Avian Pathogenic Escherichia Coli Isolates Research Articles

A comprehensive study of colisepticaemia progression in layer chickens applying novel tools elucidates pathogenesis and transmission of Escherichia coli into eggs

Colisepticaemia caused by avian pathogenic Escherichia coli (APEC) is a challenging disease due to its high economic importance in poultry, dubious pathogenesis and potential link with zoonosis and food safety. The existing in vitro studies can’t define hallmark traits of APEC isolates, suggesting a paradigm shift towards host response to understand pathogenesis. This study investigated the comprehensive pathological and microbial progression of colisepticaemia, and transmission of E. coli into eggs using novel tools. In total 48 hens were allocated into three groups and were inoculated intratracheally with ilux2-E. coli PA14/17480/5­/ovary (bioluminescent strain), E. coli PA14/17480/5­/ovary or phosphate buffered saline. Infection with both strains led to typical clinical signs and lesions of colibacillosis as in field outbreaks. Based on lung histopathology, colisepticaemia progression was divided into four disease stages as: stage I (1–3 days post infection (dpi)), stage II (6 dpi), stage III (9 dpi) and stage IV (16 dpi) that were histologically characterized by predominance of heterophils, mixed cells, pyogranuloma, and convalescence, respectively. As disease progressed, bacterial colonization in host organs also decreased, revealed by the quantification of bacterial bioluminescence, bacteriology, and quantitative immunohistochemistry. Furthermore, immunofluorescence, immunohistochemistry, and bacteria re-isolation showed that E. coli colonized the reproductive tract of infected hens and reached to egg yolk and albumen. In conclusion, the study provides novel insights into the pathogenesis of colisepticemia by characterizing microbial and pathological changes at different disease stages, and of the bacteria transmission to table eggs, which have serious consequences on poultry health and food safety.

Identification and Genomic Analyses of a Multidrug Resistant Avian Pathogenic Escherichia coli Coharboring mcr-1, blaTEM-176 and blaCTX-M-14 Genes

The emergence and transmission of the colistin-resistance gene mcr and extended-spectrum β-lactamase (ESBL) encoding genes pose a significant threat to global public health. In recent years, it has been reported that mcr-1 and ESBL genes can coexist in single bacteria strain. The objective of this study was to characterize a multidrug-resistant (MDR) avian pathogenic Escherichia coli (APEC) isolate carrying mcr and ESBL encoding genes in China. A total of 200 APEC isolates were collected for antimicrobial susceptibility testing by Kirby–Bauer (K–B) disk method. The MDR strain EC012 were then further analyzed for minimum inhibitory concentrations, antimicrobials resistance genes (ARGs) detection, conjugation, and whole-genome sequencing (WGS). Among all APEC isolates determined by K–B disk method, strain EC012 was resistant to almost all the antimicrobials, including polymyxin B, cefotaxime, and ceftazidime. Moreover, EC012 harbored ARGs mcr-1, blaTEM-176, and blaCTX-M-14. WGS analysis revealed that EC012 belonged to epidemic APEC serotype O1:H16 and multilocus sequence type ST295. EC012 consisted of one chromosome and six plasmids, encoding a broad ARGs. The blaCTX-M-14, mcr-1 or blaTEM-176 genes were located on conjugative plasmids pEC012-1 or pEC012-5, respectively. These plasmids were successfully transferred to transconjugants and resulted in the resistance to polymyxin B, cefotaxime, and ceftazidime. This study indicated that APEC was a potential reservoir of colistin-resistance gene mcr-1 and ESBL encoding genes, and highlighted the necessity for enhanced monitoring of ARGs dissemination among bacteria from different origins.

Antimicrobial resistance of avian pathogenic Escherichia coli isolated from broiler, layer, and breeder chickens.

