Search results for: hypervirulent klebsiella pneumoniae

Authors: Dingiswayo Likhona, Arko-Cobbah Emmanuel, Carolina Pohl, Nthabiseng Z. Mokoena, Jolly Musoke

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A distinct strain of Klebsiella pneumoniae (K. pneumoniae), referred to as hypervirulent (hvKp), is associated with invasive infections such as an invasive pyogenic liver abscess in young and healthy individuals. In South Africa, limited information is known about the prevalence and virulence of this hvKp strain. Thus, this study aimed to determine the prevalence of hvKp and virulence-associated factors in K. pneumoniae isolates from one of the largest Tertiary hospitals in a South African province. A total of 74 K. pneumoniae isolates were received from Pelonomi National Health Laboratory Services (NHLS), Bloemfontein. Virulence-associated genes (rmpA, capsule serotype K1/K2, iroB, and irp2) were screened, and the virulence of hvKp vs. classical Klebsiella pneumoniae (cKp) was investigated using Caenorhabditis elegans nematode model. The iutA (aerobactin transporter) gene was used as a primary biomarker of hvKp. An average of 12% (9/74) of cases were defined as hvKp. Moreover, hvKp was found to be significantly more virulent in vivo Caenorhabditis elegans relative to cKp. The virulence-associated genes (rmpA, iroB, hmv phenotype, and capsule K1/K2) were significantly (p< 0.05) associated with hvKp. Findings from this study confirm the presence of hvKp in one large Tertiary hospital in South Africa. However, the low prevalence and mild to moderate clinical presentation suggest a marginal threat to public health. Further studies in different settings are required to establish the true potential impact of hvKp in developing countries.

Keywords: hypervirulent klebsiella pneumoniae, virulence, caenorhabditis elegans, aerobactin (iutA)

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Authors: Gulcan Sahal, Nermin Hande Avcioglu, Isil Seyis Bilkay

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Klebsiella species which are natural colonizers of human upper respiratory and human gastrointestinal tracts are also responsible for every reoccurring nosocomial infections by means of having ability to form slimy layers known as biofilm on many surfaces. Therefore, in this study, investigation of biofilm formation in K. pneumoniae and K. rhinoscleromatis and examination of each Klebsiella strains’ clinical information in the light of their biofilm formation results were aimed. In this respect, biofilm formation of Klebsiella strains was analyzed via crystal violet binding assay. According to our results, biofilm formation levels of K. pneumoniae and K. rhinoscleromatis strains were different from each other. Additionally, in comparison to K. rhinoscleromatis strains, K. pneumoniae was observed to include higher amounts of strong biofilm forming strains. Besides, it was also seen that clinical information of patients from which strong biofilm forming Klebsiella strains were isolated were similar to each other. Our results indicate that there should be more precautions against K. pneumoniae which includes higher amount of strong biofilm forming strains.

Keywords: biofilm formation, Klebsiella pneumoniae, Klebsiella rhinoscleromatis, biosystems engineering

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Authors: Sweta Pandey, Samridhi Dhyani, Susmita Chaudhuri

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Klebsiella pneumoniae bacteria is a global threat to human health due to an increase in multi-drug resistance among strains. The hypervirulent strains of Klebsiella pneumoniae is a major trouble due to their association with life-threatening infections in a healthy population. One of the major virulence factors of hyper virulent strains of Klebsiella pneumoniae is the T6SS (Type six secretary system) which is majorly involved in microbial antagonism and causes interaction with the host eukaryotic cells during infections. T6SS mediates some of the crucial factors for establishing infection by the bacteria, such as cell adherence, invasion, and subsequent in vivo colonisation. The antibacterial activity and the cell invasion property of the T6SS system is a major requirement for the establishment of K. pneumoniae infections within the gut. The T6SS can be an appropriate target for developing therapeutics. The T6SS consists of an inner tube comprising hexamers of Hcp (Haemolysin -regulated protein) protein, and at the top of this tube sits VgrG (Valine glycine repeat protein G); the tip of the machinery consists of PAAR domain containing proteins which act as a delivery system for bacterial effectors. For this study, immune response to recombinant VgrG protein was generated to establish this protein as a potential immunogen for the development of therapeutic leads. The immunogenicity of the selected protein was determined by predicting the B cell epitopes by the BCEP analysis tool. The gene sequence for multiple domains of VgrG protein (phage_base_V, T6SS_Vgr, DUF2345) was selected and cloned in pMAL vector in E. coli. The construct was subcloned and expressed as a fusion protein of 203 residue protein with mannose binding protein tag (MBP) to enhance solubility and purification of this protein. The purified recombinant VgrG fusion protein was used for mice immunisation. The antiserum showed reactivity with the recombinant VgrG in ELISA and western blot. The immunised mice were challenged with K. pneumoniae bacteria and showed bacterial clearance in immunised mice. The recombinant VgrG protein can further be used for studying downstream signalling of VgrG protein in mice during infection and for therapeutic MAb development to eradicate K. pneumoniae infections.

Keywords: immune response, Klebsiella pneumoniae, multi-drug resistance, recombinant protein expression, T6SS, VgrG

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Authors: Manal Suleiman, George Abu-Aqil, Uraib Sharaha, Klaris Riesenberg, Itshak Lapidot, Ahmad Salman, Mahmoud Huleihel

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Klebsiella pneumoniae is one of the most aggressive multidrug-resistant bacteria associated with human infections resulting in high mortality and morbidity. Thus, for an effective treatment, it is important to diagnose both the species of infecting bacteria and their susceptibility to antibiotics. Current used methods for diagnosing the bacterial susceptibility to antibiotics are time-consuming (about 24h following the first culture). Thus, there is a clear need for rapid methods to determine the bacterial susceptibility to antibiotics. Infrared spectroscopy is a well-known method that is known as sensitive and simple which is able to detect minor biomolecular changes in biological samples associated with developing abnormalities. The main goal of this study is to evaluate the potential of infrared spectroscopy in tandem with Random Forest and XGBoost machine learning algorithms to diagnose the susceptibility of Klebsiella pneumoniae to antibiotics within approximately 20 minutes following the first culture. In this study, 1190 Klebsiella pneumoniae isolates were obtained from different patients with urinary tract infections. The isolates were measured by the infrared spectrometer, and the spectra were analyzed by machine learning algorithms Random Forest and XGBoost to determine their susceptibility regarding nine specific antibiotics. Our results confirm that it was possible to classify the isolates into sensitive and resistant to specific antibiotics with a success rate range of 80%-85% for the different tested antibiotics. These results prove the promising potential of infrared spectroscopy as a powerful diagnostic method for determining the Klebsiella pneumoniae susceptibility to antibiotics.

