Antibiotics modulate biofilm formation in fish pathogenic isolates of atypical Aeromonas salmonicida

Atypical Aeromonas salmonicida causes furunculosis infections of non‐salmonid fish, which requires antibiotic therapy. However, antibiotics may induce biofilm in some bacteria, which protects them against hostile conditions while allowing them to persist on surfaces, thus forming a reservoir for infection. The aim of this study was to determine whether atypical isolates of A. salmonicida increased biofilm in the presence of two antibiotics, florfenicol and oxytetracycline. A microtitre plate assay was used to quantify biofilm in the presence and absence of each antibiotic. Fifteen of 28 isolates formed biofilms under control conditions, while 23 of 28 isolates increased biofilm formation in the presence of at least one concentration of at least one antibiotic. For oxytetracycline, the most effective concentration causing biofilm to increase was one‐quarter of that preventing visible bacterial growth, whereas for florfenicol it was one‐half of this value. This is the first study to demonstrate that a bacterial pathogen of fish increases biofilm in response to antibiotics. Biofilm formation may increase the risk of re‐infection in culture systems and this lifestyle favours the transmission of genetic material, which has implications for the dissemination of antibiotic‐resistance genes and demonstrates the need for enhanced disease prevention measures against atypical A. salmonicida

Group versus individual antenatal and first year postpartum care: Study protocol for a multi-country cluster randomized controlled trial in Kenya and Nigeria [version 1; referees: 2 approved]

Background: Antenatal care (ANC) in many low- and middle-income countries is under-utilized and of sub-optimal quality. Group ANC (G-ANC) is an intervention designed to improve the experience and provision of ANC for groups of women (cohorts) at similar stages of pregnancy. Methods: A two-arm, two-phase, cluster randomized controlled trial (cRCT) (non-blinded) is being conducted in Kenya and Nigeria. Public health facilities were matched and randomized to either standard individual ANC (control) or G-ANC (intervention) prior to enrollment. Participants include pregnant women attending first ANC at gestational age <24 weeks, health care providers, and sub-national health managers. Enrollment ended in June 2017 for both countries. In the intervention arm, pregnant women are assigned to cohorts at first ANC visit and receive subsequent care together during five meetings facilitated by a health care provider (Phase 1). After birth, the same cohorts meet four times over 12 months with their babies (Phase 2). Data collection was performed through surveys, clinical data extraction, focus group discussions, and in-depth interviews. Phase 1 data collection ended in January 2018 and Phase 2 concludes in November 2018. Intention-to-treat analysis will be used to evaluate primary outcomes for Phases 1 and 2: health facility delivery and use of a modern method of family planning at 12 months postpartum, respectively. Data analysis and reporting of results will be consistent with norms for cRCTs. General estimating equation models that account for clustering will be employed for primary outcome analyzes. Results: Overall 1,075 and 1,013 pregnant women were enrolled in Nigeria and Kenya, respectively. Final study results will be available in February 2019. Conclusions: This is the first cRCT on G-ANC in Africa. It is among the first to examine the effects of continuing group care through the first year postpartum. Registration: Pan African Clinical Trials Registry PACTR201706002254227 May 02, 201

Characterisation of atypical Aeromonas salmonicida isolates from Cleaner Fish in the United Kingdom

