Resistance to Colistin Associated with a Single Amino Acid Change in Protein PmrB among Klebsiella pneumoniae Isolates of Worldwide Origin

A series of colistin-resistant Klebsiella pneumoniae isolates recovered from different countries was investigated in order to evaluate the involvement of the PmrA/PmrB two-component system in this resistance. Six isolates possessed a mutated PmrB protein, which is encoded by the pmrB gene, part of the pmrCAB operon involved in lipopolysaccharide modification. The same amino acid substitution (Thr157Pro) in PmrB was identified in the six isolates. The six isolates belonged to four distinct clonal groups, recovered in South Africa (sequence type 14 [ST14]), Turkey (ST101), and Colombia (ST258 and ST15). Three out of the four clones produced a carbapenemase, OXA-181, OXA-48, or KPC-3, while a single isolate did not produce any carbapenemase. Expression assays revealed an overexpression of the pmrA (70-fold), pmrB (70-fold), pmrC (170-fold), and pmrK (40-fold) genes in the pmrB-mutated isolate compared to expression of the pmrB wild-type isogenic K. pneumoniae isolate, confirming that the PmrB substitution was responsible for increased expression levels of those genes. Complementation assays leading to the expression of a wild-type PmrB protein restored the susceptibility to colistin in all isolates, confirming that the substitution in PmrB was responsible for the resistance phenotype. This study identified a key amino acid located in the PmrB protein as being responsible for the overexpression of pmrCAB and pmrHFIJKLM operons, leading to resistance to colistin.     

Acquisition of Broad-Spectrum Cephalosporin Resistance Leading to Colistin Resistance in Klebsiella pneumoniae

An extended-spectrum β-lactamase (ESBL)-producing and colistin-resistant Klebsiella pneumoniae clinical isolate was recovered from a patient who was treated with cefotaxime. This isolate harbored a bla_CTX-M-15 ESBL gene that was associated with an ISEcp1 insertion sequence. Transposition of that tandem occurred within the chromosomal mgrB gene, leading to inactivation of the mgrB gene and consequently to acquired resistance to colistin. We showed here a coselection of colistin resistance as a result of a broad-spectrum cephalosporin selective pressure.     

Synergistic Activity of Colistin plus Rifampin against Colistin-Resistant KPC-Producing Klebsiella pneumoniae

Infections caused by carbapenem-resistant KPC-producing Klebsiella pneumoniae are responsible for high rates of mortality and represent a major therapeutic challenge, especially when the isolates are also resistant to colistin. We used the checkerboard method to evaluate the synergistic activity of 10 antibiotic combinations against 13 colistin-resistant KPC-producing K. pneumoniae isolates (colistin MIC range of 8 to 128 mg/liter). Colistin plus rifampin was the only combination that demonstrated consistent synergistic bacteriostatic activity against 13/13 strains tested, reducing the colistin MIC below the susceptibility breakpoint (MIC ≤ 2 mg/liter) in 7/13 strains at rifampin concentrations ranging from 4 to 16 mg/liter. Bactericidal synergistic activity was also documented for 8/13 tested strains. Other antimicrobial combinations with carbapenems, gentamicin, and tigecycline showed variously synergistic results. Colistin plus rifampin also exhibited bacteriostatic synergistic activity against 4/4 colistin-susceptible KPC-producing K. pneumoniae isolates (colistin MIC range of 0.5 to 2 mg/liter) and 4/4 ertapenem-resistant extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae isolates (ertapenem MIC range of 16 to 32 mg/liter). Collectively, our data suggest that colistin plus rifampin is the most consistently synergistic combination against KPC-producing K. pneumoniae isolates, including colistin-resistant strains. Colistin-rifampin combinations may have a role in the treatment of multidrug-resistant K. pneumoniae and may possibly slow the selection of heteroresistant subpopulations during colistin therapy.     

