Genomic, transcriptomic, and phenotypic differences among archetype Shigella flexneri strains of serotypes 2a, 3a, and 6.

IMPORTANCE: Given the genomic diversity between S. flexneri serotypes and the paucity of data to support serotype-specific phenotypic differences, we applied in silico and in vitro functional analyses of archetype strains of 2457T (Sf2a), J17B (Sf3a), and CH060 (Sf6). These archetype strains represent the three leading S. flexneri serotypes recommended for inclusion in multivalent vaccines. Characterizing the genomic and phenotypic variation among these clinically prevalent serotypes is an important step toward understanding serotype-specific host-pathogen interactions to optimize the efficacy of multivalent vaccines and therapeutics. This study underpins the importance for further large-scale serotype-targeted analyses.

Clinical and Bacterial Characteristics Associated with Glove and Gown Contamination by Carbapenem-Resistant Klebsiella pneumoniae in the Health Care Setting.

Carbapenem-resistant Klebsiella pneumoniae (CRKp) is a pathogen of significant concern to public health, as it has become increasingly associated with difficult-to-treat community-acquired and hospital-associated infections. Transmission of K. pneumoniae between patients through interactions with shared health care personnel (HCP) has been described as a source of infection in health care settings. However, it is not known whether specific lineages or isolates of K. pneumoniae are associated with increased transmission. Thus, we used whole-genome sequencing to analyze the genetic diversity of 166 carbapenem-resistant K. pneumoniae isolates from five U.S. hospitals in four states as part of a multicenter study examining risk factors for glove and gown contamination by carbapenem-resistant Enterobacterales (CRE). The CRKp isolates exhibited considerable genomic diversity with 58 multilocus sequence types (STs), including four newly designated STs. ST258 was the most prevalent ST, representing 31% (52/166) of the CRKp isolates, but was similarly prevalent among patients who had high, intermediate, and low CRKp transmission. Increased transmission was associated with clinical characteristics including a nasogastric (NG) tube or an endotracheal tube or tracheostomy (ETT/Trach). Overall, our findings provide important insight into the diversity of CRKp associated with transmission from patients to the gloves and gowns of HCP. These findings suggest that certain clinical characteristics and the presence of CRKp in the respiratory tract, rather than specific lineages or genetic content, are more often associated with increased transmission of CRKp from patients to HCP. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKp) is a significant public health concern that has contributed to the spread of carbapenem resistance and has been linked to high morbidity and mortality. Transmission of K. pneumoniae among patients through interactions with shared health care personnel (HCP) has been described as a source of infection in health care settings; however, it remains unknown whether particular bacterial characteristics are associated with increased CRKp transmission. Using comparative genomics, we demonstrate that CRKp isolates associated with high or intermediate transmission exhibit considerable genomic diversity, and there were no K. pneumoniae lineages or genes that were universally predictive of increased transmission. Our findings suggest that certain clinical characteristics and the presence of CRKp, rather than specific lineages or genetic content of CRKp, are more often associated with increased transmission of CRKp from patients to HCP.

Genomic and Functional Characterization of Longitudinal Pseudomonas aeruginosa Isolates from Young Patients with Cystic Fibrosis.

