Emergence of methicillin resistance predates the clinical use of antibiotics.

The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics1. Here we show that particular lineages of methicillin-resistant Staphylococcus aureus-a notorious human pathogen-appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinacei produces two ß-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureus isolates have an advantage over susceptible isolates. Together, these results suggest that methicillin resistance emerged in the pre-antibiotic era as a co-evolutionary adaptation of S. aureus to the colonization of dermatophyte-infected hedgehogs. The evolution of clinically relevant antibiotic-resistance genes in wild animals and the connectivity of natural, agricultural and human ecosystems demonstrate that the use of a One Health approach is critical for our understanding and management of antibiotic resistance, which is one of the biggest threats to global health, food security and development.

A look at staphylococci from the one health perspective.

Staphylococcus aureus and other staphylococcal species are resident and transient multihost colonizers as well as conditional pathogens. Especially S. aureus represents an excellent model bacterium for the "One Health" concept because of its dynamics at the human-animal interface and versatility with respect to host adaptation. The development of antimicrobial resistance plays another integral part. This overview will focus on studies at the human-animal interface with respect to livestock farming and to companion animals, as well as on staphylococci in wildlife. In this context transmissions of staphylococci and of antimicrobial resistance genes between animals and humans are of particular significance.

Comparative Secretome Analyses of Human and Zoonotic Staphylococcus aureus Isolates CC8, CC22, and CC398.

The spread of methicillin-resistant Staphylococcus aureus (MRSA) in the community, hospitals and in livestock is mediated by highly diverse virulence factors that include secreted toxins, superantigens, enzymes and surface-associated adhesins allowing host adaptation and colonization. Here, we combined proteogenomics, secretome and phenotype analyses to compare the secreted virulence factors in selected S. aureus isolates of the dominant human- and livestock-associated genetic lineages CC8, CC22, and CC398. The proteogenomic comparison revealed 2181 core genes and 1306 accessory genes in 18 S. aureus isolates reflecting the high genome diversity. Using secretome analysis, we identified 869 secreted proteins with 538 commons in eight isolates of CC8, CC22, and CC398. These include 64 predicted extracellular and 37 cell surface proteins that account for 82.4% of total secretome abundance. Among the top 10 most abundantly secreted virulence factors are the major autolysins (Atl, IsaA, Sle1, SAUPAN006375000), lipases and lipoteichoic acid hydrolases (Lip, Geh, LtaS), cytolytic toxins (Hla, Hlb, PSMß1) and proteases (SspB). The CC398 isolates showed lower secretion of cell wall proteins, but higher secretion of α- and ß-hemolysins (Hla, Hlb) which correlated with an increased Agr activity and strong hemolysis. CC398 strains were further characterized by lower biofilm formation and staphyloxanthin levels because of decreased SigB activity. Overall, comparative secretome analyses revealed CC8- or CC22-specific enterotoxin and Spl protease secretion as well as Agr- and SigB-controlled differences in exotoxin and surface protein secretion between human-specific and zoonotic lineages of S. aureus.

Epidemiology of Staphylococcus aureus Nasal Carriage Patterns in the Community.

Staphylococcus aureus (S. aureus) is a Gram-positive opportunistic pathogen that colonizes frequently and asymptomatically the anterior nares of humans and animals. It can cause different kinds of infections and is considered to be an important nosocomial pathogen. Nasal carriage of S. aureus can be permanent or intermittent and may build the reservoir for autogenous infections and cross-transmission to other individuals. Most of the studies on the epidemiology of S. aureus performed in the past were focused on the emergence and dissemination of methicillin-resistant Staphylococcus aureus (MRSA) in healthcare settings. There are, however, a number of more recent epidemiological studies have aimed at analysing carriage patterns over time in the community settings providing new insights on risk factors for colonization and important data for the development of strategies to prevent infections. This chapter aims to give a review of current epidemiological studies on S. aureus carriage patterns in the general community and put them into perspective with recent, yet unpublished, investigations on the S. aureus epidemiology in the general population in northern Germany.

Evidence for Human Adaptation and Foodborne Transmission of Livestock-Associated Methicillin-Resistant Staphylococcus aureus.

