Pulsed-field Gel Electrophoresis Research Articles

Molecular epidemiology and transmission of rmtB-positive Escherichia coli among ducks and environment

This study aimed to investigate the transmission and molecular epidemiological characteristics of the rmtB gene in Escherichia coli (E. coli) strains isolated from duck farms in Guangdong Province of China from 2018 to 2021. A total of 164 (19.4%, 164/844) rmtB-positive E. coli strains were recovered from feces, viscera, and environment. We performed antibiotic susceptibility tests, pulsed-field gel electrophoresis (PFGE), and conjugation experiments. We obtained the genetic context of 46 rmtB-carrying E. coli isolates and constructed a phylogenetic tree via whole genome sequencing (WGS) and bioinformatic analysis. The isolation rate of rmtB-carrying E. coli isolates in duck farms increased yearly from 2018 to 2020 but decreased in 2021. All rmtB-harboring E. coli strains were multidrug resistant (MDR), and 99.4% of the strains were resistant to more than 10 drugs. Surprisingly, duck- and environment-associated strains similarly showed high MDR. Conjugation experiments revealed that the rmtB gene horizontally cocarried blaCTX-M and blaTEM gene dissemination via IncFII plasmids. Insertion sequences IS26, ISCR1, and ISCR3 were closely associated with the spread of rmtB-harboring E. coli isolates. WGS analysis indicated that ST48 was the most prevalent sequence type. The results of single nucleotide polymorphism (SNP) differences revealed potential clonal transmission between ducks and the environment. Based on One Health principles, we need to strictly use veterinary antibiotics, monitor the distribution of MDR strains, and evaluate the impact of plasmid-mediated rmtB gene on human, animal, and environmental health.

Evaluation of IR Biotyper for carbapenem-resistant Pseudomonas aeruginosa typing and its application potential for the investigation of nosocomial infection.

Pseudomonas aeruginosa is one of the most common opportunistic pathogens causing severe nosocomial infections for its patterns of multidrug resistance, particularly for carbapenems. Timely epidemiological surveillance could greatly facilitate infection control of P. aeruginosa and many deadly pathogens alike. IR Biotyper (IRBT), is a novel real-time typing tool, based on a Fourier-transform infrared (FTIR) spectroscopy system. It is critical to comprehensively establish and evaluate the feasibility of IRBT in P. aeruginosa strain typing. In the current study, we first established standards and schemes for its routine laboratory application, and we found that Mueller-Hinton agar plates give better discriminatory power than blood agar plates. Data showed that the cut-off value of 0.15 with an additional 0.025 range was optimal. Secondly, 27 clinically isolated carbapenem-resistant P. aeruginosa (CRPA) strains collected from October 2010 to September 2011 were evaluated for typing effectiveness by comparing IRBT to the other commonly used typing methods, such as multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS)-based typing. When using WGS-based typing as the reference method, the typing method of FTIR spectroscopy (AR = 0.757, SID = 0.749) could better cluster P. aeruginosa strains than MLST and in silico serotyping (AR = 0.544, SID = 0.470). Though PFGE showed the highest discriminatory power, low concordance was observed between PFGE and the other methods. Above all, this study demonstrates the utility of the IRBT as a quick, low-cost, real-time typing tool for the detection of CRPA strains.

Genotyping and antimicrobial resistance profiles of chicken originated Salmonella Enteritidis isolates.

Salmonellosis is a common foodborne zoonosis worldwide. The most common Salmonella serovar in humans is Salmonella enterica subsp. enterica serovar Enteritidis (50.3%) in the world. The main transmission route for S. Enteritidis is consumption of contaminated poultry products. Therefore, it is important to determine the diversity and spread of chicken-originated S. Enteritidis isolates in order to monitor and control salmonellosis. Pulsed-field gel electrophoresis (PFGE) and multiple locus variable number of tandem repeats analysis (MLVA) are frequently used for typing of S. Enteritidis isolates. This study aimed to determine the antimicrobial resistance (AMR) profiles and MLVA and PFGE genotypes of chicken-originated S. Enteritidis isolates. A total of 200 S. Enteritidis isolated from chicken broiler, layer, and breeder flocks from different locations in Turkey were investigated by Kirby-Bauer disk diffusion method, PFGE, and MLVA. The AMR test indicated that 57% of the S. Enteritidis isolates were susceptible to all antimicrobials, while 39% were resistant to at least one antimicrobial. The highest resistance (25%) was against ampicillin. Multi-drug resistance rate was low (21%) and mostly from broiler flocks (93%). All isolates were genotyped into 32 different PFGE genotypes (PT) and 34 different MLVA genotypes (MT). The dominant genotypes were PT6 (12.5%) and MT22 (50%). In specific sample groups, there was a correlation between genotypes, breeding type, geographic location, and isolation years of the isolates. There was no significant difference in the discrimination power of PFGE and MLVA. However, MLVA was more suitable for large sample groups and routine genotyping because it was easier, quicker, and less labor-intensive to use.

