Community-acquired methicillin-resistant Staphylococcus aureus colonization in healthy children attending an outpatient pediatric clinic.

BACKGROUND: We previously showed that children attending an inner city pediatric emergency department were sometimes asymptomatically colonized with clindamycin-susceptible community-acquired methicillin-resistant Staphylococcus aureus (MRSA) and borderline methicillin-resistant S. aureus (BRSA) as well. We wished to ascertain whether healthy children attending an outpatient clinic were colonized with these organisms. Therefore to estimate the prevalence of community-acquired MRSA and BRSA nasal colonization in a well child population, we cultured children attending an inner city pediatric outpatient clinic. STUDY DESIGN: This was a prospective cross-sectional study conducted from January to August, 1999, at a primary care outpatient facility at the University of Chicago. The target population was 500 healthy children < or = 16 years of age who attended this facility to receive well child care. RESULTS: One hundred twenty-two (24.4%) children were colonized with S. aureus. Three of the 122 (2.5%) S. aureus isolates were MRSA; they came from children who lacked predisposing risk factors and were susceptible to clindamycin, gentamicin, trimethoprim-sulfamethoxazole, rifampin and ciprofloxacin. Two (1.6%) additional S. aureus isolates were BRSA; both children had predisposing risk factors for MRSA colonization. The mecA gene was present in the 3 MRSA isolates and absent in both BRSA isolates. CONCLUSIONS: These data document that a reservoir of asymptomatic MRSA colonization exists among healthy children who lack traditional risk factors for MRSA infections.

Development of vancomycin and lysostaphin resistance in a methicillin-resistant Staphylococcus aureus isolate.

Glycopeptide resistance in Staphylococcus aureus is poorly understood. The diversity of change documented in cell walls of clinical glycopeptide-intermediate S. aureus (GISA) isolates is evidence that a single genetic or biochemical change cannot account for resistance in all isolates described to date. Therefore, identification of new GISA clinical isolates provides an opportunity to gain insight into the range of adaptive strategies employed by staphylococci to survive in the presence of glycopeptides. In April 1999, a GISA isolate was obtained from the blood of a 63-year-old dialysis patient in Illinois. This isolate was one of six clonally identical MRSA isolates (A-F) serially obtained from the blood of this patient who was receiving vancomycin therapy. All isolates were resistant to oxacillin (MIC > 256 mg/L). The initial isolate had an MIC of vancomycin of 1 mg/L. However, the presence of a subpopulation that could grow in the presence of 5 mg/L of vancomycin indicated that this isolate was predisposed to the acquisition of the GISA phenotype (MIC of vancomycin 10-12 mg/L), which occurred 13 days later, associated with an increased MIC of the endopeptidase lysostaphin and slightly increased cell wall thickness. The first and last isolates in the series, A and F, resisted killing when incubated in vancomycin 2 mg/L, resisted autolysis when incubated in Triton X-100 and had a decreased expression of a c. 116 kDa autolytic band, properties that were different from glycopeptide-susceptible control isolates. Lysostaphin resistance was not accompanied by alterations in the peptidoglycan pentaglycine cross-bridge or a decrease in oxacillin MIC. These data, when taken together with the demonstration of increased cross-linking in isolate F compared with isolate A, demonstrate that vancomycin resistance in these isolates probably occurred by a mechanism different from that of other GISA isolates described to date.

A spectrum of changes occurs in peptidoglycan composition of glycopeptide-intermediate clinical Staphylococcus aureus isolates.

