Azithromycin 993

Clinical isolates of Klebsiella pneumoniae resistant to carbapenems and essentially all other antibiotics (multidrug resistant) are being isolated from some hospitals in New York City with increasing frequency. A highly related pair of K. pneumoniae strains isolated on the same day from one patient in a hospital in New York City were studied for antibiotic resistance. One (KP-2) was resistant to imipenem, meropenem, and sulopenem (MICs of 16 to 32 g/ml) while the other (KP-1) was susceptible (MIC of 0.5 g/ml); both contained the bla ACT-1 , bla SHV-1 , and bla TEM-1 ␤-lactamases. bla ACT-1 in both strains was encoded on a large ϳ150-kb plasmid. Both isolates contained an identical class 1 integron encoding resistance to aminoglycosides and chloramphenicol. They each had identical insertions in ompK35 and ompK36, resulting in disruption of these key porin genes. The carbapenem-resistant and -susceptible isolates were extensively studied for differences in the structural and regulatory genes for the operons acrRAB, marORAB, romA-ramA, soxRS, micF, micC, phoE, phoBR, rpoS, and hfq. No changes were detected between the isolates except for a significant down-regulation of ompK37, phoB, and phoE in KP-2 as deduced from reverse transcription-PCR analysis of mRNA and polyacrylamide gel electrophoresis separation of outer membrane proteins. Backcross analysis was conducted using the wild-type phoE gene cloned into the vector pGEM under regulation of its native promoter as well as the lacZ promoter following transformation into the resistant KP-2 isolate. The wild-type gene reversed carbapenem resistance only when under control of the heterologous lacZ promoter. In the background of ompK35-ompK36 gene disruption, the up-regulation of phoE in KP-1 apparently compensated for porin loss and conferred carbapenem susceptibility. Down-regulation of phoE in KP-2 may represent the normal state of this gene, or it may have been selected from KP-1 in vivo under antibiotic pressure, generating the carbapenemresistant clone. This is the first study in the Enterobacteriaceae where expression of the phosphate-regulated PhoE porin has been associated with resistance to antimicrobials. Our results with this pair of Klebsiella clinical isolates highlight the complex and evolving nature of multiple drug resistance in this species.

Amid the recent attention justly focused on the potential problem of microbial sources for weapons of bioterrorism, it is also apparent that human pathogens frequently isolated from infections in patients from community and hospital sources have been growing more resistant to commonly used antibiotics. Much of the growth of multiple-drug-resistant (MDR) bacterial pathogens can be contributed to the overuse of broad-spectrum antimicrobial products. However, an equally troubling and often overlooked component of the problem involves the elegant ways in which pathogenic bacteria continually evolve complex genetic systems for acquiring and regulating an endless array of antibiotic-resistance mechanisms. Efforts to develop new antimicrobials have over the past two decades been woefully behind the rapid evolution of resistance genes developing among both gram-positive and gram-negative pathogens. Several new agents that are best suited for use in the hospital environment have been developed to combat staphylococci resistant to beta-lactam antimicrobials following acquisition of the mecA gene. However, the dramatic spread in the US of the now common community strain of Staphylococcus aureus USA300 has shifted the therapeutic need for new antibiotics useful against MRSA to the community. As the pharmaceutical industry focused on discovering new agents for use against MRSA, hospitals in many parts of the world have seen the emergence of gram-negative pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae that are clinically resistant to almost all available antimicrobials. Such MDR isolates usually contain multiple-resistance determinants, including loss of outer membrane porins via gene inactivation by chromosomally encoded insertion sequences, up-regulation of inate efflux pumps, as well as acquisition of drug-inactivating enzymes whose genes are encoded on self-transmissible plasmids, integrons, and complex transposable elements. These determinants confer a complex resistance phenotype that is often superimposed on mutations in the primary drug target in the cell. The continued evolution of such a complex array of antibiotic-resistance genes presents a formidable challenge at a time when large pharmaceutical companies have scaled down their presence in the anti-infectives arena.

Carbapenem-resistant Klebsiella strains carrying Klebsiella pneumoniae carbapenemases (KPC) are endemic to New York City and are spreading across the United States and internationally. Recent studies have indicated that the KPC structural gene is located on a 10-kb plasmid-borne element designated Tn4401. Fourteen Klebsiella pneumoniae strains and one Klebsiella oxytoca strain isolated at a New York City hospital in 2005 carrying either bla KPC-2 or bla KPC-3 were examined for isoforms of Tn4401. Ten of the Klebsiella strains contained a 100-bp deletion in Tn4401, corresponding to the Tn4401a isoform. The presence of this deletion adjacent to the upstream promoter region of bla KPC in Tn4401a resulted in a different ؊35 promoter sequence of TGGAGA than that of CTGATT present in isoform Tn4401b. Complete sequencing of one plasmid carrying bla KPC from each of three nonclonal isolates indicated the presence of genes encoding other types of antibiotic resistance determinants. The 70.6-kb plasmid from K. pneumoniae strain S9 carrying bla KPC-2 revealed two identical copies of Tn4401b inserted in an inverse fashion, but in this case, one of the elements disrupted a group II self-splicing intron. In K. pneumoniae strain S15, the Tn4401a element carrying bla KPC-2 was found on both a large 120-kb plasmid and a smaller 24-kb plasmid. Pulsed-field gel electrophoresis results indicate that the isolates studied represent a heterogeneous group composed of unrelated as well as closely related Klebsiella strains. Our results suggest that endemic KPC-positive Klebsiella strains constitute a generally nonclonal population comprised of various alleles of bla KPC on several distinct plasmid genetic backgrounds. This study increases our understanding of the genetic composition of the evolving and expanding role of KPC-producing, healthcare-associated, gram-negative pathogens.

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