Molecular characterization of the coagulase-negative staphylococcal surface flora of premature neonates.

A single point study was conducted to determine which surface sites best represent the density and composition of the coagulase-negative staphylococcal (CNS) colonizing flora in premature neonates. Five different surface sites of six randomly selected neonates hospitalized in a neonatal intensive care unit (NICU) for a month were examined. The individual strains and their clonal organization within CNS species were identified using restriction endonuclease fingerprinting of whole chromosomal DNA and ribosomal RNA genes. Cultures of the scalp, umbilicus, foot, nose and rectum were collected and quantitatively processed. Ten colonies were typed per surface culture. The most dense CNS colonization was noted on the umbilicus (mean 1.2 x 10(4) c.f.u. cm-2), foot (mean 1.6 x 10(3) c.f.u. cm-2) and nose (mean 1.7 x 10(3) c.f.u. cm-2) of NICU neonates. Scalp and rectum were scarcely colonized. Of all the CNS surface isolates, S. epidermidis accounted for 77.7% (219/282) and S. haemolyticus, S. warneri and S. capitis accounted for 20.6% (58/282), 1.4% (4/282) and 0.4% (1/282), respectively. Colonization of each surface site comprised a maximum of five different strains representing four CNS species. Overall, five clones of S. epidermidis, two of S. haemolyticus, one of S. warneri and one of S. capitis were noted among the 282 isolates. The most predominant were two clones of S. epidermidis and one of S. haemolyticus; they accounted for 94% (265/282). Cultures from the foot and scalp represented the most heterogeneous CNS colonization of the five sites examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Typing of coagulase-negative staphylococci by Southern hybridization of chromosomal DNA fingerprints using a ribosomal RNA probe.

Ribotyping consists of restriction endonuclease fingerprinting of ribosomal RNA (rRNA) genes visualized by Southern hybridization with an rRNA probe. This method was developed and compared with restriction endonuclease fingerprinting of chromosomal DNA for typing coagulase-negative staphylococci. Twenty-five American Type Culture Collection reference type strains and 53 clinical isolates were typed. Both methods clearly distinguished all 15 species of coagulase-negative staphylococci and most individual strains within each species. Except in the case of Staphylococcus warneri, ribotyping was most discriminating with the use of ClaI, one of eight endonucleases tested. HpaI and AvaI were more specific than ClaI for discrimination between strains of Staphylococcus warneri. The patterns produced by ribotyping were much simpler and thus easier to interpret than corresponding chromosomal fingerprints. However, ribotyping was slightly less discriminating. It is concluded that ribotyping offers an alternative method for molecular typing of coagulase-negative staphylococci. The application of both methods needs to be further evaluated in the clinical setting.

Evaluation of restriction endonuclease fingerprinting of chromosomal DNA and plasmid profile analysis for characterization of multiresistant coagulase-negative staphylococci in bacteremic neonates.

A procedure was developed for restriction endonuclease fingerprinting (REF) of the chromosomal DNA of coagulase-negative staphylococci. A total of 48 isolates comprising 29 Staphylococcus epidermidis and 19 Staphylococcus haemolyticus isolates from blood and mucocutaneous sites of 15 premature neonates were characterized by REF, plasmid profile (PP) analysis, antimicrobial susceptibility testing, biotyping, and slime production. On the basis of REF analysis of chromosomal DNA, the 48 coagulase-negative staphylococcal isolates were subdivided into 10 subgroups, whereas PP analysis subdivided the strains into 20 distinct subgroups. REF analysis of total DNA (i.e., chromosome plus plasmid) resulted in the same 20 subgroups as were subdivided by PP analysis. The high discriminatory power of PP analysis was associated with the variability of plasmid content in coagulase-negative staphylococcal strains isolated during the outbreak. REF patterns were found to be stable both in vitro and in vivo. Isolates carried from 2 to 10 plasmids that ranged in molecular size from 0.9 to 39.5 megadaltons. Plasmids were disseminated among the coagulase-negative staphylococci, regardless of the genetic relatedness of their chromosomal DNAs. Hence, a lack of correlation existed between the grouping of isolates by REF analysis of chromosomal DNA and the grouping by PP analysis. There were one and two distinct chromosomal patterns among 4 of 4 blood cultures and 15 of 15 mucocutaneous cultures of S. haemolyticus, respectively. In contrast, a higher proportion of distinct chromosomal patterns was found for S. epidermidis in blood cultures (7 of 11 cultures) compared with those identified for isolates in mucocutaneous cultures (6 of 18 cultures). In summary, REF analysis of chromosomal DNA, rather than total DNA, is a useful marker for epidemiological investigations of coagulase-negative staphylococci. PP analysis can also be used to provide additional epidemiological information regarding the most recent genetic events.