Intrapartum antibiotic prophylaxis selects for mutators in group B streptococci among persistently colonized patients.

Through vaginal colonization, GBS causes severe pregnancy outcomes including neonatal sepsis and meningitis. Although intrapartum antibiotic prophylaxis (IAP) has reduced early-onset disease rates, persistent GBS colonization has been observed in patients following prophylaxis. To determine whether IAP selects for genomic signatures that enhance GBS survival and persistence in the vaginal tract, whole-genome sequencing was performed on 97 isolates from 58 patients before (prenatal) and after (postpartum) IAP/childbirth. Core-gene mutation analysis identified 7,025 mutations between the paired isolates. Three postpartum isolates accounted for 98% of mutations and were classified as "mutators" because of point mutations within DNA repair systems. In vitro assays revealed stronger biofilms in two mutators. These findings suggest that antibiotics select for mutations that promote survival in vivo, which increases the likelihood of transmission to neonates. They also demonstrate how mutators can provide a reservoir of beneficial mutations that enhance fitness and genetic diversity in the GBS population.

Epidemiologic and Genomic Evidence for Zoonotic Transmission of SARS-CoV-2 among People and Animals on a Michigan Mink Farm, United States, 2020.

Farmed mink are one of few animals in which infection with SARS-CoV-2 has resulted in sustained transmission among a population and spillback from mink to people. In September 2020, mink on a Michigan farm exhibited increased morbidity and mortality rates due to confirmed SARS-CoV-2 infection. We conducted an epidemiologic investigation to identify the source of initial mink exposure, assess the degree of spread within the facility's overall mink population, and evaluate the risk of further viral spread on the farm and in surrounding wildlife habitats. Three farm employees reported symptoms consistent with COVID-19 the same day that increased mortality rates were observed among the mink herd. One of these individuals, and another asymptomatic employee, tested positive for SARS-CoV-2 by real-time reverse transcription PCR (RT-qPCR) 9 days later. All but one mink sampled on the farm were positive for SARS-CoV-2 based on nucleic acid detection from at least one oral, nasal, or rectal swab tested by RT-qPCR (99%). Sequence analysis showed high degrees of similarity between sequences from mink and the two positive farm employees. Epidemiologic and genomic data, including the presence of F486L and N501T mutations believed to arise through mink adaptation, support the hypothesis that the two employees with SARS-CoV-2 nucleic acid detection contracted COVID-19 from mink. However, the specific source of virus introduction onto the farm was not identified. Three companion animals living with mink farm employees and 31 wild animals of six species sampled in the surrounding area were negative for SARS-CoV-2 by RT-qPCR. Results from this investigation support the necessity of a One Health approach to manage the zoonotic spread of SARS-CoV-2 and underscores the critical need for multifaceted public health approaches to prevent the introduction and spread of respiratory viruses on mink farms.

Pangenomic analyses of antibiotic-resistant Campylobacter jejuni reveal unique lineage distributions and epidemiological associations.

Application of whole-genome sequencing (WGS) to characterize foodborne pathogens has advanced our understanding of circulating genotypes and evolutionary relationships. Herein, we used WGS to investigate the genomic epidemiology of Campylobacter jejuni, a leading cause of foodborne disease. Among the 214 strains recovered from patients with gastroenteritis in Michigan, USA, 85 multilocus sequence types (STs) were represented and 135 (63.1 %) were phenotypically resistant to at least one antibiotic. Horizontally acquired antibiotic resistance genes were detected in 128 (59.8 %) strains and the genotypic resistance profiles were mostly consistent with the phenotypes. Core-gene phylogenetic reconstruction identified three sequence clusters that varied in frequency, while a neighbour-net tree detected significant recombination among the genotypes (pairwise homoplasy index P<0.01). Epidemiological analyses revealed that travel was a significant contributor to pangenomic and ST diversity of C. jejuni, while some lineages were unique to rural counties and more commonly possessed clinically important resistance determinants. Variation was also observed in the frequency of lineages over the 4 year period with chicken and cattle specialists predominating. Altogether, these findings highlight the importance of geographically specific factors, recombination and horizontal gene transfer in shaping the population structure of C. jejuni. They also illustrate the usefulness of WGS data for predicting antibiotic susceptibilities and surveillance, which are important for guiding treatment and prevention strategies.

Whole-Genome Sequencing of Shiga Toxin-Producing Escherichia coli for Characterization and Outbreak Investigation.

