Comparison of two DNA microarrays for detection of plasmid-mediated antimicrobial resistance and virulence factor genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. - Related Documents




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223201.0000Comparison of two DNA microarrays for detection of plasmid-mediated antimicrobial resistance and virulence factor genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. A DNA microarray was developed to detect plasmid-mediated antimicrobial resistance (AR) and virulence factor (VF) genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. The array was validated with the following bacterial species: Escherichiacoli (n=17); Klebsiellapneumoniae (n=3); Enterobacter spp. (n=6); Acinetobacter genospecies 3 (n=1); Acinetobacterbaumannii (n=1); Pseudomonasaeruginosa (n=2); and Stenotrophomonasmaltophilia (n=2). The AR gene profiles of these isolates were identified by polymerase chain reaction (PCR). The DNA microarray consisted of 155 and 133 AR and VF gene probes, respectively. Results were compared with the commercially available Identibac AMR-ve Array Tube. Hybridisation results indicated that there was excellent correlation between PCR and array results for AR and VF genes. Genes conferring resistance to each antibiotic class were identified by the DNA array. Unusual resistance genes were also identified, such as bla(SHV-5) in a bla(OXA-23)-positive carbapenem-resistant A. baumannii. The phylogenetic group of each E. coli isolate was verified by the array. These data demonstrate that it is possible to screen simultaneously for all important classes of mobile AR and VF genes in Enterobacteriaceae and non-Enterobacteriaceae whilst also assigning a correct phylogenetic group to E. coli isolates. Therefore, it is feasible to test clinical Gram-negative bacteria for all known AR genes and to provide important information regarding pathogenicity simultaneously.201020356716
171610.9998Detection of clinically important β-lactamases by using PCR. Increasing antimicrobial resistance of nosocomial pathogens is becoming a serious threat to public health. To control the spread of this resistance, it is necessary to detect β-lactamase-producing organisms in the clinical setting. The aims of the study were to design a PCR assay for rapid detection of clinically encountered β-lactamase genes described in Enterobacteriaceae and Gram-negative non-fermenting bacteria. The functionality of proposed primers was verified using eight reference strains and 17 strains from our collection, which contained 29 different β-lactamase genes. PCR products of the test strains were confirmed by Sanger sequencing. Sequence analysis was performed using bioinformatics software Geneious. Overall, 67 pairs of primers for detecting 12 members of the class C β-lactamase family, 15 members of class A β-lactamases, six gene families of subclass B1, one member each of subclasses B2, B3 and class D β-lactamases were designed, of which 43 pairs were experimentally tested in vitro. All 29 β-lactamase genes, including 10 oxacillinase subgroups, were correctly identified by PCR. The proposed set of primers should be able to specifically detect 99.7% of analyzed β-lactamase subtypes and more than 79.8% of all described β-lactamase genes.202134100944
170920.9998High prevalence of bla(VIM-1) gene in bacteria from Brazilian soil. This study investigated bacteria from soil samples to (i) determine the main bacterial genera and species having resistance to carbapenem and other β-lactams and (ii) establish if the mechanism of resistance was due to the production of metallo-β-lactamases. The isolates were characterized by PCR for metallo-β-lactamases and integrons, by antimicrobial susceptibility testing, and by sequencing. The antimicrobial profile of 40 imipenem-resistant Gram-positive soil isolates from all Brazilian regions demonstrated that 31 (77.5%) of them were multidrug resistant. Among the 40 isolates, 19 presented the bla(VIM) gene and class 1 integrons by PCR. Six of the 19 isolates were identified as Paenibacillus sp., 12 as Bacillus sp., and just 1 was classified as Staphylococcus sp., by sequencing of the 16S rRNA gene. These results suggest that bacteria from soil can act as a source of bla(VIM-1) genes, representing a threat to public health.201627392282
