Post-discharge decolonization of patients harboring methicillin-resistant Staphylococcus aureus (MRSA) USA300 strains: secondary analysis of the CLEAR Trial

The CLEAR Trial recently found that decolonization reduced infections and hospitalizations in MRSA carriers in the year following hospital discharge. In this secondary analysis, we explored whether decolonization had a similar benefit in the subgroup of trial participants who harbored USA300, using two different definitions for the USA300 strain-type.

Free spermidine evokes superoxide radicals that manifest toxicity

Spermidine and other polyamines alleviate oxidative stress, yet excess spermidine seems toxic to Escherichia coli unless it is neutralized by SpeG, an enzyme for the spermidine N-acetyl transferase function. Besides, a specific mechanism of SpeG function conferring pathogenic fitness to Staphylococcus aureus USA300 strain is unknown. Here, we provide evidence that although spermidine mitigates oxidative stress by lowering hydroxyl radical and hydrogen peroxide levels, excess of it simultaneously triggers the production of superoxide radicals, thereby causing toxicity in the E. coli ΔspeG strain as well as naturally SpeG-deficient S. aureus RN4220 strain. However, wild-type E. coli and S. aureus USA300 with a horizontally-acquired speG gene tolerate applied exogenous spermidine stress. Furthermore, we demonstrate that while RNA-bound spermidine inhibits iron oxidation, free spermidine interacts and oxidizes the iron to evoke superoxide radicals directly. Superoxide radicals thus generated, further affects redox balance and iron homeostasis. Since iron acquisition and metabolism in the host tissues is a challenging task for S. aureus, the current findings helped us explain that the evolutionary gain of SpeG function by S. aureus USA300 strain allows it to neutralize exogenous spermidine- and spermine-mediated toxicity towards iron metabolism inside the host tissues.

Postdischarge Decolonization of Patients Harboring MRSA USA300 Strains: Secondary Analysis of the CLEAR Trial

Background: The Changing Lives by Eradicating Antibiotic Resistance (CLEAR) Trial was a trial of 2,121 recently discharged methicillin-resistant Staphylococcus aureus (MRSA) carriers randomized to MRSA education plus a 5-day decolonization regimen repeated twice monthly for the 6 months following discharge versus MRSA education alone. Decolonization resulted in a 30% reduction in MRSA infection and a 17% reduction in all-cause infection (Huang SS et al, NEJM, 2019) in the year following discharge. We pursued an evaluation of USA300 carriers to determine whether the decolonization benefit differed for this strain type. Methods: A secondary analysis of the CLEAR randomized controlled trial (RCT) was performed, limiting the cohort to participants known to harbor USA300 at or within 30 days of enrollment and who attended all follow-up visits in the year following discharge. Within this subset, we conducted a time-to-event analysis using unadjusted and adjusted Cox proportional-hazard models. Variables in adjusted analyses included demographic data, insurance type, presence of coexisting conditions or medical devices at enrollment, hospitalization or residence in a nursing home in the year before enrollment, receipt of anti-MRSA antibiotics, protocol adherence, and randomization strata. Results: USA300 was identified in 420 of the 783 participants who attended all visits and had strains genetically tested. MRSA infections occurred in 27 of 207 education group participants (0.149 per person year) and in 19 of 213 decolonization group participants (0.099 per-person year). Point estimates from the unadjusted hazard ratios of infection reduction were similar (0.59; 95% CI, 0.32–1.09) to the full trial population (0.61; 95% CI, 0.44–0.85), suggesting nondifferential benefit for the USA300 strain type. Adjusted models were highly similar. Conclusions: The reduction in MRSA infection associated with postdischarge decolonization in the subgroup of participants who harbored the USA300 strain-type was consistent with overall trial findings. Although the original trial was not powered for the evaluation of a USA300 subset, this RCT provides a valuable design for assessing the magnitude of strain-specific responsiveness to decolonization during a time when national rates of MRSA invasive disease have plateaued and USA300 is responsible for an increasing proportion of infections. These data suggest that postdischarge decolonization should be similarly effective in carriers of either USA300 or healthcare-associated MRSA strains.Funding: NoneDisclosure: Gabrielle M. Gussin, Stryker (Sage Products): Conducting studies in which antiseptic product is provided to participating hospitals and nursing homes. Clorox: Conducting studies in which antiseptic product is provided to participating hospitals and nursing homes. Medline: Conducting studies in which antiseptic product is provided to participating hospitals and nursing homes. Xttrium: Conducting studies in which antiseptic product is provided to participating hospitals and nursing homes. Mohamad Sater, Salary-Day Zero Diagnostics.

