Emily K. Crispell

Dissecting Vancomycin-Intermediate Resistance in Staphylococcus aureus Using Genome-Wide Association

Vancomycin-intermediate Staphylococcus aureus (VISA) is currently defined as having minimal inhibitory concentration (MIC) of 4–8 µg/ml. VISA evolves through changes in multiple genetic loci with at least 16 candidate genes identified in clinical and in vitro-selected VISA strains. We report a whole-genome comparative analysis of 49 vancomycin-sensitive S. aureus and 26 VISA strains. Resistance to vancomycin was determined by broth microdilution, Etest, and population analysis profile-area under the curve (PAP-AUC). Genome-wide association studies (GWAS) of 55,977 single-nucleotide polymorphisms identified in one or more strains found one highly significant association (P = 8.78E-08) between a nonsynonymous mutation at codon 481 (H481) of the rpoB gene and increased vancomycin MIC. Additionally, we used a database of public S. aureus genome sequences to identify rare mutations in candidate genes associated with VISA. On the basis of these data, we proposed a preliminary model called ECM+RMCG for the VISA phenotype as a benchmark for future efforts. The model predicted VISA based on the presence of a rare mutation in a set of candidate genes (walKR, vraSR, graSR, and agrA) and/or three previously experimentally verified mutations (including the rpoB H481 locus) with an accuracy of 81% and a sensitivity of 73%. Further, the level of resistance measured by both Etest and PAP-AUC regressed positively with the number of mutations present in a strain. This study demonstrated 1) the power of GWAS for identifying common genetic variants associated with antibiotic resistance in bacteria and 2) that rare mutations in candidate gene, identified using large genomic data sets, can also be associated with resistance phenotypes.

Antimicrobial Peptide Resistance Mechanisms of Gram-Positive Bacteria.

Antimicrobial peptides, or AMPs, play a significant role in many environments as a tool to remove competing organisms. In response, many bacteria have evolved mechanisms to resist these peptides and prevent AMP-mediated killing. The development of AMP resistance mechanisms is driven by direct competition between bacterial species, as well as host and pathogen interactions. Akin to the number of different AMPs found in nature, resistance mechanisms that have evolved are just as varied and may confer broad-range resistance or specific resistance to AMPs. Specific mechanisms of AMP resistance prevent AMP-mediated killing against a single type of AMP, while broad resistance mechanisms often lead to a global change in the bacterial cell surface and protect the bacterium from a large group of AMPs that have similar characteristics. AMP resistance mechanisms can be found in many species of bacteria and can provide a competitive edge against other bacterial species or a host immune response. Gram-positive bacteria are one of the largest AMP producing groups, but characterization of Gram-positive AMP resistance mechanisms lags behind that of Gram-negative species. In this review we present a summary of the AMP resistance mechanisms that have been identified and characterized in Gram-positive bacteria. Understanding the mechanisms of AMP resistance in Gram-positive species can provide guidelines in developing and applying AMPs as therapeutics, and offer insight into the role of resistance in bacterial pathogenesis.

Detection of Staphylococcus aureus Isolates with Heterogeneous Intermediate-Level Resistance to Vancomycin in the United States

