High Levels Of Cross-resistance Research Articles

Diversity and antibiotic sensitivity of uropathogens from a regional hospital in Rwanda

Background: Urinary tract infections are a common cause for antibiotic consumption. Empirical treatment is common for community-acquired infections owing to predictability of pathogens. Objectives: Describe sensitivity profiles of uropathogens at a regional hospital in Rwanda. Methods: Hospital-based cross-sectional study in which clean-catch urine samples were analyzed. A two-stage process involving dipstick urinalysis and urine culture was used to identify true infections. The Kirby-Bauer disc diffusion method was used for antibiotic sensitivity analysis as per EUCAST guidelines. Results: Of 198 samples studied, 107 met criterion for UTI with 94 yielding significant growth. Klebsiella pneumoniae (35%), and Escherichia coli (32%) were the most common organisms isolated. Others included Staphylococcal species, Proteus spp, Enterobacter spp and Salmonella. Very high resistance frequencies (above 70%) were observed against amoxycillin, cotrimoxazole, trimethoprim, aztreonam and fosfomycin. Resistance to fluoroquinolones, aminoglycosides and cephalosporins varied between 40-60%. High levels of cross resistance were observed for drugs of the same class. Resistance to the potentiated penicillins, meropenem, cefoxitin and nitrofurantoin were less than 30%. Piperacillintazo bactum performed best with 91% sensitivity against all organisms tested. Conclusion: High levels of resistance were observed for most antibiotics studied. For empirical treatment of community-acquired UTI in our setting, nitrofurantoin remains effective while ciprofloxacin is not. Keywords: Diversity and antibiotic sensitivity; uropathogens; Rwanda.

Investigating resistance levels to cyhalofop-butyl and mechanisms involved in Chinese sprangletop (Leptochloa chinensis L.) from Anhui Province, China

Chinese sprangletop (Leptochloa chinensis (L.) Nees) is a common grass species that severely threatens rice (Oryza sativa L.) cropping systems globally. Cyhalofop-butyl is a highly efficient acetyl-CoA carboxylase (ACCase)-inhibiting herbicide widely used for control of this species in China. However, some L. chinensis populations have gradually evolved resistance to this herbicide in recent years. To better understand the cyhalofop-butyl resistance status of L. chinensis in the major rice planting area of the middle-lower Yangtze River basin, 73 populations collected from the rice fields across Anhui Province were investigated for cyhalofop-butyl susceptibility and potential herbicide resistance-conferring mutations. Single-dose testing indicated that of the 73 populations, 25 had evolved resistance to cyhalofop-butyl and were separately classified as “RRR” and “RR” populations according to their fresh weight reductions, 8 had a high risk of evolving cyhalofop-butyl resistance and were classified as “R?” populations, and 40 were susceptible and classified as “S” populations. Whole-plant dose-response experiments showed that the resistance index (RI) of these R?, RR, and RRR populations to cyhalofop-butyl ranged from 2.47 to 36.94. Target gene sequencing identified seven ACCase resistance mutations (I1781L, W1999C, W2027S, W2027L, W2027C, I2041N, and D2078G), with W1999C and W2027C the two most common detected in about three quarters of all the resistant populations. Seven populations including LASC3, BBHY1, AQQS1, HFFD3, HFFD4, AQWJ1, and HFLJ6 each carrying a specific ACCase mutation were tested for their cross- and multiple-resistance patterns. Compared with a standard susceptible population HFLY1, the seven resistant populations showed distinct cross-resistance. All had low- to high-level cross-resistance to metamifop (RIs ranging from 6.16 to 17.65), fenoxaprop-P-ethyl (RIs ranging from 6.39 to 24.08), and quizalofop-P-ethyl (RIs ranging from 2.20 to 10.25), but responded differently to clodinafop-propargyl and clethodim. Multiple-resistance testing suggested that the seven resistant populations were all susceptible to the 4-hydroxyphenylpyruvate dioxygenase inhibitor tripyrasulfone, the protoporphyrinogen oxidase inhibitor oxyfluorfen, and the auxin mimic herbicide florpyrauxifen. In conclusion, this study has shown that cyhalofop-butyl resistance was prevalent in L. chinensis in Anhui Province, China, and target site mutation was one of the most common resistance mechanisms.

