scholarly journals Staphylococcus spp. Isolated from Bovine Subclinical Mastitis in Different Regions of Brazil: Molecular Typing and Biofilm Gene Expression Analysis by RT-qPCR

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 888
Author(s):  
Priscila Luiza Mello ◽  
Danilo Flávio Moraes Riboli ◽  
Lisiane de Almeida Martins ◽  
Maria Aparecida Vasconcelos Paiva Brito ◽  
Cassiano Victória ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Antimicrobial Agents ◽  
Bovine Mastitis ◽  
Subclinical Mastitis ◽  
The Other ◽  
Coagulase Negative Staphylococci ◽  
Expression Of Genes ◽  
Staphylococcus Spp ◽  
Sequence Types ◽  
Icaa Gene

Bovine mastitis is mainly caused by bacteria of the genus Staphylococcus spp., which possess different virulence factors, including the capacity for biofilm formation that provides enhanced protection against the action of immune system components and serves as a barrier against the penetration of antimicrobial agents. This study aimed to characterize 181 Staphylococcus spp. Strains—including Staphylococcusaureus and coagulase-negative staphylococci (CoNS) isolated from bovine subclinical mastitis in six Brazilian states—by molecular methods. RT-qPCR was used to verify the expression of genes of the ica operon—mainly responsible for biofilm formation—as well as bap and bhp. Chromosome similarity among the isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The icaA gene was detected in 79 (43.6%) isolates, icaB in 24 (13.2%), icaC in 57 (31.4%), and icaD in 127 (70.1%). The bap gene was identified in 66 (36.4%) isolates, while the bhp gene was found in nine (4.9%). RT-qPCR confirmed the expression of the icaA gene in 60 (75.9%) isolates, of icaB in six (25%), of icaC in 26 (45.6%), and of icaD in 80 (63%). Clonal typing of the isolates by PFGE permitted the identification of eight Staphylococcusaureus clusters that simultaneously included ≥3 strains, with a similarity of ≥80%. Regarding the other species studied, three clusters were observed for Staphylococcuschromogenes and four clusters for Staphylococcusepidermidis. Only one cluster each was identified for Staphylococcussaprophyticus and Staphylococcussimulans, while the other species did not form any cluster. With respect to MLST, ST126 and ST1 were the prevalent sequence types in S. aureus, while in S.epidermidis all sequence types were different. These results reveal strains with the same evolutionary origin as other isolates, which might cause infections in humans and animals, suggesting their ability to spread between these species.

scholarly journals Expression of icaA and icaD genes in biofilm formation in Staphylococcus aureus isolates from bovine subclinical mastitis

2021 ◽  
Vol 41 ◽  
Author(s):  
Viviane F. Marques ◽  
Huarrisson A. Santos ◽  
Thomas H. Santos ◽  
Dayanne A. Melo ◽  
Shana M.O. Coelho ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Phenotypic Expression ◽  
Bovine Mastitis ◽  
Subclinical Mastitis ◽  
Transcriptional Analysis ◽  
Important Species ◽  
High Prevalence ◽  
Staphylococcus Spp

ABSTRACT: Staphylococcus spp. plays a significant role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most important species due to the high prevalence and the difficulty of in vivo treatment that is related to the expression of virulence factors and biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolated from bovine mastitis and to evaluate the expression and regulation of genes involved in its production. MALDI-TOF and phenogenotypic identification assays were performed to characterize the isolates. The phenotypic biofilm production and the presence of icaA and icaD and bap genes were evaluated. The Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Furtherly, Real-time PCR (qPCR) was performed at chosen times to quantify the expression of icaA, icaD and hld genes in three selected isolates. All 20 strains were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II showed a prevalence of 70%. Transcriptional analysis revealed increased expression of ica genes at eight hours of growth. These results confirm that polysaccharides production mediated by the icaADBC operon genes is an essential mechanism to the biofilm formation and contributes to the early stages of bacterial growth.

scholarly journals Staphylococcal slime layers and biofilm from different origins

2019 ◽  
Vol 49 (5) ◽  
Author(s):  
Rodrigo Casquero Cunha ◽  
Michelle Dias Hornes da Rosa ◽  
Cleomar da Silva ◽  
Francisco Denis Souza Santos ◽  
Fábio Pereira Leivas Leite
Keyword(s):  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Susceptibility Testing ◽  
Bovine Mastitis ◽  
Diffusion Method ◽  
Slime Layer ◽  
Gentamicin Resistance ◽  
Different Origins ◽  
Staphylococcus Spp

