Detection of Anaplasma Phagocytophilum in Horses With Suspected Tick-Borne Disease in Northeastern United States by Metagenomic Sequencing

Metagenomic sequencing of clinical diagnostic specimens has a potential for unbiased detection of infectious agents, diagnosis of polymicrobial infections and discovery of emerging pathogens. Herein, next generation sequencing (NGS)-based metagenomic approach was used to investigate the cause of illness in a subset of horses recruited for a tick-borne disease surveillance study during 2017–2019. Blood samples collected from 10 horses with suspected tick-borne infection and five apparently healthy horses were subjected to metagenomic analysis. Total genomic DNA extracted from the blood samples were enriched for microbial DNA and subjected to shotgun next generation sequencing using Nextera DNA Flex library preparation kit and V2 chemistry sequencing kit on the Illumina MiSeq sequencing platform. Overall, 0.4–0.6 million reads per sample were analyzed using Kraken metagenomic sequence classification program. The taxonomic classification of the reads indicated that bacterial genomes were overrepresented (0.5 to 1%) among the total microbial reads. Most of the bacterial reads (~91%) belonged to phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria and Tenericutes in both groups. Importantly, 10–42.5% of Alphaproteobacterial reads in 5 of 10 animals with suspected tick-borne infection were identified as Anaplasma phagocytophilum. Of the 5 animals positive for A. phagocytophilum sequence reads, four animals tested A. phagocytophilum positive by PCR. Two animals with suspected tick-borne infection and A. phagocytophilum positive by PCR were found negative for any tick-borne microbial reads by metagenomic analysis. The present study demonstrates the usefulness of the NGS-based metagenomic analysis approach for the detection of blood-borne microbes

CRISPR Typing and Antibiotic Resistance Correlates with Polyphyletic Distribution in Human Isolates of Salmonella Kentucky

Although infrequently associated with reported salmonellosis in humans, Salmonella enterica, subsp. enterica serovar Kentucky (ser. Kentucky) is the most common nonclinical, nonhuman serovar reported in the United States. The goal of this study was to use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–multi-virulence-locus sequence typing (MVLST) to subtype a collection of human clinical isolates of ser. Kentucky submitted to the Pennsylvania Department of Health and to determine the extent of antibiotic resistance in these strains. This analysis highlighted the polyphyletic nature of ser. Kentucky, and separated our isolates into two groups, Group I and Group II, which were equally represented in our collection. Furthermore, antimicrobial susceptibility testing on all isolates using a National Antimicrobial Resistance Monitoring System (NARMS) panel of antibiotics demonstrated that resistance profiles could be divided into two groups. Group I isolates were resistant to cephems and penicillins, whereas Group II isolates were resistant to quinolones, gentamicin, and sulfisoxazole. Collectively, 50% of isolates were resistant to three or more classes of antibiotics and 30% were resistant to five or more classes. The correlation of antibiotic resistance with the two different lineages may reflect adaptation within two distinct reservoirs of ser. Kentucky, with differential exposure to antimicrobials

A comparison of non-typhoidal Salmonella from humans and food animals using pulsed-field gel electrophoresis and antimicrobial susceptibility patterns.

