Low occurrence of multi-antimicrobial and heavy metal resistance in Salmonella enterica from wild birds in the United States.

Wild birds are common reservoirs of Salmonella enterica. Wild birds carrying resistant S. enterica may pose a risk to public health as they can spread the resistant bacteria across large spatial scales within a short time. Here, we whole-genome sequenced 375 S. enterica strains from wild birds collected in 41 U.S. states during 1978-2019 to examine bacterial resistance to antibiotics and heavy metals. We found that Typhimurium was the dominant S. enterica serovar, accounting for 68.3% (256/375) of the bird isolates. Furthermore, the proportions of the isolates identified as multi-antimicrobial resistant (multi-AMR: resistant to at least three antimicrobial classes) or multi-heavy metal resistant (multi-HMR: resistant to at least three heavy metals) were both 1.87% (7/375). Interestingly, all the multi-resistant S. enterica (n = 12) were isolated from water birds or raptors; none of them was isolated from songbirds. Plasmid profiling demonstrated that 75% (9/12) of the multi-resistant strains carried resistance plasmids. Our study indicates that wild birds do not serve as important reservoirs of multi-resistant S. enterica strains. Nonetheless, continuous surveillance for bacterial resistance in wild birds is necessary because the multi-resistant isolates identified in this study also showed close genetic relatedness with those from humans and domestic animals.

Salmonella enterica Serovar Typhimurium Isolates from Wild Birds in the United States Represent Distinct Lineages Defined by Bird Type.

Salmonella enterica serovar Typhimurium is typically considered a host generalist; however, certain isolates are associated with specific hosts and show genetic features of host adaptation. Here, we sequenced 131 S. Typhimurium isolates from wild birds collected in 30 U.S. states during 1978-2019. We found that isolates from broad taxonomic host groups including passerine birds, water birds (Aequornithes), and larids (gulls and terns) represented three distinct lineages and certain S. Typhimurium CRISPR types presented in individual lineages. We also showed that lineages formed by wild bird isolates differed from most isolates originating from domestic animal sources, and that genomes from these lineages substantially improved source attribution of Typhimurium genomes to wild birds by a machine learning classifier. Furthermore, virulence gene signatures that differentiated S. Typhimurium from passerines, water birds, and larids were detected. Passerine isolates tended to lack S. Typhimurium-specific virulence plasmids. Isolates from the passerine, water bird, and larid lineages had close genetic relatedness with human clinical isolates, including those from a 2021 U.S. outbreak linked to passerine birds. These observations indicate that S. Typhimurium from wild birds in the United States are likely host-adapted, and the representative genomic data set examined in this study can improve source prediction and facilitate outbreak investigation. IMPORTANCE Within-host evolution of S. Typhimurium may lead to pathovars adapted to specific hosts. Here, we report the emergence of disparate avian S. Typhimurium lineages with distinct virulence gene signatures. The findings highlight the importance of wild birds as a reservoir for S. Typhimurium and contribute to our understanding of the genetic diversity of S. Typhimurium from wild birds. Our study indicates that S. Typhimurium may have undergone adaptive evolution within wild birds in the United States. The representative S. Typhimurium genomes from wild birds, together with the virulence gene signatures identified in these bird isolates, are valuable for S. Typhimurium source attribution and epidemiological surveillance.

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase.

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)(FBXW11) E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through an alternate binding site to the SCF(FBXW11) E3 ligase. We further show that disrupting the assembly of the SCF(FBXW11-NSs) E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCF(FBXW11-NSs) E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the ßTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of ßTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCF(FBXW11) complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.

Antimicrobial Resistance in Campylobacter Species: Mechanisms and Genomic Epidemiology.

