Microbiology Research

Background Antimicrobial resistance has become one of the most widespread threats to humans. Acinetobacter baumannii is one of the pathogens responsible for healthcare-associated infections (nosocomial). Colistin is considered the last resort antibiotic against infections with pandrug-resistant (PDR) pathogens. Results: Eleven isolates were detected phenotypically as PDR A. baumannii and were confirmed molecularly using 16S rDNA. The MCR-1 gene was not detected within the chromosomal DNA of the selected isolates. Plasmid bearing the MCR-1 gene was identified in 10 selected isolates of A. baumannii that had not been previously observed to carry the MCR-1 gene. Moreover, the use of colistin in combination with anionic antibiotics or natural compound pterostilbene poses a viable therapeutic alternative for PDR and revives colistin’s bactericidal effects on MCR-1-positive A. baumannii. Finally, the transmission electron microscopy studies proved the synergistic effect of these combinations and revealed the disruption of resistant A. baumannii’s outer membrane and alteration of the permeability properties that allowed overcoming the resistance of the isolates to colistin. Conclusions: Antimicrobial resistance of A. baumannii is related to the presence of the transferable plasmid-bearing MCR-1 gene. This study proved the ability of the combinations of colistin with anionic antibiotics and/or natural compound pterostilbene to restore the bactericidal effect of colistin. Overall, these combinations could be novel promising clinical alternatives against the increasing threat of the widespread multidrug-resistant A. baumannii. Full article

Alterations in faecal lactobacilli in autistic children have been reported, but little is known related to age and disorder severity. We used a culture-based method and partial 16S rRNA gene sequencing to isolate and identify lactobacilli strains from faeces of Tunisian autistic children (ASD group) and compared them with strains isolated from siblings (SIB) and children from the general population (GP). The ASD group displayed an increased number of different species compared to SIB and GP. Differences in species abundance with age accounted for a significant decrease in the abundance of Lactiplantibacillus plantarum/Lactiplantibacillus pentosus isolates in the GP at the age of 8–10 years compared to the age of 4–7 years, and to a significantly lower abundance of Lacticaseibacillus rhamnosus in the ASD group with respect to SIB and the GP at the age of 8–10 years. Simpson’s and Shannon–Wiener indices showed a more pronounced species diversity increase with age in the GP group compared to the ASD and SIB groups. Minor differences were found in lactobacilli prevalence and in species diversity between children with severe and mild-to-moderate ASD. Overall, we found substantial differences in the profile of faecal lactobacilli species in the ASD and GP groups at the age of 8–10 years. Full article

Drought has severely impacted worldwide agricultural land, resulting in substantial yield loss and abiotic pressures in crops such as Chinese cabbage. Paclobutrazol (PBZ) and plant growth-stimulating bacteria have been extensively recommended to reduce a variety of stresses in crops. Considering these facts, we used PBZ and rhizobacterium Variovorax sp. YNA59, a microbe with potential plant growth-stimulating qualities, in Chinese cabbage under drought stress and non-stressed conditions, and we observed their impacts on morphological and physiological attributes. Our findings revealed a considerable improvement in total plant biomass growth after treatment with YNA59 (28%) and PBZ (8%) under drought conditions. In contrast, negative results of PBZ were observed under normal conditions, suppressing plant growth and reducing plant biomass. The increase in physiological parameters, such as chlorophyll content, relative water content, and photochemical efficiencies of PS II, were observed in YNA59-treated plants, followed by PBZ, especially under drought. Furthermore, drought stress significantly increased the endogenous phytohormone abscisic acid level, whereas PBZ and YNA59 inoculations significantly decreased it. Antioxidant analysis revealed that PBZ and YNA increased glutathione and catalase and decreased polyphenol oxidase and peroxidase levels. These findings suggested that rhizobacterium YNA59 could be more significant for conferring water stress in the Chinese cabbage plant than PBZ. Full article

