Genomic and Transcriptomic Insights into How Bacteria Withstand High Concentrations of Benzalkonium Chloride Biocides

ABSTRACTBenzalkonium chlorides (BAC) are commonly used biocides in broad-spectrum disinfectant solutions. How microorganisms cope with BAC exposure remains poorly understood, despite its importance for disinfection and disinfectant-induced antibiotic resistance. To provide insights into these issues, we exposed two isolates of an opportunistic pathogen,Pseudomonas aeruginosa, to increasing concentrations of BAC. One isolate was preadapted to BAC, as it originated from a bioreactor fed with subinhibitory concentrations of BAC for 3 years, while the other originated from a bioreactor that received no BAC. Replicated populations of both isolates were able to survive high concentrations of BAC, up to 1,200 and 1,600 mg/liter for the non- and preadapted strains, respectively, exceeding typical application doses. Transcriptome sequencing (RNA-seq) analysis revealed upregulation of efflux pump genes and decreased expression of porins related to BAC transport as well as reduced growth rate. Increased expression of spermidine (a polycation) synthase genes and mutations in thepmrB(polymyxin resistance) gene, which cause a reduction in membrane negative charge, suggested that a major adaptation to exposure to the cationic surfactant BAC was to actively stabilize cell surface charge. Collectively, these results revealed thatP. aeruginosaadapts to BAC exposure by a combination of mechanisms and provided genetic markers to monitor BAC-resistant organisms that may have applications in the practice of disinfection.IMPORTANCEBAC are widely used as biocides in disinfectant solutions, food-processing lines, domestic households, and health care facilities. Due to their wide use and mode of action, there has been rising concern that BAC may promote antibiotic resistance. Consistent with this idea, at least 40 outbreaks have been attributed to infection by disinfectant- and antibiotic-resistant pathogens such asP. aeruginosa. However, the underlying molecular mechanisms that bacteria use to deal with BAC exposure remain poorly elucidated. Elucidating these mechanisms may be important for monitoring and limiting the spread of disinfectant-resistant pathogens. Using an integrated approach that combined genomics and transcriptomics with physiological characterization of BAC-adapted isolates, this study provided a comprehensive understanding of the BAC resistance mechanisms inP. aeruginosa. Our findings also revealed potential genetic markers to detect and monitor the abundance of BAC-resistant pathogens across clinical or environmental settings. This work contributes new knowledge about high concentrations of benzalkonium chlorides disinfectants-resistance mechanisms at the whole-cell genomic and transcriptomic level.

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PA3225 Is a Transcriptional Repressor of Antibiotic Resistance Mechanisms in Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02114-16 ◽

2017 ◽

Vol 61 (8) ◽

Cited By ~ 5

Author(s):

Clayton W. Hall ◽

Li Zhang ◽

Thien-Fah Mah

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Efflux Pump ◽

Regulatory Region ◽

Resistance Mechanisms ◽

Wild Type ◽

Type Vi Secretion System ◽

Planktonic Cells ◽

Content Type ◽

Dependent Protein

ABSTRACT The tssABC1 locus is part of the Hcp secretion island I (HSI-I) type VI secretion system (T6SS) in Pseudomonas aeruginosa. Previous work implicated the tssC1 gene in P. aeruginosa biofilm-specific antibiotic resistance, and tssC1 is preferentially expressed in biofilms compared to planktonic cells. Using a DNA-dependent protein pulldown approach, we discovered that PA3225, an uncharacterized LysR-type transcriptional regulator, specifically bound to the tssABC1 upstream regulatory region. The deletion of PA3225 led to a 2-fold decrease in tssA1 expression levels in planktonic cells compared to the wild type, and tssA1 expression was slightly reduced in ΔPA3225 biofilms compared to wild-type biofilms. Intriguingly, further investigations revealed that the ΔPA3225 mutant was less susceptible to multiple, structurally unrelated antibiotics with various mechanisms of action when grown planktonically. The ΔPA3225 mutant was additionally more resistant to ciprofloxacin when grown in a biofilm. The decreased antibiotic susceptibility of the ΔPA3225 strain was linked to the transcriptional upregulation of the MexAB-OprM efflux pump. By using transcriptome sequencing (RNA-seq), other PA3225-regulated genes were identified, and the products of these genes, such as the putative ABC transporter PA3228, may also contribute to antibiotic resistance.

