Zinc oxide nanoparticles impact the expression of the genes involved in toxin-antitoxin systems in multidrug-resistant Acinetobacter baumannii.

The aim of this study was to investigate the effect of zinc oxide nanoparticles (ZnO-NPs) on the expression of genes involved in toxin-antitoxin (TA) systems in multidrug-resistant (MDR) Acinetobacter baumannii. Seventy clinical isolates of A. baumannii were collected from variuos clinical samples. Antimicrobial susceptibility test was determined by disk diffusion. Type II TA system-related genes including GNAT, XRE-like, hipA, hipB, hicA, hicB were screened using polymerase chain reaction (PCR). ZnO-NPs prepared and characterized by field emission scanning electron microscopy and X-ray diffraction. MIC of ZnO-NPs of A. baumannii isolates was performed using the microdilution method. The expression of type II TA systems-related genes were assessed with and without exposure to ZnO-NPs using real-time PCR. The highest rate of resistance and sensitivity was observed against cefepime (77.14%), and ampicillin/sulbactam (42.85%), respectively. All A. baumannii isolates were considered as MDR. In this study, three TA loci were identified for A. baumannii including GNAT/XRE-like, HicA/HicB, and HipA/HipB and their prevalence was 100%, 42%, and 27.1%, respectively. There was no significant relationship between the prevalence of these systems and the origin of A. baumannii. Our data showed significant correlations between the presence of HicA/HicB system and resistance to ceftazidime, meropenem, imipenem, and cefepime (p < 0.05), and the presence of HipA/HipB system and resistance to ceftazidime, meropenem, imipenem, and cefepime (p < 0.05). In presence of ZnO-NPs, the expression of all studied genes decreased. GNAT and hicB showed the highest and lowest expression changes by 2.4 folds (p < 0.001) and 1.3 folds (p < 0.05), respectively. This study demonstrates the promising potential of nanoparticles to impact the expression of the genes involved in TA Systems. So, the application of ZnO-NPs may be helpful to design target-based strategies towards MDRs pathogens for empowered clinical applications by microbiologists and nanotechnologists.

Cephalosporin translocation across enterobacterial OmpF and OmpC channels, a filter across the outer membrane.

Gram-negative porins are the main entry for small hydrophilic molecules. We studied translocation of structurally related cephalosporins, ceftazidime (CAZ), cefotaxime (CTX) and cefepime (FEP). CAZ is highly active on E. coli producing OmpF (Outer membrane protein F) but less efficient on cells expressing OmpC (Outer membrane protein C), whereas FEP and CTX kill bacteria regardless of the porin expressed. This matches with the different capacity of CAZ and FEP to accumulate into bacterial cells as quantified by LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry). Furthermore, porin reconstitution into planar lipid bilayer and zero current assays suggest permeation of ≈1,000 molecules of CAZ per sec and per channel through OmpF versus ≈500 through OmpC. Here, the instant killing is directly correlated to internal drug concentration. We propose that the net negative charge of CAZ represents a key advantage for permeation through OmpF porins that are less cation-selective than OmpC. These data could explain the decreased susceptibility to some cephalosporins of enterobacteria that exclusively express OmpC porins.

Prevalence of Efflux Pump and Porin-Related Antimicrobial Resistance in Clinical Klebsiella pneumoniae in Baghdad, Iraq.

