First insights into the phylogenetic diversity of Mycobacterium tuberculosis in Nepal

Background Tuberculosis (TB) is a major public health problem in Nepal. Strain variation in Mycobacterium tuberculosis may influence the outcome of TB infection and disease. To date, the phylogenetic diversity of M. tuberculosis in Nepal is unknown. Methods and Findings We analyzed 261 M. tuberculosis isolates recovered from pulmonary TB patients recruited between August 2009 and August 2010 in Nepal. M. tuberculosis lineages were determined by single nucleotide polymorphisms (SNP) typing and spoligotyping. Drug resistance was determined by sequencing the hot spot regions of the relevant target genes. Overall, 164 (62.8%) TB patients were new, and 97 (37.2%) were previously treated. Any drug resistance was detected in 50 (19.2%) isolates, and 16 (6.1%) were multidrug-resistant. The most frequent M. tuberculosis lineage was Lineage 3 (CAS/Delhi) with 106 isolates (40.6%), followed by Lineage 2 (East-Asian lineage, includes Beijing genotype) with 84 isolates (32.2%), Lineage 4 (Euro-American lineage) with 41 (15.7%) isolates, and Lineage 1 (Indo-Oceanic lineage) with 30 isolates (11.5%). Based on spoligotyping, we found 45 different spoligotyping patterns that were previously described. The Beijing (83 isolates, 31.8%) and CAS spoligotype (52, 19.9%) were the dominant spoligotypes. A total of 36 (13.8%) isolates could not be assigned to any known spoligotyping pattern. Lineage 2 was associated with female sex (adjusted odds ratio [aOR] 2.58, 95% confidence interval [95% CI] 1.42–4.67, p = 0.002), and any drug resistance (aOR 2.79; 95% CI 1.43–5.45; p = 0.002). We found no evidence for an association of Lineage 2 with age or BCG vaccination status. Conclusions We found a large genetic diversity of M. tuberculosis in Nepal with representation of all four major lineages. Lineages 3 and 2 were dominating. Lineage 2 was associated with clinical characteristics. This study fills an important gap on the map of the M. tuberculosis genetic diversity in the Asian region.

Presence of new mecA and mph(C) variants conferring antibiotic resistance in Staphylococcus spp. isolated from the skin of horses before and after clinic admission

Because of the frequency of multiple antibiotic resistance, Staphylococcus species often represent a challenge in incisional infections of horses undergoing colic surgery. To investigate the evolution of antibiotic resistance patterns before and after preventative peri- and postoperative penicillin treatment, staphylococci were isolated from skin and wound samples at different times during hospitalization. Most staphylococci were normal skin commensals and belonged to the common coagulase-negative group. In some cases they turned out to be opportunistic pathogens present in wound infections. MICs were determined for 12 antibiotics, and antibiotic resistance genes were detected by microarray. At hospital admission, horses harbored staphylococci that were susceptible to antibiotics or resistant to one group of drugs, mainly due to the presence of new variants of the methicillin and macrolide resistance genes mecA and mph(C), respectively. After 3 days, the percentage of Staphylococcus isolates displaying antibiotic resistance, as well as the number of resistance genes per isolate, increased moderately in hospitalized horses without surgery or penicillin treatment but dramatically in hospitalized horses after colic surgery as well as penicillin treatment. Staphylococcus species displaying multiple resistance were found to harbor mainly genes conferring resistance to beta-lactams (mecA and blaZ), aminoglycosides [str and aac(6')-Ie-aph(2')-Ia], and trimethoprim [dfr(A) and dfr(D)]. Additional genes conferring resistance to macrolides [mph(C), erm(C), and erm(B)], tetracycline [tet(K) and tet(M)], chloramphenicol [cat(pC221) and cat(pC223)], and streptothricin (sat4) appeared in several strains. Hospitalization and preventive penicillin use were shown to act as selection agents for multidrug-resistant commensal staphylococcal flora.

Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria

A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.

