Role of a cryptic tRNA gene operon in survival under translational stress.

As compared to eukaryotes, bacteria have a reduced tRNA gene set encoding between 30 and 220 tRNAs. Although in most bacterial phyla tRNA genes are dispersed in the genome, many species from distinct phyla also show genes forming arrays. Here, we show that two types of arrays with distinct evolutionary origins exist. This work focuses on long tRNA gene arrays (L-arrays) that encompass up to 43 genes, which disseminate by horizontal gene transfer and contribute supernumerary tRNA genes to the host. Although in the few cases previously studied these arrays were reported to be poorly transcribed, here we show that the L-array of the model cyanobacterium Anabaena sp. PCC 7120, encoding 23 functional tRNAs, is largely induced upon impairment of the translation machinery. The cellular response to this challenge involves a global reprogramming of the transcriptome in two phases. tRNAs encoded in the array are induced in the second phase of the response, directly contributing to cell survival. Results presented here show that in some bacteria the tRNA gene set may be partitioned between a housekeeping subset, which constantly sustains translation, and an inducible subset that is generally silent but can provide functionality under particular conditions.

Strain-to-strain variability among Staphylococcus aureus causing prosthetic joint infection drives heterogeneity in response to levofloxacin and rifampicin.

INTRODUCTION: Levofloxacin and rifampicin are the preferred treatment for prosthetic joint infection (PJI) caused by Staphylococcus aureus, especially when managed with implant retention (DAIR). However, a significant variability of success has been reported, which could be related to intrinsic characteristics of the microorganism. Our aim was to evaluate the variability in the anti-biofilm response to levofloxacin and rifampicin in a clinical collection of S. aureus. MATERIAL AND METHODS: Eleven levofloxacin- and rifampicin-susceptible S. aureus isolates causing PJI managed with DAIR were included. Levofloxacin, rifampicin and levofloxacin + rifampicin were tested in an in vitro static biofilm model in microtitre plates, where 48 h biofilms were challenged with antimicrobials during 24 h. Additionally, two genetically similar strains were tested in the CDC Biofilm Reactor, where 48 h biofilms were treated during 56 h. Antimicrobial activity was assessed by viable biofilm-embedded cells recount, and by crystal violet staining. RESULTS: All antimicrobial regimens showed significant anti-biofilm activity, but a notable scattering in the response was observed across all strains (inter-strain coefficient of variation for levofloxacin, rifampicin and levofloxacin + rifampicin of 22.8%, 35.8% and 34.5%, respectively). This variability was tempered with the combination regimen when tested in the biofilm reactor. No correlation was observed between the minimal biofilm eradicative concentration and the antimicrobial activity. Recurrent S. aureus isolates exhibited higher biofilm-forming ability compared with strains from resolved infections (7.6 log10 cfu/cm2±0.50 versus 9.0 log10 cfu±0.07). CONCLUSIONS: Significant variability may be expected in response to levofloxacin and rifampicin among biofilm-embedded S. aureus. A response in the lower range, together with other factors of bad prognosis, could be responsible of treatment failure.

Mycobacterium senegalense Infection after Implant-Based Breast Reconstruction, Spain.

Bacterial infection is a well-known complication of breast implant surgery. We identified Mycobacterium senegalense, the principal pathogen of bovine farcy of cattle, in a woman after implant-based breast reconstruction. This finding indicates that unusual pathogens should be considered as an etiology of infected breast prostheses.

Predictors of Mortality in Bloodstream Infections Caused by Pseudomonas aeruginosa and Impact of Antimicrobial Resistance and Bacterial Virulence.

