High Frequency Of Resistance Research Articles

Analysis of AcrB in Klebsiella pneumoniae reveals natural variants promoting enhanced multidrug resistance

Infections caused by Klebsiella pneumoniae are often difficult to manage due to the high frequency of multidrug resistance, often conferred by efflux pumps. In this study, we analyzed sequence variations of the major RND family multidrug efflux pump AcrB from 387 assembled K. pneumoniae genomes. We confirm that AcrB is a highly-conserved efflux pump in K. pneumoniae, and identified several variants that were prevalent in clinical isolates. Molecular dynamics analyses on two of these variants (L118M and S966A) suggested conformational changes that may correlate with increased drug efflux capabilities. The L118M change resulted in enhanced protein rigidity while the flexibility of drug binding pockets was stable or increased, and the interactions between the proximal pockets and water molecules were stronger. For S966A, the significantly enlarged proximal pocket suggested higher drug accommodation ability. Consistent with these predictions, the L118M and S966A variants conferred a slightly increased ability to grow in the presence of tetracycline and to survive cefoxitin exposure when overexpressed. In summary, our results suggest that the emergence of enhanced-function AcrB variants may be a potential risk for increased antibiotic resistance in clinical K. pneumoniae isolates.

Occurrence, Phenotypic and Molecular Characteristics of Vancomycin-Resistant Enterococci Isolated from Retail Raw Milk in Egypt.

The aim of this study was to determine the occurrence, phenotypic and molecular characteristics of vancomycin-resistant enterococci (VRE), isolated from retail raw cow's milk. One hundred milk samples collected from retail shops in Egypt were examined for the occurrence of VRE by using kanamycin aesculin azide agar supplemented with 4 μg/mL vancomycin. PCR was conducted to determine enterococcal species and to screen the isolated strains for the presence of antibiotic resistance and virulence genes. All isolated strains were characterized by antimicrobial susceptibility testing for 12 antibiotics. From 24 samples (24%), we recovered 22 isolates (91.6%) classified as VRE (minimum inhibitory concentration ≥32) and 2 isolates (8.3%) classified as intermediate resistant to vancomycin (≤16). Enterococcus faecium (29.1%), Enterococcus faecalis (12.5%), Enterococcus casseliflavus (16.6%), and Enterococcus gallinarum (4.1%) were identified by using multiplex PCR. The genus Enterococcus was resistant to clindamycin (100%), linezolid (91.6%), teicoplanin (91.6%), erythromycin (87.5%), and tetracycline (29.1%). Co-resistance to vancomycin, teicoplanin, and linezolid was detected in 83.3% of isolates. Antibiotic resistance genes vanB, tet(M), tet(L), and erm(B) were identified in 29.1%, 16.6%, 8.3%, and 4.1% of isolates, respectively. Virulence genes gelE and esp were detected in 16.6% and 12.5% of isolates, respectively. In conclusion, the high occurrence of co-resistance to vancomycin, teicoplanin, and linezolid reported in this study is alarming. The high frequency of linezolid resistance prompts increased the attention of researchers to routinely perform linezolid susceptibility in food isolates. This study declares potential food safety risks from consumption and improper handling of raw milk regarding clinically important bacteria and promotes necessary legislation for forbidding the selling and consumption of retail raw milk.

Prevalence and antibiotic resistance of Escherichia coli O157:H7 in beef at a commercial slaughterhouse in Moro, Kwara State, Nigeria.

BackgroundGastroenteritis due to foodborne disease is a leading cause of death in developing countries. In Nigeria, there is an increasing demand for beef. Yet, there is no surveillance for Escherichia coli O157:H7 contamination of raw beef and little is known about the carriage of this pathogen in Nigeria’s livestock.MethodsA total of 415 samples, including 180 cow carcass swabs, 180 caecal content samples, 16 water samples, 25 hand swabs and 14 knife swabs were collected at a large abattoir in the Moro region of Kwara State, Nigeria. The samples were enriched in modified tryptone broth containing novobiocine, and plated onto Sorbitol–MacConkey agar (Oxoid SR0172E) supplemented with 0.05 mg l−1 cefixime and 2.5 mg l−1 potassium tellurite (Oxoid) (CT-SMAC). Indole-producing isolates were confirmed serologically by serotyping with antisera specific for the O157 and H7 antigens. The E. coli O157:H7 isolates were further tested for their susceptibility to antibiotic agents using the disc diffusion method. Commercially available Gram-negative multi-discs (Oxoid) comprising nitrofurantoin (30 µg), ampicillin (5 µg), ceftazidime (30 µg), gentamicin (10 µg), ciprofloxacin (5 µg), augmentin (30 µg), ofloxacin (5 µg) and cefuroxime (30 µg) were tested.ResultsOverall, 16 (3.9 %) samples were contaminated with E. coli O157:H7, of which 10 (5.6 %) were isolated from carcass swabs, 4 (2.2 %) from caecal content samples and 2 (12.5 %) from water. All isolates were multidrug-resistant (MDR), with resistance to ampicillin, ceftazidime and cefuroxime being the most common.ConclusionThis study provides evidence to suggest that E. coli O157:H7 exists in the beef production chain. The pathogen reveals a high frequency of multidrug resistance, suggesting that consumers and handlers of such meat are at risk of contracting antibiotic-resistant E. coli O157:H7-associated foodborne disease. Routine monitoring of antibiotic resistance is critical to uncovering novel therapeutic strategies that will help inform clinical practice guidelines.

