A healthy chicken\'s intestinal flora harbours a rich reservoir of Escherichia coli as part of the commensal microbiota. However, some strains, known as avian pathogenic E. coli (APEC), carry specific virulence genes (VGs) that enable them to invade and cause extraintestinal infections such as avian colibacillosis. Although several VG combinations have been identified, the pathogenic mechanisms associated with APEC are ill-defined. The current study screened a subset of 88 E. coli isolates selected from 237 pre-existing isolates obtained from commercial poultry flocks in Australia. The 88 isolates were selected based on their enterobacterial repetitive intergenic consensus (ERIC) and antimicrobial resistance (AMR) profiles and included 29 E. coli isolates cultured from chickens with colibacillosis (referred to as clinical E. coli or CEC) and 59 faecal E. coli (FEC) isolates cultured from clinically healthy chickens. The isolates were screened for the presence of 35 previously reported VGs. Of these, 34 were identified, with iucA not being detected. VGs focG, hlyA and sfa/foc were only detected in FEC isolates. Eight VGs had a prevalence of 90% or above in the CEC isolates. Specifically, astA (100%); feoB (96.6%); iutA, iss, ompT, iroN and hlyF (all 93.1%); and vat (89.7%). The prevalence of these were significantly lower in FEC isolates (astA 79.7%, feoB 77.9%, iutA 52.5%, iss 45.8%, ompT 50.9%, iroN 37.3%, hlyF 50.9% and vat 42.4%). The odds ratios that each of these eight VGs were more likely to be associated with CEC than FEC ranged from 7.8 to 21.9. These eight VGs may be used to better define APEC and diagnostically detect APEC in Australia. Further investigations are needed to identify the roles of these VGs in pathogenicity.

All organisms utilize S-adenosyl-l-methionine (SAM) as a key co-substrate for the methylation of biological molecules, the synthesis of polyamines, and radical SAM reactions. When these processes occur, 5\'-deoxy-nucleosides are formed as byproducts such as S-adenosyl-l-homocysteine, 5\'-methylthioadenosine (MTA), and 5\'-deoxyadenosine (5dAdo). A prevalent pathway found in bacteria for the metabolism of MTA and 5dAdo is the dihydroxyacetone phosphate (DHAP) shunt, which converts these compounds into dihydroxyacetone phosphate and 2-methylthioacetaldehyde or acetaldehyde, respectively. Previous work in other organisms has shown that the DHAP shunt can enable methionine synthesis from MTA or serve as an MTA and 5dAdo detoxification pathway. Rather, the DHAP shunt in Escherichia coli ATCC 25922, when introduced into E. coli K-12, enables the use of 5dAdo and MTA as a carbon source for growth. When MTA is the substrate, the sulfur component is not significantly recycled back to methionine but rather accumulates as 2-methylthioethanol, which is slowly oxidized non-enzymatically under aerobic conditions. The DHAP shunt in ATCC 25922 is active under oxic and anoxic conditions. Growth using 5-deoxy-d-ribose was observed during aerobic respiration and anaerobic respiration with Trimethylamine N-oxide (TMAO), but not during fermentation or respiration with nitrate. This suggests the DHAP shunt may only be relevant for extraintestinal pathogenic E. coli lineages with the DHAP shunt that inhabit oxic or TMAO-rich extraintestinal environments. This reveals a heretofore overlooked role of the DHAP shunt in carbon and energy metabolism from ubiquitous SAM utilization byproducts and suggests a similar role may occur in other pathogenic and non-pathogenic bacteria with the DHAP shunt.
OBJECTIVE: The acquisition and utilization of organic compounds that serve as growth substrates are essential for Escherichia coli to grow and multiply. Ubiquitous enzymatic reactions involving S-adenosyl-l-methionine as a co-substrate by all organisms result in the formation of the 5\'-deoxy-nucleoside byproducts, 5\'-methylthioadenosine and 5\'-deoxyadenosine. All E. coli possess a conserved nucleosidase that cleaves these 5\'-deoxy-nucleosides into 5-deoxy-pentose sugars for adenine salvage. The DHAP shunt pathway is found in some extraintestinal pathogenic E. coli, but its function in E. coli possessing it has remained unknown. This study reveals that the DHAP shunt enables the utilization of 5\'-deoxy-nucleosides and 5-deoxy-pentose sugars as growth substrates in E. coli strains with the pathway during aerobic respiration and anaerobic respiration with TMAO, but not fermentative growth. This provides an insight into the diversity of sugar compounds accessible by E. coli with the DHAP shunt and suggests that the DHAP shunt is primarily relevant in oxic or TMAO-rich extraintestinal environments.

