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We compared the antimicrobial susceptibility testing results generated by disk diffusion and the VITEK 2 automated system with the results of the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) reference method for 61 isolates of unusual species of Enterobacteriaceae. The isolates represented 15 genera and 26 different species, including Buttiauxella, Cedecea, Kluyvera, Leminorella, and Yokenella. Antimicrobial agents included aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, penicillins, and trimethoprim-sulfamethoxazole. CLSI interpretative criteria for Enterobacteriaceae were used. Of the 12 drugs tested by BMD and disk diffusion, 10 showed >95% categorical agreement (CA). CA was lower for ampicillin (80.3%) and cefazolin (77.0%). There were 3 very major errors (all with cefazolin), 1 major error (also with cefazolin), and 26 minor errors. Of the 40 isolates (representing 12 species) that could be identified with the VITEK 2 database, 36 were identified correctly to species level, 1 was identified to genus level only, and 3 were reported as unidentified. VITEK 2 generated MIC results for 42 (68.8%) of 61 isolates, but categorical interpretations (susceptible, intermediate, and resistant) were provided for only 22. For the 17 drugs tested by both BMD and VITEK 2, essential agreement ranged from 80.9 to 100% and CA ranged from 68.2% (ampicillin) to 100%; thirteen drugs exhibited 100% CA. In summary, disk diffusion provides a reliable alternative to BMD for testing of unusual Enterobacteriaceae, some of which cannot be tested, or produce incorrect results, by automated methods.

Background: Treatment of midface hypoplasia and forehead retrusion with monobloc advancement is associated with significant complications, including meningitis, prolonged intubation, and frontal bone flap necrosis. To see whether distraction of the monobloc segment offered decreased morbidity, the authors compared clinical outcomes of patients who underwent conventional monobloc advancement with those of patients who underwent monobloc distraction., Methods: Group 1 (conventional monobloc; n = 12) underwent traditional monobloc advancement with bone grafting. Group 2 (modified monobloc; n = 11) did not receive ventriculoperitoneal shunts and underwent the above procedures with placement of a pericranial flap and fibrin glue over the midline defect. Group 3 (monobloc distraction; n = 24) underwent advancement of the monobloc segment by distraction osteogenesis using internal distraction devices. Complications included meningitis, cerebrospinal fluid leak, frontal bone flap loss, and wound infection. Preoperative, postoperative, and follow-up lateral cephalograms were used to assess horizontal changes of the forehead, midface, and maxilla., Results: Group 3 (distraction monobloc) had the lowest complication rate (8 percent), followed by groups 2 (modified monobloc; 43 percent) and 1 (conventional monobloc; 61 percent) (p < 0.05). Group 3 achieved greater advancement (12.6 mm) than did group 2 (9.4 mm) or group 1 (9.1 mm) (p < 0.05). Relapse was least in group 3 (8 percent) compared with groups 2 (67 percent) and 1 (45 percent)., Conclusions: Monobloc advancement by distraction osteogenesis had less morbidity and achieved greater advancement with less relapse compared with conventional methods of acute monobloc advancement with bone grafting. Monobloc distraction is superior to conventional methods of acute monobloc advancement and is an alternative to staged fronto-orbital advancement followed by Le Fort III advancement.

Osteoblast biology is influenced in vivo by a 3-dimensional (3D) extracellular matrix that mediates their adhesion and interaction and by a constant state of compressive and tensile forces. To study the role of mechanical stress on osteoblasts in vitro, these parameters must be addressed. Therefore, this study describes the use of a novel, in vitro system that subjects cells to distractive and compressive forces in a 3D environment. This system, termed a microdistractor system, was used to apply linear forces to 3D collagen type I gels containing preosteoblasts. Gels were induced for up to 16 days in osteogenic medium and subjected to either constant linear distraction (distraction gels) or to repeating cycles of distraction and compression (oscillation gels). The effect of these stresses was evaluated over time by measuring proliferation rates, protein synthesis (i.e., cellular activity), and osteogenic differentiation levels. While linear forces in general appeared to increase protein synthesis, force-specific effects on proliferation and differentiation were observed. Specifically, distraction forces appeared to enhance MC3T3 proliferation while distraction/compressive forces appeared to accelerate their osteogenic differentiation program. Therefore, these results suggest that the microdistraction system may be an appropriate in vitro system for the study of mechanobiology in osteoblast phenotype.

Background: Treacher Collins and Nager syndromes may present with mandibular hypoplasia that causes posterior collapse of the tongue base and a decreased oropharyngeal airway. Mandibular distraction and orthognathic advancement are effective treatments to correct the airway, but failure may occur despite achieving class I occlusion. For this select population, the authors propose a novel procedure of genioplasty distraction and hyoid advancement to optimize epiglottal positioning., Methods: Patients diagnosed with Treacher Collins (n = 5) or Nager syndrome (n = 3) with obstructive sleep apnea or tracheostomy dependency (n = 8) underwent genioplasty distraction and hyoid advancement. Airway outcome was assessed by preoperative and 1-year follow-up comparison of (1) laryngobronchoscopy, (2) sleep studies, and (3) tracheostomy dependency. For genioplasty outcome, three groups were used: group I (distraction genioplasty, syndromic) (n = 8), group II (acute genioplasty, syndromic) (n = 7), and group III (acute genioplasty, nonsyndromic) (n = 10). Lateral cephalogram measurements were used in the preoperative, postoperative, and follow-up periods to assess horizontal and vertical advancement and relapse., Results: Epiglottal position was optimized by the procedure in all patients based on direct endoscopic assessment. All five patients with obstructive sleep apnea had resolution of symptoms, and two of three patients achieved removal of their tracheostomy. Mean advancement for groups I, II, and III was 25, 14, and 8 mm, respectively. Follow-up horizontal advancement for groups I, II, and III were 18, 4, and 6 mm, respectively. Cephalometric measurements showed a horizontal relapse for groups I, II, and III of 10, 62, and 11 percent, respectively., Conclusions: Data suggest that genioplasty distraction allows for a greater advancement and decreased relapse rate than acute procedures alone; and genioplasty distraction with hyoid advancement is a useful technique for resolution of obstructive sleep apnea or to achieve tracheostomy removal in those syndromic patients who have already undergone mandibular advancement into a class I occlusion.

Background: Augmentation of the zygomatic body enhances appearance and provides a more youthful look. Porous hydroxyapatite granules offer an alternative to alloplastic implants., Methods: Hydroxyapatite granules were placed by means of a transconjunctival approach into subperiosteal malar pockets (n = 8). From the preoperative, postoperative, and 1-year follow-up lateral views, malar projection was measured as the right angle distance from the point of malar prominence to the nasale-subnasale line., Results: Patients were either very satisfied (six of eight) or satisfied (two of eight). Malar projection was significantly improved postoperatively and was maintained after 1 year., Conclusion: This technique, performed for cosmetic and reconstructive indications, resulted in measurable improvement in malar projection, minimal complications, and optimal patient satisfaction.

