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Plasmid-mediated resistance and virulence mechanisms in the private health sector in KwaZulu-Natal, South Africa: An investigation of methicillin resistant Staphylococcus aureus (MRSA) clinical isolates collected during a three month period
Plasmid-mediated resistance and virulence mechanisms in MRSA were detected in KZN, South Africa.
•
Plasmids were extracted and MRSA confirmed by the presence of mecA gene.
•
The isolates were subjected to antimicrobial susceptibility testing.
•
Molecular characterization of common resistance genes and virulence factors were determined by PCR.
•
The genetic relatedness between the isolates was determined by PFGE giving an indication of similar circulating MRSA clones in the KZN province.
Abstract
Objectives
Due to the lack of information on the plasmid content of MRSA strains in South Africa (SA), this study investigated the resistance and virulence mechanisms of 27 clinical isolates from the private health care sector over a period of 3 months.
Methods
Plasmids were extracted and the presence of MRSA confirmed by the presence of mecA. The isolates were subjected to antimicrobial susceptibility testing and molecular characterization of common resistance encoding genes and frequently encountered virulence factors by PCR using plasmid DNA as the template. The genetic relatedness between the isolates was determined by pulsed field gel electrophoresis (PFGE).
Results
All isolates were plasmid positive, and displayed ampillicin, ciprofloxacin, gentamicin, rifampicin, tetracycline, erythromycin, and clindamycin resistance. They were all fully susceptible to daptomycin, linezolid, vancomycin, tigecycline and fusidic acid. Multidrug resistance (MDR) was found in 74.1% (20/27) of the MRSA isolates. The frequency of the resistance and virulence genes ranged from 100% to 0%. PFGE analysis revealed 10 pulsotypes, designated A–J, which showed correlation with resistance profile of the isolates in each group. Of note, 85.2% (23/27) of the isolates clustered into six major PFGE types giving an indication of similar circulating MRSA clones.
Conclusions
This study highlights the genetic diversity and resistance mechanisms in MRSA strains from the private health sector in SA hence the need for implementing effective infection control programs.
MRSA is characterized by the presence of mecA that confers resistance to methicillin. This has far reaching consequences in the public health, economic and social sectors.
These strains also harbor mobile genetic elements (MGEs), including plasmids, pathogenicity islands, transposons, integrons and prophages, which comprise 15–25% of the genome. An understanding of these MGEs will broaden our knowledge on the genetic determinants of antibiotic resistance (AR).
Although research has been conducted on MRSA in SA, information on the plasmid content is largely unknown, a study of this nature is important understanding AR patterns, comparing the plasmid profiles will help in effective infection control. The aim of this study was to ascertain the genetic relatedness, and characterize the plasmid-encoded antibiotic resistance and virulence profile of clinical MRSA isolates collected obtained from a private laboratory in Durban, SA over a three month period.
2. Methods
