Antimicrobial susceptibility of multidrug-resistant Streptococcus pneumoniae strains with penicillin MICs of 8 to 32 mg/L.

The in vitro activity of 22 antibiotics (including novobiocin) and beta-lactam/gentamicin combinations was assessed against 11 multidrug-resistant pneumococcal strains. Among orally administered drugs, only telithromycin, levofloxacin, and linezolid were active against all isolates, but their use is not indicated in pediatrics. Novobiocin could be a potential therapeutic alternative.

Antimicrobial susceptibility of Streptococcus pyogenes in Central, Eastern, and Baltic European Countries, 2005 to 2006: the cefditoren surveillance program.

The in vitro activity of penicillin, ampicillin, cefditoren, cefotaxime, erythromycin, clarithromycin, and levofloxacin against 763 clinical isolates of Streptococcus pyogenes was determined. Clinically significant isolates collected from November 2005 to December 2006 in the Czech Republic, Slovakia, Hungary, Poland, Romania, Estonia, Latvia, and Lithuania (the latter 3 analyzed as Baltic countries) were studied. No resistance to beta-lactams and levofloxacin was found. The rate of erythromycin resistance in S. pyogenes varied among countries, being low (<10%) in Romania and Baltic countries, intermediate (10-20%) in Poland and Czech Republic, and high (>25%) in Hungary and Slovakia. The predominant (75.0%) erythromycin-resistant phenotype among S. pyogenes isolates was MLS(B). The identification of the prevalence of erythromycin resistance mechanism could have impact on the choice of empiric antibiotic therapy for the clinicians in such countries.

Breakthrough in penicillin resistance? Streptococcus pneumoniae isolates with penicillin/cefotaxime MICs of 16 mg/L and their genotypic and geographical relatedness.

OBJECTIVES: To phenotypically and genotypically characterize 11 strains (isolated in four different centres) exhibiting penicillin MIC of 8-32 mg/L among isolates of the SPICE project. Nine isolates were from Romania (9/162; 5.56%) and two from Poland (2/305; 0.66%). METHODS: In vitro susceptibility was determined in triplicate by microdilution (CLSI guidelines), and additionally, MICs of penicillin, cefotaxime and amoxicillin were confirmed in triplicate by agar dilution. Multilocus sequence typing (MLST), PFGE and gene amplification and sequencing were performed. RESULTS: For the nine Romanian isolates, MICs were >/=16 mg/L for penicillin, cefotaxime and amoxicillin, >/=32 mg/L for cefuroxime and cefpodoxime, 4-8 mg/L for cefditoren and >/=128 mg/L for erythromycin and gentamicin. All isolates were non-susceptible to imipenem (MIC = 0.5-1 mg/L) and susceptible to levofloxacin (MIC = 0.5-1 mg/L) and vancomycin (MIC = 0.25-0.5 mg/L). These Romanian strains presented a new cluster in the 595-600 region of PBP2X (YSGIQL-->LSTPWF) conferring 98% homology with Streptococcus mitis PBP2X, with a new MurM allele (seven strains) with eight amino acid changes versus R6. PBP nucleotide sequences were highly conserved suggesting a common origin. Allelic profiles of two strains gave sequence type 321, three strains exhibited a single- and four a double-locus variance. MLST-predicted serotype was 23F in all but one strain (19F), but three strains were 19A by Quellung. CONCLUSIONS: The multidrug high resistance (precluding adequate oral therapy in children), its origin, the prevalence found in Romania and the presence of non-vaccine (7-valent) serotypes should worry the medical community because of a possible clonal diffusion that would limit therapeutic alternatives.

Correlation between virulence factors and in vitro biofilm formation by Escherichia coli strains.

