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UNLABELLED: Infections caused by antimicrobial-resistant Escherichia coli are the leading cause of death attributed to antimicrobial resistance (AMR) worldwide, and the known AMR mechanisms involve a range of functional proteins. Here, we employed a pan-genome wide association study (GWAS) approach on over 1,000 E. coli isolates from sick dogs collected across the US and Canada and identified a strong statistical association (empirical P < 0.01) of AMR, involving a range of antibiotics to a group 1 capsular (CPS) gene cluster. This cluster included genes under relaxed selection pressure, had several loci missing, and had pseudogenes for other key loci. Furthermore, this cluster is widespread in E. coli and Klebsiella clinical isolates across multiple host species. Earlier studies demonstrated that the octameric CPS polysaccharide export protein Wza can transmit macrolide antibiotics into the E. coli periplasm. We suggest that the CPS in question, and its highly divergent Wza, functions as an antibiotic trap, preventing antimicrobial penetration. We also highlight the high diversity of lineages circulating in dogs across all regions studied, the overlap with human lineages, and regional prevalence of resistance to multiple antimicrobial classes. IMPORTANCE: Much of the human genomic epidemiology data available for E. coli mechanism discovery studies has been heavily biased toward shiga-toxin producing strains from humans and livestock. E. coli occupies many niches and produces a wide variety of other significant pathotypes, including some implicated in chronic disease. We hypothesized that since dogs tend to share similar strains with their owners and are treated with similar antibiotics, their pathogenic isolates will harbor unexplored AMR mechanisms of importance to humans as well as animals. By comparing over 1,000 genomes with in vitro antimicrobial susceptibility data from sick dogs across the US and Canada, we identified a strong multidrug resistance association with an operon that appears to have once conferred a type 1 capsule production system.

Predatory bacteriophages have evolved a vast array of depolymerases for bacteria capture and deprotection. These depolymerases are enzymes responsible for degrading diverse bacterial surface carbohydrates. They are exploited as antibiofilm agents and antimicrobial adjuvants while rarely inducing bacterial resistance, making them an invaluable asset in the era of antibiotic resistance. Numerous depolymerases have been investigated preclinically, with evidence indicating that depolymerases with appropriate dose regimens can safely and effectively combat different multidrug-resistant pathogens in animal infection models. Additionally, some formulation approaches have been developed for improved stability and activity of depolymerases. However, depolymerase formulation is limited to liquid dosage form and remains in its infancy, posing a significant hurdle to their clinical translation, compounded by challenges in their applicability and manufacturing. Future development must address these obstacles for clinical utility. Here, after unravelling the history, diversity, and therapeutic use of depolymerases, we summarized the preclinical efficacy and existing formulation findings of recombinant depolymerases. Finally, the challenges and perspectives of depolymerases as therapeutics for humans were assessed to provide insights for their further development.

Predatory bacteriophages have evolved a vast array of depolymerases for bacteria capture and deprotection. These depolymerases are enzymes responsible for degrading diverse bacterial surface carbohydrates. They are exploited as antibiofilm agents and antimicrobial adjuvants while rarely inducing bacterial resistance, making them an invaluable asset in the era of antibiotic resistance. Numerous depolymerases have been investigated preclinically, with evidence indicating that depolymerases with appropriate dose regimens can safely and effectively combat different multidrug-resistant pathogens in animal infection models. Additionally, some formulation approaches have been developed for improved stability and activity of depolymerases. However, depolymerase formulation is limited to liquid dosage form and remains in its infancy, posing a significant hurdle to their clinical translation, compounded by challenges in their applicability and manufacturing. Future development must address these obstacles for clinical utility. Here, after unravelling the history, diversity, and therapeutic use of depolymerases, we summarized the preclinical efficacy and existing formulation findings of recombinant depolymerases. Finally, the challenges and perspectives of depolymerases as therapeutics for humans were assessed to provide insights for their further development. Phage-derived polysaccharide depolymerases are useful as antibiofilm agents and antimicrobial adjuvants. Their preclinical efficacy was reviewed, and their challenges and prospects for clinical translation were discussed. [Display omitted] [ABSTRACT FROM AUTHOR]

Haemophilus influenzae causes common and sometimes severe adult and pediatric disease including chronic obstructive respiratory disease, otitis media and infections of the central nervous system. Serotype b strains, with a b-type capsule, have been the historical cause of invasive disease, and the introduction of a serotype b-specific vaccine has led to their decline. However, unencapsulated or non-b-type H. influenzae infections are not prevented by the vaccine and appear to be increasing in frequency. Here we report two pediatric cases of severe central nervous system H. influenzae infection presenting to the same hospital in San Diego, California during the same week in January 2016. Due to good vaccine coverage in this part of the world, H. influenzae cases are normally rare and seeing two cases in the same week was unexpected. We thus suspected a recent transmission chain, and possible local outbreak. To test this hypothesis, we isolated and sequenced whole genomes from each patient and placed them in a phylogenetic tree spanning the known diversity of H. influenzae. Surprisingly, we found that the two isolates (SD2016_1 and SD2016_2) belonged to distantly related lineages, suggesting two independent transmission events and ruling out a local outbreak. Despite being distantly related, the two isolates belong to two different lineages that have exchanged capsule loci in the recent past. Therefore, as in other bacterial pathogens, capsule switching by horizontal gene transfer may be an important evolutionary mechanism of vaccine evasion in H. influenzae.

Haemophilus influenzae type b (Hib) affects 337,000 Indian children every year. A vaccine against Hib was introduced in 2011 as part of the pentavalent vaccine and scaled up nationwide. This study investigated the associations between Hib vaccination and child anthropometry, cognition, and schooling outcomes in India. We used longitudinal survey data and employed propensity score matching to control for observed systematic differences between children who reported receipt or nonreceipt of Hib vaccine before age 6 years (n = 1824). Z-scores of height-for-age (HAZ) and BMI-for-age (BMIZ), percentage scores of English, mathematics, reading, and Peabody Picture Vocabulary tests, and attained schooling grade of children were examined. Hib-vaccinated children had 0.25 higher HAZ, scored 4.09 percentage points (pp) higher on the English test and 4.78 pp higher on the mathematics test, and attained 0.16 more schooling grades than Hib-unvaccinated children at age 11-12 years. At age 14-15 years, they had 0.18 higher HAZ, scored 3.63 pp higher on the reading test and 3.22 pp higher on the mathematics test, and attained 0.15 more schooling grades than Hib-unvaccinated children. The findings indicate potential long-term health, cognitive, and schooling benefits of the Hib vaccine, subject to the effect of unobserved confounding factors.

