Shiga Toxin-Producing Escherichia coli O157:H7 Illness Outbreak Associated with Untreated, Pressurized, Municipal Irrigation Water - Utah, 2023.

During July-September 2023, an outbreak of Shiga toxin-producing Escherichia coli O157:H7 illness among children in city A, Utah, caused 13 confirmed illnesses; seven patients were hospitalized, including two with hemolytic uremic syndrome. Local, state, and federal public health partners investigating the outbreak linked the illnesses to untreated, pressurized, municipal irrigation water (UPMIW) exposure in city A; 12 of 13 ill children reported playing in or drinking UPMIW. Clinical isolates were genetically highly related to one another and to environmental isolates from multiple locations within city A's UPMIW system. Microbial source tracking, a method to indicate possible contamination sources, identified birds and ruminants as potential sources of fecal contamination of UPMIW. Public health and city A officials issued multiple press releases regarding the outbreak reminding residents that UPMIW is not intended for drinking or recreation. Public education and UPMIW management and operations interventions, including assessing and mitigating potential contamination sources, covering UPMIW sources and reservoirs, indicating UPMIW lines and spigots with a designated color, and providing conspicuous signage to communicate risk and intended use might help prevent future UPMIW-associated illnesses.

O-Antigen Diversification Masks Identification of Highly Pathogenic Shiga Toxin-Producing Escherichia coli O104:H4-Like Strains.

Shiga toxin-producing Escherichia coli (STEC) can give rise to a range of clinical outcomes from diarrhea to the life-threatening systemic condition hemolytic-uremic syndrome (HUS). Although STEC O157:H7 is the serotype most frequently associated with HUS, a major outbreak of HUS occurred in 2011 in Germany and was caused by a rare serotype, STEC O104:H4. Prior to 2011 and since the outbreak, STEC O104:H4 strains have only rarely been associated with human infections. From 2012 to 2020, intensified STEC surveillance was performed in Germany where the subtyping of ~8,000 clinical isolates by molecular methods, including whole-genome sequencing, was carried out. A rare STEC serotype, O181:H4, associated with HUS was identified, and like the STEC O104:H4 outbreak strain, this strain belongs to sequence type 678 (ST678). Genomic and virulence comparisons revealed that the two strains are phylogenetically related and differ principally in the gene cluster encoding their respective lipopolysaccharide O-antigens but exhibit similar virulence phenotypes. In addition, five other serotypes belonging to ST678 from human clinical infection, such as OX13:H4, O127:H4, OgN-RKI9:H4, O131:H4, and O69:H4, were identified from diverse locations worldwide. IMPORTANCE Our data suggest that the high-virulence ensemble of the STEC O104:H4 outbreak strain remains a global threat because genomically similar strains cause disease worldwide but that the horizontal acquisition of O-antigen gene clusters has diversified the O-antigens of strains belonging to ST678. Thus, the identification of these highly pathogenic strains is masked by diverse and rare O-antigens, thereby confounding the interpretation of their potential risk.

Epidemiology of Enteroaggregative, Enteropathogenic, and Shiga Toxin-Producing Escherichia coli Among Children Aged <5 Years in 3 Countries in Africa, 2015-2018: Vaccine Impact on Diarrhea in Africa (VIDA) Study.

BACKGROUND: To address knowledge gaps regarding diarrheagenic Escherichia coli (DEC) in Africa, we assessed the clinical and epidemiological features of enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), and Shiga toxin-producing E. coli (STEC) positive children with moderate-to-severe diarrhea (MSD) in Mali, The Gambia, and Kenya. METHODS: Between May 2015 and July 2018, children aged 0-59 months with medically attended MSD and matched controls without diarrhea were enrolled. Stools were tested conventionally using culture and multiplex polymerase chain reaction (PCR), and by quantitative PCR (qPCR). We assessed DEC detection by site, age, clinical characteristics, and enteric coinfection. RESULTS: Among 4840 children with MSD and 6213 matched controls enrolled, 4836 cases and 1 control per case were tested using qPCR. Of the DEC detected with TAC, 61.1% were EAEC, 25.3% atypical EPEC (aEPEC), 22.4% typical EPEC (tEPEC), and 7.2% STEC. Detection was higher in controls than in MSD cases for EAEC (63.9% vs 58.3%, P < .01), aEPEC (27.3% vs 23.3%, P < .01), and STEC (9.3% vs 5.1%, P < .01). EAEC and tEPEC were more frequent in children aged <23 months, aEPEC was similar across age strata, and STEC increased with age. No association between nutritional status at follow-up and DEC pathotypes was found. DEC coinfection with Shigella/enteroinvasive E. coli was more common among cases (P < .01). CONCLUSIONS: No significant association was detected between EAEC, tEPEC, aEPEC, or STEC and MSD using either conventional assay or TAC. Genomic analysis may provide a better definition of the virulence factors associated with diarrheal disease.

