The composition of planktonic prokaryotic communities in a hospital building water system depends on both incoming water and flow dynamics.

In recent years, the frequency of nosocomial infections has increased. Hospital water systems support the growth of microbes, especially opportunistic premise plumbing pathogens. In this study, planktonic prokaryotic communities present in water samples taken from hospital showers and hand basins, collected over three different sampling phases, were characterized by 16S rRNA gene amplicon sequencing. Significant differences in the abundance of various prokaryotic taxa were found through univariate and multivariate analysis. Overall, the prokaryotic communities of hospital water were taxonomically diverse and dominated by biofilm forming, corrosion causing, and potentially pathogenic bacteria. The phyla Proteobacteria, Actinobacteriota, Bacteroidota, Planctomycetota, Firmicutes, and Cyanobacteria made up 96% of the relative abundance. The α-diversity measurements of prokaryotic communities showed no difference in taxa evenness and richness based on sampling sites (shower or hand basins), sampling phases (months), and presence or absence of Vermamoeba vermiformis. However, ß-diversity measurements showed significant clustering of prokaryotic communities based on sampling phases, with the greatest difference observed between the samples collected in phase 1 vs phase 2/3. Importantly, significant difference was observed in prokaryotic communities based on flow dynamics of the incoming water. The Pielou's evenness diversity index revealed a significant difference (Kruskal Wallis, p < 0.05) and showed higher species richness in low flow regime (< 13 minutes water flushing per week and ≤ 765 flushing events per six months). Similarly, Bray-Curtis dissimilarity index found significant differences (PERMANOVA, p < 0.05) in the prokaryotic communities of low vs medium/high flow regimes. Furthermore, linear discriminant analysis effect size showed that several biofilm forming (e.g., Pseudomonadales), corrosion causing (e.g., Desulfobacterales), extremely environmental stress resistant (e.g., Deinococcales), and potentially pathogenic (e.g., Pseudomonas) bacterial taxa were in higher amounts under low flow regime conditions. This study demonstrated that a hospital building water system consists of a complex microbiome that is shaped by incoming water quality and the building flow dynamics arising through usage.

Stagnation arising through intermittent usage is associated with increased viable but non culturable Legionella and amoeba hosts in a hospital water system.

Hospital water systems are a significant source of Legionella, resulting in the potentially fatal Legionnaires' disease. One of the biggest challenges for Legionella management within these systems is that under unfavorable conditions Legionella transforms itself into a viable but non culturable (VBNC) state that cannot be detected using the standard methods. This study used a novel method (flow cytometry-cell sorting and qPCR [VFC+qPCR] assay) concurrently with the standard detection methods to examine the effect of temporary water stagnation, on Legionella spp. and microbial communities present in a hospital water system. Water samples were also analyzed for amoebae using culture and Vermamoeba vermiformis and Acanthamoeba specific qPCR. The water temperature, number and duration of water flow events for the hand basins and showers sampled was measured using the Enware Smart Flow® monitoring system. qPCR analysis demonstrated that 21.8% samples were positive for Legionella spp., 21% for L. pneumophila, 40.9% for V. vermiformis and 4.2% for Acanthamoeba. All samples that were Legionella spp. positive using qPCR (22%) were also positive for VBNC Legionella spp.; however, only 2.5% of samples were positive for culturable Legionella spp. 18.1% of the samples were positive for free-living amoebae (FLA) using culture. All samples positive for Legionella spp. were also positive for FLA. Samples with a high heterotrophic plate count (HPC ≥ 5 × 103 CFU/L) were also significantly associated with high concentrations of Legionella spp. DNA, VBNC Legionella spp./L. pneumophila (p < 0.01) and V. vermiformis (p < 0.05). Temporary water stagnation arising through intermittent usage (< 2 hours of usage per month) significantly (p < 0.01) increased the amount of Legionella spp. DNA, VBNC Legionella spp./L. pneumophila, and V. vermiformis; however, it did not significantly impact the HPC load. In contrast to stagnation, no relationship was observed between the microbes and water temperature. In conclusion, Legionella spp. (DNA and VBNC) was associated with V. vermiformis, heterotrophic bacteria, and stagnation occurring through intermittent usage. This is the first study to monitor VBNC Legionella spp. within a hospital water system. The high percentage of false negative Legionella spp. results provided by the culture method supports the use of either qPCR or VFC+qPCR to monitor Legionella spp. contamination within hospital water systems.