The mechanism of glycopeptide resistance in Staphylococcus aureus is not known with certainty. Because the target of vancomycin is the D-Ala-D-Ala terminus of the stem peptide of the peptidoglycan precursor, by subjecting muropeptides to reversed-phase high-performance liquid chromatography, we investigated peptidoglycan obtained from glycopeptide-intermediate S. aureus (GISA) isolates for changes in composition and evaluated whether any peptidoglycan structural change was a consistent feature of clinical GISA isolates. GISA isolates Mu50 and Mu3 from Japan had the large glutamate-containing monomeric peak demonstrated previously, although strain H1, a vancomycin-susceptible MRSA isolate from Japan that was clonally related to Mu3 and Mu50, and a femC mutant that we studied, did also. For the U.S. GISA isolates, strain NJ had a large monomeric peak with a retention time identical to that described for the glutamate-containing monomer in strains H1, Mu3, and Mu50. However, a much smaller corresponding peak was seen in GISA MI, and this peak was absent from both GISA PC and a recent GISA isolate obtained from an adult patient in Illinois (strain IL). These data suggest that a uniform alteration in peptidoglycan composition cannot be discerned among the GISA isolates and indicate that a single genetic or biochemical change is unlikely to account for the glycopeptide resistance phenotype in the clinical GISA isolates observed to date. Furthermore, a large monomeric glutamate-containing peak is not sufficient to confer the resistance phenotype.

Structural and topological differences between a glycopeptide-intermediate clinical strain and glycopeptide-susceptible strains of Staphylococcus aureus revealed by atomic force microscopy.

Novel cell surface topography was revealed on cocci from a glycopeptide-intermediate Staphylococcus aureus (GISA) clinical strain by using atomic force microscopy. The GISA isolate and its revertant had two parallel circumferential surface rings. One equatorial surface ring was observed in control strains. In vancomycin-susceptible strains, additional rings were formed in the presence of vancomycin. Ring depth measurements also revealed striking differences between the GISA strain and susceptible strains grown with or without vancomycin.

Reversion of the glycopeptide resistance phenotype in Staphylococcus aureus clinical isolates.

The recent identification of glycopeptide intermediate-resistant Staphylococcus aureus (GISA) clinical isolates has provided an opportunity to assess the stability of the glycopeptide resistance phenotype by nonselective serial passage and to evaluate reversion-associated cell surface changes. Three GISA isolates from the United States (MIC of vancomycin = 8 microg/ml) and two from Japan (MICs of vancomycin = 8 and 2 microg/ml) were passaged daily on nutrient agar with or without vancomycin supplementation. After 15 days of passage on nonselective medium, vancomycin- and teicoplanin-susceptible revertants were obtained from each GISA isolate as determined by broth dilution MIC. Revertant isolates were compared with parent isolates for changes in vancomycin heteroresistance, capsule production, hemolysis phenotype, coagulase activity, and lysostaphin susceptibility. Several revertants lost the subpopulations with intermediate vancomycin resistance, whereas two revertants maintained them. Furthermore, although all of the parent GISA isolates produced capsule type 5 (CP5), all but one revertant tested no longer produced CP5. In contrast, passage on medium containing vancomycin yielded isolates that were still intermediately resistant to vancomycin, had no decrease in the MIC of teicoplanin, and produced detectable CP5. No consistent changes in the revertants in hemolysis phenotype, lysostaphin susceptibility, or coagulase activities were discerned. These data indicate that the vancomycin resistance phenotype is unstable in clinical GISA isolates. Reversion of the vancomycin resistance phenotype might explain the difficulty in isolating vancomycin-resistant clinical isolates from the blood of patients who fail vancomycin therapy and, possibly, may account for some of the difficulties in identifying GISA isolates in the clinical laboratory.

Current trends in community-acquired methicillin-resistant Staphylococcus aureus at a tertiary care pediatric facility.

BACKGROUND: The prevalence of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infections increased at the University of Chicago Children's Hospital (UCCH) from 10 per 100,000 admissions from 1988 to 1990 to 259 per 100,000 admissions from 1993 to 1995. Because this increase may have represented a one time occurrence or a limited disease outbreak, we updated our previous observations at UCCH in 1998 and 1999 to see whether this trend had continued. DESIGN: Prospective observational study. RESULTS: Twenty-three hospitalized children had an MRSA isolate during the 1-year study period. Ten were community-acquired, equally distributed between children with predisposing risk factors and those without. The overall prevalence of community-acquired MRSA was 208 per 100,000 admissions. Seven of the 10 community-acquired MRSA isolates were susceptible to clindamycin. Skin and soft tissue infections predominated among the children with a community-acquired MRSA isolate. Pulsed field gel electrophoresis of the 10 community-acquired MRSA isolates revealed 8 distinct patterns; these data suggest that multiple clones were circulating at UCCH. CONCLUSION: MRSA are no longer confined to children with established risk factors. The prevalence of community-acquired MRSA among children without identified risk factors is high in our institution.