171430.9998Carbapenemase-producing enterobacteriaceae recovered from a Spanish river ecosystem. The increasing resistance to carbapenems is an alarming threat in the fight against multiresistant bacteria. The dissemination properties of antimicrobial resistance genes are supported by their detection in a diverse population of bacteria, including strains isolated from the environment. The objective of this study was to investigate the presence of carbapenemase-producing Enterobacteriaceae (CPE) collected from a river ecosystem in the Barcelona metropolitan area (Spain). Identification of β-lactamases and other resistance determinants was determined as was the antimicrobial susceptibility profile. Moreover, screening of virulence factors, plasmid addiction systems, plasmid partition systems and replicon typing was performed. The results identified 8 isolates belonging to different species (Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Klebsiella oxytoca, Raoultella ornithinolytica). The most prevalent enzyme was KPC-2 (n = 6), followed by VIM-1 (n = 2) and IMI-2 (n = 1), whereas no OXA-48-type was detected. In addition, one strain was positive for both KPC-2 and VIM-1 enzymes. All the carbapenemase-encoding plasmids carried at least one plasmid addiction or partition system, being vagCD and parAB the most frequently detected, respectively. E. coli and K. pneumoniae isolates carried a low number of virulence-associated factors and none of the detected clones has previously been identified in the clinical setting. These findings support the high dissemination potential of the carbapanemase-encoding genes and reinforce the idea that the environment is another reservoir that may play an important role in the capture, selection and dissemination of carbapenem resistance genes.201728380016
86940.9998The Prevalence of Antibiotic Resistance Phenotypes and Genotypes in Multidrug-Resistant Bacterial Isolates from the Academic Hospital of Jaén, Spain. The heterogenicity of antimicrobial resistance genes described in clinically significant bacterial isolates and their potential role in reducing the efficacy of classically effective antibiotics pose a major challenge for global healthcare, especially in infections caused by Gram-negative bacteria. We analyzed 112 multidrug-resistant (MDR) isolates from clinical samples in order to detect high resistance profiles, both phenotypically and genotypically, among four Gram-negative genera (Acinetobacter, Escherichia, Klebsiella, and Pseudomonas). We found that 9.8% of the total selected isolates were classified as extensively drug-resistant (XDR) (six isolates identified as A. baumannii and five among P. pneumoniae isolates). All other isolates were classified as MDR. Almost 100% of the isolates showed positive results for bla(OXA-23) and bla(NDM-1) genes among the A. baumannii samples, one resistance gene (bla(CTX-M)) among E. coli, and two genetic determinants (bla(CTX-M) and aac(6')-Ib) among Klebsiella. In contrast, P. aeruginosa showed just one high-frequency antibiotic resistance gene (dfrA), which was present in 68.42% of the isolates studied. We also describe positive associations between ampicillin and cefotaxime resistance in A. baumannii and the presence of bla(VEB) and bla(GES) genes, as well as between the aztreonam resistance phenotype and the presence of bla(GES) gene in E. coli. These data may be useful in achieving a better control of infection strategies and antibiotic management in clinical scenarios where these multidrug-resistant Gram-negative pathogens cause higher morbidity and mortality.202438786157
189950.9998Characteristics of plasmids in multi-drug-resistant Enterobacteriaceae isolated during prospective surveillance of a newly opened hospital in Iraq. BACKGROUND: Gram-negative multidrug-resistant (MDR) bacteria are major causes of nosocomial infections, and antibiotic resistance in these organisms is often plasmid mediated. Data are scarce pertaining to molecular mechanisms of antibiotic resistance in resource constrained areas such as Iraq. METHODOLOGY/PRINCIPAL FINDINGS: In this study, all MDR Enterobacteriaceae (n = 38) and randomly selected non-MDR counterparts (n = 41) isolated from patients, healthcare workers and environmental surfaces in a newly opened hospital in Iraq were investigated to characterize plasmids found in these isolates and determine their contribution