16S rRNA Real-time PCR Research Articles

Detection of Anaplasma phagocytophilum in Ovine Serum Samples—A Retrospective Study

Tick-borne fever in sheep is caused by Anaplasma phagocytophilum and this obligate intracellular bacterium multiplies mostly in neutrophil granulocytes of its host. In Europe, the pathogen is mainly transmitted by the tick species Ixodes ricinus. Data on the dissemination of A. phagocytophilum in the German sheep population are scarce. Hence, this pilot study aimed to investigate the presence of A. phagocytophilum in ovine serum samples retrospectively. The use of sera is beneficial because the specimens are available in large numbers and usually stored over a long period of time. Ten sheep flocks located in five federal states of Germany (Baden-Württemberg, Bavaria, Lower Saxony, North Rhine-Westphalia, Schleswig-Holstein) were included in the investigations, due to the high intra-flock seroprevalence (>63%) of antibodies against the Anaplasma species. In total, 357 serum samples from seropositive sheep were analysed by 16S rRNA real-time PCR. DNA of A. phagocytophilum was detected in two ewes from the same farm in the northern German federal state of Schleswig-Holstein and represented an overall detection rate of 0.6%. Serum samples can be used to determine A. phagocytophilum, but sensitivity might be reduced in comparison to whole blood samples. Moreover, the sampling time may influence the detection of A. phagocytophilum in sheep, due to the seasonal pattern of tick activity in Germany. Our results support the findings of previous studies and demonstrate that A. phagocytophilum is also present in sheep from northern Germany (Schleswig-Holstein).

The potential role of incorporating real-time PCR and DNA sequencing for amplification and detection of 16S rRNA gene signatures in neonatal sepsis

Objectives: This study aimed to explore whether 16S rRNA gene amplification by real time PCR and sequencing could serve as genetic-based methods in rapid and accurate diagnosis of neonatal sepsis.Patients and methods: This case control study was conducted on 40 neonates suffering from sepsis like manifestations recruited from the neonatal intensive care unit of Menoufia university hospital over a period of 6 months. Their blood samples were used for paired analysis of bacterial growth using BACTEC 9050 instrument and real time PCR assay with subsequent DNA sequencing for bacterial species identification.Results: The detection rate of culture proven sepsis was 70%. By using real time 16S r RNA PCR amplification method, the detection of bacteria was improved to 80%. Real time PCR revealed sensitivity, specificity, positive predictive value and negative predictive value of [100%, 66.7%, 87.5% and 100%] respectively. Compared to culture, the 16S rRNA real time PCR demonstrated a high negative value for ruling out neonatal sepsis. There was significant statistical difference between the PCR positive and negative cases as regards the hematological sepsis score. The results demonstrated the ability of DNA sequencing to recognize 4 pathogens which were negative by blood culture. The time consumed to detect sepsis using blood culture was up to 5 days while it took up to 16 h only by PCR and sequencing methods.Conclusion: 16S rRNA gene amplification by real time PCR and sequence analysis could be served as ideal and reliable genetic-based methods to diagnose and rule out sepsis with provision of additional data that cannot be obtained by routine laboratory tests with a shorter turnaround time than those with culture-based protocols.

Evidence of metabolically active but non-culturable Listeria monocytogenes in long-term growth at 10 °C

Cultures of Listeria monocytogenes at low temperatures (10 °C) in a broth model revealed long-term survival at about 0.04% cell density in relation to the log phase. In contrast, direct viable counts and PMA real-time PCR data suggested that 50% and 1% of the population retain membrane integrity, respectively. To elucidate the observed difference, the metabolic activity of the bacterial population was investigated by multiparametric flow cytometry, including the assessment of membrane integrity, reductase activity, as well as forward and side scatter properties. These analyses were complemented by 16S rRNA real-time PCR. The majority of the cells retained their membrane integrity and reductase activity until day 29. On day 42, 48.00 ± 4.00% (L. monocytogenes strain 3251) and 68.67 ± 3.74% (L. monocytogenes strain 535) of the cells had intact membranes, whereas 57.23 ± 1.85% (strain 3251) and 74.97 ± 3.01% (strain 535) exhibited high reductase activity. On day 42, mean 16S rRNA copy numbers of 3.98 ± 1.37 (membrane integrity) and 3.86 ± 1.32 (reductase activity) remained per intact or active cell. Our data suggest the transition of L. monocytogenes into a state of metabolic dormancy during long-term culture at low temperature.

