Various arrangements of mobile genetic elements among CC147 subpopulations of Klebsiella pneumoniae harboring blaNDM-1: a comparative genomic analysis of carbapenem resistant strains.

BACKGROUND: Certain clonal complexes (CCs) of Klebsiella pneumoniae such as CC147 (ST147 and ST392) are major drivers of blaNDM dissemination across the world. ST147 has repeatedly reported from our geographical region, but its population dynamics and evolutionary trajectories need to be further studied. METHODS: Comparative genomic analysis of 51 carbapenem-nonsusceptible strains as well as three hypervirulent K. pneumoniae (hvKp) recovered during 16-months of surveillance was performed using various bioinformatics tools. We investigated the genetic proximity of our ST147 strains with publicly available corresponding genomes deposited globally and from neighbor countries in our geographic region. RESULTS: While IncL/M plasmid harboring blaOXA-48 was distributed among divergent clones, blaNDM-1 was circulated by twenty of the 25 CC147 dominant clone and were mostly recovered from the ICU. The NDM-1 core structure was bracketed by a single isoform of mobile genetic elements (MGEs) [ΔISKpn26-NDM-TnAs3-ΔIS3000-Tn5403] and was located on Col440I plasmid in 68.7% of ST392. However, various arrangements of MGEs including MITESen1/MITESen1 composite transposon or combination of MITESen1/ISSen4/IS903B/IS5/ISEhe3 on IncFIb (pB171) were identified in ST147. It seems that ST392 circulated blaNDM-1 in 2018 before being gradually replaced by ST147 from the middle to the end of sample collection in 2019. ST147 strains possessed the highest number of resistance markers and showed high genetic similarity with four public genomes that harbored blaNDM-1 on the same replicon type. Mainly, there was a convergence between clusters and isolated neighboring countries in the minimum-spanning tree. A conserved arrangement of resistance markers/MGEs was linked to methyltransferase armA which was embedded in class 1 integron in 8 isolates of ST147/ST48 high-risk clones. CONCLUSION: Our findings highlight the dynamic nature of blaNDM-1 transmission among K. pneumoniae in Iran that occurs both clonally and horizontally via various combinations of MGEs. This is the first analysis of Iranian ST147/NDM + clone in the global context.

Arrangements of Mobile Genetic Elements among Virotype E Subpopulation of Escherichia coli Sequence Type 131 Strains with High Antimicrobial Resistance and Virulence Gene Content.

Escherichia coli sequence type 131 (ST131) is known for its contribution to multidrug resistance and the worldwide spread of this clone has become a global problem. Understanding the trends among ST131 clades will help design strategies to prevent its rapid dissemination. In this study, 72 ST131 strains were subjected to comparative genomic analysis and 64 clade C strains were compared with clade C strains reported from other regions using publicly available whole-genome sequencing data. C1 (n = 31 [48.4%]) and C2 (n = 33 [%51.5]) strains had the same prevalence in our collection, and C1-M27 (n = 22) strains were closely related, carried a unique plasmid type (F1:A2:B20), and exhibited virotype C. Removal of 11 C2 strains with varied virotype patterns and the heterogeneous IncF type identified 22 closely related virotype E/F strains with replicon type F31/F36:A4:B1, forming what we denote as the "C2-subset." In a global context, the C2-subset constituted a distinct cluster with international virotype E strains and harbored a genomic island, GI-pheU. Association of cnf1/hlyCABD genes with 1 to 7 mobile genetic elements, mostly IS682/ISKpn37 combination within GI-pheU was identified. The C2-subset accounted for excess resistance/virulence of subclade C2 relative to C1 strains. In addition, a conserved chromosomal IS26-mediated composite transposon (IS15DIV-ISEcp1-blaCTX-M-15-WbuC cupin fold metalloprotein-Tn2-IS15DIV) was observed in the C2-subset. The local spread of the C2-subset in the hospital studied, with the carriage of higher virulence/resistance markers and a peculiar F-type plasmid, demonstrates the potential for diversification of the ST131 lineage and the emergence of subpopulations with higher survival potential to cause health care-associated outbreaks. IMPORTANCE Escherichia coli sequence type 131 (ST131) is a globally dominant multidrug-resistant clone that is commonly associated with extraintestinal infections. Specific sublineages have been shown to have emerged and spread within ST131, highlighting the complex nature of ST131 epidemiology. This study systematically compared the Iranian ST131 population to those reported from other countries and found a subpopulation harboring virotype E, a homogeneous profile of plasmid Inc-F type F31/F36:A4:B1 harboring cnf1/hemolysin genes on the genomic island GI-pheU, and up to seven mobile genetic elements (MGEs) flanking cnf1/hemolysin virulence markers. The results of this study highlight the importance of MGEs for virulence gene acquisition and the formation of new subpopulations among pandemic clones such as E. coli ST131.