Selective resistance profiles emerging in patient-derived clinical isolates with cabotegravir, bictegravir, dolutegravir, and elvitegravir

BackgroundIntegrase strand transfer inhibitors (INSTIs) are recommended for first-line HIV therapy based on their relatively high genetic barrier to resistance. Although raltegravir (RAL) and elvitegravir (EVG) resistance profiles are well-characterized, resistance patterns for dolutegravir (DTG), bictegravir (BIC), and cabotegravir (CAB) remain largely unknown. Here, in vitro drug selections compared the development of resistance to DTG, BIC, CAB, EVG and RAL using clinical isolates from treatment-naïve primary HIV infection (PHI) cohort participants (n = 12), and pNL4.3 recombinant strains encoding patient-derived Integrase with (n = 5) and without (n = 5) the E157Q substitution.ResultsPatient-derived viral isolates were serially passaged in PHA-stimulated cord blood mononuclear cells in the presence of escalating concentrations of INSTIs over the course of 36–46 weeks. Drug resistance arose more rapidly in primary clinical isolates with EVG (12/12), followed by CAB (8/12), DTG (8/12) and BIC (6/12). For pNL4.3 recombinant strains encoding patient-derived integrase, the comparative genetic barrier to resistance was RAL > EVG > CAB > DTG and BIC. The E157Q substitution in integrase delayed the advent of resistance to INSTIs. With EVG, T66I/A, E92G/V/Q, T97A or R263K (n = 16, 3, 2 and 1, respectively) arose by weeks 8–16, followed by 1–4 accessory mutations, conferring high-level resistance (> 100-fold) by week 36. With DTG and BIC, solitary R263K (n = 27), S153F/Y (n = 7) H51Y (n = 2), Q146 R (n = 3) or S147G (n = 1) mutations conferred low-level (< 3-fold) resistance at weeks 36–46. Similarly, most CAB selections (n = 18) resulted in R263K, S153Y, S147G, H51Y, or Q146L solitary mutations. However, three CAB selections resulted in Q148R/K followed by secondary mutations conferring high-level cross-resistance to all INSTIs. EVG-resistant viruses (T66I/R263K, T66I/E157Q/R263K, and S153A/R263K) retained residual susceptibility when switched to DTG, BIC or CAB, losing T66I by week 27. Two EVG-resistant variants developed resistance to DTG, BIC and CAB through the additional acquisition of E138A/Q148R and S230N, respectively. One EVG-resistant variant (T66I) acquired L74M/G140S/S147G, L74M/E138K/S147G and H51Y with DTG CAB and BIC, respectively.ConclusionsSecond generation INSTIs show a higher genetic barrier to resistance than EVG and RAL. The potency of CAB was lower than BIC and DTG. The development of Q148R/K with CAB can result in high-level cross-resistance to all INSTIs.

In vitro-induced erythromycin resistance facilitates cross-resistance to the novel fluoroketolide, solithromycin, in Staphylococcus aureus.

The aim of this study was to determine whether in vitro induced erythromycin resistance facilitates the cross-resistance to the novel fluoroketolide, solithromycin, in Staphylococcus aureus. Four strains of methicillin-susceptible S. aureus strains S2, S3, S5 and S7 were successfully induced to establish erythromycin-resistant strains by continuous in vitro culture with erythromycin. Mutations at drug binding sites were shown to increase the minimal inhibitory concentrations for ketolides, including telithromycin and the novel compound solithromycin, but did not increase for lincosamides, chloramphenicols or oxazolidinones. In S2-, S5- and S7-derived strains, L22 protein mutations occurred first, resulting in a low level of cross-resistance to ketolides (≤4 μg/mL). The L4 protein mutations were dependent on the L22 protein, resulting in high-level cross-resistance to ketolides (≥8 μg/mL). In S3-derived strains, high levels of cross-resistance occurred concurrently in the 23S rRNA domains II/V and the L22 protein. Hence, long-term exposure of erythromycin results in resistance to ketolides in S. aureus through drug binding site mutations. These results demonstrate that since erythromycin has been used clinically for a long time, it is necessary to carefully evaluate the rewards and risks when prescribing solithromycin for the treatment of infectious diseases.

