Methicillin-resistant S. aureus (MRSA) has been endemic in Hospital Universitari de Bellvitge, Barcelona, since 1990. During the 1990-95 period the Iberian clone (ST-247; SCCmec-I) was dominant. Isolates of clonal complex 5 (ST-125; SCCmec-IV) gradually replaced the Iberian clone from 1996 to 2003. A new multiresistant MRSA phenotype showing rifampicin resistance emerged in 2004 and rapidly increased from 25% in 2004 to 45% in 2006. The aims of this study were i) the molecular characterisation of rifampicin resistant MRSA isolates, ii) the study of the rifampicin resistance expression by disk diffusion, microdilution and E-test, and iii) the analysis of the rpoB gene mutations involved in rifampicin resistance.
A sample of representative 108 rifampicin-resistant MRSA isolates belonged to a single PFGE genotype, ST-228, SCCmec type I and spa type t041. Of 108 isolates, 104 (96%) had a low-level rifampicin resistance (MICs, 2 to 4 mg/L) and 4 a high-level rifampicin resistance (MICs, 128 - ≥ 256 mg/L). Disk diffusion and E-test methods failed to identify a low-level rifampicin resistance in 20 and 12 isolates, respectively. A low-level rifampicin resistance was associated with amino acid substitution 481His/Asn in the beta-subunit of RNA polymerase. Isolates with a high-level rifampicin resistance carried additional mutations in the rpoB gene.
The emergence of MRSA clone ST228-SCCmecI, related to the Southern Germany clone, involved a therapeutical challenge for treating serious MRSA infections. Decreased susceptibility to rifampicin in MRSA strains of ST228-SCCmecI was associated with one or two specific mutations in the rpoB gene. One fifth of isolates with low-level rifampicin-resistance were missed by the diffusion methods.
Central dogma suggests that rifampicin resistance in Mycobacterium tuberculosis develops solely through rpoB gene mutations.
To determine whether rifampicin induces efflux pumps activation in rifampicin resistant M. tuberculosis strains thereby defining rifampicin resistance levels and reducing ofloxacin susceptibility.
Rifampicin and/or ofloxacin minimum inhibitory concentrations (MICs) were determined in rifampicin resistant strains by culture in BACTEC 12B medium. Verapamil and reserpine were included to determine their effect on rifampicin and ofloxacin susceptibility. RT-qPCR was applied to assess expression of efflux pump/transporter genes after rifampicin exposure. To determine whether verapamil could restore susceptibility to first-line drugs, BALB/c mice were infected with a MDR-TB strain and treated with first-line drugs with/without verapamil.
Measurements and Main Findings
Rifampicin MICs varied independently of rpoB mutation and genetic background. Addition reserpine and verapamil significantly restored rifampicin susceptibility (p = 0.0000). RT-qPCR demonstrated that rifampicin induced differential expression of efflux/transporter genes in MDR-TB isolates. Incubation of rifampicin mono-resistant strains in rifampicin (2 μg/ml) for 7 days induced ofloxacin resistance (MIC> 2 μg/ml) in strains with an rpoB531 mutation. Ofloxacin susceptibility was restored by exposure to efflux pump inhibitors. Studies in BALB/c mice showed that verapamil in combination with first-line drugs significantly reduced pulmonary CFUs after 1 and 2 months treatment (p < 0.05).
Exposure of rifampicin resistant M. tuberculosis strains to rifampicin can potentially compromise the efficacy of the second-line treatment regimens containing ofloxacin, thereby emphasising the need for rapid diagnostics to guide treatment. Efflux pump inhibitors have the potential to improve the efficacy of anti-tuberculosis drug treatment.
Mycobacterium tuberculosis; drug resistance; rifampicin; efflux pumps; cross resistance
Rifampicin is currently recognized as the most potent drug against Gram positive implant related infections. The use of rifampicin is limited by the emergence of bacterial resistance, which is often managed by coadministration of a second antibiotic. The purpose of this study was to determine the effectiveness of soluble rifampicin in combination with vancomycin tethered to titanium metal as a means to control bacterial growth and resistance in vitro. Bacterial growth was inhibited when the vancomycin-tethered titanium discs were treated with Staphylococcus aureus inocula of ≤2×106 CFU, however inocula greater than 2×106 CFU/disc adhered and survived. The combination of surface-tethered vancomycin with soluble rifampicin enhanced the inhibitory effect of rifampicin for an inoculum of 106 CFU/cm2 by one dilution (combination MIC of 0.008 mg/L versus 0.015 mg/L for rifampicin alone). Moreover, surface tethered vancomycin prevented the emergence of a rifampicin resistant population in an inoculum of 2×108 CFU.