Antimicrobial resistance in Neisseria gonorrhoeae and Mycoplasma genitalium isolates from the private healthcare sector in South Africa: A pilot study.

Reports have emerged globally of antimicrobial resistance (AMR) in Neisseria gonorrhoeae and Mycoplasma genitalium infections. In South Africa (SA), there are substantial differences between private and public healthcare with regard to antimicrobial drug prescribing practice, which could affect AMR patterns of private and public healthcare patients. To perform a pilot study to determine the frequency of AMR of N. gonorrhoeae and M. genitalium in patients accessing SA's private healthcare sector. In this cross-sectional study, N. gonorrhoeae-positive cultures and M. genitalium DNA samples were collected from a private healthcare reference laboratory from August 2018 to August 2019. In N. gonorrhoeae-positive cultures, antimicrobial susceptibility testing was performed, followed by N. gonorrhoeae multiantigen sequence typing (NG-MAST) to determine genetic relatedness of the isolates. To determine macrolide and fluoroquinolone resistance rates, M. genitalium-positive samples were analysed by sequencing the 23S rRNA, gyrA and parC genes. Twenty-one N. gonorrhoeae- and 27 M. genitalium-positive specimens were included in this analysis. High rates of resistance were detected among gonococcal isolates, with 90% resistance to tetracycline, 86% to penicillin and 62% to ciprofloxacin, but no resistance to azithromycin, cefixime and ceftriaxone. NG-MAST revealed genetically diverse isolates with 83% novel NG-MAST sequence types. Macrolide and fluoroquinolone resistance-associated mutations were detected in 18.5% (n=5/27) and 7.4% (n=2/27) of M. genitalium strains, respectively. We observed high frequencies of ciprofloxacin, penicillin and tetracycline resistance in N. gonorrhoeae and macrolide resistance-associated mutations in M. genitalium in private healthcare sector patients in SA. This finding highlights the need to use diagnostics for sexually transmitted infections and to include the private healthcare sector in antimicrobial surveillance and stewardship programmes.

Higher seasonal temperature enhances the occurrence of methicillin resistance of Staphylococcus aureus in house flies (Musca domestica) under hospital and environmental settings.

Antimicrobial resistance (AMR) emergence in commensal and pathogenic bacteria is a global health issue. House flies (Musca domestica) are considered as biological and mechanical vectors for pathogens causing nosocomial infections, including methicillin-resistant Staphylococcus aureus (MRSA). However, the prevalence of antimicrobial resistance and the role of temperature on the occurrence of Staphylococcus aureus and MRSA in house flies in a hospital environment have not been studied. A total of 400 house flies were collected in winter and summer from four hospital-associated areas in Mymensingh, Bangladesh. Detection of S. aureus and MRSA in flies was done by culturing, staining, and PCR methods targeting nuc and mec genes (mecA and mecC), respectively. Disc diffusion test was used to detect resistance phenotype against six antimicrobials. Logistic regression models were constructed to assess the effect of temperature on the frequency of antimicrobial resistance, and on the presence of the nuc and mecA genes, and location of samples in and around a hospital environment. By PCR, S. aureus was detected in 208 (52%) samples. High frequencies of resistance (≥ 80% of isolates) to amoxicillin, azithromycin, and oxacillin were observed by disk diffusion test. Increase in temperature had a positive effect on the occurrence of S. aureus and MRSA isolates as well as on their resistance to individual and multiple antimicrobials. Among the study areas, hospital premises had increased odds of having S. aureus. Increased temperature of summer significantly increased the occurrence of MRSA in house flies in and around the hospital environment, which might pose a human and animal health risk.

