The intrinsic macrolide resistome of Escherichia coli.

Intrinsic resistance to macrolides in Gram-negative bacteria is primarily attributed to the low permeability of the outer membrane, though the underlying genetic and molecular mechanisms remain to be fully elucidated. Here, we used transposon directed insertion-site sequencing (TraDIS) to identify chromosomal non-essential genes involved in Escherichia coli intrinsic resistance to a macrolide antibiotic, tilmicosin. We constructed two highly saturated transposon mutant libraries of >290,000 and >390,000 unique Tn5 insertions in a clinical enterotoxigenic strain (ETEC5621) and in a laboratory strain (K-12 MG1655), respectively. TraDIS analysis identified genes required for growth of ETEC5621 and MG1655 under 1/8 MIC (n = 15 and 16, respectively) and 1/4 MIC (n = 38 and 32, respectively) of tilmicosin. For both strains, 23 genes related to lipopolysaccharide biosynthesis, outer membrane assembly, the Tol-Pal system, efflux pump, and peptidoglycan metabolism were enriched in the presence of the antibiotic. Individual deletion of genes (n = 10) in the wild-type strains led to a 64- to 2-fold reduction in MICs of tilmicosin, erythromycin, and azithromycin, validating the results of the TraDIS analysis. Notably, deletion of surA or waaG, which impairs the outer membrane, led to the most significant decreases in MICs of all three macrolides in ETEC5621. Our findings contribute to a genome-wide understanding of intrinsic macrolide resistance in E. coli, shedding new light on the potential role of the peptidoglycan layer. They also provide an in vitro proof of concept that E. coli can be sensitized to macrolides by targeting proteins maintaining the outer membrane such as SurA and WaaG.

Biochemical and Structural Characterization of CRH-1, a Carbapenemase from Chromobacterium haemolyticum Related to KPC ß-Lactamases.

KPC-2 is one of the most relevant serine-carbapenemases among the carbapenem-resistant Enterobacterales. We previously isolated from the environmental species Chromobacterium haemolyticum a class A CRH-1 ß-lactamase displaying 69% amino acid sequence identity with KPC-2. The objective of this study was to analyze the kinetic behavior and crystallographic structure of this ß-lactamase. Our results showed that CRH-1 can hydrolyze penicillins, cephalosporins (except ceftazidime), and carbapenems with similar efficacy compared to KPC-2. Inhibition kinetics showed that CRH-1 is not well inhibited by clavulanic acid, in contrast to efficient inhibition by avibactam (AVI). The high-resolution crystal of the apoenzyme showed that CRH-1 has a similar folding compared to other class A ß-lactamases. The CRH-1/AVI complex showed that AVI adopts a chair conformation, stabilized by hydrogen bonds to Ser70, Ser237, Asn132, and Thr235. Our findings highlight the biochemical and structural similarities of CRH-1 and KPC-2 and the potential clinical impact of this carbapenemase in the event of recruitment by pathogenic bacterial species.

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.

Genetic background of neomycin resistance in clinical Escherichia coli isolated from Danish pig farms.

