Transfer Of Resistance Plasmids Research Articles

Effect of Zinc Oxide and Copper Sulfate on Antibiotic Resistance Plasmid Transfer in Escherichia coli.

Heavy metals such as zinc (Zn) and copper (Cu) may be associated with antibiotic resistance dissemination. Our aim was to investigate whether sub-lethal dosage of Zn and Cu may enhance plasmid transfer and subsequently resistance genes dissemination. Plasmid conjugation frequencies (PCF) were performed with Escherichia coli strains bearing IncL-blaOXA-48, IncA/C-blaCMY-2, IncI1-blaCTX-M-1, IncF-blaCTX-M-1, and IncX3-blaNDM-5 as donors. Mating-out assays were performed with sub-dosages of zinc oxide (ZnO) and Cu sulfate (CuSO4). Quantification of the SOS response-associated gene expression levels and of the production of reactive oxygen species were determined. Increased PCF was observed for IncL, IncA/C, and IncX3 when treated with ZnO. PCF was only increased for IncL when treated with CuSO4. The ROS production presented an overall positive correlation with PCF after treatment with ZnO for IncL, IncA/C, and IncX3. For CuSO4 treatment, the same was observed only for IncL. No increase was observed for expression of SOS response-associated genes under CuSO4 treatment, and under ZnO treatment, we observed an increase in SOS response-associated genes only for IncX3. Our data showed that sub-dosages of ZnO and CuSO4 could significantly enhance PCF in E. coli, with a more marked effect observed with IncL, IncA/C, and IncX3 scaffolds. Our study suggested that use of certain heavy metals is not the panacea for avoiding use of antibiotics in order to prevent the dissemination of antibiotic resistance.

Development of an antimicrobial resistance plasmid transfer gene database for enteric bacteria.

Introduction: Type IV secretion systems (T4SSs) are integral parts of the conjugation process in enteric bacteria. These secretion systems are encoded within the transfer (tra) regions of plasmids, including those that harbor antimicrobial resistance (AMR) genes. The conjugal transfer of resistance plasmids can lead to the dissemination of AMR among bacterial populations. Methods: To facilitate the analyses of the conjugation-associated genes, transfer related genes associated with key groups of AMR plasmids were identified, extracted from GenBank and used to generate a plasmid transfer gene dataset that is part of the Virulence and Plasmid Transfer Factor Database at FDA, serving as the foundation for computational tools for the comparison of the conjugal transfer genes. To assess the genetic feature of the transfer gene database, genes/proteins of the same name (e.g., traI/TraI) or predicted function (VirD4 ATPase homologs) were compared across the different plasmid types to assess sequence diversity. Two analyses tools, the Plasmid Transfer Factor Profile Assessment and Plasmid Transfer Factor Comparison tools, were developed to evaluate the transfer genes located on plasmids and to facilitate the comparison of plasmids from multiple sequence files. To assess the database and associated tools, plasmid, and whole genome sequencing (WGS) data were extracted from GenBank and previous WGS experiments in our lab and assessed using the analysis tools. Results: Overall, the plasmid transfer database and associated tools proved to be very useful for evaluating the different plasmid types, their association with T4SSs, and increased our understanding how conjugative plasmids contribute to the dissemination of AMR genes.

Outbreak of OXA-48-producing Enterobacteriaceae in a neonatal intensive care unit in Western Sweden

