Nucleotide sequence variants, gene expression and serum profile of immune and antioxidant markers associated with bacterial diarrhea susceptibility in Barki lambs.

BACKGROUND: Despite the fact that diarrhea is more accurately described as a clinical symptom than a disease. Diarrhea is one of the most important issues in ovine medicine, particularly in lambs, and because of high morbidity and mortality rate, sluggish growth performance, and veterinary costs, it is believed to be a major source of economic loss. Salmonella and enterotoxigenic Escherichia coli are the most common and commercially significant agents responsible for diarrhea. OBJECTIVE: The objective of this study was to monitor the nucleotide sequence variations, gene expression, serum inflammatory and oxidative stress biomarkers in diarrheic lambs. Another aim was to identify different pathotypes and virulence genes of Salmonella and E. coli causing diarrhea. METHODOLOGY: Blood samples were taken from 50 Barki who were diarrheal and 50 who appeared to be healthy, and then divided in 3 portions, with EDTA added to the first part for CBC, DNA and RNA extraction. The second sample received 5000 I.U. of heparin calcium, and a clean plain tube was used for the third component. The second and third sections were centrifuged to extract serum and plasma until the biochemical and immunological analysis was completed. Fecal samples were collected for bacteriological examination, and the bacteria were identified by PCR analysis. PCR-DNA sequencing was conducted for immune (SELL, JAK2, SLC11A1, IL10, FEZF1, NCF4, LITAF, SBD2, NFKB, TNF-α, IL1B, IL6, LGALS, and CATH1), antioxidant (SOD1, CAT, GPX1, GST, Nrf2, Keap1, HMOX1, and NQO1), and GIT health (CALB1, GT, and MUC2) genes in healthy and diarrheic lambs. RESULTS: Virulent genetic markers of pathogenic characteristics of E. coli (astA, Vt2e (Stx2e), CFA/I, groES and luxS) and Salmonella (invA, SopB, bcfC and avrA) were detected in all diarrheic lambs. PCR-DNA sequencing of immune, antioxidant and intestinal health genes found eleven single nucleotide polymorphisms (SNPs) linked to either diarrhea resistance or susceptibility in Barki lambs. Transcript levels of immune, antioxidant, and GIT health (CALB1, GT, and MUC2) genes varied between healthy and diarrheic lambs. Nucleotide sequence variation of the genes under inquiry between reference sequences in GenBank and those of the animals under investigation verified all identified SNPs. Significant (P = 0.001) erythrocytosis, neutrophilic leukocytosis, with lymphocytopenia were observed in diarrheic lambs. Significant (P = 0.001) increases in serum IL-1α, IL-1ß, IL-6, TNF-α (90.5 ± 1.7, 101.8 ± 1.7, 72.3 ± 6.6, 71.26 ± 4.89 Pg/ml, respectively), serum Fb, Cp, Hp, SAA (230.7 ± 12.4 mg/dl, 6.5 ± 0.07 mg/dl, 2.5 ± 0.09 g/dl, 7.4 ± 0.4 mg/L, respectively), free radicals (MDA, NO), cortisol (6.91 ± 0.18 µg/dl) and growth hormone, with significant (P = 0.001) decreases in serum IL-10 (81.71 ± 1.05 Pg/ml), antioxidants (CAT, GPx), insulin, triiodothyronine (T3) and thyroxine (T4) in diarrheic lambs. CONCLUSIONS: The study's findings provided credence to the theory that marker-assisted selection (MAS) could be used to predict and prevent diarrhea in Barki sheep by selecting lambs based on SNPs in genes linked to inflammation, antioxidants, and intestinal health. In order to establish an efficient management protocol and determine the most susceptible risk period for disease occurrence, gene expression profiles of the genes under investigation, pro-inflammatory cytokines and acute phase proteins may also be utilized as proxy biomarkers for lamb enteritis.

Rapid and persistent decrease in brain tissue oxygenation in ovine gram-negative sepsis.

