FdeC expression regulates motility and adhesion of the avian pathogenic Escherichia coli strain IMT5155.

Adaptation of avian pathogenic E. coli (APEC) to changing host environments including virulence factors expression is vital for disease progression. FdeC is an autotransporter adhesin that plays a role in uropathogenic Escherichia coli (UPEC) adhesion to epithelial cells. Expression of fdeC is known to be regulated by environmental conditions in UPEC and Shiga toxin-producing E. coli (STEC). The observation in a previous study that an APEC strain IMT5155 in which the fdeC gene was disrupted by a transposon insertion resulted in elevated adhesion to chicken intestinal cells prompted us to further explore the role of fdeC in infection. We found that the fdeC gene prevalence and FdeC variant prevalence differed between APEC and nonpathogenic E. coli genomes. Expression of the fdeC gene was induced at host body temperature, an infection relevant condition. Disruption of fdeC resulted in greater adhesion to CHIC-8E11 cells and increased motility at 42 °C compared to wild type (WT) and higher expression of multiple transporter proteins that increased inorganic ion export. Increased motility may be related to increased inorganic ion export since this resulted in downregulation of YbjN, a protein known to supress motility. Inactivation of fdeC in APEC strain IMT5155 resulted in a weaker immune response in chickens compared to WT in experimental infections. Our findings suggest that FdeC is upregulated in the host and contributes to interactions with the host by down-modulating motility during colonization. A thorough understanding of the regulation and function of FdeC could provide novel insights into E. coli pathogenesis.

Vaccines against extraintestinal pathogenic Escherichia coli (ExPEC): progress and challenges.

The emergence of antimicrobial resistance (AMR) is a principal global health crisis projected to cause 10 million deaths annually worldwide by 2050. While the Gram-negative bacteria Escherichia coli is commonly found as a commensal microbe in the human gut, some strains are dangerously pathogenic, contributing to the highest AMR-associated mortality. Strains of E. coli that can translocate from the gastrointestinal tract to distal sites, called extraintestinal E. coli (ExPEC), are particularly problematic and predominantly afflict women, the elderly, and immunocompromised populations. Despite nearly 40 years of clinical trials, there is still no vaccine against ExPEC. One reason for this is the remarkable diversity in the ExPEC pangenome across pathotypes, clades, and strains, with hundreds of genes associated with pathogenesis including toxins, adhesins, and nutrient acquisition systems. Further, ExPEC is intimately associated with human mucosal surfaces and has evolved creative strategies to avoid the immune system. This review summarizes previous and ongoing preclinical and clinical ExPEC vaccine research efforts to help identify key gaps in knowledge and remaining challenges.

Widespread prevalence of plasmid-mediated blaCTX-M type extended-spectrum beta-lactamase Escherichia coli in backyard broiler production systems in the United States.

Extended-spectrum beta-lactamase (ESBL) Escherichia coli (E. coli) is an emerging pathogen of high concern given its resistance to extended-spectrum cephalosporins. Broiler chicken, which is the number one consumed meat in the United States and worldwide, can be a reservoir of ESBL E. coli. Backyard poultry ownership is on the rise in the United States, yet there is little research investigating prevalence of ESBL E. coli in this setting. This study aims to identify the prevalence and antimicrobial resistance profiles (phenotypically and genotypically) of ESBL E. coli in some backyard and commercial broiler farms in the U.S. For this study ten backyard and ten commercial farms were visited at three time-points across flock production. Fecal (n = 10), litter/compost (n = 5), soil (n = 5), and swabs of feeders and waterers (n = 6) were collected at each visit and processed for E. coli. Assessment of ESBL phenotype was determined through using disk diffusion with 3rd generation cephalosporins, cefotaxime and ceftazidime, and that with clavulanic acid. Broth microdilution and whole genome sequencing were used to investigate both phenotypic and genotypic resistance profiles, respectively. ESBL E. coli was more prevalent in backyard farms with 12.95% of samples testing positive whereas 0.77% of commercial farm samples were positive. All isolates contained a blaCTX-M gene, the dominant variant being blaCTX-M-1, and its presence was entirely due to plasmids. Our study confirms concerns of growing resistance to fourth generation cephalosporin, cefepime, as roughly half (51.4%) of all isolates were found to be susceptible dose-dependent and few were resistant. Resistance to non-beta lactams, gentamicin and ciprofloxacin, was also detected in our samples. Our study identifies prevalence of blaCTX-M type ESBL E. coli in U.S. backyard broiler farms, emphasizing the need for interventions for food and production safety.

