Plasmid-mediated fluoroquinolone resistance in Enterobacteriaceae

In tutto il mondo si sta assistendo ad un aumento dell\u2019incidenza di isolati clinici di Enterobacteriaceae resistenti ai fluorochinoloni. La resistenza pu\uf2 essere dovuta ad un accumulo di mutazioni cromosomiche a livello dei geni che codificano le topoisomerasi, bersaglio d\u2019azione dei fluorochinoloni; mediata da determinanti plasmidici, in particolare quelli che codificano per proteine di protezione Qnr, per l\u2019enzima di acetilazione aac(6\u2019)-Ib-cr e per le pompe di efflusso QepA e OqxAB. Tutti questi determinanti da soli conferiscono al batterio bassi livelli di resistenza ma consentono un aumento della frequenza di mutazione,facilitando la selezione di alti livelli di resistenza. Lo scopo di questo lavoro \ue8 stato quello di valutare l\u2019incidenza della presenza di determinanti plasmidici che mediano la resistenza ai fluorochinoloni (PMQR), in una collezione di isolati clinici di Enterobacteriaceae provenienti da vari laboratori del nord-est Italia. Tutti i ceppi risultati positivi per la presenza di questi determinanti sono stati caratterizzati dal punto di vista plasmidico e correlati alla presenza di geni che codificano per beta-lattamasi. Sono stati raccolti 756 isolati clinici di enterobatteri: 497 Escherichia coli, 68 Klebsiella spp., 18 Citrobacter spp., 69 Proteus spp., 24 Morganella spp., 21 Providencia spp., 52 Enterobacter spp. and 7 Serratia marcescens. Per tutti i ceppi sono stati eseguiti i saggi di sensibilit\ue0 con il metodo della microdiluizione a ciprofloxacina e levofloxacina, ottenendo valori di MIC variabili da 0.06 a 128 \ub5g/ml. Lo screening dei PMQR mediante PCR ha evidenziato 108 positivi (38 qnr e 70 aac(6\u2019)-Ib-cr) su 104 isolati. L\u2019analisi delle sequenze degli ampliconi ha permesso di classificare le varianti qnr in 26 qnrS1, 2 qnrB2, 2 qnrB6, 2 qnrB8, 1 qnrB19 e 5 qnrD. Per quest\u2019ultimo determinante sono risultati positivi 4 P. mirabilis e una M. morganii verificandone la localizzazione plasmidica con Southern blot e ottenendo la sequenza completa mediante PCR inversa (2687 bp per P. mirabilis e 2684 bp per M. morganii depositate in Genbank con i numeri di accesso JN183060 and JN183061). I ceppi positivi per qnrS1 erano suddivisi in 10 E. coli, 15 Klebsiella spp. e un P. mirabilis. In questi ceppi qnrS1 era accompagnato da VIM-1, SHV-12 e LAP-2. I 4 E. coli che ospitavano sia qnrS1 che aac(6\u2019)-Ib-cr ospitavano anche CTX-M-15, OXA-1 and TEM-1. I plasmidi dei positivi sono stati caratterizzati con il metodo dell\u2019origine della replicazione. I ceppi qnrS1 ospitano questo gene su plasmidi di tipo incN. Con esperimenti di coniugazione si \ue8 visto che il plasmide con qnrS1 e VIM-1 \ue8 coniugabile. Inoltre, nei tre ceppi di K. pneumoniae ST147 con qnrS1 e blaLAP-2 si \ue8 dimostrato che questi sono integrati nello stesso contesto genetico di ISEcl2. I 70 ceppi che presentano la variante aac(6\u2019)-Ib-cr erano suddivisi in 60 E. coli, 8 Klebsiella spp., 1 P. mirabilis e 1 M. morganii. La tipizzazione dei plasmidi ha messo in evidenza i tipi incFia e incF sia in E. coli che P. mirabilis, mentre incColE era predominante in Klebsiella spp. e M. morganii era caratterizzata da un plasmide non tipizzabile. Si \ue8 inoltre evidenziata una alta correlazione fra la variante aac(6)-Ib-cr e le beta-lattamasi OXA-1 e CTX-M-15 sia in E. coli (42/60), che in Klebsiella spp. (8/8), con l\u2019eccezione di P. mirabilis dove invece con OXA-1 \ue8 presente TEM-52. Dalla nostra indagine \ue8 emersa una prevalenza di determinanti plasmidici che mediano la resistenza ai fluorochinoloni del 13.7% (104/756). Solo il 13.1% (5/38) degli isolati positivi per il qnr risultano in un range di bassa resistenza, mentre i ceppi positivi per la variante aac(6\u2019)-Ib-cr presentavano tutti alti livelli di resistenza. La nostra