The changes in brain perfusion and oxygenation in critical illness, which are thought to contribute to brain dysfunction, are unclear due to the lack of methods to measure these variables. We have developed a technique to chronically measure cerebral tissue perfusion and oxygen tension in unanesthetized sheep. Using this technique, we have determined the changes in cerebral perfusion and Po2 during the development of ovine sepsis. In adult Merino ewes, fiber-optic probes were implanted in the brain, renal cortex, and renal medulla to measure tissue perfusion, oxygen tension (Po2), and temperature, and flow probes were implanted on the pulmonary and renal arteries. Conscious sheep were infused with live Escherichia coli for 24 h, which induced hyperdynamic sepsis; mean arterial pressure decreased (from 85.2 ± 5.6 to 71.5 ± 8.7 mmHg), while cardiac output (from 4.12 ± 0.70 to 6.15 ± 1.26 L/min) and total peripheral conductance (from 48.9 ± 8.5 to 86.8 ± 11.5 mL/min/mmHg) increased (n = 8, all P < 0.001) and arterial Po2 decreased (from 104 ± 8 to 83 ± 10 mmHg; P < 0.01). Cerebral perfusion tended to decrease acutely, although this did not reach significance, but there was a significant and sustained decrease in cerebral tissue Po2 (from 32.2 ± 10.1 to 18.8 ± 11.7 mmHg) after 3 h and to 22.8 ± 5.2 mmHg after 24 h of sepsis (P < 0.02). Sepsis induced large reductions in both renal medullary perfusion and Po2 but had no effect in the renal cortex. In ovine sepsis, there is an early decrease in cerebral Po2 that is maintained for 24 h despite minimal changes in cerebral perfusion. Cerebral hypoxia may be one of the factors causing sepsis-induced malaise and lethargy.

Performance of a MALDI-TOF mass spectrometry-based method for rapid detection of third-generation oxymino-cephalosporin-resistant Escherichia coli and Klebsiella spp. from blood cultures.

We optimized and prospectively evaluated a simple MALDI-TOF MS-based method for direct detection of third-generation oxymino-cephalosporin resistance (3rd CephR) in Escherichia coli and Klebsiella spp. from blood cultures (BC). In addition, we assessed the performance of a lateral flow immunochromatographic assay (LFIC) for detecting extended-spectrum ß-lactamases (ESBL) (NG-Test CTX-M MULTI assay) using bacterial pellets from BC. A total of 168 BCs from unique patients were included. A pre-established volume of BC flagged as positive was transferred in brain heart infusion with or without ceftriaxone (2 mg/ml). After 2-h incubation, intact bacterial pellets were used for MALDI-TOF MS testing. Identification of bacterial species (index score > 2) in the presence of CRO was considered marker of 3rd CephR. The LFIC assay was evaluated in 141 BC. Bacteremia episodes were caused by E. coli (n = 115) or Klebsiella spp. (n = 53). A total of 49 strains were 3rd CephR by broth microdilution, of which 41 were ESBL producers, seven expressed ESBL and OXA-48 type D carbapenemase, and one harbored a plasmid-mediated AmpC. The MALDI-TOF MS method yielded four very major errors (false susceptibility) and two major errors (false resistance). The overall sensitivity of the assay was 91.8% and the specificity 98.3%. Concordance between the LFIC assay and the MALDI-TOF MS method for detection of ESBL-mediated 3rd CephR was 100%. Both evaluated methods may prove useful for early adjustment of empirical therapy in patients with E. coli and Klebsiella spp. bloodstream infections. Whether their use has a beneficial impact on patient outcomes is currently under investigation.

High prevalence of multidrug resistant ESBL- and plasmid mediated AmpC-producing clinical isolates of Escherichia coli at Maputo Central Hospital, Mozambique.

BACKGROUND: Epidemiological data of cephalosporin-resistant Enterobacterales in Sub-Saharan Africa is still restricted, and in particular in Mozambique. The aim of this study was to detect and characterize extended-spectrum ß-lactamase (ESBL) - and plasmid-mediated AmpC (pAmpC)-producing clinical strains of Escherichia coli at Maputo Central Hospital (MCH), a 1000-bed reference hospital in Maputo, Mozambique. METHODS: A total of 230 clinical isolates of E. coli from urine (n = 199) and blood cultures (n = 31) were collected at MCH during August-November 2015. Antimicrobial susceptibility testing was performed by the disc diffusion method and interpreted according to EUCAST guidelines. Isolates with reduced susceptibility to 3rd generation cephalosporins were examined further; phenotypically for an ESBL-/AmpC-phenotype by combined disc methods and genetically for ESBL- and pAmpC-encoding genes by PCR and partial amplicon sequencing as well as genetic relatedness by ERIC-PCR. RESULTS: A total of 75 isolates with reduced susceptibility to cefotaxime and/or ceftazidime (n = 75) from urine (n = 58/199; 29%) and blood (n = 17/31; 55%) were detected. All 75 isolates were phenotypically ESBL-positive and 25/75 (33%) of those also expressed an AmpC-phenotype. ESBL-PCR and amplicon sequencing revealed a majority of blaCTX-M (n = 58/75; 77%) dominated by blaCTX-M-15. All AmpC-phenotype positive isolates (n = 25/75; 33%) scored positive for one or more pAmpC-genes dominated by blaMOX/FOX. Multidrug resistance (resistance ≥ three antibiotic classes) was observed in all the 75 ESBL-positive isolates dominated by resistance to trimethoprim-sulfamethoxazole, ciprofloxacin and gentamicin. ERIC-PCR revealed genetic diversity among strains with minor clusters indicating intra-hospital spread. CONCLUSION: We have observed a high prevalence of MDR pAmpC- and/or ESBL-producing clinical E. coli isolates with FOX/MOX and CTX-Ms as the major ß-lactamase types, respectively. ERIC-PCR analyses revealed genetic diversity and some clusters indicating within-hospital spread. The overall findings strongly support the urgent need for accurate and rapid diagnostic services to guide antibiotic treatment and improved infection control measures.

