Background Flora Research Articles

Development and evaluation of test methods for the detection and enumeration of opportunistic waterborne pathogens from the hospital environment

Many Gram-negative bacteria other than Pseudomonas aeruginosa have been implicated in waterborne outbreaks, but standardized laboratory detection methods for these organisms have not been established. This study aimed to establish laboratory testing methodologies for six waterborne pathogens: Acinetobacter spp., Burkholderia spp., Cupriavidus spp., Delftia acidovorans, Elizabethkingia spp. and Stenotrophomonas maltophilia. Water samples were spiked by UK Health Security Agency laboratories and sent to the Glasgow Royal Infirmary laboratory for analysis. Water samples were spiked with either a pure culture of target organism or the target organism in water containing normal background flora, to ensure that the methodology could identify organisms from a mixed culture. Volumes of 100mL were filtered under negative pressure on to culture media and incubated at 30°C and 37°C. The incubation time was 7 days, with plates read on days 2, 5 and 7. Further identification of colonies was undertaken using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Optimal recovery of organisms was obtained by culturing water samples on tryptic soy agar, chocolate bacitracin agar and pseudomonas selective agar. The optimal temperature for isolation was 30°C. The optimal incubation time was 5 days, and MALDI-TOF MS identified all test species reliably. The methodology described was able to detect the six tested waterborne pathogens reliably, and can be utilized by laboratories involved in testing water samples during outbreak investigations.

Preliminary Study on the Identification of Aerobic Vaginitis by Artificial Intelligence Analysis System

To develop an artificial intelligence vaginal secretion analysis system based on deep learning and to evaluate the accuracy of automated microscopy in the clinical diagnosis of aerobic vaginitis (AV). In this study, the vaginal secretion samples of 3769 patients receiving treatment at the Department of Obstetrics and Gynecology, West China Second Hospital, Sichuan University between January 2020 and December 2021 were selected. Using the results of manual microscopy as the control, we developed the linear kernel SVM algorithm, an artificial intelligence (AI) automated analysis software, with Python Scikit-learn script. The AI automated analysis software could identify leucocytes with toxic appearance and parabasal epitheliocytes (PBC). The bacterial grading parameters were reset using standard strains of lactobacillus and AV common isolates. The receiver operating characteristic (ROC) curve analysis was used to determine the cut-off value of AV evaluation results for different scoring items were obtained by using the results of manual microscopy as the control. Then, the parameters of automatic AV identification were determined and the automatic AV analysis scoring method was initially established. A total of 3769 vaginal secretion samples were collected. The AI automated analysis system incorporated five parameters and each parameter incorporated three severity scoring levels. We selected 1.5 μm as the cut-off value for the diameter between Lactobacillus and common AV bacterial isolates. The automated identification parameter of Lactobacillus was the ratio of bacteria ≥1.5 μm to those <1.5 μm. The cut-off scores were 2.5 and 0.5, In the parameter of white blood cells (WBC), the cut-off value of the absolute number of WBC was 103 μL－1 and the cut-off value of WBC-to-epithelial cell ratio was 10. The automated identification parameter of toxic WBC was the ratio of toxic WBC toWBC and the cut-off values were 1% and 15%. The parameter of background flora was bacteria<1.5 μm and the cut-off values were 5×103 μL－1 and 3×104 μL－1. The parameter of the parabasal epitheliocytes was the ratio of PBC to epithelial cells and the cut-off values were 1% and 10%. The agreement rate between the results of automated microscopy and those of manual microscopy was 92.5%. Out of 200 samples, automated microscopy and manual microscopy produced consistent scores for 185 samples, while the results for 15 samples were inconsistent. We developed an AI recognition software for AV and established an automated vaginal secretion microscopy scoring system for AV. There was good overall concordance between automated microscopy and manual microscopy. The AI identification software for AV can complete clinical lab examination with rather high objectivity, sensitivity, and efficiency, markedly reducing the workload of manual microscopy.

Validation of the Thermo Scientific™ SureTect™ Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus PCR Assay for the Detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus in Seafood Matrixes: AOAC Performance Tested MethodsSM 022301.

