Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set

Nadkarni, Mangala A.; Martin, F. Elizabeth; Jacques, Nicholas A.; Hunter, Neil

doi:10.1099/00221287-148-1-257

Cited by 1,688 publications

(1,293 citation statements)

References 30 publications

Supporting

Mentioning

1,246

Contrasting

Unclassified

Order By: Relevance

“…This underlines the importance of basing molecular analyses such as these on an appropriate sampling system. A further consideration is the impact that differences in ribosomal operon number and growth rates between the bacteria present in the sample and those used to generate quantitative PCR calibration curves may have [22]. No significant difference in PCR amplification efficiency were identified in the calibration curves generated from S. aureus, S. pyogenes, P. aeruginosa or E. coli; however, the use of S. pyogenes, P. aeruginosa or E. coli rather than S. aureus led to log 10 changes in mean cfu/ml values of +1.10, +0.79 and −1.78 respectively.…”

Section: Discussionmentioning

confidence: 99%

“…The total bacterial load was determined using TaqMan assay, which amplified a 466-bp fragment (as determined for E. coli) of the 16S ribosomal RNA gene, using the forward primer EubF (5′-TCCTACGGGAGG CAGCAGT-3′), the reverse primer EubR (5′-GGACTAC CAGGGTATCTAATCCTGTT-3′) and the probe EubPR (5′-FAM-CGTATTACCGCGGCTGCTGGCAC-TAMRA-3′), as described previously by Nadkarni et al [22]. Quantitative PCR assays were performed using the RotorGene 6000 (Qiagen, Crawley, UK).…”

Section: Dna Extraction From Ascites Samplesmentioning

confidence: 99%

See 1 more Smart Citation

Characterisation of bacteria in ascites—reporting the potential of culture-independent, molecular analysis

Rogers

Russell

Preston

et al. 2010

Eur J Clin Microbiol Infect Dis

View full text Add to dashboard Cite

Spontaneous bacterial peritonitis (SBP) is a severe complication of liver disease. A significant proportion of patients have culture-negative ascites, despite having similar signs, symptoms and mortality to those with SBP. Therefore, empirical antibiotic treatment for infection is often started without knowledge of the causative organisms. Here, we investigated the potential of molecular techniques to provide rapid and accurate characterisation of the bacteria present in ascitic fluid. Ascites samples were obtained from 29 cirrhotic patients undergoing clinically indicated therapeutic paracentesis. Bacterial content was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis, quantitative polymerase chain reaction (PCR) and 16S ribosomal clone sequence analysis. Bacterial signal was detected in all samples, compared to three out of ten using standard methods. Bacterial loads ranged from 5.5 × 10 2 to 5.4 × 10 7 cfu/ml, with a mean value of 1.9 × 10 6 cfu/ml (standard deviation± 9.6 × 10 6 cfu/ml). In all but one instance, bacterial species identified by culture were also confirmed by molecular analyses. Preliminary data presented here suggests that culture-independent, molecular analyses could provide rapid characterisation of the bacterial content of ascites fluid, providing a basis for the investigation of SBP development and allowing early and targeted antibiotic intervention.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Dna Extraction From Ascites Samplesmentioning

confidence: 99%

Characterisation of bacteria in ascites—reporting the potential of culture-independent, molecular analysis

Rogers

Russell

Preston

et al. 2010

Eur J Clin Microbiol Infect Dis

View full text Add to dashboard Cite

show abstract

“…Following incubation, DNA was extracted from the lysed tissue samples with phenol/ chloroform. Detection of microbial 16S rDNA was performed by PCR using broad range universal bacterial primers to amplify a 466-bp region: forward primer 5 0 -T CCTACGGGAGGCAGCAGT-3 0 and reverse primer, 5 0 -G GACTACCAGGGTATCTAATCCTGTT-3 0 [26]. Amplification was performed as described above for the P. acnes identification.…”

Section: Detection Of Bacterial Dna In Tissue Samples By Pcrmentioning

confidence: 99%

Does nuclear tissue infected with bacteria following disc herniations lead to Modic changes in the adjacent vertebrae?