The Campylobacter genus is a large and diverse group of Gram-negative bacteria that are known to colonize humans and other mammals, birds, reptiles, and shellfish. While it is now recognized that several emerging Campylobacter species can be associated with human disease, two species, C. jejuni and C. coli, are responsible for the vast majority of bacterial gastroenteritis in humans worldwide. Infection with C. jejuni, in particular, has also been associated with a number of extragastrointestinal manifestations and autoimmune conditions, most notably Guillain-Barré syndrome. The antimicrobial drugs of choice for the treatment of severe Campylobacter infection include macrolides, such as erythromycin, clarithromycin, or azithromycin. Fluoroquinolones, such as ciprofloxacin, are also commonly used for empirical treatment of undiagnosed diarrheal disease. However, resistance to these and other classes of antimicrobial drugs is increasing and is a major public health problem. The US Centers for Disease Control and Prevention estimates that over 300,000 infections per year are caused by drug-resistant Campylobacter. In this chapter, we discuss the taxonomy of the Campylobacter genus, the clinical and global epidemiological aspects of Campylobacter infection, with an emphasis on C. jejuni and C. coli, and issues related to the treatment of infection and antimicrobial resistance mechanisms. We further discuss the use of next-generation sequencing for the detection and surveillance of antimicrobial resistance genes.

Use of whole-genome sequencing for Campylobacter surveillance from NARMS retail poultry in the United States in 2015.

Whole genome sequencing (WGS) has become a rapid and affordable tool for public health surveillance and outbreak detection. In this study, we used the Illuminia MiSeq® to sequence 589 Campylobacter isolates obtained in 2015 from retail poultry meats as part of the National Antimicrobial Resistance Monitoring System (NARMS). WGS data were used to identify the Campylobacter species and to compare the concordance between resistance genotypes and phenotypes. WGS accurately identified 386 C. jejuni and 203 C. coli using gyrA sequence information. Ten resistance genes, including tetO, blaOXA-61, aph(2″)-Ic, aph(2″)-If, aph(2″)-Ig, aph(3')-III, ant(6)-1a, aadE, aph(3")-VIIa, and Inu(C), plus mutations in housekeeping genes (gyrA at position 86, 23S rRNA at position 2074 and 2075), were identified by WGS analysis. Overall, there was a high concordance between phenotypic resistance to a given drug and the presence of known resistance genes. Concordance between both resistance and susceptible phenotypes and genotype was 100% for ciprofloxacin, nalidixic acid, gentamicin, azithromycin, and florfenicol. A few discrepancies were observed for tetracycline, clindamycin, and telithromycin. The concordance between resistance phenotype and genotype ranged from 67.9% to 100%; whereas, the concordance between susceptible phenotype and genotype ranged from 98.0% to 99.6%. Our study demonstrates that WGS can correctly identify Campylobacter species and predict antimicrobial resistance with a high degree of accuracy.

Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health.

Klebsiella pneumoniae is now recognized as an urgent threat to human health because of the emergence of multidrug-resistant strains associated with hospital outbreaks and hypervirulent strains associated with severe community-acquired infections. K. pneumoniae is ubiquitous in the environment and can colonize and infect both plants and animals. However, little is known about the population structure of K. pneumoniae, so it is difficult to recognize or understand the emergence of clinically important clones within this highly genetically diverse species. Here we present a detailed genomic framework for K. pneumoniae based on whole-genome sequencing of more than 300 human and animal isolates spanning four continents. Our data provide genome-wide support for the splitting of K. pneumoniae into three distinct species, KpI (K. pneumoniae), KpII (K. quasipneumoniae), and KpIII (K. variicola). Further, for K. pneumoniae (KpI), the entity most frequently associated with human infection, we show the existence of >150 deeply branching lineages including numerous multidrug-resistant or hypervirulent clones. We show K. pneumoniae has a large accessory genome approaching 30,000 protein-coding genes, including a number of virulence functions that are significantly associated with invasive community-acquired disease in humans. In our dataset, antimicrobial resistance genes were common among human carriage isolates and hospital-acquired infections, which generally lacked the genes associated with invasive disease. The convergence of virulence and resistance genes potentially could lead to the emergence of untreatable invasive K. pneumoniae infections; our data provide the whole-genome framework against which to track the emergence of such threats.

Development of real-time PCR assays for the detection of Moraxella macacae associated with bloody nose syndrome in rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques.

BACKGROUND: Moraxella macacae is a recently described bacterial pathogen that causes epistaxis or so-called bloody nose syndrome in captive macaques. The aim of this study was to develop specific molecular diagnostic assays for M. macacae and to determine their performance characteristics. METHODS: We developed six real-time PCR assays on the Roche LightCycler. The accuracy, precision, selectivity, and limit of detection (LOD) were determined for each assay, in addition to further validation by testing nasal swabs from macaques presenting with epistaxis at the Tulane National Primate Research Center. RESULTS: All assays exhibited 100% specificity and were highly sensitive with an LOD of 10 fg for chromosomal assays and 1 fg for the plasmid assay. Testing of nasal swabs from 10 symptomatic macaques confirmed the presence of M. macacae in these animals. CONCLUSIONS: We developed several accurate, sensitive, and species-specific real-time PCR assays for the detection of M. macacae in captive macaques.