Protecting the piglet gut microbiota against ETEC-mediated post-weaning diarrhoea using specific binding proteins.

Post-weaning diarrhoea (PWD) in piglets presents a widespread problem in industrial pig production and is often caused by enterotoxigenic E. coli (ETEC) strains. Current solutions, such as antibiotics and medicinal zinc oxide, are unsustainable and are increasingly being prohibited, resulting in a dire need for novel solutions. Thus, in this study, we propose and evaluate a protein-based feed additive, comprising two bivalent heavy chain variable domain (VHH) constructs (VHH-(GGGGS)3-VHH, BL1.2 and BL2.2) as an alternative solution to manage PWD. We demonstrate in vitro that these constructs bind to ETEC toxins and fimbriae, whilst they do no affect bacterial growth rate. Furthermore, in a pig study, we show that oral administration of these constructs after ETEC challenge reduced ETEC proliferation when compared to challenged control piglets (1-2 log10 units difference in gene copies and bacterial count/g faeces across day 2-7) and resulted in week 1 enrichment of three bacterial families (Prevotellaceae (estimate: 1.12 ± 0.25, q = 0.0054), Lactobacillaceae (estimate: 2.86 ± 0.52, q = 0.0012), and Ruminococcaceae (estimate: 0.66 ± 0.18, q = 0.049)) within the gut microbiota that appeared later in challenged control piglets, thus pointing to an earlier transition towards a more mature gut microbiota. These data suggest that such VHH constructs may find utility in industrial pig production as a feed additive for tackling ETEC and reducing the risk of PWD in piglet populations.

Raw meat-based diet for pets: a neglected source of human exposure to Salmonella and pathogenic Escherichia coli clones carrying mcr, Portugal, September 2019 to January 2020.

BackgroundThe pet industry is expanding worldwide, particularly raw meat-based diets (RMBDs). There are concerns regarding the safety of RMBDs, especially their potential to spread clinically relevant antibiotic-resistant bacteria or zoonotic pathogens.AimWe aimed to investigate whether dog food, including RMBD, commercially available in Portugal can be a source of Salmonella and/or other Enterobacteriaceae strains resistant to last-line antibiotics such as colistin.MethodsFifty-five samples from 25 brands (21 international ones) of various dog food types from 12 suppliers were screened by standard cultural methods between September 2019 and January 2020. Isolates were characterised by phenotypic and genotypic methods, including whole genome sequencing and comparative genomics.ResultsOnly RMBD batches were contaminated, with 10 of 14 containing polyclonal multidrug-resistant (MDR) Escherichia coli and one MDR Salmonella. One turkey-based sample contained MDR Salmonella serotype 1,4,[5],12:i:- ST34/cgST142761 with similarity to human clinical isolates occurring worldwide. This Salmonella exhibited typical antibiotic resistance (bla TEM + strA-strB + sul2 + tet(B)) and metal tolerance profiles (pco + sil + ars) associated with the European epidemic clone. Two samples (turkey/veal) carried globally dispersed MDR E. coli (ST3997-complexST10/cgST95899 and ST297/cgST138377) with colistin resistance (minimum inhibitory concentration: 4 mg/L) and mcr-1 gene on IncX4 plasmids, which were identical to other IncX4 circulating worldwide.ConclusionSome RMBDs from European brands available in Portugal can be a vehicle for clinically relevant MDR Salmonella and pathogenic E. coli clones carrying genes encoding resistance to the last-line antibiotic colistin. Proactive actions within the One Health context, spanning regulatory, pet-food industry and consumer levels, are needed to mitigate these public health risks.

Gentamicin loaded niosomes against intracellular uropathogenic Escherichia coli strains.

