ColV Virulence Plasmid Research Articles

Complete genome sequence of multidrug-resistant avian pathogenic Escherichia coli strain APEC-O2-MS1170

ObjectivesAvian pathogenic Escherichia coli (APEC) causes colibacillosis, one of the leading causes of mortality and morbidity associated with significant economic losses in the poultry industry. This study aimed to determine antimicrobial resistance and to characterise the genome sequence of a multidrug-resistant (MDR) APEC strain isolated from a broiler chicken. MethodsStrain APEC-O2-MS1170 was isolated from the broiler yolk sac of a 14-day-old broiler. Antimicrobial susceptibility testing was performed using a Sensititre National Antimicrobial Resistance Monitoring System (NARMS) Gram-negative panel. Whole-genome sequencing was performed using both the long-read sequencing approach with a Nanopore GridION sequencer and short-read sequencing with an Illumina HiSeq X-Ten sequencer to obtain a complete scaffold of the genome and an accurate sequence. ResultsThe genome size of strain APEC-O2-MS1170 is 4,993,909 bp with a GC content of 50.7% and 4,651 protein-coding sequences. Public databases were used to identify the virulence-associated gene and antimicrobial resistance gene cargo. Plasmid comparison showed that pAPEC-O2-MS1170-R is a large multidrug resistance IncB/O/K/Z plasmid, while pAPEC-O2-MS1170-ColV shares homology with the APEC ColV virulence plasmid. ConclusionThe genome sequence of APEC-O2-MS1170 provides valuable information on resistance mechanisms and virulence characteristics of pathogenic E. coli as well as information for tracing the potential spread of this MDR strain.

Whole Genome Sequencing of Escherichia coli From Store-Bought Produce.

The role of agriculture in the transfer of drug resistant pathogens to humans is widely debated and poorly understood. Escherichia coli is a valuable indicator organism for contamination and carriage of antimicrobial resistance (AMR) in foods. Whilst whole genome sequences for E. coli from animals and associated meats are common, sequences from produce are scarce. Produce may acquire drug resistant E. coli from animal manure fertilizers, contaminated irrigation water and wildlife, particularly birds. Whole genome sequencing was used to characterize 120 tetracycline (TET) resistant E. coli from store-bought, ready-to-eat cilantro, arugula and mixed salad from two German cities. E. coli were recovered on the day of purchase and after 7 days of refrigeration. Cilantro was far more frequently contaminated with TET-resistant E. coli providing 102 (85%) sequenced strains. Phylogroup B1 dominated the collection (n = 84, 70%) with multi-locus sequence types B1-ST6186 (n = 37, 31%), C-ST165 (n = 17, 14%), B1-ST58 (n = 14, 12%), B1-ST641 (n = 8, 7%), and C-ST88 (n = 5, 4%) frequently identified. Notably, seven strains of diverse sequence type (ST) carried genetic indicators of ColV virulence plasmid carriage. A number of previously identified and novel integrons associated with insertion elements including IS26 were also identified. Storage may affect the lineages of E. coli isolated, however further studies are needed. Our study indicates produce predominantly carry E. coli with a commensal phylogroup and a variety of AMR and virulence-associated traits. Genomic surveillance of bacteria that contaminate produce should be a matter of public health importance in order to develop a holistic understanding of the environmental dimensions of AMR.

Characterizing avian pathogenic Escherichia coli from diagnostic cases in Georgia, USA – comparison of gene profiles with tissue of isolation

Colibacillosis caused by Avian Pathogenic Escherichia coli (APEC) is a significant cause of morbidity, mortality and carcass condemnation to the poultry industry worldwide resulting in significant economic losses. We assessed a multiplex PCR for classifying diagnostic APEC and characterized these isolates using gene profile analysis.48 E. coli isolates collected between August and October 2018 through the Poultry Disease Research Center (PDRC) Diagnostic Laboratory were analyzed. Isolates were screened using multiplex PCR targeting genes associated with APEC chromosomal and plasmid virulence. Isolates were assessed for relationship between gene profile and host tissue of origin. Overall, isolates met the criteria for definition as well-developed pathogens with more than 90 % of isolates positive for the genes iroN, ompTp and hlyF; 78 % were positive for aerJ and 67 % for iss. A significantly lower prevalence was observed for cvaC, etsB, ireA and papC (range 5–36 %). When overall gene prevalence was examined for tissue of isolation, we found that APEC from the ovary, bone marrow, pericardium and lung had higher average numbers of genes compared to isolates recovered from skin and yolk sac. Genes associated with the ColV virulence plasmid (iss, iroN, hlyF and ompTp) were detected in 43 of 48 isolates (89.5%) further confirming the ColV plasmid is the defining trait of the APEC subpathotype. The use of a multiplex panel to screen for APEC has shown good correlation with pathogenesis, and tissue source and correlates well with invasive strains. Path panel diagnostics is available through PDRC, providing significant value to APEC screening.

