MicroScan WalkAway System Research Articles

The First Case of Septicemia Caused by Imipenem-Susceptible, Meropenem-Resistant Klebsiella pneumoniae

The First Case of Septicemia Caused by Imipenem-Susceptible, Meropenem-Resistant <i>Klebsiella pneumoniae</i>

Direct identification of bacteria in urine samples by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and relevance of defensins as interfering factors

Standard methods for the identification of uropathogens that are based on the determination of metabolic activity require cultivation on agar plates, which often takes more than 1 day. If microbial growth on agar plates is slow, or if metabolic activity is impaired by adverse interactions resulting from the patient's condition or from medical treatment, the application of standard methods may lead to delayed or erroneous identification of bacteria. In recent studies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be able to rapidly identify bacteria obtained from cultures. We tested the applicability of this analytical technique for the rapid identification of bacteria collected directly from urine samples and compared the results with those of conventional identification methods, such as the Vitek system, the MicroScan WalkAway system and the API system, and in some cases with the gas chromatographic determination of the bacterial long-chain fatty acid pattern. We analysed a total of 107 urine samples with bacterial counts ranging from 10(2) to ≥10(5) c.f.u. ml(-1). Mass spectrometric identification of bacteria was accomplished for 62 of these samples. In the mass spectra obtained from 40 of the 45 urine samples for which no identification result was achieved, a triplet of very intense peaks corresponding to the human α-defensins 1, 2 and 3 occurred at m/z values of around 3440 Da. This signal suppressed the intensity of the bacterial protein peaks and thus impaired database matching. Our results show that MALDI-TOF MS allows the reliable direct identification of bacteria in urine samples at concentrations as low as 10(3) c.f.u. ml(-1). In a subset of samples, human defensins may occur and impair the mass spectrometric identification of bacteria.

Mechanisms of resistance in clinical isolates of Pseudomonas aeruginosa less susceptible to cefepime than to ceftazidime

The MIC of cefepime determined with the MicroScan WalkAway system was ≥2 times higher than that of ceftazidime for 105 clinical isolates of Pseudomonas aeruginosa. This phenotype was confirmed by reference microdilution in 68 (64.8%) isolates, corresponding to 48 different rep-PCR patterns. The PSE-1 blactamase was identified in only 13.2% isolates, while oxacillinases were not identified in any of the 68 isolates. The level of expression of mexB, mexD and mexY was determined by real-time RT-PCR in eight clinical isolates representative of the different clones and patterns of susceptibility to cefepime and ceftazidime and in strain PAO1. All clinical strains overexpressed the mexY gene (18.3- to 152.7-fold in comparison with PAO1), although there was not a linear relationship between MIC of cefepime and level of mexY expression. Five of these strains contained mutations in the regulatory gene mexZ. mexD and mexB were also overexpressed in three and two isolates, respectively. Different mutations were observed in the regulatory genes nalD, mexR, nfxB and nalC. In conclusion, we have documented in our institution a polyclonal spread of P. aeruginosa with higher MICs of cefepime than of ceftazidime, related to overexpression of Mex-XY-OprM, coincident in some isolates with the production of PSE-1, Mex-CD-OprJ or MexAB-OprM.

Comparison of resistance in isolates of Enterococcus faecalis and Enterococcus faecium

Objectives: Enterococci are Gram positive cocci causing hospital- and community-acquired infections even though they are found in normal flora and they have low-virulence. Their most common species detected as the causative agent are Enterococcus faecalis and Enterococcus faecium. This study aimed to evaluate the differences between the antibiotic susceptibilities of the isolates of these two species isolated from various clinical specimens. Materials and methods: In this study, 68 enterococci strains (40 E.faecalis and 28 E.faecium) isolated from the specimens sent to the clinical microbiology laboratory of Zonguldak Karaelmas Practice and Research Hospital in 2007-2008 were evaluated. Identification at species level and the susceptibilities were performed by using MicroScan WalkAway system (Dade Behring, USA). Results: No resistance to glycopeptides were detected among these isolates. High rates of resistance to ciprofloxacin, rifampicin and erytromycin were observed. Resistance rates to penicillin, ampicillin, ciprofloxacin, rifampicin, erytromycin and high-level gentamicin and streptomycin in E.faecium isolates were found statistically higher than the rates of E.faecalis group (p<0.05). Conclusions: As concordant with the literature, this study observed that E.faecium isolates were significantly more resistant. In enterococcal infections, performing the identification at the species level and determining the antibiotic susceptibility will be helpful in managing of the treatment.

