Aminoglycoside cross-resistance patterns of gentamicin-resistant bacteria.

Ten strains each of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were habituated to gentamicin by serial passage in antibiotic containing medium. Complete cross-resistance to streptomycin, neomycin, kanamycin, and tobramycin in a linear proportional fashion was demonstrated at all stages of habituation. Most strains of Staph. aureus and Ps. aeruginosa showed a greater increase in resistance to gentamicin than to the other three aminoglycosides. E. coli required more transfers to reach the same degree of resistance than did the other two species. Reversion to greater susceptibility to gentamicin took place after serial passage on antibiotic-free media. 'Wild' gentamicin-resistant strains showed no such proportionality of resistance to kanamycin, neomycin, or streptomycin. But many of these strains showed a proportional increase in resistance to tobramycin.     

Comparative epidemiology of gentamicin-resistant enterobacteria: persistence of carriage and infection.

During a two-year period from January 1979, 260 patients have been involved in an outbreak of carriage and infection due to gentamicin-resistant enterobacteria. We have examined the duration of carriage of such enterobacteria and have compared the carriage of Klebsiella with that of other resistant enterobacteria. Carriage of gentamicin-resistant enterobacteria occurred most frequently and was least sporadic in the intestinal tract. Vaginal carriage was observed in 49 out of 68 patients tested and occurred more frequently in older patients. Oral carriage was noted in 36% of patients but was more sporadic than intestinal carriage. Rates of oral carriage were greater among moribund patients. Carriage at skin sites was related to their proximity to the perineum. Intestinal carriage of gentamicin-resistant Escherichia coli and Klebsiellae but not Klebsiella oxytoca nor Citrobacter persisted for long periods (half lives of 140 and 100 days respectively). Cessation of carriage of gentamicin-resistant Klebsiellae was due to loss of both the organism and its plasmid rather than a shedding of the plasmid. Chronic bacteriuria with gentamicin-resistant E coli and Klebsiellae (half life 180 days) but not Klebsiella oxytoca nor Citrobacter persisted for long periods.     

An outbreak of infection caused by a gentamicin-resistant Staphylococcus aureus.

An outbreak of infection caused by a strain of Staphylococcus aureus resistant to gentamicin and tobramycin and other antibiotics occurred in two wards in a hospital. Eight patients were colonized, of whom six had clinical infections. Previous administration of gentamicin appeared to predispose the patients to infection with the strain. Restriction of the use of gentamicin and tobramycin is essential to preserve their value in serious infections.     

含qnrA基因质粒介导不同水平环丙沙星耐药性的机制研究

目的 研究4个含qnrA基因质粒在大肠埃希菌接合子中介导环丙沙星耐药水平不同的发生机制.方法 以大肠埃希菌J53作为受体菌,通过接合试验从4株qnrA阳性的临床菌株中获得4株接合子.采用E test方法测定环丙沙星最低抑菌浓度(MIC);以PCR法检测aac(6')-Ib-cr基因,采用实时RT-PCR法测定qnrA的mRNA表达水平,通过启动子探针载体pKK232-8测定qnrA启动子强度,测定、比较启动子周边序列.结果 环丙沙星对仅含qnrA质粒pHS4及pUS5的接合子的MIC为0.094μg/ml及0.125μg/ml,同时携带qnrA及aac(6')-Ib-cr质粒pUS3及pUS6的接合子的环丙沙星MIC为0.25μg/ml及1.00μg/ml.含pUS6接合子qnrA相对表达水平较其他接合子高13～32.5倍,pUS6的qnrA上游序列启动子活性最强,比另3个质粒高12倍,序列分析发现与pUS3比较,pUS6中qnrA转录启始位点与启始密码之间有7 bp(GTYAGCA)的缺失.结论 1个质粒同时携带qnrA和aac(6')-Ib-cr 2个耐药基因及qnrA高表达是导致接合子对环丙沙星的耐药性不同的原因.     

Ampicillin tolerance of Legionella pneumophila for counter-selection of transconjugants in heterospecific matings with Escherichia coli donors.

