Tetracycline resistance transmission in Campylobacter is promoted at temperatures resembling the avian reservoir.

Campylobacter is the causal agent of campylobacteriosis in humans, a self-limiting gastroenteritis. Campylobacteriosis is a zoonosis, commonly transmitted from contaminated chicken meat by either direct consumption or cross contamination during food manipulation. Presence of plasmids encoding for resistance to antibiotics such as tetracycline is common among Campylobacter isolates. In this report, we studied the effect of the temperature in the conjugation frequency of several tet(O) carrying plasmids, providing tetracycline resistance to the recipient cells. The conjugation frequency from donor cells carrying three previously characterized plasmids (pCjA13, pCjA9 and pTet) and from two clinical isolates was determined. Two temperatures, 37 and 42 °C, mimicking the conditions encountered by C. jejuni in the human and broiler chicken gastrointestinal tracts, respectively, were assessed. Our results clearly indicate that the conjugation process is promoted at high temperature. Accordingly, the transcriptional expression of some putative conjugative apparatus genes is thermoregulated, being induced at 42 °C. The two plasmids present in the clinical isolates were sequenced and assembled. Both plasmids are highly related among them and to the pTet plasmid. The high identity of the genes putatively involved in the conjugation process among the plasmids is in agreement with the similar behavior regarding the temperature dependency of the conjugative process. This report suggest that conjugation of plasmids carrying antibiotic resistance genes occurs preferentially at temperatures that resemble the gastrointestinal tract of birds, the main reservoir of C. jejuni.

Example of antimicrobial stewardship program in a community hospital in Italy.

BACKGROUND: Inappropriate use of antibiotics has caused the emergence of resistant strains of bacteria. The hospital of Alessandria, Italy, implemented an antimicrobial stewardship (AS) pilot program between 2013 and 2015 in the intensive care units (ICUs) and internal medicine departments of Casale Monferrato and Tortona. We aimed to describe the project, results at the end of the intervention, and its strengths and weaknesses. METHODS: The protocol, designed by the local infection control committee, included three consecutive steps: local guidelines for empirical antibiotic therapy and list of prescription antibiotics with justification, monitoring of antibiotic consumption and antimicrobial resistance trend, and peer-to-peer audit sessions in the wards. RESULTS: One thousand and eighty-five observations were made, corresponding to 850 patients admitted to the ICUs (16.7%) and internal medicine departments (83.3%). Appropriate antibiotic prescriptions increased by 6.4% between 2013 and 2015. The greatest improvement in appropriate prescriptions was observed for glycopeptides and fluoroquinolones (+17.4% and +16.2%, respectively). We reported 305 inappropriate prescriptions, with the most frequent errors being absence of an infectious process (33.3%), inadequate combination therapy (12.8%), and absence of microbiological investigations (8.5%). A reduced incidence of methicillin-resistant Staphylococcusaureus (MRSA) was also observed (p<0.0037). CONCLUSIONS: Antimicrobial stewardship programs contribute to improving antibiotic prescription and can be implemented in small community hospitals. Narrower interventions, focused on a single disease or single antibiotic should be encouraged.

Burns and mental disorder.

INTRODUCTION: The study aimed to explore the prevalence of previous mental disorder and the incidence of adjustment and posttraumatic stress disorder in 60 patients with burn injuries admitted to a major burn unit in the greater Madrid area. METHODS: A total of 57 patients were assessed for serious past and present mental illness by using a semi-structured clinical interview. Current DSM-IV adjustment and posttraumatic stress disorder were assessed 6 months post burn with the structured clinical interview SCID. RESULTS: 60 patients were hospitalized for severe burns during the study period. 11 (17,9%) burns took place intentionally. 9 patients had tried to commit suicide. 33.5% of the participants had suffered any kind of mental disorder previously. 23 (52.3%) patients met criteria for adjustment disorder and 8 (18%) for posttraumatic stress disorder 6 months post burn. CONCLUSIONS: The results demonstrate the importance of routine screenings of psychological symptons in burned patients. It is also necessary to promote specific psychopharmacological measures and specific psychotherapeutic interventions in this population in order to improve their medical and psychiatric prognosis and their quality of life a long-term basis.

[The evolution over time (1960-1990) of mortality and the ratio of males in bladder cancer in Spain]. / Evolución temporal (1960-1990) de la mortalidad y la razón de masculinidad en España del cáncer vesical.