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Pleiotropy complicates a trade-off between phage resistance and antibiotic resistance

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1919888117 ◽

2020 ◽

Vol 117 (21) ◽

pp. 11207-11216 ◽

Cited By ~ 14

Author(s):

Alita R. Burmeister ◽

Abigail Fortier ◽

Carli Roush ◽

Adam J. Lessing ◽

Rose G. Bender ◽

...

Keyword(s):

Antibiotic Resistance ◽

Molecular Mechanisms ◽

Efflux Pump ◽

Resistance Mechanisms ◽

Phage Resistance ◽

Resistance Mutations ◽

Trade Off ◽

Trade Offs ◽

Evolutionary Context ◽

Structural Barrier

Bacteria frequently encounter selection by both antibiotics and lytic bacteriophages. However, the evolutionary interactions between antibiotics and phages remain unclear, in particular, whether and when phages can drive evolutionary trade-offs with antibiotic resistance. Here, we describeEscherichia coliphage U136B, showing it relies on two host factors involved in different antibiotic resistance mechanisms: 1) the efflux pump protein TolC and 2) the structural barrier molecule lipopolysaccharide (LPS). Since TolC and LPS contribute to antibiotic resistance, phage U136B should select for their loss or modification, thereby driving a trade-off between phage resistance and either of the antibiotic resistance mechanisms. To test this hypothesis, we used fluctuation experiments and experimental evolution to obtain phage-resistant mutants. Using these mutants, we compared the accessibility of specific mutations (revealed in the fluctuation experiments) to their actual success during ecological competition and coevolution (revealed in the evolution experiments). BothtolCand LPS-related mutants arise readily during fluctuation assays, withtolCmutations becoming more common during the evolution experiments. In support of the trade-off hypothesis, phage resistance viatolCmutations occurs with a corresponding reduction in antibiotic resistance in many cases. However, contrary to the hypothesis, some phage resistance mutations pleiotropically confer increased antibiotic resistance. We discuss the molecular mechanisms underlying this surprising pleiotropic result, consideration for applied phage biology, and the importance of ecology in evolution of phage resistance. We envision that phages may be useful for the reversal of antibiotic resistance, but such applications will need to account for unexpected pleiotropy and evolutionary context.

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CmeABC Multidrug Efflux Pump Contributes to Antibiotic Resistance and Promotes Campylobacter jejuni Survival and Multiplication in Acanthamoeba polyphaga

Applied and Environmental Microbiology ◽

10.1128/aem.01600-17 ◽

2017 ◽

Vol 83 (22) ◽

Cited By ~ 11

Author(s):

Ana Vieira ◽

Amritha Ramesh ◽

Alan M. Seddon ◽

Andrey V. Karlyshev

Keyword(s):

Antibiotic Resistance ◽

Campylobacter Jejuni ◽

Molecular Mechanisms ◽

Bacterial Resistance ◽

Efflux Pump ◽

Synergistic Effects ◽

Microbial Pathogens ◽

Bacterial Survival ◽

Acanthamoeba Polyphaga ◽

Content Type

ABSTRACT Campylobacter jejuni is a foodborne pathogen that is recognized as the leading cause of human bacterial gastroenteritis. The widespread use of antibiotics in medicine and in animal husbandry has led to an increased incidence of antibiotic resistance in Campylobacter. In addition to a role in multidrug resistance (MDR), the Campylobacter CmeABC resistance-nodulation-division (RND)-type efflux pump may be involved in virulence. As a vehicle for pathogenic microorganisms, the protozoan Acanthamoeba is a good model for investigations of bacterial survival in the environment and the molecular mechanisms of pathogenicity. The interaction between C. jejuni 81-176 and Acanthamoeba polyphaga was investigated in this study by using a modified gentamicin protection assay. In addition, a possible role for the CmeABC MDR pump in this interaction was explored. Here we report that this MDR pump is beneficial for the intracellular survival and multiplication of C. jejuni in A. polyphaga but is dispensable for biofilm formation and motility. IMPORTANCE The endosymbiotic relationship between amoebae and microbial pathogens may contribute to persistence and spreading of the latter in the environment, which has significant implications for human health. In this study, we found that Campylobacter jejuni was able to survive and to multiply inside Acanthamoeba polyphaga; since these microorganisms can coexist in the same environment (e.g., on poultry farms), the latter may increase the risk of infection with Campylobacter. Our data suggest that, in addition to its role in antibiotic resistance, the CmeABC MDR efflux pump plays a role in bacterial survival within amoebae. Furthermore, we demonstrated synergistic effects of the CmeABC MDR efflux pump and TetO on bacterial resistance to tetracycline. Due to its role in both the antibiotic resistance and the virulence of C. jejuni, the CmeABC MDR efflux pump could be considered a good target for the development of antibacterial drugs against this pathogen.

