aadB Gene Research Articles

Sporadic clone Escherichia coli ST615 as a vector and reservoir for dissemination of crucial antimicrobial resistance genes.

There is scarce information concerning the role of sporadic clones in the dissemination of antimicrobial resistance genes (ARGs) within the nosocomial niche. We confirmed that the clinical Escherichia coli M19736 ST615 strain, one of the first isolates of Latin America that harbors a plasmid with an mcr-1 gene, could receive crucial ARG by transformation and conjugation using as donors critical plasmids that harbor bla CTX-M-15, bla KPC-2, bla NDM-5, bla NDM-1, or aadB genes. Escherichia coli M19736 acquired bla CTX-M-15, bla KPC-2, bla NDM-5, bla NDM-1, and aadB genes, being only blaNDM-1 maintained at 100% on the 10th day of subculture. In addition, when the evolved MDR-E. coli M19736 acquired sequentially bla CTX-M-15 and bla NDM-1 genes, the maintenance pattern of the plasmids changed. In addition, when the evolved XDR-E. coli M19736 acquired in an ulterior step the paadB plasmid, a different pattern of the plasmid's maintenance was found. Interestingly, the evolved E. coli M19736 strains disseminated simultaneously the acquired conjugative plasmids in different combinations though selection was ceftazidime in all cases. Finally, we isolated and characterized the extracellular vesicles (EVs) from the native and evolved XDR-E. coli M19736 strains. Interestingly, EVs from the evolved XDR-E. coli M19736 harbored bla CTX-M-15 though the pDCAG1-CTX-M-15 was previously lost as shown by WGS and experiments, suggesting that EV could be a relevant reservoir of ARG for susceptible bacteria. These results evidenced the genetic plasticity of a sporadic clone of E. coli such as ST615 that could play a relevant transitional link in the clinical dynamics and evolution to multidrug/extensively/pandrug-resistant phenotypes of superbugs within the nosocomial niche by acting simultaneously as a vector and reservoir of multiple ARGs which later could be disseminated.

Aminoglycoside resistance genes in early members of the Acinetobacter baumannii ST78A (SMAL, Italian clone) reside in an IS26-bounded island in the chromosome.

The Acinetobacter baumannii isolate called SMAL, previously used to determine the structures of capsular polysaccharide and lipooligosaccharide, was recovered in Pavia, Italy in 2002 among the collection of aminoglycoside-resistant isolates designated as SMAL type. This type was later called the Italian clone, then ST78. ST78 isolates are now widely distributed. To establish the resistance gene complement and the location and structure of acquired resistance regions in early members of the Italian/ST78 clone. The draft genome of SMAL2002 was assembled from Illumina MiSeq reads. Contigs containing resistance genes were joined and located in the chromosome using PCR with custom primers. The resistance profile was determined using disc diffusion. SMAL2002 is an ST78A isolate and includes three aminoglycoside resistance genes, aadB (gentamicin, kanamycin, tobramycin) aphA1 (kanamycin, neomycin) and aac(6')-Ian (amikacin, kanamycin, tobramycin). The aadB gene cassette is incorporated at a secondary site in a relative of the aphA1-containing, IS26-bounded pseudo-compound transposon, PTn6020. The aac(6')-Ian gene is in an adjacent IS26-bounded structure that includes sul2 (sulphonamide) and floR (florfenicol) resistance genes. The two pseudo-compound transposons overlap and are in the chromosomal hutU gene flanked by an 8 bp target site duplication. Although aac(6')-Ian was not noticed previously, the same genes and structures were found in several available draft genomes of early ST78A isolates. This study highlights the importance of correlating resistance profiles with resistance gene content. The location of acquired resistance genes in the SMAL2002 chromosome represents the original location in the ST78A lineage of ST78.

