Removal of natural anti-αGal antibodies elicits protective immunity against Gram-negative bacterial infections.

Antibody-dependent enhancement (ADE) of bacterial infections occurs when blocking or inhibitory antibodies facilitate the infectivity of pathogens. In humans, antibodies involved in ADE of bacterial infections may include those naturally produced against Galα1-3Galß1-4GlcNAcß (αGal). Here, we investigate whether eliminating circulating anti-αGal antibodies using a soluble αGal glycopolymer confers protection against Gram-negative bacterial infections. We demonstrated that the in vivo intra-corporeal removal of anti-αGal antibodies in α1,3-galactosyltransferase knockout (GalT-KO) mice was associated with protection against mortality from Gram-negative sepsis after cecal ligation and puncture (CLP). The improved survival of GalT-KO mice was associated with an increased killing capacity of serum against Escherichia coli isolated after CLP and reduced binding of IgG1 and IgG3 to the bacteria. Additionally, inhibition of anti-αGal antibodies from human serum in vitro increases the bactericidal killing of E. coli O86:B7 and multidrug-resistant Klebsiella pneumoniae and Pseudomonas aeruginosa. In the case of E. coli O86:B7, there was also an improvement in bacteria opsonophagocytosis by macrophages. Both lytic mechanisms were related to a decreased binding of IgG2 to the bacteria. Our results show that protective immunity against Gram-negative bacterial pathogens can be elicited, and infectious diseases caused by these bacteria can be prevented by removing natural anti-αGal antibodies.

Molecular Epidemiology, Antimicrobial Susceptibility, and Clinical Features of Methicillin-Resistant Staphylococcus aureus Bloodstream Infections over 30 Years in Barcelona, Spain (1990-2019).

Methicillin-resistant Staphylococcus aureus bloodstream infections (MRSA-BSI) are a significant cause of mortality. We analysed the evolution of the molecular and clinical epidemiology of MRSA-BSI (n = 784) in adult patients (Barcelona, 1990−2019). Isolates were tested for antimicrobial susceptibility and genotyped (PFGE), and a selection was sequenced (WGS) to characterise the pangenome and mechanisms underlying antimicrobial resistance. Increases in patient age (60 to 71 years), comorbidities (Charlson's index > 2, 10% to 94%), community-onset healthcare-associated acquisition (9% to 60%), and 30-day mortality (28% to 36%) were observed during the 1990−1995 and 2014−2019 periods. The proportion of catheter-related BSIs fell from 57% to 20%. Current MRSA-BSIs are caused by CC5-IV and an upward trend of CC8-IV and CC22-IV clones. CC5 and CC8 had the lowest core genome proportions. Antimicrobial resistance rates fell, and only ciprofloxacin, tobramycin, and erythromycin remained high (>50%) due to GyrA/GrlA changes, the presence of aminoglycoside-modifying enzymes (AAC(6')-Ie-APH(2″)-Ia and ANT(4')-Ia), and mph(C)/msr(A) or erm (C) genes. Two CC22-IV strains showed daptomycin resistance (MprF substitutions). MRSA-BSI has become healthcare-associated, affecting elderly patients with comorbidities and causing high mortality rates. Clonal replacement with CC5-IV and CC8-IV clones resulted in lower antimicrobial resistance rates. The increased frequency of the successful CC22-IV, associated with daptomycin resistance, should be monitored.

Staphylococcus aureus surface protein G (sasG) allelic variants: correlation between biofilm formation and their prevalence in methicillin-resistant S. aureus (MRSA) clones.

Methicillin-resistant Staphylococcus aureus (MRSA) may persist for long periods due to biofilm formation. The objective of this study was to describe biofilm formation in association with the presence of S. aureus surface protein G (sasG) and its allelic variants in MRSA bacteraemia isolates from endemic (CC5, CC8, CC22) and sporadic clones in Spain (2008-2015). Crystal violet staining was used to assess biofilm formation; DNA microarray, RT-qPCR, and long-read whole genome sequencing were applied to determine the presence, expression and structure of sasG, respectively. The endemic CC5 and CC8 clones produced more biofilm than the sporadic clones; these endemic clones carried sasG allelic variant 1. Otherwise, sporadic clones, with less biofilm formation, showed either an absence of sasG (65%) or the presence of allelic variant 2 (35%). Variants 1 and 2 differed in the expression of sasG (1.56 ± 1.20 and 0.37 ± 0.32, respectively). The analysis of a large cohort of closed S. aureus genomes available on the NCBI database confirmed the distribution of the two allelic variants with low amino acid identity (68.1%) among endemic and sporadic clones. SasG variant 1 present in the major CC5 and CC8 clones was correlated with increased biofilm formation and may represent an important virulence determinant.

