Automated workflow for characterization of bacteriocin production in natural producers Lactococcus lactis and Latilactobacillus sakei.

BACKGROUND: Lactic acid bacteria are commonly used as protective starter cultures in food products. Among their beneficial effects is the production of ribosomally synthesized peptides termed bacteriocins that kill or inhibit food-spoiling bacteria and pathogens, e.g., members of the Listeria species. As new bacteriocins and producer strains are being discovered rapidly, modern automated methods for strain evaluation and bioprocess development are required to accelerate screening and development processes. RESULTS: In this study, we developed an automated workflow for screening and bioprocess optimization for bacteriocin producing lactic acid bacteria, consisting of microcultivation, sample processing and automated antimicrobial activity assay. We implemented sample processing workflows to minimize bacteriocin adsorption to producer cells via addition of Tween 80 and divalent cations to the cultivation media as well as acidification of culture broth prior to cell separation. Moreover, we demonstrated the applicability of the automated workflow to analyze influence of media components such as MES buffer or yeast extract for bacteriocin producers Lactococcus lactis B1629 and Latilactobacillus sakei A1608. CONCLUSIONS: Our automated workflow provides advanced possibilities to accelerate screening and bioprocess optimization for natural bacteriocin producers. Based on its modular concept, adaptations for other strains, bacteriocin products and applications are easily carried out and a unique tool to support bacteriocin research and bioprocess development is provided.

Use of a barrier membrane to repair congenital hard palate defects and to close oronasal fistulae remaining after cleft palate repair: seven dogs (2019-2022).

OBJECTIVE: To describe the use of a barrier membrane in dogs for repair of congenital hard palate defects and closure of oronasal fistulae (ONF) remaining after previous cleft palate (CFP) repair. ANIMALS: 7 client-owned dogs. METHODS: The hard palate defect was closed with medially positioned flaps (Von Langenbeck technique) or pedicle flaps (2-flap palatoplasty) and a membrane composed of autologous auricular cartilage from the pinna or allogenous fascia lata underlying the mucoperiosteal flaps. RESULTS: All palate defects were considered to have a high risk of dehiscence based on their type and size and the characteristics of the surrounding tissue. The barrier membrane was used in 5 dogs for repair of congenital hard palate defects and in 2 dogs for closure of ONF remaining after previous CFP repair. Resolution of clinical signs occurred in all cases. Complete success (ie, complete closure of the palate defect and absence of clinical signs) was achieved in 5 dogs (4 with congenital hard palate defects and 1 with an ONF remaining after previous CFP repair). The persistent ONF in 1 dog with functional success (incomplete closure, but no clinical signs) was smaller than prior to surgery. CLINICAL RELEVANCE: Barrier membranes underlying mucoperiosteal flaps may constitute an alternative technique in dogs for repair of congenital hard palate defects and closure of ONF remaining after previous CFP repair.

Etiology, clinical presentation, and outcome of temporomandibular joint luxation in cats: 21 cases (2000-2018).

This study was performed to report etiology, clinical presentation, treatment, and outcome of temporomandibular joint (TMJ) luxation in 21 cats (2000-2018). TMJ luxation was diagnosed by either dental radiography or computed tomography (CT). Causes for TMJ luxation included altercation with a dog (38%), hit-by-car (19%), unknown (19%), fall (19%), and ran into inanimate object (5%). The most common complication was development of permanent malocclusion (24%), followed by reduced vertical mandibular range of motion (5%). Reduction of TMJ luxation was successful in 94.7% of the cases. Reduction of the TMJ luxation was significantly associated with time from injury to treatment. No other significant associations were observed between cause of injury, type of TMJ stabilization, and outcome. TMJ luxation in cats carries an excellent prognosis with early joint reduction and supportive care. Concurrent dental trauma and maxillofacial injuries are common, and all patients with head trauma must be stabilized and evaluated with a thorough examination.

Surgical treatment for cleft palate in dogs yields excellent outcomes despite high rates of oronasal fistula formation: a narrative review.

Cleft palate is the most common congenital orofacial defect in dogs. Although this topic has been extensively studied in experimental dogs, current literature on clinical patients is limited to case reports and small case series studies with many individual opinions based on experience. This narrative review summarizes the literature of the last 10 years with application to clinical practice, focusing on the preparation of the patient until surgery, options for surgical management, and perioperative complications. Surgical treatment must be performed to obtain separation between the nasal/nasopharyngeal and oral/oropharyngeal passages. This will allow the dog to have independent function and reduce clinical signs associated with food, water, and other foreign material entering the respiratory tract. Surgical treatment has good to excellent outcomes when the procedure is carefully planned and appropriately executed despite the high rates of postoperative oronasal fistula reported.

