Species Interaction and Selective Carbon Addition During Antibiotic Exposure Enhances Bacterial Survival.

Biofilms are multifaceted and robust microbiological systems that enable microorganisms to withstand a multitude of environmental stresses and expand their habitat range. We have shown previously that nutritional status alters antibiotic susceptibility in a mixed-species biofilm. To further elucidate the effects of nutrient addition on inter-species dynamics and whole-biofilm susceptibility to high-dose streptomycin exposures, a CO2 Evolution Measurement System was used to monitor the metabolic activity of early steady state pure-culture and mixed-species biofilms containing Pseudomonas aeruginosa and Stenotrophomonas maltophilia, with and without added carbon. Carbon supplementation was needed for biofilm recovery from high-dose streptomycin exposures when P. aeruginosa was either the dominant community member in a mixed-species biofilm (containing predominantly P. aeruginosa and S. maltophilia) or as a pure culture. By contrast, S. maltophilia biofilms could recover from high-dose streptomycin exposures without the need for carbon addition during antibiotic exposure. Metagenomic analysis revealed that even when inocula were dominated by Pseudomonas, the relative abundance of Stenotrophomonas increased upon biofilm development to ultimately become the dominant species post-streptomycin exposure. The combined metabolic and metagenomic results demonstrated the relevance of inter-species influence on survival and that nutritional status has a strong influence on the survival of P. aeruginosa dominated biofilms.

Effect of carbon on whole-biofilm metabolic response to high doses of streptomycin.

Biofilms typically exist as complex communities comprising multiple species with the ability to adapt to a variety of harsh conditions. In clinical settings, antibiotic treatments based on planktonic susceptibility tests are often ineffective against biofilm infections. Using a CO2 evolution measurement system we delineated the real-time metabolic response in continuous flow biofilms to streptomycin doses much greater than their planktonic susceptibilities. Stable biofilms from a multispecies culture (containing mainly Pseudomonas aeruginosa and Stenotrophomonas maltophilia), Gram-negative environmental isolates, and biofilms formed by pure culture P. aeruginosa strains PAO1 and PAO1 ΔMexXY (minimum planktonic inhibitory concentrations between 1.5 and 3.5 mg/l), were exposed in separate experiments to 4000 mg/l streptomycin for 4 h after which growth medium resumed. In complex medium, early steady state multispecies biofilms were susceptible to streptomycin exposure, inferred by a cessation of CO2 production. However, multispecies biofilms survived high dose exposures when there was extra carbon in the antibiotic medium, or when they were grown in defined citrate medium. The environmental isolates and PAO1 biofilms showed similar metabolic profiles in response to streptomycin; ceasing CO2 production after initial exposure, with CO2 levels dropping toward baseline levels prior to recovery back to steady state levels, while subsequent antibiotic exposure elicited increased CO2 output. Monitoring biofilm metabolic response in real-time allowed exploration of conditions resulting in vulnerability after antibiotic exposure compared to the resistance displayed following subsequent exposures.

Impact of dryland salinity on population dynamics of vector mosquitoes (Diptera: Culicidae) of Ross River virus in inland areas of southwestern Western Australia.

Clearing of native vegetation for agriculture since European settlement has left 1.047 million ha of southwestern Australia affected by a severe form of environmental degradation called dryland salinity, characterized by secondary soil salinization and waterlogging. This area may expand by a further 1.7-3.4 million ha if current trends continue. Detailed investigations of seasonal of adult and larval mosquito population dynamics were undertaken in the region to test the hypothesis that the development of dryland salinity and waterlogging in inland southwestern Australia has led to a succession of mosquito species and increased Ross River virus (family Togaviridae, genus Alphavirus, RRV) transmission risk. Aedes (Ochlerotatus) camptorhynchus (Thomson) made up >90% of adult mosquito collections in saline regions. Nonmetric multidimensional scaling and generalized estimating equations modeling demonstrated that it was strongly associated with increasing severity of dryland salinity. This article describes the first detailed investigation of the mosquito fauna of inland southwestern Australia, and it is the first description of the influence of secondary soil salinity on mosquito population dynamics. Despite the dominant presence of Ae. camptorhynchus, RRV disease incidence is not currently a significant population health priority in areas affected by dryland salinity. Potential limiting factors include local climatic impacts on the seasonal mosquito population dynamics, vertebrate host distribution and feeding behavior of Ae. camptorhynchus, and the scarce and uneven distribution of the human population in the region.

