Evaluation of a Novel Benchtop Tool for Acceleration of Sample Preparation for MALDI-TOF Mass Spectrometry.

During the past decade, MALDI-TOF mass spectrometry (MS) has become a standard method for identification of bacteria and yeasts. Nonetheless, further optimization of the identification process is important to streamline workflows and save resources. This study evaluated the application of a multipurpose benchtop tool, MBT FAST Shuttle IVD, for accelerated drying of liquid assay components (matrix, formic acid, and/or sample) on a MALDI target. A total of 50 bacterial and fungal isolates were subjected to three different sample preparation procedures prior to the identification by MALDI-TOF MS: direct transfer (DT), extended direct transfer (eDT), and protein extraction (PE). Compared to conventional drying at room temperature, the preparation was performed with standardized heating of the MALDI target on the MBT FAST Shuttle. During DT, eDT, and PE, 56.7% (P < 0.001), 56.8% (P < 0.001), and 52.8% (P < 0.001) of time for matrix drying were saved by using the MBT FAST Shuttle, respectively. Applying the MBT FAST Shuttle, 57.5% (P < 0.001) of time for drying of formic acid were saved for eDT and 57.5% (P < 0.001) of time for sample drying were saved for PE. A significant improvement of the identification rates and scores was observed with MBT FAST Shuttle for eDT (P = 0.001) and PE (P = 0.008) methods, while the effect on identification quality for DT was not statistically significant (P = 0.16). In conclusion, the use of the MBT FAST Shuttle shortened the drying time of assay components by about a half for all preparation methods. Moreover, positive effect on identification success was observed.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Antimicrobial Susceptibility Testing.

The advent of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology has dramatically improved the accuracy and speed of diagnostics. However, this progress has mainly been limited to the identification of microorganisms, whereas the practical improvement of antimicrobial susceptibility testing (AST) still lags behind. MALDI-TOF MS-based approaches include the detection of selected resistance mechanisms and the universal phenotypic AST. This minireview focuses on the discussion of those MALDI-TOF MS methods that allow universal growth-based phenotypic AST. The method of minimal profile change concentrations (MPCC) is based on detecting proteome modification in the presence of an antimicrobial. Using stable-isotope labeling, characteristic mass shifts in the presence of an antimicrobial indicate the incorporation of the isotopic labels, and thus the viability and resistance of the microorganism. For MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA), microorganisms are incubated with or without an antimicrobial, followed by cell lysis, protein extraction, and transfer of the cell lysate onto a MALDI target plate. Using the internal standard, peak intensities are correlated to the amount of microbial proteins, and the relative microbial growth is calculated. Most recent development in the field is the direct-on-target microdroplet growth assay (DOT-MGA). Here, incubation of microorganisms with antimicrobials takes place directly on spots of a MALDI target in the form of microdroplets. After incubation, nutrient medium is removed by dabbing with absorptive material. Resistant microorganisms grow despite the presence of antimicrobial, and their amplified biomass is detected by MALDI-TOF MS. Finally, an outlook is provided for further assay improvements.

Hypervirulent Klebsiella pneumoniae Sequence Type 420 with a Chromosomally Inserted Virulence Plasmid.

BACKGROUND: Klebsiella pneumoniae causes severe diseases including sepsis, pneumonia and wound infections and is differentiated into hypervirulent (hvKp) and classic (cKp) pathotypes. hvKp isolates are characterized clinically by invasive and multiple site infection and phenotypically in particular through hypermucoviscosity and increased siderophore production, enabled by the presence of the respective virulence genes, which are partly carried on plasmids. METHODS: Here, we analyzed two K. pneumoniae isolates of a human patient that caused severe multiple site infection. By applying both genomic and phenotypic experiments and combining basic science with clinical approaches, we aimed at characterizing the clinical background as well as the two isolates in-depth. This also included bioinformatics analysis of a chromosomal virulence plasmid integration event. RESULTS: Our genomic analysis revealed that the two isolates were clonal and belonged to sequence type 420, which is not only the first description of this K. pneumoniae subtype in Germany but also suggests belonging to the hvKp pathotype. The latter was supported by the clinical appearance and our phenotypic findings revealing increased siderophore production and hypermucoviscosity similar to an archetypical, hypervirulent K. pneumoniae strain. In addition, our in-depth bioinformatics analysis suggested the insertion of a hypervirulence plasmid in the bacterial chromosome, mediated by a new IS5 family sub-group IS903 insertion sequence designated ISKpn74. CONCLUSION: Our study contributes not only to the understanding of hvKp and the association between hypervirulence and clinical outcomes but reveals the chromosomal integration of a virulence plasmid, which might lead to tremendous public health implications.

