Major Gram-positive Pathogens Research Articles

Novel motif associated with carbon catabolite repression in two major Gram-positive pathogen virulence regulatory proteins.

Carbon catabolite repression (CCR) is a widely conserved regulatory process that ensures enzymes and transporters of less-preferred carbohydrates are transcriptionally repressed in the presence of a preferred carbohydrate. This phenomenon can be regulated via a CcpA-dependent or CcpA-independent mechanism. The CcpA-independent mechanism typically requires a transcriptional regulator harboring a phosphotransferase regulatory domain (PRD) that interacts with phosphotransferase system (PTS) components. PRDs contain a conserved histidine residue that is phosphorylated by the PTS-associated HPr-His15~P protein. PRD-containing regulators often harbor additional domains that resemble PTS-associated EIIB protein domains with a conserved cysteine residue that can be phosphorylated by cognate PTS components. We noted that Mga, the PRD-containing central virulence regulator of Streptococcus pyogenes, has an EIIBGat domain containing a cysteine that, based on the presence of a similar motif in glycerol kinase, could be a target for phosphorylation. Using site-directed mutagenesis, we constructed phospho-ablative and phospho-mimetic substitutions of this cysteine and found that these substitutions modify the CCR of the Rgg2/3 quorum-sensing system. Moreover, we provide genetic evidence that the phospho-donor of this cysteine residue is likely to be ManL, the EIIA/B subunit of the mannose PTS system. Interestingly, a structurally distinct virulence gene regulator, PrfA of Listeria monocytogenes, harbors a similar cysteine-containing motif, and phospho-ablative and phospho-mimetic substitutions of the cysteine-altered CCR of PrfA-dependent virulence gene expression. Collectively, our data suggest that phosphorylation of a cysteine within the shared novel motif in Mga and PrfA may be a heretofore missing link between cellular metabolism and virulence.IMPORTANCEIn this study, we identified a novel cysteine-containing motif within the amino acid sequence of two structurally distinct transcriptional regulators of virulence in two Gram-positive pathogens that appears to link carbon metabolism with virulence gene expression. The results also highlight the potential post-translational modification of cysteine in bacterial species, a rare and understudied modification.

Emergent emm4 group A Streptococcus evidences a survival strategy during interaction with immune effector cells.

The major gram-positive pathogen group A Streptococcus (GAS) is a model organism for studying microbial epidemics as it causes waves of infections. Since 1980, several GAS epidemics have been ascribed to the emergence of clones producing increased amounts of key virulence factors such as streptolysin O (SLO). Herein, we sought to identify mechanisms underlying our recently identified temporal clonal emergence among emm4 GAS, given that emergent strains did not produce augmented levels of virulence factors relative to historic isolates. By creating and analyzing isoallelic strains, we determined that a conserved mutation in a previously undescribed gene encoding a putative carbonic anhydrase was responsible for the defective in vitro growth observed in the emergent strains. We also identified that the emergent strains survived better inside macrophages and killed macrophages at lower rates than the historic strains. Via the creation of isogenic mutant strains, we linked the emergent strain "survival" phenotype to the downregulation of the SLO encoding gene and upregulation of the msrAB operon which encodes proteins involved in defense against extracellular oxidative stress. Our findings are in accord with recent surveillance studies which found a high ratio of mucosal (i.e., pharyngeal) relative to invasive infections among emm4 GAS. Since ever-increasing virulence is unlikely to be evolutionarily advantageous for a microbial pathogen, our data further understanding of the well-described oscillating patterns of virulent GAS infections by demonstrating mechanisms by which emergent strains adapt a "survival" strategy to outcompete previously circulating isolates.

