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Multiple introductions of methicillin-resistant Staphylococcus aureus ST612 into Western Australia associated with both human and equine reservoirs
Abstract
Staphylococcus aureus is a serious human and animal pathogen. Multilocus sequence type 612 (ST612) is the dominant methicillin-resistant S. aureus (MRSA) clone in certain South African hospitals and is sporadically isolated from horses and horse-associated veterinarians in Australia. Colonisation and infection by ST612-MRSA is increasing in Western Australia. Whole-genome sequencing was performed for 51 isolates of ST612-MRSA from Western Australian patients and healthcare workers, South African hospital patients, Australian veterinarians and New South Wales horses. Core genome phylogenies suggested that Australian equine and veterinarian-associated ST612-MRSA were monophyletic. Individual Western Australian isolates grouped either with this equine-associated lineage or more diverse lineages related to those in South African hospitals. Bioinformatic analyses of the complete ST612-MRSA reference genome SVH7513 confirmed that ST612-MRSA was closely related to ST8 USA500 MRSA. Common use of rifampicin in South Africa and equine veterinarian practice may favour ST612-MRSA in these settings. Humans and horses colonised with ST612-MRSA are potential reservoirs for MRSA in Australia.
... Methicillin-resistant Staphylococcus aureus (MRSA) is one of the ESKAPE organisms [1] and is responsible for both hospitaland community-associated infections worldwide and therefore a major public health concern. Most of the reported equine hospital outbreaks in Europe are associated with MRSA clonal complex (CC) 398 [2], but in other parts of the world, other CCs and sequence types (STs) have been described [3][4][5]. MRSA carriage in horses poses a zoonotic risk and causes a risk for infection and transmission between horses and between horses and staff members in equine hospitals. The prevalence of MRSA in healthy horses ranges from 0 to 6% [3]. ...
... In ST612 isolates, notable virulence genes were present, e.g. the human-specific immune evasion cluster (scn, sak and sea), additional enterotoxins (seb, seq) and the leukocidin genes lukE and lukD together with the splA and splB genes (Table S1). Furthermore, two plasmids were present with high sequence identity with pUSA500 [18] and pSWS371 [4,19]. ...
... All horses recovered well. MRSA ST612 is widespread across South Africa associated with human infections and has been isolated from horses and incidentally in humans in Australia [4,22]. MRSA ST612 is a member of the CC8, which includes community-associated MRSA (CA-MRSA) USA300 and the closely related USA500 lineage. ...
In 2020 and 2022, nine cases of surgical site infections with a methicillin-resistant Staphylococcus aureus (MRSA) were diagnosed in horses in an equine referral clinic. Sixteen isolates (horses, n =9; environment, n =3; and staff members, n =4) were analysed retrospectively using Nanopore whole-genome sequencing to investigate the relatedness of two suspected MRSA outbreaks (2020 and 2022). The MRSA isolates belonged to ST398 and ST612. ST398 genomes from 2020 and 2022 formed three phylogenetic clusters. The first ST398 cluster from 2020 consisted of isolates from five horses and one staff member, and we suspected within clinic transmission. The second cluster of ST398 isolates from 2022 originated from two horses and two staff members but showed higher single nucleotide polymorphism (SNP) distances. One ST398 isolate from an individual staff member was not related to the other two clusters. The ST612 isolates were isolated in 2022 from two horses and three environmental samples and showed very low SNP distances ( < 7 SNPs), indicating the transmission of MRSA ST612 in this clinic in 2022. Molecular characterization revealed an abundant set of virulence genes and plasmids in the ST612 isolates in comparison to ST398 isolates. Phenotypic antimicrobial susceptibility showed that differences between the two sequence types were consistent with the genetic characteristics. MRSA ST612 has not been reported in Europe before, but it is a dominant clone in African hospitals and has been described in horses and people working with horses in Australia, indicating the importance of surveillance.
... Some additional lineages might be emerging in specific hosts: CC22-MRSA in cattle (Akkou et al., 2018;Magro et al., 2018) and possibly also horses , and ST612-MRSA in horses (Murphy et al., 2019). CC9 is a genotype predominant among MRSA from pigs in Asia (Haag et al., 2019) but has recently been found as an MSSA subtype with a high within-herd prevalence, persisting for years in dairy herds with notable resistances to pirlimycin, erythromycin and marbofloxacin (Grunert et al., 2018). ...
... Zoonotic cases involving other AMR S. aureus lineages have also been reported (Spoor et al., 2013;Cuny and Witte, 2017;Wang et al., 2018;Tomao et al., 2020). However, with both animals and humans being natural hosts of various, sometimes overlapping lineages of S. aureus, the direction of transfer is not always obvious (Juh asz- Kaszanyitzky et al., 2007;Murphy et al., 2019;Tomao et al., 2020). In addition, MRSA CC398 might actually be introduced in cattle or horses as a spillover event mostly from pigs, through the veterinarian (Krukowski et al., 2020) or owner (Locatelli et al., 2016(Locatelli et al., , 2017. ...
... mecC-MRSA, seem to have rather a sporadic potential (Barraud et al., 2013;Lozano et al., 2020). In Australia, MRSA ST612 might have a reservoir in horses, leading to colonisation of veterinarians and sporadic infections (Murphy et al., 2019). ...
Staphylococcus aureus (S. aureus) was identified among the most relevant antimicrobial-resistant (AMR) bacteria in the EU for cattle and horses in previous scientific opinions. Thus, it has been assessed according to the criteria of the Animal Health Law (AHL), in particular criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as in Article 9, and Article 8 for listing animal species related to the bacterium. The assessment has been performed following a methodology previously published. The outcome is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with uncertain outcome. According to the assessment here performed, it is uncertain whether AMR S. aureus can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (60-90% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that the bacterium does not meet the criteria in Sections 1, 2 and 4 (Categories A, B and D; 1-5%, 5-10% and 10-33% probability of meeting the criteria, respectively) and the AHAW Panel was uncertain whether it meets the criteria in Sections 3 and 5 (Categories C and E, 33-90% and 60-90% probability of meeting the criteria, respectively). The animal species to be listed for AMR S. aureus according to Article 8 criteria include mainly mammals, birds, reptiles and fish.