Circulating platelets have important functions in thrombosis and in modulating immune and inflammatory responses. However, the role of platelets in innate immunity to bacterial infection is largely unexplored. While human platelets rapidly kill Staphylococcus aureus, we found the neonatal pathogen group B Streptococcus (GBS) to be remarkably resistant to platelet killing. GBS possesses a capsule polysaccharide (CPS) with terminal α2,3-linked sialic acid (Sia) residues that mimic a common epitope present on the human cell surface glycocalyx. A GBS mutant deficient in CPS Sia was more efficiently killed by human platelets, thrombin-activated platelet releasate, and synthetic platelet-associated antimicrobial peptides. GBS Sia is known to bind inhibitory Sia-recognizing Ig superfamily lectins (Siglecs) to block neutrophil and macrophage activation. We show that human platelets also express high levels of inhibitory Siglec-9 on their surface, and that GBS can engage this receptor in a Sia-dependent manner to suppress platelet activation. In a mouse i.v. infection model, antibody-mediated platelet depletion increased susceptibility to platelet-sensitive S. aureus but did not alter susceptibility to platelet-resistant GBS. Elimination of murine inhibitory Siglec-E partially reversed platelet suppression in response to GBS infection. We conclude that GBS Sia has dual roles in counteracting platelet antimicrobial immunity: conferring intrinsic resistance to platelet-derived antimicrobial components and inhibiting platelet activation through engagement of inhibitory Siglecs. We report a bacterial virulence factor for evasion of platelet-mediated innate immunity.

Epithelial-neuronal signaling is essential for sensory encoding in touch, itch, and nociception; however, little is known about the release mechanisms and neurotransmitter receptors through which skin cells govern neuronal excitability. Merkel cells are mechanosensory epidermal cells that have long been proposed to activate neuronal afferents through chemical synaptic transmission. We employed a set of classical criteria for chemical neurotransmission as a framework to test this hypothesis. RNA sequencing of adult mouse Merkel cells demonstrated that they express presynaptic molecules and biosynthetic machinery for adrenergic transmission. Moreover, live-cell imaging directly demonstrated that Merkel cells mediate activity- and VMAT-dependent release of fluorescent catecholamine neurotransmitter analogs. Touch-evoked firing in Merkel-cell afferents was inhibited either by pre-synaptic silencing of SNARE-mediated vesicle release from Merkel cells or by neuronal deletion of β2-adrenergic receptors. Together, these results identify both pre- and postsynaptic mechanisms through which Merkel cells excite mechanosensory afferents to encode gentle touch. VIDEO ABSTRACT.

Francisella tularensis is the causative agent of tularemia and a Tier I bioterrorism agent. In the 1900s, several vaccines were developed against tularemia including the killed "Foshay" vaccine, subunit vaccines comprising F. tularensis protein(s) or lipoproteins(s) in an adjuvant formulation, and the F. tularensis Live Vaccine Strain (LVS); none were licensed in the U.S.A. or European Union. The LVS vaccine retains toxicity in humans and animals-especially mice-but has demonstrated efficacy in humans, and thus serves as the current gold standard for vaccine efficacy studies. The U.S.A. 2001 anthrax bioterrorism attack spawned renewed interest in vaccines against potential biowarfare agents including F. tularensis. Since live attenuated-but not killed or subunit-vaccines have shown promising efficacy and since vaccine efficacy against respiratory challenge with less virulent subspecies holarctica or F. novicida, or against non-respiratory challenge with virulent subsp. tularensis (Type A) does not reliably predict vaccine efficacy against respiratory challenge with virulent subsp. tularensis, the route of transmission and species of greatest concern in a bioterrorist attack, in this review, we focus on live attenuated tularemia vaccine candidates tested against respiratory challenge with virulent Type A strains, including homologous vaccines derived from mutants of subsp. holarctica, F. novicida, and subsp. tularensis, and heterologous vaccines developed using viral or bacterial vectors to express F. tularensis immunoprotective antigens. We compare the virulence and efficacy of these vaccine candidates with that of LVS and discuss factors that can significantly impact the development and evaluation of live attenuated tularemia vaccines. Several vaccines meet what we would consider the minimum criteria for vaccines to go forward into clinical development-safety greater than LVS and efficacy at least as great as LVS, and of these, several meet the higher standard of having efficacy ≥LVS in the demanding mouse model of tularemia. These latter include LVS with deletions in purMCD, sodBFt , capB or wzy; LVS ΔcapB that also overexpresses Type VI Secretion System (T6SS) proteins; FSC200 with a deletion in clpB; the single deletional purMCD mutant of F. tularensis SCHU S4, and a heterologous prime-boost vaccine comprising LVS ΔcapB and Listeria monocytogenes expressing T6SS proteins.

Antibiotic resistance has increased in recent years, especially for pathogens like Klebsiella pneumoniae. Discovering and developing new drugs is challenging due to the high resistance of pathogens. Metal nanoparticles have been widely used in recent years to overcome and treat infections. Gallic acid-coated iron oxide nanoparticles (IONPs-GA) were synthesized in a simple and cost-effective method. The morphology characteristics of synthesized IONPs-GA were analyzed using Fourier transform infrared spectroscopy (FTIR), x-ray diffraction analysis (XRD), and scanning electron microscope (SEM) analysis. IONPs were mostly spherical in shape with sizes ranging between 32 and 61 nm. All analyses used in this study confirmed the successful coating of gallic acid to iron oxide. Biological activities were studied phenotypically and on the molecular level, including antibacterial, antibiofilm, and mRNA levels of capsule-associated genes. The results showed high antimicrobial activity of the synthesized nanoparticles against different G +ve and G -ve bacteria. The highest activity was recorded against Staphylococcus aureus (43 mm) and K. pneumoniae (22 mm). The MIC of IONPs against K. pneumoniae was 3.12 mg/mL and SEM analysis showed adhering the IONPs-GA to the cell surface of K. pneumoniae resulted in disrupting the cell membrane. Different concentrations of sub-MIC inhibited K. pneumoniae biofilm formation with the highest inhibition percentage at ½ × MIC (66.86%). Also, the synthesized IONPs-GA differently affected the regulation and mRNA level of capsule-associated genes in K. pneumoniae. The results indicated that IONPs-GA could be useful in biological applications such as in drug delivery and treatment wide range of pathogens. RESEARCH HIGHLIGHTS: Gallic acid was successfully coated into iron oxide nanoparticles synthesized in a simple way. IONPs-GA was morphologically characterized using FTIR, XRD, and SEM. Evaluation the activity of IONPs-GA as antibacterial, antibiofilm, and study the potential level of mRNA affected by IONPs-GA., (© 2024 Wiley Periodicals LLC.)

Objective: The relationship between the in vitro detected virulence factors biofilm and capsule formation of Streptococcus (S.) uberis isolates of clinical mastitis in dairy cows and the bacteriological cure rate after antibiotic therapy was investigated in order to better understand the importance of these virulence factors for the bacteriological cure rate., Material and Methods: A total of 111 clinical mastitis (CM) cases were collected, in which S. uberis was bacteriologically detected. All mastitis cases were treated in accordance with the approval conditions of the antibiotic udder tubes used. Individual cow information including age, number of lactations, current lactation mastitis and antimicrobial treatment received was recorded. The microtiter plate test was used to detect biofilm formation and Anthony capsule staining was used to detect capsular capacity. Statistical analyses were performed to characterize the correlation between in vitro virulence factors and bacteriological cure (BC) rate., Results: 30.5% (n=29) of the S. uberis isolates of bacteriologically cured cases and 34.5% (n=10) of the isolates of bacteriologically non-cured mastitis cases were found to have the ability to produce capsules in vitro . 70.7% (n=58) of the S. uberis isolates from bacteriologically cured mastitis cases had the ability to produce biofilm in vitro , whereas 58.6% (n=17) of the isolates of non-cured mastitis cases showed ability in producing biofilm. No correlation was found between the in vitro ability of S. uberis to form capsules and biofilms and the BC rate after antibiotic treatment of bovine mastitis., Conclusion(s): The present work has shown that the investigated in vitro virulence factors are not associated with the BC after antibiotic therapy. Further studies on the role of S. uberis virulence factors are needed to complete the missing knowledge on the difficulties in curing S. uberis mastitis., Clinical Relevance: This study is of great clinical relevance since it enhances the understanding of the occurrence of BC in S. uberis mastitis cases. The investigated virulence factors are often addressed as possible reasons for therapy failure, although respective scientific studies are missing., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

