Additional Antibiotic Resistance Genes Research Articles

Escherichia coli novel sequence type 11873 harbours a new CTX-M-15–carrying multidrug resistance type 1/2 hybrid IncC plasmid

ObjectiveThe aim of the current study was to determine the genomic map of the resistance genes of two CTX-M-15–carrying Escherichia coli strains belonging to novel sequence type (ST) 11873. Complete, closed genome sequences of the E. coli strains were obtained by applying a combination of short-read Illumina and long-read Oxford Nanopore-based sequencing. MethodsIsolation of E. coli was performed using ECC CHROMagar and antibiotic sensitivity patterns were determined using Sensititre EUVSEC plates. Whole-genome sequencing was performed for two E. coli strains (3–338 and 5–325) using Illumina MiSeq- and Oxford Nanopore MinION-based sequencing. ResultsThe complete genome of strain 3–338 (GenBank accession no. CP130007–17) was assembled into a circular chromosome of 4.65 Mb and 10 plasmids (between 2 and 148 kb). Strain 5–325 (CP130018–27) exhibited a circular chromosome of 4.7 Mb and 9 plasmids (between 2 and 148 kb). Both strains carried an identical type 1/2 hybrid IncC plasmid (∼148 kb) harbouring multiple antibiotic resistance genes (ARGs), including blaCTX−M-15, blaOXA-1, blaTEM-1, qnrS1, sul2, aphA1, aacC2, mph(A) and floR. This plasmid also carried heavy metal resistance genes, such as chrA and arsR. Strain 5–325 carried an additional IncFIB plasmid (∼78 kb) harbouring additional ARGs, including blaTEM-1, qnrS1, tet(A), dfrA14, sul2, strA and strB. ConclusionsOur study shows the emergence of a CTX-M-15–carrying type 1/2 hybrid IncC plasmid in novel E. coli ST11873. These findings emphasise the need for population-based sewage surveillance for understanding the prevalence of antibiotic resistance in pathogens in order to mitigate the further spread of such resistance factors.

Exploring the dynamics of antibiotic resistome on plastic debris traveling from the river to the sea along a representative estuary based on field sequential transfer incubations

The environmental risks arising from ubiquitous microplastics or plastic debris (PD) acting as carriers of antibiotic resistance genes (ARGs) have attracted widespread attention. Enormous amounts of plastic waste are transported by rivers and traverse estuaries into the sea every year. However, changes in the antibiotic resistome within the plastisphere (the biofilms formed on PD) as PD travels through estuaries are largely unknown. In this study, we performed sequential migration incubations for PD along Haihe Estuary to simulate the natural process of PD floating from rivers to the ocean. Metagenomic sequencing and analysis techniques were used to track microbial communities and antibiotic resistome on migrating PD and in seawater representing the marine environment. The total relative gene copies of ARGs on traveling PD remained stable. As migration between greatly varied waters, additional ARG subtypes were recruited to the plastisphere. Above 80 % ARG subtypes identified in the plastisphere were persistent throughout the migration, and over 30 % of these persistent ARGs were undetected in seawater. The bacterial hosts composition of ARGs on PD progressively altered as transported downstream. Human pathogenic bacteria carrying ARGs (HPBs-ARG) exhibited decreasing trends in abundance and species number during transfer. Individual HPBs-ARG persisted on transferred PD and were absent in seawater samples, comprising Enterobacter cloacae, Klebsiella pneumoniae, Mycobacterium tuberculosis, and Vibrio parahaemolyticus. Based on all detected ARGs and HPBs-ARG, the Projection Pursuit model was applied to synthetically evaluate the potential risks of antibiotic resistance on migrating PD. Diminished risks on PD were observed upon the river-to-sea journey but consistently remained significantly higher than in seawater. The potential risks posed to marine environments by drifting PD as dispersal vectors for antibiotic resistance deserve greater attention. Our results provide initial insights into the dynamics or stability of antibiotic resistome on PD crossing distinct aquatic systems in field estuaries.

High throughput qPCR unveils shared antibiotic resistance genes in tropical wastewater and river water

