Whole Genome Sequencing of Invasive Neonatal Escherichia coli from Uppsala County, Sweden.

BACKGROUND: This study sought to investigate associations between a virulence factors and phylogeny in all neonatal E. coli bloodstream infections from patients admitted to the neonatal intensive care unit at Uppsala University Hospital between 2005 to 2020. METHODS: A total of 37 E. coli isolates from 32 neonates were whole genome sequenced and analysed for virulence factors related to extraintestinal E. coli, patient-related data were collected retrospectively in the medical records. RESULTS: E. coli isolates that belong to phylogroup B2 were associated with mortality (OR 26, p < 0.001), extreme prematurity with delivery before gestational week 28 (OR 9, p < 0.05) and shock (OR 9, p < 0.05) compared with isolates of non-B2 group. Female neonates were more often infected by isolates of phylogroup B2 E. coli compared with male neonates (OR 7, p = 0.05). The identification of the genotoxin determinant clb coding for colibactin exhibited strong associations with mortality (OR 67, p < 0.005), gestational age (OR 18, p < 0.005), and shock (OR 26, p < 0.005). DISCUSSION: The study highlighted the correlation between neonatal E. coli bacteraemia caused by phylogroup B2 and the role of colibactin. Moreover, it emphasised sex-based differences in bloodstream infections among the bacterial population of E. coli.

Progress toward a vaccine for extraintestinal pathogenic E. coli (ExPEC) II: efficacy of a toxin-autotransporter dual antigen approach.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a leading cause of worldwide morbidity and mortality, the top cause of antimicrobial-resistant (AMR) infections, and the most frequent cause of life-threatening sepsis and urinary tract infections (UTI) in adults. The development of an effective and universal vaccine is complicated by this pathogen's pan-genome, its ability to mix and match virulence factors and AMR genes via horizontal gene transfer, an inability to decipher commensal from pathogens, and its intimate association and co-evolution with mammals. Using a pan virulome analysis of >20,000 sequenced E. coli strains, we identified the secreted cytolysin α-hemolysin (HlyA) as a high priority target for vaccine exploration studies. We demonstrate that a catalytically inactive pure form of HlyA, expressed in an autologous host using its own secretion system, is highly immunogenic in a murine host, protects against several forms of ExPEC infection (including lethal bacteremia), and significantly lowers bacterial burdens in multiple organ systems. Interestingly, the combination of a previously reported autotransporter (SinH) with HlyA was notably effective, inducing near complete protection against lethal challenge, including commonly used infection strains ST73 (CFT073) and ST95 (UTI89), as well as a mixture of 10 of the most highly virulent sequence types and strains from our clinical collection. Both HlyA and HlyA-SinH combinations also afforded some protection against UTI89 colonization in a murine UTI model. These findings suggest recombinant, inactive hemolysin and/or its combination with SinH warrant investigation in the development of an E. coli vaccine against invasive disease.

Synergy between Group 2 capsules and lipopolysaccharide underpins serum resistance in extra-intestinal pathogenic Escherichia coli.

Escherichia coli (E. coli) is a major cause of urinary tract infections, bacteraemia, and sepsis. CFT073 is a prototypic, urosepsis isolate of sequence type (ST) 73. This laboratory, among others, has shown that strain CFT073 is resistant to serum, with capsule and other extracellular polysaccharides imparting resistance. The interplay of such polysaccharides remains under-explored. This study has shown that CFT073 mutants deficient in lipopolysaccharide (LPS) O-antigen and capsule display exquisite serum sensitivity. Additionally, O-antigen and LPS outer core mutants displayed significantly decreased surface K2 capsule, coupled with increased unbound K2 capsule being detected in the supernatant. The R1 core and O6 antigen are involved in the tethering of K2 capsule to the CFT073 cell surface, highlighting the importance of the R1 core in serum resistance. The dependence of capsule on LPS was shown to be post-transcriptional and related to changes in cell surface hydrophobicity. Furthermore, immunofluorescence microscopy suggested that the surface pattern of capsule is altered in such LPS core mutants, which display a punctate capsule pattern. Finally, targeting LPS biosynthesis using sub-inhibitory concentrations of a WaaG inhibitor resulted in increased serum sensitivity and decreased capsule in CFT073. Interestingly, the dependency of capsule on LPS has been observed previously in other Enterobacteria, indicating that the synergy between these polysaccharides is not just strain, serotype or species-specific but may be conserved across several pathogenic Gram-negative species. Therefore, using WaaG inhibitor derivatives to target LPS is a promising therapeutic strategy to reduce morbidity and mortality by reducing or eliminating surface capsule.