Legionella pneumophila, the causative agent of Legionnaires' disease is sensitive to ampicillin. However, the slowly growing bacteria are not killed even by high doses of this antibiotic. This natural tolerance was used for counter-selection of trans-conjugants in heterospecific matings with Escherichia coli as donor. This approach is useful for gentic manipulations in Legionella, as it avoids the use of antibiotic-resistant variants, which have to be tested for full virulence before use.     

Epidemiology of gentamicin-resistant, gram-negative bacillary colonization in a spinal cord injury unit.

A prospective epidemiological survey of a spinal cord injury unit for gentamicin-resistant, gram-negative bacilli was undertaken. The initial survey of the unit suggested a low level of cross-infection involving Pseudomonas aeruginosa and Providencia stuartii. However, a longitudinal study of new admissions revealed that only 13 of 52 nosocomial acquisitions could be considered to be due to cross colonization. Comparison of data on antibiotic use did not suggest selective pressure for resistant endogenous flora. Nosocomial acquisition was directly related to the length of the hospital stay. Antibiotic susceptibility testing of gentamicin-resistant, gram-negative bacilli showed only minor differences between nosocomial isolates and those present during the initial survey. Of the usual antimicrobial agents, amikacin, carbenicillin, and cefoxitin were the most active against all organisms, with the exception of Serratia spp. Of the new beta-lactams, ceftazidime and imipemide (N-formimidoyl thienamycin) were most active.     

Phenotypic factors correlated with the absence of virulence among gentamicin-resistant Pseudomonas aeruginosa strains.

Previous reports have suggested that clinical strains of gentamicin-resistant Pseudomonas aeruginosa (GRPA) generally produce only superficial infections (wounds, urinary tract infections) in contrast to their more invasive gentamicin-susceptible counterparts (GSPA). In view of this finding, a comparative study of a number of phenotypic properties of 20 GRPA and GSPA strains (10 isolates) was assessed to determine how closely related these two populations are and how their phenotypic properties might reflect virulence. GRPA isolates were found to be more adherent to buccal cells than their susceptible counterparts (P = 0.0001). Motility, however, was generally restricted in the GRPA population when compared with GSPA isolates (P = 0.02), although on the basis of zone diameters, some strains overlapped into the other group. Enzymatically, GSPA isolates produced significantly more lipase activity against C-14 lipids than GRPA strains (P = 0.04). No differences were recorded between the two populations in dye sensitivity or in their ability to grow on minimal media at 37 and 42 degrees C. Only 35% of the GRPA isolates agglutinated in 1 of the 17 monospecific antisera reactive for P. aeruginosa. The results of this study suggest that in vivo-generated GRPA strains possess phenotypic properties intermediate between those described for in vitro-derived GRPA isolates and their progenitor GSPA strains. The increased adherence of clinical GRPA isolates to buccal cells may explain their predilection to produce wound and urinary tract infections, whereas their lack of systemic dissemination may be partially due to decreased motility and to reduced lipase production.     

Combination of minocycline and rifampicin against methicillin- and gentamicin-resistant Staphylococcus aureus

Methicillin- and gentamicin-resistant Staphylococcus aureus may remain sensitive to minocycline and to rifampicin. A study of growth curves has shown that at inhibitory concentrations (0·4 μg/ml), minocycline prevents the development of mutants resistant to rifampicin.     

Molecular and epidemiological evaluation of strain replacement in patients previously harboring gentamicin-resistant MRSA