ABSTRACT: The genus Staphylococcus comprises some of the most important pathogenic bacteria for both humans and animals. It is responsible for bovine mastitis and canine otitis, besides being present in the microbiota of animals and as a contaminant in food. Its pathogenesis is related to the formation of capsule and biofilm, which contribute to its infectivity. The objective of this study was to observe the production of slime layer and formation of biofilm, which are related to the resistance to antimicrobial agents and presence of icaA and icaD genes, in 41 isolates of Staphylococcus spp. from different origins, provided by the Universidade Federal de Pelotas (UFPEL), Laboratório Regional de Diagnóstico (LRD). Strains of Staphylococcus spp. were cultivated in Congo red agar for capsule detection. Biofilm formation was detected using the 96-well microplate testing. Antimicrobial susceptibility testing was performed using the plate diffusion method. Part of the analyzed samples produced slime layer (36.6%) and formed biofilm (17.1%). However, six of those that formed biofilms were susceptible to the eight antibiotics tested in the antibiogram. In tests to determine the minimum bactericidal and inhibitory concentrations, gentamicin resistance of biofilm-forming strains was greater than that of non-forming strains. Ampicillin was the least effective antimicrobial drug (51%), followed by tetracycline (71%), neomycin (73%), and erythromycin (73%). Some isolates presented the icaA (6) and icaD (11) genes. Therefore, we suggested that the origin of an isolate can determine its expression of virulence factor and resistance to certain antibiotics.

Short Communication: Antibacterial activity of Cefquinome Sulfate in the presence of biofilm Staphylococcus spp isolated from bovine milk / Comunicação breve: Actividade antibacteriana do sulfato de Cefquinome na presença do biofilme Staphylococcus spp isolado do leite bovino

2021 ◽  
Vol 7 (7) ◽  
pp. 67261-67266
Author(s):  
Stéfani Thais Alves Dantas ◽  
Bruna Fernanda Rossi ◽  
Caroline Faganello ◽  
Ivana Giovannetti Castilho ◽  
Gousilin Leandra Rocha Da Silva ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Short Communication ◽  
Bovine Milk ◽  
Bovine Mastitis ◽  
Generation Cephalosporin ◽  
The Other ◽  
Fourth Generation ◽  
Persistent Infections ◽  
Lactating Cows ◽  
Staphylococcus Spp

Bovine mastitis is an inflammation of the mammary gland in dairy cattle usually caused by bacterial agents and cefquinome is a fourth-generation cephalosporin commonly used in treatment of this disease caused by Staphylococcus spp. in lactating cows due to a broad spectrum. On the other hand, biofilm formation provides protection for bacteria increasing the resistance to antibiotics and contributing to the persistence of infection. The objective of the present study was to assess if biofilm produced by Staphylococcus spp was able to protect the bacteria from cefquinome sulfate (CFQ) action for 24h exposure. Our findings showed that 95% (n=53/56) of Staphylococcus spp. were biofilm producer and the biofilm protected the most of species tested, except S. xylosus maybe due to the formation of a thin layer biofilm that did not interfere in the antimicrobial action. Thus, the effectiveness of cefquinome in persistent infections may be compromised due to a thicker biofilm.

scholarly journals The icaA gene in staphylococci from bovine mastitis

2013 ◽  
Vol 7 (07) ◽  
pp. 556-560 ◽  
Author(s):  
María Valeria Rumi ◽  
Miguel J Huguet ◽  
Adriana B Bentancor ◽  
Elida R Gentilini
Keyword(s):  
Staphylococcus Aureus ◽  
Bovine Mastitis ◽  
Pcr Primers ◽  
Polysaccharide Intercellular Adhesin ◽  
Coagulase Negative Staphylococci ◽  
Biofilm Production ◽  
Consensus Region ◽  
Staphylococcus Spp ◽  
First Time ◽  
Icaa Gene

Introduction: Staphylococcus aureus and coagulase-negative staphylococci (CNS) are frequently isolated from cows with mastitis. A main virulence factor of CNS is the ability to adhere and form biofilms. The intercellular gene cluster adhesion (ica) operon is one factor involved in biofilm production although ica-independent factors are also involved. Previous reports based on the results of S. epidermidis and S. aureus suggested that ica is highly conserved between species, but this detection decreases in other CNS biofilm producers. In this study we evaluated the presence of the icaA gene in strains of Staphylococcus spp. isolated from the milk of bovines with mastitis. Methodology: Thirty-seven staphylococci strains were evaluated by detecting the icaA gene. A new set of PCR primers was designed by consensus region of eight staphylococci from GenBank. Species characterization was performed using the Kloos and Schleifer scheme. Results: We identified the presence of the gene in S. aureus (n:4), S. chromogenes (n:4), and S. sciuri strains (n:2). We also, identified the presence of the gene in S. xylosus (n:5) for the first time. The icaA gene was not detected in S. capitis (n:1), S. epidermidis (n:2), S. hominis (n:2), S. saccharolyticus (n:1), S. simulans (n:4) and S. saprophyticus (n:3). The icaA gene was detected in 40.54% (15/37) of the CNS evaluated. Conclusions: Our results confirm the presence of the ica operon in various species of CNS pointing to polysaccharide intercellular adhesin (PIA) as the most important component for the formation of biofilms.

