Capsule Gene Cluster Research Articles

Epidemiological cutoff values and genetic antimicrobial resistance of Lactococcus garvieae and L. petauri

Lactococcus garvieae and L. petauri are etiological agents of lactococcosis, which cause significant losses in trout aquaculture at elevated water temperatures. Optimizing antimicrobial dosage regimens and identifying the minimum inhibitory concentration (MIC) are important prerequisites for determining clinical breakpoints and effective antimicrobial use. In the present study, epidemiological cutoff values (ECV) were established for 14 antimicrobials using L. garvieae (n = 32) and L. petauri (n = 78), isolated from rainbow trout farms in Türkiye, Spain, Italy, and Greece. The provisional ECVs were calculated using the ECOFFinder and normalized resistance interpretation (NRI) approaches. Furthermore, gene cassettes, capsule gene clusters, and the presence of fourteen antibiotic resistance genes (ARGs) that encode resistance against seven antimicrobial agents, were evaluated. The calculated ECVs of antimicrobials ranged from 1.25 μg mL−1 for amoxicillin to 80.25 μg mL−1 for streptomycin. The percentages of wild type (WT) and non-wild type (NWT) isolates differed based on the approach, and ECVs calculated based on ECOFFinder approach were generally higher. Among the ARGs, those encoding resistance against tetracycline (tetA), β-lactamase (blaSHV, blaTEM, and blaOXA), amphenicol (floR), and macrolide (ermA) were detected in <10% of the isolates. The genes encoding resistance against quinolones (gyrA and qnrA) and aminoglycosides (strB) were the most detected genes. While 40.9% of the isolates contained Class 1 integron cassette regions, 33.6% of the isolates contained Class 2 integron cassette regions, and 9.1% of the isolates contained both cassette regions. None of the screened isolates were positive for the capsule gene cluster. Significant but weak correlations were found among and between ARGs and MIC distribution of antibiotics. The results of this study could be useful for identifying WT and non-WT isolates, as well as for susceptibility surveillance.

First identification, molecular characterization, and pathogenicity assessment of Lactococcus garvieae isolated from cultured pompano in Taiwan.

Lactococcosis, caused by Lactococcus garvieae, is an acute hemorrhagic septicemia in fish recorded in marine and freshwater aquaculture during the summer months. In 2020-2021, several sea cage Pompano farms recorded sudden fish mortality events. Based on the results of phenotypic and biochemical tests, L. garvieae was predicted to be the cause. PCR with L. garvieae specific primers (pLG1 and pLG2) targeting the 16S rRNA region further confirmed the etiological agent as L. garvieae after amplifying an 1100 base pairs (bp) product. Furthermore, the 16S rRNA sequences of the two representative strains (AOD109-196-2B and AOD110-215-2B) shared 99.81% identity with L. garvieae (GenBank accession number: MT597707.1). The genetic profiles of the strains were classified using pulsed-field gel electrophoresis after digestion with SmaI and ApaI, which clustered our strains under the same pulsotype. Multiplex PCR targeting the capsule gene cluster and serotype-specific PCR collectively showed that the strains were non-capsulated; thus, they belonged to serotype I. An experimental infection was designed to fulfil Koch's postulates by infecting healthy Pompano with case-driven L. garvieae strains (AOD109-196-2B and AOD110-215-2B) with a cumulative mortality of 70%. Overall, L. garvieae infection in Pompano emphasizes the need for better monitoring and control procedures in aquaculture settings.

Extensive Diversity in Escherichia coli Group 3 Capsules Is Driven by Recombination and Plasmid Transfer from Multiple Species.

