Appl. Microbiol., Volume 1, Issue 1 (June 2021) – 12 articles

Healthcare-associated infections (HAIs) have been increasing during recent decades, leading to long hospital stays and high morbidity and mortality rates. The usage of antibiotics therapy against these infections is enhancing the emergence of more multiple-drug resistant strains, in particular in Staphylococcus epidermidis. Hence, this study focused on the resistance pattern of S. epidermidis isolates from clinical settings and its association with phenotypic and molecular traits. Our results showed that HAIs were more prevalent among infants and older adults, and the most frequent type of HAI was central line-associated bloodstream infection. Half of the patients received antibiotic therapy before laboratory diagnosis. Preceding microbiological diagnosis, the number of patients receiving antibiotic therapy increased by 29.1%. Eighty-six per cent of the clinical isolates presented a multidrug resistance (MDR) profile, and a quarter were strong biofilm producers. Furthermore, polysaccharide intercellular adhesin (PIA)-dependent biofilms presented higher biomass production (p = 0.0041) and a higher rate of antibiotic non-susceptibility than PIA-independent biofilms, emphasizing the role of icaABDC operon in infection severity. Therefore, this study suggests that a thorough understanding of the phenotypic and molecular traits of the bacterial cause of the HAIs may lead to a more suitable selection of antibiotic therapy, improving guidance and outcome assessment. Full article

Proper transcriptional regulation depends on the collaboration of multiple layers of control simultaneously. Cells tightly balance cellular resources and integrate various signaling inputs to maintain homeostasis during growth, development and stressors, among other signals. Many eukaryotes, including the budding yeast Saccharomyces cerevisiae, exhibit a non-random distribution of functionally related genes throughout their genomes. This arrangement coordinates the transcription of genes that are found in clusters, and can occur over long distances. In this work, we review the current literature pertaining to gene regulation at a distance in budding yeast. Full article

Soil amendments with organic waste products (OWPs) have been widely supported in Europe to improve soil fertility, causing wide changes in the microbial community structure and diversity, especially in the short-term period. Those changes are known to affect the volatile organic compound (VOC) emissions by soil. This work aimed to characterize, in terms of quantity and composition, the effect of green waste and sludge (GWS) application on soil VOC emissions and microbial community 49 h after the last GWS application. Two different soil samples were compared to test the effect of the soil history on VOC emissions and microbial communities. For this reason, we chose a soil that received GWS input for 20 years (GWS sample) and one that did not receive any organic input during the same period (CN sample). Furthermore, samples were manipulated to generate three microbial dilution diversity gradients (low, medium, and high). Results showed that Bacteroidetes phyla took advantage of the GWS application in all samples, increasing their relative abundance by 22% after 49 h, while the Proteobacteria phylum was penalized by the GWS amendment, passing from 58% to 49% relative abundance 49 h after the GWS application. Microbial structure differences between microbial diversity dilution levels remained even after the GWS application. GWS amendment induced a change in the emitted VOC profiles, especially in samples used to receiving GWS. GWS amendment doubled the VOC emissions from samples used to receiving GWS after 49 h. Finally, the microbial community was strongly correlated to the VOC emissions. Firmicutes, Proteobacteria, Actinobacteria, and Crenarchaeota were positively correlated (Pearson coefficient > 0.6), while other phyla, such as Bacteroidetes and Verrucomicrobia, were found to be negatively correlated (Pearson coefficient < −0.6) to the VOC emissions. After the addition of GWS, these correlations shifted from positive to negative and from negative to positive. Full article

