Biomedical subjects

Monica K Borucki

Publications and source records attributed to Monica K Borucki.

18 recordsLinked to original sources

PubMed2006

A single-nucleotide-polymorphism-based multilocus genotyping assay for subtyping lineage I isolates of Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and is the leading microbiological cause of food recalls. Lineage I isolates of L. monocytogenes are a particular public health concern because they are responsible for most sporadic cases of listeriosis and the vast majority of epidemic outbreaks. Rapid, reproducible, and sensitive methods for differentiating pathogens below the species level are required for effective pathogen control programs, and the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping food-borne pathogens. Therefore, we developed a multilocus genotyping (MLGT) assay for L. monocytogenes lineage I isolates based on nucleotide variation identified by sequencing 23,251 bp of DNA from 22 genes distributed across seven genomic regions in 65 L. monocytogenes isolates. This single-well assay of 60 allele-specific probes captured 100% of the haplotype information contained in approximately 1.5 Mb of comparative DNA sequence and was used to reproducibly type a total of 241 lineage I isolates. The MLGT assay provided high discriminatory power (Simpson's index value, 0.91), uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clone I (ECI), ECIa, and ECII. In addition, the assay included probes for a previously characterized truncation mutation in inlA, providing for the identification of a specific virulence-attenuated subtype. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses, and epidemiological investigations. DNA sequences were deposited in the GenBank database under accession numbers DQ 812146 to DQ 812517, DQ 843664 to DQ 844598, and AY 512391 to AY 512502.

Bacterial Proteins↗

PubMed2006

Role of uvrA in the growth and survival of Listeria monocytogenes under UV radiation and acid and bile stress.

Listeria monocytogenes encounters numerous stresses both in the food environment and during infection of the host. The ability to survive and tolerate bile and low pH conditions, which are two major stresses, is of particular importance for survival within the host. The uvrA gene in other bacteria is involved in the repair of acid-induced DNA damage and in adaptation to low pH. Thus, a uvrA in-frame deletion mutant was constructed to identify the role of uvrA in the growth and survival of L. monocytogenes under various environmental conditions. The uvrA mutant was highly sensitive to UV radiation. Growth under normal laboratory conditions was impaired during the exponential phase, and the time to reach the exponential phase of growth, TV(max), was significantly delayed (P < 0.05). Growth of the uvrA mutant in acidic medium (pH 5) was slightly impaired, and the TV(max) was significantly delayed (P < 0.05). Growth and the TV(max) of the mutant in the presence of 0.3% bile salts also were significantly impaired (P < 0.05). These results suggest that uvrA is needed for optimal growth and survival of L. monocytogenes under various stressful environmental conditions.

Bacterial Proteins↗

PubMed2005

Genetic diversity of Listeria monocytogenes strains from a high-prevalence dairy farm.

Listeria monocytogenes is a significant food-borne human and veterinary pathogen. Contaminated silage commonly leads to disease in livestock, but the pervasive nature of the bacterium can make it difficult to identify the source of infection. An investigation of bovine listeriosis that occurred on a Pacific Northwest dairy farm ("farm A") revealed that the clinical strain was closely related to fecal strains from asymptomatic cows, and that farm environment was heavily contaminated with a diversity of L. monocytogenes strains. In addition, the farm A clinical strain was closely related to clinical and environmental strains obtained 1 year prior from a second Northwest dairy farm ("farm B"). To investigate the source(s) of contamination on farm A, environmental samples were collected from farm A at two time points. Pulsed-field gel electrophoresis characterization of 538 isolates obtained from that farm identified 57 different AscI pulsovars. Fecal isolates obtained from individual cows were the most genetically diverse, with up to 94% of fecal samples containing more than one pulsovar. The maximum numbers of pulsovars and serotypes isolated from a fecal sample of one cow were 6 and 4, respectively. Serotype 1/2a was isolated most frequently at both time points. Microarray genotyping of bovine listeriosis, fecal, and silage strains from both farms identified four probes that differentiated listeriosis strains from environmental strains; however, no probe was common to both bovine listeriosis strains.

Animals↗

PubMed2005

Suspension microarray with dendrimer signal amplification allows direct and high-throughput subtyping of Listeria monocytogenes from genomic DNA.

