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.

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.

[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.

[Diarrhea caused by adenovirus and astrovirus in hospitalized immunodeficient patients]. / Diarrea por adenovirus y astrovirus en pacientes inmunodeficientes hospitalizados.

BACKGROUND: Acute or chronic diarrheal illness are common complications in immunosuppressed patients such as human immunodeficiency virus (HIV)-infected, bone marrow or solid organ transplanted patients and those with leukaemias or other immune deficiency disorders. Due to the importance of recognizing the feasible etiologies of diarrhea in order to give the proper antimicrobial chemotherapy or to avoid a misdiagnosis of rejection in the case of transplanted patients, we have investigated adenovirus and astrovirus antigen in faeces from different immunosuppressed patients. PATIENTS AND METHODS: Stool samples from 258 immunodeficient patients hospitalized at University Hospital Complex of Santiago of Compostela with acute or persistent diarrhea were collected between 1997-99 and assayed for astrovirus and adenovirus antigen. Viral antigen was detected by EIA. Other common enteric pathogens were also assayed. RESULTS: Adenovirus antigen was positive in 5 cases (2%) and astrovirus antigen in 12 cases (5%). The most commonly patients infected was those with haematologic disorders and premature infants. HIV-infected patients were positive for astrovirus antigen in 3 cases. The majority of the cases were related with intestinal bacterial diseases or other circumstances, such as Clostridium difficile infection, both associated with prolonged antimicrobial therapy. CONCLUSIONS: Astrovirus and adenovirus have to be considered as enteropathogens specially in immunocompromised hospitalized patients. An accurate diagnosis about diarrhea etiology is advisable in order to give a specific antimicrobial therapy, when it be necessary, or to avoid a misdiagnosis of rejection, in transplanted patients.

Stomatococcus mucilaginosus septicemia in a patient with acute lymphoblastic leukaemia.

A 16-year-old patient with acute lymphoblastic leukaemia which had relapsed for the third time developed clinical signs and symptoms of septicemia during a period of neutropenia. The patient had signs of oral mucositis, and Stomatococcus mucilaginosus was isolated from blood cultures. The patient responded well to antibiotic therapy. The biochemical characteristics and antimicrobial susceptibility patterns of 68 other pharyngeal isolates of Stomatococcus mucilaginosus from immunocompromised patients are presented.

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.

Efficacy of various non-oily adjuvants in immunization against the Aujeszky's disease (pseudorabies) virus.

Standard oil and various non-oily adjuvants were compared for use in immunization against the Aujeszky's disease (pseudorabies) virus, both in mice and swine, and using either inactivated virions or purified glycoproteins as antigen. Mineral oil, sodium alginate, aluminium hydroxide, and saponin were assayed in mice as adjuvants for inactivated virions, saponin being the most efficient. The addition of Mab anti-CD3 did not improve either immune response or protection achieved in mice using viral particles with oil or sodium alginate. When purified glycoproteins were used as antigens, the use of ISCOM greatly enhanced specific T-cell responses and protection of mice. The incorporation of Mab anti-CD3 into ISCOM conferred 100% protection of mice. Surprisingly, when an ISCOM containing glycoproteins was assayed in swine in a single-dose trial, no improvement on the protection conferred by the oily adjuvant was observed.

Regulation of prothymosin alpha mRNA levels in rat pituitary tumor cells.

Prothymosin alpha (PTA) mRNA and histone H4 (H4) mRNA levels were studied in various experimental conditions that affected GH1 pituitary tumor cell proliferation. Cell proliferation and progression through the cell cycle was assessed by counting cells, 3H-thymidine incorporation and flow cytometry. PTA mRNA levels were decreased in a time-dependent fashion following serum deprivation; when the cells were induced to grow by serum refeeding, PTA mRNA expression was greatly stimulated. Interestingly, after caprylic acid treatment (2.5 mM for 24 h) that arrested cells in the G0/G1 phase of the cell cycle, PTA mRNA and H4 mRNA levels were almost undetectable; conversely, following caprylic acid withdrawal, PTA mRNA and H4 mRNA expression were greatly stimulated. Furthermore, cells cultured in T3-deprived serum, which was found to decrease GH1 cell proliferation, had low levels of PTA and H4 mRNAs. This effect was reversed by the addition of nanomolar concentrations of T3 to the culture. On the other hand, IGF-1 addition to the culture did not substantially modify PTA mRNA levels. The present data clearly indicate that PTA mRNA expression is tied to the proliferating activity of GH1 cells and, thus, could be used as a marker of the action that various agents have on GH1 cell proliferation.

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.

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.