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Frontiers in Biology

ISSN 1674-7984

ISSN 1674-7992(Online)

CN 11-5892/Q

Front. Biol.    2015, Vol. 10 Issue (6) : 520-527    https://doi.org/10.1007/s11515-015-1380-0
RESEARCH ARTICLE
Community and hospital acquired methicillin resistant Staphylococcus aureus efficiently retain the Van A determinant
Anup Kainthola1,*(),Ajay B. Bhatt1,Ashish Gupta2
1. Laboratory of Microbiology, Department of Botany & Microbiology, HNB Garhwal Central University, Uttarakhand 246174, India
2. School of Biotechnology, Amity International University, NOIDA, UP, India
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Abstract

Dissemination of vancomycin resistance from hospital to community strains is a serious threat to public health. Our study aimed to provide evidence for transmission of Van A type resistance from the hospital to the community. Wild-type community and hospital associated methicillin resistant Staphylococcus aureus strains were studied in vitro and in a model that mimicked a natural environment to ascertain their ability to acquire and maintain the vancomycin resistance determinant (Van A gene) from vancomycin resistant Enterococcus faecalis. Fitness was assessed and the cost of Van A acquisition and retention was estimated. In vitro mating experiments were carried out using a filter mating technique and a model of a natural water body environment. Transfer of resistance was carried out in two different conditions: restricted and favorable. Transconjugants were confirmed by E test and PCR using specific primer sets. Growth kinetics and fitness measurements were done by spectrometric analysis. Using the in vitro filter mating technique, high transfer frequencies that ranged from 0.7 × 10–3(0.0006) to 3.1 × 10–4(0.00011) were recorded, with the highest transfer frequencies for CA MRSA (thermosensitively homogenous) (0.7 × 10–3), and 1.2 × 10–4 to 2.4 × 10–6 in the model. HA MRSA (homogenous) showed a greater capacity (3.6 × 10–4) to receive the Van A gene, while CA MRSA showed a reduced ability to maintain the gene after serial subcultures. CA and HA thermosensitively heterogeneous MRSA transconjugants exhibited higher growth rates. The present study provides evidence for the enhanced ability of CA and HA MRSA clones to acquire and maintain Van A type resistance.

Keywords Van A resistance      MRSA      fitness      growth kinetics      resistance transfer     
Corresponding Author(s): Anup Kainthola   
Just Accepted Date: 08 December 2015   Online First Date: 11 January 2016    Issue Date: 26 January 2016
 Cite this article:   
Anup Kainthola,Ajay B. Bhatt,Ashish Gupta. Community and hospital acquired methicillin resistant Staphylococcus aureus efficiently retain the Van A determinant[J]. Front. Biol., 2015, 10(6): 520-527.
 URL:  
https://academic.hep.com.cn/fib/EN/10.1007/s11515-015-1380-0
https://academic.hep.com.cn/fib/EN/Y2015/V10/I6/520
Characteristics of mating pair (donor+recipient) Transfer frequency (Avg.)
CA MRSA (hom. Van A )+E.faecalis ( Van A+) 0.7 × 10−3 (0.0006)
CA MRSA (hom. Van A)+E.faecalis ( Van A+) 0.8 × 10−3 (0.0013)
CA MRSA (hom. Van A)+E.faecalis ( Van A+) 3.1 × 10−4 (0.00011)
CA MRSA (hom. Van A)+E.faecalis ( Van A+) 1.4 × 10−4 (0.00009)
Tab.1  Transfer frequency was calculated as the number of transconjugants obtained per donor cell
Pairs (donor+recipient) Conditions in mimicked system Transfer frequency (average)
CA MRSA (hom. Van A) + E. faecalis ( Van A+) River water+ peptone+ minerals+ galactose+ shaking incubation 2.4 ×10−6 (0.0000012)
River water+ shaking incubation 1.1 × 10−6 (0.0000002)
CA MRSA (het. Van A) + E. faecalis ( Van A+) River water+ peptone+ minerals+ galactose+ shaking incubation 2.3 × 10−4 (0.00031)
River water+ shaking incubation 1.0 × 10−6 (0.0000013)
HA MRSA (hom. Van A) + E. faecalis ( Van A+) River water+ peptone+ minerals+ galactose+ shaking incubation 3.6 × 10−4 (0.00031)
River water+ shaking incubation 1.2 × 10−4 (0.00019)
HA MRSA (het. Van A) + E. faecalis ( Van A+) River water+ peptone+ minerals+ galactose+ shaking incubation 1.7 × 10−5 (0.000022)
River water+ shaking incubation 2.9 x× 10−5 (0.000025)
Tab.2  Average transfer frequency in mimicked model
Mating pair 12 hours 24 hours 36 hours
1 0.4 × 10−3 0.9 × 10−3 1.4 × 10−2
2 0.6 × 10−4 0.5 × 10−4 0.9 × 10−3
3 1.2 × 10−5 1.1 × 10−4 2.4 × 10−4
4 0.9 × 10−4 0.5 × 10−4 1.2 × 10−3
Tab.3  Transfer frequencies on time scale from mimicked (at optimum condition) model
Strains Van MIC (mg/L) Doubling time (min)
CA MRSA (hom.) transconjugant 8 39
CA MRSA (het.) transconjugant 8 37
E. faecalis (donor) 8 42
HA MRSA (hom.) transconjugant 8 44
HA MRSA (het.) transconjugant 8 46
Control strains CA MRSA homogenous 2928
CA MRSA heterogenous 31
HA MRSA homogenous 29
HA MRSA heterogenous 32
Tab.4  Effect of horizontal gene transfer (HGT; filter mating technique) on doubling time
Fig.1  Population analysis of transconjugants and control strains.Control strains used:control+ = VISA strain Mu50; control- = vancomycin-susceptible S. aureus strain FDA209P.
Fig.2  OD of transconjugants to determine growth kinetics after HGT.
Fig.3  Gel electrophoresis of PCR products of donor, recipients and transconjugants. Detection of Van A gene by PCR using Van A primer set. PCR products were analyzed by 1% agarose gel electrophoresis. Reference strain “R” used was Enterococcus faecium ATCC 19434. M: 100bp DNA ladder marker; (A) (Lane 1,6,7; VRE (vancomycin resistant enterococci); (B) CA (hom.) MRSA transconjugant; (C) CA (het.) MRSA transconjugant; (D) HA (het.) MRSA transconjugant; (E) HA (hom.) MRSA transconjugant); Lane 2: Plasmid DNA of VRE for van A; Lane 3: CA (het.) MRSA transconjugants grown in broth with vancomycin; Lane 4: CA (het.) MRSA transconjugants grown in broth without vancomycin; Lane 5: CA (het.) MRSA transconjugants after many subcultures; (F): HA (het.) MRSA transconjugants grown in broth with vancomycin; (G) HA (het.) MRSA transconjugants after many subcultures; (H) HA (het.) MRSA transconjugants grown in broth without vancomycin.
Fig.4  Line diagram of study area. Map not to be scaled.
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