Traditional Microbiological Methods Research Articles

Analysis of Microbial Composition of Edible Insect Products Available for Human Consumption within the United States Using Traditional Microbiological Methods and Whole Genome Sequencing

Edible insects offer a promising protein source for humans, but their food safety risks have not been previously investigated within the United States. Therefore, the aim of this study was to investigate the microbial content of processed edible insect products. A total of eight different types of edible insect products, including diving beetles, silkworms, grasshoppers, Jamaican crickets, mealworms, mole crickets, whole roasted crickets, and 100% pure cricket powder, were purchased from a large online retailer for the analysis. All the products were purchased in August 2022 and examined between August 2022 and November 2022. Traditional microbiological methods were employed to determine microbial counts for each product type using three replicates (total number of samples = 24). This included assessing aerobic bacterial spore, lactic acid bacteria, Enterobacteriaceae, total viable counts, and the presence of Salmonella. Additionally, whole genome sequencing was employed to further characterize selected colonies (n = 96). Microbial counts data were statistically analyzed using one-way ANOVA, while sequence data were taxonomically classified using Sepia.Bacilluscereusgroup isolates underwent additional characterization with Btyper3. Product type significantly influenced total viable counts, bacterial spore counts, and lactic acid bacteria counts (P = 0.00391, P = 0.0065, and P < 0.001, respectively), with counts ranging from < 1.70 to 6.01 Log10 CFU/g, <1.70 to 5.25 Log10 CFU/g, and < 1.70 to 4.86 Log10 CFU/g, respectively. Enterobacteriaceae were only detected in mole crickets (<2.30 Log10 CFU/g) and house cricket powder (<2.15 Log10 CFU/g). All samples were negative for Salmonella. Whole genome sequencing revealed the presence of 12 different bacterial genera among the analyzed isolates, with a majority belonging to the Bacillus genus. Some of the isolates of Bacillus cereus group were identified as biovar Emeticus. Overall, although edible insects offer a promising food alternative, the presence of Bacillus cereus group in some products could raise concerns regarding food safety.

Combined Mutual Learning Net for Raman Spectral Microbial Strain Identification.

Infectious diseases pose a significant threat to global health, yet traditional microbiological identification methods suffer from drawbacks, such as high costs and long processing times. Raman spectroscopy, a label-free and noninvasive technique, provides rich chemical information and has tremendous potential in fast microbial diagnoses. Here, we propose a novel Combined Mutual Learning Net that precisely identifies microbial subspecies. It demonstrated an average identification accuracy of 87.96% in an open-access data set with thirty microbial strains, representing a 5.76% improvement. 50% of the microbial subspecies accuracies were elevated by 1% to 46%, especially for E. coli 2 improved from 31% to 77%. Furthermore, it achieved a remarkable subspecies accuracy of 92.4% in the custom-built fiber-optical tweezers Raman spectroscopy system, which collects Raman spectra at a single-cell level. This advancement demonstrates the effectiveness of this method in microbial subspecies identification, offering a promising solution for microbiology diagnosis.

Comparative Molecular Virulence Typing and Antibiotic Resistance of Campylobacter Species at the Human-Animal-Environment Interface.

