MICy: a Novel Flow Cytometric Method for Rapid Determination of Minimal Inhibitory Concentration.

Early initiated adequate antibiotic treatment is essential in intensive care. Shortening the length of antibiotic susceptibility testing (AST) can accelerate clinical decision-making. Our objective was to develop a simple flow cytometry (FC)-based AST that produces reliable results within a few hours. We developed a FC-based AST protocol (MICy) and tested it on six different bacteria strains (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis) in Mueller-Hinton and Luria-Bertani broth. We monitored the bacterial growth by FC to define the optimal time of AST. All bacteria were tested against 12 antibiotics and the MIC values were compared to microdilution used as reference method. McNemar and Fleiss' kappa inter-observer tests were performed to analyze the bias between the two methods. Susceptibility profiles of the two methods were also compared. We found that FC is able to detect the bacterial growth after 4-h incubation. The point-by-point comparison of MICy and microdilution resulted in exact match above 87% (2642/3024) of all measurements. The MIC values obtained by MICy and microdilution agreed over 80% (173/216) within ±1 dilution range that gives a substantial inter-observer agreement with weighted Fleiss' kappa. By using the EUCAST clinical breakpoints, we defined susceptibility profiles of MICy that were identical to microdilution in more than 92% (197/213) of the decisions. MICy resulted 8.7% major and 3.2% very major discrepancies. MICy is a new, simple FC-based AST method that produces susceptibility profile with low failure rate a workday earlier than the microdilution method. IMPORTANCE MICy is a new, simple and rapid flow cytometry based antibiotic susceptibility testing (AST) method that produces susceptibility profile a workday earlier than the microdilution method or other classical phenotypic AST methods. Shortening the length of AST can accelerate clinical decision-making as targeted antibiotic treatment improves clinical outcomes and reduces mortality, duration of artificial ventilation, and length of stay in intensive care unit. It can also reduce nursing time and costs and the spreading of antibiotic resistance. In this study, we present the workflow and methodology of MICy and compare the results produced by MICy to microdilution step by step.

AR-12 Has a Bactericidal Activity and a Synergistic Effect with Gentamicin against Group A Streptococcus.

Streptococcus pyogenes (group A Streptococcus (GAS) is an important human pathogen that can cause severe invasive infection, such as necrotizing fasciitis and streptococcal toxic shock syndrome. The mortality rate of streptococcal toxic shock syndrome ranges from 20% to 50% in spite of antibiotics administration. AR-12, a pyrazole derivative, has been reported to inhibit the infection of viruses, intracellular bacteria, and fungi. In this report, we evaluated the bactericidal activities and mechanisms of AR-12 on GAS infection. Our in vitro results showed that AR-12 dose-dependently reduced the GAS growth, and 2.5 µg/mL of AR-12 significantly killed GAS within 2 h. AR-12 caused a remarkable reduction in nucleic acid and protein content of GAS. The expression of heat shock protein DnaK and streptococcal exotoxins was also inhibited by AR-12. Surveys of the GAS architecture by scanning electron microscopy revealed that AR-12-treated GAS displayed incomplete septa and micro-spherical structures protruding out of cell walls. Moreover, the combination of AR-12 and gentamicin had a synergistic antibacterial activity against GAS replication for both in vitro and in vivo infection. Taken together, these novel findings obtained in this study may provide a new therapeutic strategy for invasive GAS infection.

Antibacterial Activity of Thymus vulgaris L. Essential Oil Vapours and Their GC/MS Analysis Using Solid-Phase Microextraction and Syringe Headspace Sampling Techniques.

