A novel method for determining viable bacteria from a mixture of dead and viable bacteria: Delayed real-time PCR (DR-PCR) method.

Aspiration pneumonia can occur in perioperative and older patients, and various oral care methods have been used to prevent it. To validate the effective oral care methods, measuring bacterial counts before and after oral care is necessary. However, isolating and quantifying viable bacteria from those that are inactivated by agents used in oral care is not possible. In this study, we developed a novel method, Delayed real-time PCR (DR-PCR), that can quantify only viable bacteria from mixed samples of viable and dead bacteria. This method takes advantage of the fact that dead bacteria do not grow but viable bacteria do. When the samples were incubated in a liquid medium for 4 hours, the higher the percentage of viable bacteria, the higher the rate of increase in the number of bacteria. This method showed that povidone­iodine mouthwashing reduced the number of viable bacteria to approximately 1/4 of that before mouthwashing. Although DR-PCR is slightly more time consuming than real-time PCR, it is effective for studying changes in bacterial counts before and after oral care.

Pilot clinical trial: propidium monoazide PCR quantifies reduction of the viable bacterial load after antiseptic preparation of canine oral mucosa.

OBJECTIVE: A pilot clinical study to evaluate the use of propidium monoazide PCR (PMA-PCR) in quantifying a reduction of bacterial load after antiseptic use on the canine oral mucosa and skin, comparison of quantitative PCR (qPCR) to PMA-PCR, and comparison of patterns seen between PCR methods and bacterial culture. ANIMALS: Client-owned dogs (n = 10) undergoing general anesthesia and intravenous catheter placement. PROCEDURES: The oral mucosa and antebrachial skin of each dog underwent swabs for culture, qPCR, and PMA-PCR before and after antiseptic preparation of each site. Reduction in bacterial load between sampling times was evaluated for each quantification method. RESULTS: All testing methods found a significant decrease in bacterial load from oral mucosa after antiseptic preparation (culture P = .0020, qPCR P = .0039, PMA-PCR P = .0039). PMA-PCR had a significantly greater reduction of bacterial load after preparation than qPCR (P = .0494). Only culture detected a significant reduction after preparation of the skin (culture P = .0039, qPCR P = .3125, PMA-PCR P = .0703). CLINICAL RELEVANCE: PMA-PCR was able to quantify a reduction of bacterial load after antiseptic preparation of the high-bacterial load environment, with a pattern similar to that of culture, and was more specific than qPCR for detecting viable bacterial load. The results of this study support the use of PMA-PCR for antiseptic effectiveness studies performed on a high-bacterial load environment, such as canine oral mucosa.

Is skipping the definition of primary and secondary models possible? Prediction of Escherichia coli O157 growth by machine learning.

To predict bacterial population behavior in food, statistical models with specific function form have been applied in the field of predictive microbiology. Modelers need to consider the linear or non-linear relationship between the response and explanatory variables in the statistical modeling approach. In the present study, we focused on machine learning methods to skip definition of primary and secondary structure model. Support vector regression, extremely randomized trees regression, and Gaussian process regression were used to predict population growth of Escherichia coli O157 at 15 and 25 °C without defining the primary and secondary models. Furthermore, the support vector regression model was applied to predict small population of bacteria cells with probability theory. The model performance of the machine learning models were nearly equal to that of the current statistical models. Machine learning models have a potential for predicting bacterial population behavior.

A multistep enrichment process with custom growth medium improves resuscitation of chlorine-stressed coliforms from secondary sewage effluents.

Resuscitation and detection of stressed total coliforms in chlorinated water samples is needed to assess and prevent health effects from adverse exposure. In this study, we report that the addition of a growth enhancer mix consisting of trehalose, sodium pyruvate, magnesium chloride, and 1× trace mineral supplement improved growth of microorganisms from chlorinated secondary effluent in the base medium with Colilert-18. Improving growth of chlorine stressed microorganisms from secondary effluent is crucial to decreased detection time from 18 to 8 h.

Rapid cell counting and viability detection method of Escherichia coli Nissle using image cytometry.

