Acinetobacter baumannii from grass: novel but non-resistant clones.

Acinetobacter baumannii is one the most worrisome nosocomial pathogens, which has long been considered almost mainly as a hospital-associated bacterium. There have been some studies about animal and environmental isolates over the last decade. However, little effort has been made to determine if this pathogen dwells in the grass. Here, we aim to determine the evolutionary relationships and antibiotic resistance of clones of A. baumannii sampled from grass to the major human international clones and animal clones. Two hundred and forty genomes were considered in total from four different sources for this study. Our core and accessory genomic epidemiology analyses showed that grass isolates cluster in seven groups well differentiated from one another and from the major human and animal isolates. Furthermore, we found new sequence types under both multilocus sequence typing schemes: two under the Pasteur scheme and seven for the Oxford scheme. The grass isolates contained fewer antibiotic-resistance genes and were not resistant to the antibiotics tested. Our results demonstrate that these novel clones appear to have limited antibiotic resistance potential. Given our findings, we propose that genomic epidemiology and surveillance of A. baumannii should go beyond the hospital settings and consider the environment in an explicit One Health approach.

Random encounters and amoeba locomotion drive the predation of Listeria monocytogenes by Acanthamoeba castellanii.

Predatory protozoa play an essential role in shaping microbial populations. Among these protozoa, Acanthamoeba are ubiquitous in the soil and aqueous environments inhabited by Listeria monocytogenes. Observations of predator-prey interactions between these two microorganisms revealed a predation strategy in which Acanthamoeba castellanii assemble L. monocytogenes in aggregates, termed backpacks, on their posterior. The rapid formation and specific location of backpacks led to the assumption that A. castellanii may recruit L. monocytogenes by releasing an attractant. However, this hypothesis has not been validated, and the mechanisms driving this process remained unknown. Here, we combined video microscopy, microfluidics, single-cell image analyses, and theoretical modeling to characterize predator-prey interactions of A. castellanii and L. monocytogenes and determined whether bacterial chemotaxis contributes to the backpack formation. Our results indicate that L. monocytogenes captures are not driven by chemotaxis. Instead, random encounters of bacteria with amoebae initialize bacterial capture and aggregation. This is supported by the strong correlation between experimentally derived capture rates and theoretical encounter models at the single-cell level. Observations of the spatial rearrangement of L. monocytogenes trapped by A. castellanii revealed that bacterial aggregation into backpacks is mainly driven by amoeboid locomotion. Overall, we show that two nonspecific, independent mechanisms, namely random encounters enhanced by bacterial motility and predator surface-bound locomotion, drive backpack formation, resulting in a bacterial aggregate on the amoeba ready for phagocytosis. Due to the prevalence of these two processes in the environment, we expect this strategy to be widespread among amoebae, contributing to their effectiveness as predators.

Establishment of low-cost laboratory automation processes using AutoIt and 4-axis robots.

In most small laboratories, many processes are not yet automated because existing laboratory automation solutions are usually expensive and inflexible to use. Examples of this are autosamplers that are only compatible with one specific laboratory instrument or larger liquid handling stations that are expensive and usually self-contained. A flexible and inexpensive way to automate laboratory processes would be to automate existing laboratory equipment with the help of suitable robotic arms. In this study, we investigate the feasibility of such a strategy based on a low-cost 4-axis robot and freely available software. We used the scripting language AutoIt that automates any Windows-based instrument control software. Using these tools, we automated three fundamentally different laboratory processes: a pipetting process, a use as an autosampler for an atomic absorption spectroscopy instrument, and a more complex process involving the inoculation of bacterial cultures. We also integrated a conventional webcam for 2D barcode recognition. Compared to a trained professional who performed all experiments manually, all setups showed no significant differences in accuracy and precision. In summary, the tested system consisting of a 4-axis robot and freely available software is suitable for flexible automation and has potential for even more complex laboratory processes. Limitations such as a lack of collaboration and speed will be addressed in follow-up studies. The system thus represents a well-suited flexible laboratory automation system for both research and teaching purposes.

Tracing Antibiotic Resistance Genes along the Irrigation Water Chain to Chive: Does Tap or Surface Water Make a Difference?

