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.

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.

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.

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.

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.

A role for human Sp alpha as a pattern recognition receptor.

Human Sp alpha is a soluble protein belonging to group B of the scavenger receptor cysteine-rich (SRCR) superfamily for which little functional information is available. It is expressed by macrophages present in lymphoid tissues (spleen, lymph node, thymus, and bone marrow), and it binds to myelomonocytic and lymphoid cells, which suggests that it may play an important role in the regulation of the innate and adaptive immune systems. In the present study we show that recombinant human Sp alpha (rSp alpha) binds to the surface of several gram-positive and gram-negative bacterial strains. Competition studies indicated that such binding is mediated by the recognition of lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively, through nonoverlapping sites on the Sp alpha molecule. The most conserved part of LPS (2-keto-3-deoxyoctulosonic acid and lipid A) was shown to be involved in the recognition by Sp alpha. Bacterial binding studies using the SRCR domain 1 of Sp alpha showed that this domain retains both the LPS and LTA binding activities, indicating that both bacterial interacting sites are retained in a single SRCR domain. Furthermore, rSp alpha induced aggregation of gram-positive and gram-negative bacteria strains. On the other hand, rSp alpha inhibited tumor necrosis factor-alpha secretion by human monocytes stimulated with LPS or LTA. Binding of Sp alpha to conserved components of bacterial surfaces and modulation of the monocyte response indicate that this molecule is an active constituent of the innate immune response of the host.

Relationship between mutations in the gyrA gene and quinolone resistance in clinical isolates of Corynebacterium striatum and Corynebacterium amycolatum.

Quinolone susceptibility was analyzed in 17 clinical isolates of Corynebacterium striatum and 9 strains of Corynebacterium amycolatum by the E-test method in Mueller-Hinton agar plates. The C. striatum ATCC 6940 strain was used as a control strain. The amplified quinolone resistance determining regions of the gyrA genes of C. amycolatum and C. striatum were characterized. Four in vitro quinolone-resistant mutants of C. amycolatum were selected and analyzed. Both in vivo and in vitro quinolone-resistant strains of C. amycolatum showed high levels of fluoroquinolone resistance in strains with a double mutation leading to an amino acid change in positions 87 and 91 or positions 87 and 88 (unusual mutation) of GyrA, whereas the same concomitant mutations at amino acid positions 87 and 91 in GyrA of C. striatum produced high levels of resistance to ciprofloxacin and levofloxacin but only showed a moderate increase in the MIC of moxifloxacin, suggesting that other mechanism(s) of quinolone resistance could be involved in moxifloxacin resistance in C. amycolatum. Moreover, a PCR-RFLP-NcoI of the gyrA gene was developed to distinguish between C. amycolatum and C. striatum species.

In vitro activity of gemifloxacin against clinical isolates of Neisseria gonorrhoeae with and without mutations in the gyrA gene.

The MIC of gemifloxacin and five other quinolones was tested against 31 clinical isolates of Neisseria gonorrhoeae; strains were analyzed for the presence of mutations in both the gyrA and parC genes. Only seven strains were resistant to nalidixic acid due to a mutation in the gyrA gene but not in the parC gene, with six and two considered intermediate to ciprofloxacin and levofloxacin, respectively. The activity of gemifloxacin was similar to that of trovafloxacin and moxifloxacin, but was more active than nalidixic acid, ciprofloxacin or levofloxacin against the gyrA mutant strains. Gemifloxacin is a valid therapeutic alternative to treat infections with N. gonorrhoeae, retaining its activity against strains already presenting a mutation in gyrA.

Analysis of the clonal relationship among clinical isolates of Salmonella enterica serovar Infantis by different typing methods.

Salmonella Infantis has been the second most common serovar in Argentina in the last two years, being isolated mostly from paediatric hospitalised patients. In order to determine the clonal relationship among Salmonella Infantis strains, we examined 15 isolates from paediatric patient faeces in Argentina (12 geographically related and 3 geographically non-related) by using antimicrobial susceptibility, plasmid profiling, repetitive extragenic palindromic (REP) PCR, enterobacterial repetitive intergenic consensus (ERIC) PCR, and low-frequency restriction analysis of chromosomal DNA by pulsed field gel electrophoresis (PFGE). Four Spanish strains were included as controls of clonal diversity in molecular techniques. Antibiotype and plasmid profile was not useful as epidemiological tools. PFGE and REP-PCR were able to discriminate between Argentinean and Spanish isolates of Salmonella Infantis allowing to detect genetically related strains in three different cities. This finding indicates that a possible spread of a clone of this serovar in the North-eastern Region of Argentina has taken place in 1998.

Analysis of the clonal relationship among clinical isolates of Salmonella enterica serovar Infantis by different typing methods

Salmonella Infantis has been the second most common serovar in Argentina in the last two years, being isolated mostly from paediatric hospitalised patients. In order to determine the clonal relationship among Salmonella Infantis strains, we examined 15 isolates from paediatric patient faeces in Argentina (12 geographically related and 3 geographically non-related) by using antimicrobial susceptibility, plasmid profiling, repetitive extragenic palindromic (REP) PCR, enterobacterial repetitive intergenic consensus (ERIC) PCR, and low-frequency restriction analysis of chromosomal DNA by pulsed field gel electrophoresis (PFGE). Four Spanish strains were included as controls of clonal diversity in molecular techniques. Antibiotype and plasmid profile was not useful as epidemiological tools. PFGE and REP-PCR were able to discriminate between Argentinean and Spanish isolates of Salmonella Infantis allowing to detect genetically related strains in three different cities. This finding indicates that a possible spread of a clone of this serovar in the North-eastern Region of Argentina has taken place in 1998

High prevalence of nalidixic acid resistant, ciprofloxacin susceptible phenotype among clinical isolates of Escherichia coli and other Enterobacteriaceae.

Therapeutic failure of infections during their treatment with quinolones has been often described. This may be due to the development of resistance during treatment of an infecting strain which already had diminished susceptibility to quinolones, even though the initial MIC did not exceed the breakpoint. In this study the prevalence of the nalidixic acid resistant, ciprofloxacin susceptible phenotype among Enterobacteriaceae was analyzed. The results showed that 113 out of 151 (74.83%) strains of the Enterobacteriaceae with diminished susceptibility to ciprofloxacin (MICs from 0.06 to 1 microg/ml) were resistant to nalidixic acid (MICs > 32 microg/ml). The Escherichia coli strains presenting this phenotype already have a mutation in the amino acid codon Ser-83 of the gyrA gene, so that the possibility of developing a second mechanism of resistance during treatment is very high.