dfrA Genes Research Articles

Genetic Cassettes Profiling of Class I Integron and Antimicrobial Susceptibility Profiles Among Pseudomonas aeruginosa Isolates Collected from Patients in North of Iran

Background: Pseudomonas aeruginosa is an opportunistic nosocomial bacterium, especially in infection wards and among patients with burns. The present study addressed the molecular investigation of the gene cassettes of Class I integrin (intI) and its relationship with multiple drug resistance in clinical samples of P. aeruginosa isolated from Babol hospitals in north of Iran. Objectives: This study aimed to detect the frequency of intI and gene cassettes in the clinical isolates of P. aeruginosa. Methods: In this study, from 75 clinical samples, 30 strains were identified using specific biochemical methods. After determining antibiotic susceptibility using disk diffusion and agar dilution, the frequency of the intI gene and its gene cassettes were determined using the polymerase chain reaction (PCR) method. Results: The highest resistance rate was observed for cefotaxime, ampicillin, and nitrofurantoin using disk diffusion and agar dilution methods. The molecular analyses revealed that 60% of the isolates had the intI genes. The frequency of the aadB, dfrA1, and bla-OXA30 genes were 61, 66, and 33%, respectively. Conclusions: The high resistance of Pseudomonas isolates is due to the presence of intI and its gene cassettes. Considering their high resistance to cefotaxime, gentamicin, ampicillin, and imipenem in hospitals, selecting appropriate drugs or generally changing the treatment course for patients is possible to prevent the spread of resistance inducing genes and the development of nosocomial infections.

Genome Sequence of Escherichia coli Isolated from an Adult in Kibera, an Urban Informal Settlement in Nairobi, Kenya.

ABSTRACTAn Escherichia coli strain (sequence type 636) was isolated from an adult residing in an urban informal settlement in Nairobi, Kenya, and was sequenced using the Illumina MiSeq platform. The draft genome was 5,075,726 bp, with a Col(BS512) plasmid plus aph(6)-Id, blaTEM-1B, and dfrA7 genes, which encode kanamycin, ampicillin, and trimethoprim resistance proteins, respectively.

The Current Landscape of Antibiotic Resistance of Salmonella Infantis in Italy: The Expansion of Extended-Spectrum Beta-Lactamase Producers on a Local Scale.

Salmonella enterica serovar Infantis is one of the five main causes of human salmonellosis in the European Union (EU) and in recent years, has been increasingly reported to carry multiple antimicrobial resistance determinants, including extended-spectrum beta-lactamase (ESBL) genes. In our study, we used WGS-based tools to characterize S. Infantis strains circulating in the Abruzzo and Molise regions of Italy between 2017 and 2020 and compared this local dataset to the S. Infantis population present in Italy over the last two decades. Phylogenetic analyses demonstrated that the majority of strains isolated from poultry and turkeys from Abruzzo and Molise were closely related and belonged to one of the two main genetic clusters present in Italy, which were grouped predominantly as ESBL-producing strains that harbored pESI-like plasmid. We showed that 60% of the local strains carried multiple antibiotic resistance genes, including ESBL gene blaCTX–M–1 as well as aadA1, dfrA1, dfrA14, sul1, and tet(A) genes present on the pESI-like megaplasmid. The analysis of strains from Abruzzo and Molise and the publicly available Italian S. Infantis sequences revealed a dramatic increase in the number of identified AMR genes in the strains isolated after 2011. Moreover, the number of strains resistant to five or more antibiotic classes increased from 20–80% in the last decade likely due to the acquisition of the megaplasmid. The persistence of the ESBL-producing and the multidrug-resistant (MDR) clone of S. Infantis in poultry populations in Italy and in Europe requires rapid and efficient intervention strategies to prevent further expansion of the clone.

