Stephen A. Lerner

Tackling antibiotic resistance

The development and spread of antibiotic resistance in bacteria is a universal threat to both humans and animals that is generally not preventable but can nevertheless be controlled, and it must be tackled in the most effective ways possible. To explore how the problem of antibiotic resistance might best be addressed, a group of 30 scientists from academia and industry gathered at the Banbury Conference Centre in Cold Spring Harbor, New York, USA, from 16 to 18 May 2011. From these discussions there emerged a priority list of steps that need to be taken to resolve this global crisis.

Prospective Evaluation of the Effect of an Aminoglycoside Dosing Regimen on Rates of Observed Nephrotoxicity and Ototoxicity

ABSTRACT The nephrotoxicity and ototoxicity associated with once-daily versus twice-daily administration of aminoglycosides was assessed in patients with suspected or proven gram-negative bacterial infections in a randomized, double-blind clinical trial. Patients who received therapy for ≥72 h were evaluated for toxicity. Patients also received concomitant antibiotics as deemed necessary for treatment of their infection. Plasma aminoglycoside concentrations, prospective aminoglycoside dosage adjustment, and serial audiologic and renal status evaluations were performed. The probability of occurrence of a nephrotoxic event and its relationship to doses and daily aminoglycoside exposure served as the main outcome measurement. One hundred twenty-three patients were enrolled in the study, with 83 patients receiving therapy for at least 72 h. For 74 patients plasma aminoglycoside concentrations were available for analysis, and the patients formed the group evaluable for toxicity. The primary infectious diagnosis for the patients who were enrolled in the study were bacteremia or sepsis, respiratory infections, skin and soft tissue infections, or urosepsis or pyelonephritis. Of the 74 patients evaluable for toxicity, 39 received doses twice daily and 35 received doses once daily and a placebo 12 h later. Nephrotoxicity occurred in 6 of 39 (15.4%) patients who received aminoglycosides twice daily and 0 of 35 patients who received aminoglycosides once daily. The schedule of aminoglycoside administration, concomitant use of vancomycin, and daily area under the plasma concentration-time curve (AUC) for the aminoglycosides were found to be significant predictors of nephrotoxicity by multivariate logistic regression analysis (P ≤ 0.001). The time to a nephrotoxic event was significantly influenced by vancomycin use and the schedule of administration, as assessed by Cox proportional hazards modeling (P ≤ 0.002). The results of the multivariate logistic regression analysis and the Cox proportional hazards modeling demonstrate that both the probability of occurrence and the time to occurrence of aminoglycoside nephrotoxicity are influenced by the schedule on which the aminoglycoside is administered as well as by the concomitant use of vancomycin. Furthermore, this risk of occurrence is modulated by the daily AUC for aminoglycoside exposure. These data suggest that once-daily administration of aminoglycosides has a predictably lower probability of causing nephrotoxicity than twice-daily administration.

Community-acquired pneumonia

This seminar reviews the aetiology, clinical presentation, approach to diagnosis, and management of immunocompetent adults with community-acquired pneumonia (CAP). Pneumonia is a common clinical entity, particularly among the elderly. A thorough understanding of the epidemiology and microbiology of CAP is essential for appropriate diagnosis and management. Although the microbiology of CAP has remained relatively stable over the last decade, there is new information on the incidence of atypical pathogens, particularly in patients not admitted to hospital, and new information on the incidence of pathogens in cases of severe CAP and in CAP in the elderly. Recent studies have provided new data on risk factors for mortality in CAP, which can assist the clinician in decisions about the need for hospital admission. The emergence of antimicrobial resistance in Streptococcus pneumoniae, the organism responsible for most cases of CAP, has greatly affected the approach to therapy, especially in those patients who are treated empirically. Guidelines for the therapy of CAP have been published by the American Thoracic Society, the British Thoracic Society, and, most recently, the Infectious Diseases Society of America. These guidelines differ in their emphasis on empirical versus pathogenic-specific management.