A Biosensor Platform for Rapid Antimicrobial Susceptibility Testing Directly From Clinical Samples

A significant barrier to efficient antibiotic management of infection is that the standard diagnostic methodologies do not provide results at the point of care. The delays between sample collection and bacterial culture and antibiotic susceptibility reporting have led to empirical use of antibiotics, contributing to the emergence of drug resistant pathogens. As a key step toward the development of a point of care device for determining the antibiotic susceptibility of urinary tract pathogens, we report on a biosensor based antimicrobial susceptibility test. For assay development bacteria were cultured with or without antibiotics, and growth was quantitated by determining viable counts and electrochemical biosensor measurement of bacterial 16S rRNA. To determine antibiotic susceptibility directly from patient samples, urine was cultured on antibiotic plates for 2.5 hours and growth was determined by electrochemical measurement of bacterial 16S rRNA. For assay validation 252 urine samples were collected from patients at the Spinal Cord Injury Service at Veterans Affairs Palo Alto Health Care System. The biosensor based antimicrobial susceptibility test was completed for samples containing gram-negative organisms. Pathogen identification and antibiotic susceptibility results were compared between our assay and standard microbiological analysis. A direct biosensor quantitation of bacterial 16S rRNA can be used to monitor bacterial growth for a biosensor based antimicrobial susceptibility test. Clinical validation of a biosensor based antimicrobial susceptibility test with patient urine samples demonstrated that this test was 94% accurate in 368 pathogen-antibiotic tests compared to standard microbiological analysis. This biosensor based antimicrobial susceptibility test, in concert with our previously described pathogen identification assay, can provide culture and susceptibility information directly from a urine sample within 3.5 hours.

Antibiotic Susceptibility Patterns of Ocular Bacterial Flora in Patients Undergoing Intravitreal Injections

To determine the antibiotic susceptibility patterns of conjunctival flora in patients undergoing intravitreal (IVT) injection. Prospective, observational study. Patients (n = 85) scheduled to undergo 136 IVT injections at California Vitreoretinal Center at Stanford University. Conjunctival cultures were obtained on the day of the IVT injection from the injection site bulbar conjunctiva before the application of povidone-iodine or antibiotics. Bacterial isolates were identified and tested for antibiotic susceptibility using either the Kirby-Bauer disc-diffusion technique or MicroScan-WalkAway system. Our analysis included 136 samples collected from 90 eyes of 85 patients. Of those with positive cultures (n = 65), the most common bacterial isolates were coagulase-negative staphylococci (CNS), comprising 59 (83%) of the 71 bacterial strains. Among the CNS, all were susceptible to vancomycin and >80% were susceptible to gentamicin, chloramphenicol, tetracycline, and imipenem. Between 60% and 80% of the CNS were susceptible to the cephalosporins and newer generation fluoroquinolones. Fewer than 60% were susceptible to the penicillin analogs, erythromycin, and the earlier generation fluoroquinolones. Nearly half of the CNS (47%) were resistant to oxacillin/methicillin. Bacteria isolated from the injection site of patients undergoing IVT injections were mostly CNS. Most are sensitive to vancomycin, gentamicin, and chloramphenicol. Proprietary or commercial disclosure may be found after the references.

Failure of the MicroScan WalkAway System To Detect Heteroresistance to Carbapenems in a Patient with Enterobacter aerogenes Bacteremia

We report the failure of the automated MicroScan WalkAway system to detect carbapenem heteroresistance in Enterobacter aerogenes. Carbapenem resistance has become an increasing concern in recent years, and robust surveillance is required to prevent dissemination of resistant strains. Reliance on automated systems may delay the detection of emerging resistance.

