Protease inhibitors elicit anti-inflammatory effects in CF mice with Pseudomonas aeruginosa acute lung infection.

Pseudomonas aeruginosa is the major respiratory pathogen in patients with cystic fibrosis (CF). P. aeruginosa-secreted proteases, in addition to host proteases, degrade lung tissue and interfere with immune processes. In this study, we aimed at evaluating the possible anti-inflammatory effects of protease inhibitors Marimastat and Ilomastat in the treatment of P. aeruginosa infection. Lung infection with the P. aeruginosa PAO1 strain was established in wild-type and cystic fibrosis transmembrane conductance regulator (CFTR) knock-out C57BL/6 mice expressing a luciferase gene under control of bovine interleukin (IL)-8 promoter. After intratracheal instillation with 150 µM Marimastat and Ilomastat, lung inflammation was monitored by in-vivo bioluminescence imaging and bacterial load in the lungs was assessed. In vitro, the effects of protease inhibitors on PAO1 growth and viability were evaluated. Acute lung infection was established in both wild-type and CFTR knock-out mice. After 24 h, the infection induced IL-8-dependent bioluminescence emission, indicating lung inflammation. In infected mice with ongoing inflammation, intratracheal treatment with 150 µM Marimastat and Ilomastat reduced the bioluminescence signal in comparison to untreated, infected animals. Bacterial load in the lungs was not affected by the treatment, and in vitro the same dose of Marimastat and Ilomastat did not affect PAO1 growth and viability, confirming that these molecules have no additional anti-bacterial activity. Our results show that inhibition of protease activity elicits anti-inflammatory effects in cystic fibrosis (CF) mice with acute P. aeruginosa lung infection. Thus, Marimastat and Ilomastat represent candidate molecules for the treatment of CF patients, encouraging further studies on protease inhibitors and their application in inflammatory diseases.

Evaluation of plant and fungal extracts for their potential antigingivitis and anticaries activity.

The link between diet and health has lead to the promotion of functional foods which can enhance health. In this study, the oral health benefits of a number of food homogenates and high molecular mass and low molecular mass fractions were investigated. A comprehensive range of assays were performed to assess the action of these foods on the development of gingivitis and caries using bacterial species associated with these diseases. Both antigingivitis and anticaries effects were investigated by assays examining the prevention of biofilm formation and coaggregation, disruption of preexisting biofilms, and the foods' antibacterial effects. Assays investigating interactions with gingival epithelial cells and cytokine production were carried out to assess the foods' anti- gingivitis properties. Anti-caries properties such as interactions with hydroxyapatite, disruption of signal transduction, and the inhibition of acid production were investigated. The mushroom and chicory homogenates and low molecular mass fractions show promise as anti-caries and anti-gingivitis agents, and further testing and clinical trials will need to be performed to evaluate their true effectiveness in humans.

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.

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.

[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.

[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.

Molecular vs culture methods for the detection of bacterial faecal indicators in groundwater for human use.

AIMS: The current standard culture methods are unable to detect nongrowing bacteria and, thus, might not be sufficient for precise monitoring of the microbiological quality of waters. The use of a molecular method such as PCR could be a valid alternative to detect bacterial faecal contamination indicators such as Escherichia coli and Enterococcus faecalis and reveal the presence of culturable and nonculturable bacterial forms. METHODS AND RESULTS: The presence of E. coli and Ent. faecalis cells in 30 groundwater samples was evaluated with the standard culture method and compared with a specific PCR protocol. A substantial percentage (50%) of the samples not containing culturable cells proved positive in the search for Ent. faecalis DNA by PCR. Quantification by competitive PCR (cPCR) of the DNA detected allowed us to calculate the number of nonculturable cells present in water samples: the number varied from 2 to 120 cells ml(-1). Only four samples were positive for E. coli DNA and the corresponding nonculturable cells varied from 24 to 70 ml(-1). CONCLUSIONS: This study demonstrates that the standard culture methods in use are unable to detect a substantial proportion of the bacterial population which is nonculturable but, as previously demonstrated, potentially still viable and able to express those pathogenic factors needed for causing infections in humans. SIGNIFICANCE AND IMPACT OF THE STUDY: To protect human health it is necessary to develop and use methods which detect the nonculturable as well as culturable bacteria present in water.

