Enhancing sensitivity of qPCR assays targeting Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae by using a mutant Taq DNA polymerase.

AIMS: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae are important causes of bacterial meningitis. In this study, the DNA binding site of the wild type Taq DNA polymerase was modified to produce a mutant enzyme with enhanced DNA affinity and PCR performance. The engineered and the wild type enzymes were integrated into qPCR-based assays for molecular detection of S. pneumoniae, N. meningitidis, H. influenzae, and serogroups and serotypes of these three pathogens. METHODS: Bio-Speedy® Bacterial DNA Isolation Kit (Bioeksen R&D Technologies, Turkiye) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Turkiye) and CFX96 Instrument (Biorad Inc., USA) were used for all molecular analyses. Spiked negative clinical specimens were tested using the developed qPCR assays and the culture-based conventional methods for the analytical performance evaluation. RESULTS: All qPCR assays did not produce any positive results for the samples spiked with potential cross-reacting bacteria. Limit of detection (LOD) of the assays containing the mutant enzyme was 1 genome/reaction (10 cfu/mL sample) which is at least 3 times lower than the previously reported LOD levels for DNA amplification based molecular assays. LODs for the spiked serum and cerebrospinal fluid (CSF) samples decreased 2.3-4.7 and 1.2-3.5 times respectively when the mutant enzyme was used instead of the wild type Taq DNA polymerase. CONCLUSIONS: It is possible to enhance analytical sensitivity of qPCR assays targeting the bacterial agents of meningitis by using an engineered Taq DNA polymerase. These qPCR-based assays can be used for direct detection and serogrouping / serotyping of S. pneumoniae, N. meningitidis and H. influenzae at concentrations close to the lower limit of medical decision point.

Development of a new multiplex real-time PCR assay for rapid screening of hospital-acquired infection agents.

AIMS: A new multiplex real-time PCR (qPCR) assay was developed to detect antibiotic-resistant hospital-acquired infectious agents in nasal and rectal swab samples in 1.5 h without the need for nucleic acid extraction. METHODS: Spiked negative clinical specimens were used for the analytical performance evaluation. Double-blind samples were collected from 1788 patients to assess the relative clinical performance of the qPCR assay to the conventional culture-based methods. Bio-Speedy® Fast Lysis Buffer (FLB) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Istanbul, Turkey) and LightCycler® 96 Instrument (Roche Inc., Branchburg, NJ, USA) were used for all molecular analyses. The samples were transferred into 400 L FLB, homogenized and immediately used in qPCRs. The target DNA regions are vanA and vanB genes for vancomycin-resistant Enterococcus (VRE); blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-23, blaOXA-48, blaOXA-58 genes for carbapenem-resistant Enterobacteriaceae (CRE); and mecA, mecC and spa for methicillin-resistant Staphylococcus aureus (MRSA). RESULTS: No qPCR tests produced positive results for the samples spiked with the potential cross-reacting organisms. The limit of detection (LOD) of the assay for all targets was 100 colony-forming unit (cfu)/swab-sample. Results of the repeatability studies in two different centers were in 96%-100% (69/72-72/72) agreement. The relative specificity and sensitivity of the qPCR assay were respectively 96.8% and 98.8% for VRE; 94.9% and 95.1% for CRE; 99.9% and 97.1% for MRSA. CONCLUSIONS: The developed qPCR assay can screen antibiotic-resistant hospital-acquired infectious agents in infected/colonized patients with an equal clinical performance to the culture-based methods.

Effects of high-concentration influent suspended solids on aerobic granulation in pilot-scale sequencing batch reactors treating real domestic wastewater.

The aim of this study was to investigate the effect of high-influent-concentration suspended solids (SS) on the cultivation, structure and long-term stability of aerobic granular sludge (AGS). Cultivation and long-term stability of AGS were monitored in two pilot-scale sequencing batch reactors fed with raw (R1) and settled (R2) domestic wastewater, representing high and medium SS content, respectively. The real domestic wastewater had high chemical oxygen demand (COD) content (1100 ±â€¯270 mg COD L-1). Aerobic granular sludge was cultivated in 44 days (R1) and 25 days (R2) under the conditions of high settling velocity (18 m h-1) and high organic loading rate (OLR) (2.1-2.4 kg COD m3 day). The AGS in both reactors had similar structural properties during long-term operation and remained structurally and functionally stable during the last five months of operation. Comparative evaluation of the results indicated that the high influent SS content of the real domestic wastewater had a positive influence on maintaining significantly lower SVI30 and relatively lower effluent SS concentration. Moreover, a higher influent SS content resulted in smaller mature granules during the stable period. Microbial community analyses helped to understand the aerobic granular sludge structure and showed that the sludge retention time and OLR affected the granular sludge population. The high influent SS increased biomass detachment from the granular sludge surface and caused wash-out of some bacteria colonizing the exterior of the granular sludge.

Development of a fast and low-cost qPCR assay for diagnosis of acute gas pharyngitis.