et al. 2013

View full text Add to dashboard Cite

Purpose To investigate the prevalence of infected herniated nucleus material in lumbar disc herniations and to determine if patients with an anaerobic infected disc are more likely to develop Modic change (MC) (bone oedema) in the adjacent vertebrae after the disc herniation. MCs (bone oedema) in vertebrae are observed in 6 % of the general population and in 35-40 % of people with low back pain. These changes are strongly associated with low back pain. There are probably a mechanical cause and an infective cause that causes MC. Several studies on nuclear tissue from herniated discs have demonstrated the presence of low virulent anaerobic microorganisms, predominantly Propionibacterium acnes, in 7-53 % of patients. At the time of a herniation these low virulent anaerobic bacteria may enter the disc and give rise to an insidious infection. Local inflammation in the adjacent bone may be a secondary effect due to cytokine and propionic acid production. Methods Patients undergoing primary surgery at a single spinal level for lumbar disc herniation with an MRI-confirmed lumbar disc herniation, where the annular fibres were penetrated by visible nuclear tissue, had the nucleus material removed. Stringent antiseptic sterile protocols were followed. Results Sixty-one patients were included, mean age 46.4 years (SD 9.7), 27 % female. All patients were immunocompetent. No patient had received a previous epidural steroid injection or undergone previous back surgery. In total, microbiological cultures were positive in 28 (46 %) patients. Anaerobic cultures were positive in 26 (43 %) patients, and of these 4 (7 %) had dual microbial infections, containing both one aerobic and one anaerobic culture. No tissue specimens had more than two types of bacteria identified. Two (3 %) cultures only had aerobic bacteria isolated. In the discs with a nucleus with anaerobic bacteria, 80 % developed new MC in the vertebrae adjacent to the previous disc herniation. In contrast, none of those with aerobic bacteria and only 44 % of patients with negative cultures developed new MC. The association between an anaerobic culture and new MCs is highly statistically significant (P = 0.0038), with an odds ratio of 5.60 (95 % CI 1.51-21.95). Conclusion These findings support the theory that the occurrence of MCs Type 1 in the vertebrae adjacent to a previously herniated disc may be due to oedema surrounding an infected disc. The discs infected with anaerobic bacteria were more likely (P \ 0.0038) to develop MCs in the adjacent vertebrae than those in which no bacteria were found or those in which aerobic bacteria were found.

show abstract

“…To determine whether the samples differed in overall microbiota composition, permutation-based analysis of variance was applied (permanova). Quantitative PCR on total bacteria, Staphylococcus aureus and total lactobacilli were performed according to published methods (Alarcón et al, 2006;Nadkarni et al, 2002;Penders et al, 2006). The bacterial loads were compared with Mann-Whitney test.…”

Section: S Rrna Sequencing and Qpcrmentioning

confidence: 99%

Does the maternal vaginal microbiota play a role in seeding the microbiota of neonatal gut and nose?

Sakwińska

Foata

Berger

et al. 2017

View full text Add to dashboard Cite

The acquisition and early maturation of infant microbiota is not well understood despite its likely influence on later health. We investigated the contribution of the maternal microbiota to the microbiota of infant gut and nose in the context of mode of delivery and feeding. Using 16S rRNA sequencing and specific qPCR, we profiled microbiota of 42 mother-infant pairs from the GUSTO birth cohort, at body sites including maternal vagina, rectum and skin; and infant stool and nose. In our study, overlap between maternal vaginal microbiota and infant faecal microbiota was minimal, while the similarity between maternal rectal microbiota and infant microbiota was more pronounced. However, an infant's nasal and gut microbiota were no more similar to that of its own mother, than to that of unrelated mothers. These findings were independent of delivery mode. We conclude that the transfer of maternal vaginal microbes play a minor role in seeding infant stool microbiota. Transfer of maternal rectal microbiota could play a larger role in seeding infant stool microbiota, but approaches other than the generally used analyses of community similarity measures are likely to be needed to quantify bacterial transmission. We confirmed the clear difference between microbiota of infants born by Caesarean section compared to vaginally delivered infants and the impact of feeding mode on infant gut microbiota. Only vaginally delivered, fully breastfed infants had gut microbiota dominated by Bifidobacteria. Our data suggest that reduced transfer of maternal vaginal microbial is not the main mechanism underlying the differential infant microbiota composition associated with Caesarean delivery. The sources of a large proportion of infant microbiota could not be identified in maternal microbiota, and the sources of seeding of infant gut and nasal microbiota remain to be elucidated.

show abstract

Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set

Cited by 1,688 publications

References 30 publications

Characterisation of bacteria in ascites—reporting the potential of culture-independent, molecular analysis

Characterisation of bacteria in ascites—reporting the potential of culture-independent, molecular analysis

Does nuclear tissue infected with bacteria following disc herniations lead to Modic changes in the adjacent vertebrae?

Does the maternal vaginal microbiota play a role in seeding the microbiota of neonatal gut and nose?

Contact Info

Product

Resources

About