Shiga toxin-producing Escherichia coli (STEC) causes high frequencies of foodborne infections worldwide and has been linked to numerous outbreaks each year. Pulsed-field gel electrophoresis (PFGE) has been the gold standard for surveillance until the recent transition to whole-genome sequencing (WGS). To further understand the genetic diversity and relatedness of outbreak isolates, a retrospective analysis of 510 clinical STEC isolates was conducted. Among the 34 STEC serogroups represented, most (59.6%) belonged to the predominant six non-O157 serogroups. Core genome single nucleotide polymorphism (SNP) analysis differentiated clusters of isolates with similar PFGE patterns and multilocus sequence types (STs). One serogroup O26 outbreak strain and another non-typeable (NT) strain, for instance, were identical by PFGE and clustered together by MLST; however, both were distantly related in the SNP analysis. In contrast, six outbreak-associated serogroup O5 strains clustered with five ST-175 serogroup O5 isolates, which were not part of the same outbreak as determined by PFGE. The use of high-quality SNP analyses enhanced the discrimination of these O5 outbreak strains into a single cluster. In all, this study demonstrates how public health laboratories can more rapidly use WGS and phylogenetics to identify related strains during outbreak investigations while simultaneously uncovering important genetic attributes that can inform treatment practices.

Long read genome assemblers struggle with small plasmids.

Whole-genome sequencing has become a preferred method for studying bacterial plasmids, as it is generally assumed to capture the entire genome. However, long-read genome assemblers have been shown to sometimes miss plasmid sequences - an issue that has been associated with plasmid size. The purpose of this study was to investigate the relationship between plasmid size and plasmid recovery by the long-read-only assemblers, Flye, Raven, Miniasm, and Canu. This was accomplished by determining the number of times each assembler successfully recovered 33 plasmids, ranging from 1919 to 194 062 bp in size and belonging to 14 bacterial isolates from six bacterial genera, using Oxford Nanopore long reads. These results were additionally compared to plasmid recovery rates by the short-read-first assembler, Unicycler, using both Oxford Nanopore long reads and Illumina short reads. Results from this study indicate that Canu, Flye, Miniasm, and Raven are prone to missing plasmid sequences, whereas Unicycler was successful at recovering 100 % of plasmid sequences. Excluding Canu, most plasmid loss by long-read-only assemblers was due to failure to recover plasmids smaller than 10 kb. As such, it is recommended that Unicycler be used to increase the likelihood of plasmid recovery during bacterial genome assembly.

Multinational Outbreak of Listeria monocytogenes Infections Linked to Enoki Mushrooms Imported from the Republic of Korea 2016-2020.

Keeping the global food supply safe necessitates international collaborations between countries. Health and regulatory agencies routinely communicate during foodborne illness outbreaks, allowing partners to share investigational evidence. A 2016-2020 outbreak of Listeria monocytogenes infections linked to imported enoki mushrooms required a multinational collaborative investigation among the United States, Canada, Australia, and France. Ultimately, this outbreak included 48 ill people, 36 in the United States and 12 in Canada, and was linked to enoki mushrooms sourced from one manufacturer located in the Republic of Korea. Epidemiologic, laboratory, and traceback evidence led to multiple regulatory actions, including extensive voluntary recalls by three firms in the United States and one firm in Canada. In the United States and Canada, the Korean manufacturer was placed on import alert while other international partners provided information about their respective investigations and advised the public not to eat the recalled enoki mushrooms. The breadth of the geographic distribution of this outbreak emphasizes the global reach of the food industry. This investigation provides a powerful example of the impact of national and international coordination of efforts to respond to foodborne illness outbreaks and protect consumers. It also demonstrates the importance of fast international data sharing and collaboration in identifying and stopping foodborne outbreaks in the global community. Additionally, it is a meaningful example of the importance of food sampling, testing, and integration of sequencing results into surveillance databases.

Global disparities in SARS-CoV-2 genomic surveillance.

Genomic sequencing is essential to track the evolution and spread of SARS-CoV-2, optimize molecular tests, treatments, vaccines, and guide public health responses. To investigate the global SARS-CoV-2 genomic surveillance, we used sequences shared via GISAID to estimate the impact of sequencing intensity and turnaround times on variant detection in 189 countries. In the first two years of the pandemic, 78% of high-income countries sequenced >0.5% of their COVID-19 cases, while 42% of low- and middle-income countries reached that mark. Around 25% of the genomes from high income countries were submitted within 21 days, a pattern observed in 5% of the genomes from low- and middle-income countries. We found that sequencing around 0.5% of the cases, with a turnaround time <21 days, could provide a benchmark for SARS-CoV-2 genomic surveillance. Socioeconomic inequalities undermine the global pandemic preparedness, and efforts must be made to support low- and middle-income countries improve their local sequencing capacity.

