Molecular detection and antimicrobial resistance of Aeromonas from houseflies (Musca domestica) in Iran

ABSTRACT Objective. This study aimed to report the molecular detection and antimicrobial resistance of Aeromonas among houseflies (Musca domestica) in Shahrekord and Isfahan provinces of Iran. Materials and methods. Flies were caught from household kitchens, cattle farms, animal hospitals, human hospitals, slaughter house and poultry farms and put in collection separate sterile tubes. Isolation was accomplished by culture of flies in alkaline peptone water followed by identification with Aeromonas-specific Polymerase Chain Reaction (PCR). Results. Out of 600 houseflies 73 (12.2%) were infected with Aeromonas spp. Significantly higher frequencies of Aeromonas were isolated in Shahrekord province (13.0%; 39/300) than in Isfahan province (11.3%; 34/300). The recovery frequencies of the organisms were significantly lower in kitchens as compared to those in cattle farms and hospital wards which were similar. Higher proportions of infected flies were obtained during summer whereas low proportions were obtained during winter. Conclusions. It is concluded that houseflies do harbor diarrheagenic pathogens, including Aeromonas especially during summer. The carried organisms are resistant to a number of antimicrobials at different levels. Thus, future plans aimed at stemming infections caused by these organisms should take flies into account. Control efforts of infections caused by this particular bacterium should therefore take into account Musca domestica

Role of muscoid flies in the ecology of shiga toxin-producing Escherichia coli (STEC) in confined cattle environments

Master of ScienceEntomologyLudek ZurekHouse flies (Musca domestica L.) and stable flies (Stomoxys calcitrans L.) are insects of medical and veterinary importance. House flies are recognized as mechanical vectors of human foodborne pathogens and stable files are known for their painful bites resulting in reduction of body weight gain and milk production in cattle. The larval development of both fly species takes place in decaying organic materials (primarily animal manure), resulting in large fly populations in confined cattle environments. Shiga toxin-producing Escherichia coli (STEC) are a major foodborne pathogen. Cattle are the asymptomatic reservoir of STEC with bacteria being released to the environment via their feces. STEC O157 is the main serogroup causing human illness. However, infections with non-O157 STEC are increasing: more than 70% of non-O157 infections are caused by six serogroups of non-O157, referred as "Big six" (O26, O45, O103, O111, O121, and O145). In addition, there was a large 2011 outbreak in Europe caused by STEC O104. The objectives of my thesis were: 1) To assess the prevalence of seven serogroups of non-O157 STEC (O26, O45, O103, O104, O111, O121, and O145) (STEC-7) in house flies and stable flies collected from confined cattle environments; 2) To investigate the vector competence of house flies for non-O157 STEC-7. A total of 463 house flies from feedlots and dairies from six states, and 180 stable flies collected from a feedlot in Nebraska were processed for the isolation and identification of STEC-7 using a culture-based approach followed by PCR for the confirmation of serogroups, and virulence genes. A total of 34.3% of house flies and 1.1% of stable flies tested positive for at least one serogroup of E. coli of interest, and 1.5% of house flies harbored STEC with the Shiga-toxin gene (stx1). No STEC were detected in stable flies. Vector competence bioassays for non-O157 STEC revealed that house flies can carry non-O157 STEC for at least six days with the exception STEC O145. Overall, the findings of this research demonstrate that house flies, but not stable flies, likely play an important role in the ecology and transmission of non-O157 STEC in confined cattle environments

Isolation and characterisation of bacteria associated with flying insects in hospitals, with particular emphasis on Clostridium difficile

Clostridium difficile is a bacterial healthcare-associated infection, which houseflies Musca domestica may transfer due to their synanthropic nature. The aims of this thesis were to determine the ability of M. domestica to transfer C. difficile mechanically and to collect and identify flying insects in UK hospitals and classify any associated bacteria. M. domestica exposed to independent suspensions of vegetative cells and spores of C. difficile were able to mechanically transfer the bacteria on to agar for up to 4 hours following exposure. C. difficile could be recovered from fly excreta for 96hrs and was isolated from the M. domestica alimentary canal. Also confirmed was the carriage of C. difficile by M. domestica larvae, although it was not retained in the pupae or in the adults that subsequently developed. Flying insects were collected from ultra-violet light flytraps in hospitals. Flies (order Diptera) were the most commonly identified. Chironomidae were the most common flies, Calliphora vicina were the most common synanthropic fly and ‘drain flies’ were surprisingly numerous and represent an emerging problem in hospitals. External washings and macerates of flying insects were prepared and inoculated onto a variety of agars and following incubation bacterial colonies identified by biochemical tests. A variety of flying insects, including synanthropic flies (e.g. M. domestica and C. vicina) collected from UK hospitals harboured pathogenic bacteria of different species. Enterobacteriaceae were the group of bacteria most commonly isolated, followed by Bacillus spp, Staphylococci, Clostridia, Streptococci and Micrococcus spp. This study highlights the potential for M. domestica to contribute to environmental persistence and spread of C. difficile in hospitals. Also illustrated is the potential for flying insects to contribute to environmental persistence and spread of other pathogenic bacteria in hospitals and therefore the need to implement pest control as part of infection control strategies

