The Effect of Horizontal Gene Transfer on the Emergence of Multi-Drug Resistant Bacteria in Nosocomial Infections

Cara N. Wilder, Ph.D.

The discovery of antibiotics in the early twentieth century has revolutionized the treatment of infectious diseases. However in recent decades, the frequency of nosocomial infections caused by multidrug-resistant bacterial strains has steadily escalated worldwide, resulting in increased morbidity, mortality, and health-care expense^1-2. This phenomenon can be attributed to a combination of microbial evolution and clinical practices that enhance the transmission of multidrug-resistant strains, including the continuous overuse and misuse of antibiotics, the increased employment of invasive medical devices and procedures, and ineffectual infection control practices³. These selective pressures have required bacterial species to evolve an extraordinary gamut of mechanisms designed to counteract antibiotic function, including the production of antibiotic-modifying and -inactivating enzymes, efflux pumps, and genomic and ribosomal modifications of target sites^4-5.

Antibiotic resistance in bacterial strains can be achieved through either genetic mutation or by the acquisition of a laterally transmitted mobile genetic element harboring an antibiotic-resistance gene cassette⁶. This latter mechanism, termed horizontal gene transfer, can occur through cell contact-dependent DNA transfer (conjugation), the uptake of naked DNA from the surrounding environment (transformation), or by phage-dependent transfer of bacterial genetic elements (transduction). In particular, conjugation has been implicated in a vast number of reports of bacterial gene transfer in the environment, including the horizontal transfer of antibiotic resistance genes. It is predicted that this transfer system may be more predominant as the associated mobile genetic elements transferred are of a very wide host range.

Studies have shown that horizontal gene transfer can occur in both intra- and inter-species populations, with foreign DNA representing up to one-fifth of any given bacterial genome⁷. This natural selection process has been particularly prominent in the emergence of deadly multidrug-resistant strains such methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae^8-10. Numerous surveillance studies have identified the emergence of these resistant strains as a major trend in nosocomial infections among high-risk patients, and are attributed to more than 70% of these infections^11-13.

Overall, the worldwide clonal spread of multidrug-resistant organisms within and between hospitals has fueled the extensive rise in resistance, severely limiting therapeutic options and substantially increasing the incidence of incurable infection. Unless this rise in multidrug-resistance can be reversed, the effectiveness and utility of antibiotics may be a matter of years or decades, effectively plunging infection control toward a pre-antibiotic era¹⁴.

Prominent Strains of Multidrug-Resistant Bacteria

Extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae – Gram-negative bacilli within the Enterobacteriaceae family that produce extended-spectrum β-lactamase. An example of this includes strains of Escherichia coli and Klebsiella pneumoniae that produce New Delhi metallo-β-lactamase-1 (NDM-1). NDM-1 is a carbapenemase β-lactamase that inactivates carbapenem antibiotics as well as a wide range of other antibiotics through the destruction of the β-lactam ring, thus deactivating the antibacterial properties.

ATCC^® NDM-1 Strains:

·         Klebsiella pneumoniae (ATCC^® BAA-2146™)

·         Enterobacter cloacae (ATCC^® BAA-2468™)

·         Escherichia coli (ATCC^® BAA-2469™)

·         Klebsiella pneumoniae subsp. pneumoniae (ATCC^® BAA-2470™)

·         Klebsiella pneumoniae subsp. pneumoniae (ATCC^® BAA-2472™)

Methicillin-resistant Staphylococcus aureus (MRSA) – Any strain of Staphylococcus aureus that has developed resistance to β-lactam antibiotics, including methicillin. These strains harbor the SCCmec genomic island containing the mecA resistance gene, which encodes a penicillin binding protein (PBP) that does not bind methicillin or other β-lactam antibiotics. This enables the PBP to catalyze the transpeptidase reaction, consequently completing cell wall synthesis in the presence of antibiotics.

ATCC^® MRSA Strains:

·         Methicillin-Resistant Staphyloccus aureus Panel organized by SCCmec type (MP-2™)

·         Methicillin-Resistant Staphyloccus aureus Panel organized by Pulse-field type (MP-3™)

Vancomycin-resistant enterococci (VRE) – Bacterial strains of the genus Enterococcus that are resistant to the antibiotic vancomycin. This phenotype is possible through the presence of various resistance genes, including vanA, vanB, vanC, vanD, vanE, and vanF. Resistance involves the alteration of vancomycin target sequences: the terminal amino acid residues of NAM/NAG-peptide subunits. Modification of these sequences decreases the binding affinity of vancomycin, allowing antibiotic resistance.

