Capsular Polysaccharide Serotypes Research Articles

Influence of the polysaccharide capsule on virulence and fitness of Klebsiella pneumoniae

IntroductionThe capsular polysaccharide (CPS) of pathogenic bacteria is a critical virulence factor, often evading phagocytosis by host immune cells, while also interfering with the contact of the pathogen with host cells and contributing to biofilm formation. Klebsiella pneumoniae, a Gram-negative human pathogen associated with high antimicrobial resistances, produces 77 CPS serotypes. The CPS masks proteinaceous factors but also protects K. pneumoniae from uptake by host phagocytic cells and activation of the complement system. In addition to nosocomial, urinary tract and bloodstream infections or pneumonia hypervirulent strains have a highly mucoid phenotype and can cause soft tissue infections, liver abscesses, and meningitis as well. The CPS is therefore crucial for both escaping detection by the immune system and enhancing the virulence potential.MethodsIn this study, we generated a non-encapsulated mutant (Kpn2146∆wza) to observe how the CPS interferes with K. pneumoniae adhesion, survival in blood, and invasiveness in an experimental infection model.ResultsInfection of A549 lung epithelial cells showed similar adherence levels for the wild-type and non-capsulated strain, while our data showed a moderately higher internalization of Kpn2146Δwza when compared to the wild-type. In whole blood killing assays, we demonstrate that the K. pneumoniae capsule is essential for survival in human blood, protecting K. pneumoniae against recognition and clearance by the human immune system, as well as complement-mediated opsonization and killing. The non-encapsulated mutant, in contrast, was unable to survive in either whole blood or human plasma. Infections of Galleria mellonella larvae showed a significantly decreased virulence potential of the CPS-deficient mutant.DiscussionIn conclusion, our data indicate a crucial role of CPS in vivo.

Long, Synthetic Staphylococcus aureus Type 8 Capsular Oligosaccharides Reveal Structural Epitopes for Effective Immune Recognition.

Staphylococcus aureus is a Gram-positive bacterium that is responsible for severe nosocomial infections. The rise of multidrug-resistant strains, which can pose significant health threats, prompts the development of new treatment interventions, and much attention has been directed at the development of prophylactic and therapeutic vaccination strategies. Capsular polysaccharides (CPs) are key protective elements of the S. aureus cell wall and have been proposed as promising candidate antigens. Thirteen different CP serotypes have been identified to date, of which types 5 and 8 are the most prominent. CP8 is composed of trisaccharide repeating units that are built up from an N-acetyl-4-O-acetyl-β-d-mannosaminuronic acid, that carries a C-4-O-acetyl, an N-acetyl-α-d-fucosamine, and an N-acetyl-α-l-fucosamine. Synthetic oligosaccharides are valuable tools to unravel the immunogenicity of bacterial oligosaccharides at the molecular level. However, the rare monosaccharides, cis-glycosidic linkages, and O-acetylation represent significant challenges for the synthesis of CP8 fragments. Here the stereoselective assembly of well-defined CP8 fragments, comprising a trimer, hexamer, nonamer, and dodecamer, is presented. This is the first time that fragments larger than a single repeating trisaccharide, which has been proven to be insufficient for antigenic activity, have been assembled. Structural studies have revealed a linear conformation for the oligosaccharides, with each trisaccharide repeat tilted ∼90° with respect to the flanking repeats, which is stabilized by the acetyl groups that prevent rotation around the glycosidic linkages. The N-acetyl groups in each repeating unit point in the same direction, generating a hydrophobic flank in the trisaccharide repeats. We applied the oligomers to generate model glycoconjugate vaccine modalities, which we then used to raise anti-CP8 antibodies. The antibody interaction and immunization studies have revealed a clear length dependent structure-activity relationship for the oligosaccharides, with an oligosaccharide of at least three repeating units required for an adequate immune response.

