Free amino acids in the haemolymph of honey bee queens (Apis mellifera L.).

In queen honey bees the free amino acid content in the haemolymph clearly depends on the physiological function and social environment of the individual. While in drones and workers the content of free amino acids increases after emergence until it reaches a peak in 5-day-old animals and decreases afterwards, the amino acid content in queens reaches its highest level (>60 nmol/ microl haemolymph) with the onset of egg laying (10 d of age). This level is about 2.5 times more than the highest level found in workers. Queens maintain this high level also when they are older (>30 d) and continue to lay eggs in average colonies. As in drones and workers, in queens the predominant amino acid is proline, which accounts for more than 50% of the total content of free amino acids in egg-laying individuals. When 10-day-old queens are prevented from mating and do not lay eggs, their amino acid content is significantly lower compared to laying queens of the same age. Also the social environment influences the contents of free amino acids in queens. When virgin queens were kept for 6 days with 20 worker bees and sufficient honey and pollen in an incubator, they had significantly lower concentrations of amino acids than virgin queens living for the same period with about 8000 workers in a colony. Most probably, the high amino acid concentration in the haemolymph is the basis for the high protein synthesis activity of laying queens.

Importance of proline and other amino acids during honeybee flight--Apis mellifera carnica POLLMANN).

The levels of proline and other amino acids in the haemolymph and other body parts of honeybee foragers were investigated by HPLC analysis. The concentrations of proline in the blood of glucose-fed or -injected bees finishing their exhaustive tethered flights on a roundabout were significantly reduced compared to bees that were fed and rested for one hour. This indicates some utilization of proline during flight metabolism. The levels of essential amino acids and of the sum of all amino acids except proline remained roughly constant, indicating that the decrease of proline did not result from a changed haemolymph volume. 14C-labelled proline was injected into bees either shortly before starting their flight or before a resting period of equal duration in an incubator at the same temperature. Bees that rested had incorporated more proline into thorax body protein, and less of the labelled substance was unrecovered ("missing") and considered to be respired or less probably defecated. If the entire amount of missing 14C-proline is regarded as exhaled, the oxidative breakdown of proline reached higher levels after flight than in rested bees. This is another hint that proline is utilized during flight. Usually the exhaled amount did not exceed 10 microg proline in half an hour of flight. Although our data indicate involvement of proline in flight metabolism, the amount metabolized is low compared to the utilization of carbohydrates.

Amino acids and osmolarity in honeybee drone haemolymph.

In the haemolymph of honeybee drones, concentrations of free amino acids were higher than in worker haemolymph, with different relative proportions of individual amino acids. The overall concentration of free amino acids reached its highest level at the 5th day after adult drone emergence, and after the 9th day only minor changes in the concentration and distribution of free amino acids were observed. This coincides with the age when drones reach sexual maturity and change their feeding behaviour. Levels of essential free amino acids were high during the first 3 days of life and thereafter decreased. Osmolarity was lowest at emergence (334 +/- 42 mOsm), increased until the age of 3 days (423 +/- 32 mOsm) and then stayed generally constant until the 16th day of life. Only 25-day-old drones had significantly higher osmolarity (532 +/- 38 mOsm). The overall change in osmolarity during a drone's lifetime was about 40%.

Effects of cytokines, butyrate and dexamethasone on serum amyloid A and apolipoprotein A-I synthesis in human HUH-7 hepatoma cells.

Serum amyloid A (SAA) and apolipoprotein A-I (apo A-I) are secreted by the liver. As concentrations of both apolipoproteins are inversely related under normal and acute-phase conditions, human HUH-7 hepatoma cells were stimulated with interleukin (IL)-1alpha (100 and 200 U), IL-6 (50 and 100 U), butyrate (2 mM) and dexamethasone (2 x 10(-7)M and 1 x 10(-6)M), alone or in combination. Changes in SAA and apo A-I synthesis were monitored after metabolic labelling of the cells with [35S]-methionine. Intracellular and secreted SAA and apo A-I were immunoprecipitated, separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the radioactivity in the corresponding bands was counted. Intracellular apolipoprotein levels were increased by all stimuli, either alone or in combination, between 2.7- and 5.5-fold (SAA) and between 2.8- and 4.1-fold (apo A-I), respectively. In a similar manner, apolipoprotein levels secreted by HUH-7 cells were increased between 3.1- and 4.3-fold (SAA) and between 1.9- and 3. 3-fold (apo A-I). Co-administration of cytokines, butyrate and/or dexamethasone had no pronounced synergistic effect on intracellular biosynthesis and secretion of SAA and apo A-I. The results from the present study suggest that apo A-I must not necessarily be considered as a negative acute-phase reactant.