Since 2001, several studies have reported high rifampicin resistance rates (45 - 100%) among methicillin-resistant Staphylococcus aureus (MRSA) isolates from South Africa. The authors previously characterised 100 MRSA isolates from hospitals in Cape Town, South Africa; forty-five percent of these isolates were rifampicin-resistant. The majority (44/45) corresponded to ST612-MRSA-IV, which is prevalent in South Africa, but has not been reported frequently elsewhere. The remaining rifampicin-resistant isolate corresponded to ST5-MRSA-I. The aim of this study was to investigate further the prevalence and genetic basis of rifampicin-resistance in MRSA isolates from hospitals in Cape Town.
Between July 2007 and June 2011, the prevalence of rifampicin-resistant MRSA in hospitals in Cape Town ranged from 39.7% to 46.4%. Based on the results of the aforementioned study, nine ST612-MRSA-IV isolates, the rifampicin-resistant ST5-MRSA-I isolate, and two rifampicin-susceptible MRSA isolates were investigated. Four previously described ST612-MRSA-IV isolates, including two each from South Africa and Australia, were also included.
The ST5-MRSA-I isolate carried a single mutational change, H481Y, commonly associated with high-level rifampicin resistance. All ST612-MRSA-IV isolates carried an uncommon double amino acid substitution in RpoB, H481N, I527M, whilst one of the Australian ST612-MRSA-IV isolates carried an additional mutation within rpoB, representing a novel rpoB genotype: H481N, I527M, K579R. All ST612-MRSA-IV isolates also shared a unique silent single nucleotide polymorphism (SNP) within rpoB.
That local ST612-MRSA-IV isolates described here share an uncommon rpoB genotype and a unique silent SNP suggests this clone may have undergone clonal expansion in hospitals in Cape Town. Further, the data suggest that these isolates may be related to rifampicin-resistant ST612-MRSA-IV previously described in South Africa and Australia.
Rifaximin is a rifampicin derivative, poorly absorbed by the gastro-intestinal tract. We studied the in vitro susceptibility to rifamixin of 1082 Clostridium difficile isolates; among these,184 isolates from a strain collection were tested by an in-house rifaximin disc (40 µg) diffusion test, by an in-house rifaximin broth microdilution test, by rifampicin Etest and by rpoB gene sequencing. In the absence of respective CLSI or EUCAST MIC breakpoints for rifaximin and rifampicin against C. difficile we chose MIC ≥32 µg ml−1 as criterion for reduced in vitro susceptibility. To further validate the disc diffusion test 898 consecutive clinical isolates were analysed using the disc diffusion test, the Etest and rpoB gene sequence analysis for all resistant strains. Rifaximin broth microdilution tests of the 184 reference strains yielded rifaximin MICs ranging from 0.001 (n = 1) to ≥1024 µg ml−1 (n = 61); 62 isolates showed a reduced susceptibility (MIC ≥32 µg ml−1). All of these 62 strains showed rpoB gene mutations producing amino acid substitutions; the rifampicin- and rifaximin-susceptible strains showed either a wild-type sequence or silent amino acid substitutions (19 strains). For 11 arbitrarily chosen isolates with rifaximin MICs of >1024 µg ml−1, rifaximin end-point MICs were determined by broth dilution: 4096 µg ml−1 (n = 2), 8192 µg ml−1 (n = 6), 16 384 µg ml−1 (n = 2) and 32 678 µg ml−1 (n = 1). Rifampicin Etests on the 184 C. difficile reference strains yielded MICs ranging from ≤0.002 (n = 117) to ≥32 µg ml−1 (n = 59). Using a 38 mm inhibition zone as breakpoint for reduced susceptibility the use of rifaximin disc diffusion yielded 59 results correlating with those obtained by use of rifaximin broth microdilution in 98.4 % of the 184 strains tested. Rifampicin Etests performed on the 898 clinical isolates revealed that 67 isolates had MICs of ≥32 µg ml−1. There were no discordant results observed among these isolates with reduced susceptibility using an MIC of ≥32 µg ml−1 as breakpoint for reduced rifampicin susceptibility and a <38 mm inhibition zone as breakpoint for reduced rifaximin susceptibility. The prevalence of reduced susceptibility was 7.5 % for all isolates tested. However, for PCR ribotype 027 the prevalence of reduced susceptibility was 26 %. Susceptibility testing in the microbiology laboratory therefore could have an impact on the care and outcome of patients with infection. Our results show that rifaximin – despite its water-insolubility – may be a suitable candidate for disc diffusion testing.
Mutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations.
Mycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 μg/ml but remained sensitive to rifabutin (MIC<0.8 μg/ml). This mutant was subjected to a second round of selection with rifabutin.
All 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain.
First round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.
Staphylococcus aureus is an important pathogen causing a wide range of infections in the hospital and community setting. In order to have adequate information for treatment of S. aureus infections, it is crucial to understand the trends in the antibiotic-resistance patterns. In addition, the occurrence and changes in types of S. aureus, clonal identities, and their geographic spread is essential for the establishment of adequate infection control programmes. In this study, 68 S. aureus isolates obtained from clinical and non-clinical sources in Nigeria between January and April 2009 were characterized using phenotypic and molecular methods.
All the S. aureus isolates were susceptible to teicoplanin, vancomycin, phosphomycin, fusidic acid, rifampicin, daptomycin, mupirocin, linezolid and tigecycline. Sixteen percent of the isolates were resistant to oxacillin, while 55% and 72% of isolates were resistant to tetracycline and trimethoprim/sulphamethoxazole (cotrimoxazole), respectively (Table 1). There was excellent correlation between the broth microdilution assay and detection of antibiotic resistance genes by the multiplex PCR, in the determination of S. aureus resistance to erythromycin, gentamicin, methicillin and tetracycline. A total of 28 spa types were identified in the study, and the predominant spa type among the methicillin-susceptible S. aureus (MSSA) isolates was t084 (13 isolates). The t037-ST241-SCCmecIII type was the only clone identified in Maiduguri (North-East Nigeria) while in South-West Nigeria, diversity among the MRSA isolates (t451-ST8-SCCmecV; t008-ST94-SCCmecIV; t002-ST5-SCCmecV; t064-ST8-SCCmecV) was observed. The toxin genes seh and etd were detected in isolates affiliated with clonal complexes CC1, CC80 and sequence type ST25, respectively. The proportion of PVL-positive isolates among MSSA was high (40%). Most of the PVL-positive MSSA isolates were obtained from wound infections and associated with clonal complexes CC1, CC30, CC121 and with sequence type ST152.
Antibiotic resistance profile of S. aureus (MSSA and MRSA) from Nigeria
The use of phenotypic and molecular methods provided useful information on antibiotic resistance and molecular diversity of S. aureus in Nigeria. The high proportion of PVL-positive MSSA isolates affiliated to various clonal complexes and detected in all the health institutions is a major concern, both as a source of severe infections and as a potential reservoir that could lead to the emergence of PVL-positive MRSA. This study presents the first baseline information on the nature of the antibiotic resistance genes from S. aureus isolates in Nigeria. There is the need to curtail the spread and establishment of MRSA and PVL-positive MSSA clones in Nigerian health care institutions.
Genetically related Mycobacterium tuberculosis strains with alterations at codon 516 in the rpoB gene were observed amongst a substantial number of patients with drug resistant tuberculosis in the Eastern Cape Province (ECP) of South Africa. Mutations at codon 516 are usually associated with lower level rifampicin (RIF) resistance, while susceptibility to rifabutin (RFB) remains intact. This study was conducted to assess the rationale for using RFB as a substitution for RIF in the treatment of MDR and XDR tuberculosis outbreaks. Minimum inhibitory concentrations (MICs) of 34 drug resistant clinical isolates of M tuberculosis were determined by MGIT 960 and correlated with rpoB mutations. RFB MICs ranged from 0.125 to 0.25 µg/ml in the 34 test isolates thereby confirming phenotypic susceptibility as per critical concentration (CC) of 0.5 µg/ml. The corresponding RIF MICs ranged between 5 and 15 µg/ml, which is well above the CC of 1.0 µg/ml. Molecular-based drug susceptibility testing provides important pharmacogenetic insight by demonstrating a direct correlation between defined rpoB mutation and the level of RFB susceptibility. We suggest that isolates with marginally reduced susceptibility as compared to the epidemiological cut-off for wild-type strains (0.064 µg/ml), but lower than the current CC (≤0.5 µg/ml), are categorised as intermediate. Two breakpoints (0.064 µg/ml and 0.5 µg/ml) are recommended to distinguish between susceptible, intermediate and RFB resistant strains. This concept may assist clinicians and policy makers to make objective therapeutic decisions, especially in situations where therapeutic options are limited. The use of RFB in the ECP may improve therapeutic success and consequently minimise the risk of ongoing transmission of drug resistant M. tuberculosis strains.
