Evaluation of bactericidal activity of cefotaxime and other beta-lactams by a novel method.

In previous studies on Streptococcus faecium we proposed that the minimum beta-lactam concentration killing 99.9% of a bacterial population within 3 hours be defined as the minimum directly bactericidal concentration (MDBC) of that drug. In the present study we first evaluated the kinetics of cellular killing by various beta-lactams as related to penicillin-binding-protein (PBP) binding in Escherichia coli DC2, a hyperpermeable mutant. We concluded that in E. coli the MDBC for beta-lactams coincides with the minimum concentration capable of saturating PBPs 1b, 2 and 3. Of the antibacterial drugs we studied, cefsulodin, mecillinam and aztreonam had a much greater affinity for one essential PBP (PBP 1b, 2 and 3, respectively) than for all others, whereas cefotaxime had close affinities for all the above PBPs. MDBC values of greater than 500, 500, greater than 50, 10 and 1.5 mg/L were obtained for cefsulodin, mecillinam, aztreonam, ampicillin and cefotaxime, respectively. On the basis of the pharmacokinetic properties of these drugs, our results indicate that mecillinam, ampicillin and cefsulodin may be bactericidal in urine but not at other body sites; aztreonam is probably bactericidal in urine and blood, but not elsewhere; and cefotaxime is bactericidal in all the biological fluids we studied.

Inhibition of bacterial cell surface extension by various means causes blocking of macromolecular synthesis.

It has been suggested that, in rod-shaped bacteria, two sites for peptidoglycan assembly exist: one which is responsible for septum formation and the other, for lateral wall extension. The balance between the activities of these two sites enables bacteria to conserve their own morphology during cell growth. The effect of specifically inhibiting septum formation by different means (antibiotics and/or mutations), upon cell surface extension and macromolecular synthesis in rod-shaped and coccoid bacteria of various species, was studied. Inhibition of either cell wall expansion or macromolecular synthesis did not occur when septum formation was impaired in both rod-shaped bacteria and cocci possessing the two sites for peptidoglycan assembly, whereas a rapid and complete block of such synthesis was caused by inhibiting both sites in rod-shaped bacteria, or septum formation in cocci which possess only this site. These data indicate that bacteria possess a control mechanism that prevents macromolecular synthesis when envelope extension is inhibited.

Lipoteichoic acid as a target for antimicrobial action.

Daptomycin, a lipopeptide antibiotic active against gram-positive bacteria, has been found to inhibit lipoteichoic acid (LTA) synthesis as a consequence of membrane binding in the presence of Ca2+. The present study shows that among the bacterial-membrane components, daptomycin binds the protein fraction with a noncovalent bond, as suggested by the instability of the bond in the presence of an ionic detergent such as sodium dodecyl sulfate. Analysis of membrane proteins by isoelectric focusing electrophoresis reveals that 5 bands with isoelectric points ranging from 5.9 to 6.2 bind radioactive daptomycin. These proteins are therefore called daptomycin-binding proteins. In an attempt to correlate these proteins with the main inhibition observed in LTA synthesis, two-dimensional thin-layer chromatography of lipids synthesized during daptomycin treatment was performed. A 3-fold increase in diglucosyl diacylglycerol is demonstrated, while the compounds phosphatidyl-alpha-kojibiosyldiacylglycerol, glycerophosphophosphatidyl-alpha-kojibiosyldiacyl glycerol, and glycerophosphokojibiosyldiacylglycerol, which follow diglucosyl diacylglycerol in LTA synthesis, decrease progressively with time during the course of daptomycin treatment.

The gene encoding for penicillin-binding protein 5 of Enterococcus faecalis is useful for development of a species-specific DNA probe.