Keywords: urinary tract infection (UTI), Klebsiella pneumoniae, bacterial susceptibility, infrared spectroscopy, machine learning

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Authors: Lucian Mocan, Teodora Mocan, Matea Cristian, Cornel Iancu

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We present method of nanoparticle enhanced laser thermal ablation of Klebsiella pneumoniae infections, using gold nanoparticles combined with a specific growth factor and demonstrate its selective therapeutic efficacy. Ab (antibody solution) bound to GNPs (gold nanoparticles) was administered in vitro and determined the specific delivery of the nano-bioconjugate into the microorganism. The extent of necrosis was considerable following laser therapy, and at the same time, normal cells were not seriously affected. The selective photothermal ablation of the infected tissue was obtained after the selective accumulation of Ab bound to GNPs into bacteria following perfusion. These results may represent a major step in antibiotherapy treatment using nanolocalized thermal ablation by laser heating.

Keywords: gold nanoparticles, Klebsiella pneumoniae, nanoparticle functionalization, laser irradiation, antibody

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Authors: Umairah Natasya Binti Mohd Omeershffudin, Suresh Kumar

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Klebsiella pneumoniae, a Gram-negative enteric bacterium that causes nosocomial and urinary tract infections. Particular concern is the global emergence of multidrug-resistant (MDR) strains of Klebsiella pneumoniae. Characterization of antibiotic resistance determinants at the genomic level plays a critical role in understanding, and potentially controlling, the spread of multidrug-resistant (MDR) pathogens. In this study, drug-resistant Klebsiella pneumoniae strain 825795-1 was investigated with extensive computational approaches aimed at identifying novel drug targets among hypothetical proteins. We have analyzed 1099 hypothetical proteins available in genome. We have used in-silico genome subtraction methodology to design potential and pathogen-specific drug targets against Klebsiella pneumoniae. We employed bioinformatics tools to subtract the strain-specific paralogous and host-specific homologous sequences from the bacterial proteome. The sorted 645 proteins were further refined to identify the essential genes in the pathogenic bacterium using the database of essential genes (DEG). We found 135 unique essential proteins in the target proteome that could be utilized as novel targets to design newer drugs. Further, we identified 49 cytoplasmic protein as potential drug targets through sub-cellular localization prediction. Further, we investigated these proteins in the DrugBank databases, and 11 of the unique essential proteins showed druggability according to the FDA approved drug bank databases with diverse broad-spectrum property. The results of this study will facilitate discovery of new drugs against Klebsiella pneumoniae.

Keywords: pneumonia, drug target, hypothetical protein, subtractive genomics

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Authors: Sweta Pandey, Samridhi Dhyani, Susmita Chaudhuri

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Klebsiella pneumoniae causes a wide range of infections, including urinary tract infections, sepsis, bacteremia, pneumonia, and liver abscesses. The emergence of multi-drug resistance in this bacterium led to a major setback for clinical management. WHO also endorsed a need for finding alternative therapy to antibiotics for the treatment of these infections. Development of vaccines and passive antibody therapy has been proven as a potent alternative to antibiotics in the case of MDR, XDR, and PDR Klebsiella infections. Siderophore receptors have been demonstrated to be overexpressed for the internalization of iron siderophore complexes during infections in most Gram-negative bacteria. For the present study, immune response to siderophore receptors to establish this protein as a potential immunogen for the development of therapeutic leads was explored. Clinical strains of Klebsiella pneumoniae were grown in iron-deficient conditions, and the iron-regulated outer membrane proteins were extracted and characterized through mass spectrometry for specific identification. The gene for identified protein was cloned in pET- 28a vector and expressed in E. coli. The native protein and the recombinant protein were isolated and purified and used as antigens for the generation of immune response in BALB/c mice. The native protein of Klebsiella pneumoniae grown in iron-deficient conditions was identified as FepA (Ferrienterobactin receptor) and other siderophore receptors. This 80 kDa protein generated an immune response in BALB/c mice. The antiserum from mice after subsequent booster doses was collected and showed binding with FepA protein in western blot and phagocytic uptake of the K. pneumoniae in the presence antiserum from immunized mice also observed from the animal studies after bacterial challenge post immunisation in mice have shown bacterial clearance. The antiserum from mice showed binding and clearance of the Klebsiella pneumoniae bacteria in vitro and in vivo. These antigens used for generating an active immune response in mice can further be used for therapeutic monoclonal antibody development against Klebsiella pneumoniae infections.

Keywords: antiserum, FepA, Klebsiella pneumoniae, multi drug resistance, siderophore receptor

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Authors: Okafor Joan, Nwodo Uchechukwu

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Klebsiella pneumoniae (K. pneumoniae) is a significant pathogen responsible for opportunistic and nosocomial infection. One of the most significant antibiotic resistance mechanisms in K. pneumoniae isolates is efflux pumps. Our current study identified efflux genes (AcrAB, OqxAB, MacAB, and TolC) and regulatory genes (RamR and RarA) in multidrug-resistant (MDR) K. pneumoniae isolated from hospital effluents and investigated their relationship with antibiotic resistance. The sum of 145 K. pneumoniae isolates was established by PCR and screened for antibiotic susceptibility. PCR detected efflux pump genes, and their link with antibiotic resistance was statistically examined. However, 120 (83%) of the confirmed isolated were multidrug-resistant, with the largest percentage of resistance to ampicillin (88.3%) and the weakest rate of resistance to imipenem (5.5%). Resistance to the other antibiotics ranged from 11% to 76.6%. Molecular distribution tests show that AcrA, AcrB, MacA, oqxB oqxA, TolC, MacB were detected in 96.7%, 85%, 76.7%, 70.8%, 55.8%, 39.1%, and 29.1% respectively. However, 14.3% of the isolates harboured all seven genes screened. Efflux pump system AcrAB (83.2%) was the most commonly detected in K. pneumonia isolated across all the antibiotics class-tested. In addition, the frequencies of RamR and RarA were 46.2% and 31.4%, respectively. AcrAB and OqxAB efflux pump genes were significantly associated with fluoroquinolone, beta-lactam, carbapenem, and tetracycline resistance (p<0.05). The high rate of efflux genes in this study demonstrated that this resistance mechanism is the dominant way in K. pneumoniae isolates. Appropriate treatment must be used to reduce and tackle the burden of resistant Klebsiella pneumonia in hospital wastewater effluents.