Antimicrobials are extensively used in poultry production for growth promotion as well as for the treatment and control of diseases, including avian pathogenic Escherichia coli (APEC). Poor selection, overuse, and misuse of antimicrobial agents may promote the emergence and dissemination of antimicrobial resistance (AMR) in APEC. This study aimed to assess antimicrobial susceptibility patterns and detect antibiotic resistance genes (ARGs) in APEC isolated from clinical cases of colibacillosis in commercial broiler, layer, and breeder chickens. A total of 487 APEC were isolated from 539 across 300 poultry farms in various regions of Nepal. Antimicrobial susceptibility patterns was determined using the Kirby-Bauer disk diffusion and broth microdilution methods. The index of AMR, such as multiple antibiotic resistance (MAR) index, resistance score (R-score), and multidrug resistance (MDR) profile, were determined. Polymerase chain reaction was employed to detect multiple ARGs and correlations between phenotypic and genotypic resistance were analyzed. The prevalence of APEC was 91% (487/539). All of these isolates were found resistant to at least one antimicrobial agent, and 41.7% of the isolates were resistant against 8-9 different antimicrobials. The antibiogram of APEC isolates overall showed the highest resistance against ampicillin (99.4%), whereas the highest intermediate resistance was observed in enrofloxacin (92%). The MAR index and R-score showed significant differences between broiler and layers, as well as between broiler breeder and layers. The number of isolates that were resistant to at least one agent in three or more antimicrobial categories tested was 446 (91.6%) and were classified as MDR-positive isolates. The ARGs were identified in 439 (90.1%) APEC isolates, including the most detected mobilized colistin resistance (mcr1) which was detected in the highest (52.6%) isolates. Overall, resistance gene of beta-lactam (blaTEM), mcr1, resistance gene of sulphonamide (sul1) and resistance gene of tetracycline (tetB) (in broiler), were detected in significantly higher than other tested genes (p < 0.001). When examining the pair-wise correlations, a significant phenotype-phenotype correlation (p < 0.001) was observed between levofloxacin and ciprofloxacin, chloramphenicol and tetracycline with doxycycline. Similarly, a significant phenotype-genotype correlation (p < 0.001) was observed between chloramphenicol and the tetB, and colistin with blaTEM and resistance gene of quinolone (qnrA). In this study, the current state of APEC AMR in commercial chickens is revealed for the first time in Nepal. We deciphered the complex nature of AMR in APEC populations. This information of molecular surveillance is useful to combat AMR in APEC and to contribute to manage APEC associated diseases and develop policies and guidelines to enhance the commercial chicken production.

The role of AJB35136 and fdtA genes in biofilm formation by avian pathogenic Escherichia coli

BackgroundInfections caused by avian pathogenic Escherichia coli (APEC) result in significant economic losses in poultry industry. APEC strains are known to form biofilms in various conditions allowing them to thrive even under harsh and nutrient-deficient conditions on different surfaces, and this ability enables them to evade chemical and biological eradication methods. Despite knowing the whole genome sequences of various APEC isolates, little has been reported regarding their biofilm-associated genes. A random transposon mutant library of the wild-type APEC IMT 5155 comprising 1,300 mutants was analyzed for biofilm formation under nutrient deprived conditions using Videoscan technology coupled with fluorescence microscopy. Seven transposon mutants were found to have reproducibly and significantly altered biofilm formation and their mutated genes were identified by arbitrary PCR and DNA sequencing. The intact genes were acquired from the wild-type strain, cloned in pACYC177 plasmid and transformed into the respective altered biofilm forming transposon mutants, and the biofilm formation was checked in comparison to the wild type and mutant strains under the same conditions.ResultsIn this study, we report seven genes i.e., nhaA, fdeC, yjhB, lysU, ecpR, AJB35136 and fdtA of APEC with significant contribution to biofilm formation. Reintroduction of AJB35136 and fdtA, reversed the altered phenotype proving that a significant role being played by these two O-antigen related genes in APEC biofilm formation. Presence of these seven genes across nonpathogenic E. coli and APEC genomes was also analyzed showing that they are more prevalent in the latter.ConclusionsThe study has elucidated the role of these genes in APEC biofilm formation and compared them to adhesion expanding the knowledge and understanding of the economically significant pathogens.

A survey of frequency of virulence and aminoglycoside antibiotic-resistant genotypes and phenotypes in Escherichia coli in broilers in Khartoum State, Sudan.