Identification and Characterization of Corynaridin, a Novel Linaridin from Corynebacterium lactis.

Genome analysis of Corynebacterium lactis revealed a bacteriocin gene cluster encoding a putative bacteriocin of the linaridin family of ribosomally synthesized and posttranslationally modified peptides (RiPPs). The locus harbors typical linaridin modification enzymes but lacks genes for a decarboxylase and methyltransferase, which is unusual for type B linaridins. Supernatants of Corynebacterium lactis RW3-42 showed antimicrobial activity against Corynebacterium glutamicum. Deletion of the precursor gene crdA clearly linked the antimicrobial activity of the producer strain to the identified gene cluster. Following purification, we observed potent activity of the peptide against Actinobacteria, mainly other members of the genus Corynebacterium, including the pathogenic species Corynebacterium striatum and Corynebacterium amycolatum. Also, low activity against some Firmicutes was observed, but there was no activity against Gram-negative species. The peptide is resilient towards heat but sensitive to proteolytic degradation by trypsin and proteinase K. Analysis by mass spectrometry indicates that corynaridin is processed by cleaving off the leader sequence at a conserved motif and posttranslationally modified by dehydration of all threonine and serin residues, resulting in a monoisotopic mass of 3,961.19 Da. Notably, time-kill kinetics and experiments using live biosensors to monitor membrane integrity suggest bactericidal activity that does not involve formation of pores in the cytoplasmic membrane. As Corynebacterium species are ubiquitous in nature and include important commensals and pathogens of mammalian organisms, secretion of bacteriocins by species of this genus could be a hitherto neglected trait with high relevance for intra- and interspecies competition and infection. IMPORTANCE Bacteriocins are antimicrobial peptides produced by bacteria to fend off competitors in ecological niches and are considered to be important factors influencing the composition of microbial communities. However, bacteriocin production by bacteria of the genus Corynebacterium has been a hitherto neglected trait, although its species are ubiquitous in nature and make up large parts of the microbiome of humans and animals. In this study, we describe and characterize a novel linaridin family bacteriocin from Corynebacterium lactis and show its narrow-spectrum activity, mainly against other actinobacteria. Moreover, we were able to extend the limited knowledge on linaridin bioactivity in general and for the first time describe the bactericidal activity of such a bacteriocin. Interestingly, the peptide, which was named corynaridin, appears bactericidal, but without formation of pores in the bacterial membrane.

Morphometry and Morphology of the Articular Surfaces of the Medial Region of the Temporomandibular Joint in the Felis Catus (Domestic cat) - A Cone Beam Computed Tomography Study.

The articular surfaces of the temporomandibular joint (TMJ) in the cat consist of the articular head of the condylar process of the ramus of the mandible and the mandibular fossa of the petrous part of the temporal bone. Anatomic conformation of the TMJ articular surfaces can vary; however, this has not been studied in the cat. Thus, the aim of this study was to characterize the conformation of the medial region of the TMJ in mesocephalic adult cats as well as to determine the articular relationship by measurement of the degree of coverage of the mandibular fossa over the articular head. This was accomplished by assessing 60 TMJs from 30 mesocephalic adult cats by means of cone-beam computed tomography (CBCT). The width and depth of the mandibular fossa, and the degree of coverage of the mandibular fossa were evaluated using parasagittal reconstructions of the medial aspect of the TMJ by two observers. No statistically significant difference was observed during the intra and interobserver evaluation of the mandibular fossa width and depth and the degree of coverage of the mandibular fossa over the articular head. P-values for the studied variables were greater than 0.05. The resulting measurements demonstrated a concave mandibular fossa with a prominent retroarticular process and a poorly developed articular eminence; a well-defined rounded articular surface, and a high degree of coverage (171°) of the mandibular fossa over the articular head. In conclusion, the articular surfaces at the medial aspect of the TMJ displayed elevated geometric similarity, with an articular head of the condylar process deeply seated in the mandibular fossa.

Optimized recombinant production of the bacteriocin garvicin Q by Corynebacterium glutamicum.