Genomic Evaluation of Multidrug-Resistant Extended-Spectrum ß-Lactamase (ESBL)-Producing Escherichia coli from Irrigation Water and Fresh Produce in South Africa: A Cross-Sectional Analysis.

Escherichia coli, both commensal and pathogenic, can colonize plants and persist in various environments. It indicates fecal contamination in water and food and serves as a marker of antimicrobial resistance. In this context, 61 extended-spectrum ß-lactamase (ESBL)-producing E. coli from irrigation water and fresh produce from previous studies were characterized using whole genome sequencing (Illumina MiSeq). The Center for Genomic Epidemiology and Galaxy platforms were used to determine antimicrobial resistance genes, virulence genes, plasmid typing, mobile genetic elements, multilocus sequence typing (MLST), and pathogenicity prediction. In total, 19 known MLST groups were detected among the 61 isolates. Phylogroup B1 (ST58) and Phylogroup E (ST9583) were the most common sequence types. The six ST10 (serotype O101:H9) isolates carried the most resistance genes, spanning eight antibiotic classes. Overall, 95.1% of the isolates carried resistance genes from three or more classes. The blaCTX-M-1, blaCTX-M-14, and blaCTX-M-15 ESBL genes were associated with mobile genetic elements, and all of the E. coli isolates showed a >90% predicted probability of being a human pathogen. This study provided novel genomic information on environmental multidrug-resistant ESBL-producing E. coli from fresh produce and irrigation water, highlighting the environment as a reservoir for multidrug-resistant strains and emphasizing the need for ongoing pathogen surveillance within a One Health context.

Efflux of TolC protein to different antimicrobials can be replaced by other outer membrane proteins with similar ß-barrel structures in extraintestinal pathogenic Escherichia coli.

AIM: As a major efflux pump system in Gram-negative bacteria, AcrAB-TolC plays a key role in the transport of multiple drug substrates and is considered a potential target for the development of novel antimicrobials. Our previous study found that TolC inactivation compromised the resistance to different antimicrobials in porcine extraintestinal pathogenic Escherichia coli (ExPEC) strain PPECC042 (WT). This study was designed to investigate the functional substitution of TolC by other outer membrane proteins (OMPs) with similar ß-barrel structures in pumping out different antimicrobials. METHODS AND RESULTS: In this study, we found that over-expression of several OMPs with similar ß-barrel structures, OmpX, OmpC, OmpN, OmpW, and PhoE, in the ΔtolC strain restored the resistance to macrolides, quinolones, or tetracyclines to the level of WT strain. However, the introduction of any one of the five OMPs did not affect the resistance of the strains ΔacrA, ΔacrB, and ΔacrAΔtolC. Further study revealed that the efflux activity was significantly reduced in the ΔtolC strain, but not in the WT strain and the ΔtolC strains over-expressing various OMPs. Additionally, Nile red dye test and ciprofloxacin accumulation test confirmed that the lost efflux activity and drug accumulation in bacterial periplasm by TolC inactivation was restored by the over-expression of each OMP, depending on the presence of genes acrA and acrB. CONCLUSION: All five OMPs can replace the TolC protein to play the efflux role in pumping out the drugs from the periplasm to the extracellular space with the help of proteins AcrA and AcrB.