... The high rate of dfr-genes indicates a common usage of trimethoprim (in combination with sulfamethoxazole, as cotrimoxazole) in horses posing a significant selective pressure. Intriguingly, the abovementioned CC8/ST612-MRSA-IV strain that spread from humans in South Africa to horses in Australia was also resistant to trimethoprim, mediated by carriage of dfrA on a plasmid and selected for by the common usage of co-trimoxazole in Southern Africa for prophylaxis and therapy of AIDS-associated Pneumocystis jirovecii pneumonia [35]. ...
Leukocidins of Staphylococcus (S.) aureus are bicomponent toxins that form polymeric pores in host leukocyte membranes, leading to cell death and/or triggering apoptosis. Some of these toxin genes are located on prophages and are associated with specific hosts. The genes lukP/Q have been described from equine S. aureus isolates. We examined the genomes, including the lukP/Q prophages, of S. aureus strains belonging to clonal complexes CC1, CC350, CC816, and CC8115. In addition to sequencing, phages were characterised by mitomycin C induction and transmission electron microscopy (TEM). All lukP/Q prophages integrated into the lip2=geh gene, and all included also the gene scn-eq encoding an equine staphylococcal complement inhibitor. The lukP/Q prophages clustered, based on gene content and allelic variants, into three groups. One was found in CC1 and CC97 sequences; one was present mainly in CC350 but also in other lineages (CC1, CC97, CC133, CC398); and a third one was exclusively observed in CC816 and CC8115. Prophages of the latter group additionally included a rare enterotoxin A allele (sea320E). Moreover, a prophage from a CC522 goat isolate was found to harbour lukP. Its lukF component could be regarded as chimaera comprising parts of lukQ and of lukF-P83. A putative kinase gene of 1095 basepairs was found to be associated with equine strains of S. aureus. It was also localised on prophages. However, these prophages were different from the ones that carried lukP/Q, and three different integration sites of kinase-carrying phages were identified. These observations confirmed the presence of prophage-located important virulence-associated genes in equine S. aureus and that certain prophages might determine the host specificity of the staphylococcal strains they reside in.
... In addition to environmental and human samples, MRSA was found also in raw milk and cow nasal swabs raising concerns about zoonotic spread through environmental interactions [101]. Australian research linked ST612-MRSA colonization in humans and horses, identifying them as potential reservoirs, with phylogenies suggesting a monophyletic origin for equine and veterinarian-associated strains [102]. In Brazil, mecC-positive MRSA isolates were detected in milk, milker's hand swabs, and milking buckets, demonstrating MRSA's capacity to spread within dairy environments [103]. ...
... The ST612 is a double locus variant of the major clones USA500/CC8, a HA-MRSA strain (Carrel et al., 2015), and USA300/CC8, an epidemic CA-MRSA (Planet, 2017). The geographical distribution of ST612 is limited, being only described in specific regions of South Africa (Moodley et al., 2010;Jansen van Rensburg et al., 2011;Oosthuysen et al., 2014;Perovic et al., 2015Perovic et al., , 2017Lawal et al., 2022), Tanzania (Moremi et al., 2019) and Australia (Axon et al., 2011;Groves et al., 2016), frequently associated with veterinary practices (Groves et al., 2016;Murphy et al., 2018Murphy et al., , 2019Amoako et al., 2019). On the other hand, clone ST8-SCCmecIV has been frequently reported both in hospital and community settings in Angola, Cameroon, Gabon, Ghana, Madagascar, Nigeria, and São Tomé and Príncipe (Abdulgader et al., 2015). ...
Background
Staphylococcus aureus is one of the main causes of bacteraemia, associated with high mortality, mainly due to the occurrence of multidrug resistant (MDR) strains. Data on antibiotic susceptibility and genetic lineages of bacteraemic S. aureus are still scarce in Mozambique. The study aims to describe the antibiotic susceptibility and clonality of S. aureus isolated from blood cultures of children admitted to the Manhiça District Hospital over two decades (2001–2019).
Methods
A total of 336 S. aureus isolates detected in blood cultures of children aged <5 years were analyzed for antibiotic susceptibility by disk diffusion or minimal inhibitory concentration, and for the presence of resistance determinants by PCR. The clonality was evaluated by SmaI-PFGE, spa typing, and MLST. The SCCmec element was characterized by SCCmec typing.
Results
Most S. aureus (94%, 317/336) were resistant to at least one class of antibiotics, and one quarter (25%) showed a MDR phenotype. High rates of resistance were detected to penicillin (90%) and tetracycline (48%); followed by erythromycin/clindamycin (25%/23%), and co-trimoxazole (11%), while resistance to methicillin (MRSA strains) or gentamicin was less frequent (≤5%). The phenotypic resistance to distinct antibiotics correlated well with the corresponding resistance determinants (Cohen’s κ test: 0.7–1.0). Molecular typing revealed highly diverse clones with predominance of CC5 (17%, 58/336) and CC8 (16%), followed by CC15 (11%) and CC1 (11%). The CC152, initially detected in 2001, re-emerged in 2010 and became predominant throughout the remaining surveillance period, while other CCs (CC1, CC5, CC8, CC15, CC25, CC80, and CC88) decreased over time. The 16 MRSA strains detected belonged to clones t064-ST612/CC8-SCCmecIVd (69%, 11/16), t008-ST8/CC8-SCCmecNT (25%, 4/16) and t5351-ST88/CC88-SCCmecIVa (6%, 1/16). Specific clonal lineages were associated with extended length of stay and high in-hospital mortality.