Antimicrobial Susceptibility Pattern and Molecular Identification of Acinetobacter baumannii in Alex Ekwueme-Federal University Teaching Hospital Abakaliki, Nigeria

Background and Objectives: Acinetobacter baumannii, a notorious opportunistic pathogen known to seriously affect debilitated individuals especially intensive care unit (ICU) patients and others with underlying illness, have consistently jeopardized many antibiotics. This study was therefore aimed to ascertain the antimicrobial susceptibility profile and molecularly identify A. baumannii pathogens in Alex Ekwueme-Federal University Teaching Hospital, Abakaliki, Nigeria.
 Methodology: A total of 385 clinical samples were collected aseptically from debilitated patients and analyzed following standard microbiological procedures. Acinetobacter species was confirmed by Gram staining reaction and biochemical tests. All bacterial isolates were phenotypically screened for multidrug resistance using the Kirby–Bauer disc diffusion technique and results interpreted as per CLSI criteria. A. baumannii isolates were finally confirmed using 16S rRNA sequencing.
 Results: A total of 23(6%) A. baumannii isolates were recovered from 385 clinical samples collected from 87 patients comprising 48 males and 39 females admitted in various hospital wards of AE-FETHA. The age of the patients varied from 20–79 years. The commonest sites for isolation of A. baumannii pathogen were catheter urine (8/8%) and wound sores (7/8%). The highest percentage resistance was observed with cefuroxime (96%), tetracycline (96%), sulfamethoxazole/trimethoprim (96 %), and ofloxacin (91%) while meropenem (91%) and imipenem (78%) were the most effective antibiotics against A. baumannii. The isolated A. baumannii was re-confirmed genotypically by 16S rRNA gene amplification. Variations were observed in the gene sequence of all the isolated A. baumannii. 
 Conclusion: Catheter urine, wound sores, and respiratory fluids were the more easily colonized samples. Also, high frequency of multidrug resistance observed in this study further established A. baumannii as a notorious opportunistic pathogen.

Metabolic Resistance in Permethrin-Resistant Florida Aedes aegypti (Diptera: Culicidae).

Simple SummaryPyrethroid-oriented vector control programs have increased worldwide to control adult Aedes aegypti mosquitoes and quell Aedes-borne disease epidemics. Due to years of pyrethroid use, resistance to pyrethroids in Ae. aegypti has become a global issue. In Florida, permethrin is the most common pyrethroid adulticide active ingredient used to control mosquito populations. Thus far, all wild Florida Ae. aegypti populations tested against permethrin have been found to be resistant. Metabolic resistance is a major mechanism of resistance in insects in which enzyme-mediated reactions cause the degradation or sequestration of insecticides. We performed assays to investigate the presence of metabolic resistance in 20 Florida Ae. aegypti populations and found that 11 populations (55%) exhibited metabolic resistance due to the action of at least one of three classes of metabolizing enzymes: oxidases, esterases, and glutathione transferases. Additionally, we identified two metabolic enzyme inhibitors: S.S.S-tributyl phosphorotrithioate (DEF; inhibits esterase activity) and diethyl maleate (DM; inhibits glutathione transferase activity), in addition to the commonly used piperonyl butoxide (PBO; inhibits oxidase activity), which were able to increase the efficacy of permethrin against resistant Ae. aegypti populations. Pre-exposure to DEF, PBO, and DM resulted in increased mortality after permethrin exposure in eight (73%), seven (64%), and six (55%) of the Ae. aegypti populations, respectively. Increasing the effectiveness of pyrethroids is important for mosquito control, as it is the primary method used for adult control during mosquito-borne disease outbreaks. Considering that DEF and DM performed similarly to PBO, they may be good candidates for inclusion in formulated pyrethroid products to increase their efficacy against resistant mosquitoes.Aedes aegypti is the principal mosquito vector for many arthropod-borne viruses (arboviruses) including dengue, chikungunya, and Zika. In the United States, excessive permethrin use has led to a high frequency of resistance in mosquitoes. Insecticide resistance is a significant obstacle in the struggle against vector-borne diseases. To help overcome metabolic resistance, synergists that inhibit specific metabolic enzymes can be added to formulated pyrethroid products. Using modified CDC bottle bioassays, we assessed the effect of three inhibitors (piperonyl butoxide (PBO), which inhibits oxidase activity; S.S.S-tributyl phosphorotrithioate (DEF), which inhibits esterase activity; and diethyl maleate (DM), which inhibits glutathione transferase activity) + permethrin. We performed these against 20 Florida Ae. aegypti populations, all of which were resistant to permethrin. Our data indicated that 11 out of 20 populations (55%) exhibited metabolic resistance. Results revealed 73% of these populations had significant increases in mortality attributed to DEF + permethrin, 64% to PBO + permethrin, and 55% to DM + permethrin compared to permethrin alone. Currently, PBO is the only metabolic enzyme inhibitor added to formulated pyrethroid products used for adult mosquito control. Our results suggest that the DEF and DM inhibitors could also be useful additives in permethrin products, especially against metabolically resistant Ae. aegypti mosquitoes. Moreover, metabolic assays should be conducted to better inform mosquito control programs for designing and implementing integrated vector management strategies.

