Intermediate Resistance Phenotype Research Articles

Characterization of Domain V Mutations in Clinical Isolates of Helicobacter pylori in Pakistan and Their Effect on Clarithromycin MIC.

PurposeClarithromycin is commonly prescribed for H. pylori infection. Domain V mutations are responsible for clarithromycin resistance. This study was aimed to characterize the clarithromycin resistance and its associated mutations in clinical isolates of H. pylori in Pakistan.Materials and MethodsInfection was diagnosed in 93 patients’ biopsies using culture, rapid urease test, 16S rRNA, and vacA gene multiplex PCR. Clarithromycin resistance was assessed by the agar dilution method. Mutations were detected by PCR-RFLP using 46 (1.4 kb) domain V fragments. Sequencing was executed for 13 domain V fragments, of which 12 showed unusual amplicon size (1.2 kb) and 01 had a new MboII RFLP pattern.ResultsA total of 48 (83%) strains were obtained from 58 (62.3%) PCR H. pylori-positive samples. Resistance (MIC ≥ 0.001 mg/mL) and intermediate resistance phenotype (MIC = 0.0005 mg/mL) was observed in 22 (46%), and 10 (21%) isolates, respectively. The primary resistance was found in 23 (39.6%) samples. PCR-RFLP detected A2142G, A2143G, and double mutations in 19, 04, and 01 resistant strain, respectively. Sequencing of 10 amplicons obtained from intermediated resistant strains and 03 amplicons from resistant strains showed 138 new mutations. Among them, T2182C was also seen in 04 intermediated resistant isolates, whereas A2142G, A2143G, and A2143C were observed in resistant isolates. The new MboII RFLP pattern in an intermediated resistant strains was due to A1761G mutation.ConclusionH. pylori domain V mutations showed extensive diversity. Multiple mutations in domain V may give endurance to H. pylori against clarithromycin. Further investigations on the molecular mechanism of antibiotic resistance in H. pylori seem crucial at this stage.

Comparison of bacteria isolated from open fractures following debridement and subsequent infection

BackgroundOne of the major objectives for the management of open fractures is to prevent bone and soft tissue infection. Here, we identified species and drug sensitivities of bacterial isolates recovered during open fracture debridement and after infection and compared the results between the two time points. MethodsA total of 61 hospitalized patients with open fractures who developed post-operative wound infection between October 2016 and December 2017 were included in this study. The cohort included 43 males and 18 females aged between 4 and 72 years. Patients were admitted to hospital 1–14 h after injury. Samples were collected after debridement and after infection and submitted for bacterial culture. Resulting isolates were identified using a VITEK 2 Bacterial Identification System and tested for drug sensitivity using the disc diffusion method. Results from the two time points were then compared. ResultsThe positive bacterial culture rate following debridement was relatively low (14/61, 22.9%). In addition, bacteria cultured after debridement were generally inconsistent with those cultured after wound infection, with a concordance rate of only 3.3% (2/61). Gram-negative bacteria accounted for 91.3% (63/69) of isolates recovered from wound infections following surgery, among which Acinetobacter baumannii was baumannii was the predominant pathogen, accounting for 49.3% (42/69) of all isolates. Overall, 60.8% (42/69) of postoperative infections were caused by multi-drug resistant bacteria, with A. baumannii isolates accounting for 80.9% (34/42) of these cases. Rates of cefoperazone/sulbactam resistance were relatively low among the isolates (15/34, 44.1%), and most isolates showed a sensitive or intermediate resistance phenotype. ConclusionsResults of bacterial culture after debridement could not predict pathogenic bacteria causing postoperative infection. Therefore, we propose that open fracture infections are predominantly nosocomial and are mainly caused by multidrug-resistant Gram-negative bacteria. Further attention should be paid to the control of these pathogens in clinical settings.

Daptomycin Resistance in Clinical MRSA Strains Is Associated with a High Biological Fitness Cost.

