Occurrence of blaTEM and blaCTXM Genes and Biofilm-Forming Ability among Clinical Isolates of Pseudomonas aeruginosa and Acinetobacter baumannii in Yaoundé, Cameroon

Background: Pseudomonas aeruginosa (PSA) and Acinetobacter baumannii (ACB) are non-fermentative bacteria mostly associated with nosocomial infections in humans. Objective: This study aimed to determine the antimicrobial resistance profiles and virulence gene of PSA and ACB previously isolated from humans in selected health facilities in Yaoundé, Cameroon. Methods: A total of 77 and 27 presumptive PSA and ACB isolates, respectively, were collected from the Yaoundé teaching hospital. These isolates were previously isolated from various samples including pus, blood and broncho-alveolar lavage. The identities of the isolates were determined through polymerase chain reaction (PCR) amplification of PSA and ACB specific sequences. Antimicrobial susceptibility testing (AST) was performed using the Kirby–Bauer disc diffusion method. Phenotypical expression of AmpC β-lactamases (AmpC), extended spectrum β-lactamases (ESBLs) and metallo β-Lactamases (MBLs) were determined using the combined disc method. Bacterial genomes were screened for the presence of β-lactamases blaTEM and blaCTXM genes using specific PCR. The pathogenicity of PSA and ACB was assessed through amplification of the lasB, exoA, pslA and exoS as well as OmpA and csuE virulence genes, respectively. Results: Of the 77 presumptive PSA isolates, a large proportion (75 to 97.4%) were positively identified. All (100%) of the presumptive 27 ACB harbored the ACB-specific ITS gene fragment by PCR. Twenty five percent of the PSA isolates produced ESBLs phenotypically while more than 90% of these isolates were positive for the lasB, exoA, pslA and exoS genes. A large proportion (88%) of the ACB isolates harboured the OmpA and csuE genes. blaTEM and blaCTXM were detected in 17 and 4% of PSA, respectively, while a much higher proportion (70 and 29%) of the ACB isolates possessed these resistance determinants respectively. Conclusion: Our findings reveal the occurrence of both virulence and drug-resistant determinants in clinical PSA and ACB isolates from patients in health care settings in Yaoundé, Cameroon, thus suggesting their role in the pathological conditions in patients

Whole-genome sequence of multi-drug resistant Pseudomonas aeruginosa strains UY1PSABAL and UY1PSABAL2 isolated from human broncho-alveolar lavage, Yaoundé, Cameroon.

Pseudomonas aeruginosa has been implicated in a wide range of post-operation wound and lung infections. A wide range of acquired resistance and virulence markers indicate surviving strategy of P. aeruginosa. Complete-genome analysis has been identified as efficient approach towards understanding the pathogenicity of this organism. This study was designed to sequence the entire genome of P. aeruginosa UY1PSABAL and UY1PSABAL2; determine drug-resistance profiles and virulence factors of the isolates; assess factors that contribute toward stability of the genomes; and thereafter determine evolutionary relationships between the strains and other isolates from similar sources. The genomes of the MDR P. aeruginosa UY1PSABAL and UY1PSABAL2 were sequenced on the Illumina Miseq platform. The raw sequenced reads were assessed for quality using FastQC v.0.11.5 and filtered for low quality reads and adapter regions using Trimmomatic v.0.36. The de novo genome assembly was made with SPAdes v.3.13 and annotated using Prokka v.2.1.1 annotation pipeline; Rapid Annotation using Subsytems Technology (RAST) server v.2.0; and PATRIC annotation tool v.3.6.2. Antimicrobial resistance genes and virulence determinants were searched through the functional annotation data generated from Prokka, RAST and PATRIC annotation pipelines; In addition to ResFinder and Comprehensive Antibiotic Resistance Database (CARD) which were employed to determine resistance genes. The PHAge Search Tool Enhanced Release (PHASTER) web server was used for the rapid identification and annotation of prophage sequences within bacterial genome. Predictive secondary metabolites were identified with AntiSMASH v.5.0. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and cas genes regions were also investigated with the CRISPRone and CRISPRFinder server. The genome sizes of 7.0 and 6.4 Mb were determined for UY1PSABAL and UY1PSABAL2 strains with G+C contents of 66.1% and 66.48% respectively. β-lactamines resistance genes blaPAO, aminoglycoside phosphorylating enzymes genes aph(3')-IIb, fosfomycine resistance gene fosA, vancomycin vanW and tetracycline tetA were among identified resistance genes harboured in both isolates. UY1PSABAL bore additional aph(6)-Id, aph(3'')-Ib, ciprofloxacin-modifying enzyme crpP and ribosomal methylation enzyme rmtB. Both isolates were found harbouring virulence markers such as flagella and type IV pili; and also present various type III secretion systems such as exoA, exoS, exoU, exoT. Secondary metabolites such as pyochelin and pyoverdine with iron uptake activity were found within the genomes as well as quorum-sensing systems, and various fragments for prophages and insertion sequences. Only the UY1PSABAL2 contains CRISPR-Cas system. The phylogeny revealed a very close evolutionary relationship between UY1PSABAL and the similar strain isolated from Malaysia; the same trend was observed between UY1PSABAL2 and the strain from Chinese origin. Complete analyses of the entire genomes provide a wide range of information towards understanding pathogenicity of the pathogens in question