Nineteen isolates of carbapenem-resistant Klebsiella species were recovered from 7 hospitals in New York City. Most K. pneumoniae belonged to a single ribotype. Nucleotide sequencing identified KPC-2, a carbapenem-hydrolyzing beta -lactamase. In 3 strains, TEM-30, an inhibitor-resistant beta -lactamase, was detected. Carbapenem-resistant Klebsiella species possessing KPC-2 are endemic in New York City. This study documents the identification of an inhibitor-resistant TEM beta -lactamase in the United States.

Pseudomonas aeruginosa strains are less susceptible to tigecycline (previously GAR-936; MIC, 8 g/ml) than many other bacteria (P. J. Petersen, N. V. Jacobus, W. J. Weiss, P. E. Sum, and R. T. Testa, Antimicrob. Agents Chemother. 43:738-744, 1999). To elucidate the mechanism of resistance to tigecycline, P. aeruginosa PAO1 strains defective in the MexAB-OprM and/or MexXY (OprM) efflux pumps were tested for susceptibility to tigecycline. Increased susceptibility to tigecycline (MIC, 0.5 to 1 g/ml) was specifically associated with loss of MexXY. Transcription of mexX and mexY was also responsive to exposure of cells to tigecycline. To test for the emergence of compensatory efflux pumps in the absence of MexXY-OprM, mutants lacking MexXY-OprM were plated on medium containing tigecycline at 4 or 6 g/ml. Resistant mutants were readily recovered, and these also had decreased susceptibility to several other antibiotics, suggesting efflux pump recruitment. One representative carbenicillin-resistant strain overexpressed OprM, the outer membrane channel component of the MexAB-OprM efflux pump. The mexAB-oprM repressor gene, mexR, from this strain contained a 15-bp in-frame deletion. Two representative chloramphenicol-resistant strains showed expression of an outer membrane protein slightly larger than OprM. The mexCD-OprJ repressor gene, nfxB, from these mutants contained a 327-bp in-frame deletion and an IS element insertion, respectively. Together, these data indicated drug efflux mediated by MexCD-OprJ. The MICs of the narrower-spectrum semisynthetic tetracyclines doxycycline and minocycline increased more substantially than did those of tigecycline and other glycylcyclines against the MexAB-OprM-and MexCD-OprJ-overexpressing mutant strains. This suggests that glycylcyclines, although they are subject to efflux from P. aeruginosa, are generally inferior substrates for P. aeruginosa efflux pumps than are narrower-spectrum tetracyclines.Pseudomonas aeruginosa is a clinically important gram-negative opportunistic pathogen causing serious acute and chronic infections (8, 11). The exceptional array of intrinsic and acquired drug resistance mechanisms employed by P. aeruginosa renders antibiotic treatment of these infections problematic. One important resistance mechanism is efflux mediated by the so-called resistance nodulation division (RND) family of efflux pumps. Four RND pumps have been described in P. aeruginosa: MexAB-OprM (16), MexCD-OprJ (31), , and 25,40). RND pumps consist of an inner membrane transporter (MexB, MexD, MexF, and MexY), an outer membrane channel-forming component (OprM, OprJ, and OprN), and a membrane fusion protein (MexA, MexC, MexE, and MexX) (27). RND pumps show broad specificity, and their tripartite architecture allows extrusion of compounds directly from the cytoplasm to the external environment. Efflux pump action in P. aeruginosa leads to particularly high levels of drug resistance as a result of apparent synergism with the atypically impermeable outer membrane, which limits influx of antimic...

