Characterization of a colistin resistant, hypervirulent hospital isolate of Acinetobacter courvalinii from Canada.

Non-baumannii Acinetobacter spp. are becoming more prevalent in clinical settings including those that present resistance to last-resort antibiotics such as colistin. AB222-IK40 is an Acinetobacter courvalinii strain isolated from the Ottawa Hospital Research Institute located in Ottawa, Canada. To our knowledge, it is the first report of clinical A. courvalinii in Canada. Based on the susceptibility profile, AB222-IK40 is resistant to colistin and non-susceptible to ertapenem. Whole-genome sequencing allowed for genomic investigation into colistin resistance mechanisms. No previously identified mechanism(s) were observed, but a mobile colistin resistance (mcr)-like gene and a UDP-glucose dehydrogenase gene were identified. Based on phylogenomic analyses, the mcr-like gene is an intrinsic phosphoethanolamine transferase. This gene family is implicated in one of the many mechanisms responsible for colistin resistance in Acinetobacter baumannii as well as Acinetobacter modestus. UDP-glucose dehydrogenase is involved in colistin resistance in Enterobacterales and has been shown to be involved in capsule formation in A. baumannii. Global lipidomics revealed greater abundance of phosphatidyl-myo-inositol and lyso-phosphatidyl ethanolamine moieties in the membrane of A. courvalinii than in A. baumannii. Lipidomic profiles showed differences that were probably responsible for the colistin resistance phenotype in AB222-IK40. This isolate was also hypervirulent based on survival assays in Galleria mellonella. As this is the first report of A. courvalinii from a hospital in Canada, this species may be an emerging clinical pathogen, and therefore, it is important to understand this mechanism of its colistin resistance and hypervirulence.

Complete genome of Acinetobacter calcoaceticus AC001_UM from Red River soil banks in Winnipeg, Manitoba, Canada.

We report the whole-genome sequence and antibiotic-resistance gene profile of an Acinetobacter calcoaceticus isolate, designated AC001_UM, taken from soil along the Red River in Winnipeg, Manitoba, Canada. The genome comprised 3,916,544 nucleotides (GC content: 38.7%). Antibiotic-resistance gene analysis revealed a class D ß-lactamase and three efflux pump families.

Phylogenomic and phenotypic analyses highlight the diversity of antibiotic resistance and virulence in both human and non-human Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative, opportunistic pathogen that causes infections in the immunocompromised. With a high incidence of muti-drug resistance, carbapenem-resistant A. baumannii is designated as a priority 1 pathogen by the WHO. The current literature has expertly characterized clinical isolates of A. baumannii. As the challenge of these infections has recently been classified as a One Health issue, we set out to explore the diversity of isolates from human and non-clinical sources, such as agricultural surface water, urban streams, various effluents from wastewater treatment plants, and food (tank milk); and, importantly, these isolates came from a wide geographic distribution. Phylogenomic analysis considering almost 200 isolates showed that our diverse set is well-differentiated from the main international clones of A. baumannii. We discovered novel sequence types in both hospital and non-clinical settings and five strains that overexpress the resistance-nodulation-division efflux pump adeIJK without changes in susceptibility reflected by this overexpression. Furthermore, we detected a bla ADC-79 in a non-human isolate despite its sensitivity to all antibiotics. There was no significant differentiation between the virulence profiles of clinical and non-clinical isolates in the Galleria mellonella insect model of virulence, suggesting that virulence is neither dependent on geographic origin nor isolation source. The detection of antibiotic resistance and virulence genes in non-human strains suggests that these isolates may act as a genetic reservoir for clinical strains. This endorses the notion that in order to combat multi-drug-resistant infection caused by A. baumannii, a One Health approach is required, and a deeper understanding of non-clinical strains must be achieved.IMPORTANCEThe global crisis of antibiotic resistance is a silent one. More and more bacteria are becoming resistant to all antibiotics available for treatment, leaving no options remaining. This includes Acinetobacter baumannii. This Gram-negative, opportunistic pathogen shows a high frequency of multi-drug resistance, and many strains are resistant to the last-resort drugs carbapenem and colistin. Research has focused on strains of clinical origin, but there is a knowledge gap regarding virulence traits, particularly how A. baumannii became the notorious pathogen of today. Antibiotic resistance and virulence genes have been detected in strains from animals and environmental locations such as grass and soil. As such, A. baumannii is a One Health concern, which includes the health of humans, animals, and the environment. Thus, in order to truly combat the antibiotic resistance crisis, we need to understand the antibiotic resistance and virulence gene reservoirs of this pathogen under the One Health continuum.

