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1.  Molecular characterization of resistance to Rifampicin in an emerging hospital-associated Methicillin-resistant Staphylococcus aureus clone ST228, Spain 
BMC Microbiology  2010;10:68.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2180-10-68
PMCID: PMC2844403  PMID: 20202188
2.  Genetic basis of rifampicin resistance in methicillin-resistant Staphylococcus aureus suggests clonal expansion in hospitals in Cape Town, South Africa 
BMC Microbiology  2012;12:46.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2180-12-46
PMCID: PMC3364154  PMID: 22448673
3.  Leprosy Drug Resistance Surveillance in Colombia: The Experience of a Sentinel Country 
PLoS Neglected Tropical Diseases  2016;10(10):e0005041.
An active search for Mycobacterium leprae drug resistance was carried out, 243 multibacillary patients from endemic regions of Colombia were included from 2004 to 2013 in a surveillance program. This program was a World Health Organization initiative for drug resistance surveillance in leprosy, where Colombia is a sentinel country. M. leprae DNA from slit skin smear and/or skin biopsy samples was amplified and sequenced to identify mutations in the drug resistance determining region (DRDR) in rpoB, folP1, gyrA, and gyrB, the genes responsible for rifampicin, dapsone and ofloxacin drug-resistance, respectively. Three isolates exhibited mutations in the DRDR rpoB gene (Asp441Tyr, Ser456Leu, Ser458Met), two in the DRDR folP1 gene (Thr53Ala, Pro55Leu), and one isolate exhibited mutations in both DRDR rpoB (Ser456Met) and DRDR folP1 (Pro55Leu), suggesting multidrug resistance. One isolate had a double mutation in folP1 (Thr53Ala and Thr88Pro). Also, we detected mutations outside of DRDR that required in vivo evaluation of their association or not with drug resistance: rpoB Arg505Trp, folP1 Asp91His, Arg94Trp, and Thr88Pro, and gyrA Ala107Leu. Seventy percent of M. leprae mutations were related to drug resistance and were isolated from relapsed patients; the likelihood of relapse was significantly associated with the presence of confirmed resistance mutations (OR range 20.1–88.7, p < 0.05). Five of these relapsed patients received dapsone monotherapy as a primary treatment. In summary, the current study calls attention to M. leprae resistance in Colombia, especially the significant association between confirmed resistance mutations and relapse in leprosy patients. A high frequency of DRDR mutations for rifampicin was seen in a region where dapsone monotherapy was used extensively.
Author Summary
Mycobacterium leprae drug resistance is cause of surveillance due to the increase of leprosy relapsed cases. World Health Organization initiative for drug resistance surveillance in leprosy included Colombia, a country considered in post-elimination stage, as a sentinel country. During 10 years (2004–20013) an active search for M. leprae drug resistance was carried out in volunteer patients (each of whom signed a consent form) recruited from a convenience sample of patients diagnosed with leprosy in fourteen departments of Colombia: Amazonas, Antioquia, Atlántico, Bolívar, Caquetá, Cesar, Cundinamarca, Chocó, Huila, Magdalena, Norte de Santander, Santander, Tolima, and Valle. 243 multibacillary patients in various stages of multi drug therapy (MDT) were enrolled with the aim to search for primary and secondary M. leprae drug resistance: 33 new patients before treatment, 136 currently undergoing MDT with not clinical improvement of lesions after three months or more of MDT, 36 post-MDT with positive bacillary index (BI) persistence, 4 non-adherent to MDT, and 34 relapsed cases. M. leprae DNA from patient´s samples was tested to identify mutations in the drug resistance determining region (DRDR) in rpoB, folP1, gyrA, and gyrB, the genes responsible for rifampicin, dapsone and ofloxacin drug-resistance, respectively. We obtained isolates that exhibited mutations in the DRDR, three in rpoB gene, two in folP1 gene, and one isolate exhibited mutations in both rpoB and folP1, suggesting multidrug resistance. One isolate had a double mutation in folP1. Also, we detected mutations outside of DRDR that required in vivo test validation. 70% of M. leprae confirmed resistance mutations were isolated from relapsed patients (OR range 20.1–88.7, p < 0.05). Five of these relapsed patients received dapsone monotherapy as a primary treatment. The current study calls attention to M. leprae resistance in Colombia, especially the significant association between confirmed resistance mutations and relapsed in leprosy patients.
doi:10.1371/journal.pntd.0005041
PMCID: PMC5051701  PMID: 27706165
4.  Could high-concentration rifampicin kill rifampicin-resistant M. tuberculosis? Rifampicin MIC test in rifampicin-resistant isolates from patients with osteoarticular tuberculosis 
Purpose
Several studies have shown that the intralesional concentration of rifampicin in osteoarticular tuberculosis is typically at a subtherapeutic level. Sustained or controlled release by novel drug delivery systems has been investigated to maintain an effective rifampicin concentration, but the local administration of rifampicin remains controversial. Additionally, it is still unclear whether high-dose rifampicin could kill rifampicin-resistant Mycobacterium tuberculosis. The aim of this study was to assess the in vitro killing effect of high-concentration rifampicin on rifampicin-resistant M. tuberculosis isolated from patients with osteoarticular tuberculosis.
Methods
A set of 18 rifampicin-resistant M. tuberculosis isolates by the BACT/MGIT 960 system from patients with osteoarticular tuberculosis was collected for further study. The detection of rpoB gene mutations was performed using non-fluorescent, low-density DNA microarrays to determine the resistant mechanism. Following secondary culture, susceptibility to gradient concentrations of rifampicin (2 to 256 μg/ml) was tested; these concentrations are attainable for prolonged periods of local chemotherapy. The relationship between microbial killing by high-dose rifampicin and rpoB gene mutations was analyzed.
Results
Mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene were identified in 17 isolates (94.4%); one strain exhibited no mutations in this region. The most prevalent mutation sites were in codons 531 (55.56%), 516 (16.67%), 526 (11.11%), and 513 (11.11%). Isolates with mutations in the rpoB gene were highly resistant to rifampicin, 11 of which had minimal inhibitory concentrations (MICs) exceeding 256 μg/ml (not determined). The MICs for the remaining seven resistant isolates were between 32 and 256 μg/ml. Particularly in less rifampicin-resistant M. tuberculosis strains, growth was inhibited at high concentrations.
Conclusion
Increasing the rifampicin concentration may optimize this drug’s antituberculous effect, even against some rifampicin-resistant isolates, if systemic and local toxic effects can be minimized.
doi:10.1186/s13018-014-0124-1
PMCID: PMC4269955  PMID: 25467069
Rifampicin; Drug resistance; Osteoarticular tuberculosis; rpoB gene
5.  Rifampicin Reduces Susceptibility to Ofloxacin in Rifampicin-resistant Mycobacterium tuberculosis through Efflux 
Rationale
Central dogma suggests that rifampicin resistance in Mycobacterium tuberculosis develops solely through rpoB gene mutations.
