Bioterrorism: implications for the clinical microbiologist.

The specter of bioterrorism has captured the attention of government and military officials, scientists, and the general public. Compared to other sectors of the population, clinical microbiologists are more directly impacted by concerns about bioterrorism. This review focuses on the role envisioned for clinical laboratories in response to a bioterrorist event. The microbiology and clinical aspects of the biological agents thought to be the most likely tools of bioterrorists are presented. The historical background of the problem of bioterrorism and an overview of current U.S. preparedness planning, with an emphasis on the roles of health care professionals, are also included.

Isolation of Mycoplasma species from a patient with seal finger.

The etiologic agent of seal finger (speck finger) is unknown. Seal finger occurs after a seal bite, and the symptoms include acute pain, swelling, discharge, and, in some cases, there is joint involvement. The discovery of Mycoplasma species in epidemics of seal disease prompted attempts to link seal finger to mycoplasma. Mycoplasma species were isolated in cultures of a specimen from the finger of an aquarium trainer who was bitten by a seal and of a specimen from the front teeth of the biting seal. The two Mycoplasma isolates were identical biochemically; they were serum-dependent and hydrolyzed arginine. The isolates were susceptible to tetracycline but resistant to erythromycin. By growth inhibition and immunofluorescent antibody tests, both strains were identified as Mycoplasma phocacerebrale, a mycoplasma isolated in an epidemic of seal disease occurring in the Baltic Sea. The patient's infection was treated successfully with tetracycline. To our knowledge, this is the first case in which a mycoplasma has been associated with seal finger.

Treatment of experimental Staphylococcus epidermidis endophthalmitis with oral trovafloxacin.

PURPOSE: To investigate the ocular pharmacokinetics and efficacy of oral trovafloxacin, a novel fluoroquinolone antibiotic, in Staphylococcus epidermidis endophthalmitis. METHODS: Albino rabbits (n = 20) were infected with an intravitreal inoculum of S epidermidis (1.0 x 10(8) colony-forming units [CFU/0.1 ml) and 24 hours later received a single oral dose of trovafloxacin (250 mg/kg). Serum and intraocular samples from infected and control (noninfected) eyes were obtained up to 24 hours after antibiotic administration for measurement of trovafloxacin levels. A second group of rabbits (n = 72) was infected intraocularly and randomized 24 hours later to oral trovafloxacin (250 mg/kg twice a day) for 6 days or no treatment (control). Treatment efficacy was assessed by vitreous culture, clinical examination, and histopathology. RESULTS: Following a single dose of trovafloxacin, maximal vitreous levels were achieved at 8 hours in infected eyes, with a penetration ratio of 36%. Vitreous levels were greater than 15 times the minimum inhibitory concentration of the strain employed. In animals with established endophthalmitis, treated eyes were sterilized after 5 days (P = .0495) compared with control eyes, which autosterilized at 14 days. Clinical and histologic examination revealed significant amelioration of anterior segment inflammation in treated eyes, although severe destruction of posterior segment structures occurred in both groups after 6 days of therapy. CONCLUSIONS: These data support trovafloxacin as a potential oral agent for treatment or prophylaxis of S epidermidis endophthalmitis, although retinal alterations that occur over the period required for vitreous sterilization suggest that it will not replace intravitreal therapy in established endophthalmitis.

Performance of a PCR assay for detection of Pneumocystis carinii from respiratory specimens.

This study evaluates the performance of a PCR assay for the detection of Pneumocystis carinii from respiratory specimens that has been designed for use in the clinical microbiology laboratory. The test includes a simple method for nucleic acid extraction and amplification, a colorimetric probe hybridization technique for detection of amplicons, and an internal control to evaluate for the presence of inhibitors of amplification. Two hundred thirty-two clinical specimens (120 induced-sputum [IS] and 112 bronchoalveolar lavage [BAL] specimens) from 168 patients were tested by both immunofluorescent (direct fluorescent-antibody [DFA]) staining and PCR. Of the 112 BAL specimens, 17 were positive for P. carinii by DFA staining and PCR. An additional two specimens were DFA negative and PCR positive. For BAL specimens, the sensitivity and specificity of PCR compared to DFA were 100 and 98%, respectively. Eighteen IS specimens were positive for P. carinii by DFA, and 27 were positive by PCR. One of the 18 DFA-positive IS specimens was negative by PCR; this patient had just completed therapy for P. carinii pneumonia. Of the 10 specimens that were PCR positive and DFA negative, 4 were from patients who had a subsequent BAL specimen that was positive by DFA and PCR. For IS specimens, the sensitivity of DFA and PCR was 82 and 95%, respectively. The specificity of PCR for IS specimens was 94%. Due to the high sensitivity of PCR for the detection of P. carinii from IS specimens, a PCR-based diagnostic test may be a useful screening test and may alleviate the need for bronchoscopy in some patients.

