ESBLs and resistance to ceftazidime/avibactam and ceftolozane/tazobactam combinations in Escherichia coli and Pseudomonas aeruginosa

To evaluate the efficacy of the recently launched β-lactam/β-lactamase inhibitor combinations ceftazidime/avibactam and ceftolozane/tazobactam against ESBL- producing Escherichia coli and Pseudomonas aeruginosa strains.Methods: A series of ESBL-encoding genes (blaTEM, blaSHV, blaCTX-M, blaVEB, blaPER, blaGES and blaBEL) was cloned and expressed in E. coli or P. aeruginosa recipient strains. Cultures of E. coli TOP10 harbouring recombinant plasmids and therefore producing the different ESBLs tested were grown in order to perform measurements of catalytic activities, using benzylpenicillin, ceftazidime and ceftolozane as substrates. IC50s were additionally determined for clavulanic acid, tazobactam and avibactam.Results: We showed here an overall better activity of ceftazidime/avibactam compared with ceftolozane/tazobactam toward ESBL-producing E. coli and P. aeruginosa. Several ESBLs of the GES, PER and BEL types conferred resistance to ceftolozane/tazobactam in E. coli and P. aeruginosa. For GES-6 and PER-1 producers, resistance to ceftolozane/tazobactam could be explained by a high hydrolysis of ceftolozane and a low activity of tazobactam as an inhibitor. On the other hand, PER-producing P. aeruginosa also exhibited resistance to ceftazidime/avibactam.Conclusions: Altogether, the results show that the ESBL PER- 1, which is widespread worldwide, may be a source of resistance to both ceftolozane/tazobactam and ceftazidime/avibactam. Excellent activity of ceftazidime/avibactam was highlighted for both ESBL-producing E. coli and ESBL- producing P. aeruginosa

Phylogeny, Resistome, and Virulome of Escherichia coli Causing Biliary Tract Infections

Escherichia coli is the most frequent Gram-negative bacilli involved in intra-abdominal infections. However, despite high mortality rates associated with biliary tract infections due to E. coli, there is no study focusing on this pathogen. In this study, we have characterized a group of 15 E. coli isolates obtained from 12 patients with biliary tract infections. Demographic and clinical data of the patients were recovered. Phylogeny, resistome, and virulome analysis through whole genome sequencing and biofilm formation were investigated. Among the 15 E. coli isolates, no predominant sequence type (ST) was identified, although 3 of them belonged to unknown STs (20%). Resistance to ampicillin, amoxicillin/clavulanic acid, cotrimoxazole, and quinolones was more present in these isolates; whereas, third and fourth generation cephalosporins, carbapenems, amikacin, tigecycline, and colistin were highly active. Moreover, high diversity of virulence factors has been found, with sfa, fimH, and gad the most frequently detected genes. Interestingly, 26.6% of the E. coli isolates were high biofilm-producers. Altogether, our data characterized for the first time E. coli isolates associated with biliary tract infections in terms of genomic relationship, resistome, and virulome.España, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades (CP15/00132)España, Plan Nacional de I+D+i 2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (RD16/0016/0009

CTX-M-33 Is a CTX-M-15 derivative conferring reduced susceptibility to carbapenems

CTX-M-type extended-spectrum β-lactamases (ESBLs) are widespread among Enterobacterales strains worldwide. The most common variant is CTX-M-15, which hydrolyzes ceftazidime at a high rate but spares carbapenems. Here, we identified CTX-M-33, a point mutation derivative of CTX-M-15 (Asp to Ser substitution at Ambler position 109) that exhibited low carbapenemase activity. β-Lactamase CTX-M-33 was identified in a Klebsiella pneumoniae isolate, belonging to sequence type 405 and lacking the outer membrane protein OmpK36, that was resistant to broad-spectrum cephalosporins and β-lactam/β-lactamase inhibitor combinations and displayed decreased susceptibility to carbapenems. Comparative hydrolytic activity assays showed that CTX-M-33 hydrolyzed ceftazidime at a lower level than CTX-M-15 but significantly hydrolyzed meropenem. In addition, CTX-M-33 showed higher mutant prevention concentration values and a wider mutant selection window in the presence of meropenem, in accordance with its observed hydrolytic properties. Here, we identified the very first CTX-M enzyme possessing weak carbapenemase activity, which may correspond to an emerging phenomenon, considering its possible evolution from the widespread ESBL CTX-M-15