Resistance and virulence distribution in enterococci isolated from broilers reared in two farming systems

BackgroundThe impact of enterococci in human health has been growing for the last decades, mainly due to their resistance to several antimicrobial agents. Human consumption of contaminated meat, especially poultry, has been identified as a possible route of transmission. The aim of the present study was to evaluate and compare the antimicrobial resistance profiles and virulence genes of enterococci isolated from Portuguese conventional and free-range broiler farms.ResultsAntibiotic susceptibility testing showed high frequencies of resistance to tetracycline in both farming systems. Resistance to erythromycin and gentamicin were detected in about half of the isolates. Resistance to penicillin was the less frequently observed and no vancomycin resistant isolates were identified. The majority of the enterococcal isolates, from either farming systems, were resistant to more than one antibiotic, and no statistical associations were found, except for penicillin resistance which associated with the genetic clusters. No differences were found between farming systems regarding the prevalence of tet(M), erm(B), aac (6′)-Ie-aph (2″)-Ia and pbp5 genes, nevertheless pbp5 prevalence was associated with the different genetic clusters. Hemolytic activity was identified in 26.47% of all isolates and gelatinase activity in 50%. The gelE gene was identified in the majority of the isolates, whereas esp and agg genes were rarely detected. The cylA determinant was not detected in any of the isolates.ConclusionsOverall, results suggest that similar resistance patterns and virulence genes can be found in both farming systems, though enterococci in free-range conditions should be less prone to acquire further resistance genes.

Identification of antibiotic pairs that evade concurrent resistance via a retrospective analysis of antimicrobial susceptibility test results

SummaryBackgroundSome antibiotic pairs display a property known as collateral sensitivity in which the evolution of resistance to one antibiotic increases sensitivity to the other. Alternating between collaterally sensitive antibiotics has been proposed as a sustainable solution to the problem of antibiotic resistance. We aimed to identify antibiotic pairs that could be considered for treatment strategies based on alternating antibiotics.MethodsWe did a retrospective analysis of 448 563 antimicrobial susceptibility test results acquired over a 4-year period (Jan 1, 2015, to Dec 31, 2018) from 23 hospitals in the University of Pittsburgh Medical Center (Pittsburgh, PA, USA) hospital system. We used a score based on mutual information to identify pairs of antibiotics displaying disjoint resistance, wherein resistance to one antibiotic is commonly associated with susceptibility to the other and vice versa. We applied this approach to the six most frequently isolated bacterial pathogens (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa, and Proteus mirabilis) and subpopulations of each created by conditioning on resistance to individual antibiotics. To identify higher-order antibiotic interactions, we predicted rates of multidrug resistance for triplets of antibiotics using Markov random fields and compared these to the observed rates.FindingsWe identified 69 antibiotic pairs displaying varying degrees of disjoint resistance for subpopulations of the six bacterial species. However, disjoint resistance was rarely conserved at the species level, with only 6 (0·7%) of 875 antibiotic pairs showing evidence of disjoint resistance. Instead, more than half of antibiotic pairs (465 [53·1%] of 875) exhibited signatures of concurrent resistance, whereby resistance to one antibiotic is associated with resistance to another. We found concurrent resistance to extend to more than two antibiotics, with observed rates of resistance to three antibiotics being higher than predicted from pairwise information alone.InterpretationThe high frequency of concurrent resistance shows that bacteria have means of counteracting multiple antibiotics at a time. The almost complete absence of disjoint resistance at the species level implies that treatment strategies based on alternating between antibiotics might require subspecies level pathogen identification and be limited to a few antibiotic pairings.FundingUS National Institutes of Health.

The Molecular Epidemiology of Resistance to Antibiotics among Klebsiella pneumoniae Isolates in Azerbaijan, Iran.

