Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) Isolates of Swine Origin Form Robust Biofilms

Methicillin-resistant Staphylococcus aureus (MRSA) colonization of livestock animals is common and prevalence rates for pigs have been reported to be as high as 49%. Mechanisms contributing to the persistent carriage and high prevalence rates of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) strains in swine herds and production facilities have not been investigated. One explanation for the high prevalence of MRSA in swine herds is the ability of these organisms to exist as biofilms. In this report, the ability of swine LA-MRSA strains, including ST398, ST9, and ST5, to form biofilms was quantified and compared to several swine and human isolates. The contribution of known biofilm matrix components, polysaccharides, proteins and extracellular DNA (eDNA), was tested in all strains as well. All MRSA swine isolates formed robust biofilms similar to human clinical isolates. The addition of Dispersin B had no inhibitory effect on swine MRSA isolates when added at the initiation of biofilm growth or after pre-established mature biofilms formed. In contrast, the addition of proteinase K inhibited biofilm formation in all strains when added at the initiation of biofilm growth and was able to disperse pre-established mature biofilms. Of the LA-MRSA strains tested, we found ST398 strains to be the most sensitive to both inhibition of biofilm formation and dispersal of pre-formed biofilms by DNaseI. Collectively, these findings provide a critical first step in designing strategies to control or eliminate MRSA in swine herds.     

Effectiveness of Hospital-Wide Methicillin-Resistant Staphylococcus aureus (MRSA) Infection Control Policies Differs by Ward Specialty

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of preventable nosocomial infections and is endemic in hospitals worldwide. The effectiveness of infection control policies varies significantly across hospital settings. The impact of the hospital context towards the rate of nosocomial MRSA infections and the success of infection control is understudied. We conducted a modelling study to evaluate several infection control policies in surgical, intensive care, and medical ward specialties, each with distinct ward conditions and policies, of a tertiary public hospital in Sydney, Australia. We reconfirm hand hygiene as the most successful policy and find it to be necessary for the success of other policies. Active screening for MRSA, patient isolation in single-bed rooms, and additional staffing were found to be less effective. Across these ward specialties, MRSA transmission risk varied by 13% and reductions in the prevalence and nosocomial incidence rate of MRSA due to infection control policies varied by up to 45%. Different levels of infection control were required to reduce and control nosocomial MRSA infections for each ward specialty. Infection control policies and policy targets should be specific for the ward and context of the hospital. The model we developed is generic and can be calibrated to represent different ward settings and pathogens transmitted between patients indirectly through health care workers. This can aid the timely and cost effective design of synergistic and context specific infection control policies.     

Nasal Colonization of Humans with Methicillin-Resistant Staphylococcus aureus (MRSA) CC398 with and without Exposure to Pigs

Background

Studies in several European countries and in North America revealed a frequent nasal colonization of livestock with MRSA CC398 and also in humans with direct professional exposure to colonized animals. The study presented here addresses the question of further transmission to non exposed humans.

Methods

After selecting 47 farms with colonized pigs in different regions of Germany we sampled the nares of 113 humans working daily with pigs and of their 116 non exposed family members. The same was performed in 18 veterinarians attending pig farms and in 44 of their non exposed family members. For investigating transmission beyond families we samples the nares of 462 pupils attending a secondary school in a high density pig farming area. MRSA were detected by direct culture on selective agar. The isolates were typed by means of spa-sequence typing and classification of SCCmec elements. For attribution of spa sequence types to clonal lineages as defined by multi locus sequence typing we used the BURP algorithm. Antibiotic susceptibility testing was performed by microbroth dilution assay.

Results

At the farms investigated 86% of humans exposed and only 4.3% of their family members were found to carry MRSA exhibiting spa-types corresponding to clonal complex CC398. Nasal colonization was also found in 45% of veterinarians caring for pig farms and in 9% of their non exposed family members. Multivariate analysis revealed that antibiotic usage prior to sampling beard no risk with respect to colonization. From 462 pupils only 3 were found colonized, all 3 were living on pig farms.

