Efficient in vivo antitumor effect of an immunotoxin based on ribotoxin α-sarcin in nude mice bearing human colorectal cancer xenografts

Tagging of RNases, such as the ribotoxin α-sarcin, with the variable domains of antibodies directed to surface antigens that are selectively expressed on tumor cells endows cellular specificity to their cytotoxic action. A recombinant single-chain immunotoxin based on the ribotoxin α-sarcin (IMTXA33αS), produced in the generally regarded as safe (GRAS) yeast Pichia pastoris, has been recently described as a promising candidate for the treatment of colorectal cancer cells expressing the glycoprotein A33 (GPA33) antigen, due to its high specific and effective cytotoxic effect on in vitro assays against targeted cells. Here we report the in vivo antitumor effectiveness of this immunotoxin on nude mice bearing GPA33-positive human colon cancer xenografts. Two sets of independent assays were performed, including three experimental groups: control (PBS) and treatment with two different doses of immunotoxin (50 or 100 μg/ injection) (n = 8). Intraperitoneal administration of IMTXA33αS resulted in significant dose-dependent tumor growth inhibition. In addition, the remaining tumors excised from immunotoxin-treated mice showed absence of the GPA33 antigen and a clear inhibition of angiogenesis and proliferative capacity. No signs of immunotoxin-induced pathological changes were observed from specimens tissues.Overall these results show efficient and selective cytotoxic action on tumor xenografts, combined with the lack of severe side effects, suggesting that IMTXA33αS is a potential therapeutic agent against colorectal cancer.

Klebsiella pneumoniae sequence type 11 from companion animals bearing ArmA methyltransferase, DHA-1 β-lactamase, and QnrB4

Seven Klebsiella pneumoniae isolates from dogs and cats in Spain were found to be highly resistant to aminoglycosides, and ArmA methyltransferase was responsible for this phenotype. All isolates were typed by multilocus sequence typing (MLST) as ST11, a human epidemic clone reported worldwide and associated with, among others, OXA-48 and NDM carbapenemases. In the seven strains, armA was borne by an IncR plasmid, pB1025, of 50 kb. The isolates were found to coproduce DHA-1 and SHV-11 β-lactamases, as well as the QnrB4 resistance determinant. This first report of the ArmA methyltransferase in pets illustrates their importance as a reservoir for human multidrug-resistant K. pneumoniae.

Molecular organization of small plasmids bearing blaTEM-1 and conferring resistance to β-lactams in Haemophilus influenzae

TEM-1 is the dominant β-lactamase of Haemophilus influenzae and can be located on small plasmids. Three distinct plasmids with sizes from 4,304 to 5,646 nucleotides (nt) were characterized: pA1606, pA1209, and pPN223. In addition to TEM-1 and a replication enzyme of the Rep 3 superfamily, pA1606 carries a Tn3 resolvase gene and pA1606 and pA1209 carry an open reading frame (ORF) similar to a plasmid recombination enzyme gene described in Gram-positive bacteria. The plasmids transformed strain Rd to the ampicillin-resistant phenotype.

Haemophilus influenzae clinical isolates with plasmid pB1000 bearing blaROB-1: fitness cost and interspecies dissemination

Plasmid pB1000 is a mobilizable replicon bearing the bla(ROB-1) beta-lactamase gene that we have recently described in Haemophilus parasuis and Pasteurella multocida animal isolates. Here we report the presence of pB1000 and a derivative plasmid, pB1000', in four Haemophilus influenzae clinical isolates of human origin. Pulsed-field gel electrophoresis showed unrelated patterns in all strains, indicating that the existence of pB1000 in H. influenzae isolates is not the consequence of clonal dissemination. The replicon can be transferred both by transformation and by conjugation into H. influenzae, giving rise to recipients resistant to ampicillin and cefaclor (MICs, > or =64 microg/ml). Stability experiments showed that pB1000 is stable in H. influenzae without antimicrobial pressure for at least 60 generations. Competition experiments between isogenic H. influenzae strains with and without pB1000 revealed a competitive disadvantage of 9% per 10 generations for the transformant versus the recipient. The complete nucleotide sequences of nine pB1000 plasmids from human and animal isolates, as well as the epidemiological data, suggest that animal isolates belonging to the Pasteurellaceae act as an antimicrobial resistance reservoir for H. influenzae. Further, since P. multocida is the only member of this family that can colonize both humans and animals, we propose that P. multocida is the vehicle for the transport of pB1000 between animal- and human-adapted members of the Pasteurellaceae.

Novel blaROB-1-bearing plasmid conferring resistance to β-lactams in Haemophilus parasuis isolates from healthy weaning pigs

Haemophilus parasuis, the causative agent of Glässer's disease, is one of the early colonizers of the nasal mucosa of piglets. It is prevalent in swine herds, and lesions associated with disease are fibrinous polyserositis and bronchopneumonia. Antibiotics are commonly used in disease control, and resistance to several antibiotics has been described in H. parasuis. Prediction of H. parasuis virulence is currently limited by our scarce understanding of its pathogenicity. Some genes have been associated with H. parasuis virulence, such as lsgB and group 1 vtaA, while biofilm growth has been associated with nonvirulent strains. In this study, 86 H. parasuis nasal isolates from farms that had not had a case of disease for more than 10 years were obtained by sampling piglets at weaning. Isolates were studied by enterobacterial repetitive intergenic consensus PCR and determination of the presence of lsgB and group 1 vtaA, biofilm formation, inflammatory cell response, and resistance to antibiotics. As part of the diversity encountered, a novel 2,661-bp plasmid, named pJMA-1, bearing the blaROB-1 β-lactamase was detected in eight colonizing strains. pJMA-1 was shown to share a backbone with other small plasmids described in the Pasteurellaceae, to be 100% stable, and to have a lower biological cost than the previously described plasmid pB1000. pJMA-1 was also found in nine H. parasuis nasal strains from a separate collection, but it was not detected in isolates from the lesions of animals with Glässer's disease or in nontypeable Haemophilus influenzae isolates. Altogether, we show that commensal H. parasuis isolates represent a reservoir of β-lactam resistance genes which can be transferred to pathogens or other bacteria.