STUDENT POSTERS (eligible for judging at the meeting)

The Best Undergraduate Poster winner was Leah Bauer
and co-presenters David Sadler and Luiza Assis from CMU
("Genes Associated with E. coli Pathogenicity in Freshwater Beach Sand".

MIASM_sp07 062.jpg (682933 bytes)

The Best Graduate Poster winner was Anne Kizy from WSU
("Trolling for Streptococcus pyogenes virulence genes using a zebrafish host model").

MIASM_sp07 063.jpg (682480 bytes)

This was a great display of excellent research by all the presentors.
Congratulations to everyone who submitted posters.

Effects of Secreted Haemophilus influenzae Modulins on Respiratory Epithelial Cell Interleukin (IL)-8 Production

Ranjeeta Kaur, Steve Rhoades, Sreelatha Ponnaluri, Patricia Sinawe, and Dr. Daniel L. Clemans
Eastern Michigan University, Ypsilanti, MI

The pathogenesis of nontypeable Haemophilus influenzae (NTHi) can be attributed to a variety of molecules such as lipooligosaccharide
(LOS), several adhesins and outer membrane proteins (e.g., HMW1, HMW2, P2, P5), and proteins involved with iron and heme acquisition. We
hypothesize that secreted, non-LOS NTHi molecules (i.e., modulins) mediate cellular interactions with respiratory epithelial cells
leading to the production of proinflammatory cytokines. To address this hypothesis, we exposed human tracheal epithelial cells to
subcellular fractions of NTHi clinical isolates and compared the resulting profiles of IL-8 secretion using enzyme-linked immunosorbent
assays (ELISA). Previous work in this laboratory showed that the secreted fraction of NTHi clinical isolates and H.influenzae Rd was
responsible for approximately 60 to 80 % of the modulin activity. Proteomic analysis of the secreted modulin fraction revealed twenty-
seven unique proteins ranging in size from 25-kDA to 110-kDA. Database searches revealed four groups of candidate modulins including nutrient
transport proteins, biosynthetic proteins, stress proteins and hypothetical proteins. A preliminary analysis of a subset of these
proteins suggested that ClpB, OmpP2, TonB, and RelA may play a role in the IL-8 response. Current efforts in the laboratory are focused on
evaluating the remaining twenty-three candidate modulins for their ability to stimulate an IL-8 response from respiratory epithelial
cells using an Escherichia coli expression system. Further, modulin-deficient mutants of H. influenzae Rd are being constructed to confirm
their role in stimulating a proinflammatory response. In conclusion, our results suggest that secreted factors other than LOS contribute to
the NTHi stimulation of respiratory epithelial cell IL-8 secretion.

Streptococcus iniae exploits the host environment to cause a systemic infection

Wayne State University School of Medicine, Detroit, MI

Streptococcal systemic pathogens such as Streptococcus agalactiae and Streptococcus pneumoniae cause serious morbidity and mortality worldwide. Although a great deal of valuable work has been reported on streptococcal pathogenesis, achieving conclusive results on virulence mechanisms has been problematic because many streptococcal species are human specific and lack a good animal model. Our lab employs a zebrafish host model to study systemic infections caused by S. iniae, a natural systemic pathogen of both fish and humans. We have demonstrated that S. iniae systemic infections in zebrafish closely mimic the clinical pathologies caused by S. agalactiae and S. pneumoniae infections in humans. One virulence factor found in common in systemic pathogens is polysaccharide capsule. The capsule is important in circumventing the normal innate immune response of the host. In an S. iniae infection of zebrafish, the bacteria quickly disseminate from the site of injection, the dorsal muscle, to all organs of the fish. Host inflammatory cells aid in this process by engulfing and transporting the bacteria from the muscle. We hypothesize that the capsule is protecting the bacteria in this environment, and, that this mechanism requires a high level of regulation of capsule expression. The specifics of regulation of capsule and its role in dissemination are currently being investigated. The use of the zebrafish-S. iniae infection model will provide an understanding of S. iniae pathogenesis, as well as insight into the mechanisms used by streptococcal systemic pathogens to disseminate and cause disease.