A One Health Approach Molecular Analysis of Staphylococcus aureus Reveals Distinct Lineages in Isolates from Miranda Donkeys (Equus asinus) and Their Handlers

Donkeys (Equus asinus) are in decline in Europe. Occupational exposure to farm animals has been associated with increased staphylococci carriage. We aimed to isolate S. aureus and coagulase-negative staphylococci (CoNS) from donkeys and handlers and characterize the antimicrobial resistance profiles and genetic lineages of S. aureus strains. Oral and nasal swab samples were collected from 49 Miranda donkeys and 23 handlers from 15 different farms. Staphylococci species were identified by MALDI-TOF MS. The presence of antimicrobial resistance genes and virulence factors was investigated by PCR. Molecular typing was performed in S. aureus isolates. From the 49 donkey samples, 4 S. aureus (8.2%) and 21 CoNS (42.9%) were isolated. Ten handlers (43.5%) were carriers of S. aureus and 4 (17.4%) carried CoNS. The CoNS isolates showed resistance to several classes of antimicrobials encoded by the mecA, aph (3′)-IIIa, ant (4′)-Ia, tetM, tetK, lnuA, ermB, ermC, dfrA and dfrG genes. S. aureus isolates were resistant to penicillin, aminoglicosides and tetracycline harboring the blaZ, aph (3′)-IIIa, tetL, tetM and tetK genes. All S. aureus isolates from donkeys belonged to ST49 and spa-type t208 while the strains isolated from the handlers were ascribed to 3 STs and 7 spa-types. However, human isolates were from different STs than the donkey isolates. Donkeys are mainly colonized by methicillin-resistant S. sciuri. S. aureus transmission between donkeys and their handlers appears not to have occurred since the isolates belonged to different genetic lineages.

Prevalence of trimethoprim/sulfamethoxazole resistance genes among Stenotrophomonas maltophilia clinical isolates in Egypt.

Stenotrophomonas maltophilia is an important multidrug resistant nosocomial pathogen. Trimethoprim/sulfamethoxazole (TMP/SMX) is considered the drug of choice for treatment of S. maltophilia infections, thus emerging resistance to TMP/SMX poses a serious threat. In the present study we aimed to investigate the frequency of TMP/SMX resistance genes (sul1, sul2, dfrA), and to evaluate their relatedness with integron 1 (int1), and insertion sequence common regions (ISCR) among 100 S. maltophilia from different clinical isolates in Egypt. Isolates were identified biochemically and confirmed by VITEK2. Detection of sul1, sul2, and dfrA genes, int1 and ISCR elements was performed by PCR. Among the 16 TMP/SMX resistant isolates, sul1 gene was detected in all of them, and it was associated with int1 gene presence in all resistant isolates. The sul2 gene was detected in 6 out of 16 resistant isolates (37.5%), and only 2 of the 16 resistant isolates (12.5%) harboured dfrA gene. ISCR was detected in 10 of the resistant isolates (62.5%) and in 4 of them it was associated with the presence of sul2 gene. Among the 84 TMP/SMX sensitive isolates, sul1 gene was detected in 15 (17.8%), int1 in 16 (19%) and ISCR in 6 (7.1%). None of the susceptible isolates had sul2 or dfrA genes. These findings point out an increasing frequency of TMP/SMX resistance genes among S. maltophilia clinical isolates in our region, so the adoption of prudent use of S. maltophilia antimicrobial agents and the establishment of a surveillance system are desperately needed.

Genomic characterization of multidrug-resistant Salmonella serovar Kentucky ST198 isolated in poultry flocks in Spain (2011-2017).

Salmonella Kentucky is commonly found in poultry and rarely associated with human disease. However, a multidrug-resistant (MDR) S. Kentucky clone [sequence type (ST)198] has been increasingly reported globally in humans and animals. Our aim here was to assess if the recently reported increase of S. Kentucky in poultry in Spain was associated with the ST198 clone and to characterize this MDR clone and its distribution in Spain. Sixty-six isolates retrieved from turkey, laying hen and broiler in 2011–2017 were subjected to whole-genome sequencing to assess their sequence type, genetic relatedness, and presence of antimicrobial resistance genes (ARGs), plasmid replicons and virulence factors. Thirteen strains were further analysed using long-read sequencing technologies to characterize the genetic background associated with ARGs. All isolates belonged to the ST198 clone and were grouped in three clades associated with the presence of a specific point mutation in the gyrA gene, their geographical origin and isolation year. All strains carried between one and 16 ARGs whose presence correlated with the resistance phenotype to between two and eight antimicrobials. The ARGs were located in the Salmonella genomic island (SGI-1) and in some cases (bla SHV-12 , catA1, cmlA1, dfrA and multiple aminoglycoside-resistance genes) in IncHI2/IncI1 plasmids, some of which were consistently detected in different years/farms in certain regions, suggesting they could persist over time. Our results indicate that the MDR S. Kentucky ST198 is present in all investigated poultry hosts in Spain, and that certain strains also carry additional plasmid-mediated ARGs, thus increasing its potential public health significance.

