Staphylococcal Cassette Chromosome mec Typing and Multilocus Variable Number Tandem Repeat Analysis of Methicillin Resistant Staphylococcus aureus Clinical Isolates with Vancomycin Creep Phenomenon

Abstract

Background: The association of treatment failure and mortality with vancomycin minimum inhibitory concentration creep (MIC) is a matter of serious concern in patients with severe methicillin resistant Staphylococcus aureus (MRSA) infections. The purpose of the study was to identify and characterize staphylococcal cassette chromosome mec (SCCmec) and clonal types of MRSA strains, exhibiting the vancomycin MIC creep phenomenon.

Methods: A total of 3305 S. aureus strains were isolated from various clinical samples of Lahore General Hospital, Lahore, Pakistan. MRSA strains were identified by cefoxitin resistant (≤21mm) followed by mecA and mecC gene genotyping. Vancomycin MIC creep was determined by E-test. Isolates having MIC values >1.5 µg/mL were further subjected for SCCmec typing (I-V and XI) and multiple-locus variable number tandem repeat analysis (MLVA) by amplification of spa, sspA, clfA, clfB, and sdrCDE genes. A dendrogram was created based on the similarity index using bioneumerics software.

Results: About 13.3% (440/3305) isolates were MRSA with 99.3% (437/440) and 0.7% (3/440) carried mecA and mecC genes, respectively. In 120 MRSA isolates, the MIC of vancomycin was >1.5µg/mL. In MRSA isolates with high vancomycin MIC (>1.5µg/mL), the most common SCCmec type was SCCmec III (38.3%), followed by SCCmec IVa (15.8%), SCCmec IIIa (13.3%,), SCCmec IVc (7.5%), SCCmec IVe (5.8%), SCCmec IVd (5.8%), SCCmec IVb (4.2%), SCCmec II (2.5%), SCCmec V (1.7%), SCCmec I (1.7%) and SCCmec XI (1.7%). MLVA revealed 60 genotypic groups of MRSA isolates having a 92% similarity index.

Conclusion: SCCmec III was the most common type in genetically related MRSA isolates showing vancomycin MIC creep. The presence of SCCmec XI may further add burden to infection control measures.

Keywords: methicillin resistant Staphylococcus aureus; multilocus variable number tandem repeat analysis; staphylococcal chromosomal cassette.

Figure 1

Distribution trend of vancomycin MIC values (2016–2020) (n = 440). MRSA isolates with vancomycin MIC values of 0.5µg/mL and 0.75µg/mL remained at very low levels throughout the years (lower two lines). Isolates with MIC 1µg/mL show an evident decline as indicated with the grey line while isolates with MIC value of 2µg/mL show a clear rising trend (dark blue line). At the top of the figure, the yellow line depicts the highest percentage of MRSA isolates with vancomycin MICs values of 1.5 µg/mL.

Figure 2

Gel electrophoresis pattern for identification of SCCmec types and subtypes by multiplex PCR for clinical isolates of MRSA. Lane 1 is negative control. Lanes 2 represents SCCmec type IVe (175bp); lane 3 represents SCCmec type IVb (493bp); lane 4 represents SCCmec type IVd (881bp); lane 5 represents SCCmec type Iva (776bp); lane 6 represents SCCmec type I (613bp); lane 7 represents SCCmec type IVc (200bp); lane 8 represents SCCmec type II (398bp). SCCmec type III a (257bp); lane 10 represents SCCmec III type (280bp) and lane 11 represents SCCmec type V (325bp). Lane 12 is empty. M is the 100 bp DNA ladder (Thermo Scientific).

Figure 3

Gel electrophoresis pattern for identification of SCCmec type XI by multiplex PCR for clinical isolates of MRSA. M is a 100 bp DNA ladder (Thermo Scientific). Lane 1: negative control; lane 2 represents positive control of SCCmec XI ATCC- BAA 23122, Lane 3: MRSA strain represents SCC mec type XI carrying mec1 (344bp); mecR1 (710bp); blaZ (809bp); ccrA1 (932bp) and ccrB3 (1499bp).

Figure 4

Dendrogram based on the clustering of strains according to similarity index (by Dice coefficient and UPGMA approach) through MLVA analysis of MRSA strains. By MLVA analysis 120 isolates were clustered into 60 haplotypes based on similarity index due to slight variations in VNTRs using Bionumerics Software version 8.0. A dendrogram was produced with the settings of position tolerance (optimization 1% and band position tolerance 0.75%). All strains were shuffled according to their similarity index and clustered into genotypes.

Figure 5

Minimum Spanning Tree analysis of MRSA isolates depicting their phylogenetic relationship according to VNTRs (clfA, clfB, sdrD, sdrC, sdrE, spa and sspa) distribution index. It is based on slight base pair variations leading to a new MLVA type strain using Bionumerics software version 8.0. In this MST analysis total 60 clusters were found. Circles are representing MRSA isolates and the same colored circles belong to one genotype. Furthermore closely related genotypes were connected with bold lines while distantly related were connected with thin lines.