Sequence the gene that codes for 16S ribosomal RNA (rRNA)

My Experiment

The objective of my experiment was to sequence the gene that codes for 16S ribosomal RNA (rRNA) from a patient and to use that sequence to identify the bacteria present. The sample was grown on a solid medium culture dish, digested — to dissolve the cell wall — and centrifuged to collect the DNA. The collected DNA was prepared and inserted into a Polymerase Chain Reaction (PCR) machine to produce many copies of the desired piece of DNA. The entire PCR product was purified by treating it with a buffer solution and placing it into a microconcentrator. To sequence the purified PCR product, twelve primers were used (6 for each strand of DNA), which reduces error by creating redundant data. Each of the twelve tubes contains DNA polymerases, buffers, nucleotides, fluorescence-tagged terminators, a different primer, and the purified PCR product. These tubes are again placed into the PCR machine but this time, the products are copies of the DNA with variable length. The complete order of the DNA is determined by separating the contents of each tube by gel electrophoresis, sequencing them using an automated sequencer and putting every sequence together. Using a BLAST search, the nucleotide sequence was identified as the bacteria Salmonella Typhimurium.

Annotation #1

Kumar S, Rizvit M and Berry N (2008). Rising prevalence of enteric fever due to multidrug-resistant Salmonella: an epidemiological study. J Med Microbiol 57: 1247-1250

The purpose of this study was to investigate the growth of salmonella species in various growth inhibiting mediums. The study was conducted from January to December of three non-consecutive years, 1999, 2002 and 2005.

Patients in the New Delhi hospital, Lok Nayak, developing enteric fever were sampled for blood before administration of antibiotics. Blood samples of 5-10 ml from adults and 2-3 ml from children where directly placed into bottles containing a highly nutritious brain heart infusion broth. Sixteen different antibiotics were used at various amounts ranging from 5 to 30µg. The Kirby-Bauer procedure was used to test antibiotic resistance. Many bacteria produce enzymes called extended spectrum β-lactamase (ESBL) which break down antibiotics rendering them useless.

Of all the patients that took part in the study, 174 (3.1%) contained bacteraemia due to Salmonella subspecies. Most (69%) of the incidences of enteric fever occurred in the hotter months of April, May and June and in the monsoon season (July-September). In 52.9% of the occurrences, Salmonella Typhi (S. Typhi) showed resistance to ampicillin, chloramphenicol and co-trimoxazole. This multidrug resistance rose from 34% of incidences in 1999 to 66% in 2005. Ampicillin did not work 100% of the time towards S. Typhimurium. Over the years studied, there has been a significant increase of resistance to amoxicillin/clavulanic acid, chloramphenicol, co-trimoxazole, ampicillin, ofloxacin, ciprofloxacin and incremental resistance for the remainder of drugs. The amount of ESBL producing cases rose from none in 1999 to 8% in 2005 for S. Typhi. In treating typhoid fever, ceftiaxone was much less effective than ciprofloxacin; when ciprofloxacin was used first (42% of the time) no treatment failure occurred. Other than S. Typhi, no other Salmonella serogroups observed triple drug resistance.

The authors' conclusion about the complete resistance of S. Typhimurium toward ampicillin and the use of sparfloxacin and ofloxacin over ciprofloxacin and cephalosporins will be cited in my discussions to explain which antibiotic would be best to treat a patient with S. Typhimurium.

Annotation #2

Migard S and Flandrois JP (2006). 16S rRNA sequencing in routine bacterial idenentification: A 30-month experiment. J Microbiol Methods 67: 574-581

The authors' aim in this study was to evaluate the use of identifying bacteria, which is normally difficult to do using conventional methods, with 16S rDNA sequencing. The ribosome's small sub unit is universal among bacteria but contains regions of with specific variable. This makes identification of bacteria possible.

From June 2002 to December 2004, 683 bacterial strains were sequenced from blood cultures, urine cultures, broncho-pulmonary specimens and from other normally sterile body sites. These bacteria all have not yet been identified by phenotypic methods. Samples were plated onto trypticase soy agar, 5% sheep blood agar or chocolate agar. DNA present in these cultures was extracted using the Relman technique. The DNA was denatured for 3 min at 95°C then 40 cycles of amplification of 1 min at 94°C, 1 min at 55°C and 2 min at 72°C. Using the primers 91E (5'-GGAATTCAAAKGAATTGACGGGGGC-3') and 13B (5'-CGGGATCCCAGGCCCGGGAACGTATTCAC-3') the 16S rDNA portion corresponding to Escherichia coli positions 930 to 1370 were amplified. These fragments DNA were sequenced using only one strand (for financial reasons) by a firm called Biofidal in France. Using the sequences available in the BIBI Database, the identity of the 16S rDNA sequences of our isolates were identified. The phylogenies of the species were taken into account, and not only the percentage similarity of the sequences and sequence length.

Of the 683 isolates, 592 were rod bacteria (411 Gram-positive and 181 Gram-negative) and 91 cocci (83 Gram-positive and 8 Gram-negative). Sequencing the 16S rDNA made it possible to identify 568 samples to the species level and 108 to the genus level — which are sublevels of different families of taxonomic groups — with 7 samples remaining unidentified even to the genus level. But 4 of the 7 unidentified isolates were assigned a genus level using a more up-to-date database, and the remaining 3 were not because their sequences were not in the database. Within the gram- negative rods, 134 (74%) were identified unambiguously to the species level, and within the gram-positive rods, 353 (85.8%) were assigned to a species. Among the gram-negative cocci, 6 (75%) and 75 (90.3%) among the gram-positive were assigned to the species level. For 73 (67%) samples of the ones with only genus identification, no distinction was possible since their sequence was highly correlated with more than two species in the same genus. For 13 isolates, the quality of the sequence was not reputable, which could have been fixed by sequencing both strands. For 14 isolates, designation to a species was difficult since the sequence had not been entered into the database. The authors' found that analysis of the 16S rDNA sequence provides better labelling to the species level of both Gram-positive cocci and Gram-positive rods.

The authors' statement that identification of bacteria based on 16S rDNA sequence is universal and also variable in some regions will be cited in my discussion to explain why we sequenced that part of the genome in the beginning.

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