Culture Collection Research Articles

Screening of a Microbial Culture Collection: Empowering Selection of Starters for Enhanced Sensory Attributes of Pea-Protein-Based Beverages.

Pea-protein-based ingredients are gaining attention in the food industry due to their nutritional benefits and versatility, but their bitter, astringent, green, and beany off-flavors pose challenges. This study applied fermentation using microbial cultures to enhance the sensory qualities of pea-protein-based beverages. Using UHPLC-TOF-MS analyses along with sensory profile comparisons, microbial species such as Limosilactobacillus fermentum, Lactococcus lactis, Lactobacillus johnsonii, Lacticaseibacillus rhamnosus, and Bifidobacterium longum were preselected from an entire culture collection and found to be effective in improving the overall flavor impression by reducing bitter off-notes and enhancing aroma profiles. Notably, L. johnsonii NCC533 and L. fermentum NCC660 exhibited controlled proteolytic activities after 48 h of fermentation, enriching the matrix with taste-active amino acids, nucleotides, and peptides and improving umami and salty flavors while mitigating bitterness. This study has extended traditional volatile analyses, including nonvolatile metabolomic, proteomic, and sensory analyses and offering a detailed view of fermentation-induced biotransformations in pea-protein-based food. The results highlight the importance of combining comprehensive screening approaches and sensoproteomic techniques in developing tastier and more palatable plant-based protein products.

Identification of Bacteria Producing Red Pigments and Their Application in the Textile Industry

AbstractIn this study, bacterial strains previously isolated and maintained in a culture collection were evaluated for their pigment production capabilities. The pigments produced by the bacteria were using the methanol extraction method and characterized by thin-layer chromatography, UV–Vis, and Fourier transform infrared spectroscopy (FTIR). In addition, the potential use of these pigments as textile dyes in silk fabrics was investigated. Four of the 270 isolates screened (AS-54, AS-55, AS-67, and AS-75) showed superior pigment production. These isolates were further identified using both classical and molecular methods. Based on the analysis of the 16S rRNA gene region, AS-54, AS-55, and AS-67 were identified as Serratia plymuthica and, AS-75 as Serratia marcescens. Characterization results showed that the pigments exhibited a red coloration, indicating prodigiosin. The dyeing of silk fabrics with these pigments resulted in various shades of pink, highlighting their potential as textile dyes. These results suggest that pigments derived from these bacterial isolates hold great promise for applications in the textile industry.

Differentiation of Saccharomyces boulardii from Saccharomyces cerevisiae Strains Using the qPCR-HRM Technique Targeting AAD15 Gene

ABSTRACT S. boulardii, the probiotic variety of S. cerevisiae, is known for its different characteristics, even though the two strains share more than 99% genomic relatedness. Therefore, S. boulardii has been examined and evaluated for differential characteristics in many studies. This study aims to explain the differentiation of S. boulardii from well-identified S. cerevisiae strains by biochemical characteristics and the qPCR-HRM. Initially, S. cerevisiae isolates from the HUF Culture Collection, were screened for basic biochemical characteristics such as growth temperature, glucose, and galactose utilization tests. Out of all selected cultures, the viability at gastric pH was investigated by spectrophotometric readings. The results showed that one isolate was more resistant than other strains when exposed to gastric acids. After sequencing and phylogeny steps using LR0R universal primer, there was no significant differentiation for S. boulardii strains and therefore HRM analysis was performed to interrogate sequence variation and differentiate S. boulardii strains. HRM analysis has successfully distinguished even highly closely related yeast strains.

Hospital mortality and length of stay associated with Enterobacterales positive blood cultures: a multicenter analysis.

