Survival mechanisms of Burkholderia cepacia complex cells grown in biofilms

Burkholderia cepacia. Photo courtesy of Janice Haney Carr
and CDC

Cara N. Wilder, Ph.D.

The Burkholderia cepacia complex (Bcc) is a group of Gram-negative bacteria composed of 17 closely related species. Of these strains, Burkholderia cenocepacia is an opportunistic pathogen frequently associated with high rates of transmission and mortality among immune-compromised people, such as those suffering from cystic fibrosis. Unfortunately, infections caused by B. cenocepacia and other Bcc strains are very difficult to eradicate due to a variety of  intrinsic antibiotic-resistance mechanisms including the expression of multidrug efflux pumps, inducible β-lactamases, altered penicillin-binding proteins, and the ability to form biofilms.

In particular, biofilms are multicellular microbial communities that can form on various environmental, clinical, and abiotic surfaces. These groups of sessile cells are often more tolerant to antibiotics than free-living, planktonic cells due to decreased growth rates, differential gene expression, and reduced penetration of the biofilm. Thus, upon exposure to antibiotic therapies, a small subpopulation of cells within the biofilm is able to persist by entering a dormant multidrug-tolerant state. Following the removal of the antibiotic, these “persister cells” are then able to reestablish growth and create a new biofilm.

To elucidate the mechanisms behind the emergence of persister cells in Bcc biofilms, Acker et al. analyzed B. cenocepacia biofilms following treatment with Tobramycin, a bactericidal antibiotic known to induce the formation of harmful reactive oxygen species (ROS). Through the use of transcriptome analysis, flow cytometry, ROS-staining, and inhibitor studies, the group discovered that surviving persister cells were able to escape cell death through the down-regulation of the tricarboxylic acid (TCA) cycle, allowing cells to avoid ROS production, and through the activation of the glyoxylate shunt, which is an anaplerotic pathway of the TCA cycle. This finding may provide novel approaches for the treatment of Bcc biofilms as the glyoxylate shunt is absent in humans, and inhibition of this pathway prior to treatment with Tobramycin was found to decrease the number of persisters. Thus, this pathway may be an ideal target for combination therapy.

Read the published article now

 

Preventing Acinetobacter baumannii infection through active and passive immunization

Cara N. Wilder, Ph.D.

Multidrug-Resistance -- A growing concern throughout the world

Within the last two decades, multidrug-resistant strains have emerged as a common cause of drug-resistant infection throughout the world, resulting in prolonged hospitalizatin and high mortality rates. ATCC understands the danger and growing concern behind the spread of these strains, and we would like to help support your research in this field. For the several weeks, we will highlight various emerging multidrug-resistant strains, and some of the current research being performed with these superbugs. For more information, on these strains, subscribe to our eNewsletter or visit our website at www.atcc.org.

 

Preventing Acinetobacter baumannii infection through active and passive immunization

Acinetobacter baumannii is an opportunistic pathogen typically associated with a wide variety of complications, including pneumonia, localized tissue infections, septicemia, and death. This bacterium commonly infects individuals with a compromised immune system, and has become a predominant cause of infection in intensive care units, healthcare settings, and combat zones.


Within the last two decades, multidrug-resistant strains of A. baumannii have emerged as a common cause of drug-resistant infection throughout the world, resulting in prolonged hospitalization, extensive healthcare cost, and high mortality rates despite treatment. More troubling is the recent evolution of clinical variants that are pan-drug resistant, demonstrating resistance to every FDA-approved antibiotic. In response to these concerns, government agencies and research laboratories have focused their efforts on the development of new strategies targeted toward the prevention and treatment of such infections.




A CDC microbiologist working with Acinetobacter baumannii.
Photo courtesy of James Gathany and CDC.

