Tropheryma whipplei in the environment: survey of sewage plant influxes and sewage plant workers

We studied the prevalence of Tropheryma whipplei in influxes to 46 sewage treatment plants and in stool, mouthwash fluids, and dental plaques of 64 healthy workers in those facilities and 146 disease control patients. T. whipplei was found in sewage water, in stool of healthy individuals, and significantly more often in stool of workers exposed to sewage water.     

Tropheryma whipplei: a common bacterium in rural Senegal

Background

Tropheryma whipplei is known as the cause of Whipple''s disease, but it is also an emerging pathogen, detected in stool, that causes various chronic localized infections without histological digestive involvement and is associated with acute infections, including gastroenteritis and bacteremia.

Methods/Principal Findings

We conducted a study in 2008 and 2009 using 497 non-diarrheic and diarrheic stool samples, 370 saliva samples, 454 sera samples and 105 samples obtained from water samples in two rural Sine-Saloum villages (Dielmo and Ndiop) in Senegal. The presence of T. whipplei was investigated by using specific quantitative PCR. Genotyping was performed on positive samples. A serological analysis by western blotting was performed to determine the seroprevalence and to detect seroconversion. Overall, T. whipplei was identified in 31.2% of the stool samples (139/446) and 3.5% of the saliva samples (13/370) obtained from healthy subjects. The carriage in the stool specimens was significantly (p<10⁻³) higher in children who were between 0 and 4 years old (60/80, 75%) compared to samples obtained from individuals who were between 5 to 10 years old (36/119, 30.2%) or between 11 and 99 years old (43/247, 17.4%). The carriage in the stool was also significantly more common (p = 0.015) in subjects with diarrhea (25/51, 49%). We identified 22 genotypes, 16 of which were new. Only one genotype (#53) was common to both villages. Among the specific genotypes, one (#52) was epidemic in Dielmo (15/28, 53.4%, p<10⁻³) and another (#49) in Ndiop (27.6%, p = 0.002). The overall seroprevalence was estimated at 72.8% (291/400). Seroconversion was detected in 66.7% (18/27) of children for whom PCR became positive in stools between 2008 and 2009.

Conclusions/Significance

T. whipplei is a common bacterium in the Sine-Saloum area of rural Senegal that is contracted early in childhood. Epidemic genotypes suggest a human transmission of the bacterium.     

Persistence, Transmission, and Virulence Characteristics of Aeromonas Strains in a Duckweed Aquaculture-Based Hospital Sewage Water Recycling Plant in Bangladesh

The persistence and transmission of Aeromonas in a duckweed aquaculture-based hospital sewage water treatment plant in Bangladesh was studied. A total of 670 samples from different sites of the hospital sewage water treatment plant, from feces of hospitalized children suffering from diarrhea, from environmental control ponds, and from feces of healthy humans were collected over a period of three years. In total, 1,315 presumptive Aeromonas isolates were biochemically typed by the PhenePlate rapid screening system (PhP-AE). A selection of 90 representative isolates was further analyzed with PhenePlate (PhP) extended typing (PhP-48), fatty acid methyl ester analysis, and amplified fragment length polymorphism (AFLP) fingerprinting. In addition, the prevalence of the putative virulence factors hemolysin and cytotoxin and the presence of the cytolytic enterotoxin gene (AHCYTOEN) were analyzed. Aeromonas was found at all sites of the treatment plant, in 40% of the samples from environmental control ponds, in 8.5% of the samples from hospitalized children suffering from diarrhea, and in 3.5% of samples from healthy humans. A significantly high number of Aeromonas bacteria was found in duckweed, which indicates that duckweed may serve as a reservoir for these bacteria. PhP-AE typing allowed identification of more than 192 distinct PhP types, of which 18 major PhP types (MTs) were found in multiple sites and during several occasions. AFLP fingerprinting revealed the prevalence of genotypically indistinguishable Aeromonas isolates among certain PhP MTs recovered from different sampling occasions and/or at multiple sites. Hemolytic and cytotoxic activities were observed in 43% of the tested strains, whereas 29% possessed the cytolytic enterotoxin gene AHCYTOEN. Collectively, two specific MTs associated with diarrhea were shown to exhibit high cytotoxicity. Furthermore, all tested isolates of these major types were positive for the cytolytic enterotoxin gene. In conclusion, our data indicate that certain phenotypically and genotypically stable clonal lineages of Aeromonas have persisted in the treatment system for a prolonged period and might spread from the hospitalized children suffering from diarrhea to fish produced for human consumption through the sewage water treatment system.     