Genomic characterization of two carbapenem-resistant Serratia marcescens isolates causing bacteremia: Emergence of KPC-2-encoding IncR plasmids.

The occurrence and transmission of carbapenemase-producing-Enterobacterales (CPE) on a global scale has become a major issue. Clinical reports are rarely providing information on the genomic and plasmid features of carbapenem-resistant Serratia marcescens. Our objective was to investigate the resistance and transmission dynamics of two carbapenem-resistant S. marcescens that are resistant to carbapenem and have caused bacteremia in China. Blood specimens were taken from two individuals with bacteremia. Multiplex PCR was employed to identify genes that code for carbapenemase. Antimicrobial susceptibility tests and plasmid analysis were conducted on S. marcescens isolates SM768 and SM4145. The genome of SM768 and SM4145 were completely sequenced using NovaSeq 6000-PE150 and PacBio RS II platforms. Antimicrobial resistance genes (ARGs) were predicted using the ResFinder tool. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and southern blotting were employed to analyze plasmids. Two S. marcescens that produced KPC-2 were identified from bloodstream infections. The antimicrobial susceptibility testing demonstrated that both of the isolates had a resistance to various antibiotics. The whole-genome sequence (WGS) and plasmid analysis revealed the presence of bla KPC-2-bearing IncR plasmids and multiple plasmid-borne antimicrobial resistance genes in the isolates. Our comparative plasmid analysis suggested that the two IncR plasmids identified in this study could be derived from a common ancestor. Our findings revealed the emergence of bla KPC-2-bearing IncR plasmid in China, which could be a hindrance to the transmission of KPC-2-producing S. marcescens in clinical settings.

Phenotypic and genetic alterations of Burkholderia pseudomallei in patients during relapse and persistent infections.

The bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a severe tropical disease associated with high mortality and relapse and persistent infections. Treatment of melioidosis requires prolonged antibiotic therapy; however, little is known about relapse and persistent infections, particularly the phenotypic and genetic alterations of B. pseudomallei in patients. In this study, we performed pulsed-field gel electrophoresis (PFGE) to compare the bacterial genotype between the initial isolate and the subsequent isolate from each of 23 suspected recurrent and persistent melioidosis patients in Northeast Thailand. We used whole-genome sequencing (WGS) to investigate multilocus sequence types and genetic alterations of within-host strain pairs. We also investigated the bacterial phenotypes associated with relapse and persistent infections, including multinucleated giant cell (MNGC) formation efficiency and intracellular multiplication. We first identified 13 (1.2%) relapse, 7 (0.7%) persistent, and 3 (0.3%) reinfection patients from 1,046 survivors. Each of the 20 within-host strain pairs from patients with relapse and persistent infections shared the same genotype, suggesting that the subsequent isolates arise from the infecting isolate. Logistic regression analysis of clinical data revealed regimen and duration of oral antibiotic therapies as risk factors associated with relapse and persistent infections. WGS analysis demonstrated 17 within-host genetic alteration events in 6 of 20 paired isolates, including a relatively large deletion and 16 single-nucleotide polymorphism (stocktickerSNP) mutations distributed across 12 genes. In 1 of 20 paired isolates, we observed significantly increased cell-to-cell fusion and intracellular replication in the second isolate compared with the initial isolate from a patient with persistent infection. WGS analysis suggested that a non-synonymous mutation in the tssB-5 gene, which encoded an essential component of the type VI secretion system, may be associated with the increased intracellular replication and MNGC formation efficiency of the second isolate of the patient. This information provides insights into genetic and phenotypic alterations in B. pseudomallei in human melioidosis, which may represent a bacterial strategy for persistent and relapse infections.

