Efficacy of an alpha-hydroxy acid (AHA)-based cream, even in monotherapy, in patients with mild-moderate acne.

AIM AND METHODS: The treatment of mild-moderate acne with topical drugs in association with appropriate cosmetics is currently the golden standard. The tolerability and efficacy of a cream formulated with a new mix of alpha-hydroxy acids (Hyseac AHA cream) in 248 patients with mild-moderate acne (comedonic, inflammatory, or mixed) have been investigated in a multicenter, non-randomized, open study by 10 dermatologists from different Italian areas during their routine practice. The medication with Hyseac AHA cream was prescribed at first consultation for 60 days, twice a day, either as a monotherapy (49.2% patients) or in association with a pharmacological treatment (50.2%). RESULTS: The tolerability was good to excellent in 92.3% patients, without significant differences between patients using AHA cream in monotherapy (90.0%) or associated with concomitant pharmacological treatment (97.6%). The efficacy was overall high in 64.2% patients, again without significant differences related to concomitant pharmacological treatment or not (64.8% vs. 63.3%) and/or the acne clinical type (comedonic vs. inflammatory vs. mixed: 69.2% vs. 66.7% vs. 58%). CONCLUSION: The results of this study, while confirming the high tolerability and efficacy of this AHA cream in the treatment of mild/moderate acne, reasonably suggest its possible use also in monotherapy. Furthermore, its use can be reasonably hypothesized as a maintenance treatment after specific pharmacological treatment even in more severe acne types.

Characterization of antimicrobial resistance and class 1 integrons in Enterobacteriaceae isolated from Mediterranean herring gulls (Larus cachinnans).

Mediterranean herring gulls (Larus cachinnans) were investigated as a possible reservoir of antibiotic resistant bacteria and of cassette-borne resistance genes located in class 1 integrons. Two hundred and fourteen isolates of the family Enterobacteriaceae were collected from cloacal swabs of 92 chicks captured in a natural reserve in the North East of Italy. They showed high percentages of resistance to ampicillin and streptomycin. High percentages of resistance to trimethoprim/sulfamethoxazole were found in Proteus and Citrobacter and to chloramphenicol in Proteus. Twenty-two (10%) isolates carried the intI1 gene. Molecular characterization of the integron variable regions showed a great diversity, with the presence of 11 different cassette arrays and of one integron without integrated cassettes. The dfrA1-aadA1a and aadB-aadA2 cassette arrays were the most frequently detected. Also the estX cassette, alone or in combination with other cassettes, was detected in many isolates. From this study it is concluded that the enteric flora of Mediterranean herring gulls may act as a reservoir of resistant bacteria and of resistance genes. Due to their feeding habits and their ability to fly over long distances, these free-living birds may facilitate the circulation of resistant strains between waste-handling facilities, crops, waters, and urban areas.

Acquisition of different carbapenem resistance mechanisms by an epidemic clonal lineage of Pseudomonas aeruginosa.

Multidrug-resistant isolates of a clonal lineage of Pseudomonas aeruginosa producing the VIM-2 metallo-beta-lactamase (MBL), involved in a large outbreak in an Italian hospital, were compared with MBL-negative strains that had caused outbreaks in two French hospitals. Although the isolates had different carbapenem MICs, the VIM-2-producing isolates from Italy carried identical, or very similar, allelic forms of the oprD gene, harboured a common class 1 integron, belonged to the same multilocus sequence type (ST111), and showed macrorestriction profiles that were related to those of the MBL-negative French strains. These results support the concept of independent acquisition of resistance determinants by members of a widespread clonal lineage of P. aeruginosa.

IL-16 serum level in children with atopic dermatitis.

IL-16 is a natural ligand of CD4 molecules and induces chemotaxis in CD4-expressing cells. It amplifies the inflammatory reaction by stimulating cytokine production in monocytes and activating T-cells. There is evidence that IL-16 plays a role in the pathogenesis of atopic dermatitis, and increased serum levels of IL-16 have been detected in allergic diseases. However, few data are available on IL-16 serum levels in atopic dermatitis. The aim of our study is to measure IL-16 serum levels in childhood atopic dermatitis before and after treatment and to evaluate a possible correlation between IL-16 serum levels and disease severity. IL-16 serum levels were measured by an ELISA approach in 34 children (19 males and 15 females; mean age 6.8 years) with moderate to severe atopic dermatitis, at their first visit and after 3 months of treatment, and in 10 non-atopic healthy controls of the same age group. The severity of atopic dermatitis was measured by SCORAD index. IL-16 serum levels were significantly higher in patients affected by atopic dermatitis than in controls before and after treatment with tacrolimus ointment. No clear correlation was found between IL-16 serum levels and atopic dermatitis severity. IL-16 serum levels are increased in atopic dermatitis but do not seem to correlate with disease severity.

