Detection of Mycoplasma pulmonis in experimentally infected laboratory rats by 16S rRNA amplification.

Recently, an rRNA-based polymerase chain reaction (PCR) has been developed for the detection of murine mycoplasmas at both the genus and species level (F. J. M. van Kuppeveld, J. T. M. van der Logt, A. F. Angulo, M. J. van Zoest, W. G. V. Quint, H. G. Niesters, J. M. D. Galama, and W. J. G. Melchers, Appl. Environ. Microbiol. 58:2606-2615, 1992). In this study, the diagnostic value of this PCR assay for the detection of Mycoplasma pulmonis in infected rats was studied. For this purpose, 25 Wistar rats were infected intranasally with M. pulmonis strain M72-138 and investigated for the presence of this pathogen by both in vitro isolation and PCR. Five rats were monitored longitudinally by screening of throat swabs at several time points for up to 248 days postinfection. The remaining 20 rats were killed between 3 and 87 days postinfection, and organism recovery from both throat and urogenital tract specimens was attempted. M. pulmonis could be detected in the throat for up to 248 days postinfection but not in the urogenital tract, either by culture or by PCR. PCR proved to be the optimal method for testing throat samples. All samples in which M. pulmonis was detected by culture were also positive by PCR. By PCR, M. pulmonis was also detected in 3.7% of the samples which were culture negative and in 9.9% of the samples from which cultures were overgrown with bacteria. The results of this study demonstrate the suitability of PCR for the detection of mycoplasmal infection in rodents.     

Detection of Mycoplasma hyopneumoniae in nose swabs from pigs by in vitro amplification of the 16S rRNA gene.

We report the use of PCR to detect DNA from Mycoplasma hyopneumoniae, the etiological agent of enzootic porcine pneumonia. A primer set was designed for the amplification of a 649-bp fragment of the 16S rRNA gene from M hyopneumoniae. The PCR product was identified by ethidium bromide staining after gel electrophoresis and by Southern hybridization with an M. hyopneumoniae-specific oligonucleotide probe. No amplification was observed from any other porcine bacteria tested, including several closely related mycoplasmas. It was also possible to demonstrate the presence of M. hyopneumoniae in bronchial lavage samples and lung tissue samples from experimentally infected pigs. Furthermore, the PCR system was used for analysis of nasal samples obtained from pigs in a fattening herd. By this method, we were able to detect M. hyopneumoniae in nose swabs from naturally infected pigs. However, our results suggest that M. hyopneumoniae can be detected in the nasal cavities only during a limited time period.     

Detection of Balamuthia mitochondrial 16S rRNA gene DNA in clinical specimens by PCR

Balamuthia mandrillaris is a free-living ameba that causes granulomatous amebic encephalitis in both immunocompromised and immunocompetent individuals. Because of a lack of pathognomonic symptoms and the difficulty in recognizing amebas in biopsied tissues, most cases are not diagnosed or effectively treated, leading to a >95% mortality. We report here on five cases of balamuthiasis that were diagnosed by indirect immunofluorescence (IIF) staining of serum for anti-Balamuthia antibodies (titer > or = 1:128) and confirmed by IIF of unstained brain tissue sections and/or detection of amebas in hematoxylin-eosin-stained slides. Additionally, we have used the PCR for the detection of mitochondrial 16S rRNA gene DNA from the ameba in clinical specimens such as brain tissue and cerebrospinal fluid (CSF) from individuals with Balamuthia encephalitis. Balamuthia DNA was successfully detected by the PCR in clinical samples from all five individuals. It was detected in brain tissue from three cases, in CSF from three cases, and in one of two samples of lung tissue from two individuals, but not in two samples of kidney tissue tested. One sample of unfixed brain tissue was culture positive for Balamuthia. In order to test the sensitivity of the PCR for detection of Balamuthia DNA, CSF specimens from two individuals negative for amebic infection were spiked with Balamuthia amebas. We found that it was possible to detect Balamuthia DNA in the PCR mixtures containing mitochondrial DNA from 1 to as little as 0.2 ameba per reaction mixture. A single Balamuthia ameba contains multiple mitochondrial targets; thus, 0.2 ameba represents multiple targets for amplification and is not equivalent to 0.2 of an ameba as a target.     