Use of Large-Scale Genomics to Identify the Role of Animals and Foods as Potential Sources of Extraintestinal Pathogenic Escherichia coli That Cause Human Illness

Extraintestinal pathogenic Escherichia coli (ExPEC) cause urinary tract and potentially life-threatening invasive infections. Unfortunately, the origins of ExPEC are not always clear. We used genomic data of E. coli isolates from five U.S. government organizations to evaluate potential sources of ExPEC infections. Virulence gene analysis of 38,032 isolates from human, food animal, retail meat, and companion animals classified the subset of 8142 non-diarrheagenic isolates into 40 virulence groups. Groups were identified as low, medium, and high relative risk of containing ExPEC strains, based on the proportion of isolates recovered from humans. Medium and high relative risk groups showed a greater representation of sequence types associated with human disease, including ST-131. Over 90% of food source isolates belonged to low relative risk groups, while >60% of companion animal isolates belonged to medium or high relative risk groups. Additionally, 18 of the 26 most prevalent antimicrobial resistance determinants were more common in high relative risk groups. The associations between antimicrobial resistance and virulence potentially limit treatment options for human ExPEC infections. This study demonstrates the power of large-scale genomics to assess potential sources of ExPEC strains and highlights the importance of a One Health approach to identify and manage these human pathogens.

Delayed lactose utilization among Shiga toxin-producing Escherichia coli of serogroup O121.

Two outbreaks of Shiga toxin-producing Escherichia coli O121:H19 associated with wheat flour, in the United States of America and Canada, involved strains with an unusual phenotype, delayed lactose utilization (DLU). These strains do not ferment lactose when initially cultured on MacConkey agar (MAC), but lactose fermentation occurs following subculture to a second plate of MAC. The prevalence of DLU was determined by examining the ß-galactosidase activity of 49 strains of E. coli O121, and of 37 other strains of E. coli. Twenty four of forty three O121:H19 and one O121:NM displayed DLU. Two strains (O121:NM and O145:H34) did not have detectable ß-galactosidase activity. ß-glucuronidase activity of O121 strains was also determined. All but six DLU strains had normal ß-glucuronidase activity. ß-glucuronidase activity was suppressed on MAC for 17 of 23 O121 non-DLU strains. Genomic analysis found that DLU strains possessed an insertion sequence, IS600 (1267 bp), between lacZ (ß-galactosidase) and lacY (ß-galactoside permease), that was not present in strains exhibiting normal lactose utilization. The insert might reduce the expression of ß-galactoside permease, delaying import of lactose, resulting in the DLU phenotype. The high probability of DLU should be considered when using lactose-containing media for the isolation of STEC O121.

Agritourism and Kidding Season: A Large Outbreak of Human Shiga Toxin-Producing Escherichia coli O157 (STEC O157) Infections Linked to a Goat Dairy Farm-Connecticut, 2016.

The objective of this study was to determine sources of Shiga toxin-producing Escherichia coli O157 (STEC O157) infection among visitors to Farm X and develop public health recommendations. A case-control study was conducted. Case-patients were defined as the first ill child (aged <18 years) in the household with laboratory-confirmed STEC O157, or physician-diagnosed hemolytic uremic syndrome with laboratory confirmation by serology, who visited Farm X in the 10 days prior to illness. Controls were selected from Farm X visitors aged <18 years, without symptoms during the same time period as case-patients. Environment and animal fecal samples collected from Farm X were cultured; isolates from Farm X were compared with patient isolates using whole genome sequencing (WGS). Case-patients were more likely than controls to have sat on hay bales at the doe barn (adjusted odds ratio: 4.55; 95% confidence interval: 1.41-16.13). No handwashing stations were available; limited hand sanitizer was provided. Overall, 37% (29 of 78) of animal and environmental samples collected were positive for STEC; of these, 62% (18 of 29) yielded STEC O157 highly related by WGS to patient isolates. STEC O157 environmental contamination and fecal shedding by goats at Farm X was extensive. Farms should provide handwashing stations with soap, running water, and disposable towels. Access to animal areas, including animal pens and enclosures, should be limited for young children who are at risk for severe outcomes from STEC O157 infection. National recommendations should be adopted to reduce disease transmission.

