HPMCP-Coated Microcapsules Containing the Ctx(Ile21)-Ha Antimicrobial Peptide Reduce the Mortality Rate Caused by Resistant Salmonella Enteritidis in Laying Hens.

The constant use of synthetic antibiotics as growth promoters can cause bacterial resistance in chicks. Consequently, the use of these drugs has been restricted in different countries. In recent years, antimicrobial peptides have gained relevance due to their minimal capacity for bacterial resistance and does not generate toxic residues that harm the environment and human health. In this study, a Ctx(Ile21)-Ha antimicrobial peptide was employed, due to its previously reported great antimicrobial potential, to evaluate its application effects in laying chicks challenged with Salmonella Enteritidis, resistant to nalidixic acid and spectinomycin. For this, Ctx(Ile21)-Ha was synthesized, microencapsulated and coated with hypromellose phthalate (HPMCP) to be released in the intestine. Two different doses (20 and 40 mg of Ctx(Ile21)-Ha per kg of isoproteic and isoenergetic poultry feed) were included in the chick's food and administered for 28 days. Antimicrobial activity, effect and response as treatment were evaluated. Statistical results were analyzed in detail and indicate that the formulated Ctx(Ile21)-Ha peptide had a positive and significant effect in relation to the reduction of chick mortality in the first days of life. However, there was moderate evidence (p = 0.07), not considered statistically significant, in the differences in laying chick weight between the control and microencapsulation treatment groups as a function of time. Therefore, the microencapsulated Ctx(Ile21)-Ha antimicrobial peptide can be an interesting and promising option in the substitution of conventional antibiotics.

Alginate-based microparticles coated with HPMCP/AS cellulose-derivatives enable the Ctx(Ile21)-Ha antimicrobial peptide application as a feed additive.

Microencapsulation is a potential biotechnological tool, which can overcome antimicrobial peptides (AMP) instabilities and reduce toxic side effects. Thus, this study evaluates the antibacterial activities of the Ctx(Ile21)-Ha AMP against multidrug-resistant (MDR) and non-resistant bacteria and develop and characterize peptide-loaded microparticles coated with the enteric polymers hydroxypropylmethylcellulose acetate succinate (HPMCAS) and hydroxypropylmethylcellulose phthalate (HPMCP). Ctx(Ile21)-Ha was obtained by solid phase peptide synthesis (SPPS) method, purified and characterized by HPLC and Mass Spectrometry. The peptide exhibited potent antibiotic activities against Salmonella enteritidis, Salmonella typhimurium, Pseudomonas aeruginosa (MDR), Acinetobacter baumannii (MDR), and Staphylococcus aureus (MDR). Ctx(Ile21)-Ha microencapsulation was performed by ionic gelation with high efficiency, maintaining the physical-chemical stability. Ctx(Ile21)-Ha coated-microparticles were characterized by DSC, TGA, FTIR-Raman, XRD and SEM. Hemolytic activity assay demonstrated that hemolysis was decreased up to 95% compared to single molecule. In addition, in vitro release control profile simulating different portions of gastrointestinal tract was performed and showed the microcapsules' ability to protect the peptide and release it in the intestine, aiming pathogen's location, mainly by Salmonella sp. Therefore, use of microencapsulated Ctx(Ile21)-Ha can be allowed as an antimicrobial controller in monogastric animal production as an oral feed additive (antimicrobial controller), being a valuable option for molecules with low therapeutic indexes or high hemolytic rates.

Characterization of avian pathogenic Escherichia coli isolated from free-range helmeted guineafowl.

Avian pathogenic Escherichia coli (APEC) isolates from apparently healthy free range helmeted guineafowl were characterized. Most of them had a high frequency of virulence associated genes, multi drug resistance and high pathogenicity. We demonstrated that helmeted guineafowl have potential to transmit antibiotic resistant APEC to other species including humans.

Characterization of avian pathogenic Escherichia coli isolated from free-range helmeted guineafowl

Abstract Avian pathogenic Escherichia coli (APEC) isolates from apparently healthy free range helmeted guineafowl were characterized. Most of them had a high frequency of virulence associated genes, multi drug resistance and high pathogenicity. We demonstrated that helmeted guineafowl have potential to transmit antibiotic resistant APEC to other species including humans.

In-vivo evaluation of pathogenicity and antimicrobial profile susceptibility from Escherichia coli samples isolated from commercial layer hens / Avaliação da patogenicidade in vivo e do perfil de resistência antimicrobiana de amostras de Escherichia coli isoladas de galinhas de postura comercial

Antimicrobial sensitivity and pathogenicity level of 90 strains of Escherichia coli isolated from livers and intestines from commercial layer hens presenting diarrhea were analyzed. To evaluate the antimicrobial susceptibility, all samples were subjected to antimicrobial susceptibility testing using 11 commercial drugs. The results have showed none of the strains was susceptible to all antibiotics tested. All samples showed resistance to two or more drugs. According to the mortality rate of the birds, the in-vivo pathogenicity test classifies the strains into four classes: high, intermediate, low and nonpathogenic. The test has showed 23 (25.5%) of the samples were highly pathogenic, 21 (23.3%) of intermediate pathogenicity, 23 (25.5%) low pathogenic, and 23 (25.5%) nonpathogenic. When the results of the classes of pathogenicity from isolates have been associated with antimicrobial susceptibility, nonpathogenic strains were less sensitive to the antibiotic ampicillin and increased sensitive to streptomycin antimicrobial compared to the others classes of pathogenic. Nonpathogenic strains showed resistance to many antimicrobials, an alert for poultry, since these bacteria might acquire the virulence genes and infect birds, others animals and even human beings.(AU)

