RESUMO
Shotgun metagenomics is a powerful tool to identify antimicrobial resistance (AMR) genes in microbiomes but has the limitation that extrachromosomal DNA, such as plasmids, cannot be linked with the host bacterial chromosome. Here we present a comprehensive laboratory and bioinformatics pipeline HAM-ART (Hi-C Assisted Metagenomics for Antimicrobial Resistance Tracking) optimised for the generation of metagenome-assembled genomes including both chromosomal and extrachromosomal AMR genes. We demonstrate the performance of the pipeline in a study comparing 100 pig faecal microbiomes from low- and high-antimicrobial use pig farms (organic and conventional farms). We found significant differences in the distribution of AMR genes between low- and high-antimicrobial use farms including a plasmid-borne lincosamide resistance gene exclusive to high-antimicrobial use farms in three species of Lactobacilli. The bioinformatics pipeline code is available at https://github.com/lkalmar/HAM-ART.
Assuntos
Anti-Infecciosos , Microbiota , Animais , Antibacterianos , Anti-Infecciosos/farmacologia , Farmacorresistência Bacteriana/genética , Metagenômica , SuínosRESUMO
Most new pathogens of humans and animals arise via switching events from distinct host species. However, our understanding of the evolutionary and ecological drivers of successful host adaptation, expansion, and dissemination are limited. Staphylococcus aureus is a major bacterial pathogen of humans and a leading cause of mastitis in dairy cows worldwide. Here we trace the evolutionary history of bovine S. aureus using a global dataset of 10,254 S. aureus genomes including 1,896 bovine isolates from 32 countries in 6 continents. We identified 7 major contemporary endemic clones of S. aureus causing bovine mastitis around the world and traced them back to 4 independent host-jump events from humans that occurred up to 2,500 y ago. Individual clones emerged and underwent clonal expansion from the mid-19th to late 20th century coinciding with the commercialization and industrialization of dairy farming, and older lineages have become globally distributed via established cattle trade links. Importantly, we identified lineage-dependent differences in the frequency of host transmission events between humans and cows in both directions revealing high risk clones threatening veterinary and human health. Finally, pangenome network analysis revealed that some bovine S. aureus lineages contained distinct sets of bovine-associated genes, consistent with multiple trajectories to host adaptation via gene acquisition. Taken together, we have dissected the evolutionary history of a major endemic pathogen of livestock providing a comprehensive temporal, geographic, and gene-level perspective of its remarkable success.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Feminino , Humanos , Bovinos , Animais , Staphylococcus aureus/genética , Gado/genética , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/veterinária , Infecções Estafilocócicas/genética , Genoma , Especificidade de HospedeiroRESUMO
Animal welfare is usually excluded from life cycle assessments (LCAs) of farming systems because of limited consensus on how to measure it. Here, we constructed several LCA-compatible animal-welfare metrics and applied them to data we collected from 74 diverse breed-to-finish systems responsible for 5% of UK pig production. Some aspects of metric construction will always be subjective, such as how different aspects of welfare are aggregated, and what determines poor versus good welfare. We tested the sensitivity of individual farm rankings, and rankings of those same farms grouped by label type (memberships of quality-assurance schemes or product labelling), to a broad range of approaches to metric construction. We found farms with the same label types clustered together in rankings regardless of metric choice, and there was broad agreement across metrics on the rankings of individual farms. We found woodland and Organic systems typically perform better than those with no labelling and Red tractor labelling, and that outdoor-bred and outdoor-finished systems perform better than indoor-bred and slatted-finished systems, respectively. We conclude that if our goal is to identify relatively better and worse farming systems for animal welfare, exactly how LCA welfare metrics are constructed may be less important than commonly perceived.
Assuntos
Criação de Animais Domésticos , Animais Domésticos , Animais , Suínos , Fazendas , Bem-Estar do AnimalRESUMO
Klebsiella quasipneumoniae is a recently described species and often misidentified as Klebsiella pneumoniae. Here, we report the genomic characterization of Klebsiella quasipneumoniae subsp. similipneumoniae (India238 strain) isolated from fish. The annotated genome acknowledged the presence of blaCTX-M-15, blaOKP-B-1, fosA5, oqxAB and virulence genes. The strain with ST1699 and serotypes KL52 and OL103 also harboured insertion sequences (ISs): ISKpn26 and ISEc9. Three complete phage genomes were identified in contigs 1 and 6 of the bacterial genome, enhancing the prospects of genome manipulation. The study highlights the pitfall of conventional microbiological identification methods to distinguish K. pneumoniae and K. quasipneumoniae. This is the first Indian study documenting the incidence of extended-spectrum beta-lactamase (ESBL)-producing K. quasipneumoniae subsp. similipneumoniae from a non-clinical environment, equipped with virulomes and associated mobile genetic elements. Given that fish can act as a potential vector for transmission of antimicrobial resistance genes, our findings have paramount importance on human health.
