Genetic synergy between Acinetobacter baumannii undecaprenyl phosphate biosynthesis and the Mla system impacts cell envelope and antimicrobial resistance.

Acinetobacter baumannii is a Gram-negative bacterial pathogen that poses a major health concern due to increasing multidrug resistance. The Gram-negative cell envelope is a key barrier to antimicrobial entry and includes an inner and outer membrane. The maintenance of lipid asymmetry (Mla) system is the main homeostatic mechanism by which Gram-negative bacteria maintain outer membrane asymmetry. Loss of the Mla system in A. baumannii results in attenuated virulence and increased susceptibility to membrane stressors and some antibiotics. We recently reported two strain variants of the A. baumannii type strain ATCC 17978: 17978VU and 17978UN. Here, ∆mlaF mutants in the two ATCC 17978 strains display different phenotypes for membrane stress resistance, antibiotic resistance, and pathogenicity in a murine pneumonia model. Although allele differences in obgE were previously reported to synergize with ∆mlaF to affect growth and stringent response, obgE alleles do not affect membrane stress resistance. Instead, a single-nucleotide polymorphism (SNP) in the essential gene encoding undecaprenyl pyrophosphate (Und-PP) synthase, uppS, results in decreased enzymatic rate and decrease in total Und-P levels in 17978UN compared to 17978VU. The UppSUN variant synergizes with ∆mlaF to reduce capsule and lipooligosaccharide (LOS) levels, increase susceptibility to membrane stress and antibiotics, and reduce persistence in a mouse lung infection. Und-P is a lipid glycan carrier required for the biosynthesis of A. baumannii capsule, cell wall, and glycoproteins. These findings uncover synergy between Und-P and the Mla system in maintaining the A. baumannii cell envelope and antibiotic resistance.IMPORTANCEAcinetobacter baumannii is a critical threat to global public health due to its multidrug resistance and persistence in hospital settings. Therefore, novel therapeutic approaches are urgently needed. We report that a defective undecaprenyl pyrophosphate synthase (UppS) paired with a perturbed Mla system leads to synthetically sick cells that are more susceptible to clinically relevant antibiotics and show reduced virulence in a lung infection model. These results suggest that targeting UppS or undecaprenyl species and the Mla system may resensitize A. baumannii to antibiotics in combination therapies. This work uncovers a previously unknown synergistic relationship in cellular envelope homeostasis that could be leveraged for use in combination therapy against A. baumannii.

Genetic synergy in Acinetobacter baumannii undecaprenyl biosynthesis and maintenance of lipid asymmetry impacts outer membrane and antimicrobial resistance.

Acinetobacter baumannii is a Gram-negative healthcare-associated pathogen that poses a major health concern due to increasing multidrug resistance. The Gram-negative cell envelope is a key barrier to antimicrobial entry and includes an inner and outer membrane. The outer membrane has an asymmetric composition that is important for structural integrity and barrier to the environment. Therefore, Gram-negative bacteria have mechanisms to uphold this asymmetry such as the maintenance of lipid asymmetry system (Mla), which removes glycerophospholipids from the outer leaflet of the outer membrane and transports them to the inner membrane. Loss of this system in A. baumannii results in attenuated virulence and increased susceptibility to membrane stressors and some antibiotics. We recently reported two strain variants of the A. baumannii type strain ATCC 17978, 17978VU and 17978UN. We show here that ΔmlaF mutants in the two strains display different phenotypes for membrane stress resistance, antibiotic resistance, and pathogenicity in a murine pneumonia model. We used comparative genetics to identify interactions between ATCC 17978 strain alleles and mlaF to uncover the cause behind the phenotypic differences. Although allele differences in obgE were previously reported to synergize with ΔmlaF to affect growth and stringent response, we show that obgE alleles do not affect membrane stress resistance. Instead, a single nucleotide polymorphism (SNP) in the essential gene encoding undecaprenyl pyrophosphate (Und-PP) synthase, uppS, synergizes with ΔmlaF to increase susceptibility to membrane stress and antibiotics, and reduce persistence in a mouse lung infection. Und-P is a lipid glycan carrier known to be required for biosynthesis of A. baumannii capsule, cell wall, and glycoproteins. Our data suggest that in the absence of the Mla system, the cellular level of Und-P is critical for envelope integrity, antibiotic resistance, and lipooligosaccharide abundance. These findings uncover synergy between Und-P and the Mla system in maintaining the A. baumannii outer membrane and stress resistance.

Chemical shift variations in common metabolites.