Colonization of skin by Helcococcus kunzii.

In order to investigate the role of Helcococcus kunzii as a colonizer of skin and as a possible participant in diabetic foot ulcers, we used a selective medium to culture both lower- and upper-extremity skin from a study group of podiatry patients (60 diabetics and 60 nondiabetics) and a control group of 50 healthy volunteers. Although differences in colonization were not statistically significant, a trend toward higher colonization rates in the group of podiatry patients was noted. H. kunzii appears to preferentially colonize the skin of the feet, and while its pathogenic role in diabetic foot ulcers is difficult to establish, it may be a previously unrecognized component of the polymicrobial flora characteristically isolated from patients with these infections.

Implication of pneumolysin as a virulence factor in Streptococcus pneumoniae endophthalmitis.

PURPOSE: To determine if pneumolysin, a multifunctional cytotoxin produced by Streptococcus pneumoniae, may be a virulence determinant in the pathogenesis of pneumococcal endophthalmitis. METHODS: Lewis rats (n = 20) were injected intravitreally with purified recombinant pneumolysin at the following doses; 3.9 hemolytic units (HU), 39 HU, 390 HU, 3.9 x 10(3) HU, and 3.9 x 10(4) HU. After 24 hours, eyes were examined clinically and enucleated for histopathologic examination to elucidate the dose-response relationship. To determine the temporal progression of the disease model, a second group of rats (n = 8) were injected intravitreally with 390 HU of pneumolysin. At 6 and 48 hours, eyes were examined clinically and enucleated for histopathology. RESULTS: Eyes injected with pneumolysin demonstrated increasing anterior and posterior segment inflammation in response to increasing doses of administered toxin. The onset of inflammation and tissue damage occurred rapidly, and was maximal at 24 to 48 hours. The clinical and histopathologic changes observed mimicked those of S. pneumoniae endophthalmitis. Histopathologic analysis demonstrated rapid onset of iridocyclitis and vitritis with polymorphonuclear leukocyte influx, inner retinal necrosis, and retinal detachment. Retinal pigment epithelial necrosis and choroiditis were noted at the highest doses administered. Inflamed eyes were shown to be sterile. CONCLUSIONS: Pneumolysin injected intravitreally induces many of the clinical and histopathologic features of pneumococcal endophthalmitis, and may play an important role in the inflammation and tissue damage that occurs in pneumococcal endophthalmitis.

Emergence of high rates of antimicrobial resistance among viridans group streptococci in the United States.

Three hundred fifty-two blood culture isolates of viridans group streptococci obtained from 43 U.S. medical centers during 1993 and 1994 were characterized. Included were 48 isolates of "Streptococcus milleri," 219 S. mitis isolates, 29 S. salivarius isolates, and 56 S. sanguis isolates. High-level penicillin resistance (MIC, > or = 4.0 micrograms/ml) was noted among 13.4% of the strains; for 42.9% of the strains, penicillin MICs were 0.25 to 2.0 micrograms/ml (i.e., intermediate resistance). In general, amoxicillin was slightly more active than penicillin. The rank order of activity for five cephalosporins versus viridans group streptococci was cefpodoxime = ceftriaxone > cefprozil = cefuroxime >> cephalexin. The percentages of isolates resistant (MIC, > or = 2 micrograms/ml) to these agents were 15, 17, 18, 20, and 96, respectively. The rates of resistance to erythromycin, tetracycline, and trimethoprim-sulfamethoxazole were 12 to 38%. Resistance to either chloramphenicol or ofloxacin was uncommon (i.e., < 1%). In general, among the four species, S. mitis was the most resistant and "S. milleri" was the most susceptible.

