Resistance to Linezolid: Characterization of Mutations in rRNA and Comparison of Their Occurrences in Vancomycin-Resistant Enterococci

To assess the potential for emergence of resistance during the use of linezolid, we tested 10 clinical isolates of vancomycin-resistant enterococci (VRE) (four Enterococcus faecalis, five Enterococcus faecium, and one Enterococcus gallinarum) as well as a vancomycin-susceptible control (ATCC 29212) strain of E. faecalis. The enterococci were exposed to doubling dilutions of linezolid for 12 passes. After the final passage, the linezolid plate growing VRE contained a higher drug concentration with E. faecalis than with E. faecium. DNA sequencing of the 23S rRNA genes revealed that linezolid resistance in three E. faecalis isolates was associated with a guanine to uracil transversion at bp 2576, while the one E. faecium isolate for which the MIC was 16 microg/ml contained a guanine to adenine transition at bp 2505.     

Characterization of High-Level Daptomycin Resistance in Viridans Group Streptococci Developed upon In Vitro Exposure to Daptomycin

Viridans group streptococci (VGS) are part of the normal flora that may cause bacteremia, often leading to endocarditis. We evaluated daptomycin against four clinical strains of VGS (MICs = 1 or 2 μg/ml) using an in vitro-simulated endocardial vegetation model, a simulated bacteremia model, and kill curves. Daptomycin exposure was simulated at 6 mg/kg of body weight and 8 mg/kg every 24 h for endocardial and bacteremia models. Total drug concentrations were used for analyses containing protein (albumin and pooled human serum), and free (unbound) drug concentrations (93% protein bound) were used for analyses not containing protein. Daptomycin MICs in the presence of protein were significantly higher than those in the absence of protein. Despite MICs below or at the susceptible breakpoint, all daptomycin regimens demonstrated limited kill in both pharmacodynamic models. A reduction of approximately 1 to 2 log₁₀ CFU was seen for all isolates and dosages except daptomycin at 6 mg/kg, which achieved a reduction of 2.7 log₁₀ CFU/g against one strain (Streptococcus gordonii 1649) in the endocardial model. Activity was similar in both pharmacodynamic models in the presence or absence of protein. Similar activity was noted in the kill curves over all multiples of the MIC. Regrowth by 24 h was seen even at 8× MIC. Postexposure daptomycin MICs for both pharmacodynamic models increased to >256 μg/ml for all isolates by 24 and 72 h. Despite susceptibility to daptomycin by standard MIC methods, these VGS developed high-level daptomycin resistance (HLDR) after a short duration following drug exposure not attributed to modification or inactivation of daptomycin. Further evaluation is warranted to determine the mechanism of resistance and clinical implications.     

Multiple Genetic Mutations Associated with Polymyxin Resistance in Acinetobacter baumannii

We studied polymyxin B resistance in 10 pairs of clinical Acinetobacter baumannii isolates, two of which had developed polymyxin B resistance in vivo. All polymyxin B-resistant isolates had lower growth rates than and substitution mutations in the lpx or pmrB gene compared to their parent isolates. There were significant differences in terms of antibiotic susceptibility and genetic determinants of resistance in A. baumannii isolates that had developed polymyxin B resistance in vivo compared to isolates that had developed polymyxin B resistance in vitro.     

gyrA Mutations Associated with Fluoroquinolone Resistance in Eight Species of Enterobacteriaceae

Fluoroquinolone resistance (FQ-R) in clinical isolates of Enterobacteriaceae species has been reported with increasing frequency in recent years. Two mechanisms of FQ-R have been identified in gram-negative organisms: mutations in DNA gyrase and reduced intracellular drug accumulation. A single point mutation in gyrA has been shown to reduce susceptibility to fluoroquinolones. To determine the extent of gyrA mutations associated with FQ-R in enteric bacteria, one set of oligonucleotide primers was selected from conserved sequences in the flanking regions of the quinolone resistance-determining regions (QRDR) of Escherichia coli and Klebsiella pneumoniae. This set of primers was used to amplify and sequence the QRDRs from 8 Enterobacteriaceae type strains and 60 fluoroquinolone-resistant clinical isolates of Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, E. coli, K. pneumoniae, Klebsiella oxytoca, Providencia stuartii, and Serratia marcescens. Although similarity of the nucleotide sequences of seven species ranged from 80.8 to 93.3%, when compared with that of E. coli, the amino acid sequences of the gyrA QRDR were highly conserved. Conservative amino acid substitutions were detected in the QRDRs of the susceptible type strains of C. freundii, E. aerogenes, K. oxytoca (Ser-83 to Thr), and P. stuartii (Asp-87 to Glu). Strains with ciprofloxacin MICs of >2 μg/ml expressed amino acid substitutions primarily at the Gly-81, Ser-83, or Asp-87 position. Fluoroquinolone MICs varied significantly for strains exhibiting identical gyrA mutations, indicating that alterations outside gyrA contribute to resistance. The type and position of amino acid alterations also differed among these six genera. High-level FQ-R frequently was associated with single gyrA mutations in all species of Enterobacteriaceae in this study except E. coli.     