Safety, reactogenicity, and immunogenicity of different doses of 10-valent Extraintestinal Pathogenic Escherichia Coli (ExPEC10V) bioconjugate vaccine (VAC52416) in healthy Japanese adults aged 60-85 years in a randomized, double-blind, phase 1 study.

AIM: This phase 1 study (NCT04306302) evaluated the safety, reactogenicity, and immunogenicity of ExPEC10V (VAC52416) in healthy Japanese adults. METHOD: The randomized, double-blind, single-center study included 28-day screening, vaccination (Day 1), 30-day safety and immunogenicity follow-up and 181-day serious adverse events (SAEs) follow-up. Participants (60-85 years) were enrolled in dose-ascending approach and randomized to medium- and high-doses of ExPEC10V (n = 8 in each dose group) and placebo (n = 8). Incidence of adverse events: solicited AEs (until Day 15), unsolicited AEs (until Day 30), SAEs (until Day 181) and immunogenicity (electrochemiluminescent-based assay [ECL] and multiplex opsonophagocytic assay [MOPA]) were assessed on Day 15 and Day 30. RESULTS: Total of 24 participants were included (median age, 66.5 years; 50.0 % female). Incidence of solicited AEs was 81.3 % (local) and 18.8 % (systemic) for pooled ExPEC10V group (medium-dose ExPEC10V: 75.0 % [local], 12.5 % [systemic]; high-dose ExPEC10V: 87.5 % [local], 25.0 % [systemic]). One SAE, not vaccine-related, was reported in high-dose ExPEC10V group after Day 30, which was resolved during study. The ECL demonstrated increase in binding antibody titers, which was maintained from Day 15 to Day 30. For all serotypes, the geometric mean fold increases from baseline on Day 15 ranged from 2.51 to 10.60 and 1.97-5.23 for medium- and high-dose groups, respectively. The MOPA demonstrated increase in functional antibody responses for all serotypes (except O8) at Day 15 which was maintained from Day 15 to Day 30. CONCLUSIONS: ExPEC10V medium- and high-doses were well tolerated with an acceptable safety profile without any significant safety issues in healthy Japanese participants.

Can Extraintestinal Pathogenic Escherichia coli with Heat Resistance Profile Overcome Nonthermal Technologies?

Ultraviolet-C light-emitting diode (UVC-LED) and ultrasound (US) are two nonthermal technologies with the potential to destroy pathogens. However, little is known about their effectiveness in strains with a history of heat resistance. Thus, this study aimed to evaluate the phenotype and genotype of heat-resistant extraintestinal pathogenic Escherichia coli (ExPEC) with heat resistance genes after the application of US, UVC-LED, and UVC-LED+US. For this, two central composite rotatable designs were used to optimize the UVC-LED and US conditions in four ExPEC isolated from beef. From the genome of these isolates obtained in a previous study, possible genes for UVC resistance were analyzed. Results showed that US was ineffective in reducing >0.30 log colony-forming unit/mL, and that when used after UVC-LED, it showed a nonsynergic or antagonistic effect. Also, UVC-LED had the greatest effect at the maximum dose (4950 mJ/cm2 from 1.65 mW/cm2 for 50 min). However, the strains showed some recovery after that, which could be implicated in the expression of genes included in SOS system genes, some others present in the transmissible Locus of Stress Tolerance (trxBC and degP), and others (terC). Thus, ExPEC can overcome the conditions used in this study for US, UVC-LED, and UVC-LED+US, probably due to the history of resistance to other cellular damage. The result of this study will contribute to future studies that aim to find better treatment conditions for each food product.

NhaA facilitates the maintenance of bacterial envelope integrity and the evasion of complement attack contributing to extraintestinal pathogenic Escherichia coli virulence.

