Capsular Type Research Articles

Bacterial Spectrum of Acute Otitis Media in Bulgarian Children during the 10-Valent Pneumococcal Conjugate Vaccine Era

Abstract Objective The 10-valent pneumococcal conjugate vaccine (PCV10) was introduced in Bulgaria for universal childhood vaccination in 2010. The objective of this study was to describe bacterial pathogens responsible for acute otitis media (AOM) in children in the era of routine PCV10 immunization. Materials and Methods Middle ear fluid (MEF)/otorrhea or nasopharyngeal specimens were collected between May 2012 and April 2017 from 425 children aged < 12 years diagnosed with AOM; 71.5% of them were vaccinated. Capsular types of Streptococcus pneumoniae and Haemophilus influenzae and antimicrobial nonsusceptibility were determined. Results Among 240 children with “severe” AOM, the studied specimens were MEF/otorrhea, and a total of 132 (55.0%) children were culture-positive. The most frequently identified bacteria were S. pyоgenes (31.1%), followed by Staphylococcus aureus (21.2%), S. pneumoniae (20.4%), and nontypeable H. influenzae (12.1%). Among 185 nasopharyngeal specimens obtained from children at the onset of a “mild” AOM episode, 67.0% were culture-positive for otopathogens. The most common pathogens were S. pneumoniae (41.9%), followed by H. influenzae (25.8%), Moraxella catarrhalis (23.4%), and S. pyоgenes (14.5%), alone or in combinations. Among children with pneumococcal AOM (79), PCV10 serotypes (VTs) were 21.5%. A high prevalence (50%) of nonvaccine serotypes 3 (14), 19A (11), and 6C (7) was found among vaccinated children. Rates of nonsusceptibility of S. pneumoniae to penicillin, amoxicillin and erythromycin, and of multidrug resistance, were 51.2, 10.1, 51.2, and 51.2, respectively. The rate of ampicillin-non-susceptibility in H. influenzae was 25%. All M. catarrhalis isolates were β-lactamase producers, and 32.2% of S. pyogenes were erythromycin-resistant. Conclusion Following implementation of PCV10, S. pyogenes was the most prevalent pathogen in children with “severe” AOM. Numbers of S. pneumoniae recovered from MEF/otorrhea significantly decreased, as did the overall proportion of VTs among AOM patients. Streptococcus pneumoniae, H. influenzae, and M. catarrhalis were the most commonly found pathogens in the nasopharynx of children with less severe AOM episodes.

Identification of Two Depolymerases From Phage IME205 and Their Antivirulent Functions on K47 Capsule of Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) pose a significant threat to global public health. In present research, a total of 80 CRKP strains belonging to ST11 were collected with 70% (56 of 80 isolates) expressing a K47 capsular type. Thus, it is significant to prevent and control infections caused by these bacteria. Capsule depolymerases could degrade bacterial surface polysaccharides to reduce their virulence and expose bacteria to host immune attack. Previous studies have demonstrated the potential of phage-encoded depolymerases as antivirulent agents in treating CRKP infections in vitro and in vivo. Here, two capsule depolymerases (Dpo42 and Dpo43) derived from phage IME205 were expressed and characterized. Although both depolymerases act on strains with a capsular serotype K47, they are active against different subsets of strains, indicating subtle differences in capsule composition that exist within this serotype. The host range of phage IME205 matched to the sum of specificity range of Dpo42 and Dpo43. These two enzymes maintained stable activity in a relatively broad range of pH levels (pH 5.0–8.0 for Dpo42 and pH 4.0–8.0 for Dpo43) and temperatures (20–70°C). Besides, both Dpo42 and Dpo43 could make host bacteria fully susceptible to the killing effect of serum complement and display no hemolytic activity to erythrocytes. In summary, capsule depolymerases are promising antivirulent agents to combat CRKP infections.

Surface-enhanced Raman scattering as a discrimination method of Streptococcus spp. and alternative approach for identifying capsular types of S. pneumoniae isolates.