Paradoxical effect of inserting, in Enterococcus faecalis penicillin-binding protein 5, an amino acid box responsible for low affinity for penicillin in Enterococcus faecium.

Penicillin-binding proteins 5 (PBP5s) of enterococci are structurally and immunologically related proteins that are characterized by their low affinity for penicillin. For this reason, they are mainly involved in penicillin resistance, due essentially to their ability to take over the function of all other PBPs already bound and inhibited by the beta-lactam. It has been demonstrated that penicillin resistance in enterococci is acquired either by overproduction of PBP5 or by the presence of specific amino acid sequences in the protein that further decrease the affinity for penicillin. In particular, a specific amino acid box (ANNGA) previously identified in Enterococcus faecium is responsible for the high penicillin resistance displayed by this species. Here, we describe the insertion of the PBP5 amino acid box ANNGA in Enterococcus faecalis, an enterococcal species usually more sensitive to penicillin, by site-directed mutagenesis. Mutagenized PBP5 was re-introduced into a pbp5 mutant of E. faecalis obtained by insertion of transposon Tn916. Data indicate that this amino acid box brings about no reduction in penicillin sensitivity in the recipient E. faecalis strain, but, paradoxically, dramatically lowers the penicillin minimal inhibitory concentration caused by the native PBP5. We deduce that, although enterococcal PBP5s are a family of closely related proteins as far as biological function is concerned, differences exist in their three-dimensional structure that affect penicillin affinity.

Peptidoglycan synthesis by Enterococcus faecalis penicillin binding protein 5.

Low-affinity penicillin binding proteins (PBPs) are a particular class of proteins involved in beta-lactam antibiotic resistance of enterococci. The activity of these PBPs is just sufficient to allow the cells to survive in the presence of high concentrations of beta-lactams that cause saturation (and inhibition) of the other PBPs. For this reason, the low-affinity PBPs are thought to be multifunctional enzymes capable of catalyzing the entire peptidoglycan synthesis. To test the validity of this claim, we analyzed the muropeptide composition by reversed-phase high-performance liquid chromatography of the peptidoglycan synthesized by PBP5 (the low-affinity PBP) of Enterococcus faecalis, in comparison with the peptidoglycan produced normally by the concerted action of the usual PBPs (namely PBPs 1, 2, and 3). Cross-linked peptidoglycan was produced. The main difference consisted in the lack of oligomers higher than trimers, thus suggesting that this oligomer cannot be used as an acceptor/donor by the transpeptidase component of PBP5. The lack of higher oligomers had little impact on total cross-linking because of the increase observed in the dimer family. This increase was distributed among the various members of the dimer family with the result that minor dimer components figured among the prevalent ones in cells in which peptidoglycan was synthesized by PBP5. This also suggests that E. faecalis PBP5 is capable of catalyzing the synthesis of a peptidoglycan that is less precise and refined than usual, and for this reason PBP5 can be considered an enzyme endowed with poor specificity for substrates, as may be expected on the basis of its survival function.

Cell elongation and septation are two mutually exclusive processes in Escherichia coli.

Bacterial rod morphogenesis was studied in synchronously growing cells of Escherichia coli C600 during the reshaping process that follows the removal of mecillinam, a beta-lactam antibiotic that specifically inhibits lateral wall formation of gram-negative rods and causes transition to coccal shape. Removal of mecillinam after 30 min of action did not affect the timing of subsequent cell division, but removal after 50 min delayed resumption of cell division for approximately one generation time. In order to study the interplay between lateral wall elongation and septum formation in determining and maintaining the bacterial rod shape, we evaluated the effect of re-adding mecillinam or of adding aztreonam (a specific inhibitor of septum formation) at various stages of the reshaping process. We conclude that mecillinam was active only during the reshaping process, while aztreonam was active only later when the cells were close to dividing again. These results provide further evidence for our previous proposal according to which elongation and septation are two alternating and competing events of the cell cycle and are linked to each other to force bacterial rods to grow to a given length.

