Development of Quality Control Ranges for Biocide Susceptibility Testing

Every laboratory test needs validation by quality controls. For biocide susceptibility testing (BST), neither quality control (QC) strains nor QC ranges applicable to these strains are currently available. As QC strains, four well-defined laboratory reference strains (Staphylococcus aureus ATCC® 6538, Enterococcus hirae ATCC® 10541, Escherichia coli ATCC® 10536 and Pseudomonas aeruginosa ATCC® 15442), which have been used previously for biocide efficacy testing, were selected. In an interlaboratory trial with eleven participating laboratories, BST QC ranges should be developed for the aforementioned four strains and the four biocides benzalkonium chloride, chlorhexidine, octenidine and polyhexanide. The performance of three different lots of tryptic soy broth was explored using the broth microdilution method and the data were subsequently evaluated using the RangeFinder software. As a result, QC ranges were defined for all reference strain–biocide combinations, except for P. aeruginosa ATCC® 15442 with the two biocides chlorhexidine and polyhexanide. The development of the latter two QC ranges was not possible, due to the limited solubility of the biocides in the test range required for P. aeruginosa ATCC® 15442. The newly developed QC ranges comprise three to five dilution steps. The establishment of QC ranges will contribute to the validation of BST in the future

Antimicrobial and Biocide Resistance among Feline and Canine Staphylococcus aureus and Staphylococcus pseudintermedius Isolates from Diagnostic Submissions

A total of 114 Staphylococcus isolates from various infections of companion animals, including 43 feline Staphylococcus aureus, 19 canine S. aureus, 11 feline Staphylococcus pseudintermedius and 41 canine S. pseudintermedius were investigated for (i) their susceptibility to 24 antimicrobial agents and three combinations of antimicrobial agents by broth microdilution following CLSI recommendations and (ii) the corresponding resistance genes. In addition, the isolates were tested for their susceptibility to the four biocides benzalkonium chloride, chlorhexidine, polyhexanide and octenidine by a recently developed biocide susceptibility testing protocol. Penicillin resistance via blaZ was the dominant resistance property in all four groups of isolates ranging between 76.7 and 90.9%. About one quarter of the isolates (25.4%) proved to be methicillin-resistant and carried the genes mecA or mecC. Macrolide resistance was the second most prevalent resistance property (27.2%) and all isolates harbored the resistance genes erm(A), erm(B), erm(C), erm(T) or msr(A), alone or in combinations. Fluoroquinolone resistance was detected in 21.1% of all isolates tested, whereas tetracycline resistance via tet(K) and/or tet(M) occurred in 19.3% of the isolates. Resistance to last resort antimicrobial agents in human medicine was seen only in single isolates, if at all. The minimal inhibitory concentrations (MICs) of the four biocides showed unimodal distributions and were very similar for the four groups of staphylococci. Because of the large number of (multi)resistant isolates, antimicrobial susceptibility testing of feline and canine S. aureus and S. pseudintermedius isolates is highly recommended before the start of an antimicrobial chemotherapy. Moreover, no hints towards the development of biocide resistance were detected

Antimicrobial and Biocide Resistance among Canine and Feline Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii Isolates from Diagnostic Submissions

A total of 215 isolates from infections of dogs and cats, including 49 Enterococcus faecalis, 37 Enterococcus faecium, 59 Escherichia coli, 56 Pseudomonas aeruginosa, and 14 Acinetobacter baumannii, were investigated for their susceptibility to 27 (Gram-positive bacteria) or 20 (Gram-negative bacteria) antimicrobial agents/combinations of antimicrobial agents by broth microdilution according to the recommendations of the Clinical and Laboratory Standards Institute. Moreover, all isolates were analysed for their susceptibility to the biocides benzalkonium chloride, chlorhexidine, polyhexanide, and octenidine by a recently published broth microdilution biocide susceptibility testing method. While the E. faecalis isolates did not show expanded resistances, considerable numbers of the E. faecium isolates were resistant to penicillins, macrolides, tetracyclines, and fluoroquinolones. Even a single vancomycin-resistant isolate that carried the vanA gene cluster was detected. Expanded multiresistance phenotypes were also detected among the E. coli isolates, including a single carbapenem-resistant, blaOXA-48-positive isolate. In addition, multiresistant A. baumannii isolates were detected. The minimal inhibitory concentrations of the biocides showed unimodal distributions but differed with respect to the biocide and the bacterial species investigated. Although there were no indications of a development of biocide resistance, some P. aeruginosa isolates exhibited benzalkonium MICs higher than the highest test concentration

Molecular Characterization of Equine Staphylococcus aureus Isolates Exhibiting Reduced Oxacillin Susceptibility

