Essential phage component induces resistance of bacterial community.

Despite extensive knowledge on phage resistance at bacterium level, the resistance of bacterial communities is still not well-understood. Given its ubiquity, it is essential to understand resistance at the community level. We performed quantitative investigations on the dynamics of phage infection in Klebsiella pneumoniae biofilms. We found that the biofilms quickly developed resistance and resumed growth. Instead of mutations, the resistance was caused by unassembled phage tail fibers released by the phage-lysed bacteria. The tail fibers degraded the bacterial capsule essential for infection and induced spreading of capsule loss in the biofilm, and tuning tail fiber and capsule levels altered the resistance. Latent infections sustained in the biofilm despite resistance, allowing stable phage-bacteria coexistence. Last, we showed that the resistance exposed vulnerabilities in the biofilm. Our findings indicate that phage lysate plays important roles in shaping phage-biofilm interactions and open more dimensions for the rational design of strategies to counter bacteria with phage.

Genetic heterogeneity in the Salmonella Typhi Vi capsule locus: a population genomic study from Fiji.

Typhoid fever is endemic in many parts of the world and remains a major public health concern in tropical and sub-tropical developing nations, including Fiji. To address high rates of typhoid fever, the Northern Division of Fiji implemented a mass vaccination with typhoid conjugate vaccine (Vi-polysaccharide conjugated to tetanus toxoid) as a public health control measure in 2023. In this study we define the genomic epidemiology of Salmonella Typhi in the Northern Division prior to island-wide vaccination, sequencing 85% (n=419) of the total cases from the Northern and Central Divisions of Fiji that occurred in the period 2017-2019. We found elevated rates of nucleotide polymorphisms in the tviD and tviE genes (responsible for Vi-polysaccharide synthesis) relative to core genome levels within the Fiji endemic S. Typhi genotype 4.2. Expansion of these findings within a globally representative database of 12 382 S. Typhi (86 genotyphi clusters) showed evidence of convergent evolution of the same tviE mutations across the S. Typhi population, indicating that tvi selection has occurred both independently and globally. The functional impact of tvi mutations on the Vi-capsular structure and other phenotypic characteristics are not fully elucidated, yet commonly occurring tviE polymorphisms localize adjacent to predicted active site residues when overlayed against the predicted TviE protein structure. Given the central role of the Vi-polysaccharide in S. Typhi biology and vaccination, further integrated epidemiological, genomic and phenotypic surveillance is required to determine the spread and functional implications of these mutations.

Haemophilus influenzae Type b Vaccine Immunogenicity in American Indian/Alaska Native Infants.

OBJECTIVES: American Indian and Alaska Native (AI/AN) infants historically experienced a disproportionate burden of invasive Haemophilus influenzae type b (Hib) disease, especially early in life. PedvaxHIB vaccine is preferentially recommended for AI/AN infants because it elicits protective antibody levels postdose 1. Vaxelis, a hexavalent vaccine that contains the same Hib conjugate as PedvaxHIB but at lower concentration, is recommended for US children, but postdose 1 Hib immunogenicity data are needed to inform whether a preferential recommendation should be made for AI/AN infants. METHODS: We conducted a phase IV randomized, open-label, noninferiority trial comparing postdose 1 immunogenicity of Vaxelis to PedvaxHIB in AI/AN infants. Participants were randomized to receive a primary series of PedvaxHIB or Vaxelis. Serum samples collected 30 days postdose 1 were tested for anti-Hib immunoglobulin G antibody by enzyme-linked immunosorbent assay. The anti-Hib immunoglobulin G geometric mean concentration (GMC) ratio (Vaxelis/PedvaxHIB) was estimated by constrained longitudinal data analysis. Noninferiority was defined a priori as the lower bound of the 95% confidence interval (CI) of the GMC ratio ≥0.67. RESULTS: A total of 327 of the 333 infants enrolled in the study were included in the per-protocol analysis. The postdose 1 anti-Hib GMC was 0.41 µg/mL (95% CI 0.33-0.52) in the Vaxelis group (n = 152) and 0.39 µg/mL (95% CI 0.31-0.50) in the PedvaxHIB group (n = 146). The constrained longitudinal data analysis GMC ratio was 1.03 (95% CI 0.76-1.39). CONCLUSIONS: Postdose 1 immunogenicity of Vaxelis was noninferior to PedvaxHIB. Our findings support the use of Vaxelis in AI/AN children, a population with elevated risk of Hib disease.

