Non-selective Media Research Articles

The point mutation A1387G in the 16S rRNA gene confers aminoglycoside resistance in Campylobacter jejuni and Campylobacter coli.

Thermotolerant Campylobacter spp. are the most frequent cause of foodborne bacterial diarrhea and high-priority antibiotic-resistant pathogens, according to the World Health Organization (WHO). Monitoring revealed current low prevalence of gentamicin resistance in European Campylobacter spp. isolates but substantial presence of gentamicin modifying genes circulating globally. Using a combined approach of natural transformation and whole-genome sequencing, we revealed a novel gentamicin resistance mechanism, namely the point mutation A1387G in the 16S rRNA gene, originally identified in a C. coli isolate from turkey caecal content. The transformation rate of the resistance using genomic DNA of the resistant donor to sensitive recipient C. jejuni and C. coli was ~2.5 log10 lower compared to the control rpsL-A128G point mutation conferring streptomycin resistance. Antimicrobial susceptibility tests showed cross-resistance to apramycin, kanamycin, and tobramycin, with transformants exhibiting more than 4- to 8-fold increased MICs to apramycin and tobramycin and over 64-fold higher MICs to kanamycin compared to wild-type isolates. Although transformants showed 177-1,235 variations relative to the recipient, only the A1387G point mutation in the 16S rRNA was in common. This mutation was causal for resistance, as transformation of a 16S rRNA_A1387G PCR fragment into susceptible isolates also led to resistant transformants. Sanger sequencing of the 16S rRNA genes and Oxford nanopore whole-genome sequencing of transformants identified clones harboring either all three copies with A1387G or a mixed population of wild-type and mutated 16S rRNA gene alleles. Within 15 passages on non-selective medium, transformants with mixed populations of the 16S rRNA gene copies partially reverted to wild type, both geno- and phenotypically. In contrast, transformants harboring the A1387G point mutation in all three 16S rRNA gene copies kept full resistance within at least 45 passages. We speculate that partial acquisition and rapid loss of the point mutation limited its spread among C. spp. isolates. In-depth knowledge on resistance mechanisms contributes to optimal diagnosis and preventative measures.

B-019 Reduction in Culture Media Costs, without Impacting Turnaround Time, Through Implementation of an Automated Microbiology Platform and Gram Stain Interpretation

Abstract Background Automated plating instruments have been shown to improve quality in Microbiology laboratories in a number of ways including consistent streaking patterns that provide better isolation of bacterial colonies compared to manual methods. The time per culture set up and automated camera imaging is dependent upon the amount of media set, such that more individual plates slow down turn around time. Conversely, smaller media surface area and the use of non-selective media may result in mixed colonies that require sub-culture for isolation before identification and susceptibilities can be performed. Based upon these findings, we attempted to reduce our media usage per culture with the implementation of automation, with the goal of using as few plates as possible while at the same time not negatively impacting the turn around time of the culture via the need to subculture poorly isolated colonies. We conducted a pre-and post-implementation study evaluating the costs per culture and turn around time delays related to subculturing in a subset. Methods In the pre-implementation period, from April-January 2023, three individual media plates (sheep blood agar, chocolate agar and CNA/MacConkey bi-plate) were routinely set for general aerobic bacterial culture sources including swabs and body fluids. In the post implementation period, from April-January 2024, a single sheep blood/chocolate agar bi-plate was set unless the gram stain showed evidence of mixed organisms (defined as more than one morphology) at which time a secondary selective media (CNA/MacConkey) was manually set. For urine cultures, in the pre-implementation period 2 plates were set (sheep blood and CNA/MacConkey bi-plate) and in the post-implementation period a single chromogenic agar/sheep blood bi-plate was set. Total numbers of cultures in both periods were collected as well as the number of cultures for which additional media were set based on gram stain results. Orders of subculture media in the laboratory information system were compared per positive cultures. Statistical analysis was performed in GraphPad Prism. Results In the pre- versus post-implementation periods, a total of 16,454 vs 17,782 general aerobic cultures and 50,680 vs 53,916 urine cultures were set, respectively. Only 3.6% of general cultures required additional media set based on gram stain. The difference in cost between pre- and post-implementation was approximately a two-thirds reduction in cost for general cultures and a 25% reduction for urine cultures, with a total savings of approximately $34,238 in media avoidance in the post-implementation period. The frequency of positive cultures requiring subculture was not significantly different between the pre- (average 36% of positive growth) and post-implementation (average 22% of positive growth) periods for both culture types and may actually have been reduced due to automation. Conclusions The implementation of reduced media concomitant with streaking via automation resulted in significant cost savings while not negatively impacting isolation, which in turn impacts culture turn around times. The automation may have reduced need for subculture, perhaps due to better isolation, thus subsequent analyses will explore this additional finding.