The Thermo Scientific™ SureTect™ Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus PCR Assay method is a real-time PCR method for the multiplex detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus in seafood. The Thermo Scientific SureTect Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus Assay was evaluated for AOAC Performance Tested MethodsSM certification. Inclusivity/exclusivity, matrix, product consistency/stability, and robustness studies were conducted to assess the method's performance. For the matrix study, the method was validated using the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR Food Safety Instrument and the Applied Biosystems™ 7500 Fast Real-Time PCR Food Safety Instrument against the U.S. Food and Drug Administration Bacteriological Analytical Manual, Chapter 9 (2004), Vibrio and ISO 21872-1:2017 Microbiology of the food chain-Horizontal method for the determination of Vibrio spp.-Part 1: Detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio vulnificus reference methods. Matrix studies showed equivalent or superior performance of the candidate method compared to the reference method and, overall, no difference between presumptive and confirmed results, except for one matrix due to high background flora. The inclusivity/exclusivity study correctly identified/excluded all strains analyzed. Robustness testing showed no statistically significant differences in assay performance under varied test conditions. Product consistency and stability studies demonstrated no statistically significant differences between assay lots with different expiration dates. The data presented show that the assay constitutes a rapid and reliable workflow for the detection of V. cholerae, V. parahaemolyticus, and V. vulnificus in seafood matrixes. The SureTect PCR Assay method allows for fast, reliable detection of stipulated strains in seafood matrixes with results obtained in as little as 80 min post-enrichment.

Substituting Allose as the Primary Carbon Source During Enrichment Helps Improve Detection and Isolation of Lineage II Listeria monocytogenes From Food

Testing of foods for low levels of the human pathogen, Listeria monocytogenes (Lm), involves a selective enrichment procedure. A nonpathogenic species of Listeria, L. innocua (Li), is often present in foods and food-manufacturing environments and is an interference organism for Lm detection due to competition during enrichment. The present study investigated whether a novel enrichment strategy incorporating the sugar allose into the secondary enrichment broth (allose method) could improve the detection of Lm from foods when Li is present. First, Canadian food isolates of Listeria spp. were tested to confirm recent reports that lineage II Lm (LII-Lm), but not Li, could metabolize allose. All LII-Lm isolates (n = 81), but not Li (n = 36), possessed the allose genes lmo0734-lmo0739, and could efficiently metabolize allose. Next, smoked salmon was contaminated with mixtures of LII-Lm and Li and tested using different enrichment procedures to compare the ability to recover Lm. Allose broth was more effective than Fraser Broth, with Lm detected in 87% (74 of 85) compared to 59% (50 of 85) of the samples (P < 0.05), following a common preenrichment. When evaluated against a current Health Canada method (MFLP-28), the allose method was more effective, with LII-Lm detected in 88% (57 of 65) compared to 69% (45 of 65) of the samples (P < 0.05). The allose method also remarkably increased the ratio of LII-Lm to Li postenrichment, which improved the ease of obtaining isolated Lm colonies for confirmation tests. Allose may therefore provide a tool for use when the presence of background flora interferes with Lm detection. As this tool is specifically applicable to a subset of Lm, the use of this method modification may provide a working example of tailoring methodology to target the known subtype of the pathogen of interest in an outbreak investigation, or for regular monitoring activities in conjunction with a PCR screen for allose genes on preenrichment cultures.

An amplification-free, 16S rRNA test for Neisseria gonorrhoeae in urine.

An amplification-free, nanopore-based nucleic acid detection platform has been demonstrated for rapid, 16S rRNA sequence-specific detection of Neisseria gonorrhoeae at 10-100 CFU mL-1 in human urine against background bacterial flora at 1000 CFU mL-1. Gonorrhea is a very common notifiable communicable disease, antibiotic resistant strains have emerged, and the rate of reported gonococcal infections continues to increase. Since rapid clinical identification of bacterial pathogens in clinical samples is needed to guide proper antibiotic treatment and to control disease spread, it is important to engineer rapid, sensitive, selective, and inexpensive point-of-care (POC) diagnostic devices for pathogens such as N. gonorrhoeae. Our detector technology is based on straightforward conductometric detection of sustained blockage of a glass nanopore. Charge neutral, complementary peptide nucleic acid probes are conjugated to polystyrene beads to capture N. gonorrhoeae 16S rRNA selectively. In the presence of an electric field applied externally through a glass nanopore, the PNA-microbead conjugates that acquire substantial negative charge upon target hybridization are driven to the smaller diameter nanopore. At least partial blockage of the nanopore results in a sustained drop in ionic current that can be measured easily with simple electronics. The ability to detect N. gonorrhoeae over the range of 10 to 100 CFU mL-1 spiked in human urine was demonstrated successfully with estimated sensitivity and specificity of ∼98% and ∼100%, respectively. No false positives were observed for the control group of representative background flora (E. coli, K. pneumoniae, and E. faecalis) at 1000 CFU mL-1. Also, N. gonorrhoeae at 50 CFU mL-1 was successfully detected against 1000 CFU mL-1 of background flora in urine. These results suggest that this amplification-free technology may serve as the basis for rapid, inexpensive, low-power detection of pathogens in clinical samples at the POC.

