Aims:  In this study, we have evaluated the impact of methodological approaches in the determination of biofilm formation by four clinical isolates of Escherichia coli in static assays. Methods and Results:  The assays were performed in microtitre plates with two minimal and two enriched broths, with one‐ or two‐steps protocol, and using three different mathematical formulas to quantify adherent bacteria. Different biofilm formation patterns were found depending on the E. coli strain, culture medium and reading optical density on one‐ and two‐steps protocol. Strong or moderate biofilm formation occurred mostly in minimal media. The mathematical formulas used to quantify biofilm formation also gave different results and bacterial growth rate should be taken into account to quantify biofilm. Conclusions:  Escherichia coli forms biofilms on static assays in a method‐dependent fashion, depending on strain, and it is strongly modulated by culture conditions. Significance and Impact of the Study:  As verified in the studied E. coli strains, biofilm formation by any organism should be cautiously interpreted, considering all variables in the experimental settings.

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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.

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 timekill assays, the latter at concentrations of 0.253 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 23.55, 22.66 and 22.71 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 cefotaximeresistant 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.

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.New therapeutic strategies for combating infections caused by antibiotic-resistant Streptococcus pneumoniae are being sought. Lytic enzymes produced by bacteria and phages may be useful alternatives for the treatment of pneumococcal infections. The LytA amidase is the main pneumococcal autolysin (3, 6) and is responsible for the lytic effects of penicillin and other cell wall inhibitors (5, 10, 11). Cpl-1 is a lysozyme encoded by pneumococcal phage Cp-1 (1, 2, 4).The aim of this study was to compare the in vitro and in vivo activities of purified LytA and Cpl-1 with those of cefotaxime (CTX) against a ␤-lactam-resistant (penicillin MIC, 2 g/ml) meningeal pneumococcal isolate (strain MJD3693).The MICs of LytA, Cpl-1, and CTX for strain MJD3693 were 16, 32, and 4 g/ml, respectively, as determined by the broth microdilution methodology (7). LytA and Cpl-1 were overproduced and purified by affinity chromatography in DEAE-cellulose (8). One unit of enzymatic activity (5) was defined as the amount of enzyme that catalyzed the hydrolysis of 1 g (ϳ715 net cpm) of [methyl-3 H]choline-labeled pneumococcal cell walls (6) in 10 min at 37°C. Time-kill experiments were performed in three separate assays by exposing early-log-phase cultures of MJD3693 to 50 g/ml of LytA, Cpl-1, or CTX in tubes with either cation-adjusted MuellerHinton broth with 4% lysed horse blood (CA-MHB-LHB) or phosphate-buffered saline (PBS; pH 7.0). Such a concentration was over 12-fold greater than the CTX MIC but only 1.6-and 3-fold higher than the MICs of Cpl-1 and LytA, respectively. After incubation at 35°C, the bacterial titers were compared at 1, 3, and 5 h ( Table 1). The killing effects of the enzymes were very rapid and were similar in both media, and their antipneumococcal activities was remarkably higher than that of CTX, particularly in PBS. In CA-MHB-LHB at 1 and 3 h, LytA and Cpl-1 were significantly more effective than CTX (P Յ 0.018), although after 5 h incubation, only LytA demonstrated a significantly higher activity than CTX (P ϭ 0.004). In PBS, LytA and Cpl-1 exerted a profound bactericidal effect at all times, whereas, as expected, the effect of CTX in PBS was quite poor (Table 1).The plasma pharmacokinetics of purified LytA and the in vivo efficacies of the compounds were studied by using adult Swiss mice, with the approval of the Fundación Jiménez Díaz Ethics Committee. The plasma pharmacokinetics of LytA were determined by using a solution of 3.1 mg/ml (specific activity, 5.9 ϫ 10 5 U/mg). After the administration of single intravenous (i.v.) doses (via the tail vein) of LytA (25 mg/kg) to uninfected mice, blood samples were obtained from three animals per gr...

From four southern European countries (Spain, Italy, Portugal, and Greece) 877 Streptococcus pneumoniae isolates were recovered from adult patients with respiratory tract infections between September 2000 and March 2001. The antimicrobial susceptibility to 11 antibiotics was determined in a central laboratory. Penicillin resistance was high in Greece (47.1%) and Spain (25.1%) but much lower in Portugal (7.9%) and Italy (4.8%). On the other hand, erythromycin resistance was high in Italy (38.5%) and Spain (36.2%) with no statistical difference with Greece (29.4%) but reaching significance (p <0.01) with Portugal (15.7%). Resistance to levofloxacin was low (1.5%) but present in Spanish and Italian isolates. Cefditoren, a new cephem antibiotic tested, was the most potent compound (MIC90 = 0.5 microg/ml) followed by levofloxacin and cefotaxime (MIC90 = 1 microg/ml). Given the high rates of penicillin and macrolide resistance reported, there is an evident need for new drugs and continued antimicrobial surveillance of S. pneumoniae.