Resistance determinants that interfere with normal physiological processes in the bacterial cell usually cause a reduction in biological fitness. Fitness assays revealed that 17 of 18 in vitro-selected chromosomal mutations within the rpoB gene accounting for rifampin resistance in Staphylococcus aureus were associated with a reduction in the level of fitness. There was no obvious correlation between the level of resistance to rifampin and the level of fitness loss caused by rpoB mutations. Among 23 clinical rifampin-resistant S. aureus isolates from six countries, only seven different rpoB genotypes could be identified, whereby the mutation 481His→Asn was present in 21 (91%) of these 23 isolates. The mutation 481His→Asn, in turn, which confers low-level rifampin resistance on its own, was not shown to be associated with a cost of resistance in vitro. The restriction to distinct mutations that confer rifampin resistance in vivo, as demonstrated here, appears to be determined by the Darwinian fitness of the organisms.
There is evidence from previous works that bacterial secondary metabolism may be stimulated by genetic manipulation of RNA polymerase (RNAP). In this study we have used rifampicin selection as a strategy to genetically improve the erythromycin producer Saccharopolyspora erythraea.
Spontaneous rifampicin-resistant (rif) mutants were isolated from the parental strain NRRL2338 and two rif mutations mapping within rpoB, S444F and Q426R, were characterized. With respect to the parental strain, S444F mutants exhibited higher respiratory performance and up to four-fold higher final erythromycin yields; in contrast, Q426R mutants were slow-growing, developmental-defective and severely impaired in erythromycin production. DNA microarray analysis demonstrated that these rif mutations deeply changed the transcriptional profile of S. erythraea. The expression of genes coding for key enzymes of carbon (and energy) and nitrogen central metabolism was dramatically altered in turn affecting the flux of metabolites through erythromycin feeder pathways. In particular, the valine catabolic pathway that supplies propionyl-CoA for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B was strongly up-regulated in the S444F mutants, while the expression of the biosynthetic gene cluster of erythromycin (ery) was not significantly affected. In contrast, the ery cluster was down-regulated (<2-fold) in the Q426R mutants. These strains also exhibited an impressive stimulation of the nitrogen regulon, which may contribute to lower erythromycin yields as erythromycin production was strongly inhibited by ammonium.
Rifampicin selection is a simple and reliable tool to investigate novel links between primary and secondary metabolism and morphological differentiation in S. erythraea and to improve erythromycin production. At the same time genome-wide analysis of expression profiles using DNA microarrays allowed information to be gained about the mechanisms underlying the stimulatory/inhibitory effects of the rif mutations on erythromycin production.
Mutations of the rpoB gene conferring resistance to rifampin were analyzed in 40 methicillin-resistant Staphylococcus aureus isolates obtained from six countries. Interestingly, the majority of clinical isolates showed multiple mutations within rpoB. The amino acid substitution 481His→Asn was the most prevalent one, capable of conferring low-level resistance on its own. Cross-resistance to rifampin, rifabutin, and rifapentine was demonstrated for all mutants identified. The level of resistance to rifamycins correlated with both the mutation position and type of amino acid substitution.
Control of the global epidemic tuberculosis is severely hampered by the emergence of drug-resistant Mycobacterium tuberculosis strains. Molecular methods offer a more rapid means of characterizing resistant strains than phenotypic drug susceptibility testing. We have developed a molecular method for detection of rifampicin-resistant M. tuberculosis based on padlock probes and magnetic nanobeads. Padlock probes were designed to target the most common mutations associated with rifampicin resistance in M. tuberculosis, i.e. at codons 516, 526 and 531 in the gene rpoB. For detection of the wild type sequence at all three codons simultaneously, a padlock probe and two gap-fill oligonucleotides were used in a novel assay configuration, requiring three ligation events for circularization. The assay also includes a probe for identification of the M. tuberculosis complex. Circularized probes were amplified by rolling circle amplification. Amplification products were coupled to oligonucleotide-conjugated magnetic nanobeads and detected by measuring the frequency-dependent magnetic response of the beads using a portable AC susceptometer.
