Unusually high illness severity and short incubation periods in two foodborne outbreaks of Salmonella Heidelberg infections with potential coincident Staphylococcus aureus intoxication.

We describe the investigation of two temporally coincident illness clusters involving salmonella and Staphylococcus aureus in two states. Cases were defined as gastrointestinal illness following two meal events. Investigators interviewed ill persons. Stool, food and environmental samples underwent pathogen testing. Alabama: Eighty cases were identified. Median time from meal to illness was 5·8 h. Salmonella Heidelberg was identified from 27 of 28 stool specimens tested, and coagulase-positive S. aureus was isolated from three of 16 ill persons. Environmental investigation indicated that food handling deficiencies occurred. Colorado: Seven cases were identified. Median time from meal to illness was 4·5 h. Five persons were hospitalised, four of whom were admitted to the intensive care unit. Salmonella Heidelberg was identified in six of seven stool specimens and coagulase-positive S. aureus in three of six tested. No single food item was implicated in either outbreak. These two outbreaks were linked to infection with Salmonella Heidelberg, but additional factors, such as dual aetiology that included S. aureus or the dose of salmonella ingested may have contributed to the short incubation periods and high illness severity. The outbreaks underscore the importance of measures to prevent foodborne illness through appropriate washing, handling, preparation and storage of food.

Next-generation sequencing technologies and their application to the study and control of bacterial infections.

BACKGROUND: With the efficiency and the decreasing cost of next-generation sequencing, the technology is being rapidly introduced into clinical and public health laboratory practice. AIMS: The historical background and principles of first-, second- and third-generation sequencing are described, as are the characteristics of the most commonly used sequencing instruments. SOURCES: Peer-reviewed literature, white papers and meeting reports. CONTENT AND IMPLICATIONS: Next-generation sequencing is a technology that could potentially replace many traditional microbiological workflows, providing clinicians and public health specialists with more actionable information than hitherto achievable. Examples of the clinical and public health uses of the technology are provided. The challenge of comparability of different sequencing platforms is discussed. Finally, the future directions of the technology integrating it with laboratory management and public health surveillance systems, and moving it towards performing sequencing directly from the clinical specimen (metagenomics), could lead to yet another fundamental transformation of clinical diagnostics and public health surveillance.

Nationwide outbreak of Salmonella Montevideo infections associated with contaminated imported black and red pepper: warehouse membership cards provide critical clues to identify the source.

In November 2009, we initiated a multistate investigation of Salmonella Montevideo infections with pulsed-field gel electrophoresis pattern JIXX01.0011. We identified 272 cases in 44 states with illness onset dates ranging from 1 July 2009 to 14 April 2010. To help generate hypotheses, warehouse store membership card information was collected to identify products consumed by cases. These records identified 19 ill persons who purchased company A salami products before onset of illness. A case-control study was conducted. Ready-to-eat salami consumption was significantly associated with illness (matched odds ratio 8·5, 95% confidence interval 2·1-75·9). The outbreak strain was isolated from company A salami products from an environmental sample from one manufacturing plant, and sealed containers of black and red pepper at the facility. This outbreak illustrates the importance of using membership card information to assist in identifying suspect vehicles, the potential for spices to contaminate ready-to-eat products, and preventing raw ingredient contamination of these products.

Effects of mining-derived metals on riffle-dwelling benthic fishes in Southeast Missouri, USA.

We studied the ecological effects of mining-derived metals on riffle-dwelling benthic fishes at 16 sites in the Viburnum Trend lead-zinc mining district of southeast Missouri. Fish community attributes were compared to watershed features and to physical and chemical variables including metal concentrations in sediment pore water and fish. Ozark sculpin (Cottus hypselurus), rainbow darter (Etheostoma caeruleum), Ozark madtom (Noturus albater), and banded sculpin (Cottus carolinae) were the most abundant fishes collected. Species richness and density of riffle-dwelling benthic fishes were negatively correlated with metal concentrations in pore water and in fish. Sculpin densities were also negatively correlated with metal concentrations in pore water and in fish, but positively correlated with distance from mines and upstream watershed area. These findings indicate that metals associated with active lead-zinc mining adversely affect riffle-dwelling benthic fishes downstream of mining areas in the Viburnum Trend. Sculpins may be useful as a sentinel species for assessing mining-related impacts on fish communities.