To investigate the anti-bacterial activity of crude extracts of Mintha pipertia and Allium sativum against throat infection causing bacteria streptococci collected from various diagnostic laboratories and hospitals of Mingora swat

Streptococcus (S) pyogenes one of the major Gram-positive pathogens responsible for upper respiratory tract and nosocomial infections. This is a Gram Positive, aerobic, no motile, and non-spore-forming ubiquitous bacteria. The present study was conducted to evaluate the in vitro antibacterial activity of ethanol and aqueous extract of peppermint and garlic against 29 isolates of S. pyogenes recovered from the throat sample of patientsin Swat KPK-Pakistan. These isolates were purified to identify the target bacterial isolates by gram staining and biochemical tests. The Agar dilution and well method was applied for the antibacterial activity of garlic and peppermint extracts. All of the subject isolates were exposed to different concentrations of peppermint. MIC of aqua extract of peppermint was 250µL/mL and ethanol extract was 100µL/mL. The zone size was 29mm and 36mm respectively. So the ethanol extract of peppermint was found to be more effective as compared to the aqueous extract. On the other hand, Garlic has shown equal effectiveness of water and ethanol extraction with MIC value of all is 50µL/mL and zone of inhibition was 38mm. Ciprofloxacin antibiotic disk was used as reference standard.

Biology and Regulation of Staphylococcal Biofilm

Despite continuing progress in medical and surgical procedures, staphylococci remain the major Gram-positive bacterial pathogens that cause a wide spectrum of diseases, especially in patients requiring the utilization of indwelling catheters and prosthetic devices implanted temporarily or for prolonged periods of time. Within the genus, if Staphylococcus aureus and S. epidermidis are prevalent species responsible for infections, several coagulase-negative species which are normal components of our microflora also constitute opportunistic pathogens that are able to infect patients. In such a clinical context, staphylococci producing biofilms show an increased resistance to antimicrobials and host immune defenses. Although the biochemical composition of the biofilm matrix has been extensively studied, the regulation of biofilm formation and the factors contributing to its stability and release are currently still being discovered. This review presents and discusses the composition and some regulation elements of biofilm development and describes its clinical importance. Finally, we summarize the numerous and various recent studies that address attempts to destroy an already-formed biofilm within the clinical context as a potential therapeutic strategy to avoid the removal of infected implant material, a critical event for patient convenience and health care costs.

Handling the challenge of antimicrobial resistant superbugs in the clinical setting: nursing staff as a pivotal player

Bacterial resistance to antibiotics (antimicrobial resistance, AMR) is spreading globally among major Gram-positive and Gram-negative bacterial pathogens (including staphylococci, enterococci, Enterobacterales, Pseudomonas aeruginosa andBacterial resistance to antibiotics (antimicrobial resistance, AMR) is rapidly spreading globally among major Gram-positive and Gram-negative bacterial pathogens (including staphylococci, enterococci, Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii); this phenomenon has a remarkable impact on morbidity, mortality and healthcare-associated costs. Evolution and dissemination of AMR can be counteracted with a combined strategy based on i) antimicrobial stewardship programs aimed at a prudent and appropriate use of antibiotics to improve clinical outcomes and reduce the selective pressure for resistance; and ii) infection prevention and control (IPC) practices, to limit the spread of resistant pathogens within the healthcare settings. In this scenario, the nursing staff plays a pivotal role, since these figures are involved in the enforcement and supervision of IPC bundles (e.g., contact precautions, hand, and environmental hygiene, active surveillance, patient isolation, or cohorting), which are essential to limit the spread of resistant pathogens among different patients.

