Carriage Of Genes Research Articles

Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies.

Low microbial biomass and high human DNA content in nasopharyngeal aspirate samples hinder comprehensive characterization of microbiota and resistome. We obtained samples from premature infants, a group with increased risk of developing respiratory disorders and infections, and consequently frequent exposure to antibiotics. Our aim was to devise an optimal protocol for handling nasopharyngeal aspirate samples from premature infants, focusing on host DNA depletion and microbiome and resistome characterization. Three depletion and three DNA extraction protocols were compared, using RT-PCR and whole metagenome sequencing to determine the efficiency of human DNA removal, taxonomic profiling and assignment of antibiotic resistance genes. Protocols were tested using mock communities, as well as pooled and individual patient samples. The only extraction protocol to retrieve the expected DNA yield from mock community samples was based on a lytic method to improve Gram positive recovery (MasterPure™). Host DNA content in non-depleted aliquots from pooled patient samples was 99%. Only samples depleted with MolYsis™ showed satisfactory, but varied reduction in host DNA content, in both pooled and individual patient samples, allowing for microbiome and resistome characterisation (host DNA content from 15% to 98%). Other depletion protocols either retrieved too low total DNA yields, preventing further analysis, or failed to reduce host DNA content. By using Mol_MasterPure protocol on aliquots from pooled patient samples, we increased the number of bacterial reads by 7.6 to 1,725.8-fold compared to non-depleted reference samples. PCR results were indicative of achieved microbial enrichment. Individual patient samples processed with Mol_MasterPure protocol varied greatly in total DNA yield, host DNA content (from 40% to 98%), species and antibiotic resistance gene richness. Despite high human DNA and low microbial biomass content in nasopharynx aspirates of preterm infants, we were able to reduce host DNA content to levels compatible with downstream shotgun metagenomic analysis, including bacterial species identification and coverage of antibiotic resistance genes. Whole metagenomic sequencing of microbes colonizing the nasopharynx may contribute to explaining the possible role of airway microbiota in respiratory conditions and reveal carriage of antibiotic resistance genes.

885. Integrated Blood Culture and Molecular Diagnostic Device Reduces Time-to-Result to a Quarter of Traditional Methods

Abstract Background Prolonged antibiotic exposure in infants with negative blood cultures is associated with adverse outcomes, disruption of the developing microbiome, and increased carriage of antibiotic resistance genes. However, early administration of antibiotics significantly reduces risk of infant morbidity and mortality caused by infections, and therefore faster culture-to-identification is highly desirable. We combined blood culture (BC) and PCR-based pathogen identification into a single digital microfluidic (DMF) device to speed up BC results. Methods Our DMF BC+PCR device has a custom-built BC bottle incubator, which is connected to a disposable cartridge that performs sample preparation and PCR. Standard BC bottles were inoculated with K. pneumoniae, then incubated in either the DMF BC device or a traditional BC device (BD BACTEC 9050). Samples were drawn from the DMF BC bottle at 3, 4 and 5 hours and loaded in the cartridge for automated sample processing and PCR. Time-to-positivity for the DMF BC+PCR device was determined using cycle threshold and compared with the traditional method, which uses fluorescence from BC bottle followed by PCR on a separate device. Results Bacterial presence could be detected as quickly as 3 hours post inoculation (Ct values of 34, 31 and 28 at 3, 4, and 5 hrs) with five replicates of K. pneumoniae samples on the DMF BC+PCR device (growing 10x every hr), compared to > 12 hrs for the traditional method (10.5 hr culture and 2 hr PCR identification). Additionally, for 5 common pathogens that cause neonatal sepsis (K. pneumoniae, E. coli, S. aureus, S. agalactiae, or S. epidermidis), we also demonstrated that the DMF BC bottle incubator and the traditional BC device show similar growth curves using optical methods to determine growth. Conclusion The DMF BC system yields bacterial growth rates equivalent to traditional methods, but can drastically reduce time to pathogen identification, from 12 hrs to 3 hrs by leveraging PCR sensitivity to determine growth rather than fluorescence in the BC bottles. To our knowledge, this is the first demonstration of using an integrated microfluidic PCR platform to speed up culture and identification. Further work is ongoing to include antibiotic susceptibility testing and expand the panel of pathogens detected on the same cartridge. Disclosures Laura Huning, PhD, Baebies, Inc.: Stocks/Bonds Devika Varma, PhD, Baebies, Inc.: Stocks/Bonds Abbey Jackson, PhD, Baebies, Inc.: Stocks/Bonds Peter Bohlen, n/a, Baebies, Inc.: Stocks/Bonds Rainer Ng, PhD, Baebies, Inc.: Stocks/Bonds Vamsee Pamula, PhD, Baebies, Inc.: Stocks/Bonds.