Keywords: Klebsiella pneumonia, efflux pumps, regulatory genes, multidrug-resistant, hospital, PCR

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Authors: Sandeep Vasaikar, Lary Obi

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Rapid and discriminative genotyping methods are useful for determining the clonality of the isolates in nosocomial or household outbreaks. Multilocus sequence typing (MLST) is a nucleotide sequence-based approach for characterising bacterial isolates. The genetic diversity and the clinical relevance of the drug-resistant Klebsiella isolates from Mthatha are largely unknown. For this reason, prospective, experimental study of the molecular epidemiology of Klebsiella isolates from patients being treated in Mthatha over a three-year period was analysed. Methodology: PCR amplification and sequencing of the drug-resistance-associated genes, and multilocus sequence typing (MLST) using 7 housekeeping genes mdh, pgi, infB, FusAR, phoE, gapA and rpoB were conducted. A total of 32 isolates were analysed. Results: The percentages of multidrug-resistant (MDR), extensively drug-resistance (XDR) and pandrug-resistant (PDR) isolates were; MDR 65.6 % (21) and XDR and PDR with 0 % each. In this study, K. pneumoniae was 19/32 (59.4 %). MLST results showed 22 sequence types (STs) were identified, which were further separated by Maximum Parsimony into 10 clonal complexes and 12 singletons. The most dominant group was Klebsiella pneumoniae with 23/32 (71.8 %) isolates, Klebsiella oxytoca as a second group with 2/32 (6.25 %) isolates, and a single (3.1 %) K. varricola as a third group while 6 isolates were of unknown sequences. Conclusions/significance: A phylogenetic analysis of the concatenated sequences of the 7 housekeeping genes showed that strains of K. pneumoniae form a distinct lineage within the genus Klebsiella, with K. oxytoca and K. varricola its nearest phylogenetic neighbours. With the analysis of 7 genes were determined 1 K. variicola, which was mistakenly identified as K. pneumoniae by phenotypic methods. Two misidentifications of K. oxytoca were found when phenotypic methods were used. No significant differences were observed between ESBL blaCTX-M, blaTEM and blaSHV groups in the distribution of Sequence types (STs) or Clonal complexes (CCs).

Keywords: phylogenetic analysis, phylogeny, klebsiella phylogenetic, klebsiella

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Authors: Ali Fadhel Ahmed, Tuqa Abdulkareem Hameed

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Moringa oleifera (leaves and seeds) ethanolic and aqueous extracts were tested for antibacterial activity. The effect of plant extracts on three types of bacterial species: Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae, was investigated. Using the agar well diffusion method, ethanolic extracts of Moringa oleifera demonstrated a significant antibacterial effect on the forty tested bacterial strains. Seed-induced inhibition zones (ethanolic extracts)were ranged from16 to 24 mm in diameter against S. aureus, respectively, whileE. coli and K. pneumonia had no effect. Gram-positive and Gram-negative bacteria were not affected by alcoholic and aqueous plant leaf extracts. The purpose of this present study was to look at the cytotoxic effects of M.Oleifera plant (alcoholic extracts).

Keywords: moringa oleifera, escherichia coli, klebsiella pneumoniae, staphylococcus aureus

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Authors: Dawang D. N., Dasat G. S., Nden D.

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Endophyte means “in the plant” are referred to all microorganisms that live in the internal tissues of stems, petioles, roots and leaves of plants causing no apparent symptoms of disease. Secondary metabolites from fungal endophytes have an enormous potential applications as antioxidant, antimicrobial, anticancer and antidiabeties. Thus, this study aimed to determine the antimicrobial activity of these metabolites against some clinical isolates. The fungi were subjected to fermentation medium and the metabolites were extracted using ethyl acetate. The fungal extracts showed both antibacterial and antifungal activities with maximum zone of inhibition diameter of 10.5mm against Aspergillus fumigatus. Staphylococcus aureus was inhibited by all the five crude extracts with inhibition zone diameter of 4mm. Endophytic fungal crude extract2 (EDF2) exhibited antimicrobial effect against all the test organisms used, EDF4 was active against all test organisms except on Penicillium chrysogenum and Klebsiella pneumoniae. Antibacterial standard of ciprofloxacin which is 15mm is comparable to the effect of endophytic extract of EDF1 and EDF2. Klebsiella pneumoniae was resistant to EDF4 and EDF5. EDF3 showed a wide range of antimicrobial activity against all the test organisms used. The highest inhibition zone diameter of 10.50mm recorded against Aspergillus fumigatus is comparable to antifungal standard of fluconazole (15.5mm). The result of this study suggests that endophytic fungi associated with the roots of Irish potato could be a promising source of novel bioactive compounds of pharmaceutical and industrial importance.

Keywords: endophyte, fungal extract, antimicrobial, potato

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Authors: Katherine Madero Valencia, Carlos Jaramillo, Elida Dueñas, Carlos Torres, María Del Pilar Delgado

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Asthma, described as a chronic inflammatory condition of the airways, courses accompanied by episodes known as exacerbations, characterized by a worsening of symptoms. Among the triggers, some allergen-irritative and infectious agents are found, including Chlamydophila pneumoniae which seems to play an increasingly important role. In this paper a PCR was used to detect C. pneumoniae in order to estimate the frequency of infections caused by this agent in pediatric patients with asthma exacerbations. C. pneumoniae distribution throughout the study period was also evaluated. 175 nasopharyngeal aspirates from children with asthma exacerbations were analyzed by PCR and sequencing. A global prevalence of C. pneumoniae of 53.71% was obtained. This study highlights a high circulation of C. pneumoniae during the study period, in children of all ages and especially in children under 5 years old. Molecular tests applied permit a rapid detection and improved our knowledge about these infections in children with asthma.