BIChromET: A Chromogenic Culture Medium for Detection of Piperacillin/Tazobactam and Cefepime Resistance in Pseudomonas aeruginosa

Objectives: The BIChromET selective medium for detecting piperacillin-tazobactam (TZP) and cefepime (FEP) resistant Pseudomonas aeruginosa was developed. Methods: The performance of this medium was first evaluated using a collection of 100 P. aeruginosa clinical strains (70 TZP-susceptible, 30 TZP-resistant, 58 FEP-susceptible, and 42 FEP-resistant). Then, we performed clinical validation by testing 173 respiratory clinical samples. Results: The BIChromET medium showed excellent sensitivity (TZP (avg. 96.7%); FEP (avg. 92.7%)) and specificity (TZP (avg. 98.9%); FEP (avg. 98%)) in distinguishing the detection limit ranging from 104 to 108 CFU/mL. Then, testing the bronchoalveolar lavage (BAL) and tracheobronchial aspirate (TBA) clinical specimens (N = 173) revealed the excellent performance of the medium with P. aeruginosa, showing 100% and 92.6% of categorical agreements with the results obtained via the broth microdilution methods (BMD) for TZP and FEP, respectively. Conclusion: This medium allows for easy and accurate detection of TZP/FEP-resistant isolates regardless of their resistance mechanisms

Sistemas hidropónicos abierto y cerrado en la producción de tomate (Lycopersicon esculentum, Mill)

Tomato (Lycopersicum esculentum Mill.) Is one of the most important vegetables in the world and its cultivation is increasing, mainly in greenhouses and hydroponics where you have an alternative production and marketing opportunity. Hydroponic systems are open, when excess nutrient solution drained is not reused and is discarded, and are closed, when excess nutrient solution is recovered and reused. The objective of the research was to know the differences in fruit production and quality in a closed hydroponic system compared to an open one, in the tomato crop of the El Cid variety, using pots with thin perlite substrate previously used. The research was carried out in 2015 in the Academic Unit of Agronomy of the Autonomous University of Zacatecas. Plant growth (stem length and diameter) was measured; Fruit production (number, size, weight and yield of fruits); And fruit quality (electrical conductivity, hydrogen potential, acidity, total soluble solids, and fruit maturity index and fruit weight loss) in two stages of production in three maturity indices. Growth variables were maintained within normal and adequate parameters. There was no significant difference in the quality variables evaluated in the three maturity indices of the fruits measured in two stages. There was significant difference only for the equatorial diameter of fruit, however, not for polar diameter, number and weight of fruits and yield, so the closed system is a production alternative potentially comparable to the open system. With the closed hydrological system, neither the yield nor the quality of the fruits produced is affected, but it does obtain a saving of 26.81% of water and 28.9% of fertilizers, which represents a rental rate of 22.2% greater than the open hydroponic system.El tomate o jitomate (Lycopersicum esculentum Mill.) es una de las hortalizas más importantes del mundo y su cultivo va en aumento, principalmente en invernaderos e hidroponía donde se tiene una alternativa de producción y oportunidad de comercialización. Los sistemas hidropónicos son abiertos, cuando el exceso de solución nutritiva drenada no es reusado y es desechado, y son cerrados, cuando la solución nutritiva excedente es recuperada y reusada. El objetivo de investigación fue conocer las diferencias en producción y calidad de frutos en un sistema hidropónico cerrado en comparación con uno abierto, en el cultivo de tomate de variedad El Cid, utilizando macetas con sustrato perlita fina previamente utilizado. La investigación se realizó en el año 2015 en la Unidad Académica de Agronomía de la Universidad Autónoma de Zacatecas. Se midió el crecimiento de la planta (longitud y diámetro de tallo); la producción de frutos (número, tamaño, peso y rendimiento de frutos); y la calidad de los frutos (conductividad eléctrica, potencial de hidrógeno, acidez, sólidos solubles totales, e índice de madurez en el zumo y pérdida de peso de fruto) en dos etapas de la producción en tres índices de madurez. Las variables de crecimiento se mantuvieron dentro de parámetros normales y adecuados. No hubo diferencia significativa en las variables de calidad evaluadas en los tres índices de madurez de los frutos medidos en dos etapas. Hubo diferencia significativa sólo para el diámetro ecuatorial de fruto, sin embargo, no para diámetro polar, número y peso de frutos y rendimiento, por lo cual el sistema cerrado es una alternativa de producción potencialmente comparable con el sistema abierto. Con el sistema hidropónico cerrado no se afecta el rendimiento ni la calidad de los frutos producidos, pero si se obtiene un ahorro de 26.81 % de agua y 28.9 % de fertilizantes, lo cual representa una tasa de rentabilidad 22.2 % mayor respecto al sistema hidropónico abierto