Modelling the transmission dynamics of Ross River virus in Southwestern Australia.

During the 1995-1996 Australian financial year, over 1300 notifications of Ross River (RR) virus disease were notified in humans from Southwestern Australia. Due to the mild symptoms of the disease, it is difficult to diagnose and subclinical infections are common. However, these subclinical infections do give rise to immunity. For planning and control, it is important for public health authorities to estimate the true number of people who have contracted the disease and to assess the impact of key epidemiological parameters. A mathematical model was developed to describe the transmission of RR virus between its hosts (humans and kangaroos) and its vectors (mosquitoes). For this model, the threshold conditions and relative removal rates were calculated and interpreted. Finally, a computer program was written to simulate the model in order to estimate the total number, both clinical and sub clinical human infections given known and hypothetical epidemiological parameter values. Within this simulation sensitivity of the results to changes in the parameters were examined. The analysis of the threshold conditions conformed well to established principles of arboviral transmission and control. It was observed that conditions which can prevent an outbreak occuring include reducing the number of susceptibles in host and vector populations, reducing the infection rates between hosts and vectors and shortening the duration of viraemia. Results on the sensitivity analysis showed that some parameters such as the extrinsic incubation period, mosquito mortality rate in winter and the proportion of Western Grey Kangaroos in the marsupial population have little effect on human incidence. However, the transmission rate between hosts and vectors, vector-mortality rate in summer and the proportion of infectious vectors among infected vectors have pronounced effects. The simulation results on the ratio of clinical to subclinical human infections predicted a minimum ratio of 1:2 and a maximum ratio of 1:65, which is consistent with data obtained during previous sero-epidemiological studies.

Murray Valley encephalitis virus surveillance and control initiatives in Australia. National Arbovirus Advisory Committee of the Communicable Diseases Network Australia.

Mechanisms for monitoring Murray Valley encephalitis (MVE) virus activity include surveillance of human cases, surveillance for activity in sentinel animals, monitoring of mosquito vectors and monitoring of weather conditions. The monitoring of human cases is only one possible trigger for public health action and the additional surveillance systems are used in concert to signal the risk of human disease, often before the appearance of human cases. Mosquito vector surveillance includes mosquito trapping for speciation and enumeration of mosquitoes to monitor population sizes and relative composition. Virus isolation from mosquitoes can also be undertaken. Monitoring of weather conditions and vector surveillance determines whether there is a potential for MVE activity to occur. Virus isolation from trapped mosquitoes is necessary to define whether MVE is actually present, but is difficult to deliver in a timely fashion in some jurisdictions. Monitoring of sentinel animals indicates whether MVE transmission to vertebrates is actually occurring. Meteorological surveillance can assist in the prediction of potential MVE virus activity by signalling conditions that have been associated with outbreaks of Murray Valley encephalitis in humans in the past. Predictive models of MVE virus activity for south-eastern Australia have been developed, but due to the infrequency of outbreaks, are yet to be demonstrated as useful for the forecasting of major outbreaks. Surveillance mechanisms vary across the jurisdictions. Surveillance of human disease occurs in all States and Territories by reporting of cases to health authorities. Sentinel flocks of chickens are maintained in 4 jurisdictions (Western Australia, the Northern Territory, Victoria and New South Wales) with collaborations between Western Australia and the Northern Territory. Mosquito monitoring complements the surveillance of sentinel animals in these jurisdictions. In addition, other mosquito monitoring programs exist in other States (including South Australia and Queensland). Public health control measures may include advice to the general public and mosquito management programs to reduce the numbers of both mosquito larvae and adult vectors. Strategic plans for public health action in the event of MVE virus activity are currently developed or being developed in New South Wales, the Northern Territory, South Australia, Western Australia and Victoria. A southern tri-State agreement exists between health departments of New South Wales, Victoria and South Australia and the Commonwealth Department of Health and Aged Care. All partners have agreed to co-operate and provide assistance in predicting and combatting outbreaks of mosquito-borne disease in south-eastern Australia. The newly formed National Arbovirus Advisory Committee is a working party providing advice to the Communicable Diseases Network Australia on arbovirus surveillance and control. Recommendations for further enhancement of national surveillance for Murray Valley encephalitis are described.