Identification of "Streptococcus milleri" group isolates to the species level with a commercially available rapid test system.

Clinical isolates of the "Streptococcus milleri" species group were examined by conventional methods and a rapid, commercially available method for the identification of these strains to the species level. The levels of agreement between the identifications obtained with the commercially available system (Fluo-Card Milleri; KEY Scientific, Round Rock, Tex.) and conventional methods were 98% for 50 Streptococcus anginosus strains, 97% for 31 Streptococcus constellatus strains, and 88% for 17 isolates identified as Streptococcus intermedius. Patient records were also studied in order to gain information on the frequency and sites of isolation of each of the three "S. milleri" group species.

Report of cases of and taxonomic considerations for large-colony-forming Lancefield group C streptococcal bacteremia.

Traditionally, group C streptococci include four species: Streptococcus equisimilis, S. zooepidemicus, S. equi, and S. dysgalactiae, the first three of which are group C beta-hemolytic streptococci (GCBHS). However, many of the beta-hemolytic streptococci carrying Lancefield group C antigen isolated from clinical specimens are S. milleri. These organisms can be differentiated by colony size. We retrospectively collected data concerning large-colony-forming GCBHS bacteremia that occurred during a period of 8 years at the Massachusetts General Hospital. A total of 222 cases of beta-hemolytic streptococcal bacteremia were identified; data on the Lancefield grouping were available in 192 cases: 45 cases (23.6%) were group A, 96 cases (50%) were group B, 7 cases (3.6%) were group C (large colony forming), and 44 cases (22.9%) were group G. The medical records for cases of large-colony-forming GCBHS bacteremia were reviewed. In one case, the isolate was thought to be a contaminant; the other six cases are reported (five males and one female; mean age, 55 years). All patients had severe underlying conditions, and none had a history of exposure to animals. The clinical syndromes included two cases of cellulitis and one case each of endocarditis, myocardial infarction complicated by infection, pneumonia, and myofasciitis. The diagnoses for two patients with endovascular infections were delayed. Three of the six patients had fatal outcomes, and other two, after prolonged hospitalization, were transferred to a long-term rehabilitation center. We concluded that the severe outcomes reflect delay in diagnosis and treatment as well as the severity of the underlying diseases. The taxonomy of GCBHS is discussed. More reports differentiating large- and small-colony-forming GCBHS are needed.

Cost-effective, clinically relevant method for rapid identification of beta-hemolytic streptococci and enterococci.

Currently popular agglutination and coagglutination methods for the identification of beta-hemolytic streptococci, although rapid and simple to perform, are costly. Furthermore, they fail to distinguish between clinically relevant species and normal flora of the same serogroup. We investigated the use of a series of four physiologic tests to differentiate beta-hemolytic streptococci and enterococci into five clinically relevant groups. We also investigated the use of a new product, Visi-Spot, and evaluated an alternate method for the detection of beta-D-glucuronidase production. Our results suggest that for most routine processing of beta-hemolytic streptococci, physiologic tests are sufficiently rapid, more accurate, and far less costly to perform than serologic methods. The facility of our scheme is enhanced by the use of the Visi-Spot test and the substitution of a commercially available product for more traditional methods of detecting beta-D-glucuronidase.

"Streptococcus milleri" strains displaying a gliding type of motility.

Isolates belonging to the "Streptococcus milleri" species group that appear to exhibit a gliding type of motility, which is expressed as spreading growth on certain types of agar media, are described. These strains resembled a biotype of "S. milleri" that is usually isolated from genitourinary sources and is notable for its ability to ferment a wide array of carbohydrates. This biotype, which is currently included in the species Streptococcus anginosus, has been implicated in cases of neonatal infection. The "S. milleri" isolates which we studied lacked any observable organelles of motility and gave negative results when they were tested in conventional motility test medium stab cultures. Colonies growing on certain agar media, however, spread over the surfaces of plates and increased in area with increasing time of incubation. Chocolate agar supported maximum spreading, while this characteristic was barely discernible on blood agar. Electron microscopy studies revealed that there was more production of extracellular glycocalyx by motile strains than by a nonmotile isolate having a similar biotype. The results of an analysis of 16S rRNA gene sequences suggested that the motile strains are closely related to S. anginosus and represent a distinct rRNA population within the "S. milleri" species complex.

