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

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.

Download Full-text

The Effect of Antimicrobial Resistance Plasmids Carrying blaCMY-2 on Biofilm Formation by Escherichia coli from the Broiler Production Chain

Microorganisms ◽

10.3390/microorganisms9010104 ◽

2021 ◽

Vol 9 (1) ◽

pp. 104

Author(s):

Live L. Nesse ◽

Solveig S. Mo ◽

Silje N. Ramstad ◽

Ingun L. Witsø ◽

Camilla Sekse ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Resistance ◽

Biofilm Formation ◽

Monitoring Program ◽

Production Chain ◽

E Coli ◽

Microtiter Plates ◽

Broiler Production ◽

Biofilm Production ◽

Food And Feed

Extended-spectrum cephalosporin-resistant Escherichia coli (ESCR E. coli) with plasmids carrying the blaCMY-2 resistance gene have been isolated from the Norwegian broiler production chain through the Norwegian monitoring program for antimicrobial resistance in animals, food and feed, NORM-VET. The aim of the present study was to investigate the biofilm forming abilities of these strains, and in particular to see whether these might be influenced by the carriage of blaCMY-2 plasmids. The ESCR E. coli from the broiler production chain displayed relatively low biofilm forming abilities in the crystal violet biofilm assay as compared to quinolone-resistant E. coli (QREC) from the same population (mean ± SD = 0.686 ± 0.686 vs. 1.439 ± 0.933, respectively). Acquisition of two different blaCMY-2 plasmids by QREC strains reduced their biofilm production in microtiter plates, but not their biofilm production on Congo Red agar plates. Furthermore, motility was reduced, but not planktonic growth. We hypothesize that genes carried by these plasmids may have caused the observed reduction in biofilm formation, possibly mediated through changes in flagellar expression or function. Furthermore, this may help explain the different biofilm forming abilities observed between ESCR E. coli and QREC. The results also indicate that the risk of biofilm reservoirs of antimicrobial resistant E. coli on in the broiler production is lower for ESCR E. coli than for QREC.

Download Full-text

Inhibition of Bacterial Biofilm Formation by Phytotherapeutics with Focus on Overcoming Antimicrobial Resistance

Current Pharmaceutical Design ◽

10.2174/1381612826666200212121710 ◽

2020 ◽

Vol 26 (24) ◽

pp. 2807-2816 ◽

Cited By ~ 1

Author(s):

Yun Su Jang ◽

Tímea Mosolygó

Keyword(s):

Antimicrobial Resistance ◽

Biofilm Formation ◽

Antimicrobial Agents ◽

Bacterial Biofilm ◽

Experimental Investigations ◽

Resistant Bacteria ◽

Salmonella Spp ◽

Food Borne ◽

High Prevalence ◽

Scientific Attention

: Bacteria within biofilms are more resistant to antibiotics and chemical agents than planktonic bacteria in suspension. Treatment of biofilm-associated infections inevitably involves high dosages and prolonged courses of antimicrobial agents; therefore, there is a potential risk of the development of antimicrobial resistance (AMR). Due to the high prevalence of AMR and its association with biofilm formation, investigation of more effective anti-biofilm agents is required. : From ancient times, herbs and spices have been used to preserve foods, and their antimicrobial, anti-biofilm and anti-quorum sensing properties are well known. Moreover, phytochemicals exert their anti-biofilm properties at sub-inhibitory concentrations without providing the opportunity for the emergence of resistant bacteria or harming the host microbiota. : With increasing scientific attention to natural phytotherapeutic agents, numerous experimental investigations have been conducted in recent years. The present paper aims to review the articles published in the last decade in order to summarize a) our current understanding of AMR in correlation with biofilm formation and b) the evidence of phytotherapeutic agents against bacterial biofilms and their mechanisms of action. The main focus has been put on herbal anti-biofilm compounds tested to date in association with Staphylococcus aureus, Pseudomonas aeruginosa and food-borne pathogens (Salmonella spp., Campylobacter spp., Listeria monocytogenes and Escherichia coli).

Download Full-text

Motility activity, slime production, biofilm formation and genetic typing by ERIC-PCR for Pseudomonas aeruginosa strains isolated from bovine and other sources (human and environment)

Polish Journal of Veterinary Sciences ◽

10.2478/pjvs-2014-0044 ◽

2014 ◽

Vol 17 (2) ◽

pp. 321-329 ◽

Cited By ~ 2

Author(s):

K. Wolska ◽

P. Szweda ◽

K. Lada ◽

E. Rytel ◽

K. Gucwa ◽

...