Comparison between qPCR, VIDAS immunoassays, and agar streaking for the detection of Listeria monocytogenes from food and environmental surfaces containing and not containing Listeria innocua

Comparisons among a qPCR assay, VIDAS® assays and a conventional agar streaking method following the same enrichment for the detection of Listeria monocytogenes were performed under two challenging conditions. In the first comparison, L. innocua and L. monocytogenes were coinoculated into sausages at ratios (L. innocua-to-L. monocytogenes) of 10, 100, 1000, and 10 000. qPCR provided the most sensitive detection at all ratios after both 24-h and 48-h enrichments. A modified VIDAS® LMO2 assay (i.e., replacement of the kit-specified enrichment scheme with the enrichment scheme used in this study) and agar streaking yielded equivalent results when the ratio was 10 and 100; agar streaking was more sensitive when the ratio was 1000; neither method could detect L. monocytogenes at the ratio of 10 000. Enrichment duration of 48 h was needed for modified VIDAS® to detect L. monocytogenes when the ratio was 1000. Agar streaking after 24-h enrichment isolated L. monocytogenes better than after 48-h enrichment when the ratio was 100 and 1000. In the second comparison, we followed the validation guidelines of AOAC International and inoculated L. monocytogenes, without any L. innocua, onto lettuce and stainless-steel surfaces at low levels. The numbers of positive samples detected by qPCR, VIDAS® LIS assay, modified VIDAS® LMO2 assay, and agar streaking after 48-h enrichment were not statistically different. Our data showed that qPCR was the most sensitive method, while agar streaking and VIDAS® performed reasonably well. Streaking after 24-h enrichment was needed when background flora could overgrow L. monocytogenes during prolonged enrichment, and this is critical for confirming rapid screening assays. Appropriate selection of enrichment duration and rapid assays will enhance the testing of L. monocytogenes in food and environmental samples.

A study on the method and effect of the construction of a humanized mouse model of fecal microbiota transplantation.

The gestation period is critical for the health of the mother and fetus. Malnutrition or over nutrition during pregnancy may cause gestational diseases that can result in adverse pregnancy outcomes. Fecal microbiota transplantation (FMT) can be used to re-establish new gut microbiota to treat a variety of diseases and construct a model to investigate the nutritional health during pregnancy. Therefore, this study investigated whether human-derived gut microbiota during pregnancy could colonize the intestines of mice. Moreover, we determined the time and method of intervention for FMT. Based on this information, a humanized mouse model of FMT was constructed to simulate the human intestinal microecology during pregnancy, and serve as a useful animal model for the study of nutritional health and disease during pregnancy. Germ-free (GF) and specific pathogen free (SPF) C57BL/6J mice were selected for humanized gestational FMT and the transplantation outcomes were evaluated. The results demonstrated that the gestational intestinal microbiota colonized the intestines of mice, allowing researchers to construct a humanized mouse model of gestational FMT. The main intestinal flora of the gestational period were transplanted into GF mice, with the gestational flora being similar to the flora of GF mice after transplantation. However, antibiotics could not eliminate the original microbial flora in SPF mice, and the flora was complex and variable after FMT with little increase in abundance. Background flora had a significant impact on the outcomes assessment. The results were better in GF mice than in SPF mice, and after microbiota transplantation, a superior effect was observed on day 21 compared to days 7 and 14.

