Modifikasi Medium Menggunakan Saline-Water Soluble Fraction (SSF) atau Fraksi Minyak Terlarut untuk Menumbuhkan Bakteri Pendegradasi Hidrokarbon

Isolasi bakteri pendegradasi hidrokarbon memerlukan teknik yang baik dan nutrisi optimal untuk pertumbuhannya. Kendala dalam pembuatan medium dan pengamatan isolat bakteri yang mengandung hidrokarbon sering terjadi di laboratorium, sehingga dibutuhkan teknik pengembangan metode dalam proses isolasi bakteri pendegradasi hidrokarbon. Penelitian ini bertujuan untuk mengetahui dan membandingkan jumlah dan jenis koloni bakteri yang tumbuh dalam medium Zobell+saline-water soluble fraction (SSF) dibandingkan dengan medium Zobell+minyak bumi. Metode penelitian yaitu mengisolasi bakteri menggunakan medium Zobell+SSF 6 jam (A), 12 jam (B), 24 jam (C) dan sebagai kontrol adalah medium Zobell+1% v/v minyak bumi (K), perhitungan bakteri menggunakan metode TPC dan mengidentifikasi bakteri dengan alat VITEK-MS. Hasil penelitian memperlihatkan bahwa jumlah bakteri yang tumbuh pada medium Zobell+SSF 24 jam (C) adalah 6.9 x 108 CFU/ml, hal ini menunjukkan lebih baik dibandingkan dengan Zobell+1% v/v minyak bumi (K)=5.2 x 108 CFU/ml, medium Zobell+SSF 12 jam (B)=6.6 x 107 CFU/ml dan medium Zobell+SSF 6 jam (A)=1.8 x 107 CFU/ml.Kesimpulan penelitian ini adalah bahwa dari segi jumlah total bakteri medium modifikasi Zobell+SSF pengadukan selama 24 jam (C) lebih baik dalam menumbuhkan bakteri pendegradasi hidrokarbon dibandingkan dengan pengadukan 6 jam (A), 12 jam (B), dan medium Zobell+1% v/v minyak bumi (K). Sedangkan apabila berdasarkan dengan keragaman bakteri, didapatkan hasil bahwa strain bakteri yang terisolasi pada medium modifikasi Zobell+SSF perlakuan pengadukan 6, 12, 24 jam sama dengan strain bakteri yang tumbuh pada kontrol (medium Zobell +1% v/v minyak bumi. Bakteri yang teridentifikasi sebagai bakteri pendegaradsi hidrokarbon adalah bakteri Klebsiella pneumoniae, Enterobacter asburiae/Enterobacter cloacae dan Pseudomonas aeruginosa.

Aislamiento microbiológico de superficies inanimadas en contacto con pacientes en un hospital peruano

Objetivo: describir el perfil microbiológico de las superficies inanimadas en contacto con el paciente en un hospital nivel III de la seguridad social de Chiclayo, Perú. Material y métodos: Se realizó un estudio transversal, con los datos de los informes del Control microbiológico cualitativo de ambientes físicos de 5 servicios de un Hospital de Chiclayo nivel III del Perú. El método para la identificación de microorganismos fue el sistema automatizado VITEK MS. Se presentan análisis descriptivos como frecuencias y porcentajes. Resultados: Se reportaron un total de 177 aislamientos, de los cuales 97,74% (173) fueron positivos, de estos, el 50,87% (88) estuvo conformado por bacilos gramnegativos, siendo el microorganismo más aislado Acinetobacter baumannii (17 muestras) seguido de Rhizobium radiobacter (16) y Sphingomonas paucimobilis13. Conclusiones: El ambiente hospitalario se encuentra altamente contaminado, siendo la mayoría microorganismos patógenos. Estos resultados guardarían relación con el prolongado tiempo de vida de los microorganismos en las superficies inertes y el proceso de limpieza y desinfección del ambiente hospitalario, por lo que la evaluación de su eficacia y el posible desarrollo de nuevas y mejores técnicas de limpieza deben ser motivo de investigación.

A multi-country study using MALDI-TOF mass spectrometry for rapid identification of Burkholderia pseudomallei

Background Burkholderia pseudomallei is the bacterial causative agent of melioidosis, a difficult disease to diagnose clinically with high mortality if not appropriately treated. Definitive diagnosis requires isolation and identification of the organism. With the increased adoption of MALDI-TOF MS for the identification of bacteria, we established a method for rapid identification of B. pseudomallei using the Vitek MS, a system that does not currently have B. pseudomallei in its in-vitro diagnostic database. Results A routine direct spotting method was employed to create spectra and SuperSpectra. An initial B. pseudomallei SuperSpectrum was created at Shoklo Malaria Research Unit (SMRU) from 17 reference isolates (46 spectra). When tested, this initial SMRU SuperSpectrum was able to identify 98.2 % (54/55) of Asian isolates, but just 46.7 % (35/75) of Australian isolates. Using spectra (430) from different reference and clinical isolates, two additional SMRU SuperSpectra were created. Using the combination of all SMRU SuperSpectra with seven existing SuperSpectra from Townsville, Australia 119 (100 %) Asian isolates and 31 (100 %) Australian isolates were correctly identified. In addition, no misidentifications were obtained when using these 11 SuperSpectra when tested with 34 isolates of other bacteria including the closely related species Burkholderia thailandensis and Burkholderia cepacia. Conclusions This study has established a method for identification of B. pseudomallei using Vitek MS, and highlights the impact of geographical differences between strains for identification using this technique.

