Preservation of Acquired Colistin Resistance in Gram-Negative Bacteria

Colistin-resistant mutants were obtained from 17 colistin-susceptible strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli. The stability of colistin resistance in these mutants was investigated. Three of four colistin-resistant P. aeruginosa mutants recovered colistin susceptibility in colistin-free medium; however, colistin-susceptible revertants were obtained from only one strain each of A. baumannii and E. coli. No susceptible revertants were obtained from K. pneumoniae mutants.     

Molecular epidemiology and mechanisms of tigecycline resistance in carbapenem-resistant Klebsiella pneumoniae isolates

BackgroundThe emergence and transmission of tigecycline‐ and carbapenem‐resistant Klebsiella pneumoniae (TCRKP) have become a major concern to public health globally. Here, we investigated the molecular epidemiology and mechanisms of tigecycline resistance in carbapenem‐resistant K pneumoniae (CRKP) isolates.MethodsForty‐five non‐duplicate CRKP isolates were collected from January 2017 to June 2019. We performed antimicrobial susceptibility tests, multilocus sequence typing (MLST), and pulsed‐field gel electrophoresis (PFGE). PCR and DNA sequencing were performed for the detection and mutation analysis of acrR, oqxR, ramR, rpsJ, tet(A), and tet(X) genes, which are related to tigecycline resistance. The expression levels of efflux pump genes acrB and oqxB and their regulator genes rarA, ramA, soxS, and marA were assessed by quantitative real‐time PCR.ResultsThe resistance rate to tigecycline in CRKP isolates was 37.8% (17/45). K pneumoniae ST307 was a predominant clone type (70.6%, 12/17) among the TCRKP isolates. The expression levels of acrB (P < .001) and marA (P = .009) were significantly higher in the tigecycline‐resistant group than in the tigecycline‐intermediate and tigecycline‐susceptible groups. Increased expression of acrB was associated with marA expression (r = 0.59, P = .013).ConclusionsWe found that the activated MarA‐induced overexpression of AcrAB efflux pump plays an important role in the emergence of tigecycline resistance in CRKP isolates.     

Mutations That Enhance the Ciprofloxacin Resistance of Escherichia coli with qnrA1

Plasmid-mediated qnr genes provide only a modest decrease in quinolone susceptibility but facilitate the selection of higher-level resistance. In Escherichia coli strain J53 without qnr, ciprofloxacin resistance often involves mutations in the GyrA subunit of DNA gyrase. Mutations in gyrA were absent, however, when 43 mutants with decreased ciprofloxacin susceptibility were selected from J53(pMG252) with qnrA1. Instead, in 13 mutants, individual and whole-genome sequencing identified mutations in marR and soxR associated with increased expression of marA and soxS and, through them, increased expression of the AcrAB pump, which effluxes quinolones. Nine mutants had increased expression of the MdtE efflux pump, and six demonstrated increased expression of the ydhE pump gene. Many efflux mutants also had increased resistance to novobiocin, another pump substrate, but other mutants were novobiocin hypersusceptible. Mutations in rfaD and rfaE in the pathway for inner core lipopolysaccharide (LPS) biosynthesis were identified in five such strains. Many of the pump and LPS mutants had decreased expression of OmpF, the major porin channel for ciprofloxacin entry. Three mutants had increased expression of qnrA that persisted when pMG252 from these strains was outcrossed. gyrA mutations were also rare when mutants with decreased ciprofloxacin susceptibility were selected from E. coli J53 with aac(6′)-Ib-cr or qepA. We suggest that multiple genes conferring low-level resistance contribute to enhanced ciprofloxacin resistance selected from an E. coli strain carrying qnrA1, aac(6′)-Ib-cr, or qepA because these determinants decrease the effective ciprofloxacin concentration and allow more common but lower-resistance mutations than those in gyrA to predominate.     