Methicillin-resistant and borderline methicillin-resistant asymptomatic Staphylococcus aureus colonization in children without identifiable risk factors.

BACKGROUND: The recent evolution in the epidemiology of methicillin-resistant asymptomatic Staphylococcus aureus (MRSA) infections in children, whereby children without traditional risk factors for MRSA have been hospitalized in increasing numbers, prompted us to establish whether a parallel increase in "asymptomatic" MRSA colonization had occurred. METHODS: We cultured the nares and perineum of 500 children attending our Pediatric Emergency Department. RESULTS: One hundred thirty-two (26.4%) of these children were colonized with S. aureus. Eleven (8.3%) of the S. aureus isolates were MRSA; 4 (36.4%) of the 11 subjects colonized with MRSA had no risk factors. Seven (5.3%) of the 132 S. aureus isolates were borderline methicillin-resistant S. aureus (BRSA); 5 (71.4%) of the 7 subjects colonized with BRSA had no MRSA risk factors. CONCLUSIONS: These findings indicate that MRSA and BRSA isolates are circulating in the community and that MRSA isolates are no longer confined to children with frequent contact with a health care environment.

Activated cell-wall synthesis is associated with vancomycin resistance in methicillin-resistant Staphylococcus aureus clinical strains Mu3 and Mu50.

We have previously reported methicillin-resistant Staphylococcus aureus clinical strains, Mu50 and Mu3, representing two categories of vancomycin resistance: Mu50 representing vancomycin-resistant S. aureus (VRSA) with MICs > or = 8 mg/L, and Mu3 representing hetero-VRSA with MICs < or = 4 mg/L using standard MIC determination methods. The mechanisms of vancomycin resistance in these strains were investigated. These strains did not carry the enterococcal vancomycin-resistance genes, vanA, vanB, or vanC1-3, as tested by PCR using specific primers. However, both strains produced three to five times the amount of penicillin-binding proteins (PBPs) 2 and 2' when compared with vancomycin-susceptible S. aureus control strains with or without methicillin resistance; the amounts of PBP2 produced in Mu3 and Mu50 were comparable to those in the vancomycin-resistant S. aureus mutant strains selected in vitro. Incorporation of 14C-labelled Nacetyl-glucosamine into the cell was three to 20 times increased in Mu50 and Mu3, and release of the radioactive cell wall material was increased in Mu3 (and also in Mu50, though to a lesser extent), compared with control strains. The amounts of intracellular murein monomer precursor in these strains were three to eight times greater than those found in control strains. Transmission electron microscopy showed a doubling in the cell wall thickness in Mu50 compared with the control strains. Mu3 did not show obvious cell wall thickening. These data indicate that activated synthesis and an increased rate of cell wall turnover are common features of Mu3 and Mu50 and may be the prerequisite for the expression of vancomycin resistance in S. aureus.

Community-acquired methicillin-resistant Staphylococcus aureus in children with no identified predisposing risk.

CONTEXT: Community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infections in children have occurred primarily in individuals with recognized predisposing risks. Community-acquired MRSA infections in the absence of identified risk factors have been reported infrequently. OBJECTIVES: To determine whether community-acquired MRSA infections in children with no identified predisposing risks are increasing and to define the spectrum of disease associated with MRSA isolation. DESIGN: Retrospective review of medical records. PATIENTS: Hospitalized children with S aureus isolated between August 1988 and July 1990 (1988-1990) and between August 1993 and July 1995 (1993-1995). SETTING: The University of Chicago Children's Hospital. MAIN OUTCOME MEASURES: Prevalence of community-acquired MRSA over time, infecting vs colonizing isolates, and risk factors for disease. RESULTS: The number of children hospitalized with community-acquired MRSA disease increased from 8 in 1988-1990 to 35 in 1993-1995. Moreover, the prevalence of community-acquired MRSA without identified risk increased from 10 per 100000 admissions in 1988-1990 to 259 per 100000 admissions in 1993-1995 (P<.001), and a greater proportion of isolates produced clinical infection. The clinical syndromes associated with MRSA in children without identified risk were similar to those associated with community-acquired methicillin-susceptible S aureus. Notably, 7 (70%) of 10 community-acquired MRSA isolates obtained from children with an identified risk were nonsusceptible to at least 2 drugs, compared with only 6 (24%) of 25 isolates obtained from children without an identified risk (P=.02). CONCLUSIONS: These findings demonstrate that the prevalence of community-acquired MRSA among children without identified risk factors is increasing.