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Expression ofPseudomonas aeruginosaAntibiotic Resistance Genes Varies Greatly during Infections in Cystic Fibrosis Patients

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01789-18 ◽

2018 ◽

Vol 62 (11) ◽

Cited By ~ 8

Author(s):

Lois W. Martin ◽

Cynthia L. Robson ◽

Annabelle M. Watts ◽

Andrew R. Gray ◽

Claire E. Wainwright ◽

...

Keyword(s):

Gene Expression ◽

Cystic Fibrosis ◽

Antibiotic Resistance ◽

Efflux Pump ◽

Antibiotic Resistance Gene ◽

Resistance Mechanisms ◽

Laboratory Culture ◽

Bacterial Cells ◽

Content Type ◽

Key Factor

ABSTRACTThe lungs of individuals with cystic fibrosis (CF) become chronically infected withPseudomonas aeruginosathat is difficult to eradicate by antibiotic treatment. Two keyP. aeruginosaantibiotic resistance mechanisms are the AmpC β-lactamase that degrades β-lactam antibiotics and MexXYOprM, a three-protein efflux pump that expels aminoglycoside antibiotics from the bacterial cells. Levels of antibiotic resistance gene expression are likely to be a key factor in antibiotic resistance but have not been determined during infection. The aims of this research were to investigate the expression of theampCandmexXgenes during infection in patients with CF and in bacteria isolated from the same patients and grown under laboratory conditions.P. aeruginosaisolates from 36 CF patients were grown in laboratory culture and gene expression measured by reverse transcription-quantitative PCR (RT-qPCR). The expression ofampCvaried over 20,000-fold and that ofmexXover 2,000-fold between isolates. The median expression levels of both genes were increased by the presence of subinhibitory concentrations of antibiotics. To measureP. aeruginosagene expression during infection, we carried out RT-qPCR using RNA extracted from fresh sputum samples obtained from 31 patients. The expression ofampCvaried over 4,000-fold, whilemexXexpression varied over 100-fold, between patients. Despite these wide variations, median levels of expression ofampCin bacteria in sputum were similar to those in laboratory-grown bacteria. The expression ofmexXwas higher in sputum than in laboratory-grown bacteria. Overall, our data demonstrate that genes that contribute to antibiotic resistance can be highly expressed in patients, but there is extensive isolate-to-isolate and patient-to-patient variation.

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Efflux Pumps in Chromobacterium Species Increase Antibiotic Resistance and Promote Survival in a Coculture Competition Model

Applied and Environmental Microbiology ◽

10.1128/aem.00908-19 ◽

2019 ◽

Vol 85 (19) ◽

Cited By ~ 3

Author(s):

Saida Benomar ◽

Kara C. Evans ◽

Robert L. Unckless ◽

Josephine R. Chandler

Keyword(s):

