Changing molecular epidemiology of methicillin-resistant Staphylococcus aureus in an Algerian hospital

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of both hospitaland community-acquired infections worldwide. However, data about the molecular epidemiology of MRSA in North Africa are still scarce. Methodology: All MRSA isolates recovered between January 2006 and July 2011 from one Algerian hospital were genetically and phenotypically characterized. Results: The predominance of a European community-associated-MRSA (CA-MRSA) clone (ST80-SCCmec IV-PVL positive) was revealed by this analysis. Conclusion: Our data suggest that a CA-MRSA clone recently invaded the hospital setting in Algiers and replaced a typical hospitalassociated pandemic clone such as the Brazilian clone (ST239-SCCmec IIImercury-PVL negative).


Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of both hospital and community-acquired infections worldwide.However, there is a considerable epidemiological variation of MRSA at both regional and global levels [1].For example, Mediterranean countries likely represent a hyperendemic region for MRSA whereas the proportion of MRSA in northern Europe is much lower [2].In particular, the proportion of MRSA in southern Mediterranean countries (e.g., Egypt, Tunisia and Algeria) showed a recent spectacular increase [2].Nevertheless, data about the molecular epidemiology of MRSA in North Africa are still scarce compared to European countries.

Methodology
In this study, all MRSA isolates (one per patient; n = 84) recovered between January 2006 and July 2011 from the Bologhine Ibn Ziri University hospital (250 beds) located in Algiers (Algeria) were characterized.The epidemiological characteristics of these isolates are indicated in Table 1-S.Each bacterial isolate was identified with standard bacteriological procedures (i.e., Gram stain, colonial/morphological appearance, tube coagulase, catalase and DNase tests); susceptibility to 12 common antimicrobial agents (Table 1) was tested using the disc-diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [3].
The genetic diversity of this MRSA population was assessed with the highly discriminatory Double Locus Sequence Typing (DLST) method as previously described [4,5].This method is based on the analysis of partial sequences (about 500 base pairs) of the variable clfB and spa genes.Additionally, the staphylococcal cassette chromosome (SCCmec) element was determined in each strain using a multiplex PCR method described by Kondo et al.; the presence of Panton-Valentine-leukocidin (PVL) genes was tested as previously described [6,7].
A total of 13 clfB and 5 spa alleles were observed among the 84 MRSA isolates typed by DLST and these accounted for 13 different DLST types.eBURST software was used to cluster DLST types sharing at least one identical allele (http://eburst.mlst.net/).There is generally a good concordance between DLST clusters and MLST clonal complexes and it is possible to relate these clusters with international clones [8].This analysis identified two main clusters as well as three singletons (Figure 1).First, the cluster with DLST ancestor 415-26 represents 85.7% (72/84) of all isolates.This cluster corresponds to the European CA-MRSA clone (ST80) and as expected all isolates of this clone were PVL positive [5].In addition, most isolates of this clone carry the SCCmec type IV although it was not possible to identify the ccr type in two isolates.Second, the clone with DLST type 28-30 represents 8.3% (7/84) of all isolates.This cluster is related to the Brazilian clone (ST239) and as expected all the isolates of this clone carried the SCCmec IIImercury and were PVL negative [5].Finally, the remaining five isolates (6.0%) belonged to three single DLST types and could not be associated with international clones.All these isolates carried the SCCmec IV and were PVL negative.
The European CA-MRSA (ST80-IV-pos) is the most common community acquired clone in Europe [9,10].Although this clone has been recovered from many European countries, it has often been associated with patients with Middle Eastern or North African origin [9,10].This is probably explained by the predominance of the European CA-MRSA clone in Algeria and in surrounding North African countries [11][12][13][14][15].Even though this clone is generally associated with community-acquisitions, our data did not allow discrimination between hospital and community acquisitions.Nevertheless, the widespread occurrence of this clone in Algerian hospitals indicates it has invaded the hospital setting.Moreover, the proportion of isolates belonging to ST80 changed during our study period.Whereas a similar proportion of the Brazilian and European CA-MRSA clone was observed in 2006 (36% vs. 43%), the European CA-MRSA clone accounted for > 90% of the isolates in subsequent years (Table 2).This observation as well as similar previous observations suggest a recent  emergence of the ST80 clone in Algerian hospitals and indicate that this community associated clone is able to quickly replace typical hospital-associated clones such as the Brazilian clone [12,14].
To compare the antibiotic susceptibility and virulence patterns of the major clones recovered in this study with international clones, we analyzed two representative Algerian isolates of the European CA-MRSA clone and one Algerian isolate of the Brazilian clone with the Alere StaphyType DNA microarray (Alere Technologies, Jena, Germany) [16].This microarray targets approximately 170 distinct genes and it was used following protocols and procedure previously described into detail [16].Interestingly, the presence/absence of resistance and virulence genes for each major Algerian clone was similar to other isolates of the same clone as reported by Monecke (Table 2-S) [16].This confirms the general homogeneity among Algerian and European isolates of the ST80 clone [10].
In conclusion, our data confirmed the predominance of the PVL-positive European CA-MRSA (ST80-IV-pos) clone in Algeria.In addition, our findings suggest that this clone recently replaced other hospital associated pandemic clones in Algerian hospitals.

Figure 1 .
Figure1.DLST single-locus variant clustering of 84 S. aureus isolates from the Bologhine Ibn Ziri University hospital in Algeria using eBURST.Each circle represents one DLST type, and the diameter of the circle reflects the frequency (i.e., the number of isolates) of that type.Linked DLST types differ at one of the two loci (clfB or spa).SCCmec type is indicated next to each DLST type ("Un" indicates isolates with unknown ccr type) and each DLST types including PVL-positive isolates are indicated by asterisks.Two isolates had a null allele (i.e.0) at the spa locus.The name of the international clone associated with each of the main cluster is indicated under the cluster.

Table 1 .
Patterns of susceptibility of the European CA-MRSA and Brazilian clones as well as overall Algerian MRSA isolates.

Table 2 .
Number of isolates belonging to the European CA-MRSA and Brazilian clone per year.

Table 1 -
S: Characteristics of the 84 Algerian MRSA recovered in the Bologhine Ibn Ziri University hospital (Algiers, Algeria).