Three novel multiplex PCR assays for rapid detection of virulence, antimicrobial resistance, and toxin genes in Acinetobacter calcoaceticus-baumannii complex species.

The Acinetobacter calcoaceticus-baumannii (ACB) complex is an often-overlooked group of nosocomial pathogens with a significant environmental presence. Rapid molecular screening methods for virulence, antimicrobial resistance, and toxin (VAT) genes are required to investigate the potential pathogenicity of environmental isolates. This study aimed to develop and apply novel ACB complex-specific multiplex PCR (mPCR) primers and protocols for the rapid detection of eight VAT genes. We optimized three single-tube mPCR assays using reference DNA from ACB complex and other Acinetobacter species. These assays were then applied to detect VAT genes in cultured ACB complex isolates recovered from clinical and environmental sources. Widespread detection of VAT genes in environmental isolates confirmed the validity, functionality, and applicability of these novel assays. Overall, the three newly developed ACB complex species-specific mPCR assays are rapid and simple tools that can be adopted in diagnostic and clinical lab settings. The detection of VAT genes in environmental isolates suggests that environmental niches could serve as a reservoir for potentially pathogenic ACB complex and warrants further investigation. The newly developed mPCR assays are specific, sensitive, and efficient, making them well-suited for high-throughput screening in epidemiological studies and evaluating the potential pathogenicity of ACB complex recovered from various sources.

Salicylic acids and pathogenic bacteria: new perspectives on an old compound.

Salicylic acids have been used in human and veterinary medicine for their anti-pyretic, anti-inflammatory, and analgesic properties for centuries. A key role of salicylic acid-immune modulation in response to microbial infection-was first recognized during studies of their botanical origin. The effects of salicylic acid on bacterial physiology are diverse. In many cases, they impose selective pressures leading to development of cross-resistance to antimicrobial compounds. Initial characterization of these interactions was in Escherichia coli, where salicylic acid activates the multiple antibiotic resistance (mar) operon, resulting in decreased antibiotic susceptibility. Studies suggest that stimulation of the mar phenotype presents similarly in closely related Enterobacteriaceae. Salicylic acids also affect virulence in many opportunistic pathogens by decreasing their ability to form biofilms and increasing persister cell populations. It is imperative to understand the effects of salicylic acid on bacteria of various origins to illuminate potential links between environmental microbes and their clinically relevant antimicrobial-resistant counterparts. This review provides an update on known effects of salicylic acid and key derivatives on a variety of bacterial pathogens, offers insights to possible potentiation of current treatment options, and highlights cellular regulatory networks that have been established during the study of this important class of medicines.

Emergence of ADC-5 Cephalosporinase in environmental Acinetobacter baumannii from a German tank milk with a novel Sequence Type.