Comparison of DNA Extraction Methods for the Quantification of Listeria monocytogenes in Dairy Products by Real-Time Quantitative PCR>

Listeria monocytogenes is a common foodborne pathogen affecting public health. Thus, detecting L. monocytogenes, even at low levels, in food matrices is essential. However, the current culture methods used for its detection and quantification are time consuming and difficult owing to background flora and interference by food matrices. DNA-based assays depend on DNA extraction and purification techniques. No optimal DNA extraction kit has been developed for analyzing L. monocytogenes in dairy products by real-time quantitative PCR (RT-qPCR). Therefore, in this study, we aimed to determine the efficiency of three DNA extraction kits for detecting L. monocytogenes in dairy products by RT-qPCR. We tested the efficiency of three commercial kits for DNA extraction from L. monocytogenes artificially inoculated in milk and dairy products. For the PrepSEQ rapid spin sample preparation kit and Exgene Cell SV mini, the limit of detection of was 100, 100, and 101 CFU/mL L. monocytogenes in milk, processed cheese, and infant formula, respectively, whereas that of the QIAamp DNA mini kit was 101, 103, and 102 CFU/mL, respectively. In addition, the Exgene Cell SV mini was better than the PrepSEQ rapid spin sample preparation kit for obtaining a standard curve for RT-qPCR of L. monocytogenes DNA in milk and dairy products, with a high correlation coefficient and amplification efficiency. The results of this study may be valuable for diagnostic laboratories and for developing an effective extraction method for processing food samples, such as dairy products, to subsequently detect and quantify L. monocytogenes by RT-qPCR.

Gut microbiota changes and biological mechanism in hepatocellular carcinoma after transarterial chemoembolization treatment.

Background and aimsIntestinal flora is closely associated with the occurrence and development of hepatocellular carcinoma (HCC). However, gut microbial changes and biological mechanisms in HCC after transarterial chemoembolization (TACE) treatment are rarely reported.MethodsWe evaluated changes in intestinal flora after TACE in rabbit HCC models and assessed the impact of these changes on the disease. Twenty-four rabbit VX2 HCC models were established and intestinal flora structures, intestinal barrier function, changes in blood lipopolysaccharide (LPS) levels, Toll-like receptor 4 (TLR4), Cyclooxygenase-2 (COX-2), and p-signal transducer and activator of transcription 3(p-STAT3) protein expression levels were studied after TACE treatment.ResultsCompared with healthy rabbits, the intestinal flora in HCC models exhibited structural changes; intestinal barrier function was decreased, and increased LPS levels entered the circulation. A short-term follow-up after TACE showed the procedure partially reversed the intestinal microflora disorder caused by the tumor: intestinal barrier and liver functions were improved, intestinal LPS levels in the blood were reduced, and liver metabolism toward LPS was enhanced. Correlation analyses of the first 75 significantly changed bacteria with clinical factors showed that harmful bacteria had decreased and beneficial bacteria increased. Blood LPS levels and downstream signaling molecule TLR4, COX-2, and p-STAT3 protein expression levels were reduced, which correlated with tumor drug resistance and invasion capabilities.ConclusionsWe first characterized gut microbiota changes and biological mechanisms in HCC after TACE treatment. Our data provide a theoretical research basis for TACE combined with an intestinal flora intervention and systemic chemotherapy.

Trimethyllysine, a trimethylamine N-oxide precursor, predicts the presence, severity, and prognosis of heart failure

Background and aimsIntestinal flora metabolites are associated with cardiovascular (CV) diseases including heart failure (HF). The carnitine precursor trimethyllysine (TML), which participates in the generation of the atherogenic-related metabolite trimethylamine N-oxide (TMAO), was found to be related to poor prognosis in patients with CV diseases. The aim of the present study was to examine the relationship between TML and stable chronic HF.Methods and resultsIn total, 956 subjects including 471 stable chronic HF and 485 non-HF patients were enrolled in the present cohort study and subjects with stable HF were followed up for 2.0 ± 1.1 years. Serum levels of TML and TMAO were measured by liquid chromatography mass spectrometry in tandem. TML levels were significantly elevated in patients with HF compared with non-HF patients and were positively correlated with N-terminal pro-brain natriuretic peptide (NTproBNP) levels (r = 0.448, P < 0.001). TML was associated with the presence of HF after adjusting for age, sex, complications, traditional clinical factors, and TMAO (tertile 3 (T3), adjusted odds ratio (OR) 1.93, 95% confidence interval (CI) 1.19–3.13, and P = 0.007). In patients with HF, increased TML levels were associated with a composite endpoint of CV death and HF hospitalization during follow-up (T3, adjusted hazard ratio (HR) 1.93, 95% CI 1.27–2.93, and P = 0.002). Increased TML levels indicated a higher risk of CV death, re-hospitalization, and all-cause mortality.ConclusionSerum TML levels were associated with the presence and severity of HF in all subjects. High levels of TML can indicate complications and poor prognosis in HF patients.