Genomic epidemiology of Escherichia coli: antimicrobial resistance through a One Health lens in sympatric humans, livestock and peri-domestic wildlife in Nairobi, Kenya.

Livestock systems have been proposed as a reservoir for antimicrobial-resistant (AMR) bacteria and AMR genetic determinants that may infect or colonise humans, yet quantitative evidence regarding their epidemiological role remains lacking. Here, we used a combination of genomics, epidemiology and ecology to investigate patterns of AMR gene carriage in Escherichia coli, regarded as a sentinel organism. We conducted a structured epidemiological survey of 99 households across Nairobi, Kenya, and whole genome sequenced E. coli isolates from 311 human, 606 livestock and 399 wildlife faecal samples. We used statistical models to investigate the prevalence of AMR carriage and characterise AMR gene diversity and structure of AMR genes in different host populations across the city. We also investigated household-level risk factors for the exchange of AMR genes between sympatric humans and livestock. We detected 56 unique acquired genes along with 13 point mutations present in variable proportions in human and animal isolates, known to confer resistance to nine antibiotic classes. We find that AMR gene community composition is not associated with host species, but AMR genes were frequently co-located, potentially enabling the acquisition and dispersal of multi-drug resistance in a single step. We find that whilst keeping livestock had no influence on human AMR gene carriage, the potential for AMR transmission across human-livestock interfaces is greatest when manure is poorly disposed of and in larger households. Findings of widespread carriage of AMR bacteria in human and animal populations, including in long-distance wildlife species, in community settings highlight the value of evidence-based surveillance to address antimicrobial resistance on a global scale. Our genomic analysis provided an in-depth understanding of AMR determinants at the interfaces of One Health sectors that will inform AMR prevention and control.

Impact of benzalkonium chloride, benzethonium chloride and chloroxylenol on bacterial antimicrobial resistance.

This review examined 3655 articles on benzalkonium chloride (BKC), benzethonium chloride (BZT) and chloroxylenol (CHO) aiming to understand their impact on antimicrobial resistance. Following the application of inclusion/exclusion criteria, only 230 articles were retained for analysis; 212 concerned BKC, with only 18 for CHO and BZT. Seventy-eight percent of studies used MIC to measure BKC efficacy. Very few studies defined the term 'resistance' and 85% of studies defined 'resistance' as <10-fold increase (40% as low as 2-fold) in MIC. Only a few in vitro studies reported on formulated products and when they did, products performed better. In vitro studies looking at the impact of BKC exposure on bacterial resistance used either a stepwise training protocol or exposure to constant BKC concentrations. In these, BKC exposure resulted in elevated MIC or/and MBC, often associated with efflux, and at time, a change in antibiotic susceptibility profile. The clinical relevance of these findings was, however, neither reported nor addressed. Of note, several studies reported that bacterial strains with an elevated MIC or MBC remained susceptible to the in-use BKC concentration. BKC exposure was shown to reduce bacterial diversity in complex microbial microcosms, although the clinical significance of such a change has not been established. The impact of BKC exposure on the dissemination of resistant genes (notably efflux) remains speculative, although it manifests that clinical, veterinary and food isolates with elevated BKC MIC carried multiple efflux pump genes. The correlation between BKC usage and gene carriage, maintenance and dissemination has also not been established. The lack of clinical interpretation and significance in these studies does not allow to establish with certainty the role of BKC on AMR in practice. The limited literature and BZT and CHO do not allow to conclude that these will impact negatively on emerging bacterial resistance in practice.

Heightened variability observed in resistance and virulence genes across salmonella Kentucky isolates from poultry environments in British Columbia, Canada

