An oseltamivir-resistant avian H1N1 influenza A virus can transmit from mallards to chickens similarly to a wild-type strain: implications for the risk of resistance transmission to humans.

The neuraminidase inhibitor (NAI) oseltamivir is stockpiled globally as part of influenza pandemic preparedness. However, oseltamivir carboxylate (OC) resistance develops in avian influenza virus (AIV) infecting mallards exposed to environmental-like OC concentrations, suggesting that environmental resistance is a real concern. Herein we used an in vivo model to investigate if avian influenza H1N1 with the OC-resistant mutation NA-H274Y (51833/H274Y) as compared to the wild-type (wt) strain (51833 /wt) could transmit from mallards, which would potentially be exposed to environmentally contaminated environments, to and between chickens, thus posing a potential zoonotic risk of antiviral-resistant AIV. Regardless of whether the virus had the OC-resistant mutation or not, chickens became infected both through experimental infection, and following exposure to infected mallards. We found similar infection patterns between 51833/wt and 51833/H274Y such that, one chicken inoculated with 51833/wt and three chickens inoculated with 51833/H274Y were AIV positive in oropharyngeal samples more than 2 days consecutively, indicating true infection, and one contact chicken exposed to infected mallards was AIV positive in faecal samples for 3 consecutive days (51833/wt) and another contact chicken for 4 consecutive days (51833/H274Y). Importantly, all positive samples from chickens infected with 51833/H274Y retained the NA-H274Y mutation. However, none of the virus strains established sustained transmission in chickens, likely due to insufficient adaptation to the chicken host. Our results demonstrate that an OC-resistant avian influenza virus can transmit from mallards and replicate in chickens. NA-H274Y does not constitute a barrier to interspecies transmission per se, as the resistant virus did not show reduced replicative capacity compared to the wild-type counterpart. Thus, responsible use of oseltamivir and surveillance for resistance development is warranted to limit the risk of an OC-resistant pandemic strain.

Selection of Resistant Bacteria in Mallards Exposed to Subinhibitory Concentrations of Ciprofloxacin in Their Water Environment.

Emergence and selection of antibiotic resistance following exposure to high antibiotic concentrations have been repeatedly shown in clinical and agricultural settings, whereas the role of the weak selective pressures exerted by antibiotic levels below the MIC (sub-MIC) in aquatic environments due to anthropogenic contamination remains unclear. Here, we studied how exposure to sub-MIC levels of ciprofloxacin enriches for Escherichia coli with reduced susceptibility to ciprofloxacin using a mallard colonization model. Mallards were inoculated with two isogenic extended-spectrum-ß-lactamase (ESBL)-encoding E. coli strains, differing only by a gyrA mutation that results in increased MICs of ciprofloxacin, and exposed to different levels of ciprofloxacin in their swimming water. Changes in the ratios of mutant to parental strains excreted in feces over time and ESBL plasmid spread within the gut microbiota from individual birds were investigated. Results show that in vivo selection of gyrA mutants occurred in mallards during exposure to ciprofloxacin at concentrations previously found in aquatic environments. During colonization, resistance plasmids were readily transferred between strains in the intestines of the mallards, but conjugation frequencies were not affected by ciprofloxacin exposure. Our results highlight the potential for enrichment of resistant bacteria in wildlife and underline the importance of reducing antibiotic pollution in the environment.

Influenza A/H4N2 mallard infection experiments further indicate zanamivir as less prone to induce environmental resistance development than oseltamivir.

Neuraminidase inhibitors (NAIs) are the gold standard treatment for influenza A virus (IAV). Oseltamivir is mostly used, followed by zanamivir (ZA). NAIs are not readily degraded in conventional wastewater treatment plants and can be detected in aquatic environments. Waterfowl are natural IAV hosts and replicating IAVs could thus be exposed to NAIs in the environment and develop resistance. Avian IAVs form the genetic basis for new human IAVs, and a resistant IAV with pandemic potential poses a serious public health threat, as NAIs constitute a pandemic preparedness cornerstone. Resistance development in waterfowl IAVs exposed to NAIs in the water environment has previously been investigated in an in vivo mallard model and resistance development was demonstrated in several avian IAVs after the exposure of infected ducks to oseltamivir, and in an H1N1 IAV after exposure to ZA. The N1 and N2 types of IAVs have different characteristics and resistance mutations, and so the present study investigated the exposure of an N2-type IAV (H4N2) in infected mallards to 1, 10 and 100 µg l-1 of ZA in the water environment. Two neuraminidase substitutions emerged, H274N (ZA IC50 increased 5.5-fold) and E119G (ZA IC50 increased 110-fold) at 10 and 100 µg l-1 of ZA, respectively. Reversion towards wild-type was observed for both substitutions in experiments with removed drug pressure, indicating reduced fitness of both resistant viruses. These results corroborate previous findings that the development of resistance to ZA in the environment seems less likely to occur than the development of resistance to oseltamivir, adding information that is useful in planning for prudent drug use and pandemic preparedness.