Genomic variability of monkeypox virus among humans, Democratic Republic of the Congo.

Monkeypox virus is a zoonotic virus endemic to Central Africa. Although active disease surveillance has assessed monkeypox disease prevalence and geographic range, information about virus diversity is lacking. We therefore assessed genome diversity of viruses in 60 samples obtained from humans with primary and secondary cases of infection from 2005 through 2007. We detected 4 distinct lineages and a deletion that resulted in gene loss in 10 (16.7%) samples and that seemed to correlate with human-to-human transmission (p = 0.0544). The data suggest a high frequency of spillover events from the pool of viruses in nonhuman animals, active selection through genomic destabilization and gene loss, and increased disease transmissibility and severity. The potential for accelerated adaptation to humans should be monitored through improved surveillance.

Microbial water quality of recreational lakes near Tbilisi, Georgia.

Microbial safety of recreational water is one of the major human public health issues in developing countries. Three water bodies, the Tbilisi Sea, Kumisi and Lisi lakes, in the South Caucasus region near Tbilisi, Georgia, were monitored in 2006-2009 to determine microbiological quality using standard methods. Microbial pollution indicators were determined in parallel with phytoplankton abundance and measurement of a number of physical-chemical parameters. Kumisi Lake, a brackish water body in an active agricultural area, appeared to be the most polluted, whereas the Tbilisi Sea, a freshwater reservoir was the least polluted. High values for fecal indicators in all three lakes in summer and early autumn were revealed. In our study, total enterococci counts (TEC) appeared to be a better indicator than either fecal or total coliform counts for the evaluation of fresh and brackish microbial water quality. We found significant correlation between total Vibrio counts and TEC for all three water bodies. Prevalence of somatic coliphages and V. cholerae-specific phages as additional water pollution indicator significantly correlated with abundance of the host bacteria. Particular phytoplankton groups in the lakes responded to the changes of fecal indicators; however, no correlation was observed between dominant zooplankton taxonomic groups and microbial parameters.

Smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus.

Vaccination with live vaccinia virus affords long-lasting protection against variola virus, the agent of smallpox. Its mode of protection in humans, however, has not been clearly defined. Here we report that vaccinia-specific B-cell responses are essential for protection of macaques from monkeypox virus, a variola virus ortholog. Antibody-mediated depletion of B cells, but not CD4+ or CD8+ T cells, abrogated vaccine-induced protection from a lethal intravenous challenge with monkeypox virus. In addition, passive transfer of human vaccinia-neutralizing antibodies protected nonimmunized macaques from severe disease. Thus, vaccines able to induce long-lasting protective antibody responses may constitute realistic alternatives to the currently available smallpox vaccine (Dryvax).

Prevalence of Antimicrobial Resistance in Select Bacteria From Retail Seafood-United States, 2019.

In 2019, the United States National Antimicrobial Resistance Monitoring System (NARMS) surveyed raw salmon, shrimp, and tilapia from retail grocery outlets in eight states to assess the prevalence of bacterial contamination and antimicrobial resistance (AMR) in the isolates. Prevalence of the targeted bacterial genera ranged among the commodities: Salmonella (0%-0.4%), Aeromonas (19%-26%), Vibrio (7%-43%), Pseudomonas aeruginosa (0.8%-2.3%), Staphylococcus (23%-30%), and Enterococcus (39%-66%). Shrimp had the highest odds (OR: 2.8, CI: 2.0-3.9) of being contaminated with at least one species of these bacteria, as were seafood sourced from Asia vs. North America (OR: 2.7; CI: 1.8-4.7) and Latin America and the Caribbean vs. North America (OR: 1.6; CI: 1.1-2.3) and seafood sold at the counter vs. sold frozen (OR: 2.1; CI: 1.6-2.9). Isolates exhibited pan-susceptibility (Salmonella and P. aeruginosa) or low prevalence of resistance (<10%) to most antimicrobials tested, with few exceptions. Seafood marketed as farm-raised had lower odds of contamination with antimicrobial resistant bacteria compared to wild-caught seafood (OR: 0.4, CI: 0.2-0.7). Antimicrobial resistance genes (ARGs) were detected for various classes of medically important antimicrobials. Clinically relevant ARGs included carbapenemases (bla IMI-2, bla NDM-1) and extended spectrum ß-lactamases (ESBLs; bla CTX-M-55). This population-scale study of AMR in seafood sold in the United States provided the basis for NARMS seafood monitoring, which began in 2020.