Phagocytosis of Mycoplasma pneumoniae and Acholeplasma laidlawii measured as inhibition of [3H]uridine uptake by macrophages.

Many studies of the interaction between phagocytes and mycoplasmas have given controversial results. This is probably due both to the small size of the microorganisms and their ability to attach to the cell membrane, making it difficult to distinguish between adsorption and ingestion. To overcome these difficulties we took advantage of a phenomenon we noted occurring concomitantly with phase-contrast microscope-monitored phagocytosis of heat-killed C. albicans, i.e., a reduction of [3H]uridine uptake by macrophages from culture medium. This approach allowed us to measure the ability of mouse peritoneal macrophages and the macrophage-like P 388 D 1 continuous cell line to phagocytose Mycoplasma pneumoniae and Acholeplasma laidlawii. Live, UV-killed and specific antiserum-opsonized mycoplasmas were tested. A. laidlawii was ingested under all the conditions mentioned above, while live M. pneumoniae was not phagocytosed unless UV-killed. Phagocytosis of UV-killed M. pneumoniae was directly verified by transmission electron microscopy studies. Data obtained with opsonized M. pneumoniae indicated no ingestion by mouse peritoneal macrophages and incomplete phagocytosis with P388 D 1 macrophages, suggesting that different responses by different types of phagocytes can be observed. In spite of a lack of information concerning the biological meaning of the inhibition of macrophage RNA metabolism during phagocytosis, our data suggest that this phenomenon may be used to study the phagocytosis of microorganisms which are difficult to visualize.

Characterization of oligonucleotide probes for the identification of Acinetobacter spp., A. baumannii and Acinetobacter genomic species 3.

The 16S-23S intergenic spacer regions of four Acinetobacter genomic species belonging to the A. calcoaceticus-A. baumannii (Acb) complex, i.e. genomic species 1 (A. calcoaceticus), genomic species 2 (A. baumannii), genomic species 3 and Tjernberg and Ursing (TU) genomic species 13, have been cloned and sequenced. Sequence analysis led to the discovery of a single copy of IIe and Ala tRNA genes within each spacer. Sequence comparison allowed the identification of a 192-base-pair long highly conserved sequence between the 3' end of the 16S rRNA and the 5' end of the tRNA(Ala) genes. Moreover, two short regions, which were specific to, respectively, genomic species 2 and 3, could be identified. Oligonucleotides corresponding to these sequences were constructed and tested for the ability to hybridize with chromosomal DNA extracted from Acinetobacter belonging to different genomic species and with chromosomal DNA of other bacterial genera. One of these oligonucleotides was demonstrated to be useful as a sensitive and specific probe for A. baumannii. A less sensitive probe for Acinetobacter genomic species 3 was also developed.

Typing of Staphylococcus aureus by amplification of the 16S-23S rRNA intergenic spacer sequences.

The possibility of using polymorphisms in the spacer regions between 16S and 23S rRNA genes in order to type Staphylococcus aureus has been evaluated. To this purpose, DNA extracted from 74 independent isolates was amplified making use of a pair of primers complementary to conserved regions in the 16S and 23S genes. We have demonstrated that the method provides a good discrimination between unrelated isolates, giving better results when methicillin-sensitive strains are considered. Moreover, the amplification profiles were reproducible and all strains were typable. Given these results, and the technical simplicity of the process, we propose PCR-ribotyping to be taken into consideration as a method for typing S. aureus.

Mycobacterium tuberculosis isolates belonging to katG gyrA group 2 are associated with clustered cases of tuberculosis in Italian patients.

Fifty-one consecutive isolates of Mycobacterium tuberculosis, collected during a 2-year period in the north-east of Italy, were subjected to IS6110-RFLP analysis to detect the presence of clusters and assigned to one of the three genotypic groups delineated by single nucleotide polymorphisms in the genes katG and gyrA. All the isolates collected from the local population belonged to group 2 or 3, while group 1 isolates were found only in specimens collected from African immigrants. Clustered cases of tuberculosis, which are likely to be related to recently transmitted infection, were found to be significantly associated with katG gyrA group 2. In the local situation, strains belonging to this group may therefore present a higher risk of transmission.

Short-Term prophylaxis with ceftriaxone plus metronidazole in esophageal cancer surgery.

An open prospective study was carried out in 82 consecutive patients undergoing resective surgery for esophageal cancer from January 1995 to July 1996. Antimicrobial prophylaxis was done using a single dose of ceftriaxone (2 g i.v.) given at the induction of anesthesia in combination with metronidazole (0.5 g i.v.). Two further doses of metronidazole were administered 8 and 16 hours postoperatively. Fourteen patients (17%) experienced postoperative infections. This study, even though open and non-comparative, confirms that ceftriaxone given as a single-dose plus metronidazole provides adequate prophylaxis and significant cost-savings in comparison with multiple-dose prophylactic regimens in patients undergoing major surgery for esophageal cancer. Furthermore, the single-dose regimen reduces the workload for the nursing staff, the risk of side effects, and the possibility of selecting resistant strains.

