Genome editing in Acinetobacter baumannii through enhanced natural transformation.

Acinetobacter baumannii, a multidrug-resistant bacterium has become a significant cause of life-threatening infections acquired in hospitals worldwide. The existing drugs used to treat A. baumannii infections are rapidly losing efficacy, and the increasing antimicrobial resistance, which is expected to turn into a global health crisis, underscores the urgency to develop novel prevention and treatment strategies. We reasoned that the discovery of novel virulence targets for vaccine and therapy interventions requires a more enhanced method for the introduction of multiple elements of foreign DNA for genome editing than the current methods of natural transformation techniques. Herein, we employed a novel and a much-improved enhanced technique for the natural transformation of elements of the genome editing system CRISPR-Cas9 to suppress specific genomic regions linked to selectively suppress bacterial virulence. We modified the genome of the laboratory-adapted strain of A. baumannii BAA-747 by targeting the AmpC, as a marker gene, for disruption by three different genomic manipulation strategies, and created mutant strains of A. baumannii that are, at least, fourfold susceptible to ampicillin. This work has established an optimized enhanced natural transformation system that enables efficient genome editing of pathogenic bacteria in a laboratory setting, providing a valuable future tool for exploring the function of unidentified virulence genes in bacterial genomes.

Detection of FOX-AmpC-ß-lactamase gene and antibiogram of AmpC-beta-lactamase-producing pathogens isolated from chronic suppurative otitis media patients in Nigeria.

Background and Objectives: AmpC-producing Gram-negative bacterial (GNB) pathogens are distributed worldwide, especially in clinical settings. This study aimed to determine the antibiogram and the type of AmpC-ß-lactamase gene harboured by GNB pathogens implicated in chronic suppurative otitis media (CSOM) cases. Materials and Methods: Ear swab samples (300) collected from patients with active CSOM were analysed using standard microbiological techniques. Phenotypic and molecular detection of AmpC ß-lactamase production was done by cefoxitin/cloxacillin double-disk synergy test and PCR respectively. Antibiogram was determined by disk diffusion technique. Results: Among the GNB pathogens isolated from CSOM patients, P. aeruginosa was the most predominant (36.3%); followed by K. pneumoniae (22.3%), and E. coli (13.7%). Patients with active CSOM showed increased bacteria isolation rate from bilateral ear discharges than unilateral ear discharges. E. coli and P. aeruginosa were more prevalent among patients with duration of discharge >2 weeks; recording 9.0% and 20.3% respectively. AmpC ß-lactamase producers accounted for 14.0%; they were highly resistant (60%-100%) to cephalosporins, trimethoprim-sulfamethoxazole, ofloxacin, amoxicillin, and tetracycline, but very susceptible (70.4%-100%) to ciprofloxacin, imipenem, and amikacin. Multiple antibiotic resistance indices of isolates ranged from 0.7-0.8. FOX-AmpC-ß-lactamase gene was detected in 3.9% of the isolates. Conclusion: The detection of AmpC ß-lactamase-producing multidrug-resistant GNB pathogens harbouring FOX-AmpC-ß-lactamase gene among patients with CSOM infections in our study is a serious public health problem which needs urgent intervention.

Detection of Plasmid-Mediated ß-Lactamase Genes and Emergence of a Novel AmpC (CMH-1) in Enterobacter cloacae at a Medical Center in Southern Taiwan.

