Nilgai antelope display no signs of infection upon experimental challenge with a virulent Babesia bovis strain.

BACKGROUND: Bovine babesiosis is caused by infection with the protozoal parasite Babesia bovis, which is transmitted by Rhipicephalus (Boophilus) spp. It can cause mortality rates up to 90% in immunologically naive Bos taurus cattle. In south Texas, R. (B.) microplus is known to infest nilgai antelope (Boselaphus tragocamelus); however, their susceptibility to infection with B. bovis and their role in the transmission of the parasite remain unknown. In this study, we challenged nilgai antelope with B. bovis and evaluated their susceptibility to infection. METHODS: Nilgai were needle inoculated with ≈108 B. bovis-parasitized erythrocytes (merozoites) or a homogenate of B. bovis-infected larval ticks (sporozoite) delivered intravenously. Bos taurus beef calves were inoculated in parallel, as this strain of B. bovis is lethal to cattle. Temperature and hematocrit were monitored daily over the course of each study, and whole blood was collected for molecular [polymerase chain reaction (PCR)] and serological [indirect enzyme-linked immunosorbent assay (ELISA)] diagnostic evaluation. Histological sections of nilgai cerebral tissue were examined for evidence of infection. Recipient bovine calves were sub-inoculated with blood from nilgai challenged with either stage of the parasite, and they were monitored for clinical signs of infection and evaluated by a PCR diagnostic assay. Red blood cells (RBCs) from prechallenged nilgai and B. taurus beef cattle were cultured with an in vitro B. bovis merozoite culture to examine colonization of the RBCs by the parasite. RESULTS: Nilgai did not display clinical signs of infection upon inoculation with either the merozoite or sporozoite stage of B. bovis. All nilgai were PCR-negative for the parasite, and they did not develop antibodies to B. bovis. No evidence of infection was detected in histological sections of nilgai tissues, and in vitro culture analysis indicated that the nilgai RBCs were not colonized by B. bovis merozoites. Cattle subinoculated with blood from challenged nilgai did not display clinical signs of infection, and they were PCR-negative up to 45 days after transfer. CONCLUSIONS: Nilgai do not appear to be susceptible to infection with a strain of B. bovis that is lethal to cattle. Tick control on these alternative hosts remains a critical priority, especially given their potential to disseminate ticks over long distances.

Effects of tobacco smoke on the expression of virulence genes in Escherichia coli.

It is widely acknowledged that smoking exacerbates the severity of infectious diseases. A presumed mechanism involves the damage inflicted by tobacco smoke on the organs of host organisms. In this study, an alternative hypothesis was explored: smoking enhances the virulence of bacteria. This possibility was investigated using Escherichia coli as the model bacteria and Drosophila as the host organism. Our inquiry focused on the potential gene expression changes in E. coli subsequent to exposure to tobacco smoke extracts. Analysis of the transcription promoter activity of genes encoding proteins within the E. coli two-component system, a regulatory machinery governing gene expression, revealed the suppression of thirteen out of 23 promoters in response to tobacco smoke extracts. Subsequently, Drosophila was infected with E. coli exposed to tobacco smoke extracts or left untreated. Interestingly, there were no significant differences observed in the survival periods of Drosophila following infection with E. coli, whether treated or untreated with tobacco smoke extracts. Contrary to the initial hypothesis, the findings suggest that while tobacco smoke extracts alter gene expression in E. coli, these changes do not appear to impact bacterial virulence. Although this study has illuminated the influence of tobacco smoke extracts on the gene expression of E. coli, further analyses are necessary to elucidate the implications of these changes. Nevertheless, the results imply that smoking affects not only host organisms but may also exert influence on invading bacteria.

Complete telomere-to-telomere genomes uncover virulence evolution conferred by chromosome fusion in oomycete plant pathogens.

