Exploratory Metataxonomic Characterization of Bacterial Communities in Street-Vended Raw Milk from Ankara,Türkiye,Using 16S rRNA Gene Amplicon Sequencing

Abstract

Street-vended raw milk may harbor diverse bacterial communities influenced by environmental exposure, handling practices, and storage conditions. This exploratory pilot study aimed to characterize the bacterial community composition of raw milk obtained from three independent street vendors in Ankara, Türkiye, using 16S rRNA gene amplicon sequencing–based metataxonomic analysis. Following DNA extraction, the V3–V4 region of the bacterial 16S rRNA gene was sequenced, and sequence data were processed using the QIIME2 pipeline with taxonomic assignment against the SILVA 138 reference database. Sequencing generated 158,959, 160,946, and 313,696 high-quality reads for samples SS1, SS2, and SS3, respectively. The bacterial communities were predominantly composed of members of the phyla Pseudomonadota and Bacillota. At the genus level, Pseudomonas (49.75%), Acinetobacter (20.64%), Lactococcus (35.31%), Aerococcus (8.81%), and Enterococcus (7.10%) were among the predominant taxa identified across the samples. Principal coordinates analysis indicated variation in bacterial community composition among the three samples, with SS2 and SS3 exhibiting greater similarity than SS1. Overall, this exploratory pilot study provides baseline metataxonomic information on the bacterial communities associated with street-vended raw milk obtained from informal vendors in Ankara. Given the limited sample size, the findings should be regarded as preliminary descriptive observations that may support future investigations involving larger sample sizes, broader geographical coverage, and complementary functional analyses.

Keywords

16S rRNA sequencing raw milk metataxonomics bacterial community microbial diversity street-vended milk

Introduction

Raw milk is a biologically active food of animal origin that provides valuable nutrients such as proteins, essential fatty acids, vitamins, and minerals, but it can also act as a vehicle for microbial contamination within the food chain (FAO, 2023). Despite its nutritional value, raw milk may harbor foodborne pathogens and therefore represents a potential public health concern (FDA, 2023; Ntuli et al., 2023). Owing to its high nutrient content, near-neutral pH, and high water activity, raw milk creates favorable conditions for the growth of diverse microbial communities. These communities may include both beneficial microorganisms and spoilage-related bacteria. While some microorganisms contribute to natural fermentation processes, others produce extracellular enzymes such as lipases and proteases that may lead to quality deterioration during storage and processing (Glantz et al., 2020; Finton et al., 2024). With the growing demand for dairy products, maintaining hygienic conditions during raw milk production has become an important issue for the dairy industry worldwide (Yuan et al., 2022). The microbiological quality of raw milk directly influences both the safety and technological characteristics of dairy products (Naing et al., 2019; Böhnlein et al., 2021; Ntuli et al., 2023). Several factors, including animal health status, feeding practices, milking hygiene, environmental conditions, stage of lactation, and storage temperature, can affect the microbial composition of raw milk (Ahmed and Hassan, 2024; Li et al., 2024; Yap et al., 2024). Although refrigeration is commonly used to store raw milk, it does not fully prevent the growth of psychrotrophic bacteria (Ahmed and Hassan, 2024). Previous studies have reported that genera such as Acinetobacter, Pseudomonas, Aeromonas, Enterobacter, Bacillus, and Klebsiella may be present in raw milk (Griep-Moyer et al., 2022; Yuan et al., 2022). These microorganisms can produce heat-stable enzymes that may cause quality defects in milk and dairy products. In addition, disturbances in the natural microbial composition of raw milk may increase the risk of foodborne infections (Hanzelová et al., 2024).

