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The rapid and accurate identification of pathogenic bacteria is crucial for combating the growing threat of antibiotic resistance, nosocomial infections, and food safety concerns. This study presents a novel and comprehensive comparison of two vibrational spectroscopic techniques - attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and a low-cost miniature near-infrared (NIR) spectrometer - for distinguishing Gram-positive and Gram-negative bacterial samples grown using the same stock media solution. This is the first report of NIR spectroscopy being applied to differentiate Gram-positive and Gram-negative bacteria, as well as the first direct comparison of ATR-FTIR and NIR for the combined multimodal analysis of clinical bacterial isolates. Using a data set of five Gram-positive and seven Gram-negative species and recording spectra in triplicate, the study employed advanced data fusion and multivariate analysis techniques to classify the spectra and facilitate NIR band assignment. 2D correlation analysis revealed strong positive correlations between key spectral markers identified in both modalities. Partial least-squares- and support vector machine discriminant analysis models were validated using a methodology based on 100 repeated random sampling of calibration and test sets. Models demonstrated that both the standalone ATR-FTIR and the combined ATR-FTIR/NIR approach achieved exceptional classification accuracy (>98%) in differentiating the two bacterial groups. Differences observed in the spectra were attributed to the distinct cell wall compositions of Gram-Positive and Gram-negative bacteria. Notably, the low-cost NIR technique also showed promising performance, with classification accuracy values above 90%. The findings highlight the potential of these rapid, noninvasive, and cost-effective vibrational spectroscopic techniques, particularly the NIR method, for point-of-care applications in clinical microbiology and food safety monitoring. The combination of ATR-FTIR and NIR data further enhances the robustness and reliability of bacterial identification, paving the way for broader adoption of these advanced analytical tools in various healthcare and food safety settings.

Klebsiella pneumoniae is consistently ranked among the most problematic multidrug-resistant bacterial pathogens in healthcare systems. Developing novel treatments requires a better understanding of its interaction with the host environment. Although bacteria can synthesize fatty acids, emerging findings suggest a potential preference for their acquisition from the host. Fatty acid profiling of mice revealed a dramatic increase in the level of hepatic lipids during K . pneumoniae infection. The K . pneumoniae fatty acid composition and uptake capabilities were found to be largely clonally conserved. Correlations between fatty acid uptake, outer membrane vesicle production, and cell permeability were observed, but this did not translate to alterations in cell morphology, capsule production, or antimicrobial susceptibility. Importantly, hyper-capsulation did not prevent the uptake of hydrophobic fatty acids. The uptake of a saturated fatty acid by hypervirulent K . pneumoniae isolate may provide insights into the clinical association of K . pneumoniae infections with hyperlipidemic and/or obese individuals.

