Diagnostic Potential of Monoclonal Antibodies Specific to the Unique O-Antigen of Multidrug-Resistant Epidemic Escherichia coli Clone ST131-O25b:H4

Abstract

bThe Escherichia coli lineage sequence type 131 (ST131)-O25b:H4 is a globally spread multidrug-resistant clone responsible for a great proportion of extraintestinal infections. Driven by the significant medical needs associated with this successful pathogenic lineage, we generated murine monoclonal antibodies (MAbs) against its lipopolysaccharide (LPS) O25b antigen in order to develop quick diagnostic tests. Murine monoclonal antibodies were generated by immunizing mice with whole killed nonencapsulated ST131-O25b E. coli cells and screening hybridoma supernatants for binding to purified LPS molecules obtained from an E. coli ST131-O25b clinical isolate. The MAbs selected for further study bound to the surface of live E. coli O25b strains irrespective of the capsular type expressed, while they did not bind to bacteria or purified LPS from other serotypes, including the related classical O25 antigen (O25a). Using these specific MAbs, we developed a latex bead-based agglutination assay that has greater specificity and is quicker and simpler than the currently available typing methods. The high specificities of these MAbs can be explained by the novel structure of the O25b repeating unit elucidated in this article. Based on comparative analysis by nuclear magnetic resonance (NMR) and mass spectrometry, the N-acetyl-fucose in the O25a O-antigen had been replaced by O-acetylrhamnose in the O25b repeating unit. The genetic determinants responsible for this structural variation were identified by aligning the corresponding genetic loci and were confirmed by trans-complementation of a rough mutant by the subserotype-specific fragments of the rfb operons.

Bactericidal Monoclonal Antibodies Specific to the Lipopolysaccharide O Antigen from Multidrug-Resistant Escherichia coli Clone ST131-O25b:H4 Elicit Protection in Mice

“…BALB/cJRj mice were immunized three times with ϳ1 ϫ 10 7 CFU of either E. coli 81009 or E. coli 81009⌬kps (30). Mice with the highest titers against O25b antigen were boosted, and their splenocytes were subjected to hybridoma fusion as previously described (30). From five independent fusions, a total of 23 hybridoma clones were selected based on specific binding to purified O25b LPS, assessed by immunoblots and ELISA, and surface staining of live E. coli O25b cells by flow cytometry.…”

“…BALB/cJRj mice were immunized three times with ϳ1 ϫ 10 7 CFU of either E. coli 81009 or E. coli 81009⌬kps (30). Mice with the highest titers against O25b antigen were boosted, and their splenocytes were subjected to hybridoma fusion as previously described (30).…”

“…Surface staining was performed as previously described (30). Overnight cultures of bacteria were diluted 1:100 in LB broth or in a 50% depleted heat-inactivated human serum pool and grown at 37°C to mid-log phase.…”

“…Immunoblotting was performed as described previously (30). Purified and separated LPS blotted onto polyvinylidene difluoride (PVDF) was reacted with 1 g/ml of O25b-specific monoclonal antibodies or with LPS core-specific murine MAb WN1 222-5 (Hycult Biotech).…”

Alternatives to Antibiotics: Phage Lytic Enzymes and Phage Therapy

“…Pathogen-specific monoclonal antibodies are either used independently for disease prevention or in combination with antibiotics to treat bacterial infections [18]. Antibodies neutralize the effects of bacterial toxins [19][20][21][22]; they can also be directed against bacterial antigens [23][24][25] and quorum-sensing signals [26,27] inhibiting biofilm formation enhancing the effect of antibioticbased treatment and can be employed for combating persister cells insensitive to antibiotics [28,29].…”