Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance.

Many different definitions for multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR) bacteria are being used in the medical literature to characterize the different patterns of resistance found in healthcare-associated, antimicrobial-resistant bacteria. A group of international experts came together through a joint initiative by the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC), to create a standardized international terminology with which to describe acquired resistance profiles in Staphylococcus aureus, Enterococcus spp., Enterobacteriaceae (other than Salmonella and Shigella), Pseudomonas aeruginosa and Acinetobacter spp., all bacteria often responsible for healthcare-associated infections and prone to multidrug resistance. Epidemiologically significant antimicrobial categories were constructed for each bacterium. Lists of antimicrobial categories proposed for antimicrobial susceptibility testing were created using documents and breakpoints from the Clinical Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the United States Food and Drug Administration (FDA). MDR was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories, XDR was defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e. bacterial isolates remain susceptible to only one or two categories) and PDR was defined as non-susceptibility to all agents in all antimicrobial categories. To ensure correct application of these definitions, bacterial isolates should be tested against all or nearly all of the antimicrobial agents within the antimicrobial categories and selective reporting and suppression of results should be avoided.

Evaluation of methods to identify the Klebsiella pneumoniae carbapenemase in Enterobacteriaceae.

The Klebsiella pneumoniae carbapenem (KPC) beta-lactamase occurs in Enterobacteriaceae and can confer resistance to all beta-lactam agents including carbapenems. The enzyme may confer low-level carbapenem resistance, and the failure of susceptibility methods to identify this resistance has been reported. Automated and nonautomated methods for carbapenem susceptibility were evaluated for identification of KPC-mediated resistance. Ertapenem was a more sensitive indicator of KPC resistance than meropenem and imipenem independently of the method used. Carbapenemase production could be confirmed with the modified Hodge test.

Evaluation of the AdvanDx VRE EVIGENE assay for detection of vanA in vancomycin-resistant Staphylococcus aureus.

AdvanDx VRE EVIGENE, a commercial vanA/vanB DNA hybridization assay to identify vancomycin-resistant enterococci (VRE), was evaluated for the detection of vanA in Staphylococcus aureus. Performance was assessed using S. aureus, VRE, and vancomycin-intermediate and -susceptible isolates. The assay demonstrated 100% sensitivity and specificity when analyzed visually and by optical density.

Development of vancomycin and lysostaphin resistance in a methicillin-resistant Staphylococcus aureus isolate.

Glycopeptide resistance in Staphylococcus aureus is poorly understood. The diversity of change documented in cell walls of clinical glycopeptide-intermediate S. aureus (GISA) isolates is evidence that a single genetic or biochemical change cannot account for resistance in all isolates described to date. Therefore, identification of new GISA clinical isolates provides an opportunity to gain insight into the range of adaptive strategies employed by staphylococci to survive in the presence of glycopeptides. In April 1999, a GISA isolate was obtained from the blood of a 63-year-old dialysis patient in Illinois. This isolate was one of six clonally identical MRSA isolates (A-F) serially obtained from the blood of this patient who was receiving vancomycin therapy. All isolates were resistant to oxacillin (MIC > 256 mg/L). The initial isolate had an MIC of vancomycin of 1 mg/L. However, the presence of a subpopulation that could grow in the presence of 5 mg/L of vancomycin indicated that this isolate was predisposed to the acquisition of the GISA phenotype (MIC of vancomycin 10-12 mg/L), which occurred 13 days later, associated with an increased MIC of the endopeptidase lysostaphin and slightly increased cell wall thickness. The first and last isolates in the series, A and F, resisted killing when incubated in vancomycin 2 mg/L, resisted autolysis when incubated in Triton X-100 and had a decreased expression of a c. 116 kDa autolytic band, properties that were different from glycopeptide-susceptible control isolates. Lysostaphin resistance was not accompanied by alterations in the peptidoglycan pentaglycine cross-bridge or a decrease in oxacillin MIC. These data, when taken together with the demonstration of increased cross-linking in isolate F compared with isolate A, demonstrate that vancomycin resistance in these isolates probably occurred by a mechanism different from that of other GISA isolates described to date.

In vitro adherence of Staphylococcus epidermidis to silicone punctual plugs and collagen implants.