Both point mutations and low expression levels of the nicotinic acetylcholine receptor β1 subunit are associated with imidacloprid resistance in an Aphis gossypii (Glover) population from a Bt cotton field in China

Aphis gossypii Glover is a destructive pest of numerous crops throughout the world. Although the expansion of Bt cotton cultivation has helped to control some insect pests, the damage from cotton aphids has not been mitigated. The evolution of aphid resistance to imidacloprid has made its chemical control more difficult since its introduction in 1991. Field populations of A. gossypii that were collected from different transgenic (Bt) cotton planting areas of China in 2014 developed different levels of resistance to imidacloprid. The IMI_R strain has developed high resistance to imidacloprid with the resistance ratio >1200-fold. Compared with the susceptible IMI_S strain, the IMI_R strain also developed a high level cross resistance to sulfoxaflor and acetamiprid. The limited synergism with either PBO or DEF suggests that resistance may be due to the site mutation of molecular target rather than to enhanced detoxification. Three target-site mutations within the nicotinic acetylcholine receptor (nAChR) β1 subunit were detected in the IMI_R strain. The R81T mutation has been reported to be responsible for imidacloprid resistance in A. gossypii and M. persicae. Both V62I and K264E were first detected in A. gossypii. These point mutations are also present in field populations, suggesting that they play a role in the resistance to imidacloprid. Furthermore, the expression level of transcripts encoding β1 subunit was decreased significantly in the IMI_R strain compared with the IMI_S strain, suggesting that both point mutations and the down-regulation of nAChR β1 subunit expression may be involved in the resistance mechanism for imidacloprid in A. gossypii. These results should be useful for the management of imidacloprid-resistant cotton aphids in Bt cotton fields in China.

Cross-Resistance between Cry1 Proteins in Fall Armyworm (Spodoptera frugiperda) May Affect the Durability of Current Pyramided Bt Maize Hybrids in Brazil.

Genetically modified plants expressing insecticidal proteins from Bacillus thuringiensis (Bt) offer valuable options for managing insect pests with considerable environmental and economic benefits. Despite the benefits provided by Bt crops, the continuous expression of these insecticidal proteins imposes strong selection for resistance in target pest populations. Bt maize (Zea mays) hybrids have been successful in controlling fall armyworm (Spodoptera frugiperda), the main maize pest in Brazil since 2008; however, field-evolved resistance to the protein Cry1F has recently been reported. Therefore it is important to assess the possibility of cross-resistance between Cry1F and other Cry proteins expressed in Bt maize hybrids. In this study, an F2 screen followed by subsequent selection on MON 89034 maize was used to select an S. frugiperda strain (RR) able to survive on the Bt maize event MON 89034, which expresses the Cry1A.105 and Cry2Ab2 proteins. Field-collected insects from maize expressing the Cry1F protein (event TC1507) represented most of the positive (resistance allele-containing) (iso)families found. The RR strain showed high levels of resistance to Cry1F, which apparently also conferred high levels of cross resistance to Cry1A.105 and Cry1Ab, but had only low-level (10-fold) resistance to Cry2Ab2. Life history studies to investigate fitness costs associated with the resistance in RR strain revealed only small reductions in reproductive rate when compared to susceptible and heterozygous strains, but the RR strain produced 32.2% and 28.4% fewer females from each female relative to the SS and RS (pooled) strains, respectively. Consistent with the lack of significant resistance to Cry2Ab2, MON 89034 maize in combination with appropriate management practices continues to provide effective control of S. frugiperda in Brazil. Nevertheless, the occurrence of Cry1F resistance in S. frugiperda across Brazil, and the cross-resistance to Cry1Ab and Cry1A.105, indicates that current Cry1-based maize hybrids face a challenge in managing S. frugiperda in Brazil and highlights the importance of effective insect resistance management for these technologies.

Integrase inhibitor (INI) genotypic resistance in treatment-naive and raltegravir-experienced patients infected with diverse HIV-1 clades.

