Aeromonas hydrophila RIT668 and Citrobacter portucalensis RIT669—Potential Zoonotic Pathogens Isolated from Spotted Turtles

Antimicrobial resistance (AMR) is one of the biggest challenges of the 21st century, and biofilm formation enables bacteria to resist antibiotic at much higher concentrations than planktonic cells. Earlier, we showed that the Gram-negative Aeromonas hydrophila RIT668 and Citrobacter portucalensis RIT669 (closely related to C. freundii NBRC 12681) from infected spotted turtles (Clemmys guttata), formed biofilms and upregulated toxin expression on plastic surfaces, and were predicted to possess multiple antibiotic resistance genes. Here, we show that they each resist several antibiotics in the planktonic phase, but were susceptible to neomycin, and high concentrations of tetracycline and cotrimoxazole. The susceptibility of their biofilms to neomycin and cotrimoxazole was tested using the Calgary device. For A. hydrophila, the minimum inhibitory concentration (MIC) = 500–1000, and the minimum biofilm eradication concentration (MBEC) > 1000 μg/mL, using cotrimoxazole, and MIC = 32.3–62.5, and MBEC > 1000 μg/mL, using neomycin. For C. freundii MIC = 7.8–15.6, and, MBEC > 1000 μg/mL, using cotrimoxazole, and MIC = 7.8, and MBEC > 1000 μg/mL, using neomycin. Both A. hydrophila and C. portucalensis activated an acyl homoserine lactone (AHL) dependent biosensor, suggesting that quorum sensing could mediate biofilm formation. Their multidrug resistance in the planktonic form, and weak biofilm eradication even with neomycin and cotrimoxazole, indicate that A. hydrophila and C. portucalensis are potential zoonotic pathogens, with risks for patients living with implants.

Expression of a Shiga-Like Toxin during Plastic Colonization by Two Multidrug-Resistant Bacteria, Aeromonas hydrophila RIT668 and Citrobacter freundii RIT669, Isolated from Endangered Turtles (Clemmys guttata)

Aeromonas hydrophila RIT668 and Citrobacter freundii RIT669 were isolated from endangered spotted turtles (Clemmys guttata). Whole-genome sequencing, annotation and phylogenetic analyses of the genomes revealed that the closest relative of RIT668 is A. hydrophila ATCC 7966 and Citrobacter portucalensis A60 for RIT669. Resistome analysis showed that A. hydrophila and C. freundii harbor six and 19 different antibiotic resistance genes, respectively. Both bacteria colonize polyethylene and polypropylene, which are common plastics, found in the environment and are used to fabricate medical devices. The expression of six biofilm-related genes—biofilm peroxide resistance protein (bsmA), biofilm formation regulatory protein subunit R (bssR), biofilm formation regulatory protein subunit S (bssS), biofilm formation regulator (hmsP), toxin-antitoxin biofilm protein (tabA) and transcriptional activator of curli operon (csgD)—and two virulence factors—Vi antigen-related gene (viaB) and Shiga-like toxin (slt-II)—was investigated by RT-PCR. A. hydrophila displayed a >2-fold increase in slt-II expression in cells adhering to both polymers, C. freundii adhering on polyethylene displayed a >2-fold, and on polypropylene a >6-fold upregulation of slt-II. Thus, the two new isolates are potential pathogens owing to their drug resistance, surface colonization and upregulation of a slt-II-type diarrheal toxin on polymer surfaces.

A Long-Term Demographic Analysis of Spotted Turtles (Clemmys guttata) in Illinois Using Matrix Models

Matrix models and perturbation analyses provide a useful framework for evaluating demographic vital rates crucial to maintaining population growth. Determining which vital rates most influence population growth is necessary for effective management of long-lived organisms facing population declines. In Illinois, the state-endangered Spotted Turtle (Clemmys guttata) occurs in two distinct populations, and management can benefit from an understanding of its demographic behavior. We conducted a mark–recapture study on both populations in 2015 and 2016 and used historical mark–recapture data from 1988 to 2010 to determine female age-specific survival and fecundity rates. Survival increased significantly with age, and age-specific reproductive output and fecundity were >1.0. However, both populations exhibited net reproductive rates below replacement levels, and one population had a negative growth rate. Summed elasticities for all adult age classes indicate adult survival has the highest proportional impact on population growth. We found evidence of demographic divergence between the two populations, and thus the prioritization of vital rates varied somewhat between sites, with a relatively higher emphasis on juvenile and young adult survival for one population. We recommend conservation actions such as habitat management and predator control, which will have positive impacts across stage classes.

A Comparison of the Population Genetic Structure and Diversity between a Common (Chrysemys p. picta) and an Endangered (Clemmys guttata) Freshwater Turtle

The northeastern United States has experienced dramatic alteration to its landscape since the time of European settlement. This alteration has had major impacts on the distribution and abundance of wildlife populations, but the legacy of this landscape change remains largely unexplored for most species of freshwater turtles. We used microsatellite markers to characterize and compare the population genetic structure and diversity between an abundant generalist, the eastern painted turtle (Chrysemys p. picta), and the rare, more specialized, spotted turtle (Clemmys guttata) in Rhode Island, USA. We predicted that because spotted turtles have disproportionately experienced the detrimental effects of habitat loss and fragmentation associated with landscape change, that these effects would manifest in the form of higher inbreeding, less diversity, and greater population genetic structure compared to eastern painted turtles. As expected, eastern painted turtles exhibited little population genetic structure, showed no evidence of inbreeding, and little differentiation among sampling sites. For spotted turtles, however, results were consistent with certain predictions and inconsistent with others. We found evidence of modest inbreeding, as well as tentative evidence of recent population declines. However, genetic diversity and differentiation among sites were comparable between species. As our results do not suggest any major signals of genetic degradation in spotted turtles, the southern region of Rhode Island may serve as a regional conservation reserve network, where the maintenance of population viability and connectivity should be prioritized.

Record longevity of a Spotted Turtle (Clemmys guttata)

Turtles are known for their longevity, but the maximum life span for many species remains unknown. Spotted Turtle (Clemmys guttata) can live for more than 30 years in the wild, but typical or maximum longevity has not been confirmed. As part of a long-term mark–recapture project in Ottawa, Ontario, near the species’ northern limit, an adult female was captured on 27 April 2017. It had first been marked on 11 June 1983, when it was an adult with 17 growth rings on its plastron. Based on the number of growth rings at first capture, and the intervening time, this turtle is a minimum of 51 years old, setting a longevity record for the species. Ten individuals in this population were at least 30 years old when last captured, including a male at least 41 years old. Few of these turtles have grown measurably since being marked in 1983, and it is likely that these minimum ages are underestimates of actual ages.