Multidrug resistant Acinetobacter baumannii is a serious healthcare threat. In fact, the Center for Disease Control recently reported that carbapenem-resistant A. baumannii is responsible for more than 8,500 infections, 700 deaths, and $281 million in healthcare costs annually in the United States with few, if any, treatment options available, leading to its designation as a pathogen of urgent concern and a priority for novel antimicrobial development. It is hypothesized that biofilms are, at least in part, responsible for the high prevalence of A. baumannii nosocomial and recurrent infections because they frequently contaminate hospital surfaces and patient indwelling devices; therefore, there has been a recent push for mechanistic understanding of biofilm formation, maturation and dispersal. However, most research has focused on A. baumannii pneumonia and bloodstream infections, despite a recent retrospective study showing that 17.1% of A. baumannii isolates compiled from clinical studies over the last two decades were obtained from urinary samples. This highlights that A. baumannii is an underappreciated uropathogen. The following minireview will examine our current understanding of A. baumannii biofilm formation and how this influences urinary tract colonization and pathogenesis.

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Insights Into Mechanisms of Biofilm Formation in Acinetobacter baumannii and Implications for Uropathogenesis.

Chronic and recurrent bacterial infections are frequently associated with the formation of biofilms on biotic or abiotic materials that are composed of mono- or multi-species cultures of bacteria/fungi embedded in an extracellular matrix produced by the microorganisms. Biofilm formation is, among others, regulated by quorum sensing (QS) which is an interbacterial communication system usually composed of two-component systems (TCSs) of secreted autoinducer compounds that activate signal transduction pathways through interaction with their respective receptors. Embedded in the biofilms, the bacteria are protected from environmental stress stimuli, and they often show reduced responses to antibiotics, making it difficult to eradicate the bacterial infection. Besides reduced penetration of antibiotics through the intricate structure of the biofilms, the sessile biofilm-embedded bacteria show reduced metabolic activity making them intrinsically less sensitive to antibiotics. Moreover, they frequently express elevated levels of efflux pumps that extrude antibiotics, thereby reducing their intracellular levels. Some efflux pumps are involved in the secretion of QS compounds and biofilm-related materials, besides being important for removing toxic substances from the bacteria. Some efflux pump inhibitors (EPIs) have been shown to both prevent biofilm formation and sensitize the bacteria to antibiotics, suggesting a relationship between these processes. Additionally, QS inhibitors or quenchers may affect antibiotic susceptibility. Thus, targeting elements that regulate QS and biofilm formation might be a promising approach to combat antibiotic-resistant biofilm-related bacterial infections.

/pdf/targeting-the-holy-triangle-of-quorum-sensing-biofilm-2urora64.pdf

Targeting the Holy Triangle of Quorum Sensing, Biofilm Formation, and Antibiotic Resistance in Pathogenic Bacteria

Bacteria have evolved mechanisms that allow them to adapt to changes in osmolarity and some species have adapted to live optimally in high salinity environments such as in the marine ecosystem. Most bacteria that live in high salinity do so by the biosynthesis and/or uptake of compatible solutes, small organic molecules that maintain the turgor pressure of the cell. Osmotic stress response mechanisms and their regulation among marine heterotrophic bacteria are poorly understood. In this review, we discuss what is known about compatible solute metabolism and transport and new insights gained from studying marine bacteria belonging to the family Vibrionaceae.

Stressed out: Bacterial response to high salinity using compatible solute biosynthesis and uptake systems, lessons from Vibrionaceae.

Acinetobacter spp. have become of increased clinical importance as studies have shown the antimicrobial resistant potential of these species. Efflux pumps can lead to reduced susceptibility to a variety of antibiotics and are present in large number across Acinetobacter spp. There are six families of efflux pumps that have been shown to be of clinical relevance: the major facilitator superfamily (MFS), small multidrug resistance (SMR) family, ATP-binding cassette (ABC) family, multidrug and toxic compound extrusion (MATE) family, proteobacterial antimicrobial compound efflux (PACE) family, and the resistance-nodulation-division (RND) family. Much work has been done for understanding and characterizing the roles these efflux pumps play in relation to antimicrobial resistance and the physiology of these bacteria. RND efflux pumps, with their expansive substrate profiles, are a major component of Acinetobacter spp. antimicrobial resistance. New discoveries over the last decade have shed light on the complex regulation of these efflux pumps, leading to greater understanding and the potential of slowing the reduced susceptibility seen in these bacterial species.

