ESBL Genes Research Articles

Plasmid-mediated fosfomycin resistance in Escherichia coli isolates of worldwide origin

Fosfomycin is a first-line treatment for uncomplicated urinary tract infections (UTIs) in several European countries, and it is increasingly becoming the treatment of choice globally. Resistance to fosfomycin in Escherichia coli can be exerted through several mechanisms, including the acquisition of fosfomycin-modifying enzymes, of which the FosA-type enzymes are the most common. This study analysed, both phenotypically and genotypically, an international collection of E. coli strains harbouring acquired fosA genes. Thirty-one fosA-positive E. coli isolates were obtained from both clinical and environmental sources, from seven countries (Portugal (n=12), Switzerland (n=9), China (n=3), France (n=2), Nepal (n=2), South Africa (n=2), Kuwait (n=1)). MICs were determined according to EUCAST guidelines. Whole genome sequencing (WGS) was performed on 23 isolates, and complete fosA plasmid sequences were determined for 12. Conjugation assays were performed on seven isolates. All isolates exhibited high-level resistance to fosfomycin (64 to >256 mg/L). WGS of 23 isolates identified 17 sequence types (STs), and 16 harboured fosA3, four fosA4, two fosA8, and one fosA10. ESBLs, pAmpC, or carbapenemase genes were present in 15, four, and three isolates, respectively. The fosA plasmids of 12 isolates were determined and were diverse in size (∼67 kb to ∼235 kb), resistance gene carriage, and replicon types. Six fosA plasmids additionally carried ESBL or carbapenemase genes. Conjugation assays, performed on seven isolates harbouring diverse plasmids, identified that all were capable of being transmitted. This study highlights the necessity of the surveillance and close monitoring of fosfomycin resistance in E. coli, essential to maintain the optimal use of this treatment option.

Current status of β-lactam antibiotic use and characterization of β-lactam-resistant Escherichia coli from commercial farms by integrated broiler chicken operations in Korea

β-Lactam antibiotics are one of the most clinical importance in human and veterinary medicine because they are used for both preventive and therapeutic purposes against several gram-positive, gram-negative, and anaerobic organisms. In this study, it was confirmed that β-lactams (81.1%) were found to be significantly prescribed the most among 74 farms in 5 integrated broiler operations, and single prescription (84.6%), 2-day (41.5%) or 3-day (40.0%) administration, and 15 to 22 d of age (67.7%) administration was significantly higher in the farms (P < 0.05). Among the E. coli isolated from 74 farms in 5 integrated broiler operations, β-lactam-resistant E. coli isolates were detected more frequently in fecal sample (94.6%) than in dust sample (60.8%) (P < 0.05). The prevalence of MDR in β-lactam-resistant isolates, ranging from 88.1 to 96.5%, was significantly higher than that in non-β-lactam-resistant isolates (P < 0.05), without significant differences among operations. Of 466 β-lactam-resistant isolates, 432 (92.7%) isolates harbored β-lactamase genes. The non-extended-spectrum β-lactamase (ESBL) gene blaTEM-1 (81.8%) showed the highest prevalence among isolates, followed by the non-ESBL gene blaTEM-135 (6.4%) (P < 0.05). Five ESBL genes, SHV-12, OXA-1, CTX-M-27, CTX-M-55, and CTX-M-65, were found in 0.9 to 6.0% of the isolates. The pAmpC gene blaCMY-2 was detected in 17 isolates (3.6%). These results suggest that feces and dust are important reservoirs of antimicrobial-resistant bacteria, highlighting the need to strengthen farm management regulations, such as cleaning, disinfection, and litter disposal and to reduce the use of antibiotics in broiler operations.

Analysis of the stepwise acquisition of blaCTX-M-2 and subsequent acquisition of either blaIMP-1 or blaIMP-6 in highly conserved IncN-pST5 plasmids.

