Isolation and molecular characterization of toxigenic Vibrio parahaemolyticus from the Kii Channel, Japan

Studies were conducted on the ecology of potentially pathogenic Vibrio parahaemolyticus in three coastal areas of Kii Channel, Tokushima, Japan. Seawater and seaweed samples were collected seasonally between June 2003 and May 2004. Total and toxigenic strains of V. parahaemolyticus were isolated using most probable number culture and colony blot hybridization. Toxigenic strains were serotyped and further characterized by random amplified polymorphic DNA (RAPD) and ribotyping. Six thousand strains of V. parahaemolyticus were isolated and 18 were found positive for tdh. V. parahaemolyticus were detected in all samples during summer and autumn, and from some samples during winter and spring. Among the toxigenic strains seven serotypes, five ribotypes and RAPD patterns were observed. Seven strains belonged to O3:K6 clone with identical ribotypes and RAPD patterns to that of a pandemic reference strain. The presence of toxigenic V. parahaemolyticus with pandemic potential might indicate a human health risk due to consumption of marine food sources.     

Genotyping of Campylobacter coli isolated from humans and retail meats using multilocus sequence typing and pulsed-field gel electrophoresis

Aims:  To determine the antimicrobial resistant profiles and clonality of Campylobacter coli isolated from clinically ill humans and retail meats.
Methods and Results:  A total of 98 C. coli isolates (20 from humans and 78 from retail meats) were phenotypically characterized. Antimicrobial susceptibility testing was done using agar dilution method for ciprofloxacin, gentamicin, erythromycin and doxycycline. Seventy C. coli isolates including humans ( n  = 20) and retail meats ( n  = 50) were genotyped by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Resistance to ciprofloxacin was found in 29% and 15% of isolates from retail meats and humans. We observed 61 PFGE profiles using two enzymes ( Sma I, Kpn I) with an Index of discrimination of 0·99, whereas MLST generated 37 sequence types. Two clonal complexes were identified with 58 (82%) C. coli isolates clustered in the ST-828 complex.
Conclusions:  Resistance to ciprofloxacin and erythromycin was identified in C. coli obtained from retail meats and ill humans. PFGE typing of C. coli isolates was more discriminatory than MLST. Grouping of C. coli isolates (82%) by MLST in ST-828 clonal complex indicates a common ancestry.
Significance and Impact of the Study:  A high frequency of resistance found to ciprofloxacin and erythromycin is concerning from food safety perspective. PFGE using single or double restriction enzymes was found to be more discriminatory than MLST for genotyping C. coli . Overall, the C. coli populations recovered from humans and retail meats were genotypically diverse.     

Genotypic and phenotypic properties of cattle-associated Campylobacter and their implications to public health in the USA

Since cattle are a major source of food and the cattle industry engages people from farms to processing plants and meat markets, it is conceivable that beef-products contaminated with Campylobacter spp. would pose a significant public health concern. To better understand the epidemiology of cattle-associated Campylobacter spp. in the USA, we characterized the prevalence, genotypic and phenotypic properties of these pathogens. Campylobacter were detected in 181 (19.2%) out of 944 fecal samples. Specifically, 71 C. jejuni, 132 C. coli, and 10 other Campylobacter spp. were identified. The prevalence of Campylobacter varied regionally and was significantly (P<0.05) higher in fecal samples collected from the South (32.8%) as compared to those from the North (14.8%), Midwest (15.83%), and East (12%). Pulsed Field Gel Electrophoresis (PFGE) analysis showed that C. jejuni and C. coli isolates were genotypically diverse and certain genotypes were shared across two or more of the geographic locations. In addition, 13 new C. jejuni and two C. coli sequence types (STs) were detected by Multi Locus Sequence Typing (MLST). C. jejuni associated with clinically human health important sequence type, ST-61 which was not previously reported in the USA, was identified in the present study. Most frequently observed clonal complexes (CC) were CC ST-21, CC ST-42, and CC ST-61, which are also common in humans. Further, the cattle associated C. jejuni strains showed varying invasion and intracellular survival capacity; however, C. coli strains showed a lower invasion and intracellular survival potential compared to C. jejuni strains. Furthermore, many cattle associated Campylobacter isolates showed resistance to several antimicrobials including ciprofloxacin, erythromycin, and gentamicin. Taken together, our results highlight the importance of cattle as a potential reservoir for clinically important Campylobacter.     

