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Microbiology Spectrum Mar 2024Uropathogenic (UPEC) is the primary causative agent of lower urinary tract infection (UTI). UTI presents a serious health risk and has considerable secondary...
UNLABELLED
Uropathogenic (UPEC) is the primary causative agent of lower urinary tract infection (UTI). UTI presents a serious health risk and has considerable secondary implications including economic burden, recurring episodes, and overuse of antibiotics. A safe and effective vaccine would address this widespread health problem and emerging antibiotic resistance. Killed, whole-cell vaccines have shown limited efficacy to prevent recurrent UTI in human trials. We explored photochemical inactivation with psoralen drugs and UVA light (PUVA), which crosslinks nucleic acid, as an alternative to protein-damaging methods of inactivation to improve whole-cell UTI vaccines. Exposure of UPEC to the psoralen drug AMT and UVA light resulted in a killed but metabolically active (KBMA) state, as reported previously for other PUVA-inactivated bacteria. The immunogenicity of PUVA-UPEC as compared to formalin-inactivated UPEC was compared in mice. Both generated high UPEC-specific serum IgG titers after intramuscular delivery. However, using functional adherence as a measure of surface protein integrity, we found differences in the properties of PUVA- and formalin-inactivated UPEC. Adhesion mediated by Type-1 and P-fimbriae was severely compromised by formalin but was unaffected by PUVA, indicating that PUVA preserved the functional conformation of fimbrial proteins, which are targets of protective immune responses. assays indicated that although they retained metabolic activity, PUVA-UPEC lost virulence properties that could negatively impact vaccine safety. Our results imply the potential for PUVA to improve killed, whole-cell UTI vaccines by generating bacteria that more closely resemble their live, infectious counterparts relative to vaccines generated with protein-damaging methods.
IMPORTANCE
Lower urinary tract infection (UTI), caused primarily by uropathogenic , represents a significant health burden, accounting for 7 million primary care and 1 million emergency room visits annually in the United States. Women and the elderly are especially susceptible and recurrent infection (rUTI) is common in those populations. Lower UTI can lead to life-threatening systemic infection. UTI burden is manifested by healthcare dollars spent (1.5 billion annually), quality of life impact, and resistant strains emerging from antibiotic overuse. A safe and effective vaccine to prevent rUTI would address a substantial healthcare issue. Vaccines comprised of inactivated uropathogenic bacteria have yielded encouraging results in clinical trials but improvements that enhance vaccine performance are needed. To that end, we focused on inactivation methodology and provided data to support photochemical inactivation, which targets nucleic acid, as a promising alternative to conventional protein-damaging inactivation methods to improve whole-cell UTI vaccines.
Topics: Humans; Female; Animals; Mice; Aged; Uropathogenic Escherichia coli; Escherichia coli Infections; Quality of Life; Neoplasm Recurrence, Local; Urinary Tract Infections; Anti-Bacterial Agents; Vaccines; Formaldehyde; Nucleic Acids; Furocoumarins; Escherichia coli Proteins
PubMed: 38315025
DOI: 10.1128/spectrum.03661-23 -
Cell Surface (Amsterdam, Netherlands) Jun 2024Many studies reported the effects of antibiotic exposure on bacterial growth and cell modification. However, scarce descriptive information on ultrastructural effects...
BACKGROUND
Many studies reported the effects of antibiotic exposure on bacterial growth and cell modification. However, scarce descriptive information on ultrastructural effects upon exposure of commercial antibiotics.
METHODS
This study described the morphological and ultrastructural alterations caused by selected antibiotics (amoxicillin-clavulanate, ceftriaxone, polymyxin B, colistin, gentamicin, and amikacin) that targeted cell wall, plasma membrane, and cytoplasmic density, and also proteins synthesis. We determined extracellular morphological changes of exposure through scanning electron microscopy (FESEM) and intracellular activities through transmission electron microscopy (TEM) investigation.
RESULTS
FESEM and TEM micrograph of exposed with selected antibiotics shows ultrastructural changes in beta-lactam class (amoxicillin-clavulanate, ceftriaxone) elongated the cells as the cell wall was altered as it inhibits bacterial cell wall synthesis, polymyxin class (polymyxin B, colistin) had plasmid and curli-fimbriae as it breaking down the plasma/cytoplasmic membrane, and aminoglycoside class (gentamicin, and amikacin) reduced ribosome concentration as it inhibits bacterial protein synthesis by binding to 30 s ribosomes.