 

ATCC^® VRE Strains:

·         Vancomycin-Resistant Enterococci Panel (MP-1™)

References

1.     Harbarth S, et al. Control of multiply resistant cocci: do international comparisons help? Lancet Infect. Dis. 1: 251-261, 2001.

2.     Blondeau JM, Tillotson GS. Antimicrobial susceptibility patterns of respiratory pathogens – a global perspective. Semin. Respir. Infect. 15: 195-207, 2000.

3.     Jones RN, Phaller MA. Bacterial resistance; a worldwide problem. Diagn. Microbial. Infect. Dis. 31: 379-388, 1998.

4.     Levy SB. Active efflux, a common mechanism for biocide and antibiotic resistance. J. Appl. Microbial. Suppl. S1: 65S-71S, 2002.

5.     Gold HS, Moellering RC Jr. Antimicrobial-drug resistance. N. Engl. J. Med. 335: 1445-1453, 1996.

6.     Normark BH, Normark S. Evolution and spread of antibiotic resistance. J. Intern. Med. 252: 91-106, 2002.

7.     Davison J. Genetic exchange between bacteria in the environment. Plasmid 42(2): 73-91, 1999.

8.     Wielders CCL, et al. mecA Gene is Widely Disseminated in Staphylociccus aureus Population. J. Clin. Microbiol. 40(11): 3970-3975, 2002.

9.     Palmer KL, et al. Horizontal Gene Transfer and the Genomics of Enterococcal Antibiotic Resistance. Curr. Opin. Microbial. 13(5): 632-639, 2010.

10.   Bush K, Fisher JF. Epidemiological expansion, structural studies, and clinical challenges of new β-lactamases from Gram-negative bacterial. Annu. Rev. microbial. 65: 455-478, 2011.

11.   Wiener J, et al. Multiple antibiotic-resistant Klebsiella and Escherichia coli in nursing homes. JAMA 281: 517-523, 1999.

12.   Dzidic S, Bedekovic V. Horizontal gene transfer-emerging multidrug resistance in hospital bacteria. Acta. Pharmacol. Sin. 24(6): 519-526, 2003.

13.   Muto CA, et al. SHEA Guideline for Preventing Nosocomial Transmission of Multidrug-Resistant Strains of Staphylococcus aureus and Enterococcus. Infection Control and Hospital Epidemiology 24(5): 362-386, 2003.

14.   Wellington EMH, et al. The role of the natural environment in the emergence of antibiotic resistance in Gram-negative bacteria. Lancet Infec. Dis. 13(2): 155-165, 2013.

12 Food Testing Tools of Food Safety Research - A Holiday Carol

Cara N. Wilder, Ph.D.

On the first day of my food safety project my PI gave to me, a list of food testing tools from ATCC.

On the second day of my food safety project my PI gave to me, two parasitic protozoa DNA panels and a list of food testing tools from ATCC.
 
On the third day of my food safety project my PI gave to me, three species of Listeria, two parasitic protozoa DNA panels, and a list of food testing tools from ATCC.

 On the fourth day of my food safety project my PI gave to me, four cultures of Bacillus cereus, three species of Listeria, two parasitic protozoa DNA panels, and a list of food testing tools from ATCC.

On the fifth day of my food safety project my PI gave to me, five Salmonella enterica serotypes in a panel, four cultures of Bacillus cereus, three species of Listeria, two parasitic protozoa DNA panels, and a list of food testing tools from ATCC.

 On the sixth day of my food safety project my PI gave to me, Six Shiga toxin-producing Escherichia coli serotypes, five Salmonella enterica serotypes in a panel, four cultures of Bacillus cereus, three species of Listeria, two parasitic protozoa DNA panels, and a list of food testing tools from ATCC.

On the seventh day of my food safety project my PI gave to me, seven species of Staphylococcus, Six Shiga toxin-producing Escherichia coli serotypes, five Salmonella enterica serotypes in a panel, four cultures of Bacillus cereus, three species of Listeria, two parasitic protozoa DNA panels, and a list of food testing tools from ATCC.

 On the eighth day of my food safety project my PI gave to me, eight vials of Campylobacter jejuni, seven species of Staphylococcus, Six Shiga toxin-producing Escherichia coli serotypes, five Salmonella enterica serotypes in a panel, four cultures of Bacillus cereus, three species of Listeria, two parasitic protozoa DNA panels, and a list of food testing tools from ATCC.

