Sliding motility of Bacillus cereus mediates vancomycin pseudo-resistance during antimicrobial susceptibility testing.

BACKGROUND: The glycopeptide vancomycin is the antimicrobial agent-of-choice for the treatment of severe non-gastrointestinal infections with members of Bacillus cereus sensu lato (s.l.). Recently, sporadic detection of vancomycin-resistant phenotypes emerged, mostly for agar diffusion testing such as the disc diffusion method or gradient test (e.g. Etest®) method. RESULTS: In this work, we were able to disprove a preliminarily assumed high resistance to vancomycin in an isolate of B. cereus s.l. using broth microdilution and agar dilution. Microscopic imaging during vancomycin susceptibility testing showed spreading towards the inhibition zone, which strongly suggested sliding motility. Furthermore, transcriptomic analysis using RNA-Seq on the nanopore platform revealed several key genes of biofilm formation (e.g. calY, tasA, krsEABC) to be up-regulated in pseudo-resistant cells, substantiating that bacterial sliding is responsible for the observed mobility. Down-regulation of virulence (e.g. hblABCD, nheABC, plcR) and flagellar genes compared with swarming cells also confirmed the non-swarming phenotype of the pseudo-resistant isolate. CONCLUSIONS: The results highlight an insufficiency of agar diffusion testing for vancomycin susceptibility in the B. cereus group, and reference methods like broth microdilution are strongly recommended. As currently no guideline mentions interfering phenotypes in antimicrobial susceptibility testing of B. cereus s.l., this knowledge is essential to obtain reliable results on vancomycin susceptibility. In addition, this is the first report of sliding motility undermining accurate antimicrobial susceptibility testing in B. cereus s.l. and may serve as a basis for future studies on bacterial motility in susceptibility testing and its potential impact on treatment efficacy.

Analysis and Characterization of Staphylococcus aureus Small Colony Variants Isolated From Cystic Fibrosis Patients in Austria.

Cystic fibrosis (CF) is the most common hereditary lung disease in the Caucasian population, characterized by viscous bronchial secretion, consecutive defective mucociliary clearance, and unavoidable colonization with microorganisms. Besides Pseudomonas aeruginosa, Staphylococcus aureus is the most common bacterial species colonizing the CF respiratory tract. Under antibiotic pressure S. aureus is able to switch to small colony variants (SCV). These small colony variants can invade epithelial cells, overcome antibiotic therapy inside the cells and can be the starting point for extracellular recolonization. The aim of the present study was the isolation and characterization of S. aureus small colony variants from Austrian cystic fibrosis patients. Samples collected from 147 patients were screened for the presence of S. aureus wild-type and small colony variants. Antibiotic susceptibility testing and determination of the small colony variants causing auxotrophism were performed. Wild-type isolates were assigned to corresponding small colony variants with spa typing. In total, 17 different small colony variant isolates and 12 corresponding wild-type isolates were obtained. 13 isolates were determined thymidine auxotroph, 2 isolates were auxotroph for hemin, and none of the tested isolates was auxotroph for both, respectively. The presence of SCVs is directly related to a poor clinical outcome, therefore a monitoring of SCV prevalence is recommended. This study revealed rather low SCV ratios in CF patients compared to other countries.

Systemic antibiotic therapy does not significantly improve outcome in a rat model of implant-associated osteomyelitis induced by Methicillin susceptible Staphylococcus aureus.

