The development of vancomycin resistance in a patient with methicillin-resistant Staphylococcus aureus infection.
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Aged↗
Biomedical subjects
A Tomasz
Publications and source records attributed to A Tomasz.
The Staphylococcus aureus transposon Tn551: complete nucleotide sequence and transcriptional analysis of the expression of the erythromycin resistance gene.
The complete nucleotide sequence of Staphylococcus aureus transposon Tn551 was determined. The 5,266-bp sequence encoded five putative proteins. Comparison of the nucleotide sequence of Tn551 with that of the enterococcal transposon Tn917 showed that the two transposons were 99.8% identical and differed only at 11 positions along the entire sequence. The genetic organization of Tn551 was also identical to that of Tn917. Northern analysis of RNA prepared from a staphylococcal strain bearing Tn551 displayed three erm-associated transcripts that were constitutively produced. Mapping of the 5' ends of the transcripts by primer extension suggested that the constitutive transcription of erm was initiated from a nucleotide located 5 bp downstream of ORF1. A second set of three erythromycin-inducible transcripts was also detected and these showed a pattern similar to that described for Tn917. A simple and rapid method is described for the use of the Tn551 sequence information in sequencing transposon-inactivated staphylococcal genes.
Amino Acid Sequence↗
Role of transposon Tn5482 in the epidemiology of vancomycin-resistant Enterococcus faecium in the pediatric oncology unit of a New York City Hospital.
During a 36-month period between 1993 and 1995 in the Pediatric Oncology Unit of Memorial Sloan Kettering Cancer Center, 74 patients experienced episodes of infection or colonization caused by vancomycin-resistant enterococci (VRE). Characterization of the 74 bacterial isolates by microbiological and molecular techniques (pulsed-field gel electrophoresis and hybridization with DNA probes specific for the vanA and vanB genes and for IS1251) identified 73 Enterococcusfaecium and one Enterococcusfaecalis (vanB) among the primary VRE isolates. Most (69/73) of the E. faecium isolates carried vanA and four isolates, the vanB gene complex. The overwhelming majority (67/69) of the vanA -positive isolates also gave hybridization signal for IS1251, indicating the presence of the newly described conjugative transposon Tn5482. No hybridization with IS1251 was obtained with the four vanB-carrying isolates. About 30% of the vanA-positive strains (23/69) were represented by PFGE subtype variants of a single clone, most isolates of which were recovered during a 4-month period between April to June of 1994. The larger portion of the vanA-carrying VRE represented by close to 70% of the isolates (46/69) belonged to as many as 37 different clonal types, indicating tremendous genetic diversity. Among 67 of the 69 vanA-carrying isolates, the localization of the Tn5482-associated vanA gene complex could be unequivocally identified either on the chromosome (40/69) or in plasmids (27/69). Transconjugants recovered from filter mating experiments using either a chromosomally located or plasmid-borne vanA donor strain and a single vancomycin-susceptible strain of either E. faecium or E. faecalis were analyzed by molecular typing techniques. Seven out of 10 independent transconjugants recovered from the same cross showed extensive differences in PFGE pattern and also in the localization of the vanA hybridizing DNA fragment transferred from the common VRE donor with chromosomally located vanA. The observations suggest that the extensive genetic diversity observed among the clinical isolates of VRE may be generated during conjugation between vancomycin-resistant and -susceptible enterococcal isolates. The observations also suggest that the epidemic spread of VRE in the United States may be linked to the frequent presence of Tn5482 among the American isolates.
Anti-Bacterial Agents↗
Antibiotic resistance as a stress response: complete sequencing of a large number of chromosomal loci in Staphylococcus aureus strain COL that impact on the expression of resistance to methicillin.
Tn551 inactivation has identified several determinants--fem or auxiliary genes--that, in addition to the mecA gene, are also critical for the expression of high-level and homogeneous resistance to methicillin. Genetic and/or biochemical analysis has shown that of the nearly dozen aux mutations described so far most are in genes involved in cell wall synthesis (murE, pbp2, glmM, glnR, femA/B, llm, etc.) or in complex regulatory functions (sigmaB), suggesting that optimal expression of resistance may involve the cooperative functioning of a number of genes in cell wall metabolism as well as stress response. The exact mechanism of these functions is not known. In an attempt to explore this unusual aspect of methicillin resistance more fully, a Tn551 transposon library, constructed in the background of the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL, was screened for all independent insertional mutants in which the level of methicillin resistance of the parental strain (MIC, 1,600 microg/ml) was reduced by at least 15-fold and up to 500-fold. We now describe the sequencing of 21 Tn551-inactivated genes and their vicinities in 23 new auxiliary mutants that have been studied before. Using the inverted polymerase chain reaction (IPCR), we amplified fragments corresponding to the right and left junction of the Tn551 insertions, which were then sequenced by primer walking. The two largest groups of these new auxiliary genes encoded either proteins of unknown functions (6 genes) or showed homology with genes encoding proteins involved with putative sensory/regulatory activities (7 genes: protein kinases, ABC transporters, and a catabolite control protein). Sequencing upstream and downstream allowed the identification of a number of additional open reading frames, some of which may also include functions relevant for the expression of antibiotic resistance.