The role of TMS 12 in the staphylococcal multidrug efflux protein QacA.

OBJECTIVES: To elucidate the importance of a region in QacA predicted to be important in antimicrobial substrate recognition. METHODS: A total of 38 amino acid residues within or flanking putative transmembrane helix segment (TMS) 12 of QacA were individually replaced with cysteine using site-directed mutagenesis. The impact of these mutations on protein expression, drug resistance, transport activity and interaction with sulphhydryl-binding compounds was determined. RESULTS: Accessibility analysis of cysteine-substituted mutants identified the extents of TMS 12, which allowed for refinement of the QacA topology model. Mutation of Gly-361, Gly-379 and Ser-387 in QacA resulted in reduced resistance to at least one bivalent substrate. Interaction with sulphhydryl-binding compounds in efflux and binding assays demonstrated the role of Gly-361 and Ser-387 in the binding and transport pathway of specific substrates. The highly conserved residue Gly-379 was found to be important for the transport of bivalent substrates, commensurate with the role of glycine residues in helical flexibility and interhelical interactions. CONCLUSIONS: TMS 12 and its external flanking loop is required for the structural and functional integrity of QacA and contains amino acids directly involved in the interaction with substrates.

Detection and quantification of viable but non-culturable Legionella pneumophila from water samples using flow cytometry-cell sorting and quantitative PCR.

Legionella pneumophila is a waterborne pathogen and, as the causative agent of Legionnaires' disease, a significant public health concern. Exposure to environmental stresses, and disinfection treatments, promotes the formation of resistant and potentially infectious viable but non-culturable (VBNC) Legionella. The management of engineered water systems to prevent Legionnaires' disease is hindered by the presence of VBNC Legionella that cannot be detected using the standard culture (ISO11731:2017-05) and quantitative polymerase reaction (ISO/TS12869:2019) methods. This study describes a novel method to quantify VBNC Legionella from environmental water samples using a "viability based flow cytometry-cell sorting and qPCR" (VFC + qPCR) assay. This protocol was then validated by quantifying the VBNC Legionella genomic load from hospital water samples. The VBNC cells were unable to be cultured on Buffered Charcoal Yeast Extract (BCYE) agar; however, their viability was confirmed through their ATP activity and ability to infect amoeba hosts. Subsequently, an assessment of the ISO11731:2017-05 pre-treatment procedure demonstrated that acid or heat treatment cause underestimation of alive Legionella population. Our results showed that these pre-treatment procedures induce culturable cells to enter a VBNC state. This may explain the observed insensitivity and lack of reproducibility often observed with the Legionella culture method. This study represents the first time that flow cytometry-cell sorting in conjunction with a qPCR assay has been used as a rapid and direct method to quantify VBNC Legionella from environmental sources. This will significantly improve future research evaluating Legionella risk management approaches for the control of Legionnaires' disease.

Molecular screening and characterization of Legionella pneumophila associated free-living amoebae in domestic and hospital water systems.

