Antibiotic-free Environment Research Articles

High prevalence of polymyxin-heteroresistant carbapenem-resistant Klebsiella pneumoniae and its within-host evolution to resistance among critically ill scenarios.

We aimed to explore the prevalence and within-host evolution of resistance in polymyxin-heteroresistant carbapenem-resistant Klebsiella pneumoniae (PHR-CRKP) in critically ill patients. We performed an epidemiological analysis of consecutive patients with PHR-CRKP from clinical cases. Our study investigated the within-host resistance evolution and its clinical significance during polymyxin exposure. Furthermore, we explored the mechanisms underlying the dynamic evolution of polymyxin resistance at both subpopulation and genetic levels, involved population analysis profile test, time-killing assays, competition experiments, and sanger sequencing. Additionally, comparative genomic analysis was performed on 713 carbapenemase-producing K. pneumoniae strains. We enrolled 109 consecutive patients, and PHR-CRKP was found in 69.7% of patients without previous polymyxin exposure. 38.1% of PHR-CRKP isolates exhibited polymyxin resistance and led to therapeutic failure in critically ill scenarios. An increased frequency of resistant subpopulations was detected during PHR-CRKP evolution, with rapid regrowth of resistant subpopulations under high polymyxin concentrations, and a fitness cost in an antibiotic-free environment. Mechanistic analysis revealed that diverse mgrB insertions and pmrB hypermutations contributed to the dynamic changes in polymyxin susceptibility in dominant resistant subpopulations during PHR evolution, which were validated by comparative genomic analysis. Several deleterious mutations (e.g. pmrBLeu82Arg, pmrBSer85Arg) were firstly detected during PHR-CRKP evolution. Indeed, specific sequence types of K. pneumoniae demonstrated unique deletions and deleterious mutations. Our study emphasizes the high prevalence of pre-existing heteroresistance in CRKP, which can lead to polymyxin resistance and fatal outcomes. Hence, it is essential to continuously monitor and observe the treatment response to polymyxins in appropriate critically ill scenarios.

Unpredictability of the Fitness Effects of Antimicrobial Resistance Mutations Across Environments in Escherichia coli.

The evolution of antimicrobial resistance (AMR) in bacteria is a major public health concern, and antibiotic restriction is often implemented to reduce the spread of resistance. These measures rely on the existence of deleterious fitness effects (i.e. costs) imposed by AMR mutations during growth in the absence of antibiotics. According to this assumption, resistant strains will be outcompeted by susceptible strains that do not pay the cost during the period of restriction. The fitness effects of AMR mutations are generally studied in laboratory reference strains grown in standard growth environments; however, the genetic and environmental context can influence the magnitude and direction of a mutation's fitness effects. In this study, we measure how three sources of variation impact the fitness effects of Escherichia coli AMR mutations: the type of resistance mutation, the genetic background of the host, and the growth environment. We demonstrate that while AMR mutations are generally costly in antibiotic-free environments, their fitness effects vary widely and depend on complex interactions between the mutation, genetic background, and environment. We test the ability of the Rough Mount Fuji fitness landscape model to reproduce the empirical data in simulation. We identify model parameters that reasonably capture the variation in fitness effects due to genetic variation. However, the model fails to accommodate the observed variation when considering multiple growth environments. Overall, this study reveals a wealth of variation in the fitness effects of resistance mutations owing to genetic background and environmental conditions, which will ultimately impact their persistence in natural populations.

Long-term exposure of Allonais inaequalis to a mixture of antibiotics in freshwater and synthetic wastewater matrices: Reproduction, recovery, and swimming responses.