In one vaccine discovery program, Luo et al. used a screening mechanism to identify an antigenic target for the development of a candidate vaccine that provided both active and passive immunization against A. baumannii. Following the intravenous infection of mice, OmpA was identified as a potential antigenic target based on humoral immunodominance during infection. Upon the analysis of this outer membrane protein, it was found that OmpA was highly conserved among numerous clinical isolates and demonstrated minimal homology with the human proteome. Additionally, vaccination of immune-compromised mice with an emulsification of recombinant OmpA and aluminum hydroxide adjuvant induced high titers of anti-OmpA antibodies, and demonstrated improved survival and reduced bacterial burden in intravenously infected mice. The activity of anti-OmpA antibodies was further confirmed through passive transfer studies, which recapitulated protection. Overall, the results from this study indicate that recombinant OmpA is an effective immunogen and promising candidate for the development of a novel vaccine to prevent A. baumannii infections.


Read the published article now

Impact of White-Nose Syndrome

Cara N. Wilder, Ph.D.

Photo provided by Dr. Winkler and

Dr. Sikes, CDC

Bats are an essential part of our ecosystem, providing valuable services including insect control, pollination, and seed dispersal for countless plant species. In the United States, each of the 45 indigenous bat species are insectivorous, predating on nocturnal insects that are often very damaging to commercial agriculture^1-4. For example, a single colony of big brown bats has been estimated to consume as many as 1.3 million agricultural pests yearly, significantly contributing to the disruption of insect population cycles and the preservation of commercial crops⁵. In fact, scientists throughout the United States estimate that bats are worth at least $3.7-$53 billion per annum in reduced pesticide use and crop damage^4,6.

Regrettably, bat populations throughout the world are in rapid decline due to the emergence and spread of Geomyces destructans. This fungal pathogen, which causes a fungal skin infection termed white-nose syndrome (WNS), has devastated populations of cave-hibernating bats throughout North America, resulting in bat declines exceeding 75%^7,8. Since its initial documentation in 2006, WNS is estimated to have killed over 6 million bats⁷.

Presently, numerous state and federal agencies, tribes, organizations, and individual researchers are working toward further understanding the dynamics and transmission of this G. destructans in the hopes of finding a novel method to prevent the further spread of this devastating disease. In an effort to aid research efforts related to WNS, ATCC now offers the fully-sequenced G. destructans, strain 20631-21, deposited by the USGS National Wildlife Health Center, and the associated genomic DNA. Sequencing for this strain has also been published by The Broad Institute.

Help save our bats by getting started on your research today!

• ATCC^® MYA-4855™ -- Geomyces destructans strain 20631-21
• ATCC^® MYA-4855D™ -- Genomic DNA from Geomyces destructans strain 20631-21

Register to view the ATCC Webinar featuring Dr. David, Blehert, head of diagnostic microbiology at the U.S. Geological Survey – National Wildlife Health Center (NWHC; Madison, WI).

References

1. Kalka, M. B., Smith, A. R. & Kalko, E. K. Bats limit arthropods and herbivory in a tropical forest. Science 320, 71, doi:10.1126/science.1153352 (2008).
2. Williams-Guillen, K., Perfecto, I. & Vandermeer, J. Bats limit insects in a neotropical agroforestry system. Science 320, 70, doi:10.1126/science.1152944 (2008).
3. Kunz, T. H., Braun de Torrez, E., Bauer, D., Lobova, T. & Fleming, T. H. Ecosystem services provided by bats. Annals of the New York Academy of Sciences 1223, 1-38, doi:10.1111/j.1749-6632.2011.06004.x (2011).
4. Boyles, J. G., Cryan, P. M., McCracken, G. F. & Kunz, T. H. Conservation. Economic importance of bats in agriculture. Science 332, 41-42, doi:10.1126/science.1201366 (2011).
5. Whitaker Jr., J. O. Food of the big brown bat Eptesicus fuscus from maternity colonies in Indiana and Illinois. Am. Midl. Nat. 134, 346-360 (1995).
6. International, B. C. All About Bats, <www.batcon.org>
7. A Coordinated Response to the Devastating Bat Disease, <http://whitenosesyndrome.org/>
8. Blehert, D. S. et al. Bat white-nose syndrome: an emerging fungal pathogen? Science 323, 227, doi:10.1126/science.1163874 (2009).

 

 

 

 

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!