A two-component regulatory system, CsrR-CsrS, represses expression of three Streptococcus pyogenes virulence factors, hyaluronic acid capsule, streptolysin S, and pyrogenic exotoxin B.

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.

Donor-to-recipient transmission of bacteria as an unusual cause of mediastinitis in a heart transplant recipient.

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.

Examination of methicillin-resistant and methicillin-susceptible Staphylococcus aureus mutants with low-level fluoroquinolone resistance.

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.

Comparison of four antibiotics in a murine model of necrotizing cutaneous infections caused by toxigenic Streptococcus pyogenes and Staphylococcus aureus.

The ability of azithromycin, erythromycin, clarithromycin, or cefuroxime to modify the course of group A streptococcus (GAS) or Staphylococcus aureus soft-tissue infection was compared in a mouse model. In GAS-infected mice given azithromycin, fewer demonstrated dermonecrosis (P = 0.0004); the average weight gain was greater (P < 0.05) and the latency to sustained weight gain was shorter (P < 0.05) than for animals given other antibiotics. All antibiotics were effective against S. aureus infections, with no significant differences among treatments in parameters evaluated. The effectiveness of azithromycin in GAS-infected mice may be related to the high and sustained tissue concentrations achieved with this antibiotic.

Reduced virulence of group A streptococcal Tn916 mutants that do not produce streptolysin S.

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.

Mycobacterium avium complex endocarditis: spurious diagnosis resulting from laboratory cross contamination.

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.

Growth of Staphylococcus aureus with nafcillin in vitro induces alpha-toxin production and increases the lethal activity of sterile broth filtrates in a murine model.

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.

In-vitro and in-vivo selection of Staphylococcus aureus mutants resistant to ciprofloxacin.

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.

Sequence identity with type VIII and association with IS176 of type IIIc dihydrofolate reductase from Shigella sonnei.

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.

Antibiotic therapy in 1994: mechanisms of resistance.

Drug-resistant organisms are appearing with increasing frequency. Of particular concern are drug-resistant strains of enterococci, streptococci, and pneumococci. Bacteria use several adaptive mechanisms to thwart the actions of antimicrobials, including enzymes, alterations in cell membrane permeability, export of antibiotics from the cell, alteration of molecular structures, and transfer of resistance to other species. Countering the effects of resistance requires judicious use of antibiotic therapy and a clear understanding of the biologic mechanisms involved.

Geographic and temporal distribution and molecular characterization of two highly pathogenic clones of Streptococcus pyogenes expressing allelic variants of pyrogenic exotoxin A (Scarlet fever toxin).

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 rapid emergence of fluoroquinolone-methicillin-resistant Staphylococcus aureus infections in a community hospital. An in vitro look at alternative antimicrobial agents.

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.

Murine model of cutaneous infection with gram-positive cocci.

Staphylococcus aureus has remained an important cause of nosocomial wound infections, but standardized or reproducible systems for analyzing cutaneous infections caused by S. aureus do not exist. A variety of foreign materials, variable inocula, and skin traumas have been used to promote infection. To minimize these variables and ensure reproducibility, we chose a model using subcutaneous injections of a fixed quantity of dextran microbeads (Cytodex) as the foreign material added to standardized broth suspensions of S. aureus. Suspensions (0.2 ml) injected into an outbred strain of immunocompetent hairless mice generated reproducible, measurable lesions. With S. aureus Smith Diffuse, fluctuant, erythematous lesions with a peak diameter of 15 mm were observed; these lesions yielded purulent material containing gram-positive cocci and neutrophils and yielded growth of S. aureus on culture. Lesion size was proportional to the bacterial inoculum size. Histologic examination of excised lesions revealed typical abscesses. A second strain of S. aureus (SLC3) produced dermonecrosis instead of abscesses at an inoculum size of 10(7) CFU. Control injections with a sterile Cytodex suspension regularly produced nondraining, nonerythematous nodules with maximum diameters of less than or equal to 5 mm. Streptococcus pyogenes produced late-onset necrotic lesions and abscesses. Using a foreign substance, this model generates easily observed and reproducible cutaneous infection with S. aureus and streptococci that can potentially discriminate between inter- and intrastrain differences. Such a model could be used to test the pathogenicity of isogeneic strains of these bacterial species and to evaluate the efficacy of antimicrobial agents.

Molecular epidemiology of trimethoprim-resistant Shigella boydii serotype 2 strains from Bulgaria.

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.

Borderline susceptibility to antistaphylococcal penicillins is not conferred exclusively by the hyperproduction of beta-lactamase.

Staphylococcus aureus strains bearing the 17.2-kb beta-lactamase plasmid pBW15 and belonging to phage group 94/96 exhibit borderline susceptibility to the antistaphylococcal penicillins. Borderline susceptibility within phage group 94/96 is thought to be mediated by the hyperproduction of type A staphylococcal beta-lactamase. Evaluation of 84 non-94/96 phage type S. aureus strains that also produced the type A enzyme identified 7 additional hyperproducing strains. However, none of these isolates contained pBW15, and only one met the criteria for borderline susceptibility. To determine the role of pBW15 and the 94/96 phage type in the expression of borderline susceptibility, pBW15 was transformed in two plasmid-free, penicillin-susceptible strains, one of which belonged to phage group 94/96. Penicillin MICs for both transformants and quantitative beta-lactamase activity were comparable to those for the parent pBW15-containing strain. A fourfold difference in the oxacillin MICs for the 94/96 and non-94/96 phage type transformants (1.0 and 0.25 microgram/ml, respectively) was identified, and only the 94/96 phage type transformant met the criteria for borderline susceptibility. Chromosomal DNA from borderline-susceptible phage group 94/96 strains did not hybridize with a probe for mecA, and the beta-lactam binding affinity of PBPs 1, 2, 3, and 4 from a penicillin-susceptible 94/96 phage type strain and a non-94/96 phage type strain were comparable. Although hyperproduction of the type A beta-lactamase appears to be necessary for the expression of borderline susceptibility within certain phage group 94/96 strains, beta-lactamase production of a comparable magnitude by a group of S. aureus strains belonging to other phage types does not confer borderline susceptibility. These data suggest that borderline susceptibility is not solely due to the hyperproduction of beta-lactamase.

A large outbreak of antibiotic-resistant shigellosis at a mass gathering.

In July 1987, a large outbreak of shigellosis occurred among attendees at a mass gathering in a national forest, the annual Rainbow Family Gathering. Sanitation in the campsite was poor, allowing widespread transmission of disease, probably by food, water, and person-to-person spread. The attack rate may have been greater than 50% among the estimated 12,700 attendees. The outbreak was caused by Shigella sonnei, resistant to ampicillin, tetracycline, and trimethoprim-sulfamethoxazole; the organism was of colicin type 9 and contained a 90-kilobase plasmid not found in non-outbreak-related strains. The dispersal of the group resulted in nationwide dissemination of the organism, and outbreaks in three states were linked to transmission from attendees at the Gathering. This outbreak demonstrates the potential for rapid dissemination of disease in such a setting and the necessity for careful planning of mass gatherings.

Characterization of a widespread strain of methicillin-susceptible Staphylococcus aureus associated with nosocomial infections.

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.