Mpox Viral Lineage Analysis and Technique Development Using Next-generation Sequencing Approach.

BACKGROUND: In response to Mpox endemic and public health emergency, DCHHS aimed to develop NGS based techniques to streamline Mpox viral clade and lineage analysis. METHODS: The Mpox sequencing workflow started with DNA extraction and adapted Illumina's COVIDSeq assay using hMpox primer pools from Yale School of Public Health. Sequencing steps included cDNA amplification, tagmentation, PCR indexing, pooling libraries, sequencing on MiSeq, data analysis, and report generation. The bioinformatic analysis comprised read assembly and consensus sequence mapping to reference genomes and variant identification, and utilized pipelines including Illumina BaseSpace, NextClade, CLC Workbench, Terra.bio for data quality control (QC) and validation. RESULTS: In total, 171 mpox samples were sequenced using modified COVIDSeq workflow and QC metrics were assessed for read quality, depth, and coverage. Multiple analysis pipelines identified the West African clade IIb as the only clade during peak Mpox infection from July through October 2022. Analyses also indicated lineage B.1.2 as the dominant variant comprising the majority of Mpox viral genomes (77.7%), implying its geographical distribution in the United States. Viral sequences were uploaded to GISAID EpiPox. CONCLUSIONS: We developed NGS workflows to precisely detect and analyze mpox viral clade and lineages aiding in public health genomic surveillance.

Prevalence and phenotypic characterization of Salmonella enterica isolates from three species of wild marine turtles in Grenada, West Indies.

BACKGROUND AND AIM: Salmonella enterica causes enteric disease in mammals and may potentially be transmitted from marine turtles that shed the pathogen in the environment. Marine turtle-associated human salmonellosis is a potential public health concern in Grenada, as the island supports populations of leatherback turtles (Dermochelys coriacea), hawksbill turtles (Eretmochelys imbricata), and green turtles (Chelonia mydas) that interface with veterinarians and conservation workers, the local population, and the thousands of visitors that frequent the island yearly. To date, the prevalence of S. enterica has only been examined in a small subset of marine turtles in the Caribbean and no studies have been conducted in Grenada. The aim of this study was to quantify the prevalence of S. enterica in leatherback, hawksbill and green turtles in Grenada, characterize phenotypes and DNA profiles, and explore the potential risk to human health in the region. MATERIALS AND METHODS: A total of 102 cloacal swabs were obtained from nesting leatherback turtles and foraging hawksbill and green turtles. Samples were cultured on enrichment and selective media and isolates were phenotypically characterized using serotyping, pulsed-phase gel electrophoresis, and antibiotic susceptibility. Enrichment broths were additionally screened by polymerase chain reaction (PCR) using S. enterica-specific primers. RESULTS: S. enterica was cultured from 15/57 (26.3%) leatherback turtles, 0/28 hawksbill, and 0/17 green turtles. This included S. enterica serovars Montevideo, S. I:4,5,12:i:-, Salmonella Typhimurium, Salmonella Newport, S. I:6,7:-:-, and S. I:4,5,12:-:-. Five/15 leatherback turtles carried multiple serovars. Eight pulsotype groups were identified with multiple clustering; however, there was no clear association between pulsotype group and serotype profile. Five/71 isolates showed resistance to streptomycin or ampicillin. Twenty-one/57 leatherback turtles, 14/28 hawksbill turtles, and 8/17 green turtles tested positive for S. enterica by quantitative PCR. CONCLUSION: Nesting leatherback turtles actively shed S. enterica and poses a risk for zoonosis; however, the presence of viable pathogen in green and hawksbill species is unclear. These findings help elucidate the role of marine turtles as potential sources of zoonotic S. enterica and provide baseline data for one health research in Grenada and the wider Caribbean region.

Salmonella serovars associated with Grenadian tree boa (Corallus grenadensis) and their antimicrobial susceptibility.