Antibiotics from sulfonamide, fluoroquinolone, and diaminopyrimidine classes are widely used in human and veterinary medicine, and their combined occurrence in the aquatic environment is increasing around the world. In parallel, the understanding of how mixtures of these compounds affect non-target species from tropical freshwaters is scarce. Thus, this work aimed to study the long-term reproductive, recovery, and swimming effects of mixtures of 12 antibiotics from three different classes (up to 10μg L-1 ) added to freshwater (FWM) and synthetic wastewater (SWM) matrices on freshwater worm Allonais inaequalis. Results revealed that at the reproduction level, the exposure to antibiotics in the SWM matrix does not cause a significant toxic effect on species after 10days. On the other hand, exposures to initial dose mixtures (10μg L-1 each) in FWM caused a significant reduction of offspring by 19.2%. In addition, recovery bioassays (10days in an antibiotic-free environment) suggested that A. inaequalis has reduced offspring production due to previous exposure to antibiotic mixtures in both matrices. Furthermore, despite slight variation in swimming speed over treatments, no significant differences were pointed out. Regarding antibiotics in the water matrices after 10-day exposures, the highest concentrations were up to 2.7, 7.8, and 4.2μg L-1 for antibiotics from sulfonamide, fluoroquinolone, and diaminopyrimidine classes, respectively. These findings suggest that a species positioned between primary producers and secondary consumers may experience late reproductive damage even in an antibiotic-free zone, after previous 10-day exposure to antibiotic mixtures. PRACTITIONER POINTS: A mixture of sulfonamide, fluoroquinolone, and diaminopyrimidine antibiotics in freshwater affects the offspring production of A. inaequalis after 10days. After the 10-day antibiotic exposure, the reproduction of A. inaequalis remains affected in an antibiotic-free environment over the recovery period. The swimming speed of the worms does not change after 10days of exposure to the antibiotic mixture. The concentration of dissolved solids can limit the natural degradation of sulfonamide, fluoroquinolone, and diaminopyrimidine antibiotics in the aquatic environment.

Interaction with a phage gene underlie costs of a β-lactamase.

The fitness cost of an antibiotic resistance gene (ARG) can differ across host strains, creating refuges that allow the maintenance of an ARG in the absence of direct selection for its resistance phenotype. Despite the importance of such ARG-host interactions for predicting ARG dynamics, the basis of ARG fitness costs and their variability between hosts are not well understood. We determined the genetic basis of a host-dependent cost of a β-lactamase, blaTEM-116*, that conferred a significant cost in one Escherichia coli strain but was close to neutral in 11 other Escherichia spp. strains. Selection of a blaTEM-116*-encoding plasmid in the strain in which it initially had a high cost resulted in rapid and parallel compensation for that cost through mutations in a P1-like phage gene, relAP1. When the wild-type relAP1 gene was added to a strain in which it was not present and in which blaTEM-116* was neutral, it caused the ARG to become costly. Thus, relAP1 is both necessary and sufficient to explain blaTEM-116* costs in at least some host backgrounds. To our knowledge, these findings represent the first demonstrated case of the cost of an ARG being influenced by a genetic interaction with a phage gene. The interaction between a phage gene and a plasmid-borne ARG highlights the complexity of selective forces determining the maintenance and spread of ARGs and, by extension, encoding phage and plasmids in natural bacterial communities.IMPORTANCEAntibiotic resistance genes (ARGs) play a major role in the increasing problem of antibiotic resistance in clinically relevant bacteria. Selection of these genes occurs in the presence of antibiotics, but their eventual success also depends on the sometimes substantial costs they impose on host bacteria in antibiotic-free environments. We evolved an ARG that confers resistance to penicillin-type antibiotics in one host in which it did confer a cost and in one host in which it did not. We found that costs were rapidly and consistently reduced through parallel genetic changes in a gene encoded by a phage that was infecting the costly host. The unmutated version of this gene was sufficient to cause the ARG to confer a cost in a host in which it was originally neutral, demonstrating an antagonism between the two genetic elements and underlining the range and complexity of pressures determining ARG dynamics in natural populations.

Unveiling a novel mechanism for competitive advantage of ciprofloxacin-resistant bacteria in the environment through bacterial membrane vesicles

Ciprofloxacin (CIP) is a prevalent environmental contaminant that poses a high risk of antibiotic resistance. High concentrations of antibiotics can lead to the development of resistant bacteria with high fitness costs, which often face a competitive disadvantage. However, it is unclear whether low-cost resistant bacteria formed by exposure to sub-MIC CIP in the environment can evolve competitive mechanisms against sensitive Escherichia coli (SEN) other than stronger resistance to CIP. Our study exposed E. coli to sub-MIC CIP levels, resulting in the development of CIP-resistant E. coli (CIPr). In antibiotic-free co-culture assays, CIPr outcompeted SEN. This indicates that CIPr is very likely to continue to develop and spread in antibiotic-free environments such as drinking water and affect human health. Further mechanism investigation revealed that bacterial membrane vesicles (BMVs) in CIPr, functioning as substance delivery couriers, mediated a cleavage effect on SEN. Proteomic analysis identified Entericidin B (EcnB) within CIPr-BMVs as a key factor in this competitive interaction. RT-qPCR analysis showed that the transcription of its negative regulator ompR/envZ was down-regulated. Moreover, EcnB plays a crucial role in the development of CIP resistance, and some resistance-related proteins and pathways have also been discovered. Metabolomics analysis highlighted the ability of CIPr-BMVs to acidify SEN, increasing the lytic efficiency of EcnB through cationization. Overall, our study reveals the importance of BMVs in mediating bacterial resistance and competition, suggesting that regulating BMVs production may be a new strategy for controlling the spread of drug-resistant bacteria.