The goal of this study was to produce a sufficient amount of glutathione in the fermentation medium without the addition of cysteine. This would simplify and reduce the cost of its purification. In addition to reducing the cost of cysteine, it also avoids the inhibition of bacterial growth by cysteine. The gshA, gshB, and cysE genes of Escherichia coli were cloned under the control of the strong T5 promoter of the pQE-80L plasmid and introduced into an E. coli strain knocked out for the genes encoding γ-glutamyltranspeptidase and the GsiABCD glutathione transporter, which are responsible for the recycling of excreted glutathione. The overexpression of the gshA and gshB genes, genes for γ-glutamylcysteine synthetase and glutathione synthetase, and the cysE^{V95R D96P} gene, a gene for serine acetyltransferase with the V95R D96P mutation that makes it insensitive to cysteine, were effective on glutathione production. Na₂S₂O₃ was a good sulfur source for glutathione production, while the addition of Na₂SO₄ did not affect the glutathione production. With the addition of 50 mM glutamic acid and 75 mM glycine, but without the addition of cysteine, to the simplified SM1 medium, 4.6 mM and 0.56 mM of the reduced and oxidized glutathione, respectively, were accumulated in the extracellular space after 36 h of batch culture. This can eliminate the need to extract glutathione from the bacterial cells for purification. Full article

L-phenylalanine is an important aromatic amino acid that is widely used in the area of feed, food additives, and pharmaceuticals. Among the different strategies of L-phenylalanine synthesis, direct microbial fermentation from raw substrates has attracted more and more attention due to its environment friendly process and low-cost raw materials. In this study, a rational designed recombinant Escherichia coli was constructed for L-phenylalanine production. Based on wild type E. coli MG1655, multilevel engineering strategies were carried out, such as directing more carbon flux into the L-phenylalanine synthetic pathway, increasing intracellular level of precursors, blocking by-product synthesis pathways and facilitating the secretion of L-phenylalanine. During 5 L fed batch fermentation, recombinant E. coli MPH-3 could produce 19.24 g/L of L-phenylalanine with a yield of 0.279 g/g glucose. To the best of our knowledge, this is one of the highest yields of L-phenylalanine producing E. coli using glucose as the sole carbon source in fed-batch fermentation. Full article

The eutrophication of freshwater ecosystems allows the proliferation of cyanobacteria that can produce secondary metabolites such as microcystins. The main aim of this study was to explore the occurrence and concentration of microcystin and the mcyA gene in water bodies located in agricultural, urban, and recreational areas in the karst aquifer of the Yucatan peninsula of Mexico (YPM) and to analyze the water quality variables and chlorophyll-a (Chl-a) associated with their presence. Water samples were collected from 14 sites, and microcystin concentrations were quantified using antibody-based ELISA test. Total DNA was isolated from filters and used for PCR amplification of a fragment of the mcyA gene. Amplicons were cloned and sequenced to identify toxin-producing cyanobacteria present in water. Results showed that water bodies had different trophic status based on Carlson’s trophic state index. Dissolved inorganic nitrogen (DIN: NH₄⁺ + NO₃⁻ + NO₂⁻) and P-PO₄³⁻ concentrations were within a range of 0.077–18.305 mg DIN/L and 0.025–2.5 mg P-PO₄³⁻/L, respectively, per sampled site. All sampled sites presented microcystin concentrations within a range of ≥0.14 µg/L to ≥5.0 µg/L, from which 21.4% (3/14) exceeded the limit established in water quality standards for water consumption (1 µg/L). The mcyA gene fragment was detected in 28.5% (4/14) of the sites. A total of 23 sequences were obtained from which 87% (20/23) shared >95% nucleotide identity (nt) with the genus Microcystis and 13% (3/23) shared >87% nt identity with uncultured cyanobacteria. No correlation with the presence of the mcyA gene and microcystins was found; however, a positive correlation was detected between microcystin concentrations with pH and Chl-a. Full article

The aim of this study was to characterize lactic acid bacteria (LAB) isolated from cocoa mucilage and beef and evaluate their inhibitory effect in vitro against pathogenic bacteria, as well as determine their effect on beef quality. For the antagonist assay, 11 strains of LAB were selected and tested against pathogenic strains of Escherichia coli and Salmonella sp. The pathogenic bacteria were cultured in a medium, and a previously reactivated LAB bacterial pellet was added. After incubation, halos were observed around the bacterial colonies of the pathogenic strains, indicating inhibition by the LAB. It was identified that the LAB strains used belonged to the genus Lactobacillus, and the CCN-5 strain showed high percentages of inhibition against Salmonella sp. (58.33%) and E. coli (59%). The effectiveness of LAB application methods (immersion, injection, and spraying) did not present statistical differences. Furthermore, no significant changes in the physicochemical characteristics of beef were observed after the application of LAB. The results obtained demonstrate the potential of cocoa mucilage, as a biological control agent through LAB application, for beef biopreservation due to its ability to inhibit the growth of pathogenic bacteria. Full article