Establishment of the reference intervals of whole blood neutrophil phagocytosis by flow cytometry.

OBJECTIVE: To investigate the reference intervals (RIs) of the whole blood neutrophil phagocytosis by flow cytometry (FCM) and to study the application value of neutrophil phagocytosis in infectious diseases. METHODS: Pathogens (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923) cultured for 18-24 h were labeled by fluorescence probe carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), and then incubated with whole blood at 37℃. The phagocytosis of pathogens by neutrophils was detected by flow cytometry, and a reference interval was established. RESULTS: In the healthy adults, the reference interval for the neutrophil phagocytosis to Escherichia coli was 46.91%-83.09% and to Staphylococcus aureus was 33.92%-69.48%. This method showed good reproducibility. Neutrophil phagocytosis was negatively correlated with the neutrophil count, neutrophil percentage, and neutrophil-to-lymphocyte ratio (NLR, p < 0.05). CONCLUSION: We have successfully established the RIs of neutrophil phagocytosis in whole blood in healthy adults by flow cytometry (FCM), which might be of important clinical value in the diagnosis, treatment, and prognosis of infectious diseases.

Structural Determination and Genetic Identification of the O-Antigen from an Escherichia coli Strain, LL004, Representing a Novel Serogroup.

The O-antigen is the outermost component of the lipopolysaccharide layer in Gram-negative bacteria, and the variation of O-antigen structure provides the basis for bacterial serological diversity. Here, we determined the O-antigen structure of an Escherichia coli strain, LL004, which is totally different from all of the E. coli serogroups. The tetrasaccharide repeating unit was determined as →4)-ß-d-Galp-(1→3)-ß-d-GlcpNAc6OAc(~70%)-(1→3)-ß-d-GalpA-(1→3)-ß-d-GalpNAc-(1→ with monosaccharide analysis and NMR spectra. We also characterized the O-antigen gene cluster of LL004, and sequence analysis showed that it correlated well with the O-antigen structure. Deletion and complementation testing further confirmed its role in O-antigen biosynthesis, and indicated that the O-antigen of LL004 is assembled via the Wzx/Wzy dependent pathway. Our findings, in combination, suggest that LL004 should represent a novel serogroup of E. coli.

Impact of Escherichia coli infection in broiler breeder chicks: The effect of oxidative stress on weight gain.

The aim of this study was to determine whether oxidative stress occurs in Escherichia coli-infected broiler breeder chicks, as well as the impact of this infection on bird growth. Twenty birds, 25-day-old female birds were divided into two groups (n = 10 per group): an intraperitoneally-infected group (1 mL containing 1.5 × 108 CFU of E. coli) and a control group that received 1 mL of culture medium (uninfected birds). Birds were weighed individually at the beginning and at the end of the experiment, and samples were collected on days 0, 5 and 10 post-infection (PI). No clinical signs were observed throughout the experimental period; nevertheless, on day 10 PI, there was lower growth and weight gain in infected birds than in the control group. The infected birds showed pericarditis and liver congestion, as well as moderate periportal inflammatory infiltrates with predominance of neutrophils. Significantly higher numbers of total leukocytes, lymphocytes, heterophils and monocytes were observed in the infected group on days 5 and 10 PI, as well as significantly higher total protein and globulin levels; albumin values significantly decreased over the same period. Levels of serum oxidative biomarkers (lipid peroxidation (TBARS) and free radicals (ROS)) were significantly higher at 10 PI, as was glutathione S-transferase (GST) activity during the same period. Hepatic ROS and protein thiol levels were significantly higher in E. coli-infected birds, as well as activities of the antioxidant enzymes catalase, superoxide dismutase. In the spleen, only GST activity was significantly higher for the infected group, unlike the brain, where SOD activity, ROS and non-protein thiol levels were significantly higher in infected birds than in the control group. These data suggested that colibacillosis causes oxidative stress in broiler breeder chicks, negatively affecting their weight gain.