indagine ha consentito di descrivere il primo determinante qnrD isolato in Europa e la prima beta-lattamasi LAP-2 isolata in Italia.Clinical isolates of fluoroquinolone-resistant Enterobacteriaceae are emerging worldwide. The traditional resistance mechanism is the accumulation of mutations in the chromosome coding for the target molecules of fluoroquinolone. All known plasmid-mediated quinolone resistance determinants - namely, Qnr determinants, aac(6\u2019)-Ib-cr enzyme, qepA and oqxAB efflux pumps - can individually confer low-level resistance. In this condition, bacterial cells have increased mutation frequency, making it easier the selection of higher-level fluoroquinolone resistance. Our aims were to survey for plasmid-mediated quinolone resistance determinants a collection of Enterobacteriaceae clinical isolates originating from clinical microbiological laboratories of North-East Italy, and to characterize both the resistant strains and the plasmids harbouring quinolone resistance determinants and beta-lactamase genes. Altogether, 756 Enterobacteriaceae clinical isolates were collected: 497 Escherichia coli, 68 Klebsiella spp., 18 Citrobacter spp., 69 Proteus spp., 24 Morganella spp., 21 Providencia spp., 52 Enterobacter spp. and 7 Serratia marcescens. MIC values were determined by microdilution for ciprofloxacin and levofloxacin, showing values between 0.06 and 128 \ub5g/ml. Screening by PCR for plasmid-mediated quinolone resistance determinants yielded 108 positives [38 qnr and 70 aac(6\u2019)-Ib-cr] out of 104 isolates. Sequencing and analysis of PCR-positive products verified: 26 qnrS1, 2 qnrB2, 2 qnrB6, 2 qnrB8, 1 qnrB19 and 5 qnrD. Four Proteus mirabilis and one Morganella morganii isolates were qnrD positive. Plasmid extraction was performed, and Southern blot analysis verified the plasmid localization of the qnrD determinants. Inverse PCR-based sequencing of qnrD-harbouring plasmids, resulted in a 2687 bp and 2684 bp plasmid DNA for Proteus mirabilis and Morganella morganii, respectively. Both plasmid sequences are deposited at Genbank with accession numbers JN183060 and JN183061. Among the qnrS1 positive isolates, 10 E. coli, 15 Klebsiella spp. and one Proteus mirabilis were detected. Detection of beta-lactamase genes by PCR and sequencing yielded: 6 VIM-1, 7 SHV-12 and 3 LAP-2 positives. All four qnrS1 E. coli with the aac(6\u2019)-Ib-cr variant were CTX-M-15, OXA-1 and TEM-1 positive. PCR-based replicon typing found incN type plasmid to be the most prevalent. Conjugation experiment found 3 qnrS1 and VIM-1 harbouring conjugable plasmids. We could also demonstrate in three Klebsiella pneumoniae ST147 strains that qnrS1 and blaLAP-2 are integrated in the same ISEcl2 genetic context. Southern blot verified that the qnrS1 gene is localized on incN or untypable plasmids. Altogether 70 aac(6\u2019)-Ib-cr variant positive isolates were found: 60 E. coli, 8 Klebsiella spp., one P.mirabilis and one M.morganii. Replicon typing of plasmids found incFia and incF types to be common for E. coli and P. mirabilis while incColE type to be predominant in Klebsiella spp., whilst M.morganii was untypeable. A high correlation of the aac(6)-Ib-cr variant with OXA-1 and CTX-M-15 beta-lactamases was found in E. coli (42/60) and in Klebsiella spp. (8/8), whilst in P. mirabilis the aac(6)-Ib-cr variant was associated with TEM-52 and OXA-1. Our survey for plasmid-mediated quinolone resistance determinants found a 13.7% prevalence (104/756). MIC values for ciprofloxacin and levofloxacin showed uneven resistance levels among the qnr-positive isolates, with only 13.1% of strains (5/38) in the range of low-level resistance, while in case of the aac(6\u2019)-Ib-cr variant all positive isolates were resistant to fluoroquinolones. Our survey found the first qnrD determinants in Europe, and we could also demonstrate the first LAP-2 beta-lactamase in Italy