Objective
To determine whether rifampicin induces efflux pumps activation in rifampicin resistant M. tuberculosis strains thereby defining rifampicin resistance levels and reducing ofloxacin susceptibility.
Methods
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).
Conclusion
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.
doi:10.1164/rccm.201011-1924OC
PMCID: PMC3698754  PMID: 21512166
Mycobacterium tuberculosis; drug resistance; rifampicin; efflux pumps; cross resistance
6.  Sequence analysis for detection of drug resistance in Mycobacterium tuberculosis complex isolates from the Central Region of Cameroon 
BMC Microbiology  2014;14:113.
Background
The potential of genetic testing to rapidly diagnose drug resistance has lead to the development of new diagnostic assays. However, prior to implementation in a given setting, the association of specific mutations with specific drug resistance phenotypes should be evaluated. The purpose of this study was to evaluate molecular markers in predicting drug resistance in the Central Region of Cameroon.
Results
From April 2010 and March 2011, 725 smear positive pulmonary tuberculosis patients were enrolled and all positive cultures were tested for drug susceptibility. A total of 63 drug resistant and 100 drug sensitive Mycobacterium tuberculosis complex clinical isolates were screened for genetic mutations in katG, inhA, ahpC, rpoB, rpsL, rrs, gidB and embCAB loci using DNA sequencing. Of the 44 isoniazid resistant (INHR) isolates (24 high level, 1 μg/ml and 20 low level, 0.2 μg/ml), 73% (32/44) carried the katG315 and/or the -15 inhA promoter mutations. Of the 24 high level INHR, 17 (70.8%) harbored katG315 mutation, 1 a point mutation (-15C → T) in the inhA promoter and 6 were (25.0%) wild types. Thus, for INHR high level detection, katG315 mutation had a specificity and a sensitivity of 100% and 70.8% respectively. Of the 20 low level INHR, 10 (50.0%) had a -15C → T mutation in the inhA promoter region, and 1 (2.2%) a -32G → A mutation in the ahpC promoter region. All of the 7 rifampicin resistant (RIFR) isolates carried mutations in the rpoB gene (at codons Ser531Leu (71.4%), His526Asp (14.3%), and Asp516Val (14.3%)). Of the 27 streptomycin resistant (SMR) isolates, 7 carried mutations at the rpsL and the gidB genes. 1 of the 2 ethambutol resistant (EMBR) isolates displayed a mutation in embB gene.
Conclusion
This study provided the first molecular investigation assessing the correlation of phenotypic to genotypic characteristics on MTB isolates from the Central Region of Cameroon using DNA sequencing. Mutations on rpoB, katG315 and -15 point mutations in inhA promoter loci could be used as markers for RIF and INH -resistance detection respectively.
doi:10.1186/1471-2180-14-113
PMCID: PMC4017682  PMID: 24884632
Phenotype; Mutation; Drug resistance; Mycobacterium tuberculosis; Cameroon
7.  Development and characterisation of highly antibiotic resistant Bartonella bacilliformis mutants 
Scientific Reports  2016;6:33584.
The objective was to develop and characterise in vitro Bartonella bacilliformis antibiotic resistant mutants. Three B. bacilliformis strains were plated 35 or 40 times with azithromycin, chloramphenicol, ciprofloxacin or rifampicin discs. Resistance-stability was assessed performing 5 serial passages without antibiotic pressure. MICs were determined with/without Phe-Arg-β-Napthylamide and artesunate. Target alterations were screened in the 23S rRNA, rplD, rplV, gyrA, gyrB, parC, parE and rpoB genes. Chloramphenicol and ciprofloxacin resistance were the most difficult and easiest (>37.3 and 10.6 passages) to be selected, respectively. All mutants but one selected with chloramphenicol achieved high resistance levels. All rifampicin, one azithromycin and one ciprofloxacin mutants did not totally revert when cultured without antibiotic pressure. Azithromycin resistance was related to L4 substitutions Gln-66 → Lys or Gly-70 → Arg; L4 deletion Δ62–65 (Lys-Met-Tyr-Lys) or L22 insertion 83::Val-Ser-Glu-Ala-His-Val-Gly-Lys-Ser; in two chloramphenicol-resistant mutants the 23S rRNA mutation G2372A was detected. GyrA Ala-91 → Val and Asp-95 → Gly and GyrB Glu474 → Lys were detected in ciprofloxacin-resistant mutants. RpoB substitutions Gln-527 → Arg, His-540 → Tyr and Ser-545 → Phe plus Ser-588 → Tyr were detected in rifampicin-resistant mutants. In 5 mutants the effect of efflux pumps on resistance was observed. Antibiotic resistance was mainly related to target mutations and overexpression of efflux pumps, which might underlie microbiological failures during treatments.
doi:10.1038/srep33584
PMCID: PMC5035977  PMID: 27667026
8.  Mycobacterium tuberculosis Strains with Highly Discordant Rifampin Susceptibility Test Results▿  
Journal of Clinical Microbiology  2009;47(11):3501-3506.
The objectives of this study were to investigate the origin of highly discordant rifampin (rifampicin) (RMP) drug susceptibility test results obtained for Mycobacterium tuberculosis strains during proficiency testing. Nine Supra-National Tuberculosis Reference Laboratories tested the RMP susceptibilities of 19 selected M. tuberculosis strains, using standard culture-based methods. The strains were classified as definitely resistant (R) (n = 6) or susceptible (S) (n = 2) or probably resistant (PR) (n = 8) or susceptible (PS) (n = 3) based on rpoB mutations and treatment outcome. All methods yielded a susceptible result for the two S and three PS strains lacking an rpoB mutation and a resistant result for one R strain with a Ser531Leu mutation and one PR strain with a double mutation. Although the remaining 12 R and PR strains had rpoB mutations (four Asp516Tyr, three Leu511Pro, two Leu533Pro, one each His526Leu/Ser, and one Ile572Phe), they were all susceptible by the radiometric Bactec 460TB or Bactec 960 MGIT methods. In contrast, only one was susceptible by the proportion method on Löwenstein-Jensen medium and two on Middlebrook 7H10 agar. Low-level but probably clinically relevant RMP resistance linked to specific rpoB mutations is easily missed by standard growth-based methods, particularly the automated broth-based systems. Further studies are required to confirm these findings, to determine the frequency of these low-level-resistant isolates, and to identify technical improvements that may identify such strains.
doi:10.1128/JCM.01209-09
PMCID: PMC2772627  PMID: 19759221
9.  Direct sequencing for rapid detection of multidrug resistant Mycobacterium tuberculosis strains in Morocco 
Background
Tuberculosis (TB) is a major public health problem with high mortality and morbidity rates, especially in low-income countries. Disturbingly, the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) TB cases has worsened the situation, raising concerns of a future epidemic of virtually untreatable TB. Indeed, the rapid diagnosis of MDR TB is a critical issue for TB management. This study is an attempt to establish a rapid diagnosis of MDR TB by sequencing the target fragments of the rpoB gene which linked to resistance against rifampicin and the katG gene and inhA promoter region, which are associated with resistance to isoniazid.