Restrictive Streptomycin Resistance Mutations Decrease the Formation of Attaching and Effacing Lesions in Escherichia coli O157:H7 Strains

Streptomycin binds to the bacterial ribosome and disrupts protein synthesis by promoting misreading of mRNA. Restrictive mutations on the ribosomal subunit protein S12 confer a streptomycin resistance (Str^r) phenotype and concomitantly increase the accuracy of the decoding process and decrease the rate of translation. Spontaneous Str^r mutants of Escherichia coli O157:H7 have been generated for in vivo studies to promote colonization and to provide a selective marker for this pathogen. The locus of enterocyte effacement (LEE) of E. coli O157:H7 encodes a type III secretion system (T3SS), which is required for attaching and effacing to the intestinal epithelium. In this study, we observed decreases in both the expression and secretion levels of the T3SS translocated proteins EspA and EspB in E. coli O157:H7 Str^r restrictive mutants, which have K42T or K42I mutations in S12. However, mildly restrictive (K87R) and nonrestrictive (K42R) mutants showed slight or indistinguishable changes in EspA and EspB secretion. Adherence and actin staining assays indicated that restrictive mutations compromised the formation of attaching and effacing lesions in E. coli O157:H7. Therefore, we suggest that E. coli O157:H7 strains selected for Str^r should be thoroughly characterized before in vivo and in vitro experiments that assay for LEE-directed phenotypes and that strains carrying nonrestrictive mutations such as K42R make better surrogates of wild-type strains than those carrying restrictive mutations.     

CTX-M Expression and Selection of Ertapenem Resistance in Klebsiella pneumoniae and Escherichia coli

In vitro selection of mutants with decreased susceptibility to ertapenem was performed using Escherichia coli and Klebsiella pneumoniae clinical strains producing either the bla_CTX-M-2, bla_CTX-M-3, bla_CTX-M-9, or bla_CTX-M-15 gene. Frequencies of mutants with decreased susceptibilities to ertapenem were similar for all β-lactamases expressed.     

Second nationwide surveillance of bacterial pathogens in patients with acute uncomplicated cystitis conducted by Japanese Surveillance Committee from 2015 to 2016: antimicrobial susceptibility of Escherichia coli,Klebsiella pneumoniae,and Staphylococcus saprophyticus

The Japanese Surveillance Committee conducted a second nationwide surveillance of antimicrobial susceptibility patterns of uropathogens responsible for acute uncomplicated cystitis (AUC) in premenopausal patients aged 16–40 years old at 31 hospitals throughout Japan from March 2015 to February 2016. In this study, the susceptibility of causative bacteria (Escherichia coli, Klebsiella pneumoniae, Staphylococcus saprophyticus) for various antimicrobial agents was investigated by isolation and culturing of organisms obtained from urine samples. In total, 324 strains were isolated from 361 patients, including E. coli (n = 220, 67.9%), S. saprophyticus (n = 36, 11.1%), and K. pneumoniae (n = 7, 2.2%). The minimum inhibitory concentrations (MICs) of 20 antibacterial agents for these strains were determined according to the Clinical and Laboratory Standards Institute (CLSI) manual. At least 93% of the E. coli isolates showed susceptibility to fluoroquinolones and cephalosporins, whereas 100% of the S. saprophyticus isolates showed susceptibility to fluoroquinolones and aminoglycosides. The proportions of fluoroquinolone-resistant and extended-spectrum β-lactamase (ESBL)-producing E. coli strains were 6.4% (13/220) and 4.1% (9/220), respectively. The antimicrobial susceptibility of K. pneumoniae was retained during the surveillance period, while no multidrug-resistant strains were identified.In summary, antimicrobial susceptibility results of our second nationwide surveillance did not differ significantly from those of the first surveillance. Especially the numbers of fluoroquinolone-resistant and ESBL-producing E. coli strains were not increased in premenopausal patients with AUC in Japan.     

Trends in susceptibility of selected gram-negative bacilli isolated from intra-abdominal infections in North America: SMART 2005–2010

This report summarizes data from the Study for Monitoring Antimicrobial Resistance Trends 2005–2010 to identify trends in susceptibility from intra-abdominal infections in North America. Escherichia coli and Klebsiella pneumoniae were the most frequently isolated pathogens. Extended-spectrum beta-lactamase rates (%) in 2005/2010 for E. coli and K. pneumoniae were 1.7/7.3 and 3.2/13.1, respectively. Ertapenem, imipenem, amikacin, and cefoxitin showed stable susceptibility.     