Keywords: Chlamydophila pneumoniae, detection, molecular techniques, pediatric asthma

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Authors: Guna Raj Dhungana, Roshan Nepal, Apshara Parajuli, , Archana Maharjan, Shyam K. Mishra, Pramod Aryal, Rajani Malla

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Introduction: Klebsiella pneumoniae is a well-known opportunistic human pathogen, primarily causing healthcare-associated infections. The global emergence of carbapenemase-producing K. pneumoniaeis a major public health burden, which is often extensively multidrug resistant.Thus, because of the difficulty to treat these ‘superbug’ and menace and some term as ‘apocalypse’ of post antibiotics era, an alternative approach to controlling this pathogen is prudent and one of the approaches is phage mediated control and/or treatment. Objective: In this study, we aimed to isolate novel bacteriophage against carbapenemase-producing K. pneumoniaeand characterize for potential use inphage therapy. Material and Methods: Twenty lytic phages were isolated from river water using double layer agar assay and purified. Biological features, physiochemical characters, burst size, host specificity and activity spectrum of phages were determined. One most potent phage: Phage TU_Kle10O was selected and characterized by electron microscopy. Whole genome sequences of the phage were analyzed for presence/absence of virulent factors, and other lysin genes. Results: Novel phage TU_Kle10O showed multiple host range within own genus and did not induce any BIM up to 5th generation of host’s life cycle. Electron microscopy confirmed that the phage was tailed and belonged to Caudovirales family. Next generation sequencing revealed its genome to be 166.2 Kb. bioinformatical analysis further confirmed that the phage genome ‘did not’ contain any ‘bacterial genes’ within phage genome, which ruled out the concern for transfer of virulent genes. Specific 'lysin’ enzyme was identified phages which could be used as 'antibiotics'. Conclusion: Extensively multidrug resistant bacteria like carbapenemase-producing K. pneumoniaecould be treated efficiently by phages.Absence of ‘virulent’ genes of bacterial origin and presence of lysin proteins within phage genome makes phages an excellent candidate for therapeutics.

Keywords: bacteriophage, Klebsiella pneumoniae, MDR, phage therapy, carbapenemase,

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Authors: Mohamed M. Bumadian, D. James Gilmour

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Three bacterial species were isolated from the River Wye (Derbyshire, England) and identified using 16S rRNA gene sequencing as Serratia proteamaculans, Aeromonas hydrophila and Klebsiella pneumoniae. Respiration rates of the strains were measured in order to determine the metabolic activity under salt stress. The highest respiration rates of all three strains were found at 0.17 M and 0.5 M NaCl and then the respiration rate decreased with increasing concentrations of NaCl. In addition, the effect of increasing concentrations of NaCl on malate dehydrogenase activity was determined using cell-free extracts of the three strains. Malate dehydrogenase activity was stimulated at NaCl concentrations up to 0.5 M, and a small level of activity remained even at 3.5 M NaCl. The pH optimum of the malate dehydrogenase in cell-free extracts of all strains was higher than pH 7.5.

Keywords: fresh water, halotolerant pathogenic bacteria, 16S rRNA gene, cell-free extracts, respiration rates, malate dehydrogenase

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Authors: Vivek Bhat, Rohini Kelkar, Sanjay Biswas

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Introduction: Cancer patients are at increased risk of bacterial infections. This may due to the disease process itself, the effect of chemotherapeutic drugs or invasive procedures such as catheterization. A wide variety of bacteria including some emerging pathogens are increasingly being reported from these patients. The incidence of multidrug-resistant organisms particularly in the Gram negative group is also increasing, with higher resistance rates seen to cephalosporins, β-lactam/β-lactam inhibitor combinations, and the carbapenems. This study documents the bacteriological spectrum of infections and their resistance patterns in cancer patients. Methods: This study includes all bacterial isolates recovered from infections cancer patients over a period of 18 months. Samples included Blood cultures, Pus/wound swabs, urine, tissue biopsies, body fluids, catheter tips and respiratory specimens such as sputum and bronchoalveolar lavage (BAL). All samples were processed in the microbiology laboratory as per standard laboratory protocols. Organisms were identified to species level and antimicrobial susceptibility testing was performed manually by the disc diffusion technique or in the Vitek-2 (Biomereux, France) instrument. Interpretations were as per Clinical laboratory Standards Institute (CLSI) guidelines. Results: A total of 1150 bacterial isolates were cultured from 884 test samples during the study period. Of these 227 were Gram-positive and 923 were Gram-negative organisms. Staphylococcus aureus (99 isolates) was the commonest Gram-positive isolate followed by Enterococcus (79) and Gr A Streptococcus (30). Among the Gram negatives, E. coli (304), Pseudomonas aeruginosa (201) and Klebsiella pneumoniae (190) were the most common. Of the Staphylococcus aureus isolates 27.2% were methicillin resistant. Only 5.06% enterococci were vancomycin resistant. High rates of resistance to cefotaxime and ciprofloxacin were seen amongst E. coli (84.8% & 83.55%) and Klebsiella pneumoniae (71 & 62.1%) respectively. Resistance to carbapenems (meropenem) was high at 70% in Acinetobacter spp.; however all isolates were sensitive to colistin. Among the aminoglycosides, amikacin retained good efficacy against Escherichia coli (82.9%) and Pseudomonas aeruginosa (78.1%). Occasional isolates of emerging pathogens such as Chryseobacterium indologens, Roseomonas, and Achromobacter xyloxidans were also recovered. Conclusion: The common infections in cancer patients include respiratory, wound, tract infections and sepsis. The commonest isolates include Staphylococcus aureus, Enterococci, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. There is a high level of resistance to the commonly used antibiotics among Gram-negative organisms.