ESBL and carbapenemase genes in Enterobacterales spread via plasmids. Nosocomial outbreaks caused by Enterobacterales producing both CTX-M-2 and either IMP-1 or IMP-6-type carbapenemases have been reported. These organisms carry the incompatibility type N plasmid belonging to plasmid ST 5 (IncN-pST5). We investigated the construction process of the ESBL and carbapenemase genes co-carrying IncN-pST5. We retrospectively performed draft WGS analysis for blaIMP- or blaCTX-M-positive Enterobacterales in our strain collection (n = 281). We selected four types of Escherichia coli plasmids for our study: type A, which carries both blaCTX-M-2 and blaIMP-1 (n = 6); type B, which carries both blaCTX-M-2 and blaIMP-6 (n = 2); type C, which carries blaCTX-M-2 (n = 10); and type D, which carries no β-lactamase genes (n = 1). It should be noted that type D plasmid was only detected in E. coli TUM2805, which carries the blaCTX-M-14 on the IncB/O/B/Z plasmid. Long-read sequencing using MinION revealed that all types of IncN-pST5 were highly conserved and carried a class 1 integron. Integron numbers were type A for In798, type B for In1690, type C for In127 and type D for In207. Because the gene cassettes downstream of blaIMP were different between In798 and In1690, the change from blaIMP-1 to blaIMP-6 by point mutation was unlikely. Representative plasmids from types A, B and C were conjugatively transferred with quite a high frequency between 1.3 × 10-1 and 2.5 × 10-2. This study suggested that IncN-pST5 acquired blaCTX-M-2 by ISEcp1 in a stepwise manner, followed by either blaIMP-1 or blaIMP-6 into a class 1 integron.

First Emergence of NDM-5 and OqxAB Efflux Pumps Among Multidrug-Resistant Klebsiella pneumoniae Isolated from Pediatric Patients in Assiut, Egypt.

New Delhi metallo-β-lactamase (NDM)-producing K. pneumoniae poses a high risk, especially among Egyptian pediatric patients who consume carbapenems antibiotics very widely and without adequate diagnostic sources. In addition, presence of efflux pump genes such as OqxAB increases resistance against many groups of antimicrobials which exacerbates the problem faced for human health. This study aimed to determine NDM variants among K. pneumoniae strains isolated from pediatric patients in Egypt, analyze the presence of OqxAB genes, and molecular characterization ofblaNDM-5-positiveK. pneumoniae. Fifty-six K. pneumoniaeisolates were recovered from pediatric patients, and tested for carbapenemase by modified carbapenem inactivation methods (mCIM) test. Minimum inhibitory concentrations of meropenem and colistin were determined by meropenem E-test strips and broth microdilution, respectively. PCR was used for the detection of the resistant genes (ESBL gene (blaCTX-M), carbapenemase genes (blaNDM, blaKPC) colistin resistant (mcr1, mcr2)) and genes for efflux pump (oqxA and oqxB). BlaNDM was sequenced. The effect of efflux pump in NDM-5-producing isolates was assessed by measuring MIC of ciprofloxacin and meropenem before and after exposure to the carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The horizontal gene transfer ability ofblaNDM-5 was determined using liquid mating assay and PCR-based replicon typing (PBRT) was done to determine the major plasmid incompatibility group. Twenty-nine isolates were positive for blaNDM-1, nine isolates were positive for blaNDM-5, and 15 isolates were positive for blaKPC. There is a significant increase of meropenem MIC of NDM-5-positive isolates compared with NDM-1-positive isolates. In addition, 38 isolates were positive for CTX-M, and 15 isolates were positive for mcr1. Both OqxA and OqxB were detected in 26 isolates and 13 isolates were positive for OqxA while 11 isolates were positive for OqxB only. All NDM-5-producing isolates except one isolate could transfer their plasmids by conjugation to their corresponding transconjugants (E. coli J53). Plasmid replicon typing showed that FII was predominant in NDM-5-producing K. pneumoniae. Similar strains were found between the three isolates and similarity was also detected between the two isolates. The highly resistant K. pneumoniae producing blaNDM-5 type was firstly isolated from pediatric patients. The association of efflux pump genes such as OqxAB is involved in resistance to ciprofloxacin. This highlighted the severity risk ofblaNDM-5-positiveK. pneumonia as it could transfer blaNDM-5 to other bacteria and has more resistance against carbapenems. This underlines the importance of continuous monitoring of infection control guidelines, and the urgent need for a national antimicrobial stewardship plan in Egyptian hospitals.

Association between Phenotypes of Antimicrobial Resistance, ESBL Resistance Genes, and Virulence Genes of Salmonella Isolated from Chickens in Sichuan, China.

