Genomic and phenotypic studies among Clostridioides difficile isolates show a high prevalence of clade 2 and great diversity in clinical isolates from Mexican adults and children with healthcare-associated diarrhea.

Clostridioides difficile (C. difficile) is widely distributed in the intestinal tract of humans, animals, and in the environment. It is the most common cause of diarrhea associated with the use of antimicrobials in humans and among the most common healthcare-associated infections worldwide. Its pathogenesis is mainly due to the production of toxin A (TcdA), toxin B (TcdB), and a binary toxin (CDT), whose genetic variants may be associated with disease severity. We studied genetic diversity in 39 C. difficile isolates from adults and children attended at two Mexican hospitals, using different gene and genome typing methods and investigated their association with in vitro expression of toxins. Whole-genome sequencing in 39 toxigenic C. difficile isolates were used for multilocus sequence typing, tcdA, and tcdB typing sequence type, and phylogenetic analysis. Strains were grown in broth media, and expression of toxin genes was measured by real-time PCR and cytotoxicity in cell-culture assays. Clustering of strains by genome-wide phylogeny matched clade classification, forming different subclusters within each clade. The toxin profile tcdA+/tcdB+/cdt+ and clade 2/ST1 were the most prevalent among isolates from children and adults. Isolates presented two TcdA and three TcdB subtypes, of which TcdA2 and TcdB2 were more prevalent. Prevalent clades and toxin subtypes in strains from children differed from those in adult strains. Toxin gene expression or cytotoxicity was not associated with genotyping or toxin subtypes. In conclusion, genomic and phenotypic analysis shows high diversity among C. difficile isolates from patients with healthcare-associated diarrhea. IMPORTANCE: Clostridioides difficile is a toxin-producing bacterial pathogen recognized as the most common cause of diarrhea acquired primarily in healthcare settings. This bacterial species is diverse; its global population has been divided into five different clades using multilocus sequence typing, and strains may express different toxin subtypes that may be related to the clades and, importantly, to the severity and progression of disease. Genotyping of children strains differed from adults suggesting toxins might present a reduced toxicity. We studied extensively cytotoxicity, expression of toxins, whole genome phylogeny, and toxin typing in clinical C. difficile isolates. Most isolates presented a tcdA+/ tcdB+/cdt+ pattern, with high diversity in cytotoxicity and clade 2/ST1 was the most prevalent. However, they all had the same TcdA2/TcdB2 toxin subtype. Advances in genomics and bioinformatics tools offer the opportunity to understand the virulence of C. difficile better and find markers for better clinical use.

Draft genome of three uropathogenic Escherichia coli strains.

Uropathogenic Escherichia coli (UPEC) remains the main etiological agent of urinary tract infections affecting females and males. The draft genome sequence of three strains of UPEC isolated from senior citizens and pregnant women in the state of Puebla, Mexico, is reported here.

The genome sequence of Tadarida brasiliensis I. Geoffroy Saint-Hilaire, 1824 [Molossidae; Tadarida].

We present a genome assembly from an individual male Tadarida brasiliensis (The Brazilian free-tailed bat; Chordata; Mammalia; Chiroptera; Molossidae). The genome sequence is 2.28 Gb in span. The majority of the assembly is scaffolded into 25 chromosomal pseudomolecules, with the X and Y sex chromosomes assembled.

Insights on the genomic diversity, virulence and resistance profile of a Campylobacter jejuni strain isolated from a hospitalized patient in Brazil.