Bacterial capsules provide protection against environmental challenges and host immunity. Historically, Escherichia coli K serotyping scheme, which relies on the hypervariable capsules, has identified around 80 K forms that fall into four distinct groups. Based on recent work by us and others, we predicted that E. coli capsular diversity is grossly underestimated. We exploited group 3 capsule gene clusters, the best genetically defined capsule group in E. coli, to analyze publicly available E. coli sequences for overlooked capsular diversity within the species. We report the discovery of seven novel group 3 clusters that fall into two distinct subgroups (3A and 3B). The majority of the 3B capsule clusters were found on plasmids, contrary to the defining feature of group 3 capsule genes localizing at the serA locus on the E. coli chromosome. Other new group 3 capsule clusters were derived from ancestral sequences through recombination events between shared genes found within the serotype variable central region 2. Intriguingly, flanking regions 1 and 3, known to be conserved areas among capsule clusters, showed considerable intra-subgroup variation in clusters from the 3B subgroup, containing genes of shared ancestry with other Enterobacteriaceae species. Variation of group 3 kps clusters within dominant E. coli lineages, including multidrug-resistant pathogenic lineages, further supports that E. coli capsules are undergoing rigorous change. Given the pivotal role of capsular polysaccharides in phage predation, our findings raise attention to the need of monitoring kps evolutionary dynamics in pathogenic E. coli in supporting phage therapy. IMPORTANCE Capsular polysaccharides protect pathogenic bacteria against environmental challenges, host immunity, and phage predations. The historical Escherichia coli K typing scheme, which relies on the hypervariable capsular polysaccharide, has identified around 80 different K forms that fall into four distinct groups. Taking advantage of the supposedly compact and genetically well-defined group 3 gene clusters, we analyzed published E. coli sequences to identify seven new gene clusters and revealed an unexpected capsular diversity. Genetic analysis revealed that group 3 gene clusters shared closely related serotype-specific region 2 and were diversified through recombination events and plasmid transfer between multiple Enterobacteriaceae species. Overall, capsular polysaccharides in E. coli are undergoing rigorous change. Given the pivotal role capsules play in phage interactions, this work highlighted the need to monitor the evolutionary dynamics of capsules in pathogenic E. coli for effective phage therapy.

The Origins of ST11 KL64 Klebsiella pneumoniae: a Genome-Based Study.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major severe threat for human health, and its spread is largely driven by a few dominant lineages defined by sequence types (ST) and capsular (KL) types. ST11-KL64 is one such dominant lineage that is particularly common in China but also has a worldwide distribution. However, the population structure and origin of ST11-KL64 K. pneumoniae remain to be determined. We retrieved all K. pneumoniae genomes (n = 13,625, as of June 2022) from NCBI, comprising 730 ST11-KL64 strains. Phylogenomic analysis of core-genome single-nucleotide polymorphisms identified two major clades (I and II) plus an additional singleton of ST11-KL64. We performed dated ancestral reconstruction analysis using BactDating and found that clade I likely emerged in 1989 in Brazil, while clade II emerged around 2008 in eastern China. We then investigated the origin of the two clades and the singleton using a phylogenomic approach combined with analysis of potential recombination regions. We found that ST11-KL64 clade I is likely a hybrid with 91.2% (ca. 4.98 Mb) of the chromosome derived from the ST11-KL15 lineage and 8.8% (483 kb) acquired from ST147-KL64. In contrast, ST11-KL64 clade II was derived from ST11-KL47 with swapping of a 157-kb region (3% of the chromosome) containing the capsule gene cluster with clonal complex 1764 (CC1764)-KL64. The singleton also evolved from ST11-KL47 but with swapping of a 126-kb region with ST11-KL64 clade I. In conclusion, ST11-KL64 is a heterogenous lineage comprising two major clades and a singleton with different origins that emerged in different countries at different time points. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a severe threat globally and is associated with increased lengths of hospital stay and high mortality in affected patients. The spread of CRKP is largely driven by a few dominant lineages, including ST11-KL64, the dominant type in China with a worldwide distribution. Here, we tested the hypothesis that ST11-KL64 K. pneumoniae is a single genomic lineage by performing a genome-based study. However, we found that ST11-KL64 comprises a singleton and two major clades, which emerged in different countries in different years. In particular, the two clades and the singleton have different origins and acquired the KL64 capsule gene cluster from various sources. Our study underscores that the chromosomal region containing the capsule gene cluster is a hot spot of recombination in K. pneumoniae. This represents a major evolutionary mechanism employed by some bacteria for rapid evolution with novel clades that accommodate stress for survival.