This study holds significant importance due to its focus on Campylobacter, the leading bacterial cause of gastroenteritis worldwide, responsible for ∼96 million cases each year. By investigating the prevalence of both Campylobacter jejuni and Campylobacter coli in humans, animals, and the environment, this research sheds light on the broader impact of these pathogens, which can harm both human and animal populations. Traditional microbiological methods were implemented followed by optimized multiplex polymerase chain reaction targeting 16S rDNA and virulence gene markers by using specific primers. The findings revealed that a total of 219 Campylobacter isolates were recovered from 528 collected specimens from human, animal, and environmental sources. Campylobacter species showed a prevalence of 41.5%, with C. jejuni accounting for 53% and C. coli for 47%. Antimicrobial resistance rates were high, with tetracycline at 89%, ceftriaxone at 75%, cefotaxime at 70%, erythromycin at 69%, nalidixic acid at 54%, ciprofloxacin at 39%, and gentamicin at 23%. Commonly prevalent virulence-associated genes observed in the Campylobacter were cadF at 93%, flaA at 91%, cdtB at 88%, cheY at 86%, sodB at 78%, and iamA at 32%. The study confirmed multidrug-resistant Campylobacter prevalence at the human-animal-environment interface, harboring virulence-associated genes with potential harm to humans. Data analysis showed a nonsignificant (p ≥ 0.05) correlation between virulence genes and antibiotic susceptibility. To effectively manage Campylobacter infections, a multifaceted strategy incorporating preventative interventions at different levels is required. This strategy should take into account practicability, effectiveness, and sustainability while strengthening surveillance systems and addressing the economics of disease prevention.

Multicenter evaluation of BioCode GPP for syndromic molecular detection of gastrointestinal pathogens from stool specimens.

This study highlights performance of a novel technology for timely and accurate detection and differentiation of 17 common bacterial, viral, and protozoan causes of gastroenteritis. Utilizing molecular tests such as the BioCode Gastrointestinal Pathogen Panel may improve the detection of gastrointestinal pathogens and provide actionable results, particularly for patient populations at most risk.

Microbial water quality investigation through flow cytometry fingerprinting: from source to tap

Abstract Ensuring the quality of treated drinking water is crucial for preventing potential health impacts, regulatory fines, and reputation damage. Traditional culture-based microbiological methods often fail to capture the heterogeneity of the bacterial communities in drinking water. This study employed daily interstage monitoring and flow cytometry (FCM) analysis over a period of one year to investigate the dynamics of water treatment processes and service reservoirs. The objective of this study was to test the utility of FCM fingerprints for aiding in microbial event detection. We found that the chlorine concentration contact time was pivotal for microbial log reduction across the treatment works. FCM fingerprints exhibited significant deviations during operational events, such as process interruptions, but did not correlate with the presence of bacterial indicator organisms in the distributed and tap water. Furthermore, the diversity of bacterial fingerprints, quantified by the Bray–Curtis dissimilarity index, served as an indicator for identifying potentially poor microbial water quality. In chlorinated waters with low cell counts, the background signal shows potential as a metric to differentiate between different water sources, thereby offering the possibility to characterize breakthrough events in these circumstances that challenge most other microbial analytical methods. Interestingly, groundwater from simpler treatment works showed a higher occurrence of bacterial indicators, whereas surface water works had a lower incidence. These findings underpin the importance of appropriate disinfection even for “low-risk” source waters and the added value that the statistical interpretation of FCM data can offer objective decision making.

Comparative study on biochemical and molecular identification approaches of Lactobacillus species

Manufacturers’ desire to sell “healthy” food in response to the consumers’ desire to lead a healthy lifestyle has increased the use of probiotics during the past few decades. Probiotics are used in dairy products, as well as non-dairy items as a starter culture, encompassing a wide range of goods. Numerous phenotyping, physical characterisation, and genotyping techniques have been developed to identify probiotic lactobacilli to ensure quality management. These techniques are frequently precise enough to categorise probiotic strains by genus and species. Traditional microbiological methods were initially employed for genus and species identification. However, due to their numerous shortcomings as the probiotic ability is often strain-dependent, and that there is no way to differentiate between strains using simple microbiological techniques, new methods that are mostly based on the examination of nucleic acids have been developed. Therefore, the objective of the present review was to provide critical assessment on existing methods for identifying members of the genus Lactobacillus, together with newly discovered approaches. The present review aimed to give the most recent information on the scientific techniques used to measure and describe the possible probiotic properties of microorganisms. It will also emphasise molecular and non-molecular tools. Most of these tools are based on 16S ribosomal DNA sequencing, and employ PCR techniques.