Variations in chromosome number are occasionally observed among oomycetes, a group that includes many plant pathogens, but the emergence of such variations and their effects on genome and virulence evolution remain ambiguous. We generated complete telomere-to-telomere genome assemblies for Phytophthora sojae, Globisporangium ultimum, Pythium oligandrum, and G. spinosum. Reconstructing the karyotype of the most recent common ancestor in Peronosporales revealed that frequent chromosome fusion and fission drove changes in chromosome number. Centromeres enriched with Copia-like transposons may contribute to chromosome fusion and fission events. Chromosome fusion facilitated the emergence of pathogenicity genes and their adaptive evolution. Effectors tended to duplicate in the sub-telomere regions of fused chromosomes, which exhibited evolutionary features distinct to the non-fused chromosomes. By integrating ancestral genomic dynamics and structural predictions, we have identified secreted Ankyrin repeat-containing proteins (ANKs) as a novel class of effectors in P. sojae. Phylogenetic analysis and experiments further revealed that ANK is a specifically expanded effector family in oomycetes. These results revealed chromosome dynamics in oomycete plant pathogens, and provided novel insights into karyotype and effector evolution.

Regulation of Burkholderia cenocepacia virulence by the fatty acyl-CoA ligase DsfR as a response regulator of quorum sensing signal.

Quorum sensing (QS) is a cell-to-cell communication mechanism mediated by small diffusible signaling molecules. Previous studies showed that RpfR controls Burkholderia cenocepacia virulence as a cis-2-dodecenoic acid (BDSF) QS signal receptor. Here, we report that the fatty acyl-CoA ligase DsfR (BCAM2136), which efficiently catalyzes in vitro synthesis of lauryl-CoA and oleoyl-CoA from lauric acid and oleic acid, respectively, acts as a global transcriptional regulator to control B. cenocepacia virulence by sensing BDSF. We show that BDSF binds to DsfR with high affinity and enhances the binding of DsfR to the promoter DNA regions of target genes. Furthermore, we demonstrate that the homolog of DsfR in B. lata, RS02960, binds to the target gene promoter, and perception of BDSF enhances the binding activity of RS02960. Together, these results provide insights into the evolved unusual functions of DsfR that control bacterial virulence as a response regulator of QS signal.

Characterizing carbapenemase-producing Escherichia coli isolates from Spain: high genetic heterogeneity and wide geographical spread.

Introduction: Carbapenemase-Producing Escherichia coli (CP-Eco) isolates, though less prevalent than other CP-Enterobacterales, have the capacity to rapidly disseminate antibiotic resistance genes (ARGs) and cause serious difficult-to-treat infections. The aim of this study is phenotypically and genotypically characterizing CP-Eco isolates collected from Spain to better understand their resistance mechanisms and population structure. Methods: Ninety representative isolates received from 2015 to 2020 from 25 provinces and 59 hospitals Spanish hospitals were included. Antibiotic susceptibility was determined according to EUCAST guidelines and whole-genome sequencing was performed. Antibiotic resistance and virulence-associated genes, phylogeny and population structure, and carbapenemase genes-carrying plasmids were analyzed. Results and discussion: The 90 CP-Eco isolates were highly polyclonal, where the most prevalent was ST131, detected in 14 (15.6%) of the isolates. The carbapenemase genes detected were bla OXA-48 (45.6%), bla VIM-1 (23.3%), bla NDM-1 (7.8%), bla KPC-3 (6.7%), and bla NDM-5 (6.7%). Forty (44.4%) were resistant to 6 or more antibiotic groups and the most active antibiotics were colistin (98.9%), plazomicin (92.2%) and cefiderocol (92.2%). Four of the seven cefiderocol-resistant isolates belonged to ST167 and six harbored bla NDM. Five of the plazomicin-resistant isolates harbored rmt. IncL plasmids were the most frequent (45.7%) and eight of these harbored bla VIM-1. bla OXA-48 was found in IncF plasmids in eight isolates. Metallo-ß-lactamases were more frequent in isolates with resistance to six or more antibiotic groups, with their genes often present on the same plasmid/integron. ST131 isolates were associated with sat and pap virulence genes. This study highlights the genetic versatility of CP-Eco and its potential to disseminate ARGs and cause community and nosocomial infections.