Introduction Klebsiella pneumoniae (K. pneumoniae) is one of the leading causes of hospital-acquired and community-acquired infections in the world. This study was conducted to investigate the molecular epidemiology of drug resistance in clinical isolates of K. pneumoniae in Azerbaijan, Iran. Materials and Methods A total of 100 nonduplicated isolates were obtained from the different wards of Azerbaijan state hospitals, Iran, from 2019 to 2020. Antibiotic susceptibility testing was done. The DNA was extracted, and the PCR for evaluation of the resistance genes was carried out. Results The highest antibiotic resistance was shown to ampicillin (96%), and the highest susceptibility was shown to tigecycline (9%), and 85% of isolates were multidrug resistant. The most frequent ESBL gene in the tested isolates was blaSHV-1 in 58%, followed by blaCTXM-15 (55%) and blaSHV-11(42%). The qepA, oqxB, and oqxA genes were found to be 95%, 87.5%, and 70%, respectively. We detected tetB in 42%, tetA in 32%, tetD in 21%, and tetC in 16%. Seventy isolates were resistant to co-trimoxazole, and the rate of resistance genes was sul1 in 71%, followed by sul2 (43%), dfr (29%), and sul3 (7%). The most common aminoglycoside resistance genes were ant3Ia, aac6Ib, aph3Ib, and APHs in 44%, 32%, 32%, and 31.4%, respectively. The most frequent resistance gene to fosfomycin was fosA (40%) and fosX (40%) followed by fosC (20%). Conclusion The results of this study indicate the high frequency of drug resistance among K. pneumoniae isolated from hospitals of Azerbaijan state. The present study shows the presence of high levels of drug-resistant genes in various antibiotics, which are usually used in the treatment of infections due to K. pneumoniae.

Virulence associated gene profiling and antimicrobial resistance pattern of Streptococcus suis isolated from clinically healthy pigs from North East India.

This study revealed the prevalence of Streptococcus suis in 20·39% clinically healthy pigs from North East India. All these isolates were screened for the presence of virulence- associated genes such as suilysin (sly), muramidase released protein (mrp), extracellular protein factor (epf) and arginine deiminase (arcA). Of these 62 isolates, 29 isolates carried mrp gene, 17 isolates carried sly gene, 57 isolates carried arcA gene, whereas all isolates were negative for epf gene. The most prevalent genotype was mrp- sly- epf- arcA+ (45·16%) followed by genotypes mrp+ sly- epf- arcA+ (27·41%), mrp+ sly+ epf- arcA+ (19·35%) and mrp- sly+ epf- arcA- (8·06%). High frequency of resistance was observed for antimicrobials such as tetracycline (93·54%), clindamycin (91·93%), co-trimoxazole (88·70%) and erythromycin (85·48%). Antimicrobial resistance patterns of the S. suis isolates revealed 16 resistance groups (R1 to R16), where 93·54% isolates showed multi-drug resistance (≥3 antimicrobial agents). It has also been observed that 57 (91·93%) isolates were resistant to at least four antimicrobials. The most predominant resistance pattern observed was CD-COT-E-TE, which accounted for 38·70% of the isolates. The occurrence of relatively high levels of resistance of S. suis to some antimicrobials (e.g., macrolides, tetracyclines, and sulphonamides) as observed in this study may represent a human health concern. In addition, a relatively higher percentage of S. suis isolated from clinically healthy pigs indicates a carrier status with risk of dissemination to other pigs in the herd as well as to humans.

Area-wide insecticide resistance and endosymbiont incidence in the whitefly Bemisia tabaci MEAM1 (B biotype): A Neotropical context.

Agriculture insecticides are used against insect pest species, but are able to change community structure in contaminated habitats, and also the genetic pool of exposed individuals. In fact, the latter effect is a relevant tool to in situ biomonitoring of pollutant contamination and impact, besides its practical economic and management concerns. This takes place because the emergence of individuals with resistance to insecticides is particularly frequent among insect pest species and usually enhances insecticide overuse and crop losses. Pest insects of global prominence such as whiteflies are a focus of attention due to problems with insecticide resistance and association with endosymbionts, as the case of the invasive putative species Bemisia tabaci MEAM1. The scenario is particularly complex in the Neotropics, where insecticide use is ubiquitous, but whose spatial scale of occurrence is usually neglected. Here we explored the spatial-dependence of both phenomena in MEAM1 whiteflies recording resistance to two widely used insecticides, lambda-cyhalothrin and spiromesifen, and endosymbiont co-occurrence. Resistance to both insecticides was frequent exhibiting low to moderate frequency of lambda-cyhalothrin resistance and moderate to high frequency of spiromesifen resistance. Among the prevailing whitefly endosymbionts, Wolbachia, Cardinium and Arsenophonus were markedly absent. In contrast, Hamiltonella and Rickettsia prevailed and their incidence was correlated. Furthermore, Rickettsia endosymbionts were particularly associated with lambda-cyhalothrin susceptibility. These traits were spatially dependent with significant variation taking place within an area of about 700 Km2. Such findings reinforce the notion of endosymbiont-associated resistance to insecticides, and also of their local incidence allowing spatial mapping and locally-targeted mitigation.

