Real-time polymerase chain reaction for rapid detection of genes encoding SHV extended-spectrum β-lactamases

Purpose: This study aimed to develop an improved method for the detection of bacterial SHV-type extended-spectrum β-lactamases (ESBLs). Materials and Methods: Our method was based on real-time polymerase chain reaction (PCR) in which the amplification of the product was monitored with a fluorescent probe. This method enabled the detection of bla_SHV genes with high degrees of sensitivity and specificity. Results: Based on ESBL phenotyping methods and bla gene DNA sequencing, we identified 240 bla genes from 662 Enterobacteriaceae isolated from clinical culture specimens. Of these 240 isolates, 26 had the blaSHV-28 genotype and three had the blaSHV-1 genotype. With our new real-time PCR assay, we detected 29 out of 29 bla_SHV genes in ESBL-producing isolates. Conclusion: This method represents a powerful tool for epidemiological studies of SHV ESBLs. Furthermore, it has potential for use in diagnostic microbiology.     

Iberian wolf as a reservoir of extended-spectrum β-lactamase-producing Escherichia coli of the TEM, SHV, and CTX-M groups

The intensive use of antibiotics in human and veterinary medicine, associated with mechanisms of bacterial genetic transfer, caused a selective pressure that contributed to the dissemination of antimicrobial resistance in different bacteria groups and throughout different ecosystems. Iberian wolf, due to his predatory and wild nature, may serve as an important indicator of environmental contamination with antimicrobial resistant bacteria. The aim of this study was to characterize the diversity of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates within the fecal microbiota of Iberian wolf. Additionally, the identification of other associated resistance genes, phylogenetic groups, and the detection of virulence determinants were also focused on in this study. From 2008 to 2009, 237 fecal samples from Iberian wolf were collected in Portugal. E. coli isolates with TEM-52, SHV-12, CTX-M-1, and CTX-M-14-type ESBLs were detected in 13 of these samples (5.5%). This study reveals the presence of ESBL-producing E. coli isolates, in a wild ecosystem, which could be disseminated through the environment. Moreover, the presence of resistant genes in integrons and the existence of virulence determinants were shown. The association between antibiotic resistance and virulence determinants should be monitored, as it constitutes a serious public health problem.     

VIM and IMP metallo-β-lactamases and other extended-spectrum β-lactamases in Escherichia coli and Klebsiella pneumoniae from environmental samples in a Tunisian hospital

An extremely drug-resistant Enterobacteriaceae species emerged in Kasserine Hospital, Tunisia between 2009 and 2010 causing a local outbreak. We aimed to characterize extended-spectrum β-lactamase (ESBL) and metallo-β-lactamase (MBL)-producing Enterobacteriaceae from the hospital environment. Swabs were collected from ten different wards from Kasserine Hospital, Tunisia. A total of 46 isolates were cultured onto MacConkey agar supplemented with ceftazidime to select for ESBL-producing Enterobacteriaceae. Identification and susceptibility patterns were performed using Phoenix-automated phenotypic identification criteria. Extended spectrum β-lactamases (ESBLs) were detected using cefepime ESBL E-test. Colony blotting was first used to detect the occurrence of bla(SHV) , bla(CTX-M) , bla(CMY) , bla(IMP) , and bla(VIM) genes. PCR was used to amplify these genes, and the amplicons were sequenced and analyzed. Total DNA was digested with XbaI, and PFGE was used to type the major isolates that produced IMP-1. Among the 46 isolates, 63% were Klebsiella pneumoniae, 13% were Escherichia coli, 8.7% were Proteus mirabilis, 6% were Enterobacter cloaceae, 4.3% were Providencia rettgeri, 2.5% were Serratia marcescens, and 2.5% were Pantoea agglomerans. PCR amplification and DNA sequencing showed that hospital environment isolates produced SHV-125, CTX-M-15, CMY-2 ESBLs, and IMP-1 and VIM-2 MBLs. PFGE typing showed the emergence of IMP-1 MBL-producing K. pneumoniae isolates that were not clonal. In this study, we report the first characterization of IMP-1 and VIM-2 MBL-producing K. pneumoniae and E. coli isolates collected from Kasserine Hospital, Tunisia.     