Background: Correction of severe maxillary deficiency in cleft lip-cleft palate patients often results in undercorrection, relapse, and need for secondary corrective procedures. Le Fort I internal distraction osteogenesis offers an alternative to one-step orthognathic advancement, with advantages of gradual lengthening through scar and earlier treatment in growing patients., Methods: Patients with cleft lip-cleft palate deformities and maxillary deficiency were divided into three groups treated by Le Fort I advancement: group 1, mild to moderate deficiency (< 10 mm) with conventional orthognathic procedure; group 2, severe deficiency (> or = 10 mm) with conventional orthognathic procedure; and group 3, distraction procedure for severe deficiency (> or = 10 mm) (n = 51). Preoperative, postoperative, and follow-up (> 1 year) lateral cephalogram measurements were compared including angular (SNA and SNB) and linear (Deltax = horizontal and Deltay = vertical) changes. The Pittsburgh Speech Score was used to assess for velopharyngeal insufficiency (score > 3)., Results: Results demonstrated that group 1 patients had a mean SNA change from preoperatively (78.7) to postoperatively (83.8), and a horizontal change of 5.0 mm, with no relapse. Group 2 patients had a mean SNA change from preoperatively (76.3) to postoperatively (82.0) and a horizontal change of 7.2 mm, with 63 percent relapse. Group 3 patients had a mean SNA change from preoperatively (74.1) to postoperatively (84.9) and a horizontal change of 16.5 mm, with 15 percent relapse. Thus, for severe maxillary deficiency, the distraction group had 48 percent less relapse than the conventional Le Fort I group. Postoperative speech evaluation showed velopharyngeal insufficiency in the following: group 1, four of 20 patients (20 percent); group 2, nine of 11 patients (82 percent); and group 3, nine of 20 patients (45 percent)., Conclusion: These data suggest that Le Fort I internal distraction for severe cleft maxillary deficiency leads to better dental occlusion, less relapse, and better speech results.

The Phoenix 100 ID/AST system (Becton Dickinson Co., Sparks, Md.) is an automated system for the identification and antimicrobial susceptibility testing of bacterial isolates. This system with its negative identification (NID) panel was evaluated for its accuracy in the identification of 507 isolates of the family Enterobacteriaceae, 57 other nonenteric gram-negative isolates that are commonly isolated in clinical microbiology laboratories, and 138 isolates of the family Vibrionaceae. All of the isolates had been characterized by using approximately 48 conventional tube biochemicals. Of the 507 isolates of the Enterobacteriaceae, 456 (89.9%) were correctly identified to the genus and species levels. The five isolates of Proteus penneri required an off-line indole test, as suggested by the system to differentiate them from Proteus vulgaris. The identifications of 20 (3.9%) isolates were correct to the genus level but incorrect at the species level. Two (0.4%) isolates were reported as "no identification." Misidentifications to the genus and species levels occurred for 29 (5.7%) isolates of the Enterobacteriaceae. These incorrect identifications were spread over 14 different genera. The most common error was the misidentification of Salmonella species. The shortest time for a correct identification was 2 h 8 min. The longest time was 12 h 27 min, for the identification of a Serratia marcescens isolate. Of the 57 isolates of nonenteric gram-negative bacilli (Acinetobacter, Aeromonas, Burkholderia, Plesiomonas, Pseudomonas, and Stenotrophomonas spp.), 48 (84.2%) were correctly identified to the genus and species levels and 7 (12.3%) were correctly identified to the genus level but not to the species level. The average time for a correct identification was 5 h 11 min. Of the Vibrionaceae spp., 123 (89.1%) were correctly identified at the end of the initial incubation period, which averaged 4 h. Based on the findings of this study, the Phoenix 100 ID/AST system NID panel falls short of being an acceptable new method for the identification of the Enterobacteriaceae, Vibrionaceae, and gram-negative nonenteric isolates that are commonly encountered in many hospital microbiology laboratories.

Background: Distraction osteogenesis has been used to correct hypoplastic and asymmetric bony deformities in the growing patient, yet its underlying cellular mechanisms are poorly understood. Using a new in vitro model, the microdistractor, morphologic properties of preosteoblasts under mechanical strain were studied., Methods: Mouse calvarial MC3T3 cells were suspended in a polymerized three-dimensional collagen gel and stressed for 14 days as one of three groups (n = 30): (1) distraction (0.5 mm/day); (2) oscillation (1 mm/day for 2 days alternated with 1 mm/day for 2 days); and (3) control (no force). A computer modeling system, KS-300, was used to record cell shape (aspect ratio) and orientation (deviance from axis of stress)., Results: In part I of the study, morphologic cellular changes were found to be even throughout different regions of the gel (central versus peripheral, versus different vertical layers), suggesting the force was evenly applied to all cells in the gel. In addition, when linear distraction forces were applied, morphologic change occurred over time, suggesting a morphologic response to the applied stress. In part II of the study, with different forces applied, morphologic changes occurred over time such that linear distraction forces caused cells to elongate and align in a parallel direction to the force, whereas oscillation caused cells to switch from parallel (with distraction) to perpendicular (with compression) orientation relative to the force applied., Conclusion: The authors' data suggest that the microdistractor device is an effective in vitro model for studying the cellular response to distraction stresses. It may be used in future studies to optimize clinical methods of distraction.

Pansynostosis (fusion of all cranial sutures) and optic atrophy were found as incidental CT scan and ophthalmological findings in an 8-year-old who presented to the emergency room with scalp edema from tight 'cornrow' hair braiding. Cranial vault expansion was successfully performed. Ophthalmological problems have stabilized but have not reversed. Late presentation of craniosynostosis and the pathophysiology of secondary optic atrophy are discussed., (Copyright (c) 2006 S. Karger AG, Basel.)

After correction of moderate to severe hypertelorbitism (greater than 40 mm interdacryon distance) with facial bipartition or orbital box osteotomy, excess glabellar soft tissue and brow width should be addressed. Traditional methods described used a midline excision down the forehead and nasal dorsum, and left an unsightly scar. With a series of 12 patients, the authors document the K stitch technique with no external vertical scar. A mean 38.8% reduction of interbrow distance was noted using this technique. Two patients underwent revisions, and two patients had temporary eyelid ptosis. All patients reported satisfaction once the skin contracture was completed.

The cleft nose deformity in bilateral cleft lip and palate patients with severely flattened alar cartilages, a short, scarred columella, and thickened skin is a reconstructive challenge. The Wolfe double-arch tip rhinoplasty technique was compared with a cartilage release and tip grafting technique to determine the optimal modality for tip projection and columella lengthening. Patients with significant bilateral cleft nasal deformities and previous bilateral cleft lip repairs were divided into two groups (n = 22). Group 1 (double-arch) patients underwent an open rhinoplasty using conchal cartilage grafts to create a columellar strut and new lower lateral arches placed over the existing arches (n = 12). In group 2 (release and tip graft), the lower lateral cartilages were released, and nasal tip grafting was performed (n = 10). Preoperative and 6-month postoperative measurements, including (1) columellar length, (2) alar base-nasal tip-columellar base (ATC) angle, and (3) lateral tip projection, were compared. The lateral tip projection is the perpendicular distance between the nasal tip and a line created from the connection of points at the nasion to the subnasale. In group 1 (double arch), the mean columella length increased 47.2%, whereas in group 2 (release and tip graft), it only increased 14.1%. The ATC angle had a mean decrease or narrowing of 26.7 degrees in group 1, compared with a 12.5 degrees decrease in group 2. Lateral tip projection improvement was greater in group 1 (52.2% increase) compared with group 2 (19.9% increase). The authors' data showed that for the difficult bilateral cleft nasal deformity with significant tip flattening, the double-arch tip rhinoplasty provides improved nasal tip projection.

A 54-year-old ranch hand presented to the emergency room with an alleged spider bite and multiple abscesses. Both wound and blood cultures grew Photorhabdus asymbiotica, an enteric gram-negative rod that was initially misidentified by the hospital's rapid identification system. Clinical laboratories should be aware of the limitations of their rapid identification systems and always use them as an adjunct to analysis of morphological and phenotypic traits.