Tigecycline is an expanded broad-spectrum antibacterial agent that is active against many clinically relevant species of bacterial pathogens, including Klebsiella pneumoniae. The majority of K. pneumoniae isolates are fully susceptible to tigecycline; however, a few strains that have decreased susceptibility have been isolated. One isolate, G340 (for which the tigecycline MIC is 4 g/ml and which displays a multidrug resistance [MDR] phenotype), was selected for analysis of the mechanism for this decreased susceptibility by use of transposon mutagenesis with IS903kan. A tigecycline-susceptible mutant of G340, GC7535, was obtained (tigecycline MIC, 0.25 g/ml). Analysis of the transposon insertion mapped it to ramA, a gene that was previously identified to be involved in MDR in K. pneumoniae. For GC7535, the disruption of ramA led to a 16-fold decrease in the MIC of tigecycline and also a suppression of MDR. Trans-complementation with plasmid-borne ramA restored the original parental phenotype of decreased susceptibility to tigecycline. Northern blot analysis revealed a constitutive overexpression of ramA that correlated with an increased expression of the AcrAB transporter in G340 compared to that in tigecycline-susceptible strains. Laboratory mutants of K. pneumoniae with decreased susceptibility to tigecycline could be selected at a frequency of approximately 4 ؋ 10 ؊8 . These results suggest that ramA is associated with decreased tigecycline susceptibility in K. pneumoniae due to its role in the expression of the AcrAB multidrug efflux pump.Tigecycline is an expanded broad-spectrum antibiotic representing a new class called the glycylcyclines. The glycylcyclines are semisynthetic derivatives of minocycline and have activity against many bacterial pathogens (2, 14, 15). It has been noted that a few species of gram-negative bacteria, including Pseudomonas aeruginosa, Proteus spp., Providencia spp., and Morganella morganii, are intrinsically less susceptible to tigecycline. Previous studies revealed the involvement of multidrug efflux systems such as MexXY and AcrAB in the decreased tigecycline susceptibility of P. aeruginosa and Proteus mirabilis, respectively (3, 22). These pumps belong to the resistance-nodulation-division (RND) family that combines bacterial transporters with a tripartite architecture and broad substrate specificity (9, 12). Due to the broad substrate specificity of RND pumps, their overexpression usually results in the multidrug resistance (MDR) phenotype.Klebsiella pneumoniae causes infections of wounds, the urinary tract, and the respiratory system. This bacterial species is generally susceptible to tigecycline; however, a few clinical strains with decreased tigecycline susceptibility have been isolated. In this study, one such an isolate, G340, was investigated to determine the mechanism of decreased tigecycline susceptibility in K. pneumoniae.( MATERIALS AND METHODSBacterial strains and growth conditions. The bacterial strains and plasmids used in this study are shown in Table 1. Strains...

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Tigecycline has good broad-spectrum activity against many gram-positive and gram-negative pathogens with the notable exception of the Proteeae. A study was performed to identify the mechanism responsible for the reduced susceptibility to tigecycline in Proteus mirabilis. Two independent transposon insertion mutants of P. mirabilis that had 16-fold-increased susceptibility to tigecycline were mapped to the acrB gene homolog of the Escherichia coli AcrRAB efflux system. Wild-type levels of decreased susceptibility to tigecycline were restored to the insertion mutants by complementation with a clone containing a PCR-derived fragment from the parental wild-type acrRAB efflux gene cluster. The AcrAB transport system appears to be associated with the intrinsic reduced susceptibility to tigecycline in P. mirabilis.The growing threat of acquired resistance in the Enterobacteriaceae (6,10,20) indicates the crucial need for new antibiotics for continued effective treatment of bacterial infection. Tigecycline, the 9-t-butylglycylamido derivative of minocycline, is a new antimicrobial agent belonging to a novel class of tetracyclines, the glycylcyclines (24). It has good activity against gram-positive pathogens, including penicillin-resistant Streptococcus pneumoniae, vancomycin-resistant enterococci, and methicillin-resistant Staphylococcus aureus (2,5,18). Tigecycline has good activity against most gram-negative pathogens, including Klebsiella pneumoniae and Escherichia coli (4,18). Tigecycline also has good activity against organisms with a resistance determinant from the major facilitator family, including E. coli with tet(A), tet (B) , tet(C), tet(D), and tet(M); S. aureus with tet(K) and tet(M); and Enterococcus faecalis with tet(M) (18).Proteus mirabilis is a notable exception to the activity of tigecycline, which routinely shows MICs of 4 g/ml for the organism in tests.It is important to identify current and emerging resistance mechanisms. Identification and mechanistic studies of bacterial resistance mechanisms can help to further reduce health care costs due to bacterial infection. Therefore, a study was performed to identify the mechanism responsible for the reduced susceptibility of P. mirabilis to tigecycline (M. A. Visalli, E. Murphy, S. J. Projan, and P. A. Bradford, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. C1-2019, 2001). MATERIALS AND METHODSBacterial strains and growth conditions. The bacterial strains, plasmids, and transposons used in this study are listed in Table 1. A typical clinical isolate of P. mirablis, G151, was selected from a tigecycline phase II clinical trial. The E. coli strains used included Top10 (Invitrogen, Carlsbad, Calif.), Transformax EC100D pir-116 (Epicentre, Madison, Wis.), DM1 (Gibco Life Technologies, Rockville, Md.), and two laboratory strains, one wild type and the other an acrAB deletion mutant (17). The strains were grown in Luria-Bertani (LB) broth or agar in the presence of the following selective antibiotics when required: 50 g of kanamycin/ml, 50...