Susceptibility of various oral antibacterial agents against extended spectrum β-lactamase producing Escherichia coli and Klebsiella pneumoniae

With the increase in extended spectrum β-lactamase (ESBL)-producing bacteria in the community, cases are often seen in which treatment of infectious diseases with oral antimicrobial agents is difficult. Therefore, we measured the antimicrobial activities of 14 currently available oral antimicrobial agents against ESBL-producing Escherichia coli and Klebsiella pneumoniae. Based on the standard of the Clinical and Laboratory Standards Institute (CLSI), E. coli showed high susceptibility rates of 99.4% to faropenem (FRPM). In terms of fluoroquinolones, the susceptibility rate of E. coli to levofloxacin (LVFX) was low at 32.2%, whereas it showed a good susceptibility rate of 93.1% to sitafloxacin (STFX). With respect to other antimicrobial agents, susceptibility rates to fosfomycin (FOM) and colistin (CL) were more than 90% each, whereas rates of the two antimicrobial agents expected as therapeutic agents, minocycline (MINO) and sulfamethoxazole-trimethoprim (ST), were low at 62.4% and 44.3%, respectively. Based on the CLSI standard, K. pneumoniae showed high susceptibility rates to ceftibuten (CETB) (91.89%), LVFX (86.49%), and STFX (94.6%), indicating that K. pneumoniae showed higher rates than those of E. coli, particularly to fluoroquinolones.Comparison of susceptibility rates according to E. coli genotype showed that many antimicrobial agents existed to which the CTX-M-9 group showed high susceptibility rates. However, there were many agents to which the CTX-M-1 group showed low susceptibility rates, particularly to CETB (51.1%) and LVFX (17.0%). Although there was no significant difference by genotype between FRPM, STFX, and FOM, a significant difference was observed between LVFX, MINO, and ST. Antibiotic-resistant bacteria with highly pathogenic strains have spread in the community, appropriate use of oral antimicrobial agents is required.     

DNA Gyrase and Topoisomerase IV Are Dual Targets of Clinafloxacin Action in Streptococcus pneumoniae

We examined the response of Streptococcus pneumoniae 7785 to clinafloxacin, a novel C-8-substituted fluoroquinolone which is being developed as an antipneumococcal agent. Clinafloxacin was highly active against S. pneumoniae 7785 (MIC, 0.125 μg/ml), and neither gyrA nor parC quinolone resistance mutations alone had much effect on this activity. A combination of both mutations was needed to register resistance, suggesting that both gyrase and topoisomerase IV are clinafloxacin targets in vivo. The sparfloxacin and ciprofloxacin MICs for the parC-gyrA mutants were 16 to 32 and 32 to 64 μg/ml, respectively, but the clinafloxacin MIC was 1 μg/ml, i.e., within clinafloxacin levels achievable in human serum. S. pneumoniae 7785 mutants could be selected stepwise with clinafloxacin at a low frequency, yielding first-, second-, third-, and fourth-step mutants for which clinafloxacin MICs were 0.25, 1, 6, and 32 to 64 μg/ml, respectively. Thus, high-level resistance to clinafloxacin required four steps. Characterization of the quinolone resistance-determining regions of the gyrA, parC, gyrB, and parE genes by PCR, HinfI restriction fragment length polymorphism, and DNA sequence analysis revealed an invariant resistance pathway involving sequential mutations in gyrA or gyrB, in parC, in gyrA, and finally in parC or parE. No evidence was found for other resistance mechanisms. The gyrA mutations in first- and third-step mutants altered GyrA hot spots Ser-83 to Phe or Tyr (Escherichia coli coordinates) and Glu-87 to Gln or Lys; second- and fourth-step parC mutations changed equivalent hot spots Ser-79 to Phe or Tyr and Asp-83 to Ala. gyrB and parE changes produced novel alterations of GyrB Glu-474 to Lys and of Pro-454 to Ser in the ParE PLRGK motif. Difficulty in selecting first-step gyrase mutants (isolated with 0.125 [but not 0.25] μg of clinafloxacin per ml at a frequency of 5.0 × 10⁻¹⁰ to 8.5 × 10⁻¹⁰) accompanied by the small (twofold) MIC increase suggested only a modest drug preference for gyrase. Given the susceptibility of defined gyrA or parC mutants, the results suggested that clinafloxacin displays comparable if unequal targeting of gyrase and topoisomerase IV. Dual targeting and the intrinsic potency of clinafloxacin against S. pneumoniae and its first- and second-step mutants are desirable features in limiting the emergence of bacterial resistance.     

