The Widespread Multidrug-Resistant Serotype O12 Pseudomonas aeruginosa Clone Emerged through Concomitant Horizontal Transfer of Serotype Antigen and Antibiotic Resistance Gene Clusters

ABSTRACTThe O-specific antigen (OSA) inPseudomonas aeruginosalipopolysaccharide is highly varied by sugar identity, side chains, and bond between O-repeats. These differences classifiedP. aeruginosainto 20 distinct serotypes. In the past few decades, O12 has emerged as the predominant serotype in clinical settings and outbreaks. These serotype O12 isolates exhibit high levels of resistance to various classes of antibiotics. Here, we explore how theP. aeruginosaOSA biosynthesis gene clusters evolve in the population by investigating the association between the phylogenetic relationships among 83P. aeruginosastrains and their serotypes. While most serotypes were closely linked to the core genome phylogeny, we observed horizontal exchange of OSA biosynthesis genes among phylogenetically distinctP. aeruginosastrains. Specifically, we identified a “serotype island” ranging from 62 kb to 185 kb containing theP. aeruginosaO12 OSA gene cluster, an antibiotic resistance determinant (gyrAC248T), and other genes that have been transferred betweenP. aeruginosastrains with distinct core genome architectures. We showed that these genes were likely acquired from an O12 serotype strain that is closely related toP. aeruginosaPA7. Acquisition and recombination of the “serotype island” resulted in displacement of the native OSA gene cluster and expression of the O12 serotype in the recipients. Serotype switching by recombination has apparently occurred multiple times involving bacteria of various genomic backgrounds. In conclusion, serotype switching in combination with acquisition of an antibiotic resistance determinant most likely contributed to the dissemination of the O12 serotype in clinical settings.IMPORTANCEInfection rates in hospital settings by multidrug-resistant (MDR)Pseudomonas aeruginosaclones have increased during the past decades, and serotype O12 is predominant among these epidemic strains. It is not known why the MDR phenotype is associated with serotype O12 and how this clone type has emerged. This study shows that evolution of MDR O12 strains involved a switch from an ancestral O4 serotype to O12. Serotype switching was the result of horizontal transfer and genetic recombination of lipopolysaccharide (LPS) biosynthesis genes originating from an MDR taxonomic outlierP. aeruginosastrain. Moreover, the recombination event also resulted in acquisition of antibiotic resistance genes. These results impact on our understanding of MDR outbreak strain and serotype evolution and can potentially assist in better monitoring and prevention.

Development and Application of a Novel Nucleic Amplification Kit on Rapid Detection of Various Types of Staphylococci Strains

A loop-mediated isothermal amplification (LAMP) method for rapid detection of various staphylococci strains and associated antibiotic resistance determinant had been developed and evaluated in this study. Six primers, including outer primers, inner primers and loop primers, were specially designed for recognizing eight distinct sequences on four targets: 16Sr RNA,femA,mecA, andorfX. Twenty-seven reference strains, including various species of gram-negative and-positive isolates, were included in this study to evaluate and optimize LAMP assays. The optimal reaction condition was found to be 65°C for 45 min, with detection limits at 100 fg DNA/tube and 10 CFU/reaction for 16S rRNA, 100 fg DNA/tube and 10 CFU/reaction forfemA, 1 pg DNA/tube and 100 CFU/reaction formecA, 10 DNA/tube and 10 CFU/reaction fororfX, respectively. Application of LAMP assays were performed on 166 various types of staphylococci isolates, the detection rate of LAMP assays for the 16Sr RNA,femA,mecA, andorfXwas 100% (166/166), 98.5% (64/65), 94.3% (66/70), and 98.6% (69/70) and the negative predictive value (NPV) was 100%, 98.1%, 92.3%, and 92.7% respectively; with a 100% positive predictive value (PPV) for all three targets. In conclusion, LAMP assays were demonstrated to be useful and powerful tools for rapid detection of various staphylococci strains.