Functional characterization of a gene cluster responsible for inositol catabolism associated with hospital-adapted isolates of Enterococcus faecium

Microbiology ◽  
2021 ◽  
Vol 167 (9) ◽  
Author(s):  
Janetta Top ◽  
Jery Baan ◽  
Adinda Bisschop ◽  
Sergio Arredondo-Alonso ◽  
Willem van Schaik ◽  
...  
Keyword(s):  
Mouse Model ◽  
Gene Cluster ◽  
Enterococcus Faecium ◽  
Type Species ◽  
Integration Site ◽  
Functional Characterization ◽  
Donor Strain ◽  
Integrase Gene ◽  
Content Type ◽  
Link Type

Enterococcus faecium is a nosocomial, multidrug-resistant pathogen. Whole genome sequence studies revealed that hospital-associated E. faecium isolates are clustered in a separate clade A1. Here, we investigated the distribution, integration site and function of a putative iol gene cluster that encodes for myo-inositol (MI) catabolism. This iol gene cluster was found as part of an ~20 kbp genetic element (iol element), integrated in ICEEfm1 close to its integrase gene in E. faecium isolate E1679. Among 1644 E. faecium isolates, ICEEfm1 was found in 789/1227 (64.3 %) clade A1 and 3/417 (0.7 %) non-clade A1 isolates. The iol element was present at a similar integration site in 180/792 (22.7 %) ICEEfm1-containing isolates. Examination of the phylogenetic tree revealed genetically closely related isolates that differed in presence/absence of ICEEfm1 and/or iol element, suggesting either independent acquisition or loss of both elements. E. faecium iol gene cluster containing isolates E1679 and E1504 were able to grow in minimal medium with only myo-inositol as carbon source, while the iolD-deficient mutant in E1504 (E1504∆iolD) lost this ability and an iol gene cluster negative recipient strain gained this ability after acquisition of ICEEfm1 by conjugation from donor strain E1679. Gene expression profiling revealed that the iol gene cluster is only expressed in the absence of other carbon sources. In an intestinal colonization mouse model the colonization ability of E1504∆iolD mutant was not affected relative to the wild-type E1504 strain. In conclusion, we describe and functionally characterise a gene cluster involved in MI catabolism that is associated with the ICEEfm1 island in hospital-associated E. faecium isolates. We were unable to show that this gene cluster provides a competitive advantage during gut colonisation in a mouse model. Therefore, to what extent this gene cluster contributes to the spread and ecological specialisation of ICEEfm1-carrying hospital-associated isolates remains to be investigated.

Two regulatory factors of Vibrio cholerae activating the mannose-sensitive haemagglutinin pilus expression is important for biofilm formation and colonization in mice

Microbiology ◽  
2021 ◽  
Vol 167 (10) ◽  
Author(s):  
Mengting Shi ◽  
Yue Zheng ◽  
Xianghong Wang ◽  
Zhengjia Wang ◽  
Menghua Yang
Keyword(s):  
Mouse Model ◽  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Type Species ◽  
Wild Type ◽  
Expression Library ◽  
Content Type ◽  
Genomic Expression ◽  
Infant Mouse ◽  
Link Type

Vibrio cholerae the causative agent of cholera, uses a large number of coordinated transcriptional regulatory events to transition from its environmental reservoir to the host intestine, which is its preferred colonization site. Transcription of the mannose-sensitive haemagglutinin pilus (MSHA), which aids the persistence of V. cholerae in aquatic environments, but causes its clearance by host immune defenses, was found to be regulated by a yet unknown mechanism during the infection cycle of V. cholerae . In this study, genomic expression library screening revealed that two regulators, VC1371 and VcRfaH, are able to positively activate the transcription of MSHA operon. VC1371 is localized and active in the cell membrane. Deletion of vc1371 or VcrfaH genes in V. cholerae resulted in less MshA protein production and less efficiency of biofilm formation compared to that in the wild-type strain. An adult mouse model showed that the mutants with vc1371 or VcrfaH deletion colonized less efficiently than the wild-type; the VcrfaH deletion mutant showed less colonization efficiency in the infant mouse model. The findings strongly suggested that the two regulators, namely VC1371 and VcRfaH, which are involved in the regulation of MSHA expression, play an important role in V. cholerae biofilm formation and colonization in mice.

