Search Results (12)

Salmonella Typhimurium monophasic variant (STm) has emerged as a novel predominant serovar in global foodborne Salmonella outbreaks. In our previous study, the two-component regulator OmpR was significantly upregulated in STm under disinfectant stress. However, effects of ompR deletion on stress tolerance and pathogenicity of this pathogen remained unclear. Therefore, this study aimed to systematically investigate the function of ompR in the adaptation of STm to food/food processing environments (e.g., disinfectants, thermal treatment, and high-salt preservation) and virulence. An isogenic ΔompR mutant was constructed using homologous recombination, and its phenotypic changes relative to the wild-type strain were evaluated, including bacterial growth, motility, stress resistance (thermal, osmotic, acid, and oxidative stress), and in vivo virulence in ICR mice. The results revealed that the deletion of ompR significantly decreased bacterial tolerance to multiple tested stresses, including thermal (50 °C, 55 °C, 60 °C), osmotic (10%, 20%, 30% NaCl), acid (pH = 3, 4, 5), and oxidative (1 mM, 2 mM, 5 mM H₂O₂) stresses. Additionally, the ΔompR mutant exhibited markedly reduced swimming and swarming motility compared to the wild-type strain. In the ICR mice infection model, the ΔompR mutant showed significantly attenuated virulence (100% survival rate within 7 days post-infection), with significantly lower bacterial loads in the liver, spleen and intestine. Transcriptome analysis showed that compared with the wild-type strain, the ΔompR mutant strain exhibited significant upregulation of fimbriae-related genes and downregulation of flagellar biosynthesis and virulence-related genes, thereby affecting bacterial adhesion, biofilm formation, flagellar assembly and virulence expression. Collectively, these findings suggest that ompR contributes to the adaptation of STm to food/food processing environments and virulence, and this gene may provide potential targets for future research to control STm contamination in food production systems. Full article

The nucleoid-associated protein Fis functions as a global regulator that influences various cellular processes in Gram-negative bacteria. In this study, we examined the role of Fis in the transcriptional regulation of type 3 fimbriae in Klebsiella pneumoniae, a notable opportunistic pathogen associated with hospital-acquired infections. Our transcriptional analyses revealed that deleting the fis gene caused a significant upregulation of mrkA and mrkH, the genes responsible for the structure and regulation of type 3 fimbriae, respectively. Additionally, phenotypic assays demonstrated that the Δfis mutant exhibited enhanced biofilm formation and greater adherence to A549 lung epithelial cells compared to the wild-type strain. These effects were restored to wild-type levels in the cis-complemented strain. Electrophoretic mobility shift assays confirmed that Fis directly binds to the regulatory regions upstream of both mrkA and mrkH, indicating that repression occurs through direct interaction with the promoter. In summary, our findings show that Fis acts as a transcriptional repressor of mrkA and mrkH, thereby negatively regulating the expression of type 3 fimbriae, biofilm formation, and adherence. This study highlights Fis as a direct regulator of fimbrial expression and biofilm development in K. pneumoniae, deepening our understanding of its virulence regulatory network. Full article

Background: Klebsiella pneumoniae infections pose a great burden worldwide, causing high morbidity and mortality, which are worsened by the increase in multidrug-resistant strains. New therapeutic/prophylactic strategies are urgently needed to overcome antibiotic resistance and reduce the health and economic impacts of diseases caused by this pathogen. Fimbriae are important virulence factors involved in biofilm formation and adhesion to host cells. Their exposed location, conservation among clinical isolates and adjuvant properties make them interesting candidates for inclusion in protein-based vaccines. Therefore, the present work investigated the immunological potential of type 1 and 3 fimbriae subunits in a murine model of K. pneumoniae lung infection. Methods: MrkA and FimA were produced as recombinant proteins in E. coli, purified and used to immunize mice subcutaneously. The immune responses were characterized and protection against pneumonia was evaluated after intranasal challenge. Results: Subcutaneous immunization with recombinant FimA and MrkA induced high IgG1 production; the antibodies efficiently recognized the native proteins at the bacterial surface, promoted C3 deposition and reduced biofilm formation by K. pneumoniae in vitro. Mice vaccinated with the co-administered proteins reduced the bacterial loads in the lungs after intranasal challenge, less inflammation and reduced tissue damage. Conclusion: The results suggest that both type 1 and type 3 fimbriae contribute to protection against K. pneumoniae lung infection, inducing antibodies that bind to the bacteria and favoring Complement deposition and clearance by the host, while inhibiting biofilm formation. Full article