Delayed time to antimicrobial susceptibility results can impact patients' outcomes. Our study evaluated the impact of susceptibility turnaround time (TAT) and inadequate empiric antibacterial therapy (IET) in patients with bloodstream infections (BSI) caused by Enterobacterales (ENT) species on in-hospital mortality and length of stay (LOS). This retrospective, multicenter investigation which included 29,570 blood ENT-positive admissions across 161 US healthcare facilities evaluated the association between antimicrobial susceptibility testing (AST) TAT, carbapenem susceptibility, and empiric therapy on post-BSI in-hospital mortality and LOS following an ENT BSI event in adult patients. After adjusting for outcomes covariates, post-BSI in-hospital mortality was significantly higher for patients in the IET vs adequate empiric therapy (AET) group [odds ratio (OR): 1.61 (95% CI: 1.32, 1.98); P < 0.0001], and when AST TAT was >63 h [OR:1.48 (95% CI: 1.16, 1.90); P = 0.0017]. Patients with carbapenem non-susceptible (carb-NS) ENT BSI had significantly higher LOS (16.6 days, 95% CI: 15.6, 17.8) compared to carbapenem susceptible (carb-S, 12.2 days, 95% CI: 11.8, 12.6), (P < 0.0001). Extended AST TAT was significantly associated with longer LOS for TAT of 57-65 h and >65 h (P = 0.005 and P< 0.0001, respectively) compared to TAT ≤42 h (reference). Inadequate empiric therapy (IET), carb-NS, and delayed AST TAT are significantly associated with adverse hospital outcomes in ENT BSI. Workflows that accelerate AST TAT for ENT BSIs and facilitate timely and adequate therapy may reduce post-BSI in-hospital mortality rate and LOS.IMPORTANCEFor patients diagnosed with bloodstream infections (BSI) caused by Enterobacterales (ENT), delayed time to antimicrobial susceptibility (AST) results can significantly impact in-hospital mortality and hospital length of stay. However, this relationship between time elapsed from blood culture collection to AST results has only been assessed, to date, in a limited number of publications. Our study focuses on this important gap using retrospective data from 29,570 blood ENT-positive admissions across 161 healthcare facilities in the US as we believe that a thorough understanding of the dynamic between AST turnaround time, adequacy of empiric therapy, post-BSI event mortality, and hospital length of stay will help guide effective clinical management and optimize outcomes of patients with ENT infections.

Analysis of CAUTIs and Projected Effect of Increasing Pyuria Threshold in Urinalysis with Reflex to Culture

Background: Catheter-Associated Urinary Tract Infections (CAUTI) are preventable hospital acquired infections that contributes to patient morbidity, prolonged hospital stays and increased healthcare costs. Complete compliance to the CAUTI bundle is critical for preventing infection—not only reducing catheter days, but also ensuring appropriate indications are present for urine culture collection. Methods: This retrospective study included 145 patients diagnosed with CAUTI per NHSN definitions from July 1, 2020 to June 30, 2023. Data collected included laboratory data, catheter duration, catheter indication, urinalysis/culture indication and if foley was appropriately removed/changed prior to specimen collection. A urinary catheter indication order was implemented in February 2021 requiring providers to select specific criteria for catheter placement/maintenance. In July 2023, the threshold for urinalysis to reflex to culture was increased to ≥10 WBCs and this criteria was applied to these cases to estimate the effect on diagnosis of CAUTI. Results: The most common indications for urinary catheters were input and output monitoring 76 (52%) and urinary retention/obstruction 34 (23%). No indication was entered on 22 (15%) patients. No difference was seen in the number of catheters without an indication before or after the 2021 order update. Mean catheter duration was 11.5 days with a median of 7 days. The most common indications for obtaining a urine specimen were leukocytosis/fever/sepsis 91 (63%), urinary symptoms/abdominal/flank pain 13 (9%), urine appearance 6 (4%), and altered mental status 4 (3%). In 31 (21%) patients, no indication was identified. A urinalysis with reflex to culture was completed in 105 (72%) and the catheter was removed prior to culture collection in 68 (47%). Of the 127 patients with a urinalysis and culture, 11 had 0-5 WBCs, 16 had 6-9 WBCs, 15 had 10-20 WBCs and 85 had &gt;20 WBCs. Using the new pyuria criteria for urinalysis to reflex to culture, 27 (19%) CAUTIs could have been avoided. Conclusion: Review of CAUTI cases identified opportunities for improving documentation and education of appropriate indications for urinary catheters and evaluation of urinary tract infection. The majority of urine cultures were obtained due to non-specific symptoms and less than 10% had specific urinary symptoms indicating need for continued education and diagnostic stewardship. Increasing the pyuria threshold needed to reflex to culture has the potential to significantly reduce CAUTIs but additional education is needed to ensure catheters are changed prior culture collection and specimens are only sent when signs and symptoms of urinary tract infection are present.

