Adriana O. Reis

Contemporary Assessment of Antimicrobial Susceptibility Testing Methods for Polymyxin B and Colistin: Review of Available Interpretative Criteria and Quality Control Guidelines

ABSTRACT The emergence of infections caused by multidrug-resistantPseudomonas aeruginosa and Acinetobacter spp. has necessitated the search for alternative parenteral agents such as the polymyxins. The National Committee for Clinical Laboratory Standards (NCCLS) documents do not currently provide interpretative criteria for the testing of the polymyxins, colistin and polymyxin B. Therefore, an evaluation of the antimicrobial activity of colistin and polymyxin B was initiated using 200 bloodstream infection pathogens collected through the SENTRY Antimicrobial Surveillance Program. All susceptibility tests were performed according to the NCCLS recommendations. Polymyxin B and colistin displayed a nearly identical spectrum of activity, exhibiting excellent potency against P. aeruginosa (MIC90, 2 μg/ml) andAcinetobacter sp. (MIC90, 2 μg/ml). In contrast, they showed limited activity against some other nonfermentative bacilli such as Burkholderia cepacia(MIC90, ≥128 μg/ml). Excellent correlation was achieved between broth microdilution and agar dilution tests (r= 0.96 to 0.98); 94.3% of the results were ±1 log2dilution between the methods used for both compounds. At a resistance breakpoint of ≥4 μg/ml for both agents, unacceptable false-susceptible or very major errors were noted for colistin (5%) and polymyxin B (6%). Modified zone criteria for colistin (≤11 and ≥14 mm) and polymyxin B (≤10 and ≥14 mm) were suggested, but some degree of error persisted (≥3.5%). It is recommended that all susceptible disk diffusion results be confirmed by MIC tests using the preferred reference NCCLS method. The quality control (QC) ranges listed in the product package insert require an adjusted range by approximately 3 mm for both NCCLS gram-negative quality control strains. This evaluation of in vitro susceptibility test methods for the polymyxin class drugs confirmed continued serious testing error with the disk diffusion method, the possible need for breakpoint adjustments, and the recalculation of disk diffusion QC ranges. Clinical laboratories should exclusively use MIC methods to assist the therapeutic application of colistin or polymyxin B until disk diffusion test modifications are sanctioned and published by the NCCLS.

Emergence of an IMP-like metallo-enzyme in an Acinetobacter baumannii clinical strain from a Brazilian teaching hospital

Multidrug-resistant Acinetobacter spp. constitute a serious cause of nosocomial infection in Brazilian hospitals. This manuscript reports the first appearance of an IMP-like metallo-beta-lactamase encountered in a clinical isolate of A. baumannii from a Brazilian teaching hospital.

Polymyxin-Resistant Acinetobacter spp. Isolates: What is Next?

