Electron transport chain capacity expands yellow fever vaccine immunogenicity.

Vaccination has successfully controlled several infectious diseases although better vaccines remain desirable. Host response to vaccination studies have identified correlates of vaccine immunogenicity that could be useful to guide development and selection of future vaccines. However, it remains unclear whether these findings represent mere statistical correlations or reflect functional associations with vaccine immunogenicity. Functional associations, rather than statistical correlates, would offer mechanistic insights into vaccine-induced adaptive immunity. Through a human experimental study to test the immunomodulatory properties of metformin, an anti-diabetic drug, we chanced upon a functional determinant of neutralizing antibodies. Although vaccine viremia is a known correlate of antibody response, we found that in healthy volunteers with no detectable or low yellow fever 17D viremia, metformin-treated volunteers elicited higher neutralizing antibody titers than placebo-treated volunteers. Transcriptional and metabolomic analyses collectively showed that a brief course of metformin, started 3 days prior to YF17D vaccination and stopped at 3 days after vaccination, expanded oxidative phosphorylation and protein translation capacities. These increased capacities directly correlated with YF17D neutralizing antibody titers, with reduced reactive oxygen species response compared to placebo-treated volunteers. Our findings thus demonstrate a functional association between cellular respiration and vaccine-induced humoral immunity and suggest potential approaches to enhancing vaccine immunogenicity.

Alterations in cell arrangements of group B streptococcus due to virulence factor expression can bias estimates of bacterial populations based on colony count measures.

Group B streptococcus (GBS) is a chain-forming commensal bacterium and opportunistic pathogen that resides in the gastrointestinal and genitourinary tract of healthy adults. GBS can cause various infections and related complications in pregnant and nonpregnant women, adults, and newborns. Investigations of the mechanisms by which GBS causes disease pathogenesis often utilize colony count assays to estimate bacterial population size in experimental models. In other streptococci, such as group A streptococcus and pneumococcus, variation in the chain length of the bacteria that can occur naturally or due to mutation can affect facets of pathogenesis, such as adherence to or colonization of a host. No studies have reported a relationship between GBS chain length and pathogenicity. Here, we used GBS strain 874391 and several derivative strains displaying longer chain-forming phenotypes (874391pgapC, 874391ΔcovR, 874391Δstp1) to assess the impact of chain length on bacterial population estimates based on the colony-forming unit (c.f.u.) assay. Disruption of GBS chains via bead beating or sonication in conjunction with fluorescence microscopy was used to compare chaining phenotypes pre- and post-disruption to detect long- and short-chain forms, respectively. We used a murine model of GBS colonization of the female reproductive tract to assess whether chaining may affect bacterial colonization dynamics in the host during chronic infection in vivo. Overall, we found that GBS exhibiting long-chain form can significantly affect population size estimates based on the colony count assay. Additionally, we found that the length of chaining of GBS can affect virulence in the reproductive tract colonization model. Collectively, these findings have implications for studies of GBS that utilize colony count assays to measure GBS populations and establish that chain length can affect infection dynamics and disease pathogenesis for this important opportunistic pathogen.

An opportunistic pathogen under stress: how Group B Streptococcus responds to cytotoxic reactive species and conditions of metal ion imbalance to survive.

Group B Streptococcus (GBS; also known as Streptococcus agalactiae) is an opportunistic bacterial pathogen that causes sepsis, meningitis, pneumonia, and skin and soft tissue infections in neonates and healthy or immunocompromised adults. GBS is well-adapted to survive in humans due to a plethora of virulence mechanisms that afford responses to support bacterial survival in dynamic host environments. These mechanisms and responses include counteraction of cell death from exposure to excess metal ions that can cause mismetallation and cytotoxicity, and strategies to combat molecules such as reactive oxygen and nitrogen species that are generated as part of innate host defence. Cytotoxicity from reactive molecules can stem from damage to proteins, DNA, and membrane lipids, potentially leading to bacterial cell death inside phagocytic cells or within extracellular spaces within the host. Deciphering the ways in which GBS responds to the stress of cytotoxic reactive molecules within the host will benefit the development of novel therapeutic and preventative strategies to manage the burden of GBS disease. This review summarizes knowledge of GBS carriage in humans and the mechanisms used by the bacteria to circumvent killing by these important elements of host immune defence: oxidative stress, nitrosative stress, and stress from metal ion intoxication/mismetallation.