Recently, in Escherichia coli was cloned a Sau3AI 3.4-kb fragment containing the gene encoding for penicillin-binding protein 5 (PBP5) of Enterococcus faecalis. The structural gene for the PBP of E. faecalis and the flanking regions were entirely sequenced (C. Signoretto, M. Boaretti, and P. Canepari, FEMS Microbiol. Lett. 123:99-106, 1994). When the entire cloned E. faecalis DNA insert, labeled with digoxigenin, was used as a probe to detect a homology gene in enterococci, it was observed that only DNAs of all the E. faecalis strains reacted to the probe. The same results were obtained when a HindIII fragment of 0.35 kb from the entire insert of 3.4 kb was used. In this study we tested a total of 62 clinically isolated enterococcal strains, belonging to the species E. faecalis (36 strains), E. faecium (13), E. gallinarum (6), E. bovis (2) E. avium (3), E. hirae (1), and E. casseliflavus (1). The results indicate that both the entire segment and the HindIII fragment may be useful for preparing a species-specific probe for rapid identification of E. faecalis species.

Cloning, sequencing and expression in Escherichia coli of the low-affinity penicillin binding protein of Enterococcus faecalis.

Low-affinity penicillin binding proteins are particular membrane proteins, in several Gram-positive bacteria, which are involved in beta-lactam antibiotic resistance. The structural gene for the low-affinity penicillin binding protein 5 (PBP5) of Enterococcus faecalis was cloned and sequenced. From the sequence of the 3378 bp, a 2040 bp coding region was identified. From biochemical analysis it emerges that E. faecalis PBP5 is a type II membrane protein with an uncleaved N-terminal and is composed of 679 amino acids with a molecular weight of 74055. This protein showed 48 and 33% of identity with Enterococcus hirae PBP5 and Staphylococcus aureus PBP2a, both low-affinity PBPs involved in beta-lactam resistance. Anti-PBP5 antibodies cross-reacted with a membrane protein present in other species of enterococci, but the entire gene fragment cloned hybridized only with DNAs of E. faecalis strains, thus suggesting that genes coding for low-affinity PBPs of enterococci are not strictly homologous. In this experiment digoxigenin-labelled E. faecalis DNA was used.

Nonculturable Enterococcus faecalis cells are metabolically active and capable of resuming active growth.

Entry into the viable but nonculturable (VNC) state is a survival mechanism that bacteria can adopt when they find themselves in an adverse environment. When in this state, bacteria are still viable but are unable to form colonies on growth medium. The possibility of Gram-positive species entering the VNC state when environmental conditions are adverse and remaining viable and capable of resuming active growth is demonstrated for the first time in this study by using exponential-phase cultures of Enterococcus faecalis inoculated in filtered, sterilized water from Lake Garada (Italy). Over the 60-day study, the number of total cells stained with a fluorescent or counted with a Coulter Counter remained constant, while the number of cells capable of forming colonies on Tryptic Soy Agar (TSA) declined rapidly from 10(6) CFU/ml on day 0 to 10(3) CFU/ml on day 4. On day 14 no colonies could be observed when 50 ml of inoculated lake water were plated. E. faecalis cells conserved their viability while in the VNC state, as can be demonstrated by active uptake of amino acids, which are also incorporated into proteins, and by continuous detection of E. faecalis specific DNA by PCR throughout the experiment. The possibility of revival of the E. faecalis cells in the VNC state when returned to conditions supporting its cell growth has also been demonstrated. The data obtained in this study lend further support to recent criticisms of the traditional methods used to evaluate water quality based on plate counts, assessing fecal contamination indicators such as Escherichia coli and fecal streptococci.

New targets for the mechanism of action of antibiotics active against gram-positive cocci.

The main mechanisms of resistance to beta-lactams in gram-positive cocci include either the production of beta-lactamases or alterations in the molecular targets of these antibiotics, the penicillin binding proteins (PBPs). In spite of the appearance of new beta-lactams, more stable to the hydrolytic activity of beta-lactamases or with higher affinity for PBPs, no substantial progress in improving the activity against gram-positive has been achieved. In addition to the search for new beta-lactams it would be of interest to find molecules directed against new targets of the cell wall of gram-positive bacteria (i.e. teichoic acids and lipoteichoic acid) of which up to now no specific inhibitor is known. These two wall polymers are thought to be essential for cell survival within the host. Among new inhibitors a new antibiotic belonging to the class of acid lipopeptides called daptomycin (LY146032), and active against gram-positive seems of particular interest. Our studies demonstrate that daptomycin is a specific inhibitor of lipoteichoic acid synthesis.