to antibiotic resistance. Our results demonstrated that MDR E. coli and K. pneumoniae isolates harbored significantly more (≥ 3) plasmids compared to their non-MDR counterparts, which carried ≤ 2 plasmids (p<0.01). Various large plasmids (~52 to 100 kb) from representative isolates were confirmed to contain multiple resistance genes by DNA microarray analysis. Aminoglycoside (acc, aadA, aph, strA/B, and ksgA), β-lactam (bla(TEM1), bla(AMPC), bla(CTX-M-15), bla(OXA-1), bla(VIM-2) and bla(SHV)), sulfamethoxazole/trimethoprim (sul/dfr), tetracycline (tet) and chloramphenicol (cat) resistance genes were detected on these plasmids. Additionally, multiple plasmids carrying multiple antibiotic resistance genes were found in the same host strain. Genetic transfer-associated genes were identified on the plasmids from both MDR and non-MDR isolates. Seven plasmid replicon types (FII, FIA, FIB, B/O, K, I1 and N) were detected in the isolates, while globally disseminated IncA/C and IncHI1 plasmids were not detected in these isolates. CONCLUSIONS/SIGNIFICANCE: This is the first report of the characteristics of the plasmids found in Enterobacteriaceae isolated following the opening of a new hospital in Iraq. The information provided here furthers our understanding of the mechanisms of drug resistance in this specific region and their evolutionary relationship with other parts of world. The large plasmids, carrying resistance genes and transfer-associated genes, may be potential factors for regional dissemination of antibiotic resistance.201222808141
170860.9998High-level of resistance to β-lactam and presence of β-lactamases encoding genes in Ochrobactrum sp. and Achromobacter sp. isolated from soil. OBJECTIVES: Bacteria belonging to the genera Ochrobactrum and Achromobacter are bacteria considered opportunistic, causing infections mainly in immunocompromised patients. β-lactamases are the main cause of resistance to β-lactam antibiotics. This study aimed to investigate the antimicrobial resistance profile and the presence of β-lactamases encoding genes in Ochrobactrum sp. and Achromobacter sp. isolated from Brazilian soils. METHODS: Soil samples from the five regions of Brazil were collected for the isolation of bacteria, which were identified molecularly and then, the minimum inhibitory concentration and detection of β-lactamases encoding genes were performed. RESULTS: High-level of resistance to β-lactam antibiotics and different β-lactamases encoding genes were found (bla(CTX-M-Gp1), bla(SHV), bla(OXA-1-like) and bla(KPC)), including the first report of the presence of bla(KPC) in bacteria belonging to the genera Ochrobactrum and Achromobacter. CONCLUSION: The results showed that the bacteria from this study, belonging to genera Ochrobactrum and Achromobacter isolated from soil, harbor different β-lactamases encoding genes and can act as a reservoir of these genes.201729111479
170370.9998Acinetobacter baumannii clinical isolates from outbreaks in Erbil hospitals after the COVID-19 pandemic. INTRODUCTION: Acinetobacter baumannii is endemic in hospital environments, and since the coronavirus disease 2019 (COVID-19) pandemic, multidrug-resistant A. baumannii has become more potent. This potential evolution is driven by the undetectable numbers of gene resistances it has acquired. We evaluated the antibiotic-resistance genes in isolates from patients in Erbil hospitals. METHODOLOGY: This is the first study to demonstrate the antimicrobial resistance epidemic in Erbil, Iraq. A total of 570 patients, including 100 COVID-19 patients were tested. Isolate identification, characterization, antibiotics susceptibility test, polymerase chain reaction (PCR) amplification of the antibiotic resistance genes in both bacterial chromosome and plasmid, 16S-23S rRNA gene intergenic spacer (ITS) sequencing using the Sanger DNA sequencing, and phylogenetic analysis were used in this study. RESULTS: Only 13% of A. baumannii isolates were from COVID-19 patients. All isolates were multi-drug resistant due because of 24 resistance genes located in both the bacterial chromosome or the plasmid. blaTEM gene was detected in the isolates; however, aadB was not detected in the isolated bacteria. New carbapenemase genes were identified by Sanger sequencing and resistance genes were acquired by plasmids. CONCLUSIONS: The study identified metabolic differences in the isolates; although all the strains used the coumarate pathway to survive. Several resistance genes were present in the isolates' plasmids and chromosome. There were no strong biofilm producers. The role of the plasmid in A. baumannii resistance development was described based on the results.202439499748