Emergence of a plasmid-borne tigecycline resistance in Klebsiella pneumoniae in Vietnam

2021 ◽  
Author(s):  
Aki Hirabayashi ◽  
Van Thi Thu Ha ◽  
An Van Nguyen ◽  
Son Thai Nguyen ◽  
Keigo Shibayama ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Gene Cluster ◽  
Type Species ◽  
Bacterial Infections ◽  
Efflux Pump ◽  
Whole Genome Sequence ◽  
High Sequence Identity ◽  
Pump System ◽  
Content Type ◽  
Link Type

Tigecycline is a last-resort antimicrobial used to treat multidrug-resistant Gram-negative bacterial infections. One of the common antimicrobial resistance mechanisms is the efflux pump system composed of membrane protein complexes to excrete xenobiotic substrates. Recently, a novel gene cluster, tmexCD1-toprJ1, encoding the resistance–nodulation–cell division (RND) efflux pump was identified on plasmids in Klebsiella pneumoniae isolates in China. TMexCD1-TOprJ1 was found to be capable of excreting multiple antimicrobials, including tigecycline, which contributed to the strain's resistance. In this study, we identified K. pneumoniae isolates harbouring the tmexCD1-toprJ1 genes outside of China for the first time. Two tigecycline-resistant K. pneumoniae isolates belonging to ST273 by multilocus sequence typing were collected from different patients in a medical institution in Hanoi, Vietnam, in 2015. Whole-genome sequence analysis revealed that these isolates harboured a 288.0 kb tmexCD1-toprJ1–carrying plasmid with IncFIB and IncHI1B replicons. The tmexCD1-toprJ1 gene cluster was surrounded by several mobile gene elements, including IS26, and the plasmids had high sequence identity with that of K. pneumoniae isolated in China. Our finding suggests that the horizontal spread of tigecycline resistance mediated by tmexCD1-toprJ1–carrying plasmids has occurred in Vietnam and other countries, and raises concern about the further global dissemination.

scholarly journals Identification of a repressor for the two iol operons required for inositol catabolism in Geobacillus kaustophilus

Microbiology ◽  
2020 ◽  
Author(s):  
Ken-ichi Yoshida ◽  
Yusuke Shirae ◽  
Ryo Nishimura ◽  
Kaho Fukui ◽  
Shu Ishikawa
Keyword(s):  
Dna Binding ◽  
Gene Cluster ◽  
Binding Sites ◽  
Type Species ◽  
Binding Property ◽  
Promoter Regions ◽  
Content Type ◽  
Link Type ◽  
Geobacillus Kaustophilus ◽  
Inositol Dehydrogenase

Geobacillus kaustophilus HTA426, a thermophilic Gram-positive bacterium, feeds on inositol as its sole carbon source, and an iol gene cluster required for inositol catabolism has been postulated with reference to the iol genes in Bacillus subtilis . The iol gene cluster of G. kaustophilus comprises two tandem operons induced in the presence of inositol; however, the mechanism underlying this induction remains unclear. B. subtilis iolQ is known to be involved in the regulation of iolX encoding scyllo-inositol dehydrogenase, and its homologue in HTA426 was found two genes upstream of the first gene (gk1899) of the iol gene cluster and was termed iolQ in G. kaustophilus . When iolQ was inactivated in G. kaustophilus , not only cellular myo-inositol dehydrogenase activity due to gk1899 expression but also the transcription of the two iol operons became constitutive. IolQ was produced and purified as a C-terminal histidine (His)-tagged fusion protein in Escherichia coli and subjected to an in vitro gel electrophoresis mobility shift assay to examine its DNA-binding property. It was observed that IolQ bound to the DNA fragments containing each of the two iol promoter regions and that DNA binding was antagonized by myo-inositol. Moreover, DNase I footprinting analyses identified two tandem binding sites of IolQ within each of the iol promoter regions. By comparing the sequences of the binding sites, a consensus sequence for IolQ binding was deduced to form a palindrome of 5′-RGWAAGCGCTTSCY-3′ (where R=A or G, W=A or T, S=G or C, and Y=C or T). IolQ functions as a transcriptional repressor regulating the induction of the two iol operons responding to myo-inositol.