Identification of gram-negative bacilli, both enteric and nonenteric, by conventional methods is not realistic for clinical microbiology laboratories performing routine cultures in today's world. The use of commercial kits, either manual or automated, to identify these organisms is a common practice. The advent of rapid or "spot" testing has eliminated the need for some commonly isolated organisms to be identified with the systems approach. Commercially available systems provide more in-depth identification to the species level as well as detect new and unusual strains. The answers obtained from these systems may not always be correct and must be interpreted with caution. The patient demographics, laboratory workload and work flow, and technologist's skill levels should dictate the system of choice. Cost considerations introduce another variable into the equation affecting choice. Each system has its own strengths and weaknesses, and each laboratory must decide on the level of sophistication that fulfills its particular needs.

A vancomycin-resistant Staphylococcus aureus (VRSA) isolate was obtained from a patient in Pennsylvania in September 2002. Species identification was confirmed by standard biochemical tests and analysis of 16S ribosomal DNA, gyrA, and gyrB sequences; all of the results were consistent with the S. aureus identification. The MICs of a variety of antimicrobial agents were determined by broth microdilution and macrodilution methods following National Committee for Clinical Laboratory Standards (NCCLS) guidelines. The isolate was resistant to vancomycin (MIC = 32 micro g/ml), aminoglycosides, beta-lactams, fluoroquinolones, macrolides, and tetracycline, but it was susceptible to linezolid, minocycline, quinupristin-dalfopristin, rifampin, teicoplanin, and trimethoprim-sulfamethoxazole. The isolate, which was originally detected by using disk diffusion and a vancomycin agar screen plate, was vancomycin susceptible by automated susceptibility testing methods. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA indicated that the isolate belonged to the USA100 lineage (also known as the New York/Japan clone), the most common staphylococcal PFGE type found in hospitals in the United States. The VRSA isolate contained two plasmids of 120 and 4 kb and was positive for mecA and vanA by PCR amplification. The vanA sequence was identical to the vanA sequence present in Tn1546. A DNA probe for vanA hybridized to the 120-kb plasmid. This is the second VRSA isolate reported in the United States.

Six commercially available bacterial identification products were tested with Vibrio alginolyticus (12 strains), V. cholerae (30 strains), Photobacterium (Vibrio) damselae (10 strains), V. fluvialis (10 strains), V. furnissii (4 strains), V. hollisae (10 strains), V. metschnikovii (9 strains), V. mimicus (10 strains), V. parahaemolyticus (30 strains), and V. vulnificus (10 strains) to determine the accuracy of each system for identification. The products included API 20E, Crystal E/NF, MicroScan Neg ID2 and Rapid Neg ID3, and Vitek GNI+ and ID-GNB. Each product was tested only with those species that were listed in its database. Overall, the systems correctly identified 63.9, 80.9, 63.1, 73.6, 73.5, and 77.7% of the isolates to species level, respectively. Error rates ranged from 0.8% for the API 20E to 10.4% for the Rapid Neg ID3. The API 20E gave "no identification" for 13.1% of the isolates, while the Neg ID2, GNI+, ID-GNB, and Crystal were unable to identify 1.8, 2.9, 5.0, and 6.9%, respectively. For V. cholerae, specifically, accuracy ranged from 50.0 to 96.7%, with the API 20E having the worst performance and Crystal having the best. V. fluvialis presented the biggest challenge for the API 20E and the GNI+, with probabilities averaging 10%, while V. mimicus was a major problem with the Crystal E/NF, which identified none of the strains correctly. With the Neg ID2, correct answers were often obtained only after a modified inoculation of the panel with a bacterial suspension prepared with 0.85% NaCl. Additional tests required for identification often included growth in the absence of NaCl, which is not readily available in most clinical laboratories. The only product to correctly identify at least 90% of V. cholerae strains was the Crystal E/NF, and only three of the six products, the API 20E and both of the Vitek cards, correctly identified more than 90% of the V. parahaemolyticus strains. Thus, extreme care must be taken in the interpretation of answers from these six commercially available systems for the identification of Vibrio species.

We evaluated the Vitek 2 ID-GNB identification card (bioMérieux, Inc., Durham, N.C.) for its ability to identify members of the family Enterobacteriaceae and other gram-negative bacilli that are isolated in clinical microbiology laboratories. Using 482 enteric stock cultures and 103 strains of oxidase-positive, gram-negative glucose-fermenting and nonfermenting bacilli that were maintained at -70 degrees C and passaged three times before use, we inoculated cards according to the manufacturer's directions and processed them in a Vitek 2 instrument using version VT2-R02.03 software. All panel identifications were compared to reference identifications previously confirmed by conventional tube biochemical assays. At the end of the initial 3-h incubation period, the Vitek 2 instrument demonstrated an accuracy of 93.0% for the identification of enteric strains; 414 (85.9%) were correctly identified at probability levels ranging from excellent to good, and an additional 34 (7.1%) strains were correctly identified but at a low level of discrimination. Nineteen (3.9%) strains were unidentified, and 15 (3.1%) were misidentified. The 19 unidentified strains were scattered among 10 genera. Three of the 15 misidentified strains were lactose-positive Salmonella spp. and were identified as Escherichia coli; another was a lactose-positive, malonate-negative Salmonella enterica subsp. arizonae strain that was identified as E. coli. Of the 103 glucose-fermenting and nonfermenting nonenteric strains, 88 (85.4%) were correctly identified at probability levels ranging from excellent to good, and 10 (9.7%) were correctly identified, but at a low level of discrimination, for a total of 95.1% accuracy with this group. Two strains were unidentified and three were misidentified. The errors occurred for strains in three different genera. With the increased hands-off approach of the Vitek 2 instrument and accuracies of 93% for the identification of enteric organisms and 95.1% for the identification of nonenteric organisms with the ID-GNB card, use of this product presents an acceptable method for the identification of most gram-negative organisms commonly isolated in the clinical laboratory. A comparison of these results to those obtained by testing 454 of the same strains with the Vitek GNI+ card revealed no significant difference in the abilities of the two cards to identify these organisms accurately.

The MicroScan Rapid Neg ID3 panel is designed for the identification of Enterobacteriaceae and nonenteric glucose-fermenting and nonfermenting gram-negative bacilli. We evaluated this panel for its ability to identify gram-negative non-Enterobacteriaceae bacteria. A total of 134 strains, representing 26 genera and 42 species, were taken from storage at -70(o)C, passaged three times before testing, and inoculated into the panels according to the manufacturer's directions before being inserted into a Walk/Away 96 instrument loaded with version 22.28 software. At the end of the initial 2.5-h incubation period, 89 isolates (66.4%) were correctly identified at a probability level of > or =85%. After additional testing recommended by the manufacturer was completed, another 11 isolates (8.2%) were correctly identified at probability levels of > or =85%. Twenty-five (18.7%) isolates were correctly identified after additional testing, but the probability levels were less than 85%. Two isolates were unidentified, and seven (5.2%) were incorrectly identified. The seven misidentified strains were not concentrated in any one genus. With an accuracy approaching 75%, this product may be used for the identification of the commonly isolated non-Enterobacteriaceae bacteria but may present problems in identification of other non-glucose-fermenting gram-negative bacilli.

Enterobacter hormaechei was defined as a unique species in 1989. We describe six case patients of E. hormaechei bloodstream infection in three neonatal intensive care units in Rio de Janeiro, Brazil. E. hormaechei identification was performed on the Vitek system and confirmed by conventional testing. Strain relatedness was evaluated by pulsed field gel electrophoresis. All children recovered completely. Chart review for previous procedures revealed parenteral nutrition as the only common procedure.