Campylobacteriosis is currently recognized as one of the major causes of foodborne bacterial diseases worldwide. In Brazil, there is insufficient data to estimate the impact of Campylobacter in public health. The aim of this present study was to characterize a C. jejuni CJ-HBSJRP strain isolated from a hospitalized patient in Brazil by its ability to invade human Caco-2 epithelial cells, to survive in U937 human macrophages, and to assess its phenotypic antimicrobial resistance profile. In addition, prophages, virulence and antimicrobial resistance genes were search using whole-genome sequencing data. The genetic relatedness was evaluated by MLST and cgMLST analysis by comparison with 29 other C. jejuni genomes isolated from several countries. The CJ-HBSJRP strain showed an invasion percentage of 50% in Caco-2 polarized cells, 37.5% of survivability in U937 cells and was phenotypically resistant to ampicillin, ciprofloxacin and nalidixic acid. A total of 94 virulence genes related to adherence, biofilm, chemotaxis, immune modulation, invasion process, metabolism, motility and toxin were detected. The resistance genes blaOXA-605 (blaOXA-61), cmeB and mutations in the QRDR region of gyrA were also found and none prophages were detected. The MLST analysis showed 23 different STs among the strains studied. Regarding cgMLST analysis, the CJ-HBSJRP strain was genetically distinct and did not group closely to any other isolate. The results obtained reinforce the pathogenic potential of the CJHBSJRP strain and highlighted the need for more careful attention to Campylobacter spp. infections in Brazil since this pathogen has been the most commonly reported zoonosis in several countries worldwide.

Partial genomic characterization of Chromobacterium piscinae from India reveals multi drug resistance.

Species of genus Chromobacterium have been isolated from diverse geographical settings, which exhibits significant metabolic flexibility as well as biotechnological and pathogenic properties. This study describes the isolation, characterization, draft assembly, and detailed sequence analysis of Chromobacterium piscinae strain W1B-CG-NIBSM isolated from water samples from multi use community pond. The organism was characterized by biochemical tests, Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI TOF-MS) and partial genome sequencing. The partial genomic data of Chromobacterium pisciane isolate W1B NIBSM strain was submitted to GenBank with Bio project number PRJNA803347 and accession no CP092474. An integrated genome analysis of Chromobacterium piscinae has been accomplished with PATRIC which indicates good quality genome. DNA sequencing using the illumina HiSeq 4000 system generated total length of 4,155,481 bp with 63 contig with G + C content is 62.69%. This partial genome contains 4,126 protein-coding sequences (CDS), 27 repeats region and 78 transfer RNA (tRNA) genes as well as 3 ribosomal RNA (rRNA) genes. The genomic annotation of Chromobacterium W1B depicts 2,925 proteins with functional assignments and 1201 hypothetical proteins. A repertoire of specialty genes implicated in antibiotic resistance (45 genes), drug target (6 genes), Transporter (3 genes) and virulence factor (10 genes). The genomic analysis reveals the adaptability, displays metabolic varied pathways and shows specific structural complex and various virulence factors which makes this strain multi drug resistant. The isolate was found to be highly resistant to ß-lactam antibiotics whereas it showed sensitivity towards aminoglycosides and fluoroquinolone antibiotics. Hence, the recovery of Chromobacterium piscinae from community pond evidenced for uncertain hidden source of public health hazard. To the best of authors knowledge this is first report of isolation and genomic description of C. piscinae from India.

Complete genome sequence of deformed wing virus and black queen cell virus isolated from honeybees (Apis mellifera) in Argentina.

We report the complete genome sequence of deformed wing virus and black queen cell virus isolated from Argentinean's honeybees. These sequence data will be valuable for future research on the viral variants present in the country and the development of strategies to control the spread of these viruses in apiaries.

First whole-genome sequence and assembly of the Ecuadorian brown-headed spider monkey (Ateles fusciceps fusciceps), a critically endangered species, using Oxford Nanopore Technologies.

The Ecuadorian brown-headed spider monkey (Ateles fusciceps fusciceps) is currently considered one of the most endangered primates in the world and is classified as critically endangered [International union for conservation of nature (IUCN)]. It faces multiple threats, the most significant one being habitat loss due to deforestation in western Ecuador. Genomic tools are keys for the management of endangered species, but this requires a reference genome, which until now was unavailable for A. f. fusciceps. The present study reports the first whole-genome sequence and assembly of A. f. fusciceps generated using Oxford Nanopore long reads. DNA was extracted from a subadult male, and libraries were prepared for sequencing following the Ligation Sequencing Kit SQK-LSK112 workflow. Sequencing was performed using a MinION Mk1C sequencer. The sequencing reads were processed to generate a genome assembly. Two different assemblers were used to obtain draft genomes using raw reads, of which the Flye assembly was found to be superior. The final assembly has a total length of 2.63 Gb and contains 3,861 contigs, with an N50 of 7,560,531 bp. The assembly was analyzed for annotation completeness based on primate ortholog prediction using a high-resolution database, and was found to be 84.3% complete, with a low number of duplicated genes indicating a precise assembly. The annotation of the assembly predicted 31,417 protein-coding genes, comparable with other mammal assemblies. A reference genome for this critically endangered species will allow researchers to gain insight into the genetics of its populations and thus aid conservation and management efforts of this vulnerable species.