Increased copy number of 23S ribosomal RNA gene with point mutation in MRSA associated with linezolid resistance in a patient treated with long-term linezolid

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) infection is one of the most difficult infections we have to treat. Linezolid is one of the effective treatment options for refractory MRSA infections. There are cases where we are forced to use long-term linezolid treatment for refractory MRSA infections. ObjectiveTo discuss the evolution of Linezolid resistance factors in clinical isolates of MRSA. MethodsWe investigated 16 MRSA isolated from a patient treated with linezolid for a long period of 75 days. We performed antibiotic susceptibility test, 23S rRNA genes sequencing analysis, Pulsed-field gel electrophoresis. ResultsMRSA isolates were susceptible to linezolid before the start of treatment, but became less susceptible by prolonged treatment. The 23S rRNA sequencing analysis of linezolid-resistant strains that appeared 17 days after the start of treatment with linezolid revealed that all resistant MRSA had the G2576T substitution (Escherichia coli 23S rRNA gene number). The number of copies of this mutation increased with the use of linezolid. ConclusionLong-term use of linezolid in a patient or reuse of linezolid in a patient who has been previously treated with linezolid can lead to the emerging of linezolid-resistant MRSA in the host.

Sequential outbreaks due to mixed ctxB alleles of Vibrio cholerae O1 in Mayurbhanj district of Odisha, India.

Cholera is a significant threat causing outbreaks/epidemics with high morbidity and mortality in coastal and tribal districts of Odisha. A sequential cholera outbreak reported from four places in Mayurbhanj district of Odisha during June to July 2009 was investigated. Rectal swabs from diarrhea patients were analyzed for the identification, antibiogram profiles and detection of ctxB genotypes by double mismatch amplification mutation (DMAMA) polymerase chain reaction (PCR) assays and sequenced. The different virulent and drug resistant genes were detected by multiplex PCR assays. The clonality analysis on selected strains was done by pulse field gel electrophoresis (PFGE). Bacteriological analysis of rectal swabs revealed the presence of V. cholerae O1 Ogawa biotype El Tor which were resistant to co-trimoxazole, chloramphenicol, streptomycin, ampicillin, nalidixic acid, erythromycin, furazolidone and polymyxin B. DMAMA-PCR assay revealed that the cholera outbreak in Mayurbhanj district was due to both ctxB1 and ctxB7 alleles of V. cholerae O1 El Tor strains. All the V. cholerae O1 strains were positive for all virulence genes. The multiplex PCR assay on V. cholerae O1 strains revealed the presence of antibiotic resistance genes like dfrA1 (100%), intSXT (100%), sulII (62.5%) and StrB (62.5%). PFGE results on V. cholerae O1 strains exhibited two different pulsotypes with 92% similarity. This outbreak was a transition phase where both ctxB genotypes were prevalent after which the ctxB7 genotype gradually became dominant in Odisha. Therefore, close monitoring and continuous surveillance on diarrheal disorders is essential to prevent the future diarrheal outbreaks in this region.

Characterization of small plasmids carrying florfenicol resistance gene floR in Actinobacillus pleuropneumoniae and Pasteurella multocida isolates from swine in China.

Actinobacillus pleuropneumoniae and Pasteurella multocida are two important bacterial pathogens in swine industry. In the present study, resistance profiles of nine commonly used antibiotics of A. pleuropneumoniae and P. multocida isolates of swine origin from different regions of China were investigated by determination of minimum inhibitory concentrations (MICs). In addition, genetic relationship of the florfenicol-resistant A. pleuropneumoniae and P. multocida isolates was determined by pulsed-field gel electrophoresis (PFGE). The genetic basis of florfenicol resistance in these isolates were explored by floR detection and whole genome sequencing. High resistance rates (>25%) of florfenicol, tetracycline and trimethoprim- sulfamethoxazole were observed for both bacteria. No ceftiofur- and tiamulin- resistant isolates were detected. Furthermore, all the 17 florfenicol-resistant isolates (nine for A. pleuropneumoniae and eight for P. multocida) were positive for floR gene. The presence of similar PFGE types in these isolates suggested that clonal expansion of some floR-producing strains occurred in the pig farms from same regions. WGS and PCR screening showed that three plasmids, named pFA11, pMAF5, and pMAF6, were the cargos of the floR genes in the 17 isolates. Plasmid pFA11 exhibited novel structure and carried several resistance genes, including floR, sul2, aacC2d, strA, strB, and bla ROB - 1. Plasmids pMAF5 and pMAF6 were presented in A. pleuropneumoniae and P. multocida isolates from different regions, suggesting horizontal transfer of the two plasmids are important for the floR dissemination in these Pasteurellaceae pathogens. Further studies of florfenicol resistance and its transfer vectors in Pasteurellaceae bacteria of veterinary origin are warranted.

Comparison of pathogenic bacteria in the upper and lower respiratory tracts of cattle either directly transported to a feedlot or co-mingled at auction markets prior to feedlot placement.

