Serum Killing Research Articles

Evolution of Virulence, Fitness, and Carbapenem Resistance Transmission in ST23 Hypervirulent Klebsiella pneumoniae with the Capsular Polysaccharide Synthesis Gene wcaJ Inserted via Insertion Sequence Elements.

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) is recognized as a threat worldwide, but the mechanisms underlying its emergence remain unclear. As most CR-hvKP isolates are not hypermucoviscous, we speculated that the evolution of the capsule might result in the convergence of carbapenem resistance and hypervirulence. Here, 2,096 K. pneumoniae isolates were retrospectively collected to screen the ST23-K1 clone, and hypervirulence was roughly defined as being highly resistant to serum killing. The effect of wcaJ on the capsule, virulence, fitness, and resistance acquisition was further analyzed. The capsule gene wcaJ, inserted by ISKpn26/ISKpn74, was identified via whole-genome sequencing in four hvKP, but not hypermucoviscous, isolates. Uronic acid quantitation results revealed that these isolates produced significantly less capsular polysaccharides than NTUH-K2044. A significant increase in capsular production was observed in wcaJ-complemented isolates and confirmed by transmission electron microscopy. Further, all wcaJ-complemented isolates acquired greater resistance to macrophage phagocytosis, and one representative isolate resulted in a significantly higher mortality rate than the parental isolate in mice, indicating that wcaJ inactivation might compromise virulence. However, isolates with wcaJ interruption demonstrated a lower fitness cost and a high conjugation frequency of the blaKPC-2 plasmid, raising concerns about the emergence of carbapenem resistance in hvKP. IMPORTANCE Klebsiella pneumoniae is one of the most common nosocomial pathogens worldwide, and we speculated that the evolution of the capsule might result in the convergence of carbapenem resistance and hypervirulence of K. pneumoniae. The wcaJ gene was first reported to be interrupted by insertion sequence elements in ST23-K1 hypervirulent Klebsiella pneumoniae, resulting in little capsule synthesis, which plays an important role in virulence. We examined the effect of wcaJ on the capsule, virulence, and fitness. Isolates with wcaJ interruption might compromise virulence and demonstrated a lower fitness cost and a high conjugation frequency of the blaKPC-2 plasmid, highlighting its role as a potential factor facilitating hypervirulence and carbapenem resistance.

Serum Resistance of Mycoplasma agalactiae Strains and Mutants Bearing Different Lipoprotein Profiles.

In order to spread systemically, resistance against complement and other factors present in serum is an important trait in pathogenic bacteria. The variable proteins of Mycoplasma agalactiae (Vpmas) have been shown to affect differential adhesion, invasion and immune evasion, and undergo high-frequency phase-variation in expression. However, nothing is known about their involvement in M. agalactiae’s serum susceptibility. To evaluate this, the PG2 strain, the GM139 strain and the six Vpma phase-locked mutants (PLMs, PLMU to PLMZ) were tested for their ability to survive in the presence of non-sensitized and sensitized sheep serum, as well as guinea pig complement. Additionally, the reactivity of the sensitized sheep serum was analysed on the strains via western blotting. Overall data demonstrate PG2 strain to be more susceptible to sheep serum compared to the GM139 strain bearing a different Vpma profile. Significant differences were also observed between the different PLMs, with PLMU and PLMX showing the highest serum susceptibility in serum, while the other PLMs expressing longer Vpma proteins were more resistant. The results are in good correlation with previous studies where shorter lipoprotein variants contributed to a higher susceptibility to complement. Since none of the tested strains and PLMs were susceptible to non-sensitized sheep serum, antibodies seem to play an important role in serum killing.

Characterization of Novel Bacteriophage vB_KpnP_ZX1 and Its Depolymerases with Therapeutic Potential for K57 Klebsiella pneumoniae Infection.

A novel temperate phage vB_KpnP_ZX1 was isolated from hospital sewage samples using the clinically derived K57-type Klebsiella pneumoniae as a host. Phage vB_KpnP_ZX1, encoding three lysogen genes, the repressor, anti-repressor, and integrase, is the fourth phage of the genus Uetakevirus, family Podoviridae, ever discovered. Phage vB_KpnP_ZX1 did not show ideal bactericidal effect on K. pneumoniae 111-2, but TEM showed that the depolymerase Dep_ZX1 encoded on the short tail fiber protein has efficient capsule degradation activity. In vitro antibacterial results show that purified recombinant Dep_ZX1 can significantly prevent the formation of biofilm, degrade the formed biofilm, and improve the sensitivity of the bacteria in the biofilm to the antibiotics kanamycin, gentamicin, and streptomycin. Furthermore, the results of animal experiments show that 50 µg Dep_ZX1 can protect all K. pneumoniae 111-2-infected mice from death, whereas the control mice infected with the same dose of K. pneumoniae 111-2 all died. The degradation activity of Dep_ZX1 on capsular polysaccharide makes the bacteria weaken their resistance to immune cells, such as complement-mediated serum killing and phagocytosis, which are the key factors for its therapeutic action. In conclusion, Dep_ZX1 is a promising anti-virulence agent for the K57-type K. pneumoniae infection or biofilm diseases.

