Identification of bacteria in potential mutualism with toxic Alexandrium catenella in Chilean Patagonian fjords by in vitro and field monitoring.

The dinoflagellate Alexandrium catenella is a well-known paralytic shellfish toxin producer that forms harmful algal blooms, repeatedly causing damage to Chilean coastal waters. The causes and behavior of algal blooms are complex and vary across different regions. As bacterial interactions with algal species are increasingly recognized as a key factor driving algal blooms, the present study identifies several bacterial candidates potentially associated with Chilean Alexandrium catenella. This research narrowed down the selection of bacteria from the Chilean A. catenella culture using antibiotic treatment and 16S rRNA metabarcoding analysis. Subsequently, seawater from two Chilean coastal stations, Isla Julia and Isla San Pedro, was monitored for two years to detect Alexandrium species and the selected bacteria, utilizing 16S and 18S rRNA gene metabarcoding analyses. The results suggested a potential association between Alexandrium species and Spongiibacteraceae at both stations. The proposed candidate bacteria within the Spongiibacteraceae family, potentially engaging in mutualistic relationships with Alexandrium species, included the genus of BD1-7 clade, Spongiibbacter, and Zhongshania.

Environmental evaluation of the Reloncaví estuary in southern Chile based on lipophilic shellfish toxins as related to harmful algal blooms.

The Reloncaví estuary in southern Chile is famous for its aquaculture. However, recurring harmful algal blooms have adversely affected mussel production. Therefore, regular monitoring of algal toxins is urgently needed to better understand the contamination status of the estuary. In this study, we quantified 15 types of lipophilic shellfish toxins in Metri Bay in the Reloncaví estuary on a biweekly basis for 4 years. We identified algal species using microscopy and metabarcoding analysis. We also measured water temperature, salinity, chlorophyll-a, and dissolved oxygen to determine the potential relationships of these parameters with algal toxin production. Our results revealed the presence of a trace amount of pectenotoxin and the causal phytoplankton Dinophysis, as well as yessotoxin and the causal phytoplankton Protoceratium. Statistical analysis indicated that fluctuations in water temperature affected the detection of these toxins. Additionally, metabarcoding analysis detected the highly toxic phytoplankton Alexandrium spp. in some samples. Although our results suggest that the level of lipophilic shellfish toxins in Metri Bay during the study period was insignificantly low using our current LC-MS method, the confirmed presence of highly toxic algae in Metri Bay raises concerns, given that favorable environmental conditions could cause blooms.

Editorial for Special Issue: "Airborne Microbes and Their Potential Influence".

Airborne microbes, comprising a diverse range of bacteria and fungi, are a pervasive component of the atmosphere, with concentrations typically ranging from 102 to 107 cells per cubic meter [...].

Correlation between bacterial microbiome and Legionella species in water from public bath facilities by 16S rRNA gene amplicon sequencing.

Public bath facilities are a major source of Legionella infections in Japan. In this study, we performed 16S rRNA gene amplicon sequencing to characterize the bacterial community in bath and shower water from public bath facilities, along with chemical parameters, and investigated the effect of the bacterial microbiome on the presence of Legionella species. Although no significant difference in bacterial community richness was observed between bath and shower water samples, there was a remarkable difference in the bacterial community structure between them. Distance-based redundancy analysis revealed that several factors (free residual chlorine, pH, and conductivity) were correlated with the bacterial community in bath water. The most abundant bacterial genera in the samples were Pseudomonas (13.7%) in bath water and Phreatobacter (13.6%) in shower water, as indicated by the taxonomic composition, and the dominant bacteria differed between these environmental samples. Legionella pneumophila was the most frequently detected Legionella species, with additional 15 other Legionella species detected in water samples. In Legionella-positive water samples, several unassigned and uncultured bacteria were enriched together. In addition, the co-occurrence network showed that Legionella was strongly interconnected with two uncultured bacteria. Corynebacterium and Sphingomonas negatively correlated with Legionella species. The present study reveals the ecology of Legionella species, especially their interactions with other bacteria that are poorly understood to date. IMPORTANCE: Public bath facilities are major sources of sporadic cases and outbreaks of Legionella infections. Recently, 16S rRNA gene amplicon sequencing has been used to analyze bacterial characteristics in various water samples from both artificial and natural environments, with a particular focus on Legionella bacterial species. However, the relationship between the bacterial community and Legionella species in the water from public bath facilities remains unclear. In terms of hygiene management, it is important to reduce the growth of Legionella species by disinfecting the water in public bath facilities. Our findings contribute to the establishment of appropriate hygiene management practices and provide a basis for understanding the potential health effects of using bath and shower water available in public bath facilities.