The surface-enhanced Raman spectroscopy (SERS) is a method known for its effectiveness in detecting and identifying microorganisms, that was employed to differentiate various bacterial strains both at genus and species level. In this work, we have examined five species belonging to Streptococcus genus, namely S. pneumoniae, S. suis, S. pseudopneumoniae, S. oralis, and S. mitis. Additionally, we conducted SERS experiments on ten S. pneumoniae strains, representing different capsular types. In all of cases we obtained unique SERS signals being spectroscopic fingerprints of bacterial strains tested. Moreover, the principal component analysis (PCA) was performed in order to prove that the spectra of all studied strains can be well separated into five (in case of streptococcal strains) or ten (in case of pneumococcal serotypes) groups. In both investigated situations, the separation at the level of 95% was achieved, proving that SERS-PCA-based method can be used for reliable and fast identification of different strains belonging to the Streptococcus genus, including encapsulated pneumococcal isolates.

New Strategy Is Needed to Prevent Pneumococcal Meningitis.

Background:Polysaccharide conjugate vaccines (PCVs) target the pneumococcal capsular types that most commonly cause fatal pneumonia and sepsis. Because these types were eliminated by the vaccines, it became apparent that in immunized populations, most invasive pneumococcal diseases, including bacteremia, sepsis and complicated pneumonia, were greatly reduced. However, the protective effects of PCVs against another invasive disease, meningitis, has shown much less or no decrease in disease incidence.Methods:References were identified through searches of PubMed for articles published from January 1930 to the present by use of specific search terms. Relevant articles were also identified through searches in Google and Google Scholar. Relevant references cited in those articles were also reviewed.Results:Even in the presence of the PCVs, meningitis rates in children have been reported globally to be as high as 13 per 100,000 annually. Widespread use of vaccines resulted in the emergence of a broad diversity of replacement non-PCV type strains. These strains generally failed to cause sepsis, but caused meningitis of comparable severity and levels similar to, or in excess of, prior pneumococcal meningitis rates. This is probably because these non-PCV type strains do not survive well in the blood, therefore possibly entering the brain through nonhematogenous routes.Conclusions:Because virtually all cases of pneumococcal meningitis lead to either permanent neurologic sequelae or death, it would be well worth the effort to develop a new vaccine capable of preventing pneumococcal meningitis regardless of capsular type. Such a vaccine would need to protect against colonization with most, if not all, pneumococci.

Phenotypic and genotypic characterization of Streptococcus spp. isolated from tilapia (Oreochromis spp.) cultured in river-based cage and earthen ponds in Northern Thailand.

Streptococcus spp. are major pathogenic bacteria associated with massive mortality in tilapia. This study investigated the phenotypic and genotypic characterization of Streptococcus agalactiae (GBS) and Streptococcus iniae (S.iniae) isolated from tilapia in river-based floating cage and earthen pond farms in northern Thailand. Isolates were identified by biochemical and molecular analyses. Capsular typing, enterobacterial repetitive intergenic consensus polymerase chain reaction and multilocus sequence typing were performed to investigate the genetic relatedness. Six and one isolates were confirmed as GBS and S.iniae, respectively. All Streptococcus spp. isolates were obtained from 4 river-based cage farms (4/33), while samples collected from earthen pond farms (N=28) were negative for streptococcosis. All GBS with serotype Ⅲ and sequence type (ST) 283 was observed. The β-haemolytic GBS isolates were resistant to five antimicrobials, while the S.iniae was susceptible to all antimicrobials. This study indicates both GBS and S.iniae are the major bacterial pathogens responsible for streptococcosis infection in farmed tilapia of northern Thailand with GBS as dominant species. This survey highlights that the river-based cage farms seriously impact on the healthy development of the tilapia industry.