Microbiological Skip Test: Fundamentals and Evaluation of Water Activity in Pharmaceutical Ingredients

Quality control is essential for Good Manufacturing Practices in the pharmaceutical industry. The time and costs associated with microbiological quality control are well known. It is proposed the reduction of traditional microbiological methods of counting the total number of microorganisms and research of specific pathogens using the water activity (Aw) method, a practice known as the skip test. The raw material selected as a prototype for this study met the criteria for the feasibility of reductions in analyses having synthetic origins, solid physical state, low or high pH in aqueous solution, and Aw results below 0.75 at a temperature of 25°C, and absence of nonconformity records. The risk management process was implemented using risk analysis with the mode, effect, criticality, failure analysis tool, and opening change control with the replacement of microbiological standard by Aw in 20 sequential batches after complete analysis of the first batch received in the year. It was possible to certify the microbiological quality of the selected pharmaceutical ingredients using the AW results, thereby confirming the feasibility of the skip test.

ANTIMICROBIAL ACTIVITY OF SOIL BACILLI AGAINST PHYTOPATHOGENIC MICROORGANISMS ISOLATED FROM AFFECTED CEREAL PLANTS

Aim of this study was to investigate the antagonistic effect of bacilli isolated from the surface of plants and the rhizosphere zone of the soil against pathogens of barley and wheat diseases. Materials and methods. The material for the study was affected barley and wheat samples used to isolate phytopathogenic microorganisms, as well as samples of healthy plants and the rhizosphere soil zone used to isolate bacteria of the genus Bacillus. Manipulations for the isolation and investigation the biological properties of both phytopathogens and bacilli were carried out by traditional microbiological methods. The antagonistic activity of the isolated strains of bacilli against phytopathogens was studied by the block method. Results. From samples of affected wheat and barley were isolated microorganisms identified on the basis of the studied biological properties as Fusarium oxysporum, Sclerotinia sclerotiorum, Xanthomonas arboricola and Pectobacterium carotovorum. The largest proportion of strains was represented by the micromycete Fusarium oxysporum – 53.9%. From samples of the rhizosphere soil zone and leaf-stem mass of healthy wheat and barley plants, 86 strains of bacteria of the genus Bacillus were isolated by microbiological methods. Antagonistic activity was inherent in 54 strains of bacilli against 25% of phytopathogens. All pathogenic microorganisms were inhibited with different intensity by 14 strains of bacilli. Conclusion. The most active bacillus strains (Bacillus spp. 6, 9, 13, 21, 50) were selected for further research.

Intractable complicated course of tricuspid valve infective endocarditis due to non-compliance of treatment with clinical guidelines with a decisive role of molecular biological study in etiological diagnosis: a case report

A clinical observation of the treatment non-compliance consequences with clinical guidelines and principles of empirical therapy selection in a female patient with intravenous drug abuse, viral hepatitis C and HIV infection, with a history of a COVID-19 and the development of uncontrolled staphylococcal infective endocarditis (IE) of the tricuspid valve, complicated recurrence of early prosthetic IE is presented. Successful treatment was achieved only by a combination of tricuspid valve replacement and the appointment of etiotropic therapy for S. aureus (MSSA). The typical clinical scenario was not accompanied by the choice of adequate empirical antibiotic therapy, despite the high suspicion of association with MSSA, which determined the complicated course of IE. Only the polymerase chain reaction of the heart valve tissue played a key role in the etiological diagnosis. The use of valve tissue polymerase chain reaction in addition to traditional microbiological methods is a valuable diagnostic study.

