galleria-mellonella-as-an-infection-antibiotic-treatment-model-for

<em>Galleria mellonella</em> as an Infection and Antibiotic Treatment Model for <em>Acinetobacter baumannii</em>

Galleria mellonella, commonly known as the greater wax moth or waxworm, is an insect infection model that provides researchers with an informative, ...

University of Manitoba

Antibiotic resistance in  is a serious concern in healthcare systems. Among the determinants of antibiotic resistance in , 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  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  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 β-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.

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

 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 host of current strategies are available for genetic manipulations of  laboratory-strains, including ATCC 17978 and ATCC 19606, 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  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.

Recent Advances in Genetic Tools for .

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 , 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.

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

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  (~117 mg/L) and atraric acid  (~18 mg/L), from the ethyl acetate extract of the soil-derived fungus, . The structures of compounds  and  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  and  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  from a non-lichen fungal strain. We suggest the optimization of this fungal strain may exhibit elevated production of compounds  and , 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.

Isolation of Bioactive Metabolites from Soil Derived Fungus-.

The high prevalence and rapid emergence of antibiotic resistance in high-risk  ST147 clones is a global health concern and warrants molecular surveillance.

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

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  . Frequently, hospital strains of  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,  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  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  of non-clinical origin to understand the antibiotic resistance and virulence potential of environmental isolates of  as well to understand the diversity of this species.

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

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 , where salicylic acid activates the multiple antibiotic resistance () operon, resulting in decreased antibiotic susceptibility. Studies suggest that stimulation of the  phenotype presents similarly in closely related . 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.

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

 is a Gram-negative, opportunistic pathogen that causes infections in the immunocompromised. With a high incidence of muti-drug resistance, carbapenem-resistant  is designated as a priority 1 pathogen by the WHO. The current literature has expertly characterized clinical isolates of . 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 . We discovered novel sequence types in both hospital and non-clinical settings and five strains that overexpress the resistance-nodulation-division efflux pump  without changes in susceptibility reflected by this overexpression. Furthermore, we detected a   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  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 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 . 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  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,  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.

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

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.

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

We report the whole-genome sequence and antibiotic-resistance gene profile of an  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 β-lactamase and three efflux pump families.

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

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

 (ACB) complex has been identified as a group of emerging opportunistic pathogens that cause nosocomial infections. The current study investigates the prevalence, distribution, and diversity of pathogenic ACB complex in various aquatic systems with different uses. Of the total 157 agricultural, raw drinking water intake, recreational beach, and wastewater treatment plant (WWTP) effluent samples, acinetobacters were isolated, quantified, and confirmed by genus- and ACB complex-specific PCR assays. Of all agricultural surface water samples,  (65%) was more frequently detected than  (14%),  (9%), and  (3%). In WWTP effluent samples,  was more prevalent in de-chlorinated (60%) samples compared to both  and  (40%). Interestingly (43%),  (29%), and  (14%) were detected in raw drinking water intake samples, whereas  (50%) and  (25%) were detected in beach samples. Although no sampling location-specific differences were recorded, significant ( < 0.05) seasonal differences were observed when agricultural surface water samples collected in spring were compared with the summer and fall. Whereas effluent chlorination significantly impacted the degree of prevalence of  in WWTP effluent samples, overall, the prevalence of ACB complex in all sampling locations and seasons indicates that these water sources, containing human-associated ACB complex, may pose potential health risks as community-acquired opportunistic infections.IMPORTANCE (ACB) complex is a group of organisms known to cause problematic nosocomial opportunistic infections. A member of the species complex, , is becoming a global threat to infection treatment as strains are increasingly develop resistance to antibiotics. The prevalence and distribution of potentially pathogenic  complex species remain poorly understood, and there is a need to better understand the occurrence of  in non-nosocomial environments. Our research details the spatial-temporal distribution of ACB complex species in a regional watershed and highlights the presence of ACB complex in wastewater effluent that is discharged into a river. These findings deepen our understanding of this group of species in non-nosocomial environments and encourage the development of monitoring programs for these species in regional waters.

Article	Year
MexXY RND pump of PA7 effluxes bi-anionic β-lactams carbenicillin and sulbenicillin when it partners with the outer membrane factor OprA but not with OprM. Microbiology (Reading, England)\| PubMed ID: 32909933	2020
Recent Advances in Genetic Tools for . Frontiers in genetics\| PubMed ID: 33414809	2020
Density-Dependent Prophylaxis in Freshwater Snails Driven by Oxylipin Chemical Cues. Frontiers in immunology\| PubMed ID: 35173735	2022
Isolation of Bioactive Metabolites from Soil Derived Fungus-. Microorganisms\| PubMed ID: 36985164	2023
Unravelling the Evolutionary Dynamics of High-Risk ST147 Clones: Insights from Comparative Pangenome Analysis. Genes\| PubMed ID: 37239397	2023
Emergence of ADC-5 Cephalosporinase in environmental from a German tank milk with a novel Sequence Type. Access microbiology\| PubMed ID: 37424542	2023
Salicylic acids and pathogenic bacteria: new perspectives on an old compound. Canadian journal of microbiology\| PubMed ID: 37699258	2024
Phylogenomic and phenotypic analyses highlight the diversity of antibiotic resistance and virulence in both human and non-human . mSphere\| PubMed ID: 38440986	2024
Three novel multiplex PCR assays for rapid detection of virulence, antimicrobial resistance, and toxin genes in Acinetobacter calcoaceticus-baumannii complex species. Letters in applied microbiology\| PubMed ID: 38460955	2024
Complete genome of AC001_UM from Red River soil banks in Winnipeg, Manitoba, Canada. Microbiology resource announcements\| PubMed ID: 38634680	2024
Characterization of a colistin resistant, hypervirulent hospital isolate of Acinetobacter courvalinii from Canada. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology\| PubMed ID: 39073669	2024
complex prevalence, spatial-temporal distribution, and contamination sources in Canadian aquatic environments. Microbiology spectrum\| PubMed ID: 39240108	2024

Ellen M. E. Sykes

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