RESUMO
Staphylococcus aureus is a major human and veterinary pathogen worldwide. Methicillin-resistant S. aureus (MRSA) poses a significant and enduring problem to the treatment of infection by such strains. Resistance is usually conferred by the acquisition of a nonnative gene encoding a penicillin-binding protein (PBP2a), with significantly lower affinity for ß-lactams. This resistance allows cell-wall biosynthesis, the target of ß-lactams, to continue even in the presence of typically inhibitory concentrations of antibiotic. PBP2a is encoded by the mecA gene, which is carried on a distinct mobile genetic element (SCCmec), the expression of which is controlled through a proteolytic signal transduction pathway comprising a sensor protein (MecR1) and a repressor (MecI). Many of the molecular and biochemical mechanisms underlying methicillin resistance in S. aureus have been elucidated, including regulatory events and the structure of key proteins. Here we review recent advances in this area.
Assuntos
Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Infecções Estafilocócicas/microbiologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Humanos , Proteínas de Ligação às Penicilinas , Infecções Estafilocócicas/veterinária , Resistência beta-LactâmicaRESUMO
This study supports the development of predictive bacteriophage (phage) therapy: the concept of phage cocktail selection to treat a bacterial infection based on machine learning (ML) models. For this purpose, ML models were trained on thousands of measured interactions between a panel of phage and sequenced bacterial isolates. The concept was applied to Escherichia coli associated with urinary tract infections. This is an important common infection in humans and companion animals from which multidrug-resistant (MDR) bloodstream infections can originate. The global threat of MDR infection has reinvigorated international efforts into alternatives to antibiotics including phage therapy. E. coli exhibit extensive genome-level variation due to horizontal gene transfer via phage and plasmids. Associated with this, phage selection for E. coli is difficult as individual isolates can exhibit considerable variation in phage susceptibility due to differences in factors important to phage infection including phage receptor profiles and resistance mechanisms. The activity of 31 phage was measured on 314 isolates with growth curves in artificial urine. Random Forest models were built for each phage from bacterial genome features, and the more generalist phage, acting on over 20% of the bacterial population, exhibited F1 scores of >0.6 and could be used to predict phage cocktails effective against previously untested strains. The study demonstrates the potential of predictive ML models which integrate bacterial genomics with phage activity datasets allowing their use on data derived from direct sequencing of clinical samples to inform rapid and effective phage therapy.
Assuntos
Bacteriófagos , Infecções por Escherichia coli , Terapia por Fagos , Infecções Urinárias , Humanos , Animais , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Bacteriófagos/genética , Antibacterianos/farmacologia , Infecções Urinárias/tratamento farmacológicoRESUMO
Virus host shifts, where a virus transmits to and infects a novel host species, are a major source of emerging infectious disease. Genetic similarity between eukaryotic host species has been shown to be an important determinant of the outcome of virus host shifts, but it is unclear if this is the case for prokaryotes where anti-virus defences can be transmitted by horizontal gene transfer and evolve rapidly. Here, we measure the susceptibility of 64 strains of Staphylococcaceae bacteria (48 strains of Staphylococcus aureus and 16 non-S. aureus species spanning 2 genera) to the bacteriophage ISP, which is currently under investigation for use in phage therapy. Using three methods-plaque assays, optical density (OD) assays, and quantitative (q)PCR-we find that the host phylogeny explains a large proportion of the variation in susceptibility to ISP across the host panel. These patterns were consistent in models of only S. aureus strains and models with a single representative from each Staphylococcaceae species, suggesting that these phylogenetic effects are conserved both within and among host species. We find positive correlations between susceptibility assessed using OD and qPCR and variable correlations between plaque assays and either OD or qPCR, suggesting that plaque assays alone may be inadequate to assess host range. Furthermore, we demonstrate that the phylogenetic relationships between bacterial hosts can generally be used to predict the susceptibility of bacterial strains to phage infection when the susceptibility of closely related hosts is known, although this approach produced large prediction errors in multiple strains where phylogeny was uninformative. Together, our results demonstrate the ability of bacterial host evolutionary relatedness to explain differences in susceptibility to phage infection, with implications for the development of ISP both as a phage therapy treatment and as an experimental system for the study of virus host shifts.
