Filamentous prophage Pf4 promotes genetic exchange in Pseudomonas aeruginosa.

Filamentous prophages are widespread among bacteria and play crucial functions in virulence, antibiotic resistance, and biofilm structures. The filamentous Pf4 particles, extruded by an important pathogen Pseudomonas aeruginosa, can protect producing cells from adverse conditions. Contrary to the conventional belief that the Pf4-encoding cells resist reinfection, we herein report that the Pf4 prophage is reciprocally and commonly exchanged within P. aeruginosa colonies, which can repair defective Pf4 within the community. By labeling the Pf4 locus with antibiotic resistance and fluorescence markers, we demonstrate that the Pf4 locus is frequently exchanged within colony biofilms, in artificial sputum media, and in infected mouse lungs. We further show that Pf4 trafficking is a rapid process and capable of rescuing Pf4-defective mutants. The Pf4 phage is highly adaptable and can package additional DNA doubling its genome size. We also report that two clinical P. aeruginosa isolates are susceptible to the Pf4-mediated exchange, and the Pf5 prophage can be exchanged between cells as well. These findings suggest that the genetic exchanging interactions by filamentous prophages may facilitate defect rescue and the sharing of prophage-dependent benefits and costs within the P. aeruginosa community.

A response regulator controls Acinetobacter baumannii virulence by acting as an indole receptor.

Indole is an important signal employed by many bacteria to modulate intraspecies signaling and interspecies or interkingdom communication. Our recent study revealed that indole plays a key role in regulating the physiology and virulence of Acinetobacter baumannii. However, it is not clear how A. baumannii perceives and responds to the indole signal in modulating biological functions. Here, we report that indole controls the physiology and virulence of A. baumannii through a previously uncharacterized response regulator designated as AbiR (A1S_1394), which contains a cheY-homologous receiver (REC) domain and a helix-turn-helix (HTH) DNA-binding domain. AbiR controls the same biological functions as the indole signal, and indole-deficient mutant phenotypes were rescued by in trans expression of AbiR. Intriguingly, unlike other response regulators that commonly interact with signal ligands through the REC domain, AbiR binds to indole with a high affinity via an unusual binding region, which is located between its REC and HTH domains. This interaction substantially enhances the activity of AbiR in promoter binding and in modulation of target gene expression. Taken together, our results present a widely conserved regulator that controls bacterial physiology and virulence by sensing the indole signal in a unique mechanism.

An Isotope-Labeled Single-Cell Raman Spectroscopy Approach for Tracking the Physiological Evolution Trajectory of Bacteria toward Antibiotic Resistance.

Understanding evolution of antibiotic resistance is vital for containing its global spread. Yet our ability to in situ track highly heterogeneous and dynamic evolution is very limited. Here, we present a new single-cell approach integrating D2 O-labeled Raman spectroscopy, advanced multivariate analysis, and genotypic profiling to in situ track physiological evolution trajectory toward resistance. Physiological diversification of individual cells from isogenic population with cyclic ampicillin treatment is captured. Advanced multivariate analysis of spectral changes classifies all individual cells into four subsets of sensitive, intrinsic tolerant, evolved tolerant and resistant. Remarkably, their dynamic shifts with evolution are depicted and spectral markers of each state are identified. Genotypic analysis validates the phenotypic shift and provides insights into the underlying genetic basis. The new platform advances rapid phenotyping resistance evolution and guides evolution control.

Gold drugs as colistin adjuvants in the fight against MCR-1 producing bacteria.

The emergence and rapid spread of the mobile colistin resistance gene mcr-1 among bacterial species and hosts significantly challenge the efficacy of "last-line" antibiotic colistin. Previously, we reported silver nitrate and auranofin serve as colistin adjuvants for combating mcr-1-positive bacteria. Herein, we uncovered more gold-based drugs and nanoparticles, and found that they exhibited varying degree of synergisms with colistin on killing mcr-1-positive bacteria. However, pre-activation of the drugs by either glutathione or N-acetyl cysteine, thus releasing and accumulating gold ions, is perquisite for their abilities to substitute zinc cofactor from MCR-1 enzyme. X-ray crystallography and biophysical studies further supported the proposed mechanism. This study not only provides basis for combining gold-based drugs and colistin for combating mcr-1-positive bacterial infections, but also undoubtedly opens a new horizon for metabolism details of gold-based drugs in overcoming antimicrobial resistance.