Epidemiology of Carbapenemase-Producing Klebsiella pneumoniae in a Hospital, Portugal

We aimed to provide updated epidemiologic data on carbapenem-resistant Klebsiella pneumoniae in Portugal by characterizing all isolates (N = 46) recovered during 2013– 2018 in a 123-bed hospital in Lisbon. We identified blaKPC-3 (n = 36), blaOXA-181 (n = 9), and blaGES-5 (n = 8) carbapenemase genes and observed co-occurrence of blaKPC-3 and blaGES-5 in 7 isolates. A single GES-5–producing isolate co-produced the extended-spectrum β-lactamase BEL-1; both corresponding genes were co- located on the same ColE1-like plasmid. The blaOXA-181 gene was always located on an IncX3 plasmid, whereas blaKPC-3 was carried on IncN, IncFII, IncFIB, and IncFIIA plasmid types. The 46 isolates were distributed into 13 pulsotypes and 9 sequence types. All isolates remained susceptible to ceftazidime/avibactam, but some exhibited reduced antimicrobial susceptibility (MIC = 3 mg/L)

Role of blaTEM and OmpC in the piperacillin-tazobactam resistance evolution by E. coli in patients with complicated intra-abdominal infection

Piperacillin-tazobactam resistance (P/T-R) is increasingly reported among Escherichia coli isolates. Although in vitro experiments have suggested that bla gene plays a key role in the P/T-R acquisition, no clinical in vivo study has yet confirmed the role of bla or other genes. Therefore, we aimed to identify the mechanisms underlying P/T-R by following up patients with E. coli complicated intra-abdominal infections (cIAI) who experienced P/T treatment failure. Four pairs of strains, clonally related from four patients, were isolated both before and after treatment with P/T dosed at 4 g/0.5 g intravenously. The P/T MIC was tested using broth microdilution, and β-lactamase activity was determined in these isolates. Whole-genome sequencing (WGS) was performed to decipher the role of bla and other genes associated with P/T-R. Changes in the outer membrane protein (OMP) profile were analyzed using SDS-PAGE, and bla and ompC transcription levels were measured by RT-qPCR. In addition, in vitro competition fitness was performed between each pairs of strains (P/T-susceptible vs. P/T-resistant). We found a higher copy number of bla gene in P/T-R isolates, generated by three different genetic events: (1) IS26-mediated duplication of the bla gene, (2) generation of a small multicopy plasmid (ColE-like) carrying bla, and (3) adaptive evolution via reduction of plasmid size, leading to a higher plasmid copy number. Moreover, two P/T-R strains showed reduced expression of OmpC. This study describes the mechanisms involved in the acquisition of P/T-R by E. coli in patients with cIAI. The understanding of P/T-R evolution is crucial for effectively treating infected patients and preventing the spread of resistant microorganisms.This work was funded by the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad (grant PI19/01009), by Plan Nacional de I+D+i 2013–2016, and by the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (grant RD16/0016/0009), cofinanced by the European Development Regional Fund “A way to make Europe”/”Investing in your future”. A.R.V, R.A-M, J.A.L. and J.M.C. were supported (grant CB21/13/00006) by CIBERINFEC - Consorcio Centro de Investigación Biomédica en Red, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea – NextGenerationEU. L.G.B. was partly financed by a grant from the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) and is supported by the Subprograma Rio Hortega, Instituto Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain (CM22/00196). J.M.O.R. is supported by the Subprograma Sara Borrell, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain (CD21/00098). A.R.V. is supported by the Subprograma Juan Rodés, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spain (JR20/00023), and Y.S. has received a Miguel Servet Tipo II contract from the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spain (grant CPII20/00018). Funding for open access publishing: Universidad Pablo de OlavidePeer reviewe