In vivo mallard experiments indicate that zanamivir has less potential for environmental influenza A virus resistance development than oseltamivir.

Neuraminidase inhibitors are a cornerstone of influenza pandemic preparedness before vaccines can be mass-produced and thus a neuraminidase inhibitor-resistant pandemic is a serious threat to public health. Earlier work has demonstrated the potential for development and persistence of oseltamivir resistance in influenza A viruses exposed to environmentally relevant water concentrations of the drug when infecting mallards, the natural influenza reservoir that serves as the genetic base for human pandemics. As zanamivir is the major second-line neuraminidase inhibitor treatment, this study aimed to assess the potential for development and persistence of zanamivir resistance in an in vivo mallard model; especially important as zanamivir will probably be increasingly used. Our results indicate less potential for development and persistence of resistance due to zanamivir than oseltamivir in an environmental setting. This conclusion is based on: (1) the lower increase in zanamivir IC50 conferred by the mutations caused by zanamivir exposure (2-17-fold); (2) the higher zanamivir water concentration needed to induce resistance (at least 10 µg l-1); (3) the lack of zanamivir resistance persistence without drug pressure; and (4) the multiple resistance-related substitutions seen during zanamivir exposure (V116A, A138V, R152K, T157I and D199G) suggesting lack of one straight-forward evolutionary path to resistance. Our study also adds further evidence regarding the stability of the oseltamivir-induced substitution H275Y without drug pressure, and demonstrates the ability of a H275Y-carrying virus to acquire secondary mutations, further boosting oseltamivir resistance when exposed to zanamivir. Similar studies using influenza A viruses of the N2-phylogenetic group of neuraminidases are recommended.

Influenza A(H7N9) virus acquires resistance-related neuraminidase I222T substitution when infected mallards are exposed to low levels of oseltamivir in water.

Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and new human IAVs often contain gene segments originating from avian IAVs. Treatment options for severe human influenza are principally restricted to neuraminidase inhibitors (NAIs), among which oseltamivir is stockpiled in preparedness for influenza pandemics. There is evolutionary pressure in the environment for resistance development to oseltamivir in avian IAVs, as the active metabolite oseltamivir carboxylate (OC) passes largely undegraded through sewage treatment to river water where waterfowl reside. In an in vivo mallard (Anas platyrhynchos) model, we tested if low-pathogenic avian influenza A(H7N9) virus might become resistant if the host was exposed to low levels of OC. Ducks were experimentally infected, and OC was added to their water, after which infection and transmission were maintained by successive introductions of uninfected birds. Daily fecal samples were tested for IAV excretion, genotype, and phenotype. Following mallard exposure to 2.5 µg/liter OC, the resistance-related neuraminidase (NA) I222T substitution, was detected within 2 days during the first passage and was found in all viruses sequenced from subsequently introduced ducks. The substitution generated 8-fold and 2.4-fold increases in the 50% inhibitory concentration (IC50) for OC (P < 0.001) and zanamivir (P = 0.016), respectively. We conclude that OC exposure of IAV hosts, in the same concentration magnitude as found in the environment, may result in amino acid substitutions, leading to changed antiviral sensitivity in an IAV subtype that can be highly pathogenic to humans. Prudent use of oseltamivir and resistance surveillance of IAVs in wild birds are warranted.

Thirty-five years of hemodialysis: two case reports as a tribute to Nils Alwall.

Two patients with long-term (35 years) survival on hemodialysis are described. Kidney replacement therapy for these patients was initiated by a pioneer in hemodialysis, Nils Alwall, in 1968 and 1971, respectively. Kidney transplantation was attempted twice in both patients; however, the dialysis-free interval was less than 18 months in both patients. These patients represent two of the longest known survivors on hemodialysis worldwide. Factors that may have influenced their survival are discussed, and the complications that have occurred over the years are presented.