Internally Controlled Real-Time PCR Method for Quantitative Species-Specific Detection and vapA Genotyping of Rhodococcus equi

We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific as determined using 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates with known vapA⁺ and vapA-negative (including vapB⁺) plasmid genotypes. The vapA⁺ frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The choE-IAC Q-PCR could detect up to one genome equivalent using R. equi DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoalveolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to ≈10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency E of >0.94. The vapA assay had similar performance but appeared unsuitable for accurate (vapA⁺) R. equi quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of R. equi and (optional) vapA⁺ “horse-pathogenic” genotype determination.     

Rapid determination of vapA/vapB genotype in Rhodococcus equi using a differential polymerase chain reaction method

Rhodococcus equi is a facultative pathogen of foals. Infection causes an often fatal pulmonary pneumonia. The organism has also been isolated from pigs, cattle, humans and the environment. Equine virulence has a high positive correlation with the expression of a 17.4 kD polypeptide of unknown function, VapA, the product of the plasmid-encoded vapA gene. More recently an isogene of vapA, referred to as vapB and encoding an 18.2 kDa polypeptide, has been identified among pig and human isolates. The two genes share > 80% sequence identity, yet their host strains apparently exhibit different pathogenicity profiles (for example by reference to virulence in mouse model system and host specificity). In this study, a polymerase chain reaction (PCR) technique was developed that permits the selective amplification of vapA and vapB. Using this technique the distribution of the two genes among 35 randomly selected isolates of Rhodococcus equi from various animal and environmental sources was determined. Using this technique the genotype of each isolate could be unambiguously assigned as vapA+, vapB+ or vap- (i.e., scoring negative for both vapA and vapB). No isolate scored positive for both vapA and vapB. 100% of equine isolates scored vapA+, confirming the status of vapA as a reliable marker of equine virulence. All three genotypes were found among human isolates; porcine isolates scored either vapB+ or vap- and no vapA+ isolates were present in this sample. Rigorous statistical analysis using the Fisher Exact test confirmed that the high frequency of vapA+ among equine isolates is significant; however the sample size was too small to draw statistically significant conclusions regarding the distribution of genotypes among within other animal groups.     

Identification and mutagenesis by allelic exchange of choE, encoding a cholesterol oxidase from the intracellular pathogen Rhodococcus equi

The virulence mechanisms of the facultative intracellular parasite Rhodococcus equi remain largely unknown. Among the candidate virulence factors of this pathogenic actinomycete is a secreted cholesterol oxidase, a putative membrane-damaging toxin. We identified and characterized the gene encoding this enzyme, the choE monocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified in Brevibacterium sterolicum and Streptomyces spp. ChoE also exhibits significant similarities to putative cholesterol oxidases encoded by Mycobacterium tuberculosis and Mycobacterium leprae. Genetic tools for use with R. equi are poorly developed. Here we describe the first targeted mutagenesis system available for this bacterium. It is based on a suicide plasmid, a selectable marker (the aacC4 apramycin resistance gene from Salmonella), and homologous recombination. The choE allele was disrupted by insertion of the aacC4 gene, cloned in pUC19 and introduced by electroporation in R. equi. choE recombinants were isolated at frequencies between 10(-2) and 10(-3). Twelve percent of the recombinants were double-crossover choE mutants. The choE mutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-producing bacteria (Listeria ivanovii). Functional complementation was achieved by expression of choE from pVK173-T, a pAL5000 derivative conferring hygromycin resistance. Our data demonstrate that ChoE is an important cytolytic factor for R. equi. The highly efficient targeted mutagenesis procedure that we used to generate choE isogenic mutants will be a valuable tool for the molecular analysis of R. equi virulence.     