Engineered Bacteriophages Containing Anti-CRISPR Suppress Infection of Antibiotic-Resistant P. aeruginosa.

The therapeutic use of bacteriophages (phages) provides great promise for treating multidrug-resistant (MDR) bacterial infections. However, an incomplete understanding of the interactions between phages and bacteria has negatively impacted the application of phage therapy. Here, we explored engineered anti-CRISPR (Acr) gene-containing phages (EATPs, eat Pseudomonas) by introducing Type I anti-CRISPR (AcrIF1, AcrIF2, and AcrIF3) genes into the P. aeruginosa bacteriophage DMS3/DMS3m to render the potential for blocking P. aeruginosa replication and infection. In order to achieve effective antibacterial activities along with high safety against clinically isolated MDR P. aeruginosa through an anti-CRISPR immunity mechanism in vitro and in vivo, the inhibitory concentration for EATPs was 1 × 108 PFU/mL with a multiplicity of infection value of 0.2. In addition, the EATPs significantly suppressed the antibiotic resistance caused by a highly antibiotic-resistant PA14 infection. Collectively, these findings provide evidence that engineered phages may be an alternative, viable approach by which to treat patients with an intractable bacterial infection, especially an infection by clinically MDR bacteria that are unresponsive to conventional antibiotic therapy. IMPORTANCE Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic Gram-negative bacterium that causes severe infection in immune-weakened individuals, especially patients with cystic fibrosis, burn wounds, cancer, or chronic obstructive pulmonary disease (COPD). Treating P. aeruginosa infection with conventional antibiotics is difficult due to its intrinsic multidrug resistance. Engineered bacteriophage therapeutics, acting as highly viable alternative treatments of multidrug-resistant (MDR) bacterial infections, have great potential to break through the evolutionary constraints of bacteriophages to create next-generation antimicrobials. Here, we found that engineered anti-CRISPR (Acr) gene-containing phages (EATPs, eat Pseudomonas) display effective antibacterial activities along with high safety against clinically isolated MDR P. aeruginosa through an anti-CRISPR immunity mechanism in vitro and in vivo. EATPs also significantly suppressed the antibiotic resistance caused by a highly antibiotic-resistant PA14 infection, which may provide novel insight toward developing bacteriophages to treat patients with intractable bacterial infections, especially infections by clinically MDR bacteria that are unresponsive to conventional antibiotic therapy.

Whole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria.

Bacteria adapt to their constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor TF-promoter interactions in situ in living bacteria are lacking. Herein, we developed a whole-cell TF-promoter binding assay based on the intermolecular FRET between an unnatural amino acid, l-(7-hydroxycoumarin-4-yl) ethylglycine, which labels TFs with bright fluorescence through genetic encoding (donor fluorophore) and the live cell nucleic acid stain SYTO 9 (acceptor fluorophore). We show that this new FRET pair monitors the intricate TF-promoter interactions elicited by various types of signal transduction systems, including one-component (CueR) and two-component systems (BasSR and PhoPQ), in bacteria with high specificity and sensitivity. We demonstrate that robust CouA incorporation and FRET occurrence is achieved in all these regulatory systems based on either the crystal structures of TFs or their simulated structures, if 3D structures of the TFs were unavailable. Furthermore, using CueR and PhoPQ systems as models, we demonstrate that the whole-cell FRET assay is applicable for the identification and validation of complex regulatory circuit and novel modulators of regulatory systems of interest. Finally, we show that the FRET system is applicable for single-cell analysis and monitoring TF activities in Escherichia coli colonizing a Caenorhabditis elegans host. In conclusion, we established a tractable and sensitive TF-promoter binding assay, which not only complements currently available approaches for DNA-protein interactions but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria-host interface.

Ultrastrong and multifunctional aerogels with hyperconnective network of composite polymeric nanofibers.