Listeria monocytogenes is a significant cause of food-borne disease and mortality; therefore, epidemiological investigations of this pathogen require subtyping methods that are rapid, discriminatory, and reproducible. Although conventional microarray subtyping analysis has been shown to be both high resolution and genetically informative, it is still relatively low throughput and technically challenging. Suspension microarray technology eliminates the technical issues associated with planar microarrays and allows high-throughput subtyping of L. monocytogenes strains. In this study, a suspension array assay using dendrimer signal amplification allowed rapid and accurate serovar identification of L. monocytogenes strains using genomic DNA as a target. The ability to subtype genomic DNA without PCR amplification allows probes to be designed for many different regions within the bacterial genome and should allow high-resolution subtyping not possible with multiplex PCR.

Bacterial Typing Techniques↗

PubMed2004

Oral inoculation of A/J mice for detection of invasiveness differences between Listeria monocytogenes epidemic and environmental strains.

Four-week-old Harlan A/J mice were orally infected with six epidemic and six environmental strains of Listeria monocytogenes. Epidemic strains were significantly more invasive as a group than were environmental strains (P < 0.05), and the intestines of some mice infected with epidemic strains had extensive hemorrhage. Mice inoculated with epidemic strains were significantly more likely to become systemically infected than mice inoculated with environmental strains (P < 0.01).

Animals↗

PubMed2004

Intraspecific phylogeny and lineage group identification based on the prfA virulence gene cluster of Listeria monocytogenes.

Listeria monocytogenes is a serious food-borne pathogen that can cause invasive disease in humans and other animals and has been the leading cause of food recalls due to microbiological concerns in recent years. In order to test hypotheses regarding L. monocytogenes lineage composition, evolution, ecology, and taxonomy, a robust intraspecific phylogeny was developed based on prfA virulence gene cluster sequences from 113 L. monocytogenes isolates. The results of the multigene phylogenetic analyses confirm that L. monocytogenes comprises at least three evolutionary lineages, demonstrate that lineages most frequently (lineage 1) and least frequently (lineage 3) associated with human listeriosis are sister-groups, and reveal for the first time that the human epidemic associated serotype 4b is prevalent among strains from lineage 1 and lineage 3. In addition, a PCR-based test for lineage identification was developed and used in a survey of food products demonstrating that the low frequency of association between lineage 3 isolates and human listeriosis cases likely reflects rarity of exposure and not reduced virulence for humans as has been previously suggested. However, prevalence data do suggest lineage 3 isolates may be better adapted to the animal production environment than the food-processing environment. Finally, analyses of haplotype diversity indicate that lineage 1 has experienced a purge of genetic variation that was not observed in the other lineages, suggesting that the three L. monocytogenes lineages may represent distinct species within the framework of the cohesion species concept.

Animals↗

PubMed2004

Selective discrimination of Listeria monocytogenes epidemic strains by a mixed-genome DNA microarray compared to discrimination by pulsed-field gel electrophoresis, ribotyping, and multilocus sequence typing.

Listeria monocytogenes can cause serious illness in humans, and subsequent epidemiological investigation requires molecular characterization to allow the identification of specific isolates. L. monocytogenes is usually characterized by serotyping and is subtyped by using pulsed-field gel electrophoresis (PFGE) or ribotyping. DNA microarrays provide an alternative means to resolve genetic differences among isolates, and unlike PFGE and ribotyping, microarrays can be used to identify specific genes associated with strains of interest. Twenty strains of L. monocytogenes representing six serovars were used to generate a shotgun library, and subsequently a 629-probe microarray was constructed by using features that included only potentially polymorphic gene probe sequences. Fifty-two strains of L. monocytogenes were genotyped by using the condensed array, including strains associated with five major listeriosis epidemics. Cluster analysis of the microarray data grouped strains according to phylogenetic lineage and serotype. Most epidemiologically linked strains were grouped together, and subtyping resolution was the same as that with PFGE (using AscI and ApaI) and better than that with multilocus sequence typing (using six housekeeping genes) and ribotyping. Additionally, a majority of epidemic strains were grouped together within phylogenetic Division I. This epidemic cluster was clearly distinct from the two other Division I clusters, which encompassed primarily sporadic and environmental strains. Discriminant function analysis allowed identification of 22 probes from the mixed-genome array that distinguish serotypes and subtypes, including several potential markers that were distinct for the epidemic cluster. Many of the subtype-specific genes encode proteins that likely confer survival advantages in the environment and/or host.