Assuntos
Bacteriófagos , Staphylococcaceae , Fagos de Staphylococcus , Bacteriófagos/fisiologia , Especificidade de Hospedeiro , Filogenia , Reação em Cadeia da Polimerase , Staphylococcaceae/classificação , Staphylococcaceae/virologia , Staphylococcus aureus/virologia , Fagos de Staphylococcus/fisiologia , Ensaio de Placa Viral , Replicação ViralRESUMO
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
BACKGROUND: Lop-eared rabbits may be predisposed to otitis externa (OE) as a consequence of their ear conformation. Although otoscopy, otic cytological evaluation and culture are valuable tools in dogs and cats, published data on rabbits remain lacking. HYPOTHESIS/OBJECTIVES: This study aimed to assess the utility of otoscopy and cytological results in evaluating healthy rabbit external ear canals (EECs) and to characterise ear cytological and microbiological findings through culture techniques and metagenomic sequencing. ANIMALS: Sixty-three otitis-free client-owned rabbits. MATERIALS AND METHODS: All rabbits underwent otoscopy and ear cytological evaluation. In a subset of 12 rabbits, further bacterial and fungal culture, fungal DNA assessment and metagenomic sequencing were performed. RESULTS: Otic cytological results revealed yeast in 73%, cocci in 42.9% and rods in 28.6% of healthy rabbit EECs. Compared to upright-eared rabbits, lop-eared rabbits had more discharge and more bacteria per oil immersion field. Culture isolated eight different species yet metagenomic sequencing identified 36, belonging to the Bacillota (Firmicutes), Pseudomonadota and Actinomycetota phyla. Staphylococcus were the most commonly observed species with both methods. Ten of 12 rabbits were yeast-positive on cytological evaluation with only three yielding fungal growth identified as Yarrowia (Candida) lipolytica, Eurotium echinulatum and Cystofilobasidium infirmominiatum. CONCLUSIONS AND CLINICAL RELEVANCE: Healthy rabbit EECs lack inflammatory cells yet can host yeast and bacteria, emphasising the need to evaluate cytological results alongside the clinical signs. Lop-ear anatomy may predispose to bacterial overgrowth and OE. Notably, yeasts may be present despite a negative culture.
RESUMO
Keratoma is an aberrant keratin mass thought to originate from epidermal horn-producing cells interposed between the stratum medium of the hoof wall and the underlying third phalanx. The cause is unknown, although the presence of keratomas is frequently associated with chronic irritation, focal infection, or trauma. A total of 167 donkeys with keratomas were presented in this study. The diagnosis of a keratoma was based on clinical signs, radiography, and histopathologic examination. Surgical excision was attempted on all donkeys with lameness unless euthanasia was advised. Histopathologic examination, including Giemsa, periodic acid Schiff, and Young's silver special histochemical stains, was performed and showed the presence of fungal hyphae and spirochete bacteria within the degenerate keratin. Polymerase chain reaction (PCR) for treponeme bacteria was performed on 10 keratoma lesions and 9 healthy pieces of hoof (controls). All healthy donkey tissues were negative for the 3 recognized digital dermatitis (DD) treponeme phylogroups, whereas 3 of 10 (30%) donkey keratoma samples were positive for one of the DD treponeme phylogroups. Routine fungal culture and PCR for fungi were performed on 8 keratoma lesions and 8 healthy pieces of hoof (controls). Keratinopathogenic fungi were detected in 1 of 8 (12.5%) keratomas, while only non-keratinopathogenic, environmental fungi were detected in 8 control healthy hoof samples. This is the first time the DD treponemes phylogroup and keratinopathogenic fungi have been detected in keratomas. Further studies are required to assess the significance of this finding.