Gentamicin-susceptible methicillin-resistant Staphylococcus aureus (GS-MRSA) clones have gradually replaced gentamicin-resistant MRSA (GR-MRSA) clones in many European countries. We studied molecular and epidemiological aspects of MRSA strain replacement in individual patients. All patients from whom at least 2 MRSA strains showing different gentamicin susceptibility patterns were isolated between 1996 and 2008 were retrospectively identified. Staphylococcal cassette chromosome mec (SCCmec) type and clonality between isolates were determined using molecular methods. Risk factors for individual GR-MRSA SCCmec I (prevalent clone) strain replacement with GS-MRSA non-SCCmec I types were studied in a nested case-crossover study (n = 55 patients). MRSA strain replacement was observed in 127 patients, 85 (67%) of whom were initially colonized with GR-MRSA replaced subsequently by GS-MRSA. Most GS-MRSA replacement strains (50; 59%) possessed SCCmec IV. All MRSA isolate pairs from the same patient that consisted of different gentamicin susceptibility and SCCmec types were also genotypically different. Exposure to domiciliary nursing assistance (odds ratio [OR], 8.1; 95% confidence interval [CI], 1.2 to 53.7) and high Charlson scores (OR, 7.1; 95% CI, 1.1 to 46.8) were associated with individual strain replacement. In individual patients, exogenous acquisition of a different MRSA strain was responsible for strain replacement in most cases. Domiciliary nursing assistance could be a target for specific control measures to prevent transmission of GS-MRSA in our setting.     

A new methicillin- and gentamicin-resistant Staphylococcus aureus in Dublin: molecular genetic analysis

In June 1985 two new strains of methicillin- and gentamicin-resistant Staphylococcus aureus (MGRSA) were isolated in a Dublin hospital. Of these, one strain spread rapidly, affecting a total of 65 patients during the following 18 months, and subsequently spread to a second Dublin hospital. Detailed laboratory studies, including plasmid screening, plasmid restriction enzyme digest pattern analysis, hybridisation analysis, location of resistance determinants, and bacteriophage typing with a set of experimental S. aureus typing phages, demonstrated that the "new" MGRSA organisms, termed Phenotype III Dublin isolates, were completely distinct from, and unrelated to, the MGRSA strains responsible for serious nosocomial infections in Dublin hospitals during the decade before June 1985. These Phenotype III isolates were very similar to MGRSA organisms isolated in a Baghdad hospital during 1984. Data from plasmid curing, plasmid transfer and hybridisation experiments indicated that 20% of the Phenotype-III isolates expressed chromosomally encoded, high level resistance to ethidium bromide (MIC 120 micrograms/ml), and that this was possibly due to chromosomal integration of a penicillinase-like plasmid.     

Strategies for molecular characterisation of methicillin- and gentamicin-resistant Staphylococcus aureus in a Canadian nosocomial outbreak

Sixteen methicillin-resistant Staphylococcus aureus (MRSA) isolates, from a single nosocomial outbreak, were tested for molecular and phenotypic relationships. Two of the 16 outbreak strains were gentamicin resistant (Gmr) and the plasmids that they carried were characterised by reverse field electrophoresis, restriction endonuclease analysis and gene hybridisation. The gentamicin-resistant (Gmr) strains harboured two plasmids, a Gmr plasmid of 36.5 kb and a cryptic plasmid of 25.4 kb, whereas the other 14 isolates contained only the cryptic plasmid. Gentamicin resistance was encoded by a 2.5-kb HindIII fragment of the 32.8-kb plasmid and is similar to the 2.5-kb HindIII fragment also described for S. aureus Gmr plasmids from Australia and the USA. The Gmr plasmid was non-conjugal and was cured by ethidium bromide at a frequency of 4%. Two MRSA strains isolated subsequently from the same hospital were also Gmr and had identical plasmid and restriction endonuclease profiles to the two Gmr strains studied initially. Two other S. aureus isolates from the original carrier detected in this study and from his son were methicillin and gentamicin susceptible and had novel profiles. Since large plasmids show anomalous migration in agarose gels, more definitive analyses than simple plasmid identification should be considered when studying nosocomial outbreaks.     

Characterization of aminoglycoside resistance genes and class 1 integrons in porcine and bovine gentamicin-resistant Escherichia coli

