Prevalence of AmpC over-expression in bloodstream isolates of Pseudomonas aeruginosa.

This study examined the contribution of AmpC over-expression to beta-lactam resistance in clinical isolates of Pseudomonas aeruginosa obtained from a hospital in Houston, TX, USA. Seventy-six non-repeat bloodstream isolates obtained during 2003 were screened for ceftazidime resistance in the presence and absence of clavulanic acid 4 mg/L. AmpC was identified by isoelectric focusing (with and without cloxacillin inhibition); stable derepression was ascertained phenotypically by a spectrophotometric assay (with and without preceding induction by imipenem) using nitrocefin as the substrate, and was confirmed subsequently by quantitative RT-PCR of the ampC gene. The clonal relatedness of the AmpC-over-expressing isolates was assessed by pulsed-field gel electrophoresis. In addition, the ampC and ampR gene sequences were determined by PCR and sequencing. For comparison, two standard wild-type strains (PAO1 and ATCC 27853) and three multidrug-susceptible isolates were used as controls. AmpC over-expression was confirmed in 14 ceftazidime-resistant isolates (overall prevalence rate, 18.4%), belonging to seven distinct clones. The most prevalent point mutations in ampC were G27D, V205L and G391A. Point mutations in ampR were also detected in eight ceftazidime-resistant isolates. AmpC over-expression appears to be a significant mechanism of beta-lactam resistance in P. aeruginosa. Understanding the prevalence and mechanisms of beta-lactam resistance in P. aeruginosa may guide the choice of empirical therapy for nosocomial infections in hospitals.

Multiple drug-resistance in Shigella flexneri isolated from a patient with human immunodeficiency virus.

Persistent shigellosis, due to Shigella flexneri resistant to multiple antibiotics, developed in a 40-yr-old homosexual man with human immunodeficiency virus infection. The Shigella strain demonstrated resistance to ampicillin, tetracycline, and trimethoprim-sulfamethoxazole. Although Shigella flexneri isolates resistant to trimethoprim-sulfamethoxazole are uncommon in the United States, laboratories should monitor resistance patterns through routine in vitro susceptibility testing.

Mycobacterium fortuitum peritonitis in a patient undergoing chronic peritoneal dialysis.

Peritonitis, due to Mycobacterium fortuitum, developed in a 15-yr-old young man undergoing chronic peritoneal dialysis. Although of low pathogenic potential, this rapidly growing non-tuberculous mycobacterium does cause human disease particularly in the compromised host and should be considered as a potential cause of peritonitis in the chronic peritoneal dialysis patient.

Prevalence of type III secretion protein exoenzymes and antimicrobial susceptibility patterns from bloodstream isolates of patients with Pseudomonas aeruginosa bacteremia.

The purpose of this study was to determine the prevalence of two type III secretion effector proteins, exoU and exoS from bloodstream isolates of hospitalized patients with Pseudomonas aeruginosa (PSA) bacteremia, to characterize antimicrobial susceptibility patterns, and to compare mortality rates. PSA bloodstream isolates and antibiotic susceptibility profiles were collected from a university-affiliated hospital. ExoS and exoU genes were detected by polymerase chain reaction. Hospital mortality was assessed by medical chart review. 119 of 122 (97.5%) PSA bloodstream isolates contained either the exoS or exoU genes. ExoS was the most prevalent (n=86; 70.5%) followed by exoU (n=31; 25.4%), both genes (n=2; 1.6%) or neither gene (n=3; 2.5%). Isolates containing the exoU gene were significantly more likely to be resistant to cefepime, ceftazidime, piperacillintazobactam, carbapenems, fluoroquinolones, and gentamicin (p<0.05 for all). Mortality was high in patients with PSA bacteremia and did not differ among patients infected with the exoS isolates (n=37; 43%) or exoU isolates (n=11; 35%). One of two type III secretion effector proteins were almost universally present in PSA bloodstream isolates. Isolates containing the exoU gene were more likely to be resistant to multiple antibiotics.

Comparison of risk factors for candidemia versus bacteremia in hospitalized patients.