Neomycin is the first-choice antibiotic for the treatment of porcine enteritis caused by enterotoxigenic Escherichia coli. Resistance to this aminoglycoside is on the rise after the increased use of neomycin due to the ban on zinc oxide. We identified the neomycin resistance determinants and plasmid contents in a historical collection of 128 neomycin-resistant clinical E. coli isolates from Danish pig farms. All isolates were characterized by whole-genome sequencing and antimicrobial susceptibility testing, followed by conjugation experiments and long-read sequencing of eight selected representative strains. We detected 35 sequence types (STs) with ST100 being the most prevalent lineage (38.3%). Neomycin resistance was associated with two resistance genes, namely aph(3')-Ia and aph(3')-Ib, which were identified in 93% and 7% of the isolates, respectively. The aph(3')-Ia was found on different large conjugative plasmids belonging to IncI1α, which was present in 67.2% of the strains, on IncHI1, IncHI2, and IncN, as well as on a multicopy ColRNAI plasmid. All these plasmids except ColRNAI carried genes encoding resistance to other antimicrobials or heavy metals, highlighting the risk of co-selection. The aph(3')-Ib gene occurred on a 19 kb chimeric, mobilizable plasmid that contained elements tracing back its origin to distantly related genera. While aph(3')-Ia was flanked by either Tn903 or Tn4352 derivatives, no clear association was observed between aph(3')-Ib and mobile genetic elements. In conclusion, the spread of neomycin resistance in porcine clinical E. coli is driven by two resistance determinants located on distinct plasmid scaffolds circulating within a highly diverse population dominated by ST100. IMPORTANCE Neomycin is the first-choice antibiotic for the management of Escherichia coli enteritis in pigs. This work shows that aph(3')-Ia and to a lesser extent aph(3')-Ib are responsible for the spread of neomycin resistance that has been recently observed among pig clinical isolates and elucidates the mechanisms of dissemination of these two resistance determinants. The aph(3')-Ia gene is located on different conjugative plasmid scaffolds and is associated with two distinct transposable elements (Tn903 and Tn4352) that contributed to its spread. The diffusion of aph(3')-Ib is mediated by a small non-conjugative, mobilizable chimeric plasmid that likely derived from distantly related members of the Pseudomonadota phylum and was not associated with any detectable mobile genetic element. Although the spread of neomycin resistance is largely attributable to horizontal transfer, both resistance determinants have been acquired by a predominant lineage (ST100) associated with enterotoxigenic E. coli, which accounted for approximately one-third of the strains.

Driving Laboratory Standardization of Bacterial Culture and Antimicrobial Susceptibility Testing in Veterinary Clinical Microbiology in Europe and Beyond.

Globally, antimicrobial resistance is one of the most important public health challenges in which the clinical microbiology laboratory plays a critical role by providing guidance for antimicrobial treatment. Despite the recognition of its importance, there is still a real need for the standardized training of clinical microbiologists and harmonization of diagnostic procedures. This is particularly true for veterinary clinical microbiology, where additional challenges exist when microbiologists are trying to fulfill a professional role very similar to that of their colleagues working in human microbiology laboratories. The specific points that need addressing to improve the outputs of veterinary microbiology laboratories discussed here include (i) harmonization of methodologies used by veterinary laboratories for antimicrobial susceptibility testing (AST); (ii) specific guidelines for interpretation and reporting of AST results for animal pathogens; (iii) guidelines for detection of antimicrobial resistance mechanisms in animal isolates; (iv) standardization of diagnostic procedures for animal clinical specimens; and (v) the need to train more veterinary clinical microbiology specialists. However, there is now a plan to address these issues, led by the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT), which is bringing together experts in veterinary microbiology, pharmacology, epidemiology, and antimicrobial stewardship from Europe and wider afield. ENOVAT is aiming to work with project partners toward standardization and harmonization of laboratory methodologies and optimization of veterinary antimicrobial treatment. Ultimately, the project may provide a mechanism for standardization and harmonization of veterinary clinical microbiology methodologies that could then be used as a template for implementation at a wider international level.

Dogs are carriers of Clostridioides difficile lineages associated with human community-acquired infections.

There is an increasing concern about the role of animals as reservoirs of Clostridioides difficile. In this study, we investigated prevalence, antimicrobial resistance and zoonotic potential of C. difficile in dogs. Two-hundred and twenty-five dog faecal deposits were collected from trashcans in nine public gardens. C. difficile was isolated using selective plating and enrichment culture, identified by MALDI-TOF, tested for susceptibility to seven antibiotics by E-test, and sequenced on an Illumina NextSeq platform. Genome sequences were analysed to determine multilocus sequence types and resistance and toxin gene profiles. Zoonotic potential was assessed by measuring genetic variations of core genome (cg)MLST types between canine isolates and 216 temporally and spatially related human clinical isolates from a national database. C. difficile was isolated from 11 samples (4.9%). Seven isolates were toxigenic (tcdA+, tcdB+, cdtA/B-) and belonged to the sequence types ST2, ST6, ST10 and ST42. The four non-toxigenic isolates were assigned to ST15, ST26 and one novel ST. ST2, corresponding to PCR ribotype RT014/020, was the dominating lineage (n = 4) and, together with ST26 and ST42 isolates, showed close resemblance to human isolates, i.e. 2-5 allelic differences among the 1999 genes analysed by cgMLST. Three non-toxigenic isolates displayed resistance to clindamycin, erythromycin and tetracycline mediated by erm(B) and tet(M). Resistance to metronidazole, moxifloxacine, rifampicin or vancomycin was not detected. In conclusion, a small proportion of faecal deposits contained toxigenic C. difficile such as ST2 (RT014/020), which is a major cause of community-acquired infections. Our finding suggests that pathogenic strains can be exchanged between dogs and humans.