We investigated the evolution and epidemiology of a novel livestock-associated methicillin-resistant Staphylococcus aureus strain, which colonizes and infects urban-dwelling Danes even without a Danish animal reservoir. Genetic evidence suggests both poultry and human adaptation, with poultry meat implicated as a probable source.

A genomic portrait of the emergence, evolution, and global spread of a methicillin-resistant Staphylococcus aureus pandemic.

The widespread use of antibiotics in association with high-density clinical care has driven the emergence of drug-resistant bacteria that are adapted to thrive in hospitalized patients. Of particular concern are globally disseminated methicillin-resistant Staphylococcus aureus (MRSA) clones that cause outbreaks and epidemics associated with health care. The most rapidly spreading and tenacious health-care-associated clone in Europe currently is EMRSA-15, which was first detected in the UK in the early 1990s and subsequently spread throughout Europe and beyond. Using phylogenomic methods to analyze the genome sequences for 193 S. aureus isolates, we were able to show that the current pandemic population of EMRSA-15 descends from a health-care-associated MRSA epidemic that spread throughout England in the 1980s, which had itself previously emerged from a primarily community-associated methicillin-sensitive population. The emergence of fluoroquinolone resistance in this EMRSA-15 subclone in the English Midlands during the mid-1980s appears to have played a key role in triggering pandemic spread, and occurred shortly after the first clinical trials of this drug. Genome-based coalescence analysis estimated that the population of this subclone over the last 20 yr has grown four times faster than its progenitor. Using comparative genomic analysis we identified the molecular genetic basis of 99.8% of the antimicrobial resistance phenotypes of the isolates, highlighting the potential of pathogen genome sequencing as a diagnostic tool. We document the genetic changes associated with adaptation to the hospital environment and with increasing drug resistance over time, and how MRSA evolution likely has been influenced by country-specific drug use regimens.

Molecular Epidemiology of Staphylococcus aureus in the General Population in Northeast Germany: Results of the Study of Health in Pomerania (SHIP-TREND-0).

Population-based studies on Staphylococcus aureus nasal colonization are scarce. We examined the prevalence, resistance, and molecular diversity of S. aureus in the general population in Northeast Germany. Nasal swabs were obtained from 3,891 adults in the large-scale population-based Study of Health in Pomerania (SHIP-TREND). Isolates were characterized using spa genotyping, as well as antibiotic resistance and virulence gene profiling. We observed an S. aureus prevalence of 27.2%. Nasal S. aureus carriage was associated with male sex and inversely correlated with age. Methicillin-resistant S. aureus (MRSA) accounted for 0.95% of the colonizing S. aureus strains. MRSA carriage was associated with frequent visits to hospitals, nursing homes, or retirement homes within the previous 24 months. All MRSA strains were resistant to multiple antibiotics. Most MRSA isolates belonged to the pandemic European hospital-acquired MRSA sequence type 22 (HA-MRSA-ST22) lineage. We also detected one livestock-associated MRSA ST398 (LA-MRSA-ST398) isolate, as well as six livestock-associated methicillin-susceptible S. aureus (LA-MSSA) isolates (clonal complex 1 [CC1], CC97, and CC398). spa typing revealed a diverse but also highly clonal S. aureus population structure. We identified a total of 357 spa types, which were grouped into 30 CCs or sequence types. The major seven CCs (CC30, CC45, CC15, CC8, CC7, CC22, and CC25) included 75% of all isolates. Virulence gene patterns were strongly linked to the clonal background. In conclusion, MSSA and MRSA prevalences and the molecular diversity of S. aureus in Northeast Germany are consistent with those of other European countries. The detection of HA-MRSA and LA-MRSA within the general population indicates possible transmission from hospitals and livestock, respectively, and should be closely monitored.

Comprehensive molecular, genomic and phenotypic analysis of a major clone of Enterococcus faecalis MLST ST40.

BACKGROUND: Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type. RESULTS: We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro). CONCLUSION: Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.

Evolution of methicillin-resistant Staphylococcus aureus towards increasing resistance.

