16S rDNA Polymerase Chain Reaction Research Articles

Spatial Distribution and Seasonal Variation of Antibiotic-Resistant Bacteria in an Urban River in Northeast China

As the largest freshwater river flowing through Harbin, the Songhua River is a standby water source. It is very important to know the species and distribution of antibiotic-resistant bacteria (ARB) in the river. In this study, five antibiotics were selected to screen and identify ARB in spring and autumn. The results showed that the concentration of cefotaxime-resistant bacteria was the highest, and the maximum concentration at S6 in spring was up to 1.40 × 104 CFU/mL. In spring and autumn, bacteria resistant to three antibiotics were screened at S1 of the Songhua River, and bacteria resistant to five antibiotics were screened at S6. No multiple antibiotic-resistant bacteria (MARB) were screened in the other four sites in autumn, while MARB were screened in the other three samples except S2 in spring. In all sample areas in spring and autumn, the probability of screening MARB at S1 and S6 was the highest, reaching 100%. The identification results of 16S rDNA polymerase chain reaction (PCR) products of ARB showed that a total of 51 ARB strains from 15 bacterial genera were screened in the Songhua River, of which 20 ARB strains were from Pseudomonas. Among the 15 bacterial genera, bacteria from 8 bacterial genera have pathogenicity. The results of this study revealed the concentration, spatial distribution, and seasonal variation of culturable ARB in the Songhua River, providing data support for the remediation of antibiotic resistance gene (ARG) pollution in the river.

Effects of orthodontic treatment on porphyromonas gingivalis, gingipains and gingival inflammation

Purpose: To assess the effects of orthodontic treatment on the changes of Porphyromonas gingivalis ( P.g) and gingipains as well as gingival inflammation. Materials and methods: A total of 105 patients who had healthy gingivae and were treated as research subjects, the gingival crevicular fluid of whom was all collected before treatment and in the 1st, 2nd, 3rd, and 6th months after wearing an appliance. Then, the 16S ribosomal deoxyribonucleic acid (rDNA) polymerase chain reaction (PCR) technique was employed to detect the levels of P.g, Lys-gingipain (Kgp) and Arg-gingipain (Rgp) in the specimens. It was followed by a statistical analysis of the data via the SPSS 23.0 software package. Results: In P.g detection, the 16S rDNA PCR technique presented specific advantages and high sensitivity. The positive detection rate of P.g in the 1st, 2nd and 3rd month of treatment ( p < .05), that of Kgp in P.g-positive patients in the 2nd and 3rd months of treatment ( p < .05), and that of Rgp in P.g-positive patients in the 2nd and 3rd months of treatment ( p < .05) were higher than the pre-treatment results. Compared with gingival index (GI)-0 patients, GI-1 patients exhibited higher positive detection rates of P.g and Kgp and Rgp, which were further found to be positively correlated with the treatment duration and GI ( p < .05). Conclusion: In the early stage of orthodontic treatment, the levels of P.g, Kgp and Rgp rise after wearing the appliance, inducing gingival inflammation.

Aeromonas de tambaqui de cultivo do norte do Brasil: identificação molecular e potencial patogênico

ABSTRACT: The aim of this study was to molecularly identify different species of Aeromonas isolated from farmed tambaqui (Colossoma macropomum) from North Brazil, and evaluate their pathogenic potential by the presence of virulence genes. From the extraction of bacterial DNA, PCR (polymerase chain reaction) of the primers 16S rDNA, aerA (cytolytic enterotoxin), ast (cytotoxic enterotoxin) and act (cytotoxic enterotoxin) were performed. Of 24 isolates evaluated, eight amplified the ast gene, one amplified the act gene, but the areA gene was not amplified in any isolate. Sequencing of the 16S rRNA primer revealed a predominance of Aeromonas jandaei specie (92%). Aeromonas taiwanensis (4%), for the first time isolated from fish in Brazil, and Aeromonas hydrophila (4%) each appeared as just one isolate. Results showed that 32% of Aeromonas isolated from farmed tambaqui have considerable pathogenic potential for systemic damage, since the selected PCR primers are encoding the most common virulence genes in Aeromonas with high pathogenic intensity.

