[Isolation and identification of species from the genera Aeromonas, Vibrio, and Plesiomonas from extraintestinal samples in Cuba]. / Aislamiento e identificación de especies pertenecientes a los géneros Aeromonas, Vibrio y Plesiomonas procedentes de muestras extra-intestinales en Cuba.

Extraintestinal infections caused by the genera Aeromonas, Vibrio and Plesiomonas have high morbidity and mortality rates in different areas of world. From January 2002 to December 2003, the National Reference Laboratory for Acute Diarrhoeal Diseases of the Pedro Kourí Tropical Medicine Institute received 95 gram-negative, facultative anaerobic, oxidase positive bacilli strains from different extraintestinal specimen (blood, ear exudates, infected wounds, conjunctive exudates, urine, and catheters, among others) sent by different provincial laboratories along the country. Aeromonas caviae, Aeromonas veronii bv sobria, Aeromonas jandaei, Vibrio cholerae no-O1, Vibrio vulnificus, Vibrio fluvialis and Plesiomonas shigelloides were the species most frequently found in the sample analysed.

Aislamiento e identificación de especies pertenecientes a los géneros Aeromonas, Vibrio y Plesiomonas procedentes de muestras extra-intestinales en Cuba / Isolation and identification of species from the genera Aeromonas, Vibrio, and Plesiomonas from extraintestinal samples in Cuba

Las infecciones extra-intestinales producidas por los géneros Aeromonas, Vibrio y Plesiomonas presentan una elevada tasa de morbimortalidad en diferentes áreas geográficas. De enero 2002 a diciembre 2003 se recibieron en el Laboratorio Nacional de Referencia de Enfermedades Diarreicas Agudas del Instituto de Medicina Tropical Pedro Kourí, 95 cepas de bacilos gramnegativos, anaerobios facultativos, oxi-dasa positiva, procedentes de muestras extra-intestinales (hemocultivos, exudados óticos, pus de heridas, exudados conjuntivales, urocultivos, catéter, entre otras) remitidas de diferentes provincias del país. Se demostró la presencia de Aeromonas caviae, Aero-monas veronii bv sobria, Aeromonas jandaei, Vibrio cholerae no -O1, Vibrio vulnificus, Vibrio fluvialis y Plesiomonas shigelloides en las muestras estudiadas.

Extraintestinal infections caused by the genera Aeromonas, Vibrio and Plesiomonas have high morbidity and mortality rates in different areas of world. From January 2002 to December 2003, the National Reference Laboratory for Acute Diarrhoeal Diseases of the Pedro Kourí Tropical Medicine Institute received 95 gramnegative, facultative anaerobic, oxidase positive bacilli strains from different extraintestinal specimen (blood, ear exudates, infected wounds, conjunctive exudates, urine, and catheters, among others) sent by different provincial laboratories along the country. Aeromonas caviae, Aeromonas veronii bv sobria, Aeromonas jandaei, Vibrio cholerae no-O1, Vibrio vulnificus, Vibrio fluvialis and Plesiomonas shigelloides were the species most frequently found in the sample analysed.

Presumed pseudobacteremia outbreak resulting from contamination of proportional disinfectant dispenser.

Reported here are the microbiological and epidemiological details of a presumed outbreak of aerobic gram-negative bacilli infections affecting 19 hematological patients, which was traced to contaminated disinfectant. Over a 5-month period, the following organisms were isolated from the blood cultures of 19 neutropenic patients: Pseudomonas fluorescens (n = 13), Achromobacter xylosoxidans (n = 12), Comamonas testosteroni (n = 2) or Stenotrophomonas maltophilia (n = 1). The affected patients were all treated with an expensive regimen of broad-spectrum antibiotic therapy. The same bacteria were recovered from environmental samples as well as from the water pipes of an apparatus for dispensing disinfectant (didecyldimethylammonium chloride). Genotyping results indicated that many of the clinical strains were identical to strains isolated from the apparatus. It was eventually discovered that the night staff was in the habit of disinfecting the blood-culture bottles before use, thereby contaminating the bottles with bacteria contained in the disinfectant. Contamination of the apparatus resulted from faulty maintenance.

Genetic analysis of a multiresistant strain of Pseudomonas aeruginosa producing PER-1 beta-lactamase.