Pleiotropic roles of LAMMER kinase, Lkh1 in stress responses and virulence of Cryptococcus neoformans.

Dual-specificity LAMMER kinases are highly evolutionarily conserved in eukaryotes and play pivotal roles in diverse physiological processes, such as growth, differentiation, and stress responses. Although the functions of LAMMER kinase in fungal pathogens in pathogenicity and stress responses have been characterized, its role in Cryptococcus neoformans, a human fungal pathogen and a model yeast of basidiomycetes, remains elusive. In this study, we identified a LKH1 homologous gene and constructed a strain with a deleted LKH1 and a complemented strain. Similar to other fungi, the lkh1Δ mutant showed intrinsic growth defects. We observed that C. neoformans Lkh1 was involved in diverse stress responses, including oxidative stress and cell wall stress. Particularly, Lkh1 regulates DNA damage responses in Rad53-dependent and -independent manners. Furthermore, the absence of LKH1 reduced basidiospore formation. Our observations indicate that Lkh1 becomes hyperphosphorylated upon treatment with rapamycin, a TOR protein inhibitor. Notably, LKH1 deletion led to defects in melanin synthesis and capsule formation. Furthermore, we found that the deletion of LKH1 led to the avirulence of C. neoformans in a systemic cryptococcosis murine model. Taken together, Lkh1 is required for the stress response, sexual differentiation, and virulence of C. neoformans.

Nasal carriage of virulent and multidrug resistant Staphylococcus aureus: a possible comorbidity of COVID-19.

BACKGROUND: Staphylococcus aureus (S. aureus) associated with COVID-19 has not been well documented. This cross-sectional study evaluated the association between nasal S. aureus carriage and COVID-19. METHODS AND RESULTS: Nasopharyngeal samples were collected from 391 participants presenting for COVID-19 test in Lagos, Nigeria, and S. aureus was isolated from the samples. Antimicrobial susceptibility test was done by disc diffusion method. All S. aureus isolates were screened for the presence of mecA, panton-valentine leucocidin (PVL) and toxic shock syndrome toxin (TSST) virulence genes by polymerase chain reaction. Staphylococcal protein A (spa) typing was conducted for all the isolates. Participants with COVID-19 had double the prevalence of S. aureus (42.86%) compared to those who tested negative (20.54%). A significant association was seen between S. aureus nasal carriage and COVID-19 (p = 0.004). Antimicrobial sensitivity results showed resistance to oxacillin (100%), cefoxitin (53%), and vancomycin (98.7%). However, only 41% of the isolates harbored the mecA gene, with SCCmecV being the most common SCCmec type. There was no association between the carriage of virulence genes and COVID-19. A total of 23 Spa types were detected, with t13249 and t095 being the two most common spa types. CONCLUSION: This study examined the association between nasal S. aureus carriage and SARS-COV-2 infection. Further research is required to fully explore the implications of S. aureus co-infection with COVID-19.

Comparative genomic analyses provide insight into the pathogenicity of three Pseudomonas syringae pv. actinidiae strains from Anhui Province, China.

BACKGROUND: Pseudomonas syringae pv. actinidiae (Psa) is an important bacterial plant pathogen that causes severe damage to the kiwifruit industry worldwide. Three Psa strains were recently obtained from different kiwifruit orchards in Anhui Province, China. The present study mainly focused on the variations in virulence and genome characteristics of these strains based on the pathogenicity assays and comparative genomic analyses. RESULTS: Three strains were identified as biovar 3 (Psa3), along with strain QSY6 showing higher virulence than JZY2 and YXH1 in pathogenicity assays. The whole genome assembly revealed that each of the three strains had a circular chromosome and a complete plasmid. The chromosome sizes ranged from 6.5 to 6.6 Mb with a GC content of approximately 58.39 to 58.46%, and a predicted number of protein-coding sequences ranging from 5,884 to 6,019. The three strains clustered tightly with 8 Psa3 reference strains in terms of average nucleotide identity (ANI), whole-genome-based phylogenetic analysis, and pangenome analysis, while they were evolutionarily distinct from other biovars (Psa1 and Psa5). Variations were observed in the repertoire of effectors of the type III secretion system among all 15 strains. Moreover, synteny analysis of the three sequenced strains revealed eight genomic regions containing 308 genes exclusively present in the highly virulent strain QSY6. Further investigation of these genes showed that 16 virulence-related genes highlight several key factors, such as effector delivery systems (type III secretion systems) and adherence (type IV pilus), which might be crucial for the virulence of QSY6. CONCLUSION: Three Psa strains were identified and showed variant virulence in kiwifruit plant. Complete genome sequences and comparative genomic analyses further provided a theoretical basis for the potential pathogenic factors responsible for kiwifruit bacterial canker.

