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      Diagnostic Pitfalls in Identification of Elizabethkingia Meningoseptica

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          To the Editor of JCCM, Regarding the article “Emerging Infection with Elizabethkingia meningoseptica in Neonate. A Case Report” by Arbune et al. (2018) [1], there are specific facts which need clarification regarding the reporting of this organism. First of all, Arbune reported the isolation of the organism from the cerebrospinal fluid (CSF) and blood culture of one case, and that no source of infection was identified. Elizabethkingia meningoseptica, although linked to meningitis and nosocomial infections, can be an environmental contaminant as well. Repeat cultures of the samples are mandatory for the confirmation of such unusual pathogens. Secondly, at present, no antibiotic sensitivity guidelines exist for this organism. Hence, the reporting of antibiotic susceptibilities must be done along with minimum inhibitory concentration (MIC) values of the tested antimicrobials rather than merely stating they are “sensitive” or “resistant”. MIC values of the tested antibiotics may help the clinicians in deciding the drug dosage. Additionally, they may contribute to the formulation of susceptibility guidelines in the future. Thirdly, and most importantly, accurate identification is the key issue for such rare isolates. Arbune used the Vitek 2 automated system to identify Elizabethkingia meningoseptica. However, discrepancies in identification by the Vitek2 system have been reported in the published literature. In a study by Carvalho et al. (2017) [2], an isolate of Chryseobacterium indologenes was misidentified as Elizabethkingia meningoseptica by a Vitek 2 system with 99 % certainty of identification. Lau et al. (2016 ) [3] reported seventeen isolates of Elizabethkingia anopheles and one isolate of E. miricola confirmed by 16S rRNA sequencing, all having been misidentified by Vitek 2 as E. meningoseptica. These included CSF isolates from three cases of neonatal meningitis. In a study by Lau et al. (2015) [4], three isolates including two CSF samples from neonatal meningitis cases that were later confirmed as E. anophelis by whole genome sequencing were initially misidentified as E. meningoseptica by the Vitek 2 system. Lo and Chang (2014) [5] also reported a 16S rRNA confirmed Chryseobacterium gleum isolate having been misidentified as E. meningoseptica by the Vitek 2 system. The above data indicate that the Vitek 2 automated system has a high positive predictive value but also a variable number of false positives concerning the identification of Elizabethkingia meningoseptica. It can be concluded that the Vitek 2 system alone is not sufficient for confirmatory identification of this organism and more advanced techniques such as MALDI-TOF MS with its expanded database, as well as molecular techniques such as 16S rRNA gene sequencing and whole genome sequencing (WGS), should be considered for accurate identification. Further comparative studies of these molecular and microbiological techniques, with updated databases to prevent pseudo-identifications leading to false reporting of outbreaks and cases of this unusual pathogen, should be undertaken.

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          Elizabethkingia anophelis bacteremia is associated with clinically significant infections and high mortality

          Unlike Elizabethkingia meningoseptica, the clinical importance of E. anophelis is poorly understood. We determined the clinical and molecular epidemiology of bacteremia caused by Elizabethkingia-like species from five regional hospitals in Hong Kong. Among 45 episodes of Elizabethkingia-like bacteremia, 21 were caused by Elizabethkingia, including 17 E. anophelis, three E. meningoseptica and one E. miricola; while 24 were caused by other diverse genera/species, as determined by 16S rRNA gene sequencing. Of the 17 cases of E. anophelis bacteremia, 15 (88%) were clinically significant. The most common diagnosis was pneumonia (n = 5), followed by catheter-related bacteremia (n = 4), neonatal meningitis (n = 3), nosocomial bacteremia (n = 2) and neutropenic fever (n = 1). E. anophelis bacteremia was commonly associated with complications and carried 23.5% mortality. In contrast, of the 24 episodes of bacteremia due to non-Elizabethkingia species, 16 (67%) were clinically insignificant. Compared to non-Elizabethkingia bacteremia, Elizabethkingia bacteremia was associated with more clinically significant infections (P < 0.01) and positive cultures from other sites (P < 0.01), less polymicrobial bacteremia (P < 0.01), and higher complication (P < 0.05) and mortality (P < 0.05) rates. Elizabethkingia bacteremia is predominantly caused by E. anophelis instead of E. meningoseptica. Elizabethkingia bacteremia, especially due to E. anophelis, carries significant morbidity and mortality, and should be considered clinically significant unless proven otherwise.
