Effect of milk bactofugation on the counts and diversity of thermoduric bacteria.

The objective of this work was to determine the effect of milk bactofugation on the counts and microbial diversity of mesophilic (MT), psychrotrophic (PT), and thermophilic (TT) thermoduric bacteria and its potential as a technological method to remove spoilage microorganisms resistant to pasteurization. Different batches of raw milk from 69 dairy farms divided into sets in 3 bulk tanks (A, B, C) were evaluated at different times during the technological process. As the raw milk was preheated (∼55°C) immediately before bactofugation (10,000 × g), the effect of bactofugation was estimated by comparing the counts in raw, preheated, and bactofuged milk. This centrifugation was sufficient to reduce the isolation of 88% of the MT in preheated milk. For PT, it was possible to verify a reduction of 72.5% in batch C. The TT were not recovered at higher detection limits (<5 cfu/mL). For diversity, 310 isolates were identified using a molecular approach; 15 species of contaminating thermoduric bacteria were identified from raw and preheated milk, and only 6 species were recovered in bactofuged milk. Only MT were recovered from the bactofuged milk, mainly the species Lysinibacillus fusiformis (61.7%) and Bacillus licheniformis (12.3%). Both species are known to be endospore-forming psychrotrophs and have proteolytic or lipolytic activity. The bactofugation of raw milk reduced the number of isolates of B. licheniformis, Bacillus toyonensis, Micrococcus aloeverae, and Aestuariimicrobium kwangyangense by 33, 43, 86, and 92%, respectively, and reduced the isolates of Macrococcus caseolyticus, Lysinibacillus varians, Carnobacterium divergens, Microbacterium hominis, Kocuria indica, Micrococcus yunnanensis, Gordonia paraffinivorans, Bacillus invictae, and Kocuria kristinae to undetectable levels. The results of this study indicate that bactofugation can be applied by the dairy industry to reduce pasteurization-resistant microorganisms in combination with prophylactic measures to prevent the contamination of raw milk by spores and vegetative forms of bacteria.

Short communication: Molecular characterization and antimicrobial resistance of pathogenic Escherichia coli isolated from raw milk and Minas Frescal cheeses in Brazil.

The aim of this study was to quantify, identify, evaluate antimicrobial resistance, and characterize the virulence factors of enteropathogenic (EPEC), Shiga-toxigenic (STEC), and enterohemorrhagic (EHEC) Escherichia coli in raw milk (RM) and legal (LMFC) and illegal (IMFC) Minas Frescal cheeses in southern and northeast Brazil. Illegal cheeses are those made without official inspection service or sanitary surveillance. We evaluated samples of RM produced in Paraná (southern) and Maranhão (northeast) States, LMFC produced using pasteurized milk in inspected industries, and IMFC potentially produced with raw milk. Mean total coliform counts were 8.4 × 104 cfu/mL for RM, 1.4 × 107 cfu/mL for LMFC, and 2.9 × 107 cfu/mL for IMFC. Mean E. coli counts were 2.4 × 103 cfu/mL for RM, 1.9 × 102 cfu/mL for LMFC, and 1.1 × 105 cfu/mL for IMFC. Among the 205 E. coli isolates from RM, 9.75% were identified as EPEC, mainly (90%) in samples from Paraná. Of the total isolates from the cheese samples, 97.4% (n = 111) came from IMFC, of which 1.8 and 2.7% were identified as EPEC and STEC, respectively; no EHEC was detected. The phylogenetic group A (60%) and typical EPEC (68%) predominated, which confirms the possible human origin of pathogenic isolates in RM and IMFC. Of these, 50% were resistant to at least one antibiotic, and streptomycin was the antimicrobial with the highest number (8) of EPEC and STEC resistant isolates. This study reports the first isolation of serogroup O28ac in Brazilian milk. We found no predominance of a specific serogroup of EPEC or STEC in milk or cheese or clonal isolates in the same sample, indicating different origins of the contamination in these products, presumably mostly related to poor hygienic handling.

Genetic diversity of thermoduric spoilage microorganisms of milk from Brazilian dairy farms.