170480.9998Exploring virulence characteristics of Klebsiella pneumoniae isolates recovered from a Greek hospital. The objective of this study was to characterize the virulence characteristics of a collection of Klebsiella pneumoniae isolates collected from different clinical sources. A collection of 60 non-repetitive K. pneumoniae isolates, was studied. In vitro, virulence was analyzed by testing the survival of bacteria in pooled human serum. Isolates were typed by MLST. The genomes of 23 K. pneumoniae isolates, representatives of different STs and virulence profiles, were completely sequenced using the Illumina platform. Of note, 26/60 of K. pneumoniae isolates were resistant to killing by complement. Serum-resistant isolates belonged to distinct STs. Analysis of WGS data with VFDB showed the presence of several virulence genes related various virulence functions. Specifically, serum-resistant isolates carried a higher number of ORFs, which were associated with serum resistance, compared to serum-sensitive isolates. Additionally, analysis of WGS data showed the presence of multiple plasmid replicons that could be involved with the spread and acquisition of resistance and virulence genes. In conclusion, analysis of virulence characteristics showed that an important percentage (31.6%) of K. pneumoniae isolates were in vitro virulent by exhibiting resistance to serum. Thus, the presence of several virulence factors, in combination with the presence of multidrug resistance, could challenge antimicrobial therapy of infections caused by such bacteria.202540415138
169390.9998Major enzymatic factors involved in bacterial penicillin resistance in Burkina Faso. Many clinical species of bacteria were isolated from biological samples such as urines, blood and wound in Saint Camille medical centre of Ouagadougou. Among the concerned species, the most important members were Escherichia coli and Klebsiella pneumoniae. These p-lactamases producing isolates were directly screened by PCR to identify the nature of the amplified genes responsible for penicillin destroying activity. Therefore specific TEM and SHV primers were used. The PCR products were sequenced. The sequencing results indicated that the parental forms bla(TEM-1) and bla(SHV-1) were the most common determinants of beta-lactamase found, respectively in Escherichia species and Klebsiella pneumoniae. The bacterial susceptibility analysis by MICs measurement clearly correlated the presence of concerned beta-lactamase determinants and their resistance patterns. This study is part of a set of investigations carried out by our laboratory to assess the beta-lactamase incidence in the failure of beta-lactam therapy. In particular, the purpose of this study was to determine the precise nature of beta-lactamase supporting the low susceptibility of host bacteria towards penicillins.200719069526
892100.9998Sequencing analysis of tigecycline resistance among tigecycline non-susceptible in three species of G-ve bacteria isolated from clinical specimens in Baghdad. BACKGROUND: Recent emergence of high-level tigecycline resistance is mediated by tet(X) genes in Gram-negative bacteria, which undoubtedly constitutes a serious threat for public health worldwide. This study aims to identify tigecycline non-susceptible isolates and detect the presence of genes that are responsible for tigecycline resistance among local isolates in Iraq for the first time. METHODS: Thirteen clinical isolates of Klebsiella pneumonia, Acinetobacter baumannii and Pseudomonas aeruginosa tigecycline non-susceptible were investigated from blood, sputum and burns specimens. The susceptibility of different antibiotics was tested by the VITEK-2 system. To detect tigecycline resistance genes, PCR was employed. RESULTS: Strains studied in this work were extremely drug-resistant and they were resistant to most antibiotic classes that were studied. The plasmid-encoded tet(X), tet(X1), tet(X2), tet(X3), tet(X4), tet(X5), tet(M) and tet(O) genes were not detected in the 13 isolates. The results