Bacteria resistant to antibiotics arguably pose the greatest threat to human health in the twenty-first century. One such bacterium that typifies antibiotic resistance is Acinetobacter baumannii . Frequently, hospital strains of A. baumannii display multidrug resistant (MDR) or extensively drug resistant (XDR) phenotypes, often requiring the use of last resort antibiotics for treatment. In addition to hospital settings, A. baumannii has been isolated from many highly divergent sources including wastewater treatment plant effluent, soil, and agricultural run-off with global distribution. However, such isolates remain poorly characterized. In this study, we characterized a strain of A. baumannii, AB341-IK15, isolated from bulk tank milk in Germany that demonstrated resistance to ceftazidime and intermediate resistance to ceftriaxone and piperacillin/tazobactam. Further genetic characterization identified an ADC-5 cephalosporinase, first incidence in an environmental isolate; and an OXA-408 oxacillinase that may contribute to this phenotype. Interestingly, AB341-IK15 is of a novel sequence type. This research underscores the importance of studying isolates of A. baumannii of non-clinical origin to understand the antibiotic resistance and virulence potential of environmental isolates of A. baumannii as well to understand the diversity of this species.

Unravelling the Evolutionary Dynamics of High-Risk Klebsiella pneumoniae ST147 Clones: Insights from Comparative Pangenome Analysis.

BACKGROUND: The high prevalence and rapid emergence of antibiotic resistance in high-risk Klebsiella pneumoniae (KP) ST147 clones is a global health concern and warrants molecular surveillance. METHODS: A pangenome analysis was performed using publicly available ST147 complete genomes. The characteristics and evolutionary relationships among ST147 members were investigated through a Bayesian phylogenetic analysis. RESULTS: The large number of accessory genes in the pangenome indicates genome plasticity and openness. Seventy-two antibiotic resistance genes were found to be linked with antibiotic inactivation, efflux, and target alteration. The exclusive detection of the blaOXA-232 gene within the ColKp3 plasmid of KP_SDL79 suggests its acquisition through horizontal gene transfer. The association of seventy-six virulence genes with the acrAB efflux pump, T6SS system and type I secretion system describes its pathogenicity. The presence of Tn6170, a putative Tn7-like transposon in KP_SDL79 with an insertion at the flanking region of the tnsB gene, establishes its transmission ability. The Bayesian phylogenetic analysis estimates ST147's initial divergence in 1951 and the most recent common ancestor for the entire KP population in 1621. CONCLUSIONS: Present study highlights the genetic diversity and evolutionary dynamics of high-risk clones of K. pneumoniae. Further inter-clonal diversity studies will help us understand its outbreak more precisely and pave the way for therapeutic interventions.

Isolation of Bioactive Metabolites from Soil Derived Fungus-Aspergillus fumigatus.

Fungi produce numerous secondary metabolites with intriguing biological properties for the health, industrial, and agricultural sectors. Herein, we report the high-yield isolation of phenolic natural products, N-formyl-4-hydroxyphenyl-acetamide 1 (~117 mg/L) and atraric acid 2 (~18 mg/L), from the ethyl acetate extract of the soil-derived fungus, Aspergillus fumigatus. The structures of compounds 1 and 2 were elucidated through the detailed spectroscopic analysis of NMR and LCMS data. These compounds were assayed for their antimicrobial activities. It was observed that compounds 1 and 2 exhibited strong inhibition against a series of fungal strains but only weak antibacterial properties against multi-drug-resistant strains. More significantly, this is the first known instance of the isolation of atraric acid 2 from a non-lichen fungal strain. We suggest the optimization of this fungal strain may exhibit elevated production of compounds 1 and 2, potentially rendering it a valuable source for the industrial-scale production of these natural antimicrobial compounds. Further investigation is necessary to establish the veracity of this hypothesis.

Iron Acquisition Mechanisms and Their Role in the Virulence of Acinetobacter baumannii.

