Bacterial Hypoxic Responses Revealed as Critical Determinants of the Host-Pathogen Outcome by TnSeq Analysis of Staphylococcus aureus Invasive Infection

Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction.     

Hypoxia selectively inhibits respiratory burst activity and killing of Staphylococcus aureus in human neutrophils

Neutrophils play a central role in the innate immune response and a critical role in bacterial killing. Most studies of neutrophil function have been conducted under conditions of ambient oxygen, but inflamed sites where neutrophils operate may be extremely hypoxic. Previous studies indicate that neutrophils sense and respond to hypoxia via the ubiquitous prolyl hydroxylase/hypoxia-inducible factor pathway and that this can signal for enhanced survival. In the current study, human neutrophils were shown to upregulate hypoxia-inducible factor (HIF)-1α-dependent gene expression under hypoxic incubation conditions (3 kPa), with a consequent substantial delay in the onset of apoptosis. Despite this, polarization and chemotactic responsiveness to IL-8 and fMLP were entirely unaffected by hypoxia. Similarly, hypoxia did not diminish the ability of neutrophils to phagocytose serum-opsonized heat-killed streptococci. Of the secretory functions examined, IL-8 generation was preserved and elastase release was enhanced by hypoxia. Hypoxia did, however, cause a major reduction in respiratory burst activity induced both by the soluble agonist fMLP and by ingestion of opsonized zymosan, without affecting expression of the NADPH oxidase subunits. Critically, this reduction in respiratory burst activity under hypoxia was associated with a significant defect in the killing of Staphylococcus aureus. In contrast, killing of Escherichia coli, which is predominantly oxidase independent, was fully preserved under hypoxia. In conclusion, these studies suggest that although the NADPH oxidase-dependent bacterial killing mechanism may be compromised by hypoxia, neutrophils overall appear extremely well adapted to operate successfully under severely hypoxic conditions.     

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca²⁺. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSMα operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.     

Neutrophil Recruitment to Lymph Nodes Limits Local Humoral Response to Staphylococcus aureus

Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF- β1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node.     

Staphylococcus aureus Panton-Valentine Leukocidin Is a Very Potent Cytotoxic Factor for Human Neutrophils

The role of the pore-forming Staphylococcus aureus toxin Panton-Valentine leukocidin (PVL) in severe necrotizing diseases is debated due to conflicting data from epidemiological studies of community-associated methicillin-resistant S. aureus (CA-MRSA) infections and various murine disease-models. In this study, we used neutrophils isolated from different species to evaluate the cytotoxic effect of PVL in comparison to other staphylococcal cytolytic components. Furthermore, to study the impact of PVL we expressed it heterologously in a non-virulent staphylococcal species and examined pvl-positive and pvl-negative clinical isolates as well as the strain USA300 and its pvl-negative mutant. We demonstrate that PVL induces rapid activation and cell death in human and rabbit neutrophils, but not in murine or simian cells. By contrast, the phenol-soluble modulins (PSMs), a newly identified group of cytolytic staphylococcal components, lack species-specificity. In general, after phagocytosis of bacteria different pvl-positive and pvl-negative staphylococcal strains, expressing a variety of other virulence factors (such as surface proteins), induced cell death in neutrophils, which is most likely associated with the physiological clearing function of these cells. However, the release of PVL by staphylococcal strains caused rapid and premature cell death, which is different from the physiological (and programmed) cell death of neutrophils following phagocytosis and degradation of virulent bacteria. Taken together, our results question the value of infection-models in mice and non-human primates to elucidate the impact of PVL. Our data clearly demonstrate that PVL acts differentially on neutrophils of various species and suggests that PVL has an important cytotoxic role in human neutrophils, which has major implications for the pathogenesis of CA-MRSA infections.     