Klebsiella pneumoniae is an opportunistic bacterium that causes many infections, including septicemia, pneumonia, urinary tract infection, and liver abscesses. There are many mechanisms for antibiotic resistance and K. pneumonia is considered a multidrug-resistant pathogen. This study aimed to find the correlation between the susceptibility of K. pneumonia to certain antibiotics with the porin-related resistance and pumps mechanisms. In total, two genes that are responsible for porin formation were considered in the current study OmpK-35gene and OmpK-36 gene, in addition to other four genes (CfiaS, CfiaL, MFS, and MdtK genes) related to an efflux pump mechanism of antibiotic resistance. The bacterial resistance was investigated towards five cephalosporins (Cefazolin, Cefoxitin, Ceftazidime, Ceftriaxone, and Cefepime) and two carbapenems (imipenem and ertapenem). Clinical samples, including blood, swabs, and urine, consisting of 20 specimens for each group, were collected from patients who attended three hospitals in Baghdad. The VITEK-2 system and genetic tests (polymerase chain reaction and sequencing) of bacterial isolates were applied to confirm the diagnosis of K. pneumoniae and detect the antibiotic sensitivity profile. The results showed that 51 (85%) and 15 (25%) of the total 60 isolates had positive results for OmpK-35 and Omp-K36 genes, respectively. The MFS and MdtK genes were observed (70-88.3%) in cephalosporin-resistant isolates of K. pneumoniae. There were no significant variations of bacterial resistance genes of antibiotics within the specimen groups. It was concluded that the bacterial resistance of the selected antibiotics was elevated markedly with the loss of the OmpK-36 gene with a high expression of MFS and MdtK genes and a slight minimal occurrence in the new generation of carbapenems. The best antimicrobial agent was ertapenem with a percentage of 0% of resistance in all bacterial isolates.

Meropenem, Cefepime, and Piperacillin Protein Binding in Patient Samples.

BACKGROUND: The mortality rate of patients with a drug-resistant bacterial infection is high, as are the associated treatment costs. To overcome these issues, optimization of the available therapeutic options is required. Beta-lactams are time-dependent antibiotics and their efficacy is determined by the amount of time the free concentration remains above the minimum inhibitory concentration. Therefore, the aim of this study was to assess the extent and variability of protein binding for meropenem, cefepime, and piperacillin. METHODS: Plasma samples for the analysis of meropenem, cefepime, and piperacillin were collected from patients admitted to a tertiary care hospital as part of the standard care. The bound and unbound drug fractions in the samples were separated by ultrafiltration. Validated liquid chromatography-tandem mass spectrometry assays were used to quantify the total and free plasma concentrations, and the protein binding was determined. RESULTS: Samples from 95 patients were analyzed. The median (range) age of patients was 56 years (17-87) and the median (range) body mass index was 25.7 kg/m (14.7-74.2). Approximately 59% of the patients were men. The median (range) unbound fraction (fu) was 62.5% (41.6-99.1) for meropenem, 61.4% (51.6-99.2) for cefepime, and 48.3% (39.4-71.3) for piperacillin. In the bivariate analysis, as the total meropenem concentration increased, the fu increased (r = 0.37, P = 0.045). A decrease in piperacillin fu was observed as the albumin concentration increased (r = -0.56, P = 0.005). CONCLUSIONS: The average fu values were lower than those reported in the literature. There was also a large variability in fu; hence, it should be considered when managing patients administered with these drugs through direct measurements of free drug concentrations.

The Reaction Mechanism of Metallo-ß-Lactamases Is Tuned by the Conformation of an Active-Site Mobile Loop.

Carbapenems are "last resort" ß-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-ß-lactamases (MßLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all ß-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MßLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MßLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different ß-lactams in all MßLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MßLs.

Quantification of Cefepime, Meropenem, Piperacillin, and Tazobactam in Human Plasma Using a Sensitive and Robust Liquid Chromatography-Tandem Mass Spectrometry Method, Part 2: Stability Evaluation.

Although the stability of ß-lactam antibiotics is a known issue, none of the previously reported bioanalytical methods had an adequate evaluation of the stability of these drugs. In the current study, the stability of cefepime, meropenem, piperacillin, and tazobactam under various conditions was comprehensively evaluated. The evaluated parameters included stock solution stability, short-term stability, long-term stability, freeze-thaw stability, processed sample stability, and whole-blood stability. When stored at -20°C, the stock solution of meropenem in methanol was stable for up to 3 weeks, and the stock solutions of cefepime, piperacillin, and tazobactam were stable for up to 6 weeks. All four antibiotics were stable in human plasma for up to 3 months when stored at -80°C and were stable in whole blood for up to 4 h at room temperature. Short-term stability results indicated that all four ß-lactams were stable at room temperature for 2 h, but substantial degradation was observed when the plasma samples were stored at room temperature for 24 h, with the degradation rates for cefepime, meropenem, piperacillin, and tazobactam being 30.1%, 75.6%, 49.0%, and 37.7%, respectively. Because the stability information is method independent, our stability results can be used as a reference by other research groups that work with these antibiotics.