Prevalence and characterization of an integrative and conjugative element carrying tet(X) gene in Elizabethkingia meningoseptica.

OBJECTIVES: To investigate the tet(X) gene, a determinant of tigecycline resistance, in the emerging pathogen Elizabethkingia meningoseptica and its association with an integrative and conjugative element (ICE). METHODS: All E. meningoseptica genomes from the National Center for Biotechnology Information (n = 87) were retrieved and annotated for resistome searches using the CARD database. A phylogenic analysis was performed based on the E. meningoseptica core genome. The ICE was identified through comparative genomics with other ICEs occurring in Elizabethkingia spp. RESULTS: Phylogenetic analysis revealed E. meningoseptica genomes from six countries distributed across different lineages, some of which persisted for years. The common resistome of these genomes included blaBlaB, blaCME, blaGOB, ranA/B, aadS, and catB (genes associated with resistance to ß-lactams, aminoglycosides, and chloramphenicol). Some genomes also presented additional resistance genes (dfrA, ereD, blaVEB, aadS, and tet(X)). Interestingly, tet(X) and aadS were located in an ICE of 49 769 bp (ICEEmSQ101), which was fully obtained from the E. meningoseptica SQ101 genome. We also showed evidence that the other 27 genomes harboured this ICE. The distribution of ICEEmSQ101, carrying tet(X), was restricted to a single Chinese lineage. CONCLUSIONS: The tet(X) gene is not prevalent in the species E. meningoseptica, as previously stated for the genus Elizabethkingia, since it is present only in a single Chinese lineage. We identified that several E. meningoseptica genomes harboured an ICE that mobilized the Elizabethkingia tet(X) gene and exhibited characteristics similar to the ICEs of other Flavobacteria, which would favour their transmission in this bacterial family.

Imipenem reduces the efficacy of vancomycin against Elizabethkingia species.

BACKGROUND: Elizabethkingia spp. are emerging as nosocomial pathogens causing various infections. These pathogens express resistance to a broad range of antibiotics, thus requiring antimicrobial combinations for coverage. However, possible antagonistic interactions between antibiotics have not been thoroughly explored. This study aimed to evaluate the effectiveness of antimicrobial combinations against Elizabethkingia infections, focusing on their impact on pathogenicity, including biofilm production and cell adhesion. METHODS: Double-disc diffusion, time-kill, and chequerboard assays were used for evaluating the combination effects of antibiotics against Elizabethkingia spp. We further examined the antagonistic effects of antibiotic combinations on biofilm formation and adherence to A549 human respiratory epithelial cells. Further validation of the antibiotic interactions and their implications was performed using ex vivo hamster precision-cut lung sections (PCLSs) to mimic in vivo conditions. RESULTS: Antagonistic effects were observed between cefoxitin, imipenem and amoxicillin/clavulanic acid in combination with vancomycin. The antagonism of imipenem toward vancomycin was specific to its effects on the genus Elizabethkingia. Imipenem further hampered the bactericidal effect of vancomycin and impaired its inhibition of biofilm formation and the adhesion of Elizabethkingia meningoseptica ATCC 13253 to human cells. In the ex vivo PCLS model, vancomycin exhibited dose-dependent bactericidal effects; however, the addition of imipenem also reduced the effect of vancomycin. CONCLUSIONS: Imipenem reduced the bactericidal efficacy of vancomycin against Elizabethkingia spp. and compromised its capacity to inhibit biofilm formation, thereby enhancing bacterial adhesion. Clinicians should be aware of the potential issues with the use of these antibiotic combinations when treating Elizabethkingia infections.

The individual contributions of blaB, blaGOB and blaCME on MICs of ß-lactams in Elizabethkingia anophelis.

