A Clostridium difficile Lineage Endemic to Costa Rican Hospitals Is Multidrug Resistant by Acquisition of Chromosomal Mutations and Novel Mobile Genetic Elements.

The antimicrobial resistance (AMR) rates and levels recorded for Clostridium difficile are on the rise. This study reports the nature, levels, diversity, and genomic context of the antimicrobial resistance of human C. difficile isolates of the NAPCR1/RT012/ST54 genotype, which caused an outbreak in 2009 and is endemic in Costa Rican hospitals. To this end, we determined the susceptibilities of 38 NAPCR1 isolates to 10 antibiotics from seven classes using Etests or macrodilution tests and examined 31 NAPCR1 whole-genome sequences to identify single nucleotide polymorphisms (SNPs) and genes that could explain the resistance phenotypes observed. The NAPCR1 isolates were multidrug resistant (MDR) and commonly exhibited very high resistance levels. By sequencing their genomes, we showed that they possessed resistance-associated SNPs in gyrA and rpoB and carried eight to nine acquired antimicrobial resistance (AMR) genes. Most of these genes were located on known or novel mobile genetic elements shared by isolates recovered at different hospitals and at different time points. Metronidazole and vancomycin remain the first-line treatment options for these isolates. Overall, the NAPCR1 lineage showed an enhanced ability to acquire AMR genes through lateral gene transfer. On the basis of this finding, we recommend further vigilance and the adoption of improved control measures to limit the dissemination of this lineage and the emergence of more C. difficile MDR strains.

Predominance and high antibiotic resistance of the emerging Clostridium difficile genotypes NAPCR1 and NAP9 in a Costa Rican hospital over a 2-year period without outbreaks.

Clostridium difficile is the major causative agent of nosocomial antibiotic-associated diarrhea. In a 2009 outbreak of C. difficile-associated diarrhea that was recorded in a major Costa Rican hospital, the hypervirulent NAP1 strain (45%) predominated together with a local genotype variant (NAPCR1, 31%). Both strains were fluoroquinolone-resistant and the NAPCR1 genotype, in addition, was resistant to clindamycin and rifampicin. We now report on the genotypes and antibiotic susceptibilities of 68 C. difficile isolates from a major Costa Rican hospital over a 2-year period without outbreaks. In contrast to our previous findings, no NAP1 strains were detected, and for the first time in a Costa Rican hospital, a significant fraction of the isolates were NAP9 strains (n=14, 21%). The local NAPCR1 genotype remained prevalent (n=18, 26%) and coexisted with 14 strains (21%) of classic hospital NAP types (NAP2, NAP4, and NAP6), eight new genotypes (12%), four environmental strains classified as NAP10 or NAP11 (6%), three strains without NAP designation (4%) and seven non-toxigenic strains (10%). All 68 strains were resistant to ciprofloxacin, 88% were resistant to clindamycin and 50% were resistant to moxifloxacin and rifampicin. Metronidazole and vancomycin susceptibilities were universal. The NAPCR1 and NAP9 strains, which have been associated with more severe clinical infections, were more resistant to antibiotics than the other strains. Altogether, our results confirm that the epidemiology of C. difficile infection is dynamic and that A(-)B(+) strains from the NAP9 type are on the rise not only in the developed world. Moreover, our results reveal that the local NAPCR1 strains still circulate in the country without causing outbreaks but with equally high antibiotic-resistance rates and levels.

Emergence of an outbreak-associated Clostridium difficile variant with increased virulence.

