ABSTRACT
Genome sequencing and assembly of viral genomes within the Herpesviridae family, particularly herpes simplex virus (HSV), have been challenging due to the large size (~154 Kb), high GC content (68%), and nucleotide variations arising during replication. Oxford Nanopore Technology (ONT) has been successful in obtaining read lengths ranging from 100 Kb up to 2.3 Mb. We have optimized DNA extraction and sequencing with ONT to capture the whole genome of HSV-1 as a single read. Although previous studies described the presence of four different genome isomers of HSV, we provided the first report on capturing all four variants' full-length genome as single reads. These isomers were found to be present in almost equal proportion in the sequenced DNA preparation. IMPORTANCE With the advent of next-generation sequencing platforms, genome sequencing of viruses can be performed in a relatively shorter time frame in even the most austere conditions. Ultralong read sequencing platforms, such as Oxford Nanopore Technology (ONT), have made it possible to capture the full-length genome of DNA viruses as a single read. By optimizing ONT for this purpose, we captured the genome (~154 Kb) of a clinical strain of herpes simplex virus 1 (HSV-1). Additionally, we captured full-length sequences of the four isomers of lab-grown HSV-1 virus and were able to determine the frequency of each within the isogenic population. This method will open new directions in studying the significance of these isomers and their clinical relevance to HSV-1 infections. It will also improve basic studies on the recombination and replication of this virus.
Subject(s)
Herpes Simplex , Nanopore Sequencing , Humans , Simplexvirus , High-Throughput Nucleotide Sequencing/methods , Nucleotides , Sequence Analysis, DNA/methodsABSTRACT
Mycobacterium tuberculosis (Mtb) possesses five type VII secretion systems (T7SS), virulence determinants that include the secretion apparatus and associated secretion substrates. Mtb strains deleted for the genes encoding substrates of the ESX-3 T7SS, esxG or esxH, require iron supplementation for in vitro growth and are highly attenuated in vivo. In a subset of infected mice, suppressor mutants of esxG or esxH deletions were isolated, which enabled growth to high titers or restored virulence. Suppression was conferred by mechanisms that cause overexpression of an ESX-3 paralogous region that lacks genes for the secretion apparatus but encodes EsxR and EsxS, apparent ESX-3 orphan substrates that functionally compensate for the lack of EsxG or EsxH. The mechanisms include the disruption of a transcriptional repressor and a massive 38- to 60-fold gene amplification. These data identify an iron acquisition regulon, provide insight into T7SS, and reveal a mechanism of Mtb chromosome evolution involving "accordion-type" amplification.
Subject(s)
Mycobacterium tuberculosis/genetics , Type VII Secretion Systems/genetics , Animals , Bacterial Secretion Systems/genetics , Biological Evolution , Evolution, Molecular , Gene Amplification/genetics , Mice , Mycobacterium tuberculosis/metabolism , Type VII Secretion Systems/physiology , Virulence , Virulence Factors/geneticsABSTRACT
The current emergence of multi-, extensively-, extremely-, and total-drug resistant strains of Mycobacterium tuberculosis poses a major health, social, and economic threat, and stresses the need to develop new therapeutic strategies. The notion of phage therapy against bacteria has been around for more than a century and, although its implementation was abandoned after the introduction of drugs, it is now making a comeback and gaining renewed interest in Western medicine as an alternative to treat drug-resistant pathogens. Mycobacteriophages are genetically diverse viruses that specifically infect mycobacterial hosts, including members of the M. tuberculosis complex. This review describes general features of mycobacteriophages and their mechanisms of killing M. tuberculosis, as well as their advantages and limitations as therapeutic and prophylactic agents against drug-resistant M. tuberculosis strains. This review also discusses the role of human lung micro-environments in shaping the availability of mycobacteriophage receptors on the M. tuberculosis cell envelope surface, the risk of potential development of bacterial resistance to mycobacteriophages, and the interactions with the mammalian host immune system. Finally, it summarizes the knowledge gaps and defines key questions to be addressed regarding the clinical application of phage therapy for the treatment of drug-resistant tuberculosis.