Phylogenetic analysis of some Aerococcus-like organisms from clinical sources: description of Helcococcus kunzii gen. nov., sp. nov.

16S rRNA gene sequencing studies were performed on some unusual gram-positive catalase-negative cocci of unknown taxonomic position isolated from human clinical sources. Comparative analysis of the sequence data demonstrated that the clinical isolates represent a hitherto-unknown line of descent within the low-G+C-content gram-positive bacteria. On the basis of the phylogenetic findings and the phenotypic distinctiveness of the organisms, it is proposed that they be classified in a new genus, Helcococcus, as Helcococcus kunzii sp. nov. The type strain of H. kunzii is NCFB 2900.

Increasing resistance to beta-lactam antibiotics among clinical isolates of Enterococcus faecium: a 22-year review at one institution.

To identify any change in the antibiotic resistance of Enterococcus faecium, we examined the antibiotic susceptibilities of clinical strains (n = 84) isolated at one institution during the 22 years since 1968. A significant increase in resistance to penicillin was observed during the study period: the MICs of penicillin for 50 and 90% of isolates tested were 16 and 64 micrograms/ml, respectively, from 1969 to 1988 (n = 48; geometric mean MIC, 14 micrograms/ml) , whereas they were 256 and 512 micrograms/ml, respectively, from 1989 to 1990 (n = 36; geometric mean MIC, 123 micrograms/ml) (P less than 0.001). A comparable increase in resistance to ampicillin was also noted (P less than 0.001). No strains produced detectable beta-lactamase. In contrast, susceptibilities to vancomycin, teicoplanin, and ciprofloxacin remained stable. High-level resistance to gentamicin was observed in none of 48 isolates from 1969 to 1988, but was present in 22 of 36 strains (61%) from 1989 to 1990 (P less than 0.001) and was significantly associated with resistance (MIC, greater than or equal to 128 micrograms/ml) to penicillin (P less than 0.001). To assess the potential evolution of antibiotic resistance in this species, clinical isolates (n = 24) were compared with strains isolated in 1968 from a human population in the Solomon Islands that was never exposed to antibiotics. Solomon Island isolates were significantly more susceptible than all clinical strains to penicillin, ampicillin, and vancomycin (P less than 0.001 for each), but they exhibited no differences in susceptibility to teicoplanin or ciprofloxacin. The penicillin-binding affinity of penicillin-binding protein 5 (PBP 5) in penicillin-resistant clinical strains (MIC, 512 micrograms/ml) was notably lower than that in strains with more typical susceptibilities, suggesting an alteration in this PBP as a possible mechanism for increased penicillin resistance. Solomon Island strains most susceptible to penicillin demonstrated a prominent PBP 5* and the absence of PBP 5. These changes in the antibiotic resistance of E. faecium emphasize the importance of identifying this species in patients with serious enterococcal infections and the necessity of assessing its susceptibility to both beta-lactams and aminoglycosides if effective therapy is to be identified.

Comparison of Enterococcus raffinosus with Enterococcus avium on the basis of penicillin susceptibility, penicillin-binding protein analysis, and high-level aminoglycoside resistance.