Detection of the Novel optrA Gene Among Linezolid-Resistant Enterococci in Barcelona, Spain.

The purpose of this study was to describe the presence of the novel optrA gene among clinical isolates of enterococci in a Spanish teaching hospital (May 2016-April 2017). optrA and cfr genes were screened by PCR in all isolates showing linezolid minimal inhibitory concentration (MIC) ≥4 mg/L. The genetic relatedness of the isolates, the presence of resistance and virulence genes, and the genetic environment of optrA were assessed by whole-genome sequencing (WGS). Six of 1,640 enterococci had linezolid MIC ≥4 mg/L. Among them, the optrA gene was detected in five Enterococcus faecalis isolated from unrelated patients. Although none of them had received linezolid or chloramphenicol, all had antecedents of recent quinolone consumption. WGS analysis revealed the existence of two different genotypes: ST585 and ST474. cfr was not detected in any of the isolates. No mutations were detected among the 23S ribosomal RNA and the ribosomal proteins L3, L4, and L22. Both genotypes also carried genes related to aminoglycoside, lincosamide, macrolide, phenicol, and tetracycline resistance. Detection of optrA in a setting with low linezolid consumption and among patients without antecedents of oxazolidinone therapy is of concern.

High vancomycin MICs predict the development of infective endocarditis in patients with catheter-related bacteraemia due to methicillin-resistant Staphylococcus aureus.

Background: It has been suggested that there is an increased risk of treatment failure in episodes of MRSA bloodstream infection (BSI) caused by strains with high vancomycin MICs. However, it is unknown if this phenomenon may also act as a risk factor for the development of infective endocarditis (IE). Methods: We analysed 207 episodes of catheter-related (CR)-BSI recruited from June 2008 to December 2009 within a prospective study on MRSA BSI in 21 Spanish hospitals. Vancomycin susceptibility was centrally tested. The impact of high vancomycin MIC values (≥1.5 mg/L by Etest) on the subsequent development of IE was investigated by Cox regression. Results: High vancomycin MIC values were observed in 46.9% of the isolates. Initial therapy consisted of vancomycin [99 episodes (44.7%)], daptomycin [25 (12.1%)], linezolid [18 (8.7%)] and other antistaphylococcal agents [16 (7.7%)]. Haematogenous complications occurred in 41 patients (19.8%), including 10 episodes complicated by IE. Early (48 h) and late (30 day) all-cause mortality were 3.4% and 25.1%, respectively. High vancomycin MIC isolates were more common among patients that developed IE compared with those free from this complication [90.9% (9/10) versus 44.7% (88/197); P = 0.007]. This association remained significant after adjusting for multiple confounders (including initial antibiotic therapy and catheter removal) in different models (minimum hazard ratio: 9.18; 95% CI: 1.16-72.78; P = 0.036). There were no differences in mortality according to vancomycin MIC values. Conclusions: Decreased susceptibility to vancomycin acted as a predictor of the development of IE complicating MRSA CR-BSI.

Impact of multidrug resistance on the pathogenicity of Pseudomonas aeruginosa: in vitro and in vivo studies.

The biological cost of multidrug resistance in Pseudomonas aeruginosa (PA) remains unclear. This study aimed to evaluate the relationship between pathogenicity and the resistance profile of different PA strains, including the most common epidemic high-risk clones. Nine PA strains were studied, including two reference strains, PAO1 and PA14 [both susceptible to all antipseudomonals (multiS)], and seven clinical strains comprising three clinical multiS strains, a non-clonal multidrug-resistant (MDR) strain and the high-risk MDR clones ST111, ST235 and ST175. In vitro studies were performed to investigate growth rate, type III secretion system (TTSS) genotype, cytotoxicity and invasiveness. Additionally, a peritonitis/sepsis model was used in C57BL/6 mice. The in vitro bacterial duplication time was shorter in clinical multiS strains than in MDR-PA (0.42±0.08h vs. 0.55±0.14h; P=0.023). Among the clinical strains, exoU(+) genotype was observed only in the epidemic clone ST235. In the animal model, the probability of mortality at 48h was 70% for clinical multiS strains vs. 7.5% for clinical MDR-PA (P<0.001, log-rank). The high-risk clone ST235 was the only MDR strain that was able to cause mortality. Bacterial concentrations in peritoneal fluid were higher in mice inoculated with multiS strains compared with MDR-PA [log CFU/mL, 8.95 (IQR 3.42-9.32) vs. 1.98 (IQR 1.08-2.80); P<0.001]. These data indicate that MDR profiles are associated with a reduction in virulence of PA in a murine model. Further studies are needed to elucidate the clinical implications of these results.