In the US, sodium and potassium nitrite are regulated food preservatives that prevent the germination of Clostridium spores in cured and processed meats. In recent years, the use of vegetable-derived nitrite (i.e., vegetable nitrate fermented to nitrite) has been designated as ‘natural nitrite’ to accommodate natural meats that cannot use artificial ingredients, and such meat products can be labelled as having ‘no added preservatives’. This new status and labelling allowance for microbially-modified nitrite provides for a ‘clean label’ application of nitrite against the stigma of chemical ingredients and has found increased use within the processed meat industry. The objectives of this study were to examine Clostridium sporogenes as a pathogen-surrogate challenge organism and the use of vegetable (celery) nitrite to prevent spore germination in cooked meat products. A three-strain spore crop of C. sporogenes ATCC 3584, ATCC 19404 and ATCC BAA-2695 was applied during ingredient formulation of low and high-fat hotdogs that were divided into three sub-batches (control without nitrite, hotdogs with sodium nitrite, hotdogs with celery nitrite). In both low and high-fat processes, sodium nitrite was compared to hotdogs made with comparable levels of celery nitrite (156 ppm). All treatments were performed with duplicate trial replication and triplicate sample testing within each trial. Comparisons were analyzed by repeated measures analysis of variance to determine significant difference (p < 0.05) of time course treatments. In shelf-life assays, growth was inhibited at both 5 °C and 15 °C, even if nitrite was absent; however, spore germination and growth readily occurred at 35 °C. Comparison of nitrite effects was best evaluated at 35 °C as a permissive condition to examine the effects of nitrite treatments. Celery nitrite showed no significant difference from sodium nitrite when used in both low and high-fat hotdogs, and spore outgrowth was only observed after 2–3 days at 35 °C compared to hotdogs without nitrite. Application of bacteriocin preparations in the formulation that were effective against Listeria monocytogenes, and moderately inhibitory towards the 3-strain spore mixture of C. sporogenes, were not effective in spore control in manufactured hotdogs. The nitrite validation hotdog trials described herein demonstrates that (celery or sodium) nitrite may prevent Clostridium spore germination for 24–48 h even under permissive conditions to help keep processed meat safe. Full article

Foodborne pathogens are known to adhere strongly to surfaces and can form biofilms in food processing facilities; therefore, their potential to contaminate manufactured foods underscores the importance of sanitation. The objectives of this study were to (1) examine the efficacy of a new-generation sanitizer (Decon7) on Staphylococcus and Pseudomonas biofilms, (2) identify biofilm bacteria from workers’ boots in relation to previous sanitizer chemistry, (3) validate the efficacy of Decon7 on biofilm from workers’ boots from an abattoir/food processing environment, and (4) compare the sensitivity of isolated boot biofilm bacteria to new- and early (Bi-Quat)-generation QAC sanitizers. Decon7 was applied at two concentrations (5%, 10%) and was shown to be effective within 1 min of exposure against enhanced biofilms of Staphylococcus spp. and Pseudomonas spp. in 96-well microplates. Decon7 was also used to treat workers’ boots that had accumulated high levels of biofilm bacteria due to ineffective sanitization. Bacteria isolated before enzyme/sanitizer treatment were identified through 16S rRNA PCR and DNA sequencing. All treatments were carried out in triplicate and analyzed by one-way RM-ANOVA or ANOVA using the Holm–Sidak test for pairwise multiple comparisons to determine significant differences (p < 0.05). The data show a significant difference between Decon7 sanitizer treatment and untreated control groups. There was a ~4–5 log reduction in Staphylococcus spp. and Pseudomonas spp. (microplate assay) within the first 1 min of treatment and also a > 3-log reduction in the bacterial population observed in the biofilms from workers’ boots. The new next-generation QAC sanitizers are more effective than prior QAC sanitizers, and enzyme pre-treatment can facilitate biofilm sanitizer penetration on food contact surfaces. The rotation of sanitizer chemistries may prevent the selective retention of chemistry-tolerant microorganisms in processing facilities. Full article

