Neonatal Elizabethkingia anophelis meningitis originating from the water reservoir of an automated infant milk dispenser, the Netherlands, February 2024.

Elizabethkingia anophelis is a multidrug-resistant pathogen causing high mortality and morbidity in adults with comorbidities and neonates. We report a Dutch case of E. anophelis meningitis in a neonate, clonally related to samples taken from an automated infant milk dispenser located at the family's residence. We inform about the emergence of E. anophelis and suggest molecular surveillance in hospitals and other health settings. This is the first case connecting an automated formula dispenser to an invasive infection in a neonate.

Antibiotic Combination to Effectively Postpone or Inhibit the In Vitro Induction and Selection of Levofloxacin-Resistant Mutants in Elizabethkingia anophelis.

Fluoroquinolones are potentially active against Elizabethkingia anophelis. Rapidly increased minimum inhibitory concentrations (MICs) and emerging point mutations in the quinolone resistance-determining regions (QRDRs) following exposure to fluoroquinolones have been reported in E. anophelis. We aimed to investigate point mutations in QRDRs through exposure to levofloxacin (1 × MIC) combinations with different concentrations (0.5× and 1 × MIC) of minocycline, rifampin, cefoperazone/sulbactam, or sulfamethoxazole/trimethoprim in comparison with exposure to levofloxacin alone. Of the four E. anophelis isolates that were clinically collected, lower MICs of levofloxacin were disclosed in cycle 2 and 3 of induction and selection in all levofloxacin combination groups other than levofloxacin alone (all p = 0.04). Overall, no mutations were discovered in parC and parE throughout the multicycles inducted by levofloxacin and all its combinations. Regarding the vastly increased MICs, the second point mutations in gyrA and/or gyrB in one isolate (strain no. 1) occurred in cycle 2 following exposure to levofloxacin plus 0.5 × MIC minocycline, but they were delayed appearing in cycle 5 following exposure to levofloxacin plus 1 × MIC minocycline. Similarly, the second point mutation in gyrA and/or gyrB occurred in another isolate (strain no. 3) in cycle 4 following exposure to levofloxacin plus 0.5 × MIC sulfamethoxazole/trimethoprim, but no mutation following exposure to levofloxacin plus 1 × MIC sulfamethoxazole/trimethoprim was disclosed. In conclusion, the rapid selection of E. anophelis mutants with high MICs after levofloxacin exposure could be effectively delayed or postponed by antimicrobial combination with other in vitro active antibiotics.

Phylogenomics analysis of multidrug-resistant Elizabethkingia anophelis in industrial wastewater treatment plant.

AIMS: This study investigated the phylogenetic relatedness of multidrug-resistant Elizabethkingia anophelis recovered from an industrial wastewater treatment plant (WWTPi). METHODS AND RESULTS: The wastewater samples were plated in brain heart infusion agar (4 mg/L ceftazidime, 8 mg/L meropenem, and 2 mg/L polimixin). Four isolates recovered from four stages of WWTPi (influent, aeration, decantation, and treated effluent) were identified and evaluated of susceptibility profiles in the VITEK 2 system. These strains identified as E. meningoseptica were confirmed to be E. anophelis by whole genomic sequencing (Miseq-Illumina) and showed antimicrobial resistance genes of ß-lactams, aminoglycosides, and tetracycline's classes. The ribosomal multilocus sequence typing showed that they belong to the rST 65620 together with clinical strains. The phylogenomic tree revealed the similarity of our strains to those belonging to sublineage 11 and the single nucleotide polymorphism analysis confirmed that they belong to a single clade. CONCLUSIONS: To the best of our knowledge, this is the first study reporting the persistence of multidrug-resistant E. anophelis sublineage 11 along the wastewater treatment.

Genomic and phylogenetic characterization of Elizabethkingia anophelis strains: The first two cases of life-threatening infection in Japan.

