Effects of Dietary Bioactive Lipid Compounds of Acacia nilotica Bark on Productive Performance, Antioxidant Status, and Antimicrobial Activities of Growing Rabbits under Hot Climatic Conditions.

This study aimed to evaluate the efficacy of dietary Acacia nilotica bark bioactive lipid compounds (ANBBLCs) as novel feed additives on the growth performance, carcass criteria, antioxidants, and antimicrobial activities of growing male rabbits. A total of 100 California male weanling rabbits aged 35 days were divided into four nutritional treatments, each of which contained ANBBLCs at concentrations of 0 (control group), 50, 100, and 150 mg/kg diet (n = 25 per treatment, each replication consisting of one animal). The average body weight of the animals was 613 ± 14 g. The experiments lasted for 56 days. Dietary ANBBLC levels linearly improved (p < 0.05) the body weight, body weight gain, and feed conversion ratio (FCR) of rabbits. Furthermore, with increasing concentrations of ANBBLCs, the total antioxidant capacity of blood and liver tissue was linearly (p < 0.05) enhanced. Lactobacillus increased and Staphylococcus decreased (p < 0.05) in comparison to the control group when ANBBLC levels were added to the diets of rabbits. Rabbit diets supplemented with ANBBLCs increased dressing percentages and decreased abdominal fat. This study shows that ANBBLCs can be used as a feed additive to enhance the growth performance, carcass criteria, antioxidant, and antibacterial properties of growing rabbits.

Effect of substituting hydroponic barley forage with or without enzymes on performance of growing rabbits.

This study aims to evaluate the effect of hydroponic barley (HB) by substituting control diet with 25% HB with or without enzymes on rabbit performance, nutrient digestibility, and economic efficiency. A total number of 60 growing male HyPlus rabbits (average body weight 669 ± 12 g, 30 days of age) were utilized in the present study. The rabbits were randomly assigned to three groups (n = 20 rabbits per group). The first group served as a control (C). The other two groups were fed the control diet substituted with 25% hydroponic barley HB (group CHB), and the control diet substituted with 25% HB added with 0.5 g/kg enzymes (CHBE). The experiment lasted for 56 days. The results revealed that daily body weight gain improved (P < 0.05) by 18.64% and 23.94%, and feed conversion ratio improved by 3.74% and 17.91% than control, respectively, during 30-86 days of age in CHB and CHBE groups. The economic efficiency was improved (P < 0.05) by 32.17% and 39.60% in CHB and CHBE diets, respectively, compared to control; and nutrient digestibility, and mineral retention of growing rabbits were also improved (P < 0.05) by substituting HB with or without enzymes compared to control diet. Overall, the best rabbit performances were observed in both CHB and CHBE groups. In conclusion, these results suggest that substituting 25% of concentrated control diet by hydroponic barley with or without enzymes have positive effects in a sustainable way on growth performance, nutrient digestibility, and economic efficiency of growing rabbits.

Productive, physiological and nutritional responses of laying hens fed different dietary levels of turmeric powder.

This study aimed to determine the impact of supplementation of turmeric powder on laying performance, egg quality, nutrient digestibility and some blood metabolites in laying hens. A total of one hundred and twenty Bovans Brown laying hens (55 weeks old) were assigned to one of four treatment diets (n = 30) for 12 weeks including turmeric powder at 0, 2.5, 5 or 7.5 g/kg respectively. The results revealed that egg production, egg weight and egg mass were significantly increased (p < 0.05), and the feed conversion ratio was significantly improved (p < 0.012) with increasing levels of turmeric in the laying hen diet. Egg thickness and Haugh unit were linearly increased (p < 0.01) with increasing supplementation levels. Moreover, compared with the control diet, the levels of turmeric powder supplementation significantly improved nutrient digestibility (p < 0.001). Moreover, the serum metabolic profile revealed that serum total cholesterol, aspartate aminotransferase, creatinine and urea concentrations were linearly decreased with increasing turmeric powder supplementation to hen diets. In conclusion, the inclusion of turmeric powder at 2.5, 5 or 7.5 g/kg diet improved egg production, nutrient digestibility, egg quality and serum metabolic profile and may be used as a feed additive in laying hens' nutrition. However, results indicate that the best improvement was observed when 5 g/kg were supplemented.