Three-dimensional (3D) microfibrillar network represents an important structural design for various natural tissues and synthetic aerogels. Despite extensive efforts, achieving high mechanical properties for synthetic 3D microfibrillar networks remains challenging. Here, we report ultrastrong polymeric aerogels involving self-assembled 3D networks of aramid nanofiber composites. The interactions between the nanoscale constituents lead to assembled networks with high nodal connectivity and strong crosslinking between fibrils. As revealed by theoretical simulations of 3D networks, these features at fibrillar joints may lead to an enhancement of macroscopic mechanical properties by orders of magnitude even with a constant level of solid content. Indeed, the polymeric aerogels achieved both high specific tensile modulus of ~625.3 MPa cm3 g-1 and fracture energy of ~4700 J m-2, which are advantageous for diverse structural applications. Furthermore, their simple processing techniques allow fabrication into various functional devices, such as wearable electronics, thermal stealth, and filtration membranes. The mechanistic insights and manufacturability provided by these robust microfibrillar aerogels may create further opportunities for materials design and technological innovation.

Microbiome characteristics and the key biochemical reactions identified on stone world cultural heritage under different climate conditions.

The surfaces of historical stone monuments are visibly covered with a layer of colonizing microorganisms and their degradation products. In this study, a metadata analysis was conducted using the microbial sequencing data available from NCBI database to determine the diversity, biodeterioration potential and functionality of the stone microbiome on important world cultural heritage sites under four different climatic conditions. The retrieved stone microbial community composition in these metagenomes shows a clear association between climate types of the historical monuments and the diversity and taxonomic composition of the stone microbiomes. Shannon diversity values showed that microbial communities on stone monuments exposed to dry climate were more diverse than those under humid ones. In particular, functions associated with photosynthesis and UV resistance were identified from geographical locations under different climate types. The distribution of key microbial determinants responsible for stone deterioration was linked to survival under extreme environmental conditions and biochemical capabilities and reactions. Among them, biochemical reactions of the microbial nitrogen and sulfur cycles were most predominant. These stone-dwelling microbiomes on historical stone monuments were highly diverse and self-sustaining driven by energy metabolism and biomass accumulation. And metabolic products of the internal geomicrobiological nitrogen cycling on these ancient monuments play a unique role in the biodeterioration of stone monuments. These results highlight the significance of identifying the essential microbial biochemical reactions to advance the understanding of stone biodeterioration for protection management.

Detection of synergistic antimicrobial resistance mechanisms in clinical isolates of Pseudomonas aeruginosa from post-operative wound infections.

Infections caused by carbapenem-resistant Pseudomonas aeruginosa are life-threatening due to its synergistic resistance mechanisms resulting in the ineffectiveness of the used antimicrobials. This study aimed to characterize P. aeruginosa isolates for antimicrobial susceptibility, biofilm formation virulence genes, and molecular mechanisms responsible for resistance against various antimicrobials. Out of 700 samples, 91 isolates were confirmed as P. aeruginosa which were further classified into 19 non-multidrug-resistant (non-MDR), 7 multidrug-resistant (MDR), 19 extensively drug-resistant (XDR), and 8 pan drug-resistant (PDR) pulsotypes based on standard Kirby Bauer disc diffusion test and pulse field gel electrophoresis. In M9 minimal media, strong biofilms were formed by the XDR and PDR pulsotypes as compared to the non-MDR pulsotypes. The virulence genes, responsible for the worsening of wounds including LasB, plcH, toxA, and exoU, were detected among all MDR, XDR, and PDR pulsotypes. Carbapenemase activity was phenotypically detected in 45% pulsotypes and the responsible genes were found as blaGES (100%), blaVIM (58%), blaIMP (4%), and blaNDM (4%). Real-time polymerase chain reaction showed the concomitant use of multiple mechanisms such as oprD under-expression, enhanced efflux pump activity, and ampC overexpression in the resistant isolates. Polymyxin is found as the only class left with more than 80% susceptibility among the isolates which is an alarming situation suggesting appropriate measures to be taken including alternative therapies. KEY POINTS: • Multidrug-resistant P. aeruginosa isolates formed stronger biofilms in minimal media. • Only polymyxin antimicrobial was found effective against MDR P. aeruginosa isolates. • Under-expression of oprD and overexpression of ampC were found in resistant isolates.

A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation.

