Stephen E. Dietrich

Variation in virulence among clades of Escherichia coli O157:H7 associated with disease outbreaks

Escherichia coli O157:H7, a toxin-producing food and waterborne bacterial pathogen, has been linked to large outbreaks of gastrointestinal illness for more than two decades. E. coli O157 causes a wide range of clinical illness that varies by outbreak, although factors that contribute to variation in disease severity are poorly understood. Several recent outbreaks involving O157 contamination of fresh produce (e.g., spinach) were associated with more severe disease, as defined by higher hemolytic uremic syndrome and hospitalization frequencies, suggesting that increased virulence has evolved. To test this hypothesis, we developed a system that detects SNPs in 96 loci and applied it to >500 E. coli O157 clinical strains. Phylogenetic analyses identified 39 SNP genotypes that differ at 20% of SNP loci and are separated into nine distinct clades. Differences were observed between clades in the frequency and distribution of Shiga toxin genes and in the type of clinical disease reported. Patients with hemolytic uremic syndrome were significantly more likely to be infected with clade 8 strains, which have increased in frequency over the past 5 years. Genome sequencing of a spinach outbreak strain, a member of clade 8, also revealed substantial genomic differences. These findings suggest that an emergent subpopulation of the clade 8 lineage has acquired critical factors that contribute to more severe disease. The ability to detect and rapidly genotype O157 strains belonging to such lineages is important and will have a significant impact on both disease diagnosis and treatment guidelines.

An international outbreak of Salmonella infections caused by alfalfa sprouts grown from contaminated seeds

An outbreak of Salmonella serotype stanley infections occurred in the United States and Finland in 1995. The outbreak was investigated through case-control studies in Arizona, Michigan, and Finland; by isolate subtyping; and by tracing and culturing of the implicated food. Alfalfa sprout consumption was the only exposure associated with S. stanley infections in Arizona (matched odds ratio [MOR] = 11.1; 95% confidence interval [CI], 1.4-513), Michigan (MOR = 5.5; CI, 1.6-23), and Finland (MOR undefined; CI, 4.9-infinity). US and Finnish patient isolates were a unique outbreak strain distinct from S. stanley isolates not linked to the outbreak. Alfalfa sprouts eaten by patients in 6 US states and Finland were traced to seed shipped by a Dutch shipper. Thus, it was concluded that alfalfa sprouts grown from contaminated seed caused an international outbreak of > or =242 S. stanley infections in > or =17 US states and Finland. This outbreak illustrates a new mechanism through which contamination of fresh produce can cause large, widely dispersed outbreaks.

Surveillance for Shiga toxin-producing Escherichia coli, Michigan, 2001-2005.

A surveillance system used different detection methods to estimate prevalence of Shiga toxin–producing Escherichia coli during 2003–2005 and 2001–2002. More non-O157 serotypes were detected by enzyme immunoassay than by evaluation of non-sorbitol–fermenting E. coli isolates. We therefore recommend use of enzyme immunoassay and culture-based methods.

