TolC and EmrA1 contribute to Francisella novicida multidrug resistance and modulation of host cell death.

Francisella spp. are Gram-negative, facultative intracellular pathogens. Francisella tularensis causes the human disease tularemia and is considered a biological threat agent due to its high infectivity and virulence. A central aspect of Francisella virulence is its ability to dampen host immune responses. We previously identified the outer membrane channel (OMC) protein TolC as a critical F. tularensis virulence factor required for suppression of apoptotic and proinflammatory responses during macrophage infection. TolC functions as part of multidrug efflux systems and the type I secretion pathway that exports bacterial effector proteins. In these systems, TolC forms tripartite complexes together with an inner membrane transporter and periplasmic membrane fusion protein (MFP). To advance understanding of TolC function in Francisella, we analyzed OMC and MFP homologs in Francisella novicida, a widely used model species that causes a tularemia-like disease in mice. In agreement with the previous F. tularensis studies, all three OMCs present in F. novicida contributed to multidrug resistance, but only TolC was important for suppressing macrophage cell death. In addition, we identified the EmrA1 MFP as important for resisting antimicrobial compounds and dampening host cell death. In contrast to results obtained with F. tularensis, the cell death triggered during infection with the F. novicida tolC and emrA1 mutants was dominated by pyroptosis rather than apoptosis. These data expand our understanding of TolC function in Francisella and underscore both conserved and differential aspects of F. novicida and F. tularensis. IMPORTANCE: Francisella tularensis is a Gram-negative intracellular bacterial pathogen and causative agent of tularemia. We previously identified the outer membrane channel protein TolC as contributing to antimicrobial resistance and subversion of host responses by F. tularensis. To advance understanding of TolC function in Francisella and to identify components that might work together with TolC, we took advantage of a transposon mutant library in F. novicida, a model species that causes a tularemia-like disease in mice. Our findings identify TolC and the membrane fusion protein EmrA1 as important for both antimicrobial resistance and suppression of macrophage cell death. This study also revealed differences in cell death pathways triggered by F. novicida versus F. tularensis infection that may relate to differences in virulence.

Francisella sciaenopsi sp. nov. isolated from diseased red drum Sciaenops ocellatus in Florida, USA.

Piscine francisellosis is one of the most important bacterial diseases affecting various fish species worldwide. Francisella orientalis, F. noatunensis, and F. salimarina (F. marina) have been reported as etiological agents of disease in fish. A Francisella sp. was isolated from several diseased red drum Sciaenops ocellatus experiencing morbidity in Florida, USA, in 2008. In this study, molecular and phenotypic characterization of the recovered isolate was conducted. Phenotypically, the isolate showed a biochemical reaction profile distinct from that of F. orientalis and F. salimarina. Although the 16S rRNA sequence of this isolate shared 99.61% identity to the type strain of F. philomiragia O#319LT, whole genome analysis (average nucleotide identity <95%; digital DNA-DNA hybridization <70%) and a multilocus sequence analysis of 8 concatenated housekeeping genes in comparison with other Francisella spp. indicated that this isolate was a novel Francisella species, more closely related to F. orientalis. Immersion, intracoelomic injection, and co-habitation challenges using a Nile tilapia Oreochromis niloticus fingerling model of infection were done to investigate virulence in a piscine model. Variably pigmented granulomas and pigmented macrophage aggregates were observed in the kidneys and spleens of the challenged fish, but no mortality was recorded during the 15 d challenge period, suggesting that this novel Francisella sp. might be an opportunistic pathogen of fish. Based on the phenotypic and genotypic differences from other Francisella spp. observed in this study, we propose the name Francisella sciaenopsi sp. nov. for this novel isolate.

TracrRNA reprogramming enables direct PAM-independent detection of RNA with diverse DNA-targeting Cas12 nucleases.

Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA's presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage-all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection.

PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics.

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise, with a negligible affinity for mismatched substrates, but its low cellular targeting efficiency limits therapeutic use. Here, we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency, knock-in rates, and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally, we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor, highlighting its therapeutic utility.

