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139500.9923Emerging Multidrug-Resistant Hybrid Pathotype Shiga Toxin-Producing Escherichia coli O80 and Related Strains of Clonal Complex 165, Europe. Enterohemorrhagic Escherichia coli serogroup O80, involved in hemolytic uremic syndrome associated with extraintestinal infections, has emerged in France. We obtained circularized sequences of the O80 strain RDEx444, responsible for hemolytic uremic syndrome with bacteremia, and noncircularized sequences of 35 O80 E. coli isolated from humans and animals in Europe with or without Shiga toxin genes. RDEx444 harbored a mosaic plasmid, pR444_A, combining extraintestinal virulence determinants and a multidrug resistance-encoding island. All strains belonged to clonal complex 165, which is distantly related to other major enterohemorrhagic E. coli lineages. All stx-positive strains contained eae-ξ, ehxA, and genes characteristic of pR444_A. Among stx-negative strains, 1 produced extended-spectrum β-lactamase, 1 harbored the colistin-resistance gene mcr1, and 2 possessed genes characteristic of enteropathogenic and pyelonephritis E. coli. Because O80-clonal complex 165 strains can integrate intestinal and extraintestinal virulence factors in combination with diverse drug-resistance genes, they constitute dangerous and versatile multidrug-resistant pathogens.201830457551
621310.9922Use of a Dictyostelium model for isolation of genetic loci associated with phagocytosis and virulence in Klebsiella pneumoniae. Phagocytosis resistance is an important virulence factor in Klebsiella pneumoniae. Dictyostelium has been used to study the interaction between phagocytes and bacteria because of its similarity to mammalian macrophages. In this study, we used a Dictyostelium model to investigate genes for resistance to phagocytosis in NTUH-K2044, a strain of K. pneumoniae causing pyogenic liver abscess that is highly resistant to phagocytosis. A total of 2,500 transposon mutants were screened by plaque assay, and 29 of them permitted phagocytosis by Dictyostelium. In the 29 mutants, six loci were identified; three were capsular synthesis genes. Of the other three, one was related to carnitine metabolism, one encoded a subunit of protease (clpX), and one encoded a lipopolysaccharide O-antigen transporter (wzm). Deletion and complementation of these genes showed that only ΔclpX and Δwzm mutants became susceptible to Dictyostelium phagocytosis, and their complementation restored the phagocytosis resistance phenotype. These two mutants were also susceptible to phagocytosis by human neutrophils and revealed attenuated virulence in a mouse model, implying that they play important roles in the pathogenesis of K. pneumoniae. Furthermore, we demonstrated that clpP, which exists in an operon with clpX, was also involved in resistance to phagocytosis. The transcriptional profile of ΔclpX was examined by microarray analysis and revealed a 3-fold lower level of expression of capsular synthesis genes. Therefore, we have identified genes involved in resistance to phagocytosis in K. pneumoniae using Dictyostelium, and this model is useful to explore genes associated with resistance to phagocytosis in heavily encapsulated bacteria.201121173313
42420.9919Molecular analysis of bacterial cytolysins. Results of molecular and pathogenic studies of three different bacterial hemolysins (cytolysins) are presented. These exoproteins derive from the two gram-negative bacteria Escherichia coli and Aeromonas hydrophila and from the gram-positive pathogen Listeria monocytogenes. The hemolysin of E. coli is determined by an 8-kilobase (kb) region that includes four clustered genes (hlyC, hlyA, hlyB, and hlyD). This hemolysin determinant is part either of large transmissible plasmids or of the chromosome. The genes located chromosomally are found predominantly in E. coli strains that can cause pyelonephritis and/or other extraintestinal infections. A detailed analysis of the chromosomal hyl determinants of one nephropathogenic E. coli strain revealed the existence of specific, large chromosomal insertions 75 kb and 100 kb in size that carry the hly genes but that also influence the expression of other virulence properties, i.e., adhesion and serum resistance. The direct involvement of E. coli hemolysin in virulence could be demonstrated in several model systems. The genetic determinants for hemolysin (cytolysin) formation in A. hydrophila (aerolysin) and L. monocytogenes (listeriolysin) are less complex. Both cytolysins seem to be encoded by single genes, although two loci (aerB and aerC) that affect the expression and activity of aerolysin have been identified distal and proximal to the structural gene for aerolysin (aerA). Cytolysin-negative mutants of both bacteria were obtained by site-specific deletion and/or transposon mutagenesis. These mutants show a drastic reduction in the virulence of the respective bacteria.19872825323