Clonality of Lactococcus garvieae isolated from rainbow trout cultured in Spain: A molecular, immunological, and proteomic approach

This study reports the characterization of a collection of 98 strains of L. garvieae using phenotypic, antigenic, genetic, proteomic, and chemotaxonomic methods, as well as the study of their virulence. Molecular (RAPD and REP-PCR) and proteomic (MALDI-TOF-MS) techniques allowed the separation of strains of L. garvieae isolated from human and bovine from those strains of L. garvieae isolated from the aquatic environment. On the other hand, PCR typing methods showed that the L. garvieae strains tested did not belong to serotype II and did not carry the capsule gene cluster described in the genome or plasmid of Japanese L. garvieae strains. The different clonal patterns observed in L. garvieae using RAPD and REP-PCR (7 profiles) and MALDI-TOF (six patterns) were closely related to the isolation source of L. garvieae strains but not with the virulence of the strains. In addition, two protein antigenic variants (W1 and W2), differing in reactivity in the range of 35 to 70 KDa, were detected using rabbit and rainbow trout antisera. Both antisera detected two highly immunogenic protein bands of 30 and 45 KDa. These results could contribute to a better understanding of the pathogenesis of lactococcosis and could help in the selection of targets for the development of effective vaccines.

Molecular epidemiological characterization in mucoid-type Streptococcus pneumoniae isolates obtained from invasive pneumococcal disease patients in Japan

IntroductionStreptococcus pneumoniae with a mucoid-type capsule is associated with invasive pneumococcal diseases (IPDs). Despite the introduction of pneumococcal vaccines, IPDs caused by mucoid-type isolates are still prevalent. The present study aimed to characterize mucoid-type S. pneumoniae isolated from IPD patients throughout Japan in 2017 (post-vaccination era). MethodsA total of 225 mucoid-type isolates were collected. The serotype, antimicrobial susceptibility, and multilocus sequence type of these isolates were determined. ResultsThe prevalence of IPDs caused by mucoid-type isolates was high in adults, especially in the elderly (≥65 years of age), and prognosis in these patients was significantly poor. Of the mucoid-type isolates, the predominant serotype was serotype 3 (84.4%), and the remaining were serotypes 37 (15.1%) and 8 (0.4%). Antimicrobial susceptibility showed that most mucoid isolates exhibited the penicillin-intermediate resistant S. pneumoniae genotype (gPISP). However, the serotype 3 isolate exhibited the penicillin-resistant S. pneumoniae genotype (gPRSP). This gPRSP isolate was classified into ST166, which is related to serotypes 9 V and 11 strains. Sequence analysis of the capsule-coding regions and its flanking regions indicated that recombination occurred upstream and downstream of the capsule-coding region, suggesting that gPRSP (serotype 9 V/ST166) obtaining the type-3 capsule gene cluster resulted in the emergence of gPRSP (serotype 3/ST166). ConclusionsOur findings indicated that IPDs caused by mucoid-type S. pneumoniae are still a serious concern and mucoid-type S. pneumoniae with novel phenotype could emerge via capsular switching in response to environmental changes such as introduction of vaccines and improper use of antimicrobial agents.