Resisting death by metal: metabolism and Cu/Zn homeostasis in bacteria.

Metal ions such as zinc and copper play important roles in host-microbe interactions and their availability can drastically affect the survival of pathogenic bacteria in a host niche. Mechanisms of metal homeostasis protect bacteria from starvation, or intoxication, defined as when metals are limiting, or in excess, respectively. In this mini-review, we summarise current knowledge on the mechanisms of resistance to metal stress in bacteria, focussing specifically on the homeostasis of cellular copper and zinc. This includes a summary of the factors that subvert metal stress in bacteria, which are independent of metal efflux systems, and commentary on the role of small molecules and metabolic systems as important mediators of metal resistance.

A convergent evolutionary pathway attenuating cellulose production drives enhanced virulence of some bacteria.

Bacteria adapt to selective pressure in their immediate environment in multiple ways. One mechanism involves the acquisition of independent mutations that disable or modify a key pathway, providing a signature of adaptation via convergent evolution. Extra-intestinal pathogenic Escherichia coli (ExPEC) belonging to sequence type 95 (ST95) represent a global clone frequently associated with severe human infections including acute pyelonephritis, sepsis, and neonatal meningitis. Here, we analysed a publicly available dataset of 613 ST95 genomes and identified a series of loss-of-function mutations that disrupt cellulose production or its modification in 55.3% of strains. We show the inability to produce cellulose significantly enhances ST95 invasive infection in a rat model of neonatal meningitis, leading to the disruption of intestinal barrier integrity in newborn pups and enhanced dissemination to the liver, spleen and brain. Consistent with these observations, disruption of cellulose production in ST95 augmented innate immune signalling and tissue neutrophil infiltration in a mouse model of urinary tract infection. Mutations that disrupt cellulose production were also identified in other virulent ExPEC STs, Shigella and Salmonella, suggesting a correlative association with many Enterobacteriaceae that cause severe human infection. Together, our findings provide an explanation for the emergence of hypervirulent Enterobacteriaceae clones.

Does harvesting age matter? Changes in structure and rheology of a shear-thickening polysaccharide from Cyathea medullaris as a function of age.

A shear-thickening polysaccharide from the New Zealand Black tree fern (Cyathea medullaris, commonly known as mamaku) extracted from different age fronds (stage 1: young, stage 2: fully grown and stage 3: old) was characterised in terms of structure and rheological properties. Constituent sugar analysis and 1H and 13C NMR revealed a repeating backbone of -4)-ß-D-GlcpA-(1 â†’ 2)-α-D-Manp-(1→, for all mamaku polysaccharide (MP) samples from different age fronds without any alterations in molecular structure. However, the molecular weight (Mw) was reduced with increasing age, from ~4.1 × 106 to ~2.1 × 106 Da from stage 1 to stage 3, respectively. This decrease in Mw (and size) consequently reduced the shear viscosity (ηs-Stage 1 > Î·s-Stage 2 > Î·s-Stage 3). However, the extent of shear-thickening and uniaxial extensional viscosity of MP stage 2 was greater than MP stage 1, which was attributed to a greater intermolecular interaction occurring in the former. Shear-thickening behaviour was not observed in MP stage 3.

The fate of mamaku gum in the gut: effect on in vitro gastrointestinal function and colon fermentation by human faecal microbiota.

Mamaku is a fern indigenous to the Pacific Islands with a long history of use for therapeutic benefits such as to combat skin conditions and manage gastrointestinal discomfort; however, the scientific understanding is limited. In this study, we examined the effect of mamaku gum, extracted from different age fronds of the New Zealand Black tree fern (Cyathea medullaris, Mamaku) (stage 1: young, stage 2: fully grown and stage 3: old), on gut function using in vitro models of static digestion, enzyme activity and static colonic fermentation. Under simulated gastric and small intestinal conditions, mamaku polysaccharide (MP) was indigestible as there was no decrease in the molecular weight (Mw) of the polymer. Mamaku gum could reduce the activity of digestive enzymes (α-amylase, pepsin and lipase) in a concentration-dependent manner, with the stage 1 sample showing the highest inhibition and stage 3 the lowest. All three mamaku gum samples could also equally bind bile acids during intestinal digestion. During fermentation, human faecal microbiota utilised the mamaku gum and significantly increased the production of total short-chain fatty acids (SCFAs) and reduced the pH when compared with the blank. However, changes in SCFAs and pH for mamaku groups were less prominent than for inulin and guar gum control groups, suggesting lower fermentability of mamaku gum compared to the latter two. Furthermore, mamaku gum altered the composition of colonic microbiota, specifically reducing the ratio of Firmicutes to Bacteroidetes and increasing the relative abundance of Bacteroides, Enterococcus, Paraprevotella and Parabacteroides genera. No obvious difference between mamaku gum samples from stage 1, 2 and 3 was observed during fermentation. Collectively, these results suggest that mamaku gum may modulate the functionality of the host gut by reducing enzyme activity, binding bile acids, altering the colonic microbial composition and producing SCFAs.