Influence of three different manure treatments on antimicrobial resistance genes and mobile genetic elements

There is a growing concern about the potential dissemination of antimicrobial resistance into agricultural fields due to the application of manure as crop fertilizer. While reducing the usage of antibiotics in livestock production stands as the first alternative to prevent this problem, there is evidence that this may not be enough to eliminate antimicrobial resistance elements already present in manure microbiomes. This study employed a metagenomic approach to investigate the impact of common manure treatments, including aerobic storage, mesophilic anaerobic digestion (MAD), and solid-liquid separation, on the presence and abundance of antimicrobial resistance genes (ARGs), bactericides, and heavy metal resistance genes (BacMet), and mobile genetic elements (MGEs) in manure from three different farms, including one operating in an antibiotic-free environment. The results indicated that MAD was the best method to reduce the numbers of ARGs, BacMet, and MGEs, achieving reduction rates greater than 40%, 89%, and 68%, respectively. Manure storage significantly reduced BacMet levels (over 30%) and MGEs (28%) but had no significant effect on total ARG levels. Solids recovered through solid-liquid separation exhibited elevated levels of ARGs, BacMet, and MGEs, while the liquid fraction displayed levels similar to untreated manures. Correlation and co-occurrence modeling analyses indicated that changes in microbial communities, particularly fluctuations in aerobic and facultative communities belonging to Bacillota, Actinomycetota, and Pseudomonadota phyla, played a significant role in driving changes in ARGs, BacMet, and MGEs. The results also showed the presence of toxin-antitoxin and transposon systems near different ARGs. Overall, the results confirmed that genes conferring resistance to various antimicrobials and MGE capable of mobilizing them are widely spread in dairy farms; that even under the absence of antibiotics, the use of heavy metals and disinfectants may promote the maintenance of ARGs and MGEs, and; that treatment such as anaerobic digestion could reduce the risk of the spread of antimicrobial resistance.

Proteomic analysis of ceftazidime and meropenem-exposed Pseudomonas aeruginosa ATCC 9027

BackgroundPseudomonas aeruginosa is well known for its intrinsic ability to resist a wide range of antibiotics, thus complicates treatment. Thus, understanding the response of the pathogen to antibiotics is important for developing new therapies. In this study, proteomic response of P. aeruginosa to the commonly used anti-pseudomonas antibiotics, ceftazidime (Caz) and meropenem (Mem) was investigated.MethodsP. aeruginosa ATCC 9027, an antibiotic-susceptible strain, was exposed to sub-MIC values of antibiotics either Caz or Mem for 14 days to obtain E1 strains and then cultured in antibiotic-free environments for 10 days to obtain E2 strains. Proteomes of the initial and E1, E2 strains were identified and comparatively analyzed using isobaric tags for relative and absolute quantitation (iTRAQ) in cooperation with nano LC–MS/MS. Noted up and down-regulated proteins were confirmed with quantitative reverse transcriptase PCR (qRT-PCR).ResultsOverall, 1039 and 1041 proteins were identified in Caz and Mem-exposed strains, respectively. Upon antibiotic exposure, there were 7–10% up-regulated (Caz: 71, Mem: 85) and down-regulated (Caz: 106, Mem: 69) proteins (1.5-fold change cut-off). For both Caz and Mem, the DEPs were primarily the ones involved in metabolic process, membrane, virulence, protein synthesis, and antibiotic resistance in which proteins involved in antibiotics resistance tended to be up-regulated while proteins involved in protein synthesis and metabolic process were down-regulated. Noted proteins included beta-lactamase AmpC which was up-regulated and OprD which was down-regulated in both the antibiotic-exposed strains. Besides, biofilm formation related proteins TssC1 and Hcp1 in Caz- exposed strains and the membrane/ periplasmic proteins Azu and PagL in Mem-exposed strains were found significantly down-regulated. qRT-PCR results confirmed the expression change of AmpC, Hcp1 and OprD proteins.ConclusionExposure of Pseudomonas aeruginosa to sub-MIC values of Caz and Mem resulted in around 10% change in its proteome. Not only proteins with confirmed roles in antibiotic resistance mechanisms changed their expression but also virulence- associated proteins. Both Caz and Mem response involved up-regulation of AmpC and down-regulation of OprD. While TssC1 and Hcp1 were responsible for Caz response, Azu and PagL were more likely involved in Mem response.