Assuntos
Klebsiella pneumoniae , beta-Lactamases , Animais , Genômica , Índia , Klebsiella , Klebsiella pneumoniae/genética , beta-Lactamases/genéticaRESUMO
Antibiotic resistance is a sword of Damocles that hangs over humans. In regards to airborne antibiotic resistance genes (AARGs), critical knowledge gaps still exist in the identification of hotspots and quantification of exposure levels in different environments. Here, we have studied the profiles of AARGs, mobile genetic elements (MGEs) and bacterial communities in various atmospheric environments by high throughput qPCR and 16S rRNA gene sequencing. We propose a new AARGs exposure dose calculation that uses short-term inhalation (STI). Swine farms and hospitals were high-risk areas where AARGs standardised abundance was more abundant than suburbs and urban areas. Additionally, resistance gene abundance in swine farm worker sputum was higher than that in healthy individuals in other environments. The correlation between AARGs with MGEs and bacteria was strong in suburbs but weak in livestock farms and hospitals. STI exposure analysis revealed that occupational intake of AARGs (via PM10) in swine farms and hospitals were 110 and 29 times higher than in suburbs, were 1.5 × 104, 5.6 × 104 and 5.1 × 102 copies, i.e., 61.9%, 75.1% and 10.7% of the overall daily inhalation intake, respectively. Our study comprehensively compares environmental differences in AARGs to identify high-risk areas, and forwardly proposes the STI exposure dose of AARGs to guide risk assessment.
Assuntos
Antibacterianos , Genes Bacterianos , Animais , Antibacterianos/análise , Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Exposição por Inalação , RNA Ribossômico 16S/genética , SuínosRESUMO
The spread of methicillin-resistant Staphylococcus aureus (MRSA) in the community, hospitals and in livestock is mediated by highly diverse virulence factors that include secreted toxins, superantigens, enzymes and surface-associated adhesins allowing host adaptation and colonization. Here, we combined proteogenomics, secretome and phenotype analyses to compare the secreted virulence factors in selected S. aureus isolates of the dominant human- and livestock-associated genetic lineages CC8, CC22, and CC398. The proteogenomic comparison revealed 2181 core genes and 1306 accessory genes in 18 S. aureus isolates reflecting the high genome diversity. Using secretome analysis, we identified 869 secreted proteins with 538 commons in eight isolates of CC8, CC22, and CC398. These include 64 predicted extracellular and 37 cell surface proteins that account for 82.4% of total secretome abundance. Among the top 10 most abundantly secreted virulence factors are the major autolysins (Atl, IsaA, Sle1, SAUPAN006375000), lipases and lipoteichoic acid hydrolases (Lip, Geh, LtaS), cytolytic toxins (Hla, Hlb, PSMß1) and proteases (SspB). The CC398 isolates showed lower secretion of cell wall proteins, but higher secretion of α- and ß-hemolysins (Hla, Hlb) which correlated with an increased Agr activity and strong hemolysis. CC398 strains were further characterized by lower biofilm formation and staphyloxanthin levels because of decreased SigB activity. Overall, comparative secretome analyses revealed CC8- or CC22-specific enterotoxin and Spl protease secretion as well as Agr- and SigB-controlled differences in exotoxin and surface protein secretion between human-specific and zoonotic lineages of S. aureus.