Acinetobacter baumannii is one of the most prevalent causes of nosocomial infections worldwide. However, a paucity of information exists regarding the connection between metabolic capacity and in vivo bacterial fitness. Elevated lactate is a key marker of severe sepsis. We have previously shown that the putative A. baumannii lactate permease gene, lldP, is upregulated during in vivo infection. Here, we confirm that lldP expression is upregulated in three A. baumannii strains during a mammalian systemic infection. Utilising a transposon mutant disrupted for lldP in the contemporary clinical strain AB5075-UW, and a complemented strain, we confirmed its role in the in vitro utilisation of l-(+)-lactate. Furthermore, disruption of the lactate metabolism pathway resulted in reduced bacterial fitness during an in vivo systemic murine competition assay. The disruption of lldP had no impact on the susceptibility of this strain to complement mediated killing by healthy human serum. However, growth in biologically relevant concentrations of lactate observed during severe sepsis, led to bacterial tolerance to killing by healthy human blood, a phenotype that was abolished in the lldP mutant. This study highlights the importance of the lactate metabolism pathway for survival and growth of A. baumannii during infection., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Background: Recurrent vulvovaginal candidiasis (RVVC) is a recalcitrant medical condition that affects many women of reproductive age. The importance of biofilm formation by Candida in RVVC has been recently questioned. This study aimed to elucidate the fundamental growth modes of Candida in the vagina of patients with RVVC or sporadic vulvovaginal candidiasis (VVC) and to assess their roles in the persistence of RVVC., Methods: Vaginal tissues were sampled from twelve patients clinically and microbiologically diagnosed as RVVC or VVC at a post-antifungal-treatment and asymptomatic period. High-resolution scanning electron microscopy, fluorescence in situ hybridization in combination with Candida -specific 18S rRNA probes and viable fungal burden were used to qualitatively and quantitatively evaluate Candida growth in the human vagina. The presence of Candida biofilm extracellular polymeric substances was examined using confocal laser scanning microscopy and biopsy sections pre-stained with Concanavalin A. Histopathological analysis was carried out on infected vaginal tissues stained with hematoxylin and eosin. Lastly, the susceptibility of epithelium-associated Candida biofilms to fluconazole at the peak serum concentration was evaluated., Results: Candida species grew on the vaginal epithelium of RVVC patients as morphologically disparate biofilms including monolayers, microcolonies, and macro-colonies, in addition to sporadic adherent cells. Candida biofilm growth on the vaginal epithelium was associated with mild lymphocytic infiltration of the vaginal mucosa. These epithelium-based Candida biofilms presented an important characteristic contributing to the persistence of RVVC that is the high tolerance to fluconazole., Conclusions: In summary, our study provides direct evidence to support the presence of Candida biofilms in RVVC and an important role of biofilm formation in disease persistence., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Yue Qu reports financial support was provided by The Alfred Research Trusts. Yihong Pan reports financial support was provided by Zhejiang Province Natural Science Foundation. Zhenbo Xu reports financial support was provided by South China University of Technology., (© 2023 The Authors.)

Biofilm formation is an important microbial strategy for fungal pathogens, such as Fusarium , Aspergillus , and Candida , to establish keratitis in patients wearing soft contact lenses. Despite the well-documented 2006 outbreak of Fusarium keratitis that eventually led to the withdrawal of the Bausch & Lomb multipurpose lens care solution ReNu with MoistureLoc ("MoistureLoc") from the global market, contact lens care systems and solutions currently available on the market do not specifically target fungal biofilms. This is partially due to the lack of recognition and understanding of important roles that fungal biofilms play in contact lens associated fungal keratitis (CLAFK). This review aims to reemphasize the link between fungal biofilms and CLAFK, and deepen our comprehension of its importance in pathogenesis and persistence of this medical device-related infection.

We collected 163 clinical Pseudomonas aeruginosa isolates at a tertiary hospital specialising in adult cystic fibrosis (CF) and lung transplantation (LTx) in Melbourne, Australia, to explore the activity of ceftolozane-tazobactam (C/T) in populations at high-risk for antimicrobial resistance. Of these, 144 (88.3%) were collected from sputum, and 19 (11.7%) from bronchoalveolar lavage. Most (85.3%) were derived from patients with cystic fibrosis and included a subset of patients that had undergone LTx. These isolates were tested against 11 antibiotics, including C/T, using Sensititre plates for broth microdilution (BMD) testing. Sixty (36.8%) isolates were classified as multidrug resistant (MDR) and 32 (19.6%) were extensively drug resistant (XDR). Overall, 133/163 (81.6%) isolates were susceptible to C/T. For MDR and XDR isolates, 88.3% and 28.1% were C/T susceptible, respectively. Among the non-MDR/XDR isolates, 100% remained susceptible to C/T. Comparisons of C/T susceptibility were made using BioMérieux Etests and Liofilchem MIC test strips (MTS). Categorical agreement to BMD was >93% for both test strips, but essential agreement to BMD was slightly higher with Etest (89.0%) compared to Liofilchem (74.8%). In conclusion, C/T retained activity against most MDR and over a quarter of XDR P. aeruginosa isolates from complex patients with CF and post-LTx., (Copyright © 2023 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Biofilm formation and capsule production are known microbial strategies used by bacterial pathogens to survive adverse conditions in the hospital environment. The relative importance of these strategies individually is unexplored. This project aims to compare the contributory roles of biofilm formation and capsule production in bacterial survival on hospital surfaces. Representative strains of bacterial species often causing hospital-acquired infections were selected, including Acinetobacter baumannii , Klebsiella pneumoniae , Staphylococcus aureus , Staphylococcus epidermidis and Pseudomonas aeruginosa . The importance of biofilm formation and capsule production on bacterial survival was evaluated by comparing capsule-positive wild-type and capsule-deficient mutant strains, and biofilm and planktonic growth modes respectively, against three adverse hospital conditions, including desiccation, benzalkonium chloride disinfection and ultraviolet (UV) radiation. Bacterial survival was quantitatively assessed using colony-forming unit (CFU) enumeration and the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay and qualitatively by scanning electron microscopy (SEM). Correlations between capsule production and biofilm formation were further investigated. Biofilm formation contributed significantly to bacterial survival on hospital surface simulators, mediating high resistance to desiccation, benzalkonium chloride disinfection and UV radiation. The role of capsule production was minor and species-specific; encapsulated A. baumannii but not K. pneumoniae cells demonstrated slightly increased resistance to desiccation, and neither showed enhanced resistance to benzalkonium chloride. Interestingly, capsule production sensitized K. pneumoniae and A. baumannii to UV radiation. The loss of capsule in K. pneumoniae and A. baumannii enhanced biofilm formation, possibly by increasing cell surface hydrophobicity. In summary, this study confirms the crucial role of biofilm formation in bacterial survival on hospital surfaces. Conversely, encapsulation plays a relatively minor role and may even negatively impact bacterial biofilm formation and hospital survival., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Francesca Short reports financial support was provided by the 10.13039/501100000923Australian Research Council., (© 2023 The Authors.)