scholarly journals AsnB is responsible for peptidoglycan precursor amidation in Clostridium difficile in the presence of vancomycin

Microbiology ◽  
2020 ◽  
Vol 166 (6) ◽  
pp. 567-578 ◽  
Author(s):  
Fariza Ammam ◽  
Delphine Patin ◽  
Héloise Coullon ◽  
Didier Blanot ◽  
Thierry Lambert ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Gene Cluster ◽  
Enterococcus Faecalis ◽  
Type Species ◽  
Asparagine Synthetase ◽  
Structure Modification ◽  
Content Type ◽  
E Coli ◽  
Link Type ◽  
Peptidoglycan Structure

Clostridium difficile 630 possesses a cryptic but functional gene cluster vanG Cd homologous to the vanG operon of Enterococcus faecalis . Expression of vanG Cd in the presence of subinhibitory concentrations of vancomycin is accompanied by peptidoglycan amidation on the meso-DAP residue. In this paper, we report the presence of two potential asparagine synthetase genes named asnB and asnB2 in the C. difficile genome whose products were potentially involved in this peptidoglycan structure modification. We found that asnB expression was only induced when C. difficile was grown in the presence of vancomycin, yet independently from the vanG Cd resistance and regulation operons. In addition, peptidoglycan precursors were not amidated when asnB was inactivated. No change in vancomycin MIC was observed in the asnB mutant strain. In contrast, overexpression of asnB resulted in the amidation of most of the C. difficile peptidoglycan precursors and in a weak increase of vancomycin susceptibility. AsnB activity was confirmed in E. coli . In contrast, the expression of the second asparagine synthetase, AsnB2, was not induced in the presence of vancomycin. In summary, our results demonstrate that AsnB is responsible for peptidoglycan amidation of C. difficile in the presence of vancomycin.

Comparative study of multidrug-resistant Enterococcus faecium obtained from different hosts

2021 ◽  
Author(s):  
Aleksandra Trościańczyk ◽  
Aneta Nowakiewicz ◽  
Sebastian Gnat ◽  
Dominik Łagowski ◽  
Marcelina Osińska ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Host Species ◽  
Enterococcus Faecium ◽  
Type Species ◽  
Animal Species ◽  
Multidrug Resistant ◽  
Content Type ◽  
Link Type ◽  
High Level

Introduction. The possible transfer of antimicrobial resistance genes between Enterococcus faecium isolates from humans and different animal species, including those not covered by monitoring programs (e.g. pet and wildlife), poses a serious threat to public health. Hypothesis/Gap Statement. Little is known about occurrence and mechanisms of phenomenon of multidrug resistance of E. faecium isolated from various host species in Poland. Aim. The aim of the study was to characterize multidrug-resistant E. faecium isolated from humans and animals (livestock, pets and wildlife) in terms of the occurrence of genetic markers determining resistance. Methodology. Bacterial isolates were tested for phenotypic resistance and the presence of genes encoding resistance to macrolides, tetracycline, aminoglycosides, aminocyclitols and phenicols as well as efflux pump (emeA), resolvase (tndX) and integrase (Int-Tn) genes. The quinolone resistance-determining regions of gyrA and parC were sequenced. Results. Human isolates of E. faecium were characterized by high-level resistance to: ciprofloxacin, enrofloxacin, erythromycin (100 %), as well, as aminoglycosides resistance (kanamycin – 100%, streptomycin – 78 %, gentamicin – 78%). Regardless of the animal species, high level of resistance of E. faecium to tetracycline (from 88–100 %), erythromycin (from 82–94 %) and kanamycin (from 36–100 %) was observed. All E. faecium isolates from wildlife were resistant to fluoroquinolones. However, full susceptibility to vancomycin was observed in all isolates tested. Phenotypic antimicrobial resistance of E. faecium was identified in the presence of the following resistance genes: erm(B) (70%), msr(A) (50 %), tet(L) (35 %), tet(K) (34 %), tet(M) (76 %), aac(6’)-Ie-aph(2″)-Ia (25%), ant(6)-Ia (31%), aph(3)-IIIa (68 %), (tndX) (23 %), and integrase gene (Int-Tn) (34 %). A correlation between an amino acid substitution at positions 83 and 87 of gyrA and position 80 of parC and the high-level fluoroquinolone resistance in E. faecium has been observed as well. Conclusion. The level and range of antimicrobial resistance and the panel of resistance determinants is comparable between E. faecium isolates, despite host species.

