Carbapenem-resistant Enterobacter cloacae and the emergence of metallo-beta-lactamase-producing strains in a third-level hospital (Santiago de Compostela, NW Spain).

The purpose of this paper was to investigate the occurrence of carbapenem-resistant Enterobacter cloacae in our institution, to detect the carbapenemase-associated resistance and to determine the genetic relatedness of the isolates. Species identification and antimicrobial susceptibility testing were performed using the Vitek 2 system and Etest. Multiplex polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) was used for the detection of extended-spectrum beta-lactamase (ESBL)-producers. The bla (IMP) and bla (VIM) genes were amplified by PCR and sequenced. The DiversiLab System was used for strain-typing. During the period 2006-2008, 12 different isolates of carbapenem-resistant E. cloacae (2.3 %) were recovered in our laboratory. Only two positive isolates for the bla (VIM) gene were detected. The minimum inhibitory concentration (MIC) values were higher for all carbapenems in the group of non-metallo-beta-lactamase (MBL)-producers. All isolates showed MIC values

Endemic linezolid-resistant Staphylococcus epidermidis in a critical care unit.

The aim of this article was to report the emergence of patient infections with linezolid-resistant Staphylococcus epidermidis (LRSE) in a tertiary university hospital. Our objectives were to determine the molecular mechanism of the resistance, set up the genetic relationship among isolates, and analyze the relations between linezolid usage, period of treatment, and emergence of resistance in the hospital. The emergence of infection with linezolid-resistant S. epidermidis affecting 20 patients in a tertiary university hospital was investigated using repetitive sequence-based PCR (rep-PCR, DiversiLab System; BioMérieux, Inc., France). The presence of the G2576T mutation of 23S rRNA was screened by pyrosequencing. We determined the pattern of linezolid usage in the hospital as a whole and in the critical care unit that was most affected. G2576T mutation of 23S rRNA was detected in all linezolid-resistant S. epidermidis studied. Of these, 90% were genetically related and had been recovered from patients admitted to the same critical care unit. There had been an increase in linezolid usage in the hospital and in the critical care unit in the 2 years prior to the emergence of resistant strains. More strict control measures in hand washing and linezolid prescription were subsequently established, but no reduction in LRSE rates have yet been observed. Linezolid-resistant S. epidermidis emerged at our hospital, probably from a single strain originating in the critical care unit. The most likely explanation is that person-to-person spread of linezolid-resistant S. epidermidis led to skin colonization and, after linezolid treatment, this resistant staphylococci became the dominant cutaneous flora causing infection in some critical patients. In order to preserve the usefulness of this antibiotic as a therapeutic agent and to avoid a situation similar to methicillin-resistant Staphylococcus aureus, judicious use of antibiotics is essential.

[Comparison between Phoenix system and agar-based methods for antimicrobial susceptibility testing of Streptococcus spp]. / Comparación entre el sistema Phoenix y métodos basados en agar para el ensayo de sensibilidad a antibióticos en Streptococcus spp.

Introduction. Antibiotic resistance is an emerging problem among streptococcal species, especially for severe infections. Automated diagnostic systems for antimicrobial susceptibility testing, such as BD Phoenix, is a recently available instruments that makes it possible to obtain results within 12 h. Methods. Antimicrobial susceptibility testing results of the BD Phoenix system were compared to those obtained from Clinical Laboratory Standards Institute (CLSI) disk-diffusion method. Two-hundred different clinical isolates of streptococci were assayed: beta-hemolytic (n=65), viridans (n=87), S. penumoniae (n=48). Results. Overall, there was categorical agreement greater than 96.7% (94.8% for beta-hemolytic and 97.9% for viridans group) in relationship to the disk-diffusion method. The minor error rates were less than 10% for all the antibiotics. The greatest percentage of serious errors corresponded to erythromycin and clindamycin within the beta-hemolytic group (14.7%). Overall percentage of very serious errors was less 0.5%. The results for penicillin in viridans streptococci and S. pneumoniae results showed 89.7% and 91.7% of categorical agreement, respectively, using the Etest as reference. Conclusions. The automated BD Phoenix system is a very useful and effective diagnostic tool for quantitative testing of sensitivity to antibiotics in the streptococci group.

Tissue concentrations of prothymosin alpha: a novel proliferation index of primary breast cancer.