In 2015, an outbreak caused by OXA-48-producing Enterobacteriaceae affected a neonatal intensive care unit at a Swedish University Hospital. The aim was to explore the transmission of OXA-48-producing strains between infants and the transfer of resistance plasmids between strains during the outbreak. Twenty-four outbreak isolates from ten suspected cases were whole-genome sequenced. A complete assembly was created for the index isolate (Enterobacter cloacae) and used as a mapping reference to detect its plasmids in the remaining isolates (17 Klebsiella pneumoniae, 4 Klebsiella aerogenes, and 2 Escherichia coli). Strain typing was performed using core genome MLST and SNP analysis. As judged from sequencing and clinical epidemiological data, the outbreak involved nine cases (two developed sepsis) and four OXA-48-producing strains: E. cloacae ST1584 (index case), K. pneumoniae ST25 (eight cases), K. aerogenes ST93 (two cases), and E. coli ST453 (2 cases). Two plasmids from the index strain, pEclA2 and pEclA4, carrying blaOXA48 and blaCMY-4, respectively, were traced to all K. pneumoniae ST25 isolates. Klebsiella aerogenes ST93 and E. coli ST453 harboured either only pEclA2, or both pEclA2 and pEclA4. One suspected case harbouring OXA-162-producing K. pneumoniae ST37 could be excluded from the outbreak. Once initiated by an E. cloacae strain, the outbreak was caused by the dissemination of a K. pneumoniae ST25 strain and involved inter-species horizontal transfer of two resistance plasmids, one of which carried blaOXA-48. To our knowledge, this is the first description of an outbreak of OXA-48-producing Enterobacteriaceae in a neonatal setting in northern Europe.

The Soaring Increase of Resistant Microorganisms Demands Immediate Action Solutions are Available

Clinical environments provide an ideal reservoir for the growth, proliferation, and transmission of pathogenic organisms. Surfaces in hospitals e.g. hospital furniture, ECG lead wires and other cables, push buttons of infusion pumps, control knobs of ventilation machines, textiles as well as implantable biomaterials like central venous catheters, urologic catheters, endotracheal tubes are contaminated increasingly frequent with multiresistant microorganisms. These microorganisms are distributed by the hands of the nursing personnel throughout the hospital with serious, life threatening consequences. 1.8 million patients suffer from a nosocomial infection per year in Europe; approximately 180,000 deaths are attributed to these infections. The Centers for Disease Control (CDC) estimates that 2 million U.S. patients per year acquire a hospital-related infection. These infections cause 90,000 deaths each year and cost an average of $47,000 per patient to treat. The added cost to hospitals is $4.8 billion anually for extended care treatment. Microorganisms show an increasing rate of resistance against the majority of antibiotics including carbapenems as last available antibiotic. 700,000 deaths have been rеρorted worldwide, 30 000 deaths alone in Europe during the last year due to infections where no effective antimicrobial substance was available. The use of disinfectants is ostensibly intended to remove/kill pathogens on surfaces. However studies have shown that more than one-half the time, surfaces are not adequately cleaned or are re-contaminated within minutes. Much emphasis has been put therefore on hand disinfection. However there are also rеρorts of the emergence of alcohol tolerant/insensitive microorganisms e.g., vancomycin resistant enterococci. This phenomenon has the potential to undermine the effectiveness of alcohol based disinfectant standard precautions. The reason for this dramatic development of resistant microorganisms is still in debate. The indiscriminate use of antibiotics for prevention of a bacterial superinfections e.g. sinusitis, otitis media after a viral infections is frequently incriminated, however this seems to have little impact on the occurrence of multi-resistant hospital pathogens in general. However individual patients are seriously affected by the selection of multi-resistant pathogens. In contrast there is increasing evidence that the widespread use of disinfectants is responsible: disinfectants - analogous to antibiotics - must be incorporated into the metabolism of microorganisms. This is inevitably associated with induction of resistance by transfer of resistance plasmids e.g. induction of efflux pumps. 10 167 рublications (PUBMed Jan. 2023) are available in the international literature which document the resistance of microorganisms against disinfectants; 649 рublications describe the cross resistance with antibiotics, at the same time there are 11 237 publications on the toxicity of disinfectants.