Many niche-dependent barriers along the poultry production continuum favour the survival of certain Salmonella serovars over others. Historically, the presence of particular serovars has been determined by niche-specific genes which encode resistance to selective pressures such as host defenses and industrial antimicrobial practices. Over the past decade, Canada has witnessed unexplained shifts in the Salmonella landscape in the poultry sector. Several formerly minor Salmonella serovars, including S. Kentucky and S. Reading, have recently increased in prevalence in live chickens and turkeys, respectively, in British Columbia (BC). The purpose of this research was to investigate the genomic features of the top poultry-associated Salmonella spp. in BC, to probe for serovar-specific characteristics that could address the recently shifting balance of serovars along the poultry continuum. By examining the quantity and diversity of antimicrobial resistance (AMR) genes, virulence factors (VFs), Salmonella Pathogenicity Islands (SPIs), and plasmids across 50 poultry-associated S. enterica isolates using whole genome sequencing and antimicrobial resistance profiling, we have identified serovar-specific differences that likely influence niche survival. Specifically, isolates in our collection from predominantly human pathogenic serovars (S. I 4, [5], 12:i: , S. Typhimurium, and S. Enteritidis) were found to share the IncFIB(S) and IncFII(S) plasmids which carry important VFs known to aid in human host infection. Additionally, these strains held the lowest number of AMR genes, and the highest number of unique SPIs which also facilitate virulence. However, other serovars containing a greater diversity and abundance of resistance genes have been increasing across the poultry sector. S. Kentucky was found to carry unique AMR genes, VFs, SPIs, and plasmids that could bolster persistence in farm and processing environments. Overall, S. Kentucky also had comparatively high levels of intra-serovar genetic variability when compared to other prominent serovars from our collection. In addition, one of our two S. Reading isolates had high carriage of both AMR genes and VFs relative to other isolates in our collection. As the poultry-associated Salmonella landscape continues to evolve in Canada, future studies should monitor the genetic composition of prominent serovars across poultry production to maintain up-to-date risk assessments of these foodborne pathogens to consumers.

Prevalence, Infectious Characteristics and Genetic Diversity of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus (MRSA) in Two Raw-Meat Processing Establishments in Northern Greece

In the present study, we investigated the isolation frequency, the genetic diversity, and the infectious characteristics of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) from the incoming meat and the meat products, the environment, and the workers' nasal cavities, in two meat-processing establishments in northern Greece. The isolated S. aureus strains were examined for their resistance to antimicrobials, carriage of the mecA and mecC genes, carriage of genes encoding for the production of nine staphylococcal enterotoxins, carriage of the Panton-Valentine Leukocidin and Toxic Shock Syndrome genes, and the ability to form biofilm. The genetic diversity of the isolates was evaluated using Pulsed Field Gel Electrophoresis (PFGE) and spa typing. S. aureus was isolated from 13.8% of the 160 samples examined, while only one sample (0.6%) was contaminated by MRSA carrying the mecA gene. The evaluation of the antimicrobial susceptibility of the isolates revealed low antimicrobial resistance. The higher resistance frequencies were observed for penicillin (68.2%), amoxicillin/clavulanic acid (36.4%) and tetracycline (18.2%), while 31.8% of the isolates were sensitive to all antimicrobials examined. Multidrug resistance was observed in two isolates. None of the isolates carried the mecC or lukF-PV genes, and two isolates (9.1%) harbored the tst gene. Eight isolates (36.4%) carried the seb gene, one carried the sed gene, two (9.1%) carried both the sed and sei genes, and one isolate (4.5%) carried the seb, sed and sei genes. Twenty-one (95.5%) of the isolates showed moderate biofilm production ability, while only one (4.5%) was characterized as a strong biofilm producer. Genotyping of the isolates by PFGE indicates that S. aureus from different meat-processing establishments represent separate genetic populations. Ten different spa types were identified, while no common spa type isolates were detected within the two plants. Overall, our findings emphasize the need for the strict application of good hygienic practices at the plant level to control the spread of S. aureus and MRSA to the community through the end products.

Association of CRISPR-Cas System with the Antibiotic Resistance and Virulence Genes in Nosocomial Isolates of Enterococcus.