Background: Extraintestinal pathogenic Escherichia coli (ExPEC) has become a mounting public health concern. The present study was conducted to address the role of diarrheic pet animals as potential reservoirs for major human ExPEC sequence types (STs). Materials and Methods: Rectal swabs were collected from 145 diarrheic pet animals (75 dogs and 70 cats). Samples were processed for isolation and identification of E. coli by culture methods. Afterward, ExPEC isolates were identified on a molecular basis through detection of ExPEC phylogroups (B2 and D) coupled with carriage of two or more of the virulence genes associated with ExPEC (papAH, papC, sfa/focDE, afa/draBC, iutA, and kpsMT II). ExPEC STs 131, 73, 69, and 95 were identified among ExPEC isolates by quadruplex PCR and tested for their antimicrobial susceptibility. Eventually, two isolates underwent gene sequencing for the phylogenetic analysis. Results: Of 145 pet animals, 16 (11%) E. coli strains were identified as ExPEC, in which 15 (10.3%) isolates belonged to phylogroup B2 and 1 (0.69%) strain belonged to phylogroup D. The major human ExPEC STs were detected in 13 (9%) isolates, whereas the prevalence rates were 5.3% and 12.9% for dogs and cats, respectively. The isolation rates of ExPEC STs were 4.8%, 2.8%, 0.69%, and 0.69% for ST73, ST131, ST95, and ST69, respectively. Regarding the prevalence of virulence genes among ExPEC STs, the most prevalent ones were papC and sfa/focDE (92.3%), followed by papAH (76.9%), iutA (53.8%), afa/draBC (30.8%), and kpsMT II (30.8%). Moreover, 38.5% of the obtained human ExPEC STs were multidrug resistant. The phylogenetic analysis of two ExPEC ST73 gene sequences showed high genetic relatedness to those isolated from humans in different countries. Conclusions: The fecal carriage of major human ExPEC STs among diarrheic dogs and cats poses a potential zoonotic hazard with serious public health implications.

Uropathogenic Escherichia coli (UPEC) is known to cause 65-75% of human urinary tract infection (UTI) cases. Poultry meat is a reservoir of UPEC, which is suspected to cause foodborne UTIs. In the present study, we aimed to determine the growth potential of UPEC in ready-to-eat chicken breasts prepared by sous-vide processing. Four reference strains isolated from the urine of UTI patients (Bioresource Collection and Research Center [BCRC] 10,675, 15,480, 15,483, and 17,383) were tested by polymerase chain reaction assay for related genes to identify their phylogenetic type and UPEC specificity. A cocktail of these UPEC strains was inoculated into sous-vide cooked chicken breast at 103-4 colony-forming unit (CFU)/g and stored at 4°C, 10°C, 15°C, 20°C, 30°C, and 40°C. Changes in the populations of UPEC during storage were analyzed by a one-step kinetic analysis method using the U.S. Department of Agriculture [USDA] Integrated Pathogen Modeling Program-Global Fit [IPMP-Global Fit]. The results showed that the combination of the no lag phase primary model and the Huang square-root secondary model fitted well with the growth curves to obtain the appropriate kinetic parameters. This combination for predicting UPEC growth kinetics was further validated using it to study additional growth curves at 25°C and 37°C, which showed that the root mean square error, bias factor, and accuracy factor were 0.49-0.59 (log CFU/g), 0.941-0.984, and 1.056-1.063, respectively. In conclusion, the models developed in this study are acceptable and can be used to predict the growth of UPEC in sous-vide chicken breast.

Uropathogenic Escherichia coli (UPEC) is the predominant agent causing various categories of complicated urinary tract infections (cUTI). Although existing data reveals that UPEC harboured numerous virulence determinants to aid its survival in the urinary tract, the reason behind the occurrence of differences in the clinical severity of uninary tract infections (UTI) demonstrated by the UPEC infection is poorly understood. Therefore, the present study aims to determine the distribution of virulence determinants and antimicrobial resistance among different phylogroups of UPEC isolated from various clinical categories of cUTI and asymptomatic bacteriuria (ASB) E. coli isolates. The study will also attempt a relational analysis of the genotypic characteristics of cUTI UPEC and ASB E. coli isolates.
A total of 141 UPEC isolates from cUTI and 160 ASB E. coli isolates were obtained from Universiti Malaya Medical Centre (UMMC). Phylogrouping and the occurrence of virulence genes were investigated using polymerase chain reaction (PCR). Antimicrobial susceptibility of the isolates to different classes of antibiotics was determined using the Kirby Bauer Disc Diffusion method.
The cUTI isolates were distributed differentially among both Extraintestinal Pathogenic E. coli (ExPEC) and non-ExPEC phylogroups. Phylogroup B2 isolates were observed to possess the highest average aggregative virulence score (7.17), a probable representation of the capability to cause severe disease. Approximately 50% of the cUTI isolates tested in this study were multidrug resistant against common antibiotics used to treat UTI. Analysis of the occurrence of virulence genes among different cUTI categories demonstrated that UPEC isolates of pyelonephritis and urosepsis were highly virulent and had the highest average aggregative virulence scores of 7.80 and 6.89 respectively, compared to other clinical categories. Relational analysis of the occurrence of phylogroups and virulence determinants of UPEC and ASB E. coli isolates showed that 46.1% of UPEC and 34.3% of ASB E. coli from both categories were distributed in phylogroup B2 and had the highest average aggregative virulence score of 7.17 and 5.37, respectively. The data suggest that UPEC isolates which carry virulence genes from all four virulence genes groups studied (adhesions, iron uptake systems, toxins and capsule synthesis) and isolates from phylogroup B2 specifically could predispose to severe UTI involving the upper urinary tract. Therefore, specific analysis of the genotypic characteristics of UPEC could be further explored by incorporating the combination of virulence genes as a prognostic marker for predicting disease severity, in an attempt to propose a more evidence driven treatment decision-making for all UTI patients. This will go a long way in enhancing favourable therapeutic outcomes and reducing the antimicrobial resistance burden among UTI patients.