Genetic characterization of antimicrobial resistance in coagulase-negative staphylococci from bovine mastitis milk

Coagulase-negative staphylococci (CNS; n=417) were isolated from bovine milk and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Nineteen different species were identified, and Staphylococcus xylosus, Staphylococcus chromogenes, Staphylococcus haemolyticus, and Staphylococcus sciuri were the most prevalent species. Resistance to oxacillin (47.0% of the isolates), fusidic acid (33.8%), tiamulin (31.9%), penicillin (23.3%), tetracycline (15.8%), streptomycin (9.6%), erythromycin (7.0%), sulfonamides (5%), trimethoprim (4.3%), clindamycin (3.4%), kanamycin (2.4%), and gentamicin (2.4%) was detected. Resistance to oxacillin was attributed to the mecA gene in 9.7% of the oxacillin-resistant isolates. The remaining oxacillin-resistant CNS did not contain the mecC gene or mecA1 promoter mutations. The mecA gene was detected in Staphylococcus fleurettii, Staphylococcus epidermidis, Staph. haemolyticus, and Staph. xylosus. Resistance to tetracycline was attributed to the presence of tet(K) and tet(L), penicillin resistance to blaZ, streptomycin resistance to str and ant(6)-Ia, and erythromycin resistance to erm(C), erm(B), and msr. Resistance to tiamulin and fusidic acid could not be attributed to an acquired resistance gene. In total, 15.1% of the CNS isolates were multidrug resistant (i.e., resistant to 2 or more antimicrobials). The remaining CNS isolates were susceptible to antimicrobials commonly used in mastitis treatment. Methicillin-resistant CNS isolates were diverse, as determined by mecA gene sequence analysis, staphylococcal cassette chromosome mec typing, and pulsed-field gel electrophoresis. Arginine catabolic mobile element types 1 and 3 were detected in both methicillin-resistant and methicillin-susceptible Staph. epidermidis and were associated with sequence types ST59 and ST111. Because this study revealed the presence of multidrug-resistant CNS in a heterogeneous CNS population, we recommend antibiogram analysis of CNS in persistent infections before treatment with antimicrobials.

Non-phenotypic tests to detect and characterize antibiotic resistance mechanisms in Enterobacteriaceae

In the past 2 decades, we have observed a rapid increase of infections due to multidrug-resistant Enterobacteriaceae. Regrettably, these isolates possess genes encoding for extended-spectrum β-lactamases (e.g., blaCTX-M, blaTEM, blaSHV) or plasmid-mediated AmpCs (e.g., blaCMY) that confer resistance to last-generation cephalosporins. Furthermore, other resistance traits against quinolones (e.g., mutations in gyrA and parC, qnr elements) and aminoglycosides (e.g., aminoglycosides modifying enzymes and 16S rRNA methylases) are also frequently co-associated. Even more concerning is the rapid increase of Enterobacteriaceae carrying genes conferring resistance to carbapenems (e.g., blaKPC, blaNDM). Therefore, the spread of these pathogens puts in peril our antibiotic options. Unfortunately, standard microbiological procedures require several days to isolate the responsible pathogen and to provide correct antimicrobial susceptibility test results. This delay impacts the rapid implementation of adequate antimicrobial treatment and infection control countermeasures. Thus, there is emerging interest in the early and more sensitive detection of resistance mechanisms. Modern non-phenotypic tests are promising in this respect, and hence, can influence both clinical outcome and healthcare costs. In this review, we present a summary of the most advanced methods (e.g., next-generation DNA sequencing, multiplex PCRs, real-time PCRs, microarrays, MALDI-TOF MS, and PCR/ESI MS) presently available for the rapid detection of antibiotic resistance genes in Enterobacteriaceae. Taking into account speed, manageability, accuracy, versatility, and costs, the possible settings of application (research, clinic, and epidemiology) of these methods and their superiority against standard phenotypic methods are discussed.

Contribution of the collagen adhesin Acm to pathogenesis of Enterococcus faecium in experimental endocarditis.

Enterococcus faecium is a multidrug-resistant opportunist causing difficult-to-treat nosocomial infections, including endocarditis, but there are no reports experimentally demonstrating E. faecium virulence determinants. Our previous studies showed that some clinical E. faecium isolates produce a cell wall-anchored collagen adhesin, Acm, and that an isogenic acm deletion mutant of the endocarditis-derived strain TX0082 lost collagen adherence. In this study, we show with a rat endocarditis model that TX0082 Deltaacm::cat is highly attenuated versus wild-type TX0082, both in established (72 h) vegetations (P < 0.0001) and for valve colonization 1 and 3 hours after infection (P or=50-fold reduction relative to an Acm producer) were found in three of these five nonadherent isolates, including the sequenced strain TX0016, by quantitative reverse transcription-PCR, indicating that acm transcription is downregulated in vitro in these isolates. However, examination of TX0016 cells obtained directly from infected rat vegetations by flow cytometry showed that Acm was present on 40% of cells grown during infection. Finally, we demonstrated a significant reduction in E. faecium collagen adherence by affinity-purified anti-Acm antibodies from E. faecium endocarditis patient sera, suggesting that Acm may be a potential immunotarget for strategies to control this emerging pathogen.

