Resistance Genes In Clinical Isolates Research Articles

Examining the Frequency of Carbapenem Resistance Genes and Its Relationship with Different Classes of Integrons Including Classes I and II in Pseudomonas aeruginosa Isolates of Burn Patients

Background: Pseudomonas aeruginosa is one of the most important opportunistic pathogens causing hospital infections. Objectives: This study aimed to investigate the abundance of integrons and the pattern of resistance to carbapenems (metallo-β-lactamases including Spm, Imp, and Vim) and its relationship with the presence of integrons classes I and II in P. aeruginosa isolates. Methods: This study was conducted on 73 samples of P. aeruginosa isolated from burn wounds of patients admitted to the burn center of Velayat Hospital, Rasht, Iran. To confirm the phenotype of P. aeruginosa, Gram staining and diagnostic biochemical tests, including oxidation-fermentation (OF), pigment production, citrate utilization, catalase activity, oxidase test, and growth at 42°C, were used. After identification and confirmation, molecular diagnosis was conducted to identify strains producing genes classes I and II using the polymerase chain reaction (PCR) method. Results: In this research, the frequency of carbapenem resistance genes in clinical isolates of P. aeruginosa was found to be zero, 13.7%, and 21.9% for blaSpm, blaImp, and blaVim genes, respectively. Integrons of classes I and II were present in 53.4% of isolates of class I and 17.8% of isolates of class II. Also, 4.1% of the total integrin-positive isolates had both integron classes. Conclusions: There was a statistically significant relationship between class I integron and the blaImp gene. Importantly, the mechanisms of other integrons play a role in the development of resistance and the presence of these genes involved in this project.

Frequency of Aminoglycoside Resistance Genes in Clinical Isolates of Pseudomonas aeruginosa from Sistan, Iran

Frequency of Aminoglycoside Resistance Genes in Clinical Isolates of Pseudomonas aeruginosa from Sistan, Iran

Prevalence of Aminoglycoside Resistance Genes in Clinical Isolates of Pseudomonas aeruginosa from Taif, Saudi Arabia-An Emergence Indicative Study.

Hospital-acquired infections caused by P. aeruginosa contribute to global distress because of the elevated rates of microbial antibiotic resistance. Aminoglycosides are antipseudomonal agents that are effectively and frequently utilized to eradicate this infection. This current study is a retrospective study investigating plasmid-mediated aminoglycoside resistance by focusing on the prevalence of the genes encoding aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methylase among P. aeruginosa clinical isolates from Taif, Saudi Arabia. A hundred clinical isolates of P. aeruginosa were collected. The isolates were identified from February 2021 to February 2022. Antibiotic susceptibility testing and MICs were determined using (DD) and (BM-MIC) testing, respectively. AMEs and 16S rRNA methylase variants in bacterial isolates were amplified via PCR for genetic detection. A relatively high multiple antibiotic resistance rate corresponding to 10-32% was reported. Eighteen percent of P. aeruginosa isolates were gentamicin-amikacin-tobramycin resistant according to the MIC levels. The aminoglycoside-resistant strains were additionally identified via GyrA gene sequencing. The phylogenic relatedness dendrogram of the sequenced GyrA genes was performed using a neighbor-joining method via MEGAX software version 10.2.6. The most prevalent AME encoding gene was aac(6')-Ib, observed in 94.4% of resistant isolates, while a resistance gene cocktail of [aac(6')-Ib and ant(3″)-I] was a highly frequent combination (27.8%). This study updated the knowledge about aminoglycoside resistance mechanisms in P. aeruginosa, which constitutes an urgent need, especially after the COVID-19 crisis, which was associated with increased antimicrobial use and resistance rates.

