Nucleotide receptors mediate protection against neonatal sepsis and meningitis caused by alpha-hemolysin expressing Escherichia coli K1.

Neonatal meningitis-associated Escherichia coli (NMEC) is among the leading causes of bacterial meningitis and sepsis in newborn infants. Several virulence factors have been identified as common among NMEC, and have been shown to play an important role in the development of bacteremia and/or meningitis. However, there is significant variability in virulence factor expression between NMEC isolates, and relatively little research has been done to assess the impact of variable virulence factor expression on immune cell activation and the outcome of infection. Here, we investigated the role of NMEC strain-dependent P2X receptor (P2XR) signaling on the outcome of infection in neonatal mice. We found that alpha-hemolysin (HlyA)-expressing NMEC (HlyA+ ) induced robust P2XR-dependent macrophage cell death in vitro, while HlyA- NMEC did not. P2XR-dependent cell death was inflammasome independent, suggesting an uncoupling of P2XR and inflammasome activation in the context of NMEC infection. In vivo inhibition of P2XRs was associated with increased mortality in neonatal mice infected with HlyA+ NMEC, but had no effect on the survival of neonatal mice infected with HlyA- NMEC. Furthermore, we found that P2XR-dependent protection against HlyA+ NMEC in vivo required macrophages, but not neutrophils or NLRP3. Taken together, these data suggest that HlyA+ NMEC activates P2XRs which in turn confers macrophage-dependent protection against infection in neonates. In addition, our findings indicate that strain-dependent virulence factor expression should be taken into account when studying the immune response to NMEC.

Citrate utilization under anaerobic environment in Escherichia coli is under direct control of Fnr and indirect control of ArcA and Fnr via CitA-CitB system.

Most Escherichia coli (E. coli) strains do not cause disease, naturally living in the lower intestine and is expelled into the environment within faecal matter. Escherichia coli can utilize citrate under anaerobic conditions but not aerobic conditions. However, the underlying regulatory mechanisms are poorly understood. In this study, we explored regulatory mechanisms of citrate fermentation genes by global regulators ArcA and Fnr under anaerobic conditions. A gel mobility shift assay showed that the regulator proteins ArcA and Fnr binded to the promoter region localized between the citAB and citCDEFXGT operons. Subsequent assays confirmed that ArcA indirectly controled the expression of citrate fermentation genes via regulating CitA-CitB system, while Fnr directly regulated but also indirectly modulated citrate fermentation genes via controling CitA-CitB system. Deletions of arcA and fnr significantly reduced the growth of Escherichia coli in M9 medium with a citrate carbon source. We conclude that both ArcA and Fnr can indirectly control the citrate utilization via CitA-CitB system, while Fnr can also directly regulate the expression of citrate fermentation genes in E. coli under anaerobic conditions.

Phylogenomic Analysis of Extraintestinal Pathogenic Escherichia coli Sequence Type 1193, an Emerging Multidrug-Resistant Clonal Group.

The fluoroquinolone-resistant sequence type 1193 (ST1193) of Escherichia coli, from the ST14 clonal complex (STc14) within phylogenetic group B2, has appeared recently as an important cause of extraintestinal disease in humans. Although this emerging lineage has been characterized to some extent using conventional methods, it has not been studied extensively at the genomic level. Here, we used whole-genome sequence analysis to compare 355 ST1193 isolates with 72 isolates from other STs within STc14. Using core genome phylogeny, the ST1193 isolates formed a tightly clustered clade with many genotypic similarities, unlike ST14 isolates. All ST1193 isolates possessed the same set of three chromosomal mutations conferring fluoroquinolone resistance, carried the fimH64 allele, and were lactose non-fermenting. Analysis revealed an evolutionary progression from K1 to K5 capsular types and acquisition of an F-type virulence plasmid, followed by changes in plasmid structure congruent with genome phylogeny. In contrast, the numerous identified antimicrobial resistance genes were distributed incongruently with the underlying phylogeny, suggesting frequent gain or loss of the corresponding resistance gene cassettes despite retention of the presumed carrier plasmids. Pangenome analysis revealed gains and losses of genetic loci occurring during the transition from ST14 to ST1193 and from the K1 to K5 capsular types. Using time-scaled phylogenetic analysis, we estimated that current ST1193 clades first emerged approximately 25 years ago. Overall, ST1193 appears to be a recently emerged clone in which both stepwise and mosaic evolution have contributed to epidemiologic success.