Resistance to Colistin in Acinetobacter baumannii Associated with Mutations in the PmrAB Two-Component System

The mechanism of colistin resistance (Col^r) in Acinetobacter baumannii was studied by selecting in vitro Col^r derivatives of the multidrug-resistant A. baumannii isolate AB0057 and the drug-susceptible strain ATCC 17978, using escalating concentrations of colistin in liquid culture. DNA sequencing identified mutations in genes encoding the two-component system proteins PmrA and/or PmrB in each strain and in a Col^r clinical isolate. A colistin-susceptible revertant of one Col^r mutant strain, obtained following serial passage in the absence of colistin selection, carried a partial deletion of pmrB. Growth of AB0057 and ATCC 17978 at pH 5.5 increased the colistin MIC and conferred protection from killing by colistin in a 1-hour survival assay. Growth in ferric chloride [Fe(III)] conferred a small protective effect. Expression of pmrA was increased in Col^r mutants, but not at a low pH, suggesting that additional regulatory factors remain to be discovered.Among gram-negative pathogens that are reported as “multidrug resistant” (MDR), Acinetobacter baumannii is rapidly becoming a focus of significant attention (1, 7, 25, 32, 38, 39, 46, 51). In intensive care units, up to 30% of A. baumannii clinical isolates are resistant to at least three classes of antibiotics, often including fluoroquinolones and carbapenems (25).The emergence of MDR gram-negative pathogens, including A. baumannii, has prompted increased reliance on the cationic peptide antibiotic colistin (12). Regrettably, increasing colistin use has led to the discovery of resistant strains (10, 11, 22, 26). For example, in a recent study, 12% of carbapenemase-producing Enterobacteriaceae were found to be colistin resistant (Col^r) (6). Although still uncommon, A. baumannii isolates resistant to all available antimicrobial agents have been reported (26, 45) and are of enormous concern, given their potential to spread in the critical care environment.Colistin and other polymyxins are cyclic cationic peptides produced by the soil bacterium Bacillus polymyxa that act by disrupting the negatively charged outer membranes of gram-negative bacteria (37, 50). The following three distinct mechanisms that give rise to colistin resistance are known: (i) specific modification of the lipid A component of the outer membrane lipopolysaccharide, resulting in a reduction of the net negative charge of the outer membrane; (ii) proteolytic cleavage of the drug; and (iii) activation of a broad-spectrum efflux pump (13, 14, 49). The mechanism of colistin resistance in Acinetobacter spp. is not yet known. Heteroresistance to colistin in A. baumannii has been described (17, 24), but it is uncertain whether the basis for this resistance is the presence of a genetically distinct population of cells or whether variation in the regulatory program among genetically identical cells may be sufficient for the expression of resistance.In Salmonella enterica, the two-component signaling systems PmrAB and PhoPQ are involved in sensing environmental pH, Fe³⁺, and Mg²⁺ levels, leading to altered expression of a set of genes involved in lipid A modification (14, 43, 53). A small adapter protein, PmrD, serves as an interface between the two-component systems by stabilizing the activated form of PmrA in S. enterica (19), but other mechanisms of coordinated regulation are described for other species (52). Mutations causing constitutive activation of PmrA and PmrB are associated with colistin resistance (31, 33). Interestingly, the phoPQ and pmrD genes do not appear to be present in Acinetobacter spp., based on computational analysis of the genome sequences (2).PmrA-regulated resistance to colistin in S. enterica and P. aeruginosa results from modification of lipid A with 4-deoxy-aminoarabinose (Ara4N) or phosphoethanolamine via activation of ugd, the pmrF (or pbgP) operon, and pmrC, which encode UDP-glucose dehydrogenase (the first step in Ara4N biosynthesis), Ara4N biosynthetic enzymes, and lipid A phosphoethanolamine transferase, respectively (8, 15, 21, 41, 48). The Ara4N biosynthesis and attachment genes are not present in A. baumannii or Neisseria meningitidis (36, 47). N. meningitidis is intrinsically resistant to polymyxins, demonstrating that Ara4N modification of lipid A is not required for resistance. Mutations in the pmrC ortholog lptA, encoding the lipid A phosphoethanolamine transferase, reduce colistin resistance in N. meningitidis, suggesting that this modification alone may be sufficient for conferring colistin resistance (49). Here we show that the PmrAB system is involved in regulating colistin resistance in A. baumannii by identification of mutations in resistant isolates that exhibit constitutive expression of pmrA.     

Colistin Resistance Caused by Inactivation of the MgrB Regulator Is Not Associated with Decreased Virulence of Sequence Type 258 KPC Carbapenemase-Producing Klebsiella pneumoniae

Using a Galleria mellonella animal model, we compared the virulence of two sequence type 258 (ST258) KPC-producing Klebsiella pneumoniae strains, which were representative of the two clades of this clonal lineage, with that of isogenic colistin-resistant mgrB mutants. With both strains, the mgrB mutants did not exhibit modification in virulence. In the G. mellonella model, the clade 1 strain (capsular type cps-1 [wzi29, producing KPC-2]) was significantly more virulent than the clade 2 strain (capsular type cps-2 [wzi154, producing KPC-3]).     