Conclusion
We document the circulation of diverse MDR S. aureus causing paediatric bacteraemia in Manhiça district, Mozambique, requiring a prompt recognition of S. aureus bacteraemia by drug resistant clones to allow more targeted clinical management of patients.
... CC8-MSSA matching that description have been frequently observed by the authors in Uganda (Monecke et al., 2013) while there are also other observations from Africa (Aiken et al., 2014;Shittu et al., 2015). MRSA with that toxin profile have been observed by the authors in patients repatriated from Mozambique and Zimbabwe (Monecke et al., 2016), and this strain is known to be brought from South Africa to West Australia ( (Murphy et al., 2019); GenBank CP029166.1). ...
In the context of microarray-based epidemiological typing of the clonal organism Staphylococcus aureus/MRSA, a strain was identified that did not belong to known clonal complexes. The molecular analysis by microarray-based typing yielded signals suggesting that it was a mosaic or hybrid strain of two lineages. To verify this result, the isolate was sequenced with both, short-read Illumina and long-read Nanopore technologies and analysed in detail. This supported the hypothesis that the genome of this strain, ST6610-MRSA-IVg comprised of segments originating from two different clonal complexes (CC). While the backbone of the strain’s genome, i.e., roughly 2 megabases, belongs to CC8, a continuous insert of 894 kb (approx. 30% of the genome) originated from CC140. Beside core genomic markers in the normal succession and orientation, this insert also included the mecA gene, coding for PbP2a and causing methicillin resistance, localised on an SCCmec IVg element. This particular SCCmec type was also previously observed in CC140 MRSA from African countries. A second conspicuous observation was the presence of the trimethoprim resistance gene dfrG within on a prophage that occupied an attachment site normally used by Panton-Valentine Leucocidin phages. This observation could indicate a role of large-scale chromosomal recombination in the evolution of S. aureus as well as a role of phages in the dissemination of antibiotic resistance genes.
... In South Africa, ST612 (a CC8 strain) has been associated with human infections and has been identified as a dominant clone in hospitals in Cape Town and other provinces [71,104]. However, this clone has also been frequently isolated from animals in other continents such as horses in Australia [105], leading to the detection of the strains also in veterinarians working with horses and other people in contact with horses [67,[106][107][108], which indicates the possible transmission from humans to animals. This clone was also multidrug resistant, hence the importance of its spread through the food chain [109]. ...
Antimicrobial resistance has been increasing globally, which negatively affects food safety, veterinary, and human medicine. Ineffective antibiotics may cause treatment failure, which results in prolonged hospitalisation, increased mortality, and consequently, increased health care costs. Staphylococcus aureus causes a diverse range of infections including septicaemia and endocarditis. However, in food, it mainly causes food poisoning by the production of enterotoxins. With the discovery of methicillin-resistant S. aureus strains that have a separate reservoir in livestock animals, which were termed as livestock-associated methicillin-resistant S. aureus (LA-MRSA) in 2005, it became clear that animals may pose another health risk. Though LA-MRSA is mainly transferred by direct contact, food transmission cannot be excluded. While the current strains are not very pathogenic, mitigation is advisable, as they may acquire new virulence genes, becoming more pathogenic, and may transfer their resistance genes. Control of LA-MRSA poses significant problems, and only Norway has an active mitigation strategy. There is limited information about LA-MRSA, MRSA in general, and other S. aureus infections from African countries. In this review, we discuss the prevalence and characteristics of antimicrobial susceptible and resistant S. aureus (with a focus on MRSA) from meat and meat products in African countries and compare it to the situation in the rest of the world.
... Nevertheless, of the 48 clones we have observed taking ST, SCCmec type and spa type into consideration, the most common were ST612-IV-t064 (n = 8), ST612-IV-t1257 (n = 6), ST239-III-t037 (n = 6) and ST36-II-t012. Multiple introductions of ST612 was observed in Western Australia in both human and equine reservoirs [47]. ST612 was also recently observed in the clone ST612-CC8-t1257-SCCmec_ IVd(2B) obtained from the poultry food chain in South Africa [48]. ...
Background: The prevalence of Staphylococcus aureus varies depending on the healthcare facility, region and
country. To understand its genetic diversity, transmission, dissemination, epidemiology and evolution in a particular
geographical location, it is important to understand the similarities and variations in the population being studied.
This can be achieved by using various molecular characterisation techniques. This study aimed to provide detailed
molecular characterisation of South African mecA-positive S. aureus blood culture isolates by describing the SCCmec
types, spa types and to lesser extent, the sequence types obtained from two consecutive national surveillance
studies.
... Although not selected for MLST, the strain type t1257-MRSA-IV has been associated with the local clone ST612, which has been shown to be dominant in other hospitals in South Africa since 2008, but it was infrequent in this study (Jansen van Rensburg et al., 2011;Perovic et al., 2015). Interestingly, ST612 has only been described in hospitals in South Africa and Australia, suggesting possible introductions through travel between these two countries (Jansen van Rensburg et al., 2011;Murphy et al., 2019). Agr type I was the most predominant (50%), followed by types II (29%) and III (19%), while agr type IV was rare in our setting. ...
Background:
Staphylococcus aureus is a serious pathogen, able to cause life-threatening infections such as bacteraemia. The association between S. aureus microbial characteristics and clinical outcomes are under-investigated in African settings. This study aimed to determine the molecular epidemiology and virulence characteristics of S. aureus isolates from bacteraemic patients at Tygerberg Hospital, South Africa; and to investigate the associations between pathogen characteristics and clinical outcomes.
Methods:
This study included 199S. aureus isolates collected from blood cultures from February 2015 - March 2017. Methicillin resistance was determined using disc diffusion and all resistant isolates were further characterized by Staphylococcal Cassette Chromosome mec (SCCmec) typing. Genotyping was done using spa and agr typing, and agr functionality was assessed using the phenotypic δ-haemolysin assay. Logistic regression models were performed to describe the associations between strain characteristics and the clinical outcomes methicillin resistance, in-hospital mortality and length of stay (LOS).