A novel two-component signaling system facilitates uropathogenic Escherichia coli's ability to exploit abundant host metabolites.

Two-component signaling systems (TCSs) are major mechanisms by which bacteria adapt to environmental conditions. It follows then that TCSs would play important roles in the adaptation of pathogenic bacteria to host environments. However, no pathogen-associated TCS has been identified in uropathogenic Escherichia coli (UPEC). Here, we identified a novel TCS, which we termed KguS/KguR (KguS: α-ketoglutarate utilization sensor; KguR: α-ketoglutarate utilization regulator) in UPEC CFT073, a strain isolated from human pyelonephritis. kguS/kguR was strongly associated with UPEC but was found only rarely among other E. coli including commensal and intestinal pathogenic strains. An in vivo competition assay in a mouse UTI model showed that deletion of kguS/kguR in UPEC CFT073 resulted in a significant reduction in its colonization of the bladders and kidneys of mice, suggesting that KguS/KguR contributed to UPEC fitness in vivo. Comparative proteomics identified the target gene products of KguS/KguR, and sequence analysis showed that TCS KguS/KguR and its targeted-genes, c5032 to c5039, are encoded on a genomic island, which is not present in intestinal pathogenic E. coli. Expression of the target genes was induced by α-ketoglutarate (α-KG). These genes were further shown to be involved in utilization of α-KG as a sole carbon source under anaerobic conditions. KguS/KguR contributed to the regulation of the target genes with the direct regulation by KguR verified using an electrophoretic mobility shift assay. In addition, oxygen deficiency positively modulated expression of kguS/kguR and its target genes. Taken altogether, this study describes the first UPEC-associated TCS that functions in controlling the utilization of α-ketoglutarate in vivo thereby facilitating UPEC adaptation to life inside the urinary tract.

Molecular characterization and clonal relationships among Escherichia coli strains isolated from broiler chickens with colisepticemia.

This study characterized 52 Escherichia coli isolates from distinct diseased organs of 29 broiler chickens with clinical symptoms of colibacillosis in the Southern Brazilian state of Rio Grande do Sul. Thirty-eight isolates were highly virulent and 14 were virtually avirulent in 1-day-old chicks, yet all isolates harbored virulence factors characteristic of avian pathogenic E. coli (APEC), including those related to adhesion, iron acquisition, and serum resistance. E. coli reference collection phylogenetic typing showed that isolates belonged mostly to group D (39%), followed by group A (29%), group B1 (17%), and group B2 (15%). Phylogenetic analyses using the Amplified Ribosomal DNA Restriction Analysis and pulse-field gel electrophoresis methods were used to discriminate among isolates displaying the same serotype, revealing that five birds were infected with two distinct APEC strains. Among the 52 avian isolates, 2 were members of the pandemic E. coli O25:H4-B2-ST131 clone.

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.

Salmonella spp. in poultry production-A review of the role of interventions along the production continuum.

Salmonella is a significant pathogen of human and animal health and poultry are one of the most common sources linked with foodborne illness worldwide. Global production of poultry meat and products has increased significantly over the last decade or more as a result of consumer demand and the changing demographics of the world's population, where poultry meat forms a greater part of the diet. In addition, the relatively fast growth rate of birds which is significantly higher than other meat species also plays a role in how poultry production has intensified. In an effort to meet the greater demand for poultry meat and products, modern poultry production and processing practices have changed and practices to target control and reduction of foodborne pathogens such as Salmonella have been implemented. These strategies are implemented along the continuum from parent and grandparent flocks to breeders, the farm and finished broilers to transport and processing and finally from retail to the consumer. This review focuses on common practices, interventions and strategies that have potential impact for the control of Salmonella along the poultry production continuum from farm to plate.

Molecular and phenotypic characterization of Escherichia coli isolated from broiler chicken flocks in Egypt.

