IS6110 Insertion Research Articles

Influence of IS256 on Genome Variability and Formation of Small-Colony Variants in Staphylococcus aureus.

Staphylococcus aureus has acquired resistance to nearly all antibiotics used in clinical practice. Whereas some resistance mechanisms are conferred by uptake of resistance genes, others evolve by mutation. In this study, IS256 has been shown to play a role, e.g., in S. aureus strains displaying intermediate resistance to vancomycin (VISA). To characterize the IS256 insertion sites in the genomes of two closely related sequence type 247 (ST247) VISA strains, all insertions were mapped in both VISA and a susceptible control strain. The results showed that the three ST247 strains contained the highest number so far of IS256 insertions for all sequenced S. aureus strains. Furthermore, in contrast to the case with the other IS elements in these genomes, the IS256 insertion sites were not identical in the closely related strains, indicating a high transposition frequency of IS256 When IS256 was introduced into a laboratory strain which was then cultured in the presence of antibiotics, it was possible to isolate small-colony variants (SCVs) that possessed IS256 insertions in guaA and hemY that displayed increased resistance to vancomycin and aminoglycosides, respectively. For these clones, a very rapid reversion to the wild type that resembled the fast reversion of clinical SCVs was observed. The reversion was caused by excision of IS256 in a small number of fast-growing clones that quickly outcompeted the SCVs in broth cultures. In conclusion, the presence of IS256 confers a strong genomic plasticity that is useful for adaptation to antibiotic stress.

Comparative genomic analysis of Mycobacterium tuberculosis Beijing-like strains revealed specific genetic variations associated with virulence and drug resistance

Isolates of the Mycobacterium tuberculosis lineage 2/East-Asian are considered one of the most successful strains due to their increased pathogenicity, hyper-virulence associated with drug resistance, and high transmission. Recent studies in Colombia have shown that the Beijing-like genotype is associated with multidrug-resistance and high prevalence in the southwest of the country, but the genetic basis of its success in dissemination is unknown. In contribution to this matter, we obtained the whole sequences of six genomes of clinical isolates assigned to the Beijing-like genotype. The genomes were compared with the reference genome of M. tuberculosis H37Rv and 53 previously published M. tuberculosis genomes. We found that the six Beijing-like isolates belong to a modern Beijing sub-lineage and share specific genomic variants: i.e. deletion in the PPE8 gene, in Rv3806c (ubiA) responsible of high ethambutol resistance and in Rv3862c (whiB6) which is involved in granuloma formation and virulence, are some of them. Moreover, each isolated has exclusively single nucleotide polymorphisms (SNPs) in genes related with cell wall processes and cell metabolism. We identified polymorphisms in genes related to drug resistance that could explain the drug-resistant phenotypes found in the six isolates from Colombia. We hypothesize that changes due to these genetic variations contribute to the success of these strains. Finally, we analyzed the IS6110 insertion sequences finding very low variance between them, suggesting that SNPs is the major cause of variability found in Beijing-like strains circulating in Colombia.

Hopping into a hot seat: Role of DNA structural features on IS5-mediated gene activation and inactivation under stress.

Insertion sequence elements (IS elements) are proposed to play major roles in shaping the genetic and phenotypic landscapes of prokaryotic cells. Recent evidence has raised the possibility that environmental stress conditions increase IS hopping into new sites, and often such hopping has the phenotypic effect of relieving the stress. Although stress-induced targeted mutations have been reported for a number of E. coli genes, the glpFK (glycerol utilization) and the cryptic bglGFB (β-glucoside utilization) systems are among the best characterized where the effects of IS insertion-mediated gene activation are well-characterized at the molecular level. In the glpFK system, starvation of cells incapable of utilizing glycerol leads to an IS5 insertion event that activates the glpFK operon, and enables glycerol utilization. In the case of the cryptic bglGFB operon, insertion of IS5 (and other IS elements) into a specific region in the bglG upstream sequence has the effect of activating the operon in both growing cells, and in starving cells. However, a major unanswered question in the glpFK system, the bgl system, as well as other examples, has been why the insertion events are promoted at specific locations, and how the specific stress condition (glycerol starvation for example) can be mechanistically linked to enhanced insertion at a specific locus. In this paper, we show that a specific DNA structural feature (superhelical stress-induced duplex destabilization, SIDD) is associated with “stress-induced” IS5 insertion in the glpFK, bglGFB, flhDC, fucAO and nfsB systems. We propose a speculative mechanistic model that links specific environmental conditions to the unmasking of an insertional hotspot in the glpFK system. We demonstrate that experimentally altering the predicted stability of a SIDD element in the nfsB gene significantly impacts IS5 insertion at its hotspot.

