Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in broilers and workers at 'pluck shops' in Trinidad.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is a cause of zoonotic infections in many countries. People with occupational contact with food animal production are at risk of colonization. The aim of this study was to determine the prevalence of MRSA and their frequency of resistance to other antimicrobial agents from broilers and workers at the 'pluck shops' in Trinidad. For isolation of MRSA, choanal, cloacal and pharyngeal swabs taken from broilers and nasal swabs from humans were enriched then plated on CHROMagar MRSA and Brilliance MRSA. MRSA was confirmed using the PBP2a test kit, resistance to oxacillin and cefoxitin and polymerase chain reaction (PCR) for the mecA gene. Antimicrobial resistance of the MRSA isolates to 16 antimicrobial agents was determined using the disc diffusion method. Of the 287 broilers and 47 humans sampled, MRSA was isolated at a frequency of 2 (0.7%) and 0 (0.0%) respectively. All the MRSA isolates exhibited resistance to one or more of the 16 antimicrobial agents. The study demonstrated that broilers at 'pluck shops' in Trinidad harbor MRSA. This is the first isolation of MRSA from poultry in Trinidad, West Indies, and this finding is of public health significance since occupational exposure of humans can lead to increased risk of acquiring MRSA infections.

Combination of cysteine- and oligomerization domain-mediated protein immobilization on a surface plasmon resonance (SPR) gold chip surface.

Here we report an effective method for protein immobilization on a surface plasmon resonance (SPR) gold chip, describing the combination of cysteine- and oligomerization domain-mediated immobilization of enhanced green fluorescent protein (EGFP) as a model protein for the purpose of orientation-controlled surface density packing. In order to facilitate the oligomerization of EGFP, the dimeric and trimeric constructs derived from GCN4- leucine zipper domain were chosen for multimeric EGFP assembly. For orientation-controlled immobilization of the protein, EGFP modified with cysteine residues showing excellent orientation on a gold chip was used as a starting protein, as previously reported in our earlier study (Anal. Chem., 2007, 79, 2680-2687). Constructs of EGFP with oligomerization domains were genetically engineered, and corresponding fusion proteins were purified, applied to a gold chip, and then analyzed under SPR. The immobilized EGFP density on a gold chip increased according to the states of protein oligomerization, as dimeric and trimeric EGFPs displayed better adsorption capability than monomeric and dimeric forms, respectively. Fluorescence measurement corroborated the SPR results. Taken together, our findings indicated that the combination of cysteine- and oligomerization domain-mediated immobilization of protein could be used in SPR biosensor applications, allowing for an excellent orientation and high surface density simultaneously.

Effects of orange juice pH on survival, urease activity and DNA profiles of Yersinia enterocolitica and Yersinia pseudotuberculosis stored at 4 degree C.

The objective of this study was to determine the survival, growth rate and possible cellular adaptation mechanisms of Y. pseudotuberculosis and Y. enterocolitica in orange juice under different pH conditions. Yersinia was inoculated in orange juice with adjusted pH levels of 3.9, 4.0, and 7.0 and stored at 4 C for 3, 24, 72 and 168 hours (h). The inter-and intra-species variation is significant to the pH and time of incubation variables (p<0.05). At 3.9 pH the CFU (colony forming units) count decreased significantly.At pH 3.9 and 4.0, Y. enterocolitica and Y. pseudotuberculosis survived for at least 30 days and 15 days, respectively. Yersinia that survived under low pH in orange juice revealed enhanced urease activity within 12 h of incubation. The attachment gene (ail) could not be detected by PCR in Y. enterocolitica from undiluted sample incubated for 24 h or longer. Moreover, the FesI-restriction profile was altered when Y. pseudotuberculosis was stored at pH 4.0 orange juice for 7 days. These results indicate that Yersinia could survive and grow at low pH and the survival mechanisms could also enable the bacteria to survive the stomach pH barrier to cause enteric infection.

Prevalence of methicillin resistant Staphylococcus aureus (MRSA) in pigs and workers at abattoirs in Trinidad and Tobago.

