Molecular diagnosis of bloodstream infections with a new dual-priming oligonucleotide-based multiplex PCR assay.

Mortality from bloodstream infections (BSIs) correlates with diagnostic delay and the use of inappropriate empirical treatment. Early PCR-based diagnosis could decrease inappropriate treatment, improving patient outcome. The aim of the present study was to assess the clinical utility of this molecular technology to diagnose BSIs. We assessed a new dual-priming oligonucleotide-based multiplex PCR assay, the Magicplex Sepsis Test (MST) (Seegene), along with blood culture (BC). A total of 267 patients from the intensive care unit and haematology and emergency departments were enrolled. Clinical data were also used by physicians to determine the likelihood of infection. Ninety-eight (37 %) specimens were positive: 29 (11 %) by both the MST and BC, 29 (11 %) by the MST only, and 40 (15 %) by BC only. The proportion of agreement between the two methods was 73 % (Cohen's κ: 0.45; 0.28-0.6; indicating fair to moderate agreement). According to clinical assessment, 63 (64 %) positive specimens were considered BSIs: 23 (36 %) were positive by both the MST and BC, 22 (35 %) were positive only by BC, and 18 (29 %) were positive only by the MST. Thirty-eight (14 %) positive specimens by the MST and/or BC were considered as contaminants. Of 101 specimens collected from patients receiving antibiotics, 20 (20 %) were positive by the MST and 32 (32 %) by BC. Sensitivity and specificity were 65 % and 92 %, respectively, for the MST and 71 % and 88 %, respectively for BC. We concluded that the MST shows a high specificity but changes in design are needed to increase bacteraemia detection. For viability in clinical laboratories, technical improvements are also required to further automate the process.

Extended-spectrum beta-lactamase-producing Escherichia coli in Spain belong to a large variety of multilocus sequence typing types, including ST10 complex/A, ST23 complex/A and ST131/B2.

In this study, we investigated the population structure of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in Spain and determined possible associations between specific multilocus sequence typing (MLST) types and ESBL types. Ninety-two ESBL-producing E. coli isolates from 11 Spanish hospitals were studied. The predominant ESBLs in this collection were CTX-M-14 (45.7%), SHV-12 (21.7%) and CTX-M-9 (20.6%). Phylogenetic groups and MLST types were studied. Thirty-seven isolates (40.2%) belonged to phylogroup A, 26 (28.3%) to group B1, 13 (14.1%) to group B2 and 16 (17.4%) to group D. Fifty-six sequence types (STs) were identified, but ST131 (eight isolates) and ST167 (five isolates) were the most prevalent. The most common ST complexes were ST10 (13 isolates; 14.3%) and ST23 (10 isolates; 11%). Escherichia coli ST131 carried six different ESBLs (CTX-M-1, CTX-M-9, CTX-M-10, CTX-M-14, CTX-M-15 and SHV-12), E. coli ST10 complex carried five ESBLs and E. coli ST23 complex carried four ESBLs. A great diversity of MLST types was observed among Spanish ESBL-producing E. coli isolates.

Characterization of plasmids encoding blaESBL and surrounding genes in Spanish clinical isolates of Escherichia coli and Klebsiella pneumoniae.

OBJECTIVES: The aim of the study was to characterize plasmids that harbour blaESBL genes and their genetic environment in Escherichia coli and Klebsiella pneumoniae clones circulating in Spain. METHODS: The incompatibility group of plasmids within 58 strains harbouring blaCTX-M (n=45) and blaSHV (n=15) genes was determined by rep-typing-PCR and hybridization. The blaESBL genetic environment was determined by PCR and sequencing. RESULTS: The blaCTX-M-9 genes (n=14) were linked to In60 located in IncI1 (50%) or IncHI2 plasmids (28%). All blaCTX-M-14 genes (n=13) were flanked by ISEcp1 and IS903 and 12 were associated with IncK plasmids. One of two blaCTX-M-10 genes was present in an IncK plasmid, but both genes were linked to a phage-related element. Five of seven blaCTX-M-1 (71%), all three blaCTX-M-32 and one of two blaCTX-M-3 genes were linked to IncN plasmids. The other blaCTX-M-3 gene was linked to IncA/C and the remaining two blaCTX-M-1 genes to IncFII plasmids. Three blaCTX-M-15 genes were associated with IncF (repFIA) and one with IncFII plasmids. All these genes from blaCTX-M group-1 showed the ISEcp1 upstream truncated by different insertion sequences. Forty-three percent of blaSHV-12 genes (n=14) were located in IncI1 plasmids, all flanked by the IS26 and DEOR region. The only detected blaSHV-5 gene was located in an IncFII plasmid and flanked by recF and DEOR regions. CONCLUSIONS: A diversity of the plasmid incompatibility groups that harbour blaESBL genes was observed, except for the blaCTX-M-14 gene. Moreover, a high variability was confirmed in the genetic environment of these genes as a result of insertion and deletion events.

[Characterization and molecular epidemiology of ESBL in Escherichia coli and Klebsiella pneumoniae in 11 Spanish hospitals (2004)]. / Caracterización y epidemiología molecular de betalactamasas de espectro extendido en Escherichia coli y Klebsiella pneumoniae en once hospitales españoles (2004).

