Genetic traceability of Staphylococcus aureus strains isolated from primiparous dairy cows mastitis, humans and environment in the Northeast region of Brazil

Rastreabilidade genética de cepas de Staphylococcus aureus isoladas de mastite de vacas primíparas, ordenhadores e ambiente de ordenha na região Nordeste do Brasil

ABSTRACT:

This research aimed to investigate the genotypic relatedness of 18 Staphylococcus aureus strains isolated from intramammary infections in primiparous cows and extramammary sites on five dairy herds by rep-PCR using RW3A primers, and by PFGE using the endonuclease SmaI. The isolates were also evaluated in vitro for the susceptibility against beta-lactam antimicrobials drugs (penicillin and oxacillin), considering that beta-lactams are frequently used for treating staphylococcal intrammamary infections. The rep-PCR typing was highly discriminatory (D value= 0.9804) and a total of 15 patterns were detected. The PFGE method was also highly discriminatory (D value= 0.9667) and a total of 13 patterns were observed. A total of 15 out of 18 (83%) isolates were resistant to penicillin and one out of 18 (6%) to oxacillin. In conclusion, these findings confirmed the occurrence of a high genetic diversity of S. aureus strains at the herds and the presence of clonally-related strains only at the same herd, emphasizing a variety of genotypic profiles among the isolates.

Key words:
staphylococcal intramammary infection; DNA fingerprinting; drug resistance; beta-lactam; heifer

RESUMO:

Objetivou-se com este estudo investigar a correlação genética de 18 cepas de Staphylococcus aureus isoladas de infecções intramamárias em vacas primíparas e de locais extramamários em cinco propriedades leiteiras através das técnicas de PCR por sequências palindrômicas extragênicas repetitivas (rep-PCR), usando iniciadores RW3A, e de eletroforese em gel de campo pulsado (PFGE), usando a endonuclease SmaI. Os isolados também foram avaliados in vitro quanto à suscetibilidade aos antimicrobianos beta-lactâmicos (penicilina e oxacilina). A tipagem por rep-PCR foi altamente discriminatória (valor D = 0,9804) e um total de 15 padrões foram detectados. Os isolados de S. aureus foram agrupados em três grupos diferentes (A a C), com 80% de similaridade. A técnica de PFGE também foi altamente discriminatória (valor D = 0,9667) e um total de 13 padrões foi observado. A análise do dendrograma com um coeficiente de similaridade de 80% gerou dois grupos diferentes (A e B). Além disso, cepas clonais isoladas do leite foram identificadas na mesma propriedade pelos dois métodos de tipificação e, apesar da presença de cepas dominantes, nossos resultados sugerem uma alta diversidade genética dentre as cepas de S. aureus analisadas. Um total de 15, dos 18 (83%) isolados, eram resistentes à penicilina e um dos 18 (6%) à oxacilina. Assim, esses achados confirmam a ocorrência de uma alta diversidade genética de cepas de S. aureus nas propriedades e a presença de cepas clonalmente relacionadas apenas na mesma propriedade, enfatizando uma variedade de perfis genotípicos entre os isolados.

Palavras-chave:
infecção intramamária estafilocócica; impressão digital genética; resistência antimicrobiana; beta-lactâmico; primípara

INTRODUCTION:

Primiparous dairy cows are of key significance for every dairy herd as their mammary gland health status will directly impact the longevity of the animals and herd productivity. Despite of the common sense that they are not susceptible to intramammary infections (IMIs), an increasing number of recent studies have reported cases of mastitis in primiparous cows, mainly attributed to Staphylococcus spp. (ANDERSON et al., 2012; STALDER et al., 2014; BAŞTAN et al., 2015).