INTRODUCTION: Treatment of implant-associated osteomyelitis regularly involves the use of systemic antibiotics in addition to surgical intervention. However, it remains unclear if perioperative systemic application of bactericide substances can improve overall outcome in models of severe intramedullary infection. The present study investigated the use of systemic gentamicin in addition to a controlled local release from a highly lipophilic gentamicinpalmitate compound while the previous study showed efficacy of sole antibiotic implant-coating. METHODS: Forty male Sprague-Dawley rats were divided into two groups receiving an intramedullary femoral injection of 10(2) CFU of a common methicillin susceptible Staphylococcus aureus strain (MSSA Rosenbach). Group I received an uncoated implant whereas group II received a coated implant. All animals received a single shot intraperitoneal application of gentamicinsulfate directly after wound closure while the historical control group III (n = 20) had no antibiotic treatment at all. Animals were observed for 28 and 42 days. Serum haptoglobin and relative weight gain were assessed as well as roll over cultures of explanted femur nails and histological scores of periprosthetic infection in dissected femora. RESULTS: Systemic application of gentamicin combined with antibiotic-coated implant did not further reduce bacterial growth significantly compared with systemic or local antibiotic application alone. Combined local and systemic therapy reduced serum haptoglobin significantly after day 7, 28 and 42 whereas systemic application alone did not compare to controls. CONCLUSIONS: Systemic perioperative and implant-associated application of antibiotics were both comparably effective to treat implant-associated infections whereas the combined antibiotic therapy further reduced systemic signs of infection time dependent.

Coating with a novel gentamicinpalmitate formulation prevents implant-associated osteomyelitis induced by methicillin-susceptible Staphylococcus aureus in a rat model.

PURPOSE: Implant-associated osteomyelitis still represents a demanding challenge due to unfavourable biological conditions, bacterial properties and incremental resistance to antibiotic treatment. Therefore different bactericide or bacteriostatic implant coatings have been developed recently to control local intramedullary infections. Controlled local release of gentamicin base from a highly lipophilic gentamicin palmitate compound achieves extended intramedullary retention times and thus may improve its bactericide effect. METHODS: Forty male Sprague-Dawley rats were divided into two groups receiving an intramedullary femoral injection of 10(2) colony-forming units (CFU) of a common methicillin susceptible Staphylococcus aureus strain (MSSA Rosenbach) and either an uncoated femur nail (Group I) or a nail coated with gentamicin palmitate (Group II). Animals were observed for 28 and 42 days. Serum haptoglobin and relative weight gain were assessed as well as rollover cultures of explanted femur nails and histological scores of periprosthetic infection in dissected femurs. RESULTS: Implants coated with gentamicin palmitate significantly reduced periprosthetic bacterial growth as well as signs of systemic inflammation compared with uncoated implants. CONCLUSIONS: Gentamicin palmitate appears to be a viable coating for the prevention of implant-associated infections. These findings will have to be confirmed in larger animal models as well as in clinical trials.

Impact of Manual Addition of Vancomycin to Polymethylmethacrylate (PMMA) Cements.

(1) Background: The addition of antibiotics to bone cements is a common practice in the treatment of periprosthetic joint infections. In revision cases, the amount and type of antibiotic is often insufficient and additional antibiotics must be added. The addition, however, changes the product itself, and the surgeon becomes the "manufacturer" of the bone cement. PMMAe wished to clarify whether the admixture of antibiotics changes the mechanical stability of the bone cements used and if the added antibiotics were still functional and released in sufficient quantities. (2) Methods: We compared two industrially manufactured vancomycin-containing PMMA cements; the low-viscous VancogenX® (TECRES, Sommacampagna, Italy) and the high-viscous Copal® G+V (Heraeus Medical GmbH, Wehrheim, Germany), with two PMMA cements loaded with aminoglycosides, to which 2.0 g of vancomycin (Hexal CT1631) were manually added-the high-viscous Smartset® GHV and the medium-viscous Antibiotic Simplex with Tobramycin (antibiotic Simplex® T). Test specimens of the bone cements were used to determine mechanical stability (bending strength and bending module), and the release of the antibiotics was determined by HLPC and modified Kirby-Bauer assays. (3) Results: All tested bone cements showed an initial high release within the first hours. Repeated testing after 24 h showed a reduced efficacy of VancogenX® and Smartset® GHV in Kirby-Bauer assays. Long-time release over days showed a release of functional antimicrobial active ingredients over this period of time in anti-microbial assays, but no activity of VancogenX® from day 21 onward. No significant differences in the ISO bending modules could be detected, but in contrast to the bending module, the ISO bending strength was substantially reduced by 10-15 mPal in comparison to both cements of the reference group. The Simplex®T met just the ISO 5833; the Smartset® GHV did not after adding vancomycin. (4) Conclusions: In conclusion, the manual addition of 2 g of vancomycin to 40 g of PMMA powder is recommended for the treatment of methicillin-resistant staphylococci. Vancomycin is released over a period of 42 days with concentrations above the MIC for typical staphylococci. The mechanical properties of the PMMA just met, or did not fulfill, ISO mechanical specification. Copal® G+V showed a better elution than VancogenX® over time.