gyrA Mutations Associated with Quinolone Resistance in Bacteroides fragilis Group Strains

Mutations in the gyrA gene contribute considerably to quinolone resistance in Escherichia coli. Mechanisms for quinolone resistance in anaerobic bacteria are less well studied. The Bacteroides fragilis group are the anaerobic organisms most frequently isolated from patients with bacteremia and intraabdominal infections. Forty-four clinafloxacin-resistant and-susceptible fecal and clinical isolates of the B. fragilis group (eight Bacteroides fragilis, three Bacteroides ovatus, five Bacteroides thetaiotaomicron, six Bacteroides uniformis, and 22 Bacteroides vulgatus) and six ATCC strains of the B. fragilis group were analyzed as follows: (i) determination of susceptibility to ciprofloxacin, levofloxacin, moxifloxacin, and clinafloxacin by the agar dilution method and (ii) sequencing of the gyrA quinolone resistance-determining region (QRDR) located between amino acid residues equivalent to Ala-67 through Gln-106 in E. coli. Amino acid substitutions were found at hotspots at positions 82 (n = 15) and 86 (n = 8). Strains with Ser82Leu substitutions (n = 13) were highly resistant to all quinolones tested. Mutations in other positions of gyrA were also frequently found in quinolone-resistant and -susceptible isolates. Eight clinical strains that lacked mutations in their QRDR were susceptible to at least two of the quinolones tested. Although newer quinolones have good antimicrobial activity against the B. fragilis group, quinolone resistance in B. fragilis strains can be readily selected in vivo. Mutational events in the QRDR of gyrA seem to contribute to quinolone resistance in Bacteroides species.     

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.     

Resistance to Moxifloxacin in Toxigenic Clostridium difficile Isolates Is Associated with Mutations in gyrA

Clostridium difficile is the etiological agent of antibiotic-associated colitis and the most common cause of hospital-acquired infectious diarrhea. Fluoroquinolones such as ciprofloxacin are associated with lower risks of C. difficile-associated diarrhea. In this study, we have analyzed 72 C. difficile isolates obtained from patients with different clinical courses of disease, such as toxic megacolon and relapses; the hospital environment; public places; and horses. They were investigated for their susceptibilities to moxifloxacin (MXF), metronidazole (MEO), and vancomycin (VAN). Mutants highly resistant to fluoroquinolones were selected in vitro by stepwise exposure to increasing concentrations of MXF. The resulting mutants were analyzed for the presence of mutations in the quinolone resistance-determining regions of DNA gyrase (gyrA), the production of toxins A and B, and the epidemiological relationship of these isolates. These factors were also investigated using PCR-based methods. All strains tested were susceptible to MEO and VAN. Twenty-six percent of the clinical isolates (19 of 72) were highly resistant to MXF (MIC > or = 16 microg/ml). Fourteen of these 19 strains contained nucleotide changes resulting in amino acid substitutions at position 83 in the gyrA protein. Resistant strains selected in vitro did not contain mutations at that position. These findings indicate that resistance to MXF in a majority of cases may be due to amino acid substitution in the gyrA gene.     

Systematic Review of Mutations in Pyrazinamidase Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis Clinical Isolates