Complete genome sequence of Pectobacterium brasiliense strain 21PCA_AGRO2 with antimicrobial resistance isolated from napa cabbage.

We report a complete genome of Pectobacterium brasiliense strain 21PCA_AGRO2 isolated from napa cabbage, in which the genome consists of a circular chromosome comprising 4,919,671 bp with 4,399 coding DNA sequences, 22 rRNA genes, 77 tRNA genes, and 9 noncoding RNA genes.

Molecular Characterization of Salmonella Phage Wara Isolated from River Water in Brazil.

Antimicrobial resistance is increasing despite new treatments being employed, so novel strategies are required to ensure that bacterial infections remain treatable. Bacteriophages (phages; bacteria viruses) have the potential to be used as natural antimicrobial methods to control bacterial pathogens such as Salmonella spp. A Salmonella phage, Wara, was isolated from environmental water samples at the Subaé River Basin, Salvador de Bahia, Brazil. The basin has environmental impacts in its main watercourses arising from the dumping of domestic and industrial effluents and agricultural and anthropological activities. The phage genome sequence was determined by Oxford Nanopore Technologies (ONT) MinION and Illumina HiSeq sequencing, and assembly was carried out by Racon (MinION) and Unicycler (Illumina, Illumina + MinION). The genome was annotated and compared to other Salmonella phages using various bioinformatics approaches. MinION DNA sequencing combined with Racon assembly gave the best complete genome sequence. Phylogenetic analysis revealed that Wara is a member of the Tequintavirus genus. A lack of lysogeny genes, antimicrobial resistance, and virulence genes indicated that Wara has therapeutic and biocontrol potential against Salmonella species in healthcare and agriculture.

Genomic-proteomic analysis of a novel Bacillus thuringiensis strain: toxicity against two lepidopteran pests, abundance of Cry1Ac5 toxin, and presence of InhA1 virulence factor.

Bacillus thuringiensis (Bt) is a biological alternative to the indiscriminate use of chemical insecticides in agriculture. Due to resistance development on insect pests to Bt crops, isolating novel Bt strains is a strategy for screening new pesticidal proteins or strains containing toxin profile variety that can delay resistance. Besides, the combined genomic and proteomic approaches allow identifying pesticidal proteins and virulence factors accurately. Here, the genome of a novel Bt strain (Bt TOL651) was sequenced, and the proteins from the spore-crystal mixture were identified by proteomic analysis. Toxicity bioassays with the spore-crystal mixture against larvae of Diatraea saccharalis and Anticarsia gemmatalis, key pests of sugarcane and soybean, respectively, were performed. The toxicity of Bt TOL651 varies with the insect; A. gemmatalis (LC50 = 1.45 ng cm-2) is more susceptible than D. saccharalis (LC50 = 73.77 ng cm-2). Phylogenetic analysis of the gyrB gene indicates that TOL651 is related to Bt kenyae strains. The genomic analysis revealed the presence of cry1Aa18, cry1Ac5, cry1Ia44, and cry2Aa9 pesticidal genes. Virulence factor genes such as phospholipases (plcA, piplc), metalloproteases (inhA), hemolysins (cytK, hlyIII, hblA, hblC, hblD), and enterotoxins (nheA, nheB, nheC) were also identified. The combined use of the genomic and proteomic data indicated the expression of Cry1Aa18, Cry1Ac5, and Cry2Aa9 proteins, with Cry1Ac5 being the most abundant. InhA1 also was expressed and may contribute to Bt TOL651 pathogenicity. These results provide Bt TOL651 as a new tool for the biocontrol of lepidopteran pests.