The unique Legionella longbeachae capsule favors intracellular replication and immune evasion.

Legionella longbeachae and Legionella pneumophila are the most common causative agents of Legionnaires' disease. While the clinical manifestations caused by both species are similar, species-specific differences exist in environmental niches, disease epidemiology, and genomic content. One such difference is the presence of a genomic locus predicted to encode a capsule. Here, we show that L. longbeachae indeed expresses a capsule in post-exponential growth phase as evidenced by electron microscopy analyses, and that capsule expression is abrogated when deleting a capsule transporter gene. Capsule purification and its analysis via HLPC revealed the presence of a highly anionic polysaccharide that is absent in the capsule mutant. The capsule is important for replication and virulence in vivo in a mouse model of infection and in the natural host Acanthamoeba castellanii. It has anti-phagocytic function when encountering innate immune cells such as human macrophages and it is involved in the low cytokine responses in mice and in human monocyte derived macrophages, thus dampening the innate immune response. Thus, the here characterized L. longbeachae capsule is a novel virulence factor, unique among the known Legionella species, which may aid L. longbeachae to survive in its specific niches and which partly confers L. longbeachae its unique infection characteristics.

Synthesis of the pyruvic acid acetal containing tetrasaccharide repeating unit corresponding to the K82 capsular polysaccharide of Acinetobacter baumannii LUH5534 strain.

An efficient synthetic strategy has been developed to achieve a pyruvic acid acetal containing tetrasaccharide repeating unit corresponding to the K82 capsular polysaccharide of Acinetobacter baumannii LUH5534 strain in very good yield. The synthetic scheme involves the use of suitably functionalized monosaccharide thioglycosides as glycosyl donors and a combination of N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) as thiophilic glycosylation activator to furnish satisfactory yield of the products with appropriate stereochemistry at the glycosidic linkages. Incorporation of the (R)-pyruvic acid acetal in the d-galactose moiety was achieved in very good yield by the treatment of the diol derivative with methyl 2,2-bis(p-methylphenylthio)propionate in the presence of a combination of NIS and triflic acid.

Quantitative determination of C-polysaccharide in Streptococcus pneumoniae capsular polysaccharides by enzyme-linked immunosorbent assay.

Capsular polysaccharides of Streptococcus pneumoniae are used in pneumococcal polysaccharide and protein-conjugate vaccines. Cell-wall polysaccharide (C-Ps) is a critical impurity that must be kept at low levels in purified polysaccharide preparations. Hence, accurate and precise methods for determining C-Ps are needed. Currently available methods include nuclear magnetic resonance (NMR) spectroscopy and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both these methods suffer from their own limitations; therefore, we developed a simple and efficient enzyme-linked immunosorbent assay (ELISA) for accurate and precise quantification of C-Ps in samples of any serotype of pneumococcal capsular polysaccharide without interference. We quantified C-Ps in preparations of 14 serotype polysaccharides using newly developed ELISA method and compared the results with C-Ps values obtained using two previously reported methods, 1H NMR and HPAEC-PAD. The C-Ps value determined using 1H NMR for serotype 5 was 21.08%, whereas the values obtained using HPAEC-PAD and ELISA were 2.38% and 2.89% respectively, indicating some interference in 1H NMR method. The sensitivity of the ELISA method is higher because the sample is used directly unlike HPAEC-PAD method where sample is subjected to harsh treatment, such as acid digestion and quantify C-Ps based on peak area of ribitol or AAT. Furthermore, 1H NMR and HPAEC-PAD are expensive and laborious methods. Our work, underscores the simple and efficient ELISA that can be used for quantification of C-Ps in pneumococcal polysaccharide preparations.