The Class 1 type I CRISPR-Cas systems represent the most abundant and diverse CRISPR systems in nature. However, their applications for generic genome editing have been hindered due to difficulties of introducing the class-specific, multi-component effectors (Cascade) in heterologous hosts for functioning. Here we established a transferrable Cascade system that enables stable integration and expression of a highly active type I-F Cascade in heterologous bacterial hosts for various genetic exploitations. Using the genetically recalcitrant Pseudomonas species as a paradigm, we show that the transferred Cascade displayed substantially higher DNA interference activity and greater editing capacity than both the integrative and plasmid-borne Cas9 systems, and enabled deletion of large fragments such as the 21-kb integrated cassette with efficiency and simplicity. An advanced I-F-λred system was further developed to enable editing in genotypes with poor homologous recombination capacity, clinical isolates lacking sequence information, and cells containing anti-CRISPR elements Acrs. Lastly, an 'all-in-one' I-F Cascade-mediated CRISPRi platform was developed for transcription modulation by simultaneous introduction of the Cascade and the programmed mini-CRISPR array in one-step. This study provides a framework for expanding the diverse type I Cascades for widespread, heterologous genome editing and establishment of editing techniques in 'non-model' bacterial species.

Multi-target mode of action of silver against Staphylococcus aureus endows it with capability to combat antibiotic resistance.

The rapid emergence of drug resistant Staphylococcus aureus (S. aureus) poses a serious threat to public health globally. Silver (Ag)-based antimicrobials are promising to combat antibiotic resistant S. aureus, yet their molecular targets are largely elusive. Herein, we separate and identify 38 authentic Ag+-binding proteins in S. aureus at the whole-cell scale. We then capture the molecular snapshot on the dynamic action of Ag+ against S. aureus and further validate that Ag+ could inhibit a key target 6-phosphogluconate dehydrogenase through binding to catalytic His185 by X-ray crystallography. Significantly, the multi-target mode of action of Ag+ (and nanosilver) endows its sustainable antimicrobial efficacy, leading to enhanced efficacy of conventional antibiotics and resensitization of MRSA to antibiotics. Our study resolves the long-standing question of the molecular targets of silver in S. aureus and offers insights into the sustainable bacterial susceptibility of silver, providing a potential approach for combating antimicrobial resistance.

Population differentiation of Rhodobacteraceae along with coral compartments.

Coral mucus, tissue, and skeleton harbor compositionally different microbiota, but how these coral compartments shape the microbial evolution remains unexplored. Here, we sampled bacteria inhabiting a prevalent coral species Platygyra acuta and sequenced genomes of 234 isolates comprising two populations in Rhodobacteraceae, an alphaproteobacterial lineage representing a significant but variable proportion (5-50%) of the coral microbiota. The Ruegeria population (20 genomes) contains three clades represented by eight, six, and six isolates predominantly sampled from the skeleton (outgroup), mucus (clade-M), and skeleton (clade-S), respectively. The clade-M possesses functions involved in the utilization of coral osmolytes abundant in the mucus (e.g., methylamines, DMSP, taurine, and L-proline), whereas the clade-S uniquely harbors traits that may promote adaptation to the low-energy and diurnally anoxic skeleton (e.g., sulfur oxidation and swimming motility). These between-clade genetic differences were largely supported by physiological assays. Expanded analyses by including genomes of 24 related isolates (including seven new genomes) from other marine environments suggest that clade-M and clade-S may have diversified in non-coral habitats, but they also consolidated a key role of distinct coral compartments in diversifying many of the above-mentioned traits. The unassigned Rhodobacteraceae population (214 genomes) varies only at a few dozen nucleotide sites across the whole genomes, but the number of between-compartment migration events predicted by the Slatkin-Maddison test supported that dispersal limitation between coral compartments is another key mechanism diversifying microbial populations. Collectively, our results suggest that different coral compartments represent ecologically distinct and microgeographically separate habitats that drive the evolution of the coral microbiota.

Microbiome assembly for sulfonamide subsistence and the transfer of genetic determinants.