Nosocomial dissemination of hypervirulent Klebsiella pneumoniae with high-risk clones among children in Shanghai.

ObjectivesAlthough hypervirulent Klebsiella pneumoniae (hvKp) is an increasing public health problem, there remains limited epidemiological information regarding hvKp infections in children. Here, we conducted a clinical, molecular and phenotypic surveillance of hvKp strains in a pediatric population.MethodsNon-repetitive K. pneumoniae (Kp) strains consecutively collected during 2019-2020 were screened for hypervirulence genes (prmpA, prmpA2, iucA, iroB, and peg344) using PCR. Positive strains were further characterized by four phenotypic assays (string test, serum killing assay, siderophore production, Galleria mellonella lethality assay), followed by murine sepsis model to determine virulence in vitro and in vivo. Also, capsular types, sequence types, plasmid replicon types, antimicrobial resistance determinants and susceptibility were analyzed.ResultsA total of 352 isolates were collected, wherein 83 (23.6%) were hypervirulence genes-positive Kp (hgKp). A significant increase in KPC-2-producing KL47-ST11 among hgKp strains was observed, from 5.3% (1/19) in 2019 to 67.6% (25/37) in 2020 (P<.0001), suggesting the potential dissemination of the hybrid virulence and carbapenem-resistance encoding plasmid among children. Further, hgKp isolates were classified into hvKp (n = 27) and hgKp-low virulence (hgKp-Lv) (n = 56) based on virulence phenotypic assays. In hvKp, diverse genetic clones were observed and K1-ST23 or K2-ST25 strains with sensitivity to multiple antibiotics were prevalent (25.9%, 7/27). Compared with hgKp-Lv, hvKp infection had a higher propensity to involve severe pneumonia (22.2% vs. 12.5%) in elder children and significant higher mortality in mice (P = 0.0086). Additionally, either hvKp or hgKp-Lv infections were mostly healthcare-associated and hospital-acquired (74.1% vs. 91.9%).ConclusionsThese data suggest that K1-ST23 and K2-ST25 are high-risk clones of hvKp, and the genetic convergence of virulence and carbapenem-resistance is increasing among children. Control measures are needed to prevent the dissemination in clinical settings.

Genetic characterization and passage instability of a novel hybrid virulence plasmid in a ST23 hypervirulent Klebsiella pneumoniae.

Hypervirulent variants of Klebsiella pnuemoniae (hvKP), which causes life-threatening infections, is a global priority pathogen and frequently harbours virulence plasmids. The virulence plasmids have emerged as the predominant vehicles carrying the major pathogenic determinants of hypermucoviscosity and hypervirulence phenotypes. In the present study, we characterized a novel virulence plasmid in AP8555, an ST23 hvKP strain, which induced a metastatic infection and fatal septic shock in a critically ill patient. The serum killing assay, the quantitative biofilm formation assay, the G.mellonella infection model, and the mouse lethality assay demonstrated that AP8555 was almost as virulent as the hvKP strain NUTH-K2044. The plasmid pAP855 could be conjugated to Klebsiella quasipneumoniae ATCC700603 and E. coli J53 at a frequency of 7.2× 10−5 and 8.7× 10−7, respectively. Whole-genome sequencing and bioinformatics analysis confirmed that the plasmid was novel, clustered to the incompatibility type of IncHI1B/IncFIB/IncFII and presented high similarity to the pK2044 plasmid. In contrast, a 130-kb large-fragment insertion was observed on the plasmid, which introduced a genetic hybrid zone with multiple conjugation-related genes of type IV secretion systems (T4SS) and CcdAB toxin-antitoxin systems (TAS) to the plasmid. In the transconjugants, the presence of pAP855 had a negative impact on bacterial fitness, but enhancing the virulence-associated phenotypes. In vitro evolution experiments showed that pAP855 in the transconjugants could not be stably inherited after 10 days of passage. Our study not only reports a novel hybrid plasmid but also highlights the putative pathway of conjugative virulence plasmid formation and evolution by means of genetic rearrangement through sequence insertion. These findings indicate that structural versatility could contribute to the dissemination of cointegrate virulence plasmid, although the plasmid incurred a fitness cost. Therefore, continuous monitoring the acquisition of conjugative virulence plasmids may have critical value for plasmid research and increase awareness of hvKP.