Enterobacter hormaechei, one of the species within the Enterobacter cloacae complex, is a relevant agent of healthcare-associated infections. In addition, it has gained relevance because isolates have shown the capacity to resist several antibiotics, particularly carbapenems. However, knowledge regarding colonization and virulence mechanisms of E. hormaechei has not progressed to the same extent as other Enterobacteriaceae species as Escherichia coli or Klebsiella pneumoniae. Here, we describe the presence and role of the type 3 fimbria, a chaperone-usher assembled fimbria, which was first described in Klebsiella spp., and which has been detected in other representatives of the Enterobacteriaceae family. Eight Chilean E. cloacae isolates were examined, and among them, four E. hormaechei isolates were found to produce the type 3 fimbria. These isolates were identified as E. hormaechei subsp. hoffmannii, one of the five subspecies known. A mutant E. hormaechei subsp. hoffmannii strain lacking the mrkA gene, encoding the major structural subunit, displayed a significantly reduced adherence capacity to a plastic surface and to Caco-2 cells, compared to the wild-type strain. This phenotype of reduced adherence capacity was not observed in the mutant strains complemented with the mrkA gene under the control of an inducible promoter. Therefore, these data suggest a role of the type 3 fimbria in the adherence capacity of E. hormaechei subsp. hoffmannii. A screening in E. hormaechei genomes contained in the NCBI RefSeq Assembly database indicated that the overall presence of the type 3 fimbria is uncommon (5.94–7.37%), although genes encoding the structure were detected in representatives of the five E. hormaechei subspecies. Exploration of complete genomes indicates that, in most of the cases, the mrkABCDF locus, encoding the type 3 fimbria, is located in plasmids. Furthermore, sequence types currently found in healthcare-associated infections were found to harbor genes encoding the type 3 fimbria, mainly ST145, ST78, ST118, ST168, ST66, ST93, and ST171. Thus, although the type 3 fimbria is not widespread among the species, it might be a determinant of fitness for a subset of E. hormaechei representatives. Full article

Klebsiella pneumoniae is an opportunistic pathogen that frequently causes nosocomial and community-acquired (CA) infections. Until now, a limited number of studies has been focused on the analyses of changes affecting the virulence attributes. Genotypic and phenotypic methods were used to characterise the 39 clinical K. pneumoniae isolates; all belonged to the pan-drug resistant, widespread clone ST 15 and expressed the K24 capsule. PFGE has revealed that the isolates could be divided into three distinct genomic clusters. All isolates possessed allS and uge genes, known to contribute to the virulence of K. pneumoniae and 10.25% of the isolates showed hypermucoviscosity, 94.87% produced type 1 fimbriae, 92.3% produced type 3 fimbriae, and 92.3% were able to produce biofilm. In vivo persistence could be supported by serum resistance 46.15%, enterobactin (94.87%) and aerobactin (5.12%) production and invasion of the INT407 and T24 cell lines. Sequence analysis of the whole genomes of the four representative strains 11/3, 50/1, 53/2 and 53/3 has revealed high sequence homology to the reference K. pneumoniae strain HS11286. Our results represent the divergence of virulence attributes among the isolates derived from a common ancestor clone ST 15, in an evolutionary process that occurred both in the hospital and in the community. Full article

This study evaluates a possible correlation between multidrug-resistant Klebsiella pneumoniae strains and virulence markers in a Danio rerio (zebrafish) model. Whole-genome sequencing (WGS) was performed on 46 strains from three Brazilian hospitals. All of the isolates were colistin-resistant and harbored bla_KPC-2. Ten different sequence types (STs) were found; 63% belonged to CC258, 22% to ST340, and 11% to ST16. The virulence factors most frequently found were type 3 fimbriae, siderophores, capsule regulators, and RND efflux-pumps. Six strains were selected for a time-kill experiment in zebrafish embryos: infection by ST16 was associated with a significantly higher mortality rate when compared to non-ST16 strains (52% vs. 29%, p = 0.002). Among the STs, the distribution of virulence factors did not differ significantly except for ST23, which harbored a greater variety of factors than other STs but was not related to a higher mortality rate in zebrafish. Although several virulence factors are described in K. pneumoniae, our study found ST16 to be the only significant predictor of a virulent phenotype in an animal model. Further research is needed to fully understand the correlation between virulence and sequence types. Full article