Longitudinal Study of Distributions of Similar Antimicrobial-Resistant Salmonella Serovars in Pigs and Their Environment in Two Distinct Swine Production Systems

The aim of this longitudinal study was to determine and compare the prevalences and genotypic profiles of antimicrobial-resistant (AR) Salmonella isolates from pigs reared in antimicrobial-free (ABF) and conventional production systems at farm, at slaughter, and in their environment. We collected 2,889 pig fecal and 2,122 environmental (feed, water, soil, lagoon, truck, and floor swabs) samples from 10 conventional and eight ABF longitudinal cohorts at different stages of production (farrowing, nursery, finishing) and slaughter (postevisceration, postchill, and mesenteric lymph nodes [MLN]). In addition, we collected 1,363 carcass swabs and 205 lairage and truck samples at slaughter. A total of 1,090 Salmonella isolates were recovered from the samples; these were isolated with a significantly higher prevalence in conventionally reared pigs (4.0%; n = 66) and their environment (11.7%; n = 156) than in ABF pigs (0.2%; n = 2) and their environment (0.6%; n = 5) (P < 0.001). Salmonella was isolated from all stages at slaughter, including the postchill step, in the two production systems. Salmonella prevalence was significantly higher in MLN extracted from conventional carcasses than those extracted from ABF carcasses (P < 0.001). We identified a total of 24 different serotypes, with Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Anatum, Salmonella enterica serovar Infantis, and Salmonella enterica serovar Derby being predominant. The highest frequencies of antimicrobial resistance (AR) were exhibited to tetracycline (71%), sulfisoxazole (42%), and streptomycin (17%). Multidrug resistance (resistance to ≥3 antimicrobials; MDR) was detected in 27% (n = 254) of the Salmonella isolates from the conventional system. Our study reports a low prevalence of Salmonella in both production systems in pigs on farms, while a higher prevalence was detected among the carcasses at slaughter. The dynamics of Salmonella prevalence in pigs and carcasses were reciprocated in the farm and slaughter environment, clearly indicating an exchange of this pathogen between the pigs and their surroundings. Furthermore, the phenotypic and genotypic fingerprint profile results underscore the potential role played by environmental factors in dissemination of AR Salmonella to pigs.     

Podoconiosis and Soil-Transmitted Helminths (STHs): Double Burden of Neglected Tropical Diseases in Wolaita Zone,Rural Southern Ethiopia

Background

Both podoconiosis and soil-transmitted helminth (STH) infections occur among barefoot people in areas of extreme poverty; however, their co-morbidity has not previously been investigated. We explored the overlap of STH infection and podoconiosis in Southern Ethiopia and quantified their separate and combined effects on prevalent anemia and hemoglobin levels in podoconiosis patients and health controls from the same area.

Methods and Principal Findings

A two-part comparative cross-sectional study was conducted in Wolaita zone, southern Ethiopia. Data were collected from adult patients presenting with clinically confirmed podoconiosis, and unmatched adult neighborhood controls living in the same administrative area. Information on demographic and selected lifestyle factors was collected using interviewer-administered questionnaires. Stool samples were collected and examined qualitatively using the modified formalin-ether sedimentation method. Hemoglobin level was determined using two different methods: hemoglobinometer and automated hematology analyzer. A total of 913 study subjects (677 podoconiosis patients and 236 controls) participated. The prevalence of any STH infection was 47.6% among patients and 33.1% among controls (p<0.001). The prevalence of both hookworm and Trichuris trichiura infections was significantly higher in podoconiosis patients than in controls (AOR 1.74, 95% CI 1.25 to2.42, AOR 6.53, 95% CI 2.34 to 18.22, respectively). Not wearing shoes and being a farmer remained significant independent predictors of infection with any STH. There was a significant interaction between STH infection and podoconiosis on reduction of hemoglobin level (interaction p value = 0.002).

Conclusions

Prevalence of any STH and hookworm infection was higher among podoconiosis patients than among controls. A significant reduction in hemoglobin level was observed among podoconiosis patients co-infected with hookworm and ‘non-hookworm STH’. Promotion of consistent shoe-wearing practices may have double advantages in controlling both podoconiosis and hookworm infection in the study area.     

In-Feed Use of Heavy Metal Micronutrients in U.S. Swine Production Systems and Its Role in Persistence of Multidrug-Resistant Salmonellae

The study aimed to characterize the role of heavy metal micronutrients in swine feed in emergence of heavy-metal-tolerant and multidrug-resistant Salmonella organisms. We conducted a longitudinal study in 36 swine barns over a 2-year period. The feed and fecal levels of Cu²⁺ and Zn²⁺ were measured. Salmonella was isolated at early and late finishing. MICs of copper sulfate and zinc chloride were measured using agar dilution. Antimicrobial susceptibility was tested using the Kirby-Bauer method, and 283 isolates were serotyped. We amplified pcoA and czcD genes that encode Cu²⁺ and Zn²⁺ tolerance, respectively. Of the 283 isolates, 113 (48%) showed Cu²⁺ tolerance at 24 mM and 164 (58%) showed Zn²⁺ tolerance at 8 mM. In multivariate analysis, serotype and source of isolates were significantly associated with Cu²⁺ tolerance (P < 0.001). Fecal isolates were more likely to be Cu²⁺ tolerant than those of feed origin (odds ratio [OR], 27.0; 95% confidence interval [CI], 2.8 to 250; P = 0.0042) or environmental origin (OR, 5.8), implying the significance of gastrointestinal selective pressure. Salmonella enterica serotypes Typhimurium and Heidelberg, highly significant for public health, had higher odds of having >20 mM MICs of Cu²⁺ than did “other” serotypes. More than 60% of Salmonella isolates with resistance type (R-type) AmStTeKm (32 of 53) carried pcoA; only 5% with R-type AmClStSuTe carried this gene. czcD gene carriage was significantly associated with a higher Zn²⁺ MIC (P < 0.05). The odds of having a high Zn²⁺ MIC (≥8 mM) were 14.66 times higher in isolates with R-type AmClStSuTe than in those with R-type AmStTeKm (P < 0.05). The findings demonstrate strong association between heavy metal tolerance and antimicrobial resistance, particularly among Salmonella serotypes important in public health.     

