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Certain Tn916 insertions in the chromosome of an M1-type, nonmucoid Streptococcus pyogenes isolate (MGAS166) were previously shown to result in stable mucoidy with increased expression of the capsular synthetic genes. The transposon insertions in these strains are directly upstream of an apparent operon encoding a two-component regulatory system, designated csrR-csrS. Compared with MGAS166, these mucoid mutants are more hemolytic and cause significantly more tissue damage in a murine model of skin infection. To extend these observations, we constructed an in-frame deletion in the gene encoding the response regulator, csrR, and we evaluated the expression of other known S. pyogenes virulence factors. We discovered that csrR mutants have enhanced transcription of sagA, a gene associated with streptolysin S (SLS) and speB, the gene encoding pyrogenic exotoxin B (SpeB). The mutants also express substantially higher SLS activity and SpeB antigen in late-exponential-phase cultures. There is no change in expression of emm, scpA, sic, or cpa (genes encoding other S. pyogenes virulence factors). CsrR- strains but not the wild-type parental strain produce necrotizing lesions in a mouse model of subcutaneous infection. A double mutant with deletions in both csrR and the capsular synthesis genes caused fewer and smaller necrotic skin lesions than the csrR mutants. However, this nonmucoid csrR strain was more likely than the wild type to yield necrotic lesions, suggesting that mucoidy contributes to virulence in this model of infection but that there are other csrR-regulated factors involved in the production of necrotic lesions.

We present a 54-year-old male heart transplant recipient who developed mediastinitis caused by Klebsiella oxytoca and Veillonella species. Culture of the donor's bronchus also grew K. oxytoca and a Veillonella species. Pulsed-field gel electrophoresis revealed that the K. oxytoca isolates had identical banding patterns. This case illustrates transmission of pathogenic bacteria via a contaminated organ.

For Staphylococcus aureus, stepwise mutations result in high-level quinolone resistance. Methicillin-resistant and -susceptible quinolone-resistant, first-step mutants generated in vitro were obtained and found to be no different than those recovered from murine abscesses. Approximately 10% of all first-step mutants were resistant to ethidium bromide, and selected strains had mutations that mapped to flqB. NorA-mediated resistance among first-step mutants may be more prevalent than previously reported.

Streptolysin S (SLS) is a potent cytolytic toxin produced by nearly all group A streptococci (GAS). SLS-deficient Tn916 insertional mutants were generated from two clinical isolates of GAS, MGAS166s and T18Ps (M serotypes 1 and 18, respectively), by transposon mutagenesis using Tn916 donor strain Enterococcus faecalis CG110. Representative nonhemolytic transconjugants SBNH5 and SB30-2 each harbored a single Tn916 insertion in identical loci. The insertion in SBNH5 was located in the promoter region of an open reading frame, designated sagA, rendering it transcriptionally inactive. Protease, streptolysin O, and DNase activities and the production of M protein remained the same in the nonhemolytic mutants and the wild-type strains, as did the growth rates and exoprotein profiles. Transconjugants were evaluated in an established murine model by injecting the organisms subcutaneously and monitoring the mice for alterations in weight and the development of necrotic lesions. Animals infected with SBNH5, compared to those infected with MGAS166s, gained weight during the first 24 h (+1.15 versus -1.16 g; P < 0.05) and had fewer necrotic lesions (0 versus 7; P = 0.0007). Animals infected with SB30-2, compared to those infected with T18Ps, also gained weight within the first 24 h (+0.54 versus -0.66 g; P < 0.05) and produced fewer necrotic lesions (1 versus 8; P = 0.001). Revertants of the mutants in which Tn916 had been excised regained the hemolytic phenotype and the virulence profile of the wild-type strains. This study demonstrates that SLS-deficient mutants of GAS, belonging to different M serotypes and containing identical Tn916 mutations, are markedly less virulent than their isogenic parents.

