Improvements to the Success of Outbreak Investigations of Legionnaires' Disease: 40 Years of Testing and Investigation in New York State.

Since 1978, the New York State Department of Health's public health laboratory, Wadsworth Center (WC), in collaboration with epidemiology and environmental partners, has been committed to providing comprehensive public health testing for Legionella in New York. Statewide, clinical case counts have been increasing over time, with the highest numbers identified in 2017 and 2018 (1,022 and 1,426, respectively). Over the course of more than 40 years, the WC Legionella testing program has continuously implemented improved testing methods. The methods utilized have transitioned from solely culture-based methods for organism recovery to development of a suite of reference testing services, including identification and characterization by PCR and pulsed-field gel electrophoresis (PFGE). In the last decade, whole-genome sequencing (WGS) has further refined the ability to link outbreak strains between clinical specimens and environmental samples. Here, we review Legionnaires' disease outbreak investigations during this time period, including comprehensive testing of both clinical and environmental samples. Between 1978 and 2017, 60 outbreaks involving clinical and environmental isolates with matching PFGE patterns were detected in 49 facilities from the 157 investigations at 146 facilities. However, 97 investigations were not solved due to the lack of clinical or environmental isolates or PFGE matches. We found 69% of patient specimens from New York State (NYS) were outbreak associated, a much higher rate than observed in other published reports. The consistent application of new cutting-edge technologies and environmental regulations has resulted in successful investigations resulting in remediation efforts. IMPORTANCE Legionella, the causative agent of Legionnaires' disease (LD), can cause severe respiratory illness. In 2018, there were nearly 10,000 cases of LD reported in the United States (https://www.cdc.gov/legionella/fastfacts.html; https://wonder.cdc.gov/nndss/static/2018/annual/2018-table2h.html), with actual incidence believed to be much higher. About 10% of patients with LD will die, and as high as 90% of patients diagnosed will be hospitalized. As Legionella is spread predominantly through engineered building water systems, identifying sources of outbreaks by assessing environmental sources is key to preventing further cases LD.

Insights into the long-term persistence of Legionella in facilities from whole-genome sequencing.

We investigated the value of whole-genome sequencing (WGS) and single nucleotide polymorphism (SNP) analyses in determining the relationships among and evolutionary rates of Legionella species with long-term persistence in three healthcare facilities. We examined retrospective clinical and environmental isolates of Legionella micdadei and Legionella pneumophila serogroup 1 isolates with identical PFGE DNA fingerprints sampled over the course of up to 18 years. WGS analyses demonstrated that heterogeneous populations of Legionella were present within each facility despite displaying the same PFGE profiles. Additionally, clustering of some clinical isolates with those from a separate but related institution exposed a source of infection not previously detected, underscoring the importance of considering phylogenetic relationships when assessing epidemiological links. The data supported an average substitution rate of 0.80 SNPs per genome per year for L. micdadei but a reliable estimate for L. pneumophila serogroup 1 could not be obtained due to complicating factors such as non-chronological links among isolates and inadequate sampling depths. While the substitution rate for L. micdadei is consistent with previous estimates for L. pneumophila, the lack of a temporal signal in our sequence data for L. pneuomphila serogroup 1 isolates suggests either insufficient change to provide an estimate or variable evolutionary rates, which could reflect the presence of both actively dividing and viable but non-culturable Legionella spp. in the built environment. This study highlights the increased discriminatory power of WGS SNP analysis as compared to PFGE, emphasizes the need for extended sampling, and provides insight into the evolution of Legionella from longitudinal investigations.

Tracing Origins of the Salmonella Bareilly Strain Causing a Food-borne Outbreak in the United States.

