Timely and accurate detection and characterization of microbial threats is crucial for effective infection and outbreak management. Additionally, in food production, rapid microbe identification is indispensable for maintaining quality control and hygiene standards. Current methods for typing microbial strains often rely on labor-intensive, time-consuming, and expensive DNA- and sera-serotyping techniques, limiting their applicability in rapid-response scenarios. In this context, the IR Biotyper®, utilizing Fourier-transform infrared (FTIR) spectroscopy, offers a novel approach, providing specific spectra for fast strain typing within 3 h. This methodology article serves as a comprehensive resource for researchers and technicians aiming to utilize FTIR spectroscopy for microbial strain typing. It encompasses detailed guidelines on sample preparation, data acquisition, and analysis techniques, ensuring the generation of reliable and reproducible results. We highlight the IR Biotyper®\'s rapid and accurate discrimination capabilities, showcasing its potential for real-time pathogen monitoring and source-tracking to enhance public health and food safety. We propose its integration as an early screening method, followed by more detailed analysis with whole-genome sequencing, to optimize detection accuracy and response efficiency in microbial surveillance systems.

BACKGROUND: Staphylococcus aureus is a multi-host zoonotic pathogen causing human and livestock diseases. Dairy farms that make artisan cheese have distinctive concerns for S. aureus control. Antimicrobial-resistant (AMR) S. aureus is a public and animal health concern. There is a need to study the population structure of AMR S. aureus at the human-animal interface and understand the path of zoonotic transmission. This cross-sectional observational study aimed to assess the genetic diversity and AMR patterns of S. aureus isolated from cattle and humans on conventional and organic Vermont dairy farms that produce and sell farmstead cheese.
RESULTS: A convenience sample of 19 dairy farms in Vermont was enrolled, and 160 S. aureus isolates were collected from cow quarter milk (CQM), bulk tank milk (BTM), human-hand and -nasal swabs. After deduplication, 89 isolates were used for the analysis. Sequence types (STs) were determined by multilocus sequence typing and cataloged to the PubMLST database. Nine defined and five novel STs were identified. For BTM and CQM samples, six STs were identified within cow-adapted CC97 and CC151. Two human-adapted STs were isolated from BTM and CQM. Seven human-adapted clonal complexes with eight STs were identified from human samples. One cow-adapted ST was isolated from a human. Antimicrobial susceptibility of the isolates was tested using disc diffusion and broth microdilution methods. Approximately 27% of the isolates were beta-lactam resistant and blaZ gene-positive. S. aureus isolates from human swabs were more likely to carry blaZ compared to isolates from CQM or BTM. S. aureus isolated from cows and humans on the same farm belonged to different STs.
CONCLUSIONS: Humans were more likely to carry beta-lactam-resistant S. aureus compared to cows, and on organic farms only human-adapted blaZ positive STs were isolated from BTM. Moreover, we identified potential spillover events of S. aureus sequence types between host species. The presence of penicillin-resistant-human-adapted S. aureus on both organic and conventional dairy farms highlights a \"One Health\" concern at the junction of public and animal health requiring further surveillance.