The global challenge posed by rising antimicrobial resistance, and the adoption of a One Health approach, has led to the prioritisation of surveillance for antibiotic resistance genes (ARGs) in various environments. Herein lies an information gap, particularly in the context of Thailand, where there is scarce data on ARG prevalence across diverse environmental matrices and throughout different seasons. This study aimed to fill this void, analysing ARG prevalence by high-throughput qPCR in influent (n = 12) and effluent wastewater (n = 12) and river water (n = 12). The study reveals a substantial and largely uniform presence of ARGs across all water sample types (87 % similarity). Intriguingly, no ARGs were exclusive to specific water types, indicating an extensive circulation of resistance determinants across the aquatic environment. The genes intI1, tnpA, and intI3, part of the integrons and mobile genetic elements group, were detected in high relative abundance in both wastewater and river water samples, suggesting widespread pollution of rivers with wastewater. Additional high-prevalence ARGs across all water types included qepA, aadA2, merA, sul1, qacF/H, sul2, aadB, and ereA. More alarmingly, several ARGs (e.g., blaVIM, intI3, mcr-1, mexB, qepA, vanA, and vanB) showed higher relative abundance in effluent and river water than in influents, which suggests malfunctioning or inadequate wastewater treatment works and implicates this as a possible mechanism for environmental contamination. Nine genes (i.e., blaCTX-M, blaVIM, emrD, ermX, intI1, mphA, qepA, vanA, and vanB) were recovered in greater relative abundance during the dry season in river water samples as compared to the wet season, suggesting there are seasonal impacts on the efficacy of wastewater treatment practices and pollution patterns into receiving waters. This study highlights the urgency for more effective measures to reduce antibiotic resistance dissemination in water systems.

Temporal disturbance of a model stream ecosystem by high microbial diversity from treated wastewater.

Microbial communities in freshwater streams play an essential role in ecosystem functioning via biogeochemical cycling. Yet, the impacts of treated wastewater influx into stream ecosystems on microbial strain diversity remain mostly unexplored. Here, we coupled full-length 16S ribosomal RNAgene Nanopore sequencing and strain-resolved metagenomics to investigate the impact of treated wastewater on a mesocosm system (AquaFlow) run with restored river water. Over 10 days, community Bray-Curtis dissimilarities between treated and control mesocosm decreased (0.57 ± 0.058 to 0.26 ± 0.046) based on ribosomal protein S3 gene clustering, finally converging to nearly identical communities. Similarly, strain-resolved metagenomics revealed a high diversity of bacteria and viruses after the introduction of treated wastewater; these microbes also decreased over time resulting in the same strain clusters in control and treatment at the end of the experiment. Specifically, 39.2% of viral strains detected in all samples were present after the introduction of treated wastewater only. Although bacteria present at low abundance in the treated wastewater introduced additional antibiotic resistance genes, signals of naturally occurring ARG-encoding organisms resembled the resistome at the endpoint. Our results suggest that the previously stressed freshwater stream and its microbial community are resilient to a substantial introduction of treated wastewater.

Detection of an IMI-2 carbapenemase-producing Enterobacter asburiae at a Swedish feed mill.

Occurrence of multidrug resistant Enterobacteriaceae in livestock is of concern as they can spread to humans. A potential introduction route for these bacteria to livestock could be animal feed. We therefore wanted to identify if Escherichia spp., Enterobacter spp., Klebsiella spp., or Raoutella spp. with transferable resistance to extended spectrum cephalosporins, carbapenems or colistin could be detected in the environment at feed mills in Sweden. A second aim was to compare detected isolates to previous described isolates from humans and animals in Sweden to establish relatedness which could indicate a potential transmission between sectors and feed mills as a source for antibiotic resistant bacteria. However, no isolates with transferable resistance to extended-cephalosporins or colistin could be identified, but one isolate belonging to the Enterobacter cloacae complex was shown to be carbapenem-resistant and showing carbapenemase-activity. Based on sequencing by both short-read Illumina and long-read Oxford Nanopore MinIon technologies it was shown that this isolate was an E. asburiae carrying a bla IMI-2 gene on a 216 Kbp plasmid, designated pSB89A/IMI-2, and contained the plasmid replicons IncFII, IncFIB, and a third replicon showing highest similarity to the IncFII(Yp). In addition, the plasmid contained genes for various functions such as plasmid segregation and stability, plasmid transfer and arsenical transport, but no additional antibiotic resistance genes. This isolate and the pSB89A/IMI-2 was compared to three human clinical isolates positive for bla IMI-2 available from the Swedish antibiotic monitoring program Swedres. It was shown that one of the human isolates carried a plasmid similar with regards to gene content to the pSB89A/IMI-2 except for the plasmid transfer system, but that the order of genes was different. The pSB89A/IMI-2 did however share the same transfer system as the bla IMI-2 carrying plasmids from the other two human isolates. The pSB89A/IMI-2 was also compared to previously published plasmids carrying bla IMI-2, but no identical plasmids could be identified. However, most shared part of the plasmid transfer system and DNA replication genes, and the bla IMI-2 gene was located next the transcription regulator imiR. The IS3-family insertion element downstream of imiR in the pSB89A was also related to the IS elements in other bla IMI-carrying plasmids.

Do plasmids containing heavy metal resistance genes play a role in neonatal sepsis and invasive disease caused by Klebsiella pneumoniae and Klebsiella variicola?