A time series on the evolution of mortality and the masculinity attribute in vesical cancer in Spain (1960-1990) was prepared. The specific rates by age and sex by 100,000 inhabitants were calculated. To attain standardization the same study population was taken as the reference population. Overall mortality rates due to vesical cancer have increased progressively in a linearly ascending trend, though the slope in men is sharper that in women. The females cohort approaches that of males, but with a difference between them of 30 years. By age groups, the higher increase has occurred in those over 75-year old (113% men, 77% women). The masculinity attribute presents variations depending on the age group and decade considered (1.4-10.9). It is possible to establish three groups by masculinity attribute: 20-44 years (average 1.91), 45-69 years (average 7.8) and over 70 years (average 6.3).

Homologous bacterio-opsin-encoding gene expression via site-specific vector integration.

Homologous recombination in the archaebacterium Halobacterium halobium has been investigated and exploited for the wild-type (wt) level of expression of the bacterio-opsin-encoding gene (bop). The Haloferax volcanii-Escherichia coli shuttle vector, pWL102, was used to construct a shuttle-mutagenesis vector, pEF191, bearing bop and short flanking sequences. Transformation of a bacteriorhodopsin (BR)-negative H. halobium strain with pEF191 resulted in plasmid integration at the homologous bop locus. A model for this site-specific vector integration is presented which has been confirmed by determining the arrangement of the repeated homologous sequences on the chromosome. Two different configurations are obtained after integrative transformation due to the presence of an insertion element in the genomic copy of bop. In one configuration, the functional bop cluster containing the regulatory bat and brp genes was in wt arrangement. In the second configuration, the bop cluster is interrupted by 10 kb of plasmid vector sequences, and the upstream region required for bop expression was limited to 400 bp. The BR production for both configurations was determined and found to be at wt level. These results suggest that the function of the putative bop promoter does not depend on the defined upstream positions of bat and brp. The system presented here can be easily exploited for structure-function studies on BR and introduces homologous gene targeting as a powerful tool in the study of halobacterial genetics.

General mutagenesis/gene expression procedure for the construction of variant immunoglobulin domains in Escherichia coli. Production of the Bence-Jones protein REIv via fusion to beta-lactamase.

A novel mutagenesis/gene expression and protein purification scheme was established for ready construction and purification of variant immunoglobulin domains in Escherichia coli. This procedure, which has been applied to the production of the VK domain of the Bence-Jones protein REI and structural variants of it, rests on the synthesis of chimeric proteins with beta-lactamase as the amino-terminal fusion partner. The beta-lactamase not only guides the fusion protein to the periplasmic space, but also allows affinity chromatography on phenylboronate-Sepharose as an efficient and general purification procedure, independent of hypervariable loop structure. The REIv protein was released from the purified fusion protein by site-specific proteolytic cleavage. After a second passage through the same affinity column, up to 2 mg of pure REIv was obtained starting from one liter of bacterial liquid culture. A scheme of oligonucleotide-directed mutagenesis was introduced for replacement of DNA stretches encoding hypervariable loops. It exploits a colony color genetic screen and can be applied to any DNA sequence replacement. Mutations can be constructed by simple co-transformation with single-stranded template DNA and mutagenic oligonucleotide.

Primary structure of sensory rhodopsin I, a prokaryotic photoreceptor.

The gene coding for sensory rhodopsin I (SR-I) has been identified in a restriction fragment of genomic DNA from the Halobacterium halobium strain L33. Of the 1014 nucleotides whose sequence was determined, 720 belong to the structural gene of SR-I. In the 5' non-coding region two putative promoter elements and a ribosomal binding site have been identified. The 3' flanking region bears a potential terminator structure. The SR-I protein moiety carries no signal peptide and is not processed at its N terminus. The C terminus, however, lacks the last aspartic acid residue encoded by the gene. Analysis of the primary structure of SR-I reveals no consistent homology with the eukaryotic photoreceptor rhodopsin, but 14% homology with the halobacterial ion pumps, bacteriorhodopsin (BR) and halorhodopsin (HR). Residues conserved in all three proteins are discussed with respect to their contribution to secondary structure, retinal binding and ion translocation. The aspartic acid residue which mediates in BR the reprotonation of the Schiff base (D96) is replaced in SR-I by a tyrosine (Y87). This amino acid replacement is proposed to be of crucial importance in the evolution of the slow-cycling photosensing pigment SR-I.