Urinary tract infections (UTIs) are the most common bacterial infections and uropathogenic Escherichia coli (UPEC) is the main etiological agent of UTIs. UPEC can persist in bladder cells protected by immunological defenses and antibiotics and intracellular behavior leads to difficulty in eradicating the infection. The aim of this paper is to design, prepare and characterize surfactant-based nanocarriers (niosomes) able to entrap antimicrobial drug and potentially to delivery and release antibiotics into UPEC-infected cells. In order to validate the proposed drug delivery system, gentamicin, was chosen as "active model drug" due to its poor cellular penetration. The niosomes physical-chemical characterization was performed combining different techniques: Dynamic Light Scattering Fluorescence Spectroscopy, Transmission Electron Microscopy. Empty and loaded niosomes were characterized in terms of size, ζ-potential, bilayer features and stability. Moreover, Gentamicin entrapped amount was evaluated, and the release study was also carried out. In addition, the effect of empty and loaded niosomes was studied on the invasion ability of UPEC strains in T24 bladder cell monolayers by Gentamicin Protection Assay and Confocal Microscopy. The observed decrease in UPEC invasion rate leads us to hypothesize a release of antibiotic from niosomes inside the cells. The optimization of the proposed drug delivery system could represent a promising strategy to significatively enhance the internalization of antimicrobial drugs.

Synthetic phage-based approach for sensitive and specific detection of Escherichia coli O157.

Escherichia coli O157 can cause foodborne outbreaks, with infection leading to severe disease such as hemolytic-uremic syndrome. Although phage-based detection methods for E. coli O157 are being explored, research on their specificity with clinical isolates is lacking. Here, we describe an in vitro assembly-based synthesis of vB_Eco4M-7, an O157 antigen-specific phage with a 68-kb genome, and its use as a proof of concept for E. coli O157 detection. Linking the detection tag to the C-terminus of the tail fiber protein, gp27 produces the greatest detection sensitivity of the 20 insertions sites tested. The constructed phage detects all 53 diverse clinical isolates of E. coli O157, clearly distinguishing them from 35 clinical isolates of non-O157 Shiga toxin-producing E. coli. Our efficient phage synthesis methods can be applied to other pathogenic bacteria for a variety of applications, including phage-based detection and phage therapy.

Whole-genome analysis of Escherichia coli isolated from wild Amur tiger (Panthera tigris altaica) and North China leopard (Panthera pardus japonensis).

Background: Escherichia coli is an important intestinal flora, of which pathogenic E. coli is capable of causing many enteric and extra-intestinal diseases. Antibiotics are essential for the treatment of bacterial infections caused by pathogenic E. coli; however, with the widespread use of antibiotics, drug resistance in E. coli has become particularly serious, posing a global threat to human, animal, and environmental health. While the drug resistance and pathogenicity of E. coli carried by tigers and leopards in captivity have been studied intensively in recent years, there is an extreme lack of information on E. coli in these top predators in the wild environment. Methods: Whole genome sequencing data of 32 E. coli strains collected from the feces of wild Amur tiger (Panthera tigris altaica, n = 24) and North China leopard (Panthera pardus japonensis, n = 8) were analyzed in this article. The multi-locus sequence types, serotypes, virulence and resistance genotypes, plasmid replicon types, and core genomic SNPs phylogeny of these isolates were studied. Additionally, antimicrobial susceptibility testing (AST) was performed on these E. coli isolates. Results: Among the E. coli isolates studied, 18 different sequence types were identified, with ST939 (21.9%), ST10 (15.6%), and ST3246 (9.4%) being the most prevalent. A total of 111 virulence genes were detected, averaging about 54 virulence genes per sample. They contribute to invasion, adherence, immune evasion, efflux pump, toxin, motility, stress adaption, and other virulence-related functions of E. coli. Sixty-eight AMR genes and point mutations were identified. Among the detected resistance genes, those belonging to the efflux pump family were the most abundant. Thirty-two E. coli isolates showed the highest rate of resistance to tetracycline (14/32; 43.8%), followed by imipenem (4/32; 12.5%), ciprofloxacin (3/32; 9.4%), doxycycline (2/32; 6.3%), and norfloxacin (1/32; 3.1%). Conclusions: Our results suggest that E. coli isolates carried by wild Amur tigers and North China leopards have potential pathogenicity and drug resistance.

