Serotype O18 avian pathogenic and neonatal meningitis Escherichia coli strains employ similar pathogenic strategies for the onset of meningitis.

Neonatal meningitis Escherichia coli K1 (NMEC) are thought to be transmitted from mothers to newborns during delivery or by nosocomial infections. However, the source of E. coli K1 causing these infections is not clear. Avian pathogenic E. coli (APEC) have the potential to cause infection in humans while human E. coli have potential to cause colibacillosis in poultry, suggesting that these strains may lack host specificity. APEC strains are capable of causing meningitis in newborn rats; however, it is unclear whether these bacteria use similar mechanisms to that of NMEC to establish disease. Using four representative APEC and NMEC strains that belong to serotype O18, we demonstrate that these strains survive in human serum similar to that of the prototypic NMEC strain E44, a derivative of RS218. These bacteria also bind and enter both macrophages and human cerebral microvascular endothelial cells (HCMEC/D3) with similar frequency as that of E44. The amino acid sequences of the outer membrane protein A (OmpA), an important virulence factor in the pathogenesis of meningitis, are identical within these representative APEC and NMEC strains. Further, these strains also require FcγRI-α chain (CD64) and Ecgp96 as receptors for OmpA in macrophages and HCMEC/D3, respectively, to bind and enter these cells. APEC and NMEC strains induce meningitis in newborn mice with varying degree of pathology in the brains as assessed by neutrophil recruitment and neuronal apoptosis. Together, these results suggest that serotype O18 APEC strains utilize similar pathogenic mechanisms as those of NMEC strains in causing meningitis.

Avian pathogenic Escherichia coli (APEC) infection alters bone marrow transcriptome in chickens.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) is a major cause of disease impacting animal health. The bone marrow is the reservoir of immature immune cells; however, it has not been examined to date for gene expression related to developmental changes (cell differentiation, maturation, programming) after APEC infection. Here, we study gene expression in the bone marrow between infected and non-infected animals, and between infected animals with mild (resistant) versus severe (susceptible) pathology, at two times post-infection. RESULTS: We sequenced 24 bone marrow RNA libraries generated from the six different treatment groups with four replicates each, and obtained an average of 22 million single-end, 100-bp reads per library. Genes were detected as differentially expressed (DE) between APEC treatments (mild pathology, severe pathology, and mock-challenged) at a given time point, or DE between 1 and 5 days post-infection (dpi) within the same treatment group. Results demonstrate that many immune cells, genes and related pathways are key contributors to the different responses to APEC infection between susceptible and resistant birds and between susceptible and non-challenged birds, at both times post-infection. In susceptible birds, lymphocyte differentiation, proliferation, and maturation were greatly impaired, while the innate and adaptive immune responses, including dendritic cells, monocytes and killer cell activity, TLR- and NOD-like receptor signaling, as well as T helper cells and many cytokine activities, were markedly enhanced. The resistant birds' immune system, however, was similar to that of non-challenged birds. CONCLUSION: The DE genes in the immune cells and identified signaling models are representative of activation and resolution of infection in susceptible birds at both post-infection days. These novel results characterizing transcriptomic response to APEC infection reveal that there is combinatorial activity of multiple genes controlling myeloid cells, and B and T cell lymphopoiesis, as well as immune responses occurring in the bone marrow in these early stages of response to infection.

FNR regulates expression of important virulence factors contributing to pathogenicity of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is responsible for the majority of urinary tract infections (UTIs), which are some of the world's most common bacterial infections of humans. Here, we examined the role of FNR (fumarate and nitrate reduction), a well-known global regulator, in the pathogenesis of UPEC infections. We constructed an fnr deletion mutant of UPEC CFT073 and compared it to the wild type for changes in virulence, adherence, invasion, and expression of key virulence factors. Compared to the wild type, the fnr mutant was highly attenuated in the mouse model of human UTI and showed severe defects in adherence to and invasion of bladder and kidney epithelial cells. Our results showed that FNR regulates motility and multiple virulence factors, including expression of type I and P fimbriae, modulation of hemolysin expression, and expression of a novel pathogenicity island involved in α-ketoglutarate metabolism under anaerobic conditions. Our results demonstrate that FNR is a key global regulator of UPEC virulence and controls expression of important virulence factors that contribute to UPEC pathogenicity.

Porcine E. coli: virulence-associated genes, resistance genes and adhesion and probiotic activity tested by a new screening method.

We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars. Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation. In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars.

tkt1, located on a novel pathogenicity island, is prevalent in avian and human extraintestinal pathogenic Escherichia coli.

BACKGROUND: Extraintestinal pathogenic Escherichia coli are important pathogens of human and animal hosts. Some human and avian extraintestinal pathogenic E. coli are indistinguishable on the basis of diseases caused, multilocus sequence and phylogenetic typing, carriage of large virulence plasmids and traits known to be associated with extraintestinal pathogenic E. coli virulence. RESULTS: The gene tkt1 identified by a previous signature-tagged transposon mutagenesis study, was found on a 16-kb genomic island of avian pathogenic Escherichia coli (APEC) O1, the first pathogenic Escherichia coli strain whose genome has been completely sequenced. tkt1 was present in 39.6% (38/96) of pathogenic Escherichia coli strains, while only 6.25% (3/48) of E. coli from the feces of apparently healthy chickens was positive. Further, tkt1 was predominantly present in extraintestinal pathogenic E. coli belonging to the B2 phylogenetic group, as compared to extraintestinal pathogenic E. coli of other phylogenetic groups. The tkt1-containing genomic island is inserted between the metE and ysgA genes of the E. coli K12 genome. Among different extraintestinal pathogenic E. coli of the B2 phylogenetic group, 61.7% of pathogenic Escherichia coli, 80.6% of human uropathogenic E.coli and 94.1% of human neonatal meningitis-causing E. coli, respectively, harbor a complete copy of this island; whereas, only a few avian fecal E. coli strains contained the complete island. Functional analysis showed that Tkt1 confers very little transketolase activity but is involved in peptide nitrogen metabolism. CONCLUSION: These results suggest tkt1 and its corresponding genomic island are frequently associated with avian and human ExPEC and are involved in bipeptide metabolism.

papA gene of avian pathogenic Escherichia coli.