The Streptococcus pneumoniae capsule is regarded as indispensable in bacteremia. We report an infant with a ventricular septal defect and infective endocarditis caused by nontypeable S. pneumoniae. In-depth investigation confirmed a deficient capsule yet favored pneumococcal fitness for causing infective endocarditis, rather than a host immune disorder, as the cause of infective endocarditis in this case. [ABSTRACT FROM AUTHOR]

A safer and more effective vaccine than the unlicensed Francisella tularensis Live Vaccine Strain (LVS) is needed to protect against the biowarfare agent F. tularensis. Previously, we developed an LVS ΔcapB mutant that is significantly safer than LVS and provides potent protective immunity against F. tularensis respiratory challenge when administered intranasally but limited protection when administered intradermally unless as part of a prime-boost vaccination strategy. To improve the immunogenicity and efficacy of LVS ΔcapB, we developed recombinant LVS ΔcapB (rLVS ΔcapB) strains overexpressing various F. tularensis Francisella Pathogenicity Island (FPI) proteins - IglA, IglB and IglC, and a fusion protein (IglABC) comprising immunodominant epitopes of IglA, IglB, and IglC downstream of different Francisella promoters, including the bacterioferritin (bfr) promoter. We show that rLVS ΔcapB/bfr-iglA, iglB, iglC, and iglABC express more IglA, IglB, IglC or IglABC than parental LVS ΔcapB in broth and in human macrophages, and stably express FPI proteins in macrophages and mice absent antibiotic selection. In response to IglC and heat-inactivated LVS, spleen cells from mice immunized intradermally with rLVS ΔcapB/bfr-iglC or bfr-iglABC secrete greater amounts of interferon-gamma and/or interleukin-17 than those from mice immunized with LVS ΔcapB, comparable to those from LVS-immunized mice. Mice immunized with rLVS ΔcapB/bfr-iglA, iglB, iglC or iglABC produce serum antibodies at levels similar to LVS-immunized mice. Mice immunized intradermally with rLVS ΔcapB/bfr-iglABC and challenged intranasally with virulent F. tularensis Schu S4 survive longer than sham- and LVS ΔcapB-immunized mice. Mice immunized intranasally with rLVS ΔcapB/bfr-iglABC - but not with LVS - just before or after respiratory challenge with F. tularensis Schu S4 are partially protected; protection is correlated with induction of a strong innate immune response. Thus, rLVS ΔcapB/bfr-iglABC shows improved immunogenicity and protective efficacy compared with parental LVS ΔcapB and, in contrast to LVS, has partial efficacy as immediate pre- and post-exposure prophylaxis.

Objective. To investigate the efficacy of enteric-coated Bifidobacterium triple viable capsules combined with enteral nutrition in the treatment of patients with chronic critical illness (CCI) and their effects on the immune and coagulation function of patients. Methods. 106 CCI patients admitted to the intensive care unit of our hospital from December 2018 to March 2020 were selected as the research objects, and they were randomly divided into the control group (n = 53) and the observation group (n = 53). The control group was given symptomatic treatment, etiological treatment, clinical nursing, enteral nutrition support, and other conventional treatment methods according to the patient's condition. On this basis, the observation group was treated with enteric-coated Bifidobacterium triple viable capsules, and both groups were treated for 14 days. All patients were followed up for 3 months after treatment, and their death/cure prognosis was recorded. The acute physiological and chronic health (APACHE II) scoring system was used to evaluate the acute physiological and chronic health status of the two groups before and after treatment, and the organs of the patients were scored with sepsis-related organ failure assessment (SOFA) score. T lymphocyte subsets (CD3+, CD4+, CD8+, and CD4+/CD8+), prothrombin time (PT), activated partial thrombin time (APTT), fibrinogen (FIB), and D-dimer (DD) were measured before and after treatment. Results. The cure rate of the observation group was slightly higher than that of the control group, and the mortality rate was slightly lower than that of the control group, but the difference was not statistically significant (P < 0.05). After treatment, the APACHE II and SOFA scores of the two groups were lower than before treatment, and the APACHE II and SOFA scores of the observation group were lower than those of the control group, and the differences were both statistically significant (P < 0.05). After treatment, the levels of CD3+, CD4+, and CD4+/CD8+ in the two groups were higher than those before treatment, and the levels of CD8+ were lower than before treatment. The CD3+, CD4+, and CD4+/CD8+ levels of the observation group were higher than those of the control group, and the CD8+ levels were lower than the control group, and the differences were both statistically significant (P < 0.05). After treatment, the PT and APTT levels of the two groups of patients were higher than those before treatment, and the levels of FIB and DD were lower than those before treatment. The PT and APTT levels of the observation group were higher than those of the control group, and the FIB and DD levels were lower than those of the control group, and the differences were both statistically significant (P < 0.05). Conclusion. The combination of enteric-coated Bifidobacterium triple viable capsules and enteral nutrition for CCI has high cure rate, which can not only improve the patients' physiological health status and organ dysfunction but also effectively improve the patients' immune and coagulation function, which is worthy of promotion. [ABSTRACT FROM AUTHOR]

BackgroundKlebsiella pneumoniae is a leading cause of hospital-acquired urinary tract infections and pneumonia worldwide, and is responsible for many cases of pyogenic liver abscess among diabetic patients in Asia. A defining characteristic of this pathogen is the presence of a thick, exterior capsule that has been reported to play a role in biofilm formation and to protect the organism from threats such antibiotics and host immune challenge.FindingsWe constructed two knockout mutants of K. pneumoniae to investigate how perturbations to capsule biosynthesis alter the cellular phenotype. In the first mutant, we deleted the entire gene cluster responsible for biosynthesis of the extracellular polysaccharide capsule. In the second mutant, we deleted the capsule export subsystem within this cluster. We find that both knockout mutants have lower amounts of capsule but produce greater amounts of biofilm. Moreover, one of the two mutants abolishes fimbriae expression as well.ConclusionsThese results are expected to provide insight into the interaction between capsule biosynthesis, biofilm formation, and fimbriae expression in this organism.

Group A Streptococcus (GAS) commonly infects human skin and occasionally causes severe and life-threatening invasive diseases. The hyaluronan (HA) capsule of GAS has been proposed to protect GAS from host defense by mimicking endogenous HA, a large and abundant glycosaminoglycan in the skin. However, HA is degraded during tissue injury, and the functions of short-chain HA that is generated during infection have not been studied. To examine the impact of the molecular mass of HA on GAS infection, we established infection models in vitro and in vivo in which the size of HA was defined by enzymatic digestion or custom synthesis. We discovered that conversion of high molecular mass HA to low molecular mass HA facilitated GAS phagocytosis by macrophages and limited the severity of infection in mice. In contrast, native high molecular mass HA significantly impaired internalization by macrophages and increased GAS survival in murine blood. Thus, our data demonstrate that GAS virulence can be influenced by the size of HA derived from both the bacterium and host and suggest that high molecular mass HA facilitates GAS deep tissue infections, whereas the generation of short-chain HA can be protective.