Bacterial Proteins↗

PubMed2004

Dairy farm reservoir of Listeria monocytogenes sporadic and epidemic strains.

Identifying the reservoirs of a pathogen is vital for control of sporadic disease and epidemics. Listeria monocytogenes is a zoonotic foodborne pathogen that is responsible for 28% of food-related deaths in the United States annually, as well as a major cause of massive product recalls worldwide. To examine the role of the dairy farm as a potential source or reservoir for L. monocytogenes subtypes shown to cause human listeriosis, we compared the pulsed-field gel electrophoresis (PFGE) restriction enzyme digestion profiles of L. monocytogenes dairy farm-associated strains (milk, environmental, and bovine) to human sporadic and epidemic disease strains. Twenty-three percent of human sporadic strains had PFGE patterns identical to that of farm isolate(s). Additionally, three farm environmental strains and one human sporadic strain had a PFGE pattern identical to a strain of L. monocytogenes responsible for the 1985 California epidemic. These data indicate that this epidemic strain continues to cause sporadic human illness and has a potential dairy farm as a reservoir.

Animals↗

PubMed2003

Discrimination among Listeria monocytogenes isolates using a mixed genome DNA microarray.

Listeria monocytogenes can cause serious illness in humans, usually following the ingestion of contaminated food. Epidemiologic investigation requires identification of specific isolates, usually done by a combination of serotyping and subtyping using pulsed-field gel electrophoresis (PFGE). DNA microarrays provide a new format to resolve genetic differences among isolates and, unlike PFGE, to identify specific genes associated with the infecting pathogen. A 585 probe, mixed genome microarray was constructed and 24 strains of L. monocytogenes were hybridized to the array. Microarray analysis allowed discrimination among L. monocytogenes isolates within a serotype and obtained from similar geographic and epidemiologic sources. Importantly, the microarray results preserved previously described phylogenetic relationships between major serogroups and, in a limited comparison, agreed with PFGE subtypes. The association of individual probes with isolates allowed identification of specific genes. Sequencing of 10 polymorphic probes identified nine matches with previously described bacterial genes including several suspected virulence factors. These results demonstrate that mixed genomic microarrays are useful for differentiating among closely related L. monocytogenes isolates and identifying genetic markers that can be used in epidemiologic and possibly pathogenesis studies.

Animals↗

PubMed2003

Detection of bacterial pathogens in environmental samples using DNA microarrays.

Polymerase chain reaction (PCR) is an important tool for pathogen detection, but historically, it has not been possible to accurately identify PCR products without sequencing, Southern blots, or dot-blots. Microarrays can be coupled with PCR where they serve as a set of parallel dot-blots to enhance product detection and identification. Microarrays are composed of many discretely located probes on a solid substrate such as glass. Each probe is composed of a sequence that is complimentary to a pathogen-specific gene sequence. PCR is used to amplify one or more genes and the products are then hybridized to the array to identify species-specific polymorphism within one or more genes. We illustrate this type of array using 16S rDNA probes suitable for distinguishing between several salmonid pathogens. We also describe the use of microarrays for direct detection of either RNA or DNA without the aid of PCR, although the sensitivity of these systems currently limits their application for pathogen detection. Finally, microarrays can also be used to "fingerprint" bacterial isolates and they can be used to identify diagnostic markers suitable for developing new PCR-based detection assays. We illustrate this type of array for subtyping an important food-borne pathogen, Listeria monocytogenes.

Actinomycetales Infections↗

PubMed2003

Variation in biofilm formation among strains of Listeria monocytogenes.

Contamination of food by Listeria monocytogenes is thought to occur most frequently in food-processing environments where cells persist due to their ability to attach to stainless steel and other surfaces. Once attached these cells may produce multicellular biofilms that are resistant to disinfection and from which cells can become detached and contaminate food products. Because there is a correlation between virulence and serotype (and thus phylogenetic division) of L. monocytogenes, it is important to determine if there is a link between biofilm formation and disease incidence for L. monocytogenes. Eighty L. monocytogenes isolates were screened for biofilm formation to determine if there is a robust relationship between biofilm formation, phylogenic division, and persistence in the environment. Statistically significant differences were detected between phylogenetic divisions. Increased biofilm formation was observed in Division II strains (serotypes 1/2a and 1/2c), which are not normally associated with food-borne outbreaks. Differences in biofilm formation were also detected between persistent and nonpersistent strains isolated from bulk milk samples, with persistent strains showing increased biofilm formation relative to nonpersistent strains. There were no significant differences detected among serotypes. Exopolysaccharide production correlated with cell adherence for high-biofilm-producing strains. Scanning electron microscopy showed that a high-biofilm-forming strain produced a dense, three-dimensional structure, whereas a low-biofilm-forming strain produced a thin, patchy biofilm. These data are consistent with data on persistent strains forming biofilms but do not support a consistent relationship between enhanced biofilm formation and disease incidence.