Genetic characterization of clinically relevant class 1 integrons carried by multidrug resistant bacteria (MDRB) isolated from the gut microbiota of highly antibiotic treated Salmo salar

The main objective of this study was the genetic characterization of clinically relevant class 1 integrons carried by multidrug resistant bacteria isolated from the intestinal microbiota of aquaculture salmon treated with high concentrations of antibiotics. In 82 multidrug resistant bacterial isolates, the prevalence of both the conserved elements of the integrons, qacEΔ1 and sul1 genes, and the variable region (VR) was determined. Further, whole genome sequencing and complete genetic analysis was performed in VR-positive isolates. Despite the fact that 100% of the bacterial isolates presented the intI1 gene, only 12.3% carried the qacEΔ1 and sul1 genes and only two (2.4%) presented a VR with gene cassettes. In the Pseudomonas baetica 25P2F9 isolate, a VR carrying aac(6')31, qacH, and blaOXA-2 gene cassettes was described, whereas the VR of Aeromonas salmonicida 30PB8 isolate showed a dfrA14 gene cassette. The array of gene cassettes found in the Pseudomonas isolate appears with high frequency in clinically relevant pathogens such as Pseudomonas aeruginosa or Escherichia coli. Additionally, it was possible to determine that these integrons are contained in plasmids and coul be easily transferred. Resistome analysis demonstrated that both isolates carried a great diversity of antibiotic resistance genes, including many β-lactamases. Even in the Aeromonas isolate a new oxacillin-hydrolyzing beta-lactamase gene was described (blaOXA-956). The presence of multidrug resistant bacteria and clinically relevant genetic elements in the salmon intestinal microbiota make the aquaculture a hotspot in the phenomenon of antibiotic resistance; therefore, the control of antibiotics used in this activity is a key point to avoid its escalation.

Antimicrobial Resistance Dynamics in Chilean Shigella sonnei Strains Within Two Decades: Role of Shigella Resistance Locus Pathogenicity Island and Class 1 and Class 2 Integrons.

Shigellosis is an enteric infectious disease in which antibiotic treatment is effective, shortening the duration of symptoms and reducing the excretion of the pathogen into the environment. Shigella spp., the etiologic agent, are considered emerging pathogens with a high public health impact due to the increase and global spread of multidrug-resistant (MDR) strains. Since Shigella resistance phenotype varies worldwide, we present an overview of the resistance phenotypes and associated genetic determinants present in 349 Chilean S. sonnei strains isolated during the periods 1995–1997, 2002–2004, 2008–2009, and 2010–2013. We detected a great variability in antibiotic susceptibility patterns, finding 300 (86%) MDR strains. Mobile genetic elements (MGE), such as plasmids, integrons, and genomic islands, have been associated with the MDR phenotypes. The Shigella resistance locus pathogenicity island (SRL PAI), which encodes for ampicillin, streptomycin, chloramphenicol, and tetracycline resistance genes, was detected by PCR in 100% of the strains isolated in 2008–2009 but was less frequent in isolates from other periods. The presence or absence of SRL PAI was also differentiated by pulsed-field gel electrophoresis. An atypical class 1 integron which harbors the blaOXA–1-aadA1-IS1 organization was detected as part of SRL PAI. The dfrA14 gene conferring trimethoprim resistance was present in 98.8% of the 2008–2009 isolates, distinguishing them from the SRL-positive strains isolated before that. Thus, it seems an SRL-dfrA14 S. sonnei clone spread during the 2008–2009 period and declined thereafter. Besides these, SRL-negative strains harboring class 2 integrons with or without resistance to nalidixic acid were detected from 2011 onward, suggesting the circulation of another clone. Whole-genome sequencing of selected strains confirmed the results obtained by PCR and phenotypic analysis. It is highlighted that 70.8% of the MDR strains harbored one or more of the MGE evaluated, while 15.2% lacked both SRL PAI and integrons. These results underscore the temporal dynamics of antimicrobial resistance in S. sonnei strains circulating in Chile, mainly determined by the spread of MGE conferring MDR phenotypes. Since shigellosis is endemic in Chile, constant surveillance of antimicrobial resistance phenotypes and their genetic basis is a priority to contribute to public health policies.