Rapid identification of mycobacterial species by PCR amplification of hypervariable 16S rRNA gene promoter region.

A total of 0.3 to 0.4 kb of the promoter region of the 16S rRNA genes from Mycobacterium tuberculosis, M. gordonae, M. xenopi, and M. leprae was PCR amplified, cloned, and sequenced. The observed number of substitutions, insertions, and deletions exceeded those found in previously used target sequences, including the entire 16S coding region. A simple and generally applicable restriction fragment length polymorphism method that can be used to distinguish between mycobacterial species is described.     

Identification of Chlamydia pneumoniae by DNA amplification of the 16S rRNA gene.

Chlamydia pneumoniae is an important cause of respiratory disease in humans, but diagnosis of C. pneumoniae is hindered by difficulties in the in vitro growth of the organism. In order to improve detection and identification, we recently developed a polymerase chain reaction (PCR) assay which uses oligonucleotide primers specific for C. pneumoniae. The nucleic acid sequence was determined for the 16S rRNA of C. pneumoniae, and regions in which C. pneumoniae differed from both Chlamydia psittaci and Chlamydia trachomatis were identified. Oligonucleotide primers corresponding to these unique regions were then synthesized and used in a PCR for the detection of C. pneumoniae. The C. pneumoniae-specific primers permitted the identification of six isolates of C. pneumoniae, but no reaction was observed with the 15 serovars of C. trachomatis or two strains of C. psittaci. PCR should prove to be valuable in confirming the identification of C. pneumoniae and in the diagnosis of C. pneumoniae infections.     

Detection of bacteraemia in patients with fever and neutropenia using 16S rRNA gene amplification by polymerase chain reaction

Episodes of fever and neutropenia are common complications of treatment for cancer. The use of prophylactic and early empirical antibiotics has reduced mortality but decreases the sensitivity of diagnostic tests based on culture. The aim of this study was to determine the potential of a broad diagnostic approach (eubacterial) based on 16S rRNA gene amplification and sequencing to augment cultural methods of diagnosis of bacteraemia in patients with fever and neutropenia in a regional paediatric oncology centre. One hundred eleven patient-episodes of fever and neutropenia were evaluated during the study period, 17 of which were associated with positive blood cultures, as follows:Staphylococcus epidermidis (n=6 episodes),Enterococcus faecium (n=2),Streptococcus sanguis (n=3),Streptococcus mitis (n=3),Staphylococcus aureus (n=1),Micrococcus spp. (n=1), andStenotrophomonas maltophilia (n=1). Eubacterial polymerase chain reaction (PCR) detected bacterial DNA in nine of 11 blood culture-positive episodes for which a sample was available for PCR; the species identified by sequence analysis were identical to those derived from the conventional identification of the cultured isolates. Bacterial DNA was detected in 20 episodes (21 bacterial sequences) associated with negative blood cultures, 18 of which occurred in patients who were receiving antibiotics at the time of sample collection. The species presumptively identified by partial 16S rRNA gene sequencing were as follows:Pseudomonas spp. (n=6 episodes),Acinetobacter spp. (n=5),Escherichia spp. (n=3);Moraxella spp. (n=3);Staphylococcus spp. (n=2);Neisseria spp. (n=1); andBacillus spp. (n=1). The results of this study suggest that molecular techniques can augment cultural methods in the diagnosis of bacteraemia in patients who have been treated with antibiotics.     

Molecular diagnosis of Kingella kingae pericarditis by amplification and sequencing of the 16S rRNA gene

Kingella kingae is a fastidious gram-negative bacillus that is considered an emerging pathogen in pediatric settings but remains less common in adults. Here we describe a case of pericarditis in an immunocompetent adult host. The microorganism was identified directly from the clinical sample by molecular techniques, i.e., 16S rRNA gene amplification and sequencing.     