Stenotrophomonas (Xanthomonas) maltophilia: in vitro susceptibility to selected antimicrobial drugs, single and combined, with and without defibrinated human blood.

Sixteen selected isolates of Stenotrophomonas maltophilia varied in susceptibility to the combined phagocytic/serum bactericidal activity of fresh defibrinated human blood (65 vol%). Four representative isolates (X1, X11, X25, and X50), which differed in susceptibility to cefepime, ceftazidime, rifampin, and timentin, were subjected to checkerboard microtiter broth dilution tests involving combinations of cefepime plus timentin, ceftazidime plus ofloxacin, cotrimoxazole plus timentin, rifampin plus polymyxin B, and rifampin plus polymyxin B nonapeptide; all combinations yielded additive or synergistic effects against all four strains. Unexpectedly, the combination of cefepime plus timentin was bactericidally active against the two cefepime-resistant isolates. This finding was substantiated by blood/broth plus combined antimicrobial drug assays. Cefepime plus timentin effectively killed all four test strains. Ofloxacin combined with ceftazidime was bactericidally active against the test strains, including two isolates (X11, X50) with intermediate ofloxacin sensitivity. Cotrimoxazole plus timentin in blood, but not in broth, was bactericidal for the timentin-resistant isolate X25. As expected, various triple combinations of chemotherapeutic agents in blood and broth revealed polymyxin B plus rifampin, regardless of the third combination partner, to exert bactericidal activity against all test strains. Similarly, rifampin combined with ofloxacin and ceftazidime was bactericidally active in blood and broth. The observation that timentin combined with cefepime was effective against cefepime-resistant strains of S. maltophilia might prove of clinical relevance with regard to chemotherapy of nosocomial infections due to multiple-antibiotic resistant strains of this opportunistic pathogen.

Antibiotic susceptibility testing (agar disk diffusion and agar dilution) of clinical isolates of Enterococcus faecalis and E. faecium: comparison of Mueller-Hinton, Iso-Sensitest, and Wilkins-Chalgren agar media.

Forty-two isolates of Enterococcus faecalis and 56 isolates of Enterococcus faecium, including 8 vancomycin-resistant strains, were examined for comparative susceptibility to 27 antimicrobial drugs with the agar dilution method, employing Mueller-Hinton (MHA), Iso-Sensitest (ISTA), and Wilkins-Chalgren (WCA) agar. The Bauer-Kirby agar disk diffusion method was used to comparatively test 24 of the agents in parallel. The enterococci yielded better growth on ISTA and WCA. However, WCA completely antagonized co-trimoxazole and, though less, fosfomycin. Importantly, WCA slightly reduced the activities of teicoplanin (minimal inhibitory concentrations, MICs, raised up to twofold) and vancomycin (MICs raised two- to fourfold) against enterococci and staphylococcal quality control strains. Therefore, WCA was judged unsuitable for susceptibility testing of enterococci. For E. faecalis no discrepancies between agar dilution MICs and inhibition zone diameters were encountered with augmentin, ampicillin, ampicillin-sulbactam, chloramphenicol, mupirocin, oxacillin, teicoplanin, and co-trimoxazole. Overall, MHA yielded fewer very major (category I) and major (category II) discrepancies than ISTA. However, numerous minor (category III), slight (category IV), minimal (category V), and/or negligible (category VI) discrepancies were encountered with ciprofloxacin, doxycycline, erythromycin, fosfomycin, fusidic acid, meropenem, ofloxacin and rifampin. With respect to E. faecium, only cefotaxime, mupirocin, oxacillin, and teicoplanin yielded nondiscrepant results. Several very major (I) and major (II) discrepancies were observed with augmentin, ampicillin, ampicillin-sulbactam, doxycycline, fusidic acid, imipenem, and penicillin G. Minor discrepancies (categories III-VI) were particularly numerous with augmentin, chloramphenicol, ciprofloxacin, doxycycline, and piperacillin. The largest numbers of negligible (VI) discrepancies were noted with fosfomycin, fusidic acid, and ofloxacin. It is recommended to test one cephalosporin (cefuroxime or the like) in parallel for educational purposes and to exclude fosfomycin, fusidic acid, and rifampin from test batteries because of the wide scatter of test results. The large number of minimal (V) discrepancies of ciprofloxacin against E. faecalis, the numerous minor (III) and slight (IV) discrepancies of chloramphenicol against E. faecium, and the not insignificant number of very major (I) and minor (III) discrepancies observed with meropenem against isolates of E. faecalis necessitated proposals for new disk intermediate susceptibility criteria.

