Detection Of Antibiotic Resistance Research Articles

Bacteria and antibiotic resistance detection in fractures of wild birds from wildlife rehabilitation centres in Spain

Anatomic adaptations make birds more prone to open fractures with exposed bone parts losing vascularization. As a result of this exposure, fractures are colonized by different microorganisms, including different types of bacteria, both aerobic and anaerobic, causing osteomyelitis in many cases. For this reason, antibiotic treatment is common. However, carrying out antibiotic treatment without carrying out a previous antibiogram may contribute to increased resistance against antibiotics, especially in migratory wild birds. In this paper, bacterial counts regarding fracture type, bacterial identification and antibiotic resistance have been analysed in wild birds from wildlife rehabilitation centres in Spain. The results obtained showed that open fractures had higher bacterial counts (CFU/mL) than closed ones. Bacteria in family Enterobacteriaceae, identified were Escherichia spp., Enterobacter spp., Shigella spp., Hafnia alvei, Proteus mirabilis, Leclercia adecarboxylata and Pantoea agglomerans. Other bacteria present in wild birds’ fractures were Aeromonas spp., Enterococcus spp. Bacillus wiedmannii and Staphylococcus sciuri. All species found presented resistance to at least one of the antibiotics used. Wild birds can be implicated in the introduction, maintenance and global spreading of antibiotic resistant bacteria and represent an emerging public health concern. Results obtained in this paper support the idea that it is necessary to take this fact into account before antibiotic administration to wild animals, since it could increase the number of bacteria resistant to antibiotics.

Detection of Antibiotic-Resistance by MALDI-TOF Mass Spectrometry: An Expanding Area.

Several MALDI-TOF MS-based methods have been proposed for rapid detection of antimicrobial resistance. The most widely studied methods include assessment of β-lactamase activity by visualizing the hydrolysis of the β-lactam ring, detection of biomarkers responsible for or correlated with drug-resistance/non-susceptibility, and the comparison of proteomic profiles of bacteria incubated with or without antimicrobial drugs. Antimicrobial-resistance to a number of antibiotics belonging to different classes has been successfully tested by MALDI-TOF MS in a variety of clinically relevant bacterial species including members of Enterobacteriaceae family, non-fermenting Gram-negative bacteria, Gram-positive cocci, anaerobic bacteria and mycobacteria, opening this field to further clinically important developments. Early detection of drug-resistance by MALDI-TOF MS can be particularly helpful for clinicians to streamline the antibiotic therapy for a better outcome of patients with systemic infection, in all cases where a prompt and effective antibiotic treatment is essential to preserve organ function and/or patient survival.

Flow cytometry as a rapid test for detection of tetracycline resistance directly in bacterial cells in Micrococcus luteus

Correct effective doses of antibiotics are important in the treatment of infectious diseases. The most frequently used methods for determination of the antibiotic susceptibility of bacterial pathogens are slow. The detection of multidrug-resistant bacteria currently relies on primary isolation followed by phenotypic detection of antibiotic resistance by measuring bacterial growth in the presence of the antibiotic being tested. The basic requirements for methods of detection of resistance to antibiotics include speed and accuracy. We studied the speed and accuracy of flow cytometry for the detection of tetracycline resistance in the Gram-positive bacteria Micrococcus luteus. Detection of cell viability and reliability of antibiotic resistance was carried out on the Guava EasyCyte flow cytometer (Merck Millipore, Germany) with SYBR Green and PI dyes. M. luteus was exposed to tetracycline (at 30, 90, 180 and 270 μg.mL-1) over 24 hours. Concentrations of live and dead cells were measured after 4 and 24 hours of incubation. The results revealed that the use of mixed dyes PI and SYBR Green allowed the division of cells into large subpopulations of live and dead cells and the DNA of destroyed cells. After 4 h exposure to tetracycline 30 μg.mL-1, the subpopulation of live cells decreased by 47% compared to the positive control. Tetracycline at 90 μg.mL-1 decreased the subpopulation of live cells by 59% compared to the positive control. A continued increase in concentration caused a shift in the population and an increase in dead cells, indicating damage to the cells of the microorganism. Incubation of M. luteus with 180 and 270 μg.mL-1 tetracycline decreased the subpopulation of live cells by 82% and 94%, respectively, in comparison with the positive control. After incubation with 30 μg of tetracycline over 24 h the number of living cells decreased by 70% in comparison with the positive control. Tetracycline treatment (90 μg.mL-1 for 24 h) killed 71% of cells. After exposure to 90 μg.mL-1 tetracycline 29% cells were viable. The viability of living cells was confirmed by a microbiological test.