Methods
For this purpose, 133 sputum samples of TB patients from Morocco were enrolled in this study. One hundred samples were collected from new cases, and the remaining 33 were from previously treated patients (drug relapse or failure, chronic cases) and did not respond to anti-TB drugs after a sufficient duration of treatment. All samples were subjected to rpoB, katG and pinhA mutation analysis by polymerase chain reaction and DNA sequencing.
Results
Molecular analysis showed that seven strains were isoniazid-monoresistant and 17 were rifampicin-monoresistant. MDR TB strains were identified in nine cases (6.8%). Among them, eight were traditionally diagnosed as critical cases, comprising four chronic and four drug-relapse cases. The last strain was isolated from a new case. The most recorded mutation in the rpoB gene was the substitution TCG > TTG at codon 531 (Ser531 Leu), accounting for 46.15%. Significantly, the only mutation found in the katG gene was at codon 315 (AGC to ACC) with a Ser315Thr amino acid change. Only one sample harbored mutation in the inhA promoter region and was a point mutation at the −15p position (C > T).
Conclusion
The polymerase chain reaction sequencing approach is an accurate and rapid method for detection of drug-resistant TB in clinical specimens, and could be of great interest in the management of TB in critical cases to adjust the treatment regimen and limit the emergence of MDR and XDR strains.
doi:10.2147/IDR.S47724
PMCID: PMC3875366  PMID: 24399879
Morocco; Mycobacterium tuberculosis; multidrug resistance; rpoB; katG; inhA promoter
10.  Evolution of Extensively Drug-Resistant Tuberculosis over Four Decades: Whole Genome Sequencing and Dating Analysis of Mycobacterium tuberculosis Isolates from KwaZulu-Natal 
PLoS Medicine  2015;12(9):e1001880.
Background
The continued advance of antibiotic resistance threatens the treatment and control of many infectious diseases. This is exemplified by the largest global outbreak of extensively drug-resistant (XDR) tuberculosis (TB) identified in Tugela Ferry, KwaZulu-Natal, South Africa, in 2005 that continues today. It is unclear whether the emergence of XDR-TB in KwaZulu-Natal was due to recent inadequacies in TB control in conjunction with HIV or other factors. Understanding the origins of drug resistance in this fatal outbreak of XDR will inform the control and prevention of drug-resistant TB in other settings. In this study, we used whole genome sequencing and dating analysis to determine if XDR-TB had emerged recently or had ancient antecedents.
Methods and Findings
We performed whole genome sequencing and drug susceptibility testing on 337 clinical isolates of Mycobacterium tuberculosis collected in KwaZulu-Natal from 2008 to 2013, in addition to three historical isolates, collected from patients in the same province and including an isolate from the 2005 Tugela Ferry XDR outbreak, a multidrug-resistant (MDR) isolate from 1994, and a pansusceptible isolate from 1995. We utilized an array of whole genome comparative techniques to assess the relatedness among strains, to establish the order of acquisition of drug resistance mutations, including the timing of acquisitions leading to XDR-TB in the LAM4 spoligotype, and to calculate the number of independent evolutionary emergences of MDR and XDR. Our sequencing and analysis revealed a 50-member clone of XDR M. tuberculosis that was highly related to the Tugela Ferry XDR outbreak strain. We estimated that mutations conferring isoniazid and streptomycin resistance in this clone were acquired 50 y prior to the Tugela Ferry outbreak (katG S315T [isoniazid]; gidB 130 bp deletion [streptomycin]; 1957 [95% highest posterior density (HPD): 1937–1971]), with the subsequent emergence of MDR and XDR occurring 20 y (rpoB L452P [rifampicin]; pncA 1 bp insertion [pyrazinamide]; 1984 [95% HPD: 1974–1992]) and 10 y (rpoB D435G [rifampicin]; rrs 1400 [kanamycin]; gyrA A90V [ofloxacin]; 1995 [95% HPD: 1988–1999]) prior to the outbreak, respectively. We observed frequent de novo evolution of MDR and XDR, with 56 and nine independent evolutionary events, respectively. Isoniazid resistance evolved before rifampicin resistance 46 times, whereas rifampicin resistance evolved prior to isoniazid only twice. We identified additional putative compensatory mutations to rifampicin in this dataset. One major limitation of this study is that the conclusions with respect to ordering and timing of acquisition of mutations may not represent universal patterns of drug resistance emergence in other areas of the globe.
Conclusions
In the first whole genome-based analysis of the emergence of drug resistance among clinical isolates of M. tuberculosis, we show that the ancestral precursor of the LAM4 XDR outbreak strain in Tugela Ferry gained mutations to first-line drugs at the beginning of the antibiotic era. Subsequent accumulation of stepwise resistance mutations, occurring over decades and prior to the explosion of HIV in this region, yielded MDR and XDR, permitting the emergence of compensatory mutations. Our results suggest that drug-resistant strains circulating today reflect not only vulnerabilities of current TB control efforts but also those that date back 50 y. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics employed in South Africa that assess only rifampicin resistance.
Editors' Summary
Background
Tuberculosis (TB)—a contagious bacterial disease that usually infects the lungs—is a global public health problem. Every year, about 9 million people develop active TB disease, and 1.5 million people die from the disease. Mycobacterium tuberculosis, the organism that causes TB, is spread in airborne droplets when people with TB cough. The symptoms of TB include cough, weight loss, and fever. Diagnostic tests for the disease include sputum smear microscopy (microscopic analysis of mucus coughed up from the lungs) and chest X-rays. TB can be cured by taking a regimen of multiple antibiotics daily for 6 mo. However, the emergence of multidrug-resistant tuberculosis (MDR-TB, TB with resistance to both isoniazid and rifampicin) and extensively drug-resistant tuberculosis (XDR-TB, MDR-TB with additional resistance to both quinolones and second-line injectable agents), together with the spread of HIV (which increases susceptibility to TB), is now threatening TB control efforts. MDR-TB is caused by M. tuberculosis strains that have acquired mutations (genetic changes) that make them resistant to isoniazid, rifampicin, and sometimes other anti-TB drugs; XDR-TB is caused by bacteria that are resistant to isoniazid, rifampicin, one or more fluoroquinolones (for example, ofloxacin), and at least one injectable second-line drug (for example, kanamycin).