House Fly (Musca Domestica L.) Temporal and Spatial Immune Response to Streptococcus Pyogenes and Salmonella Typhimurium: Role of Pathogen Density in Bacterial Fate, Persistence and Transmission

House flies (Musca domestica L), feed and breed in decomposing organic waste and therefore are constantly in contact with different species of microorganisms. Because house flies live in close proximity to human and animal habitats, they pose a danger of transmitting pathogenic microorganisms from diseased sources to new environments. To elucidate pathogen vector potential of house flies, this study investigated dose-dependent survivability of GFPexpressing Streptococcus pyogenes and Salmonella typhimurium SR11 within the fly alimentary canal both spatially via epifluorescence microscopy and quantitatively via culture-recovery. Adult house flies were fed known amounts of bacteria (high or low dose), and were dissected to remove the entire alimentary canal for microscopy or were homogenized and cultured at intervals within 24 h post-ingestion. Excreta also were cultured to determine transmission potential. Also investigated was the dose-dependent local intestinal epithelial immune response of house flies to S. pyogenes and S. typhimurium, where the upregulation of three antimicrobial peptides Defensin, Cecropin and Diptericin were investigated. Both bacterial dose and species affected survivability of these pathogens in the house fly alimentary canal. High dose of both species survived throughout the 24 h period. The number of viable S. typhimurium increased in 2 numbers in both high and low dose, whereas S. pyogenes decreased in number with time progression. Viable S. typhimurium were recovered in large quantities from excreta as compared to S. pyogenes. Both bacterial species and dose also affected the temporal, spatial and class of AMP expression profiles in the gut. In S. pyogenes-fed flies, only Defensin was regionally produced in midgut tissue. In contrast, tissues from flies that were fed S. typhimurium expressed both Cecropin and Diptericin. In both species, the higher dose of bacterial challenge induced greater AMP expression than the low dose. The region of the gut showing AMP expression in both bacterial challenges was mainly the midgut, and peak expressions correlated with high numbers of bacteria as determined by culture-recovery. Dose-dependent effects or survival and transmission of bacteria from house flies has significant implications on vector potential

