Use of sequence-based typing and multiplex PCR to identify clonal lineages of outbreak strains of Acinetobacter baumannii

ABSTRACTRepresentatives (n = 31) of outbreak strains of Acinetobacter baumannii from five countries fell into three clear groups, designated Groups 1–3, based on their ompA (outer-membrane protein A), csuE (part of a pilus assembly system required for biofilm formation) and blaOXA-51-like (the intrinsic carbapenemase gene in A. baumannii) gene sequences. With the exception of the closely related alleles within the Group 1 clonal complex, alleles at each locus were highly distinct from each other, with a minimum of 14 nucleotide differences between any two alleles. Isolates within a group shared the same combination of alleles at the three loci, providing compelling evidence that the outbreak strains investigated belonged to three clonal lineages. These corresponded to the previously identified European clones I–III. Sequence differences among the alleles were used to design multiplex PCRs to rapidly assign isolates belonging to particular genotypes to sequence groups. In the UK, genotypes belonging to the Group 1 clonal complex have been particularly successful, accounting for the vast majority of isolates referred from hospitals experiencing problems with Acinetobacter

Intracellular trafficking and replication of Burkholderia cenocepacia in human cystic fibrosis airway epithelial cells

We investigated the trafficking of Burkholderia cenocepacia , an opportunistic respiratory pathogen of persons with cystic fibrosis (CF), in immortalized CF airway epithelial cells in vitro . Our results indicate that bacteria enter cells in a process involving actin rearrangement. Whereas both live and heat-killed bacteria reside transiently in early endosomes, only live bacteria escape from late endosomes to colocalize in vesicles positive for lysosomal membrane marker LAMP1, endoplasmic reticulum (ER) membrane marker calnexin, and autophagosome marker monodansylcadavarine (MDC). Twenty-four hours after infection, microcolonies of live bacteria were observed in the perinuclear area colocalizing with calnexin. In contrast, after ingestion, dead bacteria colocalized with late endosome marker Rab7, and lysosome markers LAMP1 and cathepsin D, but not with calnexin or MDC. Six to eight hours after ingestion of dead bacteria, degraded bacterial particles were observed in the cytoplasm and in vesicles positive for cathepsin D. These results indicate that live B. cenocepacia gain entry into human CF airway cells by endocytosis, escape from late endosomes to enter autophagosomes that fail to fuse with mature lysosomes, and undergo replication in the ER. This survival and replication strategy may contribute to the capacity of B. cenocepacia to persist in the lungs of infected CF patients.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75744/1/j.1462-5822.2006.00724.x.pd

Pseudomonas aeruginosa displays an epidemic population structure.

peer reviewedBacteria can have population structures ranging from the fully sexual to the highly clonal. Despite numerous studies, the population structure of Pseudomonas aeruginosa is still somewhat contentious. We used a polyphasic approach in order to shed new light on this issue. A data set consisting of three outer membrane (lipo)protein gene sequences (oprI, oprL and oprD), a DNA-based fingerprint (amplified fragment length polymorphism), serotype and pyoverdine type of 73 P. aeruginosa clinical and environmental isolates, collected across the world, was analysed using biological data analysis software. We observed a clear mosaicism in the results, non-congruence between results of different typing methods and a microscale mosaic structure in the oprD gene. Hence, in this network, we also observed some clonal complexes characterized by an almost identical data set. The most recent clones exhibited serotypes O1, 6, 11 and 12. No obvious correlation was observed between these dominant clones and habitat or, with the exception of some recent clones, geographical origin. Our results are consistent with, and even clarify, some seemingly contradictory results in earlier epidemiological studies. Therefore, we suggest an epidemic population structure for P. aeruginosa, comparable with that of Neisseria meningitidis, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise

Nitrates and Prussic Acid in Forages: Sampling, Testing and Management Strategies

When nitrates and prussic acid accumulate in forage, the feed may not be safe for livestock consumption

Resistance mechanisms of multiresistant serotype 012 pseudomonas aeruginosa isolated in europe