INFLUENCE OF DISINFECTANTS ON THE TOTAL BACTERIAL CONTAMINATION OF DUCK EGGSHELLS

The role of the total viable microbial count on eggshells is of significant interest, as it relates to egg safety and product shelf life.The aim of this study was to assess the influence of various disinfectants on the overall bacterial contamination of duck eggshells. For this purpose, fresh high-quality duck eggs were obtained from a farm, and solutions of disinfectants were prepared: 6% hydrogen peroxide, 4% calcium hydroxide, 4% sodium carbonate, and 3% acetic acid. When disinfecting duck or goose eggs, the dosage of substances is doubled compared to chicken eggs. The eggs were treated with prepared disinfectant solutions, with varying exposure times. The exposure times for hydrogen peroxide were 4 and 8 minutes, for calcium hypochlorite – 2 and 5 minutes, for sodium carbonate – 10 and 30 minutes, and for acetic acid – 5 and 15 minutes. Samples of washings from the eggshells were collected both before and after disinfection. Dilutions were prepared and subsequently plated on Petri dishes with non-selective media, followed by incubation at 30°C for 72 hours. As a result of colony counting, the number of mesophilic aerobic and facultatively anaerobic microorganisms (CFU/egg) was determined.The results of the study showed a direct correlation between the inactivation of microorganisms and exposure time when using acetic acid, hydrogen peroxide, and calcium hypochlorite. A longer contact period of the eggs with sodium carbonate did not result in a measurable reduction in the number of viable microorganisms. It was also found that 4% calcium hypochlorite could completely inactivate microbes on the eggshell within 5 minutes. With a 2-minute exposure, the total microbial contamination was 9.2*10¹±0.7 CFU/egg.Thus, in this study, solutions of calcium hypochlorite, hydrogen peroxide, and acetic acid demonstrated high bactericidal properties and can be considered effective agents for disinfecting duck eggshells.Most scientific articles are devoted to the study of chicken eggs. In this work, the object of research is duck eggs, the best disinfectants for processing eggshells have been identified, which is an urgent task in poultry farming.The obtained results can help farmers implement more effective strategies for combating foodborne infections.

A streamlined culturomics case study for the human gut microbiota research

Bacterial culturomics is a set of techniques to isolate and identify live bacteria from complex microbial ecosystems. Despite its potential to revolutionize microbiome research, bacterial culturomics has significant challenges when applied to human gut microbiome studies due to its labor-intensive nature. Therefore, we established a streamlined culturomics approach with minimal culture conditions for stool sample preincubation. We evaluated the suitability of non-selective medium candidates for maintaining microbial diversity during a 30-day incubation period based on 16S rRNA gene amplicon analysis. Subsequently, we applied four culture conditions (two preincubation media under an aerobic/anaerobic atmosphere) to isolate gut bacteria on a large scale from eight stool samples of healthy humans. We identified 8141 isolates, classified into 263 bacterial species, including 12 novel species candidates. Our analysis of cultivation efficiency revealed that seven days of aerobic and ten days of anaerobic incubation captured approximately 91% and 95% of the identified species within each condition, respectively, with a synergistic effect confirmed when selected preincubation media were combined. Moreover, our culturomics findings expanded the coverage of gut microbial diversity compared to 16S rRNA gene amplicon sequencing results. In conclusion, this study demonstrated the potential of a streamlined culturomics approach for the efficient isolation of gut bacteria from human stool samples. This approach might pave the way for the broader adoption of culturomics in human gut microbiome studies, ultimately leading to a more comprehensive understanding of this complex microbial ecosystem.

Evaluation of Simultaneous Growth of Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes in Ground Beef Samples in Different Growth Media.

Several multiplex approaches for the simultaneous detection of pathogens in food have been developed in recent years, but the use of a single enrichment medium remains a problem. In this study, six enrichment broths (five non-selective media, tryptic soy broth (TSB), brain heart infusion broth (BHI), buffered peptone water (BPW), universal pre-enrichment broth (UPB), no. 17 broth, and a selective, Salmonella Escherichia Listeria broth (SEL)), were studied for the simultaneous detection of E. coli O157:H7, Salmonella spp., and L. monocytogenes, to validate the suitable enrichment broth to be used for the detection methods. Different ratios of E. coli O157:H7, Salmonella spp., and L. monocytogenes were used. Almost all non-selective broths evaluated in this study showed similar growth parameters and profiles among each other. The only selective enrichment broth under analysis (SEL) showed distinct growth features compared to the non-selective media, allowing for a slower but balanced growth of the three pathogens, which could be beneficial in preventing the overgrowth of fast-growing bacteria. In addition, when tested in ground beef samples, SEL broth seems to be the most distinctive medium with a balanced growth pattern observed for the three pathogens. Overall, this study is intended to provide the basis for the selection of suitable enrichment broths according to the technology detection to be used, the desired time of enrichment, and the expected balanced concentration of pathogens.