Background and Objectives: Group B streptococcus (GBS) can cause severe and invasive infections in pregnant women, infants, and adults. This study aimed to investigate the risk factors of GBS colonization in pregnant women and determine the macrolide resistance and capsular type of isolates. Materials and Methods: In a cross-sectional study, a total of 200 pregnant women were screened for GBS colonization by phenotypic methods. Antibiotic susceptibility pattern of colonizing isolates and ermB, ermTR, mefA/E genes were detected. Also, molecular capsular types of isolates were distinguished. Results: The overall prevalence of colonization of participates with GBS was 13.5%. Statistical analysis showed that there was no association between risk factors and colonization with GBS. The highest resistance was observed to erythromycin (44.4%) followed by clindamycin (29.6%), penicillin, ampicillin, and ceftriaxone (18.5%), levofloxacin (11.1%), and 29.6% isolates were multidrug-resistant. ermTR and mefA/E genes were detected in 37% and 11.1% isolates; respectively and the ermB gene was not detected. The most common capsular type was type Ib (44.4%) followed by type III (40.7%), type II (11.1), and type Ia (3.7%). Conclusion: In the present study, the prevalence of GBS was in the medium range. Resistance to key antibiotic agents was relatively high. Also, capsular serotype Ib was the predominant serotype, which emphasizes the importance of monitoring the molecular typing of the GBS isolates regularly.

Background and Objectives: Group B streptococcus (GBS) can cause severe and invasive infections in pregnant women, infants, and adults. This study aimed to investigate the risk factors of GBS colonization in pregnant women and determine the macrolide resistance and capsular type of isolates. Materials and Methods: In a cross-sectional study, a total of 200 pregnant women were screened for GBS colonization by phenotypic methods. Antibiotic susceptibility pattern of colonizing isolates and ermB, ermTR, mefA/E genes were detected. Also, molecular capsular types of isolates were distinguished. Results: The overall prevalence of colonization of participates with GBS was 13.5%. Statistical analysis showed that there was no association between risk factors and colonization with GBS. The highest resistance was observed to erythromycin (44.4%) followed by clindamycin (29.6%), penicillin, ampicillin, and ceftriaxone (18.5%), levofloxacin (11.1%), and 29.6% isolates were multidrug-resistant. ermTR and mefA/E genes were detected in 37% and 11.1% isolates; respectively and the ermB gene was not detected. The most common capsular type was type Ib (44.4%) followed by type III (40.7%), type II (11.1), and type Ia (3.7%). Conclusion: In the present study, the prevalence of GBS was in the medium range. Resistance to key antibiotic agents was relatively high. Also, capsular serotype Ib was the predominant serotype, which emphasizes the importance of monitoring the molecular typing of the GBS isolates regularly. [ABSTRACT FROM AUTHOR]

Background: Campylobacter jejuni (C. jejuni) is a leading cause of acute gastroenteritis in human worldwide. The aim of study was to assess the distribution of sialylated lipooligosaccharide (LOS) classes and capsular genotypes in C. jejuni isolated from Iranian children with gastroenteritis. Furthermore, the level of dnaK gene expression in C. jejuni strains with selected capsular genotypes and LOS classes was intended. Moreover, a comprehensive study of C. jejuni MLST-genotypes and inclusive comparison with peer sequences worldwide was intended.Methods: Twenty clinical C. jejuni strains were isolated from fecal specimens of 280 children aged 0-5 years, suspected of bacterial gastroenteritis, which admitted to 3 children hospitals from May to October, 2018. Distribution of sialylated LOS classes and specific capsular genotypes were investigated in C. jejuni of clinical origin. The expression of dnaK in C. jejuni strains was measured by Real-Time-PCR. MLST-genotyping was performed to investigate the clonal relationship of clinical C. jejuni strains and comparison with inclusive sequences worldwide.Results: C. jejuni HS23/36c was the predominant genotype (45%), followed by HS2 (20%), and HS19 and HS4 (each 10%). A total of 80% of isolates were assigned to LOS class B and C. Higher expression level of dnaK gene was detected in strains with HS23/36c, HS2 and HS4 capsular genotypes and sialylated LOS classes B or C. MLST analysis showed that isolates were highly diverse and represented 6 different sequence types (STs) and 3 clonal complexes (CCs). CC21 and CC257 were the most dominant CCs (75%) among our C. jejuni strains. No new ST and no common ST with our neighbor countries was detected.Conclusions: The C. jejuni isolates with LOS class B or C, and capsular genotypes of HS23/36, HS2, HS4 and HS19 were dominant in population under study. The CC21 and CC257 were the largest CCs among our isolates. In overall picture, CC21 and CC353 complexes were the most frequently and widely distributed clonal complexes worldwide, although members of CC353 were not detected in our isolates. This provides a universal picture of movement of dominant Campylobacter strains worldwide. [ABSTRACT FROM AUTHOR]

The worldwide burden of disease of bacterial meningitis remains high, despite the decreasing incidence following introduction of routine vaccination campaigns.The aim of our study was to evaluate the epidemiological and bacteriological profile of paediatric bacterial meningitis (BM) in Tunisian children, during the period 2003-2019, following the implementation of Haemophilus influenzae type b (Hib) vaccine (April 2011) and before 10-valent pneumoccocal conjugate vaccine (PCV10) introduction to the childhood immunization program.All bacteriologically confirmed cases of BM admitted to children's hospital of Tunis were recorded (January 2003 to April 2019). Serogroups of Neisseria meningitidis (Nm) and serotypes of Streptococcus pneumoniae (Sp) and H. influenzae (Hi) and antibiotic resistance were determined using conventional and molecular methods.Among 388 cases, the most frequent species were Sp (51.3%), followed by Nm (27.5%) and Hi (16.8%). We observed a significant decrease in Hi BM rate during the conjugated Hib vaccine use period (P < 0.0001). The main pneumococcal serotypes were 14, 19F, 6B, 23F and 19A and the serotype coverage of PCV10, PCV13, PCV15 and PCV20 was 71.3 and 78.8%, 79.4 and 81.9% respectively. The most frequent Nm serogroup was B (83.1%). Most Hi strains were of serotype b (86.9%). High levels of resistance were found: Sp and Nm to penicillin (respectively 60.1 and 80%) and Hi to ampicillin (42.6%). All meningococcal and Hi isolates were susceptible to third-generation cephalosporins and 7.2% of pneumococcal strains had decreased susceptibility to these antibiotics.The Hib conjugate vaccine decreased the rate of BM. Sp dominated the aetiology of BM in children in Tunisia. Conjugate vaccines introducing decreases not only BM cases but also antimicrobial resistance.

