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Aims: Making a correct and rapid diagnosis is essential for managing pulmonary tuberculosis (PTB), particularly multidrug-resistant tuberculosis. We aimed to evaluate the efficacy of the combination of simultaneous amplification testing (SAT) and reverse dot blot (RDB) for the rapid detection of Mycobacterium tuberculosis (MTB) and drug-resistant mutants in respiratory samples., Methods: 225 suspected PTB and 32 non-TB pulmonary disease samples were collected. All sputum samples were sent for acid-fast bacilli smear, SAT, culture and drug susceptibility testing (DST) by the BACTEC TM MGIT TM 960 system. 53 PTB samples were tested by both RDB and DNA sequencing to identify drug resistance genes and mutated sites., Results: The SAT positive rate (64.9%) was higher than the culture positive rate (55.1%), with a coincidence rate of 83.7%. The sensitivity and specificity of SAT for diagnosing PTB were 66.7% and 100%, respectively, while those for culture were 53.9% and 84.2%, respectively. RDB has high sensitivity and specificity in identifying drug resistance genes and mutated sites. The results of RDB correlated well with those of DST and DNA sequencing, with coincidence rates of 92.5% and 98.1%, respectively., Conclusions: The combination of SAT and RDB is promising for rapidly detecting PTB and monitoring drug resistance in clinical laboratories., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Bacterial toxicity of graphene nanosheets in the form of graphene nanowalls deposited on stainless steel substrates was investigated for both gram-positive and gram-negative models of bacteria. The graphene oxide nanowalls were obtained by electrophoretic deposition of Mg(2+)-graphene oxide nanosheets synthesized by a chemical exfoliation method. On the basis of measuring the efflux of cytoplasmic materials of the bacteria, it was found that the cell membrane damage of the bacteria caused by direct contact of the bacteria with the extremely sharp edges of the nanowalls was the effective mechanism in the bacterial inactivation. In this regard, the gram-negative Escherichia coli bacteria with an outer membrane were more resistant to the cell membrane damage caused by the nanowalls than the gram-positive Staphylococcus aureus lacking the outer membrane. Moreover, the graphene oxide nanowalls reduced by hydrazine were more toxic to the bacteria than the unreduced graphene oxide nanowalls. The better antibacterial activity of the reduced nanowalls was assigned to the better charge transfer between the bacteria and the more sharpened edges of the reduced nanowalls, during the contact interaction.

A microelectronic array assay was developed to specifically genotype Helicobacter pylori versus Helicobacter heilmannii and to determine antimicrobial resistance. Helicobacter 16S rRNA and 23S rRNA genes were specifically generated with Helicobacter genus-specific primers, respectively. The single-nucleotide polymorphisms (SNPs) in 16S rRNA, 268T specific in the H. pylori sequence, and 263A specific in H. heilmannii were used as molecular markers for identification of H. pylori and H. heilmannii, respectively. A triple-base-pair resistant mutation, AGA965-967TTC in 16S rRNA, is known to be responsible for H. pylori tetracycline resistance and was detected to identify resistant strains. H. pylori macrolide resistance was determined by the identification of 3 defined mutations in the 23S rRNA gene using the same method. The assay could be directly used to detect H. pylori in feces. The assay performs multiple determinations, including identification of Helicobacter species and antibiotic resistances, on the same microelectronic platform and is highly amenable to the development of other DNA-based assays.

Random amplified polymorphic DNA (RAPD) analysis was evaluated for the selection and elimination of bacterial strains used in microbial screening. For this pilot study we used eight bacterial strains producing fragin and two Pseudomonas fragi strains, which are often isolated during the screening. A dendrogram constructed by the statistical analysis using parsimony, PAUP, based on the band patterns of RAPD with primer R28 was in good correlation with the results of DNA-DNA hybridization, HPLC analysis of metabolites, and conventional morphological and physiological characterization. RAPD was also applicable to a wide range of bacteria. This rapid selection system by RAPD was a very useful tool for excluding similar bacterial isolates encountered during screening.

For efficient fungal strain selection in microbial screening, we applied the random amplified polymorphic DNA (RAPD) method using the polymerase chain reaction (PCR). In order to evaluate this system, the genus Trichoderma was employed, because its species are difficult to distinguish from each other. We selected an appropriate oligonucleotide decamer, R28 (5'-ATGGATCCGC), determined the optimal cycles of PCR as 30 cycles, simplified the template preparation method, and determined optimal concentrations of the template and Taq DNA polymerase. We then examined 74 closely related strains of Trichoderma. The electrophoretic band patterns of the PCR products were compared. According to the statistical analysis with the phylogenetic analysis using parsimony (PAUP), the results were consistent with the morphological, physiological and ecological data on these strains. Therefore, we conclude that RAPD is a simple, efficient and reliable method for the selection of fungal strains employed in screening.

We evaluated the random amplified polymorphic DNA (RAPD) method using Streptomyces lavendulae and Streptomyces virginiae strains to eliminate duplicate actinomycete strains in our microbial screening program. The RAPD data were compared with phenotypic characteristics, DNA relatedness, HPLC analysis of metabolites and low-frequency restriction fragment analysis by pulsed-field gel electrophoresis. These results were consistent with each other. Therefore, we conclude that RAPD is a simple, efficient, and reliable method for the selection of actinomycete strains.

Introduction: Since its discovery in 1961, methicillin-resistant Staphylococcus aureus (MRSA) has been recognized as a significant healthcare-associated pathogen (HA-MRSA) and a notorious 'superbug'. Typing is crucial for surveillance, epidemiology analysis, infection control of MRSA and sequencing of the spa gene is one of the most common methods used for determining the origin of this bacterium in humans and animals. This research aimed to determine the antibiotic resistance and spa type of S. aureus strains collected from outpatients in two hospitals in the Wasit province of Iraq. Material & Methods: The study analyzed 200 outpatient MRSA isolates by collecting nasal and sputum samples from patients. Standard biochemical and molecular methods based on the nuc gene were used to identify S. aureus bacteria and amplify the mecA and spa genes. The Kirby-Bauer disc diffusion method was employed to determine the antibiotic sensitivity of the isolates using penicillin, cefoxitin, vancomycin, gentamicin, erythromycin, tetracycline, imipenem, clindamycin, chloramphenicol and rifampicin. Results: Methods. The prevalence of MRSA was more common in women than in men. Antibiogram results showed that most of the isolates were resistant to penicillin (94.2%) and sensitive to imipenem (100%), clindamycin (100%), and chloramphenicol (100%). Of these 35 isolates, 30 (87.5%) and 26 strains (74.3%) were positive for the mecA and spa genes. Typing based on spa gene sequencing revealed four different patterns: t386, t3579, U0002 and U0234. Conclusion: Variations in the spa gene among different S. aureus isolates may he of clinical importance when treating staphylococcal infections. In this study, spa typing revealed four different patterns in Iraq, representing diagnostic and therapeutic implications. [ABSTRACT FROM AUTHOR]

Background & Objective: Investigating multidrug resistance and TEM and SHV broad-spectrum beta-lactamase genes in Escherichia coli bacteria isolated from patients with urinary tract infections is very useful to improve the treatment of infection and prevent the failure of treatment of urinary tract infections.The aim of this study was to investigate multidrug resistance and TEM and SHV broadspectrum beta-lactamase genes in Escherichia coli bacteria isolated from patients with urinary tract infections. Materials & Methods: In this study, 188 strains of Escherichia coli, which cause urinary tract infections in Alborz province, were studied. Urine samples were cultured on EMB and Blood Agar media. Differential tests were performed for final identification. ESBL-producing strains were identified, PCR was performed to survey the abundance of ESBL-producing genes. Results: Based on the results of the disk diffusion and Double-disk synergy testS, 82 (43.6%) strains were determined as the final producer of ESBL. out of these isolates, the frequency of SHV, TEM, and CTX genes measured 64.3%, 55.9%, and 21.4%, respectively. These results showed that 12 (14.28%) of Escherichia coli isolated have all genes, 26 (30.95%) had 2 genes and 36 (42.85%) had one gene. Conclusion: According to the results, it was found that imipenem with the lowest resistance is the best drug in the treatment, and carbapenems are the best drug for treating diseases caused by Escherichia coli. The results of the current study may be useful in replacing ESBL enzyme resistance screening with more modern sensitivity measurement methods such as MIC and Etest. [ABSTRACT FROM AUTHOR]