Identification of a candidate gene responsible for the G locus determining chartreuse bulb color in onion (Allium cepa L.) using bulked segregant RNA-Seq.

A gene encoding a laccase responsible for chartreuse onion bulb color was identified. Markers tagging this gene showed perfect linkage with bulb colors among diverse germplasm. To identify a casual gene for the G locus determining chartreuse bulb color in onion (Allium cepa L.), bulked segregant RNA-Seq (BSR-Seq) was performed using yellow and chartreuse individuals of a segregating population. Through single nucleotide polymorphism (SNP) and differentially expressed gene (DEG) screening processes, 163 and 143 transcripts were selected, respectively. One transcript encoding a laccase-like protein was commonly identified from SNP and DEG screening. This transcript contained four highly conserved copper-binding domains known to be signature sequences of laccases. This gene was designated AcLAC12 since it showed high homology with Arabidopsis AtLAC12. A 4-bp deletion creating a premature stop codon was identified in exon 5 of the chartreuse allele. Another mutant allele in which an intact LTR-retrotransposon was transposed in exon 5 was identified from other chartreuse breeding lines. Genotypes of molecular markers tagging AcLAC12 were perfectly matched with bulb color phenotypes in segregating populations and diverse breeding lines. All chartreuse breeding lines contained inactive alleles of DFR-A gene determining red bulb color, indicating that chartreuse color appeared when both DFR-A and AcLAC12 genes were inactivated. Linkage maps showed that AcLAC12 was positioned at the end of chromosome 7. Transcription levels of structural genes encoding enzymes in anthocyanin biosynthesis pathway were generally reduced in chartreuse bulk compared with yellow bulk. Concentrations of total quercetins were also reduced in chartreuse onion. However, significant amounts of quercetins were detected in chartreuse onion, implying that AcLAC12 might be involved in modification of quercetin derivatives in onion.

Prevalence, Risk Factors, Antimicrobial Resistance and Molecular Characterization of Salmonella in Northeast Tunisia Broiler Flocks.

This study was conducted in northeastern Tunisia to estimate both the prevalence and the risk factors of Salmonella in broiler flocks as well as to characterize the isolated multidrug-resistant (MDR) Salmonella strains. In the present study, a total number of 124 farms were sampled; Salmonella isolates were identified by the alternative technique VIDAS Easy Salmonella. The susceptibility of Salmonella isolates was assessed against 21 antimicrobials using the disk diffusion method on Mueller–Hinton agar using antimicrobial discs. Some antimicrobial resistance genes were identified using PCR. The prevalence rate of Salmonella infection, in the sampled farms, was estimated at 19.9% (64/322). Moreover, a total number of 13 different serotypes were identified. High rate of resistance was identified against nalidixic acid (82.85%), amoxicillin (81.25%), streptomycin (75%), and ciprofloxacin (75%). Alarming level of resistance to ertapenem (12.5%) was noticed. A total of 87.5% (56/64) of isolated strains were recognized as MDR. Three MDR strains were extended-spectrum β-lactamases (ESBL)-producers and three MDR strains were cephalosporinase-producers. The blaCTX-M gene was amplified in all the three ESBL strains. The qnrB gene was not amplified in fluoroquinolones-resistant strains. The tetA and tetB genes were amplified in 5% (2/40) and 2.5% (1/40) of tetracycline-resistant strains, respectively. The dfrA1 gene was amplified in five of the 20 trimethoprim-resistant strains. The mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes were not amplified in any of the phenotypically colistin-resistant strains. In terms of integrase genes int1 and int2, only gene class 2 was amplified in 11% (7/64) of analyzed strains. Risk factors, such as the poor level of cleaning and disinfection, the lack of antimicrobial treatment at the start of the breeding, and a crawl space duration lower than 15 days, were associated with high Salmonella infection in birds. These data should be considered when preparing salmonellosis control programs in Tunisian broiler flocks.