Although Escherichia coli (E. coli) is considered a normal microflora in the poultry intestine, certain strains namely, Avian Pathogenic E. coli (APEC), cause colisepticaemia (fatal disease) in poultry. The aim of this study was to determine the prevalence of the virulence genes, i.e. (iroN, ompT, iss, iutA, and hlyF) and aminoglycoside-modifying enzyme (AME) genes, i.e. (strA and strB) in Escherichia coli strains in broilers in Khartoum State. A total of 25 E. coli isolates were collected from broilers farms. All isolates were screened for antimicrobial susceptibility tests using Kirby-Bauer disc diffusion method. In addition, all isolates were tested for the presence of virulence genes and modifying enzyme genes using the polymerase chain reaction (PCR). The results showed that the prevalence of positive strains to virulence genes were 14 (56%), 21 (84%), 14 (56%), 0 (0%) and 0 (0%) to iroN, iutA, hlyF, ompT and iss, respectively. Combined virulence genes include iroN, hlyF and iutA were detected in 14 (56%). The rates of resistance were as follows: Gentamycin: (32%), Kanamycin: (20%) and Streptomycin (16%). Of the genes tested, strA (72%) was the most commonly recognized gene followed by strB (56%). It could be concluded that this is the first report of molecular survey of virulence and aminoglycoside-modifying enzyme (AME) resistant genes in APEC isolates from broiler in Sudan. Therefore, prohibition of non-curative application of antibiotic, dishearten their abuse and to be frequently observant by suppling suitable research-based policy for the poultry industry is warranted.

Characterization of avian pathogenic Escherichia coli isolates based on biofilm formation, ESBL production, virulence-associated genes, and phylogenetic groups.

Escherichia coli is a part of both animal and human commensal microbiota. Avian pathogenic E. coli (APEC) is responsible for colibacillosis in poultry, an economically important disease. However, the close similarities among APEC isolates make it difficult to differentiate between pathogenic and commensal bacteria. The aim of this study was to determine phenotypic and molecular characteristics of APEC isolates and to compare them with their in vivo pathogenicity indices. A total of 198 APEC isolates were evaluated for their biofilm-producing ability and extended-spectrum β-lactamase (ESBL) production phenotypes. In addition, 36 virulence-associated genes were detected, and the isolates were classified into seven phylogenetic groups using polymerase chain reaction. The sources of the isolates were not associated with biofilms, ESBL, genes, or phylogroups. Biofilm and ESBL production were not associated with pathogenicity. Group B2 had the highest pathogenicity index. Groups B2 and E were positively associated with high-pathogenicity isolates and negatively associated with low-pathogenicity isolates. In contrast, groups A and C were positively associated with apathogenic isolates, and group B1 was positively associated with low-pathogenicity isolates. Some virulence-associated genes showed positive or negative associations with specific phylogenetic groups. None of the individual techniques produced results that correlated with the in vivo pathogenicity index. However, the combination of two techniques, namely, detection of virulence-associated genes and the phylogenetic groups, could help the classification of the isolates as pathogenic or commensal.

The genetic network underlying the evolution of pathogenicity in avian Escherichia coli.

Colibacillosis is a worldwide prevalent disease in poultry production linked to Escherichia coli strains that belong to the avian pathogenic E. coli (APEC) pathotype. While many virulence factors have been linked to APEC isolates, no single gene or set of genes has been found to be exclusively associated with the pathotype. Moreover, a comprehensive description of the biological processes linked to APEC pathogenicity is currently lacking. In this study, we compiled a dataset of 2015 high-quality avian E. coli genomes from pathogenic and commensal isolates, based on publications from 2000 to 2021. We then conducted a genome-wide association study (GWAS) and integrated candidate gene identification with available protein-protein interaction data to decipher the genetic network underlying the biological processes connected to APEC pathogenicity. Our GWAS identified variations in gene content for 13 genes and SNPs in 3 different genes associated with APEC isolates, suggesting both gene-level and SNP-level variations contribute to APEC pathogenicity. Integrating protein-protein interaction data, we found that 15 of these genes clustered in the same genetic network, suggesting the pathogenicity of APEC might be due to the interplay of different regulated pathways. We also found novel candidate genes including an uncharacterized multi-pass membrane protein (yciC) and the outer membrane porin (ompD) as linked to APEC isolates. Our findings suggest that convergent pathways related to nutrient uptake from host cells and defense from host immune system play a major role in APEC pathogenicity. In addition, the dataset curated in this study represents a comprehensive historical genomic collection of avian E. coli isolates and constitutes a valuable resource for their comparative genomics investigations.

First report of mobile colistin resistance gene mcr-1 in avian pathogenic Escherichia coli isolated from turkeys in the Gaza Strip, Palestine.