The detection of borderline oxacillin-resistant Staphylococcus aureus (BORSA) represents a challenge to both, veterinary and human laboratories. Between 2015 and 2017, 19 equine S. aureus with elevated minimal inhibitory concentrations for oxacillin were detected in routine diagnostics. The aim of this study was to characterize these isolates to identify factors possibly associated with the BORSA phenotype. All S. aureus were subjected to antimicrobial susceptibility testing and whole genome sequencing (WGS). A quantifiable β-lactamase activity assay was performed for a representative subset of 13 isolates. The WGS data analysis of the 19 BORSA isolates identified two different genomic lineages, sequence type (ST) 1 and ST1660. The core genome multilocus sequence typing (cgMLST) revealed a close relatedness of all isolates belonging to either ST1 or ST1660. The WGS analysis identified the resistance genes aadD, dfrG, tet(L), and/or blaZ and aacA-aphD. Phenotypic resistance to penicillins, aminoglycosides, tetracyclines, fluoroquinolones and sulfamethoxazole/trimethoprim was observed in the respective isolates. For the penicillin-binding proteins 1–4, amino acid substitutions were predicted using WGS data. Since neither transglycosylase nor transpeptidase domains were affected, these alterations might not explain the BORSA phenotype. Moreover, β-lactamase activity was found to be associated with an inducible blaZ gene. Lineage-specific differences regarding the expression profiles were noted.Peer Reviewe

Antimicrobial and Biocide Resistance among Feline and Canine Staphylococcus aureus and Staphylococcus pseudintermedius Isolates from Diagnostic Submissions

A total of 114 Staphylococcus isolates from various infections of companion animals, including 43 feline Staphylococcus aureus, 19 canine S. aureus, 11 feline Staphylococcus pseudintermedius and 41 canine S. pseudintermedius were investigated for (i) their susceptibility to 24 antimicrobial agents and three combinations of antimicrobial agents by broth microdilution following CLSI recommendations and (ii) the corresponding resistance genes. In addition, the isolates were tested for their susceptibility to the four biocides benzalkonium chloride, chlorhexidine, polyhexanide and octenidine by a recently developed biocide susceptibility testing protocol. Penicillin resistance via blaZ was the dominant resistance property in all four groups of isolates ranging between 76.7 and 90.9%. About one quarter of the isolates (25.4%) proved to be methicillin-resistant and carried the genes mecA or mecC. Macrolide resistance was the second most prevalent resistance property (27.2%) and all isolates harbored the resistance genes erm(A), erm(B), erm(C), erm(T) or msr(A), alone or in combinations. Fluoroquinolone resistance was detected in 21.1% of all isolates tested, whereas tetracycline resistance via tet(K) and/or tet(M) occurred in 19.3% of the isolates. Resistance to last resort antimicrobial agents in human medicine was seen only in single isolates, if at all. The minimal inhibitory concentrations (MICs) of the four biocides showed unimodal distributions and were very similar for the four groups of staphylococci. Because of the large number of (multi)resistant isolates, antimicrobial susceptibility testing of feline and canine S. aureus and S. pseudintermedius isolates is highly recommended before the start of an antimicrobial chemotherapy. Moreover, no hints towards the development of biocide resistance were detected

Antimicrobial and Biocide Resistance among Canine and Feline Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii Isolates from Diagnostic Submissions

A total of 215 isolates from infections of dogs and cats, including 49 Enterococcus faecalis, 37 Enterococcus faecium, 59 Escherichia coli, 56 Pseudomonas aeruginosa, and 14 Acinetobacter baumannii, were investigated for their susceptibility to 27 (Gram-positive bacteria) or 20 (Gram-negative bacteria) antimicrobial agents/combinations of antimicrobial agents by broth microdilution according to the recommendations of the Clinical and Laboratory Standards Institute. Moreover, all isolates were analysed for their susceptibility to the biocides benzalkonium chloride, chlorhexidine, polyhexanide, and octenidine by a recently published broth microdilution biocide susceptibility testing method. While the E. faecalis isolates did not show expanded resistances, considerable numbers of the E. faecium isolates were resistant to penicillins, macrolides, tetracyclines, and fluoroquinolones. Even a single vancomycin-resistant isolate that carried the vanA gene cluster was detected. Expanded multiresistance phenotypes were also detected among the E. coli isolates, including a single carbapenem-resistant, blaOXA-48-positive isolate. In addition, multiresistant A. baumannii isolates were detected. The minimal inhibitory concentrations of the biocides showed unimodal distributions but differed with respect to the biocide and the bacterial species investigated. Although there were no indications of a development of biocide resistance, some P. aeruginosa isolates exhibited benzalkonium MICs higher than the highest test concentration