Emerging bacterial fish pathogen Lactococcus garvieae RTCLI04, isolated from rainbow trout (Oncorhynchus mykiss): Genomic features and comparative genomics

Lactococcus garvieae is one of the emerging zoonotic bacterial pathogen, causes fatal hemorrhagic septicemia in cultured fish species, animals and humans, worldwide. Here, we report the genomic features of whole-genome sequence (WGS) of L. garvieae strain RTCLI04, recovered from lower intestine of farmed rainbow trout, Oncorhynchus mykiss in the northwest Himalayan region India. The genome of L. garvieae RTCLI04 is a single circular chromosome of 2,054,885 base pairs (bp), which encodes 1993 proteins and has G + C content of 39%. The bioinformatics analysis of WGS of RTCLI04, confirmed the presence of 51 tRNAs genes (including two pseudogenes), six rRNAs genes (four genes for 5S rRNA; one gene for 16S rRNA and one gene for 23S rRNA), five virulent domains, and twenty eight different genetic pathways. A Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) finder tool indicates that three different CRISPR and one cas system with common spacer was present in the genome of L. garvieae RTCLI04. Pan-genome analysis of RTCLI04 and all the other reference L. garvieae strains shows that pan-genome of this bacterium consisted of 2239 putative protein-coding genes in which 1850 genes are core gene, 389 genes are dispensable gene, and 221 genes are unique to RTCLI04. L. garvieae RTCLI04 lacks genomic island of 16.5 Kb capsule gene cluster. In addition, 39 virulence-associated genes (VAGs) including hly1,-2,-3; PavA, PsaA; eno; LPxTG containing surface proteins 1, 2, 3 and 4; pgm, sod and 29 antimicrobial resistant genes (ARGs) including mefE (clindamycin), srmB (lincomycin), dfrA26 (trimethoprim), gyrB (nalidixic acid), arr-3 (rifampin), otrB (tetracycline), aac(6)-Ic (tobramycin), IrgB (penicillin), mecA (oxacillin), vanRB (vancomycin) and mfpA (fluoroquinolone) were also predicted in the genome of L. garvieae RTCLI04. Our study provides new insight into understanding the virulence mechanism, antimicrobial resistance, and development of effective therapeutic measures against L. garvieae during a disease outbreak in aquaculture.

K units of the K8 and K54 capsular polysaccharides produced by Acinetobacter baumannii BAL 097 and RCH52 have the same structure but contain different di-N-acyl derivatives of legionaminic acid and are linked differently

The K8 and K54 capsular polysaccharides were isolated from Acinetobacter baumannii BAL 097 and RCH52, respectively, and studied by sugar analysis, partial acid hydrolysis and selective solvolysis with CF3CO2H in the presence of 2-methyl-1-propanol, along with 1D and 2D 1H and 13C NMR spectroscopy. The following structures of related branched tetrasaccharide repeats (K units) of the polysaccharides were established: [Display omitted] where Leg indicates 5,7-diamino-3,5,7,9-tetradeoxy-d-glycero-d-galacto-non-2-ulosonic (legionaminic) acid and R indicates (R)-3-hydroxybutanoyl or acetyl in the ratio ~2.5:1. The sequences of the KL8 and KL54 capsule gene clusters were closely related. The difference in the acyl group at O-7 on the sidechain legionaminic acid is due to differences in two genes in the legionaminic acid biosynthesis cluster. The wzy genes encoding the K unit polymerases are also different and make different linkages between the K units, allowing the first sugar of both K units to be identified as d-GlcpNAc. The shared Gtr20 glycosyltransferase, also encoded in KL63, forms the α-l-FucpNAc-(1 → 3)-d-GlcpNAc linkage, and Gtr19 was predicted to form α-d-GalpNAc-(1 → 3)-l-FucpNAc. Gtr18 from KL8 is 75% identical to Gtr108 from KL54 and both would link the Leg derivative to d-GalpNAc. Hence the genes present at the K locus were consistent with the composition and structures of the K8 and K54 capsular polysaccharides.