Use of DNA Fingerprinting To Assess Tuberculosis Infection Control

The resurgence of tuberculosis in the United States in the 1980s and early 1990s was accompanied and exacerbated by an increase in the nosocomial spread of tuberculosis [1, 2]. Several tragic and well-publicized nosocomial outbreaks of multi-drug-resistant tuberculosis led to the reevaluation of tuberculosis infection-control practices and to revision of the Centers for Disease Control and Prevention (CDC) guidelines on tuberculosis infection control [3-6]. The revised CDC guidelines propose a hierarchy of control measures, emphasizing administrative controls over engineering controls and personal respiratory devices. Since the guidelines have been available, many hospitals have implemented the recommended practices, and early studies and surveillance data have demonstrated the efficacy of these practices [7-10]. Recently, the Occupational Safety and Health Administration (OSHA) proposed new requirements for tuberculosis infection control that exceed the CDC guidelines with respect to burden on infection-control resources [11]. Critics of the OSHA proposal believe that the evidence that health care workers are at continued substantial risk for occupational tuberculosis is not sufficient to justify the increased expenditure [12]. Because it illustrates genetic relatedness among strains of M. tuberculosis, DNA fingerprinting has been used by epidemiologists and clinicians to confirm suspected outbreaks of disease and episodes of laboratory cross-contamination [3, 13, 14]. We hypothesized that routine DNA fingerprinting, done by using restriction fragment length polymorphism analysis, could be used to enhance hospital infection-control surveillance for patient-to-patient transmission of Mycobacterium tuberculosis. To test this hypothesis, we performed DNA fingerprinting on all M. tuberculosis isolates obtained from Cook County Hospital, Chicago, Illinois, over a 1-year period. During this period, no patient-to-patient transmission had been detected by routine hospital infection-control practices. Methods Setting Cook County Hospital is a 700-bed public hospital that serves a primarily indigent urban population. The prevalences of tuberculosis and HIV infection are high; the latter accounts for 9% of inpatient days. Administrative controls, including aggressive triage of patients with possible tuberculosis in the emergency department, have led to the widespread use of respiratory isolation for patients with known or suspected pulmonary tuberculosis. Patients with pulmonary tuberculosis are kept in respiratory isolation until they have clinical improvement while receiving therapy and have negative results on acid-fast smears on 3 separate days. Engineering controls consist primarily of isolation rooms that have been retrofitted with window exhaust fans to comply with recommended air-handling guidelines [6]. Health care workers wear personal respirators, approved by the National Institute for Occupational Health and Safety, while in respiratory isolation rooms; isolated patients wear surgical masks when not in negative-pressure rooms. Despite these measures, a few patients who are not isolated promptly on admission receive a diagnosis of pulmonary tuberculosis each year. In addition, the hospital has outpatient areas (including an HIV clinic) in which patients spend many hours in close proximity and where unrecognized tuberculosis can spread. Laboratory Studies During the 12-month study period (1 April 1995 to 31 March 1996), one M. tuberculosis isolate from every patient with tuberculosis at Cook County Hospital was sent to the Michigan Department of Community Health for DNA fingerprinting. All isolates were analyzed by standard methods [15] by using a 246-base pair probe representing the IS6110 sequences to the right of the PvuII site. The probe was labeled with horseradish peroxidase for detection by enhanced chemiluminescence (ECL Direct Labeling and Detection System, Amersham, Arlington Heights, Illinois) and was hybridized to DNA from M. tuberculosis isolates restricted with PvuII. PvuII-restricted DNA from M. tuberculosis strain MT14323, containing 14 copies of IS6110, was used as a DNA size marker. Autoradiographs were produced by exposing the hybridized blots to Hyperfilm ECL (Amersham). Secondary fingerprinting done by using a probe for the polymorphic guanine cytosine-rich repetitive sequence (PGRS) has proven useful for confirming the genetic relatedness of M. tuberculosis isolates [16-20]. This second assay was performed, with the recombinant plasmid pTBN12 and the methods described above, on isolates with identical IS6110 fingerprints of 5 or fewer hybridizing bands and isolates with more than 5 hybridizing bands with fragment patterns that differed by 1 to 2 bands. A 1-kilobase ladder was included as a size marker (Gibco, Gaithersburg, Maryland). The pTBN12 was donated by Don Cave (John L. McClellan Memorial Veterans Hospital, Little Rock, Arkansas). We compared