The improvement of cell enumeration methods for the counting of Escherichia coli (E. coli) is important as E. coli gains in popularity as a basis for biopharmaceutical applications. In the biopharmaceutical industry, enumerating, characterizing, and dosing the accurate number of cells is imperative. In this work, we demonstrate the utilization of a chip-based image cytometer using a thin-gap, low volume counting chamber consumable to directly enumerate E. coli in bright field and fluorescence, and measure their viability using SYTOX™ Green. The total E. coli particles can be counted accurately label-free by adjusting the focus and targeting the linear range of the instrument. The E. coli are stained with SYTOX™ Green to count the membrane-compromised dead bacterial cells in the green fluorescence channel, while the total cells are counted using the bright field channel. Optimization of the system settings, image focus, cell counting range, and staining conditions have yielded a precise, rapid, and accurate E. coli cell enumeration and viability measurement.

Determinants associated with viable genital or rectal Chlamydia trachomatis bacterial load (FemCure).

BACKGROUND: Chlamydia trachomatis (CT) is routinely diagnosed by nucleic acid amplification tests (NAATs), which are unable to distinguish between nucleic acids from viable and non-viable CT organisms. OBJECTIVES: We applied our recently developed sensitive PCR (viability PCR) technique to measure viable bacterial CT load and explore associated determinants in 524 women attending Dutch sexual health centres (STI clinics), and who had genital or rectal CT. METHODS: We included women participating in the FemCure study (Netherlands, 2016-2017). At the enrolment visit (pre-treatment), 524 were NAAT positive (n=411 had genital and rectal CT, n=88 had genital CT only and n=25 had rectal CT only). We assessed viable rectal and viable genital load using V-PCR. We presented mean load (range 0 (non-viable) to 6.5 log10 CT/mL) and explored potential associations with urogenital symptoms (coital lower abdominal pain, coital blood loss, intermenstrual bleeding, altered vaginal discharge, painful or frequent micturition), rectal symptoms (discharge, pain, blood loss), other anatomical site infection and sociodemographics using multivariable regression analyses. RESULTS: In genital (n=499) CT NAAT-positive women, the mean viable load was 3.5 log10 CT/mL (SD 1.6). Genital viable load was independently associated with urogenital symptoms-especially altered vaginal discharge (Beta=0.35, p=0.012) and with concurrent rectal CT (aBeta=1.79; p<0.001). Urogenital symptoms were reported by 50.3% of women; their mean genital viable load was 3.6 log10 CT/mL (vs 3.3 in women without symptoms). Of 436 rectal CT NAAT-positive women, the mean rectal viable load was 2.2 log10 CT/mL (SD 2.0); rectal symptoms were reported by 2.5% (n=11) and not associated with rectal viable load. CONCLUSION: Among women diagnosed with CT in an outpatient clinical setting, viable genital CT load was higher in those reporting urogenital symptoms, but the difference was small. Viable genital load was substantially higher when women also had a concurrent rectal CT. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT02694497.

Predictive Value of Swab Cultures for Cryopreserved Flaps During Delayed Cranioplasties.

OBJECTIVE: To assess the predictive value of swab cultures of cryopreserved skull flaps during cranioplasties for surgical site infections (SSIs). METHODS: A retrospective review was conducted of consecutive patients who underwent delayed cranioplasties with cryopreserved autografts between 2009 and 2017. The results of cultures obtained from swabs and infected surgical sites were assessed. The accuracy, sensitivity, and specificity of swab cultures for SSIs were evaluated. RESULTS: The study included 422 patients categorized into two groups, swab and nonswab, depending on whether swab cultures were implemented during cranioplasties. The overall infection rate was 7.58%. No difference was seen in infection rates between groups. There were 18 false-positive and no true-positive swab culture results. All bacteria between swab cultures and SSI cultures were discordant. Meanwhile, there were 19 false-negative swab cultures. The results showed high specificity but low sensitivity for swab cultures to predict SSI occurrence and the pathogens. CONCLUSIONS: Owing to low accuracy and sensitivity, swab cultures of cryopreserved autografts should not be routinely performed during delayed cranioplasties.

