Genotypic Identification Methods Research Articles

Comparison of commercial next-generation sequencing assays to conventional culture methods for bacterial identification and antimicrobial susceptibility of samples obtained from clinical cases of canine superficial bacterial folliculitis.

Bacterial identification and antimicrobial susceptibility testing is an important step in timely therapeutic decisions for canine superficial bacterial folliculitis (SBF), commonly caused by Staphylococcus pseudintermedius. Next-generation sequencing (NGS) offers the appeal of potentially expedited results with complete detection of bacterial organisms and associated resistance genes compared to culture. Limited studies exist comparing the two methodologies for clinical samples. To compare and contrast genotypic and phenotypic methods for bacterial identification and antimicrobial susceptibility from cases of canine SBF. Twenty-four client-owned dogs with lesions consistent with SBF were enrolled. A sterile culturette swab was used to sample dogs with SBF lesions. The swab was rinsed in 0.9 mL of sterile phosphate-buffered saline and vortexed to create a homogenous solution. Two swabs for NGS laboratories (Labs) and one swab for culture (Culture Lab) were randomly sampled from this solution and submitted for bacterial identification and antimicrobial susceptibility. No statistical difference regarding turnaround time for NGS Labs compared to Culture Lab was found. NGS Lab 1 identified more organisms than NGS Lab 2 and Culture Lab, which were both statistically significant. There was no statistical difference in detection frequency for Staphylococcus spp. among all laboratories. There was poor agreement for the presence of meticillin resistance and most antimicrobials among all laboratories. Utilisation of NGS as a replacement for traditional culture when sampling canine SBF lesions is not supported at this time.

Molecular insights intoexpression and silencing of resistance determinants in Staphylococcus aureus.

This study aimed to investigate the status of antimicrobial-resistant strains of Staphylococcus aureus in Pakistan, their association in terms of co-occurrence with the biofilm-forming genes, resistance profiling and associated discrepancies in diagnostic methods. A total of 384 milk samples from bovine was collected by using convenient sampling technique and were initially screened for subclinical mastitis, further preceded by isolation and confirmation of S. aureus. The S. aureus isolates were subjected to evaluation of antimicrobial resistance by phenotypic identification using Kirby-Bauer disc diffusion method, while the genotypic estimation was done by polymerase chain reaction to declare isolates as methicillin, beta-lactam, vancomycin, tetracycline, and aminoglycoside resistant S. aureus (MRSA, BRSA, VRSA, TRSA, and ARSA), respectively. The current study revealed an overall prevalence of subclinical mastitis and S. aureus to be 59.11% and 46.69%, respectively. On a phenotypic basis, the prevalence of MRSA, BRSA, VRSA, TRSA, and ARSA was found to be 44.33%, 58.49%, 20.75%, 35.84%, and 30.18%, respectively. The results of PCR analysis showed that 46.80% of the tested isolates were declared as MRSA, 37.09% as BRSA, and 36.36% as VRSA, while the occurrence of TRSA and ARSA was observed in 26.31% and 18.75%, respectively. The current study also reported the existence of biofilm-producing genes (icaA and icaD) in 49.06% and 40.57% isolates, respectively. Lastly, this study also reported a high incidence of discrepancies for both genotypic and phenotypic identification methods of resistance evaluation, with the highest discrepancy ratio for the accA-aphD gene, followed by tetK, vanB, blaZ, and mecA genes. The study concluded that different antibiotic resistance strains of S. aureus are prevalent in study districts with high potential to transmit between human populations. The study also determined that there are multiple resistance determinants and mechanisms that are responsible for the silencing and expression of antibiotic resistance genes.

Discrepancies between phenotypic and genotypic identification methods of antibiotic resistant genes harboring Staphylococcusaureus