Understanding the role of Francisella halioticida in mussel mortalities in France: an integrative approach.

Since 2014, mass mortalities of mussels Mytilus spp. have occurred in production areas on the Atlantic coast of France. The aetiology of these outbreaks remained unknown until the bacterium Francisella halioticida was detected in some mussel mortality cases. This retrospective study was conducted to assess the association between F. halioticida and these mussel mortalities. Mussel batches (n = 45) from the Atlantic coast and English Channel were selected from archived individual samples (n = 863) collected either during or outside of mortality events between 2014 and 2017. All mussels were analysed by real-time PCR assays targeting F. halioticida; in addition, 185 were analysed using histological analysis and 178 by 16S rRNA metabarcoding. F. halioticida DNA was detected by real-time PCR and 16S rRNA metabarcoding in 282 and 34 mussels, respectively. Among these individuals, 82% (real-time PCR analysis) and 76% (16S rRNA metabarcoding analysis) were sampled during a mortality event. Histological analyses showed that moribund individuals had lesions mainly characterized by necrosis, haemocyte infiltration and granulomas. Risk factor analysis showed that mussel batches with more than 20% of PCR-positive individuals were more likely to have been sampled during a mortality event, and positive 16S rRNA metabarcoding batches increased the strength of the association with mortality by 11.6 times. The role of F. halioticida in mussel mortalities was determined by reviewing the available evidence. To this end, a causation criteria grid, tailored to marine diseases and molecular pathogen detection tools, allowed more evidence to be gathered on the causal role of this bacterium in mussel mortalities.

Identification of zoonotic pathogenic bacteria from blood and ticks obtained from hares and long-eared hedgehogs (Hemiechinus megalofis) in eastern Iran.

The role of wildlife in the complex balance of tick-borne diseases within ecosystems is crucial, as they serve as hosts for tick carriers and reservoirs for the pathogens carried by these ticks. This study aimed to investigate the presence of zoonotic pathogenic bacteria in wildlife, specifically in hares and long-eared hedgehogs (Hemiechinus megalofis), in the eastern region of Iran. The focus was on the detection of Borrelia spp., Coxiella burnetii, Anaplasma spp., Francisella spp., and Leptospira spp., using the Nested-PCR method. We analyzed a total of 124 blood samples, and 196 ticks collected from hares and long-eared hedgehogs were analyzed. The Nested-PCR method was employed to identify the presence of zoonotic pathogenic bacteria DNA. Our study revealed the presence of these zoonotic pathogenic bacteria in both wildlife species, indicating their potential role as hosts and reservoirs for the ticks carrying these pathogens. The specific presence and prevalence of Borrelia spp., Coxiella burnetii, Anaplasma spp., Francisella spp., and Leptospira spp. were determined through the Nested-PCR method. This study contributes to the limited knowledge about the involvement of wild animals in the transmission of tick-borne diseases. By using the Nested-PCR method, we successfully identified the presence of zoonotic pathogenic bacteria in hares and long-eared hedgehogs. This study emphasizes the need for further research to better understand the ecological process of tick-borne diseases, particularly the role of wildlife in their spread. Such knowledge is crucial for wildlife conservation efforts and the management of tick-borne diseases, ultimately benefiting both animal and human health.

Tolfenpyrad displays Francisella-targeted antibiotic activity that requires an oxidative stress response regulator for sensitivity.

IMPORTANCE: Francisella species are highly pathogenic bacteria that pose a threat to global health security. These bacteria can be made resistant to antibiotics through facile methods, and we lack a safe and protective vaccine. Given their history of development as bioweapons, new treatment options must be developed to bolster public health preparedness. Here, we report that tolfenpyrad, a pesticide that is currently in use worldwide, effectively inhibits the growth of Francisella. This drug has an extensive history of use and a plethora of safety and toxicity data, making it a good candidate for development as an antibiotic. We identified mutations in Francisella novicida that confer resistance to tolfenpyrad and characterized a transcriptional regulator that is required for sensitivity to both tolfenpyrad and reactive oxygen species.