Evolution of the Rhodococcus equi vap pathogenicity island seen through comparison of host-associated vapA and vapB virulence plasmids

The pathogenic actinomycete Rhodococcus equi harbors different types of virulence plasmids associated with specific nonhuman hosts. We determined the complete DNA sequence of a vapB(+) plasmid, typically associated with pig isolates, and compared it with that of the horse-specific vapA(+) plasmid type. pVAPB1593, a circular 79,251-bp element, had the same housekeeping backbone as the vapA(+) plasmid but differed over an approximately 22-kb region. This variable region encompassed the vap pathogenicity island (PAI), was clearly subject to selective pressures different from those affecting the backbone, and showed major genetic rearrangements involving the vap genes. The pVAPB1593 PAI harbored five different vap genes (vapB and vapJ to -M, with vapK present in two copies), which encoded products differing by 24 to 84% in amino acid sequence from the six full-length vapA(+) plasmid-encoded Vap proteins, consistent with a role for the specific vap gene complement in R. equi host tropism. Sequence analyses, including interpolated variable-order motifs for detection of alien DNA and reconstruction of Vap family phylogenetic relationships, suggested that the vap PAI was acquired by an ancestor plasmid via lateral gene transfer, subsequently evolving by vap gene duplication and sequence diversification to give different (host-adapted) plasmids. The R. equi virulence plasmids belong to a new family of actinobacterial circular replicons characterized by an ancient conjugative backbone and a horizontally acquired niche-adaptive plasticity region.     

Quantitative TaqMan PCR for detection of Pneumocystis jiroveci

We developed a quantitative real-time PCR assay for detection and quantification of Pneumocystis jiroveci in bronchoalveolar lavage (BAL) specimens based on primers and probe targeting the gene encoding beta-tubulin. The assay was able to detect 50 DNA copies per ml of a standard plasmid containing the target sequence. The intra- and interassay coefficients of variation were 0.46%-4.27% and 0.05-2.00% over 5 log(10) values. Fifty-seven controls of human, viruses, bacteria and fungi DNA samples were amplified and found negative. Fifty-three BAL samples sent to the laboratory for diagnosis of pneumocystosis were prospectively investigated by real-time PCR and direct microscopic examinations (DME) using Giemsa stain and direct immunofluorescence. All PCR negative samples were negative by microscopy. Among the 24 (45%) BAL found PCR positive, 8 were positive by microscopy (35%). The copy numbers of the target gene were between 4.4 x 10(3) and 2.8 x 10(6) per ml for the microscopically positive samples and between 8 and 9.2 x 10(3) per ml for the microscopically negative samples. In conclusion, we developed a rapid, sensitive and specific real time PCR for the diagnosis and quantification of Pneumocystis jiroveci in BAL samples.     

Deletion of vapA encoding Virulence Associated Protein A attenuates the intracellular actinomycete Rhodococcus equi

Virulent strains of the facultative intracellular bacterium Rhodococcus equi isolated from young horses (foals) with R. equi pneumonia, carry an 80-90 kb virulence plasmid and express a highly immunogenic 15-17 kDa protein of unknown function called VapA (Virulence Associated Protein A). Recent sequencing of the virulence plasmid identified a putative pathogenicity island encoding a novel family of seven Vap proteins including VapA. These proteins exhibit a significant sequence similarity to each other but have no homologues in other organisms. In this study, we describe the construction of an R. equi mutant lacking a 7.9 kb DNA region spanning five vap genes (vapA, -C, -D, -E and -F ). This vap locus mutant was attenuated for virulence in mice as it was unable to replicate in vivo and was rapidly cleared in comparison to the virulent wild-type strain. Complementation analysis of the vap locus mutant showed that expression of vapA alone could restore full virulence, whereas expression of vapC, -D and -E could not. We subsequently constructed an R. equi strain lacking only the vapA gene and found that it was attenuated for growth in vivo to the same degree as the vap locus mutant. Unlike wild-type R. equi which replicates intracellularly, both of the mutant strains exhibited a growth defect in macrophages although their attachment to the macrophages was unaffected. These studies provide the first proof of a role for vapA in the virulence of R. equi, and demonstrate that its presence is essential for intracellular growth in macrophages.     