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne\'s Disease (JD) in ruminants, which is responsible for significant economic loss to the global dairy industry. Mixed strain infection (MSI) refers to the concurrent infection of a susceptible host with genetically distinct strains of a pathogen, whereas within-host changes in an infecting strain leading to genetically distinguishable progeny is called microevolution. The two processes can influence host-pathogen dynamics, disease progression and outcomes, but not much is known about their prevalence and impact on JD. Therefore, we obtained up to 10 MAP isolates each from 14 high-shedding animals and subjected them to whole-genome sequencing. Twelve of the 14 animals examined showed evidence for the presence of MSIs and microevolution, while the genotypes of MAP isolates from the remaining two animals could be attributed solely to microevolution. All MAP isolates that were otherwise isogenic had differences in short sequence repeats (SSRs), of which SSR1 and SSR2 were the most diverse and homoplastic. Variations in SSR1 and SSR2, which are located in ORF1 and ORF2, respectively, affect the genetic reading frame, leading to protein products with altered sequences and computed structures. The ORF1 gene product is predicted to be a MAP surface protein with possible roles in host immune modulation, but nothing could be inferred regarding the function of ORF2. Both genes are conserved in Mycobacterium avium complex members, but SSR1-based modulation of ORF1 reading frames seems to only occur in MAP, which could have potential implications on the infectivity of this pathogen. IMPORTANCE Johne\'s disease (JD) is a major problem in dairy animals, and concerns have been raised regarding the association of Mycobacterium avium subsp. paratuberculosis (MAP) with Crohn\'s disease in humans. MAP is an extremely slow-growing bacterium with low genome evolutionary rates. Certain short sequence repeats (SSR1 and SSR2) in the MAP chromosome are highly variable and evolve at a faster rate than the rest of the chromosome. In the current study, multiple MAP isolates with genetic variations such as single-nucleotide polymorphisms, and more noticeably, diverse SSRs, could simultaneously infect animals. Variations in SSR1 and SSR2 affect the products of the respective genes containing them. Since multiple MAP isolates can infect the same animal and the possibility that the pathogen undergoes further changes within the host due to unstable SSRs, this could provide a compensative mechanism for an otherwise slow-evolving pathogen to increase phenotypic diversity for overcoming host responses.

Pseudomonas aeruginosa is one of the most common opportunistic pathogens causing severe nosocomial infections for its patterns of multidrug resistance, particularly for carbapenems. Timely epidemiological surveillance could greatly facilitate infection control of P. aeruginosa and many deadly pathogens alike. IR Biotyper (IRBT), is a novel real-time typing tool, based on a Fourier-transform infrared (FTIR) spectroscopy system. It is critical to comprehensively establish and evaluate the feasibility of IRBT in P. aeruginosa strain typing. In the current study, we first established standards and schemes for its routine laboratory application, and we found that Mueller-Hinton agar plates give better discriminatory power than blood agar plates. Data showed that the cut-off value of 0.15 with an additional 0.025 range was optimal. Secondly, 27 clinically isolated carbapenem-resistant P. aeruginosa (CRPA) strains collected from October 2010 to September 2011 were evaluated for typing effectiveness by comparing IRBT to the other commonly used typing methods, such as multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS)-based typing. When using WGS-based typing as the reference method, the typing method of FTIR spectroscopy (AR = 0.757, SID = 0.749) could better cluster P. aeruginosa strains than MLST and in silico serotyping (AR = 0.544, SID = 0.470). Though PFGE showed the highest discriminatory power, low concordance was observed between PFGE and the other methods. Above all, this study demonstrates the utility of the IRBT as a quick, low-cost, real-time typing tool for the detection of CRPA strains.

Mycobacterium avium subsp. paratuberculosis (MAP) is the pathogen responsible for paratuberculosis or Johne\'s Disease (JD) in ruminants, which is responsible for substantial economic losses worldwide. MAP transmission primarily occurs through the fecal-oral route, and the introduction of an MAP infected animal into a herd is an important transmission route. In the current study, we characterized MAP isolates from 67 cows identified in 20 herds from the provinces of Quebec and Ontario, Canada. Whole genome sequencing (WGS) was performed and an average genome coverage (relative to K-10) of ∼14.9 fold was achieved. The total number of SNPs present in each isolate varied from 51 to 132 and differed significantly between herds. Isolates with the highest genetic variability were generally present in herds from Quebec. The isolates were broadly separated into two main clades and this distinction was not influenced by the province from which they originated. Analysis of 8 MIRU-VNTR loci and 11 SSR loci was performed on the 67 isolates from the 20 dairy herds and publicly available references, notably major genetic lineages and six isolates from the province of Newfoundland and Labrador. All 67 field isolates were phylogenetically classified as Type II (C-type) and according to MIRU-VNTR, the predominant type was INMV 2 (76.1%) among four distinct patterns. Multilocus SSR typing identified 49 distinct INMV SSR patterns. The discriminatory index of the multilocus SSR typing was 0.9846, which was much higher than MIRU-VNTR typing (0.3740). Although multilocus SSR analysis provides good discriminatory power, the resolution was not informative enough to determine inter-herd transmission. In select cases, SNP-based analysis was the only approach able to document disease transmission between herds, further validated by animal movement data. The presence of SNPs in several virulence genes, notably for PE, PPE, mce and mmpL, is expected to explain differential antigenic or pathogenetic host responses. SNP-based studies will provide insight into how MAP genetic variation may impact host-pathogen interactions. Our study highlights the informative power of WGS which is now recommended for epidemiological studies and to document mixed genotypes infections.