Subject(s)
Mycobacteriophages/physiology , Mycobacterium tuberculosis/virology , Phage Therapy , Tuberculosis, Multidrug-Resistant/therapy , Tuberculosis/therapy , Animals , Bacterial Load , Host Specificity , Host-Pathogen Interactions/immunology , Humans , Immune System/immunology , Immune System/metabolism , Lung/immunology , Lung/microbiology , Mycobacterium tuberculosis/immunology , Phage Therapy/methods , Treatment Outcome , Tuberculosis/immunology , Tuberculosis/microbiology , Tuberculosis, Multidrug-Resistant/immunology , Tuberculosis, Multidrug-Resistant/microbiologyABSTRACT
Phenotypic testing for drug susceptibility of Mycobacterium tuberculosis is critical to basic research and managing the evolving problem of antimicrobial resistance in tuberculosis management, but it remains a specialized technique to which access is severely limited. Here, we report on the development and validation of an improved phage-mediated detection system for M. tuberculosis We incorporated a nanoluciferase (Nluc) reporter gene cassette into the TM4 mycobacteriophage genome to create phage TM4-nluc. We assessed the performance of this reporter phage in the context of cellular limit of detection and drug susceptibility testing using multiple biosafety level 2 drug-sensitive and -resistant auxotrophs as well as virulent M. tuberculosis strains. For both limit of detection and drug susceptibility testing, we developed a standardized method consisting of a 96-hour cell preculture followed by a 72-hour experimental window for M. tuberculosis detection with or without antibiotic exposure. The cellular limit of detection of M. tuberculosis in a 96-well plate batch culture was ≤102 CFU. Consistent with other phenotypic methods for drug susceptibility testing, we found TM4-nluc to be compatible with antibiotics representing multiple classes and mechanisms of action, including inhibition of core central dogma functions, cell wall homeostasis, metabolic inhibitors, compounds currently in clinical trials (SQ109 and Q203), and susceptibility testing for bedaquiline, pretomanid, and linezolid (components of the BPaL regimen for the treatment of multi- and extensively drug-resistant tuberculosis). Using the same method, we accurately identified rifampin-resistant and multidrug-resistant M. tuberculosis strains.IMPORTANCEMycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.
Subject(s)
Antitubercular Agents/pharmacology , Drug Resistance, Bacterial , Microbial Sensitivity Tests/methods , Mycobacteriophages/genetics , Mycobacterium tuberculosis/drug effects , Rifampin/pharmacology , Humans , Luciferases/genetics , Luciferases/metabolism , Luminescent Measurements , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/virology , Tuberculosis, Multidrug-Resistant/microbiology , Tuberculosis, Pulmonary/microbiologyABSTRACT
Large genomic deletions (LGDs) (6 to 63 kbp) were observed in isoniazid-resistant Mycobacterium tuberculosis mutants derived from four M. tuberculosis strains. These LGDs had no growth defect in vitro but could be defective in intracellular growth and showed various sensitivities toward oxidative stress despite lacking katG The LGD regions comprise 74 genes, mostly of unknown function, that may be important for M. tuberculosis intracellular growth and protection against oxidative stress.
Subject(s)
Isoniazid , Mycobacterium tuberculosis , Antitubercular Agents/pharmacology , Bacterial Proteins/genetics , Catalase/genetics , Drug Resistance, Bacterial/genetics , Isoniazid/pharmacology , Mutation , Mycobacterium tuberculosis/geneticsABSTRACT
OBJECTIVE: Though several genetic variants have been recognized to be associated with susceptibility to Tuberculosis (TB) infection and disease, a recent observation on the association of TIRAP C975T (S180L) variants with TB disease severity in mice model prompted us to assess their relevance in humans. In addition, TIRAP variants have also been reported to be associated with varied circulating Interferon-gamma induced protein (IP-10) levels. We investigated the association of TIRAP variants with severity of TB disease and IP-10 production in humans, which may be useful in predicting poor clinical outcome. METHODS: Culture positive symptomatic adult pulmonary TB (PTB) patients enrolled between August 2014 and October 2017 were included in this investigation. Allelic discrimination PCR and conventional IP-10 quantification methods were employed for genotyping and IP-10 measurement followed by statistical investigations to analyse patients' variables. RESULTS: Among 211 participants, C/C allele was identified in 70% (nâ¯=â¯147); 26% (nâ¯=â¯55) and 4% (nâ¯=â¯9) had C/T and T/T alleles respectively. There was no significant association between TIRAP variants and smear grade, chest-X-ray score, symptom severity score and circulating IP-10 levels. However, significant association was observed between i) circulating IP-10 levels and time to Mycobacterium Growth Indicator Tube (MGIT) culture conversion (pâ¯=0.032); ii) smear grade among active TB patients and circulating IP-10 levels (pâ¯=0â¯.032). CONCLUSIONS: Although mice experiments showed promising results with more severe disease in C/C and T/T individuals, we did not observe any such association in humans.