Clones and Clusters of Antimicrobial-Resistant Klebsiella From Southwestern Nigeria.

Background Klebsiella pneumoniae is a World Health Organization high-priority antibiotic-resistant pathogen. However, little is known about Klebsiella lineages circulating in Nigeria.MethodsWe performed whole-genome sequencing (WGS) of 141 Klebsiella isolated between 2016 and 2018 from clinical specimens at 3 antimicrobial-resistance (AMR) sentinel surveillance tertiary hospitals in southwestern Nigeria. We conducted in silico multilocus sequence typing; AMR gene, virulence gene, plasmid, and K and O loci profiling; as well as phylogenetic analyses, using publicly available tools and Nextflow pipelines.ResultsPhylogenetic analysis revealed that the majority of the 134 K. pneumoniae and 5 K. quasipneumoniae isolates from Nigeria characterized are closely related to globally disseminated multidrug-resistant clones. Of the 39 K. pneumoniae sequence types (STs) identified, the most common were ST307 (15%), ST5241 (12%), ST15 (~9%), and ST25 (~6%). ST5241, 1 of 10 novel STs detected, is a single locus variant of ST636 carrying dfrA14, tetD, qnrS, and oqxAB resistance genes. The extended-spectrum β-lactamase (ESBL) gene blaCTX_M-15 was seen in 72% of K. pneumoniae genomes, while 8% encoded a carbapenemase. No isolate carried a combination of carbapenemase-producing genes. Four likely outbreak clusters from 1 facility, within STs 17, 25, 307, and 5241, were ESBL but not carbapenemase-bearing clones.ConclusionsThis study uncovered known and novel K. pneumoniae lineages circulating in 3 hospitals in Southwest Nigeria that include multidrug-resistant ESBL producers. Carbapenemase-producing isolates remain uncommon. WGS retrospectively identified outbreak clusters, pointing to the value of genomic approaches in AMR surveillance for improving infection prevention and control in Nigerian hospitals.

In vivo Emergence of Colistin and Tigecycline Resistance in Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae During Antibiotics Treatment.

Three carbapenem-resistant Klebsiella pneumoniae (CRKP; strains KP-426, KP-C76, and KP-CT77) were isolated from a patient with severe burns during the treatment of colistin and tigecycline. Single-nucleotide polymorphism typing showed that three ST11 CRKP were clonally related. Three isolates harbored the same set of antimicrobial resistance genes. blaKPC-2, blaSHV-12, blaTEM-1, and rmtB genes were located on the same 128,928-bp IncFII/IncR plasmid. Tet(A), catA2, sul2, and dfrA14 genes were located on a plasmid with an unknown Inc-type. blaSHV-11, fosA, and aadA2 were chromosomal genes. An IS1 and an ISKpn14 were found in the promoter region of the mgrB gene of two colistin-resistant CRKP, K. pneumoniae KP-C76, and KP-CT77, respectively. A novel amino acid substitution, G300E, was identified in the type 1 Tet(A) variant of K. pneumoniae KP-CT77 which exhibited high-level tigecycline resistance compared to strains KP-426 and KP-C76 (MIC of 32, 4, and 4mg/l, respectively). Conjugation and cloning experiments confirmed that the mutated Tet(A) resulted in a 4-fold increase in tigecycline minimal inhibitory concentration (MIC) of Escherichia coli. Three CRKP belonged to the K64 serotype and possessed a similar IncHI1B/repB virulence plasmid carrying rmpA, rmpA2, and iucABCDiutA. The survival rates of Galleria Mellonella injected with K. pneumoniae KP-426, KP-C76, and KP-CT77 were 4.2, 20.8, and 8.3%, respectively. The emergence of colistin and tigecycline resistance in carbapenem-resistant hypervirulent K. pneumoniae posed a serious threat to clinical anti-infective therapy. The type 1 Tet(A) variant carrying G300E mutation, which conferred significantly elevated tigecycline MIC and was located on a conjugative plasmid, needs attention.