The Virulence of Escherichia coli O157:H7 Isolates in Mice Depends on Shiga Toxin Type 2a (Stx2a)-Induction and High Levels of Stx2a in Stool.

In this study we compared nine Shiga toxin (Stx)-producing Escherichia coli O157:H7 patient isolates for Stx levels, stx-phage insertion site(s), and pathogenicity in a streptomycin (Str)-treated mouse model. The strains encoded stx2a, stx1a and stx2a, or stx2a and stx2c. All of the strains elaborated 105-106 cytotoxic doses 50% (CD50) into the supernatant after growth in vitro as measured on Vero cells, and showed variable levels of increased toxin production after growth with sub-inhibitory levels of ciprofloxacin (Cip). The stx2a+stx2c+ isolates were 90-100% lethal for Str-treated BALB/c mice, though one isolate, JH2013, had a delayed time-to-death. The stx2a+ isolate was avirulent. Both an stx2a and a recA deletion mutant of one of the stx2a+stx2c+ strains, JH2010, exhibited at least a three-log decrease in cytotoxicity in vitro and both were avirulent in the mice. Stool from Str-treated mice infected with the highly virulent isolates were 10- to 100-fold more cytotoxic than feces from mice infected with the clinical isolate, JH2012, that made only Stx2a. Taken together these findings demonstrate that the stx2a-phage from JH2010 induces to higher levels in vivo than does the phage from JH2012. The stx1a+stx2a+ clinical isolates were avirulent and neutralization of Stx1 in stool from mice infected with those strains indicated that the toxin produced in vivo was primarily Stx1a. Treatment of mice infected with Stx1a+Stx2a+ isolates with Cip resulted in an increase in Stx2a production in vivo and lethality in the mice. Our data suggest that high levels of Stx2a in stool are predictive of virulence in mice.

PacBio Genome Sequences of Eight Escherichia albertii Strains Isolated from Humans in the United States.

Escherichia albertii is an emerging pathogen that is closely related to Escherichia coli and can carry some of the same virulence genes as E. coli. Here, we report the release of Illumina-corrected PacBio sequences for eight E. albertii genomes. Two of these strains carry Shiga toxin 2f.

PCR-Based Method for Shigella flexneri Serotyping: International Multicenter Validation.

Shigella spp. are a leading cause of human diarrheal disease worldwide, with Shigella flexneri being the most frequently isolated species in developing countries. This serogroup is presently classified into 19 serotypes worldwide. We report here a multicenter validation of a multiplex-PCR-based strategy previously developed by Q. Sun, R. Lan, Y. Wang, A. Zhao, et al. (J Clin Microbiol 49:3766-3770, 2011) for molecular serotyping of S. flexneri This study was performed by seven international laboratories, with a panel of 71 strains (researchers were blind to their identity) as well as 279 strains collected from each laboratory's own local culture collections. This collaborative work found a high extent of agreement among laboratories, calculated through interrater reliability (IRR) measures for the PCR test that proved its robustness. Agreement with the traditional method (serology) was also observed in all laboratories for 14 serotypes studied, while specific genetic events could be responsible for the discrepancies among methodologies in the other 5 serotypes, as determined by PCR product sequencing in most of the cases. This work provided an empirical framework that allowed the use of this molecular method to serotype S. flexneri and showed several advantages over the traditional method of serological typing. These advantages included overcoming the problem of availability of suitable antisera in testing laboratories as well as facilitating the analysis of multiple samples at the same time. The method is also less time-consuming for completion and easier to implement in routine laboratories. We recommend that this PCR be adopted, as it is a reliable diagnostic and characterization methodology that can be used globally for laboratory-based shigella surveillance.

Interlaboratory Evaluation of the U.S. Food and Drug Administration Escherichia coli Identification Microarray for Profiling Shiga Toxin-Producing Escherichia coli.