Detection of Plasmid-Mediated Colistin Resistance Genes in Clinical Isolates of Klebsiella pneumoniae and Escherichia coli from Some Hospitals in Khartoum

Detection of Plasmid-Mediated Colistin Resistance Genes in Clinical Isolates of Klebsiella pneumoniae and Escherichia coli from Some Hospitals in Khartoum

Relationship between Biofilm and the Presence of Drug Resistance Genes in Clinical Isolates of Klebsiella Pneumoniae in Qom Hospitals

Background & Objectives: Klebsiella pneumoniae is a significant cause of nosocomial infections. This bacterium survives in difficult conditions by forming biofilms in hospital equipment and causes severe infections. On the other hand, the emergence and spread of carbapenem resistance among bacteria and biofilm production is a current health concern. There are controversial findings about the relevance of this issue. This study aimed to evaluate the relationship between biofilm formation and carbapenem resistance among clinical isolates.
 Materials & Methods: A total of 160 isolates of Klebsiella pneumoniae were collected. Molecular methods were used to detect resistance genes. Subsequently, the ability to produce biofilms in isolates with resistance genes was assessed. Finally, the correlation of biofilm formation among resistant isolates was calculated using χ2 test.
 Results: 79 imipenem-resistant isolates were obtained. 46 isolates (66.66%) containing VIM gene, 36 isolates (52.17%) containing OXA-48 gene, five isolates (7.24%) containing NDM gene, Six isolates (8.69%) containing gene IMP and five isolates (7.24%) also had KPC gene. The results showed a significant correlation between the ability to form biofilms and the presence of carbapenem-resistant genes.
 Conclusion: Increased carbapenem resistance in Klebsiella pneumoniae isolates and its association with biofilm formation is severe warning for basic measures to combat this phenomenon.

A Review of Global Epidemiology and Antibiotic Resistance of Staphylococcus Aureus

Background: Staphylococcus aureus is gram-positive cocci, which causes multiple complications such as bone, skin, joint and soft tissue infections. Penicillin was the first antibiotic that showed resistance to S. aureus; however, following penicillin, S. aureus became resistant to Methicillin and became a problem in many parts of the world. Worldwide epidemiology of S. aureus varies by country. In Austria, CA-MRSA accounts for 10% of all MRSA isolates. Many clones of S. aureus were discovered in Asian and as well as in European countries. Many patients in Southeast Asia are infected with various S. aureus variants. Aim: The aim of this study was to review epidemiology and antibiotic resistance of S. aureus in different countries. Results: Previous findings revealed that Panton-Valentine leukeocidin (PVL) genes were present in S. aureus strains. Furthermore, most widely accepted antibiotic resistance genes in clinical isolates of S. aureus strains are mecA. S. aureus spread throughout the hospital and into the community as well. They differ from one another in some ways, such as PVL genes and staphylococcal Chromosomal Cassette Mec (SCCmec) subtyping. Conclusion: It is concluded based on previously published research as well as review articles that different clones are of S. aureus are present in different regions of the world which further shows similar characteristics in some regions while totally different from the clones found in other countries. S. aureus infection can be treated by combining some antibiotics such as Vancomycin with Rifampin and Gentamicin. It can also be avoided by practicing good hand hygiene and avoiding intravenous drugs, which can cause a variety of infections. Keywords: Staphylococcus aureus, MRSA, CA-MRSA, HA-MRSA, Resistance

Molecular characterization of virulence (lmb, bca and rib) and macrolid resistance genes (ermB, ermTR and mefA) in clinical isolates of Streptococcus agalactiae

The aim of the study was to molecularly identify virulence and macrolide resistance genes in clinical isolates of Streptococcus agalactiae (GBS), recovered in 2019 from vaginal discharge (n=9) and urine (n=22), from two health facilities in Lima. Identification and antimicrobial susceptibility were determined by the Vitek® 2 automated system, identification was confirmed phenotypically; macrolide resistance was determined by the D-test method. Identification of virulence genes (lmb, bca and rib) and macrolide resistance genes (ermB, ermTR and mefA) was carried out by polymerase chain reaction (PCR). The predominant macrolide resistance phenotype and genotype were cMLSb (12/31) and ermB (11/31); the most frequent virulence gene was lmb (23/31). All were sensitive to penicillin, ampicillin and vancomycin. These findings show the need to implement molecular epidemiology studies that allow adequate knowledge and follow-up of GBS in Peru.