Therapeutic potential of kaempferol on Streptococcus pneumoniae infection

Co-infections with pathogens and secondary bacterial infections play significant roles during the pandemic coronavirus disease 2019 (COVID-19) pathogenetic process, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Notably, co-infections with Streptococcus pneumoniae (S. pneumoniae), as a major Gram-positive pathogen causing pneumonia or meningitis, severely threaten the diagnosis, therapy, and prognosis of COVID-19 worldwide. Accumulating evidences have emerged indicating that S. pneumoniae evolves multiple virulence factors, including pneumolysin (PLY) and sortase A (SrtA), which have been extensively explored as alternative anti-infection targets. In our study, natural flavonoid kaempferol was identified as a potential candidate drug for infection therapeutics via anti-virulence mechanisms. We found that kaempferol could interfere with the pore-forming activity of PLY by engaging with catalytic active sites and consequently inhibit PLY-mediated cytotoxicity. Additionally, exposed to kaempferol significantly reduced the SrtA peptidase activity by occupying the active sites of SrtA. Further, the biofilms formation and bacterial adhesion to the host cells could be significantly thwarted by kaempferol incubation. In vivo infection model by S. pneumoniae highlighted that kaempferol oral administration exhibited notable treatment benefits, as evidenced by decreased bacterial burden, suggesting that kaempferol has tremendous potential to attenuate S. pneumoniae pathogenicity. Scientifically, our study implies that kaempferol is a promising therapeutic option by targeting bacterial virulence factors.

Alnustone inhibits Streptococcus pneumoniae virulence by targeting pneumolysin and sortase A

Streptococcus pneumoniae (S. pneumoniae) is a major Gram-positive opportunistic pathogen that causes pneumonia, bacteremia, and other fatal infections. This bacterium is responsible for more deaths than any other single pathogen in the world. Inexplicably, these symptoms persist despite the administration of effective antibiotics. Targeting pneumolysin (PLY) and sortase A (SrtA), the major virulence factors of S. pneumoniae, this study uncovered a novel resistance mechanism to S. pneumoniae infection. Using protein phenotype assays, we determined that the small molecule inhibitor alnustone is a potent drug that inhibits both PLY and SrtA. As essential virulence factors of S. pneumoniae, PLY and SrtA play a significant role in the occurrence of infection. Furthermore, evaluation using PLY-mediated hemolysis assay demonstrated alunstone had the potential to interrupt the haemolytic activity of PLY with treatment alunstone (4 μg/ml). Co-incubation of S. pneumoniae D39 SrtA with small-molecule inhibitors decreases cell wall-bound Nan A (pneumococcal-anchored surface protein SrtA), inhibits biofilm formation, and reduces biomass significantly. The protective effect of invasive pneumococcal disease (IPD) on murine S. pneumoniae was demonstrated further. Our study proposes a comprehensive bacteriostatic mechanism for S. pneumoniae and highlights the significant translational potential of targeting both PLY and SrtA to prevent pneumococcal infections. Our findings indicate that the antibacterial strategy of directly targeting PLY and SrtA with alnustone is a promising treatment option for S. pneumoniae and that alnustone is a potent inhibitor of PLY and SrtA.

Rapid detection of major Gram-positive pathogens in ocular specimens using a novel fluorescent vancomycin-based probe

A novel probe, based on vancomycin and 4-nitrobenzoxadiazole was synthesized and characterized, and used for the rapid and specific detection of Gram positive bacteria – the major pathogens responsible for eye infections in ocular specimens.

Grad-seq identifies KhpB as a global RNA-binding protein in Clostridioides difficile that regulates toxin production.

Much of our current knowledge about cellular RNA-protein complexes in bacteria is derived from analyses in gram-negative model organisms, with the discovery of RNA-binding proteins (RBPs) generally lagging behind in Gram-positive species. Here, we have applied Grad-seq analysis of native RNA-protein complexes to a major Gram-positive human pathogen, Clostridioides difficile, whose RNA biology remains largely unexplored. Our analysis resolves in-gradient distributions for ∼88% of all annotated transcripts and ∼50% of all proteins, thereby providing a comprehensive resource for the discovery of RNA-protein and protein-protein complexes in C. difficile and related microbes. The sedimentation profiles together with pulldown approaches identify KhpB, previously identified in Streptococcus pneumoniae, as an uncharacterized, pervasive RBP in C. difficile. Global RIP-seq analysis establishes a large suite of mRNA and small RNA targets of KhpB, similar to the scope of the Hfq targetome in C. difficile. The KhpB-bound transcripts include several functionally related mRNAs encoding virulence-associated metabolic pathways and toxin A whose transcript levels are observed to be increased in a khpB deletion strain. Moreover, the production of toxin protein is also increased upon khpB deletion. In summary, this study expands our knowledge of cellular RNA protein interactions in C. difficile and supports the emerging view that KhpB homologues constitute a new class of globally acting RBPs in Gram-positive bacteria.

