Analysis of Whole-Genome Sequences for the Prediction of Penicillin Resistance and ß-Lactamase Activity in Bacillus anthracis.

Penicillin (PEN) is a low-cost option for anthrax treatment, but naturally occurring resistance has been reported. ß-Lactamase expression (bla1, bla2) in Bacillus anthracis is regulated by a sigma factor (SigP) and its cognate anti-sigma factor (RsiP). Mutations leading to truncation of RsiP were previously described as a basis for PEN resistance. Here, we analyze whole-genome sequencing (WGS) data and compare the chromosomal sigP-bla1 regions from 374 B. anthracis strains to determine the frequency of mutations, identify mutations associated with PEN resistance, and evaluate the usefulness of WGS for predicting PEN resistance. Few (3.5%) strains contained at least 1 of 11 different mutations in sigP, rsiP, or bla1. Nine of these mutations have not been previously associated with PEN resistance. Four strains showed PEN resistance (PEN-R) by conventional broth microdilution, including 1 strain with a novel frameshift in rsiP. One strain that carries the same rsiP frameshift mutation as that found previously in a PEN-R strain showed a PEN-susceptible (PEN-S) phenotype and exhibited decreased bla1 and bla2 transcription. An unexpectedly small colony size, a reduced growth rate, and undetectable ß-lactamase activity levels (culture supernatant and cell lysate) were observed in this PEN-S strain. Sequence analysis revealed mutations in genes associated with growth defects that may contribute to this phenotype. While B. anthracis rsiP mutations cannot be exclusively used to predict resistance, four of the five strains with rsiP mutations were PEN-R. Therefore, the B. anthracis sigP-bla1 region is a useful locus for WGS-based PEN resistance prediction, but phenotypic testing remains essential. IMPORTANCE Determination of antimicrobial susceptibility of B. anthracis is essential for the appropriate distribution of antimicrobial agents for postexposure prophylaxis (PEP) and treatment of anthrax. Analysis of WGS data allows for the rapid detection of mutations in antimicrobial resistance (AMR) genes in an isolate, but the presence of a mutation in an AMR gene does not always accurately predict resistance. As mutations in the anti-sigma factor RsiP have been previously associated with high-level penicillin resistance in a limited number of strains, we investigated WGS assemblies from 374 strains to determine the frequency of mutations and performed functional antimicrobial susceptibility testing. Of the five strains that contained mutations in rsiP, only four were PEN-R by functional antimicrobial susceptibility testing. We conclude that while sequence analysis of this region is useful for AMR prediction in B. anthracis, genetic analysis should not be used exclusively and phenotypic susceptibility testing remains essential.

DNA gyrase and topoisomerase IV mutations associated with fluoroquinolone resistance in Proteus mirabilis.

Mutations associated with fluoroquinolone resistance in clinical isolates of Proteus mirabilis were determined by genetic analysis of the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, and parE. This study included the P. mirabilis type strain ATCC 29906 and 29 clinical isolates with reduced susceptibility (MIC, 0.5 to 2 microg/ml) or resistance (MIC, > or =4 microg/ml) to ciprofloxacin. Susceptibility profiles for ciprofloxacin, clinafloxacin, gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, and trovafloxacin were correlated with amino acid changes in the QRDRs. Decreased susceptibility and resistance were associated with double mutations involving both gyrA (S83R or -I) and parC (S80R or -I). Among these double mutants, MICs of ciprofloxacin varied from 1 to 16 microg/ml, indicating that additional factors, such as drug efflux or porin changes, also contribute to the level of resistance. For ParE, a single conservative change of V364I was detected in seven strains. An unexpected result was the association of gyrB mutations with high-level resistance to fluoroquinolones in 12 of 20 ciprofloxacin-resistant isolates. Changes in GyrB included S464Y (six isolates), S464F (three isolates), and E466D (two isolates). A three-nucleotide insertion, resulting in an additional lysine residue between K455 and A456, was detected in gyrB of one strain. Unlike any other bacterial species analyzed to date, mutation of gyrB appears to be a frequent event in the acquisition of fluoroquinolone resistance among clinical isolates of P. mirabilis.