Iron is an essential element for survival of most organisms. One mechanism of host defense is to tightly chelate iron to several proteins to limit its extracellular availability. This has forced pathogens such as Acinetobacter baumannii to adapt mechanisms for the acquisition and utilization of iron even in iron-limiting conditions. A. baumannii uses a variety of iron acquisition strategies to meet its iron requirements. It can lyse erythrocytes to harvest the heme molecules, use iron-chelating siderophores, and use outer membrane vesicles to acquire iron. Iron acquisition pathways, in general, have been seen to affect many other virulence factors such as cell adherence, cell motility, and biofilm formation. The knowledge gained from research on iron acquisition led to the synthesis of the antibiotic cefiderocol, which uses iron uptake pathways for entry into the cell with some success as a novel cephalosporin. Understanding the mechanisms of iron acquisition of A. baumannii allows for insight into clinical infections and offer potential targets for novel antibiotics or potentiators of current drugs.

Density-Dependent Prophylaxis in Freshwater Snails Driven by Oxylipin Chemical Cues.

While animal aggregations can benefit the fitness of group members, the behaviour may also lead to higher risks of parasite infection as group density increases. Some animals are known to moderate their investment in immunity relative to the risk of infection. These animals exhibit density-dependent prophylaxis (DDP) by increasing their immune investment as group density increases. Despite being documented in many taxa, the mechanisms of DDP remain largely unexplored. Snails are known to aggregate and experience large fluctuations in density and serve as required hosts for many parasites. Further, they are known to use chemical cues to aggregate. To test whether freshwater snails exhibit DDP and investigate the role that chemical signaling compounds may play in triggering this phenomenon, we performed four experiments on the freshwater snail Stagnicola elodes, which is a common host for many trematode parasite species. First, we tested if DDP occurred in snails in laboratory-controlled conditions (control vs snail-conditioned water) and whether differences in exposure to chemical cues affected immune function. Second, we used gas chromatography to characterize fatty acids expressed in snail-conditioned water to determine if precursors for particular signaling molecules, such as oxylipins, were being produced by snails. Third, we characterized the oxylipins released by infected and uninfected field-collected snails, to better understand how differences in oxylipin cocktails may play a role in inducing DDP. Finally, we tested the immune response of snails exposed to four oxylipins to test the ability of specific oxylipins to affect DDP. We found that snails exposed to water with higher densities of snails and raised in snail-conditioned water had higher counts of haemocytes. Additionally, lipid analysis demonstrated that fatty acid molecules that are also precursors for oxylipins were present in snail-conditioned water. Trematode-infected snails emitted 50 oxylipins in higher amounts, with 24 of these oxylipins only detected in this group. Finally, oxylipins that were higher in infected snails induced naïve snails to increase their immune responses compared to sham-exposed snails. Our results provide evidence that snails exhibit DDP, and the changes in oxylipins emitted by infected hosts may be one of the molecular mechanisms driving this phenomenon.

MexXY RND pump of Pseudomonas aeruginosa PA7 effluxes bi-anionic ß-lactams carbenicillin and sulbenicillin when it partners with the outer membrane factor OprA but not with OprM.

Antibiotic resistance in Pseudomonas aeruginosa is a serious concern in healthcare systems. Among the determinants of antibiotic resistance in P. aeruginosa, efflux pumps belonging to the resistance-nodulation-division (RND) family confer resistance to a broad range of antibacterial compounds. The MexXY efflux system is widely overexpressed in P. aeruginosa isolates from cystic fibrosis (CF) patients. MexXY can form functional complexes with two different outer membrane factors (OMFs), OprA and OprM. In this study, using state-of-the-art genetic tools, the substrate specificities of MexXY-OprA and MexXY-OprM complexes were determined. Our results show, for the first time, that the substrate profile of the MexXY system from P. aeruginosa PA7 can vary depending on which OM factor (OprM or OprA) it complexes with. While both MexXY-OprA and MexXY-OprM complexes are capable of effluxing aminoglycosides, the bi-anionic ß-lactam molecules carbenicillin and sulbenicillin were found to only be the substrate of MexXY-OprA. Our study therefore shows that by partnering with different OMF proteins MexY can expand its substrate profile.

Recent Advances in Genetic Tools for Acinetobacter baumannii.