Bacterial Proteins↗
Carriage of respiratory tract pathogens and molecular epidemiology of Streptococcus pneumoniae colonization in healthy children attending day care centers in Lisbon, Portugal.
In an effort to establish the rate of carriage of antibiotic resistant respiratory pathogens in children attending urban day care centers (DCC) in Portugal, seven DCC in Lisbon were selected for determining the rate of nasopharyngeal colonization of children between the ages of 6 months to 6 years by Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Of the 586 children studied between January and March 1996, 47% carried S. pneumoniae, 72% H. influenzae, and 54% M. catarrhalis. Twenty-four percent of the pneumococci had reduced susceptibility to penicillin, and most of these belonged to serogroups 19, 23, 14, and 6. An additional 19% were fully susceptible to penicillin but showed decreased susceptibility to other antimicrobials. These isolates expressed serogroups 6, 11, 14, 18, 19, and 34. The majority (96%) of M. catarrhalis and 20% of H. influenzae were penicillin resistant due to the production of beta-lactamases. Recent antimicrobial use was associated with carriage of penicillin non-susceptible pneumococci and beta-lactamase producing H. influenzae (p < 0.05). Individual DCC differed substantially from one another in their rates of carriage of antibiotic resistant H. influenzae and S. pneumoniae. Characterization of antibiotic resistant S. pneumoniae isolates by molecular fingerprinting techniques showed that each DCC had a unique microbiological profile, suggesting little, if any, exchange of the resistant microbial flora among them. An exception to this was the presence of isolates belonging to two internationally spread epidemic clones: the multiresistant Spanish/USA clone expressing serotype 23F, and the penicillin and sulfamethoxazole-trimethoprim resistant French/Spanish clone (serotype 14) which were detected in four and three DCC, respectively.
Anti-Bacterial Agents↗
Acquisition of new capsular genes among clinical isolates of antibiotic-resistant Streptococcus pneumoniae.
Antibiotic-resistant clones of Streptococcus pneumoniae are recognizable through a combination of unique molecular, microbiological, and serological properties. In the course of surveillance of epidemic clones of S. pneumoniae, several isolates were identified that shared the clone-specific pulsed-field gel electrophoretic (PFGE) pattern and antibiotype but expressed serotypes atypical for the particular clone. A selected group of isolates belonging to the Spanish/USA clone but expressing serotypes 19, 14, or 3, instead of the expected serotype 23F, were tested using DNA probes for each of the 18 open reading frames (ORFs) of the 23F capsular locus. In no case were there any 23F-specific genes retained, with the possible exception of genes already known to be common to the capsular loci involved. Analysis of the sequence of the capsular locus of a penicillin-resistant serotype 23F isolate from Mexico showed that part of the cpsA gene of this strain, as well as genes cpsQ and cpsR, had high degrees of identity to the sequence of the homologous genes in isolates expressing serotype 19F. The capsular locus of this Mexican strain may have originated from an in vivo capsular switch event in which the original 19F locus was replaced by 23F-specific capsular genes.
Base Sequence↗
Inactivation of the methicillin resistance gene mecA in vancomycin-resistant Staphylococcus aureus.
Acquisition of high-level resistance to vancomycin in the laboratory mutant VM50 (vancomycin MIC increased from 1.5 to 100 microg/ml) was accompanied by the appearance of a heterogeneous phenotype and a virtual loss in methicillin resistance: in most cells of cultures of VM50 the methicillin MIC of the parental strain was reduced from 800 to 1.5 microg/ml with only a subpopulation (10(-5)) retaining methicillin resistance at near the parental level (MIC of 400 microg/ml). Interestingly, the vancomycin MIC of this subpopulation was less (25 microg/ml) than that of VM50 (100 microg/ml). A similar antagonism between methicillin and vancomycin resistance levels was observed upon introduction of an intact mecA into VM50 on a plasmid vector: methicillin resistance of the majority of cells increased from 1.5 to 100 microg/ml while the vancomycin MIC declined from 100 to 12/25 microg/ml. Membrane preparations from mutant VM50 showed no detectable penicillin-binding protein (PBP) 2A by the fluorographic assay. Sequencing of the mecA gene resident in mutant VM50 indicated the presence of a 19-bp duplication between nucleotide residues 280-298, leading to the generation of a stop codon TAA starting at nucleotide position 286.
Anti-Bacterial Agents↗
A high incidence of prophage carriage among natural isolates of Streptococcus pneumoniae.