BACKGROUND: The blaB, blaGOB and blaCME genes are thought to confer ß-lactam resistance to Elizabethkingia anophelis, based on experiments conducted primarily on Escherichia coli. OBJECTIVES: To determine the individual contributions of ß-lactamase genes to increased MICs in E. anophelis and to assess their impact on the in vivo efficacy of carbapenem therapy. METHODS: Scarless gene deletion of one or more ß-lactamase gene(s) was performed in three clinical E. anophelis isolates. MICs were determined by broth microdilution. Hydrolytic activity and expressions of ß-lactamase genes were measured by an enzymatic assay and quantitative RT-PCR, respectively. In vivo efficacy was determined using Galleria mellonella and murine thigh infection models. RESULTS: The presence of blaB resulted in >16-fold increases, while blaGOB caused 4-16-fold increases of carbapenem MICs. Hydrolysis of carbapenems was highest in lysates of blaB-positive strains, possibly due to the constitutionally higher expression of blaB. Imipenem was ineffective against blaB-positive isolates in vivo in terms of improvement of the survival of wax moth larvae and reduction of murine bacterial load. The deletion of blaB restored the efficacy of imipenem. The blaB gene was also responsible for a >4-fold increase of ampicillin/sulbactam and piperacillin/tazobactam MICs. The presence of blaCME, but not blaB or blaGOB, increased the MICs of ceftazidime and cefepime by 8-16- and 4-8-fold, respectively. CONCLUSIONS: The constitutionally and highly expressed blaB gene in E. anophelis was responsible for increased MICs of carbapenems and led to their poor in vivo efficacy. blaCME increased the MICs of ceftazidime and cefepime.

Molecular characterization of the multi-drug resistant Myroides odoratimimus isolates: a whole genome sequence-based study to confirm carbapenem resistance.

The bacteria belonging to the Myroides genus are opportunistic pathogens causing community or hospital-acquired infections that result in treatment failure due to antibiotic resistance. This study aimed to investigate molecular mechanisms of antibiotic resistance, clonal relatedness, and the biofilm forming capacity of the 51 multi-drug resistant Myroides odoratimimus. All isolates were screened for blaKPC, blaOXA, blaVIM, blaIMP, blaMUS, blaTUS, blaNDM, and blaB genes by using PCR amplification. Whole genome sequencing (WGS) was applied on three randomly selected isolates for further investigation of antibiotic resistance mechanisms. Clonal relatedness was analyzed by Pulsed-field gel electrophoresis (PFGE) and the microtiter plate method was used to demonstrate biofilm formation. All isolates were positive for biofilm formation. PCR analysis resulted in a positive for only the blaMUS-1 gene. WGS identified blaMUS-1, erm(F), ere(D), tet(X), and sul2 genes in all strains tested. Moreover, the genomic analyses of three strains revealed that genomes contained a large number of virulence factors (VFs). PFGE yielded a clustering rate of 96%. High clonal relatedness, biofilm formation, and multi-drug resistance properties may lead to the predominance of these opportunistic pathogens in hospital environments and make them cause nosocomial infections.

Synergistic efficacy of meropenem, ciprofloxacin and colistin antibiotics against planktonic and biofilm forms of Myroides odoratimimus bacterial isolates.

PURPOSE: In this study, it was aimed to investigate the combined synergistic efficacy of colistin (CT), meropenem (MEM), and ciprofloxacin (CIP) antibiotics on planktonic and biofilm forms in Myroidesodoratimimus strains isolated from various clinical specimens. METHODS: Antibiotic susceptibility was determined by the Kirby-Bauer disk diffusion method. In addition, minimum inhibitory concentrations (MIC) of CIP, MEM, and CT were studied using the standardized broth microdilution method. In vitro synergistic activity of antibiotics against M. odoratimimus planktonic bacteria strains was studied by the Micro Broth Checkerboard method. The microtiter plate (MtP) method was used to determine the effectiveness of antibiotics on M. odoratimimus biofilm formation. RESULTS: A zone of inhibition was not observed against other antibiotics used except amikacin and linezolid in all strains. While CT/MEM and CT/CIP combinations have a synergistic effect on all strains, the combination CIP/MEM has an additive effect. According to the biofilm inhibition results, all three antibiotics inhibited biofilm formation. However, the efficacy of MEM (60.3-76.5%) and CIP (60.2-77.8%) was approximately two times higher than that of CT (25.4-34.5%). In addition, the effectiveness of combinations of antibiotics on biofilm formation was examined and the percentage of inhibition was 30.8% when CT was used alone, while the biofilm inhibition rates of CT/MEM and CT/CIP were 92.4% and 91.7%, respectively. MEM/CIP combination was inhibited biofilm formation by 75.7%. CONCLUSIONS: This study is the first report showing the efficacy of CT, MEM and CIP antibiotics, which are frequently used in clinical practice, in combination on M. odoratimimus planktonic and biofilm forms. The findings of our study are particularly guiding for combined antibiotic treatment options in immunosuppressed patients admitted to an intensive care unit (ICU). The CT/MEM combination is currently used frequently. In addition, these results are important in terms of supporting in vitro that CT/CIP and MEM/CIP combinations can also be used as a treatment option in M. odoratimimus related infections.

