Rapid Detection of Cronobacter spp.

Image of Cronobacter sakazakii, formerly known as
Enterobacter sakazakii. Photo courtesy of CDC and Dr. JJ Farmer.

Cara N. Wilder, Ph.D.

Cronobacter sakazakii is a Gram-negative, foodborne pathogen associated with the use of powdered infant formula. In infants, this bacterium has been found to cause invasive infections with high fatality rates, including neonatal meningitis, sepsis, and necrotizing enterocolitis. Due to the severity of these infections, it is imperative that rapid and sensitive detection assays are available for the microbiological testing of powdered infant formula.

Currently, the routine procedure for the detection of Cronobacter spp. is both arduous and time intensive. To improve upon this, Cai et al., developed a novel molecular-based assay that targets the Cronobacter ompA gene through real-time PCR integrated with high resolution melting (HRM) analysis. This innovative methodology is not only faster than current procedures, reducing detection time from several days to less than 24 hours, it is also probe-free and reduces the risk of PCR carry over. To analyze the specificity and sensitivity of this assay, 11 Cronobacter isolates and 25 reference strains were examined. In this analysis, only Cronobacter spp. produced a positive signal. Moreover, the assay detection limit was found to be 10² CFU/mL, indicating that the assay is highly sensitive. Overall, this established method may provide a rapid, sensitive, and specific molecular tool for the direct detection of Cronobacter spp. in powdered infant formula.

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Identification of Enterococcus species using an automated microarray-based nucleic acid test

Image of Enterococcus sp. Photo courtesy of Janice Haney Carr.

Cara N. Wilder, Ph.D.

Enterococcus faecalis is a Gram-positive commensal bacterium known to inhabit the gastrointestinal tract of humans and animals. In immunologically compromised individuals, E. faecalis is a leading cause of urinary tract infections and nosocomial bacteraemia. This latter condition is of particular concern as it can lead to septic shock or the hematogenous spread of bacteria to other parts of the body, resulting in high rates of mortality and organ failure.

Unfortunately, the treatment of E. faecalis infection is not straightforward as a number of strains exhibit intrinsic and acquired resistance to a variety of antibiotics, including aminoglycosides, cephalosporins, and penicillins. Presently, these drug-resistant strains are commonly treated with vancomycin; however, in some instances the extended use of this antimicrobial drug has resulted in the emergence of vancomycin-resistant strains. To make matters worse, current treatment options for vancomycin-resistant strains is limited. Therefore, the rapid identification of vancomycin-resistant strains is imperative to the successful treatment of infection.

In recent years, advances in diagnostics have yielded a number of sensitive assays for the detection of E. faecalis, including fluorescent in situ hybridization (FISH), matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS), and the microarray-based Verigene Gram-Positive Blood Culture test (BC-GP) (Nanosphere). This latter method was recently validated through an examination of 12 Gram-positive targets including E. faecalis, as well as associated drug-resistance determinants. In this study, Buchan et al. used the Verigene BC-GP test to analyze 1,252 positive blood culture broths collected from five clinical centers throughout the United States. The resultant data indicated that the BC-GP test can sensitively and specifically identify E. faecalis and other leading causes of Gram-positive bacteremia directly from positive blood cultures. Further, this assay was capable of detecting the genetic markers vanA and vanB, which confer resistance to vancomycin. Overall, the Verigene BC-GP assay may prE. faecalis, ultimately enhancing public health.
ovide diagnosticians with a more sensitive means of detecting vancomycin-resistant

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Rapid and sensitive detection of blaKPC in Klebsiella pneumoniae

Cara N. Wilder, Ph.D.

Klebsiella pneumoniae is a Gram-negative bacterium found within the mouth, skin, and intestines. Although this organism is a common inhabitant of the human microflora, it can behave as an opportunistic pathogen in immunologically compromised individuals, causing a variety of clinical diseases including pneumonia, thrombophlebitis, and septicemia. Current therapies for K. pneumoniae infection include treatment with β-lactam antibiotics such as third-generation cephalosporins, carbapenems, and penicillins.