To the Editor: In Brazilian hospitals, Acinetobacter spp. has been an important etiologic agent of nosocomial infections, mainly pneumonia (1–3). In general, ampicillin/sulbactam and carbapenems remain the last therapeutic options for treatment of such infections (3,4). However, resistance rates to carbapenems have increased, reaching rates approximately 12% or higher in some Brazilian hospitals (1,3,4). Thus, more toxic agents such as polymyxins have been used as alternative therapeutic drugs against multidrug-resistant Acinetobacter infections (5,6). The clinical use of polymyxins has been based on antimicrobial susceptibility results and previous clinical experience. However, the National Committee for Clinical Laboratory Standards (NCCLS) documents do not currently provide interpretative criteria for the testing of polymyxins (7). In addition, the disk diffusion technique was reported to be an unreliable method for evaluating the susceptibility to polymyxins (8). Since Acinetobacter clinical specimens exhibiting high MICs for polymyxins (MIC, 8–32 μg/mL) were recently detected, we searched for the frequency of occurrence of Acinetobacter spp. strains exhibiting reduced susceptibility to polymyxin B among 100 bloodstream isolates of Acinetobacter spp (8). The bacterial isolates were consecutively collected between September 1999 and December 2000 from a tertiary Brazilian hospital, where Acinetobacter spp. infections have reached endemic levels and polymyxins have been frequently used. Only one isolate per patient was included in the study. The isolates were identified to the species level using the BBL Crystal System (Becton Dickinson, Sparks, MD). The susceptibility to polymyxin B and meropenem were tested by disk diffusion and agar dilution techniques according to NCCLS recommendations (9,10). The susceptibility interpretative criteria for meropenem and polymyxin B were based on the current and former NCCLS documents, respectively (7,11). The MIC was defined as the lowest antimicrobial concentration that inhibited bacterial growth. Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, and Escherichia coli ATCC 25922 were used as quality control strains. Testing errors and agreements were determined by comparing the results of the disk diffusion with the standard criterion agar dilution method. Categorical agreement was obtained when the isolates were classified within the same susceptibility category. The very major and major errors were related to false susceptibility and false resistance results, respectively. To evaluate whether the polymyxin B-resistant strains isolates were epidemiologically related, these isolates were molecularly typed by pulsed-field gel electrophoresis (PFGE) as previously described (12). PFGE patterns were considered identical if they shared every band, similar if they differed from one to three bands, and distinct if they differed by four or more bands (12). Despite the limitation of commercial systems for identifying the genus Acinetobacter at species level, Acinetobacter baumannii (80.0%) was the most commonly identified species, followed by A. lwoffi (4.0%). Sixteen percent of the Acinetobacter isolates were not identified to species level by the BBL Crystal System. Meropenem (MIC50, 1 μg/mL) and polymyxin B (MIC50, 1 μg/mL) showed similar in vitro potency. However, meropenem exhibited the highest susceptibility rate (99.0% susceptible). In contrast to previous studies, only one strain was resistant to meropenem (1–3,8), which indicates that the carbapenem-susceptibility rates among Acinetobacter spp. isolates may vary according to the period evaluated even in the same institution. By using the polymyxin B resistance breakpoint (MIC >4 μg/mL) presented by the former NCCLS document, which was recently validated, we found that five Acinetobacter spp. isolates were considered resistant to polymyxin B (MICs, 8–32 μg/mL) (8,11). All isolates were susceptible to meropenem and belonged to A. baumannnii (4) and A. lwoffi (1) species. The polymyxin B–resistant isolates were categorized as susceptible by disk diffusion (100%, very major error). The disk diffusion method is widely used in Brazil and worldwide. However, disk diffusion was confirmed to be an unreliable test for detecting Acinetobacter spp. isolates with reduced susceptibility to polymyxins. These results are in agreement with those previously reported (8). Among the five polymyxin B–resistant Acinetobacter spp., four distinct patterns were characterized by PFGE. Two polymyxin B–resistant strains, which were isolated from different units of the Sao Paulo Hospital complex, shared an identical PFGE pattern. The PFGE results suggest that the polymyxin B use may have played a role in the selection of resistant strains. On the other hand, two isolates shared an identical PFGE pattern, which raises the possibility of patient-to-patient transmission of epidemic strains. Intra- and interhospital dissemination of multidrug-resistant Acinetobacter spp. clones has already been reported in Brazilian hospitals (13). Our findings suggest that the polymyxin B–resistant strains have emerged because of antimicrobial selective pressure and dissemination of clonal strains. Further epidemiologic studies are necessary to correlate the emergence of polymyxin-resistant Acinetobacter spp. isolates to the clinical response with polymyxin B therapy. Since the emergence of polymyxin B resistance may leave no efficacious drugs for the treatment of infections caused by multidrug-resistant Acinetobacter spp. isolates, strict infection control measures must be adopted to avoid the emergence and spread of such isolates. The low accuracy of routine susceptibility tests, especially disk diffusion, may jeopardize rapid implementation of such measures.

