Amplidiff: an optimized amplicon sequencing approach to estimating lineage abundances in viral metagenomes.

BACKGROUND: Metagenomic profiling algorithms commonly rely on genomic differences between lineages, strains, or species to infer the relative abundances of sequences present in a sample. This observation plays an important role in the analysis of diverse microbial communities, where targeted sequencing of 16S and 18S rRNA, both well-known hypervariable genomic regions, have led to insights into microbial diversity and the discovery of novel organisms. However, the variable nature of discriminatory regions can also act as a double-edged sword, as the sought-after variability can make it difficult to design primers for their amplification through PCR. Moreover, the most variable regions are not necessarily the most informative regions for the purpose of differentiation; one should focus on regions that maximize the number of lineages that can be distinguished. RESULTS: Here we present AmpliDiff, a computational tool that simultaneously finds highly discriminatory genomic regions in viral genomes of a single species, as well as primers allowing for the amplification of these regions. We show that regions and primers found by AmpliDiff can be used to accurately estimate relative abundances of SARS-CoV-2 lineages, for example in wastewater sequencing data. We obtain errors that are comparable with using whole genome information to estimate relative abundances. Furthermore, our results show that AmpliDiff is robust against incomplete input data and that primers designed by AmpliDiff also bind to genomes sampled months after the primers were selected. CONCLUSIONS: With AmpliDiff we provide an effective, cost-efficient alternative to whole genome sequencing for estimating lineage abundances in viral metagenomes.

Cytotoxic Virulence Predicts Mortality in Nosocomial Pneumonia Due to Methicillin-Resistant Staphylococcus aureus.

The current study identified bacterial factors that may improve management of methicillin-resistant Staphylococcus aureus (MRSA) nosocomial pneumonia. Isolates were obtained from 386 patients enrolled in a randomized, controlled study of antibiotic efficacy. Isolates were screened for production of virulence factors and for vancomycin susceptibility. After adjustment for host factors such as severity of illness and treatment modality, cytotoxic activity was strongly and inversely associated with mortality; however, it had no effect on clinical cure. Isolates having low cytotoxicity, which were derived largely from healthcare-associated clones, exhibited a greater prevalence of vancomycin heteroresistance, and they were recovered more often from patients who were older and frailer. Additionally, a clone with low cytotoxic activity was associated with death and poor clinical improvement. Clone specificity and attenuated virulence appear to be associated with outcome. To our knowledge, these are the first correlations between MRSA virulence and mortality in nosocomial pneumonia.

Updated ASM Curriculum Guidelines describe core microbiology content to modernize the framework for microbiology education.

Curricular guidelines promote standardized approaches to coverage of essential knowledge and skills in undergraduate education. The American Society for Microbiology (ASM) Curriculum Guidelines for Undergraduate Microbiology were developed in 2012. Continuous, rapid growth of knowledge in science and a dynamic, changing world necessitate updates to these guidelines. As such, ASM formed a task force in the summer of 2022. The task force assessed the 2012 ASM Curriculum Guidelines considering advancements in technology, an understanding of an expanded role of microbes, and a broader scope addressing relevant social and environmental aspects of microbiology. Language in the updated guidelines was also modified to better include eukaryotic microbes, viruses, and other acellular microbes. The task force formed working groups, each aimed at revising specific sections of the 2012 ASM Curriculum Guidelines. The revisions to the ASM Curriculum Guidelines were reviewed by subject matter experts and education stakeholders. Feedback from this peer review was incorporated into the updated guidelines, and further comments were solicited from the ASM Conference of Undergraduate Educators (ASMCUE) attendees in November 2023 before these guidelines were finalized. In this article, we describe the rationale and development of updated ASM Curriculum Guidelines which identify foundational concepts that will serve to improve microbial literacy and that can be expanded upon to address more advanced and specialized topics.

Nasal carriage as a source of agr-defective Staphylococcus aureus bacteremia.

Inactivating mutations in the Staphylococcus aureus virulence regulator agr are associated with worse outcomes in bacteremic patients. However, whether agr dysfunction is primarily a cause or a consequence of early bacteremia is unknown. Analysis of 158 paired S. aureus clones from blood and nasal carriage sites in individual patients revealed that recovery of an agr-defective mutant from blood was usually predicted by the agr functionality of carriage isolates. Many agr-positive blood isolates produced low levels of hemolytic toxins, but levels were similar to those of colonizing strains within patients, suggesting that introduction into the blood did not select for mutations with minor functional effects. Evidently, the transition from commensalism to opportunism in S. aureus does not require full virulence in hospitalized patients. Furthermore, agr-defective mutants were found in uninfected nasal carriers in the same proportion as in carriers who develop bacteremia, suggesting low correlation between virulence and infectivity.