CONCLUSION
Morphological and ultrastructural alterations of 's mechanism of actions were translated and depicted. This study could be reference for characterization studies for morphological and ultrastructural of upon exposure to antimicrobial agents.
PubMed: 38313869
DOI: 10.1016/j.tcsw.2024.100120 -
Clinical Microbiology and Infection :... May 2024In Finland, whole cell pertussis vaccine (wP) was introduced in 1952 and was replaced by acellular pertussis vaccine (aP) without fimbrial (FIM) antigen in 2005. We...
OBJECTIVES
In Finland, whole cell pertussis vaccine (wP) was introduced in 1952 and was replaced by acellular pertussis vaccine (aP) without fimbrial (FIM) antigen in 2005. We aimed to analyse the changes in serotypes of circulating Bordetella pertussis before and after acellular vaccination and to explore the relationship between biofilm formation and serotype diversity after the introduction of aP vaccine.
METHODS
Serotyping of 1399 B. pertussis isolates collected at the Finnish National Reference Laboratory for Pertussis and Diphtheria in Turku, Finland, from 1974 to 2023 was performed by slide agglutination or indirect ELISA. Of 278 isolates collected after 2005, 53 were selected, genotyped for fim3 and fim2 alleles, and tested for biofilm formation. The selection criteria included maintaining a relatively equal distribution of isolates per time interval, ensuring approximately a 50:50 ratio of FIM2 (N = 26) and FIM3 (N = 27) serotypes. The reference strain Tohama I was used as a control.
RESULTS
During the wP era, the majority of circulating B. pertussis exhibited the FIM2 serotype. However, FIM3 strains have appeared since 1999 and become prevalent. After the implementation of aP vaccines, the distribution of serotypes has exhibited substantial variability. FIM3 isolates displayed an enhanced biofilm formation compared to FIM2 isolates (Geometric mean value (95% CI): 0.90 (0.79-1.03) vs. 0.75 (0.65-0.85); p < 0.05). Of the 27 FIM3 isolates, 8 harboured fim3-1 and 19 fim3-2 alleles. FIM3 isolates with fim3-2 allele were significantly associated with increased biofilm formation when compared to those with fim3-1 (1.07 (0.96-1.19) vs. 0.61 (0.52-0.72); p < 0.0001).
CONCLUSION
Following the implementation of aP vaccines, the distribution of serotypes in Finland has exhibited substantial variability. FIM3 isolates with the fim3-2 allele displayed an enhanced biofilm formation capability compared to FIM2 isolates.
Topics: Biofilms; Finland; Bordetella pertussis; Humans; Whooping Cough; Serogroup; Pertussis Vaccine; Vaccines, Acellular; Fimbriae Proteins; Serotyping; Genotype; Child, Preschool; Child; Infant; Vaccination; Antigens, Bacterial; Virulence Factors, Bordetella
PubMed: 38310999
DOI: 10.1016/j.cmi.2024.01.021 -
NPJ Biofilms and Microbiomes Feb 2024Otitis media (OM) is one of the most globally pervasive pediatric conditions. Translocation of nasopharynx-resident opportunistic pathogens like nontypeable Haemophilus...