Conclusion

These results indicate that so far the dissemination of MRSA CC398 to non exposed humans is infrequent and probably does not reach beyond familial communities.     

Synergism between Medihoney and Rifampicin against Methicillin-Resistant Staphylococcus aureus (MRSA)

Skin and chronic wound infections caused by highly antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) are an increasing and urgent health problem worldwide, particularly with sharp increases in obesity and diabetes. New Zealand manuka honey has potent broad-spectrum antimicrobial activity, has been shown to inhibit the growth of MRSA strains, and bacteria resistant to this honey have not been obtainable in the laboratory. Combinational treatment of chronic wounds with manuka honey and common antibiotics may offer a wide range of advantages including synergistic enhancement of the antibacterial activity, reduction of the effective dose of the antibiotic, and reduction of the risk of antibiotic resistance. The aim of this study was to investigate the effect of Medihoney in combination with the widely used antibiotic rifampicin on S. aureus. Using checkerboard microdilution assays, time-kill curve experiments and agar diffusion assays, we show a synergism between Medihoney and rifampicin against MRSA and clinical isolates of S. aureus. Furthermore, the Medihoney/rifampicin combination stopped the appearance of rifampicin-resistant S. aureus in vitro. Methylglyoxal (MGO), believed to be the major antibacterial compound in manuka honey, did not act synergistically with rifampicin and is therefore not the sole factor responsible for the synergistic effect of manuka honey with rifampicin. Our findings support the idea that a combination of honey and antibiotics may be an effective new antimicrobial therapy for chronic wound infections.     

耐甲氧西林金黄色葡萄球菌染色体盒的研究进展

耐甲氧西林金黄色葡萄球菌（MRSA）的产生是由甲氧西林敏感的金黄色葡萄球菌（MSSA）获得外源性的SCCmec所致。MRSA菌株可以产生一种新的青霉素结合蛋白PBP2a,PBP2a降低了与β-内酰胺类抗生素的亲合力,从而对β-内酰胺类抗生素产生耐药性。PBP2a由mecA基因编码,mecA基因存在于葡萄球菌盒式染色体（Staphylococcal cassette chromosome mec,SCCmec）中,SCCmec是一种可移动的遗传元件,该元件还携带除mecA基因外的其他抗菌药物的耐药基因,造成多重耐药（Multidrug-resistance,MDR）。SCCmec目前主要分为8型,其中又分为若干亚型。SCCmec的基因型与MRSA的流行背景有关,不同地区的SCCmec基因分型分布可能不同。     

Low Incidence of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Bacteraemia in The Netherlands in 2009

Methicillin-resistant Staphylococcus aureus (MRSA) is a worldwide problem in both hospitals and communities all over the world. In 2003, a new MRSA clade emerged with a reservoir in pigs and veal calves: livestock-associated MRSA (LA-MRSA). We wanted to estimate the incidence of bacteraemias due to LA-MRSA using national surveillance data from 2009 in the Netherlands. We found a low incidence of LA-MRSA and MRSA bacteraemia episodes, compared to bacteraemias caused by all S. aureus (0.04, 0.18 and 19.3 episodes of bacteraemia per 100,000 inhabitants per year, respectively). LA-MRSA and MRSA were uncommon compared to numbers from other countries as well. MRSA in general and LA-MRSA in specific does not appear to be a public health problem in the Netherlands now. The low incidence of LA-MRSA bacteraemia episodes may best be explained by differences in the populations affected by LA-MRSA versus other MRSA. However, reduced virulence of the strain involved, and the effectiveness of the search and destroy policy might play a role as well.     

