Fluorescent Amplified Fragment Length Polymorphism Probabilistic Database for Identification of Bacterial Isolates from Urinary Tract Infections

Abstract

The ability of the fluorescent amplified fragment length polymorphism (FAFLP) technique to identify bacterial isolates from urinary tract infections (UTIs) was investigated. FAFLP was carried out using the single primer combination MseI plus CT and EcoRI plus 0, and information-rich FAFLP profiles were generated from all 69 UTI isolates studied, which comprised both gram-negative and gram-positive bacteria encompassing eight genera. The genetic relatedness of these 69 bacteria was determined by cluster analysis, and this revealed eight main groups corresponding to the eight bacterial genera. Finer discrimination on the same dendrogram showed species and subspecies differentiations, thus demonstrating the potential of FAFLP for describing a wide diversity range within microbial populations. The interpretation of FAFLP profiles is often complicated because it relies upon the investigator interpreting dendrograms; this process may be subjective if the tree is complicated, particularly if it includes polytomies (unresolved nodes). Therefore, we have developed a method based on Bayes' theorem for the identification of bacteria against an FAFLP probabilistic identification matrix. Thus, FAFLP is suitable for the objective identification of causal agents of UTI, and the procedure offers great potential in the clinical laboratory.Urinary tract infections (UTIs) are of significant clinical concern worldwide. For example, the annual incidence in England and Wales is about 2.5 million, with the family doctor consultation rate for women with UTI being 63.5 consultations/1,000 women/year (37). The fact that UTIs mainly afflict women is mirrored in the United States, where 11.3 million women had at least one presumed UTI treated with antibiotics in 1995 (10). Nosocomial UTI is also common (3), as is incidence in the elderly and children, with 2% of boys and 8% of girls showing clinical symptoms by the age of 7 (6). The bacteria typically associated with UTI are members of the family Enterobacteriaceae, predominantly Escherichia coli (the causative organism of Ͼ50% of all cases) and Klebsiella spp., while gram-positive bacteria, in particular enterococci, are also a significant problem (29).Despite such clinical importance, there are considerable variations in the approaches of laboratories and physicians towards UTIs. Routine diagnosis includes a quantitative microbial count of urine. Counts of Ͼ10 5 organisms/ml are regarded as showing significant bacteriuria (25). Diagnosis is followed by characterization of the causal agent and sometimes antibiotic sensitivity tests to determine appropriate courses of treatment, because the empirical choice of an effective treatment is becoming more difficult as urinary pathogens become increasingly resistant to commonly used antibiotics (15).Further identification of the isolates may not be undertaken except in complex and acute cases (27), because conventional methods are expensive, time-consuming, and labor-intensive (11). However, recurrent infections are common, particularly in childr...

Ultra-violet resonance Raman spectroscopy for the rapid discrimination of urinary tract infection bacteria

“…Twenty clinical bacterial isolates from patients with UTI were obtained from Bronglais Hospital, Aberystwyth as previously reported [20]. Identification by API20E and subsequent sequencing by FAFLP showed the isolates to belong to: E. coli (five strains coded: Eco7, Eco13, Eco17, Eco41, Eco48), Klebsiella oxytoca (one strain coded: kox108), Klebsiella pneumoniae (four strains coded: kp51, kp52, kp59, kp61), Enterococcus spp.…”

“…Cluster analysis was performed in Matlab as previously described [1–22]. Briefly, a PC‐DFA classification model was constructed using quadruplicate spectra of four isolates from each genus under study.…”

A pith necrosis caused by Xanthomonas perforans on tomato plants

“…The PCR was performed using MseI-primer (5′-GATGAG TCCTGAGTAAC-3′) and EcoRI-primer (5′-GACTGCGTACCAATTCA-3′) labelled at the 5′ end with cy5 fluorophore. All PCR reactions were performed in GeneAmp PCR system 9600 (Perkin Elmer) following a previously described protocol (Kassama et al 2002). T4 DNA ligase, dNTPs and Taq polymerase were purchased from Invitrogen-Life Technologies (Paisley, United Kingdom) and all primers and oligos were purchased from MWG Biotech Inc. (High Point, NC, USA).…”

Occurrence of Pseudomonas syringae pv. coryli on Hazelnut Orchards in Sicily, Italy and Characterization by Fluorescent Amplified Fragment Length Polymorphism

“…All PCRs were performed in a DNA thermal cycler (GeneAmp PCR system 9600; Perkin Elmer, Norwalk, CT, USA) following a previously described protocol (Kassama et al., 2002) with some modifications: 60 s at 94°C, 30 s at 65°C and 60 s at 72°C for one cycle; a 12 cycles touch down PCR with annealing temperature reduced from 65°C by 0.7°C at each cycle; and 25 cycles of 30 s of denaturation at 94°C, 60 s of annealing at 56°C, and 60 s of extension at 72°C. This ‘touchdown’ PCR protocol was used to minimize PCR artefacts.…”

“…This technique is accurate, discriminatory, reproducible and capable of standardization. fAFLP presents as a highly sensitive rapid technique used for human and plant pathogens typing (Ticknor et al., 2001; Kassama et al., 2002; Ahmed et al., 2003; Roumagnac et al., 2004; Cirvilleri et al., 2006b).…”