Promoting RAPID Vaccine Science Education at the Onset of the COVID-19 Pandemic.

At the onset of the 2019 coronavirus disease (COVID-19) pandemic, it was clear that we needed to support public education on the science of vaccines. This project was born of that need and led to the development of comprehensive educational materials that addressed the process of science, severe acute respiratory syndrome coronavirus 2 biology, vaccine development, and science communication and outreach. Called the "Online Vaccine Science Resources for COVID-19 Education," the materials generated were designed to be implemented by educators and community groups in various contexts. They took the form of four modules and general audience informational videos available on a YouTube channel. Each module was assembled as a toolkit with instructional videos, assessments, discussion questions, assignments, synthesis activities, and guides for constructing infographics and dual poster (science and general public audience) presentations. The materials were piloted and tested in various educational settings, including 2-year and 4-year colleges. Data gathered from surveys of faculty and student participants suggested that exposure to the materials promoted student trust in vaccination and the scientific process of vaccine development, and increased the likelihood of their getting a freely available vaccine. Assessment data indicated that the materials were successful in helping students achieve the learning objectives for the modules. Our results underscored the continued need for science education strategies that address the critical problem of vaccine hesitancy as we continue to emerge from the COVID-19 pandemic.

Peer leader perspectives from a PLTL implementation in a Hispanic-serving institution.

Peer-Led Team Learning (PLTL) is a pedagogical approach that has been shown to benefit all students, especially underrepresented minority students and peer leaders in Science, Technology, Engineering, and Mathematics (STEM) disciplines. In this work, we present results from our study of the impact of PLTL on our peer leaders from a controlled implementation in general biology, general chemistry, and statistics courses at a Hispanic-serving, minority-serving institution. More specifically, we have measured our PLTL program's impact on our peer leaders' skill development, engagement with the subject material, and sense of belonging as peer leaders. Weekly peer leader reflections analyzed using the Dreyfus model exhibited a consistent set of skills, while those analyzed using the Pazos model revealed a consistent type of student-peer leader interactions, allowing for peer leaders to be assigned to specific levels in the hierarchy of each of the models. Analysis of eight skill-based Likert-scale questions on the SALG survey showed an overall positive shift at the highest level. Independent of the skill or interaction level of the peer leader, we observed several instances of peer leaders acknowledging development in their communication skills, sincere attempts at creating an engaging classroom, and a deep investment in their student's success. Peer leaders also reported improvements in understanding of the subjects they were teaching, wanting to persevere and solve problems independently, and feeling passionate about helping other students.

Wastewater surveillance of SARS-CoV-2 genomic populations on a country-wide scale through targeted sequencing.

SARS-CoV-2 surveillance of viral populations in wastewater samples is recognized as a useful tool for monitoring epidemic waves and boosting health preparedness. Next generation sequencing of viral RNA isolated from wastewater is a convenient and cost-effective strategy to understand the molecular epidemiology of SARS-CoV-2 and provide insights on the population dynamics of viral variants at the community level. However, in low- and middle-income countries, isolated groups have performed wastewater monitoring and data has not been extensively shared in the scientific community. Here we report the results of monitoring the co-circulation and abundance of variants of concern (VOCs) of SARS-CoV-2 in Uruguay, a small country in Latin America, between November 2020-July 2021 using wastewater surveillance. RNA isolated from wastewater was characterized by targeted sequencing of the Receptor Binding Domain region within the spike gene. Two computational approaches were used to track the viral variants. The results of the wastewater analysis showed the transition in the overall predominance of viral variants in wastewater from No-VOCs to successive VOCs, in agreement with clinical surveillance from sequencing of nasal swabs. The mutations K417T, E484K and N501Y, that characterize the Gamma VOC, were detected as early as December 2020, several weeks before the first clinical case was reported. Interestingly, a non-synonymous mutation described in the Delta VOC, L452R, was detected at a very low frequency since April 2021 when using a recently described sequence analysis tool (SAM Refiner). Wastewater NGS-based surveillance of SARS-CoV-2 is a reliable and complementary tool for monitoring the introduction and prevalence of VOCs at a community level allowing early public health decisions. This approach allows the tracking of symptomatic and asymptomatic individuals, who are generally under-reported in countries with limited clinical testing capacity. Our results suggests that wastewater-based epidemiology can contribute to improving public health responses in low- and middle-income countries.

