Progress towards the clinical use of antimicrobial peptides: challenges and opportunities.

INTRODUCTION: To overcome the challenge of multidrug resistance, natural and synthetic peptides are candidates to become the basis of innovative therapeutics, featuring diverse mechanisms of action. Traditionally, the time elapsed from medical discoveries to their application is long. The urgency derived from the emergence of antibiotic resistance recommends an acceleration of research to put the new weapons in the hands of clinicians. AREAS COVERED: This narrative review introduces ideas and suggestions of new strategies that may be used as a basis upon which to recommend reduced development times and to facilitate the arrival of new molecules in the fight against microbes. EXPERT OPINION: Although studies on new innovative antimicrobial treatments are being conducted, sooner rather than later, more clinical trials, preclinical and translational research are needed to promote the development of innovative antimicrobial treatments for multidrug resistant infections. The situation is worrying, no less than that generated by pandemics such as the ones we have just experienced and conflicts such as world wars. Although from the point of view of human perception, resistance to antibiotics may not seem as serious as these other situations, it is possibly the hidden pandemic that most jeopardizes the future of medicine.

Colistin Enhances Rifampicin's Antimicrobial Action in Colistin-Resistant Pseudomonas aeruginosa Biofilms.

The emergence of multidrug-resistant Pseudomonas aeruginosa infections has urged the need to find new strategies, such as the use of combinations of antibiotics. Among these, the combination of colistin with other antibiotics has been studied. Here, the action of combinations of colistin and rifampicin on both planktonic and sessile cells of colistin-resistant P. aeruginosa was studied. Dynamic biofilms were formed and treated with such a combination, resulting in an active killing effect of both colistin-resistant and colistin-susceptible P. aeruginosa in biofilms. The results suggest that the action of colistin on the outer membrane facilitates rifampicin penetration, regardless of the colistin-resistant phenotype. Based on these in vitro data, we propose a colistin-rifampicin combination as a promising treatment for infections caused by colistin-resistant P. aeruginosa.

Substantivity of mouth-rinse formulations containing cetylpyridinium chloride and O-cymen-5-ol: a randomized-crossover trial.

BACKGROUND: The efficacy of mouth-rinses strongly depends upon their substantivity. The use of natural and non-toxic products that avoid secondary effects is gaining interest in preventive dentistry. The purpose of this study was to evaluate the substantivity of two formulations of mouth-washing solutions based on cetylpyridinium (CPC) and O-cymen-5-ol. METHODS: This was a randomized, double-blind, crossover trial conducted at the Faculty of Medicine and Health Sciences of the University of Barcelona. Bacterial re-colonization was followed by live/dead (SYTOTM9 + propidium iodide) bacterial staining and measured by confocal laser scanning microscopy and fluorometry. Unstimulated saliva samples were collected from 16 healthy individuals at baseline saliva and then, at 15 min, 30 min and 1, 2, 3, and 4 h after the following mouth-rinses: (i) a single, 1-min mouth-rinse with 15 ml of placebo (negative control); (ii) a single, 1-min mouth-rinse with 15 ml of CPC (0.05%) ; (iii) a single, 1-min mouth-rinse with 15 ml of O-cymen-5-ol (0.09%); (iv) a single, 1-min mouth-rinse with 15 ml of CPC (0.05%) + O-cymen-5-ol (0.09%). RESULTS: Proportion of dead bacteria was significantly higher for all mouthrinses during the first 15 min compared to baseline (CPC = 48.0 ± 13.9; 95% CI 40.98-56.99; p < 0.001, O-cymen-5-ol = 79.8 ± 21.0; 95% CI 67.71-91.90; p < 0.05, CPC + O-cymen-5-ol = 49.4 ± 14; 95% CI 40.98-56.99; p < 0.001 by fluorometry and 54.8 ± 23.0; 95% CI 41.50-68.06; p < 0.001, 76.3 ± 17.1; 95% CI 66.36-86.14; p < 0.001, 47.4 ± 11.9; 95% CI 40.49-54.30; p < 0.001 by confocal laser scanning microscopy, respectively). Nevertheless, after 4 h, CPC + O-cymen-5-ol was the only one that obtained significant values as measured by the two quantification methods used (80.3 ± 22.8; 95% CI 67.15-93.50; p < 0.05 and 81.4 ± 13.8; 95% CI 73.45-89.43; p < 0.05). The combined use of CPC + O-cymen-5-ol increased the substantivity of the mouthrinse with respect to mouthrinses prepared with either of the two active products alone. CONCLUSION: The synergistic interaction of CPC and O-cymen-5-ol prolongs their substantivity. The resulting formulation may be as effective as other antimicrobials, such as triclosan or chlorhexidine, but without their undesirable secondary effects. Thus, mouthrinsing products based on Combinations of CPC and O-cymen-5-ol may replace in the near future Triclosan and Chlorhexidine-based mouthrinses.