This study aimed to investigate the prevalence of the CRISPR-Cas system in nosocomial isolates of Enterococcus and their possible association with antibiotic resistance and virulence genes. Identification and antimicrobial susceptibility of the microorganism were performed by the automatized VITEK 2 Compact system (bioMerieux, France). A total of 100 Enterococcus isolates were collected and identified by VITEK 2 Compact automatic microbial identification drug susceptibility analyzer. The prevalence of various CRISPR-Cas systems, antibiotic resistance genes and virulence genes were investigated by polymerase chain reaction (PCR). The prevalence of CRISPR-Cas systems associated with antibiotic resistance and virulence genes was performed by appropriate statistical tests. A total of 100 isolates of Enterococcus were identified and there were 62/100(62.0%) Enterococcus faecalis isolates and 38/100(38.0%) Enterococcus faecalis isolates. In total, 46 (46.0%) of 100 isolates had at least one CRISPR-Cas locus. CRISPR elements were more prevalent in Enterococcus faecalis isolates. The results of PCR demonstrated that CRISPR1-Cas, orphan CRISPR2, and CRISPR3-Cas were present in 23 (23.0%), 42 (42.0%) and 5 (5.0%) Enterococcus isolates, respectively. Compared with CRISPR-Casnegative isolates, the CRISPR-Cas positive isolates showed significant lower resistance rates against ampicillin, erythromycin, levofloxacin, tetracycline, vancomycin, gentamicin, streptomycin, and rifampicin. Presumably consistent with drug susceptibility, fewer CRISPR loci were identified in vanA, tetM, ermB, aac6'-aph(2"), aadE, and ant(6) positive isolates. There was a significant negative correlation between the CRISPR-Cas locus and the enterococcal virulence factors enterococcal surface protein (esp) gene. In conclusion, the results indicated that the absence of the CRISPR-Cas system was negatively associated with some antibiotic resistance in clinical isolates of Enterococcus faecalis and Enterococcus faecium. Also, there was a negative correlation with the carriage of antibiotic resistance genes. Furthermore, CRISPR-Cas may prevent some isolates from acquiring certain virulence factors.

Inflammatory bowel disease-associated adherent-invasive Escherichia coli have elevated host-defense peptide resistance.

Adherent-invasive Escherichia coli (AIEC) are isolated from inflammatory bowel disease (IBD) patients at a higher rate than from control patients. Using a collection of E. coli strains collected from Crohn's disease (CD), ulcerative colitis (UC), or non-IBD control patients, antibiotic and resistance to the antimicrobial peptides HBD-3 and LL-37 was assessed. Carriage of bacterial-encoded omptin protease genes was assessed by PCR and omptin protease activity was measured using a whole-cell based fluorescence assay. Elevated resistance to antibiotics and host defense peptides in IBD-associated AIEC were observed. IBD-associated strains showed increased (but statistically non-significant) antibiotic resistance. CD-associated strains showed greater (but statistically non-significant) resistance to HBD3-mediated killing while UC-associated strains showed statistically greater resistance to LL-37 mediated killing. High-level resistance to LL-37 was associated with carriage of omptin protease genes and with increased omptin protease activity. Antimicrobial host defense peptide resistance may be an adaptive feature of AIEC leading to enhanced pathogenesis during the initiation or progression of IBD.

Carriage of carbapenemase genes among multidrug resistant Escherichia coli isolates from contact surfaces in food vending outlets within Nasarawa State, Nigeria

Contamination of food contact surfaces by increases the risk of food-borne diseases through cross-contamination which becomes more complicated with the development of multidrug resistance by the pathogen. This study was aimed at investigating multidrug resistance and carriage of carbapenemase genes among isolates from food contact surfaces in Nasarawa State, Nigeria. : A total of 924 swab samples (522 from Lafia and 402 from Nasarawa Eggon) were collected from various food contact surfaces in food vending outlets and screened for multidrug resistance and carbapenemase genes in isolates using standard culture, modified Kirby-Bauer disk diffusion, PCR amplification and agarose gel electrophoresis techniques. Data obtained were statistically analyzed and p-value set at 0.05 confidence level. Highest overall contamination prevalence of 43.33 % was obtained from hawkers’ outlets while table top surfaces had 35.43% with highest risk (odd ratio) of 1.94. Eateries and Hotels had no contamination. The prevalence values were significantly different (p&amp;#60;0.05) among the food contact surfaces, vending outlets and the two communities. Isolates from street vendors obtained highest resistance to OFX, REF, STR, CEP, NAL, SEP, AMP antibiotics group with MDRI of 0.7. The bla, bla and bla carbapenemase genes were harbored by representative isolates. The presence of multidrug resistant with carbapenemase genes from food contact surfaces in vending outlets serves as a public health challenge and the need for personal hygiene and strict adherence to antibiotics protocols by food vendors is highly encouraged.