Development and validation of a 6-plex Luminex-based assay for measuring human serum antibodies to group B streptococcus capsular polysaccharides

ABSTRACT Six serotypes (Ia, Ib, II, III, IV, and V) cause nearly all group B streptococcal (GBS) disease globally. Capsular polysaccharide (CPS) conjugate vaccines aim to prevent GBS disease, however, licensure of a vaccine would depend on a standardized serological assay for measuring anti-CPS IgG responses. A multiplex direct Luminex-based immunoassay (dLIA) has been developed to simultaneously measure the concentration of serum IgG specific for the six prevalent GBS CPS serotypes. Assay validation was performed using serum samples obtained from human subjects vaccinated with an investigational 6-valent GBS CPS conjugate vaccine. Results for the assay are expressed as IgG concentrations (µg/mL) using a human serum reference standard composed of pooled sera from vaccinated subjects. The lower limits of quantitation (LLOQ) for all serotypes covered in the 6-plex GBS IgG dLIA fell within the range of 0.002-0.022 µg/mL IgG. Taken together, the 6-plex GBS IgG dLIA platform is specific for the six GBS serotypes included in Pfizer’s investigational vaccine, has a wide dilution adjusted assay range, and is precise (<18.5% relative standard deviation) for all serotypes, and, therefore, is suitable for quantitatively measuring vaccine-induced or naturally acquired serotype-specific anti-CPS IgG responses against GBS.

Interlaboratory comparison of a multiplex immunoassay that measures human serum IgG antibodies against six-group B streptococcus polysaccharides

ABSTRACT Measurement of IgG antibodies against group B streptococcus (GBS) capsular polysaccharide (CPS) by use of a standardized and internationally accepted multiplex immunoassay is important for the evaluation of candidate maternal GBS vaccines in order to compare results across studies. A standardized assay is also required if serocorrelates of protection against invasive GBS disease are to be established in infant sera for the six predominant GBS serotypes since it would permit the comparison of results across the six serotypes. We undertook an interlaboratory study across five laboratories that used standardized assay reagents and protocols with a panel of 44 human sera to measure IgG antibodies against GBS CPS serotypes Ia, Ib, II, III, IV, and V. The within-laboratory intermediate precision, which included factors like the lot of coated beads, laboratory analyst, and day, was generally below 20% relative standard deviation (RSD) for all six serotypes, across all five laboratories. The cross-laboratory reproducibility was < 25% RSD for all six serotypes, which demonstrated the consistency of results across the different laboratories. Additionally, anti-CPS IgG concentrations for the 44-member human serum panel were established. The results of this study showed assay robustness and that the resultant anti-CPS IgG concentrations were reproducible across laboratories for the six GBS CPS serotypes when the standardized assay was used.

Epidemiological and Antimicrobial Resistant Patterns, and Molecular Mechanisms of Carbapenem-Resistant Klebsiella pneumoniae Infections in ICU Patients.

To study the epidemiological and antimicrobial resistant patterns, clinical characteristics and risk factors of critically ill patients infected with carbapenem-resistant Klebsiella pneumoniae (CRKP) from intensive care units (ICUs). The potential molecular mechanisms of antimicrobial resistance and virulence of CRKP were investigated through evaluation of associated genes. Totally, 201 ICU patients infected with K. pneumoniae were recruited from January 2020 through January 2021. K. pneumoniae strains were collected from diverse clinical specimens and identified by microbial cultures and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Antimicrobial resistance was measured through broth micro-dilution or Kirby-Bauer assays. The carbapenemase-, virulence-, and capsular serotype-associated genes of CRKP were individually detected by PCR and sequencing. Demographic and clinical profiles were acquired from hospital databases to evaluate the correlation of CRKP infection incidence with clinical risk factors. Of the 201 K. pneumoniae strains, CRKP accounted for 41.29%. Seasonal bias existed in local prevalence of CRKP infections. CRKP strains mounted significantly strong resistance against major antimicrobial agents except ceftazidime-avibactam, tigecycline and minocycline. Recent exposure to certain antibiotics and prior treatment with invasive interventions were prone to increase CRKP infection risks with worsened infectious outcomes. The local top carbapenemase-encoding and virulence-associated genes of CRKP were blaKPC and irp2, respectively. Nearly half of CRKP isolates harbored a capsular polysaccharide serotype of K14.K64 (wzi-64) which preferentially emerged in the cohort with worse outcomes of infection. Featured epidemiology and typical clinical characteristics existed extensively in K. pneumoniae infections among ICU patients. The CRKP cohort exhibited substantially high antimicrobial resistance. Distinctive carbapenemase-, virulence-, and serotype-associated genes were intensively involved in the spread and pathogenesis of CRKP. These findings supported careful management of critically ill patients potentially infected with virulent CRKP in the ICUs.