Antibiotic subsistence in bacteria represents an alternative resistance machinery, while paradoxically, it is also a cure for environmental resistance. Antibiotic-subsisting bacteria can detoxify antibiotic-polluted environments and prevent the development of antibiotic resistance in environments. However, progress toward efficient in situ engineering of antibiotic-subsisting bacteria is hindered by the lack of mechanistic and predictive understanding of the assembly of the functioning microbiome. By top-down manipulation of wastewater microbiomes using sulfadiazine as the single limiting source, we monitored the ecological selection process that forces the wastewater microbiome to perform efficient sulfadiazine subsistence. We found that the community-level assembly selects for the same three families rising to prominence across different initial pools of microbiomes. We further analyzed the assembly patterns using a linear model. Detailed inspections of the sulfonamide metabolic gene clusters in individual genomes of isolates and assembled metagenomes reveal limited transfer potential beyond the boundaries of the Micrococcaceae lineage. Our results open up new possibilities for engineering specialist bacteria for environmental applications.

Elastic, Conductive, and Mechanically Strong Hydrogels from Dual-Cross-Linked Aramid Nanofiber Composites.

Recent research on conductive hydrogels has revealed their potential for building advanced soft bioelectronic devices. Their mechanical flexibility, water content, and porosity approach those of biological tissues, providing a compliant interface between the human body and electronic hardware. Conductive hydrogels could be utilized in many soft tools such as neural electrodes, tactile interfaces, soft actuators, and other electroactive devices. However, most of the available conductive hydrogels exhibit weak mechanical properties, which hinders their application in durable biointegrated systems. Here, we report aramid nanofiber-based hydrogels providing a combination of high elasticity, strength, and electrical conductivity. Highly branched aramid nanofibers (ANFs) provide a robust three-dimensional (3D) framework resembling those in load-bearing soft tissues. When interlaced with poly(vinyl alcohol) (PVA) and cross-linked with both noncovalent and covalent interactions, the nanofiber composites exhibit a high water content of ∼76.4 wt %, strength of ∼7.5 MPa, ductility of ∼407%, and shape recovery of ∼99.5% under cyclic tensile stress of 0.3 MPa. Mobile ions impart a conductivity of ∼2 S/m to the hydrogels, enabling large-strain sensors with stable operation. In addition, the embedded silver nanoparticles afford broad-spectrum antimicrobial activities, which is favorable for medical devices. The versatility of aramid nanofiber-based composites suggests their further possibilities for functionalization and scalable fabrication toward sophisticated bioelectronic systems.

HMD-ARG: hierarchical multi-task deep learning for annotating antibiotic resistance genes.

BACKGROUND: The spread of antibiotic resistance has become one of the most urgent threats to global health, which is estimated to cause 700,000 deaths each year globally. Its surrogates, antibiotic resistance genes (ARGs), are highly transmittable between food, water, animal, and human to mitigate the efficacy of antibiotics. Accurately identifying ARGs is thus an indispensable step to understanding the ecology, and transmission of ARGs between environmental and human-associated reservoirs. Unfortunately, the previous computational methods for identifying ARGs are mostly based on sequence alignment, which cannot identify novel ARGs, and their applications are limited by currently incomplete knowledge about ARGs. RESULTS: Here, we propose an end-to-end Hierarchical Multi-task Deep learning framework for ARG annotation (HMD-ARG). Taking raw sequence encoding as input, HMD-ARG can identify, without querying against existing sequence databases, multiple ARG properties simultaneously, including if the input protein sequence is an ARG, and if so, what antibiotic family it is resistant to, what resistant mechanism the ARG takes, and if the ARG is an intrinsic one or acquired one. In addition, if the predicted antibiotic family is beta-lactamase, HMD-ARG further predicts the subclass of beta-lactamase that the ARG is resistant to. Comprehensive experiments, including cross-fold validation, third-party dataset validation in human gut microbiota, wet-experimental functional validation, and structural investigation of predicted conserved sites, demonstrate not only the superior performance of our method over the state-of-art methods, but also the effectiveness and robustness of the proposed method. CONCLUSIONS: We propose a hierarchical multi-task method, HMD-ARG, which is based on deep learning and can provide detailed annotations of ARGs from three important aspects: resistant antibiotic class, resistant mechanism, and gene mobility. We believe that HMD-ARG can serve as a powerful tool to identify antibiotic resistance genes and, therefore mitigate their global threat. Our method and the constructed database are available at http://www.cbrc.kaust.edu.sa/HMDARG/ . Video abstract (MP4 50984 kb).

Harnessing the type I CRISPR-Cas systems for genome editing in prokaryotes.