Avian pathogenic Escherichia coli (APEC) infection is responsible for great economic losses to the poultry industry worldwide and there is increasing evidence of its zoonotic importance. In this study, 219 E. coli isolates from 84 poultry flocks in Egypt, including 153 APEC, 30 avian fecal E. coli (AFEC), and 36 environmental E. coli, were subjected to phylogenetic grouping and virulence genotyping. Additionally, 50 of these isolates (30 APEC from colisepticemia and 20 AFEC) were subjected to a more-extensive characterization which included serogrouping, antimicrobial susceptibility analysis, screening for seven intestinal E. coli virulence genes (stx1, stx2, eae, espP, KatP, hlyA, and fliCh7), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and in vivo virulence testing. More than 90% of the total APEC examined possessed iroN, ompT, hlyF, iss, and iutA, indicating that Egyptian APECs, like their counterparts from the United States, harbor plasmid pathogenicity islands (PAIs). The majority of APEC and AFEC were of phylogenetic groups A, B1, and D. For the 50-isolate subgroup, more than 70% of APEC and 80% ofAFEC were multidrug resistant. Among the subgroup of APEC, MLST analysis identified 11 sequence types (ST) while seven STs were found among AFEC. Based on PFGE, the genetic relatedness of APEC and AFEC ranged from 50%-100% and clustered into four primary groups at 50% similarity. Two of the eight APEC strains tested in chickens were able to induce 25% mortality in 1-day-old chicks. APECs were distinguished from AFECs and environmental E. coli by their content of plasmid PAI genes, whereas APEC isolated from colisepticemia and AFEC were not distinguishable based on their antimicrobial resistance patterns, as both groups were multidrug resistant. Avian E. coli strains from broiler flocks in Egypt show similar sequence types to E. coli associated with human infection.

Molecular typing of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) isolated from animals and retail meat in North Dakota, United States.

The objective of this study was to determine the prevalence and molecular typing of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in food-producing animals and retail meat in Fargo, North Dakota. A two-step enrichment followed by culture methods were used to isolate S. aureus from 167 nasal swabs from animals, 145 samples of retail raw meat, and 46 samples of deli meat. Positive isolates were subjected to multiplex polymerase chain reaction in order to identify the genes 16S rRNA, mecA, and Panton-Valentine Leukocidin. Pulsed-field gel electrophoresis and multilocus sequence typing were used for molecular typing of S. aureus strains. Antimicrobial susceptibility testing was carried out using the broth microdilution method. The overall prevalence of S. aureus was 37.2% (n=133), with 34.7% (n=58) of the animals positive for the organism, and the highest prevalence observed in pigs (50.0%) and sheep (40.6%) (p<0.05); 47.6% (n=69) of raw meat samples were positive, with the highest prevalence in chicken (67.6%) and pork (49.3%) (p<0.05); and 13.0% (n=6) of deli meat was positive. Five pork samples (7.0%) were positive for MRSA, of which three were ST398 and two were ST5. All exhibited penicillin resistance and four were multidrug resistant (MDR). The Panton-Valentine Leukocidin gene was not detected in any sample by multiplex polymerase chain reaction. The most common clones in sheep were ST398 and ST133, in pigs and pork both ST398 and ST9, and in chicken ST5. Most susceptible S. aureus strains were ST5 isolated from chicken. The MDR isolates were found in pigs, pork, and sheep. The presence of MRSA, MDR, and the subtype ST398 in the meat production chain and the genetic similarity between strains of porcine origin (meat and animals) suggest the possible contamination of meat during slaughtering and its potential transmission to humans.

Comparative genomic analysis of Campylobacter hepaticus genomes associated with spotty liver disease, Georgia, United States.

Campylobacter hepaticus has re-emerged as an important cause of disease in egg laying birds worldwide, resulting in morbidity, mortality, and significant losses in eggs for the breeding and table egg laying industries. Although birds may appear asymptomatic, the disease is characterized by spots on the liver of birds and histopathological analysis reveals multifocal fibrogranulocytic necrotizing hepatitis microscopically. The re-emergence of C. hepaticus may be linked with housing practices as the disease appears more prevalent in pasture raised birds with outside exposure. Here we describe, the whole genome sequences and comparative analysis of four C. hepaticus genomes associated with an outbreak on pasture raised breeders from a farm in Georgia, United States. All four genomes were relatively similar in size and virulence genes harbored. Using these genomes, comparison with current C. hepaticus genomes available in NCBI and other databases and other members of the Campylobacter species was carried out. Using current tools available, virulence gene factor content was compared, and it was found that different tools lead to different numbers of factors identified. The four genomes from this study were relatively similar to C. hepaticus HV10 the type strain from Australia but differed from the other sequenced US strains from Iowa and Florida. C. hepaticus was found to have an overall lower gene content for genes associated with virulence and iron acquisition compared to other Campylobacter genomes and appears to cluster differently than UK genomes on phylogenetic analysis, suggesting the emergence of two lineages of C. hepaticus. This analysis provides valuable insight into the emerging pathogen C. hepaticus, its virulence factors and traits associated with disease in poultry production in the US, potentially providing insight into targets for its control and treatment for laying birds. Our analysis also confirms genes associated with iron acquisition are limited and the presence of the multidrug efflux pump CmeABC in C. hepaticus which may promote survival and persistence in the host niche - the chicken liver/bile. One unique aspect of this study was the finding of a close genetic relationship between C. hepaticus and Campylobacter fetus species and evidence of genome reduction in relation to host niche specificity.