The Specific Capsule Depolymerase of Phage PMK34 Sensitizes Acinetobacter baumannii to Serum Killing.

The rising antimicrobial resistance is particularly alarming for Acinetobacter baumannii, calling for the discovery and evaluation of alternatives to treat A. baumannii infections. Some bacteriophages produce a structural protein that depolymerizes capsular exopolysaccharide. Such purified depolymerases are considered as novel antivirulence compounds. We identified and characterized a depolymerase (DpoMK34) from Acinetobacter phage vB_AbaP_PMK34 active against the clinical isolate A. baumannii MK34. In silico analysis reveals a modular protein displaying a conserved N-terminal domain for anchoring to the phage tail, and variable central and C-terminal domains for enzymatic activity and specificity. AlphaFold-Multimer predicts a trimeric protein adopting an elongated structure due to a long α-helix, an enzymatic β-helix domain and a hypervariable 4 amino acid hotspot in the most ultimate loop of the C-terminal domain. In contrast to the tail fiber of phage T3, this hypervariable hotspot appears unrelated with the primary receptor. The functional characterization of DpoMK34 revealed a mesophilic enzyme active up to 50 °C across a wide pH range (4 to 11) and specific for the capsule of A. baumannii MK34. Enzymatic degradation of the A. baumannii MK34 capsule causes a significant drop in phage adsorption from 95% to 9% after 5 min. Although lacking intrinsic antibacterial activity, DpoMK34 renders A. baumannii MK34 fully susceptible to serum killing in a serum concentration dependent manner. Unlike phage PMK34, DpoMK34 does not easily select for resistant mutants either against PMK34 or itself. In sum, DpoMK34 is a potential antivirulence compound that can be included in a depolymerase cocktail to control difficult to treat A. baumannii infections.

MP02-11 CHARACTERIZATION OF COMPLEMENT RESISTANCE FACTOR TRAT ENCODED IN DRUG RESISTANCE PLASMIDS OF UROPATHOGENIC ESCHERICHIA COLI

You have accessJournal of UrologyCME1 May 2022MP02-11 CHARACTERIZATION OF COMPLEMENT RESISTANCE FACTOR TRAT ENCODED IN DRUG RESISTANCE PLASMIDS OF UROPATHOGENIC ESCHERICHIA COLI Chae Hee Lim, Aryana Bhati, Katelyn Lott, Jazmyn Whitfield, John Land, and Tatyana Sysoeva Chae Hee LimChae Hee Lim More articles by this author , Aryana BhatiAryana Bhati More articles by this author , Katelyn LottKatelyn Lott More articles by this author , Jazmyn WhitfieldJazmyn Whitfield More articles by this author , John LandJohn Land More articles by this author , and Tatyana SysoevaTatyana Sysoeva More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002514.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The TraT protein is a multifunctional virulence factor that is encoded by conjugative F-like plasmids. A majority of multidrug-resistant uropathogenic Escherichia coli (UPEC) carry F-like plasmids and thus encode for this TraT factor. In non-pathogenic E. coli, TraT is a highly expressed outer membrane lipoprotein that protects the bacterial cell from excessive rounds of plasmid transfer, from a few bacteriophages, and from the host innate immune response via serum complement killing. Our objectives in this study were (1) to measure the protective effect of TraT in UPEC background and (2) to establish whether extracellular TraT domain is sufficient to provide serum resistance. METHODS: We used conjugation to transfer the model F’ plasmid in different E. coli backgrounds. Recombinant extracellular soluble domain TraT33-244 was purified from BL21(DE3) E. coli strain using Ni-affinity chromatography. Exponentially growing E. coli cultures with and without traT-encoding plasmids or the purified TraT domain were subjected to different doses of blood serum for 2 hours at 37°C. Viable cells were quantified as colony forming units by plating serial dilutions at 0 and 2 hours after addition of serum. RESULTS: The traT-encoding plasmid provides variable level of protection that depends on serum type, bacterial cells, time, and ratios of bacteria and serum, and can reach several orders of magnitude. We have developed a method of expression and purification of soluble TraT33-244 protein and showed that this extracellular domain forms stable oligomers in solution with five-to-six subunits. Testing this purified extracellular domain in serum resistance assays revealed that it is insufficient to exhibit the full protective action of TraT. CONCLUSIONS: In this study, we measured the protective effect of traT-encoding plasmid F’ in model E. coli strains, which displayed strong but variable serum resistance. The extracellular soluble domain of TraT protein oligomerizes, but does not strongly protect against serum killing when added to bacterial cells in trans. These results suggest that membrane localization, N-terminus, or lipidation itself are important for function of the TraT factor. Overall, this study sets the stage for further investigation into the mechanism of how TraT assists UPEC bacteria in evading innate immune responses within the host organism. Source of Funding: The University of Alabama in Huntsville startup funds to Tatyana Sysoeva © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e14 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Chae Hee Lim More articles by this author Aryana Bhati More articles by this author Katelyn Lott More articles by this author Jazmyn Whitfield More articles by this author John Land More articles by this author Tatyana Sysoeva More articles by this author Expand All Advertisement PDF DownloadLoading ...