Introduction: The global public health concern of Staphylococcus aureus (S. aureus) infection and its spread, particularly methicillin-resistant Staphylococcus aureus (MRSA), is exacerbated by the emergence of mec-genes, which are considered markers of MRSA. The study aims to isolate MRSA in clinical isolates and detect mec-genes using molecular assays, thereby enhancing infection control programs. Materials and Methods: The study involved 381 clinical samples processed by standard laboratory procedures and PCR was performed targeting 16S rRNA gene as a reference and then with nuc-gene to detect Staphylococcus aureus. Further, MRSA was confirmed by PCR (Polymerase Chain Reaction) amplification of S. aureus isolates by subtyping mecA and mecC gene. Sensitivity and specificity of phenotypic methods was calculated using mecA gene PCR as the gold standard. Results: In the present study, 65.85% clinical isolates were found to have similar homology with Staphylococcus aureus. Uniplex PCR results showed that all 162 isolates with S. aureus-like phenotypes were detected as MRSA carrying mecA gene, but none of them was found to carry mecC gene for MRSA. P-value for both the methods was <0.001, which is considered to be highly significant in statistical analysis. Conclusion: The study found that PCR detection of nuc and mec genes is effectively identifies MRSA from clinical isolates, aiding in infection prevention and recommending its use for confirmatory testing. The study recommends using PCR as a confirmatory test method for MRSA detection to prevent the spread of resistant strains in hospitals and communities. [ABSTRACT FROM AUTHOR]

Background and Aim:. Pseudomonas aeruginosa exotoxins are responsible for pathogenicity and antibiotic resistant. The aim of this study was to investigate the effects of the Ammi. visnaga (A. visnaga) extract on the P. aeruginosa exotoxin A and S genes expression. Materials and Methods: Antibacterial effects of the A. visnaga ethanolic extract were performed using the micro dilution technique in sequential dilutions. Then, the Real-Time- RT PCR method was used to analyze the level of expression of the exotoxin A and S genes of the bacterium. Real-Time PCR data were analyzed using Graph Pad Software Inc. San Diego (GRAPHPAD PRISM 6CA, USA). Results: Results showed that, the expression of Exotoxin A and S genes was significantly (P <0.001) reduced in comparing the expression level of the reference gene and control. It had a significant effect on the inhibition of bacterial growth at different concentrations but the lowest inhibitory concentration was observed at 3.125 µg/well. Conclusion: Accordingly, the extract of A. visnaga can decrease the expression of the Exotoxin A and S genes of P. aeruginosa and consequently it may decrease the pathogenicity of bacteria. These phenomena (exoA and exoS expression inhibition) may involve reducing the antibiotic resistance of P. aeruginosa. [ABSTRACT FROM AUTHOR]

Background and Aim: Otitis media (OM) or middle ear inflammation is an inflammatory disease caused by a bacterial infection such as Pseudomonas aeruginosa (P. aeruginosa), provided with genetic virulence factors. This study aims to detect nan1 and tox A genes of P. aeruginosa isolated from chronic OM patients. Materials and Methods: One hundred thirty OM patients were recruited from Al-Sader medical city and Al-Hakim general hospital in Najaf province from September to January 2021 to collect ear swaps. The Primary identification depends on Gram stain and biochemical tests. We also used Vitek 2 system to make another identification and examine resistance to antibiotics. A polymerase chain reaction (PCR) was used to amplify the required genes. Furthermore, gel electrophoresis was performed to show the amplified genes. Results: The results have revealed that 115 samples (88.46%) give a positive result, while 15 samples (11.54%) are negative for culture bacteria. Only 49 (42.76 %) of the 115 clinical specimens showed positive tests for P. aeruginosa. However, the toxA gene is found in 67.3% of P. aeruginosa isolates, while the nan1 gene is found in 38.77 % of the isolates. Conclusion: Our findings suggest that P. aeruginosa is one of the most common causes of OM infection in Iraqi patients. Most bacterial isolates show the presence of nan1 and tox A genes, with a high prevalence of tox A than nan1. The presence of these genes is one of the virulence factors and probably partly explains the resistance to antibiotics. [ABSTRACT FROM AUTHOR]

AimExtended-spectrum β-lactamase-producing (ESBL) Escherichia coli are a public threat worldwide. This study aimed at analyzing the genomic and functional attributes of nine ESBLs taken from rectal swabs.Materials & methodsSamples were isolated from patients admitted for gastrointestinal and urological procedures at the University Medical Center-Rizk Hospital (UMCRH) in Lebanon. Illumina paired-end libraries were prepared and sequenced.ResultsThe isolates were distributed into five lineages: ST131, ST648, ST405, ST73 and ST38, and harbored bla OXA-1, bla TEM-1B, bla TEM-1C and aac(6')Ib-cr. ST131 isolates were carriers of stx2 converting I phage.ConclusionThis is the first comprehensive genomic analysis performed on ESBLs in Lebanon.

AimExtended-spectrum β-lactamase-producing (ESBL) Escherichia coli are a public threat worldwide. This study aimed at analyzing the genomic and functional attributes of nine ESBLs taken from rectal swabs.Materials & methodsSamples were isolated from patients admitted for gastrointestinal and urological procedures at the University Medical Center-Rizk Hospital (UMCRH) in Lebanon. Illumina paired-end libraries were prepared and sequenced.ResultsThe isolates were distributed into five lineages: ST131, ST648, ST405, ST73 and ST38, and harbored bla OXA-1, bla TEM-1B, bla TEM-1C and aac(6')Ib-cr. ST131 isolates were carriers of stx2 converting I phage.ConclusionThis is the first comprehensive genomic analysis performed on ESBLs in Lebanon.

BackgroundAntimicrobial resistance (AMR) is a growing and significant threat to public health on a global scale. Escherichia coli comprises Gram-negative, fecal-borne pathogenic and commensal bacteria that are frequently associated with antibiotic resistance. AMR E. coli can be ingested via food, water and direct contact with fecal contamination.MethodsWe estimated the prevalence of AMR Escherichia coli from select drinking water sources in northern Tanzania. Water samples (n = 155) were collected and plated onto Hi-Crome E. coli and MacConkey agar. Presumptive E. coli were confirmed by using a uidA PCR assay. Antibiotic susceptibility breakpoint assays were used to determine the resistance patterns of each isolate for 10 antibiotics. Isolates were also characterized by select PCR genotyping and macro-restriction digest assays.ResultsE. coli was isolated from 71 % of the water samples, and of the 1819 E. coli tested, 46.9 % were resistant to one or more antibiotics. Resistance to ampicillin, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim was significantly higher (15-30 %) compared to other tested antibiotics (0-6 %; P