To control infections phagocytes can directly kill invading microbes. Macrophage-expressed gene 1 (Mpeg1), a pore-forming protein sometimes known as perforin-2, is reported to be essential for bacterial killing following phagocytosis. Mice homozygous for the mutant allele Mpeg1 tm1Pod succumb to bacterial infection and exhibit deficiencies in bacterial killing in vitro. Here we describe a new Mpeg mutant allele Mpeg1 tm1.1Pib on the C57BL/6J background. Mice homozygous for the new allele are not abnormally susceptible to bacterial or viral infection, and irrespective of genetic background show no perturbation in bacterial killing in vitro. Potential reasons for these conflicting findings are discussed. In further work, we show that cytokine responses to inflammatory mediators, as well as antibody generation, are also normal in Mpeg1 tm1.1Pib/tm1.1Pib mice. We also show that Mpeg1 is localized to a CD68-positive endolysosomal compartment, and that it exists predominantly as a processed, two-chain disulfide-linked molecule. It is abundant in conventional dendritic cells 1, and mice lacking Mpeg1 do not present the model antigen ovalbumin efficiently. We conclude that Mpeg1 is not essential for innate antibacterial protection or antiviral immunity, but may play a focused role early in the adaptive immune response., (© 2022 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Background: Clinical phage therapy is often delivered alongside antibiotics. However, the phenomenon of phage-antibiotic synergy has been mostly studied in vitro. Here, we assessed the in vivo bactericidal effect of a phage-antibiotic combination on Acinetobacter baumannii AB900 using phage øFG02, which binds to capsular polysaccharides and leads to antimicrobial resensitisation in vitro., Methods: We performed a two-stage preclinical study using a murine model of severe A. baumannii AB900 bacteraemia. In the first stage, with an endpoint of 11 h, mice (n = 4 per group) were treated with either PBS, ceftazidime, phage øFG02, or the combination of phage and ceftazidime. The second stage involved only the latter two groups (n = 5 per group), with a prolonged endpoint of 16 h. The primary outcome was the average bacterial burden from four body sites (blood, liver, kidney, and spleen). Bacterial colonies from phage-treated mice were retrieved and screened for phage-resistance., Findings: In the first stage, the bacterial burden (CFU/g of tissue) of the combination group (median: 4.55 × 10 5 ; interquartile range [IQR]: 2.79 × 10 5 -2.81 × 10 6 ) was significantly lower than the PBS (median: 2.42 × 10 9 ; IQR: 1.97 × 10 9 -3.48 × 10 9 ) and ceftazidime groups (median: 3.86 × 10 8 ; IQR: 2.15 × 10 8 -6.35 × 10 8 ), but not the phage-only group (median: 1.28 × 10 7 ; IQR: 4.71 × 10 6 -7.13 × 10 7 ). In the second stage, the combination treatment (median: 1.72 × 10 6 ; IQR: 5.11 × 10 5 -4.00 × 10 6 ) outperformed the phage-only treatment (median: 7.46 × 10 7 ; IQR: 1.43 × 10 7 -1.57 × 10 8 ). Phage-resistance emerged in 96% of animals receiving phages, and all the tested isolates (n = 11) had loss-of-function mutations in genes involved in capsule biosynthesis and increased sensitivity to ceftazidime., Interpretation: øFG02 reliably drives the in vivo evolution of A. baumannii AB900 towards a capsule-deficient, phage-resistant phenotype that is resensitised to ceftazidime. This mechanism highlights the clinical potential of using phage therapy to target A. baumannii and restore antibiotic activity., Funding: National Health and Medical Research Council (Australia)., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Acinetobacter baumannii is a critically important pathogen known for its widespread antibiotic resistance and ability to persist in hospital-associated environments. Whilst the majority of A. baumannii infections are hospital-acquired, infections from outside the hospital have been reported with high mortality. Despite this, little is known about the natural environmental reservoir(s) of A. baumannii and the virulence potential underlying non-clinical strains. Here, we report the complete genome sequences of six diverse strains isolated from environments such as river, soil, and industrial sites around the world. Phylogenetic analyses showed that four of these strains were unrelated to representative nosocomial strains and do not share a monophyletic origin, whereas two had sequence types belonging to the global clone lineages GC1 and GC2. Further, the majority of these strains harboured genes linked to virulence and stress protection in nosocomial strains. These genotypic properties correlated well with in vitro virulence phenotypic assays testing resistance to abiotic stresses, serum survival, and capsule formation. Virulence potential was confirmed in vivo, with most environmental strains able to effectively kill Galleria mellonella greater wax moth larvae. Using phenomic arrays and antibiotic resistance profiling, environmental and nosocomial strains were shown to have similar substrate utilisation patterns although environmental strains were distinctly more sensitive to antibiotics. Taken together, these features of environmental A. baumannii strains suggest the existence of a strain-specific distinct gene pools for niche specific adaptation. Furthermore, environmental strains appear to be equally virulent as contemporary nosocomial strains but remain largely antibiotic sensitive.