Whether multidrug resistance (MDR) is associated with mortality in patients with Pseudomonas aeruginosa bloodstream infections (BSI) remains controversial. Here, we explored the prognostic factors of P. aeruginosa BSI with emphasis on antimicrobial resistance and virulence. All P. aeruginosa BSI episodes in a 5-year period were retrospectively analyzed. The impact in early (5-day) and late (30-day) crude mortality of host, antibiotic treatment, and pathogen factors was assessed by multivariate logistic regression analysis. Of 243 episodes, 93 (38.3%) were caused by MDR-PA. Crude 5-day (20%) and 30-day (33%) mortality was more frequent in patients with MDR-PA (34.4% versus 11.3%, P < 0.001 and 52.7% versus 21.3%, P < 0.001, respectively). Early mortality was associated with neutropenia (adjusted odds ratio [aOR], 9.21; 95% confidence interval [CI], 3.40 to 24.9; P < 0.001), increased Pitt score (aOR, 2.42; 95% CI, 1.34 to 4.36; P = 0.003), respiratory source (aOR, 3.23; 95% CI,2.01 to 5.16; P < 0.001), inadequate empirical therapy (aOR, 4.57; 95% CI, 1.59 to 13.1; P = 0.005), shorter time to positivity of blood culture (aOR, 0.88; 95% CI, 0.80 to 0.97; P = 0.010), an exoU-positive genotype (aOR, 3.58; 95% CI, 1.31 to 9.79; P = 0.013), and the O11 serotype (aOR, 3.64; 95% CI, 1.20 to 11.1; P = 0.022). These risk factors were similarly identified for late mortality, along with an MDR phenotype (aOR, 2.18; 95% CI, 1.04 to 4.58; P = 0.040). Moreover, the O11 serotype (15.2%, 37/243) was common among MDR (78.4%, 29/37) and exoU-positive (89.2%, 33/37) strains. Besides relevant clinical variables and inadequate empirical therapy, pathogen-related factors such as an MDR phenotype, an exoU-positive genotype, and the O11 serotype adversely affect the outcome of P. aeruginosa BSI.

Pathogenic characteristics of Pseudomonas aeruginosa bacteraemia isolates in a high-endemicity setting for ST175 and ST235 high-risk clones.

Multidrug-resistant (MDR) Pseudomonas aeruginosa represents a major clinical concern. The interplay between antimicrobial resistance and virulence of P. aeruginosa was investigated in in vitro and in vivo studies. Thirty-eight well-characterized (21 MDR and 17 non-MDR) P. aeruginosa strains from patients with bacteraemia were analysed. Resistance phenotype, carbapenemase production, clonal relatedness, type III secretion system genotype, O-antigen serotype, cytotoxicity (ability to lyse cells) on A549 cells, and virulence (lethality in nematodes) in a Caenorhabditis elegans model were investigated. MDR strains showed lower cytotoxicity (35.4 ± 21.30% vs. 45.0 ± 18.78 %; P = 0.044) and virulence (66.7% vs. 100%; P = 0.011) than non-MDR strains. However, the pathogenicity of MDR high-risk clones varied broadly, with ST235 and ST175 clones being the most and least cytotoxic (51.8 ± 10.59% vs. 11.0 ± 1.25%; P < 0.0001) and virulent ([100% vs. 73.1; P = 0.075] and [0% vs. 93.9%; P < 0.0001], respectively). The pathogenicity of the ST235 clone was similar to that of non-MDR strains, and its ability to lyse cells and high virulence were related with the exoU-positive genotype. Furthermore, the O11 serotype was more frequent among the ST235 clone and exoU-positive genotype strains and was also essential for the pathogenicity of P. aeruginosa. Our data suggest that the pathogenicity of MDR high-risk clones is the result not only of the resistance phenotype but also of the virulence genotype. These findings have implications for the clinical management of patients and infection control programmes.

Sub-inhibitory concentrations of oxacillin modify the expression of agr locus in Staphylococcus aureus clinical strains belonging to different clonal complexes.