showed that there is a clear presence of tet(A) and tet(B) genes in tigecycline non-susceptible isolates. All 13 (100%) tigecycline non-susceptible K. pneumoniae, A. baumannii and P. aeruginosa isolates harbored the tet(B) gene. In contrast, 4 (30.77%) tigecycline non-susceptible P. aeruginosa isolates harbored the tet(A) gene and there was no tigecycline non-susceptible A. baumannii isolate harboring the tet(A) gene (0%), but one (7.69%) tigecycline non-susceptible K. pneumoniae isolate harbored the tet(A) gene. A phylogenetic tree, which is based on the nucleotide sequences of the tet(A) gene, showed that the sequence of the local isolate was 87% similar to the nucleotide sequences for all the isolates used for comparison from GenBank and the local isolate displayed genetic diversity. CONCLUSIONS: According to this study, tet(B) and tet(A) play an important role in the appearance of tigecycline non-susceptible Gram-negative isolates.202236207501
2228110.9998Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry. BACKGROUND: At present, phenotypic growth inhibition techniques are used in routine diagnostic microbiology to determine antimicrobial resistance of bacteria. Molecular techniques such as PCR are often used for confirmation but are indirect as they detect particular resistance genes. A direct technique would be able to detect the proteins of the resistance mechanism itself. In the present study targeted high resolution mass spectrometry assay was developed for the simultaneous detection of KPC, OXA-48-like, NDM, and VIM carbapenemases. METHODS: Carbapenemase specific target peptides were defined by comparing available sequences in GenBank. Selected peptide sequences were validated using 62 Klebsiella pneumoniae and Escherichia coli isolates containing: 16 KPC, 21 OXA-48-like, 16 NDM, 13 VIM genes, and 21 carbapenemase negative isolates. RESULTS: For each carbapenemase, two candidate peptides were validated. Method validation was performed in a blinded manner for all 83 isolates. All carbapenemases were detected. The majority was detected by both target peptides. All target peptides were 100% specific in the tested isolates and no peptide carry-over was detected. CONCLUSION: The applied targeted bottom-up mass spectrometry technique is able to accurately detect the four most prevalent carbapenemases in a single analysis.201931849899
2040120.9998Multidrug-resistant bacteria as intestinal colonizers and evolution of intestinal colonization in healthy university students in Portugal. Multidrug-resistant bacteria have been increasingly described in healthcare institutions, however community resistance also seems to be emerging. Escherichia coli an intestinal commensal bacteria, is also a pathogen and represents an important intestinal reservoir of resistance. Our aim was the study of the intestinal colonization and of the persistence of antibiotic resistant intestinal bacteria in healthy university students of Porto, in the north of Portugal. Samples from 30 university students were collected and analysed. Two E. coli isolates were randomly obtained from each student and Gram-negative bacilli resistant to antibiotics were studied. In addition, we evaluated changes in the Gram-negative intestinal colonization of ten university students in a short period of time. Molecular characterization showed a high presence of bla (TEM) in commensal E. coli . Gram-negative bacteria with intrinsic and extrinsic resistance were isolated, namely Pseudomonas spp., Enterobacter spp. and Pantoea spp. We isolated three ESBL-producing E. coli from two students. These isolates showed bla (CTX-M) group 1 (n=1), bla (CTX-M) group 9 (n=2), bla (TEM) (n=2), bla (SHV) (n=1) and tetA (n=2) genes. Additionally, they showed specific virulence factors and conjugational transfer of antibiotic resistance and virulence genes. One Pseudomonas spp. isolate resistant to carbapenems was detected colonizing one student. Our results confirm that healthy young adults may be colonized with commensals showing clinically relevant antibiotic resistance mechanisms, creating a risk of silent spread of these bacteria in the community.202133997613