Susceptibility of 230 penicillin- and erythromycin-susceptible and -resistant pneumococci to HMR 3647 (RU 66647), a new ketolide, was tested by agar dilution, and results were compared with those of erythromycin, azithromycin, clarithromycin, roxithromycin, rokitamycin, clindamycin, pristinamycin, ciprofloxacin, sparfloxacin, trimethoprim-sulfamethoxazole, doxycycline, chloramphenicol, cefuroxime, ceftriaxone, imipenem, and vancomycin. HMR 3647 was very active against all strains tested, with MICs at which 90% of the strains were inhibited (MIC90s) of 0.03 μg/ml for erythromycin-susceptible strains (MICs, ≤0.25 μg/ml) and 0.25 μg/ml for erythromycin-resistant strains (MICs, ≥1.0 μg/ml). All other macrolides yielded MIC90s of 0.03 to 0.25 and >64.0 μg/ml for erythromycin-susceptible and -resistant strains, respectively. The MICs of clindamycin for 51 of 100 (51%) erythromycin-resistant strains were ≤0.125 μg/ml. The MICs of pristinamycin for all strains were ≤1.0 μg/ml. The MIC90s of ciprofloxacin and sparfloxacin were 4.0 and 0.5 μg/ml, respectively, and were unaffected by penicillin or erythromycin susceptibility. Vancomycin and imipenem inhibited all strains at ≤1.0 μg/ml. The MICs of cefuroxime and cefotaxime rose with those of penicillin G. The MICs of trimethoprim-sulfamethoxazole, doxycycline, and chloramphenicol were variable but were generally higher in penicillin- and erythromycin-resistant strains. HMR 3647 had the best kill kinetics of all macrolides tested against 11 erythromycin-susceptible and -resistant strains, with uniform bactericidal activity (99.9% killing) after 24 h at two times the MIC and 99% killing of all strains at two times the MIC after 12 h for all strains. Pristinamycin showed more rapid killing at 2 to 6 h, with 99.9% killing of 10 of 11 strains after 24 h at two times the MIC. Other macrolides showed significant activity, relative to the MIC, against erythromycin-susceptible strains only.

Multidrug-resistant strains of Klebsiella pneumoniae are a problem in many hospitals. In 1999, the molecular epidemiology of K. pneumoniae with extended-spectrum beta-lactamases (ESBLs) was studied at 15 hospitals in Brooklyn. Of 824 unique patient isolates, 34% were presumptive ESBL producers. Of this subset, 34% were susceptible to cefoxitin, 42% to ciprofloxacin, 48% to ceftriaxone, 55% to piperacillin-tazobactam, 57% to amikacin, and 86% to cefepime. Ribotype analysis revealed 87 unique types. However, 2 clusters accounted for 35% of isolates and were present in most of the hospitals. One cluster was significantly more resistant to most antibiotics. Although there was a predominance of SHV-5, considerable heterogeneity of beta-lactamases was evident, even among isolates of the same cluster. A correlation was found between the use of cephalosporins and the prevalence of ESBL-producing strains of K. pneumoniae at each hospital. Our data suggest that there is an advanced outbreak of multidrug-resistant K. pneumonia infection that is affecting all Brooklyn hospitals.

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