The ability of 15 Escherichia coli strains to form biofilms on polystirene plates was studied. The strains were serotyped, and their phenotypic expression of surface virulence factors (VFs), and antibiotic susceptibility was also determined. Moreover, 30 VFs-associated genes were analysed, including 15 adhesins (papC, papG and its three alleles, sfa/focDE, sfaS, focG, afa/draBC, iha, bmaE, gafD, nfaE, fimH, fimAvMT78, agn43, F9 fimbriae and type 3 fimbriae-encoding gene clusters), four toxins (hlyA, cnf1, sat and tsh), four siderophore (iron, fyuA, iutA and iucD), five proctetins/invasion-encoding genes (kpsM II, kpsMT III, K1 kps variant- neuC, traT and ibeA), and the pathogenicity island malX and cvaC. Morphological appearance and thickness of biofilms of two strong and three weak biofilm producers were also studied by confocal laser scanning microscopy (CLSM). Seven strains were classified as strong biofilm producers and the remaining eight strains were regarded as weak biofilm producers. Mannose-resistant haemagglutination was the only phenotypically expressed surface virulence factor more frequently found in the strong biofilm group. Five virulence-associated genes were more common (p<0.05) in strong biofilm producers: papC and papG alleles, sfa/focDE, focG, hlyA and cnf1. CLSM images showed irregular biofilms with projections at the top mainly in strong biofilm.

In vitro interactions of LytA, the major pneumococcal autolysin, with two bacteriophage lytic enzymes (Cpl-1 and Pal), cefotaxime and moxifloxacin against antibiotic-susceptible and -resistant Streptococcus pneumoniae strains.

OBJECTIVES: In an innovative therapeutic exploitation against antibiotic-resistant Streptococcus pneumoniae, here we have evaluated the in vitro activity of a purified bacterially-encoded cell wall lytic enzyme, LytA (the major pneumococcal autolysin), and compared it with those of Cpl-1 and Pal (pneumococcal phage lytic enzymes) and two antibiotics versus four pneumococcal strains. METHODS: Two serotype 3, penicillin-susceptible strains and two penicillin-resistant (serotypes 19F and 19A, respectively) S. pneumoniae clinical isolates were used. The effect of several combinations of lytic enzymes and antibiotics (cefotaxime and moxifloxacin) was studied by chequerboard and time-kill assays, the latter at concentrations of 0.25 x MIC. RESULTS: LytA was more active than Cpl-1 and Pal. By the chequerboard technique, the combination of LytA and cefotaxime was synergistic for one of the two cefotaxime-resistant strains studied. The combined use of Cpl-1 and Pal was synergistic for three of the four strains, as was Cpl-1 with antibiotics for two of the three strains studied. In the time-kill assays, after 5 h of exposure to LytA, Cpl-1 or Pal, the mean differences in colony counts versus controls were -3.55, -2.66 and -2.71 log(10) cfu/mL, respectively. The combination of LytA/Pal reduced the bacterial inoculum >2 log units for three of the four strains. LytA combined with cefotaxime or moxifloxacin achieved >3 log units decrease for the strains tested. Particularly, a strong synergism was observed with LytA/cefotaxime for one cefotaxime-resistant meningeal strain. LytA/moxifloxacin was synergistic for the quinolone-resistant strain when tested by time-kill methodology, and just close to synergistic (fractional inhibitory concentration index of 0.58) by the chequerboard technique. Antagonism was not observed for any combination when assayed by either method. CONCLUSIONS: LytA, Cpl-1 or Pal, alone or in combination, might prove to be effective in combination therapy, as well as in monotherapy against S. pneumoniae. These results suggest avenues of research to study the cell wall lytic enzymes as anti-pneumococcal therapeutic agents.

Adherence of Streptococcus pneumoniae to polystyrene plates, effect of serum on adhesion, and virulence in the gerbil otitis media model.

The adherence of 11 pneumococcal strains to polystyrene was studied and expressed as the number of colony-forming units (CFU) recovered per 10(6)CFU of initial inoculum. Three strains were considered as strong adherent (>100CFU/10(6)), three as medium adherent (10-100CFU/10(6)), and five as low adherent (<10CFU/10(6)). All serotype 3 strains were low adherent whilst serotypes 23F and 19F behaved as strong or medium adherent. The impact of gerbil sera on adherence of six selected pneumococcal strains (one strong adherent, one medium adherent, and four low adherents) to abiotic material was also studied under two experimental conditions. In the presence of sera, the adherence ability of the strong, medium, and one low adherent strains decreased significantly. On the other hand, the adherence significantly increased in all strains when sera were removed following preincubation of bacteria exposed to sera, although such increase was statistically significant for five of them. Finally, the ability of two (one strong adherent and one low adherent) strains to induce otitis media in gerbils was also evaluated; the strong adherent strain behaved significantly more virulent than the less adherent in terms of ear damage and animal weight loss.

