Genes Aac Research Articles

Occurrence of virulence genes icaADBC and antibiotic resistance genes blaZ, mecA, and aac(6’)-Ie-aph (2’’)-Ia in coagulase-negative staphylococci isolates from neonates with sepsis at a regional referral hospital in Dar es Salaam, Tanzania

BackgroundCoagulase-negative staphylococci (CoNS) have been linked to severe conditions such as bloodstream infection, with biofilm formation regarded as the major virulence mechanism. This study determined virulence genes (VGs) (icaA, icaB, icaC, and icaD) responsible for biofilm formation and antibiotic resistance genes (ARGs) (blaZ, mecA, and aac(6’)-Ie-aph(2’’)-Ia) in CoNS isolated from blood samples of 70 neonates with sepsis in Dar es Salaam, Tanzania. We used VITEK®MS (BioMérieux, France) for speciation and phenotypically detected biofilm production using the Congo red agar. Polymerase chain reaction was performed for ARGs and VGs detection; principal component analysis was used to ascertain their relationship.ResultsOut of 70 CoNS, Staphylococcus epidermidis, 27 (36.8%) was the most commonly isolate, subsequently to Staphylococcus haemolyticus, 24 (34.3%). The intercellular adhesion genes (ica) were detected in all isolates, with IcaD found in 70 (100.0%), followed by icaB 69 (98.6%), icaC 69 (98.6%), and icaA 65 (92.9%). Four virulence gene combinations (icaADBC, icaBCD, icaACD, and icaABD) were detected, predominantly icaADBC 63 (90.0%). Sixty-one (87.1%) isolates harbored three ARGs, mostly the mecA gene 69 (98.6%), followed by blaZ 67 (95.7%) and aac (6’)-Ie-aph (2’’)-Ia, 65 (92.7%). We observed a moderate positive correlation between mecA and icaA genes (r = 0.4341). Sixty-five (97.0%) isolates resistant to penicillin harbored blaZ genes, and all 62 (100%) resistant to oxacillin harbored mecA genes. Of the 66 isolates resistant to gentamycin, 62 (93.9%) harbored the aac (6’)-Ie-aph (2’’)-Ia gene. The mecA gene contributed most of the resistance, followed by aac(6’)-Ie-aph(2’’)-Ia genes. However, the icaA and icaD genes significantly influenced the virulence of CoNS.ConclusionsMost CoNS isolated from blood samples of neonates with sepsis harbor a variety of clinically relevant virulence and resistance genes, indicating their ability to cause severe infections that are difficult to treat. Significantly, 59 (84.3%) isolates were phenotypically penicillin, gentamycin, and oxacillin resistant; the antibiotics recommended in the national guidelines for empiric treatment of neonatal sepsis.

Whole-Genome Sequencing Analysis of Antimicrobial Resistance, Virulence Factors, and Genetic Diversity of Salmonella from Wenzhou, China

Salmonella species are important foodborne pathogens worldwide. Salmonella pathogenicity is associated with multiple virulence factors and enhanced antimicrobial resistance. To determine the molecular characteristics and genetic correlations of Salmonella, 24 strains of Salmonella isolated from different sources (raw poultry, human stool, and food) in the Wenzhou area were investigated to determine the distribution of antimicrobial resistance and virulence determinants using whole-genome sequencing (WGS). Aminoglycoside resistance genes were detected in all samples. Over half of the samples found antimicrobial resistance genes (ARGs) and point mutations for several clinically frequently used antibiotic, beta-lactams, tetracyclines, and quinolones. Of these strains, 62.5% were predicted to be multidrug-resistant (MDR). The quinolone-modifying enzyme gene aac(6’)-Ib-cr, detected in five samples (S1–S4 and S10), was located on integrons. The analysis of Salmonella pathogenicity island (SPI) profiles suggests that serotypes with close genetic relationships share the same distribution of virulence factors, revealing a link between genotype and SPI profiles. cgMLST analysis indicated that five isolates S14–S18 were closely related to strains originating from the United Kingdom, suggesting that they may share a common origin. Data from this study may enrich the molecular traceability database for Salmonella and provide a basis for effective public health policies.