Emergence of a Hypervirulent Tigecycline-Resistant Klebsiella pneumoniae Strain Co-producing bla NDM-1 and bla KPC-2 With an Uncommon Sequence Type ST464 in Southwestern China.

Emergence of blaNDM–1 and blaKPC–2 co-producing Klebsiella pneumoniae strains is currently attracting widespread attention, but little information is available about their tigecycline resistance, virulence, and prevalence in Southwest China. In July 2021, an extensively drug-resistant K. pneumoniae strain AHSWKP25 whose genome contained both blaNDM–1 and blaKPC–2 genes was isolated from the blood of a patient with the malignant hematological disease in Luzhou, China. We investigated the resistance profiles of AHSWKP25 using microbroth dilution, agar dilution, modified carbapenemase inactivation (mCIM), and EDTA-modified carbapenemase inactivation methods (eCIM). The virulence of AHSWKP25 was assessed through string tests, serum killing assays, and a Galleria mellonella larval infection model. Conjugation and plasmid stability experiments were conducted to determine the horizontal transfer capacity of plasmids. And efflux pump phenotype test and real-time quantitative reverse transcription-PCR (RT-PCR) were used to determine its efflux pump activity. Sequencing of AHSWKP25 determined that AHSWKP25 belonged to ST464, which is resistant to antibiotics such as carbapenems, tetracycline, fluoroquinolones, tigecycline, and fosfomycin. The efflux pump phenotype tests and RT-PCR results demonstrated that efflux pumps were overexpressed in the AHSWKP25, which promoted the tigecycline resistance of the bacteria. AHSWKP25 also showed hypervirulence and serum resistance in vitro model. AHSWKP25 carried several different plasmids that contained blaNDM–1, blaKPC–2, and mutated tet(A) genes. Sequence alignment revealed that the plasmids carrying blaNDM–1 and blaKPC–2 underwent recombination and insertion events, respectively. We demonstrated that an X3 plasmid carrying blaNDM–1 was transferred from pSW25NDM1 to E. coli J53. We also identified missense mutations in the ramR, rcsA, lon, and csrD genes of AHSWKP25. Our results highlighted the potential of blaNDM–1 and blaKPC–2 co-producing K. pneumoniae strains to further develop antimicrobial resistance and hypervirulent phenotypes, but measures should be taken to closely monitor and control the spread of superbugs with multidrug-resistant phenotypes and hypervirulence.

The Role of the Two-Component QseBC Signaling System in Biofilm Formation and Virulence of Hypervirulent Klebsiella pneumoniae ATCC43816.

Hypervirulent Klebsiella pneumoniae (hvKP) is an evolving infectious pathogen associated with high mortality. The convergence of hypervirulence and multidrug resistance further challenges the clinical treatment options for K. pneumoniae infections. The QseBC two-component system (TCS) is a component of quorum-sensing regulatory cascade and functions as a global regulator of biofilm growth, bacterial motility, and virulence in Escherichia coli. However, the functional mechanisms of QseBC in hvKP have not been reported, and we aim to examine the role of QseBC in regulating virulence in hvKP strain ATCC43816. The CRISPR-Cas9 system was used to construct qseB, qseC, and qseBC knockout in ATCC43816. No significant alterations in the growth and antibiotic susceptibility were detected between wild-type and mutants. The deletion of qseC led to an increase of biofilm formation, resistance to serum killing, and high mortality in the G. mellonella model. RNAseq differential gene expression analysis exhibited that gene-associated biofilm formation (glgC, glgP, glgA, gcvA, bcsA, ydaM, paaF, ptsG), bacterial type VI secretion system (virB4, virB6, virB10, vgrG, hcp), and biosynthesis of siderophore (entC, entD, entE) were significantly upregulated in comparison with the wild-type control. In addition, qseB, ygiW (encode OB-family protein), and AraC family transcriptional regulator IT767_23090 genes showed highest expressions in the absence of QseC, which might be related to increased virulence. The study provided new insights into the functional importance of QseBC in regulating the virulence of hvKP.

