As bacterial resistance to antibacterial agents continues to spread globally, the role of infection control is increasingly important in the battle against multidrug-resistant hospital acquired infections. New developments in chromogenic culture media help clinical laboratories to screen patient samples quickly and easily for the presence of meticillin-resistant Staphylococcus aureus, Extended Spectrum Beta-Lactamase-producing organisms and Vancomycin Resistant Enterococci, allowing infections caused by these multidrug-resistant bacteria to be identified rapidly so that infection control procedures can be initiated at the earliest opportunity.
by J. E. C. Beaves
Microbiologists and infection control professionals worldwide are increasingly concerned about the growing number of clinically significant microorganisms that have developed resistance to commonly prescribed antibiotics. Such strains are a common cause of hospital-acquired infections (HAI). More and more frequently, they limit the choice of therapy available (often resulting in the empiric use of more expensive antimicrobial agents), cause increased morbidity and mortality in patients, and result in prolonged hospital stays. Measures to control such infections and to provide better outcomes are being introduced throughout the developed world.
Meticillin-resistant Staphylococcus aureus infection was, for many years, the most notorious of these HAIs, but now other, equally serious threats are emerging. Health professionals worldwide are showing particular concern about the increasing prevalence of Extended Spectrum ß-Lactamase (ESBL)-producing organisms and Vancomycin Resistant Enterococci (VRE). Anxiety about these emerging problems is now spreading to the general public as new “super bugs” increasingly feature in the news headlines.
A joint working group from the European Centre for Disease Prevention and Control (ECDC) and the European Medicines Agency (EMEA) recently produced a technical report in response to the widening gap between the increasing prevalence of multidrug-resistant bacteria and the decline in development of antibacterial agents aimed at treating infections with these organisms. The report observed that “emerging and increasing resistance has become a threat to public health in Europe and globally, so that we are now facing the possibility of a future without effective antibiotics for several types of bacteria that cause infection in humans.”
Some of the most common multidrug-resistant bacteria isolated from blood cultures (and therefore with the potential to cause serious infections) in Europe, as identified in the ECDC/EMEA report, are shown in Table 1.
The ECDC/EMEA working group reported that very few antibacterial agents with new mechanisms of action are under development to meet the challenge of multidrug-resistant bacteria and there is a particular lack of new agents to treat infections due to multidrug resistant Gram-negative bacteria, such as ESBL-producers.
Consequently, rapid detection of these resistant organisms is increasingly important in order to initiate the most appropriate treatment and the necessary infection control procedures at the earliest opportunity. Recent advances in the development of chromogenic culture media allow MRSA, VRE and ESBL-producing micro-organisms to be detected directly from clinical specimens in just 18-24 hours, providing an important rapid screening tool for use in the fight against multidrug-resistant bacteria.
MRSA
MRSA is the most common cause of antibiotic-resistant HAI in many parts of the world, including Europe, the Americas, North Africa, the Middle East and the Far East. In Europe, 31 countries participating in the European Antimicrobial Resistance Surveillance System (EARSS) reported 31,591 invasive S. aureus isolates in 2007, 22% of which were MRSA. The proportion of MRSA isolates varied across Europe, from 3% or less in northern countries to greater than 50% in some southern countries.
However, the most recent figures show that more European countries are reporting decreasing trends in MRSA rates compared with previous years, indicating that improved control efforts can have a positive effect on MRSA levels in hospitals, even in high endemic countries. In England, for example, there has been mandatory reporting of MRSA bacteraemias since 2005. Since that time, intense scrutiny of the practices of individual hospitals, improved antibiotic prescribing policies and infection control practices have assisted in reducing the number of cases reported to the Health Protection Agency (HPA) to more than half - with 2932 cases reported in the financial year 2008/2009 compared to 7096 in 2005/2006 [Figure 1].
The importance of screening as part of an effective infection control programme to limit the spread of MRSA is well recognised and compulsory MRSA screening in elective admissions was introduced in England in April of this year. The effectiveness of a screening programme relies on the speed and accuracy of results so that colonised patients can be quickly and reliably targeted for isolation, decolonisation and appropriate treatment.
Improved MRSA detection
Chromogenic MRSA culture media allow the presumptive identification of MRSA to be achieved from patient swabs or clinical isolates. Oxoid Brilliance MRSA Agar has been shown to be one of the most selective MRSA chromogenic media available and can provide reliable results in as little as 18 hours. This medium detects the phosphatase activity of MRSA using a novel chromogenic compound. When cleaved, the chromogen produces distinctive denim blue colonies. Antibacterial agents within the medium inhibit the growth of competing organisms, including MSSA, while additional compounds suppress the expression of phosphatase activity in other staphylococci. These properties result in a highly selective medium that demonstrates excellent sensitivity and specificity and allows results to be obtained up to six hours earlier than alternative chromogenic media.