Background: Dysbiosis in subgingival microbial communities, resulting from increased inflammatory transudate from the gingival tissues, is an important factor in initiation and development of periodontitis. Dysbiotic communities are characterized by increased numbers of bacteria that exploit the serum-like transudate for nutrients, giving rise to a proteolytic community phenotype. Here we investigate the contribution of interactions between members of a sub-gingival community to survival and development of virulence in a serum environment—modelling that in the subgingival pocket. Methods: Growth and proteolytic activity of three Porphyromonas gingivalis strains in nutrient broth or a serum environment were assessed using A600 and a fluorescent protease substrate, respectively. Adherence of P. gingivalis strains to serum-coated surfaces was studied with confocal microscopy and 2D-gel electrophoresis of bacterial supernatants used to investigate extracellular proteins. A model multi-species sub-gingival community containing Fusobacterium nucleatum, Streptococcus constellatus, Parvimonas micra with wild type or isogenic mutants of P. gingivalis was then created and growth and proteolytic activity in serum assessed as above. Community composition over time was monitored using culture techniques and qPCR. Results: The P. gingivalis strains showed different growth rates in nutrient broth related to the level of proteolytic activity (largely gingipains) in the cultures. Despite being able to adhere to serum-coated surfaces, none of the strains was able to grow alone in a serum environment. Together in the subgingival consortium however, all the included species were able to grow in the serum environment and the community adopted a proteolytic phenotype. Inclusion of P. gingivalis strains lacking gingipains in the consortium revealed that community growth was facilitated by Rgp gingipain from P. gingivalis. Conclusions: In the multi-species consortium, growth was facilitated by the wild-type and Rgp-expressing strains of P. gingivalis, suggesting that Rgp is involved in delivery of nutrients to the whole community through degradation of complex protein substrates in serum. Whereas they are constitutively expressed by P. gingivalis in nutrient broth, gingipain expression in the model periodontal pocket environment (serum) appeared to be orchestrated through signaling to P. gingivalis from other members of the community, a phenomenon which then promoted growth of the whole community. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a major public health concern all around the world. In the frame of this work, a set of diverse environmental P. aeruginosa isolates with various antibiotic resistance profiles were examined in a Galleria mellonella virulence model. Motility, serotypes, virulence factors and biofilm-forming ability were also examined. Molecular types were determined by pulsed-field gel electrophoresis (PFGE). Based on our results, the majority of environmental isolates were virulent in the G. mellonella test and twitching showed a positive correlation with mortality. Resistance against several antibiotic agents such as Imipenem correlated with a lower virulence in the applied G. mellonella model. PFGE revealed that five examined environmental isolates were closely related to clinically detected pulsed-field types. Our study demonstrated that industrial wastewater effluents, composts, and hydrocarbon-contaminated sites should be considered as hot spots of high-risk clones of P. aeruginosa. [ABSTRACT FROM AUTHOR]

Objective(s): Strain subtyping is an important epidemiological tool to trace contamination, determine clonal relationships between different strains, and the cause of outbreaks. Current subtyping methods, however, yield less than optimal subtype discrimination. Pulsed-field gel electrophoresis is the gold standard method for Escherichia coli and Multiple-Locus Variable-number tandem repeat Analysis (MLVA) is a rapid PCR-based method. The purpose of this study was to evaluate MLVA and pulsed-field gel electrophoresis (PFGE) methods for subtyping β -lactamase-producing E. coli strains isolated from urinary tract infections. Materials and Methods: Overall, 230 E. coli isolates from patients with urinary tract infections were examined for antimicrobial susceptibility testing. 10-loci and 7-loci MLVA and PFGE methods were used for molecular typing of β -lactamase-producing E. coli isolates. Results: Out of 230 isolates, 130 (56.5%) β -lactamase-producing E. coli isolates were found in this study. The diversity indices of the VNTR loci showed an average diversity of 0.48 and 0.54 for 7-loci and 10-loci MLVA, respectively. The discriminatory power of PFGE showed a value of 0.87. The discordance between the methods was high. Conclusion: Our study showed that PFGE is more discriminatory than MVLA. MLVA is a PCR- based method and can generate unmistakable data, in contrast to PFGE. Optimization of polymorphic VNTR is essential to improve the discriminatory power of MLVA based on geographical region. [ABSTRACT FROM AUTHOR]

Chlorhexidine and mupirocin are used in health care facilities to eradicate methicillin-resistant Staphylococcus aureus (MRSA) carriage. The objective of this study was to assess the frequency of chlorhexidine and mupirocin resistance in isolates from nares carriers in multiple nursing homes and to examine characteristics associated with resistance. Nasal swab samples were collected from approximately 100 new admissions and 100 current residents in 26 nursing homes in Orange County, CA, from October 2008 to May 2011. MRSA isolates were tested for susceptibility by using broth microdilution, disk diffusion, and Etest; for genetic relatedness using pulsed-field gel electrophoresis; and for qac gene carriage by PCR. Characteristics of the nursing homes and their residents were collected from the Medicare Minimum Data Set and Long-Term Care Focus. A total of 829 MRSA isolates were obtained from swabbing 3,806 residents in 26 nursing homes. All isolates had a chlorhexidine MIC of ≤4 μg/ml. Five (0.6%) isolates harbored the qacA and/or qacB gene loci. Mupirocin resistance was identified in 101 (12%) isolates, with 78 (9%) isolates exhibiting high-level mupirocin resistance (HLMR). HLMR rates per facility ranged from 0 to 31%. None of the isolates with HLMR displayed qacA or qacB, while two isolates carried qacA and exhibited low-level mupirocin resistance. Detection of HLMR was associated with having a multidrug-resistant MRSA isolate (odds ratio [OR], 2.69; P = 0.004), a history of MRSA (OR, 2.34; P < 0.001), and dependency in activities of daily living (OR, 1.25; P = 0.004). In some facilities, HLMR was found in nearly one-third of MRSA isolates. These findings may have implications for the increasingly widespread practice of MRSA decolonization using intranasal mupirocin.

Objectives: Increasing numbers of linezolid-resistant Enterococcus carrying optrA are being reported across different niches worldwide. We aimed to characterize the first optrA-carrying Enterococcus faecalis obtained from food-producing animals and retail meat samples in Tunisia.Methods: Seven optrA-carrying E. faecalis obtained from chicken faeces (n=3, August 2017) and retail chicken meat (n=4, August 2017) in Tunisia were analysed. Antimicrobial susceptibility was determined by disc diffusion, broth microdilution and Etest against 13 antibiotics, linezolid and tedizolid, respectively (EUCAST/CLSI). optrA stability (∼600 bacterial generations), transfer (filter mating) and location (S1-PFGE/hybridization) were characterized. WGS (Illumina-HiSeq) was done for four representatives that were analysed through in silico and genomic mapping tools.Results: Four MDR clones carrying different virulence genes were identified in chicken faeces (ST476) and retail meat (the same ST476 clone plus ST21 and ST859) samples. MICs of linezolid and tedizolid were stably maintained at 8 and 1-2 mg/L, respectively. optrA was located in the same transferable chromosomal Tn6674-like element in ST476 and ST21 clones, similar to isolates from pigs in Malaysia and humans in China. ST859 carried a non-conjugative plasmid of ∼40 kb with an impB-fexA-optrA segment, similar to plasmids from pigs and humans in China.Conclusions: The same chromosomal and transferable Tn6674-like element was identified in different E. faecalis clones from humans and animals. The finding of retail meat contaminated with the same linezolid-resistant E. faecalis strain obtained from a food-producing animal highlights the potential role of the food chain in the worrisome dissemination of optrA that can be stably maintained without selective pressure over generations. [ABSTRACT FROM AUTHOR]

Objectives: To determine the prevalence of Escherichia coli STs and associated resistance mechanisms carried by the community in North-East India.Methods: E. coli (108) were isolated from sewage collected from 19 sites across the city of Silchar by plating on MacConkey agar with/without selection (50 mg/L cefotaxime). Species identification was confirmed by MALDI-TOF MS for 82 isolates. Common resistance mechanisms were determined by WGS of pooled E. coli isolates. PFGE combined with specific probes determined the presence of common resistance mechanisms in all isolates. Phylotypes, multilocus STs, core-genome multilocus STs, resistance genes and virulence genes were determined by in silico analysis of 38 genomes.Results and Conclusions: Analysis of isolates collected without selection (n=33) indicated that cefotaxime resistance in E. coli was 42% (14/33) and estimated meropenem resistance at 9%. The remaining 58% (19/33) were additionally susceptible to ampicillin, trimethoprim, ciprofloxacin and aminoglycosides. The most common ST among the cefotaxime-resistant E. coli was ST167 (29%), followed by ST410 (17%) and ST648 (10%). E. coli ST131 was absent from the collection. Sixty-three isolates were resistant to cefotaxime and harboured blaCTX-M-15 [54% (34/63)] or blaCMY-42 [46% (29/63)], of which 10% (6/63) harboured both genes. Carbapenem resistance was due to blaNDM-5, found in 10/63 cefotaxime-resistant isolates, and/or blaOXA-181, found in 4/63 isolates. NDM-5 was encoded by IncX3 and/or IncFII plasmids and CMY-42 was mostly encoded by IncI plasmids. NDM-5 appears to have replaced NDM-1 in this region and CMY-42 appears to be in the process of replacing CTX-M-15. [ABSTRACT FROM AUTHOR]

