Mycobacteria are major pathogens of man and animals. There are approximately 10 million cases of tuberculosis world wide with an annual mortality of three million people. Leprosy, caused by Mycobacterium leprae, afflicts over ten million people, primarily in developing countries. M. tuberculosis and mycobacteria of the M. avium-intracellulare-scrofulaceum (MAIS) group are major opportunistic pathogens of patients with acquired immune deficiency syndrome (AIDS). M. paratuberculosis is the cause of Jöhne's disease in cattle. Yet, BCG (Bacille Calmette-Guerin), an avirulent strain of M. bovis, is the most widely used human vaccine in the world, having been administered to about 2.5 X 10(9) people since 1948 (ref. 4). BCG was highly protective against tuberculosis in England, but has been found not to be effective in preventing pulmonary tuberculosis in adults in Southern India. We have initiated studies to develop the methodology for efficient gene transfer in mycobacteria. We have constructed recombinant shuttle phasmids which are chimaeras containing mycobacteriophage DNA into which an E. coli cosmid is inserted. They can replicate in E. coli as plasmids and in mycobacteria as phages, and transfer DNA across both genera. These shuttle vectors permit for the first time the introduction of foreign DNA by infection into M. smegmatis and BCG. By introducing and ultimately expressing genes for protective antigens for a variety of pathogens, it may be possible to develop cultivatable mycobacteria into useful multivaccine vehicles.

Nine carbapenem-resistant Escherichia coli isolates harboring Klebsiella pneumoniae carbapenemase (KPC)-2 or KPC-3 enzymes were identified in patients residing in 7 distinct long-term care facilities. Cefotaxime-hydrolyzing (CTX-M)-type beta-lactamases were also documented in 3 isolates. The identification of these enzymes in patients staying in long-term care facilities should be of great concern to all components of health care systems.

Tigecycline, a member of the glycylcycline class of antibiotics, was designed to maintain the antibacterial spectrum of the tetracyclines while overcoming the classic mechanisms of tetracycline resistance. The current study was designed to monitor the prevalence of the tet(A), tet(B), tet(C), tet(D), tet(E), and tet(M) resistance determinants in Escherichia coli isolates collected during the worldwide tigecycline phase 3 clinical trials. A subset of strains were also screened for the tet(G), tet(K), tet(L), and tet(Y) genes. Of the 1,680 E. coli clinical isolates screened for resistance to classical tetracyclines, 405 (24%) were minocycline resistant (MIC > or = 8 microg/ml) and 248 (15%) were tetracycline resistant (MIC > or = 8 microg/ml) but susceptible to minocycline (MIC < or = 4 microg/ml). A total of 452 tetracycline-resistant, nonduplicate isolates were positive by PCR for at least one of the six tetracycline resistance determinants examined. Over half of the isolates encoding a single determinant were positive for tet(A) (26%) or tet(B) (32%) with tet(C), tet(D), tet(E), and tet(M), collectively, found in 4% of isolates. Approximately 33% of the isolates were positive for more than one resistance determinant, with the tet(B) plus tet(E) combination the most highly represented, found in 11% of isolates. The susceptibilities of the tetracycline-resistant strains to tigecycline (MIC(90), 0.5 microg/ml), regardless of the encoded tet determinant(s), were comparable to the tigecycline susceptibility of tetracycline-susceptible strains (MIC(90), 0.5 microg/ml). The results provide a current (2002 to 2006) picture of the distribution of common tetracycline resistance determinants encoded in a globally sourced collection of clinical E. coli strains.

A novel class of tetracyclines, the glycylcyclines, have been shown to be active against bacterial strains harboring genes encoding tetracycline efflux pumps. However, two veterinary Salmonella isolates that carried tetracycline resistance determinants of the tetA(A) class were found to have reduced susceptibility to glycylcyclines, especially two early investigational glycylcyclines, DMG-MINO and DMG-DMDOT. These isolates were also quite resistant to tetracycline and minocycline. The isolates, one a strain of S. cholerasuis and the other, S. typhimurium, both carried the same novel tetA(A) variant, based on DNA sequencing, with one determinant plasmid encoded and the other located on the chromosome. This tetA(A) variant was cloned and shown to provide reduced susceptibility to the glycylcycline class although GAR-936, a glycylcycline currently in clinical development, was the least affected. The novel tetA(A) gene carries two mutations in the largest cytoplasmic loop of the efflux pump, which causes a double frameshift in codons 201, 202, and 203. This "interdomain region" of the efflux pump has generally been regarded as having no functional role in the efflux of tetracycline but the double frameshift is most likely responsible for the enhanced resistance observed and points to an interaction that was previously unrecognized. Mutants of the tetA(B) class with decreased susceptibility to the glycylcyclines were also generated in vitro. These all carried mutations in the portion of the tetA(B) gene encoding a transmembrane spanning region of the efflux pump. The laboratory-generated mutants point to the tight constraints in substrate recognition of the transmembrane-spanning region and may suggest that it will be the interdomain region of the pump that is likely to be the locus of future glycylcycline resistance mutations as these compounds enter clinical use.

In concert with the development of novel ␤-lactams and broad-spectrum cephalosporins, bacterially encoded ␤-lactamases have evolved to accommodate the new agents. This study was designed to identify, at the sequence level, the genes responsible for the extended-spectrum-␤-lactamase (ESBL) phenotypes of Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates collected during the global tigecycline phase 3 clinical trials. PCR assays were developed to identify and clone the bla TEM , bla SHV , bla OXA , and bla CTX genes from clinical strains. Isolates were also screened for AmpC genes of the bla CMY , bla ACT , bla FOX , and bla DHA families as well as the bla KPC genes encoding class A carbapenemases. E. coli, K. pneumoniae, and P. mirabilis isolates with ceftazidime MICs of >2 g/ml were designated possible ESBL-producing pathogens and were then subjected to a confirmatory test for ESBLs by use of Etest. Of 272 unique patient isolates, 239 were confirmed by PCR and sequencing to carry the genes for at least one ESBL, with 44% of the positive isolates harboring the genes for multiple ESBLs. In agreement with current trends for ESBL distribution, bla CTX-M -type ␤-lactamase genes were found in 83% and 71% of the ESBL-positive E. coli and K. pneumoniae isolates, respectively, whereas bla SHV genes were found in 41% and 28% of the ESBL-positive K. pneumoniae and E. coli isolates, respectively. Ninety-seven percent of the E. coli and K. pneumoniae isolates were tigecycline susceptible (MIC 90 ‫؍‬ 2 g/ml), warranting further studies to define the therapeutic utility of tigecycline against strains producing ESBLs in a clinical setting.