Erysipelothrix spp. and other Erysipelotrichales detected by 16S rRNA microbial community profiling in samples from healthy conventionally reared chickens and their environment.

Outbreaks of erysipelas, a disease caused by infection with Erysipelothrix rhusiopathiae (ER), is a re-emerging problem in cage-free laying hen flocks. The source of ER infection in hens is usually unknown and serological evidence has also indicated the presence of ER or other antigenically related bacteria in healthy flocks. The aim of the present study was to evaluate sample collection, culture methods and DNA-based methodology to detect ER and other Erysipelotrichales in samples from healthy chickens and their environment. We used samples from a research facility with conventionally reared chickens with no history of erysipelas outbreaks where hens with high titres of IgY recognising ER previously have been observed. Microbial DNA was extracted from samples either directly or after pre-culture in nonselective or ER-selective medium. Real-time PCR was used for detection of Erysipelothrix spp. and high-throughput amplicon sequencing of 16S rRNA sequencing was used for detection of Erysipelotrichales. A pilot serological analysis of some Erysipelotrichales members with IgY from unvaccinated and ER-vaccinated high-biosecurity chickens, as well as conventionally reared chickens, was also performed. All samples were negative for ER, E. tonsillarum and E. piscisicarius by PCR analysis. However, 16S rRNA community profiling indicated the presence of several Erysipelotrichales genera in both environmental samples and chicken intestinal samples, including Erysipelothrix spp. that were detected in environmental samples. Sequences from Erysipelothrix spp. were most frequently detected in samples pre-cultured in ER-selective medium. At species level the presence of Erysipelothrix anatis and/or Erysipelothrix aquatica was indicated. Serological results indicated that IgY raised to ER showed some cross-reactivity with E. anatis. Hence, environmental samples pre-cultured in selective medium and analysis by 16S rRNA sequencing proved a useful method for detection of Erysipelotrichales, including Erysipelothrix spp., in chicken flocks. The observation of such bacteria in environmental samples offers a possible explanation for the observation of high antibody titres to ER in flocks without a history of clinical erysipelas.

Activity of Cannabidiol on Ex Vivo Amino Acid Fermentation by Bovine Rumen Microbiota

Amino-acid-fermenting bacteria are wasteful organisms within the rumens of beef cattle that remove dietary amino nitrogen by producing ammonia, which is then excreted renally. There are currently no on-label uses for the control of this microbial guild, but off-label use of broad-spectrum antimicrobials has shown efficacy, which contributes to antimicrobial resistance. Plant-derived antimicrobials supplemented into the diets of cattle may offer worthwhile alternatives. This study sought to investigate the role of cannabidiol (CBD) as a terpenophenolic antimicrobial. Ex vivo cell suspensions were harvested from the rumen fluid of Angus × Holstein steers in non-selective media with amino acid substrates. The suspensions were treated with five concentrations of CBD (860 μg mL−1–0.086 μg mL−1) and incubated (24 h), after which ammonia production and viable number of cells per substrate and treatment were measured. The data demonstrated a ~10–15 mM reduction in ammonia produced at the highest concentration of CBD and negligible changes in the viable number of amino-acid-fermenting bacteria. CBD does not appear to be a biologically or economically viable terpenophenolic candidate for the control of amino acid fermentation in beef cattle.

Metabolic engineering of Saccharomyces cerevisiae for the biosynthesis of a fungal pigment from the phytopathogenic fungus Cladosporium phlei

BackgroundCladosporium phlei is a phytopathogenic fungus that produces a pigment called phleichrome. This fungal perylenequinone plays an important role in the production of a photosensitizer that is a necessary component of photodynamic therapy. We applied synthetic biology to produce phleichrome using Saccharomyces cerevisiae.ResultsThe gene Cppks1, which encodes a non-reducing polyketide synthase (NR-PKS) responsible for the biosynthesis of phleichrome in C. phlei, was cloned into a yeast episomal vector and used to transform S. cerevisiae. In addition, a gene encoding a phosphopantetheinyl transferase (PPTase) of Aspergillus nidulans was cloned into a yeast integrative vector and also introduced into S. cerevisiae for the enzymatic activation of the protein product of Cppks1. Co-transformed yeasts were screened on a leucine/uracil-deficient selective medium and the presence of both integrative as well as episomal recombinant plasmids in the yeast were confirmed by colony PCR. The episomal vector for Cppks1 expression was so dramatically unstable during cultivation that most cells lost their episomal vector rapidly in nonselective media. This loss was also observed to a less degree in selective media. This data strongly suggests that the presence of the Cppks1 gene exerts a significant detrimental effect on the growth of transformed yeast cells and that selection pressure is required to maintain the Cppks1-expressing vector. The co-transformants on the selective medium showed the distinctive changes in pigmentation after a period of prolonged cultivation at 20 °C and 25 °C, but not at 30 °C. Furthermore, thin layer chromatography (TLC) revealed the presence of a spot corresponding with the purified phleichrome in the extract from the cells of the co-transformants. Liquid chromatography (LC/MS/MS) verified that the newly expressed pigment was indeed phleichrome.ConclusionOur results indicate that metabolic engineering by multiple gene expression is possible and capable of producing fungal pigment phleichrome in S. cerevisiae. This result adds to our understanding of the characteristics of fungal PKS genes, which exhibit complex structures and diverse biological activities.