Plasmídeos IncP-1 em Enterobacteriaceae resistentes a antibióticos

Antibiotic resistance is increasing worldwide closely linked with antibiotic misuse and overuse. Antibiotic gene resistance is commonly disseminated through MGEs (mobile genetic elements), especially plasmids. IncP-1 plasmids are BHR presumably found in several bacterial families and have been associated with antibiotic resistance and tolerance to metals. The identification of plasmid groups has been frequently done using the PBRT (PCR-based replicon typing) approach which assigns plasmids to Inc groups, including the IncP-1 plasmid group. The efforts to further characterize these structures, especially when associated with antibiotic resistant bacteria, has been lacking, and consequently, information is scarce and dispersed. Therefore, a systematic analysis was carried out to understand the occurrence, distribution, and genetic traits of IncP-1 plasmids associated with antibiotic resistant bacteria. To do so, a bibliographic search strategy was followed, where the Scopus platform was used to look for studies that used the PBRT method developed by Carattoli et al. (2005) for plasmid identification and that actually detected IncP-1 plasmids structures. Article collection for a period of 5 years resulted in 96 eligible articles, which were used to retrieve relevant information about IncP-1 plasmids occurrence and features. The articles combined reported the identification of 629 IncP-1 replicons. Altogether, the bacterial hosts of IncP-1 plasmids were found in 32 countries and were collected from a variety of environmental sources. Bacterial hosts belonged to 28 species distributed in 10 genera, of Enterobacteriaceae and Pseudomonadaceae families and were resistant to 10 different antibiotic classes, harboring 32 different resistance genes. IncP-1 plasmid-positive bacteria usually harbored at least 2 different Inc groups. Of all the IncP-1 plasmids identified, 229 (~36%) were further described, their sizes ranging from 35 to 320 kb and have been associated with medically important resistance genes and additional genetic elements that could potentiate gene dissemination. Furthermore, we studied the molecular diversity of previously reported IncP-1 plasmids occurring in 9 Escherichia coli isolates from an UWTP in Portugal. This was accomplished via PCR amplification of the 281 bp trfA gene fragment sequences and transfer to new well-known bacterial hosts for further characterizations. Amplicon sequencing showed 100% identity in all trfA fragments suggesting genetic structure conservation association to similar bacteria and environments. Similarity searching of the trfA fragment sequence was used to select closely related fully sequenced IncP-1β1 plasmids for comparisons. As a result, 63 closely related replicons were selected for comparative analysis and found to be usually large genetic structures, isolated mainly from wastewater and soil, harboring genetic determinants associated with resistance to aminoglycosides, sulphonamides, and β-lactams, and with tolerance to Hg. Attempts to transfer the 9 IncP-1 plasmids to a recipient strain were unsuccessful probably due to the chosen selective markers. The high prevalence of mer operon genes in these IncP-1β1 plasmids, suggesting mercury tolerance, could be used in a future work as a selective marker to transfer these 9 IncP-1 plasmids another bacterial host allowing for proper genomic characterization of these promiscuous structures.A resistência aos antibióticos está a aumentar em todo o mundo, intimamente associada ao uso incorreto e excessivo de antibióticos. Os genes de resistência a antibióticos são comummente disseminados por meio de EGMs (elementos genéticos móveis), especialmente plasmídeos. Os plasmídeos IncP-1 têm amplo espetro de hospedeiros, são presumivelmente encontrados em várias famílias de bactérias, e têm sido associados à resistência a antibióticos e tolerância a metais. A identificação de grupos de plasmídeos tem sido frequentemente realizada utilizando a abordagem PBRT (PCR-based replicon typing), que atribui plasmídeos a grupos de incompatibilidade, incluindo o grupo IncP-1. Os esforços para caracterizar melhor essas estruturas, principalmente quando associadas a bactérias resistentes a antibióticos, têm falhado e, consequentemente, as informações são escassas e dispersas. Portanto, uma análise sistemática foi realizada para compreender a ocorrência, distribuição e características genéticas de plasmídeos IncP-1 associados a bactérias resistentes a antibióticos. Para tal, a plataforma Scopus foi utilizada para realizar uma pesquisa bibliográfica, para selecionar estudos que identificaram plasmídeos IncP-1 através do método PBRT desenvolvido por Carattoli et al. (2005). A pesquisa abrangeu um período de 5 anos e resultou em 96 artigos elegíveis, os quais foram usados para a obtenção de informações relevantes sobre a ocorrência e características dos plasmídeos IncP-1. Os artigos combinados relataram a identificação de 629 replicões IncP-1. Ao todo, os hospedeiros bacterianos dos plasmídeos IncP-1 foram encontrados em 32 países e foram coletados de uma variedade de fontes ambientais. Os estudos identificaram hospedeiros bacterianos pertencentes a 28 espécies, distribuídas em 10 géneros das famílias Enterobacteriaceae e Pseudomonadaceae, foram identificados como resistentes a 10 classes de antibióticos, possuindo 32 genes de resistência diferentes, e pelo menos apresentando replicões de 2 grupos Inc diferentes. De todos os plasmídeos IncP-1 identificados, 229 (~ 36%) foram descritos posteriormente, e seus tamanhos variam de 35 a 320 kb, foram associados a genes de resistência clinicamente importantes e a elementos genéticos adicionais que poderiam potenciar a disseminação de genes. Além disso, foi estudada a diversidade molecular de plasmídeos IncP-1 identificados anteriormente em 9 isolados de Escherichia coli de uma UWTP em Portugal. Isso foi realizado por meio da amplificação por PCR de um fragmento do gene trfA de 281 bp, e tentativa de transferência para novos hospedeiros bacterianos bem conhecidos para posterior caracterização. A sequenciação dos amplicões mostrou 100% identidade entre fragmentos trfA sugerindo conservação das suas estruturas genéticas e associação com bactérias e ambientes semelhantes. A pesquisa por similaridade da sequência do fragmento trfA foi usada para selecionar e comparar plasmídeos IncP-1β1 totalmente sequenciados. Isso resultou na análise de 63 replicões, geralmente grandes estruturas genéticas, isolados de águas residuais e solos, possuindo determinantes genéticos associados à resistência a aminoglicosídeos, sulfonamidas e βlactâmicos, e à tolerância ao Hg. As tentativas de transferir os 9 plasmídeos IncP-1 para uma estirpe de laboratório não tiveram sucesso, provavelmente devido aos marcadores seletivos escolhidos. A ampla prevalência de genes do operão mer nestes plasmídeos IncP-1β1, sugerindo tolerância ao mercúrio, poderia ser usada em um trabalho futuro como um marcador seletivo para a transferência dos 9 plasmídeos IncP-1 de água residual para outro hospedeiro, permitindo a caracterização genética dessas estruturas.Mestrado em Microbiologi