The K90 capsular polysaccharide produced by Acinetobacter baumannii LUH5553 contains di-N-acetylpseudaminic acid and is structurally related to the K7 polysaccharide from A. baumannii LUH5533

Acinetobacter baumannii isolate LUH5553 carries the KL90 capsule gene cluster, which includes genes for three glycosyltransferases (Gtrs) and the ItrA3 initiating transferase, as well as a set of genes for synthesis of a higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic (di-N-acetylpseudaminic) acid (Pse5Ac7Ac). The K90 capsular polysaccharide (CPS) has a tetrasaccharide repeat (K90 unit), which begins with d-GlcpNAc and contains Pse5Ac7Ac. The higher sugar was cleaved by mild acid hydrolysis of the CPS, and structures of the initial and modified polysaccharides were established by 1D and 2D 1H and 13C NMR spectroscopy. K90 contains α-d-Galp-(1 → 6)-d-GlcpNAc and α-d-GlcpNAc-(1 → 3)-d-GlcpNAc fragments, and formation of these glycosidic linkages is catalysed respectively by Gtr14 and Gtr15. The gtr14 and gtr15 genes occur in several A. baumannii KL gene clusters, including KL5 and KL7 that carry itrA2 rather than itrA3. As ItrA2 introduces d-GalpNAc rather than d-GlcpNAc as the first monosaccharide, Gtr15 can transfer d-GlcpNAc to either of these amino sugars, suggesting that this enzyme has relaxed specificity. Consequently, the third, novel glycosyltransferase, Gtr163, forms the β-(2 → 3) linkage between Pse5Ac7Ac and d-Galp. Wzy polymerases encoded by KL90 and KL7 are 54% identical and form the same linkage between the K units to give branched polysaccharides with the same main chain but different disaccharide side chains, β-Pse5Ac7Ac-(2 → 3)-d-Galp in K90 and α-Leg5Ac7Ac-(2 → 6)-d-Galp in K7.

Phenotypic and molecular analysis of nontypeable Group B streptococci: identification of cps2a and hybrid cps2a/cps5 Group B streptococcal capsule gene clusters

The Group B streptococcus (GBS) can express a capsular polysaccharide (CPS). There are ten recognized CPSs (Ia, Ib, and II–IX). A GBS isolate is considered nontypeable (NT) when CPS cannot be identified as one of ten types. Two groups of GBS NT isolates were studied, isolates without surface sialic acid (sia(−)) and isolates with surface sialic acid (sia(+)). The first objective was to characterize NT sia(−) isolates that failed CPS identification by an immunodiffusion antisera typing assay and a RT-PCR capsule typing assay. NT sia(−) isolates were characterized by assaying phenotypic changes and identifying covR/S mutations that may potentially have a role in the altered phenotypes. The second objective was to characterize NT sia(+) isolates that failed to identify as one of the ten CPS types by an immundiffusion antisera-based typing assay and a RT-PCR capsule typing assay yet expressed capsule. Fifteen NT sia(−) isolates displayed increased β hemolysis/orange pigmentation, decreased CAMP activity, inability to form biofilm, and susceptibility to phagocytosis by human blood. DNA sequence analysis of the covR/S genes in the sia(−) isolates found mutations in 14 of 15 isolates assayed. These mutations in the covR/S genes may potentially contribute to lack of expression of phenotypic traits assayed in vitro. For the three NT sia(+) isolates, whole-genome sequence analyses identified two isolates with cps gene clusters identical to the recently described and uncommon CPSIIa type. The third isolate possessed a hybrid cluster containing cps genes for both CPSIIa and CPSV suggesting recombination between these two gene clusters.

Structure of the K82 Capsular Polysaccharide from Acinetobacter baumannii LUH5534 Containing a d-Galactose 4,6-Pyruvic Acid Acetal.

Type K82 capsular polysaccharide (CPS) was isolated from Acinetobacter baumannii LUH5534. The structure of a linear tetrasaccharide repeating unit of the CPS was established by sugar analysis along with one- and two-dimensional 1H and 13C NMR spectroscopy. Proteins encoded by the KL82 capsule gene cluster in the genome of LUH5534 were assigned to roles in the synthesis of the K82 CPS. In particular, functions were assigned to two new glycosyltransferases (Gtr152 and Gtr153) and a novel pyruvyltransferase, Ptr5, responsible for the synthesis of d-galactose 4,6-(R)-pyruvic acid acetal.

