Guido Werner

Multiplex PCR Assay for Simultaneous Detection of Nine Clinically Relevant Antibiotic Resistance Genes in Staphylococcus aureus

ABSTRACT In this study we describe a multiplex PCR assay for the detection of nine clinically relevant antibiotic resistance genes of Staphylococcus aureus. Conditions were optimized to amplify fragments of mecA (encoding methicillin resistance), aacA-aphD (aminoglycoside resistance), tetK, tetM (tetracycline resistance), erm(A), erm(C) (macrolide-lincosamide-streptogramin B resistance), vat(A), vat(B), and vat(C) (streptogramin A resistance) simultaneously in one PCR amplification. An additional primer pair for the amplification of a fragment of the staphylococcal 16S rDNA was included as a positive control. The multiplex PCR assay was evaluated on 30 different S. aureus isolates, and the PCR results correlated with the phenotypic antibiotic resistance data obtained by the broth microdilution assay. The multiplex PCR assay offers a rapid, simple, and accurate identification of antibiotic resistance profiles and could be used in clinical diagnosis as well as for the surveillance of the spread of antibiotic resistance determinants in epidemiological studies.

Occurrence and spread of antibiotic resistances in Enterococcus faecium

Enterococci are the second to third most important bacterial genus in hospital infections. Especially Enterococcus (E.) faecium possesses a broad spectrum of natural and acquired antibiotic resistances which are presented in detail in this paper. From medical point of view, the transferable resistances to glycopeptides (e.g., vancomycin, VAN, or teicoplanin, TPL) and streptogramins (e.g., quinupristin/dalfopristin, Q/D) in enterococci are of special interest. The VanA type of enterococcal glycopeptide resistance is the most important one (VAN-r, TPL-r); its main reservoir is E. faecium. Glycopeptide-resistant E. faecium (GREF) can be found in hospitals and outside of them, namely in European commercial animal husbandry in which the glycopeptide avoparcin (AVO) was used as growth promoter in the past. There are identical types of the vanA gene clusters in enterococci from different ecological origins (faecal samples of animals, animal feed, patients in hospitals, persons in the community, waste water samples). Obviously, across the food chain (by GREF-contaminated meat products), these multiple-resistant bacteria or their vanA gene clusters can reach humans. In hospital infections, widespread epidemic-virulent E. faecium isolates of the same clone with or without glycopeptide resistance can occur; these strains often harbour different plasmids and the esp gene. This indicates that hospital-adapted epidemic-virulent E. faecium strains have picked up the vanA gene cluster after they were already widely spread. The streptogramin virginiamycin was also used as feed additive in commercial animal husbandry in Europe for more than 20 years, and it created reservoirs for streptogramin-resistant E. faecium (SREF). In 1998/1999, SREF could be isolated in Germany from waste water of sewage treatment plants, from faecal samples and meat products of animals that were fed virginiamycin (cross resistance to Q/D), from stools of humans in the community, and from clinical samples. These isolations of SREF occurred in a time before the streptogramin combination Q/D was introduced for therapeutic purposes in German hospitals in May 2000, while other streptogramins were not used in German clinics. This seems to indicate that the origin of these SREF or their streptogramin resistance gene(s) originated from other sources outside the hospitals, probably from commercial animal husbandry. In order to prevent the dissemination of multiple antibiotic-resistant enterococci or their transferable resistance genes, a prudent use of antibiotics is necessary in human and veterinary medicine, and in animal husbandry.

Mobile genetic elements and their contribution to the emergence of antimicrobial resistant Enterococcus faecalis and Enterococcus faecium

Mobile genetic elements (MGEs) including plasmids and transposons are pivotal in the dissemination and persistence of antimicrobial resistance in Enterococcus faecalis and Enterococcus faecium. Enterococcal MGEs have also been shown to be able to transfer resistance determinants to more pathogenic bacteria such as Staphylococcus aureus. Despite their importance, we have a limited knowledge about the prevalence, distribution and genetic content of specific MGEs in enterococcal populations. Molecular epidemiological studies of enterococcal MGEs have been hampered by the lack of standardized molecular typing methods and relevant genome information. This review focuses on recent developments in the detection of MGEs and their contribution to the spread of antimicrobial resistance in clinically relevant enterococci.

Spread of ampicillin/vancomycin-resistant Enterococcus faecium of the epidemic-virulent clonal complex-17 carrying the genes esp and hyl in German hospitals.

