Comparative resistomics analysis of multidrug-resistant Chryseobacteria.

Chryseobacteria consists of important human pathogens that can cause a myriad of nosocomial infections. We isolated four multidrug-resistant Chryseobacterium bacteria from activated sludge collected at domestic wastewater treatment facilities in the New York Metropolitan area. Their genomes were sequenced with Nanopore technology and used for a comprehensive resistomics comparison with 211 Chryseobacterium genomes available in the public databases. A majority of Chryseobacteria harbor 3 or more antibiotic resistance genes (ARGs) with the potential to confer resistance to at least two types of commonly prescribed antimicrobials. The most abundant ARGs, including ß-lactam class A (blaCGA-1 and blaCIA) and class B (blaCGB-1 and blaIND) and aminoglycoside (ranA and ranB), are considered potentially intrinsic in Chryseobacteria. Notably, we reported a new resistance cluster consisting of a chloramphenicol acetyltransferase gene catB11, a tetracycline resistance gene tetX, and two mobile genetic elements (MGEs), IS91 family transposase and XerD recombinase. Both catB11 and tetX are statistically enriched in clinical isolates as compared to those with environmental origins. In addition, two other ARGs encoding aminoglycoside adenylyltransferase (aadS) and the small multidrug resistance pump (abeS), respectively, are found co-located with MGEs encoding recombinases (e.g., RecA and XerD) or transposases, suggesting their high transmissibility among Chryseobacteria and across the Bacteroidota phylum, particularly those with high pathogenicity. High resistance to different classes of ß-lactam, as well as other commonly used antimicrobials (i.e., kanamycin, gentamicin, and chloramphenicol), was confirmed and assessed using our isolates to determine their minimum inhibitory concentrations. Collectively, though the majority of ARGs in Chryseobacteria are intrinsic, the discovery of a new resistance cluster and the co-existence of several ARGs and MGEs corroborate interspecies and intergenera transfer, which may accelerate their dissemination in clinical environments and complicate efforts to combat bacterial infections.

Chryseobacterium gotjawalense sp. nov. Isolated from Soil in the Volcanic Forest Gotjawal, Jeju Island.

Strain wdc7T, a rod-shaped bacterium, was isolated from soil in the Gotjawal Forest on Jeju Island, South Korea. Strain wdc7T was Gram stain-negative, facultatively anaerobic, catalase- and oxidase positive, yellow pigmented, and non-flagellated. It grew at 4-37 °C and pH 5.0-8.0 in 0-3% (w/v) NaCl. 16S rRNA gene sequencing analysis revealed that strain wdc7T belonged to the genus Chryseobacterium and was most closely related to Chryseobacterium salivictor NBC 122T, with a sequence similarity of 98.51%. Menaquinone 6 was the sole respiratory quinone, and C15:0 anteiso, C15:0 iso, and summed feature 9 were the major fatty acids. The genome length was 3.30 Mbp, with a 37% G + C content. Average amino acid identity, average nucleotide identity, and digital DNA-DNA relatedness between strain wdc7T and C. salivictor NBC 122T were 93.52%, 92.80%, and 49.7%, respectively. Digital genomic and polyphasic analyses showed that strain wdc7T likely represented a new species of the genus Chryseobacterium. We proposed the name Chryseobacterium gotjawalense sp. nov., with wdc7T (= KCTC 92440T = JCM 35602T) as the type strain.

Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity.

The CRISPR system is an adaptive immune system found in prokaryotes that defends host cells against the invasion of foreign DNA1. As part of the ongoing struggle between phages and the bacterial immune system, the CRISPR system has evolved into various types, each with distinct functionalities2. Type II Cas9 is the most extensively studied of these systems and has diverse subtypes. It remains uncertain whether members of this family can evolve additional mechanisms to counter viral invasions3,4. Here we identify 2,062 complete Cas9 loci, predict the structures of their associated proteins and reveal three structural growth trajectories for type II-C Cas9. We found that novel associated genes (NAGs) tended to be present within the loci of larger II-C Cas9s. Further investigation revealed that CbCas9 from Chryseobacterium species contains a novel ß-REC2 domain, and forms a heterotetrameric complex with an NAG-encoded CRISPR-Cas-system-promoting (pro-CRISPR) protein of II-C Cas9 (PcrIIC1). The CbCas9-PcrIIC1 complex exhibits enhanced DNA binding and cleavage activity, broader compatibility for protospacer adjacent motif sequences, increased tolerance for mismatches and improved anti-phage immunity, compared with stand-alone CbCas9. Overall, our work sheds light on the diversity and 'growth evolutionary' trajectories of II-C Cas9 proteins at the structural level, and identifies many NAGs-such as PcrIIC1, which serves as a pro-CRISPR factor to enhance CRISPR-mediated immunity.

A Duplex PCR Assay for Rapid Detection of Klebsiella pneumoniae and Chryseobacterium in Large Yellow Croaker Fish.