The Global One Health Paradigm: Challenges and Opportunities for Tackling Infectious Diseases at the Human,Animal, and Environment Interface in Low-Resource Settings

Zoonotic infectious diseases have been an important concern to humankind for more than 10,000 years. Today, approximately 75% of newly emerging infectious diseases (EIDs) are zoonoses that result from various anthropogenic, genetic, ecologic, socioeconomic, and climatic factors. These interrelated driving forces make it difficult to predict and to prevent zoonotic EIDs. Although significant improvements in environmental and medical surveillance, clinical diagnostic methods, and medical practices have been achieved in the recent years, zoonotic EIDs remain a major global concern, and such threats are expanding, especially in less developed regions. The current Ebola epidemic in West Africa is an extreme stark reminder of the role animal reservoirs play in public health and reinforces the urgent need for globally operationalizing a One Health approach. The complex nature of zoonotic diseases and the limited resources in developing countries are a reminder that the need for implementation of Global One Health in low-resource settings is crucial. The Veterinary Public Health and Biotechnology (VPH-Biotec) Global Consortium launched the International Congress on Pathogens at the Human-Animal Interface (ICOPHAI) in order to address important challenges and needs for capacity building. The inaugural ICOPHAI (Addis Ababa, Ethiopia, 2011) and the second congress (Porto de Galinhas, Brazil, 2013) were unique opportunities to share and discuss issues related to zoonotic infectious diseases worldwide. In addition to strong scientific reports in eight thematic areas that necessitate One Health implementation, the congress identified four key capacity-building needs: (1) development of adequate science-based risk management policies, (2) skilled-personnel capacity building, (3) accredited veterinary and public health diagnostic laboratories with a shared database, and (4) improved use of existing natural resources and implementation. The aim of this review is to highlight advances in key zoonotic disease areas and the One Health capacity needs.     

Longitudinal analysis of the temporal evolution of Acinetobacter baumannii strains in Ohio, USA, by using rapid automated typing methods

Genotyping methods are essential to understand the transmission dynamics of Acinetobacter baumannii. We examined the representative genotypes of A. baumannii at different time periods in select locations in Ohio, using two rapid automated typing methods: PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS), a form of multi-locus sequence typing (MLST), and repetitive-sequence-based-PCR (rep-PCR). Our analysis included 122 isolates from 4 referral hospital systems, in 2 urban areas of Ohio. These isolates were associated with outbreaks at 3 different time periods (1996, 2000 and 2005-2007). Type assignments of PCR/ESI-MS and rep-PCR were compared to each other and to worldwide (WW) clone types. The discriminatory power of each method was determined using the Simpson's index of diversity (DI). We observed that PCR/ESI-MS sequence type (ST) 14, corresponding to WW clone 3, predominated in 1996, whereas ST 12 and 14 co-existed in the intermediate period (2000) and ST 10 and 12, belonging to WW clone 2, predominated more recently in 2007. The shift from WW clone 3 to WW clone 2 was accompanied by an increase in carbapenem resistance. The DI was approximately 0.74 for PCR/ESI-MS, 0.88 for rep-PCR and 0.90 for the combination of both typing methods. We conclude that combining rapid automated typing methods such as PCR/ESI-MS and rep-PCR serves to optimally characterize the regional molecular epidemiology of A. baumannii. Our data also sheds light on the changing sequence types in an 11 year period in Northeast Ohio.     

Occurrence, seasonality and genetic diversity of Vibrio vulnificus in coastal seaweeds and water along the Kii Channel, Japan

Vibrio vulnificus is a ubiquitous toxigenic bacterium found in a coastal environment but little is known about its occurrence and seasonality among seaweeds, which are widely consumed as seafood in Japan. Therefore, we have observed the bacterium's abundance in seawater and seaweed samples from three areas of the Kii Channel, Japan, during June 2003 to May 2004. A total of 192 samples were collected: 24 from each source in summer, autumn, winter and spring. The samples were selectively cultivated following the most probable number (MPN) technique. Vibrio vulnificus population ranged from 0 to 10(3) MPN 100 mL(-1) seawater or 10 g seaweeds; higher counts were observed during summer. The optimum temperature, salinity and pH for the bacterium were 20-24 degrees C, 24-28 p.p.t. and 7.95-8.15, respectively. However, seaweeds always contained higher V. vulnificus than seawater. Among 280 V. vulnificus strains, detected by species-specific colony hybridization and PCR, 78, 74, 11 and 16 were from seaweeds and 46, 42, 2 and 11 were from seawater during summer, autumn, winter and spring, respectively. Ribotyping of 160 selected strains revealed a higher genotypic diversity (18 patterns) among strains from seaweeds than from seawater (10 patterns). Seaweeds can thus act as a potential habitat for V. vulnificus and are more unsafe for consumption during summer.