The molecular epidemiology of Kokobera virus.

We describe herein the molecular epidemiology and phylogeny of Kokobera (KOK) virus, a flavivirus found in Australia and Papua New Guinea. We sequenced a region encompassing the 200 nucleotides of the 3' terminus of the NS5 gene, and the first 300 nucleotides of the 3' untranslated region (UTR). The study included 25 isolates of the virus, including an isolate from PNG, and several recent isolates from the south-west of Western Australia (WA), where the virus had not previously been detected. We found that the KOK isolates clustered according to geographic location and time of isolation into three distinct topotypes: one covering Queensland and New South Wales; another represented by the single isolate from PNG; and a third covering the Northern Territory and WA. This latter group was further subdivided into northern and south-west isolates. This molecular epidemiology is significantly different from other Australian flaviviruses, such as Murray Valley encephalitis (MVE) and Kunjin (KUN) viruses, which exist as single genetic types across the entire Australian continent. However, it is similar to the molecular epidemiology of the alphavirus Ross River (RR) virus. This may be explained by the fact that MVE and KUN viruses are known to have birds as their main vertebrate hosts, whereas RR virus utilises macropods, which have also been implicated as the vertebrate host for KOK virus. In addition, the south-west isolates exhibited a degree of sequence heterogeneity, including one isolate that has a nine nucleotide deletion in the 3'UTR. This suggests that KOK virus has been in the south-west of WA for some time, and was not recently introduced.

Murray Valley encephalitis in Western Australia in 2000, with evidence of southerly spread.

We describe the epidemiological and clinical features of human Murray Valley encephalitis (MVE) and Kunjin (KUN) virus infections in Western Australia (WA) during March to July 2000. A case series was performed. For laboratory-confirmed cases, travel histories and clinical details were collected from patients, family members, friends or treating physicians. Surveillance data from the sentinel chicken program and climatic conditions were reviewed. Nine encephalitic cases of MVE were recorded. Eight were non-Aboriginal adults (age range, 25 to 79 years; 5 male, 3 female) and 1 was an Aboriginal boy. Four cases acquired infection in the Murchison and Midwest regions of WA from which no human cases of MVE have been reported previously. One of the 9 cases was fatal and 3 had severe neurological sequelae. Five non-encephalitic infections were also recorded, 3 MVE and 2 KUN. Encephalitis caused by MVE virus remains a serious problem with no improvement in clinical outcomes in the last 25 years. Excessive rainfall with widespread flooding in the northern two-thirds of WA provided ideal conditions for mosquito breeding and favoured southerly spread of the virus into new and more heavily populated areas. Surveillance in WA with sentinel chickens and mosquito trapping needs expansion to define the boundaries of MVE virus activity. To enable timely warnings to the public, and to institute mosquito control where feasible, continued surveillance in all Australian areas at risk is indicated.

Geographic distribution and evolution of Sindbis virus in Australia.

The molecular epidemiology and evolution of Sindbis (SIN) virus in Australia was examined. Several SIN virus strains isolated from other countries were also included in the analysis. Two regions of the virus genome were sequenced including a 418 bp region of the E2 gene and a 484 bp region containing part of the junction region and the 5' end of the C gene. Analysis of the nucleotide and deduced amino acid sequence data from 40 SIN virus isolates clearly separated the Paleoarctic/Ethiopian and Oriental/Australian genetic types of SIN virus. Examination of the Australian strains showed a temporal rather than geographic relationship. This is consistent with the virus having migratory birds as the major vertebrate host, as it allows for movement of virus over vast areas of the continent over a relatively short period of time. The results suggest that the virus is being periodically redistributed over the continent from an enzootic focus of evolving SIN virus. However, SIN virus strains isolated from mosquitoes collected in the south-west of Australia appear to represent a new SIN virus lineage, which is distinct from the Paleoarctic/Ethiopian and Oriental/Australian lineages. Given the widespread geographic dispersal of the Paleoarctic/Ethiopian and Oriental/Australian lineages, it is surprising that the South-west genetic type is so restricted in its area of circulation. Nucleotide sequence data from the C gene of the prototype strain of the alphavirus Whataroa were also determined. This virus was found to be genetically distinct from the SIN virus isolates included in the present study; however, it is clearly SIN-like and appears to have evolved from a SIN-like ancestral virus.