INTRODUCTION: Methicillin resistant Staphylococcus aureus (MRSA), a major cause of zoonotic infections, has emerged globally in livestock, particularly pigs. People with occupational contact with food producing animals are at high risk of colonization. The aim of this study was to determine the prevalence of MRSA in pigs and abattoir workers throughout Trinidad and Tobago as well as their resistance to other antimicrobial agents. METHODOLOGY: Nasal and skin behind the ear swabs from pigs and nasal swabs from humans were enriched in Mueller Hinton broth with 6.5% sodium chloride, followed by phenol red mannitol broth with 75 mg/L aztreonam and 5 mg/L ceftizoxime. The enriched sample was then plated on both CHROMagar MRSA and Brilliance MRSA. All incubation was at 37ºC for approximately 24 h. Suspect MRSA isolates were confirmed as MRSA using the Penicillin-Binding Protein (PBP2a) test kit and polymerase chain reaction (PCR) to detect the mecA gene. Resistance of the S. aureus and MRSA isolates to 16 antimicrobial agents was determined using the disc diffusion method. RESULTS: Of the 929 pigs and 44 humans sampled, MRSA strains were isolated at a frequency of 0.9% (8/929) and 2.3% (1/44) respectively. All isolates exhibited resistance to one or more of the 16 antimicrobial agents. CONCLUSIONS: The study demonstrated that pigs and workers at slaughter houses in Trinidad and Tobago harbour multidrug resistance S. aureus and MRSA. This is of public health significance as occupational exposure of humans can lead to an increased risk of infection and therapeutic failure.

Antimicrobial Resistance of Salmonella Isolates Recovered from Chickens Sold at Retail Outlets in Trinidad.

This study determined the frequency of resistance of 135 isolates of Salmonella, including 15 serotypes recovered from chickens purchased from retail outlets (cottage processors and supermarkets) across Trinidad. Resistance to 16 antimicrobial agents was determined by using the disk diffusion method. Resistance among the isolates was related to the type of retail outlet, location of outlets, type of sample, and isolate serotype. Overall, all isolates exhibited resistance to one or more of the 16 antimicrobial agents tested. All isolates were sensitive to cefoxitin and norfloxacin, with the overall frequency of resistance ranging from 1.1% (sulfamethoxazole-trimethoprim) to 100.0% (ceftiofur and doxycycline). The frequency of resistance to tetracycline, ampicillin, ceftriaxone, amoxycillin-clavulanic acid, and chloramphenicol was significantly ( P < 0.05) higher in isolates recovered from cottage processor outlets compared with those from supermarkets. The frequency of resistance to antimicrobial agents was significantly different only to kanamycin ( P = 0.046) and enrofloxacin ( P = 0.000) across seven counties in Trinidad). Regarding sample presentation (whole versus parts), the frequency of resistance was only significantly higher to gentamicin ( P = 0.039) for chicken part isolates from cottage processor and to only tetracycline ( P = 0.034) for isolates from whole carcasses from supermarkets. All the 135 Salmonella isolates exhibited multidrug resistance patterns. The high frequency of resistance to seven antimicrobial agents (erythromycin, streptomycin, ceftiofur, doxycycline, kanamycin, tetracycline, and ampicillin), some used in the poultry industry, coupled with the occurrence of multidrug resistance, may have potential therapeutic implications for broiler farmers in Trinidad.

Prevalence and serotypes of Salmonella spp. on chickens sold at retail outlets in Trinidad.

BACKGROUND: This cross-sectional study determined the prevalence of Salmonella spp. and their serotypes on dressed chicken sold at retail outlets in Trinidad. The study also investigated the risk factors for contamination of dressed carcasses by Salmonella spp. at cottage poultry processor outlets where chickens are slaughtered and processed for sale. METHODS: A total of 133 dressed, whole chickens and 87 chicken parts from 44 cottage poultry processors and 36 dressed, whole chickens and 194 chicken parts from 46 supermarket outlets were randomly collected throughout the country. Isolation and identification of Salmonella spp. were performed using standard bacteriological techniques. Serotyping was performed by a regional reference laboratory. RESULTS: The prevalence of Salmonella spp. in chicken carcasses sampled from cottage poultry processors and supermarkets was 20.5% and 8.3% respectively (p <0.001). The frequency of isolation of Salmonella spp. at cottage poultry processors was 22.4%, 23.0%, 7.1%, and 10.0% for non-chilled whole chicken, non-chilled chicken parts, chilled whole chicken and chilled chicken parts respectively. Fresh, non-chilled chicken (22.6%) yielded a higher frequency of isolation of Salmonella spp. than chilled chickens (8.3%). For supermarket samples, the frequency of isolation of Salmonella spp. was 19.0%, 8.1%, 0.0% and 7.6% for chilled whole chickens, chill chicken parts, frozen whole chicken and frozen chicken parts respectively. The swab method of sampling yielded a statistically significantly (p = 0.029) higher frequency (3.2%) of Salmonella spp. than the rinse method (1.6%). The predominant serotypes isolated were Kentucky (30.9%) and Javiana (22.7%). Use of chilled water-bath to cool carcasses was the only risk factor significantly (p = 0.044) associated with isolation of Salmonella spp. CONCLUSION: Raw chicken carcasses purchased from cottage poultry processors pose a significantly higher risk of contamination with Salmonella spp. than those sold at supermarkets.