There are still a lot of uncertainties about the epidemiology of these IMIs and Staphylococcus aureus has been considered an important contagious agent transmitted among lactating animals (BAŞTAN et al., 2015). However, previous researches suggest humans and environment as potential sources of S. aureus causing mastitis in dairy heifers (STALDER et al., 2014; BAŞTAN et al., 2015). Furthermore, emerging antimicrobial resistance in S. aureus has posed a serious problem to the treatment of mastitis in dairy herds (DIAS et al., 2015).

Considering the important role of Staphylococcus aureus on the mastitis etiology in primiparous cows, the objective of this study was to investigate the genotypic relatedness of S. aureus isolates causing IMIs in dairy heifers and extramammary sites on five dairy herds located in the dairy region of Pernambuco State, Brazil.

MATERIALS AND METHODS:

Sample collection

The study was conducted on five medium-sized dairy farms (1, 2, 3, 4 and 5) in the dairy region of Pernambuco State, Northeastern Brazil (Figure 1), between 2018 and 2019. The number of lactating cows was over 100 cows per farm, including primiparous dairy cows. These five dairy farms had a total of 60 primiparous dairy cows in lactation, being the milk from all those sampled. A total of 240 individual quarter milk samples were aseptically collected from all primiparous dairy cows. Furthermore, samples from primiparous cows’ teat skin, dairy environment (mechanical milking equipment) and milkers (nostril and hands) were obtained by using sterile swabs. A total of 60 samples of teat swabs were collected using sterile swabs moistened with sterile saline to swab the teat skin from front and rear teats. Also, 21 samples of teat sets (forming pools of each equipment) were collected in the after milking period using sterile swabs and a total of 16 samples of nostrils (n=8) and hands (n=8) of 8 consenting milkers were sampled in the intermilking period using sterile swabs.

Figure 1:
Distribution map of the municipalities sampled, located in the dairy region of Pernambuco State, Northeastern Brazil.

Isolation and preliminary identification of Staphylococcus aureus

Isolation of Staphylococcus aureus was performed by direct plating of all samples on Mannitol salt agar (Difco Laboratories Inc., Detroit, USA) with 5% egg yolk (HiMedia, Mumbai, India) and incubated aerobically at 37ºC (±1°C) with readings after 24, and 48 hours to identify colony morphology. Bacteria were isolated and identified by their colony morphology, mannitol fermentation, Gram stain results, and biochemical test results (catalase and coagulase tests) according to NMC (1990) and Quinn et al. (2002). Subsequently, the isolates were subjected to molecular techniques in order to obtain an accurate identification and characterization.

Confirmation of Staphylococcus aureus

Species confirmation was achieved by Polymerase Chain Reaction (PCR) for amplification of the 279pb nuc gene (KATEETE et al., 2010), using the ATCC^® 43300 S. aureus subspecies aureus strain as a positive control and DNA Free Water (QIAGEN, Hilden, Germany) as a negative control. Genomic DNA extraction of all isolates was conducted according to MALIK et al., (2007), with modifications. All isolates were grown overnight at 37˚C on Mannitol Salt Agar (Difco Laboratories Inc., Detroit, USA) enriched with egg yolk emulsion (HiMedia, Mumbai, India), and then an inoculation loop (1-microliter) was used to pick up a sample of each colony and to place each one inside a tube containing 200mL of TE buffer to suspend it. The suspension was boiled at 90˚C for 15 min and was kept on ice for 30 minutes. After that, the suspension was centrifuged at 13,200 rpm for 5 min in a centrifuge and the supernatants were kept on ice. Subsequently, DNA concentration and purity were assessed using a spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA) with absorbance at 260nm (BRAKSTAD et al., 1992).

Polymerase Chain Reaction (PCR) testing for beta-lactam resistance genes

PCR was performed for amplification of blaZ gene, which encodes beta-lactamases, as previously described by SAWANT et al. (2009) and mecA and mecC genes, which are inducers of the beta-lactam site of action modification, according to NAKAGAWA et al. (2005) and PATERSON et al. (2012), respectively.

