Foodborne Pathogens and Disease

Enterococcus faecium and Enterococcus faecalis belong to the gastrointestinal flora of humans and animals. Although normally regarded harmless commensals, enterococci may cause a range of different infections in humans, including urinary tract infections, sepsis, and endocarditis. The use of avoparcin, gentamicin, and virginiamycin for growth promotion and therapy in food animals has lead to the emergence of vancomycin- and gentamicin-resistant enterococci and quinupristin/dalfopristin-resistant E. faecium in animals and meat. This implies a potential risk for transfer of resistance genes or resistant bacteria from food animals to humans. The genes encoding resistance to vancomycin, gentamicin, and quinupristin/dalfopristin have been found in E. faecium of human and animal origin; meanwhile, certain clones of E. faecium are found more frequently in samples from human patients, while other clones predominate in certain animal species. This may suggest that antimicrobial-resistant E. faecium from animals could be regarded less hazardous to humans; however, due to their excellent ability to acquire and transfer resistance genes, E. faecium of animal origin may act as donors of antimicrobial resistance genes for other more virulent enterococci. For E. faecalis, the situation appears different, as similar clones of, for example, vancomycin- and gentamicin-resistant E. faecalis have been obtained from animals and from human patients. Continuous surveillance of antimicrobial resistance in enterococci from humans and animals is essential to follow trends and detect emerging resistance.

Alkali stress is an important means of inactivating undesirable pathogens in a wide range of situations. Unfortunately, Listeria monocytogenes can launch an alkaline tolerance response, significantly increasing persistence of the pathogen in such environments. This study compared transcriptome patterns of alkali and nonalkali-stressed L. monocytogenes 10403S cells, to elucidate the mechanisms by which Listeria adapts and/or grows during short- or long-term alkali stress. Transcription profiles associated with alkali shock (AS) were obtained by DNA microarray analysis of midexponential cells suspended in pH 9 media for 15, 30, or 60 min. Transcription profiles associated with alkali adaptation (AA) were obtained similarly from cells grown to midexponential phase at pH 9. Comparison of AS and AA transcription profiles with control cell profiles identified a high number of differentially regulated open-reading frames in all tested conditions. Rapid (15 min) changes in expression included upregulation of genes encoding for multiple metabolic pathways (including those associated with Na⁺/H⁺ antiporters), ATP-binding cassette transporters of functional compatible solutes, motility, and virulence-associated genes as well as the σ^B controlled stress resistance network. Slower (30 min and more) responses to AS and adaptation during growth in alkaline conditions (AA) involved a different pattern of changes in mRNA concentrations, and genes involved in proton export.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea among infants and children in developing countries, as well as among travelers to these areas. The major virulence factors of ETEC are the colonization factor antigens (CFAs) and a heat-labile enterotoxin (LT) and/or a heat-stable enterotoxin (ST). Among Israeli recruits serving under military field conditions, 107 of all examined isolates expressed LT or ST, and CFAs could be characterized in 68% of the isolates, in which CFAs of the CFA/II group and CS6 were the most prevalent. Additionally, 31% of the 107 ETEC isolates showed resistance to three or more of the antimicrobial agents examined, and the percentage of resistant isolates expressing LT was significantly higher than those expressing ST or LT+ST. These results may be important for development of an effective vaccine and for facilitation of an empirical choice of antibiotic treatment or prophylaxis for traveler's diarrhea in this area.

This study investigated the efficacy of plant-derived antimicrobials, namely, trans-cinnamaldehyde, β-resorcylic acid, carvacrol, thymol, and eugenol or their combination, in increasing the sensitivity of Salmonella Typhimurium DT104 to five antibiotics. The subinhibitory concentrations of each antimicrobial or their combination containing concentrations lower than the individual subinhibitory concentrations were added to tryptic soy broth supplemented with antibiotics at their respective break points for resistance. Salmonella Typhimurium DT104 was inoculated into tryptic soy broth at ∼6 log CFU/mL, and growth (optical density at 600 nm) was determined before and after incubation at 37°C for 24 hours. Appropriate controls were included. Duplicate samples were assayed and the experiment was replicated three times. Trans-cinnamaldehyde increased the sensitivity of Salmonella Typhimurium DT104 (p < 0.05) toward all five antibiotics, namely, ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline, thereby making the pathogen susceptible to drugs. Thymol made the pathogen susceptible to all four antibiotics except ampicillin, whereas carvacrol increased the sensitivity to two antibiotics (chloramphenicol and sulfamethoxazole for strain H3380, and streptomycin and sulfamethoxazole for strain 43). The combination of five molecules was more effective than individual ones (p < 0.05) in rendering the pathogen susceptible to the antibiotics. Results indicate that these natural molecules individually and synergistically increased the sensitivity of Salmonella Typhimurium DT104 to all the five antibiotics, and justify future studies to control antibiotic resistance of the pathogen in food animals using these plant molecules.