Multi-locus sequence typing of Escherichia coli isolated from clinical samples in Jordan.

INTRODUCTION: Escherichia coli (E. coli) is the major cause of extraintestinal infections in the urinary tracts and bloodstream in humans in the community and health care institutions. Several studies on the genetic characterization of E. coli among clinical and environmental isolates were performed and revealed a wide diversity of sequence types (STs). In Jordan, phenotypic and genetic features of E. coli were extensively studied but there is still a need to identify the STs that inhabit the community. METHODOLOGY: In this study, multi-locus sequence typing (MLST) was performed on archived clinical E. coli isolates collected from different hospitals in Jordan and the identified STs were extensively analyzed. RESULTS: Genotyping of 92 E. coli isolates revealed 34 STs and 9 clonal complexes. The frequencies of STs ranged between 1 to 23 observations. The most frequent STs among E. coli isolates were ST131 (n = 23), ST69 (n = 19), ST998 (n = 7), ST2083 (n = 5), and ST540 (n = 4). These five ST accounted for up to 60% of the 92 E. coli isolates. Based on the MLST database, the STs reported in this work were world widely recognized in humans, animals, and in the environment. CONCLUSIONS: This study has elaborated more knowledge about the genotypes of E. coli in Jordan, with recommendations for future studies to correlate its genotypes with virulence and resistance genes.

Turbidimetric bioassays: A solution to antimicrobial activity detection in asymptomatic bacteriuria isolates against uropathogenic Escherichia coli.

Traditional bacteriocin screening methods often face limitations due to diffusion-related challenges in agar matrices, which can prevent the peptides from reaching their target organism. Turbidimetric techniques offer a solution to these issues, eliminating diffusion-related problems and providing an initial quantification of bacteriocin efficacy in producer organisms. This study involved screening the cell-free supernatant (CFS) from eight uncharacterized asymptomatic bacteriuria (ABU) isolates and Escherichia coli 83972 for antimicrobial activity against clinical uropathogenic E. coli (UPEC) strains using turbidimetric growth methods. ABU isolates exhibiting activity against five or more UPEC strains were further characterized (PUTS 37, PUTS 58, PUTS 59, S-07-4, and SK-106-1). The inhibition of the CFS by proteinase K suggested that the antimicrobial activity was proteinaceous in nature, potentially bacteriocins. The activity of E. coli PUTS 58 and SK-106-1 was enhanced in an artificial urine medium, with both inhibiting all eight UPECs. A putative microcin H47 operon was identified in E. coli SK-106-1, along with a previously identified microcin V and colicin E7 in E. coli PUTS 37 and PUTS 58, respectively. These findings indicate that ABU bacteriocin-producers could serve as viable prophylactics and therapeutics in the face of increasing antibiotic resistance among uropathogens.

Metabolism of L-arabinose converges with virulence regulation to promote enteric pathogen fitness.

Virulence and metabolism are often interlinked to control the expression of essential colonisation factors in response to host-associated signals. Here, we identified an uncharacterised transporter of the dietary monosaccharide ʟ-arabinose that is widely encoded by the zoonotic pathogen enterohaemorrhagic Escherichia coli (EHEC), required for full competitive fitness in the mouse gut and highly expressed during human infection. Discovery of this transporter suggested that EHEC strains have an enhanced ability to scavenge ʟ-arabinose and therefore prompted us to investigate the impact of this nutrient on pathogenesis. Accordingly, we discovered that ʟ-arabinose enhances expression of the EHEC type 3 secretion system, increasing its ability to colonise host cells, and that the underlying mechanism is dependent on products of its catabolism rather than the sensing of ʟ-arabinose as a signal. Furthermore, using the murine pathogen Citrobacter rodentium, we show that ʟ-arabinose metabolism provides a fitness benefit during infection via virulence factor regulation, as opposed to supporting pathogen growth. Finally, we show that this mechanism is not restricted to ʟ-arabinose and extends to other pentose sugars with a similar metabolic fate. This work highlights the importance integrating central metabolism with virulence regulation in order to maximise competitive fitness of enteric pathogens within the host-niche.

