Salmonella in Pig Farms and on Pig Meat in Suriname.

Salmonella is one of the most important food borne zoonotic pathogens. While mainly associated with poultry, it has also been associated with pigs. Compared to the high-income countries, there is much less known on the prevalence of Salmonella in low- and middle-income countries, especially in the Caribbean area. Therefore, we investigated the prevalence of Salmonella in pigs and pig meat in Suriname. A total of 53 farms and 53 meat samples were included, and Salmonella was isolated using standard protocols. Strains were subjected to whole genome sequencing. No Salmonella was found on pig meat. Five farms were found to be positive for Salmonella, and a total of eight different strains were obtained. Serotypes were S. Anatum (n = 1), S. Ohio (n = 2), a monophasic variant of S. Typhimurium (n = 3), one S. Brandenburg, and one S. Javaniana. The monophasic variant of S. Typhimurium belonged to the ST34 pandemic clone, and the three strains were very similar. A few resistance genes, located on mobile genetic elements, were found. Several plasmids were detected, though only one was carrying resistance genes. This is the first study on the prevalence of Salmonella in pigs in the Caribbean and that used whole genome sequencing for characterization. The strains were rather susceptible. Local comparison of similar serotypes showed a mainly clonal spread of certain serotypes.

One Health Genomic Study of Human and Animal Klebsiella pneumoniae Isolated at Diagnostic Laboratories on a Small Caribbean Island.

Klebsiella pneumoniae causes a variety of infections in both humans and animals. In this study, we characterised the genomes of human and animal isolates from two diagnostic laboratories on St. Kitts, a small Caribbean island inhabited by a large population of vervet monkeys. In view of the increased chances of direct or indirect contact with humans and other animal species, we used the One Health approach to assess transmission of K. pneumoniae across host species by sequencing 82 presumptive K. pneumoniae clinical isolates from humans (n = 51), vervets (n = 21), horses (n = 5), dogs (n = 4) and a cat (n = 1). Whole genome sequencing (WGS) was carried out using Illumina technology. De novo assembly was performed in CLC Genomics Workbench v.11.0. Single nucleotide polymorphisms were detected using NASP followed by phylogenetic analysis using IQ-TREE. Virulence and antimicrobial resistance gene contents were analysed using the Kleborate and CGE pipelines. WGS-based analysis showed that 72 isolates were K. pneumoniae sensu stricto and five K. quasipneumoniae and five K. variicola. K. pneumoniae isolates belonged to 35 sequence types (ST), three of which were occasionally shared between humans and animals: ST23, ST37 and ST307. The ST23 strains from vervets formed a separate cluster amongst publicly available sequenced ST23 strains, indicating the presence of a specific vervet sublineage. Animal strains harbored fewer resistance genes and displayed distinct virulence traits that appeared to be host-specific in vervet isolates. Our results show that K. pneumoniae infections on this Caribbean island are usually caused by host-specific lineages.

High Prevalence of USA300 Among Clinical Isolates of Methicillin-Resistant Staphylococcus aureus on St. Kitts and Nevis, West Indies.

Limited information is available on antimicrobial susceptibility and clonal distribution of Staphylococcus aureus in the Caribbean region. The aims of this study were to determine the prevalence of antimicrobial resistance among S. aureus isolates and to reveal the frequency and population structure of methicillin-resistant S. aureus (MRSA) in St. Kitts and Nevis, a small island country in the West Indies. A total of 152 S. aureus isolates were collected from consecutive samples submitted to the clinical microbiology laboratory of the main referral hospital from March 2017 to January 2018. Samples came from all units in the hospital and a small number came from external submissions, and comprised a total of 119 clinical specimens and 33 nasal swabs collected from staff and patients. All S. aureus isolates were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Minimal Inhibitory Concentrations (MICs) of clinically relevant antimicrobials were determined by broth microdilution, and diversity of MRSA isolates was assessed by whole genome sequencing (WGS) analysis. MRSA accounted for 45% (69/152) of the isolates. The highest rates of resistance to non-ß-lactam agents were observed for erythromycin (55%), moxifloxacin (41%), and levofloxacin (40%), whereas resistance to the other drugs tested was ≤6%. All isolates were susceptible to ceftaroline, linezolid, teicoplanin, telavancin, and vancomycin. WGS-based multilocus sequence typing (MLST) showed that approximately 88% of the MRSA isolates belonged to ST8. Phylogenetic analysis on 801 single-nucleotide polymorphisms (SNPs) identified among the MRSA ST8 isolates indicates a large degree of genetic diversity. However, all ST8 strains clustered within the distinct clade that defines the USA300 North American Epidemic lineage (Panton-Valentine Leukocidin (PVL) +, arginine catabolic mobile element (ACME) +, Staphylococcal cassettes chromosome mec IVa (SCCmec IVa)). Our data show high levels of methicillin, macrolide, and fluoroquinolone resistance among S. aureus on St. Kitts and Nevis. The USA300 North American epidemic lineage is responsible for the vast majority of MRSA infections on this Caribbean island.