Pyrazinamide (PZA) is an important first-line drug in the treatment of tuberculosis (TB) and of significant interest to the HIV-infected community due to the prevalence of TB-HIV coinfection in some regions of the world. The mechanism of resistance to PZA is unlike that of any other anti-TB drug. The gene pncA, encoding pyrazinamidase (PZase), is associated with resistance to PZA. However, because single mutations in PZase have a low prevalence, the individual sensitivities are low. Hundreds of distinct mutations in the enzyme have been associated with resistance, while some only appear in susceptible isolates. This makes interpretation of molecular testing difficult and often leads to the simplification that any PZase mutation causes resistance. This systematic review reports a comprehensive global list of mutations observed in PZase and its promoter region in clinical strains, their phenotypic association, their global frequencies and diversity, the method of phenotypic determination, their MIC values when given, and the method of MIC determination and assesses the strength of the association between mutations and phenotypic resistance to PZA. In this systematic review, we report global statistics for 641 mutations in 171 (of 187) codons from 2,760 resistant strains and 96 mutations from 3,329 susceptible strains reported in 61 studies. For diagnostics, individual mutations (or any subset) were not sufficiently sensitive. Assuming similar error profiles of the 5 phenotyping platforms included in this study, the entire enzyme and its promoter provide a combined estimated sensitivity of 83%. This review highlights the need for identification of an alternative mechanism(s) of resistance, at least for the unexplained 17% of cases.     

Characterization of Mutations Contributing to Sulfathiazole Resistance in Escherichia coli

A sulfathiazole-resistant dihydropteroate synthase (DHPS) present in two different laboratory strains of Escherichia coli repeatedly selected for sulfathiazole resistance was mapped to folP by P1 transduction. The folP mutation in each of the strains was shown to be identical by nucleotide sequence analysis. A single C→T transition resulted in a Pro→Ser substitution at amino acid position 64. Replacement of the mutant folP alleles with wild-type folP significantly reduced the level of resistance to sulfathiazole but did not abolish it, indicating the presence of an additional mutation(s) that contributes to sulfathiazole resistance in the two strains. Transfer of the mutant folP allele to a wild-type background resulted in a strain with only a low level of resistance to sulfathiazole, suggesting that the presence of the resistant DHPS was not in itself sufficient to account for the overall sulfathiazole resistance in these strains of E. coli. Additional characterization of an amplified secondary resistance determinant, sur, present in one of the strains, identified it as the previously identified bicyclomycin resistance determinant bcr, a member of a family of membrane-bound multidrug resistance antiporters. An additional mutation contributing to sulfathiazole resistance, sux, has also been identified and has been shown to affect the histidine response to adenine sensitivity displayed by these purU strains.     

Concurrent Titration and Determination of Antibiotic Resistance in Ureaplasma Species with Identification of Novel Point Mutations in Genes Associated with Resistance