Keyword(s):

Biofilm Formation ◽

Bovine Mastitis ◽

Twitching Motility ◽

Phenotypic Properties ◽

Biofilm Production ◽

Abiotic Surfaces ◽

Hospital Patients ◽

Relationship Of ◽

Initial Biofilm ◽

Different Sources

AbstractThe molecular-typing strategy, ERIC-PCR was used in an attempt to determine the genomic relationship of 28 P. aeruginosa strains isolated from faeces of healthy bovine, bovine mastitis and from faeces of hospital patients as well as from environment. ERIC-PCR fingerprinting revealed large molecular differentiation within this group of isolates. Twenty two out of 28 strains tested generated unique patterns of DNA bands and only three genotypes consisted of two isolates each were identified. We also tested the P. aeruginosa isolates for their ability to form a biofilm on abiotic surfaces including polyvinylchloride and polystyrene. Different biofilm-forming abilities were demonstrated among strains; however, most of them (64.3%) showed moderate-biofilm forming ability. The strains with increased swimming and twitching motility displayed elevated biofilm formation. However, a negative correlation was found between slime and initial biofilm production. On the basis of the results obtained, we suggest that there are no major differences in phenotypic properties between P. aeruginosa strains isolated from different sources

Download Full-text

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

Pesquisa Veterinária Brasileira ◽

10.1590/1678-5150-pvb-6645 ◽

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.

Download Full-text

Effects of Antibiotic, Nicotine and Aminoacid starvation stresses on biofilm production in respiratory Klebsiella pneumoniae

10.51429/ejmm30108 ◽

2021 ◽

Vol 30 (1) ◽

pp. 61-69

Author(s):

Rochell Davis and Paul D. Brown

Keyword(s):

Amino Acid ◽

Antimicrobial Resistance ◽

Klebsiella Pneumoniae ◽

Biofilm Formation ◽

Genetic Relatedness ◽

Amino Acid Starvation ◽

Biofilm Production ◽

Mar Index ◽

Type 3 ◽

Hospital Acquired

Background: Klebsiella pneumoniae is a major cause of hospital-acquired infections in Jamaica. Objective: We aimed to determine their antimicrobial resistance profiles and to assess biofilm formation in the presence of antibiotic, nicotine and amino acid starvation stresses. Methodology: Antimicrobial susceptibility and multiple antimicrobial resistance (MAR) index were determined for 23 K. pneumoniae strains. Biofilm production was evaluated in the presence of 50 μg/ml ceftazidime or gentamicin, 0–4 mg/ml nicotine, or 0.5 mg/ml serine hydroxamate (to induce amino acid starvation). Genetic relatedness, and the presence of type 3 fimbriae (mrkA) and determinants for extended spectrum β-lactamase and carbapenamases (bla-IMP, bla-VIM, bla-GIM and bla-SIM) were assessed by PCR-based amplification. Results: All strains were susceptible to imipenem (p<0.05); frequencies of resistance varied from 4% (for amikacin) and 8.7% (for meropenem) to over 30% for the other antimicrobials. About half of strains were resistant to ceftazidime, gentamicin and piperacillin. Mean MAR index was 0.31. The presence of antibiotics and nicotine at 2 and 4 mg/ml negatively affected biofilm formation for most strains. However, with amino acid starvation, almost 60% of strains retained medium or high biofilm production. Most strains harboured determinants for carbapenemase or metallo--lactamase, and one-third were PCRpositive for the OXA-1 gene. Strains were clustered into three groups based on ERICPCR analysis. Conclusion: These data suggest that certain antibiotics could inhibit biofilm production in K. pneumoniae even as multidrug resistance in this organism is evident. Further, this species has the propensity to harbour several genetic determinants for antimicrobial resistance.

Download Full-text

Staphylococcal slime layers and biofilm from different origins

Ciência Rural ◽

10.1590/0103-8478cr20180783 ◽

2019 ◽

Vol 49 (5) ◽

Cited By ~ 2

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.

Download Full-text

Ultrasensitive Label-Free Detection of Unamplified Multidrug-Resistance Bacteria Genes with a Bimodal Waveguide Interferometric Biosensor

Diagnostics ◽

10.3390/diagnostics10100845 ◽

2020 ◽

Vol 10 (10) ◽

pp. 845

Author(s):

Jesús Maldonado ◽

Ana Belén González-Guerrero ◽

Adrián Fernández-Gavela ◽

Juan José González-López ◽

Laura M. Lechuga

Keyword(s):