Neutrophil-derived IL-1β Is Sufficient for Abscess Formation in Immunity against Staphylococcus aureus in Mice

Neutrophil abscess formation is critical in innate immunity against many pathogens. Here, the mechanism of neutrophil abscess formation was investigated using a mouse model of Staphylococcus aureus cutaneous infection. Gene expression analysis and in vivo multispectral noninvasive imaging during the S. aureus infection revealed a strong functional and temporal association between neutrophil recruitment and IL-1β/IL-1R activation. Unexpectedly, neutrophils but not monocytes/macrophages or other MHCII-expressing antigen presenting cells were the predominant source of IL-1β at the site of infection. Furthermore, neutrophil-derived IL-1β was essential for host defense since adoptive transfer of IL-1β-expressing neutrophils was sufficient to restore the impaired neutrophil abscess formation in S. aureus-infected IL-1β-deficient mice. S. aureus-induced IL-1β production by neutrophils required TLR2, NOD2, FPR1 and the ASC/NLRP3 inflammasome in an α-toxin-dependent mechanism. Taken together, IL-1β and neutrophil abscess formation during an infection are functionally, temporally and spatially linked as a consequence of direct IL-1β production by neutrophils.     

Inactivation of Staphylococcal Phenol Soluble Modulins by Serum Lipoprotein Particles

Staphylococcus aureus virulence has been associated with the production of phenol soluble modulins (PSM). PSM are known to activate, attract and lyse neutrophils. However, the functional characterizations were generally performed in the absence of human serum. Here, we demonstrate that human serum can inhibit all the previously-described activities of PSM. We observed that serum can fully block both the cell lysis and FPR2 activation of neutrophils. We show a direct interaction between PSM and serum lipoproteins in human serum and whole blood. Subsequent analysis using purified high, low, and very low density lipoproteins (HDL, LDL, and VLDL) revealed that they indeed neutralize PSM. The lipoprotein HDL showed highest binding and antagonizing capacity for PSM. Furthermore, we show potential intracellular production of PSM by S. aureus upon phagocytosis by neutrophils, which opens a new area for exploration of the intracellular lytic capacity of PSM. Collectively, our data show that in a serum environment the function of PSM as important extracellular toxins should be reconsidered.     

Cigarette smoke-exposed neutrophils die unconventionally but are rapidly phagocytosed by macrophages

Pulmonary accumulation of neutrophils is typical for active smokers who are also predisposed to multiple inflammatory and infectious lung diseases. We show that human neutrophil exposure to cigarette smoke extract (CSE) leads to an atypical cell death sharing features of apoptosis, autophagy and necrosis. Accumulation of tar-like substances in autophagosomes is also apparent. Before detection of established cell death markers, CSE-treated neutrophils are effectively recognized and non-phlogistically phagocytosed by monocyte-derived macrophages. Blockade of LOX-1 and scavenger receptor A, but not MARCO or CD36, as well as pre-incubation with oxLDL, inhibited phagocytosis, suggesting that oxLDL-like structures are major phagocytosis signals. Specific lipid (β-carotene and quercetin), but not aqueous, antioxidants increased the pro-phagocytic effects of CSE. In contrast to non-phlogistic phagocytosis, degranulation of secondary granules, as monitored by lactoferrin release, was apparent on CSE exposure, which is likely to promote pulmonary inflammation and tissue degradation. Furthermore, CSE-exposed neutrophils exhibited a compromised ability to ingest the respiratory pathogen, Staphylococcus aureus, which likely contributes to bacterial persistence in the lungs of smokers and is likely to promote further pulmonary recruitment of neutrophils. These data provide mechanistic insight into the lack of accumulation of apoptotic neutrophil populations in the lungs of smokers and their increased susceptibility to degradative pulmonary diseases and bacterial infections.     