A total of 78 gentamicin-resistant Escherichia coli strains from swine (27) and cattle (51) were characterized by phenotypic resistance, presence of selected aminoglycoside resistance genes, class 1 integrons and gene cassettes, and pulsed-field gel electrophoresis. Gentamicin resistance was mainly encoded by the ant(2')-I gene that was found in 76% of all the strains investigated, whereas the aac(3)-IIa gene was found in 14%. The ant(2')-I gene was predominant in strains from cattle, whereas the porcine strains contained both ant(2')-I, aac(3)-IIa, and the aac(3)-IVa genes. The pulsed-field gel electrophoresis (PFGE) investigation indicated a close clonal relationship in half of the bovine strains whereas the remaining E. coli were unrelated. Among the E. coli investigated, 20% contained class 1 integrons. Genes encoding resistance to trimethoprim (dhfrI, dhfrIb, and dhfrVII), gentamicin, tobramycin, and kanamycin (ant(2')-Ia streptomycin and spectinomycin (ant(3')-Ia) and streptothricin (sat1) were identified as gene cassettes. The most prevalent gene cassettes were ant(3')-Ia (11 isolates) and the dhfrI (nine isolates).     

Presence of natural autoantibodies in hyperimmunized mice.

Mice were immunized with various antigens in complete Freund's adjuvant following various injection schedules. Hybridomas were produced from the spleens of these immunized mice and examined for production of antibodies directed against the antigen injected and against a panel of self (tubulin, actin, myosin, DNA) and non-self antigens (myoglobin, spectrin, peroxidase, trinitrobenzene). Two to five percent of the hybrids were found to secrete polyspecific antibodies able to react with two or more antigens of the panel. Several of these hybrids were subcloned and expanded into ascites. The monoclonal immunoglobulins they secreted were isolated and shown to be IgM (kappa) and to possess the polyspecific antibody function. Several hybrids were also found to secrete antibodies reacting with the immunizing antigen as well as one or more antigens of the panel. The antibody secreted by one subclone which reacts with both the immunizing antigen, prolactin and one of the panel antigens, TNP, has been isolated using a DNP-immunoadsorbent. The isolated antibody was found to be a monoclonal IgM (kappa) immunoglobulin and to react both with prolactin and TNP. The hypothesis is advanced that cells carrying polyspecific natural antibodies as receptors after a given antigenic stimulation proliferate into cells producing highly specific antibodies for epitopes of that given antigen; the cells with polyspecific receptors will be continuously replaced by new cells probably on bone-marrow origin.     

Presence of growth factors in human pituitary.

Recent reports indicate that fibroblast growth factors known to be present in the pituitary in high levels regulate the action of growth hormone and prolactin. New data also suggest a regulatory role in the pituitary for other growth factors, such as epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). Since in most systems cooperation of several growth factors is required for their optimal function, we sought to demonstrate the presence of certain growth factors in the pituitary. Acid ethanol extracts from approximately 50 autopsy-derived human pituitaries were subjected to molecular sieve chromatography and were tested for growth factors. Low molecular weight protein (10 micrograms) eluted from the molecular sieve column contained 10-20 ng material binding to the EGF/TGF-alpha receptor as determined by the EGF/TGF alpha radioreceptor binding assay which represents 11 ng EGF/TGF alpha per pituitary. By Western blotting we found EGF but could not document the presence of TGF-alpha in this material. Radioimmunoassay for insulin-like growth factor I detected 0.4-0.8 ng insulin-like growth factor-I/100 micrograms extract. TGF-beta eluted between 14,000 and 20,000 M(r) at levels of 3-4 ng/pituitary. Its ability to inhibit growth of CC164 mink lung cells was abolished by antibody to TGF-beta 1 but not by antibody raised against TGF-beta 2. The detection of platelet derived growth factor was equivocal and not fully reproducible. We have partially purified TGFe from the pituitary; it stimulated soft agar growth of carcinoma SW-13 cells, and it followed an elution pattern identical to bovine kidney TGFe on molecular sieve column and high pressure liquid and high performance electrophoretic chromatography. Our data show that in addition to fibroblast growth factors, the human pituitary contains other growth factors, such as EGF/TGF-alpha, TGF-beta, insulin-like growth factor I, and TGFe.     

Presence and characterization of lymphokines in mouse ascites tumor fluids.