Colorimetric determination of Listeria monocytogenes using aptamer and urease dual-labeled magnetic nanoparticles and cucurbit[7]uril-mediated supramolecular assembly of gold nanoparticle.

A host-guest colorimetric strategy is described for the detection of Listeria monocytogenes (L. monocytogenes). The optical probes were self-assembled based on the supramolecular interactions between the carbonyl groups of cucurbit[7]uril portals and gold nanoparticles (CB[7]-AuNPs). Aptamer and urease modified magnetic nanoparticles were used to specifically recognize and binding to L. monocytogenes, simultaneously hydrolyzing urea to produce ammonium ion (NH4+) that can reverse CB[7] induced AuNPs aggregation. In the presence of L. monocytogenes, the above-mentioned magnetic conjugates preferentially bind to the bacterial surface, which results in blocking the catalytic active sites, thus inhibiting the production of ammonium ions. The normalized absorbance ratio of A700 nm/A525 nm was proportional to the L. monocytogenes concentration ranging from 10 to 106 cfu·mL-1, and the visual determination can be done down to 10 cfu·mL-1. For spiked food samples analyzed without pre-enrichment, recoveries of 98.4% to 99.3% were achieved could be verified and RSD were less than 10%. This work may offer a broad prospect for sensitive and specific determination  of pathogens.

N, O-codoped hierarchical porous graphitic carbon for electrochemical immunosensing of Lactobacillus rhamnosus GG.

An ultrasensitive label-free electrochemical immunosensor was fabricated for quantitative detection of Lactobacillus rhamnosus GG (LGG). The N/O co-doped three-dimensional hierarchical porous graphitic (THPG) carbon was synthesized by a one-step synthesis of polyaniline hydrogel, and followed by simple carbonization and chemical activation procedures. Because of the unique structure design, the obtained THPG carbon networks possess an ultra-large specific surface area of 4859 m2 g-1 along with a class of highly graphitic carbons. The results offer an enormous surface area and excellent electrical conductivity for label-free electrochemical immunosensing of probiotic L. rhamnosus strain. Under optimal conditions, the immunosensor showed a good linear relationship between peak current and concentration of LGG (R2 = 0.9976), with a detection limit of 2 CFU mL-1. Furthermore, this label-free immunosensor also shows good specificity, long-term stability, and reliability, and could be applied to detect probiotic LGG in dairy products and drinks with satisfactory results. The present protocol was shown to be quite promising for practical screening and functional evaluation of probiotic products containing LGG. A ultrasensitive label-free electrochemical immunosensor based on THPG carbon was fabricated for detection of Lactobacillus rhamnosus GG.

Mouse pneumonia model by Acinetobacter baumannii multidrug resistant strains: Comparison between intranasal inoculation, intratracheal instillation and oropharyngeal aspiration techniques.

Infectious pneumonia induced by multidrug resistant (MDR) Acinetobacter baumannii strains is among the most common and deadly forms of healthcare acquired infections. Over the years, different strategies have been put in place to increase host susceptibility to MDR A. baumannii, since only a self-limiting pneumonia with no or limited local bacterial replication was frequently obtained in mouse models. Direct instillation into the trachea or intranasal inoculation of the bacterial suspension are the techniques used to induce the infection in most of the preclinical models of pneumonia developed to date. More recently, the oropharyngeal aspiration procedure has been widely described in the literature for a variety of purposes including pathogens administration. Aim of this study was to compare the oropharyngeal aspiration technique to the intranasal inoculation and intratracheal instillation in the ability of inducing a consistent lung infection with two MDR A. baumannii clinical isolates in immunocompromised mice. Moreover, pneumonia obtained by bacteria administration with two out of three techniques, intratracheal and oropharyngeal, was characterised in terms of histopathology of pulmonary lesions, biomarkers of inflammation level and leukocytes cells infiltration extent after mice treatment with either vehicle or the antibiotic tigecycline. The data generated clearly showed that both strains were not able to colonize the lungs when inoculated by intranasal route. By contrast, the bacterial load in lungs of mice intratracheally or oropharyngeally infected significantly increased during 26 hours of monitoring, thus highlighting the ability of these strains to generate the infection when directly instilled into the lower respiratory airways. Furthermore, the intragroup variability of mice was significantly reduced with respect to those intranasally administered. Tigecycline was efficacious in lung bacterial load and cytokines release reduction. Findings were supported by semi-quantitative histopathological evaluation of the pulmonary lesions and by inflammatory biomarkers analysis. To conclude, both intratracheal instillation and oropharyngeal aspiration techniques showed to be suitable methods for inducing a robust and consistent pneumonia infection in mice when difficult MDR A. baumannii clinical isolates were used. Noteworthy, oropharyngeal aspiration not requiring specific technical skills and dedicated equipment, was proven to be a safer, easier and faster technique in comparison to the intratracheal instillation.

