Evaluating gulls as potential vehicles of Salmonella enterica serotype Newport (JJPX01.0061) contamination of tomatoes grown on the eastern shore of Virginia.

Salmonella enterica serovar Newport pattern JJPX01.0061 has been identified as causing several multistate outbreaks in the last 10 years, primarily due to contamination of tomatoes grown in Virginia. The goal of this study was to evaluate gulls as a potential vehicle of S. Newport pattern 61 contamination for tomatoes grown on the Eastern Shore of Virginia. Gull fecal samples were collected at four sites in eastern Virginia for 3 months (May to July) in 2012, resulting in 360 samples, among which Salmonella was isolated from 62 samples. Twenty-two serotypes and 26 pulsed-field gel electrophoresis DNA fingerprint patterns, including S. Newport pattern 61, were identified. All of the patterns that were isolated multiple times, with the exception of S. Newport patterns JJPX01.0030 and JJPX01.0061, were clustered in time and geographical location. These results strongly suggest that both patterns of S. Newport are endemic to sites on the Eastern Shore where gulls were sampled. This study provides additional information regarding the epidemiology of S. Newport pattern 61 in Virginia and how tomatoes sold interstate may become contaminated in the field.

Liquid chromatography/mass spectrometry characterization of Escherichia coli and Shigella species.

Liquid chromatography/quadrupole time of flight mass spectrometry (LC/QTOF MS) utilizing electrospray ionization was employed to monitor protein expression in Escherichia coli and Shigella organisms. Comparison with MALDI/TOF-MS revealed more proteins, particularly above 15 kDa. A combination of automated charge state deconvolution, spectral mirroring, and spectral subtraction was used to reveal subtle differences in the LC/MS data. Reproducible intact protein biomarker candidates were discovered based on their unique mass, retention time, and relative intensity. These marker candidates were implemented to differentiate closely related strain types, (e.g., two distinct isolates of E. coli O157:H7) and to correctly identify unknown pathogens. This LC/MS approach is less labor-intensive than pulsed-field gel electrophoresis, affords greater specificity than real-time PCR, and requires no primers or antibodies. Additionally, this approach would be beneficial during outbreaks of foodborne disease or bioterrorism investigations by complementing methods typically used in diagnostic microbiology laboratories.

An outbreak of Escherichia coli O157:H7 infections among visitors to a dairy farm.

BACKGROUND: Outbreaks of Escherichia coli O157:H7 infections have involved direct transmission from animals and their environment to humans. We describe an outbreak among visitors to a Pennsylvania dairy and petting farm that provides public access to animals. METHODS: We conducted both a case-control study among visitors to a farm to identify risk factors for infection and a household survey to determine the rates of diarrheal illness among these visitors. We performed an extensive environmental study to identify sources of E. coli O157:H7 on the farm. RESULTS: Fifty-one patients with confirmed or suspected E. coli O157:H7 infection were enrolled in the case-control study. The median age of the patients was four years, and the hemolytic-uremic syndrome developed in eight. Contact with calves and their environment was associated with an increased risk of infection, whereas hand washing was protective. The household survey indicated that visitors to the farm during the outbreak had higher than expected rates of diarrhea. Environmental studies showed that 28 of the 216 cattle on the farm (13 percent) were colonized with E. coli O157:H7 that had the same distinct pattern on pulsed-field gel electrophoresis that was found in isolates from the patients. This organism was also recovered from surfaces that were accessible to the public. CONCLUSIONS: In a large outbreak of E. coli O157:H7 infections among visitors to a dairy farm, predominantly children, high rates of carriage of E. coli O157:H7 among calves and young cattle most likely resulted in contamination of both the animals' hides and the environment.

Spread of methicillin-resistant Staphylococcus aureus (MRSA) among household contacts of individuals with nosocomially acquired MRSA.

OBJECTIVE: To determine the frequency with which methicillin-resistant Staphylococcus aureus (MRSA) is spread from colonized or infected patients to their household and community contacts. DESIGN: Retrospective cohort study. SETTING: University hospital. PARTICIPANTS: Household and community contacts of MRSA-colonized or -infected patients for whom MRSA screening cultures were performed. RESULTS: MRSA was isolated from 25 (14.5%) of 172 individuals. Among the contacts of index patients who had at least one MRSA-colonized contact, those with close contact to the index patient were 7.5 times more likely to be colonized (53% vs 7%; 95% confidence interval, 1.1 to 50.3; P = .002). An analysis of antimicrobial susceptibility and DNA fingerprint patterns suggested person-to-person spread. CONCLUSIONS: MRSA colonization occurs frequently among household and community contacts of patients with nosocomially acquired MRSA, suggesting that transmission of nosocomially acquired MRSA outside of the healthcare setting may be a substantial source of MRSA colonization and infection in the community.