Traditional culture-based microbiological methods provide valuable information, but they are limited in their ability to detect the full range of microorganisms present in raw milk. For this reason, culture-independent approaches based on DNA analysis have become increasingly important in recent years. Metagenomic techniques allow the detection of unculturable microorganisms and provide a broader understanding of microbial diversity in raw milk samples (Wang et al., 2021; Billington et al., 2022; Rubiola et al., 2022; Santamarina-García et al., 2024; Adje et al., 2025). By analyzing microbial DNA directly from samples, metataxonomic analysis offers a more comprehensive view of microbial community structure (Zhang et al., 2022). Although 16S rRNA gene amplicon sequencing has been widely applied to characterize the microbiota of raw milk, microbiome-based information on street-vended raw milk marketed by informal vendors remains scarce. Therefore, the present study was designed as an exploratory pilot investigation to characterize the bacterial communities associated with street-vended raw milk obtained from three independent vendors in Ankara, Türkiye, using 16S rRNA gene amplicon sequencing. Rather than providing population-level inferences, this study aims to generate baseline metataxonomic information and preliminary descriptive data that may support future investigations involving larger sample sizes, longitudinal sampling, and complementary functional analyses.

Sample collection

Three raw milk samples were purchased from three independent street vendors operating in different residential districts of Ankara, Türkiye, and coded as SS1, SS2, and SS3. All samples were collected during a single sampling period, with one sample obtained from each vendor. Vendors were selected to represent common informal raw milk marketing practices in the region, where hygienic control and cold-chain management may be limited. The vendors differed in their selling environment, storage practices, and container types, thereby providing an initial representation of variability among informal street-vended raw milk sources. Following collection, all samples were transported to the laboratory under refrigerated conditions (+4°C) and processed immediately. For each sample, 250 mL of raw milk was used for DNA extraction and subsequent 16S rRNA gene amplicon sequencing. Because this work was designed as an exploratory pilot study, biological and technical replicates were not included. Likewise, temporal and seasonal variation were beyond the scope of the present investigation. These limitations have been explicitly acknowledged throughout the article, and the findings are presented as preliminary descriptive observations rather than population-level inferences.

DNA extraction, 16S rRNA gene amplification, library preparation, sequencing, and bioinformatic analysis

Total genomic DNA was extracted from 250 mL of each raw milk sample using the Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo Research, Irvine, CA) according to the manufacturer’s instructions. DNA concentration and purity were assessed using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

The bacterial community was characterized by amplification of the V3–V4 hypervariable region of the 16S rRNA gene using the universal primer pair 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′), generating an approximately 460-bp amplicon of the bacterial 16S rRNA gene. PCR amplification was performed in a total reaction volume of 25 μL containing 2.5 μL microbial DNA (5 ng/μL), 5 μL forward primer (1 μM), 5 μL reverse primer (1 μM), and 12.5 μL 2× KAPA HotStart PCR Mix. Thermal cycling consisted of an initial denaturation at 95°C for 3 min, followed by 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 5 min. PCR products were purified using AMPure XP magnetic beads (Beckman Coulter, Brea, CA). A subsequent index PCR was performed to incorporate dual indices and Illumina sequencing adapters using the Nextera XT Index Kit (Illumina Inc., San Diego, CA), followed by a second purification with AMPure XP magnetic beads. Libraries were quantified by real-time PCR, normalized, pooled at equimolar concentrations, and sequenced on an Illumina NovaSeq 6000 platform using 2 × 250 bp paired-end Sequencing-by-Synthesis chemistry.

Raw sequencing reads were initially evaluated using FastQC to assess sequence quality, and sequences were demultiplexed prior to downstream bioinformatic analyses. Primer and barcode sequences were trimmed, and reads with a Phred quality score below 20 were excluded during quality filtering. Bioinformatic analyses were performed using QIIME2 version 2021.11, in which the DADA2 plugin was used for quality filtering, denoising, chimera removal, and amplicon sequence variant (ASV) inference. Prior to denoising, forward and reverse reads were trimmed by 18 bp and 19 bp, respectively. Taxonomic assignment was performed against the SILVA release 138 reference database using a pretrained Naïve Bayes classifier. To standardize sequencing depth across samples, all datasets were rarefied to 2000 reads per sample before downstream diversity analyses.

Alpha diversity was assessed using the Shannon and Simpson diversity indices, whereas beta diversity was evaluated using weighted and unweighted UniFrac distance metrics. Rarefaction curves were generated to assess sequencing depth and sampling adequacy. For visualization, taxa with relative abundances below 2% were grouped as “other” (Bolyen et al., 2019).