Antibody enhanced HPLC for serotype-specific quantitation of polysaccharides in pneumococcal conjugate vaccine

Bacterial infection remains as one of the major healthcare issues, despite significant scientific and medical progress in this field. Infection by Streptococcus Pneumoniae (S. Pneumoniae) can cause pneumonia and other serious infectious diseases, such as bacteremia, sinusitis and meningitis. The pneumococcal capsular polysaccharides (CPS) that constitute the outermost layer of the bacterial cell are the main immunogens and protect the pathogen from host defense mechanisms. Over 90 pneumococcal CPS serotypes have been identified, among which more than 30 can cause invasive pneumococcal diseases that could lead to morbidity and mortality. Multivalent pneumococcal vaccines have been developed to prevent diseases caused by S. Pneumoniae. These vaccines employ either purified pneumococcal CPSs or protein conjugates of these CPSs to generate antigen-specific immune responses for patient protection. Serotype-specific quantitation of these polysaccharides (Ps) antigen species are required for vaccine clinical dosage, product release and quality control. Herein, we have developed an antibody-enhanced high-performance liquid chromatography (HPLC) assay for serotype-specific quantitation of the polysaccharide contents in multivalent pneumococcal conjugate vaccines (PCVs). A fluorescence-labeled multiplex assay format has also been developed. This work laid the foundation for a serotype-specific antigen assay format that could play an important role for future vaccine research and development.

Sex Influences Age-Related Changes in Natural Antibodies and CD5+ B-1 Cells.

Natural Abs are primarily produced by B-1 cells and are essential for protection against Streptococcus pneumoniae The incidence and mortality rate for pneumococcal infection increases dramatically after age 65, disproportionately affecting males in both human and murine systems. To date, there is a significant gap in our understanding of the relationship among sex, aging, natural IgM efficacy, and the natural IgM repertoire. Our investigation demonstrates that the protective capacity of serum IgM against pneumococcal infection is maintained in IgM obtained from aged female mice but absent in IgM from aged male mice. To understand this difference in protective capacity, we examined serum Ig, discovering that the protective change was not associated with shifts in levels of phosphorylcholine (PC)- or pneumococcal capsular polysaccharide serotype 3-specific IgM. Interestingly, we observed that aged females have an increase in the total number of CD5+ B-1 cells, higher serum IL-5 levels, and a larger percentage of aged female CD5+ B-1 cells that express CD86 as compared with aged males. Furthermore, single-cell IgM repertoire analysis from peritoneal PC+, splenic PC+, and bone marrow CD5+ B-1 cell subsets demonstrated greater diversity with age and a higher level of germline status in female mice than previously observed in studies of aged male mice. Aged female CD5+ B-1 cells also expressed higher levels of transcripts associated with cell activity and self-renewal, such as Nanog and Hmga2 Taken together, these data indicate that females maintain a more diverse and active CD5+ B-1 cell pool and natural IgM repertoire, which has implications for sex-related susceptibility to infection and disease.