Bacteriocins are antimicrobial peptides applied in food preservation and are interesting candidates as alternatives to conventional antibiotics or as microbiome modulators. Recently, we established Corynebacterium glutamicum as a suitable production host for various bacteriocins including garvicin Q (GarQ). Here, we establish secretion of GarQ by C. glutamicum via the Sec translocon achieving GarQ titers of about 7 mg L-1 in initial fermentations. At neutral pH, the cationic peptide is efficiently adsorbed to the negatively charged envelope of producer bacteria limiting availability of the bacteriocin in culture supernatants. A combination of CaCl2 and Tween 80 efficiently reduces GarQ adsorption to C. glutamicum. Moreover, cultivation in minimal medium supplemented with CaCl2 and Tween 80 improves GarQ production by C. glutamicum to about 15 mg L-1 but Tween 80 resulted in reduced GarQ activity at later timepoints. Using a reporter strain and proteomic analyses, we identified HtrA, a protease associated with secretion stress, as another potential factor limiting GarQ production. Transferring production to HtrA-deficient C. glutamicum K9 improves GarQ titers to close to 40 mg L-1. Applying conditions of low aeration prevented loss in activity at later timepoints and improved GarQ titers to about 100 mg L-1. This is about 50-fold higher than previously shown with a C. glutamicum strain employing the native GarQ transporter GarCD for secretion and in the range of levels observed with the native producer Lactococcus petauri B1726. Additionally, we tested several synthetic variants of GarQ and were able to show that exchange of the methionine in position 5 to a phenylalanine (GarQM5F) results in markedly increased activity against Lactococcus lactis and Listeria monocytogenes. In summary, our findings shed light on several aspects of recombinant GarQ production that may also be of relevance for production with natural producers and other bacteriocins.

Garvicin Q: characterization of biosynthesis and mode of action.

Bacteriocins are ribosomally synthesized antimicrobial peptides, that either kill target bacteria or inhibit their growth. Bacteriocins are used in food preservation and are of increasing interest as potential alternatives to conventional antibiotics. In the present study, we show that Lactococcus petauri B1726, a strain isolated from fermented balsam pear, produces a heat-stable and protease-sensitive compound. Following genome sequencing, a gene cluster for production of a class IId bacteriocin was identified consisting of garQ (encoding for the bacteriocin garvicin Q), garI (for a putative immunity protein), garC, and garD (putative transporter proteins). Growth conditions were optimized for increased bacteriocin activity in supernatants of L. petauri B1726 and purification and mass spectrometry identified the compound as garvicin Q. Further experiments suggest that garvicin Q adsorbs to biomass of various susceptible and insusceptible bacteria and support the hypothesis that garvicin Q requires a mannose-family phosphotransferase system (PTSMan) as receptor to kill target bacteria by disruption of membrane integrity. Heterologous expression of a synthetic garQICD operon was established in Corynebacterium glutamicum demonstrating that genes garQICD are responsible for biosynthesis and secretion of garvicin Q. Moreover, production of garvicin Q by the recombinant C. glutamicum strain was improved by using a defined medium yet product levels were still considerably lower than with the natural L. petauri B1726 producer strain.Collectively, our data identifies the genetic basis for production of the bacteriocin garvicin Q by L. petauri B1726 and provides insights into the receptor and mode of action of garvicin Q. Moreover, we successfully performed first attempts towards biotechnological production of this interesting bacteriocin using natural and heterologous hosts.

Surgical Treatment and Outcome of Acquired Midline Palate Defects in Cats.

Acquired midline palate defects (PDE) affect the hard palate and/or soft palate, and result from trauma, commonly falling from a height or being hit by a motor vehicle. Additional life-threating injuries and costs associated with the treatment may delay the surgical treatment. This retrospective study describes signalment, cause, and extent of the PDE, and treatment in 25 cats. In addition, the outcome of the surgical repair is described in 19 (76%) cats. All defects were repaired within 5 days of the injury. Twenty (80%) cats were 4 years of age or younger. The most common rostral extent of the PDE was to the level of the third premolar tooth (n = 8; 32%), incisor teeth (n = 7; 28%), and fourth premolar tooth (n = 5; 20%). The soft palate laceration was present in all cases. Surgical therapy was successful in all cases with follow-up. The most common techniques used for the closure of the hard palate defect were bilateral pedicle flaps with lateral releasing incisions (n = 8; 32%), direct apposition of the oral mucosa (n = 7; 28%), bilateral pedicle flaps with lateral releasing incisions and interquadrant splinting (n = 5; 20%), and unilateral pedicle flap with one lateral releasing incision (n = 4; 16%). A tension-free closure by direct apposition of the edges was possible for the soft palate laceration. No oronasal fistulae were identified at follow-up. The only complication was malocclusion. The interquadrant splinting was most often used for PDE extending to the rostral portion of the hard palate (p < 0.05). The cats that suffered postoperative malocclusion were significantly more likely to have sustained temporomandibular joint injury, underwent CT scan, or had a feeding tube placed before discharge. The results of this retrospective study indicate that the early treatment (within 5 days) of the acquired longitudinal defects in the midline of the hard and soft palates is highly successful.