Bovine cysticercosis is caused by the larval stage of the human tapeworm Taenia saginata. According to European data on meat inspection, the prevalence ranges from 0.007% to 6.8%, but the real prevalence is considered to be at least 10 times higher. Laboratory confirmation of the etiological agent is based on gross, stereomicroscopic, and histological examination of submitted specimens. False identifications may occur, possibly because of death and degeneration of cysts, or because taeniid larvae and other tissue parasites, such as Sarcocystis spp., may cause similar macroscopic morphological lesions. Therefore, tests that can warrant sure identification of taeniid lesions and calcified cysts in the muscle are needed. The focus of our study was to develop a suitable postmortem test that could be applied on putative lesions by T. saginata cysticerci, as ambiguously diagnosed after routine meat inspection. In particular, we proposed a biomolecular assay targeting the mitochondrial cytochrome c oxidase subunit I gene (COI). For developing the polymerase chain reaction assay, viable cysts of Cysticercus bovis (n = 10) were used as positive reference samples, and those of Echinococcus granulosus (n = 3), Cysticercus tenuicollis (n = 3), and Sarcocystis spp. (n = 4) as reference negative controls. Further, to evaluate the applicability of the proposed assay, 171 samples of bovine muscular tissue, obtained from local slaughterhouses and containing lesions recognized as T. saginata cysticerci by macroscopic examination, were tested. The proposed test confirmed the diagnosis at postmortem inspection in 94.7% (162/171) of samples. In conclusion, the assay developed in this study, amplifying a short fragment from the mitochondrial gene COI, showed to be suitable for samples containing both viable and degenerating T. saginata cysticerci, yielding an unequivocal diagnosis.

Contamination routes of Listeria monocytogenes were examined for 3 years in an Iberian pork-processing plant that produced high-quality ready-to-eat meat products. Molecular subtypes of L. monocytogenes were determined by polymerase chain reaction–based serotyping and pulsed-field gel electrophoresis (PFGE) restriction analysis. A total of 541 L. monocytogenes isolates were recovered from the environment and equipment (n = 165), carcasses (n = 28), raw products (n = 321), and dry-cured products (n = 27). Only 29 different PFGE types were identified, 3 of which were repeatedly found to be persistent types and accounted for 73% of the isolates. One PFGE type dominated (45% of the isolates) and was mostly recovered from intermediate manufactured products and the environment of the manufacturing area. L. monocytogenes persistence appeared strongly linked to the manufacture of products and not to its sustained entrance with the raw material. Some clones were found to survive in the manufacturing area for 3 years. Controlling the contamination of raw ingredients, improving the compartmentalization, and changing the cleaning protocols resulted in reduced prevalence rates of L. monocytogenes on products; two persistent PFGE types were eliminated from the processing plant, although eradication of other adapted strains has not been achieved.

Antibiotic-resistant Escherichia coli (n = 191) and Salmonella enterica (n = 87) isolates of human and animal origin obtained in Lithuania during 2005–2008 were characterized for the presence and diversity of class 1 and 2 integrons. E. coli isolates were obtained from patients with urinary tract infections (UTIs) (n = 59) and both healthy and diseased farm animals, including poultry (n = 54), swine (n = 35), and cattle (n = 43). Isolates of nontyphoidal S. enterica were recovered from salmonellosis patients (n = 37) and healthy animals, including poultry (n = 31) and swine (n = 19). The presence of integrons, their gene cassette structure, and genome location were investigated by polymerase chain reaction, restriction fragment-length polymorphism, DNA sequencing, Southern blot hybridization, and conjugation experiments. Forty percent of the E. coli and 11% of the S. enterica isolates carried class 1 integrons, whereas class 2 integrons were found in E. coli isolates (9%) only. The incidence of integrons in human UTIs and cattle isolates was most frequent (p < 0.01). A total of 23 different gene cassettes within 15 different variable regions were observed. Seven different integron types, all of them transferable by conjugation, were common for isolates from human infections and for one or more groups of animal isolates. The most prevalent integron types contained arrays dfrA1-aadA1 (36%), dfrA17-aadA5 (23%), and dfrA1-sat1-aadA1 (78%). Two E. coli isolates from humans with UTIs harbored class 1 integron on conjugative plasmid with the novel array type of 4800 bp/dfrA17-aadA5Δ-IS26-ΔintI1-aadB-aadA1-cmlA residing on the Tn21-like transposon. Three S. enterica isolates from swine contained class 1 integron with the newly observed array type of 1800 bp/aadA7-aadA7. Integrons of 10 different types of both classes were located on transferable plasmids in E. coli and S. enterica. Our study demonstrated the existence of a considerable and common pool of transferable integrons in E. coli and S. enterica present in clinical and livestock environment in Lithuania.