BACKGROUND: Using a novel combination of whole-genome sequencing (WGS) analysis and geographic metadata, we traced the origins of Salmonella Bareilly isolates collected in 2012 during a widespread food-borne outbreak in the United States associated with scraped tuna imported from India. METHODS: Using next-generation sequencing, we sequenced the complete genome of 100 Salmonella Bareilly isolates obtained from patients who consumed contaminated product, from natural sources, and from unrelated historically and geographically disparate foods. Pathogen genomes were linked to geography by projecting the phylogeny on a virtual globe and produced a transmission network. RESULTS: Phylogenetic analysis of WGS data revealed a common origin for outbreak strains, indicating that patients in Maryland and New York were infected from sources originating at a facility in India. CONCLUSIONS: These data represent the first report fully integrating WGS analysis with geographic mapping and a novel use of transmission networks. Results showed that WGS vastly improves our ability to delimit the scope and source of bacterial food-borne contamination events. Furthermore, these findings reinforce the extraordinary utility that WGS brings to global outbreak investigation as a greatly enhanced approach to protecting the human food supply chain as well as public health in general.

Use of copper-silver ionization for the control of legionellae in alkaline environments at health care facilities.

BACKGROUND: There are multiple treatment options for the control of legionellae in premise hot water systems. Water chemistry plays a role in the efficacy of these treatments and should be considered when selecting a treatment. This study demonstrated the efficacy of copper-silver ionization (CSI) under alkaline water conditions in 2 health care facilities. METHODS: Monitoring for copper (Cu) and silver (Ag) ions was performed, and the corresponding percentage of positive Legionella cultures was monitored. Low Legionella colony forming units (CFU), with a mean <10 CFU/100 mL, and ≤30% positive culture for each sampling period, along with no recurrent disease, were considered indicative of control. RESULTS: CSI treatment was shown to reduce both the number of CFU found and the percentage of samples found to be culture positive. After treatment was established, culture positivity was, for example, reduced from 70% (>10(3) CFU/100 mL) to consistently <30% (38 CFU/100 mL). CONCLUSION: Control of legionellae in premise water systems may be a complex process requiring long-term assessments for adequate control. This work found that CSI could be successful in controlling Legionella under alkaline water conditions, and the evidence suggests that Ag ions are responsible for the control of Legionella pneumophila 1, L pneumophila 6, and L anisa.

Two cases of adult botulism caused by botulinum neurotoxin producing Clostridium baratii.

Type F botulism occurs rarely in clinical cases. Two cases of type F botulism in elderly patients that were clustered in time and space are described. Clostridium baratii producing type F botulinum neurotoxin was isolated from both patients; molecular typing of these isolates revealed that they were unrelated strains.

Enhanced identification and characterization of non-O157 Shiga toxin-producing Escherichia coli: a six-year study.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are emerging pathogens with the potential to cause serious illness and impact public health due to diagnostic challenges. Between 2005 and 2010, the Wadsworth Center (WC), the public health laboratory of the New York State (NYS) Department of Health, requested that Shiga toxin enzyme immunoassay (EIA)-positive stool enrichment broths and/or stool specimens be submitted by clinical and commercial reference laboratories testing NYS patient specimens. A total of 798 EIA-positive specimens were received for confirmation and serotyping, and additionally a subset of STEC was assessed for the presence of six virulence genes (stx1, stx2, eaeA, hlyA, nleA, and nleB) by real-time polymerase chain reaction. We confirmed 591 specimens as STEC, 164 (28%) as O157 STEC, and 427 (72%) as non-O157 STEC. Of the non-O157 STEC serogroups identified, over 70% were O103, O26, O111, O45, O121, or O145. During this time period, WC identified and characterized a total of 1282 STEC received as E. coli isolates, stool specimens, or EIA broths. Overall, the STEC testing identified 59% as O157 STEC and 41% as non-O157 STEC; however, out of 600 isolates submitted to the WC as E. coli cultures, 543 (90%) were identified as O157 STEC. This report summarizes a 6-year study utilizing enhanced STEC testing that resulted in increased identification and characterization of non-O157 STEC in NYS. Continued utilization of enhanced STEC testing may lead to effective and timely outbreak response and improve monitoring of trends in STEC disease epidemiology.

A whole-genome single nucleotide polymorphism-based approach to trace and identify outbreaks linked to a common Salmonella enterica subsp. enterica serovar Montevideo pulsed-field gel electrophoresis type.