α-Haemolysin production, as a single factor, causes fulminant sepsis in a model of Escherichia coli-induced bacteraemia.

α-Haemolysin (HlyA) from uropathogenic Escherichia coli has been demonstrated to be a significant virulence factor for ascending urinary tract infections. Once the E. coli reach the well-vascularised kidneys, there is a high risk of bacteraemia and a subsequent septic host response. Despite this, HlyA has the potential to accelerate the host response both directly and via its ability to facilitate adenosine triphosphate release from cells. It has not been settled whether HlyA aggravates bacteraemia into a septic state. To address this, we used an E. coli strain in a model of acute urosepsis that was either transfected with a plasmid containing the full HlyA operon or one with deletion in the HlyA gene. Here, we show that HlyA accelerates the host response to E. coli in the circulation. Mice exposed to HlyA-producing E. coli showed massively increased proinflammatory cytokines, a substantial fall in circulating thrombocytes, extensive haematuria, and intravascular haemolysis. This was not seen in mice exposed to either E. coli that do not secrete HlyA or vehicle controls. Consistent with the massive host response to the bacteria, the mice exposed to HlyA-producing E. coli died exceedingly early, whereas mice exposed to E. coli without HlyA production and vehicle controls survived the entire observation period. These data allow us to conclude that HlyA is a virulence factor that accelerates a state of bacteraemia into fulminant sepsis in a mouse model.

Prognostic roles of time to positivity of blood cultures in patients with Escherichia coli bacteremia.

The time to positivity (TTP) of blood cultures has been considered a predictor of clinical outcomes for bacteremia. This retrospective study aimed to determine the clinical value of TTP for the prognostic assessment of patients with Escherichia coli bacteremia. A total of 167 adult patients with E.coli bacteremia identified over a 22-month period in a 3500-bed university teaching hospital in China were studied. The standard cut-off TTP was 11 h in the patient cohort. The septic shock occurred in 27.9% of patients with early TTP (⩽11 h) and in 7.1% of those with a prolonged TTP (>11 h) (P = 0.003). The mortality rate was significantly higher for patients in the early than in the late group (17.7% vs. 4.0%, P < 0.001). Multivariate analysis showed that an early TTP (OR 4.50, 95% CI 1.70-11.93), intensive care unit admission (OR 8.39, 95% CI 2.01-35.14) and neutropenia (OR 4.20, 95% CI 1.55-11.40) were independently associated with septic shock. Likewise, the independent risk factors for mortality of patients were an early TTP (OR 3.80, 95% CI 1.04-12.90), intensive care unit admission (OR 6.45; 95% CI 1.14-36.53), a Pittsburgh bacteremia score ⩾2 (OR 4.34, 95% CI 1.22-15.47) and a Charlson Comorbidity Index ⩾3 (OR 11.29, 95% CI 2.81-45.39). Overall, a TTP for blood cultures within 11 h appears to be associated with worse outcomes for patients with E.coli bacteremia.

Pathogenic functions and diagnostic utility of cytokines/chemokines in EHEC-HUS.

Hemolytic - uremic syndrome (HUS) is a severe complication of infection by Shiga toxin (STx)-producing enterohemorrhagic Escherichia coli. Hemolytic - uremic syndrome is defined clinically as a triad of non-immune microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injuries. Neurologic complications such as acute encephalopathy are also observed. In humans, endothelial cells, proximal tubular epithelial cells, mesangial cells, podocytes, intestinal epithelial cells, and monocytes / macrophages are susceptible to STx-mediated injury. Shiga toxin induces the secretion of inflammatory cytokines and chemokines from susceptible cells, including tumor necrosis factor-α interleukin (IL)-1, IL-6, and IL-8. These cytokines and chemokines contribute to the pathogenesis of HUS and encephalopathy by enhancing STx-induced cytotoxicity and inducing inflammatory cell infiltration. Serum cytokine/chemokine levels are therefore useful as indicators of disease activity and predictors of progression from acute kidney injury to chronic kidney disease. Anti-inflammation therapy combined with apheresis to remove excessive cytokines / chemokines and methylprednisolone pulse therapy to suppress cytokine/chemokine production may be an effective treatment regimen for severe E. coli-associated HUS. However, this regimen requires careful monitoring of potential side effects, such as infections, thrombus formation, and hypertension.

