Development of Meropenem Resistance in a Multidrug-Resistant Campylobacter coli Strain Causing Recurrent Bacteremia in a Hematological Malignancy Patient.

Campylobacter bacteremia is an uncommon disease that mainly occurs in immunocompromised patients and is associated with antibiotic resistance, particularly in Campylobacter coli. We report a patient with persistent blood infection because of a multidrug-resistant (MDR) C. coli strain over a 3-month period. Through this period monotherapy with meropenem was associated with the development of resistance to it. Improving immunity status and a combined therapy for intestinal decolonization were useful to control persistent C. coli infection in this patient.

Global Emergence of Resistance to Fluconazole and Voriconazole in Candida parapsilosis in Tertiary Hospitals in Spain During the COVID-19 Pandemic.

Background: Candida parapsilosis is a frequent cause of candidemia worldwide. Its incidence is associated with the use of medical implants, such as central venous catheters or parenteral nutrition. This species has reduced susceptibility to echinocandins, and it is susceptible to polyenes and azoles. Multiple outbreaks caused by fluconazole-nonsusceptible strains have been reported recently. A similar trend has been observed among the C. parapsilosis isolates received in the last 2 years at the Spanish Mycology Reference Laboratory. Methods: Yeast were identified by molecular biology, and antifungal susceptibility testing was performed using the European Committee on Antimicrobial Susceptibility Testing protocol. The ERG11 gene was sequenced to identify resistance mechanisms, and strain typing was carried out by microsatellite analysis. Results: We examined the susceptibility profile of 1315 C. parapsilosis isolates available at our reference laboratory between 2000 and 2021, noticing an increase in the number of isolates with acquired resistance to fluconazole, and voriconazole has increased in at least 8 different Spanish hospitals in 2020-2021. From 121 recorded clones, 3 were identified as the most prevalent in Spain (clone 10 in Catalonia and clone 96 in Castilla-Leon and Madrid, whereas clone 67 was found in 2 geographically unrelated regions, Cantabria and the Balearic Islands). Conclusions: Our data suggest that concurrently with the coronavirus disease 2019 pandemic, a selection of fluconazole-resistant C. parapsilosis isolates has occurred in Spain, and the expansion of specific clones has been noted across centers. Further research is needed to determine the factors that underlie the successful expansion of these clones and their potential genetic relatedness.

Inhibition of Mycobacterium abscessus, M. chelonae, and M. fortuitum biofilms by Methylobacterium sp.

Methylobacterium sp. is isolated from water distribution systems and has been linked in the biofilms of the systems with a lower presence of Mycobacterium avium. In this study we aimed to determine the in vitro activity of Methylobacterium sp. in the development of rapidly growing mycobacteria (RGM) biofilms. Methylobacterium sp. CECT 7805 was added as a suspension of living bacteria (LB), an autoclaved suspension (AS), and an extract obtained after sonication (ES) at different times (24, 48, and 72 h), to preformed biofilms of Mycobacterium abscessus DSM 44196, Mycobacterium chelonae ATCC 19235, and Mycobacterium fortuitum ATCC 6841, using a 96 h control of each species. The biofilms were analyzed by confocal laser scanning microscopy and by the Calgary biofilm device using the plates MBECTM Biofilm Inoculator. A statistically significant reduction in the thickness and covered surface was observed in all mycobacterial biofilms with all forms of Methylobacterium sp. A statistically significant increase in the autofluorescence was observed in M. abscessus biofilms but not in other biofilms. The increased percentage of dead mycobacteria was statistically significant in all cases. The reduced log CFU (colony-forming units)/peg recount was statistically significant in M. chelonae biofilms after treatment with AS and ES, but in M. fortuitum biofilms the recount decreased only with AS. M. abscessus biofilms were always significantly reduced with AS at 72 h and with ES. Methylobacterium sp. could inhibit RGM biofilm formation. Living cells of Methylobacterium sp. were not necessary to inhibit the growth of a preformed biofilm. M. chelonae biofilms were the most greatly reduced.

Non-pigmented rapidly growing mycobacteria smooth and rough colony phenotypes pathogenicity evaluated using in vitro and experimental models.