Over the last decade, a number of K. pneumoniae strains exhibiting multidrug resistance have emerged in numerous healthcare settings, resulting in prolonged hospitalization and increased mortality. These strains were found to produce Klebsiella pneumoniae carbapenemase (KPC), which is a β-lactamase enzyme that confers resistance to all β-lactam antibiotics. More troubling was the discovery that KPC is encoded by the Tn₃-type transposon-localized gene, bla_KPC. Mobilization of this genetic element has permitted the diffusion of bla_KPC between various plasmids, allowing for inter-species dispersion via horizontal transfer. To date, plasmids harboring bla_KPC have been isolated among Enterobacteriaceae genera commonly found in the human microbiome, including Klebsiella pneumoniae, Enterobacter spp., and Escherichia coli. Regrettably, these strains are often not detectable through routine susceptibility screening and are easily disseminated, further contributing to escalating morbidity and mortality rates.

In order to control the spread of KPC strains, a rapid and sensitive diagnostic tool is necessary. In a recent report, Mosca et al. evaluated the performance of NucliSens EasyQKPC (bioMérieux, France), which is a new molecular assay designed to rapidly detect the presence of the bla_KPC gene via real-time nucleic acid sequenced-based amplification (NASBA™). In this study, 38 non-duplicate, carbapenem-resistant K. pneumoniae isolates were first analyzed using the modified Hodge test (MHT) and the carbapenemase inhibitor test, which are traditional culture-based methods often recommended for the detection of carbapenem-resistant strains. While all strains exhibited the production of KPC when examined by the carbapenemase inhibitor test, only 84% of the strains evaluated by MHT exhibited carbapenemase-production. In contrast, NucliSens EasyQKPC was able to detect the bla_KPC gene in each of the isolates evaluated. The accuracy of this assay was confirmed when each of the isolates tested positive for the presence of the gene following PCR analysis. Overall, NucliSens EasyQKPC provides a sensitive and reliable assay for the identification of the bla_KPC gene, allowing for the rapid detection of KPC strains in clinical samples.

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Use of bacteriotherapy to resolve Clostridium difficile infection

Clostridium difficile

Cara N. Wilder, Ph.D.

Antibiotic treatment of hospitalized patients poses a serious risk for the colonization of Clostridium difficile. Infection with this anaerobic, spore-forming bacterium is characterized by a wide spectrum of symptoms including diarrhea, fulminant pseudomembranous colitis, and death. Though treatment of C. difficile with antibiotics is often effective, a number of treated patients experience a relapse in infection following the cessation of antibiotic therapy. It is believed that these recurrent infections may be linked to a condition termed dysbiosis, which is characterized by a general imbalance of the intestinal microflora resulting from continuous antibiotic exposure.

In recent years, the use of a probiotic-based approach has been proposed as a promising therapy for the treatment of C. difficile infections. To determine the ideal combination of probiotic strains that both resolves C. difficile disease and restores a healthy intestinal microflora, Lawley et al. employed the use of a C. difficile murine infection model that parallels many aspects of human disease. In the study, the murine model was infected with an epidemic strain of C. difficile that was able to out-compete health-associated intestinal bacteria, allowing for the maintenance of dysbiosis. Following the establishment of infection, the group used fecal-transplantation as a model to identify a mixture of six phylogenetically-diverse bacteria that were able to trigger the expansion of health-associated intestinal microflora and resolve C. difficile infection within the mice. Overall, the results from this study highlight the therapeutic potential of probiotic bacteriotherapy in the treatment of C. difficile and other forms of dysbiosis.

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Preventing aspergillosis through immunization

Aspergillus fumigatus. Photo courtesy of
David Gregory and Debbie Marshall

Cara N. Wilder, Ph.D.