Surveillance of Pseudomonas aeruginosa in intensive care units: clusters of nosocomial cross-infection and encounter of a multiple-antibiotic resistant strain.

Serogrouping (determination of O antigens) and bacteriocin typing (based on susceptibility to one or more of 18 bacteriocins) were employed to survey 210 isolates of Pseudomonas aeruginosa from 201 patients in 8 intensive care units (ICU) during an observation period of 18 months. Eighty-eight isolates (41.9%) were nonserogroupable (NT); most common were serogroups O1, O9, O11, and O3. All except 5 isolates (97.6%) were bacteriocin-typable. However, phenotypic variation of bacteriocin susceptibility, in particular the receptor for bacteriocin No. 13, rendered this typing method presumptive as well. Bacteriocin susceptibility profiles were not predictive of serogroup and vice versa. Workup of 19 isolates from 9 patients disclosed phenotypic variation of antibiotic susceptibility in 3 patients, superinfection by a different strain in 4 patients, and persistence (3 months) of the same strain in 2 patients, respectively. Serotyping and bacteriocin susceptibility test data revealed 15 clusters of putative cross-infection of 2 patients each, 8 clusters involving 3 patients each, one outbreak (serogroup NT, bacteriocin profile 777736) involving 4 patients in the pediatric ICU, one outbreak due to a multiple-antibiotic resistant (MAR) strain in the surgical ICU (4 patients, serogroup O12, bacteriocin profile 30400), and two putative outbreaks in the pneumonology ICU involving 6 patients (serogroup NT, bacteriocin profile 777726) and 9 patients (serogroup NT, bacteriocin profile 777736). Pulsed-field gel electrophoresis (PFGE) macrorestriction analysis (SpeI, XbaI) confirmed the pediatric and surgical ICU strains as singular strains. However, the two putative outbreaks in the pneumonology ICU were due to one particular strain which had infected 13 of the 15 patients as determined with the PFGE genotypic method. Isolates comprising the MAR strain of P. aeruginosa were susceptible only to amikacin, fosfomycin, and polymyxin B; the isolates varied in susceptibility to aztreonam and ceftazidime. This MAR strain was susceptible to the bactericidal activity of 65 vol% of fresh defibrinated human blood from donors B, L, and T. Either amikacin (16 micrograms/ ml) or fosfomycin (8 micrograms/ml) plus blood and amikacin (8 micrograms/ml) combined with fosfomycin (8 micrograms/ml) with and without blood consistently killed isolates of the MAR strain, which thus was amenable to antibiotic therapy.

Antibiotic susceptibility testing (agar disk diffusion and agar dilution) of clinical isolates of Corynebacterium jeikeium.