Acid Evolution of Escherichia coli K-12 Eliminates Amino Acid Decarboxylases and Reregulates Catabolism.

Acid-adapted strains of Escherichia coli K-12 W3110 were obtained by serial culture in medium buffered at pH 4.6 (M. M. Harden, A. He, K. Creamer, M. W. Clark, I. Hamdallah, K. A. Martinez, R. L. Kresslein, S. P. Bush, and J. L. Slonczewski, Appl Environ Microbiol 81:1932-1941, 2015, https://doi.org/10.1128/AEM.03494-14). Revised genomic analysis of these strains revealed insertion sequence (IS)-driven insertions and deletions that knocked out regulators CadC (acid induction of lysine decarboxylase), GadX (acid induction of glutamate decarboxylase), and FNR (anaerobic regulator). Each acid-evolved strain showed loss of one or more amino acid decarboxylase systems, which normally help neutralize external acid (pH 5 to 6) and increase survival in extreme acid (pH 2). Strains from populations B11, H9, and F11 had an IS5 insertion or IS-mediated deletion in cadC, while population B11 had a point mutation affecting the arginine activator adiY The cadC and adiY mutants failed to neutralize acid in the presence of exogenous lysine or arginine. In strain B11-1, reversion of an rpoC (RNA polymerase) mutation partly restored arginine-dependent neutralization. All eight strains showed deletion or downregulation of the Gad acid fitness island. Strains with the Gad deletion lost the ability to produce GABA (gamma-aminobutyric acid) and failed to survive extreme acid. Transcriptome sequencing (RNA-seq) of strain B11-1 showed upregulated genes for catabolism of diverse substrates but downregulated acid stress genes (the biofilm regulator ariR, yhiM, and Gad). Other strains showed downregulation of H2 consumption mediated by hydrogenases (hya and hyb) which release acid. Strains F9-2 and F9-3 had a deletion of fnr and showed downregulation of FNR-dependent genes (dmsABC, frdABCD, hybABO, nikABCDE, and nrfAC). Overall, strains that had evolved in buffered acid showed loss or downregulation of systems that neutralize unbuffered acid and showed altered regulation of catabolism.IMPORTANCE Experimental evolution of an enteric bacterium under a narrow buffered range of acid pH leads to loss of genes that enhance fitness above or below the buffered pH range, including loss of enzymes that may raise external pH in the absence of buffer. Prominent modes of evolutionary change involve IS-mediated insertions and deletions that knock out key regulators. Over generations of acid stress, catabolism undergoes reregulation in ways that differ for each evolving strain.

A liquid bead array for the identification and characterization of fljB-positive and fljB-negative monophasic variants of Salmonella Typhimurium

Salmonella1,4,[5],12:i:- accounts currently for one of the most common serotypes observed worldwide. These isolates do not express the FljB flagellin and mostly derive from Salmonella Typhimurium. They are therefore termed Salmonella Typhimurium monophasic variants (STMV) and are considered of comparable public health risk. Since serological identification of the somatic and flagellar antigens of STMV is not sufficient to demonstrate relatedness with Salmonella Typhimurium, additional assays detecting genetic markers unique to Salmonella Typhimurium are required. In addition, identification of the mutations affecting expression of the flagellar gene fljB can be useful to support the monophasic character observed phenotypically. Finally, genetic subtyping of the various mono- and biphasic Salmonella Typhimurium clonal groups can facilitate their epidemiological follow-up. Here, we present a home-made liquid bead array able to fulfill these requirements. This array confirmed the monophasic character of 240 STMV isolates collected in Belgium during 2014–2015 and identified 10 genetic subtypes. Microevolution in and around the fljB locus linked to IS26 insertions is probably one of the driven force accounting for STMV population diversity. Thanks to its open design, other genetic signatures could later be merged to the assay to subtype additional STMV clonal groups and to detect rare mutations.

