Foodborne Klebsiella pneumoniae: Virulence Potential, Antibiotic Resistance, and Risks to Food Safety.

ABSTRACT: Gastrointestinal carriage of Klebsiella pneumoniae is a predisposing factor for liver abscess in several Asian countries. To determine whether hypervirulent K. pneumoniae in the gut may be transmitted through food, we screened a range of raw and ready-to-eat retail food by culture and recovered K. pneumoniae in 21% (147 of 698) of samples tested. Based on PCR, no K. pneumoniae isolates carried the rmpA gene linked to community-acquired pyogenic liver abscess, providing no evidence of a link between food and liver disease. However, phenotypic resistance to multiple antibiotic classes was seen through disk diffusion tests, and carriage of genetic elements (wcaG and capsule types K1, K2, and K54) associated with increased virulence (8%, 11 of 147) was observed by PCR. Multidrug-resistant isolates were from raw vegetables, chicken or pork liver, and a ready-to-eat poultry dish; one multidrug-resistant K. pneumoniae isolate from raw bean sprouts was resistant to a third-generation cephalosporin (ceftriaxone). Although K. pneumoniae may be present in food without causing harm, we found isolates belonging to the K1 capsular serotype coexisting with the wcaG gene, one also conferring multidrug resistance. K. pneumoniae that carry antibiotic resistance genes, regardless of pathogenicity, may increase the available genetic pool of resistance along the food chain. Hygienic food handling practices are necessary to lower risks of acquiring K. pneumoniae and other opportunistic pathogens. .

Salmonella in Retail Food and Wild Birds in Singapore-Prevalence, Antimicrobial Resistance, and Sequence Types.

Non-typhoidal salmonellosis is a leading cause of foodborne zoonosis. To better understand the epidemiology of human salmonellosis, this study aimed to determine the prevalence, antimicrobial resistance and sequence types of Salmonella in retail food and wild birds (proximity to humans) in Singapore. We analyzed 21,428 cooked and ready-to-eat food and 1,510 residual faecal samples of wild birds collected during 2010-2015. Thirty-two Salmonella isolates from food and wild birds were subjected to disc diffusion and multi-locus sequence typing (MLST). Salmonella was isolated from 0.08% (17/21,428) of food and 0.99% (15/1510) of wild birds. None of the isolates from wild birds (n = 15) exhibited phenotypic resistance, while the isolates from food (47.1%, 8/17) showed a high prevalence of phenotypic resistance to, at least, one antimicrobial. These findings suggested that the avian Salmonella isolates had been subjected to less antimicrobial selection pressure than those from food samples. MLST revealed specific sequence types found in both food and wild birds. The study can guide future studies with whole-genome analysis on a larger number of isolates from various sectors for public health measures.

Exposure and risk assessment of volatile organic compounds and airborne phthalates in Singapore's Child Care Centers.

Infants and children under 6 years old spend most of daily time in Child Care Centers (CCCs), especially in the tropical regions like Singapore. Environmental exposure and associated risk during this early critical developmental stage is of great public concern. In this study, seven representative volatile organic compounds (VOCs) and five typical phthalates were analyzed in the indoor and outdoor air samples collected from 32 Singapore CCCs. The median of total VOC and phthalate concentration in indoor air was 19.03 and 5.41 µg m-3; respectively. For both indoors and outdoors environment, benzene, toluene and xylene were the dominant VOC contributors (more than 68%). For indoor air phthalates, di(2-ethylhexyl) phthalate and di-butyl phthalate (DBP) accounts for 60-76%. The level of both VOCs and phthalates in indoor environment was significantly higher than that in outdoor, with an average indoor/outdoor ratio of 1.24 and 1.45; respectively. A strong correlation (r > 0.50, p < 0.05) was observed between indoor and outdoor air compounds. VOC and phthalate levels have no significant difference between CCCs with split-unit and centrally ventilated air conditioners. Monte Carlo simulation was used to estimate exposure uncertainty and variability for the risk assessment. Overall, the concentrations of VOC were below the healthy reference values from either EPA Integrated Risk Information System (IRIS) or Singapore guideline. However, similar to other countries' report, benzene, DBP, ethylbenzene and naphthalene were at levels that could exceed the stringent standards such as Office of Environmental Health Hazard Assessment (OEHHA) cancer and reproductive health-based benchmarks.