Cloacal swabs from 45 Grenada bank tree boas (Corallus grenadensis) were sampled during a 12-month period (2011-2012) from the rain forests and scrublands of Grenada. Cloacal swabs were examined by enrichment and selective culture for the presence of Salmonella spp. In all, 16 (35.6%) of the snakes were positive for Salmonella, and six serovars of Salmonella were isolated. The most common serovar was Rubislaw (31.3%), the most frequent serovar recently isolated from green iguanas in Grenada, followed by serovar Braenderup (18.8%), and serovar IV:48:g,z51:- (formerly, S. Marina) (18.8%), also found in green iguanas in this country. The remaining three less frequent serovars were, IV:53:g,z51:-, I:6,7:e,h:- and IIIb:38:i:z. Antimicrobial susceptibility tests conducted by a disc diffusion method against amoxicillin-clavulanic acid, ampicillin, cefotaxime, ciprofloxacin, enrofloxacin, gentamicin, imipenem, nalidixic acid, streptomycin, tetracycline and trimethoprim-sulfamethoxazole showed that drug resistance is minimal, with intermediate susceptibility, only to streptomycin. This is the first report of isolation and antimicrobial susceptibilities of Salmonella serovars from wild Grenadian tree boas.

Identification of Helminth Parasites from Selar crumenophthalmus in Grenada, West Indies.

HIGHLIGHTS: Helminths in Selar crumenophthalmus fish were identified by PCR. Two helminth taxa were identified: Anisakis typica and an unknown acanthocephalan. Neither taxon of helminth identified is zoonotic. To our knowledge, this is the first report of either type of helminth in fish in Grenada.

Antimicrobial resistance patterns of commensal Escherichia coli isolated from feces of non-diarrheic dogs in Grenada, West Indies.

BACKGROUND AND AIM: There is currently no published information on the prevalence and antimicrobial susceptibility patterns of commensal Escherichia coli in dogs of Grenada origin. Monitoring antimicrobial resistance helps in the empirical selection of antibiotics. This study determined the occurrence of E. coli including the O157:H7 serotype in feces of non-diarrheic dogs of Grenada origin and the antibiotic resistance pattern of the E. coli isolates. MATERIALS AND METHODS: Fecal samples from 142 of the 144 (98.6%) dogs were culture positive for E. coli. Selection of up to three colonies from each of the 142 E. coli-positive samples yielded a total of 402 E. coli isolates, which were analyzed for the presence of non-sorbitol fermenting colonies, and O157-agglutination. RESULTS: Of the 402 E. coli isolates, 30 (7.5%) were non-sorbitol fermenters. However, none of the 402 isolates gave a positive reaction (O157:H7) to the E. coli O157:H7 latex kit. Antimicrobial susceptibility tests against 12 antibiotics revealed low resistance rates to all the tested antibiotics except for tetracycline (Te) (23.4%), cephalothin (CF) (13.2%), and ampicillin (AM) (7.7%). Thirty-nine out of the 402 (9.7%), E. coli isolates were resistant to two or more antibiotics of different classes. CONCLUSION: This is the first report of isolation and antimicrobial susceptibilities of commensal E. coli from non-diarrheic dogs in Grenada. Some of the isolates (39/402 isolates, 9.7%) were resistant to multiple antibiotics. This study showed that presently, dogs in Grenada should not be considered a reservoir for the E. coli O157:H7 serotype and for multiple antibiotic-resistant E. coli strains. Among the 402 E. coli isolates, the resistance rate to drugs other than Te, CF, and AM was very low.

Serovars and antimicrobial resistance of non-typhoidal Salmonella isolated from non-diarrhoeic dogs in Grenada, West Indies.

Non-typhoidal salmonellosis remains an important public health problem worldwide. Dogs may harbour Salmonella in their intestines and can easily shed Salmonella in the environment with the possibility of transmission to humans. Thus, monitoring is essential to understand the role of dogs in zoonotic transmission. The objectives of this study were to determine the shedding of Salmonella by owned, apparently healthy dogs in Grenada, West Indies, to identify the serovars, and to examine their antimicrobial susceptibility profiles. Faecal samples collected during August to October, 2016 from 144 non-diarrhoeic owned dogs were examined by enrichment and selective culture for the presence of Salmonella spp. Eight (5.6%) of the tested animals were culture positive, yielding 35 Salmonella isolates that belonged to six serovars of Salmonella enterica subspecies enterica. These were serovars Arechavaleta from two dogs, Arechavaleta and Montevideo from one dog, and Javiana, Rubislaw, Braenderup and Kiambu from one dog each. All these serovars have been reported as causes of human salmonellosis globally. Antimicrobial susceptibility tests on 35 isolates showed absence of resistance to the currently used drugs for cases of human salmonellosis, including ciprofloxacin and cefotaxime. One isolate (2.9%) was resistant to neomycin, two isolates (5.7%) showed intermediate susceptibility to neomycin, and another (2.9%) had intermediate susceptibility to tetracycline. This is the first report of isolation and antimicrobial susceptibilities of non-typhoidal Salmonella serovars from dogs in Grenada. This study shows that dogs in Grenada may be involved in the epidemiology of salmonellosis.