Source Tracking and Global Distribution of the Tigecycline Non-Susceptible tet(X).

The emergence of tet(X) genes has compromised the clinical use of the last-line antibiotic tigecycline. We identified 322 (1.21%) tet(X) positive samples from 12,829 human microbiome samples distributed in four continents (Asia, Europe, North America, and South America) using retrospective data from worldwide. These tet(X) genes were dominated by tet(X2)-like orthologs but we also identified 12 samples carrying novel tet(X) genes, designed tet(X45), tet(X46), and tet(X47), were resistant to tigecycline. The metagenomic analysis indicated these tet(X) genes distributed in anaerobes dominated by Bacteroidaceae (78.89%) of human-gut origin. Two mobile elements ISBf11 and IS4351 were most likely to promote the transmission of these tet(X2)-like orthologs between Bacteroidaceae and Riemerella anatipestifer. tet(X2)-like orthologs was also developed during transmission by mutation to high-level tigecycline resistant genes tet(X45), tet(X46), and tet(X47). Further tracing these tet(X) in single bacterial isolate from public repository indicated tet(X) genes were present as early as 1960s in R. anatipestifer that was the primary tet(X) carrier at early stage (before 2000). The tet(X2) and non-tet(X2) orthologs were primarily distributed in humans and food animals respectively, and non-tet(X2) were dominated by tet(X3) and tet(X4). Genomic comparison indicated these tet(X) genes were likely to be generated during tet(X) transmission between Flavobacteriaceae and E. coli/Acinetobacter spp., and ISCR2 played a key role in the transmission. These results suggest R. anatipestifer was the potential ancestral source of tet(X). In addition, Bacteroidaceae of human-gut origin was an important hidden reservoir and mutational incubator for the mobile tet(X) genes that enabled spread to facultative anaerobes and aerobes. IMPORTANCE The emergence of the tigecycline resistance gene tet(X) has posed a severe threat to public health. However, reports of its origin and distribution in human remain rare. Here, we explore the origin and distribution of tet(X) from large-scale metagenomic data of human-gut origin and public repository. This study revealed the emergency of tet(X) gene in 1960s, which has refreshed a previous standpoint that the earliest presence of tet(X) was in 1980s. The metagenomic analysis from data mining covered the unculturable bacteria, which has overcome the traditional bacteria isolating and purificating technologies, and the analysis indicated that the Bacteroidaceae of human-gut origin was an important hidden reservoir for tet(X) that enabled spread to facultative anaerobes and aerobes. The continuous monitoring of mobile tigecycline resistance determinants from both culturable and unculturable microorganisms is imperative for understanding and tackling the dissemination of tet(X) genes in both the health care and agricultural sectors.

The Integrative and Conjugative Element ICECspPOL2 Contributes to the Outbreak of Multi-Antibiotic-Resistant Bacteria for Chryseobacterium Spp. and Elizabethkingia Spp.