Indications for the use of highest priority critically important antimicrobials in the veterinary sector.

BACKGROUND: Among the measures taken to preserve the clinical efficacy of highest priority critically important antimicrobials (HP-CIAs), the WHO has recommended avoiding their use in food-producing animals. Little is known regarding the indications for which different antimicrobial classes are used in animals, even in countries where data on antimicrobial use are available. OBJECTIVES: To outline, in a narrative review, the diseases for which HP-CIAs are used in veterinary medicine, highlighting incongruences with international guidelines and disease conditions where effective alternatives to HP-CIAs are missing. METHODS: Scientific literature, national reports and expert opinion were used to describe the indications for the use of HP-CIAs in the main food-producing (pigs, cattle and poultry) and companion (horses, dogs and cats) animal species. RESULTS: The most common indications for use of HP-CIAs are enteric and respiratory infections in pigs, cattle and poultry, urogenital infections in dogs and cats and respiratory infections in horses. In some instances, no valid and convenient alternatives to colistin and macrolides are available against certain porcine enteric and bovine respiratory pathogens. Effective, legal and convenient alternatives to HP-CIAs are also lacking for managing common infections in cats, for which oral administration is difficult, Rhodococcus equi infections in horses, some enteric and respiratory infections in poultry and MDR infections in all companion animal species. CONCLUSIONS: Future research and stewardship programmes should focus on the disease conditions identified by this review to reduce the use of HP-CIAs in the veterinary sector.

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.

Impact of oral amoxicillin and amoxicillin/clavulanic acid treatment on bacterial diversity and ß-lactam resistance in the canine faecal microbiota.

BACKGROUND: Aminopenicillins with or without a ß-lactamase inhibitor are widely used in both human and veterinary medicine. However, little is known about their differential impact on the gut microbiota and development of antimicrobial resistance. OBJECTIVES: To investigate changes in the faecal microbiota of dogs treated with amoxicillin or amoxicillin/clavulanic acid. METHODS: Faeces collected from 42 dogs (21 per treatment group) immediately before, during and 1 week after termination of oral treatment with amoxicillin or amoxicillin/clavulanic acid were analysed by culture and 16S rRNA gene sequence analysis. RESULTS: In both groups, bacterial counts on ampicillin selective agar revealed an increase in the proportion of ampicillin-resistant Escherichia coli during treatment, and an increased occurrence and proportion of ampicillin-resistant enterococci during and after treatment. 16S rRNA gene analysis showed reductions in microbial richness and diversity during treatment followed by a return to pre-treatment conditions approximately 1 week after cessation of amoxicillin or amoxicillin/clavulanic acid treatment. While no significant differences were observed between the effects of amoxicillin and amoxicillin/clavulanic acid on microbial richness and diversity, treatment with amoxicillin/clavulanic acid reduced the abundance of taxa that are considered part of the beneficial microbiota (such as Roseburia, Dialister and Lachnospiraceae) and enriched Escherichia, although the latter result was not corroborated by phenotypic counts. CONCLUSIONS: Our results suggest a limited effect of clavulanic acid on selection of antimicrobial resistance and microbial richness when administered orally in combination with amoxicillin. However, combination with this ß-lactamase inhibitor appears to broaden the spectrum of amoxicillin, with potential negative consequences on gut health.

Local and Transboundary Transmissions of Methicillin-Resistant Staphylococcus aureus Sequence Type 398 through Pig Trading.