Bacterial bronchopneumonia (BP) has been associated with purchasing cattle through auction markets. However, whether auction markets are a source of BP-associated bacterial pathogens is unknown. This study evaluated prevalence, antimicrobial susceptibility, and genetic relatedness (using pulsed-field gel electrophoresis, PFGE) of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni isolated from cattle either transported to an auction market prior to feedlot placement (AUC), or directly to a feedlot from a farm (RANC). Two groups of cattle were enrolled (N = 30 per group) from two separate farms with 15 animals from an individual farm designated as AUC or RANC. Deep nasal swab (DNS) and trans-tracheal aspirates (TTA) were collected on day 0 at weaning (T0) and on day 2 at on-arrival processing at the feedlot (T1). The DNS were also collected on day 9 (T2) and day 30 (T3) after arrival at the feedlot. In both TTA and DNS, prevalence of bacteria did not differ between AUC and RANC groups (P > 0.05). None of the bacteria isolated at T0 were resistant to antimicrobials and diversity of all bacteria was greatest at T0 and T1. In Group 1 cattle, 100% of P. multocida isolated at T2 and T3 were multi-drug resistant. These isolates were highly related (>90%) according to PFGE, with most being clones. Though limited in size, results for animals evaluated in this study suggested that auction markets were not a major source of resistant BP pathogens, however, horizontal transmission of a multi-resistant strain of P. multocida occurred in a feedlot. Spread of resistant P. multocida was likely due to the selective pressures imposed by feedlot antimicrobial use and encoded resistance by the bacteria.

Molecular characterization of colistin resistance genes in a high-risk ST101/KPC-2 clone of Klebsiella pneumoniae in a University Hospital of Split, Croatia.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has become a major concern worldwide due to multidrug resistance and the ability to spread locally and globally. Infections caused by KPC-KP are great challenge in the healthcare systems because these are associated with longer hospitalization and high mortality. The emergence of colistin resistance has significantly reduced already limited treatment options. This study describes the molecular background of colistin-resistant KPC-KP isolates in the largest hospital in southern Croatia. Thirty-four non-duplicate colistin-resistant KPC-KP isolates were collected during routine work from April 2019 to January 2020 and from February to May 2021. Antimicrobial susceptibility was determined using disk diffusion, broth microdilution, and the gradient strip method. Carbapenemase was detected with an immunochromatographic test. Identification of blaKPC and mcr genes or mutations in pmrA, pmrB, mgrB, phoP, and phoQ genes were performed by polymerase chain reaction (PCR) and positive products were sequenced. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for epidemiological analysis. All isolates were multidrug-resistant, with colistin minimum inhibitory concentrations (MICs) from 4 to >16 mg/L, and all harbored blaKPC-2 and had a single point mutation in the mgrB gene resulting in a premature stop codon, with the exception of one isolate with four point mutations corresponding to stop codons. All isolates were negative for mcr genes. PFGE analysis identified a single genetic cluster, and MLST revealed that all isolates belonged to sequence type 101 (ST101). These results show emergence of the high-risk ST101/KPC-2 clone of K. pneumoniae in Croatia as well as appearance of colistin resistance due to mutations in the mgrB gene. Molecular analysis of epidemiology and possible resistance mechanisms are important to develop further strategies to combat such threats.

Genomic Characterization of Salmonella enterica serovar Weltevreden Associated with Human Diarrhea.

Salmonella Weltevreden is an emerging pathogen associated with human diarrhea, and knowledge of the genomics and epidemiology of this serovar is still limited. In this study, we performed whole-genome sequencing of 96 S. Weltevreden isolates recovered from diarrheal patients and 62 isolates from food animals in China between 2006 and 2017. Together, with an additional 199 genome sequences of S. Weltevreden published in NCBI, we performed an analysis on all 357 S. Weltevreden genome sequences. Our results demonstrated that the majority of S. Weltevreden from diarrheal patients from China (97.92%, 94/96) and the other regions in the world (94.97%, 189/199) identified in this study were sequence type (ST) 365. The remaining types were ST3771 (n = 3), ST22 (n = 1), ST155 (n = 1), and ST684 (n = 1). In addition, ST365 was also widely recovered from animals, food, and environmental samples in different regions of the world. Phylogenetic analysis and pulsed-field gel electrophoresis (PFGE) revealed that S. Weltevreden from diarrheal patients was closely related to those recovered from food and environmental specimens. We also showed that S. Weltevreden did not exhibit severe antimicrobial resistance profiles, suggesting administering antibiotics is still effective for controlling the agent. Interestingly, we found that S. Weltevreden strains carried a number of virulence factor genes, and a 100.03-kb IncFII(S) type plasmid was widely distributed in S. Weltevreden strains. Elimination of this plasmid decreased the bacterial capacity to infect both Caco-2 cells and C57BL/6 mice, suggesting the importance of this plasmid for bacterial virulence. Our results contribute to the understanding of the epidemiology and virulence of S. Weltevreden. IMPORTANCE Salmonella Weltevreden is a pathogen associated with human diarrheal diseases found across the globe. However, knowledge of the genomics and epidemiology of this pathogen is still limited. In this study, we found S. Weltevreden sequence type (ST) 365 is commonly recovered from diarrheal patients in China and many other regions of the world, and there is no major difference between the Chinese isolates and the global isolates at the phylogenetic level. We also demonstrated that ST365 was widely recovered from animal, food, and environmental samples collected in different, global regions. Importantly, we discovered an IncFII(S) type plasmid commonly carried by S. Weltevreden strains of human, animal, and food origins, and this plasmid is likely to contribute to the bacterial pathogenesis. These findings enhance our understanding of the emergence of S. Weltevreden involved in diarrheal outbreaks and the global spread of S. Weltevreden strains.