The influence of primer choice on archaeal phylogenetic analyses based on 16S rRNA gene PCR.

Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.

Rapid diagnosis of intra-amniotic infection using nanopore-based sequencing.

Early diagnosis and treatment of intra-amniotic infection is crucial. Rapid pathogen identification allows for a definite diagnosis and enables proper management. We determined whether the 16S amplicon sequencing performed by a nanopore sequencing technique make possible rapid bacterial identification at the species level in intra-amniotic infection. Five cases of confirmed intra-amniotic infection, determined by either cultivation or 16S rDNA polymerase chain reaction (PCR) Sanger sequencing, and 10 cases of women who underwent mid-trimester genetic amniocentesis were included. DNA was extracted from amniotic fluid and PCR was performed on the full-length 16S rDNA. Nanopore sequencing was performed. The results derived from nanopore sequencing were compared with those derived from cultivation and Sanger sequencing methods. Bacteria were successfully detected from amniotic fluid using nanopore sequencing in all cases of intra-amniotic infection. Nanopore sequencing identified additional bacterial species and polymicrobial infections. All patients who underwent a mid-trimester amniocentesis had negative cultures, negative 16S PCR Sanger sequencing and nanopore sequencing. Identification of the microorganisms using nanopore sequencing technique at the bacterial species level was achieved within 5-9h from DNA extraction. This is the first study demonstrating that the nanopore sequencing technique is capable of rapid diagnosis of intra-amniotic infection using fresh amniotic fluid samples.

Cerebrospinal fluid and plasma procalcitonin for the diagnosis of neonatal bacterial meningitis.

There is a paucity of data on cerebrospinal fluid (CSF) procalcitonin (PCT) to diagnose neonatal meningitis. We evaluated CSF PCT to diagnose bacterial meningitis among neonates with suspected sepsis. Neonates undergoing lumbar puncture (LP) as part of sepsis workup were included. CSF PCT, plasma PCT, CSF:plasma PCT ratio and CSF cytochemistry. Reference Standards: 'Definite meningitis' defined by positive CSF culture and/or gram stain and/or broad-based primer 16S rDNA polymerase chain reaction. 'Definite or probable' meningitis is defined as definite meningitis or abnormal cytochemistry. Of 216 eligible neonates, 18 had 'definite meningitis' and 37 'definite or probable meningitis'. Median (Q1 , Q3 ) CSF PCT level was significantly higher in 'definite meningitis' compared to 'no definite meningitis' (0.429 (0.123, 1.300) vs. 0.181 (0.119, 0.286) ng/mL respectively, P=0.028). Likewise, it was significantly higher in 'definite or probable meningitis' compared to no meningitis (0.245 (0.136, 0.675) vs. 0.170 (0.116, 0.28), P=0.01). The area under the receiver operator characteristics curve of CSF PCT level for definite meningitis was 0.656 and for 'definite or probable meningitis' 0.635. Paired comparisons of area under the receiver operator characteristics curve of CSF PCT with the other index tests showed no significant differences. Based on a priori cut-off of 0.2ng/mL, CSF PCT level had a sensitivity (95% confidence interval) of 67% (50, 80), specificity 58% (54, 61), LR+ 1.6 (1.1, 2.0) and LR- 0.6 (0.3, 0.9). Higher values of CSF PCT are associated with neonatal bacterial meningitis. However, the diagnostic performance of CSF PCT is modest and not significantly different from standard tests.

A case of a renal abscess caused by Salmonella bareilly in a previously healthy boy

BackgroundRenal abscesses are relatively uncommon in children, and usually due to Gram-negative rods or Staphylococcus aureus, whereas abscesses caused by Salmonella are very rare.Case presentationWe present the case of a previously healthy 10-year-old boy who had a renal abscess due to Salmonella bareilly. He responded well to treatment with antibiotics, and computed tomography (CT)-guided drainage of the abscess. His blood, urine and abscess aspirate cultures were sterile, but a broad-range 16S rDNA polymerase chain reaction (PCR) assay of the aspirate followed by analysis of four Salmonella genes (fliC, fliD, sopE2, and spaO) identified S. bareilly as the causative agent.ConclusionTo the best of our knowledge, this is the first report of renal abscess caused by S. bareilly.