Free-living amoebae are ubiquitous in the environment and cause both opportunistic and non-opportunistic infections in humans. Some genera of amoebae are natural reservoirs of opportunistic plumbing pathogens, such as Legionella pneumophila. In this study, the presence of free-living amoebae and Legionella was investigated in 140 water and biofilm samples collected from Australian domestic (n = 68) and hospital water systems (n = 72). Each sample was screened in parallel using molecular and culture-based methods. Direct quantitative polymerase chain reaction (qPCR) assays showed that 41% samples were positive for Legionella, 33% for L. pneumophila, 11% for Acanthamoeba, and 55% for Vermamoeba vermiformis gene markers. Only 7% of samples contained culturable L. pneumophila serogroup (sg)1, L. pneumophila sg2-14, and non-pneumophila Legionella. In total, 69% of samples were positive for free-living amoebae using any method. Standard culturing found that 41% of the samples were positive for amoeba (either Acanthamoeba, Allovahlkampfia, Stenamoeba, or V. vermiformis). V. vermiformis showed the highest overall frequency of occurrence. Acanthamoeba and V. vermiformis isolates demonstrated high thermotolerance and osmotolerance and strong broad spectrum bacteriogenic activity against Gram-negative and Gram-positive bacteria. Importantly, all Legionella positive samples were also positive for amoeba, and this co-occurrence was statistically significant (p < 0.05). According to qPCR and fluorescence in situ hybridization, V. vermiformis and Allovahlkampfia harboured intracellular L. pneumophila. To our knowledge, this is the first time Allovahlkampfia and Stenamoeba have been demonstrated as hosts of L. pneumophila in potable water. These results demonstrate the importance of amoebae in engineered water systems, both as a pathogen and as a reservoir of Legionella. The high frequency of gymnamoebae detected in this study from Australian engineered water systems identifies an issue of significant public health concern. Future water management protocols should incorporate treatments strategies to control amoebae to reduce the risk to end users.

Picrotoxin Delineates Different Transport Configurations for Malate and γ Aminobutyric Acid through TaALMT1.

Plant-derived pharmacological agents have been used extensively to dissect the structure-function relationships of mammalian GABA receptors and ion channels. Picrotoxin is a non-competitive antagonist of mammalian GABAA receptors. Here, we report that picrotoxin inhibits the anion (malate) efflux mediated by wheat (Triticum aestivum) ALMT1 but has no effect on GABA transport. The EC50 for inhibition was 0.14 nM and 0.18 nM when the ALMTs were expressed in tobacco BY2 cells and in Xenopus oocytes, respectively. Patch clamping of the oocyte plasma membrane expressing wheat ALMT1 showed that picrotoxin inhibited malate currents from both sides of the membrane. These results demonstrate that picrotoxin inhibits anion efflux effectively and can be used as a new inhibitor to study the ion fluxes mediated by ALMT proteins that allow either GABA or anion transport.

Hospital water as the source of healthcare-associated infection and antimicrobial-resistant organisms.

PURPOSE OF REVIEW: Drinking water is considered one of the most overlooked and underestimated sources of healthcare-associated infections (HAIs). Recently, the prevention and control of opportunistic premise plumbing pathogens (OPPPs) in healthcare water systems has been receiving increasing attention in infection control guidelines. However, these fail to address colonization of pathogens that do not originate from source water. Subsequently, this review explores the role of water and premise plumbing biofilm in HAIs. The potential mechanisms of contamination and transmission of antimicrobial-resistant (AMR) pathogens originating both from supply water and human microbiota are discussed. RECENT FINDINGS: OPPPs, such as Legionella pneumophila, Pseudomonas aeruginosa and Mycobacterium avium have been described as native to the plumbing environment. However, other pathogens, not found in the source water, have been found to proliferate in biofilms formed on outlets devices and cause HAI outbreaks. SUMMARY: Biofilms formed on outlet devices, such as tap faucets, showers and drains provide an ideal niche for the dissemination of antimicrobial resistance. Thus, comprehensive surveillance guidelines are required to understand the role that drinking water and water-related devices play in the transmission of AMR HAIs and to improve infection control guidelines.