Extraintestinal pathogenic Escherichia coli (ExPEC) is responsible for severe bloodstream infections in humans and animals. However, the mechanisms underlying ExPEC's serum resistance remain incompletely understood. Through the transposon-directed insertion-site sequencing approach, our previous study identified nhaA, the gene encoding a Na+/H+ antiporter, as a crucial factor for infection in vivo. In this study, we investigated the role of NhaA in ExPEC virulence utilizing both in vitro models and systemic infection models involving avian and mammalian animals. Genetic mutagenesis analysis revealed that nhaA deletion resulted in filamentous bacterial morphology and rendered the bacteria more susceptible to sodium dodecyl sulfate, suggesting the role of nhaA in maintaining cell envelope integrity. The nhaA mutant also displayed heightened sensitivity to complement-mediated killing compared to the wild-type strain, attributed to augmented deposition of complement components (C3b and C9). Remarkably, NhaA played a more crucial role in virulence compared to several well-known factors, including Iss, Prc, NlpI, and OmpA. Our findings revealed that NhaA significantly enhanced virulence across diverse human ExPEC prototype strains within B2 phylogroups, suggesting widespread involvement in virulence. Given its pivotal role, NhaA could serve as a potential drug target for tackling ExPEC infections.

Global Distribution of O Serotypes and Antibiotic Resistance in Extraintestinal Pathogenic Escherichia coli Collected From the Blood of Patients With Bacteremia Across Multiple Surveillance Studies.

BACKGROUND: Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of bacteremia worldwide, with older populations having increased risk of invasive bacterial disease. Increasing resistance to first-line antibiotics and emergence of multidrug-resistant (MDR) strains represent major treatment challenges. ExPEC O serotypes are key targets for potential multivalent conjugate vaccine development. Therefore, we evaluated the O serotype distribution and antibiotic resistance profiles of ExPEC strains causing bloodstream infections across 4 regions. METHODS: Blood culture isolates from patients aged ≥60 years collected during 5 retrospective E. coli surveillance studies in Europe, North America, Asia-Pacific, and South America (2011-2017) were analyzed. Isolates were O serotyped by agglutination; O genotyping was performed for nontypeable isolates. Antimicrobial susceptibility testing was also conducted. RESULTS: Among 3217 ExPEC blood culture isolates, the most ubiquitous O serotype was O25 (n = 737 [22.9%]), followed by O2, O6, O1, O75, O15, O8, O16, O4, O18, O77 group, O153, O9, O101/O162, O86, and O13 (prevalence of ≥1%). The prevalence of these O serotypes was generally consistent across regions, apart from South America; together, these 16 O serotypes represented 77.6% of all ExPEC bacteremia isolates analyzed. The overall MDR frequency was 10.7%, with limited variation between regions. Within the MDR subset (n = 345), O25 showed a dominant prevalence of 63.2% (n = 218). CONCLUSIONS: Predominant O serotypes among ExPEC bacteremia isolates are widespread across different regions. O25 was the most prevalent O serotype overall and particularly dominant among MDR isolates. These findings may inform the design of multivalent conjugate vaccines that can target the predominant O serotypes associated with invasive ExPEC disease in older adults.

Virulence-encoding genes related to extraintestinal pathogenic E. coli and multidrug resistant pattern of strains isolated from neonatal calves with different severity scores of umbilical infections.