Cloning of the Staphylococcus aureus ddh gene encoding NAD+-dependent D-lactate dehydrogenase and insertional inactivation in a glycopeptide-resistant isolate.

The mechanism of low-level glycopeptide resistance among staphylococci is not known. A cytoplasmic protein, provisionally called Ddh (W. M. Milewski, S. Boyle-Vavra, B. Moreira, C. C. Ebert, and R. S. Daum, Antimicrob. Agents Chemother. 40:166-172, 1996), and the RNA transcript that contains the ddh gene, which encodes Ddh, are present in increased amounts in a vancomycin-resistant isolate, 523k, compared with the susceptible parent isolate, 523. Sequence analysis had previously revealed that Ddh is related to NAD+-dependent D-lactate dehydrogenase (D-nLDH) and VanH. This latter protein is essential for high-level glycopeptide resistance in Enterococcus faecium and Enterococcus faecalis by synthesizing the D-lactate needed for biosynthesis of D-lactate-terminating peptidoglycan precursors with low affinity for vancomycin. We now provide the direct evidence that the ddh gene product is Staphylococcus aureus D-nLDH and hereafter refer to the protein as D-nLDH. However, overproduction of this protein in isolate 523k did not result in production of D-lactate-containing peptidoglycan precursors, and susceptibility testing of ddh mutants of 523k demonstrated that S. aureus D-nLDH is not necessary for glycopeptide resistance in this isolate. We conclude that the mechanism of glycopeptide resistance in this isolate is distinct from that in enterococci.

Increased production of penicillin-binding protein 2, increased detection of other penicillin-binding proteins, and decreased coagulase activity associated with glycopeptide resistance in Staphylococcus aureus.

The mechanism of glycopeptide resistance in the genus Staphylococcus is unknown. Since these antimicrobial compounds act by binding the peptidoglycan precursor terminus, the target of transglycosylase and transpeptidase enzymes, it was hypothesized that resistance might be mediated in Staphylococcus aureus by increased production or activity of these enzymes, commonly called penicillin-binding proteins (PBPs). To evaluate this possibility, glycopeptide-resistant mutants were prepared by passage of several clinical isolates of this species in nutrient broth containing successively increasing concentrations of the glycopeptide vancomycin or teicoplanin. Decreased coagulase activity and increased resistance to lysostaphin were uniformly present in the vancomycin-resistant mutants. Peptidoglycan cross-linking increased in one resistant isolate and decreased in two resistant isolates. The amounts of radioactive penicillin that bound to each PBP in susceptible and resistant strains were compared; PBP2 production was also evaluated by Western blotting. Increased penicillin labeling and production of PBP2 were found in all resistant derivatives selected by either vancomycin or teicoplanin. Moreover, the increase in PBP2 penicillin labeling occurred early in a series of vancomycin-selected derivatives and was strongly correlated (r > 0.9) with the increase in vancomycin and teicoplanin MIC. An increase in penicillin labeling also occurred, variably, in PBP1, PBP3, and/or PBP4. These data demonstrate a strong correlation between resistance to glycopeptides and increased PBP activity and/or production in S. aureus. Such an increase could allow PBPs to better compete with glycopeptides for the peptidoglycan precursor.

Antimicrobial resistance in staphylococci. Epidemiology, molecular mechanisms, and clinical relevance.