In vitro activity, beta-lactamase stability and PBP affinity of RU 51,746-2, the active metabolite of the new orally absorbed cephalosporin ester, RU 51807.

The activity of RU 51,746-2 was determined against strains of Escherichia coli producing different types of beta-lactamases or showing alterations of permeability. The production of TEM 1, OXA 2, CARB 3 and PSE 1 beta-lactamases had no influence on susceptibility to the antibiotic, whereas the synthesis of TEM 2, SHV 1 and OXA 1 beta-lactamases increased minimum inhibitory concentrations (MIC) by 2-4 times. Highly resistant to the antibiotic was a strain producing CEP 1 beta-lactamase. E. coli mutans deficient in Omp F but not in Omp C had a decreased susceptibility to RU 51,746-2. RU 51,746-2 showed a good affinity for high molecular weight penicillin binding proteins (PBPs) of E. coli. PBP 3 was found to be the target for growth inhibition, whereas the additional saturation of PBP 1 and 2 was required for obtaining the best bactericidal activity.

In vitro activity of cefpirome (HR 810) against enterococci and staphylococci.

The inhibitory activity of cefpirome (HR 810), a new cephalosporin derivative for parenteral use, was tested by agar dilution methods against Enterococcus faecalis (100 strains), Staphylococcus aureus (40 strains) and coagulase-negative staphylococcal species (60 strains) in comparison with other beta-lactam antibiotics. For E. faecalis, the cefpirome minimum inhibitory concentration (MIC) range was 2-128 micrograms/ml, with an MIC50 of 8 micrograms/ml, and an MIC90 of 64 micrograms/ml. The optimal bactericidal activity against strains with MICs of < or = 8 micrograms/ml occurred at 2-4 times the MIC, and the reduction in the initial inoculum was 99.9-99.7% after 24 h incubation at these concentrations. Mec gene-negative staphylococci (both S. aureus and coagulase-negative species) had cefpirome MICs of 0.25-2 micrograms/ml (MIC50 0.5 microgram/ml, MIC90 1 microgram/ml). Mec gene-positive strains had MICs of 0.5-128 micrograms/ml (MIC50 2 micrograms/ml, MIC90 32 micrograms/ml). Strains with borderline resistance to oxacillin which did not harbor the mec gene and which were susceptible to cefpirome maintained their susceptibility even when high-density inocula were used and after several passages in media containing the antibiotic. These studies present some potential advantages of cefpirome over other cephalosporins in the inhibitory activity against Gram-positive cocci.

In vitro activity of lomefloxacin (SC-47111) against enterobacteriaceae, enterococci and staphylococci.

The activity of lomefloxacin, a new difluorinated quinolone, was tested against 190 Enterobacteriaceae strains (belonging to 23 different species), 70 enterococci and 70 staphylococci. As regards Enterobacteriaceae, the activity of lomefloxacin was the same as that of norfloxacin in 9 out of the 23 species tested, and only slightly lower in further 8 species. Minimum inhibitory concentrations (MIC) values for 90% of strains were 0.5 microgram/ml in 2 species, 0.25 microgram/ml in 6, 0.125 microgram/ml in 4, and lower than 0.125 microgram/ml in 8. Slightly higher values were obtained for Serratia marcescens (2 micrograms/ml), whilst, as already reported for the other new quinolones, the susceptibility of the Providencia genus was very poor, with MIC values up to 128 micrograms/ml for the vast majority of strains. Lomefloxacin proved bactericidal at the MIC in all the Enterobacteriaceae strains tested but 20. In the latter strains, however, bactericidal activity could be appreciated at values slightly exceeding MIC. As regards enterococci, the MIC for 90% of strains was 32 micrograms/ml. Minimum bactericidal concentration (MBC) was the same as the MIC for 78% of the strains tested and was only twofold higher in all the others. The new drug was also active against staphylococci having an MIC50 and MIC90 of 0.5 and 2 micrograms/ml, respectively. It was bactericidal at the MIC for 62% of the strains and at twofold the MIC for all the others.