Streptococcus pneumoniae Type 1 Pilus - A Multifunctional Tool for Optimized Host Interaction.

Streptococcus pneumoniae represents a major Gram-positive human pathogen causing bacterial pneumonia, otitis media, meningitis, and other invasive diseases. Several pneumococcal isolates show increasing resistance rates against antibacterial agents. A variety of virulence factors promote pneumococcal pathogenicity with varying importance in different stages of host infection. Virulence related hair-like structures (“pili”) are complex, surface located protein arrays supporting proper host interaction. In the last two decades different types of pneumococcal pili have been identified: pilus-1 (P1) and pilus-2 (P2) are formed by the catalytic activity of sortases that covalently assemble secreted polypeptide pilin subunits in a defined order and finally anchor the resulting pilus in the peptidoglycan. Within the long pilus fiber the presence of intramolecular isopeptide bonds confer high stability to the sequentially arranged individual pilins. This mini review will focus on S. pneumoniae TIGR4 P1 molecular architecture, the subunits it builds and provides insights into P1 sortase-mediated assembly. The complex P1 architecture (anchor-/backbone-/tip-subunits) allows the specific interaction with various target structures facilitating different steps of colonization, invasion and spreading within the host. Optimized pilin subunit confirmation supports P1 function under physiological conditions. Finally, aspects of P1- host interplay are summarized, including recent insights into P1 mechanobiology, which have important implications for P1 mediated pathogenesis.

Antimicrobial Resistance and Infection Control for Gram-positive Bacteria

Antimicrobial resistance (AMR) is a serious problem worldwide. We searched for the AMR determinants of various bacteria isolated from clinical settings and studied their resistance mechanisms and molecular epidemiology. This review focuses on the AMR of Staphylococcus aureus, a major gram-positive pathogen, which has the ability to acquire resistance to antimicrobials. The resistance factors of S. aureus are frequently found on mobile elements, including plasmids and transposons. We determined the complete DNA sequence of the tetracycline-resistance plasmid and found that the inducible expression of tetK in S. aureus was regulated by a post-transcriptional attenuation mechanism. Furthermore, outbreaks of methicillin-resistant S. aureus (MRSA) in hospitals and communities have led us to study infection controls, including the antiseptic susceptibility evaluation and molecular epidemiology of MRSA. Various antiseptic resistance determinants, such as qacA/B and smr, were identified on plasmids and characterized. We demonstrated that the plasmid-mediated efflux pump QacB variant QacIII confers fluoroquinolone efflux ability to S. aureus. Studies on MRSA epidemiology had shown that community-acquired MRSA (CA-MRSA) was disseminated into hospitals and that an increased use of alcohol-based rubs could reduce the incidence of MRSA infections in such institutions. Additionally, the study of CA-MRSA collected from communities and hospitals showed an increase in Panton-Valentine leucocidin (PVL)-positive CA-MRSA, causing severe skin and soft tissue infections. Moreover, various PVL-positive CA-MRSA clones have disseminated in Japan, whereas the USA300 LV/J clone evolved in that country. Our study provides important information regarding MRSA infection control.