Inactivation of Escherichia coli, Salmonella enterica, and Listeria monocytogenes using the Contamination Sanitization Inspection and Disinfection (CSI-D) device

The Contamination Sanitization Inspection and Disinfection (CSI-D) device is a handheld fluorescence-based imaging system designed to disinfect food contact surfaces using ultraviolet-C (UVC) illumination. This study aimed to determine the optimal CSI-D parameters (i.e., UVC exposure time and intensity) for the inactivation of the following foodborne bacteria plated on non-selective media: generic Escherichia coli (indicator organism) and the pathogens enterohemorrhagic E. coli, enterotoxigenic E. coli, Salmonella enterica, and Listeria monocytogenes. Each bacterial strain was spread-plated on non-selective agar and exposed to high-intensity (10 mW/cm2) or low-intensity (5 mW/cm2) UVC for 1–5 s. Control plates were not exposed to UVC. The plates were incubated overnight at 37 °C and then enumerated. Three trials for each bacterial strain were conducted. Statistical analysis was carried out to determine if there were significant differences in bacterial growth between UVC intensities and exposure times. Overall, exposure to low or high intensity for 3–5 s resulted in consistent inhibition of bacterial growth, with reductions of 99.9–100 % for E. coli, 96.8–100 % for S. enterica, and 99.2–100 % for L. monocytogenes. The 1 s exposure time showed inconsistent results, with a 66.0–100 % reduction in growth depending on the intensity and bacterial strain. When the results for all strains within each species were combined, the 3–5 s exposure times showed significantly greater (p < 0.05) growth inhibition than the 1 s exposure time. However, there were no significant differences (p > 0.05) in growth inhibition between the high and low UVC intensities. The results of this study show that, in pure culture conditions, exposure to UVC with the CSI-D device for ≥3 s is required to achieve consistent reduction of E. coli, S. enterica, and L. monocytogenes.

Diversity and ice nucleation activity of Pseudomonas syringae in drone-based water samples from eight lakes in Austria.

Bacteria from the Pseudomonas syringae complex (comprised of at least 15 recognized species and more than 60 different pathovars of P. syringae sensu stricto) have been cultured from clouds, rain, snow, streams, rivers, and lakes. Some strains of P. syringae express an ice nucleation protein (hereafter referred to as ice+) that catalyzes the heterogeneous freezing of water. Though P. syringae has been sampled intensively from freshwater sources in the U.S. and France, little is known about the genetic diversity and ice nucleation activity of P. syringae in other parts of the world. We investigated the haplotype diversity and ice nucleation activity at -8 °C (ice+) of strains of P. syringae from water samples collected with drones in eight freshwater lakes in Austria. A phylogenetic analysis of citrate synthase (cts) sequences from 271 strains of bacteria isolated from a semi-selective medium for Pseudomonas revealed that 69% (188/271) belonged to the P. syringae complex and represented 32 haplotypes in phylogroups 1, 2, 7, 9, 10, 13, 14 and 15. Strains within the P. syringae complex were identified in all eight lakes, and seven lakes contained ice+ strains. Partial 16S rDNA sequences were analyzed from a total of 492 pure cultures of bacteria isolated from non-selective medium. Nearly half (43.5%; 214/492) were associated with the genus Pseudomonas. Five of the lakes (ALT, GRU, GOS, GOL, and WOR) were all distinguished by high levels of Pseudomanas (p ≤ 0.001). HIN, the highest elevation lake, had the highest percentage of ice+ strains. Our work highlights the potential for uncovering new haplotypes of P. syringae in aquatic habitats, and the use of robotic technologies to sample and characterize microbial life in remote settings.

