Changes in the resistance patterns among upper respiratory tract infection pathogens.
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Anti-Bacterial Agents↗
Biomedical subjects
S Stefani
Publications and source records attributed to S Stefani.
Molecular epidemiology of antibiotic resistance.
Molecular typing methods based on the analysis of the genetic structure of bacteria, are used to address many different problems such as the study of genomic organisation and evolution, the identification of patterns of infection, the identification of sources of transmission, the epidemiological surveillance of infectious diseases and for investigations into outbreaks. Of particular interest is the application of these techniques for acquiring information on the spread of micro-organisms that have become resistant to many clinically important antibiotics. The emergence of antibiotic resistance is one of the most dangerous phenomena of the last 20 years and knowledge of the mechanisms of resistant-gene exchange means fully understanding their spread into all environments. Studies on the molecular epidemiology of antibiotic-resistance in micro-organisms should make it easier to distinguish clonality with respect to horizontal transfer of the determinants of resistance.
Bacteria↗
Levofloxacin in vitro activity and time-kill evaluation of Stenotrophomonas maltophilia clinical isolates.
The in vitro activity of levofloxacin and eight other antimicrobial agents against 60 clinical isolates of Stenotrophomonas maltophilia was determined by an agar dilution method using 10(4) and 10(6) cfu/spot inocula. At the lower inoculum, 85.0% of the isolates were susceptible to levofloxacin but only 58.3% were susceptible to ofloxacin; at the higher inoculum, 78.3% were susceptible to levofloxacin and 36.7% to ofloxacin. In time-kill studies, levofloxacin exerted bactericidal activity within 4 h. With ofloxacin and ciprofloxacin bacterial regrowth was observed after 8 h. Levofloxacin may represent an alternative drug in the treatment of infections caused by S. maltophilia.
Anti-Infective Agents↗
First identification of an SHV-12 extended-spectrum beta-lactamase in Klebsiella pneumoniae isolated in Italy.
A clinical isolate of Klebsiella pneumoniae highly resistant to third- and fourth-generation cephalosporins, cephamycins and aminoglycosides, was isolated in 1991 from urine. Analysis of a crude extract showed the presence of three beta-lactamases with isoelectric points of 6.6, 7.5 and 8.2. The enzyme with pI 8.2 was transferred by conjugation into Escherichia coli K-12 J53 and was responsible for the resistance to third-generation cephalosporins and monobactams, but not to other antibiotics. Kinetic studies of partially purified beta-lactamase from the transconjugant strain confirmed that the enzyme was able to hydrolyse ceftazidime, cefotaxime and aztreonam but not cephamycins. Analysis of the transconjugant showed the presence of two small non-conjugative plasmids of 14 and 6 kb. A polymerase chain reaction was performed using primers specific for the bla(SHV) gene and a fragment of the expected size (about 961 bp) was obtained with both the K. pneumoniae clinical isolate and the transconjugant. Nucleotide sequence analysis of the fragment showed that it encoded the enzyme SHV-12, derived from SHV-5 (with Gln-35 to Leu). This is the first report of an SHV-12-like enzyme isolated in Italy.
Anti-Bacterial Agents↗
Characterization of a genetic element carrying the macrolide efflux gene mef(A) in Streptococcus pneumoniae.
The mef(A) gene from a clinical isolate of Streptococcus pneumoniae exhibiting the M-type resistance to macrolides was found to be part of the 7,244-bp chromosomal element Tn1207.1, which contained 8 open reading frames. orf2 encodes a resolvase/invertase, and orf5 is a homolog of the macrolide-streptogramin B resistance gene msr(SA).
Anti-Bacterial Agents↗
A kinetic study on the interaction between tazobactam (a penicillanic acid sulphone derivative) and active-site serine beta-lactamases.