Considerable skin-reactive and macrophage-disappearance-inducing activities were detected in cell-free fluids of 2 mouse ascites tumors (Ehrlich ascites tumor, leukemia L 1210). Fractionation of the supernatants by ammonium sulfate precipitation, gel filtration on Sephadex G-200, and DEAE-Sephadex A-50 column chromatography resulted in characterization of the proteinaceous substance which accounts for skin-reactive activity. The factor responsible bears a close physicochemical and biological resemblance to the skin-reactive factor of lymphocytic origin which is known to be generated by specific or nonspecific stimulation of lymphocytes in vitro, or to be produced spontaneously by lymphoblastoid cell lines. The biological significance of the findings is discussed.     

Presence of factor-VII and -XIII activity in ultrafiltrate during hemofiltration.

Bleeding complications during renal replacement therapies can be attributed to coagulation system and platelet function alterations in uremia, and the application of heparin in extracorporeal circulation. Small protein losses during hemofiltration are always described, however the high molecular weight of coagulation factors should significantly prevent their removal during hemofiltration. To exclude degradation of coagulation factors under conditions of spontaneous ultrafiltration, the hemofiltrate of 40 patients with acute renal failure (treated with continuous veno-venous hemofiltration, CVVH) was sampled from the filtrate line after 1 h from the beginning of treatment and in 5 patients also after 12 and 24 h. Samples were investigated with human factor deficient plasma (VII, X, XI, XII) from donors with a congenital deficiency and with human plasma depleted of factor V, VIII, IX, and protein S and C. Factor XIII was detected photometrically. Subsequently the presence of factor- XIII and -VII activity was investigated in plasma and hemofiltrate from 16 patients treated with intermittent hemofiltration before (plasma) and after (plasma, hemofiltrate) therapy. These patients also suffered from acute renal failure and needed renal replacement therapies. Quality control was carried out with a buffer solution (<1% activity in the assays according to recommended protocols). RESULTS: Factor-V, -VIII, -IX, -X, -XI, and -XII activity, and protein C and S could not be detected in the hemofiltrate from continuous hemofiltration. Factor-VII and -XIII activity was present in the hemofiltrate (mean activity in CVVH: 1.93% for factor VII and 6.9% for factor XIII, mean activity in intermittent hemofiltration: <1% for factor-VII and 7.3% for factor-XIII). Three were no significant differences (Student's t-test) in plasma activity before and after intermittent hemofiltration of factor VII (44 vs. 47%, p = 0.39) and factor XIII (44 vs. 52%, p = 0.24). The presence of factor-VII and -XIII activity in the hemofiltrate cannot influence plasma activities in intermittent hemofiltration. Rapid new synthesis and short half-life should neutralize these effects. Elimination of coagulation factor-XIII activity should be excluded by the next generation of highly permeable membranes and on-line hemodiafiltration.     

Presence of gamma interferon in human acute and congenital toxoplasmosis.

The production of gamma interferon in acute acquired and congenital toxoplasmosis was studied. Gamma interferon was produced at significant titers (P less than 0.001) in the course of both congenital toxoplasmosis and acquired toxoplasmosis at an early stage of infection, when Toxoplasma gondii was multiplying. Its presence in fetal blood was correlated with the positive inoculation of fetal blood or amniotic fluid into mice (95%). The data suggest that the fetus is able to synthesize gamma interferon as early as week 21 of pregnancy. This test, easily and rapidly performed, could be included among those useful for diagnosing fetal toxoplasmic disease.     

Presence of host-reactive T cells in lymphohaematopoietic chimeras.

The presence of autoreactive lymphocytes was investigated in murine lymphohaematopoietic chimeras. In this report we demonstrate the presence of precursors of host-reactive cytotoxic effector T lymphocytes in parent leads to F1 chimeras. Lymphocytes from these chimeras display cytotoxic activity towards the non-shared major histocompatibility complex (MHC) antigens of the host following activation with the polyclonal T-cell activator concanavalin A. These host-reactive cells were found despite the apparent absence of lymphocytes demonstrating autoreactivity in other experimental systems: mixed lymphocyte reaction, cell-mediated lympholysis, positive allogeneic effect and negative allogeneic effect. Animals possessing precursors of these cytotoxic effector cells also possess precursors of cytotoxic effector cells capable of generating an MHC-restricted anti-minor histocompatibility antigen response. The results are discussed in reference to the role of the thymus in effecting self non-self discrimination.