Rapid identification of positive blood cultures by matrix-assisted laser desorption ionization-time of flight mass spectrometry using prewarmed agar plates.

This study describes an inexpensive and straightforward method for identifying bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) directly from positive blood cultures using prewarmed agar plates. Different inoculation methods and incubation times were evaluated to determine the optimal conditions. The two methods using pelleted material from positive culture bottles performed best. In particular, the pellet streak method correctly identified 94% of the Gram negatives following 4 h of incubation and 98% of the Gram positives following 6 h of incubation.

Evaluation of FilmArray and Verigene systems for rapid identification of positive blood cultures.

The Verigene tests for Gram-positive and Gram-negative organisms in blood culture and the FilmArray blood culture identification panel were assessed for their ability to identify pathogens from positive blood cultures. Both platforms correctly identified bacteria in 92% of monomicrobial cultures analyzed, with times to identification that were significantly shorter than those for identification from subcultures.

Allergic inflammation enhances bacterial sinusitis in mice.

BACKGROUND: Although it is not proven, one factor considered important in the development of sinusitis is allergic rhinitis. OBJECTIVE: The purpose of this study was to determine whether ongoing allergic rhinitis enhances the infection and inflammation associated with Streptococcus pneumoniae acute sinus infection. METHODS: BALB/c mice were sensitized to ovalbumin by intraperitoneal injection. After infection of the sinuses by S pneumoniae, either with or without concomitant administration of ovalbumin to induce allergic inflammation, mice were killed at various times and their heads were prepared for histologic evaluation of the sinuses. RESULTS: Mice became allergic to ovalbumin and developed eosinophilia in the sinus and lung cavities in response to ovalbumin administration to each of the respective cavities. In comparison with controls, the mice with ongoing nasal allergic inflammation that were inoculated with S pneumoniae had significantly more bacteria recovered at sacrifice and had significantly more inflammation, as indicated by neutrophil, eosinophil, and mononuclear influx into the sinus mucosa. The percentage of the sinus area occupied by neutrophil clusters was also increased after infection in the allergic mice in comparison with the control mice. CONCLUSION: Our data demonstrate that mice can be sensitized to ovalbumin and develop a localized allergic reaction in the skin, nose, or lung. An ongoing local allergic response augments bacterial infection in these animals. We also demonstrate that allergic sensitization alone, allergen exposure alone, or an allergic response at a distal site, the lung, does not augment the sinus infection.

C57Bl/6 and BALB/c mice have similar neutrophil response to acute Streptococcus pneumoniae sinus infections.

BACKGROUND: Previous investigations have shown that mice with a tendency toward a T(H)1 or T(H)2 lymphocyte response manifest different reactions to inoculation with the parasite Leishmania major. BALB/c mice (with a tendency for a T(H)2 response) showed evidence of systemic infection, whereas C57Bl/6 mice (with a tendency for a T(H)1 response) showed only a local reaction. OBJECTIVE: To investigate whether BALB/c and C57Bl/6 mice respond differently to acute bacterial infection of the sinuses. METHODS: We inoculated the nasal cavities of C57Bl/6 and BALB/c mice with Streptococcus pneumoniae (type ATCC59), or with broth as a control. The mice were humanely killed 2, 5, 10, and 14 days after inoculation. Their heads were fixed, decalcified, and embedded in paraffin blocks. Sections were stained with hematoxylin and eosin, and the degree of inflammation was quantified by the number of neutrophils per square millimeter of the sinus mucosa and the percentage of the sinus cavity occupied by neutrophil clusters. RESULTS: Both groups of mice showed evidence of inflammation that was significantly greater than controls (P =.01), with no difference between groups. There was a correlation between the number of neutrophils per square millimeter in the sinus mucosa and the percentage of neutrophil clusters (C57Bl/6 mice, r = 0.37, P<.001; BALB/c mice, r = 0.20, P<.001). In the infected mice, the number of infiltrating neutrophils was significantly greater (P<.001) in anatomically lower (dependent) areas of the sinuses compared with the upper areas. CONCLUSION: Unlike leishmaniasis, acute bacterial sinusitis is not affected by the tendency of the host to favor either a T(H)1 or T(H)2 response.

Bactrim reduces the inflammatory response in a murine model of acute rhinosinusitis.