Probiotic bacteria, capable of conferring benefits to the host, can present challenges in design, development, scale-up, manufacturing, commercialization, and life cycle management. Strain identification is one of the main quality parameters; nevertheless, this task can be challenging since established methodologies can lack resolution at the strain level for some microorganisms and\\or are labor-intensive and time-consuming. Fourier transform infrared spectroscopy (FTIRS) has been largely used for the investigation of pathogenic species in the clinical field, whereas only recently has been proposed for the identification of probiotic strains. Within the probiotic industrial production, bacterial strains can be subjected to stressful conditions that may affect genomic and phenotypic characteristics; therefore, real-time monitoring of all the sequential growth steps is requested. Considering the fast, low-cost, and high-throughput features, FTIRS is an innovative and functional technology for typing probiotic strains from bench-top experiments to large-scale industrial production, allowing the monitoring of stability and identity of probiotic strains. In this study, the discriminatory power of FTIRS was assessed for four Lactiplantibacillus plantarum probiotic strains grown under different conditions, including temperatures (30 and 37°C) and medium (broth and agar), after consecutive sub-culturing steps. A comparison between the generated spectra with pulsed-field gel electrophoresis (PFGE) profiles was also performed. FTIRS was not only able to distinguish the strains of L. plantarum under different growth conditions but also to prove the phenotypic stability of L. plantarum type strain LP-CT after six growing steps. Regardless of the growth conditions, FTIRS spectra related to LP-CT constituted a unique hierarchical cluster, separated from the other L. plantarum strains. These results were confirmed by a PFGE analysis. In addition, based on FTIRS data, broth cultures demonstrated a higher reproducibility and discriminatory power with respect to agar ones. These results support the introduction of FTIRS in the probiotic industry, allowing for the step-by-step monitoring of massive microbial production while also guaranteeing the stability and purity of the probiotic strain. The proposed novel approach can constitute an impressive improvement in the probiotic manufacturing process.

Candida auris continues to be a global threat for infection and transmission in hospitals and long-term care facilities. The emergence of SARS-CoV-2 has rerouted attention and resources away from this silent pandemic to the frontlines of the ongoing COVID-19 disease. Cases of C. auris continue to rise, and clinical laboratories need a contingency plan to prevent a possible outbreak amid the COVID-19 pandemic. Here, we introduce a two-tier Candida auris surveillance program that includes, first, a rapid qualitative rt-PCR for the identification of high-risk patients and, second, a method to analyze the isolated C. auris for strain typing using the Fourier-Transform Infrared spectroscopy. We have performed this two-tier surveillance for over 700 at-risk patients being admitted into our hospital and have identified 28 positive specimens (4%) over a 1-year period. Strain typing analysis by the IR spectrum acquisition typing method, supplemented by whole genome sequencing, has shown grouping of two significant clusters. The majority of our isolates belong to circulating African lineage associated with C. auris Clade III and an isolated strain grouping differently belonging to South Asian lineage C. auris Clade I. Low numbers of genomic variation point to local and ongoing transmission within the Los Angeles area not specifically within the hospital setting. Collectively, clinical laboratories having the ability to rapidly screen high-risk patients for C. auris and to participate in outbreak investigations by offering strain typing will greatly assist in the control of C. auris transmission within the hospital setting.

