In an effort to explore the origin and/or reservoirs of the genetic determinant(s) of methicillin resistance in Staphylococcus aureus, we examined over 200 strains representing 13 different species within the genus Staphylococcus for the presence of the mecA gene, using a DNA probe internal to this gene prepared from a methicillin-resistant strain of S. aureus. Occasional mecA- positive isolates were detected among several staphylococcal species. On the other hand, each one of the 134 isolates of Staphylococcus sciuri, a species considered taxonomically the most primitive among staphylococci and found primarily on rodents and primitive mammals, gave positive reaction with the DNA probe when tested under conditions of high stringency. About two thirds (99) of these isolates, all of which belonged to S. sciuri subspecies "sciuri," as well as 9 of the 11 species carnaticum isolates, showed only marginal, if any, resistance to methicillin (minimal inhibitory concentration of 0.75-6.0 micrograms/ml), while most of the remaining isolates that belonged to the subspecies "rodentius" (13 isolates in all) expressed antibiotic resistance with a heterogeneous phenotype similar to those seen in many methicillin-resistance strains of S. aureus In SmaI digests of chromosomal DNA isolated from such "methicillin-resistant S. aureus-like" strains, the mecA probe hybridized with DNA fragments in the range of 145-180 kb, while in subspecies "sciuri" and carnaticum isolates the mecA hybridizing fragment was located in the SmaI fragment with the highest molecular size (> or = 400 kb). A DNA probe comprising an internal sequence to the regulatory gene mecI from Staphylococcus epidermidis identified the presence of sequences with low degree of homology in isolates of the three S. sciuri subspecies. The mecA-reacting sequences in these bacteria differed from mecA of S. aureus in several respects (e.g., by the absence of a ClaI restriction site from mecA of subspecies "sciuri" and carnaticum, and in some isolates of subspecies "rodentius." The uniform presence of mecA in each one of a large number of S. sciuri strains belonging to distinct ribotypes and macrorestriction patterns and recovered over a 20-year period from a wide variety of animal sources and geographic sites suggests that mecA may be a native genetic element with an as yet unidentified physiologic function in this staphylococcal species.

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.

Three hundred twenty-eight (328) penicillin-resistant Streptococcus pneumoniae isolates collected in 39 states of the United States between October, 1996, and March, 1997, from (mostly adult) patients with respiratory disease were characterized by microbiological, serological, and molecular fingerprinting techniques, including determination of chromosomal macrorestriction pattern with pulsed-field gel electrophoresis (PFGE) and hybridization with DNA probes specific for various antibiotic resistance genes. The overwhelming majority of the isolates were in five serogroups (23, 6, 19, 9, 14). All isolates had penicillin MIC values of at least 2 microg/ml, but the collection also included isolates with MIC values as high as 16 microg/ml. Virtually all isolates (96.6%) were resistant to trimethoprim/sulfamethoxazole (SXT) and many isolates were also resistant to chloramphenicol (43%), tetracycline (55%), and erythromycin (65%). Resistance to levofloxacin was extremely rare. The molecular fingerprinting methods showed that a surprisingly large proportion (167 out of 328, or 50.9%) of the isolates belonged to two international epidemic clones of S. pneumoniae: clone A (127, or 38.7%) with properties indistinguishable from that of the 23F multiresistant "Spanish/USA" clone widely spread in Europe, Asia, Latin America, and South Africa, and clone B (40, or 12.2%) belonging to the "French" serogroup 9/14 clone widely spread in Europe and South America. Virtually all members of clone A were also resistant to chloramphenicol (cat+), tetracycline (tetM+), and SXT, and about 75% were also resistant to erythromycin (mefE+ or ermB+). Close to 30% (39 out of 127) of the clone A isolates expressed anomalous serotypes (primarily serotypes 19 and 14, and nontypable) and most likely represented spontaneous capsular transformants. Most of the 40 isolates (35/40) belonging to clone B expressed serotype 9, with five of the isolates expressing serotypes 14 or 19, or were nontypable. All members of this clone were resistant to penicillin and SXT with only occasional isolates showing resistance to macrolides, tetracycline, and chloramphenicol. The combination of microbiological tests and DNA hybridizations also allowed the identification of unusual strains, for instance, isolates that reacted with the tetM or mefE DNA probes without showing phenotypic antibiotic resistance, an isolate showing phenotypic macrolide resistance without hybridizing with either the ermB or mefE DNA probes, or isolates that hybridized with both of these DNA probes. In addition to clones A and B, another large portion of the S. pneumoniae isolates (112 of 328, or 34.1%) was represented by eight clusters, each with a unique PFGE type. These clusters, together with the clone A and clone B isolates, made up 85% of all the penicillin-resistant isolates identified in this survey in the United States. Both international clones and the unique clusters showed wide geographic dispersal: Clone A was present in 30 of the 39 states and clone B in 18. The data suggest that th...