Inhibition of porcine origin Klebsiella pneumoniae capsular polysaccharide and immune escape by BY3 compounded traditional Chinese medicine residue fermentation broth.

Klebsiella pneumoniae (K. pneumoniae) is a gram-negative conditionally pathogenic bacterium that causes disease primarily in immunocompromised individuals. Recently, highly virulent K. pneumoniae strains have caused severe disease in healthy individuals, posing significant challenges to global infection control. Capsular polysaccharide (CPS), a major virulence determinant of K. pneumoniae, protects the bacteria from being killed by the host immune system, suggesting an urgent need for the development of drugs to prevent or treat K. pneumoniae infections. In this study, BY3 compounded traditional Chinese medicine residue (TCMR) was carried out using Lactobacillus rhamnosus as a fermentation strain, and BY3 compounded TCMR fermentation broth (BY3 fermentation broth) was obtained. The transcription of K. pneumoniae CPS-related biosynthesis genes after treatment with BY3 fermentation broth was detected using quantitative real-time polymerase chain reaction. The effects of BY3 fermentation broth on K. pneumoniae serum killing, macrophage phagocytosis, complement deposition and human ß-defensin transcription were investigated. The therapeutic effect of BY3 fermentation broth on K. pneumoniae-infected mice was also observed, and the major active components of BY3 fermentation broth were analysed via LC‒MS analysis, network pharmacology, and molecular docking. The results showed that BY3 fermentation broth inhibited K. pneumoniae CPS production and downregulated transcription of CPS-related biosynthesis genes, which weakened bacterial resistance to serum killing and phagocytosis, while promoting bacterial surface complement C3 deposition and human ß-defensin expression. BY3 fermentation broth demonstrated safety and therapeutic effects in vivo and in vitro, restoring body weight and visceral indices, significantly reducing the organ bacterial load and serum cytokine levels, and alleviating pathological organ damage in mice. In addition, three natural compounds-oleanolic acid, quercetin, and palmitoleic acid-were identified as the major active components in the BY3 fermentation broth. Therefore, BY3 fermentation broth may be a promising strategy for the prevention or treatment of K. pneumoniae infections.

Synergy between Group 2 capsules and lipopolysaccharide underpins serum resistance in extra-intestinal pathogenic Escherichia coli.

Escherichia coli (E. coli) is a major cause of urinary tract infections, bacteraemia, and sepsis. CFT073 is a prototypic, urosepsis isolate of sequence type (ST) 73. This laboratory, among others, has shown that strain CFT073 is resistant to serum, with capsule and other extracellular polysaccharides imparting resistance. The interplay of such polysaccharides remains under-explored. This study has shown that CFT073 mutants deficient in lipopolysaccharide (LPS) O-antigen and capsule display exquisite serum sensitivity. Additionally, O-antigen and LPS outer core mutants displayed significantly decreased surface K2 capsule, coupled with increased unbound K2 capsule being detected in the supernatant. The R1 core and O6 antigen are involved in the tethering of K2 capsule to the CFT073 cell surface, highlighting the importance of the R1 core in serum resistance. The dependence of capsule on LPS was shown to be post-transcriptional and related to changes in cell surface hydrophobicity. Furthermore, immunofluorescence microscopy suggested that the surface pattern of capsule is altered in such LPS core mutants, which display a punctate capsule pattern. Finally, targeting LPS biosynthesis using sub-inhibitory concentrations of a WaaG inhibitor resulted in increased serum sensitivity and decreased capsule in CFT073. Interestingly, the dependency of capsule on LPS has been observed previously in other Enterobacteria, indicating that the synergy between these polysaccharides is not just strain, serotype or species-specific but may be conserved across several pathogenic Gram-negative species. Therefore, using WaaG inhibitor derivatives to target LPS is a promising therapeutic strategy to reduce morbidity and mortality by reducing or eliminating surface capsule.