The interaction between tazobactam and several chromosome- and plasmid-encoded (TEM, SHV, PSE types) class A and C beta-lactamases was studied by spectrophotometry. Tazobactam behaved as a competitive inhibitor or inactivator able to restore in several cases the efficiency of piperacillin as a partner beta-lactam. A detailed kinetic analysis permitted measurement of the acylation efficiency for some cephalosporinases and broad-spectrum beta-lactamases; the presence of a turn-over of acyl-enzyme complex was also evaluated.
Acinetobacter↗
Molecular epidemiology of Pseudomonas aeruginosa from cystic fibrosis in Sicily: genome macrorestriction analysis and rapid PCR-ribotyping.
This study addresses the epidemiologic relatedness of a collection of Pseudomonas aeruginosa isolates from cystic fibrosis patients attending the Pediatric Clinic, Catania, Sicily. Genome macrorestriction analysis after pulsed field gel electrophoresis (PFGE) was used to characterise all strains. Furthermore, a rapid typing procedure, developed in this study, based on polymerase chain reaction amplified ribosomal DNA spacer polymorphisms (PCR-ribotyping), straight from bacterial cultures, was used. On the basis of macrorestriction analysis after PFGE, persistence of infection was shown in all patients; two cross-transmission episodes were identified in the nosocomial as well as in the familiar environment. PCR-ribotyping proved to be useful for a DNA-based identification test, suitable for screening purposes. The rapid amplification protocol here tested is proposed to evaluate the discriminatory power of other specific target sequences in PCR-based typing assays, for epidemiologic purposes.
Adolescent↗
Reverse transcription polymerase chain reaction method for the detection of glycopeptide resistance in enterococci.
In this work we have developed reverse transcription polymerase chain reaction (RT-PCR) methods for detecting specific mRNA from enterococci, particularly vanA and vanB genes, responsible for glycopeptide resistance in this genus. mRNA from the two genes was detected immediately after RNA extraction of a midlog phase culture, determined by growth rate analysis. Because of the short half-life associated with many bacterial RNA species (1.5-2 min), time is an important factor in obtaining RNA of good yield and high purity. Our results showed that: (i) the transcription of mRNA related to vanA ligase in enterococci showing Van A phenotype happens only after induction with both vancomycin and teicoplanin; (ii) the transcription of mRNA related to vanB ligase happens only in the presence of vancomycin and (iii) there was no transcription of mRNA in the two strains positive to vanA gene after PCR experiments. RT-PCR methodology can have numerous applications in microbiology for studying gene expression in isolated bacteria and also in nonculturable cells in environmental samples, for studies of mechanisms and/or as an indicator of viability in bacterial communities.
Anti-Bacterial Agents↗
Phylogenetic analysis of macrorestriction fragments as a measure of genetic relatedness in Staphylococcus aureus: the epidemiological impact of methicillin resistance.
The present study was undertaken for the purpose of defining the epidemiology and genetic relatedness of Staphylococcus aureus strains in a region of Italy by investigating the molecular background for which resistance to methicillin, mediated by the acquisition of another penicillin-binding protein gene, is embedded. Restriction fragment length polymorphism (RFLP) data were used for phylogenetic analyses, since genetic distance values can be used as a general measure of the number of events generating distinct clones. The percentages of methicillin-resistant S. aureus (MRSA) isolates in a six-month period from inpatients and outpatients were, respectively, 12% (22 out of 180) and 0.4% (1 out of 257). On the basis of RFLP obtained after pulsed field gel electrophoresis (PFGE), it was possible to designate isolates as indistinguishable, closely related, possibly related and unrelated. We were able to demonstrate the occurrence of at least five distinct MRSA cross-infection episodes in two hospitals, four involving two patients each and one involving four patients. Phylogenetic analyses overcame the simple pairwise comparison of common bands between strains, and provided a comprehensive epidemiological scenario, identifying three major clusters of MRSA, including different levels of genetic relatedness, while excluding the circulation of a single clone in Italy. Moreover, multidimensional scaling analysis of the obtained genetic distance confirmed that MRSA strains belong to a restricted set of clones, thus demonstrating the relatedness of broad evolutionary lineages within the species S. aureus.