Identification of Australian arboviruses in inoculated cell cultures using monoclonal antibodies in ELISA.

An ELISA using a panel of specific monoclonal antibodies was developed to identify all alpha and flaviviruses isolated from mosquitoes caught throughout Australia. This technique is sensitive and rapid and is more specific than the traditional methods used to identify flaviviruses. The ability to identify unknown virus isolates from field-caught mosquitoes quickly and accurately improves the efficiency of arbovirus surveillance programs and allows health authorities to give an early warning of an increased health risk from a mosquito-borne virus in a particular region.

A comparison of the diseases caused by Ross River virus and Barmah Forest virus.

Barmah Forest virus (BFV) and Ross River virus (RRV) are mosquito-borne viruses with similar vectors and environmental requirements. They cause diseases characterised by arthralgia, arthritis and myalgia, often accompanied by fever and rash. Arthritis is more common and more prominent in RRV disease and rash is more common and florid with BFV infection, although the diseases cannot be reliably distinguished by their clinical symptoms. Diagnosis is based on serological tests and a definite diagnosis of recent infection requires the demonstration of rising titres of IgG. Arthralgia, myalgia and lethargy may continue for at least six months in up to half of patients with RRV, but in only about 10% of patients with BFV. Both diseases are managed symptomatically.

Arboviruses in the Australian region, 1990 to 1998.

Arboviruses continue to be major human pathogens in the Australian region. This report provides a summary of the activities of these viruses over the past eight years, and comments on new findings relevant to their respective ecologies. Of particular interest and concern is the propensity of these viruses to spread. The examples discussed include the initiation of dengue epidemics in north Queensland by virus imported in viraemic travellers; the spread of Japanese encephalitis virus to the Australasian region and its probable enzootic establishment in the south-west of Papua New Guinea; the potential spread of Ross River virus to other countries, as demonstrated by the 1979-80 outbreak in the South Pacific, and the recent occurrence in military personnel from the United States of America after an exercise; and the recent spread of Barmah Forest virus into Western Australia.

Genetic stability among temporally and geographically diverse isolates of Barmah Forest virus.

An increase in the incidence of polyarthritis caused by Barmah Forest (BF) virus, and its recent emergence into Western Australia, prompted a study of the molecular epidemiology of this Australian mosquito-borne alphavirus. The nucleotide sequence of a 500-basepair region of the 3' end of the envelope (E2) gene of the prototype BF virus strain (BH2193) was compared with other members of the alphavirus genus, and to a panel of isolates of BF virus collected more for than 20 years from different geographic regions of Australia. The BF virus was shown to be genetically distinct from other members of the alphavirus genus. A high degree of sequence homology (98-100%) was found between the BF isolates, with no evidence of geographic or temporal divergence. This nucleotide homogeneity was similar to that observed with other Australian mosquito-borne viruses with avian vertebrate hosts, such as Sindbis, Murray Valley, and Kunjin viruses, but it contrasts to the heterogeneity reported for Ross River virus, an alphavirus with mammalian vertebrate hosts.

Geographic distribution and evolution of Ross River virus in Australia and the Pacific Islands.

We examined the molecular epidemiology and evolution of Ross River (RR) virus in Australia and the Pacific Islands. Nucleotide sequences of the E2 and E3 genes of five RR virus strains revealed remarkable conservation between 1959 and 1989 with a maximum divergence of only 3.3%. Sequence data from a 505-base pair fragment of the E2 gene from 51 additional strains showed that RR virus has diverged genetically into three separate groups although at least 95% sequence homology was still maintained between all 56 strains. Each genetic type predominates in a particular geographic region of Australia and can be broadly defined as occurring in the western, northeastern, and southeastern regions of Australia. However, some RR virus strains did not follow this pattern of geographic distribution indicating movement of virus by the travel of viremic humans or livestock across the continent. The Pacific Islands isolates all belong to the southeastern genotype. These findings suggest genetic divergence and independent evolution of RR virus within geographically isolated enzootic foci; however, selective pressures maintain high nucleotide conservation in nature.

Specific enzyme immunoassays for the rapid detection of Ross River virus in cell cultures inoculated with infected mosquito homogenates.