Genomic analysis of Citrobacter from Australian wastewater and silver gulls reveals novel sequence types carrying critically important antibiotic resistance genes

Antimicrobial resistance (AMR) is a major public health concern, and environmental bacteria have been recognized as important reservoirs of antimicrobial resistance genes (ARGs). Citrobacter, a common environmental bacterium and opportunistic pathogen in humans and other animals, has been largely understudied in terms of its diversity and AMR potential. Whole-genome (short-read) sequencing on a total of 77 Citrobacter isolates obtained from Australian silver gull (Chroicocephalus novaehollandiae) (n = 17) and influent wastewater samples (n = 60) was performed, revealing a diverse Citrobacter population, with seven different species and 33 sequence types, 17 of which were novel. From silver gull using non-selective media we isolated a broader range of species with little to no mobilised ARG carriage. Wastewater isolates (selected using Carbapenem- Resistant Enterobacterales (CRE) selective media) carried a heavy burden of ARGs (up to 21 ARGs, conferring resistance to nine classes of antibiotics), with several novel multidrug-resistant (MDR) lineages identified, including C. braakii ST1110, which carried ARGs conferring resistance to eight to nine classes of antibiotics, and C. freundii ST1105, which carried two carbapenemase genes, blaIMP-4 in class 1 integron structure, and blaKPC-2. Additionally, we identified an MDR C. portucalensis isolate carrying blaNDM-1, blaSHV-12, and mcr-9. We identified IncC, IncM2, and IncP6 plasmids as the likely vectors for many of the critically important mobilised ARGs. Phylogenetic analyses were performed to assess any epidemiological linkages between isolation sources, demonstrating low relatedness across sources beyond the ST level. However, these analyses did reveal some closer relationships between strains from disparate wastewater sources despite their collection some 13,000 km apart. These findings support the need for future surveillance of Citrobacter populations in wastewater and wildlife populations to monitor for potential opportunistic human pathogens.

Antimicrobial resistance of Escherichia coli in the UK: comparison of single vs. pooled samples from healthy pigs.

This study compared the antimicrobial resistance (AMR) of Escherichia coli detected from single samples vs. pooled samples at herd level. The national monitoring dataset included isolates from one sample per pig holding, whereas the research study included isolates from pooled samples of 10 pigs per holding. In both datasets, caecal samples were collected from healthy pigs randomly selected at slaughterhouses and plated on non-selective and antibiotic selective media. Resistance against a panel of nine antibiotics was compared between datasets by generalized linear mixed effects models (GLMMs) and by bootstrapped generalized linear model (GLM) to account for pooling. The highest proportion of resistant E. coli was observed against tetracycline and ampicillin in both datasets. In non-selective media, single and pooled samples showed similar results, but the bootstrapped GLM detected significantly lower resistance to ciprofloxacin and nalidixic acid in the national dataset. In selective media, a significantly greater proportion of resistant isolates was observed in the research dataset for ceftazidime (OR: 0.05, 95%CI=0.01-0.42) and nalidixic acid (OR: 0.15, 95%CI=0.05-0.51). The results suggest that one sample per holding provides similar information on AMR at herd level as pooled samples for most of the tested antibiotics, although less resistance to ciprofloxacin, ceftazidime, and nalidixic acid was detected.

THE EFFECT OF DENTAL IMPLANTS ON AEROBIC BACTERIA COLONIZATION IN THE ORAL CAVITY AND THE ANTIBIOTIC PROFILE OF COMMON ISOLATED AEROBIC BACTERIA

Background and aims: The mouth's microflora may alter as a result of dental implants. The purpose of this study was to examine the composition of aerobic bacteria in patients with dental implants and those who had natural teeth (without implants) as well as the response of those bacteria to antibiotic treatment. Methods: Bacteriological tests were performed on 72 patients (36 dental implants and 36 natural teeth) who visited dental clinics run by Sana'a University's Faculty of Dentistry and private dental clinics. Antibiotic susceptibility tests and culture trials were carried out at the National Center for Public Health Laboratories (NCPHL) in Sana'a, Yemen. Swabs were taken from the mucous membrane of the palate and the dorsum of the tongue from both groups, and cultured on selective and non-selective solid medium, in addition to culturing the swabs on medium enriched with 5% blood, then all cultured plates were incubated to microaerophilic conditions (5% CO2) and oxygenated in 37oC for 48 hours. Then bacterial growth was identified by standard methods. Results: In implant patients, the rate of bacterial isolates from the palate and tongue was slightly higher for potentially harmful bacteria such as E. coli (8.3% in tongue implant patients vs. 2.8% in non-implant patients) and Pseudomonas aeruginosa (5.6% versus 0%). While in viridians Streptococcus including S. mutans, there was a higher colonization rate in implants patients (83.3% in the palate verses, 75% in the palate of individuals without implants). A low level of oxacillin resistance (5.1%) in S. mutans isolates but S. mutans had a substantial level of tetracycline resistance (55.93%), 11.9% for co-trimoxazole, 10.2% for erythromycin, and just 1.7% for clindamycin. Conclusion: The study found that pathogenic bacteria like E. coli and Pseudomonas aeruginosa were isolated from the palate and the back of the tongue swabs at a slightly elevated rate in implant patients; also colonization rates of Streptococcus viridians, including S. mutans, were higher in implant patients compared to those without implants. There was a significant levels of antibiotics resistance in S. aureus, CoNs and S. viridians oral isolates in both groups of tested individuals. Peer Review History: Received: 14 June 2023; Revised: 13 July; Accepted: 17 August, Available online: 15 September 2023 Academic Editor: Dr. A.A. Mgbahurike, University of Port Harcourt, Nigeria, amaka_mgbahurike@yahoo.com Received file: Reviewer's Comments: Average Peer review marks at initial stage: 6.0/10 Average Peer review marks at publication stage: 8.0/10 Reviewers: Dr. Rola Jadallah, Arab American University, Palestine, rola@aauj.edu Dr. Bilge Ahsen KARA, Ankara Gazi Mustafa Kemal Hospital, Turkey, ahsndkyc@gmail.com