Evaluation of a commercial microarray as a confirmation test for the presence of extended-spectrum β-lactamases in isolates from the routine clinical setting

Since the diagnostic characteristics of the Check-KPC ESBL microarray as a confirmation test on isolates obtained in a routine clinical setting have not been determined, we evaluated the microarray in a random selection of 346 clinical isolates with a positive ESBL screen test (MIC >1 mg/L for cefotaxime or ceftazidime or an ESBL alarm from the Phoenix or Vitek-2 expert system) collected from 31 clinical microbiology laboratories in the Netherlands in 2009. Using sequencing as the reference method the sensitivity of the microarray was 97% (237/245), the specificity 98% (97/99), the positive predictive value 99% (237/239) and the negative predictive value 92% (97/105).     

Evaluation of a diagnostic flow chart for detection and confirmation of extended spectrum β-lactamases (ESBL) in Enterobacteriaceae

This study aimed to develop a modular, diagnostic algorithm for extended spectrum β-lactamase (ESBL) detection in Enterobacteriaceae. Clinical Enterobacteriaceae strains (n = 2518) were screened for ESBL production using Clinical and Laboratory Standards Institute (CLSI) breakpoints for third-generation cephalosporins and by synergy image detection (clavulanic acid/extended-spectrum cephalosporins). Isolates screening positive for ESBL (n = 242, 108 by critical CLSI diameters alone, five by double disk synergy test (DDST) alone, and 129 by both critical diameters and DDST) and 138 ESBL screening negative isolates (control group) were investigated by molecular methods considered to be the reference standard (multiplex CTX-M type PCR, TEM and SHV type sequence characterization). One hundred and twenty-four out of 242 Enterobacteriaceae isolates screening positive for ESBL were confirmed to be ESBL positive by the reference standard, the majority of them in E. coli, K. pneumoniae and E. cloacae (94, 17 and nine isolates, respectively). Prevalence of ESBL production ranged from < 1% for P. mirabilis to 4.7%, 5.1% and 6.6%, for K. pneumoniae, E. cloacae and E. coli, respectively. Combining CLSI ceftriaxone and cefpodoxime critical ESBL diameters was found to be the most sensitive phenotypic screening method (sensitivity 99.2%). Combining critical diameters of cefpodoxime and ceftriaxone with DDST for cefpodoxime resulted in a sensitivity of 100%. For phenotypic confirmation, combining the CLSI recommended combined disk test (CDT) for ceftazidime and cefotaxime amended with a cefepime CDT was highly sensitive (100%) and specific (97.5%). With respect to the studied population, the diagnostic ESBL algorithm developed would have resulted in sensitivity and specificity of 100%. The corresponding flow chart is simple, easy to use, inexpensive and applicable in the routine diagnostic laboratory.     

High rate of faecal carriage of extended-spectrum β-lactamase and OXA-48 carbapenemase-producing Enterobacteriaceae at a University hospital in Morocco

Carbapenemase-producing Enterobacteriaceae isolates are being increasingly reported, particularly from countries surrounding the Mediterranean area. We aimed to quantify the prevalence of carbapenemase- and extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in rectal swabs from hospitalized patients in a University hospital in Morocco, and to compare the performance of three screening media: ChromID ESBL (bioMérieux), Brilliance CRE (OXOID, Thermofisher) and SUPERCARBA (home made). Genetic detection and plasmid analysis were performed by PCR and sequencing. Strain comparison was performed by multi-locus sequence typing and the Diversilab technique (bioMérieux). The prevalence of multidrug-resistant Enterobacteriaceae was high, with 33 ESBL producers (42.85%, mainly CTX-M-15) and 10 OXA-48 producers (13%), corresponding to two major clones of K. pneumoniae (70%) and a clone of Enterobacter cloacae (30%). The three screening media showed the same sensitivity for detection of carbapenemase-producing Enterobacteriaceae, whereas the SUPERCARBA medium was more specific than the two other media. The average faecal carriage of ESBL or carbapenemase-producing Enterobacteriaceae varied from 1 × 10² to >1 × 10⁸ CFU/g of stools. This study shows a high prevalence of multidrug-resistant Enterobacteriaceae, and particularly of OXA-48 producers. The new carbapenem-containing medium, SUPERCARBA, was as sensitive as Brilliance CRE and ChromID ESBL, and more specific for the detection of Enterobacteriaceae expressing those carbapenemases.     