Biotechnology methods for succession of bacterial communities in polychlorinated biphenyls (PCBs) contaminated soils and isolation novel PCBs-degrading bacteria.

Polychlorinated Biphenyls (PCBs) are persistence in the contaminated sites as a result of lacking PCBs-degrading microorganisms. Cultivation-independent technique called single-strand-conformation polymorphism (SSCP) based on 16SrRNA genes was chosen to characterize the diversity of bacterial communities in PCBs polluted soil samples. The bacterial communities showed an increasing diversity from the genetic profiles using SSCP technique. 51 single products were identified from the profiles using PCR reamplification and cloning. DNA sequencing of the 51 products, it showed similarities to Acidobacteria, Actinobacteria, Betaproteobateria, Gammaproteobacteria and Alphaproteobacteria, the range of similarities were 92.3 to 100%. Pure 23 isolates were identified from PCBs contaminated sites. The identified isolates belonged to genus Bacillus, Brevibacillus, Burkholderia, Pandoraea, Pseudomonas, and Rhodococcus. The new strains have the capability to use PCBs as a source of sole carbon and harbor 2,3-dihydroxybiphenyl dioxygenase (DHBDO) which could be used as molecular marker for detection PCBs-degrading bacteria in the PCBs contaminated sites. This finding may enhance the PCBs bioremediation by monitoring and characterization of the PCBs degraders using DHBDO in PCBs contaminated sites.

Characterization and Expression Analysis of Extradiol and Intradiol Dioxygenase of Phenol-Degrading Haloalkaliphilic Bacterial Isolates.

Haloalkophilic bacteria have a potential advantage as a bioremediation organism of high oil-polluted and industrial wastewater. In the current study, Haloalkaliphilic isolates were obtained from Hamralake, Wadi EL-Natrun, Egypt. The phenotype script, biochemical characters, and sequence analysis of bacterial-16S rRNA were used to identify the bacterial isolates; Halomonas HA1 and Marinobacter HA2. These strains required high concentrations of NaCl to ensure bacterial growth, especially Halomonas HA1 strain. Notably, both isolates can degrade phenol at optimal pH values, between 8 and 9, with the ability to grow in pH levels up to 11, like what was seen in the Halomonas HA1 strain. Moreover, both isolates represent two different mechanistic pathways for phenol degradation. Halomonas HA1 exploits the 1,2 phenol meta-cleavage pathway, while Marinobacter HA2 uses the 2,3 ortho-cleavage pathway as indicated by universal primers for 1,2 and 2,3 CTD genes. Interestingly, Marinobacter HA2 isolate eliminated the added phenol within an incubation period of 72 h, while the Halomonas HA1 isolate invested 96 h in degrading 84% of the same amount of phenol. Phylogenetic analysis of these 1,2 CTD (catechol dioxygenase) sequences clearly showed an evolutionary relationship between 1,2 dioxygenases of both Halomonadaceae and Pseudomonadaceae. In comparison, 2,3 CTD of Marinobacter HA2 shared the main domains of the closely related species. Furthermore, semi-quantitative RT-PCR analysis proved the constitutive expression pattern of both dioxygenase genes. These findings provide new isolates of Halomonas sp. and Marinobacter sp. that can degrade phenol at high salt and pH conditions via two independent mechanisms.

Biochemical and genetic characterization comparison of four extradiol dioxygenases in Rhizorhabdus wittichii RW1.

Rhizorhabdus (previously Sphingomonas) wittichii RW1 uses a diverse array of aromatic organic compounds as energy and carbon sources, including some extremely recalcitrant compounds such as dibenzo-p-dioxin and dibenzofuran. Extradiol dioxygenases play a key role in the metabolism of dibenzofuran (DBF), dibenzo-p-dioxin (DBD), PCBs, and various other aromatic compounds. In this study, a detailed kinetic analysis of four extradiol dioxygenases identified in R. wittichii RW1 (DbfB, Edo2, Edo3, and Edo4) showed all of them to be typical 2,3dihydroxybiphenyl (DHB) dioxygenases with DHB as preferred substrate (kcat/Km values of 0.13-188 (µM -1 s-1)) and only slightly lower activity against trihydroxybiphenyl (THB) whereas monocyclic substrates were, to different extents, poor substrates due to high km values. All extradiol dioxygenases analyzed were subject to mechanism-based inactivation by 2,2`,3-trihydroxybiphenylether (THBE) the intermediate of DBD degradation. However, Edo4 was superior as reflected by the relatively high partition ratio and the comparably low efficiency of inactivation. Significant differences were observed with respect to their inactivation by 3-chlorocatechol. The absence of any significant mechanism-based inactivation makes Edo3 a perfect candidate for being recruited for chlorobiphenyl degradation where inactivation of extradiol dioxygenases by this intermediate creates significant metabolic problems. KEY POINTS: • Characterization of additional extradiol dioxygenases encoded by RW1 • Identification of differences in 2,2`,3-trihydroxybiphenylether transformation • Identification of differences in inhibition by 3-chlorocatechol.