A functional collagen adhesin gene, acm, in clinical isolates of Enterococcus faecium correlates with the recent success of this emerging nosocomial pathogen.

Enterococcus faecium recently evolved from a generally avirulent commensal into a multidrug-resistant health care-associated pathogen causing difficult-to-treat infections, but little is known about the factors responsible for this change. We previously showed that some E. faecium strains express a cell wall-anchored collagen adhesin, Acm. Here we analyzed 90 E. faecium isolates (99% acm(+)) and found that the Acm protein was detected predominantly in clinically derived isolates, while the acm gene was present as a transposon-interrupted pseudogene in 12 of 47 isolates of nonclinical origin. A highly significant association between clinical (versus fecal or food) origin and collagen adherence (P

Molecular epidemiology of tuberculosis in Cambodian children

SUMMARY We analysed Mycobacterium tuberculosis strains from children, hospitalized from January 2004 to July 2008 in the largest paediatric hospital complex in Cambodia. Specimens were tested for drug susceptibility and genotypes. From the 260 children, 161 strains were available. The East African-Indian genotype family was the most common (59·0%), increasing in frequency with distance from the Phnom Penh area, while the frequency of the Beijing genotype family strains decreased. The drug resistance pattern showed a similar geographical gradient: lowest in the northwest (4·6%), intermediate in the central (17·1%), and highest in the southeastern (30·8%) parts of the country. Three children (1·9%) had multidrug-resistant tuberculosis. The Beijing genotype and streptomycin resistance were significantly associated (P < 0·001). As tuberculosis in children reflects recent transmission patterns in the community, multidrug resistance levels inform about the current quality of the tuberculosis programme.

In vitro Activity of Fosfomycin Alone and in Combination with Ceftriaxone or Azithromycin Against Clinical Neisseria gonorrhoeae Isolates.

New therapeutic strategies are needed to combat the emergence of infections due to multidrug-resistant Neisseria gonorrhoeae (Ng). In this study, fosfomycin (FOS) was tested against 89 Ng using the Etest method and showing MIC50/90s of only 8/16 μg/ml (range ≤ 1-32 μg/ml). FOS in combination with ceftriaxone (CRO) or azithromycin (AZT) was then evaluated using the checkerboard method for eight strains, including F89 (CRO-resistant) and AZT-HLR (high-level AZT-resistant). All combinations including FOS gave indifferent effects (fractional inhibitory concentration [FIC] index values between 1.2-2.3 for FOS plus CRO and between 1.8-3.2 for FOS plus AZT). Time-kill experiments for FOS, CRO, AZT and their combinations (at concentrations of 0.5×, 1×, 2× and 4× MIC) were performed against ATCC 49226, one Ng of NG-MAST ST1407, F89 and AZT-HLR. For all strains, at 24 hours results indicated that: i) FOS was bactericidal at 2× MIC concentrations but after >24 hours there was re-growth of bacteria; ii) CRO was bactericidal at 0.5× MIC; iii) AZT was bactericidal at 4× MIC; iv) CRO plus AZT was less bactericidal than CRO alone; v) FOS plus AZT was bactericidal at 2× MIC; vi) CRO plus AZT and FOS plus CRO were both bactericidal at 0.5× MIC, but the latter had more rapid effects. FOS is appealing for the management of Ng infections because of its single and oral formulation. However, our results suggest its use in combination with CRO. This strategy could, after appropriate clinical trials, be implemented for the treatment of infections due to isolates possessing resistance to CRO and/or AZT.

Antibiotic resistance and phylogenetic characterization of Acinetobacter baumannii strains isolated from commercial raw meat in Switzerland.

