Freshwater and Marine Environments in California Are a Reservoir of Carbapenem-Resistant Bacteria.

Carbapenems are last-resort antibiotics used to treat multidrug-resistant bacterial infections. Resistance to carbapenems has been designated as an urgent threat and is increasing in healthcare settings. However, little is still known about the distribution and characteristics of carbapenem-resistant bacteria (CRB) outside of healthcare settings. Here, we surveyed the distribution of CRB in ten diverse freshwater and seawater environments in California, U.S., ranging from San Luis Obispo County to San Bernardino County, combining both direct isolation and enrichment approaches to increase the diversity of isolated CRB. From the locations surveyed, we selected 30 CRB for further characterization. These isolates were identified as members of the genera Aeromonas, Enterobacter, Enterococcus, Paenibacillus, Pseudomonas, Sphingobacterium, and Stenotrophomonas. These isolates were resistant to carbapenems, other ß-lactams, and often to other antibiotics (tetracycline, gentamicin, or ciprofloxacin). We also found that nine isolates belonging to the genera Aeromonas, Enterobacter (blaIMI-2), and Stenotrophomonas (blaL1) produced carbapenemases. Overall, our findings indicate that sampling different types of aquatic environments and combining different isolation approaches increase the diversity of the environmental CRB obtained. Moreover, our study supports the increasingly recognized role of natural water systems as an underappreciated reservoir of bacteria resistant to carbapenems and other antibiotics, including bacteria carrying carbapenemase genes.

The multidrug efflux pump regulator AcrR directly represses motility in Escherichia coli.

Efflux and motility are two key biological functions in bacteria. Recent findings have shown that efflux impacts flagellum biosynthesis and motility in Escherichia coli and other bacteria. AcrR is known to be the major transcriptional repressor of AcrAB-TolC, the main multidrug efflux pump in E. coli and other Enterobacteriaceae. However, the underlying molecular mechanisms of how efflux and motility are co-regulated remain poorly understood. Here, we have studied the role of AcrR in direct regulation of motility in E. coli. By combining bioinformatics, electrophoretic mobility shift assays (EMSAs), gene expression, and motility experiments, we have found that AcrR represses motility in E. coli by directly repressing transcription of the flhDC operon, but not the other flagellum genes/operons tested. flhDC encodes the master regulator of flagellum biosynthesis and motility genes. We found that such regulation primarily occurs by direct binding of AcrR to the flhDC promoter region containing the first of the two predicted AcrR-binding sites identified in this promoter. This is the first report of direct regulation by AcrR of genes unrelated to efflux or detoxification. Moreover, we report that overexpression of AcrR restores to parental levels the increased swimming motility previously observed in E. coli strains without a functional AcrAB-TolC pump, and that such effect by AcrR is prevented by the AcrR ligand and AcrAB-TolC substrate ethidium bromide. Based on these and prior findings, we provide a novel model in which AcrR senses efflux and then co-regulates efflux and motility in E. coli to maintain homeostasis and escape hazards. IMPORTANCE Efflux and motility play a major role in bacterial growth, colonization, and survival. In Escherichia coli, the transcriptional repressor AcrR is known to directly repress efflux and was later found to also repress flagellum biosynthesis and motility by Kim et al. (J Microbiol Biotechnol 26:1824-1828, 2016, doi: 10.4014/jmb.1607.07058). However, it remained unknown whether AcrR represses flagellum biosynthesis and motility directly and through which target genes, or indirectly because of altering the amount of efflux. This study reveals that AcrR represses flagellum biosynthesis and motility by directly repressing the expression of the flhDC master regulator of flagellum biosynthesis and motility genes, but not the other flagellum genes tested. We also show that the antimicrobial, efflux pump substrate, and AcrR ligand ethidium bromide regulates motility via AcrR. Overall, these findings support a novel model of direct co-regulation of efflux and motility mediated by AcrR in response to stress in E. coli.

The Multidrug Efflux Regulator AcrR of Escherichia coli Responds to Exogenous and Endogenous Ligands To Regulate Efflux and Detoxification.

