In this study, we analyzed the srtE gene from Corynebacterium glutamicum ATCC 13032, which codes for class E sortase, a transpeptidase involved in attaching surface proteins to the cell wall peptidoglycan. The surface proteins contain an N-terminal leader sequence and a C-terminal sorting signal which consist of a LAXTG motif, a transmembrane region, and a few positively charged amino acids. Sortase E deletion or its overexpression alters the attachment of the surface proteins to the cell wall peptidoglycan; however, the effects on morphology and bacterial physiology have not been studied. Thus, we constructed three C. glutamicum derivatives such as srtE deletion mutant, complemented and overexpressed strains to monitor the possible impact of the gene on cell growth, morphology, and physiological changes. Interestingly, deletion of the gene did not show any change in growth or morphology in C. glutamicum but showed a decrease in cell surface hydrophobicity and heat stress. However, the cells overexpressing the protein not only showed elongated cell morphology and a reduction in hydrophobicity when compared to wild-type and complemented strain, but also showed an increased sensitivity to heat. These results suggest that C. glutamicum sortase E deletion or overexpression causes sorting intermediates to accumulate, altering cellular morphology and physiology and adversely impacting the membrane integrity.

The extreme resistance of bacterial spores to sterilization makes them a major concern to the food industry and consumers. In this study, the effect of glucose on the inactivation of Bacillus subtilis spores by high pressure thermal sterilization (HPTS) was evaluated. The results showed that the protective effects of glucose increased with the increase in its concentration. Compared with the HPTS control (no addition of glucose), the activity of Na+/K+-ATPase was increased, the leakage of proteins and the release of 2,6-pyridine dicarboxylic acid (DPA) was decreased, and the vibrational strength of the functional group P = O was reduced by the addition of glucose. At the same time, glucose treatment increased the content of α-helix by 6%-22%, while decreased the random coil content by 5%-13% of the cellular protein. In conclusion, the addition of glucose protected the cell membrane, Na+/K+-ATPase, cellular nucleic acids and proteins of B. subtilis under HPTS treatment.

Clostridioides difficile is a spore-forming anaerobic Gram-positive bacterium responsible for a broad spectrum of intestinal symptoms and healthcare-associated diarrhoea. The hypothesis of this work was that different in vitro conditions, notably pH and human faecal microbiota composition, impact the germination and/or the growth of C. difficile. This study aimed to correlate growth kinetics of the bacterium with these two physiochemical parameters by using a static in vitro model. To better understand the initial gut colonisation, several growth curve assays were carried out to monitor the behaviour of the spores and vegetative forms of C. difficile strain 078 under different conditions mimicking the gut environment. When the faeces were added, no spore germination or growth was observed, but C. difficile spores germinated in vitro when the pH was maintained between 6.6 and 6.9 for four different faeces donors. The evolution of microbiota studied by 16S rDNA profiling showed high proportions of Enterobacteriaceae and E. coli/Shigella when C. difficile grew, regardless of the inoculated faeces. This model helped us to understand that the germination and growth of C. difficile are strongly pH dependent, and further research is needed to evaluate the potential impact of the gut microbiota composition on C. difficile.

Despite the many challenges faced by the sudden adaptation of the teaching-learning processes during the emergency remote teaching (ERT) imposed by the COVID-19 pandemic, this period allowed the exploration of innovative educational methods. Here, we report the description and evaluation of a didactic activity designed to foster an active learning environment among Veterinary Medicine undergraduate students enrolling in Microbiology classes during the ERT period at the University of Minas Gerais. The activity consisted of initial expositive classes, followed by students' active search for information, and the execution of a report and short comics covering the topic. The activity was evaluated by a voluntary postquestionnaire. The results suggest that the students had good emotional and educational perception toward the task, and that they noticed the elaboration of the comics as the most valuable tool aiding in the retention of microbiological concepts. We conclude that the proposed strategy, specially by the incorporation of the comics, helped the meaningful learning of microbiology.

Adherent-invasive Escherichia coli (AIEC) are isolated from inflammatory bowel disease (IBD) patients at a higher rate than from control patients. Using a collection of E. coli strains collected from Crohn's disease (CD), ulcerative colitis (UC), or non-IBD control patients, antibiotic and resistance to the antimicrobial peptides HBD-3 and LL-37 was assessed. Carriage of bacterial-encoded omptin protease genes was assessed by PCR and omptin protease activity was measured using a whole-cell based fluorescence assay. Elevated resistance to antibiotics and host defense peptides in IBD-associated AIEC were observed. IBD-associated strains showed increased (but statistically non-significant) antibiotic resistance. CD-associated strains showed greater (but statistically non-significant) resistance to HBD3-mediated killing while UC-associated strains showed statistically greater resistance to LL-37 mediated killing. High-level resistance to LL-37 was associated with carriage of omptin protease genes and with increased omptin protease activity. Antimicrobial host defense peptide resistance may be an adaptive feature of AIEC leading to enhanced pathogenesis during the initiation or progression of IBD.

Although probiotics have been isolated from different sources, few were isolated from traditional Chinese medicine. The current study firstly isolates Pulsatilla Radix-utilising Pediococcus pentosaceus PR-1 from human faeces. Subsequently, the tolerance of PR-1 to low pH, bile salts, simulated gastric juice and succus entericus, antioxidant activity, antimicrobial activity, cholesterol assimilation and antibiotics susceptibility were investigated. After 2 h of incubation at pH 2.0, over 80% of PR-1 survived. The cell viability of PR-1 at 2 h under 0.1% bile salt condition was 99.2%. The survival rate of PR-1 in gastric juice and succus entericus was 64.48% and 81.86%, respectively. Cell-free supernatant of PR-1 culture also showed antimicrobial activity against Escherichia coli, Staphylococcus aureus and Salmonella typhimurium. Besides, antioxidant activity of PR-1 CFS was significantly greater than cell pellet. PR-1 was shown to be resistant to kanamycin, streptomycin, vancomycin and norfloxacin and was able to lower the cholesterol level to 72.5±1.5%. In addition, PR-1 displayed γ-haemolysis and was non-pathogenic.