Genome-sequence, annotation and phylogenetic insights of the lactic acid bacterium Limosilactobacillus fermentum strain LAB1 obtained from the dairy beverage borhani

Limosilactobacillus fermentum is a promising probiotic with several documented health benefits. LAB1 is an antagonistic L. fermentum strain isolated from borhani, a traditional South Asian beverage prepared from dairy and plant ingredients. Here, I present the genome sequence of the L. fermentum LAB1 strain, its annotation, and phylogenetic features. The 2.01 Mb genome with a G+C content of 51.9% was assembled into 221 contigs and predicted to have 1,913 protein-coding genes, 98 pseudo genes, 7 rRNAs, 60 tRNAs, and 1 CRISPR array. As much as 91.1% of the coding sequences could be assigned to known functional genes. Determination of average nucleotide identity (ANI) of the genome sequence revealed 99.37% identity to that of the type strain ATCC 14931. Its 16S rRNA gene sequence extracted from the genome sequence showed close phylogenetic association with several L. fermentum strains. The genome sequence is expected to provide useful insights with regard to the phenotypic, metabolic and beneficial aspects of this lactic acid bacterium.(AU)

A fractal scaling analysis of the SARS-CoV-2 genome sequence.

An approach based on fractal scaling analysis to characterize the organization of the SARS-CoV-2 genome sequence was used. The method is based on the detrended fluctuation analysis (DFA) implemented on a sliding window scheme to detect variations of long-range correlations over the genome sequence regions. The nucleotides sequence is mapped in a numerical sequence by using four different assignation rules: amino-keto, purine-pyrimidine, hydrogen-bond and hydrophobicity patterns. The originally reported sequence from Wuhan isolates (Wuhan Hu-1) was considered as a reference to contrast the structure of the 2002-2004 SARS-CoV-1 strain. Long-range correlations, quantified in terms of a scaling exponent, depended on both the mapping rule and the sequence region. Deviations from randomness were attributed to serial correlations or anti-correlations, which can be ascribed to ordered regions of the genome sequence. It was found that the Wuhan Hu-1 sequence was more random than the SARS-CoV-1 sequence, which suggests that the SARS-CoV-2 possesses a more efficient genomic structure for replication and infection. In general, the virus isolated in the early 2020 months showed slight correlation differences with the Wuhan Hu-1 sequence. However, early isolates from India and Italy presented visible differences that led to a more ordered sequence organization. It is apparent that the increased sequence order, particularly in the spike region, endowed some early variants with a more efficient mechanism to spreading, replicating and infecting. Overall, the results showed that the DFA provides a suitable framework to assess long-term correlations hidden in the internal organization of the SARS-CoV-2 genome sequence.

Spatial scale of tuberculosis transmission in Lima, Peru.

Spatially targeted interventions may be effective alternatives to individual or population-based prevention strategies against tuberculosis (TB). However, their efficacy may depend on the mechanisms that lead to geographically constrained hotspots. Local TB incidence may reflect high levels of local transmission; conversely, they may point to frequent travel of community members to high-risk areas. We used whole-genome sequencing to explore patterns of TB incidence and transmission in Lima, Peru. Between 2009 and 2012, we recruited incident pulmonary TB patients and their household contacts, whom we followed for the occurrence of TB disease. We used whole-genome sequences of 2,712 Mycobacterial tuberculosis isolates from 2,440 patients to estimate pariwise genomic distances and compared these to the spatial distance between patients' residences. Genomic distances increased rapidly as spatial distances increased and remained high beyond 2 km of separation. Next, we divided the study catchment area into 1 × 1 km grid-cell surface units and used household spatial coordinates to locate each TB patient to a specific cell. We estimated cell-specific transmission by calculating the proportion of patients in each cell with a pairwise genomic distance of 10 or fewer single-nucleotide polymorphisms. We found that cell-specific TB incidence and local transmission varied widely but that cell-specific TB incidence did not correlate closely with our estimates of local transmission (Cohen's k = 0.27). These findings indicate that an understanding of the spatial heterogeneity in the relative proportion of TB due to local transmission may help guide the implementation of spatially targeted interventions.