scholarly journals Improved molecular characterization of the Klebsiella oxytoca complex reveals the prevalence of the kleboxymycin biosynthetic gene cluster

2021 ◽  
Vol 7 (6) ◽  
Author(s):  
Preetha Shibu ◽  
Frazer McCuaig ◽  
Anne L. McCartney ◽  
Magdalena Kujawska ◽  
Lindsay J. Hall ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Type Species ◽  
Phylogenetic Analyses ◽  
Klebsiella Oxytoca ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Genome Sequences ◽  
Content Type ◽  
Link Type

As part of the ongoing studies with clinically relevant Klebsiella spp., we characterized the genomes of three clinical GES-5-positive ST138 strains originally identified as Klebsiella oxytoca. bla OXY gene, average nucleotide identity and phylogenetic analyses showed the strains to be Klebsiella michiganensis . Affiliation of the strains to ST138 led us to demonstrate that the current multi-locus sequence typing scheme for K. oxytoca can be used to distinguish members of this genetically diverse complex of bacteria. The strains encoded the kleboxymycin biosynthetic gene cluster (BGC), previously only found in K. oxytoca strains and one strain of Klebsiella grimontii . The finding of this BGC, associated with antibiotic-associated haemorrhagic colitis, in K. michiganensis led us to carry out a wide-ranging study to determine the prevalence of this BGC in Klebsiella spp. Of 7170 publicly available Klebsiella genome sequences screened, 88 encoded the kleboxymycin BGC. All BGC-positive strains belonged to the K. oxytoca complex, with strains of four ( K. oxytoca , K. pasteurii , K. grimontii , K. michiganensis ) of the six species of complex found to encode the complete BGC. In addition to being found in K. grimontii strains isolated from preterm infants, the BGC was found in K. oxytoca and K. michiganensis metagenome-assembled genomes recovered from neonates. Detection of the kleboxymycin BGC across the K. oxytoca complex may be of clinical relevance and this cluster should be included in databases characterizing virulence factors, in addition to those characterizing BGCs.

scholarly journals Comparative genomics of global optrA-carrying Enterococcus faecalis uncovers a common chromosomal hotspot for optrA acquisition within a diversity of core and accessory genomes

2020 ◽  
Vol 6 (6) ◽  
Author(s):  
Ana R. Freitas ◽  
Ana P. Tedim ◽  
Carla Novais ◽  
Val F. Lanza ◽  
Luísa Peixe
Keyword(s):  
Enterococcus Faecalis ◽  
Type Species ◽  
De Novo ◽  
Integration Site ◽  
Medium Size ◽  
Phylogenetic Structure ◽  
Content Type ◽  
Link Type ◽  
Healthy Humans ◽  
Wide Range