Identification of trypsin-degrading commensals in the large intestine

Increased levels of proteases, such as trypsin, in the distal intestine have been implicated in intestinal pathological conditions1–3. However, the players and mechanisms that underlie protease regulation in the intestinal lumen have remained unclear. Here we show that Paraprevotella strains isolated from the faecal microbiome of healthy human donors are potent trypsin-degrading commensals. Mechanistically, Paraprevotella recruit trypsin to the bacterial surface through type IX secretion system-dependent polysaccharide-anchoring proteins to promote trypsin autolysis. Paraprevotella colonization protects IgA from trypsin degradation and enhances the effectiveness of oral vaccines against Citrobacter rodentium. Moreover, Paraprevotella colonization inhibits lethal infection with murine hepatitis virus-2, a mouse coronavirus that is dependent on trypsin and trypsin-like proteases for entry into host cells4,5. Consistently, carriage of putative genes involved in trypsin degradation in the gut microbiome was associated with reduced severity of diarrhoea in patients with SARS-CoV-2 infection. Thus, trypsin-degrading commensal colonization may contribute to the maintenance of intestinal homeostasis and protection from pathogen infection.

Genomic characterization of Staphylococcus aureus isolates causing osteoarticular infections in otherwise healthy children

Pediatric osteoarticular infections are commonly caused by Staphylococcus aureus. The contribution of S. aureus genomic variability to pathogenesis of these infections is poorly described. We prospectively enrolled 47 children over 3 1/2 years from whom S. aureus was isolated on culture-12 uninfected with skin colonization, 16 with skin abscesses, 19 with osteoarticular infections (four with septic arthritis, three with acute osteomyelitis, six with acute osteomyelitis and septic arthritis and six with chronic osteomyelitis). Isolates underwent whole genome sequencing, with assessment for 254 virulence genes and any mutations as well as creation of a phylogenetic tree. Finally, isolates were compared for their ability to form static biofilms and compared to the genetic analysis. No sequence types predominated amongst osteoarticular infections. Only genes involved in evasion of host immune defenses were more frequently carried by isolates from osteoarticular infections than from skin colonization (p = .02). Virulence gene mutations were only noted in 14 genes (three regulating biofilm formation) when comparing isolates from subjects with osteoarticular infections and those with skin colonization. Biofilm results demonstrated large heterogeneity in the isolates' capacity to form static biofilms, with healthy control isolates producing more robust biofilm formation. S. aureus causing osteoarticular infections are genetically heterogeneous, and more frequently harbor genes involved in immune evasion than less invasive isolates. However, virulence gene carriage overall is similar with infrequent mutations, suggesting that pathogenesis of S. aureus osteoarticular infections may be primarily regulated at transcriptional and/or translational levels.

Antibiotic resistance genes in the gut microbiota of mothers and linked neonates with or without sepsis from low- and middle-income countries

Early development of the microbiome has been shown to affect general health and physical development of the infant and, although some studies have been undertaken in high-income countries, there are few studies from low- and middle-income countries. As part of the BARNARDS study, we examined the rectal microbiota of 2,931 neonates (term used up to 60 d) with clinical signs of sepsis and of 15,217 mothers screening for blaCTX-M-15, blaNDM, blaKPC and blaOXA-48-like genes, which were detected in 56.1%, 18.5%, 0% and 4.1% of neonates’ rectal swabs and 47.1%, 4.6%, 0% and 1.6% of mothers’ rectal swabs, respectively. Carbapenemase-positive bacteria were identified by MALDI-TOF MS and showed a high diversity of bacterial species (57 distinct species/genera) which exhibited resistance to most of the antibiotics tested. Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae/E. cloacae complex, the most commonly found isolates, were subjected to whole-genome sequencing analysis and revealed close relationships between isolates from different samples, suggesting transmission of bacteria between neonates, and between neonates and mothers. Associations between the carriage of antimicrobial resistance genes (ARGs) and healthcare/environmental factors were identified, and the presence of ARGs was a predictor of neonatal sepsis and adverse birth outcomes.

Antimicrobial resistance and genomic analysis of staphylococci isolated from livestock and farm attendants in Northern Ghana