Objectives: The emergence of carbapenemase-positive Enterobacteriaceae poses a serious threat to public health worldwide. Here we conducted a molecular surveillance study on carbapenem-resistant Enterobacteriaceae (CRE) colonization among migratory birds at Qinghai Lake in China.Methods: A total of 420 samples from migratory birds and their surrounding environment were collected at three sites along the Qinghai Lake bird island. Carbapenem-non-susceptible isolates were identified by 16S rDNA sequencing and MALDI-TOF MS. Carbapenemase producers were determined by Carba NP testing. Antimicrobial susceptibility testing, transfer ability and PFGE were also performed, and 46 isolates from different pulsotypes were analysed by WGS.Results: Three hundred and fifty isolates were carbapenemase producers based on Carba NP testing, while 233 Klebsiella spp. and 2 Escherichia coli isolates were NDM-5-carriers. PFGE was performed and showed that the isolates were grouped into five pulsotypes; among these, type A was predominant (86.7%, n = 202) and belonged to a novel Klebsiella lineage, ST1697. WGS analysis indicated that ST1697 strains may be a hybrid of the recombination of Klebsiella quasipneumoniae subsp. similipneumoniae and Klebsiella pneumoniae genomes.Conclusions: This high frequency of carbapenemase producers in migratory birds is unexpected. These results provide new insight into the spread of antibiotic resistance, and highlight that continued vigilance for MDR carbapenemase-producing Enterobacteriaceae in migratory birds is urgently needed. [ABSTRACT FROM AUTHOR]

Objectives: To characterize the oxazolidinone resistance gene poxtA on broad-host-range Inc18 plasmids from CC17 Enterococcus faecium of pig origin.Methods: Oxazolidinone-resistant E. faecium isolates were screened for the presence of poxtA. The poxtA-carrying isolates were characterized by antimicrobial susceptibility testing, conjugation, S1-PFGE and hybridization. The poxtA-carrying plasmids were completely sequenced and their instability was verified.Results: Two individual CC17 E. faecium strains were positive for poxtA. S1-PFGE and hybridization revealed the presence of a poxtA-carrying plasmid of ∼62 kb in both WZ27-2 and the transconjugant, while poxtA-carrying plasmids of different sizes were observed in QF25-1 and the transconjugant. The two poxtA-carrying plasmids, pC25-1 and pC27-2, belonged to the broad-host-range plasmids of the Inc18 family and carried dfrG, aadE, Δsat4, aph(3')-III, erm(B), tet(M), tet(L) and fexB. Plasmid pC27-2 was virtually identical to pC25-1, with minor differences. The calculated transfer frequency was ∼0.87 × 10-8 and ∼1.03 × 10-7 per recipient to plasmids pC25-1 and pC27-2, respectively. Instability assays of the region with four adjacent IS1216Es, which forms three IS1216E translocatable units, revealed the formation of a series of mosaic circular intermediates.Conclusions: We report the emergence of the plasmid-mediated oxazolidinone resistance gene poxtA in E. faecium from different farms in China. Comparison of the poxtA genetic context suggests that IS1216E elements play an important role in the dissemination of poxtA. The co-occurrence of poxtA with other antimicrobial and heavy metal resistance genes on the broad-host-range plasmids of the Inc18 family may lead to the co-selection of poxtA, contributing to its persistence and accelerating its dissemination. [ABSTRACT FROM AUTHOR]

Objectives Carbapenem-resistant Pseudomonas aeruginosa are emerging worldwide with increasing reports of carbapenemase-producing isolates. Carbapenem-resistant isolates may also be XDR. This study characterized carbapenem-resistant and XDR P. aeruginosa isolated from patients receiving care at Canadian hospitals from 2007 to 2016. Methods Antimicrobial susceptibility testing was performed using CLSI broth microdilution methods. PCR was used to detect carbapenemases (GES, KPC, NDM, IMP, VIM, OXA-48) and other resistance markers; specific carbapenemase gene variants were identified by DNA sequencing. Genetic relatedness was assessed by MLST and PFGE. Results From 2007 to 2016, 3864 isolates of P. aeruginosa were collected; 466 (12.1%) isolates were carbapenem resistant. The prevalence of carbapenem-resistant P. aeruginosa reached a peak of 17.3% in 2014. Colistin (94% susceptible) and ceftolozane/tazobactam (92.5%) were the most active agents against carbapenem-resistant P. aeruginosa. XDR P. aeruginosa comprised 4.5% of isolates; they were found to be genetically diverse and remained susceptible to colistin and ceftolozane/tazobactam. Only 4.3% (n  = 20) of carbapenem-resistant P. aeruginosa harboured a carbapenemase; most were bla GES-5 (35%, n  = 7). Wide genetic diversity was observed among carbapenem-resistant P. aeruginosa with >200 different sequence types identified. Conclusions Although the prevalence of carbapenem-resistant P. aeruginosa in Canada spiked in 2014 and 2015, carbapenemase-producing P. aeruginosa remain rare with only 20 (4.3%) isolates identified over a 10 year period. Broad genetic diversity was observed among both carbapenem-resistant and XDR phenotypes of P. aeruginosa. Pan-drug-resistant P. aeruginosa have not yet been identified in Canada. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the occurrence, the genetic environment and the functionality of novel variants of the MDR gene cfr(C) in Campylobacter from China.Methods: A total of 370 Campylobacter isolates of porcine and chicken origin collected from three regions of China in 2015 were screened for cfr(C) by PCR. The phenotypes and genotypes of cfr(C)-positive isolates were investigated by antimicrobial susceptibility testing, PFGE, MLST, S1-PFGE, Southern blotting and WGS. Quantitative RT-PCR was used to compare the expression levels of the cfr(C) variants in their original isolate and clone constructs in Campylobacter jejuni NCTC 11168.Results: Four (1.1%) porcine Campylobacter coli isolates were positive for cfr(C). They failed to show elevated MICs of phenicols. The deduced Cfr(C) sequences identified exhibited 2-6 amino acid changes compared with the original Cfr(C) reported in the USA. Cloning of the cfr(C) variant genes into C. jejuni NCTC 11168 resulted in ≥32-fold increases in the MICs of phenicols, indicating that the cfr(C) variant genes are functional. The cfr(C)-carrying isolates belonged to three genotypes and WGS analysis revealed the cfr(C) genes were chromosomally located in MDR genomic islands, which contained multiple antibiotic resistance genes of Gram-positive origin.Conclusions: This study identified chromosomal cfr(C) genes in C. coli isolates from China. They appeared functionally dormant in the original isolates but were fully functional when cloned and expressed in C. jejuni. The cfr(C) genes were co-transferred with other antibiotic resistance genes, possibly from Gram-positive bacteria. These findings reveal new insights into the function and transmission of cfr(C) in Campylobacter. [ABSTRACT FROM AUTHOR]

Objectives: NDM-producing Enterobacteriaceae clinical isolates remain uncommon in the European region. We describe the emergence and broad dissemination of one successful NDM-1-producing Klebsiella pneumoniae clone in Greek hospitals.Methods: During a 4 year survey (January 2013-December 2016), 480 single-patient carbapenem non-susceptible K. pneumoniae isolates, phenotypically MBL positive, were consecutively recovered in eight Greek hospitals from different locations and subjected to further investigation. Antimicrobial susceptibility testing, combined-disc test, identification of resistance genes by PCR and sequencing, molecular fingerprinting by PFGE, plasmid profiling, replicon typing, conjugation experiments and MLST were performed.Results: Molecular analysis confirmed the presence of the blaNDM-1 gene in 341 (71%) K. pneumoniae isolates. A substantially increasing trend of NDM-1-producing K. pneumoniae was noticed during the survey (R2 = 0.9724). Most blaNDM-1-carrying isolates contained blaCTX-M-15, blaOXA-1, blaOXA-2 and blaTEM-1 genes. PFGE analysis clustered NDM-1 producers into five distinct clonal types, with five distinct STs related to each PFGE clone. The predominant ST11 PFGE clonal type was detected in all eight participating hospitals, despite adherence to the national infection control programme; it was identical to that observed in the original NDM-1 outbreak in Greece in 2011, as well as in a less-extensive NDM-1 outbreak in Bulgaria in 2015. The remaining four ST clonal types (ST15, ST70, ST258 and ST1883) were sporadically detected. blaNDM-1 was located in IncFII-type plasmids in all five clonal types.Conclusions: This study gives evidence of possibly the largest NDM-1-producing K. pneumoniae outbreak in Europe; it may also reinforce the hypothesis of an NDM-1 clone circulating in the Balkans. [ABSTRACT FROM AUTHOR]

