Development and early testing of a simple, low cost, fast sensor for maternal and neonatal group B Streptococcus.

Streptococcus agalactiae, (Group B Streptococcus (GBS)), is a common colonizer of the female vagina. In women giving birth it can be transmitted to the baby and cause serious illness and even death to the child. We have developed a biosensor comprising of phospholipids and fatty acids vesicles encapsulating high concentration, self-quenched carboxyfluorescein, which is released by the lysis of the vesicle by virulence factors expressed by GBS, becoming diluted and fluorescent. The microbial specificity of the sensor was tested against a number of GBS strains and other microbes including Candida albicans, Enterococcus faecalis and Staphylococcus epidermidis and a statistically significant response to GBS measured over these other microbes. To test the invivo efficacy of the biosensor, a pilot study using donated lower vaginal swabs from non-pregnant women was conducted, where 58 female adults were recruited. Participants donated two swabs, one which was used for the vesicle test and one for the 'gold standard', enriched culture media (ECM) test. An overall GBS carriage rate of 17.2% was measured using the ECM test. The vesicle biosensor test took 45 min to obtain a result, and showed a sensitivity of 83.3%, specificity of 85.7% and accuracy of 85.3%. The test accuracy is in line with current novel GBS identification tests, with the advantage of being rapid, easy to use, low-cost and able to be conducted by bedside during start of labour.

Vanillin Cross-Linked Chitosan Film with Controlled Release of Green Tea Polyphenols for Active Food Packaging.

We report a novel cross-linked chitosan composite film containing vanillin, glycerol, and green tea extract. The effects of vanillin-mediated cross-linking and the incorporation of antimicrobial green tea polyphenols were investigated. The cross-linking effect, confirmed by Fourier transform infrared (FTIR) analysis, increased the tensile strength of the biopolymer film to 20.9 ± 3 MPa. The release kinetics of polyphenols from the chitosan-vanillin matrix was studied, and we reported an initial burst release (8 h) followed by controlled release (8 to 400 h). It was found that both vanillin and green tea polyphenols were successful inhibitors of foodborne bacteria, with a minimum inhibitory concentration of the tea polyphenols determined as 0.15 mg/mL (Staphylococcus aureus). These active components also displayed strong antioxidant capacities, with polyphenols quenching >80% of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals at all concentrations tested. Degradation results revealed that there was a significant (>85%) mass loss of all samples after being buried in compost for 12 weeks. The biopolymeric films, prepared by solvent casting methods, adhere to green chemistry and waste valorization principles. The one-pot recipe reported may also be applied to other cross-linkers and active compounds with similar chemical functionalities. Based on the obtained results, the presented material provides a promising starting point for the development of a degradable active packaging material.

Improved Antibacterial Activity of 1,3,4-Oxadiazole-Based Compounds That Restrict Staphylococcus aureus Growth Independent of LtaS Function.

The lipoteichoic acid (LTA) biosynthesis pathway has emerged as a promising antimicrobial therapeutic target. Previous studies identified the 1,3,4 oxadiazole compound 1771 as an LTA inhibitor with activity against Gram-positive pathogens. We have succeeded in making six 1771 derivatives and, through subsequent hit validation, identified the incorporation of a pentafluorosulfanyl substituent as central in enhancing activity. Our newly described derivative, compound 13, showed a 16- to 32-fold increase in activity compared to 1771 when tested against a cohort of multidrug-resistant Staphylococcus aureus strains while simultaneously exhibiting an improved toxicity profile against mammalian cells. Molecular techniques were employed in which the assumed target, lipoteichoic acid synthase (LtaS), was both deleted and overexpressed. Neither deletion nor overexpression of LtaS altered 1771 or compound 13 susceptibility; however, overexpression of LtaS increased the MIC of Congo red, a previously identified LtaS inhibitor. These data were further supported by comparing the docking poses of 1771 and derivatives in the LtaS active site, which indicated the possibility of an additional target(s). Finally, we show that both 1771 and compound 13 have activity that is independent of LtaS, extending to cover Gram-negative species if the outer membrane is first permeabilized, challenging the classification that these compounds are strict LtaS inhibitors.

LoCKAmp: lab-on-PCB technology for <3 minute virus genetic detection.

