Are GES carbapenemases underdiagnosed? An allelic discrimination assay for their accurate detection and differentiation.

GES (Guiana Extended Spectrum) carbapenemases belong to "minor class A carbapenemases" and its prevalence could be underestimated due to the lack of specific tests. The aim of this study was to develop an easy PCR method to differentiate between GES ß-lactamases with or without carbapenemase activity, based on an allelic discrimination system of SNPs that encode E104K and G170S mutations, without need of sequencing. Two pair of primers and Affinity Plus probes, labeled with different fluorophores; FAM/IBFQ and YAK/IBFQ, were designed for each one of the SNPs. This allelic discrimination assay allows to detect in real time the presence of all type of GES- ß-lactamases, being able to differentiate between carbapenemases and extended-spectrum ß-lactamase (ESBL), through a quick PCR test that avoid costly sequencing approaches and could help to decrease the current underdiagnosis of minor carbapenemases that scape of phenotypic screenings.

In vivo selection of KPC-94 and KPC-95 in Klebsiella pneumoniae isolates from patients treated with ceftazidime/avibactam.

Ceftazidime/avibactam (CZA) is used to treat infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp). Resistance to CZA is commonly related to point mutations in the blaKPC gene. Here we describe the in vivo emergence of CZA resistance in clinical isolates of KPC-Kp from four patients treated with this combination therapy. Four pre-therapy and five post-therapy KPC-Kp isolates were examined. Antibiogram (microdilution and gradient strips) and whole-genome sequencing were performed. The role of KPC mutations was validated by cloning blaKPC genes into competent Escherichia coli. All KPC-Kp isolates recovered before treatment with CZA were susceptible to CZA and produced KPC-3. Five KPC-Kp isolates recovered after treatment were resistant to this combination. Three post-therapy isolates from two patients produced KPC-31 (D179Y mutation). Additionally, we identified the novel substitution LN169-170H (KPC-94) in one isolate, and the combination of two independently described mutations, D179Y and A172T (KPC-95), in another isolate. All KPC-Kp isolates belonged to sequence type 512 (ST512). All CZA-resistant isolates with blaKPC variants had restoration of carbapenem susceptibility. In conclusion, resistance to CZA was related to blaKPC mutations, including the new KPC-94 and KPC-95 alleles, which do not cause carbapenem resistance.

RedLabRA; a Spanish Network of Microbiology Laboratories for the Surveillance of Antibiotic Resistant Microorganisms.

There is an urgent need to control the clinical and public health impact that antibiotic resistance (AR) causes worldwide. Any measure for its control must be based on an up-to-date and comprehensive knowledge of the situation. However, it is difficult to determine the current dimension of AR because a large part of the available information is based on heterogeneous, insufficiently unified and retrospective data. The integration of genomic information in the surveillance of AR is another important factor for improvement. The Spanish Network of Laboratories for the Surveillance of Resistant Microorganisms (RedLabRA) is a structured network of interconnected microbiology laboratories developed within the Spanish National Plan against Antibiotic Resistance. Its main objective is to support the diagnosis of resistance to antibiotics, integrating molecular characterization in the surveillance.

Is it possible to extrapolate the rates of resistance of Escherichia coli from asymptomatic bacteriuria in pregnant women to those of E. coli in uncomplicated community-acquired UTI?

OBJECTIVE: Treatment of uncomplicated urinary tract infections in primary care is generally empirical without requesting urine culture and based on biased resistance data collected from selected patients, most of them having risk factors for the isolation of resistant microorganisms. In order to overcome the lack of information on the real resistance rates in uncomplicated UTI, we compared antimicrobial phenotype and genotype of Escherichia coli isolated from pregnant women with asymptomatic bacteriuria (culture always performed) with those from women with uncomplicated acute cystitis (culture rarely performed) of different age groups. METHODS: Between September 2017 and March 2018, 103 urines were randomly collected from pregnant women aged between 16 and 47 with asymptomatic bacteriuria (AB) (n=42), not hospitalized women in the same age range with uncomplicated acute cystitis (UAC) (n=31) and women older than 47 not hospitalized with UAC (n=30). Bacteria identification was performed using mass spectrometry and the antibiogram by broth microdilution. Genetic typification was carried out by pulsed-field gel electrophoresis. RESULTS: There are no significant differences between the groups of patients in the antibiotic susceptibility. Likewise, as expected, a wide genetic diversity is observed among the strains of E. coli studied without significant differences between the three groups. CONCLUSIONS: We propose a simple model that could provide better guidance for selection of empirical antimicrobial therapy for non-pregnant women with UAC than do generic hospital antibiogram data based on reliably extrapolating the susceptibility data of strains isolated from pregnant women with AB as representation of women with community-acquired UAC.