BackgroundThe emergence of antimicrobial resistant bacteria in food producing animals is of growing concern to food safety and health. Staphylococci are common inhabitants of skin and mucous membranes in humans and animals. Infections involving antibiotic resistant staphylococci are associated with increased morbidity and mortality, with notable economic consequences. Livestock farms may enable cross-species transfer of antibiotic resistant staphylococci. The aim of the study was to investigate antimicrobial resistance patterns of staphylococci isolated from livestock and farm attendants in Northern Ghana using phenotypic and genotypic methods. Antimicrobial susceptibility testing was performed on staphylococci recovered from livestock and farm attendants and isolates resistant to cefoxitin were investigated using whole genome sequencing.ResultsOne hundred and fifty-two staphylococci comprising S. sciuri (80%; n = 121), S. simulans (5%; n = 8), S. epidermidis (4%; n = 6), S. chromogens (3%; n = 4), S. aureus (2%; n = 3), S. haemolyticus (1%; n = 2), S. xylosus (1%; n = 2), S. cohnii (1%; n = 2), S. condimenti (1%; n = 2), S. hominis (1%; n = 1) and S. arlettae (1%; n = 1) were identified. The isolates showed resistance to penicillin (89%; n = 135), clindamycin (67%; n = 102), cefoxitin (19%; n = 29), tetracycline (15%; n = 22) and erythromycin (11%; n = 16) but showed high susceptibility to gentamicin (96%; n = 146), sulphamethoxazole/trimethoprim (98%; n = 149) and rifampicin (99%; n = 151). All staphylococci were susceptible to linezolid and amikacin. Carriage of multiple resistance genes was common among the staphylococcal isolates. Genome sequencing of methicillin (cefoxitin) resistant staphylococci (MRS) isolates revealed majority of S. sciuri (93%, n = 27) carrying mecA1 (which encodes for beta-lactam resistance) and the sal(A) gene, responsible for resistance to lincosamide and streptogramin. Most of the MRS isolates were recovered from livestock.ConclusionThe study provides insights into the genomic content of MRS from farm attendants and livestock in Ghana and highlights the importance of using whole-genome sequencing to investigate such opportunistic pathogens. The finding of multi-drug resistant staphylococci such as S. sciuri carrying multiple resistant genes is of public health concern as they could pose a challenge for treatment of life-threatening infections that they may cause.

Rectal and Tracheal Carriage of Carbapenemase Genes and Class 1 and 2 Integrons in Patients in Neurosurgery Intensive Care Unit.

The spread of multidrug-resistant Gram-negative bacteria, which is associated with the distribution of beta-lactamase genes and class 1 and 2 integrons, is a global problem. In this study, in the Moscow neurosurgery intensive care unit (neuro-ICU), the high prevalence of the above-stated genes was found to be associated with intestinal and tracheal carriage. Seven-point prevalence surveys, which included 60 patients in the neuro-ICU, were conducted weekly in the period from Oct. to Nov. 2019. A total of 293 clinical samples were analyzed, including 146 rectal and 147 tracheal swabs; 344 Gram-negative bacteria isolates were collected. Beta-lactamase genes (n = 837) were detected in the isolates, including beta-lactamase blaTEM (n = 162), blaSHV (n = 145), cephalosporinase blaCTX–M (n = 228), carbapenemase blaNDM (n = 44), blaKPC (n = 25), blaOXA–48 (n = 126), blaOXA–51–like (n = 54), blaOXA–40-like (n = 43), blaOXA–23-like (n = 8), and blaVIM (n = 2), as well as class 1 (n = 189) and class 2 (n = 12) integrons. One extensively drug-resistant Klebsiella pneumoniae strain (sequence type ST39 and capsular type K23), simultaneously carried beta-lactamase genes, blaSHV–40 and blaTEM–1B, three carbapenemase genes, blaNDM, blaKPC, and blaOXA–48, the cephalosporinase gene blaCTX–M, and two class 1 integrons. Before this study, such heavily armed strains have not been reported, suggesting the ongoing evolution of antibiotic resistance.

Dissemination of virulence and resistance genes among Klebsiella pneumoniae via outer membrane vesicle: An important plasmid transfer mechanism to promote the emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae.

Klebsiella pneumoniae is well-known opportunistic enterobacteria involved in complex clinical infections in humans and animals. The domestic animals might be a source of the multidrug-resistant virulent K. pneumoniae to humans. K. pneumoniae infections in domestic animals are considered as an emergent global concern. The horizontal gene transfer plays essential roles in bacterial genome evolution by spread of virulence and resistance determinants. However, the virulence genes can be transferred horizontally via K. pneumoniae-derived outer membrane vesicles (OMVs) remains to be unreported. In this study, we performed complete genome sequencing of two K. pneumoniae HvK2115 and CRK3022 with hypervirulent or carbapenem-resistant traits. OMVs from K. pneumoniae HvK2115 and CRK3022 were purified and observed. The carriage of virulence or resistance genes in K. pneumoniae OMVs was identified. The influence of OMVs on the horizontal transfer of virulence-related or drug-resistant plasmids among K. pneumoniae strains was evaluated thoroughly. The plasmid transfer to recipient bacteria through OMVs was identified by polymerase chain reaction, pulsed field gel electrophoresis and Southern blot. This study revealed that OMVs could mediate the intraspecific and interspecific horizontal transfer of the virulence plasmid phvK2115. OMVs could simultaneously transfer two resistance plasmids into K. pneumoniae and Escherichia coli recipient strains. OMVs-mediated horizontal transfer of virulence plasmid phvK2115 could significantly enhance the pathogenicity of human carbapenem-resistant K. pneumoniae CRK3022. The CRK3022 acquired the virulence plasmid phvK2115 could become a CR-hvKp strain. It was critically important that OMVs-mediated horizontal transfer of phvK2115 lead to the coexistence of virulence and carbapenem-resistance genes in K. pneumoniae, resulting in the emerging of carbapenem-resistant hypervirulent K. pneumoniae.