The Impact of Latrine Construction on Densities and Pathogen Infection Rates of Synanthropic Flies and Culex Quinquefasciatus Mosquitoes in Odisha, India

Diarrhoeal disease is one of the leading causes of mortality and morbidity globally. Non-biting synanthropic flies are of public health importance due to their habit of flying between faecal matter and households, potentially transmitting pathogenic bacteria by regurgitation, defecation or mechanical transmission via legs or wings. Lymphatic filariasis (LF), caused by Wuchereria bancofti and transmitted by Culex quinquefasciatus, affects approximately 120 million people in Asia. Controlling the mosquito vector can have a significant impact on LF incidence rates. It is assumed that increasing the coverage of latrines will reduce the amount of open defecation and environmental faecal contamination resulting in a healthier population. However, few studies have measured the impact of building latrines on human health and even fewer have demonstrated its impact on synanthropic fly populations or their bacterial carriage and associated diarrhoeal disease. Similarly, although Cx. quinquefasciatus has long been associated with pit latrines, the resulting impact through the construction of improved pour-flush latrines on population densities has yet to be explored. Initial experiments were conducted to determine the best methodology for trapping both synanthropic flies and Cx. quinquefasciatus, to inform the design of the entomological component of a cluster randomised control trial (cRCT). Thereafter, the focus was on determining the impact of latrine construction on the exposure of households to populations of flies and the bacteria that they carry, and on Cx. quinquefasciatus densities and W. bancofti prevalence. Results indicate that latrine construction had no impact on the density of flies within households or on the carriage of bacteria. There was no statistical difference between control and intervention arms in the population density of synanthropic flies (IRR=0.89; 95%CI=[0.76-1.03]; p=0.131). There was a significant correlation between fly numbers and rainfall. Data were analysed by season for between arm differences; in the monsoon season 40% fewer flies were caught in the intervention arm compared to the control arm (p=<0.001). Most flies caught belonged to the Muscidae family: Musca domestica or M. sorbens. Of the flies tested for bacteria, 60.3% were positive for at least one of either Escherichia coli 0157:H7, Vibrio cholerae, Salmonella spp. or Shigella spp.. Escherichia coli was detected most frequently with 46% testing positive. Numbers of Cx. quinquefasciatus caught (IRR=0.88; 95%CI=[0.74-1.06]; p=0.178). between the control and intervention villages. Less than 1% of Cx. quinquefasciatus tested positive for W. bancrofti, equivalent to an LF prevalence rate of 0.0034% within the vector population. Latrine construction was not associated with a reduction in the densities of vectors and their pathogens, compared to villages without latrines. However, it should not be concluded that latrines have little value in improving public health. Reducing open defecation is one step towards limiting fly population densities and bacterial contamination in the environment, by reducing available larval habitats. However, latrine coverage by itself is not enough to prevent open defecation; construction needs to be supported by behaviour change. These findings reflect that there are multiple pathways for the spread of bacteria in the environment, one of which is fly-borne. In environments with high bacterial transmission reducing fly numbers alone is not enough to impact the transmission of diarrhoeal pathogens

The changing epidemiology, clinical syndrome and antibiotic resistance patterns of shigellosis in Vietnamese children

Diarrhoeal diseases remain the second most common cause of death among children under-five globally, following closely behind pneumonia. In developing countries, rotavirus and Shigella spp. are thought to be the most important enteropathogens causing acute childhood diarrhoea. Systematicd ata on diarrhoea)diseases. particularly Shigella infection, in southern Viet Nam is lacking. An analysis of the historical trends in epidemiology, antibiotic susceptibility, and clinical features of Shigella infection and contemporary clinical studies of the response to antibiotic treatment in Vietnamese children were the central aims of this thesis. By analysing Shigella strains isolated from children between 1995 and 2009 I documented a transition in the dominant Shigella species causing diarrhoea from S.flexneri to S.sonnei, a change previously linked with industrialisation and economic development. During the same period there was a sharp increase of nalidixic acid resistant Shigella strains from approximately 7% to 70%. In addition, the clinical presentation appeared to be more severe and the duration of hospital stays longer. During the course of this thesis I also documented for the first time in Viet Nam Shigella spp. which harboured the plasmid-transferable drug resistance blaCTX-M genes. The occurrence of this plasmid-transferable gene may endanger the use of betalactams for this infection in the future. In a large randomised controlled trial, gatifloxacin, a new 8-methoxy-fluoroquinolone antibiotic proved comparable to ciprofloxacin for the treatment of acute dysentery including those caused by multiresistant Shigella. Although gatifloxacin has been associated with dysglycaemic side-effects in adults, this adverse effect was not observed in this study