BACKGROUND: Classic risk factors for candidemia include use of total parenteral nutrition (TPN), hospital location, use of central venous catheter, and others. Unfortunately, most of these variables are now also risk factors for antibiotic-resistant bacteria. Thus, use of these risk factors to identify patients at high risk for candidemia is difficult. The purpose of this study was to compare these classic risk factors for candidemia in patients with bloodstream infections to determine the relative strength of these predictors in differentiating patients with candidemia and bacteremia. METHODS: Clinical data were collected from the medical charts of patients who had been hospitalized between 2002 and 2004. Patients with their first episode of candidemia or bacteremia during their hospital stays were included. Risk factors were assessed using a multivariate logistic regression model and internally validated using a bootstrap analysis. A p-value < 0.05 was considered significant. RESULTS: A total of 164 patients (82 with candidemia) were evaluated. According to the logistic analysis, patients who had stayed in the intensive care unit (ICU) (OR = 6.24; 95% CI: 2.58-15.09) or had been using TPN (OR = 4.69; 95% CI: 1.76-12.48) were more likely to have candidemia than bacteremia. While patients with pulmonary (OR = 0.15; 95% CI: 0.055-0.39) or cardiac disease (OR = 0.21; 95% CI: 0.086-0.51) had a greater chance to have bacteremia than candidemia (p < 0.01 for all variables). These results were further validated using bootstrap analysis. CONCLUSION: Among classic risk factors for candidemia, the ICU location at the time of culture and TPN use were most predictive of candidemia while certain medical disorders predicted patients at the highest risk for bacteremia. These results can be used to help identify patients most likely to benefit from empiric antifungal therapy.

Evaluation of an enzyme-linked viral inducible system for the rapid detection of Herpes simplex virus.

A commercial enzyme-linked viral inducible system (ELVIS HSV; BioWhittaker, USA) was evaluated in comparison with the spin-amplified tube cell culture (SATCC) method for the rapid detection of herpes simplex virus in 1007 clinical specimens. A total of 91 (9%) specimens were positive by SATCC. The sensitivity, specificity, positive predictive value, and negative predictive value of ELVIS was 88%, >99%, 99%, and 99%, respectively. Herpes simplex virus was detected sooner by ELVIS than by SATCC in 34 of 80 (42%) specimens. Preincubated ELVIS shell vials held at room temperature for 24 h prior to reincubation and inoculation produced results similar to those of freshly preincubated shell vials, with no reduction in either the number or the staining intensity of the infected cells. The results of this study indicate that ELVIS HSV is an accurate method for the rapid detection of herpes simplex virus in a wide variety of clinical specimens.

Infection in the bone marrow transplant recipient and role of the microbiology laboratory in clinical transplantation.

Over the past quarter century, tremendous technological advances have been made in bone marrow and solid organ transplantation. Despite these advances, an enduring problem for the transplant recipient is infection. As immunosuppressive regimens have become more systematic, it is apparent that different pathogens affect the transplant recipient at different time points in the posttransplantation course, since they are influenced by multiple intrinsic and extrinsic factors. An understanding of this evolving risk for infection is essential to the management of the patient following transplantation and is a key to the early diagnosis and treatment of infection. Likewise, diagnosis of infection is dependent upon the quality of laboratory support, and services provided by the clinical microbiology laboratory play an important role in all phases of clinical transplantation. These include the prescreening of donors and recipients for evidence of active or latent infection, the timely and accurate microbiologic evaluation of the transplant patient with suspected infection, and the surveillance of asymptomatic allograft recipients for infection. Expert services in bacteriology, mycology, parasitology, virology, and serology are needed and communication between the laboratory and the transplantation team is paramount for providing clinically relevant, cost-effective diagnostic testing.

Detection of piluslike structures on clinical and environmental isolates of Vibrio vulnificus.

