GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA
Beating the Invisible Bugs
By Kathy Dix
“GRAM-NEGATIVE = A common class of bacteria normally found in the gastrointestinal tract that can be responsible for disease in man (sepsis). Bacteria are considered gram-negative because of their characteristic staining properties under the microscope, where they either do not stain or are decolorized by alcohol during Gram’s method of staining. This is a primary characteristic of bacteria that have a cell wall composed of a thin layer of peptidoglycan covered by an outer membrane of lipoprotein and lipopolysaccharide containing endotoxin.1
GRAM-POSITIVE = Bacteria that retain the stain or that are resistant to decolorization by alcohol during Gram’s method of staining. This is a primary characteristic of bacteria whose cell wall is composed of a thick layer of peptidologlycan containing teichoic and lipoteichoic acid complexed to the peptidoglycan.”1
Bacteria — ubiquitous in every location but a sterile room — bear characteristics that make each type distinct from each other. They may be gram-negative or gram-positive, aerobic or anaerobic, cocci, or rods.
Their location may even determine their potency; for example, bacteria often found in the human gastrointestinal (GI) tract may be harmless in that location, but when they infect a cut on the skin, could prove fatal.
Each bacteria type has a different preferred growth site, or hiding place. There may be different requirements to kill each type. Some grow more easily on very moist surfaces, while others get by in medium humidity. Healthcare workers (HCWs) will find different types of bacteria in various areas of a hospital or patient room.
To understand these bacteria, one must appreciate how they were differentiated in the first place.
“The Gram stain was developed by Hans C. J. Gram, a microbiologist,” says Charles Stratton, MD, associate professor of pathology and medicine, and director of the clinical microbiology laboratory at Vanderbilt University in Nashville. “The Gram stain is what differentiates the two types of bacteria, gram-negative and gram-positive. It all has to do with the cell wall of the organism. The gram-negatives have a cell wall that doesn’t retain the crystalline violet, which is what gives it the color blue, so the blue indicates gram-positive. The gram-positive organisms have a cell wall that retains the stain after you complex it with iodine, and the gram-negatives don’t. So the difference between gram-negative and gram-positive has to do with their cell wall and how they stain.”
It may be necessary to take a different approach in killing these two distinctive types of bacteria. “The bottom line is that the gram-negative cell walls are not as tough as the gram-positive cell walls,” Stratton explains. “It’s harder to kill gram-positives than gram-negatives, for the most part, because the cell walls are tougher in gram-positives; they have a thicker peptidoglycan layer.”
Although the two types of bacteria don’t have a true “preference” for a moist or dry environment, gram-negative bacteria will survive longer on a moist surface, because their cell walls are more vulnerable to drying out, he adds. “On skin, you mostly find gram-positive organisms, unless the skin is moist. If skin is moist, then you can find both gram-negatives and gram-positives. There are both gramnegatives and gram-positives in stool, but around the perineal region, unless it’s moist, the organisms that end up staying on the skin are for the most part gram-positives — Enterococcus, Staphylococcus, Streptococcus. But if the patient is obese and has a lot of moist fissures in the skin because of obesity, then you can get gram-negatives in there as well.”
Inanimate objects like instruments or environmental surfaces in the operating room (OR) or in patient rooms that have not yet been cleaned are also more likely to host gram-positives, again, because the gram-negatives don’t survive drying as well due to their less robust cell wall. “If a surface is dry, the gram-negatives aren’t apt to survive, but for example, Staphylococcus aureus survives or Enterococcus survives on a surface quite nicely,” Stratton says.
Asked about cleaning techniques for the two different types of bacteria, Stratton explains that, “there are, for example, alcohols, which work well on both, but they work better on gram-negatives than gram-positives because the cell wall is not as tough. For many of the antiseptic agents, the quaternary ammonium products, things like that, they tend to work faster and better on gram-negatives. The tougher the cell wall, the longer it takes for these surface-active drugs to work. For example, Mycobacterium tuberculosis is very tough, and hard to destroy with the surface active agents.”
For M. tuberculosis, it may be necessary to incorporate another brand of disinfectant. “If you have agents in contact longer, they’ll have time to work, but there are agents that specifically have been developed that will kill M. tuberculosis. Usually, when you look at the label of your antiseptic agents that you clean tabletops with, they’ll tell you what they’ll kill and how long it has to be in contact; it all has to do with the cell wall and how long it takes to kill that cell wall,” he says.
David Weber, MD, MPH, is a professor of medicine, pediatrics, and epidemiology at the University of North Carolina school of medicine and public health, and also serves as the medical director of hospital epidemiology for University of North Carolina Hospitals. Weber expands on the explanation of gram-negative cell walls. “Gram-negatives have a double cell wall with a periplasmic space between the cell walls, and this allows them to concentrate various enzymes in that periplasmic space. So until recently, with methicillin- resistant staphylococcus aureus (MRSA), one could say that gram-negatives were more difficult to treat than gram-positives, because this extra space made it more difficult for antibiotics to penetrate and allow them to excrete into the space various enzymes that would destroy gram-negatives that gram-positives don’t do. With MRSA and vancomycin-resistant enterococcus (VRE) being major problems in the recent past, they’ve developed other mechanisms of resistance, such as target-side alterations, where the antibiotic gets in but can’t bind to its target, or efflux pumps where the antibiotic gets in and they pump it out.”