Complete Genome Sequence of Campylobacter hepaticus RBCL71delta, Associated with Spotty Liver Disease in Organic Pasture-Raised Laying Hens in Georgia, USA.

Spotty liver disease (SLD) is as an important cause of disease in egg-producing flocks. The organisms implicated include Campylobacter hepaticus and Campylobacter bilis. Here, we report the complete genome sequence of C. hepaticus strain RBCL71delta and three additional strains, isolated from a layer flock in Georgia, USA.

Assessment of an Enterobactin Conjugate Vaccine in Layers to Protect Their Offspring from Colibacillosis.

Colibacillosis, caused by avian pathogenic Escherichia coli (APEC), is an important infectious disease in chickens and a major cause of mortality in young chicks. Therefore, protecting young chickens from colibacillosis is important for improving welfare and productivity in the poultry industry. Recently, we developed a novel enterobactin (Ent) conjugate vaccine that could induce high titers of anti-Ent immunoglobulin Y (IgY) in chicken serum and consequently mitigate the organ lesions caused by APEC infection. Considering that maternal immunization is a practical approach to confer instant immune protection to the hatchlings, in this study, we immunized breeder hens with the Ent conjugate vaccine and evaluated the maternal immune protection on the progenies challenged with APEC. Three doses of the vaccine induced high titers of anti-Ent IgY in the hens (about 16- and 64-fold higher than the control group in the sera and egg yolks, respectively), resulting in an eight-fold of increase in anti-Ent IgY in the sera of progenies. However, the anti-Ent maternal immunity did not display significant protection against APEC challenge in the young chicks as there was no significant difference in APEC load (in liver, lung, and spleen) or organ lesions (in heart, liver, spleen, lung, and air sac) between the vaccinated and control groups. In future studies, the APEC infection model needs to be optimized to exhibit proper pathogenicity of APEC, and the maternal immunization regimen can be further improved to boost the maternally derived anti-Ent IgY in the hatchlings.

Laser Capture Microdissection, Culture Analysis, and Bacterial Sequencing to Evaluate the Microbiota of Focal Duodenal Necrosis in Egg Layers.

Focal duodenal necrosis (FDN) is a common intestinal disease of table egg layers. In this research we aimed to identify the bacteria commonly found in FDN lesions as seen with histopathological analysis. Fifty-nine ethanol-fixed duodenum samples were collected from egg layers on eight FDN-affected farms, and 42 samples had typical FDN lesions. Excision of bacteria-containing lesions using laser capture microdissection was performed, followed by 16S rRNA gene sequencing of extracted DNA for bacterial identification. Bacterial sequencing analysis revealed no consistent bacterial species identified from samples with FDN. However, analysis of the relative phylum abundance revealed differences in the duodenal microbiota between layers with FDN and healthy birds. There were differences in the abundance of Proteobacteria, Firmicutes, and Actinobacteria between FDN-positive and FDN-negative control samples compatible with intestinal dysbiosis. In addition, 10 duodenal samples with FDN lesions were collected for bacteriological analysis, yielding 47 colonies on tryptone soy agar, MacConkey agar, and blood agar plates. Using 16S rRNA gene PCR, 39/47 (53.8%) colonies were identified as Escherichia coli. PCR for E. coli virulence genes identified 21/39 (53.8%) E. coli isolates as avian pathogenic E. coli-like. PCR analysis for 19 E. coli virulence genes associated with intestinal disease strains including inflammatory bowel disease found 11/39 (28.2%) isolates containing more than 10 of these virulence genes. In conclusion, FDN appears to be a multifactorial inflammatory intestinal disease associated with intestinal dysbiosis, and Gram-negative bacteria including E. coli may contribute to the pathogenesis of this disease.

