A multiplexed, indirect enzyme-linked immunoassay for the detection and differentiation of E. coli from other Enterobacteriaceae and P. aeruginosa from other glucose non-fermenters.

Gram-negative bacteria (GNB) are important causes of community (CA) and hospital (HA)- associated infections. Here we describe the development of an indirect ELISA (I-ELISA), which can be used to detect and differentiate the Enterobacteriaceae Escherichia coli, and glucose non-fermenter Pseudomonas aeruginosa from other GNB species. The I-ELISA utilizes six antibodies for bacterial speciation, which were grouped according to their bacterial targets; Enterobacteriaceae (SL-EntA and CH1810 mAb), Escherichia coli (SL-EcA and 6103-46 mAb), Pseudomonas aeruginosa (SL-PaA and SL-PaB). The six, anti-GNB antibodies were first screened against a panel of well-characterized clinical GNB isolates to optimize assay conditions and to determine individual antibody sensitivity and specificity. When tested against a diverse, blinded panel of 94 GNB clinical isolates, the I-ELISA exhibited the following sensitivity/specificity for each target: Enterobacteriaceae (94.4%/95%), E. coli (82.6%/88.7%), P. aeruginosa (83.3%/96%). An I-ELISA to detect and differentiate the most common GNB pathogens offers advantage in terms of simplicity over diagnostic tests currently used in most clinical settings.

Globally dispersed mobile drug-resistance genes in gram-negative bacterial isolates from patients with bloodstream infections in a US urban general hospital.

Mobile drug-resistance genes with identical nucleic acid sequences carried by multidrug-resistant Escherichia coli strains that cause community-acquired infections are becomingly increasingly dispersed worldwide. Over a 2-year period, we analysed gram-negative bacterial (GNB) pathogens from the blood of inpatients at an urban public hospital to determine what proportion of these isolates carried such globally dispersed drug-resistance genes. Of 376 GNB isolates, 167 (44 %) were Escherichia coli, 50 (13 %) were Klebsiella pneumoniae, 25 (7 %) were Pseudomonas aeruginosa, 25 (7 %) were Proteus mirabilis and 20 (5 %) were Enterobacter cloacae; the remainder (24 %) comprised 26 different GNB species. Among E. coli isolates, class 1 integrons were detected in 64 (38 %). The most common integron gene cassette configuration was dfrA17-aadA5, found in 30 (25 %) of 119 drug-resistant E. coli isolates and in one isolate of Moraxella morganii. Extended-spectrum ß-lactamase (ESBL) genes were found in 16 E. coli isolates (10 %). These genes with identical sequences were found in nearly 40 % of bloodstream E. coli isolates in the study hospital, as well as in a variety of bacterial species from clinical and non-clinical sources worldwide. Thus, a substantial proportion of bloodstream infections among hospitalized patients were caused by E. coli strains carrying drug-resistance genes that are dispersed globally in a wide variety of bacterial species.