The Natural History of Aerosolized Francisella tularensis Infection in Cynomolgus Macaques.

Tularemia is a severe, zoonotic infection caused by the Gram-negative bacterium Francisella tularensis. Inhalation results in a rapid, severe bacterial pneumonia and sepsis, which can be lethal. Because the cynomolgus macaque is the accepted nonhuman primate model for tularemia, we conducted a natural history study of pneumonic tularemia by exposing macaques to target inhaled doses of 50, 500, or 5000 colony forming units (CFU) of F. tularensis subsp. tularensis SCHU S4. Two animals within the 50 CFU group (calculated doses of 10 and 11 CFU) survived the challenge, while the remainder succumbed to infection. Exposure of cynomolgus macaques to aerosolized SCHU S4 resulted in fever, anorexia, increased white blood cell counts, lymphopenia, thrombocytopenia, increased liver enzymes, alterations in electrocardiogram (ECG), and pathological changes typical of infection with F. tularensis, regardless of the challenge dose. Blood pressure dropped during the febrile phase, particularly as temperature began to drop and macaques succumbed to the disease. ECG analysis indicated that in 33% of the macaques, heart rate was not elevated during the febrile phase (Faget's sign; pulse-temperature disassociation), which has been reported in a similar percentage of human cases. These results indicated that infection of cynomolgus macaques with aerosolized F. tularensis results in similar disease progression and outcome as seen in humans, and that cynomolgus macaques are a reliable animal model to test medical countermeasures against aerosolized F. tularensis.

Identification of pathogenic Vibrio species by multilocus PCR-electrospray ionization mass spectrometry and its application to aquatic environments of the former soviet republic of Georgia.

The Ibis T5000 is a novel diagnostic platform that couples PCR and mass spectrometry. In this study, we developed an assay that can identify all known pathogenic Vibrio species and field-tested it using natural water samples from both freshwater lakes and the Georgian coastal zone of the Black Sea. Of the 278 total water samples screened, 9 different Vibrio species were detected, 114 (41%) samples were positive for V. cholerae, and 5 (0.8%) samples were positive for the cholera toxin A gene (ctxA). All ctxA-positive samples were from two freshwater lakes, and no ctxA-positive samples from any of the Black Sea sites were detected.

Rapid identification of vector-borne flaviviruses by mass spectrometry.

Flaviviruses are a highly diverse group of RNA viruses classified within the genus Flavivirus, family Flaviviridae. Most flaviviruses are arthropod-borne, requiring a mosquito or tick vector. Several flaviviruses are highly pathogenic to humans; however, their high genetic diversity and immunological relatedness makes them extremely challenging to diagnose. In this study, we developed and evaluated a broad-range Flavivirus assay designed to detect both tick- and mosquito-borne flaviviruses by using RT-PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) on the Ibis T5000 platform. The assay was evaluated with a panel of 13 different flaviviruses. All samples were correctly identified to the species level. To determine the limit of detection for the mosquito-borne primer sets, serial dilutions of RNA from West Nile virus (WNV) were assayed and could be detected down to an equivalent viral titer of 0.2 plaque-forming units/mL. Analysis of flaviviruses in their natural biological background included testing Aedes aegypti mosquitoes that were laboratory-infected with dengue-1 virus. The assay accurately identified the virus within infected mosquitoes, and we determined the average viral genome per mosquito to be 2.0 x 10(6). Using human blood, serum, and urine spiked with WNV and mouse blood and brain tissues from Karshi virus-infected mice, we showed that these clinical matrices did not inhibit the detection of these viruses. Finally, we used the assay to test field-collected Ixodes scapularis ticks collected from sites in New York and Connecticut. We found 16/322 (5% infection rate) ticks positive for deer tick virus, a subtype of Powassan virus. In summary, we developed a single high-throughput Flavivirus assay that could detect multiple tick- and mosquito-borne flaviviruses and thus provides a new analytical tool for their medical diagnosis and epidemiological surveillance.