Influence of the number of visitors on the quality of bathing water at the Kravica Waterfall (Bosnia and Herzegovina)

This paper analyses the results of microbiological analyses of bathing water at the Kravica Waterfall during 2018 and 2019 and the impact of bathers on bathing water quality. The microbiological analysis of the samples was performed by the Federal Institute of Public Health in Mostar in accordance with the regulations of Directive 2006/7 EC for management of quality of bathing water. Faecal streptococci were determined using the membrane filtration method BAS EN ISO 7899-2:2003. Escherichia coli was determined using the membrane filtration method for waters with low bacterial background flora BAS EN ISO 9308-1:2015. The quality of bathing water is assessed on the basis of parameters determined by the current Directive 2006/7/EC. The number of visitors does not have a significant impact on the quality of bathing water; the reason is that of the total number of visitors there is a small percentage of bathers. During the bathing seasons in 2018 and 2019, the bathing water at the Kravica Waterfall was of excellent quality.

A New Culture Method for the Detection of Non-Tuberculous Mycobacteria in Water Samples from Heater-Cooler Units and Extracorporeal Membrane Oxygenation Machines.

The isolation of non-tuberculous mycobacteria (NTM) from cultures is particularly laborious due to the potential overgrowth of coexisting non-acid fast bacilli. To reduce the overgrowth of these non-mycobacterial organisms, a decontamination step with NaOH or cetylpyridinium chloride is highly recommended before plating the samples on the culture medium. However, due to their toxicity, decontamination solutions tend to decrease NTM recovery from clinical and environmental samples. Here, we tested an alternative method for NTM recovery based on the use of NTM Elite agar, a selective medium that does not require a decontamination step. Using NTM Elite agar, we were able to detect non-tuberculous mycobacteria in 27.7% (30/108) of water samples analyzed. The average time to NTM detection was 18 days, but some strains required longer to grow, perhaps due to the stressful environmental conditions (periodical disinfection of devices). NTM Elite agar’s effectiveness in inhibiting background flora was proven by the isolation of NTM from samples with and without background flora, showing no statistically significant differences in detection rates for different total viable counts of background flora (p = 0.4989). In conclusion, our findings indicate that effective NTM recovery from HCU- and ECMO-derived water samples can be achieved via filtration and direct culture of the filters on NTM Elite agar. This simple procedure can speed up laboratory work and provide an improved method, successfully resulting in low contamination and high detection rate, in addition to being less time-consuming. Its sensitivity and lack of a decontamination step make this protocol particularly useful for monitoring the effectiveness of device disinfection in hospital settings, even in the presence of low NTM loads. Reading timeframes should probably be extended to 7 weeks (i.e., well beyond the standard 4 weeks advised by the manufacturer), in order to isolate even the slow-growing mycobacteria. However, an extended incubation period is not necessary for exclusion of M. chimaera contamination of the devices, as M. chimaera isolation times do not generally exceed 3 weeks.

A Wash of Ethyl Acetoacetate Reduces Externally Added Salmonella enterica on Tomatoes.