Diversity and evolutionary aspects of mucin binding (MucBP) domain repeats among Lactobacillus plantarum group strains through comparative genetic analysis

Mucin binding protein (Mub) of Lactobacillus plantarum- group (LPG) is considered as a potential marker gene due to its diversity and adherence property to host intestinal layer. We recently observed strain specific diversity in mub gene of LPG strains (n=8) and grouped them into Type I, II and III based on the insertion and/or deletion of MucBP domain repeats (Devi et al., 2016). The mub gene expression pattern noted by real-time PCR revealed 67.64 increased fold change in MTCC 9483 with four MucBP repeats. Whereas, the isolates with 3 and 2 MucBP repeats showed ∼4 and 2 enhanced expression, respectively. Detection of integrase (int) and transposases (tra) in the flanking regions of mub, suggested variation in amplicon sizes and revealed strain diversity. The IS finder program identified tra nucleotide sequence similarity towards IS256 and IS5 family insertion elements that are present among other lactic acid bacteria. Phylogenetic dendrogram suggested the evolution of D7–D10 domains of L. paraplantarum MTCC 9483 from Lactobacillus namurensis, Lactobacillus hammesii and Lactobacillus spicheri. These results collectively gave information on the genetic diversity, functional MucBP domains and also facilitated in the selection of an efficient novel LPG strain with good adherence ability.

Environment-directed activation of the Escherichia coliflhDC operon by transposons.

The flagellar system in Escherichia coli K12 is expressed under the control of the flhDC-encoded master regulator FlhDC. Transposition of insertion sequence (IS) elements to the upstream flhDC promoter region up-regulates transcription of this operon, resulting in a more rapid motility. Wang and Wood (ISME J 2011;5:1517-1525) provided evidence that insertion of IS5 into upstream activating sites occurs at higher rates in semi-solid agar media in which swarming behaviour is allowed as compared with liquid or solid media where swarming cannot occur. We confirm this conclusion and show that three IS elements, IS1, IS3 and IS5, transpose to multiple upstream sites within a 370 bp region of the flhDC operon control region. Hot spots for IS insertion correlate with positions of stress-induced DNA duplex destabilization (SIDD). We show that IS insertion occurs at maximal rates in 0.24 % agar, with rates decreasing dramatically with increasing or decreasing agar concentrations. In mixed cultures, we show that these mutations preferentially arise from the wild-type parent at frequencies of up to 3×10-3 cell-1 day-1 when the inoculated parental and co-existing IS-activated mutant cells are entering the stationary growth phase. We rigorously show that the apparent increased mutation frequencies cannot be accounted for by increased swimming or by increased growth under the selective conditions used. Thus, our data are consistent with the possibility that appropriate environmental conditions, namely those that permit but hinder flagellar rotation, result in the activation of a mutational pathway that involves IS element insertion upstream of the flhDC operon.

Prevalence of ST1193 clone and IncI1/ST16 plasmid in E-coli isolates carrying blaCTX-M-55 gene from urinary tract infections patients in China

To study molecular epidemiology of CTX-M-55-carrying Escherichia coli isolates from urinary tract infections (UTIs) in China. 111 blaCTX-M-55-positive E.coli isolates from UTIs patients in China were studied. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to analyze the homologies among the strains. Conjugation experiments, S1nuclease PFGE and PCR analysis were performed to characterize plasmids harboring blaCTX-M-55 and their genetic environment. 111 isolates were clustered into 86 individual pulsotypes and three clusters by PFGE. Fifty-five (49.5%) of the isolates belonged to 8 STs. Most of the ST1193 isolates belonged to one PFGE cluster. Transconjugants (n = 45) derived from randomly selected blaCTX-M-55 donors (n = 58), were found to contain a single 90-kb conjugative plasmid, which mainly belonged to the IncI1 groups (34, 76%). Among the IncI1 plasmids, the blaCTX-M-55/IncI1/ST16 predominated (23/34, 68%). The blaTEM-1 and aac (3′)-II genes were frequently detected on the IncI1 plasmids, and the insertion of ISEcp1 or IS26 was observed at the 48 bp or 45 bp upstream of the start codon of blaCTX-M-55 gene. The dissemination of blaCTX-M-55 gene among E. coli UTI isolates, appeared to be due to both the major clonal lineage of ST1193 and the horizontal transfer of epidemic plasmid IncI1/ST16.

