Effect of hydrolysis and microwave treatment on the antibacterial activity of native bovine milk lactoferrin against Cronobacter sakazakii.

Bovine lactoferrin (bLF) is an iron-binding glycoprotein used in functional and therapeutic products due to its biological properties, the most important being its antimicrobial activity. In this study, hydrolysates of bovine lactoferrin (bLFH) obtained with pepsin, chymosin and microbial rennet were assayed against Cronobacter sakazakii (104 CFU/mL) in different media: phosphate buffered saline (PBS), bovine skim milk and whey, and reconstituted powdered infant formula (PIFM). The results obtained have shown that hydrolysis of bLF enhances its antibacterial activity against C. sakazakii. The three types of bLFH dissolved in PBS reduced C. sakazakii growth from a concentration of 0.1 mg/mL and inhibited it completely above 0.5 mg/mL, after 4 and 8 h of incubation at 37 °C. The three bLFH (1 and 2 mg/mL) did not show any antibacterial activity in skim milk, whey and reconstituted PIFM after 8 h of incubation at 37 °C. However, C. sakazakii growth was completely inhibited in whey when pepsin and chymosin bLFH (2 mg/mL) were combined with undigested bLF (2 mg/mL), after 8 h of incubation at 37 °C. On the other hand, the combination of any of the three hydrolysates with bLF showed very low activity in skim milk and practically no activity in reconstituted PIFM. Furthermore, the effect of temperature after reconstitution (4, 23 and 37 °C), on the antibacterial activity of bLF (2.5 and 5 mg/mL) in reconstituted PIFM contaminated with C. sakazakii (10-102 CFU/mL) was also investigated. bLF at 5 mg/mL significantly reduced (p < .05) the proliferation of C. sakazakii in reconstituted PIFM at 37 °C until 2 h. C. sakazakii did not grow at 4 °C for 6 days in reconstituted PIFM with or without bLF. The effect of microwave heating (450, 550 and 650 W for 5, 10 and 15 s) on the antibacterial activity and stability of bLF (2.5 mg/mL) in reconstituted PIFM contaminated with C. sakazakii (10-102 CFU/mL) was also studied. The antibacterial activity of bLF was maintained after treatments at 450 and 550 W for 5 s, which kept 94 and 89% of bLF immunoreactivity, respectively. Moreover, microwave treatments of reconstituted PIFM with or without bLF, at 650 W for 5 s, and at 450, 550 and 650 W for 10 and 15 s, completely inactivated C. sakazakii.

Antimicrobial resistance of Listeria monocytogenes and Listeria innocua from meat products and meat-processing environment.

A total of 336 Listeria isolates from ready-to-eat (RTE) meat products and meat-processing environments, consisting of 206 Listeria monocytogenes, and 130 Listeria innocua isolates, were characterized by disc diffusion assay and minimum inhibitory concentration (MIC) values for antimicrobial susceptibility against twenty antimicrobials. Resistance to one or two antimicrobials was observed in 71 L. monocytogenes isolates (34.5%), and 56 L. innocua isolates (43.1%). Multidrug resistance was identified in 24 Listeria isolates, 18 belonging to L. innocua (13.9%) and 6 to L. monocytogenes (2.9%). Oxacillin resistance was the most common resistance phenotype and was identified in 100% Listeria isolates. A medium prevalence of resistance to clindamycin (39.3% isolates) and low incidence of resistance to tetracycline (3.9% isolates) were also detected. Listeria isolates from RTE meat products displayed higher overall antimicrobial resistance (31.3%) than those from the environment (13.4%). All the strains assayed were sensitive to the preferred antibiotics used to treat listeriosis. Results showed that although antimicrobial resistance in L. monocytogenes still occurs at a low prevalence, L. innocua can form a reservoir of resistance genes which may transfer between bacterial species, including transference to organisms capable of causing disease in humans.

Comparison of sampling procedures for recovery of Listeria monocytogenes from stainless steel food contact surfaces.

A number of techniques exist for microbiological sampling of food processing environments in food industries. In the present study the efficacies of nine sampling procedures for the recovery of Listeria monocytogenes from food contact surfaces, including a new sampling device consisting of a miniroller, were evaluated and compared. A stainless steel table was inoculated with L. monocytogenes strain 935 (serovar 4b, human origin) and L. monocytogenes strain 437/07 (serovar 1/2b, food origin), at 10(5) CFU/100 cm(2). L. monocytogenes strain 935 was best recovered with the minirollers (recovery of up to 6.27%), while poor recoveries (<0.30%) were obtained with the towel (one-ply composite tissue), alginate swab, metallic swab, and Petrifilm methods. In the case of L. monocytogenes strain 437/07 the replicate organism detection and counting (RODAC) ALOA contact plates yielded the best recoveries (4.15%), followed by the minirollers (up to 1.52%). Overall, recovery percentages with the minirollers were higher with stomacher homogenization than with Vibromatic agitation. The recovery percentages obtained for the Listeria strain of human origin were higher than those obtained with the food strain for all sampling procedures except Petrifilm and RODAC ALOA. With the miniroller device coated with wool fiber, the recovery of L. monocytogenes can be improved from 2 to 17 times over recoveries obtained with the sponge and cotton swab. This is the first report of a miniroller device for microbiological sampling in the available literature. The novel sampling procedure is convenient to apply on surfaces, is cost-effective, and results in better recovery of L. monocytogenes than do the conventional methods.