Characterization of Bacterial Diversity in Aguamiel and Two Types of Pulque from the Zacatlán Region, México

Pulque is a traditional Mexican fermented beverage associated with numerous health benefits. Over time, there has been considerable interest in studying the bacterial diversity of pulque, and microbial characterization has been carried out using traditional microbiological and molecular methods. Therefore, the objective of this research was to characterize the microbiota of artisanal pulque obtained from the Zacatlán region in Puebla, México, by the means of Illumina sequencing, and to compare it with the microbial diversity of aguamiel (sap before fermentation), commercial pulque (supplemented with additives to increase its yield), and its seed (batch of pulque previously fermented). An analysis of the Shannon index showed medium diversity for both aguamiel and pulque samples (score &gt; 2), while the Chao 1 index exhibited a non-significant difference between them. On the other hand, a principal components analysis confirmed the role of the seed as an essential inoculum to define the microbial diversity of pulque, emphasizing the importance of its preservation as a quality standard during the elaboration process. In addition, results showed that the dominant phyla in artisanal and commercial pulque were Firmicutes and Bacteroidetes. As the fermentation process progressed, it was possible to observe an increase in the population of lactic acid bacteria (LAB) in both types of pulque compared to those detected in aguamiel. Of these, the species Lactobacillus, Leuconostoc, and Lactococcus represented almost 95% of the total LAB. Finally, even though the safety of pulque has been in question due to its non-aseptic manufacturing process, the present study confirmed that less than 1% of its microbiota corresponds to the genera with a pathogenic potential such as γ-proteobacteria (Enterobacter and Hafnia), which decreases as the fermentation process advances.

A New Duplex Recombinase Polymerase Amplification (D-RPA) Method for the Simultaneous and Rapid Detection of Shigella and Bacillus cereus in Food.

Shigella and Bacillus cereus are two common foodborne pathogens that cause intestinal diseases and seriously affect human life and health. Traditional microbiological culture methods are time-consuming and laborious, and polymerase chain reaction (PCR)-based methods rely on expensive thermal cyclers and lengthy reaction times. In this study, on the basis of the specific gene ipaH7 of Shigella and the virulence gene nheABC of B. cereus, a duplex detection system was established for the first time by using the recombinase polymerase amplification technique (D-RPA). After optimization, D-RPA could be effectively amplified at 42 °C for 25 min with excellent specificity, and the detection limits of D-RPA for Shigella and B. cereus in artificially contaminated samples were 2.7 × 101 and 5.2 × 102 CFU/mL, respectively. This study provides a certain research basis for multiple detection with RPA, an isothermal amplification technology. Furthermore, it lays a good foundation for high-throughput rapid detection of foodborne pathogens.

Phytochemical characterization, antibacterial, and anti-biofilm efficacy of Mangifera indica seed kernel: A preliminary study using in vitro and in silico approaches

Biofilm-formation is one of the most important virulent factors in the pathogenesis of urinary tract infections. Biofilm is a complex microbial population protected by exopolysaccharides (EPS), which presents with adaptive resistance against antimicrobials. In the current study, we aimed to evaluate the efficacy of a methanolic extract of Mangifera indica seed kernels (MISK) against uropathogenic bacteria collected from urine samples. In total, n = 21 uropathogenic Escherichia coli (UPEC) strains were identified from n = 30 urine samples. Among the n = 21 strains, n = 17 E. coli strains were biofilm-producers in different in vitro model systems (tube-based method, microtiter-plate based method, Congo Red Agar, EPS production). Uropathogens were identified by traditional microbiological methods. MISK is a potential natural source of phytochemicals such as carbohydrates, proteins, stearic and linoleic acids. In the present study, 50 μg/mL and 100 μg/mL of methanolic extract of MISK exhibited prominent antibacterial and anti-biofilm activity against the clinical UPEC strains. In addition, qualitative phytochemical characterization of methanolic extract of MISK showed the presence of bioactive compounds such as tannins, saponins, flavonoids, terpenoids, alkaloids, carbohydrates, polyphenols, and glycosides. Moreover, the characterization of the methanolic extract by UV–VIS, FTIR, and GC–MS analysis confirmed the presence of a phenolic profile, namely mangiferin (1,3,6,7-tetrahydroxyxanthone-C2 -β-D-glucoside). Furthermore, in silico analysis was also investigated to validate that mangiferin can bind to the specific active sites of CAMP LpxC in E. coli. Based on the collective findings of our in vitro and in silico analyses, mangiferin may be the principal compound responsible for the anti-bacterial and anti-biofilm effects of the extract.