Pneumococcal LytA autolysin, a potent therapeutic agent in experimental peritonitis-sepsis caused by highly beta-lactam-resistant Streptococcus pneumoniae.

The in vitro and in vivo antipneumococcal activities of the main pneumococcal autolysin (LytA) and Cpl-1, a lysozyme encoded by phage Cp-1, were studied. Intraperitoneal therapy with LytA or high-dose Cpl-1 remarkably reduced peritoneal bacterial counts (>5 log(10) CFU/ml) compared with those for the controls. After intravenous injection, LytA was the most effective treatment.

Impact of ibuprofen therapy in the outcome of experimental pneumococcal acute otitis media treated with amoxicillin or erythromycin.

The impact of ibuprofen combined with amoxicillin or erythromycin for therapy of penicillin-resistant pneumococcal acute otitis media (AOM) was evaluated in a gerbil model. Ibuprofen (at 2.5 or 7.5 mg/kg, orally) and/or amoxicillin or erythromycin (5 mg/kg each, s.c.) were administered at 5 h (early therapy, as single-dose regimen) or at 18 h (delayed therapy, five doses) postinoculation (PI). Each antibiotic alone and combined with ibuprofen was more effective administered as early regimen than as delayed treatment when evaluating the presence of otorrhea, otoscopic aspect, culture-positive and bacterial counts in middle ear (ME) samples, and loss of body weight. There was a trend for a better bacteriological outcome in animals receiving amoxicillin or erythromycin and ibuprofen, especially with the high dose. Such a dose of ibuprofen, associated with each antibiotic regimen, also preserved the animal well-being, avoiding a great weight loss in comparison to those receiving the antibiotic alone but a statistically significant difference was only observed for animals receiving delayed therapy with erythromycin and high-dose ibuprofen. In conclusion, ibuprofen combined with antibiotics seemed to improve the outcome of this experimental pneumococcal AOM.

Correlation between in vitro and in vivo activity of levofloxacin and moxifloxacin against pneumococcal strains with different susceptibilities to fluoroquinolones.

In vitro and in vivo models were developed to evaluate the efficacy of levofloxacin and moxifloxacin against three serotype 3 pneumococcal strains with different susceptibilities to fluoroquinolones (wild-type, parC mutant, and parC, parE and gyrA mutant). Levofloxacin and moxifloxacin reduced the bacterial burden in the in vitro pharmacodynamic and animal models for the wild-type strain but had very little activity against the fully resistant strain (parC, parE and gyrA mutant). Levofloxacin showed very little activity both in the in vitro pharmacodynamic model and in the animal model for the strain having a mutation in parC (levofloxacin and moxifloxacin minimum inhibitory concentrations, 2mg/L and 0.25mg/L, respectively). However, moxifloxacin still had a significant in vitro and in vivo activity against this strain.

Urinary concentrations and bactericidal activity against amoxicillin-nonsusceptible strains of Escherichia coli with single-dose, oral, sustained-release amoxicillin/clavulanic acid: a phase I, open-label, noncomparative clinical trial in healthy volunteers.