Irrigation water is well known as potential source of pathogens in fresh produce. However, its role in transferring antibiotic resistance determinants is less well investigated. Therefore, we analyzed the contribution of surface and tap water to the resistome of overhead-irrigated chive plants. Field-grown chive was irrigated with either surface water (R-system) or tap water (D-system), from planting to harvest. Water along the two irrigation chains as well as the respective plants were repeatedly sampled and screened for 264 antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), using high-capacity qPCR. Differentially abundant (DA) ARGs were determined by comparing the two systems. On R-chive, ß-lactam ARGs, multidrug-resistance (MDR) determinants, and MGEs were most abundant, while D-chive featured DA ARGs from the vancomycin class. Diversity and number of DA ARGs was the highest on young chives, strongly diminished at harvest, and increased again at the end of shelf life. Most ARGs highly enriched on R- compared to D-chive were also enriched in R- compared to D-sprinkler water, indicating that water played a major role in ARG enrichment. Of note, blaKPC was detected at high levels in surface water and chive. We conclude that water quality significantly affects the resistome of the irrigated produce.

Conjugation Dynamics of Self-Transmissible and Mobilisable Plasmids into E. coli O157:H7 on Arabidopsis thaliana Rosettes.

Many antibiotic resistance genes present in human pathogenic bacteria are believed to originate from environmental bacteria. Conjugation of antibiotic resistance conferring plasmids is considered to be one of the major reasons for the increasing prevalence of antibiotic resistances. A hotspot for plasmid-based horizontal gene transfer is the phyllosphere, i.e., the surfaces of aboveground plant parts. Bacteria in the phyllosphere might serve as intermediate hosts with transfer capability to human pathogenic bacteria. In this study, the exchange of mobilisable and self-transmissible plasmids via conjugation was evaluated. The conjugation from the laboratory strain Escherichia coli S17-1, the model phyllosphere coloniser Pantoea eucalypti 299R, and the model pathogen E. coli O157:H7 to the recipient strain E. coli O157:H7::MRE103 (EcO157:H7red) in the phyllosphere of Arabidopsis thaliana was determined. The results suggest that short-term occurrence of a competent donor is sufficient to fix plasmids in a recipient population of E. coli O157:H7red. The spread of self-transmissible plasmids was limited after initial steep increases of transconjugants that contributed up to 10% of the total recipient population. The here-presented data of plasmid transfer will be important for future modelling approaches to estimate environmental spread of antibiotic resistance in agricultural production environments.

Long-Term Persistence of blaCTX-M-15 in Soil and Lettuce after Introducing Extended-Spectrum ß-Lactamase (ESBL)-Producing Escherichia coli via Manure or Water.

The number of environmental antibiotic-resistant bacteria (ARB) has increased dramatically since the start of antibiotic mass production for broad bacterial infection treatment in 1944. Nowadays, ARB and their resistance-determining genes (ARGs) are readily detected in all environments, including the human food chain. A highly relevant food group in this context is fresh produce, frequent raw consumption of which facilitates direct transfer of ARB and ARGs to the consumer. Here, we investigate the persistence of an extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (E. coli) pEK499 and its clinically most important ARG (blaCTX-M-15), after introduction via irrigation water or manure into a lettuce-growing system. Culturable ESBL-producing E. coli persisted longest in soil and when introduced via manure (until 9 weeks after introduction), while being undetectable on lettuce beyond day 7. In contrast, qPCR detection of blaCTX-M-15 was much more frequent: introduction via water significantly increased blaCTX-M-15 on lettuce until week 4, as opposed to manure, which affected the soil in the long-term (9 weeks) while leading to blaCTX-M-15 detection on lettuce until day 7 only. Our findings demonstrate long-term persistence of undesired ARB and ARG after their introduction via both irrigation and amendment. Such an understanding of the persistence kinetics of an ESBL-producing E. coli and plasmid-encoded blaCTX-M-15 aids the determination of critical actions in order to mitigate their transfer to the consumer.

Characterization of Clostridium tyrobutyricum Strains Using Three Different Typing Techniques.

Clostridium tyrobutyricum is well known as one of the main causative agents of severe cheese spoilage. The metabolism of this anaerobic bacterium during ripening leads to textural and sensory defects in cheese and consequential loss of product value. The potential to induce cheese spoilage, however, may vary among different strains of the same species. Therefore, a better understanding of the intra-species diversity of C. tyrobutyricum may be of practical relevance for the dairy industry. In the present study, we compared the ability of three typing techniques to differentiate 95 C. tyrobutyricum strains on the subspecies level: (1) repetitive element palindromic PCR (rep-PCR) fingerprinting combined with conventional agarose gel electrophoresis, (2) hexaplex-PCR followed by an automated capillary electrophoresis and (3) matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) typing. MALDI-TOF MS fingerprinting provided only moderate reproducibility and low discriminatory power. Both PCR-based methods were highly reproducible and discriminative, with hexaplex-PCR fingerprinting being slightly more discriminative than rep-PCR typing. Overall, a high intra-species diversity was observed among the tested strains, indicating that further investigations on the strain level may be of interest.

