Partial characterization of Nocardia farcinica beta-lactamases.

The beta-lactamases obtained from culture supernatants and cell extracts of 26 clinical strains and 5 reference strains of Nocardia farcinica were partially characterized. The enzymes exhibited two patterns on isoelectric focusing (IEF). beta-Lactamases from the majority of the 31 strains (87%) including the 5 reference strains exhibited two major bands with pIs of 4.56 and 4.49. The remaining strains had two similar major bands but with slightly higher pIs. Culture supernatants and cell extracts exhibited identical patterns. The two sets of enzymes were functionally indistinguishable by substrate and inhibitor profiles and lack of inducibility. By disk testing, ampicillin, amoxicillin, ticarcillin, amoxicillin-clavulanic acid, and imipenem were highly synergistic with cefotaxime. The enzymes were primarily penicillinases and hydrolyzed cephalosporins at rates of < or = 12% of those for penicillins. N. farcinica beta-lactamases were susceptible to inhibition by clavulanic acid and BRL 42715, exhibiting 50% inhibitory concentrations of 0.025 to 0.045 micrograms/ml (0.12 to 0.22 microM) and 0.05 to 0.1 micrograms/ml (0.31 to 0.63 microM), respectively, less susceptible to tazobactam, and least susceptible to sulbactam, cloxacillin, and imipenem. The beta-lactamases of N. farcinica are believed to mediate penicillin resistance and may play a secondary role in extended-spectrum cephalosporin resistance. The close similarity among N. farcinica beta-lactamases and their distinct differences from beta-lactamases of other Nocardia species support the taxonomic identity of this species.

Chronic tenosynovitis of the hand due to Mycobacterium nonchromogenicum: use of high-performance liquid chromatography for identification of isolates.

Six cases of chronic tenosynovitis of the hand due to the Mycobacterium terrae complex were identified. All isolates from the six cases were identified as Mycobacterium nonchromogenicum by high-performance liquid chromatography and by testing for susceptibility to ofloxacin and to 5% NaCl. Ethambutol, sulfonamides (or trimethoprim-sulfamethoxazole), erythromycin, and streptomycin are the drugs most active against isolates of the M. terrae complex, and therapy with some combination of these agents plus surgical debridement offers the best current treatment of this disease. This study supports the contention arising from previous case reports of pulmonary disease that M. nonchromogenicum is the pathogenic member of the M. terrae complex.

Mercuric reductase activity and evidence of broad-spectrum mercury resistance among clinical isolates of rapidly growing mycobacteria.

Resistance to mercury was evaluated in 356 rapidly growing mycobacteria belonging to eight taxonomic groups. Resistance to inorganic Hg2+ ranged from 0% among the unnamed third biovariant complex of Mycobacterium fortuitum to 83% among M. chelonae-like organisms. With cell extracts and 203Hg(NO3)2 as the substrate, mercuric reductase (HgRe) activity was demonstrable in six of eight taxonomic groups. HgRe activity was inducible and required NADPH or NADH and a thiol donor for optimai activity. Species with HgRe activity were also resistant to organomercurial compounds, including phenylmercuric acetate. Attempts at intraspecies and intragenus transfer of HgRe activity by conjugation or transformation were unsuccessful. Mercury resistance is common in rapidly growing mycobacteria and appears to function via the same inducible enzyme systems already defined in other bacterial species. This system offers potential as a strain marker for epidemiologic investigations and for studying genetic systems in rapidly growing mycobacteria.

Clinical disease, drug susceptibility, and biochemical patterns of the unnamed third biovariant complex of Mycobacterium fortuitum.

Previous studies of Mycobacterium fortuitum identified isolates that did not fit its two recognized biovariants. Eighty-five clinical isolates of this group, the "third biovariant complex", were evaluated. They represented 16% of 410 isolates of M. fortuitum submitted to a Texas laboratory and 22% of 45 isolates in Queensland, Australia. Most infections (76%) involved skin, soft tissue, or bone and occurred after metal puncture wounds or open fractures. Isolates differed from biovar fortuitum in resistance to pipemidic acid and use of mannitol and inositol as carbon sources. Two subgroups were present, and examples were deposited in the American Type Culture Collection. Isolates were resistant to doxycycline and one-third were resistant to cefoxitin. All were susceptible to amikacin, ciprofloxacin, sulfamethoxazole, and imipenem. Surgical debridement combined with drug therapy based on in vitro susceptibilities resulted in cures of cutaneous disease or osteomyelitis. DNA homology studies are needed to determine the taxonomic status of these organisms.