The aim of this study was to explore the association between antimicrobial resistance, ESBL genes, and virulence genes of Salmonella isolates. From 2019 to 2021, a total of 117 Salmonella isolates were obtained from symptomatic chickens in Sichuan Province, China. The strains were tested for antimicrobial resistance and the presence of ESBL according to the Clinical and Laboratory Standards Institute (CLSI) instructions. The presence of ESBL genes and genes for virulence was determined using Polymerase Chain Reaction (PCR). In addition, Multilocus Sequence Typing (MLST) was applied to confirm the molecular genotyping. Moreover, the mechanism of ESBL and virulence gene transfer and the relationships between the resistance phenotype, ESBL genes, and virulence genes were explored. The isolates exhibited different frequencies of resistance to antibiotics (resistance rates ranged from 21.37% to 97.44%), whereas 68.38% and 41.03% of isolates were multi-drug resistance (MDR) and ESBL-producers, respectively. In the PCR analysis, blaCTX-M was the most prevalent ESBL genotype (73.42%, 58/79), and blaCTX-M-55 showed the most significant effect on the resistance to cephalosporins as tested by logistic regression analysis. Isolates showed a high carriage rate of invA, avrA, sopB, sopE, ssaQ, spvR, spvB, spvC, stn, and bcfC (ranged from 51.28% to 100%). MLST analysis revealed that the 117 isolates were divided into 11 types, mainly ST92, ST11, and ST3717. Of 48 ESBL-producers, 21 transconjugants were successfully obtained by conjugation. Furthermore, ESBL and spv virulence genes were obtained simultaneously in 15 transconjugants. These results highlighted that Salmonella isolates were common carriers of ESBLs and multiple virulence genes. Horizontal transfer played a key role in disseminating antimicrobial resistance and pathogenesis. Therefore, it is necessary to continuously monitor the use of antimicrobials and the prevalence of AMR and virulence in Salmonella from food animals and to improve the antibiotic stewardship for salmonellosis.

Detection and Phylogenetic Analysis of Extended-Spectrum β-Lactamase (ESBL)-Genetic Determinants in Gram-Negative Fecal-Microbiota of Wild Birds and Chicken Originated at Trimmu Barrage.

Extended-spectrum β-lactamases (ESBL) give rise to resistance against penicillin and cephalosporin antibiotics in multiple bacterial species. The present study was conducted to map genetic determinants and related attributes of ESBL-producing bacteria in three wild aquatic bird species and chickens at the "Trimmu Barrage" in district Jhang, Punjab province, Pakistan. To study the prevalence of ESBL-producing bacteria, a total of 280 representative samples were collected from wild bird species; cattle egrets (Bubulcus ibis), little egrets (Egretta garzetta) and common teals (Anas crecca) as well as from indigenous chickens (Gallus gallus domesticus) originating from a local wet market. The isolates were confirmed as ESBL producers using a double disc synergy test (DDST) and bacterial species were identified using API-20E and 20NE strips. A polymerase chain reaction (PCR) was used to detect ESBL genetic determinants and for genus identification via 16S rRNA gene amplification. A phenotypic antimicrobial susceptibility test was performed for ESBL-producing isolates against 12 clinically relevant antibiotics using the Kirby-Bauer disk diffusion susceptibility test. A phylogenetic tree was constructed for the sequence data obtained in this study and comparative sequence data obtained from GenBank. The overall prevalence of ESBL-producing bacteria was 34.64% (97/280). The highest percentage (44.28%; 31/70) of ESBL-producing bacteria was recovered from chickens (Gallus gallus domesticus), followed by little egrets (Egretta garzetta) (41.43%; 29/70), common teal (Anas crecca) (28.57%; 20/70) and cattle egrets (Bubulcus ibis) (24.28%; 17/70). Five different ESBL-producing bacteria were identified biochemically and confirmed via 16S rRNA gene sequencing, which included Escherichia coli (72; 74.23%), Enterobacter cloacae (11; 11.34%), Klebsiella pneumoniae (8; 8.25%), Salmonella enterica (4; 4.12%) and Pseudomonas aeruginosa (2; 2.06%). Based on PCR, the frequency of obtained ESBL genes in 97 isolates was blaCTX-M (51.55%), blaTEM (20.62%), blaOXA (6.18%) and blaSHV (2.06%). In addition, gene combinations blaCTX-M + blaTEM, blaTEM + blaOXA and blaCTX-M + blaSHV were also detected in 16.49%, 2.06% and 1.03% of isolates, respectively. The ESBL gene variation was significant (p = 0.02) in different bacterial species while non-significant in relation to different bird species (p = 0.85). Phylogenetic analysis of amino acid sequence data confirmed the existence of CTX-M-15 and TEM betalactamases. The average susceptibility of the antibiotics panel used was lowest for both Klebsiella pneumoniae (62.5% ± 24.42) and Salmonella enterica (62.5% ± 31.08) as compared to Enterobacter cloacae (65.90% ± 21.62), Pseudomonas aeruginosa (70.83% ± 33.42) and Escherichia coli (73.83% ± 26.19). This study provides insight into the role of aquatic wild birds as reservoirs of ESBL-producing bacteria at Trimmu Barrage, Punjab, Pakistan. Hence, active bio-surveillance and environment preservation actions are necessitated to curb antimicrobial resistance.