Characterization of Antibiotic Resistance Profile and Detection of pap and blaCTX-Genes in Escherichia coli Isolates

Aims Escherichia coli is one of the most important causes of hospital-acquired and community-acquired infections in humans and can easily gain resistance to antibiotics consumed by humans and animals. The main objectives of this study were to assess antibiotic resistance outlines and detection of antibiotic resistance and virulence genes, including the blaCTX-M and pap among the E. coli isolates recovered from the urine of patients referred to Shaheed Madani Hospital (Bejestan, Northeast of Iran). Methods & Materials This cross-sectional study was conducted from April 2016 to March 2018. A total of 100 non-duplicate isolates of E. coli were recovered from the urine of patients referred to Shaheed Madani Hospital (Bejestan, Northeast of Iran). Antimicrobial susceptibility test and extended-spectrum-beta-Lactamases (ESBLs) production were performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines. The polymerase chain reaction was carried out for the detection and distribution of the pap virulence gene and blaCTX-M antibiotic resistance gene. Findings About 100 E. coli isolates were recovered from the urine sample of patients (21 male, and 79 female). Carbapenems were the most effective antibiotic against isolates. Four strains (4%) were resistant to colistin. Twenty-seven strains (27%) were ESBL-positive. Carbapenems were the most effective antibiotic against ESBL-positive strains. Also, 82% and 89% of isolates were blaCTX-M and pap gene positive, respectively. Conclusion This study has shown that ESBL-positive strains with a high level of drug resistance and virulence factors are a potential risk for hospital wards. Colistin resistant isolates found in our study are quite alarming.

Antimicrobial Resistance Profile Among Major Bacterial Pathogens in Southern Babil, Iraq

Background: At present, drug-resistant pathogens are considered one of the major increasing causes of morbidity and mortality around the world. The data on microorganisms' resistance assist define the best available treatment for patients. Therefore, this study aimed to screen the antimicrobial-resistant profile of different drugs in major clinical pathogens of urine, ear and wound infections.
 Methods: This study was conducted in Al-Shomali General Hospital, Southern Babil, Iraq from October 2019 to May 2020. Totally 67 clinical specimens obtained from the wound, urine, and ear discharge collected from hospitalized patients as well as 30 healthy individuals participate in this study. Then, the standard microbiological methods carried outperformed to the isolated and identified bacterial species. Antimicrobial susceptibility tests were performed using different antimicrobial discs by applying the Kirby–Bauer disc diffusion method.
 Results: Totally, 67 bacterial isolates were obtained from 44 (66%) female and 23 (34%) male patients. Staphylococcus aureus and E. coli were the most common predominant organisms. All isolates were showed a high rate of resistance to evaluated cephalosporins 100% and 87% to cefotaxime and ceftriaxone respectively, while very low resistance recorded in Aminoglycosides 22% and 12% to Gentamicin and amikacin, respectively.
 Conclusion: These results suggest a constant screening for the detection of antibiotic resistance, as well as developing antimicrobial stewardship programs in Babil, Iraq. Moreover, these bacterial isolates have shown multidrug resistance, mainly to commonly administered drugs that could cause therapy ineffective. Therefore, in clinical use, appropriate treatment should be chosen based on the results obtained from antimicrobial susceptibility tests.

Detection and Profiling of Antibiotic Resistance among Culturable Bacterial Isolates in Vended Food and Soil Samples.