Evaluation of a Sepsis Alert System at a Veterans Affairs Medical Center

Background: Automated sepsis alerts have become a widely implemented screening tool aimed at early detection of clinically unstable patients. Prior research has shown mixed results depending on the type of screening tools used and the patient population studied. This study aimed to evaluate the predictive value of an alert system created for identifying patients with sepsis to determine utility in clinical practice prior to implementation. Additionally, clinical management of those with and without sepsis was compared to measure potential added benefit of this system in clinical decision making. Methods: A TheraDoc® software sepsis alert was generated for non-ICU patients meeting &gt;2 SIRS criteria within a 24-hour time period (temperature &gt;38°C or 90, respiratory rate &gt;20 or partial pressure CO2 12,000 or 10% bands/immature cells) during March 2023. Alerts were excluded if they were duplicates (using identical criteria or a second alert within 24 hours), triggered by labs collected &gt;48 hours prior, or death or discharge occurred before the time of alert. The primary outcome was positive predictive value (PPV) of sepsis identification, confirmed by ICD-10 codes and diagnostic studies (cultures, imaging). Secondary outcomes included clinical management (antibiotic utilization [AU] and choice, infectious disease [ID] consultations and culture collection). Antibiotics were categorized as broad-spectrum using National Healthcare Safety Network (NSHN) criteria. Secondary outcomes were compared between sepsis and SIRS without infection groups (SIRS) by chi-square analysis. Results: After applying exclusion criteria, 116 of 166 alerts were analyzed; 55 of 116 alerts had confirmed sepsis (PPV 47.4%). Patients with sepsis were more likely to have an ID consult (16% [9/55] vs 7% [4/61]) and cultures collected (70.9% [39/55] vs 39.3% [24/61]) compared to SIRS patients, however these differences were not statistically significant. AU was higher with confirmed infections compared to SIRS patients (94.5% [52/55] vs 32.8% [20/61], p &lt; 0 .05) along with use of broad-spectrum antibiotics (73% [38/52] vs 40% [ 8/20] p &lt; 0 .05). Conclusions: While automated alerts may enable early identification of sepsis, use of SIRS criteria alone has poor specificity, which was borne out by the low PPV in this study. Our study found that management of sepsis patients (as measured by AU and culture ordering) was better than expected and combined with the low PPV of this alert system resulted in our team rejecting widespread adoption of SIRS-based sepsis alerts.

Impact and Safety of Diagnostic Stewardship to Improve Urine Culture Testing Among Patients with Indwelling Urinary Catheters

Background: Indiscriminate urine culturing of patients with indwelling urinary catheters may lead to overdiagnosis of urinary tract infections, resulting in unnecessary antibiotic treatment and inaccurate reporting of catheter-associated urinary tract infections (CAUTIs) as a hospital quality metric. We evaluated the impact of a computerized diagnostic stewardship intervention to improve urine culture testing among patients with indwelling urinary catheters. Methods: We performed a single-center retrospective observational study at Rush University Medical Center from April 2018 – July 2023. In February 2021, we implemented a computerized clinical decision support tool to promote adherence to our internal urine culture guidelines for patients with indwelling urinary catheters. Providers were required to select one guideline criteria: 1) neutropenia, 2) kidney transplant, 3) recent urologic procedure, 4) urinary tract obstruction; or if none of the criteria were met, then an infectious diseases consultation was required for approval. We compared facility-wide CAUTI rate per 10,000 catheter days and standardized infection ratio (SIR) during baseline and intervention periods using ecologic models, controlling for time and for monthly Covid-19 hospitalizations. In the intervention period, we evaluated how providers responded to the intervention. Potential harm was defined as collection of a urine culture within 7 days of the intervention that resulted in a change in clinical management. Results: In unadjusted models, CAUTI rate decreased from 12.5 to 7.6 per 10,000 catheter days (p=0.04) and SIR decreased from 0.77 to 0.49 (p=0.09) during baseline vs intervention periods. In adjusted models, the CAUTI rate decreased from 6.9 to 5.5 per 10,000 catheter days (p=0.60) (Figure 1) and SIR decreased from 0.41 to 0.35 (p=0.65) during baseline vs intervention periods. Urine catheter standard utilization ratio (SUR) did not change (p=0.36). There were 598 patient encounters with ≥1 intervention. Selecting the first intervention for each encounter, 284 (47.5%) urine cultures met our guidelines for testing and 314 (52.5%) were averted (Figure 2). Of these, only 3 ( &lt; 1 %) had a urine culture collected in the subsequent 7 days that resulted in change in clinical management. Conclusion: We observed a trend of decreased CAUTIs over time, but effect of our diagnostic stewardship intervention was difficult to assess due to healthcare disruption caused by Covid-19. Adverse outcomes were rare among patients who had a urine culture averted. A computerized clinical decision support tool may be safe and effective as part of a multimodal program to reduce unnecessary urine cultures in patients with indwelling urinary catheters.

Back to Basics: Blood Culture Contamination Reduction Across a Multicenter Academic Health System

