ABSTRACT
Clostridioides difficile has significant clinical importance as a leading cause of healthcare-associated infections, with symptoms ranging from mild diarrhoea to severe colitis, and possible life-threatening complications. C. difficile ribotype (RT) 002, mainly associated with MLST sequence type (ST) 8, is one of the most common RTs found in humans. This study aimed at investigating the genetic characteristics of 537 C. difficile genomes of ST8/RT002. To this end, we sequenced 298 C. difficile strains representing a new European genome collection, with strains from Germany, Denmark, France and Portugal. These sequences were analysed against a global dataset consisting of 1,437 ST8 genomes available through Enterobase. Our results showed close genetic relatedness among the studied ST8 genomes, a diverse array of antimicrobial resistance (AMR) genes and the presence of multiple mobile elements. Notably, the pangenome analysis revealed an open genomic structure. ST8 shows relatively low overall variation. Thus, clonal isolates were found across different One Health sectors (humans, animals, environment and food), time periods, and geographical locations, suggesting the lineage's stability and a universal environmental source. Importantly, this stability did not hinder the acquisition of AMR genes, emphasizing the adaptability of this bacterium to different selective pressures. Although only 2.4â% (41/1,735) of the studied genomes originated from non-human sources, such as animals, food, or the environment, we identified 9 cross-sectoral core genome multilocus sequence typing (cgMLST) clusters. Our study highlights the importance of ST8 as a prominent lineage of C. difficile with critical implications in the context of One Health. In addition, these findings strongly support the need for continued surveillance and investigation of non-human samples to gain a more comprehensive understanding of the epidemiology of C. difficile.
Subject(s)
Clostridioides difficile , Clostridium Infections , Genome, Bacterial , Ribotyping , Clostridioides difficile/genetics , Clostridioides difficile/classification , Humans , Clostridium Infections/microbiology , Clostridium Infections/epidemiology , Multilocus Sequence Typing , Phylogeny , Animals , Europe , Denmark , Whole Genome Sequencing , Genomics , Drug Resistance, Bacterial/geneticsABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
The opportunistic intestinal pathogen Clostridioides difficile is the number one cause of nosocomial diarrhea in humans. In this study, C. difficile was isolated from 26.7% of potatoes and 1.9% of salads from German retail. The majority of strains possessed toxinogenic PCR-ribotypes that are associated with human clinical cases pointing towards a potential risk to human health.
Subject(s)
Clostridioides difficile , Vegetables , Clostridioides , Clostridioides difficile/genetics , Humans , Polymerase Chain Reaction , RibotypingABSTRACT
The inoculum density is an important parameter for numerous experimental approaches in bacteriology, including antimicrobial susceptibility testing (AST), biocide susceptibility testing (BST) and biocide efficacy testing (BET). Methods to determine the inoculum density commonly refer to cell counts and have been described for BET according to the German Medical Veterinary Society (Deutsche Veterinärmedizinische Gesellschaft, DVG) and for AST according to the Clinical and Laboratory Standards Institute (CLSI). In this study, the DVG method using 1000⯵L volumes of two different dilution steps and the AST method according to CLSI using a 100⯵L volume of a single dilution step from the inoculum suspension were compared. For this, each of the four reference strains, Staphylococcus aureus ATCC® 6538, Enterococcus hirae ATCC® 10541, Escherichia coli ATCC® 10536 and Pseudomonas aeruginosa ATCC® 15442, was comparatively tested 28 times using the inoculum preparation according to DVG. The results were statistically analysed using Bland-Altman plots and 95 % limits of agreement (AL). Moreover, cell counts were correlated with the optical density of the bacterial suspensions used. In comparison, the CLSI method measured lower values for colony-forming units (CFU) of -0.12 log10 compared to the DVG method. Overall, both methods returned an AL of -0.52 to 0.27 log10. Since the variations observed between the two methods were within one log10 step and the measured CFUs did not differ systematically, both methods proved to be suitable for cell count determination. Therefore, the CLSI method, which is less complex and less time-consuming, is recommended.