Detection of Pseudomonas aeruginosa producing metallo-β-lactamase VIM-2 in a central hospital from Portugal

Pseudomonas aeruginosa remains one of the most important pathogens in the nosocomial setting [1]. P. aeruginosa exhibits intrinsic resistance to several antimicrobial agents. The antipseudomonal β-lactams represent a major weapon against Pseudomonas infections, either for monotherapy or for combination therapy, for which β-lactams almost invariably represent one of the components. Therefore, resistance to these agents constitutes a major challenge for anti-Pseudomonas chemotherapy. Several mechanisms can contribute to β-lactam resistance in P. aeruginosa, including β-lactamase production, outer membrane impermeability and active efflux mediated by RND-type efflux systems [1]. During the last decade, the metallo-β-lactamases (MBLs) have emerged as new threatening mechanisms of broad-spectrum β-lactam resistance in P. aeruginosa. In fact, these enzymes can efficiently degrade virtually all antipseudomonal β-lactams (except aztreonam), while they are not susceptible to therapeutic β-lactamases inhibitors [2]. Based on amino acid sequence homology, these MBLs have been classified into four major types: IMP, VIM, SPM and GIM. Clinical isolates harbouring the MBLs IMP and VIM have been increasingly reported worldwide, mostly in European and Asian countries [2]. This increase in occurrence, types and rate of dissemination of MBLs makes early detection very critical. The benefits of such treatment include the timely implementation of strict infection control practices, as well as clinical guidance. The aim of this study was to determine the presence of these enzymes in imipenem-resistant P. aeruginosa isolates collected at the Centro Hospitalar of Coimbra (CHC), during a two-year period (April 2003 to April 2005) and to ascertain their clonal relationship. CHC is a cluster formed by a central hospital and several specialised units located at distances of less than 8 km, namely, Hospital dos Covoes (central hospital), Instituto Maternal (maternity hospital) and Hospital Pediatrico (paediatric hospital). The Microbiology Laboratory also analysed samples from another hospital (Hospital de Pombal) located 30 km away. Bacterial identification and minimal inhibitory concentrations (MIC) were performed with the MicroScan WalkAway (Dadebehring) system according to the instructions of the manufacturer and API32GN or API PSE systems (bioMerieux). All intermediate strains were considered as nonsusceptible strains. The MICs of β-lactams were also determined by E-test in isolates that presented the enzyme VIM-2. The results were interpreted on the basis of CLSI recommended breakpoints [3]. The screening of MBLs was done by the double combined disk test [4]. Polymerase chain reaction (PCR) analyses for the detection of MBLs genes (blaIMP, blaVIM, blaGIM and blaSPM-1) were carried out for all strains in which the screening test gave positive results [5]. The amplicons were directly sequenced on both DNA strands on an ABI PRISM 377 automated sequencer. The nucleotide and deduced amino acid sequences were analysed with software available online (http://blast.ncbi.nlm.nih.gov/ Eur J Clin Microbiol Infect Dis (2008) 27:1269–1271 DOI 10.1007/s10096-008-0579-2

Direct Comparison of the BD Phoenix System with the MicroScan WalkAway System for Identification and Antimicrobial Susceptibility Testing of Enterobacteriaceae and Nonfermentative Gram-Negative Organisms