Liposomal Encapsulation of Polysaccharides (LEPS) as an Effective Vaccine Strategy to Protect Aged Hosts Against S. pneumoniae Infection.

Despite the availability of licensed vaccines, pneumococcal disease caused by the bacteria Streptococcus pneumoniae (pneumococcus), remains a serious infectious disease threat globally. Disease manifestations include pneumonia, bacteremia, and meningitis, resulting in over a million deaths annually. Pneumococcal disease disproportionally impacts older adults aged ≥65 years. Interventions are complicated through a combination of complex disease progression and 100 different bacterial capsular polysaccharide serotypes. This has made it challenging to develop a broad vaccine against S. pneumoniae, with current options utilizing capsular polysaccharides as the primary antigenic content. However, current vaccines are substantially less effective in protecting the elderly. We previously developed a Liposomal Encapsulation of Polysaccharides (LEPS) vaccine platform, designed around limitations of current pneumococcal vaccines, that allowed the non-covalent coupling of polysaccharide and protein antigen content and protected young hosts against pneumococcal infection in murine models. In this study, we modified the formulation to make it more economical and tested the novel LEPS vaccine in aged hosts. We found that in young mice (2–3 months), LEPS elicited comparable responses to the pneumococcal conjugate vaccine Prevnar-13. Further, LEPS immunization of old mice (18–22 months) induced comparable antibody levels and improved antibody function compared to Prevnar-13. Importantly, LEPS protected old mice against both invasive and lung localized pneumococcal infections. In summary, LEPS is an alternative and effective vaccine strategy that protects aged hosts against different manifestations of pneumococcal disease.

A Randomized Safety Study of 13-Valent Pneumococcal Conjugated Vaccine and Post-Vaccination Immune Response to &lt;em&gt; Streptococcus pneumoniae &lt;/em&gt; Serotypes in Adult Patients with Bronchial Asthma and Chronic Obstructive Pulmonary Disease

Background. Vaccination against pneumococcal infection is one of the priorities in improving the quality of treatment and prevention measures in adults with various pathologies. The effectiveness of vaccination is directly related to the individuals ability to form an adequate specific immunity. Aims the aim of the study was to assess the level of post-vaccination antibodies to capsular polysaccharides of S. pneumoniae in adult patients with bronchial asthma (BA) or chronic obstructive pulmonary disease (COPD) after administration of 13-valent conjugated pneumococcal vaccine (PCV13). Materials and methods. The ELISA method was used to determine the level of IgG antibodies to 12 capsular polysaccharides serotypes 1, 3, 4, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F of S. pneumoniae that are part of PCV13, and 2 serotypes 9N, 15B that are not part of the vaccines using research test systems developed on the basis of the I.I. Mechnikov Research Institute of Vaccines and Sera. Groups of adult patients 32 patients with BA and 33 with COPD who received basic treatment according to accepted international standards. The comparison group consists of 20 healthy patients who do not have comorbidities. In patients, vaccination was performed outside the acute period of the disease using PCV13. Results. Vaccination of PCV13 patients with BA and COPD does not lead to the development of exacerbations of the underlying disease, while unusual symptoms in the post-vaccination period, provided for by the drugs instructions, can rarely develop. A comparative analysis of changes in IgG antibodies conducted after 6 weeks relative to the initial level of IgG antibodies to S. pneumoniae capsular polysaccharides in adult patients and healthy vaccinated PCV13 showed the same increase in specific antibodies to 12 serotypes of pneumococcus. The difference was found only in relation to IgG antibodies to a mixture of polysaccharides included in the PCV13 vaccine, which were registered higher in patients with BA and COPD in the post-vaccination period (p 0.001) than in healthy patients. Conclusions. PCV13 vaccination of patients with BA and COPD is safe and is accompanied by the synthesis of IgG antibodies to capsular polysaccharides serotypes of S. pneumoniae similarly to the healthy group.