The phylum Planctomycetota is known for having uncommon biological features. Recently, biotechnological applications of its members have started to be explored, namely in the genus Stieleria. Here, we formally describe a novel Stieleria isolate designated as strain ICT_E10.1^T, obtained from sediments collected in the Tagus estuary (Portugal). Strain ICT_E10.1^T is pink-pigmented, spherical to ovoid in shape, and 1.7 µm ± 0.3 x 1.4 µm ± 0.3 in size. Cells cluster strongly in aggregates or small chains, divide by budding, and have prominent fimbriae. Strain ICT_E10.1^T is heterotrophic and aerobic. Growth occurs from 20 to 30 °C, from 0.5 to 3% (w/v) NaCl, and from pH 6.5 to 11.0. The analysis of the 16S rRNA gene sequence placed strain ICT_E10.1^T into the genus Stieleria with Stieleria neptunia Enr13^T as the closest validly described relative. The genome size is 9,813,311 bp and the DNA G+C content is 58.8 mol%. Morphological, physiological, and genomic analyses support the separation of this strain into a novel species, for which we propose the name Stieleria sedimenti represented by strain ICT_E10.1^T as the type of strain (=CECT 30514^T= DSM 113784^T). Furthermore, this isolate showed biotechnological potential by displaying relevant biosynthetic gene clusters and potent activity against Staphylococcus aureus. Full article

Despite high level vaccination and the availability of two different types of vaccines, whole cell (wP) and acellular vaccines (aP), the resurgence of pertussis has been reported in many countries. Antigenic variation within circulating and vaccine strains is the most documented reason reported for the resurgence of pertussis. Research on genetic divergence among circulating and vaccine strains has largely been reported in countries using aP vaccines. There are inadequate data available for antigenic variation in B. pertussis from wP-using countries. India has used wP for more than 40 years in their primary immunization program. The present study reports five clinical isolates of B. pertussis from samples of pediatric patients with pertussis symptoms observed in India. Genotypic and phenotypic characterization of clinical isolates were performed by serotyping, genotyping, whole genome analyses and comparative genomics. All clinical isolates showed serotype 1, 2 and 3 based on the presence of fimbriae 2 and 3. Genotyping showed genetic similarities in allele types for five aP genes within vaccine strains and clinical isolates reported from India. The presence of the ptxP3 genotype was observed in two out of five clinical isolates. Whole-genome sequencing was performed for clinical isolates using the hybrid strategy of combining Illumina (short reads) and oxford nanopore (long reads) sequencing strategies. Clinical isolates (n = 5) and vaccine strains (n = 7) genomes of B. pertussis from India were compared with 744 B. pertussis closed genomes available in the public databases. The phylogenomic comparison of B. pertussis genomes reported from India will be advantageous in better understanding pertussis resurgence reported globally with respect to pathogen adaptation. Full article

Klebsiella pneumoniae has been the predominant pathogen of liver abscess, but ST11-K47 carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has rarely been studied as the causative organism. We identified an ST11-K47 CR-hvKP (HvKp-su1) from the drainage fluid of a liver abscess in a Chinese man who was diagnosed with liver abscess combined with diabetes, pneumonia, pleural infection, abdominal abscess, and splenic abscess. HvKp-su1 was non-hypermucoviscous and lacked the magA and rmpA genes and pLVPK plasmid but exhibited high virulence, with a high mortality rate (90%) to wax moth larvae (G. mellonella), similar to the hypervirulent Klebsiella pneumoniae ATCC43816 (91.67%). Whole-genome sequencing and bioinformatics analysis indicated that HvKp-su1 possesses a plasmid similar to a type of pLVPK-like plasmid (JX-CR-hvKP-2-P2), which is an uncommon plasmid in CR-hvKP. HvKp-su1 carried multiple resistance genes, including bla_KPC-2. bla_TEM-1, bla_SHV-55, and bla_CTX-M-65; hypervirulence genes such as aerobactin (iutA), salmochelin (iroEN), and yersiniabactin (ybtAEPQSTUX); and the type 3 fimbriae-encoding system (mrkACDF). Moreover, v_5377 and v_5429 (cofT, CFA/III (CS8)) located on plasmid 1 were simultaneously predicted to be virulence genes. After the long-term combination use of antibiotics, the patient successfully recovered. In summary, our study clarified the clinical and molecular characteristics of a rare ST11-K47 CR-hvKP (HvKp-su1), raising great concerns about the emergence of ST11-K47 CR-hvKP with multidrug resistance and hypervirulence, and providing insights into the control and treatment of liver abscess caused by ST11-K47 CR-hvKP. Full article

