Freshwater Beach Microbial Ecology, Community Dynamics and Adaptive Responses to Environmental Changes Using Metabarcoding and Transcriptomics

The Laurentian Great Lakes (LGLs) represent the single most valuable natural resource on the North American continent and are a critical source of drinking water, important aquatic species habitat, water for the industrial sector and tourism/recreational activities as well as many other ecological services. LGLs ecosystems are changing rapidly due to climate change effects and are thus highly susceptible and responsive to any added anthropogenic stressors. The aquatic bacterial community affects critical ecosystem functions, such as nutrient cycling, water quality, recreational activities, etc., in ecosystems such as the LGLs, but perturbations can alter both the composition and functionality of the bacterial community. These changes can result in negative effects on whole ecosystem health with an associated loss in economic and social values. Understanding the temporal and spatial variation in the composition, diversity and ultimately activity of the bacterial community is paramount in understanding overall ecosystem services providing by the bacterial community. Characterizing spatial and temporal variation (and the factors that contribute to it) can provide deeper insight into the processes and mechanisms operating in LGLs ecosystems, and ultimately improve our basic knowledge and ability to predict bacterial community composition, dynamics and function. Enumeration of Escherichia coli as a bioindicator of human fecal contamination is widely used to quantify recreational water quality and safety. The inclusion of microbial source tracking (MST) as part of water quality monitoring along with E. coli and waterborne pathogens in a novel monitoring tool could help to determine the specific fecal source (e.g. human, dog, cattle, wildlife, etc.) and has great potential for accurate estimation of water-related health risks. On the other hand, we still have an incomplete understanding of freshwater microbial ecology and community dynamics and their response to disturbance, particularly to human-related environmental stressors. We must be able to predict and track the sources of harmful bacterial outbreaks; this will require a clear understanding of the impact of environmental and anthropogenic stressors on microbial community diversity and function. The research comprising this dissertation was designed to characterize broad to fine-scale temporal and spatial variation in freshwater bacterial community composition and gene transcription. Temporal variation in freshwater bacterial community composition (bi-hourly, monthly and seasonal variation) and gene transcription profile (seasonally) was significant while spatial variation was significant but limited in magnitude. A novel monitoring approach (nanofluidic TaqMan qRT-PCR) was designed and optimized for rapid and reliable monitoring of freshwater quality for waterborne pathogens, MST markers and E. coli as a bioindicator of fecal contamination. Finally, an experimental bacterial microcosm study was used to study the response of adapted (pre-exposure to different levels of nutrient stress) bacteria communities to very high nutrient stress. This experiment revealed that pre-exposure to a higher level of nutrient stress provides greater protection against community change than low levels when the bacterial community is challenged with a very high level of a stressor. These cumulative insights into the temporal and spatial variation of the freshwater bacteria community composition and transcriptome, the development of a novel nanofluidic TaqMan qRT-PCR tool for detecting and quantifying harmful bacteria and our microcosm study outcomes provide baseline knowledge and tools which will be valuable for improving best management practices, monitoring and accurate prediction of changes in freshwater ecosystem function

Identification of clinical isolates of Acinetobacter baumannii from Iran and study of their heterogeneity

AbstractBackgroundAcinetobacter baumannii has become one of the most serious causative agents of nosocomial infections due to its significant ability to survive on hospital surfaces. It is mainly an emerging opportunistic pathogen infecting patients in intensive care units. This study was aimed to identify the clinical isolates of A. baumannii and to investigate their heterogeneity using polymerase chain reaction (PCR)-based typing methods.MethodsA total of 197 nonduplicate isolates recovered from a wide range of clinical samples were subjected to conventional cultural and biochemical tests. For those isolates that were preliminary identified as A. baumannii, rpoB-based PCR with subsequent restriction fragment length polymorphism (RFLP) using two restriction enzymes (TagI and HaeIII) was performed to investigate the genetic diversity of the strains and their presumptive relationships with different clinical presentation of the disease caused by this pathogen.ResultsIn total, 50 isolates (25.4%) were identified as A. baumannii using conventional phenotypic methods with subsequent confirmation by rpoB sequencing. RFLP analysis demonstrated five different restriction enzyme patterns, designated as A–E clusters. Most A. baumannii isolates were categorized under Cluster A (32%). We found no significant relationship between clinical presentation and the clustering of the isolates.ConclusionThis study showed that the rpoB region possesses high discriminatory power to identify the isolates to the species level. This marker showed high interspecies variability that might be useful for strain typing. The results also suggest the possibility of the existence of a predominant clone of A. baumannii among infected patients in Iran

Molecular Methods for Identification of Acinetobacter Species by Partial Sequencing of the rpoB and 16S rRNA Genes.