Colistin is used to treat avian pathogenic Escherichia coli (APEC), a microorganism that affects turkey meat production in the Gaza Strip and worldwide. However, the recent emergence of plasmid-borne mobile colistin resistance (mcr) genes in pathogenic E. coli strains is a serious antimicrobial resistance (AMR) challenge for both human and animal health. In December 2018, colistin was banned as a veterinary antimicrobial in the Gaza Strip. This study aimed to detect and track the prevalence of colistin-resistant APEC isolated from turkey flocks in the Gaza Strip. This study investigated 239 APEC isolates from turkey flocks in the Gaza Strip between October 2018 and December 2021 (at 6-month intervals). The colistin-resistant APEC strains were detected using the broth microdilution method. The mcr-1 gene was identified using a polymerase chain reaction. The overall colistin resistance among the isolated APECs was 32.2% during the study period. The average resistance in the first interval was 37.5%, which significantly decreased to 9.3% in the last interval. Among the 77 phenotypically resistant isolates, 32.4% were positive for mcr-1. The average abundance of mcr-1 in the first interval was 66.6%, which decreased to 25% in the last interval. To the best of our knowledge, this is the first study reporting the presence of the mcr-1 gene among the APEC isolates from turkeys in the Gaza Strip. Banned veterinary use of colistin significantly decreased the percentage of resistant APEC isolates from turkeys in Gaza Strip. Further studies are needed to investigate other colistin resistance genes and track the emergence of AMR.

Virulence genes of avian pathogenic Escherichia coli isolated from commercial chicken in Nepal

Colibacillosis is the most common bacteriological disease in poultry. The purpose of this study was to determine the recovery rate of avian pathogenic Escherichia coli (APEC) strains, the distribution, prevalence of Escherichia coli Reference (ECOR) collection and virulence associated gene (VAG) in four types of chickens infected by colibacillosis. Commercial broilers and layers had the highest percentage of positive APEC isolates (91%). We confirmed the ECOR phylogroup including B1 and E for the first time in Nepal. The prevalences of these phylogroups among chicken types were significantly different (p<0.001). Among 57 VAGs, the number of genes found per isolate ranged from 8 to 26, with the top 5 VAGs being fimH (100%), issa (92.2%), traTa (90.6%), sit chro. (86%), and ironEC (84.8%). We found significant differences in gene prevalence among the chicken types. The predominance of B1 and E, and the VAG patterns suggest considering ECOR phylogroup and VAGs while formulating strategies for the prevention and control of APEC.

Investigation of Antibiotic Resistance Pattern and Virulence Determinants in Avian Pathogenic Escherichia coli Isolated from Broiler Chickens in Egypt

Besides its zoonotic importance, avian pathogenic Escherichia coli (APEC) causes substantial financial losses in the poultry industry globally. The progress of antimicrobial resistance in APEC is mainly associated with excessive antimicrobial use and improper sanitation. Since its beginning in the 1970s, the VITEK system has developed into the VITEK 2 system, which has used an automated system to perform all the steps required for microbial identification and antibiotic susceptibility rapidly and accurately. The present study aimed to update the available circulating data about APEC isolates by phenotypic identification, sero-grouping of APEC from broilers chickens and breeders in five governates of Egypt, investigation of their antibiotic resistance pattern by VITEK 2 system, and molecular identification of their virulence determinants. The prevalence of APEC isolated from the different internal organs (liver, lung, heart, heart blood, and spleen) was 67.5%. The most prevalent serotypes were O125, 0114, O44, O127, O142, and O78. Virulence-associated genes (iutA, fimC, and papC) were detected at rates of 84.4%, 74%, and 54.8%, respectively. The highest resistance was found against ampicillin (100%), trimethoprim-sulfamethoxazole (80%), and ampicillin-sulbactam (78.5%), which indicates that the poultry farms need a surveillance and intervention system with proper accuracy and rapidity to prevent the misuse of antibiotics and APEC outbreaks.

Antimicrobial resistance pattern of avian pathogenic Escherichia coli with detection of extended-spectrum β-lactamase-producing isolates in broilers in east Algeria.