OBJECTIVES: To elucidate the evolutionary history of Staphylococcus aureus clonal complex (CC) 8, which encompasses several globally distributed epidemic lineages, including hospital-associated methicillin-resistant S. aureus (MRSA) and the highly prevalent community-associated MRSA clone USA300. METHODS: We reconstructed the phylogeny of S. aureus CC8 by mutation discovery at 112 genetic housekeeping loci from each of 174 isolates, sampled on five continents between 1957 and 2008. The distribution of antimicrobial resistance traits and of diverse mobile genetic elements was investigated in relation to the isolates' phylogeny. RESULTS: Our analyses revealed the existence of nine phylogenetic clades within CC8. We identified at least eight independent events of methicillin resistance acquisition in CC8 and dated the origin of a methicillin-resistant progenitor of the notorious USA300 clone to the mid-1970s. Of the S. aureus isolates in our collection, 88% carried plasmidic rep gene sequences, with up to five different rep genes in individual isolates and a total of eight rep families. Mapping the plasmid content onto the isolates' phylogeny illustrated the stable carriage over decades of some plasmids and the more volatile nature of others. Strikingly, we observed trends of increasing antibiotic resistance during the evolution of several lineages, including USA300. CONCLUSIONS: We propose a model for the evolution of S. aureus CC8, involving a split into at least nine phylogenetic lineages and a subsequent series of acquisitions and losses of mobile genetic elements that carry diverse virulence and antimicrobial resistance traits. The evolution of MRSA USA300 towards resistance to additional antibiotic classes is of major concern.

[Infectivity-resistotype-genotype clustering of methicillin-resistant Staphylococcus aureus strains in the Central Blacksea Region of Turkey]. / Türkiye'nin Orta Karadeniz Bölgesinde metisiline dirençli Staphylococcus aureus kökenlerinin enfeksiyözite-rezistotip-genotip kümelenmesi.

The increase in the prevalence of epidemic strains of methicillin resistant Staphylococcus aureus (MRSA) in hospitals and community requires special attention of infection control. The aim of this study was to determine the pathogenic phenotype (i.e. infectivity and resistotype) and genotypic characteristics (i.e. PFGE-pulsotyping, SLST-spa typing, MLST-sequence typing, eBURST-clonal complex detection algorithm) of clinical MRSA isolates in the Central Blacksea region of Turkey, in order to understand their short- and long-term epidemiological and evolutionary dynamics, and to investigate any probable presence of a significant clustering. This prospective study included consecutive but non-repetitive 48 MRSA isolates (of them 18 were colonized strains and 30 were causes of nosocomial infection) and seven methicillin-susceptible S.aureus (MSSA, all were isolated from nosocomial infection), collected between December 2006-February 2007 period from hospitalized patients. Identification of the isolates were performed by Vitek-2 automated system (BioMérieux, USA), and in vitro antimicrobial susceptibility testing by broth microdilution method and Vitek-2 automated system. The MRSA isolates found susceptible to erythromycin (n= 10) were further investigated for the presence of ermA gene by the PCR method. All the strains were typed by spa-typing and PFGE-pulsotyping methods. Among the isolates with different spa-types, representatives were selected (3 MRSA, 7 MSSA) and typed with MLST typing method. Among the isolates with different spa-types, representatives with different antimicrobial susceptibility patterns were selected (n= 8), and SCCmec types were determined by the multiplex PCR method. Antimicrobial resistance patterns of the isolates were digitized to get standardized antimicrobial resistance phenotypes. Clustering of MRSA isolates in pattern groups on the basis of discriminatory characteristics, namely infectivity, phenotype and genotype were statistically analyzed with specific inclusion and exclusion criteria. As a result, three different antimicrobial resistance phenotypes were found in MSSA isolates, whereas 13 were identified in MRSA isolates. In MSSA isolates, seven different PFGE-pulsotypes were detected, as compared to 14 pulsotypes in MRSA isolates. Among MRSA isolates, 10 sporadic strains with single PFGE-pulsotypes were detected. All MRSA isolates, with two exceptions (t459, t632), were of t030 spa-type; in the MLST analysis of the representatives of different spa-types (n= 3), a single type of MLST-clonal complex (CC8) and single MLST-sequence type (ST239) were identified. Each of the seven MSSA isolates yielded different spa-types, MLST-clonal complex types and MLST-sequence types (t777-ST5-CC5; t660-ST25-CC5; t153-ST34-CC30; t015-ST45-CC45; t267-ST97-CC97; t377-ST360-CC8; t084-ST15-C15). In the statistical analysis of 38 non-sporadic MRSA isolates, the isolates in Group-13 (n= 16; infectious, resistotype 14, pulsotype 4; antimicrobial resistance score= 24) displayed significant infectivity-phenotype-genotype clustering (p< 0.001). In 27 of the MRSA isolates, decreased susceptibility to teicoplanin (MIC= 4 µg/mL) was detected. Although, global MRSA isolates belonging to MLST-CC8, MLST-ST239, t030 spa-type were usually expected to be resistant to erythromycin, 10 such strains were erythromycin susceptible. However, ermA gene was found in six of these 10 strains, leading to a conclusion that the ermA gene of these isolates might be dysfunctional due to a point mutation or deletion. Selected representatives of MRSA isolates with different antimicrobial susceptibility patterns (n= 8) were detected to be SCCmec type III. In conclusion, S.aureus isolates in the patient population of our hospital representing the Central Blacksea region showed statistically significant clustering in infectivity, antimicrobial resistance phenotype and clonal genotype (p< 0.001). The dominant MRSA clone was ST239 which was one of the five major pandemic MRSA clones. Nosocomial MSSA isolates displayed long-term clonal diversity. This study produced regional evolutionary-epidemiological data that may support further regional, national and international long-term surveillance studies of S.aureus strains.