Umbilical infections in calves comprise a major cause of neonatal mortality and have been related to a variety of microorganisms. E. coli is an opportunistic enteropathogen characterized by a diversity of virulence factors (VF). Nonetheless, the gene profiles that encode VF associated with umbilical infections in calves and their effect on the clinical severity remains unclear. In this scenario, microbial identification (with an emphasis on E. coli), was carried out among 150 neonatal calves (≤30 days of age) with umbilical infections, where the omphalopathies were clinically scored as mild, moderate, or severe. Also, a panel of 16 virulence-encoding genes related to extraintestinal pathogenic E. coli (ExPEC) were investigated, i.e., fimbriae/adhesins (sfa/focDEa, papA, papC, afaBC), toxins (hlyA, sat, cnf1, cdt), siderophores (iroN, irp2, iucD, ireA), invasins (ibeA), and serum resistance (ompT, traT, kpsMT II). Bacteria and yeasts isolates were identified using mass spectrometry. Bacteria, yeasts, and fungi were isolated in 94.7% (142/150) of neonatal calves sampled. E. coli was the agent most frequently isolated (59/150 = 39.3%), in pure culture (27/59 = 45.8%) and combined infections (32/59 = 54.2%), although a great variety (n = 83) of other species of microorganisms were identified. Clinical severity scores of 1, 2, and 3 were observed in 32.2% (19/59), 23.7% (14/59), and 44.1% (26/59) of E. coli infections, respectively. The ExPEC genes detected were related to serum resistance (traT, 42/59 = 72.2%; ompT, 35/59 = 59.3%, kpsMTII, 10/59 = 17%), invasins (ibeA, 11/59 = 18.6%), siderophores (iucD, 9/59 = 15.3%; iroN, 8/59 = 13.6%), and adhesins/fimbriae (papA, 8/59 = 13.6%; papC, 15/59 = 9.6%). The presence of each virulence gene was not associated with the case's clinical score. Among all isolates, 89.8% (53/59) showed in vitro resistance to sulfamethoxazole/trimethoprim and 59.3% to ampicillin (35/59), while 94.1% (55/59) revealed a multidrug resistant profile. Great complexity of bacteria, yeast, and fungi species was identified, reinforcing the umbilical infections of neonatal calves as a polymicrobial disorder. The high occurrence of E. coli (39.3%) highlights the role of this pathogen in the etiology of umbilical infections in calves. Furthermore, a panel of ExPEC genes was investigated for the first time among calves that were clinically scored for case severity. The high prevalence of traT and ompT indicates that these serum resistance-related genes could be used as biomarkers for further investigations of ExPEC isolates from umbilical infections. Our results contribute to the etiological investigation, clinical severity scoring, antimicrobial resistance pattern, and virulence-related to ExPEC genes involved in umbilical infections of neonatal calves.

ProQ binding to small RNA RyfA promotes virulence and biofilm formation in avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is a notable subpathotype of the nonhuman extraintestinal pathogenic E. coli (ExPEC). Recognized as an extraintestinal foodborne pathogen, the zoonotic potential of APEC/ExPEC allows for cross-host transmission via APEC-contaminated poultry meat and eggs. ProQ, an RNA binding protein, is evolutionarily conserved in E. coli. However, its regulatory roles in the biofilm formation and virulence of APEC/ExPEC have not been explored. In this study, proQ deletion in the APEC strain FY26 significantly compromised its biofilm-forming ability. Furthermore, animal tests and cellular infection experiments showed that ProQ depletion significantly attenuated APEC virulence, thereby diminishing its capacity for bloodstream infection and effective adherence to and persistence within host cells. Transcriptome analysis revealed a decrease in the transcription level of the small RNA (sRNA) RyfA in the mutant FY26ΔproQ, suggesting a direct interaction between the sRNA RyfA and ProQ. This interaction might indicate that sRNA RyfA is a novel ProQ-associated sRNA. Moreover, the direct binding of ProQ to the sRNA RyfA was crucial for APEC biofilm formation, pathogenicity, adhesion, and intracellular survival. In conclusion, our findings provide detailed insight into the interaction between ProQ and sRNA RyfA and deepen our understanding of the regulatory elements that dictate APEC virulence and biofilm development. Such insights are instrumental in developing strategies to counteract APEC colonization within hosts and impede APEC biofilm establishment on food surfaces.

Genomic features and heat resistance profiles of Escherichia coli isolated from Brazilian beef.