Molecular and phenotypic characterization of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum β-lactamases with focus on CTX-M in a low-endemic area in Sweden

During the last decade increasing prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae has been detected worldwide, mainly due to dissemination of Escherichia coli and Klebsiella pneumoniae producing CTX-M-type ESBLs. CTX-M-15 is the most widespread CTX-M type, and the predominant type in various countries. Dissemination of ESBL-producing organisms is caused not only by horizontal transfer of plasmids, but also by clonal spread of ESBL-producing strains. In this study, the molecular epidemiology of class A ESBL (ESBL(A))-producing E. coli and K. pneumoniae isolated in ?rebro County, Sweden, was investigated. Out of 200 ESBL(A) -producing E. coli and K. pneumoniae isolates, collected over a 10-year period, 87% were producing CTX-M, belonging to subgroup CTX-M-1 (64%), CTX-M-9 (34%), or CTX-M-2 (2%). The remaining isolates were producing variants of SHV and TEM. Sequencing of the bla(CTX-M) genes revealed 10 different CTX-M types, with a dominance of CTX-M-15 (E. coli 54%, K. pneumoniae 50%) followed by CTX-M-14 (E. coli 28%, K. pneumoniae 27%). Phenotypic characterization of the CTX-M-producing isolates was performed using the PhenePlate system. Although a few minor clusters of CTX-M-15 and CTX-M-14 producers were identified, the majority of the isolates did not appear to be clonally related.     

Evaluation of CHROMagar™-Serratia agar,a new chromogenic medium for the detection and isolation of Serratia marcescens

European Journal of Clinical Microbiology & Infectious Diseases - A comparative analysis of the performance of the new selective chromogenic CHROMagar?-Serratia culture medium for...     

A sensitive and specific phenotypic assay for detection of metallo-β-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid,dipicolinic acid and cloxacillin

Enterobacteriaceae producing carbapenemases, such as KPC or metallo-β-lactamases (MBLs), have emerged on several continents. Phenotypic tests are urgently needed for their rapid and accurate detection. A novel carbapenemase detection test, comprising a meropenem disk, and meropenem disks supplemented with 730 μg of EDTA, 1000 μg of dipicolinic acid (DPA), 600 μg of aminophenylboronic acid (APBA), or 750 μg of cloxacillin, was evaluated against Klebsiella pneumoniae isolates with KPC (n = 34), VIM (n = 21), IMP (n = 4) or OXA-48 (n = 9) carbapenemases, and carbapenem-resistant Enterobacteriaceae with porin loss in combination with an extended-spectrum β-lactamase (ESBL) (n = 9) or AmpC hyperproduction (n = 5). Commercially available diagnostics tablets from Rosco containing meropenem and the same inhibitors as described above (except EDTA) were also evaluated. An increased meropenem inhibition zone was sought in the presence of each added β-lactamase inhibitor. APBA had excellent sensitivity for detecting K. pneumoniae with KPC enzymes. Isolates with combined AmpC hyperproduction and porin loss were also positive in the APBA test but, unlike KPC producers, showed cloxacillin synergy. Both DPA and EDTA had excellent sensitivity for detection of MBL-producing K. pneumoniae. However, EDTA showed poor specificity, with positive results noted for 1/9 ESBL-producing isolates, for 4/34 KPC-producing isolates, and for 4/9 OXA-48-producing isolates, whereas all of these were negative when DPA was used. The in-house test distinguished accurately between several different mechanisms mediating reduced susceptibility to carbapenems in Enterobacteriaceae. The commercial combination tablets from Rosco performed similarly to the in-house test, with the exception of one false-positive MBL result and one false-positive KPC result among the OXA-48 producers.     

