Rapid detection of antibiotic resistance in positive blood cultures by MALDI-TOF MS and an automated and optimized MBT-ASTRA protocol for Escherichia coli and Klebsiella pneumoniae.

Introduction: For fast and effective antibiotic therapy of serious infections like sepsis, it is crucial with rapid information about antibiotic susceptibility, especially in a time when the number of infections caused by multi resistant bacteria has escalated in the world.Methods: Here, we have used a semi-quantitative MALDI-TOF-MS based method for antibiotic resistance detection, MBT-ASTRA™, which is based on the comparison of growth rate of the bacteria cultivated with and without antibiotics. We demonstrate a new protocol where several parameters have been optimized and automated leading to reduced hands-on time and improved capacity to simultaneously analyse multiple clinical samples and antibiotics.Results: Ninety minutes of incubation at 37 °C with agitation was sufficient to differentiate the susceptible and resistant strains of E. coli and K. pneumoniae, for the antibiotics cefotaxime, meropenem and ciprofloxacin. In total, 841 positive blood culture analyses of 14 reference strains were performed. The overall sensitivity was 99%, specificity 99% and the accuracy 97%. The assay gave no errors for cefotaxime (n = 263) or meropenem (n = 289) for sensitive and resistant strains, whilst ciprofloxacin (n = 289) gave six (0.7%) major errors (false resistance) and four (0.5%) very major errors (false susceptibility). The intermediate strains showed a larger variety compared to the E-test MIC values.Conclusions: The hands-on time and the analysis time to detect antibiotic resistance of clinical blood samples can be substantially reduced and the sample capacity can be increased by using automation and this improved protocol.

Nitrogen uptake potential under different temperature-salinity conditions: Implications for nitrogen cycling under climate change scenarios.

As projected by climate change models, increase in sea surface temperature and precipitation in the future may alter nutrient cycling in the coastal regions due to potential changes in phytoplankton community structure and their ability to assimilate nitrogen (N) and carbon (C). An experiment simulating different temperature and salinity conditions (28°C-35 ambient conditions, 28ºC-31, 31ºC-35 and 31ºC-31) in mesocosms containing 1000 L of coastal water from the Arabian Sea was performed and N uptake rates were measured using 15N tracer technique on 2nd, 5th, 7th and 10th day of the experiment. The results show that, under all conditions, the total N (NO3- + NH4+) uptake rates were lower in the beginning and on the final day of the tracer experiment, while it peaked during middle, consistent with chlorophyll a concentrations. Total N uptake rate was significantly lower (p = 0.003) under ambient temperature-lower salinity condition (28ºC-31) than the others. This indicates that lowering of salinity in coastal regions due to excessive rainfall in the future may affect the N uptake potential of the phytoplankton, which may change the regional C and N budget.

Characteristics of potentially pathogenic vibrios from subtropical Mozambique compared with isolates from tropical India and boreal Sweden.

Reported outbreaks of Vibrio parahaemolyticus have increased worldwide, particularly in regions of high seafood consumption. In Mozambique, seafood constitutes an important food resource and diarrheal diseases are common among its inhabitants. Edible clams were collected in Maputo Bay during both the dry and rainy seasons, with the results showing the number of viable counts of vibrios in clams to peak during the latter. Vibrio parahaemolyticus was the predominant species identified among the isolated strains. Although only one of 109 total strains carried the tdh virulence gene, 69% of isolates showed evidence of hemolytic capacity when subjected to a functional test. Similar virulence patterns and biochemical properties were found in strains isolated from Indian and Swedish marine waters. Antibiotic resistance was, however, more pronounced in strains isolated from these latter two environments.

Water column dynamics of Vibrio in relation to phytoplankton community composition and environmental conditions in a tropical coastal area.

Vibrio abundance generally displays seasonal patterns. In temperate coastal areas, temperature and salinity influence Vibrio growth, whereas in tropical areas this pattern is not obvious. The present study assessed the dynamics of Vibrio in the Arabian Sea, 1-2 km off Mangalore on the south-west coast of India, during temporally separated periods. The two sampling periods were signified by oligotrophic conditions, and stable temperatures and salinity. Vibrio abundance was estimated by culture-independent techniques in relation to phytoplankton community composition and environmental variables. The results showed that the Vibrio density during December 2007 was 10- to 100-fold higher compared with the February-March 2008 period. High Vibrio abundance in December coincided with a diatom-dominated phytoplankton assemblage. A partial least squares (PLS) regression model indicated that diatom biomass was the primary predictor variable. Low nutrient levels suggested high water column turnover rate, which bacteria compensated for by using organic molecules leaking from phytoplankton. The abundance of potential Vibrio predators was low during both sampling periods; therefore it is suggested that resource supply from primary producers is more important than top-down control by predators.

Occurrence and potential pathogenesis of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus on the South Coast of Sweden.

