Cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) and amidase are known as catalytic domains of the bacteriophage-derived endolysin LysK and were previously reported to show lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). In the current study, the in silico design and analysis of chimeric CHAP-amidase model was applied to enhance the stability and solubility of protein, which was achieved through improving the properties of primary, secondary and tertiary structures. The coding gene sequence of the chimeric CHAP-amidase was synthesized and subcloned into the pET-22(+) expression vector, and the recombinant protein was expressed in E. coli BL21 (DE3) strain. Subsequent affinity-based purification yielded ~12 mg soluble protein per liter of E. coli culture. Statistical analysis indicated that concentrations of ≥1 μg/mL of the purified protein have significant antibacterial activity against S. aureus MRSA252 cells. The engineered chimeric CHAP-amidase exhibited 3.2 log reduction of MRSA252 cell counts at the concentration of 10 μg/mL. A synergistic interaction between CHAP-amidase and vancomycin was detected by using checkerboard assay and calculating the fractional inhibitory concentration (FIC) index. This synergistic effect was shown by 8-fold reduction in the minimum inhibitory concentration of vancomycin. The chimeric CHAP-amidase displayed strong antibacterial activity against S. aureus, S. epidermidis, and enterococcus. However, it did not indicate any significant antibacterial activity against E. coli and Lactococcus lactis. Taken together, these findings suggest that our chimeric CHAP-amidase might represent potential to be used for the development of efficient antibacterial therapies targeting MRSA and certain Gram-positive bacteria.

BackgroundMetallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa is a significant pathogen in burn patients.ObjectivesThe aim of this study was to determine the prevalence of carbapenem-resistant P. aeruginosa isolates, including those resistant to imipenemase (IMP), in a burn unit in Isfahan, Iran.Patients and MethodsOne hundred and fifty P. aeruginosa isolates from burn patients were tested for antibiotic susceptibility by the disc diffusion method in accordance with CLSI guidelines. Production of MBL was identified with the EDTA disk method. DNA was purified from the MBL-positive isolates, and detection of the blaIMP gene was performed with PCR.ResultsFifty-seven out of 150 (38%) isolates were multi-drug resistant (MDR), and 93 (62%) were extensively-drug resistant (XDR). Among all isolates, the resistance rate to ciprofloxacin, tobramycin, imipenem, meropenem, amikacin, ceftazidime, and cefepime was higher than 90%, while the resistance rates to piperacillin/tazobactam and aztreonam were 70.7% and 86%, respectively. Colistin and polymyxin B remained the most effective studied antibiotics. All of the imipenem-resistant P. aeruginosa isolates were MBL-positive, and 107 out of 144 (74.3%) of the MBL isolates were positive for the blaIMP gene.ConclusionsThe results of this study show that the rate of P. aeruginosa-caused burn wound infections was very high, and many of the isolates were resistant to three or more classes of antimicrobials. Such extensive resistance to antimicrobial classes is important because few treatment options remain for patients with burn wound infections. blaIMP-producing P. aeruginosa isolates are a rising threat in burn-care units, and should be controlled by conducting infection-control assessments.