1595. Ceftobiprole Activity against Gram-Positive Pathogens Causing Bone and Joint Infections in the United States from 2016 through 2019

BackgroundBone and joint infections (BJIs) cause serious morbidity and mortality and present significant treatment challenges. Ceftobiprole medocaril, the prodrug of ceftobiprole, is an advanced cephalosporin approved in many European and non-European countries for the treatment of adults with community- and hospital-acquired pneumonia, excluding ventilator-associated pneumonia. Ceftobiprole medocaril is not approved in the United States (USA) but has qualified infectious disease product (QIDP) status, and is being evaluated in two phase 3 clinical trials in patients with acute bacterial skin and skin structure infections (completed) or Staphylococcus aureus bacteremia (ongoing). In this study, the activity of ceftobiprole and comparators was evaluated against recent Gram-positive clinical isolates collected from BJIs in the USA.Methods306 Gram-positive pathogens were collected from patients with BJIs at 27 US medical centers from 2016 through 2019. Susceptibility to ceftobiprole and comparator agents was tested using current CLSI methods. CLSI and EUCAST interpretive criteria were applied according to current guidelines.ResultsThe major Gram-positive species and pathogen groups included S. aureus (67.0%; methicillin-resistant S. aureus [MRSA], 35.1%), β-hemolytic streptococci (BHS; 13.7%), coagulase-negative staphylococci (CoNS; 9.5%), and Enterococcus faecalis (6.9%). Ceftobiprole was highly active against S. aureus (MIC50/90 values, 0.5/1 mg/L; 100.0% susceptible by EUCAST criteria), including MRSA (MIC50/90 values, 1/2 mg/L). Ceftobiprole also exhibited potent activity against other Gram-positive cocci, including BHS (MIC50/90 values, 0.015/0.03 mg/L; 100% inhibited at ≤ 4 mg/L, which is the EUCAST PK-PD non-species-related breakpoint), CoNS (MIC50/90 values, 1/4 mg/L; 100% inhibited at ≤4 mg/L), and E. faecalis (MIC50/90 values, 0.5/2 mg/L; 100.0% inhibited at ≤ 4 mg/L).ConclusionCeftobiprole was highly active against clinical BJI isolates from the major Gram-positive pathogen groups collected at US medical centers during 2016–2019. The broad-spectrum activity of ceftobiprole, including potent activity against MRSA, supports its further evaluation for this potential indication.DisclosuresLeonard R. Duncan, PhD, A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Basilea Pharmaceutica International, Ltd. (Research Grant or Support)Dept of Health and Human Services (Research Grant or Support) Kamal Hamed, n/a, Basilea Pharmaceutica International Ltd. (Employee) Jennifer Smart, PhD, Basilea Pharmaceutica International, Ltd (Employee)Department of Health and Human Services (Research Grant or Support) Michael A. Pfaller, MD, Amplyx Pharmaceuticals (Research Grant or Support)Basilea Pharmaceutica International, Ltd (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Cidara Therapeutics (Research Grant or Support)Department of Health and Human Services (Research Grant or Support)Fox Chase Chemical Diversity Center (Research Grant or Support)Paratek Pharma, LLC (Research Grant or Support) Robert K. Flamm, PhD, A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Amplyx Pharmaceuticals (Research Grant or Support)Basilea Pharmaceutica International, Ltd (Research Grant or Support)Department of Health and Human Services (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support) Rodrigo E. Mendes, PhD, A. Menarini Industrie Farmaceutiche Riunite S.R.L. (Research Grant or Support)Allergan (Research Grant or Support)Allergan (Research Grant or Support)Basilea Pharmaceutica International, Ltd (Research Grant or Support)Cipla Ltd. (Research Grant or Support)Department of Health and Human Services (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Merck (Research Grant or Support)Merck (Research Grant or Support)Pfizer (Research Grant or Support)

Biofilm Formation on Breast Implant Surfaces by Major Gram-Positive Bacterial Pathogens.