Update on Multidrug Resistance Efflux Pumps in Acinetobacter spp.

Malaria remains a major public health problem in South America, mostly in the Amazon region. Among newly proposed ways of controlling malaria transmission to humans, paratransgenesis is a promising alternative. Paratransgenesis aims to inhibit the development of parasites within the vector through the action of genetically modified bacteria. The first step towards successful paratransgenesis in the Amazon is the identification of Anopheles darlingi symbiotic bacteria, which are transmitted vertically among mosquitoes, and are not pathogenic to humans. Culturable bacteria associated with An. darlingi and their breeding sites were isolated by conventional microbiological techniques. Isolated strains were transformed with a GFP expressing plasmid, pSPT-1-GFP, and reintroduced in mosquitoes by feeding. Their survival and persistence in the next generation was assessed by the isolation of fluorescent bacteria from eggs, larvae, pupae and adult homogenates. A total of 179 bacterial strains were isolated from samples from two locations, Coari and Manaus. The predominant genera identified in this study were Acinetobacter, Enterobacter, Klebsiella, Serratia, Bacillus, Elizabethkingia, Stenotrophomonas and Pantoea. Two isolated strains, Serratia-Adu40 and Pantoea-Ovo3, were successfully transformed with the pSPT-1-GFP plasmid and expressed GFP. The fluorescent bacteria fed to adult females were transferred to their eggs, which persisted in larvae and throughout metamorphosis, and were detected in adult mosquitoes of the next generation. Serratia-Adu40 and Pantoea-Ovo3 are promising candidates for paratransgenesis in An. darlingi. Further research is needed to determine if these bacteria are vertically transferred in nature.

/pdf/culturable-bacteria-associated-with-anopheles-darlingi-and-xvanov81vb.pdf

Culturable bacteria associated with Anopheles darlingi and their paratransgenesis potential.

Multidrug efflux pumps play an important role in antimicrobial resistance and pathogenicity in bacteria. Here, we report the functional characterization of the RND (resistance-nodulation- division) efflux pump, AcrAB, in Acinetobacter nosocomialis. An in silico analysis revealed that homologues of the AcrAB efflux pump, comprising AcrA and AcrB, are widely distributed among different bacterial species. Deletion of acrA and/or acrB genes led to decreased biofilm/pellicle formation and reduced antimicrobial resistance in A. nosocomialis. RNA sequencing and mRNA expression analyses showed that expression of acrA/B was downregulated in a quorum sensing (QS) regulator (anoR)-deletion mutant, indicating transcriptional activation of the acrAB operon by AnoR in A. nosocomialis. Bioassays showed that secretion of N-acyl homoserine lactones (AHLs) was unaffected in acrA and acrB deletion mutants; however, AHL secretion was limited in a deletion mutant of acrR, encoding the acrAB regulator, AcrR. An in silico analysis indicated the presence of AcrR-binding motifs in promoter regions of anoI (encoding AHL synthase) and anoR. Specific binding of AcrR was confirmed by electrophoretic mobility shift assays, which revealed that AcrR binds to positions -214 and -217 bp upstream of the translational start sites of anoI and anoR, respectively, demonstrating transcriptional regulation of these QS genes by AcrR. The current study further addresses the possibility that AcrAB is controlled by the osmotic stress regulator, OmpR, in A. nosocomialis. Our data demonstrate that the AcrAB efflux pump plays a crucial role in biofilm/pellicle formation and antimicrobial resistance in A. nosocomialis, and is under the transcriptional control of a number of regulators. In addition, the study emphasizes the interrelationship of QS and AcrAB efflux systems in A. nosocomialis.