Characterization of NG1684, a cell contact regulated gene of Neisseria gonorrhoeae

Hilary A. Phelps* and Cindy G. Arvidson
Department of Microbiology and Molecular Genetics
Michigan State University, East Lansing, MI

Upon introduction to a new host it is essential that the exclusively human pathogen Neisseria gonorrhoeae adapt and respond to a changing host milieu, in which the initial interactions with host epithelial cells induce critical differential regulation of bacterial factors necessary for establishment of a successful infection.  The primary goal of the research presented here is to examine NG1684, a gene that has been identified as being significantly upregulated in response to host epithelial cell contact. Transposon mutagenesis, quantitative real time PCR, and a tissue culture model of infection were employed to characterize the role of NG1684 in gonococcal infection and the regulation of NG1684 in response to host cell contact.  We have determined that a transposon insertion in NG1684 in N. gonorrhoeae strain MS11 results in a decreased ability to invade the endometrial cell line Hec1B, although the ability of this mutant to adhere to these cells is indistinguishable from the wild type strain.  Investigation of the flanking regions of NG1684 showed that this gene is divergently transcribed from farAB, which encodes an efflux pump responsible for conferring resistance to host derived antibacterial factors.  The transcriptional regulator, FarR, binds to multiple sites in the intergenic region between NG1684 and farAB and has been shown to repress farAB expression.  We have evidence to suggest that FarR also regulates NG1684 expression.  FarR expression has been shown to be regulated by MtrR, which also regulates mtrCDE.  This operon encodes a separate efflux pump responsible for resistance to a broad range of structurally diverse antimicrobial compounds.  Moreover, mtrR expression was identified as being upregulated in response to host cell contact in addition to NG1684.  This observation is consistent with our hypothesis that FarR represses NG1684, as an increase in MtrR would lead to a reduction of FarR and a subsequent increase in NG1684 expression.  Based on the fact that FarR and MtrR mediate resistance to host derived antimicrobial agents in N. gonorrhoeae, we hypothesize that NG1684 may be involved in response to and/or resistance to antimicrobial compounds.


Trolling for Streptococcus pyogenes virulence genes using a zebrafish host model
Anne E. Kizy* and Melody N. Neely
Wayne State University, School of Medicine
Department of Immunology and Microbiology

Streptococcus pyogenes is an important human pathogen that can respond and adapt to changing environments. Although in vitro analyses have proven invaluable in determining specific streptococcal virulence factors, such studies are limited in their ability to provide a natural environment in which the invading pathogen responds directly, as well as indirectly, to the host conditions and defense systems. The goal of this study is to identify S. pyogenes virulence genes important for pathogenesis within the host using signature-tagged mutagenesis. Currently there are no reports in the literature of this technique being applied to S. pyogenes. Moreover, a novel animal model, the zebrafish, is used, which has been proven to mimic the responses involved in human streptococcal infection. The importance of this study is underscored by the fact that the variety and severity of S. pyogenes infections are on the rise worldwide. Thus, the current state of antimicrobial therapy is lacking in its aim to contain or eradicate this seemingly ubiquitous human pathogen. Therefore, novel strategies are necessary and can only be accomplished with the aid of in vivo analyses in determining the molecular mechanisms involved in S. pyogenes pathogenesis.


Incidence of polyglutamine insertions (poly-Q) in Escherichia coli: A model for polyglutamine expansion in genetic diseases in humans?

Meghna Shukla, Sijana H. Dzinic, Tammy S. Ram, Ilir Mandija, and Jeffrey L. Ram
Wayne State University, School of Medicine
Department of Immunology and Microbiology

Background:  Expansions of trinucleotide regions coding for glutamines (Qs) are characteristic of several human genetic diseases, including Huntington’s disease and cancer. The mechanisms mediating poly-Q expansions in human genetic diseases are not well understood. Recently, our lab discovered trinucleotide expansions coding for Qs in the tsr gene, the serine methyl-accepting chemotaxis receptor gene.The common laboratory E. coli strain, K-12, has a sequence coding for 4 Qs that could be the core of poly-Q expansion in E. coli. This study investigates the incidence of poly-Q expansions in the tsr gene of a subset of wild E. coli strains derived from different hosts.