621230.9918Strain differences in the susceptibility and resistance of Pasteurella multocida to phagocytosis and killing by rabbit polymorphonuclear neutrophils. The interactions of 2 capsular serotype A and 4 serotype D strains of Pasteurella multocida with rabbit polymorphonuclear neutrophils (PMN) were compared in vitro, using a PMN phagocytic and bactericidal assay. Bacteria and rabbit PMN were incubated for 15 minutes. The suspensions were subjected to differential centrifugation and the percentage of phagocytosis (cell association) was determined from the number of viable noncell-associated bacteria. The cell pellets and the associated bacteria were resuspended and PMN bactericidal activity was calculated from the number of remaining viable cell-associated bacteria at 45 and 75 minutes after the start of the assay. Test bacteria were not opsonized or were opsonized with immune serum containing active complement. One type A strain was ingested and killed by PMN in the presence and absence of opsonins. The 5 remaining strains were resistant to PMN killing, but only the type A strain resisted phagocytosis. Resistance of the type A strain was attributed to the hyaluronic acid capsule, since pretreatment of the bacteria with hyaluronidase rendered opsonized bacteria susceptible to ingestion and killing. The pattern of resistance of the 4 type D strains was different from that of the resistant type A strain. Both opsonized and nonopsonized type D bacteria became cell associated, but none were killed by PMN. The mechanism of resistance of these 4 strains to PMN bactericidal activity is currently unknown.19846742581
139640.9916Genomic Characterization of hlyF-positive Shiga Toxin-Producing Escherichia coli, Italy and the Netherlands, 2000-2019. Shiga toxin-producing Escherichia coli (STEC) O80:H2 has emerged in Europe as a cause of hemolytic uremic syndrome associated with bacteremia. STEC O80:H2 harbors the mosaic plasmid pR444_A, which combines several virulence genes, including hlyF and antimicrobial resistance genes. pR444_A is found in some extraintestinal pathogenic E. coli (ExPEC) strains. We identified and characterized 53 STEC strains with ExPEC-associated virulence genes isolated in Italy and the Netherlands during 2000-2019. The isolates belong to 2 major populations: 1 belongs to sequence type 301 and harbors diverse stx(2) subtypes, the intimin variant eae-ξ, and pO157-like and pR444_A plasmids; 1 consists of strains belonging to various sequence types, some of which lack the pO157 plasmid, the locus of enterocyte effacement, and the antimicrobial resistance-encoding region. Our results showed that STEC strains harboring ExPEC-associated virulence genes can include multiple serotypes and that the pR444_A plasmid can be acquired and mobilized by STEC strains.202133622476
549250.9916Uropathogenic bacteria and deductive genomics towards antimicrobial resistance, virulence, and potential drug targets. Urinary tract infections (UTIs) are among the most prevalent bacterial infections affecting people in inpatient and outpatient settings. The current study aimed to sequence the genome of uropathogenic Escherichia coli strain CUI-B1 resourced from a woman having uncomplicated cystitis and pyelonephritis. Followed by deductive genomics towards potential drug targets using E. coli strain CUI-B1, strain O25b: H4-ST131, Proteus mirabilis strain HI4320, Klebsiella pneumoniae strain 1721, and Staphylococcus saprophyticus strain ATCC 15305 uropathogenic strains. Comparative genome analysis revealed that genes related to the survival of E. coli, P. mirabilis, K. pneumoniae, and S. saprophyticus, such as genes of metal-requiring proteins, defense-associated genes, and genes associated with general physiology, were found to be highly conserved in the genomes