Applied Microbiology

The bacterial foodborne enteropathogen Escherichia albertii, despite enjoying increased attention paid to its pathogenesis, global dissemination, and antimicrobial resistance capacity, remains difficult to identify from human foods. The primary objective of this study was to develop and test a selective and differential plating medium for the isolation of E. albertii from enteric pathogens commonly transmitted via fresh poultry meat, namely E. coli and Salmonella enterica. MacConkey agar supplemented with α-D-+-melibiose and the lactose analogue X-gal was prepared and used to differentially enumerate E. albertii, Salmonella, and E. coli from inoculated ground chicken meat. The medium, MXgMac agar, differentiated the inoculated pathogens with a greater degree of efficiency than did the previously developed E. albertii-selective medium xylose–rhamnose–melibiose (XRM) MacConkey agar, based on differential usage of the lactose analogue and melibiose. Chicken-derived feces and litter samples were subsequently tested using the medium and found not to contain E. albertii by 16S rRNA gene amplification. MXgMac agar facilitates improved differential recovery of E. albertii and other enteric pathogens from poultry meat versus other E. albertii selective/differential media. Full article

Microsomes are commonly used to perform in vitro drug metabolism, predominantly to form phase I drug metabolites. Pooled microsomes from multiple donors can contain microorganisms from underlying microbial diseases. Exposure to microbes can also occur during extraction if aseptic processing is compromised. Although microbial presence does not affect the metabolic activity of microsomes, presence of unwanted microorganisms can cause interference if the downstream application of microsomal drug metabolites is screening for antibacterial activity. In this work, traditional biochemical tests and advanced proteomics-based identification techniques were used to identify two gram-negative bacteria in pooled human liver microsomes. Several decontamination procedures were assessed to eradicate these two bacteria from the microsomes without affecting its metabolic capacity, and organic extraction was found to be the most convenient and efficient approach to decontaminate microsomes and screen drug metabolites for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Full article

Prebiotics beneficially affect the gut microbiome. Bimuno^®, a prebiotic supplement containing galactooligosaccharides (GOS), has multiple demonstrated prebiotic effects. Using short-term colonic incubations, the influence of GOS on the colonic microbiota of three healthy human adults was evaluated. Colonic reactors inoculated with fecal samples were untreated (blank) or supplemented with GOS. pH, gas pressure, short-chain fatty acids (SCFAs), lactic acid, branched SCFAs, ammonium, and microbial community composition were evaluated at 0 h, 6 h, 24 h, and 48 h. pH decreased and gas pressure increased (+29.01 kPa) with GOS treatment versus blank. Total SCFA (+22.4 mM), acetate (+14.1 mM), propionate (+5.5 mM), and butyrate (+5.8 mM) were higher for GOS than blank. Acetate and propionate production were highest earlier in the experiment, while butyrate production was highest between 24 h and 48 h. With GOS, lactic acid production increased between 0 h and 6 h (+14.4 mM) followed by apparent consumption. Levels of branched SCFAs and ammonium were low with GOS and reduced versus blank (respectively, −2.1 mM and −256.0 mg/L). GOS significantly increased the relative abundance of Bifidobacterium longum (LDA = 4; p = 0.006), and significantly increased the absolute abundance of Bifidobacteriaceae (p < 0.001), Lactobacillaceae (p < 0.05), Bifidobacterium adolescentis (LDA = 4.5; p < 0.001), and Bifidobacterium ruminantium (LDA= 3.2; p = 0.01). This in vitro model demonstrated the prebiotic potential of GOS as supplementation resulted in increased beneficial bacteria, SCFA, and lactic acid and decreased branched SCFA, pH, and ammonium. Full article