Escherichia coli sequence type 69 (ST69) are common causative agents of extraintestinal infections occurring in the bloodstream, cerebrospinal fluid, surgical sites, and, most frequently, the urinary tract. The objective of this study was to analyze the genomic characteristics of 45 antimicrobial-resistant Escherichia coli ST69 strains that were isolated from 28 calves on eight dairy farms in Pennsylvania, USA. The genomes were sequenced and the antimicrobial resistance genes (ARGs), virulence factors (VFs), and plasmid replicons were identified in silico. A phylogenetic analysis was conducted to compare these calf isolate genomes with poultry and human clinical E. coli ST69 genomes. In total, 23 ARGs, 45 VFs, and 15 plasmid replicons were identified. The majority of genomes (n = 36, 80%) had a multidrug-resistant (MDR) genotype and carried genes conferring resistance to antibiotics of human health significance. Phylogenetic analysis based on the core genomes revealed that calf isolates were nested within clades that included human and poultry isolates, indicating that they are not phylogenetically distinct. Results suggest that dairy calves are a reservoir of MDR E. coli ST69 strains with diverse ARG and VF profiles. This information will be helpful in assessing public health risks associated with E. coli ST69 in commercial dairy production systems.

Healthy poultry can be a reservoir for extraintestinal pathogenic Escherichia coli (ExPEC), some of which could be multidrug resistant to antimicrobials. These ExPEC strains could contaminate the environment and/or food chain representing thus, food safety and human health risk. However, few studies have shown the virulence of poultry-source antimicrobial-resistant (AMR) ExPEC in humans. This study characterized AMR ExPEC and investigated the virulence potential of some of their isolates in a Caenorhabditis elegans infection model. A total of 46 E. coli isolates from poultry (chicken, n = 29; turkey, n = 12) retail meats and chicken feces (n = 4), or humans (n = 1) were sequenced and identified as ExPEC. Except eight, all remaining 38 ExPEC isolates were resistant to at least one antibiotic and carried corresponding antimicrobial resistance genes (ARGs). About 27 of the 46 ExPEC isolates were multidrug-resistant (≥3 antibiotic classes). Seven ExPEC isolates from chicken or turkey meats were of serotype O25:H4 and sequence type (ST) 131 which clustered with an isolate from a human urinary tract infection (UTI) case having the same serotype and ST. The C. elegans challenge model using eight of studied ExPEC isolates harboring various ARGs and virulence genes (VGs) showed that regardless of their ARG or VG numbers in tested poultry meat and feces, ExPEC significantly reduced the life span of the nematode (P < 0.05) similarly to a human UTI isolate. This study indicated the pathogenic potential of AMR ExPEC from retail poultry meat or feces, but more studies are warranted to establish their virulence in poultry and human. Furthermore, relationships between specific resistance profiles and/or VGs in these E. coli isolates for their pathogenicity deserve investigations.

Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the leading causes of bloodstream infections in a broad spectrum of birds and mammals, thus poses a great threat to public health, while its underlying mechanism causing sepsis is not fully understood. Here we reported a high virulent ExPEC strain PU-1, which has a robust ability to colonize within host bloodstream, while induced a low level of leukocytic activation. Two serine protease autotransporters of Enterobacteriaceae (SPATEs), VatPU-1 and TshPU-1, were found to play critical roles for the urgent blood infection of strain PU-1. Although the Vat and Tsh homologues have been identified as virulence factors of ExPEC, their contributions to bloodstream infection are still unclear. In this study, VatPU-1 and TshPU-1 were verified to interact with the hemoglobin (a well-known mucin-like glycoprotein in red blood cell), degrade the mucins of host respiratory tract, and cleave the CD43 (a major cell surface component sharing similar O-glycosylated modifications with other glycoprotein expressed on leukocytes), suggesting that these two SPATEs have the common activity to cleave a broad array of mucin-like O-glycoproteins. These cleavages significantly impaired the chemotaxis and transmigration of leukocytes, and then inhibited the activation of diverse immune responses coordinately, especially downregulated the leukocytic and inflammatory activation during bloodstream infection, thus might mediate the evasion of ExPEC from immune clearance of blood leukocytes. Taken together, these two SPATEs play critical roles to cause a heavy bacterial load within bloodstream via immunomodulation of leukocytes, which provides a more comprehensive understanding how ExPEC colonize within host bloodstream and cause severe sepsis.

The dissemination of Extraintestinal pathogenic Escherichia coli (ExPEC) in food is a critical concern for human health and food safety. The present study is the first to systematically examine the diverse plant-origin foods such as cucumber, carrot, tomato, radish, chilli, fenugreek, coriander, peppermint, spring onion, cabbage, and spinach for the presence of ExPEC or specific putative ExPEC pathotypes with an in-depth assessment of their phylogenetics, virulence, and drug resistance. A total of 77 (15.9 %) ExPEC isolates were recovered from 1780 samples of the diverse plant-origin foods of distinct environments. Specific putative ExPEC pathotypes such as Uropathogenic E. coli (UPEC, 23.3 %) and Septicemia-associated E. coli (SEPEC, 24.6 %) were identified among ExPEC isolates. The Clermont revisited new phylotyping method revealed the varied distribution (1-27 %) of specific putative ExPEC pathotypes in the different phylogenetic lineages such as A, D/E, B1, and Clade 1, etc. All putative ExPEC pathotypes possess multiple genes (4.3-92.8 %) or phenotypes (3.3-100 %) associated with their virulence. In-vitro antimicrobial susceptibility testing of all putative ExPEC pathotypes demonstrated the presence of 100 % multidrug resistance with moderate to high (52-100 %) resistance to drugs used as last-resorts (chloramphenicol, colistin) or frontline (nitrofurantoin, sulfamethoxazole, ampicillin, gentamicin) in ExPEC-associated infections in humans. Overall, the present findings significantly contribute to our better understanding of the presence of ExPEC in the non-clinical niche, such as plant-origin foods with a possible consequence on human health and food safety.

The RNA chaperones, cold shock proteins CspC and CspE, are important in stress response and adaptation. We studied their role in the pathogenesis of a virulent Escherichia coli, representative of extraintestinal pathogenic E. coli (ExPEC) which are serum resistant and septicemic. We performed a global analysis to identify transcripts that interact with these cold shock proteins (CSPs), focusing on virulence-related genes. We used CLIP-seq, which combines UV cross-linking, immunoprecipitation and RNA sequencing. A large number of transcripts bound to the CSPs were identified, and many bind both CspC and CspE. Many transcripts were of genes involved in protein synthesis, transcription and energy metabolism. In addition, there were virulence-related genes, (i.e., fur and ryhB), essential for iron homeostasis. The CLIP-seq results were validated on two transcripts, clpX and tdcA, reported as virulence-associated. Deletion of either CspC or CspE significantly decreased their transcript levels and in a double deletion mutant cspC/cspE, the transcript stability of tdcA and clpX was reduced by 32-fold and 10-fold, respectively. We showed that these two genes are important for virulence, as deleting either of them resulted in loss of serum resistance, a requirement for sepsis. As several virulence-related transcripts interact with CspC or CspE, we determined the importance of these proteins for growth in serum and showed that deletion of either gene significantly reduced serum survival. This phenotype could be partially complemented by cspE and fully complemented by cspC. These results indicate that the two RNA chaperones are essential for virulence, and that CspC particularly critical. IMPORTANCE Virulent Escherichia coli strains that cause infections outside the intestinal tract-extraintestinal pathogenic E. coli (ExPEC)-constitute a major clinical problem worldwide. They are involved in several distinct conditions, including urinary tract infections, newborn meningitis, and sepsis. Due to increasing antibiotic resistance, these strains are a main factor in hospital and community-acquired infections. Because many strains, which do not cross-react immunologically are involved, developing a simple vaccine is not possible. Therefore, it is essential to understand the pathogenesis of these bacteria to identify potential targets for developing drugs or vaccines. One of the least investigated systems involves RNA binding proteins, important for stability of transcripts and global gene regulation. Two such proteins are CspC and CspE (\"cold shock proteins\"), RNA chaperones involved in stress adaptation. Here we performed a global analysis to identify the transcripts which are affected by these two chaperones, with focus on virulence-associated transcripts.