Draft Genome Sequence of Priestia sp. Strain TSO9, a Plant Growth-Promoting Bacterium Associated with Wheat (Triticum turgidum subsp. durum) in the Yaqui Valley, Mexico.

Strain TSO9 was isolated from a commercial field of wheat (Triticum turgidum L. subsp. durum) located in the Yaqui, Valley, Mexico. Here, the genome of this strain was sequenced, obtaining a total of 5,248,515 bp; 38.0% G + C content; 1,186,514 bp N50; and 2 L50. Based on the 16S rRNA gene sequencing, strain TSO9 was affiliated with the genus Priestia. The genome annotation of Priestia sp. TSO9 contains a total of 147 RNAs, 128 tRNAs, 1 tmRNA, and 5512 coding DNA sequences (CDS) distributed into 332 subsystems, where CDS associated with agricultural purposes were identified, such as (i) virulence, disease, and defense (57 CDS) (i.e., resistance to antibiotics and toxic compounds (34 CDS), invasion and intracellular resistance (12 CDS), and bacteriocins and ribosomally synthesized antibacterial peptides (10 CDS)), (ii) iron acquisition and metabolism (36 CDS), and (iii) secondary metabolism (4 CDS), i.e., auxin biosynthesis. In addition, subsystems related to the viability of an active ingredient for agricultural bioproducts were identified, such as (i) stress response (65 CDS). These genomic traits are correlated with the metabolic background of this strain, and its positive effects on wheat growth regulation reported in this work. Thus, further investigations of Priestia sp. TSO9 are necessary to complement findings regarding its application in agroecosystems to increase wheat yield sustainably.

Genomic Analysis of Carbapenem-Resistant Acinetobacter baumannii Strains Recovered from Chilean Hospitals Reveals Lineages Specific to South America and Multiple Routes for Acquisition of Antibiotic Resistance Genes.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is a public health threat accounting for a significant number of hospital-acquired infections. Despite the importance of this pathogen, there is scarce literature on A. baumannii molecular epidemiology and evolutionary pathways relevant to resistance emergence in South American strains. We analyzed the genomic context of 34 CRAb isolates recovered from clinical samples between 2010 and 2013 from two hospitals in Santiago, Chile, using whole-genome sequencing. Several Institut Pasteur scheme sequence types (STs) were identified among the 34 genomes studied here, including ST1, ST15, ST79, ST162, and ST109. No ST2 (the most widespread sequence type) strain was detected. Chilean isolates were phylogenetically closely related, forming lineages specific to South America (e.g., ST1, ST79, and ST15). The genomic contexts of the resistance genes were diverse: while genes were present in a plasmid in ST15 strains, all genes were chromosomal in ST79 strains. Different variants of a small Rep_3 plasmid played a central role in the acquisition of the oxa58 carbapenem and aacC2 aminoglycoside resistance genes in ST1, ST15, and ST79 strains. The aacC2 gene along with blaTEM were found in a novel transposon named Tn6925 here. Variants of Tn7 were also found to play an important role in the acquisition of the aadA1 and dfrA1 genes. This work draws a detailed picture of the genetic context of antibiotic resistance genes in a set of carbapenem-resistant A. baumannii strains recovered from two Chilean hospitals and reveals a complex evolutionary picture of antibiotic resistance gene acquisition events via multiple routes involving several mobile genetic elements. IMPORTANCE Treating infections caused by carbapenem-resistant A. baumannii (CRAb) has become a global challenge given that CRAb strains are also often resistant to a wide range of antibiotics. Analysis of whole-genome sequence data is now a standard approach for studying the genomic context of antibiotic resistance genes; however, genome sequence data from South American countries are scarce. Here, phylogenetic and genomic analyses of 34 CRAb strains recovered from 2010 to 2013 from two Chilean hospitals revealed a complex picture leading to the generation of resistant lineages specific to South America. From these isolates, we characterized several mobile genetic elements, some of which are described for the first time. The genome sequences and analyses presented here further our understanding of the mechanisms leading to multiple-drug resistance, extensive drug resistance, and pandrug resistance phenotypes in South America. Therefore, this is a significant contribution to elucidating the global molecular epidemiology of CRAb.