The Phoenix automated microbiology system (BD Diagnostics, Sparks, MD) is designed for the rapid identification (ID) and antimicrobial susceptibility testing (AST) of clinically significant human bacterial pathogens. We evaluated the performance of the Phoenix instrument in comparison with that of the MicroScan WalkAway system (Dade Behring, West Sacramento, CA) in the ID and AST of gram-negative clinical strains and challenge isolates of Enterobacteriaceae (n = 150) and nonfermentative gram-negative bacilli (NFGNB; 45 clinical isolates and 8 challenge isolates). ID discrepancies were resolved with the API 20E and API 20NE conventional biochemical ID systems (bioMerieux, Durham, NC). The standard disk diffusion method was used to resolve discordant AST results. The overall percentages of agreement between the Phoenix ID results and the MicroScan results at the genus and species levels for clinical isolates of Enterobacteriaceae were 98.7 and 97.7%, respectively; following resolution with conventional biochemical testing, the accuracy of the Phoenix system was determined to be 100%. For NFGNB, the levels of agreement were 100 and 97.7%, respectively. Both systems incorrectly identified the majority of the uncommon nonfermentative nonpseudomonal challenge isolates recovered from cystic fibrosis patients; these isolates are not included in the databases of the respective systems. For AST of Enterobacteriaceae, the rate of complete agreement between the Phoenix results and the MicroScan results was 97%; the rates of very major, major, and minor errors were 0.3, 0.2, and 2.7%, respectively. For NFGNB, the rate of complete agreement between the Phoenix results and the MicroScan results was 89.1%; the rates of very major, major, and minor errors were 0, 0.5, and 7.7%, respectively. Following the confirmatory testing of nine clinical isolates initially screened by the MicroScan system as possible extended-spectrum-beta-lactamase (ESBL)-producing organisms (seven Klebsiella pneumoniae isolates and two Escherichia coli isolates), complete agreement was achieved for eight isolates (one ESBL positive and seven negative); one false positive was obtained with the Phoenix instrument. The MicroScan system correctly detected the 10 ESBL challenge isolates, versus the 6 detected by the Phoenix system. Overall, there was good correlation between the Phoenix instrument and the MicroScan system for the ID and AST of Enterobacteriaceae and common NFGNB. The Phoenix system is a reliable method for the ID and AST of the majority of clinical strains encountered in the clinical microbiology laboratory. Until additional performance data are available, results for all Klebsiella pneumoniae or Klebsiella oxytoca and E. coli isolates screened and confirmed as ESBL producers by any automated system should be confirmed by alternate methods prior to the release of final results.

False susceptibility to cefotetan reported by MicroScan for DHA-type AmpC β-lactamase-producing Klebsiella pneumoniae

This study evaluated the accuracy of cefotetan susceptibility determination using the MicroScan WalkAway system for AmpC-producing Klebsiella pneumoniae. In total, 57 K. pneumoniae isolates that showed a D-shape flattening in a double-disk synergy test were studied. Cefotetan MICs were determined by the agar dilution method. The bla(DHA) gene was detected in all 57 isolates, one of which co-harboured bla(CMY-1). According to the MicroScan system, 28 isolates were susceptible, 18 were intermediately-resistant, and 11 were resistant to cefotetan. Compared with the agar dilution method, very major, minor and major error rates were 28.1% (16/57), 47.4% (27/57) and 1.8% (1/57), respectively.

P1634 Critical assessment of P. aeruginosa susceptibility testing results for six β-lactams using the BD Phoenix system

The MIC of cefepime determined with the MicroScan WalkAway system was ≥2 times higher than that of ceftazidime for 105 clinical isolates of Pseudomonas aeruginosa. This phenotype was confirmed by reference microdilution in 68 (64.8%) isolates, corresponding to 48 different rep-PCR patterns. The PSE-1 blactamase was identified in only 13.2% isolates, while oxacillinases were not identified in any of the 68 isolates. The level of expression of mexB, mexD and mexY was determined by real-time RT-PCR in eight clinical isolates representative of the different clones and patterns of susceptibility to cefepime and ceftazidime and in strain PAO1. All clinical strains overexpressed the mexY gene (18.3- to 152.7-fold in comparison with PAO1), although there was not a linear relationship between MIC of cefepime and level of mexY expression. Five of these strains contained mutations in the regulatory gene mexZ. mexD and mexB were also overexpressed in three and two isolates, respectively. Different mutations were observed in the regulatory genes nalD, mexR, nfxB and nalC. In conclusion, we have documented in our institution a polyclonal spread of P. aeruginosa with higher MICs of cefepime than of ceftazidime, related to overexpression of Mex-XY-OprM, coincident in some isolates with the production of PSE-1, Mex-CD-OprJ or MexAB-OprM.

