Development of a novel MALDI-TOF MS-based bile solubility test for rapid discrimination of Streptococcus pneumoniae.

Differentiation of Streptococcus pneumoniae from other Streptococcus mitis group streptococci (SMGS) remains challenging despite the introduction of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). While the bile solubility test (BST) provides most reliable discrimination of pneumococci, its practical implementation is limited by subjective visual interpretation and frequent inconclusive results. We aimed to develop a rapid confirmation BST based on direct-on-target MALDI-TOF MS assay. After establishment of optimal test conditions, test performance was evaluated on 36 consecutive clinical SMGS isolates. Colony material was suspended and pipetted onto a MALDI target. After drying, sodium deoxycholate in different concentrations (2%, 5%, and 10 %) was added. Incubation for 30 min (at room temperature or 35 °C) was followed by liquid removal and spot washing. After adding 70 % formic acid, spots were overlaid with matrix and measured (MALDI Biotyper smart, Bruker). The absence of microbial spectra (Biotyper score <1.7) in samples with sodium deoxycholate indicated efficient removal of bacterial biomass due to bile solubility, thus, identifying pneumococci. In contrast, scores ≥1.7 were interpreted as lack of bile solubility and confirmation as viridans streptococci other than S. pneumoniae. Highest test accuracy was achieved applying 5% sodium deoxycholate at 35 °C and 10 % sodium deoxycholate at room temperature. These test conditions provided 100 % sensitivity and 100 % specificity for discrimination of S. pneumoniae. The developed MALDI-TOF MS-based BST is an easy-to-perform assay with minimum hands-on time and objective readout. The promising results of this proof-of-principle study warrant confirmation with large collections of epidemiologically diverse strains.

Comparison of Different Phenotypic Approaches To Screen and Detect mecC-Harboring Methicillin-Resistant Staphylococcus aureus.

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.

Introducing occupational health management in the German Armed Forces.

Holistic approaches to workplace health promotion (WHP) within the military setting are challenging. In 2015, the German Ministry of Defense initiated a 6-month pilot study of WHP in the Federal Armed Forces. The pilot study was to identify organizational challenges that should be addressed before the Ministry implemented a comprehensive occupational health management policy in all departments. Eleven diverse departments were selected to participate in a WHP program that addressed physical activity, diet, stress management and addiction prevention. As part of the evaluation concept, we interviewed coordinators, and department heads focusing on transfer factors from the perspective of the implementers. All coordinators and their department heads or deputies participated in semi-structured face-to-face on-site interviews. The data were analyzed based on qualitative content analysis. The coordinators (officers with sports science degree) seemed fully prepared and capable to master the new task. They experienced difficulties in adapting WHP activities to local structures and needs, and complications in administering modular activities. Department heads described conflict regarding human resources between the military mission and the implementation of WHP. Commitment to WHP was a strong facilitator. The interviews identified various barriers related to support by middle management (supervisors) and specific work conditions (e.g. shift work). If occupational health management is to be successfully implemented on a large scale, conceptional and practically collaboration is necessary between WHP and occupational safety and health, and organization and leadership, respectively. Supervisors will benefit from open communication about compensation for the release time of their subordinates to attend WHP.

MALDI-TOF Mass Spectrometry-Based Optochin Susceptibility Testing for Differentiation of Streptococcus pneumoniae from other Streptococcus mitis Group Streptococci.

Discrimination of Streptococcus pneumoniae from other Streptococcus mitis group (SMG) species is still challenging but very important due to their different pathogenic potential. In this study, we aimed to develop a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based optochin susceptibility test with an objective read-out. Optimal test performance was established and evaluated by testing consecutively collected respiratory isolates. Optochin in different concentrations as a potential breakpoint concentration was added to a standardized inoculum. Droplets of 6 µL with optochin and, as growth control, without optochin were spotted onto a MALDI target. Targets were incubated in a humidity chamber, followed by medium removal and on-target protein extraction with formic acid before adding matrix with an internal standard. Spectra were acquired, and results were interpreted as S. pneumoniae in the case of optochin susceptibility (no growth), or as non-S. pneumoniae in the case of optochin non-susceptibility (growth). Highest test accuracy was achieved after 20 h incubation time (95.7%). Rapid testing after 12 h incubation time (optochin breakpoint 2 µg/mL; correct classification 100%, validity 62.5%) requires improvement by optimization of assay conditions. The feasibility of the MALDI-TOF MS-based optochin susceptibility test was demonstrated in this proof-of-principle study; however, confirmation and further improvements are warranted.