Whole-Genome Sequence and Interaction Analysis in the Production of Six Enzymes From the Three Bacillus Strains Present in a Commercial Direct-Fed Microbial (Norum™) Using a Bliss Independence Test.

The three Bacillus strains present in Norum™ were initially selected by their excellent to good relative enzyme activity (REA) production score for amylase, protease, lipase, phytase, cellulase, ß-glucanase, and xylanase. Further studies confirmed that the three isolates also showed an antibacterial activity, Gram-positive and Gram-negative poultry pathogens. Norum™ (Eco-Bio/Euxxis Bioscience LLC) is a Bacillus spore direct-fed microbial (DFM). The Bacillus isolates were screened and selected based on in vitro enzyme production profiles. Moreover, in chickens fed high non-starch polysaccharides, this DFM demonstrated to reduce digesta viscosity, bacterial translocation, increase performance, bone mineralization, and balance the intestinal microbiota. In the present study, we present the whole-genome sequence of each of the three isolates in Norum™, as well as the synergistic, additive, or antagonistic effects on the enzyme production behavior of the three Bacillus strains and their combinations when grown together vs. when grown individually. The whole-genome sequence identified isolate AM1002 as Bacillus subtilis (isolate 1), isolate AM0938 as Bacillus amyloliquefaciens (isolate 2), and isolate JD17 as Bacillus licheniformis (isolate 3). The three Bacillus isolates used in the present study produce different enzymes (xylanase, cellulase, phytase, lipase, protease, and ß-glucanase). However, this production was modified when two or more Bacillus strains were combined, suggesting possible synergistic, antagonistic, or additive interactions. The Bliss analysis suggested (p < 0.05) that the combination of Bacillus strains 1-2 and 1-2-3 had intermediate effects and predicted that the combination of Bacillus strains 2-3 could have better effects than the combination of all the three Bacillus strains. In summary, the current study demonstrated the need of selecting Bacillus strains based on quantitative enzyme determination and data analysis to assess the impacts of combinations to avoid antagonistic interactions that could limit treatment efficacy. These results suggest that using Bacillus strains 2-3 together could lead to a new generation of DFMs with effects superior to those already examined in Bacillus strains 1-2-3 and, therefore, a potential alternative to growth-promoting antibiotics. More research utilizing poultry models is being considered to confirm and expand the existing findings.

Bdellovibrio reynosensis sp. nov., from a Mexico soil sample.

A novel predatory bacterium, strain LBG001T, has been isolated from Reynosa, Mexico. The 16S rRNA shares approximately 97 % sequence identity with many reported strains in the genus Bdellovibrio including the type strain Bdellovibrio bacteriovorus HD100T. Phylogenetic trees based on the 16S rRNA gene and on 30 concatenated housekeeping genes or core genes showed that LBG001T is on a separate branch from the B. bacteriovorus group. LBG0001T has a genome size of 3 582 323 bp with a G+C content of 43.1 mol %. The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values with other members of the genus Bdellovibrio (<79, <72 and <17 %, respectively) qualifies the strain to represent a new species in the genus. Strain LBG001T formed visible plaques on all 10 tested Gram-negative bacterial species. The phenotypic characteristics, phylogenetic analysis and genomic taxonomic studies support the classification of the strain as representing a new species for which the name Bdellovibrio reynosensis sp. nov. is proposed. The type strain is LBG001T(=ATCC TSD-288T =CM-CNRG 0932T).

Fusarium chaquense, sp. nov, a novel type A trichothecene-producing species from native grasses in a wetland ecosystem in Argentina.