In this study, we report a whole-genome single nucleotide polymorphism (SNP)-based evolutionary approach to study the epidemiology of a multistate outbreak of Salmonella enterica subsp. enterica serovar Montevideo. This outbreak included 272 cases that occurred in 44 states between July 2009 and April 2010. A case-control study linked the consumption of salami made with contaminated black and red pepper to the outbreak. We sequenced, on the SOLiD System, 47 isolates with XbaI PFGE pattern JIXX01.0011, a common pulsed-field gel electrophoresis (PFGE) pattern associated with isolates from the outbreak. These isolates represented 20 isolates collected from human sources during the period of the outbreak and 27 control isolates collected from human, food, animal, and environmental sources before the outbreak. Based on 253 high-confidence SNPs, we were able to reconstruct a tip-dated molecular clock phylogeny of the isolates and to assign four human isolates to the actual outbreak. We developed an SNP typing assay to rapidly discriminate between outbreak-related cases and non-outbreak-related cases and tested this assay on an extended panel of 112 isolates. These results suggest that only a very small percentage of the human isolates with the outbreak PFGE pattern and obtained during the outbreak period could be attributed to the actual pepper-related outbreak (20%), while the majority (80%) of the putative cases represented background cases. This study demonstrates that next-generation-based SNP typing provides the resolution and accuracy needed for outbreak investigations of food-borne pathogens that cannot be distinguished by currently used subtyping methods.

Neisseria wadsworthii sp. nov. and Neisseria shayeganii sp. nov., isolated from clinical specimens.

An analysis of 16S rRNA gene sequences from archived clinical reference specimens has identified two novel Neisseria species. For each species, two strains from independent sources were identified. Amongst species with validly published names, the closest species to the newly identified organisms were Neisseria canis, N. dentiae, N. zoodegmatis, N. animaloris and N. weaveri. DNA-DNA hybridization studies demonstrated that the newly identified isolates represent species that are distinct from these nearest neighbours. Analysis of partial 23S rRNA gene sequences for the newly identified strains and their nearest neighbours provided additional support for the species designation. Bayesian analysis of 16S rRNA gene sequences suggested that the newly identified isolates belong to distinct but related species of the genus Neisseria, and are members of a clade that includes N. dentiae, N. bacilliformis and N. canis. The predominant cellular fatty acids [16 : 0, summed feature 3 (16 : 1ω7c and/or iso-15 : 0 2-OH) and 18 : 1ω7c], as well as biochemical and morphological analyses further support the designation of Neisseria wadsworthii sp. nov. (type strain 9715(T) =DSM 22247(T) =CIP 109934(T)) and Neisseria shayeganii sp. nov. (type strain 871(T) =DSM 22246(T) =CIP 109933(T)).

Genotypic characterization of environmental isolates of Cryptococcus gattii from Puerto Rico.

Cryptococcus neoformans and Cryptococcus gattii are found in distinct environments with some overlap around different parts of the world. However, no systematic surveys of these two pathogens have been reported from Puerto Rico, a tropical island uniquely situated between mainland USA and countries in South America. We carried out an exhaustive environmental survey in southwestern Puerto Rico for pathogenic Cryptococcus species. Twenty-two presumptive isolates of C. gattii from cacti and tree detritus were characterized in detail by physiological and molecular methods and seventeen strains were confirmed as C. gattii. Cryptococcus gattii isolates were haploid and majority of them were MATa [corrected] strains. Sixteen out of seventeen C. gattii isolates belonged to VGII/AFLP6 genotype while one isolate was a VGIV/AFLP7 genotype. The results are significant as Puerto Rico strains are distinct from VGIII/AFLP5 strains reported from Southern California, but similar to C. gattii VGII/AFLP6 molecular type implicated in recent outbreaks of cryptococcosis in Pacific Northwest and British Columbia, Canada, but different in its M13 fingerprinting, and a common genotype in South America.