While the inhalation of Thymus vulgaris L. essential oil (EO) is commonly approved for the treatment of mild respiratory infections, there is still a lack of data regarding the antimicrobial activity and chemical composition of its vapours. The antibacterial activity of the three T. vulgaris EOs against respiratory pathogens, including Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes, was assessed in both liquid and vapour phases using the broth microdilution volatilisation (BMV) method. With the aim of optimising a protocol for the characterisation of EO vapours, their chemical profiles were determined using two headspace sampling techniques coupled with GC/MS: solid-phase microextraction (HS-SPME) and syringe headspace sampling technique (HS-GTS). All EO sample vapours exhibited antibacterial activity with minimum inhibitory concentrations (MIC) ranging from 512 to 1024 µg/mL. According to the sampling technique used, results showed a different distribution of volatile compounds. Notably, thymol was found in lower amounts in the headspace-peak percentage areas below 5.27% (HS-SPME) and 0.60% (HS-GTS)-than in EOs (max. 48.65%), suggesting that its antimicrobial effect is higher in vapour. Furthermore, both headspace sampling techniques were proved to be complementary for the analysis of EO vapours, whereas HS-SPME yielded more accurate qualitative results and HS-GTS proved a better technique for quantitative analysis.

The effects of the dietary compound L-sulforaphane against respiratory pathogens.

L-sulforaphane (LSF) is an isothiocyanate derived from cruciferous vegetables that has long been known for its anticarcinogenic, antioxidant and anti-inflammatory effects. LSF also possesses antimicrobial properties, although the evidence for this is limited. Respiratory pathogens, such as Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus pyogenes and respiratory syncytial virus (RSV), are leading global causes of illness and death among children aged under five years, particularly in resource-poor countries where access to vaccines are limited or, in the case of S. pyogenes and RSV, vaccines have not been licensed for use in humans. Therefore, alternative strategies to prevent and/or treat these common infectious diseases are urgently needed. This study was conducted to investigate the antimicrobial effects of LSF against common respiratory pathogens, S. pneumoniae (serotypes 1 and 6B), H. influenzae type B (HiB), non-typeable H. influenzae (NTHi), S. pyogenes and RSV in relevant human cell-based models. LSF significantly inhibited the growth of H. influenzae, but not S. pneumoniae or S. pyogenes. LSF did not improve opsonophagocytic capacity or killing by human phagocytic cell lines (HL-60s and THP-1 macrophages) for S. pneumoniae yet showed some improved killing for H. influenzae species in THP-1 macrophages. However, LSF significantly reduced RSV infection in human lung epithelial cells, associated with increased expression of cyclin D1 (CCND1) gene as well as the antioxidant genes, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HMOX-1). Overall, LSF represents an exciting avenue for further antimicrobial research, particularly as a novel therapy against H. influenzae species and RSV.

Antibacterial, antifungal and enzyme inhibitory effects of selected plants from Turkey.

In this study, antibacterial, antifungal, antihyaluronidase, anticollagenase and antielastase activity of Hypericum bithynicum, Malva neglecta, Morus alba, Rubus discolor, Sambucus ebulus and Smilax excelsa were investigated. Methanol extracts of M. neglecta and R. discolor and all extracts of H. bithynicum were more active against Staphylococcus epidermidis. Similarly, water extracts of M. alba and S. ebulus were more active against Streptococcus pneumonia. Additionally, S. ebulus and S. excelsa had prominent antifungal activity on Candida albicans. Besides, methanol extract of M. neglecta and n-hexane extract of H. bithynicum were determined to have significant antihyaluronidase activity. Only R. discolor showed significant antielastase effect.

Hybrid donor-acceptor polymer nanoparticles and combination antibiotic for mitigation of pathogenic bacteria and biofilms.