Methods: Initially, expanded poly-Q strains were discovered during sequencing of the conserved region of the tsr gene in 20 diverse E. coli strains isolated from animal feces. Subsequently, PCR primers flanking the poly-Q expansion region were used to identify poly-Q regions of different lengths. Electrophoresis utilizing 3% agarose gels was used as a method to differentiate between the variable lengths of PCR products which are dependent upon tsr gene expansions. Searches of Genbank revealed additional tsr poly-Q variants.

Results: Survey of 37 wild E. coli strains revealed that the incidence of tsr gene expansions coding for poly-Q sequences of the same or longer length as K-12 is as follows: 4Q, 8 strains (22%); 7Q, 24 strains (65%); 13Q, 5 strains (14%). Presently, more strains are being surveyed; however, according to these results, most natural strains of E. coli have a sequence of 7 glutamines. Bioinformatic analysis revealed one 10Q strain (E. coli HS) and that O157:H7 and UTI pathogenic E. coli strains have 7Qs. Experiments are in progress to determine the stability of inheritance of the large poly-Q sequences.

Conclusion: E. coli has a naturally occurring variable poly-Q region. Studies to determine the mechanisms that bring about expansion or deletion of codons in this region may reveal mechanisms that mediate similar phenomena in human genetic diseases.


A study of common and strain-specific Mycobacterium avium subspecies paratuberculosis infection induced transcriptome changes in bovine monocyte-derived macrophages.

1Department of Biochemistry and 2Center for Animal Functional Genomics, Department of Animal Science, Michigan State University, East Lansing, MI. 3Center for Animal Health and Food Safety, University of Minnesota, Minneapolis, MN.

Mycobacterium avium subspecies paratuberculosis (MAP) is an intracellular pathogen that causes an economic burden to US dairy industries estimated at over one billion dollars annually.  MAP has also been linked to some cases of human Crohn’s disease.  A hallmark of MAP infection is survival in host macrophages, cells that normally destroy ingested microbes.   As with other mycobacteria, survival in macrophages appears to be a key determinant of pathogenesis associated with MAP infections.   Based on previous studies relating MAP infection with altered macrophage gene expression, we hypothesized that different strains of MAP would have both common and strain-specific effects on macrophage cell gene expression.   To test this hypothesis, we have now studied the effect of 10 different MAP strains on macrophage gene expression profiles, with the ultimate goal of relating gene expression differences to virulence and genetics of the MAP strains. Initial data analysis suggests that there are over 120 macrophage genes whose expression is generally altered following infection with any strain of MAP.  Upon hierarchical clustering using fold-change data, MAP strains isolated from different species showed little initial host species similarity, but two highly virulent strains clustered together, perhaps suggesting these two strains have similar effects on bovine macrophage cells.


Oral Bacteria Counts on Reused Water Bottles

Delta College, University Center, MI

Many people re-use water bottles after purchasing bottled water. This investigation was done in order to measure the numbers and types of bacteria found on the mouths of the bottles after repeated re-use. We found that even with washing, after multiple uses bacteria do accumulate on the mouths of the bottles. However, as expected, the bacteria identified were all normal resident oral flora.


Using PCR-DGGE to determine the bacterial diversity and identity of cloacal samples from Tree Swallows (Tachycineta bicolor)

Jennifer E. Klomp*, Dave Diekema, and Gregory M. Colores
Central Michigan University

Cloacal swabs were collected from 36 nestling Tree Swallows (Tachchycineta bicolor), DNA was extracted from the swabs and amplified. Amplified DNA was separated by use of denaturing gradient gel electrophoresis (DGGE). Bands of interest were picked, re-amplified, sequenced, and identified using GenBank. Gradient gel banding patterns were analyzed using Gel2K and similarity indices were calculated using a Jaccard’s Similarity Index. The similarity of bands separated by DGGE ranged drastically between individuals within a nest (4.76 - 100%). These results indicate that in some cases nest mates appear to have an identical cloacal microbial flora, whereas in other nests a uniform microbial flora was not found. DNA sequencing of DGGE bands identified two known avian pathogens present in a number of nestlings, Suttonella ornithocola and Mycoplasma sturnidae. S. ornithocola was found in 10 of the 24 birds sampled and M. sturnidae was found in 15 of the 24 birds sampled.