Livestock-associated methicillin-resistant Staphylococcus aureus sequence type (ST) 398 (LA-MRSA ST398) is a genetic lineage for which pigs are regarded as the main reservoir. An increasing prevalence of LA-MRSA ST398 has been reported in areas with high livestock density throughout Europe. In this study, we investigated the drivers contributing to the introduction and spread of LA-MRSA ST398 through the pig farming system in southern Italy. Whole-genome sequencing (WGS) of LA-MRSA ST398 isolates collected in 2018 from pigs (n = 53) and employees (n = 14) from 10 farms in the Calabria region of Italy were comparatively analyzed with previously published WGS data from Italian ST398 isolates (n = 45), an international ST398 reference collection (n = 89), and isolates from Danish pig farms (n = 283), which are the main suppliers of pigs imported to Italy. Single-nucleotide polymorphisms (SNP) were used to infer isolate relatedness, and these data were used together with data from animal trading to identify factors contributing to LA-MRSA ST398 dissemination. The analyses support the existence of two concurrent pathways for the spread of LA-MRSA ST398 in southern Italy: (i) multiple introductions of LA-MRSA ST398 through the import of colonized pigs from other European countries, including Denmark and France, and (ii) the spread of distinct clones dependent on local trading of pigs between farms. Phylogenetically related Italian and Danish LA-MRSA ST398 isolates shared extensive similarities, including carriage of antimicrobial resistance genes. Our findings highlight the potential risk of transboundary transmission of antimicrobial-resistant bacterial clones with a high zoonotic potential during import of pigs from countries with high LA-MRSA prevalence.IMPORTANCE Over the past decade, livestock-associated methicillin-resistant Staphylococcus aureus sequence type 398 (LA-MRSA ST398) has spread among pig holdings throughout Europe, in parallel with the increased incidence of infections among humans, especially in intensive pig farming regions. Despite the growing prevalence of LA-MRSA ST398 in Italian pig farms, the transmission dynamics of this clone in Italy remains unclear. This work provides genome-based evidence to suggest transboundary LA-MRSA ST398 transmission through trading of colonized pigs between European countries and Italy, as well as between farms in the same Italian region. Our findings show that both international trading and local trading of colonized pigs are important factors contributing to the global spread of LA-MRSA ST398 and underscore the need for control measures on and off the farm to reduce the dissemination of this zoonotic pathogen.

Evidence for the evolutionary steps leading to mecA-mediated ß-lactam resistance in staphylococci.

The epidemiologically most important mechanism of antibiotic resistance in Staphylococcus aureus is associated with mecA-an acquired gene encoding an extra penicillin-binding protein (PBP2a) with low affinity to virtually all ß-lactams. The introduction of mecA into the S. aureus chromosome has led to the emergence of methicillin-resistant S. aureus (MRSA) pandemics, responsible for high rates of mortality worldwide. Nonetheless, little is known regarding the origin and evolution of mecA. Different mecA homologues have been identified in species belonging to the Staphylococcus sciuri group representing the most primitive staphylococci. In this study we aimed to identify evolutionary steps linking these mecA precursors to the ß-lactam resistance gene mecA and the resistance phenotype. We sequenced genomes of 106 S. sciuri, S. vitulinus and S. fleurettii strains and determined their oxacillin susceptibility profiles. Single-nucleotide polymorphism (SNP) analysis of the core genome was performed to assess the genetic relatedness of the isolates. Phylogenetic analysis of the mecA gene homologues and promoters was achieved through nucleotide/amino acid sequence alignments and mutation rates were estimated using a Bayesian analysis. Furthermore, the predicted structure of mecA homologue-encoded PBPs of oxacillin-susceptible and -resistant strains were compared. We showed for the first time that oxacillin resistance in the S. sciuri group has emerged multiple times and by a variety of different mechanisms. Development of resistance occurred through several steps including structural diversification of the non-binding domain of native PBPs; changes in the promoters of mecA homologues; acquisition of SCCmec and adaptation of the bacterial genetic background. Moreover, our results suggest that it was exposure to ß-lactams in human-created environments that has driven evolution of native PBPs towards a resistance determinant. The evolution of ß-lactam resistance in staphylococci highlights the numerous resources available to bacteria to adapt to the selective pressure of antibiotics.