Thirty-three clinical isolates of Corynebacterium jeikeium were examined for susceptibility to 27 antimicrobial drugs with the agar dilution test. Sheep-blood-supplemented Mueller-Hinton agar performed better than Wilkins-Chalgren agar. Disk susceptibility (Bauer-Kirby) tests were carried out in parallel with 24 of the chemotherapeutic agents. All isolates were susceptible to teicoplanin and vancomycin. All isolates resisted fosfomycin, mupirocin, and trimethoprim-sulfamethoxazole. The isolates varied in susceptibility to ciprofloxacin, doxycycline, fusidic acid, ofloxacin, and tetracycline; most were susceptible to rifampin. Surprisingly few discrepancies between agar dilution and disk diffusion tests were encountered when utilizing NCCLS interpretive criteria currently valid for enterococcal isolates.

Antibiotic susceptibility of Stenotrophomonas (Xanthomonas) maltophilia: comparative (NCCLS criteria) evaluation of antimicrobial drugs with the agar dilution and the agar disk diffusion (Bauer-Kirby) tests.

Ninety-six clinical isolates of Stenotrophomonas maltophilia were examined with the agar dilution method for susceptibility to 19 antimicrobial drugs. Doxycycline, cotrimoxazole, timentin, ofloxacin, fosfomycin, and piperacillin + tazobactam, in that order, inhibited the majority of strains. All isolates were resistant to nitrofurantoin. Concurrent disk susceptibility (Bauer-Kirby method) testing, using currently valid NCCLS interpretative criteria for Pseudomonas aeruginosa, uncovered a significant incidence of very major (category I), major (category II), and minor (categories III and IV) discrepancies for aminoglycosides, cephalosporins, chloramphenicol, and piperacillin + tazobactam and ticarcillin + clavulanic acid. Therefore, new interpretative criteria indicative of intermediate (I) susceptibility of S. maltophilia to these various antibiotics were proposed. In addition, new intermediate susceptibility criteria were proposed for the two beta-lactam-beta-lactamase inhibitor combinations. It was recommended to exclude ciprofloxacin from test batteries against this microorganism due to the wide scatter of minimal inhibitory concentration values and diameters of inhibition zones; the same was true for polymyxin B. It is hoped that the proposed modified, species-specific criteria will improve the clinical utility of laboratory-generated disk antibiograms with respect to the inherently multiple antibiotic-resistant, opportunistic pathogen S. maltophilia.

Susceptibility of Moraxella catarrhalis to 21 antimicrobial drugs: validity of current NCCLS criteria for the interpretation of agar disk diffusion antibiograms.

Ninety-four clinical isolates of Moraxella catarrhalis were examined for susceptibility to 21 antimicrobial drugs; 67 isolates (= 71.3%) produced beta-lactamase(s). In terms of antibiotic resistance, the number of isolates resistant to penicillin G, ampicillin, and cotrimoxazole were 56, 32, and 1, respectively. The number of isolates with intermediate susceptibility to penicillin G, ampicillin, ciprofloxacin, ofloxacin, cotrimoxazole, and fosfomycin were 11, 34, 1, 2, 2, and 47, respectively. All 94 isolates proved susceptible to ampicillin + 10 micrograms/ml of sulbactam, amoxicillin + 4 micrograms/ml of clavulanic acid, cefuroxime, cefotaxime, cefepime, cefepime, cefixime, imipenem, meropenem, chloramphenicol, doxycycline, tetracycline, fusidic acid, erythromycin, clarithromycin, and rifampin, as based on currently valid NCCLS criteria, where applicable. There were no very major or major discrepancies between agar dilution and agar disk diffusion test results. There were only a few minor discrepancies between test results, specifically: penicillin G (category IV = 4, category VI = 1); ampicillin (category IV = 4, category V = 1, category VI = 7), amoxicillin + clavulanic acid (category III = 11), cotrimoxazole (category IV = 1, category V = 1, category VI = 1), ciprofloxacin (category V = 1), and ofloxacin (category VI = 2). The sole exception was fosfomycin, with a total of 25 minor discrepancies encountered (category III = 14, category V = 9, category VI = 2). Wilkins-Chalgren agar compared favorably with Mueller-Hinton agar following examination with 11 selected antimicrobial drugs against 31 representative isolates of M. catarrhalis.