BACKGROUND: Ross River (RR) virus is a mosquito-borne alphavirus and one of the aetiological agents of epidemic polyarthritis in humans. Early detection of increased virus activity in mosquito populations enables public health authorities to implement measures to reduce the number of human infections during epidemics. However, current surveillance techniques require a minimum of four weeks for viruses to be isolated and identified. OBJECTIVES: This study was carried out to assess the use of enzyme immunoassays (EIA) as rapid alternatives to traditional cell culture techniques for detection of RR virus in mosquitoes. STUDY DESIGN: Enzyme immunoassays and immunoperoxidase assays were developed using RR-specific monoclonal antibodies and compared to traditional methods for detection of RR virus in field-caught mosquito samples. RESULTS: By inoculation of C6/36 cell cultures with mosquito homogenates and testing monolayers and culture supernatant by EIA, RR virus was detected and identified in all infected samples within 6 days. CONCLUSIONS: The use of EIA provides a rapid, sensitive and specific alternative to traditional methods for the detection of RR virus in mosquito vectors.

Two possible mechanisms for survival and initiation of Murray Valley encephalitis virus activity in the Kimberley region of Western Australia.

Two possible mechanisms are described for the initiation of Murray Valley encephalitis (MVE) virus activity in arid, epizootic regions of tropical Australia. Virus isolations were made from mosquitoes trapped shortly after the first heavy wet season rains and flooding in the east Kimberley, which followed approximately nine months of drought. A number of isolates of MVE virus were obtained, including isolates from pools of blood-engorged Culex annulirostris mosquitoes and from a single pool of male Aedes tremulus mosquitoes. The results strongly suggested that MVE virus activity was due both to its introduction in viremic vertebrate hosts, from which first-generation mosquitoes became infected following blood meals, and also to reactivation of vertically transmitted virus from desiccation-resistant eggs of Ae. tremulus. Both mechanisms are discussed with respect to environmental conditions.

An outbreak of Barmah Forest virus disease in the south-west of Western Australia.

OBJECTIVES: To describe the first reported outbreak of Barmah Forest (BF) virus disease in the south-west of Western Australia. DESIGN: Case series correlated with results of arbovirus surveillance. All patients with clinically suspected Ross River (RR) virus infection were serologically tested for antibodies to BF and RR viruses. Home address and date of presentation of patients with serologically confirmed recent infection were recorded. Mosquitoes collected from the districts before and during the BF virus outbreak were identified to species level and tested for virus. RESULTS: Twenty-two cases of BF disease were reported from the region between August 1992 and March 1994. Most occurred in the Peel region in the spring and early summer of 1993. Eighteen isolates of BF virus were obtained from three different species of mosquito trapped between January and October 1993. Fifteen were from mosquitoes in the Peel region and a single isolate was from the Perth metropolitan area. No isolates were obtained from the region before 1993. RR virus was not isolated from mosquitoes trapped in the region during the BF virus outbreak. CONCLUSIONS: Most BF infections were acquired in the Peel region during spring and early summer of 1993. Aedes camptorhynchus mosquitoes were probably the main vectors. The lack of isolations from mosquitoes before 1993 suggests that the virus may have only recently been introduced (or reintroduced) to the region. It was transmitted under conditions that were apparently not conducive to transmission of RR virus.

Molecular epidemiology and evolution of mosquito-borne flaviviruses and alphaviruses enzootic in Australia.

Three distinct patterns in the molecular epidemiology and evolution are evident among the alphaviruses and flaviviruses enzootic in Australia. One pattern, exemplified by MVE and KUN viruses, is of a single genetic type evolving slowly and uniformly in geographically widely separated regions of Australia with no evidence of independent divergence. The second pattern, exemplified by RR virus, is of separate genotypes evolving in different geographic regions with significant nucleotide divergence between genotypes. The third pattern, exemplified by SIN virus, is of a succession of temporally related genotypes that extend over most of the Australian continent, with relatively low levels of nucleotide divergence within a genotype, and which are each replaced by the subsequent genotype. These patterns are associated in part due to the nature and dispersal of their vertebrate hosts. Nucleotide divergence rates for Australian alphaviruses are similar to those reported elsewhere. Genomic relationships between Australian flavivirus members of the JE virus serological complex and between Australian alphaviruses are discussed, and evidence is presented for a possible new genomic lineage of SIN virus.