Quinolone resistance markers in fluoroquinolone-resistant, non-ESBL- producing Escherichia coli isolated from non-human primates at selected zoological gardens and tourist centres

ABSTRACT Fluoroquinolone-resistant Escherichia coli (E. coli) from faecal matter of non-human primates (NHP) and their carriage of plasmid-mediated quinolone resistance genes were investigated in this study. Faecal samples were collected from six accredited zoological and tourism gardens in South-western and Northern Nigeria. Five fluoroquinolone-resistant E. coli were obtained from the 43 NHP sampled. Three of the isolates were from Agodi gardens, Ibadan, and one each from Osun Osogbo grove and University of Ibadan Zoological garden. The isolates were from Green monkeys (3), Mona Monkey (1) and Anubis baboon (1). They showed varying antibiotic resistance levels, with 100% resistance observed only towards sulfamethoxazole-trimethoprim. None of the isolates was positive for ESBL production. All the isolates carried qnrD, with three each carrying qnrA and qnrS. Only one isolate carried qnrB. The carriage of qnr genes by captive animals could portend serious public health risks.

Characterization of class 1, 2, 3 integrons, ESBL genes and antibiotic susceptibility of Salmonella serotypes from broiler and cattles in Turkey.

Antimicrobial resistance in Salmonella has been associated with the presence of integrons and many other resistance mechanisms contributing to the spread of antimicrobial-resistant genes within and between livestock and human populations. In this study, the presence of Salmonella serovars from broiler and cattle samples and their antimicrobial resistance, integrons, tet resistance, ESBL and resistance genes carriage were investigated. Total of 209 litter (broiler farms) and fecal samples (cattle farms) were examined by bacteriological procedures, susceptibilities against 18 antimicrobials and genes carriages were detected by singleplex and multiplex PCR. A total of 46/209 (22 %) Salmonella strains were isolated. Six different Salmonella serotypes from 46 Salmonella isolates were identified and the most common serotype was S. Infantis 38 (82.6%) from broiler litter; followed by S. Kitenge 3 (6.5 %) from fecal sample. The highest occurrence of resistance observed for penicilline (46/46, %100), lincomycin (43/46, 93.5%) and 42 isolates (43/46, 93.5%) exhibited MDR. The overall occurrence of class 1, 2 and 3 integrons carrying Salmonella in tested samples were 63.04% (29/46), 43.5% (20/46) and 84.8% (39/46) respectively. Out of the 27 isolates produced an ESBL, mostly CTX and TEM. On 46 Salmonella isolates, in 16 (34.8%) Tcr' genes were determined. Genotypic and phenotipic detection of ESBL genes found within integrons from Salmonella isolates from different sources (broiler and cattle) can provide powerful information about health and economic risk associated with transferable multidrug resistance.

High burden of ESBL- producing Klebsiella spp., Proteus mirabilis, Enterobacter cloacae and Pseudomonas aeruginosa in diagnosed cases of urinary tract infection in a Nigerian Teaching Hospital.