The pathogen Staphylococcus aureus can readily develop antibiotic resistance and evade the human immune system, which is associated with reduced levels of neutrophil recruitment. Here, we present a class of antibacterial peptides with potential to act both as antibiotics and as neutrophil chemoattractants. The compounds, which we term 'antibiotic-chemoattractants', consist of a formylated peptide (known to act as chemoattractant for neutrophil recruitment) that is covalently linked to the antibiotic vancomycin (known to bind to the bacterial cell wall). We use a combination of in vitro assays, cellular assays, infection-on-a-chip and in vivo mouse models to show that the compounds improve the recruitment, engulfment and killing of S. aureus by neutrophils. Furthermore, optimizing the formyl peptide sequence can enhance neutrophil activity through differential activation of formyl peptide receptors. Thus, we propose antibiotic-chemoattractants as an alternate approach for antibiotic development., (© 2021. The Author(s).)

We characterized two bacteriophages, ΦFG02 and ΦCO01, against clinical isolates of Acinetobacter baumannii and established that the bacterial capsule is the receptor for these phages. Phage-resistant mutants harboured loss-of-function mutations in genes responsible for capsule biosynthesis, resulting in capsule loss and disruption of phage adsorption. The phage-resistant strains were resensitized to human complement, beta-lactam antibiotics and alternative phages and exhibited diminished fitness in vivo. Using a mouse model of A. baumannii infection, we showed that phage therapy was effective.