Fate of CMY-2-Encoding Plasmids Introduced into the Human Fecal Microbiota by Exogenous Escherichia coli.

The gut is a hot spot for transfer of antibiotic resistance genes from ingested exogenous bacteria to the indigenous microbiota. The objective of this study was to determine the fate of two nearly identical blaCMY-2-harboring plasmids introduced into the human fecal microbiota by two Escherichia coli strains isolated from a human and from poultry meat. The chromosome and the CMY-2-encoding plasmid of both strains were labeled with distinct fluorescent markers (mCherry and green fluorescent protein [GFP]), allowing fluorescence-activated cell sorting (FACS)-based tracking of the strain and the resident bacteria that have acquired its plasmid. Each strain was introduced into an established in vitro gut model (CoMiniGut) inoculated with individual feces from ten healthy volunteers. Fecal samples collected 2, 6, and 24 h after strain inoculation were analyzed by FACS and plate counts. Although the human strain survived better than the poultry meat strain, both strains transferred their plasmids to the fecal microbiota at concentrations as low as 102 CFU/ml. Strain survival and plasmid transfer varied significantly depending on inoculum concentration and individual fecal microbiota. Identification of transconjugants by 16S rRNA gene sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) revealed that the plasmids were predominantly acquired by Enterobacteriaceae species, such as E. coli and Hafnia alvei Our experimental data demonstrate that exogenous E. coli of human or animal origin can readily transfer CMY-2-encoding IncI1 plasmids to the human fecal microbiota. Small amounts of the exogenous strain are sufficient to ensure plasmid transfer if the strain is able to survive the gastric environment.

Structure⁻Activity Study, Characterization, and Mechanism of Action of an Antimicrobial Peptoid D2 and Its d- and l-Peptide Analogues.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) constitutes an emerging health problem for companion animals in veterinary medicine. Therefore, discovery of novel antimicrobial agents for treatment of Staphylococcus-associated canine infections is urgently needed to reduce use of human antibiotics in veterinary medicine. In the present work, we characterized the antimicrobial activity of the peptoid D2 against S. pseudintermedius and Pseudomonas aeruginosa, which is another common integumentary pathogen in dogs. Furthermore, we performed a structure⁻activity relationship study of D2, which included 19 peptide/peptoid analogs. Our best compound D2D, an all d-peptide analogue, showed potent minimum inhibitory concentrations (MICs) against canine S. pseudintermedius (2⁻4 µg/mL) and P. aeruginosa (4 µg/mL) isolates as well as other selected dog pathogens (2⁻16 µg/mL). Time⁻kill assays demonstrated that D2D was able to inhibit MRSP in 30 min at 1× MIC, significantly faster than D2. Our results suggest that at high concentrations D2D is rapidly lysing the bacterial membrane while D2 is inhibiting macromolecular synthesis. We probed the mechanism of action at sub-MIC concentrations of D2, D2D, the l-peptide analog and its retro analog by a macromolecular biosynthesis assay and fluorescence spectroscopy. Our data suggest that at sub-MIC concentrations D2D is membrane inactive and primarily works by cell wall inhibition, while the other compounds mainly act on the bacterial membrane.

Diversity of Staphylococcus pseudintermedius in carriage sites and skin lesions of dogs with superficial bacterial folliculitis: potential implications for diagnostic testing and therapy.