Assuntos
Filogenia , Proteogenômica/métodos , Staphylococcus aureus/classificação , Staphylococcus aureus/genética , Animais , Sobrevivência Celular , Cromatografia Líquida , Bases de Dados Genéticas , Variação Estrutural do Genoma , Genótipo , Cavalos , Humanos , Proteoma/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Suínos , Espectrometria de Massas em Tandem , Virulência , Fatores de Virulência/metabolismo , Sequenciamento Completo do Genoma , ZoonosesRESUMO
OBJECTIVES: High-level ß-lactam resistance in MRSA is mediated in the majority of strains by a mecA or mecC gene. In this study, we identified 10 mec gene-negative MRSA human isolates from Austria and 11 bovine isolates from the UK showing high levels of ß-lactam resistance and sought to understand the molecular basis of the resistance observed. METHODS: Different antimicrobial resistance testing methods (disc diffusion, Etest and VITEK® 2) were used to establish the ß-lactam resistance profiles for the isolates and the isolates were further investigated by WGS. RESULTS: A number of mutations (including novel ones) in PBPs, AcrB, YjbH and the pbp4 promoter were identified in the resistant isolates, but not in closely related susceptible isolates. Importantly, a truncation in the cyclic diadenosine monophosphate phosphodiesterase enzyme, GdpP, was identified in 7 of the 10 Austrian isolates and 10 of the 11 UK isolates. Complementation of four representative isolates with an intact copy of the gdpP gene restored susceptibility to penicillins and abolished the growth defects caused by the truncation. CONCLUSIONS: This study reports naturally occurring inactivation of GdpP protein in Staphylococcus aureus of both human origin and animal origin, and demonstrates clinical relevance to a previously reported association between this truncation and increased ß-lactam resistance and impaired bacterial growth in laboratory-generated mutants. It also highlights possible limitations of genomic determination of antibiotic susceptibility based on single gene presence or absence when choosing the appropriate antimicrobial treatment for patients.
Assuntos
Doenças dos Bovinos/microbiologia , Diester Fosfórico Hidrolases/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Resistência beta-Lactâmica , beta-Lactamas/farmacologia , Alelos , Substituição de Aminoácidos , Animais , Bovinos , Genoma Bacteriano , Genômica , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Fenótipo , Diester Fosfórico Hidrolases/metabolismo , Deleção de Sequência , Staphylococcus aureus/isolamento & purificaçãoRESUMO
Similar to mecA, mecC confers resistance against beta-lactams, leading to the phenotype of methicillin-resistant Staphylococcus aureus (MRSA). However, mecC-harboring MRSA strains pose special difficulties in their detection. The aim of this study was to assess and compare different phenotypic systems for screening, identification, and susceptibility testing of mecC-positive MRSA isolates. A well-characterized collection of mecC-positive S. aureus isolates (n = 111) was used for evaluation. Routinely used approaches were studied to determine their suitability to correctly identify mecC-harboring MRSA, including three (semi)automated antimicrobial susceptibility testing (AST) systems and five selective chromogenic agar plates. Additionally, a cefoxitin disk diffusion test and an oxacillin broth microdilution assay were examined. All mecC-harboring MRSA isolates were able to grow on all chromogenic MRSA screening plates tested. Detection of these isolates in AST systems based on cefoxitin and/or oxacillin testing yielded overall positive agreements with the mecC genotype of 97.3% (MicroScan WalkAway; Siemens), 91.9% (Vitek 2; bioMérieux), and 64.9% (Phoenix, BD). The phenotypic resistance pattern most frequently observed by AST devices was "cefoxitin resistance/oxacillin susceptibility," ranging from 54.1% (Phoenix) and 83.8% (Vitek 2) to 92.8% (WalkAway). The cefoxitin disk diffusion and oxacillin broth microdilution assays categorized 100% and 61.3% of isolates to be MRSA, respectively. The chromogenic media tested confirmed their suitability to reliably screen for mecC-harboring MRSA. The AST systems showed false-negative results with varying numbers, misidentifying mecC-harboring MRSA as methicillin-susceptible S. aureus This study underlines cefoxitin's status as the superior surrogate mecC-positive MRSA marker.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Cefoxitina/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana/métodos , Oxacilina/farmacologia , Proteínas de Ligação às Penicilinas/genética , Infecções Estafilocócicas/microbiologia , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Fenótipo , Infecções Estafilocócicas/diagnósticoRESUMO
Here we describe a new species of the genus Streptococcus that was isolated from a dairy cow with mastitis in New Zealand. Strain NZ1587T was Gram-positive, coccus-shaped and arranged as chains, catalase and coagulase negative, γ-haemolytic and negative for Lancefield carbohydrates (A-D, F and G). The 16S rRNA sequence did not match sequences in the NCBI 16S rRNA or GreenGenes databases. Taxonomic classification of strain NZ1587T was investigated using 16S rRNA and core genome phylogeny, genome-wide average nucleotide identity (ANI) and predicted DNA-DNA hybridisation (DDH) analyses. Phylogeny based on 16S rRNA was unable to resolve the taxonomic position of strain NZ1587T, however NZ1587T shared 99.4â% identity at the 16S rRNA level with a distinct branch of S. pseudoporcinus. Importantly, core genome phylogeny demonstrated that NZ1587T grouped amongst the 'pyogenic' streptococcal species and formed a distinct branch supported by a 100â% bootstrap value. In addition, average nucleotide identity and inferred DNA-DNA hybridisation analyses showed that NZ1587T represents a novel species. Biochemical profiling using the rapid ID 32 strep identification test enabled differentiation of strain NZ1587T from closely related streptococcal species. In conclusion, strain NZ1587T can be classified as a novel species, and we propose a novel taxon named Streptococcus bovimastitidis sp. nov.; the type strain is NZ1587T. NZ1587T has been deposited in the Culture Collection University of Gothenburg (CCUG 69277T) and the Belgian Co-ordinated Collections of Micro-organisms/LMG (LMG 29747).
