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In Silico Identification of Three Types of Integrative and Conjugative Elements in Elizabethkingia anophelis Strains Isolated from around the World.

ABSTRACT: Elizabethkingia anophelis is an emerging global multidrug-resistant opportunistic pathogen. We assessed the diversity among 13 complete genomes and 23 draft genomes of E. anophelis strains derived from various environmental settings and human infections from different geographic regions around the world from 1950s to the present. Putative integrative and conjugative elements (ICEs) were identified in 31/36 (86.1%) strains in the study. A total of 52 putative ICEs (including eight degenerated elements lacking integrases) were identified and categorized into three types based on the architecture of the conjugation module and the phylogeny of the relaxase, coupling protein, TraG, and TraJ protein sequences. The type II and III ICEs were found to integrate adjacent to tRNA genes, while type I ICEs integrate into intergenic regions or into a gene. The ICEs carry various cargo genes, including transcription regulator genes and genes conferring antibiotic resistance. The adaptive immune CRISPR-Cas system was found in nine strains, including five strains in which CRISPR-Cas machinery and ICEs coexist at different locations on the same chromosome. One ICE-derived spacer was present in the CRISPR locus in one strain. ICE distribution in the strains showed no geographic or temporal patterns. The ICEs in E. anophelis differ in architecture and sequence from CTnDOT, a well-studied ICE prevalent in Bacteroides spp. The categorization of ICEs will facilitate further investigations of the impact of ICE on virulence, genome epidemiology, and adaptive genomics of E. anophelis IMPORTANCE Elizabethkingia anophelis is an opportunistic human pathogen, and the genetic diversity between strains from around the world becomes apparent as more genomes are sequenced. Genome comparison identified three types of putative ICEs in 31 of 36 strains. The diversity of ICEs suggests that they had different origins. One of the ICEs was discovered previously from a large E. anophelis outbreak in Wisconsin in the United States; this ICE has integrated into the mutY gene of the outbreak strain, creating a mutator phenotype. Similar to ICEs found in many bacterial species, ICEs in E. anophelis carry various cargo genes that enable recipients to resist antibiotics and adapt to various ecological niches. The adaptive immune CRISPR-Cas system is present in nine of 36 strains. An ICE-derived spacer was found in the CRISPR locus in a strain that has no ICE, suggesting a past encounter and effective defense against ICE.

SUBMITTER: Xu J

PROVIDER: S-EPMC6449604 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The incidence of Elizabethkingia anophelis bacteremia increased significantly in a tertiary hospital, Changhua Christian Hospital (CCH) since 2013. The infection density was 1.3 and 8.1 cases per 100,000 patient-days between 2005 and 2012 and 2013 and 2020, respectively (P < 0.05). During an outbreak investigation, a specific lineage of E. anophelis strains was identified by the pulsed-field gel electrophoresis analysis. To evaluate the evolution of the specific E. anophelis lineage, whole-genome sequencing was performed, and unique genomic features (GRs) were determined by comparative genomic analysis. The specific E. anophelis lineage was novel compared to worldwide strains ever reported by cg-MLST phylogenic and whole-genome comparative analysis. Multiplex PCR using primers designed from unique GRs were performed for prevalence screening among isolates from the CCH and nationwide isolates from the Taiwan surveillance of Antimicrobial Resistance (TSAR) Program. The proportion of the specific E. anophelis lineage increased from 7.9% (3/38) during 2005-2012 to 89.2% (223/250) during 2013-2020 (P < 0.05). Although E. anophelis usually confers resistance to multiple antibiotics with limited therapeutic options, the E. anophelis strains in the specific lineage had higher ciprofloxacin resistance (100% [226/226] versus 27.4% [17/62], P < 0.05) and was associated with a higher 14-day mortality rates (33.2% [37/226] versus 16.1% [10/62], P < 0.05) than other strains at CCH. A similarly increasing trend was also found in the national TSAR program during 2002-2018 (p for trend <0.05). We concluded that a novel lineage of E. anophelis strains has emerged dominantly in Taiwan. The genomic features are important for further investigations of epidemiology, resistance, virulence, and appropriate treatment. IMPORTANCE Elizabethkingia anophelis is an emerging multidrug resistant pathogen caused several global outbreaks recently. E. anophelis was frequently misidentified as E. meningoseptica in the past by conventional culture methods; therefore, the prevalence was often underestimated. Through revised identification, an increasing trend of E. anophelis infection was noted in a tertiary hospital and a dominant lineage of strains was recognized by genotyping. To our best knowledge, the dominant lineage of E. anophelis is novel in comparison to other worldwide strains by whole-genome comparative analysis and several unique genomic regions were found. The whole-genome sequencing data also demonstrated multiple putative virulence factors and genes associated with multidrug resistance. In our study, we identified a specially evolved E. anophelis in Taiwan with increasing nationwide dominance. This study will assist in further epidemiology surveillance and developing corresponsive infection control policies to restrain it potential of global dissemination.