Assuntos
Dermatite Digital , Ceratose , Infecções por Treponema , Animais , Treponema , Spirochaetales , Equidae , Ceratose/cirurgia , Ceratose/veterinária , Fungos , Infecções por Treponema/microbiologia , Infecções por Treponema/veterináriaRESUMO
Two strains, H8/1T and H16/1AT, of Gram-stain-positive, coagulase-negative staphylococci were isolated from separate healthy domestic dogs in Scotland. Both strains were genome sequenced and their inferred DNA-DNA hybridisation indicates that H8/1T and H16/1AT represent two novel species of the genus Staphylococcus. On the basis of the results of genome sequence analysis (genome blast distance phylogeny and single nucleotide polymorphism analysis) H8/1T is most closely related to Staphylococcus devriesei and H16/1AT most closely related to Staphylococcus felis. Also, average nucleotide identity distinguished H8/1T and H16/1AT from S. devriesei and S. felis as did minor phenotypic differences. On the basis of these results, it is proposed that H8/1T and H16/1AT represent novel species with the respective names Staphylococcus caledonicus and Staphylococcus canis. The type strain of S. caledonicus is H8/1T (=NCTC 14452T=CCUG 74789T). The type strain of S. canis is H16/1AT (=NCTC 14451T=CCUG 74790T).
Assuntos
Cães/microbiologia , Filogenia , Staphylococcus/classificação , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Escócia , Análise de Sequência de DNA , Staphylococcus/isolamento & purificaçãoRESUMO
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
Gastric pneumatosis is an imaging finding defined as the presence of gas foci in the gastric wall. In humans, this imaging feature can result from one of two separate clinical entities: life-threatening emphysematous gastritis or clinically benign gastric emphysema. This retrospective case series study describes the clinical and imaging features in five animals diagnosed with spontaneous gastric pneumatosis without gastric dilatation-volvulus. Three canine and two feline cases of spontaneous gastric pneumatosis were identified on radiographic and ultrasonographic examinations. In addition to gastric pneumatosis, one dog and two cats presented concomitant systemic signs such as lethargy, hematemesis, anemia, or leukocytosis. Two dogs remained asymptomatic or presented mild gastrointestinal signs. Portal gas was described in two dogs and one cat, and pneumoperitoneum in one dog. These features were not considered clinically significant. The dog and two cats with systemic signs were euthanized due to clinical deterioration and diagnosed with emphysematous gastritis. The gastric pneumatosis of both dogs without systemic signs resolved while on medical management without antibiotic therapy. These latter cases were interpreted as consistent with gastric emphysema. Findings from the current study indicated that gastric pneumatosis can occur without gastric dilatation-volvulus in cats and dogs and that a combination of clinical and imaging characteristics may help to differentiate between potentially life-threatening emphysematous gastritis and relatively benign gastric emphysema. More studies are needed to determine the etiology and risk factors associated with these conditions.
Assuntos
Doenças do Gato/diagnóstico , Doenças do Cão/diagnóstico , Enfisema/veterinária , Gastrite/veterinária , Gastropatias/veterinária , Animais , Doenças do Gato/diagnóstico por imagem , Doenças do Gato/etiologia , Gatos , Doenças do Cão/diagnóstico por imagem , Doenças do Cão/etiologia , Cães , Enfisema/diagnóstico , Enfisema/diagnóstico por imagem , Enfisema/etiologia , Feminino , Gastrite/diagnóstico , Gastrite/diagnóstico por imagem , Gastrite/etiologia , Masculino , Estudos Retrospectivos , Gastropatias/diagnóstico , Gastropatias/diagnóstico por imagem , Gastropatias/etiologiaRESUMO
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
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: 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/microbiologiaAssuntos
Doenças do Gato , Doenças do Cão , Enfisema , Gastrite , Pneumoperitônio , Animais , Gatos , CãesRESUMO
The aim of this study was to describe the pathology in seals from which Listeria monocytogenes was isolated and investigate if the lesions' nature and severity were related to the phylogeny of isolates. L. monocytogenes was isolated from 13 of 50 (26%) dead grey seal (Halichoerus grypus) pups, six (12%) in systemic distribution, on the Isle of May, Scotland. Similar fatal L. monocytogenes-associated infections were found in a grey seal pup from Carnoustie, Scotland, and a juvenile harbour seal (Phoca vitulina) in the Netherlands. Whole genome sequencing of 15 of the L. monocytogenes isolates identified 13 multilocus sequence types belonging to the L. monocytogenes lineages I and II, but with scant phenotypic and genotypic antimicrobial resistance and limited variation in virulence factors. The phylogenetic diversity present suggests there are multiple sources of L. monocytogenes, even for seal pups born in the same colony and breeding season. This is the first description of L. monocytogenes isolated from, and detected in lesions in, pinnipeds and indicates that infection can be systemic and fatal. Therefore, listeriosis may be an emerging or overlooked disease in seals with infection originating from contamination of the marine environment.