BACKGROUND: The ability of Staphylococcus aureus to invade tissues and cause an infectious disease is the result of a multi-factorial process supported by the huge number of virulence factors inherent to this microorganism tightly regulated by the accessory gene regulator (agr). During antimicrobial therapy bacteria may be exposed to sub-inhibitory concentrations (subMICs) of antibiotics that may trigger transcriptional changes that may have an impact on the pathogenesis of infection. The objective of this study was to investigate the effect of oxacillin sub-MICs on agr system expression as the key component in the regulation of virulence in methicillin-susceptible (MSSA) and -resistant S. aureus (MRSA) strains. Furthermore, we studied the genetic basis of the agr locus and their potential association with the expression levels. METHODS: We have examined the expression of RNAIII and agrA mRNA as biomarkers for agr expression in the presence and absence of oxacillin subMICs in 10 MSSA and 4 MRSA clinical strains belonging to 5 clonal complexes (CC45-agrI, CC8-agrI, CC5-agrII, CC15-agrII and CC30-agrIII) causing endovascular complications. The DNA sequences of agr locus were obtained by whole genome sequencing. RESULTS: Our results revealed that exposure to subMICs of oxacillin had an impact on agr locus expression modifying the relative levels of expression with increases in 11 strains and with decreases in 3 strains. Thereby, the exposure to subMICs of oxacillin resulted in higher levels of expression of agr in CC15 and CC45 and lower levels in CC30. We also observed the presence of mutations in agrC and agrA in 13/14 strains with similar mutation profiles among strains within individual CCs except for strains of CC5. Although, agr expression levels differed among strains within CCs, the presence of these mutations was associated with differences in agr expression levels in most cases. CONCLUSIONS: Changes in agr expression induced by exposure to oxacillin subMICs should be considered because they could lead to changes in the virulence modulation and have an adverse effect on the course of infection, especially in certain clonal complexes.

Characterization of mobile genetic elements carrying VIM-1 and KPC-2 carbapenemases in Citrobacter freundii isolates in Madrid.

Carbapenemase producing Citrobacter freundii (CPCF) infections are still uncommon in European countries. Here we report a molecular study conducted in a tertiary care facility in southern Madrid, Spain, from 2009 to 2014 to investigate the epidemiology of CPCF. The blaIMP-1,blaIMP-2,blaKPC,blaNDM,blaOXA-48,blaVIM-1 and blaVIM-2 genes were screened by PCR. Molecular typing was carried out by Pulsed-field gel electrophoresis analysis (PFGE) and multilocus sequence typing (MLST). Whole genome sequencing (WGS) was performed to characterize the resistome and the mobile genetic elements associated with the carbapenems resistance of CPCF. A total of 11/521 (2.1%) isolates had reduced susceptibility to carbapenems. PCR amplification revealed the presence of blaVIM-1 in 10 isolates and blaKPC-2 in 2 isolates. One C. freundii isolate co-harbored blaVIM-1 and blaKPC-2 genes. PFGE and MLST assigned 10 different clonal, 4 previously reported (ST11, ST18, ST22 and ST64) and 6 new STs (ST89, ST90, ST91, ST92, ST92 and ST94). The blaVIM-1 gene was part of In624 (intI1-blaVIM-1-aacA4-dfrB1-aadA1-catB2-qacEΔ1/sul1). In 3 of these isolates, plasmid-mediated quinolone resistance genes (qnrA1 and qnrB4) were present in its downstream region, taking part of a complex class 1 integron ([In624:ISCR1:qnrB4-blaDHA-1] and [In624:ISCR1:qnrA1]). On the other hand, the blaKPC-2 gene was associated with a Tn3-based transposon. The dissemination of the blaVIM-1 gene among various clones suggests a successful horizontal transfer of integron carrying elements that play a dominant role in the development of multidrug resistance in Enterobacteriaceae.

Influence of molecular characteristics in the prognosis of methicillin-resistant Staphylococcus aureus prosthetic joint infections: beyond the species and the antibiogram.

We have explored the relationship of phenotypic (antibiogram, ß-haemolysis, agr functionality, biofilm formation) and genotypic characteristics on the prognosis of 18 cases of methicillin-resistant S. aureus prosthetic joint infection (2005-2015). All isolates belonged to CC5, and had agr type II. This pilot study suggests that phage-borne genes belonging to the immune evasion cluster (chp, sak and scn) were more frequent among episodes with treatment failure (80.0 vs. 37.5%).

Microbiological methods for surveillance of carrier status of multiresistant bacteria. / Métodos microbiológicos para la vigilancia del estado de portador de bacterias multirresistentes.

