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Aeromonas hydrophila is a globally occurring, potentially virulent, gram-negative opportunistic pathogen that is known to cause water and food-borne diseases around the world. In this study, we use whole genome sequencing and in silico analyses to identify 14 putative O antigen gene clusters (OGCs) located downstream of the housekeeping genes acrB and/or oprM. We have also identified 7 novel OGCs by analyzing 15 publicly available genomes of different A. hydrophila strains. From the 14 OGCs identified initially, we have deduced that O antigen processing genes involved in the wzx/wzy pathway and the ABC transporter (wzm/wzt) pathway exhibit high molecular diversity among different A. hydrophila strains. Using these genes, we have developed a multiplexed Luminex-based array system that can identify up to 14 A. hydrophila strains. By combining our other results and including the sequences of processing genes from 13 other OGCs (7 OGCs identified from publicly available genome sequences and 6 OGCs that were previously published), we also have the data to create an array system that can identify 25 different A. hydrophila serotypes. Although clinical detection, epidemiological surveillance, and tracing of pathogenic bacteria are typically done using serotyping methods that rely on identifying bacterial surface O antigens through agglutination reactions with antisera, molecular methods such as the one we have developed may be quicker and more cost effective. Our assay shows high specificity, reproducibility, and sensitivity, being able to classify A. hydrophila strains using just 0.1 ng of genomic DNA. In conclusion, our findings indicate that a molecular serotyping system for A. hydrophila could be developed based on specific genes, providing an important molecular tool for the identification of A. hydrophila serotypes. [ABSTRACT FROM AUTHOR]

Proteus species are well-known opportunistic pathogens frequently associated with skin wound and urinary tract infections in humans and animals. O antigen diversity is important for bacteria to adapt to different hosts and environments, and has been used to identify serotypes of Proteus isolates. At present, 80 Proteus O-serotypes have been reported. Although the O antigen structures of most Proteus serotypes have been identified, the genetic features of these O antigens have not been well characterized. The O antigen gene clusters of Proteus species are located between the cpxA and secB genes. In this study, we identified 55 O antigen gene clusters of different Proteus serotypes. All clusters contain both the wzx and wzy genes and exhibit a high degree of heterogeneity. Potential functions of O antigen-related genes were proposed based on their similarity to genes in available databases. The O antigen gene clusters and structures were compared, and a number of glycosyltransferases were assigned to glycosidic linkages. In addition, an O serotype-specific suspension array was developed for detecting 31 Proteus serotypes frequently isolated from clinical specimens. To our knowledge, this is the first comprehensive report to describe the genetic features of Proteus O antigens and to develop a molecular technique to identify different Proteus serotypes. [ABSTRACT FROM AUTHOR]

Abstract Providencia is an opportunistic human pathogen that belongs to the Enterobacteriaceae family. The bacterial cell surface O-antigen is one of the most structurally variable cell constituents and serves as a basis for serotyping gram-negative bacteria. In this work, the genomes of 12 Providencia strains were sequenced, and genes driving O-antigen biosynthesis were analyzed. The O-antigen-synthesizing genes of Providencia are located in the O-antigen gene cluster (OGC) between the cpxA and yibK genes. The gene functions predicted in silico agreed with the known O-antigen structures. All clusters were found to contain both wzx and wzy and exhibit a high degree of heterogeneity. Based on the sero-specific genes, we developed a molecular serotyping system to detect 23 serotypes (from the present and previous studies) for the first time. Five Proteus strains, five Morganella strains, five uropathogenic Escherichia coli (UPEC) strains and 32 Providencia strains with other serotypes were used to assess the specificity of our multiplexed Luminex-based array. Five serogroups (O3, O8, O19, O38 and O52 strains) were used to determine the sensitivity of the suspension array. The detection sensitivity was 0.1 ng genomic DNA, 103 CFU/ml in pure culture, or 104 CFU/ml in mock urine specimens. Furthermore, 29 publicly available Providencia genomes (which have not been serotyped) were analyzed, and 23 novel putative OGC types were identified. In total, we identified 35 new OGCs and developed a molecular serotyping system based on the sero-specific genes. The established classification system can support promising applications in basic research, clinical diagnosis, and epidemiological surveillance. Highlights • Genomes of 12 Providencia strains were sequenced and characteristics of the O-antigen gene clusters (OGCs) were described. • A wzy -based suspension array to detect 23 Providencia serotypes was developed. • Twenty-nine publicly available Providencia genomes were analyzed, and 23 novel putative OGC types were identified. • This is the first molecular detection method based on the diversity of surface polysaccharides of Providencia. [ABSTRACT FROM AUTHOR]