Genetic characterization and virulence determinants of multidrug-resistant NDM-1-producing Aeromonas caviae.

The emergence of carbapenemase significantly threatens public health. It is prevalent worldwide but rare in Aeromonas caviae. Unlike most bacterial species, A. caviae has two distinct flagella systems, which are closely related to biofilm formation. The ability to form biofilms on host tissues or inert surfaces constitutes an important cause of many persistent infections, which causes difficulties in clinical treatment. Here, we report on a multidrug-resistant (MDR) A. caviae carrying bla NDM-1 with a novel sequence type 1,416. The strong ability of biofilm formation of FAHZZU2447 was verified by a crystal violet assay. The resistome profile and location of the bla NDM-1 gene were determined by antimicrobial susceptibility testing, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), and Southern blot analysis. Moreover, the strain underwent whole-genome sequencing to identify its genomic characteristics. In addition, the bla NDM-1 gene was located on a ∼243 kb plasmid with genetic context IS1R-bla NDM-1-ble-trpF-dsbD-hp-sul1-qacE. Phylogenetic analysis indicated the transmission of A. caviae in China, Japan, and Thailand. Our study aimed to elucidate the genomic features of bla NDM-1-producing A. caviae, thereby clarifying the distribution of A. caviae worldwide and emphasizing the harmfulness of biofilm formation to the clinic. Further comprehensive surveillance of this species is needed to control further dissemination.

Tracing of persistent Listeria monocytogenes contamination in ewe's milk farm.

Ewe's milk farm production is permanently associated with the risk of contamination by pathogenic bacteria, including Listeria monocytogenes. In the present study, the prevalence and diversity of L. monocytogenes strains repeatedly isolated from tank ewe's milk and the milking environment on a farm in Slovakia during a prolonged period were investigated to identify the source of potentially persistent contamination. A total of 140 samples along the milk production chain were collected during an 18-month period. From all these samples, 45 samples were found L. monocytogenes positive with 90.3% positivity of tank milk samples (28 positive samples from 31 analysed). Pulsed-field gel electrophoresis profiling resulted in strain discrimination into six profiles with one pulsotype (NS1) corresponding to MLST-ST14 being predominant. A total of 17 proportionally selected L. monocytogenes isolates, including 11 NS1/ST14 isolates, were subjected to whole genome sequencing. Resulted data were used to compare the genomes diversity and to confirm the persistent contamination when <10 allelic differences threshold in cgMLST analysis was applied. The source of persistent contamination was localized inside the milking apparatus, probably in shelters that were very difficult to clean. Despite great efforts, the ewe's milk contamination could not be eliminated during the reporting period.

Genomic insights of Leclercia adecarboxylata strains linked to an outbreak in public hospitals in Mexico.