Cutaneous Abscesses: Anaerobic and Aerobic Bacteriology and Outpatient Management

Specimens from 135 cutaneous abscesses in outpatients were cultured anaerobically and aerobically. Of these, 4% were sterile and 29% yielded pure cultures, predominantly of Staphylococcus aureus. Aerobic species were isolated from all anatomic areas. Anaerobes were found with a frequency comparable to aerobes in all nonperineal areas except the hand. In contrast, abscesses in the perineal region contained a greater variety and frequency of anaerobes. Only two patients were febrile. All abscesses were treated with incision and drainage, and all healed without complication, including those 74% that were treated without adjunctive antibiotics. Primary management of cutaneous abscesses should be incision and drainage. In general, routine culture and antibiotic therapy are not indicated for localized abscesses in patients with presumably normal host defenses.

The Clinical Use of Fluoroquinolones for the Treatment of Mycobacterial Diseases

Mycobacterial diseases often require prolonged therapy with multidrug regimens. Fluoroquinolones have excellent bactericidal activity against many mycobacteria; achieve effective serum, tissue, and intracellular levels following oral administration; and produce few adverse effects. These properties have led to the increasing use of fluoroquinolones for the treatment of mycobacterial infections. We reviewed clinical studies and reports involving the use of fluoroquinolones for mycobacterial diseases. Ofloxacin, ciprofloxacin, sparfloxacin, and pefloxacin exhibit clinical efficacy against mycobacterial diseases, especially tuberculosis and leprosy. Fluoroquinolones have generally been administered in regimens that include other agents. However, when a fluoroquinolone has been found to be the sole active agent in a multidrug regimen, the ready emergence of resistance to fluoroquinolones has been recognized, just as when they have been used as monotherapy. Therefore, to forestall the emergence of resistance to fluoroquinolones during the treatment of mycobacterial diseases, these drugs should always be used in combination with at least one other active agent, and they should be used only when effective alternative drugs are not available.

Comparative study of ototoxicity and nephrotoxicity in patients randomly assigned to treatment with amikacin or gentamicin

Fifty-four patients treated with gentamicin and 52 patients treated with amikacin were evaluated for nephrotoxicity and ototoxicity in a prospective, randomized, blinded comparative trail. According to our definition of nephrotoxicity (an increase in serum creatinine levels to at least 50 percent and 0.5 mg/dl above the baseline value), nephrotoxicity occurred in eight (15 percent) of the patients who were treated with gentamicin and none of the patients who were treated with amikacin (p = 0.006). Using several other definitions of nephrotoxicity, the differences in incidence between the treatment arms were not significant. Nephrotoxicity appeared to be associated with impaired baseline renal function, greater age, and the presence of bacteremia. Ototoxicity occurred in six (11 percent) of the 54 gentamicin-treated patients; auditory toxicity occurred in three patients, and toxic changes were observed in three of the 33 patients who could also be evaluated for vestibular toxicity. Similarly, ototoxicity was observed in seven (13 percent) of the 52 amikacin-treated patients; auditory toxicity occurred in four patients, and of the 34 patients who could also be evaluated for vestibular toxicity, three exhibited vestibular toxicity without auditory toxicity are one experienced vestibular effects in addition to those affecting the cochlea. We observed a modest association of ototoxicity with nephrotoxicity and with an elevated mean trough aminoglycoside serum level. The results of this study indicate that amikacin may be less nephrotoxic than gentamicin in humans; however, the broad applicability of this finding to other patient populations is uncertain.

Characterization of fluoroquinolone-resistant mutant strains of Mycobacterium tuberculosis selected in the laboratory and isolated from patients.

To examine the mechanism of resistance to fluoroquinolones in Mycobacterium tuberculosis, we selected spontaneous fluoroquinolone-resistant mutants from a susceptible strain, H37Rv, and studied the susceptibilities of these mutants and two fluoroquinolone-resistant clinical isolates (A-382, A-564) to various fluoroquinolones and to isoniazid and rifampin. Furthermore, since mutations within the quinolone resistance-determining region of the structural gene encoding the A subunit of DNA gyrase are the most common mechanism of acquired resistance, we amplified this region by PCR and compared the nucleotide sequences of the fluoroquinolone-resistant strains with that of the susceptible strain. Fluoroquinolone-resistant mutants of H37Rv appeared at frequencies of 2 x 10(-6) to 1 x 10(-8). For three mutants selected on ciprofloxacin, ofloxacin, and sparfloxacin, respectively, and the two clinical isolates, MICs of ciprofloxacin and ofloxacin were as high as 16 micrograms/ml, and those of sparfloxacin were 4 to 8 micrograms/ml. They displayed cross-resistance to all fluoroquinolones tested but not to isoniazid or rifampin. Sparfloxacin and FQ-A (PD 127391-0002) were the most potent fluoroquinolones. All of the fluoroquinolone-resistant strains (MICs, > or = 4 micrograms/ml) had mutations in the quinolone resistance-determining region which led to substitution of the Asp residue at position 87 (Asp-87) by Asn or Ala or the substitution of Ala-83 by Val in the A subunit of DNA gyrase. Similar mutations have been noted in other bacterial species and recently in mycobacteria. The broad resistance to fluoroquinolones that arose readily by point mutation in the laboratory and apparently during inadequate therapy, as was the case in the clinical isolates, may ultimately lead to to serious restriction of the use of these drugs in the treatment of tuberculosis.

