing an efficient PCR method. In this study, a new nested genotyping method was established and compared with various PCR-based methods using in silico, lab, and empirical tests. The in silico test showed that many PCR-based methods could not successfully align specific genotypes of Acanthamoeba, except for the newly designed nested PCR and real-time PCR method. Furthermore, 52 water samples from rivers, reservoirs, and a river basin in Taiwan were analysed by six different PCR methods and compared for genotyping and detection efficiency of Acanthamoeba. The newly developed nested-PCR-based method of genotyping was found to be significantly sensitive as it could effectively detect the occurrence of Acanthamoeba spp., which was underestimated by the JDP-PCR method. Additionally, the present results are consistent with previous studies indicating that the high prevalence of Acanthamoeba in the aquatic environment of Taiwan is attributed to the commonly found T4 genotype. Ultimately, we report the development of a small volume procedure, which is a combination of recent genotyping PCR and conventional real-time PCR for enumeration of aquatic Acanthamoeba and acquirement of biologically meaningful genotyping information. We anticipate that the newly developed detection method will contribute to the precise estimation, evaluation, and reduction of the contamination risk of pathogenic Acanthamoeba spp., which is regularly found in the water resources utilised for domestic purposes.

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Real Time PCR HBV-DNA Analysis in HBsAg Positive Patients

Journal of Armed Forces Medical College Bangladesh ◽

10.3329/jafmc.v10i2.25931 ◽

2015 ◽

Vol 10 (2) ◽

pp. 95-99

Author(s):

Wasila Rahman ◽

Muhammad Rabiul Hossain ◽

Arif Ahmed Khan ◽

Debashish Saha ◽

SM Mahbubul Alam ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Antiviral Therapy ◽

Real Time ◽

Real Time Pcr ◽

Armed Forces ◽

Hbv Dna ◽

Health Concern ◽

B Virus ◽

Pcr Method

Introduction: The hepatitis B virus is a global public health concern and leading cause of chronic liver disease in Bangladesh. For the diagnosis and monitoring of treatment of Hepatitis B virus infection, HBV-DNA detection and quantification is now extensively used worldwide.Objectives: The objective of this study was to detect HBV-DNA by real time PCR method in HBsAg positive patients, to compare the results of HBVDNA detection with HBeAg and Anti-HBe and to monitor the response after antiviral therapy in chronic hepatitis B patients and also to observe the intensity of hepatitis B infection in relation to age and sex.Methods: This was a cross sectional type of study conducted in Armed Forces Institute of Pathology (AFIP), Dhaka Cantonment. In this study, 56 sera of HBsAg positive patients were selected who all were subjected to do HBV-DNA (real time PCR) analysis during the period of 29 July to 30 0ctober, 2013.Results: Out of 56 HBsAg positive patients, HBV-DNA was detected in 34 patients. Among these, 8 (23.5%) patients were HBeAg positive, 16 (47%) patients were anti-HBe positive and 10 (29.5%) were negative for both HBeAg and anti-HBe. Age limit of patients was up to 60 years. HBV-DNA positive patients showed male predominance; 26 (76.5%) patients were male and 8 (23.5%) patients were female. Mean age of the patients was 35±14 years. Among 56 HBsAg positive patients, fifteen were receiving antiviral therapy. Out of them, HBV-DNA was decreased among 4 patients and could not be detected among 11 patients.Conclusion: Real time PCR method of detection of HBV-DNA is very important in patients who are HBeAg negative and this method is also applied to monitor treatment response to antivirals and to detect occult HBV infections immune control phase and also to detect reactivation of HBV cases.Journal of Armed Forces Medical College Bangladesh Vol.10(2) 2014

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Real-Time PCR Method for Detection of Salmonella spp. in Environmental Samples

Applied and Environmental Microbiology ◽

10.1128/aem.00644-17 ◽

2017 ◽

Vol 83 (14) ◽

Cited By ~ 21

Author(s):