Coexistence of blaOXA-48 and Truncated blaNDM-1 on Different Plasmids in a Klebsiella pneumoniae Isolate in China.

Objectives: To describe the genetic environment, transferability, and antibiotic susceptibility of one clinical Klebsiella pneumoniae isolate harboring both blaOXA-48 and blaNDM-1 on different plasmids from a Chinese hospital.Methods: The isolate was subjected to antimicrobial susceptibility testing and multilocus sequence typing using Etest and PCR. The plasmids harboring blaOXA-48 and blaNDM-1 were analyzed through conjugation experiments, S1-nuclease pulsed-field gel electrophoresis, and hybridization with specific probes. Plasmid DNA was sequenced using Pacbio RS II and annotated using RAST.Results: K. pneumoniae RJ119, carrying both blaOXA-48 and blaNDM-1, was resistant to almost all carbapenems, cephalosporins, fluoroquinolone, and aminoglycosides and belonged to ST307. blaOXA-48 was located on a 61,748-bp IncL/M conjugative plasmid, which displayed overall nucleotide identity (99%) to pKPN-E1-Nr.7. blaNDM-1 was located on a 335,317-bp conjugative plasmid, which was a fusion of a blaNDM-1-harboring InA/C plasmid pNDM-US (140,825 bp, 99% identity) and an IncFIB plasmid pKPN-c22 (178,563 bp, 99% identity). The transconjugant RJ119-1 harboring blaNDM-1 was susceptible to carbapenem, and there was an insertion of IS10 into the blaNDM-1 gene.Conclusion: This is the first report of the coexistence of blaOXA-48 and blaNDM-1 in one K. pneumoniae clinical isolate in China. OXA-48 in RJ119 contributed to the majority to its high resistance to carbapenems, whereas NDM-1 remained unexpressed, most likely due to the insertion of IS10. Our results provide new insight for the relationship between genetic diagnosis and clinical treatment. They also indicate that increased surveillance of blaOXA-48 is urgently needed in China.

Evaluation of polymerase chain reaction in space-occupying lesions of liver reported as granulomatous inflammation/tuberculosis on fine-needle aspiration cytology.

Background:Tubercular involvement of the liver is uncommon, but is a serious consideration in differential diagnosis of granulomatous conditions, especially in endemic regions like India.Objective:To assess the role of polymerase chain reaction (PCR) done on archival cytological material in diagnosing tuberculosis (TB) in cases reported as granulomatous inflammation/TB in liver lesions.Materials and Methods:This was a retrospective study including a total of 17 cases of liver space-occupying lesions (SOLs) reported as granulomatous inflammation (n = 12) and TB (n = 5). The smears were retrieved from the archives of the department and were reviewed for the cytomorphologic features. Air-dried smears stained with May–Grünwald–Giemsa (MGG) stain were assessed for the representative material in the form of epithelioid granulomas and giant cells. One/two MGG smears from each case were destained and the material was used for performing PCR for Mycobacterium tuberculosis by amplification of 123 bp fragment of the IS6110 insertion element.Results:The age of the patients ranged from 3 to 61 years. There were 12 females and 5 males. The patients presented with solitary/multiple liver SOLs. DNA could be extracted from 10/17 cases from archival MGG smears. PCR positivity was noted in 8/10 cases (including four acid-fast bacilli smear-positive cases), confirming a diagnosis of TB.Conclusion:Cytomorphology alone may not be sufficient for differentiating various granulomatous lesions reported in liver SOLs. DNA can be extracted from the archival cytological MGG-stained smears. PCR should be carried out if Ziehl–Neelsen staining is negative in granulomatous lesions, especially when material has not been submitted for culture.

Molecular Characterization of Carbapenemase-Nonproducing Clinical Isolates of Escherichia coli (from a Thai University Hospital) with Reduced Carbapenem Susceptibility.