The plasmid-mediated extended-spectrum ß-lactamases (ESBLs) and AmpC ß-lactamases in Enterobacter spp. have increasingly been reported. In this study, we investigated the prevalence of the plasmid-mediated ß-lactamases in Enterobacter cloacae from bloodstream isolates at a medical center in southern Taiwan. ESBL and ampC genes were detected by PCRs and DNA sequencing. Conjugation experiments were conducted to confirm the transferability of the genetic resistance trait. Among 41 non-repetitive blood isolates of cefuroxime-resistant E. cloacae, eight isolates exhibited ESBL phenotype confirmed by double-disk synergistic tests. Nearly all the strains were susceptible to carbapenems. The prevalence rate of the plasmid-mediated blaampC genes was 73% (30/41), including one blaDHA-1, one blaMIR-6, two novel blaCMH-1 genes and other blaACT-like genes. Coexistence of plasmid-mediated blaACT and ESBL genes (10 with blaSHV-12 and one with blaCTX-M-3) was observed. Successful transmissions of the blaACT and blaCMH-1 were demonstrated in some transconjugants. The inducible or derepressed CMH-1 had expanded activity of isolates versus ceftazidime. Enterobacterial repetitive intergenic consensus (ERIC)-PCR analysis and pulsotype showed distinct patterns suggesting non-clonal relationship. In conclusion, plasmid-mediated blaACT-like ampC genes in E. cloacae isolates have been highly prevalent in southern Taiwan and may continue genetic evolution, contributing to the complexities in antibiotic-resistant mechanisms.

Beta-lactamase Enzymes of Clinical Pseudomonas aeruginosa Strains.

OBJECTIVES: In this study, the production of extended spectrum beta-lactamase (ESBL), metallo-betalacatamase (MBL) and AmpC beta-lactamase enzymes of Pseudomonas aeruginosa (P aeruginosa) strains which were isolated from clinical samples were investigated. AmpC gene was also detected by the polymerase chain reaction (PCR) analysis. METHODS: A hundred strains of P aeruginosa were included in the study. The presence of ESBL was investigated with combined disk confirmation test, MBL was investigated with E-test method and AmpC beta-lactamase was investigated with disk induction test. In order to detect the production of AmpC betalactamase genotypically, the PCR method was used. RESULTS: Only one strain was found to be MBL positive. Four per cent of strains were found to be ESBL positive. AmpC beta-lactamase production was positive in 73% of the strains with disk induction test. AmpC gene was detected in 96% of the studied strains with the PCR method. CONCLUSION: While ESBL and MBL rates in this study were significantly lower than those found in other studies, the rate of AmpC beta-lactamase was higher. Although AmpC gene was detected in some strains (23%), they were not found to produce AmpC beta-lactamase with disk induction test.

Genotyping of AmpC Produced by Gram-negative in Infected Children / 中国药房

OBJECTIVE: To investigate the incidence rate of the ampC gene and AmpC enzyme of gram-negative(G-) bacterium in children,to analyze drug resistance of produced AmpC enzyme and un-produced AmpC enzyme strain.METHO_DS: 4 022 clinical G-isolates collected from 2002 to 2004 were identified and tested using K-B method.Selection 108 ESBLs bacterium,the ampC genes were amplified by PCR using common primers to AmpC and the AmpC enzymes were tested using the enzymatic rough extraction cefoxitin three-dimensional test.The drug resistance of bacterium produced AmpC enzymes were compared with the ones without AmpC enzymes.RESULTS: In 108 G-bacterium,the ampC genes positive bacterium were 70 strain(accounting for 64.8%),and 7 bacterium produced AmpC enzymes(accounting for 6.5%) were detected.The drug resistance of bacterium produced AmpC enzymes to ceftazidime(CAZ),ceftriaxone(CRO),piperacillin(PIP),ampicillin(AMP),aztreonam(ATM) were 85.7%,85.7%,71.4%,79.4%,79.4% respectively.The drug resistance of bacterium non-produced AmpC enzymes to CRO,PIP,gentamicin,AMP,ATM were 50.8%,55.6%,55.6%,70.3%,54.0% respectively,the drug resistance of bacterium to imipenem were the lowest,lower to ciprofloxacin.CONCLUSIONS: Detection rate of ampC gene were higher than AmpC-producing enzymes strains obviously,whereas the drug resistance to antibiotic of AmpC-producing enzymes strains were higher than non-producing enzymes strains.