Keywords: bacteria, resistance, infection, cancer

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Authors: M. E. Abalaka, O. A. Falusi, M. Galadima, D. Damisa

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The antimicrobial activity of aqueous, ethanolic and methanolic extracts of Chromolaena odorata (Siam weed) was evaluated against four wound isolates: Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae at the concentrations of 200mg/ml, 100mg/ml, 50mg/ml and 25mg/ml respectively. S. aureus and E. coli showed high susceptibility to the various extracts than the other test isolates. The aqueous extract showed activity against Staphylococcus aureus with a mean diameter of zone of inhibition of 16 ± 3.00 at concentration of 200mg/ml and as low as 8 ± 0.00 at concentration of 25mg/ml; E. coli showed susceptibility with a mean diameter of zone of inhibition of 18 ± 2.00 and 10 ± 0.00 at a concentration of 200mg/ml and 25mg/ml respectively. Pseudomonas aeruginosa and Klebsiella pneumoniae were resistant to the aqueous extract. Methanol extract showed activity against Staphylococcus aureus with a mean diameter of zone of inhibition at 28 ± 4.00 and 12 ± 2.30 at a concentration of 200mg/ml and 25mg/ml respectively; while E. coli was susceptible with mean diameter of zone of inhibition of 18 ± 2.00 and as low as 12 ± 0.00 at a concentration of 200mg/ml and 50mg/ml respectively, Pseudomonas aeruginosa showed considerable susceptibility with mean diameter of zone of inhibition of 13 ± 1.00 and 12 ± 0.00 at a concentration of 200mg/ml and 100mg/ml respectively. The ethanol extract showed activity against S. aureus with a mean diameter zone of inhibition of 15 ± 2.00 and 9 ± 0.00 at a concentration of 200mg/ml and 25mg/ml respectively: E. coli showed susceptibility with a mean diameter zone of inhibition of 20 ± 4.00 and 13 ± 2.00 at a concentration of 200mg/ml and 25mg/ml respectively. Pseudomonas aeruginosa showed considerable susceptibility with a mean diameter zone of inhibition of 13 ± 1.00 and 9 ± 0.00 at a concentration of 200mg/ml and 100mg/ml respectively. The results above indicate the efficacy and potency of the crude extracts of Chromolaena odorata leaf on the tested wound isolates.

Keywords: antibacterial, Chromolaena odorata, leaf extracts, test isolates

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Authors: Minna Chang

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Introduction: M. pneumoniae is a respiratory pathogen, which commonly causes upper and lower respiratory infections. It primarily affects children and young adults. Respiratory symptoms are well recognized, but extrapulmonary involvement is also common. Other systems that have been implicated in the disease include: skin, mucus membranes, central, peripheral nervous systems, cardiovascular, haematological, renal, and musculoskeletal systems. Here, we report a case of an otherwise healthy, young female with M. pneumonia, who presented with right upper quadrant abdominal pain. Case presentation: a healthy 25-year-old female was referred to A&E by her general practitioner, after presenting with fever, malaise, and right upper quadrant pain. M. pneumoniae was confirmed retrospectively by serology. The patient made a full recovery after a six-day course of doxycycline 100mg. Conclusion: M. pneumonia is a well-established cause of respiratory infections in children and young adults. Febrile illness with multisystem involvement, even in the absence of respiratory symptoms, should raise suspicion of M. pneumoniae infection in healthy, young adults. Our case illustrates the multi-system involvement of M. pneumoniae, which was initially missed, due to paucity of respiratory symptoms at presentation.

Keywords: infectious diseases, mycoplasma pneumoniae, respiratory infections, extra-pulmonary manifestations

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Authors: Lakmini Wijesooriya, Vicki Chalker, Jessica Day, Priyantha Perera, N. P. Sunil-Chandra

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M. pneumoniae has been identified as an etiology for exacerbation of asthma (EQA), although viruses play a major role in EOA. M. pneumoniae infection is treated empirically with macrolides, and its antibiotic sensitivity is not detected routinely. Characterization of the organism by genotyping and determination of macrolide resistance is important epidemiologically as it guides the empiric antibiotic treatment. To date, there is no such characterization of M. pneumoniae performed in Sri Lanka. The present study describes the characterization of M. pneumoniae detected from a child with EOA following a screening of 100 children with EOA. Of the hundred children with EOA, M. pneumoniae was identified only in one child by Real-Time polymerase chain reaction (PCR) test for identifying the community-acquired respiratory distress syndrome (CARDS) toxin nucleotide sequences. The M. pneumoniae identified from this patient underwent detection of macrolide resistance via conventional PCR, amplifying and sequencing the region of the 23S rDNA gene that contains single nucleotide polymorphisms that confer resistance. Genotyping of the isolate was performed via nested Multilocus Sequence Typing (MLST) in which eight (8) housekeeping genes (ppa, pgm, gyrB, gmk, glyA, atpA, arcC, and adk) were amplified via nested PCR followed by gene sequencing and analysis. As per MLST analysis, the M. pneumoniae was identified as sequence type 14 (ST14), and no mutations that confer resistance were detected. Resistance to macrolides in M. pneumoniae is an increasing problem globally. Establishing surveillance systems is the key to informing local prescriptions. In the absence of local surveillance data, antibiotics are started empirically. If the relevant microbiological samples are not obtained before antibiotic therapy, as in most occasions in children, the course of antibiotic is completed without a microbiological diagnosis. This happens more frequently in therapy for M. pneumoniae which is treated with a macrolide in most patients. Hence, it is important to understand the macrolide sensitivity of M. pneumoniae in the setting. The M. pneumoniae detected in the present study was macrolide sensitive. Further studies are needed to examine a larger dataset in Sri Lanka to determine macrolide resistance levels to inform the use of macrolides in children with EOA. The MLST type varies in different geographical settings, and it also provides a clue to the existence of macrolide resistance. The present study enhances the database of the global distribution of different genotypes of M. pneumoniae as this is the first such characterization performed with the increased number of samples to determine macrolide resistance level in Sri Lanka. M. pneumoniae detected from a child with exacerbation of asthma in Sri Lanka was characterized as ST14 by MLST and no mutations that confer resistance were detected.