Evaluation of Gentamicin Release of PMMA Cements Using Different Methods: HPLC, Elution and Inhibition Zone Testing.

(1) Background: There is an ongoing discussion on the elution efficacy of antibiotic-impregnated cements. Our experiments were intended to clarify if there are differences in the antibiotic elution of HPLC compared with inhibition zone testing using eluates or PMMA discs. (2) Materials and Methods: Two cement brands with different concentrations of the active ingredient were tested in antimicrobial Kirby-Bauer (disc diffusion) assays. Cement platelets were directly applied on the agar plates and their zone of inhibition was measured. In parallel, the platelets were incubated in phosphate buffered saline (PBS) and at distinct points of time transferred into new buffer. At these time points, 50 µL of the bone cement eluates was used for zone of inhibition testing. Standard gentamicin sulfate solutions served as a control in the same test setup. To verify the microbiological investigations, the antibiotic content of the eluates was also measured via high-performance liquid chromatography (HPLC). (3) Results: The experiments with cement eluates showed better differentiable results than the direct application of the cement discs. The results were also comparable to investigations with HPLC and gentamicin sulfate standard solutions. (4) Conclusions: The results of elution rates are influenced by the test system and the period of observation chosen. The microbial test systems reflect the results of HPLC to the same degree and give evidence of the efficacy of the antibiotics. The HPLC tests on eluates were more suitable in representing differences in release characteristics.

Antimicrobial non-porous surfaces: a comparison of the standards ISO 22196:2011 and the recently published ISO 7581:2023.

The application of antimicrobial surfaces requires the proof of their effectivity by in vitro methods in laboratories. One of the most well-known test methods is ISO 22196:2011, which represents a simple and inexpensive protocol by applying the bacterial suspension with known volume and concentration covered under a polyethylene film on the surfaces. The incubation is then done under defined humidity conditions for 24 h. Another approach for testing of non-porous surfaces is the newly published ISO 7581:2023. A "dry test" is achieved through spreading and drying 1 µL of a bacterial suspension on the surface. In this study, low alloyed carbon steel, polyethylene terephthalate (PET), and glass specimens were tested uncoated (reference) and coated with zinc according to both ISOs to compare and to evaluate the advantages and disadvantages of each one of them. Although ISO 7581:2023 allows a more realistic test environment than ISO 22196:2011, the reproducibility of the results is not given due to the low application volume. In addition, not all bacterial strains are equally suitable for this testing type. Individual adaptations to the protocols, including incubation conditions (time, temperature, or relative humidity), testing strains and volume, seem necessary to generate conditions that simulate the final application. Nevertheless, both ISOs, if used correctly, provide a good basis for estimating the antimicrobial efficacy of non-porous surfaces.

Benefits of Core-Shell Particles over Single-Metal Coatings: Mechanical and Chemical Exposure and Antimicrobial Efficacy.