Simultaneous Detection of Multiple Salmonella Serovars from Milk and Chicken Meat by Real-Time PCR Using Unique Genomic Target Regions.

A novel genomic and plasmid target-based PCR platform was developed for the detection of Salmonella serovars Heidelberg, Dublin, Hadar, Kentucky, and Enteritidis. Unique genome loci were obtained through extensive genome mining of protein databases and comparative genomic analysis of these serovars. Assays targeting Salmonella serovars Hadar, Heidelberg, Kentucky, and Dublin had 100% specificity and sensitivity, whereas those for Salmonella Enteritidis had 97% specificity and 88% sensitivity. The limits of detection for Salmonella serovars Heidelberg, Kentucky, Hadar, Enteritidis, and Dublin were 12, 9, 40, 13, and 5,280 CFU, respectively. A sensitivity assay was also performed by using milk artificially inoculated with pooled Salmonella serovars, yielding a detection limit of 1 to10 CFU/25 mL of milk samples after enrichment. The minimum DNA detected using the multiplexed TaqMan assay was 75.8 fg (1.53 × 101 genomic equivalents [GE]) for Salmonella Heidelberg, 140.8 fg (2.8 × 101 GE) for Salmonella Enteritidis, and 3.48 pg (6.96 × 102 GE) for Salmonella Dublin. PCR efficiencies were 89.8% for Salmonella Heidelberg, 94.5% for Salmonella Enteritidis, and 75.5% for Salmonella Dublin. Four types of 30 pasteurized milk samples were tested negative by culture techniques and with a genus-specific Salmonella invA gene PCR assay. Among 30 chicken samples similarly tested, 12 (40%) were positive by both culture and the invA PCR. Testing of these 12 samples with the serovar-specific PCR assay detected single and mixed contamination with Salmonella Kentucky, Salmonella Enteritidis, and Salmonella Heidelberg. Five unique primers were designed and tested by multiplex conventional PCR in conjunction with the use of the multiplex TaqMan assay with three of the primers. The diagnostic assays developed in this study could be used as tools for routine detection of these five Salmonella serovars and for epidemiological investigations of foodborne disease outbreaks.

Nanoengineered Eggshell-Silver Tailored Copolyester Polymer Blend Film with Antimicrobial Properties.

In this study, the reinforcement effect of different proportions of eggshell/silver (ES-Ag) nanomaterial on the structural and antimicrobial properties of 70/30 poly(butylene-co-adipate terephthalate)/polylactic acid (PBAT/PLA) immiscible blends was investigated. The ES-Ag was synthesized using a single step ball milling process and characterized with X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM). These results confirmed the existence of silver nanoparticles (Ag NPs) in the interstitial spaces of the eggshell particles. The thin films in this study were prepared using hot melt extrusion and 3D printing for mechanical and antimicrobial testing, respectively. These films were also characterized by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), XRD, tensile testing, and antimicrobial analysis. It was found that the incorporation of ES-Ag (0.5-2.0% content) compromised the tensile properties of the blend, due to poor interaction between the matrix and the ES-Ag in the ternary systems, but thermal analysis revealed improvement in the onset of degradation temperature and char yield at 500 °C. Though film toughness was better than that of PLA, the strength was lower, yet synergistic to those of PBAT and PLA. In general, the PBAT/PLA/ES-Ag ternary system had properties intermediate to those of the pure polymers. In vitro assessment of the antimicrobial activity of these films conducted on Listeria monocytogenes and Salmonella Enteritidis bacteria revealed that the blend composite films possessed bacteriostatic effects, due to the immobilized ES-Ag nanomaterials in the blend matrix. Atomic absorption spectroscopy (AAS) analysis of water and food samples exposed to the films showed that Ag NPs were not released in distilled water and chicken breast after 72 and 168 h, respectively.