Keywords: mycoplasma pneumoniae, Sri Lanka, characterization, macrolide resistance

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Authors: Kinza Khan, Dad Muhmmad, Asif Saleem, Nadia Mukhtar, Tahir Yaqub

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Ethomedicines are more commonly used in underdeveloped and developing countries. These medicines are sometimes more potent in controlling microbial infections than conventional medicines. Medicinal plants have been common practice to cure many diseases for centuries. Murraya koenigii is one of these plants and is commonly used in South Asian countries as a flavoring agent in food. To evaluate its anti-microbial activity, six different bacterial strains (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Bacillus cereus and Klebsiella pneumonia were used. N-hexane extract of Murraya koenigii leaves shows maximum activity against Bacillus cereus. Acetone extract of Murraya koenigii shoots showed more efficient activity against Pseudomonas aeruginosa Dichloromethane extracts showed maximum activity against Bacillus cereus. Ethanol extract exhibited maximum activity against Pseudomonas aeruginosa and Klebsiella pneumoniae. The methanol extract of Murraya koenigii shoots displayed maximum antibacterial activity against Bacillus cereus. Antifungal activity Ethanol extract was more effective against Candida albicans.

Keywords: ethnomedicines, bacteria, fungi, murraya koenigii, antimicrobial activity

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Authors: Gina Leisching, Ray-Dean Pietersen, Carel Van Heerden, Paul Van Helden, Ian Wiid, Bienyameen Baker

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The distinguishing factors that characterize the host response to infection with virulent Mycobacterium tuberculosis (M.tb) are largely confounding. We present an infection study with two genetically closely related M.tb strains that have vastly different pathogenic characteristics. The early host response to infection with these detergent-free cultured strains was analyzed through RNAseq in an attempt to provide information on the subtleties which may ultimately contribute to the virulent phenotype. Murine bone marrow-derived macrophages (BMDMs) were infected with either a hyper- (R5527) or hypovirulent (R1507) Beijing M. tuberculosis clinical isolate. RNAseq revealed 69 differentially expressed host genes in BMDMs during comparison of these two transcriptomes. Pathway analysis revealed activation of the stress-induced and growth inhibitory Gadd45 signaling pathway in hypervirulent infected BMDMs. Upstream regulators of interferon activation such as and IRF3 and IRF7 were predicted to be upregulated in hypovirulent-infected BMDMs. Additional analysis of the host immune response through ELISA and qPCR included the use of human THP-1 macrophages where a robust proinflammatory response was observed after infection with the hypervirulent strain. RNAseq revealed two early-response genes (IER3 and SAA3) and two host-defence genes (OASL1 and SLPI) that were significantly upregulated by the hypervirulent strain. The role of these genes under M.tb infection conditions are largely unknown but here we provide validation of their presence with use of qPCR and Western blot. Further analysis into their biological role under infection with virulent M.tb is required.

Keywords: host-response, Mycobacterium tuberculosis, RNAseq, virulence

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Authors: N. Meziou-Chebouti, A. Merabet, Y. Chebouti N. Behidj

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This work is a contribution to the knowledge of physicochemical characteristics of mature carob followed by evaluation of the activity, antimicrobial phenolics leaves and green pods of Ceratonia siliqua L. physicochemical study shows that mature carob it has a considerable content of sugar (50.90%), but poor in proteins (7%), fat (8%) and also has a high mineral content. The results obtained from phenolic extracts of leaves and green pods of Ceratonia siliqua L. show a wealth leaf phenolic extract especially flavonoids (0,545 mg EqQ/g) relative to the extract of green pods (0,226 mgEqQ/g). Polyphenols leaves have a slightly inhibitory effect on the growth of strains: Staphylococcus aureus, Escherichia coli, Klebsiella pneumoiae, Streptococcus sp and Sanmonella enteritidis, a strong inhibitory effect on the growth of Pseudomonas strain aerogenosa. Moreover, polyphenols pod have a slightly inhibitory effect on the growth of Streptococcus sp strains, Pseudomonas and aerogenosa Sanmonella enteritidis, a slightly inhibitory effect on the growth of Klebsiella pneumoniae strains, E. coli and Staphylococcus aureus.

Keywords: antimicrobial activity, bacteria, clove, Ceratonia siliqua, polyphenols

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Authors: Messaouda Belaid, Salima Kebbouche-Gana, Djamila Benaziza

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Our study focuses on determining the antibacterial activity of some honeys from northern Algeria. To test this activity, the agar well diffusion methods was employed. The bacterial strains tested were Staphylococcus aureus, Bacillus subtilis, Streptococcus faecalis, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeroginosae. The results showed that all the microbes tested were inhibited by all honey used in this study but Those bacteria that appear to be more sensitive to all honey tested are Staphylococcus aureus and Pseudomonas aeroginosae.

Keywords: honey, antibacterial activity, Northern Algeria, Staphylococcus aureus

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Authors: Martynova Alina, Lyubov Buzoleva

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Streptococcus pneumoniae is the causative agent of pneumonias and meningitids throught all the world. Having high genetic diversity, this microorganism can cause different clinical forms of pneumococcal infections and microbiologically it is really difficult diagnosed by routine methods. Also, epidemiological surveillance requires more developed methods of molecular typing because the recent method of serotyping doesn't allow to distinguish invasive and non-invasive isolates properly. Non-coding genome of bacteria seems to be the interesting source for seeking of highly distinguishable markers to discriminate the subspecies of such a variable bacteria as Streptococcus pneumoniae. Technically, we proposed scheme of discrimination of S.pneumoniae strains with amplification of non-coding region (SP_1932) with the following restriction with 2 types of enzymes of Alu1 and Mn1. Aim: This research aimed to compare different methods of typing and their application for molecular epidemiology purposes. Methods: we analyzed population of 100 strains of S.pneumoniae isolated from different patients by different molecular epidemiology methods such as pulse-field gel electophoresis (PFGE), restriction polymorphism analysis (RFLP) and multilolocus sequence typing (MLST), and all of them were compared with classic typing method as serotyping. The discriminative power was estimated with Simpson Index (SI). Results: We revealed that the most discriminative typing method is RFLP (SI=0,97, there were distinguished 42 genotypes).PFGE was slightly less discriminative (SI=0,95, we identified 35 genotypes). MLST is still the best reference method (SI=1.0). Classic method of serotyping showed quite weak discriminative power (SI=0,93, 24 genotypes). In addition, sensivity of RFLP was 100%, specificity was 97,09%. Conclusion: the most appropriate method for routine epidemiology surveillance is RFLP with non-coding region of Streptococcsu pneumoniae, then PFGE, though in some cases these results should be obligatory confirmed by MLST.

