MICROBIOLOGICAL AND PHYSICOCHEMICAL QUALITY OF CURD CHESSE SOLD IN THE STATE OF ALAGOAS (BRAZIL)

The present study aimed to evaluate the microbiological and physicochemical quality of curd cheese commercialized in Alagoas (BRAZIL). Thirty samples were collect during a five months period in six points of sale. It was estimated the number of coagulase-positive Staphylococcus and coliforms, as well as the presence of Salmonella spp. and Listeria monocytogenes. Regarding physicochemical characteristics, pH, fat content and humidity were evaluated. Among the thirty analyzed samples, 20 (66.6%) were in accordance with Brazilian legislation parameters for coliforms, while 10 (33.3%) surpassed those limits. For coagulase-positive Staphylococcus 15 samples (50%) showed values above the allowed by legislation. None of the samples analyzed harbored Salmonella spp. or Listeria monocytogenes. Regarding physicochemical parameters, the average values observed were of 5.89 for pH, 23.6% for fat content and 46.96 g/100 g for humidity. Overall, the results obtained reflect the poor hygienic conditions observed from manufacture to commercialization, pointing towards a potential health risk for curd cheese consumers. Furthermore is necessary to regulate physicochemical standards for curd cheese fabication.

Biofilm Production and In Vitro Susceptibility to Disinfectants of Staphylococcus Coagulase Positive Isolated from Bovine Milk

Background: Mastitis is one of the diseases that cause damage to dairy cattle, and coagulase positive Staphylococcus (CPS), are its main etiological agents. The production of biofilms gives these microorganisms a physiological advantage, being an important virulence factor, as it confers their resistance to the action of disinfectants used in hygiene procedures. Therefore, the objective of this study was to evaluate the capacity of CPS, isolated from milk samples from cases of bovine subclinical mastitis in the Alagoas dairy basin, biofilm production and its sensitivity to disinfectant agents used before and after dipping.Materials, Methods & Results: In the Alagoas Milk Basin Region, 10 properties were selected from the municipalities of Batalha, Cacimbinhas, Major Isidoro and Minador do Negrão, which used a mechanical milking system and supplied milk to a dairy under federal inspection. The California Mastitis Test (CMT) was performed on 1155 cows. The animals diagnosed with subclinical mastitis were classified as: mild (+); moderate (++) and severe (+++). CMT positive samples were collected from 2+, totaling 891 samples. After cleaning the papillary ostium, 2 to 5 mL of milk was collected from each fourth breast before milking began. Subsequently, 0.5 µL of each sample was streak plated by exhaustion on 5% Sheep Blood Agar and incubated at 37°C, with readings at 24 and 48 h. The macroscopic and microscopic characteristics of the colonies, catalase and coagulase test were evaluated. Biofilm formation was determined by the microplate adhesion test. The efficacy of the sanitizers was evaluated with the active ingredients: lactic acid (2%), allantoin (0.05%), iodine (0.5%), chlorhexidine (2.0%), chlorine (2.5%), at times 15, 30 and 60 seconds. It was observed that 91.2% of the samples were able to form biofilm; Of these, 22.2%, 29.6% and 48.2% were strong, moderate and weak producers, respectively. The sensitivity profile of chlorhexidine isolates was 79.73%, 80.41% and 83.78%; iodine was 59.46%, 65.54% and 66.22% sensitivity over the evaluated time. For allantoin, the isolates presented sensitivity of 22.30%, 27.03% and 37.74%, for lactic acid, 18.92%, 20.95% and 22.30% of the samples were sensitive, and to chlorine, 12.6%, 15.58% and 17.57% of the isolates were sensitive. Moreover, it was observed that the longer the exposure to the disinfectant, the greater the effectiveness of the same.Discussion: The results are in line with literature data. Staphylococcus spp. from milk samples of animals with subclinical mastitis are able to form biofilm, thus being an important virulence factor. The heterogeneity regarding the biofilm production potential of the isolates is due to the different species of Staphylococcus spp. that, through distinct mechanisms, triggers a greater or lesser formation of this virulent factor, as well as having their adhesion tested, end up becoming more strongly attached to the plate. Among the disinfectants tested, it was confirmed that chlorhexidine and iodine are more effective than SCP, which tend to have high levels of sensitivity to these disinfectants. However, although chlorhexidine was more sensitive to SCP, its use in the field should be done with caution, as its effectiveness may be reduced in the presence of chlorinated water or excess organic matter. It is concluded that SCP isolates from bovine milk from subclinical mastitis mammary quarters were able to produce biofilms. These isolates were also in vitro sensitive to chlorhexidine and iodine, but periodic evaluation of the efficacy of disinfectants used before and after milking is necessary