In vitro antimicrobial activity of linezolid tested against vancomycin-resistant enterococci isolated in Brazilian hospitals

The emergence of vancomycin-resistant enterococci (VRE) has been described recently in Brazil. This is in contrast to the USA and Europe, where the VRE appeared in the late 1980s. The progressive increase in VRE isolation poses important problems in the antimicrobial therapy of nosocomial infections. Treatment options and effective antimicrobial agents for VRE are often limited and the possibility of transfer of vancomycin genes to other Gram-positive microorganisms continues. In the search for antimicrobial agents for multiresistant Gram-positive cocci, compounds such as linezolid and quinupristin/dalfopristin have been evaluated. The present study was conducted to evaluate the in vitro activity of the oxazolidinone linezolid and 10 other antimicrobial agents, including quinupristin-dalfopristin, against multiresistant enterococci isolated in Brazilian hospitals. Thirty-three vancomycin resistant isolates (17 Enterococcus faecium and 16 E. faecalis), were analyzed. Strains were isolated from patients at São Paulo Hospital, Oswaldo Cruz Hospital, Hospital do Servidor Público Estadual, Santa Marcelina Hospital, Santa Casa de Misericórdia de São Paulo, and Hospital de Clínicas do Paraná. The samples were tested by a broth microdilution method following the National Committee for Clinical Laboratory Standards (NCCLS) recommendations. All isolates were molecular typed using pulsed-field gel electrophoresis (PFGE). Linezolid was the most active compound against these multiresistant enterococci, showing 100% inhibition at the susceptible breakpoints. Quinupristin/dalfopristin and teicoplanin showed poor activity against both species. The molecular typing results suggest that there has been interhospital spread of vancomycin resistant E. faecium and E. faecalis among Brazilian hospitals. The results of this study indicate that linezolid is an appropriate therapeutic option for the treatment of vancomycin-resistant enterococci infections in Brazil.

Avaliação da qualidade dos discos com antimicrobianos para testes de disco-difusão disponíveis comercialmente no Brasil

Background: The antimicrobial susceptibility test is one of the most important clinical test carried out in the microbiology laboratory. Due to the great number of antimicrobial agents and the complexity of resistance mechanisms evolved, it has become very difficult to detect problems in these tests by simply evaluating the results obtained. Consequently, a quality control program must be performed continuously. Objective: The objective of the present study was to evaluate the quality of the antimicrobial susceptibility disks used in Brazil. Methods: Eighteen antimicrobial susceptibility disks manufactured from five commercial trade marks were evaluated. These antimicrobial disks were tested against four ATCCs and following the National Committee for Clinical Laboratory Standards (NCCLS) procedures for disk-diffusion. Each test was repeated 20 times. Results: None of the trade marks presented a satisfactory performance for routine use in a microbiology laboratory. The best performance was presented by Cecon ® , with 89,6% of overall agreement. Sensifar® disks showed a similar overall concordance of 90,8%. The trade mark with the least adequate performance was Pimenta Abreu® , with only 58,6% overall agreement. Conclusion: The results from this study indicate that the disks commercialized in Brazil areIntroducao: O teste de suscetibilidade a antimicrobianos representa um dos testes de maior importância clinica realizados pelo laboratorio de microbiologia. Devido ao grande numero de antimicrobianos e a complexidade dos mecanismos de resistencia desenvolvidos pelas bacterias, fica muito dificil hoje a deteccao de problemas nos testes de suscetibilidade pela simples avaliacao dos resultados obtidos. Sendo assim, e extremamente importante que haja uma avaliacao constante da qualidade destes testes. Objetivo: O objetivo do presente estudo foi avaliar a qualidade dos discos de antimicrobianos comercializados no Brasil. Material e metodos: Foram avaliados discos de 18 antimicrobianos obtidos de cinco diferentes fontes comerciais, os quais foram testados frente a quatro cepas bacterianas oriundas da ATCC, pelo metodo de difusao em agar, seguindo as recomendacoes do National Committee for Clinical Laboratory Standards (NCCLS). Cada teste foi repetido 20 vezes. Resultados: Nenhuma das marcas apresentou desempenho satisfatorio para o uso na rotina de um laboratorio de microbiologia. O melhor desempenho foi apresentado pela marca Cecon®, com 89,6% de concordância. A marca Sensifar® apresentou taxa de concordância geral semelhante (90,8%). A marca com o pior desempenho foi a Pimenta Abreu®, com apenas 58,6% de concordância. Conclusao: Os resultados do presente estudo indicam que os discos de antimicrobianos comercializados no Brasil sao de baixa qualidade, possivelmente refletindo a falta de controle de qualidade na producao e/ou estocagem dos produtos antes da sua distribuicao. Esses dados chamam a atencao para a necessidade de implantacao de sistemas efetivos de fiscalizacao da comercializacao desses produtos e de programas criteriosos de controle de qualidade por parte dos laboratorios que os utilizam.