The continuously high numbers of food-borne disease outbreaks document that current intervention techniques are not yet satisfactory. This study describes a novel wash for tomatoes that can be used as part of the food processing chain and is designed to prevent contamination with serovars of Salmonella enterica. The wash contains ethyl acetoacetate (EAA) at a concentration of 8% in H2O. This wash reduced live bacterial counts (on Salmonella Shigella agar) of externally added S. Newport MDD14 by 2.3 log, counts of S. Typhimurium ATCC19585 by 1.5 log, and counts of S. Typhimurium FSL R6-0020 by 3.4 log. The naturally occurring background flora of the tomatoes was determined on plate count agar. The log reduction by EAA was 2.1. To mimic organic matter in the wash, we added 1% tomato homogenate to the 8% EAA solution. Prior to using the wash, the tomato homogenate was incubated with the EAA for 2 h. In the presence of the tomato homogenate, the log reductions were 2.4 log for S. Newport MDD14 and 3 log for S. Typhimurium FSL R6-0020. It seems like tomato homogenate did not reduce the efficacy of the EAA wash in the two S. enterica serovars tested. We propose the use of EAA as a wash for tomatoes to reduce bacterial counts of S. enterica well as naturally occurring background flora.

Performance Testing of Bacillus cereus Chromogenic Agar Media for Improved Detection in Milk and Other Food Samples.

In this study, the performance of four alternative selective chromogenic B. cereus agar was compared to the reference mannitol-yolk polymyxin (MYP) agar (ISO 7932) using inclusion and exclusion test strains (n = 110) and by analyzing naturally contaminated milk and other food samples (n = 64). Subsequently, the panC group affiliation and toxin gene profile of Bacillus cereus senso lato (s.l.) isolates were determined. Our results corroborate that the overall best performing media CHROMagar™ B. cereus (93.6% inclusivity; 82.7% exclusivity) and BACARA® (98.2% inclusivity, 62.7% exclusivity) are more sensitive and specific compared to Brilliance™ B. cereus, MYP and ChromoSelect Bacillus Agar. Both media allow unequivocal detection of B. cereus with low risks of misidentification. Media containing ß-D-glucosidase for the detection of presumptive B. cereus may form atypical colony morphologies resulting in a false negative evaluation of the sample. Naturally contaminated samples presented high numbers of background flora, while numbers of presumptive B. cereus were below the detection limit (<10 CFU g−1 or mL−1). Recovery after freezing resulted in the highest detection of B. cereus s.l. on BACARA® (57.8%), CHROMagar™ B. cereus (56.3%) and MYP agar (54.7%). The panC/toxin profile combination IV/A was the most abundant (33.0%), followed by III/F (21.7%) and VI/C (10.4%). More panC and toxin combinations were present in 15.6% of samples when reanalyzed after freezing. In order to improve detection and confirmation of B. cereus s.l. in food samples, we recommend the parallel use of two complementary selective media followed by molecular characterization (e.g., panC typing combined with toxin gene profiling). When determining psychrotolerant or thermophilic members of the B. cereus group, the selective agar media should additionally be incubated at appropriate temperatures (5 °C, ≥45 °C). If high-risk toxin genes (e.g., ces or cytK-1) are detected, the strain-specific ability to produce toxin should be examined to decisively assess risk.

A European multicenter evaluation study to investigate the performance on commercially available selective agar plates for the detection of carbapenemase producing Enterobacteriaceae

The European Food Safety Authority (EFSA) advised to prioritize monitoring carbapenemase producing Enterobacteriaceae (CPE) in food producing animals. Therefore, this study evaluated the performance of different commercially available selective agars for the detection of CPE using spiked pig caecal and turkey meat samples and the proposed EFSA cultivation protocol. Eleven laboratories from nine countries received eight samples (four caecal and four meat samples). For each matrix, three samples contained approximately 100 CFU/g CPE, and one sample lacked CPE. After overnight enrichment in buffered peptone water, broths were spread upon Brilliance™ CRE Agar (1), CHROMID® CARBA (2), CHROMagar™ mSuperCARBA™ (3), Chromatic™ CRE (4), CHROMID® OXA-48 (5) and Chromatic™ OXA-48 (6). From plates with suspected growth, one to three colonies were selected for species identification, confirmation of carbapenem resistance and detection of carbapenemase encoding genes, by methods available at participating laboratories. Of the eleven participating laboratories, seven reported species identification, susceptibility tests and genotyping on isolates from all selective agar plates. Agars 2, 4 and 5 performed best, with 100% sensitivity. For agar 3, a sensitivity of 96% was recorded, while agar 1 and 6 performed with 75% and 43% sensitivity, respectively. More background flora was noticed for turkey meat samples than pig caecal samples. Based on this limited set of samples, most commercially available agars performed adequately. The results indicate, however, that OXA-48-like and non-OXA-48-like producers perform very differently, and one should consider which CPE strains are of interest to culture when choosing agar type.