The recent COVID-19 outbreak highlighted the need for lab-on-chip diagnostic technology fit for real-life deployment in the field. Existing bottlenecks in multistep analytical microsystem integration and upscalable, standardized fabrication techniques delayed the large-scale deployment of lab-on-chip solutions during the outbreak, throughout a global diagnostic test shortage. This study presents a technology that has the potential to address these issues by redeploying and repurposing the ubiquitous printed circuit board (PCB) technology and manufacturing infrastructure. We demonstrate the first commercially manufactured, miniaturised lab-on-PCB device for loop-mediated isothermal amplification (LAMP) genetic detection of SARS-CoV-2. The system incorporates a mass-manufactured, continuous-flow PCB chip with ultra-low cost fluorescent detection circuitry, rendering it the only continuous-flow µLAMP platform with off-the-shelf optical detection components. Ultrafast, SARS-CoV-2 RNA amplification in wastewater samples was demonstrated within 2 min analysis, at concentrations as low as 17 gc µL-1. We further demonstrate our device operation by detecting SARS-CoV-2 in 20 human nasopharyngeal swab samples, without the need for any RNA extraction or purification. This renders the presented miniaturised nucleic-acid amplification-based diagnostic test the fastest reported SARS-CoV-2 genetic detection platform, in a practical implementation suitable for deployment in the field. This technology can be readily extended to the detection of alternative pathogens or genetic targets for a very broad range of applications and matrices. LoCKAmp lab-on-PCB chips are currently mass-manufactured in a commercial, ISO-compliant PCB factory, at a small-scale production cost of £2.50 per chip. Thus, with this work, we demonstrate a high technology-readiness-level lab-on-chip-based genetic detection system, successfully benchmarked against standard analytical techniques both for wastewater and nasopharyngeal swab SARS-CoV-2 detection.

Staph wars: the antibiotic pipeline strikes back.

Antibiotic chemotherapy is widely regarded as one of the most significant medical advancements in history. However, the continued misuse of antibiotics has contributed to the rapid rise of antimicrobial resistance (AMR) globally. Staphylococcus aureus, a major human pathogen, has become synonymous with multidrug resistance and is a leading antimicrobial-resistant pathogen causing significant morbidity and mortality worldwide. This review focuses on (1) the targets of current anti-staphylococcal antibiotics and the specific mechanisms that confirm resistance; (2) an in-depth analysis of recently licensed antibiotics approved for the treatment of S. aureus infections; and (3) an examination of the pre-clinical pipeline of anti-staphylococcal compounds. In addition, we examine the molecular mechanism of action of novel antimicrobials and derivatives of existing classes of antibiotics, collate data on the emergence of resistance to new compounds and provide an overview of key data from clinical trials evaluating anti-staphylococcal compounds. We present several successful cases in the development of alternative forms of existing antibiotics that have activity against multidrug-resistant S. aureus. Pre-clinical antimicrobials show promise, but more focus and funding are required to develop novel classes of compounds that can curtail the spread of and sustainably control antimicrobial-resistant S. aureus infections.

Recruitment of C4b-binding protein is not a complement evasion strategy employed by Staphylococcus aureus.

Complement offers a first line of defence against infection through the opsonization of microbial pathogens, recruitment of professional phagocytes to the infection site and the coordination of inflammatory responses required for the resolution of infection. Staphylococcus aureus is a successful pathogen that has developed multiple mechanisms to thwart host immune responses. Understanding the precise strategies employed by S. aureus to bypass host immunity will be paramount for the development of vaccines and or immunotherapies designed to prevent or limit infection. To gain a better insight into the specific immune evasion mechanisms used by S. aureus we examined the pathogen's interaction with the soluble complement inhibitor, C4b-binding protein (C4BP). Previous studies indicated that S. aureus recruits C4BP using a specific cell-wall-anchored surface protein and that bound C4BP limits complement deposition on the staphylococcal surface. Using flow-cytometric-based bacterial-protein binding assays we observed no interaction between S. aureus and C4BP. Moreover, we offer a precautionary warning that C4BP isolated from plasma can be co-purified with minute quantities of human IgG, which can distort binding analysis between S. aureus and human-derived proteins. Combined our data indicates that recruitment of C4BP is not a complement evasion strategy employed by S. aureus.

Extensive remodelling of the cell wall during the development of Staphylococcus aureus bacteraemia.

The bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.

Novel Antibody-independent Method to Measure Complement Deposition on Bacteria.