Presence and characterization of blaNDM-1-positive carbapenemase-producing Klebsiella pneumoniae from outpatients in Thailand.

Presently, community-associated carbapenemase-producing Enterobacterales (CPE) remains largely unknown and require public attention. This study aimed to investigate the presence of CPE from outpatients in Thailand. Non-duplicate stool (n=886) and urine (n=289) samples were collected from outpatients with diarrhea and urinary tract infection, respectively. Demographic data and characteristics of patients were collected. Isolation of CPE was performed by plating enrichment culture on agar supplemented with meropenem. Carbapenemase genes were screened by PCR and sequencing. CPE isolates were phenotypically and genotypically characterized. Fifteen samples (1.3%, 14 stool and 1 urine) yielded blaNDM-1-positive carbapenemase-producing Klebsiella pneumoniae (CPKP). Additional resistance to colistin and tigecycline was observed in 53.3% and 46.7% of isolates, respectively. Age >60 years was identified as a risk factor for patients with CPKP (P<0.001, adjusted odds ratio=11.500, 95% confidence interval=3.223-41.034). Pulsed field gel electrophoresis revealed genetic diversity of CPKP isolates; however, clonal spread has been observed. ST70 (n=4) was common, followed by ST147 (n=3). blaNDM-1 from all isolates were transferable and mainly resided on IncA/C plasmid (80%). All blaNDM-1 plasmids remained stable in bacterial host for at least 10 days in antibiotic-free environments, regardless of replicon types. This study demonstrates that the prevalence of CPE among outpatients in Thailand remains low and the spread of blaNDM-1-positive CPKP may be driven by IncA/C plasmid. Our results emphasize the need for a large-scale surveillance study to limit further spread of CPE in community.

A Streamlined Approach for Fluorescence Labelling of Low-Copy-Number Plasmids for Determination of Conjugation Frequency by Flow Cytometry.

Bacterial conjugation plays a major role in the dissemination of antibiotic resistance and virulence traits through horizontal transfer of plasmids. Robust measurement of conjugation frequency of plasmids between bacterial strains and species is therefore important for understanding the transfer dynamics and epidemiology of conjugative plasmids. In this study, we present a streamlined experimental approach for fluorescence labelling of low-copy-number conjugative plasmids that allows plasmid transfer frequency during filter mating to be measured by flow cytometry. A blue fluorescent protein gene is inserted into a conjugative plasmid of interest using a simple homologous recombineering procedure. A small non-conjugative plasmid, which carries a red fluorescent protein gene with a toxin-antitoxin system that functions as a plasmid stability module, is used to label the recipient bacterial strain. This offers the dual advantage of circumventing chromosomal modifications of recipient strains and ensuring that the red fluorescent protein gene-bearing plasmid can be stably maintained in recipient cells in an antibiotic-free environment during conjugation. A strong constitutive promoter allows the two fluorescent protein genes to be strongly and constitutively expressed from the plasmids, thus allowing flow cytometers to clearly distinguish between donor, recipient, and transconjugant populations in a conjugation mix for monitoring conjugation frequencies more precisely over time.