Twenty clinical isolates of Vibrio vulnificus were compared with 10 environmental strains by using electron microscopy and agglutination assays with human erythrocytes, guinea pig erythrocytes, and Saccharomyces cerevisiae. In addition, the isolates were tested for ability to adhere to the human epithelial cell lines HEp-2 and A549. When examined by electron microscopy, 16 (80%) of the 20 clinical isolates demonstrated the presence of piluslike structures; the composition of the bacterial populations ranged from 0 to 68% piliated cells. In contrast, only 3 (30%) of the 10 environmental isolates were piliated, with a range from 0 to 16% piliated cells. A significant association between the presence of piliated cells and the isolate source was found (P less than 0.05). None of the 30 strains agglutinated erythrocytes or yeast cells. V. vulnificus adherence results obtained with HEp-2 cells showed 10 (50%) of 20 clinical isolates and 0 (0%) of 10 environmental isolates with averages of greater than 10 adherent bacteria per cell, demonstrating a correlation between attachment and the isolate source (P less than 0.05). Selected strains were tested to determine whether methyl alpha-D-mannopyranoside, fructose, or alpha-L-(-)-fucose would inhibit bacterial adherence to HEp-2 cells. Multiple patterns of adherence inhibition were observed. Adherence to A549 cells showed 8 (40%) of 20 clinical isolates and 0 (0%) of 10 environmental strains with averages of greater than 10 adherent bacteria per cell. A statistical association between attachment and the isolate source was demonstrated (P less than 0.05). These data suggest that the presence of piluslike structures and the ability to adhere to human epithelial cell lines may be more closely associated with V. vulnificus isolates from clinical specimens than with environmental strains.

Fluconazole susceptibilities of Candida species and distribution of species recovered from blood cultures over a 5-year period.

The distribution and fluconazole susceptibilities of Candida species isolated over a 5-year period were investigated. Susceptibilities were determined by using a new microtiter procedure and the National Committee for Clinical Laboratory Standards (NCCLS) proposed standard. The new method correlated well with the NCCLS proposed standard and gave very clear end points. Results indicate that there are species-related differences in MICs as reflected in the MICs for 90% of species tested. Candida albicans is most susceptible to fluconazole, while Candida glabrata is among the least susceptible. These findings coincided with the observation of a shift in distribution of yeast species recovered from blood cultures from 1987 to 1992. C. albicans was the predominant species (87%) in the pre- or early fluconazole years but decreased to only 31% of the isolates in 1992. Thus, Candida species for which MICs of fluconazole were higher have become more prominent in recent years. Significantly, throughout this period, MICs for each species did not change appreciably.

Latex agglutination for rapid identification of methicillin-resistant Staphylococcus aureus recovered from selective media.

The accuracy of combining latex agglutination with selective media for the identification of methicillin-resistant Staphylococcus aureus (MRSA) was determined. Test strains were identified by latex agglutination on blood agar, the heat-stable thermonuclease test and broth microdilution MICs of oxacillin and included 97 MRSA, 56 methicillin-susceptible Staphylococcus aureus, 52 methicillin resistant, and 49 methicillin-susceptible Staphylococcus species. Isolates were grown on trypticase-soy agar with 5% sheep red blood cells (TSAB), Mueller-Hinton agar (MHA), mannitol-salt agar (MSA), and four media designed for the selective growth of MRSA:TSAB with clindamycin and gentamicin, MHA with oxacillin, MSA with oxacillin, and lipovitellin-salt-mannitol agar (LVSM) with 1 microgram oxacillin disks applied. The mean sensitivity, specificity, and positive predictive value for the combination of latex agglutination with selective media for the identification of MRSA was 96%, 99% and 98% respectively.

Cultivation of Nocardia spp. on chemically defined media for selective recovery of isolates from clinical specimens.

Isolation of Nocardia spp. from clinical specimens can be enhanced by the use of paraffin baiting, which relies on the selective ability of the organism to metabolize paraffin. We evaluated 44 Nocardia isolates, 18 group IV mycobacterial isolates, and 4 Streptomyces isolates for growth on blood agar (BA) and on carbon-free agar containing single or double substrates as follows: paraffin agar (PA), gelatin agar (GA), urea agar (UA), PA-gelatin (PG), and PA-urea (PU). The growth rates of Nocardia spp. on BA, PA, PU, and PG were similar; but 3-day-old colonies were larger on BA for 20 (45%) isolates. After longer incubations (7 to 14 days), some Nocardia colonies were larger on PA, PG and PU than they were on BA. Despite variable morphologies on BA, colonies on PA, PG, and PU were consistently smooth, creamy, and raised. Compared with growth on BA, the growth of mycobacteria was much slower on PA, PG, and PU, with poor growth on UA and GA. The growth of Streptomyces spp. was greatly enhanced on GA, PG, UA, and PU and was poorest on PA. Twelve sputum specimens seeded with Nocardia asteroides (10(4) CFU/ml) were inoculated onto BA and all chemically defined media. Nocardiae were recovered from 6 to 12 specimens grown on BA, GA, and UA; 11 of 12 specimens grown on PG; and 12 of 12 specimens grown on PA and PU. Only PA was able to suppress the growth of other microorganisms that were present in sputum specimens. These results suggest that chemically defined media containing PA may be useful for the selective isolation of Nocardia spp. from contaminated clinical specimens.