Using a set of 55 Staphylococcus aureus challenge organisms, we evaluated six routine methods (broth microdilution, disk diffusion, oxacillin agar screen, MicroScan conventional panels, MicroScan rapid panels, and Vitek cards) currently used in many clinical laboratories and two new rapid methods, Velogene and the MRSA-Screen, that require less than a day to determine the susceptibility of S. aureus to oxacillin. The methods were evaluated by using the presence of the mecA gene, as detected by PCR, as the "gold standard." The strains included 19 mecA-positive heterogeneously resistant strains of expression class 1 or 2 (demonstrating oxacillin MICs of 4 to >16 microg/ml) and 36 mecA-negative strains. The oxacillin MICs of the latter strains were 0.25 to 4 microg/ml when tested by broth microdilution with 2% NaCl-supplemented cation-adjusted Mueller-Hinton broth as specified by the NCCLS. However, when tested by agar dilution with 4% salt (the conditions used in the oxacillin agar screen method), the oxacillin MICs of 16 of the mecA-negative strains increased to 4 to 8 microg/ml. On initial testing, the percentages of correct results (% sensitivity/% specificity) were as follows: broth microdilution, 100/100; Velogene, 100/100; Vitek, 95/97; oxacillin agar screen, 90/92; disk diffusion, 100/89; MicroScan rapid panels, 90/86; MRSA-Screen, 90/100; and MicroScan conventional, 74/97. The MRSA-Screen sensitivity improved to 100% if agglutination reactions were read at 15 min. Repeat testing improved the performance of some but not all of the systems.

The MicroScan Rapid Neg ID3 panel (Dade Behring, Inc., West Sacramento, Calif.) is designed for the identification of gram-negative bacilli. We evaluated its ability to accurately identify Enterobacteriaceae that are routinely encountered in a clinical laboratory and glucose nonfermenting gram-negative bacilli. Using 511 stock cultures that were maintained at -70 degrees C and passaged three times before use, we inoculated panels according to the manufacturer's instructions and processed them in a Walk/Away instrument using version 22.01 software. The time to identification was 2 h and 30 min. All panel identifications were compared to reference identifications previously determined by conventional tube biochemicals. At the end of the initial 2.5-h incubation period, 405 (79.3%) identifications were correct. An additional 49 (9.6%) isolates were correctly identified after required additional off-line biochemical tests were performed. Thus, at 24 h, 88.8% of the 511 strains tested were correctly identified. Twenty-two (4.3%) were identified to the genus level only. Twenty-six (5.1%) strains were misidentified. Because the system is based on fluorogenics, there are no conventional tests readily available with which to compare possibly incorrect reactions. Of the 28 Salmonella strains that were tested, 5 were incorrectly reported. The 21 remaining errors were scattered among the genera tested. Testing on nine strains gave a result of "no identification" (very rare biotype). The Rapid Neg ID3 panel in this study approached 89% accuracy for the identification of gram-negative organisms encountered in the hospital laboratory.

This review presents the current taxonomy of the genera Proteus, Providencia, and Morganella, along with the current methods for the identification of each species within the three genera, incorporating both conventional biochemical and commercial methods. While all of these organisms are ubiquitous in the environment, individual case reports and nosocomial outbreak reports that demonstrate their ability to cause major infectious disease problems are presented. Lastly, anticipated antimicrobial susceptibility patterns are reviewed. Many of these organisms are easily controlled, but the advent of newer and more powerful antimicrobial agents has led to some problems of which laboratorians need to be aware.

Strains traditionally identified as Proteus vulgaris formed three biogroups. Biogroup 1, characterized by negative reactions for indole production, salicin fermentation and aesculin hydrolysis, is now known as Proteus penneri. Biogroup 2, characterized by positive reactions for indole, salicin and aesculin, was shown by DNA hybridization (hydroxyapatite method) to be a genetic species separate from biogroup 1 and from biogroup 3 which is positive for indole production and negative for salicin and aesculin. In this study, 52 strains were examined, of which 36 strains were Proteus vulgaris biogroup 3, which included the current type strain of the species P. vulgaris (ATCC 29905T), and compared to seven strains of Proteus vulgaris biogroup 2 and nine type strains of other species in the genera Proteus, Providencia and Morganella. By DNA hybridization, these 36 strains were separated into four distinct groups, designated as Proteus genomospecies 3, 4, 5 and 6. DNAs within each separate Proteus genomospecies were 74-99% related to each other in 60 degrees C hybridization reactions with < or = 4.5% divergence between related sequences. Proteus genomospecies 3 contained the former P. vulgaris type strain and one other strain and was negative in reactions for salicin fermentation, aesculin hydrolysis and deoxyribonuclease, unlike the reactions associated with strains considered as typical P. vulgaris which are positive in reactions for salicin, aesculin and DNase. Genomospecies 3 can be distinguished from Proteus genomospecies 4, 5 and 6 because it is negative for Jordan's tartrate. Proteus genomospecies 4, containing five strains, was differentiated from Proteus penneri, genomospecies 3 and 6 and most, but not all, strains of genomospecies 5, by its ability to ferment L-rhamnose. Proteus genomospecies 5 and 6, containing 18 and 11 strains, respectively, could not be separated from each other by traditional biochemical tests, by carbon source utilization tests or SDS-PAGE of whole-cell proteins. In an earlier publication, a request was made to the Judicial Commission that the former type strain of P. vulgaris (ATCC 13315) be replaced by P. vulgaris biogroup 2 strain ATCC 29905T, a strain considered more biochemically typical of P. vulgaris strains. This would have the effect of assigning the name P. vulgaris to P. vulgaris biogroup 2. Since this request has been acceded to, the name Proteus hauseri is herein proposed for Proteus vulgaris genomospecies 3. Its type strain is ATCC 700826T. Proteus genomospecies 4, 5 and 6 will remain unnamed until better phenotypic differentiation can be accomplished. All Proteus genomospecies were similar in their antimicrobial susceptibility patterns. Nineteen strains were isolated from urine, four from faeces, two from wounds, nine from other human sources and two from animals.

In urodele amphibian spinal cord regeneration, the ependymal cells lining the central canal remodel the lesion site to favor axonal regrowth. We profiled the production of matrix metalloproteinases by injury-reactive mesenchymal ependymal cells in vivo and in vitro and found that matrix metalloproteinases are involved in this remodeling process in the axolotl (Ambystoma mexicanum). The production of cell-associated matrix metalloproteinases in vivo was shown to be identical to that in our cultured ependymal cell model system. Activated and zymogen forms of matrix metalloproteinases were identified using zymography, chemical inhibitors of matrix metalloproteinases, and cleavage of propeptides by organomercurials. The principal cellular proteinases consisted of matrix metalloproteinase-2 (gelatinase A) and matrix metalloproteinase-1 (type I collagenase), which display characteristic shifts in molecular weight following proenzyme processing by organomercurials. In addition, ependymal cell conditioned medium contained secreted forms of the enzyme undetectable in situ. Matrix metalloproteinase-9 (gelatinase B) as well as matrix metalloproteinase-2 and matrix metalloproteinase-1 were secreted and casein substrate zymography showed the presence of a small amount of a very high molecular weight matrix metalloproteinase-3 (prostromelysin) secreted into the culture medium. Matrix metalloproteinases were still present at 4 weeks post-lesioning when the ependymal cells have just re-epithelialized, but decreased near the completion of regeneration (8 weeks post-lesioning). Zymography showed no detectable matrix metalloproteinases in unlesioned cord but the presence of tissue inhibitor of metalloproteinase-1 in intact cord was seen by Western blotting. This study shows that matrix metalloproteinases are associated with urodele spinal cord regeneration and validates the use of our ependymal cell tissue culture model system to evaluate ependymal cell behavior during spinal cord regeneration.