Microdisección por captura láser, análisis de cultivos y secuenciación bacteriana para evaluar la microbiota de la necrosis duodenal focal en aves de postura de huevo comercial. La necrosis duodenal focal (FDN) es una enfermedad intestinal común en las gallinas de postura de huevo comercial. En esta investigación, el objetivo fue identificar las bacterias que se encuentran comúnmente en las lesiones provocadas por la necrosis duodenal focal tal como se aprecian con el análisis histopatológico. Se recolectaron 59 muestras de duodeno fijadas con etanol de gallinas de postura de ocho granjas afectadas por necrosis duodenal focal, y 42 muestras tenían lesiones típicas de dicha enfermedad. Se realizó la escisión de las lesiones que contenían bacterias mediante microdisección por captura láser, seguida de la secuenciación del gene 16S rRNA del ADN extraído para la identificación bacteriana. El análisis de secuenciación bacteriana no reveló especies bacterianas consistentes identificadas a partir de muestras con necrosis duodenal focal. Sin embargo, el análisis de la abundancia relativa del phylum reveló diferencias en el microbiota duodenal entre gallinas de postura con necrosis duodenal focal y aves sanas. Hubo diferencias en la abundancia de Proteobacteria, Firmicutes y Actinobacteria entre las muestras controles positivas y negativas para la necrosis duodenal focal compatibles con disbiosis intestinal. Además, se recolectaron 10 muestras duodenales con lesiones de la necrosis duodenal focal para análisis bacteriológico, lo que produjo 47 colonias en placas de agar triptona soya, agar MacConkey y agar sangre. Utilizando un método de PCR para el gene 16S rRNA, 39/47 (53.8 %) colonias se identificaron como Escherichia coli. El método de PCR para genes de virulencia de E. coli identificó 21/39 (53.8 %) aislados de E. coli como similares a E. coli patogénica aviar. El análisis de PCR para 19 genes de virulencia de E. coli asociados con cepas que provocan enfermedades intestinales, incluida la enfermedad inflamatoria intestinal, detectó 11/39 (28.2 %) aislados que contenían más de 10 de estos genes de virulencia. En conclusión, la necrosis duodenal focal parece ser una enfermedad intestinal inflamatoria multifactorial asociada con disbiosis intestinal, y las bacterias Gramnegativas, incluida E. coli, pueden contribuir a la patogenia de esta enfermedad.

Evaluation of immunogenicity and efficacy of the enterobactin conjugate vaccine in protecting chickens from colibacillosis.

Colibacillosis is one of the most common and economically devastating infectious diseases in poultry production worldwide. Innovative universal vaccines are urgently needed to protect chickens from the infections caused by genetically diverse avian pathogenic Escherichia coli (APEC). Enterobactin (Ent) is a highly conserved siderophore required for E. coli iron acquisition and pathogenesis. The Ent-specific antibodies induced by a novel Ent conjugate vaccine significantly inhibited the in vitro growth of diverse APEC strains. In this study, White Leghorn chickens were immunized with the Ent conjugate vaccine using a crossed design with two variables, vaccination (with or without) and APEC challenge (O1, O78, or PBS control), resulting in six study groups (9 to 10 birds/group). The chickens were subcutaneously injected with the vaccine (100 µg per bird) at 7 days of age, followed by booster immunization at 21 days of age. The chickens were intratracheally challenged with an APEC strain (108 CFU/bird) or PBS at 28 days of age. At 5 days post infection, all chickens were euthanized to examine lesions and APEC colonization of the major organs. Immunization of chickens with the Ent vaccine elicited a strong immune response with a 64-fold increase in the level of Ent-specific IgY in serum. The hypervirulent strain O78 caused extensive lesions in lung, air sac, heart, liver, and spleen with significantly reduced lesion scores observed in the vaccinated chickens. Interestingly, the vaccination did not significantly reduce APEC levels in the examined organs. The APEC O1 with low virulence only caused sporadic lesions in the organs in both vaccination and control groups. The Ent conjugate vaccine altered the bacterial community of the ileum and cecum. Taken together, the findings from this study showed the Ent conjugate vaccine could trigger a strong specific immune response and was promising to confer protection against APEC infection.

Campylobacter hepaticus in the Production Environment and Stagnant Water as a Potential Source of C. hepaticus Causing Spotty Liver Disease in Free-Range Laying Hens in Georgia, United States.