Ocular infection secondary to gemella

To report four cases of ocular infection caused by Gemella species. Microbial identification was carried out using the MicroScan Walkaway system (Dade Behring) and/or Vitek-2 (bioMérieux) and susceptibility testing with either Kirby-Bauer disc diffusion and/or Vitek-2. The paper is presented as an interventional case report. Four patients developed infection due to Gemella species. All responded to vancomycin or fortified aminoglycosides and broad-spectrum antibiotics. Gemella spp. can cause opportunistic infections in the eye.

Epidemiological and microbiological aspects of acute bacterial diarrhea in children from Salvador, Bahia, Brazil

In the few cases of acute childhood diarrhea that require antimicrobial therapy, the correct choice of the drug depends on detailed previous knowledge of local strains. In order to establish such parameters in our city, we reviewed the results of all 260 positive stool cultures of children between 0 and 15 years of age during two years at a pediatric tertiary care facility in Salvador, Brazil. Bacterial strains had been presumptively identified by culturing in selective media and by biochemical testing, and their antimicrobial susceptibility patterns were automatically detected by the MicroScan Walkaway System. Data about patients' sex and age, monthly distribution of the cases, pathogens isolated and their antimicrobial resistance patterns were recorded. Males corresponded to 55.4% of our sample, and most of our patients (42.7%) were between one and four years of age. Shigella was the commonest pathogen, being found in 141 (54.3%) cultures, while Salmonella was found in 100 (38.4%) cultures and Enteropathogenic E. coli in 19 (7.3%). Salmonella was the main causal agent of diarrhea in children younger than five years old, whereas Shigella was the most frequent pathogen isolated from the stools of children between five and 15 years old. The peaks of incidence correspond to the periods of school vacations. Shigella specimens presented a very high resistance rate to trimethoprim-sulfamethoxazole (90.1%) and to ampicillin (22.0%), while Salmonella presented very low resistance rates to all drugs tested. These data are useful for practitioners and they reinforce the need for continuous microbiological surveillance.

Killing the right bug. Phenotypic and genotypic approaches for identifying infectious agents and antibiotic resistance can help to slow the increasing antibiotic resistance among infectious bacteria.

When antibiotics became available after World War II, they were little short of a miracle. Since then, their ability to fight bacterial infections quickly and effectively has literally saved the lives of millions around the globe. But their efficiency has also been their greatest weakness, as people now rely on antibiotics to fight any infection whether it makes sense to use them or not. Added to which, most clinicians and physicians, under pressure to save time and money, will prescribe a broad‐spectrum one‐size‐fits‐all antibiotic to any patient with a bacterial infection rather than choosing a narrow‐spectrum antibiotic targeted to the specific causative pathogen. In the case of life‐threatening infections this is certainly justified. But this over‐use of antibiotics in hospitals and private practices has inevitably selected for resistant bacteria that are increasingly able to withstand the very drugs designed to combat them. Public health experts therefore urge clinicians and physicians to prescribe more narrow‐spectrum antibiotics in order to slow the spread of resistance among bacterial strains. But to do so, clinicians must know as soon as possible which bug they are dealing with and to which antibiotic the strain is susceptible, and this can take up to weeks using traditional culture‐based methods. Some automated systems are available to determine the infectious strain and its susceptibility to antibiotics within hours, but they come with a price. In the long run, therefore, they will most likely be replaced by DNA‐based solutions that will cost a fraction of traditional methods and will be able to indentify an infectious agent and its resistance in less than an hour. > The over‐use of antibodies in hospitals and private practices has inevitably selected for resistant bacteria that are increasingly able to withstand the very drugs designed to combat them The problem of emerging resistance is greatest in …

Identification of motile Aeromonas strains with the MicroScan WalkAway system in conjunction with the combo negative type 1S panels.

This study was performed to compare the MicroScan WalkAway automated identification system in conjunction with the new MicroScan Combo Negative type 1S panels with conventional biochemical methods for identifying 85 environmental, clinical, and reference strains of eight Aeromonas species.