Non-pigmented rapidly growing mycobacteria (NPRGM) are widely distributed in water, soil and animals. It has been observed an increasing importance of NPRGM related-infections, particularly due to the high antimicrobial resistance. NPRGM have rough and smooth colony phenotypes, and several studies have showed that rough colony variants are more virulent than smooth ones. However, other studies have failed to validate this observation. In this study, we have performed two models, invitro and in vivo, in order to assess the different pathogenicity of these two phenotypes. We used collection and clinical strains of Mycobacteriumabscessus, Mycobacterium fortuitum and Mycobacteriumchelonae. On the invitro model (macrophages), phagocytosis was higher for M. abscessus and M. fortuitum rough colony variant strains when compared to smooth colony variants. However, we did not find differences with colonial variants of M. chelonae. Survival of Galleriamellonella larvae in the experimental model was lower for M. abscessus and M. fortuitum rough colony variants when compared with larvae infected with smooth colony variants. We did not find differences in larvae infected with M. chelonae.Results of our in vivo study correlated well with the experimental model. This fact could have implications on the interpretation of the clinical significance of the NPRGM isolate colonial variants.

Historical evolution of the diseases caused by non-pigmented rapidly growing mycobacteria in a University Hospital / Evolución histórica de las enfermedades causadas por micobacterias no pigmentadas de crecimiento rápido en un Hospital Universitario

INTRODUCTION: Non-pigmented rapidly growing mycobacteria (NPRGM) are a group of organisms of increasing interest due to the growing number of potential patients and the difficulties for a proper treatment in many of them. However, the evolution of these diseases in a long period of time and its evolutionary changes has been described only in a scanty number of reports. MATERIAL AND METHODS: We performed a retrospective study between January 1st 2004 and December 31st 2017 in order to evaluate the clinical significance and types of diseases caused by NPRGM. Patients with isolates of NPRGM during this period were selected for the study, and clinical charts were reviewed using a predefined protocol. RESULTS: During this period we identified 59 patients (76 clinical samples) with isolates of NPRGM, with 12 cases of clinical disease and one patient with doubtful significance (including 6 respiratory tract infections, 2 catheter infections, 1 skin and soft tissue infection, 1 disseminated infection, 1 conjunctivitis, 1 prosthetic joint infection and 1 mastitis). Fifty percent of M. chelonae isolates, 37.5% of M. abscessus isolates and 23.33% of M. fortuitum isolates were clinically significant. None of the isolates of other species were significant. CONCLUSIONS: Most isolates in respiratory samples were contaminants/colonizations. M. abscessus was the main etiological agent in respiratory syndromes, whereas M. chelonae and M. fortuitum were more frequently associated with other infections, especially clinical devices and skin and soft tissue infections

INTRODUCCIÓN: Las micobacterias no pigmentadas de crecimiento rápido (MNPCR) son un grupo de organismos de interés creciente debido al número cada vez mayor de pacientes potenciales y a las dificultades en el tratamiento. Sin embargo, el número de estudios que analizan la evolución de estos casos a lo largo de un periodo de tiempo largo es escaso. MATERIAL Y MÉTODOS: Se realizó un estudio retrospectivo entre el 1 de enero de 2004 y el 31 de diciembre de 2017 para evaluar el significado clínico y los tipos de enfermedades causados por MNPCR. Se seleccionaron para ello aquellos pacientes con aislamientos de MNPCR, y se revisaron las historias clínicas mediante un protocolo predefinido. RESULTADOS: Se identificaron 59 pacientes (76 muestras) con aislamientos de MNPCR, de los cuales 12 presentaron enfermedad y uno tuvo un significado dudoso (incluyendo 6 infecciones respiratorias, 2 infecciones asociadas a catéter, 1 infección de piel y partes blandas, 1 infección diseminada, 1 conjuntivitis, 1 infección de prótesis osteoarticular y 1 mastitis). El 50 % de los aislamientos de Mycobacterium chelonae, el 37,5 % de Mycobacterium abscessus y el 23,33 % de Mycobacterium fortuitum fueron clínicamente significativos. Ninguno de los aislamientos de otras especies fue significativo. CONCLUSIONES: La mayoría de los aislamientos de muestras respiratorias resultaron ser contaminantes/colonizaciones. M. abscessus fue el principal agente etiológico en las infecciones respiratorias, mientras que M. chelonae y M. fortuitum fueron asociados con mayor frecuencia a otras infecciones, especialmente infecciones de piel y partes blandas e infecciones asociadas a dispositivos biomédicos

Antimicrobial Treatment Provides a Competitive Advantage to Mycobacterium abscessus in a Dual-Species Biofilm with Pseudomonas aeruginosa.