A mathematical model of plasmid-carried antibiotic resistance transmission in two types of cells

Abstract Antibiotic resistance is a significant public health problem. When resistance genes are being carried on plasmids, the spread can be greatly accelerated. In this paper, the transmission of antibiotic resistance in two types of cells is discussed. A mathematical model is established to describe the dynamics of the transmission of plasmids. The effects of different parameters on the stable solution and sensitivity analysis are studied by numerical simulation. The conclusions show that the concentration of antibiotics must reach a certain level to kill the pathogenic bacteria. If the concentration of antibiotics is not high to a certain extent, the treatment becomes ineffective. If the cost of cells carried on plasmids and the rate of resistance plasmids segregation too high, the drug-resistant cells will gradually die out in the system. The rate of horizontal transfer of resistance plasmids is directly related to the spread of drug resistance. With the increase in the horizontal transfer rate of resistance plasmids, cells in the body gradually turn into cells with antibiotic resistance, which causes substantial difficulties in the treatment of diseases.

Paclitaxel and its derivative facilitate the transmission of plasmid-mediated antibiotic resistance genes through conjugative transfer

The rapid dissemination of antibiotic resistance by horizontal gene transfer (HGT) renders the global resistance crisis more tense and urgent as few effective antimicrobials are available to combat multidrug-resistant (MDR) pathogens at present. Conjugation is one of the most dominant and representative pathways of HGT. Antibiotic residue in environment is recognized as an important accelerator for conjugal transfer, whereas the roles of non-antibiotic pharmaceuticals in this process are not fully understood. Here we found that environmentally relevant concentrations of paclitaxel as well as its derivative docetaxel, two commonly used anticancer drugs, remarkably facilitated the conjugative transfer of resistance plasmids carrying multiple antibiotic resistance genes (ARGs). The underlying mechanisms accounting for the enhanced conjugation were investigated by detecting the activity of RpoS regulon, membrane permeability, SOS response and gene expression of conjugative transfer systems. Our results showed that paclitaxel induced a series of cellular responses, including up-regulation of rpoS expression, activated SOS response, increased cell membrane permeability, enhanced plasmid replication and mating pilus formation. Collectively, our data provide new insight on the roles of paclitaxel and its derivative in promoting the conjugal transfer of ARGs, highlighting the importance of good antimicrobial stewardship.

A Novel bla CTX-M-65-Harboring IncHI2 Plasmid pE648CTX-M-65 Isolated from a Clinical Extensively-Drug-Resistant Escherichia coli ST648.

BackgroundAn ESBL, carbapenemase- and MCR-1-producing Escherichia coli ST648 strain was isolated from the urine sample of a patient in a Chinese tertiary hospital in 2016.MethodsThe strain was fully sequenced by GridION X5 platform of Oxford Nanopore Technology.ResultsThe sequence analysis showed that the extended-spectrum β-lactamases CTX-M-65 and OXA-1, the carbapenemase NDM-5, the MCR-1 were encoded, respectively, by three different resistance plasmids. The pE648CTX-M-65-carrying blaCTX-M-65 was a novel conjugative plasmid belonging to IncHI2 type; except for the blaCTX-M-65, it also carried resistance genes ble, floR, sul1, aph(4)-Ia, aac(3)-VI, aac(6ʹ)-II, blaOXA-1, catB, arr3 and tetA. Besides, an IncX4 plasmid pE648MCR-1-carrying mcr-1 and an IncX3 plasmid pE648NDM-5-carrying blaNDM-5 were also identified.ConclusionThe three transferable resistance plasmids coexisting in the E. coli ST648 isolate indicated the high risk to disseminate the extensively-drug-resistance among Enterobacteriaceae.

Occurrence of two NDM-1-producing Raoultella ornithinolytica and Enterobacter cloacae in a single patient in China: probable a novel antimicrobial resistance plasmid transfer in vivo by conjugation

