Clostridium botulinum Type E Toxins Bind to Caco-2 Cells by a Different Mechanism from That of Type A Toxins

Cultured Clostridium botulinum strains produce progenitor toxins designated as 12S, 16S, and 19S toxins. The 12S toxin consists of a neurotoxin (NTX, 7S) and a non-toxic non-hemagglutinin (NTNH). The 16S and 19S toxins are formed by conjugation of the 12S toxin with hemagglutinin (HA), and the 19S toxin is a dimer of the 16S toxin. Type A cultures produce all 3 of these progenitor toxins, while type E produces only the 12S toxin. The 7S toxin is cleaved into heavy (H) and light (L) chains by a protease(s) in some strains, and the H chain has 2 domains, the N-terminus (Hn) and C-terminus (Hc). It has been reported that type A toxins bind to the intestinal cells or cultured cells via either HA or Hc. In this study, we investigated the binding of type A and E toxins to Caco-2 cells using Western blot analysis. Both the type E 7S and 12S toxins bound to the cells, with the 7S toxin binding more strongly, whereas, in the type A strain, only the 16S/19S toxins showed obvious binding. Pre-incubation of the type E 7S toxin with IgG against recombinant type E Hc significantly inhibited the 7S toxin binding, indicating that Hc might be a main binding domain of the type E toxin

Positivity of ExoU Gene of Type III Secretion System and Fluoroquinolone Resistance of Psedomonas aeruginosa from Sputum of Nosocomial Pneumonia Patients in Sanglah Hospital, Bali

Pseudomonas aeruginosa is one of the Gram-negative rods bacteria that frequently cause nosocomial pneumonia. One of the main virulent effector proteins on Type III secretion system (TTSS) of P. aeruginosa is Exoenzyme U ( ExoU). ExoU works as a phospholipase A2 activity and exhibits lung tissue injury effect in pneumonia. As an antibiotic that has activity against P. aeruginosa, fluoroquinolone resistance has increased as many as three fold since the last decade. Infections caused by P. aeruginosa that are fluoroquinolone resistant and positive for ExoU gene show worse clinical outcome. The aim of this study was to determine the positivity of ExoU gene TTSS and fluoroquinolone resistance of P. aeruginosa that isolated from sputum of nosocomial pneumonia patients in Sanglah Hospital, Bali. P. aeruginosa isolated from sputum of patient that diagnosed as nosocomial pneumonia, isolates had been identified phenotypically by Vitek2 Compact system (bioMérieux, Inc., Marcy-l'Etoile - France), and then continued by genotypic detection by PCR. The susceptibility testing of P. aeruginosa isolates to Ciprofloxacin were conducted by Vitek2 Compact, whereas ExoU genes were detected by PCR. Fifty-three P. aeruginosa isolates were identified in this study, in which 35 isolates (66.1%) had ExoU gene and 22 isolates (41.5%) were resistant to Ciprofloxacin. Based on nosocomial pneumonia type, the highest proportion of isolates genotipically ExoU+ and phenotypically Ciprofloxacin were on VAP group accounted for 57.1% and 54.5%, respectively. Chi-square analysis showed significant correlation between Ciprofloxacin resistance and ExoU gene (p=0.001). As a conclusion, the positivity of ExoU+ isolates were more likely found in Ciprofloxacin resistant group

Phospholipase C Produced by Clostridium botulinum Types C and D:Comparison of Gene, Enzymatic, and Biological Activities with Those of Clostridium perfringens Alpha-toxin

Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival

Application of Purified Botulinum Type A Neurotoxin to Treat Experimental Trigeminal Neuropathy in Rats and Patients with Urinary Incontinence and Prostatic Hyperplasia

Type A neurotoxin (NTX) of Clostridium botulinum was purified by a simple procedure using a lactose gel column. The toxicity of this purified toxin preparation was retained for at least 1 year at −30°C by supplementation with either 0.1% albumin or 0.05% albumin plus 1% trehalose. When purified NTX was used to treat 49 patients with urinary incontinence caused by either refractory idiopathic or neurogenic detrusor overactivity, 36 patients showed significant improvement in symptoms. These beneficial effects were also observed in cases of prostatic hyperplasia. The results obtained with NTX were similar to that of Botox. The effects of NTX on trigeminal neuralgia induced by infraorbital nerve constriction (IoNC) in rats were also studied. Trigeminal ganglion neurons from ipsilateral to IoNC exhibited significantly faster onset of FM4-64 release than sham-operated contralateral neurons. Intradermal injection of NTX in the area of IoNC alleviated IoNC-induced pain behavior and reduced the exaggerated FM4-64 release in trigeminal ganglion neurons