The StkSR Two-Component System Influences Colistin Resistance in Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic human pathogen responsible for numerous severe nosocomial infections. Genome analysis on the A. baumannii clinical isolate 04117201 revealed the presence of 13 two-component signal transduction systems (TCS). Of these, we examined the putative TCS named here as StkSR. The stkR response regulator was deleted via homologous recombination and its progeny, ΔstkR, was phenotypically characterized. Antibiogram analyses of ΔstkR cells revealed a two-fold increase in resistance to the clinically relevant polymyxins, colistin and polymyxin B, compared to wildtype. PAGE-separation of silver stained purified lipooligosaccharide isolated from ΔstkR and wildtype cells ruled out the complete loss of lipooligosaccharide as the mechanism of colistin resistance identified for ΔstkR. Hydrophobicity analysis identified a phenotypical change of the bacterial cells when exposed to colistin. Transcriptional profiling revealed a significant up-regulation of the pmrCAB operon in ΔstkR compared to the parent, associating these two TCS and colistin resistance. These results reveal that there are multiple levels of regulation affecting colistin resistance; the suggested 'cross-talk' between the StkSR and PmrAB two-component systems highlights the complexity of these systems.

Factors affecting the isolation and diversity of marine sponge-associated bacteria.

Marine sponges are an ideal source for isolating as yet undiscovered microorganisms with some sponges having about 50% of their biomass composed of microbial symbionts. This study used a variety of approaches to investigate the culturable diversity of the sponge-associated bacterial community from samples collected from the South Australian marine environment. Twelve sponge samples were selected from two sites and their bacterial population cultivated using seven different agar media at two temperatures and three oxygen levels over 3 months. These isolates were identified using microscopic, macroscopic, and 16S rRNA gene analysis. A total of 1234 bacterial colonies were isolated which consisted of four phyla: Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes, containing 21 genera. The diversity of the bacterial population was demonstrated to be influenced by the type of isolation medium, length of the incubation period and temperature, sponge type, and oxygen level. The findings of this study showed that marine sponges of South Australia can yield considerable bacterial culturable diversity if a comprehensive isolation strategy is implemented. Two sponges, with the highest and the lowest diversity of culturable isolates, were examined using next-generation sequencing to better profile the bacterial population. A marked difference in terms of phyla and genera was observed using culture-based and culture-independent approaches. This observed variation displays the importance of utilizing both methods to reflect a more complete picture of the microbial population of marine sponges. KEY POINTS: Improved bacterial diversity due to long incubations, 2 temperatures, and 3 oxygen levels. Isolates identified by morphology, restriction digests, and 16S rRNA gene sequencing. At least 70% of culturable genera were not revealed by NGS methods.

Efflux Pump Mediated Antimicrobial Resistance by Staphylococci in Health-Related Environments: Challenges and the Quest for Inhibition.

The increasing emergence of antimicrobial resistance in staphylococcal bacteria is a major health threat worldwide due to significant morbidity and mortality resulting from their associated hospital- or community-acquired infections. Dramatic decrease in the discovery of new antibiotics from the pharmaceutical industry coupled with increased use of sanitisers and disinfectants due to the ongoing COVID-19 pandemic can further aggravate the problem of antimicrobial resistance. Staphylococci utilise multiple mechanisms to circumvent the effects of antimicrobials. One of these resistance mechanisms is the export of antimicrobial agents through the activity of membrane-embedded multidrug efflux pump proteins. The use of efflux pump inhibitors in combination with currently approved antimicrobials is a promising strategy to potentiate their clinical efficacy against resistant strains of staphylococci, and simultaneously reduce the selection of resistant mutants. This review presents an overview of the current knowledge of staphylococcal efflux pumps, discusses their clinical impact, and summarises compounds found in the last decade from plant and synthetic origin that have the potential to be used as adjuvants to antibiotic therapy against multidrug resistant staphylococci. Critically, future high-resolution structures of staphylococcal efflux pumps could aid in design and development of safer, more target-specific and highly potent efflux pump inhibitors to progress into clinical use.

A Unique Sequence Is Essential for Efficient Multidrug Efflux Function of the MtrD Protein of Neisseria gonorrhoeae.