Isolation and Characterization a Novel Catabolic Gene Cluster Involved in Chlorobenzene Degradation in Haloalkaliphilic Alcanivorax sp. HA03.

Chlorobenzene (CB) poses a serious risk to human health and the environment, and because of its low degradation rate by microorganisms, it persists in the environment. Some bacterial strains can use CB as growth substrates and their degradative pathways have evolved; very little is known about these pathways and the enzymes for CB degradation in high pH and salinity environments. Alcanivorax sp. HA03 was isolated from the extremely saline and alkaline site. HA03 has the capability to degrade benzene, toluene and chlorobenzene (CB). CB catabolic genes were isolated from HA03, which have a complete gene cluster comprising α and ß subunits, ferredoxin and ferredoxin reductase (CBA1A2A3A4), as well as one gene-encoding enzyme for chlorocatechol 1,2-dioxygenase (CC12DOs). Based on the deduced amino acid sequence homology, the gene cluster was thought to be responsible for the upper and lower catabolic pathways of CB degradation. The CBA1A2A3A4 genes probably encoding a chlorobenzene dioxygenase was confirmed by expression during the growth on CB by RT-PCR. Heterologous expression revealed that CBA1A2A3A4 exhibited activity for CB transformation into 3-chlorocatechol, while CC12DOs catalyze 3-chlorocatechol, transforming it into 2-chloromucounate. SDS-PAGE analysis indicated that the sizes of CbA1 and (CC12DOs) gene products were 51.8, 27.5 kDa, respectively. Thus, Alcanivorax sp. HA03 constitutes the first bacterial strain described in the metabolic pathway of CB degradation under high pH and salinity conditions. This finding may have obvious potential for the bioremediation of CB in both highly saline and alkaline contaminated sites.

Loads of Coliforms and Fecal Coliforms and Characterization of Thermotolerant Escherichia coli in Fresh Raw Milk Cheese.

The aim of this study was to assess the hygienic status of raw milk cheese and determine the trends of virulence and antimicrobial resistance in thermotolerant Escherichia coli. Two hundred samples of karish, a popular Egyptian fresh raw milk cheese, were analyzed for coliforms and fecal coliforms using a standard most probable number (MPN) technique. Overall, 85% of samples were unsuitable for consumption, as they exceeded Egyptian standards for coliforms (10 MPN/g), and 65% of samples exhibited coliforms at 44.5 °C. Of 150 recovered thermotolerant strains, 140 (93.3%) were identified as E. coli. Importantly, one Shiga toxin-producing E. coli (STEC) strain carrying a striking virulence pattern, stx1-, stx2+, eae-, was detected. Eleven strains (7.8%, 11/140) showed resistance to third-generation cephalosporins. Antibiotic resistance genes included blaSHV, blaCTX-M, qnrS, tet(A), and tet(B), which were present in 4.3%, 2.8%, 0.71%, 2.1%, and 0.71% of isolates, respectively. In conclusion, this study indicated that hygienic-sanitary failures occurred throughout the production process of most retail karish cheese. Furthermore, our findings emphasize the need for adopting third-generation cephalosporin-resistant E. coli as an indicator for monitoring antimicrobial resistance in raw milk cheese to identify the potential public health burden associated with its consumption.

Occurrence, Phenotypic and Molecular Characteristics of Vancomycin-Resistant Enterococci Isolated from Retail Raw Milk in Egypt.