IncI1 ST3 and IncI1 ST7 plasmids from CTX-M-1-producing Escherichia coli obtained from patients with bloodstream infections are closely related to plasmids from E. coli of animal origin.

OBJECTIVES: Fully sequenced IncI1 plasmids obtained from CTX-M-1-producing Escherichia coli of human and animal origin were compared. METHODS: Twelve E. coli isolates sharing identical ESBL genes and plasmid multilocus STs sequenced on Illumina and MinION platforms were obtained from the Danish antimicrobial resistance surveillance programme, DANMAP. After de novo assembly, the sequences of plasmids harbouring blaCTX-M-1 were manually curated and ORFs annotated. Within-group comparisons were performed separately for the IncI1 ST3 plasmid type and the IncI1 ST7 plasmid type. The IncI1 ST3 plasmid group was obtained from 10 E. coli isolates (2 from patients with bloodstream infections, 6 from food and 2 from animals). The IncI1 ST7 plasmids originated from E. coli isolates obtained from a patient with bloodstream infection and from a pig. Sequences of IncI1 ST3 and IncI1 ST7 plasmids harbouring blaCTX-M-1 with determined origin were retrieved from GenBank and used for comparison within the respective group. RESULTS: The 10 IncI1 ST3 blaCTX-M-1 plasmids were highly similar in structure and organization with only minor plasmid rearrangements and differences in the variable region. The IncI1 ST7 blaCTX-M-1 plasmids also showed high similarity in structure and organization. The high level of similarity was also observed when including plasmids from E. coli of animal origin from Australia, Switzerland, the Netherlands and France. CONCLUSIONS: This study shows broad spread of a very successful CTX-M-1-producing IncI1 type plasmid among E. coli of both human and animal origin.

Biofilm formation by ESBL-producing strains of Escherichia coli and Klebsiella pneumoniae.

OBJECTIVES: Biofilm production in extended spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae provides a favourable environment for the exchange of antibiotic-resistance genes and could facilitate widespread dissemination. We aimed to assess biofilm development in ESBL-producing E. coli and K. pneumoniae isolates and determine how development relates to microbiological characteristics and clinical outcomes. METHODS: 147 ESBL-producing E. coli and 82 K. pneumoniae were genetically characterized. Biofilm formation was measured at 1.5, 4, 6, and 24 h during culture in blood heart infusion using a microbead immobilization assay (BioFilm Ring test®). Results were given as biofilm formation index (BFI) with lower values indicating increased presence of biofilm (range = 0-21). RESULTS: In total, 57.1% of strains were strong producers of biofilm (BFI < 2), whereas 13.4% lacked biofilm production (BFI > 18). Standard biofilm production (BFI < 7) was common in E. coli isolates (61.9%). For E. coli, biofilm production was less frequently observed in ST131 clones (p = 0.03) but more frequently in strains harbouring toxin (p = 0.008) or adhesin (p = 0.008) virulence factor genes. Despite almost all K. pneumoniae having standard biofilm production (90.2%), there was a 2.4-times higher odds of observing biofilm in ST29/147/323 versus other ST-types (p = 0.13). Patients with standard biofilm producing isolates were not at increased risk of transfer to intensive-care (odds-ratio=2.80, 95%CI=0.59-13.21) or death within 12-months (odds-ratio=1.61, 95%CI=0.75-3.43). CONCLUSION: In these ESBL-producing strains, biofilm production is linked to certain virulence factors in E. coli and is common in K. pneumoniae. Further exploration of whether biofilm production increases dissemination and risk of severe clinical outcomes is needed in larger collections of isolates.

Serum peptide profiling for potential biomarkers in early diagnosis of Escherichia coli bloodstream infection.