Serotype 012 Pseudomonas aeruginosa resistant to gentamicin (MIC > 4mg/l) and carbenicillin (MIC > 128 mg/l) occur widely in Europe and are homogeneous in their phenotypic and genetic properties. It has been suggested that a single multiresistant strain of this serotype has become widespread. This study examined the resistance mechanisms present in multircsistant serotype 012 P. aeruginosa isolates from Austria, Belgium, France, Greece, Italy, Holland, West Germany and the UK. Disseminated isolates produced a PSE-1 type β-lactamase, correlating with their resistance to the known substrates of this enzyme (carbenicillin, azlocillin and cefsulodin). These isolates also reacted with gene probes for the aminoglycoside modifying enzymes AAC(6′)I and ANT(3′). The probe for AAC(6′)I is known to cross-react with the gene for AAC(6′)II. The fact that the organisms were resistant to netilmicin, gentamicin, sisomicin and tobramycin, but less so to amikacin, suggested that the latter enzyme was produced rather than AAC(6′)I. PSE-1 β-lactamase and the gene for AAC(6′)I/AAC(6′)II were absent from the International Antigenic Typing Scheme 012 reference strain, which was sensitive to β-lactams and aminoglycosides, and also from a multiresistant serotype 012 strain isolated at a London burns unit in 1987. These organisms have been shown previously to be distinct from the disseminated multiresistant strain in their phenotypic properties. Two 012 isolates appeared to have additional resistance determinants to amikacin and isepamicin. © 1990 by The British Society for Antimicrobial Chemotherapy

Relationships among Pseudomonas pseudomallei isolates from patients with recurrent melioidosis

Patients with melioidosis may present with recurrent infections after clinical resolution of their primary illness. Because there has been no satisfactory typing scheme for Pseudomonas pseudomallei, recrudescence could not be distinguished from reinfection. We determined the strain identity of primary and relapse isolates of P. pseudomallei from 25 patients with culture-proven melioidosis to answer whether secondary infections were due to the initial infecting strain or to the acquisition of a new strain. Fifty- four isolates were compared by the patterns of BamHI restriction digests produced after hybridization with a cDNA copy of Escherichia coli rRNA. Twenty-three patients had primary and relapse isolates with identical or highly similar ribotype patterns. The patterns of isolates from two patients were different; the primary and relapse isolates differed by a single fragment for one, and the other had identical primary and first-relapse isolates while the second-relapse isolate was markedly different. The results indicated that recurrent infection probably resulted from endogenous relapse in most of the melioidosis patients studied, although reinfection from an exogenous source was also possible in two cases

Prevalence of a plasmid-mediated type II dihydrofolate reductase gene among trimethoprim-resistant urinary pathogens in greek hospitals

The genetic basis of trimethoprim resistance was examined in 24 Klebsiella pneumoniae, 27 Enterobacter cloacae, five Enterobacter aerogenes and nine Serratia marcescens urinary isolates from five hospitals in Greece. Analysis of the 65 isolates by serotyping and phage-typing identified 53 distinct strains. Thirty-eight isolates (15 K. pneumoniae, 19 E. cloacae, two E. aerogenes and two S. marcescens) hybridized with a probe specific for a gene encoding type II dihydrofolate reductase (DHFR). Three of the K. pneumoniae and four of the E. cloacae isolates which reacted with this probe also hybridized with probes specific for type I DHFR and transposon Tn7. Two E. cloacae isolates hybridized only with the probe for type I DHFR, while a further three isolates hybridized only with the type I DHFR and Tn7 probes. None of the isolates hybridized with a probe for type V DHFR. The plasmids in transconjugants derived from 40 isolates were analysed by digestion with restriction enzymes and Southern blotting. Eighteen (45%) of the donors (12 K. pneumoniae and 6 E. cloacae) produced transconjugants containing plasmids of about 95 kb in size, while transconjugants from the other donors had plasmids in the range 100-185 kb. Of the 18 transconjugants containing a 95 kb plasmid, 15 had similar restriction endonuclease digest patterns, although they varied in terms of the range of antimicrobial resistances which they encoded. When EcoRI digests of these 15 plasmids were hybridized with the type II DHFR probe, a 23 kb common band reacted with the probe. These findings indicate that the high level of trimethoprim resistance in these genera resulted, in part, from the spread of related plasmids encoding production of type II DHFR. © 1992 by The British Society for Antimicrobial Chemotherapy