Inhibition of mRNA nuclear export promotes SARS-CoV-2 pathogenesis.

The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2.

Isolation, identification and characteristics of Bibersteinia trehalosi from goat.

A conditionally pathogenic bacterium called Bibersteinia trehalosi inhabits the upper respiratory tract of ruminants and is becoming a significant cause of pneumonia, especially in goats. In this study, we identified a gram-negative bacteria strain isolated from dead goat's lungs, which was named M01. By integrating the outcomes of its morphological and biochemical characterization with the investigation of the 16S rRNA gene sequence analysis, the isolate was identified as B. trehalosi. Based on antibiotic susceptibility tests, the isolate was shown to be resistant to ß-lactams, tetracyclines, and amphenicols. Its genome was discovered to comprise 2115 encoded genes and a circular chromosome measuring 2,345,568 bp using whole genome sequencing. Annotation of the VFBD database revealed that isolate M01 had four virulence genes encoding three virulence factors. The CARD database revealed that its genome has two antibiotic-resistance genes. Based on pathogenicity testing, isolate M01 was highly pathogenic to mice, primarily causing pneumonia, with an LD50 of 1.31 × 107 CFU/ml. Moreover, histopathology showed loss of alveolar structure and infiltration of lung inflammatory cells. Hence, the current study could provide sufficient information for prevention and control strategies for future epidemics of B. trehalosi in goat species.

Novel target and PCR assay for identification of hypervirulent ST1 (BI/NAP1/027) Clostridioides difficile and detection of toxigenic C. Difficile.

BACKGROUND AND AIMS: The incidence of Clostridioides difficile infection and the prevalence of hypervirulent ST1 (BI/NAP1/027)strain are increasing, especially in developing countries. We aimed to develop a new PCR assay for the identification of hypervirulent ST1 strains and toxigenic C. difficile in stool samples. MATERIALS AND METHODS: We established a quadruplex TaqMan real-time PCR (pilW_4-plex PCR) assay targeting the pilW, a ST1-specific type Ⅳ minor pilin gene, and three C. difficile genes including cdtB, tcdB, and hsp. The sensitivity and specificity of the assay was tested using 403C. difficile isolates and 180 unformed stool sample. The results were compared with anaerobic culture-based conventional PCR method and MLST. RESULTS: The pilW_4-plex PCR identified toxigenic C. difficile in 333 (82.6%, 333/403) isolates with 100% sensitivity and specificity, and in 78 (43.3%, 78/180) stool samples with the sensitivity and specificity of 94.7% and 93.3%, respectively. Hypervirulent ST1 were detected in 21 strains and nine stool samples by the pilW_4-plex PCR. The pilW_4-plex PCR assay has no cross-reaction with non-toxigenic C. difficile or other bacteria. CONCLUSION: The pilW_4-plex PCR assay is an accurate and rapid method with high sensitivity and specificity for identification of ST1 and detection of toxigenic C. difficile in stool.

Lipopolysaccharide with long O-antigen is crucial for Salmonella Enteritidis to evade complement activity and to facilitate bacterial survival in vivo in the Galleria mellonella infection model.