including strain CUI-B1. However, the genes responsible for virulence and drug resistance, mainly those that are involved in bacterial secretion, fimbriae, adherence, and colonization, were found in various genomic regions and varied from one species to another or within the same species. Based on the genome sequence, virulence, and antimicrobial-resistant gene dataset, the subtractive proteomics approach revealed 22 proteins mapped to the pathogen's unique pathways and among them, entB, clbH, chuV, and ybtS were supposed to be potential drug targets and the single drug could be utilized for all above-mentioned strains. These results may provide the foundation for the optimal target for future discovery of drugs for E. coli-, P. mirabilis-, K. pneumoniae-, and S. saprophyticus-based infections and could be investigated further to employ in personalized drug development.202437553507
248560.9915Characterisation of uropathogenic Escherichia coli from children with urinary tract infection in different countries. Uropathogenic Escherichia coli (UPEC) carry many virulence factors, including those involved in long-term survival in the urinary tract. However, their prevalence and role among UPEC causing urinary tract infection (UTI) in children is not well studied. To further understand the virulence characteristics of these bacteria, we investigated the prevalence of antibiotic resistance, antigen 43 genes, curli and cellulose among UPEC in children from different countries. Isolates (n = 337) from five countries were tested for antibiotic susceptibility, phylogenetic groups, prevalence of flu, fluA(CFT073), fluB(CFT073), curli and cellulose. High prevalence of multidrug resistance and extended spectrum beta lactamase production was found among Iranian and Vietnamese isolates. Resistance was associated with phylogenetic group D while group B2 was associated with fluA(CFT073) and fluB(CFT073). Fewer Iranian isolates carried fluA(CFT073), curli and cellulose. fluB(CFT073) was most prevalent among Slovak isolates. Ampicillin and amoxicillin/clavulanic acid resistance was prevalent among fluA(CFT073)- and fluB(CFT073)-positive Australian, Iranian and Swedish isolates. Lack of curli and cellulose was associated with resistance among Vietnamese isolates. We conclude that major differences exist in the prevalence of antibiotic resistance among UPEC from different countries. Associations observed between resistance and virulence factors may, in different ways, promote the long-term survival of UPEC in the urinary tract.201121509475
249070.9915Relationship between drug resistance and the clustered, regularly interspaced, short, palindromic repeat-associated protein genes cas1 and cas2 in Shigella from giant panda dung. BACKGROUND: To detect drug resistance in Shigella obtained from the dung of the giant panda, explore the factors leading to drug resistance in Shigella, understand the characteristics of clustered, regularly interspaced, short, palindromic repeats (CRISPR), and assess the relationship between CRISPR and drug resistance. METHODS: We collected fresh feces from 27 healthy giant pandas in the Giant Panda Conservation base (Wolong, China). We identified the strains of Shigella in the samples by using nucleotide sequence analysis. Further, the Kirby-Bauer paper method was used to determine drug sensitivity of the Shigella strains. CRISPR-associated protein genes cas1 and cas2 in Shigella were detected by polymerase chain reaction (PCR), and the PCR products were sequenced and compared. RESULTS: We isolated and identified 17 strains of Shigella from 27 samples, including 14 strains of Shigella flexneri, 2 strains of Shigella sonnei, and 1 strain of Shigella dysenteriae. Further, drug resistance to cefazolin, imipenem, and amoxicillin-clavulanic acid was identified as a serious problem, as multidrug-resistant strains were detected. Further, cas1 and cas2 showed different degrees of point mutations. CONCLUSION: The CRISPR system widely exists in Shigella and shares homology with that in Escherichia coli. The cas1 and cas 2 mutations contribute to the different levels of resistance. Point mutations at sites 3176455, 3176590, and 3176465 in cas1 (a); sites 3176989, 3176992, and 3176995 in cas1 (b); sites 3176156 and 3176236 in cas2 may affect the resistance of bacteria, cause emergence of multidrug resistance, and increase the types of drug resistance.201728207509