Individuals with cystic fibrosis (CF) suffer from frequent and recurring microbial airway infections. The Gram-negative bacterium Pseudomonas aeruginosa is one of the most common organisms isolated from CF patient airways. P. aeruginosa establishes chronic infections that persist throughout a patient's lifetime and is a major cause of morbidity and mortality. Throughout the course of infection, P. aeruginosa must evolve and adapt from an initial state of early, transient colonization to chronic colonization of the airways. Here, we examined isolates of P. aeruginosa from children under the age of 3 years old with CF to determine genetic adaptations the bacterium undergoes during this early stage of colonization and infection. These isolates were collected when early aggressive antimicrobial therapy was not the standard of care and therefore highlight strain evolution under limited antibiotic pressure. Examination of specific phenotypic adaptations, such as lipid A palmitoylation, antibiotic resistance, and loss of quorum sensing, did not reveal a clear genetic basis for such changes. Additionally, we demonstrate that the geography of patient origin, within the United States or among other countries, does not appear to significantly influence genetic adaptation. In summary, our results support the long-standing model that patients acquire individual isolates of P. aeruginosa that subsequently become hyperadapted to the patient-specific airway environment. This study provides a multipatient genomic analysis of isolates from young CF patients in the United States and contributes data regarding early colonization and adaptation to the growing body of research about P. aeruginosa evolution in the context of CF airway disease. IMPORTANCE Chronic lung infection with Pseudomonas aeruginosa is of major concern for patients with cystic fibrosis (CF). During infection, P. aeruginosa undergoes genomic and functional adaptation to the hyperinflammatory CF airway, resulting in worsening lung function and pulmonary decline. All studies that describe these adaptations use P. aeruginosa obtained from older children or adults during late chronic lung infection; however, children with CF can be infected with P. aeruginosa as early as 3 months of age. Therefore, it is unclear when these genomic and functional adaptations occur over the course of CF lung infection, as access to P. aeruginosa isolates in children during early infection is limited. Here, we present a unique cohort of CF patients who were identified as being infected with P. aeruginosa at an early age prior to aggressive antibiotic therapy. Furthermore, we performed genomic and functional characterization of these isolates to address whether chronic CF P. aeruginosa phenotypes are present during early infection.

Genomic diversity of non-diarrheagenic fecal Escherichia coli from children in sub-Saharan Africa and south Asia and their relatedness to diarrheagenic E. coli.

Escherichia coli is a frequent member of the healthy human gastrointestinal microbiota, as well as an important human pathogen. Previous studies have focused on the genomic diversity of the pathogenic E. coli and much remains unknown about the non-diarrheagenic E. coli residing in the human gut, particularly among young children in low and middle income countries. Also, gaining additional insight into non-diarrheagenic E. coli is important for understanding gut health as non-diarrheagenic E. coli can prevent infection by diarrheagenic bacteria. In this study we examine the genomic diversity of non-diarrheagenic fecal E. coli from male and female children with or without diarrhea from countries in sub-Saharan Africa and south Asia as part of the Global Enteric Multicenter Study (GEMS). We find that these E. coli exhibit considerable genetic diversity as they were identified in all E. coli phylogroups and an Escherichia cryptic clade. Although these fecal E. coli lack the characteristic virulence factors of diarrheagenic E. coli pathotypes, many exhibit remarkable genomic similarity to previously described diarrheagenic isolates with differences attributed to mobile elements. This raises an important question of whether these non-diarrheagenic fecal E. coli may have at one time possessed the mobile element-encoded virulence factors of diarrheagenic pathotypes or may have the potential to acquire these virulence factors.

Spatial-temporal and phylogenetic analyses of epidemiologic data to help understand the modes of transmission of endemic typhoid fever in Samoa.