Regulation of the AcrAB Efflux System by the Quorum-Sensing Regulator AnoR in Acinetobacter Nosocomialis

Adaptation to changing environmental conditions is crucial for the survival of microorganisms. Bacteria have evolved various mechanisms to cope with osmotic stress. Here, we report the identification and functional characterization of the osmotic stress response operon, betIBA, in Acinetobacter nosocomialis. The betIBA operon encodes enzymes that are important for the conversion of choline to the osmoprotectant, glycine betaine. The betIBA operon is polycistronic and is under the regulation of the first gene, betI, of the same operon. A bioinformatics analysis revealed the presence of a BetI-binding motif upstream of the betIBA operon, and electrophoretic mobility shift assays confirmed the specific binding of BetI. An mRNA expression analysis revealed that expression of betI, betB, and betA genes is elevated in a betI-eletion mutant compared with the wild type, confirming that the autorepressor BetI represses the betIBA operon in A. nosocomialis. We further found that the betIBA operon is under the transcriptional control of the quorum-sensing (QS) regulator, AnoR in, A. nosocomialis. A subsequent analysis of the impact of BetI on expression of the QS genes, anoR and anoI, demonstrated that BetI acts as a repressor of anoR and anoI. In addition, it was noticed that the osmotic stress response regulator, OmpR might play an important role in controlling the expression of betIBA operon in A. nosocomialis. Collectively, these data demonstrate that QS and osmotic stress-response systems are correlated in A. nosocomialis and that the expression of genes in both systems is finely tuned by various feedback loops depending on osmolarity conditions.

The osmotic stress response operon betIBA is under the functional regulation of BetI and the quorum-sensing regulator AnoR in Acinetobacter nosocomialis.

Acinetobacter has emerged recently as one of the most challenging nosocomial pathogens because of its increased rate of antimicrobial resistance. The genetic complexity and genome diversity, as well as the lack of adequate knowledge on the pathogenic determinants of Acinetobacter strains often hinder with pathogenesis studies for the development of better therapeutics to tackle this nosocomial pathogen. In this study, we comparatively analyzed the whole genome sequence of a virulent Acinetobacternosocomialis strain NCTC 8102. The genomic DNA of A. nosocomialis NCTC 8102 was isolated and sequenced using PacBio RS II platform. The sequenced genome was functionally annotated and gene prediction was carried out using the program, Glimmer 3. The phylogenetic analysis of the genome was performed using Mega 6 program and the comparative genome analysis was carried out by BLAST (Basic Local Alignment Search Tool). The complete genome analysis depicted that the genome consists of a circular chromosome with an average G + C content of 38.7%. The genome comprises 3700 protein-coding genes, 96 RNA genes (18 rRNA, 74 tRNA and 4 ncRNA genes), and 91 pseudogenes. In addition, 6 prophage regions comprising 2 intact, 1 incomplete and 3 questionable ones and 18 genomic islands were identified in the genome, suggesting the possible occurrence of horizontal gene transfer in this strain. Comparative genome analysis of A. nosocomialis NCTC 8102 genome with the already sequenced A. nosocomialis strain SSA3 showed an average nucleotide identity of 99.0%. In addition, the number of prophages and genomic islands were higher in the A. nosocomialis NCTC 8102 genome compared to that of the strain SSA3. 14 of the genomic islands were unique to A. nosocomialis NCTC 8102 compared to strain SSA3 and they harbored genes which are involved in virulence, multidrug resistance, biofilm formation and bacterial pathogenesis. We sequenced the whole genome of A. nosocomialis strain NCTC 8102 followed by comparatively genome analysis. The study provides valuable information on the genetic features of A. nosocomialis strain and the data from this study would assist in further studies for the development of control measures for this nosocomial pathogen.

Complete genome sequence and phylogenetic analysis of nosocomial pathogen Acinetobacter nosocomialis strain NCTC 8102

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Regulation of the AcrAB Efflux System by the Quorum-Sensing Regulator AnoR in Acinetobacter Nosocomialis

The osmotic stress response operon betIBA is under the functional regulation of BetI and the quorum-sensing regulator AnoR in Acinetobacter nosocomialis.

Complete genome sequence and phylogenetic analysis of nosocomial pathogen Acinetobacter nosocomialis strain NCTC 8102