Antibiotic resistance genes (ARGs) and horizontal transfer of ARGs among bacterial species in the environment can have serious clinical implications as such transfers can lead to disease outbreaks from multidrug-resistant (MDR) bacteria. Infections due to antibiotic-resistant Chryseobacterium and Elizabethkingia in intensive care units have been increasing in recent years. In this study, the multi-antibiotic-resistant strain Chryseobacterium sp. POL2 was isolated from the wastewater of a livestock farm. Whole-genome sequencing and annotation revealed that the POL2 genome encodes dozens of ARGs. The integrative and conjugative element (ICE) ICECspPOL2, which encodes ARGs associated with four types of antibiotics, including carbapenem, was identified in the POL2 genome, and phylogenetic affiliation analysis suggested that ICECspPOL2 evolved from related ICEEas of Elizabethkingia spp. Conjugation assays verified that ICECspPOL2 can horizontally transfer to Elizabethkingia species, suggesting that ICECspPOL2 contributes to the dissemination of multiple ARGs among Chryseobacterium spp. and Elizabethkingia spp. Because Elizabethkingia spp. is associated with clinically significant infections and high mortality, there would be challenges to clinical treatment if these bacteria acquire ICECspPOL2 with its multiple ARGs, especially the carbapenem resistance gene. Therefore, the results of this study support the need for monitoring the dissemination of this type of ICE in Chryseobacterium and Elizabethkingia strains to prevent further outbreaks of MDR bacteria. IMPORTANCE Infections with multiple antibiotic-resistant Chryseobacterium and Elizabethkingia in intensive care units have been increasing in recent years. In this study, the mobile integrative and conjugative element ICECspPOL2, which was associated with the transmission of a carbapenem resistance gene, was identified in the genome of the multi-antibiotic-resistant strain Chryseobacterium sp. POL2. ICECspPOL2 is closely related to the ICEEas from Elizabethkingia species, and ICECspPOL2 can horizontally transfer to Elizabethkingia species with the tRNA-Glu-TTC gene as the insertion site. Because Elizabethkingia species are associated with clinically significant infections and high mortality, the ability of ICECspPOL2 to transfer carbapenem resistance from environmental strains of Chryseobacterium to Elizabethkingia is of clinical concern.

Structural characterization of a Type B chloramphenicol acetyltransferase from the emerging pathogen Elizabethkingia anophelis NUHP1.

Elizabethkingia anophelis is an emerging multidrug resistant pathogen that has caused several global outbreaks. E. anophelis belongs to the large family of Flavobacteriaceae, which contains many bacteria that are plant, bird, fish, and human pathogens. Several antibiotic resistance genes are found within the E. anophelis genome, including a chloramphenicol acetyltransferase (CAT). CATs play important roles in antibiotic resistance and can be transferred in genetic mobile elements. They catalyse the acetylation of the antibiotic chloramphenicol, thereby reducing its effectiveness as a viable drug for therapy. Here, we determined the high-resolution crystal structure of a CAT protein from the E. anophelis NUHP1 strain that caused a Singaporean outbreak. Its structure does not resemble that of the classical Type A CATs but rather exhibits significant similarity to other previously characterized Type B (CatB) proteins from Pseudomonas aeruginosa, Vibrio cholerae and Vibrio vulnificus, which adopt a hexapeptide repeat fold. Moreover, the CAT protein from E. anophelis displayed high sequence similarity to other clinically validated chloramphenicol resistance genes, indicating it may also play a role in resistance to this antibiotic. Our work expands the very limited structural and functional coverage of proteins from Flavobacteriaceae pathogens which are becoming increasingly more problematic.

Comparison of three species of Elizabethkingia genus by whole-genome sequence analysis.

Elizabethkingia are found to cause severe neonatal meningitis, nosocomial pneumonia, endocarditis and bacteremia. However, there are few studies on Elizabethkingia genus by comparative genomic analysis. In this study, three species of Elizabethkingia were found: E. meningoseptica, E. anophelis and E. miricola. Resistance genes and associated proteins of seven classes of antibiotics including beta-lactams, aminoglycosides, macrolides, tetracyclines, quinolones, sulfonamides and glycopeptides, as well as multidrug resistance efflux pumps were identified from 20 clinical isolates of Elizabethkingia by whole-genome sequence. Genotype and phenotype displayed a good consistency in beta-lactams, aminoglycosides and glycopeptides, while contradictions exhibited in tetracyclines, quinolones and sulfonamides. Virulence factors and associated genes such as hsp60 (htpB), exopolysaccharide (EPS) (galE/pgi), Mg2+ transport (mgtB/mgtE) and catalase (katA/katG) existed in all clinical and reference strains. The functional analysis of the clusters of orthologous groups indicated that 'metabolism' occupied the largest part in core genome, 'information storage and processing' was the largest group in both accessory genome and unique genome. Abundant mobile elements were identified in E. meningoseptica and E. anophelis. The most significant finding in our study was that a single clone of E. anophelis had been circulating within diversities of departments in a clinical setting for nearly 18 months.