The Effect of Prostaglandin on Candida albicans-Dendritic Cell Interactions

Wayne State University, School of Medicine
Department of Immunology and Microbiology

Candida albicans is a dimorphic (hyphal or yeast), opportunistic fungal pathogen, which poses a significant clinical threat to immunocompromised individual.  Diseases associated with this fungus ranges from systemic to superficial mucosal hypersensitivity responses. The mechanisms by which Candida persists at mucosal surfaces in the face of an adaptive response are unclear. Candida produces immunomodulatory oxylipins that cross-react functionally with host eicosanoids, which are considered to play important roles in innate and adaptive immune responses.   At the mucosal surface, dendritic cells (DC) direct the type of T-cell responses after interacting with pathogen at mucosal surface. Yeast forms induce protective DC1/Th1 responses, while more virulent hyphal forms induce non-protective DC2/Th2 responses. Interestingly, previous studies have also showed that host eicosanoid (PGE2) increases the transformation from yeast-to-hyphae which can cause the fungus to become persistent and virulent. Our objective is to characterize the role of prostaglandins produced by the host and this fungus in pathogenesis in vivo and during Candida-dendritic cell interactions. We hypothesize that production of oxylipin by both Candida and host are required for persistent infection. We are testing this hypothesis by examining effects of host and fungal prostaglandins on DC cytokine profiles and also maturation and activation markers in the presence of yeast or hyphae. To address whether the effects of prostaglandins are host-derived, we are testing responses in DCs isolated from COX-2 deficient mice (an enzyme required for synthesis of eicosanoids). Understanding the mechanisms by which Candida modulates our immune system will provide new strategies to treat infection caused by this pathogen.


Isolation of Spontaneous Suppressors of YopE in Saccharomyces cerevisiae
Western Michigan University, Kalamazoo, MI.
       Yersinia spp. are the causative agents of bubonic plague and other enteric diseases. Type III secretion is employed by Yersinia adhering at the cell surface to deliver a cocktail of effector proteins (Yops) into the cytosol where they interact with cellular proteins. YopE disrupts actin localization (GAP) at the cell periphery resulting in the inability of immune cells to destroy the bacterial invader. Our goal is to identify the cellular targets and mechanisms that YopE uses when it is injected into cells so as to develop a way to combat plague.
       For our studies, we have identified spontaneous suppressors of YopE induced lethality in the yeast, Saccharomyces cerevisiae. A vector expressing YopE under the control of the inducible GAL1 promoter was constructed and transformed into diploid WT yeast.  This vector kills yeast upon addition of galactose.  Independent colonies that arose spontaneously on galactose containing plates were selected for further study.
      A vector expressing YopE under the control of the inducible GAL1 promoter kills 90% of the yeast after 3 hrs.  Seven unique suppressors have been selected and crossed to examine the number of recombination groups identified.  We are currently examining the growth characteristics of YopE expressing strains in conjunction with each recombination group.
      Expression of YopE is lethal in yeast, with no obvious cell cycle arrest associated with cytotoxicity. In our growth assay, 10% of cells are viable 3 hours after induction compared to empty vector controls.


Establishment of a Method to Examine Plant Pathogen Effectors

Vanessa Revindran*, and J. R. Geiser
Department of Biological Sciences, Western Michigan University, Kalamazoo, MI

Our aim is to create a model system to study the action of plant pathogens when they infect plant cells. We have established an expression system in Saccharomyces cerevisiae to study HopM1. The expression vector contains an inducible GAL1 promoter for the expression of HopM1. It also contains a V5 epitope that will be used for visualization of HopM1 and 6xHIS tag for purification.

We have begun to amplify additional Hop genes using Polymerase Chain Reaction (PCR). The fragments are first cloned into the pCR-Blunt II-TOPO vector and sequenced for verification of correctness. Subsequently they are transferred to yeast plasmids for expression. HopM1 when expressed in yeast is lethal on solid media. Examination of the lethal effect using a titer assay has not duplicated the effect seen on solid media, suggesting that the effect seen is a delay or arrest of growth but not an outright cytotoxic effect. Results will be presented to show the effect of the growth rates and comparison of death, compared to WT strains containing HopM1.


Identification of a unique gene expression signature in total leukocytes from cattle with Johne’s disease

J. Brighton1, M. Romeih1, J. Steibel1, G. Rosa2, and P. Coussens1.
Center for Animal Functional Genomics, Michigan State University, East Lansing, MI, USA1. Department of Animal Science, University of Wisconsin-Madison, Madison WI, USA2.