Spotty liver disease (SLD) has emerged as an important cause of disease in egg-producing flocks in countries such as the United Kingdom and Australia and has emerged in the United States. The organisms implicated in SLD include Campylobacter hepaticus and, more recently, Campylobacter bilis. These organisms have been found to cause focal lesions on the livers of infected birds. Campylobacter hepaticus infection results in reduced egg production, decreased feed consumption resulting in reduced egg size, and increased mortality of highly valuable hens. In the fall of 2021, birds from two flocks (A and B) of organic pasture-raised laying hens were submitted to the Poultry Diagnostic Research Center at the University of Georgia with a history suspicious of SLD. Postmortem examination of Flock A found 5/6 hens had small multifocal lesions on the liver and were PCR positive for C. hepaticus from pooled swab analysis of samples of the liver and gall bladder. Necropsy of Flock B found 6/7 submitted birds had spotty liver lesions. In pooled bile swabs, 2/7 hens from Flock B were also PCR positive for C. hepaticus. A follow-up visit to Flock A was scheduled 5 days later, as well as a visit to a flock where SLD has not been reported (Flock C), which was used as a comparative control. Samples of the liver, spleen, cecal tonsil, ceca, blood, and gall bladder were collected from six hens per house. Additionally, feed, water nipples, and environmental water (stagnant water outside the house) were collected from the affected farm and the control farm. To detect the organism, all samples collected were subjected to direct plating on blood agar and enrichment in Preston broth with incubation under microaerophilic conditions. After multiple phases of bacterial culture purification from all samples, single bacterial cultures displaying characteristics of C. hepaticus were tested by PCR to confirm identity. From Flock A, liver, ceca, cecal tonsils, gall bladder, and environmental water were PCR positive for C. hepaticus. No positive samples were detected in Flock C. After another follow-up visit, 10 wk later, Flock A was PCR positive for C. hepaticus from gall bladder bile and feces and one environmental water sample displayed a weak positive reaction for C. hepaticus. Flock C was PCR negative for C. hepaticus. To gain more knowledge about C. hepaticus prevalence, a survey of 6 layer hens from 12 different layer hen flocks between the ages of 7 to 80 wk, raised in different housing systems, were tested for C. hepaticus. The 12 layer hen flocks were culture and PCR negative for C. hepaticus. Currently, there are no approved treatments for C. hepaticus and no vaccine is available. The results of this study suggest that C. hepaticus may be endemic in some areas of the United States, and free-range laying hens may be exposed from the environment/stagnant water in areas where they range.

Campylobacter hepaticus en el ambiente de producción avícola y en el agua estancada como fuente potencial de C. hepaticus que causante de la necrosis hepática focal en gallinas ponedoras de corral en Georgia, Estados Unidos. La necrosis hepática focal (SLD, por sus siglas en inglés) se ha convertido en una causa importante de enfermedad en las parvadas productoras de huevo en países como el Reino Unido y Australia y también ha surgido en los Estados Unidos. Los organismos implicados en necrosis hepática focal incluyen Campylobacter hepaticus y, más recientemente, Campylobacter bilis. Se ha encontrado que estos organismos causan lesiones focales en el hígado de las aves infectadas. La infección por C. hepaticus da como resultado una reducción en la producción de huevos, una disminución en el consumo de alimento, lo que resulta en una reducción del tamaño de los huevos y una mayor mortalidad de gallinas de alto valor económico. En el otoño del 2021, aves de dos lotes (A y B) de gallinas de postura criadas en pastos orgánicos se enviaron al Centro de Diagnóstico e Investigación Avícolas de la Universidad de Georgia con antecedentes sospechosos de necrosis hepática focal. En el examen post mortem de la parvada A se encontró que cinco de un total de seis gallinas tenían pequeñas lesiones multifocales en el hígado y fueron positivas mediante PCR para C. hepaticus a partir de un análisis de hisopos combinados de muestras del hígado y de la vesícula biliar. La necropsia de la parvada B encontró que seis de un total de siete aves enviadas tenían lesiones hepáticas irregulares. En muestras agrupadas de bilis, dos de un total de siete gallinas de la parvada B también fueron positivas a C. hepaticus por PCR. Se programó una visita de seguimiento a la Parvada A cinco días después, así como una visita a una parvada en la que no se había reportado la presencia de necrosis hepática focal (Parvada C), que se utilizó como control para propósitos de comparación. Se recolectaron muestras de hígado, bazo, tonsilas cecales, sacos ciegos, sangre y vesícula biliar de seis gallinas por gallinero. Además, se recolectó alimento, muestras de agua de bebederos de niple y agua ambiental (agua estancada fuera de la casa) de la granja afectada y la granja de control. Para detectar el organismo, todas las muestras recolectadas se sometieron a siembra directa en agar sangre y enriquecimiento en caldo Preston con incubación en condiciones microaerófilas. Después de varias fases de purificación del cultivo bacteriano de todas las muestras, se analizaron mediante PCR los cultivos bacterianos individuales que mostraban características de C. hepaticus para confirmar la identidad. De la parvada A, el hígado, el ciego, las tonsilas cecales, la vesícula biliar y el agua ambiental dieron positivo por PCR para C. hepaticus. No se detectaron muestras positivas en la parvada C. Después una segunda visita de seguimiento, 10 semanas después, la parvada A mostró resultado positivo por PCR para C. hepaticus en la bilis de la vesícula biliar y en las heces, y una muestra de agua ambiental mostró una reacción positiva débil para C. hepaticus . La parvada C resultó negativa mediante PCR para C. hepaticus. Para obtener más conocimiento sobre la prevalencia de C. hepaticus, se realizó un muestreo incluyendo seis gallinas de postura de 12 lotes diferentes de gallinas ponedoras entre las edades de 7 a 80 semanas, criadas en diferentes sistemas de alojamiento, para detectar C. hepaticus. Las doce parvadas de gallinas de postura fueron negativas por cultivo y mediante PCR para C. hepaticus. Actualmente, no hay tratamientos aprobados para C. hepaticus y no hay vacuna disponible. Los resultados de este estudio sugieren que C. hepaticus puede ser endémico en algunas áreas de los Estados Unidos, y las gallinas de postura bajo pastoreo pueden estar expuestas al medio ambiente o al agua estancada en las áreas donde están alojadas.