Studies conducted in France and Germany suggest that up to 19% of clinically identified Klebsiella sp. are actually Klebsiella planticola, an environmental species that has been attributed to two cases of septicemia, with a rare isolate of Klebsiella terrigena (0. 4%) being identified. A 1-year survey of newborns on a neonatal ward, also conducted in Germany, reported that 72% of Klebsiella sp. were Klebsiella oxytoca and 8.7% were K. planticola. The tests necessary to identify these species are not found in most clinical identification schemes or in the database matrices of most commercial identification products. To determine the incidence of unrecognized K. planticola among the Klebsiella sp. isolates in our collection, we used the battery of seven supplemental tests amended from the work of Monnet and Freney to test 352 stock isolates and 84 fresh clinical isolates from four local hospitals. After testing 436 strains of Klebsiella, only one strain was identified as a possible K. planticola and none was identified as K. terrigena. We tested an additional 43 stock strains of K. oxytoca isolated from newborns by using eight biochemical tests and found one additional strain of K. planticola. The occurrence of K. planticola in our collection is far less frequent than that observed in other countries.

Recent work describing six named species and two unnamed genomospecies within Citrobacter has enlarged the genus to 11 species. DNA relatedness and phenotypic tests were used to determine how well these species can be identified. One hundred thirty-six strains were identified to species level by DNA relatedness and then identified phenotypically in a blinded fashion. By using conventional tests, 119 of the 136 strains (88%) were correctly identified to species level. Three additional strains (2%) were identified as citrobacteria but were not identified to species level, and 14 strains (10%) were misidentified as other Citrobacter species. Carbon source utilization tests were used to identify 86 of the strains. Eighty-four strains (98%) were correctly identified, and two strains (2%) were misidentified as other Citrobacter species. Additional strains of Citrobacter genomospecies 10 and Citrobacter genomospecies 11 were identified, allowing these species to be formally named as Citrobacter gillenii sp. nov. and Citrobacter murliniae sp. nov., respectively.

OBJECTIVE: To evaluate the ID 32E bacterial identification system for accuracy in the identification of members of the family Enterobacteriaceae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter baumannii/Iwoffii. METHODS: Stock cultures of 497 Enterobacteriaceae and 27 commonly encountered non-enteric Gram-negative rods were tested in the ID 32E system. For each isolate, the resulting 11-digit profile number was converted to an identification using the APILAB Plus software (version 3.2.2). This identification was then compared to the reference identification obtained using conventional biochemicals. RESULTS: Of the 524 isolates tested, 405 (77.3%) were identified correctly; 52 (9.9%) were identified incorrectly. Sixty-seven (12.8%) identifications were either doubtful or unacceptable, and were not limited to any particular genus or species, with the exception of Ewingella americana and Serratia plymuthica, which did not grow well enough in the strip at 35 degrees C to be correctly identified. All five isolates of Acinetobacter Iwoffii were misidentified as Alcaligenes spp. CONCLUSIONS: With this challenge set of organisms, the ID 32E correctly identified 77.3% of the isolates tested. For commonly encountered isolates, the accuracy approached 90%. We conclude that the ID 32E offers an alternative for the identification of common clinical isolates.

Fluoroquinolone resistance appears to be increasing in many species of bacteria, particularly in those causing nosocomial infections. However, the accuracy of some antimicrobial susceptibility testing methods for detecting fluoroquinolone resistance remains uncertain. Therefore, we compared the accuracy of the results of agar dilution, disk diffusion, MicroScan Walk Away Neg Combo 15 conventional panels, and Vitek GNS-F7 cards to the accuracy of the results of the broth microdilution reference method for detection of ciprofloxacin and ofloxacin resistance in 195 clinical isolates of the family Enterobacteriaceae collected from six U.S. hospitals for a national surveillance project (Project ICARE [Intensive Care Antimicrobial Resistance Epidemiology]). For ciprofloxacin, very major error rates were 0% (disk diffusion and MicroScan), 0.9% (agar dilution), and 2.7% (Vitek), while major error rates ranged from 0% (agar dilution) to 3.7% (MicroScan and Vitek). Minor error rates ranged from 12.3% (agar dilution) to 20.5% (MicroScan). For ofloxacin, no very major errors were observed, and major errors were noted only with MicroScan (3.7% major error rate). Minor error rates ranged from 8.2% (agar dilution) to 18.5% (Vitek). Minor errors for all methods were substantially reduced when results with MICs within +/-1 dilution of the broth microdilution reference MIC were excluded from analysis. However, the high number of minor errors by all test systems remains a concern.

We describe the isolation and identification of Enterobacter intermedium from the gallbladder of a patient with cholecystitis. There have been only four documented isolations of this organism from humans; it normally occurs in surface water and unpolluted soils. The identification was initially made by a MicroScan Walk/Away system with a Neg Combo 18 conventional identification-susceptibility panel. The organism is susceptible to the aminoglycosides and imipenem but resistant to the cephalosporins and ciprofloxacin.

During the last several years a series of staphylococcal isolates that demonstrated reduced susceptibility to vancomycin or other glycopeptides have been reported. We selected 12 isolates of staphylococci for which the vancomycin MICs were > or =4 microg/ml or for which the teicoplanin MICs were > or =8 microg/ml and 24 control strains for which the vancomycin MICs were < or =2 microg/ml or for which the teicoplanin MICs were < or =4 microg/ml to determine the ability of commercial susceptibility testing procedures and vancomycin agar screening methods to detect isolates with reduced glycopeptide susceptibility. By PCR analysis, none of the isolates with decreased glycopeptide susceptibility contained known vancomycin resistance genes. Broth microdilution tests held a full 24 h were best at detecting strains with reduced glycopeptide susceptibility. Disk diffusion did not differentiate the strains inhibited by 8 microg of vancomycin per ml from more susceptible isolates. Most of the isolates with reduced glycopeptide susceptibility were recognized by MicroScan conventional panels and Etest vancomycin strips. Sensititre panels read visually were more variable, although with some of the panels MICs of 8 microg/ml were noted for these isolates. Vitek results were 4 microg/ml for all strains for which the vancomycin MICs were > or =4 microg/ml. Vancomycin MICs on Rapid MicroScan panels were not predictive, giving MICs of either < or =2 or > or =16 microg/ml for these isolates. Commercial brain heart infusion vancomycin agar screening plates containing 6 microg of vancomycin per ml consistently differentiated those strains inhibited by 8 microg/ml from more susceptible strains. Vancomycin-containing media prepared in-house showed occasional growth of susceptible strains, Staphylococcus aureus ATCC 29213, and on occasion, Enterococcus faecalis ATCC 29212. Thus, strains of staphylococci with reduced susceptibility to glycopeptides, such as vancomycin, are best detected in the laboratory by nonautomated quantitative tests incubated for a full 24 h. Furthermore, it appears that commercial vancomycin agar screening plates can be used to detect these isolates.

Fifty-one Rahnella aquatilis and R. aquatilis-like strains from water, snails and human sources were characterized by routine biochemical tests, carbon source utilization tests, DNA relatedness (hydroxyapatite method) and 16S rRNA sequencing. The results of the genetic methods indicated that the strains comprised three closely related species within the genus Rahnella. It was not possible to differentiate R. aquatilis from the two newly recognized species. The new species were therefore given the vernacular names Rahnella genomospecies 2 and Rahnella genomospecies 3.