Stem canker on dragon fruit caused by Neoscytalidium dimidiatum causes severe losses in production of this fruit worldwide. Biological control by Trichoderma species is widely used to control several plant diseases. However, environmental conditions affect the use of biocontrol agents in the field. The development of a new formulation may offer an alternative way to address the problem of stem canker on dragon fruit caused by N. dimidiatum. In this study, we sought to develop a Trichoderma asperelloides PSU-P1 formulation that would be effective against N. dimidiatum. Three vegetable oils, two emulsifier-dispersing agents (Tween 20 and Tween 80), and one source of carbon (dextrose) were tested for carrier additives. We assessed the viability and antifungal ability of formulations incubated at ambient temperature and at 10 °C during a storage period of 1–6 months. The formulation composed of coconut oil, DW, and tween 20 in a ratio of 30:60:10 required a mixing time of 1.14 min; this was significantly faster than the mixing times of other formulations. Application of this formulation suppressed canker development; a canker area of 0.53 cm² was recorded, compared with a control (pathogen only) area of 1.65 cm². In terms of viability, this formulation stored at ambient temperature showed a surface area percentage of T. asperelloides PSU-P1 ranging from 64.43 to 75.7%; the corresponding range for the formulation stored at cool temperature was 70.59–75.6%. For both formulations, percentage inhibition gradually decreased from 1 to 6 months, with ranges of 59.21–77% and 60.65–76.19% for formulations incubated at ambient and cool temperatures, respectively. Our findings suggest that the formulation developed in this study prolongs the viability of T. asperelloides PSU-P1 conidia by up to 6 months, effectively inhibits N. dimidiatum in vitro, and reduces stem canker in vivo. Full article

The increased emergence of multidrug-resistant Helicobacter pylori is related to many health issues. Zingiber officinale (Z. officinale) is a plant usually used in folk medicine to treat a variety of diseases. This study was conducted to evaluate the ability of Z. officinale extract to combat resistant H. pylori. The disc diffusion, microdilution, and microplate assays were performed to evaluate the susceptibility to antibiotics and the antibacterial and antibiofilm activities of the Z. officinale extracts. Using the checkerboard method, the combined effects of gentamicin and Z. officinale extract were investigated. In addition, anti-inflammatory activity and GC-MS analysis were performed according to a modified protocol. According to the findings, H. pylori isolates exhibited resistance rates of 56.33, 50.0, and 45.85 against metronidazole, gentamicin, and tetracycline, respectively. The methanolic extract of Z. officinale showed the strongest effectiveness against resistant H. pylori isolates with MICs of 20.0 to 50.0 µg/mL, including both H. pylori isolates and the standard strain NCTC 11637. Z. officinale extract suppresses the biofilm formed by H. pylori isolates with a percentage of 92.96% at 50.0 µg/mL, compared with 97.19% for gentamicin at the same concentration. According to FICI values, the combination of methanolic Z. officinale extract with gentamicin increases bacterial sensitivity to such drugs. Moreover, the Z. officinale extract exhibits strong anti-inflammatory activity, with inhibition of red blood cell membrane stabilization increasing from 49.83% to 61.47% at a concentration of 4 to 32 µg/mL. The GC-MS analysis of Z. officinale extract exhibits 17 different chemical compounds. Besides showing antibacterial properties, the extract also contains the anti-inflammatory compound gingerol as the main constituent, which inhibits the growth of H. pylori and its biofilm and is a promising natural therapeutic alternative or enhances antibiotic activity. Full article