Characterization of methicillin-resistant Staphylococcus aureus strains recovered from a phase IV clinical trial for linezolid versus vancomycin for treatment of nosocomial pneumonia.

A total of 434 methicillin-resistant Staphylococcus aureus (MRSA) baseline isolates were collected from subjects enrolled in a prospective, double-blind randomized trial comparing linezolid versus vancomycin for the treatment of nosocomial pneumonia. Isolates were susceptibility tested by broth microdilution, examined for inducible clindamycin resistance by D-test, and screened for heterogeneous resistance to vancomycin (hVISA) by the Etest macromethod. All strains were subjected to Panton-Valentine leukocidin (PVL) screening, and SCCmec, pulsed-field gel electrophoresis (PFGE), and spa typing. Selected strains were evaluated by multilocus sequence typing (MLST). Clonal complexes (CCs) were assigned based on the spa and/or MLST results. Most strains were CC5 (56.0%), which originated from North America (United States) (CC5-MRSA-SCCmec II/IV; 70.0%), Asia (CC5-MRSA-II; 14.0%) and Latin America (CC5-MRSA-I/II; 12.3%). The second- and third-most-prevalent clones were CC8-MRSA-IV (23.3%) and CC239-MRSA-III (11.3%), respectively. Furthermore, the CC5-MRSA-I/II clone predominated in Asia (50.7% within this region) and Latin America (66.7%), followed by CC239-MRSA-III (32.8% and 28.9%, respectively). The European strains were CC8-MRSA-IV (34.5%), CC22-MRSA-IV (18.2%), or CC5-MRSA-I/II/IV (16.4%), while the U.S. MRSA isolates were CC5-MRSA-II/IV (64.4%) or CC8-MRSA-IV (28.8%). Among the U.S. CC8-MRSA-II/IV strains, 73.7% (56/76 [21.2% of all U.S. MRSA strains]) clustered within USA300. One strain from the United States (USA800) was intermediate to vancomycin (MIC, 4 µg/ml). All remaining strains were susceptible to linezolid, daptomycin, vancomycin, and teicoplanin. hVISA strains (14.5%) were predominantly CC5-MRSA-II, from South Korea, and belonged to a single PFGE type. Overall, each region had two predominant clones. The USA300 rate corroborates previous reports describing increased prevalence of USA300 strains causing invasive infections. The prevalence of hVISA was elevated in Asia, and these strains were associated with CC5.

How Getting Friendly with Bacteria Can Promote Student Appreciation of Microbial Diversity and Their Civic Scientific Literacy.

ePortfolios are digital repositories where students can curate papers, projects, and reflections from individual or multiple courses across the disciplines and in a variety of formats to showcase their learning. This transparent and portable medium, which enables students to document their knowledge and abilities for assessment and career development, has been recognized by the American Association of Colleges and Universities as one of 11 high-impact practices. Using tailored rubrics, student assessment of learning gain surveys, and end-of-course exam questions, this study demonstrates how an ePortfolio assignment can be used in microbiology courses taken by majors and nonmajors to measure student learning outcomes in several course and program learning goals. Additionally, it helps students reflect on their learning and place it in a real-world context by connecting science, microbiology, and microbes with issues of social importance like cholera, gender equity, and antibiotic resistance. Writing from a first-person perspective and drawing on resources obtained in class and from their own research, students generate profiles for a chosen microbe and document the microbe's characteristics in creative ways. The ePortfolio assignment can also be partnered with creative work such as an art piece or a poem that highlights and showcases the microbe in a format that is accessible to the public to increase awareness of the role of microbes in our ecosystems. With careful design and construction of assignments, ePortfolios can also be leveraged to promote civic and scientific literacy by tying classroom content to real-world issues of civic importance.

Humidity Reduces Rapid and Distant Airborne Dispersal of Viable Viral Particles in Classroom Settings.