Comparative clinical studies of ototoxicity and nephrotoxicity of amikacin and gentamicin

A comparative study of the oto- and nephrotoxicity of amikacin and gentamicin was carried out prospectively. Twenty-six gentamicin-treated patients and 27 amikacin-treated patients were monitored for changes in auditory and renal function during and after therapy. Thirteen of those treated with gentamicin and 20 of those treated with amikacin underwent vestibular caloric testing which could be evaluated for evidence of toxicity. In four (15.4%) of the gentamicin-treated patients, nephrotoxicity developed; no such toxicity was seen in the amikacin-treated patients. This difference may have been due to a fortuitously higher incidence of pretreatment renal impairment in the gentamicin-treated group. In two gentamicin-treated patients (7.7%), ototoxicity developed (one auditory, one vestibular), and in two amikacin-treated patients (7.4%), auditory toxicity developed. Statistical analysis of oto- and nephrotoxicity and their risk factors was not attempted because of the small numbers of patients who could be evaluated. Additional patients are being studied.

Effects on Substrate Profile by Mutational Substitutions at Positions 164 and 179 of the Class A TEMpUC19 β-Lactamase from Escherichia coli

We investigated the effects of mutations at positions 164 and 179 of the TEMpUC19 β-lactamase on turnover of substrates. The direct consequence of some mutations at these sites is that clinically important expanded-spectrum β-lactams, such as third-generation cephalosporins, which are normally exceedingly poor substrates for class A β-lactamases, bind the active site of these mutant enzymes more favorably. We employed site-saturation mutagenesis at both positions 164 and 179 to identify mutant variants of the parental enzyme that conferred resistance to expanded-spectrum β-lactams by their enhanced ability to turn over these antibiotic substrates. Four of these mutant variants, Arg164 → Asn, Arg164 → Ser, Asp179 → Asn, and Asp179 → Gly, were purified and the details of their catalytic properties were examined in a series of biochemical and kinetic experiments. The effects on the kinetic parameters were such that either activity with the expanded-spectrum β-lactams remained unchanged or, in some cases, the activity was enhanced. The affinity of the enzyme for these poorer substrates (as defined by the dissociation constant, K s ) invariably increased. Computation of the microscopic rate constants (k 2 andk 3) for turnover of these poorer substrates indicated either that the rate-limiting step in turnover was the deacylation step (governed by k 3) or that neither the acylation nor deacylation became the sole rate-limiting step. In a few instances, the rate constants for both the acylation (k 2) and deacylation (k 3) of the extended-spectrum β-lactamase were enhanced. These results were investigated further by molecular modeling experiments, using the crystal structure of the TEMpUC19 β-lactamase. Our results indicated that severe steric interactions between the large 7β functionalities of the expanded-spectrum β-lactams and the Ω-loop secondary structural element near the active site were at the root of the low affinity by the enzyme for these substrates. These conclusions were consistent with the proposal that the aforementioned mutations would enlarge the active site, and hence improve affinity.

Evolution of a Catabolic Pathway in Bacteria

Genetic derepression of ribitol dehydrogenase in a mutant of Aerobacter aerogenes, strain 1033, enabled it to grow on xylitol, a substrate of the enzyme, but not its inducer. A derivative strain with an improved rate of growth on xylitol was obtained from the first mutant. The faster growth rate was made possible by the production of an altered ribitol dehydrogenase, as demonstrated by an increase in its activity on xylitol relative to ribitol and by its decreased heat stability.