 Johne's disease is a progressive, debilitating disease of all ruminant animals for which there is no treatment. It is caused by Mycobacterium paratuberculosis, which has been implicated as a cause of Crohn's disease in some humans. Control of Johne's disease has been difficult because of the lack of a gold standard test and poor accuracy of the available diagnostic tests. Many diagnostic tools, including culture, serological and molecular methods have been developed for MAP detection, but there is no single, infallible test.  As current tests may fail to detect subclinical infection, there is a need for new diagnostic approaches. In a previous study using the bovine BOTL-5 cDNA microarray, we found that gene expression profiles of bovine total leukocytes from MAP infected cows were inherently different from those of healthy control cows.  Statistical analysis revealed significant differences in expression of 77 genes in infected cattle compared to control cattle. The present study was conducted to test the generality of this apparent MAP infection “signature”.  To accomplish this, expression levels of 45 genes selected from our previous microarray results, and three control genes (-actin, GAPDH and RPL19), were analyzed using a high-throughput Q-RT-PCR system in populations of 84 subclinically infected and 21 control cattle.  Analysis of the Q-RT-PCR data was accomplished using a linear mixed model.  This analysis revealed that expression levels of genes encoding   Midnolin, CD30L, A Riken cDNA clone, Immediate early R5, IL 6ar and Chemokine C-X-C motif receptor 6 were consistently different in freshly isolated leukocytes from MAP infected relative to control cattle.  Our novel results suggest that MAP infection has a dramatic effect on peripheral leukocyte gene expression patterns that these patterns are consistently different than those observed in healthy controls, and that such an infection “signature” may have potential in developing new diagnostic procedure.  In addition, our results may provide unique insights into interactions between MAP and the host immune system.



Western Michigan University, Kalamazoo, MI

We propose to use Saccharomyces cerevisae to identify suppressors of YopO in an effort to understand YopO’s cellular targets.  We have established an expression system in Saccharomyces cerevisae to study YopO. We took advantage of the lethality imposed by YopO to screen for spontaneous revertants.  Two YopO plasmids with unique auxotrophic markers were transformed into a homozygous diploid yeast strain.   The mutation rate for our screen was consistent with the normal mutation rate in yeast, and suggests that the suppressors identified are dominant or co-dominant. 

            From the suppressor screen we identified 11 independent suppressors of the YopO imposed lethality.  Upon sporulation three of these mutations are lethal suggesting the mutation is in an essential gene. Results will be presented to show the effect of the suppressors strain on growth, viability, localization and expression of YopO. 


Genes Associated with E. coli Pathogenicity in Freshwater Beach Sand
Biology Department, Central Michigan University, Mount Pleasant, MI, 48859

This study was conducted to determine whether pathogenic strains of E. coli are present in Great Lakes beach sand. Two of the most important groups of E. coli pathogens are the enteropathogenic and the enterohemorrhagic E. coli. Pathogenicity is associated with specific attachment and toxin proteins. PCR analysis was used to determine whether the genes that code for these proteins could be detected in E. coli recovered from beach sand. Sand was collected during the summers of 2005 and 2006, filtered to collect sand-associated bacteria, and incubated on mTEC agar to recover E. coli. Total DNA was extracted from filters and used as template in the polymerase chain reaction. Over 100 sand filters have been analyzed. Shiga toxin genes were rarely detected by this method. In contrast, the genes associated with intestinal attachment are readily detectable and appear to be common in sand-associated E. coli, nearly 40% of samples were positive for bfp and 80% were positive for eae. The detection of bfp suggests the presence of E. coli of human origin in the sand. Both of these attachment genes are found on mobile genetic elements. Considering the high densities of E. coli that can be present in beach sand, the potential for horizontal exchange of these pathogenicity-associated genes to resident bacteria should be considered.