Subject(s)
Bacteria/drug effects , Cell Count/standards , Disinfectants/pharmacology , Bacteria/classification , Cell Count/methods , Enterococcus hirae/drug effects , Escherichia coli/drug effects , Microbial Sensitivity Tests , Pseudomonas aeruginosa/drug effects , Staphylococcus aureus/drug effectsABSTRACT
Biocide susceptibility testing (BST) of bacteria lacks standardised methods. Based on a recently established broth macrodilution BST method, a broth microdilution method for BST was developed. To establish the respective protocol, four reference strains Staphylococcus aureus ATCC® 6538, Enterococcus hirae ATCC® 10541, Escherichia coli ATCC® 10536 and Pseudomonas aeruginosa ATCC® 15442 were investigated for their minimal inhibitory concentrations (MICs) towards quaternary ammonium compounds (benzalkonium chloride), cationic compounds (chlorhexidine), aldehydes (glutardialdehyde) and alcohols (isopropanol) using tryptic soy broth. All combinations of (i) inoculum preparation according to the German Veterinary Medical Society (DVG) or the Clinical and Laboratory Standards Institute (CLSI) with some modifications, (ii) use of 1st subculture (SC) and 2nd SC, (iii) direct colony suspension (DCS) with/without glass beads, and (iv) incubation at 37 °C for 24 h, 48 h, and 72 h were compared using seven independent replications. Overall, the reproducibility was high for all abovementioned strain/biocide/parameter combinations. In total, 86.9 % - 100 % of the results were within ± one dilution step of the mode value. The proposed method for a standardised BST protocol comprises (i) two different inoculum densities, (ii) the use of a fresh overnight culture (1st SC or 2nd SC), (iii) the preparation of the inoculum suspension by either of the two methods using DCS with or without glass beads, and (iv) the incubation at 37 °C for 24 h. This broth microdilution method will help to harmonize BST of bacterial pathogens in routine diagnostics.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Disinfectants/pharmacology , Microbial Sensitivity Tests/methods , 2-Propanol/pharmacology , Bacteria/classification , Benzalkonium Compounds , Chlorhexidine/pharmacology , Glutaral/pharmacology , Reproducibility of ResultsABSTRACT
Surface disinfectants are regularly used in prophylactic and infection control measures. Concern has been raised whether residues of sub-inhibitory disinfectant concentrations may constitute a selective pressure and could contribute to the development of strains which are tolerant and/or resistant to biocides including antibiotics. The current study investigated whether Staphylococcus (S.) aureus ATCC® 29213™ and ATCC® 6538™ would change their growth characteristics and antimicrobial susceptibility profiles after prolonged treatment with sub-inhibitory concentrations of sodium hypochlorite (NaOCl). NaOCl is a fast-acting disinfectant with a broad-spectrum activity, inexpensive and widely used in healthcare and the food production industry. Minimum inhibitory concentration (MIC) for NaOCl was determined by broth macrodilution according to the guidelines for disinfectant efficacy testing provided by the German Veterinary Medical Society. Serial passages after 24 h and 72 h, respectively, in defined sub-inhibitory concentrations of NaOCl resulted in a number of phenotypic variants. Two of these variants, derived from S. aureus ATCC® 29213™, showed elevated MICs of oxacillin and were considered as in vitro-generated borderline oxacillin-resistant S. aureus (BORSA). Transmission electron microscopy revealed a significantly thickened cell wall in these isolates, a phenomenon that has also been described for Listeria monocytogenes after low-level exposure to NaOCl. Whole genome sequencing revealed an early stop codon in the gene coding for the GdpP protein and thereby abolishing the function of this gene. GdpP represents a phosphodiesterase that regulates gene expression, and loss of function of the GdpP protein has been described in association with borderline oxacillin resistance. Our findings suggest that a mutation in the GdpP protein gene and morphological changes of the cell wall were induced by repeated exposure to sub-lethal NaOCl concentrations, and most likely accounted for a BORSA phenotype in two variants derived from S. aureus ATCC® 29213™.