Biofilms pose a significant clinical problem in skin and soft tissue infections. Their resistance to antibiotics has spurred investigations into alternative treatments, such as nanoparticle-mediated photothermal ablation. Non-toxic Hybrid Donor- Acceptor (DA) Polymer nanoParticles (H-DAPPs) were developed for fluorescence imaging (using poly(3-hexylthiophene-2,5 diyl) (P3HT)) and rapid, near-infrared photothermal ablation (NIR- PTA) (using poly[4,4-bis(2-ethylhexyl)-cyclopenta[2,1-b;3,4-b']dithiophene-2,6-diyl-alt-2,1,3-benzoselenadiazole-4,7-diyl] (PCPDTBSe)). H-DAPPs were evaluated alone, and in combination with antibiotics, against planktonic S. aureus and S. pyogenes, and S. aureus biofilms. H-DAPPs NIR-PTA (15-700 µg/ mL) can generate rapid temperature changes of 27.6-73.1 °C, which can eradicate planktonic bacterial populations and reduce biofilm bacterial viability by more than 4- log (> 99.99%) with exposure to 60 s of 800 nm light. Reductions were confirmed via confocal analysis, which suggested that H-DAPPs PTA caused bacterial inactivation within the biofilms, but did not significantly reduce biofilm polysaccharides. SEM imaging revealed structural changes in biofilms after H-DAPPs PTA. S. aureus biofilms challenged with 100 µg/mL of H-DAPPs (H-DAPPs-100) to induce an average temperature of 55.1 °C, and the minimum biofilm eradication concentration (MBEC) of clindamycin, resulted in up to ~3- log decrease in bacterial viability compared to untreated biofilms and those administered H-DAPPs-100 PTA only, and up to ~2- log compared to biofilms administered only clindamycin. This study demonstrates that polymer nanoparticle PTA can mitigate biofilm infection and may improve antimicrobial efficacy.

Bacterial resistance to CRISPR-Cas antimicrobials.

In the age of antibiotic resistance and precise microbiome engineering, CRISPR-Cas antimicrobials promise to have a substantial impact on the way we treat diseases in the future. However, the efficacy of these antimicrobials and their mechanisms of resistance remain to be elucidated. We systematically investigated how a target E. coli strain can escape killing by episomally-encoded CRISPR-Cas9 antimicrobials. Using Cas9 from Streptococcus pyogenes (SpCas9) we studied the killing efficiency and resistance mutation rate towards CRISPR-Cas9 antimicrobials and elucidated the underlying genetic alterations. We find that killing efficiency is not correlated with the number of cutting sites or the type of target. While the number of targets did not significantly affect efficiency of killing, it did reduce the emergence of chromosomal mutations conferring resistance. The most frequent target of resistance mutations was the plasmid-encoded SpCas9 that was inactivated by bacterial genome rearrangements involving translocation of mobile genetic elements such as insertion elements. This resistance mechanism can be overcome by re-introduction of an intact copy of SpCas9. The work presented here provides a guide to design strategies that reduce resistance and improve the activity of CRISPR-Cas antimicrobials.

Effect of erythritol and xylitol on Streptococcus pyogenes causing peritonsillar abscesses.

Polyols are effective against caries-causing streptococci but the effect on oropharynx-derived pyogenic streptococci is not well characterised. We aimed to study the effect of erythritol (ERY) and xylitol (XYL) against Streptococcus pyogenes isolated from peritonsillar abscesses (PTA). We used 31 clinical isolates and 5 throat culture collection strains. Inhibition of bacterial growth by polyols at 2.5%, 5% and 10% concentrations was studied and the results were scored. Amylase levels in PTA pus were compared to polyol effectivity scores (PES). Growth curves of four S. pyogenes isolates were analysed. Our study showed that XYL was more effective than ERY inhibiting 71-97% and 48-84% of isolates, respectively, depending of concentrations. 48% of clinical and all throat strains were inhibited by polyols in all concentrations (PES 3). PES was negative or zero in 26% of the isolates in the presence of ERY and in 19% of XYL. ERY enhanced the growth of S. pyogenes isolated from pus with high amylase levels. Polyols in all concentrations inhibited the growth in exponential phase. In conclusion, ERY and XYL are potent growth inhibitors of S. pyogenes isolated from PTA. Therefore, ERY and XYL may have potential in preventing PTA in the patients with frequent tonsillitis episodes.