The transmission of airborne pathogens is considered to be the main route through which a number of known and emerging respiratory diseases infect their hosts. While physical distancing and mask wearing may help mitigate short-range transmission, the extent of long-range transmission in closed spaces where a pathogen remains suspended in the air remains unknown. We have developed a method to detect viable virus particles by using an aerosolized bacteriophage Phi6 in combination with its host Pseudomonas phaseolicola, which when seeded on agar plates acts as a virus detector that can be placed at a range of distances away from an aerosol-generating source. By applying this method, we consistently detected viable phage particles at distances of up to 18 feet away from the source within 15 min of exposure in a classroom equipped with a state of the art HVAC system and determined that increasing the relative humidity beyond 40% significantly reduces dispersal. Our method, which can be further modified for use with other virus/host combinations, quantifies airborne transmission in the built environment and can thus be used to set safety standards for room capacity and to ascertain the efficacy of interventions in closed spaces of specified sizes and intended uses.

Tracking cryptic SARS-CoV-2 lineages detected in NYC wastewater.

Tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To monitor New York City (NYC) for the presence of novel variants, we deep sequence most of the receptor binding domain coding sequence of the S protein of SARS-CoV-2 isolated from the New York City wastewater. Here we report detecting increasing frequencies of novel cryptic SARS-CoV-2 lineages not recognized in GISAID's EpiCoV database. These lineages contain mutations that had been rarely observed in clinical samples, including Q493K, Q498Y, E484A, and T572N and share many mutations with the Omicron variant of concern. Some of these mutations expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. Finally, pseudoviruses containing the spike amino acid sequence of these lineages were resistant to different classes of receptor binding domain neutralizing monoclonal antibodies. We offer several hypotheses for the anomalous presence of these lineages, including the possibility that these lineages are derived from unsampled human COVID-19 infections or that they indicate the presence of a non-human animal reservoir.

Protocol for safe, affordable, and reproducible isolation and quantitation of SARS-CoV-2 RNA from wastewater.

The following protocol describes our workflow for processing wastewater with the goal of detecting the genetic signal of SARS-CoV-2. The steps include pasteurization, virus concentration, RNA extraction, and quantification by RT-qPCR. We include auxiliary steps that provide new users with tools and strategies that will help troubleshoot key steps in the process. This protocol is one of the safest, cheapest, and most reproducible approaches for the detection of SARS-CoV-2 RNA in wastewater. Owing to a pasteurization step, it is safe for use in a BSL2 facility. In addition to making the protocol safe for the personnel involved, pasteurization had the added benefit of increasing the SARS-CoV-2 genetic signal. Furthermore, the RNA obtained using this protocol can be sequenced using both Sanger and Illumina sequencing technologies. The protocol was adopted by the New York City Department of Environmental Protection in August 2020 to monitor SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. In the future, this protocol could be used to detect a variety of other clinically relevant viruses in wastewater and serve as a foundation of a wastewater surveillance strategy for monitoring community spread of known and emerging viral pathogens.

Polyphyletic emergence of linezolid-resistant staphylococci in the United States.

Since the year 2000, linezolid has been used in the United States to treat infections caused by antimicrobial-resistant Gram-positive cocci. At present, linezolid-resistant (Linr) Staphylococcus aureus and Staphylococcus epidermidis strains are rare and the diversity of their genetic backgrounds is unknown. We performed sequence-based strain typing and resistance gene characterization of 46 Linr isolates that were collected from local and national sources between the years 2004 and 2007. Resistance was found to occur in at least three clonal complexes (CCs; lineages) of S. aureus and in at least four subclusters of a predominant, phylogenetically unstable CC of S. epidermidis. New candidate resistance mutations in 23S rRNA and the L4 riboprotein were identified among the S. epidermidis isolates. These findings suggest that linezolid resistance has emerged independently in multiple clones of S. aureus and with a variety of ribosomal mutations in multiple clones of S. epidermidis.

Identification of a novel transposon (Tn6072) and a truncated staphylococcal cassette chromosome mec element in methicillin-resistant Staphylococcus aureus ST239.