Because this study was designed as an exploratory pilot investigation, extraction blanks, PCR negative controls, and mock community controls were not included. Consequently, the potential contribution of reagent- or laboratory-derived background contamination could not be formally evaluated. Therefore, low-abundance taxa were interpreted cautiously, and the findings are presented primarily as descriptive observations rather than population-level inferences.

Statistical analyses

Due to the exploratory design of the study and the limited sample size (n = 3), inferential statistical analyses were not performed. Microbial community data were evaluated descriptively based on relative abundance and diversity indices. Alpha diversity metrics (Shannon and Simpson) were calculated to assess within-sample diversity, and rarefaction analysis was applied to evaluate sequencing depth and sampling adequacy. Observed differences among samples were interpreted qualitatively in relation to environmental and handling variability.

Results

This exploratory pilot study was conducted to characterize the bacterial communities present in raw milk samples collected from three informal street vendors in Ankara, Türkiye, using 16S rRNA gene–based metataxonomic analysis. Due to the limited number of samples, differences among microbial profiles were interpreted descriptively rather than statistically tested. Sequencing produced 158,959, 160,946, and 313,696 reads for SS1, SS2, and SS3, respectively. Taxonomic assignment using the SILVA 138 database resulted in the successful classification of many high-quality reads across all samples. ASVs were identified through high-throughput 16S rRNA gene sequencing. Shannon and Simpson diversity indices were calculated based on the relative abundance and evenness of ASVs.

Alpha diversity

Alpha diversity was evaluated using the Shannon and Simpson diversity indices based on rarefied sequence data (Fig. 1a and b). Both indices consistently showed that SS3 exhibited the highest bacterial diversity, followed by SS2 and SS1. In both rarefaction analyses, the curves approached a plateau as sequencing depth increased, indicating that the selected rarefaction depth of 2000 reads per sample provided sufficient sequencing coverage for downstream diversity analyses. Given the exploratory nature of the study and the limited sample size (n = 3), these findings are presented descriptively without inferential statistical comparisons.

Taxonomic diversity of bacterial communities (16S)

The bacterial composition of the raw milk samples was mainly represented by the phyla Pseudomonadota, Bacillota, Bacteroidota, and Actinomycetota. However, it was determined that the phyla Pseudomonadota and Bacillota were the dominant bacterial phyla. Of these dominant phyla, Pseudomonadota was found in SS1, SS2, and SS3 at 85.29%, 39.80%, and 30.33%, respectively, while the other, Bacillota, was found in SS1, SS2, and SS3 samples at 12.17%, 38.32%, and 42.04%, respectively (Fig. 2).

Genus-Level Microbial Composition

A wide diversity was observed in the raw milk samples at the genus level (Fig. 3a–c). In SS1 sample, the most dominant genus was Pseudomonas (49.75%), followed by Acinetobacter (16.28%), Shewanella (12.25%), Lactococcus (11.24%), and Paraburkholderia (4.71%) (Fig. 3a). In SS2 sample, the most dominant genus was Lactococcus (35.31%). This was followed by Acinetobacter (20.64%), Flavobacterium (10.71%), Kaistella (5.51%), Moraxella (5.15%), Epilithonimonas (3.81%), Pseudomonas (2.25%), and Aeromonas (2.10%) (Fig. 3b). In SS3 sample, the most dominant genus was Aerococcus (8.81%). This was followed by Enterococcus (7.10%), Tenebrionicola (5.39%), Ruoffia (3.49%), Corynebacterium (3.40%), Epilithonimonas (3.25%), Acinetobacter (3.04%), and Tenebrionibacter (2.63%) (Fig. 3c). Overall, the microbial profiles showed clear variation among the three raw milk samples (Fig. 3a–c). Rarefaction analysis based on observed ASVs showed that sequencing depth was sufficient to capture the majority of bacterial diversity in all three samples, with SS3 consistently exhibiting the highest observed ASV richness, followed by SS2 and SS1 (Fig. 4).