Paratyphoid Salmonella serovars in chickens: molecular detection of virulence and antimicrobial resistance genes

Paratyphoid salmonellosis is a serious disease threatens the poultry industry worldwide, besides its public health hazard. The aims of this study were characterization of paratyphoid Salmonella spp. in chicken flocks of some Egyptian governorates, demonstration of the antimicrobial susceptibility of the isolated Salmonella spp., and detection of some virulence genes and antibiotic resistance genes using recent molecular techniques. A total of 238 organ samples were collected from 52 broiler, layer, and breeder chicken flocks, representing 9 Egyptian governorates. Conventional characterization of Salmonella isolates revealed a total isolation rate of 56.3% (134/238). Moreover, the isolation rates of Salmonella spp. were (49/79; 62%), (47/81; 58%), (10/18; 55.5%), (9/20; 45%), (2/6; 33.3%), (2/3; 66.7%), and (15/82; 53.6%) from liver, yolk sac, heart, spleen, caecum, ovary, and dead-in-shell embryos, respectively. A total of 32/238 (13.44%) isolates of Salmonella were found. Serological identification revealed presence of S. enteritidis (21.9%), S. kentucky (15.6%), S. typhimurium (12.5%), S. molade (12.5%), S. takoradi (9.4%), S. wingrove (6.3%), S. infantis (6.3%), S. tsevie (6.3), S. shangani (3.1%), S. bargny (3.1%), and S. papuana (3.1%). All Salmonella strains (32/32; 100%) were resistant to streptomycin, while almost all of them (31/32; 96.9%) were susceptible to meropenem. The amplification of 16S rRNA gene of Salmonella isolates using uniplex polymerase chain reaction (PCR) generated a specific Salmonella product of approximate 550 base pair. The multiplex PCR revealed presence of invA (100%), stn (65.6%), and sopB (40.6 %) virulence-associated genes as well as aadA1 (100%), blaTEM (59.4%), aadB (18.75%), and sul1 (28.1%) antibiotic resistance genes. In conclusion, virulent paratyphoid Salmonella spp. are circulating in the Egyptian flocks, causing economic loses. Additionally, they became resistant to the most commonly used field antibiotics. Therefore, regular molecular surveillance studies on the circulating Salmonella spp. and their resistance to the used antibiotics are of significant importance.

Mobile genetic elements carrying aminoglycoside resistance genes in Acinetobacter baumannii isolates belonging to global clone 2.

Aminoglycosides are used to treat infections caused by carbapenem-resistant Acinetobacter baumannii (CRAB) strains. However, resistance to aminoglycosides has increased remarkably in the last few years. Here, we aimed to determine the mobile genetic elements (MGEs) associated with resistance to aminoglycosides in the global clone 2 (GC2) A. baumannii. Among the 315 A. baumannii isolates, 97 isolates were identified as GC2, and 52 of GC2 isolates (53.6%) were resistant to all the aminoglycosides tested. The AbGRI3s carrying armA were detected in 88 GC2 isolates (90.7%), and of them, 17 isolates (19.3%) carried a new variant of AbGRI3 (AbGRI3ABI221). aphA6 was located in TnaphA6 of 30 isolates out of 55 aphA6-harboring isolates, and 20 isolates were found to harbor TnaphA6 on a RepAci6 plasmid. Tn6020 carrying aphA1b was detected in 51 isolates (52.5%), which was located within AbGRI2 resistance islands. The pRAY* carrying the aadB gene was detected in 43 isolates (44.3%), and no isolate was found to contain a class 1 integron harboring this gene. The GC2 A. baumannii isolates contained at least one MGE carrying the aminoglycoside resistance gene, located mostly either in the chromosome within AbGRIs or on the plasmids. Thus, it is likely that these MGEs play a role in the dissemination of aminoglycoside resistance genes in GC2 isolates from Iran.

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.

Effect of aromatic oils on the expression of some virulence-associated and antimicrobial resistance genes of Escherichia coli isolated from broilers.