Antimicrobial resistance is a global issue that limits therapeutic options for infections. S. aureus being a member of the ESKAPE group is capable of “escaping” the biocidal action of antimicrobial agents. There are phenotypic and genotypic methods used for the identification of antibiotic resistant genes harboring S. aureus but these methods do not always show concordant results. To address these discrepancies, a total of 335 equine nasal swab samples from four districts of Punjab were collected using a convenient sampling technique. These samples were first subjected to common microbial techniques to identify S. aureus. The disc diffusion assay was performed for the phenotypic identification of antibiotic resistant S. aureus by using discs of oxacillin, penicillin, vancomycin, gentamycin, and tetracycline. After this, PCR was performed by targeting mecA, blaZ, vanB, aaca-aphd, and tetK genes for genotypic identification of respective antibiotic-resistant S. aureus. Phenotypic discrepancies (number of antibiotic resistant isolates found from disc diffusion who appeared to be negative for the resistant gene), and genotypic discrepancies (number of antibiotic sensitive isolates found from disc diffusion who appeared to be positive for the resistant gene) were calculated. The discrepancy ratio for mecA, blaZ, vanB, aaca-aphd, and tetK genes were 3.09, 1.96, 2.67, 1.93, and 1.67 respectively. These discrepant results indicate that the absence or presence of only one gene is not a true marker of resistant or sensitive isolates. There are multiple resistance determinants and resistance mechanisms. This study also highlighted the phenomenon of silencing of antibiotic resistance determinants.

Production, purification, and characterization of inulinase from Streptomyces anulatus.

Inulinase is an enzyme that catalyzes inulin to d-fructose. This enzyme can be extracted from plants, but it is difficult to obtain it in large quantities, so its production cost is high. Therefore, microbial inulinase has great potential for industrial needs. In the last decade, there have been very few reports on actinobacterial inulinases, especially on purification and characterization of inulinase process extraction. This study aims to select actinomycetes that possess high inulinase activity from the soil. To screen inulinase-producing bacteria, modified Czapex-Dox agar supplemented with 1% inulin powder was used. The most effective isolate was Streptomyces sp. EFBO8, morphological and genotypic identification methods, confirmed that the strain is Streptomyces anulatus and that its nucleotide sequence has been deposited in GenBank under accession number OQ073700. To optimize inulinase production, kinetics were performed by using S. anulatus strain, which proved to be most productive with a value of 24,024 EU/mL. The enzyme was purified from the culture filtrate by precipitation with ammonium sulfate (NH4 )2 SO4 , followed by column chromatography Sephadex (G-50) separation. Purified protein has a molecular mass of 3331.83 Da.

What's in a name? The importance of identity in heirloom apple tree preservation

Societal Impact StatementHistoric North American apple (Malus domestica) orchards that thrived in the late 19th and early 20th centuries, with cultivar compositions unlike today's orchards, are vanishing. There are several reasons for this loss: tree aging, cost of tree maintenance, and urbanization. Many groups have collected local knowledge regarding the history and horticulture of apples using both phenotypic and genotypic identification methods. Some of these groups have joined with scientists to form the collaborative “Historic Fruit Tree Working Group of North America” to facilitate the conservation of heirloom apple cultivars in North America through documentation, identification, collaboration, and education.

High-Resolution Melting Analysis to Detect Antimicrobial Resistance Determinants in South African Neisseria gonorrhoeae Clinical Isolates and Specimens.

Background Antimicrobial resistance is limiting treatment options for Neisseria gonorrhoeae infections. To aid or replace culture and the syndromic management approach, molecular assays are required for antimicrobial susceptibility testing to guide appropriate and rapid treatment. Objective We aimed to detect single-nucleotide polymorphisms and plasmids associated with antimicrobial resistance from N. gonorrhoeae isolates from a clinic population in South Africa, using real-time PCR as a rapid test for AMR detection. Methods N. gonorrhoeae isolates, from female and male patients presenting for care at a sexually transmitted infections clinic in Durban, South Africa, were analysed using phenotypic and genotypic methods for identification and antibiotic susceptibility testing (AST). Real-time PCR and high-resolution melting analysis were used to detect porA pseudogene (species-specific marker) and resistance-associated targets. Whole-genome sequencing was used as the gold standard for the presence of point mutations. Results The real-time porA pseudogene assay identified all N. gonorrhoeae-positive isolates and specimens. Concordance between molecular detection (real-time PCR and HRM) and resistance phenotype was ≥92% for blaTEM (HLR penicillin), rpsJ_V57M (tetracycline), tetM (tetracycline), and gyrA_S91F (ciprofloxacin). Resistance determinants 16SrRNA_C1192U (spectinomycin), mtrR_G45D (azithromycin), and penA_D545S, penA_mosaic (cefixime/ceftriaxone) correlated with the WHO control isolates. Conclusions Eight resistance-associated targets correlated with phenotypic culture results. The porA pseudogene reliably detected N. gonorrhoeae. Larger cohorts are required to validate the utility of these targets as a convenient culture-free diagnostic tool, to guide STI management in a South African population.