The Porcine Nasal Microbiota with Particular Attention to Livestock-Associated Methicillin-Resistant Staphylococcus aureus in Germany-A Culturomic Approach.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) remains a serious public health threat. Porcine nasal cavities are predominant habitats of LA-MRSA. Hence, components of their microbiota might be of interest as putative antagonistically acting competitors. Here, an extensive culturomics approach has been applied including 27 healthy pigs from seven different farms; five were treated with antibiotics prior to sampling. Overall, 314 different species with standing in nomenclature and 51 isolates representing novel bacterial taxa were detected. Staphylococcus aureus was isolated from pigs on all seven farms sampled, comprising ten different spa types with t899 (n = 15, 29.4%) and t337 (n = 10, 19.6%) being most frequently isolated. Twenty-six MRSA (mostly t899) were detected on five out of the seven farms. Positive correlations between MRSA colonization and age and colonization with Streptococcus hyovaginalis, and a negative correlation between colonization with MRSA and Citrobacter spp. were found (p < 0.05). Of 209 non-S. aureus members of the Staphylococcaceae family, 25 isolates (12.0%) from three out of the seven farms exhibited methicillin resistance, including two Macrococcus goetzii isolates carrying the mecB gene. Among 125 Enterobacterales, none tested positive for extended-spectrum beta-lactamase (ESBL) and carbapenemase production. The high frequency of methicillin-resistant staphylococci supports the need for enhanced efforts within the "One Health" concept to manage the antibiotic resistance crisis in the human and veterinary medicine sector.

Zoonotic multidrug-resistant microorganisms among non-hospitalized horses from Germany.

Colonization with multidrug-resistant organisms (MDROs) belonging to the genus Staphylococcus and the order Enterobacterales poses a particular threat to populations at risk. While previous studies focused on MDRO carriage among livestock or companion animals, respective epidemiological data on the general equine population are limited. Here, carriage of methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum beta-lactamase (ESBL) producing Enterobacteriaceae (ESBL-E) in non-hospitalized horses living on private farms in the rural area in Northwest Germany was assessed. Intranasal and perianal swab samples were cultured on solid chromogenic media directly and after enrichment in tryptic soy broth, respectively. S. aureus isolates were spa-typed, MRSA and ESBL-E were further classified by phenotypic and molecular methods. Additionally, a subgroup of the first 20 samples was used to isolate and characterize staphylococci other than S. aureus. Among 223 horses, fifteen (6.8%) carried S. aureus. Two isolates were identified as MRSA (0.9% of all horses, mecA-positive) and classified as spa types t011 and t6867, both known as members of the livestock-associated MRSA MLST clonal complex 398. Nine horses (4.0%) were colonized by ESBL-Escherichia coli positive for bla CTX-M and/or bla TEM. ESBL-E carriage was associated with prior antibiotic treatment (4/31 vs. 5/183; p = 0.0362) and veterinary examinations (4/31 vs. 5/183; p = 0.0362). In the subgroup, nine different staphylococcal species other than S. aureus were found. The high prevalence of ESBL-E. coli in non-hospitalized horses underlines the necessity to raise awareness for strain dissemination across different hosts in order to do justice to the "One Health" concept.

Zoonotic multidrug-resistant microorganisms among small companion animals in Germany.

Antimicrobial multidrug-resistant microorganisms (MDRO) can be transmitted between companion animals and their human owners. Aim of this study was to determine the prevalence of extended spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) and Staphylococcus aureus including methicillin-resistant S. aureus (MRSA) in different companion animal species. Dogs (n = 192), cats (n = 74), and rabbits (n = 17), treated in a veterinary practice and hospital or living in an animal shelter and private households, were sampled. All facilities were located in a region characterized by a high density of pig production. Nasal, buccal and perianal swabs were enriched and cultured on solid chromogenic selective media. A subgroup of 20 animals (13 dogs, 3 cats, 4 rabbits) was analyzed for the presence of staphylococci other than S. aureus. Amongst all animals (n = 283), twenty dogs (10.4%) and six cats (8.1%) carried S. aureus. MRSA was found in five dogs (2.6%) and two cats (2.7%). Isolates were of spa types t011, t034, t108 (all mecA-positive, ST398), and t843 (mecC-positive, ST130), typical for livestock-associated (LA)-MRSA. Except for one dog, MRSA-positive animals did not have direct contact to husbandry. ESBL-Escherichia coli (blaCTX-M/blaTEM/blaSHV genes) were present in seven dogs (3.6%), one cat (1.4%) possessed a cefotaxime-resistant Citrobacter freundii isolate (blaTEM/blaCMY-2 genes). MDRO carriage was associated with animals from veterinary medical settings (p<0.05). One dog and one rabbit carried methicillin-resistant coagulase-negative staphylococci. The exclusive occurrence of MRSA lineages typically described for livestock stresses the impact of MDRO strain dissemination across species barriers in regional settings. Presence of ESBL-E and LA-MRSA among pets and probable dissemination in clinical settings support the necessity of a "One Health" approach to address the potential threats due to MDRO-carrying companion animals.