Twelve nonreplicate carbapenemase-negative ertapenem (ETP)-nonsusceptible (CNENS) Escherichia coli isolates obtained at a Thai university hospital between 2010 and 2014 were characterized and compared with 2 carbapenemase-producing E. coli isolates from the same hospital. Eight unique pulsed-field gel electrophoresis patterns were obtained. All the isolates produced CTX-M-15 β-lactamase and 2 either coexpressed CMY-2 cephalosporinase or showed increased efflux pump activity. Amino acid sequence analysis revealed that an OmpF defect (in 7 isolates) due to mutations generating truncated proteins or an IS1 insertion was more prevalent than a defect in OmpC was (no truncated proteins detected). Seven out of 10 isolates possessing OmpC variants with any OmpF defect were weakly ETP-resistant (minimum inhibitory concentrations [MICs] of 1-4 μg/mL) and imipenem (IPM)- and meropenem (MEM)-susceptible (MICs 0.125-0.5 μg/mL). Two isolates with ompC PCR-negative results and an OmpF defect showed higher carbapenem MICs (8-32, 1-8, and 1-4 μg/mL for ETP, IPM, and MEM, respectively) with the highest MICs associated with the additional efflux pump activity. Both carbapenemase producers possessing CTX-M-15 and a porin background identical to that in the CNENS isolates showed ETP, IPM, and MEM MICs of 128-256, 8, and 2-32 μg/mL, respectively. These findings suggest that a porin defect combined with CTX-M-15 production is the major mechanism of low carbapenem susceptibility among our CNENS isolates, which have potential to become strongly carbapenem-resistant because of additional carbapenemase or efflux pump activities.

Diagnostic Accuracy of IS6110 Insertion Gene, <i>Hsp65</i>, and Xpert MTB/RIF for Rapid Diagnosis of Pulmonary Tuberculosis

Purpose: To evaluate diagnostic accuracy of IS6110 insertion genes, hsp65, and Xpert MTB/RIF for rapid diagnosis of pulmonary tuberculosis. Methods: Sixty patients, medically reported HIV negative, clinically suspected of having pulmonary tuberculosis, were included in this study, and consented before enrolment. Sputum samples were gathered once, and tested by smear for Acid Fast Bacilli (AFB). Cultured in the Loewenstein-Jensen (LJ) medium for M. tuberculosis growth, M. tuberculosis DNA was detected by conventional PCR targeting IS6110, and hsp65 genes using specific primers, and automated nested real-time PCR targeting rpoB gene. Sensitivity, specificity and diagnostic accuracy were calculated for each method compared to culture. Results: Compared with culture as reference method, smear, IS6110, hsp65, and Xpert MTB/RIF had sensitivity 77.14%, 100%, 100%, and 100%, specificity 92%, 96%, 96%, and 96.97%, and diagnostic accuracy 83.33%, 98.33%, 98.33% and 98.21% respectively. Molecular diagnostic methods had the highest diagnostic accuracy, whereas smear had the lowest. No statistical significance, (p value > 0.05) was detected between the patients’ demographic data and the presence or absence of TB infection. Conclusion: The diagnostic accuracy that we got from the molecular methods, confirmed the diagnostic value of molecular detection of M. tuberculosis in pulmonary cases, supporting the application of automated and conventional PCR in rapid analysis. Smear could be more efficient when used for treatment monitoring. Combination between one-molecular techniques with smear as a routine method could be valid for rapid diagnosis of TB.

Utilization and Performance of a Laboratory Developed Nucleic Acid Amplification Test for the Diagnosis of Pulmonary and Extrapulmonary Tuberculosis in a Low Prevalence Area: A 14 Year Study