ABSTRACT The prevalence of heterogeneous intermediate-level resistance to vancomycin (hVISA) in Staphylococcus aureus was assessed by screening a large collection of recent isolates. Susceptibility testing by the Clinical and Laboratory Standards Institute broth microdilution method and the Etest GRD (glycopeptide resistance detection) method (bioMérieux) was performed on 4,210 clinically significant S. aureus isolates obtained in 2009 from 43 U.S. centers. Isolates with Etest GRD-positive results for hVISA were evaluated further by repeat GRD testing and population analysis profiling–area under the curve (PAP-AUC) analysis. No VISA (vancomycin MIC, 4 to 8 μg/ml) or vancomycin-resistant (MIC ≥ 16 μg/ml) strains were detected. The Etest GRD screen for hVISA was initially positive for 68 isolates (1.6%; all by teicoplanin MIC ≥ 8 μg/ml at 24 or 48 h). Among those 68 isolates, 45 were reproducibly GRD positive. PAP-AUC testing confirmed only 11 isolates as hVISA (all had reproducible GRD-positive results). The 11 hVISA isolates were from nine medical centers and appeared genetically diverse (ten different PFGE types). The rates of resistance (including intermediate) for hVISA were as follows: oxacillin, 82%; erythromycin, 82%; clindamycin, 73%; levofloxacin, 73%; trimethoprim-sulfamethoxazole, 9%; and daptomycin, 9%. All hVISA isolates were susceptible to linezolid, tigecycline, and ceftaroline. Our data suggest that the overall prevalence of hVISA in the United States is low (0.3%). The hVISA isolates represented 10.5% of isolates with vancomycin MICs of 2 μg/ml and 0.1% of isolates with vancomycin MICs of 1 μg/ml. The positive predictive value of GRD Etest for hVISA was 16.2% for initial screen positive and 24.4% for reproducibly positive results.

Decline in Pneumococcal Nasopharyngeal Carriage of Vaccine Serotypes After the Introduction of the 13-Valent Pneumococcal Conjugate Vaccine in Children in Atlanta, Georgia.

Background: Streptococcus pneumoniae (SP) serotype distribution among nasopharyngeal (NP) carriage isolates changed significantly after the introduction of the 7-valent pneumococcal conjugate vaccine (PCV7). We evaluated the impact on NP carriage and invasive disease of SP after the introduction of the 13-valent PCV (PCV13) in March 2010. Methods: NP swabs were collected from children 6–59 months of age in an emergency department from July 2010 to June 2013. After broth enrichment, samples were cultured for SP and isolates were serotyped. Clinical and immunization records were reviewed. Findings during 6 sequential 6-month study periods were compared. Surveillance isolates of invasive disease isolates were reviewed. Results: A total of 2048 children were enrolled, and 656 (32%) were SP carriers. Mean age of carriers was 27 months, 54% were males. Carriage was higher among day-care attendees (P < 0.01) and children with respiratory tract illnesses (P < 0.5) and otitis media (P < 0.01). Commonly carried serotypes included 35B (15.2%), 15B/C (14.2%), 19A (9.6%), 11A (8%), 23B (5.6%), 6C (5.3%), 21 (5%), and 15A (5%); 13.9% were PCV13 serotypes. The proportion of children with SP carriage remained stable but the serotype distribution changed during the study period. Among carriers, PCV13 serotypes declined from 29% (36/124) to 3% (3/99; P < 0.0001), predominantly because of decline of serotype 19A from 25.8% (32/124) to 3% (3/99; P < 0.0001); non-PCV13 serotypes (excluding 6C) increased from 68.4% (78/114) to 97% (95/98; P < 0.0001); serotype 35B significantly increased from 8.9% (11/124) to 25.3% (25/99; P < 0.05). Nonsusceptibility to ceftriaxone declined from 22.6% (28/124) to 0% (0/99; P < 0.0001), with a similar decline in penicillin nonsusceptibility. Conclusions: Introduction of PCV13 for universal infant use was associated with significant reductions in nasopharyngeal carriage of PCV13 serotypes and resistant strains. Carriage of non-PCV13 serotypes increased modestly, particularly serotype 35B. Further investigation is warranted to determine whether nonvaccine pneumococcal serotypes carried in the nasopharynx are associated with significant replacement disease.