Contamination between specimens within clinical microbiology laboratories may be responsible for spurious outbreaks of mycobacterial infections. We report the case of a patient who had culture-negative endocarditis and whose cardiac tissue obtained at surgery yielded Mycobacterium avium complex (MAC). Epidemiologic investigation suggested cross contamination probably occurred during processing of the sputum specimens of a patient with pulmonary MAC disease and the cardiac samples from our patient; molecular strain typing showed the isolates from both patients to be identical. When mycobacterial infection rates increase or an unexpected case of mycobacterial infection occurs, the clinician should be alert to the possibility of cross contamination in the laboratory as a possible explanation.

The morbidity and mortality of Staphylococcus aureus infections remain high despite antibiotic therapy. To investigate further the observation that penicillins increase the hemolytic activity of staphylococcal cultures, 37 strains were grown in broth with and without subinhibitory nafcillin. Nafcillin stimulated hemolytic activity in nafcillin-susceptible and -resistant isolates. Sterile broth filtrates of nafcillin-associated cultures injected intraperitoneally in mice were more rapidly lethal than filtrates of the same strain grown without nafcillin. Lethality was neutralized by anti-alpha-toxin antisera. DNA-RNA hybridization revealed a nafcillin-associated increase in alpha-toxin mRNA during the postexponential growth phase after the activation of agr. Isolates grown in slightly inhibitory nafcillin concentrations had more alpha-toxin mRNA than did nafcillin-free cultures, whereas agr RNAIII levels were comparable. This suggests that nafcillin-induced alpha-toxin production is not entirely attributable to agr. A supplemental regulatory mechanism may be involved.

Staphylococcus aureus mutants resistant to ciprofloxacin were selected both in vitro and in vivo. In-vitro selection was achieved by incubating two strains of S. aureus (MICs of ciprofloxacin of 0.5 and 4 mg/L respectively) in the presence of ciprofloxacin in concentrations equivalent to 1/2 x MIC for 24 h and isolating the mutants on agar containing the quinolone at 1, 2 or 5 x MIC. Stably-resistant mutants of both strains were isolated, although the frequency of mutation of the susceptible strain was higher than that of the resistant strain. A murine subcutaneous abscess model was used for in-vivo selection. Mice which had been infected with a ciprofloxacin-susceptible strain of S. aureus were treated for 24 h with ciprofloxacin in a dosage which yielded concentrations in the abscess cavity equivalent to 1/2 or 1 x MIC for the pathogen. Additional groups of infected mice received ciprofloxacin for varying periods in a dosage which produced concentrations at the site of infection equivalent to 1/2 x MIC. Stably-resistant mutants were isolated from the abscesses, the number of mice from which mutants were isolated and the mutational frequency being inversely proportional to the dosage of ciprofloxacin administered and the duration of treatment. The results of this study confirm that, in the treatment of patients with infections caused by S. aureus, the dosage of ciprofloxacin should be adequate to ensure inhibitory concentrations at the site of infection.

An uncommon dihydrofolate reductase (DHFR), type IIIc, was coded for by Shigella sonnei that harbors plasmid pBH700 and that was isolated in North Carolina. The trimethoprim resistance gene carried on pBH700 was subcloned and sequenced. The nucleotide sequence of the gene encoding type IIIc DHFR was identical to the gene encoding type VIII DHFR. The type IIIc amino acid sequence was approximately 50% similar to those of DHFRs commonly found in enteric bacteria. Furthermore, this gene was flanked by IS176 (IS26), an insertion sequence usually associated with those of aminoglycoside resistance genes. The gene for type IIIc DHFR was located by hybridization within a 1,993-bp PstI fragment in each of eight conjugative plasmids from geographically diverse strains of S. sonnei. Each plasmid also conferred resistance to ampicillin, streptomycin, and sulfamethoxazole and belonged to incompatibility group M. Plasmids carrying this new trimethoprim resistance gene, which is uniquely associated with IS176, have disseminated throughout the United States.