Acquired resistance of Nocardia brasiliensis to clavulanic acid related to a change in beta-lactamase following therapy with amoxicillin-clavulanic acid.

Previous studies have demonstrated that Nocardia brasiliensis is susceptible to amoxicillin-clavulanic acid and that its beta-lactamases are inhibited in vitro by clavulanic acid. A cardiac transplant patient with disseminated infection caused by N. brasiliensis was treated with this drug combination with good response, but relapsed while still on therapy. The relapse isolate was found to be identical to the initial isolate by using genomic DNA restriction fragment patterns obtained by pulsed field gel electrophoresis, but it was resistant to amoxicillin-clavulanic acid. On isoelectric focusing, the beta-lactamase from the relapse isolate exhibited a shift in the isoelectric point (pI) of its major band from 5.10 to 5.04 compared with the enzyme from the pretreatment isolate. As determined by using values of the amount of beta-lactamase inhibitor necessary to give 50 +/- 5% inhibition of beta-lactamase-mediated hydrolysis of 50 microM nitrocefin, the beta-lactamase of the relapse isolate was also 200-fold more resistant than the enzyme from the pretreatment isolate to clavulanic acid and was more resistant to sulbactam, tazobactam, cloxacillin, and imipenem. The beta-lactamase of the relapse isolate exhibited a 10-fold decrease in hydrolytic activity for cephaloridine and other hydrolyzable cephalosporins compared with that for nitrocefin. Acquired resistance to amoxicillin-clavulanic acid in this isolate of N. brasiliensis appears to have resulted from a mutational change affecting the inhibitor and active site(s) in the beta-lactamase.

Comparison of the activity of cefixime and activities of other oral antibiotics against adult clinical isolates of Moraxella (Branhamella) catarrhalis containing BRO-1 and BRO-2 and Haemophilus influenzae.

MICs of 10 oral antibiotics were determined for 105 Moraxella catarrhalis and 96 Haemophilus influenzae isolates from adults. A two- to fourfold increase in MICs of oral cephalosporins was seen in the presence of BRO-1 but not with TEM-1 or BRO-2. The MICs of cefixime for 90% of strains of H. influenzae (0.125 microgram/ml) and M. catarrhalis (0.25 microgram/ml) were 8- to 64-fold lower than those of other oral cephalosporins.

Activities of ciprofloxacin and ofloxacin against rapidly growing mycobacteria with demonstration of acquired resistance following single-drug therapy.

The susceptibility to ciprofloxacin of 548 clinical isolates of rapidly growing mycobacteria belonging to eight subgroups or species was determined. The 170 isolates of Mycobacterium fortuitum biovar.fortuitum were most susceptible; the MIC for 90% of the organisms was 0.125 micrograms/ml. The other biovariants of M. fortuitum, M. smegmatis, and the M. chelonae-like organisms were less susceptible; the modal MIC was 0.5 micrograms/ml, and the MIC for 90% of organisms was 1.0 micrograms/ml. The two subspecies of M. chelonae were generally resistant, with only 8% of 206 isolates falling in the moderately susceptible category (MIC, 2 micrograms/ml) and only 2% falling in the susceptible category (MIC, less than or equal to 1 micrograms/ml). MICs of ofloxacin averaged 1 to 2 dilutions higher than those of ciprofloxacin for all subgroups tested. Three patients with M. fortuitum cutaneous disease relapsed after an initial response to therapy with ciprofloxacin, and their isolate was shown to have acquired drug resistance. Mutational frequencies for M. fortuitum with ciprofloxacin were relatively high (10(-5) to 10(-7), and MICs for single-step mutants were similar to those for the clinically resistant strains. Thus, despite the excellent activity of ciprofloxacin against rapidly growing mycobacterial groups other than M. chelonae, single-drug therapy should be used with caution because of the risk of development of mutational resistance.