“As a rule, gram-positives tend to live on the skin,” he adds. But there are exceptions. “Group A Streptococcus can live on the skin but also in the GI tract. In general, gram-negative organisms tend to live in the GI tract, where so-called enteric organisms include Klebsiella, E. coli, Proteus. Classically, gram-negatives are more likely — if they gain entrance into the bloodstream — to cause shock than gram-positives, but these are broad generalizations, and you can get shock with Staphylococcus aureus and Neisseria meningitides, which is a gram-negative coccus.”
Not all organisms stain well by gram stain. Legionella (which causes Legionnaire’s disease) and Rickettsia (which causes rocky mountain spotted fever) do not have standard thick cell walls, and therefore don’t stain very well by Gram’s stain. “Another one, Mycobacterium tuberculosis, does not stain well by Gram’s stain, so we use an acid fast stain,” he says.
Drying in general is not conducive to any bacteria, so very dry environments have lower counts of bacteria than a somewhat moist environment, Weber points out. “But as a general rule, the gram-positives live normally on people’s skin. For example, if you sampled the carpet or the floor, unless you were shedding on them, you’re not going to find them. The gram-negatives, in addition to living in our GI tract, will live out in the environment. Particularly Pseudomonas and Aeromonas will live in moist environments like kitchen sinks, things like that. The other gram-negatives can live there too. And of course fungi can live there,” he says.
Each organism has an ideal range of temperatures and moisture that will encourage its growth. However, nutritional requirements may be specific or very broad. “Most bacteria prefer relatively simple food sources — sugars, carbohydrates — but one of the ways to identify a specific bacteria is by which specific sugar they use, whether it’s mannitose, lactose, glucose, or if they use specific proteins or other things they survive on,” adds Weber.
Their hiding places are varied, but bacteria in general are ubiquitous. “If you culture your carpet, you’ll get about 10,000 bacteria per square inch. Even drinking water is not sterile. The environment is just covered with these bacteria,” he says.
Killing these bacteria requires a laundry list of resources. Weber first clarifies antiseptic (a chemical used on the skin to reduce its bacterial count) from disinfectant (a chemical used on an environmental surface to decrease the number of bacteria there) and from sterilization (the absence of all microbial growth).
“We can’t sterilize skin; however, we can take an endoscope and sterilize it by putting it in a chemical that would kill. Sterilization has an absolute sense; it means the absence of all microbial growth. Disinfection is not an absolute, but different sterilants will kill different bacteria at different rates. The hardest-to-kill bacteria are bacterial spores, and most disinfectants used on equipment will kill anything but bacterial spores. Those would be high-level disinfectants. A sterilant would kill the spores.”
The necessary level of reducing bacteria depends on the intended use of the item. An item that enters sterile body tissue, such as a surgical knife, or an implant, must be sterilized. A medical device touching a mucous membrane, such as an endoscope, must be high-level disinfected. A device touching intact skin, such as a blood pressure cuff, must be low-level disinfected between patients.
“That being said, we routinely disinfect environmental surfaces in patient rooms that are high-touch surfaces,” Weber explains. “Therefore, because the patient could have MRSA, then touch something that the nurse touched — and if she didn’t wash her hands when she left the room, she could carry it to another patient — things like bed rails, bedside tables, and glucose monitoring device, are generally cleaned on a regular basis, whenever they’re soiled, and before another patient uses them.”
Each sterilant or disinfectant used in the healthcare environment has disadvantages or advantages — a different contact time is required; some are not compatible with every material, or have different costs. There may be issues with disposal, or there may be issues with risk to workers. There is no ideal sterilant. “That said and done, there are better or worse products, and it depends on your need, your cost, and what you’re using it for,” Weber says.
Aerobic indicates that a bacteria grows in the presence of oxygen; anaerobic indicates that they cannot grow in the presence of oxygen.
“Facultatively anaerobic means they can grow either way, with or without oxygen. There are gram-negative and gram-positive organisms that are aerobic, and there are gram-negative and gram-positive organisms that are anaerobic. So these are different attributes, just like there are cocci and rods, which is the shape,” he adds. “The most resistant organisms are prions like those that cause Creutzfeldt- Jakob disease (CJD), followed by bacterial spores. Next would be non-enveloped viruses, and vegetative bacteria (meaning normal reproducing bacteria) are relatively easily killed. I don’t know that there’s a difference between aerobic and anaerobic, except anaerobic won’t grow on the surface of a table, generally, because they don’t grow in oxygen.”
The most contaminated areas of a hospital are the expected locations.