PURPOSE: To evaluate in vitro adherence of Staphylococcus epidermidis to silicone punctal plugs and collagen implants. SETTING: Loyola University Medical Center, Maywood, Illinois, USA. METHODS: Silicone punctal plugs and collagen implants were exposed to S epidermidis (3 x 10(8) colony forming units/mL) for 0, 5, 30, and 60 minutes, rinsed in sterile saline, and processed for light, scanning (SEM), and transmission (TEM) electron microscopy. Scanning electron microscopy (x2000) was used to quantify bacteria/mm2 adhering to the devices. RESULTS: The mean S epidermidis/mm2 +/- (SD) adhering to each device were as follows: baseline, silicone punctal plug, 1593 +/- 899, and collagen implant, 7168 +/- 2895 (P =.000, paired Student t test); 5 minutes, silicone punctal plug, 3833 +/- 537, and collagen implant, 6571 +/- 2240 (P =.008); 30 minutes, silicone punctal plug, 13 988 +/- 9076, and collagen implant, 10 404 +/- 1731 (P =.2616); and 60 minutes, silicone punctal plug, 12 644 +/- 10 402, and collagen implant, 11 748 +/- 2685 (P =.8056). CONCLUSIONS: Staphylococcus epidermidis adhered significantly more to collagen implants than to silicone punctal plugs at 0 and 5 minutes. No significant difference in bacterial adherence was seen at 30 and 60 minutes. For both devices, bacterial adherence increased with increasing exposure.

Fatal Chaetomium cerebritis in a bone marrow transplant patient.

The number of opportunistic infections in the central nervous system (CNS) has been steadily increasing because of a rising number of immunocompromised patients. A rare form of CNS infection can be caused by Chaetomium species, one of the largest genera of saprophytic ascomycetes. The CNS lesions in the present case were caused by Chaetomium atrobrunneum. The main characteristic of almost all Chaetomium species is presence of hairs or setae covering the ascomata. Microbiological studies are the only definitive way to correctly identify this fungal organism. The rapid evolvement of the cerebral infection suggests that the brain tissue provides a favorable environment for growth and proliferation of these fungi. This is the second documented case of a fatal brain abscess caused by Chaetomium atrobrunneum, and the first case report in a bone marrow transplant patient.

Amplification of rDNA loci to detect and type Neisseria meningitidis and other eubacteria.

In 1991-92, Neisseria meningitidis group C was isolated from the blood of eight students in Urbana, Illinois, USA, and from the cerebrospinal fluid of one student from a nearby community, Decatur, Illinois. These and other bacterial species were analysed by PCR fingerprinting using primers selected from the ribosomal (r)DNA loci. A rDNA primer pair spanning a region within the 16S rDNA amplified a predicted 280 base pair (bp) DNA fragment from Neisseria spp. and fragments of different sizes for other genera. This primer pair specifically detected a carrier of N. meningitidis in a small clinical battery. Identity of the fragment was confirmed by restriction endonuclease analysis. A 600 bp fragment was also amplified from the 16S-23S internal transcribed spacer (ITS) of N. meningitidis; amplification from six other genera yielded different-sized fragments. Digestion of the ITS fragment from N. meningitidis with Alu I revealed three patterns; pattern I was found only for serogroup C isolates, and it was the dominant pattern among recent isolates with the exception of the one from Decatur. The isolate from Decatur yielded pattern III which suggested a non-clonal relationship to the seven isolates from Urbana. Patterns II and III were more prevalent in isolates from the 1960's and 1980's. PCR-based analysis of these loci can complement the techniques which are currently used for the detection and typing of these and other eubacteria.

Identification of Streptococcus pneumoniae with a DNA probe.

The Accuprobe Streptococcus pneumoniae Culture Identification Test (Gen-Probe, Inc.) was evaluated with 172 isolates of S. pneumoniae and 204 nonpneumococcal isolates. The sensitivity and specificity of the Accuprobe test were 100%. Optimum results were obtained when four or more discrete colonies were selected for testing. The Accuprobe test was determined to be an accurate and rapid method for identification of S. pneumoniae.

Nutritionally variant Streptococcus pyogenes from a periorbital abscess.

A nutritionally variant Streptococcus pyogenes strain was isolated from a periorbital abscess. The organism was identified with the use of three rapid biochemical test kits, and the group A antigen was detected by conventional serology as well as direct antigen detection tests.

Clinical comparison of the Isolator 1.5 microbial tube and the BACTEC radiometric system for detection of bacteremia in children.