One of the greatest challenges worldwide is containing the spread of problematic microorganisms. A promising approach is the use of antimicrobial coatings (AMCs). The antimicrobial potential of certain metals, including copper and zinc, has already been verified. In this study, polyethylene terephthalate and aluminum (PET-Al) foils were coated with copper, zinc, and a combination of these two metals, known as core-shell particles, respectively. The resistance of the three different types of coatings to mechanical and chemical exposure was evaluated in various ways. Further, the bacteria Staphylococcus aureus and the bacteriophage ϕ6 were used to assess the antimicrobial efficacy of the coatings. The best efficacy was achieved with the pure copper coating, which was not convincing in the abrasion tests. The result was a considerable loss of copper particles on the surfaces and reduced effectiveness against the microorganisms. The core-shell particles demonstrated better adhesion to the surfaces after abrasion tests and against most chemical agents. In addition, the antimicrobial efficiency remained more stable after the washability treatment. Thus, the core-shell particles had several benefits over the pure copper and zinc coatings. In addition, the best core-shell loading for durability and efficacy was determined in this study.

Comparison of the Antibiotic Resistance of Escherichia coli Populations from Water and Biofilm in River Environments.

Antibiotic-resistant, facultative pathogenic bacteria are commonly found in surface water; however, the factors influencing the spread and stabilization of antibiotic resistance in this habitat, particularly the role of biofilms, are not fully understood. The extent to which bacterial populations in biofilms or sediments exacerbate the problem for specific antibiotic classes or more broadly remains unanswered. In this study, we investigated the differences between the bacterial populations found in the surface water and sediment/biofilm of the Mur River and the Drava River in Austria. Samples of Escherichia coli were collected from both the water and sediment at two locations per river: upstream and downstream of urban areas that included a sewage treatment plant. The isolates were subjected to antimicrobial susceptibility testing against 21 antibiotics belonging to seven distinct classes. Additionally, isolates exhibiting either extended-spectrum beta-lactamase (ESBL) or carbapenemase phenotypes were further analyzed for specific antimicrobial resistance genes. E. coli isolates collected from all locations exhibited resistance to at least one of the tested antibiotics; on average, isolates from the Mur and Drava rivers showed 25.85% and 23.66% resistance, respectively. The most prevalent resistance observed was to ampicillin, amoxicillin-clavulanic acid, tetracycline, and nalidixic acid. Surprisingly, there was a similar proportion of resistant bacteria observed in both open water and sediment samples. The difference in resistance levels between the samples collected upstream and downstream of the cities was minimal. Out of all 831 isolates examined, 13 were identified as carrying ESBL genes, with 1 of these isolates also containing the gene for the KPC-2 carbapenemase. There were no significant differences between the biofilm (sediment) and open water samples in the occurrence of antibiotic resistance. For the E. coli populations in the examined rivers, the different factors in water and the sediment do not appear to influence the stability of resistance. No significant differences in antimicrobial resistance were observed between the bacterial populations collected from the biofilm (sediment) and open-water samples in either river. The different factors in water and the sediment do not appear to influence the stability of resistance. The minimal differences observed upstream and downstream of the cities could indicate that the river population already exhibits generalized resistance.

Linking antibiotic resistance gene patterns with advanced faecal pollution assessment and environmental key parameters along 2300 km of the Danube River.