AIMS: Characterize Escherichia coli and E. coli -producing (STEC) isolates from Brazilian beef to determine heat resistance and the presence of the transmissible locus of stress tolerance (tLST). METHODS AND RESULTS: Twenty-two STEC previously isolated from beef and characterized as STEC by PCR were subjected to different heat survival challenges (60°C and 71°C). Furthermore, the three tLST-positive isolates and one tLST-negative isolate by PCR were selected for WGS analysis. Phenotypic results indicated that 3/22 (13.64%) were heat resistant, 12/22 (54.54%) were moderately resistant, and 7/22 (31.82%) were sensitive to heat treatments. WGS analyses showed that three isolates with heat resistance showed tLST with up to 80% and 42% of similarity by BLAST analysis, with the major tLST genes being responsible for the homeostasis module. However, WGS showed the absence of stx genes associated with tLST-positive isolates, albeit with virulence and resistance genes found in extraintestinal pathogenic E. coli (ExPEC). CONCLUSION: Our findings demonstrate the presence of heat-resistant E. coli as well as confirm some tLST genes in E. coli isolated from Brazilian beef.

Identification of Escherichia coli isolated from flies (Insecta: Diptera) that inhabit the environment of dairy farms harboring extraintestinal virulence markers.

AIMS: We investigate extraintestinal pathogenic genes (ExPEC) related to virulence of Escherichia coli in flies from the dairy environment. METHODS AND RESULTS: We collected 217 flies from nine dairy farms, which were submitted to microbiological culture. Fifty-one E. coli were identified using mass spectrometry. Eleven dipteran families were identified, with a predominance of Muscidae, and a minor frequency of Tachinidae, Drosophilidae, Sphaeroceridae, Ulidiidae, Syrphidae, Chloropidae, Calliphoridae, Sarcophagidae, and Piophilidae. A panel of 16 virulence-encoding genes related to ExPEC infections were investigated, which revealed predominance of serum resistance (traT, 31/51 = 60.8%; ompT, 29/51 = 56.9%), iron uptake (irp2, 17/51 = 33.3%, iucD 11/51 = 21.6%), and adhesins (papC, 6/51 = 11.8%; papA, 5/51 = 9.8%). CONCLUSIONS: Our findings reveal Dipterans from milking environment carrying ExPEC virulence-encoding genes also identified in clinical bovine E. coli-induced infections.

Antibiotic resistance profile and molecular characterization of extraintestinal pathogenic Escherichia coli (ExPEC) from human clinical samples in gaza strip, palestine.

The rates of antibiotic resistance in extraintestinal pathogenic Escherichia coli (ExPEC) have increased significantly in recent years. This study aims at studying antibiotic resistance, virulence factors (VFs), and the phylogenetic background of ExPEC isolated from Palestinian patients. A total of 42 ExPEC isolates were collected from patients with extraintestinal infections in three Palestinian hospitals. Antimicrobial susceptibility was studied by the disk diffusion method. Resistance/virulence-gene profiles, phylogenetic groups, and integron profiles of these isolates were studied by PCR. The susceptibility to carbapenems was detected in 90.5% of the isolates. Half of the isolates were resistant to ampicillin and sulfamethoxazole/trimethoprim, and 33.3% of the isolates were multidrug-resistant. BlaTEM-1 was detected in seven isolates and blaOXA-1 was identified in one isolate. Sul, qnrA, and aac(6')-Ib-cr genes were found in 12, 3, and 2 isolates, respectively. Class 1 integron has been identified in 10 isolates with the gene cassette arrangement dhfr17 + aadA5. The isolates were distributed between phylogroups B2 and D. The presence of VFs, antibiotic resistance genes, and class 1 integron associated with phylogenetic groups (B2 and D) among multidrug-resistant (MDR)-ExPEC recovered from urinary tract infections (UTIs) patients will complicate infection management and increase therapy failure. Routine screening of antibiotic resistance is important for appropriate and efficient empirical treatment.

Extraintestinal Pathogenic Escherichia Coli Causes Necrohemorrhagic Pneumonia in Multiple Research Dogs.

