Flagellar motility mediates biofilm formation in Aeromonas dhakensis.

Aeromonas dhakensis possesses dual flagellar systems for motility under different environments. Flagella-mediated motility is necessary for biofilm formation through an initial attachment of bacteria to the surface, but this has not been elucidated in A. dhakensis. This study investigates the role of polar (flaH, maf1) and lateral (lafB, lafK and lafS) flagellar genes in the biofilm formation of a clinical A. dhakensis strain WT187 isolated from burn wound infection. Five deletion mutants and corresponding complemented strains were constructed using pDM4 and pBAD33 vectors, respectively, and analyzed for motility and biofilm formation using crystal violet staining and real-time impedance-based assays. All mutants were significantly reduced in swimming (p < 0.0001), swarming (p < 0.0001) and biofilm formation using crystal violet assay (p < 0.05). Real-time impedance-based analysis revealed WT187 biofilm was formed between 6 to 21 h, consisting of early (6-10 h), middle (11-18 h), and late (19-21 h) stages. The highest cell index of 0.0746 was recorded at 22-23 h and biofilms began to disperse starting from 24 h. Mutants Δmaf1, ΔlafB, ΔlafK and ΔlafS exhibited reduced cell index values at 6-48 h when compared to WT187 which indicates less biofilm formation. Two complemented strains cmaf1 and clafB exhibited full restoration to wild-type level in swimming, swarming, and biofilm formation using crystal violet assay, hence suggesting that both maf1 and lafB genes are involved in biofilm formation through flagella-mediated motility and surface attachment. Our study shows the role of flagella in A. dhakensis biofilm formation warrants further investigations.

Aeromonas dhakensis: Clinical Isolates with High Carbapenem Resistance.

Aeromonas dhakensis is ubiquitous in aquatic habitats and can cause life-threatening septicaemia in humans. However, limited data are available on their antimicrobial susceptibility testing (AST) profiles. Hence, we aimed to examine their AST patterns using clinical (n = 94) and non-clinical (n = 23) isolates with dehydrated MicroScan microdilution. Carbapenem resistant isolates were further screened for genes related to carbapenem resistance using molecular assay. The isolates exhibited resistance to imipenem (76.9%), doripenem (62.4%), meropenem (41.9%), trimethoprim/sulfamethoxazole (11.1%), cefotaxime (8.5%), ceftazidime (6%), cefepime (1.7%) and aztreonam (0.9%), whereas all isolates were susceptible to amikacin. Clinical isolates showed significant association with resistance to doripenem, imipenem and meropenem compared to non-clinical isolates. These blacphA were detected in clinical isolates with resistance phenotypes: doripenem (67.2%, 45/67), imipenem (65.9%, 54/82) and meropenem (65.2%, 30/46). Our findings showed that the MicroScan microdilution method is suitable for the detection of carbapenem resistance in both clinical (48.9-87.2%) and non-clinical (4.3-13.0%) isolates. This study revealed that A. dhakensis isolates had relatively high carbapenem resistance, which may lead to potential treatment failure. Continued monitoring of aquatic sources with a larger sample size should be carried out to provide further insights.

Characterization of the relationship between polar and lateral flagellar genes in clinical Aeromonas dhakensis: phenotypic, genetic and biochemical analyses.

Flagellar-mediated motility is a crucial virulence factor in many bacterial species. A dual flagellar system has been described in aeromonads; however, there is no flagella-related study in the emergent human pathogen Aeromonas dhakensis. Using 46 clinical A. dhakensis, phenotypic motility, genotypic characteristics (flagellar genes and sequence types), biochemical properties and their relationship were investigated in this study. All 46 strains showed swimming motility at 30 °C in 0.3% Bacto agar and carried the most prevalent 6 polar flagellar genes cheA, flgE, flgG, flgH, flgL, and flgN. On the contrary, only 18 strains (39%) demonstrated swarming motility on 0.5% Eiken agar at 30 °C and they harbored 11 lateral flagellar genes lafB, lafK, lafS, lafT, lafU, flgCL, flgGL, flgNL, fliEL, fliFL, and fliGL. No association was found between biochemical properties and motility phenotypes. Interestingly, a significant association between swarming and strains isolated from pus was observed (p = 0.0171). Three strains 187, 277, and 289 isolated from pus belonged to novel sequence types (ST522 and ST524) exhibited fast swimming and swarming profiles, and they harbored > 90% of the flagellar genes tested. Our findings provide a fundamental understanding of flagellar-mediated motility in A. dhakensis.

Genetic relatedness and novel sequence types of clinical Aeromonas dhakensis from Malaysia.

Aeromonas dhakensis is an emergent human pathogen with medical importance. This study was aimed to determine the sequence types (STs), genetic diversity, and phylogenetic relationships of different clinical sources of 47 A. dhakensis from Malaysia using multilocus sequence typing (MLST), goeBURST, and phylogenetic analyses. The analysis of a concatenated six-gene tree with a nucleotide length of 2994 bp based on six housekeeping genes (gyrB, groL, gltA, metG, ppsA, and recA) and independent analyses of single gene fragments was performed. MLST was able to group 47 A. dhakensis from our collection into 36 STs in which 34 STs are novel STs. The most abundant ST521 consisted of five strains from peritoneal fluid and two strains from stools. Comparison of 62 global A. dhakensis was carried out via goeBURST; 94.4% (34/36) of the identified STs are novel and unique in Malaysia. Two STs (111 and 541) were grouped into clonal complexes among our strains and 32 STs occurred as singletons. Single-gene phylogenetic trees showed varying topologies; groL and rpoD grouped all A. dhakensis into a tight-cluster with bootstrap values of 100% and 99%, respectively. A poor phylogenetic resolution encountered in single-gene analyses was buffered by the multilocus phylogenetic tree that offered high discriminatory power (bootstrap value = 100%) in resolving all A. dhakensis from A. hydrophila and delineating the relationship among other taxa. Genetic diversity analysis showed groL as the most conserved gene and ppsA as the most variable gene. This study revealed novel STs and high genetic diversity among clinical A. dhakensis from Malaysia.