Antimicrobial resistance in Neisseria gonorrhoeae has reached an alarming level, severely impacting the effective treatment of gonorrhea. Belonging to the resistance-nodulation-cell division (RND) superfamily of efflux transporters, the MtrD membrane protein of N. gonorrhoeae provides resistance to a broad range of antimicrobial compounds. A unique feature of MtrD is an 11-residue sequence (from N917 to P927 [N917-P927]) that connects transmembrane helices (TMS) 9 and 10; this sequence is not present in homologous RND proteins. This study explores the structural and functional roles of the N917-P927 region by means of mutant analysis and molecular dynamics simulations. We show that N917-P927 plays a key role in modulating substrate access to the binding cleft and influences the overall orientation of the protein within the inner membrane necessary for optimal functioning. Removal of N917-P927 significantly reduced MtrD-mediated resistance to a range of antimicrobials and mutations of three single amino acids impacted MtrD-mediated multidrug resistance. Furthermore, molecular dynamics simulations showed deletion of N917-P927 in MtrD may dysregulate access of the substrate to the binding cleft and closure of the substrate-binding pocket during the transport cycle. These findings indicate that N917-P927 is a key region for interacting with the inner membrane, conceivably influencing substrate capture from the membrane-periplasm interface and thus is essential for full multidrug resistance capacity of MtrD. IMPORTANCE The historical sexually transmitted infection gonorrhea continues to be a major public health concern with an estimated global annual incidence of 86.9 million cases. N. gonorrhoeae has been identified by the World Health Organization as one of the 12 antimicrobial-resistant bacterial species that poses the greatest risk to human health. As the major efflux pump in gonococci, the MtrD transporter contributes to the cell envelope barrier in this organism and pumps antimicrobials from the periplasm and inner membrane, resulting in resistance. This study demonstrates that a unique region of the MtrD protein that connects TMS 9 and TMS 10 forms a structure that may interact with the inner membrane positioning TMS 9 and stabilizing the protein facilitating substrate capture from the inner membrane-periplasm interface. Analysis of mutants of this region identified that it was essential for MtrD-mediated multidrug resistance. Characterization of the structure and function of this unique local region of MtrD has implications for drug efflux mechanisms used by related proteins and is important knowledge for development of antibiotics that bypass efflux.

Coordination of Substrate Binding and Protonation in the N. gonorrhoeae MtrD Efflux Pump Controls the Functionally Rotating Transport Mechanism.

Multidrug resistance is a serious problem that threatens the effective treatment of the widespread sexually transmitted disease gonorrhea, caused by the Gram-negative bacterium Neisseria gonorrhoeae. The drug efflux pump primarily implicated in N. gonorrhoeae antimicrobial resistance is the inner membrane transporter MtrD, which forms part of the tripartite multiple transferable resistance (Mtr) CDE efflux system. A structure of MtrD was first solved in 2014 as a symmetrical homotrimer, and then, recently, as an asymmetrical homotrimer. Through a series of molecular dynamics simulations and mutagenesis experiments, we identify the combination of substrate binding and protonation states of the proton relay network that drives the transition from the symmetric to the asymmetric conformation of MtrD. We characterize the allosteric coupling between the functionally important local regions that control conformational changes between the access, binding, and extrusion states and allow for transition to the asymmetric MtrD conformation. We also highlight a significant rotation of the transmembrane helices caused by protonation of the proton relay network, which widens the intermonomeric gap that is a hallmark of the rotational transporter mechanism. This is the first analysis and description of the transport mechanism for the N. gonorrhoeae MtrD transporter and provides evidence that antimicrobial efflux in MtrD follows the functionally rotating transport mechanism seen in protein homologues from the same transport protein superfamily.

Antimicrobial Activities of Marine Sponge-Associated Bacteria.

