Significant variability exists in the cytotoxicity of global methicillin-resistant Staphylococcus aureus lineages

Staphylococcus aureus is a major human pathogen where the emergence of antibiotic resistant lineages, such as methicillin-resistant S. aureus (MRSA), is a major health concern. While some MRSA lineages are restricted to the healthcare setting, the epidemiology of MRSA is changing globally, with the rise of specific lineages causing disease in healthy people in the community. In the past two decades, community-associated MRSA (CA-MRSA) has emerged as a clinically important and virulent pathogen associated with serious skin and soft-tissue infections (SSTI). These infections are primarily cytotoxin driven, leading to the suggestion that hypervirulent lineages/multi-locus sequence types (STs) exist. To examine this, we compared the cytotoxicity of 475 MRSA isolates representing five major MRSA STs (ST22, ST93, ST8, ST239 and ST36) by employing a monocyte-macrophage THP-1 cell line as a surrogate for measuring gross cytotoxicity. We demonstrate that while certain MRSA STs contain highly toxic isolates, there is such variability within lineages to suggest that this aspect of virulence should not be inferred from the genotype of any given isolate. Furthermore, by interrogating the accessory gene regulator (Agr) sequences in this collection we identified several Agr mutations that were associated with reduced cytotoxicity. Interestingly, the majority of isolates that were attenuated in cytotoxin production contained no mutations in the agr locus, indicating a role of other undefined genes in S. aureus toxin regulation.

Significant Variability exists in the Toxicity of Global Methicillin-resistant Staphylococcus aureus Lineages.

Staphylococcus aureus is a major human pathogen where the emergence of antibiotic resistant lineages, such as methicillin-resistant S. aureus (MRSA), is a major health concern. While some MRSA lineages are restricted to the healthcare setting, the epidemiology of MRSA is changing globally, with the rise of specific lineages causing disease in healthy people in the community. In the past two decades, community-associated MRSA (CA-MRSA) has emerged as a clinically important and virulent pathogen associated with serious skin and soft-tissue infections (SSTI). These infections are primarily toxin driven, leading to the suggestion that hyper-virulent lineages/multi-locus sequence types (STs) exist. To examine this, we compared the toxic activity of 475 MRSA isolates representing five major MRSA STs (ST22, ST93, ST8, ST239 and ST36) by employing a monocyte-macrophage THP-1 cell line as a surrogate for measuring gross cytotoxicity. We demonstrate that while certain MRSA STs contain highly toxic isolates, there is such variability within lineages to suggest that this aspect of virulence should not be inferred from the genotype of any given isolate. Furthermore, by interrogating the accessory gene regulator (Agr) sequences in this collection we identified several Agr mutations that were associated with reduced toxicity. Interestingly, the majority of isolates that were attenuated in toxin production contained no mutations in the agr locus, indicating a role of other undefined genes in S. aureus toxin regulation.

Resistance of Maize Hybrids to Fusarium graminearum, F. culmorum, and F. verticillioides Ear Rots with Toothpick and Silk Channel Inoculation, as Well as Their Toxin Production

Testing Fusarium resistance to ear rots in maize requires a well-supported methodology and tests for toxin responses. In this study, commercial hybrids were tested for resistance to Fusarium graminearum, Fusarium culmorum, and Fusarium verticillioides (kernel and silk channel), as well as their toxin response. One third of the hybrids tested showed a similar resistance or susceptibility to the three pathogens and their toxin response, but there is no proof for their genetic background being the same or different. The performance of the remaining hybrids was highly variable and supports the idea of different genetic regulation. The mean ear rot severity of the kernel resistance was doubled compared with the silk channel resistance data. The ear rot and toxin tests displayed significant positive correlations, verifying the decisive role of resistance in toxin regulation. Several hybrids, termed toxigenic hybrids, showed significant extra toxin production, indicating an additional food safety risk. The toothpick method gave more reliable results and a better differentiation of genotypes. The resistance to different Fusarium spp. in a specific growing region should be analyzed separately in independent resistance tests. Through this, the food safety risks could be better identified. Susceptible hybrids should not be used for commercial production.