Expansion of a Subset Within the C2 Subclade of Escherichia coli Sequence Type 131 (ST131) Is Driving the Increasing Rates of Aminoglycoside Resistance.

BACKGROUND: Sequence type 131 (ST131) of Escherichia coli is a pandemic clone that drives the increasing rates of antibiotic resistance. While the pervasiveness of ST131 clade C, especially subclades C2 and C1-M27, has been demonstrated in numerous global surveys, no report about the ST131 clades and their virotypes has been published from Iran so far. METHODS: A collection of 73 consecutive ST131 isolates from extraintestinal specimens was investigated for determination of virotypes, antibiotic susceptibility patterns, resistance/virulence determinants, and clade subsets. RESULTS: Most of the isolates belonged to subclade C2 (33/73; 45.2%), which had the highest virulence factor (VF) scores and resistance rates, followed by C1-M27 (18; 24.6%), C1-non-M27 (14; 19.1%), and A (8; 10.9%). The distinctive profiles of subclade C2 virulence genes were revealed by principle coordinates analysis testing. The distribution of the hlyA virulence gene among subclade C2 was not uniform, so that positive strains (21; 63.6%) showed significantly higher rates of resistance (bla CTX-M-15, bla OXA-1, aac(6')-Ib-cr, aac(6')-Ib, aac(3)-IIa) and virulence (hra, tia/hek, K5, cnf, papGII, papC) markers and gentamicin/tobramycin resistance. Virotype C as the most common virotype (34; 46.5%) was predominant among the subclade C1 population, while virotypes E and F (21; 28.7%) were detected among subclade C2, which had the highest VF scores and aminoglycoside resistance rates. CONCLUSIONS: The appearance of virotypes E and F among subclade C2 strains with higher rates of aminoglycoside resistance/virulence gene content shows the shifting dynamics of this pandemic clone in response to antibiotic selection pressure by establishing subsets with higher survival potential.

The emergence of the hypervirulent Klebsiella pneumoniae (hvKp) strains among circulating clonal complex 147 (CC147) harbouring blaNDM/OXA-48 carbapenemases in a tertiary care center of Iran.

BACKGROUND: Klebsiella pneumoniae is a public health concern because of its ability to develop multidrug resistance and hypervirulent genotypes, of those capsular types K1 and K2 cause community and nosocomial life-threatening infections. This study aimed to determine the antibiotic susceptibility patterns and genotypic traits of a collection of Klebsiella spp. isolates. Furthermore, the clonal relatedness of blaNDM producing strains was investigated. METHODS: During a 19-months  surveillance study, 122 Klebsiella spp. isolates were cultured from extraintestinal specimens of patients admitted to the tertiary referral hospital in Semnan, Iran. Isolates were identified using biochemical tests and subjected to determination of phylogroups, capsular types and virulence/resistance genes content. Hypervirulent K. pneumoniae (hvKp) strains were detected genotypically, and Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR fingerprinting was used to determine the clonality of blaNDM producing strains. RESULTS: Multidrug resistant phenotype was detected in 75 (61.5%) isolates and amikacin was found as the most potent antibiotic with the susceptibility rate of 85.2%. The carbapenemase genes were detected in 45 (36.8%) strains, including 21 (17.2%) blaOXA-48, 7 (5.6%) blaNDM-1, 14 (11.4%) blaNDM-1/OXA-48 and 3 (2.4%) blaIMP- carrying strains, while 55 (45.08%) isolates showed carbapenem resistant phenotype. The first blaNDM-1 carrying strain was cultured from a sputum specimen on March 2015, while the last positive one was recovered from blood culture on September 2016. Most of the isolates (80.3%) belonged to phylogroup I, and blaNDM-1 was identified among all three phylogroups. The ERIC-PCR clustered the 101 blaNDM negative and 21 blaNDM-1 positive isolates into 25 and five clusters, respectively, and the latter group belonged to clonal complex 147 (CC147). One K1 and 15 K2 blaNDM-1 negative isolates were detected, of those three strains were identified as hvKp. Five K2 positive strains, including four blaOXA-48 producer and one hvKp sequence type 86 (ST86) were carbapenem resistant. Among carbapenem resistant isolates, CC147 strains harboured higher rates of siderophores iutA and ybtS. CONCLUSION: The present findings showed a hospital circulation of CC147 blaNDM-1 or blaNDM-1/OXA-48 producing strains, disseminated in different wards. The hvKp/ST86 strain expressing K2 capsular type and carbapenem resistant phenotype wasn't reported from Iran so far. So, it seems that we must be aware of the emergence and spread of new K. pneumoniae clones associated with resistant and hypermucoviscous phenotypes.