2082130.9997Rapid screening technique for class 1 integrons in Enterobacteriaceae and nonfermenting gram-negative bacteria and its use in molecular epidemiology. A screening technique for integrons in members of the family Enterobacteriaceae and nonfermenting gram-negative bacteria by real-time PCR is reported. A total of 226 isolates of gram-negative bacteria obtained from a variety of clinical specimens were screened for class 1 integrons by real-time PCR performed on a LightCycler instrument. This technique used a primer pair specific for a 300-bp conserved region at the 5' ends of class 1 integrons. The screening assay was evaluated by comparison with results obtained by the conventional, thermal-block PCR (long PCR) by using established conditions and primers for the detection of class 1 integrons, and the real-time PCR technique was thus shown to be both sensitive and specific. DNA from 50 of 226 (22%) isolates screened was identified as containing an integron by the screening PCR, and sequence data were obtained across the integron for 34 of 50 (68%) of these isolates. In an attempt to study the molecular epidemiology of antimicrobial resistance genes carried within integrons, a comparison of the types of gene cassettes carried by isolates from different patients was made. Adenyltransferase genes conferring resistance to streptomycin and spectinomycin were the predominant gene cassettes amplified in the study. Resistance to trimethoprim was also frequently found to be encoded within integrons. Furthermore, multiple bacterial isolates obtained from one patient over a 5-month period were all shown to carry an integron containing the same single adenyltransferase gene cassette, suggesting that these elements were relatively stable in this case.200111257011
2304140.9997Antimicrobial consumption and resistance in five Gram-negative bacterial species in a hospital from 2003 to 2011. BACKGROUND: The misuse of antimicrobial agents increases drug resistance in bacteria. METHODS: The correlation between antimicrobial agent consumption and related resistance in the Gram-negative bacteria Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Proteus mirabilis was analyzed during the period 2003-2011. RESULTS: Among these five bacteria, overall E. coli and K. pneumoniae were more commonly isolated from bloodstream than the other species. Regarding Enterobacteriaceae, E. coli and K. pneumoniae showed annual increases of resistance to the tested antimicrobial agents; conversely, P. mirabilis exhibited reduced resistance to cefuroxime, ceftriaxone and cefepime. In contrast to the relatively low antimicrobial resistance in P. aeruginosa, A. baumannii revealed high resistance, which was over 85% resistant rate to the tested antimicrobial agents and over 80% carbapenem resistance in 2011. E. coli, K. pneumoniae, and P. mirabilis differed in development of antimicrobial resistance after consumption of the antimicrobial agents. K. pneumoniae developed resistance to all antimicrobial groups, whereas resistance in P. mirabilis was not related to any antimicrobial consumption. P. aeruginosa developed resistance to β-lactam antimicrobials and aminoglycosides, whereas A. baumanii developed resistance to carbapenems after their use. CONCLUSION: The development of antimicrobial resistance was related to antimicrobial agents and bacterial species.201524863496
1696150.9997Assessment of the presence of Acinetobacter spp. resistant to β-lactams in commercial ready-to-eat salad samples. Acinetobacter baumannii is a well-known nosocomial infection causing agent. However, other Acinetobacter spp. have also been implicated in cases of human infection. Additionally, these bacteria are known for the development of antibiotic resistance thus making the treatment of the infections they cause, challenging. Due to their relevance in clinical setups less attention has been paid to their presence in foods, and its relation with infection/dissemination routes. In the current study commercial Ready-To-Eat (RTE) salads were analyzed seeking for antibiotic resistant Acinetobacter spp. A preliminary screening allowed us to recover Gram-negative bacteria resistant to β - lactams using cefotaxime, third generation cephalosporins, as the selective agent, and this was followed by identification with CHROMagar™ Acinetobacter and 16S rDNA sequencing. Finally, the isolates identified as Acinetobacter spp. were reanalyzed by PCR to determine the presence of nine potential Extended Spectrum β Lactamases (ESBL). Two commercial RTE salad brands were included