Prevalence and molecular characterization of cefotaxime-resistant Salmonella strains recovered from retail meat samples in Shenzhen, China, during 2014-2017.

In this work, we collected foodborne Salmonella strains in Shenzhen, China, during 2014-2017 and investigated the genetic profile of all cefotaxime-resistant isolates in the collection. The strains were subjected to antimicrobial susceptibility tests, whole-genome sequencing, bioinformatics analysis, and conjugation studies. A total of 79 cefotaxime-resistant Salmonella were identified and found to exhibit multidrug resistance. Resistance rate recorded during the study period increased from 1.9% to 9.1%. Salmonella Typhimurium was the predominant serovar, and CTX-M family genes were dominant among the ESBLs genes detected. Notably, CTX-M-bearing plasmids or transposons often contain other drug resistance genes. Furthermore, a combination of CTX-M-55 and CTX-M-65 genes was detected for the first time in foodborne Salmonella strains. Our findings reveal the prevalence and molecular characteristics of cefotaxime-resistant foodborne Salmonella strains in southern China. IMPORTANCE Cefotaxime-resistant Salmonella strains pose an increasing threat to human health by causing infections with limited treatment options. It is therefore necessary to undertake a surveillance on the prevalence of such strains and investigate the resistance and transmission mechanisms. In this work, various ESBL genes flanked by different IS located in different mobile genetic elements were detectable among cefotaxime-resistant Salmonella strains. These data show that the high prevalence and genotypic diversity of cefotaxime-resistant foodborne Salmonella strains in China are possibly attributed to the evolution and transmission of a wide range of multidrug resistance-encoding mobile genetic elements.

High frequency and molecular characterization of ESBL-producing Enterobacteriaceae isolated from wound infections in North Lebanon

ABSTRACT Background Extended-spectrum beta-lactamases producing Enterobacteriaceae (ESBL-PE) represent a major problem in wound infections. Here, we investigated the prevalence and molecular characterization of ESBL-PE associated with wound infections in North Lebanon. Research design and methods A total of 103 non-duplicated E. coli and K. pneumoniae strains isolated from 103 patients with wound infections, were collected from seven hospitals in North Lebanon. ESBL-producing isolates were detected using a double-disk synergy test. In addition, multiplex polymerase chain reaction (PCR) was used for the molecular detection of ESBLs genes. Results E. coli was the predominant bacteria (77.6%), followed by K. pneumoniae (22.3%). The overall prevalence of ESBL-PE was 49%, with a significantly higher rate among females and elderly patients. K. pneumoniae was the common MDR and ESBL-producer bacteria (86.95% and 52.17%) compared to E. coli (77.5% and 47.5%). Most of the isolated ESBL producers harbored multiple resistant genes (88%), where blaCTX-M was the most predominant gene (92%), followed by blaTEM (86%), blaSHV (64%), and blaOXA genes (28%). Conclusions This is the first data on the ESBL-PE prevalence associated with wound infections in Lebanon, showing the emergence of multidrug-resistant ESBL-PE, the dominance of multiple gene producers, and the widespread dissemination of blaCTX-M and blaTEM genes.