Clonal diversity, virulence genes content and subclone status of Escherichia coli sequence type 131: comparative analysis of E. coli ST131 and non-ST131 isolates from Iran.

BACKGROUND: The Escherichia coli sequence type 131 (ST131) is a well established clone causing significant extraintestinal infections worldwide. However, no studies have been reported the phenotypic and molecular traits of ST131 isolates in comparison to other clones of E. coli from Iran. So, we determined the differences between 69 ST131 strains collected during a one year surveillance study and 84 non-ST131 isolates, including 56 clinical fluoroquinolone resistant and 28 broiler colibacillosis isolates in terms of clonality and genetic background. RESULTS: ST131 isolates were associated with phylogroup B2 (68 out of 69 isolates, 98.4%), while clinical non-ST131 and fluoroquinolone resistant broiler isolates mainly belonged to phylogroup A. The highest virulence score was observed in ST131 clone, while they showed less diversity in virulence profiles than other clinical isolates. Almost all of the ST131 isolates (95.6%) were ExPEC and had the highest virulence scores, but their resistance scores were less than clinical non-ST131 isolates. Broiler isolates showed higher prevalence of ExPEC-associated virulence genes and CTX-M-G1/G9 resistance determinants as compared to clinical non-ST131 isolates. While blaOXA-48/NDM carbapenemases were mostly found in ST131 clone, resistance rate against ertapenem was higher among clinical non-ST131 strains. According to ERIC-based fingerprinting, the ST131 strains were more genetically similar, followed by non-ST131 and broiler isolates. CONCLUSIONS: ST131 isolates possess the ability to make a balance between clonality and extent of resistance/virulence genes content, so this phenomenon gives a fitness advantage over other E. coli clones. The broilers E. coli population poses a potential zoonotic risk which could be transmitted to the community through the food chain. A number of factors are involved in the dissemination of and infections due to ST131 clone.

Characterization of antibiotic-susceptibility patterns and virulence genes of five major sequence types of Escherichia coli isolates cultured from extraintestinal specimens: a 1-year surveillance study from Iran.

OBJECTIVES: Escherichia coli sequence types (STs) 69, 73, 95, 127, and 131 are major STs frequently causing extraintestinal infections. The prevalence of specific clones and their virulence and resistance profiles has not been described from Iran. The aim of this study was to characterize antimicrobial-susceptibility profiles and virulence traits of five major clones of E. coli recovered from human extraintestinal infections in Semnan, Iran. We compared these traits between major ST clones and also between O25b and O16 subgroups of the ST131 clone. METHODS: We characterized the five major ST clones among 335 collected E. coli isolates obtained from extraintestinal infections, and phylogenetic groups, antimicrobial susceptibility, and virulence/resistance-gene profiles of these major STs were studied. RESULTS: The highest rates of the multidrug-resistance phenotype were detected among ST131 (85.7%) and ST69 (41.7%), and trimethoprim/sulfamethoxazole resistance was detected significantly among the latter clone. Of the 151 isolates belonging to major ST clones, bla OXA-48 was detected among all except the ST127 clone, while bla NDM genes were harbored by 14 (9.2%) isolates, which all belonged to the ST131 clone. Aggregate virulence scores (median) of ST131 isolates (11) were slightly higher than ST69 (8.50) strains, but were lower than ST73 (16), ST95 (16), and ST127 (12.50) isolates. Principal-coordinate analysis revealed distinct virulence profiles with the ST131 clone. ST73, ST95 and ST131 were enriched with "urovirulence" traits, including phylogroup B2 and group B2-associated accessory traits (chuA, iutA, yfcV, papGII, usp, kpsMTII and malX) and the derived variables extraintestinal pathogenic E. coli and uropathogenic E. coli. In contrast, ST69 was depleted of these traits, but enriched with phylogroups D and E. CONCLUSION: Our data emphasize that isolates of the ST131 clone have the ability to make a balance between resistance and virulence traits to establish a wider clone in extraintestinal pathogenic E. coli.