Glaesserella parasuis (G. parasuis) can cause peritonitis in piglets. However, the pathogenesis of peritonitis remains unclear. Baicalin has been shown to possess anti-inflammatory and anti-oxidant functions. The aim of this study was to investigate the role of the PANX-1/P2X7 axis and the P2Y6 signaling pathway in peritonitis induced by G. parasuis and the effect of baicain on the PANX-1/P2X7 axis and P2Y6 pathway activation triggered by G. parasuis. A G. parasuis serovar 5 isolate SH0165 strain was obtained from the lungs of commercially produced pigs which had the typical symptoms of Glässer’s disease, namely arthritis, fibrinous polyserositis, hemorrhagic pneumonia, and meningitis. Then, 35 piglets were randomly divided into five groups, each group containing seven piglets. The groups consisted of a negative control group, an infection group, a 25 mg/kg baicalin group, a 50 mg/kg baicalin group, and a 100 mg/kg baicalin group. The results showed that G. parasuis could promote PANX-1/P2X7 axis and P2Y6 activation; induce NLRP3/caspase-1, IL-1β and IL-18 expression; trigger PLC/PKC and MLCK/MLC signaling activation; attenuate the expression of tight junction proteins ZO-1, E-cadherin, Occludins, and claudin 1; and stimulate CD14, CD24, CD36, CD47, and CD91 expression in the peritoneum as measured via Western blot (p < 0.01; PLC, p < 0.05). Baicalin could significantly inhibit PANX-1/P2X7 axis, P2Y6, and NLRP3/caspase-1 activation; reduce IL-1β and IL-18 expression; attenuate PLC/PKC and MLCK/MLC activation; promote ZO-1, E-cadherin, occludins, and claudin 1 expression; and reduce CD14, CD24, CD36, CD47, and CD91 expression in the peritoneum induced by G. parasuis as measured via Western blot. Our results deepen the understanding of the mechanism of peritonitis triggered by G. parasuis and provide some novel potential methods of controlling G. parasuis infection. Full article

Modern society is becoming more and more reluctant to use antibiotic or chemical compounds in food production and is demanding foods without what they perceive as artificial and harmful chemicals, including many used as antimicrobials and preservatives in food. Another big problem is the improper use of antibiotics, especially broad-spectrum ones, which has significantly contributed to increased antibiotic resistance in many microorganisms. As a consequence, the whole scientific world has recently concentrated numerous studies on the research of natural remedies capable of counteracting multidrug-resistant strains and fighting infections: the use of aromatic plants and their essential oils (EOs) as potential alternatives to conventional antimicrobials to extend shelf life and combat foodborne pathogens has heightened. Among EOs, sage and lavender have also been promoted for their potential antimicrobial capabilities. In this review, we summarize the latest research studies performed about sage and lavender EOs, focusing on their chemical composition and their biological and antimicrobial properties; the aim is to give an overview of the current knowledge about their major components, effectiveness, mechanisms of action, synergistic effects and use in foods to facilitate a widespread application in both food and pharmaceuticals industries. Full article

Background. Among invasive fungal infection pathogens, Candida spp. represent the most common aetiological agents. The increasing rate of severe infections and the emergence of antimicrobial resistance highlight the importance of in vitro susceptibility testing. The EUCAST and the CLSI have established reference microdilutions that are reliable but difficult to apply in a laboratory routine. Commercial microdilutions could represent a valuable alternative within a diagnostic workflow. Methods. A number of 50 Candida spp. collected from positive blood samples simultaneously underwent the Sensititre Yeast-One microdilution as a standard susceptibility test and the Micronaut-AM as an experimental method. A comparison between the two techniques was produced, evaluating the effectiveness of the Micronaut-AM compared to the extensively consolidated Sensititre Yeast-One. Results. The two techniques revealed optimal agreement rates, confirming the reliability of the commercial microdilution kits within the diagnostic workflows. The results showed remarkable concordance for both susceptible and resistant isolates, highlighting slight variations in the different identified Candida species. Conclusions. Future studies about antifungal susceptibility testing should be encouraged, including molecular confirmation of possible resistance phenotypes and extended isolate numbers for the different Candida species. Moreover, it would be interesting to plan clinical trials after the execution of the examined commercial microdilution methods. Full article

Human African Trypanosomiasis (HAT) is still endemic in the Republic of Congo. Although the incidence of cases has significantly decreased over years, the disease still persists in some active foci. Factors contributing to the maintenance of the disease such as the existence of an animal reservoir or population knowledge are still not well known. It is in this context that a study focusing on the knowledge and practices of the population with regard to HAT as well as on the prevalence of trypanosomes infecting animals was undertaken in three active HAT foci in the Republic of Congo. The study was performed using field surveys conducted from November 2019 to June 2021. Domestic animal blood was examined by microscopy and PCR to detect the presence of trypanosomes. A structured questionnaire was administered to the population to assess their knowledge and practices concerning HAT in these endemic foci. More than half of the animals examined were found to be infected with trypanosomes (51.22%). The main trypanosome species infecting animals were Trypanosoma congolense savannah (67.2%) and Trypanosoma brucei (s.l.) (32.8%). No trypanosomes infecting humans were detected. Concerning household surveys, more than half of the respondents (52.9%) were fully aware of the mode of transmission and symptoms of the disease. The majority of people preferred to wear clothes covering the whole body and to use locally made soap as repellents to protect themselves from tsetse fly bites. This study suggests frequent circulation of animal trypanosomes in domestic animals and the use of personal measures to protect against tsetse fly bites. Updating information on the HAT animal reservoir and population knowledge alongside regular monitoring of the tsetse fly populations and the use of traps to control tsetse flies are crucial to drive efforts towards the elimination of gHAT in the Republic of Congo. Full article