Studying Lipid Membrane Interactions of a Super-Cationic Peptide in Model Membranes and Living Bacteria.

The super-cationic peptide dendrimers (SCPD) family is a valuable class of antimicrobial peptide candidates for the future development of antibacterial agents against multidrug-resistant gram-negative bacteria. The deep knowledge of their mechanism of action is a major challenge in research, since it may be the basis for future modifications/optimizations. In this work we have explored the interaction between SCPD and membranes through biophysical and microbiological approaches in the case of the G1OLO-L2OL2 peptide. Results support the idea that the peptide is not only adsorbed or close to the surface of the membrane but associated/absorbed to some extent to the hydrophobic-hydrophilic region of the phospholipids. The presence of low concentrations of the peptide at the surface level is concomitant with destabilization of the cell integrity and this may contribute to osmotic stress, although other mechanisms of action cannot be ruled out.

When Combined with Colistin, an Otherwise Ineffective Rifampicin-Linezolid Combination Becomes Active in Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii.

The synergistic action of colistin, with two antibiotics active in Gram-positive bacteria but unable to kill gram negatives (linezolid and rifampicin), was investigated, since triple combinations are emerging as a tool to overtake multidrug resistance. Checkerboard determinations demonstrated that, when combined with colistin, the combination of linezolid and rifampicin turns active in multidrug-resistant Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. Thus, the presence of sublethal concentrations of colistin resulted in a strongly synergistic interaction between these two drugs. Moreover, the minimum inhibitory concentrations of linezolid-rifampicin combinations in the presence of colistin were lower than the maximal concentrations of these antimicrobials ain blood. These findings suggest the use of this triple combination as an effective treatment of multidrug-resistant (MDR) bacterial infections.

Er,Cr:YSGG Laser-Activated Irrigation and Passive Ultrasonic Irrigation: Comparison of Two Strategies for Root Canal Disinfection.

Objective: To compare the antibacterial effectiveness of 0.5% sodium hypochlorite (NaOCl) activated by the Er,Cr:YSGG laser-activated irrigation (LAI) and passive ultrasonic irrigation (PUI) against a 10-day-old intracanal Enterococcus faecalis biofilm. Background: LAI and PUI are regarded as alternative methods to release the irrigant in the inner regions of the root canal system achieving enhanced cleaning ability. Nevertheless, little evidence regarding the activation of low concentrations of NaOCl has been reported. Materials and methods: Seventy-two single-rooted teeth were instrumented, inoculated (E. faecalis ATCC 29212), and incubated for 10 days to allow biofilm formation. Specimens were randomly divided into six groups (n = 12 each): (1) 0.5% NaOCl+Er,Cr:YSGG LAI, (2) saline+Er,Cr:YSGG LAI, (3) 0.5% NaOCl+PUI, (4) saline+PUI, (5) positive control (no treatment), and (6) negative control (no bacteria). The activation time was distributed as follows: 30 sec of activation, followed by a rest phase of 30 sec, and ending with 30 sec of activation. The number of bacterial survivors was determined by plate counting. Results: Both irrigation regimens LAI and PUI reduced the number of colony-forming unit. Moreover, LAI +0.5% NaOCl and the rest of groups significantly differ (p < 0.001 for all comparisons). Conclusions: Er,Cr:YSGG LAI proved to be more effective than PUI in enhancing the antimicrobial activity of 0.5% NaOCl against 10-day-old intracanal E. faecalis biofilms.

Er,Cr:YSGG Laser-Activation Enhances Antimicrobial and Antibiofilm Action of Low Concentrations of Sodium Hypochlorite in Root Canals.