AIM: To investigate overlapping regions of the rpoB gene previously involved with rifamycin resistance in M. tuberculosis and seek correlation between rpoB mutations in clinical MAP strains with susceptibility to RIF and RFB.
METHODS: We designed a molecular-based PCR method for the evaluation of rifabutin (RFB) and rifampicin (RIF) resistance based on probable determinant regions within the rpoB gene of MAP, including the 81 bp variable site located between nucleotides 1363 and 1443. The minimum inhibitory concentration (MIC) for RIF was also determined against 11 MAP isolates in attempt to seek correlation with rpoB sequences.
RESULTS: We determined that MAP strain 18 had an MIC of > 30 mg/L and ≤ 5 mg/L for RIF and RFB respectively, and a significant and novel rpoB mutation C1367T, compared to an MIC of ≤ 1.0 mg/L for both drugs in the wild type MAP. The 30-fold increase in the MIC was a direct result of the rpoB mutation C1367T, which caused an amino acid change Thr456 to Ile456 in the drug’s binding site. In addition, MAP strain 185 contained five silent rpoB mutations and exhibited an MIC comparable to the wild-type. Moreover, our in vitro selected mutation in MAP strain UCF5 resulted in the generation of a new resistant strain (UCF5-RIF16r) that possessed T1442C rpoB mutation and an MIC > 30 mg/L and > 10 mg/L for RIF and RFB respectively. Sequencing of the entire rpoB gene in MAP strains UCF4, 18, and UCF5-RIF16r revealed an rpoB mutation A2284C further downstream of the 81 bp variable region in UCF4, accounting for observed slight increase in MIC. In addition, no other significant mutations were found in strains 18 and UCF-RIF16r.
CONCLUSION: The data clearly illustrates that clinical and in vitro-selected MAP mutants with rpoB mutations result in resistance to RIF and RFB, and that a single amino acid change in the beta subunit may have a significant impact on RIF resistance. Unconventional drug susceptibility testing such as our molecular approach will be beneficial for evaluation of antibiotic effectiveness. This molecular approach may also serve as a model for other drugs used for treatment of MAP infections.
Mycobacterium paratuberculosis; Crohn’s disease; Rifabutin; Rifampicin; rpoB; Minimum inhibitory concentration
Drug susceptibility testing (DST) remains an important concern for implementing treatment of MDR tuberculosis patients. Implementation of molecular tests for drug resistance identification would facilitate DST particularly in developing countries where culturing is difficult to perform. We have characterized multidrug resistant strains in Cambodia using MDTDRsl tests, drug target sequencing and phenotypic tests.
A total of 65 non-MDR and 101 MDR TB isolates collected between May 2007 and June 2009 were tested for resistance to fluoroquinolones and aminoglycosides/cyclic peptides using the GenoType® MTBDRsl assay and gene sequencing. Rifampicin resistance (RMP-R) was tested using gene sequencing and genotyping was assessed by spoligotyping.
A total of 95 of the 101 MDR strains were confirmed to be RMP-R by rpoB gene sequencing. Fourteen of the 101 MDR isolates (14%) carried a gyrA mutation associated with fluoroquinolone-resistance (FQ-R) (detected by the MTBDRsl assay and sequencing) compared with only 1 (1.5%) of the 65 non-MDR strains. Only 1 (1%) of the MDR isolates was found to be XDR TB. The MDR group contained a higher proportion of Beijing or Beijing like strains (58%) than the non MDR group (28%). This percentage is higher in MDR FQ-R strains (71%).
The new GenoType® MTBDRsl assay combined with molecular tests to detect RMP-R and isoniazid resistance (INH-R) represents a valuable tool for the detection of XDR TB. In Cambodia there is a low rate of XDR amongst MDR TB including MDR FQ-R TB. This suggests a low association between FQ-R and XDR TB. Strain spoligotyping confirms Beijing strains to be more prone to accumulate antibiotic resistance.
Mutations in the rifampin resistance-determining (Rif) regions of the rpoB gene of Staphylococcus aureus mutants obtained during therapy or in vitro were analyzed by gene amplification and sequencing. Each of the resistant clinical isolates, including five nonrelated clones and two strains isolated from the same patient, and of the 10 in vitro mutants had a single base pair change that resulted in an amino acid substitution in the β subunit of RNA polymerase. Eight mutational changes at seven positions were found in cluster I of the central Rif region. Certain substitutions (His481/Tyr and Asp471/Tyr [S. aureus coordinates]) were present in several mutants. Substitutions Gln468/Arg, His481/Tyr, and Arg484/His, which conferred high-level rifampin resistance, were identical or in the same codon as those described in other bacterial genera, whereas Asp550/Gly has not been reported previously. Substitutions at codon 477 conferred high- or low-level resistance, depending on the nature of the new amino acid. The levels of resistance of in vivo and one-step in vitro mutants carrying identical mutations were similar, suggesting that no other resistance mechanism was present in the clinical isolates. On the basis of these data and the population distribution of more than 4,000 clinical S. aureus isolates, we propose ≤0.5 and ≥8 μg/ml as new breakpoints for the clinical categorization of this species relative to rifampin.