Bacterial biofilm on surfaces of mammary implants is a predisposing factor for several outcomes. Because Gram-positive bacteria are potential agents of biomaterial-associated infections (BAIs), their abilities to form biofilm on breast implants should be elucidated. The aim of this study was to evaluate biofilm formation on different mammary prosthesis surfaces by major Gram-positive bacterial pathogens involved in BAIs. We initially evaluated biofilm formation on polystyrene plates with and without fibrinogen or collagen for 1 reference strain and 1 clinical isolate of Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes. We also tested the ability of clinical isolates to form biofilm on 4 different implant surfaces: polyurethane foam and smooth, microtextured, and standard textured silicone. Biofilm structure and cell viability were observed by scanning electron microscopy and confocal laser scanning microscopy. All strains showed strong biofilm formation on polystyrene. After fibrinogen or collagen treatment, biofilm formation varied. With fibrinogen, reference strains of S. aureus and S. pyogenes increased biofilm formation (P < 0.05). Reference strains of all species and the clinical isolate of S. pyogenes increased biofilm formation after collagen treatment (P < 0.05). In general, S. aureus showed higher capacity to produce biofilm. Scanning electron microscopy showed that biofilm attached to all surfaces tested, with the presence of extracellular polymeric substances and voids. Viable cells were more frequent for E. faecalis and S. pyogenes. All species produced biofilm on all prosthesis surfaces and under different conditions. Micrographies indicated thicker bacterial biofilm formation on microtextured and/or standard textured silicone by all species, except E. faecalis.

Protective Role of Peroxiredoxin I in Heat-Killed Staphylococcus Aureus-infected Mice.

Staphylococcus aureus (S. aureus) is a major gram-positive pathogen, which can cause toxic and immunogenic injuries both in nosocomial and community-acquired infections. Peroxiredoxin (Prx) I plays crucial roles in cellular apoptosis, proliferation, and signal transduction as well as in immunoregulation. The present study aimed to investigate whether Prx I protects mice from death caused by the heat-killed Staphylococcus aureus. In the present study, we challenged the wild-type and Prx I-deficient mice with heat-killed S. aureus (HKSA). The effects of Prx I were evaluated by a series of in vitro and in vivo experiments including western blot, Haematoxylin and Eosin staining, splenocyte analysis and cytokines analysis. Intra-peritoneal (ip) inoculation of HKSA resulted in increased mortality of Prx I-knockout (KO) mice with severe liver damage and highly populated spleens with lymphocytes. Furthermore, HKSA infections also bursted the production of both pro-inflammatory and anti-inflammatory serum cytokines in Prx I KO compared to wild-type mice. Enhanced mortality of S. aureus-infected mice with Prx I deficiency suggested that Prx I may protect against the infection-associated lethality of mice.

Identification of immunogenic proteins and evaluation of recombinant PDHA1 and GAPDH as potential vaccine candidates against Streptococcus iniae infection in flounder (Paralichthys olivaceus).

Streptococcus iniae is a major Gram-positive pathogen that causes invasive disease in fish worldwide. In this study, in order to identify immunogenic proteins for developing highly effective vaccine against S. iniae, whole-cell lysate proteins of S. iniae were analyzed by western blotting using flounder anti-S. iniae antibodies, and two positive protein bands of molecular weight 37 kDa and 40 kDa were screened, which were identified as pyruvate dehydrogenase E1 subunit alpha (PDHA1), BMP family ABC transporter substrate-binding protein (BMP) and L-lactate dehydrogenase (LDH), as well as ornithine carbamoyltransferase (OCT), lactate oxidas (LOx) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by mass spectrometry. Subsequently, the six recombinant proteins were produced and used to immunize healthy flounder, and the relative percent survival (RPS) value was 72.73%, 27.27%, 36.36%, 9.09%, 36.36% and 63.64% respectively after intraperitoneal challenge with live S. iniae, revealing that rPDHA1 and rGAPDH produced higher relative percent survival than formalin-killed S. iniae (36.36%). To further investigate the protective efficacy of rPDHA1 and rGAPDH, the proliferation of surface membrane immunoglobulin-positive (sIg+) lymphocytes in peripheral blood leucocytes, the total serum IgM, specific IgM against S. iniae and RPS were detected. The results showed that rPDHA1, rGAPDH and formalin-killed S. iniae significantly induced the proliferation of sIg+ lymphocytes, the production of total serum IgM and specific IgM as compared with the control group, and rGAPDH and rPDHA1 provide higher RPS (62.5% and 75%, respectively) again. These results demonstrated that rPDHA1 and rGAPDH are promising vaccine candidates against S. iniae infection in flounder.