The emergence and persistence of antibiotic resistance remain formidable health challenges. This study aimed at detecting and profiling antibiotic resistance of bacterial contaminants in vended food and the environment. Seventy antibiotic-resistant bacterial isolates were isolated from fried fish, African sausages, roasted meat, smokies, samosa, chips (potato fries), vegetable salads, and soil samples collected from Embu Town and Kangaru Market in Embu County, Kenya. The antibiotic susceptibility test, morphological and biochemical characterization, antibiosis assay, polymerase chain reaction-based detection of antibiotic resistance genes, and sequencing of the 16S rRNA gene were done. Analysis of variance on all measured data was done, and Tukey's honest test was used to compare and separate mean diameters of zones inhibition. Resistance of bacterial isolates to antibiotics was chloramphenicol (90%), cefotaxime (84.29%), nalidixic acid (81.43%), tetracycline (77.14%), amoxicillin (72.86%), gentamycin (48.57%), streptomycin (32.86%), and trimethoprim + sulphamethoxazole (30%). Isolate KMP337, Salmonella spp., exhibited highly significant antibiosis against S. aureus recording a mean inhibition diameter and standard error (SE) of 16.33 ± 0.88 mm, respectively, at P=0.001. The 70 bacterial isolates belonged to Bacillus, Paraclostridium, Lysinibacillus, Virgibacillus, and Serratia genera. The study isolated Bacillus wiedmannii (KC75) which is a risk group 2 as well as Serratia marcescens (KMP95) and Bacillus anthracis (KS606) which are risk group 3 organisms. The presence of antibiotic resistance genes Tet A, BlaTEM, StrB, Dfr A, Amp, and FloR genes was confirmed by a polymerase chain reaction. Samples from Kangaru Market recorded a higher (88.57%) proportion of resistant isolates as compared to isolates from Embu Town (11.43%). The study confirmed the presence of antibiotic-resistant bacteria in vended fast food and the soil in Embu Town and Kangaru Market. This study calls for continuous monitoring of bacterial status and hygienic handling of vended food.

Antibiotic Resistance Pattern and Detection of Enterotoxigenic and Enteroaggregative Strain of Escherichia coli among Foreign Tourist with Traveler Diarrhea in Bali using Uniplex Polymerase Chain Reaction

BACKGROUND: As one of the major tourist destinations in Southeast Asia, Bali received millions of foreign tourists each year. Diarrhea consistently placed as the most often experienced health problem among travelers. Traveler diarrhea has various etiologies. The most common was Escherichia coli. The existence of several types of E. coli that are resistant to several antibiotics causes the selection of antibiotics is crucial.
 AIM: This preliminary study aims to understand the pattern of antibiotics sensitivity and to detect the presence of enterotoxigenic and enteroaggregative strains of E. coli from fecal samples of foreign tourists with traveler’s diarrhea in Denpasar, Bali.
 METHODS: A culture examination was carried out to obtain E. coli bacterial colonies. Disk diffusion Kirby–Bauer was carried out for antibiotic sensitivity testing. The confirmed colonies were tested against several common antibiotics, including the recommended first line (ciprofloxacin and azithromycin). Uniplex polymerase chain reaction (PCR) using specific primers conducted to detect the enterotoxigenic E. coli (ETEC) (elt and estA2-4) and enteroaggregative E. coli (EAEC) (CVD432) strains.
 RESULTS: Among 48 stool culture, 14 (29.2%) were identified as E. coli colonies. All samples were still sensitive to the antibiotics meropenem, ceftazidime, and cefixime. Despite majority of the samples (78.6%) still sensitive to ciprofloxacin, large proportion of the samples have developed resistance against the other commonly used antibiotics, doxycycline (70.4%) and azithromycin (57.1%). PCR showed that 3 (21.4%) samples shown positive for CVD432 gene, 2 (14.3%) samples positive for the elt gene, and all negative for the estA2-4 gene.
 CONCLUSION: An only small proportion of E. coli was EAEC or ETEC strain. Although most E. coli still sensitive to beta-lactam antibiotics, a significant proportion had shown resistance against the commonly recommended first-line antibiotics.

Molecular detection of antibiotic resistance and virulence gene determinants of Enterococcus species isolated from coastal water in the Eastern Cape Province, South Africa

ABSTRACT Enterococci are a group of significant disease-causing bacteria, which have developed resistance to several conventionally used antibiotics. Of the 67 confirmed Enterococcus species from Kidd’s Beach, 40 (59.7%) were E. faecium, 19 (28.4%) were E. faecalis. The highest level of resistance was observed against rifampicin (83.6%) followed by erythromycin (64.2%), tetracycline (52.2%), linezolid (46.3%), tetracycline (46.3%), and vancomycin (32.8%). Ninety-six per cent of the Enterococcus spp. was found to be multi-drug resistant. MAR indices vary from 0.3 to 0.9. Two virulence determinants (ace and gelE) were detected and six resistance determinants were identified as follows: ermB (19%), tetM (30%), tetL (19%), gyrA (13%), ampC (1.5%) and Van C2/3 (4.5%). The presence of enterococci (E. faecium and E. faecalis) in Kidd’s Beach waters harbouring virulence genes that facilitate multiple antibiotic resistance signifies a possible health threat for beach goers.