Staphylococcal infections continue to pose important clinical problems in children and adults. Antibiotic resistance among the staphylococci has rendered therapy of these infections a therapeutic challenge. Despite early, uniform susceptibility to penicillin, staphylococci acquired a gene elaborating beta-lactamase that rendered penicillin inactive and that is borne by nearly all clinical isolates. "Penicillinase-resistant beta-lactams," such as methicillin, were introduced in the early 1960s, but resistance to them has become an increasing concern. The mechanism of the so-called "methicillin resistance" is complex. Moreover, once confined to the ecology of hospitals and other institutions, a recent increase in community-acquired methicillin-resistant S. aureus infections has been observed. Glycopeptides, until now the only uniformly reliable therapeutic modality, have been increasingly used for therapy of staphylococcal infections. The recent recognition of clinical isolates with reduced susceptibility to glycopeptides is of concern.

Uptake-sequence-independent DNA transformation exists in Neisseria gonorrhoeae.

A DNA transformation dose-response curve of piliated (P+) gonococci with the use of cloned DNA containing a pilE2-cat fusion showed saturation at high and low levels of transforming DNA. At low DNA concentrations, transformation of the P+ strain MS11-A was effectively inhibited by a 1000-fold molar excess of the gonococcal transformation uptake sequence (GCUS). The same molar excess of the GCUS did not inhibit transformation of MS11-A at high DNA concentrations. In MS11-B2, a nonpiliated (P-), pilin-nonproducing, isogenic variant of MS11-A, the GCUS did not inhibit transformation at any level of transforming DNA. These data suggest that two mechanisms of transformation exist in P+ cells: one which utilizes the GCUS and one which does not. In MS11-B2 P- cells, no evidence was found for the presence of the GCUS-dependent mechanism, suggesting that transformation in this background occurs solely by the GCUS-independent mechanism.

Overproduction of a 37-kilodalton cytoplasmic protein homologous to NAD+-linked D-lactate dehydrogenase associated with vancomycin resistance in Staphylococcus aureus.

We previously reported the isolation of a laboratory-derived Staphylococcus aureus mutant, 523k, that has constitutive low-level resistance to vancomycin (MIC = 5 micrograms/ml) and teicoplanin (MIC = 5 micrograms/ml) and elaborates a ca. 39-kDa cytoplasmic protein that was not detected in the parent strain 523 (MIC = 1 micrograms/ml). We have now detected the protein in strain 523 by immunoblotting with antiserum raised against the protein. Consistent with our initial observations, densitometric analysis of the immunoblots revealed an increased production of the protein in 523k compared with that of the susceptible parent 523. The 5' region of the gene encoding the protein of interest was identified by nucleotide sequencing a PCR product amplified from the genome of 523k with degenerate primers designed to encode the amino acid sequence of proteolytic peptides obtained from the protein. The remainder of the gene was identified by library screening, PCR, and nucleotide sequencing. The gene encodes a 36.7-kDa protein with homology to a family of bacterial NAD+-dependent, D-specific 2-hydroxyacid dehydrogenases which includes both D-lactate dehydrogenase and the enterococcal vancomycin resistance protein VanH and is therefore designated ddh. Increased production of the product of ddh, Ddh, was associated with increased D-lactate dehydrogenase activity in 523k, a finding which suggested that Ddh is likely to be the D-lactate dehydrogenase previously identified in S. aureus. The increased D-lactate dehydrogenase activity in strain 523k and the structural similarities among Ddh, D-lactate dehydrogenase, and VanH suggest that overproduction of Ddh might play a role in vancomycin resistance in this strain.

Shuttle mutagenesis: a mini-transposon for producing PhoA fusions with exported proteins in Neisseria gonorrhoeae.

Shuttle mutagenesis is a method for producing stable mini-transposon (mTn) insertions into the genome of Neisseria gonorrhoeae (gonococcus, Gc) and other microbes. Using an mTn3 derivative, we have produced an mTn (mTnCmPhoA) which contains a phoA' gene lacking its N-terminal signal sequence useful for isolating genes which encode exported proteins. mTnCmPhoA was characterized in Gc and Escherichia coli using a cloned target containing the Gc genes, opaE1, pilA and pilB. PhoA+ Gc containing pilB::mTnCmPhoA insertions confirm that PilB is an exported protein in Gc. This system will be useful for isolating and characterizing extracytoplasmic virulence factors from Gc and other bacterial pathogens.