Campylobacter jejuni in Poultry: Pathogenesis and Control Strategies

C. jejuni is the leading cause of human foodborne illness associated with poultry, beef, and pork consumption. C. jejuni is highly prevalent in commercial poultry farms, where horizontal transmission from the environment is considered to be the primary source of C. jejuni. As an enteric pathogen, C. jejuni expresses virulence factors regulated by a two-component system that mediates C. jejuni's ability to survive in the host. C. jejuni survives and reproduces in the avian intestinal mucus. The avian intestinal mucus is highly sulfated and sialylated compared with the human mucus modulating C. jejuni pathogenicity into a near commensal bacteria in poultry. Birds are usually infected from two to four weeks of age and remain colonized until they reach market age. A small dose of C. jejuni (around 35 CFU/mL) is sufficient for successful bird colonization. In the U.S., where chickens are raised under antibiotic-free environments, additional strategies are required to reduce C. jejuni prevalence on broilers farms. Strict biosecurity measures can decrease C. jejuni prevalence by more than 50% in broilers at market age. Vaccination and probiotics, prebiotics, synbiotics, organic acids, bacteriophages, bacteriocins, and quorum sensing inhibitors supplementation can improve gut health and competitively exclude C. jejuni load in broilers. Most of the mentioned strategies showed promising results; however, they are not fully implemented in poultry production. Current knowledge on C. jejuni's morphology, source of transmission, pathogenesis in poultry, and available preharvest strategies to decrease C. jejuni colonization in broilers are addressed in this review.

The effects of sub-MIC ciprofloxacin exposure on antibiotic susceptibility and virulence factors in Pseudomonas aeruginosa ATCC 9027

Exposure to antibiotics below their minimum inhibitory concentration (sub-MIC) is known to be a selection pressure for the development of antibiotic resistance. In this work, we aimed to provide more understanding of how antibiotic resistance and cross-resistance developed in Pseudomonas aeruginosa under sub-MIC exposure to Ciprofloxacin. Fully susceptible P. aeruginosa ATCC 9027 was serially exposed to sub-MIC level of ciprofloxacin for 14 days to generate E1 strain, then cultured in antibiotic-free environment for 10 days to obtain E2 strain. Changes in MIC values, susceptibility profile and virulence (protease, elastin, biofilm formation, motility) of these strains were evaluated compared to the original P. aeruginosa ATCC 9027. After antibiotic exposure, P. aeruginosa strains developed resistance to the ciprofloxacin with the MIC values increased by 32 times, and the exposed strains only developed resistance to ciprofloxacin and other fluoroquinolones but not to unrelated antibiotics. The resistant phenotype did not revert fully after 10 days in antibiotic-free environment. In addition, there was noticeable changes in the pathogen’s virulence factors during antibiotic exposure. In summary, sub-MICs exposure of P. aeruginosa provided positive selection for antibiotic resistant phenotype, which did not revert after 10 days in antibiotic- free environment. Exposure to sub-MIC level of antibiotics caused significant changes in virulence and antibiotic susceptibility of the pathogen.

In-silico analyses provide strong statistical evidence for intra-species recombination events of the gyrA and CmeABC operon loci contributing to the continued emergence of resistance to fluoroquinolones in natural populations of Campylobacter jejuni

The continued emergence of Campylobacter jejuni strains resistant to fluoroquinolones (FQs) has posed a significant threat to global public health, leading frequently to undesirable outcomes of human campylobacteriosis treatment. The molecular genetic mechanisms contributing to the increased retention of resistance to FQs in natural populations of this species, especially in antibiotic-free environments, are not clearly understood. This study aimed to determine whether genetic recombination could be such a mechanism. We applied a large array of algorithms, imbedded in the SplitsTree and RDP4 software packages, to analyse the DNA sequences of the chromosomal loci, including the gyrA gene and the CmeABC operon, to identify events of their genetic recombination between C. jejuni strains. The SplitsTree analyses of the above genetic loci resulted in several parallelograms with the bootstrap values being in a range of 94.7 to 100, with the high fit estimates being 99.3 to 100. These analyses were further strongly supported by the Phi test results (P ≤ 0.02715) and the RDP4-generated statistics (P ≤ 0.04005). The recombined chromosomal regions, along with the gyrA gene and CmeABC operon loci, were also found to contain the genetic loci that included, but were not limited to, the genes encoding for phosphoribosyltransferase, lipoprotein, outer membrane motility protein, and radical SAM domain protein. These findings strongly suggest that the genetic recombination of the chromosomal regions involving gyrA, CmeABC, and their adjacent loci may be an additional mechanism underlying the constant emergence of epidemiologically successful FQ-resistant strains in natural populations of C. jejuni.