During the summer of 2006, several wound infections - of which three were fatal - caused by Vibrio cholerae were reported from patients who had been exposed to water from the Baltic Sea. Before these reports, we initiated a sampling project investigating the occurrence of potential human pathogenic V. cholerae, Vibrio vulnificus and Vibrio parahaemolyticus in The Sound between Sweden and Denmark. The Blue mussel (Mytilus edulis) was used as an indicator to follow the occurrence of vibrios over time. Molecular analyses showed high frequencies of the most potent human pathogenic Vibrio spp.; 53% of mussel samples were positive for V. cholerae (although none were positive for the cholera toxin gene), 63% for V. vulnificus and 79% for V. parahaemolyticus (of which 47% were tdh(+) and/or trh(+)). Viable vibrios were also isolated from the mussel meat and screened for virulence by PCR. The mortality of eukaryotic cells when exposed to bacteria was tested in vivo, with results showing that the Vibrio strains, independent of species and origin, were harmful to the cells. Despite severe infections and several deaths, no report on potential human pathogenic vibrios in this area had been published before this study.

Improving marine water quality by mussel farming: a profitable solution for Swedish society.

Eutrophication of coastal waters is a serious environmental problem with high costs for society globally. In eastern Skagerrak, reductions in eutrophication are planned through reduction of nitrogen inputs, but it is unclear how this can be achieved. One possible method is the cultivation of filter-feeding organisms, such as blue mussels, which remove nitrogen while generating seafood, fodder and agricultural fertilizer, thus recycling nutrients from sea to land. The expected effect of mussel farming on nitrogen cycling was modeled for the Gullmar Fjord on the Swedish west coast and it is shown that the net transport of nitrogen (sum of dissolved and particulate) at the fjord mouth was reduced by 20%. Existing commercial mussel farms already perform this service for free, but the benefits to society could be far greater. We suggest that rather than paying mussel farmers for their work that nutrient trading systems are introduced to improve coastal waters. In this context an alternative to nitrogen reduction in the sewage treatment plant in Lysekil community through mussel farming is presented. Accumulation of bio-toxins has been identified as the largest impediment to further expansion of commercial mussel farming in Sweden, but the problem seems to be manageable through new techniques and management strategies. On the basis of existing and potential regulations and payments, possible win-win solutions are suggested.

Shellfish and public health: a Swedish perspective.

Bivalves are ancient animals that feed by filtering large volumes of water. In this way, phytoplankton, bacteria and viruses from the water column are greatly concentrated in the mussels. The hazards associated with the consumption of mussels are thus dependent on the occurrence and composition of toxic algae and human microbial pathogens in the areas where shellfish are grown. Diarrheic shellfish toxins have occurred regularly in Sweden during the past 27 years. Peaks of toxins in mussels are mostly recorded from October to December, but the pattern can differ significantly due to location and year, making it hard to predict toxin levels in mussels. With an expansion of aquaculture and a subsequent increase in seafood consumption, better risk management is needed to minimize the effects on humans of algal toxins and human pathogens. New control strategies that have to be implemented are: i) proper site selection of culture installations; ii) regular and cost-efficient monitoring of algae, bacteria and viruses; iii) new indicators for fecal contamination, suitable for the specific locations where shellfish grow; iv) rapid dissemination of information to the industry and public, including risk assessment and advice on how to cope with the situation.

Environmental factors influencing human viral pathogens and their potential indicator organisms in the blue mussel, Mytilus edulis: the first Scandinavian report.

This study was carried out in order to investigate human enteric virus contaminants in mussels from three sites on the west coast of Sweden, representing a gradient of anthropogenic influence. Mussels were sampled monthly during the period from February 2000 to July 2001 and analyzed for adeno-, entero-, Norwalk-like, and hepatitis A viruses as well as the potential viral indicator organisms somatic coliphages, F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, and Escherichia coli. The influence of environmental factors such as water temperature, salinity, and land runoff on the occurrence of these microbes was also included in this study. Enteric viruses were found in 50 to 60% of the mussel samples, and there were no pronounced differences between the samples from the three sites. E. coli counts exceeded the limit for category A for shellfish sanitary safety in 40% of the samples from the sites situated in fjords. However, at the site in the outer archipelago, this limit was exceeded only once, in March 2001, when extremely high levels of atypical indole-negative strains of E. coli were registered at all three sites. The environmental factors influenced the occurrence of viruses and phages differently, and therefore, it was hard to find a coexistence between them. This study shows that, for risk assessment, separate modeling should be done for every specific area, with special emphasis on environmental factors such as temperature and land runoff. The present standard for human fecal contamination, E. coli, seems to be an acceptable indicator of only local sanitary contamination; it is not a reliable indicator of viral contaminants in mussels. To protect consumers and get verification of "clean" mussels, it seems necessary to analyze for viruses as well. The use of a molecular index of the human contamination of Swedish shellfish underscores the need for reference laboratories with high-technology facilities.