[Basic competence for etiological diagnosis of lower respiratory tract infection after application of metagenomic next-generation sequencing].

In addition to clinical manifestations, medical history, and imaging, the diagnosis of low respiratory tract infection (LRTI) depends mainly on the ability of the clinical microbiology laboratory to detect the pathogens. However, conventional culture may be time-consuming, the sensitivity of microscopy is low, and nucleic acid-based targeted tests (e.g., PCR) could only cover limited range of pathogens. The use of mNGS technology has improved the diagnostic rate of LRTI, but the conventional microbiology detection has been neglected to some extent. This review addressed the appropriate use of these methods with the aim of strengthening the ability of traditional microbiology methods in LRTI diagnosis after mNGS application.

Роль бактерий рода Desulfovibrio sp. в патологии человека

Bacteria of the genus Desulfovibrio sp. They are widespread in the environment – they are found in soil, wastewater, bottom sediments. The effect of these microorganisms on the development of pathological conditions in humans has not been fully established. Bacteria of the genus Desulfovibrio sp., gram-negative, curved microorganisms,non-spore – forming, slow-growing, anaerobic. Objective. Identification of pathogenetic features of the effect of sulfate-reducing bacteria of the genus Desulfovibrio sp. on the human. Patients and methods.167 patients who signed a voluntary informed consent was conducted among patients who applied to the Federal State Budgetary Institution "Central Research Institute of Epidemiology" for an examination of intestinal microbiocenosis. Microbiological examination of the biomaterial was carried out using traditional microbiological methods. The following blood parameters were used to assess iron metabolism in the body: hemoglobin, serum iron, ferritin, transferrin, unsaturated iron binding capacity of serum and the percentage of transferrin saturation with iron. Statistical data analysis was performed using standard descriptive statistics methods using Excel (Microsoft Corporation, 2019, USA). The statistical significance of the differences in the compared indicators was assessed using the Student's t-test the significance level of p &lt; 0.05 (t &gt; 2). Results. Bacteria of the genus Desulfovibrio sp. were found in 66 patients (39.5%). In the group of patients whose examination revealed bacteria of the genus Desulfovibrio sp., the number of people with anemia was 16, without anemia was 50. Among patients with the absence of bacteria of the genus Desulfovibrio sp. in the microbiocenosis. laboratory-confirmed iron metabolism disorder with the development of anemia was detected in 11 people, the absence of anemia in 90. The calculation of the odds ratio showed that the risk of anemia increases 2.6 times [95% CI 1.13–6.08] in the presence of bacteria of the genus Desulfovibrio sp in the intestine. Conclusion. It can be assumed that bacteria of the genus Desulfovibrio sp. is one of the etiological factor of the development of iron deficiency anemia in humans. Key words: microbiocenosis, iron deficiency anemia, Desulfovibrio desulfuricans

Rapid syndromic approach to diagnosis of bacteremia – results of the first experience