Introduction. Klebsiella species are some of those most implicated in neonatal sepsis. However, many isolates from infections appear unremarkable; they are generally susceptible to antibiotics and often of sporadic types not associated with virulence.Hypothesis/Gap Statement. Investigation is needed to identify if such isolates have virulence characteristics.Aim. To sequence multiple isolates of a range of types from cases of neonatal invasive disease to identify elements that may explain their virulence, and to determine if such elements are more common among these isolates than generally.Methodology. In total, 14 isolates of K. pneumoniae/K. variicola belonging to 13 distinct types from blood or CSF from neonatal infections were sequenced using long-read nanopore technology. PCR assays were used to screen a general set of isolates for heavy metal resistance genes arsC, silS and merR.Results. Overall, 12/14 isolates carried one or more plasmids. Ten carried a large plasmid (186 to 310 kb) containing heavy metal resistance genes associated with hypervirulence plasmids, with most (nine) carrying genes for resistance to copper, silver and one other heavy metal (arsenic, tellurite or mercury), but lacking the genes encoding capsule-upregulation and siderophores. Most isolates (9/14) lacked any additional antibiotic resistance genes other than those intrinsic in the species. However, a representative of an outbreak strain carried a plasmid containing bla CTX-M-15, qnrS1, aac3_IIa, dfrA17, sul1, mph(A), tet(A), bla TEM1B and aadA5, but no heavy metal resistance genes. arsC, silS and merR were widely found among 100 further isolates screened, with most carbapenemase-gene-positive isolates (20/27) carrying at least one.Conclusion. Plasmids containing heavy metal resistance genes were a striking feature of isolates from neonatal sepsis but are widely found. They share elements in common with virulence and antibiotic resistance plasmids, perhaps providing a basis from which such plasmids evolve.

Characterization of SCCmec Instability in Methicillin-Resistant Staphylococcus aureus Affecting Adjacent Chromosomal Regions, Including the Gene for Staphylococcal Protein A (spa).

ABSTRACTStaphylococcal cassette chromosome mec (SCCmec) represents a sequence of clear clinical and diagnostic importance in staphylococci. At a minimum the chromosomal cassette contains the mecA gene encoding PBP2a but frequently also includes additional antibiotic resistance genes (e.g., ermA and aadC; macrolide and aminoglycoside resistance, respectively). Certain regions within SCCmec elements are hot spots for sequence instability due to cassette-specific recombinases and a variety of internal mobile elements. SCCmec changes may affect not only cassette stability but the integrity of adjacent chromosomal sequences (e.g., the staphylococcal protein A gene; spa). We investigated SCCmec stability in methicillin-resistant Staphylococcus aureus (MRSA) strains carrying one of four SCCmec types cultured in the absence of antimicrobial selection over a 3-month period. SCCmec rearrangements were first detected in cefoxitin-susceptible variants after 2 months of passage, and most commonly showed precise excision of the SCCmec element. Sequence analysis after 3 months revealed both precise SCCmec excision and a variety of SCCmec internal deletions, some including extensive adjacent chromosomal loss, including spa. No empty cassettes (i.e., loss of just mecA from SCCmec) were observed among the variants. SCCmec stability was influenced both by internal mobile elements (IS431) as well as the host cell environment. Genotypically similar clinical isolates with deletions in the spa gene were also included for purposes of comparison. The results indicate a role for host-cell influence and the IS431 element on SCCmec stability.

Presence and characterization of methicillin-resistant Staphylococcus aureus co-carrying the multidrug resistance genes cfr and lsa(E) in retail food in China

Staphylococcus aureus is an important food-related pathogen associated with bacterial poisoning that is difficult to treat due to its multidrug resistance. The cfr and lsa(E) genes both cause multiple drug resistance and have been identified in numerous Staphylococcus species, respectively. In this study, we found that a methicillin-resistant S. aureus (MRSA) strain, 2868B2, which was isolated from a sample of frozen dumplings in Hangzhou in 2015, co-carried these two different multidrug resistance genes. Further analysis showed that this strain was resistant to more than 18 antibiotics and expressed high-level resistance to florfenicol, chloramphenicol, clindamycin, tiamulin, erythromycin, ampicillin, cefepime, ceftazidime, kanamycin, streptomycin, tetracycline, trimethoprim-sulfamethoxazole and linezolid (MIC = 8 μg/mL). Whole genome sequencing was performed to characterize the genetic environment of these resistance genes and other genomic features. The cfr gene was located on the single plasmid p2868B2 (39,159 bp), which demonstrated considerable similarity to many plasmids previously identified in humans and animals. p2868B2 contained the insertion sequence (IS) element IS21–558, which allowed the insertion of cfr into Tn558 and played an important role in the mobility of cfr. Additionally, a novel multidrug resistance region (36.9 kb) harbouring lsa(E) along with nine additional antibiotic resistance genes (ARGs) (aadD, aadE, aacA-aphD, spc, lnu(B), lsa(E), tetL, ermC and blaZ) was identified. The multidrug resistance region harboured four copies of IS257 that were active and can mediate the formation of four circular structures containing ARGs and ISs. In addition, genes encoding various virulence factors and affecting multiple cell adhesion properties were identified in the genome of MRSA 2868B2. This study confirmed that the cfr and lsa(E) genes coexist in one MRSA strain and the presence of plasmid and IS257 in the multi-ARG cluster can promote both ARG transfer and dissemination. Furthermore, the presence of so many ARGs and virulence genes in food-related pathogens may seriously compromise the effectiveness of clinical therapy and threaten public health, its occurrence should pay public attention and the traceability of these genes in food-related samples needs further surveillance.