OBJECTIVE: Elizabethkingia anophelis causes meningitis, bloodstream infections, and respiratory infections in immunocompromised individuals. We examined two E. anophelis strains isolated from the first life-threatening cases caused by this species in Japan to determine the phylogenetic origin and genomic features of them. METHODS: We performed whole genome-based analysis to clarify the genetic relationship for the two strains (EK0004 and EK0079) and Elizabethkingia sp. strains isolated from worldwide and to characterize the genomic features such as the prevalence of virulence- and antimicrobial resistance (AMR)-related genes. PATIENTS: A 29-year-old man with hepatosplenic T-cell lymphoma and a 52-year-old man with systemic lupus erythematosus developed fatal bacteremia and meningitis due to E. anophelis, respectively. RESULTS: Two strains, EK0004 and EK0079, were genetically different but most closely related to the strains isolated from the largest outbreak in Wisconsin, USA from 2015 to 2016, and the strain isolated from cerebrospinal fluid of a patient in Florida, USA in 1982, respectively. The two strains contained AMR-related genes such as those encoding for an extended-spectrum ß-lactamase and multiple metallo-ß-lactamases and several virulence-related genes such as capsular polysaccharide synthesis gene clusters. CONCLUSIONS: Although further functional analyses are required to understand the virulence of these clones, these finding suggests that enough caution of E. anophelis infection in immunocompromised patients is required since the number of infections by this species is increasing outside Japan.

In Vitro and In Vivo Antimicrobial Activities of Vancomycin and Rifampin against Elizabethkingia anophelis.

Elizabethkingia anophelis has emerged as a critical human pathogen, and a number of isolated reports have described the successful treatment of Elizabethkingia infections with vancomycin, a drug that is typically used to target Gram-positive bacteria. This study employed in vitro broth microdilution checkerboard and time-kill assays, as well as in vivo zebrafish animal models to evaluate the individual and combination antimicrobial effects of vancomycin and rifampin against E. anophelis. The minimum inhibitory concentration ranges of vancomycin and rifampin against 167 isolates of E. anophelis were 16-256 mg/L and 0.06-128 mg/L, respectively. The checkerboard assay results revealed a synergistic effect between vancomycin and rifampin in 16.8% (28/167) of the isolates. Time-kill assays were implemented for 66 isolates, and the two-drug combination had a synergistic interaction in 57 (86.4%) isolates. In vivo zebrafish studies revealed that treatment with vancomycin monotherapy, rifampin monotherapy, or vancomycin-rifampin combination therapy yielded a higher survival rate than the control group treatment with 0.9% saline. The results of this study support the use of vancomycin to treat E. anophelis infections.

Mutant Prevention Concentrations of Ciprofloxacin and Levofloxacin and Target Gene Mutations of Fluoroquinolones in Elizabethkingia anophelis.

Fluoroquinolones are potentially effective against Elizabethkingia anophelis. We investigated the MIC, mutant prevention concentration (MPC), and target gene mutations of fluoroquinolones in E. anophelis. Eighty-five E. anophelis isolates were collected from five hospitals in Taiwan. The MIC and MPC of ciprofloxacin and levofloxacin were examined for all E. anophelis except 17 isolates, in which ciprofloxacin MPC could not be determined due to drug precipitation caused by overly high drug concentration. Mutations in the quinolone resistance-determining regions of DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) in the clinical isolates and fluoroquinolone-selected mutants were examined. Overall, 23.5% and 71.8% of the isolates tested were susceptible to ciprofloxacin and levofloxacin, respectively. The MPC50 of ciprofloxacin was 128 mg/L, and the MPC50 of levofloxacin was 51.2 mg/L. The MPC50/MIC50 ratio for ciprofloxacin was 64, whereas that for levofloxacin was 25.6. The coefficient of determination between the MPC and MIC for ciprofloxacin and levofloxacin was 0.72 and 0.56, respectively, in the linear regression analysis. Preexisting mutations in GyrA (S83I, S83R, and D87Y) were identified in 18 clinical isolates, all of which were resistant to both ciprofloxacin and levofloxacin. Additional amino acid substitutions in GyrA were identified in all ciprofloxacin- and levofloxacin-selected mutants. Furthermore, GyrB alterations (D431N or D431H) were found in nine levofloxacin-treated isolates. Given that maintaining the serum concentrations of fluoroquinolones above MPCs is impossible under presently recommended doses, the selection of mutant E. anophelis strains seems inevitable.

Molecular identification and biofilm-forming ability of Elizabethkingia species.

Recently, Elizabethkingia species have gained attention as a cause of life-threatening infections. The identification via phenotypic methods of three important species- Elizabethkingia meningoseptica, E. anophelis and E. miricola is difficult. Our objectives were to re-assess 30 archived Flavobacterium meningosepticum isolates using 16S rRNA gene sequencing, ERIC-PCR, and biofilm formation assay. Twenty-four isolates were re-identified as E. anophelis and 6 as E. miricola. All of them had the ability to form biofilm as shown in microtiter plate assay based on crystal violet staining. Overall, E. anophelis had a higher specific biofilm formation index compared to E. miricola. A total of 42% (10 out of 24) of E. anophelis were classified as strong, 29% (7 out of 24) as moderate and 29% (7 out of 24) as weak biofilm producers. E. miricola, 17% (1 out of 6) isolates were strong biofilm producers, 50% (3 out of 6) moderate and 33% (2 out of 6) were weak producers. E. anophelis from tracheal secretions were significantly associated with (p = 0.0361) strong biofilm formation. In summary, this study showed that the isolates originally identified as F. meningosepticum were re-classified using the 16S rRNA gene as one of two Elizabethkingia species. The ability of E. anophelis to form strong biofilm in endotracheal tubes indicates their probable role in the pathogenesis of Elizabethkingia infections.