New Broth Macrodilution Volatilization Method for Antibacterial Susceptibility Testing of Volatile Agents and Evaluation of Their Toxicity Using Modified MTT Assay In Vitro.

In this study, a new broth macrodilution volatilization method for the simple and rapid determination of the antibacterial effect of volatile agents simultaneously in the liquid and vapor phase was designed with the aim to assess their therapeutic potential for the development of new inhalation preparations. The antibacterial activity of plant volatiles (ß-thujaplicin, thymohydroquinone, thymoquinone) was evaluated against bacteria associated with respiratory infections (Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes) and their cytotoxicity was determined using a modified thiazolyl blue tetrazolium bromide assay against normal lung fibroblasts. Thymohydroquinone and thymoquinone possessed the highest antibacterial activity against H. influenzae, with minimum inhibitory concentrations of 4 and 8 µg/mL in the liquid and vapor phases, respectively. Although all compounds exhibited cytotoxic effects on lung cells, therapeutic indices (TIs) suggested their potential use in the treatment of respiratory infections, which was especially evident for thymohydroquinone (TI > 34.13). The results demonstrate the applicability of the broth macrodilution volatilization assay, which combines the principles of broth microdilution volatilization and standard broth macrodilution methods. This assay enables rapid, simple, cost- and labor-effective screening of volatile compounds and overcomes the limitations of assays currently used for screening of antimicrobial activity in the vapor phase.

Identification of Group A Streptococcus Genes Directly Regulated by CsrRS and Novel Intermediate Regulators.

Adaptation of group A Streptococcus (GAS) to its human host is mediated by two-component systems that transduce external stimuli to regulate bacterial physiology. Among such systems, CsrRS (also known as CovRS) is the most extensively characterized for its role in regulating ∼10% of the GAS genome, including several virulence genes. Here, we show that extracellular magnesium and the human antimicrobial peptide LL-37 have opposing effects on the phosphorylation of the response regulator CsrR by the receptor kinase CsrS. Genetic inactivation of CsrS phosphatase or kinase activity, respectively, had similar but more pronounced effects on CsrR phosphorylation compared to growth in magnesium or LL-37. These changes in CsrR phosphorylation were correlated with the repression or activation of CsrR-regulated genes as assessed by NanoString analysis. Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) revealed CsrR occupancy at CsrRS-regulated promoters and lower-affinity associations at many other locations on the GAS chromosome. Because ChIP-seq did not detect CsrR occupancy at promoters associated with some CsrR-regulated genes, we investigated whether these genes might be controlled indirectly by intermediate regulators whose expression is modulated by CsrR. Transcriptional profiling of mutant strains deficient in the expression of either of two previously uncharacterized transcription regulators in the CsrR regulon indicated that one or both proteins participated in the regulation of 22 of the 42 CsrR-regulated promoters for which no CsrR association was detected by ChIP-seq. Taken together, these results illuminate CsrRS-mediated regulation of GAS gene expression through modulation of CsrR phosphorylation, CsrR association with regulated promoters, and the control of intermediate transcription regulators. IMPORTANCE Group A Streptococcus (GAS) is an important public health threat as a cause of sore throat, skin infections, life-threatening invasive infections, and the postinfectious complications of acute rheumatic fever, a leading cause of acquired heart disease. This work characterizes CsrRS, a GAS system for the detection of environmental signals that enables adaptation of the bacteria for survival in the human throat by regulating the production of products that allow the bacteria to resist clearance by the human immune system. CsrRS consists of two proteins: CsrS, which is on the bacterial surface to detect specific stimuli, and CsrR, which receives signals from CsrS and, in response, represses or activates the expression of genes coding for proteins that enhance bacterial survival. Some of the genes regulated by CsrR encode proteins that are themselves regulators of gene expression, thereby creating a regulatory cascade.

