In vitro and in vivo susceptibility to cefalexin and amoxicillin/clavulanate in canine low-level methicillin-resistant Staphylococcus pseudintermedius.

BACKGROUND: Methicillin-resistant Staphylococcus pseudintermedius (MRSP) lineages harbouring staphylococcal cassette chromosome (SCC) mec types IV, V and ΨSCCmec57395 usually display low oxacillin MICs (0.5-2 mg/L). OBJECTIVES: To evaluate how oxacillin MICs correlate with PBP mutations and susceptibility to ß-lactams approved for veterinary use. METHODS: Associations between MICs and PBP mutations were investigated by broth microdilution, time-kill and genome sequence analyses in 117 canine MRSP strains harbouring these SCCmec types. Clinical outcome was retrospectively evaluated in 11 MRSP-infected dogs treated with ß-lactams. RESULTS: Low-level MRSP was defined by an oxacillin MIC <4 mg/L. Regardless of strain genotype, all low-level MRSP isolates (n = 89) were cefalexin susceptible, whereas no strains were amoxicillin/clavulanate susceptible according to clinical breakpoints. Exposure to 2× MIC of cefalexin resulted in complete killing within 8 h. High (≥4 mg/L) oxacillin MICs were associated with substitutions in native PBP2, PBP3, PBP4 and acquired PBP2a, one of which (V390M in PBP3) was statistically significant by multivariable modelling. Eight of 11 dogs responded to systemic therapy with first-generation cephalosporins (n = 4) or amoxicillin/clavulanate (n = 4) alone or with concurrent topical treatment, including 6 of 7 dogs infected with low-level MRSP. CONCLUSIONS: Oxacillin MIC variability in MRSP is influenced by mutations in multiple PBPs and correlates with cefalexin susceptibility. The expert rule recommending that strains with oxacillin MIC ≥0.5 mg/L are reported as resistant to all ß-lactams should be reassessed based on these results, which are highly clinically relevant in light of the shortage of effective antimicrobials for systemic treatment of MRSP infections in veterinary medicine.

Specific staphylococcal cassette chromosome mec (SCCmec) types and clonal complexes are associated with low-level amoxicillin/clavulanic acid and cefalotin resistance in methicillin-resistant Staphylococcus pseudintermedius.

BACKGROUND: Staphylococcus pseudintermedius is a common pathogen in dogs and methicillin resistance has emerged over recent decades. According to the current guidelines, S. pseudintermedius displaying oxacillin resistance should be reported as resistant to all ß-lactams. OBJECTIVES: To identify possible associations between ß-lactam resistance levels and clonal complexes (CCs) and/or staphylococcal cassette chromosome mec (SCCmec) types in methicillin-resistant S. pseudintermedius (MRSP). METHODS: MICs of oxacillin, penicillin, ampicillin, amoxicillin/clavulanic acid and cefalotin were determined by broth microdilution for 86 clinical canine MRSP isolates from Denmark and the Netherlands. PCR and sequencing were used for SCCmec typing and MLST. RESULTS: Isolates belonged to CC71 (n = 36), CC258 (n = 33), CC45 (n = 11), CC68 (n = 1) and five singleton STs. SCCmecII-III was exclusively found in CC71 and SCCmecIV was significantly associated with CC258. SCCmecV and non-typeable SCCmec types occurred in 4 and 14 isolates, respectively. SCCmecIV was associated with lower MICs of oxacillin (<2 mg/L), ampicillin (<8 mg/L) and amoxicillin/clavulanic acid (<4 mg/L) and with susceptibility to cefalotin (<4 mg/L). All isolates harbouring SCCmecV were susceptible to cefalotin as well. CONCLUSIONS: SCCmec types were associated with different CCs and with either high- or low-level resistance to different ß-lactams. The finding of amoxicillin/clavulanic acid (20%) and cefalotin (70%) in vitro susceptibility across all CCs might have clinical implications, since amoxicillin/clavulanic acid and first-generation cephalosporins are first-choice antibiotics for treatment of S. pseudintermedius infections. Pharmacokinetic/pharmacodynamic and clinical outcome studies are warranted to evaluate the in vivo efficacy of these ß-lactams for treatment of MRSP infections.

ESX-1 and phthiocerol dimycocerosates of Mycobacterium tuberculosis act in concert to cause phagosomal rupture and host cell apoptosis.

Although phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, little is known about their mechanism of action. Localized in the outer membrane of mycobacterial pathogens, DIM are predicted to interact with host cell membranes. Interaction with eukaryotic membranes is a property shared with another virulence factor of Mtb, the early secretory antigenic target EsxA (also known as ESAT-6). This small protein, which is secreted by the type VII secretion system ESX-1 (T7SS/ESX-1), is involved in phagosomal rupture and cell death induced by virulent mycobacteria inside host phagocytes. In this work, by the use of several knock-out or knock-in mutants of Mtb or Mycobacterium bovis BCG strains and different cell biological assays, we present conclusive evidence that ESX-1 and DIM act in concert to induce phagosomal membrane damage and rupture in infected macrophages, ultimately leading to host cell apoptosis. These results identify an as yet unknown function for DIM in the infection process and open up a new research field for the study of the interaction of lipid and protein virulence factors of Mtb.