Results:
Of the 199 S. aureus isolates collected, 27% were MRSA, and the overall crude in-hospital mortality rate was 29%. Seventy three different spa types were identified, including seven new types. Agr I was the most common type, in 99 (49.7%) isolates, followed by agr II, III and IV in 57 (28.6%), 37 (18.6%) and six (3%) isolates, respectively. Agr dysfunctionality was observed in 25 (13%) isolates, mostly belonging to spa-clonal complex (CC) 012. Methicillin resistance was significantly associated with HA infection (OR 4.77; 95%CI 2.09-10.87). A significant increase in mortality was observed with increasing age (OR 7.48; 95%CI 2.82-19.8), and having a HA infection (OR 2.26; 95%CI 1.12-4.55). S. aureus strains with a functional agr system showed an association with longer duration of stay (OR 1.66; 95%CI 0.93-2.99).
Conclusion:
We reported the lowest MRSA prevalence at Tygerberg hospital for the past ten years, and agr dysfunctionality was shown to be driven by certain genotype, spa-CC012. Despite the limited available clinical data, the study provided insights into associations between S. aureus epidemiology and agr related virulence characteristics, and clinical outcomes.
Objectives
Staphylococcus aureus is an important zoonotic pathogen that has often been seen in animals through the prism of the MRSA clonal complex (CC) 398 in pigs and in-contact humans. The goal of this study was first to assess the prevalence of MRSA, and second to look for MSSA CC398 in cats, dogs and horses in France.
Methods
Clinical S. aureus isolates (n = 479) were collected from 186 cats, 143 dogs and 150 horses during 2022–2023 all over the French territory. Antibiograms were performed on all isolates. MRSA and MSSA CC398 isolates were subject to WGS. Core genome (cg) MLST-based and SNP-based phylogenetic analyses were performed using published methodologies, and characterization of the isolates was performed using publicly available tools.
Results
Sixty-six MRSA isolates were identified in 14 cats (7.5%), 9 dogs (6.3%) and 43 horses (28.7%). The epidemiology of MRSA in cats and dogs remained stable since a previous study in 2015, with the presence of both CC398 and human-associated clones. In horses, in contrast, an important increase in MRSA (from 10% to 28.7%) was observed, potentially attributable to the emergence of the ST612 clones. In parallel, CC398 MSSA, a clone usually described as animal-independent, was found in 24.2% of the cat isolates.
Conclusions
Our study, which is leading the way to a genomic surveillance of S. aureus in France, revealed the emergence of both MRSA ST612 in horses and MSSA CC398 in cats. These clones should be closely monitored to avoid their zoonotic spread and to understand their dynamics of transmission between humans and animals.
Numerous questions persist regarding the role of companion animals as potential reservoirs of antimicrobial-resistant organisms that can infect humans. While relative antimicrobial usage in companion animals is lower than that in humans, certain antimicrobial-resistant pathogens have comparable colonization rates in companion animals and their human counterparts, which inevitably raises questions regarding potential antimicrobial resistance (AMR) transmission. Furthermore, the close contact between pets and their owners, as well as pets, veterinary professionals, and the veterinary clinic environment, provides ample opportunity for zoonotic transmission of antimicrobial-resistant pathogens. Here we summarize what is known about the transmission of AMR and select antimicrobial-resistant organisms between companion animals (primarily dogs, cats, and horses) and humans. We also describe the global distribution of selected antimicrobial-resistant organisms in companion animals. The impact of interspecies AMR transmission within households and veterinary care settings is critically reviewed and discussed in the context of methicillin-resistant staphylococci, extended-spectrum β-lactamase and carbapenemase-producing bacteria. Key research areas are emphasized within established global action plans on AMR, offering valuable insights for shaping future research and surveillance initiatives.
Genomic characterization was conducted on 2 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from 2 horses hospitalized during an overlapping period of time and 2 methicillin-sensitive S. aureus (MSSA) strains isolated from 2 distinct horses. Phylogenetic proximity was traced and the genotypic and phenotypic characteristics of the antimicrobial resistance of the strains were compared. Whole genome sequencing of MRSA strains for this report was similar but differed from whole genome sequencing of MSSA strains. The MRSA strains were closely related, belonging to sequence type (ST) 612, spa type t1257, and SCCmec type IVd2B. The MSSA strains were also closely related, belonging to ST1660, spa type t3043, and having no detectable staphylococcal cassette chromosome mec elements. All MSRA and MSSA strains were Panton-Valentine leukocidin negative. There were discrepancies in the genotypic analysis and the antimicrobial susceptibility testing (phenotypic analysis) of MRSA strains for rifampin, trimethoprim-sulfamethoxazole, gentamicin, amikacin, and enrofloxacin.
Functional biocompounds beneficial for animals and humans are in Mexican folk herbs. Cuphea and Eryngium species presented antimicrobial potential. Natural antibiotic uses by ethnoveterinary research with medicinal plants in equine infection or digestive diseases need more scientific evidence. Staphylococcus aureus, Escherichia coli, Salmonella enterica serotype Enteritidis are etiological agents in horses responsible for stable infections, abortions, fetal or perinatal deaths, and resistant intrahospital infections. The main objective of the present research was to evaluate the potential of antimicrobial and antioxidant activities of two Mexican medicinal plants Cuphea aequipetala var. hispida (Cav.) Koehne and Eryngium comosum Delaroche F over Listeria monocytogenes ATCC 19115, Staphylococcus sp., E. coli ATCC 25922, and S. enterica serotype Enteritidis ATCC 13076 bacterium reference strains related to equine infections. Determination of total phenol, saponins, antioxidant activity (ABTS), and antimicrobial activity with diffusion-sensitive discs was performed in triplicate. All the strains were sensitive for both extracts except for E. coli strain that was inhibited only by C. aequipetala. Staphylococcus sp. and S. enterica strains were inhibited equally by both extracts. E. comosum extracts tested have shown the highest effect over L. monocytogenes. In summary, antimicrobial activity was similar to the reported activity of Eryngium species extracts with other different solvents. Present extracts are suggested as a potential alternative antibiotic; definitely, more specific equine pathogen inhibition tests are needed in feed additives for horse nutrition research. In conclusion, antimicrobial activities of Cuphea aequipetala var. hispida (Cav.) Koehne and Eryngium comosum Delaroche F over reference strains related to equine infections suggested these medicinal plants as potential antibiotic sources for horse diseases.