Rapid detection of antibiotic resistance in Salmonella with screen printed carbon electrodes

Salmonella, one of the most common foodborne pathogens, poses a serious threat to human health. In recent years, the antibiotic resistance of Salmonella has become serious as well, which has strengthened the harm of Salmonella to human health. In this paper, a simple and effective electrochemical approach was developed to obtain profile of the antimicrobial susceptibility. Screen-printed carbon electrodes (SPCEs) were used to detect various concentrations of target bacteria at the same time, which have good stability, low cost, and easy mass production. The electroactive redox, resazurin, was used to monitor levels of metabolically active bacteria. As a demonstration, the antibacterial effect of ofloxacin and penicillin on Salmonella gallinarum (S. gallinarum) isolates was evaluated, and the minimum inhibitory concentration (MIC) was measured as well. In the real sample measurement, the MIC obtained was similar to that obtained by conventional antimicrobial susceptibility testing (AST). In this assay, bacterial activity was quantified sensitively and accurately, and the detection time was greatly reduced compared with conventional AST (16–20 h), which means that metabolic capacity of live bacteria could be observed after 1 h of incubation. We were able to clearly detect bacteria above 102 CFU/m. This method aims to nonspecific and can be widely applied to the detection of a variety of drug-resistant bacteria, providing an experimental basis for the rational use of antibiotics and bacterial resistance mechanisms.

Predicting Phenotypic Polymyxin Resistance in Klebsiella pneumoniae through Machine Learning Analysis of Genomic Data.

Polymyxins are used as treatments of last resort for Gram-negative bacterial infections. Their increased use has led to concerns about emerging polymyxin resistance (PR). Phenotypic polymyxin susceptibility testing is resource intensive and difficult to perform accurately. The complex polygenic nature of PR and our incomplete understanding of its genetic basis make it difficult to predict PR using detection of resistance determinants. We therefore applied machine learning (ML) to whole-genome sequencing data from >600 Klebsiella pneumoniae clonal group 258 (CG258) genomes to predict phenotypic PR. Using a reference-based representation of genomic data with ML outperformed a rule-based approach that detected variants in known PR genes (area under receiver-operator curve [AUROC], 0.894 versus 0.791, P = 0.006). We noted modest increases in performance by using a bacterial genome-wide association study to filter relevant genomic features and by integrating clinical data in the form of prior polymyxin exposure. Conversely, reference-free representation of genomic data as k-mers was associated with decreased performance (AUROC, 0.692 versus 0.894, P = 0.015). When ML models were interpreted to extract genomic features, six of seven known PR genes were correctly identified by models without prior programming and several genes involved in stress responses and maintenance of the cell membrane were identified as potential novel determinants of PR. These findings are a proof of concept that whole-genome sequencing data can accurately predict PR in K. pneumoniae CG258 and may be applicable to other forms of complex antimicrobial resistance.IMPORTANCE Polymyxins are last-resort antibiotics used to treat highly resistant Gram-negative bacteria. There are increasing reports of polymyxin resistance emerging, raising concerns of a postantibiotic era. Polymyxin resistance is therefore a significant public health threat, but current phenotypic methods for detection are difficult and time-consuming to perform. There have been increasing efforts to use whole-genome sequencing for detection of antibiotic resistance, but this has been difficult to apply to polymyxin resistance because of its complex polygenic nature. The significance of our research is that we successfully applied machine learning methods to predict polymyxin resistance in Klebsiella pneumoniae clonal group 258, a common health care-associated and multidrug-resistant pathogen. Our findings highlight that machine learning can be successfully applied even in complex forms of antibiotic resistance and represent a significant contribution to the literature that could be used to predict resistance in other bacteria and to other antibiotics.

Rapid Approach for Detection of Antibiotic Resistance in Bacteria Using Vibrational Spectroscopy.

Here, we applied vibrational spectroscopy to investigate the drug response following incubation of S. aureus with oxacillin. The main focus of this work was to identify the chemical changes caused by oxacillin over time and to determine the feasibility of the spectroscopic approach to detect antimicrobial resistance. The oxacillin-induced changes in the chemical composition of susceptible bacteria, preceding (and leading to) the inhibition of growth, included an increase in the relative content of nucleic acids, alteration in the α-helical/β-sheet protein ratio, structural changes in carbohydrates (observed via changes in the band at 1035 cm-1), and significant thickening of the cell wall. These observations enabled a dose-dependent discrimination between susceptible bacteria incubated with and without oxacillin after 120 min. In methicillin resistant strains, no spectral differences were observed between cells, regardless of drug exposure. These results pave the way for a new, rapid spectroscopic approach to detect drug resistance in pathogens, based on their early positive/negative drug response.