Molecular detection of multidrug-resistant Pseudomonas aeruginosa of different avian sources with pathogenicity testing and in vitro evaluation of antibacterial efficacy of silver nanoparticles against multidrug-resistant P. aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is a serious zoonotic pathogen threaten the poultry industry causing severe economic losses therefor, this study aimed to isolation, phenotypic, molecular identification of P. aeruginosa from different avian sources (chickens, turkey, pigeons, table eggs, and dead in shell chicken embryos), from different Egyptian governorates (Giza, Qalubia, Beheira, El-Minya, and Al-Sharqia) with applying of antibiotic sensitivity test on all P. aeruginosa isolates. Highly resistant isolates (n=49) were subjected to molecular identification of P. aeruginosa with detection of resistant genes including carbapenemase-encoding genes blaKPC, blaOXA-48, and blaNDM. On the base of molecular results, a highly resistant P. aeruginosa strain was tested for its pathogenicity on day old specific pathogen free (SPF) chicks. Also, in vitro experiment was adopted to evaluate the efficacy of silver nanoparticles (Ag-NPs) against highly antibiotic-resistant P. aeruginosa strains. The overall isolation percentage was from all examined samples were 36.2% (571/1,576) representing 45.2% (532/1,176) from different birds' tissues and 39/400 (9.7%) from total egg samples. Some of isolated strains showed multidrug resistance (MDR) against kanamycin, amoxicillin, amoxicillin-clavulanic acid, neomycin, chloramphenicol, vancomycin, cefotaxime clavulanic acid, lincomycin-spectinomycin, co-trimoxazole, cefoxitin, gentamycin, and doxycycline. These MDR strains were also molecularly positive for ESBL and carbapenemase-encoding genes. MDR strain showed high pathogenicity with histopathological alterations in different organs in challenged birds. Main histopathological lesions were necrosis of hepatocytes, renal tubular epithelium, and heart muscle bundles. The MDR strain showed in vitro sensitivity to Ag-NPs. In conclusion, MDR P. aeruginosa is a serious pathogen causing high morbidity, mortality, and pathological tissue alterations. Ag NPs revealed a promising in vitro antimicrobial sensitivity against MDR P. aeruginosa and further in vivo studies were recommended.

Prevalence of AmpC, ESBL, and colistin resistance genes in Enterobacterales isolated from ready-to-eat food in Algeria.

Antimicrobial resistance among bacteria present in ready-to-eat foods is an emerging concern. Hence, this study investigated the presence of extended-spectrum and AmpC β-lactamases (ESBL/AmpC)-producing Enterobacterales (ESBL-E) and the dissemination of mcr-1 in ESBL-E from ready-to-eat food samples (RTE) in Algeria. RTE food samples (n = 204) were aseptically collected and selectively cultured using MacConkey agar. The isolates were screened for ESBL production using the DDST test, confirmed ESBL-E isolates were identified using different conventional methods and MALDI-TOF MS, antibiotic susceptibility was determined using the disc diffusion and broth microdilution assay, ESBL-E isolates were analyzed for colistin and ESBL/AmpC encoding genes by PCR, and food samples were analyzed by univariate and multiple logistic regression. Overall, 48 (17.4%) of the 276 Enterobacterales were confirmed as ESBL producers, with a high prevalence in soups (40%), salads (25%), and cream-filled pastries (23.8%). Antibiotic susceptibility testing revealed that all the ESBL-E isolates were found multi-drug resistant. PCR revealed that blaTEM, blaCTX-M, blaCMY-2, blaOXA-1, and blaSHV were the most frequently detected. blaCTX-M-9 and blaCTX-M-1 were the predominant CTX-M types. Furthermore, four isolates were positive for mcr-1; three of them harbored the colistin resistance gene and ESBL/AmpC genes (2 E. cloacae and 1 S. enterica). To the best of our knowledge, this is the first report that detects the presence of the mcr-1 gene in ESBL-E strains isolated from RTE foods in Algeria. These findings suggest an urgent need for strict policies that prevent the spread and transmission of ESBL-E in food.

Characterization of Multiple Resistance Genes of Escherichia coli Strains Isolated from Food and Clinical Samples.