Ecological effects of lead mining on Ozark streams: In-situ toxicity to woodland crayfish (Orconectes hylas).

The Viburnum Trend mining district in southeast Missouri, USA is one of the largest producers of lead-zinc ore in the world. Previous stream surveys found evidence of increased metal exposure and reduced population densities of crayfish immediately downstream of mining sites. We conducted an in-situ 28-d exposure to assess toxicity of mining-derived metals to the woodland crayfish (Orconectes hylas). Crayfish survival and biomass were significantly lower at mining sites than at reference and downstream sites. Metal concentrations in water, detritus, macroinvertebrates, fish, and crayfish were significantly higher at mining sites, and were negatively correlated with caged crayfish survival. These results support previous field and laboratory studies that showed mining-derived metals negatively affect O. hylas populations in streams draining the Viburnum Trend, and that in-situ toxicity testing was a valuable tool for assessing the impacts of mining on crayfish populations.

Comparison of multiple-locus variable-number tandem repeat analysis, pulsed-field gel electrophoresis, and phage typing for subtype analysis of Salmonella enterica serotype Enteritidis.

Strain subtyping is an important tool for detection of outbreaks caused by Salmonella enterica serotype Enteritidis. Current subtyping methods, however, yield less than optimal subtype discrimination. In this study, we describe the development and evaluation of a multiple-locus variable-number tandem repeat analysis (MLVA) method for subtyping Salmonella serotype Enteritidis. The discrimination ability and epidemiological concordance of MLVA were compared with those of pulsed-field gel electrophoresis (PFGE) and phage typing. MLVA provided greater discrimination among non-epidemiologically linked isolates than did PFGE or phage typing. Epidemiologic concordance was evaluated by typing 40 isolates from four food-borne disease outbreaks. MLVA, PFGE, and, to a lesser extent, phage typing exhibited consistent subtypes within an outbreak. MLVA was better able to differentiate isolates between the individual outbreaks than either PFGE or phage typing. The reproducibility of MLVA was evaluated by subtyping sequential isolates from an infected individual and by testing isolates following multiple passages and freeze-thaw cycles. PFGE and MLVA patterns were reproducible for isolates that were frozen and passaged multiple times. However, 2 of 12 sequential isolates obtained from an individual over the course of 36 days had an MLVA type that differed at one locus and one isolate had a different phage type. Overall, MLVA typing of Salmonella serotype Enteritidis had enhanced resolution, good reproducibility, and good epidemiological concordance. These results indicate that MLVA may be a useful tool for detection and investigation of outbreaks caused by Salmonella serotype Enteritidis.

Expression analysis of the family of 14-3-3 proteins in zebrafish development.

14-3-3 proteins comprise a family of dimeric multi-functional proteins present in all eukaryotes, that are important in a whelm of ubiquitous biological processes. We have analyzed the genomic structure of all 14-3-3s from zebrafish comprising 11 genes and have analyzed their phylogeny. The gene family was cloned and its expression pattern in zebrafish embryogenesis was analyzed by whole mount in situ hybridization and microarray analysis with gene specific probes. We demonstrate that maternal mRNA of 14-3-3s is expressed evenly at the first cell division. At later stage all genes are expressed in a patterned way with, in most cases, intricate patterns in the developing brain. Our result shows distinct expression patterns of various genes. Microarray results show that differences in expression levels of highly similar 14-3-3 genes also occur in the adult stage.

Assessing contaminant sensitivity of endangered and threatened aquatic species: part II. Chronic toxicity of copper and pentachlorophenol to two endangered species and two surrogate species.