P fimbrial adhesins may be associated with the virulence of avian pathogenic Escherichia coli (APEC). However, most APECs are unable to express P fimbriae even when they are grown under conditions that favor P fimbrial expression. This failure can be explained by the complete absence of the pap operon or the presence of an incomplete pap operon in Pap-negative APEC strains. In the present study, we analyzed the pap operon, specifically the papA gene that encodes the major fimbrial shaft, to better understand the pap gene cluster at the genetic level. First, by PCR, we examined a collection of 500 APEC strains for the presence of 11 genes comprising the pap operon. Except for papA, all the other genes of the operon were present in 38% to 41.2% of APEC, whereas the papA was present only in 10.4% of the APEC tested. Using multiplex PCR to probe for allelic variants of papA, we sought to determine if the low prevalence of papA among APEC was related to genetic heterogeneity of the gene itself. It was determined that the papA of APEC always belongs to the F11 allelic variant. Finally, we sequenced the 'papA region' from two papA-negative strains, both of which contain all the other genes of the pap operon. Interestingly, both strains had an 11,104-bp contig interruptingpapA at the 281-bp position. This contig harbored a streptomycin resistance gene and a classic Tn10 transposon containing the genes that confer tetracycline resistance. However, we noted that the papA gene of every papA-negative APEC strain was not interrupted by an 11,104-bp contig. It is likely that transposons bearing antibiotic resistance genes have inserted within pap gene cluster of some APEC strains, and such genetic events may have been selected for by antibiotic use.

Sequence analysis and characterization of a transferable hybrid plasmid encoding multidrug resistance and enabling zoonotic potential for extraintestinal Escherichia coli.

ColV plasmids of extraintestinal pathogenic Escherichia coli (ExPEC) encode a variety of fitness and virulence factors and have long been associated with septicemia and avian colibacillosis. These plasmids are found significantly more often in ExPEC, including ExPEC associated with human neonatal meningitis and avian colibacillosis, than in commensal E. coli. Here we describe pAPEC-O103-ColBM, a hybrid RepFIIA/FIB plasmid harboring components of the ColV pathogenicity island and a multidrug resistance (MDR)-encoding island. This plasmid is mobilizable and confers the ability to cause septicemia in chickens, the ability to cause bacteremia resulting in meningitis in the rat model of human disease, and the ability to resist the killing effects of multiple antimicrobial agents and human serum. The results of a sequence analysis of this and other ColV plasmids supported previous findings which indicated that these plasmid types arose from a RepFIIA/FIB plasmid backbone on multiple occasions. Comparisons of pAPEC-O103-ColBM with other sequenced ColV and ColBM plasmids indicated that there is a core repertoire of virulence genes that might contribute to the ability of some ExPEC strains to cause high-level bacteremia and meningitis in a rat model. Examination of a neonatal meningitis E. coli (NMEC) population revealed that approximately 58% of the isolates examined harbored ColV-type plasmids and that 26% of these plasmids had genetic contents similar to that of pAPEC-O103-ColBM. The linkage of the ability to confer MDR and the ability contribute to multiple forms of human and animal disease on a single plasmid presents further challenges for preventing and treating ExPEC infections.

Occurrence of pathogenicity island I(APEC-O1) genes among Escherichia coli implicated in avian colibacillosis.

Colibacillosis caused by avian pathogenic Escherichia coli (APEC) is a leading cause of economic loss to the poultry industry worldwide. The ability of APEC to cause disease is determined by certain virulence markers, some of which are located on pathogenicity islands (PAls). We recently described one such PAI in an APEC O1:K1 strain (APEC-O1). This PAI, termed PAI I(APEC-O1), carries the genes of the pap operon, a region similar to the tia invasion determinant of enterotoxigenic E coli; ireA, a gene that encodes an iron-responsive element; and a novel 1.5-kb region, ORF 54. Here, the occurrence of six selected loci of PAI I(APEC-O1) (papA, papC, papG, ireA, tia, and ORF 54) among APEC and fecal E. coli strains from apparently healthy chickens (avian commensal E. coli) was determined using polymerase chain reaction (PCR) techniques. None of the commensal E. coli was positive for all six traits, whereas 7.2% of the APEC isolates were positive for all the traits. Although there was no significant difference in the occurrence of ORF 54 among APEC and commensal E. coli, tia, ireA, papC, and papG genes were predominantly present in APEC rather than in avian commensal E. coli. papA was detected in only 6.3% of APEC, perhaps because of the presence of allelic variants of the gene. Additionally, the presence of all six traits was tested with PCR in APEC isolates collected in the 1980s, and these results were compared with those obtained with the APEC isolated in the 1990s. There was no significant difference in the occurrence of tia, ireA, papC, papG, and ORF 54 between APEC isolates collected during the different decades. However, papA was more frequently present in APEC from the 1980s than it was in APEC from the 1990s. Phylogenetic group of an isolate did not correlate with pathogenicity or the presence of PAI traits, except that more APEC of the low-pathogenicity group belonged to the phylogenetic group B1. However, PAI traits occurred more frequently in isolates belonging to the intermediate- and high-pathogenicity groups than in isolates of low pathogenicity.