Clinical characteristics, treatment and outcomes of MRSA bacteraemia in the elderly.

OBJECTIVES: To compare clinical and microbiological characteristics, treatment and outcomes of MRSA bacteraemia among elderly and younger patients. MATERIAL AND METHODS: Prospective study conducted at 21 Spanish hospitals including patients with MRSA bacteraemia diagnosed between June/2008 and December/2009. Episodes diagnosed in patients aged 75 or more years old (≥75) were compared with the rest of them (<75). RESULTS: Out of 579 episodes of MRSA bacteraemia, 231 (39.9%) occurred in patients ≥75. Comorbidity was significantly higher in older patients (Charlson score ≥4: 52.8 vs. 44%; p = .037) as was the severity of the underlying disease (McCabe ≥1: 61.9 vs. 43.4%; p < .001). In this group the acquisition was more frequently health-care related (43.3 vs. 33.9%, p = .023), mostly from long-term care centers (12.1 vs. 3.7%, p < .001). An unknown focus was more frequent among ≥75 (19.9 vs. 13.8%; p = .050) while severity at presentation was similar between groups (Pitt score ≥3: 31.2 vs. 27.6%; p = .352). The prevalence of vancomycin resistant isolates was similar between groups, as was the appropriateness of empirical antibiotic therapy. Early (EM) and overall mortality (OM) were significantly more frequent in the ≥75 group (EM: 12.1 vs. 6%; p = .010 OM: 42.9 vs. 23%; p < .001). In multivariate analysis age ≥75 was an independent risk factor for overall mortality (aOR: 2.47, CI: 1.63-3.74; p < .001). CONCLUSION: MRSA bacteraemia was frequent in patients aged ≥75 of our cohort. This group had higher comorbidity rates and the source of infection was more likely to be unknown. Although no differences were seen in severity or adequacy of empiric therapy, elderly patients showed a higher overall mortality.

Methicillin-resistant Staphylococcus aureus (MRSA) catheter-related bacteraemia in haemodialysis patients.

BACKGROUND: The aim of the study was to determine clinical and microbiological differences between patients with methicillin-resistant Staphylococcus aureus (MRSA) catheter-related bacteraemia (CRB) undergoing or not undergoing haemodialysis, and to compare outcomes. METHODS: Prospective multicentre study conducted at 21 Spanish hospitals of patients with MRSA bacteraemia diagnosed between June 2008 and December 2009. Patients with MRSA-CRB were selected. Data of patients on haemodialysis (HD-CRB) and those not on haemodialysis (non-HD-CRB) were compared. RESULTS: Among 579 episodes of MRSA bacteraemia, 218 (37.7%) were CRB. Thirty-four (15.6%) were HD-CRB and 184 (84.4%) non-HD-CRB. All HD-CRB patients acquired the infection at dialysis centres, while in 85.3% of the non-HD-CRB group the infection was nosocomial (p < .001). There were no differences in age, gender or severity of bacteraemia (Pitt score); comorbidities (Charlson score ≥ 4) were higher in the HD-CRB group than in the non-HD-CRB group (73.5% vs. 46.2%, p = .003). Although there were no differences in VAN-MIC ≥ 1.5 mg/L according to microdilution, using the E-test a higher rate of VAN-MIC ≥ 1.5 mg/L was observed in HD-CRB than in non-HD-CRB patients (63.3% vs. 44.1%, p = .051). Vancomycin was more frequently administered in the HD-CRB group than in the non-HD-CRB group (82.3% vs. 42.4%, p = <.001) and therefore the appropriate empirical therapy was significantly higher in HD-CRB group (91.2% vs. 73.9%, p = .029). There were no differences with regard to catheter removal (79.4% vs. 84.2%, p = .555, respectively). No significant differences in mortality rate were observed between both groups (Overall mortality: 11.8% vs. 27.2%, p = .081, respectively), but there was a trend towards a higher recurrence rate in HD-CRB group (8.8% vs. 2.2%, p = .076). CONCLUSIONS: In our multicentre study, ambulatory patients in chronic haemodialysis represented a significant proportion of cases of MRSA catheter-related bacteraemia. Although haemodialysis patients with MRSA catheter-related bacteraemia had significantly more comorbidities and higher proportion of strains with reduced vancomycin susceptibility than non-haemodialysis patients, overall mortality between both groups was similar.

Prevalence of methicillin-resistant Staphylococcus aureus colonization in HIV-infected patients in Barcelona, Spain: a cross-sectional study.