The transcriptional repressor AcrR is the main regulator of the multidrug efflux pump AcrAB-TolC, which plays a major role in antibiotic resistance and cell physiology in Escherichia coli and other Enterobacteriaceae. However, it remains unknown which ligands control the function of AcrR. To address this gap in knowledge, this study tested whether exogenous and/or endogenous molecules identified as potential AcrR ligands regulate the activity of AcrR. Using electrophoretic mobility shift assays (EMSAs) with purified AcrR and the acrAB promoter and in vivo gene expression experiments, we found that AcrR responds to both exogenous molecules and cellular metabolites produced by E. coli. In total, we identified four functional ligands of AcrR, ethidium bromide (EtBr), an exogenous antimicrobial known to be effluxed by the AcrAB-TolC pump and previously shown to bind to AcrR, and three polyamines produced by E. coli, namely, putrescine, cadaverine, and spermidine. We found that EtBr and polyamines bind to AcrR both in vitro and in vivo, which prevents the binding of AcrR to the acrAB promoter and, ultimately, induces the expression of acrAB. Finally, we also found that AcrR contributes to mitigating the toxicity produced by excess polyamines by directly regulating the expression of AcrAB-TolC and two previously unknown AcrR targets, the MdtJI spermidine efflux pump and the putrescine degradation enzyme PuuA. Overall, these findings significantly expand our understanding of the function of AcrR by revealing that this regulator responds to different exogenous and endogenous ligands to regulate the expression of multiple genes involved in efflux and detoxification. IMPORTANCE Multidrug efflux pumps can remove antibiotics and other toxic molecules from cells and are major contributors to antibiotic resistance and bacterial physiology. Therefore, it is essential to better understand their function and regulation. AcrAB-TolC is the main multidrug efflux pump in the Enterobacteriaceae family, and AcrR is its major transcriptional regulator. However, little is known about which ligands control the function of AcrR or which other genes are controlled by this regulator. This study contributes to addressing these gaps in knowledge by showing that (i) the activity of AcrR is controlled by the antimicrobial ethidium bromide and by polyamines produced by E. coli, and (ii) AcrR directly regulates the expression of AcrAB-TolC and genes involved in detoxification and efflux of excess polyamines. These findings significantly advance our understanding of the biological role of AcrR by identifying four ligands that control its function and two novel targets of this regulator.

Urban and agricultural soils in Southern California are a reservoir of carbapenem-resistant bacteria.

Carbapenems are last-resort ß-lactam antibiotics used in healthcare facilities to treat multidrug-resistant infections. Thus, most studies on identifying and characterizing carbapenem-resistant bacteria (CRB) have focused on clinical settings. Relatively, little is still known about the distribution and characteristics of CRBs in the environment, and the role of soil as a potential reservoir of CRB in the United States remains unknown. Here, we have surveyed 11 soil samples from 9 different urban or agricultural locations in the Los Angeles-Southern California area to determine the prevalence and characteristics of CRB in these soils. All samples tested contained CRB with a frequency of <10 to 1.3 × 104  cfu per gram of soil, with most agricultural soil samples having a much higher relative frequency of CRB than urban soil samples. Identification and characterization of 40 CRB from these soil samples revealed that most of them were members of the genera Cupriavidus, Pseudomonas, and Stenotrophomonas. Other less prevalent genera identified among our isolated CRB, especially from agricultural soils, included the genera Enterococcus, Bradyrhizobium, Achromobacter, and Planomicrobium. Interestingly, all of these carbapenem-resistant isolates were also intermediate or resistant to at least 1 noncarbapenem antibiotic. Further characterization of our isolated CRB revealed that 11 Stenotrophomonas, 3 Pseudomonas, 1 Enterococcus, and 1 Bradyrhizobium isolates were carbapenemase producers. Our findings show for the first time that both urban and agricultural soils in Southern California are an underappreciated reservoir of bacteria resistant to carbapenems and other antibiotics, including carbapenemase-producing CRB.

Comparative Genomics Reveals a Well-Conserved Intrinsic Resistome in the Emerging Multidrug-Resistant Pathogen Cupriavidus gilardii.

The Gram-negative bacterium Cupriavidus gilardii is an emerging multidrug-resistant pathogen found in many environments. However, little is known about this species or its antibiotic resistance mechanisms. We used biochemical tests, antibiotic susceptibility experiments, and whole-genome sequencing to characterize an environmental C. gilardii isolate. Like clinical isolates, this isolate was resistant to meropenem, gentamicin, and other antibiotics. Resistance to these antibiotics appeared to be related to the large number of intrinsic antibiotic resistance genes found in this isolate. As determined by comparative genomics, this resistome was also well conserved in the only two other C. gilardii strains sequenced to date. The intrinsic resistome of C. gilardii did not include the colistin resistance gene mcr-5, which was in a transposon present only in one strain. The intrinsic resistome of C. gilardii was comprised of (i) many multidrug efflux pumps, such as a homolog of the Pseudomonas aeruginosa MexAB-OprM pump that may be involved in resistance to meropenem, other ß-lactams, and aminoglycosides; (ii) a novel ß-lactamase (OXA-837) that decreases susceptibility to ampicillin but not to other ß-lactams tested; (iii) a new aminoglycoside 3-N-acetyltransferase [AAC(3)-IVb, AacC10] that decreases susceptibility to gentamicin and tobramycin; and (iv) a novel partially conserved aminoglycoside 3"-adenylyltransferase [ANT(3")-Ib, AadA32] that decreases susceptibility to spectinomycin and streptomycin. These findings provide the first mechanistic insight into the intrinsic resistance of C. gilardii to multiple antibiotics and its ability to become resistant to an increasing number of drugs during therapy.IMPORTANCECupriavidus gilardii is a bacterium that is gaining increasing attention both as an infectious agent and because of its potential use in the detoxification of toxic compounds and other biotechnological applications. In recent years, however, there has been an increasing number of reported infections, some of them fatal, caused by C. gilardii These infections are hard to treat because this bacterium is naturally resistant to many antibiotics, including last-resort antibiotics, such as carbapenems. Moreover, this bacterium often becomes resistant to additional antibiotics during therapy. However, little is known about C. gilardii and its antibiotic resistance mechanisms. The significance of our research is in providing, for the first time, whole-genome information about the natural antibiotic resistance genes found in this bacterium and their conservation among different C. gilardii strains. This information may provide new insights into the appropriate use of antibiotics in combating infections caused by this emerging pathogen.