Subject(s)
Chemokine CXCL10/blood , Membrane Glycoproteins/genetics , Polymorphism, Single Nucleotide , Receptors, Interleukin-1/genetics , Tuberculosis, Pulmonary/genetics , Adult , Case-Control Studies , Female , Gene Frequency , Genetic Association Studies , Genetic Predisposition to Disease , Humans , Male , Middle Aged , Mycobacterium tuberculosis/isolation & purification , Severity of Illness Index , Tuberculosis, Pulmonary/immunology , Tuberculosis, Pulmonary/microbiologyABSTRACT
The emergence of drug resistance in Helicobacter pylori has resulted in a greater need for susceptibility-guided treatment. While the alleles associated with resistance to clarithromycin and levofloxacin have been defined, there are limited data regarding the molecular mechanisms underlying resistance to other antimicrobials. Using H. pylori isolates from 42 clinical specimens, we compared phenotypic and whole-genome sequencing (WGS)-based detection of resistance. Phenotypic resistance correlated with the presence of alleles of 23S rRNA (A2142G/A2143G) for clarithromycin (kappa coefficient, 0.84; 95% confidence interval [CI], 0.67 to 1.0) and gyrA (N87I/N87K/D91Y/D91N/D91G/D99N) for levofloxacin (kappa coefficient, 0.90; 95% CI, 0.77 to 1.0). Phenotypic resistance to amoxicillin in three isolates correlated with mutations in pbp1, pbp2, and/or pbp3 within coding regions near known amoxicillin binding motifs. All isolates were phenotypically susceptible to tetracycline, although four bore a mutation in 16S rRNA (A926G). For metronidazole, nonsense mutations and R16H substitutions in rdxA correlated with phenotypic resistance (kappa coefficient, 0.76; 95% CI, 0.56 to 0.96). Previously identified mutations in the rpoB rifampin resistance-determining region (RRDR) were not present, but 14 novel mutations outside the RRDR were found in rifampin-resistant isolates. WGS also allowed for strain lineage determination, which may be important for future studies in associating precise MICs with specific resistance alleles. In summary, WGS allows for broad analyses of H. pylori isolates, and our findings support the use of WGS for the detection of clarithromycin and levofloxacin resistance. Additional studies are warranted to better define mutations conferring resistance to amoxicillin, tetracycline, and rifampin, but combinatorial analyses for rdxA gene truncations and R16H mutations have utility for determining metronidazole resistance.
Subject(s)
Anti-Bacterial Agents , Helicobacter Infections , Helicobacter pylori , Anti-Bacterial Agents/pharmacology , Clarithromycin/pharmacology , Drug Resistance, Bacterial/drug effects , Drug Resistance, Bacterial/genetics , Female , Helicobacter Infections/drug therapy , Helicobacter Infections/genetics , Helicobacter pylori/drug effects , Helicobacter pylori/genetics , Humans , Male , Metronidazole , Microbial Sensitivity Tests , Mutation , New York , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 23S/genetics , Young AdultABSTRACT
We report here the first case of pulmonary infection due to Mycobacterium kyorinense in a 55-year-old hypertensive woman treated for pulmonary tuberculosis earlier on two occasions. She presented with productive cough, intermittent episode of left-sided chest pain, loss of appetite, low-grade fever, and breathlessness. Sputum cultures revealed non-tuberculous mycobacteria (NTM). She remained persistently symptomatic with sputum cultures positive for acid-fast bacilli even after 6 months of treatment. Hence, a 16SrRNA gene amplification and sequencing were done that revealed M. kyorinense. Based on the guidelines of the American Thoracic Society, she was started on weight-based dosing of clarithromycin, levofloxacin, ethambutol, isoniazid and injection amikacin daily. The patient improved symptomatically and became culture-negative after 3 months of therapy with the above regimen and continued to be culture negative for 12 months of treatment. She continues to remain symptom-free without evidence of any clinical or bacteriological relapse.