Why Was This Study Done?
A better understanding of the origins of drug-resistant TB is essential for effective control of TB. Public health experts need to know whether the emergence of drug-resistant TB is caused by inadequacies in TB control or related to other factors such as the spread of HIV and whether new resistant strains of M. tuberculosis repeatedly emerge during XDR-TB outbreaks or whether the transmission of a single drug-resistant strain drives these outbreaks. Here, the researchers use whole genome sequencing and dating analysis to investigate the origin and evolution of an XDR-TB outbreak identified in 2005 in Tugela Ferry, KwaZulu-Natal, South Africa. The predominant strain of XDR M. tuberculosis isolated during this large XDR-TB outbreak belongs to a subfamily called LAM4. Since the outbreak began, XDR-TB has also been reported in hospitals across KwaZulu-Natal, and some of these outbreaks have been caused by bacterial strains not falling within the LAM4 spoligotype (“spoligotyping” characterizes M. tuberculosis strains based on the presence of unique DNA sequences in a specific region of the bacterial genome).
What Did the Researchers Do and Find?
The researchers tested the antibiotic susceptibility of 337 clinical isolates of M. tuberculosis collected in KwaZulu-Natal between 2008 and 2013 and of three historical isolates—two collected in the province in the mid-1990s and a third from the Tugela Ferry XDR outbreak. They sequenced the whole genome of these isolates and used comparative techniques to assess the isolates’ relatedness and to investigate the acquisition of drug resistance. This analysis revealed a 50-member clone of XDR bacteria among the isolates collected across KwaZulu-Natal that was highly related to the LAM4 strain (a clone is defined here as a set of strains in which each member differs by no more than ten single nucleotide polymorphisms [SNPs] from at least one other member; an SNP is a type of genetic variant). Mutations that conferred isoniazid resistance in this clone were acquired in about 1957; MDR and XDR strains emerged in about 1984 and 1995, respectively. The analysis also indicates that MDR and XDR evolved de novo 56 times and nine times, respectively, and that isoniazid resistance nearly always evolved before rifampicin resistance.
What Do These Findings Mean?
These findings provide new information about the ordering and timing of the acquisition of drug-resistance mutations by M. tuberculosis in KwaZulu-Natal but do not necessarily represent the evolution of XDR-TB in other settings. Most notably, these findings indicate that the ancestral precursor of the Tugela Ferry XDR outbreak strain gained resistance to first-line antibiotics shortly after these antibiotics became available for clinical use. Subsequent stepwise accumulation of additional resistance mutations that occurred over decades led to the emergence of MDR and XDR strains. Importantly, the emergence of these strains occurred before the explosion of HIV in KwaZulu-Natal. Thus, these findings highlight the dire repercussions of the failure of historic attempts to control resistance to first-line anti-TB drugs and draw attention to the need for new anti-TB drugs to be used prudently to prevent early fixation of resistance and to protect the useful lifespan of these agents. Finally, the finding that isoniazid resistance is a key initiation event for progression to MDR and XDR suggests that TB control programs should test routinely for both isoniazid and rifampicin resistance to ensure early detection of drug-resistant TB.
Additional Information
This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001880.
The World Health Organization (WHO) provides information (in several languages) on TB and on MDR-TB; the Global Tuberculosis Report 2014 provides information about TB around the world; a supplement to the report entitled “Drug-Resistant TB—Surveillance and Response” is available
The Stop TB Partnership is working towards TB elimination and provides personal stories about TB (in English and Spanish)
The United States Centers for Disease Control and Prevention provides information about TB and about drug-resistant TB (in English and Spanish)
The US National Institute of Allergy and Infectious Diseases also has detailed information on TB, including a drug-resistant TB visual tour
TB & Me, a collaborative blogging project run by patients being treated for MDR-TB and Mèdecins sans Frontiéres, provides more patient stories
The not-for-profit organization Global Health Education provides information about TB in South Africa
MedlinePlus has links to further information about TB (in English and Spanish)
doi:10.1371/journal.pmed.1001880
PMCID: PMC4587932  PMID: 26418737
11.  Low-cost rapid detection of rifampicin resistant tuberculosis using bacteriophage in Kampala, Uganda 
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/1476-0711-6-1
PMCID: PMC1779803  PMID: 17212825
12.  Complete Reconstitution of the Vancomycin-Intermediate Staphylococcus aureus Phenotype of Strain Mu50 in Vancomycin-Susceptible S. aureus 
Complete reconstitution of the vancomycin-intermediate Staphylococcus aureus (VISA) phenotype of strain Mu50 was achieved by sequentially introducing mutations into six genes of vancomycin-susceptible S. aureus (VSSA) strain N315ΔIP. The six mutated genes were detected in VISA strain Mu50 but not in N315ΔIP. Introduction of the mutation Ser329Leu into vraS, encoding the sensor histidine kinase of the vraSR two-component regulatory (TCR) system, and another mutation, Glu146Lys, into msrR, belonging to the LytR-CpsA-Psr (LCP) family, increased the level of vancomycin resistance to that detected in heterogeneous vancomycin-intermediate S. aureus (hVISA) strain Mu3. Introduction of two more mutations, Asn197Ser into graR of the graSR TCR system and His481Tyr into rpoB, encoding the β subunit of RNA polymerase, converted the hVISA strain into a VISA strain with the same level of vancomycin resistance as Mu50. Surprisingly, however, the constructed quadruple mutant strain ΔIP4 did not have a thickened cell wall, a cardinal feature of the VISA phenotype. Subsequent study showed that cell wall thickening was an inducible phenotype in the mutant strain, whereas it was a constitutive one in Mu50. Finally, introduction of the Ala297Val mutation into fdh2, which encodes a putative formate dehydrogenase, or a 67-amino-acid sequence deletion into sle1 [sle1(Δ67aa)], encoding the hydrolase of N-acetylmuramyl-l-alanine amidase in the peptidoglycan, converted inducible cell wall thickening into constitutive cell wall thickening. sle1(Δ67aa) was found to cause a drastic decrease in autolysis activity. Thus, all six mutated genes required for acquisition of the VISA phenotype were directly or indirectly involved in the regulation of cell physiology. The VISA phenotype seemed to be achieved through multiple genetic events accompanying drastic changes in cell physiology.
doi:10.1128/AAC.00420-16
PMCID: PMC4879404  PMID: 27067329
13.  Characterization of Mutations in the rpoB Gene That Confer Rifampin Resistance in Staphylococcus aureus 
Antimicrobial Agents and Chemotherapy  1998;42(10):2590-2594.