Elizabethkingia anophelis, an emerging pathogen, inhibits RAW 264.7 macrophage function.

Elizabethkingia anophelis is a pathogen that can cause a life-threatening infection in immunocompromised patients. The first case of E. anophelis infection was reported in 2013; subsequently, an increase in its incidence has been reported globally. Additionally, a mortality rate of more than 30% was observed in the US outbreak of 2015. To date, the pathogenic mechanisms underlying E. anophelis infection, such as toxin production, remain unclear. Since tissue macrophages act as the first line of defense against pathogens, in the present study the interactions between E. anophelis and a macrophage-like cell line RAW 264.7 were examined. Although E. anophelis showed no cytotoxicity toward RAW 264.7 macrophages, the infection inhibited LPS-induced morphological changes and activation of differentiation markers for the polarization of RAW 264.7 macrophages toward an M1-like phenotype. However, when the cell contact was restricted using Transwell inserts or bacterial culture supernatants were used instead of live bacteria, no such inhibition was observed. Moreover, it was shown that E. anophelis evaded phagocytosis. Overall, the results suggest that E. anophelis infection inhibits the differentiation of RAW 264.7 macrophages to a pro-inflammatory phenotype in a contact-dependent manner.

Elizabethkingia anophelis Infection in Infants, Cambodia, 2012-2018.

We describe 6 clinical isolates of Elizabethkingia anophelis from a pediatric referral hospital in Cambodia, along with 1 isolate reported from Thailand. Improving diagnostic microbiological methods in resource-limited settings will increase the frequency of reporting for this pathogen. Consensus on therapeutic options is needed, especially for resource-limited settings.

Risk Factors for Elizabethkingia Acquisition and Clinical Characteristics of Patients, South Korea.

Elizabethkingia infections are difficult to treat because of intrinsic antimicrobial resistance, and their incidence has recently increased. We conducted a propensity score-matched case-control study during January 2016-June 2017 in South Korea and retrospectively studied data from patients who were culture positive for Elizabethkingia species during January 2009-June 2017. Furthermore, we conducted epidemiologic studies of the hospital environment and mosquitoes. The incidence of Elizabethkingia increased significantly, by 432.1%, for 2016-2017 over incidence for 2009-2015. Mechanical ventilation was associated with the acquisition of Elizabethkingia species. Because Elizabethkingia infection has a high case-fatality rate and is difficult to eliminate, intensive prevention of contamination is needed.

Elizabethkingia anophelis and Association with Tap Water and Handwashing, Singapore.

We report an Elizabethkingia anophelis case cluster associated with contaminated aerators and tap water in a children's intensive care unit in Singapore in 2017. We demonstrate a likely transmission route for E. anophelis to patients through acquisition of the bacteria on hands of healthcare workers via handwashing.

Relative Prevalence and Antimicrobial Susceptibility of Clinical Isolates of Elizabethkingia Species Based on 16S rRNA Gene Sequencing.

Some of the previously reported clinical isolates of Elizabethkingia meningoseptica may be later named species of Elizabethkingia We determined the accuracy of species identification (with two matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS] systems and the Vitek 2 GN card), relative prevalence of three Elizabethkingia spp. in clinical specimens, and antimicrobial susceptibility of the species identified by 16S rRNA gene sequencing. Specimens for culture were collected from patients in a university hospital in Seoul, South Korea, between 2009 and 2015. All 3 Elizabethkingia spp. were detected in patients; among the 86 isolates identified by 16S rRNA gene sequencing, 17 (19.8%) were E. meningoseptica, 18 (20.9%) were Elizabethkingia miricola, and 51 (59.3%) were Elizabethkingia anophelis Only the MALDI-TOF Vitek MS system with an amended database correctly identified all of the isolates. The majority (76.7%) of the isolates were from the lower respiratory tract, and 8 (9.3%) were from blood. Over 90% of E. meningoseptica and E. anophelis isolates were susceptible to piperacillin-tazobactam and rifampin. In contrast, all E. miricola isolates were susceptible to fluoroquinolones except ciprofloxacin. Further studies are urgently needed to determine the optimal antimicrobial agents for the treatment of infections due to each individual Elizabethkingia species.