Capsular polysaccharides (CPS) in Streptococcus pneumoniae are pivotal for bacterial virulence and present extensive diversity. While oral streptococci show pronounced antigenicity toward pneumococcal capsule-specific sera, insights into evolution of capsule diversity remain limited. This study reports a pneumococcal CPS-like genetic locus in Streptococcus parasanguinis , a predominant oral Streptococcus . The discovered locus comprises 15 genes, mirroring high similarity to those from the Wzy-dependent CPS locus of S. pneumoniae . Notably, S. parasanguinis elicited a reaction with pneumococcal 19B antiserum. Through nuclear magnetic resonance analysis, we ascertained that its CPS structure matches the chemical composition of the pneumococcal 19B capsule. By introducing the glucosyltransferase gene cps19cS from a pneumococcal serotype 19C, we successfully transformed S. parasanguinis antigenicity from 19B to 19C. Furthermore, substituting serotype-specific genes, cpsI and cpsJ , with their counterparts from pneumococcal serotype 19A and 19F enabled S. parasanguinis to generate 19A- and 19F-specific CPS, respectively. These findings underscore that S. parasanguinis harbors a versatile 19B-like CPS adaptable to other serotypes. Remarkably, after deleting the locus's initial gene, cpsE , responsible for sugar transfer, we noted halted CPS production, elongated bacterial chains, and diminished biofilm formation. A similar phenotype emerged with the removal of the distinct gene cpsZ , which encodes a putative autolysin. These data highlight the importance of S. parasanguinis CPS for biofilm formation and propose a potential shared ancestry of its CPS locus with S. pneumoniae ., Importance: Diverse capsules from Streptococcus pneumoniae are vital for bacterial virulence and pathogenesis. Oral streptococci show strong responses to a wide range of pneumococcal capsule-specific sera. Yet, the evolution of this capsule diversity in relation to microbe-host interactions remains underexplored. Our research delves into the connection between commensal oral streptococcal and pneumococcal capsules, highlighting the potential for gene transfer and evolution of various capsule types. Understanding the genetic and evolutionary factors that drive capsule diversity in S. pneumoniae and its related oral species is essential for the development of effective pneumococcal vaccines. The present findings provide fresh perspectives on the cross-reactivity between commensal streptococci and S. pneumoniae , its influence on bacteria-host interactions, and the development of new strategies to manage and prevent pneumococcal illnesses by targeting and modulating commensal streptococci., Competing Interests: The authors declare no conflict of interest.

Encapsulated Haemophilus influenzae strains belong to type-specific genetic lineages. Reliable capsule typing requires PCR, but a more efficient method would be useful. We evaluated capsule typing by using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Isolates of all capsule types (a−f and nontypeable; n = 258) and isogenic capsule transformants (types a−d) were investigated. Principal component and biomarker analyses of mass spectra showed clustering, and mass peaks correlated with capsule type-specific genetic lineages. We used 31 selected isolates to construct a capsule typing database. Validation with the remaining isolates (n = 227) showed 100% sensitivity and 92.2% specificity for encapsulated strains (a−f; n = 61). Blinded validation of a supplemented database (n = 50) using clinical isolates (n = 126) showed 100% sensitivity and 100% specificity for encapsulated strains (b, e, and f; n = 28). MALDI-TOF mass spectrometry is an accurate method for capsule typing of H. influenzae.

Capsule is a stable formulation wherein the bioagent is encapsulated in coatings and thus protected from extreme environmental conditions. In this study, various coating materials were tested for their stability based on the time taken for disintegration when kept under ambient conditions both in vitro and in vivo. The in vivo performance was assessed for placement in soil as well as in banana pseudostem. The coating materials tested was Hard Gelatin Transparent (HGT), Hard Gelatin Coloured (HGC) and Hydroxy Propyl Methyl Cellulose (HPMC), while the carrier materials used were talc, chitin, chitosan, sodium alginate and calcium alginate. The entomopathogen encapsulated was Beauveria bassiana (Balsamo) Vuillemin and the efficacy of product was assessed against pseudostem weevil, Odoiporus longicollis (Olivier). HGT was the best coating material as it was stable under room temperature and normal atmospheric humidity. For soil placement, chitosan based capsules in transparent gelatin coating took only 24 to 48 hr to disintegrate completely under varying soil moisture. Talc based capsules in transparent gelatin coating got disintegrated completely at the end of 24 hr when placed in banana pseudostem, owing to the receipt of sufficient humidity. Placement of capsules in leaf axils or pseudostem sheath can be adopted for prophylactic control of pseudostem weevil and placement in bore holes can be considered for curative application. The ideal moisture content of filler material for fungal encapsulation was determined as 10%. Through this research paper, we would like to disclose about the ideal coating material, carrier material and moisture content for the encapsulation of entomopathogenic fungi. [ABSTRACT FROM AUTHOR]

Pneumococcal conjugate vaccines have been successful, but their use has increased infections by nonvaccine serotypes. Oral streptococci often harbor capsular polysaccharide (PS) synthesis loci (cps). Although this has not been observed in nature, if pneumococcus can replace its cps with oral streptococcal cps, it may increase its serotype repertoire. In the current study, we showed that oral Streptococcus strain SK95 and pneumococcal strain D39 both produce structurally identical capsular PS, and their genetic backgrounds influence the amount of capsule production and shielding from nonspecific killing. SK95 is avirulent in a well-established in vivo mouse model. When acapsular pneumococcus was transformed with SK95 cps, the transformant became virulent and killed all mice. Thus, cps from oral Streptococcus strains can make acapsular pneumococcus virulent, and interspecies cps transfer should be considered a potential mechanism of serotype replacement. Our findings, along with publications from the US Centers for Disease Control and Prevention, highlight potential limitations of the 2013 World Health Organization criterion for studying pneumococcal serotypes carried without isolating bacteria. We show that an oral streptococcal strain, SK95, and a pneumococcal strain, D39, both produce chemically identical capsular PS. We also show that transferring SK95 cps into noncapsulated, avirulent pneumococcus gave it the capacity for virulence in a mouse model. [ABSTRACT FROM AUTHOR]

Isolates of Enterobacteriaceae collected from the same patient can lose carbapenem susceptibility during antimicrobial therapy, but little attention has been given to how this conversion takes place. In the current study, we retrospectively analyzed microbiological and clinical data from patients with enterobacterial infections at a tertiary hospital in Shanghai, China. After screening 4795 patients and 7120 Enterobacteriaceae isolates over the 3-year study period, we found the change from carbapenem susceptible to carbapenem resistant in 41 pairs of isolates, of which 35 pairs (85.4%) were K. pneumoniae and 25 (61.0%) were from the same anatomic sites. Thirty-six isolate pairs showed different pulsed-field gel electrophoresis patterns between the carbapenem-susceptible and the corresponding resistant strain, and 5 pairs displayed identical pulsed-field gel electrophoresis patterns. Thirty-three (91.7%) of the 36 pairs of Enterobacteriaceae isolates were carbapenem-resistant K. pneumoniae with blaKPC-2, and 28 pairs (90.3%) of K. pneumoniae isolates had different sequence types (STs), with ST11 the most common ST found in carbapenem-resistant K. pneumoniae isolates. Forty of the 41 patients had received antimicrobial therapy such as carbapenems, cephalosporins, and fluoroquinolones, before the isolation of carbapenem-resistant Enterobacteriaceae. These results demonstrated that strain replacement is the main cause of emerging carbapenem resistance in Enterobacteriaceae during hospitalization. The loss of carbapenem susceptibility was not mainly due to in vivo development of carbapenem resistance. [ABSTRACT FROM AUTHOR]