Macrolides mediate transcriptional activation of the msr(E)-mph(E) operon through histone-like nucleoid-structuring protein (HNS) and cAMP receptor protein (CRP).

The msr(E)-mph(E) operon exists widely in diverse species of bacteria and msr(E) and mph(E) genes confer high resistance to macrolides. We aimed to explore whether macrolides regulate the transcription of the operon. Antibiotic resistance genes in clinical isolates of Klebsiella pneumoniae were analysed by WGS. The transcription of the msr(E)-mph(E) operon was investigated by quantitative PCR. Construction of enhanced green fluorescent protein (eGFP) reporter plasmids, gene knockout and complementation experiments were used to further explore the induction mechanism of macrolides for the operon. Sequence analysis was finally used to investigate whether the operon exists widely in diverse species of bacteria. We originally found that the treatment of a pandrug-resistant isolate of K. pneumoniae (KP1517) with macrolides obviously up-regulated the msr(E)-mph(E) operon, which was further confirmed in another nine clinical isolates of K. pneumoniae. The induction mechanism of macrolides for the operon was partly elucidated. Macrolides could activate the operon promoter, and the J10/J35 regions (J10: 5'-AGTTATCAT-3'; J35: 5'-TTGTCT-3') of the promoter were determined. Histone-like nucleoid-structuring protein (HNS) and cAMP receptor protein (CRP) were involved in the erythromycin-mediated activation of the operon promoter. The 476 strains of bacteria carrying the msr(E)-mph(E) operon currently in the NCBI database are mainly Acinetobacter baumannii (158; 33%), K. pneumoniae (95; 20%), Escherichia coli (26; 5%) and Proteus mirabilis (25; 5%). They were mainly isolated from human clinical samples (287; 60%) and had a wide geographical distribution. Macrolides could activate transcription of the msr(E)-mph(E) operon through HNS and CRP in K. pneumoniae and E. coli, and this might occur in diverse species of bacteria.

Co-infection With Chromosomally-Located bla CTX-M-14 and Plasmid-Encoding bla CTX-M-15 in Pathogenic Escherichia coli in the Republic of Korea.

The emergence of third-generation cephalosporin resistance in Escherichia coli is increasing at an alarming rate in many countries. Thus, the aim of this study was to analyze co-infecting blaCTX-M-producing pathogenic E. coli isolates linked to three school outbreaks. Among 66 E. coli isolates, 44 were identified as ETEC O25, an ETEC isolate serotype was O2, and the other 21 were confirmed as EAEC O44. Interestingly, six patients were co-infected with EAEC O44 and ETEC O25. For these isolates, molecular analysis [antibiotic susceptibility testing, identification of the β-lactamase gene, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE)] was performed for further characterization. In addition, the transmission capacity of blaCTX-M genes was examined by conjugation experiments. Whole-genome sequencing (WGS) was performed on representative EAEC O44 and ETEC O25 isolates associated with co-infection and single-infection. All isolates were resistant to cefotaxime and ceftriaxone. All EAEC isolates carried the blaCTX-M-14 gene and all ETEC isolates the blaCTX-M-15 gene, as detected by multiplex PCR and sequencing analysis. Sequence type and PFGE results indicated three different patterns depending on the O serotype. WGS results of representative isolates revealed that the ETEC O25 strains harbored blaCTX-M-15 located on IncK plasmids associated with the ΔblaTEM-blaCTX-M-15-orf477 transposon. The representative EAEC O44 isolates carried blaCTX-M-14 on the chromosome, which was surrounded by the ISEcp1-blaCTX-M-14-IS903 transposon. To the best of our knowledge, this is the first report of co-infection with chromosomally located blaCTX-M-14 and plasmid-encoding blaCTX-M-15 in pathogenic E. coli. Our findings indicate that resistance genes in clinical isolates can spread through concurrent combinations of chromosomes and plasmids.