During infection, complement plays a critical role in inflammation, opsonisation, and destruction of microorganisms. This presents a challenge for pathogens such asStaphylococcus aureusto overcome when invading the host. Our current knowledge on the mechanisms that evolved to counteract and disable this system is limited by the molecular tools available. Present techniques utilise labelled complement-specific antibodies to detect deposition upon the bacterial surface, a method not compatible with pathogens such asS. aureus, which are equipped with immunoglobulin-binding proteins, Protein A and Sbi. This protocol uses a novel antibody-independent probe, derived from the C3 binding domain of staphylococcal protein Sbi, in combination with flow cytometry, to quantify complement deposition. Sbi-IV is biotinylated, and deposition is quantified with fluorophore-labelled streptavidin. This novel method allows observation of wild-type cells without the need to disrupt key immune modulating proteins, presenting the opportunity to analyse the complement evasion mechanism used by clinical isolates. Here, we describe a step-by-step protocol for the expression and purification of Sbi-IV protein, quantification and biotinylation of the probe, and finally, optimisation of flow cytometry to detect complement deposition using normal human serum (NHS) and bothLactococcus lactisandS. aureus.

Antimicrobial releasing hydrogel forming microneedles.

Hydrogel-forming microneedle arrays as a technique for transdermal drug delivery show promise as an alternative to traditional drug delivery methods. In this work, hydrogel-forming microneedles have been created with effective, controlled delivery of amoxicillin and vancomycin within comparable therapeutic ranges to that of oral delivered antibiotics. Fabrication using reusable 3D printed master templates enabled quick and low-cost hydrogel microneedle manufacturing through micro-molding. By 3D printing at a tilt angle of 45° the resolution of the microneedle tip was improved by double (from ca. 64 µm down to 23 µm). Amoxicillin and vancomycin were encapsulated within the hydrogel's polymeric network through a unique room temperature swell/deswell drug loading method within minutes, eliminating the need for an external drug reservoir. The hydrogel-forming microneedle mechanical strength was maintained, and successful penetration of porcine skin grafts observed with negligible damage to the needles or surrounding skin morphology. Hydrogel swell rate was tailored by altering the crosslinking density, resulting in controlled antimicrobial release for an applicable delivered dosage. The potent antimicrobial properties of the antibiotic-loaded hydrogel-forming microneedles against both Escherichia coli and Staphylococcus aureus, highlights the beneficial use of hydrogel-forming microneedles towards the minimally invasive transdermal drug delivery of antibiotics.

Novel antimicrobial strategies to treat multi-drug resistant Staphylococcus aureus infections.

Antimicrobial resistance is a major obstacle for the treatment of infectious diseases and currently represents one of the most significant threats to global health. Staphylococcus aureus remains a formidable human pathogen with high mortality rates associated with severe systemic infections. S. aureus has become notorious as a multidrug resistant bacterium, which when combined with its extensive arsenal of virulence factors that exacerbate disease, culminates in an incredibly challenging pathogen to treat clinically. Compounding this major health issue is the lack of antibiotic discovery and development, with only two new classes of antibiotics approved for clinical use in the last 20 years. Combined efforts from the scientific community have reacted to the threat of dwindling treatment options to combat S. aureus disease in several innovative and exciting developments. This review describes current and future antimicrobial strategies aimed at treating staphylococcal colonization and/or disease, examining therapies that show significant promise at the preclinical development stage to approaches that are currently being investigated in clinical trials.

Extensive re-modelling of the cell wall during the development of Staphylococcus aureus bacteraemia.

The bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.

Practical Synthesis of Polyamine Succinamides and Branched Polyamines.

Antibiotic resistance is now a growing threat to human health, further exacerbated by the lack of new antibiotics. We describe the practical synthesis of a series of substituted polyamine succinamides and branched polyamines that are potential new antibiotics against both Gram-positive and Gram-negative bacteria, including MRSA and Pseudomonas aeruginosa. They are prepared via 1,4-Michael addition of acrylonitrile and then hydrogenation of the nitrile functional groups to primary amines. They are built upon the framework of the naturally occurring polyamines thermine (3.3.3, norspermine) and spermine (3.4.3), homo- and heterodimeric polyamine succinic amides. Linking two of the same or different polyamines together via amide bonds can be achieved by introducing a carboxylic acid group on the first polyamine, then coupling that released carboxylic acid to a free primary amine in the second polyamine. If the addition of positive charges on the amino groups along the polyamine chains are a key factor in their antimicrobial activity against Gram-negative bacteria, then increasing them will increase the antimicrobial activity. Synthesising polyamine amide dimers will increase the total net positive charge compared to their monomers. The design and practical synthesis of such homo- and hetero-dimers of linear polyamines, spermine and norspermine, are reported. Several of these compounds do not display significant antibacterial activity against Gram-positive or Gram-negative bacteria, including MRSA and Pseudomonas aeruginosa. However, the most charged analogue, a branched polyamine carrying eight positive charges at physiological pH, displays antibiofilm activity with a 50 % reduction in PAO1 at 16-32 µg mL-1 .