Investigation of in vitro susceptibility and resistance mechanisms to amikacin among diverse carbapenemase-producing Enterobacteriaceae

ObjectiveThis study aims to assess the in vitro drug susceptibility of various Carbapenemase-Producing Enterobacteriaceae (CPE) genotypes and elucidate the underlying mechanisms of amikacin resistance.MethodsA total of 72 unique CPE strains were collected from the Second Hospital of Jiaxing between 2019 and 2022, including 51 strains of Klebsiella pneumoniae, 11 strains of Escherichia coli, 6 strains of Enterobacter cloacae, 2 strains of Klebsiella aerogenes, 1 strain of Citrobacter freundii, and 1strain of Citrobacter werkmanii. Among these strains, 24 carried blaKPC gene, 20 carried blaNDM gene, 23 carried blaOXA−48−like gene, and 5 carried both blaKPC and blaNDM. We measured the in vitro activity of amikacin and other common antibiotics. Strains carrying blaOXA−48-like gene were selected for whole genome sequencing (WGS) via next-generation sequencing to identify genes related to antimicrobial resistance (AMR) and virulence factor (VF).ResultsOut of the 72 CPE strains tested, 41.7% exhibited resistance to amikacin. The drug resistance rates for K. pneumoniae, E. coli, and Enterobacter spp. were 51.0%, 27.3%, and 10.0%, respectively. The majority of the CPE strains (> 90%) displayed resistance to cephalosporins and carbapenems, while most of them were sensitive to polymyxin B and tigecycline (97.2% and 94.4%). The amikacin resistance rate was 100% for strains carrying blaOXA−48, 20.8% for those with blaKPC, 5.0% for those with blaNDM, and 20.0% for those with both blaKPC and blaNDM. These differences were statistically significant (P < 0.05). Through sequencing, we detected aminoglycoside resistance genes rmtF and aac(6’)-Ib, VF genes iucABCD and rmpA2 in OXA-48-producing multidrug resistance and highly virulent strains. These genes were located on a IncFIB- and IncHI1B-type plasmid, respectively. Both plasmids were highly homologous to the plasmid from OXA-232 strains in Zhejiang province and Shanghai province. Integration of these resistance genes into the IncFIB plasmid, facilitated by the IS6 and/or Tn3 transposons, resulted in OXA232-producing K. pneumoniae with amikacin resistance.ConclusionThis study identified significant amikacin resistance in CPE strains, particularly in those carrying the blaOXA−48 gene. Resistance genes rmtF and aac(6’)-Ib were identified on plasmids. These results highlight the need for careful monitoring of amikacin resistance.

Molecular characterization of Methicillin-resistant Staphylococcus aureus isolated in ready-to-eat food sold in supermarkets in Bobo-Dioulasso: case of charcuterie products

BackgroundStaphylococcus aureus (S. aureus) is one of the most widespread bacterial pathogens in animals and humans, and its role as an important causative agent of food poisoning is well-documented. The aim of this study was to highlight and characterize the resistance patterns of methicillin-resistant S. aureus (MRSA) in charcuterie products sold in selected supermarkets (SM) in Bobo-Dioulasso, Burkina Faso.MethodsIn this study, 72 samples including ham (n = 19), merguez (n = 22), sausage (n = 15) and minced meat (n = 16) were collected from 3 supermarkets. Standard microbiology methods were utilised to characterise S. aureus isolates. Phenotypic resistance patterns were investigated using the disk diffusion method on Mueller-Hinton agar. Genotypic testing using polymerase chain reaction (PCR) was performed on the isolates to detect the 16S-23S gene. Using specific primers, the following genes PVL, TSST-1, mecA, gyrA, gyrB, qnrA, intI1 and aac(6’)-Ib-cr were identified from purified DNA by PCR.ResultsAmong the 72 ready-to-eat food samples, S. aureus was present in 51, (70.83%). The yield was highest in both the ham and merguez food products, 15/51 (29.41%) each, followed by minced meat 12/51 (23.53%) and sausage 9/51 (17.65%). A total of 35 isolates (68.63%) were confirmed as S. aureus after molecular characterization using 16–23 S primers with 05 (14.29%) strains identified as MRSA. All of the MRSA and majority of the methicillin-sensitive S.aureus (MSSA) isolates were resistant to penicillin G, ampicillin, tetracycline and erythromycin, whereas one isolate from minced meat was found in SM3-harbouring PVL, TSST-1, mecA, gyrA, gyrB and Int1 genes.ConclusionsOur study revealed a high prevalence of S. aureus in chacuterie products in Bobo-Dioulasso with antimicrobial profiles that show resistance to most antibiotics. These findings should inform and augment efforts to raise awareness among local supermarket owners on adequate food manufacturing practices as well as promoting food safety and hygiene.