Microaerobic-mediated suppression of Klebsiella pneumoniae mucoviscosity is restored by rmpD overexpression.

AIMS: Hypervirulent Klebsiella pneumoniae (hvKp) causes invasive community-acquired infections in healthy individuals, and hypermucoviscosity (HMV) is the main phenotype associated with hvKp. This study investigates the impact of microaerobic environment availability on the mucoviscosity of K. pneumoniae. METHODS AND RESULTS: By culturing 25 clinical strains under microaerobic and aerobic environments, we observed a notable reduction in mucoviscosity in microaerobic environments. RNA sequencing and qRT-PCR revealed downregulated expressions of capsule synthesis genes (galf, orf2, wzi, wza, wzb, wzc, wcaj, manC, manB, and ugd) and regulatory genes (rmpA, rmpD, and rmpC) under microaerobic conditions. Transmission electron microscopy and Indian ink staining analysis were performed, revealing that the capsular thickness of K. pneumoniae decreased by half in microaerobic conditions compared to aerobic conditions. Deletion of rmpD and rmpC caused the loss of the HMV phenotype in both aerobic and microaerobic conditions. However, compared to wild-type strain in microaerobic condition, only rmpD overexpression strain, and not rmpC overexpression strain, displayed a significant increase in capsule thickness in microaerobic conditions. CONCLUSIONS: Microaerobic conditions can suppress the mucoviscosity of K. pneumoniae, but this suppression can be overcome by altering the expression of rmpD, indicating a specific function for rmpD in the oxygen environmental adaptation of K. pneumoniae.

Insights into the recognition of hypermucoviscous Klebsiella pneumoniae clinical isolates by innate immune lectins of the Siglec and galectin families.

Klebsiella pneumoniae is an opportunistic bacterium that frequently colonizes the nasopharynx and gastrointestinal tract and can also cause severe infections when invading other tissues, particularly in immunocompromised individuals. Moreover, K. pneumoniae variants exhibiting a hypermucoviscous (HMV) phenotype are usually associated with hypervirulent strains that can produce invasive infections even in immunocompetent individuals. Major carbohydrate structures displayed on the K. pneumoniae surface are the polysaccharide capsule and the lipopolysaccharide, which presents an O-polysaccharide chain in its outermost part. Various capsular and O-chain structures have been described. Of note, production of a thick capsule is frequently observed in HMV variants. Here we examined the surface sugar epitopes of a collection of HMV and non-HMV K. pneumoniae clinical isolates and their recognition by several Siglecs and galectins, two lectin families of the innate immune system, using bacteria microarrays as main tool. No significant differences among isolates in sialic acid content or recognition by Siglecs were observed. In contrast, analysis of the binding of model lectins with diverse carbohydrate-binding specificities revealed striking differences in the recognition by galactose- and mannose-specific lectins, which correlated with the binding or lack of binding of galectins and pointed to the O-chain as the plausible ligand. Fluorescence microscopy and microarray analyses of galectin-9 binding to entire cells and outer membranes of two representative HMV isolates supported the bacteria microarray results. In addition, Western blot analysis of the binding of galectin-9 to outer membranes unveiled protein bands recognized by this galectin, and fingerprint analysis of these bands identified several proteins containing potential O-glycosylation sites, thus broadening the spectrum of possible galectin ligands on the K. pneumoniae surface. Moreover, Siglecs and galectins apparently target different structures on K. pneumoniae surfaces, thereby behaving as non-redundant complementary tools of the innate immune system.

RNA interactome of hypervirulent Klebsiella pneumoniae reveals a small RNA inhibitor of capsular mucoviscosity and virulence.