ObjectivesTo identify the general features of acquisition of drug-resistance genes in two multi-drug resistant Enterobacteriaceae strains isolated from a single patient in China. MethodsThe whole-plasmid was sequenced by Illumina Hiseq 4000 and Pacbio RSII procedures. The plasmid conjugation transfer experiment were performed by the mating-out assay. Drug-resistance genes were amplified by PCR assay. ResultsWe identified two New Delhi metallo-β-lactamase type 1(NDM-1)-producing isolates, named Raoultella ornithinolytica B1645-1 and Enterobacter cloacae B1645-2, which shared the same sulfonamide-resistant dihydropteroate synthase sul2 gene and aminoglycoside O-phosphotransferase aph(3'')-Ib gene. A novel antimicrobial resistance plasmid pCYNDM01 was first discovered from the multi-drug resistant R. ornithinolytica B1645-1. Interestingly, plasmid pCYNDM01 carried a Gifsy-2 prophage gene. The blaNDM-1 gene was located on a novel complex class 1 integron with a structure of sul1-qacEΔ1-ΔISAba125-blaNDM-1-blaMBL-trpC-ISCR1-catb8-aacA4-IS1-IS6100-dfrA14-intI1. The carrying the blaNDM-1 gene plasmid pCYNDM01 was transferred to the E. cloacae B1645-2 recipient strain. This 149.44 kb plasmid pCYNDM01 belonged to the IncFII type. ConclusionsA novel antimicrobial resistance plasmid pCYNDM01 was first recovered from a multi-drug resistance R. ornithinolytica B1645-1 isolated from China. The novel complex sul1-type class 1 integron might play an essential role in the mobilization of the blaNDM-1 gene among different enterobacterial species. The occurrence of plasmid pCYNDM01 transfer from R. ornithinolytica to E. cloacae in vitro by conjugation showed that plasmid pCYNDM01 was a self-conjugative plasmid and might cause dissemination of drug-resistance genes within different enterobacterial species from a single patient in vivo by conjugation. The novel variant F-like T4SS of plasmid pCYNDM01 might be as a tool of R. ornithinolytica B1645-1 for resistance genes transfer. The emergence of the two NDM-1-producing Enterobacteriaceae strains should be attracted China attentions and required to prevent its future prevalence.

Salmonella persisters promote the spread of antibiotic resistance plasmids in the gut.

SummaryThe emergence of antibiotic resistant bacteria by mutations or by acquisition of genetic material like resistance plasmids represents a major public health issue 1,2 (Extended Data Fig. 1a). Persisters are bacterial subpopulations surviving antibiotics by reversibly adapting their physiology 3–10. They promote the emergence of antibiotic resistant mutants 11. We asked if persisters can also promote the spread of resistance plasmids. In contrast to mutations, resistance plasmid transfer requires the co-occurrence of two different bacterial strains: a donor and a recipient (Extended Data Fig. 1a). For our experiments, we chose the facultative intracellular entero-pathogen Salmonella enterica serovar Typhimurium (S.Tm) and E. coli, a common microbiota member 12. S.Tm forms persisters surviving antibiotic therapy in several host tissues. We show that tissue-associated, S.Tm persisters account for long-lived reservoirs of plasmid donors or recipients. Persistent S.Tm reservoir formation requires Salmonella Pathogenicity Island (SPI) -1/2 in the gut-associated tissues or SPI-2 at systemic sites. Re-seeding of these bacteria into the gut lumen allows co-occurrence of donors with gut-resident recipients, thereby favouring plasmid transfer between various Enterobacteriaceae. We observe up to 99% transconjugants within 2-3 days after re-seeding. Mathematical modeling shows that rare re-seeding events may suffice for a high frequency of conjugation. Vaccination reduces tolerant reservoir formation after oral Salmonella infection and subsequent plasmid transfer. We conclude that even without selection for plasmid-encoded resistance genes, small persistent pathogen reservoirs can foster the spread of promiscuous resistance plasmids in the gut.

Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak.