Genetic Factors Associated with Elevated Carbapenem Resistance in KPC-Producing Klebsiella pneumoniae

In the United States, the most prevalent mechanism of carbapenem resistance among Enterobacteriaceae is the production of a Klebsiella pneumoniae carbapenemase (KPC). KPC-producing isolates often exhibit a range of carbapenem MICs. To better understand the factors that contribute to overall carbapenem resistance, we analyzed 27 KPC-producing K. pneumoniae isolates with different levels of carbapenem resistance, 11 with low-level (i.e., meropenem or imipenem MIC ≤ 4 μg/ml), 2 with intermediate-level (i.e., meropenem and imipenem MIC = 8 μg/ml), and 14 with high-level (i.e., imipenem or meropenem MIC ≥ 16 μg/ml) carbapenem resistance, that were received from throughout the United States. Among 14 isolates that exhibited high-level carbapenem resistance, Western blot analysis indicated that 10 produced an elevated amount of KPC. These isolates either contained an increased bla_KPC gene copy number (n = 3) or had deletions directly upstream of the bla_KPC gene (n = 7). Four additional isolates lacked elevated KPC production but had high-level carbapenem resistance. Porin sequencing analysis identified 22 isolates potentially lacking a functional OmpK35 and three isolates potentially lacking a functional OmpK36. The highest carbapenem MICs were found in two isolates that lacked both functioning porins and produced elevated amounts of KPC. The 11 isolates with low-level carbapenem resistance contained neither an upstream deletion nor increased bla_KPC copy number. These results suggest that both bla_KPC copy number and deletions in the upstream genetic environment affect the level of KPC production and may contribute to high-level carbapenem resistance in KPC-producing K. pneumoniae, particularly when coupled with OmpK36 porin loss.The occurrence of Gram-negative bacterial infections that are resistant to extended-spectrum β-lactam antimicrobial agents forces clinicians to rely on carbapenems as a “last resort” to combat these resistant pathogens. However, as carbapenems are more frequently utilized, an increasing number of bacteria with various mechanisms of resistance to this class of antimicrobial agents are identified. The most widespread resistance mechanisms include the production of a carbapenemase and the combination of porin loss with the production of either an AmpC enzyme or an extended-spectrum β-lactamase (4, 15). Klebsiella pneumoniae carbapenemase (KPC), an Ambler class A β-lactamase that can hydrolyze most β-lactam agents, including carbapenems, is now the most prevalent carbapenemase found among clinical Gram-negative isolates in the United States (22).KPC was first reported in a K. pneumoniae isolate from North Carolina in 1996 (28). However, recent reports indicate that KPC-producing Gram-negative isolates are being identified throughout the United States as well as parts of Europe, Asia, and South America (13, 20, 22). Although these β-lactamases occur most commonly in K. pneumoniae, they have also been identified in other members of the Enterobacteriaceae family and in Pseudomonas and Acinetobacter species (3, 21, 24, 26, 27). The bla_KPC gene is plasmid mediated and is carried on a Tn3-based transposon, Tn4401 (17), which may account for the high mobility of this resistance mechanism.KPC-producing isolates can exhibit a range of carbapenem MICs, thus making their detection a significant challenge for clinical laboratories. By using 2009 Clinical and Laboratory Standards Institute (CLSI) breakpoints and testing methods (1, 6), some KPC-producing isolates may be identified as susceptible to carbapenems. The clinical significance of carbapenem-susceptible isolates with elevated carbapenem MICs is unclear (6), and the cellular changes that may convert a susceptible KPC-producing isolate to one with MICs indicating resistance to carbapenem are not well described. From previous reports, we know that KPC production combined with porin loss can result in higher carbapenem MICs (10, 14, 29). This finding suggests that the KPC enzyme alone is not always sufficient to confer carbapenem resistance, as defined by the 2009 CLSI breakpoints.Other factors likely result in higher carbapenem MICs for KPC-producing isolates. For example, isolates with an increased expression of bla_KPC were previously shown to have increased rates of hydrolysis of imipenem and meropenem (14). Directly upstream of the bla_KPC gene is a nonconserved region of the Tn4401 transposon, located between the istB and the bla_KPC genes (17). Previous reports describe four isoforms in this variable region: Tn4401a contains a 100-bp deletion, Tn4401b contains no deletion (17), and isoforms with 215-bp (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"DQ989640","term_id":"116263782","term_text":"DQ989640"}}DQ989640) and 255-bp (13) deletions were recently reported. Additional studies of this variable region suggest that the 100-bp deletion may result in a different −35 promoter region of the bla_KPC gene (11). Upstream deletions that affect the promoter may impact the level of bla_KPC expression and thus would influence the overall level of carbapenem resistance. Also, KPC-producing isolates may contain different levels of bla_KPC dosage based on the presence of multiple copies of a bla_KPC-carrying plasmid, multiple bla_KPC-carrying plasmids, or multiple copies of the bla_KPC gene located within the same plasmid (11). Increasing the bla_KPC gene copy number could result in increased enzyme production and higher carbapenem MICs. Understanding the impact of these factors may help to predict the potential for KPC-producing isolates susceptible to carbapenems to convert to isolates resistant to carbapenems.In this study, we examined genetic factors that may enhance the level of carbapenem resistance. We selected 27 KPC-producing K. pneumoniae isolates that were obtained from clinical patients in different areas of the country and exhibited a range of carbapenem MICs. These isolates were characterized by determining the sequences of the two main porins, OmpK35 and OmpK36 (9), examining levels of KPC production by Western blot analysis, comparing relative bla_KPC copy numbers using quantitative real-time PCR, and analyzing sequence variations in the genetic environment directly upstream of the bla_KPC gene.     