The molecular population genetics and pathogenic potential of North American and European invasive strains of Streptococcus pyogenes were assessed. Isolates from recent invasive infections and from infections in the 1920s and 1930s were characterized for multilocus enzyme genotype and allelic variation in the gene (speA) that encodes streptococcal pyrogenic exotoxin (SPE) A (scarlet fever toxin). A subset of strains was studied for allelic variation in genes that encode SPE B and streptokinase. All contemporary strains assigned to electrophoretic types (ETs) 1 and 2 that synthesize SPE A have the speA2 and speA3 allelic variants, respectively, and their relative virulence in two mouse models is similar to that of strains of the same ET and M protein types recovered earlier. In contrast, ET 1 and 2 isolates from disease episodes in the 1920s and 1930s contain the speA1 allele. The data suggest there may be temporal and geographic variation in the occurrence of clone--virulence factor allele combinations, an observation that may in part explain fluctuations in disease frequency, severity, and character.

The introduction of ciprofloxacin on an unrestricted basis into a 900-bed community hospital resulted in the emergence of high-level fluoroquinolone resistance among methicillin-resistant Staphylococcus aureus (MRSA) during the subsequent 18 months. Susceptibility testing revealed several old and new compounds to which all the S. aureus strains were susceptible. When an MRSA strain became resistant to ciprofloxacin it also exhibited high-level resistance to ofloxacin, fleroxacin, norfloxacin, and enoxacin. Two new experimental fluoroquinolones, WIN 57273 and CI-960, exhibited good activity against all test strains. Among the glycopeptide compounds, mupirocin and teicoplanin were approximately fourfold more active than vancomycin and ramoplanin. Rifampin and trimethoprim-sulfamethoxazole (TMP/SMZ) showed good activity against most strains as did imipenem. For clindamycin, gentamicin, and tetracycline susceptibilities exhibited a bimodal distribution with at least 10% of strains having resistant MIC values. Surprisingly, the addition of sulbactam potentiated the activity of ampicillin against the ciprofloxacin-resistant MRSA strains, however, sulbactam had little effect on cefoperazone activity against these same strains. Time-kill kinetic studies of selected antimicrobials against ciprofloxacin-resistant strains indicated good killing by vancomycin, ampicillin-sulbactam, and TMP/SMZ. Teicoplanin was less bactericidal than vancomycin while these same strains rapidly developed resistance to rifampin even at concentrations 8 x MIC. These data indicate certain alternative compounds within our study warrant further investigation, especially in vivo, against multiply-resistant staphylococci.

In 1990 an increased number of strains of Shigella boydii serotype 2 were isolated from different regions of Bulgaria. Strains were reported as sporadic, although they showed identical phenotypic characteristics, including resistance to ampicillin, carbenicillin, streptomycin, sulfonamide, tetracycline, ticarcillin, and trimethoprim. The objective of this study was to determine the genetic relatedness of the strains and the mechanism of their antimicrobial resistance. Plasmid fingerprinting showed an identical pattern for 23 of 25 of the selected strains. All 25 strains tested transferred their resistances en bloc to an Escherichia coli recipient. Transconjugants contained a 112-kb R plasmid which carried all the resistance genes, including that conferring type I dihydrofolate reductase-mediated trimethoprim resistance (MIC greater than 2,000 micrograms/ml). Riboprobe analysis showed identical restriction length fragment polymorphisms, suggesting a highly conserved genome. All findings indicate that strains of S. boydii serotype 2 isolated in 1990 from different regions of Bulgaria were highly related genetically and can be considered representatives of a single bacterial clone. The presence of an R plasmid and selection pressure because of the usage of antimicrobial agents, particularly trimethoprim, have likely facilitated the spread of the clone throughout the country.