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.
PMCID: PMC105902  PMID: 9756760
14.  Rapid Change of Methicillin-Resistant Staphylococcus aureus Clones in a Chinese Tertiary Care Hospital over a 15-Year Period▿  
The incidence of methicillin-resistant Staphylococcus aureus (MRSA) has been increasing yearly at Peking Union Medical College Hospital (PUMCH). In order to understand the molecular evolution of MRSA at PUMCH, a total of 466 nonduplicate S. aureus isolates, including 302 MRSA and 164 methicillin-susceptible (MSSA) isolates recovered from 1994 to 2008 were characterized by staphylococcal cassette chromosome mec (SCCmec) typing, spa typing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). The 302 MRSA isolates were classified into 12 spa types and 9 sequence types (STs). During the years from 1994 to 2000, the most predominant MRSA clone was ST239-MRSA-III-spa t037. Since 2000, ST239-MRSA-III-spa t030 has rapidly replaced t037 and become the major clone. Another clone, ST5-MRSA-II-spa t002 emerged in 2002 and constantly existed at a low prevalence rate. The 164 MSSA isolates were classified into 62 spa types and 40 STs. ST398 was the most common MLST type for MSSA, followed by ST59, ST7, ST15, and ST1. Several MSSA genotypes, including ST398, ST1, ST121, and ST59, were identical to well-known epidemic community-acquired MRSA (CA-MRSA) isolates. MLST eBURST analysis revealed that the ST5, ST59, and ST965 clones coexisted in both MRSA and MSSA, which suggested that these MRSA clones might have evolved from MSSA by the acquisition of SCCmec. Two pvl-positive ST59-MRSA-IV isolates were identified as CA-MRSA according to the clinical data. Overall, our data showed that the ST239-MRSA-III-spa t037 clone was replaced by the emerging ST239-MRSA-III-spa t030 clone, indicating a rapid change of MRSA at a tertiary care hospital in China over a 15-year period.
doi:10.1128/AAC.01563-09
PMCID: PMC2863666  PMID: 20176895
15.  Analysis of gene mutations associated with isoniazid, rifampicin and ethambutol resistance among Mycobacterium tuberculosis isolates from Ethiopia 
Background
The emergence of drug resistance is one of the most important threats to tuberculosis control programs. This study was aimed to analyze the frequency of gene mutations associated with resistance to isoniazid (INH), rifampicin (RMP) and ethambutol (EMB) among Mycobacterium tuberculosis isolates from Northwest Ethiopia, and to assess the performance of the GenoType® MTBDRplus and GenoType® MTBDRsl assays as compared to the BacT/ALERT 3D system.
Methods
Two hundred sixty Mycobacterium tuberculosis isolates from smear positive tuberculosis patients diagnosed between March 2009 and July 2009 were included in this study. Drug susceptibility tests were performed in the Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University Hospital of Leipzig, Germany.
Results
Of 260 isolates, mutations conferring resistance to INH, RMP, or EMB were detected in 35, 15, and 8 isolates, respectively, while multidrug resistance (MDR) was present in 13 of the isolates. Of 35 INH resistant strains, 33 had mutations in the katG gene at Ser315Thr 1 and two strains had mutation in the inhA gene at C15T. Among 15 RMP resistant isolates, 11 had rpoB gene mutation at Ser531Leu, one at His526Asp, and three strains had mutations only at the wild type probes. Of 8 EMB resistant strains, two had mutations in the embB gene at Met306Ile, one at Met306Val, and five strains had mutations only at the wild type probes. The GenoType® MTBDRplus assay had a sensitivity of 92% and specificity of 99% for INH resistance, and 100% sensitivity and specificity to detect RMP resistance and MDR. The GenoType® MTBDRsl assay had a sensitivity of 42% and specificity of 100% for EMB resistance.
Conclusion
The dominance of single gene mutations associated with the resistance to INH and RMP was observed in the codon 315 of the katG gene and codon 531 of the rpoB gene, respectively. The GenoType® MTBDRplus assay is a sensitive and specific tool for diagnosis of resistance to INH, RMP and MDR. However, the GenoType® MTBDRsl assay shows limitations in detecting resistance to EMB.
doi:10.1186/1471-2334-12-37
PMCID: PMC3378438  PMID: 22325147
Mycobacterium tuberculosis; Drug resistance; Gene mutation
16.  Antibiotic resistance and molecular epidemiology of Staphylococcus aureus in Nigeria 
BMC Microbiology  2011;11:92.
Background
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.
Results
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
Conclusions
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.
doi:10.1186/1471-2180-11-92
PMCID: PMC3112067  PMID: 21545717
17.  Study of the Rifampin Monoresistance Mechanism in Mycobacterium tuberculosis 
Rifampin (RIF) susceptibility is a key factor in determining the treatment effectiveness of the standardized treatment regimens. In Mycobacterium tuberculosis, both target gene mutation and the efflux pump play major roles in the resistance to antituberculosis drugs. By eliminating RIF-resistant strains with rpoB mutation, the choice of RIF-monoresistant strains may allow us to identify the RIF-specific efflux pump genes. This study explored the RIF monoresistance mechanism in M. tuberculosis. Data from DNA sequencing and MIC measurements revealed that specific mutations, including Ser531Leu and His526Asp in RpoB, show high-level drug resistance. Three-dimensional structure modeling provided further evidence that the affinity between RIF and RpoB mutants was in accordance with the drug resistance level of the corresponding isolates. Furthermore, transcription-level analysis among the nonmutated isolates indicated that three efflux pumps (Rv0783, Rv2936, and Rv0933) might be involved in exporting RIF from the cell. Compared to 8 μg/ml for wild-type Escherichia coli, the MICs for the transgenic E. coli strains with either Rv0783 or Rv2936 were 32 and 16 μg/ml, respectively. In conclusion, our study indicated that several RpoB mutant types, including Ser531Leu and His526Asp, show high-level RIF resistance attributed to low affinity between RpoB mutant proteins and RIF. In addition, this work demonstrates that Rv2936 and Rv0783 may be responsible for low-level resistance to RIF by exporting RIF from cells. The predicted structure of RpoB and the newly identified efflux pumps in this study will provide a novel approach to design new drugs and develop novel diagnosis technologies.
doi:10.1128/AAC.01024-12
PMCID: PMC3553728  PMID: 23208715
18.  Rifampicin resistance mutations in the 81 bp RRDR of rpoB gene in Mycobacterium tuberculosis clinical isolates using Xpert® MTB/RIF in Kampala, Uganda: a retrospective study 
BMC Infectious Diseases  2014;14:481.