The spread of antibiotic-resistant bacteria through food has become a major public health concern because some important human pathogens may be transferred via the food chain. Acinetobacter baumannii is one of the most life-threatening gram-negative pathogens; multidrug-resistant (MDR) clones of A. baumannii are spreading worldwide, causing outbreaks in hospitals. However, the role of raw meat as a reservoir of A. baumannii remains unexplored. In this study, we describe for the first time the antibiotic susceptibility and fingerprint (repetitive extragenic palindromic PCR [rep-PCR] profile and sequence types [STs]) of A. baumannii strains found in raw meat retailed in Switzerland. Our results indicate that A. baumannii was present in 62 (25.0%) of 248 (CI 95%: 19.7 to 30.9%) meat samples analyzed between November 2012 and May 2013, with those derived from poultry being the most contaminated (48.0% [CI 95%: 37.8 to 58.3%]). Thirty-nine strains were further tested for antibiotic susceptibility and clonality. Strains were frequently not susceptible (intermediate and/or resistant) to third- and fourth-generation cephalosporins for human use (i.e., ceftriaxone [65%], cefotaxime [32%], ceftazidime [5%], and cefepime [2.5%]). Resistance to piperacillin-tazobactam, ciprofloxacin, colistin, and tetracycline was sporadically observed (2.5, 2.5, 5, and 5%, respectively), whereas resistance to carbapenems was not found. The strains were genetically very diverse from each other and belonged to 29 different STs, forming 12 singletons and 6 clonal complexes (CCs), of which 3 were new (CC277, CC360, and CC347). RepPCR analysis further distinguished some strains of the same ST. Moreover, some A. baumannii strains from meat belonged to the clonal complexes CC32 and CC79, similar to the MDR isolates responsible for human infections. In conclusion, our findings suggest that raw meat represents a reservoir of MDR A. baumannii and may serve as a vector for the spread of these pathogens into both community and hospital settings.

Bacterial infections in cirrhosis: a position statement based on the EASL Special Conference 2013.

Bacterial infections are very common and represent one of the most important reasons of progression of liver failure, development of liver-related complications, and mortality in patients with cirrhosis. In fact, bacterial infections may be a triggering factor for the occurrence of gastrointestinal bleeding, hypervolemic hyponatremia, hepatic encephalopathy, kidney failure, and development of acute-on-chronic liver failure. Moreover, infections are a very common cause of repeated hospitalizations, impaired health-related quality of life, and increased healthcare costs in cirrhosis. Bacterial infections develop as a consequence of immune dysfunction that occurs progressively during the course of cirrhosis. In a significant proportion of patients, infections are caused by gram-negative bacteria from intestinal origin, yet gram-positive bacteria are a frequent cause of infection, particularly in hospitalized patients. In recent years, infections caused by multidrug-resistant bacteria are becoming an important clinical problem in many countries. The reduction of the negative clinical impact of infections in patients with cirrhosis may be achieved by a combination of prophylactic measures, such as administration of antibiotics, to reduce the occurrence of infections in high-risk groups together with early identification and management of infection once it has developed. Investigation on the mechanisms of altered gut microflora, translocation of bacteria, and immune dysfunction may help develop more effective and safe methods of prevention compared to those that are currently available. Moreover, research on biomarkers of early infection may be useful in early diagnosis and treatment of infections. The current manuscript reports an in-depth review and a position statement on bacterial infections in cirrhosis.

Evolutionary history and global spread of the Mycobacterium tuberculosis Beijing lineage.

Mycobacterium tuberculosis strains of the Beijing lineage are globally distributed and are associated with the massive spread of multidrug-resistant (MDR) tuberculosis in Eurasia. Here we reconstructed the biogeographical structure and evolutionary history of this lineage by genetic analysis of 4,987 isolates from 99 countries and whole-genome sequencing of 110 representative isolates. We show that this lineage initially originated in the Far East, from where it radiated worldwide in several waves. We detected successive increases in population size for this pathogen over the last 200 years, practically coinciding with the Industrial Revolution, the First World War and HIV epidemics. Two MDR clones of this lineage started to spread throughout central Asia and Russia concomitantly with the collapse of the public health system in the former Soviet Union. Mutations identified in genes putatively under positive selection and associated with virulence might have favored the expansion of the most successful branches of the lineage.

Tuberculosis-related mortality in people living with HIV in Europe and Latin America: an international cohort study