Nasal responses to elevated temperature and Francisella noatunensis infection in Atlantic cod (Gadus morhua).

We report the histological and transcriptomic changes in the olfactory organ of Atlantic cod exposed to Francisella noatunensis. Experimental infection was performed at either 12 °C or 17 °C. Infected fish presented the classic gross pathologies of francisellosis. Nasal morpho-phenotypic parameters were not significantly affected by elevated temperature and infection, except for the number of mucus cells in the 12 °C group seven weeks after the challenge. A higher number of genes were altered through time in the group reared at 17 °C. At termination, the nasal transcriptome of infected fish in both groups was similar to the control. When both infected groups were compared, 754 DEGs were identified, many of which were involved in signalling, defence, transmembrane and enzymatic processes. In conclusion, the study reveals that elevated temperature could trigger responses in the olfactory organ of Atlantic cod and shape the nasal response to F. noatunensis infection.

Impact of endosymbionts on tick physiology and fitness.

Ticks transmit pathogens and harbour non-pathogenic, vertically transmitted intracellular bacteria termed endosymbionts. Almost all ticks studied to date contain 1 or more of Coxiella, Francisella, Rickettsia or Candidatus Midichloria mitochondrii endosymbionts, indicative of their importance to tick physiology. Genomic and experimental data suggest that endosymbionts promote tick development and reproductive success. Here, we review the limited information currently available on the potential roles endosymbionts play in enhancing tick metabolism and fitness. Future studies that expand on these findings are needed to better understand endosymbionts' contributions to tick biology. This knowledge could potentially be applied to design novel strategies that target endosymbiont function to control the spread of ticks and pathogens they vector.

Discovery of a glutathione utilization pathway in Francisella that shows functional divergence between environmental and pathogenic species.

Glutathione (GSH) is an abundant metabolite within eukaryotic cells that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular bacterial pathogens. For Francisella, GSH is thought to be a critical in vivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily (MFS) member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella, explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.

Applying next generation sequencing to detect tick-pathogens in Dermacentor nuttalli, Ixodes persulcatus, and Hyalomma asiaticum collected from Mongolia.

Ticks and tick-borne diseases represent major threats to the public health of the Mongolian population, of which an estimated 26% live a traditional nomadic pastoralist lifestyle that puts them at increased risk for exposure. Ticks were collected by dragging and removal from livestock in Khentii, Selenge, Tuv, and Umnugovi aimags (provinces) during March-May 2020. Using next-generation sequencing (NGS) with confirmatory PCR and DNA sequencing, we sought to characterize the microbial species present in Dermacentor nuttalli (n = 98), Hyalomma asiaticum (n = 38), and Ixodes persulcatus (n = 72) tick pools. Rickettsia spp. were detected in 90.4% of tick pools, with Khentii, Selenge, and Tuv tick pools all having 100% pool positivity. Coxiella spp. were detected at an overall pool positivity rate of 60%, while Francisella spp. were detected in 20% of pools and Borrelia spp. detected in 13% of pools. Additional confirmatory testing for Rickettsia-positive pools demonstrated Rickettsia raoultii (n = 105), Candidatus Rickettsia tarasevichiae (n = 65) and R. slovaca/R. sibirica (n = 2), as well as the first report of Candidatus Rickettsia jingxinensis (n = 1) in Mongolia. For Coxiella spp. reads, most samples were identified as a Coxiella endosymbiont (n = 117), although Coxiella burnetii was detected in eight pools collected in Umnugovi. Borrelia species that were identified include Borrelia burgdorferi sensu lato (n = 3), B. garinii (n = 2), B. miyamotoi (n = 16), and B. afzelii (n = 3). All Francisella spp. reads were identified as Francisella endosymbiont species. Our findings emphasize the utility of NGS to provide baseline data across multiple tick-borne pathogen groups, which in turn can be used to inform health policy, determine regions for expanded surveillance, and guide risk mitigation strategies.