Background and Aims: Many bacteria including Escherichia coli may enter into a viable but non-culturable (VBNC) state under unfavorable stresses, which are unable to be detected by culture-based methods. In this study, the use of Reverse Transcription PCR (RT-PCR) for detection of VBNC state of E. coli O157:H7 was investigated. Materials and Methods: Escherichia coli O157:H7 was inoculated in distilled water and 30 percent salt water at room temperature. The cultivability of bacteria was determined using routine culture and colony counting on MacConkey agar. The RT-PCR of 16S rRNA gene involved direct extraction and purification of RNA, DNase I treatment for removing DNA contamination, cDNA synthesis and electrophoresis of PCR products of cDNA was used to detect viable E. coli O157:H7 under studied treatments and was compared with the results of RT-PCR of 16S rRNA gene of heat- killed bacteria. Results: The cultivability of bacteria was maintained during the study period in distilled water; however, the use of 30% NaCl caused the bacteria to be non-cultivable on day 4. The RTPCR of 16S rRNA showed the positive expression of this gene in cultivable and non-cultivable bacteria during the study period, whereas heat-killed bacteria were negative for this gene, which indicated the efficacy of RT-PCR of 16S rRNA in differentiation of alive from dead bacteria. Conclusions: Escherichia coli O157:H7 entered into the VBNC under 30 percent NaCl which can be associated with serious human health problems. RT-PCR can be used to detect bacteria in the VBNC state. [ABSTRACT FROM AUTHOR]

Background Bacteremia caused by extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) is associated with inadequate empirical therapy and substantial mortality in neutropenic patients. Strategies are needed to identify neutropenic patients at high risk of these infections. Methods From April 2014 to September 2016, we collected perianal swabs, both at admission and weekly thereafter, from patients undergoing hematopoietic stem cell transplantation (HSCT). Patients received prophylactic levofloxacin while neutropenic. Swabs were plated onto selective agar, colonies were identified and underwent antimicrobial susceptibility testing, and phenotypic ESBL testing and polymerase chain reaction for β-lactamase genes were performed on ceftriaxone-resistant Enterobacteriaceae. We then determined the prevalence of pre-transplant ESBL-E colonization and risk of ESBL-E bacteremia. Colonizing and bloodstream isolates from patients with ESBL-E bacteremia underwent multilocus sequence typing and pulsed-field gel electrophoresis. Results We analyzed 312 patients, including 212 allogeneic and 100 autologous HSCT recipients. Ten percent (31/312) of patients had pre-transplant ESBL-E colonization. Susceptibility rates of colonizing ESBL-E were: levofloxacin, 25%; cefepime, 9%; piperacillin-tazobactam, 84%; and meropenem, 97%. Of 31 patients colonized with ESBL-E pre-transplant, 10 (32%) developed ESBL-E bacteremia during their transplant admission, compared to 1 (0.4%) of 281 patients not colonized with ESBL-E (P <.001). All bloodstream ESBL-E were levofloxacin-resistant and colonizing and bloodstream isolates from individual patients had identical genotypic profiles. Conclusions HSCT recipients who are colonized with levofloxacin-resistant ESBL-E pre-transplant and receive levofloxacin prophylaxis have high rates of bacteremia from their colonizing strain during neutropenia. Assessing for ESBL-E colonization in neutropenic patients could lead to optimization of empirical antibacterial therapy. [ABSTRACT FROM AUTHOR]

Objectives: To analyse VIM/IMP-type MBL-producing Enterobacteriaceae isolates identified in Poland during 2006-12.Methods: Isolates were typed by PFGE, followed by MLST. blaVIM/IMP genes were amplified and sequenced within class 1 integrons. Their plasmidic versus chromosomal location was assessed by nuclease S1 and I-CeuI plus hybridization experiments. Plasmids were characterized by transfer assays and PCR-based replicon typing.Results: One hundred and nineteen VIM/IMP-positive Enterobacteriaceae cases were reported in Poland from the first case in 2006 until 2012. The patients were in 54 hospitals and were infected or colonized by 121 organisms, including Enterobacter cloacae complex (n = 64), Klebsiella oxytoca (n = 23), Serratia marcescens (n = 20) and Klebsiella pneumoniae (n = 11). The isolates represented numerous pulsotypes and mainly original STs, and carried eight integrons with blaVIM-1-like genes (blaVIM-1/-4/-28/-37/-40; n = 101), three with blaVIM-2 variants (blaVIM-2/-20; n = 17) and one with blaIMP-19 (n = 3). Six integrons were new, and five and two formed prevalent families of In238-like (n = 96) and In1008-like (n = 16) elements, respectively. In238 (aacA4-blaVIM-4rpt) and In1008 (blaVIM-2-aacA4) had been originally observed in Polish Pseudomonas aeruginosa, suggestive of their transfer to enterobacteria, followed by spread and diversification. Four organisms have disseminated inter-regionally, i.e. Enterobacter hormaechei ST90 with plasmidic In238/In238a integrons (n = 36), K. oxytoca ST145 with a chromosomal In237-like element (n = 18) and two subclones of E. hormaechei ST89 with In1008- or In238-type variants (n = 8 and n = 7, respectively).Conclusions: The epidemiology of VIM/IMP-producing Enterobacteriaceae in Poland has revealed a remarkable number of specific or novel characteristics of the organisms, with some possible links to other mid-southern European countries. [ABSTRACT FROM AUTHOR]

Background: Staphylococcus pseudintermedius is genotypically diverse within the canine population and multiple strains may colonize individual dogs at any given time. If multiple strains with distinct antimicrobial resistance profiles are present in superficial bacterial folliculitis (SBF), sampling a single skin lesion for culture and antimicrobial susceptibility testing (AST) might be inadequate to select effective therapy. Hypothesis/Objectives: To investigate S. pseudintermedius diversity in carriage sites and lesions of dogs with SBF. Animals: Fourteen dogs with SBF. Methods: Staphylococcus pseudintermedius isolates obtained from perineum, gingiva and four to six skin lesions per dog were subjected to pulsed‐field gel electrophoresis (PFGE) and AST to assess diversity between lesions. For two dogs, 14–16 isolates per lesion were included in the analysis to assess diversity within lesions. Results: Analysis of one isolate per lesion revealed one to four strains displaying unique PFGE profiles, and up to three unique antimicrobial resistance (AMR) profiles for each dog. Multiple pustules from the same dog always harboured the same strain, whereas papules, crusts and collarettes did not. Up to four strains with distinct AMR profiles were isolated from the same lesion in two dogs. In 12 dogs, at least one carriage site strain also was represented in lesions. Conclusions and clinical importance: Lesions of SBF may harbour multiple S. pseudintermedius strains with distinct antimicrobial resistance profiles. Pustules are the best target for bacterial culture. It remains unclear whether isolation of different strains from other lesion types is a consequence of contamination or co‐infection by multiple strains. [ABSTRACT FROM AUTHOR]