Antibacterial activity of novel linear polyamines against Staphylococcus aureus.

New therapeutic options are urgently required for the treatment of Staphylococcus aureus infections. Accordingly, we sought to exploit the vulnerability of S. aureus to naturally occurring polyamines. We have developed and tested the anti-staphylococcal activity of three novel linear polyamines based on spermine and norspermine. Using a panel of genetically distinct and clinically relevant multidrug resistant S. aureus isolates, including the polyamine resistant USA300 strain LAC, compound AHA-1394 showed a greater than 128-fold increase in inhibition against specific S. aureus strains compared to the most active natural polyamine. Furthermore, we show that AHA-1394 has superior biofilm prevention and biofilm dispersal properties compared to natural polyamines while maintaining minimal toxicity toward human HepG2 cells. We examined the potential of S. aureus to gain resistance to AHA-1394 following in vitro serial passage. Whole genome sequencing of two stable resistant mutants identified a gain of function mutation (S337L) in the phosphatidylglycerol lysyltransferase mprF gene. Inactivation of mutant mprF confirmed the importance of this allele to AHA-1394 resistance. Importantly, AHA-1394 resistant mutants showed a marked decrease in relative fitness and increased generation time. Intriguingly, mprF::S337L contributed to altered surface charge only in the USA300 background whereas increased cell wall thickness was observed in both USA300 and SH1000. Lastly, we show that AHA-1394 displays a particular proclivity for antibiotic potentiation, restoring sensitivity of MRSA and VRSA isolates to daptomycin, oxacillin and vancomycin. Together this study shows that polyamine derivatives are impressive drug candidates that warrant further investigation.

Novel method for detecting complement C3 deposition on Staphylococcus aureus.

The primary host response to Staphylococcus aureus infection occurs via complement. Complement is an elegant evolutionarily conserved system, playing essential roles in early defences by working in concert with immune cells to survey, label and destroy microbial intruders and coordinate inflammation. Currently the exact mechanisms employed by S. aureus to manipulate and evade complement is not clear and is hindered by the lack of accurate molecular tools that can report on complement deposition on the bacterial surface. Current gold-standard detection methods employ labelled complement-specific antibodies and flow cytometry to determine complement deposited on bacteria. These methods are restricted by virtue of the expression of the S. aureus immunoglobulin binding proteins, Protein A and Sbi. In this study we describe the use of a novel antibody-independent C3 probe derived from the staphylococcal Sbi protein, specifically Sbi-IV domain. Here we show that biotin-labelled Sbi-IV interacts specifically with deposited C3 products on the staphylococcal surface and thus can be used to measure complement fixation on wild-type cells expressing a full repertoire of immune evasion proteins. Lastly, our data indicates that genetically diverse S. aureus strains restrict complement to different degrees suggesting that complement evasion is a variable virulence trait among S. aureus isolates.

CidA and LrgA: a "Hole" Lot More than Programmed Cell Death.

What do programmed cell death (PCD) and carbohydrate metabolism by-product transport have in common? Intriguingly, both processes involve the cidABC and lrgAB operons in the major human pathogen Staphylococcus aureus. Previously, CidA and LrgA have been studied in the context of programmed cell death, but a second function in overflow metabolism is increasingly evident. New work from J. L. Endres, S. S. Chaudhari, X. Zhang, J. Prahlad, et al. (mBio 13:e02827-21, 2022, https://doi.org/10.1128/mBio.02827-21) combining a lysis cassette, mutagenesis, and classic microbiology demonstrates that CidA and LrgA function as holins to support endolysin-induced lysis. But that's not all-the lrgAB operon also facilitates pyruvate uptake during microaerobic and anaerobic growth. This commentary highlights the main findings from this work and places them in context of the literature to date. Finally, as these proteins are highly conserved and carry out disparate functions of great importance, it is tempting to speculate future work will elucidate the link between S. aureus lysis and pyruvate metabolism.

Clinical Isolates of Acinetobacter spp. Are Highly Serum Resistant Despite Efficient Recognition by the Complement System.