BACKGROUND Acinetobacter spp. is a diverse group of Gram-negative bacteria which are ubiquitous in soil and water, and an important cause of nosocomial infections. The purpose of this study was to identify a collection of Acinetobacter spp. clinical isolates accurately and to investigate their antibiotic susceptibility patterns. MATERIALS AND METHODS A total of 197 non-duplicate clinical isolates of Acinetobacter spp. isolates identified using conventional biochemical tests. The molecular technique of PCR-RFLP and sequence analysis of rpoB and 16S rRNA genes was applied for species identification. Antimicrobial susceptibility test was performed with a disk diffusion assay. RESULTS Based on 16S rRNA and rpoB genes analysis separately, most of clinical isolates can be identified with high bootstrap values. However, the identity of the isolate 555T was uncertain due to high similarity of A. grimontii and A. junii. Identification by concatenation of 16S rRNA and rpoB confirmed the identity of clinical isolates of Acenitobacer to species level confidently. Accordingly, the isolate 555T assigned as A. grimontii due to 100% similarity to A. grimontii. Moreover, this isolate showed 98.64% to A. junii. Besides, the identity of the isolates 218T and 364T was confirmed as Genomic species 3 and A. calcoaceticus respectively. So, the majority of Acinetobacter spp. isolates, were identified as: A. baumannii (131 isolates, 66%), A. calcoaceticus (9 isolates, 4.5%), and A. genomosp 16 (8 isolates, 4%). The rest of identified species showed the lower frequencies. In susceptibility test, 105 isolates (53%), presented high antibiotic resistance of 90% to ceftriaxone, piperacillin, piperacillin tazobactam, amikacin, and 81% to ciprofloxacin. CONCLUSION Sequence analysis of the 16S rRNA and rpoB spacer simultaneously was able to do identification of Acinetobacter spp. to species level. A.baumannii was identified as the most prevalent species with high antibiotic resistance. Other species showed lower frequencies ranged from 4 to 9 strains

Identification of Mycobacterium species isolated from patients using high-performance liquid chromatography in Tehran during 2014-2015

Background: Non-tuberculous mycobacteria (NTM) are defined as mycobacteria other than Mycobacterium tuberculosis (MTB), which do not cause tuberculosis or leprosy. Early and precise diagnosis of NTM is particularly important for the correct epidemiological control and specific treatments. The aim of this study was to identify the mycobacterium species isolated from patients referred to hospitals in Tehran using the high-performance liquid chromatography (HPLC) method. Materials and Methods: In this cross-sectional descriptive study, a collection of isolates (n=20) was obtained from clinical specimens submitted to the Masoud Laboratory in Tehran, Iran, during 2014-2015. The strains were isolated from sputum, urine, blood, and various sterile body fluid specimens. Chromatography was conducted at a flow rate with a curvilinear gradient of methanol and methylene chloride, beginning at 98 methanol containing 2 methylene chloride and ending at 35 methanol contained in 65 methylene chloride. Results: From a total of 20 clinical isolates, 8 isolates (40) were identified as Mycobacterium abscessus, 6 isolates (30) M. tuberculosis, 3 isolates (15) M. intracellulare and 3 isolates (15) M. fortuitum. Conclusion: For the proper treatment, rapid differentiation between MTB and NTM should be performed in persons who are diagnosed with or are suspected of having infectious TB disease. So, the HPLC method can be suggested as a cost-effective, specific and reliable method for rapid identification of MTB and differentiation of NTM strain from positive cultures isolated from clinical specimens

Application of bacteriophage cocktails for reducing the bacterial load of nosocomial pathogens in hospital wastewater