Antibiotic Resistance ◽

Efflux Pump ◽

Gene Clusters ◽

Efflux Pumps ◽

Resistance Mechanisms ◽

Competition Model ◽

Content Type ◽

Resistance Induction ◽

Resistance Nodulation Division

ABSTRACT Members of the Chromobacterium genus include opportunistic but often-fatal pathogens and soil saprophytes with highly versatile metabolic capabilities. In previous studies of Chromobacterium subtsugae (formerly C. violaceum) strain CV017, we identified a resistance nodulation division (RND)-family efflux pump (CdeAB-OprM) that confers resistance to several antibiotics, including the bactobolin antibiotic produced by the soil saprophyte Burkholderia thailandensis. Here, we show the cdeAB-oprM genes increase C. subtsugae survival in a laboratory competition model with B. thailandensis. We also demonstrate that adding sublethal bactobolin concentrations to the coculture increases C. subtsugae survival, but this effect is not through CdeAB-OprM. Instead, the increased survival requires a second, previously unreported pump we call CseAB-OprN. We show that in cells exposed to sublethal bactobolin concentrations, the cseAB-oprN genes are transcriptionally induced, and this corresponds to an increase in bactobolin resistance. Induction of this pump is highly specific and sensitive to bactobolin, while CdeAB-OprM appears to have a broader range of antibiotic recognition. We examine the distribution of cseAB-oprN and cdeAB-oprM gene clusters in members of the Chromobacterium genus and find the cseAB-oprN genes are limited to the nonpathogenic C. subtsugae strains, whereas the cdeAB-oprM genes are more widely distributed among members of the Chromobacterium genus. Our results provide new information on the antibiotic resistance mechanisms of Chromobacterium species and highlight the importance of efflux pumps for saprophytic bacteria existing in multispecies communities. IMPORTANCE Antibiotic efflux pumps are best known for increasing antibiotic resistance of pathogens; however, the role of these pumps in saprophytes is much less well defined. This study describes two predicted efflux pump gene clusters in the Chromobacterium genus, which is comprised of both nonpathogenic saprophytes and species that cause highly fatal human infections. One of the predicted efflux pump clusters is present in every member of the Chromobacterium genus and increases resistance to a broad range of antibiotics. The other gene cluster has more narrow antibiotic specificity and is found only in Chromobacterium subtsugae, a subset of entirely nonpathogenic species. We demonstrate the role of both pumps in increasing antibiotic resistance and demonstrate the importance of efflux-dependent resistance induction for C. subtsugae survival in a dual-species competition model. These results have implications for managing antibiotic-resistant Chromobacterium infections and for understanding the evolution of efflux pumps outside the host.

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Antagonistic Interactions of Pseudomonas aeruginosa Antibiotic Resistance Mechanisms in Planktonic but Not Biofilm Growth

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00519-11 ◽

2011 ◽

Vol 55 (10) ◽

pp. 4560-4568 ◽

Cited By ~ 34

Author(s):

Xavier Mulet ◽

Bartolomé Moyá ◽

Carlos Juan ◽

María D. Macià ◽

José L. Pérez ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Efflux Pump ◽

Resistance Mechanisms ◽

Negative Regulator ◽

Intrinsic Resistance ◽

Major Drawback ◽

Biofilm Growth ◽

Content Type ◽

Bacterial Physiology

ABSTRACTPseudomonas aeruginosahas an extraordinary capacity to evade the activity of antibiotics through a complex interplay of intrinsic and mutation-driven resistance pathways, which are, unfortunately, often additive or synergistic, leading to multidrug (or even pandrug) resistance. However, we show that one of these mechanisms, overexpression of the MexCD-OprJ efflux pump (driven by inactivation of its negative regulator NfxB), causes major changes in the cell envelope physiology, impairing the backbone ofP. aeruginosaintrinsic resistance, including the major constitutive (MexAB-OprM) and inducible (MexXY-OprM) efflux pumps and the inducible AmpC β-lactamase. Moreover, it also impaired the most relevant mutation-driven β-lactam resistance mechanism (constitutive AmpC overexpression), through a dramatic decrease in periplasmic β-lactamase activity, apparently produced by an abnormal permeation of AmpC out of the cell. While these results could delineate future strategies for combating antibiotic resistance in cases of acute nosocomial infections, a major drawback for the potential exploitation of the described antagonistic interaction between resistance mechanisms came from the differential bacterial physiology characteristics of biofilm growth, a hallmark of chronic infections. Although the failure to concentrate AmpC activity in the periplasm dramatically limits the protection of the targets (penicillin-binding proteins [PBPs]) of β-lactams at the individual cell level, the expected outcome for cells growing as biofilm communities, which are surrounded by a thick extracellular matrix, was less obvious. Indeed, our results showed that AmpC produced bynfxBmutants is protective in biofilm growth, suggesting that the permeation of AmpC into the matrix protects biofilm communities against β-lactams.

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Resistance to nitrofurantoin is an indicator of extensive drug-resistant (XDR) Enterobacteriaceae

Journal of Medical Microbiology ◽

10.1099/jmm.0.001347 ◽

2021 ◽

Vol 70 (4) ◽

Author(s):

Balaram Khamari ◽

Prakash Kumar ◽

Bulagonda Eswarappa Pradeep

Keyword(s):

Antimicrobial Agents ◽

Efflux Pump ◽

Treatment Options ◽

Strong Association ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Resistant Bacteria ◽

