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Isolation of Elizabethkingia anophelis From COVID-19 Swab Kits.

ABSTRACT: Purpose: To investigate and characterize the putative Elizabethkingia anophelis contaminant isolated from throat and anal swab samples of patients from three fever epidemic clusters, which were not COVID-19 related, in Shenzhen, China, during COVID-19 pandemic. Methods: Bacteria were cultured from throat (n = 28) and anal (n = 3) swab samples from 28 fever adolescent patients. The isolated bacterial strains were identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) and the VITEK2 automated identification system. Nucleic acids were extracted from the patient samples (n = 31), unopened virus collection kits from the same manufacturer as the patient samples (n = 35, blank samples) and from unopened throat swab collection kits of two other manufacturers (n = 22, control samples). Metagenomic sequencing and quantitative real-time PCR (qPCR) detection were performed. Blood serum collected from patients (n = 13) was assessed for the presence of antibodies to E. anophelis. The genomic characteristics, antibiotic susceptibility, and heat resistance of E. anophelis isolates (n = 31) were analyzed. Results: The isolates were identified by MALDI-TOF/MS and VITEK2 as Elizabethkingia meningoseptica. DNA sequence analysis confirmed isolates to be E. anophelis. The patients' samples and blank samples were positive for E. anophelis. Control samples were negative for E. anophelis. The sera from a sub-sample of 13 patients were antibody-negative for isolated E. anophelis. Most of the isolates were highly homologous and carried multiple β-lactamase genes (bla _B, bla _GOB, and bla _CME). The isolates displayed resistance to nitrofurans, penicillins, and most β-lactam drugs. The bacteria survived heating at 56°C for 30 min. Conclusion: The unopened commercial virus collection kits from the same manufacturer as those used to swab patients were contaminated with E. anophelis. Patients were not infected with E. anophelis and the causative agent for the fevers remains unidentified. The relevant authorities were swiftly notified of this discovery and subsequent collection kits were not contaminated. DNA sequence-based techniques are the definitive method for Elizabethkingia species identification. The E. anophelis isolates were multidrug-resistant, with partial heat resistance, making them difficult to eradicate from contaminated surfaces. Such resistance indicates that more attention should be paid to disinfection protocols, especially in hospitals, to avoid outbreaks of E. anophelis infection.

SUBMITTER: Xu L

PROVIDER: S-EPMC8763855 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:Elizabethkingia anophelis has recently gained global attention and is emerging as a cause of life-threatening nosocomial infections. The present study aimed to investigate the association between antimicrobial resistance and the ability to form biofilm among E. anophelis isolated from hospitalized patients in China. Over 10 years, a total of 197 non-duplicate E. anophelis strains were collected. Antibiotic susceptibility was determined by the standard agar dilution method as a reference assay according to the Clinical and Laboratory Standards Institute. The biofilm formation ability was assessed using a culture microtiter plate method, which was determined using a crystal violet assay. Culture plate results were cross-checked by scanning electron microscopy imaging analysis. Among the 197 isolates, all were multidrug-resistant, and 20 were extensively drug-resistant. Clinical E. anophelis showed high resistance to current antibiotics, and 99% of the isolates were resistant to at least seven antibiotics. The resistance rate for aztreonam, ceftazidime, imipenem, meropenem, trimethoprim-sulfamethoxazole, cefepime, and tetracycline was high as 100%, 99%, 99%, 99%, 99%, 95%, and 90%, respectively. However, the isolates exhibited the highest susceptibility to minocycline (100%), doxycycline (96%), and rifampin (94%). The biofilm formation results revealed that all strains could form biofilm. Among them, the proportions of strong, medium, and weak biofilm-forming strains were 41%, 42%, and 17%, respectively. Furthermore, the strains forming strong or moderate biofilm presented a statistically significant higher resistance than the weak formers (p < 0.05), especially for piperacillin, piperacillin-tazobactam, cefepime, amikacin, and ciprofloxacin. Although E. anophelis was notoriously resistant to large antibiotics, minocycline, doxycycline, and rifampin showed potent activity against this pathogen. The data in the present report revealed a positive association between biofilm formation and antibiotic resistance, which will provide a foundation for improved therapeutic strategies against E. anophelis infections in the future.

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Isolation of Elizabethkingia anophelis From COVID-19 Swab Kits.

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