Transferable Extended-Spectrum ß-Lactamase (ESBL) Plasmids in Enterobacteriaceae from Irrigation Water.

Extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae are classified as serious threats to human health by the U.S. Centers for Disease Control and Prevention. Water used for irrigation of fresh produce can transmit such resistant bacteria directly to edible plant parts. We screened ESBL-producing Escherichia coli, Enterobacter cloacae, and Citrobacter freundii isolated from irrigation water for their potential to transmit resistance to antibiotic-susceptible E. coli. All strains were genome-sequenced and tested in vitro for transmission of resistance to third-generation cephalosporins on solid agar as well as in liquid culture. Of the 19 screened isolates, five ESBL-producing E. coli were able to transfer resistance with different efficiency to susceptible recipient E. coli. Transconjugant strains were sequenced for detection of transferred antibiotic resistance genes (ARGs) and compared to the known ARG pattern of their respective donors. Additionally, phenotypic resistance patterns were obtained for both transconjugant and corresponding donor strains, confirming ESBL-producing phenotypes of all obtained transconjugants.

Role of DNA repair in Bacillus subtilis spore resistance to high energy and low energy electron beam treatments.

Bacillus subtilis spore inactivation mechanisms under low energy electron beam (LEEB) and high energy electron beam (HEEB) treatment were investigated using seven mutants lacking specific DNA repair mechanisms. The results showed that most of the DNA repair-deficient mutants, including ΔrecA, ΔKu ΔligD, Δexo Δnfo, ΔuvrAB and ΔsbcDC, had reduced resistances towards electron beam (EB) treatments at all investigated energy levels (80 keV, 200 keV and 10 MeV) compared to their wild type. This result suggested DNA damage was induced during EB treatments. The mutant lacking recA showed the lowest resistance, followed by the mutant lacking Ku and ligD. These findings indicated that recA, Ku and ligD and their associated DNA repair mechanisms, namely, homologous recombination and non-homologous end joining, play important roles in spore survival under EB treatment. Furthermore, exoA, nfo, uvrAB, splB, polY1 and polY2, which are involved in nucleotide damage repair/removal, showed different levels of effects on spore resistance under EB treatment. Finally, the results suggested that HEEB and LEEB inactivate B. subtilis spores through similar mechanisms. This research will provide a better understanding of how EB technologies inactivate B. subtilis spores and will contribute to the application of these technologies as a non-thermal, gentle spore control approach.

Plant variety and soil type influence Escherichia coli O104:H4 strain C227/11ϕcu adherence to and internalization into the roots of lettuce plants.

Human disease outbreaks caused by pathogenic Escherichia coli are increasingly associated with the consumption of contaminated fresh produce. Internalization of enteroaggregative/enterohemorrhagic E. coli (EAEC/EHEC) strains into plant tissues may present a serious threat to public health. In the current study, the ability of the fluorescing Shiga toxin-negative E. coli O104:H4 strain C227/11ϕcu/pKEC2 to adhere to and to internalize into the roots of Lactuca sativa and Valerianella locusta grown in diluvial sand (DS) and alluvial loam (AL) was investigated. In parallel, the soil microbiota was analyzed by partial 16S rRNA gene sequencing. The experiments were performed in a safety level 3 greenhouse to simulate agricultural practice. The adherence of C227/11ϕcu/pKEC2 to the roots of both plant varieties was increased by at least a factor three after incubation in DS compared to AL. Compared to V. locusta, internalization into the roots of L. sativa was increased 12-fold in DS and 108-fold in AL. This demonstrates that the plant variety had an impact on the internalization ability, whereas for a given plant variety the soil type also affected bacterial internalization. In addition, microbiota analysis detected the inoculated strain and showed large differences in the bacterial composition between the soil types.

Geobacillus and Bacillus Spore Inactivation by Low Energy Electron Beam Technology: Resistance and Influencing Factors.