The U.S. Food and Drug Administration Escherichia coli Identification (FDA-ECID) microarray provides rapid molecular characterization of E. coli. The effectiveness of the FDA-ECID for characterizing Shiga toxin-producing E. coli (STEC) was evaluated by three federal laboratories and one reference laboratory with a panel of 54 reference E. coli strains from the External Quality Assurance program. Strains were tested by FDA-ECID for molecular serotyping (O and H antigens), Shiga toxin subtyping, and the presence of the ehxA and eae genes for enterohemolysin and intimin, respectively. The FDA-ECID O typing was 96% reproducible among the four laboratories and 94% accurate compared with the reference External Quality Assurance data. Discrepancies were due to the absence of O41 target loci on the array and to two pairs of O types with identical target sequences. H typing was 96% reproducible and 100% accurate, with discrepancies due to two strains from one laboratory that were identified as mixed by FDA-ECID. Shiga toxin (Stx) type 1 subtyping was 100% reproducible and accurate, and Stx2 subtyping was 100% reproducible but only 64% accurate. FDA-ECID identified most Stx2 subtypes but had difficulty distinguishing among stx2a, stx2c, and stx2d genes because of close similarities of these sequences. FDA-ECID was 100% effective for detecting ehxA and eae and accurately subtyped the eae alleles. This interlaboratory study revealed that FDA-ECID for STEC characterization was highly reproducible for molecular serotyping, stx and eae subtyping, and ehxA detection. However, the array was less useful for distinguishing among the highly homologous O antigen genes and the stx2a, stx2c, and stx2d subtypes.

DNA microarray-based assessment of virulence potential of Shiga toxin gene-carrying Escherichia coli O104:H7 isolated from feedlot cattle feces.

Escherichia coli O104:H4, a hybrid pathotype reported in a large 2011 foodborne outbreak in Germany, has not been detected in cattle feces. However, cattle harbor and shed in the feces other O104 serotypes, particularly O104:H7, which has been associated with sporadic cases of diarrhea in humans. The objective of our study was to assess the virulence potential of Shiga toxin-producing E. coli (STEC) O104:H7 isolated from feces of feedlot cattle using DNA microarray. Six strains of STEC O104:H7 isolated from cattle feces were analyzed using FDA-E. coli Identification (ECID) DNA microarray to determine their virulence profiles and compare them to the human strains (clinical) of O104:H7, STEC O104:H4 (German outbreak strain), and O104:H21 (milk-associated Montana outbreak strain). Scatter plots were generated from the array data to visualize the gene-level differences between bovine and human O104 strains, and Pearson correlation coefficients (r) were determined. Splits tree was generated to analyze relatedness between the strains. All O104:H7 strains, both bovine and human, similar to O104:H4 and O104:H21 outbreak strains were negative for intimin (eae). The bovine strains were positive for Shiga toxin 1 subtype c (stx1c), enterohemolysin (ehxA), tellurite resistance gene (terD), IrgA homolog protein (iha), type 1 fimbriae (fimH), and negative for genes that code for effector proteins of type III secretory system. The six cattle O104 strains were closely related (r = 0.86-0.98) to each other, except for a few differences in phage related and non-annotated genes. One of the human clinical O104:H7 strains (2011C-3665) was more closely related to the bovine O104:H7 strains (r = 0.81-0.85) than the other four human clinical O104:H7 strains (r = 0.75-0.79). Montana outbreak strain (O104:H21) was more closely related to four of the human clinical O104:H7 strains than the bovine O104:H7 strains. None of the bovine E. coli O104 strains carried genes characteristic of E. coli O104:H4 German outbreak strain and unlike other human strains were also negative for Shiga toxin 2. Because cattle E. coli O104:H7 strains possess stx1c and genes that code for enterohemolysin and a variety of adhesins, the serotype has the potential to be a diarrheagenic foodborne pathogen in humans.

High-Quality Whole-Genome Sequences for 77 Shiga Toxin-Producing Escherichia coli Strains Generated with PacBio Sequencing.

Shiga toxin-producing Escherichia coli (STEC) is an enteric foodborne pathogen that can cause mild to severe illness. Here, we report the availability of high-quality whole-genome sequences for 77 STEC strains generated using the PacBio sequencing platform.

High-Quality Whole-Genome Sequences for 59 Historical Shigella Strains Generated with PacBio Sequencing.

Shigella spp. are enteric pathogens that cause shigellosis. We report here the high-quality whole-genome sequences of 59 historical Shigella strains that represent the four species and a variety of serotypes.

Genetic Analysis of Virulence Potential of Escherichia coli O104 Serotypes Isolated From Cattle Feces Using Whole Genome Sequencing.