Evolution of Regions Containing Antibiotic Resistance Genes in FII-2-FIB-1 ColV-Colla Virulence Plasmids.

Three ColV virulence plasmids carrying antibiotic resistance genes were assembled from draft genome sequences of commensal ST95, ST131, and ST2705 Escherichia coli isolates from healthy Australians. Plasmids pCERC4, pCERC5, and pCERC9 include almost identical backbones containing FII-2 and FIB-1 replicons and the conserved ColV virulence region with an additional ColIa determinant. Only pCERC5 includes a complete, uninterrupted F-like transfer region and was able to conjugate. pCERC5 and pCERC9 contain Tn1721, carrying the tet(A) tetracycline resistance determinant in the same location, with Tn2 (blaTEM; ampicillin resistance) interrupting the Tn1721 in pCERC5. pCERC4 has a Tn1721/Tn21 hybrid transposon carrying dfrA5 (trimethoprim resistance) and sul1 (sulfamethoxazole resistance) in a class 1 integron. Four FII-2:FIB-1 ColV-ColIa plasmids in the GenBank nucleotide database have a related transposon in the same position, but an IS26 has reshaped the resistance gene region, deleting 2,069 bp of the integron 3'-CS, including sul1, and serving as a target for IS26 translocatable units containing blaTEM, sul2 and strAB (streptomycin resistance), or aphA1 (kanamycin/neomycin resistance). Another ColV-ColIa plasmid containing a related resistance gene region has lost the FII replicon and acquired a unique transfer region via recombination within the resistance region and at oriT. Eighteen further complete ColV plasmid sequences in GenBank contained FIB-1, but the FII replicons were of three types, FII-24, FII-18, and a variant of FII-36.

PCERC3 from a commensal ST95 Escherichia coli: A ColV virulence-multiresistance plasmid carrying a sul3-associated class 1 integron

The rare sulphonamide resistance gene sul3 was found in the commensal Escherichia coli ST95 strain 22.1-R1 that was isolated in 2010 from the faeces of a healthy Australian adult. The genome of 22.1-R1 was sequenced and a 144,344bp RepFII/FIB plasmid, pCERC3, carrying sul3 was assembled. The sul3 gene is part of a class 1 integron featuring a sul3-containing conserved segment (sul3-CS) that replaced the classic sul1-containing 3′-conserved segment (3′-CS) usually seen in class 1 integrons. The integron contained the cassette array dfrA12-orfF-aadA2-cmlA1-aadA1-qacH, conferring resistance to trimethoprim, streptomycin, spectinomycin, chloramphenicol and quaternary ammonium compound. Two additional antibiotic resistance genes, blaTEM (ampicillin resistance) and tetA(B) (tetracycline) were adjacent to the integron, forming a single resistance region. In pCERC3, the sul3-type class 1 integron was flanked by sequence derived from the tnp and mer modules of Tn21 and was in the same location as In2, the sul1-containing In5-type class 1 integron of Tn21. At one end the sequence extends into Tn2670-derived sequence and then into sequence derived from the plasmid NR1 (R100). Examination of the sequences of eleven more complete sul3-containing plasmids in GenBank confirmed the relationship between sul3-associated integrons and Tn21/Tn2670/NR1. This suggests that the events that formed sul3-associated class 1 integrons occurred within the Tn21/Tn2670 context, most likely in NR1 or a related plasmid. The backbone of pCERC3 is most closely related to the backbones of ColV virulence plasmids and contains a complete ColV operon as well as several virulence associated genes and gene clusters. Hence, pCERC3 is both an antibiotic resistance and virulence plasmid.