Antimicrobial resistance (AMR) is a global crisis with impacts on the future health and welfare of humans and animals. Determining key factors that influence veterinarians’ antimicrobial prescribing behaviours can bridge the gap between prescribing guidelines and clinical usage. Veterinarians practicing in Australia were surveyed on their frequency in prescribing different antibiotics; factors influencing their antibiotic prescribing behaviours; and their perceptions of current drivers of AMR. Antibiotics were prescribed in a third of consultations with key differences in the frequency of use of specific antibiotics by small companion animal (SCA), equine and livestock veterinarians, which broadly aligned with antibiotic registration restrictions in Australia. SCA veterinarians reported prescribing broad-spectrum antibiotics of higher importance to human health more frequently than livestock veterinarians. Factors that were reported as ‘strong’ or ‘moderate’ barriers to appropriate antibiotic prescribing were the 1) cost of culture and susceptibility testing and 2) lack of access to rapid and affordable diagnostic tests. Fear of losing clients, colleague pressure, and lack of their own understanding about antibiotics were considered to be ‘no’ or ‘somewhat’ of a barrier to appropriate prescribing by respondents. SCA veterinarians placed greater importance on the contribution of antibiotic use in livestock to AMR, than antibiotic use in companion animals. Despite reporting use of fewer, mostly narrow spectrum antibiotics of lower importance to human and animal health, livestock veterinarians were generally more aware of their potential contribution to AMR. This study provides insights into the similarities and differences in SCA, equine and livestock veterinarians practicing in Australia and informs sector-specific strategies to improve antimicrobial stewardship.
Staphylococcus aureus is a serious pathogen of humans and animals. Multilocus sequence type 612 is dominant and highly virulent in South African hospitals but relatively uncommon elsewhere. We present the complete genome sequence of methicillin-resistant Staphylococcus aureus strain SVH7513, isolated from a horse at a veterinary clinic in New South Wales, Australia.
The capacity for some pathogens to jump into different host-species populations is a major threat to public health and food security. Staphylococcus aureus is a multi-host bacterial pathogen responsible for important human and livestock diseases. Here, using a population-genomic approach, we identify humans as a major hub for ancient and recent S. aureus host-switching events linked to the emergence of endemic livestock strains, and cows as the main animal reservoir for the emergence of human epidemic clones. Such host-species transitions are associated with horizontal acquisition of genetic elements from host-specific gene pools conferring traits required for survival in the new host-niche. Importantly, genes associated with antimicrobial resistance are unevenly distributed among human and animal hosts, reflecting distinct antibiotic usage practices in medicine and agriculture. In addition to gene acquisition, genetic diversification has occurred in pathways associated with nutrient acquisition, implying metabolic remodelling after a host switch in response to distinct nutrient availability. For example, S. aureus from dairy cattle exhibit enhanced utilization of lactose-a major source of carbohydrate in bovine milk. Overall, our findings highlight the influence of human activities on the multi-host ecology of a major bacterial pathogen, underpinned by horizontal gene transfer and core genome diversification.
USA500 isolates are clonal complex 8 (CC8) Staphylococcus aureus strains closely related to the prominent community- and hospital-associated USA300 group. Despite being relatively understudied, USA500 strains cause a significant burden of disease and are the third most common methicillin-resistant S. aureus (MRSA) strains identified in the U.S. Emerging Infections Program (EIP) invasive S. aureus surveillance. To better understand the genetic relationships of the strains, we sequenced the genomes of 539 USA500 MRSA isolates from sterile site infections collected through the EIP between 2005 and 2013 in the United States. USA500 isolates fell into three major clades principally separated by their distribution across different U.S. regions. Clade C1 strains, found principally in the Northeast, were associated with multiple IS 256 insertion elements in their genomes and higher levels of antibiotic resistance. C2 was associated with Southern states, and E1 was associated with Western states. C1 and C2 strains all shared a frameshift in the gene encoding AdsA surface-attached surface protein. We propose that the term “USA500” should be used for CC8 strains sharing a recent common ancestor with the C1, C2, and E1 strains but not in the USA300 group.
IMPORTANCE In this work, we have removed some of the confusion surrounding the use of the name “USA500,” placed USA500 strains in the context of the CC8 group, and developed a strategy for assignment to subclades based on genome sequence. Our new phylogeny of USA300/USA500 will be a reference point for understanding the genetic adaptations that have allowed multiple highly virulent clonal strains to emerge from within CC8 over the past 50 years.
Plasmids are mobile genetics elements that play an important role in the environmental adaptation of microorganisms. Although plasmids are usually analyzed in cultured microorganisms, there is a need for methods that allow for the analysis of pools of plasmids (plasmidomes) in environmental samples. To that end, several molecular biology and bioinformatics methods have been developed; however, they are limited to environments with low diversity and cannot recover large plasmids. Here, we present PlasFlow, a novel tool based on genomic signatures that employs a neural network approach for identification of bacterial plasmid sequences in environmental samples. PlasFlow can recover plasmid sequences from assembled metagenomes without any prior knowledge of the taxonomical or functional composition of samples with an accuracy up to 96%. It can also recover sequences of both circular and linear plasmids and can perform initial taxonomical classification of sequences. Compared to other currently available tools, PlasFlow demonstrated significantly better performance on test datasets. Analysis of two samples from heavy metal-contaminated microbial mats revealed that plasmids may constitute an important fraction of their metagenomes and carry genes involved in heavy-metal homeostasis, proving the pivotal role of plasmids in microorganism adaptation to environmental conditions.