Beta-lactam-specific resistant mutants of Staphylococcus aureus.

In an approach to understanding the origin of methicillin resistance in clinical isolates of staphylococci, a series of Staphylococcus aureus mutants resistant to various beta-lactam antibiotics were isolated in the laboratory by antibiotic selection. Mutants with low- and intermediate-level resistance showed considerable specificity for the particular antibiotic used in the selection process (methicillin, cefotaxime, cephalexin, and amdinocillin), and resistance in such mutants also showed alterations in the antibiotic binding capacities of penicillin-binding proteins (PBPs). In each case the isolation of mutants resistant to high concentrations of antibiotics required sequential passage in gradually increasing concentrations of the drug. The acquisition of increasing levels of methicillin resistance was paralleled by a gradual decrease in the binding capacities of PBPs 2, 3, and, possibly, 1. In a highly methicillin-resistant mutant (MIC, 150 micrograms/ml), PBPs 2 and 3 were no longer detectable by the penicillin binding assay. Instead, a new PBP of poor binding capacity and anomalous molecular size (about 78 kilodaltons [kDa]) appeared in these cells. This corresponds to the molecular size of PBP 2a, the unique PBP that appears to be the biochemical correlate of resistance in clinical isolates of methicillin-resistant S. aureus. Also, similar to the case of resistant clinical isolates, high-level beta-lactam resistance was highly pH dependent in the laboratory mutants. We compared the patterns of radioactive peptides generated by partial proteolysis from the penicillin-labeled PBP 2 of antibiotic-susceptible staphylococci and from the 78-kDa PBP 2a of a resistant clinical strain. Although the patterns were clearly different, seven of the eight characteristic peptides generated from PBP 2 of the susceptible strain were also detectable among the peptides released from PBP 2a. The results suggest that the 78-kDa PBP 2a of the resistant clinical strain evolved from PBP 2 of antibiotic-susceptible staphylococci and that in PBP 2a of the clinical isolate mutational changes have resulted in extensive alterations near the beta-lactam binding site.

Enzyme-linked immunosorbent assay for detection of Mycoplasma pneumoniae antibodies.

The enzyme-linked immunosorbent assay (ELISA) described by Engvall and Perlmann has been used for the detection of antibodies against Mycoplasma pneumoniae. These organisms, grown in flat-bottom wells of microtiter plates, were used as antigen. Preliminary results suggest that ELISA is specific and that its sensitivity is somewhere in between those of the metabolic inhibition and radioimmunoprecipitation methods. Unlike other serological methods, such as metabolic inhibition and mycoplasmacidal tests, in which the presence of antibiotics in sera may give false results, ELISA provided results that were not influenced by antibiotics. Furthermore, ELISA offers the possiblity of measuring not only immunoglobulin M, but also complement-independent antibodies, especially those of the immunoglobulin A class. Theoretical aspects concerning the different sensitivities of some serological reactions are discussed.

Interaction of FCE 22101 with penicillin-binding proteins of Staphylococcus aureus.

FCE 22101 is a new penem characterized by a broad spectrum of activity which includes activity against methicillin-resistant strains of Staphylococcus aureus. The interaction of FCE 22101 with penicillin-binding protein 2a of a methicillin-resistant S. aureus strain has been investigated in the present study. In competition experiments, the penem showed a very low affinity for this protein, and a concentration more than 4 times the MIC was required for penicillin-binding protein 2a saturation. When the classical competitive procedure was modified by increasing the time of incubation of either membranes or growing cells with FCE 22101, the antibiotic showed a much higher affinity for penicillin-binding protein 2a and saturated the protein at a concentration close to the MIC, with slow kinetics.

Regulation of penicillin-binding protein activity: description of a methicillin-inducible penicillin-binding protein in Staphylococcus aureus.

The penicillin-binding proteins (PBPs) of two methicillin-resistant strains of Staphylococcus aureus (R2 and R1) were analyzed in cells grown in the absence and in the presence of methicillin. Under the former condition, strain R2 showed the typical PBP pattern of beta-lactam-susceptible strains, while strain R1 showed a markedly increased amount of PBP-3. Under the latter condition, on the other hand, a novel PBP (PBP-2a) located between PBP-2 and -3 was detected in strain R2, while strain R1 appeared to synthesize an even greater amount of PBP-3, in respect to untreated cells. Both R2 PBP-2a and R1 PBP-3 showed a very low affinity for methicillin, which was consistent with the MICs for the respective strains.

Enzyme-linked immunosorbent assay for serodiagnosis of Mycoplasma pneumoniae infections.