Daptomycin is an important antibiotic for the treatment of infections caused by Staphylococcus aureus . The emergence of daptomycin resistance in S. aureus is associated with treatment failure and persistent infections with poor clinical outcomes. Here, we investigated host innate immune responses against clinically derived, daptomycin-resistant (DAP-R) and -susceptible S. aureus paired isolates using a zebrafish infection model. We showed that the control of DAP-R S. aureus infections was attenuated in vivo due to cross-resistance to host cationic antimicrobial peptides. These data provide mechanistic understanding into persistent infections caused by DAP-R S. aureus and provide crucial insights into the adaptive evolution of this troublesome pathogen.

Staphylococcus aureus small-colony variants (SCVs) are associated with unusually chronic and persistent infections despite active antibiotic treatment. The molecular basis for this clinically important phenomenon is poorly understood, hampered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable S. aureus SCV that arose spontaneously while studying rifampicin resistance. This SCV showed no nucleotide differences across its genome compared with a normal-colony variant (NCV) revertant, yet the SCV presented the hallmarks of S. aureus linked to persistent infection: down-regulation of virulence genes and reduced hemolysis and neutrophil chemotaxis, while exhibiting increased survival in blood and ability to invade host cells. Further genome analysis revealed chromosome structural variation uniquely associated with the SCV. These variations included an asymmetric inversion across half of the S. aureus chromosome via recombination between type I restriction modification system (T1RMS) genes, and the activation of a conserved prophage harboring the immune evasion cluster (IEC). Phenotypic reversion to the wild-type-like NCV state correlated with reversal of the chromosomal inversion (CI) and with prophage stabilization. Further analysis of 29 complete S. aureus genomes showed strong signatures of recombination between hsdMS genes, suggesting that analogous CI has repeatedly occurred during S. aureus evolution. Using qPCR and long-read amplicon deep sequencing, we detected subpopulations with T1RMS rearrangements causing CIs and prophage activation across major S. aureus lineages. Here, we have discovered a previously unrecognized and widespread mechanism of reversible genomic instability in S. aureus associated with SCV generation and persistent infections., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Acinetobacter baumannii is a Gram negative opportunistic pathogen that has demonstrated a significant insurgence in the prevalence of infections over recent decades. With only a limited number of "traditional" virulence factors, the mechanisms underlying the success of this pathogen remain of great interest. Major advances have been made in the tools, reagents, and models to study A. baumannii pathogenesis, and this has resulted in a substantial increase in knowledge. This article provides a comprehensive review of the bacterial virulence factors, the host immune responses, and animal models applicable for the study of this important human pathogen. Collating the most recent evidence characterizing bacterial virulence factors, their cellular targets and genetic regulation, we have encompassed numerous aspects important to the success of this pathogen, including membrane proteins and cell surface adaptations promoting immune evasion, mechanisms for nutrient acquisition and community interactions. The role of innate and adaptive immune responses is reviewed and areas of paucity in our understanding are highlighted. Finally, with the vast expansion of available animal models over recent years, we have evaluated those suitable for use in the study of Acinetobacter disease, discussing their advantages and limitations.

Staphylococcus aureus is a notorious human bacterial pathogen with considerable capacity to develop antibiotic resistance. We have observed that human infections caused by highly drug-resistant S. aureus are more prolonged, complicated, and difficult to eradicate. Here we describe a metabolic adaptation strategy used by clinical S. aureus strains that leads to resistance to the last-line antibiotic, daptomycin, and simultaneously affects host innate immunity. This response was characterized by a change in anionic membrane phospholipid composition induced by point mutations in the phospholipid biosynthesis gene, cls2 , encoding cardiolipin synthase. Single cls2 point mutations were sufficient for daptomycin resistance, antibiotic treatment failure, and persistent infection. These phenotypes were mediated by enhanced cardiolipin biosynthesis, leading to increased bacterial membrane cardiolipin and reduced phosphatidylglycerol. The changes in membrane phospholipid profile led to modifications in membrane structure that impaired daptomycin penetration and membrane disruption. The cls2 point mutations also allowed S. aureus to evade neutrophil chemotaxis, mediated by the reduction in bacterial membrane phosphatidylglycerol, a previously undescribed bacterial-driven chemoattractant. Together, these data illustrate a metabolic strategy used by S. aureus to circumvent antibiotic and immune attack and provide crucial insights into membrane-based therapeutic targeting of this troublesome pathogen., Competing Interests: The authors declare no conflict of interest.