Keywords: molecular epidemiology typing, non-coding genome, Streptococcus pneumoniae, MLST

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Authors: Aida Kalantari, Boyang Ji, Tao Chen, Ivan Mijakovic

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3-hydroxypropanoic acid (3-HP) is one of the most important biomass-derivable platform chemicals that can be converted into a number of industrially important compounds. There have been several attempts at production of 3-HP from renewable sources in cell factories, focusing mainly on Escherichia coli, Klebsiella pneumoniae, and Saccharomyces cerevisiae. Despite the significant progress made in this field, commercially exploitable large-scale production of 3-HP in microbial strains has still not been achieved. In this study, we investigated the potential of Bacillus subtilis to be used as a microbial platform for bioconversion of glycerol into 3-HP. Our recombinant B. subtilis strains overexpress the two-step heterologous pathway containing glycerol dehydratase and aldehyde dehydrogenase from various backgrounds. The recombinant strains harboring the codon-optimized synthetic pathway from K. pneumoniae produced low levels of 3-HP. Since the enzymes in the heterologous pathway are sensitive to oxygen, we had to perform our experiments in micro-aerobic conditions. Under these conditions, the cell produces lactate in order to regenerate NAD+, and we found the lactate production to be in competition with the production of 3-HP. Therefore, based on the in silico predictions, we knocked out the glycerol kinase (glpk), which in combination with growth on glucose, resulted in improving the 3-HP titer to 1 g/L and the removal of lactate. Cultivation of the same strain in an enriched medium improved the 3-HP titer up to 7.6 g/L. Our findings provide the first report of successful introduction of the biosynthetic pathway for conversion of glycerol into 3-HP in B. subtilis.

Keywords: bacillus subtilis, glycerol, 3-hydroxypropanoic acid, metabolic engineering

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Authors: Thilivhali Emmanuel Tshikalange, Phiwokuhle Mamba

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Ten medicinal plants used traditionally in the treatment of sexually transmitted diseases (STDs) and urinary tract infections (UTIs) were selected from an ethnobotanical database developed in Mpumalanga. The plants were investigated for their antimicrobial activity against five bacterial strains (Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Neisseria gonorrhoeae and Staphylococcus aureus) and one fungal strain (Candida albicans). Eight of the plants inhibited the growth of all microorganisms at a concentration range of 0.4 mg/ml to 12.5 mg/ml. Acacia karroo showed the most promising antimicrobial activity, with a minimum inhibitory concentration (MIC) of 0.4 mg/ml on Staphylococcus aureus and 0.8 mg/ml on Neisseria gonorrhoeae. All ten plants were further investigated for their antioxidant activities using the DPPH scavenging method. Acacia karroo and Rhoicissus tridentata subsp. cuneifolia showed good antioxidant activity with IC50 values of 0.83 mg/ml and 0.06 mg/ml, respectively. The toxicity of plants was determined using the XTT reduction method against Vero cells. None of the ten plants showed toxicity on the cells. The obtained results confirmed that Acacia karroo and possibly Rhoicissus tridentata subsp. cuneifolia have the potential of being used as antimicrobial agents in the treatment of STDs and UTIs. These results support and validate traditional use of medicinal plants studied.

Keywords: antimicrobial, antioxidant, Neisseria gonorrhoeae, sexually transmitted diseases

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Authors: Belete B. Beyene, Ayenew M. Mihirteu, Misganaw T. Ayana, Amogne W. Yibeltal

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Modification of synthetic porphyrins is one of the promising strategies in an attempt to get molecules with desired properties and applications. Here in, we report synthesis, photophysical characterization and antibacterial activity of 5, 10, 15, 20-tetrakis-(4- methoxy carbonyl phenyl) porphyrin M(II); where M = Co, Fe, Ni, Zn. Metallation of the ligand was confirmed by using UV–Vis spectroscopy and ESI-Ms measurement, in which the number of Q bands in absorption spectra of the ligand decreased from four to one or two as a result of metal insertion to the porphyrin core. The antibacterial activity study of the complexes toward two Gram-positive (Staphylococcus aureus (S. aureus) and Streptococcus pyogenes (s. pyogenes)) and two Gram-negative (Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae)) bacteria by disc diffusion method showed a promising inhibitory activity. The complexes exhibited highest activities at highest concentration and were better than the activity of free base ligand, the salts, and blank solution. This could be explained on the basis of Overton's concept of cell permeability and Tweed's Chelation theory. An increased lipo-solubility enhances the penetration of the complexes into the lipid membrane and interferes with the normal activities of the bacteria. Our study, therefore, showed that the growth inhibitory effect of these metalloporphyrins is generally in order of ZnTPPCOOMe > NiTPPCOOMe > CoTPPCOOMe> FeTPPCOOMe, which may be attributed to the better lipophilicity and binding of the complex with the cellular components.

Keywords: porphyrins, metalloporphyrins, spectral property, antibacterial activity, synthesis

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Authors: Nassima Behidj-Benyounes, Nadjiba Chebouti, Thoraya Dahmane, Amina Henni

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This work examines the study of the antimicrobrial effect of oil extracted from the seeds of Argania spinosa L. (Sapotaceae) in the area of Stida (Mostaganem). This natural substance is extracted by using the Soxhlet. The antimicrobial activity of this oil is evaluated on several microorganisms. It has been tested on five bacterial strains; Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis and Staphylococcus aureus. The extract has been studied by using Candida albicans. It should be noted that these agents are characterized by a high frequency of contamination and pathogenicity. Through this study, we note that these microorganisms are moderately sensitive to the argan oil.