Caudal and middle segmental mandibulectomies for the treatment of unilateral temporomandibular joint ankylosis in cats.

Case series summary: Three cats affected by unilateral temporomandibular joint ankylosis underwent segmental mandibulectomy, while one cat with bilateral ankylosis underwent right temporomandibular joint gap arthroplasty and left segmental mandibulectomy. Minimal intraoperative complications were linked to the segmental mandibulectomies in the cases reported. All cats recovered their ability to open the mouth, and the vertical range of motion was unaltered during the postoperative period. Mandibular drift and dental malocclusion were the main postoperative complications related to the segmental mandibulectomies. Relevance and novel information: Surgical excision of ankylotic tissue at the temporomandibular joint region may be technically challenging and associated with severe iatrogenic trauma of surrounding tissues. The results of this case series suggest that middle or caudal segmental mandibulectomy is a safe, feasible and effective surgical procedure to treat unilateral temporomandibular ankylosis in cats and could be considered as an alternative surgical technique to surgical excision of ankylotic tissue at the temporomandibular joint.

Metabolic Footprinting of Microbial Systems Based on Comprehensive In Silico Predictions of MS/MS Relevant Data.

Metabolic footprinting represents a holistic approach to gathering large-scale metabolomic information of a given biological system and is, therefore, a driving force for systems biology and bioprocess development. The ongoing development of automated cultivation platforms increases the need for a comprehensive and rapid profiling tool to cope with the cultivation throughput. In this study, we implemented a workflow to provide and select relevant metabolite information from a genome-scale model to automatically build an organism-specific comprehensive metabolome analysis method. Based on in-house literature and predicted metabolite information, the deduced metabolite set was distributed in stackable methods for a chromatography-free dilute and shoot flow-injection analysis multiple-reaction monitoring profiling approach. The workflow was used to create a method specific for Saccharomyces cerevisiae, covering 252 metabolites with 7 min/sample. The method was validated with a commercially available yeast metabolome standard, identifying up to 74.2% of the listed metabolites. As a first case study, three commercially available yeast extracts were screened with 118 metabolites passing quality control thresholds for statistical analysis, allowing to identify discriminating metabolites. The presented methodology provides metabolite screening in a time-optimised way by scaling analysis time to metabolite coverage and is open to other microbial systems simply starting from genome-scale model information.

Dental Abnormalities in Immature Dogs with a History of Mandibular Fractures.

This study was performed to report the dental abnormalities encountered in immature dogs with a history of mandibular fracture. Dogs were included in the study if mandibular fractures were diagnosed by means of oral examination and diagnostic imaging, they were treated with non-invasive or minimally invasive methods, and there was a follow-up examination of at least 90 days after the initial presentation with the last follow-up visit occurring at 6 months of age or older. Eleven dogs met the inclusion criteria. Dental abnormalities occurred exclusively in those dogs that had sustained fracture of the mandibular body or fracture at the transition of mandibular body and mandibular ramus. A high number of developing permanent teeth located in or near the mandibular fracture were affected (73.5%). The most common dental abnormalities were failure of eruption or partial eruption (29.0%), resorption (22.6%), abnormal shape (19.4%), and enamel hypoplasia (16.1%). The majority of deciduous teeth (64.3%) in or near the mandibular fracture exfoliated uneventfully. Ten out of 11 dogs needed a surgical procedure to treat dental abnormalities after mandibular fracture healing. The development of the tooth germs located in or near a mandibular fracture is frequently affected. Radiographic evaluation of the area of trauma is recommended until eruption and full development of the teeth are completed.

Etiology, Clinical Presentation, and Outcome of Mandibular Fractures in Immature Dogs Treated with non-Invasive or Minimally Invasive Techniques.