Antibiotic Resistance and Pathogenomics of Staphylococci Circulating in Novosibirsk, Russia.

A total of 394 strains of staphylococci found in humans and pets in Novosibirsk, Siberian Russia, were characterized in terms of antibiotic resistance and corresponding genes. Two coagulase-positive and 17 coagulase-negative species were identified. The majority of isolates, with the exception of S. haemolyticus and hospital S. epidermidis isolates, were sensitive to most of the tested antibiotics, and isolates from pets displayed the lowest level of resistance. Nevertheless, methicillin-resistant (MRS) and/or multidrug-resistant (MDR) isolates were found in all prevailed species, including coagulase-negative. A set of genes corresponding to the detected resistance was identified: mecA (beta-lactam resistance), aac(6′)-Ie-aph(2″)-Ia, aph(3′)-IIIa, ant(4′)-Ia (aminoglycoside-modifying enzymes), ermA/ermC, and msrA (macrolide resistance). Complete genome analysis for ten MDR S. epidermidis and five MDR S. haemolyticus isolates revealed additional antibiotic resistance genes mphC, qacA/qacB, norA, dfrC/dfrG, lnuA, BseSR, and fosB. NorA, dfrC, and fosB were present in all S. epidermidis genomes, whereas mphC and msrA were identified in all S. haemolyticus ones. All investigated MDR S. epidermidis and four of five S. haemolyticus strains were moderate or strong biofilm producers, whereas multiple genes responsible for this function and for virulence and pathogenicity were identified mostly in S. epidermidis, but were less frequently represented in S. haemolyticus.

Occurrence of the Colistin Resistance Gene mcr-1 and Additional Antibiotic Resistance Genes in ESBL/AmpC-Producing Escherichia coli from Poultry in Lebanon: A Nationwide Survey.

ABSTRACTThe objective of the present study was to determine genomic characteristics of expanded-spectrum cephalosporin (ESC)-resistant Escherichia coli spreading in healthy broilers in Lebanon in 2018. Rectal swabs (n = 280) from 56 farms were screened for the presence of ESC-resistant E. coli isolates. Antimicrobial susceptibility and extended-spectrum β-lactamase (ESBL)/AmpC production were determined by the disk diffusion method. Whole-genome sequencing (WGS) of 102 representative isolates of E. coli was performed to determine their phylogenetic diversity, serotypes, sequence types (ST), acquired resistance genes, and virulence-associated genes. Fifty-two out of 56 farms housed broilers carrying ESC-resistant E. coli isolates. These farms had large and recurrent antimicrobial practices, using, for some of them, critically important antibiotics for prophylactic and therapeutic purposes. Among the 102 sequenced multidrug-resistant (MDR) E. coli isolates, the proportion of ESBL, plasmid-mediated AmpC β-lactamase (pAmpC) producers, and ESBL/pAmpC coproducers was 60%, 27.6%, and 12.4%, respectively. The most prevalent ESBL/pAmpC genes were blaCMY-2, blaCTX-M-3, blaCTX-M-15, blaCTX-M-27, and blaCTX-M-14b (n = 42, n = 31, n =15, n = 9, and n = 7, respectively). These ESBL/pAmpC producers were distributed in different STs, most being well-known avian-associated and sometimes pathogenic STs (ST-10, ST-48, ST-93, ST-115, ST-117, and ST-457). Phylogenetic single nucleotide polymorphism (SNP) analysis confirmed their genetic diversity and wide dispersion across the Lebanese territory. Most isolates were also resistant to ciprofloxacin (101/102 with 3 QRDR mutations), and 19/102 isolates from 11 unrelated STs also carried the mobile resistance gene mcr-1. This survey illustrates the alarming prevalence of MDR E. coli resistant to medically important antibiotics in broilers in Lebanon. This advocates the need for surveillance programs of antimicrobial resistance in Lebanon and the reduction of excessive use of antibiotics to limit the spread of MDR E. coli in food-producing animals.IMPORTANCE Poultry production is a main contributor of the global trend of antimicrobial resistance arising from food-producing animals worldwide. In Lebanon, inappropriate use of antibiotics is frequent in chickens for prophylactic reasons and to improve productivity, resulting in an alarming prevalence of extended-spectrum β-lactamase (ESBL)/AmpC-producing Escherichia coli, also resistant to other medically important antibiotics (i.e., colistin and ciprofloxacin). Their complex genomic epidemiology highlighted by an important genetic diversity suggests that these resistance determinants are largely spreading in enteric bacteria in Lebanese poultry. Further molecular surveillance is needed to understand the country-specific epidemiology of ESBL/AmpC and mcr-1 genes in Lebanese poultry production. In addition, decisive interventions are urgently needed in order to ban the use of critically important antibiotics for human medicine in food-producing animals and limit the spread of antibiotic resistance in Lebanon.