43680.9915The capsule is a virulence determinant in the pathogenesis of Pasteurella multocida M1404 (B:2). Capsules from a range of pathogenic bacteria are key virulence determinants, and the capsule has been implicated in virulence in Pasteurella multocida. We have previously identified and determined the nucleotide sequence of the P. multocida M1404 (B:2) capsule biosynthetic locus (J. D. Boyce, J. Y. Chung, and B. Adler, Vet. Microbiol. 72:121-134, 2000). The cap locus consists of 15 genes, which can be grouped into three functional regions. Regions 1 and 3 contain genes proposed to encode proteins involved in capsule export, and region 2 contains genes proposed to encode proteins involved in polysaccharide biosynthesis. In order to construct a mutant impaired in capsule export, the final gene of region 1, cexA, was disrupted by insertion of a tetracycline resistance cassette by allelic replacement. The genotype of the tet(M) OmegacexA mutant was confirmed by Southern hybridization and PCR. The acapsular phenotype was confirmed by immunofluorescence, and the strain could be complemented and returned to capsule production by the presence of a cloned uninterrupted copy of cexA. Wild-type, mutant, and complemented strains were tested for virulence by intraperitoneal challenge of mice; the presence of the capsule was shown to be a crucial virulence determinant. Following intraperitoneal challenge of mice, the acapsular bacteria were removed efficiently from the blood, spleen, and liver, while wild-type bacteria multiplied rapidly. Acapsular bacteria were readily taken up by murine peritoneal macrophages, but wild-type bacteria were significantly resistant to phagocytosis. Both wild-type and acapsular bacteria were resistant to complement in bovine and murine serum.200010816499
248990.9915First Isolation and Identification of Aeromonas veronii in a Captive Giant Panda (Ailuropoda melanoleuca). The objective of this study was to understand biological characteristics of one bacteria strain named as VPG which was isolated from multiple organs of a dead captive giant panda cub. Here, we use biochemical tests, 16S rRNA and gyrB genes for bacterial identification, the disk diffusion method for antibiotic resistance phenotype, smart chip real-time PCR for the antibiotic resistance genotype, multiplex PCR for determination of virulence genes, and the acute toxicity test in mice for testing the pathogenicity of isolates. The isolate was identified as A. veronii strain based on the biochemical properties and genetic analysis. We found that the strain carried 31 antibiotic resistance genes, revealed antimicrobial resistance phenotypically to several antibiotics including penicillin, ampicillin, oxacillin, amoxicillin, imipenem, and vancomycin, and carried virulence genes including aer, act, lip, exu, ser, luxs, and tapA. The main pathological changes in giant panda were congestion, necrotic lesions and a large number of bacteria in multiple organs. In addition, the LD(50) in Kunming mice infected with strain VGP was 5.14 × 10(7) CFU/mL by intraperitoneal injection. Infection with strain VGP led to considerable histological lesions such as hemorrhage of internal organs, necrosis of lymphocytes and neurons in Kunming mice. Taken together, these results suggest that infection with strain VGP would be an important causes of death in this giant panda cub.202337685043
8893100.9914Transcriptome of uropathogenic Escherichia coli during urinary tract infection. A uropathogenic Escherichia coli strain CFT073-specific DNA microarray that includes each open reading frame was used to analyze the transcriptome of CFT073 bacteria isolated directly from the urine of infected CBA/J mice. The in vivo expression profiles were compared to that of E. coli CFT073 grown statically to exponential phase in rich medium, revealing the strategies this pathogen uses in vivo for colonization, growth, and survival in the urinary tract environment. The most highly expressed genes overall in vivo encoded translational machinery, indicating that the bacteria were in a rapid growth state despite specific nutrient limitations. Expression of type 1 fimbriae, a virulence factor involved in adherence, was highly upregulated in vivo. Five iron acquisition systems were all highly upregulated during urinary tract infection, as were