Epidemiological and phylogenetic analysis reveals Flavobacteriaceae as potential ancestral source of tigecycline resistance gene tet(X).

Emergence of tigecycline-resistance tet(X) gene orthologues rendered tigecycline ineffective as last-resort antibiotic. To understand the potential origin and transmission mechanisms of these genes, we survey the prevalence of tet(X) and its orthologues in 2997 clinical E. coli and K. pneumoniae isolates collected nationwide in China with results showing very low prevalence on these two types of strains, 0.32% and 0%, respectively. Further surveillance of tet(X) orthologues in 3692 different clinical Gram-negative bacterial strains collected during 1994-2019 in hospitals in Zhejiang province, China reveals 106 (2.7%) tet(X)-bearing strains with Flavobacteriaceae being the dominant (97/376, 25.8%) bacteria. In addition, tet(X)s are found to be predominantly located on the chromosomes of Flavobacteriaceae and share similar GC-content as Flavobacteriaceae. It also further evolves into different orthologues and transmits among different species. Data from this work suggest that Flavobacteriaceae could be the potential ancestral source of the tigecycline resistance gene tet(X).

Identification of novel tetracycline resistance gene tet(X14) and its co-occurrence with tet(X2) in a tigecycline-resistant and colistin-resistant Empedobacter stercoris.

Tigecycline is one of the last-resort antibiotics to treat severe infections. Recently, tigecycline resistance has sporadically emerged with an increasing trend, and Tet(X) family represents a new resistance mechanism of tigecycline. In this study, a novel chromosome-encoded tigecycline resistance gene, tet(X14), was identified in a tigecycline-resistant and colistin-resistant Empedobacter stercoris strain ES183 recovered from a pig fecal sample in China. Tet(X14) shows 67.14-96.39% sequence identity to the other variants [Tet(X) to Tet(X13)]. Overexpression of Tet(X14) in Escherichia coli confers 16-fold increase in tigecycline MIC (from 0.125 to 2 mg/L), which is lower than that of Tet(X3), Tet(X4) and Tet(X6). Structural modelling predicted that Tet(X14) shared a high homology with the other 12 variants with RMSD value from 0.003 to 0.055, and Tet(X14) can interact with tetracyclines by a similar pattern as the other Tet(X)s. tet(X14) and two copies of tet(X2) were identified on a genome island with abnormal GC content carried by the chromosome of ES183, and no mobile genetic elements were found surrounding, suggesting that tet(X14) might be heterologously obtained by ES183 via recombination. Blasting in Genbank revealed that Tet(X14) was exclusively detected on the chromosome of Riemerella anatipestifer, mainly encoded on antimicrobial resistance islands. E. stercoris and R. anatipestifer belong to the family Flavobacteriaceae, suggesting that the members of Flavobacteriaceae maybe the major reservoir of tet(X14). Our study reports a novel chromosome-encoded tigecycline resistance gene tet(X14). The expanded members of Tet(X) family warrants the potential large-scale dissemination and the necessity of continuous surveillance for tet(X)-mediated tigecycline resistance.

Early neonatal sepsis and meningitis caused by Elizabethkingia meningoseptica in Saudi Arabia.