Leclercia adecarboxylata is a bacteria closely related to Escherichia coli according to its biochemical characteristics and is commonly considered non-pathogenic although a growing number of publications classify it as an emerging pathogen. Fosfomycin resistance is a common trait for L. adecarboxylata encoded by fosALA gene. To analyze genomic traits of sixteen L. adecarboxylata strains isolated from blood culture and a bottle of total parenteral nutrition. Twenty-eight L. adecarboxylata strains isolated from blood culture and a bottle of total parenteral nutrition were identified biochemically with a Vitek ® automated system. The strains were phenotyped by their growth on Eosin Methylene Blue agar or MacConkey agar plates. Additionally, Pulsed field gel electrophoresis (PFGE) was performed to establish the clonal relationship. The genomic DNA of sixteen strains was obtained using a Qubit ® dsDNA HS Assay Kit and sequenced on an Illumina ® MiSeq instrument. Draft genomes were assembled using PROKKA and Rast. Assemblies were submitted to Resfinder and PathogenFinder from the Center for Genomic Epidemiology in order to find resistance genes and pathogenic potential. IslandViewer4 was also used to find Pathogenicity and Phage Islands. For identification of the fosA gene, manual curation and Clustal analysis was performed. A novel FosA variant was identified. Finally, phylogenetic analysis was performed using VAMPhyRE software and Mega X. In this paper, we report the genomes of sixteen strains of Leclercia adecarboxylata causing an outbreak associated with parenteral nutrition in public hospitals in Mexico. The genomes were analyzed for genetic determinants of virulence and resistance. A high pathogenic potential (pathogenicity index 0.82) as well as multiple resistance genes including carbapenemics, colistin and efflux pumps were determined. Based on sequence analysis, a new variant of the fosALA gene was described. Finally, the outbreak was confirmed by establishing the clonal relationship among the sixteen genomes obtained. Commensal strains of L. adecarboxylata may acquire genetic determinants that provide mechanisms of host damage and go unnoticed in clinical diagnosis. L. adecarboxylata can evolve in a variety of ways including the acquisition of resistance and virulence genes representing a therapeutic challenge in patient care.

Escherichia coli virulence genes and clonality in strains from diarrhoea in goat kids

Escherichia coli can cause a range of diseases either extraintestinal or intestinal, from which colibacillosis outstands among the leading causes of goat kids’ mortality. However, the related pathotypes are still unknown. The aim of this study was to determine the clonality of 41 strains of E. coli obtained from the goat kids with diarrhoea from different regions, using pulsed-field gel electrophoresis (PFGE). In addition, the presence of virulence genes of diarrhoeagenic pathotypes: EAEC, STEC/EHEC, ETEC, EPEC, and EIEC/Shigella was determined by PCR amplification. Phylogenetic grouping was determined by PCR, and serotypes were determined by conventional serology. Two clusters presented 100 % similarity, the same serotype (O25:H8), and almost the same virulence genes. The remaining were heterogeneous with a percentage of similarity from 82.8 % to 50.6 %, and one strain was non-typable by PFGE. All strains had at least one virulence gene and most of the strains showed a combination of virulence genes belonging to the STEC and ETEC pathotype. The most frequent pathotypes were those combinations of virulence genes of the STEC/ETEC with 30 strains, 7 strains with virulence genes of the STEC pathotype, and 4 strains with virulence genes of the ETEC pathotype. Regarding phylogenetic groups, a preponderance of B1 (65.85 %) and A (19.51 %) was found. The main serotypes found were O25:H8 (12.19 %), O39: H49 (4.87 %), O76: H19 (7.31 %), and OR:H? (4.87 %). In this identification, a heterogeneous E. coli population with predominant virulence genes of STEC and ETEC prevailed, and the more frequent phylogenetic groups were B1 and A.

Emergence and clonal spread of KPC-2-producing clinical Klebsiella aerogenes isolates in a hospital from northwest Argentina.

Introduction. Klebsiella aerogenes is a nosocomial pathogen associated with drug resistance and healthcare-associated infections.Gap Statement. K. aerogenes is associated with hospital-acquired infections with the ability to acquire mechanisms of resistance to reserve antimicrobials; its clinical behaviour has been poorly documented.Objective. We proposed to investigate an outbreak of carbapenem-resistant K. aerogenes in a hospital that persisted for 4 months.Methods. The primary aim was to evaluate the molecular characteristics and the clonal relationships among the isolates. We characterized isolates by polymerase chain reaction (PCR) and pulsed-field gel electrophoresis (PFGE). The information was integrated with clinical and epidemiological data.Results. Fourteen strains were disseminated in an intensive care unit and different wards at the hospital. The overall mortality was 42.8 %, and mortality attributed to infection was 21.4 %; strains showed high rates of resistance to most of the antimicrobials tested and carried bla KPC-2, bla SHV-2 and bla CTXM-15 genes. PFGE analysis indicated 2 PFGE groups; 12/14 isolates were associated with subgroup A and were likely to be primarily responsible for the first isolation and subsequent dissemination. The outbreak characteristics data showed prolonged hospitalization and previous use of antibiotics as potential risk factors.Conclusion. We consider that it is essential to perform phenotypic and genotypic identification of early genetic resistance mechanisms in K. aerogenes isolates, not only from infection sites but also from colonization, to prevent the spread of these multidrug-resistant (MDR) isolates.