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.

Comparative in vitro activity of bacteriophage endolysin HY-133 against Staphylococcus aureus attached to vascular graft surface.

Novel strategies are needed for combating Staphylococcus aureus biofilm in vascular graft infections. We investigated the in vitro activity of bacteriophage endolysin HY-133, daptomycin and rifampin against S. aureus attached to vascular graft surface. Daptomycin showed rapid bactericidal effect on surface-associated S. aureus, while the activity of HY-133 on graft surface-adherent cells was moderate and rifampin did not achieve bactericidal effect. Even in the highest concentrations, all antimicrobials used failed in a complete eradication of the surface-adherent bacteria.

Molecular typing and in vitro resistance of Cryptococcus neoformans clinical isolates obtained in Germany between 2011 and 2017.

Cryptococcosis is a fungal infection of the central nervous system predominantly caused by Cryptococcus neoformans in immunocompromised patients. In several countries worldwide, up to 50% of isolates show in vitro resistance to clinically used antifungals including fluconazole. No prospective data on susceptibility to antifungal drugs are available for Germany. In this study, we characterised all C. neoformans isolates collected from individual patients' samples at the German reference laboratory for cryptococcosis 2011 and 2017 (n = 133) by multi-locus sequence typing and phenotypic drug susceptibility testing. We identified serotype A/genotype VNI isolates belonging to clonal complexes previously described from Europe, Africa, Asia and South America as the most prevalent agents of cryptococcosis in Germany. Overall, we observed minimal inhibitory concentrations (MICs) above the epidemiological cut-offs (ECVs) in 1.6% of isolates regarding fluconazole and 2.3% of isolates regarding 5-flucytosine. Here, two C. neoformans var. grubii isolates displayed decreased drug susceptibility to fluconazole, one of them additionally to 5-flucytosine. We also found 5-flucytosine MICs above the ECV for two C. neoformans var. neoformans isolates. We identified a novel mutation in the ERG11 gene which might be associated with the elevated fluconazole MIC in one of the isolates. The clinical importance of the detected in vitro resistance is documented by patient histories showing relapsed infection or primary fatal disease. Of note, sertraline demonstrated antifungal activity comparable to previous reports. Systematic collection of susceptibility data in combination with molecular typing of C. neoformans is important to comprehensively assess the spread of isolates and to understand their drug resistance patterns.

In Vitro Activity of the Bacteriophage Endolysin HY-133 against Staphylococcus aureus Small-Colony Variants and Their Corresponding Wild Types.

Nasal carriage of methicillin-susceptible (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) represents both a source and a risk factor for subsequent infections. However, existing MRSA decolonization strategies and antibiotic treatment options are hampered by the duration of administration and particularly by the emergence of resistance. Moreover, beyond classical resistance mechanisms, functional resistance as the formation of the small-colony variant (SCV) phenotype may also impair the course and treatment of S. aureus infections. For the recombinant bacteriophage endolysin HY-133, rapid bactericidal and highly selective in vitro activities against MSSA and MRSA has been shown. In order to assess the in vitro efficacy of HY-133 against the SCV phenotype, minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) were evaluated on clinical SCVs, their isogenic wild types, as well as on genetically derived and gentamicin-selected SCVs. For all strains and growth phases, HY-133 MIC and MBC ranged between 0.12 and 1 mg/L. Time-kill studies revealed a fast-acting bactericidal activity of HY-133 resulting in a ≥3 - log10 decrease in CFU/mL within 1 h compared to oxacillin, which required 4⁻24 h. Since the mode of action of HY-133 was independent of growth phase, resistance pattern, and phenotype, it is a promising candidate for future S. aureus decolonization strategies comprising rapid activity against phenotypic variants exhibiting functional resistance.

Plasmid-Encoded Transferable mecB-Mediated Methicillin Resistance in Staphylococcus aureus.