Foram verificados a sensibilidade antimicrobiana e o índice de patogenicidade de 90 amostras de Escherichia coli isoladas do fígado e do intestino de pintainhas de postura comercial com diarreia. Para avaliar a sensibilidade antimicrobiana, todas as amostras foram submetidas ao teste de susceptibilidade antimicrobiana por meio de 11 drogas comerciais. Os resultados demonstraram que nenhuma das estirpes foi sensível a todos os antimicrobianos testados. Todas as amostras apontaram resistência a duas ou mais drogas. De acordo com o índice de mortalidade das aves, o teste de patogenicidade in vivo classificou as estirpes em quatro classes: alta, intermediária, baixa e não patogênica. O teste revelou que 23 (25,5%) das amostras foram de alta patogenicidade, 21 (23,3%) de patogenicidade intermediária, 23 (25,5%) de baixa patogenicidade e 23 (25,5%) não patogênicas. Quando os resultados das classes de patogenicidade dos isolados foram associados à sensibilidade antimicrobiana, estirpes não patogênicas apresentaram menor sensibilidade ao antimicrobiano ampicilina e maior sensibilidade ao antimicrobiano estreptomicina, quando comparadas com as estirpes das demais classes de patogenicidade. Estirpes não patogênicas exibiram resistência a vários antimicrobianos, representando um alerta para a avicultura, uma vez que essas bactérias podem adquirir genes de virulência e, assim, infectar aves, outros animais e até mesmo o seres humanos.(AU)

Characterization of avian pathogenic Escherichia coli isolated from free-range helmeted guineafowl

Abstract Avian pathogenic Escherichia coli (APEC) isolates from apparently healthy free range helmeted guineafowl were characterized. Most of them had a high frequency of virulence associated genes, multi drug resistance and high pathogenicity. We demonstrated that helmeted guineafowl have potential to transmit antibiotic resistant APEC to other species including humans.

Variants of astA gene among extra-intestinal Escherichia coli of human and avian origin.

Many Escherichia coli strains harbour astA, which is the gene encoding the enteroaggregative E. coli heat-stable enterotoxin (EAST1). This gene is embedded in a putative transposase (ORF1) and presents polymorphism in diarrheagenic strains. Although astA and orf1 are detected in extraintestinal strains, little is known about polymorphism and differential gene transcription in this pathotype. In the present work, extraintestinal E. coli from humans (ExPEC - Extraintestinal Pathogenic E. coli) and poultry (APEC - Avian Pathogenic E. coli) were assayed to verify the presence of astA/orf1 and possible polymorphisms in these genes. Three astA/orf1 patterns were detected via Sanger sequencing. Pattern 1 was novel and represented an astA pseudogene. Pattern 2 and pattern 3 presented distinct amino acids within the reading frame encoding astA and were identical to the sequences found in EAEC 17-2 and EAEC 042, respectively. Regarding the frame encoding ORF1, all mutations detected in the three patterns were neutral. The transcripts of astA/orf1 in vitro were underregulated in strains possessing the pattern 1 sequence. The results demonstrate that the same astA sequences may be detected in diarrheagenic and extra-intestinal E. coli. However, extraintestinal isolates may also present an astA pseudogene that has not been reported in diarrheagenic E. coli.

Overlapped sequence types (STs) and serogroups of avian pathogenic (APEC) and human extra-intestinal pathogenic (ExPEC) Escherichia coli isolated in Brazil.

Avian pathogenic Escherichia coli (APEC) strains belong to a category that is associated with colibacillosis, a serious illness in the poultry industry worldwide. Additionally, some APEC groups have recently been described as potential zoonotic agents. In this work, we compared APEC strains with extraintestinal pathogenic E. coli (ExPEC) strains isolated from clinical cases of humans with extra-intestinal diseases such as urinary tract infections (UTI) and bacteremia. PCR results showed that genes usually found in the ColV plasmid (tsh, iucA, iss, and hlyF) were associated with APEC strains while fyuA, irp-2, fepC sitDchrom, fimH, crl, csgA, afa, iha, sat, hlyA, hra, cnf1, kpsMTII, clpVSakai and malX were associated with human ExPEC. Both categories shared nine serogroups (O2, O6, O7, O8, O11, O19, O25, O73 and O153) and seven sequence types (ST10, ST88, ST93, ST117, ST131, ST155, ST359, ST648 and ST1011). Interestingly, ST95, which is associated with the zoonotic potential of APEC and is spread in avian E. coli of North America and Europe, was not detected among 76 APEC strains. When the strains were clustered based on the presence of virulence genes, most ExPEC strains (71.7%) were contained in one cluster while most APEC strains (63.2%) segregated to another. In general, the strains showed distinct genetic and fingerprint patterns, but avian and human strains of ST359, or ST23 clonal complex (CC), presented more than 70% of similarity by PFGE. The results demonstrate that some "zoonotic-related" STs (ST117, ST131, ST10CC, ST23CC) are present in Brazil. Also, the presence of moderate fingerprint similarities between ST359 E. coli of avian and human origin indicates that strains of this ST are candidates for having zoonotic potential.