Calprotectin Increases the Activity of the SaeRS Two Component System and Murine Mortality during Staphylococcus aureus Infections

Calprotectin, the most abundant cytoplasmic protein in neutrophils, suppresses the growth of Staphylococcus aureus by sequestering the nutrient metal ions Zn and Mn. Here we show that calprotectin can also enhance the activity of the SaeRS two component system (TCS), a signaling system essential for production of over 20 virulence factors in S. aureus. The activity of the SaeRS TCS is repressed by certain divalent ions found in blood or neutrophil granules; however, the Zn bound-form of calprotectin relieves this repression. During staphylococcal encounter with murine neutrophils or staphylococcal infection of the murine peritoneal cavity, calprotectin increases the activity of the SaeRS TCS as well as the production of proinflammatory cytokines such as IL-1β and TNF-α, resulting in higher murine mortality. These results suggest that, under certain conditions, calprotectin can be exploited by S. aureus to increase bacterial virulence and host mortality.     

Effects of gentamycin on interactions of neutrophils with Staphylococcus

The authors studied the effect of gentamicin on abilities of neutrophils to uptake and killing of extracellular Staphylococcus. Gentamicin had not changed naturally the ability of healthy men and burn patients neutrophils to uptake Staphylococcus, but intensified the ability of neutrophils to killing of these bacteria.     

Mycobacterium abscessus Induces a Limited Pattern of Neutrophil Activation That Promotes Pathogen Survival

Mycobacterium abscessus is a rapidly growing mycobacterium increasingly detected in the neutrophil-rich environment of inflamed tissues, including the cystic fibrosis airway. Studies of the immune reaction to M. abscessus have focused primarily on macrophages and epithelial cells, but little is known regarding the neutrophil response despite the predominantly neutrophillic inflammation typical of these infections. In the current study, human neutrophils released less superoxide anion in response to M. abscessus than to Staphylococcus aureus, a pathogen that shares common sites of infection. Exposure to M. abscessus induced neutrophil-specific chemokine and proinflammatory cytokine genes. Although secretion of these protein products was confirmed, the quantity of cytokines released, and both the number and level of gene induction, was reduced compared to S. aureus. Neutrophils mediated killing of M. abscessus, but phagocytosis was reduced when compared to S. aureus, and extracellular DNA was detected in response to both bacteria, consistent with extracellular trap formation. In addition, M. abscessus did not alter cell death compared to unstimulated cells, while S. aureus enhanced necrosis and inhibited apoptosis. However, neutrophils augment M. abscessus biofilm formation. The response of neutrophils to M. abscessus suggests that the mycobacterium exploits neutrophil-rich settings to promote its survival and that the overall neutrophil response was reduced compared to S. aureus. These studies add to our understanding of M. abscessus virulence and suggest potential targets of therapy.     

The mechanism underlying the appearance of late apoptotic neutrophils and subsequent TNF-α production at a late stage during Staphylococcus aureus bioparticle-induced peritoneal inflammation in inducible NO synthase-deficient mice

During inflammation, neutrophils infiltrate into the involved site and undergo apoptosis. Early apoptotic neutrophils are then cleared by phagocytes, leading to resolution of the inflammation, whereas if late apoptotic neutrophils are accumulated for some reason, they provoke proinflammatory responses such as TNF-α production. To determine how endogenously produced nitric oxide (NO) regulates neutrophil apoptosis and the resolution of inflammation, we compared peritoneal inflammation induced by Staphylococcus aureus bioparticles in wild type mice with that in inducible NO synthase (iNOS)-deficient ones. In this model, NO production was largely dependent on iNOS, the NO level peaking at 24 h. There were increases in the numbers of neutrophils and late apoptotic ones at 24 h in iNOS-deficient mice as compared with in wild type ones, and consequently TNF-α production at 36 h in iNOS-deficient mice. On the other hand, the administration of a NO donor to iNOS-deficient mice at 12 h decreased the numbers of neutrophils and late apoptotic ones at 24 h, and thereafter TNF-α production at 36 h. In addition, coculturing of macrophages with late apoptotic neutrophils caused TNF-α production and a NO donor inhibited the transmigration of neutrophils in a dose-dependent manner. Collectively, these results suggest a novel mechanism that endogenously produced NO suppresses neutrophil accumulation at a late stage of inflammation, thereby preventing the appearance of late apoptotic neutrophils and subsequent proinflammatory responses.     