The presence of colonised patients is one of the main routes for the spread of multiresistant bacteria, and its containment is a clinical and public health priority. Surveillance studies are essential for early detection of colonisation by these bacteria. This article discusses the different microbiological methods, both based on culturing and molecular methods, for detection of carriers of multiresistant bacteria. Those species with a high clinical/epidemiological impact or generating therapeutic difficulties are included: Methicillin-resistant Staphylococcus aureus, Enterococcus spp. resistant to glycopeptides, enterobacteriaceae producing extended spectrum ß-lactamases and plasmid-mediated AmpC, carbapenemases producing enterobacteriaceae, Acinetobacter baumannii and multiresistant Pseudomonas aeruginosa. The information in this document should be considered as a structure matrix to be tailored to the specific needs of each centre.

Clinical, epidemiological and microbiological characteristics of relapse and re-infection in Clostridium difficile infection.

Recurrent diarrhea is a common complication of Clostridium difficile infection (CDI). Recurrent CDI (r-CDI) may be produced by the persistence of spores (relapse) or by the acquisition of a new strain (reinfection). In this study, we analyze epidemiological, clinical, microbiological and laboratory data from patients with r-CDI, relapse, and reinfection-CDI over 5 years and compared with a control group (non r-CDI). Among 60 patients with r-CDI, 36 patients had stool samples collected from two or more episodes, which were molecularly analyzed. Based on ribotyping, 63.9% of the samples were relapse, and 36.1% reinfection. In a multivariable logistic regression analysis, previous antibiotic exposure was found to be a risk factor for r-CDI (OR: 2.23; 95% CI: 1.0-4.9; p = 0.04). Patients with relapse had previous antibiotic exposure more frequently than did patients with reinfection (p = 0.03), and patients with reinfection suffered more frequently from chronic liver disease (p = 0.02) than did relapse patients. Relapse patients compared with the control group had a higher percentage of previous antibiotic exposure, although the difference was statistically no significant (73.9% vs. 91.3 p = 0.06). No significant differences for the selected variables were observed between the reinfection and control groups, although we observed a higher percentage of patients with chronic liver disease (30.8% vs 13.3%; p = 0.08). All isolates were sensitive to metronidazole and vancomycin. No significant differences in antibiotic susceptibility were found between the different groups. Sporulation and germination frequency of r-CDI were higher than non r-CDI (p = 0.02 and p < 0.01, respectively). Nevertheless, there were statistically not significant differences between the relapse and reinfection groups. Both frequencies were compared between the first and second episode of CDI for the relapse and reinfection groups, but differences were not observed to be statistically significant. In conclusion, our study showed that the recurrence of CDI was associated with antibiotic use and sporulation/germination frequency, regardless of relapse or reinfection. The use of antibiotics would produce a dysbiosis and favor the persistence of the C. difficile spores and relapse. A possible alteration of the intestinal microbiota and the bile salts produced by chronic liver disease could favor reinfection.

High MICs for Vancomycin and Daptomycin and Complicated Catheter-Related Bloodstream Infections with Methicillin-Sensitive Staphylococcus aureus.

We investigated the prognostic role of high MICs for antistaphylococcal agents in patients with methicillin-sensitive Staphylococcus aureus catheter-related bloodstream infection (MSSA CRBSI). We prospectively reviewed 83 episodes from 5 centers in Spain during April 2011-June 2014 that had optimized clinical management and analyzed the relationship between E-test MICs for vancomycin, daptomycin, oxacillin, and linezolid and development of complicated bacteremia by using multivariate analysis. Complicated MSSA CRBSI occurred in 26 (31.3%) patients; MICs for vancomycin and daptomycin were higher in these patients (optimal cutoff values for predictive accuracy = 1.5 µg/mL and 0.5 µg/mL). High MICs for vancomycin (hazard ratio 2.4, 95% CI 1.2-5.5) and daptomycin (hazard ratio 2.4, 95% CI 1.1-5.9) were independent risk factors for development of complicated MSSA CRBSI. Our data suggest that patients with MSSA CRBSI caused by strains that have high MICs for vancomycin or daptomycin are at increased risk for complications.

Comparison of local features from two Spanish hospitals reveals common and specific traits at multiple levels of the molecular epidemiology of metallo-ß-lactamase-producing Pseudomonas spp.