The global spread of antibiotic resistance among Enterobacteriaceae is largely due to multidrug resistance plasmids that can transfer between different bacterial strains and species. Horizontal gene transfer of resistance plasmids can complicate hospital outbreaks and cause problems in epidemiological tracing, since tracing is usually based on bacterial clonality. We have developed a method, based on optical DNA mapping combined with Cas9-assisted identification of resistance genes, which is used here to characterize plasmids during an extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae outbreak at a Swedish neonatal intensive care unit. The outbreak included 17 neonates initially colonized with ESBL-producing Klebsiella pneumoniae (ESBL-KP), some of which were found to carry additional ESBL-producing Escherichia coli (ESBL-EC) in follow-up samples. We demonstrate that all ESBL-KP isolates contained two plasmids with the blaCTX-M-15 gene located on the smaller one (~80 kbp). The same ESBL-KP clone was present in follow-up samples for up to 2 years in some patients, and the plasmid carrying the blaCTX-M-15 gene was stable throughout this time period. However, extensive genetic rearrangements within the second plasmid were observed in the optical DNA maps for several of the ESBL-KP isolates. Optical mapping also demonstrated that even though other bacterial clones and species carrying blaCTX-M group 1 genes were found in some neonates, no transfer of resistance plasmids had occurred. The data instead pointed toward unrelated acquisition of ESBL-producing Enterobacteriaceae (EPE). In addition to revealing important information about the specific outbreak, the method presented is a promising tool for surveillance and infection control in clinical settings.IMPORTANCE This study presents how a novel method, based on visualizing single plasmids using sequence-specific fluorescent labeling, could be used to analyze the genetic dynamics of an outbreak of resistant bacteria in a neonatal intensive care unit at a Swedish hospital. Plasmids are a central reason for the rapid global spread of bacterial resistance to antibiotics. In a single experimental procedure, this method replaces many traditional plasmid analysis techniques that together provide limited details and are slow to perform. The method is much faster than long-read whole-genome sequencing and offers direct genetic comparison of patient samples. We could conclude that no transfer of resistance plasmids had occurred between different bacteria during the outbreak and that secondary cases of ESBL-producing Enterobacteriaceae carriage were instead likely due to influx of new strains. We believe that the method offers potential in improving surveillance and infection control of resistant bacteria in hospitals.

Bacterial exposure to ZnO nanoparticles facilitates horizontal transfer of antibiotic resistance genes

Bacterial exposure to ZnO nanoparticles (nZnO) at sublethal concentrations (1 to 10mg/l) for 24h significantly increased the conjugative frequency of antibiotic resistance plasmid RP4. A 24.3-fold increase was observed in E. coli pure cultures, and an 8.3-fold increase occurred in a mixed culture of indigenous aquatic microbiota. In addition, nZnO increased by three fold the transformation efficiency of E. coli via the uptake of naked plasmid pGEX4T-1, which was confirmed by confocal fluorescence microscopy. Enhanced horizontal transfer of resistance plasmids was a nanoparticle-specific effect, since 5.3mg/l Zn2+ (representing the maximum release of Zn2+ from 10mg/l nZnO) did not enhance RP4 transfer or pGEX4T-1 uptake frequency. This phenomenon was attributed to increased bacterial cell permeability after exposure to nZnO (but not to Zn2+), which was confirmed by flow cytometry with propidium iodide staining. Overall, this study forewarns that accidental or incidental release of nZnO may facilitate environmental dissemination and propagation of antibiotic resistance genes through horizontal gene transfer.

Emergence of a multidrug-resistant Citrobacter freundii ST8 harboring an unusual VIM-4 gene cassette in Poland

ObjectivesThe growing incidence of multidrug-resistant (MDR) bacteria is an emerging challenge in modern medicine. The utility of carbapenems, which are considered ‘last-line’ agents, is being diminished by the growing incidence of various resistance mechanisms in Gram-negative bacteria. A molecular investigation was performed of an MDR carbapenem-resistant Citrobacter freundii of sequence type 8 (ST8) isolated from a hematology patient with acute myeloid leukemia. MethodsMultilocus sequence typing and analysis of the nucleotide sequence of the class I integron were performed using PCR and Sanger sequencing. Transformation of the resistance plasmid isolated following the alkaline lysis method was performed using chemically competent E. coli TOP10. ResultsMolecular analysis of the carbapenem-resistant C. freundii revealed the presence of the VIM-4 isoenzyme located on the ∼55-kb transferable resistance plasmid. Interestingly, the blaVIM-4 gene was inserted into an unusual gene cassette containing a 169-bp direct repeat of the 3′ segment of the blaVIM-4 gene. ConclusionsAll unusual gene cassettes containing VIM-DR (direct repeat) described thus far have been harbored by non-fermenters, i.e., Acinetobacter and Pseudomonas, underscoring the importance of resistance determinant mobility, which may go even beyond genus, family, and order boundaries. Great efforts need to be taken to explore pathways of resistance to ‘last-resort’ antimicrobials, especially among clinically relevant pathogens.

