Kinetic Study of Laboratory Mutants of NDM-1 Metallo-ß-Lactamase and the Importance of an Isoleucine at Position 35.

Two laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1(I35T)and NDM-1(I35S)enzymes. These mutants were well characterized, and their kinetic parameters were compared with those of the NDM-1 wild type. Thekcat,Km, andkcat/Kmvalues calculated for the two mutants were slightly different from those of the wild-type enzyme. Interestingly, thekcat/Kmof NDM-1(I35S)for loracarbef was about 14-fold higher than that of NDM-1. Far-UV circular dichroism (CD) spectra of NDM-1 and NDM-1(I35T)and NDM-1(I35S)enzymes suggest local structural rearrangements in the secondary structure with a marked reduction of α-helix content in the mutants.

Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems.

Site-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-ß-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

Identification of New Natural CphA Metallo-ß-Lactamases CphA4 and CphA5 in Aeromonas veronii and Aeromonas hydrophila Isolates from Municipal Sewage in Central Italy.

Two new natural CphA metallo-ß-lactamases, the CphA4 and CphA5 enzymes, were identified in water samples from municipal sewage in central Italy. Compared to CphA, the CphA4 and CphA5 enzymes showed numerous point mutations. These enzymes have a narrow spectrum of substrates focused on carbapenems only. CphA5 showed kcat values about 40-, 12-, and 97-fold higher than those observed for CphA4 versus imipenem, ertapenem, and biapenem, respectively.

Evaluation of Commercial Meat Products of Red Chicken Reared under LED Lights.

The objective of our study was to investigate the role of three different light-color temperatures of Light-Emitting Diodes (LEDs) [Neutral (K=3300-3700); Warm (K=3000-2500) and Cool (K=5500-6000)] on the qualitative attributes of breast meat obtained from male AZ Extra Heavy Red chickens. The comparison was made with meat deriving from chickens reared in the presence of classic neon lighting (Control). The meat was analyzed for the determination of both physical and chemical properties (cooking loss, moisture, total lipids and fatty acid composition). Furthermore, meat samples subjected to cooking were also analyzed for the identification of volatile compounds produced during the process; such evaluation was performed both immediately after cooking (T0) and after 7 days (T7) of cooked-meat storage at 4 °C. Cooking-loss values were higher for samples from chickens raised with Neutral LED (p < 0.05) compared to the other groups. For the fatty acid profiles of the meat, higher values were found for monounsaturated fatty acids (MUFAs) such as C18:1, C9 and C16:1 in Cool LED compared to the Control. Regarding the volatile profile of cooked meat, compounds belonging to the families of aldehydes, alcohols, ketones, and aromatic compounds were identified. Compounds belonging to the aldehyde family, such as hexanal, increased in Cool LED samples at T0 in comparison to the Control. On the other hand, the amounts of 1-Pentanol, 1-Octanol and 2-Octen-1-ol, which belong to the alcohol family, increased at T7 in Cool LED samples compared to the Warm LED. In conclusion, LED lighting showed to be effective in inducing significant variations on chicken breast meat ready to be introduced to the market, in particular regarding fatty acid profiles and the accumulation of volatile compounds. However more in-depth evaluation is needed for the identification of modifications regarding the sensorial sphere, which could have an impact on the consumer acceptability of the product.

Impact of different shades of light-emitting diode on fecal microbiota and gut health in broiler chickens.

OBJECTIVE: The aim of this study was to characterize the fecal microbiota of broiler chickens reared in the presence of different shades of light-emitting diode (LED) lights, correlating this information with biochemical and molecular evidence that allowed drawing conclusions on the state of health of the animals. METHODS: Overall, the metagenomic approach on fecal samples was associated with evaluations on enzymes involved in the cellular response to oxidative stress: glutathione peroxidase (GPX), superoxide dismutase and catalase; while the inflammatory aspect was studied through the dosage of a proinflammatory cytokine, the interleukin 6 (IL-6), and the evaluation of the matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9). Specifically, analysis was performed on distinct groups of chickens respectively raised in the presence of neutral (K = 3,300 to 3,700), cool (K = 5,500 to 6,000), and warm (K = 3,000 to 2,500) LED lightings, and a direct comparison was performed with animals reared with traditional neon lights. RESULTS: The metagenomic analysis highlighted the presence of two most abundant bacterial phyla, the Firmicutes and the Bacteroidetes, with the latter characterized by a greater relative abundance (p<0.05) in the group of animals reared with Neutral LED light. The analysis on the enzymes involved in the antioxidant response showed an effect of the LED light, regardless of the applied shade, of reducing the expression of GPX (p<0.01), although this parameter is not correlated to an effective reduction in the tissue amount of the enzyme. Regarding the inflammatory state, no differences associated with IL-6 and MMP-9 were found; however, is noteworthy the significant reduction of MMP-2 activity in tissue samples obtained from animals subjected to illumination with neutral LED light. CONCLUSION: This evidence, combined with the metagenomic findings, supports a potential positive effect of neutral LED lighting on animal welfare, although these considerations must be reflected in more targeted biochemical evaluations.