Genetic analysis is crucial to the understanding, exploitation, and control of microorganisms. The advent of CRISPR-Cas-based genome-editing techniques, particularly those mediated by the single-effector (Cas9 and Cas12a) class 2 CRISPR-Cas systems, has revolutionized the genetics in model eukaryotic organisms. However, their applications in prokaryotes are rather limited, largely owing to the exceptional diversity of DNA homeostasis in microorganisms and severe cytotoxicity of overexpressing these nuclease proteins in certain genotypes. Remarkably, CRISPR-Cas systems belonging to different classes and types are continuously identified in prokaryotic genomes and serve as a deep reservoir for expansion of the CRISPR-based genetic toolkits. ~90% of the CRISPR-Cas systems identified so far belong to the class 1 system which hinges on multi-protein effector complexes for DNA interference. Harnessing these widespread native CRISPR-Cas systems for 'built-in' genome editing represents an emerging and powerful genetic tool in prokaryotes, especially in the genetically recalcitrant non-model species and strains. In this progress review, we introduce the general workflow of this emerging editing platform and summarize its establishment in a growing number of prokaryotes by harnessing the most widespread, diverse type I CRISPR-Cas systems present in their genomes. We also discuss the various factors affecting the success and efficiency of this editing platform and the corresponding solutions.

Repurposing the Native Type I-F CRISPR-Cas System in Pseudomonas aeruginosa for Genome Editing.

Repurposing the broadly distributed native CRISPR-Cas systems in prokaryotes for genome editing is emerging as a new strategy for genetic manipulations. We recently reported the establishment of a single plasmid-mediated, one-step genome-editing technique in a multidrug-resistant genotype of the opportunistic pathogen Pseudomonas aeruginosa by harnessing its endogenous type I-F CRISPR-Cas system. The platform is readily applicable in additional type I-F CRISPR-containing clinical and environmental P. aeruginosa isolates. Herein, we provide the detailed protocol for the methodology. For complete details on the establishment and exploitation of this protocol, please refer to Xu et al. (2019).

Comparative Peptidomic and Metatranscriptomic Analyses Reveal Improved Gamma-Amino Butyric Acid Production Machinery in Levilactobacillus brevis Strain NPS-QW 145 Cocultured with Streptococcus thermophilus Strain ASCC1275 during Milk Fermentation.

The high-gamma-amino butyric acid (GABA)-producing bacterium Levilactobacillus brevis strain NPS-QW 145, along with Streptococcus thermophilus (one of the two starter bacteria used to make yogurt for its proteolytic activity), enhances GABA production in milk. However, a mechanistic understanding of how Levilactobacillus brevis cooperates with S. thermophilus to stimulate GABA production has been lacking. Comparative peptidomic and metatranscriptomic analyses were carried out to unravel the casein and lactose utilization patterns during milk fermentation with the coculture. We found that particular peptides hydrolyzed by S. thermophilus ASCC1275 were transported and biodegraded with peptidase in Lb. brevis 145 to meet the growth needs of the latter. In addition, amino acid synthesis and metabolism in Lb. brevis 145 were activated to further support its growth. Glucose, as a result of lactose hydrolysis by S. thermophilus 1275, but not available lactose in milk, was metabolized as the main carbon source by Lb. brevis 145 for ATP production. In the stationary phase, under acidic conditions due to the accumulation of lactic acid produced by S. thermophilus 1275, the expression of genes involved in pyridoxal phosphate (coenzyme of glutamic acid decarboxylase) metabolism and glutamic acid decarboxylase (Gad) in Lb. brevis 145 was induced for GABA production.SIGNIFICANCE A huge market for GABA-rich milk as a dietary therapy for the management of hypertension is anticipated. The novelty of this work lies in applying peptide profiles supported by metatranscriptomics to elucidate (i) the pattern of casein hydrolysis by S. thermophilus 1275, (ii) the supply of peptides and glucose by S. thermophilus 1275 to Lb. brevis 145, (iii) the transportation of peptides in Lb. brevis and the degradation of peptides by this organism, which was reported to be nonproteolytic, and (iv) GABA production by Lb. brevis 145 under acidic conditions. Based on the widely reported contribution of lactic acid bacteria (LAB) and GABA to human health, the elucidation of interactions between the two groups of bacterial communities in the production of GABA-rich milk is important for promoting the development of functional dairy food and may provide new insight into the development of industrial GABA production.