Microbial Quality and Safety of Sushi Prepared with Gloved or Bare Hands: Food Handlers' Impact on Retail Food Hygiene and Safety.

Food handlers play an important role in retail food hygiene and safety. This study was conducted to better understand the impact of food handlers on the microbiological quality and safety of sushi and ingredients handled by gloved and bare hands. At retail premises, food handlers were asked to prepare a batch of sushi with raw fish followed by a batch of sushi with cooked ingredients. Food (sushi and ready-to-eat ingredients), hand, and glove samples were collected for analysis of overall microorganisms (standard plate counts) and targeted foodborne bacteria: Staphylococcus aureus, Bacillus cereus, Escherichia coli, Salmonella, and Listeria monocytogenes. Results suggested that cross-contamination was more prevalent at premises where bare hands were used to prepare sushi. When bare hands were used, significantly higher standard plate counts were obtained from samples of cooked rice (2.3 to 4.9 log CFU/g) and sushi (2.8 to 6.9 log CFU/g) and the prevalence of S. aureus in samples was higher on food (21.7%, 28 of 129 samples) and hands (30%, 18 of 60 samples) ( P < 0.05). Glove changing in combination with hand washing minimized cross-contamination during sushi preparation as indicated by the lower prevalence of S. aureus (0%, 0 of 28 samples) and total targeted foodborne bacteria (3.6%, 1 of 28 samples) on the gloves of food handlers who changed gloves and washed their hands compared with those handlers who did not don new gloves. Repeated use of dishcloths could be a cause of cross-contamination, and the prevalence of total targeted foodborne bacteria was significantly higher on hands dried with dishcloths (64.7%, 11 of 17 samples) than on hands dried with paper towels (12.5%, 1 of 8 samples) ( P < 0.05). The prevalences of B. cereus, L. monocytogenes, and Salmonella in the 356 food samples were 5.1% (18 samples), 0.8% (3 samples), and 0%, respectively. Improvements to hand washing, hand drying, and glove changing practices are needed to lower the occurrence of cross-contamination during sushi preparation.

Sick pets as potential reservoirs of antibiotic-resistant bacteria in Singapore.

An analysis of 186 diagnostic reports collected from a veterinary clinic in Singapore between 2014 to 2016 showed that sick companion animals can carry bacteria that are of significance to human health. Among the 186 specimens submitted, 82 showed polymicrobial growth (45%, 82/186) and in total, 359 bacteria were isolated. Of the 359 bacteria reported, 45% (162/359) were multi-drug resistant and 18% (66/359) were extended-spectrum-beta-lactamase species. Resistance to broad-spectrum antibiotics were also observed among individual species. Namely, methicillin-resistance among Staphylococcus pseudintermedius (63%, 32/51) and Staphylococcus aureus (50%, 4/8); fluoroquinolone-resistance among Escherichia coli (40%, 17/42) and carbapenem-resistance among Klebsiella pneumoniae (7%, 2/30) were noted. Our analysis suggests that sick pets may contribute to the pool of clinically relevant antibiotic-resistant bacteria and play a role in the spread of antibiotic resistance in Singapore. A more extensive study to better understand the extent of distribution and the factors affecting transmission of antibiotic-resistant bacteria to and from pets is necessary.

Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in retail food in Singapore.

