Microbiology Research

Introduction: Invading extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-PE), non-ESBL E. coli, and other bacteria form a complex environment in the gut. The duration and dynamics of ESBL-PE colonization varies among individuals. Understanding the factors associated with colonization may lead to decolonization strategies. In this study, we aim to identify (i) single E. coli strains and (ii) microbiome networks that correlate with retention or decline of colonization, and (iii) pan-sensitive E. coli strains that potentially could be used to displace ESBL-PE during colonization. Methods and analysis: We recruit healthy travellers to Southeast Asia for a one-year prospective observational follow-up study. We collect and biobank stool, serum, and peripheral blood mononuclear cells (PBMCs) at predefined timepoints. Additional information is collected with questionnaires. We determine the colonization status with ESBL-PE and non-ESBL E. coli and quantify cell densities in stools and ratios over time. We characterize multiple single bacterial isolates per patient and timepoint using whole genome sequencing (WGS) and 16S/ITS amplicon-based and shotgun metagenomics. We determine phylogenetic relationships between isolates, antimicrobial resistance (AMR; phenotypic and genotypic), and virulence genes. We describe the bacterial and fungal stool microbiome alpha and beta diversity on 16S/ITS metagenomic data. We describe patterns in microbiome dynamics to identify features associated with protection or risk of ESBL-PE colonization. Ethics and dissemination: The study is registered (clinicaltrials.gov; NCT04764500 on 09/02/2019) and approved by the Ethics Committee (EKNZ project ID 2019-00044). We will present anonymized results at conferences and in scientific journals. Bacterial sequencing data will be shared via publicly accessible databases according to FAIR principles. Full article

This study investigated the presence of Aeromonas spp. on a dairy farm in central Texas that employed a free-stall management system. A total of 140 samples were collected from areas of two different barns. Twenty-two presumptive Aeromonas isolates were cultured. Phenotypic analysis identified five Aeromonas spp. Twenty isolates exhibited β-lactam and one displayed tetracycline resistance. Phylogenetic analysis of the WGS data suggested only four Aeromonas spp. All isolates possessed at least one β-lactam resistance gene and one isolate possessed tet(E). No plasmids were identified from sequence alignments. Virulence genes were identified in all four Aeromonas spp. Mobility elements were identified in three of these, with the exception being A. dhakensis. Four of the transposons identified in this study have been associated with multidrug resistance in Italy, Sweden, and Singapore. There was no significant difference in the proportion of isolates from either barn. The absence of plasmids suggests mobility elements and virulence genes were localized to the chromosome. On a dairy farm of healthy cattle, these 22 Aeromonas isolates were considered normal environmental flora while illustrating the ubiquitous nature of Aeromonas spp. globally. Full article

E. coli O157:H7 is a known Shiga toxin-producing Escherichia coli (STEC), causing foodborne disease globally. Cattle are the main reservoir and consumption of beef and beef products contaminated with E. coli O157:H7 is an important source of STEC infections in humans. To emphasize the cattle-to-human transmission through the consumption of contaminated beef in Bishoftu, Ethiopia, whole-genome sequencing (WGS) was performed on E. coli O157 strains isolated from three sources (cattle, beef, and humans). Forty-four E. coli O157:H7 isolates originating from 23 cattle rectal contents, three cattle hides, five beef carcasses, seven beef cuts at retail shops, and six human stools in Bishoftu between June 2017 and May 2019 were included. This study identified six clusters of closely related E. coli O157:H7 isolates based on core genome multilocus sequence typing (cgMLST) by targeting 2,513 loci. A genetic linkage was observed among the isolate genomes from the cattle rectal contents, cattle hides, beef carcasses at slaughterhouses, beef at retail shops, and human stool within a time frame of 20 months. All the strains carried practically the same repertoire of virulence genes except for the stx2 gene, which was present in all but eight of the closely related isolates. All the strains carried the mdfA gene, encoding for the MdfA multi-drug efflux pump. CgMLST analysis revealed genetically linked E. coli O157:H7 isolates circulating in the area, with a potential transmission from cattle to humans through the consumption of contaminated beef and beef products. Full article