Genetic analyses of mutations contributing to fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae.

Twenty-one clinical isolates of Streptococcus pneumoniae showing reduced susceptibility or resistance to fluoroquinolones were characterized by serotype, antimicrobial susceptibility, and genetic analyses of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE. Five strains were resistant to three or more classes of antimicrobial agents. In susceptibility profiles for gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, ofloxacin, sparfloxacin, and trovafloxacin, 14 isolates had intermediate- or high-level resistance to all fluoroquinolones tested except gemifloxacin (no breakpoints assigned). Fluoroquinolone resistance was not associated with serotype or with resistance to other antimicrobial agents. Mutations in the QRDRs of these isolates were more heterogeneous than those previously reported for mutants selected in vitro. Eight isolates had amino acid changes at sites other than ParC/S79 and GyrA/S81; several strains contained mutations in gyrB, parE, or both loci. Contributions to fluoroquinolone resistance by individual amino acid changes, including GyrB/E474K, ParE/E474K, and ParC/A63T, were confirmed by genetic transformation of S. pneumoniae R6. Mutations in gyrB were important for resistance to gatifloxacin but not moxifloxacin, and mutation of gyrA was associated with resistance to moxifloxacin but not gatifloxacin, suggesting differences in the drug-target interactions of the two 8-methoxyquinolones. The positions of amino acid changes within the four genes affected resistance more than did the total number of QRDR mutations. However, the effect of a specific mutation varied significantly depending on the agent tested. These data suggest that the heterogeneity of mutations will likely increase as pneumococci are exposed to novel fluoroquinolone structures, complicating the prediction of cross-resistance within this class of antimicrobial agents.

Activities of clinafloxacin, gatifloxacin, gemifloxacin, and trovafloxacin against recent clinical isolates of levofloxacin-resistant Streptococcus pneumoniae.

The activities of two investigational fluoroquinolones and three fluoroquinolones that are currently marketed were determined for 182 clinical isolates of Streptococcus pneumoniae. The collection included 57 pneumococcal isolates resistant to levofloxacin (MIC >/= 8 microg/ml) recovered from patients in North America and Europe. All isolates were tested with clinafloxacin, gatifloxacin, gemifloxacin, levofloxacin, and trovafloxacin by the National Committee for Clinical Laboratory Standards broth microdilution and disk diffusion susceptibility test methods. Gemifloxacin demonstrated the greatest activity on a per gram basis, followed by clinafloxacin, trovafloxacin, gatifloxacin, and levofloxacin. Scatterplots of the MICs and disk diffusion zone sizes revealed a well-defined separation of levofloxacin-resistant and -susceptible strains when the isolates were tested against clinafloxacin and gatifloxacin. DNA sequence analyses of the quinolone resistance-determining regions of gyrA, gyrB, parC, and parE from 21 of the levofloxacin-resistant strains identified eight different patterns of amino acid changes. Mutations among the four loci had the least effect on the MICs of gemifloxacin and clinafloxacin, while the MICs of gatifloxacin and trovafloxacin increased by up to six doubling dilutions. These data indicate that the newer fluoroquinolones have greater activities than levofloxacin against pneumococci with mutations in the DNA gyrase or topoisomerase IV genes. Depending upon pharmacokinetics and safety, the greater potency of these agents could provide improved clinical efficacy against levofloxacin-resistant pneumococcal strains.

Activities of newer fluoroquinolones against Streptococcus pneumoniae clinical isolates including those with mutations in the gyrA, parC, and parE loci.