OBJECTIVE: To determine whether treatment with an antibiotic (trimethoprim-sulfamethoxazole) reduced the inflammatory response in a murine form of Streptococcus pneumoniae-induced rhinosinusitis. DESIGN: We randomized 18 C57BL/6 mice to either treatment with intraperitoneal trimethoprim-sulfamethoxazole (Bactrim, 30 mg/kg) or no treatment (control). After 2 days, we inoculated all C57BL/6 mice intranasally with a Bactrim-susceptible strain of Streptococcus pneumoniae, ATCC 49619, suspended in Trypticase soy broth. At day 5 after bacterial inoculation, we sacrificed the mice and prepared histopathologic sections of their sinuses after culturing their nasal cavities by lavage. SETTING: Animal care facility at a tertiary, academic institution. METHODS: The histopathologic sections of the sinuses were examined in a blind manner for the percent of sinus cavity area occupied by neutrophil clusters, and for the number of neutrophils per square millimeter of sinus mucosa. RESULTS: The Bactrim group had a significantly smaller sinus area occupied by neutrophil clusters (1.58% +/- 1.13 vs 4.38% +/- 3.41; P < 0.05), significantly fewer neutrophils infiltrating the mucosa (58.81 +/- 29.63/mm2 vs 105.85 +/- 48.49/mm2; P < 0.05), and significantly less growth of Streptococcus pneumoniae colonies in the intranasal cultures (8 few and 1 moderate vs 3 few, 3 moderate, and 1 many; P = 0.05) compared to the control group. CONCLUSION: In our murine model of acute rhinosinusitis, Bactrim decreased the number of neutrophil clusters in the sinus cavities, the number of neutrophils infiltrating the sinus mucosa, and the growth of Streptococcus pneumoniae. We propose that our murine model can be used for the study of the pathophysiology and treatment of acute rhinosinusitis.

A mouse model of acute bacterial rhinosinusitis.

OBJECTIVE: To develop a mouse model of acute bacterial rhinosinusitis. DESIGN: Study mice (C57BL6/J) were inoculated intranasally with Streptococcus pneumoniae, ATCC 49619 suspended in trypticase soy broth, and controls were inoculated with trypticase soy broth alone. After 2, 5, or 14 days, intranasal cultures were obtained and mice were killed. The sinuses were prepared for histological investigation. SETTING: Animal care facility at a tertiary, academic institution. METHOD: The histological sections of the sinuses were examined in a blinded manner for the percentage of sinus cavity occupied by neutrophil clusters, and for the number of neutrophils per square millimeter of sinus mucosa. RESULTS: Infected mice killed at 5 days had significantly more sinus area occupied by neutrophil clusters, significantly more neutrophils within the mucosa, and significantly more S pneumoniae growth in the intranasal cultures compared with controls (15/15 vs 0/6; P<.01). The amount of inflammation had decreased at 2 weeks. CONCLUSION: Streptococcus pneumoniae induces acute bacterial rhinosinusitis in C57BL6/4 mice as measured by culture and influx of neutrophils, and can be used as a model of acute bacterial rhinosinusitis.

Detection of high-level aminoglycoside resistance in enterococci other than Enterococcus faecalis.

The ability of six screening methods to detect high-level aminoglycoside resistance in enterococcal species other than Enterococcus faecalis was investigated. The 85 Enterococcus isolates, which included 55 E. faecium, 11 E. gallinarum, 9 E. casseliflavus, 5 E. raffinosus, 4 E. avium, and 1 E. mundtii, were tested by using aminoglycoside-supplemented brain heart infusion agar (BHI), Remel EF Synergy Quad plates, high-content aminoglycoside diffusion disks, standard (prepared in-house) microdilution panels, Pasco MIC Gram Positive microdilution panels, and Vitek GPS-TA cards. When tested on BHI, 32 and 35 strains showed resistance to gentamicin and streptomycin, respectively. Resistance profiles obtained with Remel EF Synergy Quad plates were in complete agreement with those obtained on BHI. However, growth on Mueller-Hinton agar-based plates was not as heavy. Some isolates showed only weak growth and required 48 h for resistance to become evident, especially with swab inoculation of quadrants containing 2,000 micrograms of gentamicin per ml. Profiles obtained by use of the agar-based screens were used as the basis for evaluating the other methods. Disk diffusion showed complete agreement. No false resistance occurred by either microdilution method, but 48 h of incubation was needed for detection of some gentamicin-resistant isolates, and 14% of the streptomycin-resistant strains were not detected by standard microdilution. The Vitek GPS-TA card detected 81 and 100% of the gentamicin- and streptomycin-resistant isolates, respectively. In general, most methods used to detect high-level aminoglycoside resistance in E. faecalis appear to be reliable for the testing of the other enterococcal species. However, further investigations with a greater number of resistant E. raffinosus, E. avium, and E. mundtii isolates, when they are available, will be useful for establishing the full range of enterococci that can reliably be tested by the various methods.

Factors influencing determination of high-level aminoglycoside resistance in Enterococcus faecalis.