Antibiotic susceptibility of alpha- and nonhemolytic streptococci from patients and healthy adults to 24 antimicrobial drugs.

A total of 278 alpha- and nonhemolytic streptococcal isolates (patients, n = 116; healthy adults, n = 162) were examined for susceptibility to 23 and 24 antimicrobial drugs with the Bauer-Kirby agar disk diffusion and the agar dilution method, respectively. Wilkins-Chalgren medium compared favorably with sheep blood Mueller-Hinton agar, the reference medium, for 58 representative streptococcal isolates. In terms of minimal inhibitory concentrations (MICs), all 278 isolates were susceptible to teicoplanin and vancomycin. None of the isolates revealed high-level gentamicin resistance. All isolates were resistant to fusidic acid. Twelve Streptococcus mitis isolates, all from patients, were resistant to penicillin G and variably to other antibiotics. Oxacillin disks failed to predict penicillin G susceptibility. In general, patient isolates were more frequently resistant to beta-lactam antibiotics and fluoroquinolones; conversely, isolates from healthy carriers were slightly more resistant to macrolide antibiotics. Specifically, susceptibility rates were: penicillin G 79.1%, oxacillin 87.8%, ampicillin 66.9%, piperacillin 98.2%, cefoxitin 76.6%, cefuroxime 96.8%, cefotaxime 98.6%, ceftriaxone 98.6%, cefepime 98.6%, impipenem 98.2%, ciprofloxacin 59.7%, ofloxacin 89.2%, doxycycline 65.8%, tetracycline 56.8%, clindamycin 87.8%, erythromycin 59%, clarithromycin 74.9%, chloramphenicol 98.9%, cotrimoxazole 97.9%, rifampin 97.5%, and fosfomycin 2.2%. On the basis of numerous minor discrepancies between disk diffusion and agar dilution test results for certain antibiotics, it is proposed that the current NCCLS inhibition zone (diameter, mm) criteria indicative of intermediate susceptibility of alpha- and nonhemolytic streptococci be changed for the following antimicrobial drugs: ampicillin = I = 22-27 mm; ciprofloxacin = I = 16-18 mm; clindamycin = I = 15-18 mm; doxycycline = I = 17-19 mm; tetracycline = I = 17-19 mm; erythromycin = I = 14-19 mm, and cotrimoxazole = I = 11-13 mm. It is recommended to exclude both cefoxitin and doxycycline (substitute = tetracycline) disks from test batteries for non-group A, B beta-hemolytic and alpha-/nonhemolytic streptococcal isolates.

Macrorestriction analysis (PFGE) of serologically cross-reactive serovars of Acinetobacter baumannii and genospecies 3.

Forty-two serovar reference strains of Acinetobacter baumannii and genospecies 3, which yielded major or minor, one-way or two-way (reciprocal) serological cross-reactions, were subjected to macrorestriction (SmaI, ApaI) analysis with the aid of pulsed-field gel electrophoresis (PFGE). The PFGE patterns of serovars 3 and 21 of genospecies 3 differed by 3 (SmaI) and 2-4 (ApaI) DNA fragments and thus were closely/possibly related in their genotype. Serovars 13 and 26 of genospecies 3 differed by only 2 DNA fragments (SmaI), suggesting close genetic relatedness; however, these two particular serovars of genospecies 3 appeared to be genotypically indistinguishable following restriction with ApaI. Serovars 2, 4, and 12 of genospecies 3 appeared to be unrelated to serovar 13 of genospecies 3 (SmaI); however, restriction with ApaI indicated a possible relatedness. Genospecies 3 serovars 2 and 26 differed by 5 DNA fragments (SmaI and ApaI), implying a possible relatedness. All other cross-reactive serovars examined proved to be genotypically unrelated, i.e., differed by > or = 7 DNA fragments (SmaI restriction).

Comparative susceptibility of clinical group A, B, C, F, and G beta-hemolytic streptococcal isolates to 24 antimicrobial drugs.