Resistomes in freshwater bioaerosols and their impact on drinking and recreational water safety: A perspective.

Antibiotic resistance genes (ARGs) are widespread environmental pollutants of biological origin that pose a significant threat to human, animal, and plant health, as well as to ecosystems. ARGs are found in soil, water, air, and waste, and several pathways for global dissemination in the environment have been described. However, studies on airborne ARG transport through atmospheric particles are limited. The ARGs in microorganisms inhabiting an environment are referred to as the "resistome". A global search was conducted of air-resistome studies by retrieving bioaerosol ARG-related papers published in the last 30 years from PubMed. We found that there is no dedicated methodology for isolating ARGs in bioaerosols; instead, conventional methods for microbial culture and metagenomic analysis are used in combination with standard aerosol sampling techniques. There is a dearth of information on the bioaerosol resistomes of freshwater environments and their impact on freshwater sources used for drinking and recreational activities. More studies of aerobiome freshwater environments are needed to ensure the safe use of water and sanitation. In this review we outline and synthesize the few studies that address the freshwater air microbiome (from tap water, bathroom showers, rivers, lakes, and swimming pools) and their resistomes, as well as the likely impacts on drinking and recreational waters. We also discuss current knowledge gaps for the freshwater airborne resistome. This review will stimulate new investigations of the atmospheric microbiome, particularly in areas where both air and water quality are of public health concern.

Potential airborne human pathogens: A relevant inhabitant in built environments but not considered in indoor air quality standards.

Potential airborne human pathogens (PAHPs) may be a relevant component of the air microbiome in built environments. Despite that PAHPs can cause infections, particularly in immunosuppressed patients at medical centers, they are scarcely considered in standards of indoor air quality (IAQ) worldwide. Here, we reviewed the current information on microbial aerosols (bacteria, fungal and viruses) and PAHPs in different types of built environments (e.g., medical center, industrial and non-industrial), including the main factors involved in their dispersion, the methodologies used in their study and their associated biological risks. Our analysis identified the human occupancy and ventilation systems as the primary sources of dispersal of microbial aerosols indoors. We also observed temperature and relative humidity as relevant physicochemical factors regulating the dispersion and viability of some PAHPs. Our analysis revealed that some PAHPs can survive and coexist in different environments while other PAHPs are limited or specific for an environment. In relation to the methodologies (conventional or molecular) the nature of PAHPs and sampling type are pivotal. In this context, indoors air-borne viruses are the less studies because their small size, environmental lability, and absence of efficient sampling techniques and universal molecular markers for their study. Finally, it is noteworthy that PAHPs are not commonly considered and included in IAQ standards worldwide, and when they are included, the total abundance is the single parameter considered and biological risks is excluded. Therefore, we propose a revision, design and establishment of public health policies, regulations and IAQ standards, considering the interactions of diverse factors, such as nature of PAHPs, human occupancy and type of built environments where they develop.

Monitoring bacterial composition and assemblage in the Gulf of Corcovado, southern Chile: Bacteria associated with harmful algae.

Harmful Algal Blooms (HABs) have caused damage to the marine environment in Isla San Pedro in the Gulf of Corcovado, Chile. While rising water temperature and artificial eutrophication are the most discussed topics as a cause, marine bacteria is a recent attractive parameter as an algal bloom driver. This study monitored algal and bacterial compositions in the water of Isla San Pedro for one year using microscopy and 16S rRNA metabarcoding analysis, along with physicochemical parameters. The collected data were analyzed with various statistical tools to understand how the particle-associated bacteria (PA) and the free-living (FL) bacteria were possibly involved in algal blooms. Both FL and PA fractions maintained a stable bacterial composition: the FL fraction was dominated by Proteobacteria (α-Proteobacteria and γ-Proteobacteria), and Cyanobacteria dominated the PA fraction. The two fractions contained equivalent bacterial taxonomic richness (c.a. 8,000 Operational Taxonomic Units) and shared more than 50% of OTU; however, roughly 20% was exclusive to each fraction. The four most abundant algal genera in the Isla San Pedro water were Thalassiosira, Skeletonema, Chaetoceros, and Pseudo-nitzchia. Statistical analysis identified that the bacterial species Polycyclovorans algicola was correlated with Pseudo-nitzschia spp., and our monitoring data recorded a sudden increase of particle-associated Polycyclovorans algicola shortly after the increase of Pseudo-nitzschia, suggesting that P. algicola may have regression effect on Pseudo-nitzschia spp. The study also investigated the physicochemical parameter effect on algal-bacterial interactions. Oxygen concentration and chlorophyll-a showed a strong correlation with both FL and PA bacteria despite their assemblage differences, suggesting that the two groups had different mechanisms for interacting with algal species.