When correctly pasteurized, packaged, and stored, milk with low total bacterial counts (TBC) has a longer shelf life. Therefore, microorganisms that resist heat treatments are especially important in the deterioration of pasteurized milk and in its shelf life. The aim of this work was to quantify the thermoduric microorganisms after the pasteurization of refrigerated raw milk samples with low TBC and to identify the diversity of these isolates with proteolytic or lipolytic potential by RFLP analysis. Twenty samples of raw milk were collected in bulk milk tanks shortly after milking in different Brazilian dairy farms and pasteurized. The mean thermoduric count was 3.2 (±4.7) × 102 cfu/mL (2.1% of the TBC). Of the 310 colonies obtained, 44.2% showed milk spoilage potential, 32.6% were proteolytic and lipolytic simultaneously, 31% were exclusively proteolytic, and 48 (36.4%) were only lipolytic. Regarding the diversity, 8 genera were observed (Bacillus, Brachybacterium, Enterococcus, Streptococcus, Micrococcus, Kocuria, Paenibacillus, and Macrococcus); there was a predominance of endospore-forming bacteria (50%), and Bacillus licheniformis was the most common (34.1%) species. Considering the RFLP types, it was observed that the possible clonal populations make up the microbiota of different milk samples, but the same milk samples contain microorganisms of a single species with different RFLP types. Thus, even in milk with a high microbiological quality, it is necessary to control the potential milk-deteriorating thermoduric microorganisms to avoid the risk of compromising the shelf life and technological potential of pasteurized milk.

Acute Toxicity of Fresh and Aged Residues of Pesticides to the Parasitoid Tamarixia radiata and to the HLB-Bacteria Vector Diaphorina citri.

One method for controlling the Asian citrus psyllid (ACP) Diaphorina citri Kuwayama, the vector of the putative causal agent of Huanglongbing, uses the parasitoid Tamarixia radiata (Waterston). However, the general intensive use of insecticides has reduced the numbers of this parasitoid. This study evaluated the effect of the residual action of 24 insecticides on T. radiata and also determined the differential toxicity of insecticides to D. citri and T. radiata, using three bioassays. In the first, when adults of the parasitoid were exposed to residues of the 24 insecticides, ten were considered short-life (class 1), six slightly persistent (class 2), five moderately persistent (class 3), and three insecticides were considered persistent (class 4), under the IOBC/WPRS classification system. The second bioassay evaluated the sublethal concentrations of the persistent insecticides (formetanate, dimethoate, spinosad). Increasing the concentrations of the insecticides increased the number that were classified as persistent. In the third bioassay, evaluation of the differential toxicity of eight insecticides to the ACP and the parasitoid showed that chlorpyrifos and bifenthrin were more harmful to T. radiata. Therefore, these two insecticides are not recommended for application at the time of parasitoid release. Cypermethrin, imidacloprid, and dimethoate caused higher mortality of D. citri and are most often recommended in IPM programs. The choice of an insecticide for the control of citrus pests must be made with care, aiming to preserve the natural enemies in the ecosystem, and thereby contribute to the success of biological control.

The main spoilage-related psychrotrophic bacteria in refrigerated raw milk.

Refrigerated raw milk may contain psychrotrophic microorganisms that produce thermoresistant exoproteases and lipases, which may compromise the quality of processed fluid milk and dairy products during storage. The aim of this work was to quantify and identify the deteriorating psychrotrophic microbiota in Brazilian refrigerated raw milk using genetic diversity analysis. The mean psychrotrophic count was 1.1 × 104 cfu/mL. Of the total isolates, 47.8 and 29.8% showed deteriorating activity at 35°C within 48 h and 7°C within 10 d, respectively. Among the proteolytic species, more isolated by this study were Lactococcus lactis (27.3%), Enterobacter kobei (14.8%), Serratia ureilytica (8%), Aerococcus urinaeequi (6.8%), and Bacillus licheniformis (6.8%). Observed among lipolytics were E. kobei (17.7%), L. lactis (15.6%), A. urinaeequi (12.5%), and Acinetobacter lwoffii (9.4%). The isolates S. ureilytica, E. kobei, Pseudomonas spp., and Yersinia enterocolitica potentially produced alkaline metalloprotease (aprX). Despite the low counts, a considerable portion of the psychrotrophic microbiota presented spoilage potential, which reaffirms the need for rigor in the control of contamination and the importance of rapid processing as factors that maintain the quality of milk and dairy products.