BACKGROUND: Staphylococcus pseudintermedius is genotypically diverse within the canine population and multiple strains may colonize individual dogs at any given time. If multiple strains with distinct antimicrobial resistance profiles are present in superficial bacterial folliculitis (SBF), sampling a single skin lesion for culture and antimicrobial susceptibility testing (AST) might be inadequate to select effective therapy. HYPOTHESIS/OBJECTIVES: To investigate S. pseudintermedius diversity in carriage sites and lesions of dogs with SBF. ANIMALS: Fourteen dogs with SBF. METHODS: Staphylococcus pseudintermedius isolates obtained from perineum, gingiva and four to six skin lesions per dog were subjected to pulsed-field gel electrophoresis (PFGE) and AST to assess diversity between lesions. For two dogs, 14-16 isolates per lesion were included in the analysis to assess diversity within lesions. RESULTS: Analysis of one isolate per lesion revealed one to four strains displaying unique PFGE profiles, and up to three unique antimicrobial resistance (AMR) profiles for each dog. Multiple pustules from the same dog always harboured the same strain, whereas papules, crusts and collarettes did not. Up to four strains with distinct AMR profiles were isolated from the same lesion in two dogs. In 12 dogs, at least one carriage site strain also was represented in lesions. CONCLUSIONS AND CLINICAL IMPORTANCE: Lesions of SBF may harbour multiple S. pseudintermedius strains with distinct antimicrobial resistance profiles. Pustules are the best target for bacterial culture. It remains unclear whether isolation of different strains from other lesion types is a consequence of contamination or co-infection by multiple strains.

Cephem Potentiation by Inactivation of Nonessential Genes Involved in Cell Wall Biogenesis of ß-Lactamase-Producing Escherichia coli.

Reversal of antimicrobial resistance is an appealing and largely unexplored strategy in drug discovery. The objective of this study was to identify potential targets for "helper" drugs reversing cephem resistance in Escherichia coli strains producing ß-lactamases. A CMY-2-encoding plasmid was transferred by conjugation to seven isogenic deletion mutants exhibiting cephem hypersusceptibility. The effect of each mutation was evaluated by comparing the MICs in the wild type and the mutant harboring the same plasmid. Mutation of two genes encoding proteins involved in cell wall biosynthesis, dapF and mrcB, restored susceptibility to cefoxitin (FOX) and reduced the MICs of cefotaxime and ceftazidime, respectively, from the resistant to the intermediate category according to clinical breakpoints. The same mutants harboring a CTX-M-1-encoding plasmid fell into the intermediate or susceptible category for all three drugs. Individual deletion of dapF and mrcB in a clinical isolate of CTX-M-15-producing E. coli sequence type 131 (ST131) resulted in partial reversal of ceftazidime and cefepime resistance but did not reduce MICs below susceptibility breakpoints. Growth curve analysis indicated no fitness cost in a ΔmrcB mutant, whereas a ΔdapF mutant had a 3-fold longer lag phase than the wild type, suggesting that drugs targeting DapF may display antimicrobial activity, in addition to synergizing with selected cephems. DapF appeared to be a potential FOX helper drug target candidate, since dapF inactivation resulted in synergistic potentiation of FOX in the genetic backgrounds tested. The study showed that individual inactivation of two nonessential genes involved in cell wall biogenesis potentiates cephem activity according to drug- and strain-specific patterns.

Evolutionary Origin of the Staphylococcal Cassette Chromosome mec (SCCmec).

Several lines of evidence indicate that the most primitive staphylococcal species, those of the Staphylococcus sciuri group, were involved in the first stages of evolution of the staphylococcal cassette chromosome mec (SCCmec), the genetic element carrying the ß-lactam resistance gene mecA However, many steps are still missing from this evolutionary history. In particular, it is not known how mecA was incorporated into the mobile element SCC prior to dissemination among Staphylococcus aureus and other pathogenic staphylococcal species. To gain insights into the possible contribution of several species of the Staphylococcus sciuri group to the assembly of SCCmec, we sequenced the genomes of 106 isolates, comprising S. sciuri (n = 76), Staphylococcus vitulinus (n = 18), and Staphylococcus fleurettii (n = 12) from animal and human sources, and characterized the native location of mecA and the SCC insertion site by using a variety of comparative genomic approaches. Moreover, we performed a single nucleotide polymorphism (SNP) analysis of the genomes in order to understand SCCmec evolution in relation to phylogeny. We found that each of three species of the S. sciuri group contributed to the evolution of SCCmec: S. vitulinus and S. fleurettii contributed to the assembly of the mec complex, and S. sciuri most likely provided the mobile element in which mecA was later incorporated. We hypothesize that an ancestral SCCmec III cassette (an element carried by one of the most epidemic methicillin-resistant S. aureus clones) originated in S. sciuri possibly by a recombination event in a human host or a human-created environment and later was transferred to S. aureus.