Conjugation Assay for Testing CRISPR-Cas Anti-plasmid Immunity in Staphylococci.

CRISPR-Cas is a prokaryotic adaptive immune system that prevents uptake of mobile genetic elements such as bacteriophages and plasmids. Plasmid transfer between bacteria is of particular clinical concern due to increasing amounts of antibiotic resistant pathogens found in humans as a result of transfer of resistance plasmids within and between species. Testing the ability of CRISPR-Cas systems to block plasmid transfer in various conditions or with CRISPR-Cas mutants provides key insights into the functionality and mechanisms of CRISPR-Cas as well as how antibiotic resistance spreads within bacterial communities. Here, we describe a method for quantifying the impact of CRISPR-Cas on the efficiency of plasmid transfer by conjugation. While this method is presented in Staphylococcus species, it could be more broadly used for any conjugative prokaryote.

CRISPR-Cas and Restriction-Modification Act Additively against Conjugative Antibiotic Resistance Plasmid Transfer in Enterococcus faecalis.

Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections. Conjugative pheromone-responsive plasmids are narrow-host-range mobile genetic elements (MGEs) that are rapid disseminators of antibiotic resistance in the faecalis species. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification confer acquired and innate immunity, respectively, against MGE acquisition in bacteria. Most multidrug-resistant E.faecalis isolates lack CRISPR-Cas and possess an orphan locus lacking cas genes, CRISPR2, that is of unknown function. Little is known about restriction-modification defense in E.faecalis. Here, we explore the hypothesis that multidrug-resistant E.faecalis strains are immunocompromised. We assessed MGE acquisition by E.faecalis T11, a strain closely related to the multidrug-resistant hospital isolate V583 but which lacks the ~620kb of horizontally acquired genome content that characterizes V583. T11 possesses the E.faecalis CRISPR3-cas locus and a predicted restriction-modification system, neither of which occurs in V583. We demonstrate that CRISPR-Cas and restriction-modification together confer a 4-log reduction in acquisition of the pheromone-responsive plasmid pAM714 in biofilm matings. Additionally, we show that the orphan CRISPR2 locus is functional for genome defense against another pheromone-responsive plasmid, pCF10, only in the presence of cas9 derived from the E.faecalis CRISPR1-cas locus, which most multidrug-resistant E.faecalis isolates lack. Overall, our work demonstrated that the loss of only two loci led to a dramatic reduction in genome defense against a clinically relevant MGE, highlighting the critical importance of the E.faecalis accessory genome in modulating horizontal gene transfer. Our results rationalize the development of antimicrobial strategies that capitalize upon the immunocompromised status of multidrug-resistant E.faecalis. IMPORTANCE Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E.faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E.faecalis populations. The loss of these systems in high-risk E.faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics.

Antimicrobial Resistance status and prevalence rates of Extended Spectrum Beta-Lactamase (ESBL) producers isolated from a mixed human population.