Antibiotyping, RAPD- and ERIC-PCR fingerprinting of Klebsiella pneumoniae clinical isolates at a tertiary reference hospital in Denpasar, Bali, Indonesia

Background and Objectives: Klebsiella pneumoniae is a healthcare-associated infections agent and could be an extended spectrum β-lactamase (ESBL) producer. Understanding the transmission of this bacterium in a hospital setting needs accurate typing methods. An antibiogram is used to detect the resistance pattern of the isolates. Random Amplified Polymorphic DNA (RAPD) and Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR are rapid, technically simple, and easy-to-interpret DNA typing methods. This study aimed to evaluate the use of antibiotyping, RAPD-, and ERIC-PCR to investigate the heterogeneity of K. pneumoniae isolated from clinical specimens. Materials and Methods: The antibiograms of 46 K. pneumoniae clinical isolates were determined by Vitek® 2 Compact. All isolates underwent RAPD-PCR using AP4 primer and ERIC-PCR using ERIC-2 primer. The dendrogram was generated using the GelJ software and analyzed to determine its similarity. The analysis of antibiogram and the molecular typing diversity index was calculated using the formula of the Simpson’s diversity index. Results: About 71.7% of the isolates were ESBL-producers, and more than 80% of isolates were susceptible to amikacin, ertapenem, and meropenem. The antibiotyping produced 32 diverse types with DI = 0.964. In Conclusion: Antibiotyping, RAPD- and ERIC-PCR showed powerful discrimination power among the isolates, supported the diversity of K. pneumoniae isolates in current study. These combination could be promising tools for clonal relationship determination, including in tracking the transmission of the outbreak’s agent in hospital setting

POLA KEPEKAAN Methicillin-Resistant Staphylococcus aureusTERHADAP ANTIBIOTIKA DI RSUP SANGLAH PADA AGUSTUS 2013 - OKTOBER 2013

Staphylococcus aureus merupakan bakteri gram positif yang dapat menyebabkan beragam penyakit mulai dari yang ringan seperti infeksi kulit hingga penyakit yang dapat membahayakan nyawa seperti pneumonia, meningitis dan toxic shock syndrome. Kemampuan adaptasi S.aureus terhadap antibiotika menimbulkan peningkatan resistensi antibiotika oleh S. aureus. Pada masa kini, prevalensi Methicillin-Resistant Staphylococcus aureus (MRSA) cenderung meningkat tidak hanya di lingkungan rumah sakit, yang disebut hospital-associatedMRSA (HA-MRSA),tetapi juga di komunitas, yang disebut dengan community-acquired MRSA (CA-MRSA).Uji kepekaan isolat MRSA terhadap antibiotikadibutuhkan untuk menyediakan suatu pola kepekaan yang dapat digunakan sebagai referensidalampemilihan antibiotika yang lebih rasional.Penelitian ini menggunakan metode potong lintang dimana sampel penelitian adalah isolat MRSAyang diidentifikasi dengan Vitek 2 (Biomérieux) di Laboratorium Instalasi Mikrobiologi Klinik RSUP Sanglah. Dari 41 isolatS. aureusyang terisolasi pada periode Agustus hingga Oktober 2013, terdapat 6 sampel (14,6%) yang merupakan MRSA. MRSA terisolasilebih sensitifterhadap antibiotika quinupristin/dalfopristin, linezolide, vancomycin, tigecycline dan nitrofurantoin, namun resisten terhadap antibiotika penicillin, cephalosporin dan carbapenems. Penelitian seperti ini penting untuk dilaksanakan secara berkelanjutan di masa yang akan datang dengan sampel dan metode identifikasi MRSA yang lebih baik seperti latex agglutination test atau dengan Polymerase Chain Reaction (PCR).   </p

In-House RT-PCR Assay for Detection of Human Immunodeficiency Virus Type 1 Infection

Serologic assays are commonly used for screening (ELISA) and for confirmation (Western blot) of HIV-1 infection; however, both assays have potentially yielded the false-positive or false-negative results. In this study, a diagnostic RT-PCR assay as an alternative test for detection of HIV-1 was developed. Forty-six plasma specimens from highly risky groups, who visited a voluntary counseling and testing for HIV (VCT) in Sanglah Clinic of General Hospital, Denpasar, Bali, were tested by RT-PCR assay with specific primers for Pol region of HIV-1 genome. The results of the RT-PCR tests were then compared with those of serologic tests to obtain the sensitivity and specificity of RT-PCR assay. The results of this study showed that the RT-PCR assay could detect 17 (sensitivity: 65.4%) of 26 serologically positive specimens was unexpectedly able to detect 2 (specificity: 90%) of 20 serologically negative specimens. Thus, the RT-PCR assay developed in this study is potential to be used as an alternative test, even though there are numerous aspects, particularly the sensitivity, that need to be improved in further research