Assuntos
Mastite Bovina/microbiologia , Filogenia , Infecções Estreptocócicas/veterinária , Streptococcus/classificação , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , Bovinos , DNA Bacteriano/genética , Feminino , Nova Zelândia , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Streptococcus/genética , Streptococcus/isolamento & purificaçãoRESUMO
Passerine salmonellosis is a well-recognized disease of birds in the order Passeriformes, which includes common songbirds such as finches and sparrows, caused by infection with Salmonella enterica serovar Typhimurium. Previous research has suggested that some subtypes of S Typhimurium-definitive phage types (DTs) 40, 56 variant, and 160-are host adapted to passerines and that these birds may represent a reservoir of infection for humans and other animals. Here, we have used the whole-genome sequences of 11 isolates from British passerines, five isolates of similar DTs from humans and a domestic cat, and previously published S Typhimurium genomes that include similar DTs from other hosts to investigate the phylogenetic relatedness of passerine salmonellae to other S Typhimurium isolates and investigate possible genetic features of the distinct disease pathogenesis of S Typhimurium in passerines. Our results demonstrate that the 11 passerine isolates and 13 other isolates, including those from nonpasserine hosts, were genetically closely related, with a median pairwise single nucleotide polymorphism (SNP) difference of 130 SNPs. These 24 isolates did not carry antimicrobial resistance genetic determinants or the S Typhimurium virulence plasmid. Although our study does not provide evidence of Salmonella transmission from passerines to other hosts, our results are consistent with the hypothesis that wild birds represent a potential reservoir of these Salmonella subtypes, and thus, sensible personal hygiene precautions should be taken when feeding or handling garden birds. IMPORTANCE: Passerine salmonellosis, caused by certain definitive phage types (DTs) of Salmonella Typhimurium, has been documented as a cause of wild passerine mortality since the 1950s in many countries, often in the vicinity of garden bird feeding stations. To gain better insight into its epidemiology and host-pathogen interactions, we sequenced the genomes of a collection of 11 isolates from wild passerine salmonellosis in England and Wales. Phylogenetic analysis showed these passerine isolates to be closely related to each other and to form a clade that is distinct from other strains of S Typhimurium, which included a multidrug-resistant isolate from invasive nontyphoidal Salmonella disease that shares the same phage type as several of the passerine isolates. Closely related to wild passerine isolates and within the same clade were four S Typhimurium isolates from humans as well as isolates from horses, poultry, cattle, an unspecified wild bird, and a domestic cat and dog with similar DTs and/or multilocus sequence types. This suggests the potential for cross-species transmission, and the genome sequences provide a valuable resource to investigate passerine salmonellosis further.
