Detection of Spore Germination for Sterilization Processes

by Florine C. Cleary

freshman at Moses Brown High School Providence, Rhode Island

In the Headlines
US$ 9000 8000 7000 per patient 6000 5000 4000 3000 2000 1000 0

“No population is more vulnerable to multi drug-resistance than those admitted to hospital wards”. “Of the resistant organisms now proliferating around the world, none carry more potential for destruction and threaten existing medical interventions than the emergence of hospital-acquired "super-infections". “In the United States alone, some 14,000 individuals are infected and die each year from drug-resistant microbes picked up in hospital”.
Source: World Health Organization's Report on Infectious Disease


1st line 2nd line 3rd line

Source: Farmer et al. The Global Impact of Drug Resistant TB, Harvard Med School and Open Society Institute: pp. 168,1999

“So far, current preventive methods emphasizing hygiene and aggressive infection-control measures have reaped only dubious benefits and at best, only slowed the spread of resistant bacteria.”
“This means that commonplace medical procedures once previously taken for granted – hip replacements, dental surgery and cyst removals – could conceivably be consigned to medical limbo. The repercussions are almost unimaginable.” “An added concern is that hospitalacquired infections rarely stay put. Ample evidence would suggest that many resistant infections erupted in hospital settings before migrating to the community at large.”
Source: World Health Organization's Report on Infectious Disease

Percentage

35 30 25 20 15 10 5 0

89 90 91 92 93 94 95 96 97

Source: ReacherMH at al. BMI 2000,320: 213-216

A Global Problem
• “Inadequately cleaned equipment is also a major determinant in the spread of infectious disease.” “In one study, researchers surveying health clinics in United Republic of Tanzania discovered that some 40% of presumed sterile reusable needles and syringes were contaminated with bacteria.” “Inadequate training, monitoring and education on basic hygiene has serious implications, not only for the hospital population itself, but also for the community at large.”
Source: World Health Organization's Report on Infectious disease.

Hospitals are a breeding ground for antibiotic resistant bacteria.
Costs (US$) include:
- USA = $10 billion per year - Mexico = $450 million per year - Thailand = $40 million per year
Source: World Health Organization/CDS. Data from published sources

The Reason
With the number of antibiotic-resistant strains rising dramatically, the emergence of new virulent strains, and concerns about food contamination, it is clear that something must be done to improve current methods of determining the presence of bacteria. The current methods are subject to human error and often require days to culture samples - days that some facilities don’t have, nor do they have the ability to keep the equipment sterile while they wait for the results. So often they don’t. In view of the risk to human life, this uncertainty is unacceptable.

Goals
1) To find faster, more reliable methods for determining bacterial contamination. 3) To develop technology to use these new methods for easy and quick detection of bacteria.

The Science
• All living cells need to perform respiration to produce energy from food. The first step in cellular respiration is glycolysis where glucose [sugar] is converted to pyruvate and energy is released. Once glycolysis has begun, there are ten steps with products [metabolites] produced along the way. Two of these metabolites are NADH (reduced nicotinic adenine dinucleotide) and FP (oxidized flavoproteins).

How We Use It
• live cells perform respiration, producing NADH and FP both of these metabolites fluoresce
input light

fluorescence light is given off at a different color than the input light

color of input light needed and color of fluorescence depends on the chemical composition of the metabolite each metabolite fluoresces at a different color

fluorescence

metabolite fluorescence can be used to determine the presence of bacterial contamination

sample

Light Microscopy Bacterial Cells
X 1000

NADH fluorescence

flavoproteins fluorescence

white light

Confocal Microscope
• • clearer, better-defined image observe the structure of cells
out-of-focus fluorescence is eliminated by a shallow depth-of-field successive views along the Z-axis allows construction of 3-D models of the sample

ideal tool for quantitative studies of the fluorescence of cells

laser beam, special optics, and high signal-to-noise ratio of the photodetector provide the capability for sensitive and quantitative measurements

Confocal Microscopy Bacterial Cells
combined

flavoproteins fluorescence µm porphyrins fluorescence

680nm fluorescence

Germination of Bacterial Spores
140 120 100

flavoproteins porphyrins 680nm

8-bit gray scale

80 60 40 20 0 0

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minutes after addition of minimal media

Applications
This method can be used to determine • • • • • effectiveness of sterilization and decontamination procedures presence of infection in spinal fluid and urine abnormal cell function food and water contamination presence of extraterrestrial life and life in extreme environments

Future Efforts
• • study metabolite behavior during sporulation and cell death design and build a device (about the size of a flashlight) which uses these methods to detect bacteria test this device in a variety of conditions where bacterial contamination is present