Acinetobacter baumannii is classified as a top priority pathogen by the World Health Organization (WHO) because of its widespread resistance to all classes of antibiotics. This makes the need for understanding the mechanisms of resistance and virulence critical. Therefore, tools that allow genetic manipulations are vital to unravel the mechanisms of multidrug resistance (MDR) and virulence in A. baumannii. A host of current strategies are available for genetic manipulations of A. baumannii laboratory-strains, including ATCC® 17978TM and ATCC® 19606T, but depending on susceptibility profiles, these strategies may not be sufficient when targeting strains newly obtained from clinic, primarily due to the latter's high resistance to antibiotics that are commonly used for selection during genetic manipulations. This review highlights the most recent methods for genetic manipulation of A. baumannii including CRISPR based approaches, transposon mutagenesis, homologous recombination strategies, reporter systems and complementation techniques with the spotlight on those that can be applied to MDR clinical isolates.

Maternal MEMI Promotes Female Meiosis II in Response to Fertilization in Caenorhabditis elegans.

In most animals, female meiosis completes only after fertilization. Sperm entry has been implicated in providing a signal for the initiation of the final meiotic processes; however, a maternal component required for this process has not been previously identified. We report the characterization of a novel family of three highly similar paralogs (memi-1, memi-2, memi-3) that encode oocyte-specific proteins. A hyper-morphic mutation memi-1(sb41) results in failure to exit female meiosis II properly; however, loss of all three paralogs results in a "skipped meiosis II" phenotype. Mutations that prevent fertilization, such as fer-1(hc1), also cause a skipped meiosis II phenotype, suggesting that the MEMI proteins represent a maternal component of a postfertilization signal that specifies the meiosis II program. MEMI proteins are degraded before mitosis and sensitive to ZYG-11, a substrate-specific adapter for cullin-based ubiquitin ligase activity, and the memi-1(sb41) mutation results in inappropriate persistence of the MEMI-1 protein into mitosis. Using an RNAi screen for suppressors of memi-1(sb41), we identified a sperm-specific PP1 phosphatase, GSP-3/4, as a putative sperm component of the MEMI pathway. We also found that MEMI and GSP-3/4 proteins can physically interact via co-immunoprecipitation. These results suggest that sperm-specific PP1 and maternal MEMI proteins act in the same pathway after fertilization to facilitate proper meiosis II and the transition into embryonic mitosis.

The KLP-7 Residue S546 Is a Putative Aurora Kinase Site Required for Microtubule Regulation at the Centrosome in C. elegans.

Regulation of microtubule dynamics is essential for many cellular processes, including proper assembly and function of the mitotic spindle. The kinesin-13 microtubule-depolymerizing enzymes provide one mechanism to regulate microtubule behaviour temporally and spatially. Vertebrate MCAK locates to chromatin, kinetochores, spindle poles, microtubule tips, and the cytoplasm, implying that the regulation of kinesin-13 activity and subcellular targeting is complex. Phosphorylation of kinesin-13 by Aurora kinase inhibits microtubule depolymerization activity and some Aurora phosphorylation sites on kinesin-13 are required for subcellular localization. Herein, we determine that a C. elegans deletion mutant klp-7(tm2143) causes meiotic and mitotic defects that are consistent with an increase in the amount of microtubules in the cytoplasmic and spindle regions of meiotic embryos, and an increase in microtubules emanating from centrosomes. We show that KLP-7 is phosphorylated by Aurora A and Aurora B kinases in vitro, and that the phosphorylation by Aurora A is stimulated by TPXL-1. Using a structure-function approach, we establish that one putative Aurora kinase site, S546, within the C-terminal part of the core domain is required for the function, but not subcellular localization, of KLP-7 in vivo. Furthermore, FRAP analysis reveals microtubule-dependent differences in the turnover of KLP-7(S546A) and KLP-7(S546E) mutant proteins at the centrosome, suggesting a possible mechanism for the regulation of KLP-7 by Aurora kinase.