Heterogeneity among isolates of rapidly growing mycobacteria responsible for infections following augmentation mammaplasty despite case clustering in Texas and other southern coastal states.

Thirty-seven cases of rapidly growing mycobacterial wound infections following augmentation mammaplasty were identified between 1979 and 1988. The infections were usually unilateral and had a narrow geographic distribution: almost 60% were from Texas and 92% from five southern coastal states. In Texas a seasonal incidence was observed; 45% of all previously reported and current patients had undergone mammaplasty in April, May, or June. Although these findings suggested a possible common source, analysis of 35 available isolates revealed 19 different phenotype patterns. Five different taxonomic groups were represented, although most isolates (70%) were Mycobacterium fortuitum biovar fortuitum. Plasmid bands were identified in 10 of 15 strains studied, with nine different profiles. An additional 11 cases of breast infection due to rapidly growing mycobacteria not associated with augmentation were also identified, of which nine came from the same states that contributed mammaplasty cases. Rapidly growing mycobacterial infections of the breast are endemic in Texas and other southern coastal states, and the heterogeneity of the isolates suggests that most cases are unrelated.

Diversity and sources of rapidly growing mycobacteria associated with infections following cardiac surgery.

Eighty-nine isolates of rapidly growing mycobacteria associated with cardiac bypass-related infections were characterized. Isolates from sporadic infections belonged to eight taxonomic groups and displayed numerous multilocus enzyme genotypes, plasmid profiles, and heavy metal and antibiotic resistance patterns. Compared with 449 noncardiac wound isolates, 45 sporadic cardiac isolates were more likely to be Mycobacterium fortuitum and M. smegmatis and less likely to be M. chelonae. About 80% of cardiac and noncardiac isolates were from southern coastal states. Eight outbreaks of cardiac bypass-related infections were identified. Strains from each outbreak were genotypically distinctive, and five outbreaks involved more than one strain. In two outbreaks, isolates from environmental sources and noncardiac infections were similar or identical to isolates from sternal wound infections. The heterogeneity of these isolates suggests that most isolates are unrelated and are derived from local environmental sources rather than from contaminated commercial surgical materials or devices.

Antimicrobial susceptibility patterns of Nocardia asteroides.

Testing of the susceptibility to 12 antimicrobial agents, including beta-lactams, aminoglycosides, ciprofloxacin, and erythromycin, was performed by broth microdilution on 78 consecutive clinical isolates of Nocardia asteroides. Surprisingly, a limited number of patterns of susceptibility were identified that included all drug classes, with 95% of isolates exhibiting one of five patterns. One group (17%) exhibited resistance to the broad-spectrum cephalosporins, one group (18%) was susceptible to both ampicillin and erythromycin, one group (17%) was susceptible to ampicillin and carbenicillin but intermediate in susceptibility to imipenem, and the most common group (35%) was resistant to ampicillin but susceptible to the broad-spectrum cephalosporins and imipenem. The most active parenteral agents were amikacin (95%), imipenem (88%), ceftriaxone (82%), and cefotaxime (82%), while the most active oral agents were the sulfonamides (100%), minocycline (100%), and ampicillin (40%). Additional studies are needed to determine whether differences in beta-lactamases relate to varying beta-lactam resistance and whether taxonomic differences that correlate with the different susceptibility groups can be identified.

Ampicillin, tetracycline, and chloramphenicol resistant Haemophilus influenzae in adults with chronic lung disease. Relationship of resistance to prior antimicrobial therapy.