The medium demonstrates exceptional positive and negative predictive values (98.1% and 99.2% respectively). These are higher than those claimed by many PCR-based systems and yet are achieved at a fraction of the cost. In circumstances where patient isolation facilities are in short supply, leading to the cohorting of MRSA-positive patients, good positive and negative predictive values are essential. False-positive results could lead to MRSA-negative patients being put at increased risk of infection by prolonged stays in MRSA cohorted wards, in addition to the unnecessary and improper use of ‘last resort’ antibiotics. False-negative results could prevent an MRSA-positive patient from being isolated and receiving appropriate treatment. In an additional comparative trial, it was concluded that the excellent selectivity of Oxoid Brilliance MRSA Agar required fewer confirmatory tests, concluding that it is a reliable and economical option for clinical laboratories.
ESBL-producing micro-organisms
Extended Spectrum ß-Lactamase (ESBL)-producing microorganisms are another class of resistant bacteria that have been on the rise since they were first characterised in Europe in 1983. The relevant enzymes are found in a family of organisms known as the Enterobacteriaceae, which includes common commensal flora, such as Klebsiella pneumoniae and Escherichia coli.
Enterobacteriaceae are a significant cause of nosocomial and community-acquired infections. Due to consistent reporting over the last 26 years, it has been possible to identify a number of factors that have contributed to the increase of these infections, including the overuse of antibiotics in humans and animals, hospital cross-infection, human migration and changes in the food chain [1].
The main therapeutic options for the treatment of Enterobacteriaceae infections are ß-lactam antibiotics (broad spectrum penicillins and cephalosporins). However, ESBLs confer resistance to these compounds. Furthermore, ESBL resistance genes are encoded on freely transmissible genetic elements, greatly increasing the risk of spread of resistance to other organisms. The European Antibiotic Resistance Surveillance System has been monitoring trends in the numbers of bacteria producing these enzymes since 2000.
Previously, ESBLs were mostly found in Klebsiella species, with infections restricted to certain vulnerable patient groups, often in intensive care. However, a new class of ESBL (referred to as CTX-M enzymes) has emerged, which is widely detected among E. coli and is most frequently found in hospital- and community-acquired urinary tract infections.
The latest EARSS report (2006) showed that there has been a continuous increase in the number of invasive E. coli and K. pneumoniae isolates that are resistant to third generation cephalosporins and contain the ESBL enzymes. The report included data from 800 laboratories in 31 countries and showed a higher than 10% occurrence in half of the enrolled countries [1].
As with MRSA, there is a definite split between the northern and southern European countries. The occurrence of ESBL isolates is still considered low in the most northern European countries compared to southern and eastern countries. The lowest occurrence of ESBL clinical isolates is in the Nordic countries. However, even there, awareness has heightened recently as an outbreak of ESBL-producing Klebsiella has been reported amongst newborns and children in a Swedish hospital. Although the outbreak has been relatively small (to date; four children infected and an additional four identified as carriers), the effects have been devastating, claiming the lives of three newborn babies. In southern and eastern Europe, there is an increasingly high occurrence of ESBLs in both nosocomial and community settings [1].
ESBL-producing isolates are normally resistant to a number of different antibiotic families (including beta-lactams, fluoroquinolones, aminoglycosides and trimetoprim-sulfametoxazole), which severely limits treatment options and increases the multidrug-resistant ESBL strains in both medical and social settings. Rapid detection of ESBL production is, therefore, extremely important, so that an effective antibiotic can be prescribed.
Rapid detection of ESBL-production
A new chromogenic medium, Oxoid Brilliance ESBL Agar, has been developed for the presumptive identification of ESBL-producing micro-organisms direct from clinical samples, in just 24 hours. This carefully formulated medium distinguishes between ESBL-producing E. coli and ESBL production in the KESC group of bacteria (Klebsiella, Enterobacter, Serratia and Citrobacter).
Differentiation is achieved by using two chromogenic compounds which target galactosidase and glucuronidase activity. The KESC group of organisms produce galactosidase, resulting in green colonies on the agar, while E. coli express galactosidase and glucuronidase, producing easily distinguished blue colonies [Figure 2]. Occasionally, E. coli will be galactosidase negative, but these organisms produce pink colonies. Proteus, Morganella and Providencia do not utilise either chromogen, but produce tan-coloured colonies with a brown halo due to the deamination of tryptophan. The semiopaque background of the medium contrasts with the brightly coloured colonies and allows clear and easy identification of target organisms.
The medium contains cefpodoxime and additional antibacterial agents to inhibit non-ESBL-producing Enterobacteriaceae and to suppress the growth of less resistant AmpC organisms and other non-ESBL flora, including Stenotrophomonas maltophilia. Inhibition of these organisms reduces the incidence of false-positive results compared to traditional culture media, minimising the need for confirmatory testing.
An evaluation of the medium was performed using a variety of isolates, including CTX-M, TEM, SHV and K1-hyper-producing strains, from clinical* and other sources. Results indicated that K1-hyper-producing (non-ESBL) strains were inhibited while all representative ESBL strains grew. The agar was selected by MOSAR (Mastering Hospital Antimicrobial Resistance in Europe) for use in a pioneering European ESBL prevalence study (for further information visit www.mosar-sic.org).