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Pesquisa de Staphylococcus aureus enterotoxigênicos formadores de biofilmes isolados de bovinos com mastite em rebanhos leiteiros e perfil de resistência frente a desinfetantes

A produção leiteira no Brasil se destaca por ser uma das atividades mais importantes para pecuária brasileira, sendo responsável pela fonte de renda de milhões de brasileiros. Entretanto, essa atividade é constantemente influenciada pela ocorrência da mastite, principalmente na forma subclínica, que ocorre nos rebanhos. Staphylococcus spp constitui o principal gênero de bactérias causadoras de mastite em bovinos. Objetivou-se com este estudo pesquisar Staphylococcus aureus enterotoxigênicos formadores de biofilmes isolados de bovinos com mastite em rebanhos leiteiros e traçar o perfil de resistência frente à desinfetantes. Foram selecionadas 10 fazendas vinculadas à uma Usina de beneficiamento de leite sob registro federal. Foi realizado o exame físico da glândula mamária e do leite dos animais e, em seguida, realizado o California Mastitis Test. Foram coletadas amostras de 960 animais para o exame microbiológico após antissepsia do óstio dos tetos com álcool 70°GL. No laboratório, alíquotas de 10 μL de leite foram semeadas em ágar sangue ovino a 5% e, em seguida, as placas incubadas a 37°C por 48 horas. Posteriormente, 213 isolados Staphylococcus spp. classificados como fortes e moderados produtores de biofilmes foram selecionados para caracterização quanto ao perfil fenotípico e presença do gene femA para identificação de S. aureus. Destes, foram confirmados 152 isolados, que foram submetidos a detecção de genes capazes de produzir biofilmes (icaA, icaD e Bap), genes que codificam as enterotoxinas (SeA, SeB, SeC, SeD, SeE), e genes relacionados a resistência (Tet(k), Vana). Testou-se a ação de desinfetantes (clorexidine e ácido láctico) utilizados na rotina de ordenha sobre o biofilme em formação e consolidado em 86 destas amostras. Em 84,16% (134/152) das amostras foram constatados o gene BAP, em 78,29% (119/152) o gene IcaD e, em apenas 1,32% (2/152) o gene IcaA. Apenas as enterotoxinas Sec em 17,76% (27/152) das amostras e See em 0,66% (1/152). Foram encontrados genes de resistência TetK em 48,68% (74/152) das amostras. Observou-se resultado satisfatório dos dois desinfetantes testados sobre o biofilme em formação, em 94,2% (81/86) dos isolados submetidos ao clorexidine e 100% dos submetidos ao ácido láctico. Entretanto, para o biofilme consolidado o percentual de ação foi bem inferior, o ácido láctico apresentou baixa eficácia sobre o biofilme já formado, com apenas 3,5% da taxa de redução, enquanto a clorexidine conseguiu reduzir 43% do biofilme consolidado. O efeito dos desinfetantes na redução da adesão de biofilmes foi bastante significativo. Sugere-se a investigação de produtos mais eficazes, que atuem tanto nas células bacterianas livres, como nos microrganismos na forma de biofilmes.Dairy production in Brazil stands out as one of the most important activities for Brazilian livestock, being responsible for the source of income of millions of Brazilians. However, this activity is constantly influenced by the occurrence of mastitis, especially in the subclinical form, which occurs in herds. Staphylococcus spp is the main genus of mastitis-causing bacteria in cattle. The aim of this study was to investigate enterotoxigenic Staphylococcus aureus biofilm-forming isolates from bovine mastitis in dairy herds and to profile resistance against disinfectants. Ten farms linked to a federally registered Milk Processing power plant were selected. Physical examination of the mammary gland and milk of the animals was performed and then the California Mastitis Test. Samples were collected from 960 animals for microbiological examination after antisepsis of the roof ostium with 70 ° GL alcohol. In the laboratory, aliquots of 10 μl of milk were seeded on 5% sheep blood agar and then plates incubated at 37 ° C for 48 hours. Subsequently, 213 isolates Staphylococcus spp. classified as strong and moderate biofilm producers were selected for characterization regarding the phenotypic profile and presence of the femA gene for identification of S. aureus. Of these, 152 isolates were confirmed, which were subjected to detection of genes capable of producing biofilms (icaA, icaD and Bap), genes encoding enterotoxins (SeA, SeB, SeC, SeD, SeE), and resistance related genes (Tet (k), Vana). The action of disinfectants (chlorhexidine and lactic acid) used in the milking routine on biofilm in formation and consolidated in 86 of these samples was tested. In 84.16% (134/152) of the samples the BAP gene was found, in 78.29% (119/152) the IcaD gene and in only 1.32% (2/152) the IcaA gene. Only enterotoxins Sec in 17.76% (27/152) of the samples and See in 0.66% (1/152). TetK resistance genes were found in 48.68% (74/152) of the samples. Satisfactory results were observed for the two disinfectants tested on the biofilm in formation, in 94.2% (81/86) of chlorhexidine isolates and 100% of lactic acid isolates. However, for the consolidated biofilm the action percentage was much lower, the lactic acid showed low efficacy over the already formed biofilm, with only 3.5% of reduction rate, while chlorhexidine was able to reduce 43% of the consolidated biofilm. The effect of disinfectants on reducing biofilm adhesion was quite significant. Research into more effective products that act on both free bacterial cells and microorganisms in the form of biofilms is suggested