Phage-Mediated Molecular Detection (PMMD): A Novel Rapid Method for Phage-Specific Bacterial Detection.

Bacterial infections pose a challenge to human health and burden the health care system, especially with the spread of antibiotic-resistant populations. To provide effective treatment and improved prognosis, effective diagnostic methods are of great importance. Here we present phage-mediated molecular detection (PMMD) as a novel molecular method for the detection and assessment of bacterial antibiotic resistance. This technique consists of a brief incubation, of approximately ten minutes, of the biological sample with a natural bacteriophage (phage) targeting the bacteria of interest. This is followed by total RNA extraction and RT-PCR. We applied this approach to Staphylococcus aureus (SA), a major causative agent of human bacterial infections. PMMD demonstrated a high sensitivity, rapid implementation, and specificity dependent on the phage host range. Moreover, due to the dependence of the signal on the physiological state of the bacteria, PMMD can discriminate methicillin-sensitive from methicillin-resistant SA (MSSA vs. MRSA). Finally, we extended this method to the detection and antibiotic sensitivity determination of other bacteria by proving PMMD efficacy for Bacillus anthracis.

Detection of antibiotic resistance and virulence genes of Vibrio strains isolated from ready-to-eat shrimps in Delta and Edo States, Nigeria.

The current study was conducted to determine the incidence, antibiotic resistance and virulence genes of Vibrio strains isolated from ready-to-eat shrimps in Edo and Delta States, Nigeria. A total of 1440 ready-to-eat shrimp samples were obtained from open markets from November 2016 to October 2017 and analysed using standard culture-based procedures. Overall, our result showed that the ready-to-eat shrimp samples had high mean aerobic mesophilic bacterial count between 3·543 and 7·489 log10 CFU per gram. Vibrio cell densities ranged between 0·663 and 6·761 log10 CFU per gram. From the total samples, 1343/1440 (93·3%) were positive for Vibrio species where 120 Vibrio isolates were randomly selected and confirmed using genus- and species-specific PCR approach. The PCR identification revealed the presence of Vibrio parahaemolyticus 46 (38·33%), Vibrio vulnificus 14 (11·67%), Vibrio fluvialis 12 (10%), Vibrio alginolyticus 8 (6·67%), Vibrio cholerae 2 (1·67%), Vibrio mimicus 10 (8·33%), Vibrio harveyi 3 (2·5%) and other Vibrio sp. 25 (20·83%). All Vibrio isolates were sensitive to colistin and gentamycin with varying percentage of resistance to other antibiotics used in the study. Multiple antibiotic-resistant (MAR) index ranged from 0·08 to 0·83. The tcp, tdh and trl virulence genes were identified in 95 (79·2%), 92 (76·7%) and 95 (79·2%) of the examined isolates respectively. Antibiotic-resistant genes also revealed the presence of class 1 integrase 75 (62·5%), sul2 87 (72·5%), strB 94 (78·3%) and catB3 68 (56·7%). Our findings revealed that the ready-to-eat shrimps may serve as potential reservoirs and medium in the dissemination of prospective MAR pathogens to the consumers and thus constitute a potential risk to public health. The findings from this study represent the first comprehensive report of Vibrio isolates from ready-to-eat shrimps in Edo and Delta States, Nigeria. Incessant monitoring of Vibrio strains and their predisposition to antimicrobials is a necessity to guarantee seafood safety and guarantee the best treatment regimen for patients with gastroenteritis.