Prevalence of gyrA and parE mutations in clinical isolates of Streptococcus pneumoniae with decreased susceptibilities to different Fluoroquinolones

Introduction: Streptococcus pneumoniae is a major Gram-positive pathogen responsible for pneumonia, bactermia, otitis media, and meningitis leading to considerable morbidity and mortality among children and elderly individuals. The primary goals of antibiotic treatment of respiratory tract infections are clinical efficacy of treatment, pathogen eradication, and prevention of resistance development. Resistance to fluoroquinolones in S. pneumoniae arises in a stepwise fashion and results from alterations in the target binding site due to the acquisition of spontaneous mutations in the quinolone resistance-determining regions (QRDRs) of the topoisomerase IV and DNA gyrase genes. Although mutations usually occur in the QRDRs of parC and gyrA, a role for mutations in the parE subunit in low-level resistance has been reported. Aim of the work: The aim of this study was to determine the prevalence of fluoroquinolone resistance Streptococcus pneumoniae (FQRSP) and to examine the genetic relatedness of pneumococcal isolates with parE and gyrA genes mutations in different specimens in Sohag University Hospital. Patients and Methods: This study was prospectively conducted over a period of 24 months between October 2015 and September 2017, at Sohag university hospital. During the study period, 78 patients hospitalized for a syndrome consistent with a diagnosis of community acquired pneumonia (CAP ) included in this study with a mean age of 34.5 years (range, 2 to 67), 60% of whom were males. A CAP syndrome was defined as a newly recognized pulmonary infiltrate together with 2 of the following findings: subjective fever or documented temperature 37.4 °C, increased cough, sputum production, or shortness of breath, pleuritic chest pain, confusion, rales, leukocytosis, (according to age) (1). Patients who had taken antibiotic treatment within 3 days prior to initial visit were excluded from this study. Results: Our study illustrate the role of mutation in the gyrA&parE genes and the effect of mutations in the both genes in fluoroquinolone resistance among S. pneumoniae isolates. Conclusion: The present study provide an opportunity to view the predominant mutations conferring reduced susceptibility to FQs in clinical pneumococcal isolates. There is a strong relationship between these mutations and decrese susceptibility to the most fameous FQs to some extent, although this varies between strains and for each drug.

Validity of somatic cell count as indicator of pathogen-specific intramammary infections.

The objective of this study was to determine whether somatic cell count (SCC) was an effective test, with a sensitivity exceeding 85%, to determine species-specific bacterial infections. In addition, the relation between the SCC and various udder pathogen groups was investigated. SCC thresholds of greater than 200 000 cells/mL were used in quarter and greater than 150 000 cells/mL in composite milk samples. A retrospective study was conducted on a data set for 89 635 quarter and 345 467 composite cow milk samples. Eleven SCC threshold values were used to evaluate the diagnostic efficacy for the following bacteria: Gram-positive major pathogens: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis; Gram-negative major pathogens: Escherichia coli, Klebsiella pneumonia and Serratia spp.; minor pathogens: coagulase-negative staphylococci, Micrococcus spp., Staphylococcus pseudintermedius, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus canis, Trueperella pyogenes and other Enterobacteriaceae. Sensitivity and specificity were calculated taking the effect of clustering into account with quarter milk samples. Most samples yielding major Gram-positive pathogens (88.9% in quarter and 79.9% in composite samples) and minor pathogens (61.4% in quarter and 51.7% in composite samples) had SCC greater than 200 000 cells/mL. Sensitivity of the SCC test to detect major pathogens at an SCC threshold of greater than 200 000 cells/mL in quarter samples and greater than 150 000 cells/mL in composite milk samples was 88.2% and 84.2%, respectively, but specificity was low (57.7% and 52.8%, respectively).