Improved Broth Microdilution Method for Antimicrobial Susceptibility Testing of Francisella Noatunensis Orientalis.

In this project we optimized a minimal inhibitory concentration testing protocol for Francisella noatunensis orientalis. Thirty-three F. noatunensis orientalis isolates recovered from different fish species and locations were tested, and Escherichia coli ATCC 25922 was used as a quality control reference strain. A modified cation-adjusted Mueller Hinton broth supplemented with 2% IsoVitalex and 0.1% glucose (MMH) was tested at a pH of 6.4 ± 0.1, 7.1 ± 0.1, and 7.3 ± 0.1. Growth curves generated for F. noatunensis orientalis indicated that MMH at a pH of 6.4 ± 0.1 provided optimal growth. There were no significant differences in the growth curves obtained from isolates recovered from different fish species or from fresh or marine water. The pH of 6.4 ± 0.1 in the MMH media interfered with the inhibitory properties of the potentiated sulfonamides (ormetoprim-sulfadimethoxine and trimethoprim-sulfamethoxazole) when using the E. coli ATCC reference strain. Minimal inhibitory concentrations of eight antimicrobials (gentamicin, enrofloxacin, ampicillin, oxytetracycline, erythromycin, florfenicol, flumequine, and oxolinic acid) were similar for all F. noatunensis orientalis isolates. The in vitro susceptibility data provided here can provide a baseline for monitoring the development of antimicrobial resistance among F. noatunensis orientalis isolates, as well as provide valuable data in the development of potential therapeutics. Received October 27, 2015; accepted April 13, 2016.

Biofilm formation of Francisella noatunensis subsp. orientalis.

Francisella noatunensis subsp. orientalis (Fno) is an emergent fish pathogen in both marine and fresh water environments. The bacterium is suspected to persist in the environment even without the presence of a suitable fish host. In the present study, the influence of different abiotic factors such as salinity and temperature were used to study the biofilm formation of different isolates of Fno including intracellular growth loci C (iglC) and pathogenicity determinant protein A (pdpA) knockout strains. Finally, we compared the susceptibility of planktonic and biofilm to three disinfectants used in the aquaculture and ornamental fish industry, namely Virkon(®), bleach and hydrogen peroxide. The data indicates that Fno is capable of producing biofilms within 24 h where both salinity as well as temperature plays a role in the growth and biofilm formation of Fno. Mutations in the iglC or pdpA, both known virulence factors, do not appear to affect the capacity of Fno to produce biofilms, and the minimum inhibitory concentration, and minimum biocidal concentration for the three disinfectants were lower than the minimum biofilm eradication concentration values. This information needs to be taken into account if trying to eradicate the pathogen from aquaculture facilities or aquariums.

Resistance of Klebsiella pneumoniae to the innate immune system of African green monkeys.

In recent years, an emergent Klebsiella pneumoniae hypermucoviscosity (HMV) phenotype has been associated with increased invasiveness and pathogenicity in primates. In this project, bacteria recovered from infected African green monkeys (AGM) (Chlorocebus aethiops sabaeus) were screened for HMV phenotype, and were compared to non-HMV isolates in in vitro, serum, and oxidative-mediated killing assays. Complement-mediated killing was assessed utilizing freshly collected serum from healthy AGM. Oxidative-mediated killing was investigated utilizing sodium hypochlorite and hydrogen peroxide. Compared to non-HMV isolates, HMV isolates were more resistant to serum-mediated and oxidative killing (p<0.05). Phagocytosis resistance was evaluated using AGM peripheral blood monocytes (PBMC), and results indicated that non-HMV isolates associated with the AGM PBMC to a greater extent than HMV isolates (p<0.001). Measurement of lactate dehydrogenase release showed that HMV isolates were more cytotoxic to AGM PBMC than non-HMV isolates (p<0.001). Thus, the hypermucoid phenotype appears to be an important virulence factor that promotes evasion of innate immune defenses.