Gram-negative bacteria from the genus Acinetobacter are responsible for life-threating hospital-related infections such as pneumonia, septicemia, and meningitis, especially in immunocompromised patients. Worryingly, Acinetobacter have become multi- and extensively drug resistant (MDR/XDR) over the last few decades. The complement system is the first line of defense against microbes, thus it is highly important to increase our understanding of evasion mechanisms used by Acinetobacter spp. Here, we studied clinical isolates of Acinetobacter spp. (n=50), aiming to characterize their recognition by the complement system. Most isolates tested survived 1 h incubation in 30% serum, and only 8 isolates had a lower survival rate, yet none of those isolates were fully killed. Intriguingly, four isolates survived in human whole blood containing all cell component. Their survival was, however, significantly reduced. Flow cytometry analyses revealed that most of the isolates were detected by human IgG and IgM. Interestingly, we could not detect any significant concentration of deposited C1q, despite observing C4b deposition that was abolished in C1q-deficient serum, indicating transient binding of C1q to bacteria. Moreover, several isolates were recognized by MBL, with C4b deposition abolished in MBL-deficient serum. C3b was deposited on most isolates, but this was not, however, seen with respect to C5b and formation of the membrane attack complex (MAC), indicating that many isolates could avoid complement-mediated lysis. India ink staining showed that isolates were capsulated, and capsule thickness varied significantly between isolates. Studies performed on a wild-type strain and capsule mutant strains, demonstrated that the production of a capsular polysaccharide is one mechanism that mediates resistance to complement-mediated bactericidal activity by preventing MAC deposition and lysis. Our data showed that most clinical Acinetobacter spp. isolates are highly serum resistant despite being efficiently recognized by the complement system.

Optimisation of a lozenge-based sensor for detecting impending blockage of urinary catheters.

Catheter-associated urinary tract infections resulting from urease-positive microorganisms are more likely to cause a urinary catheter blockage owing to the urease activity of the microbes. Catheter blockage can be dangerous and increases the risk of severe infections, such as sepsis. Ureases, a virulence factor in Proteus mirabilis, cause an increase in urine pH - leading to blockage. An optimised biosensor "lozenge" is presented here, which is able to detect impending catheter blockage. This lozenge has been optimised to allow easy manufacture and commercialisation. It functions as a sensor in a physiologically representative model of a catheterised urinary tract, providing 6.7 h warning prior to catheter blockage. The lozenge is stable in healthy human urine and can be sterilized for clinical use by ethylene oxide. Clinically, the lozenge will provide a visible indication of impending catheter blockage, enabling quicker clinical intervention and thus reducing the morbidity and mortality associated with blockage.

Practical Synthesis of Antimicrobial Long Linear Polyamine Succinamides.

There are many severe bacterial infections notorious for their ability to become resistant to clinically relevant antibiotics. Indeed, antibiotic resistance is a growing threat to human health, further exacerbated by the lack of new antibiotics. We now describe the practical synthesis of a series of substituted long linear polyamines that produce rapid antibacterial activity against both Gram-positive and Gram-negative bacteria, including meticillin-resistant Staphylococcus aureus. These compounds also reduce biofilm formation in Pseudomonas aeruginosa. The most potent analogues are thermine, spermine, and 1,12-diaminododecane homo- and heterodimeric polyamine succinic acid amides. They are of the order of activity of the aminoglycoside antibiotics kanamycin and tobramycin as positive controls. Their low human cell toxicity is demonstrated in ex vivo hemolytic assays where they did not produce even 5% hemolysis of human erythrocytes. These long, linear polyamines are a new class of broad-spectrum antibacterials active against drug-resistant pathogens.

Significant variability exists in the cytotoxicity of global methicillin-resistant Staphylococcus aureus lineages.

Staphylococcus aureus is a major human pathogen where the emergence of antibiotic resistant lineages, such as methicillin-resistant S. aureus (MRSA), is a major health concern. While some MRSA lineages are restricted to the healthcare setting, the epidemiology of MRSA is changing globally, with the rise of specific lineages causing disease in healthy people in the community. In the past two decades, community-associated MRSA (CA-MRSA) has emerged as a clinically important and virulent pathogen associated with serious skin and soft-tissue infections (SSTI). These infections are primarily cytotoxin driven, leading to the suggestion that hypervirulent lineages/multi-locus sequence types (STs) exist. To examine this, we compared the cytotoxicity of 475 MRSA isolates representing five major MRSA STs (ST22, ST93, ST8, ST239 and ST36) by employing a monocyte-macrophage THP-1 cell line as a surrogate for measuring gross cytotoxicity. We demonstrate that while certain MRSA STs contain highly toxic isolates, there is such variability within lineages to suggest that this aspect of virulence should not be inferred from the genotype of any given isolate. Furthermore, by interrogating the accessory gene regulator (Agr) sequences in this collection we identified several Agr mutations that were associated with reduced cytotoxicity. Interestingly, the majority of isolates that were attenuated in cytotoxin production contained no mutations in the agr locus, indicating a role of other undefined genes in S. aureus toxin regulation.