Salmonella enterica serovar Typhi (S. Typhi) is either widely distributed or proximally transmitted via fecally-contaminated food or water to cause typhoid fever. In Samoa, where endemic typhoid fever has persisted over decades despite water quality and sanitation improvements, the local patterns of S. Typhi circulation remain unclear. From April 2018-June 2020, epidemiologic data and GPS coordinates were collected during household investigations of 260 acute cases of typhoid fever, and 27 asymptomatic shedders of S. Typhi were detected among household contacts. Spatial and temporal distributions of cases were examined using Average Nearest Neighbor and space-time hotspot analyses. In rural regions, infections occurred in sporadic, focal clusters contrasting with persistent, less clustered cases in the Apia Urban Area. Restrictions to population movement during nationwide lockdowns in 2019-2020 were associated with marked reductions of cases. Phylogenetic analyses of isolates with whole genome sequences (n = 186) revealed one dominant genotype 3.5.4 (n = 181/186) that contains three Samoa-exclusive sub-lineages: 3.5.4.1, 3.5.4.2, and 3.5.4.3. Variables of patient sex, age, and geographic region were examined by phylogenetic groupings, and significant differences (p<0.05) associated genetically-similar isolates in urban areas with working ages (20-49 year olds), and in rural areas with age groups typically at home (<5, 50+). Isolates from asymptomatic shedders were among all three sub-lineages. Whole genome sequencing provided evidence of bacterial genetic similarity, which corroborated 10/12 putative epidemiologic linkages among cases and asymptomatic shedders, as well as 3/3 repeat positives (presumed relapses), with a median of one single nucleotide polymorphism difference. These findings highlight various patterns of typhoid transmission in Samoa that differ between urban and rural regions as well as genomic subtypes. Asymptomatic shedders, detectable only through household investigations, are likely an important reservoir and mobile agent of infection. This study advances a "Samoan S. Typhi framework" that supports current and future typhoid surveillance and control efforts in Samoa.

Persistence of Rare Salmonella Typhi Genotypes Susceptible to First-Line Antibiotics in the Remote Islands of Samoa.

For decades, the remote island nation of Samoa (population ~200,000) has faced endemic typhoid fever despite improvements in water quality, sanitation, and economic development. We recently described the epidemiology of typhoid fever in Samoa from 2008 to 2019 by person, place, and time; however, the local Salmonella enterica serovar Typhi (S. Typhi) population structure, evolutionary origins, and genomic features remained unknown. Herein, we report whole genome sequence analyses of 306 S. Typhi isolates from Samoa collected between 1983 and 2020. Phylogenetics revealed a dominant population of rare genotypes 3.5.4 and 3.5.3, together comprising 292/306 (95.4%) of Samoan versus 2/4934 (0.04%) global S. Typhi isolates. Three distinct 3.5.4 genomic sublineages were identified, and their defining polymorphisms were determined. These dominant Samoan genotypes, which likely emerged in the 1970s, share ancestry with other 3.5 clade isolates from South America, Southeast Asia, and Oceania. Additionally, a 106-kb pHCM2 phenotypically cryptic plasmid, detected in a 1992 Samoan S. Typhi isolate, was identified in 106/306 (34.6%) of Samoan isolates; this is more than double the observed proportion of pHCM2-containing isolates in the global collection. In stark contrast with global S. Typhi trends, resistance-conferring polymorphisms were detected in only 15/306 (4.9%) of Samoan S. Typhi, indicating overwhelming susceptibility to antibiotics that are no longer effective in most of South and Southeast Asia. This country-level genomic framework can help local health authorities in their ongoing typhoid surveillance and control efforts, as well as fill a critical knowledge gap in S. Typhi genomic data from Oceania. IMPORTANCE In this study, we used whole genome sequencing and comparative genomics analyses to characterize the population structure, evolutionary origins, and genomic features of S. Typhi associated with decades of endemic typhoid fever in Samoa. Our analyses of Samoan isolates from 1983 to 2020 identified a rare S. Typhi population in Samoa that likely emerged around the early 1970s and evolved into sublineages that are presently dominant. The dominance of these endemic genotypes in Samoa is not readily explained by genomic content or widespread acquisition of antimicrobial resistance. These data establish the necessary framework for future genomic surveillance of S. Typhi in Samoa for public health benefit.

Comparative Genomics Identifies Features Associated with Methicillin-Resistant Staphylococcus aureus (MRSA) Transmission in Hospital Settings.