BackgroundTuberculosis (TB) is a significant global health problem. Nucleic acid amplification tests (NAATs) are valuable in reducing delays to initiation of therapy and infection control protocols. A retrospective study was performed to assess the utilization and performance of a laboratory developed Mycobacterium tuberculosis complex (MTBC) PCR assay (TBPCR) for diagnosis of pulmonary (PTB) and extrapulmonary (EPTB) tuberculosis.MethodsStudy site was a 4 hospital system in suburban Chicago. All culture confirmed TB specimens with complete laboratory data from January 2002 to December 2016 were included. Patient records were accessed using an electronic data warehouse, following approval from Institutional Review Board. Standard microbiology procedures were followed for smear and culture of MTBC. A lab-developed real time PCR targeting a 123 bp region of the IS6110 insertion sequence of MTBC was performed on smear positive specimens or if ordered by physician. Clinical and laboratory data was compared with TBPCR results for all culture confirmed cases.ResultsThere were 151 culture positive patients and 2186 TBPCR performed. Median age of patients at diagnosis was 49 years (IQR 33–66), 74 (49%) were female and 14 were on immunosupressive therapy. The mean number of samples tested per patient was 2. Of culture positive specimens, 59% were from a respiratory source and 3 were MDR; ordering of TBPCR was higher in specimens from PTB source (58.4%) as compared with EPTB source (37%). Combined sensitivity of the TBPCR on all specimen types was 86.6% (95% CI 76.3–93.1); 90.3% for PTB specimens alone (95% CI 78.2–96.4). Specificity was 100% (95% CI 99.5–100), PPV 100% (95% CI 90.5–100%) and NPV 99.5% (95% CI 98.8–99.8%), and were similar for all specimen types. Sensitivity of TBPCR was 97% in smear positive and 79% in smear negative PTB specimens. The median time to culture positivity was 7 days longer in specimens that were TBPCR negative compared with those that were positive (P = 0.14, NS), however, TBPCR shortened time to diagnosis by 13 days.ConclusionWe found TBPCR to be underutilized in both PTB and EPTB although it was found to be a rapid and reliable method for early diagnosis. Education regarding utility of NAATs could be useful in low burden areas where paucibacillary disease is more common, especially in EPTB.Disclosures All authors: No reported disclosures.

The mechanisms of linezolid resistance in 43 strains of Staphylococcus

Objective To investigate the prevalence of linezolid-resistant Staphylococcus in China and analyze the resistance mechanism of linezolid-resistant isolates to provide the evidence of rational use of antibiotics. Methods Forty-three non-duplicate linezolid-resistant isolates were collected from 9 hospitals throughout China over a period of 4 years (2009-2012). The MIC of each antimicrobial agent was determined by the broth microdilution method. Presence of cfr and mutations in 23S rRNA were detected by PCR and sequencing. Presence of cfr in plasmid was determined by Southern blot. The genetic environment of the cfr was determined by whole plasmid genome sequencing and analysis. Results Most of the 43 linezolid-resistant Staphylococcus isolates showed a multidrug-resistant phenotype, and most of isolates harboured the cfr gene and mutation of 23S rRNA G2576T. Southern blott indicated that the cfr gene of these isolates resided on plasmids, which contained IS256 or IS21-558 insertion sequence and Tn558 transposon mobile genetic elements. Conclusions Acquired the cfr gene or the mutation of 23S rRNA G2576T in Staphylococcus can lead to the linezolid resistance. The cfr gene was located on the plasmid and surrounded by the mobile genetic elements, which could result in the spread of the cfr gene among the different isolates and species.(Chin J Lab Med, 2016, 39: 848-851) Key words: Staphylococcus; Acetamides; Bacterial proteins

Complete Circular Genome Sequence of Successful ST8/SCCmecIV Community-Associated Methicillin-Resistant Staphylococcus aureus (OC8) in Russia: One-Megabase Genomic Inversion, IS256's Spread, and Evolution of Russia ST8-IV.

ST8/SCCmecIV community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has been a common threat, with large USA300 epidemics in the United States. The global geographical structure of ST8/SCCmecIV has not yet been fully elucidated. We herein determined the complete circular genome sequence of ST8/SCCmecIVc strain OC8 from Siberian Russia. We found that 36.0% of the genome was inverted relative to USA300. Two IS256, oppositely oriented, at IS256-enriched hot spots were implicated with the one-megabase genomic inversion (MbIN) and vSaβ split. The behavior of IS256 was flexible: its insertion site (att) sequences on the genome and junction sequences of extrachromosomal circular DNA were all divergent, albeit with fixed sizes. A similar multi-IS256 system was detected, even in prevalent ST239 healthcare-associated MRSA in Russia, suggesting IS256’s strong transmission potential and advantage in evolution. Regarding epidemiology, all ST8/SCCmecIVc strains from European, Siberian, and Far Eastern Russia, examined had MbIN, and geographical expansion accompanied divergent spa types and resistance to fluoroquinolones, chloramphenicol, and often rifampicin. Russia ST8/SCCmecIVc has been associated with life-threatening infections such as pneumonia and sepsis in both community and hospital settings. Regarding virulence, the OC8 genome carried a series of toxin and immune evasion genes, a truncated giant surface protein gene, and IS256 insertion adjacent to a pan-regulatory gene. These results suggest that unique single ST8/spa1(t008)/SCCmecIVc CA-MRSA (clade, Russia ST8-IVc) emerged in Russia, and this was followed by large geographical expansion, with MbIN as an epidemiological marker, and fluoroquinolone resistance, multiple virulence factors, and possibly a multi-IS256 system as selective advantages.