Lethal and Sublethal Impacts of Acaricides on Tamarixia radiata (Hemiptera: Eulophidae), an Important Ectoparasitoid of Diaphorina citri (Hemiptera: Liviidae).

The use of synthetic acaricides for management of pest mites may alter the efficacy of the ectoparasitoid Tamarixia radiata (Waterston) in biological control of Diaphorina citri Kuwayama, the vector of the bacteria associated with huanglongbing (HLB) in citrus orchards. We evaluated the toxicity of 16 acaricides that are recommended for the control of citrus-pest mites to T. radiata. Acrinathrin, bifenthrin, carbosulfan, and fenpropathrin caused high acute toxicity and were considered harmful (mortality >77%) to T. radiata. Abamectin, diflubenzuron, etoxazole, fenbutatin oxide, fenpyroximate, flufenoxuron, hexythiazox, propargite, spirodiclofen, and sulfur caused low acute toxicity and affected the parasitism rate and emergence rate of adults (F1 generation), and were considered slightly harmful to T. radiata. Dicofol and pyridaben did not affect the survival and action of the ectoparasitoid, and were considered harmless. In addition to its acute toxicity, carbosulfan caused mortality higher than 25% for >30 d after application, and was considered persistent. Acrinathrin, bifenthrin, fenpropathrin, fenpyroximate, propargite, and sulfur caused mortalities over 25% until 24 d after application and were considered moderately persistent; abamectin was slightly persistent, and fenbutatin oxide was short lived. Our results suggest that most acaricides used to control pest mites in citrus affect the density and efficacy of T. radiata in the biological control of D. citri. However, further evaluations are needed in order to determine the effect of these products on this ectoparasitoid under field conditions.

Estafilococos em alimentos / Staphilococci in food

Este artigo de revisão tem como objetivo descrever as principais características do Staphylocococus aureus e a relação deste micro-organismo com as doenças transmitidas por alimentos. S. aureus é a principal espécie do gênero e o homem e os animais são os reservatórios. Este micro-organismo se multiplica com facilidade em vários alimentos e produz enterotoxinas (EE) termorresistentes. Leite cru e derivados, creme, tortas recheadas com creme, salada de batata, atum, carne de frango, presunto, carnes e produtos a base de ovos já foram incriminados nos surtos alimentares envolvendo os estafilococos. Os principais sintomas são náuseas, vômito, cãibras abdominais e diarréia. O período de incubação varia de 2 a 4 horas. São conhecidos 20 tipos diferentes de EE e sua produção é influenciada pela temperatura, pH, atividade de água, tamanho do inóculo, fonte de carbono e nitrogênio, concentração de sal e condições atmosféricas do substrato. A maioria das intoxicações é produzida por EEA e EED. As EE são detectáveis nos alimentos que apresentam populações de S. aureus acima de 105 UFC/mL de alimento. A dose mínima de EE ingerida para causar intoxicação é 100 ng. Para detecção de EE, existem métodos como Sensibilidade Ótima em Placas (OSP- Optimun Sensitivity Place), microslide, Aglutinação Reversa Passiva em Látex (RPLA- Reversed Passive Latex Agglutination) e ELISA. A Reação em Cadeia pela Polimerase (PCR- Polimerase Chain Reaction), apesar de não diferenciar células viáveis de não viáveis, pode ser aplicada para detecção de diversos tipos de estafilococos enterotoxigênicos.