The physiological factors that contribute to Mycobacterium abscessus lung infections remain unclear. We determined whether antibiotic treatment targeting a major cystic fibrosis pathogen (i.e., Pseudomonas aeruginosa) could provide the ideal conditions for the establishment of M. abscessus infection. Our data showed that P. aeruginosa inhibited M. abscessus biofilm formation under control conditions and that antimicrobial therapy selectively targeting P. aeruginosa diminished this competitive interaction, thereby increasing M. abscessus survival.

Influence of three-dimensional lung epithelial cells and interspecies interactions on antibiotic efficacy against Mycobacterium abscessus and Pseudomonas aeruginosa.

Mycobacterium abscessus lung infection is a major health problem for cystic fibrosis (CF) patients. Understanding the in vivo factors that influence the outcome of therapy may help addressing the poor correlation between in vitro and in vivo antibiotic efficacy. We evaluated the influence of interspecies interactions and lung epithelial cells on antibiotic efficacy. Therefore, single and dual-species biofilms of M. abscessus and a major CF pathogen (Pseudomonas aeruginosa) were cultured on a plastic surface or on in vivo-like three-dimensional (3-D) lung epithelial cells, and the activity of antibiotics (colistin, amikacin, clarithromycin, ceftazidime) in inhibiting biofilm formation was evaluated. Using the most physiologically relevant model (dual-species biofilms on 3-D cells), we observed that treatment with antibiotics during biofilm development inhibited P. aeruginosa but not M. abscessus biofilms, resulting in a competitive advantage for the latter. Clarithromycin efficacy against P. aeruginosa was inhibited by 3-D lung cells. In addition, biofilm induction of M. abscessus was observed by certain antibiotics on plastic but not on 3-D cells. Pseudomonas aeruginosa influenced the efficacy of certain antibiotics against M. abscessus, but not vice versa. In conclusion, these results suggest a role of host cells and interspecies interactions in bacterial responses to antimicrobials.

Non-Tuberculous Mycobacteria multispecies biofilms in cystic fibrosis: development of an in vitro Mycobacterium abscessus and Pseudomonas aeruginosa dual species biofilm model.

Lung disease in cystic fibrosis (CF) is characterized by the progressive colonization of the respiratory tract by different bacteria, which develop polymicrobial biofilms. In the past decades, there has been an increase in the number of CF patients infected with Non-Tuberculous Mycobacteria (NTM). Although Mycobacterium abscessus is the main NTM isolated globally, little is known about M. abscessus multispecies biofilm formation. In the present study we developed an in vitro model to study the phenotypic characteristics of biofilms formed by M. abscessus and Pseudomonas aeruginosa, a major pathogen in CF. For that purpose, dual species biofilms were grown on polycarbonate membranes with a fixed concentration of P. aeruginosa and different inoculums of M. abscessus. The biofilms were sampled at 24, 48, and 72 h and bacteria were quantified in specific media. The results revealed that the increasing initial concentration of M. abscessus in dual species biofilms had an effect on its population only at 24 and 48 h, whereas P. aeruginosa was not affected by the different concentrations used of M. abscessus. Time elapsed increased biofilm formation of both species, specially between 24 and 48 h. According to the results, the conditions to produce a mature dual species biofilm in which the relative species distribution remained stable were 72 h growth of the mixed microbial culture at a 1:1 ratio. A significant decrease in mycobacterial population in dual compared to single species biofilms was found, suggesting that P. aeruginosa has a negative influence on M. abscessus. Finally, in a proof of concept experiment, young and mature dual species biofilms were exposed to clarithromycin.

Evaluation of a commercial multiplex PCR (Unyvero i60(R)) designed for the diagnosis of bone and joint infections using prosthetic-joint sonication / Evaluación de una PCR múltiple comercial (Unyvero i60(C)) diseñada para el diagnóstico de infecciones osteoarticulares utilizando prótesis articulares sonicadas