Campylobacter Species Isolated from Pigs in Grenada Exhibited Novel Clones: Genotypes and Antimicrobial Resistance Profiles of Sequence Types.

Infections caused by Campylobacter species pose a severe threat to public health worldwide. However, in Grenada, the occurrence and characteristics of Campylobacter in food animals, including pigs, remain mostly unknown. In this study, we identified the sequence types (STs) of Campylobacter from young healthy pigs in Grenada and compared the results with previous studies in Grenada and other countries. Antimicrobial resistance patterns and diversity of the Campylobacter clones were evaluated. Ninety-nine Campylobacter isolates (97 Campylobacter coli and 2 Campylobacter jejuni) were analyzed by multilocus sequence typing. Eighteen previously reported STs and 13 novel STs were identified. Of the 18 previously reported STs, eight STs (ST-854, -887, -1068, -1096, -1445, -1446, 1556, and -1579) have been associated with human gastroenteritis in different geographical regions. Among these 18 previously reported STs, ST-1428, -1096, -1450, and -1058 predominated and accounted for 18.2%, 14.1%, 11.1%, and 8.1% of all isolates, respectively. Of the 13 novel STs, ST-7675 predominated and accounted for 20% (4 of 20 isolates), followed by ST-7678, -7682, and -7691, each accounting for 10% (2 of 20 isolates). Antimicrobial resistance testing using Epsilometer test revealed a low resistance rate (1-3%) of all C. coli/jejuni STs to all antimicrobials except for tetracycline (1-10.1%). Some of the C. coli STs (13 STs, 24/99 isolates, 24.2%) were resistant to multiple antimicrobials. This is the first report on antimicrobial resistance and multidrug resistance patterns associated with Campylobacter STs recovered from swine in Grenada. This study showed that pigs in Grenada are not major reservoirs for STs of C. coli and C. jejuni that are associated with human gastroenteritis worldwide.

Influence of land use and climate on Salmonella carrier status in the small Indian mongoose (Herpestes auropunctatus) in Grenada, West Indies.

Invasive mammals can be important reservoirs for human pathogens. A recent study showed that 12% of mongooses carried Salmonella spp. in their large intestines. We investigated whether anthropogenic, environmental and climatic variables predicted Salmonella status in mongooses (Herpestes auropunctatus) in Grenada. Using multivariate logistic regression and contingency table analysis, we found that increased human density, decreased distance from roads, and low monthly precipitation were associated with increased probability of Salmonella carriage. Areas with higher human density likely support a higher abundance of mongooses because of greater food availability. These areas also are a likely source for infection to mongooses due to high densities of livestock and rodents shedding Salmonella. The higher probability of Salmonella carriage in mongooses during drier months and closer to roadsides is likely due to water drainage patterns and limited water availability. Although the overall prevalence of Salmonella in mongooses was moderate, the strong patterns of ecologic correlates, combined with the high density of mongooses throughout Grenada suggest that the small Indian mongoose could be a useful sentinel for Salmonella surveillance. Its affinity for human-associated habitats suggests that the small Indian mongoose is also a risk factor in the maintenance and possible spread of Salmonella species to humans and livestock in Grenada.

Prevalence and antimicrobial susceptibility of Salmonella spp. in small Indian mongooses (Herpestes auropunctatus) in Grenada, West Indies.

Intestinal samples from 156 small Indian mongooses (Herpestes auropunctatus) collected island-wide in Grenada from April 2011 to March 2013 were examined for the presence of Salmonella enterica spp. Nineteen (12%) mongooses were culture-positive for S. enterica spp. of which five serotypes were identified. Salmonella javiana and S. Montevideo were the most commonly isolated serotypes. The other serotypes isolated were S. Rubislaw, S. Panama and S. Arechavaleta. All isolates were susceptible to amoxicillin-clavulanic acid, ampicillin, cefotaxime, ceftazidime, ciprofloxacin, enrofloxacin, gentamicin, nalidixic acid, imipenem and trimethoprim-sulfamethoxazole. One isolate (S. Montevideo) showed resistance to tetracycline and intermediate resistance to streptomycin. The five isolated Salmonella serotypes are potential human pathogens suggesting that the mongoose may play a role in the epidemiology of human salmonellosis in Grenada.