Control of simultaneous outbreaks of carbapenemase-producing enterobacteriaceae and extensively drug-resistant Acinetobacter baumannii infection in an intensive care unit using interventions promoted in the Centers for Disease Control and Prevention 2012 carbapenemase-resistant Enterobacteriaceae Toolkit.

OBJECTIVE: We describe the efficacy of enhanced infection control measures, including those recommended in the Centers for Disease Control and Prevention's 2012 carbapenem-resistant Enterobacteriaceae (CRE) toolkit, to control concurrent outbreaks of carbapenemase-producing Enterobacteriaceae (CPE) and extensively drug-resistant Acinetobacter baumannii (XDR-AB). DESIGN: Before-after intervention study. SETTING: Fifteen-bed surgical trauma intensive care unit (ICU). METHODS: We investigated the impact of enhanced infection control measures in response to clusters of CPE and XDR-AB infections in an ICU from April 2009 to March 2010. Polymerase chain reaction was used to detect the presence of blaKPC and resistance plasmids in CRE. Pulsed-field gel electrophoresis was performed to assess XDR-AB clonality. Enhanced infection-control measures were implemented in response to ongoing transmission of CPE and a new outbreak of XDR-AB. Efficacy was evaluated by comparing the incidence rate (IR) of CPE and XDR-AB before and after the implementation of these measures. RESULTS: The IR of CPE for the 12 months before the implementation of enhanced measures was 7.77 cases per 1,000 patient-days, whereas the IR of XDR-AB for the 3 months before implementation was 6.79 cases per 1,000 patient-days. All examined CPE shared endemic blaKPC resistance plasmids, and 6 of the 7 XDR-AB isolates were clonal. Following institution of enhanced infection control measures, the CPE IR decreased to 1.22 cases per 1,000 patient-days (P = .001), and no more cases of XDR-AB were identified. CONCLUSIONS: Use of infection control measures described in the Centers for Disease Control and Prevention's 2012 CRE toolkit was associated with a reduction in the IR of CPE and an interruption in XDR-AB transmission.

Development of a multiplex polymerase chain reaction assay for diarrheagenic Escherichia coli and Shigella spp. and its evaluation on colonies, culture broths, and stool.

Detection of diarrheagenic Escherichia coli (DEC) typically depends on identification of virulence genes from stool cultures, not on stool itself. We developed a multiplex polymerase chain reaction (PCR) assay that detects key DEC virulence genes (stx1, stx2, eae, bfpA, ipaH, LT, STh, aaiC, aatA). The assay involved a multiplex PCR reaction followed by detection of amplicon(s) using Luminex beads. The assay was evaluated on over 100 colony and broth specimens. We then evaluated the assay using DNA extracted from stool, colony pools, and Gram-negative broths, using stool spiked with known quantities of DEC. Performance of the assay on stool DNA was most quantitative, while stool broth DNA offered the lowest limit of detection. The assay was prospectively evaluated on clinical specimens in Tanzania. Stool DNA yielded higher sensitivity than colony pools compared with broth DNA as the standard. We propose using this assay to screen for DEC directly in stool or stool broths.

Characterization of Clostridium species utilizing liquid chromatography/mass spectrometry of intact proteins.

A method for biomarker candidate discovery and strain level pathogen characterization using liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization is described. This method was applied to two pathogenic Clostridium species: C. difficile and C. perfringens. Seven marker proteins per species (fourteen total) were successfully implemented to speciate unknowns during a blind study and could enhance serological and genetic approaches by serving as new targets for detection. Two sets of C. perfringens isolates that were 100% similar by pulsed-field gel electrophoresis (PFGE) were distinguished using LC/MS, demonstrating the high specificity of this approach. The use of LC/MS is less labor intensive than PFGE, affords greater specificity than real-time PCR, and requires no primers or antibodies.

Identification of a Naegleria fowleri membrane protein reactive with anti-human CD59 antibody.

Naegleria fowleri, the causative agent of primary amebic meningoencephalitis, is resistant to complement lysis. The presence of a complement regulatory protein on the surface of N. fowleri was investigated. Southern blot and Northern blot analyses demonstrated hybridization of a radiolabeled cDNA probe for CD59 to genomic DNA and RNA, respectively, from pathogenic N. fowleri. An 18-kDa immunoreactive protein was detected on the membrane of N. fowleri by Western immunoblot and immunofluorescence analyses with monoclonal antibodies for human CD59. Complement component C9 immunoprecipitated with the N. fowleri "CD59-like" protein from amebae incubated with normal human serum. In contrast, a gene or protein similar to CD59 was not detected in nonpathogenic, complement-sensitive N. gruberi amebae. Collectively, our studies suggest that a protein reactive with antibodies to human CD59 is present on the surface of N. fowleri amebae and may play a role in resistance to lysis by cytolytic proteins.