Biofilms in water, its role and impact in human disease transmission.

Understanding the mechanism of biofilm formation is the first step in determining its function and, thereby, its impact and role in the environment. Extensive studies accomplished during the past few years have elucidated the genetics and biochemistry of biofilm formation. Cell-to-cell communication, that is, quorum sensing, is a key factor in the initiation of biofilm. Occurrence of viable but nonculturable bacteria, including Vibrio cholerae in biofilms has been reported and most likely such cells were overlooked previously because appropriate methods of detection were not employed. For this reason discovery and investigation of this important bacterial ecological niche in the environment were impeded.

Assay for and replication of Karshi (mammalian tick-borne flavivirus group) virus in mice.

Little is known about the replication of Karshi virus, a member of the mammalian tick-borne flavivirus group, in its rodent hosts. Therefore, we developed a quantitative real-time RT-PCR assay and measured the amount of viral RNA in selected tissues of infected Swiss Webster mice. Two-day-old mice were highly susceptible, with 100% fatality 9 to 12 days after infection, whereas infection in 9-day-old mice was less virulent, with death occurring only rarely. In nearly all cases, mice inoculated when 2 days old contained similar numbers of viral genome equivalents from blood and liver samples from any given mouse, with titers declining after day 7. In contrast, the amount of viral RNA in the brain began to rise rapidly 4 days after exposure, peaked at about 6 days after virus exposure (titer of > 10(13) genome equivalents/g), and remained at that level until euthanasia or death. Viral profiles were similar in needle-inoculated or tick-exposed mice.

Presence and Persistence of Salmonella in Water: The Impact on Microbial Quality of Water and Food Safety.

Salmonella ranks high among the pathogens causing foodborne disease outbreaks. According to the Centers for Disease Control and Prevention, Salmonella contributed to about 53.4% of all foodborne disease outbreaks from 2006 to 2017, and approximately 32.7% of these foodborne Salmonella outbreaks were associated with consumption of produce. Trace-back investigations have suggested that irrigation water may be a source of Salmonella contamination of produce and a vehicle for transmission. Presence and persistence of Salmonella have been reported in surface waters such as rivers, lakes, and ponds, while ground water in general offers better microbial quality for irrigation. To date, culture methods are still the gold standard for detection, isolation and identification of Salmonella in foods and water. In addition to culture, other methods for the detection of Salmonella in water include most probable number, immunoassay, and PCR. The U.S. Food and Drug Administration (FDA) issued the Produce Safety Rule (PSR) in January 2013 based on the Food Safety Modernization Act (FSMA), which calls for more efforts toward enhancing and improving approaches for the prevention of foodborne outbreaks. In the PSR, agricultural water is defined as water used for in a way that is intended to, or likely to, contact covered produce, such as spray, wash, or irrigation. In summary, Salmonella is frequently present in surface water, an important source of water for irrigation. An increasing evidence indicates irrigation water as a source (or a vehicle) for transmission of Salmonella. This pathogen can survive in aquatic environments by a number of mechanisms, including entry into the viable but nonculturable (VBNC) state and/or residing within free-living protozoa. As such, assurance of microbial quality of irrigation water is critical to curtail the produce-related foodborne outbreaks and thus enhance the food safety. In this review, we will discuss the presence and persistence of Salmonella in water and the mechanisms Salmonella uses to persist in the aquatic environment, particularly irrigation water, to better understand the impact on the microbial quality of water and food safety due to the presence of Salmonella in the water environment.

Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing.

The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.

Draft Genome Sequences of Bifidobacterium Strains Isolated from Dietary Supplements and Cultured Food Products.

Here, we present the genome sequences of 23 Bifidobacterium isolates from several commercially available dietary supplements and cultured food products. Strains of this genus are natural inhabitants of the mammalian mouth, gastrointestinal tract, and vagina. Some species are considered beneficial to human health.

Fifty-Six Draft Genome Sequences of 10 Lactobacillus Species from 22 Commercial Dietary Supplements.

Here, we present the genome sequences of 56 isolates of 10 species of the genus Lactobacillus that are considered beneficial components of the gut microbiota. The isolates examined were found in commercially available dietary supplements in the U.S. market.