Extraintestinal pathogenic Escherichia coli expressing cytotoxic necrotizing factor (CNF) 1 and 2 virulence factors is a rarely reported cause of acute, fatal necrohemorrhagic pneumonia in canines. A review of cases of necrohemorrhagic pneumonia in beagles at our facility between 2013 and 2021 revealed 21 dogs that died or were euthanized after acute onset lethargy, dyspnea, and hemorrhage. Some affected animals had recently been transported to the facility. In all dogs, lung lobes were discolored dark red and consolidated. Histologic lesions in 17 of these included alveolar necrosis, hemorrhage, edema, fibrin, acute inflammation, and intralesional colonies of bacilli. Lung was cultured for 10 dogs with E. coli isolated and CNF1 identified by virulence factor PCR in 7 of those. Based on these findings, extraintestinal E. coli should be considered an important cause of acute fatal necrohemorrhagic pneumonia in purpose-bred beagle research dogs and may be associated with a recent history of transport.

Distribution of extraintestinal pathogenic Escherichia coli O-serotypes and antibiotic resistance in blood isolates collected from patients in a surveillance study in Japan.

INTRODUCTION: Invasive extraintestinal pathogenic Escherichia coli (ExPEC) disease (IED), characterised by sepsis and bacteraemia, is a major global healthcare concern worsened by emerging multidrug resistant (MDR) strains. The development of multivalent prophylactic vaccines targeting E. coli strains of IED-associated O-serotypes could address this. A better understanding of O-serotype distribution is required for this purpose. Here, we characterised O-serotype prevalence and drug resistance among ExPEC bacteraemia isolates in Japan. METHODS: E. coli blood isolates from patients aged ≥60 years with bacteraemia were obtained from a retrospective surveillance study in Japan (2015-2017). O-serotyping was performed by agglutination; for isolates non-typeable by agglutination, O-genotyping was performed. Antimicrobial susceptibility was evaluated by broth microdilution using a 21-antibiotic panel. The frequency of drug resistant (DR) isolates was evaluated by antimicrobial susceptibility testing. RESULTS: Of 401 ExPEC bacteraemia isolates evaluated, the most prevalent O-serotype (≥1%) was O25 (28.7% [n = 115]), followed by O1 (14.2% [n = 57]), O2 (8.5% n = 34]), O6 (5.5% [n = 22]), O75, O18, O13, O16, O15, O4, O46/O134, O86, O8 and O83 (each <5% prevalence). These 14 O-serotypes accounted for 81.5% of isolates collected. In total, 19% (n = 77) of isolates were DR ≥ 3, of which 59.7% were O25. Fluoroquinolone-resistance among all and O25 isolates was most prevalent (35.7% and 84.3%, respectively). Almost all (98%) isolates identified as O25 were of subtype O25B. CONCLUSIONS: E. coli serotype O25B showed the highest prevalence and highest multidrug resistance among ExPEC bacteraemia isolates from patients ≥60 years in Japan. Our data may inform development of multivalent glycoconjugate vaccines to prevent IED.

Third-generation cephalosporin (3GC) resistance and its association with Extra-intestinal pathogenic Escherichia coli (ExPEC). Focus on broiler carcasses.

The worldwide spread of Extra-intestinal Pathogenic Escherichia coli (ExPEC), together with the antimicrobial resistance linked with extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases (pAmpCs) are pressing threats for public health. This study aimed to investigate the presence of ExPEC genes in third-generation cephalosporin (3 GC)-resistant E. coli and to study their distribution in broiler carcasses at the slaughterhouse after the chilling process. To this purpose, isolates from a collection of 3 GC-resistant E. coli from carcasses of broilers originating from twelve broiler farms and three production chains were investigated. Several multivariate statistical approaches were adopted to elucidate the relationships among features. Phylogroup F was predominant in all broiler batches and was mainly associated with blaTEM and ESBL genes but less correlated to ExPEC genes. Another remarkable finding was the predominance of ExPEC strains assigned to uncommon phylogroups, such as B2, D, E and Clade I, commonly found into the environment. This study represents a first step for a comprehensive characterization of ExPEC genes harboured by 3 GC-resistant E. coli. These findings may be valuable for the identification of potential risks associated to broiler carcasses as source of uncommon E. coli phylogroups.

