Rapid detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water using peptide nucleic acid probes

A new chemiluminescent in situ hybridization (CISH) method that provides simultaneous detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water within 1 working day has been developed. Individual micro-colonies of P. aeruginosa were detected directly on membrane filters following 5 h of growth by use of soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeted to a species-specific sequence in P. aeruginosa rRNA. Within each micro-colony, reaction of the peroxidase with a chemiluminescent substrate generated light that was subsequently captured by film or with a digital camera system. Each spot of light represented one micro-colony of P. aeruginosa. Sensitivity and specificity for the identification of P. aeruginosa were 100% as determined by testing 28 P. aeruginosa strains and 17 other bacterial species that included closely related Pseudomonas species. Furthermore, the number of micro-colonies of P. aeruginosa represented by light spots correlated with counts of visible colonies following sustained growth. We conclude that PNA CISH speeds up traditional membrane filtration techniques and adds the specificity of PNA probe technology to generate fast and definitive results.     

The significance of testing for Pseudomonas aeruginosa in recreational seawater beaches.

The occurrence of Pseudomonas aeruginosa and faecal coliforms in Mediterranean sea water from beaches was investigated. Water samples (1,598 in toto) were tested and P. aeruginosa was found in 222 samples (14%). In 31% of samples where P. aeruginosa was detected, faecal coliforms of less than ten bacteria per 100 ml were found. In a group of 98 samples which had > 500 faecal coliforms per 100 ml, 41% had no detectable P. aeruginosa. The inclusion of P. aeruginosa as an additional parameter for approving beaches for recreational activity is recommended.     

Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution

We have constructed light-up probes for nucleic acid detection. The light-up probe is a peptide nucleic acid (PNA) oligonucleotide to which the asymmetric cyanine dye thiazole orange (TO) is tethered. It combines the excellent hybridization properties of PNA and the large fluorescence enhancement of TO upon binding to DNA. When the PNA hybridizes to target DNA, the dye binds and becomes fluorescent. Free probes have low fluorescence, which may increase almost 50-fold upon hybridization to complementary nucleic acid. This makes the light-up probes particularly suitable for homogeneous hybridization assays, where separation of the bound and free probe is not necessary. We find that the fluorescence enhancement upon hybridization varies among different probes, which is mainly due to variations in free probe fluorescence. For eight probes studied the fluorescence quantum yield at 25 degrees C in the unbound state ranged from 0.0015 to 0.08 and seemed to depend mainly on the PNA sequence. The binding of the light-up probes to target DNA is highly sequence specific and a single mismatch in a 10-mer target sequence was readily identified.     

Occurrence of Staphylococcus aureus and Pseudomonas aeruginosa in Israeli coastal water.

The occurrence of Pseudomonas aeruginosa and coagulase-positive Staphylococcus aureus in seawater from beaches of central Israel was investigated from June 1983 until June 1985. P. aeruginosa was monitored in 652 samples of seawater from 34 beaches, and S. aureus was monitored in 628 samples. P. aeruginosa was found in 44.8% of samples (6.5% with 1 bacterium per 100 ml of water), and S. aureus was recovered from 60.7% of samples (5.3% with 1 organism per 100 ml), compared with 91.6% of samples with total coliforms (TC) and 82.2% with fecal coliforms (FC). The correlation between the presence of P. aeruginosa to that of TC and FC was 99.1 and 98.3%, respectively, while S. aureus was found in 4.3 and 8% of samples where TC and FC, respectively, were absent. Monitoring of S. aureus as a supplementary indicator in populated beaches is recommended because it will add valuable information on the sanitary quality of the seawater.     

Design and evaluation of 16S rRNA-targeted peptide nucleic acid probes for whole-cell detection of members of the genus Listeria

Six fluorescein-labeled peptide nucleic acid oligomers targeting Listeria-specific sequences on the 16S ribosomal subunit were evaluated for their abilities to hybridize to whole cells by fluorescence in situ hybridization (FISH). Four of these probes yielded weak or no fluorescent signals after hybridization and were not investigated further. The remaining two FISH-compatible probes, LisUn-3 and LisUn-11, were evaluated for their reactivities against 22 Listeria strains and 17 other bacterial strains belonging to 10 closely related genera. Hybridization with BacUni-1, a domain-specific eubacterial probe, was used as a positive control for target accessibility in both Listeria spp. and nontarget cells. RNase T1 treatment of select cell types was used to confirm that positive fluorescence responses were rRNA dependent and to examine the extent of nonspecific staining of nontarget cells. Both LisUn-3 and LisUn-11 yielded rapid, bright, and genus-specific hybridizations at probe concentrations of approximately 100 pmol ml(-1). LisUn-11 was the brightest probe and stained all six Listeria species. LisUn-3 hybridized with all Listeria spp. except for L. grayi, for which it had two mismatched bases. A simple ethanolic fixation yielded superior results with Listeria spp. compared to fixation in 10% buffered formalin and was applicable to all cell types studied. This study highlights the advantages of peptide nucleic acid probes for FISH-based detection of gram-positive bacteria and provides new tools for the rapid detection of Listeria spp. These probes may be useful for the routine monitoring of food production environments in support of efforts to control L. monocytogenes.     