Variable resistance to zinc intoxication among Streptococcus agalactiae reveals a novel IS1381 insertion element within the zinc efflux transporter gene czcD.

Streptococcus agalactiae, also known as group B Streptococcus, is an important human and animal pathogen. Zinc (Zn) is an essential trace element for normal bacterial physiology but intoxicates bacteria at high concentrations. Molecular systems for Zn detoxification exist in S. agalactiae, however the degree to which Zn detoxification may vary among different S. agalactiae isolates is not clear. We measured resistance to Zn intoxication in a diverse collection of clinical isolates of S. agalactiae by comparing the growth of the bacteria in defined conditions of Zn stress. We found significant differences in the ability of different S. agalactiae isolates to resist Zn intoxication; some strains such as S. agalactiae 18RS21 were able to survive and grow at 3.8-fold higher levels of Zn stress compared to other reference strains such as BM110 (6.4mM vs 1.68mM Zn as inhibitory, respectively). We performed in silico analysis of the available genomes of the S. agalactiae isolates used in this study to examine the sequence of czcD, which encodes an efflux protein for Zn that supports resistance in S. agalactiae. Interestingly, this revealed the presence of a mobile insertion sequence (IS) element, termed IS1381, in the 5' region of czcD in S. agalactiae strain 834, which was hyper-resistant to Zn intoxication. Interrogating a wider collection of S. agalactiae genomes revealed identical placement of IS1381 in czcD in other isolates from the clonal-complex-19 (CC19) 19 lineage. Collectively, these results show a resistance spectrum among S. agalactiae isolates enables survival in varying degrees of Zn stress, and this phenotypic variability has implications for understanding bacterial survival in metal stress.

Effects of Incorporating Alkaline Hydrogen Peroxide Treated Sugarcane Fibre on The Physical Properties and Glycemic Potency of White Bread.

The consumption of dietary fibres can affect glycemic power and control diabetes. Sugarcane fibre (SCF) is known as insoluble dietary fibre, the properties of which can be affected by physical, chemical, and enzymatic treatments. In this study, alkaline hydrogen peroxide (AHP) treatments were conducted over time (0.5, 1, 3, and 5 h) at 12.6% (w/v) SCF and the effects on the physicochemical and structural properties of the SCF were evaluated. After making dough and bread with the SCF, with and without AHP treatments, the glycemic responses of the bread samples were evaluated. Shorter durations of AHP treatment (0.5 and 1 h) reduced lignin effectively (37.3 and 40.4%, respectively), whereas AHP treatment at 1 and 3 h duration was more effective in increasing particle sizes (50.9 and 50.1 µm, respectively). The sugar binding capacity, water holding capacity (from 2.98 to 3.86 g water/g SCF), and oil holding capacity (from 2.47 to 3.66 g oil/g SCF) increased in all AHP samples. Results from Fourier-transform infrared spectroscopy (FTIR) confirmed the polymorphism transition of cellulose (cellulose I to cellulose II). The morphology of SCF detected under scanning electron microscopy (SEM) indicated the conversion of the surface to a more porous, rough structure due to the AHP treatment. Adding SCF decreased dough extensibility but increased bread hardness and chewiness. All SCF-incorporated bread samples have reduced glycemic response. Incorporation of 1, 3, and 5 h AHP-treated SCF was effective in reducing the glycemic potency than 0.5 h AHP-treated SCF, but not significantly different from the untreated SCF. Overall, this study aims to valorize biomass as AHP is commonly applied to bagasse to produce value-added chemicals and fuels.

Insights into the Mechanism of Aluminum-Catalyzed Halodefluorination.