A Mutation in the PP2C Phosphatase Gene in a Staphylococcus aureus USA300 Clinical Isolate with Reduced Susceptibility to Vancomycin and Daptomycin

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin (MIC of 4 to 8 μg/ml) are referred to as vancomycin-intermediate S. aureus (VISA). In this study, we characterized two isogenic USA300 S. aureus isolates collected sequentially from a single patient with endocarditis where the S. aureus isolate changed from being susceptible to vancomycin (VSSA) (1 μg/ml) to VISA (8 μg/ml). In addition, the VISA isolate lost beta-lactamase activity and showed increased resistance to daptomycin and linezolid. The two strains did not differ in growth rate, but the VISA isolate had a thickened cell wall and was less autolytic. Transcriptome sequencing (RNA-seq) analysis comparing the two isolates grown to late exponential phase showed significant differences in transcription of cell surface protein genes (spa, SBI [second immunoglobulin-binding protein of S. aureus], and fibrinogen-binding proteins), regulatory genes (agrBCA, RNAIII, sarT, and saeRS), and others. Using whole-genome shotgun resequencing, we identified 6 insertion/deletion mutations between the VSSA and VISA isolates. A protein phosphatase 2C (PP2C) family phosphatase had a 6-bp (nonframeshift) insertion mutation in a highly conserved metal binding domain. Complementation of the clinical VISA isolate with a wild-type copy of the PP2C gene reduced the vancomycin and daptomycin MICs and increased autolytic activity, suggesting that this gene contributed to the reduced vancomycin susceptibility phenotype acquired in vivo. Creation of de novo mutants from the VSSA strain resulted in different mutations, demonstrating that reduced susceptibility to vancomycin in USA300 strains can occur via multiple routes, highlighting the complex nature of the VISA phenotype.

Activities of Vancomycin, Ceftaroline, and Mupirocin against Staphylococcus aureus Isolates Collected in a 2011 National Surveillance Study in the United States

ABSTRACT Forty-two medical centers from throughout the United States participating in a longitudinal surveillance program were asked to submit 100 consecutive Staphylococcus aureus isolates during July to December 2011. Susceptibility testing using CLSI broth microdilution and mecA detection by PCR analysis was performed on the 4,131 isolates collected. Methods employing Etest glycopeptide resistance detection (GRD; bioMérieux) and brain heart infusion agar containing 4 μg/ml vancomycin (BHIV) were used to screen methicillin-resistant S. aureus (MRSA) isolates for heterogeneous intermediate-level resistance to vancomycin (hVISA). Isolates with positive hVISA screen results were confirmed by population analysis profiling-area under the curve (PAP-AUC) determinations. The genetic relatedness of hVISA, ceftaroline-nonsusceptible, or high-level (HL) mupirocin resistance MRSA isolates was assessed by pulsed-field gel electrophoresis (PFGE). Among 2,093 MRSA isolates, the hVISA screen results were positive with 47 isolates by Etest GRD and 30 isolates by BHIV agar screen. Twenty-five of the GRD- or BHIV screen-positive isolates were confirmed as hVISA by PAP-AUC testing. Results of the current study were compared to results obtained from prior surveillance performed in 2009. The prevalence of hVISA among MRSA isolates was higher in 2011 than in 2009 (1.2% versus 0.4%, P = 0.003), especially for isolates with a vancomycin MIC of 2 (45.4% versus 14.3%, P = 0.01). The overall rate of ceftaroline susceptibility in the current study was 99.4% (one hVISA isolate had an intermediate ceftaroline MIC). HL mupirocin resistance increased from 2.2% in 2009 to 3.2% in 2011 (P = 0.006). Although overall rates of hVISA and HL mupirocin resistance are low, they have increased since 2009.

Prevalence of blaZ gene types and the inoculum effect with cefazolin among bloodstream isolates of methicillin-susceptible Staphylococcus aureus.

ABSTRACT We sought to define the prevalence of blaZ gene types and the inoculum effect to cefazolin among methicillin-susceptible Staphylococcus aureus (MSSA) bloodstream infections. The blaZ gene was present in 142/185 (77%) isolates. A total of 50 (27%) isolates had a ≥4-fold increase in the cefazolin MIC from a standard to a high inoculum, and 8 (4%) demonstrated a nonsusceptible cefazolin MIC, all type A blaZ strains. The efficacy of cefazolin in the presence of the inoculum effect requires further study.