Evolution of Bacterial Persistence to Antibiotics during a 50,000-Generation Experiment in an Antibiotic-Free Environment.

Failure of antibiotic therapies causes > 700,000 deaths yearly and involves both bacterial resistance and persistence. Persistence results in the relapse of infections by producing a tiny fraction of pathogen survivors that stay dormant during antibiotic exposure. From an evolutionary perspective, persistence is either a ‘bet-hedging strategy’ that helps to cope with stochastically changing environments or an unavoidable minimal rate of ‘cellular errors’ that lock the cells in a low activity state. Here, we analyzed the evolution of persistence over 50,000 bacterial generations in a stable environment by improving a published method that estimates the number of persister cells based on the growth of the reviving population. Our results challenged our understanding of the factors underlying persistence evolution. In one case, we observed a substantial decrease in persistence proportion, suggesting that the naturally observed persistence level is not an unavoidable minimal rate of ‘cellular errors’. However, although there was no obvious environmental stochasticity, in 11 of the 12 investigated populations, the persistence level was maintained during 50,000 bacterial generations.

Rapid Decline of Ceftazidime Resistance in Antibiotic-Free and Sublethal Environments Is Contingent on Genetic Background.

Trade-offs of antibiotic resistance evolution, such as fitness cost and collateral sensitivity (CS), could be exploited to drive evolution toward antibiotic susceptibility. Decline of resistance may occur when resistance to other drug leads to CS to the first one and when compensatory mutations, or genetic reversion of the original ones, reduce fitness cost. Here we describe the impact of antibiotic-free and sublethal environments on declining ceftazidime resistance in different Pseudomonas aeruginosa resistant mutants. We determined that decline of ceftazidime resistance occurs within 450 generations, which is caused by newly acquired mutations and not by reversion of the original ones, and that the original CS of these mutants is preserved. In addition, we observed that the frequency and degree of this decline is contingent on genetic background. Our results are relevant to implement evolution-based therapeutic approaches, as well as to redefine global policies of antibiotic use, such as drug cycling.

Spreading Advantages of Coresident Plasmids blaCTX-M-Bearing IncFII and mcr-1-Bearing IncI2 in Escherichia coli.

ABSTRACTTwo diverse conjugative plasmids can interact within bacterial cells. However, to the best of our knowledge, the interaction between blaCTX-M-bearing IncFII plasmid and mcr-1-carrying IncI2 plasmid colocated on the same bacterial host has not been reported. This study was initiated to explore the interaction and to analyze the reasons that these two plasmids are often coresident in multidrug-resistant Escherichia coli. To assess the interactions on plasmid stabilities, fitness costs, and transfer rates, we constructed two groups of isogenic derivatives, C600FII, C600I2, and C600FII+I2 of E. coli C600 and J53FII, J53I2, and J53FII+I2 of E. coli J53, respectively. We found that carriage of FII and I2 plasmids, independently and together, had not impaired the growth of the bacterial host. It was difficult for the single plasmid FII or I2 in E. coli C600 to reach stable persistence for a long time in an antibiotic-free environment, while the stability would be striking improved when they coresided. Meanwhile, plasmids FII and I2, whether together or apart, could notably enhance the fitness advantage of the host; moreover, E. coli coharboring plasmids FII and I2 presented more obvious fitness advantage than that carrying single plasmid FII. Coresident plasmids FII and I2 could accelerate horizontal cotransfer by conjugation. The transfer rates from a strain carrying coresident FII and I2 plasmids increased significantly when it mated with a recipient cell carrying one of them. Our findings highlight the advantages of coinhabitant FII and I2 plasmids in E. coli to drive the persistence and spread of plasmid-carried blaCTX-M and mcr-1 genes, although the molecular mechanisms of their coresidence warrant further study.IMPORTANCE More and more Enterobacteriaceae carry both blaCTX-M and mcr-1, which are usually located on IncFII-type and IncI2-type plasmids in the same bacterial host, respectively. However, the study on advantages of coresident plasmids in bacterial host is still sparse. Here, we investigated the stability, fitness cost, and cotransfer traits associated with coresident IncFII-type and IncI2-type plasmids in E. coli. Our results show that coinhabitant plasmids in E. coli are more stable, confer more fitness advantages, and are easier to transfer and cotransfer than a single plasmid IncFII or IncI2. Our findings confirm the advantages of coresident plasmids of blaCTX-M-bearing IncFII and mcr-1-bearing IncI2 in clinical E. coli, which will pose a serious threat to clinical therapy and public health.