IS6110 and PGRS fingerprints by using Whole Band Analyzer software, version 3.2.2 (BioImage, Inc., Ann Arbor, Michigan). The average linkage clustering method was used to match patterns with an SD of 2.5%. Matching patterns were compared visually to ensure similarity. For PGRS fingerprinting, only hybridizing bands greater than 1.6 kilobases were analyzed. Isolates were considered to be genetically related (that is, clustered) if they had identical IS6110 patterns of more than 5 bands, had IS6110 patterns of more than 5 bands that differed by 1 to 2 bands and had identical PGRS patterns, or had IS6110 patterns of 5 or fewer bands and had identical PGRS patterns. Susceptibility testing was done at the Cook County Hospital laboratory. Susceptibilities on any isolate found to be resistant to one or more first-line drugs (isoniazid, rifampin, ethambutol, or streptomycin) were confirmed by the Illinois Department of Public Health laboratory. Clinical Data Collection Demographic information and disease characteristics were gathered by review of the medical record for the first admission during which tuberculosis was diagnosed. This record was available for 166 of 173 patients whose isolates were fingerprinted (96%), and data were available from other sources [such as the outpatient record or the Department of Health tuberculosis database] for most of the other 7. Screening for laboratory cross-contamination was done for all clustered isolates. Laboratory cross-contamination was considered likely when 1) only one culture was positive for tuberculosis, 2) the specimen was negative on an acid-fast smear, 3) the clinical syndrome was not consistent with tuberculosis, and 4) the specimen was processed on the same day as the specimen of a patient whose isolate had a matching DNA fingerprint [13, 14]. To determine whether nosocomial transmission of M. tuberculosis had occurred between patients with clustered isolates, the times and locations of all outpatient visits and inpatient admissions from 1 April 1993 to 31 March 1996 were reviewed. If two or more patients who had isolates in a cluster had been in any inpatient or outpatient area of the hospital on the same day before they had both received a diagnosis of tuberculosis, the medical records were reviewed to determine the likelihood of M. tuberculosis transmission. Data were gathered on hospital or clinic location, use of and compliance with respiratory isolation, acid-fast smear status, HIV infection status, tuberculin skin-test status, and susceptibility patterns. Records were also reviewed to determine whether the patients had concurrently undergone laboratory or radiographic procedures or had obtained medication at the same pharmacy. Data on hospital location, time of registration, and time and location of laboratory and radiographic procedures were obtained from a computerized database that is complete and accurate; data on drug susceptibility were available for all patients with clustered isolates who were on hospital grounds concurrently with another patient with clustered isolates. If patients were registered in the emergency department on sequential days, the records were reviewed to determine whether temporal overlap existed. Contact Tracing The results of hospital contact tracing were reviewed. Tracing is done when a patient who has not been appropriately isolated receives a diagnosis of acid-fast smear-positive pulmonary tuberculosis. Employees who may have been exposed to this patient are identified by their supervisors and undergo tuberculin skin testing with chest radiography, if appropriate. All employees receive routine tuberculin skin testing annually. Compliance with this testing is a condition of continued employment. The results of community contact tracing performed by the Chicago Department of Public Health were reviewed to identify out-of-hospital links between patients whose isolates were clustered. Contact tracing is done for all cases of pulmonary tuberculosis and consists of interview with the patient to identify close contacts. Contacts undergo tuberculin skin testing and, if conversions are detected, the circle of contact tracing is widened by repeated interviews to include more casual contacts, who are also tested. Community contact tracing beyond that performed by the Chicago Department of Public Health was not done in our study. Statistical Analysis Chi-square tests (or the Fisher exact test, when expected cell sizes were <5) were used to test the association of clustering with patient demographic and clinical characteristics. Two-tailed P values are presented with relative risks (RRs) and 95% CIs. All variables found to be significantly associated with clustering on univariate analysis were entered into a logistic model by using the statistical software package SPS, version 8.0 (SPS, Inc., Chicago, Illinois). The output of the model, which was the ln(odds) of clustering, was converted to the probability of clustering. Results Mycobacterium tuberculosis was recovered f