A genetic cluster of MDR Enterobacter cloacae complex ST78 harbouring a plasmid containing bla VIM-1 and mcr-9 in the Netherlands.

BackgroundCarbapenemases produced by Enterobacterales are often encoded by genes on transferable plasmids and represent a major healthcare problem, especially if the plasmids contain additional antibiotic resistance genes. As part of Dutch national surveillance, 50 medical microbiological laboratories submit their Enterobacterales isolates suspected of carbapenemase production to the National Institute for Public Health and the Environment for characterization. All isolates for which carbapenemase production is confirmed are subjected to next-generation sequencing.ObjectivesTo study the molecular characteristics of a genetic cluster of Enterobacter cloacae complex isolates collected in Dutch national surveillance in the period 2015–20 in the Netherlands.MethodsShort- and long-read genome sequencing was used in combination with MLST and pan-genome MLST (pgMLST) analyses. Automated antimicrobial susceptibility testing (AST), the Etest for meropenem and the broth microdilution test for colistin were performed. The carbapenem inactivation method was used to assess carbapenemase production.ResultspgMLST revealed that nine E. cloacae complex isolates from three different hospitals in the Netherlands differed by <20 alleles and grouped in a genetic cluster termed EclCluster-013. Seven isolates were submitted by one hospital in 2016–20. EclCluster-013 isolates produced carbapenemase and were from ST78, a globally disseminated lineage. EclCluster-013 isolates harboured a 316 078 bp IncH12 plasmid carrying the blaVIM-1 carbapenemase and the novel mcr-9 colistin resistance gene along with genes encoding resistance to different antibiotic classes. AST showed that EclCluster-013 isolates were MDR, but susceptible to meropenem (<2 mg/L) and colistin (<2 mg/L).ConclusionsThe EclCluster-013 reported here represents an MDR E. cloacae complex ST78 strain containing an IncH12 plasmid carrying both the blaVIM-1 carbapenemase and the mcr-9 colistin resistance gene.

Genomic and phenotypic analyses of multidrug-resistant Acinetobacter baumannii NCCP 16007 isolated from a patient with a urinary tract infection

ABSTRACT Polymyxin B (PMB) is increasingly used as a last-line antibiotic; however, the emergence of PMB resistance is a serious threat to global health. Here, a total of 40 Acinetobacter baumannii clinical isolates were collected to screen for PMB-resistant strains. Several clinical isolates including NCCP 16007 were far more resistant to PMB (MIC: 128–256 μg/ml) than the ATCC 17978 strain (MIC: 2 μg/ml) and appeared to possess resistance to broad-spectrum antibiotics including meropenem and 12 others. Four highly PMB-resistant strains possessed point mutations in the histidine kinase PmrB, leading to an increased expression of pmrC encoding a phosphoethanolamine transferase. Whole-genome analyses revealed that the NCCP 16007 stain had acquired two additional copies of the pmrC gene with phage integrase and 13 antibiotic resistance genes (ARGs) from other pathogens, including Klebsiella pneumoniae and Pseudomonas aeruginosa. The GC ratios of the ARGs (50–60%) were higher than that of the chromosomal backbone (39.06%), further supporting the horizontal gene transfer of ARGs. Comparative genomics with other multidrug-resistant A. baumannii strains revealed that the NCCP 16007 strain has many additional ARGs and has lost several virulence factors including Csu pili and heme oxygenase but exhibited high pathogenicity in Galleria mellonella-infection models. The observation of condensed biofilm through confocal and scanning electron microscopy suggested that the NCCP 16007 strain may possess high adhesion capacity during urinary tract infection. Therefore, our genomic and phenotypic analyses suggested that the multidrug-resistant A. baumannii NCCP 16007 strain possesses high genome plasticity, natural transformation ability, and pathogenicity.