Subject(s)
Antitubercular Agents/therapeutic use , Mycobacterium Infections, Nontuberculous/diagnosis , Mycobacterium Infections, Nontuberculous/drug therapy , Mycobacterium/genetics , Respiratory Tract Infections/diagnosis , Amikacin/therapeutic use , Clarithromycin/therapeutic use , Ethambutol/therapeutic use , Female , Humans , Isoniazid/therapeutic use , Levofloxacin/therapeutic use , Middle Aged , Mycobacterium/isolation & purification , Mycobacterium Infections, Nontuberculous/microbiology , RNA, Ribosomal, 16S/genetics , Respiratory Tract Infections/drug therapy , Respiratory Tract Infections/microbiologyABSTRACT
Acinetobacter baumannii is an important human pathogen and considered as a major threat due to its extreme drug resistance. In this study, the genome of a hyper-virulent MDR strain PKAB07 of A. baumannii isolated from an Indian patient was sequenced and analyzed to understand its mechanisms of virulence, resistance and evolution. Comparative genome analysis of PKAB07 revealed virulence and resistance related genes scattered throughout the genome, instead of being organized as an island, indicating the highly mosaic nature of the genome. Many intermittent horizontal gene transfer events, insertion sequence (IS) element insertions identified were augmenting resistance machinery and elevating the SNP densities in A. baumannii eventually aiding in their swift evolution. ISAba1, the most widely distributed insertion sequence in A. baumannii was found in multiple sites in PKAB07. Out of many ISAba1 insertions, we identified novel insertions in 9 different genes wherein insertional inactivation of adeN (tetR type regulator) was significant. To assess the significance of this disruption in A. baumannii, adeN mutant and complement strains were constructed in A. baumannii ATCC 17978 strain and studied. Biofilm levels were abrogated in the adeN knockout when compared with the wild type and complemented strain of adeN knockout. Virulence of the adeN knockout mutant strain was observed to be high, which was validated by in vitro experiments and Galleria mellonella infection model. The overexpression of adeJ, a major component of AdeIJK efflux pump observed in adeN knockout strain could be the possible reason for the elevated virulence in adeN mutant and PKB07 strain. Knocking out of adeN in ATCC strain led to increased resistance and virulence at par with the PKAB07. Disruption of tetR type regulator adeN by ISAba1 consequently has led to elevated virulence in this pathogen.
Subject(s)
Acinetobacter Infections/microbiology , Acinetobacter baumannii/genetics , Acinetobacter baumannii/pathogenicity , Bacterial Proteins/metabolism , Mutagenesis, Insertional , Trans-Activators/metabolism , Acinetobacter baumannii/growth & development , Acinetobacter baumannii/metabolism , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Humans , Interspersed Repetitive Sequences , Trans-Activators/genetics , VirulenceABSTRACT
PURPOSE: The objective of the present study is to investigate the diverse resistance determinants, their association with insertion sequence mobile elements and predilection of a particular clone for such associations in Acinetobacter baumannii. METHODOLOGY: Fifty-four consecutive isolates collected during 2011-2012 from a tertiary care hospital were subjected to susceptibility testing followed by PCR screening of commonly reported ß-lactamases and 16S rRNA methyltransferase encoding genes. The integrity of resistance-nodulation-cell division efflux pump-related genes in their respective operons was also investigated. RESULTS: ß-Lactamase genes such as blaADC (100â%), blaOXA-23 (81â%), blaPER-1 (81â%), blaIMP-1 (31â%) and blaNDM-1 (15â%) were found to be present more frequently while blaVIM-2 and blaOXA-24 were not observed in our study population. ISAba1 was associated only with blaOXA-51-like like (30â%), blaOXA-23-like (55â%) and blaADC-like (33â%). armA was found in 87â% of isolates and ISAba1 linked with one novel variant of ADC, namely blaADC-82, which was identified to have 15 nucleotide differences with blaADC-79, and this finding is of much significance. In many isolates, efflux pump genes were not intact, resulting in severely altered effluxing functions. For the first time, we have identified ISAba1-mediated disruption of adeN among the isolates of ST 195B, which would have led to overexpression of AdeIJK efflux pump causing elevated resistance. Multilocus sequence typing revealed the predominance of CC 92B (IC-IIB) and CC 447B clonal complexes. CONCLUSION: High incidence of IC-II clones, novel resistance determinants (ADC-82) and elevated resistance mediated by ISAba1 reported here will be of enormous importance while assessing the emergence of extremely resistant A. baumannii in India.