Heterosigma akashiwo is a golden-brown unicellular phytoflagellate with a high potential to create harmful algal blooms (HABs) and kill fish in many coastal regions worldwide, resulting in significant economic losses. Climate change and global warming have been introduced as triggers that impact the frequency and severity of H. akashiwo and other bloom-forming species in the past decades. In this review paper, the author tried to briefly discuss the morphology and taxonomy of H. akashiwo and show how environmental parameters can influence the physiology and toxicity of this species. Although the toxin production and mechanisms are still a conundrum, the proposed fish-killing mechanisms will be reviewed in the next step. Full article

Vector-borne diseases spread from wild animals and their associated ectoparasites to humans and domesticated animals. Wildlife markets are recognized as important areas where this transfer can take place. We assessed the potential for spreading vector-borne diseases in two live and wet markets in Myanmar (Mong La, on the Myanmar-China border) and Indonesia (Sukahaji in Bandung on the island of Java) by making an inventory of all live and freshly killed wild mammals for sale. For eight mammal families, we quantified the number of animals on offer, and we used a heatmap cluster analysis to map vector-borne diseases that these families may carry. In Myanmar, we observed large numbers of wild pigs and deer (potentially carrying West Nile and various encephalitis viruses) whereas in Indonesia we observed Old World fruit bats (potentially carrying Chikungunya and encephalitis viruses) and squirrels (potentially carrying West Nile and encephalitis viruses). The trade in Indonesia was dominated by live mammals offered for sale as pets, and only Old World fruit bats and squirrels traded for traditional Asian medicine were killed in the markets. The trade in Myanmar was more geared towards wild meat (e.g., wild pigs, deer, primates) and traditional Asian medicine (squirrels). The combined risks of vector-borne diseases spreading from traded animals to human health highlight the need for an integrated approach protecting public health, economic interests and biodiversity. Full article

Between 2018 and 2021, several mastic trees (Pistacia lentiscus var. Chia) sampled in the field and the nursery of the Chios Mastiha Growers Association (CMGA) were analyzed to determine the cause of dominant diseases. Symptoms included defoliation, leaf, and twig blight, wilting and/or apoplexy of trees and apoplexy of young hardwood cuttings. Moreover, brown discoloration had also been observed on older woody parts of the trees such as branches and tree trunks. Several pathogens have been isolated and identified as the causing agents. Neopestalotiopsis and Alternaria species were isolated consistently from necrotic tissues of mastic trees (branches, twigs, and leaves) in the field and the nursery. All fungal isolates’ pathogenicity was confirmed by applying Koch’s postulates on young mastic trees under glasshouse conditions. Fungal pathogens were identified by sequence analyses of the ITS, β-tubulin, and histone gene regions. Alternaria species were analyzed further by sequencing the endopolygalacturonase (endoPG) and the Alternaria major allergen (Alta1) genes. More specifically, the isolates were identified as Neopestalotiopsis clavispora, Alternaria arborescens, and A. alternata based on morphological features and sequence analyses. This is the first report of N. clavispora, A. arborescens, and A. alternata on P. lentiscus var. Chia. Full article

Increasing pollution has significantly threatened the Antarctic ecosystem. The contamination of hydrocarbons has drawn a considerable amount of attention owing to their toxicity, recalcitrance, and persistence. Considering the Antarctic Treaty, only indigenous species are allowed to bioremediate the contaminated environment. However, the knowledge of the ecological role, physiology, function, and genomics of endemic hydrocarbon consumers is still limited. Here, we investigated the dynamics of phenanthrene-consuming communities derived from the Antarctic soil and found that Variovorax, Rhodocyclaceae, and Hydrogenophaga were differentiated in all the phenanthrene-consuming subcultures. We isolated a pure culture of the key hydrocarbon consumer Variovorax sp. strain N23. Moreover, the result of the polyphasic approach suggested that strain N23 represents a novel species of the genus Variovorax. In addition, the genomic characteristics of this strain revealed incomplete degradation pathways for diverse hydrocarbons. Overall, this study reveals the relatively weak hydrocarbon-degrading potential of the indigenous bacteria and suggests the need for more careful protection of the Antarctic ecosystem. Full article