High Prevalence of Vancomycin-Resistant Enterococci in Swedish Sewage

In Europe the use of the growth promoter avoparcin is considered to have selected for vancomycin-resistant enterococci (VRE). Sweden ceased using avoparcin in 1986, and only occasional cases of VRE from hospitals have been reported since 1995. Within the framework of a European study, samples from urban raw sewage, treated sewage, surface water, and hospital sewage in Sweden (n = 118) were screened for VRE. Surprisingly, VRE were isolated from 21 of 35 untreated sewage samples (60%), from 5 of 14 hospital sewage samples (36%), from 6 of 32 treated sewage samples (19%), and from 1 of 37 surface water samples. Thirty-five isolates from 33 samples were further characterized by geno- and phenotyping, MIC determination, and PCR analysis. Most isolates (30 of 35) carried the vanA gene, and the majority (24 of 35) of the isolates were Enterococcus faecium. Most of the VRE were multiresistant. The typing revealed high diversity of the isolates. However, one major cluster with seven identical or similar isolates was found. These isolates came from three different sewage treatment plants and were collected at different occasions during 1 year. All VRE from hospital sewage originated from one of the two hospitals studied. That hospital also had vancomycin consumption that was 10-fold that of the other. We conclude that VRE were commonly found in sewage samples in Sweden. The origin might be both healthy individuals and individuals in hospitals. Possibly, antimicrobial drugs or chemicals released into the sewage system may sustain VRE in the system.     

High Prevalence of Tropheryma whipplei in Lao Kindergarten Children

Background

Tropheryma whipplei is a bacterium commonly found in feces of young children in Africa, but with no data from Asia. We estimated the prevalence of T. whipplei carriage in feces of children in Lao PDR (Laos).

Methods/Principal Findings

Using specific quantitative real-time PCR, followed by genotyping for each positive specimen, we estimated the prevalence of T. whipplei in 113 feces from 106 children in Vientiane, the Lao PDR (Laos). T. whipplei was detected in 48% (51/106) of children. Those aged ≤4 years were significantly less frequently positive (17/52, 33%) than older children (34/54, 63%; p< 0.001). Positive samples were genotyped. Eight genotypes were detected including 7 specific to Laos. Genotype 2, previously detected in Europe, was circulating (21% of positive children) in 2 kindergartens (Chompet and Akad). Genotypes 136 and 138 were specific to Chompet (21% and 15.8%, respectively) whereas genotype 139 was specific to Akad (10.55%).

Conclusions/Significance

T. whipplei is a widely distributed bacterium, highly prevalent in feces of healthy children in Laos. Further research is needed to identify the public health significance of this finding.     

Distribution of Coliphages Against Four <Emphasis Type="Italic">E. Coli</Emphasis> Virotypes in Hospital Originated Sewage Sample and a Sewage Treatment Plant in Bangladesh

The distribution of coliphages infecting different Escherichia coli virotypes (EHEC, EIEC, EPEC, ETEC) and an avirulent strain (K-12) in sewage system of a hospital and a sewage treatment plant (STP) was investigated by culture-based agar overlay methods. Coliphages were found in all the samples except stool dumping site in the sewage system of the hospital and lagoon of the STP. Bacteriophage count (pfu/ml) infecting E. coli strains showed the following ascending pattern (EHEC < EIEC < EPEC < ETEC < E coli K-12) in all the collected samples except one. Phages capable of infecting avirulent E. coli K-12 strains were present in the highest number among all the examined locations. Phages specific for E. coli K-12 presented high diversity in plaque size on the bacterial lawn. Virulent E. coli specific coliphages rarely produced plaques with diameter of 1–2 mm or over. Conventional agar overlay method was found to be not satisfactory for phage community analysis from primary stool dumping site of the hospital, probably due to the presence of high concentration of antimicrobial substances. The gradual decrease seen in the five groups of coliphage quantity with the ongoing treatment process and then the absolute absence of coliphages in the outlet of the examined treatment plant is indicative of the usefulness of the treatment processes practiced there.     

Occurrence of Cytophagas in Sewage Plants

With the application of plate count methods and of the KOH-flexirubin test, bacteria belonging to the Cytophaga group were proved to occur regularly in samples from biological sewage treatment facilities. Generally, the percentage of Cytophaga colonies of the total heterotrophic colonies was lowest in the inflow sewage water as compared with the values found in activated sludge, trickling filter, and effluent samples. During an observation period of 16 months, the highest percentages of cytophagas were found in winter samples from activated sludge and trickling filters. Furthermore, cytophagas were shown to have high percentages of the bacteria lytic to polymeric substrates such as cellulose, chitin, dextran, pectin, xylan, and gelatin. Thus, it is suggested that cytophagas may contribute to sewage purification, especially at cold temperatures and by polymer breakdown. Cytophaga strains isolated were shown to have gliding motility, flexirubin pigmentation, and a low guanine plus cytosine base ratio in common. The strains were roughly subdivided into a spreading, a nonspreading, and a cellulolytic group.     