RETRACTED: Safety of Commercially Available Beef Burger in Saudi Arabia

The safety of meat has been at the forefront of societal concerns in recent years, and indications exist that challenges to meat safety will continue in the future. Major meat safety issues and related challenges include the need to control traditional and emerging pathogenic microorganisms, such as increased virulence and low infectious doses or resistance to antibiotics or food-related stresses. This study aimed to recognize microbial contamination and heavy metals content. Thirty-eight frozen and freshly prepared burger (local and imported) samples were collected from randomly selected supermarkets and fast-food restaurants in Jeddah. Yeasts/Molds had the highest count (204.3 CFU/mL) followed by total aerobic mesophiles (69.5 CFU/mL), total coliforms (16.2 CFU/mL) and Escherichia coli (10.0 CFU/mL). Salmonella species were positive in 39.5% of samples. Fresh burgers had more counts of TVC, total coliforms, Escherichia coli, and Bacillus cereus. Amoxicillin-clavulanate and Ampicillin had a high frequency of resistance in the studied sample. None of the studied samples had detectable traces of heavy metals’ elements. This research provides valid data to protect consumers from different health risks related to burgers in Saudi Arabia.

Antimicrobial susceptibility profile of Streptococcus agalactiae strains isolated from bovine mastitis

Bovine mastitis is the most important disease of dairy herds worldwide. Its main etiologic agents are bacteria, including Streptococcus agalactiae. The importance of this agent in bovine mastitis is because it is highly contagious and has a high impact on the occurrence of clinical mastitis cases and in the increase of the bulk milk somatic cell counts. The dry cow therapy and the treatment of the clinical mastitis cases stand out among the measures to control intramammary infections in cows. However, these strategies require knowledge about the antimicrobial susceptibility of the causal microorganisms. Thus, this study aimed to evaluate the antimicrobial susceptibility of 89 S. agalactiae strains isolated from bovine mastitis between the years 2004 and 2008 in dairy herds from Campo das Vertentes region, Minas Gerais State, Brazil. The disc diffusion technique was used and the antimicrobials currently used in mastitis therapy were tested. The isolates tested showed 100% susceptibility to chloramphenicol, ceftiofur, cefotaxime, enrofloxacin, and cefquinome. High frequencies of susceptibility (>95%) were also observed for the beta-lactams (penicillin G, ampicillin, and oxacillin), cephalosporins (cephalotin, ceftiofur, cefotaxime, cefoperazone, and cefquinome), florfenicol, gentamicin, lincomycin, nitrofurantoin, and sulfamethoprim. The strains showed high frequencies of resistance to neomycin (15.74%), and tetracycline (21.35%). Multidrug resistance was detected in 2.25% of the tested isolates. The results pointed to variations in the antimicrobial susceptibility profiles of the studied strains and the importance of the use of the susceptibility tests to determine the correct antimicrobial to be applied in the treatment of bovine mastitis caused by S. agalactiae. The high frequencies of resistance observed to some antimicrobials, such as neomycin and tetracycline, commonly used in the treatment of mastitis and other pathologies, highlighted the need for more judicious use of antimicrobials on dairy farms.

Prevalence and Antimicrobial Resistance of Listeria monocytogenes in Raw Milk in Tehran, Iran

Background and Objective: One of the major sources of Listeria monocytogenes, as the causative agent of invasive listeriosis, is raw milk. The aim of the present study was to detect Listeria monocytogenes in raw milk samples collected from dairy stores in Tehran, Iran, 2019. Material and Methods: A total of 100 raw milk samples were assessed using cultural techniques. Furthermore, antimicrobial resistance profiles of the Listeria isolates were assessed against eight antimicrobials using disc diffusion method. Results and Conclusion: Listeria spp., including Listeria grayi (5%), Listeria ivanovii (3%) and Listeria monocytogenes (2%), were detected in 10% of the samples. Listeria monocytogenes isolates were susceptible to major antimicrobials used for the treatment of listeriosis with no multidrug resistances. The highest frequencies of resistance were seen against streptomycin (60%), gentamicin (50%) and tetracycline (50%). In conclusion, potential risks of listeriosis still threaten consumers of the raw milk in Tehran. Conflict of interest: The authors declare no conflict of interest.

Comparative proteomics and variations in extracellular matrix of Candida tropicalis biofilm in response to citral.

