Genomic analysis of the initial dissemination of carbapenem-resistant Klebsiella pneumoniae clones in a tertiary hospital.

Carbapenem-resistant Klebsiella pneumoniae is a major cause of hospital-acquired infections and the fastest-growing pathogen in Europe. Carbapenem resistance was detected at the Consorcio Hospital General Universitario de Valencia (CHGUV) in early 2015, and there has been a significant increase in carbapenem-resistant isolates since then. In this study, we collected carbapenem-resistant isolates from this hospital during the period of increase (from 2015 to 2019) and studied how K. pneumoniae carbapenem-resistant isolates emerged and spread in the hospital. A total of 225 isolates were subjected to whole-genome sequencing with Illumina NextSeq. We characterized the isolates by identifying lineages and antimicrobial resistance genes and plasmids, especially those related to reduced carbapenem susceptibility. Our findings show that the initial carbapenem resistance emergence and dissemination at the CHGUV occurred during a short period of 1 year. Furthermore, it was complex, involving six different lineages of types ST307, ST11, ST101 and ST437, different resistance-determinant factors, including OXA-48, NDM-1, NDM-23 and DHA-1, and different plasmids.

Tracking the Emergence and Dissemination of a blaNDM-23 Gene in a Multidrug Resistance Plasmid of Klebsiella pneumoniae.

Since the discovery of blaNDM-1, NDM ß-lactamases have become one of the most widespread carbapenemases worldwide. To date, 43 different NDM variants have been reported but some, such as blaNDM-23, have not been characterized in detail yet. Here, we describe the emergence of a novel blaNDM-23 allele from a blaNDM-1 ancestor and the multidrug resistance plasmid that has disseminated it through a Klebsiella pneumoniae ST437 clone in several Spanish hospitals. Between 2016 and 2019, 1,972 isolates were collected in an epidemiological survey for extended-spectrum-ß-lactamase (ESBL)-producing Klebsiella pneumoniae in the Comunitat Valenciana (Spain). Three carbapenem-resistant strains failed to be detected by carbapenemase-producing Enterobacteriaceae (CPE) screening tests. These isolates carried a blaNDM-23 gene. To characterize this gene, its emergence, and its dissemination, we performed antimicrobial susceptibility tests, hybrid sequencing with Illumina and Nanopore technologies, and phylogenetic analyses. The MICs of the blaNDM-23 allele were identical to those of the blaNDM-1 allele. The blaNDM-23 allele was found in 14 isolates on a 97-kb nonmobilizable, multidrug-resistant plasmid carrying 19 resistance genes for 9 different antimicrobial families. In this plasmid, the blaNDM-23 gene is in the variable region of a complex class 1 integron with a singular genetic environment. The small genetic distance between blaNDM-23-producing isolates reflects a 5-year-long clonal dispersion involving several hospitals and interregional spread. We have characterized the genomic and epidemiological contexts in the emergence and community spread of a new blaNDM-23 allele in a multidrug resistance (MDR) plasmid of Klebsiella pneumoniae. IMPORTANCE At a time when antimicrobial resistance has become one of the biggest concerns worldwide, the emergence of novel alleles and extremely drug-resistant plasmids is a threat to public health worldwide, especially when they produce carbapenem resistance in one of the most problematic pathogens, such as Klebsiella pneumoniae. We used genomic epidemiology to describe the emergence of a novel NDM-23 allele and identify it in a MDR plasmid that has disseminated through a K. pneumoniae ST437 clone in several hospitals in Spain. Using bioinformatic and phylogenetic analyses, we have traced the evolutionary and epidemiological route of the new allele, the hosting plasmid, and the strain that carried both of them from Pakistan to Spain. A better understanding of the NDM-producing K. pneumoniae populations and plasmids has made evident the spread of this clone through the region, enhancing the importance of genomic surveillance in the control of antimicrobial resistance.

Evaluation of a mass spectrometry and Vitek 2 combined protocol for rapid identification and susceptibility testing of Enterobacterales directly from positive blood cultures / Evaluación de la combinación de espectrometría de masas y el sistema Vitek 2 para la identificación rápida y el estudio de sensibilidad de Enterobacterales directamente desde frascos de hemocultivo positivos