Avian pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis, one of the most prevalent bacterial diseases responsible for significant economic losses in the poultry industry worldwide. This study aimed to assess the antimicrobial resistance (AMR) patterns of APEC isolates recovered from poultry in east Algeria and estimate the prevalence of extended-spectrum β-lactamase (ESBL)-producing isolates. In the slaughterhouse of Batna City (Algeria), livers indicating colibacillosis were sampled from 204 suspected carcasses with growth retardation and generalized congestion. Escherichia coli isolation and identification were performed on MacConkey agar using conventional methods and the API 20E system. Antimicrobial resistance susceptibility was tested by the disk diffusion method according to the Clinical Laboratory Standards Institute Guidelines. Extended-spectrum β-lactamase detection was carried out using the double-disk confirmation test. One hundred sixty E. coli isolates were recovered (one isolate per sample). Avian pathogenic Escherichia coli isolates showed high levels of resistance to ampicillin and tetracycline (100%), nalidixic acid (95%), ofloxacin (93.75%), doxycycline (91.87), ciprofloxacin (87.50%), trimethoprim/sulfamethoxazole (62.50%), gentamycin (32.50%), chloramphenicol (27.50%), amoxicillin/clavulanic acid (16.25%), colistin (14.37%), and nitrofurantoin (10.62%). All strains were multidrug-resistant to at least three antibiotics, and more than half (52.52%) of the isolates were resistant to at least seven antibiotics. All isolates were susceptible to ceftriaxone, ceftazidime, and aztreonam. Two E. coli isolates were ESBL producers (1.25%). Avian pathogenic Escherichia coli resistance to most antimicrobial agents used in poultry may lead to antimicrobial therapy failure.

Occurrence of ESBL-producing avian pathogenic Escherichia coli (APEC) isolates in spiced chicken meat in Goias, Brazil.

Some extraintestinal pathogenic Escherichia coli isolates (ExPEC), obtained from humans and chickens avian pathogenic E. coli (APEC), share similar virulence genes. Thus, products of avian origin can be a source of human infection. Moreover, these APEC isolates are resistant to antimicrobials and can spread in the environment through the chicken feces. Although the development of multidrug-resistant (MDR) microorganisms in poultry is on the rise, healthcare entities have raised concerns since MDRs can horizontally transfer resistance genes to other microorganisms and complicate the management of human infections by MDR APEC. The results of our study showed that of 80 investigated spiced chicken meat samples, 55% were contaminated with E. coli, of which 34% (15/44) contaminate with APEC. No diarrheagenic E. coli (DEC) pathotypes were found. Twenty-six isolates were MDR E. coli. Among the APEC isolates, 87% (13/15) produced extended-spectrum beta-lactamase (ESBL). The emergence of MDR/ESBL-producing APEC with zoonotic potential for humans is extremely worrying. Therefore, further studies are required to identify the prevalence of MDR/ESBL-producing APEC in the entire chicken production chain from creation, slaughter, processing, and butchery.

High resistance of fluoroquinolone and macrolide reported in avian pathogenic Escherichia coli isolates from the humid subtropical regions of Pakistan

ObjectivesThis study aimed to assess the antimicrobial resistance profile, virulence potential, and genetic characterization of avian pathogenic Escherichia coli (APEC) that cause colibacillosis in poultry. MethodsAntibiotic susceptibility testing (AST) was measured via the Kirby-Bauer disc diffusion method against 27 commonly used antibiotics. Phylogrouping, virulence-associated gene detection, and hybrid strain detection via multiplex polymerase chain reaction (PCR) and genetic diversity were analysed via ERIC-PCR fingertyping method. ResultsAST analysis showed 100% of isolates were multidrug-resistant (MDR) and highest resistance was against penicillin, tetracycline, and macrolide classes of antibiotics. The mcr-1 gene was present in 40% of the isolates, though only 4% of isolates were showing phenotypic resistance. Despite the scarce use of fluoroquinolone, carbapenem, and cephalosporin in the poultry sector, resistance was evident because of the high prevalence of extended-spectrum β-lactamase (ESBL) (53.7%) and other β-lactamases in APEC isolates. β-lactamase genotyping of APEC isolates revealed that 85.7% of isolates contained either blaCTX or blaTEM and around 38% of isolates were complement resistant. Growth in human urine was evident in 67.3% of isolates. Phylogroup B1 (51%) was the most prevalent group followed by phylogroups A (30.6%), D (13.61%), and B2 (4.76%). The most prevalent virulence-associated genes were fimH, iss, and tatT. Results showed that 26 isolates (17.69%) can be termed hybrid strains and APEC/EHEC (enterohemorrhagic E. coli) was the most prevalent hybrid E. coli pathotype. ERIC-PCR fingerprinting genotype analysis clustered APEC isolates in 40 groups (E1-E40). This study provides insights into the antibiotic resistance and virulence profiling of the APEC isolates in Pakistan. ConclusionsThe findings of this study provide insights into that the antibiotic resistance and virulence profiling of the APEC isolates in Pakistan. This data can inform future studies designed to better estimate the severity of the colibacillosis in poultry farms.