Cluster Analysis↗
Recurrent mutations in the iron regulatory element of L-ferritin in hereditary hyperferritinemia-cataract syndrome.
BACKGROUND AND OBJECTIVE: Hereditary hyperferritinemia-cataract syndrome (HHCS) is an autosomal dominant disorder characterized by bilateral cataracts and increased serum and tissue L-ferritin, in the absence of iron overload. The deregulation of ferritin production is caused by heterogeneous mutations in the iron regulatory element (IRE) of L-ferritin that interfere with the binding of iron regulatory proteins. DESIGN AND METHODS: We have identified several patients from three unrelated Italian families with HHCS. Iron parameters were assessed by standard methods. The IRE element of L-ferritin was amplified by PCR using appropriate primers and directly sequenced. RESULTS: Ferritin levels ranged from 918 microg/L to 2490 microg/L in the patients studied. In one family bilateral cataracts were diagnosed early in life, whereas in the others cataracts were diagnosed around 40-50 years. The female proband of family 3 presented with a severe iron deficiency anemia, which was unrecognized because of the increased ferritin values. Sequencing of the IRE element of L-ferritin in the probands of the three families identified three different nucleotide substitutions (+32 GAE A, +40 AAE G and +39 CT) in the IRE of L-ferritin. These mutations have already been reported in unrelated subjects of different ethnic origins. INTERPRETATION AND CONCLUSIONS: Our findings are consistent with recurrent mutations associated with HHCS and underline the importance of this syndrome in the differential diagnosis of unexplained hyperferritinemia. In addition, the findings highlight the role played by transferrin saturation in the diagnosis of iron deficiency in these patients.
Adult↗
A Kinetic Study on the Interaction between Tazobactam (A Penicillanic Acid Sulphone Derivative) and Active-Site Serine beta-Lactamases.
The interaction between tazobactam and several chromosome- and plasmid-encoded (TEM, SHV, PSE types) class A and C beta-lactamases was studied by spectrophotometry. Tazobactam behaved as a competitive inhibitor or inactivator able to restore in several cases the efficiency of piperacillin as a partner beta-lactam. A detailed kinetic analysis permitted measurement of the acylation efficiency for some cephalosporinases and broad-spectrum beta-lactamases; the presence of a turn-over of acyl-enzyme complex was also evaluated.
Journal Article↗
Cloning and sequencing of a 16S/23S ribosomal spacer from Haemophilus parainfluenzae reveals an invariant, mosaic-like organisation of sequence blocks.
A 16S/23S ribosomal spacer from a Haemophilus parainfluenzae rrn locus was cloned and sequenced. Analysis of PCR-amplified genomic fragments showed that this region is strongly conserved among unrelated isolates; computer analysis of database homologies showed that the spacer consists of sequence blocks, arranged in a mosaic-like structure, with strong homologies with analogous blocks present in the spacer regions of Haemophilus influenzae, Haemophilus ducreyi and Actinobacillus spp. It also contains a tRNA(Glu) gene, which is highly homologous to tRNA(Glu) genes found in spacers of other species. These data strongly support the hypothesis that recombination events are involved in the organisation of the sequence of the spacer, as a result of horizontal gene transfer.
Blotting, Southern↗
Polymerase chain reaction-mediated typing of microorganisms: tracking dissemination of genes and genomes.
The polymerase chain reaction (PCR) is a powerful molecular biology tool which can be used for the identification of species and strains of diverse microorganisms. By aimed amplification of characteristic genes (i.e., genes encoding ribosomal RNA molecules) and subsequent genetic analysis of amplified fragments, information on microbiological systematics and phylogeny can be obtained in a fast and efficient manner. Similar types of gene identification can be used to verify or detect genes responsible for phenotypic characteristics, whereas modified forms of the PCR enable whole genome searches for genetic polymorphisms among strains of a given species. In medical sciences, both strategies, gene and genome variability analysis by PCR, have an increasing impact on the study of the spread of especially those microbes that are multiply resistant to clinically used antibiotics. In this communication we will exemplify the usefulness of PCR-mediated typing of microorganisms from a clinical perspective while focusing on gene- versus genome-scanning. Special emphasis will be placed on analysis of the dissemination and characteristics of methicillin-resistant Staphylococcus aureus (MRSA) strains and bacterial factors providing resistance to penicillin and other beta-lactam antibiotics. Technical limitations and possibilities for improvement will be discussed.