Methicillin-resistant coagulase-positive staphylococci (CoPS) have become increasingly recognised as opportunistic pathogens that limit therapeutic options in companion animals. The frequency of methicillin resistance amongst clinical isolates on an Australia-wide level is unknown. This study determined antimicrobial susceptibility patterns for CoPS isolated from clinical infections in companion animals (dogs, cats and horses) as part of the first nation-wide survey on antimicrobial resistance in animal pathogens in Australia for a one-year period (January 2013 to January 2014). Clinical Staphylococcus spp. isolates (n = 888) obtained from 22 veterinary diagnostic laboratories were identified by MALDI-TOF mass spectrometry and subjected to antimicrobial susceptibility testing for 16 antimicrobials, representing 12 antimicrobial classes. Potential risk factors associated with methicillin resistance in Staphylococcus pseudintermedius isolates from dogs were analysed based on demographic factors and clinical history, including gender, age, previous antimicrobial treatment, chronic and/or recurrent diseases and site of infections. The most commonly identified CoPS were S. pseudintermedius (70.8%; dogs n = 616, cats n = 13) and S. aureus (13.2%, horses n = 53, dogs n = 47 and cats n = 17). Overall, the frequency of methicillin resistance among S. pseudintermedius (MRSP) and S. aureus (MRSA) was 11.8% and 12.8%, respectively. MRSP isolates were strongly associated with resistance to fluoroquinolones (OR 287; 95%CI 91.2–1144.8) and clindamycin (OR 105.2, 95%CI 48.5–231.9). MRSA isolates from dogs and cats were also more likely to be resistant to fluoroquinolones (OR 5.4, 95%CI 0.6–252.1), whereas MRSA from horses were more likely to be resistant to rifampicin. In multivariate analysis, MRSP-positive status was significantly associated with particular infection sites, including surgical (OR 8.8; 95%CI 3.74–20.7), and skin and soft tissue (OR 3.9; 95%CI 1.97–7.51). S. pseudintermedius isolated from dogs with surgical site infections were three times more likely to be methicillin-resistant if cases had received prior antimicrobial treatment. Whilst the survey results indicate the proportion of CoPS obtained from Australian companion animals that are methicillin-resistant is currently moderate, the identified risk factors suggest that it could rapidly increase without adequate biosecurity and infection control procedures in veterinary practice.
This work investigated the molecular epidemiology and antimicrobial resistance of methicillin-resistant Staphylococcus aureus (MRSA) isolated from veterinarians in Australia in 2009. The collection (n = 44) was subjected to extensive molecular typing (MLST, spa, SCCmec, dru, PFGE, virulence and antimicrobial resistance genotyping) and antimicrobial resistance phenotyping by disk diffusion. MRSA was isolated from Australian veterinarians representing various occupational emphases. The isolate collection was dominated by MRSA strains belonging to clonal complex (CC) 8 and multilocus sequence type (ST) 22. CC8 MRSA (ST8-IV [2B], spa t064; and ST612-IV [2B], spa variable,) were strongly associated with equine practice veterinarians (OR = 17.5, 95% CI = 3.3-92.5, P < 0.001) and were often resistant to gentamicin and rifampicin. ST22-IV [2B], spa variable, were strongly associated with companion animal practice veterinarians (OR = 52.5, 95% CI = 5.2-532.7, P < 0.001) and were resistant to ciprofloxacin. A single pig practice veterinarian carried ST398-V [5C2], spa t1451. Equine practice and companion animal practice veterinarians frequently carried multiresistant-CC8 and ST22 MRSA, respectively, whereas only a single swine specialist carried MRSA ST398. The presence of these strains in veterinarians may be associated with specific antimicrobial administration practices in each animal species.
During the past 25 years an increase in the prevalence of methicillin-resistant Staphylococcus aureus (HA-MRSA) was recorded worldwide. Additionally, MRSA infections may occur outside and independent of hospitals, caused by community associated MRSA (CA-MRSA). In Germany, we found that at least 10% of these sporadic infections are due to livestock-associated MRSA (LA-MRSA), which is initially associated with livestock. The majority of these MRSA cases are attributed to clonal complex CC398. LA-MRSA CC398 colonizes the animals asymptomatically in about half of conventional pig farms. For about 77%–86% of humans with occupational exposure to pigs, nasal carriage has been reported; it can be lost when exposure is interrupted. Among family members living at the same farms, only 4%–5% are colonized. Spread beyond this group of people is less frequent. The prevalence of LA-MRSA in livestock seems to be influenced by farm size, farming systems, usage of disinfectants, and in-feed zinc. LA-MRSA CC398 is able to cause the same kind of infections in humans as S. aureus and MRSA in general. It can be introduced to hospitals and cause nosocomial infections such as postoperative surgical site infections, ventilator associated pneumonia, septicemia, and infections after joint replacement. For this reason, screening for MRSA colonization at hospital admittance is recommended for farmers and veterinarians with livestock contacts. Intrahospital dissemination, typical for HA-MRSA in the absence of sufficient hygiene, has only rarely been observed for LA-MRSA to date. The proportion of LA-MRSA among all MRSA from nosocomial infections is about 3% across Germany. In geographical areas with a comparatively high density of conventional farms, LA-MRSA accounts for up to 10% of MRSA from septicemia and 15% of MRSA from wound infections. As known from comparative genome analysis, LA-MRSA has evolved from human-adapted methicillin-susceptible S. aureus, and the jump to livestock was obviously associated with several genetic changes. Reversion of the genetic changes and readaptation to humans bears a potential health risk and requires tight surveillance. Although most LA-MRSA (>80%) is resistant to several antibiotics, there are still sufficient treatment options.