Prevalence, risk factor analysis, and hematological findings of hemoplasma infection in domestic cats from Valdivia, Southern Chile.

Four distinct cat hemoplasma species are recognized worldwide. However, this is the first study to investigate the prevalence, risk factors, and hematological findings of hemoplasmas in cats from Chile. Complete blood count and 16S rRNA real-time PCR for cat hemoplasma species were performed in 384 blood samples from domestic cats in Valdivia, Chile. Among the 384 samples the species-specific prevalence was as follows: 'Candidatus Mycoplasma haemominutum' (7.8%), Mycoplasma haemofelis (4.4%), 'Candidatus Mycoplasma turicensis' (1%), 'Ca. M. haemominutum'+M. haemofelis (0.78%), 'Ca. M. haemominutum'+'Ca. M. turicensis' (0.52%), 'Ca. M. haemominutum'+'Candidatus Mycoplasma haematoparvum' (0.26%) and 'Ca. M. haemominutum'+M. haemofelis+'Ca. M. haematoparvum' (0.26%). Male sex, older age, outdoor access, and FIV status were risk factors for hemoplasmosis. Mycoplasma haemofelis-positive cats had higher mean corpuscular volume and monocyte count. Four hemoplasma species circulate in the cat population of Valdivia. 'Candidatus M. turicensis' and 'Ca. M. haematoparvum' have been reported for the first time in Chilean cats.

PO-0536 Circulating Bacterial Dna And Lipopolysaccharide Preceding Necrotising Enterocolitis In Preterm Infants

Introduction Loss of barrier function of the intestinal wall resulting in translocation of bacteria and bacterial toxins is regularly cited in the pathogenesis of necrotising enterocolitis (NEC) but objective evidence before the onset of NEC is limited. Objective To make serial measurements of bacterial DNA and LPS endotoxin in a population of preterm babies and compare those who developed NEC with those who did not. Methods 16SrRNA Real-time PCR and ELISA were used to detect the presence of bacterial DNA and LPS endotoxin in blood and plasma from infants Results Eighty six infants were enrolled [median (range) gestation 26.5 (23–30) weeks and median (range) birth-weight 859 (572–1800) g]; 23 infants (26%) developed NEC (any NEC) of whom 13 (15%) had ≥Bells Stage II NEC. Bacterial DNA was detected in 5 infants, 3 of whom subsequently developed NEC. Babies with bacterial DNA were more likely to have ≥Bells Stage II NEC (p = 0.03) but not to have any NEC. Fourteen babies were positive for LPS, 7 of whom developed NEC. LPS endotoxinaemia was associated with development of any NEC (p = 0.04) but not with ≥Bells StageII NEC. Only 2 babies were positive for both16S rRNA and LPS all of whom developed ≥Bells Stage II NEC. Conclusion Circulating bacterial DNA and endotoxin, probably entering the blood stream via the intestinal wall, were detected in a cohort of preterm babies and associated with the subsequent development of NEC.

Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from Norway: evaluation of a PCR test targeting the chromosomal flaB gene

