Data from the European Antimicrobial Resistance Surveillance System (EARSS) [www.earss.rivm] indicate that the highest levels of antibiotic resistance have been found within the Mediterranean countries participating in the system. On the other hand, information about the prevalence of antimicrobial resistance in the non-European countries of the southern and eastern Mediterranean has, in the past, been sparse. Nevertheless, high levels of resistance have been reported in Streptococcus pneumoniae [1,2], Staphylococcus aureus [3] as well as within species of the Enterobacteriacae [4,5]. Unfortunately, besides being few in number, these studies have been totally unrelated, using different methodologies and, as a result, are difficult to compare [6].

This deficiency has been addressed by the Antibiotic Resistance Surveillance & Control in the Mediterranean Region (ARMed) project [www.slh.gov.mt/armed] which began in January 2003,7 and is funded by the European Commission under the INCOMED programme of the DG Research Fifth Framework Protocol (ICA3-CT-2002-10015). Over its four year funding period, this study is documenting the prevalence of antibiotic resistance in southern and eastern Mediterranean countries, as well as attempting to investigate potential factors such as antibiotic consumption and infection control. We report on the midway findings of ARMed-EARSS, the resistance epidemiology subcomponent of the project.

Methodology

ARMed-EARSS collects susceptibility test results from invasive isolates of Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Enterococcus faecium and faecalis routinely isolated from clinical samples of blood and cerebrospinal fluid in the participating laboratories situated in Algeria, Cyprus, Egypt, Jordan, Lebanon, Malta, Morocco, Tunisia and Turkey. These laboratories are asked to send information only about the first isolate of each organism from each patient. ARMed-EARSS uses almost identical protocols to those adopted and validated by the EARSS project, enabling comparison between data from the two projects. The laboratories follow their routine procedures and breakpoints, which in 86.8% of the participants were based on CLSI (formerly NCCLS) guidelines.

S. aureus testing determines oxacillin susceptibility by an oxacillin screen plate (6 mg/l) or alternatively, by an oxacillin disk test (1 µg or 5 µg) and a cefoxitin disk. ARMed laboratories screen invasive S. pneumoniae isolates for oxacillin resistance which, when found to be non-susceptible, is confirmed and determined to intermediate or high-level resistance to penicillin by determination of minimum inhibitory concentration (MIC) using E-test (AB Biodisk, Solna, Sweden). The protocol for E. coli susceptibility testing requires disc diffusion testing, amongst others, of a fluoroquinolone (ciprofloxacin and/or ofloxacin) and a third-generation cephalosporin (cefotaxime or ceftriaxone and/or ceftazidime). E-test confirmation is requested in cases of resistance to third generation cephalosporins in E. coli and vancomycin in enterococci. ARMed-EARSS protocols are accessible at the ARMed website (http://www.slh.gov.mt/armed/earss.asp). The website also includes an interactive function where maps for specific drug-bug combinations for any of the participating countries can be generated, as specified by the user.

To assess the reliability and comparability of susceptibility test results, two external quality assurance (EQA) exercises were performed in September 2003 and 2004 by all ARMed participating laboratories. These exercises were performed in collaboration with UK NEQAS (United Kingdom National External Quality Assessment Service) and undertaken concurrently with those run by EARSS for their laboratories.

Results
A total of 5883 isolates were investigated, as reported in the first 21 months of the project by the 53 participating ARMed laboratories, which in turn serve 60 hospitals. Of these, 3017 (51.3%) were S. aureus, 1567 (26.6%) were E. coli, 745 (12.7%) were S. pneumoniae, 390 (6.6%) were E. faecalis, and 164 (2.8%) were E. faecium. Resistance to penicillin in isolates of S. pneumoniae was reported from all the collaborating countries except for Malta and Cyprus (Fig. 1). When only data from countries reporting at least 10 isolates during the study period were evaluated, the prevalence of penicillin non-susceptibility S. pneumoniae ranged from 0% (Malta) to 36% (Algeria) (median: 29% [interquartile range 18% – 33%]). Except for Tunisia, macrolide non-susceptibility in S. pneumoniae was generally lower than penicillin non-susceptibility for each country, particularly in Turkey, where the difference was statistically significant (P=0.001).

There was considerably greater variability for resistance within S. aureus isolates [FIGURE 2]. Percentages of oxacillin resistance - used as a marker for methicillin resistant Staphylococcus aureus (MRSA) - varied from 10% in Lebanon to 65% in Jordan (median: 43% [IQR: 20 – 47%]). The most common resistance pattern showed co-resistance to methicillin, erythromycin and gentamicin. These isolates were particularly evident in Turkey where they accounted for more than a third of all S. aureus isolates.

Even greater disparity was seen in the resistance patterns for E. coli, mainly for fluoroquinolones and third generation cephalosporins [FIGURE 3]. Resistance to third generation cephalosporins varied from 3% in Malta to 72% in Egypt (median 18% [16 – 26%]) and in the fluoroquinolones ranged from 5% in Algeria to 40% in Turkey (median 27% [18 – 33%]). Multiresistant isolates were also particularly evident. In fact, simultaneous resistance to four major antimicrobial classes (aminoglycosides, third generation cephalosporins, aminopenicillins, fluoroquinolones) was the second most common resistance pattern seen and accounted for 13.7% of all isolates reported. Multiresistant E. coli isolates were most commonly found in Egypt, comprising more than half of all resistant isolates from that country, followed by Turkey and Lebanon where around 30% were similarly multiply resistant to three or more antibiotic groups.