Comparative genomic analysis and identification of pathogenicity islands of hypervirulent ST-17 Streptococcus agalactiae Brazilian strain

Streptococcus agalactiae are important pathogenic bacteria that cause severe infections in humans, especially neonates. The mechanism by which ST-17 causes invasive infections than other STs is not well understood. In this study, we sequenced the first genome of a S. agalactiae ST-17 strain isolated in Brazil using the Illumina HiSeq 2500 technology. S. agalactiae GBS90356 ST-17 belongs to the capsular type III and was isolated from a neonatal with a fatal case of meningitis. The genome presented a size of 2.03 Mbp and a G + C content of 35.2%. S. agalactiae has 706 genes in its core genome and an open pan-genome with a size of 5.020 genes, suggesting a high genomic plasticity. GIPSy software was used to identify 10 Pathogenicity islands (PAIs) which corresponded to 15% of the genome size. IslandViewer4 corroborated the prediction of six PAIs. The pathogenicity islands showed important virulence factors genes for S. agalactiae e.g. neu, cps, dlt, fbs, cfb, lmb. SignalP detected 20 proteins with signal peptides among the 352 proteins found in PAIs, which 60% were located in the SagPAI_5. SagPAI_2 and 5 were mainly detected in ST-17 strains studied. Moreover, we identified 51 unique genes, 9 recombination regions and a large number of SNPs with an average of 760.3 polymorphisms, which can be related with high genomic plasticity and virulence during host-pathogen interactions. Our results showed implications for pathogenesis, evolution, concept of species and in silico analysis value to understand the epidemiology and genome plasticity of S. agalactiae.

Isolation of Four Lytic Phages Infecting Klebsiella pneumoniae K22 Clinical Isolates from Spain.

The emergence of multi-drug-resistant bacteria represents a major public-health threat. Phages constitute a promising alternative to chemical antibiotics due to their high host specificity, abundance in nature, and evolvability. However, phage host specificity means that highly diverse bacterial species are particularly difficult to target for phage therapy. This is the case of Klebsiella pneumoniae, which presents a hypervariable extracellular matrix capsule exhibiting dozens of variants. Here, we report four novel phages infecting K. pneumoniae capsular type K22 which were isolated from environmental samples in Valencia, Spain. Full genome sequencing showed that these phages belong to the Podoviridae family and encode putative depolymerases that allow digestion of specific K22 K. pneumoniae capsules. Our results confirm the capsular type-specificity of K. pneumoniae phages, as indicated by their narrow infectivity in a panel of K. pneumoniae clinical isolates. Nonetheless, this work represents a step forward in the characterization of phage diversity, which may culminate in the future use of large panels of phages for typing and/or for combating multi-drug-resistant K. pneumoniae.

Isolation and Characterization of a Novel Bacteriophage Infecting Carbapenem-Resistant Klebsiella pneumoniae

A novel virulent phage, vB_KpnP_IME337, isolated from a hospital sewage in Beijing, China, that infects carbapenem-resistant Klebsiella pneumoniae KN2 capsular type was identified and characterized. Next-generation sequencing and genome analysis revealed that vB_KpnP_IME337 had a linear double-stranded genome with a length of 44,266 base pairs and G+C content of 53.7%. Fifty-two putative open reading frames were identified, and no transfer RNA-encoding genes were detected. BLASTn analysis revealed that phage vB_KpnP_IME337 had the highest sequence similarity with Klebsiella phage phiBO1E, with genome coverage of 79%. Based on morphology, phage vB_KpnP_IME337 was determined to belong to the family Podoviridae of the order Caudovirales. It was shown that phage vB_KpnP_IME337 had an infection duration of ~ 90min and 10min latent period, and a highly specific to host strain. In conclusion, phage vB_KpnP_IME337 may be a promising alternative candidate to antibiotic treatment for controlling diseases caused by drug-resistant K. pneumoniae.

Draft Genome Sequences of Clinical K1-Type Klebsiella pneumoniae Strains Isolated in Russia.

Klebsiella pneumoniae of capsular type K1 is the most common causative agent of both health care-associated and community-acquired infections. Here, we report the draft genome sequences of 10 K1-type K. pneumoniae strains isolated from patients in an infectious disease hospital and neurosurgical intensive care unit in Russia.