Livestock associated MRSA (LA-MRSA) and its relevance for humans in Germany.

Livestock-associated Staphylococcus aureus (LA-MRSA) are mainly associated with the clonal complex (CC) 398. Although having its main reservoir as MRSA in livestock such as pigs, poultry or cattle LA-MRSA CC398 has no pronounced host specificity and can colonize or infect other animals such as horses and dogs and also humans. In German conventional farming systems nasal colonization of the animals and of humans occupationally exposed to them (up to 86%) are frequent. Further human-to-human dissemination in households occurs more rarely in general (∼4% of humans living on farms but without occupational exposition). Nasal colonization with LA-MRSA of humans at hospital admission is found in 0.08-0.2% for Germany in general. However, this proportion is higher in areas with a high density of livestock production such as in northwestern North Rhine-Westphalia or Lower Saxony. LA-MRSA CC398 is not less pathogenic for humans than S. aureus in general. Hence, LA-MRSA accounts for ∼15% of all MRSA isolates from deep-seated skin and soft-tissue infections in the community and for about 0.8-2% of all MRSA isolated from clinical specimens obtained in hospital settings. When introduced into the hospital it can cause postoperative wound infections and even septicemia. Differently from hospital-associated MRSA clones, LA-MRSA CC398 has obviously limited capacity to spread in the nosocomial setting so far (proportion of ∼1.8% among MRSA from nosocomial infections, the proportion among MRSA from blood cultures is ∼1%).

Complete genome sequence of the porcine isolate Enterococcus faecalis D32.

The complete and annotated genome sequence of Enterococcus faecalis D32, a commensal strain isolated from a Danish pig, suggests putative adaptation to the porcine host and absence of distinct virulence-associated traits.

Emergence of OXA-48-type carbapenemase-producing Enterobacteriaceae in German hospitals.

Nine carbapenem-resistant Enterobacteriaceae isolates collected from eight patients in five German hospitals were investigated. Six isolates produced the OXA-48 carbapenemase, and three isolates produced OXA-162, which is a point mutant form of OXA-48. Both carbapenemase genes were located on IncL/M-type conjugative plasmids. Insertion sequence IS1999 (truncated or not by IS1R) was located upstream of the bla(OXA-48) and bla(OXA-162) genes in all of the isolates. Pulsed-field gel electrophoresis typing indicated the clonal transmission of an OXA-48-producing Klebsiella pneumoniae strain in two hospitals.