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious public health concern in the United States. Patients colonized and/or infected can transmit MRSA to healthcare workers and subsequent patients However, the components of this transmission chain are just becoming evident, including certain patient factors, specific patient-healthcare worker interactions, and microbial factors. We conducted a comparative genomic analysis of 388 isolates from four hospitals in three states: Maryland, California, and New York. Isolates from nasal surveillance or clinical cultures were categorized as high, moderate, or low transmission surrogate outcomes based on the number of times the species was identified on the gloves or gowns of healthcare providers. The comparative analyses included a single gene, multigene, and core genome phylogenetic analysis, as well as a genome-wide association analysis to identify molecular signatures associated with the observed transmission surrogate outcomes, geographic origin, or sample source of isolation. Based on the phylogenetic analysis, 95% (n = 372) of the MRSA isolates were from four well-described genomic clades, with most of the isolates being part of the USA300 containing clade (n = 187; 48%). Genome-wide association studies also identified genes that were exclusive or prevalent among specific geographic locations. The identified genes provide insights into the transmission dynamics of MRSA isolates providing additional insights into the basis of the geographical differences of MRSA for molecular diagnostics. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is considered a serious threat to public health and contributes to the dissemination of S. aureus in both the healthcare and community setting. Transmission of MRSA between patients via healthcare worker (HCW) has been described. However, what is not understood are the genetic determinants that contribute to the transmission of MRSA from patients to HCWs. In this study, we demonstrated that certain genes may be associated with transmission in the hospital setting.

Draft Genome Sequences of Two Enteroinvasive Escherichia coli Strains Representative of Major Enteroinvasive E. coli Clades.

There are six described pathotypes of Escherichia coli that cause significant clinical illness in humans. Enteroinvasive E. coli (EIEC) strains have been shown to be separated into three phylogenomic clades. To add to a limited body of EIEC genomic data, we report two high-quality draft genome sequences representing different EIEC phylogenomic clades.

Comparative Genomics of Atypical Enteropathogenic Escherichia coli from Kittens and Children Identifies Bacterial Factors Associated with Virulence in Kittens.

Typical enteropathogenic Escherichia coli (tEPEC) is a leading cause of diarrhea and associated death in children worldwide. Atypical EPEC (aEPEC) lacks the plasmid encoding bundle-forming pili and is considered less virulent, but the molecular mechanism of virulence is poorly understood. We recently identified kittens as a host for aEPEC where intestinal epithelial colonization was associated with diarrheal disease and death. The purposes of this study were to (i) determine the genomic similarity between kitten aEPEC and human aEPEC isolates and (ii) identify genotypic or phenotypic traits associated with virulence in kitten aEPEC. We observed no differences between kitten and human aEPEC in core genome content or gene cluster sequence identities, and no distinguishing genomic content was observed between aEPEC isolates from kittens with nonclinical colonization (NC) versus those with lethal infection (LI). Variation in adherence patterns and ability to aggregate actin in cultured cells mirrored descriptions of human aEPEC. The aEPEC isolated from kittens with LI were significantly more motile than isolates from kittens with NC. Kittens may serve as a reservoir for aEPEC that is indistinguishable from human aEPEC isolates and may provide a needed comparative animal model for the study of aEPEC pathogenesis. Motility seems to be an important factor in pathogenesis of LI associated with aEPEC in kittens.

Draft Genome Sequence of Pseudomonas aeruginosa Strain PA14-UM.

Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen that is a leading cause of morbidity and mortality in cystic fibrosis patients and immunocompromised individuals worldwide. The isolate examined in this study, PA14-UM, is a well-characterized isolate utilized in studies from the University of Maryland.

Draft Genome Sequence of Escherichia coli Strain UMD142.

Escherichia coli can be a harmless commensal organism or cause a range of diseases in humans, including diarrhea, urinary tract infections, meningitis, sepsis, and skin and soft tissue infections. Here, we describe the genome of an isolate that was associated with necrotizing fasciitis and the decompensation of previously undiagnosed cirrhosis.

Genome Sequencing of Escherichia coli and Klebsiella pneumoniae Isolates That Harbor the FOX-5 ß-Lactamase Gene.