PGN-027 - Tigecycline heteroresistance and resistance mechanism in clinical isolates of Acinetobacter baumannii

Tigecycline is regarded as a last-resort treatment for multidrug-resistant Acinetobacter baumannii. However, tigecycline resistance in A. baumannii has increased worldwide. In this study, we investigated tigecycline heteroresistance in A. baumannii isolates from South Korea. Antibiotic susceptibility testing was performed on 323 nonduplicated A. baumannii isolates. Among 260 and 37 tigecycline-susceptible and -intermediate-resistant A. baumannii isolates, 146 (56.2%) and 22 (59.5%) isolates were identified as heteroresistant to tigecycline through a disk diffusion assay and population analysis profiling. For selected isolates, an in vitro time-kill assay was performed, and survival rates were measured after preincubation with diverse concentrations of tigecycline. Heteroresistant isolates showed regrowth after 12 h of 2× MIC of tigecycline treatment, and resistant subpopulations were selected by preexposure to tigecycline. Furthermore, genetic alterations in adeABC, adeRS, and rpsJ were assessed, and the relative mRNA expression levels of adeB and adeS were compared. The tigecycline resistance in subpopulations might be due to the insertion of ISAba1 in adeS, leading to the overexpression of the AdeABC efflux pump. However, the tigecycline resistance of subpopulations was not stable during serial passages in antibiotic-free medium. The reversion of tigecycline susceptibility by antibiotic-free passages might occur by additional insertions of ISAba10 in adeR and nucleotide alterations in adeS in some mutants. Tigecycline heteroresistance is prevalent in A. baumannii isolates, which results in treatment failure. Tigecycline resistance is mainly due to the overexpression of the AdeABC efflux pump, which is associated with genetic mutations, but this resistance could be reversed into susceptibility by additional mutations in antibiotic-free environments. IMPORTANCE The evidence that antibiotic heteroresistance is responsible for treatment failure in clinical settings is increasing. Thus, detection and characterization of heteroresistance would be important for appropriate therapeutic guidance to treat bacterial infections. However, data on tigecycline heteroresistance in Gram-negative bacteria is currently limited, although tigecycline is regarded as a last-line antibiotic against infections caused by antibiotic-resistant pathogens. In this study, we investigated the tigecycline heteroresistance in Acinetobacter baumannii, which has been listed by the WHO as a priority for research and development of new antibiotics. We found very high prevalence of tigecycline-heteroresistant A. baumannii clinical isolates, which may result in treatment failure due to the selection of resistant subpopulations. We also identified the main resistance mechanism in tigecycline-resistant subpopulations, that is, upregulation of AdeABC efflux pumps due to ISAba1 insertion in adeS.

Fitness Cost Evolution of Natural Plasmids of Staphylococcus aureus.

ABSTRACTPlasmids have largely contributed to the spread of antimicrobial resistance genes among Staphylococcus strains. Knowledge about the fitness cost that plasmids confer on clinical staphylococcal isolates and the coevolutionary dynamics that drive plasmid maintenance is still scarce. In this study, we aimed to analyze the initial fitness cost of plasmids in the bacterial pathogen Staphylococcus aureus and the plasmid-host adaptations that occur over time. For that, we first designed a CRISPR (clustered regularly interspaced palindromic repeats)-based tool that enables the removal of native S. aureus plasmids and then transferred three different plasmids isolated from clinical S. aureus strains to the same-background clinical cured strain. One of the plasmids, pUR2940, obtained from a livestock-associated methicillin-resistant S. aureus (LA-MRSA) ST398 strain, imposed a significant fitness cost on both its native and the new host. Experimental evolution in a nonselective medium resulted in a high rate pUR2940 loss and selected for clones with an alleviated fitness cost in which compensatory adaptation occurred via deletion of a 12.8-kb plasmid fragment, contained between two ISSau10 insertion sequences and harboring several antimicrobial resistance genes. Overall, our results describe the relevance of plasmid-borne insertion sequences in plasmid rearrangement and maintenance and suggest the potential benefits of reducing the use of antibiotics both in animal and clinical settings for the loss of clinical multidrug resistance plasmids.