This review article aims to describe the main characteristics of Staphylocococus aureus and the relation of this microorganism with food-borne disease. S. aureus is the main species of this genus, and man and animals are the main reservoirs. These microorganisms can easily grow in food and produce thermo-resistant enterotoxins. Whole milk and dairy products, cream, cream-filled pies, potato salad, tuna, chicken, ham, meat and egg-based products have been incriminated in food-borne outbreaks involving Staphylococcus. The main symptoms are nausea, vomit, abdominal cramps and diarrhea. The incubation period varies from 2 to 4 hours. Twenty different enterotoxins are known and their production is influenced by temperature, pH, water activity, inoculum size, carbon and nitrogen availability, salt concentration and atmospheric conditions. The majority of food poisoning is caused by enterotoxins A and E. The enterotoxins are detectable in food containing S. aureus populations above 105 CFU/mL. The minimum dose of enterotoxin necessary to cause staphylococcal poisoning is 100 ng. To detect enterotoxins, methods like optimum sensitivity plate (OSP), microslide, reversed passive latex agglutination (RPLA) and ELISA can be applied. Although polimerase chain reaction (PCR) cannot differentiate viable and nonviable cells, it can be used to detect different kinds of enterotoxigenic staphylococci.

Listeria monocytogenes and Salmonella spp. in raw milk produced in Brazil: occurrence and interference of indigenous microbiota in their isolation and development.

This study aimed to verify the occurrence of Listeria monocytogenes and Salmonella spp. in raw milk produced in Brazil. On account of the poor microbiological quality of this product, possible interference from the indigenous microbiota in these pathogens was also evaluated. Two-hundred and ten raw milk samples were collected in four important milk-producing areas in Brazil, tested for L. monocytogenes and Salmonella spp. presence, and for enumeration of indicator microorganisms: mesophilic aerobes, total coliforms and Escherichia coli. The interference of the indigenous microbiota in the isolation procedures was also tested, as well the frequency of naturally occurring raw milk strains with antagonistic activity against both pathogens. The pathogens were not isolated in any raw milk sample, but poor microbiological quality was confirmed by the high levels of indicator microorganisms. When present at high levels, the indigenous microbiota generated an evident interference in the methodologies of L. monocytogenes and Salmonella spp. isolation, mainly when the pathogens appeared at low levels. Three-hundred and sixty raw milk strains were tested for antagonistic activity against both pathogens, and 91 (25.3%) showed inhibitory activity against L. monocytogenes and 33 (9.2%) against Salmonella spp. The majority of the antagonistic strains were identified as Lactic Acid Bacteria species, mainly Lactococcus lactis subsp. lactis and Enterococcus faecium, known by antimicrobial substance production.

Resistance and plasmid profile of coagulase negative staphylococci isolated from bovine subclinical mastitis in Brazil

Estudou-se a resistência a agentes antimicrobianos e o perfil plasmidial de amostras de estafilococos coagulase negativos (ECN) isolados de mastite subclínica bovina. Dos 33 ECN isolados, 57 por cento foram resistentes a sulfametoxazol, 42 por cento ao trimetoprim, 33 por cento à tetraciclina, 27 por cento à lincomicina e 18 por cento à penicilina. Nenhuma amostra foi resistente a tetraciclina, 27 por cento à lincomicina e 18 por cento à penicilina. Nenhuma amostra foi resistente a amicacina, cefalotina, neocina, oxacilina e vancomicina. Detectaram-se plasmídios em 24 (72 por cento) destas amostras, variando em número de um a nove por cultura, totalizando 76 plasmídios com 48 diferentes pesos moleculares. Em algumas amostras, mediante experimentos de cura e transformaçäo, concluiu-se que o determinante genético de resistência à tetraciclina, eritomicina e cloranfenicol está localizado em plasmídios específicos

Plasmid profile of Staphylococcus hyicus isolated from swine exudative epidermitis in Brazil

Staphylococcus hyicus isolados de leitöes com lesöes de pele foram estudados no que se refere aos seus plasmídios e resistência a agentes antimicrobianos. Plasmídios de diferentes pesos moleculares foram detectados nas seis amostras de Staphylococcus hyicus. Todas as amostras mostraram resistência à tetraciclina e um pequeno plasmídio de 4.0 Kb, com uma exceçäo. Experimentos de cura e transformaçäo demonstraram que este plasmídio abriga o determinante genético para resistência à tetraciclina nas amostras estudadas