scholarly journals Etiology, antimicrobial susceptibility profile of Staphylococcus spp. and risk factors associated with bovine mastitis in the states of Bahia and Pernambuco

2013 ◽  
Vol 33 (5) ◽  
pp. 601-606 ◽  
Author(s):  
Carina C. Krewer ◽  
Izabela P. de S. Lacerda ◽  
Evandro S. Amanso ◽  
Noelly B. Cavalcante ◽  
Rodolfo de M. Peixoto ◽  
...  
Keyword(s):  
Risk Factors ◽  
Antimicrobial Susceptibility ◽  
Antimicrobial Agents ◽  
Bovine Milk ◽  
Bovine Mastitis ◽  
Subclinical Mastitis ◽  
Control Measures ◽  
Factors Associated ◽  
Antimicrobial Susceptibility Profile ◽  
Staphylococcus Spp

The purpose of this paper was to study the etiology of mastitis, determine the antimicrobial susceptibility profile of Staphylococcus spp. and to identify the risk factors associated with infection in dairy cows in the states of Bahia and Pernambuco, Brazil. From the 2,064 milk samples analyzed, 2.6% were associated with cases of clinical mastitis and 28.2% with subclinical mastitis. In the microbiological culture, Staphylococcus spp. (49.1%) and Corynebacterium spp. (35.3%) were the main agents found, followed by Prototheca spp. (4.6%) and Gram negative bacilli (3.6%). In the antimicrobial susceptibility testing, all 218 Staphylococcus spp. were susceptible to rifampicin and the least effective drug was amoxicillin (32.6%). Multidrug resistance to three or more drugs was observed in 65.6% of Staphylococcus spp. The risk factors identified for mastitis were the extensive production system, not providing feed supplements, teat drying process, not disinfecting the teats before and after milking, and inadequate hygiene habits of the milking workers. The presence of multiresistant isolates in bovine milk demonstrates the importance of the choice and appropriate use of antimicrobial agents. Prophylactic and control measures, including teat antisepsis and best practices for achieving hygienic milking should be established in order to prevent new cases of the disease in herds.

Biofilm-formation by Staphylococcus aureus and Staphylococcus epidermidis isolates from subclinical mastitis in conditions mimicking the udder environment

2015 ◽  
Vol 18 (4) ◽  
pp. 787-792 ◽  
Author(s):  
R. Seixas ◽  
D. Varanda ◽  
R. Bexiga ◽  
L. Tavares ◽  
M. Oliveira
Keyword(s):  
Biofilm Formation ◽  
Antimicrobial Agents ◽  
Bovine Mastitis ◽  
Subclinical Mastitis ◽  
Protective Role ◽  
Dynamic Conditions ◽  
Biofilm Production ◽  
Static Conditions

AbstractStaphylococcusis the genus most commonly isolated from bovine mastitis in many countries. It may express several virulence factors including biofilm formation, which may protect the bacterial community from antimicrobials’ action, preventing these compounds from reaching its interior, where they reach subinhibitory concentrations (subMIC).Most biofilm production assays are performed in static conditions, while studies regarding antimicrobial resistance usually do not resemble the udder environment because they are performed at high concentrations. In this study we evaluated the influence of dynamic conditions and media, including Mueller Hinton Broth (MHB) and UHT whole milk (WM), as well as the effect of subMIC concentrations of five different antimicrobial agents on biofilm formation by staphylococci isolated from subclinical mastitis. Results suggest that dynamic conditions and media may influence biofilm formation and revealed that milking simulation may significantly increase biofilm production. Sub-MIC concentrations decrease biofilm formation in MHB but increase in WM, suggesting a protective role of milk against antimicrobial compounds’ action. Therefore,in vitroconditions that simulate the udder environment andin vivoconditions should be included as one of the parameters in evaluation of biofilm producing strains, in order to provide more reliable results.

scholarly journals Expression of genes encoding resistance in Staphylococcus spp. isolated from bovine subclinical mastitis in Brazil

