Investigating Conjugation-Mediated Spread of ESBLs and Carbapenemases Genes In Clinical Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae in Yaounde, Cameroon.

<jats:p>Antibiotic resistance is a major threat to public health, especially in opportunistic bacteria, such as Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae. Horizontal gene transfer is a key mechanism for the rapid dissemination of resistance genes, including extended-spectrum β-lactamases (ESBLs) and carbapenemases. This study aimed to evaluate the potential for horizontal transfer of resistance genes between multidrug-resistant clinical strains of P. aeruginosa, E. coli and K. pneumoniae. A total of 110 isolates of Pseudomonas aeruginosa ; 208 Escherichia coli and 30 Klebsiella pneumoniae were collected in five hospitals in the city of Yaoundé. Bacterial identification was performed using the Api 20E and API 20NE galleries. The resistance profile was determined by solid antibiogram susceptibility testing, including the detection of ESBL and carbapenemases according to the recommendations of CASFM and CLSI, 2023. Genomic DNA was extracted by the boiling method, while plasmid DNA was isolated using the Pure Link® Quick Plasmid Miniprep Kit (Invitrogen, Thermo Fisher Scientific, 2025). Resistance genes were visualized by PCR followed by agarose gel electrophoresis. In vitro conjugation experiments were performed using the filter mating method, using a recombinant plasmid pBBR1-MCS-2 and a helper plasmid pRK2013 to facilitate plasmid transfer. For crosses : 25 multidrug-resistant strains of P. aeruginosa were used as donors and 5 susceptible strains of E. coli and K. pneumoniae as receptors. Cross-breeding 1 allowed the mobilization of the two plasmid vectors (pBBR1-MCS-2 and pRK2013) to P. aeruginosa via a double transformation in the E. coli strain DH5α, used as an intermediate host. Cross-breeding 2 then evaluated the horizontal transfer of these plasmids carrying resistance genes from P. aeruginosa to susceptible E. coli and K. pneumoniae. Resistance genes were dominated by carbapenemases in P. aeruginosa (bla OXA-50 and bla VIM, 45.8 % each) and ESBLs in E. coli (bla TEM, 53.3% and bla SHV, 48.9%), and in K. pneumoniae (bla SHV, 46.2 %). Plasmid analyses showed high mobility of some genes, including bla VIM (45.8%) and bla OXA-50 (37.5%) in P. aeruginosa and bla TEM (35.5%) and bla SHV (33.5%) in E. coli. In vitro conjugation  showed that the cross from P. aeruginosa to K. pneumoniae had the highest mean conjugation frequency, reaching 1.1 × 10 -3. In contrast, crosses from E. coli to P. aeruginosa and from P. aeruginosa to E. coli showed significantly lower conjugation frequencies, in the range of 2.2 × 10-6 and 6.0 × 10-6, respectively. These results highlight the central role of horizontal transfer and plasmids in the spread of antibiotic resistance and the need to monitor these transfers to prevent the emergence of multidrug-resistant strains.</jats:p>