2-O-Sulfated Domains in Syndecan-1 Heparan Sulfate Inhibit Neutrophil Cathelicidin and Promote Staphylococcus aureus Corneal Infection

Ablation of syndecan-1 in mice is a gain of function mutation that enables mice to significantly resist infection by several bacterial pathogens. Syndecan-1 shedding is induced by bacterial virulence factors, and inhibition of shedding attenuates bacterial virulence, whereas administration of purified syndecan-1 ectodomain enhances virulence, suggesting that bacteria subvert syndecan-1 ectodomains released by shedding for their pathogenesis. However, the pro-pathogenic functions of syndecan-1 ectodomain have yet to be clearly defined. Here, we examined how syndecan-1 ectodomain enhances Staphylococcus aureus virulence in injured mouse corneas. We found that syndecan-1 ectodomain promotes S. aureus corneal infection in an HS-dependent manner. Surprisingly, we found that this pro-pathogenic activity is dependent on 2-O-sulfated domains in HS, indicating that the effects of syndecan-1 ectodomain are structure-based. Our results also showed that purified syndecan-1 ectodomain and heparan compounds containing 2-O-sulfate motifs inhibit S. aureus killing by antimicrobial factors secreted by degranulated neutrophils, but does not affect intracellular phagocytic killing by neutrophils. Immunodepletion of antimicrobial factors with staphylocidal activities demonstrated that CRAMP, a cationic antimicrobial peptide, is primarily responsible for S. aureus killing among other factors secreted by degranulated neutrophils. Furthermore, we found that purified syndecan-1 ectodomain and heparan compounds containing 2-O-sulfate units potently and specifically inhibit S. aureus killing by synthetic CRAMP. These results provide compelling evidence that a specific subclass of sulfate groups, and not the overall charge of HS, permits syndecan-1 ectodomains to promote S. aureus corneal infection by inhibiting a key arm of neutrophil host defense.     

Neutrophil Crawling in Capillaries; A Novel Immune Response to Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA), particularly the USA300 strain, is a highly virulent pathogen responsible for an increasing number of skin and soft tissue infections globally. Furthermore, MRSA-induced soft tissue infections can rapidly progress into life-threatening conditions, such as sepsis and necrotizing fasciitis. The importance of neutrophils in these devastating soft tissue infections remains ambiguous, partly because of our incomplete understanding of their behaviour. Spinning disk confocal microscopy was used to visualize the behaviour of GR1-labelled neutrophils in subcutaneous tissue in response to GFP-expressing MRSA attached to a foreign particle (agarose bead). We observed significant directional neutrophil recruitment towards the S. aureus agarose bead but not a control agarose bead. A significant increase in neutrophil crawling within the capillaries surrounding the infectious nidus was noted, with impaired capillary perfusion in these vessels and increased parenchymal cell death. No neutrophils were able to emigrate from capillaries. The crawling within these capillaries was mediated by the β₂ and α₄ integrins and blocking these integrins 2 hours post infection eliminated neutrophil crawling, improved capillary perfusion, reduced cell death and reduced lesion size. Blocking prior to infection increased pathology. Neutrophil crawling within capillaries during MRSA soft tissue infections, while potentially contributing to walling off or preventing early dissemination of the pathogen, resulted in impaired perfusion and increased tissue injury with time.     