Prevalence and characterization of carbapenem-resistant bacteria in water bodies in the Los Angeles-Southern California area.

Carbapenems are ß-lactam antibiotics used in healthcare settings as last resort drugs to treat infections caused by antibiotic-resistant bacteria. Carbapenem-resistant bacteria are increasingly being isolated from healthcare facilities; however, little is known about their distribution or prevalence in the environment, especially in the United States, where their distribution in water environments from the West Coast has not been studied before. The aim of this study was to determine the prevalence of carbapenem-resistant bacteria and carbapenemase genes in water bodies from the Los Angeles area (California, USA). All samples that were analyzed contained carbapenem-resistant bacteria with a frequency of between 0.1 and 324 carbapenem-resistant cfu per 100 mls of water. We identified 76 carbapenem-resistant or -intermediate isolates, most of which were also resistant to noncarbapenem antibiotics, as different strains of Enterobacter asburiae, Aeromonas veronii, Cupriavidus gilardii, Pseudomonas, and Stenotrophomonas species. Of them, 52 isolates were carbapenemase-producers. Furthermore, PCR and sequence analysis to identify the carbapenemase gene of these carbapenemase-producing isolates revealed that all Enterobacter asburiae isolates had a blaIMI-2 gene 100% identical to the reference sequence, and all Stenotrophomonas maltophlia isolates had a blaL1 gene 83%-99% identical to the reference blaL1 . Our findings indicate that water environments in Southern California are an important reservoir of bacteria-resistant to carbapenems and other antibiotics, including bacteria carrying intrinsic and acquired carbapenemase genes.

A mutant with aberrant extracellular LcrV-YscF interactions fails to form pores and translocate Yop effector proteins but retains the ability to trigger Yop secretion in response to host cell contact.

The plasmid-encoded type three secretion system (TTSS) of Yersinia spp. is responsible for the delivery of effector proteins into cells of the innate immune system, where these effectors disrupt the target cells' activity. Successful translocation of effectors into mammalian cells requires Yersinia to both insert a translocon into the host cell membrane and sense contact with host cells. To probe the events necessary for translocation, we investigated protein-protein interactions among TTSS components of the needle-translocon complex using a chemical cross-linking-based approach. We detected extracellular protein complexes containing YscF, LcrV, and YopD that were dependent upon needle formation. The formation of these complexes was evaluated in a secretion-competent but translocation-defective mutant, the YscFD28AD46A strain (expressing YscF with the mutations D28A and D46A). We found that one of the YscF and most of the LcrV and YopD cross-linked complexes were nearly absent in this mutant. Furthermore, the YscFD28AD46A strain did not support YopB insertion into mammalian membranes, supporting the idea that the LcrV tip complex is required for YopB insertion and translocon formation. However, the YscFD28AD46A strain did secrete Yops in the presence of host cells, indicating that a translocation-competent tip complex is not required to sense contact with host cells to trigger Yop secretion. In conclusion, in the absence of cross-linkable LcrV-YscF interactions, translocon insertion is abolished, but Yersinia still retains the ability to sense cell contact.

Identification and characterization of small-molecule inhibitors of Yop translocation in Yersinia pseudotuberculosis.

Type three secretion systems (TTSSs) are virulence factors found in many pathogenic Gram-negative species, including the family of pathogenic Yersinia spp. Yersinia pseudotuberculosis requires the translocation of a group of effector molecules, called Yops, to subvert the innate immune response and establish infection. Polarized transfer of Yops from bacteria to immune cells depends on several factors, including the presence of a functional TTSS, the successful attachment of Yersinia to the target cell, and translocon insertion into the target cell membrane. Here we employed a high-throughput screen to identify small molecules that block translocation of Yops into mammalian cells. We identified 6 compounds that inhibited translocation of effectors without affecting synthesis of TTSS components and secreted effectors, assembly of the TTSS, or secretion of effectors. One compound, C20, reduced adherence of Y. pseudotuberculosis to target cells. Additionally, the compounds caused leakage of Yops into the supernatant during infection and thus reduced polarized translocation. Furthermore, several molecules, namely, C20, C22, C24, C34, and C38, also inhibited ExoS-mediated cell rounding, suggesting that the compounds target factors that are conserved between Pseudomonas aeruginosa and Y. pseudotuberculosis. In summary, we have identified 6 compounds that specifically inhibit translocation of Yops into mammalian cells but not Yop synthesis or secretion.