Owing to the increasing epidemiological and therapeutic challenges associated with infections due to ESBL producers, ESBL prevalence rate among some bacteria isolates from healthy and non-healthy human population in a metropolitan Nigerian setting was evaluated. A total of one hundred and forty-five (145) bacteria strains were isolated from a total of four hundred and sixty (460) samples collected from urine, wound, throat and anal swabs of 220 healthy volunteers in the community and from 240 patients in 2 secondary and 2 tertiary hospitals (altogether, 4) in Enugu metropolis. The presumptive confirmatory test used for ESBL detection was the Double Disc Synergy Test (DDST) method. Conjugation and plasmid curing studies were also done for resistance factor determination. Of the 145 isolates, 20 were ESBL producers with 35% of these ESBL producers being of community origin and 65% from hospitals. This translates to 4.8% and 9% incidences (comparably higher than established prevalence of 4.4% and 7.5 respectively) for community and hospital infections respectively. The ESBL isolates showed high resistance to tetracycline, gentamicin, pefloxacin, ceftriaxone, cefuroxime, ciprofloxacin and Augmentin(®) (Amoxicilin and clavulanic acid combination). Conjugation studies for Resistance plasmid transfer showed non-transference of resistance determinants between the ESBL transconjugants and recipient strains. Correspondingly, the plasmid curing studies revealed that the acridine orange could not effect a cure on the isolates as they still retained high resistance to the antibiotics after the treatment. This study confirms the growing incidences/pool of ESBL strains in Nigeria and call for widespread and continuous monitoring towards an effective management of the potential therapeutic hurdle posed by this trend.

Increased detection of extended spectrum beta-lactamase producing Salmonella enterica and Escherichia coli isolates from poultry

To gain more information on the genetic basis of the rapid increase in the number of isolates exhibiting non-wild type Minimum Inhibitory Concentrations (MICs) for cefotaxime observed since 2003, beta-lactamase genes of 22 Salmonella enterica and 22 Escherichia coli isolates from broilers in 2006 showing this phenotype were characterized by miniaturized micro-array, PCR and DNA-sequencing. Presence and size of plasmids were determined by S1-digest pulsed-field gel electrophoresis and further characterized by PCR-based replicon typing. Transfer of resistance plasmids was tested by conjugation and transformation experiments. To link resistance genes and plasmid type, Southern blot hybridization experiments were conducted. In 42 isolates, five ( bla CTX-M-1, bla CTX-M-2, bla TEM-20, bla TEM-52, bla SHV-2) different extended spectrum beta-lactamase (ESBL)-genes and two ( bla ACC-1, bla CMY-2) AmpC-genes were present. Three of the detected ESBL-genes ( bla CTX-M-1, bla TEM-52 and bla CTX-M-2) were located on similar types of plasmids (IncI1 and IncHI2/P) in both E. coli and Salmonella. Two other detected ESBL- and AmpC-genes bla SHV-2 and bla CMY-2 respectively (on IncK plasmids), were only found in E. coli, whereas the AmpC-gene bla ACC-1 (on non-typable plasmids), and the ESBL-gene bla TEM-20 (on IncI1 plasmids), were only detected in Salmonella. In two isolates, no ESBL- or AmpC-gene could be detected through these methods. The increase in the number of E. coli and S. enterica isolates from the gastro-intestinal tract of broilers exhibiting non-wild type MICs for cefotaxime is mainly due to an increase in IncI1 plasmids containing bla CTX-M-1. The reason for the successful spread of this plasmid type in these species is not yet understood.

Comment on: Emergence of multidrug-resistant Gram-negative bacteria during selective decontamination of the digestive tract on an intensive care unit

Objectives: During treatment with selective decontamination of the digestive tract (SDD), four multidrugresistant (MDR) strains, three different Escherichia coli and one Klebsiella pneumoniae, were isolated from four patients not known as carriers of such MDR strains before their admission to the intensive care unit (ICU) in the Academic Medical Center (AMC) in Amsterdam. These isolates were extended-spectrum b-lactamase (ESBL)-positive. We investigated whether this was due to interspecies transfer of resistance genes. Methods: The MDR strains were typed by amplified fragment length polymorphism (AFLP) analysis. The plasmids from these strains were characterized by restriction fragment length polymorphism and the resistance genes were characterized by PCR and sequence analysis. Results: The strains were genetically unrelated and contained identical plasmids with ESBL genes. Conclusions: We identified an outbreak of plasmid-mediated ESBL genes during SDD treatment in the ICU. The use of third-generation cephalosporins in SDD is associated with the emergence of ESBLs. We conclude that identification of emerging MDR Gram-negative bacteria and recognition of resistance plasmid transfer during SDD treatment are crucial for optimal application of this regimen in ICUs.