Daptomycin remains as one of the main treatment options for Methicillin-Resistant Staphylococcus aureus (MRSA). Sporadic resistance cases reported in patients treated with either daptomycin or glycopeptides are a growing concern. In a previous study, we described a clinical case of a patient with a community-acquired MRSA infection resistant to daptomycin and with intermediate resistance to vancomycin who developed a recurrent infection with a susceptible isogenic strain. In the present work, we further investigated the sequential events to determine whether the switch from a daptomycin resistance to a susceptible phenotype was due to a phenomenon of resistance reversion or recurrent infection with a susceptible strain. Pairwise competition experiments showed that the susceptible clinical recurrent SA6850 strain had increased fitness when compared to the resistant counterpart SA6820 strain. In fact, although we have demonstrated that reversion of daptomycin resistance to daptomycin susceptible can occur in vitro after serial passages in drug-free media, phylogenetic analysis suggested that the in vivo process was the result of a recurrent infection with a previous susceptible isolate carried by the patient rather than a resistance reversion of the strain. Whole genome sequence of evolved strains showed that daptomycin resistance in MRSA is associated with a high fitness cost mediated by mutations in mprF gene, revealed as a key element of the biological cost. Moreover, we determined that daptomycin resistance-associated fitness cost was independent of vancomycin intermediate resistance phenotype, as demonstrated in additional clinical MRSA vancomycin susceptible strains. This study highlights important observations as, despite daptomycin offers a useful treatment option for the patients with persistent infections, it has to be carefully monitored. The high fitness cost associated to daptomycin resistance may explain the reduced dissemination of daptomycin resistance and the absence of daptomycin reported outbreaks.

Development of secondary mutations in wild-type and mutant EZH2 alleles cooperates to confer resistance to EZH2 inhibitors.

The histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2) is frequently dysregulated in cancers, and gain-of-function (GOF) EZH2 mutations have been identified in non-Hodgkin lymphomas. Small-molecule inhibitors against EZH2 demonstrated anti-tumor activity in EZH2-mutated lymphomas and entered clinical trials. Here, we developed models of acquired resistance to EZH2 inhibitor EI1 with EZH2-mutated lymphoma cells. Resistance was generated by secondary mutations in both wild-type (WT) and GOF Y641N EZH2 alleles. These EZH2 mutants retained the substrate specificity of their predecessor complexes but became refractory to biochemical inhibition by EZH2 inhibitors. Resistant cells were able to maintain a high level of H3K27Me3 in the presence of inhibitors. Interestingly, mutation of EZH2 WT alone generated an intermediate resistance phenotype, which is consistent with a previously proposed model of cooperation between EZH2 WT and Y641N mutants to promote tumorigenesis. In addition, the findings presented here have implications for the clinical translation of EZH2 inhibitors and underscore the need to develop novel EZH2 inhibitors to target potential resistance emerging in clinical settings.

CARRIAGE OF ANTIBIOTIC-RESISTANT ENTERIC BACTERIA VARIES AMONG SITES IN GALÁPAGOS REPTILES

Increased overlap between humans and wildlife populations has increased the risk for novel disease emergence. Detecting contacts with a high risk for transmission of pathogens requires the identification of dependable measures of microbial exchange. We evaluated antibiotic resistance as a molecular marker for the intensity of human-wildlife microbial connectivity in the Galápagos Islands. We isolated Escherichia coli and Salmonella enterica from the feces of land iguanas (Conolophus sp.), marine iguanas (Amblyrhynchus cristatus), giant tortoises (Geochelone nigra), and seawater, and tested these bacteria with the use of the disk diffusion method for resistance to 10 antibiotics. Antibiotic-resistant bacteria were found in reptile feces from two tourism sites (Isla Plaza Sur and La Galapaguera on Isla San Cristóbal) and from seawater close to a public use beach near Puerto Baquerizo Moreno on Isla San Cristóbal. No resistance was detected at two protected beaches on more isolated islands (El Miedo on Isla Santa Fe and Cape Douglas on Isla Fernandina) and at a coastal tourism site (La Lobería on Isla San Cristóbal). Eighteen E. coli isolates from three locations, all sites relatively proximate to a port town, were resistant to ampicillin, doxycycline, tetracycline, and trimethoprin/sulfamethoxazole. In contrast, only five S. enterica isolates showed a mild decrease in susceptibility to doxycycline and tetracycline from these same sites (i.e., an intermediate resistance phenotype), but no clinical resistance was detected in this bacterial species. These findings suggest that reptiles living in closer proximity to humans potentially have higher exposure to bacteria of human origin; however, it is not clear from this study to what extent this potential exposure translates to ongoing exchange of bacterial strains or genetic traits. Resistance patterns and bacterial exchange in this system warrant further investigation to understand better how human associations influence disease risk in endemic Galápagos wildlife.