Detection of Bartonella henselae and Bartonella quintana by a simple and rapid procedure using broad-range PCR amplification and direct single-strand sequencing of part of the 16S rRNA gene

Objective: To detect directly Bartonella henselae and Bartonella quintana using culture-independent, molecular techniques, and to evaluate a simple and rapid procedure that allows uncultivable bacteria to be detected in usually sterile clinical specimens in a diagnostic laboratory.
Method: From four clinical specimens proven to contain B. henselae (n=3) or B. quintana (n=1) DNA, part of the 16S rRNA gene was amplified using the polymerase chain reaction (PCR) and broad-range bacterial primers followed by reamplification and direct, single-strand sequencing. The partial 16S rRNA sequences were compared to reference sequences in databases.
Results: Similarities between sequences derived from clinical samples and those of B. henselae and B. quintana , respectively, were in the range 98.7-100%, indicating a strong association to the genus Bartonella. Intraspecies sequence variations within the B. henselae sequences were observed.
Conclusions: The method described is a rapid, sensitive and reliable tool to generate partial 16S rRNA sequences of B. henselae and B. quintana directly from normally sterile clinical specimens. It is compatible with adequate prevention of contamination as is needed for diagnostic purposes, and it possesses the potential to detect other pathogens, including those as yet unknown.     

Bacterial taxa associated with the hematophagous mite Dermanyssus gallinae detected by 16S rRNA PCR amplification and TTGE fingerprinting

Dermanyssus gallinae (Arthropoda, Mesostigmata) is suspected to be involved in the transmission of a wide variety of pathogens, but nothing is known about its associated non-pathogenic bacterial community. To address this question, we examined the composition of bacterial communities in D. gallinae collected from standard poultry farms in Brittany, France. Genetic fingerprints of bacterial communities were generated by temporal temperature gradient gel electrophoresis (TTGE) separation of individual polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments, followed by DNA sequence analysis. Most of the sequences belonged to the Proteobacteria and Firmicute phyla, with a majority of sequences corresponding to the Enterobacteriales order and the Staphylococcus genus. By using statistical analysis, we showed differences in biodiversity between poultry farms. We also determined the major phylotypes that compose the characteristic microbiota associated with D. gallinae. Saprophytes, opportunistic pathogens and pathogenic agents such as Pasteurella multocida, Erysipelothrix rhusiopathiae and sequences close to the genus Aerococcus were identified. Endosymbionts such as Schineria sp., Spiroplasma sp. Anistosticta, “Candidatus Cardinium hertigii” and Rickettsiella sp. were also present in the subdominant bacterial community. Identification of potential targets within the symbiont community may be considered in the future as a means of ectoparasite control.     

细菌直接PCR和双引物策略鉴定16S rRNA基因技术的建立

目的探索一种基于16S rRNA基因的快速鉴定细菌方法,为临床诊断治疗及耐药菌的分子遗传分析提供科学依据。方法对卫生部室间质评菌株和临床病人标本分离培养纯菌落,用双蒸水稀释菌落,然后直接以菌液为模板优化反应体系PCR扩增16S rRNA基因片段,再测序扩增片段。将测序结果在细菌Ribosomal数据库中进行同源性比对,根据序列同源性鉴定病原细菌。结果本实验鉴定的卫生部质评菌株结果与卫生部报告结果一致。本实验能够一次性鉴定出临床病人标本分离的菌株。结论本研究优化了一种免纯化细菌核酸直接PCR鉴定细菌16S rRNA基因的方法。本研究建立的基于病原细菌16S rRNA基因鉴定方法可用于细菌的快速诊断。     

Identification of Balamuthia mandrillaris by PCR assay using the mitochondrial 16S rRNA gene as a target

Balamuthia mandrillaris is an opportunistic pathogen that causes granulomatous amebic meningoencephalitis in animals, including humans. Based on sequence analysis of mitochondrial small-subunit-rRNA genes, we developed primers that amplify a Balamuthia-specific PCR product. These primers will be useful for retrospective analyses of fixed tissues and possible identification of Balamuthia in vivo.     

Identification of a novel, invasive, not-yet-cultivated Treponema sp. in the large intestine of pigs by PCR amplification of the 16S rRNA gene

Laser capture microdissection in combination with fluorescent in situ hybridization was used to identify an unknown species of spirochetes from the pig colonic mucosa. The 16S rRNA gene was PCR amplified, and the closest related type strain was Treponema bryantiiT (90.1%). The spirochete, here named "Candidatus Treponema suis," was associated with colitis, including invasion of the surface epithelium as well as superficial parts of the mucosa.     