Description and Comparative Genomics of Macrococcus caseolyticus subsp. hominis subsp. nov., Macrococcus goetzii sp. nov., Macrococcus epidermidis sp. nov., and Macrococcus bohemicus sp. nov., Novel Macrococci From Human Clinical Material With Virulence Potential and Suspected Uptake of Foreign DNA by Natural Transformation.

The genus Macrococcus is a close relative of the genus Staphylococcus. Whilst staphylococci are widespread as human pathogens, macrococci have not yet been reported from human clinical specimens. Here we investigated Gram-positive and catalase-positive cocci recovered from human clinical material and identified as Macrococcus sp. by a polyphasic taxonomic approach and by comparative genomics. Relevant phenotypic, genotypic and chemotaxonomic methods divided the analyzed strains into two separate clusters within the genus Macrococcus. Comparative genomics of four representative strains revealed enormous genome structural plasticity among the studied isolates. We hypothesize that high genomic variability is due to the presence of a com operon, which plays a key role in the natural transformation of bacilli and streptococci. The possible uptake of exogenous DNA by macrococci can contribute to a different mechanism of evolution from staphylococci, where phage-mediated horizontal gene transfer predominates. The described macrococcal genomes harbor novel plasmids, genomic islands and islets, as well as prophages. Capsule gene clusters, intracellular protease, and a fibronectin-binding protein enabling opportunistic pathogenesis were found in all four strains. Furthermore, the presence of a CRISPR-Cas system with 90 spacers in one of the sequenced genomes corresponds with the need to limit the burden of foreign DNA. The highly dynamic genomes could serve as a platform for the exchange of virulence and resistance factors, as was described for the methicillin resistance gene, which was found on the novel composite SCCmec-like element containing a unique mec gene complex that is considered to be one of the missing links in SCC evolution. The phenotypic, genotypic, chemotaxonomic and genomic results demonstrated that the analyzed strains represent one novel subspecies and three novel species of the genus Macrococcus, for which the names Macrococcus caseolyticus subsp. hominis subsp. nov. (type strain CCM 7927T = DSM 103682T), Macrococcus goetzii sp. nov. (type strain CCM 4927T = DSM 103683T), Macrococcus epidermidis sp. nov. (type strain CCM 7099T = DSM 103681T), and Macrococcus bohemicus sp. nov. (type strain CCM 7100T = DSM 103680T) are proposed. Moreover, a formal description of Macrococcus caseolyticus subsp. caseolyticus subsp. nov. and an emended description of the genus Macrococcus are provided.

Variation in the Integration Site for Capsule Gene Cluster in the Genome among Strains of &lt;i&gt;Lactococcus garvieae&lt;/i&gt;

Variation in the Integration Site for Capsule Gene Cluster in the Genome among Strains of &lt;i&gt;Lactococcus garvieae&lt;/i&gt;

Three tandem promoters, together with IHF, regulate growth phase dependent expression of the Escherichia coli kps capsule gene cluster

In this study we characterise three tandem promoters (PR1-1, PR1-2 and PR1-3) within the PR1 regulatory region of the Escherichia coli kps capsule gene cluster. Transcription from promoter PR1-2 was dependent on the activity of the upstream promoter PR1-1, which activated PR1-2 via transcription coupled DNA supercoiling. During growth at 37 °C a temporal pattern of transcription from all three promoters was observed with maximum transcriptional activity evident during mid-exponential phase followed by a sharp decrease in activity as the cells enter stationary phase. The growth phase dependent transcription was regulated by Integration Host Factor (IHF), which bound within the PR1 region to repress transcription from PR1-2 and PR1-3. This pattern of transcription was mirrored by growth phase dependent expression of the K1 capsule. Overall these data reveal a complex pattern of transcriptional regulation for an important virulence factor with IHF playing a role in regulating growth phase expression.