Reactions were assembled separately for each gene in a final volume of 15μL per well, containing 100ng of DNA template, 10pmol of each oligonucleotide (Table 1), Taq buffer (10mM Tris, 50mM KCl, 2.5mM MgCl2), 200mM dNTPs and 1U Taq DNA polymerase (Cenbiot, Taq DNA polymerase, Ludwig Biotec, Porto Alegre, RS, Brazil). Thermal profiles of amplifications were 4 min. at 94 °C, followed by 32 cycles of denaturation at 94 °C for 30 sec., annealing at 50.5 °C for 30 sec. (blaZ gene) or 55 °C (mecA and mecC genes) and extended at 72 °C for 30 sec., with final extension at 72 °C for 5 min. Then 10μL of each reaction was electrophoresed for 40 minutes at 100V in 1.5% agarose gel stained with BlueGreen, visualized and photodocumented under ultraviolet light.

Table 1
Sequences of the oligonucleotides used in this study and sizes of the amplified fragments, in base pairs (bp).

Primer	Sequence (5'-3')	Amplicon (bp)	Thermocycling conditionsa	Reference
blaZ	F-AAGAGATTTGCCTATGCTTC R-GCTTGACCACTTTTATCAGC	517	1	Sawantet al. 2009
mecA	F-TGGTATGTGGAAGTTAGATTGGGAT R-CTAATCTCATATGTGTTCCTGTATTGGC	155	1	Nakagawa et al. 2005
mecC	F-CATTAAAATCAGAGCGAGGC R-TGGCTGAACCCATTTTTGAT	188	2	Paterson et al. 2012

• ^a1 = 4 min. at 94 °C, followed by 32 cycles of denaturation at 94 °C for 30 sec., annealing at 50.5°C for 30 sec. and extended at 72 °C for 30 sec., with final extension at 72°C for 5 min.; 2 = 4 min. at 94 °C, followed by 32 cycles of denaturation at 94 °C for 30 sec., annealing at 55 °C for 30 sec. and extended at 72 °C for 30 sec., with final extension at 72 °C for 5 min.

Beta-lactam susceptibility test

Isolates were suspended in brain-heart infusion (BHI) broth and the suspension was adjusted to a turbidity equivalent to 0.5 McFarland standards. Drug susceptibility testing was performed by Minimum Inhibitory Concentration (MIC) technique using microdilutions of penicillin and oxacillin (CLSI, 2018). Afterwards, the isolates were categorized as susceptible, intermediate and resistant based upon interpretative criteria developed by the National Committee of Clinical Laboratory Standards. The ATCC^® 43300 S. aureus subspecies aureus strain was used as a recommended quality control reference organism to be run with each group of unknowns.

DNA fingerprinting by rep-PCR

Repetitive element palindromic-polymerase chain reaction (rep-PCR) assay was carried out as described by VAN DER ZEE et al.(1999) to identify possible transmission routes of S. aureus among herds. Genomic DNA was extracted (CHAPAVAL et al., 2006) and 25-μL reactions contained Taq DNA polymerase buffer (1X), MgCl2 (3 mM), Taq DNA polymerase (1 U; Invitrogen, USA), dNTPs (200 μM each; Ludwing Biotec, Brazil), primer RW3A (VAN DER ZEE et al., 1999) (1 pMol; Invitrogen, USA), and DNA template (100 ng). Amplification cycles included 1 min at 94 °C, 1 min at 50 °C, and 2 min at 72 °C. Amplified products were separated by electrophoresis in a 1.5% agarose gel in 0.5X TBE buffer at a constant voltage of 4 V/cm and stained using ethidium bromide (0.5 μg/ml). The PCR pattern analysis was performed as was described previously. Dendograms was built by an unweighted pair group method with arithmetic mean clustering algorithm (UPGMA) and the genetic similarity between isolates was calculated using the Jaccard’s coefficient (1% tolerance), using BioNumerics ^® software (Version 7.1, Applied Maths, Belgium).