In 71 patients with classic invasive ductal carcinomas, levels of prothymosin alpha (PT alpha), as assayed by a radioimmunoassay that detects thymosin alpha 1 (the NH2-terminal fragment of PT alpha), were significantly greater in tumour samples than in normal breast tissue. PT alpha levels were correlated with (a) the number of positive axillary lymph nodes (rs = 0.5384, P < 0.01), and (b) the percentage of tumour cells in the S or G2/M phase as assessed by flow cytometry (rs = 0.5027, P < 0.01). Since the beginning of this study in 1989, 21 patients have presented distant metastases, all of whom were previously shown to have tumour PT alpha levels greater than 124 ng of thymosin alpha 1/mg protein. The present report indicates that PT alpha might be used to identify breast cancer patients at high risk for distant metastases.

Prothymosin alpha mRNA is expressed in competent and proliferating rat thyroid cells (FRTL-5) but is not sufficient to elicit cell progression through the cell cycle.

Using flow cytometry we observed the effects that different hormonal treatments had on the progression of rat thyroid (FRTL-5) cells through the cell cycle. The absence of hormones or the addition of TSH (6 mU/ml) did not induce DNA synthesis; however, the addition of IGF-I (30 ng/ml) promoted cell proliferation. The number of cells recruited by IGF-I was lower than when IGF-I and TSH were used. We therefore concluded that we had a model with three different types of cells: (1) quiescent cells, cells cultured in the absence of hormones, considered to be G0-arrested cells, (2) competent cells, TSH-treated cells that did not proliferate (being arrested in a cycle phase different from G0) and (3) actively proliferating cells, cells treated with TSH plus IGF-I. Prothymosin alpha (PTA) mRNA levels were almost undetectable in cells cultured without hormones at all times studied, i.e. 8, 14 and 24 h. On the contrary, TSH and/or IGF-I greatly increased PTA mRNA. These data indicate that G0-arrested quiescent cells do not express PTA mRNA and that PTA mRNA is induced when FRTL-5 cells are committed to proliferate by the addition of TSH, in spite of being arrested by the lack of IGF-I. We therefore conclude that PTA mRNA expression may be an event that is necessary for cells to proliferate, but that it is not sufficient for the promotion of cell progression through the cell cycle.

Milk progesterone radioimmunoassay using radioiodinated tracers: a rapid and reliable assay system.

Both the validity and practicability of a direct progesterone radioimmunoassay based on radioiodinated progesterone tracers were studied. The results obtained show the reliability of the assay; when compared with assays based on 3H-progesterone tracers there are fewer steps for assay execution, saving time and reducing the number of reagents used. Various commercially available 125I-progesterone tracers were assayed, and only those with an 11 alpha-hemisuccinate bridge were suitably bound by antisera raised against progesterone-bovine serum albumin conjugates having identical bridge structure. The bridge effect caused no observable alteration in validity parameters. Finally, our results support the utility of this assay as a practical method of early diagnosis of pregnancy and as a reliable experimental technique to monitor cow ovarian function.

Diorganotin dihalide complexes of 2,2'-biimidazole. A preliminary study of their inhibitory effects on cell division.

We report the synthesis of new complexes with the general formula (R2SnX2)y.H2BiIm, where y = 1 or 2; R = Me, Et, Bun; X = Cl or Br (for R = Et) and H2BiIm = 2,2'-Biimidazole. The complexes have been characterized by elemental analysis and Mössbauer, infra-red and 1H n.m.r. spectroscopy and tested (like the ligand, Me2SnCl2 and Et2SnCl2) against P388D1 leukemic cells.

Comparison of the protective efficacy of Aujeszky's disease (pseudorabies) virus glycoproteins obtained from different sources.

The immunogenic properties of a series of glycoprotein preparations are compared using inactivated conventional vaccines as reference. Serological response and protective efficacy of vaccination of mice and pigs are evaluated for glycoprotein immunogens obtained from various sources. BHK-21 cell cultures were infected with Aujeszky's disease virus and used as antigenic source. Glycoproteins were obtained from (i) the whole culture (ii) the cell sediment and (iii) the clarified supernatant. Both in pigs and in mice, protection was greater with glycoproteins purified from infected-cell membranes than with viral mature particle glycoproteins. The specific profiles of humoral responses were basically identical regardless of the source of glycoprotein. Bartha strain, one of the gI- strains most commonly used as an attenuated vaccine, was also used as a glycoprotein source. Immunogens obtained from this strain were protective in challenge trials with the virulent E-974 strain of the Aujeszky's disease virus. Glycoproteins did not induce detectable delayed type hypersensitivity in mice but conferred greater protection than particulate antigens (which, conversely, did induce a detectable delayed type hypersensitivity reaction). Until the recent proposal of the potency criterion delta 7, no objective method was available to evaluate the degree of protection conferred by Aujeszky's disease vaccines. In this study, we thus used the protection index, a quantitative parameter designed to evaluate potency of vaccines against Aujeszky's disease virus.

