The main spoilage-related psychrotrophic bacteria in refrigerated raw milk

Staphylococcus aureus is one of the most important pathogens of humans and animals. Livestock production contributes a significant proportion to the South African Gross Domestic Product. Consequently, the aim of this study was to determine for the first time the prevalence, virulence, antibiotic and heavy metal resistance in livestock-associated S. aureus isolated from South African livestock production systems. Microbial phenotypic methods were used to detect the presence of antibiotic and heavy metal resistance. Furthermore, molecular DNA based methods were used to genetically determine virulence as well as antibiotic and heavy metal resistance determinants. Polymerase chain reaction (PCR) confirmed 217 out of 403 (53.8%) isolates to be S. aureus. Kirby-Bauer disc diffusion method was conducted to evaluate antibiotic resistance and 90.8% of S. aureus isolates were found to be resistant to at least three antibiotics, and therefore, classified as multidrug resistant. Of the antibiotics tested, 98% of the isolates demonstrated resistance towards penicillin G. High resistance was shown against different heavy metals, with 90% (196/217), 88% (192/217), 86% (188/217) and 84% (183/217) of the isolates resistant to 1500 µg/mL concentration of Cadmium (Cd), Zinc (Zn), Lead (Pb) and Copper (Cu) respectively. A total of 10 antimicrobial resistance and virulence genetic determinants were screened for all livestock associated S. aureus isolates. Methicillin-resistant S. aureus (MRSA) isolates were identified, by the presence of mecC, in 27% of the isolates with a significant relationship (p < 0.001)) with the host animal. This is the first report of mecC positive LA-MRSA in South Africa and the African continent. The gene for tetracycline resistance (tetK) was the most frequently detected of the screened genes with an overall prevalence of 35% and the highest prevalence percentage was observed for goats (56.76%) followed by avian species (chicken, duck and wild birds) (42.5%). Virulence-associated genes were observed across all animal host species. The study reports the presence of luks/pv, a gene encoding the PVL toxin previously described to be a marker for community acquired-MRSA, suggesting the crossing of species between human and livestock. The high prevalence of S. aureus from the livestock indicates a major food security and healthcare threat. This threat is further compounded by the virulence of the pathogen, which causes numerous clinical manifestations. The phenomenon of co-selection is observed in this study as isolates exhibited resistance to both antibiotics and heavy metals. Further, all the screened antibiotic and heavy metal resistance genes did not correspond with the phenotypic resistance.

https://www.mdpi.com/2076-0817/8/2/79/pdf

Isolation and Molecular Identification of Virulence, Antimicrobial and Heavy Metal Resistance Genes in Livestock-Associated Methicillin-Resistant Staphylococcus aureus.

Thirty-three (33) isolates of methicillin-resistant Staphylococcus aureus (MRSA) from healthy edible marine fish harvested from two aquaculture settings and the Kariega estuary, South Africa, were characterised in this study. The phenotypic antimicrobial susceptibility profiles to 13 antibiotics were determined, and their antibiotic resistance determinants were assessed. A multiplex PCR was used to determine the epidemiological groups based on the type of SCCmec carriage followed by the detection of staphylococcal enterotoxin-encoding genes sea-sed and the Panton Valentine leucocidin gene (pvl). A high antibiotic resistance percentage (67-81%) was observed for Erythromycin, Ampicillin, Rifampicin, and Clindamycin, while maximum susceptibility to Chloramphenicol (100%), Imipenem (100%), and Ciprofloxacin (94%) was recorded. Nineteen (58%) of the MRSA strains had Vancomycin MICs of ≤2 μg/mL, 4 (12%) with MICs ranging from 4-8 μg/mL, and 10 (30%) with values ≥16 μg/mL. Overall, 27 (82%) isolates were multidrug-resistant (MDR) with Erythromycin-Ampicillin-Rifampicin-Clindamycin (E-AMP-RIP-CD) found to be the dominant antibiotic-resistance phenotype observed in 4 isolates. Resistance genes such as tetM, tetA, ermB, blaZ, and femA were detected in two or more resistant strains. A total of 19 (58%) MRSA strains possessed SCCmec types I, II, or III elements, characteristic of healthcare-associated MRSA (HA-MRSA), while 10 (30%) isolates displayed SCCmec type IVc, characteristic of community-associated MRSA (CA-MRSA). Six (18%) of the multidrug-resistant strains of MRSA were enterotoxigenic, harbouring the see, sea, or sec genes. A prevalence of 18% (6/33) was also recorded for the luk-PVL gene. The findings of this study showed that marine fish contained MDR-MRSA strains that harbour SCCmec types, characteristic of either HA-MRSA or CA-MRSA, but with a low prevalence of enterotoxin and pvl genes. Thus, there is a need for continuous monitoring and implementation of better control strategies within the food chain to minimise contamination of fish with MDR-MRSA and the ultimate spread of the bug.