Association of capsular types with carbapenem resistance, disease severity, and mortality in Acinetobacter baumannii

ABSTRACT Acinetobacter baumannii emerged as one of the most important pathogens that causes nosocomial infections due to its increased multidrug resistance. Identifying capsular epidemiology in A. baumannii can aid in the development of effective treatments and preventive measures against this emerging pathogen. Here we established a wzc-based method, and combined it with wzy-PCR to determine capsular types of A. baumannii causing nosocomial bacteraemia collected at two medical centres in Taiwan from 2015 to 2017. Among the 237 patients with A. baumannii bacteraemia, 98 (41.4%) isolates were resistant to carbapenems. Four prevalent capsular types (KL2, KL10, KL22, and KL52) accounted for 84.7% of carbapenem-resistant A. baumannii (CRAB) and 12.2% of non-CRAB. The rate of pneumonia, intensive care unit admission, APACHE II score, and Pitt bacteraemia score were higher in patients with KL2/10/22/52 infection than in those with non-KL2/10/22/52 infection. Patients with KL2/10/22/52 infection and patients with CRAB infection have a higher cumulative incidence of attributable and all-cause in-hospital 30-day mortality. On multivariate analysis, appropriate empirical antimicrobial therapy within 24 h was associated with a lower risk of 30-day attributable mortality in the KL2/10/22/52 isolates (odds ratio = 0.19, 95% CI: 0.06–0.66, p = 0.008) but not in non-KL2/10/22/52 isolates. Early recognition of carbapenem resistance-associated capsular types may help clinicians to promptly implement appropriate antimicrobial therapy for improving the outcomes in patients with CRAB bacteraemia.

Capsular types, LPS genotypes, and virulence-associated genes of Pasteurella multocida strains isolated from pigs

Pasteurella multocida (P. multocida), an opportunistic zoonotic pathogen associated with high morbidity and mortality in livestock, shows significant temporal and geographical variation in its serotype distributions and phenotypic characteristics. The aim of this study was To investigate capsular types, lipopolysaccharide (LPS) genotypes, and virulence-associated genes of P. multocida strains isolated from pigs. A total of 801 samples (lungs, tonsils, nasal swabs) were collected from slaughterhouses and various regions of the Hunan province. P. multocida strains were isolated from various samples, classified, and virulence-associated genes were detected by polymerase chain reaction (PCR). 124 P. multocida strains were assigned to six groups based on both capsular type and LPS genotype, namely A: L3 (capsular type A and LPS genotype 3, 64/124); A: L6 (16/124); D: L6 (38/124); F: L3 (4/124); L3 (1/124) and 1 untypable strain. Of the 23 virulence-associated genes investigated in this study, 14 were highly expressed in 98% to 100% of the 124 strains. While tbpA was undetectable in any of the isolated strains, hsf-1, pfhA, tadD, toxA, pmHAS, hgbA, hgbB, and nanB showed differential distribution among the strain groups. Interestingly, pfhA (Mutation or inactivation of pfhA was reported to decrease the virulence of P. multocida) was found in 46% of group A: L3 strains and in 100% of group F: L3 strains, but not found in other groups. Further investigation is needed to determine whether strains in group A: L3show greater virulence than the A: L6 P. multocida strains.

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.

Molecular Characterization of Hospital- and Community-Acquired Streptococcus agalactiae Isolates among Nonpregnant Adults in Isfahan, Iran.

Background:The increasing incidence of Group B Streptococcus (GBS) infection among nonpregnant adults has become of growing clinical and public health concern. The current study investigated the distribution of important virulence determinants and antibiotic susceptibility of GBS isolates causing community acquired (CA) and hospital acquired (HA) infections among nonpregnant adults.Materials and Methods:A total of 62 GBS, including 31 CA GBS and 31 HA GBS, were collected from a teaching hospital in Isfahan, Iran. Capsular polysaccharide genotypes (CPS), PI 1, PI 2a, PI 2b, and hypervirulent GBS adhesin (hvgA) virulence genes and antibiotic resistance profiling were determined.Results:There were 19 (30.6%) cases of underlying disease that diabetes mellitus (20.9%) was most common. The rate of multidrug resistant GBS strains was accounted for 29%. Distribution of macrolide resistant phenotypes was as follows: constitutive macrolides, lincosamides, and streptogramin B (MLSB) (15 isolates); inducible resistance to MLSB; and L phenotype (each 5 isolates) and M phenotype (1 isolate). V and Ia serotypes were the most predominant capsular type in HA GBS and CA GBS isolates, respectively. The most frequent pilus types were PI 1, PI 1+PI 2a, PI 1+PI 2b, and PI 2a. PI 1 and PI 1+PI 2a had significantly different distributions between CA and HA GBS isolates. Three CA GBS isolates (9.6%) were positive for hvgA gene that belonged to clonal complex 17/sequence type 17/CPS III/PI 1+PI 2b lineage.Conclusion:There was a significant difference in the distribution of PIs among CA GBS and HA GBS isolates in our region.