Transmission and Persistence of Livestock-Associated Methicillin-Resistant Staphylococcus aureus among Veterinarians and Their Household Members

After the first isolation of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in 2003, this MRSA variant quickly became the predominant MRSA obtained from humans as part of the Dutch national MRSA surveillance. Previous studies have suggested that human-to-human transmission of LA-MRSA, compared to that of other MRSA lineages, rarely occurs. However, these reports describe the transmission of LA-MRSA based on epidemiology and limited molecular characterization of isolates, making it difficult to assess whether transmission actually occurred. In this study, we used whole-genome maps (WGMs) to identify possible transmission of LA-MRSA between humans. For this, we used LA-MRSA isolates originating from a 2-year prospective longitudinal cohort study in which livestock veterinarians and their household members were repeatedly sampled for the presence of S. aureus. A considerable degree of genotypic variation among LA-MRSA strains was observed. However, there was very limited variability between the maps of the isolates originating from the same veterinarian, indicating that each of the veterinarians persistently carried or had reacquired the same LA-MRSA strain. Comparison of WGMs revealed that LA-MRSA transmission had likely occurred within virtually every veterinarian household. Yet only a single LA-MRSA strain per household appeared to be involved in transmission. The results corroborate our previous finding that LA-MRSA is genetically diverse. Furthermore, this study shows that transmission of LA-MRSA between humans occurs and that carriage of LA-MRSA can be persistent, thus posing a potential risk for spread of this highly resistant pathogen in the community.     

Clinical Significance of Methicillin-Resistant Staphylococcus aureus Colonization on Hospital Admission: One-Year Infection Risk

Background

Methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization among inpatients is a well-established risk factor for MRSA infection during the same hospitalization, but the long-term risk of MRSA infection is uncertain. We performed a retrospective cohort study to determine the one-year risk of MRSA infection among inpatients with MRSA-positive nasal polymerase chain reaction (PCR) tests confirmed by positive nasal culture (Group 1), patients with positive nasal PCR but negative nasal culture (Group 2), and patients with negative nasal PCR (Group 3).

Methodology/Principal Findings

Subjects were adults admitted to a four-hospital system between November 1, 2006 and March 31, 2011, comprising 195,255 admissions. Patients underwent nasal swab for MRSA PCR upon admission; if positive, nasal culture for MRSA was performed; if recovered, MRSA was tested for Panton-Valentine Leukocidin (PVL). Outcomes included MRSA-positive clinical culture and skin and soft tissue infection (SSTI). Group 1 patients had a one-year risk of MRSA-positive clinical culture of 8.0% compared with 3.0% for Group 2 patients, and 0.6% for Group 3 patients (p<0.001). In a multivariable model, the hazard ratios for future MRSA-positive clinical culture were 6.52 (95% CI, 5.57 to 7.64) for Group 1 and 3.40 (95% CI, 2.70 to 4.27) for Group 2, compared with Group 3 (p<0.0001). History of MRSA and concurrent MRSA-positive clinical culture were significant risk factors for future MRSA-positive clinical culture. Group 1 patients colonized with PVL-positive MRSA had a one-year risk of MRSA-positive clinical culture of 10.1%, and a one-year risk of MRSA-positive clinical culture or SSTI diagnosis of 21.7%, compared with risks of 7.1% and 12.5%, respectively, for patients colonized with PVL-negative MRSA (p = 0.04, p = 0.005, respectively).

Conclusions/Significance

MRSA nasal colonization is a significant risk factor for future MRSA infection; more so if detected by culture than PCR. Colonization with PVL-positive MRSA is associated with greater risk than PVL-negative MRSA.     