The onset and persistence of endodontic infections due to residual biofilm after chemical disinfection promotes secondary bacterial infection. Alternative methods to disinfect operated root canals are a matter of great interest. The aim was to evaluate the antibacterial effectiveness of sodium hypochlorite (NaOCl) at low concentrations activated by the Er,Cr:YSGG laser-activated irrigation (LAI) against 10-day-old intracanal Enterococcus faecalis biofilm. Biofilms were formed inside the root canals and divided into 7 groups (n13): 0.5% NaOCl + Er,Cr:YSGG; Saline + Er,Cr:YSGG; 0.5% NaOCl + syringe irrigation(SI); 2.5% NaOCl + SI; 5% NaOCl + SI; positive and negative controls. Bacterial survivors were counted and specimens visualized under scanning electron and confocal laser scanning microscopy. Treatments with 0.5% NaOCl + Er,Cr:YSGG and 2.5% NaOCl + SI gave a significant reduction in the number of CFU/mm2. Moreover, scanning electron microscopy and confocal laser scanning microscopy imaging confirmed and reinforced bacteriological data. Thus, Er,Cr:YSGG LAI proved to be able to improve the intracanal distribution of 0.5% NaOCl after 60 s of activation, reaching the same level of effectiveness than 2.5% NaOCl. This is regarded as of clinical interest, since working with lower concentrations may contribute to reduce undesired effects.

Pulsed electric fields reduce bacterial attachment to stainless steel plates.

The purpose of this study was to evaluate the capacity of pulsed bilateral electric fields to control bacterial attachment on stainless steel plates. Previously sterilized circular metal plates of stainless steel were submerged in a liquid medium with a known concentration of Staphylococcus epidermidis and incubated for 1 hour at 36oC while a 200 Hz pulsed electric field of 18 V/cm was applied for 2.5 µseg and then sonicated for 5 minutes in 10 ml of saline. Three different models were cultured and compared: 1) negatively-charged plate, 2) positively-charged plate, and 3) control plate without electric current. A total of 39 metal plates were processed. The median adherence in the control group and the electric field group was 312 CFU/mm2 and 16,2 CFU/mm2 respectively (p < 0.001, reduction of 95% of bacterial attachment). Bilateral pulsed electric field is able to reduce bacterial attachment on stainless steel plates in in vitro conditions.

Novel synthetic polymyxins kill Gram-positive bacteria.

Background: Staphylococcus aureus, including 'superbug' MRSA, is a major cause of nosocomial infections. In the European Union, up to 171 200 new nosocomial MRSA infections are acquired annually, and in the USA S. aureus causes more deaths than HIV/AIDS and tuberculosis combined. MRSA is also the first group of pathogens that infect the pulmonary tract in young patients with cystic fibrosis. Objectives: We describe two newly developed and synthesized colistin (polymyxin E)-inspired molecules. Methods: A collection of several isolates of S. aureus [including MRSA and vancomycin-resistant S. aureus (VRSA)] was tested. To check the antimicrobial activity, we performed time-kill curves, growth curves, biofilm eradication, toxicity and isothermal titration calorimetry. Results: Both peptides showed high antimicrobial activities (MIC 4 mg/L) and low relative toxicities (selectivity index close to 23). Conclusions: Successful production of polymyxin-scaffold molecules active against S. aureus, both MRSA and VRSA, opens up new approaches to the treatment of these complicated infections.

New and old tools to evaluate new antimicrobial peptides.

The emergence of antimicrobial resistance due to the overuse of antimicrobials together with the existence of naturally untreatable infections well demonstrates the need for new instruments to fight microbes. Antimicrobial peptides (AMPs) are a promising family of molecules in this regard, because they abundantly occur in nature and the results of preliminary studies of their clinical potential have been encouraging. However, further progress will benefit from the standardization of research methods to assess the antimicrobial properties of AMPs. Here we review the diverse methods used to study the antimicrobial power of AMPs and recommend a pathway to explore new molecules. The use of new methodologies to quantitatively evaluate the physical effect on bacterial biofilms such as force spectroscopy and surface cell damage evaluation, constitute novel approaches to study new AMPs.

An overview of antimicrobial peptides and the latest advances in their development.