ObjectivesThe aim of this study was to characterize the prevalence and patterns of genotypic integrase inhibitor (INI) resistance in relation to HIV-1 clade.MethodsThe cohort comprised 533 INI-naive subjects and 255 raltegravir recipients with viraemia who underwent integrase sequencing in routine care across Europe, including 134/533 (25.1%) and 46/255 (18.0%), respectively, with non-B clades (A, C, D, F, G, CRF01, CRF02, other CRFs, complex).ResultsNo major INI resistance-associated mutations (RAMs) occurred in INI-naive subjects. Among raltegravir recipients with viraemia (median 3523 HIV-1 RNA copies/mL), 113/255 (44.3%) had one or more major INI RAMs, most commonly N155H (45/255, 17.6%), Q148H/R/K + G140S/A (35/255, 13.7%) and Y143R/C/H (12/255, 4.7%). In addition, four (1.6%) raltegravir recipients showed novel mutations at recognized resistance sites (E92A, S147I, N155D, N155Q) and novel mutations at other integrase positions that were statistically associated with raltegravir exposure (K159Q/R, I161L/M/T/V, E170A/G). Comparing subtype B with non-B clades, Q148H/R/K occurred in 42/209 (20.1%) versus 2/46 (4.3%) subjects (P = 0.009) and G140S/A occurred in 36/209 (17.2%) versus 1/46 (2.2%) subjects (P = 0.005). Intermediate- to high-level cross-resistance to twice-daily dolutegravir was predicted in 40/255 (15.7%) subjects, more commonly in subtype B versus non-B clades (39/209, 18.7% versus 1/46, 2.2%; P = 0.003). A glycine (G) to serine (S) substitution at integrase position 140 required one nucleotide change in subtype B and two nucleotide changes in all non-B clades.ConclusionsNo major INI resistance mutations occurred in INI-naive subjects. Reduced occurrence of Q148H/R/K + G140S/A was seen in non-B clades versus subtype B, and was explained by the higher genetic barrier to the G140S mutation observed in all non-B clades analysed.

The R263K substitution in HIV-1 subtype C is more deleterious for integrase enzymatic function and viral replication than in subtype B

Dolutegravir is an integrase strand-transfer inhibitor that has shown unprecedented robustness against the emergence of HIV drug-resistant strains in treatment-naive individuals. The R263K substitution in integrase was identified through culture selection as a resistance-associated substitution for dolutegravir and was recently detected in two treatment-experienced participants in the SAILING clinical trial, who experienced dolutegravir-based treatment failure, one of whom was infected by a subtype C virus. The objective of this study was to characterize the R263K substitution in HIV-1 subtype C integrase. We used cell-free strand transfer assays and tissue culture experiments to characterize the R263K substitution in HIV-1 subtype C integrase in comparison with subtype B. Cell-free biochemical assays showed that the R263K substitution diminished subtype C integrase strand-transfer activity by decreasing the affinity of integrase for target DNA. Similarly, both viral infectiousness and replication capacity were reduced by the R263K substitution in tissue culture. Decrease in enzyme activity and viral infectiousness exceeded 35 and 50%, respectively - significantly more than in HIV-1 subtype B. R263K in HIV-1 subtype C also conferred low levels of resistance against dolutegravir and high levels of cross-resistance against elvitegravir, but not raltegravir. The R263K substitution is more deleterious to integrase strand-transfer activity and viral infectiousness in HIV-1 subtype C than in subtype B. Our results suggest that cross-resistance may prevent treatment-experienced individuals who are experiencing treatment failure with dolutegravir from being subsequently treated with elvitegravir.

Over-expression of CYP6A2 is associated with spirotetramat resistance and cross-resistance in the resistant strain of Aphis gossypii Glover

A laboratory-selected spirotetramat-resistant strain (SR) of cotton aphid developed 579-fold and 15-fold resistance to spirotetramat in adult aphids and 3rd instar nymphs, respectively, compared with a susceptible strain (SS) [26]. The SR strain developed high-level cross-resistance to alpha-cypermethrin and bifenthrin and very low or no cross-resistance to the other tested insecticides. Synergist piperonyl butoxide (PBO) dramatically increased the toxicity of spirotetramat and alpha-cypermethrin in the resistant strain. RT-qPCR results demonstrated that the transcriptional levels of CYP6A2 increased significantly in the SR strain compared with the SS strain, which was consistent with the transcriptome results [30]. The depletion of CYP6A2 transcripts by RNAi also significantly increased the sensitivity of the resistant aphid to spirotetramat and alpha-cypermethrin. These results indicate the possible involvement of CYP6A2 in spirotetramat resistance and alpha-cypermethrin cross-resistance in the cotton aphid. These together with other cross-resistance results have implications for the successful implementation of resistance management strategies for Aphis gossypii.

Genotypic susceptibility testing of Mycobacterium tuberculosis isolates for amikacin and kanamycin resistance by use of a rapid sloppy molecular beacon-based assay identifies more cases of low-level drug resistance than phenotypic Lowenstein-Jensen testing.