Objective. To describe results of the first experience of using a syndromic approach to the diagnosis of bacteremia using multiplex panels for real-time polymerase chain reaction (real-time PCR). Materials and Methods. The prospective study included 10 consecutive positive blood cultures obtained from 10 patients in the intensive care unit after cardiac surgery. Hemocultures were carried out in BacT/ ALERT FA Plus vials using a BacT/ALERT 3D 120 incubator (bioMérieux, France). After short subcultivation on blood agar (4-6 hours), monocultures were identified using a MALDI-ToF mass spectrometer Vitek MS (bioMérieux, France) with sensitivity to antimicrobial drugs determined on a Vitek-2 compact analyzer (bioMérieux, France). The production of carbapenemases was detected phenotypically using a modified carbapenem inactivation method (mCIM test); the molecular type of enzymes was determined using immunochromatographic tests (NG-Test CARBA 5, NG Biotech, France). In parallel with the described process of identifying microorganisms and determining their sensitivity to antibiotics, positive blood cultures were analyzed using a FilmArray 2.0 analyzer and multiplex real-time PCR panels BioFire FilmArray BCID2 (bioMérieux, France). Results. Using MALDI-ToF mass spectrometry, the following microorganisms were identified in the studied blood cultures: K. pneumoniae (n = 5), E. faecalis (n = 2), A. baumannii (n = 1), Raoultella ornithinolytica (n = 1) and S. aureus (n = 1). 4⁄5 (80%) of K. pneumoniae isolates were resistant to carbapenems; another 1 isolate produced an ESBL and remained sensitive to carbapenems. All carbapenem-resistant K. pneumoniae gave a positive result of the mCIM test, while the immunochromatographic method detected the production of carbapenemases of the molecular types NDM (n = 1), KPC (n = 1), as well as combinations of KPC + OXA-48 (n = 1) and NDM + OXA-48 (n = 1). All E. faecalis were sensitive to ampicillin, isolates of A. baumannii and R. ornithinolytica remained sensitive to carbapenems, S. aureus was sensitive to cefoxitin. Using the real-time PCR, 9⁄10 (90%) pathogens were identified to species level in 10 positive blood cultures. In the remaining 1 case (R. ornithinolytica, not included in the list of detected species), the microorganism was assigned to the order Enterobacterales. The data obtained by the traditional method completely coincided with the results of real-time PCR analysis, while the time to obtain results was statistically significantly shorter compared to traditional microbiological method (22 hours versus 49 hours, p &lt; 0.001). In 7⁄10 (70%) cases, based on the results of real-time PCR analysis, a decision was made to change the tactics of antibiotic therapy. Conclusions. Real-time PCR analysis using BCID2 panels is an effective and reliable tool for the etiological diagnosis of bacteremia.

Advancing Microbe Detection for Lower Respiratory Tract Infection Diagnosis and Management with Metagenomic Next-Generation Sequencing.

Due to limitations of traditional microbiological methods and the presence of the oropharyngeal normal flora, there are still many pathogens that cause lower respiratory tract infections (LRTIs) cannot be detected. Metagenomic next-generation sequencing (mNGS) has the potential capacity to solve this problem. This retrospective study successively reviewed 77 patients with LRTI and 29 patients without LRTI admitted to Tianjin Medical University General Hospital, China from August 2020 to June 2021. Pathogens in bronchoalveolar lavage fluid (BALF) specimens were detected adopting mNGS and traditional microbiological assays. The diagnostic performance of pathogens was compared between mNGS and BALF culture. The value of mNGS for aetiological and clinical impact investigation in LRTI was also evaluated. Among 77 patients with LRTI, 22.1%, 40.3%, and 65.0% of cases were detected as definite or probable pathogens by culture, all conventional microbiological tests, and mNGS, respectively. Using the final diagnosis as a gold standard, mNGS exhibited a sensitivity of 76.6% (95% confidence interval [CI], 65.6-85.5%), which was considerably superior to that of BALF culture (76.6% vs 18.2%; P < 0.01); specificity of 79.3% (95% CI, 60.3-92.0%), which was similar (79.3% vs 89.7%; P = 0.38); positive-predictive value of 90.8% (95% CI, 81.0-96.5%), and negative-predictive value of 56.1% (95% CI, 39.7-71.5%). According to our data, mNGS identified potential microorganisms in 66.7% (42/63) of culture-negative samples. Among 59 patients with pathogens identified by mNGS, conventional microbiological methods confirmed pathogenic infections in less than half (28/59) cases. Within the 77 patients, 34 (44.2%) patients received pathogen-directed therapy, 7 (9.1%) patients underwent antibiotic adjustment, and 3 (3.9%) patients stopped using antibiotics due to mNGS results. mNGS exhibits high accuracy in diagnosing LRTI, and combine with traditional microbiological tests, causative pathogens can be detected in approximately 70.0% of cases, thus yields a positive effect on antibiotic application.