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            Evidence for Elizabethkingia anophelis Transmission from Mother to Infant, Hong Kong

            Microbial genome sequencing can enhance diagnosis and control of infectious diseases ( 1 , 2 ). Its ultimate molecular resolution is superior to other phenotypic and genotypic tests and enables not only rapid microbial identification but also characterization of transmission events. The technique has been applied in large-scale infectious disease outbreaks such as those caused by Escherichia coli O104:H4, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecium, Pseudomonas aeruginosa, Vibrio cholerae, and mycobacteria ( 3 – 14 ). However, the routine application of this method in diagnostic microbiology and infection control, especially for less well-defined, emerging pathogens, is yet to be explored. Elizabethkingia anophelis is a recently discovered bacterium isolated from the midgut of the Anopheles gambiae mosquito in 2011 ( 15 ). The genus Elizabethkingia also includes E. meningoseptica (previously named Chryseobacterium/Flavobacterium meningosepticum) and E. miricola ( 16 ). E. meningoseptica causes neonatal sepsis and infections in immunocompromised persons. E. anophelis has also recently been reported to cause neonatal meningitis in the Central African Republic, and a nosocomial outbreak was reported in an intensive care unit in Singapore ( 17 – 19 ). However, the role of mosquitoes or other sources in the transmission of E. anophelis remains unclear. In 2012, we encountered 3 cases of Elizabethkingia sepsis associated with meningitis in 2 neonates and chorioamnionitis in a neonate’s mother in a hospital in Hong Kong. Three strains of Elizabethkingia-like, gram-negative bacilli sharing similar phenotypic characteristics were isolated from the 3 patients, but confident identification results were not obtained by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry and 16S rRNA gene sequencing. Moreover, clinical and microbiological data did not provide adequate clues about the possible transmission route. We therefore attempted to use draft genome sequencing to rapidly dissect transmission pathways and confirm the identity of the species. Materials and Methods Setting and Patients The 3 patients were hospitalized in an acute regional hospital, Pamela Youde Nethersole Eastern Hospital, which is situated in the eastern area of Hong Kong Island. This study was approved by the Institute Review Board, Hospital Authority, Hong Kong (reference HKEC-2013-051). Microbiological Methods Bacterial cultures and phenotypic identification were performed according to standard protocols by using the Vitek II system (bioMérieux, Marcy l’Etoile, France). Antimicrobial drug susceptibility testing was performed by E-test method for vancomycin and Kirby-Bauer disk diffusion for other drugs; because interpretative criteria for Elizabethkingia were lacking, results were interpreted according to Clinical and Laboratory Standards Institute for Pseudomonas aeruginosa ( 20 ). MALDI-TOF mass spectrometry was performed by the direct transfer method as described previously ( 21 ), with modifications by using the Bruker Daltonics microflex LT system with Reference Library Biotyper version 3.1 (Bruker Daltonik GmbH, Leipzig, Germany). Full 16S rRNA gene amplification and sequencing were performed according to previously published protocols with modifications ( 22 , 23 ). Pulsed-field gel electrophoresis (PFGE) was performed by using the CHEF Mapper XA system (Bio-Rad, Hercules, CA, USA) and restriction endonuclease XbaI as described previously ( 8 , 22 ). Draft Genome Sequencing and Analysis The draft genome sequences of the 3 E. anophelis strains were determined by high-throughput sequencing with the Illumina HiSeq 2500 system (Illumina, San Diego, CA, USA). Samples of 50 ng of genomic DNA were extracted by using a genomic DNA purification kit (QIAGEN, Hilden, Germany) from cultures grown overnight on blood agar at 37°C, as described previously ( 24 , 25 ). Each sample was sequenced by 151-bp paired-end reads with mean library size of 350 bp. Sequencing errors were corrected by k-mer frequency spectrum analysis using SOAPec (http://soap.genomics.org.cn/about.html). De novo assembly was performed in SOAPdenovo2 (http://soap.genomics.org.cn/soapdenovo.html). Prediction of protein coding regions and automatic functional annotation was performed by using Glimmer3 ( 26 ) and the