Genetic Characteristics and Multiple-PCR Development for Capsular Identification of Specific Serotypes of Campylobacter jejuni.

The polysaccharide capsule (CPS) of Campylobacter jejuni is a virulence factor linked to cell surface carbohydrate diversity which mainly determines the serotypes. Thirty-four CPS gene cluster structures have been published and some of them can be distinguished by multiple-PCR. Penner serotypes HS1/44c, HS2, HS4c, HS19, HS23/36c and HS41 are markers for Guillain—Barré syndrome (GBS). The capsules may contribute to GBS susceptibility. Analysis of 18 CPS loci revealed high gene content diversity and a mosaic nature of the capsule loci, which are possibly due to gene gain/loss events, and demonstrated a high degree of conservation of genes within serotypes/serotype complexes. A method of multiple-PCR was developed to distinguish five specific serotypes and three GBS-related serotypes. Primers specific for each capsule type were designed on the basis of paralogs or a unique DNA region of the CPS locus. The multiple-PCR can distinguish the eight serotypes in two PCRs with sensitivity and specificity of 100% using 227 strains of known Penner type. The multiple-PCR method will help to distinguish serotypes simply and rapidly.

Related structures of neutral capsular polysaccharides of Acinetobacter baumannii isolates that carry related capsule gene clusters KL43, KL47, and KL88

Capsular polysaccharides were recovered from four Acinetobacter baumannii isolates, and the following related structures of oligosaccharide repeating units were established by sugar analyses along with 1D and 2D 1H and 13C NMR spectroscopy:NIPH 60 and LUH5544 (K43) [Display omitted] NIPH 601 (K47)The K locus for capsule biosynthesis in the genome sequences available for NIPH 60 and LUH5544, designated KL43, was found to be related to gene clusters KL47 in NIPH 601 and KL88 in LUH5548. The three clusters share most gene content differing in only a small portion that includes an additional glycosyltransferase genes in KL47 and KL88, as well as genes encoding distinct Wzy polymerases that were found to form the same α-d-GlcpNAc-(1 → 6)-α-d-GlcpNAc linkage in K43 and K47.

K19 capsular polysaccharide of Acinetobacter baumannii is produced via a Wzy polymerase encoded in a small genomic island rather than the KL19 capsule gene cluster

Polymerization of the oligosaccharides (K units) of complex capsular polysaccharides (CPSs) requires a Wzy polymerase, which is usually encoded in the gene cluster that directs K unit synthesis. Here, a gene cluster at the Acinetobacter K locus (KL) that lacks a wzy gene, KL19, was found in Acinetobacter baumannii ST111 isolates 28 and RBH2 recovered from hospitals in the Russian Federation and Australia, respectively. However, these isolates produced long-chain capsule, and a wzy gene was found in a 6.1 kb genomic island (GI) located adjacent to the cpn60 gene. The GI also includes an acetyltransferase gene, atr25, which is interrupted by an insertion sequence (IS) in RBH2. The capsule structure from both strains was →3)-α-d-GalpNAc-(1→4)-α-d-GalpNAcA-(1→3)-β-d-QuipNAc4NAc-(1→, determined using NMR spectroscopy. Biosynthesis of the K unit was inferred to be initiated with QuiNAc4NAc, and hence the Wzy forms the β-(1→3) linkage between QuipNAc4NAc and GalpNAc. The GalpNAc residue is 6-O-acetylated in isolate 28 only, showing that atr25 is responsible for this acetylation. The same GI with or without an IS in atr25 was found in draft genomes of other KL19 isolates, as well as ones carrying a closely related CPS gene cluster, KL39, which differs from KL19 only in a gene for an acyltransferase in the QuiNAc4NR synthesis pathway. Isolates carrying a KL1 variant with the wzy and atr genes each interrupted by an ISAba125 also have this GI. To our knowledge, this study is the first report of genes involved in capsule biosynthesis normally found at the KL located elsewhere in A. baumannii genomes.