genes responsible for capsular polysaccharide and lipopolysaccharide synthesis, drug resistance, and microcin secretion. Surprisingly, other fimbrial genes, such as pap and foc/sfa, and genes involved in motility and chemotaxis were downregulated in vivo. E. coli CFT073 grown in human urine resulted in the upregulation of iron acquisition, capsule, and microcin secretion genes, thus partially mimicking growth in vivo. On the basis of gene expression levels, the urinary tract appears to be nitrogen and iron limiting, of high osmolarity, and of moderate oxygenation. This study represents the first assessment of any E. coli pathotype's transcriptome in vivo and provides specific insights into the mechanisms necessary for urinary tract pathogenesis.200415501767
2097110.9914Effective Photodynamic Therapy with Ir(III) for Virulent Clinical Isolates of Extended-Spectrum Beta-Lactamase Klebsiella pneumoniae. BACKGROUND: The extended-spectrum beta-lactamase (ESBL) Klebsiella pneumoniae is one of the leading causes of health-associated infections (HAIs), whose antibiotic treatments have been severely reduced. Moreover, HAI bacteria may harbor pathogenic factors such as siderophores, enzymes, or capsules, which increase the virulence of these strains. Thus, new therapies, such as antimicrobial photodynamic inactivation (aPDI), are needed. METHOD: A collection of 118 clinical isolates of K. pneumoniae was characterized by susceptibility and virulence through the determination of the minimum inhibitory concentration (MIC) of amikacin (Amk), cefotaxime (Cfx), ceftazidime (Cfz), imipenem (Imp), meropenem (Mer), and piperacillin-tazobactam (Pip-Taz); and, by PCR, the frequency of the virulence genes K2, magA, rmpA, entB, ybtS, and allS. Susceptibility to innate immunity, such as human serum, macrophages, and polymorphonuclear cells, was tested. All the strains were tested for sensitivity to the photosensitizer PSIR-3 (4 µg/mL) in a 17 µW/cm(2) for 30 min aPDI. RESULTS: A significantly higher frequency of virulence genes in ESBL than non-ESBL bacteria was observed. The isolates of the genotype K2+, ybtS+, and allS+ display enhanced virulence, since they showed higher resistance to human serum, as well as to phagocytosis. All strains are susceptible to the aPDI with PSIR-3 decreasing viability in 3log10. The combined treatment with Cfx improved the aPDI to 6log10 for the ESBL strains. The combined treatment is synergistic, as it showed a fractional inhibitory concentration (FIC) index value of 0.15. CONCLUSIONS: The aPDI effectively inhibits clinical isolates of K. pneumoniae, including the riskier strains of ESBL-producing bacteria and the K2+, ybtS+, and allS+ genotype. The aPDI with PSIR-3 is synergistic with Cfx.202133922077
6234120.9913Impact of the rpoS genotype for acid resistance patterns of pathogenic and probiotic Escherichia coli. BACKGROUND: Enterohemorrhagic E. coli (EHEC), a subgroup of Shiga toxin (Stx) producing E. coli (STEC), may cause severe enteritis and hemolytic uremic syndrome (HUS) and is transmitted orally via contaminated foods or from person to person. The infectious dose is known to be very low, which requires most of the bacteria to survive the gastric acid barrier. Acid resistance therefore is an important mechanism of EHEC virulence. It should also be a relevant characteristic of E. coli strains used for therapeutic purposes such as the probiotic E. coli Nissle 1917 (EcN). In E. coli and related enteric bacteria it has been extensively demonstrated, that the alternative sigma factor sigmaS, encoded by the rpoS gene, acts as a master regulator mediating resistance to various environmental stress factors. METHODS: Using rpoS deletion mutants of a highly virulent EHEC O26:H11 patient isolate and the sequenced prototype EHEC EDL933 (ATCC 700927) of serotype O157:H7 we investigated the impact of a functional rpoS gene for orchestrating a satisfactory response to acid stress in these strains. We then functionally characterized rpoS of probiotic EcN and five rpoS genes selected from STEC isolates pre-investigated for acid resistance. RESULTS: First, we found out that ATCC isolate 700927 of EHEC EDL933 has a point mutation in rpoS, not present in the published sequence, leading to a premature stop codon. Moreover, to our surprise, one STEC strain as well as EcN was acid sensitive in our test environment, although their cloned rpoS genes could effectively complement acid sensitivity of an rpoS deletion mutant. CONCLUSION: The attenuation of sequenced EHEC EDL933 might be of importance for anyone planning to do either in vitro or in vivo studies with this prototype strain. Furthermore our data supports recently published observations, that individual E. coli isolates are able to significantly modulate their acid resistance phenotype independent of their rpoS genotype.200717386106