Horizontal Gene Transfer of a ColV Plasmid Has Resulted in a Dominant Avian Clonal Type of Salmonella enterica Serovar Kentucky

Salmonella enterica continues to be a significant cause of foodborne gastrointestinal illness in humans. A wide variety of Salmonella serovars have been isolated from production birds and from retail poultry meat. Recently, though, S. enterica subsp. enterica serovar Kentucky has emerged as one of the prominent Salmonella serovars isolated from broiler chickens. Recent work suggests that its emergence apparently coincides with its acquisition of a ColV virulence plasmid. In the present study, we examined 902 Salmonella isolates belonging to 59 different serovars for the presence of this plasmid. Of the serovars examined, the ColV plasmid was found only among isolates belonging to the serovars Kentucky (72.9%), Typhimurium (15.0%) and Heidelberg (1.7%). We demonstrated that a single PFGE clonal type of S. Kentucky harbors this plasmid, and acquisition of this plasmid by S. Kentucky significantly increased its ability to colonize the chicken cecum and cause extraintestinal disease. Comparison of the completed sequences of three ColV plasmids from S. Kentucky isolated from different geographical locales, timepoints and sources revealed a nearly identical genetic structure with few single nucleotide changes or insertions/deletions. Overall, it appears that the ColV plasmid was recently acquired by a single clonal type S. Kentucky and confers to its host enhanced colonization and fitness capabilities. Thus, the potential for horizontal gene transfer of virulence and fitness factors to Salmonella from other enteric bacteria exists in poultry, representing a potential human health hazard.

Examination of the Source and Extended Virulence Genotypes ofEscherichia coliContaminating Retail Poultry Meat

Extraintestinal pathogenic Escherichia coli (ExPEC) are major players in human urinary tract infections, neonatal bacterial meningitis, and sepsis. Recently, it has been suggested that there might be a zoonotic component to these infections. To determine whether the E. coli contaminating retail poultry are possible extraintestinal pathogens, and to ascertain the source of these contaminants, they were assessed for their genetic similarities to E. coli incriminated in colibacillosis (avian pathogenic E. coli [APEC]), E. coli isolated from multiple locations of apparently healthy birds at slaughter, and human ExPEC. It was anticipated that the retail poultry isolates would most closely resemble avian fecal E. coli since only apparently healthy birds are slaughtered, and fecal contamination of carcasses is the presumed source of meat contamination. Surprisingly, this supposition proved incorrect, as the retail poultry isolates exhibited gene profiles more similar to APEC than to fecal isolates. These isolates contained a number of ExPEC-associated genes, including those associated with ColV virulence plasmids, and many belonged to the B2 phylogenetic group, known to be virulent in human hosts. Additionally, E. coli isolated from the crops and gizzards of apparently healthy birds at slaughter also contained a higher proportion of ExPEC-associated genes than did the avian fecal isolates examined. Such similarities suggest that the widely held beliefs about the sources of poultry contamination may need to be reassessed. Also, the presence of ExPEC-like clones on retail poultry meat means that we cannot yet rule out poultry as a source of ExPEC human disease.

Complete DNA Sequence of a ColBM Plasmid from Avian Pathogenic Escherichia coli Suggests that It Evolved from Closely Related ColV Virulence Plasmids

Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli causing colibacillosis in birds, is responsible for significant economic losses for the poultry industry. Recently, we reported that the APEC pathotype was characterized by possession of a set of genes contained within a 94-kb cluster linked to a ColV plasmid, pAPEC-O2-ColV. These included sitABCD, genes of the aerobactin operon, hlyF, iss, genes of the salmochelin operon, and the 5' end of cvaB of the ColV operon. However, the results of gene prevalence studies performed among APEC isolates revealed that these traits were not always linked to ColV plasmids. Here, we present the complete sequence of a 174-kb plasmid, pAPEC-O1-ColBM, which contains a putative virulence cluster similar to that of pAPEC-O2-ColV. These two F-type plasmids share remarkable similarity, except that they encode the production of different colicins; pAPEC-O2-ColV contains an intact ColV operon, and pAPEC-O1-ColBM encodes the colicins B and M. Interestingly, remnants of the ColV operon exist in pAPEC-O1-ColBM, hinting that ColBM-type plasmids may have evolved from ColV plasmids. Among APEC isolates, the prevalence of ColBM sequences helps account for the previously observed differences in prevalence between genes of the "conserved" portion of the putative virulence cluster of pAPEC-O2-ColV and those genes within its "variable" portion. These results, in conjunction with Southern blotting and probing of representative ColBM-positive strains, indicate that this "conserved" cluster of putative virulence genes is primarily linked to F-type virulence plasmids among the APEC isolates studied.