Modified peptidoglycan chemical composition in shape-altered Escherichia coli.

Peptidoglycan synthesis and its fine chemical composition were studied in dividing cocci of Escherichia coli carrying the lov-1 mutation and in which the coccal shape was obtained either by mecillinam treatment or by transferring a pbpA mutation (penicillin-binding protein 2- phenotype), as compared to normal rods and non-dividing cocci. Synchronously dividing cocci showed peptidoglycan synthesis only in the cell cycle phase corresponding to cell septation. During the phase corresponding to lateral wall elongation, peptidoglycan synthesis was strongly reduced. This type of synthesis suggests that the dividing cocci consisted only of the two poles. Analysis of the muropeptide composition revealed a specific fourfold increase in the tetra-tetra-tetra trimer in dividing cocci as compared to non-dividing cocci or parental rods. We postulate that, in E. coli, the chemical composition of septal peptidoglycan differs from that of lateral wall peptidoglycan.

Cell wall chemical composition of Enterococcus faecalis in the viable but nonculturable state.

The viable but nonculturable (VBNC) state is a survival mechanism adopted by many bacteria (including those of medical interest) when exposed to adverse environmental conditions. In this state bacteria lose the ability to grow in bacteriological media but maintain viability and pathogenicity and sometimes are able to revert to regular division upon restoration of normal growth conditions. The aim of this work was to analyze the biochemical composition of the cell wall of Enterococcus faecalis in the VBNC state in comparison with exponentially growing and stationary cells. VBNC enterococcal cells appeared as slightly elongated and were endowed with a wall more resistant to mechanical disruption than dividing cells. Analysis of the peptidoglycan chemical composition showed an increase in total cross-linking, which rose from 39% in growing cells to 48% in VBNC cells. This increase was detected in oligomers of a higher order than dimers, such as trimers (24% increase), tetramers (37% increase), pentamers (65% increase), and higher oligomers (95% increase). Changes were also observed in penicillin binding proteins (PBPs), the enzymes involved in the terminal stages of peptidoglycan assembly, with PBPs 5 and 1 being prevalent, and in autolytic enzymes, with a threefold increase in the activity of latent muramidase-1 in E. faecalis in the VBNC state. Accessory wall polymers such as teichoic acid and lipoteichoic acid proved unchanged and doubled in quantity, respectively, in VBNC cells in comparison to dividing cells. It is suggested that all these changes in the cell wall of VBNC enterococci are specific to this particular physiological state. This may provide indirect confirmation of the viability of these cells.

mRNA detection by reverse transcription-PCR for monitoring viability over time in an Enterococcus faecalis viable but nonculturable population maintained in a laboratory microcosm.

The viable but nonculturable (VBNC) state is a survival strategy adopted by bacteria when they are exposed to hostile environmental conditions. It has been shown that VBNC forms of bacteria are no longer capable of growing on conventional bacteriological media but conserve pathogenic factors and/or genes. It is thus necessary to develop methods capable of detecting nonculturable bacteria and of establishing their viability when the microbiological quality of environments is monitored. In this study we demonstrated that a gene was expressed during the VBNC state in a low-nutrient-concentration microcosm through detection of Enterococcus faecalis pbp5 mRNA by reverse transcription-PCR over a 3-month period. The presence of mRNA correlated with metabolic activity and resuscitation capability, indicating the viability of the VBNC cells.

Resuscitation rate in different enterococcal species in the viable but non-culturable state.