A total of 27 consecutive non-repetitive MRSA isolates were obtained from June to August 2015, from a pathology laboratory that caters for the private healthcare sector. The isolates were identified using Vitek 2 (bioMerieux, Durham, NC, USA) and confirmed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/MS). The cefoxitin disc diffusion (CDD) test was used to identify putative MRSA, which was then confirmed by PCR detection of mecA. The MIC was determined for 12 antibiotics by the broth microdilution method. The Clinical and Laboratory Standards Institute (CLSI) Guideline
was used for interpreting the results, and Staphylococcus aureus ATCC 29213 was used as the control. Isolates resistant to β-lactams, and at least three classes of antibiotics, were defined as multidrug resistant (MDR). A plasmid DNA extraction kit (GeneJET Plasmid Miniprep kit, Thermoscentific) was used to purify the plasmid DNA from all 27 MRSA strains, according to the manufacturer's instructions. The presence of resistance genes conferring resistance to ampicillin-penicillin (blaZ), aminoglycoside (aac (6′)–aph (2″)), macrolide-lincosamide-streptogramins B [MLSB] (ermC) and tetracycline (tetK) were determined using PCR. The virulence determinants encoding the bio-component Panton-Valentine leukocidin (LukS/F-PV gene), exfoliative toxin (eta), alpha and delta hemolysin genes (hla and hld) were also ascertained by PCR. PFGE was used to predict the genetic relatedness of the MRSA isolates. Clusters were defined using the criterion of a difference of ≤6 bands, as described by Tenover et al
The demographic data and resistance patterns of the isolates and MICs are summarized in Tables 1 and S1. Ampicillin showed no activity against MRSA isolates, while 85.2% (23/27) were resistant to ciprofloxacin, 74.1% (20/27) to gentamicin, 70.4% (19/27) to rifampicin, 66.7% (18/27) to tetracycline, 63.0% (17/27) to erythromycin, and 11.1% (3/27) to clindamycin. Multidrug resistance (MDR) was determined in 74.1% (20/27) of the MRSA isolates. Resistance rates in this study varied compared to those seen in another KZN study on 61 confirmed MRSA isolates by Shittu et al
, particularly for gentamicin (74.1% vs. 96.7%), rifampicin (70.4% vs. 73.8%), tetracycline (66.7% vs. 90.2%) and erythromycin (63.0% v. 82.0%). Resistance to clindamycin of 11.1% was also much lower in this investigation than the rates of 82%
reported in other studies conducted on MRSA isolates in KZN and SA. Only the ciprofloxacin resistance rate in our study was notably higher (85.2% v. 18%
in KZN with a rate of 79% on 19 clinical MRSA. All MRSA isolates were susceptible to daptomycin, vancomycin, linezolid, fusidic acid and tigecycline, ampillicin. The susceptibility patterns of the isolates in this study were comparable to studies conducted on MRSA in South Africa
which were totally susceptible to daptomycin, vancomycin, linezolid, fusidic acid and tigecycline. The susceptibility of MRSA to these antibiotics observed in this study confirms their use as treatment options for infections in SA.
Table 1Clinical data, minimum inhibitory concentrations (MIC), and results of PCR for 27 MRSA isolates
ETT, Endotracheal tube; CVP, Central venous catheter; ICU, Intensive/High care unit; LW, Labour ward: OPD, outpatient department, NB, Newborn (day 0), -, No information.
AP
CP
GT
ET
RF
TT