Genetics and pathology associated with Klebsiella pneumoniae and Klebsiella spp. isolates from North American Pacific coastal marine mammals

Southern sea otters (SSO: Enhydra lutris nereis) are a federally-listed threatened subspecies found almost exclusively in California, USA. Despite their zoonotic potential and lack of host specificity, K. pneumoniae and Klebsiella spp. have largely unknown epizootiology in SSOs. Klebsiella pneumoniae is occasionally isolated at necropsy, but not from live SSOs. Hypermucoviscous (HMV) K. pneumoniae strains are confirmed pathogens of Pacific Basin pinnipeds, but have not been previously isolated from SSOs. We characterized the virulence profiles of K. pneumoniae isolates from necropsied SSOs, evaluated killing of marine mammal K. pneumoniae following in vitro exposure to California sea lion (CSL: Zalophanus californianus) whole blood and serum, and characterized lesion patterns associated with Klebsiella spp. infection in SSOs. Four of 15 SSO K. pneumoniae isolates were HMV and all were recovered from SSOs that stranded during 2005. Many K. pneumoniae infections were associated with moderate to severe pathology as a cause of death or sequela. All HMV infections were assessed as a primary cause of death or as a direct result of the primary cause of death. Klebsiella-infected SSOs exhibited bronchopneumonia, tracheobronchitis and/or pleuritis, enteritis, Profilicollis sp. acanthocephalan peritonitis, septic peritonitis, and septicemia. All SSO HMV isolates were capsular type K2, the serotype most associated with HMV infections in CSLs. Multiplex PCR revealed two distinct virulence gene profiles within HMV isolates and two within non-HMV isolates. In vitro experiments investigating CSL whole blood and serum killing of K. pneumoniae suggest that HMV isolates are more resistant to serum killing than non-HMV isolates.

Population analysis of Legionella pneumophila reveals a basis for resistance to complement-mediated killing

Legionella pneumophila is the most common cause of the severe respiratory infection known as Legionnaires’ disease. However, the microorganism is typically a symbiont of free-living amoeba, and our understanding of the bacterial factors that determine human pathogenicity is limited. Here we carried out a population genomic study of 902 L. pneumophila isolates from human clinical and environmental samples to examine their genetic diversity, global distribution and the basis for human pathogenicity. We find that the capacity for human disease is representative of the breadth of species diversity although some clones are more commonly associated with clinical infections. We identified a single gene (lag-1) to be most strongly associated with clinical isolates. lag-1, which encodes an O-acetyltransferase for lipopolysaccharide modification, has been distributed horizontally across all major phylogenetic clades of L. pneumophila by frequent recent recombination events. The gene confers resistance to complement-mediated killing in human serum by inhibiting deposition of classical pathway molecules on the bacterial surface. Furthermore, acquisition of lag-1 inhibits complement-dependent phagocytosis by human neutrophils, and promoted survival in a mouse model of pulmonary legionellosis. Thus, our results reveal L. pneumophila genetic traits linked to disease and provide a molecular basis for resistance to complement-mediated killing.

Anti-LPS IgA and IgG Can Inhibit Serum Killing of Pseudomonas aeruginosa in Patients with Cystic Fibrosis.

Pseudomonas aeruginosa is one of the principal pathogens implicated in respiratory infections of patients with cystic fibrosis (CF) and non-CF bronchiectasis. Previously, we demonstrated that impaired serum-mediated killing of P. aeruginosa was associated with increased severity of respiratory infections in patients with non-CF bronchiectasis. This inhibition was mediated by high titers of O-antigen-specific IgG2 antibodies that cloak the surface of the bacteria, blocking access to the membrane. Infection-related symptomatology was ameliorated in patients by using plasmapheresis to remove the offending antibodies. To determine if these inhibitory "cloaking antibodies" were prevalent in patients with CF, we investigated 70 serum samples from patients with P. aeruginosa infection and 5 from those without P. aeruginosa infection. Of these patients, 32% had serum that inhibited the ability of healthy control serum to kill P. aeruginosa. Here, we demonstrate that this inhibition of killing requires O-antigen expression. Furthermore, we reveal that while IgG alone can inhibit the activity of healthy control serum, O-antigen-specific IgA in patient sera can also inhibit serum-killing. We found that antibody affinity, not just titer, was also important in the inhibition of serum-mediated killing. These studies provide novel insight into cloaking antibodies in human infection and may provide further options in CF and other diseases for treatment of recalcitrant P. aeruginosa infections.