Bacterial dynamics of the plastisphere microbiome exposed to sub-lethal antibiotic pollution

BackgroundAntibiotics and microplastics are two major aquatic pollutants that have been associated to antibiotic resistance selection in the environment and are considered a risk to human health. However, little is known about the interaction of these pollutants at environmental concentrations and the response of the microbial communities in the plastisphere to sub-lethal antibiotic pollution. Here, we describe the bacterial dynamics underlying this response in surface water bacteria at the community, resistome and mobilome level using a combination of methods (next-generation sequencing and qPCR), sequencing targets (16S rRNA gene, pre-clinical and clinical class 1 integron cassettes and metagenomes), technologies (short and long read sequencing), and assembly approaches (non-assembled reads, genome assembly, bacteriophage and plasmid assembly).ResultsOur results show a shift in the microbial community response to antibiotics in the plastisphere microbiome compared to surface water communities and describe the bacterial subpopulations that respond differently to antibiotic and microplastic pollution. The plastisphere showed an increased tolerance to antibiotics and selected different antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs). Several metagenome assembled genomes (MAGs) derived from the antibiotic-exposed plastisphere contained ARGs, virulence factors, and genes involved in plasmid conjugation. These include Comamonas, Chryseobacterium, the opportunistic pathogen Stenotrophomonas maltophilia, and other MAGs belonging to genera that have been associated to human infections, such as Achromobacter. The abundance of the integron-associated ciprofloxacin resistance gene aac(6’)-Ib-cr increased under ciprofloxacin exposure in both freshwater microbial communities and in the plastisphere. Regarding the antibiotic mobilome, although no significant changes in ARG load in class 1 integrons and plasmids were observed in polluted samples, we identified three ARG-containing viral contigs that were integrated into MAGs as prophages.ConclusionsThis study illustrates how the selective nature of the plastisphere influences bacterial response to antibiotics at sub-lethal selective pressure. The microbial changes identified here help define the selective role of the plastisphere and its impact on the maintenance of environmental antibiotic resistance in combination with other anthropogenic pollutants. This research highlights the need to evaluate the impact of aquatic pollutants in environmental microbial communities using complex scenarios with combined stresses.3sW_WiGiki6wh61j1XWikeVideo

Antimicrobial Resistance Profiles of Enterococcus Faecalis and Enterococcus Faecium Isolated from Clinical Specimens at Kitwe Teaching Hospital in Zambia

This research examined the occurrence of Enterococcus faecalis and Enterococcus faecium in clinical samples processed at Kitwe Teaching Hospital Laboratory, along with their resistance to antimicrobials and presence of virulence determinants. A total of 230 clinical specimens were analyzed to identify suspected Enterococcus strains. Standard laboratory techniques were employed to isolate, characterize, and confirm the species of Enterococcus, followed by testing their susceptibility to antimicrobials and detection of resistance and virulence genes using polymerase chain reaction. Among the 230 cultured specimens, 89 were stool samples, 3 were swabs, and 138 were urine specimens. Out of these, 71 isolates were confirmed Enterococcus by PCR using genus specific primers. The resulting prevalence rate of 30.9% was thus obtained. The most prevalent species was Enterococcus faecalis, accounting for 56.3% of the 71 Enterococcus isolates. The prevalence of E. faecium was 14.1%. All tested E. faecalis isolates displayed resistance to ciprofloxacin and erythromycin. Furthermore, high rates of resistance were observed among E. faecalis isolates for chloramphenicol and tetracycline (97.5%), vancomycin (90.0%), nitrofurantoin (75.0%), penicillin (75.0%) and ampicillin (67.5%). Only two E. faecium isolates exhibited susceptibility to tetracycline and vancomycin, while the remaining isolates were 100% resistant to all other tested antimicrobials. Multidrug resistance was detected in all E. faecium isolates. In addition, various antibiotic resistance genes (aac(6′)-Ie-aph(2″)-LA, ermA, ermB, tetK, tetL, tetM, tetX, vanA) were identified in both E. faecalis and E. faecium isolates. A positive association between phenotype and genotype was found for tetracycline and erythromycin.

The Abundance of Plasmid-Mediated Quinolone Resistance Genes in Enterobacter cloacae Strains Isolated from Clinical Specimens in Kermanshah, Iran.

Enterobacter cloacae (E. cloacae) is one of the most common Enterobacteriaceae causing nosocomial infections. Plasmid-mediated quinolone resistance (PMQR) determinants have been considered recently. This study evaluated the abundance of PMQR genes in strains of E. cloacae obtained from clinical samples in Kermanshah, Iran. In this descriptive cross-sectional study, after collecting 113 isolates of E. cloacae, their identity was confirmed using specific biochemical tests. After determining their drug resistance patterns using disc diffusion, the phenotypic frequency of extended-spectrum beta-lactamase (ESBL)-producing isolates was measured by the double-disk synergy test (DDST) method. The isolates were examined for the presence of qnrA, qnrB, qnrS, and aac(6')-Ib-cr genes by the polymerase chain reaction (PCR) assay. The antibiotic resistance rate of E. cloacae isolates varied from 9.7% to 60.2%; among them, 78% were multidrug-resistant (MDR). The highest quinolone resistance was observed in ESBL-producing strains of E. cloacae. The frequency of positive isolates for PMQR and ESBL was 79.6% and 57.5%, respectively. The genes aac(6')-ib-cr (70.8%) and qnrB (38.1%) had the highest frequency among other genes. The number of isolates simultaneously carrying 2 and 3 genes was 64 and 5 isolates, respectively. The obtained results indicate a high degree of quinolone resistance among ESBL-producing E. cloacae strains. Nevertheless, there was a significant relationship between the PMQR gene and ESBL-positive isolates. Therefore, special attention should be paid to molecular epidemiological studies on antibiotic resistance to quinolones and beta-lactamases in these strains.