Aluminum has been reported to catalyze halodefluorination reactions, where aliphatic fluorine is substituted with a heavier halogen. Although it is known that stoichiometric aluminum halide can perform this reaction, the role of catalytic aluminum halide and organyl alane reagents is not well understood. We investigate the mechanism of the halodefluorination reaction using catalytic aluminum halide and stoichiometric trimethylsilyl halide. We explore the use of B(C6F5)3 as a catalyst to benchmark pathways where aluminum acts either as a Lewis acid catalyst in cooperation with trimethylsilyl halide or as an independent halodefluorination reagent which is subsequently regenerated by trimethylsilyl halide. Computational and experimental results indicate that aluminum acts as an independent halodefluorination reagent and that reactivity trends observed between different halide reagents can be attributed to relative barriers in halide delivery to the organic fragment, which is the rate-limiting step in both the aluminum halide- and B(C6F5)3-catalyzed pathways.

Dual RNA sequencing of group B Streptococcus-infected human monocytes reveals new insights into host-pathogen interactions and bacterial evasion of phagocytosis.

Streptococcus agalactiae, also known as Group B Streptococcus (GBS) is a frequent cause of infections, including bacteraemia and other acute diseases in adults and immunocompromised individuals. We developed a novel system to study GBS within human monocytes to define the co-transcriptome of intracellular GBS (iGBS) and host cells simultaneously using dual RNA-sequencing (RNA-seq) to better define how this pathogen responds to host cells. Using human U937 monocytes and genome-sequenced GBS reference strain 874,391 in antibiotic protection assays we validated a system for dual-RNA seq based on measures of GBS and monocyte viability to ensure that the bacterial and host cell co-transcriptome reflected mainly intracellular (iGBS) rather than extracellular GBS. Elucidation of the co-transcriptome revealed 1119 dysregulated transcripts in iGBS with most genes, including several that encode virulence factors (e.g., scpB, hvgA, ribD, pil2b) exhibiting activation by upregulated expression. Infection with iGBS resulted in significant remodelling of the monocyte transcriptome, with 7587 transcripts differentially expressed including 7040 up-regulated and 547 down-regulated. qPCR confirmed that the most strongly activated genes included sht, encoding Streptococcal Histidine Triad Protein. An isogenic GBS mutant strain deficient in sht revealed a significant effect of this gene on phagocytosis of GBS and survival of the bacteria during systemic infection in mice. Identification of a novel contribution of sht to GBS virulence shows the co-transcriptome responses elucidated in GBS-infected monocytes help to shape the host-pathogen interaction and establish a role for sht in the response of the bacteria to phagocytic uptake. This study provides comprehension of concurrent transcriptional responses that occur in GBS and human monocytes that shape the host-pathogen interaction.

Enhanced Monitoring System to Better Monitor High-Risk COVID-19 Patients.

Telemedicine (TM) is a useful tool to extend medical care during a pandemic. TM was extensively utilized in Singapore during the COVID-19 pandemic as part of the Nation's COVID-19 healthcare strategy. Patients were risk stratified to prioritize limited healthcare resources and the Telemedicine Allocation Reconciliation System (TMARS) was adapted to monitor and manage limited TM resources. High-Risk patients (Protocol 1) had an escalation rate of 4.87%, compared to the non-High-Risk patients' 0.002% and TM doctors spent an average of six hours to complete one tele-consultation. In order to optimize the efficiency of the TM system, an enhanced monitoring system was implemented in March 2022. The intent was to focus monitoring efforts on the High-Risk patients. High-Risk patients reporting sick for the first time were prioritized to receive tele-consultations through this system. With the aid of a data-driven dashboard, the Operations Control and Monitoring team (OCM) was able to closely monitor the performance of the various TM providers (TMPs), sent them timely reminders and re-assigned patients to other TMPs when the requisite turnaround time was not met. Implementing the enhanced monitoring system resulted in a significant reduction in the average time taken to provide tele-consultations. After 3 months of implementation, the percentages of consultations completed within two hours were raised from 75.7% (February 2022) to 96.8% (May 2022), greatly increasing productivity and efficiency.

High-Protein Foods for Dysphagia: Manipulation of Mechanical and Microstructural Properties of Whey Protein Gels Using De-Structured Starch and Salts.