High prevalence of multiple resistance to antibiotics in Salmonella serovars isolated from a poultry slaughterhouse in Spain.

Salmonellosis is a major foodborne infection in Spain, and strains that are resistant to a great variety of antibiotics have become a major public health concern. The aim of this study was to determine the level of antibiotic resistance in 133 Salmonella isolates obtained from a poultry slaughterhouse in Zaragoza (NE Spain). Antimicrobial resistance testing was performed by disk diffusion method using 19 antibiotics. Results were interpreted following the NCCLS criteria. Overall, the highest percentage of resistance was found to the following antimicrobial agents: sulfadiazine (96.2%), neomycin (53.4%), tetracycline (21.8%), and streptomycin (11.3%). All isolates were found to be resistant to one or more of the antibiotics tested. Multiple resistance was observed in 87 strains (65.4%). We found 23 different patterns of resistance in Salmonella Enteritidis. Resistance to sulfadiazine was the most common single resistance. The most frequent patterns of multiresistant strains were neomycin+sulfadiazine and neomycin+tetracycline+sulfadiazine. S. 4,5,12:b: showed the highest percentages of resistance to the tested drugs, with five different resistance patterns found. Ampicillin+chloramphenicol+streptomycin+sulphonamides+tetracycline (ACSSuT) resistance pattern, commonly associated with S. Typhimurium DT 104, was not detected in strains of the same phage type from broilers. The appearance of substantial multiresistance in foodborne Salmonella isolates suggests the need for more prudent use of antibiotics by farmers, veterinarians, and physicians.

Salmonella Incidence and Distribution of Serotypes throughout Processing in a Spanish Poultry Slaughterhouse.

A survey of contamination with Salmonella spp. was done at 11 sampling sites in a poultry slaughter establishment in Spain for a total of 192 samples. Samples included fecal material, utensils, water, and poultry carcasses and livers at several stages of processing. Salmonella incidence rates increased from 30% in fecal material collected from incoming birds to 60% in air-chilled carcasses and 80% in cold-stored livers, indicating that cross-contamination occurred. The rate of incidence of Salmonella organisms on carcasses averaged 56.7% through post-picking to post-air chilling and reached a maximum of 70% on carcasses at the post-spray wash site. Poultry livers were more heavily contaminated with salmonellae, as 55% and 80% samples after evisceration and cold storage, respectively, were positive for those pathogenic bacteria. From a total of 112 strains isolated, 87 (77.6%) were S. enteritidis , 7 (6.2%) Salmonella serotype 4,5,12:b:-(II), and 6 (5.4%) Salmonella serotype 4,12:b:-(II), and the remaining 12 strains were equally distributed among S. typhimurium . S. virchow , and S. blockley (3.6% each). Serotypes isolated from feces were later detected in matched carcasses and livers indicating a cross-contamination of carcasses by endogenous microflora in bird feces. The incidence of Salmonella serotype 4,5, 12:b:-(II) and that of S. typhimurium were significantly higher (P < 0.05) in samples obtained prior to evisceration than in those collected after that particular step. The situation with S. enteritidis was quite the reverse, since this serotype was more frequently detected in samples taken after the evisceration step (P < 0.01).

High Prevalence of Multiple Resistance to Antibiotics in 144 Listeria Isolates from Spanish Dairy and Meat Products.

The resistance to 12 commonly used antimicrobial agents of 144 foodborne isolates belonging to the genus Listeria (23 L. monocytogenes , 54 L. innocua , 66 L. seeligeri , and 1 L. welshimeri ) was tested by using the agar disc-diffusion assay. The Listeria strains were isolated from dairy products (different varieties of unripened fresh and bacteria-ripened hard cheeses made from ewe's, cow's, and goat's milk) and meat products ( longaniza , a type of pork sausage). A total of 84 (93%) and 54 (100%) Listeria strains isolated from cheese and pork sausage, respectively, were resistant to multiple antimicrobial agents. More than 80% of the Listeria strains of both food origins were found to be susceptible to penicillin G and ampicillin, whereas the proportion of isolates resistant to the cephalosporins cefotaxime and cefoxitin was nearly 100%. The prevalence of resistance was much higher for isolates from pork sausage (73.8% on average) than for isolates from cheese (20.9%). This marked difference was statistically significant (P < 0.05; chi-square test) for all antibiotics except ampicillin, cefotaxime, and cefoxitin. The strains of the foodborne pathogen L. monocytogenes isolated from cheese were all susceptible to 9 of the 12 antimicrobial agents evaluated. In contrast, more than 80% of the L. monocytogenes strains isolated from pork sausage exhibited resistance to cefotaxime, cefoxitin, tobramycin, amikacin, chloramphenicol, tetracycline, and erythromycin. The appearance of substantial resistance to antibiotics in foodborne Listeria isolates suggests the need for more prudent use of antibiotics by farmers, veterinarians, and physicians.