Resistance to amikacin (AMK) and kanamycin (KAN) in clinical Mycobacterium tuberculosis strains is largely determined by specific mutations in the rrs gene and eis gene promoter. We developed a rapid, multiplexed sloppy molecular beacon (SMB) assay to identify these mutations and then evaluated assay performance on 603 clinical M. tuberculosis DNA samples collected in South Korea. Assay performance was compared to gold-standard phenotypic drug susceptibility tests, including Lowenstein-Jensen (LJ) absolute concentration, mycobacterial growth indicator tubes (MGIT), and TREK Sensititre MycoTB MIC plate (MycoTB) methods. Target amplicons were also tested for mutations by Sanger sequencing. The SMB assay correctly detected 115/116 mutant and mixed sequences and 487/487 wild-type sequences (sensitivity and specificity of 99.1 and 100%, respectively). Using the LJ method as the reference, sensitivity and specificity for AMK resistance were 92.2% and 100%, respectively, and sensitivity and specificity for KAN resistance were 87.7% and 95.6%, respectively. Mutations in the rrs gene were unequivocally associated with high-level cross-resistance to AMK and KAN in all three conventional drug susceptibility testing methods. However, eis promoter mutations were associated with KAN resistance using the MGIT or MycoTB methods but not the LJ method. No testing method associated eis promoter mutations with AMK resistance. Among the discordant samples with AMK and/or KAN resistance but wild-type sequence at the target genes, we discovered four new mutations in the whiB7 5' untranslated region (UTR) in 6/22 samples. All six samples were resistant only to KAN, suggesting the possible role of these whiB7 5' UTR mutations in KAN resistance.

High-level cross-resistance to didanosine observed in South African children failing an abacavir- or stavudine-based 1st-line regimen.

BackgroundThe knowledge-base of emerging drug resistance profiles in children exposed to abacavir-based antiretroviral regimens in South Africa is very limited. This study investigated the suitability of didanosine-based 2nd-line regimens for children in the context of antiretroviral drug resistance patterns emerging after 1st-line virologic failure.MethodsA retrospective dataset of 354 antiretroviral drug resistant genotypes from children failing either abacavir (n = 81) or stavudine (n = 273) based 1st-line regimens, was analysed. Samples were sent to the HIV genotyping laboratory at Charlotte Maxeke Johannesburg Academic Hospital, for routine testing. Pol sequences were submitted to the Stanford HIV drug resistance database for genotypic predictions.ResultsChildren were exposed to abacavir or stavudine-based 1st-line regimens for an average of 21 and 36 months, respectively. The frequency of reduced susceptibility to didanosine was substantial in the abacavir-exposed group (69.1%).This reduced susceptibility was commonly attributed to L74V/I (n = 44) and to a lesser extent K65R (n = 10) mutations. Didanosine resistance was observed in 43.2% of patients exposed to stavudine-based regimens. In contrast, most children remained susceptible to stavudine regardless of exposure to abacavir (77.8%) or stavudine (74.7%). At least 80% of children remained susceptible to zidovudine irrespective of stavudine or abacavir-exposure. The presence of the K65R mutation was more common after abacavir pressure (12.3% vs 1.8%).ConclusionAnalysis revealed that didanosine-based 2nd-line regimens have limitations for South African children, given the high frequency of mutations that confer cross-resistance to didanosine; especially after abacavir-exposure. This data has influenced South African paediatric treatment guidelines, which now recommend zidovudine-based 2nd-line regimens.

Mechanisms of Anti-retroviral Drug Resistance: Implications for Novel Drug Discovery and Development

Anti-retroviral drug resistance evolves as an inevitable consequence of expanded combination Anti-retroviral Therapy (cART). According to each drug class, resistance mutations may occur due to the infidel nature of HIV reverse transcriptase (RT) and inadequate drug pressures. Correspondingly, resistance to Nucleoside Reverse Transcriptase Inhibitors (NRTIs) occurs due to incorporation impairment of the agent or its removal from the elongating viral DNA chain. With regard to Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), resistance mutations may alter residues of the RT hydrophobic pocket and demonstrate high level of cross resistance. However, resistance to Protease Inhibitors requires complex accumulation of primary and secondary mutations that substitute amino acids in proximity to the viral protease active site. Resistance to novel entry inhibitors may also evolve as a result of mutations that affect the interactions between viral glycoprotein and CD4 or the chemokine receptors. According to the current studies, future drug initiative programs should consider agents that possess higher genetic barrier toward resistance for ascertaining adequate drug efficacy among patients who have failed first-line regimens.