Objectives:This study aimed to prove the effects of Escherichia coli isolates isolated from diseased broilers to form biofilms, describe their antimicrobial sensetivity, and determine the effect of allicin and cinnamon essential oils on the expression of some genes (fimH, int1, and luxS) through quantitative polymerase chain reaction (q-PCR).Materials and Methods:140 samples were obtained from diseased broilers in Beni-Suef Governorate, Egypt. These samples were examined by conventional bacteriology methods to detect the causative agent. The antimicrobial susceptibility of the isolated bacteria was assessed using the disc diffusion method, The ability of yeast extract-casamino acids Congo Red Agar to generate phenotypic biofilms was next tested. The presence of resistance and virulence genes in some multidrug resistant isolates was genotypically investigated. The antibacterial effects of allicin and cinnamon oil were evaluated against the growth of multidrug-resistant E. coli. Finally, q-PCR was utilized to assess changes in some genes’ expression.Results:Escherichia coli was isolated from 61 samples (43.6%). An antimicrobial susceptibility test revealed that multidrug-resistance (MDR) (could resist more than three antimicrobial classes) E. coli prevalence was 100%. 40.8% of isolates phenotypically produce biofilms. The detection of resistance and virulence genes by PCR showed that all tested isolates carry aadB, fimH, int1, qnrS, and luxS genes, while only 40% harbor iss genes. q-PCR showed that after treatment with allicin and cinnamon oils, gene expression went down.Conclusion:This investigation highlights that E. coli showed resistance against most of the tested antimicrobials; all isolates were MDR. The study showed wide dissemination of virulence and resistance genes among E. coli. Allicin and cinnamon oils have antimicrobial activities and could be used as alternatives to synthetic antimicrobial agents.

Evaluation of efflux pumps overexpression and β-lactamase genes among colistin resistant Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) is one of the major factors in severe opportunistic and hospital-acquired infections. Structural mechanisms such as resistance–nodulation–division (RND) efflux pumps and drug resistance genes such as extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) are factors causing drug resistance. Colistin is the prominent therapeutic strategy with an excellent inhibitory role for combating multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates. Wide usage of colistin and genetics interactions leads to the emergence of colistin-resistant (CoR) PA isolates which is a dangerous occurrence in XDR-PA isolates. The current study investigates the presence and expression of PA-related RND genes and harboring the antibiotic resistance genes such as ESBL and MBL genes in CoR-PA strains.70 clinical isolates were obtained from different clinical specimens during 2019–2020. The antimicrobial susceptibility testing was performed by the both Kirby-Bauer disk agar diffusion method and the minimum inhibitory concentration (MIC). Presence of blaTEM-1, blaSHV, blaCTX-M, blaIMP, blaVIM and blaNDM was measured in all CoR-PA isolates. Besides, screening of mcr-1, aadA, aadB and efflux pump genes (mexA, mexD, mexE and mexY) was conducted for CoR-PA isolates. To determine the expression level of efflux pump genes, Quantitative real-time PCR (qRT-PCR) reactions were performed.According to the standard antimicrobial susceptibility methods 39 isolates, (55.7%) and 31 isolates (44.3%) were considered as MDR-PA and XDR-PA strains, respectively. 12 isolates (17.1%) displayed resistance to colistin using MIC assessment. All CoR-PA isolates were negative for mcr-1 gene. The blaSHV (n = 11, 91.6%) and blaNDM (n = 7, 58.3%) were the most prevalent ESBL and MBL genes among CoR-PA, respectively. PCR results showed all CoR-PA isolates were positive for all 4 efflux pumps genes. On the other hand, overexpression of MexA, MexD, MexE and MexY genes was observed in 5 (41.6%), 5 (41.6%), 5 (41.6%) and 6 (50%) CoR-PA isolates by qRT-PCR, respectively. 9 (75%) CoR-PA isolates overexpressed at least one of the four efflux pumps and overexpression of altogether 4 efflux pumps was shown in 2 (16.6%) CoR-PA isolates. Overexpression of MexA, MexD, MexE and MexY genes was associated to the resistance toward imipenem, ciprofloxacin, ceftazidime and aminoglycosides, respectively.The current research demonstrated the co-existence of CoR-PA with ESBL and MBL genes besides overexpression of efflux pumps. The distribution of such isolates can lead to a burden on the healthcare system by the incidence of the most crucial situation named pan-drug resistant PA strains.

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.