Bacterial Typing Techniques↗
Repeated epidemics caused by extended-spectrum beta-lactamase-producing Serratia marcescens strains.
An outbreak of Serratia marcescens involving 42 patients admitted to the general intensive care unit of the Hospital of Varese, Italy, occurred from March 1994 to August 1995. The causative strains were resistant to oxyimino-cephalosporins and monobactams due to their production of an extended-spectrum beta-lactamase. Another outbreak caused by Serratia marcescens strains had occurred in the same unit a few months earlier, from February to October 1993, with the strains involved producing a novel TEM-derived extended-spectrum beta-lactamase. In order to verify whether there were any relationships between isolates from the two epidemics, the strains and their enzymes were characterized. Biochemical data and gene amplification experiments showed that the isolates of the second outbreak harbored a non-conjugative plasmid of approximately 48 kb, codifying for the production of an SHV-derived extended-spectrum beta-lactamase with pI 8.2. Restriction fragment length polymorphism analysis of total genomic DNA by pulsed-field gel electrophoresis of Serratia marcescens isolates unambiguously identified two different bacterial clones responsible for the two epidemics. Epidemiological and microbiological investigations demonstrated the long persistence of Serratia marcescens strains and their circulation in other hospital wards, thus suggesting their possible role as a long-term reservoir for further epidemic spread.
Cross Infection↗
The systemic treatment of AIDS-related Kaposi's sarcoma.
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Acquired Immunodeficiency Syndrome↗
Molecular epidemiology and phylogenetic analysis of Haemophilus parainfluenzae from chronic obstructive pulmonary disease exacerbations.
This study was undertaken to characterize serial Haemophilus parainfluenzae strains from epidemiologically unrelated chronic obstructive pulmonary disease (COPD) patients and from healthy carriers. A comprehensive approach was used including different phenotypical and molecular typing methods: biotyping, antibiotyping, conventional ribotyping, pulsed field gel electrophoresis (PFGE) assay, and PCR-ribotyping. Conventional ribotyping and PFGE analysis were confirmed as excellent procedures to differentiate isolates of the same species and biotype. Conversely, in our study, PCR-ribotyping proved to be suitable for taxonomic purposes, unambiguously identifying H. parainfluenzae from H. influenzae but not discriminating strains at the intraspecific level for epidemiological typing. Phylogenetic analysis of restriction fragment length polymorphism (RFLP) data of sequences related to the rrn operon demonstrated that H. parainfluenzae strains associated to COPD are spread among many diverging lineages.
Bacterial Typing Techniques↗
Antibiotic resistance in Pseudomonas aeruginosa: an Italian survey.
In order to assess the current level of resistance to widely used antipseudomonal antibiotics in clinical isolates of Pseudomonas aeruginosa, a national survey was undertaken. Fifteen hospitals throughout Italy participated in the study. The University of Catania tested the antibiotic susceptibility of 1005 consecutive clinically significant P. aeruginosa collected from March to June 1995. Lack of susceptibility, according to NCCLS breakpoints, was at the following rates: meropenem, 9.1%; imipenem, 19.3%; ceftazidime, 13.4%; carbenicillin, 27.3%; piperacillin, 12%; ticarcillin/clavulanic acid, 22.8%; amikacin, 10.6%; and ciprofloxacin, 31.9%. About half of the isolates (44.4%) were not susceptible to at least one of the antibiotics tested.
Amikacin↗
Study on the in-vitro activity of LY333328 against gram-positive cocci.
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Animals↗