Subject(s)
Acinetobacter baumannii/genetics , Bacterial Proteins/genetics , Drug Resistance, Multiple, Bacterial/genetics , beta-Lactamases/genetics , Acinetobacter baumannii/enzymology , Adolescent , Adult , Aged , Alleles , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Carbapenems/pharmacology , Child , Child, Preschool , Cloning, Molecular , Female , Hospitalization , Humans , India , Infant , Infant, Newborn , Male , Methyltransferases/genetics , Methyltransferases/metabolism , Microbial Sensitivity Tests , Middle Aged , Multilocus Sequence Typing , RNA, Ribosomal, 16S/genetics , Young Adult , beta-Lactamases/classificationABSTRACT
Fusobacterium nucleatum plays a key role in several diseases such as periodontitis, gingivitis, appendicitis, and inflammatory bowel disease (IBD). The development of antibiotic resistance by this bacterium demands novel therapeutic intervention. Our recent study has reported UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA) as one of the potential target proteins in F. nucleatum. In this study, we proposed two novel MurA inhibitors through in silico screening and evaluated their mode of inhibition by in vitro experiments. It was found that MurA structural arrangement (inside-out α/ß barrel) was stabilized by L/FXXXG(A) motif-based interactions. The protein was maintained in an open or substrate-free conformation due to repulsive forces between two parallelly arranged positively charged residues of domain I and II. In this conformation, we identified six best compounds that held key interactions with the substrate-binding pocket via a structure-based virtual screening of natural and chemical compound libraries. However, among these, only orientin and quercetin-3-O-d-glucuronide (Q3G) showed better interaction capability through consistent H-bond occupancy and lowest binding free energy during molecular dynamic simulations. In vitro inhibition studies evidenced the mixed and uncompetitive mode of inhibition by orientin and Q3G, respectively, with purified MurA protein. This explains the binding of orientin in both open and closed (substrate-bound) conformations of MurA, and Q3G binding in only closed conformation. Therefore, the Q3G binding mode was predicted on a MurA-substrate complex, which highlighted its constant H-bond with Cys118, a phosphoenolpyruvate (PEP) interacting residue. This suggests that Q3G may interrupt the PEP binding, thereby inhibiting the MurA activity. Thus, the current study discusses the structure of MurA and demonstrates the inhibitory action of two novel compounds.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Computational Biology/methods , Enzyme Inhibitors/pharmacology , Fusobacterium nucleatum/enzymology , Alkyl and Aryl Transferases/chemistry , Bacterial Proteins/antagonists & inhibitors , Computer Simulation , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Flavonoids/chemistry , Flavonoids/pharmacology , Glucosides/chemistry , Glucosides/pharmacology , Hydrogen Bonding , Models, Molecular , Molecular Dynamics Simulation , Protein Conformation , Quercetin/analogs & derivatives , Quercetin/chemistry , Quercetin/pharmacology , Structure-Activity RelationshipABSTRACT
Secretory N-acyl homoserine lactones (AHLs) mediate quorum sensing (QS) in bacteria. AHLs are shown to be inhibitory for an unrelated group of bacteria and might mimic host signalling elements, thereby subverting the regulatory events in host cells. This study investigated the AHL produced by Acinetobacter baumannii and analysed its effect on other bacterial species and mammalian cells. Chemically characterized AHL had an m/z value of 325 with a molecular formula C18H31NO4 and showed its inhibitory potential against Staphylococcus aureus. Molecular docking studies identified D-alanine-D-alanine synthetase A, a cell wall synthesizing enzyme of S. aureus having a strong binding affinity towards AHL. Electron microscopy showed the disruption and sloughing off of the S. aureus cell wall when treated with AHL. In vitro experiments revealed that this bacteriostatic AHL showed time-dependent activity and induced apoptosis in cancer cell lines. This compound could be a potential structural backbone for constructing new AHL analogues against S. aureus. The findings emphasize the need to re-evaluate all previously characterized AHLs for any additional new biological functions other than QS.