Amplification of P1 and 16S rRNA genes by nested PCR for detection of Mycoplasma pneumoniae in paediatric patients

Mycoplasma (M.) pneumoniae is the most frequent atypical pathogen responsible for community-acquired respiratory infection in children and adults. The etiologic diagnosis of these infections still remains difficult. This is mainly due to the absence of characteristic clinical findings, and to the available detection methods (serology and culture) which are time consuming, insensitive and non-specific. To improve the detection of this infectious agent, nested polymerase chain reaction (PCR) analysis was developed. A total of 46 nasal aspirates, from children hospitalised with severe lower respiratory tract infection and in whom M. pneumoniae was suspected, were analysed for the presence of M. pneumoniae DNA by PCR. Routine microbiological investigations revealed no virus in these 46 samples. Using nested PCR, two targets were amplified: the sequences of 16S ribosomal (r) RNA gene (rDNA) and P1 adhesin gene. Evidence of M. pneumoniae infection was identified in four paediatric patients. The amplification of 16S rDNA was found to be more sensitive for the detection of M. pneumoniae. Our results suggest that amplification of the 16S rDNA by nested PCR and detection of the amplification products by visual inspection of the polyacrylamide gel should allow the rapid diagnosis of M. pneumoniae in respiratory tract infection in paediatric patients.     

Species-specific identification of Leptospiraceae by 16S rRNA gene sequencing

The genus Leptospira is classified into 13 named species and 4 genomospecies based upon DNA-DNA reassociation studies. Phenotypic tests are unable to distinguish between species of Leptospira, and there is a need for a simplified molecular approach to the identification of leptospires. 16S rRNA gene sequences are potentially useful for species identification of Leptospira, but there are a large number of sequences of various lengths and quality in the public databases. 16S rRNA gene sequences of near full length and bidirectional high redundancy were determined for all type strains of the species of the Leptospiraceae. Three clades were identified within the genus Leptospira, composed of pathogenic species, nonpathogenic species, and another clade of undetermined pathogenicity with intermediate 16S rRNA gene sequence relatedness. All type strains could be identified by 16S rRNA gene sequences, but within both pathogenic and nonpathogenic clades as few as two or three base pairs separated some species. Sequences within the nonpathogenic clade were more similar, and in most cases < or =10 bp distinguished these species. These sequences provide a reference standard for identification of Leptospira species and confirm previously established relationships within the genus. 16S rRNA gene sequencing is a powerful method for identification in the clinical laboratory and offers a simplified approach to the identification of Leptospira species.     

Comparison of two Mycoplasma genitalium real-time PCR detection methodologies

Established in-house quantitative PCR (qPCR) assays to detect the Mycoplasma genitalium adhesion protein (MgPa) and the 16S rRNA gene were found to be comparable for screening purposes, with a kappa value of 0.97 (95% confidence interval [CI], 0.94 to 1.01) and no difference in bacterial load quantified (P = 0.4399).     

Detection of a Mixed Infection in a Culture-Negative Brain Abscess by Broad-Spectrum Bacterial 16S rRNA Gene PCR

We describe the identification of two bacterial pathogens from a culture-negative brain abscess by the use of broad-spectrum 16S rRNA gene PCR. Simultaneous detection of Fusobacterium nucleatum and Porphyromonas endodontalis was possible due to a 24-bp length difference of their partially amplified 16S rRNA genes, which allowed separation by high-resolution polyacrylamide gel electrophoresis.Broad-spectrum 16S rRNA gene PCR is a universal and highly versatile technology for the detection and identification of bacterial pathogens (11). Microbiological examination of life-threatening brain infections relies on microscopic examination and culture of abscess material. A recent study by Al Masalma et al. (1) explored an expanded technology applying multiple 16S rRNA fragment cloning and subsequent sequencing to analyze mixed floras in brain abscesses and found that the number of species associated with brain abscesses is much larger than previously noted. Based on a recent case and an in silico feasibility study, we present a modified technique for the molecular analysis of polymicrobial infections. After broad-spectrum 16S rRNA gene amplification, amplicons are separated electrophoretically and excised from a polyacrylamide gel for subsequent sequence identification. In comparison to the previously described labor-intensive cloning and sequencing strategies used to resolve polymicrobial infections by PCR (1, 4), the technique presented herein is less time-consuming and less expensive, further allowing its introduction in the routine diagnostic laboratory.     