Antibiotic resistance determination of Ureaplasma spp. (Ureaplasma parvum and Ureaplasma urealyticum) usually requires predetermination of bacterial titer, followed by antibiotic interrogation using a set bacterial input. This 96-well method allows simultaneous quantification of bacteria in the presence and absence of antibiotics. A method for determining precise MICs and a method for screening against multiple antibiotics using breakpoint thresholds are detailed. Of the 61 Ureaplasma-positive clinical isolates screened, one (1.6%) was resistant to erythromycin (MIC, >64 mg/liter) and clarithromycin (MIC, 4 mg/liter), one to ciprofloxacin (1.6%), and one to tetracycline/doxycycline (1.6%). Five isolates were also consistently found to have an elevated MIC of 8 mg/liter for erythromycin, but this may not represent true antibiotic resistance, as no mutations were found in the 23S rRNA operons or ribosome-associated L4 and L22 proteins for these strains. However, two amino acids (R66Q67) were deleted from the L4 protein of the erythromycin-/clarithromycin-resistant strain. The tetM genetic element was detected in the tetracycline-resistant clinical isolate as well as in the positive control Vancouver strain serotype 9. The tetM gene was also found in a fully tetracycline-susceptible Ureaplasma clinical isolate, and no mutations were found in the coding region that would explain its failure to mediate tetracycline resistance. An amino acid substitution (D82N) was found in the ParC subunit of the ciprofloxacin-resistant isolate, adjacent to the S83L mutation reported by other investigators in many ciprofloxacin-resistant Ureaplasma isolates. It is now possible to detect antibiotic resistance in Ureaplasma within 48 h of positive culture without prior knowledge of bacterial load, identifying them for further molecular analysis.Ureaplasmas and mycoplasmas are eubacteria belonging to the class Mollicutes. These unique organisms are the smallest self-replicating cells that lack a cell wall. Lack of a rigid cell wall prevents reactivity with Gram staining and makes them insusceptible to antibiotics that target bacterial cell walls (i.e., β-lactams and glycopeptides), while imparting a frailty that mostly limits them to a parasitic existence in association with the eukaryotic cells of their host (39). Shepard first described ureaplasmas, or T-mycoplasmas, in the 1950s, following isolation from a male patient with nongonococcal urethritis (31). This remains the most widely recognized patient group; however, they are also commonly found as commensals in the genital tract in as many as 80% of women of child-bearing age (30). Although the factors leading to intrauterine infection are unclear, detection of Ureaplasma in the amniotic fluid is frequently associated with chorioamnionitis, spontaneous abortion, and premature birth (13, 14, 40). It has been suggested that the rate of vertical transmission is inversely proportional to gestational age at time of delivery (1). Furthermore, the presence of Ureaplasma in the lungs of very premature neonates has been associated with the development of chronic lung disease (or bronchopulmonary dysplasia) and long-term hospitalization (7, 19, 29, 30).Ureaplasma spp. are susceptible to bacteriostatic agents such as protein synthesis-inhibiting tetracyclines and macrolides as well as bactericidal agents, including fluoroquinolones. For premature neonatal patients, however, the removal of a microbe must be balanced against the potential (and often unknown) toxicity of the antibiotics in these patients (38). For this reason, physicians routinely favor macrolide antibiotics such as erythromycin when deciding to treat Ureaplasma infections in premature neonates.Only a limited number of reports have been made regarding the trends in resistance among Ureaplasma isolates from neonates (18, 34, 37), and all of these comment on the lack of a standardized methodology, which hinders comparison of results. Factors such as inoculum size, pH of media, and time of incubation are known to dramatically alter the MIC, and standardized means of detecting resistance are needed.Here, we describe a modified breakpoint analysis in a 96-well broth microdilution format that enables a concurrent determination of bacterial load in a sample simultaneously with the determination of resistance without prior knowledge of bacterial load. For isolates showing resistance within the breakpoint concentration, full MICs can be determined using a similar full-plate methodology. The breakpoint method was used to screen 15 strains isolated during 2006 and 2007 from bronchoalveolar lavage samples from neonates of various gestational ages at the University Hospital of Wales (UHW), Cardiff, United Kingdom, in addition to 46 frozen isolates from a reference repository of samples submitted to the Health Protection Agency at Colindale (London, United Kingdom) from 2003 to present. Within these 61 isolates, we identified one erythromycin-/clarithromycin-resistant, one ciprofloxacin-resistant, and one tetracycline-resistant Ureaplasma strain. Further PCR and sequence analyses were performed to determine the mechanism of resistance.     

Analysis of embCAB Mutations Associated with Ethambutol Resistance in Multidrug-Resistant Mycobacterium tuberculosis Isolates from China

Ethambutol (EMB) plays a pivotal role in the chemotherapy of drug-resistant tuberculosis (TB), including multidrug-resistant tuberculosis (MDR-TB). Resistance to EMB is considered to be caused by mutations in the embCAB operon (embC, embA, and embB). In this study, we analyzed the embCAB mutations among 139 MDR-TB isolates from China and found a possible association between embCAB operon mutation and EMB resistance. Our data indicate that 56.8% of MDR-TB isolates are resistant to EMB, and 82.2% of EMB-resistant isolates belong to the Beijing family. Overall, 110 (79.1%) MDR-TB isolates had at least one mutation in the embCAB operon. The majority of mutations were present in the embB gene and the embA upstream region, which also displayed significant correlations with EMB resistance. The most common mutations occurred at codon 306 in embB (embB306), followed by embB406, embA(−16), and embB497. Mutations at embB306 were associated with EMB resistance. DNA sequencing of embB306–497 was the best strategy for detecting EMB resistance, with 89.9% sensitivity, 58.3% specificity, and 76.3% accuracy. Additionally, embB306 had limited value as a candidate predictor for EMB resistance among MDR-TB infections in China.     

Mutations in Ribosomal Protein L3 Are Associated with Oxazolidinone Resistance in Staphylococci of Clinical Origin