Transcriptional control of gonococcal pilE expression: involvement of an alternate sigma factor.

The pilE gene encoding Neisseria gonorrhoeae (Gc) pilin contains two putative promoter sequences 5' to the transcription start point (tsp), a Pribnow box and an RpoN-binding consensus sequence. Sequence analysis shows that the nucleotide (nt) sequence of the pilE promoter region is completely conserved among eight different Gc isolates. Using a pilE::lacZ transcriptional fusion, we demonstrate that the RpoN sigma factor can function in Escherichia coli to increase pilE transcription when the NifA activator from Klebsiella is present in trans. In addition, over-production of the native pilin protein using RpoN and NifA is lethal to E. coli. Finally, we show that the RpoN sigma factor decreases the basal expression of pilE when an activator is not present. These results suggest that, in Gc, pilE transcription may be regulated by RpoN in conjunction with an activator and that sigma 70 can also act to direct transcription of pilE.

Shuttle mutagenesis: two mini-transposons for gene mapping and for lacZ transcriptional fusions in Neisseria gonorrhoeae.

Shuttle mutagenesis is a system we developed for producing stable transposon insertions in Saccharomyces cerevisiae [Seifert et al., Proc. Natl. Acad. Sci. USA 83 (1986) 735-739; Hoekstra et al., Methods Enzymol. 194 (1991) 329-342] and Neisseria gonorrhoeae (Gc) [Seifert et al., J. Bacteriol. 172 (1990) 40-46] by transposition in Escherichia coli and transformation into yeast or Gc. In developing the system for use in Gc, a series of mini-transposons (mTn) were derived from mTn3 which confer resistance to chloramphenicol in Gc (mTnCm) (Seifert et al., 1990). Herein, we describe the creation of two mTnCm derivatives for use in Gc. One of these transposons, mTnCmNS, contains the infrequently occurring NheI and SpeI restriction sites to localize genes on the gonococcal macro-restriction map which was recently developed using these restriction sites [Bihlmaier et al., Mol. Microbiol. 5 (1991) 2529-2539; Dempsey et al., J. Bacteriol. 173 (1991) 5476-5486]. The mTnCmLac was developed to generate lacZ transcriptional fusions using transposition. It contains at its end a promoterless lacZ gene which is expressed once the element has transposed downstream from a promoter in a cloned gene. In adapting the use of mTnCmLac to the shuttle mutagenesis system, we have identified some factors which affect the transformation of Gc using cloned chromosomal fragments containing the large heterologous insertion, mTnCmLac. Using mTnCmLac, we have created Gc variants containing a pilE::mTnCmLac fusion to determine that pilE transcription in Gc is not auto-regulated.

Eosinophils and mast cell homogeneity of the guinea pig eyelid skin, conjunctiva, and ileum.

Mast cell heterogeneity has been described on the basis of differential staining reactions, light microscopic morphology, anatomic location, degranulation after polyamines, biochemical contents, growth requirements, and reactions to lymphokines. We have demonstrated typical "connective-tissue mast cells" by using anatomic criteria, histological staining reactions, electron microscopy, and reaction to compound 48/80 in the guinea pig conjunctiva, eyelid skin, and ileum. A second, much larger population of cells in the ileal mucosa and the conjunctiva, and rarely in the eyelid skin stained reddish-blue with acid toluidine blue in tissue fixed in ethanol-acetate-lead subacetate (BLA) and with alkaline Giemsa in formaldehyde-fixed tissue, did not stain with ethanolic or acid toluidine blue in formaldehyde-fixed tissue or with alkaline Giemsa in BLA-fixed tissue, and did not degranulate after 48/80 treatment. These are features of the rat intestinal "mucosal mast cells"; however, ultrastructural and light microscopic studies with the orcein Giemsa stain demonstrated these cells in the guinea pig to be eosinophils. Tissue culture, biochemical, and immunological studies indicate the existence of a second type of mast cell (bone-marrow-derived mast cell), ultrastructurally almost indistinguishable from the connective tissue mast cell. Our studies demonstrate only one mast cell type in the guinea pig and support the contention that other forms of mast cells are immature forms or variants of the connective-tissue mast cell.