Molecular Characterization of Plasmid-Mediated Quinolone resistance in Urinary Escherichia coli from some Healthcentres in Nasarawa-West Senatorial District, Nasarawa State, Nigeria

The most common bacteria associated with urinary tract infections (UTIs) worldwide Escherichia coli. To treat UTIs, Fluoroquinolones are widely used especially Ciprofloxacin. The honey moon did not last long due to the development of resistance acquired by the bacteria as a result of resistance gene. This study investigated ciprofloxacin resistant urinary E. coli resistance via plasmid-mediated quinolone resistance (PMQR) genes rather than the chromosomal mutation of patients attending some Healthcentres in Nasarawa-West Senatorial District, Nasarawa State, Nigeria. Ninety-Two (92) 42% confirmed ciprofloxacin urinary resistant E. coli from patients attending the three (3) study Centres were screened for the presence of PMQR resistant genes (aac(6’)-Ib-cr, oqxA, oqxB, qnrA, qnrB, qnrS) using polymerase chain reaction (PCR) method. The screening detected the presence of QnrA 4(44.4%), QnrB and QnrS both with 5(55.6%) occurrence, meaning aac(6’)-Ib-cr, QnrVC ,GyrA, ParC and others were not detected. It is clear that these isolates harbored some PMQR genes both singly and in combination. This is a strong warning to both the prescribers and users of Antibiotic of the consequences of negligence of strict adherence to the usage of antimicrobials, since resistance to antibiotics has led to death of great number of People worldwide.

In-depth characterization of multidrug-resistant NDM-1 and KPC-3 co-producing Klebsiella pneumoniae bloodstream isolates from Italian hospital patients.

Bloodstream infection (BSI) caused by carbapenem-resistant Klebsiella pneumoniae (KP) poses significant challenges, particularly when the infecting isolate carries multiple antimicrobial resistance (AMR) genes/determinants. This study, employing short- and long-read whole-genome sequencing, characterizes six New Delhi metallo-β-lactamase (NDM) 1 and KP carbapenemase (KPC) 3 co-producing KP isolates, the largest cohort investigated in Europe to date. Five [sequence type (ST) 512] and one (ST11) isolates were recovered from patients who developed BSI from February to August 2022 or February 2023 at two different hospitals in Rome, Italy. Phylogenetic analysis revealed two distinct clusters among ST512 isolates and a separate cluster for the ST11 isolate. Beyond blaNDM-1 and blaKPC-3, various AMR genes, indicative of a multidrug resistance phenotype, including colistin resistance, were found. Each cluster-representative ST512 isolate harbored a blaNDM-1 plasmid (IncC) and a blaKPC-3 plasmid [IncFIB(pQil)/IncFII(K)], while the ST11 isolate harbored a blaNDM-1 plasmid [IncFII(pKPX1)] and a blaKPC-3 plasmid [IncFIB(K)/IncFII(K)]. The blaNDM-1 plasmids carried genes conferring resistance to clinically relevant antimicrobial agents, and the aminoglycoside resistance gene aac(6')-Ib was found on different plasmids. Colistin resistance-associated mgrB/pmrB gene mutations were present in all isolates, and the yersiniabactin-encoding ybt gene was unique to the ST11 isolate. In conclusion, our findings provide insights into the genomic context of blaNDM-1/blaKPC-3 carbapenemase-producing KP isolates.IMPORTANCEThis study underscores the critical role of genomic surveillance as a proactive measure to restrict the spread of carbapenemase-producing KP isolates, especially when key antimicrobial resistance genes, such as blaNDM-1/blaKPC-3, are plasmid borne. In-depth characterization of these isolates may help identify plasmid similarities contributing to their intra-hospital/inter-hospital adaptation and transmission. Despite the lack of data on patient movements, it is possible that carbapenem-resistant isolates were selected to co-produce KP carbapenemase and New Delhi metallo-β-lactamase via plasmid acquisition. Studies employing long-read whole-genome sequencing should be encouraged to address the emergence of KP clones with converging phenotypes of virulence and resistance to last-resort antimicrobial agents.