Development of a pneumococcal conjugate vaccine based on chemical conjugation of polysaccharide serotype 6B to PspA

The use of conjugate vaccines remains an effective intervention to prevent pneumococcal diseases. In order to expand vaccine coverage, the inclusion of pneumococcal proteins as carriers is a propitious alternative that has been explored over the past few years. In this study, pneumococcal surface protein A (PspA) clade 1, family 1 (PspA1) and clade 3, family 2 (PspA3) were used as carrier proteins for pneumococcal capsular polysaccharide serotype 6B (Ps6B). Employing an improved reductive amination chemistry, 50% of Ps6B was incorporated to each protein, PspA1 and PspA3. The effect of chemical modifications in Ps6B and PspA was assessed by an antigenicity assay and circular dichroism, respectively. Fragmentation and oxidation decreased the antigenicity of Ps6B while conjugation improved antigenicity. In the same manner, introduction of adipic acid dihydrazide (ADH) reduced PspA secondary structure content, which was partially restored after conjugation. Immunization of Ps6B-PspA1 and Ps6B-PspA3 conjugates in mice induced specific IgG antibodies against the Ps6B and the protein; and anti-PspA antibodies had functional activity against two pneumococcal strains with different serotypes. These results suggest that chemical coupling between Ps6B and PspA did not affect antigenic epitopes and support the further development of PspA as a carrier protein in pneumococcal conjugate vaccines to provide broader protection.

Prevalence of Capsular Serotype, Pilus Island Distribution, and Antibiotic Resistance in Pediatric and Adult Invasive Group B Streptococcus Isolates: Data From a Nationwide Prospective Surveillance Study in Germany.

For neonates, group B Streptococcus is life threatening. Current prevention strategies remain insufficient, especially for cases of late-onset sepsis, where intrapartum antibiotic prophylaxis has demonstrated no benefit. One promising approach is the vaccination of pregnant women, which offers protective immunity via transplacental transmission of neutralizing antibodies. Our nationwide, prospective surveillance study aimed to characterize the prevalence of pilus antigen, capsular polysaccharide serotypes, and antibiotic resistance from invasive GBS infections in neonates and compare these results with those from children and adults in Germany. Our study includes 173 neonatal isolates of a total of 450 reported cases during the study period (incidence: 0.34/1000 live births), in addition to 2 pediatric and 803 adult isolates. The comparison between neonatal and adult isolates reveals age-dependent differences in capsular serotype and pilus type distribution and differences in antibiotic resistance patterns.

Group B streptococcus vaccines: one step further

In The Lancet Infectious Diseases, Judith Absalon and colleagues1 report on a phase 1/2 clinical trial evaluating the safety, tolerability, and immunogenicity of a vaccine composed of capsular polysaccharide conjugated to cross-reactive material 197 (CRM197) and directed against six capsular polysaccharide serotypes of group B streptococcus (GBS). These serotypes (Ia, Ib, II, III, IV, and V) account for the vast majority of isolates causing invasive GBS disease worldwide—in neonates as well as in adults.

Vaccines to Protect Older Adults against Pneumococcal Disease.

Determining the optimal vaccination strategy for the protection of the elderly population against pneumococcal disease remains a challenge. Older adults are, second to young infants, most susceptible to become colonized and invaded by Streptococcus pneumoniae, causing serious disease such as bacteremic pneumonia, sepsis, and meningitis. In an era with increasing antimicrobial resistance and the growing susceptible population of aged adults, S. pneumoniae is a priority bacterial pathogen for research and development of new intervention strategies. While elderly indirectly profit from infant immunization programs through herd immunity, vaccination of older age groups can offer more direct protection. Two types of pneumococcal vaccines for adults, both based on capsular polysaccharide serotypes, are currently available but have limitations, such as short-lived protection or limited serotype coverage. These vaccine limitations and the biological aging of the immune system call for novel vaccination strategies for the older adults. Here, we highlight how host-pathogen interactions, immune protection, and effectiveness of currently available vaccines shift with increasing age, and how future pneumococcal vaccine strategies could be tailored for the elderly.

