Rapid and quantitative detection of blood<i>Serratia marcescens</i>by a real-time PCR assay: Its clinical application and evaluation in a mouse infection model

Iwaya, Akira; Nakagawa, Saori; Iwakura, Nobuhiro; Taneike, Ikue; Kurihara, Mizuki; Kuwano, Tomoko; Gondaira, Fumio; Endo, Miyoko; Hatakeyama, Katsuyoshi; Yamamoto, Tatsuo

doi:10.1016/j.femsle.2005.05.041

Cited by 31 publications

(25 citation statements)

References 18 publications

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“…It has recently been demonstrated that high bacterial loads in blood correlate with disease severity in patients with infections caused by Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis (4,22,35,41), as well as in a murine model of Serratia marcescens infection (25). In the case of S. aureus-related infections, it was reported that positive blood cultures during follow-up and the persistence of fever were suggestive of a complicated course (6,15,37).…”

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confidence: 99%

High Levels of mecA DNA Detected by a Quantitative Real-Time PCR Assay Are Associated with Mortality in Patients with Methicillin-Resistant Staphylococcus aureus Bacteremia

Chang

Lin

et al. 2009

J Clin Microbiol

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Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is known to be a poor prognostic factor. While several PCR assays for the detection of MRSA in various clinical samples were recently reported, the possibility that a quantitative PCR assay could be used to quantify and monitor MRSA bacteremia has not been explored. In this study, we established a quantitative real-time PCR assay for the mecA gene using known copy numbers of a plasmid containing mecA DNA as a standard and the previously described mecA-specific primers and probe (P. Francois et al., J. Clin. Microbiol. 41:254-260, 2003). We employed this assay to examine 250 sequential whole-blood samples from 20 adult patients, including 13 survivors and 7 nonsurvivors, with culture-proven MRSA bacteremia at the intensive care units of National Taiwan University Hospital between 1 July 2006 and 31 January 2007. The levels of mecA DNA in the nonsurvivors were significantly higher than those in the survivors during the three periods of bacteremia examined (days 0 to 2, 3 to 5, and 6 to 8) (P ‫؍‬ 0.003 by two-tailed Mann-Whitney U test). Moreover, the nonsurvivors had higher mecA DNA levels than the survivors after 3 days and 7 days of anti-MRSA therapy (medians for nonsurvivors and survivors at 3 days, 5.86 and 4.30 log copies/ml, respectively; medians for nonsurvivors and survivors at 7 days, 5.21 and 4.36 log copies/ml, respectively; P ‫؍‬ 0.02 and P ‫؍‬ 0.04, respectively, by two-tailed Mann-Whitney U test). Together, these findings suggest that the level of mecA DNA in blood could potentially be used to monitor MRSA bacteremia and evaluate responses to therapy.Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen that is one of the most common causes of both community-acquired and nosocomial infections worldwide, especially in intensive care units (ICUs) (5,8,33,49). In the past two decades, the proportion of MRSA infections has increased dramatically, and up to 60% of S. aureus isolates from ICUs were reported to be methicillin resistant (26). Compared with cases of bacteremia caused by methicillin-susceptible S. aureus (MSSA) strains, cases of bacteremia caused by MRSA strains have been shown to be associated with more persistent infections, more recurrent episodes, longer hospital stays, and higher rates of mortality (3,6,11,13,14,27). Because of the high rates of mortality and the refractoriness of MRSA bacteremia to treatment, MRSA bacteremia has become a very challenging infectious disease (18,21,38,39,42).It has recently been demonstrated that high bacterial loads in blood correlate with disease severity in patients with infections caused by Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis (4,22,35,41), as well as in a murine model of Serratia marcescens infection (25). In the case of S. aureus-related infections, it was reported that positive blood cultures during follow-up and the persistence of fever were suggestive of a complicated course (6,15,37). However, the drastic decrease in the sen...