The aim of this study was to determine the occurrence, phenotypic and molecular characteristics of vancomycin-resistant enterococci (VRE), isolated from retail raw cow's milk. One hundred milk samples collected from retail shops in Egypt were examined for the occurrence of VRE by using kanamycin aesculin azide agar supplemented with 4 µg/mL vancomycin. PCR was conducted to determine enterococcal species and to screen the isolated strains for the presence of antibiotic resistance and virulence genes. All isolated strains were characterized by antimicrobial susceptibility testing for 12 antibiotics. From 24 samples (24%), we recovered 22 isolates (91.6%) classified as VRE (minimum inhibitory concentration ≥32) and 2 isolates (8.3%) classified as intermediate resistant to vancomycin (≤16). Enterococcus faecium (29.1%), Enterococcus faecalis (12.5%), Enterococcus casseliflavus (16.6%), and Enterococcus gallinarum (4.1%) were identified by using multiplex PCR. The genus Enterococcus was resistant to clindamycin (100%), linezolid (91.6%), teicoplanin (91.6%), erythromycin (87.5%), and tetracycline (29.1%). Co-resistance to vancomycin, teicoplanin, and linezolid was detected in 83.3% of isolates. Antibiotic resistance genes vanB, tet(M), tet(L), and erm(B) were identified in 29.1%, 16.6%, 8.3%, and 4.1% of isolates, respectively. Virulence genes gelE and esp were detected in 16.6% and 12.5% of isolates, respectively. In conclusion, the high occurrence of co-resistance to vancomycin, teicoplanin, and linezolid reported in this study is alarming. The high frequency of linezolid resistance prompts increased the attention of researchers to routinely perform linezolid susceptibility in food isolates. This study declares potential food safety risks from consumption and improper handling of raw milk regarding clinically important bacteria and promotes necessary legislation for forbidding the selling and consumption of retail raw milk.

Monitorización de la capacidad de degradación de las nuevas bacterias haloalcalifílicas resistentes a cloruro de tributiltina (TBTCl) en una ubicación contaminada por butiltina / Monitoring the degradation capability of novel haloalkaliphilic tributyltin chloride (TBTCl) resistant bacteria from butyltin-polluted site

Tributyltin (TBT) is recognized as a major environmental problem at a global scale. Haloalkaliphilic tributyltin (TBT)-degrading bacteria may be a key factor in the remediation of TBT polluted sites. In this work, three haloalkaliphilic bacteria strains were isolated from a TBT-contaminated site in the Mediterranean Sea. After analysis of the 16S rRNA gene sequences the isolates were identified as Sphingobium sp. HS1, Stenotrophomonas chelatiphaga HS2 and Rhizobium borbori HS5. The optimal growth conditions for biodegradation of TBT by the three strains were pH 9 and 7% (w/v) salt concentration. S. chelatiphaga HS2 was the most effective TBT degrader and has the ability to transform most TBT into dibutyltin and monobutyltin (DBT and MBT). A gene was amplified from strain HS2 and identified as TBTB-permease-like, that encodes an ArsB-permease. A reverse transcription polymerase chain reaction analysis in the HS2 strain confirmed that the TBTB-permease-like gene contributes to TBT resistance. The three novel haloalkaliphilic TBT degraders have never been reported previously.

Se considera a la tributiltina (TBT) como un problema medioambiental serio a escala global. Las bacterias haloalcalifílicas degradadoras de TBT pueden constituir un factor clave para remediar áreas contaminadas con dicho xenobiótico. En este estudio se aislaron 3 cepas de bacterias haloalcalifílicas procedentes de un sitio contaminado con TBT en el mar Mediterráneo. Tras analizar las secuencias del gen de 16S del ARNr, se identificaron los aislados como Sphingo-bium sp. HS1, Stenotrophomonas chelatiphaga HS2 y Rhizobium borbori HS5. Las condiciones de crecimiento óptimas para la biodegradación de TBT por parte de las 3 cepas fueron pH 9 y 7% (p/v) de concentración de sal. S. chelatiphaga HS2 fue el degradador de TBT más efectivo, con capacidad de transformar la mayor parte de ese compuesto en dibutiltina y monobutiltina (DBT y MBT). Se amplificó un gen de la cepa HS2, que fue identificado como tipo TBTB-permeasa, que codifica para una ArsB permeasa. Un análisis de la cepa HS2 por reacción en cadena de la polimerasa con transcriptasa inversa (RT PCR) confirmó que el gen TBTB-permeasa contribuye a la resistencia al TBT. Estos 3 nuevos degradadores haloalcalifílicos de TBT no habían sido reportados previamente.