Global disparities in SARS-CoV-2 genomic surveillance.

Genomic sequencing provides critical information to track the evolution and spread of SARS-CoV-2, optimize molecular tests, treatments and vaccines, and guide public health responses. To investigate the spatiotemporal heterogeneity in the global SARS-CoV-2 genomic surveillance, we estimated the impact of sequencing intensity and turnaround times (TAT) on variant detection in 167 countries. Most countries submit genomes >21 days after sample collection, and 77% of low and middle income countries sequenced <0.5% of their cases. We found that sequencing at least 0.5% of the cases, with a TAT <21 days, could be a benchmark for SARS-CoV-2 genomic surveillance efforts. Socioeconomic inequalities substantially impact our ability to quickly detect SARS-CoV-2 variants, and undermine the global pandemic preparedness.

Antibiotic Susceptibility Profiles and Frequency of Resistance Genes in Clinical Shiga Toxin-Producing Escherichia coli Isolates from Michigan over a 14-Year Period.

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen that contributes to over 250,000 infections in the United States each year. Because antibiotics are not recommended for STEC infections, resistance in STEC has not been widely researched despite an increased likelihood for the transfer of resistance genes from STEC to opportunistic pathogens residing within the same microbial community. From 2001 to 2014, 969 STEC isolates were collected from Michigan patients. Antibiotic susceptibility profiles to clinically relevant antibiotics were determined using disc diffusion, while epidemiological data were used to identify factors associated with resistance. Whole-genome sequencing was used for serotyping, examining genetic relatedness, and identifying genetic determinants and mechanisms of resistance in the non-O157 isolates. Increasing frequencies of resistance to at least one antibiotic were observed over the 14 years (P = 0.01). While the non-O157 serogroups were more commonly resistant than O157 (odds ratio, 2.4; 95% confidence interval,1.43 to 4.05), the frequency of ampicillin resistance among O157 isolates was significantly higher in Michigan than the national average (P = 0.03). Genomic analysis of 321 non-O157 isolates uncovered 32 distinct antibiotic resistance genes (ARGs). Although mutations in genes encoding resistance to ciprofloxacin and ampicillin were detected in four isolates, most of the horizontally acquired ARGs conferred resistance to aminoglycosides, ß-lactams, sulfonamides, and/or tetracycline. This study provides insight into the mechanisms of resistance in a large collection of clinical non-O157 STEC isolates and demonstrates that antibiotic resistance among all STEC serogroups has increased over time, prompting the need for enhanced surveillance.

Population structure and genetic diversity of non-O157 Shiga toxin-producing Escherichia coli (STEC) clinical isolates from Michigan.

Non-O157 STEC are increasingly linked to foodborne infections, yet little is known about the diversity and molecular epidemiology across locations. Herein, we used whole genome sequencing to examine genetic variation in 894 isolates collected from Michigan patients between 2001 and 2018. In all, 67 serotypes representing 69 multilocus sequence types were identified. Serotype diversity increased from an average of four (2001-2006) to 17 (2008-2018) serotypes per year. The top six serogroups reported nationally caused > 60% of infections in 16 of the 18 years; serogroups O111 and O45 were associated with hospitalization as were age ≥ 65 years, diarrhea with blood and female sex. Phylogenetic analyses of seven multilocus sequence typing (MLST) loci identified three clades as well as evidence of parallel evolution and recombination. Most (95.5%) isolates belonged to one clade, which could be further differentiated into seven subclades comprising isolates with varying virulence gene profiles and serotypes. No association was observed between specific clades and the epidemiological data, suggesting that serogroup- and serotype-specific associations are more important predictors of disease outcomes than lineages defined by MLST. Molecular epidemiological studies of non-O157 STEC are important to enhance understanding of circulating strain distributions and traits, genetic variation, and factors that may impact disease risk and severity.

Genetic and Phenotypic Factors Associated with Persistent Shedding of Shiga Toxin-Producing Escherichia coli by Beef Cattle.

Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne infections. Cattle are an important STEC reservoir, although little is known about specific pathogen traits that impact persistence in the farm environment. Hence, we sought to evaluate STEC isolates recovered from beef cattle in a single herd in Michigan. To do this, we collected fecal grabs from 26 cattle and resampled 13 of these animals at 3 additional visits over a 3-month period. In all, 66 STEC isolates were recovered for genomics and biofilm quantification using crystal violet assays. The STEC population was diverse, representing seven serotypes, including O157:H7, O26:H11, and O103:H2, which are commonly associated with human infections. Although a core genome analysis of 2,933 genes grouped isolates into clusters based on serogroups, some isolates within each cluster had variable biofilm levels and virulence gene profiles. Most (77.8%; n = 49) isolates harbored stx2a, while 38 (57.5%) isolates formed strong biofilms. Isolates belonging to the predominant serogroup O6 (n = 36; 54.5%) were more likely to form strong biofilms, persistently colonize multiple cattle, and be acquired over time. A high-quality single nucleotide polymorphism (SNP) analysis of 33 O6 isolates detected between 0 and 13 single nucleotide polymorphism (SNP) differences between strains, indicating that highly similar strain types were persisting in this herd. Similar findings were observed for other persistent serogroups, although key genes were found to differ among strong and weak biofilm producers. Together, these data highlight the diversity and persistent nature of some STEC types in this important food animal reservoir.IMPORTANCE Food animal reservoirs contribute to Shiga toxin-producing Escherichia coli (STEC) evolution via the acquisition of horizontally acquired elements like Shiga toxin bacteriophages that enhance pathogenicity. In cattle, persistent fecal shedding of STEC contributes to contamination of beef and dairy products and to crops being exposed to contaminated water systems. Hence, identifying factors important for STEC persistence is critical. This longitudinal study enhances our understanding of the genetic diversity of STEC types circulating in a cattle herd and identifies genotypic and phenotypic traits associated with persistence. Key findings demonstrate that multiple STEC types readily persist in and are transmitted across cattle in a shared environment. These dynamics also enhance the persistence of virulence genes that can be transferred between bacterial hosts, resulting in the emergence of novel STEC strain types. Understanding how pathogens persist and diversify in reservoirs is important for guiding new preharvest prevention strategies aimed at reducing foodborne transmission to humans.

Genetic Diversity of Non-O157 Shiga Toxin-Producing Escherichia coli Recovered From Patients in Michigan and Connecticut.

Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens and non-O157 serotypes have been gradually increasing in frequency. The non-O157 STEC population is diverse and is often characterized using serotyping and/or multilocus sequence typing (MLST). Although spacers within clustered regularly interspaced repeat (CRISPR) regions were shown to comprise horizontally acquired DNA elements, this region does not actively acquire spacers in STEC. Hence, it is useful for further characterizing non-O157 STEC and examining relationships between strains. Our study goal was to evaluate the genetic relatedness of 41 clinical non-O157 isolates identified in Michigan between 2001 and 2005 while comparing to 114 isolates from Connecticut during an overlapping time period. Whole genome sequencing (WGS) was performed, and sequences were extracted for serotyping, MLST and CRISPR analysis. Phylogenetic analysis of MLST and CRISPR data was performed using the Neighbor joining and unweighted pair group method with arithmetic mean (UPGMA) algorithms, respectively. In all, 29 serogroups were identified; eight were unique to Michigan and 13 to Connecticut. "Big-six" serogroup frequencies were similar by state (Michigan: 73.2%, Connecticut: 81.6%), though STEC O121 was not found in Michigan. The distribution of sequence types (STs) and CRISPR profiles was also similar across states. Interestingly, big-six serogroups such as O103 and O26, grouped into different STs located on distinct branches of the phylogeny, further confirming that serotyping alone is not adequate for evaluating strain relatedness. Comparatively, the CRISPR analysis identified 361 unique spacers that grouped into 80 different CRISPR profiles. CRISPR spacers 231 and 317 were isolated from 79.2% (n = 118) and 59.1% (n = 88) of strains, respectively, regardless of serogroup and ST. Spacer profiles clustered according to the MLST analysis, though some discrepancies were noted. Indeed, use of both MLST and CRISPR typing enhanced the discriminatory power when compared to the use of each tool separately. These data highlight the genetic diversity of clinical STEC from different locations and show that CRISPR profiling can be used alongside MLST to discriminate related strains. Use of targeted sequencing approaches are particularly helpful for sites without WGS capabilities and can help define which strains require additional characterization using more discriminatory methods.