Vancomycin-resistant enterococci (VRE) are nosocomial pathogens with genetic plasticity and widespread antimicrobial resistance (AMR). To prevent the spread of VRE in the hospital setting, molecular epidemiological approaches such as pulsed-field gel electrophoresis and multilocus sequence typing have been implemented for pathogen outbreak surveillance. However, due to the insufficient discriminatory power of these methods, whole-genome sequencing (WGS), which enables high-resolution analysis of entire genomic sequences, is being used increasingly. Herein, we performed WGS of VRE using both short-read next-generation sequencing (SR-NGS) and long-read next-generation sequencing (LR-NGS). Since standardized workflows and pipelines for WGS-based bacterial epidemiology are lacking, we established three-step pipelines for SR- and LR-NGS, as a standardized WGS-based approach for strain typing and AMR profiling. For strain typing, we analyzed single-nucleotide polymorphisms (SNPs) of VRE isolates and constructed SNP-based maximum-likelihood phylogenies. The phylogenetic trees constructed using short and long reads showed good correspondence. Still, SR-NGS exhibited higher sensitivity for detecting nucleotide substitutions of bacterial sequences. During AMR profiling, we examined AMR genes and resistance-conferring mutations. We also assessed the concordance between genotypic and phenotypic resistance, which was generally better for LR-NGS than SR-NGS. Further validation of our pipelines based on outbreak cases is necessary to ensure the overall performance of pipelines.

IR Biotyper (IRBT), which is a spectroscopic system for microorganism typing based on Fourier transform infrared (FTIR) technology, has been used to detect the spread of clones in clinical microbiology laboratories. However, the use of IRBT to detect probiotics has rarely been reported. Herein, we evaluated the discriminatory power of IRBT to type Lactiplantibacillus plantarum isolates at the strain level and explored its application potential in probiotic preliminary selection. Twenty Lactiplantibacillus isolates collected from pickled radishes during successive fermentation were used to test the robustness of IRBT at the strain level. IRBT was then compared with genotyping methods such as whole-genome sequencing (WGS), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) to evaluate its discrimination power. IRBT distributed the 20 isolates into five clusters, with L. argentoratensis isolate C7-83 being the most distant from the other isolates, which belonged to L. plantarum. IRBT showed good reproducibility, although deviation in the discriminative power of IRBT was found at the strain level across laboratories, probably due to technical variance. All examined methods allowed bacterial identification at the strain level, but IRBT had higher discriminatory power than MLST and was comparable to the WGS and PFGE. In the phenotypic comparison study, we observed that the clustering results of probiotic physiological attributes (e.g., sensitivity to acid and bile salts, hydrophobicity of the cell surface, and resistance to antibiotics) were consistent with the typing results of IRBT. Our results indicated that IRBT is a robust tool for L. plantarum strain typing that could improve the efficiency of probiotic identification and preliminary screening, and can potentially be applied in probiotic traceability and quality control.

To study the contamination of microorganisms in the food industry, pharmaceutical industry, clinical diagnosis, or bacterial taxonomy, accurate identification of species is a key starting point of further investigation. The conventional method of identification by the 16S rDNA gene or other marker gene comparison is not accurate, because it uses a tiny part of the genomic information. The average nucleotide identity calculated between two whole bacterial genomes was proven to be consistent with DNA-DNA hybridization and adopted as the gold standard of bacterial species delineation. Furthermore, there are more bacterial genomes available in public databases recently. All of those contribute to a genome era of bacterial species identification. However, wrongly labeled and low-quality bacterial genome assemblies, especially from type strains, greatly affect accurate identification. In this study, we employed a multi-step strategy to create a type-strain genome database, by removing the wrongly labeled and low-quality genome assemblies. Based on the curated database, a fast bacterial genome identification platform (fIDBAC) was developed (http://fbac.dmicrobe.cn/). The fIDBAC is aimed to provide a single, coherent, and automated workflow for species identification, strain typing, and downstream analysis, such as CDS prediction, drug resistance genes, virulence gene annotation, and phylogenetic analysis.