Aerobic Vaginitis Diagnosis Criteria Combining Gram Stain with Clinical Features: An Establishment and Prospective Validation Study.

Wet-mount microscopy aerobic vaginitis (AV) diagnostic criteria need phase-contrast microscopy and keen microscopists, and the preservation of saline smears is less common in clinical practice. This research work developed new AV diagnostic criteria that combine Gram stain with clinical features. We enrolled 325 AV patients and 325 controls as a study population to develop new AV diagnostic criteria. Then, an independent group, which included 500 women, was used as a validation population. AV-related microscopic findings on Gram-stained and wet-mount smears from the same participants were compared. The accuracy of bacterial indicators from the two methods was verified by bacterial 16S rRNA V4 sequencing (n = 240). Logistic regression was used to analyse AV-related clinical features. The screened clinical features were combined with Gram-stain microscopic indicators to establish new AV diagnostic criteria. There were no significant differences in the leukocyte counts or the parabasal epitheliocytes (PBC) proportion between the Gram-stain and wet-mount methods (400×). Gram stain (1000×) satisfied the ability to identify bacteria as verified by 16S rRNA sequencing but failed to identify toxic leukocytes. The new criteria included: Lactobacillary grades (LBG) and background flora (Gram stain, 1000×), leukocytes count and PBC proportion (Gram stain, 400×), and clinical features (vaginal pH > 4.5, vagina hyperemia, and yellow discharge). These criteria satisfied the accuracy and reliability for AV diagnosis (Se = 86.79%, Sp = 95.97%, and Kendall’s W value = 0.899) in perspective validation. In summary, we proposed an alternative and valuable AV diagnostic criteria based on the Gram stain, which can make it possible to diagnose common vaginitis like AV, BV, VVC, and mixed infections on the same smear and can be available for artificial intelligence diagnosis in the future.

A duplex real-time NASBA assay targeting a serotype-specific gene for rapid detection of viable Salmonella Paratyphi C in retail foods of animal origin.

Salmonella enterica serovar Paratyphi C is highly adapted to humans and can cause a typhoid-like disease with high mortality rates. In this study, three serovar-specific genes were identified by comparative genomics for Salmonella Paratyphi C, SPC_0871, SPC_0872, and SPC_0908. Based on the SPC_0908 and xcd genes for testing Salmonella spp., we developed a duplex real-time nucleic acid sequence-based amplification (real-time NASBA) with a molecular beacon approach for the simultaneous detection of viable cells of Salmonella spp. and serotype Paratyphi C. The test selectively and consistently detected 53 Salmonella spp. (representing 31 serotypes) and 18 non-Salmonella strains. Additionally, the method showed high resistance to interference from natural background flora in pork and chicken samples. The sensitivity of the established approach was determined to be 4.89 cfu/25 g in artificially contaminated pork and chicken samples after pre-enrichment. We propose this NASBA-based protocol as a potential detection method for Salmonella spp. and serotype Paratyphi C in foods of animal origin.

Inhibitory Effects of Cinnamaldehyde Derivatives on Biofilm Formation and Virulence Factors in Vibrio Species.

Vibrio parahaemolyticus is considered one of the most relevant pathogenic marine bacteria with a range of virulence factors to establish food-related gastrointestinal infections in humans. Cinnamaldehyde (CNMA) and some of its derivatives have antimicrobial and antivirulence activities against several bacterial pathogens. This study examined the inhibitory effects of CNMA and its derivatives on biofilm formation and the virulence factors in Vibrio species, particularly V. parahaemolyticus. CNMA and ten of its derivatives were initially screened against V. parahaemolyticus biofilm formation, and their effects on the production of virulence factors and gene expression were studied. Among the CNMA derivatives tested, 4-nitrocinnamaldehyde, 4-chlorocinnamaldehyde, and 4-bromocinnamaldehyde displayed antibacterial and antivirulence activities, while the backbone CNMA had weak effects. The derivatives could prevent the adhesion of V. parahaemolyticus to surfaces by the dose-dependent inhibition of cell surface hydrophobicity, fimbriae production, and flagella-mediated swimming and swarming phenotypes. They also decreased the protease secretion required for virulence and indole production, which could act as an important signal molecule. The expression of QS and biofilm-related genes (aphA, cpsA, luxS, and opaR), virulence genes (fliA, tdh, and vopS), and membrane integrity genes (fadL, and nusA) were downregulated in V. parahaemolyticus by these three CNMA analogs. Interestingly, they eliminated V. parahaemolyticus and reduced the background flora from the squid surface. In addition, they exhibited similar antimicrobial and antibiofilm activities against Vibrio harveyi. This study identified CNMA derivatives as potential broad-spectrum antimicrobial agents to treat biofilm-mediated Vibrio infections and for surface disinfection in food processing facilities.

