This study substantially extends our knowledge of the diversity within the B1 subclass of metallo-β-lactamases. By exploring 2906 bacterial genomes, 9230 plasmid sequences and more than 5 terabases of metagenomic data, we predicted 76 previously uncharacterized B1BL genes with less than 70% amino acid identity to previously known metallo-β-lactamases. These genes were divided into 59 novel families, revealing that the diversity within the B1BL subclass of metallo-β-lactamases is far greater than what has previously been described. Experimental validation of 21 selected genes showed that 18 (86%) were able to hydrolyze imipenem when expressed in an E. coli host. This provides strong confidence to the applied computational method. In addition, the predicted enzymatic activity was also confirmed for the gene present in the strain P. stutzeri DSM 10701. The phylogenetic analysis revealed that all but one (G38, family 30) of the novel B1BL genes identified in this study, together with the previously known B1BL genes, could be divided into five main groups, which could be largely explained by the taxonomic affiliation of their corresponding hosts. Proteobacteria, which were represented by two large groups in the phylogenetic tree, dominated, followed by Bacteroidetes. All mobile B1BL families, except for SPM, were located in the two Proteobacteria-associated groups, indicating that the clinically important mobile B1BL genes originated in Proteobacteria and that, in the future, the host range for mobile B1BL genes acquired by pathogens may be limited. Interestingly, Bacteroidetes was found to be highly overrepresented in relation to the number of genomes present in the databases, suggesting that they more frequently carry B1BLs. Furthermore, we also found that B1BLs were significantly overrepresented among Gram-negative bacteria compared to Gram-positive. However, we did not find any overrepresentation of B1BL in pathogens or human-associated bacteria. These results substantially increase the number of gene families of subclass B1 [39] and provides a more detailed view of the evolutionary history of this important subclass of metallo-β-lactamases.

The genetic contexts of two of the novel B1BL genes suggest that they may be mobile. First, the B1BL gene G67 (family 50) (in this study named PST-1), found on the chromosome of P. stutzeri DSM 10701, phylogenetically clustered with the previously characterized mobile B1BL families KHM, SIM, and IMP. The gene was located on a structurally variable region that was absent in the five other P. stutzeri strains present in the RefSeq database. In addition, no homology could be found with other species within the Pseudomonas genus, and no known integrons or transposons could be detected in the up- or downstream regions of the gene. There was, however, weak homology to two other genes located on the same insert as the predicted β-lactamase. These genes, NERD and Fic, have previously been found on plasmids, phages, and associated with integrons and have been suggested to code for mechanisms related to the horizontal transfer of genetic material [30, 40]. Pseudomonas is notorious for carrying mobile class B β-lactamases [41, 42], and several of the known mobile B1BL families were initially identified in this genus (IMP, VIM, SPM, and GIM) [43]. Thus, our results indicate that the production of PST-1 by P. stutzeri DSM 10701 may be the result of a recent gene acquisition event and that this gene could therefore, potentially, be a member of a previously uncharacterized mobile B1BL family.

Second, the gene G04 (in this study named MYO-1) in family 4 was located, together with a tetracycline resistance gene, on a plasmid in the genome of the opportunistic pathogen M. odoratimimus strain PR63039. This gene phylogenetically belonged to group B1–3, along with the chromosomal Bacteroidetes BLB1 genes, and was the only mobile gene in this cluster. Furthermore, the plasmid also contained parts of a class B conjugation system, which is known to be predominantly found in the Bacteroidetes phylum [33]. Thus, our results suggest that this gene was potentially mobilized from a species within the Bacteroidetes phylum and that its host-range may be limited at present. However, when validated in E. coli, the gene showed enzymatic activity despite no codon optimization, demonstrating that it is functional in a Proteobacterial host. Gene transfer events between Bacteroidetes and Gram-positive bacteria have been previously suggested to occur [44], and therefore, it cannot be excluded that this gene may be, or become, transferable to pathogens outside Bacteroidetes. This gene may thus constitute an important factor to be considered for clinically relevant carbapenem resistance in the future. The remaining B1BL genes predicted in this study did not have any clear indications regarding their mobility with respect to their genetic context or their location in the phylogenetic tree. However, the majority of the tested B1BLs exhibited enzymatic activity in E. coli. Thus, if these genes were to become mobilized and spread to pathogenic bacteria, they may constitute a threat to human health in the future [45]. Furthermore, the large increase in known chromosomal B1BL genes will enable more detailed phylogenetic studies of the B1 subclass of metallo-β-lactamases [23], an essential step to further elucidate the evolution and origins of mobile B1BL genes.