Background: Blood culture contamination is common in healthcare and contributes to diagnostic uncertainty, unnecessary treatments and follow-up testing, increased length of stay, higher rates of reportable healthcare-associated infections and events, over utilization of resources and staff including consultative care, and undue emotional stress to patients. The national benchmark for institutional blood culture contamination rates as recommended by The American Society for Microbiology (ASM) and the Clinical Laboratory Standards Institute (CLSI) is &lt; 3 %. Our institution’s overall rate was 8.9% with the highest burden being from our Emergency Department (ED) locations. We formed a multidisciplinary team aimed to reduce these rates through efforts centered around education and simplification of the collection process. Method: Working closely with and gathering feedback from nursing staff, nurse educators, and supply chain, we developed a blood culture collection kit that included all items necessary for blood culture collection thus eliminating the need for nurses to gather these items individually prior to collection. Additionally, simple step-by-step educational materials (Fig. 1) detailing the collection technique were provided. Education was presented at nursing huddles and skills fairs prior to kit roll-out. Blood culture kits were then stocked in place of individual blood culture collection bottles in all ED stock rooms in August 2022. Result: The 12-week pre-intervention period found 249 contamination events from 4265 total collections deriving a contamination rate of 5.8% across our health system’s four ED locations. During a 12-week post-intervention period following kit roll-out, 116 contamination events occurred from 3629 total collections deriving a contamination rate of 3.2% across our four ED locations. Given our results, we ultimately rolled this out to all units in all locations of our health system. When including all time from kit rollout to present (August 2022 to November 2023, 16 months), there were 1077 contamination events from 43379 total collections deriving an overall contamination rate of 2.5%. When compared to the 16 months prior to the kit rollout (April 2021 to July 2022) there were 1803 contamination events in 49335 total collections (3.7% contamination rate) deriving an overall percent reduction of 32.1%. Conclusion: We were able to decrease our health system’s blood culture contamination rate through simple interventions aimed at reducing the mental burden on nursing staff by developing a blood culture collection kit and educational materials. Since implementation of the kits, we have continued to maintain lower contamination rates as evident by our 16 month follow up period.

A New Method for Determination of Rifampicin and Isoniazid Resistance in Mycobacterium tuberculosis complex Isolates: Capillary Tube Method

Tuberculosis continues to be an important public health problem worldwide. Culture methods are still considered the gold standard in the diagnosis of tuberculosis and the determination of drug resistance. The most important limitation of these methods is their long turnaround time. Commercial culture systems developed to shorten the duration are emerging as an economic problem, especially for developing countries. Therefore, cheap, fast, easy to apply and objectively evaluable tests are needed. In this study, in addition to culture-based methods for determining RIF and INH resistance in Mycobacterium tuberculosis complex isolates, it was aimed to develop the capillary tube method to accelerate the evaluation process. The study included 27 RIF-resistant, 36 RIF -sensitive, 30 INH-resistant, and 33 INH-sensitive isolates obtained from the mycobacteriology laboratory culture collection, for which susceptibility testing to firstline drugs were previously performed using the BACTEC MGIT 960 system (BD, USA) and were stored. H37Rv standard strain and an external quality control strain (IDT3) with known RIF and INH resistance were used as quality control isolates in the study. As a new testing method, the capillary tube method for detecting rifampicin and isoniazid resistance was compared to the standard BACTEC MGIT 960 system. In the determination of RIF and INH resistance, the sensitivity of the capillary tube method compared to the reference method was determined as 85% and 80%, respectively; however, the specificity values (25% and 45.5%, respectively) for both drugs were found to be low in the studies. The time to detect resistance with the capillary tube method varied between 4-9 days. Capillary tube method, which was developed especially for the rapid identification and treatment of multidrug-resistant isolates, is promising in that it detects resistant strains in a short time with a relatively high sensitivity, although its specificity is very low. It is thought that it would be beneficial to continue the study with a larger number of samples and even improve the method with studies conducted directly from clinical samples.

Impacts of Hospital Acquired Bloodstream Infections in Patients Undergoing Hemodialysis Through a Central Venous Catheter

Background: Hospital acquired infections (HAI) are of interest given their resultant morbidity, mortality, and hospital utilization. Among HAIs, central line associated bloodstream infections result in the highest rates of mortality and additional costs. While all central venous catheters (CVC) carry risk for BSI, long-term catheter use is at increased risk. One population that utilize CVCs for extended durations are those undergoing hemodialysis. While data are available characterizing BSI impacts on outpatient hemodialysis patients, little data exist describing inpatients. The purpose of this study was to characterize the demographics, outcomes, and economics associated with the development of hospital acquired BSI (HA-BSI) in patients undergoing hemodialysis through a CVC (HD-CVC). Methods: All admissions of adult patients in the Premier Healthcare Database with hospital stays including HD-CVC with discharge dates during 2020-2022 were retrospectively evaluated. BSIs were identified by ICD-10 codes and blood culture collection dates. A BSI was deemed hospital acquired if the blood culture date was ≥3 calendar days after admission. Descriptive analyses were undertaken for HA-BSI patients including: baseline demographics, clinical characteristics, and outcomes. Length of stay (LOS), ICU utilization, and estimated costs were evaluated for HAI-BSI and non-BSI populations. Results: 166,394 admissions from 91,448 patients were identified. Of these, 5,722 patients (6.3%) had 5,842 admissions with a HA-BSI. These patients were 58.9% white, 28.3% black, 56.8% male, and 62.9% were aged ≥60years. Patients had considerable comorbidities at baseline with 88.9% having ≥2 Charlson comorbid conditions and 46.9% with ≥6. During the study period, all-cause mortality was 27.8% for HA-BSI patients with 85.5% of deaths occurring while inpatient. Median LOS for patients with HA-BSI was 25 days compared with 6 days for HD-CVC without BSI; patients with HA-BSI were also more likely to require the ICU (65.6% vs. 27.6%). The median ICU LOS was 12 days for HA-BSI versus 34 days for HD-CVC without BSI. Greater intensity of healthcare utilization was reflected in median costs of $402K for HA-BSI, compared with $43K for HD-CVC without BSI. Discussion: We described the characteristics of HD-CVC patients that developed HA-BSI. These patients had many comorbidities and relatively high rates of all-cause in-hospital mortality. Patients were likely to have long LOS, both in-hospital and within the ICU. Collectively, care of these patients was associated with considerable healthcare costs, particularly as compared with HD-CVC patients not developing a HA-BSI. Future studies should characterize risk factors and evaluate potential prevention strategies for this high-risk population.