RAST (Rapid Annotations using Subsystem Technology) server ( 27 ). Antibiotic resistomes were identified by using the Antibiotic Resistance Genes Database ( 28 ). BLASTn comparisons were run in BLAST+ (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with an E-value cutoff of 10.0. In addition, manual annotation was performed on putative virulence and antibiotic resistance genes by protein domain predictions and multiple sequence alignments with orthologous genes. Intergenomic distance was calculated by using Genome-to-Genome Distance Calculator 2.0 (http://ggdc.dsmz.de/distcalc2.php) ( 29 ). Results Patients In July 2012, a 21-day-old male neonate (patient 1) was admitted to Pamela Youde Nethersole Eastern Hospital for fever of 1 day’s duration. He was born at the same hospital 21 days earlier at 41 weeks’ gestation by vaginal delivery and was discharged on day 3. Physical examination did not show obvious infective focus. Serum C-reactive protein (CRP) was elevated to 109 mg/L. Lumbar puncture was performed; analysis of cerebrospinal fluid (CSF) showed polymorph pleocytosis, elevated protein levels, and low glucose levels (Table). Treatment was initiated for bacterial meningitis with empirical intravenous ampicillin and cefotaxime. Blood and CSF cultures recovered a gram-negative bacillus, designated HKU36. Antimicrobial drugs were changed to vancomycin, piperacillin, and rifampin on day 3. The patient was discharged after 3 weeks of intravenous drug treatment, without neurologic sequelae (Figure 1). The neonate’s mother was admitted to the same hospital 1 day after the infant’s admission for postpartum fever, chills, rigor, and abdominal pain. Transvaginal ultrasound showed no retained gestational products. Serum CRP level was elevated to 109 mg/L; however, blood cultures were negative. She was treated with intravenous cefuroxime and metronidazole and discharged on day 6 with oral cefuroxime and metronidazole. Table Clinical characteristics and results of testing for 3 patients infected with Elizabethkingia anophelis, Hong Kong, 2012* Characteristics Patient 1 Patient 2† Patient 3 Patient age/sex 21 d/M 33y/F 0 d/F Signs/symptoms Fever Fever, PPROM Apnea at birth Blood test results Total leukocytes, × 109 cells/L 16.0 (5.0–19.5) 15.2 (3.7–9.3) 5.1 (10.0–27.0) Neutrophils, × 109 cells/L 6.8 (2.0–9.5) 12.5 (1.8–6.2) 1.2 (5.0–17.0) Lymphocytes, × 109 cells/L 6.8 (2.5–11.0) 1.7 (1.0–3.2) 3.4 (3.0–10.0) Monocytes, × 109 cells/L 2.3 (0.2–1.2) 0.8 (0.2–0.7) 0 (0.5–2.0) Hemoglobin, g/dL 14.0 (11.0–19.0) 10.7 (11.5–15.4) 16.1 (13.5–19.5) Platelets, × 109/L 180 (180–460) 241 (160–420) 186 (100–300) C-reactive protein, mg/L 109 ( 500 bp) contigs (EMBL accession nos. CBYD010000001–CBYD010000042, CBYE010000001–CBYE010000032, CBYF010000001–CBYF010000038; Technical Appendix Table 2). These contigs contained 3,654–3,667 predicted protein-coding genes (Figure 2, panel A). Using Genome-to-Genome Distance Calculator for intergenomic distance estimation, which enabled genome-based species delineation analogous to traditional DNA–DNA hybridization method, we found that these genomes shared 78.3%–85.4% nucleotide identities to the draft genome sequence of E. anophelis type strain R26T, the initial isolate from an Anopheles gambiae mosquito (GenBank accession no. NZ_ANIW00000000.1). However, the genomes shared only 23.6%–23.7% nucleotide identities to the draft genome sequence of E. meningoseptica type strain ATCC 13253T (GenBank accession no. BARD00000000.1) (Figure 2, panel B). Phylogenetic analysis using the draft genomes and concatenated sequences of 69 housekeeping genes also supported the identification of the 3 strains as E. anophelis (Figure 3; Technical Appendix Figure 2). Figure 2 Comparison of draft genome sequence data of the 3 Elizabethkingia anophelis strains from patients in Hong Kong (HKU36–38), E anophelis type strain R26T, and E. meningoseptica type strain ATCC 13253T. A) Distributions of predicted coding sequence function in genomes of E. anophelis strains HKU36–38, E. anophelis type strain R26T, and E. meningoseptica type strain ATCC 13253T according to SEED Subsystems are shown. The columns indicate the number of proteins in different subsystems. B) Circular representation of sequence comparison between the draft genome of strain HKU37 and other draft genomes as labeled. Comparison generated in Rapid Annotations using Subsystem Technology ( 27 ). Intensity of color indicates degree of protein identity. Figure 