Assuntos
Doenças das Aves/microbiologia , Reservatórios de Doenças/microbiologia , Genoma Bacteriano , Salmonelose Animal/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/isolamento & purificação , Pardais/microbiologia , Animais , Animais Selvagens/microbiologia , Tipagem de Bacteriófagos , Doenças das Aves/epidemiologia , Gatos , Farmacorresistência Bacteriana/genética , Inglaterra/epidemiologia , Genômica , Humanos , Tipagem de Sequências Multilocus , Filogenia , Plasmídeos , Polimorfismo de Nucleotídeo Único , Infecções por Salmonella/microbiologia , Salmonelose Animal/epidemiologia , Salmonelose Animal/prevenção & controle , Salmonelose Animal/transmissão , Salmonella typhimurium/classificação , Salmonella typhimurium/patogenicidade , Sorogrupo , Sorotipagem , Virulência/genética , País de Gales/epidemiologiaRESUMO
ß-Lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) is mediated by the expression of an alternative penicillin-binding protein 2a (PBP2a) (encoded by mecA) with a low affinity for ß-lactam antibiotics. Recently, a novel variant of mecA, known as mecC, was identified in MRSA isolates from both humans and animals. In this study, we demonstrate that mecC-encoded PBP2c does not mediate resistance to penicillin. Rather, broad-spectrum ß-lactam resistance in MRSA strains carrying mecC (mecC-MRSA strains) is mediated by a combination of both PBP2c and the distinct ß-lactamase encoded by the blaZ gene of strain LGA251 (blaZLGA251), which is part of mecC-encoding staphylococcal cassette chromosome mec (SCCmec) type XI. We further demonstrate that mecC-MRSA strains are susceptible to the combination of penicillin and the ß-lactam inhibitor clavulanic acid in vitro and that the same combination is effective in vivo for the treatment of experimental mecC-MRSA infection in wax moth larvae. Thus, we demonstrate how the distinct biological differences between mecA- and mecC-encoded PBP2a and PBP2c have the potential to be exploited as a novel approach for the treatment of mecC-MRSA infections.
Assuntos
Ácido Clavulânico/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Proteínas de Ligação às Penicilinas/genética , Penicilinas/farmacologia , Animais , Proteínas de Bactérias/genética , Interações Medicamentosas , Larva/efeitos dos fármacos , Larva/microbiologia , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Mariposas/efeitos dos fármacos , Mariposas/microbiologia , Mutação , Resistência às Penicilinas/efeitos dos fármacos , Resistência às Penicilinas/genética , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/genéticaRESUMO
OBJECTIVES: MDR methicillin-resistant Staphylococcus pseudintermedius (MRSP) strains have emerged rapidly as major canine pathogens and present serious treatment issues and concerns to public health due to their, albeit low, zoonotic potential. A further understanding of the genetics of resistance arising from a broadly susceptible background of S. pseudintermedius is needed. METHODS: We sequenced the genomes of 12 S. pseudintermedius isolates of varied STs and resistance phenotypes. RESULTS: Nine distinct clonal lineages had acquired either staphylococcal cassette chromosome (SCC) mec elements and/or Tn5405-like elements carrying up to five resistance genes [aphA3, sat, aadE, erm(B), dfrG] to generate MRSP, MDR methicillin-susceptible S. pseudintermedius and MDR MRSP populations. The most successful and clinically problematic MDR MRSP clones, ST68 SCCmecV(T) and ST71 SCCmecII-III, have further accumulated mutations in gyrA and grlA conferring resistance to fluoroquinolones. The carriage of additional mobile genetic elements (MGEs) was highly variable, suggesting that horizontal gene transfer is frequent in S. pseudintermedius populations. CONCLUSIONS: Importantly, the data suggest that MDR MRSP evolved rapidly by the acquisition of a very limited number of MGEs and mutations, and that the use of many classes of antimicrobials may co-select for the spread and emergence of MDR and XDR strains. Antimicrobial stewardship will need to be comprehensive, encompassing human medicine and veterinary disciplines to successfully preserve antimicrobial efficacy.