Dye-polluted wastewater poses a serious threat to humans’, animals’ and plants’ health, and to avoid these health risks in the future, the treatment of wastewater containing dyes is necessary before its release to environment. Herein, a biological approach is used; the textile azo dye brown 703 is degraded utilizing Pseudomonas aeruginosa. The bacterial strain was isolated from textile wastewater dumping sites in Mingora, Swat. The optimization for bacterial degradation was carried out on the nutrient broth medium, which was then subjected to a variety of environmental physicochemical conditions and nutritional source supplementation before being tested. Under micro-aerophilic circumstances, the maximum decolorization and degradation of dye occurred at a 20 ppm dye concentration within 3 days of incubation at a neutral pH and 38 °C. The decrease in the intensity of the absorbance peak in the UV–Vis spectrum was used to measure the extent of decolorization. Initially, 15 bacterial strains were isolated from the textile effluent. Out of these strains, Pseudomonas aeruginosa was found to be the most potent degrading bacteria, with a degradation extent of around 71.36% at optimum conditions. The appearance and disappearance of some new peaks in the FT-IR analysis after the degradation of brown 703 showed that the dye was degraded by Pseudomonas aeruginosa. The GC–MS analysis performed helped in identifying the degraded compounds of azo dye that were utilized in illustrating the under-study process of brown 703 degradation. The biodegradation brought about by Pseudomonas aeruginosa can be employed successfully in the future as an eco-friendly approach with far reaching results. Full article

The genus Laurencia, a category of marine red algae, is well recognized for producing a large variety of natural products (NPs) that are both chemically intriguing and structurally distinct. The aim of this research was to identify NPs with potential anti-SARS-CoV-2 activity. The crystals of the proteins RdRp and nsp15 were obtained from the RCSB protein database. About 300 NPs were discovered using the PubChem, ChemSpider, and CMNPD databases. The program Autodock Vina was used to conduct the molecular docking procedure once the proteins and ligands were prepared. Before running MD simulations using the CABS-flex 2.0 website, binding affinity assessments and interactions between amino acids were carefully reviewed. Only nine NPs were shortlisted to be examined further. Bromophycolide R, S, and bromophycoic acid C show the tendency to inhibit RdRp by β-hairpin motif binding at the N-terminal known as Active site 2 (AS2), whereas the other four NPs, bromophycolide E, H, P, and thyrsenol A, may effectively inhibit RdRp through interactions via C-terminal, also known as the Active site 1 (AS1). For the enzyme nsp15, bromophycoic B, C, and floridoside showed plausible interactions. In conclusion, out of nine, seven candidates shortlisted for RdRp exhibited strong interactions with the key residues in the AS1 and AS2 regions. Bromophycoic acid C may work as a dual inhibitor due to its favorable interactions with the nsp15 protein and RdRp’s N-terminal, with affinities of −8.5 and −8.2 kcal/mol, respectively. Full article

Biomolecules of marine origin have many applications in the field of biotechnology and medicine, but still hold great potential as bioactive substances against different diseases. The purification or total synthesis of marine metabolites is expensive, and requires a reliable selection method to reveal their pharmaceutical potential prior to clinical validation. This study aimed to explore the hidden potential of natural products from the gorgonian genus Antillogorgia as anti-SARS-CoV-2 agents, via binding affinity assessments and molecular dynamics (MDs) simulations. The three-dimensional protein structures of the nsp16–nsp10 complex, nsp13, and nsp14 were acquired from the RCSB PDB database. All 165 natural products (NPs) were discovered using the PubChem, ChemSpider, and CMNPD databases. The freeware Autodock Vina was used to conduct the molecular docking procedure, once the proteins and ligands were prepared using BIOVIA discovery studio and Avogadro software v1.95. Before running MDs simulations using the CABS-flex 2.0 website, the binding affinity assessments and amino acid interactions were carefully examined. Just twelve NPs were selected, and five of those NPs interacted optimally with the catalytic amino acids of proteins. To conclude, pseudopterosin A (−8.0 kcal/mol), seco-pseudopterosin A (−7.2 kcal/mol), sandresolide B (−6.2 kcal/mol), elisabatin A (−7.0 kcal/mol), and elisapterosin A (−10.7 kcal/mol) appeared to be the most promising candidates against the nsp16–nsp10, nsp13, and nsp14 proteins. Full article