This study was performed to report etiology, clinical presentation, and outcome of mandibular fractures in immature dogs treated with non-invasive or minimally invasive techniques. Immature dogs diagnosed with mandibular fractures from 2001 to 2016 were included in this study. Diagnosis of the mandibular fracture was achieved by oral examination and diagnostic imaging in the anesthetized dog. Twenty-nine immature dogs with 54 mandibular fractures were selected. Within the mandibular body, the regions of the developing permanent canine and first molar teeth were most commonly involved (46.4% and 35.0%, respectively). Within the mandibular ramus, 53.8% of the fractures were located in its ventral half, and 38.5% in the condylar process. Muzzling was applied in 72.4% of the dogs. All dogs had clinical healing with resolution of signs of pain and recovery of mandibular function. Mean time for clinical healing was 21 ± 9 days. Age of the dog and duration of muzzling were significantly associated with the time needed for clinical healing. In immature dogs, fractures of the mandibular body occur most commonly in the regions of the developing permanent canine and first molar teeth, while fractures of the mandibular ramus are most commonly found in its ventral half and the condylar process. Non-invasive or minimally invasive management of mandibular fractures in immature dogs carries a good prognosis regarding clinical healing and recovery of mandibular function. Dogs should be monitored for the development of dental abnormalities and/or skeletal malocclusion until permanent teeth have erupted and jaw growth is completed.

Oral and Maxillofacial Tumor Management - From Biopsy to Surgical Removal.

The main objective of oral and maxillofacial (OMF) tumor resection is to get local control of the disease. Many OMF tumors can be cured with wide or radical surgery, whereas others might only achieve temporary local control of the disease by removing infection and the source of pain, thereby improving the quality of life of the patient while keeping masticatory function. The standard of care on managing OMF tumors includes the diagnosis and identification of the local and distant extension of the disease to establish an appropriate treatment plan tailored for each patient. In this article, we provide a practical review of the current information related to staging, biopsy, and main surgical techniques for OMF tumor removal.

Radiographic Assessment of the Feline Temporomandibular Joint with Oblique Projections: A Preliminary Ex Vivo Study.

Radiographic assessment of the temporomandibular joint in the domestic cat using conventional radiographic views can be challenging due to superimposition of overlying structures and the complex anatomy of the skull. The use of computed tomography, magnetic resonance imaging, and cone beam computed tomography to assess the temporomandibular joint in the cat has increased, but these modalities are not always available in general veterinary practices. Conventional radiography is still commonly used for first line assessment of the temporomandibular joint. The aim of this preliminary study was to determine optimal angle of obliquity of lateroventral-laterodorsal and laterorostral-laterocaudal (nose up lateral oblique) oblique radiographic views in the assessment of the temporomandibular joints in five feline mesaticephalic dry skulls. Visibility of the mandibular head, mandibular fossa, retroarticular process, and temporomandibular joint space were evaluated and scored by two veterinary radiologists. The results of this study identified that the dependent temporomandibular joint anatomy was best seen on the latero-10°-ventral-laterodorsal, latero-15°-ventral-laterodorsal, and latero-20°-ventral-laterodorsal, oblique views, and opposite lateral oblique views at these angulations may be helpful in characterization of this anatomy in clinical patients. The results also indicate that the laterorostral-laterocaudal (nose up lateral oblique) oblique view did not allow adequate discrimination of all TMJ anatomy at any angle, and is not recommended.

Metabolic engineering of Pseudomonas putida for production of the natural sweetener 5-ketofructose from fructose or sucrose by periplasmic oxidation with a heterologous fructose dehydrogenase.

5-Ketofructose (5-KF) is a promising low-calorie natural sweetener with the potential to reduce health problems caused by excessive sugar consumption. It is formed by periplasmic oxidation of fructose by fructose dehydrogenase (Fdh) of Gluconobacter japonicus, a membrane-bound three-subunit enzyme containing FAD and three haemes c as prosthetic groups. This study aimed at establishing Pseudomonas putida KT2440 as a new cell factory for 5-KF production, as this host offers a number of advantages compared with the established host Gluconobacter oxydans. Genomic expression of the fdhSCL genes from G. japonicus enabled synthesis of functional Fdh in P. putida and successful oxidation of fructose to 5-KF. In a batch fermentation, 129 g l-1 5-KF were formed from 150 g l-1 fructose within 23 h, corresponding to a space-time yield of 5.6 g l-1 h-1 . Besides fructose, also sucrose could be used as substrate for 5-KF production by plasmid-based expression of the invertase gene inv1417 from G. japonicus. In a bioreactor cultivation with pulsed sucrose feeding, 144 g 5-KF were produced from 358 g sucrose within 48 h. These results demonstrate that P. putida is an attractive host for 5-KF production.