A multiresistant strain of Pseudomonas aeruginosa, PA2345, belonging to serotype O:1, was isolated at the Teaching Hospital of Besançon, France. Resistance to beta-lactams, including third-generation cephalosporins, depended upon a chromosomally-located composite transposon carrying the bla(PER-1) gene encoding extended-spectrum beta-lactamase PER-1. PA2345 was unrelated genotypically to two previous PER-1-producing isolates of P. aeruginosa. Sequence analysis of the transposon in PA2345 revealed the presence of two insertion sequences (ISPa23 and ISPa24) with very different predicted transposases (TnpA1, TnpA2), which were both bordered by closely related 16-bp inverted repeats. High resistance of PA2345 to aminoglycosides was caused, in part, by a chromosomal class-I integron containing gene cassettes aadB, encoding an ANT(2'') enzyme, and aadA11, encoding a new ANT(3'') enzyme with 281 amino-acids that conferred elevated resistance to streptomycin and spectinomycin. Stable overproduction of efflux system MexXY contributed to resistance to amikacin, while mutations in the quinolone resistance-determining regions of gyrA and parC accounted for the high resistance of PA2345 to fluoroquinolones. The study indicates that multidrug resistance in P. aeruginosa might arise from sequential acquisition of a variety of mechanisms provided by both horizontal gene transfers and mutations in chromosomal genes.

[Two efflux systems expressed simultaneously in clinical Pseudomonas aeruginosa]. / Deux systèmes d'efflux exprimés simultanément chez des souches cliniques de Pseudomonas aeruginosa.

Active efflux systems MexAB-OprM and MexXY were found to be overexpressed simultaneously in 12 multiresistant clinical isolates of Pseudomonas aeruginosa. Nine of these strains (agrZ mutants) harbored mutations in gene mexZ, the product of which down-regulates expression of operon mexXY. Eight of the 12 strains exhibited mutations in genes known to control transcription of operon mexAB-oprM, such as mexR (four nalB mutants) or PA3721 (three nalC mutants). One strain was a nalB/nalC double mutant. For MexAB-OprM as well as for MexXY, no clear correlation could be established between (i) the types of mutations, (ii) the over-expression levels of genes mexA or mexX, and (iii) the resistance levels to effluxed antibiotics. Finally, three and four isolates overproduced MexXY (agrW mutants) or MexAB-OprM (nalD mutants), respectively, without any mutation in the known regulator genes. These data show that clinical isolates are able to broaden their drug resistance profiles by coexpressing two Mex efflux pumps and suggest the existence of additional regulators for MexAB-OprM and MexXY.

Propagation of TEM- and PSE-type beta-lactamases among amoxicillin-resistant Salmonella spp. isolated in France.

A survey conducted between 1987 and 1994 at the University Hospital of Besançon, France, demonstrated a dramatic increase (from 0 to 42. 5%) in the prevalence of amoxicillin resistance among Salmonella spp. Of the 96 resistant isolates collected during this period (including 77 Typhimurium), 54 were found to produce TEM-1 beta-lactamase, 40 produced PSE-1 (equivalent to CARB-2), one produced PSE-1 plus TEM-2, and one produced OXA-1 in isoelectric focusing and DNA hybridization experiments. Plasmids coding for these beta-lactamases were further characterized by (i) profile analysis, (ii) restriction fragmentation pattern analysis, (iii) hybridization with an spvCD-orfE virulence probe, and (iv) replicon typing. In addition, isolates of S. typhimurium were genotypically compared by pulsed-field gel electrophoresis of XbaI-macrorestricted chromosomal DNA. Altogether, these methods showed that 40 of the 41 PSE-1 producers were actually the progeny of a single epidemic S. typhimurium strain lysotype DT104. Isolates of that strain were found to harbor RepFIC virulence plasmids with somewhat different restriction profiles, but which all carried the bla(PSE-1) gene. Of these virulence/resistance plasmids, 15 were transmissible to Escherichia coli. TEM-1-producing S. typhimurium displayed much greater genotypic and plasmidic diversities, suggesting the acquisition of the bla(TEM-1) gene from multiple bacterial sources by individual strains. In agreement with this, 32 of the 35 S. typhimurium plasmids encoding TEM-1 were found to be conjugative. These data show that development of amoxicillin resistance among Salmonella, especially in serovar Typhimurium, results from both gene transfers and strain dissemination.

In vivo emergence of multidrug-resistant mutants of Pseudomonas aeruginosa overexpressing the active efflux system MexA-MexB-OprM.