The Isolator 1.5 microbial tube (E. I. du Pont de Nemours & Co., Inc., Wilmington , Del.) was compared with the BACTEC radiometric detection system (Johnston Laboratories, Inc., Cockeysville, Md.) for the detection of bacteremia in children. The Isolator 1.5 is a blood culture system designed for small volumes of blood (0.5 to 1.5 ml). The method involves lysis of the cells of the patient and the direct plating of the entire blood lysate on agar media appropriate for the growth of fastidious microorganisms. Of 1,500 paired samples inoculated into the two systems, 68 were positive for 73 clinically significant organisms. The Isolator 1.5 recovered 81% of the positive cultures compared with 84% recovered by the BACTEC system. When paired blood samples with disproportionate volumes were excluded, the Isolator 1.5 detected 3% more positive cultures. More isolates of Streptococcus pneumoniae and Neisseria meningitidis were recovered by the Isolator 1.5, whereas Haemophilus influenzae was recovered most often in the BACTEC bottles (P greater than 0.1). The contamination rates were 8.7 and 3.1% for the Isolator 1.5 and the BACTEC system, respectively. In cultures positive by both systems, the mean time to detection was 4.1 h faster with the Isolator 1.5. The mean time to obtain isolated colonies was 26.6 h faster with the Isolator 1.5. These data indicate the potential value of the Isolator 1.5 microbial tube as a simple, rapid, and sensitive method for the detection of bacteremia in children.

Quantitative determination in human sera of vaccine-induced antibody to type-specific polysaccharides of group B streptococci using an enzyme-linked immunosorbent assay.

The humoral immune response of human volunteers vaccinated with highly purified type II-or type III-specific polysaccharide of group B streptococci was evaluated using an enzyme-linked immunosorbent assay standardized with quantitative precipitin analysis, a method which permits calculation of the micrograms of specific antibody protein per milliliter of serum, rather than expression of the data as titers. By inhibition studies, the assays were shown to be specific for antibody to the undegraded type II or III polysaccharide antigen. Purity of the antigens and the specificity of the immune response to them were evidenced by an increase in level of antibody only to the type-specific antigen used for immunization. The isotype of the antibody raised in the sera of immunized volunteers was primarily IgG, thus confirming the potential utility of vaccination against group B streptococci using polysaccharide vaccines to induce antibodies which will cross the human placenta.

Soluble group- and type-specific antigens from type III group B Streptococcus.

Two soluble polysaccharide antigens of a type III group B Streptoccus were isolated from the culture medium after growth of strain M732 in a chemically defined broth supplemented with acid-hydrolyzed casein. The type- and group- specific antigens were isolated from the culture supernatant by anion-exchange chromatography with diethylaminoethyl-Sephacel. Two carbohydrate-containing peaks, which had serological reactivity with group B or type III antiserum, respectively, were eluted with a linear NaCl gradient and further purified by gel filtration. The type III polysaccharide was found to contain glucose, galactose, glucosamine, and sialic acid, whereas the group B polysaccharide contained galactose, glucosamine, and rhamnose. For the type III polysaccharide, sialic acid was shown to be the major immunodeterminant, and for the group B polysaccharide, rhamnose was the immunodominant sugar. Both the type III and group B polysaccharides were obtained in high yields without employing harsh physical or chemical treatment and both were immunologically distinct. By immunoelectrophoresis or counterimmunoelectrophoresis, type III antigen failed to react with group-specific antiserum and the group B antigen failed to react with type III antiserum.

Antimicrobial therapy of vitamin B6-dependent streptococcal endocarditis.

Vitamin B6-dependent viridans streptococci were isolated from two patients with microbial endocarditis. Because of their unique requirement for pyridoxal hydrochloride, these organisms did not grow normally in the media usually used in diagnostic laboratories. When tested in supplemented media, both strains were resistant to penicillin G and relatively sensitive to streptomycin. Penicillin-streptomycin synergy was demonstrated in vitro as well as in experimental endocarditis. These laboratory findings confirmed the clinical observations in these two patients that penicillin-streptomycin therapy should be used in vitamin B6-dependent streptococcal endocarditis. Nutritionally varient streptococci may be important pathogens in microbial endocarditis and must be considered in patients with suggestive clinical findings but negative blood cultures.

Vitamin B6-dependent Streptococcus mitior (mitis) isolated from patients with systemic infections.

Strains of nutritional-variant viridans Streptococcus were isolated from two patients with bacterial endocarditis and from one patient with a pancreatic abscess. All three strains grew as satellite colonies of other bacteria but did not grow as pure cultures in media lacking sufficient thiol compounds. For one-half maximal growth these organisms required the addition of one of the active forms of the B6 vitamin, pyridoxal with HCl (0.16-1.45 mug/ml) or pyridoxamine dihydrochloride (0.67-2.0 mug/ml), or addition of l-cysteine (0.235-0.425 mg/ml) to routine bacteriological media. The active forms of vitamin B6 are essential coenzymes in the synthesis of l-cysteine and of other thiol compounds. Use of media supplemented with 0.001 percent pyridoxal with HCL led to identification of the strains as Streptococcus mitior (mitis). Incorporation of pyridoxal with HCl, pyridoxamine dihydrochloride, or l-cysteine into media is recommended for the isolation and identification of vitamin B6-dependent viridans streptococci.