Characterization of Oral Microbiota in Cats: Novel Insights on the Potential Role of Fungi in Feline Chronic Gingivostomatitis.

Previous studies have suggested the involvement of viral and bacterial components in the initiation and progression of feline chronic gingivostomatitis (FCGS), but the role of fungi remains entirely unknown. This pilot study aimed to investigate the bacteriome and mycobiome in feline oral health and disease. Physical exams, including oral health assessment, of privately owned, clinically healthy (CH) cats (n = 14) and cats affected by FCGS (n = 14) were performed. Using a sterile swab, oral tissue surfaces of CH and FCGS cats were sampled and submitted for 16S rRNA and ITS-2 next-generation DNA sequencing. A high number of fungal species (n = 186) was detected, with Malassezia restricta, Malassezia arunalokei, Cladosporium penidielloides/salinae, and Aspergillaceae sp. being significantly enriched in FCGS samples, and Saccharomyces cerevisiae in CH samples. The bacteriome was significantly distinct between groups, and significant inter-kingdom interactions were documented. Bergeyella zoohelcum was identified as a potential biomarker of a healthy feline oral microbiome. These data suggest that fungi might play a role in the etiology and pathogenesis of FCGS, and that oral health should not simply be regarded as the absence of microbial infections. Instead, it may be viewed as the biological interactions between bacterial and fungal populations that coexist to preserve a complex equilibrium in the microenvironment of the mouth. Additional investigations are needed to improve our understanding of the feline oral ecosystem and the potential interactions between viruses, bacteria, and fungi in FCGS.

Immunohistochemical evaluation of suspected oral malignant melanoma in cats.

Oral malignant melanoma (OMM) is considered the third most common oral malignant neoplasm in cats, but its variable morphology and frequent lack of melanin pigment make it a diagnostic challenge. Twenty-two cases of cats with malignant oral neoplasms that were diagnosed as OMM or listed OMM as a suspected differential diagnosis on the biopsy report were examined using an immunohistochemistry (IHC) panel for S100, melan-A, PNL2, laminin, CD34, and pan-cytokeratin. Although OMM was suspected (n = 14) or previously diagnosed (n = 8), only 2 cases were immunohistochemically confirmed as OMM. Seven cases were classified as soft tissue sarcoma based on positive expression of CD34 or laminin, and one was classified as carcinoma based on positive expression of pan-cytokeratin. The majority of cases (n = 12) were categorized as unclassified malignant neoplasms because they did not express melan-A, PNL2, laminin, CD34, or pan-cytokeratin; however, a proportion of these did express S100 (n = 7). Long-term prognosis of all 22 cats was poor, with a median survival time of 87 days (range = 2-249 days). Cases with longer survival times (>100 days) were treated with surgery, radiation therapy, or a combination. For feline oral malignant neoplasms thought to be OMM, routine use of IHC is required for an accurate diagnosis.

Need for speed: evaluation of dilute and shoot-mass spectrometry for accelerated metabolic phenotyping in bioprocess development.

With the utilization of small-scale and highly parallelized cultivation platforms embedded in laboratory robotics, microbial phenotyping and bioprocess development have been substantially accelerated, thus generating a bottleneck in bioanalytical bioprocess sample analytics. While microscale cultivation platforms allow the monitoring of typical process parameters, only limited information about product and by-product formation is provided without comprehensive analytics. The use of liquid chromatography mass spectrometry can provide such a comprehensive and quantitative insight, but is often limited by analysis runtime and throughput. In this study, we developed and evaluated six methods for amino acid quantification based on two strong cation exchanger columns and a dilute and shoot approach in hyphenation with either a triple-quadrupole or a quadrupole time-of-flight mass spectrometer. Isotope dilution mass spectrometry with 13C15N labeled amino acids was used to correct for matrix effects. The versatility of the methods for metabolite profiling studies of microbial cultivation supernatants is confirmed by a detailed method validation study. The methods using chromatography columns showed a linear range of approx. 4 orders of magnitude, sufficient response factors, and low quantification limits (7-443 nM) for single analytes. Overall, relative standard deviation was comparable for all analytes, with < 8% and < 11% for unbuffered and buffered media, respectively. The dilute and shoot methods with an analysis time of 1 min provided similar performance but showed a factor of up to 35 times higher throughput. The performance and applicability of the dilute and shoot method are demonstrated using a library of Corynebacterium glutamicum strains producing L-histidine, obtained from random mutagenesis, which were cultivated in a microscale cultivation platform.