CM
DP
VM
LZ
FA
TG
mecA
blaZ
ermC
aac-aph
tetK
hla
hld
eta
lukS/F-PV
B11970
1
Blood
Neo ICU
F
NB
>512
0.5
32
8
≤0.25
2
≤0.25
1
1
2
≤0.25
≤0.25
+
+
+
+
-
+
+
-
-
P10781
15
Nasal
OPD
M
86
>512
256
64
32
512
256
≤0.25
0.5
1
2
≤0.25
≤0.25
+
+
+
+
-
+
+
-
-
P10747
2
CVP
ICU
F
66
>512
4
>64
64
512
128
≤0.25
0.5
0.5
1
≤0.25
≤0.25
+
+
+
+
-
+
+
-
-
S37938
-
-
-
-
-
>512
256
16
32
256
64
2
0.5
1
2
≤0.25
≤0.25
+
+
+
+
-
+
+
-
-
S18155
3
ETT
ICU
F
76
>512
256
64
64
256
128
≤0.25
0.25
0.5
2
≤0.25
≤0.25
+
+
-
+
-
+
-
-
-
B13178
5
Blood
LW
F
26
>512
256
>64
64
512
128
≤0.25
0.5
1
2
≤0.25
≤0.25
+
+
+
+
-
+
+
-
-
440260
-
-
-
-
-
>512
>512
>64
64
256
128
≤0.25
0.5
1
2
≤0.25
≤0.25
+
+
+
+
-
+
+
-
-
S18970
-
-
-
-
-
>512
256
64
32
512
64
≤0.25
0.5
0.5
2
≤0.25
≤0.25
+
+
-
+
-
+
+
-
-
P11520
6
Pus
OPD
M
62
512
>512
0.25
0.5
≤0.25
≤0.25
≤0.25
0.5
1
2
≤0.25
≤0.25
+
+
-
-
-
+
+
-
-
T5683
7
Nasal
OPD
F
43
>512
8
0.5
0.5
256
32
≤0.25
0.5
1
1
≤0.25
≤0.25
+
+
-
-
-
+
+
-
-
B15227
1
Blood
Neo ICU
F
NB
>512
4
64
8
≤0.25
≤0.25
≤0.25
1
1
1
≤0.25
≤0.25
+
+
+
+
-
+
+
-
-
P13563
-
-
-
M
49
>512
128
>64
0.5
128
16
≤0.25
0.5
1
2
≤0.25
≤0.25
+
+
-
+
-
+
+
-
-
S22589
4
Sputum
ICU
M
49
>512
128
>64
0.5
128
64
≤0.25
1
1
2
≤0.25
≤0.25
+
+
-
+
-
+
+
-
-
B15612
8
Blood
ICU
M
46
>512
128
>64
16
512
256
≤0.25
1
1
2
≤0.25
≤0.25
+
+
-
+
-
+
-
-
-
B15810
5
Pus
Surgical
M
41
>512
256
32
16
128
64
≤0.25
0.5
1
2
0.5
≤0.25
+
+
+
+
-
+
+
-
-
B15583
1
Blood
ICU
F
37
>512
16
>64
2
64
2
≤0.25
0.5
1
2
≤0.25
≤0.25
+
+
-
+
-
+
+
-
-
S24463
10
ETT
ICU
F
59
512
1
32
1
≤0.25
≤0.25
1
0.5
0.5
2
≤0.25
≤0.25
+
+
+
+
-
+
+
-
-
P15045
1
Wound
Surgical
F
47
>512
64
64
16
256
64
≤0.25
0.25
0.25
2
≤0.25
≤0.25
+
+
-
+
-
+
+
-
-
P15028
10
Eye
Nursery
F
NB
512
4
16
0.5
0.5
≤0.25
≤0.25
0.25
1
2
≤0.25
≤0.25
+
+
-
+
-
-
+
-
-
P14890
11
Wound
ICU
F
41
512
256
64
0.5
256
128
≤0.25
0.5
1
2
≤0.25
≤0.25
+
+
-
+
-
+
+
-
-
P15558
1
CVP
Medical
F
94
512
>512
0.12
1
256
≤0.25
>512
0.5
0.5
1
≤0.25
≤0.25
+
+
+
+
-
+
+
-
-
P15469
12
Humerus
General
F
68
128
64
1
0.5
≤0.25
0.5
≤0.25
0.25
0.5
2
≤0.25
≤0.25
+
+
-
+
-
+
+
-
-
P15490
13
Bone
General
M
63
>512
128
32
16
128
64
≤0.25
0.25
0.5
2
≤0.25
≤0.25
+
+
+
+
-
+
+
-
-
P15742
6
cheek
Trauma
M
29
256
0.5
0.5
16
≤0.25
64
≤0.25
0.25
0.5
2
≤0.25
≤0.25
+
+
+
+
-
+
+
-
-
P15825
14
Buttock
Paediatri
M
5
512
1
0.5
0.5
≤0.25
0.25
≤0.25
0.25
1
2
≤0.25
≤0.25
+
+
-
+
-
+
+
-
-
P15793
2
Head
Surgical
M
10
512
256
64
32
256
32
≤0.25
0.5
0.5
2
≤0.25
≤0.25
+
+
+
+
-
+
+
-
-
T8060
-
-
-
-
-
512
4
4
16
128
64
≤0.25
0.5
0.5
2
≤0.25
≤0.25
+
+
-
+
-
+
+
-
-
a ETT, Endotracheal tube; CVP, Central venous catheter; ICU, Intensive/High care unit; LW, Labour ward: OPD, outpatient department, NB, Newborn (day 0), -, No information.
b AP, ampicillin; CP, ciprofloxacin; GT, gentamicin; ET, erythromycin; RF, rifampicin; TT, tetracycline; CM, clindamycin; DP, daptomycin; VM, vancomycin; LZ, linezolid; FA, fusidic acid; TG, tigecycline.
c The numbers 1–15 indicates codes of the hospital centers where the MRSA isolates were collected.
The structural component of mecA encodes the penicillin-binding protein 2a (PBP2a) that establishes resistance to methicillin, other semisynthetic penicillinase-resistant beta-lactams that are frequently co-carried with genes conferring resistance to aminoglycosides, macrolide-lincosamide-streptogramin B [MLSB] and spectinomycin.