Prevalence of virulence factor, antibiotic resistance, and serotype genes of Pasteurella multocida strains isolated from pigs in Vietnam.

Aim:The study was conducted to determine the prevalence and characterization of the Pasteurella multocida isolates from suspected pigs in Vietnam.Materials and Methods:A total of 83 P. multocida strains were isolated from lung samples and nasal swabs collected from pigs associated with pneumonia, progressive atrophic rhinitis, or reproductive and respiratory symptoms. Isolates were subjected to multiplex polymerase chain reaction (PCR) for capsular typing, detection of virulence-associated genes and antibiotic resistance genes by PCR. The antimicrobial sensitivity profiles of the isolates were tested by disk diffusion method.Results:All the isolates 83/83 (100%) were identified as P. multocida by PCR: serogroup A was obtained from 40/83 (48.19%), serogroup D was detected from 24/83 strains (28.91%), and serogroup B was found in 19/83 (22.35%) isolates. The presence of 14 virulence genes was reported including adhesins group (ptfA – 93.97%, pfhA – 93.97%, and fimA – 90.36%), iron acquisition (exbB – 100%, and exbD – 85.54%), hyaluronidase (pmHAS – 84.33%), and protectins (ompA – 56.62%, ompH 68.67%, and oma87 – 100%). The dermonecrotoxin toxA had low prevalence (19.28%). The antimicrobial susceptibility testing revealed that cephalexin, cefotaxime, ceftriaxone, ofloxacin, pefloxacin, ciprofloxacin, and enrofloxacin were the drugs most likely active against P. multocida while amoxicillin and tetracycline were inactive. The usage of PCR revealed that 63/83 isolates were carrying at least one of the drug resistance genes.Conclusion:Unlike other parts of the word, serotype B was prevalent among Vietnamese porcine P. multocida strains. The high antibiotic resistance detected among these isolates gives us an alert about the current state of imprudent antibiotic usage in controlling the pathogenic bacteria.

Molecular Capsular Typing and Multi Locus Sequence Typing of Invasive, Non-invasive and Commensal Streptococcus pneumoniae Isolates from North India

AbstractPurpose: The purpose of this study is to determine the antimicrobial susceptibility pattern, serotype distribution and sequence type (ST) of Streptococcus pneumoniae isolates from invasive and non-invasive infection and correlate it with isolates from commensal nasopharyngeal flora to ascertain their role in infection. Materials and Methods:S. pneumoniae isolates from blood, cerebrospinal fluid, pleural fluid and respiratory secretions (sputum, bronchoalveolar lavage and nasopharyngeal swab/throat swab) were analysed to determine ST, serotype and antimicrobial susceptibility pattern. Serotyping was performed by multiplex polymerase chain reactions as well as by quellung reaction. Antimicrobial susceptibility testing was determined using Kirby Bauer’s disc diffusion method as per the Clinical Laboratory Standards Institute guidelines. Minimum inhibitory concentration was determined using E-test for penicillin. Multilocus sequence typing (MLST) was done to understand genetic relatedness and evolutionary relationship among strains. Results: A total of 125 S. pneumoniae isolates were collected, including 25 from invasive pneumococcal disease, 25 from non-invasive and 75 from nasopharyngeal swab of healthy children (Commensal). Resistance to penicillin, erythromycin, and co-trimoxazole was observed in 14.4%, 12% and 81.6% of the isolates, respectively, by KirbyBauer’s disc diffusion method. Serotype 14 was found to be the most prevalent in invasive and non-invasive isolates, while serotype 6 was the most common in commensal isolates. New STs were found among invasive (ST13826, ST13827), non-invasive (ST13823, ST13824, and ST13961) and commensal (ST13825) isolates. Conclusion: MLST sequence analysis shows that invasive isolates were found to be clustered with non-invasive and commensal isolates. Analysis of MLST suggests the possibility of genetic relatedness and exchange of genetic material between invasive, non-invasive and commensal isolates.