Perspectives on Sampling and New Generation Sequencing Methods for Low-Biomass Bioaerosols in Atmospheric Environments.

Bioaerosols play essential roles in the atmospheric environment and can affect human health. With a few exceptions (e.g., farm or rainforest environments), bioaerosol samples from wide-ranging environments typically have a low biomass, including bioaerosols from indoor environments (e.g., residential homes, offices, or hospitals), outdoor environments (e.g., urban or rural air). Some specialized environments (e.g., clean rooms, the Earth's upper atmosphere, or the international space station) have an ultra-low-biomass. This review discusses the primary sources of bioaerosols and influencing factors, the recent advances in air sampling techniques and the new generation sequencing (NGS) methods used for the characterization of low-biomass bioaerosol communities, and challenges in terms of the bias introduced by different air samplers when samples are subjected to NGS analysis with a focus on ultra-low biomass. High-volume filter-based or liquid-based air samplers compatible with NGS analysis are required to improve the bioaerosol detection limits for microorganisms. A thorough understanding of the performance and outcomes of bioaerosol sampling using NGS methods and a robust protocol for aerosol sample treatment for NGS analysis are needed. Advances in NGS techniques and bioinformatic tools will contribute toward the precise high-throughput identification of the taxonomic profiles of bioaerosol communities and the determination of their functional and ecological attributes in the atmospheric environment. In particular, long-read amplicon sequencing, viability PCR, and meta-transcriptomics are promising techniques for discriminating and detecting pathogenic microorganisms that may be active and infectious in bioaerosols and, therefore, pose a threat to human health. Supplementary Information: The online version contains supplementary material available at 10.1007/s41745-023-00380-x.

Unique genomic sequences in a novel Mycobacterium avium subsp. hominissuis lineage enable fine scale transmission route tracing during pig movement.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most prevalent mycobacteria causing non-tuberculous mycobacterial disease in humans and animals. Of note, MAH is a major cause of mycobacterial granulomatous mesenteric lymphadenitis outbreaks in pig populations. To determine the precise source of infection of MAH in a pig farm and to clarify the epidemiological relationship among pig, human and environmental MAH lineages, we collected 50 MAH isolates from pigs reared in Japan and determined draft genome sequences of 30 isolates. A variable number of tandem repeat analysis revealed that most pig MAH isolates in Japan were closely related to North American, European and Russian human isolates but not to those from East Asian human and their residential environments. Historical recombination analysis revealed that most pig isolates could be classified into SC2/4 and SC3, which contain MAH isolated from pig, European human and environmental isolates. Half of the isolates in SC2/4 had many recombination events with MAH lineages isolated from humans in East Asia. To our surprise, four isolates belonged to a new lineage (SC5) in the global MAH population. Members of SC5 had few footprints of inter-lineage recombination in the genome, and carried 80 unique genes, most of which were located on lineage specific-genomic islands. Using unique genetic features, we were able to trace the putative transmission route via their host pigs. Together, we clarify the possibility of species-specificity of MAH in addition to local adaptation. Our results highlight two transmission routes of MAH, one exposure on pig farms from the environment and the other via pig movement. Moreover, our study also warns that the evolution of MAH in pigs is influenced by MAH from patients and their residential environments, even if the MAH are genetically distinct.

DSF inactivator RpfB homologous FadD upregulated in Bradyrhizobium japonicum under iron limiting conditions.