Evaluation of the MicroScan system for identification of staphylococci

Conventional biochemical tests were compared with reactions in a multiple test system, MicroScan Walkaway (Dade Diagnostic Inc. MicroScan Divison, West Sacramento, California) in conjugation with the Combo Pos ID Panels (Dade Diagnostic Inc. MicroScan Divison, West Sacramento, California), in order to evaluate the accuracy for the identification of 99 clinical isolates of Staphylococcus spp. and five reference strains. False-negative or positive reactions were detected from Voges-Proskauer, urease and mannose tests. A good correlation was found among the two identification systems for the fermentation of trehalose, lactose, raffinose, as well as for arginine dyhydrolase, esculin hydrolisis and nitrate reduction. From the results of the present study, it is concluded that the MicroScan Walkaway system is a reliable method for identification of staphylococci (94.23%), although 8.2% could be identified to the species level only after use of additional test.

Identification of Hafnia alvei with the MicroScan WalkAway system.

Hafnia alvei is a gram-negative facultatively anaerobic bacillus that belongs to the family Enterobacteriaceae. This organism is a causative agent of intestinal disorders and is found in different environments. H. alvei has received increased clinical attention as a cause of different infections in humans. This study was performed to compare the MicroScan WalkAway automated identification system in conjunction with the new MicroScan Combo Negative type 1S panels with conventional biochemical methods for identification of 21 H. alvei strains. The MicroScan WalkAway system was found capable of correctly identifying 20 of the 21 strains tested.

Antimicrobial susceptibility of coagulase-negative staphylococci and characterization of isolates with reduced susceptibility to glycopeptides

The antimicrobial susceptibility of 239 coagulase-negative staphylococci (CNS) isolates consecutively collected from blood culture in patients admitted in a 600-bed teaching hospital was evaluated. The isolates were identified to the species level by conventional methods and the MicroScan Positive Combo Panel type 6 system, and their susceptibility to vancomycin, teicoplanin, and oxacillin were tested by agar dilution, disk diffusion, and MicroScan-WalkAway system. The species distribution was as follows: Staphylococcus epidermidis 120 (50.2%), S. hominis 29 (12.1%), S. haemolyticus 24 (10.0%), S. cohnii 14 (5.9%), and isolates from other CNS species 52 (21.8%). The percentage of resistance to oxacillin was 74.5% by agar dilution. The highest percentages of oxacillin resistance were found among S. haemolyticus (95.8%) and S. epidermidis (80.8%). Teicoplanin resistance (MIC ≥ 32 μg/mL) was detected in five S. haemolyticus isolates, whereas intermediate resistance (MIC = 16 μg/mL) was detected in nine strains. These isolates with reduced susceptibility to teicoplanin were resistant to oxacillin, but remained susceptible to vancomycin (MIC ≤ 4 μg/mL). Two isolates, one S. haemolyticus and one S. epidermidis, showed a vancomycin MIC of 8 μg/mL, and both MicroScan and disk diffusion methods classified these isolates as susceptible. Our results showed that glycopeptide resistance is emerging among CNS isolates in our institution and the disk diffusion method may not detect isolates with decreased susceptibility to these antimicrobial agents.

Comparison of MicroScan WalkAway system and Vitek system for identification of gram-negative bacteria

In a prospective side-by-side comparison conducted from September through November 1994, we compared the MicroScan WalkAway system, a conventional biochemical identification system (Dade MicroScan, Inc., Sacramento, Calif.), with the Vitek system (bioMerieux Vitek, Hazelwood, Mo. [analysis software version AMS-RO8.2] for the identification of gram-negative bacteria. Three-hundred thirty-one nonurine isolates and 493 urine isolates were tested. For nonurinary isolates, there was 91.5% agreement between the two methods. For urinary isolates, there was 97.4% agreement between the two methods. Overall, there was 95% agreement between the two systems. The results suggest that the current version of the MicroScan WalkAway system with conventional panels is essentially comparable to the current Vitek system.