A Natural Lipotrisaccharide and Its Derivatives Selectively Lyse Streptococcus pneumoniae via Interaction with Cell Membrane.

A natural lipotrisaccharide (NP000778, 1a), a new triglycosidic tri-O-substituted glycolipid isolated from the Morinda citrifolia plant, and its chemical derivatives were identified to be active against major Gram-positive pathogens, particularly Streptococcus pneumoniae. Additional evidence indicated that 1a and its synthetic derivatives exerted their bactericidal activities against S.pneumoniae by selectively targeting the bacterial membrane, leading to the rapid lysis of the pneumococci. Efficient synthesis of 1a and its derivatives was performed using an application of the intramolecular aglycon delivery (IAD) reaction to establish its structure-activity relationships (SARs). SAR analysis indicated that trisaccharide glycolipid compounds showed good selectivity and high potency against S.pneumoniae. These compounds contain a linear chain with a chain length from C3 to C9 at the 2-position (R1) and 4'-position (R3), as well as a 2-methyl butyryl group at the 3'-position (R2), without an aza substitution in the lipid chain. This is the first lipotrisaccharide identified with potent bactericidal activity via interaction with cell membrane. The results reported herein offer a valuable guideline for the design of glycolipid derivatives that selectively target pathogenic bacteria.

Antibiofilm activity of cashew juice pulp against Staphylococcus aureus, high performance liquid chromatography/diode array detection and gas chromatography-mass spectrometry analyses, and interference on antimicrobial drugs

The epidemiology of Staphylococcus aureus infections has evolved in recent years, as this species is a major Gram-positive pathogen associated with healthcare services. The antimicrobial resistance of this species raises an urgent need for new treatment strategies. Fruits play important nutritional and economic roles in society, but their biological and pharmacological features are poorly explored when compared to nonedible parts of plants such as barks and leaves. In this study, we show that the cashew apple juice [cashew juice pulp (CJP)] extract is active against the planktonic cells of S. aureus strains, and for the first time, we show that CJP is also active against S. aureus biofilms. High performance liquid chromatography and gas chromatography-mass spectrometry analyses were conducted to prospect for polyphenols and free carbohydrates, respectively. Cashew apple juice, which is rich in nutrients, is widely consumed in Brazil; therefore, the quality attributes of CJPs were investigated. Samples were evaluated for pH, total titratable acidity, vitamin C levels, and total soluble solids. We also detected an antagonistic interference of CJP when it was combined with different antimicrobial drugs.

Multiplex identification of drug-resistant Gram-positive pathogens using stuffer-free MLPA system.

Early detection of pathogens from blood and identification of their drug resistance are essential for sepsis management. However, conventional culture-based methods require relatively longer time to identify drug-resistant pathogens, which delays therapeutic decisions. For precise multiplex detection of drug-resistant Gram-positive pathogens, we developed a method by using stuffer-free multiplex ligation-dependent probe amplification (MLPA) coupled with high-resolution CE single-strand conformation polymorphisms (CE-SSCP) system. We designed three probe sets for genes specific to Gram-positive species (Staphylococcus aureus: nuc, Enterococcus faecium: sodA, and Streptococcus pneumoniae: lytA) and two sets for genes associated with drug resistance (mecA and vanA) to discriminate major Gram-positive pathogens with the resistance. A total of 94 different strains (34 reference strains and 60 clinical isolates) were used to validate this method and strain-specific peaks were successfully observed for all the strains. To improve sensitivity of the method, a target-specific preamplification step was introduced and, consequently, the sensitivity increased from 10 pg to 100 fg. We also reduced a total assay time to 8 h by optimizing hybridization time without compromising test sensitivity. Taken together, our multiplex detection system can improve detection of drug-resistant Gram-positive pathogens from sepsis patients' blood.