Arboviruses causing human disease in the Australasian zoogeographic region.

Over 65 arboviruses have been reported from countries in the Australasian zoogeographic region, but only a few have been implicated in human disease. These include the flaviviruses Murray Valley encephalitis (MVE), Kunjin (KUN), Kokobera (KOK), and dengue, particularly types 1 and 2; the alphaviruses Ross River (RR), Barmah Forest (BF), and Sindbis (SIN); and the bunyaviruses, Gan Gan and Trubanaman. In this paper recent epidemiological and clinical results pertaining to these viruses are reviewed, with major emphasis on MVE and RR viruses. The extensive early studies of Australian arboviruses have been reviewed by Doherty [49, 50], and their ecology and vectors more recently by Kay and Standfast [87]. In addition, the biology of MVE and KUN [113] and RR [87, 114] viruses have been the subjects of more detailed reviews. The Australasian zoogeographic region is defined as countries east of the Wallace and Weber lines, two hypothetical lines in the Indo-Australian archipelago where the fauna of the Australasian and Oriental regions meet. Seroepidemiological studies of human arboviral infections have suggested that the Japanese encephalitis flavivirus and the chikungunya alphavirus occur only in the Oriental region, whereas the related MVE and RR viruses, respectively, are restricted to the Australasian region [85, 148]. Serological results from Wallacea, the zone between the Wallace and Weber lines, are not so clear-cut [85]. This review is therefore restricted to countries east of Wallacea, specifically New Guinea and Australia.

Ross River virus isolations from mosquitoes in arid regions of Western Australia: implication of vertical transmission as a means of persistence of the virus.

Outbreaks of mosquito-borne Ross River (RR) virus disease (epidemic polyarthritis) occur suddenly in the arid north and interior of the State of Western Australia, often within a few weeks of heavy rainfall. Between outbreaks, these regions may undergo long periods of drought, with little or no mosquito or arbovirus activity. The means by which RR virus is reintroduced or reactivated in these areas when environmental conditions favor mosquito-borne virus activity are unknown. In this paper, we describe isolations of RR virus from eight mosquito species trapped at two different locations, one coastal and one inland, in the arid Pilbara region of Western Australia, prior to outbreaks of epidemic polyarthritis. The isolation of RR virus has not been previously reported for five of these species and the isolations from the other three species are new records for Western Australia. The timing and number of isolations of RR virus from Aedes (Ochlerotatus) vigilax (Skuse, 1889) implicate that species as a vector of the virus on the Pilbara coast. Significantly, RR virus was isolated from pools of male Ae. vigilax and male Ae. (Macleaya) tremulus (Theobald, 1903) mosquitoes. This is the first report of RR virus (or other Australian arbovirus) isolates from wild-caught male mosquitoes. Both Ae. vigilax and Ae. tremulus have desiccation-resistant eggs that can survive long periods of drought, making them ideal candidates for the overwintering of arboviruses. The findings implicate vertical transmission as a means of persistence of RR virus in arid regions of Australia and therefore offer a likely explanation for the sudden recurrence of virus activity after heavy rains.

Genetic heterogeneity among isolates of Ross River virus from different geographical regions.

The RNase T1 maps of 80 isolates of Ross River virus from different regions of mainland Australia and the Pacific Islands were compared. Four different clusters of isolates with greater than an estimated 5 to 6% diversity at the nucleotide level were found. There was a pattern of differences between eastern and western Australian strains; however, the pattern was disturbed by overlaps and incursants. Pacific Islands isolates belonged to the eastern Australian topotype. Our findings suggest that certain genetic types of Ross River virus predominate in different geographical regions. In contrast, populations of other important Australian arboviruses (Murray Valley encephalitis, Kunjin, and Sindbis viruses) are distributed across the Australian continent as minor variants of one strain. Our data also show that in one region, strains of Ross River virus with identical RNase T1 maps circulate during both years when epidemics occur and years when they do not. This finding suggests that Ross River virus epidemics are not dependent on the introduction or evolution of new strains of the virus. Two strains, belonging to the eastern Australian topotype, were isolated in Western Australia. It is likely that viremic humans or possibly domestic livestock travelling by aircraft were responsible for this movement.