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confidence: 99%

High Levels of mecA DNA Detected by a Quantitative Real-Time PCR Assay Are Associated with Mortality in Patients with Methicillin-Resistant Staphylococcus aureus Bacteremia

Chang

Lin

et al. 2009

J Clin Microbiol

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“…The Q-PCR primers (16S_F and 16S_R) used for the 16S rRNA gene of S. marcescens have been described (25). The specificity of the primers (16S_F and 16S_R) has been tested previously (25) and in the present study.…”

Section: Methodsmentioning

confidence: 91%

“…The specificity of the primers (16S_F and 16S_R) has been tested previously (25) and in the present study.…”

Section: Methodsmentioning

confidence: 94%

“…Several Q-PCR assays have been developed and validated for the detection and quantification of BW agents (e.g., Y. pestis, B. anthracis, F. tularensis, and smallpox virus) (17,18,21,23,24,33,43,48,57). However, to date, only one Q-PCR assay for the BW surrogate B. atrophaeus and one Q-PCR assay for the BW surrogate S. marcescens have been reported (4,5,25). While Buttner et al (4,5) reported the detection of B. atrophaeus using Q-PCR targeting the B. atrophaeus recA gene, the specificity of the assay was not reported.…”

mentioning

confidence: 99%

“…In addition, the sequence of the target gene could not be identified in published sequences in the GenBank, DNA Data Bank of Japan (DDBJ), and European Molecular Biology Laboratory (EMBL) databases. Iwaya et al (25) used the 16S rRNA gene for designing a Q-PCR assay for detecting S. marcescens in blood. Although the 16S rRNA gene has commonly been used for modern species classification, it has limitations in discriminating species of closely related taxa because of high 16S rRNA sequence similarities (13).…”

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Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate

Saikaly

Barlaz

Reyes

2007

Appl Environ Microbiol

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Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R 2 > 0.98) over a 7-log-unit dynamic range down to 10 1 B. atrophaeus cells or spores. Quantification of S. marcescens (R 2 > 0.98) was linear over a 6-log-unit dynamic range down to 10 2 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.The first recorded attempt to use pathogens as biological warfare (BW) agents was in the 14th century when the Mongols catapulted plague-infected victims into the city of Kaffa (Feodosiya, Ukraine) to spread the disease (33, 53). During and after World War II, the development and use of pathogens, such as Yersinia pestis (plague), Bacillus anthracis (anthrax), and Francisella tularensis (tularemia), as BW agents intensified (41, 53). Recently, there has been concern about the potential "weaponization" of pathogens for bioterrorism use, with the October 2001 bioterrorist attack with B. anthracis in the United States as the most prominent example (17, 41).The 2001 event has sparked renewed interest in the development of detection platforms for BW agents (8,17,24,33,48,57), methods for inactivation (16,39,61) and decontamination of Bacillus spores (4, 44), sampling protocols for recovery of Bacillus spores from surfaces (...

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PCR Detection of Serratia spp. Using Primers Targeting pfs and luxS Genes Involved in AI-2-Dependent Quorum Sensing

2008

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Quorum sensing is the ability of bacteria to communicate and coordinate behavior emitting signaling molecules. A series of primers for PCR detection of Serratia spp. has been designed using as targets the pfs and luxS genes involved in AI-2-dependent quorum sensing. The identities of the PCR products (193 and 102 bp) were confirmed by commercial sequencing. Twenty-seven Serratia strains (representing 10 different species) tested positive for the presence of the pfs and luxS genes, while a total of 7 different species of non-Serratia (25 strains) were tested and gave negative results. The sensitivity and specificity of the pfs- and luxS-based PCR assay were also checked in artificially contaminated bacterial samples. In this study we established a novel method to detect Serratia using quorum-sensing genes as diagnostic markers.

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Rapid and quantitative detection of bloodSerratia marcescensby a real-time PCR assay: Its clinical application and evaluation in a mouse infection model

Cited by 31 publications

References 18 publications

High Levels of mecA DNA Detected by a Quantitative Real-Time PCR Assay Are Associated with Mortality in Patients with Methicillin-Resistant Staphylococcus aureus Bacteremia

High Levels of mecA DNA Detected by a Quantitative Real-Time PCR Assay Are Associated with Mortality in Patients with Methicillin-Resistant Staphylococcus aureus Bacteremia

Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate

PCR Detection of Serratia spp. Using Primers Targeting pfs and luxS Genes Involved in AI-2-Dependent Quorum Sensing

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