Phytopathogenic bacteria Xanthomonas campestris pv. campestris (Xcc) causes black rot and other plant diseases. Xcc senses diffusible signal factor (DSF) as a quorum-sensing (QS) signal that mediates mainly iron uptake and virulence. RpfB deactivates DSF in this DSF-QS circuit. We examined differential gene expression profiles of Bradyrhizobium japonicum under low versus high iron conditions and found that fadD and irr were upregulated under low iron (log2 fold change 0.825 and 1.716, respectively). In addition to having similar protein folding patterns and functional domain similarities, FadD shared 58% sequence similarity with RpfB of Xcc. The RpfB-DSF and FadD-DSF complexes had SWISSDock molecular docking scores of - 8.88 kcal/mol and - 9.85 kcal/mol, respectively, and the 100 ns molecular dynamics simulation results were in accord with the docking results. However, significant differences were found between the binding energies of FadD-DSF and RpfB-DSF, indicating possible FadD-dependent DSF turnover. The protein-protein interaction network showed that FadD connected indirectly with ABC transporter permease (ABCtp), which was also upregulated (log2 fold change 5.485). We speculate that the low iron condition may be a mimetic environmental stimulus for fadD upregulation in B. japonicum to deactivate DSF, inhibit iron uptake and virulence of DSF-producing neighbors. This finding provides a new option of using B. japonicum or a genetically improved B. japonicum as a potential biocontrol agent against Xcc, with the added benefit of plant growth-promoting properties.

Learning from mistakes: challenges in finding holobiont factors from environmental samples and the importance of methodological consistency.

The cause of harmful algal blooms has been a mystery, but research to elucidate its mechanism has progressed over the years thanks to genetic technologies. We have monitored toxic algae and its associated bacteria as a community, the so-called 'holobiont' in Chilean coastal waters for years from the perspective of bacteria as an algal bloom driver. This review describes the challenges of holobiont monitoring, specifically with respect to standardizing and compliance with the monitoring protocols to collect reliable and sustainable data. Further, we suggest adopting the high-throughput sequencing (HTS) standard operating procedure (SOP) by the International Human Microbiome to improve the quality and consistency of holobiont monitoring in the harmful algal world.

Whole-Genome and Plasmid Comparative Analysis of Campylobacter jejuni from Human Patients in Toyama, Japan, from 2015 to 2019.

Campylobacter jejuni is a major causative agent of food poisoning, and increasing antimicrobial resistance is a concern. This study investigated 116 clinical isolates of C. jejuni from Toyama, Japan, which were isolated from 2015 to 2019. Antimicrobial susceptibility testing and whole-genome sequencing were used for phenotypic and genotypic characterization to compare antimicrobial resistance (AMR) profiles and phylogenic linkage. The multilocus sequence typing approach identified 37 sequence types (STs) and 15 clonal complexes (CCs), including 7 novel STs, and the high frequency CCs were CC21 (27.7%), CC48 (10.9%), and CC354 (9.9%). The AMR profiles and related resistant factors were as follows: fluoroquinolones (51.7%), mutation in quinolone resistance-determining region (QRDRs) (GyrA T86I); tetracyclines (27.6%), acquisition of tet(O); ampicillin (7.8%), harboring blaOXA184 or a promoter mutation in blaOXA193; aminoglycosides (1.7%), acquisition of ant(6)-Ia and aph(3')-III; chloramphenicol (0.9%), acquisition of cat. The acquired resistance genes tet(O), ant(6)-Ia, aph(3')-III, and cat were located on pTet family plasmids. Furthermore, three pTet family plasmids formed larger plasmids that incorporated additional genes such as the type IV secretion system. Sequence type 4526 (ST4526; 10.9%), which is reported only in Japan, was the most predominant, suggesting continued prevalence. This study reveals the sequences of the pTet family plasmids harbored by C. jejuni in Japan, which had been unclear, and the acquisition of the insertion sequences in a part of the pTet family plasmids. Because pTet family plasmids can be horizontally transmitted and are a major factor in acquired resistance in Campylobacter, the risk of spreading pTet that has acquired further resistance should be considered. IMPORTANCE Campylobacter jejuni is among the major causes of enteritis and diarrhea in humans in many countries. Drug-resistant Campylobacter is increasing in both developing and developed countries, and in particular, fluoroquinolone-resistant Campylobacter was one of the species included on the priority list of antibiotic-resistant bacteria. Campylobacter drug resistance surveillance is important and has been conducted worldwide. In this study, we performed whole-genome analysis of Campylobacter jejuni isolated from diarrhea patients at a hospital in Toyama, Japan. This revealed the continued prevalence of Campylobacter jejuni ST4526, which has been reported to be prevalent in Japan, and the acquisition of resistance and virulence factors in the pTet family plasmids. The diversity of pTet family plasmids, the major resistance transmission factor, is expected to potentially increase the risk of Campylobacter. The usefulness of whole-genome sequencing in Campylobacter surveillance was also demonstrated.

