Molecular Characterization of Plasmids Harbored by Actinomycetes Isolated From the Great Salt Plains of Oklahoma Using PFGE and Next Generation Whole Genome Sequencing

One of the unique features of actinomycetes, especially the genus Streptomyces, is the presence of linear plasmids. These range in size from 12 to 600 kb, and are often termed mega-plasmids. While many of the genes involved in secondary metabolite production reside in clusters on the chromosome, several studies have identified biosynthetic clusters on large linear plasmids that produce important secondary metabolites, including antibiotics. In this study, Pulse Field Gel Electrophoresis (PFGE) was used to screen 176 actinomycete isolates for the presence of plasmids; these bacterial strains were previously isolated from the Great Salt Plains of Oklahoma. Seventy-eight of the 176 actinomycete isolates (44%) contained plasmids. Several strains contained more than one plasmid, accounting for a total of 109 plasmids. Ten isolates showed extrachromosomal DNA larger than 200 kb, thus falling into the category of mega-plasmids. A subset of plasmids from 55 isolates was treated with S1 nuclease to determine topology; all plasmids examined appeared to be linear and ranged from ~55 to 400 kb. Eleven isolates were chosen for Whole Genome Next Generation Sequencing. From the 11 sequenced isolates, seven plasmids were partially assembled. While the majority of the genes identified on the plasmids coded for hypothetical proteins, others coded for general functions, stress response, and antibiotic and heavy metal resistance. Draft genome sequences of two mega-plasmid-bearing Streptomyces sp. strains, BF-3 and 4F, revealed the presence of genes involved in antibiotic production, antibiotic, and heavy metal resistance, osmoregulation, and stress response, which likely facilitate their survival in this extreme halophilic environment. To our knowledge, this is the first study to explore plasmids harbored by actinomycetes isolated from the Great Salt Plains of Oklahoma

Campylobacter coli From Retail Liver and Meat Products Is More Aerotolerant Than Campylobacter jejuni

Aerotolerance in the microaerophilic species Campylobacter was previously reported and could increase bacterial survival and transmission in foods during stressful processing and storage conditions. In this study, 167 Campylobacter isolates (76 C. jejuni and 91 C. coli) were screened for aerotolerance; these strains were previously isolated from retail chicken meat, chicken livers, chicken gizzards, turkey, pork, and beef liver samples. Bacterial cultures were incubated aerobically in Mueller Hinton broth with agitation and viable cell counts were taken at 0, 6, 12, and 24 h. Approximately 47% of the screened Campylobacter isolates were aerotolerant (viable after a 12-h aerobic incubation period), whereas 24% were hyper-aerotolerant (viable after a 24-h aerobic incubation). A greater prevalence of aerotolerant strains (80%) was found among C. coli isolates as compared to C. jejuni isolates (6%). Differences in the oxidative stress response related genes were detected among C. jejuni and C. coli isolates when comparative genomics was used to analyze 17 Whole Genome Sequenced (WGS) strains from our laboratory. Genes encoding putative transcriptional regulator proteins and a catalase-like heme binding protein were found in C. coli genomes, but were absent in the genomes of C. jejuni. PCR screening showed the presence of a catalase-like protein gene in 75% (68/91) of C. coli strains, which was absent in all tested C. jejuni strains. While about 79% (30/38) of the hyper-aerotolerant C. coli strains harbored the catalase-like protein gene, the gene was also present in a number of the aerosensitive strains. The Catalase like protein gene was found to be expressed in both aerobic and microaerobic conditions with a 2-fold higher gene expression detected in aerobic conditions for an aerosensitive strain. However, the exact function of the gene remains unclear and awaits further investigation. In conclusion, aerotolerant Campylobacter strains (especially C. coli) are prevalent in various retail meats. Further studies are needed to investigate whether the genes encoding catalase-like heme binding protein and putative transcriptional regulators in C. coli strains are involved in stress response

Prevalence, Serotyping, Molecular Typing, and Antimicrobial Resistance of Salmonella Isolated From Conventional and Organic Retail Ground Poultry

Ground poultry is marketed as a healthier alternative to ground beef despite the fact that poultry is a major source of foodborne Salmonella. The objectives of this study were to determine the prevalence of Salmonella in Oklahoma retail ground poultry and to characterize representative isolates by serotyping, antimicrobial resistance, PFGE patterns, and large plasmid profiling. A total of 199 retail ground poultry samples (150 ground turkey and 49 ground chicken) were investigated. The overall prevalence of Salmonella in ground poultry was 41% (82/199), and the incidence in conventional samples (47%, 66/141) was higher than in organic samples (27%, 16/58). The prevalence of Salmonella in organic ground chicken and organic ground turkey was 33% (3/9) and 26% (13/49), respectively. Twenty six Salmonella isolates (19 conventional and 7 organic) were chosen for further characterization. The following six serotypes and number of isolates per serotype were identified as follows: Tennessee, 8; Saintpaul, 4; Senftenberg, 4; Anatum, 4 (one was Anatum_var._15+); Ouakam, 3; and Enteritidis, 3. Resistance to 16 tested antimicrobials was as follows: gentamycin, 100% (26/26); ceftiofur, 100% (26/26); amoxicillin/clavulanic acid, 96% (25/26); streptomycin, 92% (24/26); kanamycin, 88% (23/26); ampicillin, 85% (22/26); cephalothin, 81% (21/26); tetracycline, 35% (9/26); sulfisoxazole, 27% (7/26); nalidixic acid, 15% (4/26); and cefoxitin, 15% (4/26). All isolates were susceptible to amikacin, chloramphenicol, ceftriaxone, and trimethoprim/sulfamethoxazole. All screened isolates were multidrug resistant (MDR) and showed resistance to 4–10 antimicrobials; isolates from organic sources showed resistance to 5–7 antimicrobials. PFGE was successful in clustering the Salmonella isolates into distinct clusters that each represented one serotype. PFGE was also used to investigate the presence of large plasmids using S1 nuclease digestion. A total of 8/26 (31%) Salmonella isolates contained a ∼100 Kb plasmid that was present in all Anatum and Ouakam isolates. In conclusion, the presence of multidrug resistant Salmonella with various serotypes, PFGE profiles, and large plasmids in ground poultry stresses the importance of seeking novel interventions to reduce the risk of this foodborne pathogen. Multidrug resistance (MDR) is considered a high additional risk and continued surveillance at the retail level could minimize the risk for the consumer