We characterised 227 Staphylococcus aureus isolates from retail food and food handlers' gloves samples obtained through food surveillance and risk assessment studies between 2011 and 2014. Of 227 isolates, five (2.2%) were methicillin-resistant and belonged to sequence types ST80 (n = 3) and ST6 (n = 2). All five isolates belonged to SCCmec type IV, were Panton-Valentine leukocidin (pvl)-negative and staphylococcal enterotoxin genes-positive. Resistance to azithromycin was found in ST80 isolates, in addition to resistance to beta-lactams. Our finding of two clinically relevant methicillin-resistant S. aureus (MRSA) strains (ST80 and ST6) in ready-to-eat food and food contact surfaces at retail in Singapore suggests food and food contact surfaces as potential environmental sources of MRSA in the community.

Microbial survey of ready-to-eat salad ingredients sold at retail reveals the occurrence and the persistence of Listeria monocytogenes Sequence Types 2 and 87 in pre-packed smoked salmon.

BACKGROUND: As the preparation of salads involves extensive handling and the use of uncooked ingredients, they are particularly vulnerable to microbial contamination. This study aimed to determine the microbial safety and quality of pre-packed salads and salad bar ingredients sold in Singapore, so as to identify public health risks that could arise from consuming salads and to determine areas for improvement in the management of food safety. RESULTS: The most frequently encountered organism in pre-packed salad samples was B. cereus, particularly in pasta salads (33.3%, 10/30). The most commonly detected organism in salad bar ingredients was L. monocytogenes, in particular seafood ingredients (44.1%, 15/34), largely due to contaminated smoked salmon. Further investigation showed that 21.6% (37/171) of the pre-packed smoked salmon sold in supermarkets contained L. monocytogenes. Significantly higher prevalence of L. monocytogenes and higher Standard Plate Count were detected in smoked salmon at salad bars compared to pre-packed smoked salmon in supermarkets, which suggested multiplication of the organism as the products move down the supply chain. Further molecular analysis revealed that L. monocytogenes Sequence Type (ST) 2 and ST87 were present in a particular brand of pre-packed salmon products over a 4-year period, implying a potential persistent contamination problem at the manufacturing level. CONCLUSIONS: Our findings highlighted a need to improve manufacturing and retail hygiene processes as well as to educate vulnerable populations to avoid consuming food prone to L. monocytogenes contamination.

Diarrheagenic pathogens in adults attending a hospital in Singapore.

BACKGROUND: Singapore's diarrhoeal notification system is based on specific pathogens. Official data may thus be skewed towards notifiable diseases. Limited information is available on the profiles of aetiological agents responsible for acute gastroenteritis (AGE) cases, especially among the adult population. To understand the frequency and distribution of potential causative agents of diarrheal disease in Singapore, we screened adults' stool samples collected from a large public hospital. METHODS: The stool samples were screened for 18 diarrheagenic pathogens using a combination of commercial multiplex polymerase chain reaction (PCR), in-house singleplex PCR and immunochromatographic assays. One hundred adult faecal samples that were collected from October 2013 to January 2014 for routine diagnostic purposes and submitted for culture at Tan Tock Seng Hospital, Singapore were used. RESULTS: Pathogens were detected in 32% of the samples. The predominant organisms encountered were norovirus genogroup II (11%), Aeromonas spp. (9%) and Campylobacter spp. (5%). One sample was positive for both verocytotoxigenic E. coli (VTEC) and E. coli O157:H7. Two other samples were positive for VTEC only, and one other sample was positive for E. coli O157:H7 only. Astrovirus, C. perfringens, Shigella spp. and toxigenic C. difficile were each detected in 2% of the samples. Cryptosporidium parvum, Giardia lamblia, group A rotavirus, Salmonella spp. and Vibrio spp. were each detected in 1% of the samples. No L. monocytogenes, Y. enterocolitica, enteric adenovirus, or norovirus genogroup I were detected. CONCLUSION: Our preliminary findings suggest that pathogens causing non-notifiable diseases might have contributed considerably to the adult hospitalised AGE cases. However, as the samples were from an adult hospital, the data obtained may not be representative of the whole community. Thus, a larger study to collect clinical samples and risk exposure data from primary healthcare clinics and children hospital is planned for, to gain a more holistic perspective on the epidemiology of AGE in Singapore. A larger study may also offer valuable insights for improving the approach of microbiological surveillance of food, as well as strategizing inspection efforts along the food supply chain by public health authorities.