BACKGROUND: Colonization by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has been found to be markedly more common in HIV-infected individuals in the USA. Studies evaluating the prevalence MRSA colonization in HIV-infected populations in Europe are scarce. The aim of this study was to investigate the prevalence of MRSA colonization in a cohort of HIV-infected patients in Barcelona, Spain. METHODS: Nasal and pharyngeal S. aureus carriage was assessed in a random sample of 190 patients from an outpatient HIV clinic. Nasal and pharyngeal swab specimens were obtained for staphylococcal culture from 190 and 110 patients respectively. All MRSA isolates were screened for Panton-Valentine leukocidin (PVL) genes by PCR. Molecular characterization of MRSA isolates was performed by multilocus sequence typing. Data related to HIV infection, healthcare exposure, and previously described risk factors for MRSA were collected from medical records and a questionnaire administered to each patient. RESULTS: The patients' characteristics were as follows: male, 83 %; median (IQR) age, 45 (39-49) years; intravenous drug users, 39 %; men who have sex with men, 32 %; heterosexual, 26 %; CD4 count, 528/µL (IQR 351-740); on antiretroviral therapy, 96 %; and undetectable plasma viral load, 80 %. Sixty-five patients (34 %) were colonized by S. aureus. MRSA colonization was found in 1 % and 2 % of nasal and pharyngeal samples respectively. No PVL positive MRSA strains were detected and all the MRSA isolates belonged to typical hospital-acquired clones. CONCLUSIONS: Our data suggest that CA-MRSA colonization is not currently a problem in HIV-infected individuals in our area.

Prospective observational study of prior rectal colonization status as a predictor for subsequent development of Pseudomonas aeruginosa clinical infections.

The potential role of Pseudomonas aeruginosa (PA) intestinal colonization in the subsequent development of infections has not been thoroughly investigated. The aims of this study were to assess the role of PA intestinal colonization as a predictor of subsequent infections and to investigate the risk factors associated with the development of PA infection in patients in the intensive care unit (ICU). For this purpose, a prospective study was conducted that included (i) active surveillance of PA rectal colonization at ICU admission and weekly until ICU discharge, (ii) detection of PA clinical infections, and (iii) genotypic analysis by pulsed-field gel electrophoresis (PFGE). A total of 414 patients were included, of whom 179 (43%) were colonized with PA. Among the 77 patients who developed PA infection, 69 (90%) had prior PA colonization, and 60 (87%) of these showed genotyping concordance between rectal and clinical isolates. The probability of PA infection 14 days after ICU admission was 26% for carriers versus 5% for noncarriers (P < 0.001). Cox regression analysis identified prior PA rectal colonization as the main predictor of PA infection (hazard ratio [HR], 15.23; 95% confidence interval [CI], 6.9 to 33.7; P < 0.001). Prior use of nonantipseudomonal penicillins was also identified as an independent variable associated with PA infection (HR, 2.15; 95% CI, 1.3 to 3.55; P < 0.003). Our study demonstrated that prior PA rectal colonization is a key factor for developing PA infection.

Antibiotic pressure is a major risk factor for rectal colonization by multidrug-resistant Pseudomonas aeruginosa in critically ill patients.

The intestinal reservoir is central to the epidemiology of Pseudomonas aeruginosa, but the dynamics of intestinal colonization by different phenotypes have been poorly described. To determine the impact of antimicrobial exposure on intestinal colonization by multidrug-resistant (MDR) and extensively drug-resistant (XDR) P. aeruginosa, we screened intensive care unit (ICU) patients for rectal colonization on admission and at weekly intervals. During an 18-month study period, 414 ICU patients were enrolled, of whom 179 (43%) were colonized; 112 (63%) of these were identified at ICU admission and 67 (37%) during their ICU stay. At 10 days after ICU admission, the probabilities of carriage were 44%, 24%, and 24% for non-MDR, MDR-non-XDR, and XDR P. aeruginosa strains, respectively (log rank, 0.02). Pulsed-field gel electrophoresis showed 10 pairs of non-MDR P. aeruginosa and subsequent MDR-non-XDR strains isolated from the same patients to be clonally identical and another 13 pairs (8 MDR-non-XDR and 5 XDR) to be unrelated. There was one specific clone between the 8 MDR-non-XDR strains and an identical genotype in the 5 XDR isolates. The Cox regression analysis identified MDR P. aeruginosa acquisition as associated with the underlying disease severity (adjusted hazard ratio [aHR], 1.97; 95% confidence interval [CI], 1.22 to 3.18; P = 0.006) and prior use of fluoroquinolones (aHR, 1.02; 95% CI, 1.00 to 1.04; P = 0.039), group 2 carbapenems (aHR, 1.03; 95% CI, 1.00 to 1.07; P = 0.041), and ertapenem (aHR, 1.08; 95% CI, 1.02 to 1.14; P = 0.004). The epidemiology of MDR P. aeruginosa is complex, and different clusters may coexist. Interestingly, ertapenem was found to be associated with the emergence of MDR isolates.