2020 ◽  
Vol 14 (07) ◽  
pp. 772-780
Author(s):  
Eveline Zuniga ◽  
Nilson Roberti Benites ◽  
Aline Santana da Hora ◽  
Priscila Luiza Mello ◽  
Marco Antonio Laes ◽  
...  
Keyword(s):  
Methicillin Resistance ◽  
Meca Gene ◽  
Bovine Mastitis ◽  
Subclinical Mastitis ◽  
Gene Encoding ◽  
Resistance Factors ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Staphylococcus Spp

Introduction: Staphylococci are the most important agents associated with bovine mastitis. This study aimed at characterizing resistance factors to antimicrobials in Staphylococcus spp. isolated from the milk of cows with subclinical mastitis. Methodology: In vitro resistance of 243 Staphylococcus spp. isolates to antimicrobials commonly used in clinical practice was evaluated. The detection and expression of genes encoding resistance mecA (gene encoding penicillin binding protein 2a) mecALGA251 (mecA homologue), blaZ (gene encoding penicillin resistance), femA and femB (genes encoding essential factors - A and B - for the expression of methicillin resistance) and aacA-aphD (gene encoding for a bifunctional enzyme that confers resistance to gentamicin) using PCR and RT-PCR was investigated. Results: One or more genes encoding resistance to different antimicrobials were detected in 184 Staphylococcus spp. samples. The femA and femB genes were the most frequent. Regarding the variables’ detection (N = number of strains) and expression (% of strains), the following results were obtained: blaZ (N = 40 – 82.5%), femA (N = 147 – 47.6%), aacAaphD (N = 30 – 43.3%), femB (N = 138 – 29.7%), mecA (N = 33 – 27.3%), mecALGA251 (N = 01 – 0.0%). There was a higher occurrence of phenotypic resistant strains for amoxicillin, ampicillin and penicillin in isolates positive for detection and/or expression of blaZ gene when compared with the other genes. Conclusions: The present study provides new information on genotypic traits of Staphylococcus isolates from bovine subclinical mastitis especially regarding the evaluation of expression of genes associated with antimicrobial resistance in Staphylococcus spp. using molecular tools.

scholarly journals Phenotypic and Genotypic Characteristics and Resistance Profile of Staphylococcus spp. from Bovine Mastitis

2020 ◽  
Vol 48 ◽  
Author(s):  
Érica Chaves Lucio ◽  
Gisele Veneroni Gouveia ◽  
Mateus Matiuzzi Da Costa ◽  
Mário Baltazar De Oliveira ◽  
Rinaldo Aparecido Mota ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Milk Production ◽  
Biofilm Formation ◽  
Negative Impact ◽  
Meca Gene ◽  
Bovine Mastitis ◽  
Resistant Bacteria ◽  
Production Chain ◽  
Biofilm Production ◽  
Staphylococcus Spp

Background: Bovine mastitis remains one of the health problems that cause the most damage to milk producers. The negative impact of mastitis is due to reduced milk production, early slaughter of females, reduced commercial value of the affected animals, losses in the genetic potential of the herd, expenses with medicines and veterinary medical assistance. Staphylococcus spp. stands out as the cause of this disease and has been able to remain in the mammary gland, becoming resistant to several antimicrobials. The aims of the present study were to characterize the phenotypes, genotypes and resistance profiles of Staphylococcus spp. isolates from bovine mastitis cases in the state of Pernambuco, Brazil.Materials, Methods & Results: These isolates were classified according to biochemical tests and the presence of the nuc gene.  The polymerase chain reaction (PCR) for amplification of the mecA and blaZ genes was used to analyze the genetic potentials of antimicrobial resistance. Isolates were also phenotypically tested for resistance to nine antimicrobials (ampicillin, doxicillin, erythromycin, gentamicin, rifampicin, cephalothin, amoxicillin, nalidixic acid and oxacillin). The genetic potentials for biofilm production were evaluated by the amplifications of the icaD, icaA and bap genes. The phenotypic test of gentian violet was used for biofilm formation analyzes. Ninety-three (93.0%) of the isolates among the Staphylococcus spp. samples were classified as Staphylococcus aureus. The lowest percentage of sensitivity observed was for amoxicillin (28.0%). All of the isolates were sensitive to erythromycin and gentamicin, and 15 (15%) exhibited sensitivity to all of the drugs tested. All of the isolates were negative for the mecA gene, and 36 (36%) were positive for blaZ. In the adhesion microplate tests, 44 (44%) of the isolates were capable of biofilm formation. Of these, seven (15.9%) were strong formers, whereas 16 (36.3%) and 21 (47.8%) were moderate and weak formers, respectively. The icaD gene was confirmed in 89 (89%) of the isolates. The icaA gene was confirmed in 61 (61%) samples, and the bap gene in 52 (52%) samples. One of the samples did not possess icaA, icaD or bap and exhibited moderate biofilm formation according to the microplate adherence test. Sixteen isolates simultaneously exhibited the three genes tested for biofilm production (icaA, icaD and bap) and were negative according to the microplate adherence test.Discussion:  The indiscriminate use of antibiotics to treat mastitis is a common practice in the study area, which may have contributed to the high proportion of herds (88.23%; 15/17) with multi-resistant isolates, constituting a selection factor for the dissemination of resistant bacteria among herds.  The absence of the mecA gene in the present study may be associated with the development of resistant bacteria through another mechanism, such as the overproduction of beta-lactamases. The results demonstrate that antimicrobial resistance occurs in Staphylococcus spp. that cause bovine mastitis in herds of Pernambuco and that these isolates have the a great capacity for biofilm formation. It is necessary to sensitize the professionals involved in the milk production chain of Brazil regarding the importance of the adequate use of antimicrobials for the treatment and control of mastitis, since studies in the country indicate the dissemination of resistant bacterial strains.