On the ninth day of my food safety project my PI gave to me, nine anaerobic cultures of Clostridium perfringens, eight vials of Campylobacter jejuni, seven species of Staphylococcus, Six Shiga toxin-producing Escherichia coli serotypes, five Salmonella enterica serotypes in a panel, four cultures of Bacillus cereus, three species of Listeria, two parasitic protozoa DNA panels, and a list of food testing tools from ATCC.

 On the tenth day of my food safety project my PI gave to me, ten flasks of Giardia lamblia, nine anaerobic cultures of Clostridium perfringens, eight vials of Campylobacter jejuni, seven species of Staphylococcus, Six Shiga toxin-producing Escherichia coli serotypes, five Salmonella enterica serotypes in a panel, four cultures of Bacillus cereus, three species of Listeria, two parasitic protozoa DNA panels, and a list of food testing tools from ATCC.

On the eleventh day of my food safety project my PI gave to me, eleven cultures of Saccharomyces cerevisiae, ten flasks of Giardia lamblia, nine anaerobic cultures of Clostridium perfringens, eight vials of Campylobacter jejuni, seven species of Staphylococcus, Six Shiga toxin-producing Escherichia coli serotypes, five Salmonella enterica serotypes in a panel, four cultures of Bacillus cereus, three species of Listeria, two parasitic protozoa DNA panels, and a list of food testing tools from ATCC.

 On the twelfth day of my food safety project my PI gave to me, twelve strains of Enterococcus, eleven cultures of Saccharomyces cerevisiae, ten flasks of Giardia lamblia, nine anaerobic cultures of Clostridium perfringens, eight vials of Campylobacter jejuni, seven species of Staphylococcus, Six Shiga toxin-producing Escherichia coli serotypes, five Salmonella enterica serotypes in a panel, four cultures of Bacillus cereus, three species of Listeria, two parasitic protozoa DNA panels, and a list of food testing tools from ATCC.

'Twas the Night Before the ATCC Delivery



Cara N. Wilder, Ph.D.

 

 

 

'Twas the night before the ATCC delivery, when all through the lab

Not a creature was stirring, not even the first year grads.

The petri dishes and test tubes were placed on the lab benches with care,

In hopes that the ATCC delivery soon would be there.

The grad students were nestled all snug in their beds,

While visions of authenticated microbes danced in their heads.

And I in my old college sweatshirt and coffee stained pants,

Had just settled down to finish writing a grant.
When out in the parking lot there arose such a clatter,

I sprang from my office to see what was the matter.

Away to the loading dock I ran with much haste,

Tore open the doors, there was no time to waste.

The street lamps reflecting off the new-fallen snow
Provided a beautiful shine off the garbage bins below.

When, what to my wondering eyes should arrive,
But a giant delivery truck driving up College Drive.
With an energetic driver, so lively and spry,

I knew in a moment it must be the ATCC delivery guy.

With precision driving he drove his truck up the road,

And he parked, opened his truck, and described his load;

"Here is your Listeria! Your, E. coli! Your, Enterococcus and Salmonella!
Your enteric protozoa! Your Bacillus cereus! Your Giardia and Vorticella!

To the top of the loading ramp! To your lab down the hall!

Now dash away! To the freezer! Preserve them all!"

Quick as a bunny I ran to the lab,

And carefully placed the strains in the coldest freezer I had.

Satisfied with my purchase I let out a sigh,

Then headed back to the loading dock to say goodbye.

The delivery guy pulled a clipboard from the front seat,

I signed my name slowly to make sure it was neat.

He sprang to his truck, turned the key in the ignition,

And drove back to ATCC to prepare for his next mission.

But I heard him exclaim, as he drove out of sight,

"Authenticated ATCC strains for all, and to all a good-night."

Analyzing Novel Methods to Test Antimicrobial Drugs

Cara N. Wilder, Ph.D.



Since the discovery of penicillin, the first medically-employed antibiotic, scientists have strived to further unearth and synthesize novel antibiotics to treat infections. However, in recent years, the overuse of antibiotics has led to the emergence of numerous antibiotic-resistant microbial strains. This complication has further spurred scientists to develop novel therapeutic agents, such as antimicrobial peptides, to complement current antibiotic treatments. With the development of these novel antimicrobial therapies, traditional drug analysis methods must now be re-evaluated to determine their applicability.  

Currently, a standardized approach used to evaluate the efficacy of novel antimicrobial therapies is the time-kill study. This method, which was proposed by the Clinical and Laboratory Standards Institute (CLSI), is used to examine dynamically the bactericidal rate of an antimicrobial agent at varying concentrations and time intervals. Unfortunately, this contemporary time-kill testing procedure requires subcultures from broth macrodilutions prepared in flasks, thus necessitating the excessive use of the antimicrobial test compound. This is problematic as most antimicrobial therapies are expensive to produce.