Droplet digital polymerase chain reaction (ddPCR) is a method used to detect and quantify nucleic acids even when present in exceptionally low numbers. While it has proven to be valuable for clinical studies, it has failed to be widely adopted for environmental studies but despite some limitations, ddPCR may represent a better option than classical qPCR for environmental samples. Due to the complexity of the chemical and biological composition of environmental samples, protocols tailored to clinical studies are not appropriate, and results are difficult to interpret. We used environmental DNA samples originating from field studies to determine a protocol for environmental samples. Samples included field soils which had been inoculated with the soil fungus Rhizophagus irregularis (environmental positive control), field soils that had not been inoculated and the targeted fungus was not naturally present (environmental negative control), and root samples from both field categories. To control for the effect of soil inhibitors, we also included DNA samples of an organismal control extracted from pure fungal spores (organismal positive control). Finally, we included a no-template control consisting only of the PCR reaction reagents and nuclease free water instead of template DNA. Using original data, we examined which factors contribute to poor resolution in root and soil samples and propose best practices to ensure accuracy and repeatability. Furthermore, we evaluated manual and automatic threshold determination methods and we propose a novel protocol based on multiple controls that is more appropriate for environmental samples. Full article

Biopesticides, which consist of antagonistic microorganisms, or the bioactive compounds they produce offer attractive alternatives to synthetic agrochemicals as a means of reducing plant diseases in crop production systems. In our previous study, a strain of Pseudomonas entomophila 23S was reported to have a strong antagonistic activity toward Clavibacter michiganensis subsp. michiganensis (Cmm), a pathogen of tomato bacterial canker disease, which causes serious damage to the tomato production industry. This study focused on purification and characterization of the anti-Cmm compound(s) produced by P. entomophila 23S. The cell-free supernatant was first subjected to butanol extraction, fractioned by solid-phase extraction (SPE), and purified by high-performance liquid-chromatography (HPLC). The semi-preparative purified compounds retained anti-Cmm activity. These compounds were then characterized by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) and nuclear magnetic resonance (NMR). Two anti-Cmm compounds, C₁₅H₁₆N₂O and C₁₆H₁₈N₂O, were isolated by this procedure. From the LC/MS spectra, the compounds were predicted to have an isoquinoline ring, the presence of which was confirmed by NMR. The compounds differed with respect to the presence of a methyl group in a side chain. In addition, P. entomophila 23S grown in different culture media produced the greatest amounts of these anti-Cmm compounds when in Nutrient Broth (NB), where bacterial growth was the slowest. NB composition is more favorable in stimulating P. entomophila 23S to produce higher amounts of these anti-Cmm compounds. Full article

Brevundimonas is a genus of freshwater bacteria belonging to the family Caulobacteraceae. The present study describes two novel species of the genus Brevundimonas (LVF1^T and LVF2^T). Both were genomically, morphologically, and physiologically characterized. Average nucleotide identity analysis revealed both are unique among known Brevundimonas strains. In silico and additional ProphageSeq analyses resulted in two prophages in the LVF1^T genome and a remnant prophage in the LVF2^T genome. Bacterial LVF1^T cells form an elliptical morphotype, in average 1 µm in length and 0.46 µm in width, with a single flagellum. LVF2^T revealed motile cells approximately 1.6 µm in length and 0.6 µm in width with a single flagellum, and sessile cell types 1.3 µm in length and 0.6 µm in width. Both are Gram-negative, aerobic, have optimal growth at 30 °C (up to 0.5 to 1% NaCl). Both are resistant towards erythromycin, meropenem, streptomycin, tetracycline and vancomycin. Anaerobic growth was observed after 14 days for LVF1^T only. For LVF1^T the name Brevundimonas pondensis sp. nov. and for LVF2^T the name Brevundimonas goettingensis sp. nov. are proposed. Type strains are LVF1^T (=DSM 112304^T = CCUG 74982^T = LMG 32096^T) and LVF2^T (=DSM 112305^T = CCUG 74983^T = LMG 32097^T). Full article