Management of serious infections caused by metallo β-lactamases with or without OXA-48-like expressing Enterobacterales with aztreonam and ceftazidime/avibactam combination: Dosing strategy for better clinical outcome

Serious infections caused by MBLs with or without OXA-48-like expressing Enterobacterales remain challenging to treat. Since aztreonam is stable to MBLs, it can be combined with ceftazidime/avibactam to protect against concurrently expressed ESBLs and class C β-lactamases in MBL pathogens. However, in the light of dose-limiting hepatotoxicity of aztreonam, short half life of avibactam, significant protein binding of aztreonam, appropriate dosing and method of administration to optimize PK/PD and toxicodynamics for this combination is being debated. Based on in-vitro PK/PD studies, simultaneous administration of 6/1.5 g of ceftazidime/avibactam and 8 g of aztreonam per day has been recently suggested.     

Evaluation of the Bio-Rad Dx CT/NG/MG® assay for simultaneous detection of Chlamydia trachomatis,Neisseria gonorrhoeae and Mycoplasma genitalium in urine

We evaluated the performance of the Bio-Rad real-time Dx CT/NG/MG® assay for detection of C. trachomatis, N. gonorrhoeae and M. genitalium on a collection of 441 urine samples from sexually transmitted infections, or travellers consultations and from anonymous sperm donors that were previously analysed with the Abbott RealTime CT/NG assay. Samples positive for C. trachomatis or N. gonorrhoeae with the Abbott assay had all previously been confirmed with an in-house real-time PCR assay. Samples positive for M. genitalium with the Bio-Rad assay were subsequently analysed by an in-house real-time PCR. On a total of 441 urines, 104 samples were positive for C. trachomatis, 12 were positive for N. gonorrhoeae and seven were positive for M. genitalium. After retesting of discrepant results, the test results were completely concordant, resulting in a calculated sensitivity and specificity of the Bio-Rad assay of 98.1 % and 100 % for C. trachomatis and of 91.7 % and 100 % for N. gonorrhoeae. Results for M. genitalium with the Bio-Rad assay were also concordant with the results of an in house PCR. We also evaluated the performance of automated nucleic acid extractions of urine samples with the NucliSENS easyMAG (bioMérieux) compared to the manual DNA extraction prescribed by the insert of the kit. The easyMAG extraction gave lower Ct values, relieved inhibition and had a lower hands-on time.     

Real-time PCR assay and a synthetic positive control for the rapid and sensitive detection of the emerging resistance gene New Delhi Metallo-β-lactamase-1 (<Emphasis Type="Italic">bla</Emphasis><Subscript>NDM-1</Subscript>)

Carbapenems are important last-line antibiotics for the treatment of hospital infections. Enterobacteriaceae (such as Klebsiella pneumoniae or Escherichia coli) expressing the “New Delhi Metallo-β-lactamase” gene bla _NDM-1 are resistant to carbapenems and were predicted to become a major global health problem. To cope with this emerging threat, there is a need for rapid and sensitive molecular assays to detect bla _NDM-1 in carbapenem-resistant Enterobacteriaceae from clinical isolates. In diagnostic laboratories, real-time PCR is the current gold standard for the sensitive and rapid detection of pathogens. We describe a real-time PCR assay as well as two conventional PCR assays to detect bla _NDM-1. Only minute amounts of total DNA extracted from one bacterial colony are sufficient to allow detection of bla _NDM-1 by real-time PCR within less than 1 h. We also introduce a chemically synthesized bla _NDM-1 gene as a convenient positive control for those laboratories wishing to setup in-house assays for bla _NDM-1 detection. Importantly, our study represents a proof of principle for the usefulness of rapidly synthesized genes serving as positive controls for novel diagnostic PCR assays of emerging pathogens during the initial phase after their discovery when biological isolates are still rare and not commonly available.