In light of high production costs associated with manufacturing new antimicrobial drugs, a more cost-effective and convenient method for analyzing drug efficacy is desired. The use of a 96-well microplate approach has been of recent interest as it was successfully employed in the examination of minimum inhibitory concentration (MIC). When used in these assays, the microplate procedure proved to be quick, reproducible, and cost-effective. To determine if a microplate-based tactic was also applicable in a time-kill assay, Zhou et al. compared this method to the conventional CLSI macrodilution approach.

To analyze the validity of the microplate time-kill assay, Zhou et al. tested the bactericidal activity of conventional antibiotics, antimicrobial peptides, and antisense peptide nucleic acids against Escherichia coli (ATCC^® 25922™) and Staphylococcus epidermidis (ATCC^® 14990™). The bacterial growth inhibition rates of each antimicrobial therapy were analyzed to compare directly the microplate and macrodilution time-kill assays. From these assays, they found that results from the microplate-based method were directly comparable to the contemporary macrodilution method. Additionally, the microplate-based method provided a high-throughput screening approach that required significantly smaller volumes of drugs, conveniently allowing for direct measurements of microbial turbidity, and provided reproducibility among replicate experiments. Thus, this technique provides a more economical, effective, and expedient approach to test the antimicrobial efficacy over current standardized protocols.

Overall, with the synthesis of novel antimicrobial agents, it is imperative that accurate and applicable antimicrobial efficacy tests are available. As demonstrated by Zhou et al., this can be possible through the use of standardized reference materials and the adaptation of currently used methods that have demonstrated past success.

Happy culturing!

Just for Fun: Surviving a viral-induced zombie apocalypse – Part 2

Cara N. Wilder, Ph.D.

Below is the second entry of the journal we found in an old decrepit lab that was used during the “zombie” outbreak of 2035. Though the journal is decades old, we managed to preserve the entries so that they can be shared with the world.

(Note: this is a fictional story that is meant for fun, it is not a real story, there is no zombie apocalypse)

Photo Provider: CDC

November, 2035

For days we worked silently in the BSL3 zone, carefully handling the virus in Class III biosafety cabinets, working to isolate the contagion and discover its secrets. Over time, we found that the virus was similar to Rabies virus genotype 1, commonly found in carnivores and human rabies cases. Using ATCC^® VR-138™ and ATCC^® VR-137™ rabies strains as references, we found that the viral contagion had a single-stranded, negative-sense RNA genome, a bullet-like morphology, and affected the limbic and peripheral nervous systems similar to our standards. However, upon sequencing analysis, we found that though the virus was genotypically similar to rabies, there several mutational changes in the genomic material. After further examination, we discovered that these mutations resulted in more localized infections of amygdala, resulting in extreme agitation; the hippocampus, resulting is the loss of human memories; and the orbitofrontal cortex, resulting in the loss of cognitive decision making. These mutations also appeared to contribute to the lack of hydrophobia or paralysis commonly seen in rabies victims, thus allowing for the infected subjects to survive for longer time periods.

Using this data, we managed to develop a vaccine generated from the inactivated virus, which was confirmed to offer protection against the contagion. Since then, we’ve been working towards the production and distribution of the vaccine to reach people throughout the world. So far, we’ve managed to keep parts of Europe and Australia free from infection. I can only hope that one day we will be able to develop an antiviral therapy to save those that remain infected.

But until then, I can only leave this one piece advice; get vaccinated, and never stop running!

Just for Fun: Surviving a viral-induced zombie apocalypse – Part 1

Cara N. Wilder, Ph.D.

Here is an excerpt from a journal we found in the filing cabinets of an old decrepit lab that was used during the “zombie” outbreak of 2035. Though the journal is decades old, we managed to preserve the entries so that they can be shared with the world. Below is the first entry of the journal.

(Note: this is a fictional story that is meant for fun, it is not a real story, there is no zombie apocalypse)

October, 2035

It is amazing that any of us survived this past year. So many lost their faith, calling this the reckoning or the end of days, others believed it was a zombie apocalypse like in the movies. Only a few of us saw the situation for what it really was…. the worse viral outbreak the world has ever seen. If it wasn’t for the ingenuity and determination of a small group of scientists, I don’t know how much longer the human race would have survived.

Who would have believed that this literal hell on earth began with a dog? Looking back on old news reports, it seemed like a straight forward case of rabies; family dog runs away, gets rabies, comes home frothing at the mouth, bites one of its owners, then the man goes to the hospital for a rabies vaccination while the dog is put down. Quick fix, everyone stays healthy, right? But this wasn’t like a normal rabies infection, and the vaccine had no effect.