Prevalence of avian-pathogenic Escherichia coli strain O1 genomic islands among extraintestinal and commensal E. coli isolates.

Escherichia coli strains that cause disease outside the intestine are known as extraintestinal pathogenic E. coli (ExPEC) and include pathogens of humans and animals. Previously, the genome of avian-pathogenic E. coli (APEC) O1:K1:H7 strain O1, from ST95, was sequenced and compared to those of several other E. coli strains, identifying 43 genomic islands. Here, the genomic islands of APEC O1 were compared to those of other sequenced E. coli strains, and the distribution of 81 genes belonging to 12 APEC O1 genomic islands among 828 human and avian ExPEC and commensal E. coli isolates was determined. Multiple islands were highly prevalent among isolates belonging to the O1 and O18 serogroups within phylogenetic group B2, which are implicated in human neonatal meningitis. Because of the extensive genomic similarities between APEC O1 and other human ExPEC strains belonging to the ST95 phylogenetic lineage, its ability to cause disease in a rat model of sepsis and meningitis was assessed. Unlike other ST95 lineage strains, APEC O1 was unable to cause bacteremia or meningitis in the neonatal rat model and was significantly less virulent than uropathogenic E. coli (UPEC) CFT073 in a mouse sepsis model, despite carrying multiple neonatal meningitis E. coli (NMEC) virulence factors and belonging to the ST95 phylogenetic lineage. These results suggest that host adaptation or genome modifications have occurred either in APEC O1 or in highly virulent ExPEC isolates, resulting in differences in pathogenicity. Overall, the genomic islands examined provide targets for further discrimination of the different ExPEC subpathotypes, serogroups, phylogenetic types, and sequence types.

Genotypic and phenotypic traits that distinguish neonatal meningitis-associated Escherichia coli from fecal E. coli isolates of healthy human hosts.