Alterations in the chemical composition of peptidoglycan of Escherichia coli DH5 alpha induced by the expression of Enterococcus faecalis penicillin binding protein 5.

Low-affinity penicillin binding proteins (PBPs) are a particular class of membrane proteins involved in penicillin resistance in Enterococci and other micro-organisms. This PBP is thought to be capable of taking over the activity of all other PBPs during peptidoglycan synthesis. Unfortunately, nothing is known about the enzymatic activity catalyzed by this PBP, but a transpeptidase/transglycosylase action can be postulated to allow complete peptidoglycan synthesis. Recently, we cloned and expressed in Escherichia coli the PBP5 (a low-affinity PBP) of Enterococcus faecalis (Signoretto, C., Boaretti, M., and Canepari, P.: FEMS Microbiol. Lett. 123, 99-106, 1994). Here we describe some of the effects of this PBP when expressed in E. coli, in terms of increased growth rate and autolysis, and particularly its effects on the fine chemical composition of the E. coli peptidoglycan. A distinct increase in the di- and tripeptide monomers and a parallel decrease in the tetrapeptide monomer are described. The results presented here are explained in terms of a partial action of the postulated transpeptidase/ transglycosylase enzymatic complex which leads to the cleavage of one, two or three amino-acids from the pentapeptide monomer, but is incapable of performing the cross-linking between two side-chains due to lack of the natural substrate which is different from that of E. coli.

Simpler peptidoglycan chemical composition in the highly transformant Escherichia coli DH5 alpha strain.

Artificial transformation of Escherichia coli is obtainable by treating the culture with CA2+ and other substances known to increase the permeability of the outer membrane. Nevertheless, particular strains of E. coli are more useful for transformation since the number of transformants obtained is far higher. We postulate that an additional layer of the envelope may play an important role comparable to that of the outer membrane. The chemical composition of the peptidoglycan of a highly efficient transformant E. coli strain (DH5 alpha) was analyzed in comparison with a normal and poorly transformant E. coli strain (KN126) revealing a simpler peptidoglycan chemical composition in the DH5 alpha strain. This may be responsible for the simpler architecture of the peptidoglycan which, in turn, may interfere less with the passage of the DNA across the bacterial envelope.

Purification of daptomycin binding proteins (DBPs) from the membrane of Enterococcus hirae.

Daptomycin binding proteins (DBPs) are membrane proteins which act as daptomycin targets. Daptomycin is a cyclic lipopeptide antibiotic which is active against Gram-positive bacteria and was shown to be the first inhibitor of lipoteichoic acid (LTA) synthesis. It was found that the antibiotic did not penetrate the bacterial cytoplasm but bound membranes with a non-covalent bond and in particular some proteins which were called DBPs. DBPs were indicated as enzymes involved in LTA synthesis whose binding and inhibition by daptomycin is responsible for the observed effect on bacterial LTA synthesis. The purification of DBPs will make it possible not only to shed light on the biosynthesis of the cell wall polymer but will also provide innovative targets for selection of new antibacterial compounds. In this study, the purification of DBPs is described. Affinity chromatography was used with daptomycin as the ligand. Final elution of DBPs from daptomycin-coupled resin was performed using either 0.1% SDS or 3 M NaCl. Polyacrylamide gel electrophoresis of the eluted protein fractions consistently showed four protein bands (ranging from 55 to 66 kDa) in denaturating conditions and two protein bands (60 and 66 kDa) in non-denaturating conditions. Isoelectrofocusing analysis of the same sample consistently revealed two bands with pIs around 5. That these purified proteins were really the desired DBPs is demonstrated by the retention of daptomycin-binding capability they displayed.