We reidentified our laboratories' collections of 57 enterococcal isolates previously classified as Enterococcus avium by the API Rapid Strep identification system (Analytab Products, Plainview, N.Y.) with the identification criteria recommended by Facklam and Collins (R. R. Facklam and M. D. Collins, J. Clin. Microbiol. 27: 731-734, 1989). Thirty isolates were identified as true E. avium, 25 isolates were identified as E. raffinosus, and 2 isolates were identified as E. pseudoavium. E. raffinosus could be differentiated from E. avium on the basis of penicillin susceptibility, as follows: MIC for 50% of E. raffinosus isolates tested (MIC50), 32 micrograms/ml; MIC90, 64 micrograms/ml (range, 4 to 64 micrograms/ml); E. avium MIC50, 1 microgram/ml; MIC90, 2 micrograms/ml (range, 0.5 to 2 micrograms/ml). No strains produced detectable beta-lactamase. Penicillin-binding protein (PBP) analysis of all E. raffinosus isolates demonstrated the unique pattern reported previously (M. D. Collins, R. R. Facklam, J. A. E. Farrow, and R. Williamson, FEMS Microbiol. Lett. 57:283-288, 1989); however, a number of newly identified PBPs were noted. Of 25 isolates, 13 had an additional PBP of 77 kDa (designated PBP 6*), while all isolates possessed a 52-kDa PBP (PBP 7) and a 46-kDa PBP (PBP 8). The presence or absence of PBP 6* did not correlate with penicillin susceptibility; however, PBP 7 demonstrated many features suggestive of low penicillin-binding affinity and may represent a possible mechanism for the relative resistance of this species to penicillin, although this hypothesis remains speculative since attempts to develop a penicillin-hypersusceptible E. raffinosus mutant were unsuccessful. E. raffinosus isolates were significantly more likely to exhibit high-level resistance to kanamycin than E. avium strains were (P < 0.001; chi-square); however, no strains demonstrated high-level resistance to gentamicin. No trend toward increasing penicillin resistance was noted among this collection of E. avium and E. raffinosus isolates collected over the past 35 and 14 years, respectively. Relative resistance to penicillin may be a helpful differentiating feature between E. avium and E. raffinosus when assessment of raffinose metabolism is not possible or is indeterminant.

Nutritionally variant streptococci.

Streptococci requiring either pyridoxal or L-cysteine for growth were first observed 30 years ago as organisms forming satellite colonies adjacent to colonies of "helper" bacteria. Although they were previously considered nutritional mutants of viridans streptococcal species, the nutritionally variant streptococci (NVS) are currently thought to belong to distinct species of the genus Streptococcus. NVS strains may display pleomorphic cellular morphologies, depending on their growth conditions, and are distinguished from most other streptococci by enzymatic and serological characteristics and the presence of a cell wall chromophore. NVS are found as normal inhabitants of the oral cavity, and in addition to their participation in endocarditis, they have been isolated from a wide range of clinical specimens. Endocarditis caused by NVS is often difficult to eradicate; combinations of penicillin and an aminoglycoside are recommended for treatment. The unique physiological features of the NVS contribute to the difficulties encountered in their recovery from clinical specimens and may play a role in the problems associated with successful treatment of NVS endocarditis.

Infectious crystalline keratopathy. Role of nutritionally variant streptococci and other bacterial factors.

Infectious crystalline keratopathy (ICK) is a chronic corneal infection characterized by interlamellar plaques of gram-positive coccal bacteria in the absence of inflammatory cells. It generally occurs within a corneal graft. Viridans streptococci are usually isolated, but the clinical response to antibiotics is poor and disparate with the in vitro antimicrobial sensitivities. These features suggest the possibility of unusual bacterial factors in pathogenesis. Four cases caused by nutritionally variant viridans streptococci are described. The organisms were fully characterized. They have a rare nutritional requirement for pyridoxal and require defined culture conditions and specific identification. Nutritional variant streptococci (NVS) are principally described as causing endocarditis, another infection involving an avascular collagenous tissue, and exhibiting similar biologic behavior. Electronmicrographic evidence is also adduced that suggests the possible importance of intracorneal glycocalyx deposition. Such factors might explain the anomalous clinical characteristics of this condition.

Species identities of enterococci isolated from clinical specimens.

Conventional tests and commercially available systems were used to determine the species identities of clinical isolates of enterococci. Strict adherence to the conventional test scheme of Facklam and Collins (R. R. Facklam and M. D. Collins, J. Clin. Microbiol. 27:731-734, 1989) resulted in the misidentification of lactose-negative Enterococcus faecalis isolates as Enterococcus solitarius, but this problem was overcome by the application of additional tests. The commercially available systems tested were unable to recognize some of the more recently described enterococcal species. E. faecalis accounted for 87.1% of 302 consecutive isolates. Enterococcus faecium (8.6%), Enterococcus avium (0.7%), Enterococcus durans (0.3%), Enterococcus gallinarum (1.0%), Enterococcus casseliflavus (1.0%), Enterococcus hirae (0.3%), and Enterococcus raffinosus (0.3%) isolates were also identified. None of the isolates produced beta-lactamase, but 15.4% of 235 isolates tested, including 1 strain of E. gallinarum, displayed high-level resistance to gentamicin.