Over the last 20 years, Aspergillus fumigatus has become one of the most frequent causes of invasive fungal infection in immunologically compromised patients. This infection, termed aspergillosis, is associated with a wide spectrum of symptoms, including allergic reactions, organ failure, and lung infection. In most patients, A. fumigatus predominantly affects the lungs, resulting in an often fatal illness termed invasive pulmonary aspergillosis (IPA).

Current therapies for aspergillosis include treatment with antifungal drugs such as Amphotericin B or Triazole medications (Voriconazole, Posaconazole, Itraconazole). Unfortunately, these therapies have had limited success in treating IPA, and are often associated with serious toxicities. This is further compounded by the growing number A. fumigatus strains that have developed resistance against antifungal drugs, thus making the eradication of aspergillosis increasing more difficult. In response to these concerns, many research laboratories have focused their efforts on the development of new strategies targeted toward the prevention and treatment of such infections.

In one vaccine discovery program, Ito et al. used an immunochemical and mass spectrometric approach to identify an antigenic target for the development of a candidate vaccine that provides immunization against A. fumigatus. Following nasopulmonary inoculation of immunocompetent mice with viable A. fumigatus conida, Asp f 3 was identified as a potential antigenic target based on immunodominance during infection. Upon the analysis of this allergen, it was determined that subcutaneous immunization with full-length and truncated recombinant Asp f 3 offered protection against A. fumigatus infection. This was further confirmed following the examination of the lungs of vaccinated survivors, which were deemed free of hyphae and exhibited minimal infiltration of mononuclear cells. Overall, the findings from this study indicate that recombinant Asp f 3 is an effective immunogen and promising candidate for the development of a novel vaccine to prevent aspergillosis.



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Response of Campylobacter jejuni to Erythromycin Exposure

Campylobacter jejuni. Photo courtesy of Dr. Patricia Fields
and Dr. Collette Fitzgerald.

Cara N. Wilder, Ph.D.

Campylobacter jejuni is a Gram-negative, motile bacterium that is commonly present in the intestinal tract of both domestic and wild animals. In humans, C. jejuni causes a foodborne infection termed campylobacteriosis, which results in symptoms that range from mild enteritis to fever, headache, and bloody diarrhea. In some cases, Campylobacter infection has been associated with Guillain-Barré syndrome, which is a post-infection autoimmune disorder that damages nerve tissue.

Although most cases of campylobacteriosis are self-limiting, antibiotic treatment may be necessary for patients that are either immunologically compromised, or exhibit severe or persistent infection. The most common antimicrobial therapy used to treat campylobacteriosis is erythromycin, which is a macrolide antibiotic that inhibits bacterial protein translation. However, due the regular use of this antibiotic in animal production and veterinary medicine, an increasing number of C. jejuni isolates have become drug-resistant. In many of these strains, the mechanism of resistance is frequently conferred by target modification or through the expression of antibiotic efflux pumps.

Though the genetic basis of erythromycin resistance has been well-studied, the initial response and adaptive mechanisms directly following erythromycin exposure is not well understood. To analyze this, Xia et al. performed a competitive microarray hybridization study that examined the genome-wide transcriptional response of a sensitive and resistant strain of C. jejuni upon exposure to inhibitory and sub-inhibitory doses of erythromycin. Following treatment with erythromycin, the resistant strain of C. jejuni exhibited little to no differential gene expression. In contrast, a number of genes were differentially regulated in the sensitive strain, including the up-regulation of genes associated with motility, and the down-regulation of genes associated with energy production and conversion. Moreover, the inactivation of several differentially expressed genes appeared to negatively affect host colonization and the ability to tolerate high levels of oxygen. Overall, these results provide new insight into the adaptive response of C. jejuni to antibiotic treatment, and my help provide further understanding into the mechanisms underlying the emergence of antibiotic resistance.



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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.

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