A novel real-time PCR for Listeria monocytogenes that monitors analytical performance via an internal amplification control

We describe a novel quantitative real-time (Q)-PCR assay for Listeria monocytogenes based on the coamplification of a target hly gene fragment and an internal amplification control (IAC). The IAC is a chimeric double-stranded DNA containing a fragment of the rapeseed BnACCg8 gene flanked by the hly-specific target sequences. This IAC is detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the hly and IAC signals. The hly-IAC assay had a specificity and sensitivity of 100%, as assessed using 49 L. monocytogenes isolates of different serotypes and 96 strains of nontarget bacteria, including 51 Listeria isolates. The detection and quantification limits were 8 and 30 genome equivalents, and the coefficients for PCR linearity (R2) and efficiency (E) were 0.997 and 0.80, respectively. We tested the performance of the hly-IAC Q-PCR assay using various broth media and food matrices. Fraser and half-Fraser media, raw pork, and raw or cold-smoked salmon were strongly PCR-inhibitory. This Q-PCR assay for L. monocytogenes, the first incorporating an IAC to be described for quantitative detection of a food-borne pathogen, is a simple and robust tool facilitating the identification of false negatives or underestimations of contamination loads due to PCR failure.     

Epidemiology of Rhodococcus equi strains on Thoroughbred horse farms

Pulsed-field gel electrophoresis of restriction endonuclease-digested genomic DNA from a large collection of clinical isolates of Rhodococcus equi, an important pathogen of foals, was used to compare strain distribution between farms and over time. Forty-four strains were found among 209 isolates, with 5 of these accounting for over half the isolates and the 22 strains isolated more than once accounting for 90% of the isolates. The average genotypic diversity on each farm and in each year was found to be less than the genotypic diversity of the isolates taken as a whole, with 5.2% of the total diversity being due to differences between farms and 5.5% to differences between years. A small number of strains on each farm were found to have caused at least half the clinical cases of disease, and these varied between farms and, to a lesser extent, years. Most strains were found on more than one farm, and some very similar restriction patterns were found among isolates from different continents, indicating that strains can be very widespread. Multiple strains were isolated in five of the six cases in which more than one isolate from a single foal was examined, indicating that disease may commonly be caused by simultaneous infection with multiple strains. It was concluded that there are a number of different strains of R. equi which carry the vapA gene, and these strains tend to be widespread, but individual farms tend to have particular strains associated with them.     

用聚合酶链反应（PCR）检测支气管肺泡灌洗（BAL）液中的嗜肺军团杆菌

本文研究了由嗜肺军团杆菌的巨噬细胞感染性增效子（ｍｉｐ）基因设计的一对引物,用ＰＣＲ扩增嗜肺军团杆菌３、５、７、８血清型的４个标准菌株的特异ＤＮＡ序列,研究了用该引物扩增ＢＡＬ液中嗜肺军团菌特异ＤＮＡ序列的方法、灵敏度及特异性。结果表明：用上述引物扩增嗜肺军团菌４个标准菌株的ＤＮＡ,均可得到２０７ｂｐ的特异扩增产物,ＢＡＬ液中的军团菌经离心及裂解液裂解后,可直接进行ＤＮＡ扩增,当ＢＡＬ与液中军团菌量为２×１０３ＣＦＵ／ｍｌ时,即可检测出特异扩增带（电泳法）,除军团菌外,其它受试细菌均无此特异性扩增,用本法对４２例临床非典型肺炎患者的ＢＡＬ液进行嗜肺军团菌的检测,在４２份嗜肺军团菌培养均为阴性的ＢＡＬ液中,其中一例ＰＣＲ检测军团菌为阳性。本研究提示：用ＰＣＲ检测ＢＡＬ液中的军团菌是可行的,并有快速、灵敏、特异之忧点。     

Rhodococcus equi human clinical isolates enter and survive within human alveolar epithelial cells

Rhodococcus equi is an emerging opportunistic human pathogen associated with immunosuppressed people, especially those infected with the human immunodeficiency virus (HIV). This pathogen resides primarily within lung macrophages of infected patients, which may explain in part its ability to escape normal pulmonary defense mechanisms. Despite numerous studies as a pulmonary pathogen in foals, where a plasmid seems to play an important role in virulence, information on the pathogenesis of this pathogen in humans is still scarce. In this study, fluorescence microscopy and vancomycin protection assays were used to investigate the ability of R. equi human isolates to adhere to and to invade the human alveolar epithelial cell line A549. Our findings indicate that some R. equi clinical strains are capable of adhering, entering and surviving within the alveolar cell line, which may contribute to the pathogen persistence in lung tissues.     