Multi-drug resistance (MDR), which is formed by the development of antimicrobial resistance, which is one of the defense mechanisms in bacteria, has pushed human beings to constantly seek new antimicrobial agents in the fight against these microorganisms. Speed and precision are very important in identifying resistance in strains with MDR that reach humans in an open system through the consumption of contaminated food or water. The main aim of this study was the molecular characterization of ESBL gene variants (blaTEM, blaOXA, blaSHV, and blaCTX-M), integron genes (int1, int2, and int3), and sulphonamide (sul1, sul2, and sul3) resistance genes by PCR from E. coli isolates originated from food and clinical samples. A total of 17 sets of primers were used for phylogenetic identification, molecular detection, and resistance and integron gene characterization of phenotypically identified E. coli isolates. The 45 red meat samples were collected from local markets, which are located in four (Adıyaman, Gaziantep, Kahramanmaraş, and Hatay) different provinces, clinical isolates were obtained from urine samples of patients with UTI from Şanlıurfa Mehmet Akif İnan Training and Research Hospital. Three gene groups were screened with 3 multiplex PCR applications in 63 food and 33 clinical isolates found to be E. coli by molecular identification. In terms of the 3 gene groups screened in food samples, the highest rates were found in the blaSHV gene at 44.44%, the sul1 gene at 69.84% and the int2 gene at 73.02%; in clinical samples, it is listed as blaCTX-M gene at 15.15%, sul2 gene at 81.82% and int1 gene at 54.55%. In terms of 3 gene groups scanned, the presence of 3 or more genes, including at least one gene from each gene group, was detected in 31 isolates from food samples and 2 isolates from clinical samples. Overall, it can be said that the high frequency of MDR genes isolated from E. coli-contaminated red meat samples and clinical samples gives a clue about the overuse of antibiotics in Türkiye.

Detection and genetic characterization of blaESBL-carrying plasmids of cloacal Escherichia coli isolates from white stork nestlings (Ciconia ciconia) in Spain

This study aimed to characterize Escherichia coli isolates from cloacal samples of white stork nestlings, with a special focus on extended-spectrum β-lactamases (ESBLs)-producing E. coli isolates and their plasmid content. Cloacal samples of 88 animals were seeded on MacConkey-agar and chromogenic-ESBL plates to recover E. coli and ESBL-producing E. coli. Antimicrobial susceptibility was screened using the disc diffusion method, and the genotypic characterization was performed by polymerase chain reaction (PCR) and subsequent sequencing. S1 nuclease Pulsed-Field-Gel-Electrophoresis (PFGE), Southern blotting, and conjugation essays were performed on ESBL-producing E. coli, as well as whole-genome sequencing by short- and long-reads. The four blaESBL-carrying plasmids were completely sequenced. A total of 113 non-ESBL-producing E. coli isolates were collected on antibiotic-free MacConkey-agar, of which 27 (23.9%) showed a multidrug-resistance (MDR) phenotype, mainly associated with β-lactam-phenicol-sulfonamide resistance (blaTEM/cmlA/floR/sul1/sul2/sul3). Moreover, four white stork nestlings carried ESBL-producing E. coli (4.5%) with the following characteristics: blaSHV-12/ST38-D, blaSHV-12/ST58-B1, blaCTX-M-1/ST162-B1, and blaCTX-M-32/ST155-B1. Whole-genome sequencing followed by Southern blot hybridizations on S1-PFGE gels in ESBL-positive isolates proved that the blaCTX-M-1 gene and one of the blaSHV-12 genes were carried by IncI1/pST3 plasmids, while the second blaSHV-12 gene and the blaCTX-M-32 gene were located on IncF plasmids. The two blaSHV-12 genes and the two blaCTX-M genes had similar but non-identical close genetic environments, as all four genes were flanked by a variety of insertion sequences. The role played by several genetic platforms in the mobility of ESBL genes allows for interchangeability on a remarkably small scale (gene-plasmid-clones), which may support the spread of ESBL genes.

Carriage of ESBL-producing Klebsiella pneumoniae and Escherichia coli among children in rural Ghana: a cross-sectional study