Catheter-related Microbacterium bacteremia identified by 16S rRNA gene sequencing

We describe the application of 16S rRNA gene sequencing in defining two cases of catheter-related Microbacterium bacteremia. In the first case, a gram-positive bacillus was isolated from both the blood culture and central catheter tip of a 39-year-old woman with chronic myeloid leukemia. The API Coryne system identified the isolate as 98.9% Aureobacterium or Corynebacterium aquaticum. In the second case, a gram-positive bacillus was recovered from five sets of blood cultures from both central catheter and percutaneous venipuncture of a 5-year-old girl with acute myeloid leukemia. The isolate was identified by the API Coryne system as 99.7% Cellulomonas or Microbacterium species. Further phenotypic tests failed to identify the two isolates. 16S rRNA gene sequencing showed 99.4% similarity between the first isolate and Microbacterium oxydans and 98.7% similarity between the second isolate and Microbacterium trichotecenolyticum, indicating that both isolates were Microbacterium species. Microbacterium infections are rarely reported in the literature. Although the central venous catheter was previously proposed to be a source of bacteremia, the first case in this report represents the first culture-documented case of catheter-related Microbacterium bacteremia.     

Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification

Here we determined the analytical sensitivities of broad-range real-time PCR-based assays employing one of three different genomic DNA extraction protocols in combination with one of three different primer pairs targeting the 16S rRNA gene to detect a panel of 22 bacterial species. DNA extraction protocol III, using lysozyme, lysostaphin, and proteinase K, followed by PCR with the primer pair Bak11W/Bak2, giving amplicons of 796 bp in length, showed the best overall sensitivity, detecting DNA of 82% of the strains investigated at concentrations of < or =10(2) CFU in water per reaction. DNA extraction protocols I and II, using less enzyme treatment, combined with other primer pairs giving shorter amplicons of 466 bp and 342 or 346 bp, respectively, were slightly more sensitive for the detection of gram-negative but less sensitive for the detection of gram-positive bacteria. The obstacle of detecting background DNA in blood samples spiked with bacteria was circumvented by introducing a broad-range hybridization probe, and this preserved the minimal detection limits observed in samples devoid of blood. Finally, sequencing of the amplicons generated using the primer pair Bak11W/Bak2 allowed species identification of the detected bacterial DNA. Thus, broad-spectrum PCR targeting the 16S rRNA gene in the quantitative real-time format can achieve an analytical sensitivity of 1 to 10 CFU per reaction in water, avoid detection of background DNA with the introduction of a broad-range probe, and generate amplicons that allow species identification of the detected bacterial DNA by sequencing. These prerequisites are important for its application to blood-containing patient samples.     

Comparison of 16S rRNA gene PCR and BACTEC 9240 for detection of neonatal bacteremia

Ten percent of infants born in the United States are admitted to neonatal intensive care units (NICU) annually. Approximately one-half of these admissions are from term infants (>34 weeks of gestation) at risk for systemic infection. Most of the term infants are not infected but rather have symptoms consistent with other medical conditions that mimic sepsis. The current standard of care for evaluating bacterial sepsis in the newborn is performing blood culturing and providing antibiotic therapy while awaiting the 48-h preliminary result of culture. Implementing a more rapid means of ruling out sepsis in term newborns could result in shorter NICU stays and less antibiotic usage. The purpose of this feasibility study was to compare the utility of PCR to that of conventional culture. To this end, a total of 548 paired blood samples collected from infants admitted to the NICU for suspected sepsis were analyzed for bacterial growth using the BACTEC 9240 instrument and for the bacterial 16S rRNA gene using a PCR assay which included a 5-h preamplification culturing step. The positivity rates by culture and PCR were 25 (4.6%) and 27 (4.9%) positive specimens out of a total of 548 specimens, respectively. The comparison revealed sensitivity, specificity, and positive and negative predictive values of 96.0, 99. 4, 88.9, and 99.8%, respectively, for PCR. In summary, this PCR-based approach, requiring as little as 9 h of turnaround time and blood volumes as small as 200 microl, correlated well with conventional blood culture results obtained for neonates suspected of having bacterial sepsis.