The available literary data suggest the general applicability and benefits of the Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) in the field of microbiological identification. Due to its high reliability, MALDI-TOF might generally be the alternative to the the16s-rRNA sequence-based and serological-based methods. The essence of the technique is to map the unique protein pattern of microbes that contributes to characterizing a wide variety of microorganisms, including bacteria, fungi, and viruses. With its application, the well-known bacterial and fungal species can be quickly identified, thus saving time in clinical diagnostics. In recent years, new protocols have appeared for directly identifying pathogenic strains from patient samples (blood, urine, feces), a major milestone in healthcare applications. On the other hand, these applications only have reliable results under certain conditions (homogeneous infection, adequate cell count, appropriate separation technique). This review aims to introduce and summarize those developments that have been enabled for routine application in the field of clinical diagnosis. Full article

Background: D-dimer, generated upon the degradation of fibrin, is extensively used to detect thrombosis in various diseases. It is also explored as a marker for thrombosis in cases with COVID-19 disease. Few studies have confirmed its utility as a marker for assessing disease severity. Objectives: The current research was undertaken to determine the role of D-dimer in patients with COVID-19 and to investigate any association with the progression and severity of the disease in the Saudi population. Methods: Clinical indices in confirmed COVID-19 patients were collected from tertiary care hospitals in Aljouf and Qassim regions. The plasma D-dimer levels were quantified directly in the samples collected from COVID-19 patients (n = 148) using an immunofluorescence assay, and the data were presented in Fibrinogen Equivalent Units (mg/L). The collected data of D-dimer were analyzed based on COVID-19 severity, age, and the gender of patients. Results: The findings show that the plasma D-dimer concentrations were significantly (p = 0.0027) elevated in COVID-19 cases (n = 148), compared to in the normal healthy uninfected controls (n = 309). Moreover, the D-dimer levels were analyzed according to the severity of the disease in the patients. The data revealed that D-dimer concentrations were significantly increased in patients with mild infection to moderate disease, and the levels were the highest in patients with severe COVID-19 disease (p < 0.05). Our analysis demonstrates that the D-dimer levels have no association with the age or gender of COVID-19 patients (p > 0.05) in the study population. Conclusions: D-dimer can serve as a biomarker not only for the detection of COVID-19 infection, but also for determining the severity of infection of COVID-19 disease. Full article

Sampling animals for carriage of meticillin-resistant, coagulase-positive staphylococci (MRCoPS), considered zoonotic pathogens, can be challenging and time-consuming. Developing methods to identify mecA from non-invasive samples, e.g., faeces, would benefit AMR surveillance and management of MRS carrier animals. This study aimed to distinguish MRS carriers from non-carriers from faecal samples using quantitative polymerase chain reaction (qPCR) for mecA. Paired faecal and nasal swab samples (n = 86) were obtained from 13 dogs and 20 humans as part of a longitudinal study. Nasal MRCoPS carriage (either MR-Staphylococcus aureus or MR-Staphylococcus pseudintermedius was confirmed by identification of species (nuc) and meticillin resistance (mecA) (PCR). Faecal DNA (n = 69) was extracted and a qPCR method was optimised to provide a robust detection method. The presence of faecal mecA was compared between MRS carriers and non-carriers (Kruskal–Wallis test). Nasal swabbing identified seven canine and four human MRCoPS carriers. mecA was detected in 13/69 faecal samples, including four MRCoPS carriers and nine non-carriers. For dogs, there was no significant association (p = 1.000) between carrier status and mecA detection; for humans, mecA was more commonly detected in MRCoPS carriers (p = 0.047). mecA was detected in faeces of MRCoPS carriers and non-carriers by qPCR, but larger sample sizes are required to determine assay sensitivity. This rapid method enables passive surveillance of mecA in individuals and the environment. Full article