Target-site resistance to pyrethroid insecticides in German populations of the cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae)

Cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae) is a major pest of winter oilseed rape in several European countries particularly attacking young emerging plants in autumn. Over the last several decades, pyrethroid insecticides have been foliarly applied to control flea beetle outbreaks. Recent control failures in northern Germany suggested pyrethroid resistance development in cabbage stem flea beetles, which were confirmed by resistance monitoring bioassays using lambda-cyhalothrin in an adult vial test. The purpose of this study was to investigate the presence of polymorphisms in the para-type voltage-gated sodium channel gene of P. chrysocephala known to be involved in knock-down resistance (kdr). By using a degenerate primer approach we PCR amplified part of the para-type sodium channel gene and identified in resistant flea beetles a single nucleotide polymorphism resulting in an L1014F (kdr) mutation within domain IIS6 of the channel protein, known as one of the chief pyrethroid target-site resistance mechanisms in several other pest insects. Twenty populations including four archived museum samples collected between 1945 and 1958 were analyzed using a newly developed pyrosequencing diagnostic assay. The assay revealed a kdr allele frequency of 90–100% in those flea beetle populations expressing high-level cross-resistance in discriminating dose bioassays against different pyrethroids such as lambda-cyhalothrin, tau-fluvalinate, etofenprox and bifenthrin. The presence of target-site resistance to pyrethroids in cabbage stem flea beetle is extremely worrying considering the lack of effective alternative modes of action to control this pest in Germany and other European countries, and is likely to result in major control problems once it expands to other geographies. The striking fact that cabbage stem flea beetle is next to pollen beetle, Meligethes aeneus the second coleopteran pest in European winter oilseed rape resisting pyrethroid treatments by expressing a target-site mutation, underpins the importance of diversity in available chemistry for resistance management tactics based on mode of action rotation in order to guarantee sustainable winter oilseed rape cultivation in Europe.

Cross-resistance, inheritance and stability of resistance to acetamiprid in cotton whitefly, Bemisia tabaci Genn (Hemiptera: Aleyrodidae)

The cotton whitefly Bemisia tabaci, (Genn.) is an important pest of field crops, vegetables and ornamentals worldwide. Neonicotinoids are considered an important group of insecticides being used against B. tabaci for several years . B. tabaci has developed resistance to some of the compounds of the group. This study was designed to investigate if the selection of B. tabaci with acetamiprid would give a broad-spectrum of cross-resistance and to genetically classify the resistance. At G 1 a low level of resistance to acetamiprid, imidacloprid, thiamethoxam, thiacloprid and nitenpyram was observed with resistance ratios of 3-fold, 8-, 9-, 6- and 5-fold, respectively, compared with a laboratory susceptible population. After selection for eight generations with acetamiprid, resistance to acetamiprid increased to 118-fold compared with the laboratory susceptible population. Selection also increased resistance to imidacloprid, thiamethoxam, thiacloprid, nitenpyram, endosulfan and bifenthrin but no change in susceptibility to fipronil was observed. Furthermore resistance in a field population was stable in the absence of acetamiprid selection pressure. Genetic crosses between resistant and susceptible populations indicated autosomal and incompletely recessive resistance. Further genetic analysis suggested that resistance could be controlled by a single factor. The high level of cross-resistance and stability of incomplete resistance in the field population is of some concern. However, lack of cross-resistance between acetamiprid and fipronil or unstable resistance in the resistant population could provide options to use alternative products which could reduce acetamiprid selection pressure.

Documentation of In Vivo and In Vitro Aerobic Resistance of Feline Tritrichomonas foetus Isolates to Ronidazole

The mainstays of treatment for clinically important trichomonad infections are the 5-nitroimidazoles. Metronidazole resistance of feline Tritrichomonas foetus is presumed because of common treatment failure, and tinidazole does not consistently eradicate infection. To date, ronidazole is the only drug demonstrated as effective for treatment of cats infected with T. foetus. To document in vivo treatment failure and identify underlying causes and in vitro conditions of resistance of feline T. foetus to ronidazole. Two intact male Abyssinians failing>or=5 courses of treatment with increasing doses of 5-nitroimidazole drugs. An intact male Abyssinian documented to clear infection after treatment with a single course of ronidazole. T. foetus isolates were cultured from feces and tested in vitro for susceptibility to ronidazole under aerobic and anaerobic culture conditions. A urogenital nidus of T. foetus infection was investigated by culture, polymerase chain reaction, or immunohistochemical testing of urogenital specimens. Resistance to ronidazole under aerobic conditions was uniquely identified in T. foetus isolated from cats with well-documented treatment failure. Treatment failure could not be attributed to reinfection, inappropriate treatment protocol, or presence of a urogenital nidus of infection. Clinical resistance to metronidazole, low efficacy of tinidazole, and present documentation of in vivo and in vitro resistance to ronidazole in some cats are consistent with a high level of cross resistance of feline T. foetus to 5-nitroimidazole drugs. Current lack of alternative drugs with clinical efficacy against feline T. foetus suggests that active investigation of other treatment approaches is warranted.