INTRODUCTION: The recent dramatic increase in the incidence of antimicrobial resistance has been recognized by organizations such as the United Nations and World Health Organization as well as the governments of the USA and several European countries. A relatively new weapon in the fight against severe infections caused by multi-drug resistant bacteria is antimicrobial peptides (AMPs). These include colistin, currently regarded as the last line of antimicrobial therapy against multi-drug resistant microorganisms. Areas covered: Here, the authors provide an overview of the current research on AMPs. The focus is AMPs currently being developed for the treatment of recalcitrant bacterial infections, the synergies of AMPs and antibiotics, and the activity of AMPs against biofilm. This review also includes a brief introduction into the use of AMPs in infections caused by Mycobacterium, fungi, and parasites. Expert opinion: In research into new antimicrobials, AMPs are gaining increasing attention. While many are natural and are produced by a wide variety of organisms, others are being newly designed and chemically synthesized in the laboratory to achieve novel antimicrobial agents. The same strategy to fight infections in nature is thus being effectively exploited to safeguard human and animal health.

Adherence of S. epidermidis on different metals. A comparative in vitro study.

AIM: Staphylococcus epidermidis is the most common cause of orthopedic infections. Adhesion and biofilm formation on orthopedic implant surfaces play an important role in the physiopathology of these infections. The aim of our study was to evaluate the adhesion of S. epidermidis on the surface of metals usually used in orthopedics. METHODS: Previously sterilized circular metal plates of titanium (Ti), porous titanium (p-Ti), cobalt chromium (CoCr) and stainless steel (SS) were hung completely submerged in a liquid medium with a known concentration of S. epidermidis (RP62A). They were incubated for 1 h or 24 h at 36°C. After incubation, each plate was washed with PBS and sonicated during 5 minutes in 10 mL of saline. Different dilutions were performed and 100 µL from each sample was cultured on agar plates. RESULTS: 26 metal plates were incubated for 1 h and other 55 metal plates for 24 h. The lowest bacterial count (cfu/mm2) at 1 h was observed in CoCr plates while in p-Ti it was 6 times higher. At 24 h the highest bacterial count was observed in SS plates while the lowest in Ti. However, these differences were not statistically significant. CONCLUSIONS: After 1 h and 24 h of exposure, the lowest adherence was observed in CoCr and Ti plates, respectively. However, bacterial attachment occurred with all materials. It is necessary to further investigate new materials able to avoid bacterial attachment.

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.

Linezolid limits burden of methicillin-resistant Staphylococcus aureus in biofilm of tracheal tubes.

OBJECTIVE: To evaluate the effects of systemic treatment with linezolid compared with vancomycin on biofilm formation in mechanically ventilated pigs with severe methicillin-resistant Staphylococcus aureus-induced pneumonia. DESIGN: Prospective randomized animal study. SETTING: Departments of Pneumology, Microbiology, and Pharmacy of the Hospital Clínic, Barcelona, and Scientific and Technological Services of the University of Barcelona. SUBJECTS: We prospectively analyzed 70 endotracheal tube samples. Endotracheal tubes were obtained from pigs either untreated (controls, n=20), or treated with vancomycin (n=32) or linezolid (n=18). INTERVENTIONS: The endotracheal tubes were obtained from a previous randomized study in tracheally intubated pigs with methicillin-resistant Staphylococcus aureus severe pneumonia, and mechanically ventilated for 69±16 hrs. MEASUREMENTS AND MAIN RESULTS: Distal and medial hemisections of the endotracheal tube were assessed to quantify methicillin-resistant Staphylococcus aureus burden, antibiotic biofilm concentration by high-performance liquid chromatography or bioassay, and biofilm thickness through scanning electron microscopy. We found a trend toward a significant variation in biofilm methicillin-resistant Staphylococcus aureus burden (log colony-forming unit/mL) among groups (p=.057), and the lowest bacterial burden was found in endotracheal tubes treated with linezolid (1.98±1.68) in comparison with untreated endotracheal tubes (3.72±2.20, p=.045) or those treated with vancomycin (2.97±2.43, p=.286). Biofilm linezolid concentration was 19-fold above the linezolid minimum inhibitory concentration, whereas biofilm vancomycin concentration (1.60±0.91 µg/mL) was consistently below or close to the vancomycin minimum inhibitory concentration. Biofilm was thicker in the vancomycin group (p=.077). CONCLUSIONS: Systemic treatment with linezolid limits endotracheal tube biofilm development and methicillin-resistant Staphylococcus aureus burden. The potential clinical usefulness of linezolid in decreasing the risk of biofilm-related respiratory infections during prolonged tracheal intubation requires further investigation.