Escherichia coli O104:H4, a Shiga toxin-producing hybrid pathotype that was implicated in a major foodborne outbreak in Germany in 2011, has not been detected in cattle. However, serotypes of O104, other than O104:H4, have been isolated from cattle feces, with O104:H7 being the most predominant. In this study, we investigated, based on whole genome sequence analyses, the virulence potential of E. coli O104 strains isolated from cattle feces, since cattle are asymptomatic carriers of E. coli O104. The genomes of ten bovine E. coli O104 strains (six O104:H7, one O104:H8, one O104:H12, and two O104:H23) and five O104:H7 isolated from human clinical cases were sequenced. Of all the bovine O104 serotypes (H7, H8, H12, and H23) that were included in the study, only E. coli O104:H7 serotype possessed Shiga toxins. Four of the six bovine O104:H7 strains and one of the five human strains carried stx1c. Three human O104 strains carried stx2, two were of subtype 2a, and one was 2d. Genomes of stx carrying bovine O104:H7 strains were larger than the stx-negative strains of O104:H7 or other serotypes. The genome sizes were proportional to the number of genes carried on the mobile genetic elements (phages, prophages, transposable elements and plasmids). Both bovine and human strains were negative for intimin and other genes associated with the type III secretory system and non-LEE encoded effectors. Plasmid-encoded virulence genes (ehxA, epeA, espP, katP) were also present in bovine and human strains. All O104 strains were negative for antimicrobial resistance genes, except one human strain. Phylogenetic analysis indicated that bovine E. coli O104 strains carrying the same flagellar antigen clustered together and STEC strains clustered separately from non-STEC strains. One of the human O104:H7 strains was phylogenetically closely related to and belonged to the same sequence type (ST-1817) as the bovine O104:H7 STEC strains. This suggests that the bovine feces could be a source of human illness caused by E. coli O104:H7 serotype. Because bovine O104:H7 strains carried virulence genes similar to human clinical strains and one of the human clinical strains was phylogenetically related to bovine strains, the serotype has the potential to be a diarrheagenic pathogen in humans.

High-Quality Whole-Genome Sequences for 21 Enterotoxigenic Escherichia coli Strains Generated with PacBio Sequencing.

Enterotoxigenic Escherichia coli (ETEC) is an important diarrheagenic pathogen. We report here the high-quality whole-genome sequences of 21 ETEC strains isolated from patients in the United States, international diarrheal surveillance studies, and cruise ship outbreaks.

High-Quality Draft Genome Sequences for Four Drug-Resistant or Outbreak-Associated Shigella sonnei Strains Generated with PacBio Sequencing and Whole-Genome Maps.

Drug-resistant Shigella sonnei poses a clinical and public health challenge. We report here the high-quality draft whole-genome sequences of four outbreak-associated S. sonnei isolates; three were resistant to two or more antibiotics, and one was resistant to streptomycin only.

Draft Genome Sequences of Escherichia coli O104 Strains of Bovine and Human Origin.

Cattle harbor and shed in their feces several Escherichia coli O104 serotypes. All O104 strains examined were intimin negative and belonged to the B1 phylogroup, and some were Shiga toxigenic. We report here the genome sequences of bovine O104:H7 (n = 5), O104:H23 (n = 2), O104:H8 (n = 1), and O104:H12 (n = 1) isolates and human clinical isolates of O104:H7 (n = 5).

Investigation of Escherichia coli Harboring the mcr-1 Resistance Gene - Connecticut, 2016.

The mcr-1 gene confers resistance to the polymyxins, including the antibiotic colistin, a medication of last resort for multidrug-resistant infections. The mcr-1 gene was first reported in 2015 in food, animal, and patient isolates from China (1) and is notable for being the first plasmid-mediated colistin resistance mechanism to be identified. Plasmids can be transferred between bacteria, potentially spreading the resistance gene to other bacterial species. Since its discovery, the mcr-1 gene has been reported from Africa, Asia, Europe, South America, and North America (2,3), including the United States, where it has been identified in Escherichia coli isolated from three patients and from two intestinal samples from pigs (2,4-6). In July 2016, the Pathogen Detection System at the National Center for Biotechnology Information (Bethesda, Maryland) identified mcr-1 in the whole genome sequence of an E. coli isolate from a Connecticut patient (7); this is the fourth isolate from a U.S. patient to contain the mcr-1 gene.