Production of an Attenuated Phenol-Soluble Modulin Variant Unique to the MRSA Clonal Complex 30 Increases Severity of Bloodstream Infection

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of morbidity and death. Phenol-soluble modulins (PSMs) are recently-discovered toxins with a key impact on the development of Staphylococcus aureus infections. Allelic variants of PSMs and their potential impact on pathogen success during infection have not yet been described. Here we show that the clonal complex (CC) 30 lineage, a major cause of hospital-associated sepsis and hematogenous complications, expresses an allelic variant of the PSMα3 peptide. We found that this variant, PSMα3N22Y, is characteristic of CC30 strains and has significantly reduced cytolytic and pro-inflammatory potential. Notably, CC30 strains showed reduced cytolytic and chemotactic potential toward human neutrophils, and increased hematogenous seeding in a bacteremia model, compared to strains in which the genome was altered to express non-CC30 PSMα3. Our findings describe a molecular mechanism contributing to attenuated pro-inflammatory potential in a main MRSA lineage. They suggest that reduced pathogen recognition via PSMs allows the bacteria to evade elimination by innate host defenses during bloodstream infections. Furthermore, they underscore the role of point mutations in key S. aureus toxin genes in that adaptation and the pivotal importance PSMs have in defining key S. aureus immune evasion and virulence mechanisms.     

Contribution of NOD2 to lung inflammation during Staphylococcus aureus-induced pneumonia

Staphylococcus aureus is the most commonly found Gram-positive bacterium in patients admitted in intensive-care units, causing septicaemia or pneumonia. In this work, we investigated the role of NOD2 in S. aureus-induced pneumonia. We found that the absence of NOD2 affected weight loss and recovery speed. Nod2?/? mice showed a reduced lung inflammation in comparison to wild-type animals, with lower presence of cytokines in broncho-alveolar lavage fluids and reduced recruitment of neutrophils. Furthermore, histological analysis of the lungs revealed less severe lesions in Nod2?/? mice at day 2 and day 7 post-infection. In conclusion, we demonstrated that NOD2 is not a crucial receptor to fight S. aureus-induced pneumonia, but that it contributes to the inflammatory response in the lungs. Interestingly, the absence of NOD2 led to a lesser inflammation and was finally beneficial for the animal recovery.     

The Loss of RGS Protein-Gαi2 Interactions Results in Markedly Impaired Mouse Neutrophil Trafficking to Inflammatory Sites

Neutrophils are first responders rapidly mobilized to inflammatory sites by a tightly regulated, nonredundant hierarchy of chemoattractants. These chemoattractants engage neutrophil cell surface receptors triggering heterotrimeric G-protein Gα_i subunits to exchange GDP for GTP. By limiting the duration that Gα_i subunits remain GTP bound, RGS proteins modulate chemoattractant receptor signaling. Here, we show that neutrophils with a genomic knock in of a mutation that disables regulator of G-protein signaling (RGS)-Gα_i2 interactions accumulate in the bone marrow and mobilize poorly to inflammatory sites. These defects are attributable to enhanced sensitivity to background signals, prolonged chemoattractant receptor signaling, and inappropriate CXCR2 downregulation. Intravital imaging revealed a failure of the mutant neutrophils to accumulate at and stabilize sites of sterile inflammation. Furthermore, these mice could not control a nonlethal Staphylococcus aureus infection. Neutrophil RGS proteins establish a threshold for Gα_i activation, helping to coordinate desensitization mechanisms. Their loss renders neutrophils functionally incompetent.     

Oxidation of calprotectin by hypochlorous acid prevents chelation of essential metal ions and allows bacterial growth: Relevance to infections in cystic fibrosis