5190130.9913Genomic Analysis of Cronobacter condimenti s37: Identification of Resistance and Virulence Genes and Comparison with Other Cronobacter and Closely Related Species. Cronobacter condimenti are environmental commensals that have not been associated with any clinical infections. To date, they are the least understood and described Cronobacter species within the genus. The objective of this study was to use a draft genome sequence (DGS) of the Cronobacter condimenti strain s37 to screen for genes encoding for antibiotic resistance, virulence, response to environmental stress, and biofilm formation. The strain was isolated in Poland from commercial small radish sprouts. This is the second genome of this species available in the GenBank database. The comparative genome analysis (cgMLST) of C. condimenti s37 with other Cronobacter spp. including the pathogenic species C. sakazakii and the plant-associated closely related genera Franconibacter and Siccibacter was also performed. The assembled and annotated genome of the C. condimenti s37 genome was 4,590,991 bp in length, with a total gene number of 4384, and a GC content of 55.7%. The s 37 genome encoded for genes associated with resistance to stressful environmental conditions (metal resistance genes: zinc, copper, osmotic regulation, and desiccation stress), 17 antimicrobial resistance genes encoding resistance to various classes of antibiotics and 50 genes encoding for the virulence factors. The latter were mainly genes associated with adhesion, chemotaxis, hemolysis, and biofilm formation. Cg-MLST analysis (3991 genes) revealed a greater similarity of C. condimenti s37 to S. turicensis, F. pulveris, and C. dublinensis than to other species of the genus Cronobacter. Studies on the diversity, pathogenicity, and virulence of Cronobacter species isolated from different sources are still insufficient and should certainly be continued. Especially the analysis of rare strains such as s37 is very important because it provides new information on the evolution of these bacteria. Comparative cgMLST analysis of s37 with other Cronobacter species, as well as closely related genera Franconibacter and Siccibacter, complements the knowledge on their adaptability to specific environments such as desiccation.202439201307
421140.9913Effect of pap copy number and receptor specificity on virulence of fimbriated Escherichia coli in a murine urinary tract colonization model. Escherichia coli FN506 containing pap genes that encode two different P fimbriae adherence specificity types were tested for virulence in a murine urinary colonization model. Strains containing adherence genes on either high copy or low copy plasmids were compared. Bacteria that harbored the adherence genes on high copy plasmids colonized mouse kidneys less well than bacteria with the same adherence genes in low copy even though the high copy strains exhibited greater hemagglutination capacity. Bacteria with either type of P fimbriae were able to colonize but pap-2+ bacteria showed increased colonizing capacity when strains containing pap-1 or pap-2 genes on low copy plasmids were compared. Bacteria containing plasmids with both adherence specificities had a similar colonizing capacity as bacteria with either type separately.19947861959
425150.9913A novel ColV plasmid encoding type IV pili. Many septicaemic Escherichia coli strains harbour ColV virulence plasmids. This paper describes pO78V, a conjugative ColV plasmid from an avian pathogenic E. coli strain that encodes type IV pili in addition to other virulence-related genes and tetracycline resistance. Plasmid location of type IV pili genes was demonstrated using Southern hybridization and expression of the pili was demonstrated using RT-PCR and phage sensitivity assays. This is a first report of a ColV plasmid encoding type IV pili. Plasmid pO78V is a mosaic plasmid containing replicons and other genes typical to both IncI1 and IncFII groups. As type IV pili of Gram-negative bacteria are involved in several stages of infection, their presence on a ColV virulence plasmid could expand the repertoire of pathogenesis-related genes.200312576591