Evaluation of complementary diagnostic tools for bovine tuberculosis detection in dairy herds from India.

Aim:A cross-sectional study was undertaken to know the herd prevalence and evaluate the single intradermal tuberculin testing (SITT), culture isolation, and polymerase chain reaction (PCR) analysis for the diagnosis of bovine tuberculosis (TB).Materials and Methods:A total of 541 cows of three dairy farms of Bareilly and Mukteshwar were screened by SITT followed by collection of pre-scapular lymph node (PSLN) aspirates (71), milk (54), and blood (71) samples from reactor animals. These clinical samples were processed for culture isolation and direct PCR-based identification and species differentiation.Results:Out of 541 cows screened by SITT, 71 (13.12%) animals were found positive. Mycobacteria were isolated from 3 (4.22%) PSLN aspirate but not from any cultured milk and blood samples. 28 (39.43%) PSLN aspirate and 5 (9.25%) milk samples were positive for Mycobacterium TB (MTB) complex (MTC) by PCR amplification for the IS6110 insertion sequence; however, blood samples were found negative. For species differentiation, multiplex-PCR using 12.7 kb primers was conducted. Out of 28 PSLN aspirate, Mycobacterium bovis was detected in 18 (64.28%) and MTB in 8 (28.57%), whereas 2 aspirate samples (7.14%) were positive for both the species. All the five milk positive samples were positive for M. bovis.Conclusion:Direct detection of bovine TB by a molecular-based method in dairy animals after preliminary screening was appeared to be more sensitive and specific compared to the conventional method (i.e., culture isolation). Its application in form of serial testing methodology for the routine diagnosis and thereafter, culling of infected stock may be suggested for the control programs in dairy herds. The PSLN aspirate was found to be the most suitable specimen for culture isolation and PCR-based detection of Mycobacterium spp. among live infected animals.

Factors Determining Staphylococcus aureus Susceptibility to Photoantimicrobial Chemotherapy: RsbU Activity, Staphyloxanthin Level, and Membrane Fluidity.

Photoantimicrobial chemotherapy (PACT) constitutes a particular type of stress condition, in which bacterial cells induce a pleiotropic and as yet unexplored effect. In light of this, the key master regulators are of putative significance to the overall phototoxic outcome. In Staphylococcus aureus, the alternative sigma factor σB controls the expression of genes involved in the response to environmental stress. We show that aberration of any sigB operon genes in S. aureus USA300 isogenic mutants causes a pronounced sensitization (>5 log10 reduction in CFU drop) to PACT with selected photosensitizers, namely protoporphyrin diarginate, zinc phthalocyanine and rose bengal. This effect is partly due to aberration-coupled staphyloxanthin synthesis inhibition. We identified frequent mutations in RsbU, a σB activator, in PACT-vulnerable clinical isolates of S. aureus, resulting in σB activity impairment. Locations of significant changes in protein structure (IS256 insertion, early STOP codon occurrence, substitutions A230T and A276D) were shown in a theoretical model of S. aureus RsbU. As a phenotypic hallmark of PACT-vulnerable S. aureus strains, we observed an increased fluidity of bacterial cell membrane, which is a result of staphyloxanthin content and other yet unidentified factors. Our research indicates σB as a promising target of adjunctive antimicrobial therapy and suggests that enhanced cell membrane fluidity may be an adjuvant strategy in PACT.