The use of bacteriophages against pathogenic bacteria in health care and in the food industry is now being advocated as an alternative to the use of antibiotics. But what is the evolutionary response for a bacterial population if both antibiotics and phages are used in combination? We employ an experimental evolution approach to address these questions and exposed Pseudomonas fluorescens SBW25 and a related hypermutator strain (mutS−) to the action of the antibiotic rifampicin and the lytic bacteriophage SBW25ϕ2. We then compared the densities, growth rates, and the mutations at the rpoB locus leading to rifampicin resistance of the evolved bacterial populations. We observed that the evolutionary response of populations under different treatments varied depending on the order in which the antimicrobials were added and whether the bacterium was a hypermutator. We found that wild-type rifampicin-resistant populations involved in biofilm formation often reverted to rifampicin sensitivity when stresses were added sequentially. In contrast, when the mortality agents were added simultaneously, phage populations frequently went extinct and the bacteria evolved antibiotic resistance. However, populations of the hypermutator mutS− converged to a single genotype at the rpoB locus. Future investigation on other bacteria and using different antibiotics and bacteriophage are needed to evaluate the generality of our findings.
antibiotic resistance; bacteria; experimental evolution; hypermutator; phage therapy; Pseudomonas
Multiply- and methicillin-resistant Staphylococcus aureus (MRSA) strains have been isolated from five small outbreaks of nosocomial infection in five different hospitals. The MRSA were typed by phage patterns, biochemical traits, resistance phenotypes and plasmid patterns. Three different groups of strains can be distinguished. The MRSA from three outbreaks in one country share identical characters. Phage typing by the use of the International Basic Set for Phage Typing staphylococci as well as experimental phages does not completely discriminate between the strains. Attribution of several resistance determinants to plasmids in two of the described strain groups proved valuable for strain differentiation. These multiply-resistant strains are sensitive to vancomycin and to rifampicin.
Resistance to anti-tuberculosis drugs is a serious public health problem. Multi-drug resistant tuberculosis (MDR-TB), defined as resistance to at least rifampicin and isoniazid, has been reported in all regions of the world. Current phenotypic methods of assessing drug susceptibility of M. tuberculosis are slow. Rapid molecular methods to detect resistance to rifampicin have been developed but they are not affordable in some high prevalence countries such as those in sub Saharan Africa. A simple multi-well plate assay using mycobacteriophage D29 has been developed to test M. tuberculosis isolates for resistance to rifampicin. The purpose of this study was to investigate the performance of this technology in Kampala, Uganda.
In a blinded study 149 M. tuberculosis isolates were tested for resistance to rifampicin by the phage assay and results compared to those from routine phenotypic testing in BACTEC 460. Three concentrations of drug were used 2, 4 and 10 μg/ml. Isolates found resistant by either assay were subjected to sequence analysis of a 81 bp fragment of the rpoB gene to identify mutations predictive of resistance. Four isolates with discrepant phage and BACTEC results were tested in a second phenotypic assay to determine minimal inhibitory concentrations.
Initial analysis suggested a sensitivity and specificity of 100% and 96.5% respectively for the phage assay used at 4 and 10 μg/ml when compared to the BACTEC 460. However, further analysis revealed 4 false negative results from the BACTEC 460 and the phage assay proved the more sensitive and specific of the two tests. Of the 39 isolates found resistant by the phage assay 38 (97.4%) were found to have mutations predictive of resistance in the 81 bp region of the rpoB gene. When used at 2 μg/ml false resistant results were observed from the phage assay. The cost of reagents for testing each isolate was estimated to be 1.3US$ when testing a batch of 20 isolates on a single 96 well plate. Results were obtained in 48 hours.
The phage assay can be used for screening of isolates for resistance to rifampicin, with high sensitivity and specificity in Uganda. The test may be useful in poorly resourced laboratories as a rapid screen to differentiate between rifampicin susceptible and potential MDR-TB cases.