Cleaner fish such as ballan wrasse (Labrus bergylta) and lumpfish (Cyclopterus lumpus) are currently used as a biological approach to controlling sea lice (Lepeophtheirus salmonis) in Atlantic salmon (Salmo salar) in the United Kingdom and elsewhere. However, these newly domesticated fish species are susceptible to infection by various bacterial pathogens, including atypical strains of A. salmonicida (aAs) that causes atypical furunculosis. Little is known of the diversity of aAs strains causing problems and no commercially licensed vaccine is presently available. Instead, multivalent autogenous vaccines (which include aAs strains) are used to control disease incidences, though outbreaks do still occur and these must be treated with antibiotics. For vaccines to be effective, the formulation must include isolates that confer protection against the prevalent circulating strains. Monitoring of the susceptibility of isolates to antibiotics is critical for these drugs to remain efficacious. Therefore, the aim of this thesis was to investigate the diversity of aAs strains causing disease problems in the UK and determine their antibiotic susceptibility. Seventy-seven aAs isolates collected from six different cleaner fish sites in the UK were characterised by phenotypic and genotypic methods. Two strain lineages were identified within the UK isolates by pulsed-field gel electrophoresis (PFGE), with a predominant group composed of ballan wrasse isolates and a minor cluster of mainly lumpsucker isolates. One pulsotype (B2) was detected at five UK sites and this accounted for 73% of isolates. The existence of two genetic groups was further supported by whole-genome sequencing (WGS) and virulence array protein gene typing of the A-layer (vapA). PFGE group B correlated with vapA type V isolates from ballan wrasse and PFGE group A with genotype vapA type VI typically from lumpsuckers. Additionally, the substrate, B-methyl-D-Glucoside from biolog assay differentiated between vapA type V and VI of aAs. Immuno-proteomic profiling of the aAs was performed by 1D and 2D SDS-PAGE to determine the protein profiles and the antigenic variation between the isolates. Six protein profiles (P1, P3-P6) were identified in the 77 aAs UK isolates. An association was noted between the isolate genotypes and the protein profiles, with five different protein profiles derived from pulsotype group B and vapA type V originating from wrasse. The remaining three related protein profiles originated from both cleaner fish species and the minor pulsotype group A and vapA type VI. Two key immuno-reactive proteins, enolase and vapA, were identified by MALDI-TOF-MS in SAIC-24 isolate. This will be useful in determining the function of aAs immune-related proteins and potential antigens for vaccine development. Antibiotic susceptibility of the aAs isolates was determined by disc diffusion assay and minimum inhibitory concentration (MIC) testing. The disc diffusion assay was of limited use in determining reduced susceptibility due to slow growth of the isolates and large zone sizes of growth inhibition. Conversely, provisional MIC cut-off values were successfully generated for seven antibiotics; florfenicol (FLO), oxytetracycline (OTC), enrofloxacin (ENR), flumequine (FLUQ), oxolinic acid (OXO), ormetoprim/sulphadimethoxine (ORS) and trimethoprim/sulphamethoxazole (TRS) by normalised resistance interpretation (NRI) analysis. The provisional MIC cut-off values of four antibiotics (FLO, OTC, OXO and ORS) were within the acceptable limits of precision and allowed for the setting of epidemiological cut-off values (ECV) of aAs strains and typical strains. There was evidence for reduced susceptibility to the antibiotics commonly used in the UK to treat atypical furunculosis, with 28 isolates (34%) having reduced susceptibility to multiple antibiotics. The prevalence of reduced susceptibility to the commonly used antibiotics, FLO, OTC and OXO increased between 2013 - 2019 Non-wild type (NWT) phenotypes were observed at all the UK farm sites, suggesting widespread presence of NWT aAs isolates within the UK. Gel-based plasmid profiling separated the aAs isolates into two main plasmid profiles that correlated with the vapA and PFGE genotype and host origin. Plasmid sequencing confirmed homology to typical A. salmonicida (tAs) plasmids associated with the type three secretion system (T3SS). A correlation was also found between acquired plasmids associated with reduced antibiotic susceptibility to OTC (pKp589-231, 2.9 kb) and extended spectrum beta lactamases (pAm08WL, 3.1 kb). Additionally, homology to a large conjugative plasmid pEIB202 (43.7 kb) encoding multidrug resistance determinants consistent with NWT phenotype of six aAs isolates was observed. Furthermore, WGS revealed chromosomal genes encoding resistance to erythromycin (ERY) and ampicillin (AMP) in all the 13 aAs genomes studied. In conclusion, this present study has provided insight into the diversity of aAs strains affecting cleaner fish in the UK and their susceptibility to antibiotics. These findings will assist in the prevention of atypical furunculosis and control strategies of cleaner fish management. Indeed, some of the isolates characterised herein were used in the development of a commercial whole-cell autogenous vaccine for use in Scotland that has shown to be successful in controlling outbreaks. In the absence of a licensed vaccine and selection of conserved antigenic targets, the antibiotic susceptibility patterns characterised in this present study will help inform on effective treatment regimes for aAs and assist to monitor the development of antibiotic resistance in UK aquaculture

The use of non-pharmaceutical interventions for the prevention and control of schistosomiasis in sub-Saharan Africa: A systematic review