Otitis media (OM) is one of the most globally pervasive pediatric conditions. Translocation of nasopharynx-resident opportunistic pathogens like nontypeable Haemophilus influenzae (NTHi) assimilates into polymicrobial middle ear biofilms, which promote OM pathogenesis and substantially diminish antibiotic efficacy. Oral or tympanostomy tube (TT)-delivered antibiotics remain the standard of care (SOC) despite consequences including secondary infection, dysbiosis, and antimicrobial resistance. Monoclonal antibodies (mAb) against two biofilm-associated structural proteins, NTHi-specific type IV pilus PilA (anti-rsPilA) and protective tip-region epitopes of NTHi integration host factor (anti-tip-chimer), were previously shown to disrupt biofilms and restore antibiotic sensitivity in vitro. However, the additional criterion for clinical relevance includes the absence of consequential microbiome alterations. Here, nine chinchilla cohorts (n = 3/cohort) without disease were established to evaluate whether TT delivery of mAbs disrupted nasopharyngeal or fecal microbiomes relative to SOC-OM antibiotics. Cohort treatments included a 7d regimen of oral amoxicillin-clavulanate (AC) or 2d regimen of TT-delivered mAb, AC, Trimethoprim-sulfamethoxazole (TS), ofloxacin, or saline. Fecal and nasopharyngeal lavage (NPL) samples were collected before and several days post treatment (DPT) for 16S sequencing. While antibiotic-treated cohorts displayed beta-diversity shifts (PERMANOVA, P < 0.05) and reductions in alpha diversity (q < 0.20) relative to baseline, mAb antibodies failed to affect diversity, indicating maintenance of a eubiotic state. Taxonomic and longitudinal analyses showed blooms in opportunistic pathogens (ANCOM) and greater magnitudes of compositional change (P < 0.05) following broad-spectrum antibiotic but not mAb treatments. Collectively, results showed broad-spectrum antibiotics induced significant fecal and nasopharyngeal microbiome disruption regardless of delivery route. Excitingly, biofilm-targeting antibodies had little effect on fecal and nasopharyngeal microbiomes.
Topics: Animals; Child; Humans; Anti-Bacterial Agents; Chinchilla; Standard of Care; Otitis Media; Ear, Middle; Biofilms; Nasopharynx
PubMed: 38310144
DOI: 10.1038/s41522-024-00481-0 -
IScience Feb 2024Biofilm formation, a major concern for healthcare systems, is initiated when bacteria adhere to surfaces. adhesion is mediated by appendages, including type-1 fimbriae...
Biofilm formation, a major concern for healthcare systems, is initiated when bacteria adhere to surfaces. adhesion is mediated by appendages, including type-1 fimbriae and curli amyloid fibers. Antifouling surfaces prevent the adhesion of bacteria to combat biofilm formation. Here, we used single-cell force-spectroscopy to study the interaction between and glass or two antifouling surfaces: the tripeptide DOPA-Phe(4F)-Phe(4F)-OMe and poly(ethylene glycol) polymer-brush. Our results indicate that both antifoulants significantly deter initial adhesion. By using two mutant strains expressing no type-1 fimbriae or curli amyloids, we studied the adhesion mechanism. Our results suggest that the bacteria adhere to different antifoulants via separate mechanisms. Finally, we show that some bacteria adhere much better than others, illustrating how the variability of bacterial cultures affects biofilm formation. Our results emphasize how additional study at the single-cell level can enhance our understanding of bacterial adhesion, thus leading to novel antifouling technologies.
PubMed: 38303698
DOI: 10.1016/j.isci.2024.108803 -
PLoS Biology Feb 2024Cells must access resources to survive, and the anatomy of multicellular structures influences this access. In diverse multicellular eukaryotes, resources are provided...
Cells must access resources to survive, and the anatomy of multicellular structures influences this access. In diverse multicellular eukaryotes, resources are provided by internal conduits that allow substances to travel more readily through tissue than they would via diffusion. Microbes growing in multicellular structures, called biofilms, are also affected by differential access to resources and we hypothesized that this is influenced by the physical arrangement of the cells. In this study, we examined the microanatomy of biofilms formed by the pathogenic bacterium Pseudomonas aeruginosa and discovered that clonal cells form striations that are packed lengthwise across most of a mature biofilm's depth. We identified mutants, including those defective in pilus function and in O-antigen attachment, that show alterations to this lengthwise packing phenotype. Consistent with the notion that cellular arrangement affects access to resources within the biofilm, we found that while the wild type shows even distribution of tested substrates across depth, the mutants show accumulation of substrates at the biofilm boundaries. Furthermore, we found that altered cellular arrangement within biofilms affects the localization of metabolic activity, the survival of resident cells, and the susceptibility of subpopulations to antibiotic treatment. Our observations provide insight into cellular features that determine biofilm microanatomy, with consequences for physiological differentiation and drug sensitivity.
Topics: Humans; Anti-Bacterial Agents; Pseudomonas aeruginosa; Biofilms; Pseudomonas Infections; Fimbriae, Bacterial
PubMed: 38300958
DOI: 10.1371/journal.pbio.3002205 -
ACS Applied Materials & Interfaces Feb 2024Antimicrobial peptides (AMPs) can kill bacteria by destabilizing their membranes, yet translating these molecules' properties into a covalently attached antibacterial...