Whole-genome sequencing reveals misidentification of a multidrug-resistant urine clinical isolate as Corynebacterium urealyticum

ObjectivesCorynebacterium urealyticum is a non-diphtherial urease-producing clinically relevant corynebacterium associated with urinary tract infections. Most clinical C. urealyticum isolates are multidrug-resistant. Whole-genome sequencing (WGS) of C. urealyticum VH4248 isolated from a clinical urine sample at Hospital Universitario Marqués de Valdecilla, Santander, Spain, was performed to predict its antimicrobial resistance profile and to compare it with results of culture-based phenotypic antimicrobial susceptibility testing. MethodsClassical microbiological methods and VITEK® MS were used for isolation and initial identification of strain VH4248. Draft genome sequencing was performed on an Illumina HiSeq 2500 platform, followed by assembly and annotation using SPAdes and RAST. Resistance genes were identified through PATRIC, the Pathosystems Resource Integration Center. Average nucleotide identity (ANI) analysis was done using the EDGAR and OrthoANI databases. Antimicrobial susceptibility was determined by Etest. ResultsIsolate VH4248 was initially identified asC. urealyticum. Its genome size is 2 261 231 bp with 64.4% GC content. Genome-based identification tools showed an average 93.7% similarity between VH4248 and C. urealyticum genomes deposited in public databases. Therefore, this isolate must be classified as Corynebacterium sp. The blaA and ermX genes as well as a class 1 integron including the aadB and sul1 genes are present in the VH4248 genome. This isolate is highly resistant to ampicillin, erythromycin and trimethoprim/sulfamethoxazole, and moderately resistant to gentamicin and kanamycin. ConclusionsWGS is a powerful tool forCorynebacterium identification to species level and for detection of unusual resistance determinants, such as that encoded by the class 1 integron in isolate VH4248.

First identification of blaNDM-1 carbapenemase in blaOXA-94-producing Acinetobacter baumannii ST85 in Spain

IntroductionNDM-1 carbapenemase is spreading rapidly all over the world, but this metallo-beta-lactamase has just been detected for the first time in an Acinetobacter baumannii (Ab) isolate of the ST85 clone in Spain. The aim of this study was to characterize a NDM-1-producing carbapenem-resistant A. baumannii (CR-Ab) isolate submitted to the Andalusian PIRASOA [infection prevention program] referral laboratory. MethodsCarbapenemases were detected by PCR and Sanger DNA sequencing. Whole genome sequencing was performed by NGS (Miseq, Illumina). Resistance genes were identified with RESfinder, while MLSTfinder was used for sequence typing (ST). The genetic location of blaNDM-1 was determined by nuclease S-1/PFGE/hybridization with specific probe. ResultsThe isolate was susceptible to amikacin and tigecycline and belonged to the ST85 clone. blaOXA-94 and blaNDM-1 were identified by PCR and Sanger DNA sequencing, respectively. The resistance genes aadB, blaADC-25, blaNDM-1, blaOXA-94, msr(E), mph(E) and floR,sul2 were identified by NGS. The chromosome of the isolate contained a defective Tn125 transposon with blaNDM-1 flanked by the insertion sequences ISAbA125 and ISAba14. The blaNDM-1 gene was only detected in the chromosomal DNA. ConclusionThis is the first time that blaNDM-1 has been detected and characterized in a blaOXA-94-producing CR-Ab isolate belonging to the ST85 clone in Spain.

Detection of some antibiotic resistant genes within Salmonella serovars isolated from broiler chickens

This study aimed to examine the sensitivity of different antibiotics against 10 Salmonella serovars isolated previously from broiler farms. After that, all resistant serovas were detected genetically for the presence of qnrA, blaTEM, tetA (A) and aadB resistant genes. Two S. Typhimurium, one S. Kentucky, one S. Newport, one S. Tamale, one S. Enteritidis, one S. Molade, one S. Takoradi, one S. Virchow and one S. Inganda were examined sensitivity against different antibiotics by disc diffusion method. The resistant serovars to quinolones, β lactams, gentamycin and doxycyclin were confirmed genetically. S. Kentucky, S. Tamale, S. Molade, S. Typhimurium and S. Takoradi were resistant to quinolones and carrying qnrAgene. S. Tamale, S. Inganda, S. Typhimurium, S. Newport, S. Molade, S. Typhimurium, S. Enteritidis, S. Takoradi and S. Virchow were resistant to β lactams and carrying blaTEM gene. S.Tamale, S. Inganda, S. Typhimurium, S. Newport, S. Molade, S. Enteritidis and S. Virchow were resistant to doxycyclin and carrying tetA (A) gene. S. Typhimurium, S. Enteritidis and S. Takoradi were resistant togentamycin and carrying aadB gene. The results showed that emerging of multidrug resistant strains to common antibiotics in the poultry field required newly discovered herbal compounds for control the disease and prevent the resistant phenomena.