3 Phylogenetic trees constructed by using draft genome sequences and concatenated sequences of 69 housekeeping genes of 3 Elizabethkingia anophelis strains from patients in Hong Kong (HKU36–38). A) Neighbor-joining tree constructed on the basis of draft genome sequences using by using Genome-to-Genome Distance Calculator 2.0 (http://ggdc.dsmz.de/distcalc2.php; formula 1) and Chryseobacterium gleum ATCC 35910 as the root. Arrow indicates route of mother-to-neonate transmission. B) Maximum-likelihood tree constructed on the basis of 69 housekeeping genes, showing the relationship of E. anophelis strains HKU36–38 to related bacterial species, using RAxML version 7.2.8 (http://sco.h-its.org/exelixis/software.html) and Weeksella virosa DSM 16922 as the root. A total of 78,520 nt positions were included in the analysis. Bootstrap values were calculated from 1,000 replicates. Scale bars indicate mean number of nucleotide substitutions per site on the respective branches. Gene names and accession numbers are given as cited in GenBank (Technical Appendix Table 2). ‘E. meningoseptica’ strain 502 is a misidentified isolate that actually belongs to E. anophelis on the basis of draft genome sequencing. The sequences from 52 contigs of strain HKU37 demonstrated 99.4% nucleotide identity to those from 46 contigs of strain HKU38, indicating that these draft genomes are essentially identical (Figure 2, panel B, and Figure 3). The small intergenomic distance can be explained by slight differences in coverage or contig assembly; sequences of 2,000 high-coverage protein-coding genes were identical between HKU37 and HKU38. In contrast, these sequences demonstrated only 78.6% nucleotide identity to those from the 42 contigs of strain HKU36, indicating that strain HKU36 is genetically divergent (Figure 2, panel B, and Figure 3), consistent with PFGE patterns (Figure 4). Moreover, a potential genetic island consisting of conjugative transposable elements was found in strains HKU37 and HKU38 but not in HKU36. Our results exclude a clonal outbreak, but the extremely close genetic relatedness between strains HKU37 and HKU38 provides evidence for vertical transmission from patient 2 to patient 3 (mother to infant). Figure 4 Pulsed-field gel electrophoresis (PFGE) analysis of samples from patients in Hong Kong showing 3 Elizabethkingia anophelis strains compared with reference Elizabethkingia isolates. A) PFGE performed by using CHEF Mapper XA system (Bio-Rad, Hercules, CA, USA) and restriction endonuclease XbaI shows that isolates from patient 2 and patient 3 are indistinguishable, wheras isolates from patient 1 possess distinct PFGE patterns. Lane 1, E. anophelis strain HKU37 from uterine swab specimen of patient 2; lane 2, placental swab specimen from patient 2; lane 3, E. anophelis strain HKU38 from blood of patient 3; lane 4, cerebrospinal fluid from patient 3; lane 5, E. anophelis strain HKU36 from blood of patient 1; lane 6, cerebrospinal fluid from patient 1; lane 7, E. anophelis type strain R26T; lane 8, E. meningoseptica type strain ATCC 13253T; lane 9, E. miricola type strain LMG22470T. B) Dendrogram constructed with PFGE data by similarity and clustering analysis using the Dice coefficient (1% tolerance and 0.5% optimization) and the unweighted pair-group method using average linkages with GelCompar II (Applied Maths, Sint-Martens-Latem, Belgium). Potential Virulence Factors and Resistance Genes in E. anophelis The association of E. anophelis with neonatal meningitis in this and previous reports ( 17 , 18 ) suggests that the bacterium may possess virulence factors that enable it to invade the central nervous system. The 3 draft genomes we identified contain homologs of several virulence genes found in Listeria monocytogenes, which also causes neonatal meningitis. These genes include cell wall hydrolase A, which enables host cell invasion; phosphatidylinositol-specific phospholipase (PlcA) and listeriolysin O (LLO), which enable escape from the primary vacuole of macrophages, and genes that enable survival in the secondary vacuole of macrophages; and virulence cluster protein B (VclB). Phosphatidylinositol-specific phospholipase, listeriolysin O, and virulence cluster protein B are located in the Listeria pathogenicity island LIPI-1 ( 30 , 31 ). Moreover, the 3 genomes we identified contain homologs of arylsulfatase and genes that enable invasion of brain endothelial cells, which contribute to the ability of Escherichia coli to cross