During cefoxitin-based nasal screening, phenotypically categorized methicillin-resistant Staphylococcus aureus (MRSA) was isolated and tested negative for the presence of the mecA and mecC genes as well as for the SCCmec-orfX junction region. The isolate was found to carry a mecB gene previously described for Macrococcus caseolyticus but not for staphylococcal species. The gene is flanked by ß-lactam regulatory genes similar to mecR, mecI, and blaZ and is part of an 84.6-kb multidrug-resistance plasmid that harbors genes encoding additional resistances to aminoglycosides (aacA-aphD, aphA, and aadK) as well as macrolides (ermB) and tetracyclines (tetS). This further plasmidborne ß-lactam resistance mechanism harbors the putative risk of acceleration or reacceleration of MRSA spread, resulting in broad ineffectiveness of ß-lactams as a main therapeutic application against staphylococcal infections.

In Vitro Susceptibility of Clinical Staphylococcus aureus Small-Colony Variants to ß-Lactam and Non-ß-Lactam Antibiotics.

The Staphylococcus aureus small-colony variant (SCV) phenotype has been associated with relapsing and antibiotic-refractory infections. However, little is known about the activities of antibiotics on clinical SCVs. Here, we demonstrated that SCVs without detectable auxotrophies were at least as susceptible to most ß-lactam and non-ß-lactam antibiotics in vitro as their corresponding clonally identical strains with a normal phenotype. After prolonged incubation, a regrowth phenomenon has been observed in gradient diffusion inhibition zones irrespective of the strains' phenotype.

The Novel Phage-Derived Antimicrobial Agent HY-133 Is Active against Livestock-Associated Methicillin-Resistant Staphylococcus aureus.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) isolates are increasingly migrating from livestock into human and animal health care settings. Alternative substances are needed to overcome the drawbacks of currently available drugs used for MRSA eradication. The recombinant bacteriophage endolysin HY-133 has proved to be an active agent against S. aureus Here, the in vitro activity of HY-133 was studied against a large collection of genetically diverse LA-MRSA isolates revealing its high activity against mecA-, mecB-, and mecC-positive LA-MRSA.

Rapid Direct Susceptibility Testing from Positive Blood Cultures by the Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry-Based Direct-on-Target Microdroplet Growth Assay.

The recently developed direct-on-target microdroplet growth assay (DOT-MGA) allows rapid universal antimicrobial susceptibility testing (AST) using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Here, we investigated a direct application of this method on positive blood cultures (BCs) for the acceleration of sepsis diagnostics. Blood samples spiked with meropenem-nonsusceptible and meropenem-susceptible Enterobacterales isolates were inoculated into Bactec Plus Aerobic/F bottles and incubated in the Bactec automated system. Positive-BC broth was processed using four different methods, filtration/dilution, dilution, lysis/centrifugation, and differential centrifugation. For both dilution-based methods, AST was performed from 1:100, 1:1,000, and 1:10,000 dilutions of positive-BC broth in cation-adjusted Mueller-Hinton broth (CA-MHB). For both centrifugation-based methods, a 0.5 McFarland standard turbidity suspension was prepared from a bacterial pellet and adjusted to a final inoculum of 5 × 105 CFU/ml in CA-MHB. Six-microliter microdroplets with or without meropenem at the breakpoint concentration were spotted in triplicate onto a MALDI-TOF MS target, followed by incubation in a humidity chamber for 3 or 4 h and subsequent broth removal. Spectra were evaluated by MALDI Biotyper software. The test was considered valid if the growth control without antibiotic achieved an identification score of ≥1.7. For samples with meropenem, successful identification (score, ≥1.7) was interpreted as a nonsusceptible result, whereas failed identification (score, <1.7) defined susceptibility. The best test performance was achieved with the lysis/centrifugation method after a 4-h incubation. At this time point, 96.3% validity, 91.7% sensitivity, and 100% specificity were reached. This study demonstrated the feasibility and accuracy of a rapid DOT-MGA from positive BCs. Parallel to susceptibility determination, this method provides simultaneous species identification.

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.

Staphylococcus aureus-Associated Musculoskeletal Infections.

Musculoskeletal infections caused by Staphylococcus aureus are among the most difficult-to-treat infections. S. aureus osteomyelitis is associated with a tremendous disease burden through potential for long-term relapses and functional deficits. Although considerable advances have been achieved in diagnosis and treatment of osteomyelitis, the management remains challenging and impact on quality of life is still enormous. S. aureus acute arthritis is relatively seldom in general population, but the incidence is considerably higher in patients with predisposing conditions, particularly those with rheumatoid arthritis. Rapidly destructive course with high mortality and disability rates makes urgent diagnosis and treatment of acute arthritis essential. S. aureus pyomyositis is a common disease in tropical countries, but it is very seldom in temperate regions. Nevertheless, the cases have been increasingly reported also in non-tropical countries, and the physicians should be able to timely recognize this uncommon condition and initiate appropriate treatment. The optimal management of S. aureus-associated musculoskeletal infections requires a strong interdisciplinary collaboration between all involved specialists.