MICROBIOLOGICAL SAFETY ASSESSMENT AND RISK MITIGATION OF INDIAN ROJAK (DEEP FRIED READYTO-EAT FOOD) IN SINGAPORE.

We conducted a microbiological assessment of Indian Rojak, a popular deep fried food in Singapore to evaluate its overall microbial quality, assess the effectiveness of reheating and identify key food items that could contribute to the microbial load of the dish. In 2009, an outbreak of foodborne illness associated with this food led to 154 reported cases of acute gastroenteritis, 48 were hospitalized and 2 died. Vibrio parahaemolyticus was isolated from the patients. We evaluated 455 Indian Rojak ingredients from 35 stalls; no Salmonella spp, Vibrio cholerae/parahaemolyticus or Escherichia coli O157:H7 were recovered from the studied samples. The reheating by the food handlers significantly reduced the overall median Standard Plate Count (SPC) of food from 4.5 to 2.7 log colony forming units (CFU)/g (p<0.05). The cooked ingredients with the highest microbial loads were tofu and fish cake, with those purchased from wet markets having significantly higher bacterial loads than those purchased from supermarkets (p<0.05). The Rojak gravy had the lowest median bacterial load (1.9 log CFU/g). Raw, ready-to-eat vegetables, namely green chillis, cucumbers and onions had higher levels ranging from 5.9 to 6.1 log CFU/g. Contamination with E. coli, Staphylococcus aureus, and Bacillus cereus was seen with some of the ready-to-eat raw vegetables. Repeated education of food handlers with emphasis on good hygiene practices should be conducted to reduce the risk of foodborne illnesses.

Biased mutational pattern and quasispecies hypothesis in H5N1 virus.

Like other RNA viruses, influenza viruses are subject to high mutation rates. Carrying segmented RNA genomes, their genetic variability is even higher. We aimed at analyzing the mutational events occurring during the infection of chickens by the Highly Pathogenic Avian Influenza (HPAI) H5N1 virus. We therefore studied the different sequences of two surface proteins, hemagglutinin (HA) and neuraminidase (NA), as well as two internal proteins, PB2 and NS. Three organs (lung, spleen, brain) were obtained from a chicken, experimentally infected with a lethal dose of HPAI H5N1 virus. Cloning these PCR fragments enabled us to investigate the mutations undergone by the virus after several replicative cycles. The first outcome is the presence of a strong mutational bias, resembling host-driven ADAR1 adenosine deamination, which is responsible for 81% of all mutations. Whereas the frequency of RNA dependent RNA polymerase-related mutations is compatible with the survival of the virus, the ADAR1-like activity usually strongly increases the mutation frequency into a level of "error catastrophe" in theory incompatible with virus survival. Nevertheless, the virus was successfully infective. HPAI H5N1 virus displayed traits in agreement with the quasispecies theory. The role of this quasispecies structure in successful infection and the superposition with the ADAR1-like response is discussed.

Eurasian Tree Sparrows, risk for H5N1 virus spread and human contamination through Buddhist ritual: an experimental approach.