Elizabethkingia meningoseptica (E. meningoseptica ) are Gram-negative bacteria commonly associated with nosocomial infections in neonates. This is a case study of E. meningoseptica, presented as meningitis and sepsis in a term baby. The female infant was born by vaginal delivery at 37 weeks gestational age. The case was peculiar because the baby was neither premature nor immuno-compromised, which are known risk factors for E. meningoseptica infection. The onset began on the second day of the neonate's life. On day 3, peripheral blood culture and cerebrospinal fluid findings isolated a gram-negative bacteria identified as E. meningoseptica. The first-line antibiotics therapy was changed to ciprofloxacin, vancomycin, and rifampicin, based on the laboratory determination of antimicrobial sensitivity. The patient's clinical condition improved, although post hemorrhagic ventricular dilatation was revealed by imaging studies. Clinicians should possess proper awareness of the antibiotic sensitivity of E. meningoseptica, as it is important in preventing high rates of morbidity and mortality.

Vancomycin improves Plasmodium yoelii malaria parasite in vitro liver stage cultures by controlling Elizabethkingia anophelis, a bacterium in the microbiome of lab-reared Anopheles mosquitoes.

Studies of Plasmodium sporozoites and liver stages require dissection of Anopheles mosquitoes to obtain sporozoites for experiments. Sporozoites from the rodent parasite P. yoelii are routinely used to infect hepatocytes for liver stage culture, but sometimes these cultures become contaminated. Using standard microbiological techniques, a single colony type of Gram-negative rod-shaped bacteria was isolated from contaminated cultures. Mass spectrometry and sequencing of the bacterial 16S ribosomal RNA gene identified the contaminant as Elizabethkingia spp. Based on sequence comparison and published studies of the Anopheles microbiome, the best match was E. anophelis. Culture contamination was not ameliorated by density gradient purification of sporozoites. However, the addition of vancomycin to the culture media consistently reduced contamination and improved culture outcomes as measured by liver stage parasite size. Thus, mosquito salivary gland-derived E. anophelis is identified a potential contaminant of Plasmodium liver stage cultures that can be mitigated by the addition of antibiotics.

Molecular diversity of chromosomal metallo-ß-lactamase genes in Elizabethkingia genus.

Elizabethkingia genus is an opportunistic life-threatening pathogen with an intrinsic multidrug-resistant phenotype. It is the only known microorganism with multi-chromosome-borne metallo-ß-lactamase (MBL) genes. To determine the diversity and distribution of MBLs BlaBlaB and BlaGOB in this genus, comprehensive bioinformatic screening was applied in 109 available Elizabethkingia genomes. A total of 23 and 32 novel BlaBlaB and BlaGOB variants were found in Elizabethkingia spp., respectively; 12 and 15 clusters were assigned in these BlaBlaB and BlaGOB based on the amino acid identities and phylogenetic studies. Clustering of some variants did not conform to species-specific clades, which indicated potential inter-species dissemination of MBL genes among Elizabethkingia species. Cloning of representative blaBlaB and blaGOB into E. coli DH5α resulted in increased and diverse minimum inhibitory concentrations (MICs) to most ß-lactams, including cephalosporins, carbapenems, and ß-lactams-inhibitors. This study extends the database of class B carbapenemases, emphasizing the diversity of different MBL genes in the genus Elizabethkingia, which may represent potential reservoirs of acquired MBLs.

Joint infection due to Elizabethkingia miricola / Infección articular debida a Elizabethkingia miricola

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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.

Association of Myroides odoratimimus in immunocompromized piglets with post weaning multisystemic wasting syndrome.