Coagulase-negative staphylococci (CoNS) represent one of the major causes of health care- and medical device-associated infections. Emerging antimicrobial resistance has complicated the treatment of systemic infections caused by CoNS. Here, we describe the prevalence of antimicrobial resistance in clinical CoNS strains from a tertiary care hospital over a 4-year period, and we observed a significant increase in resistance to daptomycin. Notably, Staphylococcus capitis accounted for the majority of these daptomycin-resistant (DAP-R) CoNS. To further investigate the mechanisms of daptomycin resistance in CoNS, daptomycin-susceptible clinical strains of S. capitis and Staphylococcus epidermidis underwent in vitro daptomycin exposure to generate DAP-R CoNS mutants. Unlike that seen with Staphylococcus aureus , alteration of cell surface charge was not observed in the DAP-R CoNS strains, but biofilm formation was compromised. Whole-genome sequencing analysis of the DAP-R CoNS strains identified single nucleotide polymorphisms (SNPs) in walKR , the essential two-component regulatory system controlling cell wall biogenesis. PCR and sequencing of walK and walR from 17 DAP-R CoNS clinical isolates identified seven nonsynonymous mutations. The results were confirmed by the recreation of the walK SNP in S. epidermidis , which resulted in reduced susceptibility to daptomycin and vancomycin. This study highlights the significance of CoNS in evolving daptomycin resistance and showed that walKR is shared among the staphylococcal species and is involved in antibiotic resistance development. Notably, we did not observe mutations in genes responsible for phospholipid biosynthesis or an altered cell surface charge, suggesting that reduced daptomycin susceptibility in CoNS may emerge in a fashion distinct from that in S. aureus ., (Copyright © 2019 American Society for Microbiology.)

Acinetobacter baumannii is a common causative agent of hospital-acquired infections and a leading cause of infection in burns patients. Carbapenem-resistant A. baumannii is considered a major public-health threat and has been identified by the World Health Organization as the top priority organism requiring new antimicrobials. The most common mechanism for carbapenem resistance in A. baumannii is via horizontal acquisition of carbapenemase genes. In this study, we sampled 20 A. baumannii isolates from a patient with extensive burns, and characterized the evolution of carbapenem resistance over a 45 day period via Illumina and Oxford Nanopore sequencing. All isolates were multidrug resistant, carrying two genomic islands that harboured several antibiotic-resistance genes. Most isolates were genetically identical and represented a single founder genotype. We identified three novel non-synonymous substitutions associated with meropenem resistance: F136L and G288S in AdeB (part of the AdeABC efflux pump) associated with an increase in meropenem MIC to ≥8 µg ml -1 ; and A515V in FtsI (PBP3, a penicillin-binding protein) associated with a further increase in MIC to 32 µg ml -1 . Structural modelling of AdeB and FtsI showed that these mutations affected their drug-binding sites and revealed mechanisms for meropenem resistance. Notably, one of the adeB mutations arose prior to meropenem therapy but following ciprofloxacin therapy, suggesting exposure to one drug whose resistance is mediated by the efflux pump can induce collateral resistance to other drugs to which the bacterium has not yet been exposed.

The treatment of infections caused by methicillin-resistant Staphylococcus aureus is complicated by the emergence of strains with intermediate-level resistance to vancomycin (termed VISA). We have characterised a molecular pathway involved in the in vivo evolution of VISA mediated by the regulatory proteins YycH and YycI. In contrast to their function in other bacterial species, we report a positive role for these auxiliary proteins in regulation of the two-component regulator WalRK. Transcriptional profiling of yycH and yycI deletion mutants revealed downregulation of the 'WalRK regulon' including cell wall hydrolase genes atlA and sle1, with functional autolysis assays supporting these data by showing an impaired autolytic phenotype for each deletion strain. Using bacterial-two hybrid assays, we showed that YycH and YycI interact, and that YycHI also interacts with the sensor kinase WalK, forming a ternary protein complex. Mutation to YycH or YycI associated with clinical VISA strains had a deleterious impact on the YycHI/WalK complex, suggesting that the interaction is important for the regulation of WalRK. Taken together, we have described a novel antibiotic resistance strategy for the human pathogen S. aureus, whereby YycHI mutations are selected for in vivo leading to reduced WalRK activation, impaired cell wall turnover and ultimately reduced vancomycin efficacy.