The incidence of vancomycin-resistant Enterococcus faecium isolation was low (≤5%) in German hospitals before 2003. Within the second half of 2003 and the first half of 2004, however, increasing frequencies of up to 14% were noticed in several hospitals in southwestern Germany. This increase was attributed mainly to the occurrence and spread of epidemic-virulent ampicillin/vancomycin-resistant, vanA- and vanB-positive E. faecium clones, most of which exhibited the virulence factors enterococcal surface protein (esp) and bacteriocin activity and some which exhibited hyaluronidase (hyl). E. faecium possessing hyaluronidase was initially found in U.S. hospitals and recently detected in several European hospitals and, subsequently, in German hospitals as well. Ampicillin/vancomycin-resistant E. faecium clones originating mainly from southwestern German hospitals were characterized by multilocus sequence typing since different sequence types (STs) belonging to the clonal complex-17 are currently disseminated worldwide. Multilocus sequence typing revealed that, in 1998 and 1999, ampicillin/vancomycin-resistant E. faecium clone ST-117 was prevalent in various German hospitals, while in 2003 and 2004, clone ST-203 dominated in several hospitals located in southwestern Germany. Both sequence types display single-locus variants of ST-78, which was frequently recorded in various Italian hospitals between 2000 and 2003, and all of these STs belong to the clonal complex-17. Expression of linezolid resistance was observed in ampicillin/glycopeptide-resistant E. faecium strains (VanA type) from two tertiary hospitals in southwestern Germany due to mutations in domain V of the 23S rDNA (G2576T). While in one hospital the resistance emerged during linezolid therapy, in the other hospital resistance was caused by transfer of an identical linezolid/ampicillin/glycopeptide-resistant E. faecium strain. In conclusion, it is very important to monitor the occurrence of epidemic-virulent clonal complex-17 strains of E. faecium to prevent their spread in hospitals, especially if they are resistant to glycopeptides and linezolid.

Emergence of methicillin-resistant Staphylococcus aureus with Panton-Valentine leukocidin genes in central Europe.

The aim of the present study was to investigate strains of methicillin-resistant Staphylococcus aureus (MRSA) for the presence of the lukS–lukF determinant of Panton–Valentine leukocidin and to further characterize strains found to contain the genes. During the past 2 years, MRSA containing the lukS–lukF genes for Panton–Valentine leukocidin, particularly those emerging outside of hospitals, have become of interest. MRSA strains sent to the national reference center in Germany were investigated for lukS–lukF by polymerase chain reaction (PCR). If the presence of lukS–lukF was demonstrated, strains were further characterized by molecular typing (determination of SmaI pattern, spa sequence, and multilocus sequence type), PCR demonstration of resistance genes, and characterization of the SCCmec element. Since the end of 2002, MRSA containing Panton–Valentine leukocidin genes have been demonstrated as the causative agent of 28 cases of infection (9 community-acquired cases, 19 sporadic nosocomial cases) in different areas of Germany. Twenty-seven of these 28 isolates exhibited a unique pattern of genomic typing: all exhibited multilocus sequence type 80, spa sequence type 44, and a SmaI macrorestriction pattern that corresponds to a community-acquired strain of MRSA from France and Switzerland. In addition to resistance to oxacillin, the strains exhibited resistance to ciprofloxacin, tetracycline (tetM), and fusidic acid, the last of which is encoded by the far-1 gene. The far-1 gene was shown to be located on the plasmid. One isolate corresponded to community MRSA (cMRSA) of multilocus sequence type 1 from the USA.

Emergence and spread of antibiotic-resistant Gram-positive bacterial pathogens

Development of multiple resistances to antibiotics in staphylococci, enterococci and pneumococci became a health threat during the past 20 years, not only with respect to nosocomial infections. This resistance development is based on acquisition of resistance genes by predominant epidemic subpopulations (clonal complexes). Although emergence and spread of methicillin-resistant Staphylococcus aureus is associated with a limited number of epidemic clones which have been widely disseminated, acquisition of SCCmec elements by susceptible ancestors has taken place at different times and at different locations. Among Staphylococcus epidermidis and Enterococcus faecium, one clonal complex, which had acquired resistance genes at several occasions, is widely disseminated in hospitals. Also in Streptococcus pneumoniae, antibiotic resistance is preferentially associated with clonal lineages which have a capacity for spreading. They became, however, more rare after introduction of the 7-valent conjugate vaccine.

Obvious lack of association between dynamics of epidemic methicillin-resistant Staphylococcus aureus in central Europe and agr specificity groups.