A compilation of virulence-associated genes that are frequently reported in avian pathogenic Escherichia coli (APEC) compared to other E. coli.

Escherichia coli (E. coli) survive in various hosts and environments due to their highly diversified genome. These bacteria have co-evolved with human, colonized a broad range of hosts and survive as a commensal organism or pathogen. E. coli that adopted a pathogenic lifecycle in avian hosts typically belong to phylogroups B2 and D. Phylogenic investigations discovered these E. coli are noticeably overlapped with the phylogroup of E. coli infecting humans. This overlapping is possibly due to a parallel evolution in both hosts from a common ancestor, which indicates a high zoonotic potential of avian pathogenic E. coli (APEC). However, some contrasting evidence of other phylogroups infecting the avian host has also been reported in recent studies indicating phylogroups of E. coli are not definitive, only suggestive to their virulence in chickens. Furthermore, virulence-associated genes that contribute to bacterial features necessary to establish APEC infection, are predominantly located in plasmids. Therefore, phylogenetic classification based on chromosomal markers is often inadequate to identify APEC. Moreover, E. coli can obtain virulent plasmids from other bacteria which further complicates the link between phylogenetic classification and pathotype. Previous research has reported an array of virulence-associated genes highly prevalent only in APEC isolates. Function of these genes are possibly a prerequisite to establishing APEC infections in chickens. Consequently, these genes can be used to distinguish APEC from environmental, commensal, intestinal and other extra-intestinal E. coli. Therefore, we have extensively reviewed previous literature to compile the virulence-associated genes that are highly prevalent in APEC compared to other E. coli. From this review, we have identified 10 key virulence-associated genes (iss, tsh, iroN, episomal/chromosomal ompT, iutA, cvaC, hlyF, iucD, papG allel (II/III) and papC) that are frequently reported in APEC isolates than non-pathogenic E. coli. A compilation of these research findings can be crucial to the molecular identification of APEC. Furthermore, it can serve as a guideline for future investigation and aid in formulation of intervention strategies.

A molecular epidemiological study on Escherichia coli in young chicks with colibacillosis identified two possible outbreaks across farms

Avian pathogenic Escherichia coli (APEC) is the cause of colibacillosis outbreaks in young poultry chicks, resulting in acute to peracute death. The high morbidity and mortality caused by colibacillosis results in poor animal welfare, reduced sustainability and economical loss worldwide. To advance the understanding of the molecular epidemiology, genomic relatedness and virulence traits of APEC, we performed systematic sampling from 45 confirmed colibacillosis broiler flocks with high first week mortality (FWM) during 2018–2021. From these flocks, 219 APEC isolates were whole genome sequenced (WGS) and bioinformatic analyses were performed. The bioinformatic analyses included sequence typing (ST), serotyping, detection of virulence-associated genes (VAGs) and phylogenetic analysis. Our results showed a high prevalence of ST23, ST429 and ST95 among APEC isolates from Norwegian broiler flocks, and identified ST23, ST429, ST117 and ST371 to cause disease more often alone, compared to ST95, ST69 and ST10. Phylogenetic analyses, together with associated metadata, identified two distinct outbreaks of colibacillosis across farms caused by ST429 and ST23 and gave insight into expected SNP distances within and between flocks identified with the same ST. Further, our results highlighted the need for combining two typing methods, such as serotyping and sequence typing, to better discriminate strains of APEC. Ultimately, systematic sampling of APEC from multiple birds in a flock, together with WGS as a diagnostic tool is important to identify the disease-causing APEC within a flock and to detect outbreaks of colibacillosis across farms.