Inhibition of germ tube development of Aspergillus fumigatus in cell-free transudate produced in subcutaneous chambers in rabbits.

The interaction of Aspergillus fumigatus conidia with host factors produced in rabbits was studied by means of subcutaneous, perforated plastic chambers. Transudate fluid from spore-free chambers, sampled 30 days after implantation, supported germ tube development and rapid hyphal growth of A. fumigatus in an assay in vitro. Inoculation of spores into chambers implanted 30 days previously produced a rapid infiltration of leukocytes, predominately neutrophils, into the chamber fluid. Cell-free supernatants, prepared from transudates collected 5-6 days after inoculation, inhibited germ tube development in vitro. This inhibition was also demonstrated using lysates derived from 3.6 X 10(6) leukocytes obtained from chambers 5 days after inoculation with 1 X 10(7) spores. However, lysates derived from greater than 10(7) leukocytes obtained from pre-inoculated chambers as well as peritoneal exudate cells did not inhibit germ tube development in vitro. The inhibitory activity of cell free supernatants was not altered by heating at 56 degrees C for 30 min but was destroyed by pronase treatment as well as boiling. These results provide evidence for a host defense mechanism against rapid hyphal extension mediated by the extracellular release of inhibitory factors by leukocytes.

Destructive native valve endocarditis caused by Staphylococcus lugdunensis.

Coagulase-negative staphylococci are uncommon causes of native valve endocarditis, and the clinical course after valvular infection with these organisms is variable. In clinical practice, species identification is frequently not done, and possible differences in the pathogenicity of various species may be unrecognized. We report a case of Staphylococcus lugdunensis native valve endocarditis associated with valve leaflet perforation and cerebral embolization. This recently described species appears to be more virulent when infecting native cardiac valves than other species of coagulase-negative staphylococci. We review S lugdunensis native valve endocarditis.

Latex agglutination-negative methicillin-resistant Staphylococcus aureus recovered from neonates: epidemiologic features and comparison of typing methods.

An unusual strain of methicillin-resistant Staphylococcus aureus (MRSA) was repeatedly isolated from infants in a newborn special care unit (NBSC) and a newborn intensive care unit. Between January 1989 and March 1990, approximately 100 isolates from infected or colonized infants were recovered. Surveillance cultures taken during this time revealed a 20% colonization rate, which was defined as recovery of MRSA from the nares, umbilicus, or groin. Isolates were identified as S. aureus by tube coagulase reactivity and heat-stable nuclease production but were unreactive in a latex agglutination assay. Representative isolates that were collected during the outbreak and that were found to share the latex agglutination assay-negative phenotype were compared by antibiogram (12 isolates), bacteriophage typing (20 isolates), capsular polysaccharide typing (30 isolates), and plasmid as well as chromosomal DNA analyses (20 isolates). All isolates known to be associated with the outbreak had nearly identical antibiograms and were notably susceptible to clindamycin. Staphylococcal bacteriophage typing was not useful in determining the relatedness of the isolates, since the majority were nontypeable. Plasmid pattern analysis revealed one large plasmid (approximately 100 kb) of equivalent size among the isolates. Capsular polysaccharide typing revealed that 14 of 30 isolates tested were type 5. Isolates identified in children at two other hospitals in the city which were also unreactive by the latex agglutination assay and clindamycin susceptible had plasmid and antibiogram patterns identical to those of isolates from the NBSC. Pulsed-field gel electrophoresis of restriction enzyme-digested genomic DNAs from the outbreak isolates demonstrated identical patterns which could be clearly differentiated from those of other unrelated MRSA. The strain from the NBSC is, therefore, unique and underscores the need for caution in interpreting the latex agglutination reactivities of MRSA isolates.