The Gram-negative bacterium Klebsiella pneumoniae is an opportunistic pathogen, which can cause life-threatening infections such as sepsis. Worldwide, emerging multidrug resistant K. pneumoniae infections are challenging to treat, hence leading to increased mortality. Therefore, understanding the interactions between K. pneumoniae and the immune system is important to develop new treatment options. We characterized ten clinical K. pneumoniae isolates obtained from blood of bacteremia patients. The interaction of the isolates with human serum was investigated to elucidate how K. pneumoniae escapes the host immune system, and how complement activation by K. pneumoniae changed the capsule structure. All K. pneumoniae isolates activated the alternative complement pathway despite serum resistance of seven isolates. One serum sensitive isolate activated two or all three pathways, and this isolate was lysed and had numerous membrane attack complexes in the outer membrane. However, we also found deposition of complement components in the capsule of serum resistant isolates resulting in morphological capsule changes and capsule shedding. These bacteria did not lyse, and no membrane attack complex was observed despite deposition of C5b-9 within the capsule, indicating that the capsule of serum resistant K. pneumoniae isolates is a defense mechanism against complement-mediated lysis. [ABSTRACT FROM AUTHOR]

Endothelial cell (EC) metabolism is thought to be one of the driving forces for angiogenesis. Here we report the identification of the hexosamine D-mannosamine (ManN) as an EC mitogen and survival factor for bovine and human microvascular EC, with an additivity with VEGF. ManN inhibits glycosylation in ECs and induces significant changes in N-glycan and O-glycan profiles. We further demonstrate that ManN and two N-glycosylation inhibitors stimulate EC proliferation via both JNK activation and the unfolded protein response caused by ER stress. ManN results in enhanced angiogenesis in a mouse skin injury model. ManN also promotes angiogenesis in a mouse hindlimb ischemia model, with accelerated limb blood flow recovery compared to controls. In addition, intraocular injection of ManN induces retinal neovascularization. Therefore, activation of stress pathways following inhibition of protein glycosylation can promote EC proliferation and angiogenesis and may represent a therapeutic strategy for treatment of ischemic disorders.

The TP-84 bacteriophage, which infects Geobacillus stearothermophilus strain 10 ( G. stearothermophilus ), has a genome size of 47.7 kilobase pairs (kbps) and contains 81 predicted protein-coding ORFs. One of these, TP84&#95;26 encodes a putative tail fiber protein possessing capsule depolymerase activity. In this study, we cloned the TP84&#95;26 gene into a high-expression Escherichia coli ( E. coli ) system, modified its N-terminus with His-tag, expressed both the wild type gene and His-tagged variant, purified the recombinant depolymerase variants, and further evaluated their properties. We developed a direct enzymatic assay for the depolymerase activity toward G. stearothermophilus capsules. The recombinant TP84&#95;26 protein variants effectively degraded the existing bacterial capsules and inhibited the formation of new ones. Our results provide insights into the novel TP84&#95;26 depolymerase with specific activity against thermostable G. stearothermophilus and its role in the TP-84 life cycle. The identification and characterization of novel depolymerases, such as TP84&#95;26, hold promise for innovative strategies to combat bacterial infections and improve various industrial processes.

Background Bacterial infections caused by group A Streptococcus (GAS) are common in childhood. Few study reports have provided data on pediatric-specific trends in the epidemiology and bacterial strain characteristics of GAS infections. Methods We prospectively collected GAS isolates from the clinical microbiology laboratory at Texas Children's Hospital between July 1, 2013, and June 30, 2017. Patient characteristics and GAS disease categories were determined through chart review. GAS isolates were obtained from patients in either the inpatient or outpatient setting, and cases were defined as pharyngeal disease, skin and soft-tissue infection (SSTI), or invasive disease on the basis of predefined criteria. All isolates were emm typed to determine trends over time. Results We identified 930 cases over the 4-year period, including 432 (46.4%) pharyngeal, 235 (25.3%) SSTI, and 263 (28.3%) invasive disease types. The most frequently encountered emm types were emm1 (21.4%), emm12 (15.7%), emm89 (14.6%), emm4 (9.2%), and emm3 (8.2%). We observed significant changes over the 4-year period in the relative frequency of infections caused by emm1 (−17.7%; P =.046), emm4 (8.7%; P =.023), or emm6 (−7.9%; P =.024). Using bioinformatic analyses and targeted gene sequencing, we also discovered that all GAS emm28 and emm87 types harbored mutations that rendered them incapable of producing capsule. The relative frequency of GAS disease cases caused by capsule-negative GAS emm types (emm4 , emm22 , emm28 , emm87 , and emm89) increased over the 4-year period (32.2%–44.4%), although the difference was statistically significant for only nonpharyngeal disease types (27.1%–43.9%; P =.038). Conclusions Our data suggest an evolving epidemiology of GAS in the Houston pediatric population characterized by an increase in the frequency of capsule-negative emm types. [ABSTRACT FROM AUTHOR]

Summary: E. coli strains responsible for elevated counts (blooms) in freshwater reservoirs in Australia carry a capsule originating from Klebsiella. The occurrence of Klebsiella capsules in E. coli was about 7% overall and 23 different capsule types were detected. Capsules were observed in strains from phylogroups A, B1 and C, but were absent from phylogroup B2, D, E and F strains. In general, few A, B1 or C lineages were capsule‐positive, but when a lineage was encapsulated multiple different capsule types were present. All Klebsiella capsule‐positive strains were of serogroups O8, O9 and O89. Regardless of the phylogroup, O9 strains were more likely to be capsule‐positive than O8 strains. Given the sequence similarity, it appears that both the capsule region and the O‐antigen gene region are transferred to E. coli from Klebsiella as a single block via horizontal gene transfer events. Pan genome analysis indicated that there were only modest differences between encapsulated and non‐encapsulated strains belonging to phylogroup A. The possession of a Klebsiella capsule, but not the type of capsule, is likely a key determinant of the bloom status of a strain. [ABSTRACT FROM AUTHOR]

Soft-rot Enterobacteriaceae (SRE) typified by Pectobacterium and Dickeya genera are phytopathogenic bacteria inflicting soft-rot disease in crops worldwide. By combining genomic information from 100 SRE with whole-transcriptome datasets, we identified novel genomic and transcriptional associations amongst key pathogenicity themes in this group. Comparative genomics revealed solid linkage between the type I secretion system (T1SS) and the carotovoricin bacteriophage (Ctv) conserved in 96.7% of Pectobacterium genomes. Moreover, their co-activation during infection might indicate a novel functional association involving T1SS/Ctv. Another bacteriophage-borne genomic region mostly confined in less than 10% of Pectobacterium organisms was found, presumably comprising a novel lineage-specific prophage in the genus. We also detected the transcriptional co-regulation of a previously predicted toxin/immunity pair (WHH and SMI1_KNR4 families) along with type VI secretion system (T6SS) and hcp/vgrG genes suggesting a role in disease development as T6SS-dependent effectors. Further, we showed that another predicted T6SS-dependent endonuclease (AHH-family) exhibited toxicity in ectopic expression assays indicating antibacterial activity. Additionally, we report the striking conservation of group-4-capsule (GFC) cluster in 100 SRE strains which consistently features adjacently conserved serotype-specific gene-arrays comprising a previously unknown organization in GFC clusters. Also, extensive sequence variations found in gfcA orthologs suggest a serotype-specific role in the GfcABCD machinery. [ABSTRACT FROM AUTHOR]

