Carlos Quesada-Gómez

Emergence of Clostridium difficile NAP1 in Latin America.

The NAP1 and NAP2 strains of Clostridium difficile have been linked to nosocomial outbreaks of antibiotic-associated diarrhea (AAD) and pseudomembranous colitis in North American and European countries (4, 5). We found these strains, together with seven additional pulsed-field gel electrophoresis (PFGE) patterns, among 37 isolates recently recovered from patients with AAD in a Costa Rican hospital. Herein we present the macrorestriction patterns of the isolates as well as data regarding their toxin genotypes and susceptibility to selected antibiotics. The isolates were recovered by inoculating a loopful of diarrheic stool samples onto cefoxitin-cycloserine fructose agar plates (CCFA; Oxoid). They were identified with the rapid ID32A system (bioMerieux) and a PCR targeting the triose phosphate isomerase gene (9). We typed the isolates by PFGE (1) and amplified fragments of the tcdA, tcdB, tcdC, and cdtB genes by PCR with oligonucleotides and conditions reported elsewhere (3, 9, 10). These genes code for toxin A, toxin B, the negative regulator of the pathogenicity locus, and the binding domain of the binary toxin, respectively. MICs of clindamycin, metronidazole, vancomycin, moxifloxacin, ciprofloxacin, and amoxicillin-clavulanate were determined using Etest strips (AB bioMerieux). C. difficile ATCC 700057 and Bacteroides fragilis ATCC 25285 were used as reference strains. The breakpoints recommended by the CLSI (2) and Pelaez et al. (6) were used for antimicrobial susceptibility interpretation. Nine different PFGE types were identified in the collection (Fig. ​(Fig.1).1). All isolates were positive for both tcdA and tcdB and susceptible to vancomycin and metronidazole, which are the first antibiotics to be prescribed for this type of infection. They were also susceptible to amoxicillin-clavulanic acid. FIG. 1. Pulsed-field gel electrophoresis. SmaI dendrogram of 37 isolates of Clostridium difficile recovered from patients with antibiotic-associated diarrhea in a Costa Rican hospital. More than half the isolates exhibited the macrorestriction pattern of the NAP1 strain (n = 20; 54%). All these bacteria had the gene for the binding domain of the binary toxin and a deletion in tcdC (Table ​(Table1).1). Only one of the NAP1 strains was susceptible to clindamycin. In fact, 10 (50%) were categorized as intermediate and another 9 (45%) as resistant to this antibiotic. One clindamycin-resistant NAP1 strain had a MIC of ≥256 μg ml−1. In agreement with recent data (7), all NAP1 isolates were highly resistant to the two fluoroquinolones tested (Table ​(Table11). TABLE 1. SmaI macrorestriction patterns, toxin genotype, and MICs of clindamycin, metronidazole, vancomycin, moxifloxacin, ciprofloxacin, and amoxicillin-clavulanic acid of 37 isolates of C. difficile recovered from patients with antibiotic-associated diarrhea ... The 13 isolates with the SmaI patterns 447, 448, 449, and 452 clustered together (Fig. ​(Fig.1).1). These 13 isolates lacked the binary toxin and had a deletion in the tcdC gene (Table ​(Table1).1). In addition, they were without exception categorized as highly resistant to clindamycin, moxifloxacin, and ciprofloxacin (Table ​(Table1).1). One strain with the PFGE pattern 447 and the strains with the PFGE patterns 449 and 452 exhibited the highest MICs to vancomycin overall. The isolates with the PFGE patterns 100, 450, and 451 (NAP2) and 247 (NAP9) were negative for the binary toxin and did not have deletions in the tcdC gene (Table ​(Table1).1). The strains with the PFGE patterns 100, 450, and 247 (NAP9) were resistant to clindamycin, moxifloxacin, and ciprofloxacin (Table ​(Table1).1). In contrast, the isolate with the PFGE pattern 451 (NAP2) was moderately resistant to clindamycin (MIC = 16 μg ml−1) and highly resistant to ciprofloxacin (MIC > 32 μg ml−1) but susceptible to moxifloxacin (MIC = 2 μg ml−1). NAP9 strains have been recently isolated from retail meat (8). The finding of the NAP1 strain in Latin American countries is novel and deserves attention from infectious disease specialists and epidemiologists to prevent its dissemination.

Emergence of an Outbreak-Associated Clostridium difficile Variant with Increased Virulence

ABSTRACT 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.

Spread of epidemic Clostridium difficile NAP1/027 in Latin America: case reports in Panama.

The rate and severity of Clostridium difficile infection (CDI) have been linked to the emergence and spread of the hypervirulent toxigenic strain NAP1/027. This strain has been responsible for large outbreaks in healthcare facilities in North America and Europe and most recently in Latin America. This is the first report of the NAP1 strain in Panama. It suggests that the spread of C. difficile NAP1 throughout Latin America could be a possibility as evidenced in the following case reports. Five isolates typed as NAP1 had tcdA, tcdB, binary toxin gene cdtB and tcdC deletion. All isolates were resistant to clindamycin, fluoroquinolones and rifampicin. Under this scenario, surveillance programmes for CDI should be implemented in public health facilities in Latin America and diagnosis of CDI should be considered, especially in patients with predisposing factors.

Isolation of a toxigenic and clinical genotype of clostridium difficile in retail meats in Costa Rica.