Twenty-seven well-characterized metallo-ß-lactamase (MBL)-producing Pseudomonas strains from two distantly located hospitals were analyzed. The results revealed specific features defining the multilevel epidemiology of strains from each hospital in terms of species, clonality, predominance of high-risk clones, composition/diversity of integrons, and linkages of Tn402-related structures. Therefore, despite the global trends driving the epidemiology of MBL-producing Pseudomonas spp., the presence of local features has to be considered in order to understand this threat and implement proper control strategies.

Relationship between agr dysfunction and reduced vancomycin susceptibility in methicillin-susceptible Staphylococcus aureus causing bacteraemia.

OBJECTIVES: Limited data exist regarding the role of agr dysfunction in reducing susceptibility to vancomycin in methicillin-susceptible Staphylococcus aureus (MSSA). This study investigated the clinical and molecular epidemiology of MSSA causing bacteraemia, with emphasis on the reduced susceptibility to vancomycin (RSV) phenotype (MIC ≥ 1.5 mg/L) and its relationship with agr dysfunction. METHODS: All MSSA bloodstream isolates obtained at our hospital during 2010 were analysed. Antimicrobial susceptibility was determined and time-kill experiments were performed for oxacillin. Multilocus sequence type and agr genotype were determined and DNA microarray analysis of virulence factors was performed. agr dysfunction was assessed phenotypically and by RT-PCR quantification of RNAIII. RESULTS: Of 84 MSSA, 55 (65.5%) exhibited the RSV phenotype, comprising 13 clonal complexes. agr II polymorphism was more prevalent in RSV than non-RSV isolates (41.8% versus 17.2%, P = 0.023) and average levels of RNAIII gene expression were higher in RSV than non-RSV isolates (ΔCt 4.05 ± 3.29 versus 1.5 ± 2.11, P = 0.005), implying greater agr dysfunction in RSV MSSA. CONCLUSIONS: We demonstrated a correlation between RSV phenotype in MSSA and reduced agr expression, particularly in association with the agr II genotype. These results may help to understand the role of agr dysfunction in the increased mortality in MSSA infections.

Molecular epidemiology and virulence of Escherichia coli O16:H5-ST131: comparison with H30 and H30-Rx subclones of O25b:H4-ST131.

The present study was carried out to evaluate the prevalence of the clonal subgroup O16:H5-ST131 and the H30 and H30-Rx subclones among E. coli isolates causing extraintestinal infections and to know their virulence potential. The ST131 clonal group accounted for 490 (16%) of the 2995 isolates obtained from clinical samples in five Spanish hospitals during the study period (2005-2012). Among those 490 ST131 isolates, 456 belonged to serotype O25b:H4, 27 to O16:H5 and seven were O-non-typeable:H4 (ONT:H4). All 27 O16:H5 isolates showed fimH41, whereas fimH30 and fimH22 alleles were the most frequently detected among O25b:H4 isolates. The majority (381/490; 78%) of ST131 isolates belonged to H30 subclone, and 302 of 381 (79%) H30 isolates belonged to the H30-Rx subclone. Of the 27 O16:H5 isolates, 48% produced CTX-M-14; however, none produced CTX-M-15. In contrast, 46% of O25b:H4 isolates produced CTX-M-15 while only 2% produced CTX-M-14. More than a half of the O16:H5 isolates (56%) showed the ExPEC status which was significantly more prevalent within O25b:H4 isolates (81%) (P<0.01), especially among H30-Rx (97%) isolates. In the present study, a modified virotype scheme was applied within which approximately half (52%) of the O16:H5 isolates showed the C1 specific virotype. Despite their low virulence-gene score (mean of virulence genes 6.4 versus 8.5 in O25b:H4 isolates), six out of the 10 O16:H5 isolates assayed showed high virulence in the mouse model of sepsis (killed 90-100% of mice challenged). Furthermore, four O16:H5 isolates of virotypes A and C1, carrying K2 variant of group II capsule, showed lethality at 24h. Thus, certain O16:H5 fimH41 isolates show a similar in vivo virulence to that reported with the highly virulent O25b:H4 H30-Rx isolates (Mora et al., PLOS ONE 2014, e87025), supporting their potential virulence for humans.