Loss of Hypermucoviscosity and Increased Fitness Cost in Colistin-Resistant Klebsiella pneumoniae Sequence Type 23 Strains

In this study, we investigated the effects of colistin resistance on virulence and fitness in hypermucoviscous (HV) Klebsiella pneumoniae sequence type 23 (ST23) strains. Colistin-resistant mutants were developed from three colistin-susceptible HV K. pneumoniae ST23 strains. The lipid A structures of strains were analyzed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. Changes in HV were investigated using the string test, and extracellular polysaccharide production was quantified. The expression levels of the phoQ, pmrD, pmrB, pbgP, magA, and p-rmpA2 genes, serum resistance, and biofilm-forming activity were determined. The fitness of colistin-resistant mutants compared to that of the parental strains was examined by determining the competitive index (CI). The colistin-resistant mutants exhibited reduced HV, which was accompanied by decreased formation of capsular polysaccharides (CPS) and reduced expression of genes (magA and p-rmpA2). While there was enhanced expression of pmrD and pbgP in all colistin-resistant derivatives, there were differences in the expression levels of phoQ and pmrB between strains. MALDI-TOF analysis detected the addition of aminoarabinose or palmitate to the lipid A moiety of lipopolysaccharide in the colistin-resistant derivatives. In addition, survival rates in the presence of normal human serum were decreased in the mutant strains, and CI values (0.01 to 0.19) indicated significant fitness defects in the colistin-resistant derivatives compared to the respective parental strains. In hypervirulent HV K. pneumoniae strains, the acquisition of colistin resistance was accompanied by reduced CPS production, impaired virulence, and a significant fitness cost.     

Antimicrobial Activities of Ceftaroline and Comparator Agents against Bacterial Organisms Causing Bacteremia in Patients with Skin and Skin Structure Infections in U.S. Medical Centers, 2008 to 2014

We evaluated the antimicrobial susceptibility of 1,454 organisms consecutively collected from patients with bacteremia associated with skin and skin structure infections. The most common organisms obtained were Staphylococcus aureus (670 organisms [46.1%]), Escherichia coli (200 organisms [13.8%]), β-hemolytic streptococci (βHS) (138 organisms [9.5%]), and Klebsiella pneumoniae (109 organisms [7.5%]). The susceptibility rates for ceftaroline were 97.9% for S. aureus (95.9% among methicillin-resistant S. aureus [MRSA]), 100.0% for βHS, 86.5% for E. coli, and 89.0% for K. pneumoniae. Ceftaroline and tigecycline provided the best overall coverage.     

Amdinocillin (Mecillinam) Resistance Mutations in Clinical Isolates and Laboratory-Selected Mutants of Escherichia coli

Amdinocillin (mecillinam) is a β-lactam antibiotic that is used mainly for the treatment of uncomplicated urinary tract infections. The objectives of this study were to identify mutations that confer amdinocillin resistance on laboratory-isolated mutants and clinical isolates of Escherichia coli and to determine why amdinocillin resistance remains rare clinically even though resistance is easily selected in the laboratory. Under laboratory selection, frequencies of mutation to amdinocillin resistance varied from 8 × 10⁻⁸ to 2 × 10⁻⁵ per cell, depending on the concentration of amdinocillin used during selection. Several genes have been demonstrated to give amdinocillin resistance, but here eight novel genes previously unknown to be involved in amdinocillin resistance were identified. These genes encode functions involved in the respiratory chain, the ribosome, cysteine biosynthesis, tRNA synthesis, and pyrophosphate metabolism. The clinical isolates exhibited significantly greater fitness than the laboratory-isolated mutants and a different mutation spectrum. The cysB gene was mutated (inactivated) in all of the clinical isolates, in contrast to the laboratory-isolated mutants, where mainly other types of more costly mutations were found. Our results suggest that the frequency of mutation to amdinocillin resistance is high because of the large mutational target (at least 38 genes). However, the majority of these resistant mutants have a low growth rate, reducing the probability that they are stably maintained in the bladder. Inactivation of the cysB gene and a resulting loss of cysteine biosynthesis are the major mechanism of amdinocillin resistance in clinical isolates of E. coli.     

Influence of the yjiL-mdtM Gene Cluster on the Antibacterial Activity of Proline-Rich Antimicrobial Peptides Overcoming Escherichia coli Resistance Induced by the Missing SbmA Transporter System