Antibiotyping and genotyping of extensively drug-resistant (XDR) Salmonella sp. isolated from clinical samples of Lahore, Pakistan.

Antibiotic resistance is a major problem in Salmonella enterica serovar Typhi. The objective of this study was to evaluate the prevalence of XDR Salmonella among local population of Lahore and genotyping of isolates for antibiotic-resistant genes. A total of 200 blood samples from suspected typhoid fever patients were collected. One hundred and fifty-seven bacterial samples were confirmed as Salmonella Typhi and 23 samples were confirmed as Salmonella Paratyphi after biochemical, serological and PCR based molecular characterization. Antibiogram analysis classified 121 (67·2%) Salmonella isolates as MDR and 62 isolates (34·4%) as XDR. The predominant resistance gene was ampC with 47·7% prevalence, followed by gyrA, catA1, tet(A), aac (3)-la, qnrS, blaNDM-1 and blaCTX-M-15 genes in 45·5, 40, 21·6, 18·3, 11·6, 2·2 and 0·5% isolates respectively. Sequence analysis showed the presence of sul1 and dfrA7 gene cassette arrays in 12 class 1 integron integrase positive isolates. Large number of clinical XDR S. Typhi-resistant against third generation cephalosporins have been reported. The current study highlights the possible emergence of clinical XDR S. Typhi cases in Lahore, Pakistan. Potential attribution of phenotypic and genotypic XDR cases may help to contribute targeted therapy.

DfrA trimethoprim resistance genes found in Gram-negative bacteria: compilation and unambiguous numbering

To track the spread of antibiotic resistance genes, accurate identification of individual genes is essential. Acquired trimethoprim resistance genes encoding trimethoprim-insensitive homologues of the sensitive dihydrofolate reductases encoded by the folA genes of bacteria are increasingly found in genome sequences. However, naming and numbering in publicly available records (journal publications or entries in the GenBank non-redundant DNA database) has not always been unambiguous. In addition, the nomenclature has evolved over time. Here, the changes in nomenclature and the most commonly encountered problems and pitfalls affecting dfrA gene identification arising from historically incorrect or inaccurate numbering are explained. The complete set of dfrA genes/DfrA proteins found in Gram-negative bacteria for which readily searchable sequence information is currently available has been compiled using less than 98% identity for both the gene and the derived protein sequence as the criteria for assignment of a new number. In most cases, trimethoprim resistance has been demonstrated. The gene context, predominantly in a gene cassette or near the ori end of CR1 or CR2, is also covered. The RefSeq database that underpins the programs used to automatically identify resistance genes in genome data sets has been curated to assign all sequences listed to the correct number. This led to the assignment of corrected or new gene numbers to several mis-assigned sequences. The unique numbers assigned for the dfrA/DfrA set are now listed in the RefSeq database, which we propose provides a way forward that should end future duplication of numbers and the confusion that causes.

Analysis of Antibiotic Resistance of <i>Vibrio Сholerae</i> Isolated From Environmental Objects in Russia in 2019

The aim of the study was to analyze the resistance to antibacterial drugs of Vibrio cholerae strains isolated from environmental water bodies on the territory of Russia in 2019.V.cholerae O1 El Tor (14) and V.cholerae nonO1/nonO139 strains were used in this work. Sensitivity/resistance to 11 antibacterial drugs was determined using the method of serial dilutions in a solid nutrient medium. The presence of drug resistance genes was determined using real-time PCR. Fluctuations in sensitivity/resistance of V.cholerae were found in various years. The phenotypic resistance of the strains to tetracycline and trimethoprim/sulfamethoxazole correlated with the presence of the tetR and dfrA1 genes in them. The presence of ICE was not detected in V.cholerae strains containing the tetR and qnrVC1 genes. The variability and wide spectrum of V.cholerae resistance require close attention to the problem of antibiotic resistance of cholera. The detection of ICE in the studied V.cholerae strains, as well as antibiotic resistance genes not associated with ICE elements, emphasize the need for molecular genetic monitoring of V.cholerae antibiotic resistance.