Out of the five countries (Cyprus, Malta, Morocco, Tunisia and Turkey) that provided data on more than 10 isolates of each of the enterococcal species under investigation, only two reported vancomycin non-susceptibility (intermediate or resistant): Turkey, with 0.9% (95% CI 0%–2%) E. faecalis and 3.8% (2–7 %) E. faecium; and Cyprus, with 2% (0%–14%) E. faecalis.

Discussion
ARMed results have provided, for the first time, a standardised, comparable snapshot on the prevalence of resistance in important clinically relevant pathogens within hospitals in the southeastern Mediterranean. A median of 43% for methicillin resistance within isolates of S. aureus confirms that the Mediterranean region indeed constitutes a high prevalence region for MRSA. It also correlates well with previous sporadic reports from individual countries within the region [8-10]. It appears that MRSA seems to be more widespread in the eastern Mediterranean than in the south. Overall data from the hospitals in Cyprus, Egypt, Jordan and Turkey all showed MRSA proportions in excess of 40%. Results from the EARSS network, using the same methodology, have reported MRSA proportions in excess of 30% from Croatia, France, Greece, Israel and Portugal [11,12]. This would therefore indicate that the whole of the Mediterranean region is a high prevalence region for MRSA.

On the other hand, resistance within E. coli to third generation cephalosporins, and by association possibly extended-spectrum beta-lactamase (ESBL) and/or Amp-C enzyme production, appears to be more significant to those previously reported in the region. This seems especially the case in the eastern Mediterranean where the centres in Turkey, Lebanon, Jordan and Egypt reported average proportions in excess of 20%. The figure of 72% resistance to third generation cephalosporins reported from Egypt is one of the highest figures recorded for this resistance trait. Nevertheless indications of high level resistance within Gram negative pathogens in this region are not new. Bouchillon and colleagues, studying isolates from 38 centres in 17 countries, reported the incidence of ESBL production in Enterobacteriacae to be at its highest in their Egyptian centres at 38.5% [13]. El Kholy et al noted that 62% of E. coli isolated from blood cultures in three Cairo hospitals were non-susceptible to ceftazidime [14]. The ARMed results seem to confirm these previous reports and indicate that the situation may be even more acute and widespread throughout the Mediterranean region than previously indicated. In addition the presence of co-resistance to other antibiotic groups further compounds the challenge posed by such pathogens.

In contrast, the proportions of penicillin non-susceptibility in Streptococcus pneumoniae (PNSP) isolates from the ARMed laboratories are broadly in line with those already reported in centres within other Mediterranean and Middle Eastern countries [15,16]. PNSP levels seem to be uniform throughout the region and the apparent differences seen between eastern and southern Mediterranean centres in S. aureus and E. coli were not found in pneumococci. It is also interesting to note that, contrary to reports from the European countries of the region where macrolide resistance in pneumococci often exceeds that of penicillin [15, 16], erythromycin non-susceptibility appears to be less prevalent in the southeastern Mediterranean countries. Finally, the presence of vancomycin resistance in enterococci from ARMed laboratories in Turkey supports previous reports from this country [17].

The use of routinely collected clinical laboratory data provides the advantage that epidemiological conclusions mirror the day-to-day situation in the participating institutions. Furthermore, the choice of blood culture isolates minimises sample bias and reflects the clinical situation in the more severe infections. Unlike patients with less critical infections, such as those of the respiratory and urinary tract, most patients with signs of sepsis or meningitis will probably have a microbiological sample taken. In addition, the major strength of the ARMed study resides in the common methodology used throughout the different centres in the nine participating countries. Any such study has the limitation of depending on the accuracy and validity of the individual participating laboratories, which can vary, especially in limited resource countries. It is also hampered by the inability to perform third party reference laboratory verification and do more detailed investigation in results of particular significance. Nevertheless, the concurrent satisfactory quality assurance results would suggest that this potential limitation did not prejudice the conclusions reached from the preliminary data collected by ARMed-EARSS to any significant degree [18].

In addition to the direct repercussions for the countries of the Mediterranean region [19], the epidemiology of resistance in the southern and eastern Mediterranean also has European implications. Human mobility in this region is highly significant, in terms of both recognised travel (particularly tourism) and the results of migration. The importation of multiresistant organisms to European hospitals via patients arriving from countries within the Mediterranean region is well documented [20,21]. Such an occurrence may result in the possibility of subsequent intra-institutional spread with the potential for an outbreak [22,23]. Prior knowledge of the epidemiology of resistance in the Mediterranean region will therefore facilitate the introduction of effective interventions on initial contact with patients originating from this region and who may be potentially colonised or infected with multiresistant organisms. It should also prove beneficial for stakeholders in the countries involved to plan and implement correct antibiotic stewardship programmes to attempt control and possibly reduction of the incidence of antimicrobial resistance in pathogens of critical importance [24].

Conclusion
The preliminary results from the ARMed project have started to shed new light on the incidence of antimicrobial resistance in the south and east of the Mediterranean. They appear to support and accentuate previous sporadic reports suggesting a high prevalence in this region and indicate that this is particularly the case in the eastern region where multiresistance in S. aureus and E. coli seem to be especially high, and higher than that reported in the other countries of the Mediterranean. This picture will become clearer once the full duration of the study is completed and a more comprehensive isolate database is established.

Acknowledgements
This study was undertaken as part of project ARMed (Antibiotic Resistance Surveillance & Control in the Mediterranean region) which is funded by the European Commission under the INCOMED programme of the DG Research Fifth Framework Protocol (ICA3-CT-2002-10015).