Preventing Biofilm Formation with Antimicrobial Peptide-Functionalized Surface Coatings: Recognizing the Dependence on the Bacterial Binding Mode Using Live-Cell Microscopy.
Antimicrobial peptides (AMPs) can kill bacteria by destabilizing their membranes, yet translating these molecules' properties into a covalently attached antibacterial coating is challenging. Rational design efforts are obstructed by the fact that standard microbiology methods are ill-designed for the evaluation of coatings, disclosing few details about why grafted AMPs function or do not function. It is particularly difficult to distinguish the influence of the AMP's molecular structure from other factors controlling the total exposure, including which type of bonds are formed between bacteria and the coating and how persistent these contacts are. Here, we combine label-free live-cell microscopy, microfluidics, and automated image analysis to study the response of surface-bound challenged by the same small AMP either in solution or grafted to the surface through click chemistry. Initially after binding, the grafted AMPs inhibited bacterial growth more efficiently than did AMPs in solution. Yet, after 1 h, on the coated surfaces increased their expression of type-1 fimbriae, leading to a change in their binding mode, which diminished the coating's impact. The wealth of information obtained from continuously monitoring the growth, shape, and movements of single bacterial cells allowed us to elucidate and quantify the different factors determining the antibacterial efficacy of the grafted AMPs. We expect this approach to aid the design of elaborate antibacterial material coatings working by specific and selective actions, not limited to contact-killing. This technology is needed to support health care and food production in the postantibiotic era.
Topics: Antimicrobial Peptides; Escherichia coli; Microscopy; Biofilms; Anti-Bacterial Agents; Bacteria; Coated Materials, Biocompatible
PubMed: 38294883
DOI: 10.1021/acsami.3c16004 -
Journal of Global Antimicrobial... Mar 2024The recent worldwide spread of New Delhi metallo-beta-lactamase-producing Klebsiella pneumoniae (NDM-KP) in health-care settings remains a concern. The aim of the study...
Genomic surveillance of carbapenem-resistant Klebsiella pneumoniae reveals a prolonged outbreak of extensively drug-resistant ST147 NDM-1 during the COVID-19 pandemic in the Apulia region (Southern Italy).
OBJECTIVES
The recent worldwide spread of New Delhi metallo-beta-lactamase-producing Klebsiella pneumoniae (NDM-KP) in health-care settings remains a concern. The aim of the study was to describe an outbreak of extensively drug-resistant ST147 NDM-1-KP in the Apulia region of Southern Italy that occurred between 2020 and 2022 through genomic surveillance of carbapenem-resistant Enterobacterales.
METHODS
A total of 459 carbapenem-resistant KP isolates collected from patients hospitalised with bloodstream infections were tested using a commercial multiplex real-time polymerase chain reaction to identify carbapenemase genes. A subset of 27 isolates was subjected to whole-genome sequencing. Core-genome multilocus sequence typing was performed by analysing a panel of 4884 genes.
RESULTS
Molecular testing revealed that 104 (22.6%) isolates carried the carbapenemase NDM gene. Phylogenetic analysis of the 27 isolates subjected to whole-genome sequencing revealed high genetic relatedness among strains. All isolates were resistant to all first-line antibiotics. Virulome analysis identified the ybt locus, the two well-recognised virulence factors iucABCDiutA and rmpA, and the genes encoding the type 3 pilus virulence factor. Plasmids IncFIB(pkPHS1), IncFIB(pNDM-Mar), IncFIB(pQil), IncHI1B(pNDM-MAR), IncR, and Col(pHAD28) were identified in all isolates. Moreover, further analysis identified the IncFIB-type plasmid carrying the NDM-1 genes.
CONCLUSION
The increasing circulation of extensively drug-resistant NDM-1 ST147 KP strains in Southern Italy in recent years is worrisome, because these clones pose a real risk, particularly in hospital settings. Genomic surveillance is a crucial tool for early identification of emerging threats such as the spread of high-risk pathogens. Rapid infection control measures and antimicrobial stewardship are key to preventing further spread of hypervirulent KP strains.