Effects of Subinhibitory Concentrations of Ceftaroline on Methicillin-Resistant Staphylococcus aureus (MRSA) Biofilms

Ceftaroline (CPT) is a novel cephalosporin with in vitro activity against Staphylococcus aureus. Ceftaroline exhibits a level of binding affinity for PBPs in S. aureus including PBP2a of methicillin-resistant S. aureus (MRSA). The aims of this study were to investigate the morphological, physiological and molecular responses of MRSA clinical strains and MRSA biofilms to sub-MICs (1/4 and 1/16 MIC) of ceftaroline by using transmission, scanning and confocal microscopy. We have also used quantitative Real-Time PCR to study the effect of sub-MICs of ceftaroline on the expression of the staphylococcal icaA, agrA, sarA and sasF genes in MRSA biofilms. In one set of experiments, ceftaroline was able to inhibit biofilm formation in all strains tested at MIC, however, a strain dependent behavior in presence of sub-MICs of ceftaroline was shown. In a second set of experiments, destruction of preformed biofilms by addition of ceftaroline was evaluated. Ceftaroline was able to inhibit biofilm formation at MIC in all strains tested but not at the sub-MICs. Destruction of preformed biofilms was strain dependent because the biofilm formed by a matrix-producing strain was resistant to a challenge with ceftaroline at MIC, whereas in other strains the biofilm was sensitive. At sub-MICs, the impact of ceftaroline on expression of virulence genes was strain-dependent at 1/4 MIC and no correlation between ceftaroline-enhanced biofilm formation and gene regulation was established at 1/16 MIC. Our findings suggest that sub-MICs of ceftaroline enhance bacterial attachment and biofilm formation by some, but not all, MRSA strains and, therefore, stress the importance of maintaining effective bactericidal concentrations of ceftaroline to fight biofilm-MRSA related infections.     

The Role of MRSA (Methicillin-Resistant Staphylococcus aureus) Adherence and Colonization in the Upper Respiratory Tract of Geriatric Patients in Nosocomial Pulmonary Infections

The mechanism of nosocomial respiratory infections caused by MRSA (methicillin-resistant Staphylococcus aureus) in geriatric patients was investigated. Seriously ill patients (SIP) undergoing naso-gastric tube feeding or intravenous hyperalimentation and moderately ill patients (MIP) who were orally fed, were examined for their colonization and infection by Staphylococcus aureus (S. aureus) in the respiratory tract. Colonization of MRSA in the upper respiratory tract in SIP was from six to ten times higher than that in MIP and was associated with a high incidence of MRSA pulmonary infections. In vitro S. aureus adherence to nasal or oropharyngeal cells demonstrated that bacteria binding to nasal cells was higher, which probably can be interpreted as an elevated occurrence of S. aureus colonization in the nasal cavity than in the throat. The binding activity of MRSA was not superior to that of MSSA (methicillin-sensitive S. aureus). Though MRSA binding to the nasal cells from SIP was not higher than those from MIP, MRSA colonization in the upper respiratory tract was more frequently seen in SIP (P < 0.01). A higher incidence of total infectious episodes (P < 0.02-0.001) and more frequent use of antibiotics (P < 0.02-0.001), which were potent against MSSA might be the basis for selection of MRSA in these patients. In fact, the rate of MRSA colonization on the skin (pressure sores) was also higher in SIP (P < 0.01). A low nutritional state in SIP (P < 0.01-0.02) might also be associated with MRSA colonization. The present results indicate that the high frequency of infections, antibiotic administration, MRSA skin colonization and low nutritional condition, are enhancing factors of MRSA acquisition in the respiratory tract for SIP undergoing artificial feeding, in a geriatric hospital.     

Whole-Genome Sequence of Livestock-Associated ST398 Methicillin-Resistant Staphylococcus aureus Isolated from Humans in Canada

Despite reports of high colonization rates of ST398 livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) among pigs and pig farmers, the incidence of LA-MRSA infection in the general population in Canada appears to be rare in comparison to that in some European countries. In this study, the complete genome sequence of a Canadian representative LA-MRSA isolate (08BA02176) from a human postoperative surgical site infection was acquired and compared to the sequenced genome of an LA-MRSA isolate (S0385) from Europe to identify genetic traits that may explain differences in the success of these particular strains in some locales.     

In-Silico Repurposing of Anticancer Drug (5-FU) as an Antimicrobial Agent Against Methicillin-Resistant Staphylococcus aureus (MRSA)

International Journal of Peptide Research and Therapeutics - The Centers for Disease Control and Prevention (CDC) reported earlier that more than 11,000 people died from a methicillin-resistant...     