Coexistence of blaNDM-5, blaCTX-M-15, blaOXA-232, blaSHV-182 genes in multidrug-resistant K. pneumoniae ST437-carrying OmpK36 and OmpK37 porin mutations: First report in Italy

K. pneumoniae is a common cause of severe hospital-acquired infections. In the present study, we have characterised the whole-genome of two K. pneumoniae ST437 belonging to the clonal complex CC258. The whole-genome sequencing was performed by MiSeq Illumina, with a 2 × 300bp paired-end run. ResFinder 4.4.2 was used to detect acquired antimicrobial resistance genes (ARGs) and chromosomal mutations. Mobile genetic elements (plasmids and ISs) were identified by MobileElementFinder v1.0.3. The genome was also assigned to ST using MLST 2.0.9. Virulence factors were detected using the Virulence Factor Database (VFDB). K. pneumoniae KPNAQ_1/23 and KPNAQ_2/23 strains, isolated from urine samples of hospitalised patients, showed resistance to most antibiotics, including ceftazidime-avibactam, ceftolozane-tazobactam, and meropenem-vaborbactam combinations. Both strains were susceptible only to cefiderocol. Multiple mechanisms of resistance were identified. Resistance to β-lactams was due to the presence of NDM-5, OXA-232, CTX-M-15, SHV-182 β-lactamases, and OmpK36 and OmpK37 porin mutations. Resistance to fluoroquinolones was mediated by chromosomal mutations in acrR, oqxAB efflux pumps, and the bifunctional gene aac(6')-Ib-cr. The presence of different virulence genes makes these KPNAQ_1/23 and KPNAQ_2/23 high-risk clones.

Ciprofloxacin- and levofloxacin-loaded nanoparticles efficiently suppressed fluoroquinolone resistance and biofilm formation in Acinetobacter baumannii

The spread of fluoroquinolone (FQ) resistance in Acinetobacter baumannii represents a critical health threat. This study aims to overcome FQ resistance in A. baumannii via the formulation of polymeric nanoFQs. Herein, 80 A. baumannii isolates were obtained from diverse clinical sources. All A. baumannii isolates showed high resistance to most of the investigated antimicrobials, including ciprofloxacin (CIP) and levofloxacin (LEV) (97.5%). FQ resistance-determining regions of the gyrA and parC genes were the most predominant resistant mechanism, harbored by 69 (86.3%) and 75 (93.8%) of the isolates, respectively. Additionally, plasmid-mediated quinolone resistance genes aac(6′)-Ib and qnrS were detected in 61 (76.3%) and 2 (2.5%) of the 80 isolates, respectively. The CIP- and LEV-loaded poly ε-caprolactone (PCL) nanoparticles, FCIP and FLEV, respectively, showed a 1.5–6- and 6–12-fold decrease in the MIC, respectively, against the tested isolates. Interestingly, the time kill assay demonstrated that MICs of FCIP and FLEV completely killed A. baumannii isolates after 5–6 h of treatment. Furthermore, FCIP and FLEV were found to be efficient in overcoming the FQ resistance mediated by the efflux pumps in A. baumannii isolates as revealed by decreasing the MIC four-fold lower than that of free CIP and LEV, respectively. Moreover, FCIP and FLEV at 1/2 and 1/4 MIC significantly decreased biofilm formation by 47–93% and 69–91%, respectively. These findings suggest that polymeric nanoparticles can restore the effectiveness of FQs and represent a paradigm shift in the fight against A. baumannii isolates.

Distribution of bacterial community structures and spread of antibiotic resistome at industrially polluted sites of Mini River, Vadodara, Gujarat, India.

The influence of anthropogenic pollution on the distribution of bacterial diversity, antibiotic-resistant bacteria (ARBs), and antibiotic resistance genes (ARGs) was mapped at various geo-tagged sites of Mini River, Vadodara, Gujarat, India. The high-throughput 16S rRNA gene amplicon sequencing analysis revealed a higher relative abundance of Planctomycetota at the polluted sites, compared to the pristine site. Moreover, the relative abundance of Actinobacteriota increased, whereas Chloroflexi decreased in the water samples of polluted sites than the pristine site. The annotation of functional genes in the metagenome samples of Mini River sites indicated the presence of genes involved in the defence mechanisms against bacitracin, aminoglycosides, cephalosporins, chloramphenicol, streptogramin, streptomycin, methicillin, and colicin. The analysis of antibiotic resistome at the polluted sites of Mini River revealed the abundance of sulfonamide, beta-lactam, and aminoglycoside resistance. The presence of pathogens and ARB was significantly higher in water and sediment samples of polluted sites compared to the pristine site. The highest resistance of bacterial populations in the Mini River was recorded against sulfonamide (≥ 7.943 × 103CFU/mL) and ampicillin (≥ 8.128 × 103CFU/mL). The real-time PCR-based quantification of ARGs revealed the highest abundance of sulfonamide resistance genes sul1 and sul2 at the polluted sites of the Mini River. Additionally, the antimicrobial resistance genes aac(6')-Ib-Cr and blaTEM were also found abundantly at polluted sites of the Mini River. The findings provide insights into how anthropogenic pollution drives the ARG and ARB distribution in the riverine ecosystem, which may help with the development of antimicrobial resistance mitigation strategies.

