Hemolysins of <i>Staphylococcus aureus</i>—An Update on Their Biology, Role in Pathogenesis and as Targets for Anti-Virulence Therapy

Staphylococcus aureus is a dangerous gram positive bacterial pathogen which, not only evades the host’s immune system but also can destroy the leucocytes especially neutrophils. It has an embodiment of virulence factors most of which are secreted. Staphylococcus aureus secretes a number of toxins which cause tissue damage and facilitate spreading and nutrients uptake. Among the toxins, hemolysins α, β, γ, δ and Panton Valentine Leukocidin (PVL) are unique that they drill pores in the membrane, leading to the efflux of vital molecules and metabolites. Hemolysins also help in the scavenging of iron, although many of them also have leucolytic properties. α-hemolysin, also known as α-toxin, is the most prominent cytotoxin which damages a wide range of host cells including epithelial cells, endothelial cells, erythrocytes, monocytes, keratinocytes and it damages cell membrane and induces apoptosis. β-Hemolysin significantly affects human immune cell function. It has Mg2+ dependent sphingomyelinase activity and degrades sphingomyelin of plasma membrane into phosphorylcholine and ceramides. The bi-component leukocidins, which include γ-hemolysin and PVL, attack human phagocytic cells and greatly contribute to immune evasion. Delta toxin is a low molecular weight exotoxin with a broad cytolytic activity. Virulence determinants, quorum sensing and biofilm synthesis provide some attractive targets for design and development of a new group of antimicrobial compounds. This review provides an update on the structure, biological functions of hemolysins and their role in quorum sensing/biofilm synthesis (if any) and as effective therapeutic targets for anti-virulence drug development. We have tried to bring together information available on various aspects of hemolysins and highlighted their distribution among all species of Staphylococcus and other bacteria. We have updated the status of development of candidate drugs targeting the hemolysins for anti-virulence therapy as it offers an additional strategy to reduce the severity of infection and which would, through quorum quenching, delay the development biofilms leading to drug resistance.

Serum Procalcitonin and Neutrophil Toxic Granules Guided Management of Post-Operative <i>K. pneumoniae</i>Septic-Shock in Laminectomy—A Case Report

Introduction: We still rely on clinical diagnosis for initiating empirical antibiotic therapy and await blood culture for confirmation of infection, species identification and drug sensitivity. Differential blood cell count (WBC and neutrophils) and recording of toxic granules in the neutrophils are established methods for indirect diagnosis of infection though they are not used in the diagnosis of sepsis per se. Serum Procalcitonin is considered as a good biomarker in the management of sepsis. Materials and Methods: Whole blood and serum were used for laboratory analysis. We have combined the detection of toxic granules in the peripheral blood smear and serum PCT levels for diagnosis and monitoring the recovery of a patient with septic shock. A 63 year old laminectomy patient was transferred 2 days after the surgery to our hospital with several co-morbidities and complications. He was in septic shock and was put on Continuous Renal Replacement Therapy, with ionotropic support and IV fluids via nasogastric feeding and administration of Activated Protein C. Blood culture and daily measurements of serum Procalcitonin, differential blood cells count, and observation of toxic granules in neutrophils were done. Results: The blood culture showed infection due to K. pneumoniae resistant to carbapenems. WBC and Neutrophil counts were quite variable and showed incoherent response to treatment. Serum PCT was 24.57 ng/mL on the next day of admission and it peaked at 30.2 ng/mL on day 3. Its level started decreasing from the 4th day. Toxic granules disappeared when serum PCT levels reached < 1 ng/mL. The patient responded well to treatment and he was discharged on the 16th day upon request. Conclusion: This case is presented as an example of managing sepsis with a combination of a conventional hematology marker and a modern biomarker. Resource poor hospitals with inadequate microbiology services, may evaluate and use these two biomarkers not only for the total management of sepsis but also to reduce the cost of critical care.

A Simple Method for Direct Detection and Discrimination of <i>A. baumannii</i>in Tracheal Aspirates without Culture Isolation

Currently, available phenotyping and commercial methods report A. baumannii only as Acinetobacter calcoaceticus-baumannii complex (ACB) and do not identify individual members of the complex. This is a single blind study aimed to evaluate certain commonly used species-specific genetic markers namely, Intergenic Transcribed Spacer region in 16S rRNA of A. baumannii (Ab-ITS) and gyrB, for identification of ACB members. These molecular targets were first validated on clinical isolates (n = 200) and subsequently on uncultured tracheal aspirates (n = 172). Among the clinical isolates, 183/200 (91.5%) were positive for Ab-ITS. The clinical isolates 17 (17/200) which are failed to amplify in Ab-ITS PCR were subsequently diagnosed by gyrB PCR as A. calcoaceticus (n = 2), A. pitti (n = 6) and A. nosocomialis (n = 9) but not A. baumannii. Among the tracheal aspirates, 62 samples were reported as sterile in Advanced Expert System of VITEK-2, among the remaining 110 samples, 68.1% (75/110) samples contained Ab-ITS target. Twenty-five of the sterile samples (25/62) were found to contain Ab-ITS target sequence. Since, our sample processing method enabled identification of all the species of ACB complex by PCR even in uncultured tracheal aspirates, adaptation of our protocol would enable same day (6 - 8 h) reporting and help the clinician make evidence based therapeutic decision quickly.

A Simple Sample Processing Protocol and Multiplex PCR for Direct Detection of MRSA from Uncultured Clinical Samples—A Pilot Study

Phenotypic tests have limited discrimatory power to identify closely related members of genus Staphylococcus and particularly for identification of S. aureus. 157 isolates of S. aureus obtained from different clinical specimens were included in our study. To present a demonstration of our method’s sensitivity and ability to correctly detect S. aureus from uncultured clinical specimen, 30 known S. aureus positive but leftover uncultured clinical specimens from clinical microbiology laboratory were processed by our protocol and analyzed. All the 30 clinical specimens were confirmed as S. aureus among which 26 specimen were identified as MRSA and the remaining 4 as MSSA. These 30 clinical specimens used in the study showed 100% correlation with coagulase test and Cefoxitin disc diffusion method. Though commercial molecular diagnostic kits are available for detecting MRSA from swabs, this is probably the first time that multiplex PCR is being demonstrated directly on a variety of uncultured clinical specimens.

Heterogeneity in <i>femA</i>in the Indian Isolates of <i>Staphylococcus aureus</i>Limits Its Usefulness as a Species Specific Marker

Increase in prevalence of MRSA worldwide and hence the need for rapid detection, have led to use of molecular methods for confirmation of the species and also MRSA. Species specific markers like fem or nuc along with the methicillin-resistance determinant, mecA, have been used by several investigators worldwide for the identification of MRSA. In the current study, we have screened 54 microbiologically confirmed (MRSA, MSSA and CoNS) isolates for the presence of mecA, 16S rRNA, femA and nuc markers. While mecAPCR and 16S rRNAPCR results were consistent with other studies, femA and nuc showed dramatic variation in detection rate (sensitivity) of S. aureus 29.6% and 53.7% respectively. Evidences are presented to demonstrate the absence of femA. Our attempt to amplify the complete femA gene using sequences flanking femA further confirmed these results and also indicated that variations exist even in the genomic sequences around femA. Our data reveals the need for exercising care while using primers designed on sequences of constitutive genes like femA and nuc for PCR based identification of S. aureus species. Though geographic variations in the genome of S. aureus have previously been reported from around the world, we present here evidence for the first time from India for absence of femA and also for probable variations in the sequences around the femA gene in clinical isolates of S. aureus.