In vitro antimicrobial activity of the aminoglycoside arbekacin tested against oxacillin-resistant Staphylococcus aureus isolated in Brazilian hospitals.

Arbekacin is an aminoglycoside used in Japan for treating infections caused by gentamicin and oxacillin-resistant S. aureus (ORSA). The objective of this study was to determine the in vitro antimicrobial activity of arbekacin against 454 clinical isolates of ORSA. The isolates were consecutively collected between January and July, 2000, from patients hospitalized in 8 Brazilian medical centers. The antimicrobial susceptibility testing was performed by disk diffusion method according to NCCLS recommendations. The vast majority of the isolates, 453 strains (99.8%), were considered susceptible to arbekacin based on the criteria proposed by the Requirements for Antibiotic Products of Japan. Only 1 isolate (0.2%) was classified as resistant. On the other hand, high rates of resistance were demonstrated for other aminoglycosides, such as gentamicin (97.6% resistance) and amikacin (97.0% resistance). Resistance rate was also high for ciprofloxacin (98.0%). All isolates were considered susceptible to vancomycin. The excellent in vitro antimicrobial activity of arbekacin demonstrated in this study indicates that this antimicrobial agent may play an important role in the treatment of severe ORSA infections, especially those that show poor clinical response with vancomycin monotherapy. Since the aminoglycosides should not be used as monotherapy to treat Gram positive infections, further studies evaluating in vitro and in vivo synergistic activity of arbekacin combinations are necessary to clarify the clinical role of this aminoglycoside.

Sensibilidade a antimicrobianos de bactérias isoladas do trato respiratório de pacientes com infecções respiratórias adquiridas na comunidade: resultados brasileiros do Programa SENTRY de Vigilância de Resistência a Antimicrobianos dos anos de 1997 e 1998

Background: Antimicrobial treatment of community-acquired respiratory tract infections (CARTI) is usually empiric and antibiotics are chosen on the basis of surveillance studies. The SENTRY Program was designed to monitor antimicrobial resistance via a worldwide surveillance network of sentinel laboratories. Three sites in Brazil participated in the 1997 and 1998 SENTRY Program. Methods: A total of 344 bacterial isolates, collected from patients with CARTI in 1997 and 1998, were tested against more than 20 antimicrobial agents by the broth microdilution method. Results: Among S. pneumoniae (176 isolates), 71.6% were susceptible to penicillin. High level resistance to penicillin and resistance to cefotaxime was found in 2.3 and 4.0%, respectively. The newer quinolones, levofloxacin, (MIC90 of 2 mg/mL) and gatifloxacin (MIC90 of 0.5 mg/mL) were active against 100% of the isolates tested. Among the other non-b-lactam drugs tested, the rank order of pneumococci activity was (% susceptible): chloramphenicol (97.5%) > clindamycin (94.0%) > azithromycin (90.3%) > clarithromycin (89.4%) > tetracycline (76.4%) > trimethoprim/sulfamethoxazole (60.2%). The percentage of Haemophilus influenzae (101 isolates) susceptible to amoxicillin was 90.1%, whereas among Moraxella catarrhalis (67 isolates) only 9.0% were susceptible. Clavulanic acid restored the activity of amoxicillin against both H. influenzae and M. catarrhalis. However, H. influenzae showed increased levels of resistance to trimethoprim/sulfametoxazole (55.1% susceptibility), clarithromycin (80.4% susceptibility), and cefaclor (88.2%) susceptibility). All H. influenzae and M. catarrhalis isolates were susceptible to levofloxacin (MIC90, < 0.5 mg/mL for both) and gatifloxacin (MIC90, < 0.06 mg/mL for both) with very low MICs. Conclusion: Results indicate that the rate of S. pneumoniae showing high-level penicillin resistance is still low in Brazil. However, intermediate resistance to penicillin associated with resistance to other classes of antimicrobial agents was relatively high. On the other hand, the new quinolones were highly active against 100% of the respiratory pathogens tested.