The Microbiota and Abundance of the Class 1 Integron-Integrase Gene in Tropical Sewage Treatment Plant Influent and Activated Sludge

Bacteria are assumed to efficiently remove organic pollutants from sewage in sewage treatment plants, where antibiotic-resistance genes can move between species via mobile genetic elements known as integrons. Nevertheless, few studies have addressed bacterial diversity and class 1 integron abundance in tropical sewage. Here, we describe the extant microbiota, using V6 tag sequencing, and quantify the class 1 integron-integrase gene (intI1) in raw sewage (RS) and activated sludge (AS). The analysis of 1,174,486 quality-filtered reads obtained from RS and AS samples revealed complex and distinct bacterial diversity in these samples. The RS sample, with 3,074 operational taxonomic units, exhibited the highest alpha-diversity indices. Among the 25 phyla, Proteobacteria, Bacteroidetes and Firmicutes represented 85% (AS) and 92% (RS) of all reads. Increased relative abundance of Micrococcales, Myxococcales, and Sphingobacteriales and reduced pathogen abundance were noted in AS. At the genus level, differences were observed for the dominant genera Simplicispira and Diaphorobacter (AS) as well as for Enhydrobacter (RS). The activated sludge process decreased (55%) the amount of bacteria harboring the intI1 gene in the RS sample. Altogether, our results emphasize the importance of biological treatment for diminishing pathogenic bacteria and those bearing the intI1 gene that arrive at a sewage treatment plant.     

Higher Isolation of NDM-1 Producing Acinetobacter baumannii from the Sewage of the Hospitals in Beijing

Multidrug resistant microbes present in the environment are a potential public health risk. In this study, we investigate the presence of New Delhi metallo-β-lactamase 1 (NDM-1) producing bacteria in the 99 water samples in Beijing City, including river water, treated drinking water, raw water samples from the pools and sewage from 4 comprehensive hospitals. For the bla _NDM-1 positive isolate, antimicrobial susceptibility testing was further analyzed, and Pulsed Field Gel Electrophoresis (PFGE) was performed to determine the genetic relationship among the NDM-1 producing isolates from sewage and human, as well as the clinical strains without NDM-1. The results indicate that there was a higher isolation of NDM-1 producing Acinetobacter baumannii from the sewage of the hospitals, while no NDM-1 producing isolates were recovered from samples obtained from the river, drinking, or fishpond water. Surprisingly, these isolates were markedly different from the clinical isolates in drug resistance and pulsed field gel electrophoresis profiles, suggesting different evolutionary relationships. Our results showed that the hospital sewage may be one of the diffusion reservoirs of NDM-1 producing bacteria.     

Enterococcus faecalis Gene Transfer under Natural Conditions in Municipal Sewage Water Treatment Plants

The ability of Enterococcus faecalis to transfer various genetic elements under natural conditions was tested in two municipal sewage water treatment plants. Experiments in activated sludge basins of the plants were performed in a microcosm which allowed us to work under sterile conditions; experiments in anoxic sludge digestors were performed in dialysis bags. We used the following naturally occurring genetic elements: pAD1 and pIP1017 (two so-called sex pheromone plasmids with restricted host ranges, which are transferred at high rates under laboratory conditions); pIP501 (a resistance plasmid possessing a broad host range for gram-positive bacteria, which is transferred at low rates under laboratory conditions); and Tn916 (a conjugative transposon which is transferred under laboratory conditions at low rates to gram-positive bacteria and at very low rates to gram-negative bacteria). The transfer rate between different strains of E. faecalis under natural conditions was, compared to that under laboratory conditions, at least 10⁵-fold lower for the sex pheromone plasmids, at least 100-fold lower for pIP501, and at least 10-fold lower for Tn916. In no case was transfer from E. faecalis to another bacterial species detected. By determining the dependence of transfer rates for pIP1017 on bacterial concentration and extrapolating to actual concentrations in the sewage water treatment plant, we calculated that the maximum number of transfer events for the sex pheromone plasmids between different strains of E. faecalis in the municipal sewage water treatment plant of the city of Regensburg ranged from 10⁵ to 10⁸ events per 4 h, indicating that gene transfer should take place under natural conditions.     