Background
Introduction of Xpert® MTB/RIF assay has revolutionalised the diagnosis of tuberculosis (TB) by simultaneously detecting the bacteria and resistance to rifampicin (rif), a surrogate marker for multi-drug resistant TB (MDR-TB) as well as one of the principal first-line anti-tuberculosis drugs. In general, rpoB mutations can be found in 96.1% of rif-resistant Mycobacterium tuberculosis (MTB) strains worldwide and these mutations usually are located in a region at the 507-533rd amino acid residuals (81 bp) in the MTB rpoB gene, which is referred to as Rifampicin-resistance-determining region (RRDR). In this study, we determined the frequency of MDR-TB in Kampala using Xpert® MTB/RIF in comparison with the agar proportion method using Middlebrook 7H11and further determined the frequency of probes for different rpoB gene mutations using Xpert® MTB/RIF assay in the 81 bp RRDR.
Methods
A total of 1501 specimens received at Mycobacteriology laboratory, Makerere University for Xpert testing between May 2011 and May 2014 were analysed by Xpert® MTB/RIF assay. Specimens that were positive for both MTB and rifampicin resistance were further subjected to a complete first line anti-mycobacterial drug susceptibility testing using Middlebrook 7H11 agar proportion method (APM).
Results
Xpert® MTB/RIF assay detected 313 MTB positive specimens and out of which 12 specimens had both MTB and rifampicin- resistance conferred by four different rpoB gene mutations in the 81 bp-RRDR of MTB, further one (1/12), specimen was found to be rifampicin mono-resistant on APM while the 11 were found to be MDR-TB. Probes associated with the observed rif- resistance were as follows: E (7/12), B (3/12), A (1/12), D (1/12) and no rif-resistance was associated with probe C. No specimen yielded rif-resistance associated with more than one probe failure (mutation combinations). Probe D was associated with rifampicin mono-resistant.
Conclusions
MDR-TB was at 3.5% in the studied population. Mutations associated with Probe E (58%) also known as codons 531and 533 are the commonest rpoB gene mutation identified by Xpert® MTB/RIF assay in this setting and mutations identified by probe E of the assay, turned out to be MDR-TB strains by agar proportion method antimicrobial susceptibility testing. No mutation was detected in the codon 522.
doi:10.1186/1471-2334-14-481
PMCID: PMC4164707  PMID: 25190040
Rifampicin-resistance-determining region; RNA polymerase B gene; Multi-drug Resistant tuberculosis; Xpert® MTB/RIF
19.  Dissemination of fusidic acid resistance among Staphylococcus aureus clinical isolates 
BMC Microbiology  2015;15:210.
Background
A significant trend towards increased fusidic acid (FA) resistance among Staphylococcus aureus with increased duration of use is of concern. The aim of the present study is to investigate the dissemination of fusidic acid resistance among Staphylococcus aureus clinical isolates.
Methods
The susceptibility of S. aureus isolates to antimicrobial agents was determined by disc-diffusion method. The minimal inhibitory concertrations(MICs) of fusidic acid and vacomycin for fusidic acid-resisitant isolates were determined by ager dillution method. FA resistance determinants were determined by PCR and DNA sequencing. SCCmec typing, spa typing and multi-locus sequence typing were used for the determination of molecular characteristics for S. aureus isolates.
Results
A total of 392 non-duplicate S. aureus isolates including 181 methicillin-resistant S. aureus (MRSA) isolates, which were isolated from the clinical specimens of patients at a Chinese tertiary hospital from January, 2012 to September, 2013, were collected for investigating FA resistance. Among 392 S. aureus clinical isolates tested, 56 (14.3 %) with FA MIC values ranging from 2 μg/ml to ≥128 μg/ml were resistant to FA. The proportions of FA resistance among MRSA and MSSA isolates were 27.1 % (49/181) and 3.3 % (7/211). There was a trend of rapidly increased FA resistance among S. aureus and MRSA isolates from 5.2 % and 8.9 % in 2012 to 24.9 % and 45.1 % in 2013. Acquired FA resistance gene, fusB, was present in 73.2 % (41/56) of FA-resistant S. aureus isolates. fusC and fusA mutation were not found in any of tested isolates. A total of 9 sequence types (STs) and 12 spa types were identified among the 56 FA-resistant S. aureus isolates. ST5 accounting for 66.1 % (37/56) was the most prevalent ST. The majority (92.9 %, 52/56) of the isolates tested belonged to clonal complex 5(CC5). t2460 was the most prevalent spa type, accounting for 67.9 % (38/56) . ST5-MRSA- II-t2460 was predominant clone, accounting for 75.5 % (37/49) of FA-resistant MRSA isolates and 66.1 % (37/56) of FA-resistant S. aureus isolates. Five of 7 FA-resistant MSSA isolates belonged to ST630-MSSA.
Conclusion
Increased FA resistance among S. aureus isolates was found in China. fusB was predominant FA resistance determinant. The spread of CC5 clone, especially novel ST5-MRSA- II-t2460 clone with high-level resistance to FA, was responsible for the increase of FA resistance.
doi:10.1186/s12866-015-0552-z
PMCID: PMC4604626  PMID: 26463589
Staphylococcus aureus; Fusidic acid; Resistance; Resistance determinants; Molecular characteristic
20.  Evaluation of Multidrug Resistant Staphylococcus aureus and their Association with Biofilm Production in a Tertiary Care Hospital, Tripura, Northeast India 
Background
High morbidity and mortality rates are associated with Methicillin-resistant Staphylococcus aureus (MRSA) because of development of multidrug resistance. Staphylococcus aureus (S. aureus) has the ability to colonize and form biofilms on biomaterials which is causing resistance towards antimicrobials and thus making them difficult to eradicate from the infected hosts.
Materials and Methods
Culture isolation, identification was done following standard protocol and antibiogram of the isolates were done. The detection of MRSA, Macrolide-Lincosamide-Streptogramin B resistance (MLSB), vancomycin resistance phenotypes were done by using cefoxitin disc diffusion test, D zone test and vancomycin E test. Biofilm was detected by Congo red agar method.