Co-occurrence of Klebsiella variicola and Klebsiella pneumoniae Both Carrying bla KPC from a Respiratory Intensive Care Unit Patient.

ObjectiveThe aim of this study was to use whole-genome sequencing to characterize Klebsiella pneumoniae SKp2F and Klebsiella variicola SKv2E, both carrying blaKPC, co-isolated from the same sputum specimen.MethodsAntimicrobial susceptibility testing was performed using microbroth dilution. Biofilm formation was determined by crystal violet staining and virulence was measured by a serum killing assay. Whole-genome sequencing of SKp2F and SKv2E was performed using an Illumina sequencer and the genetic characteristics were analyzed by computer.ResultsSKp2F and SKv2E were sensitive only to tigecycline and polymyxin among the tested antibiotics. The biofilm-forming ability of SKv2E is stronger than that of SKp2F. The grades of serum resistance of SKp2F and SKv2E are 4 and 3. MLST analysis of the 6,115,610 bp and 5,403,687 bp of SKv2E and SKp2F showed associations with ST1615 and ST631, respectively. SKv2E carried 13 resistance genes (blaKPC-2, blaTEM-1A, blaLEN17, aadA16, arr-3, qnrB4, oqxA/B, dfrA27, sul1, tetD, fosA, qacEΔ1) and SKp2F carried 23 (blaKPC-2, blaCTX-M-3, blaTEM-1B, blaCTX-M-65, blaSHV-27, aac(6ʹ)-IIa, rmtB, arr-3, aph(3ʹ)-Ia, aadA16, qnrS1, aac(6ʹ)-Ib-cr, qnrB91, oqxA/B, mph(A), tet(A), fosA, dfrA27, and two copies of qacEΔ1-sul1). Most of them were carried by various mobile genetic elements, such as IncFIB(K)/IncFII(K)/IncFII(Yp), IncFII(K) plasmid, Tn6338, and In469. Both SKv2E and SKp2F carried a large number of virulence factors, including type 1 and 3 fimbriae, capsule, aerobactin (iutA), ent siderophore (entABCDEFS, fepABCDGfes), and salmochelin (iroE/iroEN). SKv2E also carried type IV pili (pilW), fimbrial adherence (steB, stfD), and capsule biosynthesis gene (glf).ConclusionblaKPC-2-carrying K. variicola and K. pneumoniae, which carried multiple resistance genes, virulence factors, and highly similar mobile genetic elements, were identified from the same specimen, indicating that clinical samples may carry multiple bacteria. We should avoid misidentification, and bear in mind that resistance genes carrying mobile genetic elements can be transmitted or integrated between bacteria in the same host.

FBXO2/SCF ubiquitin ligase complex directs xenophagy through recognizing bacterial surface glycan.

Xenophagy, also known as antibacterial selective autophagy, degrades invading bacterial pathogens such as group A Streptococcus (GAS) to defend cells. Although invading bacteria are known to be marked with ubiquitin and selectively targeted by xenophagy, how ubiquitin ligases recognize invading bacteria is poorly understood. Here, we show that FBXO2, a glycoprotein-specific receptor for substrate in the SKP1/CUL1/F-box protein (SCF) ubiquitin ligase complex, mediates recognition of GlcNAc side chains of the GAS surface carbohydrate structure and promotes ubiquitin-mediated xenophagy against GAS. FBXO2 targets cytosolic GAS through its sugar-binding motif and GlcNAc expression on the GAS surface. FBXO2 knockout resulted in decreased ubiquitin accumulation on intracellular GAS and xenophagic degradation of bacteria. Furthermore, SCF components such as SKP1, CUL1, and ROC1 are required for ubiquitin-mediated xenophagy against GAS. Thus, SCFFBXO2 recognizes GlcNAc residues of GAS surface carbohydrates and functions in ubiquitination during xenophagy.

Factor H binding protein (fHbp)-mediated differential complement resistance of a serogroup C Neisseria meningitidis isolate from cerebrospinal fluid of a patient with invasive meningococcal disease.