Bacterial resistance to serum is a key virulence factor for the development of systemic infections. The amount of lipopolysaccharide (LPS) and the O-antigen chain length distribution on the outer membrane, predispose Salmonella to escape complement-mediated killing. In Salmonella enterica serovar Enteritidis (S. Enteritidis) a modal distribution of the LPS O-antigen length can be observed. It is characterized by the presence of distinct fractions: low molecular weight LPS, long LPS and very long LPS. In the present work, we investigated the effect of the O-antigen modal length composition of LPS molecules on the surface of S. Enteritidis cells on its ability to evade host complement responses. Therefore, we examined systematically, by using specific deletion mutants, roles of different O-antigen fractions in complement evasion. We developed a method to analyze the average LPS lengths and investigated the interaction of the bacteria and isolated LPS molecules with complement components. Additionally, we assessed the aspect of LPS O-antigen chain length distribution in S. Enteritidis virulence in vivo in the Galleria mellonella infection model. The obtained results of the measurements of the average LPS length confirmed that the method is suitable for measuring the average LPS length in bacterial cells as well as isolated LPS molecules and allows the comparison between strains. In contrast to earlier studies we have used much more precise methodology to assess the LPS molecules average length and modal distribution, also conducted more subtle analysis of complement system activation by lipopolysaccharides of various molecular mass. Data obtained in the complement activation assays clearly demonstrated that S. Enteritidis bacteria require LPS with long O-antigen to resist the complement system and to survive in the G. mellonella infection model.

Measuring Urease and Phospholipase Secretion in Cryptococcus neoformans.

Urease and phospholipase are enzymes that are important virulence factors for Cryptococcus neoformans. These are two of the most studied enzymes involved in how C. neoformans breaches the blood-brain barrier. Additionally, phospholipase secretion also supports dissemination from the lungs. This chapter describes the methods used to measure the secretion of these enzymes, which may be used to characterize strain invasiveness and virulence.

Virulent and Multi-drug-Resistant Edwardsiella tarda Infection in Oscar Fish: Unveiling the Threat of Mass Mortality and AMR Dissemination.

The Oscar fish (Astronotus ocellatus) is among the most commonly domesticated and exported ornamental fish species from Kerala. The ornamental fish industry faces a significant challenge with the emergence of diseases caused by multi-drug-resistant bacteria. In the present study, six isolates were resolved from the diseased Oscar fish showing haemorrhages, necrosis, and loss of pigmentation. After phenotypic and genotypic characterization, the bacteria were identified as Edwardsiella tarda, Klebsiella pneumoniae, Enterococcus faecalis, Escherichia coli, Brevibacillus borstelensis, and Staphylococcus hominis. Experimental challenge studies in healthy Oscar fish showed that E. tarda caused 100% mortality within 240 h with 6.99 × 106 CFU/fish as LD50 and histopathology revealed the typical signs of infection. The pathogen was re-recovered from the moribund fish thereby confirming Koch's postulates. E. tarda was confirmed through the positive amplification of tarda-specific gene and virulence genes viz., etfD and escB were also detected using PCR. Antibiotic susceptibility tests using disc diffusion displayed that the pathogen is multi-drug-resistant towards antibiotics belonging to aminoglycosides, tetracyclines, and quinolones categories with a MAR index of 0.32, which implicated the antibiotic pressure in the farm. Plasmid curing studies showed a paradigm shift in the resistance pattern with MAR index of 0.04, highlighting the resistance genes are plasmid-borne except for the chromosome-borne tetracycline resistance gene (tetA). This study is the first of its kind in detecting mass mortality caused by E. tarda in Oscar fish. Vigilant surveillance and strategic actions are crucial for the precise detection of pathogens and AMR in aquaculture.

Escherichia coli isolated from pyometra and cystitis in the same animal exhibit a wide phenotypic similarity.