Hypervirulent Klebsiella pneumoniae (HvKP) is an emerging bacterial pathogen causing invasive infection in immune-competent humans. The hypervirulence is strongly linked to the overproduction of hypermucoviscous capsule, but the underlying regulatory mechanisms of hypermucoviscosity (HMV) have been elusive, especially at the post-transcriptional level mediated by small noncoding RNAs (sRNAs). Using a recently developed RNA interactome profiling approach iRIL-seq, we interrogate the Hfq-associated sRNA regulatory network and establish an intracellular RNA-RNA interactome in HvKP. Our data reveal numerous interactions between sRNAs and HMV-related mRNAs, and identify a plethora of sRNAs that repress or promote HMV. One of the strongest HMV repressors is ArcZ, which is activated by the catabolite regulator CRP and targets many HMV-related genes including mlaA and fbp. We discover that MlaA and its function in phospholipid transport is crucial for capsule retention and HMV, inactivation of which abolishes Klebsiella virulence in mice. ArcZ overexpression drastically reduces bacterial burden in mice and reduces HMV in multiple hypervirulent and carbapenem-resistant clinical isolates, indicating ArcZ is a potent RNA inhibitor of bacterial pneumonia with therapeutic potential. Our work unravels a novel CRP-ArcZ-MlaA regulatory circuit of HMV and provides mechanistic insights into the posttranscriptional virulence control in a superbug of global concern.

The GE296_RS03820 and GE296_RS03830 genes are involved in capsular polysaccharide biosynthesis in Riemerella anatipestifer.

Riemerella anatipestifer is a pathogenic bacterium that causes duck serositis and meningitis, leading to significant harm to the duck industry. To escape from the host immune system, the meningitis-causing bacteria must survive and multiply in the bloodstream, relying on specific virulence factors such as capsules. Therefore, it is essential to study the genes involved in capsule biosynthesis in R. anatipestifer. In this study, we successfully constructed gene deletion mutants Δ3820 and Δ3830, targeting the GE296_RS03820 and GE296_RS03830 genes, respectively, using the RA-LZ01 strain as the parental strain. The growth kinetics analysis revealed that these two genes contribute to bacterial growth. Transmission and scanning electron microscopy (TEM and SEM) and silver staining showed that Δ3820 and Δ3830 produced the altered capsules and compounds of capsular polysaccharides (CPSs). Serum resistance test showed the mutants also exhibited reduced C3b deposition and decreased resistance serum killing. In vivo, Δ3820 and Δ3830 exhibited markedly declining capacity to cross the blood-brain barrier, compared to RA-LZ01. These findings indicate that the GE296_RS03820 and GE296_RS03830 genes are involved in CPSs biosynthesis and play a key role in the pathogenicity of R. anatipestifer. Furthermore, Δ3820 and Δ3830 mutants presented a tendency toward higher survival rates from RA-LZ01 challenge in vivo. Additionally, sera from ducklings immunized with the mutants showed cross-immunoreactivity with different serotypes of R. anatipestifer, including 1, 2, 7 and 10. Western blot and SDS-PAGE assays revealed that the altered CPSs of Δ3820 and Δ3830 resulted in the exposure of some conserved proteins playing the key role in the cross-immunoreactivity. Our study clearly demonstrated that the GE296_RS03820 and GE296_RS03830 genes are involved in CPS biosynthesis in R. anatipestifer and the capsule is a target for attenuation in vaccine development.

In Vivo Evolution of a Klebsiella pneumoniae Capsule Defect With wcaJ Mutation Promotes Complement-Mediated Opsonophagocytosis During Recurrent Infection.

BACKGROUND: Klebsiella pneumoniae carbapenemase-producing K pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. METHODS: We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct enzyme-linked immunosorbent assay, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. RESULTS: We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In 5 genetically related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsonophagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. CONCLUSIONS: Loss of function in wcaJ led to increased complement resistance, complement binding, and opsonophagocytosis, which may promote KPC-Kp persistence by enabling coexistence of increased bloodstream fitness and reduced tissue virulence.