Control of Multidrug-Resistant Pseudomonas aeruginosa in Allogeneic Hematopoietic Stem Cell Transplant Recipients by a Novel Bundle Including Remodeling of Sanitary and Water Supply Systems.

Background: Infections by multidrug-resistant Pseudomonas aeruginosa (MDRPa) are an important cause of morbidity and mortality in patients after allogeneic hematopoietic stem cell transplantation (HSCT). Humid environments can serve as a reservoir and source of infection by this pathogen. To minimize the risk of infection from these reservoirs, we performed extensive remodeling of sanitation and water installations as the focus of our hygiene bundle. Methods: During the reconstruction of our transplantation unit (April 2011-April 2014) we implemented several technical modifications to reduce environmental contamination by and subsequent spreading of MDRPa, including a newly designed shower drain, disinfecting siphons underneath the sinks, and rimless toilets. During a 3-year study period (2012-2014), we tracked the number of patients affected by MDRPa (colonized and/or infected) and the outcome of infected patients, and monitored the environmental occurrence of this pathogen. We further performed whole-genome sequencing of nosocomial MDRPa strains to evaluate genotypic relationships between isolates. Results: Whereas 31 (9.2%; 18 colonized, 13 infected) patients were affected in 2012 and 2013, the number decreased to 3 in 2014 (17%; 3 colonized, 0 infected). Lethality by MDRPa similarly decreased from 3.6% to 0%. Environmental detection of MDRPa decreased in toilets from 18.9% in 2012-2013 to 6.1% in the following year and from 8.1% to 3.0%, respectively, in shower outlets. Whole-genome sequencing showed close relationships between environmental and patient-derived isolates. Conclusions: Hospital construction measures aimed at controlling environmental contamination by and spread of MDRPa are effective at minimizing the risk of highly lethal MDRPa infections.

Rapid Detection and Identification of Candidemia by Direct Blood Culturing on Solid Medium by Use of Lysis-Centrifugation Method Combined with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS).

Candida sepsis is a life-threatening condition with increasing prevalence. In this study, direct blood culturing on solid medium using a lysis-centrifugation procedure enabled successful Candida species identification by matrix-assisted laser desorption-ionization time of flight mass spectrometry on average 3.8 h (Sabouraud agar) or 7.4 h (chocolate agar) before the positivity signal for control samples in Bactec mycosis-IC/F or Bactec Plus aerobic/F bottles, respectively. Direct culturing on solid medium accelerated candidemia diagnostics compared to that with automated broth-based systems.

Susceptibility of MDR Pseudomonas aeruginosa to ceftolozane/tazobactam and comparison of different susceptibility testing methods.

BACKGROUND: Infections caused by MDR Pseudomonas aeruginosa are on the rise, particularly in critically ill patients. Therefore, there is a need to evaluate new antimicrobial regimens. The objectives of this study were to investigate the ceftolozane/tazobactam resistance rates of MDR and XDR P. aeruginosa, the underlying resistance genes, the clonal structure and different antimicrobial susceptibility testing (AST) methods regarding their accuracy for ceftolozane/tazobactam testing. METHODS: In total, 112 MDR and XDR P. aeruginosa (from infection and colonization) from one German tertiary care hospital were included (2013-16). AST was done using broth microdilution (BMD), gradient diffusion test strips and disc diffusion. Resistance genes were screened by PCR. A randomly selected subset of 77 isolates was subjected to WGS to assess the clonal structure. RESULTS: In total, 38 isolates (33.9%) were resistant to ceftolozane/tazobactam according to the BMD reference method. Resistance was significantly lower in MDR P. aeruginosa (4.8%) compared with XDR P. aeruginosa (50%, P < 0.0001). The underlying mechanism in carbapenemase-positive ceftolozane/tazobactam-resistant isolates (n = 38) was blaIMP (n = 25), blaVIM (n = 4) and blaGES (n = 1). The resistance mechanism of the remaining eight ceftolozane/tazobactam-resistant isolates remained unclear. Although our strain collection was diverse, resistance to ceftolozane/tazobactam was almost exclusively associated with MLST ST235. The disc diffusion method was accurate for ceftolozane/tazobactam AST (no false-susceptible results, categorical agreement = 92.9%). CONCLUSIONS: Ceftolozane/tazobactam resistance was low in MDR P. aeruginosa, but higher in XDR P. aeruginosa. The disc diffusion method showed an acceptable accuracy for ceftolozane/tazobactam AST.