Innate cellular immune responses are a critical first-line defense against invading bacterial pathogens. Leukocyte migration from the bloodstream to a site of infection is mediated by chemotactic factors that are often host-derived. More recently, there has been a greater appreciation of the importance of bacterial factors driving neutrophil movement during infection. Here, we describe the development of a zebrafish infection model to study Acinetobacter baumannii pathogenesis. By using isogenic A. baumannii mutants lacking expression of virulence effector proteins, we demonstrated that bacterial drivers of disease severity are conserved between zebrafish and mammals. By using transgenic zebrafish with fluorescent phagocytes, we showed that a mutation of an established A. baumannii global virulence regulator led to marked changes in neutrophil behavior involving rapid neutrophil influx to a localized site of infection, followed by prolonged neutrophil dwelling. This neutrophilic response augmented bacterial clearance and was secondary to an impaired A. baumannii phenylacetic acid catabolism pathway, which led to accumulation of phenylacetate. Purified phenylacetate was confirmed to be a neutrophil chemoattractant. These data identify a previously unknown mechanism of bacterial-guided neutrophil chemotaxis in vivo, providing insight into the role of bacterial metabolism in host innate immune evasion. Furthermore, the work provides a potentially new therapeutic paradigm of targeting a bacterial metabolic pathway to augment host innate immune responses and attenuate disease., Competing Interests: The authors declare no conflict of interest.

Multidrug-resistant (MDR) Acinetobacter baumannii is an opportunistic human pathogen that has become highly problematic in the clinical environment. Novel therapies are desperately required. To assist in identifying new therapeutic targets, the antagonistic interactions between A. baumannii and the most common human fungal pathogen, Candida albicans, were studied. We have observed that the C. albicans quorum-sensing molecule, farnesol, has cross-kingdom interactions, affecting the viability of A. baumannii. To gain an understanding of its mechanism, the transcriptional profile of A. baumannii exposed to farnesol was examined. Farnesol caused dysregulation of a large number of genes involved in cell membrane biogenesis, multidrug efflux pumps (AcrAB-like and AdeIJK-like), and A. baumannii virulence traits such as biofilm formation (csuA, csuB, and ompA) and motility (pilZ and pilH). We also observed a strong induction in genes involved in cell division (minD, minE, ftsK, ftsB, and ftsL). These transcriptional data were supported by functional assays showing that farnesol disrupts A. baumannii cell membrane integrity, alters cell morphology, and impairs virulence characteristics such as biofilm formation and twitching motility. Moreover, we showed that A. baumannii uses efflux pumps as a defense mechanism against this eukaryotic signaling molecule. Owing to its effects on membrane integrity, farnesol was tested to see if it potentiated the activity of the membrane-acting polymyxin antibiotic colistin. When coadministered, farnesol increased sensitivity to colistin for otherwise resistant strains. These data provide mechanistic understanding of the antagonistic interactions between diverse pathogens and may provide important insights into novel therapeutic strategies., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Acinetobacter baumannii is one of the most notorious hospital-acquired pathogens, and novel treatment strategies are desperately required. Two-component regulatory systems represent potential therapeutic targets as they mediate microorganism adaptation to changing environments, often control virulence, and are specific to bacteria. Here we describe the first global virulence regulator in A. baumannii., Methods and Results: Using transcriptional profiling and functional assays of a deletion mutant in the A. baumannii sensor kinase gene, A1S_0574 (termed as gacS), we show that this sensor kinase regulates key virulence characteristics, including pili synthesis, biofilms, and motility, resulting in virulence attenuation in a mammalian septicemia model. Notably, we also identified that GacS regulates an operon novel to A. baumannii (paa operon), which is responsible for the metabolism of aromatic compounds. Deletion of paaE (A1S_1340) confirmed the role of this operon in A. baumannii virulence. Finally, we identified the cognate response regulator (A1S_0236) for GacS and confirmed their interaction. A1S_0236 was shown to regulate 75% of the GacS transcriptome and the same virulence phenotypes. Overexpression of A1S_0236 restored virulence in the gacS mutant., Conclusions: Our study characterizes a global virulence regulator, which may provide an alternate therapeutic target, in one of the most troublesome hospital-acquired pathogens.