<i>In vitro</i> Antibacterial Activity of <i>Terminalia avicennioides </i>Extracts Against Multidrug Resistant <i>Staphylococcus aureus </i>Strains

Infections caused by multidrug resistance bacteria are now alarming globally, and the increasing rates of antimicrobial resistance are resulting in fewer treatment options. The search for new phytochemicals that could be developed as useful drugs for treatment of infectious diseases consequently increased with medicinal plants extracts receiving greater attention. This study was carried out to determine in vitro antimicrobial activity of <i>Terminalia avicennioides</i> extracts against multidrug resistant <i>Staphylococcus aureus</i> strains isolated from wound infections. Wound swab samples were collected from patients attending Barau Dikko Teaching Hospital Kaduna, Nigeria. Isolation and characterization of <i>Staphylococcus aureus</i> was carried out using standard phenotypic and genotypic identification methods. Antimicrobial susceptibility profile of <i>Staphylococcus aureus</i> isolates was carried out using standard procedures. Also, <i>Terminalia avicennioides</i> extracts were prepared and their in vitro antimicrobial activities tested against multidrug resistant <i>Staphylococcus aureus</i> using standard procedures. The results of the susceptibility profile showed <i>Staphylococcus aureus</i> isolates to be resistant to a ranged of 8.18% to 100% conventional antibiotics used. However, the isolates were 100% sensitive to imipenem. Qualitative and quantitative phytochemical analysis of the extracts revealed the presence of tannin, alkaloids, flavonoids, cardiac glycoside, phenols, saponins and terpenoids and absent of anthraquinones in all extracts. Antimicrobial activity of <i>Terminalia avicennioides</i> extracts against multidrug resistant <i>Staphylococcus aureus</i> isolates showed zones of growth inhibition ranged from 16.28±10.45 – 23.81±6.69 mm and showed significant difference (P < 0.05). Minimum inhibitory concentration (MIC) of the extracts ranged from 56.2500 ± 29.1241 – 31.2500 + 22.16013 gm/ml and showed no significant difference (p > 0.05). The minimum bactericidal concentration (MBC) of the extracts ranged from 175.000 ± 64.2910 – 68.7500 ± 45.8063 mg/ml and showed no significant difference (p > 0.05). Remarkably, the antimicrobial activity of the <i>Terminalia avicennioides</i> extracts exhibit higher inhibitory effects against the multidrug resistant <i>Staphylococcus aureus</i> strains, hence, can further be study and developed for wound infection therapeutic purpose.

Comparative Study of CTX-M-15 Producing Escherichia coli ST131 Clone Isolated from Urinary Tract Infections and Acute Diarrhoea.

Background and PurposeThe alarming increase in the prevalence of CTX-M-15 extended-spectrum β-lactamase (ESBL) producing E. coli has been significantly linked to the clonal expansion of emerging sequence type (ST131). This study aimed to screen for the O16/O25-ST131 clones among different phylogenetic types of E. coli strains isolated from urinary and diarrhoeal samples.MethodsA total of 205 E. coli strains isolated from patients with UTI and acute diarrhoea were investigated by phenotypic and genotypic methods for ESBL identification. Molecular methods were used for identification of O25/O16-ST131 clone and phylogenetic typing of E. coli isolates.ResultsO25-ST131 clone was detected in 89/105 (84.8%) and 47/100 (47%) of urinary and intestinal E. coli isolates, respectively, with a significant difference (P-value<0.001). There was a significant high rate of occurrence of ESBLs, MDR, and antibiotic resistance to most antibiotic classes among O25-ST131 than non-O25-ST131 isolates. CTX-M-15 gene was detected in 64/71 (90%) of ESBLs producing intestinal isolates and 54/79 (68.4%) of urinary ESBLs producing isolates. The O25-ST131 clone was reported among all phylogenetic groups. The O16-ST131 clone serotype was not detected in the study isolates.ConclusionHigh prevalence of the O25-ST131 clone was reported among extraintestinal and intestinal E. coli isolates. First detection of the O25-ST131 clone among phylogenetic groups other than group B2 draws attention of the ability of this clone to transfer among commensal groups. An increasing in the prevalence of CTX-M-15 among E. coli strains especially of intestinal origin is alarming as the intestine is the main reservoir for ExPEC strains causing UTI.