Livestock-Associated Methicillin-Resistant Staphylococcus aureus in Patients Admitted to Kuwait Hospitals in 2016-2017.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has been reported to colonize and cause infections in animals as well as in humans. LA-MRSA isolates have only recently been identified in patients admitted to Kuwait hospitals. This study was conducted to characterize LA-MRSA isolates obtained from patients admitted to Kuwait hospitals. A total of 202 (7.1%) of 2,823 MRSA isolates obtained from clinical samples in 2016 and 2017 in 11 public Kuwait hospitals were assigned to lineages previously known to be associated with livestock. They were characterized using antibiogram, spa typing, and DNA microarray for the assignment of clonal complexes (CCs) and detection of antibiotic resistance and virulence determinants. Identification as putative LA-MRSA clones was based on the molecular definition inferred from DNA microarray. The LA-MRSA isolates consisted of CC96 (N = 31), CC97 (N = 169), and CC398 (N = 2). Isolates belonging to CC96 and CC398 were resistant to erythromycin and clindamycin mediated by erm(A) and erm(C). CC97 isolates were multiresistant to gentamicin, kanamycin, erythromycin, clindamycin, tetracycline, chloramphenicol, fusidic acid, trimethoprim, and ciprofloxacin and harbored aacA-aphD, erm(A), erm(C), msr(A), tet(K), cat, fusC, and dfrS1. In total, 35 spa types were identified among the isolates. CC398 isolates consisted of t899 and t034. Ten spa types were identified among CC96 with t11822 (N = 13) as the most prevalent. CC97 consisted of 26 spa types with most belonging to t267 (N = 73) followed by t359 (N = 39). CC398 was composed of CC398-MRSA-IV and CC398-MRSA-V (PVL+). CC96 belonged to CC96-MRSA-IV and CC96-MRSA-IV (PVL+) Central Asian caMRSA/WA MRSA-119. CC97 consisted of six strains including CC97-MRSA-V (fusC+), CC97-MRSA-IV WA MRSA-54/63, CC97-MRSA-V, CC97-MRSA-(V+fus), CC97-MRSA-(mec VI+fus), and CC97-MRSA (mecV/VT+fus+ccrAB2). Whereas CC96 and CC97 isolates were identified in 2016 and 2017, CC398 isolates were detected only in 2016. This study identified four LA-MRSA clones among MRSA isolated from patients in Kuwait hospitals in 2016–2017 with CC97-MRSA-V (fusC+) as the dominant clone. The presence of LA-MRSA with different genetic backgrounds suggests its independent acquisition from different sources.

Detection of Biofilm Formation and Antibiotic Resistance in Klebsiella Oxytoca and Klebsiella Pneumoniae from Animal Origin Foods

Biofilms are surface-attached microbial communities with distinct properties, which have a tremendous impact on our health and food safety. The study was aimed to detect biofilm formation and antibiotic resistance by Klebsiella oxytoca and Klebsiella pneumoniae from animal origin foods. In this study100 food samples were examined for the presence of Klebsiella oxytoca, Klebsiella pneumoniae, and other Enterobacteriaceae family members. In this study, 19 Klebsiella oxytoca and 5 Klebsiella pneumoniae isolates were isolated from cheese and minced meat samples using standard biochemical tests and identification kit. Biofilm formation in these isolates was detected by using microplate, Congo red agar, and tube adherence methods. Antibiotic susceptibility testing was performed using the disk diffusion method on Muller-Hinton agar. Using a microplate method strong biofilm formation was observed in 16 (84%) and 5 (100%) isolates of Klebsiella oxytoca and Klebsiella pneumoniae after 24 hours of incubation on Tryptic Soy Broth medium containing 2% of glucose respectively. After 24 and 48 hours of incubation on Tryptic Soy Broth without glucose strong biofilm formation was detected in 10 (52.6%) and 2 (40%) isolates of Klebsiella oxytoca and Klebsiella pneumoniae respectively. After 24 hours of incubation on Congo red agar 14 (73.7%) and 3 (60%), isolates of Klebsiella oxytoca and Klebsiella pneumoniae were slime factor positive respectively. In tube adherence method 13 (68.4%) and 4 (80%), isolates of Klebsiella oxytoca and Klebsiella pneumoniae were seen to have adhered strongly after 24 hours of incubation on Tryptic Soy Broth medium containing 2% of glucose. In general, strong biofilm formation by these strains was seen on Tryptic Soy Broth medium when supplemented with glucose. Among all the Klebsiella oxytoca, the highest rates of susceptibility were seen toward Trimethoprim-Sulfamethoxazole (100%) and Imipenem (94.7%) followed by Chloramphenicol (73.7%) and gentamicin (68.4%). Among 19 Klebsiella oxytoca isolates, the highest rates of resistance were seen in streptomycin (73.7%) and Kanamycin (73.7%) followed by ampicillin (63.2%). The majority of Klebsiella pneumoniae isolates were resistant to Kanamycin (80%) and Streptomycin (80%) followed by Amikacin (60%). On the other hand, 80% of Klebsiella pneumoniae were susceptible to imipenem, chloramphenicol, Trimethoprim-Sulfamethoxazole, and cefotaxime. Generally, majority of Klebsiella oxytoca, and Klebsiella pneumoniae isolates showed strong biofilm production on different growth conditions and majority of the isolates were also resistance for antibiotics. Therefore, biofilm production by these nosocomial bacteria has an implication public health and pave the way for increased resistance of biofilm-associated organisms to antimicrobial agents.