Project description:Flavobacteria (members of the family Flavobacteriaceae) dominate the bacterial community in the Anopheles mosquito midgut. One such commensal, Elizabethkingia anophelis, is closely associated with Anopheles mosquitoes through transstadial persistence (i.e., from one life stage to the next); these and other properties favor its development for paratransgenic applications in control of malaria parasite transmission. However, the physiological requirements of E. anophelis have not been investigated, nor has its capacity to perpetuate despite digestion pressure in the gut been quantified. To this end, we first developed techniques for genetic manipulation of E. anophelis, including selectable markers, reporter systems (green fluorescent protein [GFP] and NanoLuc), and transposons that function in E. anophelis. A flavobacterial expression system based on the promoter PompA was integrated into the E. anophelis chromosome and showed strong promoter activity to drive GFP and NanoLuc reporter production. Introduced, GFP-tagged E. anophelis associated with mosquitoes at successive developmental stages and propagated in Anopheles gambiae and Anopheles stephensi but not in Aedes triseriatus mosquitoes. Feeding NanoLuc-tagged cells to A. gambiae and A. stephensi in the larval stage led to infection rates of 71% and 82%, respectively. In contrast, a very low infection rate (3%) was detected in Aedes triseriatus mosquitoes under the same conditions. Of the initial E. anophelis cells provided to larvae, 23%, 71%, and 85% were digested in A. stephensi, A. gambiae, and Aedes triseriatus, respectively, demonstrating that E. anophelis adapted to various mosquito midgut environments differently. Bacterial cell growth increased up to 3-fold when arginine was supplemented in the defined medium. Furthermore, the number of NanoLuc-tagged cells in A. stephensi significantly increased when arginine was added to a sugar diet, showing it to be an important amino acid for E. anophelis. Animal erythrocytes promoted E. anophelis growth in vivo and in vitro, indicating that this bacterium could obtain nutrients by participating in erythrocyte lysis in the mosquito midgut.

Project description:Elizabethkingia anophelis bacteria encounter fluxes of iron in the midgut of mosquitoes, where they live as symbionts. They also establish bacteremia with severe clinical manifestations in humans, and live in water service lines in hospitals. In this study, we investigated the global gene expression responses of E. anophelis to iron fluxes in the midgut of female Anopheles stephensi mosquitoes fed sucrose or blood, and in iron-poor or iron-rich culture conditions. Of 3,686 transcripts revealed by RNAseq technology, 218 were upregulated while 112 were down-regulated under iron-poor conditions. Most of these differentially expressed genes were enriched in functional groups assigned within “biological process,” “cell component” and “molecular function” categories. E. anophelis possessed 4 iron/heme acquisition systems. Hemolysin gene expression was significantly repressed when cells were grown under iron-rich or high temperature (37℃) conditions. Furthermore, hemolysin gene expression was down-regulated after a blood meal, indicating that E. anophelis cells responded to excess iron and its associated physiological stress by limiting iron loading. By contrast, genes encoding respiratory chain proteins were up-regulated under iron-rich conditions, allowing these iron-containing proteins to chelate intracellular free iron. In vivo studies showed that growth of E. anophelis cells increased 3-fold in blood-fed mosquitoes over those in sucrose-fed ones. Deletion of aerobactin synthesis genes led to impaired cell growth in both iron-rich and iron-poor media. Mutants showed more susceptibility to H2O2 toxicity and less biofilm formation than did wild-type cells. Mosquitoes with E. anophelis experimentally colonized in their guts produced more eggs than did those treated with erythromycin or left unmanipulated, as controls. Results reveal that E. anophelis bacteria respond to varying iron concentration in the mosquito gut, harvest iron while fending off iron-associated stress, contribute to lysis of red blood cells, and positively influence mosquito host fecundity.

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In Silico Identification of Three Types of Integrative and Conjugative Elements in Elizabethkingia anophelis Strains Isolated from around the World.

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