Assuntos
Antibacterianos/farmacologia , Evolução Biológica , Farmacorresistência Bacteriana Múltipla , Staphylococcus/efeitos dos fármacos , Animais , DNA Bacteriano/química , DNA Bacteriano/genética , Transferência Genética Horizontal , Genoma Bacteriano , Humanos , Sequências Repetitivas Dispersas , Dados de Sequência Molecular , Mutação , Análise de Sequência de DNARESUMO
OBJECTIVES: To determine whether extending prophylactic antimicrobial administration into the postoperative period would decrease the surgical site infection (SSI) rate in clean canine orthopedic surgery associated with a metal implant. STUDY DESIGN: Randomized prospective clinical study. SAMPLE POPULATION: Consecutive procedures (n = 400) on dogs that had clean orthopedic surgery using a metal implant. METHODS: Cases were randomly allocated to 1 of 2 groups. Group 1 was only administered perioperative antimicrobial drugs whereas group 2 was administered perioperative and 5 days of postoperative antimicrobial therapy. Owners were questioned or dogs were examined at 2 and 6 weeks after surgery to identify any SSI. Long term follow-up by questionnaire of the referring veterinary surgeon ≥1 year after surgery was obtained. RESULTS: Ten of 191 dogs (5.24%) in group 1 developed SSI within 6 weeks compared with 7 of 198 (3.54%) in group 2; 7.22% of dogs in group 1 and 8.24% in group 2 developed infections more than 6 weeks after surgery. CONCLUSIONS: SSI rates in this population of dogs were similar where antimicrobial prophylaxis was administered perioperatively over 3 hours or as a course continued for 6 days.
Assuntos
Anti-Infecciosos/administração & dosagem , Cefuroxima/administração & dosagem , Doenças do Cão/prevenção & controle , Infecção da Ferida Cirúrgica/veterinária , Administração Oral , Animais , Antibioticoprofilaxia/veterinária , Cães , Feminino , Infusões Intravenosas , Masculino , Procedimentos Ortopédicos/veterinária , Período Perioperatório , Período Pós-Operatório , Estudos Prospectivos , Próteses e Implantes/veterinária , Infecção da Ferida Cirúrgica/prevenção & controle , Resultado do TratamentoRESUMO
OBJECTIVES: Methicillin resistance in Staphylococcus spp. results from the expression of an alternative penicillin-binding protein 2a (encoded by mecA) with a low affinity for ß-lactam antibiotics. Recently, a novel variant of mecA known as mecC (formerly mecALGA251) was identified in Staphylococcus aureus isolates from both humans and animals. In this study, we identified two Staphylococcus sciuri subsp. carnaticus isolates from bovine infections that harbour three different mecA homologues: mecA, mecA1 and mecC. METHODS: We subjected the two isolates to whole-genome sequencing to further understand the genetic context of the mec-containing region. We also used PCR and RT-PCR to investigate the excision and expression of the SCCmec element and mec genes, respectively. RESULTS: Whole-genome sequencing revealed a novel hybrid SCCmec region at the orfX locus consisting of a class E mec complex (mecI-mecR1-mecC1-blaZ) located immediately downstream of a staphylococcal cassette chromosome mec (SCCmec) type VII element. A second SCCmec attL site (attL2), which was imperfect, was present downstream of the mecC region. PCR analysis of stationary-phase cultures showed that both the SCCmec type VII element and a hybrid SCCmec-mecC element were capable of excision from the genome and forming a circular intermediate. Transcriptional analysis showed that mecC and mecA, but not mecA1, were both expressed in liquid culture supplemented with oxacillin. CONCLUSIONS: Overall, this study further highlights that a range of staphylococcal species harbour the mecC gene and furthers the view that coagulase-negative staphylococci associated with animals may act as reservoirs of antibiotic resistance genes for more pathogenic staphylococcal species.
Assuntos
Genes Bacterianos , Infecções Estafilocócicas/veterinária , Staphylococcus/genética , Animais , Bovinos , DNA Bacteriano/química , DNA Bacteriano/genética , Perfilação da Expressão Gênica , Ordem dos Genes , Genoma Bacteriano , Genótipo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Infecções Estafilocócicas/microbiologia , Staphylococcus/isolamento & purificaçãoRESUMO
OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) is an important global health problem. MRSA resistance to ß-lactam antibiotics is mediated by the mecA or mecC genes, which encode an alternative penicillin-binding protein (PBP) 2a that has a low affinity to ß-lactam antibiotics. Detection of mec genes or PBP2a is regarded as the gold standard for the diagnosis of MRSA. We identified four MRSA isolates that lacked mecA or mecC genes, but were still phenotypically resistant to pencillinase-resistant ß-lactam antibiotics. METHODS: The four human S. aureus isolates were investigated by whole genome sequencing and a range of phenotypic assays. RESULTS: We identified a number of amino acid substitutions present in the endogenous PBPs 1, 2 and 3 that were found in the resistant isolates but were absent in closely related susceptible isolates and which may be the basis of resistance. Of particular interest are three identical amino acid substitutions in PBPs 1, 2 and 3, occurring independently in isolates from at least two separate multilocus sequence types. Two different non-conservative substitutions were also present in the same amino acid of PBP1 in two isolates from two different sequence types. CONCLUSIONS: This work suggests that phenotypically resistant MRSA could be misdiagnosed using molecular methods alone and provides evidence of alternative mechanisms for ß-lactam resistance in MRSA that may need to be considered by diagnostic laboratories.