High Level of Cross-Resistance between Kanamycin, Amikacin, and Capreomycin among Mycobacterium tuberculosis Isolates from Georgia and a Close Relation with Mutations in the rrs Gene

The aminoglycosides kanamycin and amikacin and the macrocyclic peptide capreomycin are key drugs for the treatment of multidrug-resistant tuberculosis (MDR-TB). The increasing rates of resistance to these drugs and the possible cross-resistance between them are concerns for MDR-TB therapy. Mutations in the 16S rRNA gene (rrs) have been associated with resistance to each of the drugs, and mutations of the tlyA gene, which encodes a putative rRNA methyltransferase, are thought to confer capreomycin resistance in Mycobacterium tuberculosis bacteria. Studies of possible cross-resistance have shown variable results. In this study, the MICs of these drugs for 145 clinical isolates from Georgia and the sequences of the rrs and tlyA genes of the isolates were determined. Of 78 kanamycin-resistant strains, 9 (11.5%) were susceptible to amikacin and 16 (20.5%) were susceptible to capreomycin. Four strains were resistant to capreomycin but were susceptible to the other drugs, whereas all amikacin-resistant isolates were resistant to kanamycin. Sequencing revealed six types of mutations in the rrs gene (A514C, C517T, A1401G, C1402T, C1443G, T1521C) but no mutations in the tlyA gene. The A514C, C517T, C1443G, and T1521C mutations showed no association with resistance to any of the drugs. The A1401G and C1402T mutations were observed in 65 kanamycin-resistant isolates and the 4 capreomycin-resistant isolates, respectively, whereas none of the susceptible isolates showed either of those mutations. The four mutants with the C1402T mutations showed high levels of resistance to capreomycin but no resistance to kanamycin and amikacin. Detection of the A1401G mutation appeared to be 100% specific for the detection of resistance to kanamycin and amikacin, while the sensitivities reached 85.9% and 94.2%, respectively.

On the relationship between drug’s size, cell membrane mechanical properties and high levels of multi drug resistance: a comparison to published data

Multi drug resistance (MDR) or cross resistance to drugs was initially explained on the basis that MDR cells express drug transporters that expel membrane-embedded drugs. However, it is now clear that transporters are a single piece from a complex puzzle. An issue that has been solved recently is, given that these transporters have to handle drugs, why should a membrane-embedded drug and a transporter meet? To solve this problem, a theory has been suggested considering the interaction between the cell membrane mechanical properties and the size of drugs. In simple terms, this theory proposes that an excess in the packing of lipid in the inner leaflet of the membrane of MDR cells is responsible for blocking drugs mechanically as a function of their sizes at the membrane level, thus impairing their flux into the cytosol. In turn it is expected that this would allow any membrane embedded drug to diffuse toward transporters. The study concluded that the size of drugs is necessarily important regarding the mechanical interaction between the drug and the membrane, and likely to be central to MDR. Remarkably, an experimental study based on MDR and published years ago concluded that the molecular weight (MW) of drugs was central to MDR (Biedler and Riehm in Cancer Res 30:1174-1184, 1970). Given that size and MW are linked together, I have compared the former theory to the latter experimental data and demonstrate that, indeed, basic membrane mechanics is involved in high levels of cross resistance to drugs in Pgp expressing cells.