Diversity of aminoglycoside modifying enzyme genes among multidrug resistant Acinetobacter baumannii genotypes isolated from nosocomial infections in Tehran hospitals and their association with class 1 integrons

The aim of the present study was to investigate, for the first time, the diversity of the genes encoding aminoglycoside-modifying enzymes (AME) and their association with class 1 integrons in Iranian Acinetobacter baumannii strains. A total of 100 multidrug resistant A. baumannii, isolated from eight distinct hospitals in Tehran, were enrolled in this study. Susceptibility of these isolates to antimicrobial agents including gentamicin and amikacin was determined by E-test. Aminoglycoside resistant isolates were then tested by PCR for AME genes, including aphA6, aacC1, aacC2, aacA4, aadB, aadA1, classes 1 integron, 5'-CS-3' and typed by RAPD PCR. The rate of resistance to imipenem, meropenem, gentamicin and amikacin were 39%, 39%, 38% and 32%, respectively. Intermediate resistance phenotype to gentamicin and amikacin was observed in 2% and 5% of all the isolates, respectively. After aph6 with 90% (n = 36/40), aadA1, aacC1 and aadB with 82.5% (n = 33/40), 65% (n = 26/40) and 20% (n = 8/40) were the most prevalent AME genes among aminoglycosides resistant A. baumannii isolates. A combination of two to four different resistance genes was observed in 39 of 40 strains (97.5%), with a total of 7 different combinations. PCR of integrase genes revealed that AME gene was associated with 67% of class 1 integrons. RAPD analysis showed three predominant genotypes A (n = 20), B (n = 10) and 10 unrelated genotypes. The occurrence of identical resistance genes, gene combinations and class 1 integrons associated with these genes in clonally distinct strains indicates that horizontal gene transfer plays a major role in the dissemination of aminoglycoside resistance in A. baumannii.

Interaction of the GraRS Two-Component System with the VraFG ABC Transporter To Support Vancomycin-Intermediate Resistance in Staphylococcus aureus

Current treatment for serious infections caused by methicillin-resistant Staphylococcus aureus relies heavily upon the glycopeptide antibiotic vancomycin. Unfortunately, this practice has led to an intermediate resistance phenotype that is particularly difficult to treat in invasive staphylococcal diseases, such as septicemia and its metastatic complications, including endocarditis. Although the vancomycin-intermediate resistance phenotype has been linked to abnormal cell wall structures and autolytic rates, the corresponding genetic changes have not been fully elucidated. Previously, whole-genome array studies listed numerous genes that are overexpressed in vancomycin-intermediate sensitive strains, including graRS (SACOL0716 to -0717), encoding a two-component regulatory system (TCRS), as well as the adjacent vraFG (SACOL0718 to -0720), encoding an ATP-binding cassette (ABC) transporter; but the exact contribution of these genes to increased vancomycin resistance has not been defined. In this study, we showed that isogenic strains with mutations in genes encoding the GraRS TCRS and the VraFG ABC transporter are hypersensitive to vancomycin as well as polymyxin B. Moreover, GraRS regulates the expression of the adjacent VraFG pump, reminiscent of gram-positive bacteriocin-immunity regulons. Mutations of graRS and vraFG also led to increased autolytic rates and a more negative net surface charge, which may explain, in part, to their increased sensitivity to cationic antimicrobial peptides. Taken together, these data reveal an important genetic mediator to the vancomycin-intermediate S. aureus phenotype and may hold clues to the selective pressures on staphylococci upon exposure to selective cationic peptide antibiotics used in clinical practice.

