Application of Whole-Genome Sequencing Data for O-Specific Antigen Analysis and In Silico Serotyping of Pseudomonas aeruginosa Isolates

Abstract

Accurate typing methods are required for efficient infection control. The emergence of whole-genome sequencing (WGS) technologies has enabled the development of genome-based methods applicable for routine typing and surveillance of bacterial pathogens. In this study, we developed the Pseudomonas aeruginosa serotyper (PAst) program, which enabled in silico serotyping of P. aeruginosa isolates using WGS data. PAst has been made publically available as a web service and aptly facilitates highthroughput serotyping analysis. The program overcomes critical issues such as the loss of in vitro typeability often associated with P. aeruginosa isolates from chronic infections and quickly determines the serogroup of an isolate based on the sequence of the O-specific antigen (OSA) gene cluster. Here, PAst analysis of 1,649 genomes resulted in successful serogroup assignments in 99.27% of the cases. This frequency is rarely achievable by conventional serotyping methods. The limited number of nontypeable isolates found using PAst was the result of either a complete absence of OSA genes in the genomes or the artifact of genomic misassembly. With PAst, P. aeruginosa serotype data can be obtained from WGS information alone. PAst is a highly efficient alternative to conventional serotyping methods in relation to outbreak surveillance of serotype O12 and other high-risk clones, while maintaining backward compatibility to historical serotype data. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and a major cause of mortality and morbidity among hospitalized and compromised patients, including those with cystic fibrosis (CF). P. aeruginosa is well known for its ability to cause chronic and extensively drug-resistant infections (1). The outer membrane lipopolysaccharide (LPS) layer is a major virulence factor of P. aeruginosa (2). LPS has been linked to antibiotic resistance and immune evasion. Furthermore, LPS is one of the receptors that determines the susceptibility of the bacterium to bacteriophages and pyocins (2-4). Our ability to control P. aeruginosa infections depends on the availability of accurate typing methods. Previously, serotyping was a benchmark typing method for P. aeruginosa. In the 1980s, the International Antigenic Typing Scheme (IATS) was established to classify the species P. aeruginosa into 20 serotypes (O1 to O20) (5-7). Today, serotyping is infrequently used in the clinic for typing purposes, mainly because of the time-consuming protocol, the need for a continuous supply of serotype-specific antisera, and a high prevalence of polyagglutinating or nontypeable isolates.The loss of P. aeruginosa typeability has been known for decades and has often been linked to bacteria isolated from chronic infections, where typeability is lost over time during the course of infection (8, 9). A study performed by Pirnay et al. (10) showed that 65% of all P. aeruginosa isolates examined were either non-or multitypeable and therefore assigning a particular serotype to these strains would be difficult. The occur...

The Role of Pseudomonas aeruginosa Lipopolysaccharide in Bacterial Pathogenesis and Physiology

“…Serotyping of P. aeruginosa was originally developed using immunochemical assays but has now been supplemented with genetic methods such as PCR and sequencing. [27,29]. We guide the reader to Lam et al [27] for a perspective on the history, benefits, and challenges of different serotyping methods in P. aeruginosa.…”

Heterogenous susceptibility to R-pyocins in populations ofPseudomonas aeruginosasourced from cystic fibrosis lungs

“…Sam files generated from mapping each isolate to PAO1 were used to visualize coverage with BRIG (version 0.95) (118) and CGView (119). Sequences were compiled into de novo assemblies using SPAdes (120) through KBASE (119) for serotype prediction with PAst 1.0 (122) and strain typing with PubMLST (123). Table S1 Supplementary Data 1 R-type specificity is determined by the sequence on the foot of the tail fiber, which is believed to recognize glycosylated structures on the outer membrane (such as the LPS).…”

A multidrug resistant clinical P. aeruginosa isolate in the MLST550 clonal complex: uncoupled quorum sensing modulates the interplay of virulence and resistance

“…2A), but the complete genome sequences of these two strains are unavailable. PAst analysis revealed that PA154197 belongs to the Serotype O9 (26) (Table 1). Analyzing its accessory genome identified 21 genome islands (GIs) including 3 prophages (Table S1).…”

“…The multiple locus sequence type (MLST) and serotype of PA154197 was predicted based on the PubMLST database (www.pumlst.org) and the Pseudomonas aeruginosa serotyper (PAst) tool (26).…”