Drug Resistant ◽

Content Type ◽

Link Type

Introduction. Nitrofurantoin is one of the preferred antibiotics in the treatment of uropathogenic multidrug-resistant (MDR) infections. However, resistance to nitrofurantoin in extensively drug-resistant (XDR) bacteria has severely limited the treatment options. Gap statement. Information related to co-resistance or collateral sensitivity (CS) with reference to nitrofurantoin resistant bacteria is limited. Aim. To study the potential of nitrofurantoin resistance as an indicator of the XDR phenotype in Enterobacteriaceae . Methods. One hundred (45 nitrofurantoin-resistant, 21 intermediately resistant and 34 nitrofurantoin-susceptible) Enterobacteriaceae were analysed in this study. Antibiotic susceptibility testing (AST) against nitrofurantoin and 17 other antimicrobial agents across eight different classes was performed by using the Vitek 2.0 system. The isolates were screened for the prevalence of acquired antimicrobial resistance (AMR) and efflux pump genes by PCR. Results. In total, 51 % of nitrofurantoin-resistant and 28 % of intermediately nitrofurantoin resistant isolates exhibited XDR characteristics, while only 3 % of nitrofurantoin-sensitive isolates were XDR (P=0.0001). Significant co-resistance was observed between nitrofurantoin and other tested antibiotics (β-lactam, cephalosporin, carbapenem, aminoglycoside and tetracycline). Further, the prevalence of AMR and efflux pump genes was higher in the nitrofurantoin-resistant strains compared to the susceptible isolates. A strong association was observed between nitrofurantoin resistance and the presence of bla PER-1, bla NDM-1, bla OXA-48, ant(2) and oqxA-oqxB genes. Tigecycline (84 %) and colistin (95 %) were the only antibiotics to which the majority of the isolates were susceptible. Conclusion. Nitrofurantoin resistance could be an indicator of the XDR phenotype among Enterobacteriaceae , harbouring multiple AMR and efflux pump genes. Tigecycline and colistin are the only antibiotics that could be used in the treatment of such XDR infections. A deeper understanding of the co-resistance mechanisms in XDR pathogens and prescription of AST-based appropriate combination therapy may help mitigate this problem.

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Multidrug Adaptive Resistance of Pseudomonas aeruginosa Swarming Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01999-19 ◽

2019 ◽

Vol 64 (3) ◽

Cited By ~ 2

Author(s):

Shannon R. Coleman ◽

Travis Blimkie ◽

Reza Falsafi ◽

Robert E. W. Hancock

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Efflux Pump ◽

Iron Acquisition ◽

Polymyxin B ◽

Rna Seq ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Content Type ◽

Adaptive Resistance

ABSTRACT Swarming surface motility is a complex adaptation leading to multidrug antibiotic resistance and virulence factor production in Pseudomonas aeruginosa. Here, we expanded previous studies to demonstrate that under swarming conditions, P. aeruginosa PA14 is more resistant to multiple antibiotics, including aminoglycosides, β-lactams, chloramphenicol, ciprofloxacin, tetracycline, trimethoprim, and macrolides, than swimming cells, but is not more resistant to polymyxin B. We investigated the mechanism(s) of swarming-mediated antibiotic resistance by examining the transcriptomes of swarming cells and swarming cells treated with tobramycin by transcriptomics (RNA-Seq) and reverse transcriptase quantitative PCR (qRT-PCR). RNA-Seq of swarming cells (versus swimming) revealed 1,581 dysregulated genes, including 104 transcriptional regulators, two-component systems, and sigma factors, numerous upregulated virulence and iron acquisition factors, and downregulated ribosomal genes. Strain PA14 mutants in resistome genes that were dysregulated under swarming conditions were tested for their ability to swarm in the presence of tobramycin. In total, 41 mutants in genes dysregulated under swarming conditions were shown to be more resistant to tobramycin under swarming conditions, indicating that swarming-mediated tobramycin resistance was multideterminant. Focusing on two genes downregulated under swarming conditions, both prtN and wbpW mutants were more resistant to tobramycin, while the prtN mutant was additionally resistant to trimethoprim under swarming conditions; complementation of these mutants restored susceptibility. RNA-Seq of swarming cells treated with subinhibitory concentrations of tobramycin revealed the upregulation of the multidrug efflux pump MexXY and downregulation of virulence factors.