AcrAB-TolC directs efflux-mediated multidrug resistance in Salmonella enterica serovar typhimurium DT104.

Multidrug-resistant Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) strains harbor a genomic island, called Salmonella genomic island 1 (SGI1), which contains an antibiotic resistance gene cluster conferring resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides, and tetracyclines. They may be additionally resistant to quinolones. Among the antibiotic resistance genes there are two, i.e., floR and tet(G), which code for efflux pumps of the major facilitator superfamily with 12 transmembrane segments that confer resistance to chloramphenicol-florfenicol and the tetracyclines, respectively. In the present study we determined, by constructing acrB and tolC mutants, the role of the AcrAB-TolC multidrug efflux system in the multidrug resistance of several DT104 strains displaying additional quinolone resistance or not displaying quinolone resistance. This study shows that the quinolone resistance and the decreased fluoroquinolone susceptibilities of the strains are highly dependent on the AcrAB-TolC efflux system and that single mutations in the quinolone resistance-determining region of gyrA are of little relevance in mediating this resistance. Overproduction of the AcrAB efflux pump, as determined by Western blotting with an anti-AcrA polyclonal antibody, appeared to be the major mechanism of resistance to quinolones. Moreover, chloramphenicol-florfenicol and tetracycline resistance also appeared to be highly dependent on the presence of AcrAB-TolC, since the introduction of mutations in the respective acrB and tolC genes resulted in a susceptible or intermediate resistance phenotype, according to clinical MIC breakpoints, despite the presence of the FloR and Tet(G) efflux pumps. Resistance to other antibiotics, ampicillin, streptomycin, and sulfonamides, was not affected in the acrB and tolC mutants of DT104 strains harboring SGI1. Therefore, AcrAB-TolC appears to direct efflux-mediated resistance to quinolones, chloramphenicol-florfenicol, and tetracyclines in multidrug-resistant S. enterica serovar Typhimurium DT104 strains.

Involvement of thempooperon in resistance to class IIa bacteriocins inListeria monocytogenes

High resistance to class IIa bacteriocins in Listeria monocytogenes has been clearly linked to lack of expression of the mptACD operon, encoding the EIIt Man mannose PTS permease. Also, intermediate resistance has been associated with membrane phospholipid modifications in the spontaneous mutants L. monocytogenes B73-V1 and B73-V2. We constructed a new mutant of L. monocytogenes that was interrupted in mpoA, and which also exhibited an intermediate resistance phenotype. The mpoABCD operon putatively encodes a PTS permease of the mannose family similar to that encoded by the mpt operon. In silico analysis indicated that mpo transcription might be dependent on sigma54. Our study demonstrated that the three intermediate resistant mutants have a slight decrease in mptACD expression, showing that the level of sensitivity is correlated to the level of mpt expression. We show a cross-regulation between mpo and mpt. In particular, the mpo mutant has a defect in mpt expression that possibly could explain its intermediate resistance phenotype.

Involvement of the mpo operon in resistance to class IIa bacteriocins in Listeria monocytogenes

High resistance to class IIa bacteriocins in Listeria monocytogenes has been clearly linked to lack of expression of the mptACD operon, encoding the EII t Man mannose PTS permease. Also, intermediate resistance has been associated with membrane phospholipid modifications in the spontaneous mutants L. monocytogenes B73-V1 and B73-V2. We constructed a new mutant of L. monocytogenes that was interrupted in mpoA, and which also exhibited an intermediate resistance phenotype. The mpoABCD operon putatively encodes a PTS permease of the mannose family similar to that encoded by the mpt operon. In silico analysis indicated that mpo transcription might be dependent on σ 54. Our study demonstrated that the three intermediate resistant mutants have a slight decrease in mptACD expression, showing that the level of sensitivity is correlated to the level of mpt expression. We show a cross-regulation between mpo and mpt. In particular, the mpo mutant has a defect in mpt expression that possibly could explain its intermediate resistance phenotype.