Distribution of aminoglycoside resistance genes in clinical isolates of Pseudomonas aeruginosa in north of Iran

BackgroundP. aeruginosa is a nosocomial pathogen of global concern due to its hospital-acquired infections and antibacterial resistance. Aminoglycoside is a common treatment alternative for infections caused by this bacterium. This study investigated the distribution of aminoglycoside modifying enzymes (AMEs) and 16S rRNA methylase in P. aeroginosa isolated from Guilan province, Iran. Materials and methodsDuring the eight months of study, 122 isolates of P. aeruginosa were collected from clinical specimens. Three common aminoglycoside susceptibility tests were performed and minimum inhibitory concentration (MIC) was determined using disk diffusion method and broth-microdilution testing. AMEs and 16S rRNA methylase variants in test bacteria were detected by PCR assay. ResultsIn this study, 51 (41.8%) P. aeruginosa isolates were pan aminoglycoside resistant and according to MIC level, 40 (32.8%) isolates showed high levels of resistance. The most prevalent aminoglycoside resistance genes in test isolates were ant(2″)-Ia and aac(6′)-Ib, which were detected in 85% and 71.2% of the resistant isolates, respectively. rmtC and ant(4′)Ia were not detected in any isolates. All high-level aminoglycoside-resistant isolates, multiple modifying enzyme genes, were investigated with 23 different gene combinations comprised of 2–5 AMEs and 16S rRNA methyltransferases. ConclusionHigh-level carriage of different aminoglycoside resistance genes in P. aeruginosa isolates indicates the reduced effectiveness of this class of antibacterials, hence the necessity of producing effective antibacterial agents stable in the presence of these resistance genes.

Acquired fluoroquinolone resistance genes in corneal isolates of Pseudomonas aeruginosa.

Fluoroquinolones are widely used as an empirical therapy for pseudomonal ocular infections. Based on increasing reports on acquired fluoroquinolone resistance genes in clinical isolates of Pseudomonas aeruginosa, we investigated 33 strains of P. aeruginosa isolated from the cornea of microbial keratitis patients in India and Australia between 1992 and 2018 to understand the prevalence of acquired fluoroquinolone resistance genes in ocular isolates and to assess whether the possession of those genes was associated with fluoroquinolone susceptibility. Fourteen out of 33 strains were resistant to at least one fluoroquinolone. We obtained the whole genome sequence of 33 isolates using Illumina MiSeq platform and investigated the prevalence of two fluoroquinolone resistance genes crpP and qnrVC1. To examine the associated mobile genetic elements of qnrVC1 positive strains, we obtained long read sequences using Oxford Nanopore MinION and performed hybrid assembly to combine long reads with Illumina short sequence reads. We further assessed mutations in quinolone resistance determining regions (QRDRs) and antibiotic susceptibilities to ciprofloxacin, levofloxacin and moxifloxacin to examine the association between resistance genes and phenotype. Twenty strains possessed crpP in genetic islands characterised by possession of integrative conjugative elements. The qnrVC1 gene was carried by four isolates on class I integrons and Tn3 transposons along with aminoglycoside and beta-lactam resistance genes. We did not observe any evidence of plasmids carrying fluoroquinolone resistance genes. Resistance to fluoroquinolones was observed in those strains which possessed crpP, qnrVC1 and that had QRDRs mutations. The presence of crpP on its own was not associated with increased resistance to fluoroquinolones.