Emergence of multidrug-resistant Gram-negative bacteria during selective decontamination of the digestive tract on an intensive care unit

During treatment with selective decontamination of the digestive tract (SDD), four multidrug-resistant (MDR) strains, three different Escherichia coli and one Klebsiella pneumoniae, were isolated from four patients not known as carriers of such MDR strains before their admission to the intensive care unit (ICU) in the Academic Medical Center (AMC) in Amsterdam. These isolates were extended-spectrum beta-lactamase (ESBL)-positive. We investigated whether this was due to interspecies transfer of resistance genes. The MDR strains were typed by amplified fragment length polymorphism (AFLP) analysis. The plasmids from these strains were characterized by restriction fragment length polymorphism and the resistance genes were characterized by PCR and sequence analysis. The strains were genetically unrelated and contained identical plasmids with ESBL genes. We identified an outbreak of plasmid-mediated ESBL genes during SDD treatment in the ICU. The use of third-generation cephalosporins in SDD is associated with the emergence of ESBLs. We conclude that identification of emerging MDR Gram-negative bacteria and recognition of resistance plasmid transfer during SDD treatment are crucial for optimal application of this regimen in ICUs.

Evolution of multi-resistance plasmids in Australian clinical isolates of Escherichia coli

Plasmids allow the movement of genetic material, including antimicrobial resistance genes, between bacterial species and genera. They frequently mediate resistance to multiple antimicrobials and can result in the acquisition by a pathogen of resistance to all or most clinically relevant antimicrobials. Unfortunately, there are still large gaps in our understanding of how new multi-resistance plasmids evolve. Five Australian clinical institutions collaborated in this study of multi-resistance plasmids in clinical isolates of Escherichia coli. We characterized 72 resistance plasmids in terms of the antimicrobial resistance profile they conferred, their size and their incompatibility group. Restriction fragment length polymorphisms were used to determine the genetic relationships between the plasmids. Relationships between the host cells were determined using multi-locus enzyme electrophoresis. A lack of correlation between the evolutionary history of the host cells and their plasmids suggests that the horizontal transfer of resistance plasmids between strains of E. coli is common. The resistance plasmids were very diverse, with a wide range of resistance profiles and a lack of discrete evolutionary lineages. Multi-resistance plasmids did not evolve via the co-integrative capture of smaller resistance plasmids; rather, the roles of recombination and the horizontal movement of mobile genetic elements appeared to be most important.

Inhibition of resistance plasmid transfer in Escherichia coli by ionophores, chlortetracycline, bacitracin, and ionophore/antimicrobial combinations.

Medicinal feed additives bacitracin, chlortetracycline (CTC), laidlomycin, lasalocid, and salinomycin inhibited the transfer of multiresistance-conferring plasmid pBR325 (Tet(r) Amp(r) Cp(r), 6.0 kb) into selected gram-negative strains with the use of an in vitro model. High concentrations of ampicillin-sensitive competence-pretreated Escherichia coli HB 101 cells were exposed to 10% (v/v) of 1:10 dimethyl sulfoxide/agent : water containing test mixtures for 0.5 hr prior to plasmid addition and transforming conditions. Transformation was inhibited for all antimicrobials and showed a positive association wich higher concentration. Additional testing of ionophore compounds separately and in combination with bacitracin, chlortetracycline, lincomycin, roxarsone, tylosin, and virginiamycin at representative feed concentrations demonstrated 80.6% to >99.9% inhibition (P < 0.001) of resistance transfer. Bacitracin alone inhibited transformation within the range of 50-500 ppm. No increase in resistance transfer was observed when poultry-derived and reference gram-negative isolates having low or no transformation efficiency were additionally tested. The results suggest that these compounds, at relevant concentrations used in animal feed, may interfere with cell envelope-associated DNA uptake channels or other transformation competence mechanisms. Through these mechanisms, ionophores and cell membrane-interactive feed agents such as CTC and bacitracin may act to inhibit resistance transfer mechanisms within poultry and livestock.