Zooplankton act as cruise ships promoting the survival and pathogenicity of pathogenic bacteria.

Bacteria in general interact with zooplankton in aquatic ecosystems. These zooplankton-bacterial interactions help to shape the bacterial community by regulating bacterial abundances. Such interactions are even more significant and crucially in need of investigation in the case of pathogenic bacteria, which cause severe diseases in humans and animals. Among the many associations between a host metazoan and pathogenic bacteria, zooplankton provide nutrition and protection from stressful conditions, promote the horizontal transfer of virulence genes, and act as a mode of pathogen transport. These interactions allow the pathogen to survive and proliferate in aquatic environments and to endure water treatment processes, thereby creating a potential risk to human health. This review highlights current knowledge on the contributions of zooplankton to the survival and pathogenicity of pathogenic bacteria. We also discuss the need to consider these interactions as a risk factor in water treatment processes.

Protocol of living cell separation using the microfluidic dielectrophoresis integrated chip.

This protocol demonstrates the separation of living cells with the microfluidic dielectrophoresis chip, using the Jurkat cell as a model. The successful living cell separation lies in familiarity with the detailed tips, which are aided by this stepwise protocol. The knowledge of correct chip installation, sample and buffer filling, flow rate and cell concentration adjustments, and contamination sources increases the efficiency of target viable cell collection. Such instructions, although trivial, are critical for achieving cell separation. For complete details on the use and execution of this protocol, please refer to Oshiro et al. (2022).

Direct Attachment with Erythrocytes Augments Extracellular Growth of Pathogenic Mycobacteria.

Pathogenic intracellular mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium avium, which cause lung diseases, can grow in macrophages. Extracellular mycobacteria have been reported in the lungs, blood, and sputum of patients, indicating the involvement of these pathogens in disease progression. Erythrocytes are involved in the symptoms associated with pulmonary mycobacterial diseases, such as bloody sputum and hemoptysis; however, little attention has been paid to the role of erythrocytes in mycobacterial diseases. Herein, we found that Mycobacterium avium subsp. hominissuis (MAH) and Mycobacterium intracellulare colocalized with erythrocytes at the sites of lung infection, inside capillaries and necrotic areas of granulomas, using histopathological examinations. Electron microscopy showed that MAH adhered and entered human erythrocytes when they were cocultured in vitro. MAH adhered to erythrocytes through complement receptor 1 and cell-surface sialo-glycoproteins. Importantly, MAH grew vigorously without causing any pronounced damage to erythrocytes. This erythrocyte-mediated enhancement of MAH growth occurred extracellularly depending on its direct attachment to erythrocytes. In contrast, MAH failed to multiply inside erythrocytes. Similarly, erythrocytes augmented the growth of other pathogenic mycobacteria, such as M. intracellulare and M. tuberculosis. THP-1 cell-derived human macrophages preferentially phagocytosed erythrocytes that were attached to mycobacteria (compared to bacteria alone), suggesting that erythrocyte-attached mycobacteria are an efficient infectious source for macrophages. Our findings provide new insights into the pathogenesis of mycobacterial diseases and offer an alternative and useful strategy for treating mycobacterial disease. IMPORTANCE Pathogenic mycobacteria, such as Mycobacterium tuberculosis, Mycobacterium avium subsp. hominissuis (MAH), and Mycobacterium intracellulare, cause pulmonary infections as intracellular parasites of lung macrophages and epithelial cells. Here, using histopathological examinations we found that MAH and M. intracellulare colocalized with erythrocytes in lung infection sites. Subsequent studies demonstrated that direct interaction with erythrocytes enhances the extracellular proliferation of mycobacteria based on the following results: 1. MAH adhered and invaded human erythrocytes upon coculture in vitro; 2. MAH adhered to erythrocytes through complement receptor 1 and cell-surface sialo-glycoproteins; 3. MAH rapidly proliferated when directly attached to erythrocytes but not within them; 4. other mycobacteria, such as M. intracellulare and M. tuberculosis, also proliferated in the same way as MAH. The finding that pathogenic mycobacteria grow extracellularly in an erythrocyte-dependent manner is of considerable clinical importance for understanding disease progression and latent infection.

Fabrication of a new all-in-one microfluidic dielectrophoresis integrated chip and living cell separation.