Enterococcal Bacteremia outbreaks during SARS-COV-2 in Intensive Care Unit (ICU): The role of Strain Identification

Background: An unprecedented burden of morbidity and mortality has been reported in patients admitted to healthcare facilities with SARS-COV-2 infection globally since March 2020. A higher incidence of ICU-acquired bloodstream infection has been described with the cumulative risk increased with the length of ICU stay, use of steroids, anti-inflammatory agents, and indwelling catheters. Additionally, SARS-COV-2 infection may increase the risk of bacteremia from gastrointestinal flora such as Enterococcus species by disrupting the gastrointestinal barrier and microbiome. Methods: We aimed to investigate the outbreaks of Enterococcus bacteremia in patients with SARS-COV-2 infection and included patients aged&gt;18 years admitted to the ICU at the Oklahoma City Veterans Affairs Medical Center who were infected with SARS-COV-2 and were identified as having positive blood cultures for Enterococcus faecalis, Enterococcus faecium including vancomycin-resistant species and other bacterial or candida species between March 1, 2020, to March 9, 2022. We collected the following data: duration of hospitalization including ICU stay, unit and room location during the hospital stay, blood culture collection date and results, duration, and site of central line placement, duration of ventilation, administration of antimicrobials and COVID-19 directed therapeutics using computerized patient record system (CPRS). We sent 10 E. faecalis and 12 E. Faecium isolates for genetic analysis. DNA was sequestered from each isolate and multi-locus sequence typing (MLST) was performed by amplifying 7 regions of the E. faecalis genome and 7 regions of the E. faecium genome by polymerase chain reaction (PCR). Resulting amplicons were sequenced and allele type for each gene region and overall sequence type was determined using MLST database (http://pubmlst.org). Results: There were 22 episodes of enterococcal bacteremia in a 3-month duration in the ICU in 20 patients of which 17 were associated with another episode with the same strain (Figure-1-2). Central line placement was noted in 18/22 episodes. Genetic analysis by multi-locus sequence typing of enterococcal bacterial strains was performed by the Public Health Reference Laboratory, Palo Alto. Similar strains were localized to patients in the same geographical region in the ICU (Figure 3). The isolates from 2 other patients who presented with the same strain of Enterococcus but were never hospitalized at the same time during the COVID-19 pandemic were localized to another hospital where both had received care at different times. Conclusion: Higher rates of enterococcal bacteremia were reported during the SARS-COV-2 pandemic. Geographical proximity with strained infection control measures accounted for ICU outbreaks seen in our tertiary care setup.

Reducing Blood Culture Contamination in Adult Patients with Cancer Presenting to the Emergency Department

Background: Infection is one of the most common complications of cancer and cancer treatment. Most patients admitted for fever or infection come through the Emergency Department (ED), which is a primary site for blood culture collection. Contamination of blood cultures complicates the diagnoses, compromises quality of care, leads to unnecessary antibiotic exposure and increases financial burdens. It may also lead to unnecessary removal of central venous access devices or delay of critical therapy or procedures. At our institution, the contamination rate of blood cultures drawn in the ED was over twice that of the remainder of the hospital (2.8 versus 0.8), prompting this quality improvement project. Unlike on hospital floors, nurses, instead of phlebotomists, draw most blood cultures due to the urgency of managing suspected sepsis. Our aim was to decrease the ED contamination rate by 20 percent after the first PDSA cycle, and ultimately bring it on par with the remainder of the hospital. Methods: First, we compared ED contamination rates versus other hospital inpatient floors and outpatient centers over a three-month period. We then evaluated the contamination rates of ED nurses versus ED phlebotomists and peripheral versus central line blood draws. Process mapping and fishbone analysis helped identify practices contributing to higher contamination rates. Key drivers of these practices were diagrammed, and potential interventions were ranked on a prioritization matrix. Results: We identified use of alcohol rather than chlorhexidine swabs for peripheral disinfection and inconsistent techniques of blood draw by nurses as critical contributors to increased contamination rates in the ED. Our intervention was creating premade blood culture kits promoting the use of chlorhexidine swabs through availability and easy access in the fast-paced ED environment. Ten cubic centimeter (cc) syringes in the kits encouraged withdrawal of adequate blood samples in compliance with the 7-10 cc guideline. Designated nursing team leaders checked off ED nurses at the bedside, implementing education and adherence in using the blood culture collection kits. The average number of blood cultures in the emergency department was 1,400. A reduction in blood culture contamination from 2.46 percent to 1.89 percent was seen after two months. Conclusions: A guideline-driven, standardized blood culture collection process followed by ED nurses is vital to reducing blood culture contamination. Chlorhexidine is necessary to maintain the lowest contamination rates. Readily available premade blood culture kits improve compliance with materials and techniques associated with best practices.