Clonal Expansion of a Streptococcus pneumoniae Serotype 3 Capsule Variant Sequence Type 700 With Enhanced Vaccine Escape Potential After 13-Valent Pneumococcal Conjugate Vaccine Introduction.

BACKGROUND: Streptococcus pneumoniae serotype 3 remains a problem globally. Malawi introduced 13-valent pneumococcal conjugate vaccine (PCV13) in 2011, but there has been no direct protection against serotype 3 carriage. We explored whether vaccine escape by serotype 3 is due to clonal expansion of a lineage with a competitive advantage. METHODS: The distribution of serotype 3 Global Pneumococcal Sequence Clusters (GPSCs) and sequence types (STs) globally was assessed using sequences from the Global Pneumococcal Sequencing Project. Whole-genome sequences of 135 serotype 3 carriage isolates from Blantyre, Malawi (2015-2019) were analyzed. Comparative analysis of the capsule locus, entire genomes, antimicrobial resistance, and phylogenetic reconstructions were undertaken. Opsonophagocytosis was evaluated using serum samples from vaccinated adults and children. RESULTS: Serotype 3 GPSC10-ST700 isolates were most prominent in Malawi. Compared with the prototypical serotype 3 capsular polysaccharide locus sequence, 6 genes are absent, with retention of capsule polysaccharide biosynthesis. This lineage is characterized by increased antimicrobial resistance and lower susceptibility to opsonophagocytic killing. CONCLUSIONS: A serotype 3 variant in Malawi has genotypic and phenotypic characteristics that could enhance vaccine escape and clonal expansion after post-PCV13 introduction. Genomic surveillance among high-burden populations is essential to improve the effectiveness of next-generation pneumococcal vaccines.

Structure characterization and immunological activity of capsular polysaccharide from live and heat-killed Lacticaseibacillus paracasei 6235.

Capsular polysaccharide (CPS) as a probiotic component has the ability to regulate the function of the host's immune system. However, how the structure and function of heat-killed CPS are altered remains unclear. In the present study, CPS were isolated and purified from live (LCPS) and heat-killed (HCPS) Lacticaseibacillus paracasei 6235. The differences in structure and immunomodulation between LCPS and HCPS were compared and analyzed. The results demonstrate that after heat killed, the molecular weight of CPS decreased from 23.4 kDa to 17.5 kDa, with the disappearance of galactosamine in the monosaccharide composition, and changes in the microstructure. Methylation analysis and nuclear magnetic resonance analysis revealed that the LCPS and HCPS are similar in structure, which main units of →3,4)-α-D-Glcp-(1→4)-α-D-Galp-(1→3)-ß-L-Rhap-(1→6)-ß-D-Galp-(1→, and repeating units of →3,4)-α-D-Glcp-(1→, →3)-ß-L-Rhap-(1→, and →4)-α-D-Galp-(1→ residues. Furthermore, both LCPS and HCPS significantly downregulated the expression of pro-inflammatory cytokines in RAW264.7 cells induced by LPS. Specifically, HCPS reduced the levels of IL-6 and IL-1ß by 79.38 % and 88.42 %, respectively, compared to LCPS. Concurrently, both LCPS and HCPS effectively mitigated inflammatory responses through the NF-κB and MAPK signaling pathways. Moreover, compared to LCPS, HCPS increased the protein expression levels of NF-κB/p-NF-κB and IκB/p-IκB by 26.14 % and 28.92 %, respectively. These results suggest that CPS has a role in modulating immune responses and that HCPS is more effective. This study can be further developed into new products related to postbiotics.

[The effect of wza gene deletion in Klebsiella pneumoniae on capsule formation and bacteriophage sensitivity].