Ticks are amongst the important ectoparasites where livestock are concerned, as they adversely affect the animals through bloodsucking. In tropical and subtropical countries, they transmit pathogens such as babesiosis, theileriosis, ehrlichiosis, and anaplasmosis in cattle, causing a reduction in production rate and significant concomitant economic losses. Ticks affect 80% of the cattle population across the world, with an estimated economic loss of USD 20–30 billion per year. In South Africa, economic losses in the livestock industry caused by ticks and tick-borne diseases are estimated to exceed USD 33 million per year (ZAR 500 million). There are seven major genera of ixodid ticks in Southern Africa (i.e., Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus). The environment in which a tick lives is made up of all the various biological and abiotic factors that are either necessary or unnecessary for its life. The areas where various ticks have been found have been documented in many publications. Using these data, maps of possible species’ habitats can be made. Historical records on tick distribution may be incorrect due to identification mistakes or a change in the tick’s name. All the sources used to generate the maps for this review were unpublished and came from a wide range of sources. To identify tick species and the accompanying microbial ecosystems, researchers are increasingly adopting tick identification methods including 16S and 18S rDNA gene sequencing. Indeed, little is known about the genetic alterations that give important traits, including the predilection for tick hosts, transmission, and acaricide resistance. Opportunities for exploring these changes in tick populations and subpopulations are provided by advancements in omics technologies. The literature on the variety of ixodid ticks, their direct and indirect effects, and control methods in South Africa is compiled in this review. Full article

Treating type 2 diabetes (T2D) patients with sodium–glucose cotransporter 2 (SGLT2) inhibitors may be associated with an increased risk of urinary tract infections (UTIs), such as diabetes-induced asymptomatic bacteriuria. Pyuria—a condition wherein leukocytes are detected in the urine—is a predictor of UTIs. The aim of this study was to examine the urinary microbiome of Taiwanese T2D patients, with or without pyuria, undergoing SGLT2 treatment. We recruited seven T2D patients, recorded their clinical and biochemical characteristics, and collected their urine samples for 16S metagenomic sequencing. The primary outcomes were the diversity of urinary microbiota and the relative abundance of different species. We found that the microbiome of the pyuria group was significantly less diverse than the non-pyuria group (0.24 ± 0.04 vs. 2.21 ± 0.28, p = 0.002), while the number of operational taxonomic units did not differ significantly (763.5 ± 78.67 and 747 ± 141.3, p = 0.92). Escherichia-Shigella spp. dominated the microbiome of the pyuria group (97.4%–99.4%), and these patients tended to have more comorbidities. In conclusion, pyuria is associated with urinary microbiota dysbiosis in T2D patients being treated with SGLT2 inhibitors. Full article

Serological diagnosis provides a robust and effective approach to monitoring and controlling small ruminant brucellosis. Brucella melitensis Rev. 1 is a live vaccine strain used in prophylactic vaccination against small ruminant brucellosis. Because the vaccine strain shares identical serological antigens with the corresponding field strains, differentiating infected from vaccinated animals (DIVA) serological responses hamper surveillance campaigns and interventions that involve vaccination. We have developed a serum PCR-based approach in which we amplify and sequence Brucella omp2a as a DIVA solution and tRNA (uracil-5-)-methyltransferase as a species marker in the serum samples to determine the etiological agent involved in brucellosis field cases. Using this method, we identified the involvement of both the Rev. 1 vaccine strain and a field strain in an outbreak of brucellosis in a flock. This method represents a novel approach in studying the etiology of brucellosis using serum samples as a source of the pathogen’s DNA. Full article

In the last ten years, Stenotrophomonas maltophilia has gained increasing interest as an important agent of infection, which is why it has come to be recognized as a serious cause of nosocomial infections related to bloodstream infections, pneumonia, and cancer, mainly in patients with intensive care, and is associated with high mortality rates in immunocompromised patients, with prolonged hospital stays and extensive use of antimicrobials. The importance of this microorganism lies in its low pathogenicity, high multiresistance to various antibiotics, and frequent and persistent isolation in predisposed patients. In addition, few studies have evaluated its epidemiology and clinical relevance. The pathogenesis of biofilms lies mainly in the fact that they can generate persistent chronic infections that are difficult to eradicate. To this extent, it is important to make the characteristics of the biofilm formation behavior of Stenotrophomonas maltophilia known and generate more knowledge about its colonization or infection in humans through this review, which discusses more recent information. Full article