Comparative evaluation of phenotypic,automated VITEK-2 compact system & genotypic methods for identification of Acinetobacter baumannii: An analysis of 78 isolates

Comparative evaluation of phenotypic,automated VITEK-2 compact system & genotypic methods for identification of Acinetobacter baumannii: An analysis of 78 isolates

Genotypic Characterization of Clinical Klebsiella spp. Isolates Collected From Patients With Suspected Community-Onset Sepsis, Sweden.

Klebsiella is a genus of Gram-negative bacteria known to be opportunistic pathogens that may cause a variety of infections in humans. Highly drug-resistant Klebsiella species, especially K. pneumoniae, have emerged rapidly and are becoming a major concern in clinical management. Although K. pneumoniae is considered the most important pathogen within the genus, the true clinical significance of the other species is likely underrecognized due to the inability of conventional microbiological methods to distinguish between the species leading to high rates of misidentification. Bacterial whole-genome sequencing (WGS) enables precise species identification and characterization that other technologies do not allow. Herein, we have characterized the diversity and traits of Klebsiella spp. in community-onset infections by WGS of clinical isolates (n = 105) collected during a prospective sepsis study in Sweden. The sequencing revealed that 32 of the 82 isolates (39.0%) initially identified as K. pneumoniae with routine microbiological methods based on cultures followed by matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) had been misidentified. Of these, 23 were identified as Klebsiella variicola and nine as other members of the K. pneumoniae complex. Comparisons of the number of resistance genes showed that significantly fewer resistance genes were detected in Klebsiella oxytoca compared to K. pneumoniae and K. variicola (both values of p < 0.001). Moreover, a high proportion of the isolates within the K. pneumoniae complex were predicted to be genotypically multidrug-resistant (MDR; 79/84, 94.0%) in contrast to K. oxytoca (3/16, 18.8%) and Klebsiella michiganensis (0/4, 0.0%). All isolates predicted as genotypically MDR were found to harbor the combination of β-lactam, fosfomycin, and quinolone resistance markers. Multi-locus sequence typing (MLST) revealed a high diversity of sequence types among the Klebsiella spp. with ST14 (10.0%) and ST5429 (10.0%) as the most prevalent ones for K. pneumoniae, ST146 for K. variicola (12.0%), and ST176 for K. oxytoca (25.0%). In conclusion, the results from this study highlight the importance of using high-resolution genotypic methods for identification and characterization of clinical Klebsiella spp. isolates. Our findings indicate that infections caused by other members of the K. pneumoniae complex than K. pneumoniae are a more common clinical problem than previously described, mainly due to high rates of misidentifications.

Bioinformatics in the identification of microorganisms

Rapid and accurate identification of microorganisms can be of great value for clinical management. For many fastidious and slow-growing microorganisms, the conventional method used for detection is time-consuming, costly and labour-intensive. Hence, the development of new and improved microbial identification methods are necessary to overcome this bottleneck. Current trend has shifted towards the use of new molecular technologies in genomics and proteomics for bacterial identification and characterization. This mini review will focus on summarizing different types of genotypic and proteomics identification methods, as well as bioinformatics tools used for rapid identification and characterization of microorganisms from various specimens.

Mobile genetic elements responsible for discordant Staphylococcus aureus phenotypes and genotypes in the same blood culture bottle

Approximately 15–20% of the S. aureus genome contains mobile genetic elements that can cause discrepancies between phenotypic and genotypic identification methods. Three blood culture bottles (each from a different patient) that showed discordant results, were shown to contain 2 S. aureus isolates after additional subcultures. One bottle had MRSA and MSSA that by DNA sequence analysis differed only by 31 kb; however, the deletions encompassed parts of SCCmec including mecA and SCCM1. The second bottle contained MRSA and MSSA that differed by 124 kb; the MSSA was missing the entire SCCmec and spa regions. The last bottle contained 2 MRSA, one with ACME II disrupting SCCmec and a 24 bp spa deletion. The deletions in SCCmec and the other elements gave rise to the discrepancies between molecular and the original culture results. Such discrepancies should prompt a search for additional strains in the blood culture bottle.