The global spread of antimicrobial resistance (AMR) in the environment is a growing health threat. Large rivers are of particular concern as they are highly impacted by wastewater discharge while being vital lifelines serving various human needs. A comprehensive understanding of occurrence, spread and key drivers of AMR along whole river courses is largely lacking. We provide a holistic approach by studying spatiotemporal patterns and hotspots of antibiotic resistance genes (ARGs) along 2311 km of the navigable Danube River, combining a longitudinal and temporal monitoring campaign. The integration of advanced faecal pollution diagnostics and environmental and chemical key parameters allowed linking ARG concentrations to the major pollution sources and explaining the observed patterns. Nine AMR markers, including genes conferring resistance to five different antibiotic classes of clinical and environmental relevance, and one integrase gene were determined by probe-based qPCR. All AMR targets could be quantified in Danube River water, with intI1 and sul1 being ubiquitously abundant, qnrS, tetM, blaTEM with intermediate abundance and blaOXA-48like, blaCTX-M-1 group, blaCTX-M-9 group and blaKPC genes with rare occurrence. Human faecal pollution from municipal wastewater discharges was the dominant factor shaping ARG patterns along the Danube River. Other significant correlations of specific ARGs were observed with discharge, certain metals and pesticides. In contrast, intI1 was not associated with wastewater but was already established in the water microbiome. Animal contamination was detected only sporadically and was correlated with ARGs only in the temporal sampling set. During temporal monitoring, an extraordinary hotspot was identified emphasizing the variability within natural waters. This study provides the first comprehensive baseline concentrations of ARGs in the Danube River and lays the foundation for monitoring future trends and evaluating potential reduction measures. The applided holistic approach proved to be a valuable methodological contribution towards a better understanding of the environmental occurrence of AMR.

Fiber-based food packaging materials in view of bacterial growth and survival capacities.

Understanding interactions of bacteria with fiber-based packaging materials is fundamental for appropriate food packaging. We propose a laboratory model to evaluate microbial growth and survival in liquid media solely consisting of packaging materials with different fiber types. We evaluated food contaminating species (Escherichia coli, Staphylococcus aureus, Bacillus cereus), two packaging material isolates and bacterial endospores for their growth abilities. Growth capacities differed substantially between the samples as well as between bacterial strains. Growth and survival were strongest for the packaging material entirely made of recycled fibers (secondary food packaging) with up to 10.8 log10 CFU/ml for the packaging isolates. Among the food contaminating species, B. cereus and E. coli could grow in the sample of entirely recycled fibers with maxima of 6.1 log10 and 8.6 log10 CFU/mL, respectively. Escherichia coli was the only species that was able to grow in bleached fresh fibers up to 7.0 log10 CFU/mL. Staphylococcus aureus perished in all samples and was undetectable after 1-6 days after inoculation, depending on the sample. The packaging material strains were isolated from recycled fibers and could grow only in samples containing recycled fibers, indicating an adaption to this environment. Spores germinated only in the completely recycled sample. Additionally, microbial digestion of cellulose and xylan might not be a crucial factor for growth. This is the first study describing bacterial growth in food packaging materials itself and proposing functionalization strategies toward active food packaging through pH-lowering.

Inactivation of Bacteriophage ɸ6 and SARS-CoV-2 in Antimicrobial Surface Tests.

Due to the COVID-19 pandemic, researchers have focused on new preventive measures to limit the spread of SARS-CoV-2. One promising application is the usage of antimicrobial materials on often-touched surfaces to reduce the load of infectious virus particles. Since tests with in vitro-propagated SARS-CoV-2 require biosafety level 3 (BSL-3) laboratories with limited capacities and high costs, experiments with an appropriate surrogate like the bacteriophage ɸ6 are preferred in most studies. The aim of this study was to compare ɸ6 and SARS-CoV-2 within antiviral surface tests. Different concentrations of copper coatings on polyethylene terephthalate (PET) were used to determine their neutralizing activity against ɸ6 and SARS-CoV-2. The incubation on the different specimens led to similar inactivation of both SARS-CoV-2 and ɸ6. After 24 h, no infectious virus particles were evident on any of the tested samples. Shorter incubation periods on specimens with high copper concentrations also showed a complete inactivation. In contrast, the uncoated PET foils resulted only in a negligible reduced inactivation during the one-hour incubation. The similar reduction rate for ɸ6 and SARS-CoV-2 in our experiments provide further evidence that the bacteriophage ɸ6 is an adequate model organism for SARS-CoV-2 for this type of testing.

Single- and Multilayer Build-Up of an Antibacterial Temperature- and UV-Curing Sol-Gel System with Atmospheric Pressure Plasma.