Relationship between the degree of osteolysis and cultures obtained by sonication of the prostheses in patients with aseptic loosening of a hip or knee arthroplasty.

INTRODUCTION: The pathogenesis of prosthesis loosening is not well understood. The aim of our study was to sonicate components of joint prostheses removed due to aseptic loosening, culture the sonicate fluid, and to correlate these results with the degree of radiological osteolysis. METHODS: From January 2008 to June 2009 all consecutive patients who underwent a revision of hip or knee prosthesis due to aseptic loosening were included in the study. Aseptic loosening was established when the patient had radiological signs of loosening without symptoms or signs of infection. The diagnosis was confirmed when histology was negative, and ≥ 5 out of 6 standard cultures of periprosthetic tissue were negative. Bone lysis was measured according to the Paprosky or Engh classifications without knowing the result of sonication cultures. Removed components were placed in sterile bags and immediately transported to the microbiology laboratory and sonicated. Sonicate fluid was cultured and the results were correlated with the degree of bone lysis. The proportion of components with positive sonication culture according to the bone lysis classification was compared using χ(2) test. RESULTS: A total of 52 patients were included and 123 components were sonicated. In 30 patients at least 1 sonicated component was positive (57.7%) and 44 out of 123 (35.8%) components were positive. The proportion of positive sonication cultures was significantly higher in the group of components with a higher degree of bone lysis of 3 (76.5%) than in those with lower degrees (33.9% for 1 and 24% for 2) (χ(2) test, p = 0.0004). CONCLUSIONS: Sonication cultures were positive in 57% of patients who underwent revision arthroplasty for aseptic loosening. The percentage of positive sonication cultures was significantly higher in patients with severe osteolysis. LEVEL OF EVIDENCE: level I of Prognostic Studies-Investigating the Effect of a Patient Characteristic on the Outcome of Disease.

Blood culture flasks for culturing synovial fluid in prosthetic joint infections.

BACKGROUND: Identifying the etiologic microorganism is essential to guide antimicrobial therapy in prosthetic joint infection. QUESTIONS/PURPOSE: We (1) compared the frequency of positive cultures with synovial fluid inoculated in blood culture flasks (SF) with those of periprosthetic tissues or swabs in traditional cultures from patients with acute and chronic prosthetic joint infections (PJI) and (2) determined the sensitivity, specificity, and predictive values of the three methods. PATIENTS AND METHODS: We retrospectively reviewed 87 patients with PJIs (54 knees, 33 hips) and 63 patients with aseptic loosening (34 knees, 29 hips). Two SF, periprosthetic tissue, and swab samples were taken for culture in all 150 patients except for 14 in whom only one SF fluid sample was obtained. Synovial fluid was inoculated in blood culture flasks and periprosthetic tissue and swab samples in standard media. Positive cultures were identified with standard biochemical procedures. RESULTS: SF samples were positive in 78 of 87 infected cases (90%), periprosthetic tissue samples were positive in 71 (82%), and swab samples were positive in 59 (68%). SF, periprosthetic tissue, and swab samples were positive more frequently in acute than in chronic infections (96% versus 82% for SF, 87% versus 74% for periprosthetic tissue, and 87% versus 44% for swabs). The sensitivity, specificity, and positive and negative predictive values of SF were 91, 100, 100, and 93 for acute infections and 79, 100, 100, and 88 for chronic infections, respectively. CONCLUSIONS: SF samples cultured in flasks had higher sensitivity, specificity, and positive and negative predictive values for diagnosis of PJI when compared with standard tissue and swab samples. The usefulness of all samples was less in chronic than in acute infections. LEVEL OF EVIDENCE: Level II, diagnostic study. See Guidelines for Authors for a complete description of levels of evidence.

Outcome of acute prosthetic joint infections due to gram-negative bacilli treated with open debridement and retention of the prosthesis.