A comparative study for optimization of MALDI-TOF MS identification of filamentous fungi.

To evaluate and compare the performance of three commercial culture media, two filamentous fungi libraries, and two different protein extraction procedures in MALDI-TOF MS fungal identification. A total of 21 quality control samples were cultured on Sabouraud dextrose agar (SDA), ID fungi plate medium (IDFP), and Sabouraud gentamicin chloramphenicol 2 agar (SGC2). For four consecutive days, fungal growths were inoculated on a MALDI target plate both by using a direct transfer technique (DT) and by using a formic acid-ethanol protein extraction procedure (EEP). The MALDI-TOF MS-generated spectra were identified by the MBT Bruker library and the MSI database. Selective culture media (IDFP and SGC2) significantly outperformed the non-selective SDA medium. IDFP was superior to the SGC2 medium for dermatophyte identification. The EEP only demonstrated a benefit over DT in the underperforming SDA medium. The MBT Bruker library outperformed the MSI database in Aspergillus identification while the MSI database outperformed the MBT library in dermatophyte identification. For non-Aspergillus fungi, the libraries performed comparably. The results of our study show the necessity of using selective culture media (IDFP and SGC2) for fungal identification with MALDI-TOF MS and demonstrate no significant benefit of the formic acid-ethanol protein extraction technique in these media. Given the relative strengths and weaknesses of the MBT library and the MSI database, it might currently be beneficial to consider these libraries as complementary and employ both databases to achieve optimal fungal identification.

Combination of the electronic nose with microbiology as a tool for rapid detection of Salmonella

Salmonella is one of the most important foodborne pathogens and its analysis in raw and processed products is mandatory in the food industry. Although microbiological analysis is the standard practice for Salmonella determination, these assays are commonly laborious and time-consuming, thus, alternative techniques based on easy operation, few manipulation steps, low cost, and reduced time are desirable. In this paper, we demonstrate the use of an e-nose based on ionogel composites (ionic liquid + gelatine + Fe3O4 particles) as a complementary tool for the conventional microbiological detection of Salmonella. We used the proposed methodology for differentiating Salmonella from Escherichia coli, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Staphylococcus aureus in nonselective medium: pre-enrichment in brain heart infusion (BHI) (incubation at 35 °C, 24 h) and enrichment in tryptone soy agar (TSA) (incubation at 35 °C, 24 h), whereas Salmonella differentiation from E. coli and P. fluorescens was also evaluated in selective media, bismuth sulfite agar (BSA), xylose lysine deoxycholate agar (XLD), and brilliant green agar (BGA) (incubation at 35 °C, 24 h). The obtained data were compared by principal component analysis (PCA) and different machine learning algorithms: multilayer perceptron (MLP), linear discriminant analysis (LDA), instance-based (IBk), and Logistic Model Trees (LMT). For the nonselective media, under optimized conditions, taking merged data of BHI + TSA (total incubation time of 48 h), an accuracy of 85% was obtained with MLP, LDA, and LMT, while five separated clusters were presented in PCA, each cluster corresponding to a bacterium. In addition, for evaluation of the e-nose for discrimination of Salmonella using selective media, considering the combination of BSA + XLD and total incubation of 72 h, the PCA showed three separated and well-defined clusters corresponding to Salmonella, E. coli, and P. fluorescens, and an accuracy of 100% was obtained for all classifiers.