Genomic profiling of antimicrobial resistance genes in clinical isolates of Salmonella Typhi from patients infected with Typhoid fever in India

The development of multidrug resistance in Salmonella enterica serovar Typhi currently forms a major roadblock for the treatment of enteric fever. This poses a major health problem in endemic regions and extends to travellers returning from developing countries. The appearance of fluoroquinolone non-susceptible strains has resulted in use of ceftriaxone as drug of choice with azithromycin being recommended for uncomplicated cases of typhoid fever. A recent sporadic instance of decreased susceptibility to the latest drug regime has necessitated a detailed analysis of antimicrobial resistance genes and possible relationships with their phenotypes to facilitate selection of future treatment regimes. Whole genome sequencing (WGS) was conducted for 133 clinical isolates from typhoid patients. Sequence output files were processed for pan-genome analysis and prediction of antimicrobial resistance genes. The WGS analyses disclosed the existence of fluoroquinolone resistance conferring mutations in gyrA, gyrB, parC and parE genes of all strains. Acquired resistance determining mechanisms observed included catA1 genes for chloramphenicol resistance, dfrA7, dfrA15, sul1 and sul2 for trimethoprim-sulfamethoxazole and blaTEM-116/blaTEM-1B genes for amoxicillin. No resistance determinants were found for ceftriaxone and cefixime. The genotypes were further correlated with their respective phenotypes for chloramphenicol, ampicillin, co-trimoxazole, ciprofloxacin and ceftriaxone. A high correlation was observed between genotypes and phenotypes in isolates of S. Typhi. The pan-genome analysis revealed that core genes were enriched in metabolic functions and accessory genes were majorly implicated in pathogenesis and antimicrobial resistance. The pan-genome of S. Typhi appears to be closed (Bpan = 0.09) as analysed by Heap’s law. Simpson’s diversity index of 0.51 showed a lower level of genetic diversity among isolates of S. Typhi. Overall, this study augments the present knowledge that WGS can help predict resistance genotypes and eventual correlation with phenotypes, enabling the chance to spot AMR determinants for fast diagnosis and prioritize antibiotic use directly from sequence.

Survey on Genetic Diversity, Biofilm Formation, and Detection of Colistin Resistance Genes in Clinical Isolates of Acinetobacter baumannii.

IntroductionAcinetobacter baumannii is an opportunistic pathogen responsible for nosocomial infections. The emergence of colistin-resistant A. baumannii is a significant threat to public health. The aim of this study was to investigate the molecular characterization and genotyping of clinical A. baumannii isolates in Southwestern Iran.MethodsA total of 70 A. baumannii isolates were collected from patients admitted to Imam Khomeini Hospital in Ahvaz, Southwestern Iran. Minimum inhibitory concentration test was conducted by using Vitek 2 system. The presence of biofilm-forming genes and colistin resistance-related genes were evaluated by PCR. The isolates were also examined for their biofilm formation ability and the expression of pmrA and pmrB genes. Finally, multilocus sequence typing (MLST) and PCR-based sequence group were used to determine the genetic relationships of the isolates.ResultsOverall, 61 (87.1%) and 9 (12.8%) isolates were multidrug-resistant (MDR) and extensively drug-resistant (XDR), respectively. Colistin and tigecycline with 2 (2.8%) and 32 (45.7%) resistance rates had the highest effect. Among all the isolates, 55 (78.5%), 7 (10%), and 3 (4.3%) were strong, moderate, and weak biofilm producers, respectively. The frequency rates of biofilm-related genes were 64 (91.4%), 70 (100%), 56 (80%), and 22 (31.42%) for bap, ompA, csuE, and blaPER1, respectively. Overexpression of pmrA and pmrB genes was observed in two colistin-resistance isolates, but the expression of these genes did not change in colistin-sensitive isolates. Additionally, 37 (52.8%) and 8 (11.4%) isolates belonged to groups 1 (ICII) and 2 (IC I), respectively. MLST analysis revealed a total of nine different sequence types that six isolates belonged to clonal complex 92 (corresponding to ST801, ST118, ST138, ST 421, and ST735). Other isolates were belonging to ST133 and ST216, and two colistin-resistant (Ab4 and Ab41) isolates were belonging to ST387 and ST1812.ConclusionThe present study revealed the presence of MDR and XDR A. baumannii isolates harboring biofilm genes and emergence of colistin-resistant isolates in Southwestern Iran. These isolates had high diversity, which was affirmed by typing techniques. The control measures and regular surveillance are urgently needed to preclude the spread of these isolates.