In vitro induction and selection of fluoroquinolone-resistant mutants in Elizabethkingia anophelis.

For 29 parent strains, recognized by pulsed-field gel electrophoresis, the MICs multiplied significantly in the ciprofloxacin group than levofloxacin group, following the first and third induction cycle. Ser83Arg in GyrA was the most common site of mutations. No mutation in ParC nor ParE was identified in the selected mutants.

Central Line-Associated Bloodstream Infection Due to Elizabethkingia anophelis: Case Report and Literature Review on Pediatric Infections.

Elizabethkingia anophelis is an opportunistic pathogen causing lifethreatening infections in humans, particularly in immunocompromised patients, neonates and the elderly. We report a case of central line-associated bloodstream infection by E. anophelis in a 2.5-year-old girl with acute lymphoblastic leukemia successfully treated with a combination of piperacillin/tazobactam and amikacin. The literature was also reviewed on pediatric infections caused by E. anophelis, focusing on clinical manifestations, underlying medical conditions, treatment and outcome. Accurate identification with MALDI-TOF, or using molecular techniques, is of the utmost importance because treatment and prognosis differ depending on the species. Considering that E. anophelis is multiresistant to antibiotics and that inappropriate antimicrobial therapy is an independent risk factor for mortality, the early, accurate identification of bacterial species and prompt effective treatment are essential to achieve optimal therapeutic outcomes.

A Genetic Locus in Elizabethkingia anophelis Associated with Elevated Vancomycin Resistance and Multiple Antibiotic Reduced Susceptibility.

The Gram-negative Elizabethkingia express multiple antibiotic resistance and cause severe opportunistic infections. Vancomycin is commonly used to treat Gram-positive infections and has also been used to treat Elizabethkingia infections, even though Gram-negative organisms possess a vancomycin permeability barrier. Elizabethkingia anophelis appeared relatively vancomycin-susceptible and challenge with this drug led to morphological changes indicating cell lysis. In stark contrast, vancomycin growth challenge revealed that E. anophelis populations refractory to vancomycin emerged. In addition, E. anophelis vancomycin-selected mutants arose at high frequencies and demonstrated elevated vancomycin resistance and reduced susceptibility to other antimicrobials. All mutants possessed a SNP in a gene (vsr1 = vancomycin-susceptibility regulator 1) encoding a PadR family transcriptional regulator located in the putative operon vsr1-ORF551, which is conserved in other Elizabethkingia spp as well. This is the first report linking a padR homologue (vsr1) to antimicrobial resistance in a Gram-negative organism. We provide evidence to support that vsr1 acts as a negative regulator of vsr1-ORF551 and that vsr1-ORF551 upregulation is observed in vancomycin-selected mutants. Vancomycin-selected mutants also demonstrated reduced cell length indicating that cell wall synthesis is affected. ORF551 is a membrane-spanning protein with a small phage shock protein conserved domain. We hypothesize that since vancomycin-resistance is a function of membrane permeability in Gram-negative organisms, it is likely that the antimicrobial resistance mechanism in the vancomycin-selected mutants involves altered drug permeability.

Transcriptome dataset of Caenorhabditis elegans responses to varied microbial pathogens.

Transcriptome analysis through next-generation sequencing (NGS) is an invaluable tool for investigating changes in gene expression across diverse organisms. The nematode Caenorhabditis elegans (C. elegans) serves as an excellent model organism for dissecting host responses to bacterial infections. Here, our dataset obtained from bulk RNA-sequencing (RNA-seq) can be used to provide in-depth characterization of the mRNA transcriptome profiles of wild-type N2 animals and null mutants of the cytoskeletal regulatory gene unc-53/Nav2 following exposure to distinct bacterial environments: their natural laboratory food source, Escherichia coli OP50, the human and nematode pathogen Pseudomonas aeruginosa PA14, and the emerging pathogen Elizabethkingia anophelis Ag1. As proof of the dataset quality, downstream differential gene expression analysis reveals significant shifts in gene expression patterns within N2 and unc-53 mutants under varying bacterial conditions that will be useful for our companion studies investigating these pathways. These data provide an effective methodological framework for future investigators to investigate the interplay between cytoskeletal proteins and the innate immune response. The raw FASTQ files generated from our transcriptome experiment is deposited in the publicly available NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA1010192, for further exploration and validation by the C. elegans research community.