Screening of Lactic Acid Bacteria for Silage Inoculant Candidate

Abstract Silage fermentation is generally assisted by lactic acid bacteria (LAB). The aim of this study was to screen selected LABs for silage inoculant. A total of 60 LAB isolates from the Indonesian Culture Collection (InaCC) were used in this study. Screening was carried out in an MRS broth medium using three different substrates, which were glucose, sucrose, and carboxyl methyl cellulose (CMC). The supernatant from LAB cultures was tested for pH, lactic acid (LA) concentration, antimicrobial activity, and cellulolytic activity. The results of the screening obtained 4 LAB isolates to be used as silage inoculant. Three LAB isolates selected from the glucose substrate were Lactobacillus fermentum InaCC-B1024, Lactobacillus plantarum InaCC-B1028, and L. brevis InaCC-B1052. Lactobacillus fermentum InaCC B1024 has a pH, LA concentration, cellulolytic activity, and percentage inhibition of 3.71, 318.931 mM, and 0.806 U/mL, &gt; 95% antibacterial inhibition against B. subtilis, S. auerus, K. michiganensis, S. lloydii, and S. gallinarum, and &lt;35% antifungal activity against M. ramosissimus, A. flavus, F. oxysporum, and P. griseofulvum, respectively. L. plantarum InaCC-B1028 has a pH, LA concentration, cellulolytic activity, and percentage activity of 3.61, 318.833 mM, 0.866 U/ml, &gt; 95% antibacterial activity against B. subtilis, E. coli, M. luteus, S.auerus, K. michiganensis, S.lloydii, S. gallinarum, and &lt; 40% antifungal activity against M. ramosissimus, A. flavus, F. oxysporum, and P. griseofulvum, respectively. L. brevis InaCC-B1052 has a pH, LA concentration, cellulolytic activity, and percentage activity of 3.67, 301.676 mM, no cellulolytic activity, &gt; 95% antibacterial activity against E. coli, M. luteus, S. auerus, K. michiganensis, S. lloydii, S. gallinarum, and &lt; 25% antifungal activity against M. ramosissimus and A. flavus, respectively. One LAB isolate selected from sucrose was L. kimchii InaCC-B982. Its pH, LA concentration, cellulolytic activity, and percentage activity were 3.77, 285.5 mM, 7.288 U/mL, &gt; 95% antibacterial activity against E. coli, M. luteus, S. auerus, K. michiganensis, S. lloydii, S. gallinarum, and &lt;15% antifungal activity against M. ramosissimus, A. flavus, F. oxysporum, and P. griseofulvum, respectively. Based on the results, the four LABs could be used as silage inoculant candidates.

IsolateR: an R package for generating microbial libraries from Sanger sequencing data.

Sanger sequencing of taxonomic marker genes (e.g. 16S/18S/ITS/rpoB/cpn60) represents the leading method for identifying a wide range of microorganisms including bacteria, archaea, and fungi. However, the manual processing of sequence data and limitations associated with conventional BLAST searches impede the efficient generation of strain libraries essential for cataloging microbial diversity and discovering novel species. isolateR addresses these challenges by implementing a standardized and scalable three-step pipeline that includes: (1) automated batch processing of Sanger sequence files, (2) taxonomic classification via global alignment to type strain databases in accordance with the latest international nomenclature standards, and (3) straightforward creation of strain libraries and handling of clonal isolates, with the ability to set customizable sequence dereplication thresholds and combine data from multiple sequencing runs into a single library. The tool's user-friendly design also features interactive HTML outputs that simplify data exploration and analysis. Additionally, in silico benchmarking done on two comprehensive human gut genome catalogues (IMGG and Hadza hunter-gather populations) showcase the proficiency of isolateR in uncovering and cataloging the nuanced spectrum of microbial diversity, advocating for a more targeted and granular exploration within individual hosts to achieve the highest strain-level resolution possible when generating culture collections. isolateR is available at: https://github.com/bdaisley/isolateR.