Keywords: Argania spinosa, oil, several microorganisms, almonds, antimicrobial activity

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Authors: Ankita Baidya, Vanishri Ganakumar, Ranveer S. Jadon, Piyush Ranjan, Rita Sood

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Septic embolism can have varied presentations and clinical considerations. Infected central venous catheters are commonly associated with septic emboli but peripheral vascular catheters are rarely implicated. We describe a rare case of septic pulmonary emboli related to infected peripheral venous cannulation caused by an unusual etiological agent. A young male presented with complaints of fever, productive cough, sudden onset shortness of breath and cellulitis in both the upper limbs. He was recently hospitalised for dengue fever and administered intravenous fluids through peripheral venous line. The patient was febrile, tachypneic and in respiratory distress, there were multiple pus filled bullae in left hand alongwith swelling and erythema involving right forearm that started at the site of cannulation. Chest examination showed active accessory muscles of respiration, stony dull percussion at the base of right lung and decreased breath sounds at right infrascapular, infraaxillary and mammary area. Other system examination was within normal limits. Chest X-ray revealed bilateral multiple patchy heterogenous peripheral opacities and infiltrates with right-sided pleural effusion. Contrast-enhanced computed tomography (CECT) chest showed feeding vessel sign confirming the diagnosis as septic emboli. Venous Doppler and 2D-echocardiogarm were normal. Laboratory findings showed marked leucocytosis (22000/mm3). Pus aspirate, blood sample, and sputum sample were sent for microbiological testing. The patient was started empirically on ceftriaxone, vancomycin, and clindamycin. The Pus culture and sputum culture showed Klebsiella pneumoniae sensitive to cefoperazone-sulbactum, piperacillin-tazobactum, meropenem and amikacin. The antibiotics were modified accordingly to antimicrobial sensitivity profile to Cefoperazone-sulbactum. Bronchoalveolar lavage (BAL) was done and sent for microbiological investigations. BAL culture showed Klebsiella pneumoniae with same antimicrobial resistance profile. On day 6 of starting cefoperazone-sulbactum, he became afebrile. The skin lesions improved significantly. He was administered 2 weeks of cefoperazone–sulbactum and discharged on oral faropenem for 4 weeks. At the time of discharge, TLC was 11200/mm3 with marked radiological resolution of infection and healed skin lesions. He was kept in regular follow up. Chest X-ray and skin lesions showed complete resolution after 8 weeks. Till date, only couple of case reports of septic emboli through peripheral intravenous line have been reported in English literature. This case highlights that a simple procedure of peripheral intravenous cannulation can lead to catastrophic complication of septic pulmonary emboli and widespread cellulitis if not done with proper care and precautions. Also, the usual pathogens in such clinical settings are gram positive bacteria, but with the history of recent hospitalization, empirical therapy should also cover drug resistant gram negative microorganisms. It also emphasise the importance of appropriate healthcare practices to be taken care during all procedures.

Keywords: antibiotics, cannula, Klebsiella pneumoniae, septic emboli

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Authors: Aliya Tokbergenova, Maida Tusupbekova, Daulet Dzhangaliyev, Alena Lavrinenko

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Background: Odontogenic phlegmons are ranked the first among pyoinflammatory processes in the frequency of hospitalization in maxillofacial surgery in the post-Soviet countries. The main role in etiology is played by obligate anaerobes and aerobes. According to numerous data, the structure of aerobic pathogens is dominated by staphylococci and gram-negative bacteria. Aim: The research aim is to study the microflora of the purulent discharge odontogenic inflammatory processes. Materials and methods: A total of 220 patients have been examined, of which 120 patients aged 25-59 years have been included in the research who did not have comorbidity hospitalized in the maxillofacial hospital in Karaganda (Kazakhstan) from January 2016 to July 2017. The bacteriological research has been carried out on the basis of the multiaccess laboratory of the KSMU, through the Matrix Assisted Laser Desorption/Ionization (MALDI) apparatus. The material sample was pus from the inflammation focus, taken during the operating period. Results: According to the research among 120 patients (100%), 15 patients (12.5%) have had microorganisms not grown. From 105 (87.5%) bacteriological results, it has been revealed the following 1) Streptococcus: 51 (42.5%): Streptococcus beta-haemolytic: 17 (14.2%), Streptococcus pneumoniae: 12 (10%), Streptococcus anginosus: 8 (6.6%), Streptococcus oralis: 8 (6.6%), Streptococcus constellatus: 6 (5.0%); 2) Staphylococci: 27 (22.5%): Staphylococci aureus: 14 (11.7%) and Staphylococci epidermidis: 13 (10.8%); 3) Pseudomonas aeruginosa: 12 (10%); 4) Neisseria: 11 (9.1%): Neisseria mucosa: 5 (4.1%) and Neisseria macacae: 6 (5.0%); 5) Klebsiella pneumoniae: 2 (1.7%); 6) Stenotrophomonas maltophilia: 2 (1.7%). 15 patients (12.5%) experienced complications in the form of 1) The dissemination of the process in 10 patients (8.4%). 2) Osteomyelitis in 3 (2.5%). 3) Mediastinitis in 1 (0.8%). 4) Sinusitis in 1 (0.8%). 15 patients (100%) were carried out repeated bacteriological examination, the following was revealed: 1) Streptococcus: 10 (66.7%): Streptococcus beta-haemolytic: 4 (26.7%), Streptococcus pneumoniae: 2 (13.3%), Streptococcus аnginosus: 2 (13.3%), Streptococcus oralis: 1 (6.7%), Streptococcus constellatus: 1 (6.7%); 2) Staphylococci: 4 (26.7%): Staphylococci aureus: 3 (20%) and Staphylococci epidermidis: 1 (6.7%); 3) Pseudomonas aeruginosa: 1 (6.7%). Conclusions: Thus, according to our research data, streptococci predominate in the odontogenic processes microflora in aerobic flora in the central Kazakhstan region, which refutes the leading role of staphylococci in the development of odontogenic inflammatory processes, thus creating prerequisites for studying new treatment approaches.

Keywords: maxillofacial surgery, microflora, odontogenic phlegmons, pyo-inflammatory

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Authors: M. Lamamra, H. Laouer, A. Adjaoud, Sahli Farida

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Essential oils can have significant antimicrobial activities and can successfully replace antibiotics that show their ineffectiveness against resistant germs. The chemical composition of the essential oil obtained by hydrodistillation from the aerial part of Daucus aristidis (Apiaceae) at the pre-flowering stage was investigated for the first time, by GC and GC-MS and evaluated for in vitro antimicrobial activity by the disk diffusion method. The Main components of D. aristidis oil were α-pinene (20.13%), cedrol (20.11%), and E- asarone (18.53%). The oil exhibited an antibacterial activity against almost strains tested except for Klebsiella pneumoniae ATCC 700603 K6 and Enterococcus faecalis ATCC 49452, the oil of D. aristidis had no activity against all fungi tested.

Keywords: α-pinene, antimicrobial activity, Daucus aridtidis, essential oil

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