Subject(s)
Acinetobacter baumannii/metabolism , Acyl-Butyrolactones/pharmacology , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/pharmacology , Quorum Sensing/drug effects , Staphylococcus aureus/drug effects , A549 Cells , Acinetobacter baumannii/genetics , Acinetobacter baumannii/physiology , Acyl-Butyrolactones/isolation & purification , Acyl-Butyrolactones/metabolism , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/metabolism , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/metabolism , Biofilms/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , HeLa Cells , Humans , Molecular Docking Simulation , Staphylococcus aureus/metabolismABSTRACT
BACKGROUND: Extended spectrum betalactamase (ESBL)-producing organisms are a major cause of hospital-acquired infections. ESBL-producing Escherichia coli (E. coli) have been recovered from the hospital environment. These drug-resistant organisms have also been found to be present in humans as commensals. The present investigation intended to isolate ESBL-producing E. coli from the gut of already infected patients; to date, only a few studies have shown evidence of the gut microflora as a major source of infection. AIMS: This study aimed to detect the presence of ESBL genes in E.coli that are isolated from the gut of patients who have already been infected with the same organism. METHODS: A total of 70 non-repetitive faecal samples were collected from in-patients of our hospital. These in-patients were clinically diagnosed and were culture-positive for ESBL-producing E. coli either from blood, urine, or pus. Standard microbiological methods were used to detect ESBL from clinical and gut isolates. Genes coding for major betalactamase enzymes such as bla CTX-M , bla TEM, and bla SHV were investigated by polymerase chain reaction (PCR). RESULTS: ESBL-producing E. coli was isolated from 15 (21 per cent) faecal samples of the 70 samples that were cultured. PCR revealed that out of these 15 isolates, the bla CTX-M gene was found in 13 (86.6 per cent) isolates, the bla TEM was present in 11 (73.3 per cent) isolates, and bla SHV only in eight (53.3 per cent) isolates. All 15 clinical and gut isolates had similar phenotypic characters and eight of the 15 patients had similar pattern of genes (bla TEM, bla CTX-M, and bla SHV) in their clinical and gut isolates. CONCLUSION: Strains with multiple betalactamase genes that colonise the gut of hospitalised patients are a potential threat and it may be a potential source of infection.
ABSTRACT
The molecular epidemiology and carbapenem resistance mechanisms of clinical isolates of Acinetobacter baumannii obtained from a south Indian tertiary care hospital were investigated by repetitive extragenic palindromic sequence PCR (REP-PCR) and multi-locus sequence typing (MLST). Analysis of resistant determinants was achieved by PCR screening for the presence of genes encoding OXA-carbapenemases, metallo-ß-lactamases (MBLs) and efflux pumps. REP-PCR generated around eight clusters of high heterogeneity; of these, two major clusters (I and V) appeared to be clonal in origin. Analysis of representative isolates from different clusters by MLST revealed that most of the isolates belonged to sequence type 103 of CC103(B) . Second most prevalent ST belonged to clonal complex (CC) 92(B) which is also referred to as international clone II. Most of the isolates were multi-drug resistant, being susceptible only to polymyxin-B and newer quinolones. Class D ß-lactamases such as blaOXA-51-like (100%), blaOXA-23-like (56.8%) and blaOXA-24-like (14.8%) were found to be predominant, followed by a class B ß-lactamase, namely blaIMP-1 (40.7%); none of the isolates had blaOXA-58 like, blaNDM-1 or blaSIM-1 . Genes of efflux-pump adeABC were predominant, most of isolates being biofilm producers that were PCR-positive for autoinducer synthase gene (>94%). Carbapenem non-susceptible isolates were highly diverse and present throughout the hospital irrespective of type of ward or intensive care unit. Although previous reports have documented diverse resistant mechanisms in A. baumannii, production of MBL and OXA-type of carbapenamases were found to be the predominant mechanism(s) of carbapenem resistance identified in strains isolated from Southern India.