Potent In Vitro Bactericidal Activity of Polymyxin B against Methicillin-Resistant Staphylococcus aureus (MRSA)

Eradication of methicillin-resistant Staphylococcus aureus (MRSA) carried by inpatients or healthy hospital personnel by topical use of antibiotics is an important step for preventing outbreak of MRSA nosocomial infection. In the screening of the antibiotic best suited for this purpose, we have found that polymyxin B, a commonly used antibiotic for gram-negative infection, had an unexpected strong cytokilling activity towards MRSA clinical strains, which was more potent than that of vancomycin or gentamicin. The data suggested that polymyxin B could be an antibiotic of choice in the treatment of topical carriage of or infection caused by MRSA.     

耐甲氧西林金黄色葡萄球菌皮肤脓肿小鼠感染模型的建立与评估

目的建立耐甲氧西林金黄葡萄球菌（methicillin-resistant Staphylococcus aureus,MRSA）小鼠皮肤脓肿感染模型,观察脓肿形成动态变化及药物对脓肿愈合的影响。方法 45只SPF级裸鼠随机分为PBS对照组、感染组及给药组,用临床分离鉴定的ST-239型MRSA菌株皮下注射感染裸鼠,对脓肿的形成过程进行时相性观察,测定脓肿体积的变化,并通过H.E染色观察皮肤的组织病理学改变。结果 PBS处理组小鼠皮肤无脓肿形成,显微镜下皮肤各层结构清楚;感染组和给药组可见到典型的脓肿,临床症状的时相性过程明显,感染组小鼠皮肤真皮层的胶原纤维消失,可见大量炎性细胞浸润,给药组小鼠的脓肿体积在整个实验周期内低于感染组,且在第5天时与感染组相比有差异。结论成功建立MRSA小鼠皮肤脓肿感染模型,该模型的建立可为进一步研究来源于临床MRSA菌株的病原特性、发病机制、治疗方法等提供可靠的动物模型。     

Characterization of Insertion Sequence ISSau2 in the Human and Livestock-Associated Staphylococcus aureus

Mobile genetic elements play important roles in evolution and diversification of bacterial genomes. ISSau2 is 1660bp in length with terminal 5’-TG and CA-3’ dinucleotides and has two overlapping reading frames orfA and orfB. It has been found in a wide range of S. aureus, such as HA-MRSA252, LGA251, MRSA S0385 and ED133. To determine distribution of ISSau2, 164 S. aureus isolates from milk samples of mastitic cows from our laboratory and all the S. aureus strains from the National Center for Biotechnology Information (NCBI) database were screened for the presence of ISSau2. Next, in order to explore a potential relationship among S. aureus ISSau2-containing strains and isolates, a relationship among 10 ISSau2-positive S. aureus isolates and 27 ISSau2-positive S. aureus strains was investigated by a phylogenetic analysis. These ISSau2 isolates and strains could be classified into four groups (A, B, C and D). The strains or isolates in Group D were all isolated from mammary glands, suggesting tissue specificity. All strains in Group B had an identical ISSau2 derivative, termed ISSau2₁₆₂₈, with 32bp deletion at the 3’ terminus. ISSau2₁₆₂₈ in strain ST398 from Group B was closely related to ISSau2 in strain LGA251 from Group D.     

Serum Stable and Low Hemolytic Temporin-SHa Peptide Analogs Disrupt Cell Membrane of Methicillin-Resistant Staphylococcus aureus (MRSA)

Probiotics and Antimicrobial Proteins - Anti-microbial peptides (AMPs) have attracted major attention due to their potential bio-activities against some multidrug resistant pathogens. The present...     