The versatility of sol-gel systems makes them ideal for functional coatings in industry. However, existing coatings are either too thin or take too long to cure. To address these issues, this paper proposes using an atmospheric pressure plasma source to fully cure and functionalize thicker sol-gel coatings in a single step. The study explores coating various substrates with sol-gel layers to make them scratch-resistant, antibacterial, and antiadhesive. Microparticles like copper, zinc, or copper flakes are added to achieve antibacterial effects. The sol-gel system can be sprayed on and quickly functionalized on the substrate. The study focuses on introducing and anchoring particles in the sol-gel layer to achieve an excellent antibacterial effect by changing the penetration depth. Overall, this method offers a more efficient and effective approach to sol-gel coatings for industrial applications. In order to achieve a layer thickness of more than 100 µm, the second part of the study proposes a multilayer system comprising 15 to 30 µm thick monolayers that can be modified by introducing fillers (such as TiO2) or scratch-resistant chemicals like titanium isopropoxide. This system also allows for individual plasma functionalization of each sol-gel layer. For instance, the top layer can be introduced with antibacterial particles, while another layer can be enhanced with fillers to increase wear resistance. The study reveals the varying antibacterial effects of spherical particles versus flat flakes and the different scratch hardnesses induced by changes in pH, number of layers, and particle introduction.

Modelling and Determination of Parameters Influencing the Transfer of Microorganisms from Food Contact Materials.

The transfer of microorganisms on packaging materials to a contact surface has only been investigated in the context of laboratory-produced spiked packaging products and agar surfaces in small quantities (0.03-0.10%) so far. Correspondingly, this study focused on the localization of microorganisms on/in industrially produced packaging materials and on the establishment of an experimental laboratory set-up to determine and quantify the parameters influencing the microbial transport from surfaces and different layers of packaging materials to contact agar media. We established a simple model to determine the transfer of microorganisms from packaging materials to microbiological agar plates. In order to clarify the transfer of microorganisms within the material, the samples were split horizontally in their z-dimension, and so produced layers (inner layers) were investigated for their microbial transfer. The parameters incubation time, applied weight and bacterial load for the samples were investigated in more detail in the outer layers (front/back) and the inner layers. No significant difference in the microbial transfer was observed between the outer and inner layers of all samples. We indicate a time-dependent transfer to the media and an independence of the transfer from the applied weight. Moreover, the number of transferred microorganisms is not dependent on the bacterial load of the samples.

Analysis of Extended Spectrum Beta Lactamase (ESBL) Genes of Non-Invasive ESBL Enterobacterales in Southeast Austria in 2017.

Extended spectrum beta lactamases producing Enterobacteriaceae are a major player in the antibiotic resistance challenge. In general, the situation regarding antibiotic resistance in Austria is very good compared to many other countries. Perhaps this is why there is a lack of data on the distribution of ESBL genes in the clinical setting. The aim of this study was to collect data on ESBL genes from a larger sample of human non-invasive clinical isolates from one region in Austria. In total, 468 isolates from different sample materials isolated at the Medical University of Graz from 2017 were examined. The most frequent organisms were Escherichia coli and Klebsiella pneumoniae. Among the enzymes produced, CTX-M-15 was clearly dominant, exotic ESBLs were only represented by three Proteus mirabilis isolates harboring genes for VEB-6 and one P. mirabilis for CTX-M-2, respectively. Compared to other countries, the results are in line with the expectations. The data help to better classify the many studies from the non-clinical field in Austria and to shift the focus slightly away from the exotic results and sample sites.

Antimicrobial activity of gentamicin palmitate against high concentrations of Staphylococcus aureus.