Antibiotic resistance in 1,003 sputum isolates of Haemophilus influenzae from adults with chronic lung disease was assessed from January 1983 through June 1986. The incidence of resistance was 3.2% for tetracycline, 0.6% for chloramphenicol, and 12.5% for ampicillin. Resistance to ampicillin or tetracycline usually occurred alone, while 100% of chloramphenicol resistant isolates were co-resistant to tetracycline or ampicillin. More than 90% of antibiotic resistant isolates were nontypable and belonged to biotypes II, III, or V. Chart reviews of 331 patients revealed that patients with ampicillin resistant isolates were more likely than control subjects to have received ampicillin in the prior 6 wk (33% versus 6%, p less than 0.0001), whereas patients with isolates resistant to tetracycline or chloramphenicol plus tetracycline were more likely to have received tetracycline than control subjects (24% and 50%, respectively, versus 5%, p less than 0.005). The incidence of ampicillin resistance and the reluctance of physicians caring for adults to use chloramphenicol suggests that a newer cephalosporin such as cefotaxime may be the initial therapy of choice for severe H. influenzae disease in our patient population.

Susceptibility testing of Nocardia species for the clinical laboratory.

Not all patients are able to tolerate or show a favorable response to the treatment of choice for Nocardia, the sulfonamides. Many new drugs are available with good activity against N. asteroides, the most common pathogenic species, although susceptibility to these agents, including amikacin, amoxicillin/clavulanic acid, and the third generation cephalosporins, is variable. For these and other reasons, we recommend routine susceptibility testing of Nocardia. Disk diffusion testing on Mueller Hinton agar is the best currently available clinical method. A suggested control strain and tentative susceptible and resistant breakpoints for 12 antimicrobial agents are provided. This includes agents not previously evaluated, including cefotaxime, amikacin, ciprofloxacin, and doxycycline. The zones of inhibition were all larger than those currently used by the NCCLS for rapidly growing bacteria, and the disk breakpoints generally fit best with the MIC breakpoints used with the dilution susceptibility method (M7-T). A broth microdilution MIC method is described that showed good correlation with disk diffusion results, but need additional study. Because of limited experience in most laboratories with this species, reliance on a good reference laboratory for confirmatory susceptibility testing is recommended. Beta-lactamase testing is not helpful, as almost all Nocardia produce beta-lactamase, although many isolates retain susceptibility to selected beta-lactams.

Beta-lactam resistance in Nocardia brasiliensis is mediated by beta-lactamase and reversed in the presence of clavulanic acid.

Forty clinical isolates and the type strain of Nocardia brasiliensis were screened for susceptibility to 20 beta-lactams. Isolates exhibited a single pattern of resistance, with large zones of inhibition by disk diffusion and low MICs by broth and agar dilutions only to cefotaxime, ceftriaxone, ceftizoxime, Augmentin, and Timentin. All strains produced beta-lactamase, with five different enzyme patterns by isoelectric focusing. Despite the differences in their isoelectric points, the enzymes had the same substrate profiles, with equivalent activity against penicillin, ampicillin, cefamandole, cephalothin, and cephalordine. In an in vitro assay, the enzymes were highly susceptible to clavulanic acid. The MIC50 and MIC90 for the combination of amoxicillin and clavulanic acid (Augmentin) was 2 and 4 micrograms/ml, respectively, compared with 16 micrograms/ml for both values for amoxicillin alone. These studies suggest that beta-lactamase is the major mechanism of beta-lactam resistance in this species and that Augmentin is the first oral beta-lactam with good potential for treating infections due to N. brasiliensis.

Ability of ciprofloxacin but not pipemidic acid to differentiate all three biovariants of Mycobacterium fortuitum from Mycobacterium chelonae.

When tested against 312 clinical isolates of rapidly growing mycobacteria, the quinolone pipemidic acid correctly separated Mycobacterium chelonae (M. chelonei) from Mycobacterium fortuitum biovar fortuitum but not from biovar peregrinum or the third biovar complex. The new 4-quinolone ciprofloxacin correctly separated all three biovars of M. fortuitum from M. chelonae and appears to provide a better taxonomic test.

Sulfonamide-containing regimens for disease caused by rifampin-resistant Mycobacterium kansasii.