The phylogenetic analysis showed that 9 out of the 10 known mobile B1BL gene families clustered together with chromosomal B1BL genes in Proteobacteria (groups B1–1 and B1–2; the only exception was SPM-1). Interestingly, the B1–2 group, which contained 6 of the known mobile B1BLs, was highly overrepresented by chromosomal genes from the Shewanella genus. Shewanella has previously been hypothesized to be the origin of mobile antibiotic resistance genes, including the class D carbapenemase OXA-48 (Shewanella oneidensis, [46]) and the fluoroquinolone resistance gene qnrA (Shewanella algae [47]). Thus, our results suggest that many of the known mobile B1BL genes likely originated from hosts associated with the phylum Proteobacteria, especially Shewanella spp. In addition, our results suggest that clinically relevant mobile B1BL genes originating from hosts outside Proteobacteria are rare. This could potentially be due to the many barriers preventing genes from being efficiently transferred between phyla. The beta-lactamase protein needs to be transported outside the cytoplasmic membrane of the cell to induce a resistance phenotype, and its signal peptides therefore need to be recognized by the host [48, 49]. β-Lactamases also need to be highly expressed to provide high levels of resistance, which requires a codon distribution that is suitable for the host cell [50]. In fact, all of the predicted genes that showed negative Carba NP tests were located in the Bacteroidetes group B1–3, and two of them contained codons that are rare in E. coli. Since the experimental validation was based on the identified protein sequences and no codon optimization was applied, rare codons could potentially explain the lack of observed enzymatic activity. Another explanation could be that their signal peptides were not recognized by E. coli. Thus, it is plausible that many B1BL genes from distant phyla may be less suitable for Proteobacterial hosts and may therefore not yield any substantial resistance phenotype. Furthermore, many forms of conjugative elements, which are the primary mechanisms for the transfer of antibiotic resistance genes between cells, have a limited host range that can prevent sharing of genes between phylogenetically distant species [51, 52]. Indeed, the identified bacterial hosts carrying the mobile B1BLs IMP and VIM all belong to the phylum Proteobacteria. Nevertheless, our validation showed that 6 of 8 (75%) of the chromosomally located genes from phyla other than Proteobacteria were able to hydrolyze imipenem in an E. coli host, suggesting that no further evolution is needed for them to be functional should they be horizontally transferred. This suggests that, at least in theory, the recruitment of B1BL genes outside of Proteobacteria should be possible. It should, however, be pointed out that such genes may pose a significant risk if present in, or transferred to, pathogens belonging to their original phylum. Furthermore, we noted that none of the known mobile B1BL genes had a close match to a chromosomal gene in a known pathogenic or non-pathogenic bacterial species. This suggests that the known mobile B1BL genes either were not recently mobilized or originated from species with yet uncharacterized genomes. Given that a large proportion of the pathogenic- and human-associated bacteria have been sequenced to date [53], it is plausible that many of the mobile B1BL genes have an environmental origin. Nevertheless, our results underscore the fact that Proteobacteria, in particular Shewanella, constitutes an important reservoir for the mobilization of B1BLs.

In B1 enzymes, the Zn(II) ion at the first binding site is coordinated to the residues His116, His118, and His196, according to the standard BBL numbering scheme [27], while the second binding site for the second Zn(II) ion is coordinated by the residues Asp120, Cys221, and His263. Interestingly, two of the novel B1BL genes, G09 (here named SPS-1, in family 9) and G76 (in family 59), which were both located in the fifth group (B1–5), had the conserved His116 replaced by a glycine, which has previously not been reported in any class B β-lactamase. One of these enzymes was selected for validation (SPS-1 in family 9; see Table 3 and Additional file 2: Table S1), and its imipenem hydrolyzing activity was confirmed when expressed in E. coli. Metallo-β-lactamases in the closely related B2 subclass are known to have the His116 replaced by asparagine [3]. Mutational analysis of the subclass B2 gene CphA has shown that the replacement of asparagine with histidine at position His116 resulted in a 12- and 260-fold increase in activity against penicillins and cephalosporins, respectively [54]. Thus, it cannot be excluded that the changes in the zinc-binding sites of SPS-1 and G76, and potentially other yet to be described β-lactamases within this group, may have significantly different enzymatic activities and substrate spectra. It should be noted that, based on the current paradigm of three separate B subclasses (B1, B2, and B3), our study clearly classified SPS-1 and G76 as B1 β-lactamases. Thus, our results suggest that the B1 subclass has a higher diversity of zinc-binding sites than what has previously been described in the literature.

Thirteen of the predicted B1BL genes were identified from metagenomic data, all of which were assembled from environmental samples, primarily polluted ones. The abundance of B1BL gene fragments was also found to be three-fold higher in the environmental metagenomes compared to metagenomes from the human microbiome, although the variation between the environmental metagenomes was high. This further strengthens the hypotheses that the environment serves as an unexplored reservoir of class B β-lactamases, which is in agreement with previous studies [11, 20]. However, there were B1BL fragments found in the human microbiome, especially in stool samples, but no assembly of full-length genes was possible. To investigate if this was due to lack of sequencing depth, we attempted to pool the B1BL gene fragments from different individuals based on body site, but no significant improvement was observed. One likely reason for the inability to identify new B1BLs in the human-associated metagenomes is the combination of the low abundance of β-lactam resistance genes with the high diversity of the microbiome, both between individuals and body sites [53]. In contrast to the environmental metagenomes, where replicated samples were taken from similar, often spatially close, communities, the human-associated data used in this study were comprised of samples from a large number of individuals, each sequenced at a limited depth. Thus, deeper sequencing of the individual samples is likely necessary to identify potential variants only present in smaller proportions of individuals. However, our results indicate that many of the identified fragments are false positives and that there is likely no single widespread previously uncharacterized B1BL gene in the human microbiomes investigated.

The identification of novel β-lactamases was based on a probabilistic profile hidden Markov model (HMM) designed to identify novel B1BL gene families. Compared to more standard bioinformatics approaches to homology searches, such as BLAST, profile HMMs take the position-specific sequence conservation into account and therefore offer much higher sensitivity [55]. The model was optimized using cross-validation where the probability to identify a B1BL gene excluded from the model was maximized. This enabled us to identify novel B1BL genes, even in highly fragmented sequence data, with high accuracy. This is underscored by the experimental validation where imipenem hydrolysis was verified in 86% (18 of 21 tested genes) of the validated genes. In addition, two of the genes that showed no activity both contained several codons that are considered rare in E. coli, suggesting that they may not have been translated with sufficient efficiency and could be functional in other hosts. Taken together, it is therefore likely that the majority of the genes predicted in this study that were not subjected to validation are also novel, functional B1BLs.