Since 2001, several studies have reported high rifampicin resistance rates (45 - 100%) among methicillin-resistant Staphylococcus aureus (MRSA) isolates from South Africa. The authors previously characterised 100 MRSA isolates from hospitals in Cape Town, South Africa; forty-five percent of these isolates were rifampicin-resistant. The majority (44/45) corresponded to ST612-MRSA-IV, which is prevalent in South Africa, but has not been reported frequently elsewhere. The remaining rifampicin-resistant isolate corresponded to ST5-MRSA-I. The aim of this study was to investigate further the prevalence and genetic basis of rifampicin-resistance in MRSA isolates from hospitals in Cape Town.
Between July 2007 and June 2011, the prevalence of rifampicin-resistant MRSA in hospitals in Cape Town ranged from 39.7% to 46.4%. Based on the results of the aforementioned study, nine ST612-MRSA-IV isolates, the rifampicin-resistant ST5-MRSA-I isolate, and two rifampicin-susceptible MRSA isolates were investigated. Four previously described ST612-MRSA-IV isolates, including two each from South Africa and Australia, were also included.The ST5-MRSA-I isolate carried a single mutational change, H481Y, commonly associated with high-level rifampicin resistance. All ST612-MRSA-IV isolates carried an uncommon double amino acid substitution in RpoB, H481N, I527M, whilst one of the Australian ST612-MRSA-IV isolates carried an additional mutation within rpoB, representing a novel rpoB genotype: H481N, I527M, K579R. All ST612-MRSA-IV isolates also shared a unique silent single nucleotide polymorphism (SNP) within rpoB.
That local ST612-MRSA-IV isolates described here share an uncommon rpoB genotype and a unique silent SNP suggests this clone may have undergone clonal expansion in hospitals in Cape Town. Further, the data suggest that these isolates may be related to rifampicin-resistant ST612-MRSA-IV previously described in South Africa and Australia.
Visualisation of genome comparisons is invaluable for helping to determine genotypic differences between closely related prokaryotes. New visualisation and abstraction methods are required in order to improve the validation, interpretation and communication of genome sequence information; especially with the increasing amount of data arising from next-generation sequencing projects. Visualising a prokaryote genome as a circular image has become a powerful means of displaying informative comparisons of one genome to a number of others. Several programs, imaging libraries and internet resources already exist for this purpose, however, most are either limited in the number of comparisons they can show, are unable to adequately utilise draft genome sequence data, or require a knowledge of command-line scripting for implementation. Currently, there is no freely available desktop application that enables users to rapidly visualise comparisons between hundreds of draft or complete genomes in a single image.
BLAST Ring Image Generator (BRIG) can generate images that show multiple prokaryote genome comparisons, without an arbitrary limit on the number of genomes compared. The output image shows similarity between a central reference sequence and other sequences as a set of concentric rings, where BLAST matches are coloured on a sliding scale indicating a defined percentage identity. Images can also include draft genome assembly information to show read coverage, assembly breakpoints and collapsed repeats. In addition, BRIG supports the mapping of unassembled sequencing reads against one or more central reference sequences. Many types of custom data and annotations can be shown using BRIG, making it a versatile approach for visualising a range of genomic comparison data. BRIG is readily accessible to any user, as it assumes no specialist computational knowledge and will perform all required file parsing and BLAST comparisons automatically.
There is a clear need for a user-friendly program that can produce genome comparisons for a large number of prokaryote genomes with an emphasis on rapidly utilising unfinished or unassembled genome data. Here we present BRIG, a cross-platform application that enables the interactive generation of comparative genomic images via a simple graphical-user interface. BRIG is freely available for all operating systems at http://sourceforge.net/projects/brig/.
Clin Microbiol Infect 2011; 17: 785–792
There is currently limited information available on the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in South Africa. A molecular characterization of 100 MRSA from five hospitals in Cape Town was carried out in this study. The strains were separated into six clusters by pulsed-field gel electrophoresis, indicating transmission of MRSA between local hospitals. None of the strains carried the Panton-Valentine Leukocidin gene. SCCmec typing, multilocus sequence typing and spa typing were used to further characterize the MRSA. Three clones corresponded to frequently described pandemic clones: ST239-MRSA-III, ST36-MRSA-II and ST5-MRSA-I. ST239-MRSA-III and ST36-MRSA-II were minor clones and collectively accounted for 16% of the isolates. ST5-MRSA-I was the second-most prevalent clone and accounted for 37% of the isolates. The dominant local clone was the infrequently described ST612-MRSA-IV (44% of isolates), which has only been described in South Africa and Australia.
We recently described FastTree, a tool for inferring phylogenies for alignments with up to hundreds of thousands of sequences. Here, we describe improvements to FastTree that improve its accuracy without sacrificing scalability.
Where FastTree 1 used nearest-neighbor interchanges (NNIs) and the minimum-evolution criterion to improve the tree, FastTree 2 adds minimum-evolution subtree-pruning-regrafting (SPRs) and maximum-likelihood NNIs. FastTree 2 uses heuristics to restrict the search for better trees and estimates a rate of evolution for each site (the "CAT" approximation). Nevertheless, for both simulated and genuine alignments, FastTree 2 is slightly more accurate than a standard implementation of maximum-likelihood NNIs (PhyML 3 with default settings). Although FastTree 2 is not quite as accurate as methods that use maximum-likelihood SPRs, most of the splits that disagree are poorly supported, and for large alignments, FastTree 2 is 100-1,000 times faster. FastTree 2 inferred a topology and likelihood-based local support values for 237,882 distinct 16S ribosomal RNAs on a desktop computer in 22 hours and 5.8 gigabytes of memory.
FastTree 2 allows the inference of maximum-likelihood phylogenies for huge alignments. FastTree 2 is freely available at http://www.microbesonline.org/fasttree.