NON-STUDENT POSTERS (not eligible for judging)

In Vitro Antibacterial Activity of a Novel Trifluorophenyl Oxazolidinone Compared to Linezolid and Other Antibacterial Agents Against 1220 Recent Bacterial Clinical Isolates
M.D. Huband, P.J. Pagano, J. W. Gage, D.L. Hanna, L.A. Skerlos, J.V.N. Vara Prasad, J. Brieland
Presenter at the meeting: Joe Penzien
Pfizer Global R&D, Ann Arbor, MI

Background: The continuing emergence of resistance in Gram-positive bacterial species (multi-drug resistant S. pneumoniae, vancomycin-R Enterococci (VRE), community acquired MRSA (CAMRSA), Vancomycin-intermediate S. aureus (VISA)) has created the need for new antibacterial compounds. PF-7296 was developed as part of a program to introduce an orally active oxazolidinone to treat infections caused by susceptible and resistant Gram-positive bacterial strains. This study investigated the antimicrobial activity of PF-7296, linezolid, and conventional antibacterials against 1220 geographically diverse recent bacterial clinical isolates.

Methods: Microbroth dilution MICs (expressed in ug/mL) and their interpretation followed CLSI guidelines.


_________________________________           PF-7296 MICs (ug/mL)_______

Organism                                   No. Isolates       MIC50               MIC90               Range

S. aureus MSSA                       20                     4                       4                       2-4

S. aureus MRSA                       81                     4                       4                       2-4

S. aureus VISA                          4                     8                       --                      8

S. epidermidis MRSE               23                     1                       1                       0.5-2

S. pneumoniae PSSP                24                     2                       2                       1-2

S. pneumoniae PISP                 28                     2                       2                       1-4

S. pneumoniae PRSP               36                     2                       2                       1-4

S. pneumoniae Levo-R             26                     2                       2                       1-2

E. faecalis Van A                     14                     2                       2                       1-4

E. faecalis Van B                      22                     2                       4                       2-4

E. faecium                                13                     4                       4                       2-4

E. faecium Van A                     45                     2                       4                       1-4


PF-7296 MIC90s ranged 1-4 ug/mL versus clinically significant Gram-positive bacterial pathogens. At 2x MIC, PF-7296 displayed a very low frequency of spontaneous resistance development (<6.7 x 10 -11) against both Staphylococcus aureus (SA-1) and Streptococcus pneumoniae (SP-3).

Conclusions: This study confirms the in vitro antibacterial potency of PF-7296 against clinically significant Gram-positive organisms and a low frequency of spontaneous resistance development. (received February 15)

In Vitro Antibacterial Activity of Novel Hygromycin A Analogs Compared to Levofloxacin and Other Antibacterial Agents Against 1220 Recent Clinical Isolates

M.D. Huband1, K. Brighty2, R. Monahan2, S. Brickner2, S. Wade2, P. Le3
Presenter at the meeting: Joe Penzien
1Pfizer Global R&D, Ann Arbor, MI,  2Pfizer Global R&D, Groton, CT, 3Pfizer Global R&D, La Jolla, CA.

Background: The continuing emergence of resistance in Gram-positive bacterial species (multi-drug resistant S. pneumoniae, vancomycin-R Enterococci (VRE), community acquired MRSA (CAMRSA), Vancomycin-intermediate S. aureus (VISA)) has created a need for new antibacterial compounds. CE-6811, CP-9474, and CP-9898 were evaluated as part of an effort to treat serious bacterial infections caused by susceptible and multi-drug resistant Gram-positive organisms.  This study investigated the in vitro antimicrobial activity of CE-6811, CP-9474, CP-9898, levofloxacin, and conventional antibacterials against 1220 geographically diverse recent clinical isolates.

Methods: Microbroth dilution MIC90s (expressed in ug/mL) and their interpretation followed CLSI guidelines.


________________________________           MIC90s (ug/mL)_____                              _

Organism (# tested)                     CE-6811          CP-9474         CP-9898        Levofloxacin

Staphylococcus aureus (105)               2                       4                       8                       32

S. epidermidis (30)                              1                       2                       2                       64

Coag. neg. Staphylococci (23)              2                       4                       4                       32

Streptococcus pneumoniae (117)         0.5                    1                       2                       16

Streptococcus spp. (87)                        1                       2                       2                       2

Enterococcus faecalis (59)                   2                       4                       4                       32

E. faecium (65)                                    2                       4                       8                       64

Corynebacterium spp. (20)                  1                       4                       4                       32