The misuse and overuse of antibiotics have led to the emergence of multidrug-resistant microorganisms, which decreases the chance of treating those infected with existing antibiotics. This resistance calls for the search of new antimicrobials from prolific producers of novel natural products including marine sponges. Many of the novel active compounds reported from sponges have originated from their microbial symbionts. Therefore, this study aims to screen for bioactive metabolites from bacteria isolated from sponges. Twelve sponge samples were collected from South Australian marine environments and grown on seven isolation media under four incubation conditions; a total of 1234 bacterial isolates were obtained. Of these, 169 bacteria were tested in media optimized for production of antimicrobial metabolites and screened against eleven human pathogens. Seventy bacteria were found to be active against at least one test bacterial or fungal pathogen, while 37% of the tested bacteria showed activity against Staphylococcus aureus including methicillin-resistant strains and antifungal activity was produced by 21% the isolates. A potential novel active compound was purified possessing inhibitory activity against S. aureus. Using 16S rRNA, the strain was identified as Streptomyces sp. Our study highlights that the marine sponges of South Australia are a rich source of abundant and diverse bacteria producing metabolites with antimicrobial activities against human pathogenic bacteria and fungi.

Water as a Source of Antimicrobial Resistance and Healthcare-Associated Infections.

Healthcare-associated infections (HAIs) are one of the most common patient complications, affecting 7% of patients in developed countries each year. The rise of antimicrobial resistant (AMR) bacteria has been identified as one of the biggest global health challenges, resulting in an estimated 23,000 deaths in the US annually. Environmental reservoirs for AMR bacteria such as bed rails, light switches and doorknobs have been identified in the past and addressed with infection prevention guidelines. However, water and water-related devices are often overlooked as potential sources of HAI outbreaks. This systematic review examines the role of water and water-related devices in the transmission of AMR bacteria responsible for HAIs, discussing common waterborne devices, pathogens, and surveillance strategies. AMR strains of previously described waterborne pathogens including Pseudomonas aeruginosa, Mycobacterium spp., and Legionella spp. were commonly isolated. However, methicillin-resistant Staphylococcus aureus and carbapenem-resistant Enterobacteriaceae that are not typically associated with water were also isolated. Biofilms were identified as a hot spot for the dissemination of genes responsible for survival functions. A limitation identified was a lack of consistency between environmental screening scope, isolation methodology, and antimicrobial resistance characterization. Broad universal environmental surveillance guidelines must be developed and adopted to monitor AMR pathogens, allowing prediction of future threats before waterborne infection outbreaks occur.

Legionella pneumophila and Protozoan Hosts: Implications for the Control of Hospital and Potable Water Systems.

Legionella pneumophila is an opportunistic waterborne pathogen of public health concern. It is the causative agent of Legionnaires' disease (LD) and Pontiac fever and is ubiquitous in manufactured water systems, where protozoan hosts and complex microbial communities provide protection from disinfection procedures. This review collates the literature describing interactions between L. pneumophila and protozoan hosts in hospital and municipal potable water distribution systems. The effectiveness of currently available water disinfection protocols to control L. pneumophila and its protozoan hosts is explored. The studies identified in this systematic literature review demonstrated the failure of common disinfection procedures to achieve long term elimination of L. pneumophila and protozoan hosts from potable water. It has been demonstrated that protozoan hosts facilitate the intracellular replication and packaging of viable L. pneumophila in infectious vesicles; whereas, cyst-forming protozoans provide protection from prolonged environmental stress. Disinfection procedures and protozoan hosts also facilitate biogenesis of viable but non-culturable (VBNC) L. pneumophila which have been shown to be highly resistant to many water disinfection protocols. In conclusion, a better understanding of L. pneumophila-protozoan interactions and the structure of complex microbial biofilms is required for the improved management of L. pneumophila and the prevention of LD.

Characterization of a Halotolerant Fungus from a Marine Sponge.