Developing Two Rapid Protein Extraction Methods Using Focused-Ultrasonication and Zirconia-Silica Beads for Filamentous Fungi Identification by MALDI-TOF MS

Filamentous fungi identification by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been challenging due to the lack of simple and rapid protein extraction methods and insufficient species coverage in the database. In this study, we created two rapid protein extraction methods for filamentous fungi: a one-step zirconia-silica beads method (ZSB) and a focused-ultrasonication method (FUS). The identification accuracy of two methods were evaluated with the VITEK MS, as well as number of spectra peaks and signal-to-noise ratio (S/N) with M-Discover 100 MALDI-TOF MS compared to the routine method. The better method was applied to build a filamentous fungi in-house spectra library for the M-Discover 100 MS, and then another one and routine method were performed in parallel to verify the accuracy and commonality of the in-house library. Using the two optimized methods, the dedicated operating time before MALDI-TOF MS analysis was reduced from 30 min to 7 (ZSB) or 5 (FUS) min per sample, with only a few seconds added for each additional strain. And both two methods identified isolates from most mold types equal to or better than the routine method, and the total correct identification rate using VITEK MS was 79.67, 76.42, and 76.42%, respectively. On the other hand, the two rapid methods generally achieved higher maximum and minimum S/N ratios with these isolates tested as compared to the routine method. Besides, the ZSB method produced overall mean of maximum and minimum S/N ratio higher than that by FUS. An in-house library of M-Discover MS was successfully built from 135 isolates from 42 species belonging to 18 genera using the ZSB method. Analysis of 467 isolates resulted in 97.22% correctly identified isolates to the species level by the ZSB method versus 95.50% by the routine method. The two novel methods are time- and cost-effective and allow efficient identification of filamentous fungi while providing a simplified procedure to build an in-house library. Thus, more clinical laboratories may consider adopting MALDI-TOF MS for filamentous fungi identification in the future.

Molecular Characterization and Biofilm Formation Study of Contaminant Bacteria Isolated from Domiaty and Hungarian Cheeses in Jeddah City

The aim was to study the microbiological quality of Domiaty and Hungarian cheeses, molecular identification and biofilm formation of some selected contaminant bacteria. Samples were collected from two M and P big markets in Jeddah City through the period from February to October 2018, nine visits for two types of natural cheese. Results showed that the total bacterial counts (CFU/ml) from Domiaty cheese from two markets (M and P) were 0.1 x 105, 8 x 105 and 1 x 10 5 CFU/ml respectively (3 visits of M market) and 4 x 106, 0.4 x 106, 6.5 x 103, 1 x 103, 0.1 x 103 and 0.1 x 103 CFU/ml respectively (six samples from 6 visits from P market). Results showed that the total bacterial counts (CFU/ml) from Hungarian cheese were 1.5 x 10 5, 1x 10 4, 11 x 10 4 and 4 x10 6 CFU/ml respectively from (4 visits of M market) and 0.18 x 104, 3 x 106, 22 x 106, 6 x 106 and 5 x 104 CFU/ml respectively (5 visits from P market).Different bacterial isolates from cheese were identified by morphology and biochemical test. Bacterial isolates from cheeses were identified by VITEK MS as follow: Serratia liquefaciens (D6-1, D6-2, D14-1, D13-1 and D13-2), and Pseudomonas fluorescens (D14-2) were isolated from Domiaty cheese while Enterococcus faecium (H11-2), Serratia liquefaciens (H15-1) and Streptococcus thermophilus (H14-1) were isolated from Hungarian cheese. Some selected bacterial isolates were identified by 16S rRNA. Isolates were belong to MK757978 (Raoultilla terrigena (D15-1)), MK757979 (Bacillus cereus (D16-1)), MK757980 (Enterococcus faecalis (H10-2)), MK757982 (Enterococcus fiscalism (H11-1)), MK757981 (Serratia liquefactions (H13-1)), MK757984 (Anoxybacillus flavithermus (H17-1). All bacterial isolates have been tested for the formation of biofilm using a Tissue Culture Plate (TCP). Results revealed 12.5% and 46.15% of high biofilm formation respectively for bacterial isolates of Domiaty and Hungarian cheeses.