The majority (591 of 791, or 76%) of Streptococcus pneumoniae clinical isolates examined showed the presence of two or more chromosomal SmaI fragments that hybridized with the lytA-specific DNA probe. Only one of these fragments, frequently having an approximate molecular size of 90 kb, was shown to carry the genetic determinant of the pneumococcal autolysin (N-acetylmuramic acid-L-alanine amidase). Strains carrying multiple copies of lytA homologues included both antibiotic-susceptible and -resistant isolates as well as a number of different serotypes and strains recovered from geographic sites on three continents. Mitomycin C treatment of strains carrying several lytA-hybridizing fragments caused the appearance of extrachromosomal DNA hybridizing to the lytA gene, followed by lysis of the bacteria. Such lysates contained phage particles detectable by electron microscopy. The findings suggest that the lytA-hybridizing fragments in excess of the host lytA represent components of pneumococcal bacteriophages. The high proportion of clinical isolates carrying multiple copies of lytA indicates the widespread occurrence of lysogeny, which may contribute to genetic variation in natural populations of pneumococci.
Bacteriolysis↗
Gradual alterations in cell wall structure and metabolism in vancomycin-resistant mutants of Staphylococcus aureus.
In five vancomycin-resistant laboratory step mutants selected from the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL (MIC of methicillin, 800 microg/ml; MIC of vancomycin, 1.5 microg/ml), the gradually increasing levels of resistance to vancomycin were accompanied by parallel decreases in the levels of methicillin resistance and abnormalities in cell wall metabolism. The latter included a gradual reduction in the proportion of highly cross-linked muropeptide species in peptidoglycan, down-regulation of the production of penicillin-binding protein 2A (PBP2A) and PBP4, and hypersensitivity to beta-lactam antibiotics each with a relatively selective affinity for the various staphylococcal PBPs; the PBP2-specific inhibitor ceftizoxime was particularly effective.
Bacterial Proteins↗
Heterogeneously vancomycin-resistant Staphylococcus epidermidis strain causing recurrent peritonitis in a dialysis patient during vancomycin therapy.
Methicillin-resistant Staphylococcus epidermidis (MRSE) was recovered over a 2-month period from the dialysis fluid of a peritoneal dialysis (PD) patient who experienced recurrent episodes of peritonitis during therapeutic and prophylactic use of vancomycin. Characterization of five consecutive MRSE isolates by molecular and microbiological methods showed that they were representatives of a single strain, had reduced susceptibility to vancomycin, did not react with DNA probes specific for the enterococcal vanA or vanB gene, and showed characteristics reminiscent of the properties of a recently described vancomycin-resistant laboratory mutant of Staphylococcus aureus. Cultures of these MRSE isolates were heterogeneous: they contained-with a frequency of 10(-4) to 10(-5)-bacteria for which vancomycin MICs were high (25 to 50 microg/ml) which could easily be selected to "take over" the cultures by using vancomycin selection in the laboratory. In contrast, the five consecutive MRSE isolates recovered from the PD patient during virtually continuous vancomycin therapy showed no indication for a similar enrichment of more resistant subpopulations, suggesting the existence of an "occult" infection site in the patient (presumably at the catheter exit site) which was not accessible to the antibiotic.
Adult↗
Prophage carriage as a molecular epidemiological marker in Streptococcus pneumoniae.
The great majority of clinical isolates of Streptococcus pneumoniae carry prophages that may be identified through their hybridization with a DNA probe specific for the pneumococcal lytA gene (M. Ramirez, E. Severina, and A. Tomasz, J. Bacteriol. 181:3618-3625, 1999). We now show that the lytA hybridization pattern of chromosomal SmaI digests is stable for a given strain during extensive serial culturing in the laboratory; the pattern is specific for the strain's clonal type, as defined by pulsed-field gel electrophoretis (PFGE) pattern, and variations in PFGE subtypes may be explained by changes in the number and chromosomal localization of this prophage(s). These observations indicate that the lytA hybridization pattern may be used as a molecular epidemiological marker that offers additional resolution of the genetic background of S. pneumoniae strains.
Bacteriophages↗
Molecular characterization of penicillin-resistant Streptococcus pneumoniae isolates from Bulgaria.