Occurrence of Staphylococcus aureus and Pseudomonas aeruginosa in Israeli coastal water

The occurrence of Pseudomonas aeruginosa and coagulase-positive Staphylococcus aureus in seawater from beaches of central Israel was investigated from June 1983 until June 1985. P. aeruginosa was monitored in 652 samples of seawater from 34 beaches, and S. aureus was monitored in 628 samples. P. aeruginosa was found in 44.8% of samples (6.5% with 1 bacterium per 100 ml of water), and S. aureus was recovered from 60.7% of samples (5.3% with 1 organism per 100 ml), compared with 91.6% of samples with total coliforms (TC) and 82.2% with fecal coliforms (FC). The correlation between the presence of P. aeruginosa to that of TC and FC was 99.1 and 98.3%, respectively, while S. aureus was found in 4.3 and 8% of samples where TC and FC, respectively, were absent. Monitoring of S. aureus as a supplementary indicator in populated beaches is recommended because it will add valuable information on the sanitary quality of the seawater.     

Fluorescence in situ hybridization using peptide nucleic acid probes for rapid detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in potable-water biofilms

Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybridization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive and grow in drinking-water biofilms and possibly transmit via drinking water. The designed PNA probe (MAV148) specificity was tested with several bacterial species, including other mycobacteria and mycolic acid-containing bacteria. From the range of bacterial strains tested, only M. avium subsp. avium and M. avium subsp. paratuberculosis strains were hybridized. The PNA FISH method was applied successfully to detect M. avium subsp. avium spiked in water samples and biofilm established within a Propella biofilm reactor fed with potable water from a distribution supply.     

Synthetic enterotoxin B DNA probes for detection of enterotoxigenic Staphylococcus aureus strains.

DNA-DNA colony hybridization experiments with three different synthetic probes were carried out with 210 strains of Staphylococcus aureus. The synthetic probes encoded the amino acids 15 to 29 (probe 1), 179 to 192 (probe 2), and 207 to 219 (probe 3) of staphylococcal enterotoxin B (SEB). The amino acid sequences of these parts of SEB are identical to those of SEC1. All 21 SEB-producing strains tested reacted with each of the three probes. Of the 69 SEC-producing strains, 21 reacted with probe 1, none reacted with probe 2, and all 69 reacted with probe 3. With other strains no hybridization signals were obtained. The results presented here show that with a single synthetic DNA probe (probe 3) both SEB- and SEC-producing strains are detectable.     

Modified Pseudomonas agar: new differential medium for the detection/enumeration of Pseudomonas aeruginosa in mineral water.

Pseudomonas aeruginosa has been implicated as a foodborne and waterborne pathogen and is now considered a primary infectious agent. In the present study, the survival of P. aeruginosa inoculated in mineral water was evaluated by drop counts on Pseudomonas Agar Base (PAB), PAB with CN supplement X107, PAB with cetrimide, PAB with nalidixic acid, and these media with added FeSO(4). Initial counts, before starvation, were the same in all media tested. Following this period, P. aeruginosa became sensitive to PAB with added cetrimide. The addition of FeSO(4) did not improve the recovery of stressed P. aeruginosa but gave colonies a typical dark brown colour being easily differentiated from other species that can grow at 42 degrees C. The modified Pseudomonas agar medium was also tested with several P. aeruginosa strains, other species of Pseudomonas, and other genera. Only P. aeruginosa strains (pyocyanin positive) produced the typical colonies. Our results demonstrate that Pseudomonas agar with ferrous sulphate, used for the differentiation of P. aeruginosa colonies, and nalidixic acid, used as an inhibitor of Gram-positive bacteria, might be a useful medium for the detection of injured P. aeruginosa in mineral water.     

呼吸道病毒核酸快速检测方案的建立与应用

本研究旨在建立一套便携、准确、操作简便的呼吸道病毒核酸快速检测方案。通过实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)验证免提取的呼吸道病毒处理试剂(extraction-free respiratory virus treatment reagent,RTU)对病毒核酸处理的效果以及超快速荧光定量PCR仪(FQ-8A)对核酸扩增的效果;将RTU和FQ-8A结合构建呼吸道病毒核酸快速检测方案,通过荧光定量PCR仪中Ct值判断阳性检出率,以验证该方案检测临床样本时的准确性。结果表明,RTU与全自动核酸提取仪在提取效果上灵敏度相当;RTU在提取不同病毒类型样本时,与其他3种提取方法效果相当,但RTU提取时间少于5 min;FQ-8A检测呼吸道合胞病毒(respiratory syncytial virus,RSV)及腺病毒(adenovirus,ADV)与对照仪器ABI-7500具有良好一致性,kappa系数分别为0.938(P<0.001)和0.887(P<0.001),但FQ-8A耗时更短,扩增时间仅在0.5 h左右;RTU和FQ-8A相结合的快检方案与常规检测方案具有高度一致的检出率,其灵敏度为91.70%,特异度为100%,kappa系数为0.944(P<0.001)。总之,通过RTU与FQ-8A的结合构建了一套可在35 min内完成全部流程的呼吸道病毒核酸快速检测方案。该方案准确性高、操作简便,可为呼吸道病毒快速诊断和治疗提供重要支持。     

Factors influencing detection and enumeration of Pseudomonas aeruginosa by most-probable-number and membrane filtration techniques.

