Production of β-lactamase enzymes such as AmpC β-lactamases and Extended-Spectrum β-Lactamases (ESBLs) is among the main mechanisms for resistance to expanded-spectrum cephalosporins. The present study was conducted to investigate prevalence and molecular epidemiology of plasmid mediated AmpC beta-lactamase in ESBL co-producing Escherichia coli (E.coli) and Klebsiella spp. (Klebsiella pneumoniae and Klebsiella oxytoca) clinical isolates in northeast of Iran. A total of 602 E.coli and Klebsiella spp. clinical isolates were collected from three hospitals in Mashhad (northeast of Iran). Combination disk test was performed for phenotypic detection of ESBLs. Screening for detection of AmpC β-lactamases was performed by susceptibility test to cefoxitin disc among ESBL producing isolates. Confirmatory test for AmpC β-lactamases was performed by The Mast® D68C test. Identification of plasmid mediated AmpC cluster genes was done by multiplex PCR. Among 336 ESBL-producing strains, 230 (68.4%) isolates were resistant to cefoxitin. Results of the Mast® D68C test showed that 30% (69/230) of cefoxitin resistant isolates simultaneously exhibited ESBL and AmpC activity and 22% (51/230) of isolates probably showed MDR phenotype. Results of multiplex PCR among ESBL-positive isolates showed that, 16.7% (56/336) of isolates were positive for plasmid-borne ampC cluster genes, and CMY (38%) was the most frequent genotype of plasmid mediated AmpC. Findings of the study revealed that an increase in the prevalence of ESBL and AmpC co-producer in E. coli and Klebsiella spp strains may become an important public health issue. Therefore, there is a vital need for surveillance of spread of these clinical isolates.

One of the most critical challenges of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression systems from bacteria to mammalian tissue culture cells are available for the production of recombinant proteins for medical and industrial purposes. Among various choices, yeast expression systems such as Pichia pastoris are promising candidates. The P. pastoris expression system is a standard tool for the production of biopharmaceuticals and industrial enzymes. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnology. Using P. pastoris as an expression system for heterologous proteins, this article provides detailed basic protocols for cloning of a recombinant cassette into a suitable expression vector, the transformation of foreign vector DNAs into the yeast by electroporation, and expression and purification of desired recombinant protein. © 2021 Wiley Periodicals LLC.Basic Protocol 1: Cloning of a recombinant cassette into a suitable expression vectorBasic Protocol 2: Transformation of P. pastoris and selection of transformantsBasic Protocol 3: Optimization and large‐scale expression of recombinant proteinsBasic Protocol 4: Purification of recombinant proteins