Resuscitation of viable but nonculturable bacteria promoted by ATP-mediated NAD+ synthesis

IntroductionEntry into the viable but nonculturable (VBNC) state is a survival strategy adopted by bacteria to survive harsh environment. Although VBNC cells still have metabolic activity, they lose the ability to form colonies on nonselective culture media. Thus, conventional bacterial detection methods, such as plate counting, are unable to detect the presence of VBNC cells. When the environmental conditions are appropriate, VBNC cells can initiate resuscitation, posing a great risk to the safety of public health. The study of the VBNC resuscitation mechanism could provide new insights into the prevention and control of VBNC resuscitation. ObjectivesUncovering the molecular mechanism of VBNC cell resuscitation by investigating the role of O-antigen ligase (RfaL) in inhibiting the resuscitation of Escherichia coli O157:H7 in the VBNC state. MethodsRfaL was screened and verified as a resuscitation inhibitor of VBNC Escherichia coli O157:H7 by detecting resuscitation curve and time-lapse microscopy. The mechanism of RfaL impacts VBNC E. coli resuscitation was investigated by detecting the single cell ATP content, metabolomic changes, NAD(H) content and new protein biosynthesis of WT and ΔrfaL at different stage of resuscitation. ResultsMutation of rfaL, which encoded an O-antigen ligase, markedly shortened the resuscitating lag phase. Further studies indicated that ΔrfaL VBNC cells contained higher ATP levels, and ATP consumption during the resuscitating lag phase was highly correlated with resuscitation efficiency. Metabolomic analysis revealed that ATP was utilized to activate the Handler and salvage pathways to synthesize NAD+, balancing redox reactions to recover cell activity and promote cell resuscitation. ConclusionOur findings revealed a strategy employed by VBNC cells for revival, that is, using residual ATP to primarily recover metabolic activity, driving cells to exit dormancy. The synthesis pathway of lipopolysaccharide (LPS) in rfaL null mutant was inhibited and could supply more ATP to synthesis NAD+ and promote resuscitation.

Impact of preculture temperature on peracetic acid-induced inactivation and sublethal injury of L. monocytogenes and subsequent growth potential of single cells

The disinfectant peracetic acid (PAA) that is used in the food industry can cause sublethal injury in L. monocytogenes. The effect of preculture temperature on the inactivation and sublethal injury of L. monocytogenes cells due to PAA was evaluated by plating on non-selective and selective agar medium supplemented with 5 % (w/v) NaCl. L. monocytogenes cells were precultured at 30 °C, 20 °C or 4 °C, and the former was used as reference temperature. Preculture of cells at 20 °C or 4 °C and subsequent exposure to PAA at the respective growth temperatures caused higher injury compared to cells grown at 30 °C and exposed to PAA 20 °C and PAA 4 °C, respectively. Survival was also affected by the preculture temperature; 20 °C-grown cultures resulted in lower survival at PAA 20 °C. Nevertheless, preculture at 4 °C resulted in a similar number of surviving cells when exposed to PAA 4 °C compared to cells precultured at 30 °C and exposed to PAA at 4 °C. Flow cytometry was subsequently used to quantify outgrowth capacity of stressed and sublethal damaged populations following sorting of single cells in nutrient rich medium (Tryptone soy broth supplemented with yeast extract [TSBY]). PAA treatment affected the outgrowth of L. monocytogenes at single-cell level resulting in increased outgrowth-times reflecting higher single cell heterogeneity. To conclude, the response of L. monocytogenes when exposed to PAA depended on the preculture conditions, and the highly heterogeneous outgrowth potential of PAA-injured cells may affect their detection accuracy and pose a food safety risk.

Bacterial contamination of water used as thermal transfer fluid in fluid-warming devices

Recent reports implied heater-cooler units (HCU), which are used for warming of infusions, blood or in ECMO devices, as possible origin of healthcare-associated infections (HAIs) with potentially pathogenic bacteria like nontuberculous mycobacteria [1]. This represents a source of contamination in a usually sterile setting. Aim of this study is to analyse water from infusion heating devices (IHDs) for bacterial contamination and to raise the question if IHDs act as a possible source in the transmission of HAIs. 300-500 ml of thermal transfer fluid (TTF) derived from the reservoirs of 22 independent IHDs were collected and processed on different selective and non-selective media for colony count and identification of bacteria. Strains of Mycobacterium species (spp.) were further analysed by whole genome sequencing. Bacterial growth was observed in each of the 22 collected TTFs after cultivation at 22°C and 36°C. Pseudomonas aeruginosa was the most frequent pathogen identified, being present in 13.64% (3/22) at > 100 CFU/100mL. Colonization with Mycobacterium chimaera, Ralstonia pickettii and Ralstonia mannitolilytica was detectable in 9.09% (2/22) of the isolates. Primary sequencing of the detected M. chimaera suggests a close relationship to a M. chimaera strain detected in an outbreak in Switzerland, which led to the death of two patients. A contamination of the TTF represents a germ reservoir in a sensitive setting. Handling errors of the IHD may lead to distribution of opportunistic or facultative bacterial pathogens, increasing the risk of nosocomial infection transmission.