Resistance to fluoroquinolone (FQ) antibiotics in Streptococcus pneumoniae has been attributed primarily to specific mutations in the genes for DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). Resistance to some FQs can result from a single mutation in one or more of the genes encoding these essential enzymes. A group of 160 clinical isolates of pneumococci was examined in this study, including 36 ofloxacin-resistant isolates (MICs, > or = 8 micrograms/ml) recovered from patients in North America, France, and Belgium. The susceptibilities of all isolates to clinafloxacin, grepafloxacin, levofloxacin, sparfloxacin, and trovafloxacin were examined by the National Committee for Clinical Laboratory Standards reference broth microdilution and disk diffusion susceptibility testing methods. Among the ofloxacin-resistant strains, 32 of 36 were also categorized as resistant to levofloxacin, 35 were resistant to sparfloxacin, 29 were resistant to grepafloxacin, and 19 were resistant to trovafloxacin. In vitro susceptibility to clinafloxacin appeared to be least affected by resistance to the other FQs. Eight isolates with high- and low-level resistance to the newer FQs were selected for DNA sequence analysis of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE. The DNA and the inferred amino acid sequences of the resistant strains were compared with the analogous sequences of reference strain S. pneumoniae ATCC 49619 and FQ-susceptible laboratory strain R6. Reduced susceptibilities to grepafloxacin and sparfloxacin (MICs, 1 to 2 micrograms/ml) and trovafloxacin (MICs, 0.5 to 1 microgram/ml) were associated with either a mutation in parC that led to a single amino acid substitution (Ser-79 to Phe or Tyr) or double mutations that involved the genes for both GyrA (Ser-81 to Phe) and ParE (Asp-435 to Asn). High-level resistance to all of the compounds except clinafloxacin was associated with two or more amino acid substitutions involving both GyrA (Ser-81 to Phe) and ParC (Ser-79 to Phe or Ser-80 to Pro and Asp-83 to Tyr). No mutations were observed in the gyrB sequences of resistant strains. These data indicate that mutations in pneumococcal gyrA, parC, and parE genes all contribute to decreased susceptibility to the newer FQs, and genetic analysis of the QRDR of a single gene, either gyrA or parC, is not predictive of pneumococcal resistance to these agents.

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 microg/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.

Characterization of outer membranes isolated from Treponema pallidum, the syphilis spirochete.

Previous freeze-fracture electron microscopy (EM) studies have shown that the outer membrane (OM) of Treponema pallidum contains sparse transmembrane proteins. One strategy for molecular characterization of these rare OM proteins involves isolation of T. pallidum OMs. Here we describe a simple and extremely gentle method for OM isolation based upon isopycnic sucrose density gradient ultracentrifugation of treponemes following plasmolysis in 20% sucrose. Evidence that T. pallidum OMs were isolated included (i) the extremely low protein/lipid ratio of the putative OM fraction, (ii) a paucity of antigenic and/or biochemical markers for periplasmic, cytoplasmic membrane, and cytosolic compartments, and (iii) freeze-fracture EM demonstrating that the putative OMs contained intramembranous particles highly similar in size and density to those in native T. pallidum OMs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the OMs contained a relatively small number of treponemal proteins, including several which did not appear to correspond to previously characterized T. pallidum antigens. Interestingly, these candidate rare OM proteins reacted poorly with syphilitic sera as determined by both conventional immunoblotting and enhanced chemiluminescence. Compared with whole cells, T. pallidum OMs were deficient in cardiolipin, the major lipoidal antigen reactive with antibodies in syphilitic sera. Also noteworthy was that other lipoidal constituents of OMs, including the recently discovered glycolipids, did not react with human syphilitic sera. These latter observations suggest that the poor antigenicity of virulent T. pallidum is a function of both the lipid composition and the low protein content of its OM.

The 47-kDa major lipoprotein immunogen of Treponema pallidum is a penicillin-binding protein with carboxypeptidase activity.