Occurrence of cross-resistance and β-lactam seesaw effect in glycopeptide-, lipopeptide- and lipoglycopeptide-resistant MRSA correlates with membrane phosphatidylglycerol levels

Background Glycopeptides (GPs), lipopeptides (LPs) and lipoglycopeptides (LGPs) are related antimicrobials important for the management of invasive MRSA infections. Cross-resistance among these antibiotics in MRSA is well documented, as is the observation that susceptibility of MRSA to β-lactams increases as susceptibility to GPs and LPs decreases (i.e. the seesaw effect). Efforts to understand the relationship between GP/LP/LGP cross-resistance and the seesaw effect have focused on the PBPs, but the role of lipid metabolism has not been investigated. Objectives Since the cell membrane is structurally and metabolically integrated with the cell wall and anchors associated proteins, including PBPs, we examined the relationship between membrane lipid composition and the phenomena of cross-resistance among GPs/LPs/LGPs and the β-lactam seesaw effect. Methods We selected for daptomycin, vancomycin and dalbavancin resistance using the USA300 strain JE2 and evaluated the resulting mutants by WGS, MS-based lipidomics and antimicrobial susceptibility testing to assess the relationship between membrane composition, cross-resistance, and the seesaw effect. Results We observed cross-resistance to GPs/LPs/LGPs among the selected strains and the seesaw effect against various β-lactams, depending on the PBP targets of the particular β-lactam. We found that modification of membrane composition occurs not only in daptomycin-selected strains, but also vancomycin- and dalbavancin-selected strains. Significantly, we observed that the abundance of most phosphatidylglycerols positively correlates with MICs of GPs/LPs/LGPs and negatively correlates with the MICs of β-lactams. Conclusions These studies demonstrate a major association between membrane remodelling, cross-resistance and the seesaw effect.

Systematic evasion of the restriction-modification barrier in bacteria

Bacteria that are recalcitrant to genetic manipulation using modern in vitro techniques are termed genetically intractable. Genetic intractability is a fundamental barrier to progress that hinders basic, synthetic, and translational microbiology research and development beyond a few model organisms. The most common underlying causes of genetic intractability are restriction-modification (RM) systems, ubiquitous defense mechanisms against xenogeneic DNA that hinder the use of genetic approaches in the vast majority of bacteria and exhibit strain-level variation. Here, we describe a systematic approach to overcome RM systems. Our approach was inspired by a simple hypothesis: if a synthetic piece of DNA lacks the highly specific target recognition motifs for a host’s RM systems, then it is invisible to these systems and will not be degraded during artificial transformation. Accordingly, in this process, we determine the genome and methylome of an individual bacterial strain and use this information to define the bacterium’s RM target motifs. We then synonymously eliminate RM targets from the nucleotide sequence of a genetic tool in silico, synthesize an RM-silent “SyngenicDNA” tool, and propagate the tool as minicircle plasmids, termed SyMPL (SyngenicDNA Minicircle Plasmid) tools, before transformation. In a proof-of-principle of our approach, we demonstrate a profound improvement (five orders of magnitude) in the transformation of a clinically relevant USA300 strain of Staphylococcus aureus. This stealth-by-engineering SyngenicDNA approach is effective, flexible, and we expect in future applications could enable microbial genetics free of the restraints of restriction-modification barriers.

SyngenicDNA: stealth-based evasion of restriction-modification barriers during bacterial genetic engineering

Restriction-modification (RM) systems hinder the use of genetic approaches in the vast majority of bacteria. Here, we describe a systematic approach to adapt genetic tools for use in bacteria that are genetically intractable or poorly tractable owing to active RM defenses. In this process, we determine the genome and methylome of a bacterial strain and use this information to define the bacterium’s RM target motifs. We then synonymously eliminate RM targets from the nucleotide sequence of a genetic toolin silico,synthesize an RM-silent ‘SyngenicDNA’ tool and propagate the tool as novel minicircle plasmids, termed SyMPL tools, before transformation. Using SyngenicDNA and SyMPL tools, we achieved a profound, >100,000- fold, improvement in the transformation of a clinically relevant USA300 strain ofStaphylococcus aureusdemonstrating the efficacy of these approaches for evading RM systems. The SyngenicDNA and SyMPL approaches are effective, flexible, and should be broadly applicable in microbial genetics. We expect these will facilitate a new era of microbial genetics free of the restraints of restriction-modification barriers.