Neonatal meningitis Escherichia coli (NMEC) is one of the top causes of neonatal meningitis worldwide. Here, 85 NMEC and 204 fecal E. coli isolates from healthy humans (HFEC) were compared for possession of traits related to virulence, antimicrobial resistance, and plasmid content. This comparison was done to identify traits that typify NMEC and distinguish it from commensal strains to refine the definition of the NMEC subpathotype, identify traits that might contribute to NMEC pathogenesis, and facilitate choices of NMEC strains for future study. A large number of E. coli strains from both groups were untypeable, with the most common serogroups occurring among NMEC being O18, followed by O83, O7, O12, and O1. NMEC strains were more likely than HFEC strains to be assigned to the B2 phylogenetic group. Few NMEC or HFEC strains were resistant to antimicrobials. Genes that best discriminated between NMEC and HFEC strains and that were present in more than 50% of NMEC isolates were mainly from extraintestinal pathogenic E. coli genomic and plasmid pathogenicity islands. Several of these defining traits had not previously been associated with NMEC pathogenesis, are of unknown function, and are plasmid located. Several genes that had been previously associated with NMEC virulence did not dominate among the NMEC isolates. These data suggest that there is much about NMEC virulence that is unknown and that there are pitfalls to studying single NMEC isolates to represent the entire subpathotype.

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.

Recombinant Iss as a potential vaccine for avian colibacillosis.

Avian pathogenic Escherichia coli (APEC) cause colibacillosis, a disease which is responsible for significant losses in poultry. Control of colibacillosis is problematic due to the restricted availability of relevant antimicrobial agents and to the frequent failure of vaccines to protect against the diverse range of APEC serogroups causing disease in birds. Previously, we reported that the increased serum survival gene (iss) is strongly associated with APEC strains, but not with fecal commensal E. coli in birds, making iss and the outer membrane protein it encodes (Iss) candidate targets for colibacillosis control procedures. Preliminary studies in birds showed that their immunization with Iss fusion proteins protected against challenge with two of the more-commonly occurring APEC serogroups (O2 and O78). Here, the potential of an Iss-based vaccine was further examined by assessing its effectiveness against an additional and widely occurring APEC serogroup (O1) and its ability to evoke both a serum and mucosal antibody response in immunized birds. In addition, tissues of selected birds were subjected to histopathologic examination in an effort to better characterize the protective response afforded by immunization with this vaccine. Iss fusion proteins were administered intramuscularly to four groups of 2-wk-old broiler chickens. At 2 wk postimmunization, chickens were challenged with APEC strains of the O1, O2, or O78 serogroups. One week after challenge, chickens were euthanatized, necropsied, any lesions consistent with colibacillosis were scored, and tissues from these birds were taken aseptically. Sera were collected pre-immunization, postimmunization, and post-challenge, and antibody titers to Iss were determined by enzyme-linked immunosorbent assay (ELISA). Also, air sac washings were collected to determine the mucosal antibody response to Iss by ELISA. During the observation period following challenge, 3/12 nonimmunized chickens, 1/12 chickens immunized with 10 microg of GST-Iss, and 1/12 chickens immunized with 50 microg of GST-Iss died when challenged with the O78 strain. No other deaths occurred. Immunized chickens produced a serum and mucosal antibody response to Iss and had significantly lower lesion scores than nonimmunized chickens following challenge, regardless of the challenge strain. This study expands on our previous report of the value of Iss as an immunoprotective antigen and demonstrates that immunization with Iss can provide significant protection of chickens against challenge with three different E. coli strains.

Associations between multidrug resistance, plasmid content, and virulence potential among extraintestinal pathogenic and commensal Escherichia coli from humans and poultry.

The emergence of plasmid-mediated multidrug resistance (MDR) among enteric bacteria presents a serious challenge to the treatment of bacterial infections in humans and animals. Recent studies suggest that avian Escherichia coli commonly possess the ability to resist multiple antimicrobial agents, and might serve as reservoirs of MDR for human extraintestinal pathogenic Escherichia coli (ExPEC) and commensal E. coli populations. We determined antimicrobial susceptibility profiles for 2202 human and avian E. coli isolates, then sought for associations among resistance profile, plasmid content, virulence factor profile, and phylogenetic group. Avian-source isolates harbored greater proportions of MDR than their human counterparts, and avian ExPEC had higher proportions of MDR than did avian commensal E. coli. MDR was significantly associated with possession of the IncA/C, IncP1-α, IncF, and IncI1 plasmid types. Overall, inferred virulence potential did not correlate with drug susceptibility phenotype. However, certain virulence genes were positively associated with MDR, including ireA, ibeA, fyuA, cvaC, iss, iutA, iha, and afa. According to the total dataset, isolates segregated significantly according to host species and clinical status, thus suggesting that avian and human ExPEC and commensal E. coli represent four distinct populations with limited overlap. These findings suggest that in extraintestinal E. coli, MDR is most commonly associated with plasmids, and that these plasmids are frequently found among avian-source E. coli from poultry production systems.