Type I Interferon Induction Is Detrimental during Infection with the Whipple's Disease Bacterium,Tropheryma whipplei

Macrophages are the first line of defense against pathogens. Upon infection macrophages usually produce high levels of proinflammatory mediators. However, macrophages can undergo an alternate polarization leading to a permissive state. In assessing global macrophage responses to the bacterial agent of Whipple''s disease, Tropheryma whipplei, we found that T. whipplei induced M2 macrophage polarization which was compatible with bacterial replication. Surprisingly, this M2 polarization of infected macrophages was associated with apoptosis induction and a functional type I interferon (IFN) response, through IRF3 activation and STAT1 phosphorylation. Using macrophages from mice deficient for the type I IFN receptor, we found that this type I IFN response was required for T. whipplei-induced macrophage apoptosis in a JNK-dependent manner and was associated with the intracellular replication of T. whipplei independently of JNK. This study underscores the role of macrophage polarization in host responses and highlights the detrimental role of type I IFN during T. whipplei infection.     

Depletion of Cryptosporidium parvum Oocysts from Contaminated Sewage by Using Freshwater Benthic Pearl Clams (Hyriopsis schlegeli)

The freshwater benthic pearl clam, Hyriopsis schlegeli, was experimentally exposed to Cryptosporidium parvum oocysts, and it was verified that the oocysts were eliminated predominantly via the fecal route, retaining their ability to infect cultured cells (HCT-8). The total fecal oocyst elimination rate was more than 90% within 5 days after exposure to the oocysts. H. schlegeli was able to survive in the final settling pond of a sewage plant for long periods, as confirmed by its pearl production. In the light of these findings, the clam was placed in the final settling pond in a trial to test its long-term efficacy in depleting oocysts contaminating the pond water. The number of clams placed was set to ensure a theoretical oocyst removal rate of around 50%, and the turbidity and the density of feed microbes in the overflow trough water of the pond were about 35% and 40 to 60% lower, respectively, than in the control water throughout the year. It was found that the clam feces containing oocysts were sufficiently heavy for them to settle to the bottom of the pond, despite the upward water flow. From these results, we concluded that efficient depletion of oocysts in the sewage water of small or midscale sewage treatment plants can be achieved by appropriate placement of H. schlegeli clams.     

Real-Time PCR for Quantification of Giardia and Cryptosporidium in Environmental Water Samples and Sewage

The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the β-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method.     

Occurrence of Cryptosporidium Oocysts and Giardia Cysts in Sewage in Norway

Samples of sewage influent from 40 sewage treatment works (STW) throughout Norway were examined for Cryptosporidium oocysts and Giardia duodenalis cysts. Both parasites were detected frequently (80% of STW were Cryptosporidium positive; 93% of STW were Giardia positive) and at maximum concentrations of >20,000 parasites/liter. The data suggest giardiasis is more widespread, and/or occurs with greater infection intensity, than cryptosporidiosis in Norway. STW serving higher person equivalents were more likely to be positive and had higher parasite concentrations. Parasite concentrations were used to estimate the proportion of contributing populations that could be clinically infected. For Cryptosporidium, the highest estimates were up to 5 per 100,000 individuals for two populations in eastern Norway. For Giardia, the highest estimate was 40 infected per 100,000 persons (approximately five times the usual national annual average) contributing to an STW in western Norway. As this population experienced a large waterborne giardiasis outbreak 6 months after sampling, it can be speculated that regular challenge with Giardia may occur here. Most Giardia isolates in sewage influent were assemblage A, although some assemblage B isolates were detected. There was substantial heterogeneity, but most samples contained isolates similar to genotype A3. Removal efficiencies at two STW with secondary treatment processes were estimated to be approximately 50% for Cryptosporidium and >80% for Giardia. An STW with minimal treatment had negligible removal of both parasites. Many STW in Norway have minimal treatment and discharge effluent into rivers and lakes, thus, risk of contamination of water courses by Cryptosporidium and Giardia is considerable.     

Human Enteropathogen Load in Activated Sewage Sludge and Corresponding Sewage Sludge End Products

This study demonstrated a significant reduction in the concentrations of Cryptosporidium parvum and Cryptosporidium hominis oocysts, Giardia lamblia cysts, and spores of human-virulent microsporidia in dewatered and biologically stabilized sewage sludge cake end products compared to those of the respective pathogens in the corresponding samples collected during the sludge activation process.     