Transferrable Resistance to Tobramycin in Klebsiella pneumoniae and Enterobacter cloacae Associated with Enzymatic Acetylation of Tobramycin

Among gram-negative bacilli isolated from burn wound cultures, some strains of Enterobacteriaceae were resistant to tobramycin (minimal inhibitory concentration [MIC]≥ 20 μg/ml) but susceptible to gentamicin (MIC ≤ 5 μg/ml). One Klebsiella pneumoniae and two Enterobacter cloacae strains were selected for studies on their mechanisms of resistance to aminoglycoside antibiotics. Resistance to high concentrations of tobramycin (MICs of 25 to 50 μg/ml) was conjugally transferred to a susceptible Escherichia coli strain at rates of 1.2 × 10⁻⁴ to 2.8 to 10⁻⁴ per donor cell, suggesting that resistance is controlled by R factors. Resistances to tobramycin, kanamycin, and neomycin were cotransferred. Enzymatic activities were present that acetylated tobramycin, gentamicin, and kanamycin in osmotic lysates from the donor and transcipient strains. Enzymatic adenylylation of these aminoglycosides was not observed. The aminoglycoside-acetylating activities from K. pneumoniae and E. cloacae resembled kanamycin acetyltransferase (KAT) in their specificity for aminoglycoside substrates. Not all isolates of bacteria that produce KAT are resistant to tobramycin, but the factors that determine susceptibility or resistance to tobramycin in KAT-producing bacteria have not yet been established.     

Comparative Evaluation of Colistin Susceptibility Testing Methods among Carbapenem-Nonsusceptible Klebsiella pneumoniae and Acinetobacter baumannii Clinical Isolates

We compared six colistin susceptibility testing (ST) methods on 61 carbapenem-nonsusceptible Klebsiella pneumoniae (n = 41) and Acinetobacter baumannii (n = 20) clinical isolates with provisionally elevated colistin MICs by routine ST. Colistin MICs were determined by broth microdilution (BMD), BMD with 0.002% polysorbate 80 (P80) (BMD-P80), agar dilution (AD), Etest, Vitek2, and MIC test strip (MTS). BMD was used as the reference method for comparison. The EUCAST-recommended susceptible and resistant breakpoints of ≤2 and >2 μg/ml, respectively, were applied for both K. pneumoniae and A. baumannii. The proportions of colistin-resistant strains were 95.1, 77, 96.7, 57.4, 65.6, and 98.4% by BMD, BMD-P80, AD, Etest, MTS, and Vitek2, respectively. The Etest and MTS methods produced excessive rates of very major errors (VMEs) (39.3 and 31.1%, respectively), while BMD-P80 produced 18% VMEs, AD produced 3.3% VMEs, and Vitek2 produced no VMEs. Major errors (MEs) were rather limited by all tested methods. These data show that gradient diffusion methods may lead to inappropriate colistin therapy. Clinical laboratories should consider the use of automated systems, such as Vitek2, or dilution methods for colistin ST.     