Molecular detection of antibiotic resistance and virulence genes in staphylococcus species isolated from human and poultry

Staphylococcus species are the most precarious micro-organisms in poultry meat, because they cause health problems to consumers and the economy of the country. Therefore, the current study was aimed to investigate the incidence rate of Staphylococcus species isolated from broiler chicken and hospitalized patient, detection the antimicrobial susceptibility of these isolates by using Vitek2 system and confirmation the presence of genes encoding for pathogenicity and antimicrobial resistance in detected isolates by PCR. To achieve these 200 samples were collected from poultry farms and hospitalized patient (100 for each) in dissimilar districts in Assiut province, to be subjected to bacteriological examination. The results showed that the incidence of Staphylococcus species was 35% and 45% in poultry and human samples respectively on mannitol agar. Vitek2 system divided poultry isolates to 11 isolates as coagulase-positive Staphylococci (CoPS) and 24 isolates as coagulase-negative staphylococci (CoNS). Whereas 20 human isolates alienated as CoPS and 25 isolates assigned as CoNS. Antibiogram cleared that 45.7% and 53.3% of Staphylococcus isolates from poultry and human were identified as methicillin-resistant Staphylococci respectively, also Staphylococcal spp. cleared a resistance to different types of antimicrobials such as penicillin, tetracycline clindamycin. Vitek2 system showed a significant ability to differentiate among Staphylococcus species and distinguish its antimicrobial susceptibility which was complicated by conventional method. PCR results showed that the Staphylococcal isolates of poultry and human were harboring genes encoding for pathogenicity (coa, hld and pvl) and antimicrobial resistance (mecA, vanA, cfr and BlaZ) while none of the isolates harbored sei and seh genes.

Conventional culture diagnostics vs. multiplex PCR for the detection of causative agents of vascular graft infections - results of a single centre observational pilot study.

Background: Timely diagnosis of vascular graft infections is of major importance in vascular surgery. The detection of causative microorganisms is needed for specific medical treatment, but conventional culture is often slow, insensitive and inconclusive due to antibiotic pre-treatment. Detection of bacterial DNA by polymerase chain reaction (PCR) might bypass these problems. We hypothesised that multiplex PCR (mPCR) is feasible, fast and sensitive to detect causative microorganisms in vascular graft infections. Patients and methods: We performed a pilot observational prospective study comparing conventional culture and a commercial mPCR. Inclusion criteria were: confirmed graft infection, suspicious imaging, clinical suspicion, anastomotic aneurysm and repeated graft occlusion. Diagnostic methods were performed using identical samples. Time to result, microorganisms and antibiotic resistance in both groups were compared using Student's t-test or nonparametric tests. Results: 22 samples from 13 patients were assessed and 11 samples were negative for bacteria. Some showed multiple germs. In total, we found 15 different organisms. 13 samples matched, 9 had non-concordant results. Out of the mismatches 3 microorganisms identified in PCR were not detected by culture. Time to result with PCR was shorter (median 5h vs. 72h, p<0.001) than with culture. No resistance genes were detected by mPCR, but conventional culture allowed susceptibility testing and revealed resistance in 5 samples. Conclusions: mPCR seems to be a feasible and quick tool to detect causes of vascular graft infections within 24h and might be helpful in antibiotic pre-treated patients. The detection of antibiotic resistance with mPCR needs improvement for clinical practice.

Combination of mass spectrometry and DNA sequencing for detection of antibiotic resistance in diagnostic laboratories.