Caracterização fenotípica de biofilme e resistência antimicrobiana de estafilococos coagulase positiva presentes em leite pasteurizado sob inspeção estadual

Milk is an alternative source of proteins accessible to the low income population, which is generally lacking in animal protein, proving the socioeconomic importance of this food. In public health, particularly in the area of sanitary food surveillance, Staphylococcus aureus, the main staphylococci of positive coagulase, is considered one of the most frequent cause of outbreaks of foodborne diseases. Milk and milk products are among the foods most involved in the outbreaks identified and studied. The objective of this study was to evaluate the quality of pasteurized milk with state inspection by the positive coagulase staphylococci, its antimicrobial resistance and the ability to form biofilm. The study was carried out in Alagoas and 75L of pasteurized milk were collected from five processing plants and three different lots in the municipalities of Maceió, Coruripe, Capela, Chã Preta and Quebrangulo. Positive coagulase staphylococci (PCS) was performed using Baird-Parker Agar medium, rabbit plasma and brain-heart infusion broth. The PCS were submitted to the antibiogram by means of inoculation in Müller Hinton broth and the discs were applied with the drugs: gentamicin (5μg), amoxicillin (10mcg), polymyxin (300mg), erythromycin (10μg), penicillin (10μg), ampicillin (10μg), tetracycline (30μg), sulfazotrim (25μg) and cephalexin (30μg). The phenotypic characterization of PCS was also performed by the ELISA plate adhesion method for biofilm formation. Of the 75 samples, 4 (5.3%) samples were identified with PCS. The count ranged from 1,2x104 a 2,6x105CFU/mL-¹. Isolated PCS showed 100% sensitivity to all antimicrobials tested and none were biofilm forming. The quality of pasteurized milk with state inspection of the study in relation to the presence of coagulase positive staphylococci is inadequate for consumption. The sensitivity of PCS to all antimicrobials and non-biofilm formation reduces the risk that these microorganisms could cause to public health.O leite é uma fonte alternativa de proteínas acessíveis à população de baixa renda, que geralmente é carente em proteína de origem animal, comprovando a importância socioeconômica deste alimento. Em saúde pública, em particular na área de vigilância sanitária de alimentos, o S. aureus, o principal dos estafilococos coagulase positiva, é considerado um dos mais frequentes causadores de surtos de doenças transmitidas por alimentos. O leite e derivados estão entre os alimentos mais envolvidos nos surtos identificados e estudados. Objetivou-se com este estudo avaliar a qualidade de leite