Low energy electron beam (LEEB) treatment is an emerging non-thermal technology that performs surface decontamination with a minimal influence on food quality. Bacterial spore resistance toward LEEB treatment and its influencing factors were investigated in this study. Spores from Geobacillus and Bacillus species were treated with a lab-scale LEEB at energy levels of 80 and 200 keV. The spore resistances were expressed as D-values (the radiation dose required for one log10 reduction at a given energy level) calculated from the linear regression of log10 reduction against absorbed dose of the sample. The results revealed that the spore inactivation efficiency by LEEB is comparable to that of other ionizing radiations and that the inactivation curves are mostly log10-linear at the investigated dose range (3.8 - 8.2 kGy at 80 keV; 6.0 - 9.8 kGy at 200 keV). The D-values obtained from the wildtype strains varied from 2.2 - 3.0 kGy at 80 keV, and from 2.2 - 3.1 kGy at 200 keV. Bacillus subtilis mutant spores lacking α/ß-type small, acid-soluble spore proteins showed decreased D-values (1.3 kGy at 80 and 200 keV), indicating that spore DNA is one of the targets for LEEB spore inactivation. The results revealed that bacterial species, sporulation conditions and the treatment dose influence the spore LEEB inactivation. This finding indicates that for the application of this emerging technology, special attention should be paid to the choice of biological indicator, physiological state of the indicator and the processing settings. High spore inactivation efficiency supports the application of LEEB for the purpose of food surface decontamination. With its environmental, logistical, and economic advantages, LEEB can be a relevant technology for surface decontamination to deliver safe, minimally processed and additive-free food products.

Antibiotic-resistant indicator bacteria in irrigation water: High prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli.

Irrigation water is a major source of fresh produce contamination with undesired microorganisms including antibiotic-resistant bacteria (ARB), and contaminated fresh produce can transfer ARB to the consumer especially when consumed raw. Nevertheless, no legal guidelines exist so far regulating quality of irrigation water with respect to ARB. We therefore examined irrigation water from major vegetable growing areas for occurrence of antibiotic-resistant indicator bacteria Escherichia coli and Enterococcus spp., including extended-spectrum ß-lactamase (ESBL)-producing E. coli and vancomycin-resistant Enterococcus spp. Occurrence of ARB strains was compared to total numbers of the respective species. We categorized water samples according to total numbers and found that categories with higher total E. coli or Enterococcus spp. numbers generally had an increased proportion of respective ARB-positive samples. We further detected high prevalence of ESBL-producing E. coli with eight positive samples of thirty-six (22%), while two presumptive vancomycin-resistant Enterococcus spp. were vancomycin-susceptible in confirmatory tests. In disk diffusion assays all ESBL-producing E. coli were multidrug-resistant (n = 21) and whole-genome sequencing of selected strains revealed a multitude of transmissible resistance genes (ARG), with blaCTX-M-1 (4 of 11) and blaCTX-M-15 (3 of 11) as the most frequent ESBL genes. Overall, the increased occurrence of indicator ARB with increased total indicator bacteria suggests that the latter might be a suitable estimate for presence of respective ARB strains. Finally, the high prevalence of ESBL-producing E. coli with transmissible ARG emphasizes the need to establish legal critical values and monitoring guidelines for ARB in irrigation water.

Adherence factors of enterohemorrhagic Escherichia coli O157:H7 strain Sakai influence its uptake into the roots of Valerianella locusta grown in soil.

Increasing numbers of outbreaks caused by enterohemorrhagic Escherichia coli (EHEC) are associated with the consumption of contaminated fresh produce. The contamination of the plants may occur directly on the field via irrigation water, surface water, manure or fecal contamination. Suggesting a low infectious dose of 10 to 102 cells, internalization of EHEC into plant tissue presents a serious public health threat. Therefore, the ability of EHEC O157:H7 strain Sakai to adhere to and internalize into root tissues of the lamb's lettuce Valerianella locusta was investigated under the environmental conditions of a greenhouse. Moreover, the influence of the two adherence and colonization associated genes hcpA and iha was surveyed regarding their role for attachment and invasion. Upon soil contamination, the number of root-internalized cells of EHEC O157:H7 strain Sakai exceeded 102 cfu/g roots. Deletion of one or both of the adherence factor genes did not alter the overall attachment of EHEC O157:H7 strain Sakai to the roots, but significantly reduced the numbers of internalized bacteria by a factor of between 10 and 30, indicating their importance for invasion of EHEC O157:H7 strain Sakai into plant roots. This study identified intrinsic bacterial factors that play a crucial role during the internalization of EHEC O157:H7 strain Sakai into the roots of Valerianella locusta grown under the growth conditions in a greenhouse.