Enzyme-linked immunosorbent assay (ELISA) was used for detection of antibodies against Mycoplasma pneumoniae. The results of ELISA were compared with those of other serological methods, such as complement fixation, metabolic inhibition, mycoplasmacidal test, and radioimmunoprecipitation. ELISA and mycoplasmacidal test showed greater sensitivity than did complement fixation and metabolic inhibition. Radioimmunoprecipitation showed serum titers 2- to 16-fold higher. Specificity of ELISA was determined by testing other human mycoplasmas. A simplified method based on the determination of ELISA antibody end-point titer by a single serum dilution is proposed.

In vitro response to bactericidal activity of cell wall-active antibiotics does not support the general opinion that enterococci are naturally tolerant to these antibiotics.

The incidence of tolerance and paradoxical response to bactericidal activity of penicillin was investigated in 50 clinical isolates of Enterococcus faecalis. Of the isolates tested, 86% exhibited the paradoxical phenomenon whereby there were more survivors at high than at low concentrations above the MIC. Low penicillin concentrations caused decreases equal to or higher than 99.9% in 11 strains, from 99.9 to 99.5% in 23 strains, and lower than 99.5% in 9 strains. Of the total strains, 14% were killed to the same extent by all concentrations above the MIC. The bactericidal activities of other beta-lactams (ampicillin and piperacillin) and other cell wall inhibitors (vancomycin and daptomycin) were also tested against some of these strains. In general, beta-lactams exhibited the best bactericidal activity at 2 x MIC. Piperacillin was the most active, as at 2 x MIC it reduced the original inoculum by 99.9% or more in most of the strains. No concentration of vancomycin above the MIC caused 99.9% killing of the strains, whereas daptomycin was bactericidal at 8 x MIC in most cases. Paradoxical response to bactericidal activity of beta-lactams was abolished by incubation of the inoculum with 2 x MIC before exposure to higher antibiotic concentrations. These findings suggest that enterococci are not always tolerant to cell wall-active antibiotics and that accurate in vitro bactericidal tests may be useful for the choice of appropriate therapy for infections caused by these microorganisms.

Nonradioactive DNA probe for detection of gene for methicillin resistance in Staphylococcus aureus.

A DNA probe was developed by inserting, in the SmaI site of pBluescript sK, a 528-bp fragment of the gene responsible for intrinsic resistance to beta-lactam antibiotics in Staphylococcus aureus (mec determinant). The mec probe provided a useful tool for the rapid identification of the intrinsic resistance trait and for establishing guidelines for testing the in vitro susceptibility of S. aureus to beta-lactams.

Identification of Acinetobacter isolates in the A. calcoaceticus-A. baumannii complex by restriction analysis of the 16S-23S rRNA intergenic-spacer sequences.

Members of the genus Acinetobacter are reported to be involved in hospital-acquired infections with an increasing frequency. However, clinical laboratories still lack simple methods that allow complete identification of some pathogenic species, i.e., those corresponding to A. baumannii (DNA group or genospecies 2), unnamed genospecies 3 and 13, and two new genospecies that have recently been described. In fact, a complete discrimination between these species is possible only by DNA-DNA hybridization or ribotyping. Both of these techniques are complex and time-consuming and cannot be performed in most clinical laboratories. As a consequence, isolates belonging to these genospecies are often not differentiated and included, together with the environmental genospecies 1, in the A. calcoaceticus-A. baumannii complex. In this report, a simple and rapid method for the identification of the genospecies belonging to the A. calcoaceticus-A. baumannii complex is proposed. It is based on the combined digestion by the restriction endonuclease AluI and NdeII of the DNA fragments resulting from the amplification of the 16S-23S rRNA intergenic spacer sequences. The analysis of 36 strains characterized by DNA-DNA hybridization in previous studies showed that the restriction profiles obtained are highly reproducible and characteristic for each genospecies. Moreover, extending this study to 68 clinical strains, which were assigned to the A. calcoaceticus-A. baumannii complex by phenotypic tests, confirmed the existence of a panel of limited and well-conserved restriction patterns and allowed the identification of the strains tested. This study thus proposes the detection of restriction length polymorphism in the spacer sequences between the 16S and 23S rRNA genes as a method for the identification of isolates in the A. calcoaceticus-A. baumannii complex.

Further characterization of borderline methicillin-resistant Staphylococcus aureus and analysis of penicillin-binding proteins.

Eighty-nine Staphylococcus aureus strains were grouped according to their susceptibility or resistance to methicillin and oxacillin. The role of beta-lactamase in borderline methicillin resistance was confirmed by tests with beta-lactamase inhibitors, particularly when salt-supplemented medium was used. A penicillin-binding protein assay indicated that borderline methicillin-resistant S. aureus strains do not produce PBP 2a.