Molecular characteristics, antimicrobial resistance profiles, and antibiotic resistance determinants in uropathogenic fluoroquinolone resistant-Escherichia coli isolates

The concern about multidrug resistance (MDR) in the bacterial pathogen is significant. The antimicrobial susceptibility testing performed in forty Escherichia coli isolates responsible for urinary tract infections (UTIs) to following antibiotics: ceftazidime, cefotaxime, ceftriaxone, aztreonam, co-trimoxazole, gentamicin, amikacin, nitrofurantoin, tobramycin, ciprofloxacin, and nalidixic acid performed by disk diffusion method. The presence of resistance sul1, aacC1, aadB, aphA6, qnrA, qnrB, qnrS, and ESBL genes were investigated in clinical isolates by PCR-based sequencing assay. Molecular fingerprinting of UTI isolates characterized by Pulsed-Field Gel Electrophoresis for clonal distribution and determined of predominant pulsotypes. The highest and least active agents against uropathogenic E. coli (UPEC) isolates were ciprofloxacin (100%), and tobramycin (7.5%). Fifty-percent (20/40) of the UPEC isolates were identified as ESBL-producers by phenotype. The most prevalent ESBL genes were related to bla TEM (100%; 20/20). The presence of sul1, aadB, qnrB, and qnrS genes detected in 67.5% (n = 27), 5% (n = 2), 47.5% (n = 19), and 2.5% (n = 1) of E. coli isolates, respectively. None of isolates harbor of aacC1, aphA6, and qnrA genes. The results of molecular characterizations confirmed that 31 pulsotypes found among 40 UPEC isolates. Of 31 pulsotypes, 26 types were unique and five types found in more than one isolate. Only two isolates (2/40) were not typeable. A high prevalence of plasmid-mediated quinolone resistance and ESBL genes in E. coli detected in urine samples. Data in this study suggested that these multiple pulsotypes patterns showed no clonal association between UPEC isolates.

Multidrug resistance genes are associated with a 42-kb island TGI1 carrying a complex class 1 integron in Trueperella pyogenes

ObjectivesThis research was conducted to ascertain the context and location of the antibiotic resistance determinants in a multiple antibiotic-resistant Trueperella pyogenes isolate TP1. MethodsThe genome was sequenced using PacBio RS II, and the filtered data were assembled using Canu. Sequences were annotated on the basis of those in GenBank, and the genomic island (GI) of the TP1 was predicted by IslandPath-DIMOB. ResultsTP1 as a multiple antibiotic-resistant isolate was recovered at Jilin Province (China) in 2017 from a dairy cow with pneumonia. TP1 exhibited resistance to aminoglycosides (gentamicin and amikacin), macrolides (erythromycin), lincosamides (clindamycin), sulfonamides (sulfamonomethoxine), tetracyclines (tetracycline and doxycycline) and chloramphenicols (chloramphenicol and florfenicol). An antibiotic resistance gene clustered together with the aadB, aadA1, cmlA5 and cmlA6 resistance genes located on a 7-kilobase (kb) multidrug-resistant (MDR) region, constituting a complex class 1 integron. The MDR region was located at one end of a 42-kb GI, and IS6100Δ1 mediated a genetic rearrangement with the complex class 1 integron-like SGI1 and formed a composite transposon. Furthermore, the tetW gene was located outside the four GIs consistent with tetracycline and doxycycline resistance. The ermD gene positioned in the front end of the 42-kb GI played an important role in mediating acquired erythromycin and clindamycin resistance. ConclusionsMultiple resistance genes are located in a complex class 1 integron within a 42-kb T. pyogenes genomic island (TGI1), leading to TP1 multiple drug resistance. In comparison with SG1 families, TGI1 possesses versatile gene distribution and specific gene context for it upstream and downstream, and it represents a new lineage of genomic resistance islands.