A total of 312 clinical beta-hemolytic streptococcal isolates (Streptococcus pyogenes, group A = 63; Streptococcus agalactiae, group B = 145; group C = 50; group F = 27; group G = 27) were examined for susceptibility to 23 and 24 antimicrobial drugs with the Bauer-Kirby agar disk diffusion and the agar dilution method, respectively. Sheep blood Mueller-Hinton agar served as the reference medium. Wilkins-Chalgren agar supported optimal growth of group A and B, but not of all group C, F, and G streptococci. The group A streptococci were susceptible to all beta-lactam antibiotics, clindamycin, chloramphenicol, rifampin, teicoplanin, and vancomycin, but resistant to cotrimoxazole, fusidic acid, and, except for 2 strains, to fosfomycin. Resistance (R)/intermediate susceptibility (I) rates (R/I%) to ciprofloxacin (0/2%), ofloxacin (1/2%), erythromycin (1.6/0%), and clarithromycin (0/1%) were low. Higher resistance rates were noted with tetracyclines (doxycycline 23.8/15.9%; tetracycline 39.7/3.2%). Among the group B streptococcal isolates, one strain was resistant against oxacillin and of intermediate susceptibility to penicillin G and cefoxitin. All isolates were susceptible to teicoplanin and rifampin. Conversely, all group B isolates were resistant to cotrimoxazole and fusidic acid; 69% and 51% of these isolates were susceptible to fosfomycin and rifampin, respectively. R/I rates of the group B streptococcal isolates were low for ciprofloxacin and ofloxacin (0/0.7%), clindamycin (0.7/0%), erythromycin (1.4/ 3.5%), clarithromycin (1.4/0%), and chloramphenicol (0.7/0%). Resistance to tetracyclines was significant (doxycycline: 72.4/2.1%; tetracycline; 74.5/1.4%). Among the non-A, non-B beta-hemolytic streptococci, 2 group C strains were resistant to oxacillin and showed intermediate susceptibility to penicillin G. All isolates were susceptible to third and fourth-generation cephalosporins, imipenem, chloramphenicol, rifampin, teicoplanin, and vancomycin. R/I rates to the other antimicrobial drugs were: ciprofloxacin (3.9/1.9%), ofloxacin (2.9/1.9%), clindamycin (2.9/1%), erythromycin (5.8/0%), clarithromycin (3.8/2.9%), and cotrimoxazole (16.4/3.9%). Resistance against tetracyclines was more frequent (doxycycline: 18.3/2.9%; tetracycline: 20.2/6.7%). On the basis of various minor discrepancies between MIC and disk diffusion test results, it is proposed that the current NCCLS inhibition zone (diameter, mm) criteria indicative of intermediate susceptibility of beta-hemolytic streptococci be changed for the following antimicrobial drugs: ampicillin: 22-27 mm (only for group A and B beta-hemolytic streptococci); ciprofloxacin: 16-18 mm; clindamycin: 15-18 mm; doxycycline: 17-19 mm; tetracycline: 17-19 mm, and erythromycin: 14-19 mm.

Role of endothelin, nitric oxide and L-arginine release in ischaemia/reperfusion injury of rat heart.

OBJECTIVES: We tested the hypothesis that endothelin-1 (ET-1) aggravates ischaemia/reperfusion injury by stimulating cellular L-arginine depletion, which would result in reduced synthesis of nitric oxide (NO) and withdrawal of cardioprotection. METHODS: Five groups of rat hearts (n = 5 each) were perfused at 9 ml/min per g for 45 min, subjected to 15 min total global ischaemia and reperfused for 30 min; they received, from 5 min pre-ischaemia to end of reperfusion, either vehicle, L-arginine (1 mmol/l), the NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP; 200 mumol/l), the inhibitor of NO formation NG-nitro-L-arginine (L-NNA; 200 mumol/l), or the ET receptor antagonist PD 142893 (200 nmol/l). Cardiac function and release of L-arginine, cyclic GMP and lactate dehydrogenase (LDH) into coronary effluent were measured. RESULTS: Systolic, diastolic, and coronary reperfusion function were consistently improved by L-arginine, SNAP, or PD 142893, but worsened by L-NNA (P < 0.05 in each case). L-arginine release was transiently increased up to 25-fold on reperfusion (vehicle); release was reduced by SNAP (mean: 68%) and entirely prevented by PD 142893. Despite the increased outflow of L-arginine, formation of cyclic GMP was not reduced, but enhanced in reperfusion (11-fold; vehicle), and SNAP further augmented this release, but L-NNA had no significant effect. Release of LDH was decreased by L-arginine, SNAP, and PD 142893 in reperfusion. Finally, release of ET-1 was inhibited by NO in normoxia as well as throughout reperfusion as evident from the stimulatory effect of L-NNA. CONCLUSION: In ischaemia, ET-1 cause cell necrosis and L-arginine outflow without compromising NO synthesis in this model.