INTRODUCTION: The epidemiological distribution of extended-spectrum beta-lactamase (ESBL) types in Escherichia coli and Klebsiella pneumoniae was evaluated in various hospitals in Spain and compared with previous studies. METHODS: A total of 11 Spanish hospitals participated in this study. Each center collected the first 15 isolates of E. coli and the first 5 of K. pneumoniae suspected of being ESBL-producers and isolated during the first quarter of 2004. Clonal study was done by PFGE after total DNA digestion with XbaI and by ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus Sequences-Polymerase Chain Reaction), typing. ESBL-producers were characterized by isoelectric focusing (IEF), PCR and sequencing. RESULTS: A total of 124 strains were collected. PFGE restriction patterns showed considerable diversity among E. coli strains; 4 clusters of 2 strains each were detected. ESBL characterization of 92 E. coli strains showed a predominance of CTX-M-14 (45.7%), CTX-M-9 (20.6%) and SHV-12 (21.7%). Clonal diversity among the 32 K. pneumoniae strains was less pronounced than in E. coli; 3 clusters included 53.1% of strains. The ESBL detected in these strains included a CTX-M type in 20 cases (62.5%) (CTX-M-1, CTX-M-9, CTX-M-14 and CTX-M-15); a SHV type in 11 (34.4%) (SHV-12 and SHV-5) and TEM-4 (3.1%) in 1 case. CONCLUSION: The E. coli and K. pneumoniae strains analyzed in this period displayed a greater diversity of ESBL than has been observed in previous epidemiological studies. Analysis of clonal relationships revealed a greater diversity in E. coli than in K. pneumoniae.

Caracterización y epidemiología molecular de betalacta masas de espectro extendido en Escherichia coli y Klebsiella pneumoniae en once hospitales españoles (2004) / Characterization and molecular epidemiology of ESBL in Escherichia coli and Klebsiella pneumoniae in 11 Spanish hospitals (2004)

INTRODUCCIÓN. Se analizó la distribución epidemiológica de los diferentes tipos de betalacta masas de espectro extendido (BLEE) en Escherichia coli y Klebsiella pneumoniae en distintos hospitales de España y se comparó con estudios previos. MÉTODOS. En 11 hospitales españoles se recogieron los15 primeros aislamientos de E. coli y los 5 primeros de K. pneumoniae con sospecha de ser portadores de BLEE, aislados en el primer trimestre de 2004. Los estudios de clonalidad se realizaron mediante electroforesis en gel de campos pulsantes (PFGE) tras digestión del ADN total con XbaI y mediante ERIC-PCR (Entero bacterial Repetitive Intergenic Concensus Secuences-Polimerase Chain Reaction). Las BLEE se caracterizaron mediante isoelectro enfoque, PCR y secuenciación. RESULTADOS. Se estudiaron 124 aislamientos. El análisis de los patrones de restricción obtenidos por PFGE mostró una gran diversidad clonal entre los aislamientos de E. coli, observándose cuatro agrupaciones de dos cepas en cada una de ellas. En las 92 cepas de E. coli, la caracterización de las BLEE mostró un predominio de CTX-M-14 (45,7%),CTX-M-9 (20,6%) y SHV-12 (21,7%). En las 32 cepas de K. pneumoniae se observó una menor diversidad clonal, detectándose tres agrupaciones que incluían el 53,1%de los aislamientos. Las BLEE detectadas en estas cepas fueron del tipo CTX-M en 20 casos (62,5%) (CTX-M-1,CTX-M-9, CTX-M-14 y CTX-M-15), de tipo SHV en 11(34,4%) (SHV-12 y SHV-5) y TEM-4 (3,1%) en una. CONCLUSIÓN. Las cepas de E. coli y K. pneumoniae analizadas en ese período presentan una mayor diversidad de BLEE que la observada en estudios epidemiológicos realizados con anterioridad. Además, el análisis de la relación clonal definió una gran diversidad en E. coliy menor en K. Pneumoniae (AU)

INTRODUCTION. The epidemiological distribution ofextended-spectrum beta-lactamase (ESBL) typesin Escherichia coli and Klebsiella pneumoniae wasevaluated in various hospitals in Spain and comparedwith previous studies.METHODS. A total of 11 Spanish hospitals participatedin this study. Each center collected the first 15 isolatesof E. coli and the first 5 of K. pneumoniae suspected ofbeing ESBL-producers and isolated duringthe first quarter of 2004. Clonal study was doneby PFGE after total DNA digestion with XbaI andby ERIC-PCR (Enterobacterial Repetitive IntergenicConcensus Secuences-Polimerase Chain Reaction),typing. ESBL-producers were characterized byisoelectric focusing (IEF), PCR and sequencing.RESULTS. A total of 124 strains were collected. PFGErestriction patterns showed considerable diversityamong E. coli strains; 4 clusters of 2 strains each weredetected. ESBL characterization of 92 E. coli strainsshowed a predominance of CTX-M-14 (45.7%),CTX-M-9 (20.6%) and SHV-12 (21.7%). Clonal diversityamong the 32 K. pneumoniae strains was lesspronounced than in E. coli; 3 clusters included 53.1%of strains. The ESBL detected in these strainsincluded a CTX-M type in 20 cases (62.5%)(CTX-M-1, CTX-M-9, CTX-M-14 and CTX-M-15);a SHV type in 11 (34.4%) (SHV-12 and SHV-5)and TEM-4 (3.1%) in 1 case.CONCLUSION. The E. coli and K. pneumoniae strainsanalyzed in this period displayed a greater diversityof ESBL than has been observed in previousepidemiological studies. Analysis of clonalrelationships revealed a greater diversity in E. colithan in K. pneumoniae