BACKGROUND Management of tuberculosis in patients with HIV in eastern Europe is complicated by the high prevalence of multidrug-resistant tuberculosis, low rates of drug susceptibility testing, and poor access to antiretroviral therapy (ART). We report 1 year mortality estimates from a multiregional (eastern Europe, western Europe, and Latin America) prospective cohort study: the TB:HIV study. METHODS Consecutive HIV-positive patients aged 16 years or older with a diagnosis of tuberculosis between Jan 1, 2011, and Dec 31, 2013, were enrolled from 62 HIV and tuberculosis clinics in 19 countries in eastern Europe, western Europe, and Latin America. The primary endpoint was death within 12 months after starting tuberculosis treatment; all deaths were classified according to whether or not they were tuberculosis related. Follow-up was either until death, the final visit, or 12 months after baseline, whichever occurred first. Risk factors for all-cause and tuberculosis-related deaths were assessed using Kaplan-Meier estimates and Cox models. FINDINGS Of 1406 patients (834 in eastern Europe, 317 in western Europe, and 255 in Latin America), 264 (19%) died within 12 months. 188 (71%) of these deaths were tuberculosis related. The probability of all-cause death was 29% (95% CI 26-32) in eastern Europe, 4% (3-7) in western Europe, and 11% (8-16) in Latin America (p<0·0001) and the corresponding probabilities of tuberculosis-related death were 23% (20-26), 1% (0-3), and 4% (2-8), respectively (p<0·0001). Patients receiving care outside eastern Europe had a 77% decreased risk of death: adjusted hazard ratio (aHR) 0·23 (95% CI 0·16-0·31). In eastern Europe, compared with patients who started a regimen with at least three active antituberculosis drugs, those who started fewer than three active antituberculosis drugs were at a higher risk of tuberculosis-related death (aHR 3·17; 95% CI 1·83-5·49) as were those who did not have baseline drug-susceptibility tests (2·24; 1·31-3·83). Other prognostic factors for increased tuberculosis-related mortality were disseminated tuberculosis and a low CD4 cell count. 18% of patients were receiving ART at tuberculosis diagnosis in eastern Europe compared with 44% in western Europe and 39% in Latin America (p<0·0001); 12 months later the proportions were 67% in eastern Europe, 92% in western Europe, and 85% in Latin America (p<0·0001). INTERPRETATION Patients with HIV and tuberculosis in eastern Europe have a risk of death nearly four-times higher than that in patients from western Europe and Latin America. This increased mortality rate is associated with modifiable risk factors such as lack of drug susceptibility testing and suboptimal initial antituberculosis treatment in settings with a high prevalence of drug resistance. Urgent action is needed to improve tuberculosis care for patients living with HIV in eastern Europe. FUNDING EU Seventh Framework Programme.

BlaB-15, a new BlaB metallo-β-lactamase variant found in an Elizabethkingia miricola clinical isolate

A multidrug-resistant strain of Elizabethkingia miricola was isolated from the urine of a 2-year-old boy hospitalized for severe clinical conditions. The strain produces 2 metallo-β-lactamases belonging to subclasses B1 and B3: a new BlaB variant (BlaB-15) and a GOB-7–like enzyme.

Analysis of Mycobacterium tuberculosis in the state of Texas for rifampin resistance using molecular beacons

Mycobacterium tuberculosis, a bacillus known to cause disease in humans since ancient times, is the etiological agent of tuberculosis (TB). The infection is primarily pulmonary, although other organs may also be affected. The prevalence of pulmonary TB disease in the US is highest along the US-Mexico border, and of the four US states bordering Mexico, Texas had the second highest percentage of cases of TB disease among Mexico-born individuals in 1999 (CDC, 2001). Between the years of 1993 and 1998, the prevalence of drug-resistant (DR) TB was 9.1% among Mexican-born individuals and 4.4% among US-born individuals (CDC, 2001). In the same time period, the prevalence of multi-drug resistant (MDR) TB was 1.4% among Mexican-born individuals and 0.6% among US-born individuals (CDC, 2001). There is a renewed urgency in the quest for faster and more effective screening, diagnosis, and treatment methods for TB due to the resurgence of tuberculosis in the US during the mid-1980s and early 1990s (CDC, 2007a), and the emergence of drug-resistant, multidrug-resistant, and extremely drug-resistant tuberculosis worldwide. Failure to identify DR and MDR-TB quickly leads to poorer treatment outcomes (CDC, 2007b). The recent rise in TB/HIV comorbidity further complicates TB control efforts. The gold standard for identification of DR-TB requires mycobacterial growth in culture, a technique taking up to three weeks, during which time DR/MDR-TB individuals harboring resistant organisms may be receiving inappropriate treatment. The goal of this study was to determine the sensitivity and specificity of real-time quantitative polymerase chain reaction (qPCR) using molecular beacons in the Texas population. qPCR using molecular beacons is a novel approach to detect mycobacterial mutations conferring drug resistance. This technique is time-efficient and has been shown to have high sensitivity and specificity in several populations worldwide. Rifampin (RIF) susceptibility was chosen as the test parameter because strains of M. tuberculosis which are resistant to RIF are likely to also be MDR. Due to its status as a point of entry for many immigrants into the US, control efforts against TB and drug-resistant TB in Texas is a vital component of prevention efforts in the US as a whole. We show that qPCR using molecular beacons has high sensitivity and specificity when compared with culture (94% and 87%, respectively) and DNA sequencing (90% and 96%, respectively). We also used receiver operator curve analysis to calculate cutoff values for the objective determination of results obtained by qPCR using molecular beacons. ^