Review on infection control strategies to minimize outbreaks of the emerging pathogen Elizabethkingia anophelis.

BACKGROUND: Elizabethkingia anophelis is a multi-drug resistant emerging opportunistic pathogen with a high mortality rate, causing healthcare-associated outbreaks worldwide. METHODS: We report a case of E. anophelis pleuritis, resulting from transmission through lung transplantation, followed by a literature review of outbreak reports and strategies to minimize E. anophelis transmission in healthcare settings. RESULTS: From 1990 to August 2022, 14 confirmed E. anophelis outbreak cohorts and 21 cohorts with suspected E. anophelis outbreaks were reported in literature. A total of 80 scientific reports with recommendations on diagnostics and infection control measures were included and summarized in our study. CONCLUSION: Strategies to prevent and reduce spread of E. anophelis include water-free patient rooms, adequate hygiene and disinfection practices, and optimized diagnostic techniques for screening, identification and molecular typing.

Elizabethkingia anophelis outer membrane vesicles as a novel vaccine candidate against infection: insights into immune response and potential for passive immunity.

IMPORTANCE: Elizabethkingia anophelis, a Gram-negative pathogen, causes infections such as bacteraemia, pneumonia, and neonatal meningitis. The pathogen resists most antimicrobial classes, making novel approaches urgently needed. In natural settings, Gram-negative bacteria secrete outer membrane vesicles (OMVs) that carry important molecules in the bacterial life cycle. These OMVs are enriched with proteins involved in virulence, survival, and carbohydrate metabolism, making them a promising source for vaccine development against the pathogen. This study investigated the efficacy of imipenem-induced OMVs (iOMVs) as a vaccine candidate against E. anophelis infection in a mouse pneumonia model. Mice immunized with iOMVs were completely protected during lethal-dose challenges. Passive immunization with hyperimmune sera and splenocytes conferred protection against lethal pneumonia. Further investigation is needed to understand the mechanisms underlying the protective effects of iOMV-induced passive immunity, such as the action on specific antibody subclasses or T cell subsets.

Identification of potential novel therapeutic drug target against Elizabethkingia anophelis by integrative pan and subtractive genomic analysis: An in silico approach.

Elizabethkingia anophelis is a human pathogen responsible for severe nosocomial infections in neonates and immunocompromised patients. The significantly higher mortality rate from E. anophelis infections and the lack of available regimens highlight the critical need to explore novel drug targets. The current study investigated effective novel drug targets by employing a comprehensive in silico subtractive genomic approach integrated with pangenomic analysis of E. anophelis strains. A total of 2809 core genomic proteins were found by pangenomic analysis of non-paralogous proteins. Subsequently, 156 pathogen-specific, 442 choke point, 202 virulence factor, 53 antibiotic resistant and 119 host-pathogen interacting proteins were identified in E. anophelis. By subtractive genomic approach, at first 791 proteins were found to be indispensable for the survival of E. anophelis. 558 and 315 proteins were detected as non-homologous to human and gut microflora respectively. Following that 245 cytoplasmic, 245 novel, and 23 broad-spectrum targets were selected and finally four proteins were considered as potential therapeutic targets of E. anophelis based on highest degree score in PPI network. Among those, three proteins were subjected to molecular docking and subsequent MD simulation as one protein did not contain a plausible binding pocket with sufficient surface area and volume. All the complexes were found to be stable and compact in 100 ns molecular dynamics simulation studies as measured by RMSD, RMSF, and Rg. These three short-listed targets identified in this study may lead to the development of novel antimicrobials capable of curing infections and pave the way to prevent and control the disease progression caused by the deadly agent E. anophelis.

The First Reported Case of Elizabethkingia anophelis From Nepal.

Elizabethkingia anophelis, a gram-negative bacillus belonging to the Flavobacteriaceae family, is found in various environmental sources and has been associated with community and hospital outbreaks. Correct identification is crucial, guided by advanced genomic techniques, i.e., matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system with an updated database. The case fatality rate, ranging from 24 to 60%, underscores the need for timely recognition and appropriate management. Additionally, Elizabethkingia presents a challenge due to its recent discovery, misidentification history, and drug resistance. Here, we present a case of fatal infection in a 30-year-old male, who presented with pneumonia. It gradually progressed and ultimately proved fatal underscoring the virulence of the pathogen involved. It was a diagnostic challenge as it likely is the first reported instance of Elizabethkingia anophelis infection from Nepal.