DNA fingerprinting by PFGE

Genomic DNA from 2 ml of overnight cultures was prepared in low-melting-point agarose plugs as described by ANDRÉ et al. (2008), and digested with the endonuclease SmaI (New England Biolabs, France) according to the manufacturer’s instructions. The PFGE pattern analysis was performed as described by TENOVER et al. (1995). Dendrograms was built by an unweighted pair group method with arithmetic mean clustering algorithm (UPGMA) and the genetic similarity between isolates was calculated using the Dice’s coefficient (5% tolerance), using BioNumerics software (Version 7.1, Applied Maths, Belgium).

Discriminatory power of the typing methods

The discriminatory power was assessed using the Simpson’s index of diversity (D) that indicates the average probability that a typing system will assign a different type to two unrelated strains randomly sampled from a population (HUNTER, 1990).

RESULTS AND DISCUSSION:

In this study, a total of 18 S. aureus strains were obtained from the five dairy farms, named Farm 1 (n=12; 11 from milk and 1 from teat swab), Farm 2 (n=1; from human nostril), Farm 3 (n=1; from human nostril), Farm 4 (n=2; 1 from milk and 1 from human nostril) and Farm 5 (n=2; 1 from human hand and 1 from human nostril). All 18 isolates were confirmed to be S. aureus by PCR for nuc gene and the presence of the blaZ gene was detected in 100% (18/18) of them; however, mecA and mecC were not detected among the isolates. Considering the antimicrobial susceptibility testing results, approximately 83% (15/18) of the strains were resistant to penicillin and 6% (1/18) to oxacillin.

The detection of blaZ gene in all S. aureus isolates was mostly compatible to the phenotypic resistance profiles of penicillin and this usually occurs due to beta-lactamases action, whose production is induced by blaZ gene (DIAS et al., 2015). Also, the high occurrence of blaZ gene is in agreement with a study conducted by KREWER et al. (2015) in the Brazilian Northeast region, strongly suggesting its great epidemiological importance as a mechanism of beta-lactam resistance in staphylococci in dairy herds.

The identification of one S. aureus isolate (from milk sample) showing phenotypic resistance to oxacillin and lacking both mecA and mecC genes could be explained by other resistance mechanisms, such as the occurrence of putative homologous genes (MENDONÇA et al., 2012), the production of other classes of Penicillin-binding proteins (MENDONÇA et al., 2012), due to a beta-lactamase hyperproduction (MENDONÇA et al., 2012), or mediated by efflux pumps (JANG, 2016). These mechanisms may be used by the bacteria as a defense strategy, avoiding drug effectiveness and allowing phenotypic resistance.

The rep-PCR method was highly discriminatory (D value= 0.9804) and a total of 15 patterns were observed. The S. aureus isolates were grouped in three different clusters (A to C) at 80% similarity (Figure 2). The isolates from human origin were not related to those from animal and environmental sources. Conversely, SILVA et al. (2020) reported the isolation of clonally-related strains of S. aureus in milk, milker’s hand swabs and milking bucket samples from the same herd. At a 100% similarity level, most of the isolates showed unique genotypic patterns and only three clusters (I to III) containing clonal isolates were identified. A highly diversity of genotypic patterns were observed among isolates within the same farm. On the contrary, REINOSO et al. (2007) reported that genotypic patterns were similar in the different herds assessed during their study, suggesting a close genetic relationship between herds.

Figure 2:
Dendrogram showing the genotypic relatedness of 18 Staphylococcus aureus isolated from different sources in five dairy herds in Pernambuco State, Northeastern Brazil, which shows three clusters (A-C) at 80% similarity among the band profiles. Dendrogram was built based on the UPGMA and genetic similarity using the Jaccard’s coefficient (1% tolerance) of the genotypic band patterns generated by rep-PCR, using the RW3A primer.