Detection of putative virulence genes of Lactococcus garvieae.

Lactococcus garvieae is the causative agent of lactococcosis and has been isolated from a wide variety of animals. In the present study, 34 strains of L. garvieae isolated from fish from different sources and locations were tested for the presence or absence of the following putative virulence genes: a capsule gene cluster (CGC), hemolysins 1, 2, and 3 (hly1, -2, -3), NADH oxidase, superoxide dismutase (sod), phosphoglucomutase (pgm), adhesin Pav (adhPav), adhesin PsaA (adhPsaA), enolase (eno), LPxTG-containing surface proteins 1, 2, 3, and 4 (LPxTG-1, LPxTG-2, LPxTG-3, LPxTG-4; where LPxTG means Leu-Pro-any-Thr-Gly), adhesin clusters 1 and 2 (adhCI, adhCII), and adhesin (adh). To determine the presence of the CGC, we developed a multiplex PCR. All strains of L. garvieae had the hly1, -2, -3, NADH oxidase, pgm, adhPav, LPxTG-2, LPxTG-3, sod, eno, adhPsaA, adhCII, and adhCII genes, while only the Lg2 strain contained the CGC. The virulent Lg2 strain contained all 17 virulent genes. All Turkish, Spanish, Italian, and French strains did not contain the CGC. The multiplex PCR assay was useful for the detection of the CGC genes. In conclusion, the CGC is not the only virulent factor in L. garvieae because strains that lack the CGC are virulent to rainbow trout. Single genes also might not be responsible for the virulence of L. garvieae.

Phylogenetic study of clonal complex (CC)198 capsule null locus (cnl) genomes: A distinctive group within the species Neisseria meningitidis

Capsule null locus (cnl) strains, one type of specific unencapsulated Neisseria spp., only have regions D and E of the capsule gene cluster which encodes the genes for capsule biosynthesis, modification, and transportation. Compared with encapsulated strains, regions A and C of cnl strains have been replaced by 113 or 114bp conserved non-coding sequences. Cnl strains include multiple clonal complexes (CC). According to previous studies, CC198 is the major clonal lineage in both cnl patients and healthy cnl carriers. We hypothesized that CC198 possesses different genome characteristics compared with other cnl strains. In this study, we obtained the draft genomes of two CC198 strains from healthy carriers. Using 75071 single nucleotide polymorphisms located in 1163 core genes, we constructed the phylogenetic relationships between a batch of representative Neisseria meningitidis genomes. CC198 and CC1136 clustered together, but apart from other N. meningitidis strains including CC53. We also aligned the sequences of genes located in regions D and E of the capsule gene locus from encapsulated and unencapsulated strains. A number of possible recombination events were identified in the galE and tex genes between different serogroups of encapsulated N. meningitidis and CC53 strains, especially in tex. In contrast, there is almost no recombination in N. meningitidis CC198 strains. These results showed that CC198 belongs to a phylogenetically distinct group within the species N. meningitidis, which may be directly derived from the cnl-type ancestor of N. meningitidis. The encapsulated strains may acquire other necessary genes for capsule formation by horizontal transfer.

Draft Genome Sequence of Human-Pathogenic Lactococcus garvieae LG-ilsanpaik-gs201105 That Caused Acute Acalculous Cholecystitis.

Lactococcus garvieae, which is generally known as a marine and freshwater fish pathogen, is now considered to be an emerging zoonotic pathogen in both human and veterinary medicine. In recent years, we have reported the infection of L. garvieae LG-ilsanpaik-gs201105 in the gallbladder of an old fisherman. In this study, we present the draft genome sequence of L. garvieae LG-ilsanpaik-gs201105, with a total genome size of 1,960,261 bp in 53 contigs and a 38.1% average G+C content. Interestingly, the capsule gene cluster, which was known as one of the crucial virulence factors in L. garvieae, was not detected in our isolate. This is the first genome sequence of human-pathogenic L. garvieae, which caused acute acalculous cholecystitis.