in the study (2 batches per brand and 8 samples of each batch making a total of 32 independent samples), and compared against an organic lettuce. High concentrations of β - lactam, resistant bacteria were found in all the samples tested (5 log CFU/g). Additionally, 209 isolates were phenotypically characterized on CHROMagar Acinetobacter. Finally, PCR analysis identified the presence of different ESBL genes, being positive for blaACC, blaSHV, blaDHA and blaVEB; out of these, blaACC was the most prevalent. None of the isolates screened were positive for more than one gene. To conclude, it is important to highlight the fact that pathogenic species within the genus Acinetobacter spp., other than A. baumannii, have been identified bearing resistance genes not typically associated to these microorganisms highlight the importance of continuous surveillance.202438049272
1685160.9997Molecular epidemiology and mechanisms of carbapenem and colistin resistance in Klebsiella and other Enterobacterales from treated wastewater in Croatia. Among the most problematic bacteria with clinical relevance are the carbapenem-resistant Enterobacterales (CRE), as there are very limited options for their treatment. Treated wastewater can be a route for the release of these bacteria into the environment and the population. The aim of this study was to isolate CRE from treated wastewater from the Zagreb wastewater treatment plant and to determine their phenotypic and genomic characteristics. A total of 200 suspected CRE were isolated, 148 of which were confirmed as Enterobacterales by MALDI-TOF MS. The predominant species was Klebsiella spp. (n = 47), followed by Citrobacter spp. (n = 40) and Enterobacter cloacae complex (cplx.) (n = 35). All 148 isolates were carbapenemase producers with a multidrug-resistant phenotype. Using multi-locus sequence typing and whole-genome sequencing (WGS), 18 different sequence types were identified among these isolates, 14 of which were associated with human-associated clones. The virulence gene analysis of the sequenced Klebsiella isolates (n = 7) revealed their potential pathogenicity. PCR and WGS showed that the most frequent carbapenemase genes in K. pneumoniae were bla(OXA-48) and bla(NDM-1), which frequently occurred together, while bla(KPC-2) together with bla(NDM-1) was mainly detected in K. oxytoca, E. cloacae cplx. and Citrobacter spp. Colistin resistance was observed in 40% of Klebsiella and 57% of Enterobacter isolates. Underlying mechanisms identified by WGS include known and potentially novel intrinsic mechanisms (point mutations in the pmrA/B, phoP/Q, mgrB and crrB genes) and acquired mechanisms (mcr-4.3 gene). The mcr-4.3 gene was identified for the first time in K. pneumoniae and is probably located on the conjugative IncHI1B plasmid. In addition, WGS analysis of 13 isolates revealed various virulence genes and resistance genes to other clinically relevant antibiotics as well as different plasmids possibly associated with carbapenemase genes. Our study demonstrates the important role that treated municipal wastewater plays in harboring and spreading enterobacterial pathogens that are resistant to last-resort antibiotics.202438479059
1648170.9997Molecular characterization of the multi-drug resistant Myroides odoratimimus isolates: a whole genome sequence-based study to confirm carbapenem resistance. The bacteria belonging to the Myroides genus are opportunistic pathogens causing community or hospital-acquired infections that result in treatment failure due to antibiotic resistance. This study aimed to investigate molecular mechanisms of antibiotic resistance, clonal relatedness, and the biofilm forming capacity of the 51 multi-drug resistant Myroides odoratimimus. All isolates were screened for bla(KPC), bla(OXA), bla(VIM), bla(IMP), bla(MUS), bla(TUS), bla(NDM), and bla(B) genes by using PCR amplification. Whole genome sequencing (WGS) was applied on three randomly selected isolates for further investigation of antibiotic resistance mechanisms. Clonal relatedness was analyzed by Pulsed-field gel electrophoresis (PFGE) and the microtiter plate method was used to demonstrate biofilm formation. All isolates were positive for biofilm formation. PCR analysis resulted in a positive for only the bla(MUS-1) gene. WGS identified bla(MUS-1), erm(F), ere(D), tet(X), and sul2 genes in all strains tested. Moreover, the genomic analyses of three strains revealed that genomes contained a large number of virulence factors (VFs). PFGE yielded a clustering rate of 96%. High clonal relatedness, biofilm formation, and multi-drug resistance properties may lead to the predominance of these opportunistic pathogens in hospital environments and make them cause nosocomial infections.202438127105