The recent model of Treponema pallidum molecular architecture proposes that the vast majority of the bacterium's integral membrane proteins are lipoprotein immunogens anchored in the cytoplasmic membrane while the outer membrane contains only a limited number of surface-exposed transmembrane proteins. This unique model explains, in part, the organism's remarkable ability to evade host immune defenses and establish persistent infection. Our strategy for refining this model involves demonstrating that the physiological functions of treponemal membrane proteins are consistent with their proposed cellular locations. In this study, we used an ampicillin-digoxigenin conjugate to demonstrate by chemiluminescence that the 47-kDa lipoprotein immunogen of T. pallidum (Tpp47) is a penicillin-binding protein. Reexamination of the Tpp47 primary sequence revealed the three amino acid motifs characteristic of penicillin-binding proteins. A recombinant, nonlipidated, soluble form of Tpp47 was used to demonstrate that Tpp47 is a zinc-dependent carboxypeptidase. Escherichia coli expressing Tpp47 was characterized by cell wall abnormalities consistent with altered peptidoglycan biosynthesis. Though the inability to cultivate T. pallidum in vitro and the lack of genetic exchange systems continue to impede treponemal research, this study advances strategies for utilizing E. coli molecular genetics as a means of elucidating the complex relationships between syphilis pathogenesis and T. pallidum membrane biology.

Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence.

This paper describes a highly sensitive new method for the identification of penicillin-binding proteins (PBPs) that is based on the use of an ampicillin-digoxigenin conjugate (DIG-AMP conjugate) which is detected by immunoblotting and chemiluminescence. The sensitivity of chemiluminescence permitted X-ray film exposure times to be decreased to minutes, as opposed to the days or weeks which are requisite when conventionally radiolabeled beta-lactams are used. Coupling of ampicillin to digoxigenin yielded a product containing digoxigenin (detected by chemiluminescence) which also was inhibitory for Staphylococcus aureus and Escherichia coli. Unconjugated digoxigenin at concentrations of up to 100 micrograms/ml was not inhibitory for either organism. For S. aureus the MICs of DIG-AMP (0.7 microgram of conjugated ampicillin per ml) and of free ampicillin (0.5 microgram/ml) were comparable, indicating that ampicillin retained its bioactivity when coupled to digoxigenin. However, for E. coli the MICs of DIG-AMP (70 micrograms of conjugated ampicillin per ml) and of free ampicillin (8 micrograms/ml) were widely disparate, suggesting that the DIG-AMP conjugate was too large and/or hydrophobic to traverse the E. coli outer membrane via porins. DIG-AMP binding assays with E. coli and S. aureus cell envelopes revealed profiles of PBPs similar to those detected with 125I-ampicillin or [3H]penicillin. DIG-AMP binding to PBPs was completely inhibited in competition experiments with free ampicillin or penicillin, supporting the specificity of the DIG-AMP conjugate for PBPs. DIG-AMP thus represents an advantageous alternative to radioactive beta-lactams for the identification and analysis of PBPs.

Analysis of the N-terminal region of the 47-kilodalton integral membrane lipoprotein of Treponema pallidum.

The 47-kDa lipoprotein is an abundant integral membrane protein and dominant immunogen of Treponema pallidum subsp. pallidum. Previous DNA sequencing of the 47-kDa-lipoprotein gene did not reveal consensus features representative of other bacterial lipoprotein genes; this prompted further analyses with emphasis on elucidation of the N terminus of the molecule. To assist in localizing start signals for the protein, the transcription initiation site for the 47-kDa-antigen gene was determined. RNA isolated from both T. pallidum and recombinant Escherichia coli expressing the 47-kDa antigen was used as a template in reverse transcriptase primer extension. Upon analysis of cDNA products, transcription initiation was localized to one nucleotide in T. pallidum and to two adjacent nucleotides in E. coli. When various primers were used in DNA sequencing reactions for these analyses, a previously undetected nucleotide (G) was found in the purported 5' untranslated region; this altered the upstream reading frame to reveal plausible sites for ribosome binding (GGAGG), translation initiation (GTG start codon), and signal peptidase II processing (Val-Val-Gly-Cys). Discounting acylation, the molecular weight of the mature polypeptide is 45,756 (approximately 46,600 with acylation). To derive nonacylated 47-kDa antigen for further structure-function studies, the 47-kDa-antigen gene was subcloned without acylation signals as a genetic construct encoding a glutathione S-transferase fusion protein; following cleavage with thrombin, the nonacylated 47-kDa protein was hydrophilic rather than amphiphilic but retained its antigenicity when tested against 116 human serum samples from patients with various stages of syphilis.