Influence of tick sex and geographic region on the microbiome of Dermacentor variabilis collected from dogs and cats across the United States.

As tick-borne diseases continue to increase across North America, current research strives to understand how the tick microbiome may affect pathogen acquisition, maintenance, and transmission. Prior high throughput amplicon-based microbial diversity surveys of the widespread tick Dermacentor variabilis have suggested that life stage, sex, and geographic region may influence the composition of the tick microbiome. Here, adult D. variabilis ticks (n = 145) were collected from dogs and cats from 32 states with specimens originating from all four regions of the United States (West, Midwest, South, and Northeast), and the tick microbiome was examined via V4-16S rRNA gene amplification and Illumina sequencing. A total of 481,246 bacterial sequences were obtained (median 2924 per sample, range 399-11,990). Fifty genera represented the majority (>80%) of the sequences detected, with the genera Allofrancisella and Francisella being the most abundant. Further, 97%, 23%, and 5.5% of the ticks contained sequences belonging to Francisella spp., Rickettsia spp., and Coxiella spp., respectively. No Ehrlichia spp. or Anaplasma spp. were identified. Co-occurrence analysis, by way of correlation coefficients, between the top 50 most abundant genera demonstrated five strong positive and no strong negative correlation relationships. Geographic region had a consistent effect on species richness with ticks from the Northeast having a significantly greater level of richness. Alpha diversity patterns were dependent on tick sex, with males exhibiting higher levels of diversity, and geographical region, with higher level of diversity observed in ticks obtained from the Northeast, but not on tick host. Community structure, or beta diversity, of tick microbiome was impacted by tick sex and geographic location, with microbiomes of ticks from the western US exhibiting a distinct community structure when compared to those from the other three regions (Northeast, South, and Midwest). In total, LEfSe (Linear discriminant analysis Effect Size) identified 18 specific genera driving these observed patterns of diversity and community structure. Collectively, these findings highlight the differences in bacterial diversity of D. variabilis across the US and supports the interpretation that tick sex and geographic region affects microbiome composition across a broad sampling distribution.

First insights into the microbiome of Tunisian Hyalomma ticks gained through next-generation sequencing with a special focus on H. scupense.

Ticks are one of the most important vectors of several pathogens affecting humans and animals. In addition to pathogens, ticks carry diverse microbiota of symbiotic and commensal microorganisms. In this study, we have investigated the first Tunisian insight into the microbial composition of the most dominant Hyalomma species infesting Tunisian cattle and explored the relative contribution of tick sex, life stage, and species to the diversity, richness and bacterial species of tick microbiome. In this regard, next generation sequencing for the 16S rRNA (V3-V4 region) of tick bacterial microbiota and metagenomic analysis were established. The analysis of the bacterial diversity reveals that H. marginatum and H. excavatum have greater diversity than H. scupense. Furthermore, microbial diversity and composition vary according to the tick's life stage and sex in the specific case of H. scupense. The endosymbionts Francisella, Midichloria mitochondrii, and Rickettsia were shown to be the most prevalent in Hyalomma spp. Rickettsia, Francisella, Ehrlichia, and Erwinia are the most common zoonotic bacteria found in Hyalomma ticks. Accordingly, Hyalomma ticks could represent potential vectors for these zoonotic bacterial agents.

Recently Evolved Francisella-Like Endosymbiont Outcompetes an Ancient and Evolutionarily Associated Coxiella-Like Endosymbiont in the Lone Star Tick (Amblyomma americanum) Linked to the Alpha-Gal Syndrome.