Comparison of Two Commercial PCR Methods for Methicillin-Resistant Staphylococcus aureus (MRSA) Screening in a Tertiary Care Hospital

Nose/throat-swabs from 1049 patients were screened for MRSA using CHROMagar MRSA, LightCycler Advanced MRSA, and Detect-Ready MRSA. Results were compared to the CHROMagar MRSA results, which was set as reference system. MRSA was detected in 3.05% of the patients with CHROMagar MRSA. LightCycler MRSA Advanced showed a higher clinical sensitivity (84.38%) than Detect-Ready MRSA (57.69%).The negative predictive values were high for both tests (>98%). The specificity and the positive predictive value were higher for the Detect-Ready MRSA test than for the LightCycler MRSA test (99.59% and 78.95% versus 98.52% and 64.29%). For routine screening LightCycler MRSA Advanced proved to be more efficient in our clinical setting as the clinical sensitivity was much higher than the sensitivity of Detect-Ready MRSA. CHROMagar MRSA detected more MRSA positive samples than both PCR methods, leading to the conclusion that the combination of PCR with cultural screening is still the most reliable way for the detection of MRSA. LightCycler MRSA Advanced was faster and needed less hands-on time. The advantage of Detect-Ready MRSA was the additional identification of methicillin-sensitive S.aureus (here in 34.63% of the samples), an information which can be possibly used for reducing the risk of postoperative infections in surgical patients in future.     

Phenol Soluble Modulin (PSM) Variants of Community-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) Captured Using Mass Spectrometry-Based Molecular Networking