BACKGROUND: Group A streptococci (GAS) are the most common bacterial cause of acute pharyngitis and account for 15-30 % of cases of acute pharyngitis in children and 5-10 % of cases in adults. In this study, a real-time quantitative PCR (qPCR) based GAS detection assay in pharyngeal swab specimens was developed. METHODS: The qPCR assay was compared with the gold standard bacterial culture and a rapid antigen detection test (RADT) to evaluate its clinical performance in 687 patients. The analytical sensitivity of the assay was 240 cfu/swab. Forty-five different potential cross-reacting organisms did not react with the test. Four different laboratories for the reproducibility studies were in 100 % (60/60) agreement for the contrived GAS positive and negative swab samples. RESULTS: The relative sensitivities of the RADT and the qPCR test were 55.9 and 100 %; and the relative specificities were 100 and 96.3 %, respectively. Duration of the total assay for 24 samples including pre-analytical processing and analysis changed between 42 and 55 min depending on the type of qPCR instrument used. A simple DNA extraction method and a low qPCR volume made the developed assay an economical alternative for the GAS detection. CONCLUSION: We showed that the developed qPCR test is rapid, cheap, sensitive and specific and therefore can be used to replace both antigen detection and culture for diagnosis of acute GAS pharyngitis.

Effect of nitrogen deficiency during SBR operation on PHA storage and microbial diversity.

In this study, changes in microbial diversity and polyhydroxyalkanoate storage ability of activated sludge under aerobic dynamic feeding conditions were investigated. Two sequencing batch reactors were operated with and without nitrogen limitation, by applying a moderate sludge retention time. Polymer storage abilities of the biomasses were improved significantly under dynamic conditions, in terms of specific polymer storage rate, polymer storage yield and polymer content of activated sludge. Moreover, aerobic dynamic feeding conditions together with nutrient limitation further improved the storage ability of the mixed population. Polymer storage yields of the biomass enriched under nitrogen-sufficient and nitrogen-deficient conditions were 0.43 and 0.61 Cmmol PHA/Cmmol substrate, respectively. This study also contributes to the knowledge of activated sludge microbiology, providing detailed information about temporal changes in community structure under dynamic conditions. Microbial community structure was determined by 16S rDNA clone library construction. Also changes in communities under different operating conditions were monitored by DGGE analysis based on bacterial 16S rDNA. The beta subclass of Proteobacteria was the most abundant phylum in both reactors during the operation periods. Changes in the community structure occurred in terms of relative abundance of the operational taxonomic units (OTUs) rather than the OTU types present in the system.

Increment in anaerobic hydrocarbon degradation activity of Halic Bay sediments via nutrient amendment.

In this study, hydrocarbon (HC) degradation activity of a HC-rich marine sediment was assessed in anaerobic microcosms during a 224 days incubation period. Natural TOC/N/P ratio of the sediment porewater (1,000/5/1) was gradually decreased to 1,000/40/6 which resulted in approximately ninefold increase in gas production (CH(4)+CO(2)) and HC removal. Addition of external HCs to the microcosms was also resulted in approximately twofold higher gas production and HC removal. A high proportion (92%) of aromatic HCs and all n-alkanes were removed from the microcosms under unlimited nutrient supply conditions without external HC addition. The microorganisms of the sediment degraded a wide range of aliphatic (n-C(9-31) alkanes and acyclic isoprenoids) and aromatic (18 different one- to five-ring aromatics) HCs. Monitoring functional gene and transcript abundances revealed that methanogenesis and dissimilatory sulfate reduction took place simultaneously during the first 126 days, afterwards, only the syntrophic methanogenic consortium was active. Genes and transcripts related to initial activation of HCs were highly abundant throughout the incubation period showing that fumarate addition was the main pathway of anaerobic HC degradation. In conclusion, biostimulation of highly polluted anoxic marine sediments via nutrient amendment is effective and may constitute a suitable and cost-effective field-scale bioremediation strategy.

Biogeographical distribution and diversity of bacterial and archaeal communities within highly polluted anoxic marine sediments from the Marmara Sea.

Physicochemical and microbiological characterization of anoxic sediments taken from seven highly polluted sites of the Marmara Sea was carried out. The 16S rRNA based microbial community structure analyses were performed using domain-specific PCR followed by denaturant gradient gel electrophoresis (DGGE) and sequencing of characteristic bands. The results showed that the microbial communities in these sediments were diverse and evenly distributed. Relating the prokaryotic and geochemical variables through statistical tools revealed that the microbial diversity in the sediments significantly related to depth, and S, Mn and Fe content of the sediments. Fermentative bacteria, denitrifying bacteria and hydrogenotrophic methanogens were dominant whereas sulfate reducing bacteria were absent in the DGGE patterns. This unusual microbial community structure implied that the newly discovered anaerobic methane oxidation coupled to denitrification process may occur in these subseafloor environments.

Toluene inhibition on an anaerobic reactor sludge in terms of potential activity and composition of acetoclastic methanogens.

The aim of this study was to determine the effect of toluene on an anaerobic sludge taken from a full-scale upflow anaerobic sludge blanket (UASB) reactor in terms of potential activity and composition of acetoclastic methanogens. Specific methanogenic activity (SMA) test results showed that 5%, 9.5%, 14%, 24%, 29%, 38% and 62% inhibition occurred in the potential methane production (PMP) rate of the sludge at toluene concentrations of 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM and 1 mM, respectively. Fluorescence in situ hybridization (FISH) results showed that relative abundance of archaeal cells was approx. 19% throughout the SMA tests. The anaerobic sludge was dominated by acetoclastic genus Methanosaeta which were slightly affected by increasing toluene concentrations do not have any effect on relative abundance of Methanosaeta spp., which was between 73% +/- 1.6 and 68% +/- 2.1 of the archaeal population.