Identification of novel genes involved in the biofilm formation process of Avian Pathogenic Escherichia coli (APEC).

Avian pathogenic Escherichia coli (APEC) is the etiological agent of avian colibacillosis, a leading cause of economic loss to the poultry industry worldwide. APEC causes disease using a diverse repertoire of virulence factors and has the ability to form biofilms, which contributes to the survival and persistence of APEC in harsh environments. The objective of this study was to identify genes most widespread and important in APEC that contribute to APEC biofilm formation. Using the characterized APEC O18 as the template strain, a total of 15,660 mutants were randomly generated using signature tagged mutagenesis and evaluated for decreased biofilm formation ability using the crystal violet assay. Biofilm deficient mutants were sequenced, and a total of 547 putative biofilm formation genes were identified. Thirty of these genes were analyzed by PCR for prevalence among 109 APEC isolates and 104 avian fecal E. coli (AFEC) isolates, resulting in nine genes with significantly greater prevalence in APEC than AFEC. The expression of these genes was evaluated in the wild-type APEC O18 strain using quantitative real-time PCR (qPCR) in both the exponential growth phase and the mature biofilm phase. To investigate the role of these genes in biofilm formation, isogenic mutants were constructed and evaluated for their biofilm production and planktonic growth abilities. Four of the mutants (rfaY, rfaI, and two uncharacterized genes) displayed significantly decreased biofilm formation, and of those four, one (rfaI) displayed significantly decreased growth compared to the wild type. Overall, this study identified novel genes that may be important in APEC and its biofilm formation. The data generated from this study will benefit further investigation into the mechanisms of APEC biofilm formation.

Genetic characterization of third- or fourth-generation cephalosporin-resistant avian pathogenic Escherichia coli isolated from broilers.

The third- or fourth-generation cephalosporins (3GC or 4 GC) are classified as "critically important antimicrobials for human medicine" by WHO, but resistance to these drugs is increasing rapidly in avian pathogenic E. coli (APEC). This study investigated the distribution and genetic characteristics of 3GC- or 4 GC-resistant APEC isolates from five major integrated broiler operations in Korea. The prevalence of 3GC- or 4GC-resistant APEC isolates in 1-week-old broilers was the highest in farms of operation C (53.3%); however, the highest prevalence of these isolates in 4-week-old broilers was the highest on the farms of operation A (60.0%), followed by operations E (50.0%) and C (35.7%). All 49 3GC- or 4GC-resistant APEC isolates had at least one β-lactamase-encoding gene. The most common β-lactamase-encoding genes was extended-spectrum β-lactamase gene, bla CTX-M-15, detected in 24 isolates (49.0%), followed by bla TEM-1 (32.7%). Sixteen isolates (32.7%) harbored class 1 integrons, and four isolates (8.2%) showed different gene cassette-arrangements. However, only 1 of 26 isolates harboring class 2 integrons carried a gene cassette. Furthermore, both CRISPR 1 and 2 arrays were detected in most isolates (36 isolates; 73.5%), followed by CRISPR 2 (18.4%) and CRISPR 1 (4.1%). Interestingly, CRISPR 2 was significantly more prevalent in multidrug resistant (MDR)-APEC isolates than in non-MDR APEC isolates, whereas CRISPR 3 and 4 were significantly more prevalent in non-MDR APEC isolates (each 11.1%; p < 0.05). None of the protospacers of CRISPR arrays were directly associated with antimicrobial resistance. Our findings indicate that the distribution and characteristics of 3GC or 4GC-resistant APEC isolates differed among the integrated broiler operations; moreover, improved management protocols are needed to control the horizontal transmission of 3GC or 4GC-resistant APEC isolates.