During an outbreak of invasive meningococcal disease (IMD) at the University of Southampton, UK, in 1997, two Neisseria meningitidis serogroup C isolates were retrieved from a student (‘Case’), who died of IMD, and a close contact (‘Carrier’) who, after mouth-to-mouth resuscitation on the deceased, did not contract the disease. Genomic comparison of the isolates demonstrated extensive nucleotide sequence identity, with differences identified in eight genes. Here, comparative proteomics was used to measure differential protein expression between the isolates and investigate whether the differences contributed to the clinical outcomes. A total of six proteins were differentially expressed: four proteins (methylcitrate synthase, PrpC; hypothetical integral membrane protein, Imp; fructose-1,6-bisphosphate aldolase, Fba; aldehyde dehydrogenase A, AldA) were upregulated in the Case isolate, while one protein (Type IV pilus-associated protein, PilC2) was downregulated. Peptides for factor H binding protein (fHbp), a major virulence factor and antigenic protein, were only detected in the Case, with a single base deletion (ΔT366) in the Carrier fHbp causing lack of its expression. Expression of fHbp resulted in an increased resistance of the Case isolate to complement-mediated killing in serum. Complementation of fHbp expression in the Carrier increased its serum resistance by approximately 8-fold. Moreover, a higher serum bactericidal antibody titre was seen for the Case isolate when using sera from mice immunized with Bexsero (GlaxoSmithKline), a vaccine containing fHbp as an antigenic component. This study highlights the role of fHbp in the differential complement resistance of the Case and the Carrier isolates. Expression of fHbp in the Case resulted in its increased survival in serum, possibly leading to active proliferation of the bacteria in blood and death of the student through IMD. Moreover, enhanced killing of the Case isolate by sera raised against an fHbp-containing vaccine, Bexsero, underlines the role and importance of fHbp in infection and immunity.

OmpC, a novel factor H-binding surface protein, is dispensable for the adherence and virulence of Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium utilizes a series of strategies to evade host innate immune defenses, including the serum complement system. Many microbial pathogens have evolved the ability to bind the complement regulatory protein factor H (FH) through their surface factor H-binding proteins (FHBPs) to circumvent the complement-mediated bactericidal effect. However, the roles of FHBPs in Salmonella pathogenesis are not well understood. In this study, we demonstrated that the survival of S. Typhimurium in human serum was decreased in a time and concentration dependent manner. Pre-incubation with FH attenuated the sensitivity of S. Typhimurium strain χ3761 to complement-mediated serum killing, suggesting FH binding enhance survival in serum. We aimed to identify novel S. Typhimurium FHBPs and characterize their biological functions. Here, six potential FHBPs were identified by two-dimensional (2D)-Far-western blot, and three of them were further confirmed to bind FH by Far-western blot and dot blot. We found that deletion of ompC (ΔompC) significantly inhibited the survival of S. Typhimurium strain χ3761 in human serum. Our results indicated that the ompC mutation does not affect χ3761 adhesion to HeLa cells. Furthermore, a mice infection model showed that deletion of ompC had no significant effect on the histopathological lesions or viability compared with the wild-type strain χ3761. In summary, these results suggested that OmpC is an important FHBP, but not a critical virulence factor of S. Typhimurium.

β-lactam resistance associated with β-lactamase production and porin alteration in clinical isolates of E. coli and K. pneumoniae.

β-lactam resistance represents a worldwide problem and a serious challenge for antimicrobial treatment. Hence this research was conducted to recognize several mechanisms mediating β-lactam resistance in E. coli and K. pneumoniae clinical isolates collected from Mansoura University hospitals, Egypt. A total of 80 isolates, 45 E. coli and 35 K. pneumoniae isolates, were collected and their antibiotic susceptibility was determined by the Disc diffusion method followed by phenotypic and genotypic detection of extended-spectrum β-lactamases (ESBLs), AmpC β-lactamase, carbapenemase enzymes. The outer membrane protein porins of all isolates were analyzed and their genes were examined using gene amplification and sequencing. Also, the resistance to complement-mediated serum killing was estimated. A significant percentage of isolates (93.8%) were multidrug resistance and showed an elevated resistance to β-lactam antibiotics. The presence of either ESBL or AmpC enzymes was high among isolates (83.75%). Also, 60% of the isolated strains were carbapenemase producers. The most frequently detected gene of ESBL among all tested isolates was blaCTX-M-15 (86.3%) followed by blaTEM-1 (81.3%) and blaSHV-1 (35%) while the Amp-C gene was present in 83.75%. For carbapenemase-producing isolates, blaNDM1 was the most common (60%) followed by blaVIM-1 (35%) and blaOXA-48 (13.8%). Besides, 73.3% and 40% of E. coli and K. pneumoniae isolates respectively were serum resistant. Outer membrane protein analysis showed that 93.3% of E. coli and 95.7% of K. pneumoniae isolates lost their porins or showed modified porins. Furthermore, sequence analysis of tested porin genes in some isolates revealed the presence of frameshift mutations that produced truncated proteins of smaller size. β-lactam resistance in K. pneumoniae and E. coli isolates in our hospitals is due to a combination of β-lactamase activity and porin loss/alteration. Hence more restrictions should be applied on β-lactams usage to decrease the emergence of resistant strains.