Involvement of lipid microdomains in human endothelial cells infected by Streptococcus agalactiae type III belonging to the hypervirulent ST-17.

BACKGROUND Streptococcus agalactiae capsular type III strains are a leading cause of invasive neonatal infections. Many pathogens have developed mechanisms to escape from host defense response using the host membrane microdomain machinery. Lipid rafts play an important role in a variety of cellular functions and the benefit provided by interaction with lipid rafts can vary from one pathogen to another.OBJECTIVES This study aims to evaluate the involvement of membrane microdomains during infection of human endothelial cell by S. agalactiae. METHODS The effects of cholesterol depletion and PI3K/AKT signaling pathway activation during S. agalactiae-human umbilical vein endothelial cells (HUVEC) interaction were analysed by pre-treatment with methyl-β-cyclodextrin (MβCD) or LY294002 inhibitors, immunofluorescence and immunoblot analysis. The involvement of lipid rafts was analysed by colocalisation of bacteria with flotillin-1 and caveolin-1 using fluorescence confocal microscopy.FINDINGS In this work, we demonstrated the importance of the integrity of lipid rafts microdomains and activation of PI3K/Akt pathway during invasion of S. agalactiae strain to HUVEC cells. Our results suggest the involvement of flotillin-1 and caveolin-1 during the invasion of S. agalactiae strain in HUVEC cells.CONCLUSIONS The collection of our results suggests that lipid microdomain affects the interaction of S. agalactiae type III belonging to the hypervirulent ST-17 with HUVEC cells through PI3K/Akt signaling pathway.

First characterization of K. pneumoniae ST11 clinical isolates harboring blaKPC-3 in Latin America

Antimicrobial resistance due to carbapenemase production in Enterobacteriaceae clinical isolates is a global threat. Klebsiellapneumoniae harboring the blaKPC gene is one of the major concerns in hospital settings in Latin America.The aim of this study was to characterize the antibiotic resistance mechanisms and to typify four carbapenem-resistant K. pneumoniae clinical isolates from the city of Manizales, Colombia.We identified blaKPC-3 in all four isolates by polymerase chain reaction and subsequent sequencing. The plasmid-mediated quinolone resistance genes qnrB19-like and aac(6′)Ib-cr; fosfomycin resistance gene fosA and an insertion sequence IS5-like in mgrB (colistin resistance) were also detected. Sequence types ST11 with capsular type wzi75, and ST258 with wzi154, were characterized. The blaKPC-3 gene was mobilized in a 100-kb IncFIB conjugative plasmid with vagCD toxin–antitoxin system.This work reports multiple resistance genes in blaKPC-producing K. pneumoniae and the first occurrence of ST11 clinical isolates harboring blaKPC-3 in Latin America.

Differences in pathogenicity and virulence-associated gene expression among Pasteurella multocida strains with high and low virulence in a lung tissue model

Pasteurella multocida capsular type A can cause a pulmonary infection, leading to serious pecuniary losses in cattle. The heterogeneity of infection outcome of P. multocida strains showing different virulence may be related to divergent expression of virulence genes. In this study, we compared the transcriptional response of virulence-associated genes in high (PMPAN001) and low (PMPAN007) virulence P. multocida capsular type A strains in lung tissues and in vitro. These clinical isolates differ in their organ bacterial loads, mRNA abundance of the same virulence genes between lung and culture medium, and extent of lung damage. Among the eight virulence-associated genes (fimA, tbpA, exbD, fur, oma87, pmHAS, nanH, and tonB), seven genes showed higher expression in lung compared with in vitro at 16 h (P ≤ 0.05) in PMPAN001, but not in PMPAN007. FimA, exbD, fur, oma87, pmHAS, and tonB gene transcripts showed significantly higher expression in PMPAN001 than in PMPAN007 in the lung tissues at 16 h post-infection (P ≤ 0.05). Specially, the virulence gene, nanH, in both strains was associated with poor expression in vitro and lung tissue (mean relative mRNA abundance values < 0.6). Strain PMPAN001 had a higher proliferation rate in vivo than strain PMPAN007. The bacterial loads of PMPAN001 in the organs increased from 12 h post-infection, with maximum bacteria count ranging from 1 million to 20 million/mg. In addition, lungs treated with PMPAN001 produced serious and extensive lesions marked with inflammation at 20 h. Overall, our results reveal that the highly expressed virulence-associated genes, fimA, exbD, fur, oma87, pmHAS, and tonB can be used as markers for assessing the virulence of P. multocida capsular type A strains.