During a 6-month period, 21 pairs of Pseudomonas aeruginosa isolates susceptible (pretherapy) and resistant (posttherapy) to antipseudomonal beta-lactam antibiotics were isolated from hospitalized patients. In vivo emergence of beta-lactam resistance was associated with the overexpression of AmpC beta-lactamase in 10 patients. In the other 11 patients, the posttherapy isolates produced only low, basal levels of beta-lactamase and had increased levels of resistance to a variety of non-beta-lactam antibiotics (e.g., quinolones, tetracyclines, and trimethoprim) compared with the levels of beta-lactamase production and resistance of their pretherapy counterparts. These data suggested the involvement of the MexA-MexB-OprM active efflux system in the multidrug resistance phenotype of the posttherapy strains. Immunoblotting of the outer membrane proteins of these 11 bacterial pairs with a specific polyclonal antibody raised against OprM demonstrated the overexpression of OprM in all the posttherapy isolates. To determine whether mutations in mexR, the regulator gene of the mexA-mexB-oprM efflux operon, could account for the overproduction of the efflux system, sequencing experiments were carried out with the 11 bacterial pairs. Eight posttherapy isolates were found to contain insertions or deletions that led to frameshifts in the coding sequences of mexR. Two resistant strains had point mutations in mexR that yielded single amino acid changes in the protein MexR, while another strain did not show any mutation in mexR or in the promoter region upstream of mexR. Introduction of a plasmid-encoded wild-type mexR gene into five posttherapy isolates partially restored the susceptibility of the bacteria to selected antibiotics. These results indicate that in the course of antimicrobial therapy multidrug-resistant active efflux mutants overexpressing the MexA-MexB-OprM system may emerge as a result of mutations in the mexR gene.

The effect of ethanol and/or food restriction on coenzyme Q in liver in rats.

The individual and combined effects of ethanol and lovastatin on rats and their prevention by supplemental coenzyme Q10 (CoQ10) was studied. The ethanol and lovastatin findings are reported elsewhere. This paper focuses on the food restriction which occurred in rats fed 35% of energy as ethanol and those control rats pair-fed to the 35% of energy as ethanol group. Six groups of rats received 35% of energy as ethanol (with or without lovastatin and/or CoQ10 treatment). One group served as a 0% ethanol ad libitum control and one 0% ethanol control group was pair-fed to the 35% ethanol group. Rats receiving 35% of energy as ethanol and their pair-fed controls consumed 83% of the energy/day consumed by the ad libitum controls. This was consistent regardless of lovastatin or CoQ10 treatment. Weight gains were 84% of control. The energy reduction was consistently associated with a substantial (48%+) increase in liver CoQ9 concentrations. Reports by others of associations between food restriction and increased longevity in rodents has focused on a decrease in oxidant damage in tissues of food restricted animals. The increase in CoQ levels in the food restricted animals would result in an increase in antioxidant protection and might explain the observed increases in longevity.

Molecular analysis of the replication elements of the broad-host-range RepA/C replicon.

RepA/C is a replicon specific to the IncA/C incompatibility group of plasmids and was isolated recently from plasmid RA1. The sequence of this autoreplicative region was established; it contains 13 repeats, suggesting that the replicon uses iterons to control its copy number. The sequence contains two ORFs, one potentially coding for a 33-kDa protein (ORF1) and a second potentially coding for a 14-kDa protein (ORF2) (Llanes et al., 1994b). In this work, using an in vitro transcription/translation system, we detected a polypeptide whose size corresponded well to that of the deduced product of ORF1. Deletion and insertion mutation analysis showed that ORF1 is essential for replication; it encodes an initiator protein (called RepA). ORF2 was not essential for replication in Escherichia coli and its function remains to be determined. Using complementation experiments, the replication origin (ori) of RepA/C was defined. The ori was located in a 600-bp fragment downstream from repA, containing 10 direct repeats. To study the control of repA expression, a transcriptional fusion PrepA::lacZ was constructed. Its analysis showed that repA is transcriptionally autoregulated as are most repA genes of replicons controlled by iterons.

Acetylcholinesterase expression in NTera 2 human neuronal cells: a model for developmental expression in the nervous system.

Acetylcholinesterase (AChE; EC 3.1.1.7) is expressed in the central nervous system in multiple molecular forms that may subserve multiple functions and may be selectively lost in neurodegenerative illnesses such as Alzheimer's disease. AChE expression has been studied in primary cultures of developing vertebrate nervous system, but investigation has been limited by the lack of a suitable human CNS surrogate cell model system for in vitro studies and the inability of primary brain cultures to provide large numbers of pure neurons. To develop an in vitro model for studies of neuronal AChE expression and function, we utilized a neuronally committed human teratocarcinoma cell line, NTera 2, that can be induced to differentiate to a post-mitotic CNS neuronal phenotype. We found that NTera 2 cells express multiple molecular forms of AChE that are similar to CNS-derived AChE isoforms in velocity sedimentation profile, anion exchange elution profile, and sensitivity to inhibitors. At least two forms of AChE are expressed (G1 and G4), similar to human and rodent brain, and induction of NTera 2 cell differentiation results in an increased G4/G1 ratio, which is characteristic of mature neurons. As in primary CNS neurons, AChE is present in NTera 2 cells in both the cytosolic fraction and in the outer membrane, and is also released in a soluble form. These observations indicate that NTera 2 cells provide a useful human model system for studies of expression of cell-associated and soluble cell-free AChE in developing and mature human neurons and for elucidating the potential role(s) of acetylcholinesterase metabolism in both normal development and neurodegenerative disease states.

