Preventing the emergence and spread of MDR/XDR High-Risk Pseudomonas aeruginosa clones

Pseudomonas aeruginosa (P. aeruginosa) is a major contributor of nosocomial infections. The large and diverse gene pool and plastic genome enable it to exhibit significant diversity in its antibiotic resistance profile. As a common opportunistic pathogen, types of antibiotics that can effectively treat P. aeruginosa infection are still limited, among which carbapenems are broad-spectrum-β-lactam antibiotics. However, the prevalence of Carbapenem-Resistant P. aeruginosa (CRPA) has increased worldwide owing to the overuse and abuse of antibiotics. It is a critical clinical problem due to these drugs have long been recognized as the most effective treatments for infections caused by multidrug-resistant bacteria.

The ever-increasing prevalence of infections caused by multidrug-resistant (MDR) or extensively drug-resistant (XDR) P. aeruginosa is commonly linked to a limited number of aptly-named epidemical ‘high-risk clones´ (HiRiCs) that are widespread among and within hospitals worldwide. HiRiCs are generally associated with poor clinical outcomes and contain large repertories of horizontally acquired resistance determinants. However, why and how specific clone types (but not others) emerge as epidemic drug-resistant HiRiCs is currently not well understood. The emergence of new potential HiRiC strains in hospitals highlights the need to better and further understand the underlying genetic mechanisms for their emergence and success. Although P. aeruginosa-related HiRiCs have been sporadically found in China, genome sequences of these clonal types have rarely been described. The chief purpose of the previous project is to fill the gap related to cultures of P. aeruginosa HiRiCs from China and their associated genome sequences. Therefore, collecting enough multidrug-resistance HiRiC strains to identify and understand their genome mechanism can help us detect the P. aeruginosa HiRiCs evolution and transmission in China. The summary in detail is listed below:
1. Guangdong Province was chosen as the studied region. A total of 416 unduplicated strains were isolated from 10 tertiary hospitals with susceptibility testing results and clinical information. The Single Tube Long Fragment Reads (stLFR) based whole genome sequencing (WGS) was first used for P. aeruginosa genome assembly and antibiotic resistance analysis.
2. The various isolates were assigned to 149 known and 72 novel clone types. No predominant STs were present within the collected strains and even for the identified XDR strains. Among these strains, the most commonly identified clone types were ST244 (n=17, 4.1%) and ST274 (n=13, 3.1%), followed by ST1971 (n=12, 2.9%). More importantly, there were six global HiRiCs identified among these P. aeruginosa strains, including ST111, ST235, ST244, ST277, ST298, and ST357. The phylogenetic network showed that most of the sequence types found belonged to already-existing P. aeruginosa clonal complexes. Through the SNP comparison analysis, the ST1338 was identified belong to the clonal complex (CC)244. Furthermore, one particular disseminated clone, the ST1971 strains were only found in China and exhibited multidrug resistance and high virulence, which alarmed further surveillance of this high virulent and high antibiotic-resistant HiRiC.
3. A total of twelve serotypes that contained various O-antigens or O-polysaccharides were identified in this study. Out of all the isolates that were assigned specific serotypes, serotype O11 was the most prevalent in collected strains (n=109, 26.2%), followed by O6 (n=85, 20.4%), O1 (n=53, 12.7%), and O5 (n=41, 9.9%). Most of the clone types have only one corresponding serotype, even though the same clone recovered from different hospitals, except for the strains of the global HiRiC ST244 type exhibited genetic difference, with three O-antigens being identified among such strains (O2 (n=12), O5 (n=4) and O12 (n=1)). Normally, ExoU and ExoS are not secreted by the same strain, yet nine strains carrying both exoU and exoS genes were observed in this study. These nine strains were serotyped as O2 (n=1), O3 (n=2), and O4 (n=6) and belonged to seven different STs.
4. WGS analysis showed that 13.0% (54/416) strains harbored carbapenemase-encoding genes. We showed that imipenem and meropenem resistance in isolated strains was mainly due to mutations in the porin D gene oprD as well as over expression of specific efflux pumps. The mutational changes in oprD included complete inactivation of the oprD gene by frameshift mutation, insertion of a large fragment, and mutations that lead to the creation of a stop codon, but not missense mutations. The mutations in the mexAB and oprM genes of the MexAB-OprM efflux systems were less closely associated with meropenem resistance. The regulatory genes, mexR and nalD of MexAB-OprM efflux systems appear to play a critical role in mediating the development of meropenem resistance, as mutations in these two regulatory genes were found in 53.2% of the meropenem-resistant strains.
5. The core-pan and phylogenetic analysis were performed based on the combination dataset of newly sequenced Guangdong (GD) 416 strains and published 282 P. aeruginosa data in China. The phylogenetic distribution and clades coverage of the newly sequenced strains showed the representativeness of GD strains in China. We speculate that the result is related to the fact that Guangdong Province is the most populous province with the largest population inflow in China. We conducted an evolutionary and comparative genomics study of GD HiRiCs from a global perspective and 868 global high-risk clone data retrieved from public databases. The linkage relationship between P. aeruginosa in China and the world was identified. The phylogenetic analyses revealed a close relationship between GD and America ST244 strains. The GD HiRiC ST244-specific genes were also discovered. The most recent common ancestor (MRCA) analysis result illustrated that the origin of GD HiRiC ST1971 - the ancestral strain of China ST1971, which evolved from Europe, initially started in Africa, then to North America and Europe, and finally from Europe to China.

The current study significantly increased the strains volume and clonal diversity of P. aeruginosa sequences from China, providing some novel insights into the mechanism of HiRiCs emergency and dissemination. The epidemiology and genetic characteristics analysis of CRPA HiRiCs can expand our knowledge of the prevalence of CRPA background mechanisms. Through the comparative genomic analysis together with public data, we can have a deeper understanding of the population and evolution of China HiRiCs under a global context.