Isolation of a CTX-M-55 (ESBL)-Producing Escherichia coli Strain of the Global ST6448 Clone from a Captive Orangutan in the USA.

The purpose of this study was to determine if orangutans (Pongo spp.) living in captivity at a zoo in Wisconsin were colonized with antimicrobial-resistant bacteria and, if found, to identify underlying genetic mechanisms contributing to their resistant phenotypes. We hypothesize that since antimicrobial-resistant bacteria are so prevalent within humans, the animals could also be carriers of such strains given the daily contact between the animals and the zoo staff that care for them. To test this theory, fecal samples from two orangutans were examined for resistant bacteria by inoculation on HardyCHROM™ ESBL and HardyCHROM™ CRE agars. Isolates were identified using MALDI-TOF mass spectrometry and antimicrobial susceptibility testing was performed using a Microscan autoSCAN-4 System. An isolate was selected for additional characterization, including whole genome sequencing (WGS). Using the Type (Strain) Genome Server (TYGS) the bacterium was identified as Escherichia coli. The sequence type identified was (ST/phylogenetic group/ß-lactamase): ST6448/B1/CTX-M-55.

Escherichia coli isolated from pyometra and cystitis in the same animal exhibit a wide phenotypic similarity.

AIMS: Pyometra and cystitis caused by Escherichia coli are common diseases identified in canine or feline females. The origin of pyometra infection remains uncertain, and effective prevention strategies for this disease are still unknown. This study aimed to provide a phenotypic characterization, including antimicrobial resistance and virulence profiles, of endometrial pathogenic (EnPEC) and uropathogenic (UPEC) E. coli strains isolated simultaneously from the same animal. METHODS AND RESULTS: Sixteen E. coli strains, from eight different animals, were analyzed in this study. The antimicrobial susceptibility profile of EnPEC and UPEC strains was determined using the disc diffusion method, which showed a similar susceptibility profile among strains (EnPEC and UPEC) from the same animal. The virulence profile of the strains was assessed through biofilm formation, as well as serum resistance abilities. EnPEC and UPEC strains from the same animal exhibited slight variations in their virulence and antimicrobial resistance capabilities. Overall, most of the strain pairs showed a high similarity in their ability to establish biofilms and survive in serum complement activity. CONCLUSIONS: Overall, strains of E. coli isolated from both pyometra and cystitis in the same animal, despite presenting distinct clinical diseases, exhibit a wide phenotypic similarity, suggesting a common origin for the strains.

Sporadic clone Escherichia coli ST615 as a vector and reservoir for dissemination of crucial antimicrobial resistance genes.

There is scarce information concerning the role of sporadic clones in the dissemination of antimicrobial resistance genes (ARGs) within the nosocomial niche. We confirmed that the clinical Escherichia coli M19736 ST615 strain, one of the first isolates of Latin America that harbors a plasmid with an mcr-1 gene, could receive crucial ARG by transformation and conjugation using as donors critical plasmids that harbor bla CTX-M-15, bla KPC-2, bla NDM-5, bla NDM-1, or aadB genes. Escherichia coli M19736 acquired bla CTX-M-15, bla KPC-2, bla NDM-5, bla NDM-1, and aadB genes, being only blaNDM-1 maintained at 100% on the 10th day of subculture. In addition, when the evolved MDR-E. coli M19736 acquired sequentially bla CTX-M-15 and bla NDM-1 genes, the maintenance pattern of the plasmids changed. In addition, when the evolved XDR-E. coli M19736 acquired in an ulterior step the paadB plasmid, a different pattern of the plasmid's maintenance was found. Interestingly, the evolved E. coli M19736 strains disseminated simultaneously the acquired conjugative plasmids in different combinations though selection was ceftazidime in all cases. Finally, we isolated and characterized the extracellular vesicles (EVs) from the native and evolved XDR-E. coli M19736 strains. Interestingly, EVs from the evolved XDR-E. coli M19736 harbored bla CTX-M-15 though the pDCAG1-CTX-M-15 was previously lost as shown by WGS and experiments, suggesting that EV could be a relevant reservoir of ARG for susceptible bacteria. These results evidenced the genetic plasticity of a sporadic clone of E. coli such as ST615 that could play a relevant transitional link in the clinical dynamics and evolution to multidrug/extensively/pandrug-resistant phenotypes of superbugs within the nosocomial niche by acting simultaneously as a vector and reservoir of multiple ARGs which later could be disseminated.