From what we’ve been able to put together, based on subsequent cases and eye witness reports, within 36 hours following the initial wound the man began to exhibit flu-like symptoms. By the 60 hour mark, he began to demonstrate fever, paranoia, agitation, and terror. After 72 hours post-infection, he was a monster. His eyes were severely blood shot, he foamed at the mouth; but it was his uncontrollable rage that was the most terrifying symptom. It was as if everything that had ever made him a conscious human being was gone; that there was nothing left but a crazed predator with only the basest of instincts. He was Subject Zero.

It was after Subject Zero became violent that things went from bad to worse. The outbreak began with Subject Zero’s family after he bit and tore at their flesh in a frenzied rage, transmitting the contagion through his infectious saliva. Those that managed to escape alive eventually became one of the infected, and in turn spread the virus like wildfire.

Photo Provider: CDC

I was among the scientists called upon to stop the spread of the virus. By that time, the east coast was already overrun, with the outbreak rapidly extending throughout the Midwest. The federal and local military forces were doing the best they could to quarantine the infected, but there were just too many of them.  Bodies of the infected and those who were attacked littered the streets; intense fear and the stench of blood permeated the air.

 It was our job to take samples from the infected to try to identify the virus in hopes of developing a vaccine and eventually some form of antiviral therapy. Because the virus presented as rabies in animals, and appeared to be transmitted by saliva, we hypothesized that the contagion was some form of mutated rabies virus that resulted in extreme symptoms in humans. We began our intellectual journey by gathering samples from some of the quarantined infected subjects. We donned our biological safety suits and entered the quarantine zone. It seemed cruel that the infected were chained down like common animals, writhing in pain; but it was the only way we could get samples without becoming infected ourselves. Following our acquisition of the samples, we quickly left the room and begin the viral identification process.

To be continued…..

A Night Out with Mycology – How Yeast and Fungi Add Culture to Our Lives

Cara N. Wilder, Ph.D.

It is a cool, crisp autumn evening. You are sitting in a downtown restaurant, gazing dreamily out the window, admiring the changing leaves and observing the occasional pedestrian hurrying by, bundled up from the chill. You begin to relax as you casually sip a glass of red wine, letting the smooth, rich liquid excite your taste buds and gradually warm your blood. In the background, a single violin begins to play a soft melody that sounds strangely familiar. As you ponder the origins of the musical piece, you are momentarily distracted by the waiter that has arrived at your table, presenting you with a warm Portobello sandwich. As you take your first bite, you savor every flavor; the subtle yeasty hue of the bun, the rich earthy tones of the Portobello mushroom, and the piquant bite of the bleu cheese dressing. You chuckle softly to yourself as you realize that your evening would not have been possible without Mycology.

Penicillium sp. Photo courtesy of the CDC

Yeast and fungi play a much larger role in our lives than commonly imagined by most individuals. For centuries, yeasts and fungi have been used in the generation of various food products and alcoholic beverages. In the preparation of the exquisite meal described above, yeast contributed to the production of the bread and wine, the Portobello mushroom is a fungus, and mold was used in the preparation of the bleu cheese dressing. Products such as bread, wine, and beer are commonly produced using various species of Saccharomyces, among other genera, which convert present fermentable sugars into ethanol and carbon dioxide. The flavor and consistency of these products are affected by the ingredients and the strain of yeast used. In contrast, bleu cheeses are commonly produced from cow’s milk, sheep’s milk, or goat’s milk cheeses that have cultures of the mold, Penicillium roqueforti or Penicillium glaucum. These molds give bleu cheeses their pungent flavor and characteristic blue tinge.

In addition to their role in fine dining, fungi have recently been implicated in shaping the musical world.  Fungal infections of trees caused by the species Physisporinus vitreus and Xylaria longipes, among others, are found to cause a decrease in wood density, ideal for creating a violin with superb acoustics.  Violins produced in this fashion, known colloquially as bioviolins, were found to be equivalent if not superior in sound quality as compared to Stradivarius violins. These latter violins are known to the musical world as the highest quality instruments made. In a recent sound quality experiment, over 90 individuals in a panel of 180 people ranked the bioviolin sound quality above that of the Stradivarius. However, only time will tell if this technique will continue to be used for the construction of violins or other wood-based instruments.

So, the next time you go out on the town for a nice meal or listen to music echoing wistfully from the body of a violin, think of Mycology. Remember, your microflora isn’t the only culture you have!