BACKGROUND: The Highly Pathogenic Avian Influenza H5N1 virus has dramatically spread throughout Southeast Asia since its first detection in 1997. Merit Release Birds, such as the Eurasian Tree Sparrow, are believed to increase one's positive karma when kissed and released during Buddhist rituals. Since these birds are often in close contact with both poultry and humans, we investigated their potential role in the spread of H5N1 virus. METHODOLOGY/PRINCIPAL FINDINGS: Seven series of experiments were conducted in order to investigate the possible interactions between inoculated and exposed birds, including sparrow/sparrow, sparrow/chicken, duck/sparrow. Daily and post-mortem samples collected were tested for H5N1 virus by real-time RT-PCR and egg inoculation. When directly inoculated, Eurasian Tree Sparrows were highly susceptible to the H5N1 virus, with a fatality rate approaching 100% within 5 days post-inoculation. Although transmission of fatal infection between sparrows did not occur, seroconversion of the exposed birds was observed. Up to 100% chickens exposed to inoculated sparrows died of H5N1 infection, depending on the caging conditions of the birds, while a fatality rate of 50% was observed on sparrows exposed to infected ducks. Large quantities of H5N1 virus were detected in the sparrows, particularly in their feathers, from which infectious particles were recovered. CONCLUSIONS/SIGNIFICANCE: Our study indicates that under experimental conditions, Eurasian Tree Sparrows are susceptible to H5N1 infection, either by direct inoculation or by contact with infected poultry. Their ability to transmit H5N1 infection to other birds is also demonstrated, suggesting that the sparrows may play a role in the dissemination of the virus. Finally, the presence of significant quantities of H5N1 virus on sparrows' feathers, including infectious particles, would suggest that Merit Release Birds represent a risk for human contamination in countries where avian influenza virus is circulating and where this religious ritual is practiced.

Superior neutralizing antibody response and protection in mice vaccinated with heterologous DNA prime and virus like particle boost against HPAI H5N1 virus.

BACKGROUND: Although DNA plasmid and virus-like particle (VLP) vaccines have been individually tested against highly pathogenic avian influenza (HPAI) H5N1 viruses, the combination of both vaccines into a heterologous prime-boost strategy against HPAI H5N1 viruses has not been reported before. METHODOLOGY/PRINCIPAL FINDINGS: We constructed DNA plasmid encoding H5HA (A/Shenzhen/406H/06, subclade 2.3.4) and generated VLP expressing the same H5HA and N1NA. We then compared neutralizing antibody responses and immune protection elicited with heterologous DNA-VLP, homologous DNA-DNA and VLP-VLP prime-boost strategies against HPAI H5N1 viruses in mice. We demonstrate that DNA-VLP elicits the highest neutralizing antibody titers among the three prime-boost strategies, whereas DNA-DNA elicits higher neutralizing antibody titers than VLP-VLP. We show that although all three prime-boost strategies protect mice from death caused by 10 MLD(50) of homologous and heterologous H5N1 challenge, only DNA-VLP and DNA-DNA protect mice from infection as manifested by no weight loss and no lung pathology. In addition, we show that although DNA-VLP and DNA-DNA protect mice from death caused by 1,000 MLD(50) of homologous H5N1 challenge, only DNA-VLP protects mice from infection. Moreover, we show that after 1,000 MLD(50) of heterologous H5N1 challenge, while all mice in PBS, VLP-VLP and DNA-DNA died, 3 of 6 mice in DNA-VLP actually survived. Finally, we show that DNA-VLP completely protects mice from infection after 1,000 MLD(50) of homologous H5N1 challenge even when the challenge was administrated at 60 days post the boost. CONCLUSIONS/SIGNIFICANCE: These results provide strong support for clinical evaluation of heterologous DNA-VLP prime-boost strategy as a public health intervention against a possible H5N1 pandemic.

A(H5N1) Virus Evolution in South East Asia.

Highly Pathogenic Avian Influenza (HPAI) H5N1 virus is an ongoing public health and socio-economic challenge, particularly in South East Asia. H5N1 is now endemic in poultry in many countries, and represents a major pandemic threat. Here, we describe the evolution of H5N1 virus in South East Asia, the reassortment events leading to high genetic diversity in the region, and factors responsible for virus spread. The virus has evolved with genetic variations affecting virulence, drug-resistance, and adaptation to new host species. The constant surveillance of these changes is of primary importance in the global efforts of the scientific community.