Some of the goals of meat production systems are to improve feed efficiency, reduce costs, provide proper nutrition and avoid metabolic disorders. Hence, the aim of this work was to compare a rabbit diet based on untreated sorghum (T1) with a second one that included the cereal biotransformed by the co-cultivation of two basidiomycetes (T2). Their effects on the cereal, diets, and fecal structural fiber composition, as well as on rabbit intestinal morphology, were evaluated. A completely randomized design was applied, employing 24 New Zealand rabbits (21 days old). The fiber chemical composition results indicated that the sorghum treated with the co-culture had lower amounts of neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin, hemicellulose and cellulose, as did rabbits fed the T2 diet. Decreases in the NDF, ADF and cellulose contents were detected in T2-nourished rabbit excrements at day 21 (p ≤ 0.05), while lignin percentages diminished at days 35 and 49 (p ≤ 0.05). These rabbits also showed longer jejunal and cecal villi (p ≤ 0.05). The results obtained suggest a positive effect on the bioavailability of structural fibers after sorghum fungal co-fermentation, as their content decreased in rabbit fecal matter without damaging the intestinal morphology. Full article

Lead (Pb) pollution from local mines and industrial use increases risks for human, animal, and plant health. Pectin is an effective chelator of Pb, and it has been shown that a unicellular green alga, Penium margaritaceum, synthesizes pectin in the cell wall. In this study, the ability of P. margaritaceum to remove Pb from an aqueous solution was investigated. Energy dispersive X-ray spectroscopy revealed that two strains of P. margaritaceum accumulated Pb on the cell surface. Hence, P. margaritaceum cells were immobilized on cellulose filter paper. The immobilized algal cells were soaked in 1.0 mg/L Pb solution with gentle shaking for 8 h, and Pb in the solution and on the filter paper was measured by flame atomic absorption spectrometry. The immobilized algal cells continuously decreased the Pb concentration to less than 0.5 mg/L and recovered 31.8–32.7% of added Pb. The specific decrease in Pb and increase in Ca were observed in the presence of 1.0 mg/L each of Ca, Mg, Na, and K. Fourier transform infrared spectra suggested that the carboxylic acid group would be responsible for the adsorption of Pb. This study is the first to demonstrate the effectiveness of the immobilized P. margaritaceum cell in removing Pb from aqueous solutions with simple solid–liquid separation. Full article

Although clothes washing machines remove dirt, microorganisms are not reliably removed by modern cold-water machine-washing practices. Microbial bioburden on clothing originates from the wearer’s skin, the environment (indoor and outdoor), and the washing machine itself. While most clothing microbes are commensals, microbes causing odors and opportunistic pathogens may also be present. Understanding the extent of microbial transfer from washing machines to clothes may inform strategies for odor control and for mitigating the transmission of microbes through the laundering process. This study was designed to quantify and identify bacteria/fungi transferred from laundromat machines to sentinel cotton washcloths under standard cold-water conditions. Bacterial 16S rRNA and fungal ITS sequencing enabled identification of microorganisms in the washcloths following laundering. Total plate-based enumeration of viable microorganisms also was performed, using growth media appropriate for bacteria and fungi. Opportunistic human bacterial pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., were recovered. The fungal bioburden was ~two-fold lower than the bacterial bioburden. Most sequences recovered were assigned to non-pathogenic fungi, such as those from genera Malassezia and Ascomycota. These results suggest that public washing machines represent a source of non-pathogenic and pathogenic microbial contamination of laundered garments. Full article

Cryptococcosis, a life-threatening fungal infection, frequently occurs in patients suffering from AIDS. The treatment of the disease is hampered by the limited number of the effective drugs and the increasing resistance; therefore, to find new active substances is needed. As meningitis is the most serious infection affecting the AIDS patients, effective drugs have to be capable of entering to the central nervous system. Ecdysteroids are natural bioactive molecules with considerable anabolic activity and without toxic side effects on humans. The aim of this work was to study the anti-cryptococcal activity of a natural ecdysteroid, 20E, and its three semi-synthetic derivatives obtained by structural modification of the original molecule. We established the minimum inhibitory concentration of the compounds with microdilution method and demonstrated their fungicidal activity by flow cytometry and cultivation of the drug-treated cells. The interaction of the compounds with each other and efflux transporter inhibitors was assessed by checkerboard titration method. Two derivatives, 20E-EOx and 20E-ZOx, inhibited the growth of Cryptococcus neoformans with minimum inhibitory concentration 2 mg/mL and 1 mg/mL, respectively; both compounds possess fungicidal effect. A combination of the ecdysteroids with each other and verapamil resulted in additive interaction. This study confirmed that structural modification of an originally non-antimicrobial molecule can enhance its effectiveness. Full article