Biofilm Formation and Phenotypic Detection of ESBL, MBL, KPC and AmpC Enzymes and Their Coexistence in Gram negative bacteria Isolated from different hospitals in Gaza Strip - Palestine

Gram-negative bacilli, most commonly Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii are responsible for causing various pathological diseases like UTIs, septicemia, pneumonia, nosocomial infections, opportunistic infections etc..

Beta-lactams were wonder drugs until the dissemination of beta-lactamases (ESBL and MBL) producing strains were detected. “extended-spectrum beta-lactamases (ESBLs) may be defined as plasmid-mediated enzymes that hydrolyze oxyimino-cephalosporins (ceftriaxone, cefotaxime, and ceftazidime) and monobactams (aztreonam) but not cephamycins or carbapenems.

They are inhibited in vitro by clavulanate”. Metallo-beta-lactamases (MBLs) are carbapenems hydrolyzing enzymes; inhibited by metal chelating agents like EDTA.

Biofilms are the bacterial aggregates firmly lodged in the extracellular matrices of polysaccharides, proteins, enzymes, and nucleic acids; thereby, facilitating anchorage to any surfaces irreversibly.

The matrix confers antibiotic resistance through processes such as expression of chromosomally encoded resistant genes, restriction of antibiotics, reduction in growth rate, and even counteracting the host immunity.

The biofilm formation and beta-lactamases production synergistically contribute for extensive dissemination of multi-drug resistant strains of gram-negative bacilli.

They are responsible for implicating chronicity, persistence, and relapse of infections leading to high morbidity and mortality; thus, posing a serious health crisis.

Objectives: The aim of this research is to study Biofilm Formation and Phenotypic Detection of ESBL, MBL, KPC and AmpC Enzymes and Their Coexistence in Gram-negative bacteria Isolated from different hospitals in Gaza Strip - Palestine Methods: Antibiotic susceptibility test : It was performed on Muller Hinton agar by Kirby Bauer disc diffusion method following CLSI guidelines.

Ofloxacin (5 μg), levofloxacin (5 μg), ceftazidime (30 μg), cefepime (30 μg), amikacin (30 μg), gentamicin (10 μg), piperacillin (100 μg), piperacillin-tazobactam (100/10 μg), imipenem (10 μg), and colistin (10 μg) were tested as common antibiotics for all strains. Polymyxin B (300 units), tobramycin (10 μg) and carbenicillin (100 μg) will be added for Pseudomonas spp.

Phenotypic detection of ESBL and MBL production The ceftazidime resistant strains were screened for ESBL production by using disc diffusion test.

The increase in the zone of the diameter of ≥ 5-mm between ceftazidime (30 μg) and ceftazidime-clavulanate (30/10 μg) was considered ESBL positive.

Whereas, imipenem-resistant strains were tested for MBL production by combined disc diffusion assay using two imipenem discs, one with added 10 μl of 0.5 M EDTA.

The increased zone of inhibition of > 7 mm around the imipenem-EDTA disc in comparison to zone Biofilm detection by tube adherence and Congo red agar Tube adherence method : A growth organism was inoculated into trypticase soy broth and incubated for 24h at 35 °C. After discarding the supernatant, the tube was washed with phosphate buffer saline.

It was treated with 0.1% crystal violet for staining and then washed with water and dried. The appearance of visible biofilm lining the bottom and wall of the tube was considered positive. Congo red agar method : The organisms were inoculated on Congo red agar plate and incubated at 37 °C for 24 h.

The formation of black colonies indicated biofilm production. Genotypic characterization of biofilm formation using pulsed field gel electrophoresis