Objective: To investigate the effects of wza gene deletion in Klebsiella pneumoniae on capsule formation ability and bacteriophage sensitivity. Methods: The wza deletion mutant strain was constructed through a temperature-sensitive plasmid-mediated homologous recombination. The growth curves of W14 and Δwza were detected by measuring the optical density OD600. In order to analyze the effect of gene wza on bacterial capsule formation, wild-type strain W14 and Δwza mutant strain were detected by transmission electron microscope, and their capsule contents were measured by quantifying the uronic acid contents. The plaque assay was used to detect bacterial sensitivity to bacteriophage in wild-type strain W14 and Δwza mutant strain. The t test was used to compare whether there were differences in the contents of uronic acid in the capsules of wild-type strain W14 and Δwza mutant strain. Results: The PCR results revealed that the Δwza mutant strain was successfully constructed. Compared with wild-type strain W14, the growth curves of Δwza on the solid plates demonstrated a slightly slower growth. However, no difference in growth was observed among wild-type strain W14 and Δwza mutant strains in LB broth. The transmission electron microscope results showed that wza gene deletion resulted in the loss of capsule in bacteria. The uronic acid content assay suggested that the capsule content was significantly decreased in Δwza mutant strain (45.963±2.795) µg/ml compared with wild-type strain W14 (138.800±5.201) µg/ml. There was a statistical difference between the two groups (t=27.233, P<0.001). The plaque assay indicated that bacteria lost its sensitivity to bacteriophage when gene wza was deleted. Conclusion: Deletion of the wza gene impairs bacterial capsule formation ability and can affect bacterial sensitivity to bacteriophage phiW14.

Evolutionary genomic analyses of canine E. coli infections identify a relic capsular locus associated with resistance to multiple classes of antimicrobials.

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.

Quorum sensing positively regulates CPS-dependent Autographiviridae phage infection in Vibrio alginolyticus.

Quorum sensing (QS) orchestrates many bacterial behaviors, including virulence and biofilm formation, across bacterial populations. Nevertheless, the underlying mechanism by which QS regulates capsular polysaccharide (CPS)-dependent phage-bacterium interactions remains unclear. In this study, we report that QS upregulates the expression of CPS-dependent phage receptors, thus increasing phage adsorption and infection rates in Vibrio alginolyticus. We found that QS upregulated the expression of the ugd gene, leading to increased synthesis of Autographiviridae phage receptor CPS synthesis in V. alginolyticus. The signal molecule autoinducer-2 released by Vibrio from different sources can potentially enhance CPS-dependent phage infections. Therefore, our data suggest that inhibiting QS may reduce, rather than improve, the therapeutic efficacy of CPS-specific phages. IMPORTANCE: Phage resistance is a direct threat to phage therapy, and understanding phage-host interactions, especially how bacteria block phage infection, is essential for developing successful phage therapy. In the present study, we demonstrate for the first time that Vibrio alginolyticus uses quorum sensing (QS) to promote capsular polysaccharide (CPS)-specific phage infection by upregulating ugd expression, which is necessary for the synthesis of Autographiviridae phage receptor CPS. Although increased CPS-specific phage susceptibility is a novel trade-off mediated by QS, it results in the upregulation of virulence factors, promoting biofilm development and enhanced capsular polysaccharide production in V. alginolyticus. This suggests that inhibiting QS may improve the effectiveness of antibiotic treatment, but it may also reduce the efficacy of phage therapy.

The mycobacterial glycoside hydrolase LamH enables capsular arabinomannan release and stimulates growth.

Mycobacterial glycolipids are important cell envelope structures that drive host-pathogen interactions. Arguably, the most important are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are trafficked from the bacterium to the host via unknown mechanisms. Arabinomannan is thought to be a capsular derivative of these molecules, lacking a lipid anchor. However, the mechanism by which this material is generated has yet to be elucidated. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH (Rv0365c in Mycobacterium tuberculosis) which specifically cleaves α-1,6-mannoside linkages within LM and LAM, driving its export to the capsule releasing its phosphatidyl-myo-inositol mannoside lipid anchor. Unexpectedly, we found that the catalytic activity of this enzyme is important for efficient exit from stationary phase cultures, potentially implicating arabinomannan as a signal for growth phase transition. Finally, we demonstrate that LamH is important for M. tuberculosis survival in macrophages.