Both Klebsiella pneumoniae and Chryseobacterium cause an increasing number of diseases in fish, resulting in great economic losses in aquaculture. In addition, the disease infected with Klebsiella pneumoniae or Chryseobacterium exhibited the similar clinical symptoms in aquatic animals. However, there is no effective means for the simultaneous detection of co-infection and discrimination them for these two pathogens. Here, we developed a duplex polymerase chain reaction (PCR) method based on the outer membrane protein A (ompA) gene of Klebsiella pneumoniae and Chryseobacterium. The specificity and validity of the designed primers were confirmed experimentally using simplex PCR. The expected amplicons for Klebsiella pneumoniae and Chryseobacterium had a size of 663 and 1404 bp, respectively. The optimal condition for duplex PCR were determined to encompass a primer concentration of 0.5 µM and annealing temperature of 57°C. This method was analytical specific with no amplification being observed from the genomic DNA of Escherichia coli, Vibrio harveyi, Pseudomonas plecoglossicida, Aeromonas hydrophila and Acinetobacter johnsonii. The limit of detection was estimated to be 20 fg of genomic DNA for Chryseobacterium and 200 fg for Klebsiella pneumoniae, or 100 colony-forming units (CFU) of bacterial cells in both cases. The duplex PCR was capable of simultaneously amplifying target fragments from genomic DNA extracted from the bacteria and fish liver. For practical validation of the method, 20 diseased fish were collected from farms, among which 4 samples were PCR-positive for Klebsiella pneumoniae and Chryseobacterium. The duplex PCR method developed here is time-saving, specific, convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of Klebsiella pneumoniae and Chryseobacterium in the field of aquaculture.

Whole-genome sequencing and analysis of Chryseobacterium arthrosphaerae from Rana nigromaculata.

Chryseobacterium arthrosphaerae strain FS91703 was isolated from Rana nigromaculata in our previous study. To investigate the genomic characteristics, pathogenicity-related genes, antimicrobial resistance, and phylogenetic relationship of this strain, PacBio RS II and Illumina HiSeq 2000 platforms were used for the whole genome sequencing. The genome size of strain FS91703 was 5,435,691 bp and GC content was 37.78%. A total of 4,951 coding genes were predicted; 99 potential virulence factors homologs were identified. Analysis of antibiotic resistance genes revealed that strain FS91703 harbored 10 antibiotic resistance genes in 6 categories and 2 multidrug-resistant efflux pump genes, including adeG and farA. Strain FS91703 was sensitive to ß-lactam combination drugs, cephem, monobactam and carbapenems, intermediately resistant to phenicol, and resistant to penicillin, aminoglycosides, tetracycline, fluoroquinolones, and folate pathway inhibitors. Phylogenetic analysis revealed that strain FS91703 and C. arthrosphaerae CC-VM-7T were on the same branch of the phylogenetic tree based on 16 S rRNA; the ANI value between them was 96.99%; and the DDH values were 80.2, 72.2 and 81.6% by three default calculation formulae. These results suggested that strain FS91703 was a species of C. arthrosphaerae. Pan-genome analysis showed FS91703 had 566 unique genes compared with 13 other C. arthrosphaerae strains, and had a distant phylogenetic relationship with the other C. arthrosphaerae strains of the same branch in phylogenetic tree based on orthologous genes. The results of this study suggest that strain FS91703 is a multidrug-resistant and highly virulent bacterium, that differs from other C. arthrosphaerae strains at the genomic level. The knowledge about the genomic characteristics and antimicrobial resistance of strain FS91703 provides valuable insights into this rare species, as well as guidance for the treatment of the disease caused by FS91703 in Rana nigromaculata.

Parallel evolution of alternate morphotypes of Chryseobacterium gleum during experimental evolution with Caenorhabditis elegans.

Microbial evolution within polymicrobial communities is a complex process. Here, we report within-species diversification within multispecies microbial communities during experimental evolution with the nematode Caenorhabditis elegans. We describe morphological diversity in the target species Chryseobacterium gleum, which developed a novel colony morphotype in a small number of replicate communities. Alternate morphotypes coexisted with original morphotypes in communities, as well as in single-species experiments using evolved isolates. We found that the original and alternate morphotypes differed in motility and in spatial expansion in the presence of C. elegans. This study provides insight into the emergence and maintenance of intraspecies diversity in the context of microbial communities.

Elucidating doxycycline biotransformation mechanism by Chryseobacterium sp. WX1: Multi-omics insights.

Doxycycline (DOX) represents a second-generation tetracycline antibiotic that persists as a challenging-to-degrade contaminant in environmental compartments. Despite its ubiquity, scant literature exists on bacteria proficient in DOX degradation. This study marked a substantial advancement in this field by isolating Chryseobacterium sp. WX1 from an activated sludge enrichment culture, showcasing its unprecedented ability to completely degrade 50 mg/L of DOX within 44 h. Throughout the degradation process, seven biotransformation products were identified, revealing a complex pathway that began with the hydroxylation of DOX, followed by a series of transformations. Employing an integrated multi-omics approach alongside in vitro heterologous expression assays, our study distinctly identified the tetX gene as a critical facilitator of DOX hydroxylation. Proteomic analyses further pinpointed the enzymes postulated to mediate the downstream modifications of DOX hydroxylation derivatives. The elucidated degradation pathway encompassed several key biological processes, such as the microbial transmembrane transport of DOX and its intermediates, the orchestration of enzyme synthesis for transformation, energy metabolism, and other gene-regulated biological directives. This study provides the first insight into the adaptive biotransformation strategies of Chryseobacterium under DOX-induced stress, highlighting the potential applications of this strain to augment DOX removal in wastewater treatment systems containing high concentrations of DOX.