Understanding the impacts of herbicides on soil microbial communities is important, as these organisms mediate a wide range of ecosystem services. Here, we investigated whether the diversity and function of soil microbial communities were significantly influenced by one-off applications of atrazine, diuron, fluazifop-P-butyl, haloxyfop-P-methyl and pendimethalin as pure compounds at their recommended doses over multiple time points (1, 3, 7, 14, 30 and 60 days). Phylogenetic marker gene sequencing revealed that none of the herbicides influenced the numbers of bacterial and archaeal taxa or the evenness of their abundances. Similarly, none of the herbicides influenced the composition of bacterial and archaeal communities, except for diuron, fluazifop-P-methyl and pendimethalin, which were associated with larger relative abundances of a small number of OTUs on day 30 only. Functionally, none of the herbicides significantly influenced fluorescein diacetate hydrolysis (FDA) and beta-glucosidase activities or the induced respiratory responses of soil microbial communities to a range of substrates. These data indicate that the active herbicide ingredients tested may have minimal non-target effects when applied once at their recommended dose. Given their frequent use, it is important to next consider whether these herbicides have more pronounced effects at higher doses and application frequencies. Full article

An increasing prevalence and spread of Clostridioides difficile infection (CDI) caused by DH/NAP11/106/ST-42 has been observed worldwide, probably fostered by its great capacity to produce spores or by the higher resistance rates observed for some strains. Based on the results of our previous study where RT106 showed higher recurrence rates than other relevant ribotypes, a genetic analysis by whole-genome sequencing (WGS) of primary and recurrent RT106 isolates from ten patients was performed to determine whether the higher rate of recurrence associated with RT106 is due to relapses, caused by the same strain, or reinfections, caused by different strains. MLST profiles, resistance mutations, and phylogenetic relatedness were determined by comparative single nucleotide variant (SNV) analysis. All isolates were classified as ST42, and those belonging to the same patient were isogenic, with one exception; strains belonging to different patients were not with two exceptions, pointing to putative transmission events. Phylogenetic analysis also suggested the presence of similar local epidemic lineages associated with moxifloxacin resistance, except for one patient whose isolates clustered with different nonresistant US strains. Our results show that recurrent CDIs caused by RT06/ST42 are mainly due to relapses caused by the primary strains, showing the higher capacity of RT106/ST42 to persist and cause recurrences as compared to other ribotypes. Full article

Abandoned and flooded ore mines are examples of hostile environments (cold, dark, oligotrophic, trace metal) with a potential vast diversity of microbial communities rarely characterized. This study aimed to understand the effects of depth, the source of water (surface or groundwater), and abiotic factors on the communities present in the old Forsyth iron mine in Quebec (Canada). Water and biofilm samples from the mine were sampled by a team of technical divers who followed a depth gradient (0 to 183 m deep) to study the planktonic and sessile communities’ diversity and structure. We used 16S/18S rRNA amplicon to characterize the taxonomic diversity of Bacteria, Archaea, and Eukaryotes. Our results show that depth was not a significant factor explaining the difference in community composition observed, but lifestyle (planktonic/sessile) was. We discovered a vast diversity of microbial taxa, with taxa involved in carbon- and sulfur-cycling. Sessile communities seem to be centered on C₁-cycling with fungi and heterotrophs likely adapted to heavy-metal stress. Planktonic communities were dominated by ultra-small archaeal and bacterial taxa, highlighting harsh conditions in the mine waters. Microbial source tracking indicated sources of communities from surface to deeper layers and vice versa, suggesting the dispersion of organisms in the mine, although water connectivity remains unknown. Full article

The recent description and characterization of several novel and unique lumpy skin disease virus (LSDV) strains have revealed the inadequacy of current techniques for differentiating between vaccine- and wild-type viruses. The lack of reliable sequencing targets for promptly distinguishing circulating recombinant vaccine-like strains (RVLSs) highlights the need to develop a single and simple differentiation tool. In this study, we analyzed the available LSDV whole-genome sequences and identified a 705-bp region in open reading frame (ORF) LW134. Based on a single run of nucleotide sequencing and phylogenetic analysis, the region with 13 informative single nucleotide polymorphisms (SNPs) was capable of accurately segregating the novel RVLSs into the same five clusters previously confirmed by whole-genome sequencing. In addition, archived RVLSs from Russia were analyzed for further characterization using the newly described single PCR and sequencing assay. The ORF LW134 assay identified one archived RVLS as a novel cluster distinct from the previously described five clusters, while clustering the remaining samples into previously designated lineages, demonstrating the reliability of the assay. The novel PCR and sequencing assays described in this study have great potential for accurately delineating the molecular and evolutionary affiliation of circulating RVLSs. Full article