AIMS: The viable but non-culturable (VBNC) state is a survival strategy adopted by bacteria when exposed to environmental stress. When in this state bacteria are no longer culturable on conventional growth media, but cells display metabolic activity and maintain pathogenicity factors/genes and, in some cases, resuscitation from the VBNC state has been shown. This state has been described for both human pathogens and faecal pollution indicators. In this study, we present evidence for entry of different enterococcal species into the VBNC state in an oligotrophic microcosm. METHODS AND RESULTS: The duration of the viability of the cells in the VBNC state was measured either by detecting the presence of pbp5 mRNA or by quantifying their resuscitation capability. Enterococci showed different behaviours. Enterococcus faecalis and Enterococcus hirae entered into the VBNC state within 2 weeks and remained in that state for 3 months. In the experiments described the resuscitation rate was 1:10 000 cells as soon as the cells entered the VBNC state and decreased gradually to undetectable levels over the following 3 months. Enterococcus faecium, however, remained culturable up to 4 weeks. After this time period, when the population was totally unculturable, the cells were far less resuscitable than other enterococci and only over a narrow time interval (2 weeks). CONCLUSIONS: These results suggest that Ent. faecalis and Ent. hirae enter the VBNC state but that Ent. faecium, in an oligotrophic laboratory environment, tends to die instead of entering the VBNC state. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when enterococci are released by humans and animals in natural environments.

Lactobacillus plantarum reduces infection of pancreatic necrosis in experimental acute pancreatitis.

BACKGROUND: Infection is the commonest cause of death in acute pancreatitis. Early reduction of commensal flora (particularly Lactobacillus species) and, at the same time, overgrowth of Enterobacteriaceae, especially Escherichia coli, have recently been described during acute pancreatitis. Lactobacillus plantarum has been shown to be effective in reducing the egress of endotoxin and microbial translocation in several experimental models such as chemically induced hepatitis and ulcerative colitis. AIM: The aim of the study was to determine whether L. plantarum 299v (Lp 299v) is capable of effectively reducing microbial translocation in experimental pancreatitis. METHODS: Acute pancreatitis was induced by isolation and ligation of the biliopancreatic duct in Lewis rats weighing 250-350 g. The animals were divided into 3 groups: group A, sham operation; group B, induction of pancreatitis and no further treatment, and group C, induction of pancreatitis + daily administration by gavage of a 5-ml/day suspension of Lp 299v at 0.5-1.0 x 10(9) bacteria/ml for 8 days, 4 days before and 4 days after induction of pancreatitis. All animals were sacrificed after 96 h. Histological studies and microbiological analyses were performed. RESULTS: At sacrifice, 40/55 animals showed signs of severe pancreatitis. Since acute pancreatitis was the specific disease investigated, only these animals were subjected to further study. In group B, we found pathogenic micro-organisms in the mesenteric lymph nodes in 14/20 animals and in the pancreatic tissue in 10/20. The bacterial flora consisted predominantly of E. coli, Enterococcus faecalis, Pseudomonas and Proteus species. In contrast, when the animals were kept under an 'umbrella' of Lp 299v, growth of E. faecalis or E. coli were detected only in 4/20 mesenteric lymph node cultures and in 3/20 pancreatic tissue cultures. CONCLUSIONS: Lp 299v is effective in reducing microbial translocation in experimental pancreatitis. Treatment with probiotic bacteria seems to be a promising alternative to antibiotic therapy.

Competitive polymerase chain reaction for quantification of nonculturable Enterococcus faecalis cells in lake water.

Among the survival strategies developed by bacteria when faced with adverse environmental conditions, the viable but nonculturable (VNC) state has been described. In this state, bacteria are unable to form colonies but are still alive and capable of metabolic activity. The VNC state has been described in numerous Gram-negative species, but recently also in Enterococcus faecalis, a Gram-positive species which can be found in the environment. In this study we describe a competitive PCR (cPCR) protocol to detect and quantify a specific sequence of DNA from culturable and nonculturable E. faecalis cells present in water samples. The protocol was found to be specific and capable of detecting amounts of DNA up to 0.1 pg corresponding to approximately 2 cells ml(-1). Moreover, it allows an internal standard to be used to quantify the amount of specific DNA present in samples from different environments. The application of this cPCR method to water samples from Lake Garda enabled us to demonstrate the presence of nonculturable forms of E. faecalis in lake water and to quantify their DNA and the corresponding concentration of nonculturable cells.