Most-probable-number (MPN) and membrane filtration (mF) techniques were evaluated with respect to selectivity, sensitivity, and efficiency in recovering Pseudomonas aeruginosa strains in hospital fluids and extramural water environments. Known numbers of cells of a naturally occurring strain of P. aeruginosa maintained in distilled water or cells subcultured on Standard Methods agar were added to test samples containing various types and levels of background microbial contamiants. Environmental samples containing unknown numbers of P. aeruginosa strains also were tested. Asparagine and acetamide broths were employed as presumptive media in MPN tests, and mPA and Pseudosel agars were used in mF assays. Statistical analyses of data showed the superiority and comparability of the asparagine-MPN and mPA-mF systems. Greater precision and accuracy were consistently obtained in either assay technique by the use of naturally occurring cells as test organisms. The type of filter and nature of diluents employed, as well as pH of assay media, were found to greatly influence both recovery and developemnt of characteristic colonial morphology in the mPA-mF system.     

An improved amperometric immunosensor for the detection and enumeration of protein A-bearing Staphylococcus aureus

An amperometric immunosensor specific to the protein A of Staphylococcus aureus, was developed using the direct electrochemical detection of phenol produced by alkaline phosphatase from phenylphosphate. The immunosensor could detect protein A at 0.01 ng/ml and could reliably detect and quantify pure cultures of protein A-bearing Staph. aureus above 10(3) cfu/ml. A similar sensitivity of detection was obtained with samples of beef and milk.     

Development and application of a triplex-PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus from pigs

A triplex-PCR assay was developed and evaluated for rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) recovered from various biological samples of pig. Three sets of primers were designed to target mecA, 16S rRNA and nuc genes of MRSA. The specific amplification generated three bands on agarose gel, with sizes 280 bp for mecA, 654 bp for 16S rRNA and 481 bp for nuc, respectively. A potential advantage of the PCR assay is its sensitivity with a detection limit of 10² CFU per ml of bacteria. In all, 79 MRSA isolates recovered from various samples of pigs were subjected to the amplification by the triplex-PCR assay and all the isolates yielded three bands corresponding to the three genes under this study. No false-positive amplification was observed, indicating the high specificity of the developed triplex-PCR assay. This assay will be a useful and powerful method for differentiation of MRSA from methicillin-sensitive S. aureus, coagulase-negative methicillin-resistant staphylococci and coagulase-negative methicillin-sensitive staphylococci.     

Application of a lanthanide fluorescent chelate label for detection of single-nucleotide mutations with peptide nucleic acid probes

We developed the approach to detect single-nucleotide mutation with peptide nucleic acid (PNA) probes and time-resolved fluorometry using a fluorescence lanthanide chelate label, {2,2',2',2'-{4'-{[(4,6-dichloro-1,3,5-triazin-2-yl)amino]biphenyl-4-yl}-2,2': 6',2'-terpyridine-6,6'-diyl}bis(methylenenitrilo)}tetrakis(acetato)}europium(III) (DTBTA-Eu3+). Compared with DNA probes, PNA probes showed lower mismatch signals and gave higher signal/noise (S/N) ratios. Using the system, we examined the single-nucleotide mutations of codon 12 in the c-Ha-ras gene of PCR amplicons of genome DNAs isolated from human umbilical vein endothelial cells (HUVECs) and T24 cells.     

Structure analysis of peptide deformylases from Streptococcus pneumoniae,Staphylococcus aureus,Thermotoga maritima and Pseudomonas aeruginosa: snapshots of the oxygen sensitivity of peptide deformylase

Peptide deformylase (PDF) has received considerable attention during the last few years as a potential target for a new type of antibiotics. It is an essential enzyme in eubacteria for the removal of the formyl group from the N terminus of the nascent polypeptide chain. We have solved the X-ray structures of four members of this enzyme family, two from the Gram-positive pathogens Streptococcus pneumoniae and Staphylococcus aureus, and two from the Gram-negative bacteria Thermotoga maritima and Pseudomonas aeruginosa. Combined with the known structures from the Escherichia coli enzyme and the recently solved structure of the eukaryotic deformylase from Plasmodium falciparum, a complete picture of the peptide deformylase structure and function relationship is emerging. This understanding could help guide a more rational design of inhibitors. A structure-based comparison between PDFs reveals some conserved differences between type I and type II enzymes. Moreover, our structures provide insights into the known instability of PDF caused by oxidation of the metal-ligating cysteine residue.     

Accelerated procedure for the enumeration and identification of food-borne Staphylococcus aureus.

A procedure was developed for accelerating to 29 h the enumeration and identification of both healthy and stressed cells of Staphylococcus aureus in foods. Baird-Parker agar medium was incubated for 24 h; S. aureus was identified within 5 additional h by using a simplified thermonuclease test.