Animals↗

PubMed2003

Listeria monocytogenes serotype identification by PCR.

Serotyping is a universally accepted subtyping method for Listeria monocytogenes. Identification of the strain serotype permits differentiation between important food-borne strains (1/2a, 1/2b, and 4b) and provides a "gold standard" for comparing isolates analyzed in different labs and with different techniques. Although an efficient enzyme-linked immunosorbent assay serotyping protocol was described recently, identification of PCR serotyping primers would further increase the ease and accessibility of this classification system. Serotyping PCR primers were designed from variable regions of the L. monocytogenes genome. Three primer sets were used in conjunction with a previously described Division III primer set in order to classify 122 L. monocytogenes strains into five serotype groups [1/2a(3a), 1/2b, 1/2c(3c), 4b(d,e), and 4a/c]. Results of the PCR method agreed with those of the conventional slide agglutination method for 97, 100, 94, and 91% of strains belonging to serotypes 1/2a, 1/2b, 1/2c, and 4b, respectively.

Animals↗

PubMed2003

Serotyping of Listeria monocytogenes by enzyme-linked immunosorbent assay and identification of mixed-serotype cultures by colony immunoblotting.

Routine analysis of Listeria monocytogenes by serotyping using traditional agglutination methods is limited in use because of the expense and limited availability of commercially prepared antisera and intra- and interlaboratory discrepancies arising from differences in antiserum preparation and visual determination of agglutination. We have adapted a commercially available set of L. monocytogenes antisera to an enzyme-linked immunosorbent assay (ELISA) format for high-throughput, low-cost serotype determination. Rather than subjective visualization of agglutination, positive antigen and antiserum reactions were scored by a quantitative, colorimetric reaction. ELISA serotyping of 89 of 101 L. monocytogenes isolates agreed with slide agglutination serotyping data, and 100 previously uncharacterized isolates were serotyped unambiguously by the ELISA method. In addition, mixed-serotype cultures of L. monocytogenes were identified by a colony immunoblot procedure, in which serogroup 1/2 and serogroup 4 colonies were discriminated by differential staining.

Agglutination↗

PubMed2003

Mixed-genome microarrays reveal multiple serotype and lineage-specific differences among strains of Listeria monocytogenes.

Epidemiological studies and analysis of putative virulence genes have shown that Listeria monocytogenes has diverged into several phylogenetic divisions. We hypothesize that similar divergence has occurred for many genes that influence niche-specific fitness and virulence and that identifying these differences may offer new opportunities for the detection, treatment, and control of this important pathogen. To explore this issue further, we developed a microarray composed of fragmented DNA taken from 10 strains of L. monocytogenes. We then hybridized genomic DNA from 50 different strains to replicate arrays and analyzed the resulting hybridization patterns. A simple Euclidean distance metric permitted the reconstruction of previously described genetic relationships between serotypes, and only four microarray probes were needed to discriminate between the most important serotypes (1/2a, 1/2b, 1/2c, and 4). We calculated an index of linkage equilibrium from the microarray data and confirmed that L. monocytogenes has a strongly clonal population structure (I(A) = 3.85). Twenty-nine informative probes were retrieved from the library and sequenced. These included genes associated with repairing UV-damaged DNA, salt tolerance, biofilm formation, heavy metal transport, ferrous iron transport, and teichoic acid synthesis. Several membrane-bound lipoproteins and one internalin were identified, plus three phage sequences and six sequences with unknown function. Collectively, these data confirm that many genes have diverged between lineages of L. monocytogenes. Furthermore, these results demonstrate the value of mixed-genome microarrays as a tool for deriving biologically useful information and for identifying and screening genetic markers for clinically important microbes.