the blood–brain barrier in neonatal meningitis ( 32 ). Vertical transmission of E. anophelis from mother to infant also suggests that the bacterium may be able to colonize the vagina before causing ascending chorioamnionitis in the mother and neonatal infection through transplacental spread. A homologof the gene encoding agmatine deiminase, AgDI, which mediates acid tolerance in L. monocytogenes ( 33 ), was found in the E. anophelis genomes. Further studies may investigate the possible role of AgDI and potential adherence factors for vaginal colonization in E. anophelis. Similar to E. meningoseptica, the 3 E. anophelis isolates we identified are resistant to multiple antimicrobial drugs. We found various antimicrobial resistance genes consistent with their resistance phenotypes, including metallo-β-lactamase (bla GOB-1 and blaB14 in strain HKU36 and a novel bla GOB and blaB1 in strains HKU37 and HKU38) and extended-spectrum β-lactamase (blaA CME-1 in strains HKU37 and HKU38 and a potential novel blaA CME-1 variant in strain HKU36). A comparison of these β-lactamases to their corresponding orthologs in E. meningoseptica genomes revealed only 74%–85% amino acid identities, indicating that E. anophelis and related bacteria are potential reservoirs of novel β-lactamase genes ( 19 , 34 , 35 ). Other antimicrobial resistance genes found included multidrug-resistance efflux pumps (ATP binding cassette superfamily, major facilitator superfamily, resistance-nodulation-division families, multidrug and toxic-compound extrusion family) that potentially carry resistance to a variety of compounds; chloramphenicol acetyltransferase; aminoglycoside 6-adenyltransferase; and tetracycline resistant gene. Moreover, a putative tetX gene was also identified; this gene encodes a predicted flavin-dependent monooxygenase with tetracycline/tigecycline-degrading activity, although the 3 strains we identified are only resistant to tetracycline but remained susceptible to other related drugs, including tigecycline. Comparison of Genomes from Human and Mosquito E. anophelis Strains E. anophelis strains R26T and Ag1 were isolated from mosquitoes ( 35 ). Compared with those strains, the genomes of the 3 strains we identified possessed 33 unique hypothetical proteins. Moreover, the genetic island consisting of conjugative transposable elements found in strains HKU37 and HKU38 was also absent in the mosquito strains. In contrast to the mosquito strains, which possessed genes encoding for xylose isomerase (XylA) and xylulose kinase (XylB), these 2 genes were absent in the 3 strains we identified. This finding may reflect different requirements for sugar metabolism in E. anophelis under different environments. Notably, despite the presence of XylA and XylB, E. anophelis mosquito strain R26T did not produce acid from xylose ( 15 ). However, this finding does not exclude the strain’s ability to metabolize xylose, as D-xylulose 5-phosphate, the product of XylA and XylB, can be used as a substrate for the pentose-phosphate pathway. XylA and XylB were also absent in the genome of E. meningoseptica type strain ATCC 13253T, which suggests that mosquito strains of E. anophelis may be evolutionarily distinct from clinical strains of E. anophelis and E. meningoseptica. More genome sequence data from other clinical and environmental strains of E. anophelis may shed light on the ecology, biology, and pathogenesis of E. anophelis. Discussion This study demonstrates the power of draft genome sequencing to rapidly dissect transmission pathways for emerging bacterial infections. Our results showed that vertical perinatal transmission had occurred from patient 2, a pregnant woman who had chorioamnionitis, to patient 3, a neonate who had early onset neonatal meningitis. The infective source for patients 2 and 3 was unlikely to have been patient 1 or his mother. However, we speculate that the mother of patient 1 might also have had E. anophelis chorioamnionitis, as evidenced by postpartum fever and abdominal pain, which resulted in late-onset meningitis in her son owing to fastidious bacterial growth. Although strain HKU36 did not belong to the same clone as strains HKU37/38, a polyclonal outbreak of E. anophelis sepsis in the labor ward, in which case an environmental source is likely, could not be excluded. The discovery of E. anophelis in mosquito gut has raised suspicion that mosquitoes are the source of neonatal meningitis cases in Africa ( 17 ). Although