Subject(s)
Acinetobacter baumannii/drug effects , Acinetobacter baumannii/isolation & purification , Anti-Bacterial Agents/pharmacology , Carbapenems/pharmacology , Drug Resistance, Multiple, Bacterial , beta-Lactamases/metabolism , Acinetobacter baumannii/classification , Acinetobacter baumannii/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Cluster Analysis , Female , Genotype , Humans , India/epidemiology , Infant , Male , Middle Aged , Molecular Epidemiology , Molecular Sequence Data , Multilocus Sequence Typing , Polymerase Chain Reaction , Tertiary Care Centers , Young Adult , beta-Lactamases/geneticsABSTRACT
BACKGROUND & OBJECTIVES: The nosocomial human pathogen Acinetobacter baumannii has high propensity to develop resistance to antimicrobials and to become multidrug resistant (MDR), consequently complicating the treatment. This study was carried out to investigate the presence of resistant plasmids (R-plasmids) among the clinical isolates of A. baumannii. In addition, the study was performed to check the presence of common ß-lactamases encoding genes on these plasmids. METHODS: A total of 55 clinical isolates of A. baumannii were included in the study and all were subjected to plasmid DNA isolation, followed by PCR to check the presence of resistance gene determinants such as blaOXA-23, blaOXA-51, blaOXA-58 and blaIMP-1 on these plasmids that encode for oxacillinase (OXA) and metallo-ß-lactamase (MBL) type of carbapenemases. Plasmid curing experiments were carried out on selected isolates using ethidium bromide and acridine orange as curing agents and the antibiotic resistance profiles were evaluated before and after curing. RESULTS: All the isolates were identified as A. baumannii by 16SrDNA amplification and sequencing. Plasmid DNA isolated from these isolates showed the occurrence of multiple plasmids with size ranging from 500bp to ≥25 kb. The percentage of blaOXA-51 and blaOXA-23 on plasmids were found to be 78 and 42 per cent, respectively and 20 isolates (36%) carried blaIMP-1 gene on plasmids. Significant difference was observed in the antibiograms of plasmid cured isolates when compared to their parental ones. The clinical isolates became susceptible to more than two antibiotic classes after curing of plasmids indicating plasmid borne resistance. INTERPRETATION & CONCLUSIONS: Our study determined the plasmid mediated resistance mechanisms and occurrence of different resistance genes on various plasmids isolated from MDR A. baumannii. The present findings showed the evidence for antibiotic resistance mediated through multiple plasmids in A. baumannii clinical isolates. This indicates towards a need for preventive measures to avert the dissemination of plasmid resistance determinants in clinical environments.
Subject(s)
Acinetobacter baumannii/genetics , Bacterial Proteins/metabolism , Cross Infection/microbiology , Drug Resistance, Multiple, Bacterial/genetics , R Factors/genetics , beta-Lactamases/metabolism , Acinetobacter baumannii/enzymology , Base Sequence , DNA Primers/genetics , DNA, Bacterial/genetics , Humans , India , Microbial Sensitivity Tests , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNAABSTRACT
Acinetobacter baumannii has emerged as one of the most common nosocomial pathogens and is considered to be a significant threat to public health worldwide. Here, we present the draft genome sequence of a multidrug-resistant clinical strain of A. baumannii PKAB07 isolated from a wound infection in India during 2011 to 2012.
ABSTRACT
Although not previously known to cause human infections, Kocuria species have now emerged as human pathogens, mostly in compromised hosts with severe underlying disease. Recently, there has been an increasing incidence of different types of Kocuria infections reported, most likely due to the adoption of better identification methods. Here, we report a case of peritonitis caused by Kocuria rosea in a diabetic nephropathy patient who was on continuous ambulatory peritoneal dialysis. Sepsis and peritonitis caused by K. rosea in our case yielded two identical Kocuria isolates from the peritoneal dialysate fluid within a period of three days. The infection was subsequently resolved by antibiotic treatment and catheter removal. In addition to reporting this case, we herein review the literature concerning the emergence of Kocuria as a significant human pathogen. The majority of cases were device-related, acquired in the hospital or endogenous, and different Kocuria species appear to share a common etiology of peritonitis. The overall disease burden associated with Kocuria appears to be high, and the treatment guidelines for diseases associated with Kocuria have not yet been clearly defined.
ABSTRACT
[This corrects the article DOI: 10.1038/emi.2013.71.].