Results
A total of 100 (31.7%) S. aureus strains were isolated from 315 clinical specimens. The prevalence of MRSA was 47% (47/100) with 85.1% were homogeneous MRSA and 14.9% were heterogeneous. Out of 47 MRSA strains, 63.8% were Hospital acquired-MRSA (HA-MRSA) infections whereas rests 36.2% were caused by Community acquired-MRSA (CA-MRSA) strains. Maximum number of MRSA isolates belonged to group A biotype (34%). A 14.9% isolates were of nontypeable group. Out of 100 S. aureus isolates, the prevalence of Vancomycin resistant S. aureus (VRSA) was found to be 3%. The MLSB phenotypes showed that the rates of inducible MLSB (iMLSB), constitutive MLSB (cMLSB) and Macrolide-Streptogramin B (MSB) in case of MRSA to be 19.1%, 31.9% and 12.8%. Prevalence of low-level (MUPL) and high-level mupirocin resistance (MUPH) among MRSA was 19.1% and 6.4%. Biofilm production was found in 55% strains of S. aureus. Out of 47 MRSA strains 76.6%were producing biofilm in comparison to 38.8% in methicillin-sensitive S. aureus (MSSA). Higher degree of antibiotic resistance in biofilm producers was seen especially in case of ciprofloxacin, co-trimoxazole, rifampicin, kanamycin, erythromycin and clindamycin whereas gentamycin, tetracycline and penicillin resistance was more in non-biofilm producers.
Conclusion
This study shows high rate of circulating MRSA with a majority of these isolates are multi-drug resistant of which mostly are biofilm producers in our hospital setup. Development of antimicrobial stewardship program based on the local epidemiological data and national guidelines is the need of the hour.
doi:10.7860/JCDR/2015/13965.6417
PMCID: PMC4606231  PMID: 26500902
Congo red agar method; Methicillin resistant Staphylcoccus aureus; Vancomycin resistant Staphylcoccus aureus
21.  Rifampicin resistance mutations in the 81 bp RRDR of rpoB gene in Mycobacterium tuberculosis clinical isolates using Xpert MTB/RIF in Khyber Pakhtunkhwa, Pakistan: a retrospective study 
BMC Infectious Diseases  2016;16:413.
Background
Multi-drug resistant tuberculosis (MDR-TB) is a major public health problem especially in developing countries. World Health Organization (WHO) recommends use of Xpert MTB/RIF assay to simultaneously detecting Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance. The primary objective of this study was to determine the frequency of MDR-TB in patients suspected to have drug resistance in Khyber Pakhtunkhwa. The frequency of probes for various rpoB gene mutations using Xpert MTB/RIF assay within 81 bp RRDR (Rifampicin Resistance Determining Region) was the secondary objective.
Methods
A total of 2391 specimens, received at Programmatic Management of Drug Resistant TB (PMDT) Unit, Lady Reading Hospital (LRH) Peshawar, Pakistan, between October 2011 and December 2014, were analyzed by Xpert MTB/RIF test. MTB positive with rifampicin resistance were further analyzed to first line anti-mycobacterial drug susceptibility testing (DST) using middle brook 7H10 medium. The data was analyzed using statistical software; SPSS version 18.
Results
Out of 2391 specimens, 1408 (59 %) were found positive for MTB and among them, 408 (29 %) showed rifampicin-resistance with four different rpoB gene mutations within 81 bp RRDR. The frequency of various probes among RIF-resistant isolates was observed as: probe E, in 314 out of 408 isolates; B, 44 out of 408; A, 5 out of 408; D, 34 out of 408; and probe C was observed among 6 out of 408 RIF-resistant isolates. The probe A&B and E&D mutation combination was found in only 1 isolate in each case, while B&D mutation combination was detected among 3 out of 408 RIF-resistant isolates.
Conclusions
Hence, it is concluded from our study on a selected population, 29 % of patients had MDR-TB. Probe E related mutations (also known as codon 531and 533) were the most common rpoB genetic mutation [314 (77 %)], acknowledged by Xpert MTB/RIF assay. Least mutation was detected within the sequence 511 (1.2 %).
doi:10.1186/s12879-016-1745-2
PMCID: PMC4983047  PMID: 27519406
RRDR; RNA polymerase B gene; MDR-TB; Xpert MTB/RIF
22.  Antimicrobial susceptibility patterns and characterization of clinical isolates of Staphylococcus aureus in KwaZulu-Natal province, South Africa 
Background
Antimicrobial resistance of Staphylococcus aureus especially methicillin-resistant S. aureus (MRSA) continues to be a problem for clinicians worldwide. However, few data on the antibiotic susceptibility patterns of S. aureus isolates in South Africa have been reported and the prevalence of MRSA in the KwaZulu-Natal (KZN) province is unknown. In addition, information on the characterization of S. aureus in this province is unavailable. This study investigated the susceptibility pattern of 227 S. aureus isolates from the KZN province, South Africa. In addition, characterization of methicillin-sensitive S. aureus (MSSA) and MRSA are reported in this survey.
Methods
The in-vitro activities of 20 antibiotics against 227 consecutive non-duplicate S. aureus isolates from clinical samples in KZN province, South Africa were determined by the disk-diffusion technique. Isolates resistant to oxacillin and mupirocin were confirmed by PCR detection of the mecA and mup genes respectively. PCR-RFLP of the coagulase gene was employed in the characterization of MSSA and MRSA.
Results
All the isolates were susceptible to vancomycin, teicoplanin and fusidic acid, and 26.9% of isolates studied were confirmed as MRSA. More than 80% of MRSA were resistant to at least four classes of antibiotics and isolates grouped in antibiotype 8 appears to be widespread in the province. The MSSA were also susceptible to streptomycin, neomycin and minocycline, while less than 1% was resistant to chloramphenicol, ciprofloxacin, rifampicin and mupirocin. The inducible MLSB phenotype was detected in 10.8% of MSSA and 82% of MRSA respectively, and one MSSA and one MRSA exhibited high-level resistance to mupirocin. There was good correlation between antibiotyping and PCR-RFLP of the coagulase gene in the characterization of MRSA in antibiotypes 1, 5 and 12.
Conclusion
In view of the high resistance rates of MRSA to gentamicin, erythromycin, clindamycin, rifampicin and trimethoprim, treatment of MRSA infections in this province with these antibacterial agents would be unreliable. There is an emerging trend of mupirocin resistance among S. aureus isolates in the province. PCR-RFLP of the coagulase gene was able to distinguish MSSA from MRSA and offers an attractive option to be considered in the rapid epidemiological analysis of S. aureus in South Africa. Continuous surveillance on resistance patterns and characterization of S. aureus in understanding new and emerging trends in South Africa is of utmost importance.
doi:10.1186/1471-2334-6-125
PMCID: PMC1564024  PMID: 16875502
23.  Xpert® Mtb/Rif assay for pulmonary tuberculosis and rifampicin resistance in adults 
Background
Accurate, rapid detection of tuberculosis (TB) and TB drug resistance is critical for improving patient care and decreasing TB transmission. Xpert® MTB/RIF assay is an automated test that can detect both TB and rifampicin resistance, generally within two hours after starting the test, with minimal hands-on technical time. The World Health Organization (WHO) issued initial recommendations on Xpert® MTB/RIF in early 2011. A Cochrane Review on the diagnostic accuracy of Xpert® MTB/RIF for pulmonary TB and rifampicin resistance was published January 2013. We performed this updated Cochrane Review as part of a WHO process to develop updated guidelines on the use of the test.