Assuntos
Caniformia , Listeria monocytogenes , Phoca , Focas Verdadeiras , Animais , Listeria monocytogenes/genética , Filogenia , GenótipoRESUMO
Recently, a novel variant of mecA known as mecC (mecA(LGA251)) was identified in Staphylococcus aureus isolates from both humans and animals. In this study, we identified a Staphylococcus xylosus isolate that harbors a new allotype of the mecC gene, mecC1. Whole-genome sequencing revealed that mecC1 forms part of a class E mec complex (mecI-mecR1-mecC1-blaZ) located at the orfX locus as part of a likely staphylococcal cassette chromosome mec element (SCCmec) remnant, which also contains a number of other genes present on the type XI SCCmec.
Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus/genética , Staphylococcus/isolamento & purificação , Animais , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Bovinos , Loci Gênicos , Humanos , Resistência a Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Proteínas de Ligação às Penicilinas , Isoformas de Proteínas/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Staphylococcus/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
A mecC (mecALGA251)-adapted multiplex PCR-based methicillin-resistant Staphylococcus aureus (MRSA) detection assay was evaluated using an international, spa-typed Staphylococcus aureus collection comprising 51 mecC-positive MRSA, 240 mecA-positive MRSA, and 50 mecA- and mecC-negative methicillin-susceptible S. aureus (MSSA) isolates. The assay showed 100% sensitivity and specificity for S. aureus species identification as well as for mecA and mecC detection.
Assuntos
Técnicas Bacteriológicas/métodos , DNA Bacteriano/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Infecções Estafilocócicas/microbiologia , Proteínas de Bactérias/genética , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Sensibilidade e Especificidade , Infecções Estafilocócicas/diagnósticoRESUMO
The emergence of mecC methicillin-resistant Staphylococcus aureus (MRSA) poses a diagnostic challenge for clinical microbiology laboratories. Using the Vitek 2 system, we tested a panel of 896 Staphylococcus aureus isolates and found that an oxacillin-sensitive/cefoxitin-resistant profile had a sensitivity of 88.7% and a specificity of 99.5% for the identification of mecC MRSA isolates. The presence of the mecC gene, determined by bacterial whole-genome sequencing, was used as the gold standard. This profile could provide a zero-cost screening method for identification of mecC-positive MRSA strains.
Assuntos
Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana/métodos , Sensibilidade e EspecificidadeRESUMO
Streptococcus pneumoniae is an important cause of fatal pneumonia in humans. These bacteria express virulence factors, such as the toxins pneumolysin and autolysin, that drive host inflammatory responses. In this study we confirm loss of pneumolysin and autolysin function in a group of clonal pneumococci that have a chromosomal deletion resulting in a pneumolysin-autolysin fusion gene Δ(lytA'-ply')593. The Δ(lytA'-ply')593 pneumococci strains naturally occur in horses and infection is associated with mild clinical signs. Here we use immortalized and primary macrophage in vitro models, which include pattern recognition receptor knock-out cells, and a murine acute pneumonia model to show that a Δ(lytA'-ply')593 strain induces cytokine production by cultured macrophages, however, unlike the serotype-matched ply+lytA+ strain, it induces less tumour necrosis factor α (TNFα) and no interleukin-1ß production. The TNFα induced by the Δ(lytA'-ply')593 strain requires MyD88 but, in contrast to the ply+lytA+ strain, is not reduced in cells lacking TLR2, 4 or 9. In comparison to the ply+lytA+ strain in a mouse model of acute pneumonia, infection with the Δ(lytA'-ply')593 strain resulted in less severe lung pathology, comparable levels of interleukin-1α, but minimal release of other pro-inflammatory cytokines, including interferon-γ, interleukin-6 and TNFα. These results suggest a mechanism by which a naturally occurring Δ(lytA'-ply')593 mutant strain of S. pneumoniae that resides in a non-human host has reduced inflammatory and invasive capacity compared to a human S. pneumoniae strain. These data probably explain the relatively mild clinical disease in response to S. pneumoniae infection seen in horses in comparison to humans.