AIMS: Pyometra and cystitis caused by Escherichia coli are common diseases identified in canine or feline females. The origin of pyometra infection remains uncertain, and effective prevention strategies for this disease are still unknown. This study aimed to provide a phenotypic characterization, including antimicrobial resistance and virulence profiles, of endometrial pathogenic (EnPEC) and uropathogenic (UPEC) E. coli strains isolated simultaneously from the same animal. METHODS AND RESULTS: Sixteen E. coli strains, from eight different animals, were analyzed in this study. The antimicrobial susceptibility profile of EnPEC and UPEC strains was determined using the disc diffusion method, which showed a similar susceptibility profile among strains (EnPEC and UPEC) from the same animal. The virulence profile of the strains was assessed through biofilm formation, as well as serum resistance abilities. EnPEC and UPEC strains from the same animal exhibited slight variations in their virulence and antimicrobial resistance capabilities. Overall, most of the strain pairs showed a high similarity in their ability to establish biofilms and survive in serum complement activity. CONCLUSIONS: Overall, strains of E. coli isolated from both pyometra and cystitis in the same animal, despite presenting distinct clinical diseases, exhibit a wide phenotypic similarity, suggesting a common origin for the strains.

Deletion of codY impairs Staphylococcus epidermidis biofilm formation, generation of viable but non-culturable cells and stimulates cytokine production in human macrophages.

Introduction. Staphylococcus epidermidis biofilms are one of the major causes of bloodstream infections related to the use of medical devices. The diagnosis of these infections is challenging, delaying their treatment and resulting in increased morbidity and mortality rates. As such, it is urgent to characterize the mechanisms employed by this bacterium to endure antibiotic treatments and the response of the host immune system, to develop more effective therapeutic strategies. In several bacterial species, the gene codY was shown to encode a protein that regulates the expression of genes involved in biofilm formation and immune evasion. Additionally, in a previous study, our group generated evidence indicating that codY is involved in the emergence of viable but non-culturable (VBNC) cells in S. epidermidis.Gap statement/Hypothesis. As such, we hypothesized that the gene codY has have an important role in this bacterium virulence.Aim. This study aimed to assess, for the first time, the impact of the deletion of the gene codY in S. epidermidis virulence, namely, in antibiotic susceptibility, biofilm formation, VBNC state emergence and in vitro host immune system response.Methodology. Using an allelic replacement strategy, we constructed and then characterized an S. epidermidis strain lacking codY, in regards to biofilm and VBNC cell formation, susceptibility to antibiotics as well as their role in the interaction with human blood and plasma. Additionally, we investigate whether the codY gene can impact the activation of innate immune cells by evaluating the production of both pro- and anti-inflammatory cytokines by THP-1 macrophages.Results. We demonstrated that the deletion of the gene codY resulted in biofilms with less c.f.u. counts and fewer VBNC cells. Furthermore, we show that although WT and mutant cells were similarly internalized in vitro by human macrophages, a stronger cytokine response was elicited by the mutant in a toll-like receptor 4-dependent manner.Conclusion. Our results indicate that codY contributes to S. epidermidis virulence, which in turn may have an impact on our ability to manage the biofilm-associated infections caused by this bacterium.

Circulating hypervirulent Marek's disease viruses in vaccinated chicken flocks in Taiwan by genetic analysis of meq oncogene.

Marek's disease (MD) is an important neoplastic disease caused by serotype 1 Marek's disease virus (MDV-1), which results in severe economic losses worldwide. Despite vaccination practices that have controlled the MD epidemic, current increasing MD-suspected cases indicate the persistent viral infections circulating among vaccinated chicken farms in many countries. However, the lack of available information about phylogeny and molecular characterization of circulating MDV-1 field strains in Taiwan reveals a potential risk in MD outbreaks. This study investigated the genetic characteristics of 18 MDV-1 strains obtained from 17 vaccinated chicken flocks in Taiwan between 2018 and 2020. Based on the sequences of the meq oncogene, the phylogenetic analysis demonstrated that the circulating Taiwanese MDV-1 field strains were predominantly in a single cluster that showed high similarity with strains from countries of the East Asian region. Because the strains were obtained from CVI988/Rispens vaccinated chicken flocks and the molecular characteristics of the Meq oncoprotein showed features like vvMDV and vv+MDV strains, the circulating Taiwanese MDV-1 field strains may have higher virulence compared with vvMDV pathotype. In conclusion, the data presented demonstrates the circulation of hypervirulent MDV-1 strains in Taiwan and highlights the importance of routine surveillance and precaution strategies in response to the emergence of enhanced virulent MDV-1.