BACKGROUND: Bacterial bloodstream infection (BSI) remains an important cause of morbidity and mortality, which is a widespread and uncontrolled inflammatory response. There are some cytokines for the auxiliary diagnosis, such as procalcitonin (PCT), C reactive protein (CRP), and interleukin 6 (IL-6), which are not sufficient. This study was aimed to explore a new method of diagnosing bacterial BSI and to find some new biomarkers that could differentiate bloodstream infected patients from healthy people. METHODS: An animal model was used to find relevant changes of peptides in the serum and was validated in clinical samples. Mice (25-27 g) were randomized to infection with Escherichia coli ATCC25922 or phosphate buffer saline. The serum samples were purified by weak cation exchange beads and the serum peptide profiling was established by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Statistical analysis and diagnostic modeling were conducted on BioExplorer. Amino acid sequences of the candidate peptides were identified by nano-liquid chromatography electrospray ionization-tandem mass spectrometry and relevant proteins were recognized on the Uniprot database. The identified proteins were confirmed via enzyme-linked immunosorbent assay on clinical samples. RESULTS: Five peptide peaks (m/z 1941, 2924.1, 3962.1, 4126.9 and 5514) were found as candidate biomarkers for E. coli infection, and the diagnostic model discriminated E. coli infected patients from healthy controls with an accuracy of 92.2%. Peptide peaks m/z 1941, 2924.1 and 4126.9 were identified as the fragments of Serotransferrin (TRF), Complement C3 and Serum amyloid A-1 protein (SAA1), respectively, but only C3 and SAA1 showed significant difference in clinical samples. CONCLUSION: MALDI-TOF MS could be a new method to find the changes of serum peptides after infection, C3 and SAA1 could be new biomarkers in diagnosing BSI.

Uropathogenic Escherichia coli strains harboring tosA gene were associated to high virulence genes and a multidrug-resistant profile.

TosA, a putative repeats-in-toxin protein that has recently gained importance as an antigenic molecule, has characteristics of nonfimbrial adhesins and can act as a virulence marker in uropathogenic Escherichia coli (UPEC) strains; however, little is known about the association of this protein with antibiotic resistance profiles in UPEC tosA+ clinical strains. The aim of this study was to evaluate UPEC tosA+ strains, including examining genetic diversity, associations with phylogenetic groups, resistance profiles, virulence genes, adherence assays, integrons, and extended-spectrum beta-lactamase phenotypes. Pulsed-field gel electrophoresis analysis grouped these strains into eight clusters with 62% genetic diversity. These strains were mainly associated with the multidrug-resistant profiles, together with an association with class 1 integron and the extended-spectrum beta-lactamase phenotype. Additionally, the strains exhibited a distribution of ≥96% for core-associated genes, while a variable distribution was identified for pathogenic islands-associated genes. Strong associations between UPEC tosA+ strains and two phylogenetic groups (B2 and D) were identified, including resistance to ß-lactam and non-ß-lactam antibiotics. The UPEC tosA+ clinical strains exhibited major adherence, which was related to the fitness and virulence genes. A recombinant TosA protein reacted with antibodies from the sera of urinary tract infection patients, and anti-recombinant TosA polyclonal antibodies also detected TosA expression in these strains. In conclusion, strains of UPEC tosA+ belonging to phylogenetic group B2 had a high frequency of fitness and virulence genes associated with class 1 integrons and the extended-spectrum beta-lactamase phenotype, which exhibited a high adherence profile. The TosA protein is expressed during infection with UPEC and is considered an immunogenic molecule.

Co-carrying of KPC-2, NDM-5, CTX-M-3 and CTX-M-65 in three plasmids with serotype O89: H10 Escherichia coli strain belonging to the ST2 clone in China.

Carbapenem-resistant Enterobacteriaceae strains as a new serious threat for the public health have been increasingly reported worldwide. In this study, one multi-resistant Escherichia coli strain ZSH6 which co-carried blaKPC-2, blaNDM-5 and blaCTX-M, was isolated from human blood sample. By using plasmid conjugation experiments, ZSH6 was found to harbor three plasmids carrying the blaNDM-5 gene, the blaKPC-2 and blaCTX-M gene, respectively. Whole-genome sequencing of ZSH6 yielded 122 scaffolds of chromosomal DNA and three circular plasmids including pZSH6-blaKPC-2 (46,319 bp), pZSH6-blaNDM-5 (46,161bp) and pZSH6-blaCTX-M (184,723). The isolate was classified to Sequence Type 2 and to the O89: H10 serotype. The results of genome analyses revealed that ZSH6 carried three virulence factors (capU, gad and iss) and twenty resistance genes [blaKPC-2blaNDM-5, blaCTX-M-3, blaCTX-M-65, blaTEM-1, floR, tet(A), tet(B), dfrA17, aadA5, sul1, mdf(A), mph(A), erm(B), aph(3')-Ia, aph(3')-Ib, aph(4)-Ia, aph(6)-Id, aac(3)-Iva, aac(3)-IId]. Therefore, the co-existence of such a large number of resistance genes in multiple plasmids making ZSH6 highly resistant to almost all kinds of commonly used antibiotics, and brings a serious challenge for resistance control and clinical treatment of infections caused by this bacterium.