Conclusions: This study confirms the high in vitro antibacterial potency of CE-6811, CP-9474, and CP-9898 relative to levofloxacin. This activity was maintained against multi-drug resistant isolates including: penicillin- and levofloxacin-R S. pneumoniae, VRE, MRSA, CAMRSA, VISA, and MRSE.  (received Feb 15)


Laser-Induced Breakdown Spectroscopy: A Novel Technology for the Rapid Detection, Identification, and Discrimination of Biological Agents

1Department of Physics and Astronomy and 2Department of Immunology and Microbiology
Wayne State University, Detroit, Michigan

Laser-Induced Breakdown Spectroscopy (LIBS) is an atomic spectroscopic technique that utilizes an intense laser pulse to ablate a target and seed the constituent atoms into a high temperature microplasma.  Characteristic optical emission from the plasma uniquely identifies the elemental composition of the vaporized target.  Due to its inherent speed, accuracy, portability, and simplicity, LIBS is currently being investigated as an early-warning technology for the real-time detection and identification of harmful biological agents.

We have utilized LIBS to rapidly identify and discriminate between four strains of Escherichia coli, one strain of environmental mold and one strain of Candida albicans yeast.  This was the first ever demonstration of a rapid, efficient discrimination between different strains of a single bacteria species based on the LIBS technology.  The effects of the bacteria growth environment were investigated by preparing samples of Pseudomonas aeruginosa on three different nutrient media.  Nearly identical spectra were obtained from P. aeruginosa grown on TS agar and blood agar plates, while the bacteria grown on a MacConkey plate exhibited easily distinguishable differences indicating a chemical composition change, most likely in the outer membrane of the bacteria.  All samples of P. aeruginosa were easily discriminated from all E. coli strains.   


Isolation of Highly Beta-Hemolytic Bacteria for Undergraduate Education

G. Pocialik, and J. Graves*
University of Detroit Mercy, Department of Biology, Detroit, MI

Beta-hemolytic bacteria were isolated from the environment as a pedagogical exercise. Microbes in aqueous soil suspension were separated by streak plate inoculation on sheep blood agar. After incubation at 37oC, among colonies of various sizes and pigmentation were some demonstrating a high degree of beta-hemolysis. The appearance of selected colonies was recorded by a low power computer microscope (Intel). Computer enhancement increased contrast and opportunity for learning. The Gram stain revealed gram-positive bacilli and staining of aged culture with malachite green showed spores. The hanging drop slide revealed motility. An antibiogram was prepared by the antibiotic disc technique. Resistance to carbenicillin, sensitivity to kanamycin and standard biochemical tests (glucose, citrate, mannitol, indole and catalase) provided results consistent with those for Bacillus cereus. Identification was supported by the Biolog automated redox-based system. The organism is recognized as a significant cause of food poisoning. Isolation of beta-hemolytic bacteria from nature could acquaint students with hemolysis and the characteristics of Bacillus species of interest to public health.


Antimicrobial Resistance in Escherichia coli Isolates from Urinary Tract Infections in the Detroit, Michigan Area

1Oakland University, Rochester, MI, 2William Beaumont Hospital, Royal Oak, MI

Background: The most common cause of urinary tract infections (UTIs) is Uropathogenic Escherichia coli (UPEC). Antimicrobial resistance in UPEC isolates is highly varied and increasing. Objective: To study UPEC strains in the Detroit area to assess antimicrobial resistance, which may lead to improved treatment of UTIs. Methods: Antimicrobial resistance data from 500 isolates of UPEC strains from UTIs were collected from William Beaumont Hospital Laboratories in Royal Oak, Michigan during 2006. The antimicrobial susceptibilities were performed on a Dade-Behring Microscan Walkaway, with interpretation of susceptibility testing according to CLSI guidelines. Results: Of the 500 culture isolates, 52% showed no resistance to any of the drugs tested, and 48% were resistant to at least one drug. The resistant isolates showed a range of resistance from 1 to as many as 20 different drugs each. 77% of the resistant isolates were resistant to more than 1 drug, and 24% were resistant to more than 5 drugs. 52% of the resistant isolates were resistant to 1 or more of the combination drugs (Trimethoprim/Sulfamethoxazole, Ampicillin/Sulbactam). Conclusions: Clearly, these results show resistance patterns among local UPEC strains that are disturbing; which will require further study of their resistance patterns to distinguish strains with highly abnormal patterns.