Assuntos
Resistência a Meticilina , Staphylococcus aureus Resistente à Meticilina/enzimologia , Staphylococcus aureus Resistente à Meticilina/genética , Proteínas de Ligação às Penicilinas/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Humanos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Proteínas Mutantes/genética , Análise de Sequência de DNA , Infecções Estafilocócicas/microbiologiaRESUMO
OBJECTIVE: To evaluate the quasi-isometric points (nearest isometric points) between the distal aspect of the femur and fabella and the proximal aspect of the tibia for placement of a lateral suture in cats. STUDY DESIGN: Radiographic study. ANIMALS: Cadaveric cat stifles (n = 7 cats; 14 stifles). METHODS: Specimens were secured in a mounting frame to maintain rigid fixation of the femur and allow free range of motion of the stifle joint and proximal tibia. Two anatomic landmarks were identified: the center of the lateral fabella (Ff) and a point 4 mm proximal to the insertion of the patellar tendon adjacent to the tibial cortex (Tt). Radiopaque spheres were placed at predefined landmarks in the femur (caudal aspect of the lateral femoral condyle distal [F1] and proximal [F2] to the lateral fabella) and in the tibia (caudal to the proximal aspect of the extensor groove [T1]; cranial to the proximal aspect of the extensor groove [T2]; 2 mm proximal and caudal to the insertion of the patellar tibial tendon [T3] and 3 mm caudal to the insertion of the patellar tibial tendon [T4]. For each stifle, 4 radiographic projections were made: in extension (166°), in flexion (45°), and 2 intermediate stance phases (90°, 130°). ANOVA was used to compare means of the distance between the point pairs and means of the percent change in variation of distance (VOD%) using the 45° measurement as a reference. RESULTS: Mean VOD% nearest to zero, over all the different angles tested, was produced by Ff-Tt, which was statistically significantly different from each of the other point pairs. CONCLUSION: Ff-Tt provides the best quasi-isometric points for placement of lateral sutures in cats, compared with all combinations tested. Further assessments with biomechanical studies are needed to evaluate the reproducibility of these landmarks for stabilization of CCL rupture in cats.
Assuntos
Gatos/cirurgia , Joelho de Quadrúpedes/cirurgia , Técnicas de Sutura/veterinária , Animais , Fenômenos Biomecânicos , CadáverRESUMO
Farming externalities are believed to co-vary negatively, yet trade-offs have rarely been quantified systematically. Here we present data from UK and Brazilian pig production systems representative of most commercial systems across the world ranging from 'intensive' indoor systems through to extensive free range, Organic and woodland systems to explore co-variation among four major externality costs. We found that no specific farming type was consistently associated with good performance across all domains. Generally, systems with low land use have low greenhouse gas emissions but high antimicrobial use and poor animal welfare, and vice versa. Some individual systems performed well in all domains but were not exclusive to any particular type of farming system. Our findings suggest that trade-offs may be avoidable if mitigation focuses on lowering impacts within system types rather than simply changing types of farming.
Assuntos
Criação de Animais Domésticos , Animais , Suínos , Criação de Animais Domésticos/métodos , Brasil , Reino Unido , Bem-Estar do Animal , Gases de Efeito Estufa , Agricultura/economiaRESUMO
Carbapenem-resistant Escherichia coli (CREC) ST410 has recently emerged as a major global health problem. Here, we report a shift in CREC prevalence in Chinese hospitals between 2017 and 2021 with ST410 becoming the most commonly isolated sequence type. Genomic analysis identifies a hypervirulent CREC ST410 clone, B5/H24RxC, which caused two separate outbreaks in a children's hospital. It may have emerged from the previously characterised B4/H24RxC in 2006 and has been isolated in ten other countries from 2015 to 2021. Compared with B4/H24RxC, B5/H24RxC lacks the blaOXA-181-bearing X3 plasmid, but carries a F-type plasmid containing blaNDM-5. Most of B5/H24RxC also carry a high pathogenicity island and a novel O-antigen gene cluster. We find that B5/H24RxC grew faster in vitro and is more virulent in vivo. The identification of this newly emerged but already globally disseminated hypervirulent CREC clone, highlights the ongoing evolution of ST410 towards increased resistance and virulence.
Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos , Infecções por Escherichia coli , Criança , Humanos , Escherichia coli/genética , Infecções por Escherichia coli/epidemiologia , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Células Clonais , Carbapenêmicos/farmacologia , Antibacterianos/farmacologia , beta-Lactamases/genética , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: DNA sequencing could become an alternative to in vitro antibiotic susceptibility testing (AST) methods for determining antibiotic resistance by detecting genetic determinants associated with decreased antibiotic susceptibility. Here, we aimed to assess and improve the accuracy of antibiotic resistance determination from Enterococcus faecium genomes for diagnosis and surveillance purposes. METHODS: In this retrospective diagnostic accuracy study, we first conducted a literature search in PubMed on Jan 14, 2021, to compile a catalogue of genes and mutations predictive of antibiotic resistance in E faecium. We then evaluated the diagnostic accuracy of this database to determine susceptibility to 12 different, clinically relevant antibiotics using a diverse population of 4382 E faecium isolates with available whole-genome sequences and in vitro culture-based AST phenotypes. Isolates were obtained from various sources in 11 countries worldwide between 2000 and 2018. We included isolates tested with broth microdilution, Vitek 2, and disc diffusion, and antibiotics with at least 50 susceptible and 50 resistant isolates. Phenotypic resistance was derived from raw minimum inhibitory concentrations and measured inhibition diameters, and harmonised primarily using the breakpoints set by the European Committee on Antimicrobial Susceptibility Testing. A bioinformatics pipeline was developed to process raw sequencing reads, identify antibiotic resistance genetic determinants, and report genotypic resistance. We used our curated database, as well as ResFinder, AMRFinderPlus, and LRE-Finder, to assess the accuracy of genotypic predictions against phenotypic resistance. FINDINGS: We curated a catalogue of 228 genetic markers involved in resistance to 12 antibiotics in E faecium. Very accurate genotypic predictions were obtained for ampicillin (sensitivity 99·7% [95% CI 99·5-99·9] and specificity 97·9% [95·8-99·0]), ciprofloxacin (98·0% [96·4-98·9] and 98·8% [95·9-99·7]), vancomycin (98·8% [98·3-99·2] and 98·8% [98·0-99·3]), and linezolid resistance (after re-testing false negatives: 100·0% [90·8-100·0] and 98·3% [97·8-98·7]). High sensitivity was obtained for tetracycline (99·5% [99·1-99·7]), teicoplanin (98·9% [98·4-99·3]), and high-level resistance to aminoglycosides (97·7% [96·6-98·4] for streptomycin and 96·8% [95·8-97·5] for gentamicin), although at lower specificity (60-90%). Sensitivity was expectedly low for daptomycin (73·6% [65·1-80·6]) and tigecycline (38·3% [27·1-51·0]), for which the genetic basis of resistance is not fully characterised. Compared with other antibiotic resistance databases and bioinformatic tools, our curated database was similarly accurate at detecting resistance to ciprofloxacin and linezolid and high-level resistance to streptomycin and gentamicin, but had better sensitivity for detecting resistance to ampicillin, tigecycline, daptomycin, and quinupristin-dalfopristin, and better specificity for ampicillin, vancomycin, teicoplanin, and tetracycline resistance. In a validation dataset of 382 isolates, similar or improved diagnostic accuracies were also achieved. INTERPRETATION: To our knowledge, this work represents the largest published evaluation to date of the accuracy of antibiotic susceptibility predictions from E faecium genomes. The results and resources will facilitate the adoption of whole-genome sequencing as a tool for the diagnosis and surveillance of antimicrobial resistance in E faecium. A complete characterisation of the genetic basis of resistance to last-line antibiotics, and the mechanisms mediating antibiotic resistance silencing, are needed to close the remaining sensitivity and specificity gaps in genotypic predictions. FUNDING: Wellcome Trust, UK Department of Health, British Society for Antimicrobial Chemotherapy, Academy of Medical Sciences and the Health Foundation, Medical Research Council Newton Fund, Vietnamese Ministry of Science and Technology, and European Society of Clinical Microbiology and Infectious Disease.