Rapid detection and molecular survey of blaVIM, blaIMP and blaNDM genes among clinical isolates of Acinetobacter baumannii using new multiplex real-time PCR and melting curve analysis

BackgroundAcinetobacter baumannii is a cosmopolitan bacterium that is frequently reported from hospitalized patients, especially those patients who admitted in the intensive care unit. Recently, multiplex real-time PCR has been introduced for rapid detection of the resistance genes in clinical isolates of bacteria. The current study aimed to develop and evaluate multiplex real-time PCR to detect common resistance genes among clinical isolates of A. baumannii.ResultsMultiplex real-time PCR based on melting curve analysis showed different Tm corresponding to the amplified fragment consisted of 83.5 °C, 93.3 °C and 89.3 °C for blaIMP, blaVIM and blaNDM, respectively. Results of multiplex real-time PCR showed that the prevalence of blaIMP, blaVIM and blaNDM among the clinical isolates of A. baumannii were 5/128(3.9%), 9/128(7.03%) and 0/128(0%), respectively. Multiplex real-time PCR was able to simultaneously identify the resistance genes, while showed 100% concordance with the results of conventional PCR.ConclusionsThe current study showed that blaVIM, was the most prevalent MBL gene among the clinical isolates of A. baumannii while no amplification of blaNDM was seen. Multiplex real-time PCR can be sensitive and reliable technique for rapid detection of resistance genes in clinical isolates.

Targeting antimicrobial resistance genes in clinical isolates from healthcare-associated infections using CRISPR-Cas9

Of the numerous approaches to counteracting the spread of antimicrobial resistance (AMR), one that is receiving increased attention is the use of CRISPR-Cas-based gene editing technology to remove resistance-conferring sequences from bacteria. Proof-of-principle studies have shown that CRISPR-Cas is able to successfully remove AMR genes from monocultures or very simple bacterial communities in the laboratory, but these constructs and their delivery must be adapted to be used in real-world medical settings. One area where such a technology may be implemented is tackling healthcare-associated infections. Development of a CRISPR-Cas9 cassette that is able to target particular resistance genes would be a powerful tool in addressing these infections. CRISPR-Cas9 will therefore be implemented to target AMR genes in clinical isolates of vancomycin-resistant Enterococcus faecium, and extended-spectrum β-lactamase (CTX-M) or carbapenemase (Oxa-48)-producing Klebsiella pneumoniae and Escherichia coli, by utilising a broad host range conjugative plasmid to deliver this construct via conjugation. Preliminary work using E. coli MG1655 as plasmid donor have shown that all strains can receive the plasmid, and there is significant variation in conjugation frequency between E. coli recipients.

Detection of antibiotic resistance genes in culturable bacteria isolated from soils around mines in Hamedan, Iran

Antibiotic resistance genes are considered to be emerging contaminants. Considering the limited number of papers on detection of antibiotic resistance genes in metal-polluted soils of Iran and due to the fact that nonclinical strains carrying resistance determinants can be the origin of resistance genes in clinical isolates, the present study was conducted to detect some resistance genes (i.e., blaTEM, vanA, tetB, strA, and aac(3)-II) in the most prevalent culturable bacteria isolated from soils around mines. A total of 70 species of Pseudomonas, Azotobacter, Enterobacter, and Bacillus were isolated from soils under different land uses. Polymerase chain reaction was used to detect resistance genes in the isolates. The blaTEM gene was the most abundant gene detected in the isolates (45.71%). The number of Azotobacter, Pseudomonas, and Enterobacter isolates containing blaTEM was higher in the agricultural and pasture soils than in the mining waste soils (28.57%, 57.14%, and 20%, respectively), but the pasture and mining waste soils proved to harbor more Bacillus species containing blaTEM compared to the agricultural soils (64.28%, 50%, and 42.86%, respectively). The vanA gene was found in 5.71% of all the strains, and only one Pseudomonas isolates harbored aac(3)-II. The tetB and strA genes were not detected in any of the isolates. More than 77% of the isolates were phenotypically resistant to β-lactams, and 28.57%, 40%, and 31.43% of them were resistant to streptomycin, vancomycin, and tetracyclines, respectively. Overall, the high number of bacteria containing at least one resistance gene isolated from the samples indicated the persistence of environmental reservoirs of resistance genes in the metal-polluted soils.