Detection of Phylogenetic Groups and Drug Resistance Genes of Escherichia coli Causing Urinary Tract Infection in Southwest Iran

Background: Many bacteria can cause urinary tract infections (UTIs), among which Escherichia coli is the most common causative agent. Escherichia coli strains are divided into eight phylogenetic groups based on the new Quadroplex-PCR method, which are different in terms of patterns of resistance to antibiotics, virulence, and environmental characteristics. Objectives: This study aimed to determine the phylogenetic groups and the prevalence of drug resistance genes in E. coli strains causing UTIs. Methods: In this descriptive cross-sectional study, 129 E. coli isolates obtained from the culture of patients with UTIs were evaluated for phylogenetic groups using the new method of Clermont et al. The identification of phylogenetic groups and antibiotic resistance genes was performed using the multiplex polymerase chain reaction (PCR) method. Results: In this study, concerning the distribution of phylogenetic groups among E. coli isolates, the phylogenetic group B2 (36.4%) was the most common phylogenetic group, followed by phylogroups C (13.2%), clade I (10.1%), D (9.3%), and A (3.1%) while groups B1 and F were not observed in any of the isolates, and 20.2% had an unknown state. Also, out of 129 E. coli isolates, the total frequency of tetA, tetB, sul1, sul2, CITM, DfrA, and qnr resistance genes was 59.7%, 66.7, 69, 62, 30.2, 23.3, and 20.2%, respectively. In this study, there was a significant relationship between antibiotics (P = 0.026), cefotaxime (P = 0.003), and nalidixic acid (P = 0.044) and E. coli phylogenetic groups. No significant relationship was observed between E. coli phylogenetic groups and antibiotic resistance genes. Conclusions: The results of this study showed that strains belonging to group B2 had the highest prevalence among other phylogroups, and also, the frequency of antibiotic resistance genes and drug-resistant isolates had a higher prevalence in this phylogroup. These results show that phylogroup B2 has a more effective role in causing urinary tract infections compared to other phylogroups, and this phylogroup can be considered a genetic reservoir of antibiotic resistance.

Multidrug-resistant Klebsiella pneumoniae harboring extended spectrum β-lactamase encoding genes isolated from human septicemias.

Klebsiella pneumoniae is a major pathogen implicated in nosocomial infections. Extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae isolates are a public health concern. We aim to characterize the type of β-lactamases and the associated resistance mechanisms in ESBL-producing K. pneumoniae isolates obtained from blood cultures in a Portuguese hospital, as well as to determine the circulating clones. Twenty-two cefotaxime/ceftazidime-resistant (CTX/CAZR) K. pneumoniae isolates were included in the study. Identification was performed by MALDI-TOF MS and the antimicrobial susceptibility testing by disk-diffusion. The screening test for ESBL-production was performed and ESBL-producer isolates were further characterized. The presence of different beta-lactamase genes (blaCTX-M, blaSHV, blaTEM, blaKPC, blaNDM, blaVIM, blaOXA-48, blaCMY-2, blaDHA-1, blaFOX, blaMOX, and blaACC) was analyzed by PCR/sequencing in ESBL-producer isolates, as well as the presence of other resistance genes (aac(6’)-Ib-cr, tetA/B, dfrA, qnrA/B/S, sul1/2/3) or integron-related genes (int1/2/3). Multilocus-sequence-typing (MLST) was performed for selected isolates. ESBL activity was detected in 12 of the 22 CTX/CAZR K. pneumoniae isolates and 11 of them carried the blaCTX-M-15 gene (together with blaTEM), and the remaining isolate carried the blaSHV-106 gene. All the blaCTX-M-15 harboring isolates also contained a blaSHV gene (blaSHV-1, blaSHV-11 or blaSHV-27 variants). Both blaSHV-27 and blaSHV-106 genes correspond to ESBL-variants. Two of the CTX-M-15 producing isolates carried a carbapenemase gene (blaKPC2/3 and blaOXA-48) and showed imipenem resistance. The majority of the ESBL-producing isolates carried the int1 gene, as well as sulphonamide-resistance genes (sul2 and/or sul3); the tetA gene was detected in all eight tetracycline-resistant isolates. Three different genetic lineages were found in selected isolates: ST348 (one CTX-M-15/TEM/SHV-27/KPC-2/3-producer isolate), ST11 (two CTX-M-15/TEM/SHV-1- and CTX-M-15-TEM-SHV-11-OXA-48-producer isolates) and ST15 (one SHV-106/TEM-producer isolate). ESBL enzymes of CTX-M-15 or SHV-type are detected among blood K. pneumoniae isolates, in some cases in association with carbapenemases of KPC or OXA-48 type.