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Pan-β-Lactam Resistance Development in Pseudomonas aeruginosa Clinical Strains: Molecular Mechanisms, Penicillin-Binding Protein Profiles, and Binding Affinities

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00680-12 ◽

2012 ◽

Vol 56 (9) ◽

pp. 4771-4778 ◽

Cited By ~ 83

Author(s):

Bartolomé Moyá ◽

Alejandro Beceiro ◽

Gabriel Cabot ◽

Carlos Juan ◽

Laura Zamorano ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Molecular Mechanisms ◽

Efflux Pump ◽

Binding Protein ◽

Efflux Pumps ◽

Binding Affinities ◽

Penicillin Binding Protein ◽

Content Type ◽

Reverse Transcription Pcr

ABSTRACTWe investigated the mechanisms leading toPseudomonas aeruginosapan-β-lactam resistance (PBLR) development during the treatment of nosocomial infections, with a particular focus on the modification of penicillin-binding protein (PBP) profiles and imipenem, ceftazidime, and ceftolozane (former CXA-101) PBP binding affinities. For this purpose, six clonally related pairs of sequential susceptible-PBLR isolates were studied. The presence ofoprD,ampD, anddacBmutations was explored by PCR followed by sequencing and the expression ofampCand efflux pump genes by real-time reverse transcription-PCR. The fluorescent penicillin Bocillin FL was used to determine PBP profiles in membrane preparations from all pairs, and 50% inhibitory concentrations (IC50s) of ceftolozane, ceftazidime, and imipenem were analyzed in 3 of them. Although a certain increase was noted (0 to 5 2-fold dilutions), the MICs of ceftolozane were ≤4 μg/ml in all PBLR isolates. All 6 PBLR isolates lacked OprD and overexpressedampCand one or several efflux pumps, particularlymexBand/ormexY. Additionally, 5 of them showed modified PBP profiles, including a modified pattern (n= 1) or diminished expression (n= 1) of PBP1a and a lack of PBP4 expression (n= 4), which correlated with AmpC overexpression driven bydacBmutation. Analysis of the essential PBP IC50s revealed significant variation of PBP1a/b binding affinities, both within each susceptible-PBLR pair and across the different pairs. Moreover, despite the absence of significant differences in gene expression or sequence, a clear tendency toward increased PBP2 (imipenem) and PBP3 (ceftazidime, ceftolozane, imipenem) IC50s was noted in PBLR isolates. Thus, our results suggest that in addition to AmpC, efflux pumps, and OprD, the modification of PBP patterns appears to play a role in thein vivoemergence of PBLR strains, which still conserve certain susceptibility to the new antipseudomonal cephalosporin ceftolozane.

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IS5Element Integration, a Novel Mechanism for RapidIn VivoEmergence of Tigecycline Nonsusceptibility in Klebsiella pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03053-14 ◽

2014 ◽

Vol 58 (10) ◽

pp. 6151-6156 ◽

Cited By ~ 24

Author(s):

Lindsey E. Nielsen ◽

Erik C. Snesrud ◽

Fatma Onmus-Leone ◽

Yoon I. Kwak ◽

Ricardo Avilés ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Efflux Pump ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Insertion Element ◽

Content Type ◽

Reverse Transcription Quantitative Pcr ◽

Antimicrobial Susceptibility Tests ◽

Susceptibility Tests

ABSTRACTTigecycline nonsusceptibility is concerning because tigecycline is increasingly relied upon to treat carbapenem- or colistin-resistant organisms. InEnterobacteriaceae, tigecycline nonsusceptibility is mediated by the AcrAB-TolC efflux pump, among others, and pump activity is often a downstream effect of mutations in their transcriptional regulators, cognate repressor genes, or noncoding regions, as demonstrated inEnterobacteriaceaeandAcinetobacterisolates. Here, we report the emergence of tigecycline nonsusceptibility in a longitudinal series of multidrug-resistant (MDR) and extensively drug-resistant (XDR)Klebsiella pneumoniaeisolates collected during tigecycline therapy and the elucidation of its resistance mechanisms. Clinical isolates were recovered prior to and during tigecycline therapy of a 2.5-month-old Honduran neonate. Antimicrobial susceptibility tests to tigecycline determined that the MIC increased from 1 to 4 μg/ml prior to the completion of tigecycline therapy. Unlike other studies, we did not find increased expression oframA,ramR,oqxA,acrB,marA, orrarAgenes by reverse transcription-quantitative PCR (qRT-PCR). Whole-genome sequencing revealed an IS5insertion element in nonsusceptible isolates 85 bp upstream of a putative efflux pump operon, here namedkpgABC, previously unknown to be involved in resistance. Introduction of thekpgABCgenes in a non-kpgABCbackground increased the MIC of tigecycline 4-fold and is independent of a functional AcrAB-TolC pump. This is the first report to propose a function forkpgABCand identify an insertion element whose presence correlated with thein vivodevelopment of tigecycline nonsusceptibility inK. pneumoniae.

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