Following recent reports of ribosomal protein L3 mutations in laboratory-derived linezolid-resistant (LZD^r) Staphylococcus aureus, we investigated whether similar mutations were present in LZD^r staphylococci of clinical origin. Sequence analysis of a variety of LZD^r isolates revealed two L3 mutations, ΔSer145 (S. aureus NRS127) and Ala157Arg (Staphylococcus epidermidis 1653059), both occurring proximal to the oxazolidinone binding site in the peptidyl transferase center. The oxazolidinone torezolid maintained a ≥8-fold potency advantage over linezolid for both strains.Oxazolidinone resistance in clinical staphylococci is most often associated with mutations in 23S rRNA domain V, in particular G2576T (Escherichia coli numbering) (24, 30). Other 23S rRNA mutations, such as G2447T, until recently (19) were strictly associated with laboratory-derived strains (28). Methylation of 23S rRNA (A2503) by the horizontally transmitted Cfr methyltransferase also confers resistance to linezolid (LZD) as well as phenicols, lincosamides, pleuromutilins, and streptogramin A (12, 29). Incidences of LZD resistance in strains lacking 23S rRNA mutations or the cfr gene have prompted analysis of other structural components of the ribosome which may have the potential to influence oxazolidinone binding.A number of 50S large-subunit ribosomal proteins have regions which interact closely with the oxazolidinone binding site in the peptidyl transferase center (PTC), most notably L3 and L4. In rare cases, mutations in L4 have been implicated in LZD nonsusceptibility in clinical Streptococcus pneumoniae isolates (32) and in laboratory-derived Staphylococcus aureus strains (17). Mutations in L3 have typically been associated with resistance to pleuromutilins (whose binding site overlaps with that of oxazolidinones in the PTC) such as tiamulin (TIA) and retapamulin (1, 2, 8, 13, 20, 22). However, we recently described a variety of L3 mutations in S. aureus following in vitro selection with oxazolidinones LZD and torezolid (TR-700) (17) a novel oxazolidinone with enhanced potency against a broad range of gram-positive pathogens, including strains resistant to LZD (11, 14, 26, 27).To investigate the relevance of L3 mutations to clinical oxazolidinone resistance, we sequenced L3-encoding rplC genes in 11 Lzd^r clinical isolates, 2 of which included the uncharacterized Staphylococcus epidermidis strain 1653059 (cfr negative; methicillin [meticillin]-resistant S. epidermidis [MRSE]; Eurofins Medinet, Inc., Chantilly, VA) and S. aureus strain NRS127 (cfr negative; methicillin resistant; Network of Antimicrobial Resistance in Staphylococcus aureus [NARSA] collection, Chantilly, VA), previously reported as having an unknown, non-23S rRNA-based resistance mechanism (27).(Portions of this work were presented at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy [16], San Francisco, CA, 12 to 15 September 2009.)Chromosomal DNA was isolated, and PCR amplification of the six S. aureus rrn alleles was performed as previously described (17, 21). The 3′ portions of S. epidermidis 23S rRNA genes were amplified using the VdomainF primer in conjunction with 23S rRNA allele-specific downstream flanking reverse primers (Se_rrlA-FR) designed using the S. epidermidis RP62A genome sequence (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_002976","term_id":"57865352","term_text":"NC_002976"}}NC_002976) (Table ​(Table1).1). Genes encoding ribosomal proteins L3 (rplC), L4 (rplD), and L22 (rplV) were amplified as a single amplicon (∼3.3 kb) from S. aureus (rplCF/rplVR) (17) and S. epidermidis (rplCF/Se_rplVR) (Table ​(Table1).1). Sequencing of PCR products (Retrogen, Inc., San Diego, CA) was performed with primers flanking the 23S rRNA domain V region (21) and individual ribosomal protein genes (Table ​(Table11).

TABLE 1.

Primers used to amplify and sequence 23S rRNA and ribosomal protein genes

Sulfonamide Resistance in Streptococcus pyogenes Is Associated with Differences in the Amino Acid Sequence of Its Chromosomal Dihydropteroate Synthase

Sulfonamide resistance in recent isolates of Streptococcus pyogenes was found to be associated with alterations of the chromosomally encoded dihydropteroate synthase (DHPS). There were 111 different nucleotides (13.8%) in the genes found in susceptible and resistant isolates, respectively, resulting in 30 amino acid changes (11.3%). These substantial changes suggested the possibility of a foreign origin of the resistance gene, in parallel to what has already been found for sulfonamide resistance in Neisseria meningitidis. The gene encoding DHPS was linked to at least three other genes encoding enzymes of the folate pathway. These genes were in the order GTP cyclohydrolase, dihydropteroate synthase, dihydroneopterin aldolase, and hydroxymethyldihydropterin pyrophosphokinase. The nucleotide differences in genes from resistant and susceptible strains extended from the beginning of the GTP cyclohydrolase gene to the end of the gene encoding DHPS, an additional indication for gene transfer in the development of resistance. Kinetic measurements established different affinities for sulfathiazole for DHPS enzymes isolated from resistant and susceptible strains.     