Bacillus spore enumeration using flow cytometry: A proof of concept for probiotic application.

Use of flow cytometry (FCM) for bacteria quantification is growing in the food industry. We report here a FCM method using a double-staining LDS751/SYTO24 for the quantification of probiotic Bacillus viable cells and its spores, with potential application for the control of commercial product specifications.

Measuring the absolute abundance of the microbiome by adding yeast containing 16S rRNA gene from a hyperthermophile.

High-throughput sequencing (HTS) of 16S rRNA gene amplicons provides compositional information regarding the microbial community, but not the absolute abundance of the bacteria. We aimed to develop a standardized method for quantifying the absolute abundance of bacteria in microbiome studies. To demonstrate the utility of our approach, we quantified the number of bacteria from the compositional data of the fecal and cecal microbiomes. The 16S rRNA gene of a hyperthermophile, Thermus aquaticus, was cloned into Pichia pastoris (yeast) genome, and an equivalent amount of the yeast was added to the stool and cecal samples of mice before DNA extraction. 16S rRNA gene library construction and HTS were performed after DNA extraction. The absolute abundances of bacteria were calculated using T. aquaticus reads. The average relative abundances of T. aquaticus in the five stool and five cecal samples were 0.95% and 0.33%, respectively, indicating that the number of bacteria in a cecum sample is 2.9 times higher than that in a stool sample. The method proposed for quantifying the absolute abundance of the bacterial population in this study is expected to overcome the limitation of showing only compositional data in most microbiome studies.

Evaluation of the Genmark ePlex® and QIAstat-Dx® respiratory pathogen panels in detecting bacterial targets in lower respiratory tract specimens.

BACKGROUND: The ePlex® and QIAstat-Dx® respiratory pathogen panels detect multiple respiratory pathogens, mainly viruses but also Legionella pneumophila, Mycoplasma pneumoniae and Bordetella pertussis. The assays have been marketed for use in nasopharyngeal swab specimens. For diagnosing bacterial pneumonia, lower respiratory tract (LRT) specimens are indicated. Aim of this study was to evaluate the performance of these syndromic panels for these three bacterial targets in samples from the LRT. Fifty-six specimens were collected from our repositories, five negative samples and fifty-one samples which had been previously tested positive with the routine diagnostic real-time PCR assays for Legionella spp. (N = 20), Bordetella spp. (N = 16) or M. pneumoniae (N = 15). RESULTS: The QIAstat-Dx Respiratory Panel V2 (RP) assay detected all of the L. pneumophila and B. pertussis positive samples but only 11/15 (73.3 %) of the M. pneumoniae targets. The ePlex Respiratory Pathogen Panel (RPP) assay detected 10/14 (71.4 %) of the L. pneumophila targets, 8/12 (66.7 %) of the B. pertussis positive samples and 13/15 (86.7 %) of the M. pneumoniae targets. CONCLUSIONS: No false-positive results were reported for all three bacterial pathogens by both assays. The clinical performance of both assays depended highly on the bacterial load in the sample and the type of specimen under investigation.

Measurement uncertainty evaluation and risk of false conformity assessment for microbial enumeration tests.