Genetic relatedness of Burkholderia (Pseudomonas) cepacia isolates from five cystic fibrosis centers in Michigan

Burkholderia cepacia isolates from patients with cystic fibrosis (CF) attending five CF centers were studied for relatedness by cellular fatty acid methyl esters (FAME) and by chromosomal DNA restriction analysis. Twenty-eight of 32 (87.5%) isolates tested were grouped in cluster group 1 based on their FAME profiles. DNA analysis revealed that 29 of 32 (90.6%) B. cepacia isolates from five CF centers had one closely related DNA pattern. To examine strain variation over a time period, FAME profiles and DNA patterns of isolates from serial cultures on seven patients from center D were studied. For four patients, all serial B. cepacia isolates belonged to a single FAME cluster group; for the remaining three patients, all serial isolates belonged to any two of the four cluster groups. On serial culture isolates, a single DNA pattern (pattern A) was found in 31 of 32 isolates demonstrating a close genetic relatedness. These data corroborate the observations that the majority of patients colonised with B. cepacia in a CF center harbor strains genetically closely related as determined by FAME profiles and DNA patterns.

Escherichia coli O157 cluster evaluation.

We investigated a multistate cluster of Escherichia coli O157:H7 isolates; pulsed-field gel electrophoresis subtyping, using a single enzyme, suggested an epidemiologic association. An investigation and additional subtyping, however, did not support the association. Confirmating E. coli O157 clusters with two or more restriction endonucleases is necessary before public health resources are allocated to follow-up investigations.

Within-Farm Changes in Dairy Farm-Associated Salmonella Subtypes and Comparison to Human Clinical Isolates in Michigan, 2000-2001 and 2009

ABSTRACT Temporal changes in the distribution of Salmonella subtypes in livestock populations may have important impacts on human health. The first objective of this research was to determine the within-farm changes in the population of subtypes of Salmonella on Michigan dairy farms that were sampled longitudinally in 2000-2001 and again in 2009. The second objective was to determine the yearly frequency (2001 through 2012) of reported human illnesses in Michigan associated with the same subtypes. Comparable sampling techniques were used to collect fecal and environmental samples from the same 18 Michigan dairy farms in 2000-2001 and 2009. Serotypes, multilocus sequence types (STs), and pulsed-field gel electrophoresis (PFGE) banding patterns were identified for isolates from 6 farms where >1 Salmonella isolate was recovered in both 2000-2001 and 2009. The distribution of STs was significantly different between time frames (P < 0.05); only two of 31 PFGE patterns were identified in both time frames, and each was recovered from the same farm in each time frame. Previously reported within-farm decreases in the frequency of multidrug-resistant (MDR) Salmonella were due to recovery of MDR subtypes of S. enterica serotypes Senftenberg and Typhimurium in 2000-2001 and genetically distinct, pansusceptible subtypes of the same serotypes in 2009. The annual frequency of human illnesses between 2001 and 2012 with a PFGE pattern matching a bovine strain decreased for patterns recovered from dairy farms in 2000-2001 and increased for patterns recovered in 2009. These data suggest important changes in the population of Salmonella on dairy farms and in the frequency of human illnesses associated with cattle-derived subtypes.

Persistent tuberculosis or specimen contamination

Cross-contamination during sequential processing of sputum specimens from different patients causes false-positive growth of Mycobacterium tuberculosis in culture. We describe an unusual case of cross-contamination in a 36-year-old man with acquired immunodeficiency syndrome and possible persistent tuberculosis. Culture with 1 of 3 sputum specimens was positive for rifampin-susceptible M tuberculosis. Review of processing revealed that his single culture-positive sputum specimen had followed a sputum specimen from another patient with active pulmonary tuberculosis that was positive in culture for M tuberculosis resistant to rifampin. Molecular strain typing by restriction fragment length polymorphism demonstrated the 2 isolates to be an identical strain of M tuberculosis. Agar proportion susceptibility testing of the rifampin-resistant isolate revealed low numbers of resistant organisms in a range of 1.5% to 3.3%. It was concluded that rifampin-susceptible organisms that constituted approximately 98% of the resistant isolate contaminated sputum from the patient with possible persistent tuberculosis. His culture result was, therefore, considered false positive, not an indication of tuberculosis.