Molecular genetic analysis indicates that the problematic human bacterial pathogen methicillin-resistant Staphylococcus aureus possesses more than 2000 open reading frames in its genome. This number of potential gene products, coupled with intrinsic mechanisms of posttranslational modification, endows methicillin-resistant Staphylococcus aureus with a highly complex biochemical repertoire. Recent proteomic and metabolomic advances have provided methodologies to better understand and characterize the biosynthetic factors released by microbial organisms. Here, the emerging tool of mass spectrometry-based molecular networking was used to visualize and map the repertoire of biosynthetic factors produced by a community-associated methicillin-resistant Staphylococcus aureus strain representative of the epidemic USA300 clone. In particular, the study focused on elucidating the complexity of the recently discovered phenol soluble modulin family of peptides when placed under various antibiotic treatment stresses. Novel PSM truncated variant peptides were captured, and the type of variants that were clustered by the molecular networks platform changed in response to the different antibiotic treatment conditions. After discovery, a group of the peptides were selected for functional analysis in vitro. The peptides displayed bioactive properties including the ability to induce proinflammatory responses in human THP-1 monocytes. Additionally, the tested peptides did not display antimicrobial activity as previously reported for other phenol soluble modulin truncated variants. Our findings reveal that the PSM family of peptides are quite structurally diverse, and suggest a single phenol soluble modulin parent peptide can functionally spawn differential bioactivities in response to various external stimuli.A significant percentage of the human population harbors Staphylococcus aureus as a natural resident of their microbial flora (1). In non-infected individuals, S. aureus maintains a homeostatic coexistence with neighboring microbes and with the host (2). However, when equilibrium is disrupted, S. aureus is capable of establishing diseases ranging from minor superficial infections to potentially life-threatening invasive conditions (3, 4). Through the discovery and administration of antibiotics, clinicians have significantly reduced the morbidity and mortality associated with S. aureus (5, 6). In recent years, the consequences of persistent antibiotic exposure have become increasingly manifest through the emergence of resistant strains, including those left unperturbed by methicillin and related β-lactam antibiotics (MRSA)¹ or those with intermediate or full resistance to vancomycin (VISA/VRSA) (7). Certain drug-resistant S. aureus strains, such as the USA300 clone, have spread beyond the hospital setting and into the community, producing serious infections even in previously healthy individuals (8, 9). Both community-associated (CA) and hospital-associated (HA) multi-drug resistant S. aureus are highly evolved, multifaceted pathogens that pose an increasing threat to the health of not only our own, but future generations as well.The pathogenic mechanisms of S. aureus have been examined extensively through different stages of infection (10, 11), with an improved understanding of specific functions that various staphylococcal components contribute to virulence (12). The ability to outcompete other microflora for colonization, or to circumvent the host immune response during infectious spread, largely reflects a complex network of proteins and non-protein molecules that S. aureus secretes into its surroundings-which may collectively be termed biosynthetic release factors. One component of this network is the newly studied and multifunctional phenol soluble modulin (PSM) family of peptides. Associated with biofilm formation and possessing bioactivity toward host cells, PSMs are produced more abundantly by CA-MRSA compared with HA-MRSA strains (13, 14). PSMs are biosynthesized and released with formylated initiator methionine, allowing binding and activation of host neutrophils via the formyl peptide receptor 2 (FPR2) (15). A number of PSMs show cytolytic properties toward host cell lines, and supporting this, isogenic PSM-knockout mutants have decreased virulence in murine models of invasive infection (13). Recently, it was found that many in vitro PSMs phenotypes (e.g. neutrophil lysis, FPR2 binding) were strongly inhibited by serum lipoproteins (16). The same study showed up-regulation of PSM biosynthesis following phagocytosis, suggesting an important role in intracellular survival. Additionally, N-terminal truncated derivative forms of PSMs (dPSMs) with antibacterial properties were identified (17, 18). The finding that dPSMs possess antibacterial activities that are absent in the corresponding full-length parent PSMs, inspired a hypothesis that dPSMs endow CA-MRSA strains with a competitive advantage against resident microbiota upon colonizing host epithelia (i.e. once truncated PSMs gain antimicrobial properties).The invaluable tool of MS has played a major role in characterizing biosynthetic release factors for a diverse number of organisms (19). Over the past decade, the field has spawned several innovative methods to optimize data collection, processing, and organization. The advancement of MS processing methods has improved the capabilities for extracting significant information regarding an organism''s biochemical repertoire. One emerging tool to study biosynthetic release factor potential is the tandem MS data processing program, Spectral Networks. In conjunction with Cytoscape graphical mapping, Spectral Networks allows for the construction of a molecular network map that gives a visualization of tandem mass spectra grouping, based on similar scoring of overlapping ion fragmentation patterns (20, 21). The matching of spectra, termed spectral pairing, is unlike the conventional mode of processing MS data by matching against a reference library or specifying specific posttranslational modifications for identification (22). Instead, within a molecular network map, ion fragmentation patterns can be represented by correlation points, displayed as circular nodes, connected with a straight line to other nodes containing high similarity scores. The power of molecular networking is the information provided on tandem MS subpopulation clusters, which represent structurally matching but variant protein or non-protein biosynthetic release factors.In this work, the MS-based molecular networking platform was applied to a representative strain of the epidemic CA-MRSA USA300 clone. The study sought to provide insight into the biosynthetic release factor map of this pre-eminent human bacterial pathogen under normal growth and antibiotic challenged conditions. Biologically relevant pharmacological stimuli studied included sub-inhibitory concentrations of commonly prescribed commercial antibiotics as well as the innate human defense peptide LL-37 (23–25). In particular, our study goal was to monitor any variations in the type of dPSMs clustered within the molecular network map in response to these stimuli. We show an enriched extract derived from CA-MRSA alone or under these specific challenge conditions provides a highly complex biosynthetic release factor map. Analysis of subpopulation node clusters associated with PSMs prompted the discovery of several previously unreported α, β, and γ type dPSMs. The antibiotic stimuli modulated the type of dPSMs clustered, allowing for differential subclusters to be visualized that were absent in the untreated sample; including nodes that did not sequence to proteogenic PSMs. A group of the newly discovered PSM variants were assessed for functional properties and showed the induction of proinflammatory responses in THP-1 cells. In addition, in contrast to previous reports (17, 18), the tested dPSMs did not display antimicrobial activity. We conclude MS-based molecular networking can be used as a targeted discovery tool with the ability to identify previously unreported bioactive peptides.