Characterization of the IncHI2 plasmid pTW4 harboring tet(M) from an isolate of Escherichia coli ST162.

To investigate the genetic features and biological costs of the plasmid pTW4 harboring tet(M) in an isolate of Escherichia coli ST162 from a duck. The complete nucleotide sequence of plasmid pTW4 was determined. The characteristics of plasmid pTW4 in E. coli were investigated by stability and direct competition assays. pTW4 is an IncHI2-type plasmid that contained the resistant genes tet(M), floR, strAB, sul2, rmtB, and blaCMY-2. Tet(M) is located in the composite transposon Tn6539 within the multidrug resistant (MDR) region on this plasmid. Furthermore, the resistance gene rmtB and blaCMY-2 were found outside the MDR region. The plasmid pTW4 remained stable in the host strain E. coli J53 after passage under an antibiotic-free environment for 7 days. However, the strain E. coli J53/pTW4 showed a fitness disadvantage of 6% per ten generations in the process of growth competition with E. coli J53. In conclusion, the plasmid pTW4, a mobile MDR vehicle, may promote the dissemination of tet(M), floR, rmtB, strAB, sul2, and blaCMY-2 among bacteria and then, but it appears to confer growth disadvantage to the host.

Rapid decline of bacterial drug-resistance in an antibiotic-free environment through phenotypic reversion.

Antibiotic resistance typically induces a fitness cost that shapes the fate of antibiotic-resistant bacterial populations. However, the cost of resistance can be mitigated by compensatory mutations elsewhere in the genome, and therefore the loss of resistance may proceed too slowly to be of practical importance. We present our study on the efficacy and phenotypic impact of compensatory evolution in Escherichia coli strains carrying multiple resistance mutations. We have demonstrated that drug-resistance frequently declines within 480 generations during exposure to an antibiotic-free environment. The extent of resistance loss was found to be generally antibiotic-specific, driven by mutations that reduce both resistance level and fitness costs of antibiotic-resistance mutations. We conclude that phenotypic reversion to the antibiotic-sensitive state can be mediated by the acquisition of additional mutations, while maintaining the original resistance mutations. Our study indicates that restricting antimicrobial usage could be a useful policy, but for certain antibiotics only.

Variability of the Ability of Complex Microbial Communities to Exclude Microbes Carrying Antibiotic Resistance Genes in Rabbits.

Reducing antibiotic use is a necessary step toward less antibiotic resistance in livestock, but many antibiotic resistance genes can persist for years, even in an antibiotic-free environment. In this study, we investigated the potential of three fecal complex microbial communities from antibiotic-naive does to drive the microbiota of kits from antibiotic-exposed dams and outcompete bacteria-carrying antibiotic-resistant genes. The fecal complex microbial communities were either orally delivered or simply added as fresh fecal pellets in four to five nests that were kept clean from maternal feces. Additionally, four nests were cleaned for the maternal feces and five nests were handled according to the common farm practice (i.e., cleaning once a week) as controls. At weaning, we measured the relative abundance of 26 antibiotic resistance genes, the proportion of Enterobacteriaceae resistant to tetracycline and sulfonamide antibiotics, and the taxonomic composition of the microbiota by sequencing the 16S rRNA genes of one kit per nest. Changing the surrounding microbes of the kits can hinder the transmission of antibiotic resistance genes from one generation to the next, but the three communities widely differed in their ability to orient gut microbes and in their impact on antibiotic resistance genes. The most efficient delivery of the microbial community reduced the proportion of resistant Enterobacteria from 93 to 9%, decreased the relative abundance of eight antibiotic resistance genes, and changed the gut microbes of the kits at weaning. The least efficient did not reduce any ARG or modify the bacterial community. In addition, adding fecal pellets was more efficient than the oral inoculation of the anaerobic suspension derived from these fecal pellets. However, we were unable to predict the outcome of the exclusion from the data of the donor does (species composition and abundance of antibiotic resistance genes). In conclusion, we revealed major differences between microbial communities regarding their ability to exclude antibiotic resistance genes, but more work is needed to understand the components leading to the successful exclusion of antibiotic resistance genes from the gut. As a consequence, studies about the impact of competitive exclusion should use several microbial communities in order to draw general conclusions.