Early life-stage toxicity tests with copper and pentachlorophenol (PCP) were conducted with two species listed under the United States Endangered Species Act (the endangered fountain darter, Etheostoma fonticola, and the threatened spotfin chub, Cyprinella monacha) and two commonly tested species (fathead minnow, Pimephales promelas, and rainbow trout, Oncorhynchus mykiss). Results were compared using lowest-observed effect concentrations (LOECs) based on statistical hypothesis tests and by point estimates derived by linear interpolation and logistic regression. Sublethal end points, growth (mean individual dry weight) and biomass (total dry weight per replicate) were usually more sensitive than survival. The biomass end point was equally sensitive as growth and had less among-test variation. Effect concentrations based on linear interpolation were less variable than LOECs, which corresponded to effects ranging from 9% to 76% relative to controls and were consistent with thresholds based on logistic regression. Fountain darter was the most sensitive species for both chemicals tested, with effect concentrations for biomass at < or = 11 microg/L (LOEC and 25% inhibition concentration [IC25]) for copper and at 21 microg/L (IC25) for PCP, but spotfin chub was no more sensitive than the commonly tested species. Effect concentrations for fountain darter were lower than current chronic water quality criteria for both copper and PCP. Protectiveness of chronic water-quality criteria for threatened and endangered species could be improved by the use of safety factors or by conducting additional chronic toxicity tests with species and chemicals of concern.

Identification of a mutation associated with erythromycin resistance in Bordetella pertussis: implications for surveillance of antimicrobial resistance.

Erythromycin treatment failures and in vitro resistance of Bordetella pertussis have been reported on several occasions in the past few years, but the mechanism of resistance has not been described. One potential mechanism, genetic modification of the erythromycin-binding site on the 23S rRNA of the 50S ribosomal subunit, has been observed in other bacteria. To explore this possibility, we amplified the portion of the 23S rRNA gene encoding the central loop of domain V. DNA sequencing and restriction fragment length polymorphism of the PCR products showed that each of the four erythromycin-resistant B. pertussis strains tested contained an A-to-G transition mutation at position 2058 (Escherichia coli numbering) of the 23S rRNA gene. The mutation was not found in seven erythromycin-susceptible isolates tested. Two of the resistant isolates were heterozygous, containing at least one mutant copy and one wild-type copy of the 23S rRNA gene. These results indicate that erythromycin resistance in these strains is likely due to a mutation of the erythromycin-binding site in the 23S rRNA gene. Identification of the resistance mechanism will facilitate development of molecular susceptibility testing methods that can be used directly on clinical specimens in the absence of an isolate.

Molecular analysis of a hospital cafeteria-associated salmonellosis outbreak using modified repetitive element PCR fingerprinting.

A hospital cafeteria-associated outbreak of gastroenteritis due to Salmonella enterica serotype Infantis was retrospectively evaluated using modified repetitive element PCR (rep-PCR) fingerprinting with the ERIC2 and BOXA1R primers and computer-assisted gel analysis and dendrogram construction. Rep-PCR yielded objective between-cycler, same-strain similarity values of from 92% (composite fingerprints) to 96% (ERIC2 fingerprints). The 70 Salmonella isolates (which included 19 serotype Infantis isolates from the hospital outbreak, 10 other serotype Infantis isolates, and 41 isolates representing 14 other serotypes) were resolved well to the serotype level with each of the three fingerprint types (ERIC2, BOXA1R, and composite). Rep-PCR typing uncovered several historical serotyping errors and provided presumptive serotype assignments for other isolates with incomplete or undetermined serotypes. Analysis of replicate fingerprints for each isolate, as generated on two different thermal cyclers, indicated that most of the seeming subserotype discrimination noted in single-cycler dendrograms actually represented assay variability, since it was not reproducible in combined-cycler dendrograms. Rep-PCR typing, which would have been able to identify the presence of the hospital-associated serotype Infantis outbreak after the second outbreak isolate, could be used as a simple surrogate for serotyping by clinical microbiology laboratories that are equipped for diagnostic PCR.

Epidemiology and clonality of community-acquired methicillin-resistant Staphylococcus aureus in Minnesota, 1996-1998.