Staphylococcus aureus capsular polysaccharides: a structural and synthetic perspective.

The S. aureus bacterium is surrounded by capsular polysaccharides. These capsular polysaccharides are important in the pathogenesis of staphylococcal infection. There are 11 serotypes of capsular polysaccharides that have been identified, and a majority of strains express capsular polysaccharides type 5 (CP5) or 8 (CP8). The main focus of this review is to describe recent advances in the area of the chemical synthesis of monosaccharide components of S. aureus CP, oligosaccharide assembly and functionalization. Chemical routes to obtain oligosaccharides derived from CP1, CP5 and CP8 represent a compendium of modern classics of the total synthesis of challenging glycan sequences.

Persistence of Immune Responses Through 36 Months in Healthy Adults After Vaccination With a Novel Staphylococcus aureus 4-Antigen Vaccine (SA4Ag).

Background Staphylococcus aureus causes serious health care– and community-associated disease, requiring improved preventive measures such as vaccines. The investigational S. aureus 4-antigen vaccine (SA4Ag), comprising capsular polysaccharide serotypes 5 and 8 (CP5 and CP8) conjugated to CRM197, recombinant mutant clumping factor A (rmClfA), and recombinant manganese transporter protein C (rP305A or rMntC), was well tolerated, inducing robust functional immune responses to all 4 antigens through 12 months postvaccination. This is a serological extension study through 36 months postvaccination.MethodsIn 2 previous studies, healthy adults received SA4Ag, SA3Ag (without rMntC), or placebo; serology was also assessed at ~24 and ~36 months postvaccination. Functional immune responses (antibody responses that facilitate killing of S. aureus or neutralize S. aureus virulence mechanisms) were assessed with opsonophagocytic activity killing assays (CP5 or CP8) and a fibrinogen-binding inhibition assay (ClfA). A competitive Luminex immunoassay assessed ClfA and rMntC responses. Adverse events within 48 hours of blood draw were recorded.ResultsFour hundred forty subjects (18–64 years old, 255; 65–85 years old, 185) were enrolled. At 24 and 36 months postvaccination, subjects receiving SA4Ag had substantially higher geometric mean titers (GMTs) for CP5, CP8, and ClfA vs baseline; geometric mean fold rises (GMFRs) from baseline to month 36 were 2.7–8.1. For rMntC, 36-month GMTs declined from peak levels but remained above baseline for all SA4Ag groups; GMFRs from baseline to month 36 were 1.8 and 1.5 in the younger and older cohorts, respectively.ConclusionsPersistent functional immune responses to S. aureus antigens were observed through 36 months in healthy adults.ClinicalTrials.govNCT01643941 and NCT01364571.