The Importance of Gram-stained Smears in the Diagnosis of Nocardia Infections

Nocardia species are low virulence bacteria found in nature. They can be an infectious agent, especially in patients with risk factors such as underlying immunosuppression, chronic lung disease, and malignancy. They can be easily overlooked because they are not seen frequently and has no pathognomonic symptoms. With this study, it was aimed to draw attention to the importance of microscopic examination of Gram-stained smears in the diagnosis of Nocardia infections in routine microbiology laboratories. Cases in which Nocardia spp. were detected in their clinical samples between November 2014-December 2015 in Hacettepe University Medical Faculty Hospital were included in the study. In the direct microscopic examination of Gram-stained smears of the samples arriving to the laboratory, the incubation periods of the cultures of the samples compatible with Nocardia spp. were extended. Then relevant colonies were identified by conventional microbiological methods and also by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS, bioMerieux, France) automated system. Species-level identification of Nocardia isolates was performed by 16S rRNA gene sequence analysis. To demonstrate the genetic relationship between Nocardia isolates, pulsed-field gel electrophoresis (PFGE) was performed. In vitro susceptibility of the isolates against amoxicillin-clavulanate (AMC), linezolid, moxifloxacin, trimethoprim-sulfamethoxazole (TMP-SXT), amikacin, imipenem, clarithromycin, cefepime, cefotaxime, ceftriaxone, and ciprofloxacin was determined using the gradient strip method (E-test). A total of 19 Nocardia spp. strains were isolated from eight patients. Four cases exhibited repeated growth of Nocardia spp. up to a period of nine months. The most frequently isolated species was N.cyriacigeorgica, which was identified in four cases. Other species isolated from patients were N.asteroides, N.transvalensis, N.farcinicia, and N.asiatica/arthritidis. When the results obtained with DNA sequence analysis and MALDI-TOF MS were compared, 16 (84.2%) of 19 isolates were correctly identified to the genus level and 9 (47.4%) to the species level with MALDI-TOF MS, while three (15.8%) isolates could not be identified, and seven (36.8%) isolates were misidentified. According to the PFGE results, it was determined that the strains isolated from the same patient were genetically identical. All isolates were susceptible to amikacin, cefepime, cefotaxime, ceftriaxone, imipenem, linezolid, and except one isolate to TMP-SXT. Among the study isolates, the most common resistance was against ciprofloxacin (62.5%), followed by clarithromycin (37.5%). N.cyriacigeorgica was determined as the most frequently detected and the most resistant species to antibiotics in the study population. Direct microscopic examination of clinical specimens is one of the most valuable methods for the identification of Nocardia-type bacteria, which is difficult to isolate in microbiology laboratories. With this study, the importance of examining Gram-stained clinical samples was emphasized in the identification of Nocardia species, which can emerge with a wide variety of clinical forms and can be easily overlooked. In addition, antibiotic susceptibility profiles of the isolated bacteria were determinedto contribute to species-specific susceptibility profiles. Accurate identification of Nocardia species will contribute to clinical and epidemiological studies.

First Report of IMI-2-Producing Enterobacter bugandensis and CTX-M-55-Producing Escherichia coli isolated from Healthy Volunteers in Tunisia.

The aim of this study was to characterize the prevalence of fecal carriage of extended-spectrum beta-lactamases and carbapenemase-producing Gram-negative bacteria among healthy humans in Tunisia. Fifty-one rectal swabs of healthy volunteers were plated on MacConkey agar plates supplemented with cefotaxime or imipenem. The occurrences of resistance genes, integrons, and phylogroup typing were investigated using PCR and sequencing. The genetic relatedness of isolates was determined by pulsed-field-gel-electrophoresis (PFGE) and multilocus-sequence-typing (MLST). Whole-genome-sequencing (WGS) was performed for the carbapenem-resistant isolate. Sixteen ESBL-producing Escherichia coli isolates and one carbapenem-resistant Enterobacter bugandensis were detected out of the fifty-one fecal samples. The ESBL-producing E. coli strains contained genes encoding CTX-M-15 (n = 9), CTX-M-1 (n = 3), CTX-M-27 (n = 3), and CTX-M-55 (n = 1). Three CTX-M-1-producers were of lineages ST131, ST7366, and ST1158; two CTX-M-15-producers belonged to lineage ST925 and ST5100; one CTX-M-27-producer belonged to ST2887, and one CTX-M-15-producer belonged to ST744. Six isolates contained class 1 integrons with the following four gene cassette arrangements: dfrA5 (two isolates), dfrA12-orf-aadA2 (two isolates), dfrA17-aadA5 (one isolate), and aadA1 (one isolate). E. bugandensis belonged to ST1095, produced IMI-2 carbapenemase, and contained qnrE1 and fosA genes. A genome-sequence analysis of the E. bugandensis strain revealed new mutations in the blaACT and qnr genes. Our results reveal an alarming rate of ESBL-E. coli in healthy humans in Tunisia and the first description of IMI-2 in E. bugandensis.