Fourteen wild strains and 14 relapse or treatment failure isolates of Mycobacterium kansasii were tested and found to be highly susceptible to sulfamethoxazole (SMX), with 26 of 28 isolates having minimal inhibitory concentrations (MIC) of less than or equal to 4 micrograms/ml), using a broth microdilution method. Treatment failure isolates frequently exhibited resistance to rifampin (RMP) (greater than 2 micrograms/ml), isoniazid (INH) (greater than 4 micrograms/ml), and ethambutol (EMB) (greater than 4 micrograms/ml) not seen among the wild strain isolates. Eight patients with cavitary disease caused by RMP-resistant M. kansasii were treated with SMX-containing regimens that also included high dose INH (900 mg), EMB (25 mg/kg), and an aminoglycoside (either streptomycin or amikacin). Patients were treated initially in the hospital for 4 to 10 wk. In 7 of the 8 patients, sputum cultures became negative in a mean of 10 wk (range, 7 to 14 wk). Acquired drug resistance to INH, RMP, and EMB can be demonstrated in M. kansasii, and SMX in combination with other agents chosen on the basis of MIC determinations are effective in the treatment of disease caused by RMP-resistant M. kansasii.

Characterization of beta-lactamases in Mycobacterium fortuitum including a role in beta-lactam resistance and evidence of partial inducibility.

The beta-lactamases from the 3 biovariants of M. fortuitum were compared on the basis of substrate profiles, susceptibility to enzyme inhibitors, and inducibility in the presence of selected beta-lactams. Despite differences in the distribution of beta-lactamase bands observed when enzymes from different isolates were subjected to isoelectric focusing, substrate profiles for the 3 biovariants were similar. All demonstrated a comparable broad spectrum hydrolytic activity for both cephalosporins and penicillins. The MIC for amoxicillin were reduced 4- to 16-fold when combined with the beta-lactamase inhibitor clavulanic acid, but not to a clinically susceptible range. The degree of reduction in MIC for amoxicillin correlated well with the susceptibility of enzyme to inhibition by clavulanic acid as determined in an in vitro assay. Although all M. fortuitum strains produce beta-lactamase under routine growth conditions, 90% of strains demonstrated an increase in the amount of this enzyme when cultured in the presence of selected beta-lactams as potential inducers. Quantitative assays and isoelectric focusing further indicated that this apparent induction of beta-lactamase is a simple enhancement of the same enzyme(s) produced in the absence of a known inducer. This is the first demonstration of any inducibility among mycobacterial beta-lactamases and suggests that synthesis of these enzymes in M. fortuitum is under some form of regulatory control. These results indicate that the beta-lactamases have a role in resistance of M. fortuitum to the beta-lactams. Other factors, such as permeability and penicillin-binding proteins, were not evaluated.

Susceptibility testing of slowly growing mycobacteria by a microdilution MIC method with 7H9 broth.

Based on previous success with rapidly growing mycobacteria, a microdilution MIC system was devised for slowly growing mycobacterial species using 7H9 broth. Test drugs included isoniazid, rifampin, ethambutol, streptomycin, clofazamine, and sulfamethoxazole. Sixty isolates of four mycobacterial species, including Mycobacterium tuberculosis, from patients who had never received drug therapy were evaluated in the system, as well as 25 drug-resistant isolates and 11 control strains. MICs were read when good macroscopic control growth was evident, a period which varied with each species. Most species exhibited a narrow range of MICs with easily discernible growth endpoints. The aminoglycosides, ethambutol, clofazamine, and sulfamethoxazole were the only drugs with activity against all species at clinically achievable levels in serum. Correlation between susceptibilities by the proportion method in agar with single drug concentrations and the broth method were excellent for M. tuberculosis, M. kansasii, and M. marinum for isoniazid, rifampin, and ethambutol. Isolates of the M. avium complex were much more susceptible in broth than in agar for rifampin, ethambutol, and streptomycin. Given the successful transition of most microbiology laboratories to MIC plates for other bacterial species, this method would allow for testing of multiple drugs at multiple concentrations and has good potential for evaluation of drug combinations and drug-resistant isolates.

Isoelectric focusing of beta-lactamases in Mycobacterium fortuitum. Association of a single enzyme pattern with cefoxitin resistance.