Phenotypic and genotypic characterization of clinically recovered presumptive Acinetobacter baumannii isolates which failed to grow at 44 degrees C.

Eleven clinical isolates of Acinetobacter, which exhibited an identical biochemical profile compatible with genospecies 3 and failed to grow at 44 degrees C, were not agglutinated by polyclonal rabbit immune sera against 26 serovars of genospecies 3. Rather, all 11 isolates reacted strongly with antiserum against serovar 18 of A. baumannii. Macrorestriction (SmaI) analysis of genomic DNA revealed that only one isolate was genotypically different, whereas the remaining ones were either closely related or identical. However, the genomic DNA of the A. baumannii serovar 18 reference strain proved to be genotypically unrelated.

Antagonism of teicoplanin and vancomycin activity by chocolatized Mueller-Hinton agar.

Chocolatized (80 degrees C, 13 min) Mueller-Hinton agar antagonized the inhibitory activities of teicoplanin and vancomycin against reference strains of Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Staphylococcus aureus, and Enterococcus faecalis. This antagonism was due to heat-exposed sheep erythrocytes, sheep hemoglobin, and the supernatant fluid from lysed sheep erythrocytes, but not to sheep serum. Neither water-soluble cholesterol, bovine albumin, bovine serum, hematin, hemin nor egg yolk suspension antagonized teicoplanin and vancomycin.

Antibiotic susceptibility of clinical isolates of Streptococcus pneumoniae.

Ninety-three representative, recent clinical isolates of Streptococcus pneumoniae were examined for susceptibility to 9 antimicrobial drugs utilizing Mueller-Hinton agar (MHA) enriched with sheep blood and a hypercapnic atmosphere of incubation. One isolate was resistant to penicillin G (minimum inhibitory concentration, MIC = 2 micrograms/ml) and 6 isolates were of intermediate susceptibility to penicillin G (MICs = 0.125-0.25 microgram/ml). The penicillin-G-resistant isolate was also resistant to cefuroxime (MIC = 4 micrograms/ml) and of intermediate susceptibility to cefotaxime (MIC = 1 microgram/ml). This isolate was resistant to chloramphenicol (MIC = 16 micrograms/ml) as well. All 93 isolates were susceptible to teicoplanin and vancomycin. Two isolates each were resistant (MICs = 16 micrograms/ml) or moderately susceptible (MICs = 8 micrograms/ml) to chloramphenicol. Eight isolates were resistant to doxycycline (MICs > or = 8 micrograms/ml), whereas 2 isolates were of intermediate susceptibility to this antibiotic (MICs = 4 micrograms/ml). Three isolates were resistant to erythromycin (MICs > or = 4 micrograms/ml), and 2 isolates showed reduced susceptibility to erythromycin (MICs = 2 micrograms/ml). Chocolatized MHA antagonized the activity of teicoplanin and vancomycin against pneumococcal isolates. Haemophilus test and Wilkins-Chalgren media failed to support optimal growth of all pneumococcal isolates.

Typing of nosocomial strains of Serratia marcescens: comparison of restriction enzyme cleaved genomic DNA fragment (PFGE) analysis with bacteriocin typing, biochemical profiles and serotyping.