Anopheles mosquitoes are not found in Hong Kong, the role of local mosquitoes as reservoirs for E. anophelis remains unknown. Nonetheless, the vertical transmission demonstrated in 1 neonate makes mosquitoes unlikely as vehicles of transmission in our cases. Our report provides genomic evidence for vertical transmission in neonatal meningitis. Whereas we cannot ascertain how the mother(s) acquired the infection, our results prompt further work to assess the importance of maternal source in neonatal meningitis caused by E. anophelis and other bacterial agents. Maternal colonization with Lancefield group B streptococcus (GBS) during pregnancy is the primary risk factor for early onset neonatal disease. However, direct microbiological evidence for vertical transmission is seldom available, especially for bacterial agents other than GBS. Further genomic studies may help investigate the role of vertical transmission in neonatal meningitis caused by other bacteria. Current indications for intrapartum antimicrobial drugs prophylaxis have been determined on the basis of risk factors for early onset GBS disease; therefore, intravenous penicillin G or ampicillin is often the standard empirical regimen used. However, if further research determines that the mother may also be a source of transmission for other bacterial agents, broader-spectrum antimicrobial drugs may need to be considered as treatment for intrapartum fever or prolonged rupture of membranes. E. anophelis is likely an underreported bacterium because it can be easily misidentified as E. meningoseptica, which shares a similar phenotypic profile ( 17 , 19 ). The E. anophelis isolates from the recent outbreak reported in Singapore were initially mistakenly identified as E. meningoseptica ( 19 , 36 ). Of the 3 strains we identified, 2 were misidentified as E. meningoseptica with MALDI-TOF mass spectrometry, the state-of-the-art technology, which is replacing conventional phenotypic identification in diagnostic laboratories. The reason for failure of MALDI-TOF mass spectrometry to identify these strains was that reference E. anophelis strains are lacking in existing diagnostic spectrum databases, as is the case with other less commonly encountered organisms ( 21 ). Although 16S rRNA gene sequencing should provide sufficient resolution, some strains indexed as E. meningoseptica, such as strains G3-1-08 and 502, were actually more closely related to E. anophelis than to E. meningoseptica in their 16S rRNA sequences (Figure 3; Technical Appendix Figure 2) ( 37 ). These ambiguous, potentially misidentified strains may cause incorrect interpretations in suspected E. anophelis infections. For example, the sequence of strain HKU36 possessed 99.8% nucleotide identity to that of E. meningingoseptica strain G3-1-08 but only 99.1% nucleotide identity to that of E. anophelis strain R26T. Furthermore, phenotypic tests such as acid production from cellobiose and citrate utilization, previously proposed to be useful for identification of E. anophelis ( 15 ), are probably unreliable in differentiating among Elizabethkingia species. For example, E. anophelis strain R26T produces acid from cellobiose, but the 3 strains we identified do not; in addition, E. anophelis strains R26T, HKU37, and HKU38, but not strain HKU36, utilize citrate (Technical Appendix Table 1). Strain HKU36 displayed higher MIC of vancomycin than did strains HKU37 and HKU38 and type strains of E. anophelis, E. meningoseptica, and E. miricola, which correlates with previous reports on variable vancomycin susceptibilities in Elizabethkingia ( 38 , 39 ). The species identity of the 3 strains we identified was only resolved by intergenomic comparison. Inclusion of E. anophelis in MALDI-TOF MS databases and rectification of 16S rRNA gene sequences of Elizabethkingia strains deposited in databases will enable accurate diagnosis of more E. anophelis infections. The draft genome sequences we identified have enabled rapid exploration of novel β-lactamase and other antimicrobial drug resistance genes and possible virulence genes in E. anophelis, highlighting the potential of genome sequencing in identifying novel drug-resistance mechanisms and guiding treatment regimens for emerging, multidrug-resistant bacteria ( 25 , 34 , 40 ). Because previous cases of E. anophelis neonatal meningitis have been associated with poor outcomes ( 17 , 18 ), further work to elucidate the pathogenesis and antimicrobial drug resistance patterns of