A novel ColV plasmid encoding type IV pili.

Many septicaemic Escherichia coli strains harbour ColV virulence plasmids. This paper describes pO78V, a conjugative ColV plasmid from an avian pathogenic E. coli strain that encodes type IV pili in addition to other virulence-related genes and tetracycline resistance. Plasmid location of type IV pili genes was demonstrated using Southern hybridization and expression of the pili was demonstrated using RT-PCR and phage sensitivity assays. This is a first report of a ColV plasmid encoding type IV pili. Plasmid pO78V is a mosaic plasmid containing replicons and other genes typical to both IncI1 and IncFII groups. As type IV pili of Gram-negative bacteria are involved in several stages of infection, their presence on a ColV virulence plasmid could expand the repertoire of pathogenesis-related genes.

Relationship between the Tsh autotransporter and pathogenicity of avian Escherichia coli and localization and analysis of the Tsh genetic region.

The temperature-sensitive hemagglutinin Tsh is a member of the autotransporter group of proteins and was first identified in avian-pathogenic Escherichia coli (APEC) strain chi7122. The prevalence of tsh was investigated in 300 E. coli isolates of avian origin and characterized for virulence in a 1-day-old chick lethality test. Results indicate that among the tsh-positive APEC isolates, 90.6% belonged to the highest virulence class. Experimental inoculation of chickens with chi7122 and an isogenic tsh mutant demonstrated that Tsh may contribute to the development of lesions within the air sacs of birds but is not required for subsequent generalized infection manifesting as perihepatitis, pericarditis, and septicemia. Conjugation and hybridization experiments revealed that the tsh gene is located on a ColV-type plasmid in many of the APEC strains studied, including strain chi7122, near the colicin V genes in most of these strains. DNA sequences flanking the tsh gene of strain chi7122 include complete and partial insertion sequences and phage-related DNA sequences, some of which were also found on virulence plasmids and pathogenicity islands present in various E. coli pathotypes and other pathogenic members of the Enterobacteriaceae. These results demonstrate that the tsh gene is frequently located on the ColV virulence plasmid in APEC and suggest a possible role of Tsh in the pathogenicity of E. coli for chickens in the early stages of infection.

Envelope protein changes, autoagglutination, sensitivity to hydrophobic agents and a conditional division lesion in Escherichia coli strains carrying ColV virulence plasmids

The presence of the virulence plasmids ColV,I-K94 or ColV-K30 in Escherichia coli produces a number of cell membrane and envelope changes. The most striking of these are (1) the presence of the 33K VmpA outer membrane protein and (2) the ColV-associated occurrence of autoagglutination. The VmpA protein is a plasmid-encoded outer membrane protein which is synthesized from a larger precursor. It is distinct from the chromosomally-encoded OmpA protein but resembles it in a few respects. The VmpA protein does not appear to be involved in colicin synthesis or immunity, or in plasmid transfer. This protein was found in 6 out of 8 new ColV+ isolates, but not in 2 ColIa+ strains. ColV-induced autoagglutination occurred for strains grown in static culture at 37 but not at 25 degrees C. Detergents prevented agglutination, as did the presence in a ColV+ strain of a fi+ plasmid, ColB. Autoagglutination may be a virulence phenotype. Associated with the ability of ColV+ bacteria to agglutinate was inhibition of motility. ColV+ bacteria also showed changes in envelope permeability indicated by inhibitor sensitivity and by a ColV-associated suppression of the lac Y lesion. Some ColV,I-K94+ strains showed a mucoid colonial phenotype and this ability to form mucoid colonies was efficiently transferred with ColV but apparently not without it. The mucoid ColV+ strains resembled lon mutants in UV-sensitivity, division behaviour and sensitivity to lambda phage.