Microbial enumeration tests are widely used to assess the microbiological quality of non-sterile pharmaceutical products. Despite of all efforts to guarantee the reliability of microbial enumeration tests, there will always be an uncertainty associated with the measured values, which can lead to false conformity/non-conformity decisions. In this work, we evaluated the measurement uncertainty using a bottom-up approach and estimate the consumer's or producer's risk due to the measurement uncertainty. Three main sources of uncertainty were identified and quantified: dilution factor, plated volume, and microbial plate counts. The contribution of these sources of uncertainty depends on the measured value of microbial load in pharmaceutical products. The contribution of dilution factor and plated volume uncertainties increase with an increase of measured value, while the contribution of microbial plate count uncertainty decreases with an increase of measured value. The overall uncertainty values were expressed as uncertainty factors, which provide an asymmetric 95% level confidence level of microbial load in pharmaceutical products. In addition, the risk of false conformity/non-conformity decisions due to measurement uncertainty was assess using Monte Carlo method. When the measured value is close to the upper specification limit and/or the measurement uncertainty is large, the risk of false conformity/non-conformity decisions may be significantly high. Thus, we conclude that the use of uncertainty factor in the conformity/non-conformity assessment is important to guarantee the reliability of microbial enumeration test results and to support decision-making.

Replication Dynamics for Six Gram-Negative Bacterial Species during Bloodstream Infection.

Bloodstream infections (BSI) are a major public health burden due to high mortality rates and the cost of treatment. The impact of BSI is further compounded by a rise in antibiotic resistance among Gram-negative species associated with these infections. Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Enterobacter hormaechei, Citrobacter freundii, and Acinetobacter baumannii are all common causes of BSI, which can be recapitulated in a murine model. The objective of this study was to characterize infection kinetics and bacterial replication rates during bacteremia for these six pathogens to gain a better understanding of bacterial physiology during infection. Temporal observations of bacterial burdens of the tested species demonstrated varied abilities to establish colonization in the spleen, liver, or kidney. K. pneumoniae and S. marcescens expanded rapidly in the liver and kidney, respectively. Other organisms, such as C. freundii and E. hormaechei, were steadily cleared from all three target organs throughout the infection. In situ replication rates measured by whole-genome sequencing of bacterial DNA recovered from murine spleens demonstrated that each species was capable of sustained replication at 24 h postinfection, and several species demonstrated <60-min generation times. The relatively short generation times observed in the spleen were in contrast to an overall decrease in bacterial burden for some species, suggesting that the rate of immune-mediated clearance exceeded replication. Furthermore, bacterial generation times measured in the murine spleen approximated those measured during growth in human serum cultures. Together, these findings provide insight into the infection kinetics of six medically important species during bacteremia. IMPORTANCE Bloodstream infections are a global public health problem. The goal of this work was to determine the replication characteristics of Gram-negative bacterial species in the host following bloodstream infection. The number of bacteria in major organs is likely determined by a balance between replication rates and the ability of the host to clear bacteria. We selected a cohort of six species from three families that represent common causative agents of bloodstream infections in humans and determined their replication rates in a murine bacteremia model. We found that the bacteria grow rapidly in the spleen, demonstrating that they can obtain the necessary nutrients for growth in this environment. However, the overall number of bacteria decreased in most cases, suggesting that killing of bacteria outpaces their growth. Through a better understanding of how bacteria replicate during bloodstream infections, we aim to gain insight into future means of combating these infections.

Baseline Xpert MTB/RIF ct values predict sputum conversion during the intensive phase of anti-TB treatment in HIV infected patients in Kampala, Uganda: a retrospective study.