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10 years; 78.2% included were male with a median age of 37 years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Arsenic Resistance and Prevalence of Arsenic Resistance Genes in Campylobacter jejuni and Campylobacter coli Isolated from Retail Meats

Studies that investigate arsenic resistance in the foodborne bacterium Campylobacter are limited. A total of 552 Campylobacter isolates (281 Campylobacter jejuni and 271 Campylobacter coli) isolated from retail meat samples were subjected to arsenic resistance profiling using the following arsenic compounds: arsanilic acid (4–2,048 μg/mL), roxarsone (4–2048 μg/mL), arsenate (16–8,192 μg/mL) and arsenite (4–2,048 μg/mL). A total of 223 of these isolates (114 Campylobacter jejuni and 109 Campylobacter coli) were further analyzed for the presence of five arsenic resistance genes (arsP, arsR, arsC, acr3, and arsB) by PCR. Most of the 552 Campylobacter isolates were able to survive at higher concentrations of arsanilic acid (512–2,048 μg/mL), roxarsone (512–2,048 μg/mL), and arsenate (128–1,024 μg/mL), but at lower concentrations for arsenite (4–16 μg/mL). Ninety seven percent of the isolates tested by PCR showed the presence of arsP and arsR genes. While 95% of the Campylobacter coli isolates contained a larger arsenic resistance operon that has all of the four genes (arsP, arsR, arsC and acr3), 85% of the Campylobacter jejuni isolates carried the short operon (arsP, and arsR). The presence of arsC and acr3 did not significantly increase arsenic resistance with the exception of conferring resistance to higher concentrations of arsenate to some Campylobacter isolates. arsB was prevalent in 98% of the tested Campylobacter jejuni isolates, regardless of the presence or absence of arsC and acr3, but was completely absent in Campylobacter coli. To our knowledge, this is the first study to determine arsenic resistance and the prevalence of arsenic resistance genes in such a large number of Campylobacter isolates

Incidence, Antimicrobial Susceptibility, and Toxin Genes Possession Screening of Staphylococcus aureus in Retail Chicken Livers and Gizzards

Few recent outbreaks in Europe and the US involving Campylobacter and Salmonella were linked to the consumption of chicken livers. Studies investigating Staphylococcus aureus in chicken livers and gizzards are very limited. The objectives of this study were to determine the prevalence, antimicrobial resistance, and virulence of S. aureus and MRSA (Methicillin-Resistant Staphylococcus aureus) in retail chicken livers and gizzards in Tulsa, Oklahoma. In this study, 156 chicken livers and 39 chicken gizzards samples of two brands were collected. While one of the brands showed very low prevalence of 1% (1/100) for S. aureus in chicken livers and gizzards, the second brand showed prevalence of 37% (31/95). No MRSA was detected since none harbored the mecA or mecC gene. Eighty seven S. aureus isolates from livers and 28 from gizzards were screened for antimicrobial resistance to 16 antimicrobials and the possession of 18 toxin genes. Resistance to most of the antimicrobials screened including cefoxitin and oxacillin was higher in the chicken gizzards isolates. While the prevalence of enterotoxin genes seg and sei was higher in the gizzards isolates, the prevalence of hemolysin genes hla, hlb, and hld was higher in the livers ones. The lucocidin genes lukE-lukD was equally prevalent in chicken livers and gizzards isolates. Using spa typing, a subset of the recovered isolates showed that they are not known to be livestock associated and, hence, may be of a human origin. In conclusion, this study stresses the importance of thorough cooking of chicken livers and gizzards since it might contain multidrug resistant enterotoxigenic S. aureus. To our knowledge this is the first study to specifically investigate the prevalence of S. aureus in chicken livers and gizzards in the US

Sequence Variation in the Outer Membrane Protein-Encoding Gene cmeC, Conferring Multidrug Resistance among Campylobacter jejuni and Campylobacter coli Strains Isolated from Different Hosts▿

A novel PCR primer pair was used to detect the presence of cmeC in 131 Campylobacter jejuni and C. coli strains isolated from various hosts (cattle, turkeys, humans, and pigs). DNA sequence analysis revealed a high degree of genetic variation between the two species, while extremely limited genetic variation among isolates of the same species was detected