Tracing back multidrug-resistant bacteria in fresh herb production: from chive to source through the irrigation water chain.

Environmental antibiotic-resistant bacteria (ARB) can be transferred to humans through foods. Fresh produce in particular is an ideal vector due to frequent raw consumption. A major contamination source of fresh produce is irrigation water. We hypothesized that water quality significantly affects loads of ARB and their diversity on fresh produce despite various other contamination sources present under agricultural practice conditions. Chive irrigated from an open-top reservoir or sterile-filtered water (control) was examined. Heterotrophic plate counts (HPC) and ARB were determined for water and chive with emphasis on Escherichia coli and Enterococcus spp. High HPC of freshly planted chive decreased over time and were significantly lower on control- vs. reservoir-irrigated chive at harvest (1.3 log (CFU/g) lower). Ciprofloxacin- and ceftazidime-resistant bacteria were significantly lower on control-irrigated chive at harvest and end of shelf life (up to 1.8 log (CFU/g) lower). Escherichia coli and Enterococcus spp. repeatedly isolated from water and chive proved resistant to up to six or four antibiotic classes (80% or 49% multidrug-resistant, respectively). Microbial source tracking identified E. coli-ST1056 along the irrigation chain and on chive. Whole-genome sequencing revealed that E. coli-ST1056 from both environments were clonal and carried the same transmissible multidrug-resistance plasmid, proving water as source of chive contamination. These findings emphasize the urgent need for guidelines concerning ARB in irrigation water and development of affordable water disinfection technologies to diminish ARB on irrigated produce.

Inactivation of Antibiotic Resistant Bacteria and Resistance Genes by Ozone: From Laboratory Experiments to Full-Scale Wastewater Treatment.

Ozone, a strong oxidant and disinfectant, seems ideal to cope with future challenges of water treatment, such as micropollutants, multiresistant bacteria (MRB) and even intracellular antibiotic resistance genes (ARG), but information on the latter is scarce. In ozonation experiments we simultaneously determined kinetics and dose-dependent inactivation of Escherichia coli and its plasmid-encoded sulfonamide resistance gene sul1 in different water matrixes. Effects in E. coli were compared to an autochthonous wastewater community. Furthermore, resistance elimination by ozonation and post-treatment were studied in full-scale at a wastewater treatment plant (WWTP). Bacterial inactivation (cultivability, membrane damage) and degradation of sul1 were investigated using plate counts, flow cytometry and quantitative real-time PCR. In experiments with E. coli and the more ozone tolerant wastewater community disruption of intracellular genes was observed at specific ozone doses feasible for full-scale application, but flocs seemed to interfere with this effect. At the WWTP, regrowth during postozonation treatment partly compensated inactivation of MRB, and intracellular sul1 seemed unaffected by ozonation. Our findings indicate that ozone doses relevant for micropollutant abatement from wastewater do not eliminate intracellular ARG.

MiniTn7-transposon delivery vectors for inducible or constitutive fluorescent protein expression in Enterobacteriaceae.

Here we present the generation and function of two sets of bacterial plasmids that harbor fluorescent genes encoding either blue, cyan, yellow or red fluorescent proteins. In the first set, protein expression is controlled by the strong and constitutive nptII promoter whereas in the second set, the strong tac promoter was chosen that underlies LacI(q) regulation. Furthermore, the plasmids are mobilizable, contain Tn7 transposons and a temperature-sensitive origin of replication. Using Escherichia coli S17-1 as donor strain, the plasmids allow fast and convenient Tn7-transposon delivery into many enterobacterial hosts, such as the here-used E. coli O157:H7. This procedure omits the need of preparing competent recipient cells and antibiotic resistances are only transiently conferred to the recipients. As the fluorescence proteins show little to no overlap in fluorescence emission, the constructs are well suited for the study of multicolored synthetic bacterial communities during biofilm production or in host colonization studies, e.g. of plant surfaces. Furthermore, tac promoter-reporter constructs allow the generation of so-called reproductive success reporters, which allow to estimate past doublings of bacterial individuals after introduction into environments, emphasizing the role of individual cells during colonization.

Bacillus cereus Group-Type Strain-Specific Diagnostic Peptides.