The rise of a new hypervirulent variant of Klebsiella pneumoniae (hvKp) was recently reported, mainly linked to the ST23 lineage. The hvKp variants can cause severe infections, including hepatic abscesses, bacteremia, and meningitis, with a particularly disconcerting propensity to cause community-acquired, life-threatening infection among young and otherwise healthy individuals. The present study aimed to report the clinical characteristics of a hypermucoviscous K. pneumoniae strain isolated in Italy and sustaining recurrent meningitis in a patient of Peruvian origin. A further objective was to retrospectively investigate, by means of whole-genome sequencing (WGS) analysis, the genomic features of such an isolate. The hypermucoviscosity phenotype of the strain (sk205y205t) was determined using the string test. Genomic information was obtained by WGS (Illumina) and bioinformatic analysis. Strain sk205y205t was susceptible to most antibiotics, despite the presence of some resistance genes, including bla_SHV-11, bla_SHV-67, fosA, and acrR. The isolate belonged to ST65 and serotype K2, and exhibited several virulence factors related to the hvKp variant. Among these, were the siderophore genes entB, irp2, iroN, iroB, and iucA; the capsule-regulating genes rmpA and rmpA2; and the type 1 and 3 fimbriae fimH27 and mrkD, respectively. A further operon, encoding the genotoxin colibactin (clbA-Q), was also identified. The virulence plasmids pK2044, pRJA166b, and pNDM. MAR were also detected. Phylogenetic investigation showed that this Italian strain is highly similar to a Chinese isolate, suggesting a hidden circulation of this hvKp ST65 K2 lineage. Full article

Bordetella pertussis whole-cell vaccines (wP) caused a spectacular drop of global pertussis incidence, but since the replacement of wP with acellular pertussis vaccines (aP), pertussis has resurged in developed countries within 7 to 12 years of the change from wP to aP. In the mouse infection model, we examined whether addition of further protective antigens into the aP vaccine, such as type 2 and type 3 fimbriae (FIM2/3) with outer membrane lipooligosaccharide (LOS) and/or of the adenylate cyclase toxoid (dACT), which elicits antibodies neutralizing the CyaA toxin, could enhance the capacity of the aP vaccine to prevent colonization of the nasal mucosa by B. pertussis. The addition of the toxoid and of the opsonizing antibody-inducing agglutinogens modestly enhanced the already high capacity of intraperitoneally-administered aP vaccine to elicit sterilizing immunity, protecting mouse lungs from B. pertussis infection. At the same time, irrespective of FIM2/3 with LOS and dACT addition, the aP vaccination ablated the natural capacity of BALB/c mice to clear B. pertussis infection from the nasal cavity. While wP or sham-vaccinated animals cleared the nasal infection with similar kinetics within 7 weeks, administration of the aP vaccine promoted persistent colonization of mouse nasal mucosa by B. pertussis. Full article

Enterobacter cloacae complex has been increasingly recognized as a nosocomial pathogen representing the third major Enterobacteriaceae species involved with infections. This study aims to evaluate virulence and antimicrobial susceptibility of subpopulations generated from macrocolonies of NDM-1 producing Enterobacter hormaechei clinical isolates. Biofilm was quantified using crystal violet method and fimbrial genes were investigated by PCR. Susceptibility of antimicrobials, alone and combined, was determined by minimum inhibitory concentration and checkerboard assays, respectively. Virulence and efficacy of antimicrobials were evaluated in Galleria mellonella larvae. Importantly, we verified that some subpopulations that originate from the same macrocolony present different biofilm production ability and distinct susceptibility to meropenem due to the loss of bla_NDM-1 encoding plasmid. A more in-depth study was performed with the 798 macrocolony subpopulations. Type 3 fimbriae were straightly related with biofilm production; however, virulence in larvae was not statistically different among subpopulations. Triple combination with meropenem–rifampicin–polymyxin B showed in vitro synergistic effect against all subpopulations; while in vivo this treatment showed different efficacy rates for 798-1S and 798-4S subpopulations. The ability of multidrug resistant E. hormaechei isolates in generating bacterial subpopulations presenting different susceptible and virulence mechanisms are worrisome and may explain why these infections are hardly overcome. Full article