The aim of our study was to evaluate the outcome of acute prosthetic joint infections (PJIs) due to gram-negative bacilli (GNB) treated without implant removal. Patients with an acute PJI due to GNB diagnosed from 2000 to 2007 were prospectively registered. Demographics, comorbidity, type of implant, microbiology data, surgical treatment, antimicrobial therapy, and outcome were recorded. Classification and regression tree analysis, the Kaplan-Meier survival method, and the Cox regression model were applied. Forty-seven patients were included. The mean age was 70.7 years, and there were 15 hip prostheses and 32 knee prostheses. The median number of days from the time of arthroplasty was 20. The most frequent pathogens were members of the Enterobacteriaceae family in 41 cases and Pseudomonas spp. in 20 cases. Among the Enterobacteriaceae, 14 were resistant to ciprofloxacin, while all Pseudomonas aeruginosa isolates were susceptible to ciprofloxacin. The median durations of intravenous and oral antibiotic treatment were 14 and 64 days, respectively. A total of 35 (74.5%) patients were in remission after a median follow-up of 463 days (interquartile range, 344 to 704) days. By use of the Kaplan-Meier survival curve, a C-reactive protein (CRP) concentration of < or = 15 mg/dl (P = 0.03) and receipt of a fluoroquinolone, when all GNB isolated were susceptible (P = 0.0009), were associated with a better outcome. By use of a Cox regression model, a CRP concentration of < or = 15 mg/dl (odds ratio [OR], 3.57; 95% confidence interval [CI], 1.05 to 12.5; P = 0.043) and receipt of a fluoroquinolone (OR, 9.09; 95% CI, 1.96 to 50; P = 0.005) were independently associated with better outcomes. Open debridement without removal of the implant had a success rate of 74.5%, and the factors associated with good prognosis were a CRP concentration at the time of diagnosis < or = 15 mg/dl and treatment with a fluoroquinolone.

Group A streptococcal infections in injection drug users in Barcelona, Spain: epidemiologic, clinical, and microbiologic analysis of 3 clusters of cases from 2000 to 2003.

An unexplained resurgence of Group A streptococci (GAS) infections has been observed since the mid-1980s in the United States and Europe, particularly among intravenous drug users (IDUs). Several risk factors have been identified. Mutations in the capsule synthesis regulator genes (csrRS) have been associated with an increase in virulence. From January 1998 to December 2003, we conducted a prospective and retrospective descriptive analysis of invasive GAS soft-tissue infections in IDUs in Barcelona, Spain. Clinical features were collected, and we conducted a surveillance study to identify risk factors associated with GAS soft-tissue infections. We analyzed chromosomal DNA by low cleavage restriction enzymes and used pulsed-field gel electrophoresis (PFGE) and variable gene sequence typing (VGST) of the emm gene to disclose the epidemiologic relationship between the strains. We analyzed the influence of clonality (M-type) and mutations in csrRS genes of these strains on clinical features. We identified 44 cases, all of which were grouped in 3 clusters: fall 2000, fall 2002, and fall 2003. Cellulitis with or without abscesses (75%) and fever (90.9%) were the most common clinical manifestations. Distant septic complications were infrequent (18.2%). Although all patients had severe infections (mainly bacteremic needle abscesses), their outcome with antibiotic therapy, usually beta-lactam, was successful in all cases. However, surgery was needed in 40.9% of patients. Through the surveillance study we found that infected patients had a higher number of drug injections per day (odds ratio [OR], 18.84; 95% confidence interval [CI], 4.83-79.4; p<0.00001), shared paraphernalia for drug use more frequently (OR, 11.11; 95% CI, 3.24-39.04; p<0.0001), were in a higher proportion both currently unemployed and homeless (OR, 4.22; 95% CI, 1.5-12.15; p<0.0001), were not in a methadone maintenance program (OR, 0.03; 95% CI, 0-0.19; p<0.00001), and more often bought drugs at a specific site (OR, 33.92; 95% CI, 7.44-174.93; p<0.00001) and from a specific dealer (OR, 72; 95% CI, 8-3090; p<0.00001), compared with patients not infected. The fall 2000 cluster was polyclonal, whereas the other 2 clusters were mainly due to the same strain of GAS (emm 25.2), and were defined as epidemic outbreaks. Clinically, the cases due to the clonal strain presented abscesses and needed surgery more frequently (p<0.001 and p=0.005, respectively). On the other hand, mutations in the csrRS genes were not associated with invasive GAS soft-tissue infection. There has been an increase in the number of cases of invasive GAS soft-tissue infections in IDUs in Barcelona, which seems to be related to drug users' habits and their socioeconomic status. Clonality (emm 25.2) but not mutations in the csrRS genes was associated with more severe GAS soft-tissue infections.