Validation of Genetic Markers for AmpC, AAC, and RNR3 Genes in Potentially Present Pathogens Organisms in Fecal Sludge

The issue of sanitation and water pollution due to fecal waste has become a serious concern in Indonesia, particularly after it was revealed that approximately 70% of the community's drinking water sources are contaminated with human feces. Determining safe waste management requirements necessitates indicators confirming the contamination-free nature of the waste management methods. Pathogen testing indicators for fecal waste are highly diverse. The current standard testing is the culture-based method for microorganisms and microscopy observation for helminths. However, molecular detection tools for these pathogens can give higher sensitivity and specificity in a relatively shorter time of technical processing. In this research, we designed primers for molecular-based diagnostics on three pathogens exhibiting extreme survival capabilities under various environmental conditions. The species-coupled genes are AmpC for Escherichia coli, AAC for Mycobacterium tuberculosis, and RNR3 for Ascaris lumbricoides. The design of molecular-based biomarkers and their specificity tests were conducted using web-based bioinformatics programs to prevent unintended dimers. Ultimately, primers were validated by conventional gradient PCR on available positive controls and a sample from processed fecal sludge. The PCR validation test showed that the designed primers were validated to amplify the intended target with the right size without unspecific bands on the positive controls. Meanwhile, the AAC gene for E. coli was detected in the sample from the fecal sludge.

Evolution and genomic profile of Salmonella enterica serovar Gallinarum biovar Pullorum isolates from Brazil.

Salmonella enterica subspecies enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is a pathogenic bacterium that causes Pullorum disease (PD). PD is an acute systemic disease that affects young chickens, causing white diarrhea and high mortality. Although many sanitary programs have been carried out to eradicate S. Pullorum, PD outbreaks have been reported in different types of birds (layers, broilers, breeders) worldwide. This study aimed to evaluate the evolution and genetic characteristics of S. Pullorum isolated from PD in Brazil. Phylogenetic analysis of S. Pullorum genomes sequenced in this study and available genomic databases demonstrated that all isolates from Brazil are from sequence type 92 (ST92) and cluster into two lineages (III and IV). ColpVC, IncFIC(FII), and IncFII(S) were plasmid replicons frequently found in the Brazilian lineages. Two resistance genes (aac(6')-Iaa, conferring resistance to aminoglycoside, disinfecting agents, and antiseptics (mdf(A)) and tetracycline (mdf(A)) were detected frequently. Altogether, these results are important to understand the circulation of S. Pullorum and, consequently, to develop strategies to reduce losses due to PD.

Frequency of Vancomycin and Aminoglycoside Resistance Genes in Enterococcus spp. Isolated from Chicken Raw Meat Samples

Background: Enterococci are main normal microbial flora of both humans and animals and can survive in a diverse range of environments. These bacteria carry out aminoglycoside and vancomycin resistance genes and spread them in environment by many routs such as chicken meat products. The present study was aimed to determine the frequency of aminoglycoside and vancomycin resistant genes in Enterococcus species isolated from chicken meat specimens.&#x0D; Methods: A total of 250 chicken raw meat specimens was prepared from slaughterhouses at Zanjan province, cultured at BHI broth and incubated at 37°C for 24h. The positive cultures were sub-cultured in blood agar plates and grown colonies identified using phenotypical and biochemical tests. Antibiotic susceptibility was determined according to the Clinical and Laboratory Standards Institute (CLSI) standards and PCR assays were performed to detect vanA, vanB, aph (2")1c, aph (2")1b, aph (2")1d, ant(3'), aph(3')IIIa, ant(4')1a, ant(6') and aac(6') genes.&#x0D; Results: In total, 100 Enterococcus species isolated from 250 specimens and 35% of them belonged to E. faecalis and the others were E. faecium (65%). The prevalence of the vanA, vanB, aph (2")1c, aph (2")1b, aph (2")1d, ant(3'), aph(3')IIIa, ant(4')1a, ant(6') and aac(6') genes among the 100 Enterococcus species was 14%, 12%, 10%, 1%, 2%, 50%, 26%, 9%, 18% and 22%, respectively.&#x0D; Conclusion: The current study revealed that the rate of antimicrobial resistance genes to aminoglycosides and vancomycin was worrying and health measurements in meat products industries must be performed to prevent spread of antimicrobial resistance elements among bacteria.

Comparative genomics analysis of Stenotrophomonas maltophilia strains from a community.