INTRODUCTION: Marine sponges have established symbiotic interactions with a large number of microorganisms including fungi. Most of the studies so far have focussed on the characterization of sponge-associated bacteria and archaea with only a few reports on sponge-associated fungi. During the isolation and characterization of bacteria from marine sponges of South Australia, we observed multiple types of fungi. One isolate in particular was selected for further investigation due to its unusually large size and being chromogenic. Here, we report on the investigations on the physical, morphological, chemical, and genotypic properties of this yeast-like fungus. METHODS AND MATERIALS: Sponge samples were collected from South Australian marine environments, and microbes were isolated using different isolation media under various incubation conditions. Microbial isolates were identified on the basis of morphology, staining characteristics, and their 16S rRNA or ITS/28S rRNA gene sequences. RESULTS: Twelve types of yeast and fungal isolates were detected together with other bacteria and one of these fungi measured up to 35 µm in diameter with a unique chromogen compared to other fungi. Depending on the medium type, this unique fungal isolate appeared as yeast-like fungi with different morphological forms. The isolate can ferment and assimilate nearly all of the tested carbohydrates. Furthermore, it tolerated a high concentration of salt (up to 25%) and a range of pH and temperature. ITS and 28S rRNA gene sequencing revealed a sequence similarity of 93% and 98%, respectively, with the closest genera of Eupenidiella, Hortaea, and Stenella. CONCLUSIONS: On the basis of its peculiar morphology, size, and genetic data, this yeast-like fungus possibly constitutes a new genus and the name Magnuscella marinae, gen nov., sp. nov., is proposed. This study is the first of its kind for the complete characterization of a yeast-like fungus from marine sponges. This novel isolate developed a symbiotic interaction with living hosts, which was not observed with other reported closest genera (they exist in a saprophytic relationship). The observed unique size and morphology may favour this new isolate to establish symbiotic interactions with living hosts.

Multidrug Resistance in Neisseria gonorrhoeae: Identification of Functionally Important Residues in the MtrD Efflux Protein.

A key mechanism that Neisseria gonorrhoeae uses to achieve multidrug resistance is the expulsion of structurally different antimicrobials by the MtrD multidrug efflux protein. MtrD resembles the homologous Escherichia coli AcrB efflux protein with several common structural features, including an open cleft containing putative access and deep binding pockets proposed to interact with substrates. A highly discriminating N. gonorrhoeae strain, with the MtrD and NorM multidrug efflux pumps inactivated, was constructed and used to confirm and extend the substrate profile of MtrD to include 14 new compounds. The structural basis of substrate interactions with MtrD was interrogated by a combination of long-timescale molecular dynamics simulations and docking studies together with site-directed mutagenesis of selected residues. Of the MtrD mutants generated, only one (S611A) retained a wild-type (WT) resistance profile, while others (F136A, F176A, I605A, F610A, F612C, and F623C) showed reduced resistance to different antimicrobial compounds. Docking studies of eight MtrD substrates confirmed that many of the mutated residues play important nonspecific roles in binding to these substrates. Long-timescale molecular dynamics simulations of MtrD with its substrate progesterone showed the spontaneous binding of the substrate to the access pocket of the binding cleft and its subsequent penetration into the deep binding pocket, allowing the permeation pathway for a substrate through this important resistance mechanism to be identified. These findings provide a detailed picture of the interaction of MtrD with substrates that can be used as a basis for rational antibiotic and inhibitor design.IMPORTANCE With over 78 million new infections globally each year, gonorrhea remains a frustratingly common infection. Continuous development and spread of antimicrobial-resistant strains of Neisseria gonorrhoeae, the causative agent of gonorrhea, have posed a serious threat to public health. One of the mechanisms in N. gonorrhoeae involved in resistance to multiple drugs is performed by the MtrD multidrug resistance efflux pump. This study demonstrated that the MtrD pump has a broader substrate specificity than previously proposed and identified a cluster of residues important for drug binding and translocation. Additionally, a permeation pathway for the MtrD substrate progesterone actively moving through the protein was determined, revealing key interactions within the putative MtrD drug binding pockets. Identification of functionally important residues and substrate-protein interactions of the MtrD protein is crucial to develop future strategies for the treatment of multidrug-resistant gonorrhea.

MITE Aba12 , a Novel Mobile Miniature Inverted-Repeat Transposable Element Identified in Acinetobacter baumannii ATCC 17978 and Its Prevalence across the Moraxellaceae Family.

