Application of real-time PCR array to the multiple detection of antibiotic resistant genes in glacier ice samples

Ushida, Kazunari; Segawa, Tomio; Kohshima, Shiro; Takeuchi, Nozomu; Fukui, Kotaro; Li, Zhongqin; Kanda, Hiroshi

doi:10.2323/jgam.56.43

Cited by 24 publications

(20 citation statements)

References 45 publications

(36 reference statements)

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“…UPL probes have been used successfully in a variety of qPCR studies, including the quantification of bacterial antibiotic resistance genes in environmental samples (42,43). Using UPL probes, we can easily design TaqMan qPCR assays that can be run in identical PCR conditions, which is essential when applying qPCR assays to microfluidic qPCR.…”

Section: Discussionmentioning

confidence: 99%

Simultaneous Quantification of Multiple Food- and Waterborne Pathogens by Use of Microfluidic Quantitative PCR

Ishii

Segawa

Okabe

2013

Appl Environ Microbiol

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122

159

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bThe direct quantification of multiple pathogens has been desired for diagnostic and public health purposes for a long time. In this study, we applied microfluidic quantitative PCR (qPCR) technology to the simultaneous detection and quantification of multiple food-and waterborne pathogens. In this system, multiple singleplex qPCR assays were run under identical detection conditions in nanoliter-volume chambers that are present in high densities on a chip. First, we developed 18 TaqMan qPCR assays that could be run in the same PCR conditions by using prevalidated TaqMan probes. Specific and sensitive quantification was achieved by using these qPCR assays. With the addition of two previously validated TaqMan qPCR assays, we used 20 qPCR assays targeting 10 enteric pathogens, a fecal indicator bacterium (general Escherichia coli), and a process control strain in the microfluidic qPCR system. We preamplified the template DNA to increase the sensitivity of the qPCR assays. Our results suggested that preamplification was effective for quantifying small amounts of the template DNA without any major impact on the sensitivity, efficiency, and quantitative performance of qPCR. This microfluidic qPCR system allowed us to detect and quantify multiple pathogens from fecal samples and environmental water samples spiked with pathogens at levels as low as 100 cells/liter. These results suggest that the routine monitoring of multiple pathogens in food and water samples is now technically feasible. This method may provide more reliable information for risk assessment than the current fecal contamination indicator approach.

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Section: Discussionmentioning

confidence: 99%

Simultaneous Quantification of Multiple Food- and Waterborne Pathogens by Use of Microfluidic Quantitative PCR

Ishii

Segawa

Okabe

2013

Appl Environ Microbiol

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122

159

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“…However, we should note that some resistance genes were not detected by traditional PCR detection, probably due to the low copy number of target genes. In contrast, real-time PCR arrays are more sensitive to resistance genes in the environment, even in glacier and permafrost sediments (Ushida et al, 2010;D'Costa et al, 2011). Thus, because ARGs exist naturally, the quantitative characteristics of the pristine area may be appropriate for setting background levels of resistance to compare with affected sites.…”

Section: Occurrence Of Resistance In Four Diverse Soil Typesmentioning

confidence: 99%

Identification of Super Antibiotic-resistant Bacteria in Diverse Soils

Zhang¹,

ChangPing²,

Shamsi³

et al. 2015

IJAB

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Environmental bacteria have been revealed to be a reservoir of antibiotic resistance genes and a potential pool of novel resistance genes in clinical pathogens. Recently, the soils, which have never been treated with antibiotics before have proved to contain the resistant bacterial strains. In this study, we assessed whether the soil that had not previously been influenced by antibiotics contained super-resistant bacteria or not. We identified super penicillin-resistant strains in four diverse soils containing up to 1000 mg/L penicillin compared to 100 mg/L, but none of super tetracycline resistant strains were detected in four soils containing up to 1000 mg/L tetracycline, which is the highest concentration of exogenous penicillin or tetracycline reported to date. Twenty bacterial isolates were selected, representing a diverse set of species in the genera Pseudomonas (85%) and Variovorax (15%); these bacteria showed multiple antibiotic resistance patterns for four or more antibiotics. Using polymerase chain reaction and sequence, we carefully examined the existence of antibiotic-resistance genes and integron genes in soils and strains. It was observed that the resistance genes tet(C) and blaTEM were fairly widely distributed in these super penicillin-resistant isolates, and 20%-50% of tested genes were found in the study soils. All the study sites provided great opportunities for horizontal resistance transfer. The antibiotic-resistant genes pool, which is potentially large and diverse, may have considerable implications for ecology and health.

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“…In addition to such local transmission, a worldwide transmission of resistant bacteria can occur. Research on the distribution of antibiotic-resistance genes in remote areas such as glaciers suggests that antibiotic-resistant bacteria can be transmitted by global atmospheric circulation (Ushida et al 2010b, Segawa et al 2013. However, resistant bacteria may not be preserved in the intestines of animals for a long period if antibiotics are not applied.…”

Section: Antibiotic Resistancementioning

confidence: 99%

“…These show the limitation of DNA-based meta-analyses. Accordingly, we think that bacterial isolates should be obtained for further analyses from wild animals as well as hu- ＊ ampC and blaACT were amplified by different primer set (Ushida et al 2010b) mans living adjacent to wildlife area. In usual cases, cultivation, isolation, and purification of bacteria should be manipulated under equippedlaboratory conditions.…”

Section: Development Of Field Bacteriologymentioning

confidence: 99%

Bacteria culturing and isolation under field conditions of Moukalaba-Doudou National Park, Gabon, and preliminary survey on bacteria carrying antibiotic resistance genes

Nguema

Tsuchida

Ushida

2015

Tropics

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Culturing the intestinal bacteria of wild animals is a difficult task under field conditions, although isolation and characterization of bacteria are absolutely necessary to evaluate the transmission of bacteria from human to animal or vice versa. We have developed a protocol for intestinal bacteria culturing from feces of wild animals such as gorillas in Moukalaba-Doudou National Park in Gabon, where no laboratory settings were available. The prevalence of resistance genes in Enterobacteriaceae isolates estimated by real time PCR array was higher for aminoglycosides resistance, followed by tetracycline resistance, except for possible naturally occurring β-lactam resistance. The detection level of resistance genes was higher for isolates from humans than those from gorillas. Occasional monitoring of this prevalence may help to measure the intensity of introduction of humanborne bacteria to wildlife.

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Application of real-time PCR array to the multiple detection of antibiotic resistant genes in glacier ice samples

Cited by 24 publications

References 45 publications

Simultaneous Quantification of Multiple Food- and Waterborne Pathogens by Use of Microfluidic Quantitative PCR

Simultaneous Quantification of Multiple Food- and Waterborne Pathogens by Use of Microfluidic Quantitative PCR

Identification of Super Antibiotic-resistant Bacteria in Diverse Soils

Bacteria culturing and isolation under field conditions of Moukalaba-Doudou National Park, Gabon, and preliminary survey on bacteria carrying antibiotic resistance genes

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