OBJECTIVE: The aim was to evaluate a rapid method which would combine identification and susceptibility testing directly from positive blood cultures for Gram-negative bacilli of the Enterobacterales. MATERIAL AND METHODS: Gram-negative rods from blood cultures were directly identified by MALDI-TOF. Samples with Enterobacterales were selected for direct antimicrobial susceptibility testing by Vitek 2. The results were compared to those obtained with our laboratory's standard method. RESULTS: MALDI-TOF directly from blood cultures identified correctly 83% of the samples. Enterobacterales (n = 68) were identified at gender and species level in 85% of blood cultures with a score >1.7. In general, MICs were obtained after 7 h. MICs of amoxicillin-clavulanate, amikacin and ciprofloxacin showed in almost 50% of the cases after 5 h. CONCLUSIONS: A simple procedure with low cost and reduced working time makes it possible to integrate both identification and susceptibility testing directly from blood cultures. Thus, this protocol could offer advantages when it comes to selection and cost of treatment and patients' clinical outcomes

OBJETIVO: Evaluar un método rápido de identificación y estudio de sensibilidad directamente desde hemocultivos positivos para bacilos gramnegativos del orden Enterobacterales. MATERIAL Y MÉTODOS: Los hemocultivos con bacilos gramnegativos fueron utilizados para identificación mediante MALDI-TOF, seleccionándose aquellos con Enterobacterales para estudio de sensibilidad con Vitek 2. Los resultados fueron comparados con el método estándar del laboratorio. RESULTADOS: MALDI-TOF identificó correctamente el 83% de las muestras obtenidas directamente de hemocultivos. En caso de Enterobacterales (n = 68), el 85% se identificaron a nivel de género y especie con un score > 1,7. El tiempo necesario para la obtención de CMIs fue de 7 horas, reduciéndose a 5 en amoxicilina-clavulánico, amicacina y ciprofloxacino, casi en el 50% de los casos. CONCLUSIONES: Un protocolo simplificado, con bajo coste y reducido tiempo de trabajo, combinando identificación y estudio de sensibilidad directamente desde hemocultivos, podría proporcionar beneficios en la elección y coste del tratamiento, y mejora clínica del paciente

Molecular epidemiology and drug-resistance mechanisms in carbapenem-resistant Klebsiella pneumoniae isolated in patients from a tertiary hospital in Valencia, Spain.

OBJECTIVES: The aim of this study has been to characterize carbapenem-resistant Klebsiella pneumoniae isolates and to determine the resistance mechanisms involved, the clonal relationship between strains and clinical and demographical data of the infected patients. METHODS: Clinical and demographical data from patients were collected and statistically analysed. Antimicrobial susceptibility testing was performed and resistance genes were detected both phenotypically and genotypically. Conjugation assays were performed to show horizontal transferability of resistance genes. Clonal relationship was also studied. Next-generation sequencing (NGS) was performed to obtain information regarding resistance genes, sequence types, virulence factors and plasmid types. RESULTS: Statistical significance was shown by the presence of an infection if there had been a previous hospital stay; urinary catheter carriage and chronic renal disease also indicated higher probabilities of being infected. More than 95% of the isolates were non-susceptible to third-generation cephalosporins, and more than 90% were non-susceptible to quinolones. Phenotypic and genotypic methods for resistance detection were concordant and later confirmed by NGS. This is the first detection of OXA-48, NDM-1 and CTX-M-15 co-production in the area. No plasmid-mediated colistin resistance was found. Tetracycline, sulfonamides and aminoglycoside resistance genes were found in almost all the isolates studied. No virulence factors were detected. Multilocus sequence typing showed more than 15 different sequence types, with ST101, ST307 and ST11 being the most prevalent. CONCLUSIONS: This study is the first to report such a large group of OXA-48 carbapenemases with clonal dissemination among carbapenem-resistant K. pneumoniae in Valencia. This is also the first detection of OXA-48, NDM-1 and CTX-M-15 co-production in the area.

Evaluation of a mass spectrometry and Vitek 2 combined protocol for rapid identification and susceptibility testing of Enterobacterales directly from positive blood cultures.

OBJECTIVE: The aim was to evaluate a rapid method which would combine identification and susceptibility testing directly from positive blood cultures for Gram-negative bacilli of the Enterobacterales. MATERIAL AND METHODS: Gram-negative rods from blood cultures were directly identified by MALDI-TOF. Samples with Enterobacterales were selected for direct antimicrobial susceptibility testing by Vitek 2. The results were compared to those obtained with our laboratory's standard method. RESULTS: MALDI-TOF directly from blood cultures identified correctly 83% of the samples. Enterobacterales (n=68) were identified at gender and species level in 85% of blood cultures with a score >1.7. In general, MICs were obtained after 7h. MICs of amoxicillin-clavulanate, amikacin and ciprofloxacin showed in almost 50% of the cases after 5h. CONCLUSIONS: A simple procedure with low cost and reduced working time makes it possible to integrate both identification and susceptibility testing directly from blood cultures. Thus, this protocol could offer advantages when it comes to selection and cost of treatment and patients' clinical outcomes.