Shift in the Dominant Sequence Type of Carbapenem-Resistant Klebsiella pneumoniae Bloodstream Infection from ST11 to ST15 at a Medical Center in Northeast China, 2015-2020.

ObjectiveTo investigate the clinical characteristics and molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) bloodstream infection at a medical center in northeast China, especially after coronavirus disease (COVID-19) pandemic.MethodsFifty-one patients were diagnosed with CRKP bloodstream infection between January 2015 and December 2020, among which 42 isolates were available for further study. Species identification and antibiotic susceptibilities were tested with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and VITEK 2 systems. Carbapenemase genes, virulence genes and MLST genes were detected by polymerase chain reaction. Moreover, the string test and serum killing assay were performed to evaluate the virulence of the CRKP isolates.ResultsDuring the six-year period, the detection rate of CRKP in bloodstream infection showed an increasing trend, with the intensive care unit, hematology and respiratory medicine wards mainly affected. Molecular epidemiology analyses showed that KPC-2 was the dominant carbapenemase gene. In addition, the dominant sequence type (ST) of CRKP shifted from ST11 to ST15 strains, which were all sensitive to amikacin in contrast to the ST11 stains. Furthermore, ST15 CRKP strains were positive for the KfuB virulence gene and more resistant to serum killing compared to the ST11 CRKP strains. Nonetheless, the mortality rate of patients infected with ST11 and ST15 CRKP did not show any significant differences.ConclusionA shift in the dominant sequence type of CRKP bloodstream infections from ST11 to ST15 was observed during the years 2015–2020. Compared to ST11, the ST15 CRKP strains showed amikacin sensitivity, positivity for KfuB gene, and serum resistance, which may indicate stronger virulence.

Negative correlation between virulence and multidrug resistance in intrahospital and community acquired infections by Proteus mirabilis, in Eastern Venezuela

This is the first report for Venezuela of virulence/pathogenicity and resistance factors in intrahospital (HCAI) and community-acquired infections (CAI) by P. mirabilis in two main hospitals from Eastern Venezuela. Virulence factors such as motility, biofilms, and resistance to serum killing (RSK) were determined. Antimicrobial susceptibility allowed classifying the isolates into resistant, multidrug resistant (MDR) and extensively drug-resistant (XDR). P. mirabilis was identified in HCAI in both hospitals mostly from secretions, while some CAI were identified from urine and secretions. Twitching, swarming, biofilm and RSK were identified in many isolates. Eleven antimicrobials showed resistance frequencies from 22-54% in one or both hospitals. A high frequency of MDR isolates was found in these hospitals (60.6 to 56.5%). Strains carrying both blaCTX-M and blaTEM genes were found in one hospital in a frequency of 27.0%. We also found that the frequency of MDR was lower in strains with three or more virulence factors compared to those with fewer factors. Bacteria with swarming showed 5.85 times lower probability of being MDR, and those with twitching, 7.52 times lower probability. Infections by MDR/XDR P. mirabilis strains in HCAI and CAI represent a public health problem that requires effective control and prevention measures to reduce their potential spread and persistence in the population.

A Protective and Pathogenic Role for Complement During Acute Toxoplasma gondii Infection.

The infection competence of the protozoan pathogen Toxoplasma gondii is critically dependent on the parasite’s ability to inactivate the host complement system. Toxoplasma actively resists complement-mediated killing in non-immune serum by recruiting host-derived complement regulatory proteins C4BP and Factor H (FH) to the parasite surface to inactivate surface-bound C3 and limit formation of the C5b-9 membrane attack complex (MAC). While decreased complement activation on the parasite surface certainly protects Toxoplasma from immediate lysis, the biological effector functions of C3 split products C3b and C3a are maintained, which includes opsonization of the parasite for phagocytosis and potent immunomodulatory effects that promote pro-inflammatory responses and alters mucosal defenses during infection, respectively. In this review, we discuss how complement regulation by Toxoplasma controls parasite burden systemically but drives exacerbated immune responses locally in the gut of genetically susceptible C57BL/6J mice. In effect, Toxoplasma has evolved to strike a balance with the complement system, by inactivating complement to protect the parasite from immediate serum killing, it generates sufficient C3 catabolites that signal through their cognate receptors to stimulate protective immunity. This regulation ultimately controls tachyzoite proliferation and promotes host survival, parasite persistence, and transmissibility to new hosts.