Resistance and Virulence Features in Carbapenem-resistant Acinetobacter baumannii Community Acquired and Nosocomial Isolates in Romania

We aimed to identify the virulence and antimicrobial resistance features in Carbapenem Resistant Acinetobacter baumannii (CRAB) strains isolated from hospital settings and compare them with those isolated in the same period of time from community acquired (CA) infections in Bucharest, south of Romania. A total number of 93 A. baumannii strains were isolated in majority from hospitalized patients and from CA infections. The resistance and virulence mechanisms of the strains were characterized by phenotypic and genotypic methods. The antibiotic resistance profiles in H and CA A. baumannii isolates revealed high percentages of carbapenem-resistance in both H and CA isolates. The ciprofloxacin resistance was found very closed in both types of isolates (84%/83.33%). CRAB H and CA isolates revealed the intrinsec carbapenemase OXA-51and the acquired carbapenemases OXA-23, OXA-24, IMP,and VIM-2. The blaOXA-23 gene was identified in different plasmid types (GR2-Aci1, GR6-pACICU2). rep135040, p3S18 and Aci6 in H A. baumannii isolates. The most frequently expressed virulence factor was lipase and DN-ase. OXA-51-like alleles corresponding to the two main sequence groups were identified as blaOXA66 (63.63% of the isolates) and respectively, blaOXA-69 (38.39%) and revealed the corresponding type of ompAand csuE sequence grouping. AphA6 (24%/16.6%), AphA1 (16%/16.6%) and aadB (9.3%/5.5%) genes were responsible for aminoglycosides resistance. Our survey revealed a high drug resistance in A. baumannii isolates. Different plasmid groups containing CRAB isolates may facilitate the blaOXA23 dissemination.

Resistance and Virulence Features in Carbapenem-resistant Acinetobacter baumannii Community Acquired and Nosocomial Isolates in Romania

We aimed to identify the virulence and antimicrobial resistance features in Carbapenem Resistant Acinetobacter baumannii (CRAB) strains isolated from hospital settings and compare them with those isolated in the same period of time from community acquired (CA) infections in Bucharest, south of Romania. A total number of 93 A. baumannii strains were isolated in majority from hospitalized patients and from CA infections. The resistance and virulence mechanisms of the strains were characterized by phenotypic and genotypic methods. The antibiotic resistance profiles in H and CA A. baumannii isolates revealed high percentages of carbapenem-resistance in both H and CA isolates. The ciprofloxacin resistance was found very closed in both types of isolates (84%/83.33%). CRAB H and CA isolates revealed the intrinsec carbapenemase OXA-51and the acquired carbapenemases OXA-23, OXA-24, IMP,and VIM-2. The blaOXA-23 gene was identified in different plasmid types (GR2-Aci1, GR6-pACICU2). rep135040, p3S18 and Aci6 in H A. baumannii isolates. The most frequently expressed virulence factor was lipase and DN-ase. OXA-51-like alleles corresponding to the two main sequence groups were identified as blaOXA66 (63.63% of the isolates) and respectively, blaOXA-69 (38.39%) and revealed the corresponding type of ompAand csuE sequence grouping. AphA6 (24%/16.6%), AphA1 (16%/16.6%) and aadB (9.3%/5.5%) genes were responsible for aminoglycosides resistance. Our survey revealed a high drug resistance in A. baumannii isolates. Different plasmid groups containing CRAB isolates may facilitate the blaOXA23 dissemination. Keywords: carbapenem resistance, virulence, community acquired, nosocomial infections

First identification of blaNDM-1 carbapenemase in blaOXA-94-producing Acinetobacter baumannii ST85 in Spain