Eighty-eight selected clinical isolates of Serratia marcescens, representing 27 putative outbreaks of nosocomial cross-infection encountered during 1980-1995, were tested comparatively by bacteriocin typing, carbon source assimilation tests, serotyping (O and H antigens), and restriction pattern (RFLP) analysis of restriction cleaved (SpeI, XbaI) genomic DNA fragments after pulsed-field gel electrophoresis (PFGE). Serotyping served as the "gold standard" of the phenotypic methods. One pseudo-outbreak (bacteriocin typing incriminated type 26) was uncovered through serotyping as well as the biochemical profile and confirmed by PFGE analysis of genomic DNA. Bacteriocin typing and determination of biochemical profiles disclosed several instances of phenotypic variation; serotyping revealed two episodes of shifts from motility (H12) to nonmotility. Resolution of restricted genomic DNA fragments with the PFGE procedure permitted detection of 27 PFGE patterns (A-M, N-1-N-3, O-1, O-2, P-1-P-3, Q-1-Q-3, R-1-R-3, S-1, and T). Based on the analysis of PFGE patterns against the background of epidemiological data, the number of nosocomically significant strains of S. marcescens could be reduced to 16 (PFGE patterns A-M, N-2, O-1, P-2, and T). It was concluded that PFGE analysis of restricted genomic DNA of S. marcescens was superior to the three phenotypic methods.

Clusters of nosocomial cross-infection due to Acinetobacter baumannii and genospecies 3: comparison of serotyping with macrorestriction analysis of genomic DNA with pulsed-field gel electrophoresis.

Triplets of isolates representing 20 putative clusters of nosocomial cross-infection due to Acinetobacter baumannii and genospecies 3 were examined comparatively using serotyping and analysis of restriction fragments (SmaI and ApaI) of genomic DNA with the aid of pulsed-field gel electrophoresis. Carbon source assimilation tests disclosed phenotypic variation among 6 to 20 triplets of isolates. Two misleading results of serotyping were encountered. With respect to the presumptive cluster No. 9, one of the genospecies 3 (originally serovar 4) isolates proved to be polyagglutinable upon repeat examination; this particular putative cluster was shown to be a pseudocluster by comparison of the macrorestriction profiles of the respective triple isolates. A strain of A. baumannii serovar 15 had infected 8 patients in a surgical intensive care unit, while a second, genotypically totally different strain of identical serovar had caused infection in one additional patient. With this exception, the correlation between serotyping and analysis of macrorestriction profiles was excellent.

Phenotypic variation of clinical Serratia marcescens isolates repeatedly recovered from individual patients.

A total of 129 selected isolates of Serratia marcescens which had been recovered from 50 patients during the 1980-1995 period and which revealed phenotypic variation in terms of bacteriocin (phage tail) susceptibility, carbon source assimilation, or serotype, were reexamined with these three phenotypic methods. Seven isolates (5.4%) were bacteriocin nontypable; all 129 isolates utilized carbon sources and could be serotyped. Fourty-eight isolates from 20 patients yielded unambiguous results with these 3 phenotypic methods and were excluded from further analysis. Among the remaining 81 isolates from 30 patients, isolates from 2 patients revealed phenotypic variation in bacteriocin susceptibility only, whereas isolates from 6 patients showed variant bacteriocin types and variant biochemical profiles, but were of identical serotype. Isolates from 20 patients revealed variant biochemical profiles only. Three patients had become superinfected with strains of S. marcescens of different phenotype and genotype. In 4 patients, previously motile (H12) isolates had become nonmotile (H-). PFGE analysis of XbaI and SpeI-restricted genomic DNA of the 81 isolates of the 30 patients demonstrated the isolates of 22 patients to be genotypically identical. The isolates from 3 patients were closely related by genotype, and those from an additional patient proved to be possibly related. PFGE analysis demonstrated one patient to have become infected by two genotypically different strains of S. marcescens of identical serotype, which, however, differed in bacteriocin type and biochemical profile. It was concluded that PFGE analysis of restricted genomic S. marcescens DNA was superior to the three phenotypic methods examined comparatively. Serotyping was more reliable than bacteriocin typing, and the latter technique yielded fewer phenotypic variants than determination of biochemical profiles among consecutively recovered isolates from patients with long-lasting S. marcescens infection.