The ability of seven methods to detect high-level gentamicin (58 strains) and streptomycin resistance (56 strains) among 107 Enterococcus faecalis isolates was investigated at the University of Chicago Medical Center and the University of Nebraska Medical Center. Methods included a standard agar screen plate, high-content disk diffusion, Remel (Lenexa, Kans.) EF Synergy Quad plates, standard microdilution panels prepared in house, Pasco MIC Gram-Positive panels (Difco Laboratories, Detroit, Mich.), MicroScan MIC Type 5 dry panels (Baxter Healthcare Corp., MicroScan Div., West Sacramento, Calif.), and Vitek GPS-TA cards (Vitek Systems Inc., Hazelwood, Mo.). Results indicating false resistance were not obtained by any method, and there was 100% agreement between the results of the disk diffusion and standard agar screen methods. Prolonging incubation from 24 to 48 h increased resistance detection for both agar and microdilution screens. EF Synergy Quad plates inoculated with micropipettes detected 100% of the streptomycin- and gentamicin-resistant isolates. Resistance detection for streptomycin and gentamicin, respectively, was 93 and 96% by standard microdilution, 93 and 98% by Pasco panels, 88 and 89% by MicroScan panels, and 88 and 91% by Vitek GPS-TA cards. False susceptibility occurred more frequently with streptomycin-resistant isolates than it did with gentamicin-resistant strains and appeared to be strain related in some instances. The use of an increased inoculum size enhanced resistance detection with these strains, but it complicated interpretation of results and led to the selection of streptomycin-resistant mutants. Until results of further studies delineate optimum test conditions, a delay in the final interpretation of agar and microdilution screen results until 48 h for isolates showing no or light growth at 24 h may help to minimize the occurrence of false susceptibility reporting.

Direct susceptibility testing of blood culture isolates with the AutoMicrobic System (AMS).

To decrease the time needed to obtain preliminary antimicrobial susceptibility results with blood culture isolates, we inoculated a suspension of centrifuged organisms from blood culture broth directly into the AutoMicrobic System Gram-Positive (GPS) and Gram-Negative (GSC+) susceptibility cards (AMS, Vitek Systems Inc., Hazelwood, MO). Interpretive category results (susceptible, moderately susceptible, resistant) obtained by this direct method (DAMS) were then compared with results obtained by conventional inoculation (i.e., using 18-hr subcultures) of both AMS cards (CAMS method) and broth microdilution panels (MIC method, Micro-Media Systems Inc., Potomac, MD). Ninety-six Gram-positive cocci (951 antimicrobial agent--organism combinations) and 112 Gram-negative bacilli (1006 antimicrobial agent-organism combinations) were tested. When only very major (false susceptible DAMS results) and major (false resistant DAMS results) discrepancies were considered, 95% of the DAMS results for Gram-positive cocci agreed with CAMS results and 93% agreed with MIC results. Most discrepancies were observed when staphylococci were tested against oxacillin and when enterococci were tested against several antimicrobial agents. For Gram-negative bacilli, 94% of DAMS results agreed with CAMS results and 93% agreed with MIC results. Most discrepancies occurred when Enterobacter spp. and Serratia marcescens were tested against ampicillin and cefamandole. The DAMS method provides accurate and rapid preliminary susceptibility test results, usually within 6 to 7 hr of the time a positive blood culture is first detected.

Detection of Mycoplasma hominis septicemia by radiometric blood culture.

The ease with which Mycoplasma hominis can be recovered and the frequency of its occurrence in septicemia may not be fully appreciated. We detected the growth of M. hominis radiometrically with an automated blood culture instrument. The organism grew in both aerobic and anaerobic culture media, but the cultures were not visibly positive. It was necessary to stain the cultures with acridine orange to visualize M. hominis and to subculture them on Columbia base sheep blood agar to confirm the positive growth index indicated by the instrument. Sodium polyanetholesulfonate inhibited the growth of M. hominis and is not recommended for use as the anticoagulant when blood is cultured for Mycoplasma spp.

Cryopreservation of human granulocytes: study of granulocyte function and ultrastructure.

Human granulocytes can be cryopreserved with dimethyl sulfoxide (DMSO) at--80 degrees C. However, the percent recovery of functional cells has been unsatisfactory to date. Throughout this study we have been able to prepare relatively pure granulocytes approximately equal to 85%), cryopreserve them with 5%--10% DMSO with and without serum, and store them at--80 degrees C for up to 4 mo. The parameters studied were absolute cell counts and viability determination, myeloperoxidase activity, phagocytosis, candidacidal activity, bactericidal activity, nitroblue tetrazolium reduction, chemiluminescence, and cell morphology by transmission and scanning electron microscopy. Based on our investigation, granulocytes cryopreserved without serum showed an intact membrane of superior integrity as compared with those preserved with serum. At least 50% of the cells recovered were functional after 2 mo of storage, but there was a progressive loss of viability and function on prolonged storage. The property of phagocytosis was the best preserved after storage for 4 mo, whereas myeloperoxidase activity, killing activity, nitroblue tetrazolium reduction, and chemiluminescence were maintained less efficiently. Morphological studies of cryopreserved granulocytes revealed that the nuclear, cytoplasmic, and cell surface architectures were altered by storage. Depletion of nuclear and cytoplasmic material, as well as changes in configuration, were also noted.