Determination of Antibiotic Susceptibility of Avian Pathogenic Escherichia coli by Phenotypic and Genotypic Methods

tEscherichia coli infection is one of the most important infections, which causes heavy economic losses in the poultry industry. It is caused by avian pathogenic E.coli (APEC), which belongs to the group Extra-intestinal Pathogenic E. coli (ExPEC). In this study, a total of 60 APEC isolates from various poultry farms located in and around Anand were evaluated for antibiotics susceptibility by disc diffusion (phenotypic) method for 21 different antibiotics. The E. coli isolates showed 100% resistance against pefloxacin, moxifloxacin and tetracycline followed by ampicillin (86.67%), levofloxacin (81.67%), amoxiclav and ciprofloxacin (71.67% each), co-trimoxazole (56.67%), sulphadiazine (53.34%) and erythromycin (33.33%). The study also revealed that E. coli isolates were highly susceptible to colistin (100.00%), followed by ceftriaxone and spectinomycin (85.00% each), cefixime (81.67%), amikacin (80.00%) and gentamicin (76.67%). All these isolates were also screened for the presence of 20 different antibiotic-resistant genes (ARGs) by genotypic method, i.e., polymerase chain reaction. PCR revealed presence of cmlA gene responsible for chloramphenicol resistance in cent percent isolates. The distribution of other ARGs in the E. coli isolates were qnrS (85.00%), aac(3)-IV (56.66%), strB (53.33%), aadA1 (51.66%), dhfrI (50.00%), tetB (41.66%) sulI and tetA (33.33% each), blaOXA (31.66%), cat1 and blaCMY (21.66% each), strA (6.67%), blaSHV (3.33%) and dhfrV (1.66%), while all the isolates were negative for blaTEM, ere(A), qnrA, qnrB and mcr-1 genes.

High Rates of Multidrug-Resistant Escherichia coli in Great Cormorants (Phalacrocorax carbo) of the German Baltic and North Sea Coasts: Indication of Environmental Contamination and a Potential Public Health Risk.

Antimicrobial-resistant bacteria pose a serious global health risk for humans and animals, while the role of wildlife in the dynamic transmission processes of antimicrobial resistance in environmental settings is still unclear. This study determines the occurrence of antimicrobial-resistant Escherichia coli in the free-living great cormorants (Phalacrocorax carbo) of the North and Baltic Sea coasts of Schleswig-Holstein, Germany. For this, resistant E. coli were isolated from cloacal or faecal swabs and their antimicrobial resistance pheno- and genotypes were investigated using disk diffusion tests and PCR assays. The isolates were further assigned to the four major phylogenetic groups, and their affiliation to avian pathogenic E. coli (APEC) was tested. Resistant E. coli were isolated from 66.7% of the 33 samples, and 48.9% of all the resistant isolates showed a multidrug resistance profile. No spatial differences were seen between the different sampling locations with regard to the occurrence of antimicrobial resistance or multidrug resistance. Most commonly, resistance percentages occurred against streptomycin, followed by tetracycline and sulfonamides. More than half of the isolates belonged to the phylogenetic group B1. Of all the isolates, 24.4% were classified as APEC isolates, of which almost 82% were identified as multidrug-resistant. These results add information on the dispersal of antimicrobial-resistant bacteria in wild birds in Germany, thereby allowing conclusions on the degree of environmental contamination and potential public health concerns.

Isolation, Molecular Characterization, and Antibiotic Resistance of Avian Pathogenic Escherichia coli in Eastern China.

Avian pathogenic Escherichia coli (APEC) causes colibacillosis in avians, resulting in considerable losses in the poultry industry. APEC showed zoonotic potential initially related to the fact that APEC serves as the reservoir of virulence genes and antibiotic resistance genes for other E. coli. Thus, we determine the serotypes, phylogenetic groups, virulence genes distribution, and antibiotic resistance profiles of APEC isolates in eastern China. A total of 230 APEC were isolated from diseased chicken and duck with typical colibacillosis symptoms. Serotyping identified that O78 (44.78%) was the predominant serotype. The majority of APEC isolates were classified into B2 (29.57%), A (26.96%), D (20.00%), and B1 (18.26%), respectively. Among the 15 virulence genes, a high prevalence of ibeB (99.57%), fimC (91.74%), mat (91.30%), ompA (83.04%), and iss (80.43%) genes was observed. Except for low resistance rates for imipenem (1.7%) and polymyxin B (0.4%), most of the APEC isolates were resistant to erythromycin (98.7%), enrofloxacin (96.1%), tetracycline (95.2%), doxycycline (93.9%), lincomycin (90.0%), and streptomycin (90.0%). Moreover, all APEC exhibit multi-drug resistance. This study indicated that APEC isolates harbor a variety of virulence genes and showed multi-antibiotic resistance profiles, providing proof for understanding the epidemiological background and zoonotic potential of APEC in poultry farms.