Thirty Staphylococcus aureus isolates from five hospitals were determined to exhibit borderline susceptibility to the antistaphylococcal penicillins. Of the isolates submitted for phage typing, 96% belonged to phage group 94/96, and 96% possessed a common plasmid, pBW15. Also, four reference borderline-susceptible isolates from the Centers for Disease Control belonged to phage group 94/96 and possessed pBW15. Screening of 43 other phage group 94/96 isolates demonstrated that 36 (84%) contained pBW15 and exhibited the borderline phenotype. In contrast, pBW15 was not identified among 10 penicillin-susceptible, 10 methicillin-resistant, and 40 penicillin-resistant but non-borderline-susceptible S. aureus. These data show a close association between pBW15, phage group 94/96, and the borderline-susceptible phenotype. Furthermore, these isolates were associated with infections in multiple institutions, suggesting the widespread dissemination of a clinically important and pathogenic strain of S. aureus.

Members of the family Enterobacteriaceae that elaborate aminoglycoside 3-5-acetyltransferase [AAC(3)-5] caused a nosocomial outbreak at the Vanderbilt University Medical Center and have persisted. To see whether the gene for AAC(3)-5 was present in the community, stool cultures of newly admitted patients and ambulatory persons were examined with a specific gene probe. AAC(3)-5-positive strains were present in the intestinal flora examined.

Two strains of trimethoprim-resistant Shigella sonnei bearing R plasmids pBH600 and pBH700 each elaborated a dihydrofolate reductase (DHFR) and were moderately resistant to trimethoprim (minimum inhibitory concentrations, 128 and 256 micrograms/ml, respectively). Neither plasmid hybridized to probes for DHFR types I, II, or III. The trimethoprim resistance genes from the R plasmids resided on a 1600-base pair (bp) PstI fragment of pBH600 and an 1800-bp PstI fragment of pBH700. Isoelectric focusing showed distinct isoelectric points for the enzymes coded for on pBH600 (5.3) and pBH700 (5.6-5.7). Trimethoprim-resistant S. sonnei from 10 locations in nine states were examined. Isolates from 8 locations hybridized only to a pBH700-derived probe and one isolate hybridized to a pBH600-derived probe. These two trimethoprim resistance genes appear novel. The gene on plasmid pBH700 codes for an enzyme that seems widespread among S. sonnei isolates in the USA.

A young woman with varicella complicated by streptococcal cellulitis developed an intracerebral bleed in the absence of thrombocytopenia, serious coagulopathy, or a detectable vascular lesion. We postulate that during the acute infection with varicella, a cerebral endothelial lesion was produced which later caused the intracerebral bleed.

The clinical and microbiologic features of group A streptococcal bacteremia are described in 40 patients, all of whom were seen between January 1982 and June 1983 and all of whom were intravenous drug abusers. Eleven patients had endocarditis (two with left-sided and nine with right-sided), and 29 patients had bacteremia without endocardial involvement. Twenty-seven of the 29 patients without endocarditis had soft tissue infections, primarily groin abscesses. Constitutional symptoms were more severe in patients with endocarditis. The two patients with left-sided endocarditis died despite antimicrobial therapy; all nine patients with right-sided endocarditis and all 29 patients without endocarditis were cured of their infection. A predominant strain of group A streptococcus was identified by serologic typing, suggesting a common source for these cases.

A recent surveillance study at Vanderbilt University Medical Center indicated that 8.5% of the gram-negative bacilli isolated were resistant to gentamicin. To determine what proportion of current gentamicin-resistant isolates elaborated aminoglycoside 3-V-acetyltransferase [AAC(3)-V], a probe for this gene was constructed from a 975-base-pair PstI-SalI fragment of the nonconjugative R plasmid pCER954b. This plasmid was first isolated at Vanderbilt University Medical Center from epidemic strains of Serratia marcescens. Nineteen isolates determined to produce AAC(3)-V by MIC profile all reacted with the probe. The probe did not hybridize with DNA from organisms producing 10 other aminoglycoside-modifying enzyme types. With this probe, 30 (36%) of 84 gentamicin-resistant, gram-negative bacilli elaborated AAC(3)-V. Of these 30 isolates, 25 contained a conjugative plasmid that transferred gentamicin resistance. In contrast to other medical centers, at Vanderbilt a sizable number of gentamicin-resistant, gram-negative bacilli produced AAC(3)-V. This resistance determinant, initially identified in an epidemic Serratia strain, has persisted and become incorporated into currently isolated endemic strains of gram-negative bacilli.