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged among patients in the general population who do not have established risk factors for MRSA. Records from 10 Minnesota health facilities were reviewed to identify cases of MRSA infection that occurred during 1996-1998 and to identify which cases were community acquired. Susceptibility testing and pulsed-field gel electrophoresis (PFGE) subtyping were performed on available isolates. A total of 354 patients (median age, 16 years) with community-acquired MRSA (CAMRSA) infection were identified. Most case patients (299 [84%]) had skin infections, and 103 (29%) were hospitalized. More than 90% of isolates were susceptible to all antimicrobial agents tested, with the exception of beta-lactams and erythromycin. Of 334 patients treated with antimicrobial agents, 282 (84%) initially were treated with agents to which their isolates were nonsusceptible. Of 174 Minnesota isolates tested, 150 (86%) belonged to 1 PFGE clonal group. CAMRSA infections were identified throughout Minnesota; although most isolates were genetically related and susceptible to multiple antimicrobials, they were generally nonsusceptible to initial empirical therapy.

Population-based incidence of pertussis among adolescents and adults, Minnesota, 1995-1996.

To estimate the incidence of pertussis, a prospective study was done among members of a managed care organization in Minneapolis/St. Paul, Minnesota. Of 212 patients 10-49 years old enrolled from January 1995 through December 1996, 8 were found to be culture positive, 10 were found to be positive by polymerase chain reaction assay, 13 had a > or =2-fold increase in IgG or IgA to pertussis toxin (PT), and 18 had IgG to PT in a single serum specimen > or =3 SD above the mean of an age-matched control group. At least 1 positive laboratory test result for pertussis infection was found in 27 (13%) patients, among whom the duration of cough illness was a median of 42 days (range, 27-66 days). On the basis of any positive laboratory result, the estimated annual incidence of pertussis was 507 cases per 100,000 person-years (95% confidence interval, 307-706 cases). Bordetella pertussis infection may be a more common cause of cough illness among adolescents and adults than was recognized previously.

Use of molecular subtyping in surveillance for Salmonella enterica serotype typhimurium.

BACKGROUND: Because Salmonella enterica serotype typhimurium is the most common serotype isolated from persons with salmonellosis in the United States, it is difficult to detect unusual clusters or outbreaks. To determine whether molecular subtyping could be useful in public health surveillance for S. enterica serotype typhimurium, the Minnesota Department of Health initiated the routine use of pulsed-field gel electrophoresis (PFGE) of isolates. METHODS: Beginning in 1994, all S. enterica serotype typhimurium isolates submitted by clinical laboratories to the Department of Health were subtyped by PFGE. A standard questionnaire was used to interview patients about possible sources of infection. RESULTS: From 1994 through 1998, 998 cases of infection with S. enterica serotype typhimurium were reported to the Minnesota Department of Health (4.4 cases per 100,000 person-years). PFGE was performed on 958 of the isolates (96 percent), and 174 different patterns were identified. Sixteen outbreaks with a common source were identified, accounting for 154 cases. PFGE subtyping made it possible to confirm 10 outbreaks that involved small numbers of cases in institutional settings. Of six larger, community-based outbreaks, four would probably not have been recognized without PFGE subtyping. These four outbreaks accounted for 96 of the 154 culture-confirmed outbreak cases (62 percent). Fifty-six of 209 isolates tested for antimicrobial susceptibility (27 percent) were resistant to at least five antimicrobial agents. The multidrug-resistant isolates identified had unique PFGE patterns. CONCLUSIONS: Routine molecular subtyping of S. enterica serotype typhimurium by PFGE can improve the detection of outbreaks and aid in the identification of multidrug-resistant strains. Combining routine molecular subtyping with a method of rapid communication among public health authorities can improve surveillance for S. enterica serotype typhimurium infections.

Evaluation of pulsed-field gel electrophoresis in epidemiological investigations of meningococcal disease outbreaks caused by Neisseria meningitidis serogroup C.