ABSTRACT
Based on antimicrobial susceptibility testing (AST), correct classifications as susceptible, intermediate or resistant are challenging for some antimicrobial agent-bacterial species combinations. In this study, we investigated 19 equine Staphylococcus aureus isolates for their susceptibility to the combination sulfamethoxazole/trimethoprim (SXT) by using broth microdilution (BMD), agar disk diffusion (DD) and automated test systems. To elucidate the presence of the corresponding genetic resistance properties among the isolates, whole genome sequence analysis was performed and the genomes were screened for trimethoprim (TMP) resistance genes and mutations in the deduced FolP amino acid (aa) sequences, known to confer sulfonamide resistance. To check for hetero-resistance, zone diameters in DD were screened after 18 and 42 h of incubation. All 19 isolates harboured one of the TMP resistance genes dfrG or dfrS1. Three isolates had an aa exchange in their FolP aa sequence (F17L), which has previously been described to result in sulfonamide resistance. These isolates were classified as SXT-resistant by all methods. The remaining 16 isolates were classified as SXT-susceptible or -intermediate (BMD and/or DD) or SXT-resistant (mainly automated test systems). None of the isolates had relevant aa variations in their FolP aa sequences. All 19 isolates showed slight growth within their SXT inhibition zone by DD, pointing towards hetero-resistance. Overall, automated test systems classified isolates lacking genetic resistance determinants more frequently as SXT-resistant than DD and BMD. Therefore, further studies are needed to define a reliable method for SXT susceptibility testing.
Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Staphylococcal Infections/veterinary , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Trimethoprim, Sulfamethoxazole Drug Combination/pharmacology , Animals , Bacterial Proteins/genetics , Horses/microbiology , Microbial Sensitivity Tests , Mutation , Staphylococcal Infections/microbiology , Whole Genome SequencingABSTRACT
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 ß-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, ß-lactamase activity was found to be associated with an inducible blaZ gene. The lineage-specific differences regarding the expression profiles were noted.
Subject(s)
Horse Diseases/microbiology , Staphylococcal Infections/microbiology , Staphylococcus aureus/isolation & purification , Animals , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Horses , Microbial Sensitivity Tests , Oxacillin/pharmacology , Phylogeny , Staphylococcal Infections/veterinary , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism , Virulence Factors/genetics , beta-Lactamases/metabolismABSTRACT
In comparison to biocide efficacy testing, biocide susceptibility testing of bacteria so far lacks standardized methods for routine use. The aims of the present study were to develop a broth macrodilution method to test bacterial pathogens for their biocide susceptibility and to evaluate this method in an interlaboratory trial. Staphylococcus aureus ATCC®6538 was tested for its susceptibility to benzalkonium chloride, chlorhexidine and isopropanol comparing test strain suspension preparations, test volumes and incubation times. The use of 2â¯mL volumes for the testing and an incubation time of 24â¯h were proposed. Ten German laboratories participated in the interlaboratory trial. Four reference strains (S. aureus ATCC®6538, Enterococcus hirae ATCC®10541, Escherichia coli ATCC®10536 and Pseudomonas aeruginosa ATCC®15442) commonly used for biocide activity testing, were included. Strains were tested three times at independent occasions for their susceptibility to benzalkonium chloride, glutardialdehyde and isopropanol. In total, 360 data points were obtained (30 per strain/biocide combination). The modal minimal inhibitory concentration ± one dilution step was defined as acceptable range. For the four reference strains and the three biocides 80-100% of the values were considered as acceptable. The deviations within the laboratories for a strain/biocide combination were rather consistent. In general, the testing was performed without difficulties by the laboratories. Although inoculum plate counts of four laboratories were outside the acceptable range, this did not have a large impact on the results. The proposed method was stable and easy to perform. It may contribute to a harmonization and standardization of biocide susceptibility testing.