426160.9913Plasmid-determined resistance to serum bactericidal activity: a major outer membrane protein, the traT gene product, is responsible for plasmid-specified serum resistance in Escherichia coli. Resistance to the bactericidal activity of serum appears to be an important virulence property of invasive bacteria. The conjugative multiple-antibiotic-resistance plasmid R6-5 was found to confer upon Escherichia coli host bacteria increased resistance against rabbit serum. Gene-cloning techniques were used to localize the serum resistance determinant of R6-5 to a segment of the plasmid that encodes conjugal transfer functions, and a pACYC184 hybrid plasmid, designated pKT107, that contains this segment was constructed. The generation and analysis of deletion and insertion mutant derivatives of the pKT107 plasmid that no longer specify serum resistance permitted precise localization of the serum-resistance cistron on the R6-5 map and demonstrated that this locus is coincident with that of traT, one of the two surface exclusion genes of R6-5. Examination of the proteins synthesized in E. coli minicells of pKT107 and its serum-sensitive mutant derivative plasmids confirmed that the serum-resistance gene product of R6-5 is the traT protein and showed that this protein is a major structural component (about 21,000 copies per cell) of the bacterial outer membrane.19806995306
1704170.9913Exploring virulence characteristics of Klebsiella pneumoniae isolates recovered from a Greek hospital. The objective of this study was to characterize the virulence characteristics of a collection of Klebsiella pneumoniae isolates collected from different clinical sources. A collection of 60 non-repetitive K. pneumoniae isolates, was studied. In vitro, virulence was analyzed by testing the survival of bacteria in pooled human serum. Isolates were typed by MLST. The genomes of 23 K. pneumoniae isolates, representatives of different STs and virulence profiles, were completely sequenced using the Illumina platform. Of note, 26/60 of K. pneumoniae isolates were resistant to killing by complement. Serum-resistant isolates belonged to distinct STs. Analysis of WGS data with VFDB showed the presence of several virulence genes related various virulence functions. Specifically, serum-resistant isolates carried a higher number of ORFs, which were associated with serum resistance, compared to serum-sensitive isolates. Additionally, analysis of WGS data showed the presence of multiple plasmid replicons that could be involved with the spread and acquisition of resistance and virulence genes. In conclusion, analysis of virulence characteristics showed that an important percentage (31.6%) of K. pneumoniae isolates were in vitro virulent by exhibiting resistance to serum. Thus, the presence of several virulence factors, in combination with the presence of multidrug resistance, could challenge antimicrobial therapy of infections caused by such bacteria.202540415138
4788180.9912Acinetobacter baumannii Clinical Isolates Resist Complement-Mediated Lysis by Inhibiting the Complement Cascade and Improperly Depositing MAC. INTRODUCTION: Acinetobacter baumannii is a gram-negative opportunistic bacterium that causes life-threatening infections in immunocompromised hosts. The complement system is a critical mechanism of innate immunity that protects the human body from bacterial infections. Complement activation leads to the deposition of the membrane attack complex (MAC), which can directly lyse gram-negative bacteria. However, A. baumannii has developed evasion mechanisms to protect itself from complement. METHODS: Complement deposition was investigated by flow cytometry and Western blotting. Soluble MAC formation was assessed by ELISA. Bacterial serum resistance was determined by the SYTOX Green Assay. Galleria mellonella was used as an infection model. Genome sequencing revealed virulence genes carried by isolates. RESULTS: We examined clinical isolates of A. baumannii and found 11 isolates with MAC deposition and 5 isolates without deposition. Trypsinization of MAC-positive isolates significantly reduced