Stenotrophomonas maltophilia is a multidrug-resistant (MDR) opportunistic pathogen with high resistance to most clinically used antimicrobials. The dissemination of MDR S. maltophilia and difficult treatment of its infection in clinical settings are global issues. To provide more genetic information on S. maltophilia and find a better treatment strategy, we isolated five S. maltophilia, SMYN41-SMYN45, from a Chinese community that were subjected to antibiotic susceptibility testing, biofilm formation assay, and whole-genome sequencing. Whole-genome sequences were compared with other thirty-seven S. maltophilia sequences. The five S. maltophilia strains had similar antibiotic resistance profiles and were resistant to β-lactams, aminoglycosides, and macrolides. They showed similar antimicrobial resistance (AMR) genes, including various efflux pumps, β-lactamase resistance genes (blaL1/2), aminoglycoside resistance genes [aac(6'), aph(3'/6)], and macrolide-resistant gene (MacB). Genome sequencing analysis revealed that SMYN41-SMYN45 belonged to sequence type 925 (ST925), ST926, ST926, ST31, and ST928, respectively, and three new STs were identified (ST925, ST926, and ST928). This study provides genetic information by comparing genome sequences of several S. maltophilia isolates from a community of various origins, with the aim of optimizing empirical antibiotic medication and contributing to worldwide efforts to tackle antibiotic resistance.

Outbreak of vancomycin-resistant Enterococcus faecium ST1133 in paediatric patients with acute lymphoblastic leukaemia from southern Brazil

We aimed to investigate an outbreak of vancomycin-resistant Enterococcus faecium (VREfm) in paediatric patients from Hospital Pequeno Príncipe. The susceptibility profile was determined, and whole-genome sequencing (WGS) was used to analyse the genetic context of the strains. Five VREfm isolates were recovered from sterile sites and surveillance cultures of two paediatric patients with acute lymphoblastic leukaemia. Species identification was performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the minimum inhibitory concentration (MIC) was assessed according to the European Committee for Antimicrobial Susceptibility Testing (EUCAST). WGS was performed to analyse the genetic context of virulence and resistance genes, and in silico multilocus sequence typing was performed to identify the sequence typing of the strains. High-level vancomycin resistance was observed in all isolates (≥256 mg/L). WGS revealed the presence of mobile genetic elements, such as plasmids (rep2, rep11a, repUS15, rep17, and rep18a), insertion sequences, and phages. Multiple resistance genes (aac(6')-aph(2"), dfrG, ermB, and vanA) and virulence genes (acm and efaAfm) were identified. All the isolates were assigned to ST117 (ST1133 - via a novel MLST), an important epidemic lineage associated with nosocomial infections and outbreaks. Our results show that the ST117 (ST1133) VREfm isolates are circulating in paediatric patients, which raises a great concern. The development of new drugs as well as the implementation of an antimicrobial stewardship program are necessary for their correct management, limiting the spread of resistance in oncohematological patients.

Molecular characterization of Escherichia coli producing extended-spectrum β-lactamase CTX-M-14 and CTX-M-28 in Mexico.

The spread of ESBL-producing Escherichia coli has constantly increased in both clinical and community infections. Actually, the main ESBL reported is the CTX-M family, which is widely disseminated between the Enterobacteriaceae family. The epidemiology of the CTX-M family shows the CTX-M-15 variant dominating worldwide, followed by CTX-M-14 and CTX-M-27. The specific ESBL-producing E. coli clones included mainly the sequence types ST131, ST405, and ST648. In this report, we present the molecular characterization of ESBL-producing E. coli clinical isolates from eight hospitals in Mexico. From a collection of 66 isolates, 39 (59%) were identified as blaCTX-M-14 and blaCTX-M-27 belonging to the group CTX-M-9. We identified 25 (38%) isolates, producing blaCTX-M-28 belonging to the group CTX-M-1. blaCTX-M-2 and blaTEM-55 were identified in one isolate, respectively. Fourteen isolates (21%) were positive for blaCTX-M-14 (13%) and blaCTX-M-28 (7.3%) that were selected for further analyses; the antimicrobial susceptibility showed resistance to ampicillin (> 256µg/mL), cefotaxime (> 256µg/mL), cefepime (> 64µg/mL), and ceftazidime (16µg/mL). The ResFinder analysis showed the presence of the antimicrobial resistance genes aacA4, aadA5, aac(3)lla, sul1, dfrA17, tet(A), cmlA1, and blaTEM-1B. PlasmidFinder analysis identified in all the isolates the replicons IncFIB, which were confirmed by PCR replicon typing. The MLST analysis identified isolates belonging to ST131, ST167, ST405, and ST648. The ISEcp1B genetic element was found at 250 pb upstream of blaCTX-M-14 and flanked by the IS903 genetic element at 35 pb downstream. The IS1380-like element ISEc9 family transposase was identified at 250 pb upstream of blaCTX-M-14 and flanked downstream by the IS5/IS1182 at 80 pb. Our study highlights the significant prevalence of CTX-M-14 and CTX-M-28 enzymes as the second-most common ESBL-producing E. coli among isolates in Mexican hospitals. The identification of specific sequence types in different regions provides valuable insights into the correlation between ESBL and E. coli strains. This contribution to understanding their epidemiology and potential transmission routes is crucial for developing effective strategies to mitigate the spread of ESBL-producing E. coli in healthcare settings.