Targeting bacterial nickel transport with aspergillomarasmine A suppresses virulence-associated Ni-dependent enzymes.

Microbial Ni2+ homeostasis underpins the virulence of several clinical pathogens. Ni2+ is an essential cofactor in urease and [NiFe]-hydrogenases involved in colonization and persistence. Many microbes produce metallophores to sequester metals necessary for their metabolism and starve competing neighboring organisms. The fungal metallophore aspergillomarasmine A (AMA) shows narrow specificity for Zn2+, Ni2+, and Co2+. Here, we show that this specificity allows AMA to block the uptake of Ni2+ and attenuate bacterial Ni-dependent enzymes, offering a potential strategy for reducing virulence. Bacterial exposure to AMA perturbs H2 metabolism, ureolysis, struvite crystallization, and biofilm formation and shows efficacy in a Galleria mellonella animal infection model. The inhibition of Ni-dependent enzymes was aided by Zn2+, which complexes with AMA and competes with the native nickelophore for the uptake of Ni2+. Biochemical analyses demonstrated high-affinity binding of AMA-metal complexes to NikA, the periplasmic substrate-binding protein of the Ni2+ uptake system. Structural examination of NikA in complex with Ni-AMA revealed that the coordination geometry of Ni-AMA mimics the native ligand, Ni-(L-His)2, providing a structural basis for binding AMA-metal complexes. Structure-activity relationship studies of AMA identified regions of the molecule that improve NikA affinity and offer potential routes for further developing this compound as an anti-virulence agent.

Streptococcus suis serotype 4: a population with the potential pathogenicity in humans and pigs.

Streptococcus suis is a major bacterial pathogen in pigs and an emerging zoonotic pathogen. Different S. suis serotypes exhibit diverse characteristics in population structure and pathogenicity. Surveillance data highlight the significance of S. suis serotype 4 (SS4) in swine streptococcusis, a pathotype causing human infections. However, except for a few epidemiologic studies, the information on SS4 remains limited. In this study, we investigated the population structure, pathogenicity, and antimicrobial characteristics of SS4 based on 126 isolates, including one from a patient with septicemia. We discovered significant diversities within this population, clustering into six minimum core genome (MCG) groups (1, 2, 3, 4, 7-2, and 7-3) and five lineages. Two main clonal complexes (CCs), CC17 and CC94, belong to MCG groups 1 and 3, respectively. Numerous important putative virulence-associated genes are present in these two MCG groups, and 35.00% (7/20) of pig isolates from CC17, CC94, and CC839 (also belonging to MCG group 3) were highly virulent (mortality rate ≥ 80%) in zebrafish and mice, similar to the human isolate ID36054. Cytotoxicity assays showed that the human and pig isolates of SS4 strains exhibit significant cytotoxicity to human cells. Antimicrobial susceptibility testing showed that 95.83% of strains isolated from our labs were classified as multidrug-resistant. Prophages were identified as the primary vehicle for antibiotic resistance genes. Our study demonstrates the public health threat posed by SS4, expanding the understanding of SS4 population structure and pathogenicity characteristics and providing valuable information for its surveillance and prevention.

Loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing K1-ST23 hypervirulent Klebsiella pneumoniae.

OBJECTIVES: This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent Klebsiella pneumoniae (hvKp). METHODS: Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted. RESULTS: Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes blaCTX-M-15 and blaCTX-M-71, respectively, but KP30086 lost both. Cloning of gene blaCTX-M-71 and conjugation experiment of blaCTX-M-71-carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105. CONCLUSIONS: In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying blaCTX-M-71 and mutated OmpK36 had a synergetic effect on the resistance.