An Infected Bicuspid Aortic Valve, an Anomalous Coronary Artery, and a Dog-Bitten Postman

Risk factors for infective endocarditis (IE) include congenital heart defects, poor dentition, immunosuppression, or recent instrumentation. The occupational hazard of a dog bite, combined with bicuspid aortic valve (BAV) led to IE. 16S ribosomal DNA was able to pinpoint the causative organism. A healthy 33-year-old postman presented in profound heart failure and sepsis due to aortic regurgitation and an aortic root abscess. He underwent emergency aortic valve replacement and was found to have a BAV and anomalous right coronary artery. Blood cultures remained negative. 16S ribosomal DNA polymerase chain reaction (PCR) revealed the causative organism was Capnocytophaga canimorsus. On review, he recalled receiving a dog bite followed by a febrile illness a few days later. Congenital BAVs may become infected by seemingly innocuous injuries. 16S rDNA PCR is a more sensitive and specific diagnostic test than culture. This case demonstrates its utility in providing appropriate antimicrobial management for IE.

O11 Neisseria Meningitidis as a cause of isolated bilateral polyarticular native knee joint septic arthritis

O11 <i>Neisseria Meningitidis</i> as a cause of isolated bilateral polyarticular native knee joint septic arthritis

Effect of Horticultural Mineral Oil on Huanglongbing Transmission by Diaphorina citri Kuwayama (Hemiptera: Psyllidae) Population in a Commercial Citrus Orchard in Sarawak, Malaysia, Northern Borneo.

Simple SummaryIncrease in abundance of the vector Diaphorina citri Kuwayama population is mostly influenced by the cyclic flushing of citrus. Relative degree of aggregation index for D. citri adults increased during periods of cyclic production of new flush as females aggregated to lay eggs, and the adult migration and dispersal was influenced by flushing cycles. Horticultural mineral oil (HMO) can reduce the spread of HLB as HMO produced significantly lower level of 11.43% of diseased citrus trees compared to 42.20% in untreated control plots.Diaphorina citri Kuwayama transmits a destructive citrus disease caused by a fastidious bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) designated as Huanglongbing (HLB) which posed a risk of detrimental threat to the Malaysian citrus industry. All D. citri life stages show a lumped habit on young flushes and its population fluctuations was closely related to accessibility of young flushes. The study aimed to investigate if the appearance of young flush shoots on citrus influences ACP population fluctuation and if horticultural mineral oil (HMO) could reduce spread of HLB transmission by ACP in a commercial healthy orchard. Field research was carried out from 1 April 2011 to 1 December 2014 in a 2-year-old 1 ha citrus farm that consisted of 200 PCR-certified disease-free grafted non-bearing honey tangerine (Citrus reticulata L.) in southwestern Sarawak, Malaysia. The experiment had two treatments namely control (unsprayed) and nC24 HMO with four replications arranged in a simple randomized block design. ACP eggs, nymphs, and adults per flush shoot was assessed and HLB incidence was monitored for visual inspection of the citrus trees for the current existence of usual signs of characteristic symptoms of HLB such as yellowing shoots, leaf mottling, and corky or enlarged veins on leaves. HLB-specific primer was employed in 16S rDNA polymerase chain reaction to detect the CLas gene in diseased trees. Increase in abundance of D. citri is mainly affected by the citrus flushing cycles and their life stages are completed on these flush shoots. Relative degree of aggregation index for D. citri adults increased during periods of cyclic production of new flush. HMO-treated plots produced a significantly lower percentage up to 11.43% of diseased trees against 42.20% in untreated control plots. HMO is effective against D. citri and recommended to be incorporated in the IPM program to prevent infection and reduce the spread of HLB.