Detection and homology analysis of carbapenem resistant Acinetobacter baumannii resistance gene.

To explore the carrying status and homology of carbapenem resistant Acinetobacter baumannii (CRAB) in our hospital. From January 2015 to December 2017, 52 strains of acinetobacter baumannii isolated from the bacteria room of the clinical laboratory of Baogang hospital in Inner Mongolia were selected as the research object. K-B disk diffusion method and Vitek-2 were used to determine the drug sensitivity of Acinetobacter baumannii. The drug resistance gene was detected by polymerase chain reaction (PCR) and its homology was analyzed by pulsed field gel electrophoresis (PFGE). Except for Cefoperazone/sulbactam, other antibiotics were resistant to ab. The detection rate of drug resistance gene class C β-lactamases (ADC) was 100%, and the higher detection rates of other drug resistance genes were class D β-lactamases (OXA)-51 (36 strains, 90.0%),disinfectant gene qacE△1-sull (32 strains, 80.0%), and klebsiella pneumoniae carbapenemase (KPC) gene was not detected. 2-8 drug resistance genes were detected in each CRAB strain, and the strains with 6 drug resistance genes were the most (15 strains, 37.5%); Among the detected drug-resistant gene combinations, ADC+OXA-23 + OXA-51 gene was detected at the same time (29 strains, 72.5%), followed by ADC+ intl1 + qacE △ 1-sull gene (26 strains, 65.0%), ADC + qacE △ 1-sull + ant (3 '') -i gene (19 strains, 47.5%), and 11 strains (27.5%). There were 19 different types in PFGE homology test, each type was 1-9 strains, including 9 strains of A5 type and 8 strains of A18 type, mainly from intensive care unit. CRAB in the hospital is highly resistant to common clinical antibiotics. OXA-23 and OXA-51 genes are most likely to be the main factors causing drug resistance of Acinetobacter baumannii in the hospital. Homology analysis showed that there was CRAB nosocomial infection transmission in different wards of the hospital.

Molecular Detection and Characterization of the Staphylococcus epidermidis and Staphylococcus haemolyticus Isolated from Hospitalized Patients and Healthcare Workers in Iran.

The present study is aimed at surveying the antibiotics resistance profile, biofilm formation ability, staphylococcal cassette chromosome mec (SCCmec) types, and molecular epidemiology of Staphylococcus epidermidis and Staphylococcus haemolyticus isolated from hospitalized patients and healthcare workers in four teaching hospitals in Iran. In total, 43 Staphylococcus epidermidis and 12 Staphylococcus haemolyticus were isolated from hospitalized patients, and 19 Staphylococcus epidermidis and 7 Staphylococcus haemolyticus isolated from healthcare workers were included in the present study. The antimicrobial resistance profile of isolates was determined using the disk diffusion method. Moreover, the resistance of isolates to methicillin was identified using the cefoxitin disk diffusion test. The microtiter-plate test was used for quantifying biofilm formation. Moreover, the frequency of icaA and icaD genes was determined using PCR assay. The molecular epidemiology of methicillin-resistant isolates was determined using SCCmec typing and pulsed-field gel electrophoresis methods. Among all coagulase-negative staphylococci isolates, the highest resistance rate (81.5%) was seen for cefoxitin and cotrimoxazole. All of the isolates were susceptible to linezolid. Out of the 66 mecA-positive isolates, the most common SCCmec type was the type I (n = 23; 34.8%) followed by type IV (n = 13; 19.7%). Using pulsed-field gel electrophoresis (PFGE) assay, 27 PFGE types including 14 common types and 13 singletons were obtained among 51 methicillin-resistant S. epidermidis (MRSE) isolates. Moreover, among 12 methicillin-resistant S. haemolyticus (MRSH) isolates, 8 PFGE types were detected, of which 5 PFGE types were singletons. The high rate of resistance to antibiotics as well as the possibility of cross-infection shows the importance of a pattern shift in the management and controlling programs of coagulase-negative staphylococci, especially in healthcare centers. Clinical trial registration. The present study is not a clinical trial study. Thus, a registration number is not required.