Antibiotic failure mediated by a resistant subpopulation in Enterobacter cloacae.

Antibiotic resistance is a major public health threat, further complicated by unexplained treatment failures caused by bacteria that appear antibiotic susceptible. We describe an Enterobacter cloacae isolate harbouring a minor subpopulation that is highly resistant to the last-line antibiotic colistin. This subpopulation was distinct from persisters, became predominant in colistin, returned to baseline after colistin removal and was dependent on the histidine kinase PhoQ. During murine infection, but in the absence of colistin, innate immune defences led to an increased frequency of the resistant subpopulation, leading to inefficacy of subsequent colistin therapy. An isolate with a lower-frequency colistin-resistant subpopulation similarly caused treatment failure but was misclassified as susceptible by current diagnostics once cultured outside the host. These data demonstrate the ability of low-frequency bacterial subpopulations to contribute to clinically relevant antibiotic resistance, elucidating an enigmatic cause of antibiotic treatment failure and highlighting the critical need for more sensitive diagnostics.

The Atypical Occurrence of Two Biotin Protein Ligases in Francisella novicida Is Due to Distinct Roles in Virulence and Biotin Metabolism

ABSTRACT The physiological function of biotin requires biotin protein ligase activity in order to attach the coenzyme to its cognate proteins, which are enzymes involved in central metabolism. The model intracellular pathogen Francisella novicida is unusual in that it encodes two putative biotin protein ligases rather than the usual single enzyme. F. novicida BirA has a ligase domain as well as an N-terminal DNA-binding regulatory domain, similar to the prototypical BirA protein in E. coli. However, the second ligase, which we name BplA, lacks the N-terminal DNA binding motif. It has been unclear why a bacterium would encode these two disparate biotin protein ligases, since F. novicida contains only a single biotinylated protein. In vivo complementation and enzyme assays demonstrated that BirA and BplA are both functional biotin protein ligases, but BplA is a much more efficient enzyme. BirA, but not BplA, regulated transcription of the biotin synthetic operon. Expression of bplA (but not birA) increased significantly during F. novicida infection of macrophages. BplA (but not BirA) was required for bacterial replication within macrophages as well as in mice. These data demonstrate that F. novicida has evolved two distinct enzymes with specific roles; BplA possesses the major ligase activity, whereas BirA acts to regulate and thereby likely prevent wasteful synthesis of biotin. During infection BplA seems primarily employed to maximize the efficiency of biotin utilization without limiting the expression of biotin biosynthetic genes, representing a novel adaptation strategy that may also be used by other intracellular pathogens. IMPORTANCE Our findings show that Francisella novicida has evolved two functional biotin protein ligases, BplA and BirA. BplA is a much more efficient enzyme than BirA, and its expression is significantly induced upon infection of macrophages. Only BplA is required for F. novicida pathogenicity, whereas BirA prevents wasteful biotin synthesis. These data demonstrate that the atypical occurrence of two biotin protein ligases in F. novicida is linked to distinct roles in virulence and biotin metabolism. Our findings show that Francisella novicida has evolved two functional biotin protein ligases, BplA and BirA. BplA is a much more efficient enzyme than BirA, and its expression is significantly induced upon infection of macrophages. Only BplA is required for F. novicida pathogenicity, whereas BirA prevents wasteful biotin synthesis. These data demonstrate that the atypical occurrence of two biotin protein ligases in F. novicida is linked to distinct roles in virulence and biotin metabolism.

Cepheid Xpert MRSA Cycle Threshold in Discordant Colonization Results and as a Quantitative Measure of Nasal Colonization Burden

ABSTRACT We analyzed the cycle threshold (CT ) of PCR surveillance MRSA swabs obtained from veterans. Lower CT on admission was associated with a positive culture from nasal swabs at discharge. Compared to PCR, direct plating of nasal swabs performed poorly, especially for patients with an elevated CT . The CT is strongly correlated with quantitative nasal cultures. Clinical and infection control applications of the CT have yet to be defined and warrant further evaluation.