Multistate outbreak of Pseudomonas fluorescens bloodstream infection after exposure to contaminated heparinized saline flush prepared by a compounding pharmacy.

BACKGROUND: Pharmaceutical compounding, the manipulation of ingredients to create a customized medication, is a widespread practice. In January 2005, the Centers for Disease Control and Prevention was notified of 4 cases of Pseudomonas fluorescens bacteremia that were traced to contaminated heparinized saline intravenous flush syringes prepared as a compounded medical product. PATIENTS AND METHODS: We reviewed medical records of symptomatic patients with P. fluorescens-positive cultures of blood specimens or sections of explanted catheters, reviewed the production process of syringes, performed syringe cultures, compared isolates by pulsed-field gel electrophoresis (PFGE), and examined catheters by scanning electron microscopy. RESULTS: We identified 80 patients in 6 states with P. fluorescens-positive cultures during December 2004-March 2006. Sixty-four patients (80%) had received a diagnosis of cancer. Seventy-four (99%) of 75 patients for whom information about catheter type was available had long-term indwelling catheters. Thirty-three (41%) of 80 cases were diagnosed 84-421 days after the patient's last potential exposure to a contaminated flush (delayed-onset cases). Compared with patients with early infection onset, more patients with delayed infection onset had venous ports (100% versus 50%; P <.001). By PFGE, clinical isolates from 50 (98%) of 51 patients were related to isolates cultured from unopened syringes. Scanning electron microscopy of explanted catheters revealed biofilms containing organisms morphologically consistent with P. fluorescens. CONCLUSION: This outbreak underscores important challenges in ensuring the safety of compounded pharmaceuticals and demonstrates the potential for substantially delayed infections after exposures to contaminated infusates. Exposures to compounded products should be considered when investigating outbreaks. Patients exposed to contaminated infusates require careful follow-up, because infections can occur long after exposure.

Pulsed-field gel electrophoresis (PFGE) analysis of temporally matched Listeria monocytogenes isolates from human clinical cases, foods, ruminant farms, and urban and natural environments reveals source-associated as well as widely distributed PFGE types.

A total of 495 temporally and geographically matched Listeria monocytogenes isolates from human clinical cases, foods, ruminant farms, and urban and natural environments were used to investigate L. monocytogenes pulsed-field gel electrophoresis (PFGE) type diversity. Two-enzyme (AscI and ApaI) PFGE discriminated 310 PFGE types and exhibited higher overall discriminatory power (Simpson's index of discrimination [D] = 0.995) than either EcoRI ribotyping (D = 0.950) or AscI or ApaI single-enzyme PFGE (D = 0.992 for both). Seven PFGE types showed significant associations with specific sources, including one and four PFGE types, respectively, associated with human clinical cases and foods. Spatial analysis of 13 PFGE types occurring >5 times showed that two PFGE types were specific to a single processing facility each, where they appear to have persisted over time. Nine PFGE types were geographically widespread and occurred among isolates from multiple sources. For example, a PFGE type that matched isolates from listeriosis outbreaks in Los Angeles and Switzerland occurred among isolates from farms (n = 7), human clinical cases (n = 4), environmental sources (n = 3), and foods (n = 1). Our data indicate that (i) PFGE is highly discriminatory for the subtyping of L. monocytogenes, (ii) some L. monocytogenes PFGE types are associated with specific sources, and (iii) some L. monocytogenes PFGE types are widely distributed and appear to be stable and pandemic. Large PFGE type databases representing isolates from different sources are thus needed to appropriately interpret subtype data in epidemiological investigations and to identify common as well as source-specific PFGE types.

A large, multiple-restaurant outbreak of infection with Shigella flexneri serotype 2a traced to tomatoes.