Triptans and troponin: a case report

We report a patient who presented with inflammatory back pain due to multisegmental spondylitis. Following a vertebral biopsy which failed to detect an infectious organism, the patient was treated with etanercept, a tumor necrosis factor (TNF)-α inhibitor, for suspected undifferentiated spondyloarthritis. The back pain worsened and the spondylitic lesions increased. Only in a vertebral rebiopsy with polymerase chain reaction (PCR) amplification of Tropheryma whipplei, the causative agent of Whipple's disease was identified. Tropheryma whipplei should be considered as a cause of spondylitis even with multisegmental involvement and in the absence of gastrointestinal symptoms. In this clinical setting, routine PCR for Tropheryma whipplei from vertebral biopsies is recommended.     

Intervening Sequence Acquired by Lateral Gene Transfer in Tropheryma whipplei Results in 23S rRNA Fragmentation

Completion of Tropheryma whipplei genome sequencing may provide insights into the evolution of the molecular mechanisms underlying the pathogenicity of this microorganism. The first postgenomic application was the successful design of a comprehensive culture medium that allows axenic growth of this bacterium, which is particularly recalcitrant to cultivation. This achievement in turn permitted analysis of T. whipplei RNA without contaminating eukaryotic nucleic acids. To obtain high-quality RNA, several extraction methods were compared, but under all conditions tested an atypical profile was observed. By using a Northern blot assay we demonstrated that an insertion sequence previously described in T. whipplei 23S rRNA is in fact an intervening sequence excised during maturation. This cleavage could involve an RNase III identified in the genome of this microorganism. Among the bacteria with a 23S rRNA insertion sequence, T. whipplei is the only gram-positive microorganism. We present phylogenetic evidence that this mobile genetic element was acquired by lateral gene transfer from another enteric bacterium.     

Detection of Small Numbers of Campylobacter jejuni and Campylobacter coli Cells in Environmental Water, Sewage, and Food Samples by a Seminested PCR Assay

A rapid and sensitive assay was developed for detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples. Water and sewage samples were filtered, and the filters were enriched overnight in a nonselective medium. The enrichment cultures were prepared for PCR by a rapid and simple procedure consisting of centrifugation, proteinase K treatment, and boiling. A seminested PCR based on specific amplification of the intergenic sequence between the two Campylobacter flagellin genes, flaA and flaB, was performed, and the PCR products were visualized by agarose gel electrophoresis. The assay allowed us to detect 3 to 15 CFU of C. jejuni per 100 ml in water samples containing a background flora consisting of up to 8,700 heterotrophic organisms per ml and 10,000 CFU of coliform bacteria per 100 ml. Dilution of the enriched cultures 1:10 with sterile broth prior to the PCR was sometimes necessary to obtain positive results. The assay was also conducted with food samples analyzed with or without overnight enrichment. As few as ≤3 CFU per g of food could be detected with samples subjected to overnight enrichment, while variable results were obtained for samples analyzed without prior enrichment. This rapid and sensitive nested PCR assay provides a useful tool for specific detection of C. jejuni or C. coli in drinking water, as well as environmental water, sewage, and food samples containing high levels of background organisms.     

New insights into Whipple’s disease and Tropheryma whipplei infections

Whipple’s disease is a rare multi-systemic disease associated with the ubiquitous environmental bacterium Tropheryma whipplei. Over the last 10 years, since the isolation of the bacterium, recent advances in medical microbiology, epidemiology and cellular biology have provided major insights into the understanding of the pathophysiology of T. whipplei infections that may result in Whipple’s disease.     

Near-Bottom Pelagic Bacteria at a Deep-Water Sewage Sludge Disposal Site

The epibenthic bacterial community at deep-ocean sewage sludge disposal site DWD-106, located approximately 106 miles (ca. 196 km) off the coast of New Jersey, was assessed for changes associated with the introduction of large amounts of sewage sludge. Mixed cultures and bacterial isolates obtained from water overlying sediment core samples collected at the deep-water (2,500 m) municipal sewage disposal site were tested for the ability to grow under in situ conditions of temperature and pressure. The responses of cultures collected at a DWD-106 station heavily impacted by sewage sludge were compared with those of samples collected from a station at the same depth which was not contaminated by sewage sludge. Significant differences were observed in the ability of mixed bacterial cultures and isolates from the two sites to grow under deep-sea pressure and temperature conditions. The levels of sludge contamination were established by enumerating Clostridium perfringens, a sewage indicator bacterium, in sediment samples from the two sites. The results of hybridization experiments in which DNAs extracted directly from the water overlying sediment core samples were used indicate that the reference site epibenthic community, the disposal site epibenthic community, and the community in a surface sludge plume share many members. Decreased culturability of reference site mixed cultures in the presence of sewage sludge was observed. Thus, the culturable portions of both the autochthonous and allochthonous bacterial communities at the disposal site may be inhibited in situ, the former by sewage sludge and the latter by high pressure and low temperature.