Insertion sequences (IS) are fundamental mediators of genome plasticity with the potential to generate phenotypic variation with significant evolutionary outcomes. Here, a recently active miniature inverted-repeat transposon element (MITE) was identified in a derivative of Acinetobacter baumannii ATCC 17978 after being subjected to stress conditions. Transposition of the novel element led to the disruption of the hns gene, resulting in a characteristic hypermotile phenotype. DNA identity shared between the terminal inverted repeats of this MITE and coresident ISAba12 elements, together with the generation of 9-bp target site duplications, provides strong evidence that ISAba12 elements were responsible for mobilization of the MITE (designated MITE Aba12 ) within this strain. A wider genome-level survey identified MITE Aba12 in 30 additional Acinetobacter genomes at various frequencies and one Moraxella osloensis genome. Ninety MITE Aba12 copies could be identified, of which 40% had target site duplications, indicating recent transposition events. Elements ranged between 111 and 114 bp; 90% were 113 bp in length. Using the MITE Aba12 consensus sequence, putative outward-facing Escherichia coli σ70 promoter sequences in both orientations were identified. The identification of transcripts originating from the promoter in one direction supports the proposal that the element can influence neighboring host gene transcription. The location of MITE Aba12 varied significantly between and within genomes, preferentially integrating into AT-rich regions. Additionally, a copy of MITE Aba12 was identified in a novel 8.5-kb composite transposon, Tn6645, in the M. osloensis CCUG 350 chromosome. Overall, this study shows that MITE Aba12 is the most abundant nonautonomous element currently found in AcinetobacterIMPORTANCE One of the most important weapons in the armory of Acinetobacter is its impressive genetic plasticity, facilitating rapid genetic mutations and rearrangements as well as integration of foreign determinants carried by mobile genetic elements. Of these, IS are considered one of the key forces shaping bacterial genomes and ultimately evolution. We report the identification of a novel nonautonomous IS-derived element present in multiple bacterial species from the Moraxellaceae family and its recent translocation into the hns locus in the A. baumannii ATCC 17978 genome. The latter finding adds new knowledge to only a limited number of documented examples of MITEs in the literature and underscores the plastic nature of the hns locus in A. baumannii MITE Aba12 , and its predicted parent(s), may be a source of substantial adaptive evolution within environmental and clinically relevant bacterial pathogens and, thus, have broad implications for niche-specific adaptation.

Resistance to pentamidine is mediated by AdeAB, regulated by AdeRS, and influenced by growth conditions in Acinetobacter baumannii ATCC 17978.

In recent years, effective treatment of infections caused by Acinetobacter baumannii has become challenging due to the ability of the bacterium to acquire or up-regulate antimicrobial resistance determinants. Two component signal transduction systems are known to regulate expression of virulence factors including multidrug efflux pumps. Here, we investigated the role of the AdeRS two component signal transduction system in regulating the AdeAB efflux system, determined whether AdeA and/or AdeB can individually confer antimicrobial resistance, and explored the interplay between pentamidine resistance and growth conditions in A. baumannii ATCC 17978. Results identified that deletion of adeRS affected resistance towards chlorhexidine and 4',6-diamidino-2-phenylindole dihydrochloride, two previously defined AdeABC substrates, and also identified an 8-fold decrease in resistance to pentamidine. Examination of ΔadeA, ΔadeB and ΔadeAB cells augmented results seen for ΔadeRS and identified a set of dicationic AdeAB substrates. RNA-sequencing of ΔadeRS revealed transcription of 290 genes were ≥2-fold altered compared to the wildtype. Pentamidine shock significantly increased adeA expression in the wildtype, but decreased it in ΔadeRS, implying that AdeRS activates adeAB transcription in ATCC 17978. Investigation under multiple growth conditions, including the use of Biolog phenotypic microarrays, revealed resistance to pentamidine in ATCC 17978 and mutants could be altered by bioavailability of iron or utilization of different carbon sources. In conclusion, the results of this study provide evidence that AdeAB in ATCC 17978 can confer intrinsic resistance to a subset of dicationic compounds and in particular, resistance to pentamidine can be significantly altered depending on the growth conditions.