We generated draft genome assemblies of three Escherichia coli and seven Klebsiella pneumoniae isolates that harbor the FOX-5 ß-lactamase-encoding gene.

Draft Genome Sequences of Five Diverse Klebsiella Species Isolates from Intensive Care Unit Patients.

We have examined the draft genomes of five isolates from two different Klebsiella species obtained from intensive care unit patients at two geographically distributed hospitals to examine the genomic diversity of hospital-acquired organisms in this understudied population.

Genome Sequences of Four Shigella boydii Strains Representative of the Major S. boydii Clades.

There are four bacterial species in the genus Shigella that cause shigellosis or dysentery. Shigella boydii is one of the least studied Shigella species but has been shown to be separated into three phylogenomic clades. Here, we report four complete reference sequences of the S. boydii phylogenomic clades.

Examination of Staphylococcus aureus Isolates from the Gloves and Gowns of Intensive Care Unit Health Care Workers.

Interactions with health care workers are often thought to be associated with the spread of microbes in the hospital setting. We have examined the genomic diversity of methicillin-resistant Staphylococcus aureus isolates from the gloves and gowns of health care workers from four hospitals in three states.

Examination of 189 Campylobacter Species Isolates from the Global Enteric Multicenter Study.

We have examined the draft genomes of 189 Campylobacter species isolates from the Global Enteric Multicenter Study, in which Campylobacter species were identified as significant pathogens.

Comparative genomic analysis provides insight into the phylogeny and virulence of atypical enteropathogenic Escherichia coli strains from Brazil.

BACKGROUND: Atypical enteropathogenic Escherichia coli (aEPEC) are one of the most frequent intestinal E. coli pathotypes isolated from diarrheal patients in Brazil. Isolates of aEPEC contain the locus of enterocyte effacement, but lack the genes of the bundle-forming pilus of typical EPEC, and the Shiga toxin of enterohemorrhagic E. coli (EHEC). The objective of this study was to evaluate the phylogeny and the gene content of Brazilian aEPEC genomes compared to a global aEPEC collection. METHODOLOGY: Single nucleotide polymorphism (SNP)-based phylogenomic analysis was used to compare 106 sequenced Brazilian aEPEC with 221 aEPEC obtained from other geographic origins. Additionally, Large-Scale BLAST Score Ratio was used to determine the shared versus unique gene content of the aEPEC studied. PRINCIPAL FINDINGS: Phylogenomic analysis demonstrated the 106 Brazilian aEPEC were present in phylogroups B1 (47.2%, 50/106), B2 (23.6%, 25/106), A (22.6%, 24/106), and E (6.6%, 7/106). Identification of EPEC and EHEC phylogenomic lineages demonstrated that 42.5% (45/106) of the Brazilian aEPEC were in four of the previously defined lineages: EPEC10 (17.9%, 19/106), EPEC9 (10.4%, 11/106), EHEC2 (7.5%, 8/106) and EPEC7 (6.6%, 7/106). Interestingly, an additional 28.3% (30/106) of the Brazilian aEPEC were identified in five novel lineages: EPEC11 (14.2%, 15/106), EPEC12 (4.7%, 5/106), EPEC13 (1.9%, 2/106), EPEC14 (5.7%, 6/106) and EPEC15 (1.9%, 2/106). We identified 246 genes that were more frequent among the aEPEC isolates from Brazil compared to the global aEPEC collection, including espG2, espT and espC (P<0.001). Moreover, the nleF gene was more frequently identified among Brazilian aEPEC isolates obtained from diarrheagenic patients when compared to healthy subjects (69.7% vs 41.2%, P<0.05). CONCLUSION: The current study demonstrates significant genomic diversity among aEPEC from Brazil, with the identification of Brazilian aEPEC isolates to five novel EPEC lineages. The greater prevalence of some virulence genes among Brazilian aEPEC genomes could be important to the specific virulence strategies used by aEPEC in Brazil to cause diarrheal disease.