scholarly journals An Overview of Specific Pathogens in Goat Mastitis

2019 ◽  
Vol 76 (2) ◽  
pp. 118
Author(s):  
Cristiana Ștefania NOVAC ◽  
Sanda ANDREI ◽  
Nicodim Iosif FIȚ
Keyword(s):  
Somatic Cell Count ◽  
Goat Milk ◽  
Subclinical Mastitis ◽  
Clinical Mastitis ◽  
Economic Consequences ◽  
The Other ◽  
Aetiological Agent ◽  
Dairy Goats ◽  
Coagulase Negative Staphylococci ◽  
Intramammary Infections

Goat milk ranks fourth in terms of global milk production and lately it has become increasingly popular among consumers. Unfortunately, mastitis is one of the most common diseases that affects dairy goats, with serious economic consequences and food safety matters. The prevalence of clinical mastitis is lower than 5% and the main aetiological agent is S. aureus. On the other hand, the prevalence of subclinical mastitis is between 5-30%, with coagulase negative staphylococci (CNS) representing the most often isolated microorganisms.The aim of this paper is to highlight the main aspects regarding the aetiology of goat mastitis, as well as the importance of the milk somatic cell count (MSCC) in the diagnosis process. Although the inflammation of the mammary gland in goats is not as frequently diagnosed compared to cow mastitis, there are several aspects worth discussing in order to fully understand the pathogenesis of intramammary infections.

scholarly journals Risk factors associated with the occurrence of multiresistant Staphylococcus spp. isolated from bovine subclinical mastitis in northern Brazil

2022 ◽  
Vol 43 (2) ◽  
pp. 901-910
Author(s):  
Lilian Bernardina Ferreira ◽  
◽  
Larissa de Freitas Santiago Israel ◽  
Renata Fernandes Rabello ◽  
Guilherme Nunes de Souza ◽  
...  
Keyword(s):  
Risk Factors ◽  
Multidrug Resistance ◽  
Phenotypic Expression ◽  
Bovine Mastitis ◽  
Subclinical Mastitis ◽  
Isolation And Identification ◽  
Phenotypic Profile ◽  
Milk Samples ◽  
California Mastitis Test ◽  
Staphylococcus Spp

Staphylococcus bacteria are often associated with subclinical bovine mastitis. This study aimed to identify multiresistant Staphylococcus spp. associated with subclinical mastitis and the associated risk factors. Twenty-three dairy farms with a history of decrease in milk production, located in the lower Acre region, Brazil, were selected. An epidemiological questionnaire was provided in all farms. All animals were examined using the California Mastitis Test (CMT) and their milk samples were collected for bacterial culture. After isolation and identification, the disk diffusion antimicrobial susceptibility test was performed against nine classes of antimicrobials. Of the 339 cows examined using the CMT, 108 had mastitis. A total of 229 milk samples were collected from individual teats. MALDI-TOF MS found isolates belonging to eight species of Staphylococcus, in 101 of these samples. S. chromogenes (58.4%) demonstrated strongest resistance to the nine classes of antimicrobial active principles. Nineteen isolates with multidrug resistance phenotypic profile were identified. This phenotypic expression indicates wide circulation of resistant genes in this species. The presence of multidrug resistance in Staphylococcus spp. in this study was correlated with lack of water for cleaning the corral, which is a preventive factor, minimizing the transmission and persistence of pathogens in the farms.

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