Increase in bloodstream infection due to vancomycin-susceptible Enterococcus faecium in cancer patients: risk factors, molecular epidemiology and outcomes.

We conducted a prospective study to assess the risk factors, molecular epidemiology and outcome of bloodstream infection (BSI) due to Enterococcus faecium in hospitalized cancer patients. Between 2006 and 2012, a significant increase in vancomycin-susceptible E. faecium BSI was observed among cancer patients. Comparison of 54 episodes of BSI due to E. faecium with 38 episodes of BSI due to E. faecalis showed that previous use of carbapenems was the only independent risk factor for E. faecium acquisition (OR 10.24; 95% CI, 1.35-77.66). All E. faecium isolates were susceptible to glycopeptides, whereas 97% showed high-level resistance to ampicillin and ciprofloxacin. All 30 isolates available for genotyping belonged to the hospital-associated E. faecium lineages 17, 18 and 78. After 2009, most of the isolates belonged to ST117 (lineage 78). Patients with E. faecium BSI were more likely to receive inadequate initial empirical antibiotic therapy than patients with E. faecalis BSI, and time to adequate empirical antibiotic therapy was also longer in the former group. No significant differences were found between the two groups regarding early and overall case-fatality rates. Independent risk factors for overall case-fatality were current corticosteroids (OR 4.18; 95% CI, 1.34-13.01) and intensive care unit admission (OR 9.97; 95% CI, 1.96-50.63). The emergence of E. faecium among cancer patients is a concern since there are limited treatment options and it may presage the emergence of vancomycin-resistant enterococci. A rationale approach that combines infection control with antimicrobial stewardship.

Prevalence and molecular characterization of methicillin-resistant Staphylococcus aureus ST398 resistant to tetracycline at a Spanish hospital over 12 years.

Methicillin-resistant Staphylococcus aureus (MRSA) ST398, associated with livestock animals, was described in 2003 as a new lineage infecting or colonizing humans. We evaluated the prevalence and molecular characteristics of MRSA ST398 isolated in the Hospital Universitari de Bellvitge from January 2000 to June 2011. Tetracycline resistant (Tet-R) MRSA isolates from single patients (pts) were screened by SmaI-pulsed field gel electrophoresis (PFGE). Nontypable MRSA strains by SmaI (NT Sma I)-MRSA were further analysed by ApaI-PFGE, spa, SCCmec, agr, MLST typing, and by DNA microarray hybridization. Among 164 pts harboring Tet-R MRSA, NT Sma I-MRSA ST398-agrI was found in 33 pts (20%). Although the first pt was detected in 2003, 22/33 pts (67%) were registered in the 2010-2011 period. Ten pts (30%) were infected and cancer was the most frequent underlying disease. In one case, death was due to MRSA-ST398-related infection. Five pulsotypes (A-E) were detected using ApaI-PFGE, with type A accounting for 76% of the strains. The majority of the studied isolates presented spa type t011 (70%) and SCCmec type V (88%). One strain was spa negative both by PCR and microarray analysis. Forty-nine percent of the studied isolates showed resistance to 3 or more antibiotic classes, in addition to beta-lactams. Ciprofloxacin resistance was 67%. Tet-R was mediated by tet(M) and tet(K) in 26 isolates. All isolates lacked Panton-Valentine Leukocidin production, as well as other significant toxins. This study displays the molecular features of MRSA-ST398 clone and shows the increase in tetracycline resistance together with arise in MRSA-ST398 isolates infecting or colonizing patients in our clinical setting.

Low prevalence of Cfr-mediated linezolid resistance among methicillin-resistant Staphylococcus aureus in a Spanish hospital: case report on linezolid resistance acquired during linezolid therapy.

Linezolid is an effective antimicrobial agent to treat methicillin-resistant Staphylococcus aureus (MRSA). Resistance to linezolid due to the cfr gene is described worldwide. The present study aimed to analyze the prevalence of the cfr-mediated linezolid resistance among MRSA clinical isolates in our area. A very low prevalence of cfr mediated linezolid resistance was found: only one bacteremic isolate out of 2 215 screened isolates. The only linezolid resistant isolate arose in a patient, previously colonized by MRSA, following linezolid therapy. Despite the low rate of resistance in our area, ongoing surveillance is advisable to avoid the spread of linezolid resistance.