Infections of the urinary tract have been on the rise globally and these are also worsened by the increasing rate of antibiotic resistance in uropathogens. This study aimed to determine the susceptibility profile of extended spectrum β-lactamase (ESBL)- producing uropathogens to selected antibiotics and their carriage of ESBL genes. Bacterial uropathogens were obtained from the urine bench of a Microbiology laboratory in a Teaching Hospital in South-West Nigeria. Susceptibility to antibiotics was tested using the disc diffusion method, while detection of ESBL production was done using the double disc synergy test (DDST). Detection of ESBL genes was performed by PCR. A total of 21 ESBL- producing uropathogens were obtained namely: Klebsiella pneumoniae (11), Klebsiella oxytoca (6), Proteus mirabilis (2), Enterobacter cloacae (1) and Pseudomonas aeruginosa (1). The resistance to antibiotics in the uropathogens was: imipenem (0%), gentamicin (38.1%), sulfamethoxazole-trimethoprim (52.4%), amoxicillin-clavulanate (61.9%), aztreonam (66.7%), ceftazidime (66.7%), tetracycline (90.5%), cefpodoxime (100%) and cefotaxime (100%). Altogether, 90.5% (19/21) of the isolates were multidrug resistant (MDR). Of the 21 uropathogens, 61.9% (13/21) carried bla CTX-M, 52.4% (11/21) carried bla TEM while bla SHV was detected in 47.6% (10/21) of the isolates. There was co-carriage of ESBL genes in 12 uropathogens. This study showed a high prevalence of multidrug resistance and a high carriage of ESBL genes in the ESBL- producing isolates obtained over the study period. There is a need for a review of antibiotic options in the treatment of UTI to clamp down on the ever-increasing tide of antibiotic resistance in uropathogens.

Phenotypic and Molecular Detection of Slime Producing Staphylococcus Spp. Obtained from Blood Samples of Patients Undergoing Hematopoietic Stem-Cell Transplantation

Abstract Aim: to investigate the slime production in isolates of Staphylococcus spp., associated with bacteremia in patients after hematopoietic stem-cell transplantation (HSCT) and to determine the relationship between the slime production and ica genes carriage, as well as the correlation of ica and methicillin resistance. Materials and methods: Between 2019 and 2020, twenty-one clinically significant Staphylococcus spp. isolates were obtained from blood cultures of 17 patients after HSCT. The species identification and the susceptibility to cefoxitin were determined by BD Phoenix M50. Two phenotypic tests (Congo red agar, CRA; Christensen’s method, TT) and PCR for icaA and icaD were used to detect slime production. A PCR method was also used to detect the mecA, mecC genes. Results: In the studied group of 21 isolates (S. epidermidis, n = 12; S. haemolyticus, n = 4; S. hominis, n = 2; S. aureus, n = 3), the phenotypic tests were positive in 13 isolates. Ten isolates (47.6%) were identified as carriers of ica genes (S. epidermidis, n = 9, and S. haemolyticus, n = 1). Five isolates (23.8%) were detected as slime producers by all three methods. The mecA gene was identified in 18 isolates (85.7%). All ica positive isolates were also mecA carriers. Conclusion: A relatively high proportion of the blood isolates of Staphylococcus spp. were slime producers, associ-ated with ica genes. A combination of both phenotypic and genetic methods should be used to detect alternative routes of slime production. The co-expression of ica and mecA is associ-ated with the occurrence of difficult-to-eradicate isolates.

Prevalence, Antibiotic-Resistance, and Replicon-Typing of Salmonella Strains among Serovars Mainly Isolated from Food Chain in Marche Region, Italy.

Nontyphoidal salmonellosis (NTS) is the second most commonly reported gastrointestinal infection in humans and an important cause of food-borne outbreaks in Europe. The use of antimicrobial agents for animals, plants, and food production contributes to the development of antibiotic-resistant Salmonella strains that are transmissible to humans through food. The aim of this study was to investigate the presence and the potential dissemination of multidrug-resistant (MDR) Salmonella strains isolated in the Marche Region (Central Italy) via the food chain. Strains were isolated from different sources: food, human, food animal/livestock, and the food-processing environment. Among them, we selected MDR strains to perform their further characterization in terms of resistance to tetracycline agent, carriage of tet genes, and plasmid profiles. Tetracycline resistance genes were detected by PCR and plasmid replicons by PCR-based replicon typing (PBRT). A total of 102 MDR Salmonella strains were selected among the most prevalent serovars: S. Infantis (n = 36/102), S. Derby (n = 20/102), S. Typhimurium (n = 18/102), and a monophasic variant of S. Typhimurium (MVST, n = 28/102). Resistance to sulfisoxazole (86%) and tetracycline (81%) were the most common, followed by ampicillin (76%). FIIS was the most predominant replicon (17%), followed by FII (11%) and FIB (11%) belonging to the IncF incompatibility group. Concerning the characterization of tet genes, tetB was the most frequently detected (27/89), followed by tetA (10/89), tetG (5/89), and tetM (1/89). This study showed the potential risk associated with the MDR Salmonella strains circulating along the food chain. Hence, epidemiological surveillance supported by molecular typing could be a very useful tool to prevent transmission of resistant Salmonella from food to humans, in line with the One Health approach.