Antibiotic resistance pattern of Pseudomonas spp. from patients in a tertiary hospital in South-West Nigeria.

Pseudomonads constitute critical agents of opportunistic infections in hospital settings particularly in immunocompromised patients and Pseudomonas aeruginosa is a major flagship member of these infectious agents. This study assessed the distribution of Pseudomonas spp. associated with infections in patients and their antibiotic resistance patterns as part of an antibiotic stewardship intervention program and resistance surveillance. One hundred and fifty Pseudomonas spp. from different clinical specimens were obtained from the Obafemi Awolowo University Teaching Hospitals Complex Ile-Ife. Culture was carried out on MacConkey and blood agar while phenotypic characterization was done by Gram staining, oxidase, and catalase test. Species identification was done using MICROBACTTM 24E bacterial identification kit and confirmed by 16S rDNA polymerase chain reaction (PCR) assay. Antibiotic susceptibility testing to eight antibiotics in four classes was done. Pseudomonas aeruginosa was the most frequently occurring species (96.0%); P. putida (2.67%) and P. fluorescens (0.67%) were also identified as well as an isolate of Burkholderia pseudomallei (0.67%). The highest resistance rate among isolates was observed towards gentamicin (35.4%); piperacillin/tazobactam was the most active antibiotic. Multidrug-resistant (MDR) strains constituted 12.8% of the isolates and most MDR strains also displayed a high multiple antibiotic resistance index (MAR). Pseudomonas aeruginosa is emerging as a highly MDR pathogen in our hospital setting. This calls for the establishment of a surveillance system and antimicrobial stewardship programme in place. Furthermore, we propose a review of the current antibiotics prescription policy, and infection control programmes (ICPs) if we must control the spread of MDR-P. aeruginosa in this environment.

Long-Term Use of Oral Hygiene Products Containing Stannous and Fluoride Ions: Effect on Viable Salivary Bacteria.

The aim of this randomized, controlled clinical trial was to isolate and identify viable microorganisms in the saliva of study participants that continuously used a stannous and fluoride ion (F/Sn)-containing toothpaste and mouth rinse over a period of three years in comparison to a control group that used stannous ion free preparations (noF/Sn) over the same time period. Each group (F/Sn and noF/Sn) included 16 participants that used the respective oral hygiene products over a 36-month period. Stimulated saliva samples were collected at baseline (T0) and after 36 months (T1) from all participants for microbiological examination. The microbial composition of the samples was analyzed using culture technique, matrix-assisted laser desorption ionization–time of flight (MALDI–TOF) mass spectrometry, and 16S rDNA Polymerase Chain Reaction (PCR). There were only minor differences between both groups when comparing the absolute values of viable microbiota and bacterial composition. The treatment with F/Sn led to a slight decrease in disease-associated and a slight increase in health-associated bacteria. It was shown that the use of stannous ions had no negative effects on physiological oral microbiota even after prolonged use. In fact, a stabilizing effect of the oral hygiene products containing stannous ions on the health-associated oral microbiota could be expected.

Molecular identification and genetic characterization of tick-borne pathogens in sheep and goats at two farms in the central and southern regions of Malawi