The reshaping process of Klebsiella pneumoniae cells after removal of mecillinam, an antibiotic that causes transition from rod to coccal shape.

The process of bacterial morphogenesis that leads to rod shape formation was studied in synchronous cells during the reshaping process after removal of mecillinam, a beta-lactam antibiotic which, by specifically inhibiting lateral wall formation of rods, cause rod-to-sphere transition in Gram-negative rods. The addition of mecillinam for 50 min of the cell cycle made the cells to skip a division, while the addition of the antibiotic for 30 min (or less), allowed the cells to divide regularly. In order to study the interplay between lateral wall elongation and septum formation in reacquisition of rod shape, we evaluated the effect of re-adding mecillinam or adding piperacillin, a specific inhibitor of septum formation, at various stages of the reshaping process. It was found that mecillinam was active only when added within the first 30 min of the reshaping process, while piperacillin was active only after 30 min when the cells were close to starting to divide again. These findings provide further support for our previous proposal that, in bacterial rods, elongation and septation are two alternating and competing events of the cell cycle, and are linked to each other in such a way as to force bacterial rods to grow to a given length.

Rapid detection of PCR products of hepatitis C virus (HCV) with a non-radioactive oligoprobe.

Dot-blot for rapid detection of PCR amplification products of hepatitis C virus (HCV) using a digoxigenin (DIG)-labelled oligoprobe was developed and its sensitivity compared with Southern blot hybridization. The specificity and sensitivity of the DIG-labelled probe were identical to those of the 32P-labelled when the DIG-labelled nucleic acids were detected by enzyme-catalyzed chemiluminescent reaction. The lack of radioactivity makes this procedure suitable for routine use in diagnostic laboratories.

Peptidoglycan synthesis and its fine chemical composition in dividing and not dividing Klebsiella pneumoniae cocci.

Peptidoglycan synthesis and its fine chemical composition were studied in dividing and in non-dividing Klebsiella pneumoniae cocci and compared with rods. The beta-lactam mecillinam, a specific inhibitor of lateral wall elongation which causes rod-to-sphere transition in rods, showed 50% inhibition of the peptidoglycan in normal rods of the parent Mir A12 only if added at an early stage of the cell cycle and no effect if added later or during septation. In the rods of the mutant Mir M7, mecillinam was shown to inhibit 50% of peptidoglycan synthesis until rods become cocci, and thereafter to be absolutely devoid of effects. On the contrary, piperacillin, a specific inhibitor of septum formation, was active on all strains regardless of their cell shape, only if added at 20 and removed at 40 min of the cell cycle. As regards the analysis of peptidoglycan fine chemical composition, bacteria dividing as cocci showed alterations in the muropeptide composition consisting in a 50-fold increase in the tetramer family. This alteration was not seen in the cocci that did not divide as such. These results confirm our previous claim that septum formation and lateral wall elongation are mutually exclusive in normal rods and that septum formation requires the synthesis of a peptidoglycan of different chemical composition.

[A probiotic as an antagonist of bacterial translocation in experimental pancreatitis]. / Un probiotico come antagonista della traslocazione batterica nella pancreatite sperimentale.

Infection is the most common cause of death in acute pancreatitis. Earlier studies have demonstrated that early enteral nutrition decreases microbial translocation, upregulates the immune function and reduces septic complications and mortality. Lactobacillus plantarum (Lp) has been shown to be effective in reducing egress of endotoxin and microbial strain that showed very high adherence power to gut mucosa. We adopted a model of acute pancreatitis induced by isolation and ligation of biliopancreatic duct in adult Lewis rats. Three groups were studied: A. control group (sham operation); B. induced pancreatitis, no further treatment; C. Induced pancreatitis + gavage with 5 ml/day of a suspension of Lp 299 v in a dose of 0.5-1.0 x 10(9)/ml during 4 days before and 4 days after induction of pancreatitis. All animals were sacrificed after 96 hours. Histological studies and microbiological analyses were performed. Forty out of 55 animals showed signs of severe pancreatitis on sacrifice after 96 hours. Only these animals were further studied. In group A, we found only 1/20 bacteria in mesenteric nodes (MN). Pathogenic microrganisms were found in the non-treated group in MN in 14/20 and in the pancreatic tissue in 10/20. In contrast, when kept on an umbrella of Lp 299 v, only 4/20 animals demonstrated growth of enteric bacteria in MN and 3/20 in pancreatic tissue. All of these results showed a significant reduction of infection in the treated groups. In our model, Lp 299 v is effective in preventing microbial translocation in experimental pancreatitis. Treatment with probiotic bacteria, such as Lactobacillus spp, seems to be a promising alternative as problems with antibiotic-resistant bacteria seem to accumulate.