The reduction of implant related infections plays a pivotal role in orthopaedic surgery as an increasing number of people require implants (up to 200,000 per year in the United States (source: Joint Implant Surgery & Research Foundation 2010)). The aim of the current study is to prevent and thus decrease the number of bacterial infections. Both pre and post operative systemic antibiotic treatment and gentamicin containing bone cements (polymethylmethacrylate, PMMA) are commonly used strategies to overcome infections. In this study, the antimicrobial efficacy of gentamicin sulfate loaded bone cement was compared with titan discs coated with a new form of gentamicin, gentamicin palmitate. Adherence prevention, killing rates and killing kinetics were compared in an in vitro model, using Staphylococcus aureus (S. aureus), which together with Staphylococcus epidermidis (S. epidermidis) represents 60% of bacteria found responsible for hip implant infections (An and Friedman, 1996, J Hosp Infect 33(2):93-108). In our experiments gentamicin, which was applied as gentamicin palmitate on the surface of the implants, showed a high efficacy in eliminating bacteria. In contrast to gentamicin sulfate containing bone cements, gentamicin palmitate is released over a shorter period of time thus not inducing antibiotic resistance. Another benefit for clinical application is that it achieves high local levels of active ingredient which fight early infections and minimize toxic side effects. Furthermore, the short term hydrophobic effect of gentamicin palmitate can successfully impede biofilm formation. Thus, the use of self-adhesive antibiotic fatty acid complexes like gentamicin palmitate represents a new option for the anti-infective coating of cementless titan implants.

Bacillus cereus in Packaging Material: Molecular and Phenotypical Diversity Revealed.

The Bacillus cereus group has been isolated from soils, water, plants and numerous food products. These species can produce a variety of toxins including several enterotoxins [non-hemolytic enterotoxin (Nhe), hemolysin BL (Hbl), cytotoxin K, and enterotoxin FM], the emetic toxin cereulide and insecticidal Bt toxins. This is the first study evaluating the presence of B. cereus in packaging material. Among 75 different isolates, four phylogenetic groups were detected (II, III, IV, and VI), of which the groups III and IV were the most abundant with 46.7 and 41.3%, respectively. One isolate was affiliated to psychrotolerant group VI. Growth experiments showed a mesophilic predominance. Based on PCR analysis, nhe genes were detectable in 100% of the isolates, while hbl genes were only found in 50.7%. The cereulide encoding gene was found in four out of 75 isolates, no isolate carried a crystal toxin gene. In total, thirteen different toxin gene profiles were identified. We showed that a variety of B. cereus group strains can be found in packaging material. Here, this variety lies in the presence of four phylogenetic groups, thirteen toxin gene profiles, and different growth temperatures. The results suggest that packaging material does not contain significant amounts of highly virulent strains, and the low number of cereulide producing strains is in accordance with other results.

Effects of Ribosomal Protein S10 Flexible Loop Mutations on Tetracycline and Tigecycline Susceptibility of Escherichia coli.

Tigecycline is a tetracycline derivative that is being used as an antibiotic of last resort. Both tigecycline and tetracycline bind to the small (30S) ribosomal subunit and inhibit translation. Target mutations leading to resistance to these antibiotics have been identified both in the 16S ribosomal RNA and in ribosomal proteins S3 and S10 (encoded by the rpsJ gene). Several different mutations in the S10 flexible loop tip residue valine 57 (V57) have been observed in tigecycline-resistant Escherichia coli isolates. However, the role of these mutations in E. coli has not yet been characterized in a defined genetic background. In this study, we chromosomally integrated 10 different rpsJ mutations into E. coli, resulting in different exchanges or a deletion of S10 V57, and investigated the effects of the mutations on growth and tigecycline/tetracycline resistance. While one exchange, V57K, decreased the minimal inhibitory concentration (MIC) (Etest) to tetracycline to 0.75 µg/ml (compared to 2 µg/ml in the parent strain) and hence resulted in hypersensitivity to tetracycline, most exchanges, including the ones reported previously in resistant isolates (V57L, V57D, and V57I) resulted in slightly increased MICs to tigecycline and tetracycline. The strongest increase was observed for the V57L mutant, with a MIC (Etest) to tigecycline of 0.5 µg/ml (compared to 0.125 µg/ml in the parent strain) and a MIC to tetracycline of 4.0 µg/ml. Nevertheless, none of these exchanges increased the MIC to the extent observed in previously described clinical tigecycline-resistant isolates. We conclude that, next to S10 mutations, additional mutations are necessary in order to reach high-level tigecycline resistance in E. coli. In addition, our data reveal that mutants carrying S10 V57 exchanges or deletion display growth defects and, in most cases, also thermosensitivity. The defects are particularly strong in the V57 deletion mutant, which is additionally cold-sensitive. We hypothesize that the S10 loop tip residue is critical for the correct functioning of S10. Both the S10 flexible loop and tigecycline are in contact with helix h31 of the 16S rRNA. We speculate that exchanges or deletion of V57 alter the positioning of h31, thereby influencing both tigecycline binding and S10 function.