Evaluation of the commercial combined disk test and minimum inhibitory concentration (MIC) determination for detection of carbapenemase producers among Gram-negative bacilli isolated in a region with high prevalence of blaOXA-48 and blaNDM

Carbapenem-resistant Gram-negative bacilli (GNB) are a concern in the Middle East and worldwide. Simple screening methods have been sought to detect carbapenemase producers to determine appropriate therapeutic measures and implement infection control interventions. In this study, we evaluated the efficiency of agar disc diffusion, commercial combined disc test (Rosco), and carbapenem MIC determination in comparison to molecular detection of carbapenemase genes among 82 carbapenem non-susceptible Enterobacteriaceae (CNSE) and 37 Acinetobacter/Pseudomonas isolates. The blaOXA-48, blaNDM, blaNDM/OXA-48, and blaIMP were detected in 68 out of 82 CNSE isolates. All of the Acinetobacter baumannii isolates were positive for the blaOXA-51 (n=23), of those some were positive for blaOXA-48 (n=13) and blaNDM (n=3). Sensitivities and specificities of combined disc test for detection of blaNDM and blaOXA-48 carrying Enterobacteriaceae isolates were 92.5% and 100%, and 58.5% and 100%, respectively, while those for Acinetobacter/Pseudomonas isolates were 100%, 81.8% and 96.2%, 89%, respectively. While carbapenem MIC values had excellent concordance with phenotypic combined disc test for detection of blaOXA-48 producers (area under curve >90%), only ertapenem MIC's could precisely detect blaOXA-48 PCR-positive Enterobacteriaceae isolates (AUC 70%, sensitivity 70%, specificity 50%). The phenotypic commercial test showed excellent sensitivity for detection of blaNDM producers, but had poor sensitivity for blaOXA-48-producing Enterobacteriaceae. Ertapenem MIC values had low sensitivity and specificity for detection of the blaOXA-48-carrying Enterobacteriaceae. This is the first report of A. baumannii isolates co-harbored the blaOXA-48/blaNDM carbapenemases from Iran

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Evaluation of the commercial combined disk test and minimum inhibitory concentration (MIC) determination for detection of carbapenemase producers among Gram-negative bacilli isolated in a region with high prevalence of blaOXA-48 and blaNDM.

Carbapenem-resistant Gram-negative bacilli (GNB) are a concern in the Middle East and worldwide. Simple screening methods have been sought to detect carbapenemase producers to determine appropriate therapeutic measures and implement infection control interventions. In this study, we evaluated the efficiency of agar disc diffusion, commercial combined disc test (Rosco), and carbapenem MIC determination in comparison to molecular detection of carbapenemase genes among 82 carbapenem non-susceptible Enterobacteriaceae (CNSE) and 37 Acinetobacter/Pseudomonas isolates. The blaOXA-48, blaNDM, blaNDM/OXA-48, and blaIMP were detected in 68 out of 82 CNSE isolates. All of the Acinetobacter baumannii isolates were positive for the blaOXA-51 (n = 23), of those some were positive for blaOXA-48 (n = 13) and blaNDM (n = 3). Sensitivities and specificities of combined disc test for detection of blaNDM and blaOXA-48 carrying Enterobacteriaceae isolates were 92.5% and 100%, and 58.5% and 100%, respectively, while those for Acinetobacter/Pseudomonas isolates were 100%, 81.8% and 96.2%, 89%, respectively. While carbapenem MIC values had excellent concordance with phenotypic combined disc test for detection of blaOXA-48 producers (area under curve > 90%), only ertapenem MIC's could precisely detect blaOXA-48 PCR-positive Enterobacteriaceae isolates (AUC 70%, sensitivity 70%, specificity 50%). The phenotypic commercial test showed excellent sensitivity for detection of blaNDM producers, but had poor sensitivity for blaOXA-48-producing Enterobacteriaceae. Ertapenem MIC values had low sensitivity and specificity for detection of the blaOXA-48-carrying Enterobacteriaceae. This is the first report of A. baumannii isolates co-harbored the blaOXA-48/blaNDM carbapenemases from Iran.

Is it true that gut microbiota is considered as panacea in cancer therapy?