Background: Ticks are hematophagous arthropods that transmit various bacterial, viral, and protozoan pathogens of public health significance. The lone star tick (Amblyomma americanum) is an aggressive human-biting tick that transmits bacterial and viral pathogens, and its bites are suspected of eliciting the alpha-gal syndrome, a newly emerged delayed hypersensitivity following consumption of red meat in the United States. While ongoing studies have attempted to investigate the contribution of different tick-inherent factors to the induction of alpha-gal syndrome, an otherwise understudied aspect is the contribution of the tick microbiome and specifically obligate endosymbionts to the establishment of the alpha-gal syndrome in humans. Materials and Methods: Here we utilized a high-throughput metagenomic sequencing approach to cataloging the entire microbial communities residing within different developmental stages and tissues of unfed and blood-fed ticks from laboratory-maintained ticks and three new geographical locations in the United States. The Quantitative Insights Into Microbial Ecology (QIIME2) pipeline was used to perform data analysis and taxonomic classification. Moreover, using a SparCC (Sparse Correlations for Compositional data) network construction model, we investigated potential interactions between members of the microbial communities from laboratory-maintained and field-collected ticks. Results: Overall, Francisellaceae was the most dominant bacteria identified in the microbiome of both laboratory-raised and field-collected Am. americanum across all tissues and developmental stages. Likewise, microbial diversity was seen to be significantly higher in field-collected ticks compared with laboratory-maintained ticks as seen with a higher number of both Operational Taxonomic Units and measures of species richness. Several potential positive and negative correlations were identified from our network analysis. We observed a strong positive correlation between Francisellaceae, Rickettsiaceae, and Midichloriaceae in both developmental stages and tissues from laboratory-maintained ticks, whereas ovarian tissues had a strong positive correlation of bacteria in the family Xanthobacteraceae and Rhizobiaceae. A negative interaction was observed between Coxiellaceae and Francisellaceae in Illinois, and all the bacteria detected from ticks from Delaware were negatively correlated. Conclusion: This study is the first to catalog the microbiome of Am. americanum throughout its developmental stages and different tissue niches and report the potential replacement of Coxiellaceae by Francisellaceae across developmental stages and tissues tested except in ovarian tissues. These unique and significant findings advance our knowledge and open a new avenue of research to further understand the role of tick microbiome in tick-borne diseases and develop a holistic strategy to control alpha-gal syndrome.

The type IV pili component PilO is a virulence determinant of Francisella novicida.

Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. While its ability to replicate within cells has been studied in much detail, the bacterium also encodes a less characterised type 4 pili (T4P) system. T4Ps are dynamic adhesive organelles identified as major virulence determinants in many human pathogens. In F. tularensis, the T4P is required for adherence to the host cell, as well as for protein secretion. Several components, including pilins, a pili peptidase, a secretin pore and two ATPases, are required to assemble a functional T4P, and these are encoded within distinct clusters on the Francisella chromosome. While some of these components have been functionally characterised, the role of PilO, if any, still is unknown. Here, we examined the role of PilO in the pathogenesis of F. novicida. Our results show that the PilO is essential for pilus assembly on the bacterial surface. In addition, PilO is important for adherence of F. novicida to human monocyte-derived macrophages, secretion of effector proteins and intracellular replication. Importantly, the pilO mutant is attenuated for virulence in BALB/c mice regardless of the route of infection. Following intratracheal and intradermal infection, the mutant caused no histopathology changes, and demonstrated impaired phagosomal escape and replication within lung liver as well as spleen. Thus, PilO is an essential virulence determinant of F. novicida.

The first case of Francisella novicida infection in Taiwan: bacteraemic pneumonia in a haemodialysis adult.

Tularaemia is a zoonotic disease caused by Francisella tularensis (F. tularensis). Human infection is rare and can be life-threatening. F. tularensis subsp. novicida used to be a subspecies of F. tularensis, is now considered a different species, F. novicida. Though less virulent, F. novicida can cause morbidity and mortality among debilitated or immunocompromised patients. We reported that an adult with end-stage renal disease undergoing haemodialysis and a history of melioidotic aortic aneurysm developed F. novicida bacteraemic pneumonia, which was uneventfully treated by antimicrobial therapy. The microbiological confirmation of F. novicida infection relies on 16S rRNA sequencing. It is the first case of F. novicida infection in Taiwan.

Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA.

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5'-scaffold moiety and variable 3'-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.

A dual endosymbiosis supports nutritional adaptation to hematophagy in the invasive tick Hyalomma marginatum.

Many animals are dependent on microbial partners that provide essential nutrients lacking from their diet. Ticks, whose diet consists exclusively on vertebrate blood, rely on maternally inherited bacterial symbionts to supply B vitamins. While previously studied tick species consistently harbor a single lineage of those nutritional symbionts, we evidence here that the invasive tick Hyalomma marginatum harbors a unique dual-partner nutritional system between an ancestral symbiont, Francisella, and a more recently acquired symbiont, Midichloria. Using metagenomics, we show that Francisella exhibits extensive genome erosion that endangers the nutritional symbiotic interactions. Its genome includes folate and riboflavin biosynthesis pathways but deprived functional biotin biosynthesis on account of massive pseudogenization. Co-symbiosis compensates this deficiency since the Midichloria genome encompasses an intact biotin operon, which was primarily acquired via lateral gene transfer from unrelated intracellular bacteria commonly infecting arthropods. Thus, in H. marginatum, a mosaic of co-evolved symbionts incorporating gene combinations of distant phylogenetic origins emerged to prevent the collapse of an ancestral nutritional symbiosis. Such dual endosymbiosis was never reported in other blood feeders but was recently documented in agricultural pests feeding on plant sap, suggesting that it may be a key mechanism for advanced adaptation of arthropods to specialized diets.

Francisella philomiragia bacteremia in an immunocompromised patient: a rare case report.

BACKGROUND: Francisella philomiragia is a very rare opportunistic pathogen of humans which causes protean diseases such as pneumonia and other systemic infections. Subsequent failure of prompt treatment may result in poor prognosis with mortality among infected patients. CASE PRESENTATION: The present report describes a case of F. philomiragia bacteraemia first reported in Malaysia and Asian in a 60-year-old patient with underlying end-stage renal disease (ESRF) and diabetes mellitus. He presented with Acute Pulmonary Oedema with Non-ST-Elevation Myocardial Infarction (NSTEMI) in our hospital. He was intubated in view of persistent type I respiratory failure and persistent desaturation despite post haemodialysis. Blood investigation indicated the presence of ongoing infection and inflammation. The aerobic blood culture growth of F. philomiragia was identified using the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (Score value: 2.16) and confirmed by 16S Ribosomal DNA (16S rDNA) sequencing. He was discharged well on day 26 of admission, after completing one week of piperacillin/tazobactam and two weeks of doxycycline. CONCLUSION: Clinical suspicion should be raised if patients with known risk factors are presenting with pneumonia or pulmonary nodules especially as these are the most common manifestations of F. philomiragia infection. Early diagnosis via accurate laboratory identification of the organism through MALDI-TOF mass spectrometry and molecular technique such as 16S rDNA sequencing are vital for prompt treatment that results in better outcomes for the afflicted patients.

Francisella FlmX broadly affects lipopolysaccharide modification and virulence.

The outer membrane protects Gram-negative bacteria from the host environment. Lipopolysaccharide (LPS), a major outer membrane constituent, has distinct components (lipid A, core, O-antigen) generated by specialized pathways. In this study, we describe the surprising convergence of these pathways through FlmX, an uncharacterized protein in the intracellular pathogen Francisella. FlmX is in the flippase family, which includes proteins that traffic lipid-linked envelope components across membranes. flmX deficiency causes defects in lipid A modification, core remodeling, and O-antigen addition. We find that an F. tularensis mutant lacking flmX is >1,000,000-fold attenuated. Furthermore, FlmX is required to resist the innate antimicrobial LL-37 and the antibiotic polymyxin. Given FlmX's central role in LPS modification and its conservation in intracellular pathogens Brucella, Coxiella, and Legionella, FlmX may represent a novel drug target whose inhibition could cripple bacterial virulence and sensitize bacteria to innate antimicrobials and antibiotics.