During the past 8 years, changes in the prevalence and spread of different epidemic methicillin-resistant Staphylococcus aureus (MRSA) have been observed in central Europe, with the emergence of new strains possessing fewer resistance characters. This has also been demonstrated at the level of particular hospitals. Since variation in agr specificity type has been proposed as a possible reason for population dynamics in Staphylococcus aureus, the agr specificity groups of different epidemic MRSA strains were investigated by PCR using agr group-specific primers. Four of the “old” as well as two “new” epidemic strains exhibited agr specificity group I. One group of epidemic MRSA strains, which has been observed since the beginning of the 1990s, exhibited the agr specificity group II. Sequencing the variable part (agrB-D-C) of the agr locus revealed only six relevant nucleotide changes within this region, with three of them modifying the Shine-Dalgarno sequence region of agrC. On the basis of the results obtained, it is proposed that the dynamics observed in the population of MRSA in Germany is not due to different agr group specificities in “old” and “new” epidemic clones.

Tigecycline-resistant Enterococcus faecalis strain isolated from a German intensive care unit patient

Institute for LaboratoryMedicine, Vinzenz von Paul Clinics, MarienhospitalStuttgart, Bo¨heimstr. 37, D-70199 Stuttgart, GermanyKeywords: reserpine, omeprazole, glycylcyclines, tetX*Corresponding author. Tel: þ49-3943-679210; Fax: þ49-3943-679207; E-mail: wernerg@rki.de†Present address. Centre for Diagnostic Medicine, Robert BoschHospital Stuttgart, Auerbachstr. 110, D-70376 Stuttgart,Germany.Sir,Tigecycline is a member of the new group of glycylcyclines anda promising new antibiotic of last resort, active against manybacteria including Enterococcus spp.

Antibiotic resistant enterococci—Tales of a drug resistance gene trafficker

Enterococci have been recognized as important hospital-acquired pathogens in recent years, and isolates of E. faecalis and E. faecium are the third- to fourth-most prevalent nosocomial pathogen worldwide. Acquired resistances, especially against penicilin/ampicillin, aminoglycosides (high-level) and glycopeptides are therapeutically important and reported in increasing numbers. On the other hand, isolates of E. faecalis and E. faecium are commensals of the intestines of humans, many vertebrate and invertebrate animals and may also constitute an active part of the plant flora. Certain enterococcal isolates are used as starter cultures or supplements in food fermentation and food preservation. Due to their preferred intestinal habitat, their wide occurrence, robustness and ease of cultivation, enterococci are used as indicators for fecal pollution assessing hygiene standards for fresh- and bathing water and they serve as important key indicator bacteria for various veterinary and human resistance surveillance systems. Enterococci are widely prevalent and genetically capable of acquiring, conserving and disseminating genetic traits including resistance determinants among enterococci and related Gram-positive bacteria. In the present review we aimed at summarizing recent advances in the current understanding of the population biology of enterococci, the role mobile genetic elements including plasmids play in shaping the population structure and spreading resistance. We explain how these elements could be classified and discuss mechanisms of plasmid transfer and regulation and the role and cross-talk of enterococcal isolates from food and food animals to humans.

Methicillin-Resistant, Quinupristin-Dalfopristin-Resistant Staphylococcus aureus with Reduced Sensitivity to Glycopeptides

ABSTRACT Of 3,052 Staphylococcus aureus strains collected by the European SENTRY surveillance study, 35 were found to be nonsusceptible to quinupristin-dalfopristin (MIC of ≥2 mg/liter). These isolates originated from four hospitals in France and one in Spain. In isolates from two Parisian hospitals exhibiting the sameSmaI macrorestriction pattern, streptogramin resistance was based on vatA and vgbA. One isolate from a hospital in Lyon and 22 from a hospital in Lille were of thevatB vgaB streptogramin A resistance genotype and possessed ermA and/or ermC. As deduced from the loss of either streptogramin A or streptogramin B resistance determinants in particular isolates, resistance to quinupristin-dalfopristin requires mechanisms conferring resistance to both compounds. The SmaI macrorestriction patterns of strains from hospitals in Lille and Lyon were different; however, similarity analysis suggested a relatedness of 20 methicillin-resistantS. aureus strains from the Lille hospital, a finding confirmed by PCR typing based on three different genomic polymorphisms. These groups of isolates were found to be hetero-glycopeptide-intermediate susceptible S. aureus. Information about the failure of glycopeptide chemotherapy has not been available.