The prevalence of Clostridium difficile infections has increased due to the emergence of epidemic variants from diverse genetic lineages. Here we describe the emergence of a novel variant during an outbreak in a Costa Rican hospital that was associated with severe clinical presentations. This C. difficile variant elicited higher white blood cell counts and caused disease in younger patients than did other strains isolated during the outbreak. Furthermore, it had a recurrence rate, a 30-day attributable disease rate, and disease severity as great as those of the epidemic strain NAP1. Pulsed-field gel electrophoresis genotyping indicated that the outbreak strains belong to a previously undescribed variant, designated NAPCR1. Whole-genome sequencing and ribotyping indicated that the NAPCR1 variant belongs to C. difficile ribotype 012 and sequence type 54, as does the reference strain 630. NAPCR1 strains are resistant to fluoroquinolones due to a mutation in gyrA, and they possess an 18-bp deletion in tcdC that is characteristic of the epidemic, evolutionarily distinct, C. difficile NAP1 variant. NAPCR1 genomes contain 10% more predicted genes than strain 630, most of which are of hypothetical function and are present on phages and other mobile genetic elements. The increased virulence of NAPCR1 was confirmed by mortality rates in the hamster model and strong inflammatory responses induced by bacteria-free supernatants in the murine ligated loop model. However, NAPCR1 strains do not synthesize toxin A and toxin B at levels comparable to those in NAP1 strains. Our results suggest that the pathogenic potential of this emerging C. difficile variant is due to the acquisition of hypothetical functions associated with laterally acquired DNA.

Desarrollo y evaluación de un método de obtención de larvas estériles de Lucilia eximia para su uso en terapia larval / Development and evaluation of a method to obtain sterile Lucilia eximia larvae for use in larval therapy

Objetivo: desarrollar un método de tratamiento de huevos de Lucilia eximia para la obtención de larvas estériles y evaluar la capacidad de supervivencia de dichas larvas en condiciones de refrigeración a 4°C. Métodos: se estableció un sistema de crianza para L. eximia. Los huevos fueron colectados en el sustrato de oviposición, se lavaron con solución salina estéril (0,85%), se trataron con hipoclorito de sodio (0,5%) y finalmente se esterilizaron con formalina, evaluando tres diferentes concentraciones (2,5%; 5,0% y 10,0%). Se verificó la esterilidad de los huevos empleando medios de cultivo bacteriológicos y la eclosión de los huevos esterilizados fue expresada mediante el cálculo de un índice de eclosión (IE). Además, se evaluó la supervivencia de las larvas de segundo y tercer estadio temprano (L2 y L3, respectivamente) en condiciones de refrigeración, durante las 4 primeras horas y luego a las 24 y 48 horas. Resultados: todas las concentraciones de formalina evaluadas fueron capaces de esterilizar los huevos. No se encontró una correlación entre el IE y las concentraciones de formalina utilizadas (R Spearman = -0,030, p = 0,848). Durante las primeras 4 horas a 4°C, un 100% de las L2 y las L3 sobrevivieron. Sin embargo, en ambos estadios larvales hubo un marcado descenso de la supervivencia a las 24 y 48 horas, siendo las L2 las más sensibles a las condiciones de refrigeración. Conclusiones: los resultados mostraron que la obtención de larvas estériles de L. eximia con el método utilizado es un proceso sencillo, pero la supervivencia de las larvas en condiciones de refrigeración es limitada.

Objective: develop a method to treat eggs of Lucilia eximia to obtain sterile larvae and evaluate the survival capacity of those larvae under refrigeration at 4°C. Methods: a rearing system was set up for L. eximia. The eggs were collected from the oviposition substrate, washed with sterile saline solution (0.85%), treated with sodium hypochlorite (0.5%), and sterilized with formalin. Three concentrations were evaluated: 2.5%; 5.0% and 10.0%. Egg sterility was verified by bacteriological culture. Eclosion of sterilized eggs was expressed by estimation of an eclosion rate (ER). An evaluation was conducted of the survival of larvae from the second and third early stages (L2 and L3, respectively) under refrigeration during the first 4 hours and then at 24 and 48 hours. Results: all the formalin concentrations evaluated were capable of sterilizing the eggs. No correlation was found between the ER and the formalin concentrations used (Spearman's Rho = -0.030, p = 0.848). During the first 4 hours at 4°C, 100% L2 and L3 survived. However, both larval stages showed a marked decrease in survival at 24 and 48 hours, L2 being the most sensitive to refrigeration. Conclusions: results show that obtaining sterile L. eximia larvae by this method is a simple process. However, larval survival under refrigeration is limited.