Erwinia persicinus was first described in 1990 after being isolated from a variety of fruits and vegetables, including bananas, cucumbers, and tomatoes. In 1994, it was shown to be the causative agent of necrosis of bean pods. We now report the first human isolate of E. persicinus. The strain was isolated from the urine of an 88-year-old woman who presented with a urinary tract infection. By the hydroxyapatite method, DNA from this strain was shown to be 94.5% related at 60 degrees C and 86% related at 75 degrees C to the type strain of E. persicinus. The biochemical profile of E. persicinus is most similar to those of Erwinia rhapontici, Pantoea agglomerans, and Enterobacter species. It is negative in tests for lysine, arginine, ornithine, dulcitol, and urea. It is motile and positive in tests for D-sorbitol and sucrose. It is susceptible to the expanded-spectrum cephalosporins, aminoglycosides, and fluoroquinolones, but it is resistant to ampicillin, ticarcillin, and cefazolin.

We evaluated the Vitek GNI+ and Becton Dickinson Crystal E/NF identification systems for their ability to accurately identify 619 and 626 strains, respectively, of members of the family Enterobacteriaceae and other glucose-fermenting and non-glucose-fermenting gram-negative rods. All strains tested were taken from a stock collection and passed three times on 5% sheep blood agar prior to testing. These strains represented a more rigorous challenge to both systems than one resulting from the testing of consecutive clinical isolates. Testing with both systems was done according to the manufacturers' instructions, and tests were repeated in duplicate when errors occurred. Vitek version 5.01 and Crystal version 3.0 softwares were used for identifications. The identification results from each system were compared with identifications previously determined with reference biochemicals. At the completion of the appropriate incubation period, the GNI+ and Crystal systems correctly identified 80.1 and 71.1% of the total isolates, respectively. After additional tests suggested by the software programs were completed, the GNI+ had an accuracy of 87.6% and the Crystal system's accuracy had improved to 87.9%. The error rates for the GNI+ and Crystal systems were 6.5 and 5.3%, respectively. A report of "no identification" was given for 6.0 and 6.9% of the isolates, respectively, and was associated with no particular organism group. One isolate each of Acinetobacter lwoffii and Vibrio alginolyticus would not grow in the Vitek card. The average times to detection for correct enteric identifications in the GNI+ system were 4.1 and 6.8 h for nonenteric identifications, while the Crystal results were routinely read at 18 h. We conclude that there was no significant difference (P > 0.05) between the results of the GNI+ card and those of the Crystal E/NF system after additional testing was performed with the group of organisms tested, but the overall accuracy for both systems in this study was below 90%.

Previously, we showed that both D2 and D4 dopamine receptors inhibited adenylate cyclase in a pertussis toxin (Ptx)-sensitive manner in the dopamine-producing MN9D cell line, whereas only D2 receptors did so in a fibroblast cell line, CCL1.3. Of the known Ptx-sensitive G proteins, MN9D cells expressed G alpha i2, G alpha oA and G alpha oB, whereas CCL1.3 cells expressed only G alpha i2. Here we cotransfected MN9D and CCL1.3 cells with either the long form of the D2 receptor (D2L) or the D4 receptor and a mutant Ptx-resistant G protein alpha-subunit. When cotransfected CCL1.3 cell lines were tested for the ability of Ptx to block receptor-mediated inhibition of cyclic AMP accumulation, D2 receptors were found to couple to mutant G alpha i2 and G alpha i3 but not G alpha i1 or G alpha oA. D2 also coupled to mutant G alpha i2 but not G alpha oA in MN9D cells. In contrast, D4 receptors did not couple to either mutant G alpha i2 or G alpha oA subunits in MN9D cells. These data suggest that D4 receptor-mediated inhibition of adenylate cyclase is not coupled via the same mechanisms used by D2 receptors. D2L receptors are capable of coupling to more than one G protein in the modulation of cyclic AMP.

The goal of the current study was to determine which of the D2-like receptors (D2, D3 or D4) are involved in autoreceptor regulation of dopamine synthesis. We have derived a model system utilizing a mouse mesencephalic cell line, MN9D, which both synthesizes and releases dopamine, to characterize the modulation of tyrosine hydroxylase activity, the rate limiting enzyme in the conversion of tyrosine to dopamine, by the D2-like receptors. Previously, we have shown that stimulation of D2 and D3, but not D4, dopamine receptors transfected into MN9D cells inhibited the release of dopamine. In the current study, we show that quinpirole stimulation of transfected D2 and D3, but not D4, dopamine receptors inhibited K+-stimulated tyrosine hydroxylase activity in a pertussis toxin-sensitive manner, strongly suggesting G-protein coupling as a mechanistic pathway. The D2 receptor effect could be maintained for at least 60 min, whereas the D3 receptor effect desensitized. Treatment with 10 microM forskolin, which raises cyclic AMP levels or with 100 nM okadaic acid, a potent phosphatase inhibitor, had no effect on the D2-or D3-mediated inhibition, suggesting that these effects may be independent of both cyclic AMP- and okadaic acid-sensitive phosphatase activity. Taken together, these data confirm the hypothesis that dopamine D2 and D3 receptors can perform dual roles in autoreceptor regulation.

The current type strain of Proteus vulgaris, NCTC 4175 (= ATCC 13315), differs substantially from typical strains of this species both biochemically and chemotaxonomically. DNA relatedness studies revealed that strains previously classified as P. vulgaris belong to six genomospecies. One of these genomospecies contains strains that are negative in indole, salicin, and esculin reactions (biogroup 1) and has been named Proteus penneri. A second genomospecies, which is most frequently isolated from human urine, contains typical P. vulgaris strains that are positive in indole, salicin, and esculin reactions (biogroup 2). The members of the remaining four genomospecies are indole positive and negative in salicin and esculin reactions (biogroup 3). Of 36 biogroup 3 strains studied, only strain NCTC 4175T (T = type strain) and one other strain, CDC 1732-80, belong to genomospecies 3. To retain NCTC 4175 as the type strain of P. vulgaris would restrict this species to these two strains, whose origins are unknown. This would mean that hundreds of strains for which the description of P. vulgaris was written and which have been representatives of this species for the past 50 years would have to be renamed as members of a new species. To prevent this confusion, we request that biogroup 2 reference strain ATCC 29905 (= CDC PR1) replace NCTC 4175 as the type strain of P. vulgaris.

An unusual bacterial pathogen of laboratory mice has been previously classified as an atypical biotype of Citrobacter freundii. Designated C. freundii biotype 4280, this bacterium is the etiologic agent of transmissible murine clonic hyperplasia. An eaeA gene has been shown to be present in this organism and to be necessary for virulence in laboratory mice. However, other biotypes of C. freundii lack DNA homology with the eaeA gene. Because of the recent reclassification in which five named species and three unnamed species, all previously considered C. freundii, were described, we determined the taxonomic status of C. freundii biotype 4280. With a battery of biochemical tests and DNA relatedness studies, three isolates of C. freundii biotype 4280 were shown to be members of an unnamed Citrobacter species, designated species 9. In total, six isolates of Citrobacter species 9, but none of the type strains of the other eight named species or of the two remaining unnamed species of Citrobacter, were shown to possess DNA homology with both the eaeA and the eaeB genes. Species 9 was named Citrobacter rodentium sp. nov.