Calprotectin provides nutritional immunity by sequestering manganese and zinc ions. It is abundant in the lungs of patients with cystic fibrosis but fails to prevent their recurrent infections. Calprotectin is a major protein of neutrophils and composed of two monomers, S100A8 and S100A9. We show that the ability of calprotectin to limit growth of Staphylococcus aureus and Pseudomonas aeruginosa is exquisitely sensitive to oxidation by hypochlorous acid. The N-terminal cysteine residue on S100A9 was highly susceptible to oxidation which resulted in cross-linking of the protein monomers. The N-terminal methionine of S100A8 was also readily oxidized by hypochlorous acid, forming both the methionine sulfoxide and the unique product dehydromethionine. Isolated human neutrophils formed these modifications on calprotectin when their myeloperoxidase generated hypochlorous acid. Up to 90% of the N-terminal amine on S100A8 in bronchoalveolar lavage fluid from young children with cystic fibrosis was oxidized. Oxidized calprotectin was higher in children with cystic fibrosis compared to disease controls, and further elevated in those patients with infections. Our data suggest that oxidative stress associated with inflammation in cystic fibrosis will stop metal sequestration by calprotectin. Consequently, strategies aimed at blocking extracellular myeloperoxidase activity should enable calprotectin to provide nutritional immunity within the airways.     

Oxygen-dependent killing of Staphylococcus aureus by human neutrophils

Luminol-dependent chemiluminescence was used as a monitor of reactive oxidant generation during phagocytosis of Staphylococcus aureus by human neutrophils. Reactive oxidants play a crucial role in the killing of this organism because: (a) S. aureus was killed most rapidly when the rate of increase of chemiluminescence was greatest; (b) neutrophils which had been activated to generate reactive oxidants by re-aeration of anaerobic suspensions killed this bacterium more efficiently than control suspensions; and (c) neutrophils from a patient with chronic granulomatous disease could neither generate reactive oxidants nor kill S. aureus.     

Intravenously administered cloxacillin-induced neutropenia with eosinophilia in a patient with infective endocarditis: a case report

Background

Bacteremia following Staphylococcus aureus is a serious clinical condition which is often associated with distant metastatic infections. One of the most dreaded complications of Staphylococcus aureus bacteremia is infective endocarditis. Cloxacillin is a common antibiotic prescribed for suspected staphylococcal infections and confirmed methicillin-sensitive Staphylococcus aureus infections. Prolonged use of cloxacillin may lead to neutropenia.

Case presentation

A 38-year-old Sinhalese man presented to Teaching Hospital Kurunegala, Sri Lanka, complaining of a 3-week history of fever; he was found to have a pansystolic murmur over the apex of his heart. He had leukocytosis with predominant neutrocytosis. His C-reactive protein was 231?mg/l and erythrocyte sedimentation rate was 100?mm/first hour. Transthoracic two-dimensional echocardiography revealed prolapsed mitral valve with 7?×?13?mm vegetation over the posterior mitral valve. On the following day, three blood cultures became positive and were subsequently identified as Staphylococcus aureus. Intravenously administered cloxacillin 3?g 6 hourly was started. Following day 24 of intravenously administered cloxacillin, our patient developed high spike fever. His total white blood cells were: 990/mm³ with 34% neutrophils and 22% eosinophils. His hemoglobin concentration was 9.5?g/dL and platelet count remained normal (202?×?10⁶/mm³). His C-reactive protein was 78?mg/l, erythrocyte sedimentation rate was 95?mm/first hour, and he was otherwise comfortable, showing no signs of sepsis beside the high grade fever. His serum was negative for filarial and Toxoplasma antibodies while stool was negative for oocytes and amoebic cysts. Further, his serum was negative for dengue virus, Epstein–Barr virus, cytomegalovirus, and hepatitis B antibodies. He was clinically well on day 6 after stopping cloxacillin with 44% neutrophils and 18% eosinophils. His C-reactive protein and erythrocyte sedimentation rate became normal, and there was no further plan for cardiothoracic intervention or administration of antimicrobials. He was discharged from hospital and remained well 6?months later.

Conclusion

This case report signifies the potential fatal adverse effect of cloxacillin in methicillin-sensitive Staphylococcus aureus infections. Leukopenia is associated with prolonged use of high doses of cloxacillin. In addition to transthoracic two-dimensional echocardiography and inflammatory markers, sequential white blood cells and differential counts would help clinicians to assess the prognosis of patients with infective endocarditis.