Co-Occurrence of the mcr-1.1 and mcr-3.7 Genes in a Multidrug-Resistant Escherichia coli Isolate from China.

ObjectiveA colistin-resistant Escherichia coli strain isolated from dog feces was characterized in this study.Methods and ResultsA multiplex PCR assay was used to detect the presence of colistin-resistant mcr genes; it was found that E. coli QDFD216 co-harbored the mcr-1 and mcr-3 genes. Whole-genome sequencing and further bioinformatics analysis revealed that E. coli QDFD216 belonged to serotype O176:H11, fimH1311 type and ST132. The resistance genes blaCTX-M-14, mdfA, dfrA3, acrA, acrB, tolc, and sul3 were present in the chromosome. The mcr-1.1 and mcr-3.7 genes were located in two plasmids of different incompatibility groups. mcr-1.1 was carried by a IncX4-type plasmid within an typical IS26-parA-mcr-1.1-pap2 cassette, while mcr-3.7 was encoded by an IncP1-type plasmid with a genetic structure of TnAs2-mcr-3.7-dgkA-IS26. No additional antibiotic resistance genes were carried by either plasmid.ConclusionThis is the first report of an E. coli isolate co-harboring a mcr-1.1-carrying IncX4 plasmid and a mcr-3.7-carrying IncP1 plasmid. The evolution and mechanism of mcr gene co-existence need further study to assess its impact on public health.

Metadata Analysis of mcr-1-Bearing Plasmids Inspired by the Sequencing Evidence for Horizontal Transfer of Antibiotic Resistance Genes Between Polluted River and Wild Birds.

We sequenced the whole genomes of three mcr-1-positive multidrug-resistant E. coli strains, which were previously isolated from the environment of egret habitat (polluted river) and egret feces. The results exhibit high correlation between antibiotic-resistant phenotype and genotype among the three strains. Most of the mobilized antibiotic resistance genes (ARGs) are distributed on plasmids in the forms of transposons or integrons. Multidrug-resistant (MDR) regions of high homology are detected on plasmids of different E. coli isolates. Therefore, horizontal transfer of resistance genes has facilitated the transmission of antibiotic resistance between the environmental and avian bacteria, and the transfer of ARGs have involved multiple embedded genetic levels (transposons, integrons, plasmids, and bacterial lineages). Inspired by this, systematic metadata analysis was performed for the available sequences of mcr-1-bearing plasmids. Among these plasmids, IncHI2 plasmids carry the most additional ARGs. The composition of these additional ARGs varies according to their geographical distribution. The phylogenetic reconstruction of IncI2 and IncX4 plasmids provides the evidence for their multiregional evolution. Phylogenetic analysis at the level of mobile genetic element (plasmid) provides important epidemiological information for the global dissemination of mcr-1 gene. Highly homologous mcr-1-bearing IncI2 plasmids have been isolated from different regions along the East Asian-Australasian Flyway, suggesting that migratory birds may mediate the intercontinental transportation of ARGs.

Fitness costs of methicillin resistance in Staphylococcus aureus

Staphylococcus aureus is a Gram-positive bacteria, and a common human pathogen. It is a leading cause of healthcare-associated infections worldwide, causing potentially fatal bacteraemia. Infections can be treated using antibiotics, but many strains have quickly developed resistance to the most common antibiotics. Methicillin resistance in Staphylococcus is acquired through the uptake of the Staphylococcal Cassette Chromosome mec (SCCmec). SCCmec is a large mobile genetic element, which can integrate into the Staphylococcus genome. It carries the mecA gene, which confers resistance to broad-spectrum β-lactam antibiotics, including methicillin. Several different SCCmec elements have been described, some of which can carry additional antibiotic resistance genes, alongside mecA. These can include resistance to common antibiotics or resistance to heavy metals. Smaller SCCmec types do not encode any additional antibiotic resistance genes. These have been shown to confer no fitness cost to the bacteria. Because of this, smaller SCCmec types are becoming increasingly more common, particularly in community-associated MRSA infections.

A Pan-Genomic Approach to Understand the Basis of Host Adaptation in Achromobacter.