CagA phosphorylation EPIYA-C motifs and the vacA i genotype in Helicobacter pylori strains of asymptomatic children from a high-risk gastric cancer area in northeastern Brazil.

Helicobacter pylori infection is one of the most common infections worldwide and is associated with gastric diseases. Virulence factors such as VacA and CagA have been shown to increase the risk of these diseases. Studies have suggested a causal role of CagA EPIYA-C in gastric carcinogenesis and this factor has been shown to be geographically diverse. We investigated the number of CagA EPIYA motifs and the vacA i genotypes in H. pylori strains from asymptomatic children. We included samples from 40 infected children (18 females and 22 males), extracted DNA directly from the gastric mucus/juice (obtained using the string procedure) and analysed the DNA using polymerase chain reaction and DNA sequencing. The vacA i1 genotype was present in 30 (75%) samples, the i2 allele was present in nine (22.5%) samples and both alleles were present in one (2.5%) sample. The cagA-positive samples showed distinct patterns in the 3’ variable region of cagA and 18 of the 30 (60%) strains contained 1 EPIYA-C motif, whereas 12 (40%) strains contained two EPIYA-C motifs. We confirmed that the studied population was colonised early by the most virulent H. pylori strains, as demonstrated by the high frequency of the vacA i1 allele and the high number of EPIYA-C motifs. Therefore, asymptomatic children from an urban community in Fortaleza in northeastern Brazil are frequently colonised with the most virulent H. pylori strains.

Molecular characterization of Achromobacter isolates from cystic fibrosis and non-cystic fibrosis patients in Madrid, Spain.

Multilocus sequence typing and nrdA sequence analysis identified 6 different species or genogroups and 13 sequence types (STs) among 15 Achromobacter isolates from cystic fibrosis (CF) patients and 7 species or genogroups and 11 STs among 11 isolates from non-CF patients. Achromobacter xylosoxidans was the most frequently isolated species among CF patients.

Clonal diversity among Burkholderia cepacia complex isolates from cystic fibrosis patients in a reference unit.

INTRODUCTION: The epidemiology of Burkholderia cepacia complex (Bcc) in cystic fibrosis (CF) is not widely known. METHODS: All CF patients with Bcc between 2002 and 2011 were reviewed, and a molecular analysis of isolates was performed. RESULTS: The prevalence of Bcc infection was 7.2% (18/250). Molecular analysis of 16 Bcc isolates showed 5 species (7 B. contaminans, 6 B. cepacia, 1 B. cenocepacia, 1 B. multivorans, and 1 B. stabilis) and 13 sequence types. There were no cases of cross-transmission. CONCLUSION: A high diversity of Bcc species was found in infected CF patients.

[Staphylococcus aureus nasal colonization in medical students: importance in nosocomial transmission]. / Colonización nasal por Staphylococcus aureus en estudiantes de medicina: importancia en la transmisión hospitalaria.

BACKGROUND: Staphylococcus aureus is the main pathogen causing nosocomial infections. Health professionals, including medical students, could be a source of transmission. The aims of the study were to determine the rate of nasal carriage of S.aureus susceptible and resistant to methicillin (MRSA) and evaluate the knowledge and adherence that students had about hand hygiene. METHODS: The study included medical students attached to the Hospital Universitario 12 de Octubre (Madrid, Spain). We collected samples from both nasal vestibules, and the antimicrobial susceptibility was determined on all isolates. Data collection was performed using a self-administered questionnaire that included risk factors for colonization, hygiene habits and knowledge of hand hygiene protocols. RESULTS: Of the 140 students included, 55 (39.3%) were colonized by S.aureus, and 3 (2,1%) by MRSA. The exposure to antibiotics in the last 3 months was lower in colonized students (12.3% vs. 25.9%, P=.03). Self-assessment showed that 56.4% of students almost never washed their hands before to attending to the first patient, and only 38.6% always washed after examining patients. More than a third (35.7%) ignored the hand hygiene protocol, and 38.6% had not received specific formation. CONCLUSIONS: Medical students should be included in hospital infection control programs. Hand hygiene training should be given to students before they begin their practices in the hospital.