Study of the effect of protein synthesis inhibitors on growing &lt;i&gt;Escherichia coli&lt;/i&gt; bacteria using electrochemical sensors

   Background. The study of the mechanisms of action of antibiotics requires the integrated use of traditional microbiological and physicochemical methods.   The aim. To study the response of Escherichia coli bacteria to the action of four antibiotics, inhibitors of protein synthesis, using combined approach.   Methods. Bacteria were grown under aerobic conditions on minimal M9 medium with glucose. Tetracycline, kanamycin, streptomycin and chloramphenicol have been tested. The effect of antibiotics on survival (CFU) and growth rate was determined. Respiratory activity, sulfide production, extracellular potassium, as well as pH and Eh of the medium were measured using electrochemical sensors directly in a growing culture in the “real time”, membrane potential was measured using a DiBAC dye and a Leica DM2000 fluorescent microscope.   Results. The tested antibiotics were divided into two groups according to their properties. Tetracycline and chloramphenicol showed a pronounced bacteriostatic effect, growth inhibition began immediately after the addition of antibiotics and occurred at a high rate. Both antibiotics inhibited respiration, stimulated sulfide production and an Eh jump. Bacteria treated with tetracycline and chloramphenicol retained the ability to maintain membrane potential and intracellular potassium better. Inhibition of respiration led to a decrease in glucose catabolism, as evidenced by a lower rate of acidification of the medium compared to the control. Growth inhibition with streptomycin and kanamycin was initiated with a 30-minute delay. Both antibiotics showed a bactericidal effect, did not stimulate sulfide production and Eh jump, did not inhibit respiration, but caused a drop in membrane potential and intracellular potassium. High respiratory activity promoted glucose catabolism, as evidenced by the rapid acidification of the medium. Of interest is the detection of kanamycin-induced sulfide production during E. coli growth on MOPS medium.   Conclusion. An analysis of the data obtained indicates that the use of electrochemical sensors in combination with traditional methods is a promising approach to studying the mechanisms of action of antibiotics.

168. Closing Time: A Quasi-experiment Comparing Time to Optimal Therapy using Traditional Identification and Susceptibility Methods, Rapid Identification, or Rapid Identification with Phenotype for Gram-negative Bloodstream Infections

Abstract Background Bloodstream infections (BSI) are a major cause of morbidity and mortality. Compared to traditional microbiology methods, rapid diagnostic testing (RDT) provides prompt organism identification and antibiotic susceptibility results. This leads to earlier opportunities to optimize therapy, which may improve patient outcomes. Methods This was a single center quasi-experimental study of hospitalized adults with Gram-negative (GN) BSI, comparing the management and outcomes across three periods: pre-RDT (traditional identification and susceptibilities with BD Phoenix®, December 2014 to September 2016), RDT1 (Nanosphere Verigene®, December 2016 to December 2019), and RDT2 (Accelerate Pheno™, February 2020 to September 2021). The primary outcome was time to optimal therapy (TTOT). Secondary outcomes included time to oral antibiotic step-down, hospital length of stay (LOS), incidence of Clostridioides difficile infection, and 30-day inpatient mortality. Chi-squared and Kruskal-Wallis tests were used to compare categorical and continuous variables. Significance values for the primary endpoint were adjusted with Bonferroni correction to account for multiple pairwise comparisons. Results Of 296 included patients, 100 were in the pre-RDT, 98 in the RDT1, and 98 in the RDT2 arms. The most common organisms were Escherichia coli (64.5%) and Klebsiella species (20.3%). The TTOT (median, interquartile range) in the pre-RDT, RDT1, and RDT2 groups were 46 (7-61), 30 (0-53), and 12 (0-28) hours (P&amp;lt; 0.001), respectively. The time to oral antibiotic step-down and hospital LOS was similar between groups. There was no difference in C. difficile infection or 30-day inpatient mortality (Table 1). Table 1.Comparison of Outcomes Conclusion In patients with GN BSI the TTOT in RDT1 was shorter when compared to traditional susceptibility methods. Compared to both rapid identification alone and traditional identification and susceptibility methods, RDT2 improved time to optimal therapy. Larger, controlled studies are needed to examine the clinical impact of different RDT methods for Gram negative bloodstream infections. Disclosures Rachel Perry, PharmD, Accelerate Diagnostics: Investigator initiated funding for research presentation; no funding for study design or data analysis Minkey Wungwattana, PharmD, BCIDP, Accelerate Diagnostics: Investigator initiated funding for research presentation; no funding for study design or data analysis.