Schistosomiasis remains a major cause of global parasitic morbidity. Current control strategies focus on pharmaceutical approaches using Mass Drug Administration (MDA) to distribute praziquantel in endemic areas of sub-Saharan Africa. Our paper systematically reviewed the literature on non-pharmaceutical interventions for enhanced schistosomiasis control. We conducted a systematic review of peer-reviewed English language literature using PubMed, Embase and Web of Science. Our search terms were limited to the year 2000 to March 2019 to reflect the period of the Millennium and Sustainable Development Goals. We initially identified 1733 publications, which were reduced to 1324 after screening by title and abstract. After the inclusion and exclusion criteria, a total of 1312 studies were excluded. Following this, we had a total of 12 articles, which we later screened by full text. Out of the twelve articles, seven were excluded for being systematic reviews or examining clinical and nutritional aspects of schistosomiasis control. We finally remained with five studies that met our inclusion criteria. Our paper indicates a gap in non-pharmaceutical based interventions for schistosomiasis control. We propose that future research addresses this gap by engaging communities in participatory approaches such as environmental sanitation, Water, Sanitation and Hygiene (WASH), health education and economic empowerment

Enforcement approaches against illegal fishing in national fisheries legislation

Illegal, unreported and unregulated (IUU) fishing activities are amongst the most serious and persisting global concerns that negatively impact the environment, economy, and livelihoods. The concept of IUU fishing is elaborated under Para 3 of the 2001 International Plan of Action to Prevent, Deter and Eliminate Illegal, Unreported and Unregulated Fishing (IPOA-IUU) of the Food and Agriculture Organization of the United Nations (FAO). In this article, the authors narrow the focus of the discussion herein on ‘illegal fishing’ activities, which generally fall under Para 3.1 of the IPOA-IUU, particularly examining the enforcement approaches against illegal fishing activities in national fisheries legislation. We explore a few overarching questions underpinning the scholarly debate on illegal fishing and crimes in the fisheries sector. First, whether criminalising illegal fishing or subjecting such fishing to criminal law processes lead to better compliance with fisheries legislation or is a more effective approach to tackling illegal fishing. Second, whether the problem of illegal fishing persists due to the lack of its criminalization or the resistance by States to criminalizing illegal fishing activities. Our assessment analyses the primary fisheries legislation of States and the European Union (EU) to better understand the enforcement approaches adopted therein, the responses used to empower national authorities, establish processes, delineate liability, and fix the sanction scheme, including the level of sanctions in terms of severity for illegal fishing. We ultimately aim to demonstrate that the options used to combat illegal fishing set out in national fisheries legislation are not limited to a single type of enforcement approach. Indeed, our assessment of national fisheries legislation shows that most States seem to follow a dual enforcement approach, which includes provisions enabling the use of both administrative and criminal processes and sanctions to enforce against illegal fishing and fishing related activities. We support a multipronged approach to address illegal fishing, which may include legal solutions such as criminalizing serious fisheries violations

Enforcement approaches against illegal fishing in national fisheries legislation

Illegal, unreported and unregulated (IUU) fishing activities are amongst the most serious and persisting global concerns that negatively impact the environment, economy, and livelihoods. The concept of IUU fishing is elaborated under Para 3 of the 2001 International Plan of Action to Prevent, Deter and Eliminate Illegal, Unreported and Unregulated Fishing (IPOA-IUU) of the Food and Agriculture Organization of the United Nations (FAO). In this article, the authors narrow the focus of the discussion herein on ‘illegal fishing’ activities, which generally fall under Para 3.1 of the IPOA-IUU, particularly examining the enforcement approaches against illegal fishing activities in national fisheries legislation. We explore a few overarching questions underpinning the scholarly debate on illegal fishing and crimes in the fisheries sector. First, whether criminalising illegal fishing or subjecting such fishing to criminal law processes lead to better compliance with fisheries legislation or is a more effective approach to tackling illegal fishing. Second, whether the problem of illegal fishing persists due to the lack of its criminalization or the resistance by States to criminalizing illegal fishing activities. Our assessment analyses the primary fisheries legislation of States and the European Union (EU) to better understand the enforcement approaches adopted therein, the responses used to empower national authorities, establish processes, delineate liability, and fix the sanction scheme, including the level of sanctions in terms of severity for illegal fishing. We ultimately aim to demonstrate that the options used to combat illegal fishing set out in national fisheries legislation are not limited to a single type of enforcement approach. Indeed, our assessment of national fisheries legislation shows that most States seem to follow a dual enforcement approach, which includes provisions enabling the use of both administrative and criminal processes and sanctions to enforce against illegal fishing and fishing related activities. We support a multipronged approach to address illegal fishing, which may include legal solutions such as criminalizing serious fisheries violations