This study focuses on understanding the effect of ionic strength on the mechanical and microstructural properties of novel composite gels containing 13% whey protein isolate (WPI) and 4% de-structured waxy potato starch (DWPS). The DWPS is a physically modified waxy potato starch treated at 140 °C for 30 min under constant shear. Thermodynamic incompatibility between WPI and DWPS was observed upon the addition of NaCl (~75 mM) or CaCl2 (10-75 mM). The combined effects of such thermodynamic incompatibility with the changes in protein connectivity induced by varied ionic strength led to the formation of distinctive gel structures (inhomogeneous self-supporting gels with a liquid centre and weak gels with paste-like consistency) that were different from thermodynamic compatible homogeneous self-supporting gels (pure WPI and WPI + maltodextrin gels). At ≥ 250 mM NaCl, instead of a paste-like texture, a recovered soft and creamy self-supporting gel structure was observed when using DWPS. The ability to generate a range of textures in WPI gelation-based foods by using DWPS under different ionic conditions, is a feasible strategy for formulating high-protein foods for dysphagia-aimed to be either thickened fluids or soft solids. Additionally, this acquired knowledge is also relevant when formulating food gels for 3-D printing.

Complexation of Anthocyanin-Bound Blackcurrant Pectin and Whey Protein: Effect of pH and Heat Treatment.

A complexation study between blackcurrant pectin (BCP) and whey protein (WP) was carried out to investigate the impact of bound anthocyanins on pectin−protein interactions. The effects of pH (3.5 and 4.5), heating (85 °C, 15 min), and heating sequence (mixed-heated or heated-mixed) were studied. The pH influenced the color, turbidity, particle size, and zeta-potential of the mixtures, but its impact was mainly significant when heating was introduced. Heating increased the amount of BCP in the complexes­especially at pH 3.5, where 88% w/w of the initial pectin was found in the sedimented (insoluble) fraction. Based on phase-separation measurements, the mixed-heated system at pH 4.5 displayed greater stability than at pH 3.5. Heating sequence was essential in preventing destabilization of the systems; mixing of components before heating produced a more stable system with small complexes (<300 nm) and relatively low polydispersity. However, heating WP before mixing with BCP prompted protein aggregation­producing large complexes (>400 nm) and worsening the destabilization. Peak shifts and emergence (800−1200 cm−1) in infrared spectra confirmed that BCP and WP functional groups were altered after mixing and heating via electrostatic, hydrophobic, and hydrogen bonding interactions. This study demonstrated that appropriate processing conditions can positively impact anthocyanin-bound pectin−protein interactions.

Regulatory cross-talk supports resistance to Zn intoxication in Streptococcus.

Metals such as copper (Cu) and zinc (Zn) are important trace elements that can affect bacterial cell physiology but can also intoxicate bacteria at high concentrations. Discrete genetic systems for management of Cu and Zn efflux have been described in several bacterial pathogens, including streptococci. However, insight into molecular cross-talk between systems for Cu and Zn management in bacteria that drive metal detoxification, is limited. Here, we describe a biologically consequential cross-system effect of metal management in group B Streptococcus (GBS) governed by the Cu-responsive copY regulator in response to Zn. RNAseq analysis of wild-type (WT) and copY-deficient GBS subjected to metal stress revealed unique transcriptional links between the systems for Cu and Zn detoxification. We show that the Cu-sensing role of CopY extends beyond Cu and enables CopY to regulate Cu and Zn stress responses that effect changes in gene function for central cellular processes, including riboflavin synthesis. CopY also supported GBS intracellular survival in human macrophages and virulence during disseminated infection in mice. In addition, we show a novel role for CovR in modulating GBS resistance to Zn intoxication. Identification of the Zn resistome of GBS using TraDIS revealed a suite of genes essential for GBS growth in metal stress. Several of the genes identified are novel to systems that support bacterial survival in metal stress and represent a diverse set of mechanisms that underpin microbial metal homeostasis during cell stress. Overall, this study reveals a new and important mechanism of cross-system complexity driven by CopY in bacteria to regulate cellular management of metal stress and survival.

Ucl fimbriae regulation and glycan receptor specificity contribute to gut colonisation by extra-intestinal pathogenic Escherichia coli.

Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.

Effects of Xanthan Gum, Lambda-Carrageenan and Psyllium Husk on the Physical Characteristics and Glycaemic Potency of White Bread.