In view of increasing health threats from multiresistant pathogens, antimicrobial peptides (AMPs) and, specifically, proline-rich AMPs (PrAMPs) have been investigated in animal models. PrAMPs enter bacteria via the ABC transporter SbmA and inhibit intracellular targets. We used phage transduction (Tn10 insertion) to screen by random mutagenesis for alternative uptake mechanisms for analogs of apidaecin 1b, a honeybee-derived PrAMP. All 24 apidaecin-resistant mutants had the Tn10 insertion in the sbmA gene. These sbmA::Tn10 insertion mutants and the Escherichia coli BW25113 ΔsbmA (JW0368) strain were still susceptible to the bactenecin PrAMP Bac7(1-35) and oncocin PrAMPs Onc18 and Onc112, as well as to Chex1-Arg20, despite significantly reduced internalizations. In a second round of random mutagenesis, the remaining susceptibility was linked to the yjiL-mdtM gene cluster. E. coli BW25113 and its ΔyjiL null mutant (JW5785) were equally susceptible to all PrAMPs tested, whereas the BW25113 ΔmdtM mutant was less susceptible to oncocins. The JW0368 yjiL::Tn10 transposon mutant (BS2) was resistant to all short PrAMPs and susceptible only to full-length Bac7 and A3-APO. Interestingly, PrAMPs appear to enter bacteria via MdtM, a multidrug resistance transporter (drug/H⁺ antiporter) of the major facilitator superfamily (MFS) that can efflux antibiotics, biocides, and bile salts. In conclusion, PrAMPs enter bacteria via ABC and MFS transporters that efflux antibiotics and cytotoxic compounds from the cytoplasm to the periplasm.     

Toughened chitosan-based composite membranes with antibiofouling and antibacterial properties via incorporation of benzalkonium chloride

Biofouling due to biofilm formation is a major problem in ultrafiltration membrane applications. In this work, a potential approach to solve this issue has been developed by functionalization of chitosan-based membranes with benzalkonium chloride (BKC). The chitosan composite membranes consisting of poly(ethylene glycol) (PEG), multiwalled carbon nanotubes (MWCNT), and BKC were synthesized by mixing the membrane precursors and the antibacterial solution, and casting via an inversed phase technique. The effects of the BKC content on the morphology and performance of the membranes are investigated by varying the BKC feed compositions. The composite membranes demonstrate better antibacterial efficacy against Staphylococcus aureus than Escherichia coli. The permeability and selectivity performances of the composites as filter membranes are examined by employing a dead-end filtration system. Interestingly, enhanced toughness of the membranes is observed as a function of the BKC content. Mechanisms of the structural formation are investigated. The results from SEM, XRD, and FTIR spectroscopy revealed that MWCNT/BKC are located as nanoclusters with π–π stacking interactions, and are covered by PEG chains. The shape of the dispersed domains is spherical at low BKC contents, but becomes elongated at high BKC contents. These act as soft domains with an anisotropic shape with toughening of the brittle chitosan matrix, leading to enhanced durability of the membranes, especially in ultrafiltration applications. The composite membranes also demonstrate improved rejection in dead-end ultrafiltration systems due to high porosity, high hydrophilicity, and the positive charges of the membrane surface.

Chitosan/PEG/MWCNT/BKC membranes exhibit enhanced antibiofouling properties against S. aureus and E. coli. MWCNT/BKC are located as dispersed nano-clusters with π–π stacking interactions in the chitosan matrix, and are coved by PEG chains.     

Mutations in Bartonella bacilliformis gyrB Confer Resistance to Coumermycin A1

This study describes the first isolation and characterization of spontaneous mutants conferring natural resistance to an antibiotic for any Bartonella species. The Bartonella bacilliformis gyrB gene, which encodes the B subunit of DNA gyrase, was cloned and sequenced. The gyrB open reading frame (ORF) is 2,079 bp and encodes a deduced amino acid sequence of 692 residues, corresponding to a predicted protein of ~77.5 kDa. Sequence alignment indicates that B. bacilliformis GyrB is most similar to the GyrB protein from Bacillus subtilis (40.1% amino acid sequence identity) and that it contains the longest N-terminal tail (52 residues) of any GyrB characterized to date. The cloned B. bacilliformis gyrB was expressed in an Escherichia coli S30 cell extract and was able to functionally complement a temperature-sensitive E. coli Cou^r gyrB mutant (strain N4177). We isolated and characterized spontaneous mutants of B. bacilliformis resistant to coumermycin A₁, an antibiotic that targets GyrB. Sequence analysis of gyrB from 12 Cou^r mutants of B. bacilliformis identified single nucleotide transitions at three separate loci in the ORF. The predicted amino acid substitutions resulting from these transitions are Gly to Ser at position 124 (Gly124→Ser), Arg184→Gln, and Thr214→Ala or Thr214→Ile, which are analogous to mutated residues found in previously characterized resistant gyrB genes from Borrelia burgdorferi, E. coli, Staphylococcus aureus, and Haloferax sp. The Cou^r mutants are three to five times more resistant to coumermycin A₁ than the wild-type parental strain.