We undertook a prospective randomized observer-blinded study comparing the ability of preoperative showers with chlorhexidine gluconate (Hibiclens), povidone-iodine (Betadine), and a lotion soap (Safe 'N Sure) to diminish the staphylococcal skin flora of patients. By block randomization, patients scheduled for an elective cardiac operation or coronary artery angioplasty were assigned to shower with one of the study skin cleansers either once (evening only) or twice (both evening and morning) before the procedure. Semiquantitative samples for culture were obtained from the subclavian and inguinal sites on the evening before the procedure (baseline culture) and again the next morning before the operation. The chlorhexidine skin cleanser consistently reduced staphylococcal colony counts at both the subclavian and inguinal sites before the procedure. This reduction was significant for patients showering both evening and morning (p less than 0.05). The use of the povidone-iodine skin cleanser inconsistently affected skin flora. Patients using lotion soap either experienced no change or had an increase in colony counts. Chlorhexidine is more effective than povidone-iodine in diminishing skin colonization with staphylococci in patients before operation. Repeated applications of chlorhexidine are superior to a single shower with this agent.

By use of techniques that have been developed to detect small numbers of methicillin-resistant staphylococci, we cultured samples from the nares and subclavian and inguinal areas of 29 patients before and after cardiac surgery and 10 patients before and after coronary angioplasty. Methicillin-resistant coagulase-negative staphylococci were recovered before the surgical or angioplasty procedure from 74% of patients. The quantitative recovery of methicillin-resistant isolates before cardiac surgery or coronary angioplasty was compared with the number of methicillin-resistant staphylococci detected at the same site 3 days after the procedure. In cardiac surgery patients (who received antibiotic prophylaxis), 17 of the 28 sites (61%) in which low-level colonization with methicillin-resistant strains was detected preoperatively contained high levels of methicillin-resistant staphylococci postoperatively. In contrast, coronary angioplasty patients (who did not receive antibiotic prophylaxis) did not have any of the 14 sites containing low levels of methicillin-resistant strains before angioplasty emerge to harbor high levels of methicillin-resistant staphylococci after angioplasty. Methicillin-resistant coagulase-negative staphylococci from each site in which high levels of methicillin-resistant staphylococci emerged postoperatively were paired with preoperative isolates from the same site. Identical antibiograms and plasmid profile patterns were demonstrated for seven of the pre- and postoperative isolate pairs, suggesting that the high levels of methicillin-resistant coagulase-negative staphylococci detected on the skin or in the nares after cardiac surgery were derived from methicillin-resistant organisms present at the site preoperatively in much smaller numbers.

A patient with methicillin-resistant Staphylococcus aureus bacteremia received vancomycin (MIC = 0.8 microgram/ml, MBC = 15 micrograms/ml) and heparin simultaneously through the same intravenous line to treat a septic deep venous thrombosis. Bacteremia persisted for 7 days. Bacteremia terminated when the simultaneous infusion of heparin and vancomycin through the same line was stopped. This suggested that an interaction between vancomycin and heparin may have occurred, which resulted in a reduction in vancomycin activity. To test for such an interaction, mixtures of heparin and vancomycin in various concentrations were made and tested for antimicrobial activity against the organisms in the patient. A precipitate formed at the concentrations achieved in the intravenous lines, and when the vancomycin concentrations were measured by bioassay, a 50 to 60% reduction in activity was noted. In contrast, when these solutions were prepared and mixed at microgram concentrations, a precipitate was no longer observed, and antimicrobial activity was not reduced. Heparin appeared to interact unfavorably with vancomycin at the concentrations in the intravenous lines when these drugs were administered simultaneously to patients. This may be the cause of poor therapeutic responses to vancomycin in some patients, especially those infected with tolerant organisms.