The Recombinant Bacteriophage Endolysin HY-133 Exhibits In Vitro Activity against Different African Clonal Lineages of the Staphylococcus aureus Complex, Including Staphylococcus schweitzeri.

HY-133 is a recombinant bacteriophage endolysin with bactericidal activity againstStaphylococcus aureus Here, HY-133 showedin vitroactivity against major African methicillin-susceptible and methicillin-resistantS. aureuslineages and ceftaroline/ceftobiprole- and borderline oxacillin-resistant isolates. HY-133 was also active againstStaphylococcus schweitzeri, a recently described species of theS. aureuscomplex. The activity of HY-133 on the tested isolates (MIC50, 0.25 µg/ml; MIC90, 0.5 µg/ml; range, 0.125 to 0.5 µg/ml) was independent of the species and strain background or antibiotic resistance.

Detection of mecA- and mecC-Positive Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates by the New Xpert MRSA Gen 3 PCR Assay.

An advanced methicillin-resistant Staphylococcus aureus (MRSA) detection PCR approach targeting SCCmec-orfX along with mecA and mecC was evaluated for S. aureus and coagulase-negative staphylococci. The possession of mecA and/or mecC was correctly confirmed in all cases. All methicillin-susceptible S. aureus strains (n = 98; including staphylococcal cassette chromosome mec element [SCCmec] remnants) and 98.1% of the MRSA strains (n = 160, including 10 mecC-positive MRSA) were accurately categorized.

Missense mutations of PBP2a are associated with reduced susceptibility to ceftaroline and ceftobiprole in African MRSA.

OBJECTIVES: Ceftaroline and ceftobiprole are new cephalosporins, which are active against MRSA by inhibiting PBP2a. Recently, high rates of resistance to ceftaroline were reported from Ghana. The objective of this study was to assess rates of resistance to ceftaroline and ceftobiprole in MRSA from Africa and to describe potential missense mutations of PBP2a. METHODS: MRSA isolates derived from Staphylococcus aureus colonization (n = 37) and infection (n = 23) and were collected in Côte d'Ivoire (n = 17), DR Congo (n = 6), Gabon (n = 21) and Nigeria (n = 16). The MICs were determined by the broth microdilution method. The mecA gene was sequenced and missense mutations were associated with the corresponding MLST ST. RESULTS: In total, 16.7% (n = 10) and 15% (n = 9) of isolates were resistant to ceftaroline and ceftobiprole, respectively. The corresponding MICs of ceftaroline and ceftobiprole correlated significantly (r = 0.92). Isolates belonging to ST241 harboured a triple mutation of PBP2a (N146K-N204K-G246E), which was associated with high rates of resistance to ceftaroline (90.9%) and ceftobiprole (81.8%). CONCLUSIONS: Resistances to ceftaroline and ceftobiprole were only detected in Nigeria and were associated with ST241 and a triple mutation of PBP2a.

[Multidrug-resistant bacteria in Germany. The impact of sources outside healthcare facilities]. / Antibiotika-resistente Erreger in Deutschland. Die Rolle von nicht nosokomialen Ansteckungsquellen.

BACKGROUND: Currently, there is an ongoing discussion about the question whether the emergence of multidrug-resistant microorganisms (MDRO) among humans is due to transfer of these bacteria from animals. OBJECTIVES: This review summarizes data on the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum beta-lactamase (ESBL) producing enterobacteria in animals and humans, and describes knowledge about transmission pathways. MATERIAL AND METHODS: After a scientific literature analysis, relevant articles were identified by screening of titles and abstracts, amended by publications of infection control authorities and the respective reference lists. RESULTS: MDRO are both transmitted in the nosocomial setting and are increasingly detected as sources of infection outside healthcare facilities. CONCLUSIONS: Due to new transmission pathways of MDRO an inter-disciplinary approach towards prevention is necessary, involving medical, pharmaceutical and veterinary expertise.