Comparison of Updated Methods for Legionella Detection in Environmental Water Samples.

The difficulty of cultivation of Legionella spp. from water samples remains a strenuous task even for experienced laboratories. The long incubation periods for Legionellae make isolation difficult. In addition, the water samples themselves are often contaminated with accompanying microbial flora, and therefore require complex cultivation methods from diagnostic laboratories. In addition to the recent update of the standard culture method ISO 11731:2017, new strategies such as quantitative PCR (qPCR) are often discussed as alternatives or additions to conventional Legionella culture approaches. In this study, we compared ISO 11731:2017 with qPCR assays targeting Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1. In samples with a high burden of accompanying microbial flora, qPCR shows an excellent negative predictive value for Legionella pneumophila, thus making qPCR an excellent tool for pre-selection of negative samples prior to work-intensive culture methods. This and its low limit of detection make qPCR a diagnostic asset in Legionellosis outbreak investigations, where quick-risk assessments are essential, and are a useful method for monitoring risk sites.

Comparison of elution characteristics and mechanical properties of acrylic bone cements with and without superficial vancomycin coating (SVC) in the late phase of polymerization.

INTRODUCTION: Antibiotic-loaded bone cements (ALBCs) are used as spacers in two-stage revision arthroplasty for periprosthetic joint infection. We previously described a new technique applying vancomycin powder coating to custom-made cements. To our best knowledge, this method of superficial vancomycin coating (SVC) has not been assessed before. We therefore performed an in-vitro study to determine: (1) whether manually applied SVC strengthened the cements' antibiotic effect; and (2) whether the mechanical requirements for the cements were fulfilled. HYPOTHESIS: SVC increases the antibiotic effect of cement within the first 24hours. METHODS: Cuboid blocks were produced from two commercially available acrylic ALBCs (Palacos R+G and Copal G+V) with and without SVC. Each block was eluted in phosphate-buffered saline at 37°C. Eluates obtained at 1, 2, 3, 4, 5, 10, 15, 30 and 60minutes and 3, 6 and 24hours were evaluated against Staphylococcus aureus (Palacos, Copal) and methicillin-resistant Staphylococcus aureus (MRSA) (Copal) using zone of inhibition tests. Mechanical test results (bending modulus, bending strength) were compared to ISO requirements (≥1800MPa, ≥50MPa). RESULTS: Palacos with SVC produced significantly greater zones of inhibition against Staphylococcus aureus than Palacos without SVC (p=0.002). Copal with SVC showed greater zones of inhibition against both Staphylococcus aureus and MRSA (p=0.002). The antibiotic effect was enhanced by SVC in both cements at every time point within 24hours. The bending modulus and bending strength of Palacos with SVC (2089±166MPa, 60.8±2.6 MPA) and Copal with SVC (2283±195MPa, 56.9±2.4MPa) were significantly above ISO requirements. CONCLUSION: SVC boosts the antibiotic effect of ALBCs in the first 24hours, while maintaining sufficient stability. These findings endorse SVC as a promising additive in septic revision surgery. LEVEL OF EVIDENCE: III; case control study.