Contribution ofhlaRegulation by SaeR toStaphylococcus aureusUSA300 Pathogenesis

ABSTRACTThe SaeRS two-component system inStaphylococcus aureusis critical for regulation of many virulence genes, includinghla, which encodes alpha-toxin. However, the impact of regulation of alpha-toxin by Sae onS. aureuspathogenesis has not been directly addressed. Here, we mutated the SaeR-binding sequences in thehlaregulatory region and determined the contribution of this mutation tohlaexpression and pathogenesis in strain USA300 JE2. Western blot analyses revealed drastic reduction of alpha-toxin levels in the culture supernatants of SaeR-binding mutant in contrast to the marked alpha-toxin production in the wild type. The SaeR-binding mutation had no significant effect on alpha-toxin regulation by Agr, MgrA, and CcpA. In animal studies, we found that the SaeR-binding mutation did not contribute to USA300 JE2 pathogenesis using a rat infective endocarditis model. However, in a rat skin and soft tissue infection model, the abscesses on rats infected with the mutant were significantly smaller than the abscesses on those infected with the wild type but similar to the abscesses on those infected with asaeRmutant. These studies indicated that there is a direct effect ofhlaregulation by SaeR on pathogenesis but that the effect depends on the animal model used.

Accessory Gene Regulator-1 Locus Is Essential for Virulence and Pathogenesis ofClostridium difficile

ABSTRACTClostridium difficileinfection (CDI) is responsible for most of the definable cases of antibiotic- and hospital-associated diarrhea worldwide and is a frequent cause of morbidity and mortality in older patients.C. difficile, a multidrug-resistant anaerobic pathogen, causes disease by producing toxins A and B, which are controlled by an accessory gene regulator (Agr) quorum signaling system. SomeC. difficilestrains encode two Agr loci in their genomes, designatedagr1andagr2. Theagr1locus is present in all of theC. difficilestrains sequenced to date, whereas theagr2locus is present in a few strains. The functional roles ofagr1andagr2inC. difficiletoxin regulation and pathogenesis were unknown until now. Using allelic exchange, we deleted components of bothagrloci and examined the mutants for toxin production and virulence. The results showed that theagr1mutant cannot produce toxins A and B; toxin production can be restored by complementation with wild-typeagr1. Furthermore, theagr1mutant is able to colonize but unable to cause disease in a murine CDI model. These findings have profound implications for CDI treatment because we have uncovered a promising therapeutic target for the development of nonantibiotic drugs to treat this life-threatening emerging pathogen by targeting the toxins directly responsible for disease.IMPORTANCEWithin the last decade, the number of cases ofC. difficileinfections has been increasing exponentially in the United States, resulting in about 4.8 billion U.S. dollars in health care costs annually. As a multidrug-resistant, spore-forming, anaerobic pathogen,C. difficileoverpopulates the colon after the gut microbiota has been altered by antibiotic therapy. With increasing resistance to antibiotic treatment ofC. difficileinfections, patients are experiencing higher costs of health care and a lower quality of life as treatment options decrease. During infection,C. difficileproduces toxins A and B, which directly cause disease. As a result, the toxins have become promising nonantibiotic treatment targets. Here, we have identified a pathway responsible for activating the production of the toxins. This important finding opens up a unique therapeutic target for the development of a novel nonantibiotic therapy forC. difficileinfections.

Mutagenesis and Functional Characterization of the RNA and Protein Components of the toxIN Abortive Infection and Toxin-Antitoxin Locus of Erwinia

ABSTRACT Bacteria are constantly challenged by bacteriophage (phage) infection and have developed multiple adaptive resistance mechanisms. These mechanisms include the abortive infection systems, which promote “altruistic suicide” of an infected cell, protecting the clonal population. A cryptic plasmid of Erwinia carotovora subsp. atroseptica, pECA1039, has been shown to encode an abortive infection system. This highly effective system is active across multiple genera of gram-negative bacteria and against a spectrum of phages. Designated ToxIN, this two-component abortive infection system acts as a toxin-antitoxin module. ToxIN is the first member of a new type III class of protein-RNA toxin-antitoxin modules, of which there are multiple homologues cross-genera. We characterized in more detail the abortive infection phenotype of ToxIN using a suite of Erwinia phages and performed mutagenesis of the ToxI and ToxN components. We determined the minimal ToxI RNA sequence in the native operon that is both necessary and sufficient for abortive infection and to counteract the toxicity of ToxN. Furthermore, site-directed mutagenesis of ToxN revealed key conserved amino acids in this defining member of the new group of toxic proteins. The mechanism of phage activation of the ToxIN system was investigated and was shown to have no effect on the levels of the ToxN protein. Finally, evidence of negative autoregulation of the toxIN operon, a common feature of toxin-antitoxin systems, is presented. This work on the components of the ToxIN system suggests that there is very tight toxin regulation prior to suicide activation by incoming phage.