A consensus TaqMan real-time PCR test targeting the chromosomal flaB gene of Borrelia burgdorferi sensu lato was constructed. The test was compared with a recently published generic Light Upon eXtension (LUX) 16S rRNA real-time PCR test (Wilhelmsson et al. in J Clin Microbiol 48:4169–4176, 2010) on material consisting of 242 Ixodes ricinus ticks collected from dogs and cats in Northern Norway (n = 139) and Telemark County in Southern Norway (n = 103). Ticks positive in either test were further tested by nested PCR amplification of the 5S-23S rRNA intergenic-spacer region followed by sequencing for species identification. A tick was defined as Borrelia positive if two of three tests were positive. Thirty-four of the 242 (14 %) ticks satisfied this definition of positivity. Of these ticks 32 were positive both in the rRNA and flaB test, while two were positive only in the rRNA test. One tick was positive only in the rRNA test and was considered false positive since PCR for sequencing failed. The sensitivity of the flaB test was 94 % and the specificity 100 %. It was possible to determine the species present using Tm analysis. Among ticks from Northern Norway the prevalence of Borrelia was 13 %, whereas the prevalence in Telemark was 16 %. Among identified species (n = 33) B. afzelii was found in 16 (47 %), B. garinii in 15 (44 %) and B. valaisiana in 2 (6 %) ticks, respectively. The flaB test is a rapid, sensitive and specific test for detection and quantification of Borrelia burgdorferi s.l. in I. ricinus ticks. This is the first report on Borrelia prevalence in I. ricinus in Northern Norway.

Development of Real-Time PCR Assays for Detection of the Streptococcus milleri Group from Cystic Fibrosis Clinical Specimens by Targeting the cpn60 and 16S rRNA Genes

Cystic fibrosis (CF) is a multiorgan disease, with the majority of mortalities resulting from pulmonary failure due to repeated pulmonary exacerbations. Recently, members of the Streptococcus anginosus group (S. anginosus, S. constellatus, and S. intermedius), herein referred to as the "Streptococcus milleri group" (SMG) have been implicated as important etiological pathogens contributing to pulmonary exacerbations in CF patients. This is partly due to better microbiological detection of the SMG species through the development of a novel specific medium termed "McKay agar." McKay agar demonstrated that SMG has been an underreported respiratory pathogen contributing to lung exacerbations. Our aim was to develop a real-time PCR assay to expedite the detection of SMG within diagnostic samples. The cpn60 gene was chosen as a target, with all three members amplified using a single hybridization probe set. SMG strain analysis showed that speciation based on melting curve analysis allowed for the majority of the S. constellatus (96%), S. intermedius (94%), and S. anginosus (60%) strains to be correctly identified. To increase specificity for S. anginosus, two 16S rRNA real-time PCR assays were developed targeting the 16S rRNA gene. The 16s_SA assay is specific for S. anginosus (100%), while the 16s_SCI assay is specific for S. constellatus and S. intermedius (100%). These assays can detect <10 genome equivalents in pure culture and >10(4) genome equivalents in sputum samples, making this a great tool for assessment of the presence of SMG in complex polymicrobial samples. Novel molecular methods were developed providing detection ability for SMG, an emerging opportunistic pathogen.

Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples ( n = 162) from retail market from Ribeirão Preto, São Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L. monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTE™ QPCR SYBR ® Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method ( p < 0.05). Moreover, RTi-PCR was fast and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method.

Parachlamydia acanthamoebaeInfection and Abortion in Small Ruminants

<i>Parachlamydia acanthamoebae</i>Infection and Abortion in Small Ruminants

Prospective Clinical Evaluation of the Accuracy of 16S rRNA Real-Time PCR Assay for the Diagnosis of Melioidosis

The accuracy of a Burkholderia pseudomallei 16s rRNA real-time PCR assay was evaluated against culture for the diagnosis of melioidosis in Thailand. A total of 846 samples were obtained from 383 patients with suspected melioidosis. One or more specimens were PCR positive for 47 of 77 patients with culture-proven melioidosis (sensitivity 61.0%, 95% CI: 49.2-72.0%). PCR was negative for all 306 patients who were culture negative for B. pseudomallei (specificity 100%, 95% CI: 98.8-100%). Diagnostic sensitivity of PCR was 22.7% for patients who were culture positive for blood only, compared with 79.4% for patients who were culture positive for samples other than blood. The median (interquartile range) B. pseudomallei colony count in blood for 44 of 77 patients with positive blood cultures was 2.4 CFU/ml (0.2-13.5 CFU/ml); this may explain the low sensitivity of PCR for this specimen. The PCR assay described here is not sufficiently sensitive to replace culture in our clinical setting.