Evaluating the Clinical Impact of Species-Level Identification in Coagulase-Negative Staphylococci Positive Blood Cultures

Background: Coagulase-negative staphylococci (CoNS) are often considered contaminants when isolated from blood cultures. While criteria exist to distinguish true bacteremia from contamination (Table 1), clinical judgement is often necessary. Clinical microbiology laboratories have traditionally identified CoNS to the species level only if present in multiple cultures. There have been concerns that blood cultures positive for rare or less familiar CoNS species might be misinterpreted as true bacteremia. Tufts Medical Center (TMC) clinical microbiology laboratory started reporting all CoNS to the species level in January 2023. We studied the impact of species-level identification of CoNS on clinically relevant outcomes following this change. Methods: The study evaluated inpatients at TMC aged ≥ 18 years with CoNS isolated from blood cultures between July 2022 and June 2023. The primary outcome was the difference in anti-staphylococcal antibiotic utilization between the pre- and post-intervention groups. Secondary outcomes included differences in true bacteremia diagnosis, length of hospital stay, and mortality between the two groups. We also compared the performance of Souvenir’s criteria with clinical judgement at distinguishing contamination from true bacteremia. A total of 100 patients were included in the pre- and post-intervention groups to detect an estimated effect size of 15% with a power of 81%. Results: Most patients were male, White, and English speaking (Table 2). No differences were found between the two groups in terms of infectious disease consultation frequency, blood culture collection department, or the presence of central venous catheters (Table 2). Staphylococcus epidermidis was the predominant CoNS in the post-intervention group. Blood cultures were repeated before and after starting antibiotics in 24% and 74% (pre-intervention) and 18% and 84% (post-intervention) of cases, respectively. Anti-staphylococcus antibiotic use was the same in both groups (82%). The median antibiotic therapy duration was 4.5 days pre- vs 3 days post-intervention (p =0.39). There were no differences in hospital length of stay or mortality between the two groups (Table 3). The clinical diagnosis of true bacteremia was established in 28% of cases in the pre- vs 25% in the post-intervention group (p= 0.63). Compared to clinical judgement, Souvenir’s true bacteremia criteria demonstrated a sensitivity of 80.3%, negative PV of 91.9%, and positive PV of 55.2%. Conclusions: Species-level identification of CoNS positive blood cultures did not impact antibiotic utilization, diagnosis of true bacteremia, length of hospital stay, or mortality. Further studies with larger cohorts and prospective designs are needed to validate these findings and assess the long-term implications in patients.

Back to the future: Using herbarium specimens to isolate nodule-associated bacteria.

Herbarium specimens are increasingly being used as sources of information to understand the ecology and evolution of plants and their associated microbes. Most studies have used specimens as a source of genetic material using culture-independent approaches. We demonstrate that herbarium specimens can also be used to culture nodule-associated bacteria, opening the possibility of using specimens to understand plant-microbe interactions at new spatiotemporal scales. We used historic and contemporary nodules of a common legume, Medicago lupulina, to create a culture collection. We were able to recover historic bacteria in 15 genera from three specimens (collected in 1950, 2004, and 2015). This work is the first of its kind to isolate historic bacteria from herbarium specimens. Future work should include inoculating plants with historic strains to see if they produce nodules and if they affect plant phenotype and fitness. Although we were unable to recover any Ensifer, the main symbiont of Medicago lupulina, we recovered some other potential nodulating species, as well as many putative growth-promoting bacteria.

DESIGN THINKING FOR BUSINESS INNOVATION IN ASIA: THE CULTURAL PERSPECTIVE

As a human-centered innovation approach, design thinking (DT) has been widely applied in a variety of industries and spheres to create new and enhance existing products, services and processes by organizations around the globe. DT is highly collaborative, involves direct communication/feedback and requires cross-functional/cross-hierarchical participation. DT is also iterative, necessitating embracing risk and ambiguity of the experiment and instead of penalizing ‘failures’, learning promptly from them. Nevertheless, Asian countries are often described as high power distance, high uncertainty avoidance, collectivist cultures in which high context and indirect communication are implied. This conceptual article includes analysis of academic research articles, reports, interviews and books by design thinking experts to analyze advantages of design thinking for business innovations, discuss major cultural characteristics of Asian countries using Hofstede’s cultural dimensions and respective challenges in applying design thinking. It concludes by providing practical recommendations, limitations of the analysis and suggesting aspects for future research.

A New Guanidine-Core Small-Molecule Compound as a Potential Antimicrobial Agent against Resistant Bacterial Strains.