[GB virus C: lack of association with transaminases levels, CD4 and HIV viral load in aids patients]. / Virus GB-C: ausencia de asociación con niveles de transaminasas, CD4 y carga vírica en pacientes con sida.

OBJECTIVE: To study the prevalence of GBV-C-RNA in sera of HIV-infected patients and determine whether differences in immunological condition and hepatic disease exist between GBV-C positive and negative patients. METHODS: The presence of GBV-C-RNA was determined in sera of 222 HIV-positive patients by semi-automated RT-PCR. A comparison of GBV-C-RNA positive and negative patients was made by studying a series of clinical and analytical parameters. This same comparison was made in particular between those coinfected with HCV and GBV-C and those who only presented GBV-C. RESULTS: Prevalence of GBV-C-RNA was 28.8%. The most frequent hepatotropic virus was HCV, appearing in 71.6% of cases. Coinfection with HCV and HGV was present in 17% and 8.6% only had GBV-C. Patients positive for GBV-C-RNA showed clinical and analytical characteristics similar to those found in GBV-C-RNA negative patients. Among the HCV-GBV-C coinfected and those presenting HGV as the only virus it was observed that the coinfected group presented alterations in transaminases and predominance of parenteral transmission as a risk factor for HIV, whereas the GBV-C group presented normal transaminases and predominance of sexual transmission. No differences were perceived in mean CD4 and HIV-RNA values in both groups. CONCLUSIONS: Being positive for GBV-C in HIV-positive patients does not influence the presence of hepatic disease that in these patients is frequently accompanied by coinfection with other hepatotropic viruses. Moreover, it does not seem to influence the viremia of the HIV nor the CD4 cell counts.

Nosocomial infection by VIM-2 metallo-beta-lactamase-producing Pseudomonas putida.

Nosocomial infections caused by multidrug-resistant and carbapenem-resistant Pseudomonas putida isolates have been reported occasionally in severely ill or immunocompromised patients. Here we report the microbiological characteristics of what are believed to be the two first carbapenem-resistant VIM metallo-beta-lactamase (MBL)-producing P. putida strains in Spain, which were isolated from patients at the University Hospital Complex of Santiago de Compostela. Both patients were immunocompromised with severe underlying diseases and had been hospitalized for more than 15 days. One of them had previously been treated with a broad-spectrum therapy. Antimicrobial susceptibility testing showed that both strains were resistant to piperacillin/tazobactam, ceftazidime, cefepime, imipenem, meropenem, gentamicin, tobramycin, aztreonam, trimethoprim/sulfamethoxazole and ciprofloxacin, but sensitive to amikacin and colistin. For both isolates PCR and sequencing was positive for the bla(VIM-2) gene. Fingerprinting analysis revealed these were two different strains. One patient recovered clinically and one died; no direct link could be established between the isolation of P. putida and death. Our data expose the emergence of multidrug-resistant P. putida VIM-2 MBL, probably arising by independent horizontal transfer of resistance genes. So, although P. putida is not frequently isolated, it may survive easily in the hospital setting and occasionally cause difficult-to-treat nosocomial infections in severely ill patients.

Effects of antigen and internal environment on anti-phosphorylcholine immune responses of autoimmune aged NZB/W F1 mice.