A novel composite transposon (Tn6072) resembling staphylococcal cassette chromosome mercury (SCCHg) was identified in a collection of sequence type (ST) 239 methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates from Romanian hospitals. Tn6072 is homologous to the 5' region of SCCHg found in staphylococcal cassette chromosome mec (SCCmec) type III prototype strain 85/2082 but lacks the characteristic mer operon. SCCHg has previously been reported to integrate downstream of orfX, at the same chromosomal location as SCCmec. Tn6072, by contrast, is demarcated by two IS431 elements, flanked by 8-bp direct repeats, and inserted upstream of the origin of replication, within an open reading frame homologous to SAR2700 of S. aureus strain MRSA252. Analysis of a geographically and temporally diverse collection of 111 strains from the ST239 clonal group uncovered 11 additional strains harboring Tn6072, demonstrating a lineage-specific insertion pattern. Complete sequence analysis of the SCCmec regions of two representative Romanian strains (BK16704, BK16691) revealed two additional novel structures derived from a type III SCCmec background. BK16704 possesses an SCCmec 3A.1.4 structure, with an IS256 insertion downstream of the right chromosomal junction. In contrast, the SCCmec element of BK16691 is truncated downstream of the mec gene complex, with a 24-kb deletion encompassing the right chromosomal junction and an inverted downstream IS256 element. This structure, tentatively named "psiSCCmec16691," confers methicillin resistance but lacks most of the J1/J2 region, including the ccr gene complex. Taken together, these findings provide evidence for the continuing evolution of SCC elements, as well as the ST239 clonal group.

Biological sex influences susceptibility to Acinetobacter baumannii pneumonia in mice.

Acinetobacter baumannii (A. baumannii) is an extremely versatile multidrug-resistant pathogen with a very high mortality rate; therefore, it has become crucial to understand the host response during its infection. Given the importance of mice for modeling infection and their role in preclinical drug development, equal emphasis should be placed on the use of both sexes. Through our studies using a murine model of acute pneumonia with A. baumannii, we observed that female mice were more susceptible to infection. Likewise, treatment of male mice with estradiol increased their susceptibility to infection. Analysis of the airway compartment revealed enhanced inflammation and reduced neutrophil and alveolar macrophage numbers compared with male mice. Depletion of either neutrophils or alveolar macrophages was important for bacterial clearance; however, depletion of alveolar macrophages further exacerbated female susceptibility because of severe alterations in metabolic homeostasis. Our data highlight the importance of using both sexes when assessing host immune pathways.

Loop-Mediated Isothermal Amplification (LAMP) as a Rapid, Affordable and Effective Tool to Involve Students in Undergraduate Research.

Undergraduate research (UR) is a high-impact practice (HIP) to engage undergraduate student in science, technology, engineering and mathematics (STEM), especially from underrepresented groups. UR experiences (UREs) can be integrated into the classroom, making authentic research experiences inclusive and available to all students. However, developing UR pedagogy can be challenging for faculty in resource-limited labs, such as community colleges and small liberal arts colleges. Often molecular biology research methods are expensive, time-consuming and need equipment not readily available or affordable in small schools. Polymerase chain reaction (PCR) is one of the most commonly used techniques in research labs and many UREs. We have investigated loop-mediated isothermal amplification (LAMP) as an inexpensive, accessible alternative to PCR for DNA amplification enabling the identification of microorganisms in the context of UREs. LAMP does not require expensive instrumentation or reagents and uses equipment commonly found in teaching labs. By performing the technique, students learn several key scientific skills that will be useful in their undergraduate or graduate STEM careers. We designed guided independent research experiences for several undergraduates that included the use of LAMP. Students successfully applied the technique to culture samples of common environmental bacteria, including Escherichia coli, Salmonella spp., Staphylococcus aureus, and Enterococcus, and were in addition, able to detect both Salmonella and Enterococcus in directly sampled environmental waters. To highlight the accessibility and affordability of this URE, a simple boiling method was used for DNA preparation from environmental samples. Student response data show positive attitudes toward UR when LAMP is utilized as a research tool to tackle relevant biological questions. The feasibility of using simplified LAMP in UREs points to a potential, more expanded application to public engagement with science and broader and more inclusive interactions with the research community.

Integrative and sequence characteristics of a novel genetic element, ICE6013, in Staphylococcus aureus.

A survey of chromosomal variation in the ST239 clonal group of methicillin-resistant Staphylococcus aureus (MRSA) revealed a novel genetic element, ICE6013. The element is 13,354 bp in length, excluding a 6,551-bp Tn552 insertion. ICE6013 is flanked by 3-bp direct repeats and is demarcated by 8-bp imperfect inverted repeats. The element was present in 6 of 15 genome-sequenced S. aureus strains, and it was detected using genetic markers in 19 of 44 diverse MRSA and methicillin-susceptible strains and in all 111 ST239 strains tested. Low integration site specificity was discerned. Multiple chromosomal copies and the presence of extrachromosomal circular forms of ICE6013 were detected in various strains. The circular forms included 3-bp coupling sequences, located between the 8-bp ends of the element, that corresponded to the 3-bp direct repeats flanking the chromosomal forms. ICE6013 is predicted to encode 15 open reading frames, including an IS30-like DDE transposase in place of a Tyr/Ser recombinase and homologs of gram-positive bacterial conjugation components. Further sequence analyses indicated that ICE6013 is more closely related to ICEBs1 from Bacillus subtilis than to the only other potential integrative conjugative element known from S. aureus, Tn5801. Evidence of recombination between ICE6013 elements is also presented. In summary, ICE6013 is the first member of a new family of active, integrative genetic elements that are widely dispersed within S. aureus strains.