The guanidine core has been one of the most studied functional groups in medicinal chemistry, and guanylation reactions are powerful tools for synthesizing this kind of compound. In this study, a series of five guanidine-core small molecules were obtained through guanylation reactions. These compounds were then evaluated against three different strains of Escherichia coli, one collection strain from the American Type Culture Collection (ATCC) of E. coli ATCC 35218, and two clinical extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates (ESBL1 and ESBL2). Moreover, three different strains of Pseudomonas aeruginosa were studied, one collection strain of P. aeruginosa ATCC 27853, and two clinical multidrug-resistant isolates (PA24 and PA35). Among Gram-positive strains, three different strains of Staphylococcus aureus, one collection strain of S. aureus ATCC 29213, and two clinical methicillin-resistant S. aureus (MRSA1 and MRSA2) were evaluated. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) experiments were reported, and the drop plate (DP) method was used to determine the number of viable suspended bacteria in a known beaker volume. The results from this assessment suggest that guanidine-core small molecules hold promise as therapeutic alternatives for treating infections caused by clinical Gram-negative and Gram-positive bacteria, highlighting the need for further studies to explore their potential. The results from this assessment suggest that the chemical structure of CAPP4 might serve as the basis for designing more active guanidine-based antimicrobial compounds, highlighting the need for further studies to explore their potential.

Cowboy as a Symbol of Individualism: A Pragmatic Analysis of Red Dead Redemption 2

As cultures are mainly divided into collectivistic and individualistic, members tend to emphasize, through communication, either their position as part of their group or their independence from the group. This emphasis is manifested in using the pragmatic concepts of positive politeness and negative politeness. The present study looks into the reflection of these two cultures in Rockstar’s renowned video game, Red Dead Redemption 2 (2018). It aims at identifying the two cultures as present in the game and showing their significance to its narrative. It fills the gap in the studies of language used within video games as well as its cultural reflections. The study addresses the following question: What are the positive and negative politeness strategies used in Red Dead Redemption 2 (2018), and what are the implications of using these strategies in its narrative? By adopting Brown and Levinson’s (1987) politeness theory, the researcher analyzed the positive and negative politeness used in the scripted dialogue of the in-game cutscenes through the mixed methods approach. The researcher hypothesized that the protagonist shows adherence to a collectivistic culture using positive politeness, and he begins transitioning towards an individualistic culture using negative politeness. The results showed that 75% of the protagonist’s interactions contained positive politeness while only 25% contained negative politeness. This transition explains the protagonist’s loss of trust and loyalty to his group as he begins to witness the futility of maintaining such loyalty. It also supports the overall narrative of the game as well as its cultural foundations.

Mining biosynthetic gene clusters in Paenibacillus genomes to discover novel antibiotics

BackgroundBacterial antimicrobial resistance poses a severe threat to humanity, necessitating the urgent development of new antibiotics. Recent advances in genome sequencing offer new avenues for antibiotic discovery. Paenibacillus genomes encompass a considerable array of antibiotic biosynthetic gene clusters (BGCs), rendering these species as good candidates for genome-driven novel antibiotic exploration. Nevertheless, BGCs within Paenibacillus genomes have not been extensively studied.ResultsWe conducted an analysis of 554 Paenibacillus genome sequences, sourced from the National Center for Biotechnology Information database, with a focused investigation involving 89 of these genomes via antiSMASH. Our analysis unearthed a total of 848 BGCs, of which 716 (84.4%) were classified as unknown. From the initial pool of 554 Paenibacillus strains, we selected 26 available in culture collections for an in-depth evaluation. Genomic scrutiny of these selected strains unveiled 255 BGCs, encoding non-ribosomal peptide synthetases, polyketide synthases, and bacteriocins, with 221 (86.7%) classified as unknown. Among these strains, 20 exhibited antimicrobial activity against the gram-positive bacterium Micrococcus luteus, yet only six strains displayed activity against the gram-negative bacterium Escherichia coli. We proceeded to focus on Paenibacillus brasilensis, which featured five new BGCs for further investigation. To facilitate detailed characterization, we constructed a mutant in which a single BGC encoding a novel antibiotic was activated while simultaneously inactivating multiple BGCs using a cytosine base editor (CBE). The novel antibiotic was found to be localized to the cell wall and demonstrated activity against both gram-positive bacteria and fungi. The chemical structure of the new antibiotic was elucidated on the basis of ESIMS, 1D and 2D NMR spectroscopic data. The novel compound, with a molecular weight of 926, was named bracidin.ConclusionsThis study outcome highlights the potential of Paenibacillus species as valuable sources for novel antibiotics. In addition, CBE-mediated dereplication of antibiotics proved to be a rapid and efficient method for characterizing novel antibiotics from Paenibacillus species, suggesting that it will greatly accelerate the genome-based development of new antibiotics.