Topics: Humans; Klebsiella pneumoniae; Phylogeny; Pandemics; Klebsiella Infections; Drug Resistance, Multiple, Bacterial; COVID-19; Carbapenems; Carbapenem-Resistant Enterobacteriaceae; Genomics; Disease Outbreaks; Italy; Virulence Factors
PubMed: 38280719
DOI: 10.1016/j.jgar.2024.01.015 -
Frontiers in Cellular and Infection... 2023strain 17978 is an opportunistic pathogen possessing a DNA damage response (DDR) in which multiple error-prone polymerase genes are co-repressed by a UmuD homolog,...
INTRODUCTION
strain 17978 is an opportunistic pathogen possessing a DNA damage response (DDR) in which multiple error-prone polymerase genes are co-repressed by a UmuD homolog, UmuDAb, and the small -specific protein DdrR. Additionally, these regulators coactivate nine other genes. We identified the DNA damage-inducible transcriptome for wildtype, , and strains, and later established the DDR transcriptome. However, the ATCC 17978 reference genome had several assembly errors and lacked the 44 kb virulence locus, AbaAL44, that is present in the strain 17978 UN.
METHODS
For this project, we combined our earlier single-end read RNAseq data with the paired-end reads and aligned these data to the improved 17978 UN genome assembly that resembled our laboratory strain, 17978 JH.
RESULTS
New DESeq2 analyses verified previous differentially expressed genes (DEGs) but also found 339 genes in 17978 JH that were not annotated or physically present in the older genome assembly. Sixty-three were differentially expressed after DNA damage, and 182 had differential basal expression when comparing , , or strains to wildtype, with 94 genes' expression unchanged. This work identified and characterized the 55 gene DNA damage-repressible transcriptome, 98% of which required either or for repression. Two-thirds of these DEGs required both regulators. We also identified 110 genes repressed only in the strain, ~50% of which were due to increased basal expression levels. Basal gene expression in the mutant was further dysregulated independent of the DDR. Over 800 genes were upregulated, and over 1200 genes were downregulated compared to wildtype expression. Half of essential genes were upregulated in the strain, including cell division genes, and two-thirds of these were downregulated in the strain.
DISCUSSION
The mutant upregulated genes enriched in translation, RNA metabolism, protein metabolism, AA/FA/cell-structure synthesis, and transport, while downregulating genes enriched in quorum sensing, biofilm production, secretion systems, pilus production, cell adhesion, and aromatics and chlorine degradation. Our data underscore the need for accurate and appropriately matched genome assemblies and indicate that affects approximately 60% of the genome, rendering it a potential target for infection treatment.
Topics: Acinetobacter baumannii; DNA Damage; Transcriptome; DNA Repair; Bacterial Proteins
PubMed: 38274737
DOI: 10.3389/fcimb.2023.1324091 -
FEBS Open Bio Mar 2024Citrobacter braakii (C. braakii) is an anaerobic, gram-negative bacterium that has been isolated from the environment, food, and humans. Infection by C. braakii has...
Citrobacter braakii (C. braakii) is an anaerobic, gram-negative bacterium that has been isolated from the environment, food, and humans. Infection by C. braakii has been associated with acute mucosal inflammation in the intestine, respiratory tract, and urinary tract. However, the pathogenesis of C. braakii in the gastric mucosa has not yet been clarified. In this study, the bacterium was detected in 35.5% (61/172) of patients with chronic gastritis (CG) and was closely associated with the severity of mucosal inflammation. Citrobacter braakii P1 isolated from a patient with CG exhibited urease activity and acid resistance. It contained multiple secretion systems, including a complete type I secretion system (T1SS), T5aSS and T6SS. We then predicted the potential pilus-related adhesins. Citrobacter braakii P1 diffusely adhered to AGS cells and significantly increased lactate dehydrogenase (LDH) release; the adhesion rate and LDH release were much lower in HEp-2 cells. Strain P1 also induced markedly increased mRNA and protein expression of IL-8 and TNF-α in AGS cells, and the fold increase was much higher than that in HEp-2 cells. Our results demonstrate proinflammatory and cytotoxic role of C. braakii in gastric epithelial cells, indicating the bacterium is potentially involved in inducing gastric mucosa inflammation.
Topics: Humans; Citrobacter; Stomach; Inflammation
PubMed: 38268325
DOI: 10.1002/2211-5463.13770