First case of Chryseobacterium gallinarum bloodstream infection: a diagnostic and therapeutic challenge for an emerging pathogen.

Chryseobacterium spp. belongs to the Flavobacteriaceae family and is a rod-shaped gram-negative, glucose non-fermenting, non-motile bacterium ubiquitous in the environment. In humans, Chryseobacterium may be responsible for infections such as urinary tract infections (UTI) and ventriculitis with a pathogenic burden increasing in recent years. Chryseobacterium gallinarum was isolated for the first time in 2014 in a pharyngeal scrape sample of chicken and, until now, only one case of human UTI has been described in a pregnant 20-year-old Indian patient. Herein, we report the first case of bloodstream infection caused by C. gallinarum in a 67-year-old female burn patient, correctly identified by 16S-rRNA sequencing and successfully treated with cefepime and fosfomycin.

Genome analysis deciphered Chryseobacterium indicum is a distinct species associated with freshwater pufferfish.

A bacterium, strain PS-8T of the genus Chryseobacterium, was isolated from the skin of freshwater pufferfish (Tetraodon cutcutia). Strain PS-8T is a Gram-negative, aerobic, non-motile, and rod-shaped bacterium. Colonies appear in yellowish-orange colors. The major cellular fatty acids were C15:0 iso, C17:0 iso 3OH, C15:0 iso 3OH, and C11:0 anteiso. The predominant polar lipids were phosphatidylethanolamine and amino lipids. The genome size is 4.83 Mb. The G + C content was 35.6%. The in silico dDDH homology, ANI, and AAI were below the cutoff value, 70% and 95% to 96%, respectively, suggesting that strain PS-8T represents a defined species. The phylogenetic tree based on core and the non-recombinant genes showed the strain PS-8T clustered with Chryseobacterium gambrini DSM 18014T. Genome-wide analysis decodes several virulence factors of the genus Chryseobacterium, including genes for adherence, biofilm and stability, proliferation, resistance to immune response, and host-defense evasion system. The cladogram of the virulence genes showed a phylogenetic relationship among the Chryseobacterium species. Knowledge of the association of Chryseobacterium with freshwater pufferfish adds a new ecological niche to this bacterium.

Draft genome sequence of Chryseobacterium limigenitum SUR2T (LMG 28734T) isolated from dehydrated sludge

ABSTRACT The type strain SUR2 of the novel species Chryseobacterium limigenitum was isolated from a dehydrated sludge of the municipal sewage treatment plant in Dogoše near Maribor in Slovenia. The draft genome, with 60 contigs, 4,697,725 bp, 34.4% of G+C content, was obtained using the Illumina HiSeq 2500-1 platform. Joint Genome Institute Microbial Genome Annotation Pipeline (MGAP v.4) has identified 4322 protein-coding sequences including resistance genes against arsenic and other heavy metals. In addition, a subclass B3 metallo-β-lactamase, which confers resistance to penicillins, cephalosporins and carbapenems, was also present in the genome. The genome sequence provides important information regarding bioremediation potential and pathogenic properties of this newly identified species.

Polyphasic characterization of bacteria obtained from upland rice cultivated in Cerrado soil

ABSTRACT This work aimed to characterize 20 isolates obtained from upland rice plants, based on phenotypic (morphology, enzymatic activity, inorganic phosphate solubilization, carbon source use, antagonism), genotypic assays (16S rRNA sequencing) and plant growth promotion. Results showed a great morphological, metabolic and genetic variability among bacterial isolates. All isolates showed positive activity for catalase and protease enzymes and, 90% of the isolates showed positive activity for amylase, catalase and, nitrogenase. All isolates were able to metabolize sucrose and malic acid in contrast with mannitol, which was metabolized only by one isolate. For the other carbon sources, we observed a great variability in its use by the isolates. Most isolates showed antibiosis against Rhizoctonia solani (75%) and Sclerotinia sclerotiorum (55%) and, 50% of them showed antibiosis against both pathogens. Six isolates showed simultaneous ability of antibiosis, inorganic phosphate solubilization and protease activity. Based on phylogenetic analysis of the 16S rRNA gene all the isolates belong to Bacillus genus. Under greenhouse conditions, two isolates (S4 and S22) improved to about 24%, 25%, 30% and 31% the Total N, leaf area, shoot dry weight and root dry weight, respectively, of rice plants, indicating that they should be tested for this ability under field conditions.