Multidrug-resistant phenotypes of genetically diverse Escherichia coli isolates from healthy domestic cats.

Β-lactamases-producing Escherichia coli are a widely distributed source of antimicrobial resistance (AMR), for animals and humans. Little is known about the sensitivity profile and genetic characteristics of E. coli strains isolated from domestic cats. We report a cross-sectional study that evaluated E. coli strains isolated from domestic cats in Panama. For this study the following antibiotics were analyzed: ampicillin, amoxicillin-clavulanate cefepime, cefotaxime, cefoxitin, ceftazidime, aztreonam, imipenem, gentamicin, kanamycin, streptomycin, tetracycline, ciprofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, and chloramphenicol. The data obtained were classified as resistant, intermediate, or sensitive. MDR strains were established when the strain presented resistance to at least one antibiotic from three or more antimicrobial classes. Forty-eight E. coli isolates were obtained, of which 80% presented resistance to at least one of the antibiotics analyzed, while only 20% were sensitive to all (p = 0.0001). The most common resistance was to gentamicin (58%). Twenty-nine percent were identified as multidrug-resistant isolates and 4% with extended spectrum beta-lactamase phenotype. The genes blaTEM (39%), blaMOX(16%), blaACC (16%) and blaEBC (8%) were detected. Plasmid-mediated resistance qnrB (25%) and qnrA (13%) are reported. The most frequent sequence types (STs) being ST399 and we reported 5 new STs. Our results suggest that in intestinal strains of E. coli isolated from domestic cats there is a high frequency of AMR.

In vitro evaluation of two novel Escherichia bacteriophages against multiple drug resistant avian pathogenic Escherichia coli.

BACKGROUND: In recent years, there has been a growing interest in phage therapy as an effective therapeutic tool against colibacillosis caused by avian pathogenic Escherichia coli (APEC) which resulted from the increasing number of multidrug resistant (MDR) APEC strains. METHODS: In the present study, we reported the characterization of a new lytic bacteriophage (Escherichia phage AG- MK-2022. Basu) isolated from poultry slaughterhouse wastewater. In addition, the in vitro bacteriolytic activity of the newly isolated phage (Escherichia phage AG- MK-2022. Basu) and the Escherichia phage VaT-2019a isolate PE17 (GenBank: MK353636.1) were assessed against MDR- APEC strains (n = 100) isolated from broiler chickens with clinical signs of colibacillosis. RESULTS: Escherichia phage AG- MK-2022. Basu belongs to the Myoviridae family and exhibits a broad host range. Furthermore, the phage showed stability under a wide range of temperatures, pH values and different concentrations of NaCl. Genome analysis of the Escherichia phage AG- MK-2022. Basu revealed that the phage possesses no antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and any E. coli virulence associated genes. In vitro bacterial challenge tests demonstrated that two phages, the Escherichia phage VaT-2019a isolate PE17 and the Escherichia phage AG- MK-2022. Basu exhibited high bactericidal activity against APEC strains and lysed 95% of the tested APEC strains. CONCLUSIONS: The current study findings indicate that both phages could be suggested as safe biocontrol agents and alternatives to antibiotics for controlling MDR-APEC strains isolated from broilers.

The resistance patterns and molecular characteristics of ESBL/AmpC-producing Escherichia coli from captive panda ecosystem in China.