Over the past decade, there has been a rising interest in Achromobacter sp., an emerging opportunistic pathogen responsible for nosocomial and cystic fibrosis lung infections. Species of this genus are ubiquitous in the environment, can outcompete resident microbiota, and are resistant to commonly used disinfectants as well as antibiotics. Nevertheless, the Achromobacter genus suffers from difficulties in diagnosis, unresolved taxonomy and limited understanding of how it adapts to the cystic fibrosis lung, not to mention other host environments. The goals of this first genus-wide comparative genomics study were to clarify the taxonomy of this genus and identify genomic features associated with pathogenicity and host adaptation. This was done with a widely applicable approach based on pan-genome analysis. First, using all publicly available genomes, a combination of phylogenetic analysis based on 1,780 conserved genes with average nucleotide identity and accessory genome composition allowed the identification of a largely clinical lineage composed of Achromobacter xylosoxidans, Achromobacter insuavis, Achromobacter dolens, and Achromobacter ruhlandii. Within this lineage, we identified 35 positively selected genes involved in metabolism, regulation and efflux-mediated antibiotic resistance. Second, resistome analysis showed that this clinical lineage carried additional antibiotic resistance genes compared with other isolates. Finally, we identified putative mobile elements that contribute 53% of the genus’s resistome and support horizontal gene transfer between Achromobacter and other ecologically similar genera. This study provides strong phylogenetic and pan-genomic bases to motivate further research on Achromobacter, and contributes to the understanding of opportunistic pathogen evolution.

QnrS1- and Aac(6')-Ib-cr-Producing Escherichia coli among Isolates from Animals of Different Sources: Susceptibility and Genomic Characterization.

Salmonella enterica and Escherichia coli can inhabit humans and animals from multiple origins. These bacteria are often associated with gastroenteritis in animals, being a frequent cause of resistant zoonotic infections. In fact, bacteria from animals can be transmitted to humans through the food chain and direct contact. In this study, we aimed to assess the antibiotic susceptibility of a collection of S. enterica and E. coli recovered from animals of different sources, performing a genomic comparison of the plasmid-mediated quinolone resistance (PMQR)-producing isolates detected. Antibiotic susceptibility testing revealed a high number of non-wild-type isolates for fluoroquinolones among S. enterica recovered from poultry isolates. In turn, the frequency of non-wild-type E. coli to nalidixic acid and ciprofloxacin was higher in food-producing animals than in companion or zoo animals. Globally, we detected two qnrS1 and two aac(6′)-Ib-cr in E. coli isolates recovered from animals of different origins. The genomic characterization of QnrS1-producing E. coli showed high genomic similarity (O86:H12 and ST2297), although they have been recovered from a healthy turtle dove from a Zoo Park, and from a dog showing symptoms of infection. The qnrS1 gene was encoded in a IncN plasmid, also carrying blaTEM−1−containing Tn3. Isolates harboring aac(6′)-Ib-cr were detected in two captive bottlenose dolphins, within a time span of two years. The additional antibiotic resistance genes of the two aac(6′)-Ib-cr-positive isolates (blaOXA−1, blaTEM−1,blaCTX−M−15, catB3, aac(3)-IIa, and tetA) were enclosed in IncFIA plasmids that differed in a single transposase and 60 single nucleotide variants. The isolates could be assigned to the same genetic sublineage—ST131 fimH30-Rx (O25:H4), confirming clonal spread. PMQR-producing isolates were associated with symptomatic and asymptomatic hosts, which highlight the aptitude of E. coli to act as silent vehicles, allowing the accumulation of antibiotic resistance genes, mobile genetic elements and other relevant pathogenicity determinants. Continuous monitoring of health and sick animals toward the presence of PMQR should be strongly encouraged in order to restrain the clonal spread of these antibiotic resistant strains.

PCERC3 from a commensal ST95 Escherichia coli: A ColV virulence-multiresistance plasmid carrying a sul3-associated class 1 integron

The rare sulphonamide resistance gene sul3 was found in the commensal Escherichia coli ST95 strain 22.1-R1 that was isolated in 2010 from the faeces of a healthy Australian adult. The genome of 22.1-R1 was sequenced and a 144,344bp RepFII/FIB plasmid, pCERC3, carrying sul3 was assembled. The sul3 gene is part of a class 1 integron featuring a sul3-containing conserved segment (sul3-CS) that replaced the classic sul1-containing 3′-conserved segment (3′-CS) usually seen in class 1 integrons. The integron contained the cassette array dfrA12-orfF-aadA2-cmlA1-aadA1-qacH, conferring resistance to trimethoprim, streptomycin, spectinomycin, chloramphenicol and quaternary ammonium compound. Two additional antibiotic resistance genes, blaTEM (ampicillin resistance) and tetA(B) (tetracycline) were adjacent to the integron, forming a single resistance region. In pCERC3, the sul3-type class 1 integron was flanked by sequence derived from the tnp and mer modules of Tn21 and was in the same location as In2, the sul1-containing In5-type class 1 integron of Tn21. At one end the sequence extends into Tn2670-derived sequence and then into sequence derived from the plasmid NR1 (R100). Examination of the sequences of eleven more complete sul3-containing plasmids in GenBank confirmed the relationship between sul3-associated integrons and Tn21/Tn2670/NR1. This suggests that the events that formed sul3-associated class 1 integrons occurred within the Tn21/Tn2670 context, most likely in NR1 or a related plasmid. The backbone of pCERC3 is most closely related to the backbones of ColV virulence plasmids and contains a complete ColV operon as well as several virulence associated genes and gene clusters. Hence, pCERC3 is both an antibiotic resistance and virulence plasmid.