The objectives of this study were to report an outbreak of highly drug-resistant Salmonella enterica serotype Oranienburg in dairy calves, and conduct an epidemiological investigation of Oranienburg identified on a dairy herd during a study to determine whether discontinuing feeding medicated milk replacer to preweaned dairy calves resulted in increased antimicrobial susceptibility in enteric bacteria. Calf fecal samples and swabs of calf and maternity pens were collected monthly over 18 months. Samples were streaked onto XLT-4 agar and characteristic colonies were subjected to biochemical tests to confirm Salmonella. Strain relatedness was examined by Xbal and BlnI pulsed-field gel electrophoresis analysis on 62 randomly selected isolates. Antimicrobial susceptibility testing, using automated microbroth dilution, was conducted using a panel containing tetracycline, amikacin, amoxicillin-clavulanic acid, ampicillin, ceftiofur, ceftriaxone, cephalothin, chloramphenicol, ciprofloxacin, cefoxitin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole, and trimethoprim-sulfamethoxazole. A total of 190 Salmonella spp. were isolated from 604 calf and 36 pen samples, of which 86% were Oranienburg and 97% were resistant to at least 9 agents. Environmental isolates had lower levels of resistance than fecal isolates. Pulsed-field gel electrophoresis analysis identified three strains: the most common strain was consistently present before the outbreak and at its peak. One strain was exclusively an environmental isolate, with little antimicrobial resistance. Multiresistant isolates with resistance to ciprofloxacin appeared early in the outbreak, and were replaced by multiresistant isolates with resistance to cephalothin. The differences in strains and resistance patterns suggest that the strains of Oranienburg found in fecal isolates may have different origins from environmental isolates.

Campylobacteriosis in humans is primarily caused by handling or consuming contaminated poultry or their products. The aims of this study were to estimate the prevalence of Campylobacter spp. in domestic and imported poultry meat in Korea and to further characterize the obtained isolates. From 2004 to 2008, a total of 475 domestic and 867 imported raw poultry meat samples were examined for the presence of Campylobacter spp. Among 475 domestic poultry meat samples, Campylobacter jejuni and Campylobacter coli were isolated from 219 (46.1%) and 156 (32.8%), respectively. Relative prevalence of C. jejuni and C. coli was higher in meat from Brazil (39/78, 50.0% and 7/78, 8.9%) and France (32/96, 33.3% and 8/96, 8.3%), whereas lower in meat from Denmark (72/516, 14.0% and 12/516, 2.3%) and Thailand (5/39, 12.8% and 3/39, 7.6%). The resistance to ampicillin and tetracycline was highly prevalent in Campylobacter spp. from most countries investigated, whereas lower in meat from Denmark. On the other hand, the prevalence of erythromycin and gentamicin resistance was less than 10% in most countries. The resistance rate to nalidixic acid, ciprofloxacin, and enrofloxacin ranged from 11.9% to 87.5%. The use of fla-polymerase chain reaction–restriction fragment length polymorphism for epidemiological analysis found that some pattern types were considerably more frequent and distinct in meat from each country. In conclusion, we report the presence of high contamination in domestic and imported poultry meat in Korea and the antimicrobial and genetic diversity of Campylobacter spp. between each country.

Although considerable research has been carried out on a range of environmental factors that impact on the survival of Campylobacter jejuni, there is limited information on the effects of violet/blue light on this pathogen. This investigation was carried out to determine the effects of high-intensity 405-nm light on C. jejuni and to compare this with the effects on two other important Gram-negative enteric pathogens, Salmonella enteritidis and Escherichia coli O157:H7. High-intensity 405-nm light generated from an array of 405-nm light-emitting diodes was used to inactivate the test bacteria. The results demonstrated that while all three tested species were susceptible to 405-nm light inactivation, C. jejuni was by far the most sensitive organism, requiring a total dose of 18 J cm⁻² of 405-nm light to achieve a 5-log₁₀ reduction. This study has established that C. jejuni is particularly susceptible to violet/blue light at a wavelength of 405 nm. This finding, coupled with the safety-in-use advantages of this visible (non-ultraviolet wavelength) light, suggests that high-intensity 405-nm light may have applications for control of C. jejuni contamination levels in situations where this type of illumination can be effectively applied.