AMR Escherichia coli and its temporal and spatial variability within the aquatic environment

Phenotypic and genotypic identification methods have been used to determine the temporal and spatial dynamics of AMR-Escherichia coli in a mainly rural watercourse that receives WWTP-effluent compared to a parallel river which does not. We aimed to investigate the incidence of plasmid-mediated mcr-1and β-lactamase-genes in E. coli recovered from both water and Asellus aquaticus samples throughout two-calendar-years. Samples of the water and A. aquaticus were recovered from the relevant locations each month. CHROMagar ESBL agar was used throughout to isolate and identify ESBL-E. coli. The presence of AMR-genes was confirmed using the ‘BSAC’ antibiotic-disk-synergy method and PCR analysis to confirm the presence of mcr-1and ESBL-genes. The CHROMagar ESBL agar was found to be 99.7% (n=578) accurate when confirmed with a PCR analysis of the ESBL-genes. Seventy-six-point-six percent (n=449) of the isolated ESBL-E. coli were correctly identified as ESBL-producing organisms using the ‘BSAC’ method. Interestingly 61.9% (n=358) of the ESBL-E. coli were also found to carry the mcr-1 gene. Our data shows that AMR levels were highest at the WWTP-effluent throughout the two-years for both water and A. aquaticus samples. The incidence of AMR-E. coli 1km downstream of the effluent discharge was equivalent to the parallel river sites, suggesting that the dispersal of AMR from the WWTP-effluent is limited, although AMR-E. coli were found in relatively high numbers at the WWTP-effluent. We argue that the presence of AMR in the freshwater invertebrate A. aquaticus could represent an important route by which AMR can spread from the aquatic environment to the terrestrial environment.

Comparing conventional, biochemical and genotypic methods for accurate identification of Klebsiella pneumoniae in Sudan.

Klebsiella pneumoniae is recognized as one of the most important healthcare-associated pathogens worldwide due to its tendency to develop antibiotic resistance and cause fatal outcomes. Bacterial identification methods such as culture and biochemical tests are routinely used with limited accuracy in many low- and middle-income countries, including Sudan. The aim of this study was to test the accuracy of identification of K. pneumoniae in Khartoum, Sudan. Two hundred and fifty K. pneumoniae isolates were collected and identified using conventional phenotypic methods, biochemically using API 20E and genotypically by amplification of 16S−23S rDNA and sequencing of rpoB, gapA and pgi. Only 139 (55.6 %) of the isolates were confirmed as K. pneumoniae genotypically by PCR and 44.4 % were identified as non- K. pneumoniae . The results demonstrate that the identification panels used by the hospitals were inaccurately identifying K. pneumonia and led to overestimation of the prevalence of this organism. The current identification methods used in Khartoum hospitals are highly inaccurate, and therefore we recommend the use of a comprehensive biochemical panel or molecular methods, when possible, for accurate identification of K. pneumoniae .

Identification of Candida Species from Clinical Samples in a Honduran Tertiary Hospital.

Candida species are one of the most important causes of human infections, especially in hospitals and among immunocompromised patients. The correct and rapid etiological identification of yeast infections is important to provide adequate therapy, reduce mortality, and control outbreaks. In this study, Candida species were identified in patients with suspected fungal infection, and phenotypic and genotypic identification methods were compared. A total of 167 axenic fungal cultures and 46 clinical samples were analyzed by HardyCHROM®, MicroScan®(Omron Microscan Systems Inc, Renton, WA, USA), and PCR-RFLP (Restriction Fragment Length Polymorphisms). The species of the C. albicans complex were the most frequent, followed by C. tropicalis and C. glabrata. Less common but clinically relevant species of Candida were also isolated. The comparison between the three methods was concordant, especially for the most common Candida species. Fungal DNA amplification was successful in all clinical samples.

Phenotypic and genotypic methods for identification of slime layer production, efflux pump activity, and antimicrobial resistance genes as potential causes of the antimicrobial resistance of some mastitis pathogens from farms in Menoufia, Egypt.