High Frequency of Integron-Associated Cassette Arrays Containing dfrA and aad Genes in Urinary Isolates of Escherichia coli in the Southwest of Iran

High Frequency of Integron-Associated Cassette Arrays Containing dfrA and aad Genes in Urinary Isolates of Escherichia coli in the Southwest of Iran

Dissemination of antibiotic resistance genes, mobile genetic elements, and efflux genes in anthropogenically impacted riverine environments

Anthropogenically impacted surface waters are an important reservoir for multidrug-resistant bacteria and antibiotic-resistant genes. The present study aimed at MDR, ESBL, AmpC, efflux genes, and heavy metals resistance genes (HMRGs) in bacterial isolates from four Indian rivers belonging to different geo-climatic zones, by estimating the mode of resistance transmission exhibited by the resistant isolates. A total 71.27% isolates exhibited MDR trait, showing maximum resistance towards β-lactams (P = 66.49%; AMX = 59.04%), lincosamides (CD = 65.96%), glycopeptides (VAN = 25.19%; TEI = 56.91%), cephalosporins (CF = 53.72%; CXM = 30.32%) sulphonamide (COT = 43.62%; TRIM = 12.77%), followed by macrolide and tetracycline. The dfrA1 and dfrB genes were detected in total 37.5% isolates whereas; dfrA1 genes were detected in 33.34%. The sul1 gene was detected in 9.76% and sul2 gene was detected in 2.44% isolates. A total of 69.40% MDR integron positive isolates were detected with intI1and intI2 detected at 89.25% and 1.07%, respectively; encoding class 1 and class 2 integron-integrase. ESBL production was confirmed in 73.13% isolates that harboured the genes blaTEM (96.84%), blaSHV (27.37%), blaOXA (13.68%) and blaCTXM (18.95%) while the frequency of HMRGs; 52.24% (zntB), 33.58% (chrA), and 6.72% (cadD). Efflux activity was confirmed in 96.26% isolates that harbored the genes acrA (93.02%), tolC (88.37%), and acrB (86.04%). AmpC (plasmid-mediated) was detected in 20.9% of the riverine isolates. Detection of such hidden molecular modes of antibiotic resistance in the rivers is alarming that requires urgent and stringent measures to control the resistance threats.

Class I integrons among multidrug resistant Enterobacter spp. isolates from hospitalized patients in Babol, North of Iran.

Background:Multidrug resistance (MDR) in Enterobacter spp. has created therapeutic challenges all over the world. The present study was conducted for evaluating the prevalence of class I integron, determining the gene cassettes and antimicrobial resistance profile of Enterobacter spp. isolates from clinical samples in Babol, North of Iran.Methods:During a 13-month period, 30 Enterobacter spp. isolates were collected from Ayatollah Rouhani Hospital, Babol, Iran. Various types of antimicrobial agents were used to determine the resistance pattern. Class I integron and associated gene cassettes were detected by PCR assay.Results:The resistance rates to AP, CPM, CTX, TM, NI, IMI, AK, CIP and GM antimicrobials were 100%, 93.3%, 33.3%, 33.3%, 30%, 20%, 20%, 20% and 13.3%, respectively. The distribution results of int genes showed that 63.3% of isolates carried the intI genes. Also, the prevalence of aadB, dfrA1, blaOXA30 and blaPSE1 genes were estimated at 36.6%%, 33.3%, 6.6% and 0%, respectively.Conclusion:Our results showed that class I integrons have a widespread distribution among the Enterobacter spp. isolates and have clinical relevance to MDR isolates. The results confirmed the necessity for uninterrupted monitoring to prevent distribution of multidrug resistance among Enterobacter spp. strains in Iran.