Genotypic and phenotypic relationships among methicillin-resistant Staphylococcus aureus from three multicentre bacteraemia studies.

BACKGROUND: At a time when the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) was changing, we sought to characterize several genotypic markers and glycopeptide susceptibility features of clinical isolates from patients with bacteraemia. METHODS: One hundred and sixty-eight MRSA bloodstream isolates obtained from three multicentre clinical trials were microbiologically and genotypically characterized. RESULTS: All isolates were susceptible to vancomycin (MIC < or = 2 mg/L); 38% belonged to accessory gene regulator (agr) group I, 52% belonged to group II and 10% belonged to group III. Typing of the staphylococcal cassette chromosome mec (SCCmec) showed that 67% were type II and 33% were type IV. The agr group II polymorphism was associated with SCCmec II (P < 0.001). Fifty-three percent of SCCmec II and 27% of SCCmec IV isolates had vancomycin MICs > or =1 mg/L (P = 0.001). One hundred percent of agr II strains were predicted to be members of clonal complex 5. SCCmec II was the genetic marker most predictive of vancomycin MICs of > or =1 mg/L. SCCmec IV isolates were more likely to have vancomycin MICs < or =0.5 mg/L. CONCLUSIONS: Given that SCCmec IV is a marker for a community-based organism for which less prior vancomycin exposure is predicted, we conclude that prior antibiotic exposure in agr group II organisms may account for their increased vancomycin MICs.

Associations between enterotoxin gene cluster types egc1, egc2 and egc3, agr types, enterotoxin and enterotoxin-like gene profiles, and molecular typing characteristics of human nasal carriage and animal isolates of Staphylococcus aureus.

Twenty genes encoding enterotoxin and enterotoxin-like proteins have been described in Staphylococcus aureus strains. Five of these occur commonly in the enterotoxin gene cluster (egc: selo, selm, sei, seln and seg). In the sei-seln intergenic region, two pseudogenes, psient1 and psient2, can be present or an additional gene designated selu or a variant selu(v). Whilst frequencies of loci bearing pseudogenes (egc1) or the selu gene (egc2) have been reported, the distinction between selu-bearing and selu(v)-bearing (egc3) loci has rarely been made. A PCR-RFLP procedure involving cleavage of the sei-seln intergenic region by restriction endonuclease BbvI or TseI was developed that allowed differentiation of selu(+) and selu(v)(+) loci. In addition, PCR primers were designed to yield a 203 bp amplimer for sequencing of a selu or selu(v) intragenic region, which encompassed ten signature nucleotide differences. A total of 43 egc(+) human nasal isolates and 53 egc(+) bovine, ovine, caprine, leporine and gallinaceous isolates were egc typed and agr typed. None of the animal isolates was of agr type III. A total of 12 out of 17 egc3(+) human nasal isolates were of agr type III, the other 5 being agr type I. On the basis of representative multilocus sequence typing, agr type III/egc3(+) strains belonged to CC30. Human nasal isolates bearing an egc1 locus were distributed evenly across agr types I, II and III. Only two nasal isolates had an egc2 locus. All 14 agr type IV isolates, only 1 of which was of human origin, possessed an egc2 locus. The agr IV nasal isolate was fusidic acid sensitive and was found to be ST123 (CC121). There were strong associations between bovine, leporine and gallinaceous S. aureus clonal types and egc locus types. The PCR-RFLP procedure was used to screen an additional 45 S. aureus isolates from dogs, cats, rats, pigs and horses for egc locus types. Of these, 33 were egc(-). Six equine isolates were selu(+). One canine and three porcine isolates possessed pseudogenes psient1 and psient2. One porcine and one canine isolate each had the selu(v) gene. Putative relationships between disease-causing propensity and egc type need (re-)evaluation.