Evaluation of Veterinary-Specific Interpretive Criteria for Susceptibility Testing of Streptococcus equi Subspecies with Trimethoprim-Sulfamethoxazole and Trimethoprim-Sulfadiazine.

Antimicrobial susceptibility test results for trimethoprim-sulfadiazine with Streptococcus equi subspecies are interpreted based on human data for trimethoprim-sulfamethoxazole. The veterinary-specific data generated in this study support a single breakpoint for testing trimethoprim-sulfamethoxazole and/or trimethoprim-sulfadiazine with S. equi This study indicates trimethoprim-sulfamethoxazole as an acceptable surrogate for trimethoprim-sulfadiazine with S. equi.

Recommendations for approaches to meticillin-resistant staphylococcal infections of small animals: diagnosis, therapeutic considerations and preventative measures.: Clinical Consensus Guidelines of the World Association for Veterinary Dermatology.

BACKGROUND: Multiple drug resistance (MDR) in staphylococci, including resistance to the semi-synthetic penicillinase-resistant penicillins such as meticillin, is a problem of global proportions that presents serious challenges to the successful treatment of staphylococcal infections of companion animals. OBJECTIVES: The objective of this document is to provide harmonized recommendations for the diagnosis, prevention and treatment of meticillin-resistant staphylococcal infections in dogs and cats. METHODS: The authors served as a Guideline Panel (GP) and reviewed the literature available prior to September 2016. The GP prepared a detailed literature review and made recommendations on selected topics. The World Association of Veterinary Dermatology (WAVD) provided guidance and oversight for this process. A draft of the document was presented at the 8th World Congress of Veterinary Dermatology (May 2016) and was then made available via the World Wide Web to the member organizations of the WAVD for a period of three months. Comments were solicited and posted to the GP electronically. Responses were incorporated by the GP into the final document. CONCLUSIONS: Adherence to guidelines for the diagnosis, laboratory reporting, judicious therapy (including restriction of use policies for certain antimicrobial drugs), personal hygiene, and environmental cleaning and disinfection may help to mitigate the progressive development and dissemination of MDR staphylococci.

Diagnostic microbiology in veterinary dermatology: present and future.

BACKGROUND: The microbiology laboratory can be perceived as a service provider rather than an integral part of the healthcare team. OBJECTIVES: The aim of this review is to discuss the current challenges of providing a state-of-the-art diagnostic veterinary microbiology service including the identification (ID) and antimicrobial susceptibility testing (AST) of key pathogens in veterinary dermatology. METHODS: The Study Group for Veterinary Microbiology (ESGVM) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) identified scientific, technological, educational and regulatory issues impacting the predictive value of AST and the quality of the service offered by microbiology laboratories. RESULTS: The advent of mass spectrometry has significantly reduced the time required for ID of key pathogens such as Staphylococcus pseudintermedius. However, the turnaround time for validated AST methods has remained unchanged for many years. Beyond scientific and technological constraints, AST methods are not harmonized and clinical breakpoints for some antimicrobial drugs are either missing or inadequate. Small laboratories, including in-clinic laboratories, are usually not adequately equipped to run up-to-date clinical microbiologic diagnostic tests. CONCLUSIONS AND CLINICAL IMPORTANCE: ESGVM recommends the use of laboratories employing mass spectrometry for ID and broth micro-dilution for AST, and offering assistance by expert microbiologists on pre- and post-analytical issues. Setting general standards for veterinary clinical microbiology, promoting antimicrobial stewardship, and the development of new, validated and rapid diagnostic methods, especially for AST, are among the missions of ESGVM.

A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli.

The envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measure Escherichia coli envelope permeability to a ß-galactosidase chromogenic substrate. The signal produced by cytoplasmic ß-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds and E. coli gene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinct E. coli strains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 µM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R > 0.5 µg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.