BACKGROUND: In resource-limited settings, sputum smear conversion is used to document treatment response. Many People living with HIV (PLHIV) are smear-negative at baseline. The Xpert MTB/RIF test can indirectly measure bacterial load through cycle threshold (ct) values. This study aimed to determine if baseline Xpert MTB/RIF could predict time to culture negativity in PLHIV with newly diagnosed TB. METHODS: A subset of 138 PLHIV from the 'SOUTH' study on outcomes related to TB and antiretroviral drug concentrations were included. Bacterial load was estimated by Mycobacterium Growth Indicator Tubes (MGIT) culture time-to-positivity (TTP) and Lowenstein Jensen (LJ) colony counts. Changes in TTP and colony counts were analyzed with Poisson Generalised Estimating Equations (GEE) and multilevel ordered logistic regression models, respectively, while time to culture negativity analysed with Cox proportional hazard models. ROC curves were used to explore the accuracy of the ct value in predicting culture negativity. RESULTS: A total of 81 patients (58.7%) were males, median age 34 (IQR 29  ̶ 40) years, median CD4 cell count of 180 (IQR 68  ̶ 345) cells/µL and 77.5% were ART naive. The median baseline ct value was 25.1 (IQR 21.0  ̶ 30.1). A unit Increase in the ct value was associated with a 5% (IRR = 1.05 95% CI 1.04  ̶ 1.06) and 3% (IRR = 1.03 95% CI 1.03  ̶ 1.04) increase in TTP at week 2 and 4 respectively. With LJ culture, a patient's colony grade was reduced by 0.86 times (0R = 0.86 95% CI 0.74  ̶ 0.97) at week 2 and 0.84 times (OR = 0.84 95% CI 0.79  ̶ 0.95 P = 0.002) at week 4 for every unit increase in the baseline ct value. There was a 3% higher likelihood of earlier conversion to negativity for every unit increase in the ct value. A ct cut point ≥28 best predicted culture negativity at week 4 with a sensitivity of 91. 7% & specificity 53.7% while a cut point ≥23 best predicted culture negativity at week 8. CONCLUSION: Baseline Xpert MTB/RIF ct values predict sputum conversion in PLHIV on anti-TB treatment. Surrogate biomarkers for sputum conversion in PLHIV are still a research priority.

Integrating programmable DNAzymes with electrical readout for rapid and culture-free bacterial detection using a handheld platform.

The detection and identification of bacteria currently rely on enrichment steps such as bacterial culture and nucleic acid amplification to increase the concentration of target analytes. These steps increase assay time, cost and complexity, making it difficult to realize a truly rapid point-of-care test. Here we report the development of an electrical assay that uses electroactive RNA-cleaving DNAzymes (e-RCDs) to identify specific bacterial targets and subsequently release a DNA barcode for transducing a signal onto an electrical chip. Integrating e-RCDs into a two-channel electrical chip with nanostructured electrodes provides the analytical sensitivity and specificity needed for clinical analysis. The e-RCD assay is capable of detecting 10 CFU (equivalent to 1,000 CFU ml-1) of Escherichia coli selectively from a panel containing multiple non-specific bacterial species. Clinical evaluation of this assay using 41 patient urine samples demonstrated a diagnostic sensitivity of 100% and specificity of 78% at an analysis time of less than one hour compared with the several hours needed for currently used culture-based methods.

Managing Contamination and Diverse Bacterial Loads in 16S rRNA Deep Sequencing of Clinical Samples: Implications of the Law of Small Numbers.

In this article, we investigate patterns of microbial DNA contamination in targeted 16S rRNA amplicon sequencing (16S deep sequencing) and demonstrate how this can be used to filter background bacterial DNA in diagnostic microbiology. We also investigate the importance of sequencing depth. We first determined the patterns of contamination by performing repeat 16S deep sequencing of negative and positive extraction controls. This process identified a few bacterial species dominating across all replicates but also a high intersample variability among low abundance contaminant species in replicates split before PCR amplification. Replicates split after PCR amplification yielded almost identical sequencing results. On the basis of these observations, we suggest using the abundance of the most dominant contaminant species to define a threshold in each clinical sample from where identifications with lower abundances possibly represent contamination. We evaluated this approach by sequencing of a diluted, staggered mock community and of bile samples from 41 patients with acute cholangitis and noninfectious bile duct stenosis. All clinical samples were sequenced twice using different sequencing depths. We were able to demonstrate the following: (i) The high intersample variability between sequencing replicates is caused by events occurring before or during the PCR amplification step. (ii) Knowledge about the most dominant contaminant species can be used to establish sample-specific cutoffs for reliable identifications. (iii) Below the level of the most abundant contaminant, it rapidly becomes very demanding to reliably discriminate between background and true findings. (iv) Adequate sequencing depth can be claimed only when the analysis also picks up background contamination. IMPORTANCE There has been a gradual increase in 16S deep sequencing studies on infectious disease materials. Management of bacterial DNA contamination is a major challenge in such diagnostics, particularly in low biomass samples. Reporting a contaminant species as a relevant pathogen may cause unnecessary antibiotic treatment or even falsely classify a noninfectious condition as a bacterial infection. Yet, there are few studies on how to filter contamination in clinical microbiology. Here, we demonstrate that sequencing of extraction controls will not reveal the full spectrum of contaminants that could occur in the associated clinical samples. Only the most abundant contaminant species were consistently detected, and we present how this can be used to set sample specific thresholds for reliable identifications. We believe this work can facilitate the implementation of 16S deep sequencing in diagnostic laboratories. The new data we provide on the patterns of microbial DNA contamination is also important for microbiome research.