Vancomycin Resistant Enterococci (VRE)
Glycopeptide resistance in enterococcal species is also increasing and, in particular, Vancomycin Resistant Enterococci (VRE) have emerged as significant nosocomial pathogens. This is thought to be due to the increased use of vancomycin for treatment of MRSA, particularly in the United States, and the use of a vancomycin-like glycopeptide (avoparcin) as a growth promoter in animal husbandry in Europe [2].
There are a number of vancomycin resistance mechanisms, of which the most genetically mobile and widespread are VanA and the less common VanB. The first report of VRE occurred in the early 1980s in Europe. Most VRE strains are confined to Enterococcus faecium, although a few cases have been confirmed as originating in Enterococcus faecalis. Typing has confirmed that there are distinct differences between the subpopulations that are found in hospitals, and human commensal and animal strains. The hospital-acquired strains have additional genomic content with associated virulent factors (and ampicillin resistance in European strains). Resistance to ampicillin tends to precede an increased VRE rate, which emerges within several years [3].
Tracking VRE rates has become even more important now that a link between ampicillin resistance and VRE rates in hospital isolates has become apparent. Within Europe, there is much variability between surveillance systems that have been set up to collect data on vancomycin resistance in enterococci, and in some countries no data is collected. Due to these missing and variable data, it is difficult to perform reliable statistical analysis. However, the EARSS report for 2007 suggests that some trends have emerged. There have been increasing VRE rates in some countries, including Ireland, Germany and Greece, whereas in the Nordic countries and the Netherlands there is low VRE prevalence. Austria, Portugal and Italy have seen decreases, but there is insufficient evidence to link this to measures that these countries took to contain outbreaks [3].
VRE-related infections are usually seen in hospital patients who are already very ill, in particular those who are immunocompromised, those who have had previous treatment with certain antibiotics (particularly cephalosporins and glycopeptides), those on a prolonged hospital stay, or those in specialist units, such as intensive care, oncology or renal units. Prompt identification of infection in these vulnerable patient groups is extremely important in order to improve outcomes and to prevent the spread of infection to other patients.
Enhanced detection of VRE
Recently, Oxoid Brilliance VRE agar, a new chromogenic screening plate for the detection of VRE, has become available. This medium provides presumptive identification of vancomycin resistant Enterococcus faecium and Enterococcus faecalis, from a faecal sample, swab, isolate or suspension, within just 24 hours. Differentiation between the two species is achieved using two chromogenic compounds - one that targets phosphatase activity (present in both species) and another that targets α-galactosidase activity (present in E. faecium). This results in light blue E. faecalis colonies, which are easily distinguished from the indigo/purple colonies of E. faecium [Figure 3].
The growth of competing flora, including E. gallinarum and E. casseliflavus (both of which are intrinsically resistant to vancomycin) is suppressed by the inclusion of vancomycin and additional antibiotics in the medium.
The medium was evaluated in a clinical trial, using a panel of 120 clinical isolates, and demonstrated a sensitivity of 94.7% and 100% at 24 and 48 hours respectively.
Routine screening
Unlike some other rapid methods for the identification of resistant organisms, which require expensive or specialised equipment, chromogenic culture media can be adopted easily by clinical laboratories of any size. Brilliance MRSA, VRE and ESBL media are supplied in pre-poured agar plates that are ready to inoculate, and give easy-to-read results within 24 hours or less, directly from clinical samples. This speed, convenience and ease of use make chromogenic media a valuable option for routine screening for significant resistant micro-organisms by clinical laboratories.
As resistance mechanisms, such as those described above, continue to challenge health professionals around the world, it is important to have such reliable and easily accessible tools for local screening programmes, which not only help clinicians to provide the most effective care, but can contribute to more widespread epidemiological studies and to our understanding of these important pathogens.
References
1. Coque TM, Baquero F, Canton R. Increasing Prevalence of ESBL-producing Enterobacteriaceae in Europe. Eurosurveillance 2008; 13:47.
2. Bell JM, Paton JC, Turnidge J. Emergence of Vancomycin Resistant Enteroccocci in Australia: Phenotypic and Genotypic Characteristic of Isolates. J. Clin. Microbiol 1998; 36, 2187-2190.
3. Werner G, Coque TM et al. Emergence and spread of Vancomycin Resistance Among Enterococci in Europe. Eurosurveillance 2008; 13:47.
A full list of references is availble from the author.
* Clinical isolates were provided by Dr. Maurine A. Leverstein-van-Hall, Clinical Microbiologist, University Medical Centre Utrecht (UMCU)/National Institute for Public Health and Environment (RIVM), Netherlands, and Professor Youri Glupczynski, University Clinic of the Catholic University of Louvain (UCL) Mont-Godinne, Belgium.
The author
James E. C. Beaves BSc.(Hons)
Clinical Applications Manager Oxoid,
Thermo Fisher Scientific
Basingstoke, Hants, UK.
Email: james.beaves@thermofisher.com