According to current knowledge, a properly colonized human microbiota contributes to the proper functioning of the body. The composition of the natural flora changes depending on age, health, living conditions, and the use of antimicrobial agents: antibiotics, disinfectants, and some cosmetics. The human body is diversely populated with microorganisms and undergoes constant changes under the influence of various factors, and its proper composition is extremely important for the proper functioning of the body. Given the above, it was decided that we would review current scientific research that explains the cause–effect relationship between the composition of microorganisms populating the human body and health, focusing on women’s health. As a result, an overview paper was prepared based on 109 scientific sources from 2009–2022. Special attention was paid to the most recent scientific studies of the last five years, which account for more than 75% of the cited sources. Full article

The oyster industry is a significant component of United States aquaculture and is vulnerable to various food frauds. In addition to species substitution, mislabeling of oyster geographical origin is performed for economic gains. The geographical origin misrepresentations are performed to claim a famed region of origin known for its unique flavor profile. DNA barcoding is the gold standard method for identifying seafood species but has limited resolution to the species level. This pilot study was conducted to characterize and compare the oyster gill microbiome as an alternative approach for tracking oysters’ origin. Commercially available raw east coast oysters (Crassostrea virginica) from two distinct geographical locations were purchased. Genomic DNA isolated from the gills was processed for microbiome analysis. The data revealed distinct microbiome signatures among the two sample sets. Oysters from Louisiana showed the presence of eighteen unique bacterial genera, whereas Maryland oysters showed a higher abundance of twelve genera. Findings from this study demonstrate the applicability of microbiome analysis as an emerging alternative approach for identifying geographical origin misrepresentations. Full article

The Green Revolution (GR) involved selective breeding of cereals and the use of high fertilizer inputs with the goal of increasing crop yields to alleviate hunger. As a result of both greater use of inorganic fertilizers and the introduction of semi-dwarf cultivars, grain yield increased globally and hunger was alleviated in certain areas of the world. However, these changes in varietal selection and fertilization regimes have impacted soil fertility and the root-associated microbiome. Higher rates of inorganic fertilizer application resulted in reduced rhizosphere microbial diversity, while semi-dwarf varieties displayed a greater abundance of rhizosphere microbes associated with nitrogen utilization. Ultimately, selection for beneficial aboveground traits during the GR led to healthier belowground traits and nutrient uptake capabilities. Full article

Grapevine crown gall (GCG), which is caused by tumorigenic Allorhizobium vitis (=Rhizobium vitis), is the most important bacterial disease in grapevine, and its economic impact on grapevine is very high. When young vines develop GCG, they often die, whereas older vines may show stress and poor growth depending on the severity of GCG, because GCG interferes with the vascular system of the grapevine trunk and prevents nutrient flow, leading to inferior growth and death. Viticultural practices and chemical control designed to inhibit GCG are only partially effective presently; thus, a biocontrol procedure could be a desirable and effective approach for GCG prevention. This article reviews the practical use of biocontrol options for GCG inhibition that involve using nonpathogenic and antagonistic A. vitis strains. In these studies, screening tests of biocontrol agents discovered nonpathogenic A. vitis strains VAR03-1, ARK-1, ARK-2, and ARK-3. After dipping grapevine roots in a suspension of candidate strains prior to planting in the field, treatment using ARK-1 was shown to significantly reduce the number of plants with GCG. A meta-analysis indicated that ARK-1 is very useful for controlling crown gall in various plant species, including grapevine. It was reported that when a mixture of ARK-1 and a tumorigenic strain was examined in grapevines, the expression levels of several virulence genes of the virulent strain were significantly lower. ARK-1 can reduce the pathogen population in grapevines and gall incidence. Moreover, ARK-1 can prime the induction of certain defense genes of grapevine. These results indicate that ARK-1 has a unique biocontrol mechanism and that it is a promising new biocontrol agent to control GCG. Full article