Mastitis caused by multi- or pan-drug resistant bacteria is a growing health concern. A total of 110 milk samples were collected: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Enterococcus faecalis, and Escherichia coli were present in 54/110 (49.09%), 37/110 (33.63%), 25/110 (22.72%), 7/110 (6.36%), and 50/110 (45.45%) samples, respectively. A total of 20 methicillin-resistant S. aureus (MRSA) isolates, 19 Streptococcus sp. isolates, and 15 E. coli isolates were selected, and 100% were positive for (coagulase and hemolysins), streptokinase, and hemolytic activity, respectively. A number of 11 E. coli isolates were serotyped, and the serotypes were: O26, O55, O111, O119, O124, O125, O127, and O158. The antimicrobial resistance index ranges for MRSA, Streptococcus sp., and E. coli were 0.49-0.83, 0.39-0.83, and 0.56-1, respectively. The most effective antimicrobials on Gram-positive isolates were cephradine, ciprofloxacin, doxycycline, norfloxacin, and vancomycin, while doxycycline and norfloxacin were effective on E. coli serotypes. All of the selected isolates exhibited slime layer production. The efflux pumps of the 12 MRSA, 12 Streptococcus sp., and 11 E. coli isolates exhibited activity with ethidium bromide concentrations of 1, 1.5, and 0.5µg/ml, respectively. There was a simultaneous antimicrobial activity of the efflux pump inhibitor chlorpromazine with amoxicillin/clavulanic acid, erythromycin, and oxacillin, to which the isolates were resistant. The 12 MRSA isolates harboured the methicillin resistance genes mec(A,A1, and A2), mecA1, and mecC at frequencies of 9/12 (75%), 9/12 (75%), and 8/12 (66.7%), respectively, and the penicillin resistance gene BlaZ was present at a frequency of 5/12 (41.7%). The distributions of erm(A), erm(B), erm(C), erm(F), erm(G), and erm(Q) were 8/12 (66.7%), 5/12 (41.7%), 12/12 (100%), 2/12 (16.7%), 0/12 (0.0%), and 8/12 (66.7%), respectively. The 12 Streptococcus sp. isolates harboured mec(A, A1, and A2), mecA1, mecC, and blaZ at rates of 4/12 (33.33%), 4/12 (33.33%), 5/12 (41.7%), and 4/12 (33.33%), respectively. The frequencies of erm(A) and erm(F) were 4/12 (33.33%), and 9/12 (75%), respectively. The 11 E. coli isolates harboured the extended-spectrum β-lactamases integrase1, integrase2, blaCTX-M, blaCTX-M-1, and blaTEM at frequencies of 10/11 (90.90%), 11/11 (100%), 9/11 (81.81%), 6/11 (54.54%), and 10/11 (90.90%), respectively. Moreover, the frequencies of erm(A), erm(B), erm(C), erm(F), erm(G), and erm(Q) were 7/11 (63.63%), 4/11 (36.36%), 4/11 (36.36%), 5/11 (45.45%), 10/11 (90.90%), and 10/11 (90.90%), respectively. Our results demonstrated the high antimicrobial resistance of the investigated isolates and confirmed the existence of multiple mechanisms underlying multidrug resistance.

Enhancement of clavulanic acid production by Streptomyces sp MU-NRC77 via mutation and medium optimization

Purpose: To enhance clavulanic acid production using UV-mutagenesis on Streptomyces sp. NRC77.Methods: UV-mutagenesis was used to study the effect of Streptomyces sp. NRC77 on CA production. Phenotypic and genotypic identification methods of the promising mutant strain were characterized. Optimization of the fermentation medium and culture conditions were investigatedResults: Out of the screened mutants, 120A3 mutant isolate was selected as promising. The phenotypic properties of 120A3 mutant showed culture characteristics similar to those of Streptomyces species. Phylogenetic analysis of 16S rRNA gene sequence indicate that this strain has similarity (99 %) to Streptomyces sp.T2-7; therefore it was suggested as Streptomyces sp. MU-NRC77 and has Gen Bank accession no. KT953342.Conclusion: Improvement of CA yield by 48 % was obtained from fermentation medium and culture condition optimization. Further optimization by addition of H2O2 and activated charcoal to the production medium increased CA yield to 646.12 and 682.94 mg/L respectively, i.e., 83 % more than that obtained prior to addition.Keywords: Clavulanic acid, Medium optimization, Phenotypic and Genotypic identification, Streptomyces sp. MU-NRC77, UV-Mutagenesis

Detection and Characterization of a Novel Lytic Bacteriophage (vB-KpneM-Isf48) Against Klebsiella pneumoniae Isolates from Infected Wounds Carrying Antibiotic-Resistance Genes (TEM, SHV, and CTX-M)