this emerging pathogen may help improve clinical management of illness. The findings of potential genes related to neuroinvasion and acid tolerance and the unique genetic characteristics in clinical strains of E. anophelis compared with mosquito strains may also provide insights on the ability of E. anophelis to adapt to different ecologic niches and cause neonatal infection through vertical transmission. In conclusion, the genome data we obtained for these cases offered superior discriminatory power that supported appropriate infection control measures. The ability to distinguish different bacterial isolates often has critical implications on practical infection-control management, but different strains of the same bacterial species may not be distinguishable by their phenotypes because they reflect a tiny portion of the microbial genome. With better automation and lower costs, draft genome sequencing, which offers a short turnaround time, may replace existing typing methods such as PFGE or multilocus sequence typing for outbreak investigations. Technical Appendix Results of matrix-assisted laser desorption ionization/time-of-flight mass spectrometry identification of 3 Elizabethkingia anophelis strains from patients in Hong Kong (HKU36–38), phylogenetic tree showing the relationship of HKU36–38 to closely related bacterial species using 16S rRNA gene sequence analysis, phenotypic characteristics and vancomycin susceptibilities of HKU36–38 compared with those of closely related bacterial species, results of draft genome assembly for HKU36–38, and protein names and accession numbers of the 69 housekeeping genes used for phylogenetic analysis.
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              Identification, characterization, and biofilm formation of clinical Chryseobacterium gleum isolates.

              Chryseobacterium gleum is not commonly isolated from clinical source(s). Using 16S rRNA gene sequencing, we identified 15 C. gleum isolates from the Central Region Hospital Alliance, Taiwan, which were all misidentified: 14 as Chryseobacterium indologenes and 1 as Elizabethkingia meningoseptica using the Vitek 2 GN card. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a rapid and clinically applicable method, was evaluated for the identification of C. gleum, and the rate of species or probable species level identification reached 13.3% and 86.6%, respectively. Using pulsed-field gel electrophoresis analysis, all C. gleum isolates from central Taiwan were found to be epidemiologically unrelated. The most prevalent sample was urine (35.7%, 5/14), followed by sputum (28.6%, 4/14), whereas 1 isolate was from an unknown source. All of the isolates were susceptible to minocycline, 93.3% susceptible to trimethoprim/sulfamethoxazole, but were completely or highly resistant to the other drugs examined. Biofilm-forming ability was observed in 40.0% (6/15) isolates using the Luria-Bertani broth. To the best of our knowledge, this is the first focusing on exploring clinical C. gleum isolates.
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                Author and article information

                Journal
                J Crit Care Med (Targu Mures)
                J Crit Care Med (Targu Mures)
                jccm
                jccm
                The Journal of Critical Care Medicine
                Sciendo
                2393-1809
                2393-1817
                October 2018
                01 October 2018
                : 4
                : 4
                : 149-150
                Affiliations
                [1 ]deptMicrobiology Department , Shah Satnam Ji Speciality Hospitals , Sirsa, Haryana, India
                [2 ]deptDepartment of Medicine , Shah Satnam Ji Speciality Hospitals , Sirsa, Haryana, India
                Author notes
                [* ] Neha Gupta, Microbiology lab, Department of Microbiology, Shah Satnam Ji Speciality Hospitals, Near Shah SatnamJi Dham, Dera Sacha Sauda, Bhadra Road, Sirsa-125055, Haryana, India. nehagupta0606@ 123456gmail.com
                Article
                jccm-2018-0021
                10.2478/jccm-2018-0021
                6296276
                © 2018 Gurmeet Ram Rahim, Neha Gupta, Gaurav Aggarwal published by Sciendo

                This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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                Pages: 2
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