Capsules allow bacteria to colonize novel environments, to withstand numerous stresses, and to resist antibiotics. Yet, even though genetic exchanges with other cells should be adaptive under such circumstances, it has been suggested that capsules lower the rates of homologous recombination and horizontal gene transfer. We analysed over one hundred pan-genomes and thousands of bacterial genomes for the evidence of an association between genetic exchanges (or lack thereof) and the presence of a capsule system. We found that bacteria encoding capsules have larger pan-genomes, higher rates of horizontal gene transfer, and higher rates of homologous recombination in their core genomes. Accordingly, genomes encoding capsules have more plasmids, conjugative elements, transposases, prophages, and integrons. Furthermore, capsular loci are frequent in plasmids, and can be found in prophages. These results are valid for Bacteria, independently of their ability to be naturally transformable. Since we have shown previously that capsules are commonly present in nosocomial pathogens, we analysed their co-occurrence with antibiotic resistance genes. Genomes encoding capsules have more antibiotic resistance genes, especially those encoding efflux pumps, and they constitute the majority of the most worrisome nosocomial bacteria. We conclude that bacteria with capsule systems are more genetically diverse and have fast-evolving gene repertoires, which may further contribute to their success in colonizing novel niches such as humans under antibiotic therapy. [ABSTRACT FROM AUTHOR]

A novel concept is proposed in which alginate capsules containing a model probiotic Lactobacillus plantarum strain are coated with different surfactants with the aim to enhance cell survival during passage initially through simulated gastric (SGF) and then intestinal (SIF) fluid. The surfactants investigated included the anionic sodium dodecyl sulphate (SDS) and ammonium lauryl sulphate (ALS), the cationic dimethyldioctadecylammonium chloride (DDAC), benzalkonium chloride (BZK) and hexadecyltrimethylammonium bromide (CTAB), and the zwitterionic lecithin. Coating the alginate capsules with CTAB, BZK, ALS and SDS resulted in worst survival (∼4–9 log CFU/g decrease) compared to uncoated capsules (∼3 log CFU/g decrease), after 1 h exposure to SGF and two hours in SIF, which was most likely associated with their gradual penetration inside the microcapsules, as shown by confocal microscopy, and their antimicrobial effects. Coating the alginate capsules with DDAC improved cell survival compared to uncoated capsules (∼1.2 CFU/g decrease), whereas coating with lecithin improved cell survival considerably, resulting in almost complete recovery of viable cells in SGF and SIF (∼0.3 log CFU/g decrease). Although the interaction between alginate and lecithin was relatively weak as demonstrated by turbidity and contact angle measurements, it is likely that the protection was associated with the fact that lecithin was able to penetrate into the capsule rapidly, an observation that was supported by the fact that lecithin enhanced the viability of free cells in SGF and SIF. Lecithin has significant potential of being used as a coating material for probiotic containing capsules. [ABSTRACT FROM AUTHOR]

The constitutively expressed hyaluronic acid capsule is an important virulence factor of Streptococcus equi , the cause of equine strangles. Study of the genomic sequence of CF22caps–, a non-encapsulated mutant of S. equi CF22 generated by gamma (Co 60 ) irradiation revealed a non-sense mutation in fasC (SEQ_0302), a sensor kinase gene in FasBCAX an operon with an important regulatory role in expression of streptococcal secreted virulence and matrix binding proteins. The mutation was associated with a significant (p < .05) decrease in transcription of hasA, the synthase gene essential for hyaluronic acid synthesis and, conversely, with small increases in transcription of skc, covR and seM . The early growth phase of CF22caps– was also delayed compared to the CF22caps + parent. In contrast to the human pathogen, S. pyogenes, capsule synthesis in S. equi therefore appears to be controlled by FasBCAX and not by CovRS. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) has been associated with illnesses and outbreaks linked to fresh vegetables, prompting a growing public health concern. Most studies regarding interactions of STEC on fresh produce focused on E. coli O157:H7. Limited information is available about survival or fitness of E. coli O104:H4, non-O157 pathogen that was linked to one of the largest outbreaks of hemolytic uremic syndrome in 2011. In this study, survival of E. coli O104:H4 was evaluated on Arabidopsis thaliana plant and lettuce for 5 days compared with E. coli O157:H7, and expression of pathogenesis-realted gene ( PR1 ; induction of plant defense response) was examined by reverse transcription quantitative PCR, and potential influence of capsular polysaccharide (CPS) on the bacterial fitness on plant was investigated. Populations of E. coli O104:H4 strains (RG1, C3493, and LpfA) on Arabidopsis and lettuce were significantly ( P  < 0.05) greater than those of E. coli O157:H7 strains (7386 and sakai) at day 5 post-inoculation, indicating E. coli O104:H4 may have better survival ability on the plants. In addition, the E. coli O104:H4 strains produced significantly ( P  < 0.05) higher amounts of CPS compared with the E. coli O157:H7 strains. RG1 strain (1.5-fold) initiated significantly ( P  < 0.05) lower expression of PR1 gene indicating induction of plant defense response compared with E. coli O157:H7 strains 7386 (2.9-fold) and sakai (2.7-fold). Collectively, the results in this study suggests that different level of CPS production and plant defense response initiated by each STEC strain might influence the bacterial survival or persistence on plants. The present study provides better understanding of survival behavior of STEC, particularly E. coli O104:H4, using a model plant and vegetable under pre-harvest conditions with plant defense response. [ABSTRACT FROM AUTHOR]

Cosmic dust samples from the surface of the illuminator of the International Space Station (ISS) were collected by a crew member during his spacewalk. The sampler with tampon in a vacuum container was delivered to the Earth. Washouts from the tampon's material and the tampon itself were analyzed for the presence of bacterial DNA by the method of nested PCR with primers specific to DNA of the genus Mycobacteria, DNA of the strains of capsular bacteria Bacillus, and DNA encoding 16S ribosomal RNA. The results of amplification followed by sequencing and phylogenetic analysis indicated the presence of the bacteria of the genus Mycobacteria and the extreme bacterium of the genus Delftia in the samples of cosmic dust. It was shown that the DNA sequence of one of the bacteria of the genus Mycobacteria was genetically similar to that previously observed in superficial micro layer at the Barents and Kara seas' coastal zones. The presence of the wild land and marine bacteria DNA on the ISS suggests their possible transfer from the stratosphere into the ionosphere with the ascending branch of the global electric circuit. Alternatively, the wild land and marine bacteria as well as the ISS bacteria may all have an ultimate space origin. [ABSTRACT FROM AUTHOR]