Salmonella is one of the most important foodborne and zoonotic pathogens, and monophasic S. Typhimurium is ranked among the top-five Salmonella serovars causing animal and human infections worldwide. Resistance to the third- and higher-generation cephalosporins in Salmonella has attracted great attention. Bacteria are frequently exposed to sub-minimal inhibitory concentrations (sub-MICs) of antimicrobials that can trigger diverse adaptive responses such as biofilm formation. Biofilms can promote bacterial defense to external and internal harsh conditions. This study aimed to investigate the effect of sub-MICs of cefotaxime, one of the third-generation cephalosporins, on biofilm formation by non-clinical S. enterica strains. Crystal violet staining demonstrated that cefotaxime at 1/8 MIC enhanced biofilm formation by two monophasic S. Typhimurium strains. Confocal laser scanning microscopy and enzymatic treatment assay revealed that cellulose was the most dominant extracellular matrix component contributing to Salmonella biofilm formation. Scanning electron microscopy demonstrated that cefotaxime treatment led to bacterial incomplete cell division and filamentous morphology during the whole process of biofilm formation. Our study is the first to report the enhancement effect of cefotaxime on non-clinical, monophasic S. Typhimurium by affecting bacterial morphology. The results will contribute to conducting risk assessments of Salmonella in the pork production chain and guiding the rational use of antimicrobial agents to reduce the risk of biofilm formation.

Many Gram-negative bacteria coordinate their gene expression via quorum sensing (QS) mediated by small diffusible autoinducer molecules such as Acyl Homoserine Lactones (AHL). The degradation of AHLs or quorum quenching (QQ) imparts less selection pressure on the target organisms leading to a possible alternative to antimicrobial agents and traditional biofilm control methods. Here, a novel strain of Bacillus velezensis, strain PM7, exhibiting extracellular QQ activity against Gram-negative bacteria has been isolated. Analytical studies of the AHL degradation using LC-MS as well as bioreporters revealed the mechanism of QQ as homoserine lactone ring hydrolysis. Using molecular techniques, the presence of an aiiA homologues gene, specific for acyl homoserine lactonase enzyme, was confirmed in the bacterium. A biofilm inhibitory activity in the range of 36.9%-77.4% was achieved by the PM7 against three different Gram-negative bacteria. Moreover, a significant reduction (P < .05) in the QS-controlled traits such as violacein production in Chromobacterium violaceum (73%), swarming motility and prodigiosin production (67.2%) in Serratia marcescens, and exopolysaccharide production (97.9%) in Pseudomonas aeruginosa, was also observed. The extracellular and broad-spectrum lactonase of this novel strain can be beneficial for its use in QQ applications.

Microbes produce and respond to a range of structurally and functionally diverse volatiles. Many microbial volatiles have antimicrobial properties. Since volatiles can diffuse through complex 3D systems like spider nests, they are promising pathogen protection for social arthropods. Here, we analyzed the volatilomes of five nest microbiome members of the Namibian, social spider Stegodyphus dumicola, namely the bacteria Massilia sp. IC2-278, Massilia sp. IC2-477, Sphingomonas sp. IC-11, Streptomyces sp. IC-207, and the fungus Aureobasidium sp. CE_32, and tested their antimicrobial activity against two putative spider pathogens, namely Bacillus thuringiensis and Purpureocillium lilacinum. Most nest microbiome members released volatilomes with antibacterial and/or antifungal activities under in vitro conditions. The analysis of their volatilomes using GC/Q-TOF revealed that they include numerous antimicrobial volatiles. We tested the antimicrobial activity of five pure volatile compounds found in the volatilomes and revealed that all of them were antibacterial and/or antifungal. We could not identify the same antimicrobial volatiles as in a previous in situ study, but our results indicate that social spider-associated microorganisms as a source of antimicrobial volatiles are important for pathogen inhibition. Additionally, we showed the influence of the volatilomes on the antibiotic sensitivity of B. thuringiensis offering novel approaches to counter antibiotic resistance.

Fungi often experience oxidative stress in response to the environment during growth. In this study, Aspergillus niger HY2, whose presence easily results in paddy mildew, was used to investigate the effects of different carbon sources on morphological development, reactive oxygen species (ROS) metabolites, and antioxidant enzymes activities. Morphological development mainly includes the changes in conidial production and colony diameter. ROS metabolites production include the content of hydrogen peroxide (H2O2), superoxide anion (O2·-), and malondialdehyde (MDA). The results indicated that A. niger HY2 adapted to H2O2 exposure by decreasing growth and increasing the activities of some antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Different carbon sources also affected the expression of the developmental-specific gene flbA and the oxidative stress tolerance-related gene cat. When incubated with glucose, sucrose, and xylose as carbon sources, A. niger exhibited stronger oxidative stress tolerance, but when incubated with maltose as a carbon source, A. niger exhibited relatively poor oxidative stress tolerance. Our results can provide a theoretical basis for further understanding mechanisms of metabolic adaptation and developing targeted strategies to control the spoilage caused by A. niger.