Objectives: We evaluated the antimicrobial susceptibility and ribotypes of Clostridium difficile isolates from participants in a Phase 2 study of ridinilazole, a novel targeted-spectrum agent for treatment of C. difficile infection.Methods: Participants received ridinilazole (200 mg twice daily) or vancomycin (125 mg four times daily) for 10 days (ClinicalTrials.gov: NCT02092935). The MICs of ridinilazole and comparators for C. difficile isolates from stool samples were determined by agar dilution. Toxin gene profiling was performed by multiplex PCR and ribotype identification by capillary electrophoresis.Results: Eighty-nine isolates were recovered from 88/100 participants (one participant had two strains at baseline). The median colony count (cfu/g stool) was 1.9 × 104 (range: 2.5 × 102-7.0 × 106). Twelve participants (three received ridinilazole and nine received vancomycin) experienced recurrence, confirmed by immunoassays for free toxin in stool samples. The ribotype of eight out of nine isolates obtained at recurrence matched those of the initial isolates. All isolates, including those obtained at recurrence, were susceptible to ridinilazole within the expected range [median (range) MIC: 0.12 (0.06-0.5) mg/L]. The median (range) vancomycin MIC was 1 (0.5-4.0) mg/L. At baseline, 13.6% and 13.3% of samples in the ridinilazole and vancomycin groups were positive for VRE, increasing to 23.7% and 29.7% by day 40, respectively. Common ribotypes included 014-20 (14 isolates), 027 (13), 106 (7), 002 (7), 078-126 (4), 001 (4), 087 (3) and 198 (3). Toxin gene profiling of nearly all baseline isolates (98.9%) revealed a binary toxin gene (cdtA/cdtB) prevalence of 35%.Conclusions: Ridinilazole potently inhibited recovered C. difficile isolates. Recurrence was not associated with altered susceptibility. [ABSTRACT FROM AUTHOR]

Objectives: This study characterized the 11 most predominant serotypes of invasive Streptococcus pneumoniae infections collected by the annual SAVE study in Canada, between 2011 and 2015.Methods: A subset of the 11 most predominant serotypes (7F, 19A, 22F, 3, 12F, 11A, 9N, 8, 33F, 15A and 6C) collected by the SAVE study was analysed using PFGE and MLST, as well as PCR to identify pilus-encoding genes. WGS analyses were performed on a subset of the above isolates plus a random selection of background strains.Results: Of the predominant serotypes analysed, 7F, 33F and 19A were obtained more commonly from children <6 years of age, whereas 15A, 6C, 22F and 11A were more common in adults >65 years of age. Pneumococcal pilus PI-1 was identified in antimicrobial-susceptible serotype 15A (61/212) and <10% of 6C isolates (16/188). PI-2 was found in serotype 7F (683/701) and two-thirds of 11A isolates (162/241). Only serotype 19A-ST320 possessed both pili. Molecular and phylogenetic analyses identified serotypes 19A, 15A, 6C, 9N and 33F as highly diverse, whereas 7F, 22F and 11A demonstrated clonality. Antimicrobial resistance determinants were common within diverse serotypes, and usually similar within a clonal complex.Conclusions: Despite successful use of conjugate vaccines, S. pneumoniae remains a highly diverse organism in Canada. Several predominant serotypes, both antimicrobial susceptible and MDR, have demonstrated rapid clonal expansion or an increase in diversity. As S. pneumoniae continues to evolve in Canada, WGS will be a necessary component in the ongoing surveillance of antimicrobial-resistant and expanding clones. [ABSTRACT FROM AUTHOR]

Objectives: To discover the Escherichia coli STs and associated resistance mechanisms in the community in Islamabad, Pakistan by analysis of E. coli isolates in sewage.Methods: One hundred and ten E. coli were isolated from sewage across the city of Islamabad without antibiotic bias and confirmed as E. coli by MALDI-TOF MS. Isolates were characterized by fumC/fimH (CH) typing and core-genome MLST. Resistance mechanisms, virulence genes, phylotypes and plasmid incompatibility types were determined in a subset of isolates by in silico analysis. The genomic position of blaCTX-M-15 was determined using S1-PFGE, probing and Nanopore MinION sequencing.Results and conclusions: The most prevalent STs were ST394, ST10 and ST648, accounting for 39% of all isolates collected and were found at many sites across Islamabad. Carbapenemase genes were absent and only a single isolate of ST131 was found. The most prevalent resistance mechanisms were qnrS1 and blaCTX-M-15, with blaCTX-M-15 penetrating many STs and found in 31% of all collected isolates. However, the majority of the successful STs were blaCTX-M-15 negative indicating that resistance is not the main driver of prevalence. Twenty-three percent of blaCTX-M-15 genes were chromosomally encoded and large ISEcp1-mediated insertions included qnrS1 and several plasmid genes. In all chromosomally encoded isolates no plasmid copies of blaCTX-M-15 were found. The most prevalent ST (ST394) contained many enteroaggregative E. coli virulence genes and the fimH30 variant allele previously linked to the success of ST131. [ABSTRACT FROM AUTHOR]

Background: Streptococcus gallolyticus ssp. gallolyticus (Sgg) is a commensal bacterium and an opportunistic pathogen. In humans it has been clinically associated with the incidence of colorectal cancer (CRC) and epidemiologically recognized as an emerging cause of infective endocarditis (IE). The standard therapy of Sgg includes the administration of a penicillin in combination with an aminoglycoside. Even though penicillin-resistant isolates have still not been reported, epidemiological studies have shown that this microbe is a reservoir of multiple acquired genes, conferring resistance to tetracyclines, aminoglycosides, macrolides and glycopeptides. However, the underlying antibiotic resistance mobilome of Sgg remains poorly understood.Objectives: To investigate the mobile genetic basis of antibiotic resistance in multiresistant clinical Sgg.Methods: Isolate NTS31106099 was recovered from a patient with IE and CRC at Nantes University Hospital, France and studied by Illumina WGS and comparative genomics. Molecular epidemiology of the identified mobile element(s) was performed using antibiotic susceptibility testing (AST), PCR, PFGE and WGS. Mobility was investigated by PCR and filter mating.Results: Two novel conjugative transposons, Tn6263 and Tn6331, confer aminoglycoside/macrolide co-resistance in clinical Sgg. They display classical family Tn916/Tn1545 modular architecture and harbour an aph(3')-III→sat4→ant(6)-Ia→erm(B) multiresistance gene cluster, related to pRE25 of Enterococcus faecium. These and/or closely related elements are highly prevalent among genetically heterogeneous clinical isolates of Sgg.Conclusions: Previously unknown Tn916-like mobile genetic elements conferring aminoglycoside/macrolide co-resistance make Sgg, collectively with other gut Firmicutes such as enterococci and eubacteria, a potential laterally active reservoir of these antibiotic resistance determinants among the mammalian gastrointestinal microbiota. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the presence and molecular characteristics of oxazolidinone resistance genes cfr and optrA in staphylococci from household animals in rural China.Methods: Various samples were collected from household animals in 12 rural villages. Staphylococcal isolates showing florfenicol MICs ≥10 mg/L were identified and screened for the presence of cfr and/or optrA. PCR-positive isolates were characterized by antimicrobial susceptibility testing, S1 nuclease PFGE and Southern blotting. WGS data were analysed to identify the core-genome phylogenetic profile of each isolate as well as the genetic environment of cfr and/or optrA.Results: Nine optrA-positive (seven Staphylococcus sciuri and two Staphylococcus simulans) and 10 cfr-positive staphylococci were identified from eight and five villages, respectively. The gene optrA was chromosomally encoded in all nine isolates, whereas cfr was located on a plasmid in one S. sciuri and three Staphylococcus saprophyticus and in the chromosomal DNA of single Staphylococcus cohnii and Staphylococcus lentus isolates and two S. sciuri isolates. The remaining two cfr-carrying Staphylococcus haemolyticus isolates were indistinguishable by PFGE. Most optrA- or cfr-carrying staphylococci also harboured phenicol, tetracycline and/or macrolide-lincosamide-streptogramin B resistance genes. Genetic environment analysis showed that, for the first time, optrA was associated with transposon Tn6261, while cfr was adjacent to both a tnp (transposase) gene and a Tn558 transposon.Conclusions: The current study reveals for the first time the wide distribution of oxazolidinone resistance genes optrA and cfr in household animals in rural areas of China and is the first identification of optrA in S. simulans isolates. [ABSTRACT FROM AUTHOR]