Phenol-Soluble Modulin Peptides Contribute to Influenza A Virus-Associated Staphylococcus aureus Pneumonia

ABSTRACT Influenza A virus (IAV) infection is often followed by secondary bacterial lung infection, which is a major reason for severe, often fatal pneumonia. Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains such as USA300 cause particularly severe and difficult-to-treat cases of IAV-associated pneumonia. CA-MRSA strains are known to produce extraordinarily large amounts of phenol-soluble modulin (PSM) peptides, which are important cytotoxins and proinflammatory molecules that contribute to several types of S. aureus infection. However, their potential role in pneumonia has remained elusive. We determined the impact of PSMs on human lung epithelial cells and found that PSMs are cytotoxic and induce the secretion of the proinflammatory cytokine interleukin-8 (IL-8) in these cells. Both effects were boosted by previous infection with the 2009 swine flu pandemic IAV H1N1 strain, suggesting that PSMs may contribute to lung inflammation and damage in IAV-associated S. aureus pneumonia. Notably, the PSM-producing USA300 strain caused a higher mortality rate than did an isogenic PSM-deficient mutant in a mouse IAV-S. aureus pneumonia coinfection model, indicating that PSMs are major virulence factors in IAV-associated S. aureus pneumonia and may represent important targets for future anti-infective therapies.

Evolving Epidemiology ofStaphylococcus aureusBacteremia

BACKGROUNDMethicillin-resistantStaphylococcus aureus(MRSA) infections due to USA300 have become widespread in community and healthcare settings. It is unclear whether risk factors for bloodstream infections (BSIs) differ by strain type.OBJECTIVETo examine the epidemiology ofS. aureusBSIs, including USA300 and non-USA300 MRSA strains.DESIGNRetrospective observational study with molecular analysis.SETTINGLarge urban public hospital.PATIENTSIndividuals withS. aureusBSIs from January 1, 2007 through December 31, 2013.METHODSWe used electronic surveillance data to identify cases ofS. aureusBSI. Available MRSA isolates were analyzed by pulsed-field gel electrophoresis. Poisson regression was used to evaluate changes in BSI incidence over time. Risk factor data were collected by medical chart review and logistic regression was used for multivariate analysis of risk factors.RESULTSA total of 1,015 cases ofS. aureusBSIs were identified during the study period; 36% were due to MRSA. The incidence of hospital-onset (HO) MRSA BSIs decreased while that of community-onset (CO) MRSA BSIs remained stable. The rate of CO– and HO– methicillin-susceptibleS. aureusinfections both decreased over time. More than half of HO-MRSA BSIs were due to the USA300 strain type and for 4 years, the proportion of HO-MRSA BSIs due to USA300 exceeded 60%. On multivariate analysis, current or former drug use was the only epidemiologic risk factor for CO- or HO-MRSA BSIs due to USA300 strains.CONCLUSIONSUSA300 MRSA is endemic in communities and hospitals and certain populations (eg, those who use illicit drugs) may benefit from enhanced prevention efforts in the community.Infect. Control Hosp. Epidemiol.2015;36(12):1417–1422

Pediatric Staphylococcus aureus Isolate Genotypes and Infections from the Dawn of the Community-Associated Methicillin-Resistant S. aureus Epidemic Era in Chicago, 1994 to 1997

Widespread infections with community-associated (CA) methicillin-resistantStaphylococcus aureus(MRSA) have occurred in the United States with the dissemination of the USA300 strain beginning in 2000. We examined 105 isolates obtained from children treated at the University of Chicago from 1994 to 1997 (75 methicillin-susceptibleS. aureus[MSSA] and 30 MRSA isolates) in order to investigate for possible evidence of USA300 during this period. Infections were defined epidemiologically based on medical record review. The isolates underwent multilocus sequence typing (MLST), as well as assays for the Panton-Valentine leukocidin (PVL) genes, the protein A gene (spa), andarcAandopp3, proxy markers for the arginine catabolic mobile element (ACME), characteristic of USA300 MRSA. MRSA isolates also underwent staphylococcal cassette chromosomemec(SCCmec) typing and pulsed-field gel electrophoresis (PFGE) subtyping. MSSA isolates belonged to 17 sequence type (ST) groups. The 12 epidemiologically defined CA-MRSA infection isolates were either ST1 (n= 4) or ST8 (n= 8). They belonged to 3 different PFGE types: USA100 (n= 1), USA400 (n= 5), and USA500 (n= 6). Among the CA-MRSA infection isolates, 8 (67%) were PVL+. None of the MRSA or MSSA isolates containedarcAoropp3. Only one MRSA isolate was USA300 by PFGE. This was a health care-associated (HA) MRSA isolate, negative for PVL, that carried SCCmectype II. USA300 with its characteristic features was not identified in the collection from the years 1994 to 1997.