“In hospitals, like houses, the most contaminated areas tend to be things like sinks and toilets and food preparation areas, because, for example, lettuce is heavily contaminated. Hospitals, of course, are cleaner than homes, because we use higher level disinfectants, and we clean much more frequently. The air is much cleaner because we change it more frequently, and we filter it better. But of course the main source of pathogenic bacteria in the hospital is not the environment — it’s patients. The trick is to keep it from going from patient to patient. We can’t get rid of it in patients, but if we do things effectively, like washing our hands with an antiseptic, we can prevent it from going from patient to patient.”
“There are a group of microorganisms called ‘water bugs,’ because they like to live in water, but particularly they like to live on ‘scale’ inside of pipes,” says Peter H. Gilligan, PhD, D(ABMM), director of clinical microbiology-immunology laboratories at University of North Carolina Hospitals, and professor of microbiology, immunology, and pathology laboratory medicine at the University of North Carolina school of medicine.
“If you have aerators on the faucet at home and take them off and feel the inside of the pipe, that slime is the ‘scale.’ Microorganisms like to grow in scale, in biofilm. There are lots of organisms that like biofilms, but the one we know the most about is Pseudomonas aeruginosa, one of the most common causes of hospital-acquired infections. Another that we see in scale — but not all the time — is Legionella pneumophilia, an organism that can cause pneumonia in adults. That tends not to be in all hospitals, but some hospitals will have it in the water system, also in showerheads. Again, it grows in that scale, and when people take showers, the organism is aerosolized, and there are a lot of interventions that need to be taken.
“There are other organisms like Stenotrophomonas maltophila, Acinetobacter calcoaceticus, or Acinetobacter baumannii, Serratia marcescens, and these all cause nosocomial infections that you can get in the environment from the water. The other organisms we’re really worried about in the hospital are two that are found in the bathroom — and they’re both gram-positives. One is vancomycin-resistant Enterococcus (VRE), and the other is Clostridium difficile,” Gilligan adds.
“C. difficile is a very interesting organism, because it produces spores, which can remain viable for months. There’s a famous study done by the University of Michigan, by Bob Fekety and his colleagues. Essentially, they had a room of a patient with C. difficile. The room was in an area of the hospital that was being renovated, so they sealed the room for six months, then came back six months later and were able to recover the organism from that room.
“It lived there so well because of the spores, which are structures that microorganisms make that allow them to survive in very hostile environments. Not all microorganisms produce spores, but Clostrid ium do. They’re soil organisms; that’s probably where they first came from. In soil, there’s tremendous competition among the microorganisms for nutrients, so if there aren’t sufficient nutrients, there are certain signals sent to the organism saying, ‘There’s not enough stuff to eat; we need to go into our survival mode,’ which is making spores. When the environment is favorable again, these spores germinate and become viable bacteria again. It’s not hibernation, because there is no metabolic activity; spores are metabolically inert. They’re protected from drying and heat and from many disinfectants, so that’s what makes them so problematic,” Gilligan adds.
“These spores can remain viable on any variety of surfaces that someone who has this disease touches, especially if they haven’t done a good job of washing their hands. Spores are resistant to alcohol, so if you’re trying to control infection with C. difficile, it’s important that when caregivers come in contact with people with this disease, they wash their hands with old-fashioned soap and water,” he says.
“Studies show that C. difficile is mainly found in the bathroom, but it can be found on bedrails, on bedside tables, on the telephone,” states Gilligan. “It’s an organism that we have a lot of respect for, because it can cause very severe diarrheal disease, and actually can cause pseudomembranous colitis, which can result in the death of patients. One of the things that we’ve seen recently is there are ‘super-strains’ that seem to cause more severe disease and have higher mortality associated with them.”
C. difficile can be found in the GI tracts of some patients, but it is likely they carry it in spore form. When a patient is given antibiotics, which disturbs the normal gut flora, the positive organisms that typically keep C. difficile in check may be killed off, allowing the spores to vegetate and produce toxin.
Gilligan observes that the main “bugs” that concern him are the aforementioned water bugs, mainly because they’re often resistant to many different antibiotics. “VRE is found in hospital environment in the same types of places you find C. difficile. It’s not going to survive for months at a time, but if people with this organism in their GI tract don’t wash their hands, or if their bedpans are not cleaned up as carefully as might be, the organism can remain viable in the environment for some period of time.”
A hallway setting, in contrast to patient rooms, may be host to fungi rather than bacteria. Many hospitals no longer allow windows to be opened for fear of mold spores entering the environment.
Moreover, patients with compromised immune systems should not receive flowers or plants, “because Pseudomonas — which can affect them — is found all over the surface of flowers and in the water. If you send them plants, it’s in the soil, so people who are immuno-suppressed should only be sent artificial flowers without water. Also, people who are immuno-suppressed can’t have salads, because salads have Pseudomonas on their surface. It’s very difficult to get soil off the lettuce in such a way that the microorganisms in the soil are eliminated, so raw vegetables, especially lettuce, are problematic,” Gilligan emphasizes.
“Our immune systems are miracles,” he says. “Microorganisms are everywhere. We walk through a soup of microorganisms every day. We are exposed to microorganisms that in the hospital kill people, and we are exposed to them every day with no ill effect.”