MAC, indicating incorrect insertion, consistent with a lack of lysis of these strains. MAC-negative isolates inhibited alternative pathway activation and were significantly more serum-resistant. These strains were also more virulent in a G. mellonella infection model. Whole genome sequencing revealed that MAC-negative isolates carried more virulence genes, and both MAC-negative and MAC-positive A. baumannii significantly differed in capsule type. Importantly, a correlation was observed between complement inhibition and capsule type (e.g., capsule locus KL171) of MAC-negative bacteria, while the capsule type (e.g., KL230) of MAC-positive A. baumannii was associated with increased sensitivity to MAC-mediated lysis. CONCLUSION: Our findings suggest a relationship between capsule type, complement resistance, and host virulence in A. baumannii. INTRODUCTION: Acinetobacter baumannii is a gram-negative opportunistic bacterium that causes life-threatening infections in immunocompromised hosts. The complement system is a critical mechanism of innate immunity that protects the human body from bacterial infections. Complement activation leads to the deposition of the membrane attack complex (MAC), which can directly lyse gram-negative bacteria. However, A. baumannii has developed evasion mechanisms to protect itself from complement. METHODS: Complement deposition was investigated by flow cytometry and Western blotting. Soluble MAC formation was assessed by ELISA. Bacterial serum resistance was determined by the SYTOX Green Assay. Galleria mellonella was used as an infection model. Genome sequencing revealed virulence genes carried by isolates. RESULTS: We examined clinical isolates of A. baumannii and found 11 isolates with MAC deposition and 5 isolates without deposition. Trypsinization of MAC-positive isolates significantly reduced MAC, indicating incorrect insertion, consistent with a lack of lysis of these strains. MAC-negative isolates inhibited alternative pathway activation and were significantly more serum-resistant. These strains were also more virulent in a G. mellonella infection model. Whole genome sequencing revealed that MAC-negative isolates carried more virulence genes, and both MAC-negative and MAC-positive A. baumannii significantly differed in capsule type. Importantly, a correlation was observed between complement inhibition and capsule type (e.g., capsule locus KL171) of MAC-negative bacteria, while the capsule type (e.g., KL230) of MAC-positive A. baumannii was associated with increased sensitivity to MAC-mediated lysis. CONCLUSION: Our findings suggest a relationship between capsule type, complement resistance, and host virulence in A. baumannii.202539842423
2467190.9912Whole-genome sequencing of multidrug-resistant Klebsiella pneumoniae with capsular serotype K2 isolates from mink in China. BACKGROUND: Klebsiella pneumoniae is a zoonotic opportunistic pathogen, and also one of the common pathogenic bacteria causing mink pneumonia. The aim of this study was to get a better understanding of the whole-genome of multi-drug resistant Klebsiella pneumoniae with K2 serotype in China. This study for the first time to analyze Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, resistance and virulence genes of Klebsiella pneumoniae in mink. RESULTS: The isolate was Klebsiella pneumoniae with serotype K2 and ST6189 by PCR method. The string test was positive and showed high mucus phenotype. There was one plasmid with IncFIB replicons in the genome. The virulence factors including capsule, lipopolysaccharide, adhesin, iron uptake system, urease, secretory system, regulatory gene (rcsA, rcsB), determinants of pili adhesion, enolase and magnesium ion absorption related genes. The strain was multi-drug resistant. A total of 26  resistance genes, including beta-lactam, aminoglycosides, tetracycline, fluoroquinolones, sulfonamides, amide alcohols, macrolides, rifampicin, fosfomycin, vancomycin, diaminopyrimidines and polymyxin. Multidrug-resistant efflux protein AcrA, AcrB, TolC, were predicted in the strain. CONCLUSION: It was the first to identify that serotype K2 K. pneumonia with ST6189 isolated from mink in China. The finding indicated that hypervirulent and multi-drug resistant K. pneumoniae was exist in Chinese mink. The whole-genome of K. pneumoniae isolates have importance in mink farming practice.202439127663