A new case report of urinary tract infection due to KPC-3-producing klebsiella pneumoniae (ST258) in Spain. / A propósito de un nuevo caso de infección del tracto urinario por klebsiella pneumoniae (ST258) productor de KPC-3 en España.

OBJECTIVE: We describe the characterization of a new isolated in Spain of Klebsiella pneumoniae ST258 producing KPC-3, carbapenems non-susceptible, recovered from a sample of urine from a patient with urinary tract infection and no history of carbapenems exposure. METHODS: After the isolation, identification of K. pneumoniae was performed by biochemical tests and mass spectrometry. The carbapenems susceptibility testing was performed by microdilution and E-test in cation-adjusted Mueller-Hinton. The study was completed by Rapidec® Carba NP. In order to determine the genetic basis of resistance to carbapenems we used Xpert® Carba-R for carbapenemase type and subtype was subsequently analyzed by amplification by PCR and sequencing. RESULT: We demonstrated by MLST that the strain belonged to the clone of high-risk ST258. CONCLUSIONS: This is the first characterization, in our media, of a clinical isolated of K. pneumoniae ST258 producing KPC-3 and no history of carbapenems exposure.

A propósito de un nuevo caso de infección del tracto urinario por klebsiella pneumoniae (ST258) productor de KPC-3 en España / A new case report of urinary tract infection due to KPC-3-producing klebsiella pneumoniae (ST258) in Spain

OBJETIVO: Presentamos la caracterización de un nuevo aislado en España de Klebsiella pneumoniae ST258 productor de KPC-3, no sensible a carbapenémicos, recuperado de una muestra de orina de una paciente con infección del tracto urinario y sin antecedentes de exposición previa a carbapenémicos. MÉTODOS: Tras el aislamiento, la identificación de K. pneumoniae fue realizada mediante pruebas bioquímicas y espectrometría de masas y la prueba de sensibilidad a carbapenémicos se realizó mediante microdilución y E-test en Mueller-Hinton ajustado para cationes. El estudio se completó mediante Rapidec® Carba NP. Con el fin de determinar las bases genéticas de la resistencia a carbapenémicos se analizó el tipo de carbapenemasa mediante Xpert® Carba-R, posteriormente se subtipo mediante amplificación por PCR y secuenciación. RESULTADO: Mediante MLST, se demostró que la cepa pertenecía al clon de alto riesgo ST258. CONCLUSIONES: Esta es la primera caracterización en nuestro medio de un aislado clínico de K. pneumoniae ST258 productor de KPC-3, sin antecedentes de exposición previa a carbapenémicos

OBJECTIVE: We describe the characterization of a new isolated in Spain of Klebsiella pneumoniae ST258 producing KPC-3, carbapenems non-susceptible, recovered from a sample of urine from a patient with urinary tract infection and no history of carbapenems exposure. METHODS: After the isolation, identification of K. pneumoniae was performed by biochemical tests and mass spectrometry. The carbapenems susceptibility testing was performed by microdilution and E-test in cation-adjusted Mueller-Hinton. The study was completed by Rapidec® Carba NP. In order to determine the genetic basis of resistance to carbapenems we used Xpert® Carba-R for carbapenemase type and subtype was subsequently analyzed by amplification by PCR and sequencing. RESULT: We demonstrated by MLST that the strain belonged to the clone of high-risk ST258. CONCLUSIONS: This is the first characterization, in our media, of a clinical isolated of K. pneumoniae ST258 producing KPC-3 and no history of carbapenems exposure