Fluoroquinolone resistance determinants in carbapenem-resistant Escherichia coli isolated from urine clinical samples in Thailand.

Escherichia coli is the most common cause of urinary tract infections and has fluoroquinolone (FQ)-resistant strains, which are a worldwide concern. To characterize FQ-resistant determinants among 103 carbapenem-resistant E. coli (CREc) urinary isolates using WGS. Antimicrobial susceptibility, biofilm formation, and short-read sequencing were applied to these isolates. Complete genome sequencing of five CREcs was conducted using short- and long-read platforms. ST410 (50.49%) was the predominant ST, followed by ST405 (12.62%) and ST361 (11.65%). Clermont phylogroup C (54.37%) was the most frequent. The genes NDM-5 (74.76%) and CTX-M-15 (71.84%) were the most identified. Most CREcs were resistant to ciprofloxacin (97.09%) and levofloxacin (94.17%), whereas their resistance rate to nitrofurantoin was 33.98%. Frequently, the gene aac(6')-Ib (57.28%) was found and the coexistence of aac(6')-Ib and blaCTX-M-15 was the most widely predominant. All isolates carried the gyrA mutants of S83L and D87N. In 12.62% of the isolates, the coexistence was detected of gyrA, gyrB, parC, and parE mutations. Furthermore, the five urinary CREc-complete genomes revealed that blaNDM-5 or blaNDM-3 were located on two plasmid Inc types, comprising IncFI (60%, 3/5) and IncFI/IncQ (40%, 2/5). In addition, both plasmid types carried other resistance genes, such as blaOXA-1, blaCTX-M-15, blaTEM-1B, and aac(6')-Ib. Notably, the IncFI plasmid in one isolate carried three copies of the blaNDM-5 gene. This study showed FQ-resistant determinants in urinary CREc isolates that could be a warning sign to adopt efficient strategies or new control policies to prevent further spread and to help in monitoring this microorganism.

Determination of Antimicrobial Resistance and the Impact of Imipenem + Cilastatin Synergy with Tetracycline in Pseudomonas aeruginosa Isolates from Sepsis.

Pseudomonas aeruginosa is among the most ubiquitous bacteria in the natural world, exhibiting metabolic and physiological versatility, which makes it highly adaptable. Imipenem + cilastatin and tetracycline are antibiotic combinations commonly used to treat infections caused by P. aeruginosa, including serious infections such as sepsis. In the context of bacterial infections, biofilm, formed by bacterial cells surrounded by extracellular substances forming a matrix, plays a pivotal role in the resistance of P. aeruginosa to antibiotics. This study aimed to characterize a representative panel of P. aeruginosa isolates from septicemias, assessing their susceptibility to various antibiotics, specifically, imipenem + cilastatin and tetracycline, and the impact of these treatments on biofilm formation. Results from antibiotic susceptibility tests revealed sensitivity in most isolates to six antibiotics, with four showing near or equal to 100% sensitivity. However, resistance was observed in some antibiotics, albeit at minimal levels. Notably, tetracycline showed a 100% resistance phenotype, while imipenem + cilastatin predominantly displayed an intermediate phenotype (85.72%), with some resistance (38.1%). Microdilution susceptibility testing identified effective combinations against different isolates. Regarding biofilm formation, P. aeruginosa demonstrated the ability to produce biofilms. The staining of microtiter plates confirmed that specific concentrations of imipenem + cilastatin and tetracycline could inhibit biofilm production. A significant proportion of isolates exhibited resistance to aminoglycoside antibiotics because of the presence of modifying genes (aac(3)-II and aac(3)-III), reducing their effectiveness. This study also explored various resistance genes, unveiling diverse resistance mechanisms among P. aeruginosa isolates. Several virulence genes were detected, including the las quorum-sensing system genes (lasI and lasR) in a significant proportion of isolates, contributing to virulence factor activation. However, genes related to the type IV pili (T4P) system (pilB and pilA) were found in limited isolates. In conclusion, this comprehensive study sheds light on the intricate dynamics of P. aeruginosa, a remarkably adaptable bacterium with a widespread presence in the natural world. Our findings provide valuable insights into the ongoing battle against P. aeruginosa infections, highlighting the need for tailored antibiotic therapies and innovative approaches to combat biofilm-related resistance.