BackgroundExtended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae (ESBL-KP) and Escherichia coli (ESBL-EC) present a high burden in both communities and healthcare sectors, leading to difficult-to-treat infections. Data on intestinal carriage of ESBL-KP and ESBL-EC in children is scarce, especially in sub-Saharan African countries. We provide data on faecal carriage, phenotypic resistance patterns, and gene variation of ESBL-EC and ESBL-KP among children in the Agogo region of Ghana.MethodsFrom July to December 2019, fresh stool samples were collected within 24 h from children < 5 years with and without diarrhoea attending the study hospital. The samples were screened for ESBL-EC and ESBL-KP on ESBL agar and confirmed using double-disk synergy testing. Bacterial identification and an antibiotic susceptibility profile were performed using the Vitek 2 compact system (bioMérieux, Inc.). ESBL genes, blaSHV, blaCTX-M, and blaTEM were identified by PCR and further sequencing.ResultsOf the 435 children recruited, stool carriage of ESBL-EC and ESBL-KP was 40.9% (n/N = 178/435) with no significant difference in prevalence between children with diarrhoea and non-diarrhoea. No association between ESBL carriage and the age of the children was found. All isolates were resistant to ampicillin and susceptible to meropenem and imipenem. Both ESBL-EC and ESBL-KP isolates showed over 70% resistance to tetracycline and sulfamethoxazole-trimethoprim. Multidrug resistance was observed in over 70% in both ESBL-EC and ESBL-KP isolates. The blaCTX-M-15 was the most prevalent ESBL gene detected. blaCTX-M-27, blaCTX-M-14, and blaCTX-M-14b were found in non-diarrhoea stools of children, whereas blaCTX-M-28 was found in both the diarrhoea and non-diarrhoea patient groups.ConclusionsThe carriage of ESBL-EC and ESBL-KP among children with and without diarrhoea in the Agogo community with a high prevalence of blaCTX-M-15 is noteworthy, highlighting the importance of both the population as a possible reservoir. This study reports for the first time the ESBL gene blaCTX-M-28 among the studied populations in Ghana.

Phenotypic and molecular characterization of enteropathogenic Escherichia coli and Salmonella spp. causing childhood diarrhoea in Awka, South-Eastern Nigeria

BackgroundDiarrhoea is a major cause of childhood disease in the developing countries. This experimental study investigated the prevalence of ESBL and MBL genes in enteropathogenic strains of Escherichia coli and Salmonella spp. isolated from diarrheagenic children in Awka, Nigeria.MethodsTwo hundred stool samples were collected from diarrhea patients in three paediatric hospitals within Awka metropolis, Nigeria. All E. coli and Salmonella spp. isolated through standard bacteriological methods were subjected to antibiotic-susceptibility testing. Double disc synergy and imipenem-EDTA combined disc tests were used to phenotypically confirm the presence of ESBL and MBL respectively. PCR amplification of β-lactamase genes was done.ResultsThe prevalence of E. coli and Salmonella species in this study were 54% and 24.5% respectively. The organisms were highly resistant to metronidazole, cefuroxime and ceftazidime, and also showed a high sensitivity to nitrofurantoin and gentamicin. ESBL production was recorded in E. coli (49%) and Salmonella spp. (51.1%) while 27 isolates of E. coli (25%) and 7 isolates of Salmonella spp. were confirmed MBL positive by the combined disk diffusion technique. Eleven E. coli and 4 Salmonella spp. co-harbored both ESBL and MBL production. The most prevalent MBL gene in this study is the blaVIM gene (18.8%) which mediate MBL production in Gram negative bacteria; and this was followed by blaSHV (12.5%), blaTEM and blaCTX-M (6.3% each) for E. coli isolates. Salmonella spp. was recorded to have blaVIM (28.8%), blaSHV (28.8%), blaTEM (14.3%) and blaCTX-M (14.3%) genes.ConclusionsThis study reveals the prevalence of enteropathogenic E. coli and Salmonella strains bacteriologically recovered from diarrheic children in Awka, Nigeria, and which were found to be multiple resistant to clinically-relevant antibiotics because they co-express ESBL and MBL genes which mediate multidrug resistance in Gram negative bacteria.

Colonization of Residents and Staff of an Italian Long-Term Care Facility and an Adjacent Acute Care Hospital Geriatrics Unit by Multidrug-Resistant Bacteria.