Multiple Benefits of Plasmid-Mediated Quinolone Resistance Determinants in Klebsiella pneumoniae ST11 High-Risk Clone and Recently Emerging ST307 Clone

International high-risk clones of Klebsiella pneumoniae are among the most common nosocomial pathogens. Increased diversity of plasmid-encoded antimicrobial resistance genes facilitates spread of these clones causing significant therapeutic difficulties. The purpose of our study was to investigate fluoroquinolone resistance in extended-spectrum beta-lactamase (ESBL)-producing strains, including four K. pneumoniae and a single K. oxytoca, isolated from blood cultures in Hungary. Whole-genome sequencing and molecular typing including multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed in selected strains. Gene expression of plasmid-mediated quinolone resistance determinants (PMQR) was investigated by quantitative-PCR. MLST revealed that three K. pneumoniae strains belonged to ST11 and one to ST307 whereas K. oxytoca belonged to ST52. The isolates harbored different β-lactamase genes, however, all K. pneumoniae uniformly carried blaCTX-M-15. The K. pneumoniae isolates exhibited resistance to fluoroquinolones and carried various PMQR genes namely, two ST11 strains harbored qnrB4, the ST307 strain harbored qnrB1 and all K. pneumoniae harbored oqxAB efflux pump. Levofloxacin and moxifloxacin MIC values of K. pneumoniae ST11 and ST307 clones correlated with qnr and oqxAB expression levels. The qnrA1 carrying K. oxytoca ST52 exhibited reduced susceptibility to fluoroquinolones. The maintained expression of qnr genes in parallel with chromosomal mutations indicate an additional protective role of Qnr proteins that can support dissemination of high-risk clones. During development of high-level fluoroquinolone resistance, high-risk clones retain fitness thus, enabling them for dissemination in hospital environment. Based on our knowledge this is the first report of ST307 clone in Hungary, that is emerging as a potential high-risk clone worldwide. High-level fluoroquinolone resistance in parallel with upregulated PMQR gene expression are linked to high-risk K. pneumoniae clones

Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage

Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use meta-genomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.Peer reviewe

Setting a baseline for global urban virome surveillance in sewage

The rapid development of megacities, and their growing connectedness across the world is becoming a distinct driver for emerging disease outbreaks. Early detection of unusual disease emergence and spread should therefore include such cities as part of risk-based surveillance. A catch-all metagenomic sequencing approach of urban sewage could potentially provide an unbiased insight into the dynamics of viral pathogens circulating in a community irrespective of access to care, a potential which already has been proven for the surveillance of poliovirus. Here, we present a detailed characterization of sewage viromes from a snapshot of 81 high density urban areas across the globe, including in-depth assessment of potential biases, as a proof of concept for catch-all viral pathogen surveillance. We show the ability to detect a wide range of viruses and geographical and seasonal differences for specific viral groups. Our findings offer a cross-sectional baseline for further research in viral surveillance from urban sewage samples and place previous studies in a global perspective

Alternative stabilities of a proline-rich antibacterial peptide in vitro and in vivo

The proline-rich designer antibacterial peptide dimer A3-APO is currently under preclinical development for the treatment of systemic infections caused by antibiotic-resistant Gram-negative bacteria. The peptide showed remarkable stability in 25% mouse serum in vitro, exhibiting a half-life of ∼100 min as documented by reversed-phase chromatography. Indeed, after a 30-min incubation period in undiluted mouse serum ex vivo, mass spectrometry failed to identify any degradation product. The peptide was still a major peak in full blood ex vivo, however, with degradation products present corresponding to amino-terminal cleavage. When injected into mice intravenously, very little, if any unmodified peptide could be detected after 30 min. Nevertheless, the major early metabolite, a full single-chain fragment, was detectable until 90 min, and this fragment exhibited equal or slightly better activity in the broth microdilution antimicrobial assay against a panel of resistant Enterobactericeae strains. The Chex1-Arg20 metabolite, when administered three times at 20 mg/kg to mice infected with a sublethal dose (over LD50) of an extended spectrum β-lactamase-producing Escherichia coli strain, completely sterilized the mouse blood, similar to imipenem added at a higher dose. The longer and presumably more immunogenic prodrug A3-APO, injected subcutaneously twice over a 3-wk period, did not induce any antibody production, indicating the suitability of this peptide or its active metabolite for clinical development

Setting a baseline for global urban virome surveillance in sewage

The rapid development of megacities, and their growing connectedness across the world is becoming a distinct driver for emerging disease outbreaks. Early detection of unusual disease emergence and spread should therefore include such cities as part of risk-based surveillance. A catch-all metagenomic sequencing approach of urban sewage could potentially provide an unbiased insight into the dynamics of viral pathogens circulating in a community irrespective of access to care, a potential which already has been proven for the surveillance of poliovirus. Here, we present a detailed characterization of sewage viromes from a snapshot of 81 high density urban areas across the globe, including in-depth assessment of potential biases, as a proof of concept for catch-all viral pathogen surveillance. We show the ability to detect a wide range of viruses and geographical and seasonal differences for specific viral groups. Our findings offer a cross-sectional baseline for further research in viral surveillance from urban sewage samples and place previous studies in a global perspective.</p