We evaluated the abilities of 10 commercially available antimicrobial susceptibility testing methods and four reference methods (agar dilution, broth microdilution, disk diffusion, and the agar screen plate) to classify enterococci correctly as vancomycin susceptible or resistant using 50 well-characterized strains of enterococci. There was a high level of agreement of category classification data obtained with broth-based systems (Sceptor, MicroMedia, Pasco, and Sensititre), agar dilution, and an antibiotic gradient method (E test) with data obtained by reference broth microdilution; no very major or major errors were seen, and minor errors were < or = 6%. Increased minor error rates were observed with disk diffusion (12%), Alamar (16%), Uniscept (16%), and conventional (overnight) MicroScan panels (16%). The errors were primarily with Enterococcus casseliflavus strains and organisms containing the vanB vancomycin resistance gene. Very major error rates of 10.3 and 20.7% were observed with Vitek and MicroScan Rapid (MS/Rapid) systems, respectively; however, only the MS/Rapid system produced major errors (13.3%). On repeat testing of discrepant isolates, the very major error rate with the Vitek system dropped to 3.4%, while the very major error rate with the MS/Rapid system increased to 27.6%; major errors with the MS/Rapid system were not resolved. Many of the commercial systems had only 4 dilutions of vancomycin, which resulted in up to 84% of values being off scale (e.g., Uniscept). Of the methods tested, most conventional broth- and agar-based methods proved to be highly accurate when incubation was done for a full 24 h, although several of the tests had high minor error rates. Automated systems continued to demonstrate problems in detecting low-level resistance.

The genus Citrobacter was recently determined to contain 11 genetically distinct species. In addition, the International Committee on Systematic Bacteriology no longer recognizes C. diversus and has, instead, validated the name C. koseri in its place. The 11 species are C. freundii, C. koseri, C. amalonaticus, C. farmeri, C. youngae, C. braakii, C. werkmanii, C. sedlakii, and three unnamed groups, genomospecies 9, 10, and 11. To determine the ease with which some identification systems could respond to these changes, we evaluated five systems for their potential ability to recognize current species in the genus Citrobacter. A simple dichotomous key using conventional biochemicals is presented that may be helpful to presumptively identify Citrobacter strains.

Six chronic hemodialysis patients acquired bloodstream infections (BSIs) with Klebsiella pneumoniae of the same serotype and similar plasmid profile during an 11-day period. The 6 case-patients were more likely than noncase-patients to have received dialysis during the fourth shift (p < 0.05) and to have their dialyzers reprocessed for reuse after those of the noncase-patients (p = 0.05). Investigation identified a patient during the same shift with an arteriovenous fistula infected with K. pneumoniae. The dialyzer reprocessing technician did not change gloves between contacting patients and their dialyzers in the treatment area and reprocessing the case-patients' dialyzers at the end of the fourth shift. We conclude that the outbreak of BSIs was caused by cross-contamination of the case-patients' dialyzers with bacteria from the gloves of the reprocessing technician and by inadequate dialyzer disinfection. After revised dialyzer reprocessing techniques and glove-changing policies were instituted, no further clusters of BSIs occurred.

Injury-reactive ependymal cells from regenerating axolotl spinal cord can be maintained in their mesenchymal outgrowth phase in culture (O'Hara et al., 1992). To address the ability of specific growth factors in stimulating or maintaining migration and proliferation, mesenchymal ependymal cell cultures derived from injured axolotl spinal cord at 2 weeks post-lesioning were used to determine the potential effects of epidermal growth factor, platelet-derived growth factor and transforming growth factor-beta 1. In our cultures, medium containing epidermal growth factor alone or in combination with the other growth factors permitted significant migration and proliferation from ependymal explants. Platelet-derived growth factor alone was shown to have a small positive effect on ependymal cell migration and no effect on proliferation. Transforming growth factor-beta 1 alone did not support cell migration and was found to be inhibitory towards cellular proliferation. Lastly, medium containing platelet-derived growth factor and transforming growth factor-beta 1, but not epidermal growth factor, caused ependymal cell explants to break apart and migrate on the dish as cords. Migration and proliferation of injury-reactive ependymal cells was shown to be dependent on epidermal growth factor in vitro. These results suggest that epidermal growth factor may be a critical component in vivo during the initiation of ependymal migration and proliferation following transection of the axolotl spinal cord. The reorganization of cultured ependymal cells in response to the combination of platelet-derived growth factor and transforming growth factor-beta shows that ependymal organization can be modulated by growth factors. This suggests that the progressive changes observed during regeneration may be under the control of growth factors.

Background: Nosocomial bloodstream infection is an important cause of morbidity and mortality among neonates. From September 1 through December 5, 1990 (epidemic period), gram-negative bacteremia developed in 26 neonates after their admission to the neonatal intensive care unit (NICU) of Hospital General, a 1000-bed public teaching hospital in Guatemala with a 16-bed NICU. Twenty-three of the 26 patients (88%) died., Methods: To determine risk factors for and modes of transmission of gram-negative bacteremia in the NICU, we conducted a cohort study of NICU patients who had at least one blood culture drawn at least 24 hours after admission to the NICU and performed a microbiologic investigation in the NICU., Results: The rate of gram-negative bacteremia was significantly higher among patients born at Hospital General, delivered by cesarian section, and exposed to selected intravenous medications and invasive procedures in the NICU during the 3 days before the referent blood culture was obtained. During the epidemic period, the hospital's chlorinated well-water system malfunctioned; chlorine levels were undetectable and tap water samples contained elevated microbial levels, including total and fecal coliform bacteria. Serratia marcescens was identified in 81% of case-patient blood cultures (13/16) available for testing and from 57% of NICU personnel handwashings (4/7). Most S. marcescens blood isolates were serotype O3:H12 (46%) or O14:H12 (31%) and were resistant to ampicillin (100%) and gentamicin (77%), the antimicrobials used routinely in the NICU., Conclusions: We hypothesize that gram-negative bacteremia occurred after invasive procedures were performed on neonates whose skin became colonized through bathing or from hands of NICU personnel.

Aerosol pentamidine (AP) is an FDA-approved prophylaxis against pneumocystis carinii pneumonia (PCP) in HIV-infected individuals who have a CD4+ lymphocyte count less than 200/mm3, constitutional symptoms, or a previous history of the pneumonia. The University of Washington Medical Center, a 450-bed tertiary care center, established a successful aerosol pentamidine treatment program, providing treatment in its special procedure nit. The authors present an overview of AP and discuss the role of interdisciplinary teamwork, staff training, patient teaching, and the provision of safety measures for patients and healthcare providers.

We compared the API 20E (21 h) (API; bioMérieux Vitek, Hazelwood, Mo.), the Vitek GNI card (4 to 18 h) (Vitek; bioMérieux Vitek), the identification portion of the MicroScan Walk/Away Rapid Neg Combo 3 panel (2 h) (W/A; Baxter Diagnostics, Inc., West Sacramento, Calif.), and the Becton Dickinson Cobas Micro ID-E/NF rotor (21 h) (Cobas; Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.), versus conventional biochemicals for their abilities to identify accurately 252 strains of biochemically typical and atypical members of the family Enterobacteriaceae and common non-glucose-fermenting gram-negative bacilli. All strains used were included in the data base of each product. At the end of the initial incubation, 194 (77.0%), 213 (84.5%), 198 (78.6%), and 192 (76.2%) strains were correct to the genus and species levels with the API, Vitek, W/A, and Cobas systems, respectively. After additional biochemical tests were performed, as directed by each manufacturer's protocol, the numbers of strains correctly identified to the genus and species levels were 241 (95.6%), 234 (92.8%), 243 (96.4%), and 230 (91.3%) with the four systems, respectively. The errors were random in all systems, with the exception of two atypical Salmonella enteritidis strains, each of which was misidentified by three systems. After the initial recommended incubation period, both API and Cobas were significantly less accurate than Vitek (Yates' corrected P < 0.05). No significant differences were noted between the results of Vitek and W/A or between the results of API and W/A. After additional tests were completed, Cobas was significantly less accurate than W/A (P < 0.05) but was equal in accuracy to Vitek and API. API, Vitek, and W/A were equal in accuracy after these same additional tests. All four systems were significantly more accurate after additional biochemical testing than after the initial reporting period (194 of 252 versus 241 of 252 for API, 213 of 252 versus 234 of 252 for Vitek, 198 of 252 versus 243 or 252 for W/A, and 192 of 252 versus 230 of 252 for Cobas [P<0.05]).