The plant pathogen Botrytis cinerea (B. cinerea) causes severe plant diseases worldwide. Trichoderma is widely used as a biocontrol agent against B. cinerea through multiple biocontrol mechanisms. However, Trichoderma spp. with high biocontrol efficiency against B. cinerea under low-temperature conditions are barely reported. This study aimed to find potential low-temperature resistance biocontrol Trichoderma spp. against B. cinerea, and to characterize the biological principles underlying the activity of Trichoderma. Trichoderma koningiopsis (T. koningiopsis) C5-9 could fully overgrow a B. cinerea colony at 16 °C in a dual-culture assay. Treatment of cucumber leaves with T. koningiopsis C5-9 fermentation broth using the dipping method prior to B. cinerea inoculation significantly reduced the necrotic lesion diameter, with an inhibition rate of 55.30%. T. koningiopsis C5-9 could be successfully cultivated using the mycelia of B. cinerea as a carbon source at 16 °C. Transcriptomic analysis indicated that the origin recognition complex, organic substance catabolic process, and peroxisome were involved in the responses of T. koningiopsis C5-9 to B. cinerea. The findings of this study not only identified T. koningiopsis C5-9 as a potential biological control agent inhibiting B. cinerea under low-temperature conditions, but also provided new insights to develop a deeper understanding of the activity of Trichoderma against B. cinerea for plant protection. Full article

L-methioninase is an enzyme that has recently gained significant interest in the scientific community because of its potential as a targeted therapy for cancer. This study aims to isolate and identify extremophilic bacteria that could produce L-methioninase and to access the enzymatic potential of isolated bacteria under stress conditions, specifically in agro-industrial waste. In this study, a rare marine bacterium, Alcaligenes aquatilis BJ-1, exhibited the highest specific activity of 4.61 U/mg at an optimum pH of 8.3. The L-methioninase was purified 4.3-fold and 7.15-fold by acetone precipitation and Sephadex G-100 gel filtration chromatography, which revealed a molecular weight of 46 kDa. In addition, agriculture waste materials such as cottonseed oil cake had the highest L-methioninase production. Moreover, A. aquatilis BJ-1 can tolerate and produce enzymes in the presence of 10% NaCl, 6% KCl, and 4% MgSO₄. Similarly, substrates such as L-asparagine, L-glutamine, L-alanine, and L-tyrosine were found suitable to increase enzyme production. The strain produced L-methioninase in the presence of various heavy metals. Maximum enzyme activity was found in Zn²⁺ at 0.1% (2.52 U/mL), Li²⁺ at 0.03% (2.90 U/mL), and Ni²⁺ at 0.01% (2.78 U/mL), as compared to the control (2.23 U/mL) without metal. Enzyme production was also observed at a high temperature (60 °C), with the produced enzymes possessing antioxidant properties. In addition, no hemolytic activity was observed. The results indicate that A. aquatilis BJ-1 is an appropriate bacterium for metal bioremediation procedures in unfavorable circumstances. Full article

When worn, clothing acquires a microbiome of bacteria and fungi derived from the wearer’s skin and from the environment. The types of bacteria and fungi that may be recovered from clothing in healthcare settings have been well characterized, but less is known regarding the microbiome of clothing worn in non-healthcare settings and the possible roles that such clothing may play in microbial exchange. Culture-based methods and culture-independent genomic sequencing were used to enumerate and identify bacteria and fungi recovered from T-shirts, baby onesies, socks, and underwear worn for a single day after having been purchased new, washed, and dried. The highest bacterial loads were recovered from socks, underwear, and onesies (>10⁶ colony-forming units [cfu]/sample) and the highest fungal loads were obtained from socks and underwear (>5 × 10² cfu/sample). The sequencing method identified opportunistic pathogens present in the samples, including members of genus Staphylococcus and Corynebacterium, as well as anaerobic members of the family Clostridiales. The opportunistic fungal pathogen Candida parapsilosis was identified in a high proportion of worn clothing samples. These results suggest that clothing may represent a pathogen reservoir and a vector for microbial exchange between household occupants or the community outside of the home. Full article