Composition of Vaginal Microbiota in Pregnant Women With Aerobic Vaginitis.

Vaginal dysbiosis, such as bacterial vaginosis (BV) and aerobic vaginitis (AV), is an important cause of premature birth in pregnant women. However, there is very little research on vaginal microbial distribution in AV compared to that in BV. This study aimed to analyze the composition of the vaginal microbiota of pregnant women with AV using microbial community analysis and identify the causative organism using each criterion of the AV scoring system. Also, we compared the quantification of aerobic bacteria using quantitative polymerase chain reaction (qPCR) and their relative abundances (RA) using metagenomics. This prospective case–control study included 228 pregnant Korean women from our previous study. A wet mount test was conducted on 159 women to diagnose AV using the AV scoring system. Vaginal samples were analyzed using metagenomics, Gram staining for Nugent score determination, conventional culture, and qPCR for Staphylococcus spp., Streptococcus spp., and Enterobacteriaceae. The relative abundances (RAs) of eleven species showed significant differences among the three groups (Normal flora (NF), mild AV, and moderate AV). Three species including Lactobacillus crispatus were significantly lower in the AV groups than in the NF group, while eight species were higher in the AV groups, particularly moderate AV. The decrease in the RA of L. crispatus was common in three criteria of the AV scoring system (Lactobacillary, WBC, and background flora grades), while it did not show a significant difference among the three grade groups of the toxic leukocyte criterion. Also, the RAs of anaerobes, such as Gardnerella and Megasphaera, were higher in the AV groups, particularly moderate AV, while the RAs of aerobes were very low (RA < 0.01). Therefore, qPCR was performed for aerobes (Staphylococcus spp., Streptococcus spp., and Enterobacteriaceae); however, their quantification did not show a higher level in the AV groups when compared to that in the NF group. Therefore, AV might be affected by the RA of Lactobacillus spp. and the main anaerobes, such as Gardnerella spp. Activation of leukocytes under specific conditions might convert them to toxic leukocytes, despite high levels of L. crispatus. Thus, the pathogenesis of AV can be evaluated under such conditions.

Development of a new chromogenic medium for the enumeration of Bacillus cereus in various ready-to-eat foods

Enumeration of Bacillus cereus in ready-to-eat food with high background flora has been challenging due to the low selectivity and recoverability of the selective media. In this study, a new medium for detection of B. cereus was developed. This medium, B. cereus chromogenic agar (BCCA), contains an egg yolk emulsion for detection of lecithinase activity, a chromogenic substrate specific for detecting alkaline phosphatase activity, and three antibiotics for selectivity. The aim of this study was to compare the performance of BACCA with that of two conventional selective media (MYPA and PEMBA) and two commercialized chromogenic media (CBC agar and Bacara medium). The newly developed media has been evaluated for its inclusivity/exclusivity and recoverability in pure culture and in artificially inoculated ready-to-eat foods, respectively. BCCA exhibited similar or better recoverability and inclusivity/exclusivity for enumeration of B. cereus and B. thuringiensis from pure cultures than two conventional [mannitol yolk polymyxin B agar (MYPA) and polymyxin pyruvate egg yolk mannitol bromothymol blue agar (PEMBA)] and two commercial chromogenic media [chromogenic B. cereus (CBC) agar and Bacara medium]. Furthermore, BCCA yielded similar or enhanced levels of recoverability and superior selectivity of B. cereus and B. thuringiensis from ready-to-eat food products, particularly radish sprouts and a sprout mix, compared with the other media tested. These findings indicate that BCCA comprises a useful option for the isolation and enumeration of B. cereus from commercial foods.