Stability, Entrapment and Variant Formation of Salmonella Genomic Island 1

<div><h3>Background</h3><p>The <em>Salmonella</em> genomic island 1 (SGI1) is a 42.4 kb integrative mobilizable element containing several antibiotic resistance determinants embedded in a complex integron segment In104. The numerous SGI1 variants identified so far, differ mainly in this segment and the explanations of their emergence were mostly based on comparative structure analyses. Here we provide experimental studies on the stability, entrapment and variant formation of this peculiar gene cluster originally found in <em>S</em>. Typhimurium.</p> <h3>Methodology/Principal Findings</h3><p>Segregation and conjugation tests and various molecular techniques were used to detect the emerging SGI1 variants in <em>Salmonella</em> populations of 17 <em>Salmonella enterica</em> serovar Typhimurium DT104 isolates from Hungary. The SGI1s in these isolates proved to be fully competent in excision, conjugal transfer by the IncA/C helper plasmid R55, and integration into the <em>E. coli</em> chromosome. A trap vector has been constructed and successfully applied to capture the island on a plasmid. Monitoring of segregation of SGI1 indicated high stability of the island. SGI1-free segregants did not accumulate during long-term propagation, but several SGI1 variants could be obtained. Most of them appeared to be identical to SGI1-B and SGI1-C, but two new variants caused by deletions via a short-homology-dependent recombination process have also been detected. We have also noticed that the presence of the conjugation helper plasmid increased the formation of these deletion variants considerably.</p> <h3>Conclusions/Significance</h3><p>Despite that excision of SGI1 from the chromosome was proven in SGI1⁺<em>Salmonella</em> populations, its complete loss could not be observed. On the other hand, we demonstrated that several variants, among them two newly identified ones, arose with detectable frequencies in these populations in a short timescale and their formation was promoted by the helper plasmid. This reflects that IncA/C helper plasmids are not only involved in the horizontal spreading of SGI1, but may also contribute to its evolution.</p> </div

Presumed pseudobacteremia outbreak resulting from contamination of proportional disinfectant dispenser

International audienceReported 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

Molecular identification of blaSHV, blaLEN and blaOKP β-lactamase genes in Klebsiella pneumoniae by bi-directional sequencing of universal SP6- and T7-sequence-tagged blaSHV-PCR amplicons

Plasmid encoded blaSHV enzymes represent an important sub-group of class A β-lactamases causing an ESBL-phenotype which is increasingly found in Enterobacteriaceae including Klebsiella pneumoniae. The chromosomally encoded β-lactamase blaLEN and blaOKP enzymes, which so far only have been found in K. pneumoniae, do not hydrolyse extended-spectrum cephalosporins. In the present study, multiple displacement amplified DNA derived from 20 K. pneumoniae clinical isolates with a blaSHV-like genotype was used in a universal SHV PCR assay using SP6- (forward) and T7- (reverse) sequence-tagged primers. Identification and differentiation of blaSHV, blaLEN and blaOKP genes was obtained by bi-directional amplicon sequencing using SP6- and T7-specific primers. Three well characterised K. pneumoniae strains having a SHV-genotype were included in the study. The bi-directional amplicon sequencing, covering 800 bp (93%) of the blaSHV, blaLEN and blaOKP enzyme encoding sequences, allowed for an unequivocal discrimination of SHV, LEN and OKP genes. Moreover, sequencing revealed the presence of blaSHV allelic variants in six K. pneumoniae isolates in which the amplicons had to be cloned accordingly. Based on deduced amino-acid sequences, a dendrogram was constructed. Seventeen out of 20 K. pneumoniae isolates with an ESBL-phenotype formed a SHV-like cluster, two were LEN-like, and one isolate was OKP-like. The PCR-based molecular typing method described here enables a rapid, reliable and cost-effective identification and differentiation of blaSHV, blaOKP and blaLEN genes.Original Publication: Maria Tärnberg, Lennart E. Nilsson and Hans-Jürg Monstein, Molecular identification of blaSHV, blaLEN and blaOKP β-lactamase genes in Klebsiella pneumoniae by bi-directional sequencing of universal SP6- and T7-sequence-tagged blaSHV-PCR amplicons, 2009, Molecular and Cellular Probes, (23), 3-4, 195-200. http://dx.doi.org/10.1016/j.mcp.2009.01.005 Copyright: Elsevier Science B.V., Amsterdam http://www.elsevier.com/</p