IntroductionNDM-1 carbapenemase is spreading rapidly all over the world, but this metallo-beta-lactamase has just been detected for the first time in an Acinetobacter baumannii (Ab) isolate of the ST85 clone in Spain. The aim of this study was to characterize a NDM-1-producing carbapenem-resistant A. baumannii (CR-Ab) isolate submitted to the Andalusian PIRASOA [infection prevention program] referral laboratory. MethodsCarbapenemases were detected by PCR and Sanger DNA sequencing. Whole genome sequencing was performed by NGS (Miseq, Illumina). Resistance genes were identified with RESfinder, while MLSTfinder was used for sequence typing (ST). The genetic location of blaNDM-1 was determined by nuclease S-1/PFGE/hybridization with specific probe. ResultsThe isolate was susceptible to amikacin and tigecycline and belonged to the ST85 clone. blaOXA-94 and blaNDM-1 were identified by PCR and Sanger DNA sequencing, respectively. The resistance genes aadB, blaADC-25, blaNDM-1, blaOXA-94, msr(E), mph(E) and floR,sul2 were identified by NGS. The chromosome of the isolate contained a defective Tn125 transposon with blaNDM-1 flanked by the insertion sequences ISAbA125 and ISAba14. The blaNDM-1 gene was only detected in the chromosomal DNA. ConclusionThis is the first time that blaNDM-1 has been detected and characterized in a blaOXA-94-producing CR-Ab isolate belonging to the ST85 clone in Spain.

Class 1 Integron, Sulfonamide and Florfenicol Resistance Genes in Bacteria from Three Unsanitary Landfills, Ibadan, Nigeria

Objective: Municipal solid waste (MSW) landfills recently emerged as potential reservoirs of antibiotic resistance. However, little is known about the potentials of unsanitary landfill sites which are common in developing countries as reservoir of antibiotic resistance. In this study, we investigated the antibiotic resistance pattern and presence of selected resistance genes in bacteria from three MSW landfills in Ibadan, southwestern Nigeria. Methodology: Fifty six antibiotic- and metal-resistant bacteria isolated from leachate and leachate-contaminated surface and groundwater collected from three MSW landfills in Ibadan, Southwestern Nigeria, were searched for sulfonamides, florfenicol and beta-lactams resistance genes and integrons by PCR. Results: None of the 27 β-lactamase genes tested was detected in the isolates. sul1 , sul2 and floR were however detected in 12 (21.4%), 2 (3.6%) and 12 (21.4%) bacteria species identified as Alcaligenes faecalis , Advenella kashmirensis , Brevundimonas spp. and Ralstonia pickettii . The 12 bacteria carrying sul1 / floR gene combination were also positive for class 1 integron. Analysis of the gene cassettes in the variable regions of the class 1 integrons (>1.5kb) of the 12 intl1-positive isolates revealed the presence of aadB and unknown genes. The deduced amino acid sequence of the unknown portion of the gene cassettes shared 42-65% identity with sequences of Class D β-lactamase and OXA-2-like protein in the GenBank. Conclusions: Findings of this study suggest that the landfill ecosystem is a potential site for the evolution of novel resistance genes and are hence important reservoir of antibiotic resistant bacteria. J Microbiol Infect Dis 2019; 9(1): 34-42.

Antibacterial Activity of Gold Nanoparticles Conjugated by Aminoglycosides Against A. baumannii Isolates from Burn Patients.

Today, resistance to multiple classes of antibiotics, and notably to the β-lactam and aminoglycosides in A. baumannii is becoming a great problem and it necessitates to make a new approach to combat with multidrug-resistant (MDR), extensive drug-resistance (XDR) or Pandrug-resistant (PDR) isolates. In this case, a new strategy and ways should be designed and introduced against such infections. Therefore the aim of the present study was the evaluation of antibacterial activity of nanoconjugate gentamicin and amikacin with gold against clinical isolates of A. baumannii that were collected from burn wound infection. There are some patents of gold nanoparticles that are conjugated with antibiotics (WO2017161296A1, US20090181101A1). Eighteen A. baumannii were collected from burn wound infections. For confirmation and detection of aminoglycoside-resistant genes, PCR was carried out. Gold nanoparticles and nanoconjugates were prepared according to the protocol. For the evaluation of the nanoconjugate, Dynamic light cattering, Transmission electron microscopy and FTIR Analysis were carried out. Then, the antibacterial activity of nanoconjugates was conducted by using micro broth dilution method. Prevalence of aminoglycoside-resistant genes was aacC1, aphA6, aadA1, aadB genes 55.5%, 22.2%, 38.8% and 22.2% respectively. Synthesis of bare nanoconjugates resulted in nanoparticle in a size of 10 nm. Amikacin bound to Gnps showed excellent antibacterial activity (94.5%) and just one isolate showed intermediate resistance. Also, gentamicin bound to Gnps had a good antimicrobial effect (50%) in contrast to gentamicin alone. Our study showed that a combination of amikacin and gentamicin with Gnps has a significant antibacterial efficiency against clinical isolates of A. baumannii. Gnps can be used as extraordinary molecular carriers for targeting, and delivery of the antibiotic molecules to the specific infection.