Bile Salts Regulate Zinc Uptake and Capsule Synthesis in a Mastitis-Associated Extraintestinal Pathogenic Escherichia coli Strain.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are major causes of urinary and bloodstream infections. ExPEC reservoirs are not completely understood. Some mastitis-associated E. coli (MAEC) strains carry genes associated with ExPEC virulence, including metal scavenging, immune avoidance, and host attachment functions. In this study, we investigated the role of the high-affinity zinc uptake (znuABC) system in the MAEC strain M12. Elimination of znuABC moderately decreased fitness during mouse mammary gland infections. The ΔznuABC mutant strain exhibited an unexpected growth delay in the presence of bile salts, which was alleviated by the addition of excess zinc. We isolated suppressor mutants with improved growth in bile salts, several of which no longer produced the K96 capsule made by strain M12. The addition of bile salts also reduced capsule production by strain M12 and ExPEC strain CP9, suggesting that capsule synthesis may be detrimental when bile salts are present. To better understand the role of the capsule, we compared the virulence of mastitis strain M12 with that of its unencapsulated ΔkpsCS mutant in two models of ExPEC disease. The wild-type strain successfully colonized mouse bladders and kidneys and was highly virulent in intraperitoneal infections. Conversely, the ΔkpsCS mutant was unable to colonize kidneys and was unable to cause sepsis. These results demonstrate that some MAEC strains may be capable of causing human ExPEC illness. The virulence of strain M12 in these infections is dependent on its capsule. However, capsule may interfere with zinc homeostasis in the presence of bile salts while in the digestive tract.

Comparative Pathogenomics of Escherichia coli: Polyvalent Vaccine Target Identification through Virulome Analysis.

Comparative genomics of bacterial pathogens has been useful for revealing potential virulence factors. Escherichia coli is a significant cause of human morbidity and mortality worldwide but can also exist as a commensal in the human gastrointestinal tract. With many sequenced genomes, it has served as a model organism for comparative genomic studies to understand the link between genetic content and potential for virulence. To date, however, no comprehensive analysis of its complete "virulome" has been performed for the purpose of identifying universal or pathotype-specific targets for vaccine development. Here, we describe the construction of a pathotype database of 107 well-characterized completely sequenced pathogenic and nonpathogenic E. coli strains, which we annotated for major virulence factors (VFs). The data are cross referenced for patterns against pathotype, phylogroup, and sequence type, and the results were verified against all 1,348 complete E. coli chromosomes in the NCBI RefSeq database. Our results demonstrate that phylogroup drives many of the "pathotype-associated" VFs, and ExPEC-associated VFs are found predominantly within the B2/D/F/G phylogenetic clade, suggesting that these phylogroups are better adapted to infect human hosts. Finally, we used this information to propose polyvalent vaccine targets with specificity toward extraintestinal strains, targeting key invasive strategies, including immune evasion (group 2 capsule), iron acquisition (FyuA, IutA, and Sit), adherence (SinH, Afa, Pap, Sfa, and Iha), and toxins (Usp, Sat, Vat, Cdt, Cnf1, and HlyA). While many of these targets have been proposed before, this work is the first to examine their pathotype and phylogroup distribution and how they may be targeted together to prevent disease.

Surviving Serum: the Escherichia coli iss Gene of Extraintestinal Pathogenic E. coli Is Required for the Synthesis of Group 4 Capsule.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains constitute a serious and emerging clinical problem, as they cause a variety of infections and are usually highly antibiotic resistant. Many ExPEC strains are capable of evading the bactericidal effects of serum and causing sepsis. One critical factor for the development of septicemia is the increased serum survival (iss) gene, which is highly correlated with complement resistance and lethality. Although it is very important, the function of the iss gene has not been elucidated so far. We have been studying the serum survival of a septicemic strain of E. coli serotype O78, which has a group 4 capsule. Here, we show that the iss gene is required for the synthesis of capsules, which protect the bacteria from the bactericidal effect of complement. Moreover, we show that the deletion of the iss gene results in significantly increased binding of the complement proteins that constitute the membrane attack complex to the bacterial surface.