White bread contains a high proportion of easily digestible starch, which contributes to an undesirable rapid increase in blood glucose concentration. This study investigated the effects of nonstarch polysaccharides (NSP) -xanthan gum, lambda-carrageenan and psyllium husk on the physical functionality and glycaemic potency of white bread. The amount of water for each formulation was adjusted based on DoughLab set at a target torque value of ~500 FU for sufficient dough development. Adding NSP generally resulted in significantly increased loaf volumes and decreased hardness. The glycaemic potency (glycaemic glucose equivalents (GGE) g) of bread was found to be reduced with the addition of NSP at all levels (1, 3 and 5% w/w based on flour weight). Increasing the concentration of xanthan gum and lambda-carrageenan did not show any further decrease in the glycaemic potency. Notably, adding 5% w/w psyllium husk significantly reduced the glycaemic potency from ~49 GGE/100 g in the reference bread to 32 GGE/100 g. The reduction in the glycaemic potency was attributed to viscosity effects (for xanthan) and starch-NSP interactions (for psyllium husk). Overall, the 5% w/w psyllium husk bread sample was most promising in terms of both physical characteristics and its effect on in vitro glucose release.

The Copper Resistome of Group B Streptococcus Reveals Insight into the Genetic Basis of Cellular Survival during Metal Ion Stress.

In bacteria, copper (Cu) can support metabolic processes as an enzymatic cofactor but can also cause cell damage if present in excess, leading to intoxication. In group B Streptococcus (GBS), a system for control of Cu efflux based on the prototypical cop operon supports survival during Cu stress. In some other bacteria, genetic systems additional to the cop operon are engaged during Cu stress and also contribute to the management of cellular Cu homeostasis. Here, we examined genetic systems beyond the cop operon in GBS for regions that contribute to survival of GBS in Cu stress using a forward genetic screen and probe of the entire bacterial genome. A high-density mutant library, generated using pGh9-ISS1, was used to expose GBS to Cu stress and compare it to nonexposed controls en masse. Eight genes were identified as essential for GBS survival in Cu stress, whereas five genes constrained GBS growth in Cu stress. The genes encode varied factors including enzymes for metabolism, cell wall synthesis, transporters, and cell signaling factors. Targeted mutation of the genes validated their roles in GBS resistance to Cu stress. Excepting copA, the genes identified are new to the area of bacterial metal ion intoxication. We conclude that a discrete and limited suite of genes beyond the cop operon in GBS contributes to a repertoire of mechanisms used to survive Cu stress in vitro and achieve cellular homeostasis. IMPORTANCE Genetic systems for copper (Cu) homeostasis in bacteria, including streptococci, are vital to survive metal ion stress. Genetic systems that underpin survival of GBS during Cu stress, beyond the archetypal cop operon for Cu management, are undefined. We show that Streptococcus resists Cu intoxication by utilizing a discrete and limited suite of genes beyond the cop operon, including several genes that are new to the area of bacterial cell metal ion homeostasis. The Cu resistome of GBS defined here enhances our understanding of metal ion homeostasis in GBS.

Quantification of Fosfomycin in Combination with Nine Antibiotics in Human Plasma and Cation-Adjusted Mueller-Hinton II Broth via LCMS.

Fosfomycin-based combination therapy has emerged as an attractive option in our armamentarium due to its synergistic activity against carbapenem-resistant Gram-negative bacteria (CRGNB). The ability to simultaneously measure fosfomycin and other antibiotic drug levels will support in vitro and clinical investigations to develop rational antibiotic combination dosing regimens against CRGNB infections. We developed an analytical assay to measure fosfomycin with nine important antibiotics in human plasma and cation-adjusted Mueller-Hinton II broth (CAMHB). We employed a liquid-chromatography tandem mass spectrometry method and validated the method based on accuracy, precision, matrix effect, limit-of-detection, limit-of-quantification, specificity, carryover, and short-term and long-term stability on U.S. Food & Drug Administration (FDA) guidelines. Assay feasibility was assessed in a pilot clinical study in four patients on antibiotic combination therapy. Simultaneous quantification of fosfomycin, levofloxacin, meropenem, doripenem, aztreonam, piperacillin/tazobactam, ceftolozane/tazobactam, ceftazidime/avibactam, cefepime, and tigecycline in plasma and CAMHB were achieved within 4.5 min. Precision, accuracy, specificity, and carryover were within FDA guidelines. Fosfomycin combined with any of the nine antibiotics were stable in plasma and CAMHB up to 4 weeks at -80 °C. The assay identified and quantified the respective antibiotics administered in the four subjects. Our assay can be a valuable tool for in vitro and clinical applications.