Objectives
To assess the diagnostic accuracy of Xpert® MTB/RIF for pulmonary TB (TB detection), where Xpert® MTB/RIF was used as both an initial test replacing microscopy and an add-on test following a negative smear microscopy result.
To assess the diagnostic accuracy of Xpert® MTB/RIF for rifampicin resistance detection, where Xpert® MTB/RIF was used as the initial test replacing culture-based drug susceptibility testing (DST).
The populations of interest were adults presumed to have pulmonary, rifampicin-resistant or multidrug-resistant TB (MDR-TB), with or without HIV infection. The settings of interest were intermediate- and peripheral-level laboratories. The latter may be associated with primary health care facilities.
Search methods
We searched for publications in any language up to 7 February 2013 in the following databases: Cochrane Infectious Diseases Group Specialized Register; MEDLINE; EMBASE; ISI Web of Knowledge; MEDION; LILACS; BIOSIS; and SCOPUS. We also searched the metaRegister of Controlled Trials (mRCT) and the search portal of the WHO International Clinical Trials Registry Platform to identify ongoing trials.
Selection criteria
We included randomized controlled trials, cross-sectional studies, and cohort studies using respiratory specimens that allowed for extraction of data evaluating Xpert® MTB/RIF against the reference standard. We excluded gastric fluid specimens. The reference standard for TB was culture and for rifampicin resistance was phenotypic culture-based DST.
Data collection and analysis
For each study, two review authors independently extracted data using a standardized form. When possible, we extracted data for subgroups by smear and HIV status. We assessed the quality of studies using QUADAS-2 and carried out meta-analyses to estimate pooled sensitivity and specificity of Xpert® MTB/RIF separately for TB detection and rifampicin resistance detection. For TB detection, we performed the majority of analyses using a bivariate random-effects model and compared the sensitivity of Xpert® MTB/RIF and smear microscopy against culture as reference standard. For rifampicin resistance detection, we undertook univariate meta-analyses for sensitivity and specificity separately to include studies in which no rifampicin resistance was detected.
Main results
We included 27 unique studies (integrating nine new studies) involving 9557 participants. Sixteen studies (59%) were performed in low- or middle-income countries. For all QUADAS-2 domains, most studies were at low risk of bias and low concern regarding applicability.
As an initial test replacing smear microscopy, Xpert® MTB/RIF pooled sensitivity was 89% [95% Credible Interval (CrI) 85% to 92%] and pooled specificity 99% (95% CrI 98% to 99%), (22 studies, 8998 participants: 2953 confirmed TB, 6045 non-TB).As an add-on test following a negative smear microscopy result, Xpert®MTB/RIF pooled sensitivity was 67% (95% CrI 60% to 74%) and pooled specificity 99% (95% CrI 98% to 99%; 21 studies, 6950 participants).
For smear-positive, culture-positive TB, Xpert® MTB/RIF pooled sensitivity was 98% (95% CrI 97% to 99%; 21 studies, 1936 participants).
For people with HIV infection, Xpert® MTB/RIF pooled sensitivity was 79% (95% CrI 70% to 86%; 7 studies, 1789 participants), and for people without HIV infection, it was 86% (95% CrI 76% to 92%; 7 studies, 1470 participants).
Comparison with smear microscopy
In comparison with smear microscopy, Xpert® MTB/RIF increased TB detection among culture-confirmed cases by 23% (95% CrI 15% to 32%; 21 studies, 8880 participants).
For TB detection, if pooled sensitivity estimates for Xpert® MTB/RIF and smear microscopy are applied to a hypothetical cohort of 1000 patients where 10% of those with symptoms have TB, Xpert® MTB/RIF will diagnose 88 cases and miss 12 cases, whereas sputum microscopy will diagnose 65 cases and miss 35 cases.
Rifampicin resistance
For rifampicin resistance detection, Xpert® MTB/RIF pooled sensitivity was 95% (95% CrI 90% to 97%; 17 studies, 555 rifampicin resistance positives) and pooled specificity was 98% (95% CrI 97% to 99%; 24 studies, 2411 rifampicin resistance negatives). Among 180 specimens with nontuberculous mycobacteria (NTM), Xpert® MTB/RIF was positive in only one specimen that grew NTM (14 studies, 2626 participants).
For rifampicin resistance detection, if the pooled accuracy estimates for Xpert® MTB/RIF are applied to a hypothetical cohort of 1000 individuals where 15% of those with symptoms are rifampicin resistant, Xpert® MTB/RIF would correctly identify 143 individuals as rifampicin resistant and miss eight cases, and correctly identify 833 individuals as rifampicin susceptible and misclassify 17 individuals as resistant. Where 5% of those with symptoms are rifampicin resistant, Xpert® MTB/RIF would correctly identify 48 individuals as rifampicin resistant and miss three cases and correctly identify 931 individuals as rifampicin susceptible and misclassify 19 individuals as resistant.
Authors' conclusions
In adults thought to have TB, with or without HIV infection, Xpert® MTB/RIF is sensitive and specific. Compared with smear microscopy, Xpert® MTB/RIF substantially increases TB detection among culture-confirmed cases. Xpert® MTB/RIF has higher sensitivity for TB detection in smear-positive than smear-negative patients. Nonetheless, this test may be valuable as an add-on test following smear microscopy in patients previously found to be smear-negative. For rifampicin resistance detection, Xpert® MTB/RIF provides accurate results and can allow rapid initiation of MDR-TB treatment, pending results from conventional culture and DST. The tests are expensive, so current research evaluating the use of Xpert® MTB/RIF in TB programmes in high TB burden settings will help evaluate how this investment may help start treatment promptly and improve outcomes.
doi:10.1002/14651858.CD009593.pub3
PMCID: PMC4470349  PMID: 24448973
24.  Biological Cost of Rifampin Resistance from the Perspective of Staphylococcus aureus 
Antimicrobial Agents and Chemotherapy  2002;46(11):3381-3385.
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.
doi:10.1128/AAC.46.11.3381-3385.2002
PMCID: PMC128759  PMID: 12384339
25.  Correlation between rpoB gene mutation in Mycobacterium avium subspecies paratuberculosis and clinical rifabutin and rifampicin resistance for treatment of Crohn’s disease 
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.
doi:10.3748/wjg.14.2723
PMCID: PMC2709052  PMID: 18461657
Mycobacterium paratuberculosis; Crohn’s disease; Rifabutin; Rifampicin; rpoB; Minimum inhibitory concentration

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