Application of Green Algal Planktochlorella nurekis Biomasses to Modulate Growth of Selected Microbial Species.

As microalgae are producers of proteins, lipids, polysaccharides, pigments, vitamins and unique secondary metabolites, microalgal biotechnology has gained attention in recent decades. Microalgae can be used for biomass production and to obtain biotechnologically important products. Here, we present the application of a method of producing a natural, biologically active composite obtained from unicellular microalgae of the genus Planktochlorella sp. as a modulator of the growth of microorganisms that can be used in the cosmetics and pharmaceutical industries by exploiting the phenomenon of photo-reprogramming of metabolism. The combination of red and blue light allows the collection of biomass with unique biochemical profiles, especially fatty acid composition (Patent Application P.429620). The ethanolic and water extracts of algae biomass inhibited the growth of a number of pathogenic bacteria, namely Enterococcus faecalis, Staphylococcus aureus PCM 458, Streptococcus pyogenes PCM 2318, Pseudomonas aeruginosa, Escherichia coli PCM 2209 and Candida albicans ATCC 14053. The algal biocomposite obtained according to our procedure can be used also as a prebiotic supplement. The presented technology may allow the limitation of the use of antibiotics and environmentally harmful chemicals commonly used in preparations against Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Escherichia coli or Candida spp.

Characterization of a putative maltodextrin-binding protein of Streptococcus pyogenes, SPs0871 and the development of a VHH inhibitor.

Streptococcus pyogenes causes a wide range of human infections. Currently, antibiotics are the main treatment for S. pyogenes infection, but serious anti-microbial resistance requires alternative treatment options. To develop a novel strategy for treatment, we physicochemically characterized SPs0871, a putative maltose/maltodextrin-binding protein that is thought to have important roles in the pathogenesis of invasive streptococci. We obtained a variable domain of heavy chain of heavy-chain antibody, the smallest unit of an antibody, which specifically binds to SPs0871. Although the VHH completely inhibited the binding of maltodextrins to SPs0871, the inhibition did not lead to growth suppression of the bacteria. Our results provide important insights for development of VHH as an anti-streptococcal therapeutic.

Characterization of the antibacterial activity from ethanolic extracts of the botanical, Larrea tridentata.

BACKGROUND: ß-lactam antibiotics are a class of broad-spectrum antibiotics consisting of all antibiotic agents that contain a ß-lactam ring in their molecular structures. ß-lactam antibiotics are only known to be isolated from fungi (e.g. Acremonium chrysogenum, Penicillium chrysogenum and Aspergillus nidulans) and bacteria (e.g. Streptomyces clavuligerus). We have shown that botanical extracts prepared from Larrea tridentata have strong antimicrobial activity against several bacteria, including members of Staphylococcus and Streptococcus genera. METHODS: Through resistance studies, inhibitor assays, and ELISA testing, we demonstrated L. tridentata extracts may contain a ß-lactam type antibiotic activity. RESULTS: Based on the estimated ß-lactam concentration within the extract, the antimicrobial activity of the L. tridentata extract was approximately 2000-8000-fold greater against Staphylococcus as compared to other ß-lactams, penicillin or ampicillin. In the L. tridentata extract, this increased activity was found to be associated with the likely presence of a cofactor leading to increased potentiation of the ß-lactam activity. This potentiation activity was also observed to enhance the activity of exogenously added natural penicillin antibiotics. CONCLUSIONS: Although constituents were not isolated in this study, the results obtained strongly support the presence of ß-lactam type antibiotic activity and antibiotic potentiation activity present in ethanolic extracts prepared from L. tridentata.

Effectiveness of antibacterial agents against cell-invading bacteria such as Streptococcus pyogenes and Haemophilus influenzae.