CXCL5 regulates chemokine scavenging and pulmonary host defense to bacterial infection.

The chemokine sink hypothesis pertaining to erythrocyte Duffy Antigen Receptor for Chemokines (DARC) during inflammation has received considerable attention, but lacks direct in vivo evidence. Here we demonstrate, using mice with a targeted deletion in CXCL5, that CXCL5 bound erythrocyte DARC and impaired its chemokine scavenging in blood. CXCL5 increased the plasma concentrations of CXCL1 and CXCL2 in part through inhibiting chemokine scavenging, impairing chemokine gradients and desensitizing CXCR2, which led to decreased neutrophil influx to the lung, increased lung bacterial burden and mortality in an Escherichia coli pneumonia model. In contrast, CXCL5 exerted a predominant role in mediating neutrophil influx to the lung during inflammation after LPS inhalation. Platelets and lung resident cells were the sources of homeostatic CXCL5 in blood and inflammatory CXCL5 in the lung respectively. This study presents a paradigm whereby platelets and red cells alter chemokine scavenging and neutrophil-chemokine interaction during inflammation.

Glyco-iELISA: a highly sensitive and unambiguous serological method to diagnose STEC-HUS caused by serotype O157.

BACKGROUND: Providing proof of presence of Shiga toxin-producing E. coli (STEC) infection forms the basis for differentiating STEC-hemolytic uremic syndrome (HUS) and atypical HUS. As the gold standard to diagnose STEC-HUS has limitations, using ELISA to detect serum antibodies against STEC lipopolysaccharides (LPS) has proven additional value. Yet, conventional LPS-ELISA has drawbacks, most importantly presence of cross-reactivity due to the conserved lipid A part of LPS. The newly described glyco-iELISA tackles this issue by using modified LPS that eliminates the lipid A part. Here, the incremental value of glyco-iELISA compared to LPS-ELISA is assessed. METHODS: A retrospective study was performed including all pediatric patients (n = 51) presenting with a clinical pattern of STEC-HUS between 1990 and 2014 in our hospital. Subsequently, the diagnostic value of glyco-iELISA was evaluated in a retrospective nationwide study (n = 264) of patients with thrombotic microangiopathy (TMA). LPS- and glyco-iELISA were performed to detect IgM against STEC serotype O157. Both serological tests were compared with each other and with fecal diagnostics. RESULTS: Glyco-iELISA is highly sensitive and has no cross-reactivity. In the single-center cohort, fecal diagnostics, LPS-ELISA, and glyco-iELISA identified STEC O157 infection in 43%, 65%, and 78% of patients, respectively. Combining glyco-iELISA with fecal diagnostics, STEC infection due to O157 was detected in 89% of patients. In the nationwide cohort, 19 additional patients (8%) were diagnosed with STEC-HUS by glyco-iELISA. CONCLUSION: This study shows that using glyco-iELISA to detect IgM against STEC serotype O157 has clear benefit compared to conventional LPS-ELISA, contributing to optimal diagnostics in STEC-HUS.

Genome-Wide Identification by Transposon Insertion Sequencing of Escherichia coli K1 Genes Essential for In Vitro Growth, Gastrointestinal Colonizing Capacity, and Survival in Serum.