Microfluidic dielectrophoresis (DEP) technology has been applied to many devices to perform label-free target cell separation. Cells separated by these devices are used in laboratories, mainly for medical research. The present study designed a microfluidic DEP device to fabricate a rapid and semiautomated cell separation system in conjunction with microscopy to enumerate the separated cells. With this device, we efficiently segregated bacterial cells from liquid products and enriched one cell type from two mixed eukaryotic cell types. The device eliminated sample pretreatment and established cell separation by all-in-one operation in a lab-on-chip, requiring only a small sample volume (0.5-1 mL) to enumerate the target cells and completing the entire separation process within 30 min. Such a rapid cell separation technique is in high demand by many researchers to promptly characterize the target cells.

The effect of iron on Chilean Alexandrium catenella growth and paralytic shellfish toxin production as related to algal blooms.

The dinoflagellate Alexandrium catenella is a well-known paralytic shellfish toxin producer that forms harmful algal blooms (HABs) worldwide. Blooms of this species have repeatedly brought severe ecological and economic impacts to Chile, especially in the southern region, where the shellfish and salmon industries are world-famous. The mechanisms of such HABs have been intensively studied but are still unclear. Nutrient overloading is one of the often-discussed drivers for HABs. The present study used the A. catenella strain isolated from southern Chile to investigate how iron conditions could affect their growth and toxin production as related to HAB. Our results showed that an optimum concentration of iron was pivotal for proper A. catenella growth. Thus, while excess iron exerted a toxic effect, low iron media led to iron insufficiency and growth inhibition. In addition, the study shows that the degree of paralytic shellfish toxin production by A. catenella varied depending on the iron concentration in the culture media. The A. catenella strain from southern Chile produced GTX1-4 exclusively in the fmol cell-1 scale. Based on these findings, we suggest that including iron and paralytic shellfish toxin measurements in the fields can improve the current HAB monitoring and contribute to an understanding of A. catenella bloom dynamics in Chile.

A Standardized Procedure for Monitoring Harmful Algal Blooms in Chile by Metabarcoding Analysis.

Harmful algae blooms (HABs) monitoring has been implemented worldwide, and Chile, a country famous for its fisheries and aquaculture, has intensively used microscopic and toxin analyses for decades for this purpose. Molecular biological methods, such as high-throughput DNA sequencing and bacterial assemblage-based approaches, are just beginning to be introduced in Chilean HAB monitoring, and the procedures have not yet been standardized. Here, 16S rRNA and 18S rRNA metabarcoding analyses for monitoring Chilean HABs are introduced stepwise. According to a recent hypothesis, algal-bacterial mutualistic association plays a critical synergetic or antagonistic relationship accounting for bloom initiation, maintenance, and regression. Thus, monitoring HAB from algal-bacterial perspectives may provide a broader understanding of HAB mechanisms and the basis for early warning. Metabarcoding analysis is one of the best suited molecular-based tools for this purpose because it can detect massive algal-bacterial taxonomic information in a sample. The visual procedures of sampling to metabarcoding analysis herein provide specific instructions, aiming to reduce errors and collection of reliable data.

CRISPR loci-PCR as Tool for Tracking Azospirillum sp. Strain B510.

Azospirillum-based plant and soil inoculants are widely used in agriculture. The inoculated Azospirillum strains are commonly tracked by both culture-dependent and culture-independent methods, which are time-consuming or expensive. In this context, clustered regularly interspaced short palindromic repeats (CRISPR) loci structure is unique in the bacterial genome, including some Azospirillum species. Here, we investigated the use of CRISPR loci to track specific Azospirillum strains in soils systems by PCR. Primer sets for Azospirillum sp. strain B510 were designed and evaluated by colony and endpoint PCR. The CRISPRloci-PCR approach was standardized for Azospirillum sp. strain B510, and its specificity was observed by testing against 9 different Azospirillum strains, and 38 strains of diverse bacterial genera isolated from wheat plants. The CRISPRloci-PCR approach was validated in assays with substrate and wheat seedlings. Azospirillum sp. strain B510 was detected after of two weeks of inoculation in both sterile and nonsterile substrates as well as rhizosphere grown in sterile substrate. The CRISPRloci-PCR approach was found to be a useful molecular tool for specific tracking of Azospirillum at the strain level. This technique can be easily adapted to other microbial inoculants carrying CRISPR loci and can be used to complement other microbiological techniques.