The occurrence of mutations in methicillin-resistant Staphylococcus aureus (MRSA) during persistent infection leads to antimicrobial resistance but may also impact host-pathogen interactions. Here, we investigate the host-pathogen consequences of 2 mutations arising in clinical MRSA during persistent infection: RpoB H₄₈₁Y, which is linked to rifampicin resistance, and RelA F₁₂₈Y, which is associated with an active stringent response. Allelic exchange experiments showed that both mutations cause global transcriptional changes, leading to upregulation of capsule production, with attenuated virulence in a murine bacteremia model and reduced susceptibility to both antimicrobial peptides and whole-blood killing. Disruption of capsule biosynthesis reversed these impacts on innate immune function. These data clearly link MRSA persistence and reduced virulence to the same mechanisms that alter antimicrobial susceptibility. Our study highlights the wider consequences of suboptimal antimicrobial use, where drug resistance and immune escape mechanisms coevolve, thus increasing the likelihood of treatment failure.

The genetic mechanisms that contribute to reduced susceptibility to vancomycin in Staphylococcus aureus are complex and heterogeneous. In addition, debate is emerging as to the true effect of reduced susceptibility to vancomycin on staphylococcal virulence. To investigate this, comparative genomics was performed on a collection of vancomycin-exposed isogenic S. aureus pairs (14 strains in total). Previously described mutations were observed in genes such as vraG, agrA, yvqF, and rpoB; however, a new mechanism was identified involving a serine/threonine phosphatase, Stp1. After constructing an stp1 deletion mutant, we showed that stp1 is important in vancomycin susceptibility and cell wall biosynthesis. Gene expression studies showed that stp1 also regulates virulence genes, including a hemolysin, superantigen-like protein, and phenol-soluble modulin, and that the deletion mutant is attenuated in virulence in vivo. Stp1 provides a new link between vancomycin susceptibility and virulence in S. aureus.

Purpose: Periostin is a secreted extracellular matrix protein that is differentially expressed in the developing kidney. We analyzed the temporal-spatial expression of periostin in the developing kidney and ureter as well as its roles in ureter branching morphogenesis, nephrogenesis and ureter development., Materials and Methods: RNA in situ hybridization and immunofluorescence histochemistry were used to investigate the expression of periostin, αv integrin and α-smooth muscle actin during mouse renal and ureteral development. Metanephric explants were cultured in the presence of recombinant periostin, and ureteral branch points/tips and the glomerular number were quantified. Explants were also cultured in the presence of exogenous bone morphogenetic protein 4 and the effect on periostin mRNA levels was determined by quantitative real-time polymerase chain reaction., Results: Periostin expression was observed in the mesenchyme surrounding the kidney and ureter, renal stroma, metanephric mesenchyme, ureter epithelium and developing nephrons. At embryonic day 15.5 periostin and αv integrin, a common subunit of periostin receptors, were co-expressed in smooth muscle cells of the ureter, renal artery and intrarenal arteries. Bone morphogenetic protein 4 up-regulated periostin mRNA expression and exogenous periostin inhibited branching morphogenesis and glomerular number., Conclusions: Bone morphogenetic protein 4 which inhibits ureteral branching morphogenesis and promotes smooth muscle cell migration in the ureter up-regulated periostin mRNA expression in the developing kidney. Ureteral smooth muscle cells express periostin and αv integrin. Periostin inhibited ureteral branching morphogenesis and glomerular number. Together these results suggest that periostin and bone morphogenetic protein 4 may have a role in branching morphogenesis, nephrogenesis and possibly smooth muscle cell migration., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.