Background and Objectives: In the third world and developing countries, hospital sewage is mixed with municipal wastewater. The treated effluent contains dangerous bacteria released into the environment and used in the irrigation of agricultural products, and eventually these bacteria may endanger the human health through foods. Antibiotic-resistant bacteria are mostly found in hospital wastewater. In water and wastewater treatment plants, large amounts of toxic and polluting substances are removed and destroyed, but this process does not eliminate bacteria. Materials and Methods: Wastewater samples from 22 hospitals in Iran were collected and in the meantime specific phages (against drug-resistant pathogenic bacteria) extracted using the bilayer agar technique. Phage amplification was performed by employing a fermenter after phage identification. Amplified phages were added to the primary sedimentation pond using New-Brunwick biofermenter BioFlo/Celligen®115 and the bacterial count was evaluated for the desired bacteria. Results: Our phage cocktail was able to reduce 99.8%, 99.4%, 99.5%, 99.8%, 99.7%, 99.8%, 99.6% and 99.9% of E. coli, E. faecium, E. faecalis, K. pneumoniae, A. baumannii, P. aeruginosa, S. maltophilia and S. aureus counts respectively. Conclusion: The application of phage cocktails can remarkably help improve personal hygiene, the environment, and the optimization of surface wate

Phage Therapy for Mycobacterium Abscessus and Strategies to Improve Outcomes

Members of Mycobacterium abscessus complex are known for causing severe, chronic infections. Members of M. abscessus are a new “antibiotic nightmare” as one of the most resistant organisms to chemotherapeutic agents. Treatment of these infections is challenging due to the either intrinsic or acquired resistance of the M. abscessus complex to the available antibiotics. Recently, successful phage therapy with a cocktail of three phages (one natural lytic phage and two engineered phages) every 12 h for at least 32 weeks has been reported against a severe case of the disseminated M. abscessus subsp. massiliense infection, which underlines the high value of phages against drug-resistant superbugs. This report also highlighted the limitations of phage therapy, such as the absence of lytic phages with a broad host-range against all strains and subspecies of the M. abscessus complex and also the risk of phage resistant bacteria over treatment. Cutting-edge genomic technologies have facilitated the development of engineered phages for therapeutic purposes by introducing new desirable properties, changing host-range and arming the phages with additional killing genes. Here, we review the available literature and suggest new potential solutions based on the progress in phage engineering that can help to overcome the present limitations of M. abscessus treatment

Phage Therapy for Mycobacterium Abscessus and Strategies to Improve Outcomes

Members of complex are known for causing severe, chronic infections. Members of are a new "antibiotic nightmare" as one of the most resistant organisms to chemotherapeutic agents. Treatment of these infections is challenging due to the either intrinsic or acquired resistance of the complex to the available antibiotics. Recently, successful phage therapy with a cocktail of three phages (one natural lytic phage and two engineered phages) every 12 h for at least 32 weeks has been reported against a severe case of the disseminated subsp. infection, which underlines the high value of phages against drug-resistant superbugs. This report also highlighted the limitations of phage therapy, such as the absence of lytic phages with a broad host-range against all strains and subspecies of the complex and also the risk of phage resistant bacteria over treatment. Cutting-edge genomic technologies have facilitated the development of engineered phages for therapeutic purposes by introducing new desirable properties, changing host-range and arming the phages with additional killing genes. Here, we review the available literature and suggest new potential solutions based on the progress in phage engineering that can help to overcome the present limitations of treatment

Plague in Iran: its history and current status

OBJECTIVES: Plague remains a public health concern worldwide, particularly in old foci. Multiple epidemics of this disease have been recorded throughout the history of Iran. Despite the long-standing history of human plague in Iran, it remains difficult to obtain an accurate overview of the history and current status of plague in Iran. METHODS: In this review, available data and reports on cases and outbreaks of human plague in the past and present in Iran and in neighboring countries were collected, and information was compiled regarding when, where, and how many cases occurred. RESULTS: This paper considers the history of plague in Persia (the predecessor of today’s Iran) and has a brief review of plague in countries in the World Health Organization Eastern Mediterranean Region, including a range of countries in the Middle East and North Africa. CONCLUSIONS: Since Iran has experienced outbreaks of plague for several centuries, neighboring countries have reported the disease in recent years, the disease can be silent for decades, and the circulation of Yersinia pestis has been reported among rodents and dogs in western Iran, more attention should be paid to disease monitoring in areas with previously reported human cases and in high-risk regions with previous epizootic and enzootic activity