The rapid spread of multidrug-resistant bacterial pathogens necessitates the search for alternative antibacterial agents. We examined the efficacy of the antibiotic nisin against 56 multidrug-resistant isolates of Streptococcus pneumoniae, 33 Staphylococcus aureus and 29 vancomycin-resistant Enterococcus faecium and Enterococcus faecalis isolates. The test strains represented a large variety of clonal types (as determined by a combination of DNA fingerprints) isolated from a variety of geographic sources, and included some of the major internationally-spread multiresistant epidemic clones of S. pneumoniae and methicillin-resistant S. aureus (MRSA), MRSA strains resistant to over 16 generically distinct antibacterial agents, and enterococcal strains resistant to all currently available chemotherapeutic agents including glycopeptides. In the overwhelming majority of cases, treatment of growing cultures with nisin at 1 mg/L (S. pneumoniae) or 10-20 mg/L (in MRSA and enterococci) caused extensive (10(3)- to 10(4)-fold) loss of viable titre accompanied by various degrees of loss in the optical density of the cultures, which was most extensive in pneumococci (>90%) and least extensive (40-50%) in enterococci. Nevertheless, extensive variation in rates of nisin-induced autolysis was observed in each bacterial species. Serial exposure of a penicillin-susceptible strain of S. pneumoniae to nisin (1 mg/L) in liquid culture resulted in the rapid appearance of stable nisin-resistant mutants in which the MIC increased from 0.4 to 6.4 mg/L and the resistance trait was transferable by genetic transformation.

The Pan American Health Organization (PAHO) has conducted a study of Streptococcus pneumoniae in six Latin-American countries: Argentina, Brazil, Chile, Colombia, Mexico, and Uruguay. Sterile site isolates from children aged < or =5 years showing clinical symptoms of pneumonia (as defined by the clinical criteria of WHO), meningitis, sepsis or bacteremia (without infectious foci), arthritis, and peritonitis were the source of most of the invasive pneumococcal isolates collected between the end of 1993 and 1996 in the six participating countries. Partial characterization of these isolates (antibiotic resistance and serotyping) have already been described (Microbial Drug Resistance 3:(2):131-163, 1997). In the next phase of the study, 326 S. pneumoniae isolates with reduced penicillin susceptibility were transferred to the Laboratory of Microbiology at The Rockefeller University for molecular characterization, and a summary and overview of the findings is described in this article. Some of the most interesting findings were as follows: (1) There was a surprisingly high representation of two internationally spread clones, which made up >80% of the strains with penicillin MIC of 1 microg/ml or higher; most of these isolates were recovered in large cities, supporting the likelihood that the source of these clones is through international travel. (2) The frequency of resistance to trimethoprim/sulfamethoxazole was extremely high (present in 85% of all isolates with decreased penicillin susceptibility). (3) None of these isolates was resistant to ofloxacin, and macrolide resistance was rare (present in 6.4% of the isolates). (4) There was an apparent inverse relationship between level of penicillin resistance and genetic diversity. (5) There were striking differences in the "microbiologic profiles" of the six different Latin-American countries.

Young age and intrafamilial transmission were important risk factors for carriage of both susceptible and resistant S pneumoniae. In contrast, previous cephalosporin use was linked specifically to resistant pneumococcal carriage, which suggests that modifications in antibiotic usage patterns may have salutary effects on antimicrobial resistance. These results extend previous observations in large cities regarding the penetration of multiple-drug-resistant clones of pneumococci into community populations.