A Novel Real-Time PCR for Listeria monocytogenes That Monitors Analytical Performance via an Internal Amplification Control

We describe a novel quantitative real-time (Q)-PCR assay for Listeria monocytogenes based on the coamplification of a target hly gene fragment and an internal amplification control (IAC). The IAC is a chimeric double-stranded DNA containing a fragment of the rapeseed BnACCg8 gene flanked by the hly-specific target sequences. This IAC is detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the hly and IAC signals. The hly-IAC assay had a specificity and sensitivity of 100%, as assessed using 49 L. monocytogenes isolates of different serotypes and 96 strains of nontarget bacteria, including 51 Listeria isolates. The detection and quantification limits were 8 and 30 genome equivalents, and the coefficients for PCR linearity (R²) and efficiency (E) were 0.997 and 0.80, respectively. We tested the performance of the hly-IAC Q-PCR assay using various broth media and food matrices. Fraser and half-Fraser media, raw pork, and raw or cold-smoked salmon were strongly PCR-inhibitory. This Q-PCR assay for L. monocytogenes, the first incorporating an IAC to be described for quantitative detection of a food-borne pathogen, is a simple and robust tool facilitating the identification of false negatives or underestimations of contamination loads due to PCR failure.     

Quantitative culture of Rhodococcus equi from the feces of horse

The selective isolation of R. equi in NANC medium was made by quantitative culture of the organism in the feces. R. equi was observed in the feces of all the mares and foals investigated. The mean viable count of R. equi organisms in 1 gram of feces was 8.42 X 10(2) in the mares and 7.57 X 10(2) in the foals, and its 95% confidence limits were in a range of 6.48 X 10(2) to 1.09 X 10(3) and 4.19 X 10(2) to 1.37 X 10(3), respectively. This result indicates that R. equi is a member of the normal intestinal flora in the horse.     

Cloning and nucleotide sequence analysis of a chloramphenicol acetyltransferase gene from Vibrio anguillarum.

The chloramphenicol resistant gene (cat) encoding chloramphenicol acetyltransferase (CAT) in a transferable R plasmid (pJA7324) isolated from the fish pathogen Vibrio anguillarum strain PT24 was cloned into the plasmid vector pUC19. The nucleotide sequence analysis of 1,348 base pair DNA identified an open reading frame encoding a protein of 216 amino acid residues with a calculated molecular mass of 25,471 daltons. The predicted amino acid sequences for this cat gene are 37-69% homologous with other CAT proteins of both Gram-negative and -positive bacteria. Colony hybridization performed with a PvuII-BamHI fragment including this cat gene as a probe, revealed that the same or similar chloramphenicol resistance genes existed among V. anguillarum isolates.     

Demonstration of tra+ plasmid activity in bacteria indigenous to the phyllosphere of sugar beet; gene transfer to a recombinant pseudomonad

Abstract The presence of transfer proficient plasmids in bacteria isolated from the leaves of sugar beet ( Beta vulgaris L.) was studied. Of 435 bacteria sampled 79 (18%) contained plasmids. Pseudomonads (30%), Erwinia (12%) and Klebsiella (9%) were the largest populations sampled of which 22%, 33% and 29%, respectively, contained plasmids. The ability of these plasmids to self-transfer or mediate the mobilization of the tra⁻ mob⁺ broad host range IncQ plasmid R300B was determined. R300B was maintained in 61/79 natural plasmid containing isolates, the Gram positive isolates could not support R300B. Pseudomonas aureofaciens SBW25, isolated from sugar beet leaves, was chromosomally marked with a tetracycline resistance gene and used as a recipient (SBW25ETc). Five isolates of Erwinia herbicola and one of Erwinia salicis containing natural plasmids were able to mobilize R300B into the recombinant, SBW25ETc. These mobolizing ( tra⁺ ) plasmids were not maintained in transconjugant SBW25 cells. Analysis of the fragment patterns of Pst I digested plasmid DNA demonstrated that four (pSB139, pSB140, pSB142, pSB146; 110 kb) were identical, one (pSB153; 65 kb) was common to a subset of fragments in these four and another (pSB169; 100 kb) was unique. Other natural isolates were able to transfer copper resistance ( Erwinia rhapontici , 2 strains) or mercury resistance ( Pseudomonas fluorescens SBW340) to a rifampicin resistant recipient Pseudomonas putida UWC1 but not to SBW25ETc. These self-transferable plasmids were not able to mobilize R300B. These data demonstrate that the phyllosphere supports indigenous microbial populations which have the capacity to transfer genetic material between bacteria of different genera.     