The Chaco wetland is among the most biologically diverse regions in Argentina. In collections of fungi from asymptomatic native grasses (Poaceae) from the wetlands, we identified isolates of Fusarium that were morphologically similar to F. armeniacum, but distinct from it by their production of abundant microconidia. All the isolates had identical, or nearly identical, partial sequences of TEF1 and RPB2. But they were distinct from reference sequences from F. armeniacum and Fusarium species closely related to it. Phylogenetic analysis of 34 full-length housekeeping gene sequences retrieved from whole genome sequences of three Chaco wetland isolates, 29 genes resolved the isolates as an exclusive clade within the F. sambucinum species complex. Based on results of the morphological and phylogenetic analysis, we concluded that the Chaco wetland isolates are a distinct and novel species, herein described as Fusarium chaquense, sp. nov., which is closely related to F. armeniacum. F. chaquense in culture can produce the trichothecenes T-2 and HT-2 toxin, neosolaniol, diacetoxyscirpenol, and monoacetoxyscirpenol, as well as beauvericin and the pigment aurofusarin. Genome sequence analysis also revealed the presence of three previously described loci required for trichothecene biosynthesis. This research represents the first study of Fusarium in a natural ecosystem in Argentina.

Genetic description of VanD phenotype vanA genotype in vancomycin-resistant Enterococcus faecium isolates from a Bone Marrow Transplantation Unit.

BACKGROUND: Vancomycin-resistant Enterococcus faecium (VREfm) is an important agent of hospital-acquired infection. VanA phenotype is characterized by resistance to high levels of vancomycin and teicoplanin and is encoded by the vanA gene, whereas VanD phenotype is characterized by resistance to vancomycin and susceptibility or intermediate resistance to teicoplanin; however, some isolates carry a VanD phenotype with a vanA genotype, but there are many gaps in the knowledge about the genetic mechanisms behind this pattern. OBJECTIVE: To characterize the genetic structure, clonality, and mobile genetic elements of VRE isolates that display a VanD-vanA phenotype. RESULTS: All vanA VRE-fm isolates displayed minimum inhibitory concentration (MIC) for vancomycin > 32µg/mL and intermediate or susceptible MIC range for teicoplanin (8-16µg/mL). The isolates were not clonal, and whole-genome sequencing analysis showed that they belonged to five different STs (ST478, ST412, ST792, ST896, and ST1393). The absence of some van complex genes were observed in three isolates: Ef5 lacked vanY and vanZ, Ef2 lacked vanY, and Ef9 lacked orf1 and orf2; moreover, another three isolates had inverted positions of orf1, orf2, vanR, and vanS genes. IS1542 was observed in all isolates, whereas IS1216 in only five. Moreover, presence of other hypothetical protein-encoding genes located downstream the vanZ gene were observed in six isolates. CONCLUSION: VRE isolates can display some phenotypes associated to vanA genotype, including VanA and VanB, as well as VanD; however, further studies are needed to understand the exact role of genetic variability, rearrangement of the transposon Tn1546, and presence of insertion elements in isolates with this profile.

Genomic analysis revealed conserved acid tolerance mechanisms from native micro-organisms in fermented feed.

AIMS: Fermented feed is an agricultural practice used in many regions of the world to improve the growth performance of farm animals. This study aimed to identify and evaluate the lactic acid bacteria and yeast involved in the production of fermented feed. METHODS AND RESULTS: We isolated and described two micro-organisms from autochthonous microbiota origin present in a regional feed product, Lactobacillus paracasei IBR07 (Lacticaseibacillus paracasei) and Kazachstania unispora IBR014 (Saccharomyces unisporum). Genome sequence analyses were performed to characterize both micro-organisms. Potential pathways involved in the acid response, tolerance and persistence were predicted in both genomes. Although L. paracasei and K. unispora are considered safe for animal feed, we analysed the presence of virulence factors, antibiotic resistance and pathogenicity islands. Furthermore, the Galleria mellonella model was used to support the safety of both isolates. CONCLUSIONS: We conclude that IBR07 and IBR014 strains are good candidates to be used as starter cultures for feed fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented here will be helpful to explore other biotechnological aspects and constitute a starting point for further studies to establish the consumption benefit of fermented feed in farm animal production.