Lack of correlation between salivary Streptococcus mutans and lactobacilli counts and caries in IDDM children.

In a previous clinical study regarding the incidence of caries and the periodontal health, a group of young patients with various levels of glyco-metabolic control was studied and the results showed that the decayed-missing-filled teeth (DMFT) index was higher in insulin dependent diabetes mellitus (IDDM) type 1 patients with a poor glyco-metabolic balance than in a control group or in IDDM patients with sufficient glyco-metabolic balance. In light of these results, the purpose of this study was to find an explanation for these clinical observations by searching at a microbiological level. The results indicate that salivary counts of Streptococcus mutans and lactobacilli were higher in patients with active caries whether or not they be diabetic, than in people with no active caries, but the count of S. mutans was not directly correlated to the DMFT index. No significant alterations were found in salivary flow, pH, buffer capacity and glucose concentration in all the groups in this study. We conclude that the salivary count of S. mutans is not sufficient alone to account for the higher susceptibility to active caries of young IDDM patients with poor glyco-metabolic control.

[Microbiological contamination in the dental office and its possible decrease]. / L'inquinamento microbiologico nell'ambulatorio odontoiatrico ed il suo possibile abbattimento.

A microbiological analysis of the environment after dental work is presented in this paper. Detection of oral streptococci in the air is used as an index of the presence of salivary aerosol in consequence of the use of dental tools at high spin. This salivary aerosol may be considered a very important cause for the transmission of infectious diseases in the dental surgery. The real efficacy of a tool for the production of a dry aerosol of phenols or clorexidine with the purpose of environmental disinfection, is evaluated. Among possible parameters has been considered both the spray ability of the tool and the bactericidal activity of the aerosol at variable length from the source. Data here presented demonstrate the real utility of such an instrument for the disinfection of the dental surgery to be applied daily at the end of the work, not only in reducing environment microbial counts but also in totally eliminating salivary microorganisms.

[Effect of saliva on resorption time of some resorbable membranes]. / L'influenza della saliva sul tempo di riassorbimento di alcune membrane riassorbibili.

Since resorbable membranes have been introduced their resorption time has been always an important topic of discussion. The current literature does not cover very accurately the contributing factors associated with this biologic process in the oral cavity. The clinical experience shows that the influence of saliva may be an important factor during the resorption of synthetic resorbable membranes. Six experiments are described in this article in which four synthetic resorbable membranes are tested (Vicryl periodontal mesh, Vicryl collagene, Guidor and Resolut). The membranes are plated in Petri dishes precoated with Agar in contact with saliva. Experiment number 1 and 2 demonstrated that saline solution and Agar do not alter the resorption time of the membranes. Experiment 3 and 4 showed that a dilution of saliva to 1:10 and a non diluted saliva accelerate their resorption time of two of the tested membranes. The Vicryl periodontal mesh and the Vicryl collagene disappeared respectively after 7 and 9 days of contact with the not diluted saliva and after 10 and 12 days of contact with the 1:10 diluted saliva. The experiment 5 and 6 indicated that both salivas (diluted and not diluted) deprived of bacteria do not alter the resorption time of the membranes. In conclusion the pattern of resorption of the synthetic membranes, in this in vitro study, is recognized in the contact between the membrane and the bacterial enzymes present in saliva, and in the mechanical structure of the membrane design.