BACKGROUND: The development of sonication protocols over the last few years has improved the sensitivity of conventional cultures for the diagnosis of prosthetic-joint infection (PJI). However, the development of a new, specifically designed kit for the molecular diagnosis of PJI could provide a major improvement in this field. METHODS: Prostheses retrieved from patients who underwent implant removal from May 2014 to May 2015 were sent for culture, and processed according to a previously defined protocol that included sonication. Furthermore, 180 microlitres of sonication fluid were used to carry out the multiplex PCR test (Unyvero i60 system®). A comparison of the sensitivity, specificity, positive (PPV) and negative (NPV) predictive value, was performed. The study was approved by the Clinical Research Ethics Committee. RESULTS: The analysis included 88 prostheses from 68 patients (1.29 prostheses/patient). The type of prostheses studied were knee (n=55), total hip (n=26), partial hip (n=5), and shoulder (n=2). Twenty-nine patients were diagnosed with a PJI (15 delayed, 12 acute, and 2 haematogenous infections). In 24 cases, the result of the PCR was positive, all but 1 corresponding to patients with clinical criteria of PJI. Nine resistance mechanisms were detected from 5 samples. The Unyvero i60 system® showed slightly better results than traditional culture in terms of specificity and PPV. CONCLUSIONS: The Unyvero i60 system® may play a role in rapid diagnosis of PJI, due to its high specificity and PPV. However, despite these results, cultures have to be performed to detect organisms not detected by the system

INTRODUCCIÓN: El desarrollo de la sonicación durante los pasados años ha incrementado la sensibilidad de los cultivos convencionales para el diagnóstico de Infecciones de Prótesis Articulares (IPA). Sin embargo, el desarrollo de un nuevo kit, diseñado específicamente para el diagnóstico de las IPA podría suponer un avance significativo en este campo. MÉTODOS: Todas las prótesis retiradas de pacientes entre mayo 2014 y mayo 2015 fueron enviadas para cultivo mediante un protocolo de procesamiento que incluye la sonicación del implante. Además, se emplearon 180 microlitros del líquido de sonicado en la realización de una PCR múltiple (Unyvero i60®). Se realizó una comparación de la sensibilidad, especificidad, valor predictivo positivo (VPP) y negativo (VPN). El estudio fue aprobado por el Comité de Ética en Investigación Clínica. RESULTADOS: Se analizaron 88 prótesis de 68 pacientes (1,29 prótesis/paciente). Las prótesis estudiadas fueron rodillas (n=55), total de cadera (n=26), parcial de cadera (n=5), y hombro (n=2). Veintinueve pacientes fueron diagnosticados de IPA (15 crónicas, 12 agudas y 2 hematógenas). En 24 casos, el resultado de la PCR fue positivo, siendo todas menos 1 de estas de pacientes con criterios de IPA. Se detectaron además 9 mecanismos de resistencia en 5 muestras. El sistema Unyvero i60® mostró resultados ligeramente superiores al cultivo tanto en especificidad como en VPP. CONCLUSIONES: El sistema Unyvero i60® puede tener un papel en el diagnóstico rápido de IPA debido a su elevada especificidad y VPP. Sin embargo, a pesar de estos resultados, debe realizarse cultivo para detectar organismos no detectados por el sistema

Evaluation of a commercial multiplex PCR (Unyvero i60®) designed for the diagnosis of bone and joint infections using prosthetic-joint sonication.

BACKGROUND: The development of sonication protocols over the last few years has improved the sensitivity of conventional cultures for the diagnosis of prosthetic-joint infection (PJI). However, the development of a new, specifically designed kit for the molecular diagnosis of PJI could provide a major improvement in this field. METHODS: Prostheses retrieved from patients who underwent implant removal from May 2014 to May 2015 were sent for culture, and processed according to a previously defined protocol that included sonication. Furthermore, 180 microlitres of sonication fluid were used to carry out the multiplex PCR test (Unyvero i60 system®). A comparison of the sensitivity, specificity, positive (PPV) and negative (NPV) predictive value, was performed. The study was approved by the Clinical Research Ethics Committee. RESULTS: The analysis included 88 prostheses from 68 patients (1.29 prostheses/patient). The type of prostheses studied were knee (n=55), total hip (n=26), partial hip (n=5), and shoulder (n=2). Twenty-nine patients were diagnosed with a PJI (15 delayed, 12 acute, and 2 haematogenous infections). In 24 cases, the result of the PCR was positive, all but 1 corresponding to patients with clinical criteria of PJI. Nine resistance mechanisms were detected from 5 samples. The Unyvero i60 system® showed slightly better results than traditional culture in terms of specificity and PPV. CONCLUSIONS: The Unyvero i60 system® may play a role in rapid diagnosis of PJI, due to its high specificity and PPV. However, despite these results, cultures have to be performed to detect organisms not detected by the system.