AIM: To study the association of opportunistic infection due to Myroides odoratimimus in piglets immunocompromised by porcine circovirus type 2 (PCV2) infection. METHODS AND RESULTS: The clinical samples (n = 101) were analysed bacteriologically. The isolates were identified by their phenotypes and MALDI TOF-MS analysis as Myroides species. The phylogram constructed based on nucleotide sequences of the 16S rRNA gene showed identity (~99%) with the M. odoratimimus isolates. The minimum inhibitory concentration values for antibiotics revealed M. odoratimimus to be resistant against carbapenem, cephalosporins, aminoglycosides and fluoroquinolones. The presence of PCV2 in affected tissue samples was confirmed by amplification of the 565 bp region of ORF2 of the PCV2 genome. The topology of the phylogenetic tree grouped the PCV2 with cluster-2d. CONCLUSIONS: PCV2 being immunosuppressive in nature might have impaired the immunity thereby increasing the susceptibility of immunocompromised piglets to opportunistic pathogens such as M. odoratimimus leading to disease severity and high mortality. The M. odoratimimus isolates were found to be multidrug resistant and evidenced for uncertain clinical relevance and hence could act as hidden source of public health hazard. SIGNIFICANCE AND IMPACT OF THE STUDY: Myroides odoratimimus is a rarely reported human pathogen. We reported the incidence of infection due to seemingly rare isolates of M. odoratimimus causing an outbreak of pneumonia in piglets. This appears, to the best of authors' knowledge, to be the first outbreak due to Myroides recorded in animal clinical cases described in the literature.

Microbiology of Elizabethkingia spp. isolates in hospitalized patients.

An increasing number of infections due to Elisabethkingia spp. have been observed and reported in recent years. Here we report the microbiological aspects of 13 cases with positive cultures for Elizabethkingia spp. from 1998 to 2017. Elizabethkingia isolates were identified using the Vitek 2 Compact 60 (AES software) Gram-Negative Identification test (GNI) card (bioMerieux. Marcy-l'Etoile, France). The MICs were determined using Vitek 2 and interpreted according to CLSI guidelines based on the interpretive MIC breakpoints for Acinetobacter spp. Vitek 2 susceptibility results were confirmed using the manual E-test and the colistin result was confirmed using the broth dilution method. Twelve cases were observed between 2010 and 2017 with four cases in 2017. Of the 2017 samples, three out of four isolates were obtained outside the intensive care units compared to one out of eight samples between 2010 and 2016. There was variable susceptibility to trimethoprim/sulfamethoxazole (58.3%), ciprofloxacin (41.7%), piperacillin/tazobactam and gentamicin (16.6% each), and all were resistant to colistin. There is a high rate of drug resistance and further studies to identify the source of the infection are needed to build up a profile of Elizabethkingia spp. to inform public health policy in this context.

Sulfated steroids of Halichondriidae family sponges - Natural inhibitors of polysaccharide-degrading enzymes of bacterium Formosa algae, inhabiting brown alga Fucus evanescens.

Inhibiting effects of sulfated steroids from marine sponges of Halichondriidae family: halistanol sulfate, topsentiasterol sulfate D and chlorotopsentiasterol sulfate D were investigated on three different types of enzymes degrading polysaccharides of brown algae: endo-1,3-ß-d-glucanase GFA, fucoidan hydrolase FFA2 and bifunctional alginate lyase ALFA3 from marine bacterium Formosa algae KMM 3553T, inhabiting thalli of brown alga Fucus evanescens. This is the first research, devoted to influence of a marine natural compound on three functionally related enzymes that make up the complex of enzymes, necessary to degrade unique carbohydrate components of brown algae. Alginic acid, 1,3-ß-D-glucan (laminaran) and fucoidan jointly constitute practically all carbohydrate biomass of brown algae, so enzymes, able to degrade such polysaccharides, are crucial for digesting brown algae biomass as well as for organisms surviving and proliferating on brown algae thalli. Halistanol sulfate irreversibly inhibited native endo-1,3-ß-D-glucanases of marine mollusks, but reversibly competitively inhibited recombinant endo-1,3-ß-d-glucanase GFA. This fact indicates that there are significant structural differences between the enzymes of practically the same specificity. For alginate lyase and fucoidan hydrolase halistanol sulfate was irreversible inhibitor. Topsentiasterol sulfate D was less active inhibitor whereas chlorotopsentiasterol sulfate D was the strongest inhibitor of enzymes under the study. Chlorotopsentiasterol sulfate D caused 98% irreversible inhibition of GFA. Chlorotopsentiasterol sulfate D also caused reversible and 100% inhibition of ALFA3, which is unusual for reversible inhibitors. Inhibition of FFA2 was complete and irreversible in all cases.