In Vitro Activity of Fosfomycin against blaKPC-Containing Klebsiella pneumoniae Isolates,Including Those Nonsusceptible to Tigecycline and/or Colistin

In vitro activity of fosfomycin was evaluated against 68 bla_KPC-possessing Klebsiella pneumoniae (KpKPC) isolates, including 23 tigecycline- and/or colistin-nonsusceptible strains. By agar dilution, 93% of the overall KpKPC were susceptible (MIC_50/90 of 16/64 μg/ml, respectively). The subgroup of 23 tigecycline- and/or colistin-nonsusceptible strains showed susceptibility rates of 87% (MIC_50/90 of 32/128 μg/ml, respectively). Notably, 5 out of 6 extremely drug-resistant (tigecycline and colistin nonsusceptible) KpKPC were susceptible to fosfomycin. Compared to agar dilution, disk diffusion was more accurate than Etest.The worldwide spread of bla_KPC-possessing Klebsiella pneumoniae (KpKPC) isolates represents a serious threat to our health care systems (20). KpKPC isolates are responsible for hospital outbreaks in the United States, Israel, and Greece with mortality rates of approximately 35% (14, 18, 23, 26).KpKPC isolates are nonsusceptible (NS) in vitro to all β-lactams, including β-lactam/β-lactamase inhibitor combinations and carbapenems, quinolones, and frequently, aminoglycosides (10, 22). Thus, our therapeutic options against infections due to KpKPC are often limited to tigecycline and colistin. Unfortunately, an increasing number of colistin- and/or tigecycline-NS KpKPC isolates have been observed recently, primarily in the New York City area (1, 15). KpKPC isolates that are both colistin and tigecycline NS are defined as “extremely drug-resistant” (XDR) strains because all available standard antibiotics are ineffective in vitro (22). The spread of these XDR K. pneumoniae isolates may have devastating effects on patient outcomes (7).As a result of this urgency, new therapeutic strategies against KpKPC isolates need to be rapidly devised and implemented. Thus far, there are a few novel compounds in development that promise to be active (9, 11). However, these new drugs are currently in clinical trials and it will take a few years to see if their promise holds true in the clinic.Fosfomycin is an “old” antimicrobial that inhibits the first step of peptidoglycan synthesis and shows potent bactericidal action against many Gram-negative and Gram-positive pathogens (17). Fosfomycin tromethamine, an oral formulation, is approved in the United States and other countries for the treatment of uncomplicated urinary tract infections (UTIs) caused by Escherichia coli or Enterococcus faecalis (21, 27). In some European countries and Japan, fosfomycin disodium is also available for parenteral use. The drug shows little toxicity, and very high peak levels can be achieved in serum and urine (12, 13). Interestingly, fosfomycin rapidly penetrates tissues (12, 24), a property that is highly desirable in the treatment of serious infections. Unfortunately, resistance develops rapidly when fosfomycin is used as monotherapy (12, 19). Since fosfomycin shows synergistic action with other antimicrobials (2), it is used in combination to treat a wide range of infections, including pneumonia and septicemia. Overall, cure rates of >80% are observed (12, 13). Surprisingly, data regarding in vitro and in vivo activity of fosfomycin against KpKPC isolates are lacking.In the present work, we analyzed the in vitro activity of fosfomycin against a collection of 68 KpKPC clinical isolates. Forty-two isolates were previously characterized strains collected in the Eastern United States (8, 10, 11), whereas the remaining 26 were recently (January to July 2009) isolated in institutions located in New York City (n = 17) and Cleveland, OH (n = 9).MICs for tigecycline and colistin were obtained using the Etest method (AB bioMérieux) on Mueller-Hinton agar (MHA; BBL, Becton Dickinson). The results for tigecycline were interpreted according to the U.S. FDA criteria, whereas those for colistin were interpreted according to Clinical and Laboratory Standards Institute (CLSI) criteria established for organisms that are not members of the Enterobacteriaceae (i.e., susceptibility of ≤2 μg/ml for both antimicrobials) (5).Susceptibility to fosfomycin was determined using three methods: agar dilution (AD), disk diffusion (DD), and Etest. AD was performed using fosfomycin disodium salt (Sigma-Aldrich Co.) on MHA containing 25 μg/ml of glucose-6-phosphate (G6P; Roche Diagnostics), employing a Steers replicator that delivered 10⁴ CFU/10-μl spot (4, 5). Disk diffusion was carried out on MHA with disks (BBL, Becton Dickinson) containing 200 μg of fosfomycin and 50 μg of G6P (5). Etest was performed on MHA containing G6P, following the manufacturer''s instructions. Since CLSI criteria to evaluate fosfomycin susceptibility in K. pneumoniae are not available, results were interpreted according to guidelines approved for Escherichia coli in UTIs (i.e., susceptible at MICs of ≤64 μg/ml or with zones of ≥16 mm) (5); these breakpoints have been used by authors of similar studies (6, 16, 17, 25). ATCC strains Escherichia coli 25922 and Pseudomonas aeruginosa 27853 were used as controls for all experiments.Among the 68 KpKPC isolates analyzed, 23 were tigecycline and/or colistin NS (i.e., 5 tigecycline NS, 12 colistin NS, and 6 XDR). In Fig. ​Fig.1,1, we present the susceptibility results for fosfomycin obtained with the AD, Etest, and DD. By AD, an overall susceptibility of 92.6% (MIC₅₀ and MIC₉₀ of 16 and 64 μg/ml, respectively) was found. The subgroup of tigecycline- and/or colistin-NS isolates showed susceptibility rates of 87.0% (MIC₅₀ and MIC₉₀ of 32 and 128 μg/ml, respectively). Notably, fosfomycin was active in vitro against five of the six XDR KpKPC isolates (i.e., two with MICs of 32 μg/ml, three with MICs of 64 μg/ml, and one with a MIC of 256 μg/ml). By Etest and DD, overall susceptibility rates of approximately 60% were recorded (Fig. ​(Fig.11).Open in a separate windowFIG. 1.MIC distributions for fosfomycin were obtained using agar diffusion and Etest (MICs were adjusted up to the next highest doubling concentration, e.g., from 48 to 64 μg/ml). Inhibition zone (IZ) diameter distribution was obtained with disk diffusion. Results were interpreted according to CLSI criteria for E. coli (5). The number of KpKPC isolates that had each result is presented above the bars. Dashed line, susceptible cutoff; solid line, resistant cutoff; S, susceptible; I, intermediate; R, resistant.The results of Etest and DD were compared with those obtained from the AD, used as the reference method, to establish their ability to characterize fosfomycin susceptibility. Essential agreement (EA), categorical agreement (CA), minor errors (MiE), major errors (MaE), and very major errors (VME) were calculated (see definitions in Table ​Table1).1). CLSI recommends that <10% MiE, <3% MaE, and <1.5% VME should be obtained to approve the performance of susceptibility tests (3).