2618. Determination of the Chemical Structure of a Novel Pneumococcal Serotype, 39X

Background Streptococcus pneumoniae produces a diverse group of capsular polysaccharides (serotypes) that are important for the virulence of the organism and for the serotype-specific prevention of pneumococcal disease. As a consequence of widespread PCV usage and pneumococcal genome plasticity, the distribution of pneumococcal serotypes is changing with an increase in non-vaccine serotypes post-vaccine introduction, a phenomenon known as serotype replacement. Recently, a potentially novel serotype was described and was provisionally named as serotype 39X. Genetic studies suggest that this novel serotype may be a hybrid of serotypes 6C and 39/10A.MethodsThree 39X strains with the distinct serological and genetic description of the cps biosynthetic loci were obtained from the Global Pneumococcal Sequencing project (www.pneumogen.net). Capsular polysaccharide from one (Camb.853/MNZ2334) of the 39X strains was purified by sequential ethanol precipitation followed by ion-exchange chromatography. To detect polysaccharide fractions during purification, an inhibition ELISA assay was developed using factor serum 10d. The chemical structure of the 39X repeating unit was determined using one-dimensional (1D) and 2D nuclear magnetic resonance (NMR).ResultsAll three isolates were confirmed to have the 39X genotype by PCR amplification and sequencing of the 39X specific region (wciN6c-wcrO-wcrC39) of the cps locus. (Figure 1). The 39X capsule PS fractions were detected during purification and pooled for structural studies (Figure 2). 1D-NMR for 39X showed it to be chemically distinct (Figure 3). 2D-NMR studies revealed that five of the sugar residues in 39X PS are identical to those in 39 PS, except the acetylation (Figure 4). The remaining part of the structure is being investigated.ConclusionThe 39X capsular PS has a distinct chemical structure in addition to its distinct serologic and genetic properties. Given that serotype 39X is a new serotype, it becomes the 100th pneumococcal serotype. The chemical structure supports the genetic depiction of serotype evolution as a result of recombination between well-characterized and unrelated serotypes. Structural elucidation of the 39X capsule PS will help facilitate our understanding of serotype replacement and vaccine development. Disclosures All authors: No reported disclosures.

Genomic characterization of multidrug-resistant ESBL-producing Klebsiella pneumoniae isolated from a Ghanaian teaching hospital

This study delineated the clonal lineages, antibiotic resistome and plasmid replicon types in multidrug-resistant K. pneumoniae isolates from a teaching hospital in Ghana. Identification and antibiotic susceptibility testing were done using the MALDI-TOF MS and Vitek-2 automated system. Genomic DNA extraction was carried out using the NucliSens easyMAG® (BioMérieux) kits and the DNA was subjected to whole genome sequencing (WGS) using the Illumina MiSeq platform. Of the 200 isolates obtained, 37 were identified as K. pneumoniae of which 9 were resistant to all second and third-generation cephalosporins. These 9 isolates selected for further genomic analysis were characterized by the presence of 8 diverse sequence types (STs), capsular polysaccharide serotypes (K types and wzi allelic types) and multiple genes encoding resistance to β-lactams (blaCTX-M-15, blaSHV-11,blaTEM-1B,blaOXA-1), aminoglycosides (aac(3)-IIa, strB, strA, aadA16), fluoroquinolones/quinolones (qnrB66, oqxA, oqxB) and other antibiotic classes. Resistance genes were associated with plasmids, predominantly IncFIB(K) and ColRNAI. Multiple and diverse mutations in quinolone resistance-determining regions of gyrA (S83Y, D87A) and parC (S80I, N304S) in isolates resistant to ciprofloxacin (MIC ≥ 4 mg/mL) were found. Global phylogenomic analysis affirmed the diverse clonal clustering and origin of these isolates. The varied clonal clusters and resistome identified in the multidrug-resistant K. pneumoniae isolates is a major threat to the management of infections in Ghana. The molecular characterization of antibiotic resistance is thus imperative to inform strategies for containment.

Whole genome sequencing of NDM-1-producing serotype K1 ST23 hypervirulent Klebsiella pneumoniae in China.

The emergence and spread of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) is causing worldwide concern, whereas NDM-producing hvKP is still rare. Here we report the complete genome sequence characteristics of an NDM-1-producing ST23 type clinical hvKP in PR China. Capsular polysaccharide serotyping was performed by PCR. The complete genome sequence of isolate 3214 was obtained using both the Illumina Hiseq platform and Pacbio RS platform. Multilocus sequence type was identified by submitting the genome sequence to mlst 2.0 and the antimicrobial resistance genes and plasmid replicons were identified using ResFinder and PlasmidFinder, respectively. Transferability of the blaNDM-1-bearing plasmid was determined by conjugation experiment, S1 pulsed-field gel electrophoresis and Southern hybridization. Isolate 3214 was classified to ST23 and belonged to the K1 capsular serotype. The isolate's total genome size was 6 171 644 bp with a G+C content of 56.39 %, consisting of a 5 448 209 bp chromosome and seven plasmids. The resistome included 18 types of antibiotic resistance genes. Fourteen resistance genes including blaNDM-1 and blaCTX-M-14 were located on plasmids and five also including blaCTX-M-14 were in the chromosome. Plasmid pNDM_3214 carrying blaNDM-1 harboured six types of resistance genes surrounded by insertion sequences and was conjugative. The worldwide pLVPK-like virulence plasmid harbouring rmpA2 and rmpA was also found in this isolate. This study provides basic information of phenotypic and genomic features of ST23 CR-hvKP isolate 3214. Our data highlights the potential risk of spread of NDM-1-producing ST23 hvKP.