As part of an ongoing surveillance program of antibiotic-resistant Streptococcus pneumoniae in Sofia, Bulgaria, 120 penicillin-resistant strains (PRSP) (most of them recovered from children hospitalized with pneumococcal disease) were analyzed by microbiological and molecular methods. Several unique features of this collection are of particular interest. (i) Most isolates (112 of 120) were also resistant to trimethoprim-sulfamethoxazole (SXT) (97 of 120 isolates, or 80%), and over 70% (86 of 120) of the isolates were resistant to at least three antibiotics in addition to penicillin. (ii) Close to 80% of all isolates were represented by large clusters of bacteria, each with a unique serotype, antibiotype, and chromosomal macrorestriction pattern (determined by pulsed-field gel electrophoresis), as well as unique restriction fragmentation length polymorphisms of the penicillin-binding protein genes pbp1a, pbp2x, and pbp2b. (iii) A large proportion (45 of 120, or 38%) of the strains belonged to two internationally spread epidemic clones of S. pneumoniae, the first expressing capsular type 23F and the second expressing serotype 9. (iv) A unique Bulgarian cluster composed of eight serotype 19F isolates was resistant to tetracycline, SXT, cefotaxime, and extremely high levels of penicillin and erythromycin. Nevertheless, this clone did not react with either the erm or the mef DNA probes, and thus the mechanism of macrolide resistance in this group of PRSP remains to be elucidated.
Adolescent↗
Recurrent peritonitis in a patient on dialysis and prophylactic vancomycin.
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Adult↗
Low-level teicoplanin resistance and heteroresistance to vancomycin.
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Anti-Bacterial Agents↗
Accelerated evolution: emergence of multidrug resistant gram-positive bacterial pathogens in the 1990's.
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Drug Resistance, Multiple↗
Capsular transformation of a multidrug-resistant Streptococcus pneumoniae in vivo.
Several multidrug-resistant (MDR) isolates of Streptococcus pneumoniae recovered from a variety of clinical sources and expressing serotypes 9N, 14, 19F, and 3 could not be distinguished from the capsular type 23 Spanish/USA epidemic clone in antibiotype, pulsed-field gel electrophoretic pattern, and restriction fragment length polymorphism types of their penicillin binding protein genes 1A, 2X, and 2B. When tested in a mouse model of virulence, isolates expressing capsular type 3 were lethal for 100% of infected animals within 2 days after intraperitoneal injection of 10(2) cfu/mouse. In contrast, several capsular type 23F isolates belonging to the MDR Spanish/USA clone and recovered from the same site were not lethal, even when injected at the dose of 10(7) cfu/mouse. These data suggest that the pneumococcal isolates expressing serotypes 9N, 14, 19F, and 3 represent products of in vivo capsular transformation events in which the MDR epidemic capsular type 23F Spanish/USA clone was the recipient.
Animals↗
Molecular epidemiology of methicillin-resistant Staphylococcus aureus in 12 New York hospitals. MRSA Collaborative Study Group.
Consecutive single-patient methicillin-resistant Staphylococcus aureus (MRSA) isolates (270) from 12 hospitals (8217 beds) in metropolitan New York City were collected during May 1996. In 11 of 12 hospitals, MRSA was most frequent in the general medical services. DNA typing ("fingerprinting") revealed that mecA:Tn554:PFGE (pulsed-field gel electrophoresis) type I:A:A accounted for 113 (42%) of 270 isolates, was detected in all hospitals, and was the predominant clone in 9. Thirteen of 15 I:E:F isolates were from 1 hospital, and the remaining 2 were from another hospital of the same health system. Type V:NH:E was isolated from 22 (79%) of the 28 patients with AIDS, including 8 of 9 patients from an additional hospital. Subtype V:NH:E2 was recovered from 11 patients, 9 of whom had AIDS, including all 5 AIDS patients from one floor of a nursing home affiliated with a third hospital. By using both mecA:Tn554 probes and PFGE, MRSA clusters and outbreaks may be detected and provide a rationale for appropriate infection control intervention.
Adolescent↗
Why have group A streptococci remained susceptible to penicillin? Report on a symposium.
In spite of 50 years of extensive use of penicillin, group A streptococci remain exquisitely susceptible to this antibiotic. This observation that continuing susceptibility has occurred despite the development of resistance to other antimicrobial agents prompted a day-long meeting at Rockefeller University (New York) in October 1996. Among the most likely explanations for this remarkable state of continued susceptibility to penicillin are that beta-lactamase may not be expressed or may be toxic to the organism and/or that low-affinity penicillin-binding proteins either are not expressed or render organisms nonviable. Other potential explanations are that circumstances favorable for the development of resistance have not yet occurred and/or that there are inefficient mechanisms for or barriers to genetic transfer. Recommended future actions include (1) additional laboratory investigations of gene transfer, penicillin-binding proteins, virulence factors, and homeologous recombination and mismatch repair; (2) increased surveillance for the development of penicillin resistance; (3) application of bioinformatics to analyze streptococcal genome sequences; and (4) development of vaccines and novel antimicrobial agents. Thus far the susceptibility of group A streptococci to penicillin has not been a major clinical or epidemiological problem. A similar observation, however, could have been made decades ago about Streptococcus pneumoniae. It is therefore vital for the scientific community to closely examine why penicillin has remained uniformly highly active against group A streptococci in order to maintain this desirable state.
Humans↗