The effect of low-dose irradiation (0.75 and 1.5 kGy) in combination with acidified sodium chlorite (ASC) on the reduction of Escherichia coli O157:H7 on mung bean seeds was examined. Washing with ASC (0.2, 0.5, 0.8, and 1.2 g/L sodium chlorite and 1.0 g/L citric acid) for 2 h reduced the E. coli O157:H7 population from 5.2 to 2.3–3.3 log CFU/g, depending on the concentrations of sodium chlorite. Gamma ray irradiation at 0.75 and 1.5 kGy resulted in reductions of about 1.8 and 2.8 log CFU/g, respectively. Therefore, a single treatment with ASC washing or gamma ray irradiation at 0.75 or 1.5 kGy could not achieve the complete elimination of E. coli O157:H7 on mung bean seeds. Conversely, low-dose irradiation (0.75 and 1.5 kGy) followed by washing with ASC (0.5–1.2 g/L) reduced the population of E. coli O157:H7 to below the detection limit (<1 log CFU/g). However, E. coli O157:H7 was detected in most samples in the enrichment and germination studies. When the treatment order was reversed (ASC washing followed by low-dose irradiation), the E. coli O157:H7 population was also observed to be below the detection limit. Under this treatment, fewer samples (16.7%) were shown to be positive in the enrichment and germination studies, and complete elimination was not achieved. The germination rates of mung bean seeds were not affected by ASC washing and gamma irradiation; however, the yield and length of sprouts were decreased by gamma irradiation.

This study aims to determine the serogroup profiles of randomly collected 46 chicken meat and 15 turkey meat samples, following the U.S. Food and Drug Administration's (FDA) Bacteriological Analytical Manual Chapter 5: Salmonella and International Organization for Standardization (ISO) Method 6579 culture methods. The total number of poultry meat samples with more than one serogroup isolated by the FDA and ISO culture methods were 10 (37.0%) and 21 (77.8%) of 27, respectively. Presence of multiple serogroups per sample was more frequently observed in chicken meat samples than in turkey meat samples. The profile of Salmonella serogroup isolates of chicken meat samples in descending order were serogroups D and E4 (15.8%), B and C2 (8.8%), C1 (5.3%), G (3.5%), and E1 and F (1.7%). The serogroup distribution of turkey meat sample isolates were serogroups B (27.2%), E4 (18.2%), and C2 (9.1%). On the basis of our findings that a selective plate in Salmonella culture method can harbor more than one serogroup, and that the FDA and ISO methods could detect different serogroups from chicken and turkey meats, we suggest screening multiple suspect colonies from each plate, if possible, and considering the collective and comparative use of the FDA and ISO culture methods and/or including several selective and differential media to ensure the detection of Salmonella and the possible detection of multiple serogroups from samples.

The prevalence and concentration of many foodborne pathogens exhibit seasonal patterns at different stages of the farm-to-table continuum. Escherichia coli O157:H7 is one such pathogen. While numerous studies have described the seasonal trend of E. coli O157:H7 in live cattle, ground beef, and human cases, it is difficult to relate the results from these different studies and determine the interrelationships that drive the seasonal pattern of beef-related human illnesses. This study uses a common modeling approach, which facilitates the comparisons across data sets, to relate prevalence in live cattle to raw ground beef and human illness. The results support an intuitive model where a seasonal rise of E. coli O157:H7 in cattle drives increased ground beef prevalence and a corresponding rise in the human case rate. We also demonstrate the use of these models to assess the public health impact of consumer behaviors. We present an example that suggests that the probability of illness, associated with summertime cooking and handling practices, is not substantially higher than the baseline probability associated with more conventional cooking and handling practices during the remainder of the year.