Kuppuswamy N. Kasturi ◽

Tomas Drgon

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Environmental Samples ◽

False Negative ◽

Screening Method ◽

Reference Method ◽

Rapid Screening ◽

Content Type ◽

Pcr Method ◽

Group D

ABSTRACT The methods currently used for detecting Salmonella in environmental samples require 2 days to produce results and have limited sensitivity. Here, we describe the development and validation of a real-time PCR Salmonella screening method that produces results in 18 to 24 h. Primers and probes specific to the gene invA, group D, and Salmonella enterica serovar Enteritidis organisms were designed and evaluated for inclusivity and exclusivity using a panel of 329 Salmonella isolates representing 126 serovars and 22 non-Salmonella organisms. The invA- and group D-specific sets identified all the isolates accurately. The PCR method had 100% inclusivity and detected 1 to 2 copies of Salmonella DNA per reaction. Primers specific for Salmonella-differentiating fragment 1 (Sdf-1) in conjunction with the group D set had 100% inclusivity for 32 S. Enteritidis isolates and 100% exclusivity for the 297 non-Enteritidis Salmonella isolates. Single-laboratory validation performed on 1,741 environmental samples demonstrated that the PCR method detected 55% more positives than the Vitek immunodiagnostic assay system (VIDAS) method. The PCR results correlated well with the culture results, and the method did not report any false-negative results. The receiver operating characteristic (ROC) analysis documented excellent agreement between the results from the culture and PCR methods (area under the curve, 0.90; 95% confidence interval of 0.76 to 1.0) confirming the validity of the PCR method. IMPORTANCE This validated PCR method detects 55% more positives for Salmonella in half the time required for the reference method, VIDAS. The validated PCR method will help to strengthen public health efforts through rapid screening of Salmonella spp. in environmental samples.

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Application of a novel pathogenicity marker in a multiplex real-time PCR method to assess total and pathogenic Vibrio vulnificus in food and environmental samples

Food Control ◽

10.1016/j.foodcont.2013.07.007 ◽

2014 ◽

Vol 35 (1) ◽

pp. 274-283 ◽

Cited By ~ 5

Author(s):

Alejandro Garrido-Maestu ◽

María-José Chapela ◽

Belén Román ◽

Juan M. Vieites ◽

Ana G. Cabado

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Vibrio Vulnificus ◽

Pathogenic Vibrio ◽

Pcr Method

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118. Real-Time PCR Method for Detecting and Enumerating Legionella from Environmental Samples

10.3320/1.2766033 ◽

2002 ◽

Author(s):

R. Miller ◽

C. Martinez-Miller

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Pcr Method

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Rapid-Viability PCR Method for Detection of Live, Virulent Bacillus anthracis in Environmental Samples

Applied and Environmental Microbiology ◽

10.1128/aem.00623-11 ◽

2011 ◽

Vol 77 (18) ◽

pp. 6570-6578 ◽

Cited By ~ 22

Author(s):

Sonia E. Létant ◽

Gloria A. Murphy ◽

Teneile M. Alfaro ◽

Julie R. Avila ◽

Staci R. Kane ◽

...

Keyword(s):

Bacillus Anthracis ◽

Real Time ◽

Incubation Time ◽

Real Time Pcr ◽

Environmental Samples ◽

Limit Of Detection ◽

Turnaround Time ◽

Pcr Analysis ◽

Content Type ◽

Pcr Method

ABSTRACTIn the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulentBacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for theB. anthracischromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulentB. anthracisin environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.

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Detection of Streptococcus pneumoniae in Sputum Samples by Real- Time PCR.

Anti-Infective Agents ◽

10.2174/2211352518999200629165108 ◽

2020 ◽

Vol 18 ◽

Author(s):

Pegah Shakib ◽

Mohammad Reza Zolfaghari

Keyword(s):

Streptococcus Pneumoniae ◽

Real Time ◽

Real Time Pcr ◽

False Negative ◽

Cross Sectional Study ◽

Laboratory Culture ◽

Cross Sectional ◽

Negative Results ◽

False Negative Results ◽

Pcr Method

Background: Conventional laboratory culture-based methods for diagnosis of Streptococcus pneumoniae are time-consuming and yield false negative results. Molecular methods including real-time (RT)-PCR rapid methods and conventional PCR due to higher sensitivity and accuracy have been replaced instead traditional culture assay. The aim of the current study was to evaluate lytA gene for detection of Streptococcus pneumoniae in the cerebrospinal fluid of human patients with meningitis using real-time PCR assay. Material and Methods: In this cross-sectional study, a total of 30 clinical specimens were collected from patients in a period from September to December 2018. In order to evaluate the presence of lytA gene, conventional and real-time PCR methods were used without culture. Results: From 30 sputum samples five (16.66%) isolates were identified as S. pneumoniae by lytA PCR and sequencing. Discussion: In this research, an accurate and rapid real-time PCR method was used, which is based on lytA gene for diagnosis of bacteria so that it can be diagnosed. Based on the sequencing results, the sensitivity for detection of lytA gene was 100% (5/5).

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Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

Applied and Environmental Microbiology ◽

10.1128/aem.69.12.7430-7434.2003 ◽

2003 ◽

Vol 69 (12) ◽

pp. 7430-7434 ◽

Cited By ~ 77

Author(s):

Trevor G. Phister ◽

David A. Mills

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Primers ◽

Rrna Gene ◽

Dekkera Bruxellensis ◽

Pcr Assay ◽

Specific Pcr ◽

Spoilage Yeast ◽

Pcr Method ◽

26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

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