The Bacillus cereus group consists of eight very closely related species and comprises both harmless and human pathogenic species such as Bacillus anthracis, Bacillus cereus, and Bacillus cytotoxicus. Numerous efforts have been undertaken to allow presumptive differentiation of B. cereus group species from one another. However, methods to rapidly and accurately distinguish these species are currently lacking. We confirmed that classical matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) biotyping cannot achieve reliable identification of each type strain. We therefore assigned type strain-specific diagnostic peptides to the B. cereus group based on comparisons of their proteomic profiles. The number of diagnostic peptides varied remarkably in a type strain-dependent manner. The accuracy of the reference database was crucial to validate candidate diagnostic peptides and led to a noteworthy reduction of verified diagnostic peptides. Diagnostic peptides ranged from one for B. weihenstephanensis to 62 for B. pseudomycoides and were associated with proteins involved in diverse biological processes, e.g. amino acid biosynthesis, cell envelope, cellular processes, energy metabolism, and transport processes. However, 45.6% of all diagnostic peptides comprised currently unclassified proteins or proteins of unknown function. In addition, a phylogenetic tree based on clustering of theoretical precursor masses deduced from in silico-generated tryptic peptides was reconstructed.

Antimicrobial Resistance in Agriculture.

In this article, the current knowledge and knowledge gaps in the emergence and spread of antimicrobial resistance (AMR) in livestock and plants and importance in terms of animal and human health are discussed. Some recommendations are provided for generation of the data required in order to develop risk assessments for AMR within agriculture and for risks through the food chain to animals and humans.

Complete genome sequence of Pseudomonas citronellolis P3B5, a candidate for microbial phyllo-remediation of hydrocarbon-contaminated sites.

Pseudomonas citronellolis is a Gram negative, motile gammaproteobacterium belonging to the order Pseudomonadales and the family Pseudomonadaceae. We isolated strain P3B5 from the phyllosphere of basil plants (Ocimum basilicum L.). Here we describe the physiology of this microorganism, its full genome sequence, and detailed annotation. The 6.95 Mbp genome contains 6071 predicted protein coding sequences and 96 RNA coding sequences. P. citronellolis has been the subject of many studies including the investigation of long-chain aliphatic compounds and terpene degradation. Plant leaves are covered by long-chain aliphates making up a waxy layer that is associated with the leaf cuticle. In addition, basil leaves are known to contain high amounts of terpenoid substances, hinting to a potential nutrient niche that might be exploited by P. citronellolis. Furthermore, the isolated strain exhibited resistance to several antibiotics. To evaluate the potential of this strain as source of transferable antibiotic resistance genes on raw consumed herbs we therefore investigated if those resistances are encoded on mobile genetic elements. The availability of the genome will be helpful for comparative genomics of the phylogenetically broad pseudomonads, in particular with the sequence of the P. citronellolis type strain PRJDB205 not yet publicly available. The genome is discussed with respect to a phyllosphere related lifestyle, aliphate and terpenoid degradation, and antibiotic resistance.

Beyond the matrix-assisted laser desorption ionization (MALDI) biotyping workflow: in search of microorganism-specific tryptic peptides enabling discrimination of subspecies.

A well-accepted method for identification of microorganisms uses matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) coupled to analysis software which identifies and classifies the organism according to its ribosomal protein spectral profile. The method, called MALDI biotyping, is widely used in clinical diagnostics and has partly replaced conventional microbiological techniques such as biochemical identification due to its shorter time to result (minutes for MALDI biotyping versus hours or days for classical phenotypic or genotypic identification). Besides its utility for identifying bacteria, MS-based identification has been shown to be applicable also to yeasts and molds. A limitation to this method, however, is that accurate identification is most reliably achieved on the species level on the basis of reference mass spectra, making further phylogenetic classification unreliable. Here, it is shown that combining tryptic digestion of the acid/organic solvent extracted (classical biotyping preparation) and resolubilized proteins, nano-liquid chromatography (nano-LC), and subsequent identification of the peptides by MALDI-tandem TOF (MALDI-TOF/TOF) mass spectrometry increases the discrimination power to the level of subspecies. As a proof of concept, using this targeted proteomics workflow, we have identified subspecies-specific biomarker peptides for three Salmonella subspecies, resulting in an extension of the mass range and type of proteins investigated compared to classical MALDI biotyping. This method therefore offers rapid and cost-effective identification and classification of microorganisms at a deeper taxonomic level.