BACKGROUND: Recurrent tonsillitis is one of the most common otolaryngological disorders caused by cell-invading bacteria, such as Streptococcus pyogenes (S. pyogenes) and Haemophilus influenzae. The aim of this study was to investigate the effect of antibacterial agents against cell-invading bacteria. METHODS: The intracellular invasion of Detroit 562 cells by five strains of nontypeable Haemophilus influenzae (NTHi) and four strains of S. pyogenes was investigated. The antibacterial agents used were garenoxacin (GRNX), clarithromycin (CAM), amoxicillin (AMPC), cefditoren pivoxil (CDTR-PI), and levofloxacin (LVFX). RESULTS: Both NTHi and S. pyogenes fully invaded Detroit 562 cells in 6 h and were less sensitive to CAM. GRNX, CAM, and LVFX were effective against bacteria invading the cells, but AMPC and CDTR-PI were not effective. GRNX was the most effective. CONCLUSION: GRNX was the most effective agent against bacteria invading cells.

Synergistic effects of Cu-doped ZnO nanoantibiotic against Gram-positive bacterial strains.

A viable hydrothermal technique has been explored for the synthesis of copper doped Zinc oxide nanoparticles (Cu-doped ZnO-NPs) based on the precursor's mixture of Copper-II chloride dihydrate (CuCl2.2H2O), Zinc chloride (ZnCl2), and potassium hydroxide (KOH). X-ray diffraction (XRD) reported the hexagonal wurtzite structure of the synthesized Cu-doped ZnO-NPs. The surface morphology is checked via field emission scanning electron microscopy (FE-SEM), whereas, the elemental compositions of the samples were confirmed by Raman, and X-ray photoelectron spectroscopy (XPS), respectively. The as-obtained ZnO-NPs and Cu-doped ZnO-NPs were then tested for their antibacterial activity against clinical isolates of Gram-positive (Staphylococcus aureus, Streptococcus pyogenes) and Gram-negative (Escherichia coli, Klebsiella pneumonia) bacteria via agar well diffusion method. The zone of inhibition (ZOI) for Cu-doped ZnO-NPs was found to be 24 and 19 mm against S. Aureus and S. pyogenes, and 18 and 11 mm against E. coli and K. pneumoniae, respectively. The synthesized Cu-doped ZnO-NPs can thus be found as a potential nano antibiotic against Gram-positive multi-drug resistant bacterial strains.

Invasive Streptococcus pyogenes disease in Spain: a microbiological and epidemiological study covering the period 2007-2019.

The aim of this study is to present the first nationwide microbiological and epidemiological study of invasive group A Streptococcus (iGAS) disease in Spain. One thousand eight hundred ninety-three iGAS isolates were analyzed over 2007-2019. emm typing was performed by sequencing the gene's variable 5' end, exotoxin genes were identified by PCR, and antimicrobial susceptibility explored via the E test and disk diffusion. Five hundred twenty-three isolates were associated with sepsis, 292 with cellulitis, 232 with scarlet fever, 153 with pneumonia, 141 with streptococcal toxic shock syndrome, and 94 with necrotizing fasciitis. The most prevalent emm types were emm1 (449/1893 isolates), emm89 (210/1893), emm3 (208/1893), emm4 (150/1893), emm12 (112/1893) emm6 (107/1893), emm87 (89/1893), emm28 (88/1893), emm75 (78/1893), emm77 (78/1893), emm11 (58/1893), and emm22 (35/1893). emm1, emm3, emm4, and emm6 were the predominant types affecting children (mostly respiratory infections), while emm11, emm77, and emm89 prevailed in the elderly (mostly skin infections). Each emm type was associated with one or more exotoxin gene (spe, sme, and ssa) profiles. speA was detected in 660 isolates, speB in 1829, speC in 1014, speF in 1826, speG in 1651, speJ in 716, speH in 331, smeZ in 720, and ssa in 512. Isolates with speA were associated with the most severe infections. Penicillin susceptibility was universal. Two hundred twenty-four isolates were resistant to tetracycline, 169 to erythromycin, and 81 to clindamycin. Tetracycline, erythromycin, and clindamycin resistance rates declined over the study period. The above information could serve as the basis for continued surveillance efforts designed to control disease cause by this bacterium.