Antimicrobial Resistance ◽

Negative Impact ◽

Multidrug Resistant ◽

Therapeutic Interventions ◽

Great Promise ◽

Resistant Bacteria ◽

Label Free ◽

Multidrug Resistant Bacteria ◽

Label Free Detection ◽

Encoding Gene

Infections by multidrug-resistant bacteria are becoming a major healthcare emergence with millions of reported cases every year and an increasing incidence of deaths. An advanced diagnostic platform able to directly detect and identify antimicrobial resistance in a faster way than conventional techniques could help in the adoption of early and accurate therapeutic interventions, limiting the actual negative impact on patient outcomes. With this objective, we have developed a new biosensor methodology using an ultrasensitive nanophotonic bimodal waveguide interferometer (BiMW), which allows a rapid and direct detection, without amplification, of two prevalent and clinically relevant Gram-negative antimicrobial resistance encoding sequences: the extended-spectrum betalactamase-encoding gene blaCTX-M-15 and the carbapenemase-encoding gene blaNDM-5 We demonstrate the extreme sensitivity and specificity of our biosensor methodology for the detection of both gene sequences. Our results show that the BiMW biosensor can be employed as an ultrasensitive (attomolar level) and specific diagnostic tool for rapidly (less than 30 min) identifying drug resistance. The BiMW nanobiosensor holds great promise as a powerful tool for the control and management of healthcare-associated infections by multidrug-resistant bacteria.

Download Full-text

Biofilm formation by Prototheca zopfii isolated from clinical and subclinical bovine mastitis in distinct growth conditions under different dyes

Ciência Rural ◽

10.1590/0103-8478cr20180574 ◽

2019 ◽

Vol 49 (2) ◽

Cited By ~ 1

Author(s):

Valessa Lunkes Ely ◽

Letícia Trevisan Gressler ◽

Fernando Jonas Sutili ◽

Márcio Garcia Ribeiro ◽

Mateus Matiuzzi da Costa ◽

...

Keyword(s):

Crystal Violet ◽

Biofilm Formation ◽

Bovine Mastitis ◽

Growth Conditions ◽

Blue Dye ◽

Prototheca Zopfii ◽

Biofilm Production ◽

Cotton Blue ◽

Dynamic Growth

ABSTRACT: Prototheca spp. have been reported as an emergent environmental mastitis pathogen in several countries. Biofilm formation is a significant factor associated with different degrees of virulence developed by many microorganisms, including Prototheca spp. The present study aimed to compare two growth conditions and two staining dyes to determine which combination was more appropriate to evaluate qualitatively and quantitatively the production of biofilm by P. zopfii. Biofilm formation was evaluated in polystyrene microplates under static and dynamic growth conditions and staining with crystal violet or cotton blue dye. All P. zopfii isolates from cows with mastitis were classified as biofilm-producers in all growth conditions and staining. The cotton blue dye proved to be more appropriate method to classify the intensity of P. zopfii biofilm production.

Download Full-text

Temporal profile of antimicrobial resistance exhibited by strains of Staphylococcus spp. isolated from cases of bovine mastitis for 20 years (1992-2011)

Ciência Rural ◽

10.1590/0103-8478cr20140928 ◽

2015 ◽

Vol 45 (6) ◽

pp. 1035-1041

Author(s):

Ananda Paula Kowalski ◽

Grazieli Maboni ◽

Julia Pires Espindola ◽

Ariane Foletto ◽

Guerino Bandeira Junior ◽

...

Keyword(s):

Regression Analysis ◽

Antimicrobial Resistance ◽

Bovine Mastitis ◽

The Other ◽

In Vitro Susceptibility ◽

Resistance Patterns ◽

Susceptibility Tests ◽

Resistance Rates ◽

Staphylococcus Spp

Records of in vitro susceptibility tests performed between 1992 and 2011 were retrospectively reviewed in order to evaluate the dynamic profiles of possible changes in antimicrobial resistance of Staphylococcus spp. isolated from milk samples of cows with mastitis during two decades. The results of 2,430 isolates tested by disk diffusion technique for susceptibility to oxacillin, penicillin, ampicillin, cephalexin, norfloxacin, tetracycline, sulfazotrim, gentamicin, and neomycin were analysed. Comparisons were performed between the percentages of resistance to antimicrobials and their classes and also between the decades studied. Additionally, the possible tendency or changes in the behaviour of these pathogens against the major drugs used in the last two decades were evaluated using regression analysis. The highest rates of resistance (P<0.0001) were observed for the beta-lactams (34.3%), with exception of cephalexin (6.9%), and for the tetracyclines (28%). Similar resistance rates (7.6% to 15.7%) were observed among the other drugs. Regression analysis showed a reduction in resistance to penicillin and ampicillin throughout the period, whilst for oxacillin and neomycin a decrease in the resistance was observed during the first decade, followed by an increase. A trend towards decreased resistance was found for sulfazotrim, whereas for the other antimicrobials no decrease was observed. The results indicated no trend towards increased resistance for most antimicrobials tested. Nevertheless, it is necessary to monitor the resistance patterns of these pathogens in order to save these drugs as a therapeutic reserve

Download Full-text

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

Brazilian Journal of Development ◽

10.34117/bjdv7n7-144 ◽

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.

Download Full-text