Recent studies demonstrated that a combination of the gut microbiome has the vital effect on the efficacy of anticancer immune therapies. Regulatory effects of microbiota have been shown in different types of cancer therapies such as chemotherapy and immunotherapy. Immune-checkpoint-blocked therapies are the recent efficient cancer immunotherapy strategies. The target of immune-checkpoint blocking is cytotoxic T lymphocyte protein-4 (CTLA-4) or blockade of programmed death-1 (PD-1) protein and its ligand programmed death ligand 1 (PD-L1) that they have been considered as cancer immunotherapy in recent years. In the latest studies, it have been demonstrated that several gut bacteria such as Akkermansia muciniphila, Bifidobacterium spp., Faecalibacterium spp., and Bacteroides fragilis have the regulatory effects on PD-1, PD-L1, and CTLA-4 blocked anticancer therapy outcome.

A multicenter study of ß-lactamase-producing Klebsiella pneumoniae isolated from university teaching hospitals of Urmia, Iran.

INTRODUCTION: Klebsiella pneumoniae is an opportunistic pathogen accounting for 5-7% of hospital acquired infections. The emergence of carbapenem-resistant Klebsiella pneumoniae has been increasing rapidly over recent years causing many therapeutic problems worldwide. This study aimed to research the antimicrobial resistance profile, detect ß-lactamase genes among clinical isolates of K. pneumoniae, and determine their clonal relatedness. METHODOLOGY: All Klebsiella pneumoniae isolates were obtained from teaching hospitals in Urmia, Iran. Antimicrobial susceptibility testing was done by the disk diffusion method. Furthermore, minimum inhibitory concentrations of imipenem were determined by applying Etest strips. Screening of ß-lactamase-producing isolates was performed by the combined disk method and modified Hodge test. The detection of ß-lactamase genes was conducted by polymerase chain reaction (PCR), and isolates' clonal relatedness was evaluated by random amplified polymorphic DNA (RAPD)-PCR. RESULTS: Overall, 45 out of 182 (24.7%) K. pneumoniae isolates were non-susceptible to imipenem. The combined disk method and modified Hodge test revealed that 93.3% and 71.1% of the imipenem non-susceptible isolates were ß-lactamase producers, respectively. The presence of blaVIM, blaNDM, blaKPC, and blaIMP genes was confirmed in 48.9%, 15.6%, 11.1%, and 6.7% of the ß-lactamase-producing isolates, respectively. RAPD-PCR revealed that 73% of these isolates were classified into six different clusters. CONCLUSIONS: A relatively high prevalence of ß-lactamase genes was seen among multidrug-resistant isolates of K. pneumoniae. Most patients infected with ß-lactamase-producing isolates had a history of long-term hospitalization and nosocomial infections. The predominance of ß-lactamase genes in intensive care unit and internal units alarm clinicians to the growth of hospitalization and mortality rates.

Probiotics importance and their immunomodulatory properties.

Mammalian intestine contains a large diversity of commensal microbiota, which is far more than the number of host cells. Probiotics play an insecure and protective role against the colonization of intestinal pathogenic microbes and increase mucosal integrity by stimulating epithelial cells. Probiotics have innate capabilities in many ways, including receptor antagonism, receptor expression, binding and expression of adapter proteins, expression of negative regulatory signal molecules, induction of microRNAs, endotoxin tolerance, and ultimately secretion of immunomodulatory proteins, lipids, and metabolites to modulate the immune system. Probiotic bacteria can affect homeostasis, inflammation, and immunopathology through direct or indirect effects on signaling pathways as immunosuppressant or activators. Probiotics suppress inflammation by inhibiting various signaling pathways such as the nuclear factor-κB (NF-κß) pathway, possibly related to alterations in mitogen-activated protein kinases and pattern recognition receptors pathways. Probiotics can also inhibit the binding of lipopolysaccharides to the CD14 receptor, thereby reducing the overall activation of NF-κß and producing proinflammatory cytokines. Some effects of modulation by probiotics include cytokine production by epithelial cells, increased mucin secretion, increased activity of phagocytosis, and activation of T and natural killer T cells, stimulation of immunoglobulin A production and decreased T cell proliferation. Intestinal microbiota has a major impact on the systemic immune system. Specific microbiota controls the differentiation of cells in lamina propria, in which Th17 cells secrete interleukin 17. The presence of Th17 and Treg cells in the small intestine is associated with intestinal microbiota, with the preferential Treg differentiation and the absence of Th17 cells, possibly reflecting alterations in the lamina propria cytokines and the intestinal gut microbiota.