Evaluation of a Novel Culture System for Rapid Pathogen Identification and Detection of Cephalosporin Resistance in Neonatal Gram-negative Sepsis at a Tertiary Referral Unit in Harare, Zimbabwe.

BACKGROUND: Neonatal sepsis accounts for a large proportion of neonatal deaths in sub-Saharan Africa. The lack of access to diagnostic testing and excessively long turnaround times to result contributes to delays in sepsis identification and initiation of appropriate treatment. This study aims to evaluate the novel InTrays COLOREX Screen and extended-spectrum beta-lactamase for rapid identification of bacterial pathogens causing sepsis and detection of resistance. METHODS: Neonates with suspected sepsis admitted to the Harare Central Hospital were prospectively enrolled. One blood culture was collected and incubated using the BacT/ALERT automated system. Positive blood cultures with potential pathogens identified by Gram stain were inoculated on the InTray COLOREX Screen and extended-spectrum beta-lactamase culture plates. RESULTS: A total of 216 neonates with suspected sepsis were recruited. Pathogens were isolated from blood cultures in 56 (25.9%) neonates of which 54 were Klebsiella pneumoniae. All K. pneumoniae were resistant to ceftriaxone and 53 (98%) were resistant to gentamicin. Sensitivity and specificity for ceftriaxone-resistant K. pneumoniae detection using InTrays were 100%. InTrays results were interpretable as early as 5-10 hours (median 7 hours, interquartile range 7-7) post blood culture positivity enabling rapid identification and notification of result and leading to a 60% reduction in time to result from blood culture collection. CONCLUSIONS: This study shows that the implementation of a novel culture method was feasible and reduced turnaround times for results by 60% compared with standard microbiologic techniques. An impact on patient outcomes and cost-effectiveness of this method needs to be demonstrated.

Fabrication of gold/silver nanodimer SERS probes for the simultaneous detection of Salmonella typhimurium and Staphylococcus aureus.

Salmonella typhimurium (S. typhimurium) and Staphylococcus aureus (S. aureus) are the two most important foodborne pathogens which can easily cause disease infections. Here, the aptamer-facilitated gold/silver nanodimer SERS probes were built for the simultaneous detection of the two bacteria with the help of magnetic separation enrichment. First, two nanodimer SERS signal probes and two magnetic capture probes each connected with the specific aptamer were fabricated. The distance between gold and silver nanoparticles in the dimer can amplify the Raman signal (Cy3 and Rox) at the junction but modified in the aptamer sequence. Then, after the addition of S. typhimurium and S. aureus, the sandwich-like composite structures "SERS signal probes-target-magnetic capture probes" formed because of the high affinity between aptamer sequences and their target bacteria. Under the optimal experimental conditions, the linear correlations between Raman intensity and the logarithm of the concentration of bacteria were y = 876.95x-67.84 (R2 = 0.9865) for S. typhimurium and y = 1280.43x-1752.6 (R2 = 0.9883) for S. aureus. The SERS detection showed the nanodimer probe had high selectivity. Besides, the recovery experiment in milk sample indicated good accuracy compared with the traditional plate counting method.