Cloning and characterization of the Inc A/C plasmid RA1 replicon.

The Inc A/C plasmids, like Inc P and Inc Q plasmids, have a broad host range. However, their maintenance functions remain to be studied. An autoreplicative region of 2.79 kb named RepA/C, able to replicate both in the family Enterobacteriaceae and in Pseudomonas spp., was isolated and sequenced. The stability, copy number, and incompatibility expression of this replicon were determined. RepA/C and a nonautoreplicative fragment of 16 kb of this replicon were used as probes and showed specific hybridizations with the Inc P3-A/C plasmids from Pseudomonas spp. and members of the Enterobacteriaceae. These probes could be used as tools for identification of the plasmids of this epidemiologically important Inc group.

Replicon typing of 71 multiresistant Serratia marcescens strains.

Replicon typing is the identification of plasmids by hybridization with specific DNA probes which contain the genes involved in plasmid maintenance. This new method has been used to classify plasmids into replicon (rep) groups which can often be correlated with incompatibility (Inc) groups. We studied 71 multiresistant Serratia marcescens strains with 19 rep probes constructed from reference plasmid replicons belonging to known Inc groups. These probes are known to react with enteric bacterial plasmids. However, they did not represent the totality of the thirty known Inc groups. For 52% of the studied strains, plasmids were identified and classified into groups FIB, FIC, FIIA, HI2, L/M, N, B/O, P, W, Y and Com9. Most (79%) of the plasmid preparations hybridized with a single rep probe, and 21% hybridized with two different probes. Electrophoretic analysis of DNA suggested that double hybridization could result from the presence of either two different Inc plasmids in the same strain (e.g. S37) or one single plasmid with a multireplicon (e.g. S113).

Natural killer (NK) cell stimulatory factor increases the cytotoxic activity of NK cells from both healthy donors and human immunodeficiency virus-infected patients.

Natural killer cell stimulatory factor (NKSF), or interleukin 12 (IL-12), is a heterodimeric lymphokine produced by B cells that has multiple effects on T and NK cell functions. NKSF at concentrations as low as 0.4 pM enhances the spontaneous cytotoxic activity of peripheral blood lymphocytes (PBL) against a variety of tumor-derived target cell lines and virus-infected target cells. The combined treatment of PBL with NKSF and IL-2 results in a less than additive enhancement of cytotoxicity. NKSF enhances the cytotoxic activity of spontaneously cytotoxic CD16+CD5- NK cells and does not confer cytotoxic activity to CD16-CD5+ T cells. PBL from patients infected with human immunodeficiency virus (HIV) have significantly lower cytotoxic activity against tumor-derived target cells and virus-infected target cells than PBL from control healthy donors. Treatment of PBL from HIV-infected patients with NKSF and/or IL-2 results in an increase of NK cell cytotoxicity against both types of target cells to levels similar to or higher than those of untreated PBL from healthy donors. PBL from HIV-infected patients produce interferon gamma in response to NKSF and/or IL-2, although at levels 5- or 10-fold lower than those produced by PBL from healthy donors. The multiple biological effects of NKSF, its activity at very low molar concentrations, and its ability to synergize with other physiological stimuli suggest that NKSF/IL-12 is a lymphokine likely to have physiological importance and considerable therapeutic potential.

Stability of conjugative and non-conjugative R-plasmids from Serratia marcescens to gyrase inhibitors.

The stability of conjugative and non-conjugative R-plasmids was compared using two gyrase inhibitors (novobiocin and ciprofloxacin). Conjugative R-plasmids from eighteen ticarcillin resistant Serratia marcescens were more stable than non-conjugative R-plasmids from eleven ticarcillin resistant bacteria of the same species. Moreover, novobiocin (gyrase B sub-unit inhibitor) is a better curing agent than ciprofloxacin (A sub-unit inhibitor).

[Antibiotic resistance plasmids from Serratia marcescens and their elimination by DNA-gyrase inhibitors]. / Les plasmides de résistance aux antibiotiques de Serratia marcescens et leur élimination par les inhibiteurs de l'ADN-gyrase.

We studied the effects of ciprofloxacin on 73 strains of Serratia marcescens. In the first place, we have tested their plasmidic content: 76% of Serratia marcescens strains contained plasmids by electrophoresis, and 29% of these plasmids were self-transmissible by conjugation. Secondly, we studied the plasmid stability with regard to ciprofloxacin. We obtained a spontaneous cure with 19% of plasmids, and ciprofloxacin, at very low concentrations (0.4 mg/l), increased the rate of cure and more efficiently than novobiocin, a compound used as a known curing agent.