Barotolerance of Acid-adapted and Cold-adapted Bacterial Isolates of E. coli O157:H7, Salmonella spp., and L. monocytogenes in an Acidic Buffer Model

The fruit and vegetable juice industry has shown a growing trend in minimally processed juices. A frequent technology used in the production of functional juices is cold pressure, which refers to the application of high pressure processing (HPP) at low temperatures to inactivate foodborne pathogens. HPP juice manufacturers are required to demonstrate a 5-log reduction of the pertinent microorganism to comply with FDA Juice HACCP. However, there is no consensus on validation study approaches for bacterial strain selection or their preparation. Individual bacterial strains were grown using three different growth conditions: neutral, cold-adapted, and acid-adapted. Approximately 6.0–7.0 log CFU/mL of the matrix-adapted bacterial strains were inoculated individually into buffered peptone water (BPW) at pH 3.50 ± 0.10 (HCl adjusted) and treated at sublethal pressures of 500 MPa for Escherichia coli O157:H7 and 200 MPa for Salmonella spp. and Listeria monocytogenes (180 s, 4°C). Analyses were conducted at 0, 24, and 48 h (4°C storage) post-HPP on nonselective media. E. coli O157:H7 exhibited greater barotolerance than Salmonella spp. and L. monocytogenes. In neutral growth conditions, E. coli O157:H7 strain TW14359 demonstrated the greatest resistance (2.94 ± 0.64 log reduction), and E. coli O157:H7 strain SEA13B88 was significantly more sensitive (P < 0.05). Salmonella isolates, neutral and acid-adapted, expressed similar barotolerance to one another. Cold-adapted S. Cubana and S. Montevideo showed greater resistance compared to other cold-adapted strains. Acid-adapted L. monocytogenes strain MAD328 had <1.00 ± 0.23 log reduction while acid-adapted L. monocytogenes strains CDC and Scott A were significantly more sensitive (P < 0.05) with reductions of 2.13 ± 0.48 and 3.43 ± 0.50 log CFU/mL, respectively. These results suggested, under the conditions tested, bacterial strain and preparation methods influence HPP efficacy and should be considered when conducting validation studies.

Evaluation of four environmental sampling methods for the recovery of multidrug-resistant organisms

Background: Environmental contamination is a major risk factor for multidrug-resistant organism (MDRO) exposure and transmission in the healthcare setting. Sponge-stick sampling methods have been developed and validated for MDRO epidemiological investigations, leading to their recommendation by public health agencies. However, similar bacteriological yields with more readily available methods that require less processing time or specialized equipment have also been reported. We compared the ability of 4 sampling methods to recover a variety of MDRO taxa from a simulated contaminated surface. Methods: We assessed the ability of (1) cotton swabs moistened with phosphate buffer solution (PBS), (2) e-swabs moistened with e-swab solution, (3) cellulose-containing sponge sticks (CSS), and (4) non–cellulose-containing sponge sticks (NCS) to recover extended-spectrum β-lactamase (ESBL)–producing Escherichia coli, carbapenem-resistant Pseudomonas aeruginosa (CRPA), carbapenem-resistant Acinetobacter baumannii (CRAB), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), and a mixture that contained VRE, MRSA, and ESBL organisms. A solution of known bacterial inoculum (~105 CFU/mL) was made for each MDRO. Then, 1 mL solution was pipetted on a stainless-steel surface (8 × 12 inch) in 5 µL dots and allowed to dry for 1 hour. All samples were collected by 1 individual to minimize variation in technique. Sponge sticks were expressed in PBS containing 0.02% Tween 80 using a stomacher, were centrifuged, and were then resuspended in PBS. Cotton and e-swabs were spun in a vortexer. Then, 1 mL of fluid from each method was plated to selective and nonselective media in duplicate and incubated at 35°C for 24 hours (MRSA plates, 48 hours) (Fig. 1). CFU per square inch and percentage recovery were calculated. Results: Table 1 shows the CFU per square inch and percentage recovery for each sampling method–MDRO taxa combination. The percentage recovery varied across MDRO taxa. Across all methods, the lowest rate of recovery was for CRPA and the highest was for VRE. Regardless of MDRO taxa, the percentage recovery was highest for the sponge stick (CSS and NCS) compared to swab (cotton and E-swab) methods across all taxa (Table 1 and Fig. 2).Conclusions: These findings support the preferential use of sponge sticks for the recovery of MDROs from the healthcare environment, despite the additional processing and equipment time needed for sponge sticks. Further studies are needed to assess the robustness of these findings in noncontrived specimens as well as the comparative effectiveness of different sampling methods for non–culture-based MDRO detection.Disclosure: None