Rifampin and trimethoprim were used alone and in combination in the treatment of chronic osteomyelitis due to Staphylococcus aureus in rabbits. Rifampicin levels in infected bone were well above the minimum inhibitory concentration of the infecting strain of S. aureus for at least 4 h after injection. In contrast, trimethoprim levels in diseased bone were below the minimum inhibitory concentration as early as 1 h after injection. Trimethoprim or rifampin, administered alone for 14 days, were ineffective in sterilizing infected rabbit bones. The combination of rifampin plus trimethoprim was significantly more effective (P less than 0.005) than either agents given alone for a comparable duration of time. Staphylococci isolated from the bones of rabbits treated with rifampin alone or rifampin plus trimethoprim were uniformly resistant to rifampin, but retained their susceptibility to trimethoprim.
In this study, genotyping techniques including staphylococcal chromosomal cassette mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and restriction-modification tests were used to compare the molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered at two times within a 10-year interval (1998 and 2008) from a tertiary Brazilian hospital. In addition, the antimicrobial susceptibility profiles were analyzed. All 48 MRSA isolates from 1998 and 85.7% from 2008 (48/56 isolates) displayed multidrug-resistance phenotypes and SCCmec III. All but one of the 13 representative SCCmec III isolates belonged to CC8 and had PFGE patterns similar to that of the BMB9393 strain (Brazilian epidemic clone of MRSA; BEC). In 2008, we found an increased susceptibility to rifampicin and chloramphenicol among the SCCmec III isolates. In addition, we detected the entrance of diverse international MRSA lineages susceptible to trimethoprim-sulfamethoxazole (SXT), almost all belonging to CC5. These non-SCCmec III isolates were related to the USA300 (ST8-SCCmec IV; PFGE-type B), USA800 (ST5-SCCmec IV; subtype D1), USA100 (ST5-SCCmec II; subtype D2), and EMRSA-3/Cordobes (ST5-SCCmec I, type C) clones. To the best of our knowledge, this is the first report of the emergence of isolates genetically related to the EMRSA-3/Cordobes clone in southeast Brazil. In this regard, these isolates were the most common non-SCCmec III MRSA in our institution, accounting for 8.9% of all isolates recovered in 2008. Thus, despite the supremacy of BEC isolates in our country, significant changes may occur in local MRSA epidemiology, with possible consequences for the rationality of MRSA empiric therapy.
MRSA; Antimicrobial susceptibility; CC5; Cordobes clone; Trimethoprim-sulfamethoxazole
The aim of this study was to investigate the significance of multiple mutations in the rpoB gene as well as predominant nucleotide changes and their correlation with high levels of resistance to rifampin (rifampicin) in Mycobacterium tuberculosis isolates that were randomly collected from the sputa of 46 patients with primary and secondary cases of active pulmonary tuberculosis from the southern region (Afghanistan border) of Iran where tuberculosis is endemic. Drug susceptibility testing was performed using the CDC standard conventional proportional method. DNA extraction, rpoB gene amplification, and DNA sequencing analysis were performed. Thirty-five (76.09%) isolates were found to have multiple mutations (two to four) in the rpoB (β-subunit) gene. Furthermore, we demonstrate that the combination of mutations with more prevalent nucleotide changes were observed in codons 523, 526, and 531, indicating higher frequencies of mutations among patients with secondary infection. In this study, 76.08% (n = 35) of all isolates found to have mutation combinations involving nucleotide changes in codons 523 (GGG→GCG), 531 (TCG→TTG or TTC), and 526 (CAC→CGC, TTC, AAC, or CAA) demonstrated an association with higher levels of resistance to rifampin (MIC, ≥100 μg/ml).