Research on Thermal Imaging Usage as a Method for Detecting Bacillus subtilis Bacteria in Mortadella

Meat and meat products are important sources of valuable proteins and other nutrients, but they are also a conducive environment for microorganisms’ growth and can easily corrupt. In order to ensure the safety and quality of these products throughout its entire shelf-life, reliable microbial contamination assessment techniques must be used. Despite their effectiveness, traditional diagnostics methods are expensive and time consuming. Therefore, new timesaving, reliable techniques are searched for. Infrared thermography could be a good alternative method in this area. As a quick technique to detect microorganisms, it can overcome the limitations of traditional microbiological food-quality assessment methods. It has numerous advantages, such as the possibility of taking immediate temperature measurements and short processing times for obtaining a thermal image, non-contact and non-destructive measurements, and, unlike other methods, real-time measurement monitoring. Real-time monitoring is particularly important for modern production systems. The purpose of this research study is to develop a methodology for microbiological quality control of mortadella inoculated with Bacillus subtilis ATCC 6633 bacteria using a thermal imaging camera with an uncooled microbolometric detector. It was found that the thermal imaging measurements used in this research study enabled the distinction of contaminated samples (min. 106 CFU/g) from sterile samples. The tests should only record the temperature of the samples during the first 25 s after previously performing activations at −18 °C ± 1 °C for 60 s. This is where differences between the samples are most pronounced. Estimating the trend line of the sample’s cooling process is advisable.

Quantitative PCR reveals the frequency and distribution of 3 indigenous yeast species across a range of specialty cheeses.

Indigenous microorganisms are important components of the complex ecosystem of many dairy foods including cheeses, and they are potential contributors to the development of a specific cheese's sensory properties. Among these indigenous microorganisms are the yeasts Cyberlindnera jadinii, Pichia kudriavzevii, and Kazachstania servazzii, which were previously detected using traditional microbiological methods in both raw milk and some artisanal specialty cheeses produced in the province of Québec, Canada. However, their levels across different cheese varieties are unknown. A highly specific and sensitive real-time quantitative PCR assay was developed to quantitate these yeast species in a variety of specialty cheeses (bloomy-rind, washed-rind, and natural-rind cheeses from raw, thermized, and pasteurized milks). The specificity of the quantitative PCR assay was validated, and it showed no cross-amplification with 11 other fungal microorganisms usually found in bloomy-rind and washed-rind cheeses. Cyberlindnera jadinii and P. kudriavzevii were found in the majority of the cheeses analyzed (25 of 29 and 24 of 29 cheeses, respectively) in concentrations up to 104 to 108 gene copies/g in the cheese cores, which are considered oxygen-poor environments, and 101 to 104 gene copies/cm2 in the rind. However, their high abundance was not observed in the same samples. Whereas C. jadinii was present and dominant in all core and rind samples, P. kudriavzevii was mostly present in cheese cores. In contrast, K. servazzii was present in the rinds of only 2 cheeses, in concentrations ranging from 101 to 103 gene copies/cm2, and in 1 cheese core at 105 gene copies/g. Thus, in the ecosystems of specialty cheeses, indigenous yeasts are highly frequent but variable, with certain species selectively present in specific varieties. These results shed light on some indigenous yeasts that establish during the ripening of specialty cheeses.