Direct detection and genotyping of Klebsiella pneumoniae carbapenemases from urine by use of a new DNA microarray test.

Klebsiella pneumoniae carbapenemases (KPCs) are considered a serious threat to antibiotic therapy, as they confer resistance to carbapenems, which are used to treat extended-spectrum beta-lactamase (ESBL)-producing bacteria. Here, we describe the development and evaluation of a DNA microarray for the detection and genotyping of KPC genes (bla(KPC)) within a 5-h period. To test the whole assay procedure (DNA extraction plus a DNA microarray assay) directly from clinical specimens, we compared two commercial DNA extraction kits (the QIAprep Spin miniprep kit [Qiagen] and the urine bacterial DNA isolation kit [Norgen]) for the direct DNA extraction from urine samples (dilution series spiked in human urine). Reliable single nucleotide polymorphism (SNP) typing was demonstrated using 1 × 10(5) CFU/ml urine for Escherichia coli (Qiagen and Norgen) and 80 CFU/ml urine, on average, for K. pneumoniae (Norgen). This study presents, for the first time, the combination of a new KPC microarray with commercial sample preparation for detecting and genotyping microbial pathogens directly from clinical specimens; this paves the way toward tests providing epidemiological and diagnostic data, enabling better antimicrobial stewardship.

A timescale for evolution, population expansion, and spatial spread of an emerging clone of methicillin-resistant Staphylococcus aureus.

Due to the lack of fossil evidence, the timescales of bacterial evolution are largely unknown. The speed with which genetic change accumulates in populations of pathogenic bacteria, however, is a key parameter that is crucial for understanding the emergence of traits such as increased virulence or antibiotic resistance, together with the forces driving pathogen spread. Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of hospital-acquired infections. We have investigated an MRSA strain (ST225) that is highly prevalent in hospitals in Central Europe. By using mutation discovery at 269 genetic loci (118,804 basepairs) within an international isolate collection, we ascertained extremely low diversity among European ST225 isolates, indicating that a recent population bottleneck had preceded the expansion of this clone. In contrast, US isolates were more divergent, suggesting they represent the ancestral population. While diversity was low, however, our results demonstrate that the short-term evolutionary rate in this natural population of MRSA resulted in the accumulation of measurable DNA sequence variation within two decades, which we could exploit to reconstruct its recent demographic history and the spatiotemporal dynamics of spread. By applying Bayesian coalescent methods on DNA sequences serially sampled through time, we estimated that ST225 had diverged since approximately 1990 (1987 to 1994), and that expansion of the European clade began in 1995 (1991 to 1999), several years before the new clone was recognized. Demographic analysis based on DNA sequence variation indicated a sharp increase of bacterial population size from 2001 to 2004, which is concordant with the reported prevalence of this strain in several European countries. A detailed ancestry-based reconstruction of the spatiotemporal dispersal dynamics suggested a pattern of frequent transmission of the ST225 clone among hospitals within Central Europe. In addition, comparative genomics indicated complex bacteriophage dynamics.

Surface-associated motility, a common trait of clinical isolates of Acinetobacter baumannii, depends on 1,3-diaminopropane.

While flagella-independent motility has long been described in representatives of the genus Acinetobacter, the mechanism of motility remains ambiguous. Acinetobacter baumannii, a nosocomial pathogen appearing increasingly multidrug-resistant, may profit from motility during infection or while persisting in the hospital environment. However, data on the frequency of motility skills among clinical A. baumannii isolates is scarce. We have screened a collection of 83 clinical A. baumannii isolates of different origin and found that, with the exception of one isolate, all were motile on wet surfaces albeit to varying degrees and exhibiting differing morphologies. Screening a collection of transposon mutants of strain ATCC 17978 for motility defects, we identified 2 akinetic mutants carrying transposon insertions in the dat and ddc gene, respectively. These neighbouring genes contribute to synthesis of 1,3-diaminopropane (DAP), a polyamine ubiquitously produced in Acinetobacter. Supplementing semi-solid media with DAP cured the motility defect of both mutants. HPLC analyses confirmed that DAP synthesis was abolished in ddc and dat mutants of different A. baumannii isolates and was re-established after genetic complementation. Both, the dat and ddc mutant of ATCC 17978 were attenuated in the Galleria mellonella caterpillar infection model. Taken together, surface-associated motility is a common trait of clinical A. baumannii isolates that requires DAP and may play a role in its virulence.