Salmonellosis is one of the most important foodborne diseases affecting humans. To characterize the relationship between Salmonella causing human infections and their food animal reservoirs, we compared pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility patterns of non-typhoidal Salmonella isolated from ill humans in Pennsylvania and from food animals before retail. Human clinical isolates were received from 2005 through 2011 during routine public health operations in Pennsylvania. Isolates from cattle, chickens, swine and turkeys were recovered during the same period from federally inspected slaughter and processing facilities in the northeastern United States. We found that subtyping Salmonella isolates by PFGE revealed differences in antimicrobial susceptibility patterns and, for human Salmonella, differences in sources and invasiveness that were not evident from serotyping alone. Sixteen of the 20 most common human Salmonella PFGE patterns were identified in Salmonella recovered from food animals. The most common human Salmonella PFGE pattern, Enteritidis pattern JEGX01.0004 (JEGX01.0003ARS), was associated with more cases of invasive salmonellosis than all other patterns. In food animals, this pattern was almost exclusively (99%) found in Salmonella recovered from chickens and was present in poultry meat in every year of the study. Enteritidis pattern JEGX01.0004 (JEGX01.0003ARS) was associated with susceptibility to all antimicrobial agents tested in 94.7% of human and 97.2% of food animal Salmonella isolates. In contrast, multidrug resistance (resistance to three or more classes of antimicrobial agents) was observed in five PFGE patterns. Typhimurium patterns JPXX01.0003 (JPXX01.0003 ARS) and JPXX01.0018 (JPXX01.0002 ARS), considered together, were associated with resistance to five or more classes of antimicrobial agents: ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline (ACSSuT), in 92% of human and 80% of food animal Salmonella isolates. The information from our study can assist in source attribution, outbreak investigations, and tailoring of interventions to maximize their impact on prevention

Molecular Characterization of Cephalosporin-Resistant Salmonella enterica Serotype Newport Isolates from Animals in Pennsylvania

Multidrug-resistant (MDR) strains of Salmonella enterica serotype Newport have been described for many years. However, the recognition of Newport strains with resistance to cephalosporin antibiotics is more recent. Plasmid-mediated CMY-2 AmpC β-lactamases have been identified in Salmonella in the United States, and the bla(CMY-2) gene has been shown to be present in Salmonella serotype Newport. This organism is currently undergoing epidemic spread in both animals and humans in the United States, and this is to our knowledge the first description of the molecular epidemiology of this Salmonella strain in animals. Forty-two isolates were included in this study. All isolates were characterized by pulsed-field gel electrophoresis, plasmid analysis, and antibiogram. Four pulsed-field profiles with XbaI were observed. Plasmid analyses showed that although the majority of isolates harbored a single plasmid of 140 kb, this plasmid was not identical in all strains. All isolates showed the presence of the bla(CMY) gene by PCR. Integrons were detected in 16 of the 42 isolates; a fragment of approximately 1,000 bp, amplified with the intI-F and aadAI-R primers, confirmed the presence of the aadAI gene cassette within an integron in these 16 isolates. The potential for coselection of the bla(CMY) gene, if located on an MDR replicon, may not be dependent on any particular antibiotic but rather may be the result of more general antimicrobial use. If this replicon is mobile, it is to be expected that similar MDR strains of additional Salmonella serotypes will be recognized in due course

Food Animal Sources of <i>Salmonella</i> Recovered at Federally Inspected Slaughter and Processing Facilities in the Northeastern U.S.¹ with Human Isolates Shown for Reference.

<p>Food Animal Sources of <i>Salmonella</i> Recovered at Federally Inspected Slaughter and Processing Facilities in the Northeastern U.S.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077836#nt101" target="_blank">1</a>} with Human Isolates Shown for Reference.</p

Antimicrobial resistance associated with the 16 most common human <i>Salmonella</i> PFGE patterns shared by food animal <i>Salmonella</i>.

<p>Antimicrobial resistance associated with the 16 most common human <i>Salmonella</i> PFGE patterns shared by food animal <i>Salmonella</i>.</p

Pennsylvania’s Most Common Human <i>Salmonella</i> PFGE Patterns Also Found in <i>Salmonella</i> Recovered from Food Animals Processed in the Northeastern U.S.

<p>Pennsylvania’s Most Common Human <i>Salmonella</i> PFGE Patterns Also Found in <i>Salmonella</i> Recovered from Food Animals Processed in the Northeastern U.S.</p

DNA fingerprints of the 20 most common human <i>Salmonella</i> patterns.

<p>PFGE was conducted as described in Materials and Methods, with restriction endonucleases XbaI and BlnI. BlnI patterns represented at least three times in the dataset are shown.</p