The reliability and robustness of metabolite assignments in 1H NMR is complicated by numerous factors including variations in temperature, pH, buffer choice, ionic strength, and mixture composition that led to peak overlap and spectral crowding. As sample conditions fluctuate, peak drift and line broadening further complicate peak deconvolution and subsequent chemical assignment. We present a collection of 1D 1H NMR spectra of 54 common metabolites at varied pH (6.0 to 8.0 in 0.5 step increments) and temperature (290 K to 308 K) to quantify chemical shift variability to facilitate automated metabolite assignments. Our results illustrate the fundamental challenges with accurately assigning NMR peaks under varied environmental conditions prevalent in complex mixtures. Phosphorylated metabolites showed a larger variation in chemical shifts due to pH, whereas; amino acids showed a higher variation due to temperature. Mixtures of phosphorous compounds showed a consistently poor reliability in achieving an accurate assignment. Phosphorylated cholines, amino acids, and glycerols yielded a 40 % false negative rate for 7 out of 9 mixture conditions. Amino acids had a false negative rate of 57 % at 298 K and pH 8. Our results demonstrate that the automated assignments of complex biofluid mixtures require an expert to intervene to confirm the accuracy of metabolite assignments. Our analysis also indicates the need for reference databases to include spectra under a variety of conditions that includes mixtures and a range of pH and temperature to improve the accuracy and reproducibility of metabolite assignments.

Mobile genetic elements in Acinetobacter antibiotic-resistance acquisition and dissemination.

Pathogenic Acinetobacter species, most notably Acinetobacter baumannii, are a significant cause of healthcare-associated infections worldwide. Acinetobacter infections are of particular concern to global health due to the high rates of multidrug resistance and extensive drug resistance. Widespread genome sequencing and analysis has determined that bacterial antibiotic resistance is often acquired and disseminated through the movement of mobile genetic elements, including insertion sequences (IS), transposons, integrons, and conjugative plasmids. In Acinetobacter specifically, resistance to carbapenems and cephalosporins is highly correlated with IS, as many ISAba elements encode strong outwardly facing promoters that are required for sufficient expression of ß-lactamases to confer clinical resistance. Here, we review the role of mobile genetic elements in antibiotic resistance in Acinetobacter species through the framework of the mechanism of resistance acquisition and with a focus on experimentally validated mechanisms.

Identification of Two Variants of Acinetobacter baumannii Strain ATCC 17978 with Distinct Genotypes and Phenotypes.

Acinetobacter baumannii is a nosocomial pathogen that exhibits substantial genomic plasticity. Here, the identification of two variants of A. baumannii ATCC 17978 that differ based on the presence of a 44-kb accessory locus, named AbaAL44 (A. baumannii accessory locus 44 kb), is described. Analyses of existing deposited data suggest that both variants are found in published studies of A. baumannii ATCC 17978 and that American Type Culture Collection (ATCC)-derived laboratory stocks comprise a mix of these two variants. Yet, each variant exhibits distinct interactions with the host in vitro and in vivo. Infection with the variant that harbors AbaAL44 (A. baumannii 17978 UN) results in decreased bacterial burdens and increased neutrophilic lung inflammation in a mouse model of pneumonia, and affects the production of interleukin 1 beta (IL-1ß) and IL-10 by infected macrophages. AbaAL44 harbors putative pathogenesis genes, including those predicted to encode a type I pilus cluster, a catalase, and a cardiolipin synthase. The accessory catalase increases A. baumannii resistance to oxidative stress and neutrophil-mediated killing in vitro. The accessory cardiolipin synthase plays a dichotomous role by promoting bacterial uptake and increasing IL-1ß production by macrophages, but also by enhancing bacterial resistance to cell envelope stress. Collectively, these findings highlight the phenotypic consequences of the genomic dynamism of A. baumannii through the evolution of two variants of a common type strain with distinct infection-related attributes.

Clustering of adolescent health concerns: a latent class analysis of school students in New Zealand.

AIMS: The aims of this study are to identify clinically meaningful groups of adolescents based on their engagement in high levels of risk behaviours or severe emotional health concerns and to describe the demographic characteristics of these groups in two populations of school students in New Zealand. METHODS: A nationally representative sample of secondary school students was surveyed in 2007; alternative education (AE) students in Auckland and Northland were surveyed in 2009. A total of 9107 secondary school students and 335 AE students completed a youth health questionnaire using Internet tablets. Latent class analysis (LCA) was used to identify groups of students on the basis of distinct profiles of their risk behaviours and mental health concerns. RESULTS: The majority (80%) of students in secondary schools are 'healthy' and report few health concerns, 16% are considered 'risky' or 'distressed', and 4% report 'multiple' risk behaviour profiles or emotional health concerns. In AE, only 21% of students were considered 'healthy' with most featuring in the 'risky' or 'multiple' groups. Females were more likely to be 'distressed', whereas males were more likely to feature in the 'risky' or 'multiple' groups. CONCLUSIONS: Clinically-concerning health risk behaviours and emotional health concerns 'cluster' in up to 20% of students in secondary schools and up to 79% of students in AE. Gender, ethnic and socio-economic disparities are also observed. This highlights the importance of comprehensive psychosocial assessment and appropriate service provision, particularly for at-risk groups.