Principal Coordinates Analysis

Principal coordinates analysis (PCoA) based on beta-diversity metrics indicated differences in bacterial community composition among the three street-vended raw milk samples (Fig. 5). The first principal coordinate (PC1) explained 65.23% of the total variation, whereas the second principal coordinate (PC2) accounted for the remaining 34.77%. The ordination plot showed that SS2 and SS3 were positioned closer to each other than to SS1, suggesting greater similarity in their bacterial community composition. Given the exploratory design of the present study and the limited sample size (n = 3), these observations should be regarded as descriptive and interpreted with appropriate caution.

Discussion

Pseudomonas was detected in SS1 (49.75%) and SS2 (2.25%) samples and constituted the dominant bacterial genus in SS1 raw milk sample. Pseudomonas may originate from various environmental sources such as farm silage, cow udders, and equipment surfaces (Badawy et al., 2023). Due to the high nutrient content, water activity, and near-neutral pH of milk, this environment may provide favorable conditions for the growth of Pseudomonas (Oikonomou et al., 2020; Narvhus et al., 2021; Ryu et al., 2021; Hassan et al., 2024). However, the occurrence and relative abundance of Pseudomonas in street-vended raw milk may also reflect environmental exposure and handling-associated microbial variability. Members of this genus are known to produce heat-stable proteases and lipases that may contribute to spoilage in raw milk and dairy products (Chang et al., 2024).

Lactococcus, an important genus in the dairy industry, was present in 11.24% of SS1 and 35.31% of SS2. Members of the genus Lactococcus are Gram-positive cocci typically occurring in pairs or chains and are widely used as starter cultures in dairy fermentation, particularly L. lactis. Through the production of lactic acid and bacteriocins such as nisin, these bacteria contribute to microbial stability and shelf life of dairy products (Teshome et al., 2022; Elsaadany et al., 2024). Previous studies have also reported the presence of Lactococcus in raw milk (Fusco et al., 2020; Kondrotiene et al., 2020). However, under conditions such as cold-chain disruption or storage at room temperature, lactose fermentation by Lactococcus may lead to acidification, potentially affecting flavor and quality of milk (Basar and Heperkan, 2021; Luo et al., 2024). Therefore, the detection of Lactococcus in raw milk should not necessarily be interpreted solely as a positive indicator.

Acinetobacter was detected in SS1, SS2, and SS3 at relative abundances of 16.28%, 20.64%, and 3.04%, respectively. Members of this Gram-negative genus are widely distributed in environmental sources and have also been isolated from raw milk and dairy products (Cao et al., 2018; Guo et al., 2021). A previous study has suggested that Acinetobacter in milk may originate from multiple environmental and dairy-associated sources, including milking environments and water systems (Malta et al., 2020). Members of this genus are capable of producing heat-stable enzymes such as lipases and proteases that contribute to spoilage in milk and dairy products (Hoque et al., 2019). In addition, some Acinetobacter species have been described as opportunistic pathogens in the literature (Du et al., 2020; Chen et al., 2024). However, interpretations regarding potential health implications should be made cautiously, as genus-level 16S rRNA gene data do not provide species- or strain-level resolution.

Interestingly, Shewanella was identified only in sample SS1 (12.25%) and could not be detected in the other raw milk samples. According to Vaz-Moreira et al. (2017), Shewanella has been associated with environmental and aquatic habitats. The presence of Shewanella in SS1 may reflect environmental variation among samples; however, the specific source of occurrence cannot be inferred from genus-level metataxonomic data alone.

Flavobacterium (Bacteroidota phylum, Flavobacteriaceae family) was detected only in sample SS2 (10.71%) and was not observed in the other raw milk samples. Members of this genus are Gram-negative, aerobic bacteria widely distributed in aquatic and environmental habitats (Kämpfer et al., 2020). Previous studies have also reported Flavobacterium among psychrotrophic bacteria associated with raw milk spoilage (Mikulec et al., 2024).