All the isolated plasmids of the MRSA isolates contained the mecA and blaZ resistance genes, showing the correlation between MICs and the presence of genes encoding resistance against beta-lactams. The gentamicin resistance gene aac (6′)–aph (2″) was identified in 25 (92.6%) of the isolates, which varied from the phenotypic resistance profile of 74.1%, indicating that gene carriage does not necessarily translate into the resistance phenotype. The ermC gene responsible for macrolide-lincosamide-streptogramins B [MLSB] resistance was amplified in 48.2% (13/27) of the MRSA isolates, while it was not found in those that were susceptible to both erythromycin and clindamycin. The 23.5% (4/17) with phenotypic resistance to MLSB that did not contain the ermC gene indicates the occurrence of other resistance mechanism, ermA, ermB and msrA, which was not investigated in this study but have been previously reported.
Although there was high tetracycline resistance, the tetK gene was not detected, indicating that this may be due to different mechanisms and not mediated by active drug efflux, as tetK resistance has so far not been reported in clinical MRSA studies in South Africa.
The prevalence of virulence factors in all isolated plasmids showed a similar trend, with the hla and hld being the most abundant genes, with frequencies of 96.3% (26/27) and 92.6% (25/27) respectively. Comparatively, this was similar to other studies conducted from Uganda
with either hla being more frequent than the hld genes, or both showing 100% co-dominance. The prevalence rate of eta in our study was 0%, however, the prevalence of eta differed among studies, which could be associated with a variety of geographical and health conditions.
LukS/F-PV was not detected in any of the 27 clinical MRSA isolates, which was comparable to a study conducted in South Africa on 320 clinical MRSA isolates with only one positive LuKS/F-PV gene being detected.
The PFGE profiles and the dendrogram of the MRSA isolates are shown in Figure 1. PFGE analysis grouped the 27 isolates into 10 pulsotypes designated A-J, displaying 70.0% similarity, and correlating with their resistance profile and the genetic determinants tested in this study (Fig. 1). Of note, 85.2% of the isolates were clustered into six major PFGE types: pulsotypes F (8/27 strains; 29.6%), G (5/27; 18.5%), C, I (3/27; 11.1%) and A, H (2/27; 7.4%). Pulse types B, D, E and J were each represented by single isolates. Although the sample size was too small to show a definite correlation, the assertion of similar circulating clones in the province was supported by our study, as the PFGE analysis revealed some form of association between pulsotypes and the centers of sample collection. Centers 1 and 10 were found to contain pulsotypes C and H, while identical pulsotypes F and G were spread across nine of the 15 centers, intimating the possibility of similar clones of MRSA within the health care centers in the province as predicted by Shittu et al
Figure 1PFGE SmaI genotypic types generated from clinical MRSA isolates from private sector in KZN. Pretested Salmonella serotype Braenderup strain H9812 was used as the reference control strain. The R and S indicate resistance or susceptibility for ciprofloxacin, gentamicin, erythromycin, tetracycline and rifampicin respectively. The alphabets A–J shows the main pulsotype and subtype of each isolate. The numbers 1–15 indicates codes of the hospital centers where the MRSA isolates were collected.
To the best of our knowledge this is the first study characterizing the plasmid-mediated resistance and virulence genes of clinical MRSA isolates in the private sector in Durban. The study provides a private sector perspective of antibotic resistance patterns and genetic relatedness of MRSA highlighting the need for implementing efficient and effective infection control programs.
Acknowledgement
We thank the South African National Research Foundation and the College of Health Sciences, UKZN, for supporting and funding this project. The funders had no role in the study design, data collection and interpretation, nor the decision to submit the work for publication.
We express our gratitude to Professor Olarniran Ademola and Mr. Collins Odjadjare of the School of Life Sciences, University of Kwa-Zulu Natal (UKZN), Durban, South Africa for granting us access to the Bionumerics software.
Ethical approval: Permission to carry out this study was granted by the Biomedical Research Ethics Committee (BREC) (REF/No: BE394/15) of the University of KwaZulu-Natal (UKZN).
Conflict of interest: Professor SY Essack is a member of the Global Respiratory Infection Partnership sponsored by Reckitt and Benckiser.
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