Efficacy of ceftiofur N-acyl homoserine lactonase niosome in the treatment of multi-resistant Klebsiella pneumoniae in broilers.

In this study, the efficiency of the ceftiofur N-acyl homoserine lactonase niosome against multi-resistant Klebsiella pneumoniae in broilers was evaluated. Fifty-six K. pneumoniae isolates previously recovered from different poultry and environmental samples were screened for the ahlK gene. The lactonase enzyme was extracted from eight quorum-quenching isolates. The niosome was formulated, characterized, and tested for minimal inhibitory concentration (MIC) and cytotoxicity. Fourteen-day-old chicks were assigned to six groups: groups Ӏ and П served as negative and positive controls, receiving saline and K. pneumoniae solutions, respectively. In groups Ш and IV, ceftiofur and niosome were administrated intramuscularly at a dose of 10 mg/kg body weight for five consecutive days, while groups V and VI received the injections following the K. pneumoniae challenge. Signs, mortality, and gross lesions were recorded. Tracheal swabs were collected from groups П, V, and VI for counting K. pneumoniae. Pharmacokinetic parameters were evaluated in four treated groups at nine-time points. The niosome was spherical and 56.5 ± 4.41 nm in size. The viability of Vero cells was unaffected up to 5 × MIC (2.4 gml-1). The niosome-treated challenged group showed mild signs and lesions with lower mortality and colony count than the positive control group. The maximum ceftiofur serum concentrations in treated groups were observed 2 h following administration. The elimination half-life in niosome-treated groups was longer than that reported in ceftiofur-treated groups. This is the first report of the administration of N-acyl homoserine lactonase for the control of multi-resistant K. pneumoniae infections in poultry.

Detection of Quorum Sensing N-Acyl-Homoserine Lactone Molecules Produced by Different Resistant Klebsiella pneumoniae Isolates Recovered from Poultry and Different Environmental Niches.

This study aimed to detect and identify the N-acyl-homoserine lactones molecules (AHLs) produced by different resistant Klebsiella pneumoniae isolates recovered from poultry and environmental samples using a modified validated high-performance liquid chromatography method. A total of 56 K. pneumoniae isolates were recovered, investigated for their antibiotic susceptibility, and screened for AHLs production using the Agrobacterium tumefaciens NTL4 biosensor system and a validated high-performance liquid chromatography method. The results revealed the detection of different short- and long-chain AHLs molecules among 39 K. pneumoniae isolates recovered from poultry and environmental samples. All environmental isolates produced nine peaks with retention times for C4-HSL, C6-HSL, C12-HSL, C8-HSL, C14-HSL, C8-oxo-HSL, C10-HSL, C6-oxo-HSL, and C7-HSL. The most quantifiable AHL signal molecules in poultry isolates were C4-HSL, C6-HSL, and C12-HSL. No statistical correlation between the AHL-producing ability of K. pneumoniae isolates and antibiotic resistance was reported. To the best of our knowledge, this study provides the first detailed report on the detection and identification of AHLs in K. pneumoniae isolates recovered from poultry and environmental samples. Furthermore, it provides a new insight available tool other than LC-MS/MS for detection and identification of AHL molecules.

Evaluation of a modified method of extraction, purification, and characterization of lipopolysaccharide (O antigen) from Salmonella Typhimurium.

BACKGROUND AND AIM: Lipopolysaccharide (LPS) is an integral part of the outer cell membrane complex of Gram-negative bacteria. It plays an important role in the induction and stimulation of the immune system. Various LPS purification protocols have been developed. However, analysis of their efficacy is limited by contamination during downstream applications or the public health hazard of LPS. The aim of this study was to evaluate a modified method for extracting LPS as well as assess the purity of the extracted LPS by high-performance liquid chromatography (HPLC) analysis. Further, we evaluated its immunopotentiating function by measuring the relative RNA expression levels of splenic immune-related genes such as interleukin 1ß (IL-1ß) and interferon-γ (IFN-γ), after intramuscular injection of increasing concentrations of the extracted LPS in specific pathogen-free (SPF) chick. MATERIALS AND METHODS: Isolation, identification, and serotyping of Salmonella Typhimurium were performed using chicken flocks. We then performed molecular typing of Salmonella isolates using conventional polymerase chain reaction (PCR). A new protocol for purification of LPS from Salmonella isolate (S. Typhimurium) was conducted. HPLC analysis of the extracted LPS in the current study was compared to existing methods. An in vivo study was performed to evaluate the ability of LPS to induce an immune response by measuring relative IFN-γ and IL-1ß gene expression after injecting increasing concentrations of the extracted LPS into SPF chicks. RESULTS: Isolation and serotyping revealed that Salmonella enterica was of the serovar Typhimurium. Confirmation was conducted by molecular typing through conventional PCR. Fractionation of the LPS extract by HPLC revealed a high degree of purity comparable with standard commercial LPS. These results demonstrate the high purity of extracted LPS based on our modified method using propanol and sodium hydroxide mixture. Intramuscular injection of the extracted LPS in 22 day-old SPF chicks, compared to the negative control, revealed significant upregulation of IFN-γ and slight downregulation of IL-1ß. CONCLUSION: The new modified method can be used for high purity LPS extraction and demonstrates effective immunopotentiating activity.

Prophylactic impact of nano-selenium on performance, carcasses quality, and tissues' selenium concentration using reversed-phase high-performance liquid chromatography during microbial challenge in broiler chickens.

BACKGROUND AND AIM: Nano-selenium (NS) supplementation contributes in improving productivity, performance, and meat quality while reducing public health concern. Influence of NS and inorganic selenium (Se) water additive on performance, carcass quality, immunoglobulin concentration, intestinal microbiota, Se tissue concentrations, and tissue architecture was studied. MATERIALS AND METHODS: Two-hundred and sixty 1-day-old Hubbard chicks were randomly grouped into five groups (5×52) and supplemented with 0.5 and 1.0 mL of NS and inorganic Se (100 mg.L-1). G1, G2, G3, and G4 were challenged with Escherichia coli O157: H7 2.6×108 on the 14th day. A total of 2250 samples, including 250 sera, 250 intestinal swabs, and 1500 organ and tissue samples as liver, spleen, heart, bursa, intestine, and breast muscles, and 250 eviscerated carcasses were collected. RESULTS: The results revealed a highly significant increase (p<0.01) in live body weights, weight gains, performance indices, carcasses, and organs weights, whereas immunoglobulin G and M concentrations in broilers treated with 0.5 and 1.0 mL NS, respectively, synchronized reveal a highly significant decline (p<0.01) in total bacterial and Enterobacteriaceae counts of intestinal swabs and breast muscles, final pH24, and drip loss in broilers treated with 0.5 and 1.0 mL NS, respectively. Meanwhile, water holding capacity revealed no significant differences between all groups. Reversed-phase high-performance liquid chromatography examination revealed the earlier disappearance of NS residues than inorganic Se from the broiler's liver and muscles. Histopathological photomicrographs of the liver, spleen, bursa of Fabricius, and intestine, as well as, the immunohistochemistry of intestinal sections revealed superior tissue architecture in broilers treated with NS contrary to inorganic Se. CONCLUSION: The study showed significant stimulation actions of NS on performance, immunity, carcass and meat quality, intestinal and muscles' bacterial load as well as short withdrawal period and nearly normal cellular architecture compared to inorganic Se.