Tick-borne diseases (TBDs) caused by pathogens belonging to the genera Anaplasma, Ehrlichia, Babesia and Theileria in small ruminants are widespread in the tropical and sub-tropical countries. The epidemiology of tick-borne pathogens (TBPs) in small ruminants is less understood compared to those infecting cattle in general. This study was carried out to investigate and characterize TBPs in sheep and goats using molecular tools. A total of 107 blood samples from sheep (n = 8) and goats (n = 99) were collected from animals that were apparently healthy from two farms in the central and the southern regions of Malawi. The V4 hypervariable region of the 18S ribosomal RNA gene (rDNA) and the V1 hypervariable region of the 16S rDNA polymerase chain reaction (PCR) assays were used for detection of tick-borne piroplasms and Anaplasmataceae, respectively. Almost the full-length 18S rDNA and the heat shock protein (groEL) gene sequences were used for genetic characterization of the piroplasms and Anaplasmataceae, respectively. The results showed that 76.6 % of the examined animals (n = 107) were positive for at least one TBP. The overall co-infection with at least two TBPs was observed in forty-eight animals (45 %). The detected TBPs were Anaplasma ovis (65 %), Ehrlichia ruminantium (4%), Ehrlichia canis (2%), Babesia strain closely related to Babesia gibsoni (1%), Theileria ovis (52 %), Theileria mutans (3%), Theileria separata (2%), Anaplasma sp. (1%) and Theileria sp. strain MSD-like (17 %). To the authors knowledge this is the first molecular study of TBPs in sheep and goats in Malawi. These results have therefore provided a significant milestone in the knowledge of occurrence of TBPs in sheep and goats in Malawi, which is prerequisite to proper diagnosis and control.

Evaluation of Procalcitonin, C-Reactive Protein, and Interleukin-6 as Early Markers for Diagnosis of Neonatal Sepsis.

Background Neonatal sepsis diagnosis is a challenge because of its nonspecific presentation together with low sensitivity of the time-consuming bacterial cultures. So, many sepsis markers, like C-reactive protein (CRP), procalcitonin (PCT), and interleukin-6 (IL-6), are emerging to improve its diagnosis. Aim This study was done to investigate the role of CRP, PCT, and IL-6 in promoting the early diagnosis of neonatal sepsis in an attempt to decrease morbidity and mortality. Methods This cross-sectional study was conducted on 50 neonates suspected with sepsis enrolled from the neonatal intensive care unit (NICU) of Zagazig University Hospitals, Egypt. Blood cultures for these neonates were done before starting antibiotics. Also, bacterial DNA was revealed from the blood by broad-range 16S rDNA polymerase chain reaction (PCR). Measurements of CRP using the immunoturbidimetry method, PCT using fluorescence immunoassay quantitative method, and IL-6 using commercially available ELISA kit were done to all enrolled neonates. Results Forty-one neonates with proved sepsis were found to be positive in blood culture and/or PCR for bacterial 16S rDNA. The most common isolated organisms were Klebsiella (61.3%), followed by E. coli (9.7%) and CONS (9.7%). We detected much significant higher levels of PCT, CRP, and IL-6 in the proved sepsis group than the suspected neonatal sepsis cases (p ≤ 0.001, 0.001, and 0.004, respectively). Serum PCT levels showed the highest sensitivity, specificity, PPV, NPV, and accuracy of 97.6%, 89%, 97%, 88.9%, and 96% than other studied sepsis markers. Conclusion PCT has satisfactory characteristics as a good marker than IL-6 and CRP for the diagnosis of neonatal sepsis.

Broad-range 16s rDNA PCR in synovial fluid does not improve the diagnostic performance of septic arthritis in native joints in adults: cross-sectional single-center study in 95 patients.

To evaluate the diagnostic performance of bacterial identification by broad-range polymerase chain reaction (PCR) of ribosomal DNA (rDNA) 16s (16S rDNA PCR) for the diagnosis of septic arthritis on native joints. Patients with acute mono or oligoarthritis who underwent synovial fluid puncture and prospective follow-up allowing definitive diagnosis (septic arthritis, crystal related disease, chronic inflammatory arthritis, undifferentiated arthritis) were recruited in this single-center study. Systematic analysis of synovial fluid included leukocytes count, search for urate and pyrophosphate crystals with polarized light microscopy, direct bacteriological examination (gram staining), bacteriological culture, and 16S rDNA PCR. Ninety-five patients were included, 34 of which (35.8%) had septic arthritis. Nineteen (20.0%) patients had received probabilistic antibiotic therapy prior to joint puncture. Gram + cocci infection accounted for 79.4% of septic arthritis, of which nearly half (47.1%) was caused by Staphylococcus aureus. Eight (23.5%) septic arthritis patients had a 16S rDNA PCR positive in the synovial fluid with an AUC of 0.618 (95% CI, 0.493-0.742), a sensitivity of 0.24 (95% CI, 0.12-0.40), and a specificity of 1.00 (95% CI 0.94-1.00). The diagnostic performance of 16S rDNA PCR was lower than that of direct examination (AUC at 0.691, CI 95%, 0.570-0.812), blood cultures (AUC at 0.727, CI 95%, 0.610-0.844), and culture (0.925, CI 95%, 0.856-0.994) for the diagnosis of septic arthritis. There was no difference in the positivity of 16S rDNA PCR according to previous exposure to antibiotics. 16s rDNA PCR in the synovial fluid does not improve the diagnostic performance of septic arthritis on native adult joints, particularly for Gram-positive cocci infections.