Escherichia coli K1 strains are major causative agents of invasive disease of newborn infants. The age dependency of infection can be reproduced in neonatal rats. Colonization of the small intestine following oral administration of K1 bacteria leads rapidly to invasion of the blood circulation; bacteria that avoid capture by the mesenteric lymphatic system and evade antibacterial mechanisms in the blood may disseminate to cause organ-specific infections such as meningitis. Some E. coli K1 surface constituents, in particular the polysialic acid capsule, are known to contribute to invasive potential, but a comprehensive picture of the factors that determine the fully virulent phenotype has not emerged so far. We constructed a library and constituent sublibraries of ∼775,000 Tn5 transposon mutants of E. coli K1 strain A192PP and employed transposon-directed insertion site sequencing (TraDIS) to identify genes required for fitness for infection of 2-day-old rats. Transposon insertions were lacking in 357 genes following recovery on selective agar; these genes were considered essential for growth in nutrient-replete medium. Colonization of the midsection of the small intestine was facilitated by 167 E. coli K1 gene products. Restricted bacterial translocation across epithelial barriers precluded TraDIS analysis of gut-to-blood and blood-to-brain transits; 97 genes were required for survival in human serum. This study revealed that a large number of bacterial genes, many of which were not previously associated with systemic E. coli K1 infection, are required to realize full invasive potential.IMPORTANCEEscherichia coli K1 strains cause life-threatening infections in newborn infants. They are acquired from the mother at birth and colonize the small intestine, from where they invade the blood and central nervous system. It is difficult to obtain information from acutely ill patients that sheds light on physiological and bacterial factors determining invasive disease. Key aspects of naturally occurring age-dependent human infection can be reproduced in neonatal rats. Here, we employ transposon-directed insertion site sequencing to identify genes essential for the in vitro growth of E. coli K1 and genes that contribute to the colonization of susceptible rats. The presence of bottlenecks to invasion of the blood and cerebrospinal compartments precluded insertion site sequencing analysis, but we identified genes for survival in serum.

Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo.

Activated protein C (APC) is a multifunctional serine protease with anticoagulant, cytoprotective, and anti-inflammatory activities. In addition to the cytoprotective effects of APC on endothelial cells, podocytes, and neurons, APC cleaves and detoxifies extracellular histones, a major component of neutrophil extracellular traps (NETs). NETs promote pathogen clearance but also can lead to thrombosis; the pathways that negatively regulate NETosis are largely unknown. Thus, we studied whether APC is capable of directly inhibiting NETosis via receptor-mediated cell signaling mechanisms. Here, by quantifying extracellular DNA or myeloperoxidase, we demonstrate that APC binds human leukocytes and prevents activated platelet supernatant or phorbol 12-myristate 13-acetate (PMA) from inducing NETosis. Of note, APC proteolytic activity was required for inhibiting NETosis. Moreover, antibodies against the neutrophil receptors endothelial protein C receptor (EPCR), protease-activated receptor 3 (PAR3), and macrophage-1 antigen (Mac-1) blocked APC inhibition of NETosis. Select mutations in the Gla and protease domains of recombinant APC caused a loss of NETosis. Interestingly, pretreatment of neutrophils with APC prior to induction of NETosis inhibited platelet adhesion to NETs. Lastly, in a nonhuman primate model of Escherichia coli-induced sepsis, pretreatment of animals with APC abrogated release of myeloperoxidase from neutrophils, a marker of neutrophil activation. These findings suggest that the anti-inflammatory function of APC at therapeutic concentrations may include the inhibition of NETosis in an EPCR-, PAR3-, and Mac-1-dependent manner, providing additional mechanistic insight into the diverse functions of neutrophils and APC in disease states including sepsis.

Synergistic Antimicrobial Activity of Colistin in Combination with Rifampin and Azithromycin against Escherichia coli Producing MCR-1.

The lack of available antibiotics is a global public health problem due to the emergence of antimicrobial resistance. Effective therapeutic regimens are urgently needed against Escherichia coli strains that produce the colistin resistance gene mcr-1 and to inhibit the emergence of resistance. In this study, we assessed the antimicrobial activity of a series of concentrations of colistin-based combinations with rifampin and/or azithromycin against three strains of Escherichia coli, including colistin-resistant isolate MZ1501R, isolate HE1704R that produces MCR-1, and colistin-susceptible isolate MZ1509S Experiments were conducted with a medium inoculum of ∼107 CFU/ml over 48 h. Subsequently, the in vivo therapeutic effect was investigated using a neutropenic mouse thigh infection model. Almost all monotherapies showed unsatisfactory antibacterial activity against E. coli isolates producing MCR-1. In contrast, colistin in combination with rifampin or azithromycin resulted in an obvious decrease in the bacterial burden albeit with regrowth. More obviously, synergistic antimicrobial activity of colistin-based triple-combination therapy with rifampin and azithromycin was observed, resulting in a rapid and exhaustive antibacterial effect. In vivo treatments confirmed these findings, where mean decreases of 0.38 to 0.90 log10 CFU and 1.27 to 1.78 log10 CFU were noted after 24 h and 48 h of treatment, respectively, against colistin-resistant E. coli strains when 5 mg/kg of body weight of colistin was combined with rifampin and azithromycin. Colistin-based combinations with rifampin and azithromycin provide a more active therapeutic regimen than monotherapy or colistin-based double combinations against E. coli producing MCR-1.