Background: Approximately 80% of nosocomial infections are caused by strains of Klebsiella pneumoniae. Resistance to β-lactam antibiotics is a result of expression of extended-spectrum β-lactamase (ESBL) genes. Recently, phage therapy has gained increasing attention due to its many advantages over chemotherapy. Objectives: The aim of this study was to isolate ESBL-positive Klebsiella pneumoniae strains from different types of wounds, and a lytic bacteriophage against them. Methods: During a two-year period from January 2013 to February 2015, in a cross-sectional study, 41 K. pneumoniae strains were isolated from 193 categories of infected wounds at three hospitals in Isfahan, Iran. Phenotypic and genotypic methods were used to detect the ESBL-positive strains. A lytic phage against K. pneumoniae was isolated, and its host range, morphology, thermal and pH stability, saline stress, and estimated genome size were determined. Results: Of the 41 K. pneumoniae isolates, 18 were ESBL-producing and 36 carried antibiotic-resistance genes. A total of 36 out of 41 isolated samples carried one or more resistance genes. The results showed that the differences between phenotypic and genotypic identification methods were significant (P = 0.0001). The SHV, CTX-M, and TEM genes were detected in 29, 10, and 9 isolates of the tested bacteria, respectively. No bacteria contained both the SHV and the CTX-M genes. The frequency of the SHV gene was significantly higher than that of the other genes (P = 0.0001). The phage’s morphology features placed it in the Myoviridae family. Only 38 out of 41 clinical isolates were susceptible to the phage. Phage titers were completely preserved after one hour of incubation at 30°C and 40°C, and they were stable at different pH values. The phage’s survival decreased when the salt concentration was increased. Conclusions: The high rate of isolation of antibiotic-resistant strains of K. pneumoniae was consistent with other studies. As the phage was virulent and specific for K. pneumoniae, and was stable and active at different pH values, salt concentrations, and temperatures, its application in phage therapy of infected wounds is suggested.

LytA-based identification methods can misidentify Streptococcus pneumoniae

During surveillance studies we detected, among over 1500 presumptive pneumococci, 11 isolates displaying conflicting or novel results when characterized by widely accepted phenotypic (optochin susceptibility and bile solubility) and genotypic (lytA-BsaAI-RFLP and MLST) identification methods. We aimed to determine the genetic basis for the unexpected results given by lytA-BsaAI-RFLP and investigate the accuracy of the WHO recommended lytA real-time PCR assay to classify these 11 isolates. Three novel lytA-BsaAI-RFLP signatures were found (one in pneumococcus and two in S. mitis). In addition, one pneumococcus displayed the atypical lytA-BsaAI-RFLP signature characteristic of non-pneumococci and two S. pseudopneumoniae displayed the typical lytA-BsaAI-RFLP pattern characteristic of pneumococci. lytA real-time PCR misidentified these three isolates. In conclusion, identification of pneumococci by lytA real-time PCR, and other lytA-based methodologies, may lead to false results. This is of particular relevance in the increasingly frequent colonization studies relying solely on culture-independent methods.

Sporothrix schenckii complex in Iran: Molecular identification and antifungal susceptibility.

Sporotrichosis is a global subcutaneous fungal infection caused by the Sporothrix schenckii complex. Sporotrichosis is an uncommon infection in Iran, and there have been no phenotypic, molecular typing or antifungal susceptibility studies of Sporothrix species. This study aimed to identify nine Iranian isolates of the S. schenckii complex to the species level using colony morphology, carbohydrate assimilation tests, and PCR-sequencing of the calmodulin gene. The antifungal susceptibilities of these Sporothrix isolates to five antifungal agents (amphotericin B (AMB), voriconazole (VRC), itraconazole (ITC), fluconazole (FLC), and terbinafine (TRB)) were also evaluated according to the M27-A3 and M38-A2 protocols of the Clinical and Laboratory Standards Institute for yeast and mycelial phases, respectively. Five of seven clinical isolates were identified as S. schenckii, and two clinical and two environmental isolates were identified as S. globosa. This is the first report of S. globosa in Iran. There was significant agreement (73%) between the results of the phenotypic and genotypic identification methods. TRB and ITC were the most effective antifungals against the Sporothrix isolates. The minimum inhibitory concentration (MIC) values of TRB for the yeast and mycelial phases of S. schenckii differed significantly. There was also a significant difference in the minimum fungicidal concentration (MFC) values of AMB and TRB for the two phases. Considering the low efficacy of VRC and FLC and the wide MIC ranges of AMB (1-16 μg/ml and 1-8 μg/ml for yeast and mycelial forms, respectively) observed in the present study, in vitro antifungal susceptibility testing should be performed to determine appropriate therapeutic regimens.