The uninduced culture supernatants and cell extracts from 58 strains of the 3 biovariants (biovar) of Mycobacterium fortuitum were all positive for beta-lactamase with the chromogenic cephalosporin substrate. By analytical isoelectric focusing (IEF), 29 of 30 strains of biovar fortuitum exhibited an identical beta-lactamase pattern with 1 major band. In contrast, the beta-lactamases of biovar peregrinum and the unnamed third biovar were heterogeneous, with multiple bands and a variety of patterns. The pH range of isoelectric points for the beta-lactamases was relatively narrow, however, with most bands appearing between pH 4.3 and 5.2. Although additional genetic studies are required, these enzymes appear to be chromosomal, as they are present in all strains including some without detectable plasmids. Repeat isolates from the same patient obtained up to six months apart always had the same beta-lactamase pattern by IEF. Of the third biovar complex, 30% are cefoxitin resistant with minimal inhibitory concentrations greater than 32 micrograms/ml. All 9 cefoxitin-resistant isolates tested had the same unique beta-lactamase pattern by IEF, although this enzyme failed to hydrolyze cefoxitin while hydrolyzing cephalothin and benzylpenicillin. Thus, despite the association of cefoxitin-resistance with a single enzyme pattern, the role of this beta-lactamase in resistance is not known.

Mutational resistance as the mechanism of acquired drug resistance to aminoglycosides and antibacterial agents in Mycobacterium fortuitum and Mycobacterium chelonei. Evidence is based on plasmid analysis, mutational frequencies, and aminoglycoside-modifying enzyme assays.

Possible mechanisms of drug resistance of Mycobacterium fortuitum and Mycobacterium chelonei to antibacterial agents were investigated. Single-step mutational frequencies were low (generally less than or equal to 10(-7] for cefoxitin, doxycycline, erythromycin, and sulfamethoxazole and relatively high (10(-4) to 10(-7] for kanamycin and amikacin. Aminoglycoside-susceptible strains of both species contained an aminoglycoside acetyltransferase (3)-III or IV. No additional enzymes were seen with laboratory or clinically acquired aminoglycoside resistance. Plasmids of several sizes were present in some susceptible isolates of both species, but acquired resistance was not associated with a change in the apparent size or number of these plasmids. Isolates with acquired resistance to amikacin were resistant to the other 2-deoxystreptamine aminoglycosides but showed little or no change in minimal inhibitory concentrations to streptomycin, suggesting either a difference in cellular uptake between the 2 groups of drugs or, more likely, different binding sites on the ribosome. In 59 patients treated with 63 courses of therapy with a single agent for 1 month or longer, the development of resistance was observed only twice (3%). Both isolates had high mutational frequencies (10(-4) and 10(-5]. These studies support mutational resistance as the mechanism of acquired resistance to antibacterial agents in M. fortuitum and M. chelonei.

Amoxicillin-clavulanic acid in the treatment of lower respiratory tract infections caused by beta-lactamase-positive Haemophilus influenzae and Branhamella catarrhalis.

Twenty-one adult patients hospitalized with lower respiratory tract infections due to Branhamella catarrhalis or Haemophilus influenzae or both were treated with the combination of oral amoxicillin and potassium clavulanate (Augmentin) in an open, noncomparative clinical trial. Diseases included pneumonia, empyema, and exacerbations of bronchiectasis and chronic lung disease. Thirteen of 16 B. catarrhalis and six of nine H. influenzae isolates were beta-lactamase positive. The patients with B. catarrhalis were treated for a mean of 5.3 days, and those with H. influenzae were treated for a mean of 7.0 days. The overall response to therapy was excellent, with 18 of 19 beta-lactamase-producing strains eradicated on therapy. One patient secondarily infected with Pseudomonas aeruginosa was a clinical failure, and two patients with H. influenzae who became culture positive again after therapy were considered microbiologic failures. Gastrointestinal side effects (especially nausea) were common, although all patients completed a course of therapy. Sputum levels of amoxicillin were surprisingly low (less than 0.05 to 0.54 micrograms/ml), a finding which may explain the high relapse rate (22%) seen with H. influenzae, as these are below the usual MICs of amoxicillin for this organism. The combination of amoxicillin plus potassium clavulanate appears to be an excellent drug for treatment of beta-lactamase-producing strains of these two species, although mild gastrointestinal side effects are common.