Since 1990, the frequency of Neisseria meningitidis serogroup C (NMSC) outbreaks in the United States has increased. Based on multilocus enzyme electrophoresis (MEE), the current molecular subtyping standard, most of the NMSC outbreaks have been caused by isolates of several closely related electrophoretic types (ETs) within the ET-37 complex. We chose 66 isolates from four well-described NMSC outbreaks that occurred in the United States from 1993 to 1995 to evaluate the potential of pulsed-field gel electrophoresis (PFGE) to identify outbreak-related isolates specific for each of the four outbreaks and to differentiate between them and 50 sporadic isolates collected during the outbreak investigations or through active laboratory-based surveillance from 1989 to 1996. We tested all isolates collected during the outbreak investigations by four other molecular subtyping methods: MEE, ribotyping (ClaI), random amplified polymorphic DNA assay (two primers), and serotyping and serosubtyping. Among the 116 isolates, we observed 11 clusters of 39 NheI PFGE patterns. Excellent correlation between the PFGE and the epidemiological data was observed, with an overall sensitivity of 85% and specificity of 71% at the 95% pattern relatedness breakpoint using either 1.5 or 1.0% tolerance. For all four analyzed outbreaks, PFGE would have given public health officials additional support in declaring an outbreak and making appropriate public health decisions.

Bioavailability of metals in stream food webs and hazards to brook trout (Salvelinus fontinalis) in the upper Animas River watershed, Colorado.

The water quality, habitats, and biota of streams in the upper Animas River watershed of Colorado, USA, are affected by metal contamination associated with acid drainage. We determined metal concentrations in components of the food web of the Animas River and its tributaries-periphyton (aufwuchs), benthic invertebrates, and livers of brook trout (Salvelinus fontinalis)-and evaluated pathways of metal exposure and hazards of metal toxicity to stream biota. Concentrations of the toxic metals cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn) in periphyton, benthic invertebrates, and trout livers from one or more sites in the upper Animas River were significantly greater than those from reference sites. Periphyton from sites downstream from mixing zones of acid and neutral waters had elevated concentrations of aluminum (Al) and iron (Fe) reflecting deposition of colloidal Fe and Al oxides, and reduced algal biomass. Metal concentrations in benthic invertebrates reflected differences in feeding habits and body size among taxa, with greatest concentrations of Zn, Cu, and Cd in the small mayfly Rhithrogena, which feeds on periphyton, and greatest concentrations of Pb in the small stonefly Zapada, a detritivore. Concentrations of Zn and Pb decreased across each trophic linkage, whereas concentrations of Cu and Cd were similar across several trophic levels, suggesting that Cu and Cd were more efficiently transferred via dietary exposure. Concentrations of Cu in invertebrates and trout livers were more closely associated with impacts on trout populations and invertebrate communities than were concentrations of Zn, Cd, or Pb. Copper concentrations in livers of brook trout from the upper Animas River were substantially greater than background concentrations and approached levels associated with reduced brook trout populations in field studies and with toxic effects on other salmonids in laboratory studies. These results indicate that bioaccumulation and transfer of metals in stream food webs are significant components of metal exposure for stream biota of the upper Animas River watershed and suggest that chronic toxicity of Cu is an important factor limiting the distribution and abundance of brook trout populations in the watershed.

Characterization of gyrA mutations associated with fluoroquinolone resistance in Campylobacter coli by DNA sequence analysis and MAMA PCR.

Increasing numbers of fluoroquinolone-resistant Campylobacter coli isolates received at the Minnesota State Public Health Laboratory and at the Centers for Disease Control and Prevention have been a cause for concern. The gyrA quinolone resistance-determining regions of several fluoroquinolone-resistant isolates were sequenced to examine the mechanism of resistance. Ciprofloxacin-resistant C. coli isolates examined by DNA sequencing had a Thr-86 to Ile (ACT-->ATT) gyrA mutation, leading to resistance to fluoroquinolone antibiotics. A mismatch amplification mutation assay polymerase chain reaction protocol was developed to detect this gyrA mutation.