Six bacteria (Bacillus velezensis 13, Bacillus subtillis 42, Pseudomonas fluorescens E221, Pseudomonas Poae EE12, Rahnella sp. EM1, and Serratia sp. EM2) isolated from the soil and litter of Mexican oak forests were characterized by identifying their ability to acquire phosphorus from different sources, analyzed for their biocontrol capabilities against two different phytopathogenic fungi, and finally tested for their ability to stimulate the germination of maize seeds and promotion of maize seedling growth. The greatest capacity to biocontrol the mycelial growth of phytopathogenic fungi Botrytis cinerea and Fusarium oxysporum was found in B. velezensis 13 and B. subtillis 42. P. poae EE12 and P. fluorescens E221 significantly promoted germination and the length of the primary root in Zea mays. Rahnella sp. EM1 and Serratia sp. EM2 could produce indole compounds related to auxin synthesis and increased the fresh weight of the maize seedlings. Together, these isolates represent an alternative to reduce the use of agrochemicals in maize cultivation. In general, soil microorganisms from Mexican oak forests represent a source of genetic resources for the sustainable management and conservation of soils for agricultural use. Full article

Sheep pox (SPP) constitutes a global animal health scourge, despite the numerous efforts targeting the eradication of the disease implemented in affected countries. An efficient control and eradication strategy incorporates the use of live attenuated vaccines, which in turn requires a method for differentiation between vaccinated and infected sheep. The NISKHI live attenuated SPP vaccine (LAV) is abundantly used in Russia, Kazakhstan and other Central Asian countries. This study describes the development and evaluation of a real-time PCR with a high-resolution melting assay, capable of differentiating the NISKHI vaccine virus from circulating virulent field strains. The RNA polymerase subunit RPO132 gene contains a unique single nucleotide polymorphism (SNP) capable of altering the melting curves of amplicons from LAV and virulent field isolates circulating in the region. The melting temperature (Tm) of field isolates ranged from 75.47 °C ± 0.04 to 75.86 °C ± 0.08, while the vaccine strain averaged 76.46 °C ± 0.12. Subsequent evaluation of this assay demonstrated that the recent SPP outbreaks in central Russia may be attributed to virulent field isolates. This robust assay was proven to consistently and differentially detect the NISKHI LAV strain when analyzing clinical samples from affected sheep. Full article

Histoplasmosis is a major cause of mortality in individuals with advanced human immunodeficiency virus (HIV) disease (AHD). We evaluated in patients with AHD a lateral flow assay (LFA) developed by MiraVista^® Diagnostics (MVD LFA). Histoplasmosis was defined based on the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) case definitions. We also compared the results of this LFA with those obtained using a commercial enzyme immunoassay (EIA) developed by IMMY, Clarus Histoplasma GM EIA, IMMY (HGM EIA). A retrospective observational study was conducted at Hospital Juan A. Fernández, located in Buenos Aires, Argentina. The study included 48 urine specimens from patients aged >18 years with AHD. Urine specimens included 17 patients with disseminated histoplasmosis and 31 specimens from patients without evidence of histoplasmosis. Specimens were tested using the MVD LFA and the HGM EIA. The MVD LFA and the HGM EIA had similar analytical performance, with a sensitivity of 94%, specificity of 100%, positive predictive value of 100%, negative predictive value of 97%, and an accuracy of 98%. Comparison of the MVD LFA with the HGM EIA demonstrated a Kappa agreement index of 0.906. The LFA evaluated in this study had high analytical performance; it provided rapid diagnosis of histoplasmosis with minimal requirements for laboratory training, equipment, and laboratory infrastructure. Full article