We investigated the activity of telavancin, a novel lipoglycopeptide, alone and combined with gentamicin or rifampin (rifampicin) against strains of Staphylococcus aureus with various vancomycin susceptibilities. Strains tested included methicillin (meticillin)-resistant S. aureus (MRSA) 494, methicillin-sensitive S. aureus (MSSA) 1199, heteroresistant glycopeptide-intermediate S. aureus (hGISA) 1629, which was confirmed by a population analysis profile, and glycopeptide-intermediate S. aureus (GISA) NJ 992. Regimens of 10 mg/kg telavancin daily and 1 g vancomycin every 12 h were investigated alone and combined with 5 mg/kg gentamicin daily or 300 mg rifampin every 8 h in an in vitro model with simulated endocardial vegetations over 96 h. Telavancin demonstrated significantly greater killing than did vancomycin (P < 0.01) for all isolates except MRSA 494 (P = 0.07). Telavancin absolute reductions, in log10 CFU/g, at 96 h were 2.8 ± 0.5 for MRSA 494, 2.8 ± 0.3 for MSSA 1199, 4.2 ± 0.2 for hGISA 1629, and 4.1 ± 0.3 for GISA NJ 992. Combinations of telavancin with gentamicin significantly enhanced killing compared to telavancin alone against all isolates (P < 0.001) except MRSA 494 (P = 0.176). This enhancement was most evident against hGISA 1629, where killing to the level of detection (2 log10 CFU/g) was achieved at 48 h (P < 0.001). The addition of rifampin to telavancin resulted in significant (P < 0.001) enhancement of killing against only MSSA 1199. No changes in telavancin susceptibilities were observed. These results suggest that telavancin may have therapeutic potential, especially against strains with reduced susceptibility to vancomycin. Combination therapy, particularly with gentamicin, may improve bacterial killing against certain strains.
Twenty isolates ofMycobacterium tuberculosis resistant to rifampicin(RIF), isoniazid(INH) and streptomycin(STR) were analysed by Polymerase Chain Reaction (PCR) amplification of rpoB, katG and rrs genes to evaluate comparative diagnostic significance of genetic assays. Mutations were identified by single strand conformation polymorphism (SSCP) and cleavase fragment length polymorphism (CFLP) and were confirmed by DNA sequencing. SSCP of 4 RIF resistant and 14 INH resistant isolates showed an extra peak at the level of 75-bp and 85-bp respectively, while 2 STR resistant isolates showed 2 peaks with 9 bases difference. CFLP showed a different pattern among RIF, INH and STR sensitive and resistant isolates Thus SSCP and CFLP can be used as alternative diagnostic methods for identification of mutations in RIF, INH and STR resistant strains of M.tuberculosis.
Single strand conformation polymorphism; Cleavase fragment length polymorphism; Mycobacterium tuberculosis; Drug resistance
The objective in this study was to develop an osteoconductive, biodegradable and rifampicin releasing bone filling composite material for the treatment of osteomyelitis, a bacterial infection of bone that is very difficult and expensive to treat. The composite material will be used together with a ciprofloxacin releasing composite, because of the rapid development of resistant bacteria when rifampicin is used alone. Three composites were manufactured by twin-screw extrusion. The polymer matrix for the composites was poly(L-lactide-co-ε-caprolactone) 70/30 and all the composites contained 8 wt% (weight percent) of rifampicin antibiotic. The β-TCP contents of the composites were 0 wt%, 50 wt% and 60 wt%. The composites were sterilized by gamma irradiation before in vitro degradation and drug release tests. The hydrolytical degradation of the studied composites proceeded quickly and the molecular weight of the polymer component of the composites decreased rapidly. Rifampicin release occurred in four phases in which the high β-TCP content of the samples, polymer degradation and mass loss all played a role in determining the phases. The ceramic component was seen to have a positive effect on the drug release. The composite with 50 wt% of β-TCP showed the most promising rifampicin release profile and it also showed activity against a common osteomyelitis causing bacteria Pseudomonas aeruginosa. A clear inhibition zone was formed in 16 h incubation. Overall, the tested materials showed great potential to be developed into a bone filler material for the treatment of osteomyelitis or other bone related infections in combination with the ciprofloxacin releasing materials.
drug release; antibiotic; rifampicin; biodegradable; poly(L-lactide-co-caprolactone); polylactide
The resistance of majority ofM. tuberculosis strains to rifampicin a key drug in the tuberculosis therapy, has been cause of major alarm world-wide. Such an association of strain with the drug takes a long time i.e. 2–3 months. We have modified a method for rapid detection of rifampicin resistant strains by screening them through PCR-SSCP.
We have identified mutations in rpo, β region of the gene encoding, β sub-unit of RNA polymerase in rifampicin resistant 67 clinical isolates ofM. tuberculosis. Mutations were screened in these isolates by single strand confirmation polymorphism (SSCP). When DNA sequencing data was compared with the result of SSCP analysis, direct PCR sequencing results were more easily interpreted and contatined more sequence dependent information. These findings provide the basis for rapid detection of rifampicin esistance, a marker of multidrug resistance.
Genotypic detection; Rifampicin resistance; mutation; Mycobacterium tuberculosis