2227180.9997Prophylactic application of antibiotics selects extended-spectrum β-lactamase and carbapenemases producing Gram-negative bacteria in the oral cavity. Prophylactic administration of broad-spectrum antibiotics in surgery can change the oral microbiome and induce colonization of oral cavity with Gram-negative bacteria including multidrug (MDR) or extensively drug resistant (XDR) organisms which can lead to lower respiratory tract infections. The aim of the study was to analyse the Gram-negative isolates obtained from oral cavity of the mechanically ventilated patients in ICUs, after prophylactic application of antibiotics and their resistance mechanisms and to compare them with the isolates obtained from tracheal aspirates from the same patients. The antibiotic susceptibility was determined by broth dilution method. PCR was applied to detect genes encoding β-lactamases. Marked diversity of Gram-negative bacteria and resistance mechanisms was found. High resistance rates and high rate of bla(CTX-M) and carbapenemase encoding genes (bla(VIM-1) , bla(OXA-48) ) were found among Klebsiella pneumoniae. Pseudomonas aeruginosa was found to harbour bla(VIM) and in one strain bla(PER-1) gene, whereas Acinetobacter baumannii produced OXA-23-like and OXA-24/40-like oxacillinases and was XDR in all except one case. All XDR isolates belong to international clonal lineage II (IC II). The main finding of the study is that the prophlylactic application of antibiotics in surgery intensive care units (ICUs) is associated with the colonization of oral cavity and lower respiratory tract with Gram-negative bacteria. The identity of Gram-negative bacteria in oral cavity reflected those found in endotracheal aspirates leading to conclusion that oral swab as non-invasive specimen can predict the colonization of lower respiratory tract with resistant Gram-negative organisms and the risk for development of ventilator-associated pneumonia.202133896011
1713190.9997Conjugative plasmidic AmpC detected in Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae human clinical isolates from Portugal. AmpC is a type of β-lactamase enzyme produced by bacteria; these enzymes are classified in Class C and Group 1, and these confer resistance to cephamycin. Enterobacterales producing AmpC are reported worldwide and have great clinical importance due to therapeutic restriction and epidemiological importance once the easy dissemination by plasmidic genes to other bacteria is a real threat. These genes are naturally found in some enterobacteria as Enterobacter cloacae, Morganella morganii, and Citrobacter freundii, but other species have demonstrated similar resistance phenotype of AmpC production. Genes carried in plasmids have been described in these species conferring resistance to cefoxitin and causing therapeutic failure in some bacterial infections. This work detected and described five clinical strains of Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae that presented plasmid ampC (pAmpC) isolated from the north of Portugal collected in 2009. AmpC production was confirmed by inhibition of the enzyme by cloxacillin and boronic acid in agar diffusion tests. Also, PCR (polymerase chain reaction) was performed for the detection of gene universal to AmpC, bla(ampC), and others to AmpC group: bla(ACC), bla(CIT), bla(CMY), bla(DHA), and bla(EBC). The conjugation in liquid medium for 24 h was realized to determine if gene is localized in chromosome or plasmid. The isolates and their conjugants showed phenotypic characteristics and bla(CMY) and bla(CIT) were detected by PCR corroborating the AmpC characteristics observed in these bacteria. Confirmation of transfer of plasmid containing genes encoding AmpC is of high epidemiological relevance to the hospital studied and demonstrated the importance of AmpC surveillance and studies in hospital and community environments in order to choose the appropriate therapy for bacterial infections.202032740783