/pdf/antibiotic-resistance-and-virulence-gene-characteristics-of-4t4127ghlo.pdf

Antibiotic Resistance and Virulence Gene Characteristics of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated from Healthy Edible Marine Fish.

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

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

The spore-forming microbiota is mainly responsible for the deterioration of pasteurized milk with long shelf life in the United States. The identification of these microorganisms, using molecular tools, is of particular importance for the maintenance of the quality of milk. However, these molecular techniques are not only costly but also labor-intensive and time-consuming. The aim of this study was to compare the efficiency of boiling in conjunction with four other methods for the genomic DNA extraction of sporulated bacteria with proteolytic and lipolytic potential isolated from raw milk in the states of Parana and Maranhao, Brazil. Protocols based on cellular lysis by enzymatic digestion, phenolic extraction, microwave-heating, as well as the use of guanidine isothiocyanate were used. This study proposes a method involving simple boiling for the extraction of genomic DNA from these microorganisms. Variations in the quality and yield of the extracted DNA among these methods were observed. However, both the cell lysis protocol by enzymatic digestion (commercial kit) and the simple boiling method proposed in this study yielded sufficient DNA for successfully carrying out the Polymerase Chain Reaction (PCR) of the rpoB and 16S rRNA genes for all 11 strains of microorganisms tested. Other protocols failed to yield sufficient quantity and quality of DNA from all microorganisms tested, since only a few strains have showed positive results by PCR, thereby hindering the search for new microorganisms. Thus, the simple boiling method for DNA extraction from sporulated bacteria in spoiled milk showed the same efficacy as that of the commercial kit. Moreover, the method is inexpensive, easy to perform, and much less time-consuming.

Efficiency of boiling and four other methods for genomic DNA extraction of deteriorating spore-forming bacteria from milk

Microbiota psicrotrófica deteriorante proteolítica e lipolítica do leite cru da região de castro-pr

A presença de Escherichia coli em um alimento indica contaminação microbiana de origem fecal, demonstrando condições higiênicas insatisfatórias. Outro aspecto a ser considerado é que alguns isolados de E. coli apresentam fatores de virulência que os tornam capazes de causar infecção intestinal. Esses fatores precisam ser pesquisados para que seja possível relacionar um alimento contaminado com E. coli como causa de infecção gastrointestinal. E. coli produtora de toxina Shiga (STEC) é uma importante causa de doença gastrintestinal em humanos, e pode resultar em sequelas com risco de vida como síndrome hemolítica urêmica (SHU) e púrpura trombocitopênica trombótica (PATON E PATON, 1998). E. coli enteropatogênica atípica (EPEC) para humanos carregam o gene eae, responsável pela produção de uma adesina conhecida como intima, causadora da lesão chamada attaching and effacing (A/E) (PATON E PATON, 1998), que causa uma dramática perda das vilosidades dos enterócitos (DONNENBERG et. al., 1992). E. coli enterohemorrágica (EHEC), que pode causar colite hemorrágica (CH), é produtora de toxina Shiga, portadora do gene eae e também apresenta gene para produção de hemolisina (PATON E PATON, 1998). O sorotipo de EHEC mais importante é O157:H7, mas outros sorotipos já foram descritos como produtores de citotoxinas (FRANCO E LANDGRAF, 2008). O objetivo do presente trabalho foi avaliar a presença dos genes stx1, stx2 e eae de cepas de E. coli isoladas de amostras de leite cru refrigerado.

Nayara Assis Augusto

Papers

Efficiency of boiling and four other methods for genomic DNA extraction of deteriorating spore-forming bacteria from milk

Microbiota psicrotrófica deteriorante proteolítica e lipolítica do leite cru da região de castro-pr

Pesquisa de genes de virulência de escherichia coli isoladas de leite cru refrigerado da região central do paraná