We isolated a regional toxigenic genotype of Clostridium difficile, previously found in human infection in 4 of 200 (2%) samples of retail meats for human consumption: 1 of 67 samples of beef, 2 of 66 of pork, and 1 of 67 of poultry meat. These four isolates were positive for the tcdA and tcdB genes but negative for deletion of the tcdC and cdtB genes. All strains induced cytopathic effects in HeLa cells. However, they were susceptible to some antibiotics to which clinical isolates are often resistant. All strains were susceptible to vancomycin, metronidazole, moxifloxacin, and rifampicin but resistant to clindamycin and ciprofloxacin. This first report of isolation of C. difficile in foodstuff from Latin America lends support to the notion that animal products serve as a reservoir for clinical strains of this pathogen in the community.

Community-acquired Clostridium difficile NAP1/027-associated diarrhea in an eighteen month old child

Clostridium difficile infection (CDI), characterized by symptoms varying from diarrhea to life-threatening colitis, is a major complication of antibiotic therapy. Studies suggested that CDI is emerging as an important cause of childhood diarrhea in community and hospital settings. This work is the first report of a documented case of community-acquired CDI by a NAP1 hypervirulent strain in an eighteen month old child from Latin America.

Analysis of TcdB Proteins within the Hypervirulent Clade 2 Reveals an Impact of RhoA Glucosylation on Clostridium difficile Proinflammatory Activities

ABSTRACT Clostridium difficile strains within the hypervirulent clade 2 are responsible for nosocomial outbreaks worldwide. The increased pathogenic potential of these strains has been attributed to several factors but is still poorly understood. During a C. difficile outbreak, a strain from this clade was found to induce a variant cytopathic effect (CPE), different from the canonical arborizing CPE. This strain (NAP1V) belongs to the NAP1 genotype but to a ribotype different from the epidemic NAP1/RT027 strain. NAP1V and NAP1 share some properties, including the overproduction of toxins, the binary toxin, and mutations in tcdC. NAP1V is not resistant to fluoroquinolones, however. A comparative analysis of TcdB proteins from NAP1/RT027 and NAP1V strains indicated that both target Rac, Cdc42, Rap, and R-Ras but only the former glucosylates RhoA. Thus, TcdB from hypervirulent clade 2 strains possesses an extended substrate profile, and RhoA is crucial for the type of CPE induced. Sequence comparison and structural modeling revealed that TcdBNAP1 and TcdBNAP1V share the receptor-binding and autoprocessing activities but vary in the glucosyltransferase domain, consistent with the different substrate profile. Whereas the two toxins displayed identical cytotoxic potencies, TcdBNAP1 induced a stronger proinflammatory response than TcdBNAP1V as determined in ex vivo experiments and animal models. Since immune activation at the level of intestinal mucosa is a hallmark of C. difficile-induced infections, we propose that the panel of substrates targeted by TcdB is a determining factor in the pathogenesis of this pathogen and in the differential virulence potential seen among C. difficile strains.

Scarce detection of mobile erm genes associated with tetQ in Bacteroides and Parabacteroides from Costa Rica.

The frequency of finding of clindamycin-resistant anaerobic bacteria in clinical samples has doubled from 2008 to 2010 in Costa Rica. To determine whether this increase is due to dissemination of erm genes aided by tetQ elements, we analyzed 100 isolates of Bacteroides or Parabacteroides from a regional hospital, a national hospital, and the community. Antimicrobial susceptibilities were recorded with a broth micro-dilution method and erm genes were detected by PCR and Southern blotting. In addition, plasmid isolation and mating experiments were performed to clarify the location and mobility of the detected erm genes. Resistance to clindamycin was by far more frequent in the regional hospital (72%) than in the national hospital (29%) and the community (26%). Resistance to tetracycline was even more common, with the community (85%) outweighing the hospitals (71-72%). While MIC of clindamycin were higher in the hospitals than in the community (P < 0.05), the opposite was seen for tetracycline (P < 0.0001). Of the sought-after genes, only ermG (n = 2), ermA (n = 1), and ermF (n = 1) were detected in the hospitals and ermF in the community (n = 2). In opposition to the low frequency of finding of erm genes, 71% of the isolates were positive for tetQ. None of the detected genes were encoded on plasmids. Only three isolates from the hospitals transferred their erm genes laterally. By contrast, 13 hospital isolates and two community isolates transferred tetQ. Despite the widespread finding of tetracycline-resistant tetQ-positive bacteria, mobile erm genes were rare in our bacterial collection. We conclude that the detected erm genes are likely not included in typical conjugative transposons of Bacteroides and Parabacteroides.

Phenotypic identification of over 1000 isolates of anaerobic bacteria recovered between 1999 and 2008 in a major Costa Rican hospital.

Because of limitations in infrastructure, the aetiology of infections caused by anaerobic bacteria is seldom determined in clinical laboratories of developing countries. This study reports on the identification of 1010 anaerobic bacterial isolates collected between 1999 and 2008 in a major Costa Rican hospital with the use of two commercial phenotypic systems (RapID 32A and API 20A). Approximately 60% of the isolates were Gram-positive and, among the 35 species of Gram-positive bacteria found, the genera Clostridium, Propionibacterium and Eggerthella, and anaerobic cocci predominated. Twenty eight species were found among 395 isolates of Gram-negative bacteria. Species of Bacteroides were very frequent, followed by species of Prevotella, Veillonella, Fusobacterium and Porphyromonas.

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.

A Clostridium difficile lineage endemic to Costa Rican hospitals is multidrug-resistant by acquisition of chromosomal mutations and novel mobile genetic elements

ABSTRACT 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.