Candida tropicalis is an opportunistic human pathogen with an ability to cause superficial as well as systemic infections in immunocompromised patients. The formation of biofilm by C. tropicalis can cause dreadful and persistent infections which are difficult to treat due to acquired resistance. Presently, available anti-Candida drugs exhibit a high frequency of resistance, low specificity and toxicity at a higher dosage. In addition, the discovery of natural or synthetic anti-Candida drugs is slow paced and often does not pass clinical trials. Citral, a monoterpene aldehyde, has shown effective antimicrobial activities against various microorganisms. However, only few studies have elaborated the action of citral against the biofilm of C. tropicalis. In the present work, the aim was to study the fungicidal effect, differential expression of proteome and changes in extracellular matrix in response to the sub-lethal concentration (16µg/mL) of citral. The administration of citral on C. tropicalis biofilm leads to a fungicidal effect. Furthermore, the differential expression of proteome has revealed twenty-five proteins in C. tropicalis biofilm, which were differentially expressed in the presence of citral. Among these, amino acid biosynthesis (Met6p, Gln1p, Pha2p); nucleotide biosynthesis (Xpt1p); carbohydrate metabolism (Eno1p, Fba1p, Gpm1p); sterol biosynthesis (Mvd1p/Erg19p, Hem13p); energy metabolism (Dnm1p, Coa1p, Ndk1p, Atp2p, Atp4p, Hts1p); oxidative stress (Hda2p, Gre22p, Tsa1p, Pst2p, Sod2p) and biofilm-specific (Adh1p, Ape1p, Gsp1p) proteins were identified. The overexpression of oxidative stress-related proteins indicates the response of biofilm cell to combating oxidative stress during citral treatment. Moreover, the upregulation of Adh1p is of particular interest because it subsidizes the biofilm inhibition through ethanol production as a cellular response. The augmented expression of Mvd1p/Erg19p signifies the effect of citral on ergosterol biosynthesis. The presence of citral has also shown an increment in hexosamine and ergosterol component in extracellular matrix of C. tropicalis biofilm. Hence, it is indicated that the cellular response towards citral acts through multifactorial processes. This study will further help in the interpretation of the effect of citral on C. tropicalis biofilm and development of novel antifungal agents against these potential protein targets.

Molecular detection of Extended-Spectrum β-lactamases (ESBLs) and biofilm formation in uropathogen Klebsiella pneumoniae in Iran.

Background: Uropathogenic Klebsiella pneumoniae is one of the well-kown uropathogens that have the main rule in biofilm formation. Increased prevalence of ESBL enzyme is one of the therapeutic problems. However, the aims of this study were to characterize the ability of biofilm formation and ESBL-producing isolates produced by urinary tract infection’s K. pneumoniae to identify the prevalence of this type of infection in the studied area. Methods: Between the 500 nonrepetitive clinical isolates, 128 isolates were detected as K. pneumoniae. Biofilm production of these isolates was showed by Merrit and Christensen method. The standard Kirby-Bauer disk diffusion method was used for antimicrobial susceptibility testing. The phenotype ESBL was confirmed by double disc synergy test (DDST). Genotypic identification of ESBLs did by molecular detection. The statistical analysis was done using software IBM SPSS Statistics (SPSS Inc) and chi-square and Fisher exact tests. Results: The result of microtiter plate was observed and it was found that 86 (67.2%) isolates had weak biofilm, 24 (18.8%) moderate biofilm, and 18 (14.1%) strong biofilm. Also, 57 (44.5%) out of 128 isolates were diagnosed as MDR. The highest frequency of resistance was identified for cefotaxime 60 (46.9%) and tetracycline 60 (46.9%), and the lowest rate was for amikacin 16 (12.5%). The results of DDST showed 55 of 128 (43%) produced ESBL enzymes. PCR detection in ESBL-producing isolates showed contained blaTEM 33 of 55(63.1%), and blaVEB 13 of 55 (23% ). Also, 1 of 55 (2%) had both blaTEM and blaVEB. Also, 5 of 13 (38.4%) isolates that had the blaVEB gene were also MDR and had weak biofilm (8/13; 61.5%), intermediate biofilm (3/13; 23%), and strong biofilm (2/13; 15.4%). Conclusion: To decrease treatment complications and mortality rate of drug-resistant bacterial infections, rapid detection of β-lactamases genes and evaluation of these properties and infection management programs can help to prevent the transmission of drug resistant-strains.

DNA Breaks-Mediated Fitness Cost Reveals RNase HI as a New Target for Selectively Eliminating Antibiotic-Resistant Bacteria.