The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins.

BACKGROUND: The Cry6 family of proteins from Bacillus thuringiensis represents a group of powerful toxins with great potential for use in the control of coleopteran insects and of nematode parasites of importance to agriculture. These proteins are unrelated to other insecticidal toxins at the level of their primary sequences and the structure and function of these proteins has been poorly studied to date. This has inhibited our understanding of these toxins and their mode of action, along with our ability to manipulate the proteins to alter their activity to our advantage. To increase our understanding of their mode of action and to facilitate further development of these proteins we have determined the structure of Cry6Aa in protoxin and trypsin-activated forms and demonstrated a pore-forming mechanism of action. RESULTS: The two forms of the toxin were resolved to 2.7 Å and 2.0 Å respectively and showed very similar structures. Cry6Aa shows structural homology to a known class of pore-forming toxins including hemolysin E from Escherichia coli and two Bacillus cereus proteins: the hemolytic toxin HblB and the NheA component of the non-hemolytic toxin (pfam05791). Cry6Aa also shows atypical features compared to other members of this family, including internal repeat sequences and small loop regions within major alpha helices. Trypsin processing was found to result in the loss of some internal sequences while the C-terminal region remains disulfide-linked to the main core of the toxin. Based on the structural similarity of Cry6Aa to other toxins, the mechanism of action of the toxin was probed and its ability to form pores in vivo in Caenorhabditis elegans was demonstrated. A non-toxic mutant was also produced, consistent with the proposed pore-forming mode of action. CONCLUSIONS: Cry6 proteins are members of the alpha helical pore-forming toxins - a structural class not previously recognized among the Cry toxins of B. thuringiensis and representing a new paradigm for nematocidal and insecticidal proteins. Elucidation of both the structure and the pore-forming mechanism of action of Cry6Aa now opens the way to more detailed analysis of toxin specificity and the development of new toxin variants with novel activities.

Within-Household Transmission and Bacterial Diversity of Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius can be transmitted between dogs and their owners and can cause opportunistic infections in humans. Whole genome sequencing was applied to identify the relatedness between isolates from human infections and isolates from dogs in the same households. Genome SNP diversity and distribution of plasmids and antimicrobial resistance genes identified related and unrelated isolates in both households. Our study shows that within-host bacterial diversity is present in S. pseudintermedius, demonstrating that multiple isolates from each host should preferably be sequenced to study transmission dynamics.

Absence of Host-Specific Genes in Canine and Human Staphylococcus pseudintermedius as Inferred from Comparative Genomics.

Staphylococcus pseudintermedius is an important pathogen in dogs that occasionally causes infections in humans as an opportunistic pathogen of elderly and immunocompromised people. This study compared the genomic relatedness and antimicrobial resistance genes using genome-wide association study (GWAS) to examine host association of canine and human S. pseudintermedius isolates. Canine (n = 25) and human (n = 32) methicillin-susceptible S. pseudintermedius (MSSP) isolates showed a high level of genetic diversity with an overrepresentation of clonal complex CC241 in human isolates. This clonal complex was associated with carriage of a plasmid containing a bacteriocin with cytotoxic properties, a CRISPR-cas domain and a pRE25-like mobile element containing five antimicrobial resistance genes. Multi-drug resistance (MDR) was predicted in 13 (41%) of human isolates and 14 (56%) of canine isolates. CC241 represented 54% of predicted MDR isolates from humans and 21% of predicted MDR canine isolates. While it had previously been suggested that certain host-specific genes were present the current GWAS analysis did not identify any genes that were significantly associated with human or canine isolates. In conclusion, this is the first genomic study showing that MSSP is genetically diverse in both hosts and that multidrug resistance is important in dog and human-associated S. pseudintermedius isolates.

Comparative genomics of phenotypic antimicrobial resistances in methicillin-resistant Staphylococcus pseudintermedius of canine origin.

Staphylococcus pseudintermedius is an important pathogen in dogs. Since 2004, methicillin- resistant S. pseudintermedius (MRSP) isolates, often multidrug resistant, have been observed in dogs in the Netherlands. This study aims to link the observed resistance phenotypes in canine MRSP to genotypic antimicrobial resistance markers, and to study the phylogeny of MRSP by genomic comparisons. The genomes of fifty clinical isolates of MRSP from dogs from the Netherlands were sequenced. The resistance genes were identified, and for twenty one different antimicrobials their presence and sequence were associated with the resistance phenotypes. In case of observed discrepancies, the genes were aligned with reference genes. Of the phenotypic resistances, 98.3% could be explained by the presence of an associated resistance gene or point mutation. Discrepancies were mainly resistance genes present in susceptible isolates; 43.8% (7/16) were explained by an insertion, deletion or mutation in the gene. In relation with the resistance gene presence or absence, a single-nucleotide polymorphism (SNP) based phylogeny was constructed to define the population dynamics. The resistance gene content differed according to clonal complex, from very conserved (CC45), to partly conserved (CC71) to highly diverse (CC258) resistance gene patterns. In conclusion, this study shows that the antimicrobial genotype from whole genome sequencing is highly predictive of the resistance phenotype in MRSP. Interestingly, the observed clonal complexes of MRSP isolates were linked with resistance gene patterns.