By PFGE, using SmaI as the restriction enzyme, samples 15 and 18, both from human nostrils, could not be lysed. These isolates were; therefore, excluded from the analysis. Furthermore, the PFGE method was highly discriminatory (D value = 0.9667) and a total of 13 patterns were observed. The dendrogram analysis with an 80% similarity coefficient generated two different clusters (A and B), all from Farm 1 (Figure 3). At a 100% similarity level, most of the isolates showed unique genotypic patterns and only two pulsotypes (I and II) containing clonal isolates were identified. The majority of the strains were grouped in pulsotype I. These findings are in agreement with the results reported by GIRARDINI et al., (2016) indicating that S. aureus strains are more presumably to be unique to a herd than to be discovered in several herds.

Figure 3:
Dendrogram showing the genotypic relatedness of 16 Staphylococcus aureus isolated from different sources in five dairy herds in Pernambuco State, Northeastern Brazil, which shows two clusters (A-B) at 80% similarity among the band profiles. Dendrogram was built based on the UPGMA and genetic similarity using the Dice’s coefficient (5% tolerance) of the genotypic band patterns generated by PFGE, using the restriction enzyme SmaI.

Interestingly, both molecular typing methods demonstrated quite different genotypic profiles between human and milk samples. Additionally, there was no isolation of S. aureus in human sources in the farms with higher mastitis index, and there were no cases of heifer mastitis in the farms with higher isolation of S. aureus in human sources. Hence, S. aureus isolates associated with heifer mastitis were not of human origin.

Based on the cluster analysis, clonally-related strains isolated from milk were identified at the same farm (Farm 1) by both typing methods, and despite the presence of dominant strains, our results suggested a high genetic diversity ofS. aureusstrains exist at the farms. This is in concordance with recent studies that showed genetically diverse and heterogeneous S. aureus strains isolated from bovine, environment and humans (KÜMMEL et al., 2016; PAPADOPOULOS et al., 2018).

No agreement between antimicrobial patterns, rep-profiles and PFGE-profiles was observed for the majority of the isolates in this study, which is in consonance with the results of REINOSO et al. (2007) and LI et al. (2009). Therefore, beta-lactam resistance patterns alone were reported to be of limited value in differentiating closely related strains.

These findings revealed a significant diversity within the strain collection using rep-PCR and PFGE typing methods. Thus, the rep-PCR technique is a useful method for the epidemiologic characterization ofS. aureus, the results being comparable to those obtained with the PFGE technique, both with a high discriminatory power.

All these observations open new perspectives for detailed knowledge on the epidemiology of intramammary infections in primiparous cows caused by S. aureus, which might help in creating new approaches to reduce infection spread. Also, a better knowledge of drug-resistant S. aureus strains distribution in dairy herds might help in formulating strategies to control of infection.

CONCLUSION:

This report clearly demonstrates the occurrence of a high genetic diversity ofS. aureusstrains at the herds and the presence of clonally-related strains only at the same herd, emphasizing a variety of genotypic profiles among the isolates.

ACKNOWLEDGEMENTS

This work was supported by Fundação de Amparo e Pesquisa do Estado de Pernambuco (FACEPE, process number 0500-5.05/17) and by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES, financing code 001).

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  » http://www.ncbi.nlm.nih.gov/pmc/articles/pmc84303/

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  CR-2020-0679.R1

BIOETHICS AND BIOSSECURITY COMMITTEE APPROVAL

• BIOETHICS AND BIOSSECURITY COMMITTEE APPROVAL

  The experimental procedures were approved by the Ethics Committee on the Use of Animals (CEUA) of Federal Rural University of Pernambuco (UFRPE), Recife, Brazil (License No. 037/2018) and to the Human Research Ethics Committee at University of Pernambuco (UPE), Recife, Brazil (License No. 88030518.0.0000.5207).

Publication Dates

• Publication in this collection
  22 Feb 2021
• Date of issue
  2021

History

• Received
  22 July 2020
• Accepted
  29 Sept 2020
• Reviewed
  13 Nov 2020