Pyrimidine nucleotides play an important role in the biosynthesis of activated nucleotide sugars (NDP-sugars). NDP-sugars are the precursors of structural polysaccharides in bacteria, including capsule, which is a major virulence factor of the human pathogen S. pneumoniae. In this work, we identified a spontaneous non-reversible mutant of strain D39 that displayed a non-producing capsule phenotype. Whole-genome sequencing analysis of this mutant revealed several non-synonymous single base modifications, including in genes of the de novo synthesis of pyrimidines and in the -10 box of capsule operon promoter (Pcps). By directed mutagenesis we showed that the point mutation in Pcps was solely responsible for the drastic decrease in capsule expression. We also demonstrated that D39 subjected to uracil deprivation shows increased biomass and decreased Pcps activity and capsule amounts. Importantly, Pcps expression is further decreased by mutating the first gene of the de novo synthesis of pyrimidines, carA. In contrast, the absence of uracil from the culture medium showed no effect on the spontaneous mutant strain. Co-cultivation of the wild-type and the mutant strain indicated a competitive advantage of the spontaneous mutant (non-producing capsule) in medium devoid of uracil. We propose a model in that uracil may act as a signal for the production of different capsule amounts in S. pneumoniae. [ABSTRACT FROM AUTHOR]

Encapsulated Haemophilus influenzae strains belong to type-specific genetic lineages. Reliable capsule typing requires PCR, but a more efficient method would be useful. We evaluated capsule typing by using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Isolates of all capsule types (a-f and nontypeable; n = 258) and isogenic capsule transformants (types a-d) were investigated. Principal component and biomarker analyses of mass spectra showed clustering, and mass peaks correlated with capsule type-specific genetic lineages. We used 31 selected isolates to construct a capsule typing database. Validation with the remaining isolates (n = 227) showed 100% sensitivity and 92.2% specificity for encapsulated strains (a-f; n = 61). Blinded validation of a supplemented database (n = 50) using clinical isolates (n = 126) showed 100% sensitivity and 100% specificity for encapsulated strains (b, e, and f; n = 28). MALDI-TOF mass spectrometry is an accurate method for capsule typing of H. influenzae. [ABSTRACT FROM AUTHOR]

Invasive infections caused by Streptococcus pneumoniae, a commensal in the nasopharynx, pose significant risk to human health. Limited serotype coverage by the available polysaccharide-based conjugate vaccines coupled with increasing incidence of antibiotic resistance complicates therapeutic strategies. Bacterial physiology and metabolism that allows pathogens to adapt to the host are a promising avenue for the discovery of novel therapeutics. Intracellular polyamine concentrations are tightly regulated by biosynthesis, transport and degradation. We previously reported that deletion of cadA, a gene that encodes for lysine decarboxylase, an enzyme that catalyzes cadaverine synthesis results in an attenuated phenotype. Here, we report the impact of cadA deletion on pneumococcal capsule and protein expression. Our data show that genes for polyamine biosynthesis and transport are downregulated in DcadA. Immunoblot assays show reduced capsule in DcadA. Reduced capsule synthesis could be due to reduced transcription and availability of precursors for synthesis. The capsule is the predominant virulence factor in pneumococci and is critical for evading opsonophagocytosis and its loss in DcadA could explain the reported attenuation in vivo. Results from this study show that capsule synthesis in pneumococci is regulated by polyamine metabolism, which can be targeted for developing novel therapies. [ABSTRACT FROM AUTHOR]

Objectives: The bacterial pathogen Neisseria meningitidis is able to escape the currently available capsule-based vaccines by undergoing capsule switching. In this study, we investigated whether capsule switching has occurred in a recently emerged sequence type (ST) 7 serogroup X isolate in China, for which currently no vaccine is available.Methods: To identify capsule switching breakpoints, the capsule locus and flanking regions of the ST-7 serogroup X isolate and three endemic ST-7 serogroup A isolates were sequenced and compared. To obtain further insight into capsule switching frequency and length of DNA fragments involved, capsule switching assays were performed using genomic DNA containing combinations of antibiotic selection markers at various locations in the capsule locus and flanking regions.Results: Sequence analyses showed that capsule switching has occurred and involved a 8450 bp serogroup X DNA fragment spanning the region from galE to ctrC. Capsule switching assays indicate that capsule switching occurs at a frequency of 6.3 × 10-6 per bacterium per μg of DNA and predominantly involved DNA fragments of about 8.1-9.6 kb in length.Conclusions: Our results show that capsule switching in N. meningitidis occurs at high frequency and involves recombination in the flanking regions of the capsule biosynthesis genes. [ABSTRACT FROM AUTHOR]

Lactobacillus strains can synthesize capsular polysaccharides (CPS), which are important substances in the dairy industry-they exhibit many important technological as well as health-promoting properties. Technological advancements have made it possible to detect bacterial capsules using costly and labor-intensive methods, such as serological reactions, molecular genetic techniques, and electron microscopy. Light microscopy, which is the method of interest in this paper, is one of the most widely accessible and cheapest techniques. CPS may be observed under a light microscope after staining bacterial cells and the background with a basic die and an acidic die, respectively (negative-positive staining), with the capsules remaining transparent. The literature offers many polysaccharide staining methods, but due to the considerable structural diversity of CPS and possible dye-capsule interactions, a suitable staining technique should be carefully selected for each strain. The current study showed that not all methods adequately reveal Lactobacillus CPS, with the most effective ones being those proposed by Hiss and Maneval. [ABSTRACT FROM AUTHOR]

The gram-negative bacterium Francisella tularensis (Ft) is both a potential biological weapon and a naturally occurring microbe that survives in arthropods, fresh water amoeba, and mammals with distinct phenotypes in various environments. Previously, we used a number of measurements to characterize Ft grown in Brain-Heart Infusion (BHI) broth as (1) more similar to infection-derived bacteria, and (2) slightly more virulent in naïve animals, compared to Ft grown in Mueller Hinton Broth (MHB). In these studies we observed that the free amino acids in MHB repress expression of select Ft virulence factors by an unknown mechanism. Here, we tested the hypotheses that Ft grown in BHI (BHI-Ft) accurately displays a full protein composition more similar to that reported for infection-derived Ft and that this similarity would make BHI-Ft more susceptible to pre-existing, vaccine-induced immunity than MHB-Ft. We performed comprehensive proteomic analysis of Ft grown in MHB, BHI, and BHI supplemented with casamino acids (BCA) and compared our findings to published “omics” data derived from Ft grown in vivo. Based on the abundance of ~1,000 proteins, the fingerprint of BHI-Ft is one of nutrient-deprived bacteria that—through induction of a stringent-starvation-like response—have induced the FevR regulon for expression of the bacterium's virulence factors, immuno-dominant antigens, and surface-carbohydrate synthases. To test the notion that increased abundance of dominant antigens expressed by BHI-Ft would render these bacteria more susceptible to pre-existing, vaccine-induced immunity, we employed a battery of LVS-vaccination and S4-challenge protocols using MHB- and BHI-grown Ft S4. Contrary to our hypothesis, these experiments reveal that LVS-immunization provides a barrier to infection that is significantly more effective against an MHB-S4 challenge than a BHI-S4 challenge. The differences in apparent virulence to immunized mice are profoundly greater than those observed with primary infection of naïve mice. Our findings suggest that tularemia vaccination studies should be critically evaluated in regard to the growth conditions of the challenge agent.