Case Report and Cohort Analysis of Drug-Induced Liver Injury Associated with Daptomycin

A patient receiving daptomycin developed asymptomatic transaminitis and hyperbilirubinemia without concurrent multiorgan dysfunction or elevation of his creatinine kinase level. After ruling out other etiologies, the liver injury was attributed to daptomycin and was subsequently resolved. A single-center retrospective cohort analysis of baseline and follow-up liver function panels (n = 614) from all admissions from 2008 to 2013 during which daptomycin was administered did not reveal any other cases of probable or definite drug-induced liver injury associated with daptomycin.     

Compensatory Mutations of Rifampin Resistance Are Associated with Transmission of Multidrug-Resistant Mycobacterium tuberculosis Beijing Genotype Strains in China

Mycobacterium tuberculosis can acquire resistance to rifampin (RIF) through mutations in the rpoB gene. This is usually accompanied by a fitness cost, which, however, can be mitigated by secondary mutations in the rpoA or rpoC gene. This study aimed to identify rpoA and rpoC mutations in clinical M. tuberculosis isolates in northern China in order to clarify their role in the transmission of drug-resistant tuberculosis (TB). The study collection included 332 RIF-resistant and 178 RIF-susceptible isolates. The majority of isolates belonged to the Beijing genotype (95.3%, 486/510 isolates), and no mutation was found in rpoA or rpoC of the non-Beijing genotype strains. Among the Beijing genotype strains, 27.8% (89/320) of RIF-resistant isolates harbored nonsynonymous mutations in the rpoA (n = 6) or rpoC (n = 83) gene. The proportion of rpoC mutations was significantly higher in new cases (P = 0.023) and in strains with the rpoB S531L mutation (P < 0.001). In addition, multidrug-resistant (MDR) strains with rpoC mutations were significantly associated with 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat clustering (P = 0.016). In summary, we believe that these findings indirectly suggest an epistatic interaction of particular mutations related to RIF resistance and strain fitness and, consequently, the role of such mutations in the spread of MDR M. tuberculosis strains.     

Chlorhexidine Induces VanA-Type Vancomycin Resistance Genes in Enterococci

Chlorhexidine is a bisbiguanide antiseptic used for infection control. Vancomycin-resistant E. faecium (VREfm) is among the leading causes of hospital-acquired infections. VREfm may be exposed to chlorhexidine at supra- and subinhibitory concentrations as a result of chlorhexidine bathing and chlorhexidine-impregnated central venous catheter use. We used RNA sequencing to investigate how VREfm responds to chlorhexidine gluconate exposure. Among the 35 genes upregulated ≥10-fold after 15 min of exposure to the MIC of chlorhexidine gluconate were those encoding VanA-type vancomycin resistance (vanHAX) and those associated with reduced daptomycin susceptibility (liaXYZ). We confirmed that vanA upregulation was not strain or species specific by querying other VanA-type VRE. VanB-type genes were not induced. The vanH promoter was found to be responsive to subinhibitory chlorhexidine gluconate in VREfm, as was production of the VanX protein. Using vanH reporter experiments with Bacillus subtilis and deletion analysis in VREfm, we found that this phenomenon is VanR dependent. Deletion of vanR did not result in increased chlorhexidine susceptibility, demonstrating that vanHAX induction is not protective against chlorhexidine. As expected, VanA-type VRE is more susceptible to ceftriaxone in the presence of sub-MIC chlorhexidine. Unexpectedly, VREfm is also more susceptible to vancomycin in the presence of subinhibitory chlorhexidine, suggesting that chlorhexidine-induced gene expression changes lead to additional alterations in cell wall synthesis. We conclude that chlorhexidine induces expression of VanA-type vancomycin resistance genes and genes associated with daptomycin nonsusceptibility. Overall, our results indicate that the impacts of subinhibitory chlorhexidine exposure on hospital-associated pathogens should be further investigated in laboratory studies.     

分子生物学方法在肠球菌耐药性研究中的应用

目的：为了及时准确地检测细菌的耐药性,该文将肠球菌耐药性研究中的分子生物学方法进行综述。方法对近年来肠球菌耐药性研究的相关文献进行整理、分析与归纳。结果一些方法比较常见,如 PCR,PFGE,MSLT和 Southern杂交等,而一些方法如焦磷酸测序和基因芯片技术,目前在细菌耐药机制研究中的应用并不广泛,属于交叉学科,但是稍加利用会有一定的发展前景。结论任何一个方法都有优点,也存在一些局限性,应取长补短,发挥每种方法的最大优势。