Prevalence and antibiotics susceptibility profiles of Streptococcus pyogenes among pediatric patients with acute pharyngitis at Felege Hiwot Comprehensive Specialized Hospital, Northwest Ethiopia.

BACKGROUND: Streptococcus pyogenes (S. pyogenes) is a Gram positive bacterium which is a leading cause of pharyngitis, skin and soft tissue infection and post streptococcal syndromes. Due to lack of ß-lactamase enzyme production, it was considered universally susceptible to penicillin group and later generation of ß-lactam antibiotics. As such, empirical treatment was common which might leads to development of antibiotics resistance. Therefore, the aims of this study were to determine the prevalence, antibiotics susceptibility profile; and associated factors of S. pyogenes among pediatric patients with acute pharyngitis in Felege Hiwot Comprehensive Specialized Hospital (FHCSH), Northwest Ethiopia. METHODS: Hospital based cross-sectional study was carried out on 154 pediatric patients, whose age ranged from 0 to 18 years old using consecutive convenient sampling technique from 1st February to 19th June 2020 at FHCSH. S. pyogenes were identified by throat swab culture on 5% sheep blood agar with an overnight incubation at 37 °C in candle jar containing 5% CO2. Gram stain, catalase test and bacitracin test were used to identify S. pyogenes. Then,the data were entered into EpiData version 3.1 and analyzed by SPSS version 20 software. Finally, stepwise, bivariable and multivariable logistic regressions were carried out for identifyying factors having significant ssociation (p<0.05) with  acute pharyngitis.  RESULTS: From the total throat swabs, 14 (9.1%) with (95% CI; 4.5-14.3) were culture positive for S. pyogenes. From these, all isolates were sensitive to penicillin and ampicillin. On the otherhand, 4 (35.7%), 4 (35.5%), 3 (21.4%), 2 (14.3%), 1 (7.1%), 7 (50.0%) and 1 (7.1%) isolates were resistant for ceftriaxone, vancomycin, erythromycin, tetracycline, chloramphenicol, clindamycin and levofloxacin, respectively. The presence of any smoker in home showed significant association with S. pyogenes acute pharyngitis. Furthermore, having tender lymphadenopathy and recurrence were clinical predictors for S. pyogenes acute pharyngitis (P < 0.05). CONCLUSION: The prevalence of S. pyogenes was guaged at 9.1% which is considered as low prevalence. All S. pyogenes isolats remain sensitive to penicillin. However, resistance was reported to clindamycin 7 (50.0%), ceftriaxone 5 (35.7%) and erythromycin 3 (21.4%). The current practice of giving erythromycin, clindamycin instead of penicillin and ampicillin is againest the microbiology result. Therefore, current empirical treatment of acute pharyngitis shall take in to account the current evidences. Continuous surveillance of antibiotics resistance pattern of S. pyogenes for acute pharyngitis must be strengthen to improve the use of antibiotics in hospitals.

Revisiting the inoculum effect for Streptococcus pyogenes with a hollow fibre infection model.