Linezolid-resistant Enterococcus faecalis (LREfs) carrying optrA are increasingly reported globally from multiple sources, but we lack a comprehensive analysis of human and animal optrA-LREfs strains. To assess if optrA is dispersed in isolates with varied genetic backgrounds or with common genetic features, we investigated the phylogenetic structure, genetic content [antimicrobial resistance (AMR), virulence, prophages, plasmidome] and optrA-containing platforms of 27 publicly available optrA-positive E. faecalis genomes from different hosts in seven countries. At the genome-level analysis, an in-house database with 64 virulence genes was tested for the first time. Our analysis showed a diversity of clones and adaptive gene sequences related to a wide range of genera from Firmicutes . Phylogenies of core and accessory genomes were not congruent, and at least PAI-associated and prophage genes contribute to such differences. Epidemiologically unrelated clones (ST21, ST476-like and ST489) obtained from human clinical and animal hosts in different continents over eight years (2010–2017) could be phylogenetically related (3–126 SNPs difference). optrA was located on the chromosome within a Tn6674-like element (n=10) or on medium-size plasmids (30–60 kb; n=14) belonging to main plasmid families (RepA_N/Inc18/Rep_3). In most cases, the immediate gene vicinity of optrA was generally identical in chromosomal (Tn6674) or plasmid (impB-fexA-optrA) backbones. Tn6674 was always inserted into the same ∆radC integration site and embedded in a 32 kb chromosomal platform common to strains from different origins (patients, healthy humans, and animals) in Europe, Africa, and Asia during 2012–2017. This platform is conserved among hundreds of E. faecalis genomes and proposed as a chromosomal hotspot for optrA integration. The finding of optrA in strains sharing common adaptive features and genetic backgrounds across different hosts and countries suggests the occurrence of common and independent genetic events occurring in distant regions and might explain the easy de novo generation of optrA-positive strains. It also anticipates a dramatic increase of optrA carriage and spread with a serious impact on the efficacy of linezolid for the treatment of Gram-positive infections.

scholarly journals Functional characterization of the transcription regulator WhiB1 from Gordonia sp. IITR100

Microbiology ◽  
2020 ◽  
Vol 166 (12) ◽  
pp. 1181-1190
Author(s):  
Pooja Murarka ◽  
Aditi Keshav ◽  
Bintu Kumar Meena ◽  
Preeti Srivastava
Keyword(s):  
Type Species ◽  
Functional Characterization ◽  
Rhodococcus Erythropolis ◽  
Multiple Sequence ◽  
Content Type ◽  
Transcription Regulator ◽  
Link Type ◽  
Family Protein ◽  
The Family

WhiB is a transcription regulator which has been reported to be involved in the regulation of cell morphogenesis, cell division, antibiotic resistance, stress, etc., in several members of the family Actinomycetes . The present study describes functional characterization of a WhiB family protein, WhiB1 (protein ID: WP_065632651.1), from Gordonia sp. IITR100. We demonstrate that WhiB1 affects chromosome segregation and cell morphology in recombinant Escherichia coli , Gordonia sp. IITR100 as well as in Rhodococcus erythropolis . Multiple sequence alignment suggests that WhiB1 is a conserved protein among members of the family Actinomycetes . It has been reported that overexpression of WhiB1 leads to repression of the biodesulfurization operon in recombinant E. coli , Gordonia sp. IITR100 and R. erythropolis . A WhiB1-mut containing a point mutation Q116A in the DNA binding domain of WhiB1 led to partial alleviation of repression of the biodesulfurization operon. We show for the first time that the WhiB family protein WhiB1 is also involved in repression of the biodesulfurization operon by directly binding to the dsz promoter DNA.

scholarly journals Enterococcus faecium genome dynamics during long-term asymptomatic patient gut colonization

2019 ◽  
Vol 5 (7) ◽  
Author(s):  
Jumamurat R. Bayjanov ◽  
Jery Baan ◽  
Malbert R. C. Rogers ◽  
Annet Troelstra ◽  
Rob J. L. Willems ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Type Species ◽  
Multidrug Resistant ◽  
Gene Gain ◽  
Nucleotide Polymorphisms ◽  
Nosocomial Pathogen ◽  
Content Type ◽  
Link Type ◽  
Gut Colonization ◽  
Genome Dynamics