Monitoring the degradation capability of novel haloalkaliphilic tributyltin chloride (TBTCl) resistant bacteria from butyltin-polluted site.

Tributyltin (TBT) is recognized as a major environmental problem at a global scale. Haloalkaliphilic tributyltin (TBT)-degrading bacteria may be a key factor in the remediation of TBT polluted sites. In this work, three haloalkaliphilic bacteria strains were isolated from a TBT-contaminated site in the Mediterranean Sea. After analysis of the 16S rRNA gene sequences the isolates were identified as Sphingobium sp. HS1, Stenotrophomonas chelatiphaga HS2 and Rhizobium borbori HS5. The optimal growth conditions for biodegradation of TBT by the three strains were pH 9 and 7% (w/v) salt concentration. S. chelatiphaga HS2 was the most effective TBT degrader and has the ability to transform most TBT into dibutyltin and monobutyltin (DBT and MBT). A gene was amplified from strain HS2 and identified as TBTB-permease-like, that encodes an ArsB-permease. A reverse transcription polymerase chain reaction analysis in the HS2 strain confirmed that the TBTB-permease-like gene contributes to TBT resistance. The three novel haloalkaliphilic TBT degraders have never been reported previously.

Isolation and characterization of three novel catechol 2,3-dioxygenase from three novel haloalkaliphilic BTEX-degrading Pseudomonas strains.

Benzene, toluene, ethylbenzene, and xylene (BTEX) are highly water soluble, hence can contaminate a large volume of groundwater and soil, exhibiting a serious negative impact on human health. To get efficient biodegradation and bioremediation of BTEX in the highly salt and pH contaminated sites, this study captured, investigated and identified three novel haloalkaliphilic bacterial strains HA10, HA12 and HA14 belong to genus Pseudomonas that have strong capability to degrade BTEX at 7% NaCl (w/v) and pH 9. Study of enzymes in halophiles will help understanding the mechanism of BTEX degradation in saline and alkaline environments. Three novel catechol 2,3-dioxygenase genes C23O10, C23O12 and C23O14 were amplified, cloned and overexpressed from the three obtained haloalkaliphilic strains HA10, HA12 and HA14 respectively. Phylogenetic tree analysis for the three novel C23Os and their relatives formed a new branch. C23O12 and C23O14 showed activity with only catechol, while the activity was observed in C23O10 on catechol and 2,3-dihydroxybiphenyl. Kinetic properties analysis for C23O10 indicated that its preferred substrates were catechol and 2,3-Dihydroxybiphenyl. C23O10 activity severely affected and rapidly inactivated by 3-Chlorocatechol. This finding may be necessary for developing in-site bioremediation of BTEX contaminated sites in both highly saline and alkaline environments.

SugE belongs to the small multidrug resistance (SMR) protein family involved in tributyltin (TBT) biodegradation and bioremediation by alkaliphilic Stenotrophomonas chelatiphaga HS2.

Tributyltin (TBT) used in a variety of industrial processes, subsequent discharge into the environment, its fate, toxicity and human exposure are topics of current concern. TBT degradation by alkaliphilic bacteria may be a key factor in the remediation of TBT in high pH contaminated sites. In this study, Stenotrophomonas chelatiphaga HS2 were isolated and identified from TBT contaminated site in Mediterranean Sea. S. chelatiphaga HS2 has vigor capability to transform TBT into dibutyltin and monobutyltin (DBT and MBT) at pH 9 and 7% NaCl (w/v). A gene was amplified and characterized from strain HS2 as SugE protein belongs to SMR protein family, a reverse transcription polymerase chain reaction analysis confirmed that SugE protein involved in the TBT degradation by HS2 strain. TBT bioremediation was investigated in stimulated TBT contaminated sediment samples (pH 9) using S chelatiphaga HS2 in association with E. coli BL21 (DE3)-pET28a(+)-sugE instead of S chelatiphaga HS2 alone reduced significantly the TBT half-life from 12d to 5d, although no TBT degradation appeared using E. coli BL21 (DE3)-pET28a(+)-sugE alone. This finding indicated that SugE gene increased the rate and degraded amount of TBT and is necessary in enhancing TBT bioremediation.