A pilot study of occupational exposure to pathogenic microorganisms through lip cosmetics among dental hygienists.

ObjectivesIn dental clinics, dental hygienists are exposed to aerosolized pathologic bacteria, which can be transmitted to the oral cavity via lip cosmetics. Accordingly, such contamination poses a consistent health risk among staffs. Our study examined the bacterial contamination of lip cosmetics used by dental hygienists while in a clinic setting.MethodsSixteen dental hygienists were surveyed regarding their job assignments and habits associated with lip cosmetic. Subsequently, microorganisms were analyzed in collected samples of the hygienists' lip cosmetics using colony‐forming unit (CFU) assays, 16s‐rDNA polymerase chain reaction, and DNA sequencing.ResultsNotably, 81.3% of the submitted lip cosmetic samples were contaminated, with bacterial CFUs ranging from undetectable to innumerable. Many samples (43.8%) exceeded the microbial limits of cosmetic contamination. Of the lip cosmetic used for more than 6 months, 60% exceeded the microbial limit. When wearing a mask every time, only one of the six samples exceeded the microbial limit. More frequent dental mask changing was associated with a lower likelihood that the cosmetic sample would exceed the microbial limit. No samples from hygienists who changed their masks four times a day exceeded the microbial limit, compared to 33.3% from hygienists who only changed the mask when it became wet. Most isolated bacteria were gram‐positive, facultative anaerobic, asporogenic, and opportunistically pathogenic, and the most prevalent species were Staphylococcus aureus, Streptococcus salivarius, and Staphylococcus epidermidis.ConclusionOur findings indicate that dental staff, including dental hygienists, should exercise more careful workplace habits, particularly with regard to infection control and cosmetic use.

Bacterial Profile of Middle Ear Fluid with Recurrent Acute Otitis Media Infection Using Culture Independent 16S rDNA Gene Sequencing

Background Recurrent otitis media is one of the common infections of childhood. The causative bacterial pathogen is one of the major risk factors of recurrent infection. With limited availability of Indian data, we performed this study to identify the bacterial pathogens. Materials and Methods Otitis media cases were diagnosed based on clinical criteria. Thirty-six middle ear fluid (MEF) samples were collected by tympanocentesis and cultured for pathogens. Seventy-eight per cent of the cases had three previous episodes of otitis media in the past 6 months; the remaining 22% had four episodes in the preceding 6 months. At the time of sample collection, all patients were on antibiotic coverage. Genomic DNA was extracted from MEF samples using Qiagen DNA mini Kit. The 16s rDNA polymerase chain reaction (PCR) and quantitative multiplex (qmPCR) for Streptococcus pneumoniae was performed on these samples. Streptococcus pneumoniae–positive samples were serotyped using PCRSeqTyping. Results None of the 36 samples showed growth by conventional culture. The 16s rDNA PCR identified bacterial pathogens in 33 samples. Four samples gave mixed reads. The organisms identified were Neisseria spp. other than Neisseria meningitidis (n = 7), N. meningitidis (n = 8), Lactococcus spp. (n = 5), S. pneumoniae (n = 2), Pseudomonas aeruginosa (n = 2), Haemophilus influenzae (n = 1), Salmonella infantis (n = 1), Staphylococcus epidermidis (n = 1), Staphylococcus auricularis (n = 1), and Streptococcus sp. (n = 1). The qmPCR detected the presence of S. pneumoniae in six samples. PCRSeqTyping was able to identify Serotype 19A in two samples positive for S. pneumoniae. Conclusion The study demonstrates the usefulness of 16s rDNA PCR protocol to identify the bacterial pathogens in MEF by a culture-independent method. Neisseria spp. were the predominant species identified followed by Lactococcus spp. and S. pneumoniae. Detection of pneumococci by 16s rDNA PCR correlated well with qmPCR-based detection and PCRSeqTyping.

