Fernando Pilotto

Miniaturized most probable number for the enumeration of Salmonella sp in artificially contaminated chicken meat

Salmonella is traditionally identified by conventional microbiological tests, but the enumeration of this bacterium is not used on a routine basis. Methods such as the most probable number (MPN), which utilize an array of multiple tubes, are time-consuming and expensive, whereas miniaturized most probable number (mMPN) methods, which use microplates, can be adapted for the enumeration of bacteria, saving up time and materials. The aim of the present paper is to assess two mMPN methods for the enumeration of Salmonella sp in artificially-contaminated chicken meat samples. Microplates containing 24 wells (method A) and 96 wells (method B), both with peptone water as pre-enrichment medium and modified semi-solid Rappaport-Vassiliadis (MSRV) as selective enrichment medium, were used. The meat matrix consisted of 25g of autoclaved ground chicken breast contaminated with dilutions of up to 106 of Salmonella Typhimurium (ST) and Escherichia coli (EC). In method A, the dilution 10-5 of Salmonella Typhimurium corresponded to >57 MPN/mL and the dilution 10-6 was equal to 30 MPN/mL. There was a correlation between the counts used for the artificial contamination of the samples and those recovered by mMPN, indicating that the method A was sensitive for the enumeration of different levels of contamination of the meat matrix. In method B, there was no correlation between the inoculated dilutions and the mMPN results.

Salmonella spp. isolated by miniaturized most probable number and conventional microbiology in poultry slaughterhouses

Background: Salmonella spp. are frequently isolated from fowls, and their detection in poultry products varies according to the breeding system and the slaughtering process, bringing risks to the consumer and compromising the marketability. The control of Salmonella in poultry slaughterhouses is based on the detection of bacteria, but the quantification of the agent would be important in assessing risk, as well as in obtaining data to determine the capacity of each step of the process to decrease or increase bacterial contamination. The aims of this study were to propose a method for the quantification of Salmonella in poultry slaughterhouses, frequency of isolation and serovars identified. Materials, Methods & Results: Twenty-one broiler flocks from seven federally inspected slaughterhouses in southern Brazil, totaling 1,071 samples, were assessed by miniaturized most probable number (mMPN) and conventional microbiology. The samples were collected in triplicate at 17 points, which included cloacae, transportation cages before and after sanitization, water (scald tank, supply, pre-chiller and chiller), and carcasses (before and after scalding, defeathering, rinsing, evisceration, final rinsing, chilling at 4oC, and freezing at -12°C for 24 h, 30 and 60 days). Typical Salmonella colonies were submitted to TSI, LIA, SIM, urea, and polyvalent anti-O antiserum tests, and to final identification by Microarray by Check&Trace. Nine of the 1,071 (0.83%) samples analyzed by mMPN and by conventional microbiology were positive for Salmonella and the following serovars were identified: Anatum, Brandenburg, Agona, Tennessee, Bredeney, Schwarzengrund and Infantis. Discussion: This positive rate was lower than that described by other authors, whose rates ranged from 3% and 39% for the isolation of Salmonella spp. from different sources, such as slaughterhouses and retail sales in samples collected in Brazil. The low frequency of isolation of Salmonella in this study can be attributed to the efficiency of control systems used from the field to the slaughterhouse, such as Good Manufacturing Practices (GMP) and Sanitation Standard Operating Procedures (SSOP), which are HACCP requirements. Also, when slaughtering technology actions are properly managed, such as water replacement and temperatures lower than 4oC in the chiller, the initial contamination by Salmonella spp. can be reduced, with a decline in contamination from 70% to 20%, and with a reduction in the contamination of broiler carcasses after chilling from 15.8% to 3.3%. On the other hand the contamination of carcasses by Salmonella before pre-chilling and in post-chilling might be due to the automated system, inadequate temperatures during chilling, and inappropriate water chlorination in the assessed meat-packing plant. Of the 17 points evaluated, seven were positive for Salmonella, especially the cages after sanitization and frozen carcasses. The contamination by Salmonella spp. in transportation cages after sanitization indicates inefficiency of the automated system as well as possible bacterial resistance to the sanitizers used in SSOP while the isolation in carcasses frozen for 24 h and 60 days demonstrates the thermal resistance of the bacterium to a conservation method widely used in the food industry. In this work, just one of the nine positive samples for Salmonella was identified by conventional methods (CM) and mMPN. The discrepancy between methods can be explained by the heterogeneous distribution of Salmonella and other bacteria in naturally contaminated samples. Samples that were positive in the qualitative test but negative in the mMPN protocol could have had a number of Salmonella below the detection amount.

Effect of Antimicrobials on Salmonella Spp. Strains Isolated from Poultry Processing Plants

The routine use of antimicrobials in animal production for the treatment of infections, disease prevention, or as growth promoters is a predisposing factor for the development and dissemination of antimicrobial resistance. In food industries, sanitizers are used for the control of microbial colonization, and their efficacy depends on contact time and on the dilution of the products used. The present study assessed the effect of 12 antimicrobials and four commercial sanitizers on 18 Salmonella spp. strains isolated from poultry processing plants. None of the evaluated antimicrobials was 100% effective against the tested Salmonella spp. strains; however, 94% of the isolates were susceptible to ciprofloxacin, 77% to amoxicillin + clavulanic acid and to ampicillin, and 72% to enrofloxacin, whereas 100% of the isolates were resistant to penicillin G, 16% to tetracycline, and 11% to sulfonamide. The tested Salmonella spp. strains were 100% inhibited by peracetic acid after five minutes of contact, 0.5% by quaternary ammonium after 15 minutes, and 85.7% by chlorhexidine after 15 minutes. The results indicate the importance of testing of efficacy of antimicrobials used in animal production and in public health to monitor their action and the development of resistance.

Development and Performance of a Mechanical Egg Collection System Versus the Hand-Operated Dutch Nest

The aim of this study was to design a new mechanical nesting system based on scientific works on the behavior of laying hens, and to compare its performance with that of a hand-operated Dutch nest. The equipment was assessed in two poultry houses; in the first one, the eggs were collected manually, whereas in the second one, the newly designed mechanical nest was used for the collection. A total of 7,800 hens and 800 males were housed in each poultry house, and the percentages of eggs produced, nest dirty eggs, floor eggs, and cracked and discarded eggs were determined between weeks 25 and 52. The percentage of eggs with microcracks, infertility, embryo mortality, bacterial contamination, fungal contamination, chicks with stunting syndrome and hatching from both types of nest was assessed in the hatchery on a fortnightly basis. The mechanical nest designed herein proved to be a good alternative to the hand-operated Dutch nest, but it still has to be improved in order to have the same performance, especially with regard to reduction in the incidence of floor eggs.