Objectives: This study identified and characterized mcr-1-positive Enterobacteriaceae (MCRPE) and carbapenemase-producing Enterobacteriaceae (CPE) in hospital sewage water.Methods: Influent and effluent sewage samples were collected from five tertiary hospitals in Beijing in December 2016. Samples were screened for MCRPE and CPE using antibiotic selection media. Results were confirmed by PCR amplification of β-lactamase and colistin resistance (mcr-1 and mcr-2) genes and by sequencing. Antimicrobial susceptibility testing, MLST, conjugation and plasmid typing and S1-nuclease-PFGE/Southern blotting were performed for all MCRPE and CPE isolates.Results: Nine MCRPE and 12 CPE isolates were obtained. All mcr-1-positive isolates (n = 9) were Escherichia coli and belonged to eight different STs. The blaKPC-2-positive Enterobacteriaceae included Klebsiella pneumoniae (n = 4), Enterobacter cloacae (n = 4) and Citrobacter freundii (n = 1) isolates. Two C. freundii isolates and one E. cloacae isolate harboured the blaNDM-1 gene. MLST analysis revealed distinct genetic relatedness among all ST11 K. pneumoniae but not among any other carbapenemase-producing isolates. Conjugation and plasmid typing confirmed that three MCRPE isolates harboured mcr-1 on the self-transmissible IncX4 plasmid and the blaNDM-1 gene on the IncX3 plasmid. The sizes of the plasmids harbouring mcr-1, blaNDM-1 and blaKPC-2 were ∼33 to ∼240, ∼40 to ∼75 and ∼30 to ∼90 kb, respectively.Conclusions: To the best of our knowledge, this is the first report of mcr-1-positive E. coli and blaNDM-1-carrying E. cloacae and C. freundii in hospital sewage water. These findings, especially the diversity of MCRPE and K. pneumoniae ST11 that harbour the blaKPC-2 gene, suggest that monitoring and management of hospital sewage water should be enhanced. [ABSTRACT FROM AUTHOR]

Objectives: To investigate mechanisms for the decreased susceptibility to ceftazidime/avibactam in KPC-producing Klebsiella pneumoniae (KPC-KP).Methods: A total of 24 isolates, 8 each with ceftazidime/avibactam MICs of 4-8, 1-2 and ≤0.5 mg/L, were randomly selected from 214 clinical isolates of KPC-KP, and the β-lactamase hydrolysis activity and porin expression profiles were determined. Plasmid profile and relative expression and copy number of the bla KPC gene were also analysed.Results: Ceftazidime/avibactam MIC 50 and MIC 90 were 2 and 4 mg/L, respectively, for the 214 KPC-KP isolates. The hydrolysis activities of nitrocefin and ceftazidime in both of the ceftazidime/avibactam MIC 4-8 and 1-2 mg/L groups were significantly higher than those of the MIC ≤0.5 mg/L group, while the hydrolysis activities were 4-4.6-fold higher in the MIC 4-8 mg/L group than in the other two groups when 4 mg/L avibactam was added. The relative expression and copy number of the bla KPC gene in the MIC 4-8 mg/L group were 4.2-4.8-fold higher than in the other two groups. Meanwhile, SDS-PAGE showed that all isolates in the two groups with MIC ≥1 mg/L lacked OmpK35, which had either an early frameshift with a premature stop codon ( n  =   15, ST11) or overexpression of the negative regulation genes, micF and ompR ( n  =   1, ST15), whereas OmpK35 and OmpK36 could both be observed in all isolates with MIC ≤0.5 mg/L.Conclusions: Decreased ceftazidime/avibactam susceptibility in KPC-KP clinical isolates is caused by high ceftazidime hydrolysis activity and OmpK35 porin deficiency and the majority of isolates belong to ST11. [ABSTRACT FROM AUTHOR]

Objectives: Vancomycin-intermediate Staphylococcus aureus (VISA) strains have spread globally. We previously isolated an ST239 VISA (XN108) with a vancomycin MIC of 12 mg/L. The mechanism for XN108 resistance to vancomycin was investigated in this study.Methods: Genome comparison was performed to characterize mutations that might contribute to the XN108 resistance phenotype. The novel mutation WalK(S221P) was identified and investigated using allelic replacement experiments. Vancomycin susceptibilities, autolytic activities and morphologies of the strains were examined. Autophosphorylation activities of WalK and the WalK(S221P) mutant were determined in vitro with [λ- 32 P]ATP, and binding activity of WalK(S221P)-activated WalR to the promoter region of its target gene lytM was determined by electrophoretic mobility shift assay.Results: Genome comparison revealed three mutations, GraS(T136I), RpoB(H481N) and WalK(S221P), which might be responsible for vancomycin resistance in XN108. The introduction of WalK(S221P) to the vancomycin-susceptible strain N315 increased its vancomycin MIC from 1.5 to 8 mg/L, whereas the allelic replacement of WalK(S221P) with the native N315 WalK allele in XN108 decreased its vancomycin MIC from 12 to 4 mg/L. The VISA strains have thickened cell walls and decreased autolysis, consistent with observed changes in the expression of genes involved in cell wall metabolism and virulence regulation. WalK(S221P) exhibited reduced autophosphorylation, which may lead to reduced phosphorylation of WalR. WalK(S221P)-phosphorylated WalR also exhibited a reduced capacity to bind to the lytM promoter.Conclusions: The naturally occurring WalK(S221P) mutation plays a key role in vancomycin resistance in XN108. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is a protozoan parasitic disease and a global health problem. The aim of this study is to diagnose the parasitic infection in humans for epidemiological identification and providing control programs using proprietary co-designed primers in three species of Leishmania. Materials and Methods: 30 common Leishmania isolates were gathered from different centers in Iran. Having been cultured in RPMI-1640 Medium, DNA was extracted and the gene ITS2-rRNA was amplified by PCR. The amplicons were examined by electrophoresis on agarose gel 2%. Also, in FLASH PCR method, a specific probe and florence colour were used to investigae the amplicon existence on sample. Results: The results of the investigations by PCR and FLASH PCR methods show that these methods are sensitive and specific for diagnosis of Leishmania Conclusion: In this study, identification of Leishmania parasite using specific primer pairs was successful and TaqMan could be one of the most sensitive diagnostic methods to identify parasite load for the ITS2 region of Leishmania [ABSTRACT FROM AUTHOR]

Introduction. Infections due to extended spectrum β-lactamase producing Enterobacteriaceae are on the rise. They pose serious public health problems due to their resistance to large number of antibiotics. However, little is known about the genotypes of ESBL from Nepal. Therefore, the study presents results of phenotypic and molecular characterization of ESBL producing Escherichia coli and Klebsiella spp. isolated from various clinical specimens in a tertiary care teaching hospital of Nepal. Methods. A total of 172 Enterobacteriaceae clinical isolates recovered from various clinical specimens were analyzed for their antibiotic susceptibility test. Detection of ESBLs was carried out using combination disk test and multiplex PCR for their genotypes (CTX-M, SHV, and TEM). Results. Out of 172 clinical isolates, 70 (40.6%) of them were found ESBL producers. The major source of ESBL producers was urinary tract samples and the highest ESBL production was observed in Escherichia coli (46.5%). Among ESBL genotypes, CTX-M (91.4%) was most predominant, followed by TEM (65.7%) and SHV (11.4%) in both of the isolates. Conclusions. High level of drug resistance and ESBL production was observed among the clinical isolates. There is a need for longitudinal and nationwide surveillance for drug resistance in clinical isolates and antimicrobial stewardship is necessary to guide the appropriate and judicious antibiotic use. [ABSTRACT FROM AUTHOR]