Enterococcus faecium is a gut commensal of humans and animals. In addition, it has recently emerged as an important nosocomial pathogen through the acquisition of genetic elements that confer resistance to antibiotics and virulence. We performed a whole-genome sequencing-based study on 96 multidrug-resistant E. faecium strains that asymptomatically colonized five patients with the aim of describing the genome dynamics of this species. The patients were hospitalized on multiple occasions and isolates were collected over periods ranging from 15 months to 6.5 years. Ninety-five of the sequenced isolates belonged to E. faecium clade A1, which was previously determined to be responsible for the vast majority of clinical infections. The clade A1 strains clustered into six clonal groups of highly similar isolates, three of which consisted entirely of isolates from a single patient. We also found evidence of concurrent colonization of patients by multiple distinct lineages and transfer of strains between patients during hospitalization. We estimated the evolutionary rate of two clonal groups that each colonized single patients at 12.6 and 25.2 single-nucleotide polymorphisms (SNPs)/genome/year. A detailed analysis of the accessory genome of one of the clonal groups revealed considerable variation due to gene gain and loss events, including the chromosomal acquisition of a 37 kbp prophage and the loss of an element containing carbohydrate metabolism-related genes. We determined the presence and location of 12 different insertion sequence (IS) elements, with ISEfa5 showing a unique pattern of location in 24 of the 25 isolates, suggesting widespread ISEfa5 excision and insertion into the genome during gut colonization. Our findings show that the E. faecium genome is highly dynamic during asymptomatic colonization of the human gut. We observed considerable genomic flexibility due to frequent horizontal gene transfer and recombination, which can contribute to the generation of genetic diversity within the species and, ultimately, can contribute to its success as a nosocomial pathogen.

scholarly journals Genomic rearrangements uncovered by genome-wide co-evolution analysis of a major nosocomial pathogen, Enterococcus faecium

2020 ◽  
Vol 6 (12) ◽  
Author(s):  
Janetta Top ◽  
Sergio Arredondo-Alonso ◽  
Anita C. Schürch ◽  
Santeri Puranen ◽  
Maiju Pesonen ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Type Species ◽  
Hospitalized Patients ◽  
Genomic Islands ◽  
Hospital Environment ◽  
Genomic Rearrangements ◽  
Nosocomial Pathogen ◽  
Content Type ◽  
Link Type ◽  
Genome Wide

Enterococcus faecium is a gut commensal of the gastro-digestive tract, but also known as nosocomial pathogen among hospitalized patients. Population genetics based on whole-genome sequencing has revealed that E. faecium strains from hospitalized patients form a distinct clade, designated clade A1, and that plasmids are major contributors to the emergence of nosocomial E. faecium . Here we further explored the adaptive evolution of E. faecium using a genome-wide co-evolution study (GWES) to identify co-evolving single-nucleotide polymorphisms (SNPs). We identified three genomic regions harbouring large numbers of SNPs in tight linkage that are not proximal to each other based on the completely assembled chromosome of the clade A1 reference hospital isolate AUS0004. Close examination of these regions revealed that they are located at the borders of four different types of large-scale genomic rearrangements, insertion sites of two different genomic islands and an IS30-like transposon. In non-clade A1 isolates, these regions are adjacent to each other and they lack the insertions of the genomic islands and IS30-like transposon. Additionally, among the clade A1 isolates there is one group of pet isolates lacking the genomic rearrangement and insertion of the genomic islands, suggesting a distinct evolutionary trajectory. In silico analysis of the biological functions of the genes encoded in three regions revealed a common link to a stress response. This suggests that these rearrangements may reflect adaptation to the stringent conditions in the hospital environment, such as antibiotics and detergents, to which bacteria are exposed. In conclusion, to our knowledge, this is the first study using GWES to identify genomic rearrangements, suggesting that there is considerable untapped potential to unravel hidden evolutionary signals from population genomic data.

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