Total Syntheses of Conjugation-Ready Trisaccharide Repeating Units of Pseudomonas aeruginosa O11 and Staphylococcus aureus Type 5 Capsular Polysaccharide for Vaccine Development.

Pseudomonas aeruginosa belongs to the group of three "critical priority" multi-drug-resistant pathogens listed by WHO and is responsible for severe and often deadly infections such as bloodstream infections and pneumonia. Staphylococcus aureus is also a "high priority" pathogen which is a major cause of serious nosocomial infections such as bacteremia, sepsis, and endocarditis. Owing to their ability to adapt resistance to almost any antibiotics, vaccines against these pathogens are urgently required. These pathogens express structurally unique and densely functionalized glycans on their surfaces which are absent on the host cells. Such carbohydrate antigens are valuable targets for the development of glycoconjugate vaccines and diagnostics. Here, we report the first total synthesis of the conjugation-ready trisaccharide repeating unit of Pseudomonas aeruginosa O11 via a highly stereoselective and efficient assembly of a rare l-fucosamine- and d-fucosamine-containing 1,2-cis-linked disaccharide motif and its regioselective glycosylation at O3. A systematic study was conducted for the notoriously difficult glycosylation with the most unreactive axial 4-OH of the rare disaccharide, and the successful outcome was utilized to accomplish the total synthesis of an aminopropyl linker-attached trisaccharide repeating unit of Staphylococcus aureus capsular polysaccharide type 5, which is also a potential antigen for immunotherapy and vaccine development. The judicious selection of protecting groups and reaction conditions allowed the stereoselective assembly and selective functional group interconversions to access the structurally complex linker-attached trisaccharide repeating units, which are valuable tools for immunological evaluation and vaccine development. The strategy is useful for the synthesis of other structurally related complex glycans.

Characterization of Staphylococcus aureus isolates from mastitic milk, udder surfaces and milkers' hands from arid and semi arid regions of India for capsular (cap5K and cap8K) and collagen adhesin (cna) genes

Staphylococcus aureus (S. aureus) is an important cause of bovine mastitis. The antiphagocytic capsule of bacteria promotes colonization and persistence on mucosal surfaces while the presence of collagen adhesin (cna gene) is associated with bacterial adhesion in mammary tissues; hence both are regarded as important determinants of virulence. In the present study, 197 samples consisting of mastitic milk, milkers' hand swabs and udder surface swabs were collected which yielded 107 S. aureus isolates. These S. aureus isolates (107) from different sources of sampling were characterized for their capsular types targeting cap5K and cap8K genes in a duplex PCR along with amplification of cna gene. Majority of the isolates (67.6%) possessed cap5K gene with a lower percentage (22.9%) of isolates carrying cap8K gene and 9.5% carrying both the genes. Moreover, cap5K gene was predominant in isolates from milkers’ hands (85.7%) while cap8K gene was more common in bovine udder isolates (41.2%). cna gene was observed in 27.6% isolates with highest occurrence in milk isolates (44.8%) compared to udder (37.9%) and milkers' hand (17.2%). cna positive isolates carried cap8K gene (66.7%) and were significantly associated with both cap5k and cap8k genes. In conclusion, the majority of the S. aureus isolates of mastitis obtained from arid and semi arid zones of India possessed the genes for capsule production, and cap5K was the predominant gene. The cna gene for collagen adhesion was observed in fewer isolates with significant association with cap8K gene.