Development and application of molecular tools in the study of IncN-related plasmids from lakewater sediments

Homology to IncN, P, Q and W inc regions was investigated amongst 114 Hg(2+)-resistant or antibiotic-resistant bacteria isolated from lakewater sediments. No hybridisation signals were found with Inc P, Q and W probes, and only one plasmid, pLV1402, hybridised to the IncN probe. PCR primers designed to conserved regions in the replicon of the IncN plasmid pCU1 and the related beta replicon from pGSH500 were used to amplify a 978-bp fragment from pLV1402, with sequence analysis showing a close relationship (99.2% identity) between their replication genes. A 387-bp region from the pLV1402 rep gene was used to re-screen the isolates and identified another related plasmid, pLV1403. A 3.7-kb probe containing the alpha replicon from pGSH500 hybridised to both pLV1402 and pLV1403, suggesting that both are multi-replicon plasmids. The PCR primers and probes described will be useful in future studies of plasmid diversity.     

银离子抗性细菌质粒的分离与鉴定

通过对不同化工厂废水处理池的活性污泥进行富集、不同浓度银离子驯化后,分离得到30株银离子抗性细菌,最大硝酸银耐受量可达80 mg·ml^-1.对这些细菌进行质粒抽提与鉴定,质粒检出率为76.67％.40 mmol·L^-1的苯甲酸钠对HAg4细菌质粒的消除率可达98.75％;而350 μg· ml^-1的吖啶橙对HAg4细菌质粒的消除率只有77.78％.细菌质粒与银离子抗性密切相关.     

Re-Emergence of the Apicomplexan Theileria equi in the United States: Elimination of Persistent Infection and Transmission Risk

Arthropod-borne apicomplexan pathogens that cause asymptomatic persistent infections present a significant challenge due to their life-long transmission potential. Although anti-microbials have been used to ameliorate acute disease in animals and humans, chemotherapeutic efficacy for apicomplexan pathogen elimination from a persistently infected host and removal of transmission risk is largely unconfirmed. The recent re-emergence of the apicomplexan Theileria equi in U.S. horses prompted testing whether imidocarb dipropionate was able to eliminate T. equi from naturally infected horses and remove transmission risk. Following imidocarb treatment, levels of T. equi declined from a mean of 10(4.9) organisms/ml of blood to undetectable by nested PCR in 24 of 25 naturally infected horses. Further, blood transfer from treated horses that became nested PCR negative failed to transmit to na?ve splenectomized horses. Although these results were consistent with elimination of infection in 24 of 25 horses, T. equi-specific antibodies persisted in the majority of imidocarb treated horses. Imidocarb treatment was unsuccessful in one horse which remained infected as measured by nested PCR and retained the ability to infect a na?ve recipient via intravenous blood transfer. However, a second round of treatment eliminated T. equi infection. These results support the utility of imidocarb chemotherapy for assistance in the control and eradication of this tick-borne pathogen. Successful imidocarb dipropionate treatment of persistently infected horses provides a tool to aid the global equine industry by removing transmission risk associated with infection and facilitating international movement of equids between endemic and non-endemic regions.     

Isolation of rare opportunistic pathogens in Hungary: case report and short review of the literature. Rhodococcus equi

Rhodococcus equi is a well-established pathogen in foal pneumonia and is increasingly recognized as a pathogen in immunocompromised humans. We have isolated a Gram-positive coccobacillus from 8 blood samples and lung tissues of a renal transplant patient. Colony morphology, growth in Lowenstein-Jensen medium, 21 biochemical reactions, the characteristic morphological cycle (coccus-rod-coccus) and the CAMP test established the R. equi diagnosis. Histological studies of 2 lung biopsy specimens revealed numerous microabscesses with aggregates of polymorphonuclear leukocytes surrounded by abundant foamy macrophages. Our isolates proved to be sensitive to majority of antibacterial drugs. The appropriate therapy (amoxicillin-clavulanate) proved to be effective, however six months later a relapse was observed. Data show that in spite of its rare occurrence, R. equi infection represents a diagnostic and therapeutic challenge. The taxonomical, epidemiological, clinico-pathological, diagnostic and therapeutic data of R. equi are discussed.