BACKGROUND: Foodborne outbreaks of Shigella infection are uncommon and tomatoes are an unusual vehicle. We describe a large, multiple-restaurant outbreak of Shigella flexneri serotype 2a infection that was associated with tomatoes. METHODS: We conducted nationwide surveillance and a case-control study, collected fecal specimens for culture, and measured the survival of the outbreak strain of S. flexneri in tomatoes. RESULTS: We interviewed 306 of 886 ill restaurant patrons and 167 control subjects. Matched univariate analysis showed that several food items were associated with illness, but only tomatoes remained significant in multivariate models. Illness peaked at each restaurant within 24 h after the arrival of hand-sorted bruised and overripe tomatoes from a new distributor; all patient isolates that were tested were indistinguishable by PFGE. Sliced tomatoes from the distributor were inoculated with the outbreak strain, and viable S. flexneri were recovered for 72 h. CONCLUSION: To prevent such outbreaks, persons with shigellosis should be excluded from handling food at all points along the distribution chain.

Multistate outbreak of Listeriosis linked to turkey deli meat and subsequent changes in US regulatory policy.

BACKGROUND: Listeriosis, a life-threatening foodborne illness caused by Listeria monocytogenes, affects approximately 2500 Americans annually. Between July and October 2002, an uncommon strain of L. monocytogenes caused an outbreak of listeriosis in 9 states. METHODS: We conducted case finding, a case-control study, and traceback and microbiological investigations to determine the extent and source of the outbreak and to propose control measures. Case patients were infected with the outbreak strain of L. monocytogenes between July and November 2002 in 9 states, and control patients were infected with different L. monocytogenes strains. Outcome measures included food exposure associated with outbreak strain infection and source of the implicated food. RESULTS: Fifty-four case patients were identified; 8 died, and 3 pregnant women had fetal deaths. The case-control study included 38 case patients and 53 control patients. Case patients consumed turkey deli meat much more frequently than did control patients (P = .008, by Wilcoxon rank-sum test). In the 4 weeks before illness, 55% of case patients had eaten deli turkey breast more than 1-2 times, compared with 28% of control patients (odds ratio, 4.5; 95% confidence interval, 1.3-17.1). Investigation of turkey deli meat eaten by case patients led to several turkey processing plants. The outbreak strain was found in the environment of 1 processing plant and in turkey products from a second. Together, the processing plants recalled > 30 million pounds of products. Following the outbreak, the US Department of Agriculture's Food Safety and Inspection Service issued new regulations outlining a L. monocytogenes control and testing program for ready-to-eat meat and poultry processing plants. CONCLUSIONS: Turkey deli meat was the source of a large multistate outbreak of listeriosis. Investigation of this outbreak helped guide policy changes designed to prevent future L. monocytogenes contamination of ready-to-eat meat and poultry products.

Multistate outbreak of Listeria monocytogenes infection linked to delicatessen turkey meat.

BACKGROUND: Despite a decreasing incidence of listeriosis in the United States, molecular subtyping has increased the number of recognized outbreaks. In September 2000, the New York City Department of Health identified a cluster of infections caused by Listeria monocytogenes isolates with identical molecular subtypes by pulsed-field gel electrophoresis (PFGE) and ribotyping. METHODS: To determine the magnitude of the outbreak and identify risk factors for infection, we notified state health departments and conducted a case-control study. A case was defined as a patient or mother-infant pair infected with Listeria monocytogenes whose isolate yielded the outbreak PFGE pattern. Controls were patients infected with Listeria monocytogenes whose isolate yielded a different PFGE pattern. Patients were asked about food and drink consumed during the 30 days before the onset of illness. RESULTS: Between May and December 2000, there were 30 clinical isolates of Listeria monocytogenes with identical PFGE patterns identified in 11 US states. Cases of infection caused by these isolates were associated with 4 deaths and 3 miscarriages. A case-control study implicated sliced processed turkey from a delicatessen (Mantel-Haenszel odds ratio, 8.0; 95% confidence interval, 1.2-43.3). A traceback investigation identified a single processing plant as the likely source of the outbreak, and the company voluntarily recalled 16 million pounds of processed meat. The same plant had been identified in a Listeria contamination event that had occurred more than a decade previously. CONCLUSIONS: Prevention of persistent L. monocytogenes contamination in food processing plants presents a critical challenge to food safety professionals.