Escherichia coli (E. coli) plays an important ecological role, and is a useful bioindicator to recognize the evolution of resistance in human, animal and environment. Recently, extended-spectrum ß-lactamases (ESBL) producing E.coli has posed a threat to public health. Generally, captive healthy giant pandas are not exposed to antibiotics; however, they still acquire antimicrobial resistant bacteria. In order to understand whether there is an exchange of resistance genes within the ecosystems of captive giant pandas, this study explored resistance characteristics of 330 commensal E. coli isolates from feces of giant pandas, the surroundings, and breeders. Isolates from different sources showed similar resistance phenotype, and ESBL/AmpC-producing isolates showed more profound resistance to antibiotics than non-ESBL/AmpC-producing isolates (P<0.05). Furthermore, the occurrence of broad-spectrum ß-lactamase related resistance genes and colistin resistance genes was detected, and isolates phylogenetic typing and multilocus sequence typing (MLST) were applied in this study. Seven different ß-lactamase resistance genes (blaCTX-M-55, blaCTX-M-15, blaCTX-M-27, blaCTX-M-65, blaTEM-1, blaOXA-1 and blaCMY) and mcr-1 were found in 68 ESBL/AmpC-producing isolates. blaCTX-M-55 (48.53 %) was found the most predominant resistance genes, followed by blaTEM-1 (19.12 %) and blaCTX-M-27 (16.18 %). Nonetheless, blaCTX-M-55 was commonly detected in the isolates from giant pandas (63.16 %), the surroundings (43.48 %), and breeders (33.33 %). However, there were no carbapenemase genes detected in this study. mcr-1 was harbored in only one isolate from giant panda. Forty-five tansconjugants were successfully obtained in the conjugation experiments. The presence of antimicrobial resistance and related resistance genes tested were observed in the transconjugants. The results indicated that 52.63 % of the isolates from giant panda 73.91 % of the isolates from surroundings, and 100 % of the isolates from breeders were phylogroup A. Total of 27 sequence types (ST) were recognized from the isolate by MLST and found that ST48 (19/68; 27.94 %) was the predominant ST type, especially in the isolates from giant pandas and the surroundings. In conclusion, commensal ESBL/AmpC-producing E. coli becomes a reservoir of ESBL resistance genes, which is a potential threaten to health of giant pandas. The interaction between giant pandas, surroundings and breeders contribute to development of resistant phenotypes and genotypes which might transfer across species or the surroundings easily; hence, strict monitoring based on a "One Health" approach is recommended.

Quantifying combined effects of colistin and ciprofloxacin against Escherichia coli in an in silico pharmacokinetic-pharmacodynamic model.

Co-administering a low dose of colistin (CST) with ciprofloxacin (CIP) may improve the antibacterial effect against resistant Escherichia coli, offering an acceptable benefit-risk balance. This study aimed to quantify the interaction between ciprofloxacin and colistin in an in silico pharmacokinetic-pharmacodynamic model from in vitro static time-kill experiments (using strains with minimum inhibitory concentrations, MICCIP 0.023-1 mg/L and MICCST 0.5-0.75 mg/L). It was also sought to demonstrate an approach of simulating concentrations at the site of infection with population pharmacokinetic and whole-body physiologically based pharmacokinetic models to explore the clinical value of the combination when facing more resistant strains (using extrapolated strains with lower susceptibility). The combined effect in the final model was described as the sum of individual drug effects with a change in drug potency: for ciprofloxacin, concentration at half maximum killing rate (EC50) in combination was 160% of the EC50 in monodrug experiments, while for colistin, the change in EC50 was strain-dependent from 54.1% to 119%. The benefit of co-administrating a lower-than-commonly-administrated colistin dose with ciprofloxacin in terms of drug effect in comparison to either monotherapy was predicted in simulated bloodstream infections and pyelonephritis. The study illustrates the value of pharmacokinetic-pharmacodynamic modelling and simulation in streamlining rational development of antibiotic combinations.

A novel chaperone-effector-immunity system identified in uropathogenic Escherichia coli UMN026.