Inactivation of the recently identified murMN operon in penicillinresistant strains of Streptococcus pneumoniae was shown already to cause two major effects: elimination of branched-structured muropeptides from the cell wall and complete loss of penicillin resistance. We now show that cells with inactivated murMN also have a third phenotype: an increased susceptibility to lysis when exposed to low concentrations of fosfomycin, D-cycloserine, vancomycin, and nisin, indicating a wide-spectrum hypersensitivity to inhibitors of both early and late stages of cell wall biosynthesis. Mutants of murMN also lysed faster than the parental strain when treated with the detergent deoxycholate. Several different alleles of murM cloned in plasmid pLS578 and introduced into a murM deletion mutant of the penicillin-resistant strain Pen6 were able to reconstitute each one of the three mutant phenotypes: the highly branched cell wall structure, original high level of penicillin resistance, and normal sensitivity to lysis. In a penicillin-susceptible strain the same experiments caused increased concentration of cell wall branched peptides and suppression of sensitivity to antibiotic induced lysis. The observations suggest that the murMN operon plays a key role in the regulation of a stress-response pathway that can be triggered by perturbation of cell wall biosynthesis in S. pneumoniae. S everal penicillin-resistant clones of Streptococcus pneumoniaeproduce abnormal cell walls in which a high proportion of the stem peptide lysine residues carry short (seryl alanine or alanyl alanine) branches (1). The genetic determinants involved with the branching process, the murMN operon, were identified recently (2). Penicillin-resistant isolates with highly branched cell walls were shown to carry murM alleles that contained divergent sequences as compared with the murM genes of penicillin-susceptible pneumococci (3). The two proteins, MurM (407 amino acids) and MurN (411 amino acids), are involved with the addition of the first and second amino acid residues, respectively, to the cell wall precursor stem peptide at the lipid II stage of biosynthesis (4, 5). Inactivation of murMN by insertion-duplication mutagenesis did not interfere with growth of the bacteria but caused two phenotypes: elimination of branched peptides from the cell wall and complete loss of penicillin resistance (2). The purpose of the studies described in this communication was to follow up these earlier findings by the use of a deletion mutant of murM and complementation experiments. The surprising observation made was that inactivation of murM also caused a third phenotype: increased sensitivity of the bacteria to lysis by cell wall inhibitors. Experimental ProceduresStrains and Growth Conditions. All strains and plasmids used in this study are listed in Table 1. S. pneumoniae strains were grown in a casein-based semisynthetic medium (C ϩ Y) at 37°C without aeration as described (11). S. pneumoniae strains containing pLS578 (10) or its derivatives were grown in the presen...

OBJECTIVE: To determine the genetic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from six provincial hospitals in Hungary between 1993 and 1994. METHODS: Molecular fingerprinting methods were used: hybridization with a mecA-specific DNA probe after ClaI restriction; hybridization with a probe for Tn554; and pulsed-field gel electrophoresis after Smal digestion of chromosomal DNA. RESULTS: All strains were resistant to penicillin, oxacillin, erythromycin, gentamicin, tetracycline, imipenem, and cephalosporins, and variably resistant to ofloxacin, clindamycin and tobramycin; all isolates were susceptible to vancomycin. Forty-eight of the 51 isolates carried the mecA gene as determined by Southern hybridization, using a mecA-specific DNA probe, indicating that the methodology used for initial identification may have been in error in three of the cases. Forty-seven of the 48 mecA-positive isolates showed very similar genetic backgrounds as defined by pulsed-field gel electrophoresis (PFGE) patterns after Smal digestion of chromosomal DNAs: a unique PFGE pattern was seen in 32 isolates and minor variants of it in 15 additional isolates. All the 47 isolates carried the same mecA polymorph (Clal type III), as determined by DNA hybridization after Clal digestion of chromosomal DNA. Only one of the MRSA isolates had a completely different PFGE pattern and a novel mecA polymorph. CONCLUSIONS: The findings demonstrate the existence of a unique, epidemic MRSA clone, in both invasive and colonizing strains, which is widely dispersed in Hungarian hospitals hundreds of kilometers apart.