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Julie’s Biggest TakeawaysNosocomial infections are a type of opportunistic infection: one that wouldn’t normally cause disease in healthy individuals. Once the immune system is compromised due to other infection or treatment, the opportunist bacteria take advantage of the conditions to grow to higher numbers and cause disease.
How are different pathogens transmitted in the hospital? Previously, transmission was considered to occur from one patient to a second patient, perhaps via a healthcare worker. When patients from very different parts of the hospital began to come down with the same resistant strain of bacteria, without interacting through the same space or staff, researchers began to look at a different reservoir: the hospital wastewater.
How does the bacteria get from the sink to the patients? The bacteria, existing in a biofilm in the pipe right below the drain, can be transferred in droplets when the water is run. These droplets can fall as far as 36 inches from the drain plate and can contaminate the sink bowl or patient care items next to the sink.
Some of the solutions to decrease bacterial dispersion from hospital sinks are very simple: for example, offsetting the drain from the tap, which keeps the water from directly running onto the drain, helps decrease the force with which the water hits the drain and therefore decreases bacterial dispersion.
The Sink Lab at University of Virginia couldn’t replicate the bacterial growth patterns seen in the rest of the building; in particular, there were fewer protein nutrients that promoted bacterial growth. By setting up a camera observation of sink stations used in the hospital, the team realized that the waste thrown down the sink (extra soda, milk, soup, etc) was feeding the microbial biofilm. This helps the CRE in the biofilms in the sinks thrive.
“A lot of times, hospital acquired infections are carried in the gut of patients who are in the hospital and don't cause any problems for the patient and are carried on or the skin of these patients. Because the bacteria are there in a vulnerable patient, the bacteria takes advantage of the opportunity — either a break in the skin or the patient being on a ventilator and not being up and mobile — to get into the wrong spot and start multiplying and causes an infection of varying severity.”
“When I started thinking about wastewater in 2013, there were about 30 publications describing hospital drains or wastewater potentially at play with drug resistant pathogens in the hospital. Now there’s over 100 and counting. I think a lot of that has happened because sequencing means we can make these links more definitively.”
“I worry a lot about creating new bacteria in that biofilm with sort of the perfect storm — the antibiotics in wastewater of hospital systems, potential nutrients, water — that selects for more antibiotic resistance.”
“I think Klebsiella is a very interesting pathogen and opportunistic. It’s an opportunist for the modern world...because it’s good at picking up antibiotic resistant genes, because it’s good at causing many types of infections, and because it’s good at colonizing human hosts, it’s a pathogen for the modern era.”
“I really do favor treating antibiotics as a precious resource, almost like a carbon-type approach to the world: that there’s a finite amount. We really, really need antibiotics for patients that need them.”
Links for This Episode
- MTM Listener Survey, only takes 3 minutes. Thanks!
- Amy Mathers website at University of Virginia
- The Sink Lab at UVA
- Kotay SM et al. Droplet- Rather than Aerosol-Mediated Dispersion is the Primary Mechanism of Bacterial Transmission from Contaminated Hand-Washing Sink Traps. Applied and Environmental Microbiology. 2018.
- Mather AJ et al. Klebsiella quasipneumoniae Provides a Windo into Carbapenemase Gene Transfer, Plasmid Rearrangements, and Patient Interactions within the Hospital Environment. Antimicrobial Agents and Chemotherapy. 2018.
- Kotay S et al. Spread from the Sink to the Patient: in situ Study Using Green Fluorescent Protein (GFP)-Expressing Escherichia coli to Model Bacteral Dispersion from Hand-Washuing Sink-Trap Reservoirs. Applied and Environmental Microbiology. 2016.
History of Microbiology Tidbit
For today’s history of microbiology tidbit, I want to discuss the history of visualizing microbes. There have been all sorts of milestones in microscopy worthy of highlight on our show, but I want to go the simplest route and examine negative staining especially when performed using India Ink. Why? I think most microbiology lab courses still teach a number of different staining techniques, from Gram staining to acid-fast staining, and I remember looking through the microscope at a slide stained with India Ink to differentiate different gram-negative bacilli in my first microbiology lab class. Because Klebsiella has the polysaccharide capsule that Amy mentioned, it excludes the India Ink and you can see a clear ring around the bacterial cell. That dark liquid being excluded is India Ink.
India Ink has been used in India since at least the 4th century BCE, when it was made of a mixture of hide glue, carbon black, lampblack, and bone black pigments ground with a mortar and pestle. Of course, 2400 years ago, people weren’t using India ink for microscopy, but this substance has a lot of other uses, including ink in the more traditional sense, used on paper or for the black ink in tattoos. India ink has even been used to tattoo corneas of people with opaque corneas due to keratitis or cataracts! Today, India ink is more commonly composed of the molecule nigrosin or acid black 2 and is one of the ingredients in artist pens, such as those sold by Faber-Castell. India ink plays additional scientific roles too, for visualizing blood vessels and labeling tissues, in part because the ink can be fixed using acetic acid and then survive tissue processing.
The negative staining procedure works by both size exclusion, as the stain is too large to intercalate the polysaccharide strands of the capsule, and by charged interactions. Nigrosin, a mix of synthetic black dyes, is an acidic stain, which means in a neutral pH, it will give up a proton and become negatively charged. The negative charge is repelled by the bacterial cell surface; both the lipid membrane and the polysaccharide capsule are negatively charged.
All of this background will help you understand why a new procedure for negative staining introduced in 1991 was able to work. In an article published in Applied and Environmental Microbiology in 1991, Steven Woeste and Paul Demchick proposed a new version of the negative stain. The suggested using a very similar protocol to traditional negative staining: the bacteria were first air dried and heat fixed onto a glass slide. Then, instead of applying the india ink in a drop-wise fashion, they applied a single coat of a blunt-tipped black marker, brushed lightly over the smear. They tested a variety of water-soluble and water-resistant ink markers, and found that the best marker was the Mighty Mark 7000, made by Faber Castell.
Sadly, these markers are no longer sold by Faber Castell, but hopefully you’ve put 2 and 2 together to realize that rather than discovering a new dye, these two scientists found a new way to apply the same India ink using a marker instead of a dropper! I should add that developing a new technique, especially one that introductory students often have trouble with due to flooding their slides with too much ink, is always a valuable addition and this marker-negative-stain technique seems to have been picked up by a number of teaching labs at the time, from what I can tell.
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