The idiotypic profile of anti-phosphorylcholine plaque-forming cell responses and their evolution with ageing were studied in (NZB X NZW) F1 mice. Our results showed that the anti-phosphorylcholine plaque-forming cell response induced by phosphorylcholine coupled to keyhole limpet haemocyanin and, paralleling, the T15 idiotype clonal dominance declined with ageing. This loss of immune competence was also observed with another thymus-dependent (phosphorylcholine coupled to egg globulin) as well as thymus-independent (capsular polysaccharide of Streptococcus pneumoniae strain R36a) antigens. In contrast, old mice challenged with an antigenic preparation of Neisseria meningitidis showed an immune response not significantly different from that elicited by the same antigen in young mice. The hapten-augmentable plaque-forming cells were assayed to determine whether a putative auto-antiidiotypic regulation underlies this loss of immune competence. Only minimal numbers and non-significant differences between young and old mice immunized with any antigen could be detected. Further studies using an adoptive transfer system demonstrated that cells from aged mice were able to support a normal anti-phosphorylcholine response when transferred into lethally irradiated young recipients. Our results suggest that no permanent cellular defects, but rather internal environment or/and radioresistant suppressor cells, are involved in this loss of immune competence. The role played by these factors and their effect on distinct subpopulations of B cells are discussed.

Immunoresponses to Neisseria meningitidis epitopes: in vivo analysis of immunocompetent cells involved in suppression of secondary response to phosphorylcholine.

The immune response to phosphorylcholine (PC) antigens has been extensively studied in recent years. Neisseria meningitidis serogroup B M986 (NMB) was recently reported to induce a PC-specific plaque-forming cell (PFC) immuno-response in mice, a characteristic useful for the study of immunomodulating properties of N. meningitidis. With this technique, priming mice with low doses of NMB has been shown greatly to impair their ability, one month after priming, to mount an anti-PC response induced by NMB; this suppression is permanent, does not involve switching from IgM to another immunoglobulin class, transiently affects the T15 idiotype expression and is carrier specific. We report, based on an analysis of spleen cells from NMB-primed mice in an adoptive transfer model, that this suppression does not appear to be mediated by B lymphocytes nor does it seem to be under the direct control of T lymphocytes; rather, it involves radio-resistant cells. Additionally, our results show that NMB modulates the idiotype composition of the anti-phosphorylcholine response, probably by enhancing the expression of so called hapten-augmentable PFC. These results demonstrate that NMB can interfere effectively with the immune response in a variety of ways.

Antigens involved in vaccination of swine against Aujeszky's disease (pseudorabies) virus.

The polypeptide and glycopolypeptide composition of a local virulent Aujeszky's disease virus (suid herpesvirus 1, SHV-1) strain (E-974) was determined in order to characterize the individual SHV-1 antigens inducing the serological responses in immunized and non-immunized animals. A commercially available inactivated vaccine of known efficacy and three experimental immunogen preparations (whole inactivated SHV-1 particles, lectin-purified glycoproteins from SHV-1 culture, and a combination of both) were used for immunization. Sera of two-month old immunized and non-immunized animals were analyzed by ELISA, seroneutralization and Western immunoblotting prior to and following challenge with E-974. Sera of 7- to 30-day-old piglets littered by immunized and non-immunized sows were likewise analyzed by immunoblotting. The following variables were determined: the total level of anti-SHV-1 antibodies, the level of neutralizing antibodies, the IgG responses to individual SHV-1 antigens, and the clinical parameters and degree of protection of the animals. The whole-particle experimental immunogen conferred greatest protection, but correlation between antibody levels and the degree of protection was imperfect. Serological responses seemed to be directed against certain structural polypeptides and viral envelope glycoproteins. The glycoprotein immunogen caused a selective response to bands which closely resemble the glycopolypeptides gII and gIII. A 71 kDa component of uncertain location within the viral structure appeared to be one of the main antigens involved in porcine serological response to SHV-1 and colostral protection of piglets.

Prothymosin alpha mRNA levels are invariant throughout the cell cycle.

Prothymosin alpha (PT-alpha) mRNA levels were evaluated at different stages during the cell cycle. NIH 3T3 cells were synchronized: (a) by serum deprivation, (b) by mitotic shake off after nocodazole arrest, and (c) by double thymidine block. Cell synchronism was estimated by flow cytometry. In cells grown in serum-free medium, PT-alpha mRNA levels were almost undetectable. 14 h after serum restoration PT-alpha mRNA was induced as had been described by others (Eschendfeldt, W. H., and Berger, S. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 9403-9407). PT-alpha mRNA induction seems to require the synthesis of proteic factor(s) since PT-alpha mRNA response to serum restoration was abolished in the presence of cycloheximide. Interestingly, cycling cells that were synchronized at different stages of the cycle by means of mitotic shake off after nocodazole arrest or double thymidine block did not show variations in the levels of PT-alpha mRNA when progressed synchronously through the cycle. On the contrary, histone H4 mRNA was expressed only during the S phase. These data indicate that PT-alpha mRNA was present in roughly the same amount through all phases of the cell cycle, arguing against the concept that PT-alpha is a cell cycle-regulated gene.