In the last two decades, microbiology laboratories have radically changed by the introduction of novel technologies, like Next-Generation Sequencing (NGS) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Nevertheless, emergence of antibiotic-resistant microorganisms represents a global threat of current medicine, being responsible for increasing mortality and health-care direct and indirect costs. In addition, the identification of antibiotic-resistant microorganisms, like OXA-48 carbapenemase-producing Enterobacteriaceae, has been changeling for clinical microbiology laboratories. Even the cost of NGS technology and MALDI-TOF MS equipment is relatively high, both technologies are increasingly used in diagnostic and research protocols. Therefore, the aim of this review is to present applications of these technologies used in clinical microbiology, especially in detection of antibiotic resistance and its surveillance, and to propose a combinatory approach of MALDI-TOF MS and NGS for the investigation of microbial associated infections.

Coagulase negative staphylococci in Anti-Cancer Center, Batna, Algeria: antibiotic resistance pattern, biofilm formation, and detection of mecA and icaAD genes

Background : Coagulase-negative staphylococci (CoNS) are normal microbial flora found on the skin and mucous membranes of mammals. Considered for a long time as avirulent commensals, these bacteria are now recognized as opportunistic pathogens by virtue of their high resistance to multiple antibiotics and capacity for biofilm formations, which made them important agents of nosocomial and community-acquired infections. The objectives of this study are to determine the antibiotic resistance pattern and biofilm formation, and to detect mecA and icaAD genes in clinical CoNS isolates from Batna’s Anti-Cancer Center (ACC) in Algeria. Methods : A total of 66 CoNS were isolated from different samples and identified by API Staph system. In vitro antibiotic susceptibility testing (AST) of each isolate to selected antibiotics was determined by the disk diffusion method, and minimum inhibitory concentrations (MICs) of oxacillin and vancomycin were determined by E-test. Biofilm formation was assessed by Tissue Culture Plate (TCP) and Congo Red Agar (CRA) methods. The polymerase chain reaction (PCR) was used to amplify mecA gene in 9 oxacillin-resistant and 1 oxacillinsensitive CoNS, and icaAD gene in 9 biofilm forming and 1 non-biofilm forming CoNS. Sequencing of the 16S rDNA of 1 mecA and 1 icaAD positive isolates was performed by the Sanger method. Results : Nine species of CoNS were identified, with Staphylococcus epidermidis (n=29, 44%) and Staphylococcus haemolyticus (n=15, 22.7%) constituting the largest proportion, and isolated mainly from the onco-haematology service unit of the center. The isolates were resistant to penicillin G (98.5%), cefoxitin (80.3%) and oxacillin (72.2%). The TCP method was more sensitive (89.4%) than CRA method (31.8%) in detecting biofilm formation. The mecA gene was detected in 66.7% (6/9) of oxacillin resistant CoNS and the icaAD gene in 55.6% (5/9) of TCP positive CoNS isolates Conclusion : Invitro resistance to methicillin (oxacillin) and biofilm formation were high among the CoNS isolates in this study, but the association of these with respective carriage of mecA and icaAD genes was low. Keywords : Coagulase negative staphylococci, identification, antibiotic resistance, biofilm, PCR

Rapid detection of antibiotic resistance in positive blood cultures by MALDI-TOF MS and an automated and optimized MBT-ASTRA protocol for Escherichia coli and Klebsiella pneumoniae

Introduction: For fast and effective antibiotic therapy of serious infections like sepsis, it is crucial with rapid information about antibiotic susceptibility, especially in a time when the number of infections caused by multi resistant bacteria has escalated in the world.Methods: Here, we have used a semi-quantitative MALDI-TOF-MS based method for antibiotic resistance detection, MBT-ASTRA™, which is based on the comparison of growth rate of the bacteria cultivated with and without antibiotics. We demonstrate a new protocol where several parameters have been optimized and automated leading to reduced hands-on time and improved capacity to simultaneously analyse multiple clinical samples and antibiotics.Results: Ninety minutes of incubation at 37 °C with agitation was sufficient to differentiate the susceptible and resistant strains of E. coli and K. pneumoniae, for the antibiotics cefotaxime, meropenem and ciprofloxacin. In total, 841 positive blood culture analyses of 14 reference strains were performed. The overall sensitivity was 99%, specificity 99% and the accuracy 97%. The assay gave no errors for cefotaxime (n = 263) or meropenem (n = 289) for sensitive and resistant strains, whilst ciprofloxacin (n = 289) gave six (0.7%) major errors (false resistance) and four (0.5%) very major errors (false susceptibility). The intermediate strains showed a larger variety compared to the E-test MIC values.Conclusions: The hands-on time and the analysis time to detect antibiotic resistance of clinical blood samples can be substantially reduced and the sample capacity can be increased by using automation and this improved protocol.