Cluster Analysis↗

PubMed2002

Complete nucleotide sequence of a Chilean hantavirus.

We have determined the genomic sequence of an Andes virus (ANDV) strain isolated from an infected Oligoryzomys longicaudatus rodent trapped in Chile in 1997. This strain, for which we propose the designation Chile R123, reproduces essential attributes of hantavirus pulmonary syndrome (HPS) when injected intramuscularly into laboratory hamsters (Hooper et al., Virology 289 (2001) 6-14). The L, M, and S segment sequences of Chile R123 are 6562, 3671, and 1871 nt long, respectively, with an overall G+C content of 38.5%. These respective genome segments could encode a 247 kd RNA-dependent RNA polymerase (RdRP), 126 kd glycoprotein precursor (GPC), and 48 kd nucleocapsid (N) protein, in line with other Sigmodontine rodent-associated hantaviruses. Among hantaviruses for which complete genomic sequences are available, Chile R123 is most closely related to Sin Nombre virus (SNV) strain NM R11, with greater than 85% amino acid identity between translated L and S segments and 78% amino acid identity between translated M segments. Because Chile R123 shares essentially 100% amino acid identity in regions of overlap with partially sequenced Argentinian and Chilean ANDV strains, Syrian hamster pathogenicity and the potential for interhuman transmission are features likely common to all ANDV strains.

Animals↗

PubMed2002

La Crosse virus: replication in vertebrate and invertebrate hosts.

La Crosse virus is maintained in a cycle involving mosquitoes and small mammals. Vertebrate cell infection is generally cytolytic; vector cell infection results in persistent infection. Features of La Crosse virus replication that may permit the virus to traffic between vector and vertebrate hosts and condition different infection outcomes are described.

Aedes↗

PubMed2001

The effect of mosquito passage on the La Crosse virus genotype.

The genetic consequences of passing three different strains of La Crosse (LAC) virus orally and transovarially in Aedes triseriatus mosquitoes were examined. Two of the LAC strains (WT LAC and LAC ORI) had been passaged numerous times in cell culture; the third strain (SM1-78) had been passaged only once in suckling mice. Genetic changes were monitored in three regions of the LAC genome after oral infection and dissemination in the mosquito, and transovarial transmission (TOT) of the virus to progeny. Sequence analyses were used to characterize the genetic changes occurring in regions of G1, G2 and N open reading frames (ORFs) during passage. Only one mutation was detected in the G1 ORF of SM1-78 virus after mosquito passage; however, numerous nucleotide and amino acid substitutions were detected in the G1 ORF of WT LAC and LAC ORI (cell culture-adapted viruses). In contrast to G1, the N and G2 ORF sequences examined were stable. Mutations introduced into viral genomes during replication in parental mosquitoes were expressed in progeny mosquitoes following TOT. Genetic diversity of virus populations from a single mosquito was examined by single-strand conformation polymorphisms analysis of the variable region of glycoprotein G1. LAC virus RNA genotype diversity was greatest in virus that infected and replicated in the midgut, and declined as virus disseminated from the midgut and infected ovaries and salivary glands.

Amino Acid Substitution↗

PubMed1999

Bunyavirus superinfection and segment reassortment in transovarially infected mosquitoes.

Rapid evolution of bunyaviruses may occur by RNA segment reassortment between closely related viruses. Reassortment between viruses occurs in dually infected mosquitoes when two different viruses are simultaneously ingested or when the second virus is ingested within 2 days of the first virus. By 3 days after oral infection, interference to superinfection occurs, thus limiting the potential for evolution. Aedes triseriatus mosquitoes can also be transovarially infected (TI+) with LaCrosse (LAC) virus. In these studies, the potential for oral superinfection of TI+ mosquitoes was assessed. Approximately 20% of mosquitoes TI+ with either a temperature-sensitive LAC virus or wild-type (wt) LAC virus became superinfected by ingesting blood meals containing wt LAC or snowshoe hare (SSH) viruses. LAC virus TI+ mosquitoes superinfected with SSH virus were detected by blot hybridization or RT-PCR. Viruses from these mosquitoes were plaque purified and genotyped using RT-PCR. Reassortant genomes were detected in 2.3% of the viruses genotyped, and 4.0% of the genomes tested were diploid for one genome segment.

Aedes↗