Frozen White-Leg Shrimp (Litopenaeus vannamei) in Korean Markets as a Source of Aeromonas spp. Harboring Antibiotic and Heavy Metal Resistance Genes.

As the most consumed shrimp variety, white-leg shrimp (Litopenaeus vannamei) owns a high market demand in Korea. This study sought to screen the frozen white-leg shrimp for Aeromonas spp. harboring antimicrobial and heavy metal resistance characteristics. A total of 44 Aeromonas spp. strains were isolated and tested for antibiotic susceptibility and heavy metal tolerance followed by PCR-based detection of resistance genes and integrons. It was observed that resistance to ampicillin and oxacillin was 100% among isolates. Besides, 95%, 89%, 86%, 80%, 66%, and 43% of the isolates were resistant to nalidixic acid, tetracycline, cephalothin, streptomycin, trimethoprim-sulfamethoxazole, and imipenem, respectively, and less resistance to other antibiotics was also observed. Cr resistance was the highest (91%) among five heavy metals tested, whereas 57%, 32%, 20%, and 9% of the isolates were tolerant to Cu, Pb, Cd, and Hg, respectively. The PCR assays implied the presence of qnrB, qnrS, tetA, tetE, aac(6')-Ib, and aphAI-IAB, and intI1 genes among 80%, 77%, 18%, 30%, 9%, 0.25%, and 82% of the isolates, respectively. There were 35 (80%) integron 1-positive isolates harboring qacE2, dfrA1, orfC, orfD, aadB, catB3, oxa-10, and aadA1 genes in varying combinations. In addition, heavy metal resistance genes, CopA, merA, and CzcA were positive in 9%, 7%, and 27% of the isolates, respectively. According to these outcomes, the frozen white-leg shrimp in Korean markets can be suggested as a source of multidrug and heavy metal-resistant Aeromonas spp. that carries genetic determinants.

Insertional inactivation of oprD in carbapenem-resistant Pseudomonas aeruginosa strains isolated from burn patients in Tehran, Iran.

In this study, we report the insertion sequence ISPpu21 in the oprD porin gene of carbapenem-resistant Pseudomonas aeruginosa isolates from burn patients in Tehran, Iran. Antibiotic susceptibility tests for P. aeruginosa isolates were determined. Production of metallo-β-lactamases (MBLs) and carbapenemase was evaluated and the β-lactamase-encoding and aminoglycoside-modifying enzyme genes were investigated by PCR and sequencing methods. The mRNA transcription level of oprD and mex efflux pump genes were evaluated by real-time PCR. The outer membrane protein profile was determined by SDS–PAGE. The genetic relationship between the P. aeruginosa isolates was assessed by random amplified polymorphic DNA PCR. In all, 10.52% (10/95) of clinical isolates of P. aeruginosa harboured the ISPpu21 insertion element in the oprD gene. The extended-spectrum β-lactamase-encoding gene in ISPpu21-carrying isolates was blaTEM. PCR assays targeting MBL and carbapenemase-encoding genes were also negative in all ten isolates. The rmtA, aadA, aadB and armA genes were positive in all ISPpu21 harbouring isolates. The relative expression levels of the mexX, mexB, mexT and mexD genes in ten isolates ranged from 0.1- to 1.4-fold, 1.1- to 3.68-fold, 0.3- to 8.22-fold and 1.7- to 35.17-fold, respectively. The relative expression levels of the oprD in ten isolates ranged from 0.57- to 35.01-fold, which was much higher than those in the control strain P. aeruginosa PAO1. Evaluation of the outer membrane protein by SDS–PAGE suggested that oprD was produced at very low levels by all isolates. Using random amplified polymorphic DNA PCR genotyping, eight of the ten isolates containing ISPpu21 were shown to be clonally related. The present study describes a novel molecular mechanism, ISPpu21 insertion of the oprD gene, associated with carbapenem resistance in clinical P. aeruginosa isolates.