One hundred thirty-nine beta-lactamases in this study exhibited ≥ 70% amino acid identity with known beta-lactamases. The distribution of potential beta-lactamases across various regions and hosts is shown in Figure 1. In total, we identified 109 bacterial genera and one Candidatus Ornithobacterium species (see Table S1), comprising the following bacterial classes (Actinomycetes, Alphaproteobacteria, Bacilli, Bacteroidia, Betaproteobacteria, Cyanophyceae, Cytophagia, Deltaproteobacteria, Epsilonproteobacteria, Flavobacteriia, Gammaproteobacteria, Chitinophagia, Chlorobiia, and Sphingobacteriia), in which the following novel potential beta-lactamases of group A (53 times), group B (27 times), group C (12 times) and group D (47 times) were detected.

The highest numbers of potential beta-lactamases were found in the genera Chryseobacterium, Enterobacter, Aeromonas and Chitinophaga, in which six, four and three enzymes (twice), respectively, were detected. For example, two potential class A beta-lactamases (83.2% and 80.5% amino acid identity with CGA-1), three class B beta-lactamases (87.9% identity with ESP-1; 71.6% and 70.7% identity with GOB-like enzymes), and one class D beta-lactamase (89% identity with OXA-209) were detected in the genus Chryseobacterium. Further, in the genus Enterobacter, there were two potential class A beta-lactamases (89.1% and 82.5% amino acid identity with LAP-1 and PLA-6, respectively) and two class C beta-lactamases (87.1% and 85.6% identity with ACT-96 and CDA-1, respectively). In the case of the genus Aeromonas, there was one potential beta-lactamase of class A (70.6% amino acid identity with BlaP-2), class C (86.4% identity with PAC-1), and class D (86.2% identity with OXA-12).

Identification of mobile genetic elements revealed the presence of genes for transposases, integrases/recombinases, and mobilization proteins in 14 cases (Table 1), with a higher abundance of transposases, which may lead to overproduction of beta-lactamases and facilitate gene transfer, spreading beta-lactamase genes. In addition, two potential beta-lactamases were detected nearby in some cases (Figure 2A/B).

The study analyzed the presence of potential beta-lactamases in bacterial isolates from various countries and demonstrated a notable distribution of these enzymes across different regions. China had the highest number of potential beta-lactamases, with 39 cases detected. In addition, there were 31 cases where the source country was not specified. The USA reported 19 cases, Japan had seven cases, and France, Germany, and South Korea each had five cases. Additionally, we explored the presence of potential beta-lactamases across different isolation environments. The majority were detected in environmental samples, accounting for 76 cases, followed by 22 cases where the origin was not determined. Furthermore, 19 cases were identified in animal and 17 in human hosts.

Our detailed study of potential beta-lactamases binding to mobile genetic elements resulted in a significant discovery. Specifically, we identified two potential class A beta-lactamases with identical amino acid sequences in Enterobacter sp. (AKXM01000000) and Cedecea sp. (NZ_JBBCPU010000048), both of which were found in mosquitoes and had an insertion sequence located nearby. This finding is crucial as it highlights potential sources of antibiotic resistance, with one case originating from the USA and the other from an unspecified location.

An insertion sequence, a short DNA segment that acts as a simple transposable element, was also found around potential class C beta-lactamase described in Aeromonas veronii (AP022282) isolated from a wastewater treatment plant effluent in Japan. This insertion sequence has also been detected in other bacterial species of this genus, such as Aeromonas caviae (AP022242, AP019196), all isolated from the same source and country.

The most worrying situation seems to be related to potential class A beta-lactamases, which show 99.9% nucleotide and 99.7-100% amino acid identity (change in one amino acid) with each other and have been detected in Bacteroides faecis (CP103274), Parabacteroides distasonis (CP103286) and Alistipes sp. (RKBL01000025), and these enzymes were located near the mobilization protein. More detailed in silico analysis indicated their occurrence in other species of the above genera, such as Bacteroides ovatus (CP103071), Bacteroides fragilis (CP036539, CP119600), Bacteroides salyersiae (CP072243), Bacteroides sp. (CP040630), Parabacteroides distasonis (CP072231), and Parabacteroides goldsteinii (CP081906). In addition, these enzymes have also been detected in other bacterial species, such as Phocaeicola vulgatus (CP096965) and Prevotella sp. (AP035786). These enzymes originated from humans and were found in India, the USA, Canada, Denmark, and the Netherlands, and from horses (Equus caballus) in Japan. This fact illustrates the seriousness of the situation, as the presence of beta-lactamases with a mobilization protein has been detected in many genera and species, indicating their widespread distribution and potential for the spread of antibiotic resistance.

Our study highlights the significant distribution of potential beta-lactamases across different bacterial genera, regions, and hosts. The presence of these enzymes, with exact amino acid identities with known beta-lactamases, in genera such as Cedecea, Enterobacter, Alistipes, Bacteroides, and Parabacteroides, suggests that horizontal gene transfer may play a role in spreading antibiotic resistance. This is supported by the identification of mobile genetic elements surrounding these genes, which may facilitate their transfer. China had the highest number of potential beta-lactamases, with the majority detected in environmental samples. The detection of these enzymes in various environmental and clinical settings underscores the need for comprehensive monitoring and international surveillance. To combat antibiotic resistance, global cooperation is needed. Mobile genetic elements, such as transposases, highlight the potential for these resistance genes to spread. This is crucial for predicting treatment failures and understanding the spread of resistance within and across bacterial species. The presence of beta-lactamases in diverse environments suggests that natural habitats may serve as reservoirs for these resistance genes, potentially allowing them to be transferred to clinical settings. Environmental strains provide a significant reservoir of new resistance genes, including carbapenemases, which can be disseminated through the food chain, accelerating global antibiotic resistance. Human activities facilitate the spread of resistance from various reservoirs such as washing machines and urban beaches, contributing to the complexity of combating antibiotic resistance. This broad distribution underscores the severity of the issue and the need for further research to understand and tackle antimicrobial resistance. This is also why we are convinced that we will soon witness the discovery of numerous new types of beta-lactamases. Many of these previously undescribed beta-lactamases may also play a role in the resulting resistance to various beta-lactam antibiotics. This emphasizes the urgent need for continued surveillance and research to combat antibiotic resistance and protect public health. Addressing this challenge requires the integration of clinical and environmental perspectives to effectively monitor and mitigate the impact of beta-lactamases on global health.