Antibiotic resistance often generates defects in bacterial growth called fitness cost. Understanding the causes of this cost is of paramount importance, as it is one of the main determinants of the prevalence of resistances upon reducing antibiotics use. Here we show that the fitness costs of antibiotic resistance mutations that affect transcription and translation in Escherichia coli strongly correlate with DNA breaks, which are generated via transcription–translation uncoupling, increased formation of RNA–DNA hybrids (R-loops), and elevated replication–transcription conflicts. We also demonstrated that the mechanisms generating DNA breaks are repeatedly targeted by compensatory evolution, and that DNA breaks and the cost of resistance can be increased by targeting the RNase HI, which specifically degrades R-loops. We further show that the DNA damage and thus the fitness cost caused by lack of RNase HI function drive resistant clones to extinction in populations with high initial frequency of resistance, both in laboratory conditions and in a mouse model of gut colonization. Thus, RNase HI provides a target specific against resistant bacteria, which we validate using a repurposed drug. In summary, we revealed key mechanisms underlying the fitness cost of antibiotic resistance mutations that can be exploited to specifically eliminate resistant bacteria.

PREVALENCE OF ANTIMICROBIAL RESISTANCE AND MOLECULAR CHARACTERIZATION OF CARBAPENEMASE ENCODING GENE IN ESCHERICHIA COLI ISOLATES IN HOSPITAL OF HUE UNIVERSITY OF MEDICINE AND PHARMACY

Background: Currently, the existence of Extended-spectrum beta-lactamases (ESBL) and carbapenemase genes as well as genetic diversity has led to the rapid spread of resistance between E. coli species and the emergence of bacteria with pan-resistance. Objectives: To investigate the prevalence of antimicrobial resistance, prevalence of ESBL-producing E. coli, and carbapenemase-coding gene in E. coli isolates at Hospital of Hue University of Medicine and Pharmacy. Materials and method: The susceptibility to 13 antimicrobials of 246 E. coli isolates was detemined by the Kirby-Bauer method. ESBL-producing potential E. coli isolates were confirmed phenotypic by the Double Disc Synergy Test (DDST) and Combination Disc Test (CDT). Using multiplex PCR to detect genes encoding carbapenemase belonging to class B (Metallo β-lactamase), class D (oxacillinase), and blaKPC gene. Sequencing of carbapenemase genes were used to identify carbapenemase variants. Results: 91.46% of E. coli strains were identified as multidrug-resistant; most of them resistant to penicillins, fluoroquinolone, sulfonamide; high frequency of resistance to 3rd generation cephalosporin, chloramphenicol, gentamycin. The high isolation rate of ESBL-producing E. coli (50%) was detected. The resistance to carbapenems was largely mediated by the expression of acquired carbapenemases: blaKPC-2, blaNDM-4, and blaOXA-48. This study first reported blaNDM-4-harboring E. coli isolates in Vietnam. Conclusion: The high prevalence of multidrug-resistant and ESBL-producing E. coli, as well as the coexistence of ESBLs and carbapenemase genes, could seriously limit options for clinical treatment.

Clonal Lineages, Antimicrobial Resistance, and PVL Carriage of Staphylococcus aureus Associated to Skin and Soft-Tissue Infections from Ambulatory Patients in Portugal.

Staphylococcus aureus (S. aureus) is a leading cause of skin and soft-tissue infections (SSTIs) in the community. In this study, we characterized a collection of 34 S. aureus from SSTIs in ambulatory patients in Portugal and analyzed the presence of Panton–Valentine leucocidin (PVL)-encoding genes and antibiotic-resistance profile, which was correlated with genetic determinants, plasmid carriage, and clonal lineage. Nearly half of the isolates (15, 44.1%) were methicillin-resistant Staphylococcus aureus (MRSA) and/or multidrug resistant (MDR). We also detected resistance to penicillin (33/34, 97.1%), fluoroquinolones (17/34, 50.0%), macrolides and lincosamides (15/34, 44.1%), aminoglycosides (6/34, 17.6%), and fusidic acid (2/34, 5.9%), associated with several combinations of resistance determinants (blaZ, erm(A), erm(C), msr(A), mph(C), aacA-aphD, aadD, aph(3′)-IIIa, fusC), or mutations in target genes (fusA, grlA/gyrA). The collection presented a high genetic diversity (Simpson’s index of 0.92) with prevalence of clonal lineages CC5, CC22, and CC8, which included the MRSA and also most MDR isolates (CC5 and CC22). PVL-encoding genes were found in seven isolates (20.6%), three methicillin-susceptible Staphylococcus aureus (MSSA) (ST152-agrI and ST30-agrIII), and four MRSA (ST8-agrI). Plasmid profiling revealed seventeen distinct plasmid profiles. This work highlights the high frequency of antimicrobial resistance and PVL carriage in SSTIs-related S. aureus outside of the hospital environment.