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has recently emerged worldwide. The United States, in particular, is experiencing a serious epidemic of CA-MRSA that is almost entirely caused by an extraordinarily infectious strain named USA300. However, the molecular determinants underlying the pathogenic success of CA-MRSA are mostly unknown. To gain insight into the evolution of the exceptional potential of USA300 to cause disease, we compared the phylogeny and virulence of USA300 with that of closely related MRSA clones. We discovered that the sublineage from which USA300 evolved is characterized by a phenotype of high virulence that is clearly distinct from other MRSA strains. Namely, USA300 and its progenitor, USA500, had high virulence in animal infection models and the capacity to evade innate host defense mechanisms. Furthermore, our results indicate that increased virulence in the USA300/USA500 sublineage is attributable to differential expression of core genome-encoded virulence determinants, such as phenol-soluble modulins and alpha-toxin. Notably, the fact that the virulence phenotype of USA300 was already established in its progenitor indicates that acquisition of mobile genetic elements has played a limited role in the evolution of USA300 virulence and points to a possibly different role of those elements. Thus, our results highlight the importance of differential gene expression in the evolution of USA300 virulence. This finding calls for a profound revision of our notion about CA-MRSA pathogenesis at the molecular level and has important implications for design of therapeutics directed against CA-MRSA.
The staphylococcal pathogenicity islands (SaPIs) are highly mobile 15kb genomic islands that carry superantigen genes and other virulence factors and are mobilized by helper phages. Helper phages counteract the SaPI repressor to induce the SaPI replication cycle, resulting in encapsidation in phage like particles, enabling high frequency transfer. The SaPIs split from a protophage lineage in the distant past, have evolved a variety of novel and salient features, and have become an invaluable component of the staphylococcal genome. This review focuses on recent studies describing three different mechanisms of SaPI interference with helper phage reproduction and other studies demonstrating that helper phage mutations to resistance against this interference impact phage evolution. Also described are recent results showing that SaPIs contribute in a major way to lateral transfer of host genes as well as enabling their own transfer. SaPI-like elements, readily identifiable in the bacterial genome, are widespread throughout the Gram-positive cocci, though functionality has thus far been demonstrated for only a single one of these.
Staphylococcus aureus has evolved as a pathogen that causes a range of diseases in humans. There are two dominant modes of evolution thought to explain most of the virulence differences between strains. First, virulence genes may be acquired from other organisms. Second, mutations may cause changes in the regulation and expression of genes. Here we describe an evolutionary event in which transposition of an IS element has a direct impact on virulence gene regulation resulting in hypervirulence. Whole genome analysis of a methicillin-resistant S. aureus (MRSA) strain USA500 revealed acquisition of a transposable element (IS256) that is absent from close relatives of this strain. Of the multiple copies of IS256 found in the USA500 genome, one was inserted in the promoter sequence of repressor of toxins (Rot), a master transcriptional regulator responsible for the expression of virulence factors in S. aureus. We show that insertion into the rot promoter by IS256 results in the derepression of cytotoxin expression and increased virulence. Taken together, this work provides new insight into evolutionary strategies by which S. aureus is able to modify its virulence properties and demonstrates a novel mechanism by which horizontal gene transfer directly impacts virulence through altering toxin regulation.
To evaluate if methicillin-resistant Staphylococcus aureus (MRSA) is present in the horse population in Australia.
A two-part retrospective study of laboratory submissions of microbial culture results from horses.
Part A: medical records of 216 horses that had MRSA screening performed on nasal swabs collected over a 30-day period at admission to the Scone Equine Hospital Clovelly Intensive Care Unit were retrieved. Part B: laboratory records from 2004 to 2009 of culture submissions to the Scone Veterinary Laboratory were reviewed and cultures that grew MRSA were identified. The MRSA isolates from Parts A and B were genotyped over an 18-month period.
MRSA screening of 216 horses identified eight (3.7%) positive samples. MRSA was isolated from cultures of 80 (0.002%) clinical bacteriology samples over a 6-year period. Genotypic analysis was performed on 36 isolates. All MRSA characterised had the same pulse field gel electrophoresis pattern (type 1), with eight closely related subtypes identified (subtypes A-F and H) and 66% of isolates classified as subtype D, which multilocus sequence and staphylococcal cassette chromosome mec typing analysis identified as ST612-MRSA-IVa, a clonal complex (CC) 8 S. aureus strain. Antimicrobial resistance to more than two classes of antimicrobials was common.
MRSA was present in a population of horses in Australia. Genotypic analysis of the isolates identified the MRSA strain as CC8 S. aureus. Further research needs to be undertaken to evaluate MRSA infection and colonisation of horses and personnel in Australia.
Clinical specimens of small animals (n=869) were screened for the occurrence of methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA; MRSA) during routine microbiological examinations, and results were confirmed by a multiplex PCR strategy. The genetic relatedness of all mecA-positive S. aureus isolates was further investigated by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), PCR for Panton-Valentine leukocidine genes (PVL) and staphylococcal cassette chromosome mec-typing (SCCmec). A total of 61 S. aureus isolates were found during a 20-month period of investigation, 27 (44.3%) of them harbouring the mecA gene for methicillin-resistance. The majority of MRSA were isolated in specimens from dogs (n=18) and cats (n=4). One guinea pig and one rabbit were found to be positive for an MRSA infected site. Similarly, three exotic animals, a turtle, a bat and a parrot, were found to be infected with MRSA. PFGE and MLST analysis revealed a certain genotype ("A" and "A-1") dominating the isolate collection (23 of 27). Furthermore, one isolate showed homologous PFGE pattern to the German epidemic strain Barnim ("BE") and another one ("BE-1") was considered to be closely related. A third genotype ("B") was detected in two cases. Two different sequence types (ST) were identified among the 27 MRSA isolates. PFGE type "A" and both strains related to the Barnim epidemic strain were assigned to ST22, whereas ST239 was associated to PFGE profile "B". The present data show that certain MRSA genotypes are capable of infecting a wide spectrum of small and exotic animals, especially in clinical facilities.
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Zoonotic transmission of the methicillin-resistant Staphylococcus aureus ST612-IV equine strain
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