Genomic and Functional Characterization of qnr-Encoding Plasmids from Municipal Wastewater Biosolid Klebsiella pneumoniae Isolates.

Municipal wastewater treatment facilities are considered to be “hotspots” for antibiotic resistance, since they conjoin high densities of environmental and fecal bacteria with selective pressure in the form of sub-therapeutic concentrations of antibiotics. Discharged effluents and biosolids from these facilities can disseminate antibiotic resistant genes to terrestrial and aquatic environments, potentially contributing to the increasing global trend in antibiotic resistance. This phenomenon is especially pertinent when resistance genes are associated with mobile genetic elements such as conjugative plasmids, which can be transferred between bacterial phyla. Fluoroquinolones are among the most abundant antibiotic compounds detected in wastewater treatment facilities, especially in biosolids, where due to their hydrophobic properties they accumulate to concentrations that may exceed 40 mg/L. Although fluoroquinolone resistance is traditionally associated with mutations in the gyrA/topoisomerase IV genes, there is increasing evidence of plasmid-mediated quinolone resistance, which is primarily encoded on qnr genes. In this study, we sequenced seven qnr-harboring plasmids from a diverse collection of Klebsiella strains, isolated from dewatered biosolids from a large wastewater treatment facility in Israel. One of the plasmids, termed pKPSH-11XL was a large (185.4 kbp), multi-drug resistance, IncF-type plasmid that harbored qnrB and 10 additional antibiotic resistance genes that conferred resistance to five different antibiotic families. It was highly similar to the pKPN3-like plasmid family that has been detected in multidrug resistant clinical Klebsiella isolates. In contrast, the six additional plasmids were much smaller (7–9 Kbp) and harbored a qnrS -type gene. These plasmids were highly similar to each other and closely resembled pGNB2, a plasmid isolated from a German wastewater treatment facility. Comparative genome analyses of pKPSH-11XL and other pKPN3-like plasmids concomitant to phylogenetic analysis of housekeeping genes from host Klebsiella strains, revealed that these plasmids are limited to a predominantly human-associated sub-clade of Klebsiella, suggesting that their host range is very narrow. Conversely, the pGNB2-like plasmids had a much broader host range and appeared to be associated with Klebsiella residing in natural environments. This study suggests that: (A) qnrB-harboring multidrug-resistant pKPN3-like plasmids can endure the rigorous wastewater treatment process and may therefore be disseminated to downstream environments; and (B) that small qnrS-harboring pGNB2-like plasmids are ubiquitous in wastewater treatment facilities and are most likely environmental in origin.

Whole-genome assembly of Akkermansia muciniphila sequenced directly from human stool.

BackgroundAlterations in gut microbiota composition under antibiotic pressure have been widely studied, revealing a restricted diversity of gut flora, including colonization by organisms such as Enterococci, while their impact on bacterial load is variable. High-level colonization by Akkermansia muciniphila, ranging from 39% to 84% of the total bacterial population, has been recently reported in two patients being treated with broad-spectrum antibiotics, although attempts to cultivate this microorganism have been unsuccessful.ResultsHere, we propose an original approach of genome sequencing for Akkermansia muciniphila directly from the stool sample collected from one of these patients. We performed and assembly using metagenomic data obtained from the stool sample. We used a mapping method consisting of aligning metagenomic sequencing reads against the reference genome of the Akkermansia muciniphila MucT strain, and a De novo assembly to support this mapping method. We obtained draft genome of the Akkermansia muciniphila strain Urmite with only 56 gaps. The absence of particular metabolic requirement as possible explanation of our inability to culture this microorganism, suggests that the bacterium was dead before the inoculation of the stool sample. Additional antibiotic resistance genes were found following comparison with the reference genome, providing some clues pertaining to its survival and colonization in the gut of a patient treated with broad-spectrum antimicrobial agents. However, no gene coding for imipenem resistance was detected, although this antibiotic was a part of the patient’s antibiotic regimen.ConclusionsThis work highlights the potential of metagenomics to facilitate the assembly of genomes directly from human stool.ReviewersThis article was reviewed by Eric Bapteste, William Martin and Vivek Anantharaman.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-015-0041-1) contains supplementary material, which is available to authorized users.