Immunoresponses to Neisseria meningitidis epitopes: immunomodulation by meningococcus B acts on more than one meningococcal surface epitope.

Neisseria meningitidis group B strain M986 (serotypes 2a, 7) (NMB) elicits a specific primary antiphosphorylcholine immune response in mice but not a secondary response. The ability of other serotype and serogroup meningococci to induce similar primary responses in mice was studied, as was the immunogenicity of trinitrophenyl coupled NMB (TNP-NMB) in primary and secondary antitrinitrophenyl responses. Except for NMB, all other strains tested (three serogroup B and one serogroup A meningococcal strains) were found to be very poor phosphorylcholine immunogens. TNP-NMB, however, though proving to be a very good TNP antigen, was only a weak phosphorylcholine antigen. Priming NBF1 female mice with TNP-NMB one month or more before challenging them with the same antigen induced a strong depression of anti-TNP response in the subsequent challenge. However, this effect was not observed with Xid NBF1 male mice. Furthermore, priming mice with NMB weakly affected the anti-TNP response, but greatly depressed the antiphosphorylcholine response, after TNP-NMB challenge. In addition, whereas apparently only one TNP-specific B cell subpopulation was responding in unprimed mice challenged simultaneously with TNP-NMB and TNP-Ficoll (non-additive response), priming mice with NMB appeared to facilitate the independent activation of two different TNP-specific B cell subpopulations (additive response).

[Anti-phosphorylcholine response of autoimmune NZB/W mice: variations with age of serum titers and homogeneity]. / Respuesta antifosforilcolina de ratones autoinmunes NZB/W: variaciones con la edad del título y homogenidad de los sueros.

Antibody titers and avidity of sera of autoimmune NZB/W mice in responses induced by different antigens were determined. Results show an age-dependent decrease of the antibody titer in sera from female mice immunized with phosphorylcholine coupled to keyhole limpet haemocyanin. This decrease was not detected when using as immunogen an antigenic preparation of Neisseria meningitidis that naturally induces anti-phosphorylcholine antibodies, but was detected with a modification of this antigen (heat inactivation and further coupling with the hapten). Determinations of inhibition profiles of antisera suggest that this loss of immune competence is paralleled by a decrease in avidity and homogeneity of antisera. This finding may be related to the loss of idiotypic clonal dominance recently reported to occur in these mice.

The antiphosphorylcholine plaque-forming cell responses induced by the nematode Trichinella in BWF1 mice.

We have tested the ability of the nematode Trichinella to infect young and old (NZB x NZW) F1 (BWF1) mice. We report the capacity of these mice to respond to the parasite antigens containing the epitope phosphorylcholine. The values obtained in adult worm intestinal retention rates and in the number of encysted larvae on the 35th day postinfection showed that the old BWF1 mice are more susceptible than young BWF1 and control (BALB/c x CBA/j)F1 (BCF1) mice to Trichinella infection. However, unlike the BCF1 mice, young BWF1 mice were unable to produce a good anti-phosphorylcholine plaque-forming cell response after the killing of Trichinella larvae by the anthelminthic mebendazole. Old BWF1 mice presented a discrete response which is discussed. Finally, our results seem to indicate that the lack of anti-phosphorylcholine response in young BWF1 mice after mebendazole treatment may be related to the high susceptibility of these mice to the suppressive properties of encysted Trichinella larvae against their own antigens.

Effect of clofibrate on the growth-kinetics of the murine P 1798(sc) lymphoma.

Clofibrate (CPIB) is a drug applied as an antilipidaemic agent in mammals. In this work we have tested its efficacy in vivo on the growth kinetics of P 1798(sc) lymphoma transplanted to recipient (BALB/c x AKR)F1 mice. Our results show a facilitation of the tumour growth rate in treated recipients. This fact may be related to an effect of the agent on the recipient which produces a decrease in the immune response as was confirmed on testing CPIB on thymus-dependent antigens in haemolytic plaque assays.