Whole Blood Transcriptome Profiling Reveals Positive Effects of Olive Leaves-Supplemented Diet on Cholesterol in Goats.

Agro-industrial by-products represent an important source of compounds credited with high biotechnological potential. In the last decade, considerable interest has developed toward the use of these matrices as dietary supplements in the zootechnical field, paying particular attention to the qualitative aspects associated with animal products. However, less is known about the effect of these matrices on gene expression and thus on animal metabolism. Therefore, the aim of this study was to analyze the whole blood transcriptome of lactating goats fed a dietary supplementation with 10% olive leaves (OL), one of the main by-products deriving from the olive oil chain supply. By applying a false discovery rate (FDR) < 0.05 and a Log2 Fold change (Log2Fc) lower than -0.5 or higher than +0.5, it was possible to identify the differential regulation of gene coding for the apolipoprotein B (apoB) mRNA editing enzyme catalytic subunit 2 (APOBEC2), which showed downregulation in goats that received the dietary supplementation. An evaluation of both blood and milk cholesterol was performed, taking into account the strong association between plasma apoB and low-density lipoprotein (LDL). Results showed significantly lower concentrations of circulating cholesterol and cholesterol released into the milk through the mammary gland, demonstrating positive effects of OL feeding on animal welfare and potential health benefits for consumers.

Interaction of carbapenems and ß-lactamase inhibitors towards CTX-M-15 and CTX-M-15G238C mutant.

OBJECTIVES: The aim of this study was to evaluate the role of residue 238 in CTX-M-15 and CTX-M-15G238C mutant with respect to carbapenems and various ß-lactamase inhibitors. METHODS: A CTX-M-15G238C laboratory mutant was generated by site-directed mutagenesis from CTX-M-15 enzyme by replacing glycine 238 with cysteine. Thiol titration and p-chloromercuribenzoate (PCMB) inactivation assays were used to ascertain the presence of a disulfide bridge in the active site of CTX-M-15G238C. Kinetic parameters were determined both for CTX-M-15 and CTX-M-15G238C enzymes by analysing either the complete hydrolysis time courses or under initial rate conditions. RESULTS: In CTX-M-15G238C mutant, the two cysteines (C69 and C238) located in the enzyme active site were unable to form a disulfide bridge. CTX-M-15 and thermostable CTX-M-15G238C were used to study the kinetic interaction with carbapenems, which behaved as poor substrates for both enzymes. Meropenem and ertapenem acted as transient inactivators for CTX-M-15 and CTX-M-15G238C, and for these compounds the variation of kobs versus the inactivator concentration was linear. Imipenem behaved as a transient inactivator for CTX-M-15 and as an inactivator (with k+3=0) for CTX-M-15G238C. In any case, the k+2/K values for CTX-M-15G238C were higher than those for CTX-M-15. CONCLUSIONS: Compared with CTX-M-15, CTX-M-15G238C mutant appears to have a more favourable conformation for carbapenem acylation and higher activity against cefotaxime, which could be due to the presence of free -SH groups in the enzyme active site.

BlaB-15, a new BlaB metallo-ß-lactamase variant found in an Elizabethkingia miricola clinical isolate.

A multidrug-resistant strain of Elizabethkingia miricola was isolated from the urine of a 2-year-old boy hospitalized for severe clinical conditions. The strain produces 2 metallo-ß-lactamases belonging to subclasses B1 and B3: a new BlaB variant (BlaB-15) and a GOB-7-like enzyme.