Absence of livestock-associated methicillin-resistant staphylococcus aureus clonal complex CC398 as a nasal colonizer of pigs raised in an alternative system.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST398 isolated from pigs raised in conventional farms was previously reported. Here we report a study on 25 farms adhering to an alternative system. LA-MRSA ST398 was not detected in nasal swabs from 178 pigs or from 89 humans working and living on these farms.

Colonization of Dogs and Their Owners with Staphylococcus aureus and Staphylococcus pseudintermedius in Households, Veterinary Practices, and Healthcare Facilities.

There are uncertainties with respect to the transmission of methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA) and Staphylococcus pseudintermedius between dogs and humans. In this study, we investigated concomitant nasal colonization of dogs and humans in three cohorts. Cohort I, households owning dogs: In 42 of 84 households, 66 humans (36.9%) and 10 dogs (8.9%) carried S. aureus. MRSA, attributed to sequence type (ST) 22 and ST130, were detected in two (1.1%) of the humans but in none of the dogs. Typing by means of spa-typing and whole-genome sequencing (WGS) indicated eight transmissions of S. aureus between humans and dogs in 8 of 42 (19.0%) households with human S. aureus carriers, whereas in 11 of 38 (29.0%) households with ≥two persons and S. aureus colonization of humans, 15 human-to-human transmissions were observed (p = 0.43). S. pseudintermedius was isolated from 42 dogs (37.5%), but from only one human (0.6%). In this case, WGS-based typing indicated strong relatedness of this isolate with a canine isolate from the same household. Cohort II, dogs and their owners visiting a veterinary practice: Among 17 humans and 17 dogs attending a veterinary practice, MSSA was detected in three humans and two dogs, and S. pseudintermedius in only six dogs. Cohort III, dogs used for animal-assisted interventions in human healthcare facilities and their owners: MSSA was obtained in 1 of 59 dogs (1.7%) and in 17 of 60 (28.3%) of the dog owners, while S. pseudintermedius was isolated from seven (12%) dogs and one (1.7%) human owner. We conclude that the risk of exchanging S. aureus/MRSA between humans and dogs is higher than that for S. pseudintermedius.

Molecular characterization of blaNDM-1 in an Acinetobacter baumannii strain isolated in Germany in 2007.

OBJECTIVES: To investigate the genetic environment of the metallo-ß-lactamase gene bla(NDM-1) in an Acinetobacter baumannii isolated in 2007 in a German hospital. METHODS: Antimicrobial susceptibility testing was performed and resistance genes were characterized by PCR amplification and sequencing. Transferability of ß-lactam resistance was tested by broth mating assays and transformation of plasmids. The genetic background of bla(NDM-1) was analysed by primer walking. Typing of the A. baumannii strain was performed by repetitive extragenic palindromic sequence-based PCR (rep-PCR) using the DiversiLab system. RESULTS: The multidrug-resistant A. baumannii isolate harboured ß-lactamase genes bla(NDM-1) and intrinsic bla(OXA-64), but without the insertion sequence ISAba1 often located upstream. Transfer of carbapenem resistance by conjugation and transformation failed. Hybridization of isolated plasmid DNA with bla(NDM) probes was not successful. Shotgun cloning of whole genomic DNA and sequence analyses revealed that bla(NDM-1) was located between two insertion elements of ISAba125. Furthermore, this bla(NDM-1)-containing transposon structure was integrated into a chromosomal gene encoding a putative A. baumannii major facilitator superfamily (MFS) metabolite/H+ symporter. CONCLUSIONS: The metallo-ß-lactamase gene bla(NDM-1) in this A. baumannii strain was integrated in the chromosome on a new transposon structure composed of two copies of insertion sequence ISAba125. The variability of the genetic environment of bla(NDM-1) likely facilitates the rapid dissemination of this gene within many Gram-negative bacterial species.