BACKGROUND: Urinary tract infections (UTIs) account for 5 to 6 million medical consultations in the United States each year. Worldwide, an estimated 20% to 30% of women aged 20 to 40 years have at least 1 episode of the most common type of UTI (uncomplicated cystitis) in their lifetime, with Escherichia coli being the causative pathogen in 80% of cases. Antibacterial activity in urine has been shown to be correlated with outcomes of uncomplicated cystitis. OBJECTIVE: The objectives of this study were to determine urinary concentrations and ex vivo bactericidal activity of sustained-release (SR) amoxicillin/clavulanic acid 2000/125 mg against intermediately resistant and resistant strains of E coli over 72 hours and compare them with those of a susceptible strain. This study also investigated whether urinary concentrations obtained after dosing cover E coli strains categorized as intermediately resistant and resistant based on current Clinical and Laboratory Standards Institute (formerly NCCLS) breakpoints in uncomplicated cystitis. METHODS: This Phase I, open-label, noncomparative study in healthy male volunteers was conducted at the Pharmacology Unit, Autónoma University, Madrid, Spain. Subjects received a single oral dose of amoxicillin/clavulanic acid 2000/125 mg SR. The amoxicillin/clavulanic acid MICs were 8/4 microg/mL (susceptible), 16/8 microg/mL (intermediately resistant), and 32/16 and 64/32 microg/mL (resistant). Urine samples were collected before (baseline; time 0) and at the following intervals: 0-2, 2-4, 4-8, 8-12, 12-16, 16-24, 24-36, 36-48, 48-60, and 60-72 hours after dosing. Killing curves with urine samples were performed with initial inocula of approximately 10(7) colony-forming units (CFU)/mL, and bactericidal activity (defined as >3 log(10) CFU/mL and >99.9% reduction in bacterial counts) was calculated as the difference between log(10) initial inoculum and log(10) CFU/mL after 4 hours of incubation of each sample. RESULTS: Twelve volunteers were included (mean [SD] age, 24.83 [5.64] years; mean [SD] height, 175.75 [7.56] cm; and mean [SD] weight, 73.55 [9.19] kg). No statistically significant differences between the activities in the 12-16-hour interval compared with baseline were found in any of the strains tested. Bactericidal activity against the susceptible and intermediately resistant strains (MICs 8/4 and 16/8 g/mL, respectively) was obtained up to 8 hours after dosing. Bactericidal activity against the resistant strains (MICs 32/16 and 64/32 microg/mL) was obtained in the 2-4-hour interval. CONCLUSIONS: In this Phase I study in healthy volunteers, urinary concentrations after dosing with amoxicillin/clavulanic acid 2000/125 mg SR showed bactericidal activity against the amoxicillin-susceptible and intermediately resistant strains of E coli.

Pulmonary damage and bacterial load in assessment of the efficacy of simulated human treatment-like amoxicillin (2,000 milligrams) therapy of experimental pneumococcal pneumonia caused by strains for which amoxicillin MICs differ.

An experimental rat pneumonia model using two amoxicillin-susceptible (MICs, < or =0.015 and 2 microg/ml) and two non-amoxicillin-susceptible (MIC, 4 microg/ml) Streptococcus pneumoniae strains was developed for testing the efficacy of amoxicillin administered to simulate human serum kinetics after treatment with amoxicillin-clavulanate (2,000 and 125 mg, respectively, twice a day, for 2.5 days). The end points for efficacy were reductions in bacterial loads in the lungs and reductions in levels of pulmonary damage. For the amoxicillin-susceptible strains (serotypes 23F and 14), a decrease greater than 4.5 log(10) CFU/pair of lungs was obtained, and the time for which the serum antibiotic concentration (SAC) was higher than the MIC (T(S)(A)(C)(>)(MIC)) was greater than 60% of the dosing interval. For non-amoxicillin-susceptible strains, the decrease in bacterial load was 1.34 to 1.75 log(10) CFU/pair of lungs, with a T(S)(A)(C)(>)(MIC) of 46.7% of the dosing interval. An in vitro study showed that serotype 9V non-amoxicillin-susceptible strains behaved as tolerant-like to concentrations similar to those in the in vivo model. The high and maintained SACs (T(S)(A)(C)(>)(MIC), >46% for all strains) significantly diminished lung injury (affected area of the lung and lung weight), compared to that in controls, by all strains, regardless of the MIC, bactericidal behavior in in vitro killing curves, or the serotype of the infecting strain. These results show the importance of host therapeutic end points in the evaluation of antibiotic efficacy. The antibiotic was more efficacious, for one nonsusceptible strain tested, when the treatment was started early (1 h postinoculation [p.i.]) than when treatment was delayed (24 h p.i.).