The β-Lactamase Gene Profile and a Plasmid-Carrying Multiple Heavy Metal Resistance Genes of Enterobacter cloacae.

In this work, by high-throughput sequencing, antibiotic resistance genes, including class A (bla CTX-M, bla Z, bla TEM, bla VEB, bla KLUC, and bla SFO), class C (bla SHV, bla DHA, bla MIR, bla AZECL-29, and bla ACT), and class D (bla OXA) β-lactamase genes, were identified among the pooled genomic DNA from 212 clinical Enterobacter cloacae isolates. Six bla MIR-positive E. cloacae strains were identified, and pulsed-field gel electrophoresis (PFGE) showed that these strains were not clonally related. The complete genome of the bla MIR -positive strain (Y546) consisted of both a chromosome (4.78 Mb) and a large plasmid pY546 (208.74 kb). The extended-spectrum β-lactamases (ESBLs) (bla SHV-12 and bla CTX-M-9a) and AmpC (bla MIR) were encoded on the chromosome, and the pY546 plasmid contained several clusters of genes conferring resistance to metals, such as copper (pco), arsenic (ars), tellurite (ter), and tetrathionate (ttr), and genes encoding many divalent cation transporter proteins. The comparative genomic analyses of the whole plasmid sequence and of the heavy metal resistance gene-encoding regions revealed that the plasmid sequences of Klebsiella pneumoniae (such as pKPN-332, pKPN-3967, and pKPN-262) shared the highest similarity with those of pY546. It may be concluded that a variety of β-lactamase genes present in E. cloacae which confer resistance to β-lactam antibiotics and the emergence of plasmids carrying heavy metal resistance genes in clinical isolates are alarming and need further surveillance.

Characterization of resistance genes to macrolides, lincosamides and streptogramins (MLS) among clinical isolates of Staphylococcus aureus in North Lebanon

Background. – Staphylococcus aureus is one of the most significant pathogens causing significant morbidity and mortality. Moreover, the incidence of MLS-resistant Staphylococcus aureus infections continues to grow globally.Objective. – The aim of this study is to examine the expression of resistance of Staphylococcus aureus (S. aureus) isolates to MLS and the prevalence of genes involved in this resistance by PCR.Methods. – 38 strains of S. aureus MLS-resistant (resistant at least for one macrolide) were isolated in the sector of microbiology at Nini Hospital in North Lebanon. The disk diffusion method was used to determine the phenotype of the MLS resistance. The resistance genes involved were detected by PCR using specific gene primers for ermA, ermB, ermC, msrA, linA, mefA, vat and vgb genes.Results. – A total of 55.3% of the isolates were positive for inducible phenotype (iMLSB), 15.8% for the constitutive phenotype (cMLSB), 23.7% for MSB phenotype and 5.2% for L phenotype. The ermC gene was the most prevalent (52.6%), while ermA, ermB, msrA and linA genes were observed with lower prevalence. However, a combination of several of these genes was detected. The vgb, vat and mefA genes were not detected in any of the clinical isolates.Conclusion. – To our knowledge, this study is the first investigation regarding characterization of MLS resistance genes in clinical isolates of S. aureus in Lebanon. The study revealed a high prevalence of the inducible resistance to lincosamides (iMLSB phenotype) and the most prevalent resistance determinants was ermC.

Characterization of extensively drug-resistant Mycobacterium tuberculosis isolates circulating in Siberia.