The skin microbiome is an important part of amphibian immune defenses and protects against pathogens such as the chytrid fungus Batrachochytrium dendrobatidis (Bd), which causes the skin disease chytridiomycosis. Alteration of the microbiome by anthropogenic factors, like pesticides, can impact this protective trait, disrupting its functionality. Chlorothalonil is a widely used fungicide that has been recognized as having an impact on amphibians, but so far, no studies have investigated its effects on amphibian microbial communities. In the present study, we used the amphibian Lithobates vibicarius from the montane forest of Costa Rica, which now appears to persist despite ongoing Bd-exposure, as an experimental model organism. We used 16S rRNA amplicon sequencing to investigate the effect of chlorothalonil on tadpoles’ skin microbiome. We found that exposure to chlorothalonil changes bacterial community composition, with more significant changes at a higher concentration. We also found that a larger number of bacteria were reduced on tadpoles’ skin when exposed to the higher concentration of chlorothalonil. We detected four presumed Bd-inhibitory bacteria being suppressed on tadpoles exposed to the fungicide. Our results suggest that exposure to a widely used fungicide could be impacting host-associated bacterial communities, potentially disrupting an amphibian protective trait against pathogens. Full article

In the US, sodium nitrate is used as a preservative and curing agent in processed meats and is therefore a regulated ingredient. Nitrate reducing bacteria (NRB) can convert vegetable nitrate into nitrite allowing green/clean label status in the US as per the USDA-FSIS definition of ‘natural nitrite’. The current ‘in-liquid’ test tube assay for detecting nitrite is not suitable for screening mixtures of bacteria nor is commercial nitrate broth suitable for growth of many Gram (+) bacteria. M17 broth was therefore used to develop M17-nitrate broth to be inclusive of Gram (+) bacteria. An ‘on-agar’ colony-screening assay was developed to detect the conversion of nitrate to nitrite on agar plates and could detect one NRB⁺ colony among ~300–500 colonies on a single plate. Samples that might have NRB were spread-plated on M17 agar plates, sandwiched with nitrate agar, and after incubation followed with sequential agar overlays containing the reagents used in the nitrate reduction assay; the appearance of red color zones above colonies indicated the presence of nitrite. NRB derived from various samples were confirmed for nitrate conversion and both nitrate and nitrite were quantified by C₈ reversed-phase (RP) ion-pairing high performance liquid chromatography (HPLC) analysis (1 ppm limit of detection). Staphylococcus carnosus, a strain commonly used for nitrate reduction, was able to convert 1100 ppm M17-nitrate broth to 917 ppm nitrite. Staphylococcus caprae and Panteoa agglomerans, NRB isolated using the M17-nitrate agar assay, were also able to ferment the same broth to 916 ppm and 867 ppm nitrite, respectively. This is the first report of an on-agar colony screening assay for the detection and isolation of nitrite reducing bacteria allowing NRB to be readily isolated. This may allow for the identification of new bacteria that may have a more efficient process to generate nitrite, and possibly concomitant with production of additional natural antimicrobials, as vegetable nitrite becomes more widely used to prevent spore germination. Full article

The aim of the present study was to assess the commercial potential of the Origanum vulgare ssp. hirtum essential oil (OEO) as a natural intrinsic hurdle against common spoilage and pathogenic microbes in tomato juice. The main volatile compounds of the OEO identified by gas chromatography mass spectrometry (GC/MS) analysis were thymol and carvacrol, accounting for approximately 48% and 27%, respectively. Its activity against common food spoilage and pathogenic microbes was confirmed and the minimum inhibitory concentration (MIC), non-inhibitory concentration (NIC), and minimum lethal concentration (MLC) values were determined. OEO effectiveness was further validated in commercial tomato juice. Supplementation of tomato juice with OEO at concentrations lower than the MIC (350 ppm) resulted in significant delay of food spoilage and extension of the product’s shelf-life, as well as in inhibition of Listeria monocytogenes, Clostridium difficile, Saccharomyces cerevisiae, and Aspergillus niger growth after deliberate inoculation in both room and refrigerated temperatures. In conclusion, the results suggested that OEO may be used as an efficient intrinsic inhibitor of food spoilage and growth of pathogenic microbes in tomato juice. Full article