High-throughput screening of bacterial pathogens in clinical specimens using 16S rDNA qPCR and fragment analysis

Molecular-based detection of bacterial pathogens directly from clinical specimens permits rapid initiation of effective antimicrobial treatment and adequate patient management. Broad-range polymerase chain reaction (PCR) amplification of the 16S rRNA gene (16S rDNA qPCR) is used in many diagnostic laboratories as a complement to cultural identification of bacterial pathogens. However, efforts for automation of 16S rDNA PCR workflows are needed in order to reduce turnaround times and to enhance reproducibility and standardization of the technique. In this retrospective method evaluation study, clinical specimens (N = 499) from patients with suspected bacterial infections were used to evaluate 2 diagnostic semiautomated workflows for rapid bacterial pathogen detection. The workflows included automated DNA extraction (QIASymphony), 16S rDNA qPCR, fragment or melting curve analysis, and amplicon sequencing. Our results support the use of the 16S rDNA qPCR and fragment analysis workflow as it enabled rapid and accurate identification of bacterial pathogens in clinical specimens.

No evidence of Chlamydia pneumoniae in the synovia of patients with osteoarthritis.

ObjectiveOsteoarthritis (OA) is a common cause of disability affecting millions of people of all ages worldwide. The pathogenesis involves an inflammatory component, but the cause of the inflammation remains incompletely understood. The intracellular bacteria Chlamydia trachomatis and C. pneumoniae have been demonstrated in patients with reactive arthritis. Both of these microorganisms can cause chronic and persistent infections, with C. trachomatis being the most common cause of reactive arthritis. This study was performed to investigate the presence of C. pneumoniae in a large number of patients with primary OA.MethodsThe study included 75 patients who underwent total knee arthroplasty. During surgery, a synovial biopsy was performed and synovial fluid drawn. Real-time polymerase chain reaction (PCR) of C. pneumoniae was run on all patients, and real-time PCR of bacterial 16S rDNA was conducted on 30 of the 75 patients to screen for the presence of other bacteria.ResultsReal-time PCR showed no evidence of the presence of C. pneumoniae in the patients’ specimens, nor were other bacteria detected.ConclusionsAlthough an inflammatory component is part of the pathogenesis of OA, we found no evidence indicating that C. pneumoniae is a stimulator of that inflammation.

Characterization of moderately thermostable α-amylase-producing Bacillus licheniformis from decaying potatoes and sweet potatoes

Bacillus licheniformis is an endospore-forming bacterium that is commonly present in soil. The aim of the present study was to isolate and characterize local strains of α-amylase producing B. licheniformis. Soil samples were collected from the decaying surfaces of potatoes and sweet potatoes. The samples were identified by Gram staining, spore staining, and motility testing under aerobic conditions. Twenty-three isolates were found to be from the Bacillus genus and six of those (26%) were found to be B. licheniformis. Two representative samples were run on API 20E and API 50 CH biochemical kits, and 16S rDNA polymerase chain reaction. The isolates were confirmed to be B. licheniformis by the API-web program and molecular detection. Partially purified α-amylase was characterized to determine the effect of the incubation period, temperature tolerance, and pH stability. The activity peaked at 740 mU/mL after 42 h of culturing. The relative activity reached a maximum at 55 °C and a pH of 8.0. The decaying surfaces of potatoes and sweet potatoes are promising sources of α-amylase-producing strains of B. licheniformis that can tolerate both a high temperature and drastic pH level.