Aims: The increasing usage of antibiotics can cause resistance to the treatment of infections, which can caused by bacteria, e.g. Pseudomonas aeruginosa. The aim of this study was to trace the class I, II and III integrons in isolates of P. aeruginosa of nosocomial infection and determining the antibiotic resistance pattern of the bacteria. Instrument & Methods: In this cross-sectional study, 100 Pseudomonas aeruginosa clinical isolates of infected wounds, bedsores, burns, urinary tract infections and respiratory tract infections were collected from patients of 3 Isfahan City hospitals, Iran (Al Zahra, Kashani, Shariati) in 2015. After identification tests and antibiogram, integrons class I, II and III were detected by M-PCR method. Data analysis were performed in SPSS 16 software using Chi-square and Fisher exact tests and the relationship between the presence of class III, II, I was calculated by M-PCR test. Findings: All isolates had multiple antibiotic resistances. The highest antibiotic resistance was to Tetracycline (85%) and the lowest to Norfloxacin (12.5%). There were significant differences between class I and the two other classes of integrons (p=0.036). There was a statistically significant difference between the presence of blaTEM gene in Pseudomonas aeruginosa with other coding genes for antibiotic resistance (p=0.029). Conclusion: Pseudomonas aeruginosa isolates are multi-drug resistant and almost all isolates from clinical infections have class I, II and III Integrons. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the molecular mechanisms of ertapenem resistance among Enterobacteriaceae isolates in a clinical microbiology laboratory at a tertiary university hospital. A total of 40 clinical isolates including 20 resistant and 20 intermediate isolates were collected from August 2012 to July 2013. Ertapenem susceptibility was confirmed by the broth microdilution method. PCR and sequencing analysis of carbapenemase, AmpC β-lactamase, and extended-spectrum β-lactamase (ESBL) genes were performed. Outer membrane proteins (OMPs) were examined by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Molecular epidemiology studies were performed by pulsed-field gel electrophoresis (PFGE). AmpC β-lactamases and ESBLs were found in 32 (80.0%) and 20 (50.0%) of the 40 isolates with ertapenem non-susceptibility, respectively. Distributions of β-lactamase genes differed among the species. One Citrobacter freundii isolate among the 40 isolates with ertapenem non-susceptibility carrying the blaIMP-1 associated class 1 integron was detected. SDS-PAGE of OMPs showed altered or greatly diminished expression of porins in all isolates of Klebsiella pneumoniae (n=5) and Enterobacter cloacae (n=11) with ertapenem resistance. Porin alterations were less common among the isolates with intermediate susceptibility (4/19). Integration of the results of molecular analysis of β-lactamases and OMP analysis revealed that most of the isolates with ertapenem resistance exhibited β-lactamase activity and porin alteration. PFGE revealed that most isolates were epidemiologically unrelated. Ertapenem resistance in clinical Enterobacteriaceae isolates was associated with β-lactamase activity and porin alteration. Even though carbapenemase-producing Enterobacteriaceae are still rare, continuous monitoring and infection control for carbapenem-resistant Enterobacteriaceae are necessary. [ABSTRACT FROM AUTHOR]

Objectives Due to the lack of information on the plasmid content of MRSA strains in South Africa (SA), this study investigated the resistance and virulence mechanisms of 27 clinical isolates from the private health care sector over a period of 3 months. Methods Plasmids were extracted and the presence of MRSA confirmed by the presence of mec A. The isolates were subjected to antimicrobial susceptibility testing and molecular characterization of common resistance encoding genes and frequently encountered virulence factors by PCR using plasmid DNA as the template. The genetic relatedness between the isolates was determined by pulsed field gel electrophoresis (PFGE). Results All isolates were plasmid positive, and displayed ampillicin, ciprofloxacin, gentamicin, rifampicin, tetracycline, erythromycin, and clindamycin resistance. They were all fully susceptible to daptomycin, linezolid, vancomycin, tigecycline and fusidic acid. Multidrug resistance (MDR) was found in 74.1% (20/27) of the MRSA isolates. The frequency of the resistance and virulence genes ranged from 100% to 0%. PFGE analysis revealed 10 pulsotypes, designated A–J, which showed correlation with resistance profile of the isolates in each group. Of note, 85.2% (23/27) of the isolates clustered into six major PFGE types giving an indication of similar circulating MRSA clones. Conclusions This study highlights the genetic diversity and resistance mechanisms in MRSA strains from the private health sector in SA hence the need for implementing effective infection control programs. [ABSTRACT FROM AUTHOR]

Background: Plasmid mediated quinolone resistance (PMQR) has been shown to play an important role in resistance not only to quinolones, but also β-lactams and aminoglycosides. In fact, qnr genes are frequently carried along with β-lactamase determinants on the same plasmids. We studied the prevalence of qnrA , qnrB , qnrS and aac(6')-Ib-cr genes among quinolone and cephalosporin resistant clinical isolates of Klebsiella pneumoniae (K. pneumoniae) , as well as the association between PMQR genes with resistance to quinolones, cephalosporins and aminoglycosides. Methods: The study was conducted on 79 K. pneumoniae clinical isolates collected from Imam Hussein hospital in Tehra n between July 2010 and January 2011, based on their resistance to quinilones and cephalosporins. Antibacterial susceptibility was determined to 15 antibiotics by disc diffusion. Presence of qnrA , qnrB , qnrS and aac(6')-Ib-cr genes were investigated using specific primers and PCR. Results: Of the 79 K. pneumoniae isolates, 47 (59.5%) carried the PMQR determinants. Among these, 42 (89.4%) carried aac(6')-Ib-cr of which, 21 (50%) also harbored qnrB . Three isolates carried qnrB alone, two (4.2%) harbored qnrS and none had qnrA . Resistance to aminoglycosides and cephalosporins was significantly higher in the isolates carrying both qnrB and aac(6')-Ib-cr genes compared to aac(6')-Ib-cr alone. Conclusion: This study showed a high prevalence of aac(6')- Ib-cr and qnrB genes among the Iranian K. pneumoniae clinical isolates as well as co-carriage of the two genes. Ther e was a significant association between qnrB gene carriage and resistance to quinolones, cephalosporins, and aminoglycosides. [ABSTRACT FROM AUTHOR]

Delftia acidovorans is an opportunistic agent in several types of infections, both in immunocompromised and immune-competent individuals; its resistance to aminoglycosides and polymyxin, choice drugs for empirical treatment of Gram-negative infections, is remarkable. We report the antimicrobial susceptibility and the genetic relatedness of 24 D. acidovorans strains recovered from tracheal aspirates of 21 adult inpatients hospitalized in an intensive care unit at a Brazilian hospital, from 2012 to 2013. All of the isolates were recovered as pure cultures and in counts above 1,000,000 CFU/mL. None of them were susceptible to polymyxin B, amikacin, gentamicin, or tobramycin; quinolones and trimethoprim-sulfamethoxazole presented varied activities against the isolates, while β-lactam resistance was not detected. Four clusters were verified in pulsed-field gel electrophoresis analysis, and a major pulsotype comprised 10 strains. A possible, but undetermined common source, can be responsible for this strain dissemination, underscoring the need of reinforcing the adherence to disinfection and infection control standard techniques. [ABSTRACT FROM AUTHOR]

Objective: To characterize the microbial etiology of chronic suppurative otitis media comparing the methods of classical bacteriological culture and polymerase chain reaction. Design/Setting/Patients: Bacteriological analysis by classical culture and by molecular polymerase chain reaction of 35 effusion otitis samples from patients with cleft lip and palate attending the Hospital for Rehabilitation of Craniofacial Anomalies of the University of São Paulo, Bauru, Brazil. Interventions: Collection of clinical samples of otitis by effusion through the external auditory tube. Main Outcome Measure: Otolaryngologic diagnosis of chronic suppurative otitis media. Results: Positive cultures were obtained from 83% of patients. Among the 31 bacterial lineages the following were isolated. In order of decreasing frequency: Pseudomonas aeruginosa (54.9%), Staphylococcus aureus (25.9%), and Enterococcus faecalis (19.2%). No anaerobes were isolated by culture. The polymerase chain reaction was positive for one or more bacteria investigated in 97.1% of samples. Anaerobe lineages were detected by the polymerase chain reaction method, such as Fusobacterium nucleatum, Bacteroides fragilis, and Peptostreptococcus anaerobius. Conclusions: Patients with cleft lip and palate with chronic suppurative otitis media presented high frequency of bacterial infection in the middle ear. The classical bacteriological culture did not detect strict anaerobes, whose presence was identified by the polymerase chain reaction method. [ABSTRACT FROM AUTHOR]