Characterization of ST258 Colistin-Resistant, blaKPC-Producing Klebsiella pneumoniae in a Greek Hospital.

The emergence of colistin resistance may further contribute to treatment failure of infection caused by multidrug-resistant (MDR) Klebsiella pneumoniae. The colistin resistance rates were determined and colistin-resistant carbapenemase-producing K. pneumoniae (COL-R CP-Kp) were characterized over an 18-month period in a Greek hospital. Out of 135 carbapenemase producers, 19 isolates (14%) were categorized as resistant to colistin. Phenotypic and molecular characterization of the COL-R CP-Kp isolates revealed that all were MDR blaKPC producers and, excluding one isolate of MLST ST383, belonged to the international clonal lineage ST258. Furthermore, PCR amplification and sequencing of the mgrB locus revealed nucleotide sequences of different sizes and insertions of IS1- and IS5-like mobile elements. The majority (63%) of the COL-R blaKPC producers was recovered from patients in the intensive care unit (ICU) and clinical data indicated that all patients should have acquired these isolates in the ICU. The findings of the present study underscore a concerning evolution of colistin resistance in a setting of high K. pneumoniae carbapenemase (KPC)-Kp endemicity, such as Greece. Thus, continuous surveillance, molecular characterization, prudent use of antibiotics, and implementation of infection control measures for K. pneumoniae are urgent.

Typing Method for the QUB11a Locus of Mycobacterium tuberculosis: IS6110 Insertions and Tandem Repeat Analysis.

QUB11a is used as a locus for variable number of tandem repeats (VNTR) analysis of Mycobacterium tuberculosis Beijing lineage. However, amplification of QUB11a occasionally produces large fragments (>1,400 bp) that are not easily measured by capillary electrophoresis because of a lack of the typical stutter peak patterns that are used for counting repeat numbers. IS6110 insertion may complicate VNTR analysis of large QUB11a fragments in M. tuberculosis. We established a method for determining both tandem repeat numbers and IS6110 insertion in the QUB11a locus of M. tuberculosis using capillary electrophoresis analysis and BsmBI digestion. All 29 large QUB11a fragments (>1,200 bp) investigated contained IS6110 insertions and varied in the number of repeats (18 patterns) and location of IS6110 insertions. This method allows VNTR analysis with high discrimination.

Identification and prevalence of tetracycline resistance in enterococci isolated from poultry in Ilishan, Ogun State, Nigeria.

Background:Tetracycline is one of the most frequently used antibiotics in Nigeria both for human and animal infections because of its cheapness and ready availability. The use of tetracycline in animal husbandry could lead to horizontal transfer of tet genes from poultry to human through the gut microbiota, especially enterococci. Therefore, this study is designed to identify different enterococcal species from poultry feces in selected farms in Ilishan, Ogun State, Nigeria, determine the prevalence of tetracycline resistance/genes and presence of IS256 in enterococcal strains.Materials and Methods:Enterococci strains were isolated from 100 fresh chicken fecal samples collected from seven local poultry farms in Ilishan, Ogun State, Nigeria. The strains were identified by partial sequencing of 16S rRNA genes. Antibiotic susceptibility of the isolates to vancomycin, erythromycin, tetracycline, gentamicin, amoxycillin/claulanate, and of loxacin were performed by disc diffusion method. Detection of tet, erm, and van genes and IS256 insertion element were done by polymerase chain reaction amplification.Results:Sixty enterococci spp. were identified comprising of Enterococcus faecalis 33 (55%), Enterococcus casseliflavus 21 (35%), and Enterococcus gallinarium 6 (10%). All the isolates were resistant to erythromycin (100%), followed by tetracycline (81.67%), amoxicillin/clavulanic acid (73.33%), ofloxacin (68.33%), vancomycin (65%), and gentamicin (20%). None of the enterococcal spp. harbored the van and erm genes while tet(M) was detected among 23% isolates and is distributed mostly among E. casseliflavus. IS256 elements were detected only in 33% of E. casseliflavus that were also positive for tet(M) gene.Conclusion:This study provides evidence that tetracycline resistance gene is present in the studied poultry farms in Ilishan, Ogun State, Nigeria and underscores the need for strict regulation on tetracycline usage in poultry farming in the studied location and consequently Nigeria.