A multicenter trial of the Sensititre AP80 panel read on the Sensititre AutoReader (Radiometer America, Westlake, Ohio) for the automated identification of gram-negative bacilli was conducted with 1,023 clinical isolates (879 members of the family Enterobacteriaceae plus 144 nonenteric organisms). Assignment of taxa was based on the computer-assisted interpretation of the results of a series of reactions with fluorogenic enzyme substrates after 5 h of incubation, with an incubation interval of approximately 18 h used when indicated. Accuracy was determined initially by comparison with the results obtained with the API 20E or Rapid NFT system (Analytab Products, Plainview, N.Y.). Isolates showing discrepancies were identified by using conventional biochemical profiles. Identifications were available after 5 h of incubation for 918 isolates (90%). Agreements with reference results for members of the family Enterobacteriaceae were 95.3 and 92.5% at the genus and species levels, respectively, and for the nonmembers of the family Enterobacteriaceae, the agreements with reference results were 95.1 and 84.7%, respectively. The Sensititre AP80 panel was found to be simple and convenient to use, allowed for the testing of three isolates per panel, required minimal supplementary testing for completion of identification, performed in a reproducible fashion, and demonstrated an accuracy of same-day identification comparable to that reported for other automated systems. The AP80 panel appears well suited for routine use in the clinical microbiology laboratory as an automated means of identifying both members of the family Enterobacteriaceae and nonenteric gram-negative bacilli.

We evaluated the ability of the Baxter autoSCAN-W/A System (MicroScan Division, Baxter Diagnostics, Inc., West Sacramento, Calif.) to use the rapid (2-h) gram-negative identification panel for accurate identification of members of the family Enterobacteriaceae. At 2 h, 353 of 467 (75.6%) strains in a challenge set of biochemically typical and atypical stock cultures were correctly identified to genus and species. Another 76 (16.3%) strains were correctly identified to genus and species after the performance of recommended additional biochemical testing. Thus, at 24 h, 91.9% of the 467 strains were correctly identified. Twenty-two strains (4.7%) were identified to the correct genus but the incorrect species, and 16 strains (3.4%) were misidentified. Of these 16 strains, 9 were incorrect at 2 h, and 7 were incorrect after the additional testing. Because the system is based on fluorogenic substrates, no conventional tests were readily available with which to compare aberrant reactions. These results suggest that the autoSCAN-W/A with its rapid gram-negative panels is acceptable for the identification of the Enterobacteriaceae in a clinical microbiology laboratory.

Changes in intermediate filament content and extracellular matrix material showed that the injury response of ependymal cells in lesioned axolotl spinal cord involves an epithelial-to-mesenchymal transformation, and that fibrous astrocytes are excluded from the remodeling lesion site. Antibody localization was used to visualize cytokeratin-, vimentin-, and glial fibrillary acidic protein- (GFAP-) containing intermediate filaments, as well as the adhesive glycoprotein fibronectin. In normal axolotl spinal cord cytokeratins were found near the apical surface of the ependymal cells. Transmission electron microscopic examination suggested that these cytokeratins were in tonofilaments. Cytokeratin expression was lost and vimentin production was initiated in ependymal cells 2-3 weeks following spinal cord injury. There was a period of approximately 1-2 weeks when cytokeratins and vimentin were co-expressed in vivo. This co-expression was maintained in vitro by culture on a fibronectin-coated substratum. As the central canal reformed, vimentin expression was lost. Ependymal cells lacked GFAP intermediate filaments, but GFAP was present in fibrous astrocytes of the neuropil and white matter. Following injury, GFAP localization showed that fibrous astrocytes disappeared from the remodeling lesion site and reappeared only after the ependymal epithelium reformed and newly myelinated axons were found. Fibronectin expression closely followed the expression of vimentin during mesenchymal ependymal cell outgrowth. These results suggest that the ependymal cell outgrowth requires changes in cell shape followed by changes in production of extracellular matrix.

The API 20E bacterial identification system has been used for 19 years, often as the standard with which other identification systems are compared. Because the accuracy of this system compared with conventional biochemical tests has not been determined in many years, we evaluated the API 20E linear strip by using 291 typical and atypical strains of the family Enterobacteriaceae taken from a culture collection. At 24 h, the API 20E correctly identified by genus and species 229 of 291 (78.7%) of the strains, using Salmonella and Shigella serotyping where indicated. At 48 h, 95.2% were correctly identified by using additional biochemical tests as recommended by the manufacturer. The API 20E misidentified eight (2.7%) strains; these strains were not limited to any particular genus. When 81 of these Enterobacteriaceae strains were arranged into a weighted assortment correlating to the frequency with which they might be found in a clinical laboratory, the API 20E correctly identified 71 (87.7%) at 24 h and 78 (96.3%) at 48 h. This evaluation concluded that the accuracy of the identification of Enterobacteriaceae strains at 24 h (78.7%) may be significantly lower than that of earlier evaluations. However, there is no significant difference in the ability of the API 20E to correctly identify "challenge" type organisms (229 of 291) versus routine hospital isolates (71 of 81) (P greater than 0.05), but the system is not as accurate as the conventional biochemical method of identification.

Two unrelated hospital outbreaks of Enterobacter sakazakii, involving meningitis, bacteremia, and colonization of neonates, were investigated. In each of these outbreaks, E. sakazakii was isolated from both patients and dried infant formula. In previous outbreaks, the source and mode of transmission of E. sakazakii in neonatal infections was not determined. In this study, we used a combination of typing methods (plasmid analysis, antibiograms, chromosomal restriction endonuclease analysis, ribotyping, and multilocus enzyme electrophoresis) to evaluate the isolates from each outbreak as to their relatedness. The typing results differed among outbreaks, but in each one, patient and formula isolates shared the same typing pattern. The only exceptions were disk antibiograms, which often varied among colonies selected from each of the isolates. Plasmid analysis, chromosomal restriction endonuclease analysis, ribotyping, and multilocus enzyme electrophoresis all were effective as epidemiological typing methods for E. sakazakii, especially when used in combination. By using this typing scheme, we have confirmed that E. sakazakii from intrinsically contaminated dried infant formula was the source of neonatal infection.

The UniScept API system was evaluated for agreement of visual versus automated readings of both its identification panels and its antimicrobial susceptibility panels. The biochemical responses of 340 oxidase-negative and oxidase-positive fermentative bacterial cultures were read both visually and automatically in the UniScept API 20E system. Automated and visual readings agreed with 99.3% of the biochemicals. Of the 45 tests that disagreed, the tests for indole and citrate were most often in disagreement. A total of 470 fermentative and nonfermentative cultures were used in the UniScept MIC system to compare visual and automated readings of susceptibility results with 17 antimicrobial agents. Agreement within +/- 1 dilution occurred with 94.1% of the enteric fermenters and with 91.7% of the other cultures. Comparison of visual and automated readings resulted in very major discrepancies in 0.95% of the readings, with the largest percentage of discrepancies associated with glucose nonfermenters (1.8%). It was felt that an automated reading is an acceptable alternative to a visual reading of the biochemicals but that 0.95% was just within the acceptable range of the 1% allowable very major discrepancies in the automated reading of susceptibilities.