Second generation HIV protease inhibitors against resistant virus

Background: The AIDS epidemic has spread around the world at an alarming rate. Although the first generation of HIV protease inhibitors, including indinavir, nelfinavir, saquinavir, ritonavir and amprenavir, were initially effective against HIV infection, the fast emerging resistance to these agents has been a substantial and persistent problem in the treatment of AIDS. Attempts to address the resistance issue with ‘salvage therapy’ consisting of high doses of multiple protease inhibitors have only been moderately successful owing to the high level of cross-resistance and toxicities associated with the protease inhibitors. Objective: To study the second generation HIV protease inhibitors against resistant virus. Method: This review highlights new developments achieved by various organizations to address the challenge of high level resistance of current therapies since 2000. Conclusion: All second generation protease inhibitors used in patients who experienced extensive treatment require ritonavir as a pharmacological boosting agent to increase the drug level in the plasma, but there is toxicity associated with such a practice. Accordingly, there remains a need for new protease inhibitors with improved effectiveness against the resistant viral variants. A third generation protease inhibitor will require no boosting agent while maintaining high potency against resistant virus.

Application of normalised resistance interpretation to disc diffusion data on the susceptibility of Aeromonas salmonicida to three quinolone agents

The susceptibilities of 106 strains of Aeromonas salmonicida to oxolinic acid (OXA), flumequine (FLU) and enrofloxacin (ENR) were established in two independent laboratories, using the M42-A disc diffusion protocols of the Clinical and Laboratory Standards Institute. The zone sizes obtained for OXA and FLU by both laboratories showed a bi-modal distribution. For OXA there was clear separation of the two modal classes but for FLU the zones for the two classes showed a degree of overlap. In contrast, there was no clear evidence of bi-modality in the data obtained for ENR.Normalised resistance interpretation (NRI) was used to calculate laboratory-specific epidemiological cut-off values for wild type (WT) strains for all three agents. For OXA these cut-off values were 30 mm and 33 mm for the two laboratories and their application resulted in a complete concordance in the classification of the strains by the two laboratories. For FLU the NRI cut-off values were 39 mm and 40 mm and these resulted in an agreement between the laboratories in the classification of 95% of the 106 strains. The NRI cut-off values for ENR were 36 m and 40 mm and resulted in an agreement with the classification of 90% of these strains. When the zone sizes for the two modal classes overlap some strains with non wild type (NWT) phenotypes would generate zones larger than the WT cut-off value set by NRI analysis. Thus, definitely for ENR and possibly for FLU, the use of NRI cut-off values as provisional breakpoints could lead to some NWT strains being reported as sensitive.Analysis of the data obtained in this work demonstrated that the frequency of cross-resistance between the three agents was high and this is consistent with other reports in the literature. This high level of cross resistance provides a method by which the problems with the cut-off values set by NRI analysis can be avoided. It is argued that the use of OXA as a reporter agent represent an effective method of reducing error in reporting susceptibility to ENR and FLU. In this approach all strains shown to be NWT with respect to OXA would also be reported as NWT with respect to the other quinolones. The validity of this approach is dependent on there being a very low frequency of strains that are resistant to ENR and FLU but sensitive to OXA.It is recommended that, at the present state of our knowledge, the most effective method of reducing errors in determining the susceptibility of strains of A. salmonicida to quinolone agents is to employ OXA disc data as a proxy measure for susceptibility to the other agents in this class.

Development of Culex quinquefasciatus resistance to Bacillus sphaericus strain IAB59 needs long term selection pressure

Abstract Culex quinquefasciatus may exhibit resistance to the biolarvicide Bacillus sphaericus (Bsp), when subjected to high selection pressure. The major insecticidal factor present in most Bsp products is the binary (Bin) toxin. The IAB59 strain, in particular, has attracted attention, since it shows another toxic factor, besides the Bin toxin, and this feature is likely to decrease the risk for selecting resistance. The aim of this study was to investigate whether it was possible to select a Cx. quinquefasciatus colony (CqRL2/IAB59) for high level resistance towards Bsp strain IAB59, under laboratory conditions. Selection performed by treating groups of about 15,000 third and fourth instar larvae from each generation with high concentrations of Bsp IAB59 yielded a 70% average pre-imaginal mortality. The CqRL2/IAB59 colony reached a high level of resistance (≈40,000-fold), after 72 generations. This colony also displayed a high level of cross-resistance to strain 2362, close to 70,000-fold, while larvae remained susceptible to Bti. Cross-resistance to strain 2362 was observed before the colony had attained a high level of resistance to strain IAB59. Data from the reciprocal crosses, between individuals from the susceptible and the resistant colonies, showed that resistance is inherited as a recessive trait, since the LC50 of Bsp IAB59 towards F1 larvae were similar to that of the susceptible parental colony. Data from this study showed that continuous selection pressure with Bsp IAB59 can select Culex resistant populations, although the evolution of resistance is much slower, than formerly observed for the commercial 2362 strain.