BackgroundThe spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis compromises effective control of tuberculosis (TB) in Siberia. Early identification of drug-resistant isolates is, therefore, crucial for effective treatment of this disease. The aim of this study was to conduct drug susceptibility testing and identify mutations in drug resistance genes in clinical isolates of M. tuberculosis from some TB patients presenting for treatment in Siberia.MethodsThirty randomly selected clinical isolates of M. tuberculosis were obtained from the Novosibirsk Research Institute of Tuberculosis, Russia. Isolates were screened for drug resistance and characterized by variable number of tandem repeats (VNTR)-typing using 15 standard and four additional loci. Deligotyping on multiple large sequences was performed using 10 loci.ResultsTwenty-nine of the isolates were assigned XDR status. Twenty-eight isolates belonged to the M. tuberculosis Beijing family, from which 11 isolates were considered the M11 type (39%), two the M2 type (7%), and one the M33 type (3%). Seventeen isolates (60.7%) from this family exhibited unique genetic patterns. The remaining two isolates belonged to the Latino-American Mediterranean family. Gene sequences (rpoB, katG, rrs, rpsL, tlyA, gidB, gyrA, gyrB) were analyzed to identify mutations that confer resistance to rifampicin, isoniazid, amikacin, kanamycin, capreomycin, and ofloxacin. The most common mutations among the XDR isolates were S531L in RpoB, S315T in KatG, various codon 94 mutations in gyrA, A90V in GyrA, K43R in RpsL, and 1401 A → G in rrs; these confer resistance to rifampicin, isoniazid, ofloxacin, streptomycin and kanamycin/capreomycin, respectively. There was high congruence between the two typing methods (VNTR typing and deligotyping) and RD105, RD149, RD152, RD181, and RD207 regions of difference were absent from the 28 Beijing family isolates.ConclusionsDeligotyping can be used for rapid and reliable screening of M. tuberculosis isolates, followed by more in-depth genotyping. Identification of Beijing family isolates with extensive drug resistance confirms that such strains have epidemiological importance in Siberia. Rapid detection of mutations that lead to drug resistance should facilitate selection of effective drug therapies, and the development of early prevention strategies to combat this infection.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2334-14-478) contains supplementary material, which is available to authorized users.

Analysis of plasmid-mediated quinolone resistance genes in clinical isolates of the tribe Proteeae from Argentina: First report of qnrD in the Americas.

To analyse the occurrence and prevalence of plasmid-mediated quinolone resistance (PMQR) genes in the tribe Proteeae, 81 isolates (65 Proteus spp., 12 Morganella morganii and 4 Providencia stuartii) consecutively collected in 66 hospitals belonging to the WHONET-Argentina Resistance Surveillance Network were studied. Of the 81 isolates, 50 (62%) were susceptible to quinolones [43/65 (66%) Proteus spp. and 7/12 (58%) M. morganii). The remaining 31 isolates (22 Proteus spp., 5 M. morganii and all P. stuartii) showed high-level resistance to nalidixic acid (NAL) and decreased susceptibility or resistance to ciprofloxacin. All NAL-resistant isolates harboured mutations associated with quinolone resistance (MAQRs) in both gyrA (S83I/R) and parC (S80I/R), and some also had MAQRs in gyrB (S464Y/F). The unique PMQR gene detected was qnrD, which was found in 2/81 isolates (Proteus mirabilis Q1084 and Proteus vulgaris Q5169), giving a prevalence of 2.5% in Proteeae. These two isolates were from different geographical regions and both harboured MAQRs in gyrA and parC. The qnrD genes were located on the related plasmids pEAD1-1 (2683bp) and pEAD1-2 (2669bp). Plasmid pEAD1-1 was 100% identical to pCGH15 and differed in only three nucleotides from pDIJ09-518a, which were previously found in clinical isolates of P. mirabilis (China) and Providencia rettgeri (France), respectively, whilst pEAD1-2 was not previously described. The extended-spectrum β-lactamase CTX-M-2 was found in 27% (22/81) of the isolates and was significantly associated with quinolone resistance but not with qnrD (only P. mirabilis Q1084 expressed CTX-M-2). This is the first report of qnrD in the Americas.