Antimicrobial resistance is a disseminated global health challenge because many of the genes that cause resistance can transfer horizontally between bacteria. Despite the central role of extrachromosomal DNA elements called plasmids in driving the spread of resistance, the detection and surveillance of plasmids remains a significant barrier in molecular epidemiology. We assessed two DNA sequencing platforms alone and in combination for laboratory diagnostics in Botswana by annotating antibiotic resistance genes and plasmids in extensively drug resistant bacteria from diarrhea in Botswana. Long-read Nanopore DNA sequencing and high accuracy basecalling effectively estimated the architecture and gene content of three plasmids in Escherichia coli HUM3355 and two plasmids in Klebsiella pneumoniae HUM7199. Polishing the assemblies with Illumina reads increased base calling precision with small improvements to gene prediction. All five plasmids encoded one or more antibiotic resistance genes, usually within gene islands containing multiple antibiotic and metal resistance genes, and four plasmids encoded genes associated with conjugative transfer. Two plasmids were almost identical to antibiotic resistance plasmids sequenced in Europe and North America from human infection and a pig farm. These One Health connections demonstrate how low-, middle-, and high-income countries collectively benefit from increased whole genome sequencing capacity for surveillance and tracking of infectious diseases and antibiotic resistance genes that can transfer between animal hosts and move across continents. Full article

Listeria monocytogenes is a pathogenic, gram-positive bacterium causing foodborne infections and listeriosis, an infection responsible for serious medical conditions, especially for pregnant women, newborns, or people with a weak immune system. Even after antibiotic treatment, 30% of clinical infections result in death. L. monocytogenes is able to enter and multiply in mammalian cells. Invasion into epithelial cells in the human intestine is mediated by the interaction of the bacterial surface protein internalin A (InlA) with the host cell receptor E-cadherin (E-cad). We have used phage display to select InlA-specific peptides consisting of 12 amino acids using a randomized, recombinant peptide library. We could demonstrate that the selected peptides bound to recombinant InlA protein as well as to L. monocytogenes cells. In vitro, some of the peptides inhibited the interaction between recombinant InlA and human E-cad. As far as we know, this is the first publication on the development of InlA-specific peptide ligands. In the future, our peptides might be used for the development of innovative diagnostic tools or even therapeutic approaches. Full article

Deciphering the key factors underlying individual responses to a habitual diet is important in implementing personalized nutrition strategies to treat allergic diseases. This prospective randomized cohort study aimed to identify key factors determining individual pollen allergy (PA) trajectories in a natural setting. Baseline data on fecal microbiota composition, lifestyle activities, and diet habits of 190 participants with PA and 92 healthy controls were collected, followed by a SOMPO-guided intestinal activity program. Three enterotypes enriched in Bacteroides, Prevotella, and Ruminococcus and four subenterotypes for enterotypes Bacteroides and Prevotella enriched with Faecalibacterium, Megamonas, and Fusobacterium were identified at baseline. PA severity was significantly negatively correlated with the daily intake of fermented plants and no weekly intake of meat, but positively correlated with poor sleep quality. Interactions between enterotype and lifestyle factors affected PA severity, and intestinal activity intervention based on the baseline enterotype reduced the PA severity score. In conclusion, the findings of this study demonstrated that the baseline gut enterotype plays a crucial role in PA. This study suggests combining enterotype data with habitual diet can improve PA severity. Full article

Pangolins are the only animals other than bats proposed to have been infected with SARS-CoV-2 related coronaviruses (SARS2r-CoVs) prior to the COVID-19 pandemic. Here, we examine the novel SARS2r-CoV we previously identified in game animal metatranscriptomic datasets sequenced by the Nanjing Agricultural University in 2022, and find that sections of the partial genome phylogenetically group with Guangxi pangolin CoVs (GX PCoVs), while the full RdRp sequence groups with bat-SL-CoVZC45. While the novel SARS2r-CoV is found in 6 pangolin datasets, it is also found in 10 additional NGS datasets from 5 separate mammalian species and is likely related to contamination by a laboratory researched virus. Absence of bat mitochondrial sequences from the datasets, the fragmentary nature of the virus sequence and the presence of a partial sequence of a cloning vector attached to a SARS2r-CoV read suggests that it has been cloned. We find that NGS datasets containing the novel SARS2r-CoV are contaminated with significant Homo sapiens genetic material, and numerous viruses not associated with the host animals sampled. We further identify the dominant human haplogroup of the contaminating H. sapiens genetic material to be F1c1a1, which is of East Asian provenance. The association of this novel SARS2r-CoV with both bat CoV and the GX PCoV clades is an important step towards identifying the origin of the GX PCoVs. Full article