Genetic Diversity of Streptococcus agalactiae from Multiple Host Species in China

Background: Streptococcus agalactiae is a serious zoonotic pathogen that causes disease in humans and other animals worldwide. Objectives: Due to the increasing prevalence of S. agalactiae, as well as the transmission risk of S. agalactiae from animals to human, it was important to identify the serotype diversity and molecular typing population structure of S. agalactiae isolated from human, bovine, rabbit, and fish. Methods: Capsular polysaccharide serotypes (CPS), multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE) technologies were used to compare the genetic diversity of 56 S. agalactiae isolates from human, rabbit, bovine, and fish sources in China. Results: We found that two serotypes (Ia, III) existed, with serotype Ia being the most common (85.7%). The 56 isolates were assigned to nine sequence types (STs) and seven clonal complexes (CCs) using MLST. Themost common ST was ST891 (35.7%). The ST19 and ST103 were found in two different isolates, while the remaining STs were found in one isolate. A total of 18 PFGE clusters were identified, however, only cluster P was found in both human and bovine isolates. Conclusions: Our findings suggest that serotypes, STs and PFGE, had no obvious correlation with particular host species. However, the similarity of genotypes of S. agalactiae isolated from different host species, speculating cross-infection possibility between them at genetic level.

Microbiological and clinical characteristics of Group B Streptococcus isolates causing materno-neonatal infections: high prevalence of CC17/PI-1 and PI-2b sublineage in neonatal infections.

Group B Streptococcus (GBS) is one of the major pathogens in severe materno-neonatal infections. We aimed to describe the clinical and molecular characteristics of GBS isolates causing infections in 45 maternal and 50 neonatal subjects, collected from eight healthcare centres in mainland China over the period 2010- 2017. The phenotypic and genotypic features of the GBS isolates, including capsular polysaccharide (cps) serotypes, pilus island (PI) genes and antibiotic resistance profiles and genes, were characterized by both conventional and molecular methods. The clonal relationship between these strains was investigated using multilocus sequence typing (MLST). Of the 95 isolates, the predominant serotypes were III (51, 53.7 %), V (13, 13.7 %) and Ib (13, 13.7 %). All GBS strains carried at least one pilus island, with 32.6 % carrying PI-2b and 67.4 % PI-2a, singly or in combination. The most frequently-detected pilus island pattern was the combination of PI-1 and PI-2a, accounting for 56.8 % (54 isolates), followed by PI-1 combined with PI-2b (28, 29.5 %), PI-2a (10, 10.5 %) and PI-2b (3, 3.2 %). The strains were classified into 17 individual sequence types, and further clustered into six clonal complexes (CCs). A high prevalence of CC17/PI-1 and PI-2b (17, 34.0 %) was detected in 50 GBS isolates causing neonatal infections. No strain was resistant to penicillin, ampicillin, ceftriaxone or vancomycin, whereas 78.9, 76.8 and 81.5 % were resistant to erythromycin, clindamycin and tetracycline, respectively. Our study highlights the high genotypic diversity of GBS strains causing materno-neonatal infections, and the CC17/PI-1 and PI-2b sublineages should be noted in neonatal infections.