Novel ampC Beta-Lactamase Genes from Morganella Morganii

Antibiotic resistance becomes a global threat to human health due to a rise of bacterial infections and a decline of antibiotic discovery rate. Beta-lactams represent the most effective and most prescribed antibiotics, but some bacteria, such as Morganella morganii, are intrinsically resistant to Beta-lactams, such as cephamycins and oxyimino-cephalosporins, due to the chromosomal ampC gene. Even more alarming is that AmpC enzymes can induce resistance to other Beta-lactam antibiotics, including those of the latest generation, and that ampC gene can be mobilized on plasmids and passed to other bacteria. The aims of this study were two-fold: 1) determine if inducible Beta-lactamases are present from Morganella isolates from natural environments; 2) identify and characterize genetic determinants of AmpC Beta-lactamases at molecular level. The 24 isolates used in this study were obtained from aquatic sites in Southern California. Identification was conducted via MALDI-TOF mass spectrometry, according to a species-specific protein profile. Disk diffusion tests were conducted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Polymerase chain reaction (PCR) amplification alongside with agarose gel electrophoresis were used to find out genes conferring antibiotic resistance. Once detected, the genes were sequenced, assembled using Lasergene software, and BLASTed against GenBank to confirm their authenticity. All 24 isolates were susceptible to cefotaxime and ceftazidime while 16 (67%) were resistant to cefoxitin. 23 out of 24 (96%) M. morganella isolates exhibited pattern consistent with inducible beta-lactamases. 12 of 18 (67%) DHA genes found are new variants encoding proteins that differ from the closest known ones (deposited in GenBank) by 2 to 18 amino acids; therefore, this study more than doubles the number of known DHA variants. A novel DHA-like gene was found that is 89% similar to known DHAs, with a divergence at the amino acid level ranging from 7-8%. It is the most diverged DHA reported to date, reinforcing the hypothesis that plasmid-borne DHA descended from M. morganii ampC; moreover, the data shows that there were at least five independent mobilization events involving transfer of the ampC genes from M. morganella chromosome onto plasmids, with subsequent spread of the plasmids among diverse bacterial species. Only 11 accessible DHA gene variants have been described so far worldwide. In conclusion, combination of phenotypic and genotypic approaches found new highly diverged DHA variants within M. morganii.