Therapeutic LysK-CHAP is a potent anti-staphylococcal protein that could be utilized as an antibiotic substitute. Intein-mediated protein purification is a reasonable and cost-effective method that is most recently used for recombinant therapeutic protein production. Intein (INT) is the internal parts of the protein that can be separated from the immature protein during protein splicing process. This sequence requires no specific enzyme or cofactor for separation. INT sequence and their characteristic of self-cleavage by thiol induction, temperature, and pH changes are used for protein purification. The current study presents the expression of CHAP domain of LysK gene that is fused with INT/chitin-binding sequence while evaluating its purification procedure and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The coding gene sequence of LysK-CHAP (CHAP) in pET22-b was amplified with polymerase chain reaction (PCR); the digested product was then cloned into the pTXB1 vector. Electrophoresis confirmed the cloning accuracy of the gene. The pTXB1-CHAP plasmid was transformed to the Escherichia coli ER (E. coli ER) expression strain and analyzed for expression of the recombinant protein by SDS-PAGE and Western blotting methods. Finally, CHAP was purified by chitin affinity column using INT tag technology and confirmed by SDS-PAGE. Lytic activity of the purified protein was investigated by disk diffusion method. Cloning of CHAP into the pTXB1 vector, which comprised INT/chitin-binding sequence, was successfully achieved. The SDS-PAGE data also revealed successful expression of the CHAP-INT fusion protein and Western blotting method validated the accuracy of the protein. Moreover, purification of CHAP protein was induced by dithiothreitol (DTT) and confirmed by SDS-PAGE. Finally, inhibition zone in MRAS culture medium confirmed antibacterial activity of the protein. Application of intein-mediated antibacterial protein is an appropriate and streamlined method for one-step purification of CHAP as a therapeutic and antibacterial protein. Self-cleaving tags like intein are cost-effective and could be used as a proper purification method for industrial purposes.

Context: Diagnosis of human brucellosis still challenges clinicians and scientists with several considerable aspects, particularly in endemic countries. The current study aimed at reviewing laboratory tests in the diagnosis of human brucellosis. Evidence Acquisition: A literature search was conducted in PubMed, Scopus, Thompson Reuters, and Mesh databases using keywords for articles published until December 2018. Seventy studies were selected for data collection. Results: The current inclusive review included information about the currently used advanced diagnostic tests to confirm the detection of human brucellosis. Conclusions: The article reviewed the methods for the diagnosis of human brucellosis and summarized developments for the future.

Acinetobacter baumannii (A. baumannii) is an important nosocomial pathogen in healthcare institutions. β-Lactamase-mediated resistance is the most common mechanism for carbapenem resistance in A. baumannii. The aim of this study was to determine the antibiotic resistance pattern, to detect OXA encoding genes, class A, bla PER-1, and to detect the presence of ISAba1. A total of 124 A. baumannii isolates were collected from hospitalized patients in a teaching hospital in Kashan, Iran. The susceptibility of isolates to different antibiotics was determined by disk-diffusion method. PCR was used to detect bla PER-1, bla OXA-23, bla OXA-24, bla OXA-51, bla OXA-58, and ISAba1 genes. All isolates were resistant to ceftazidime, ceftriaxone, and cefotaxime. All of the isolates revealed susceptibility to polymyxin B and colistin. Ninety-six percent of the isolates were extensive drug resistance (XDR), 5.6% extended spectrum beta-lactamase (ESBL), and 54.8% metallo-beta-lactamase (MBL). All isolates were positive for bla OXA-51 and ISAba1. bla OXA-23,  bla OXA-24, and bla OXA-58 were found in 79.8%, 25%, and 3.2%, respectively. The frequency rate of bla PER-1 gene was 52.4%. Multidrug resistant A. baumannii isolates are increasing in our setting and extensively limit therapeutic options. The high rate presence of class D carbapenemase-encoding genes, mainly bla OXA-23 carbapenemases, is worrying and alarming as an emerging threat in our hospital.

The intestinal tract is a host to various types of bacteria that are essential to health. Interactions between intestinal bacteria, i.e. the normal microbiota of the host's intestine, have been a subject of intensive research, as they may influence disease cycles. Recent studies of selected probiotic species and their therapeutic benefits have suggested a potential efficacy in treatment of several gastrointestinal illnesses, including Helicobacter pylori infection. The increasing evidence from these clinical studies supports the promising role of probiotics in improving the treatment of H. pylori by increasing eradication rates as well as decreasing the adverse effects of current medication therapy. However, many unsolved questions remain which require high quality trials on specific probiotic strains in the future. The main part of this review will focus on the effects of supplementary probiotic products during standard triple H. pylori therapy.