Salmonella infection of the biliary and intestinal tract of wild opossums.

Bacteriologic cultures were taken from the mesenteric lymph nodes, biliary tract, blood, liver, spleen and pancreas of opossums (Didelphis virginiana) obtained directly from the wild for use as research animals. The overall incidence of salmonellosis outside the intestinal tract was 61% among 18 opossums. Salmonella was recovered from the gallbladder of six (33%) animals, indicating chronic biliary tract infection. Among these six animals, translocation of Salmonella to regional lymph nodes was observed in five animals, bacteremia in three animals, and spread to liver or spleen in five animals, respectively. The biliary tract was sterile in 12 opossums (67%). In these 12 animals, bacteria were isolated from the celiac and superior mesenteric lymph nodes of five animals, the blood of two animals, and the liver and spleen of one animal, respectively. Bacteriologic cultures were obtained from the intestinal tract and from extraintestinal sites in nine opossums. Salmonella were found in the small bowel of two animals, both of which had biliary salmonellosis. In addition, Salmonella was isolated from extraintestinal organs of three animals with negative cultures from the gut. All isolates identified were: S. enterica subsp houtenae. These data establish the biliary tract of wild opossums as a reservoir for Salmonella enterica subsp houtenae which may be particularly important when opossums are used in research laboratories.

Comparison of E test with standard broth microdilution for determining antibiotic susceptibilities of penicillin-resistant strains of Streptococcus pneumoniae.

We compared the E test (AB Biodisk North America, Inc., Culver City, Calif.) with the National Committee for Clinical Laboratory Standards broth microdilution method for the determination of MICs of penicillin and cefotaxime for 108 isolates of Streptococcus pneumoniae. The E test was performed following manufacturer's recommendations with Mueller-Hinton blood agar, and the broth microdilution procedure was performed with lysed horse blood-supplemented Mueller-Hinton broth. The microdilution method classified 26 isolates as highly penicillin resistant (MIC, > or = 2 micrograms/ml), 33 as intermediately resistant to penicillin (MIC, > or = 0.1 < 2.0 micrograms/ml), and 49 as susceptible to penicillin (MIC, < 0.1 micrograms/ml). Discordant results obtained with the E test for penicillin susceptibility testing compared with broth microdilution occurred for 19 of the 108 isolates tested. Cefotaxime MICs for 90% of isolates found highly resistant, intermediately resistant, and susceptible to penicillin by broth microdilution were 2.0, 0.5, and 0.06 micrograms/ml, respectively. There were 16 susceptibility category changes when the E test was used to determine cefotaxime MICs. All of the discrepancies in the penicillin and cefotaxime MICs determined by the E test occurred at the susceptibility category breakpoints, and all represented differences of only one twofold dilution factor. Properly performed and controlled, the E test should be a reliable quantitative procedure for more accurately predicting the susceptibility of S. pneumoniae to several antibiotics.

Protein fingerprinting for the determination of relatedness in Acinetobacter calcoaceticus subspecies anitratus isolated from patients in a surgical intensive care unit.

The use of protein fingerprinting for the establishment of relatedness among isolates of Acinetobacter calcoaceticus subspecies anitratus, isolated from patients in a surgical intensive care unit was examined. Polyacrylamide gel electrophoresis was used to analyze the cellular proteins from whole cell lysates of 14 intensive care unit A calcoaceticus subspecies anitratus isolates and 11 control strains. Antimicrobial susceptibilities and plasmid profiles were also determined for all isolates. All intensive care unit A calcoaceticus subspecies anitratus isolates exhibited identical cellular protein fingerprints, no detectable plasmids, and minimum inhibitory concentrations within +/- 1 log2 dilution of the mode value for each of the antimicrobial agents tested. The other clinical isolates demonstrated a range of antimicrobial susceptibilities, various numbers and sizes of plasmids, and distinctly different protein fingerprints. These data indicate that protein fingerprinting may be useful as an epidemiologic tool in A calcoaceticus subspecies anitratus outbreaks and this method deserves further study.