Severe, invasive Streptococcus pyogenes (Strep A) infections result in greater than 500,000 deaths annually. First line treatment for such infections is benzylpenicillin, often with the addition of clindamycin, but treatment failure can occur with this regimen. This failure has been partially attributed to the inoculum effect, which presents as reduced antibiotic susceptibility during high bacterial density and plateau-phase growth. Hollow fibre infection models (HFIM) have been proposed as an in vitro alternative to in vivo research to study these effects. To re-evaluate the inoculum effect for benzylpenicillin, clindamycin, linezolid, and trimethoprim-sulfamethoxazole using a Strep A HFIM. Differential antibiotic susceptibility of Strep A was measured in a HFIM starting from low- and high-density inocula with an average difference in bacterial concentration of 56-fold. Dynamic antibiotic concentrations were delivered over 48 h to simulate in vivo human pharmacokinetics in an in vitro model. Differences in antibiotic susceptibility were measured by plate count of colony-forming units over time. Inoculum effects were seen in benzylpenicillin and linezolid at 24 h, and benzylpenicillin, linezolid, and clindamycin at 48 h. The effect size was greatest for continuously infused benzylpenicillin at 48 h with a log10-fold difference of 4.02 between groups. No inoculum effect was seen in trimethoprim-sulfamethoxazole, with a maximal log10-fold difference of 0.40. Inoculum effects were seen using benzylpenicillin, linezolid, and clindamycin, which may predict reduced clinical efficacy following treatment delay. The model has proven robust and largely in agreeance with published data, recommending it for further Strep A study.

In-vitro and in-vivo evaluation of the antibacterial potential of Typha elephantina.

The present study was aimed to evaluate the in-vitro and in-vivo antibacterial effects of the Typha elephantina aqueous extract (TE.AQ), ethanolic extract (TE.ET) and T. elephantina methanolic extract (TE.ME) against eight selected clinical pathogens. The test samples were tested for in-vitro analysis (by disc diffusion method) at different concentrations of 5, 15, 25, 50 and 100 mg/dL against both gram positive and gram-negative strains. The highest potential was observed in TE.ME at a concentration of 100 mg/dL against Pseudomonas aeruginosa exhibiting 19.67 ± 0.577 mm zone of inhibition (ZOI). The same fraction also showed good activity against Staphylococus aureus with ZOI of 17.50 ± 0.70 mm. The TE.ET was found most active against P. aeruginosa and Streptococcus pyogenes having ZOI of 18.53 ± 0.503 and 16.2 ± 1.55 mm respectively at a concentration of 100 mg/dL. The most sensitive bacteria P. aeruginosa was selected for in-vivo study (using poultry chicks) for induction of infection in chicks. The effects of TE.AQ, TE.ET and TE.ME were determined at concentrations of 300 mg/kg body weight based on hematological parameters, liver enzymes and gross pathological findings of lungs and livers. The findings of the in-vivo study in chick's model showed that treatment of experimental animals with TE.ME significantly restored the hematological parameters, liver enzymes and architecture of lungs and livers. Based on scientific evidence, the current study suggests that TE.ME may serve as a best and new natural antibacterial agent and can be used against infections caused by P. aeruginosa.

Amplify antimicrobial photo dynamic therapy efficacy with poly-beta-amino esters (PBAEs).

Light-activated antimicrobial agents (photosensitisers) are promising alternatives to antibiotics for the treatment of skin infections and wounds through antimicrobial photo dynamic therapy (aPDT); utilisation of this technique is still restricted by general low efficacy requiring long exposure time (in the order of tens of minutes) that make the treatment very resource intensive. We report for the first time the possibility of harvesting the cell penetrating properties of poly-beta-amino esters (PBAEs) in combination with toluidine blue O (TBO) to shorten aPDT exposure time. Candidates capable of inactivation rates 30 times quicker than pure TBO were discovered and further improvements through PBAE backbone optimisation could be foreseen. Efficacy of the complexes was PBAE-dependent on a combination of TBO uptake and a newly discovered and unexpected role of PBAEs on reactive species production. Chemometric approach of partial least square regression was employed to assess the critical PBAE properties involved in this newly observed phenomenon in order to elicit a possible mechanism. The superior antimicrobial performance of this new approach benefits from the use of well established, low-cost and safe dye (TBO) coupled with inexpensive, widely tested and biodegradable polymers also known to be safe. Moreover, no adverse cytotoxic effects of the PBAEs adjuvated TBO delivery have been observed on a skin cells in vitro model demonstrating the safety profile of this new technology.