TABLE 1.

Agreement of the Etest and disk diffusion methods with agar dilution in testing susceptibility to fosfomycin

Heteroresistance to Fosfomycin Is Predominant in Streptococcus pneumoniae and Depends on the murA1 Gene

Fosfomycin targets the first step of peptidoglycan biosynthesis in Streptococcus pneumoniae catalyzed by UDP-N-acetylglucosamine enolpyruvyltransferase (MurA1). We investigated whether heteroresistance to fosfomycin occurs in S. pneumoniae. We found that of 11 strains tested, all but 1 (Hungary^19A) displayed heteroresistance and that deletion of murA1 abolished heteroresistance. Hungary^19A differs from the other strains by a single amino acid substitution in MurA1 (Ala₃₆₄Thr). To test whether this substitution is responsible for the lack of heteroresistance, it was introduced into strain D39. The heteroresistance phenotype of strain D39 was not changed. Furthermore, no relevant structural differences between the MurA1 crystal structures of heteroresistant strain D39 and nonheteroresistant strain Hungary^19A were found. Our results reveal that heteroresistance to fosfomycin is the predominant phenotype of S. pneumoniae and that MurA1 is required for heteroresistance to fosfomycin but is not the only factor involved. The findings provide a caveat for any future use of fosfomycin in the treatment of pneumococcal infections.     

KPC酶在肺炎克雷伯菌碳青霉烯类耐药中的研究

目的运用RNA干扰技术探讨blaKPC表达与碳青霉烯类耐药的关系。方法用电转移法将双链小RNA（SiRNA）转入到2株产肺炎克雷伯菌碳青霉烯酶-2（KPC-2）的肺炎克雷伯菌中,检测RNA干扰前后菌株blaKPC的RNA水平和亚胺培南、厄他培南的最低抑菌浓度（MIC）值的变化。结果菌株在RNA干扰前后blaKPC-2的RNA水平有差异,但干扰前后亚胺培南和厄他培南的MIC值没有变化。结论 SiRNA虽然能在RNA水平上抑制blaKPC的表达,但对产KPC-2菌株碳青霉烯类耐药的影响不明显,证明革兰阴性杆菌对碳青霉烯类耐药由多种因素共同引起。