In the present study, the bacteriocins produced by Staphylococcus aureus 4185, a strain isolated from bovine mastitis, were purified and partially characterized. After purification by ammonium sulfate precipitation, cation-exchange chromatography, and five runs of high-performance liquid chromatography (HPLC), antimicrobial activity was recovered with 40% and 80% isopropanol, suggesting that more than one antimicrobial peptide, named aureocins 4185, is produced by S. aureus 4185. Mass spectrometry analyses revealed three peptides eluted with 40% isopropanol: peptide A (2,305.3 ± 1.5 Da), peptide B (2,327.3 ± 1.5 Da), and peptide C (3,005.5 ± 1.5 Da), and two peptides eluted with 80% isopropanol: peptide D (6,413.5 ± 1.5 Da) and peptide E (12,834.5 ± 1.5 Da). Although five peptides have been detected, only four small peptide sequences were obtained by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF)/TOF mass spectrometry analyses: SLLEQFTGK (eluted with 40% isopropanol), ALLYDER, NNTSHNLPLGWFNVK, and NNLAQGTFNATK (eluted with 80% isopropanol). The sequences SLLEQFTGK and ALLYDER revealed identity with hypothetical peptides with unknown function. The sequences NNTSHNLPLGWFNVK and NNLAQGTFNATK showed similarity to a segment of a precursor of staphylococcal autolysins. The antimicrobial activity detected in the supernatant of strain 4185 proved to be resistant to heat treatment at 65°C; however, treatment at 80°C abolished completely its antimicrobial properties. The concentrated supernatant containing aureocins 4185 exhibited a strong bacteriolytic activity toward Micrococcus luteus ATCC 4698. Additionally, aureocins 4185 exhibited antagonistic activity against important foodborne pathogens, including Listeria monocytogenes, thus showing a potential application in food preservation.

In this study, the effects of the incorporation of some herbal teas at different concentrations into the ice cream mix on the population of Listeria monocytogenes were studied using Taguchi method. The ice cream mix samples flavored with herbal teas were prepared using green tea and sage at different concentrations. Afterward, fresh culture of L. monocytogenes was inoculated into the samples and the L. monocytogenes was counted at different storage periods. Taguchi method was used for experimental design and analysis. In addition, some physicochemical properties of samples were examined. Results suggested that there was some effect, although little, on the population of L. monocytogenes when herbal tea was incorporated into the ice cream mix. Additionally, the use of herbal tea caused a decrease in the pH values of the samples and significant changes in the color values.

The objective of this study was to determine the frequency of nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) in slaughter pigs, to characterize the recovered isolates, and to investigate their genomic relatedness. Nasal swabs were collected from 53 finishing-pigs (F-pigs) and 53 suckling-piglets (S-piglets) at two different abattoirs in La Rioja (Northern Spain) coming from six production holdings. MRSA isolates were characterized by spa−, agr−, SCCmec−, and multilocus sequence typing, pulsed-field gel electrophoresis (PFGE)-ApaI, toxin gene profiling, antimicrobial susceptibility, and determination of antimicrobial resistance genes. MRSA isolates were recovered from 11 F-pigs (14 isolates) and 26 S-piglets (30 isolates). Forty of the 44 MRSA presented the spa-types t011, t108, t1197, and t2346, which corresponded to the sequence type ST398 and to the clonal complex CC398. Interestingly, the remaining four isolates from F-pigs presented the spa-type t3992, and they were ascribed to a new sequence type named ST1379 (a single-locus variant of ST97), which was included in clonal complex CC97. Five PFGE-ApaI clusters with up to nine individual patterns detected among our MRSA and low genomic relatedness was observed between F-pig and S-piglet isolates. All MRSA were positive for hla, hld, and hlg hemolysin genes. ST1379 isolates harbored eta, lukE/D, and hlg-2 toxin genes, whereas ST398 isolates were positive for hlb. A great variety of distinct resistance gene patterns were observed, most of them coming from F-pig isolates. MRSA virulence properties seem to be dependent of the isolate clonal lineage. This study showed that slaughter pigs are frequently colonized by MRSA CC398; moreover, the detection of strains belonging to CC97 underlines that other lineages are also able to spread in livestock. Further studies should assess the risk of CC398 and non-CC398 MRSA to enter the food chain as well as the human health implications.

Coffee berry borer (CBB, Hypothenemus hampei Ferrari) is a common insect pest in coffee plantations and is a suspected vector of various mycotoxin-producing molds. In the present study, field trials were undertaken consecutively for 3 years to evaluate the impact of CBB on the microbial contamination of Arabica and Robusta coffee bean varieties, with emphasis laid toward ochratoxin A (OTA)-producing fungi. Results revealed higher microbial contamination in CBB-infested beans in both the varieties of coffee with the presence of toxigenic molds (such as Aspergillus niger, Aspergillus ochraceus). The “timely harvested” coffee, which was infested with CBB, was found to possess comparatively lesser OTA levels than those berries left in soil or on coffee plants. Studies carried out on coffee beans collected from nine curing factories indicated the presence of OTA in almost all the CBB-infested coffee beans, irrespective of the variety. Results of the present study provide sufficient baseline information and evidence to understand and correlate the role of CBB with various OTA-producing molds in coffee beans. Understanding the role of CBB might be useful and applicable in the coffee-growing regions of the world, especially in plantations for production of quality coffee.