Aerococcus was detected only in SS3 (8.81%) and was not observed in the other raw milk samples. Members of this genus have been reported in environmental sources such as soil, air, and the microbiota of mammals (Yabes et al., 2018). Some species, particularly A. viridans, have also been reported in dairy-associated environments (Murugesan et al., 2018). Similar to Shewanella (detected only in SS1), the occurrence of Aerococcus exclusively in SS3 may reflect sample-to-sample microbial variability potentially influenced by environmental conditions. However, the specific source of occurrence cannot be inferred from genus-level metataxonomic data alone.

Enterococcus, one of the lactic acid bacteria genera, was detected only in SS3 (7.10%) and was not observed in the other samples. Members of this Gram-positive genus are commonly found in environmental sources and foods of animal origin (Nasiri and Hanifian, 2022; Sakoui et al., 2022; Abarquero et al., 2024). However, the occurrence and origin of Enterococcus in raw milk remain controversial and may be influenced by multiple environmental and dairy-associated factors (Oikonomou et al., 2020). Although some Enterococcus species have been investigated for potential probiotic properties (Kanak et al., 2022; Badr et al., 2024), others have raised safety concerns and are not included in the Qualified Presumption of Safety list (Terzić-Vidojević et al., 2021; Dapkevicius et al., 2021). Therefore, the detection of Enterococcus in the present study should be interpreted cautiously, as genus-level 16S rRNA gene amplicon sequencing does not allow reliable inference regarding contamination routes, hygienic conditions, or strain-level characteristics.

Epilithonimonas, commonly found in natural environments such as soil and freshwater ecosystems, was present in 3.81% of SS2 and 3.25% of SS3, whereas it was not detected in SS1. This genus has also been reported in raw cow and camel milk (Sun et al., 2024).

However, the findings of the present study should be interpreted within the context of its exploratory pilot design, limited sample size, and restricted geographical coverage. Although 16S rRNA gene amplicon sequencing provides a robust and widely accepted approach for characterizing bacterial community composition, its taxonomic resolution is generally limited to the genus level and does not permit reliable species- or strain-level identification or direct functional characterization. Consequently, the present study was not designed to evaluate foodborne pathogenic species, antimicrobial resistance determinants, microbial spoilage potential, contamination sources, or other functional characteristics of the detected bacterial communities. Furthermore, the absence of extraction blanks, PCR negative controls, and mock community controls should be acknowledged as an additional limitation when interpreting low-abundance taxa in this low-biomass matrix. Therefore, the findings should be regarded as preliminary baseline metataxonomic information describing the bacterial communities associated with street-vended raw milk rather than as a comprehensive assessment of its microbiological quality or safety. Future investigations incorporating larger and geographically more representative sampling, seasonal sampling, appropriate contamination controls, and complementary culture-based, genomic, metagenomic, resistome, and other multi-omics approaches will provide a more comprehensive understanding of the microbial ecology of street-vended raw milk and its implications for food quality and public health.

Conclusion

This exploratory pilot study provides baseline metataxonomic insights into the bacterial communities associated with street-vended raw milk obtained from three independent vendors in Ankara, Türkiye. Genus-level differences among the samples indicate considerable heterogeneity in bacterial community composition, which may reflect variations in environmental conditions and postmilking handling practices. The detection of psychrotrophic and environmentally associated genera further illustrates the microbial complexity of street-vended raw milk and demonstrates the value of culture-independent molecular approaches for the descriptive characterization of bacterial communities.

Nevertheless, the findings should be interpreted within the limitations of the present study, including the limited sample size, restricted geographical coverage, the absence of contamination controls, and the inherent taxonomic resolution of 16S rRNA gene amplicon sequencing. Consequently, the results should be regarded as preliminary baseline observations rather than a comprehensive assessment of the microbiological quality or safety of street-vended raw milk. Future studies incorporating larger and geographically broader sampling strategies, seasonal sampling, appropriate contamination controls, and complementary culture-based, genomic, metagenomic, and other multi-omics approaches are warranted to achieve a more comprehensive understanding of the microbial ecology and food safety implications of street-vended raw milk.

Authors’ Contributions

The author conceived and designed the study, performed the investigation, analyzed and interpreted the data, wrote the manuscript, and approved the final version of the manuscript.

Footnotes

Disclosure Statement

The author declares that there are no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

Funding Information

This research received no external funding.

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