Background: Urinary tract infections (UTIs) are very common worldwide. According to their symptomatology, these infections are classified as pyelonephritis, cystitis, or asymptomatic bacteriuria (AB). Approximately 75-95% of UTIs are caused by uropathogenic Escherichia coli (UPEC), which is an extraintestinal bacterium that possesses virulence factors for bacterial adherence and invasion in the urinary tract. In addition, UPEC possesses type 6 secretion systems (T6SS) as virulence mechanisms that can participate in bacterial competition and in bacterial pathogenicity. UPEC UMN026 carries three genes, namely, ECUMN_0231, ECUMN_0232, and ECUMN_0233, which encode three uncharacterized proteins related to the T6SS that are conserved in strains from phylogroups B2 and D and have been proposed as biomarkers of UTIs. Aim: To analyze the frequency of the ECUMN_0231, ECUMN_0232, ECUMN_0233, and vgrG genes in UTI isolates, as well as their expression in Luria Bertani (LB) medium and urine; to determine whether these genes are related to UTI symptoms or bacterial competence and to identify functional domains on the putative proteins. Methods: The frequency of the ECUMN and vgrG genes in 99 clinical isolates from UPEC was determined by endpoint PCR. The relationship between gene presence and UTI symptomatology was determined using the chi2 test, with p < 0.05 considered to indicate statistical significance. The expression of the three ECUMN genes and vgrG was analyzed by RT-PCR. The antibacterial activity of strain UMN026 was determined by bacterial competence assays. The identification of functional domains and the docking were performed using bioinformatic tools. Results: The ECUMN genes are conserved in 33.3% of clinical isolates from patients with symptomatic and asymptomatic UTIs and have no relationship with UTI symptomatology. Of the ECUMN+ isolates, only five (15.15%, 5/33) had the three ECUMN and vgrG genes. These genes were expressed in LB broth and urine in UPEC UMN026 but not in all the clinical isolates. Strain UMN026 had antibacterial activity against UPEC clinical isolate 4014 (ECUMN-) and E. faecalis but not against isolate 4012 (ECUMN+). Bioinformatics analysis suggested that the ECUMN genes encode a chaperone/effector/immunity system. Conclusions: The ECUMN genes are conserved in clinical isolates from symptomatic and asymptomatic patients and are not related to UTI symptoms. However, these genes encode a putative chaperone/effector/immunity system that seems to be involved in the antibacterial activity of strain UMN026.

Multidrug-resistant Escherichia coli causing canine pyometra and urinary tract infections are genetically related but distinct from those causing prostatic abscesses.

Despite extensive characterisation of uropathogenic Escherichia coli (UPEC) causing urinary tract infections (UTIs), the genetic background of non-urinary extraintestinal pathogenic E. coli (ExPEC) in companion animals remains inadequately understood. In this study, we characterised virulence traits of 104 E. coli isolated from canine pyometra (n = 61) and prostatic abscesses (PAs) (n = 38), and bloodstream infections (BSIs) in dogs (n = 2), and cats (n = 3). A stronger association with UPEC of pyometra strains in comparison to PA strains was revealed. Notably, 44 isolates exhibited resistance to third-generation cephalosporins and/or fluoroquinolones, 15 were extended-spectrum ß-lactamase-producers. Twelve multidrug-resistant (MDR) strains, isolated from pyometra (n = 4), PAs (n = 5), and BSIs (n = 3), along with 7 previously characterised UPEC strains from dogs and cats, were sequenced. Genomic characteristics revealed that MDR E. coli associated with UTIs, pyometra, and BSIs belonged to international high-risk E. coli clones, including sequence type (ST) 38, ST131, ST617, ST648, and ST1193. However, PA strains belonged to distinct lineages, including ST12, ST44, ST457, ST744, and ST13037. The coreSNPs, cgMLST, and pan-genome illustrated intra-clonal variations within the same ST from different sources. The high-risk ST131 and ST1193 (phylogroup B2) contained high numbers of ExPEC virulence genes on pathogenicity islands, predominating in pyometra and UTI. Hybrid MDR/virulence IncF multi-replicon plasmids, containing aerobactin genes, were commonly found in non-B2 phylogroups from all sources. These findings offer genomic insights into non-urinary ExPEC, highlighting its potential for invasive infections in pets beyond UTIs, particularly with regards to high-risk global clones.