pasteurizado com inspeção estadual pela pesquisa de estafilococos coagulase positiva, sua resistência antimicrobiana e a capacidade de formar biofilme. O estudo foi realizado em Alagoas e foram colhidos 75L de leite pasteurizado, de três estabelecimentos e de três lotes diferentes, nos municípios Maceió, Coruripe, Capela, Chã Preta e Quebrangulo. Realizou-se a pesquisa de estafilococos coagulase positiva (ECP) utilizando o meio Agar Baird-Parker e BHI. Das 75 amostras, foram identificadas 4(5,3%) amostras com ECP.  A contagem variou de 1,2x104 a 2,6x105 UFC/mL-¹. Os ECP foram submetidos ao antibiograma por meio da inoculação em Caldo Müeller Hinton e aplicados os discos com as drogas: gentamicina (5µg), amoxicilina (10mcg), polimixina (300mg), eritromicina (10µg), penicilina (10µg), ampicilina (10 µg), tetraciclina (30µg), sulfazotrim (25µg) e cefalexina (30µg). Realizou-se também a caracterização fenotípica dos ECP pelo método de aderência em placa de ELISA para formação de biofilme. Os estafilococos coagulase positiva isolados apresentaram 100% sensibilidade a todos os antimicrobianos testados e nenhum foi formador de biofilme. A qualidade do leite pasteurizado com inspeção estadual do estudo com relação à presença de estafilococos coagulase positiva não é adequada para o consumo. A sensibilidade dos estafilococos a todos antimicrobianos e a não formação de biofilme diminui o risco que esses microrganismos poderiam ocasionar à saúde pública

Frequência de genes codificadores de toxinas em Staphylococcus aureus isolados de leite de tanques expansão comunitários

RESUMO: A capacidade de produção de toxinas pelo Staphylococcus aureus no leite e produtos derivados está relacionado com surtos de intoxicação alimentar. Objetivou-se nesta pesquisa, estudar a ocorrência de genes que codificam para enterotoxinas estafilocócicas (sea, seb, sed, seg, seh e sei) e toxinas α e β hemolítica (hla e hlb) em S. aureus isolados de 53 amostras de leite de tanques expansão comunitários no Estado de Alagoas, Brasil. Foram identificados 27 isolados (50,94%) como S. aureus pela amplificação do gene nuc. 13/27 isolados (48,1%) foram positivos para pelo menos um gene das enterotoxinas estudadas, sendo as frequências dos genes sea 33,3%, seh 18,5%, sei 11,1% e sed 7,4%; não entanto não foram identificados os genes seb e seg nestas bactérias. Para as toxinas hemolíticas, 51,9% dos isolados portavam ambos genes (hla e hlb), sendo a frequência para o gene hla de 81,5% e para o gene hlb de 51,9%. A frequência de genes das toxinas avaliadas é alta o que constitui um risco potencial para a saúde pública em especial, as enterotoxinas por serem termoestáveis e estarem asssociados com surtos de intoxicação alimentar