In 2022, we undertook a point prevalence screening study for Enterobacterales with extended-spectrum β-lactamases (ESBLs), high-level AmpC cephalosporinases and carbapenemases, and also methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) in a long-term care facility (LTCF) and the associated acute-care hospital Geriatrics unit in Bolzano, Northern Italy. Urine samples and rectal, inguinal, oropharyngeal, and nasal swabs were plated on selective agar plates. Metadata of the patients, including demographic data, were collected, and risk factors for colonization were determined. ESBL, AmpC, carbapenemase, and quinolone resistance genes were investigated by the HybriSpot 12 PCR AUTO System. The following colonization percentages by multidrug-resistant (MDR) bacteria have been found in LTCF residents: all MDR organisms, 59.5%; ESBL producers, 46.0% (mainly CTX-M-type enzymes); carbapenemase producers, 1.1% (one Klebsiella pneumoniae with KPC-type); MRSA, 4.5%; VRE, 6.7%. Colonization by MDR bacteria was 18.9% for LTCF staff and 45.0% for Geriatrics unit patients. Peripheral vascular disease, the presence of any medical device, cancer, and a Katz Index of 0 were significant risk factors for colonization of LTCF residents by MDR bacteria in univariate and/or multivariate regression analysis. To conclude, the ongoing widespread diffusion of MDR bacteria in the LTCF suggests that efforts should be strengthened on MDR screening, implementation of infection control strategies, and antibiotic stewardship programs targeting the unique aspects of LTCFs. ClinicalTrials.gov ID: 0530250-BZ Reg01 30/08/2022.

Phenotypic and molecular characterization of beta-lactam resistant Multidrug-resistant Enterobacterales isolated from patients attending six hospitals in Northern Nigeria

Infections caused by multi-drug resistant Enterobacterales (MDR-E) are difficult to treat and cause significant mortality, especially in developing countries. This study characterized the phenotypic and genotypic profiles of 49 randomly selected beta-lactam resistant MDR-E previously isolated from patients being managed in hospitals in Nigeria using whole genome sequencing. The study isolates exhibited 85.5% resistance to 3rd generation cephalosporins and 65.3% resistance to carbapenems. The blaTEM-1B (29, 59.2%),blaCTX-M-15 (38, 77.6%), and blaNDM-1 (17, 51.5%) were the most common penicillinase, ESBL, and carbapenem resistant genes across isolates, respectively. Seventeen (45%) of blaCTX-M-15 was carried on the insertion sequence ISEc9 while blaNDM-1 (11, 64.7%) were associated with ISEc33. None of the 21 plasmids detected were associated with β-lactamase genes. Higher resistance rates were found in E. coli ST-88 (n = 2) and the high-risk ST-692 (n = 2). For Klebsiella species, the high-risk clones ST-476 (n = 8) and ST-147 (n = 3) predominated and had higher phenotypic resistance rates and higher number of AMR genes. The mechanisms and pattern of antibiotic resistance differ from patterns previously described with isolates harbouring a wide range of AMRGs. The detection of several chromosomally mediated carbapenemases in our study also represents a significant finding that warrants further investigation to better understand its’ implications for clinical practice and public health. The selected MDR-Es were found to be pan-susceptible to tigecycline and had very low resistance to fosfomycin, suggesting a potential for these as empiric treatments. A surveillance approach incorporating both conventional laboratory techniques and modern molecular techniques is essential for the comprehensive characterization of the emergence and dissemination of antimicrobial resistance in Enterobacterales infections within Nigeria.

Whole strains vs MGEs in short and longterm transmission of ESBL genes between healthcare and community settings in Uganda

Multidrug-resistant ESBL-producing Escherichia coli are a leading cause of infections in hospital and community settings. Based on samples from two hospitals in Uganda and households of inpatients we tested the hypothesis that ESBL E. coli and/or their resistance determinants could spread within the healthcare and community settings through discharged patients that were still colonized. We used bacterial culture, susceptibility testing whole genome sequencing and detailed bioinformatics analysis to test the above hypothesis. Genome analysis revealed presence of predominantly blaCTX-M-15 and blaOXA-1 genes with a total resistome with genes belonging to 14 different classes of antimicrobials. Short-term cases of strain sharing were reported within each setting and strains from the two settings were found to cluster together based on their overall resistome. Long-term horizontal transfer of ESBL genes by various IncF and IncY types of plasmids shared between healthcare and community settings was demonstrated. Based on hybrid assembly, plasmid reconstruction and phylogenetic analyses, our study suggests that while the dissemination of AMR between healthcare and community settings in the short-term is possible at whole strain level, the long-term transmission between healthcare and communities is sustained by the transfer of plasmids circulating across niches and disseminating related resistomes.

Genotypic Determination of Extended Spectrum β-Lactamases and Carbapenemase Production in Clinical Isolates of Klebsiella pneumoniae in Southwest Nigeria

Introduction: Klebsiella pneumoniae is a major pathogen implicated in healthcare-associated infections. Extended-spectrum β-lactamase (ESBL) and carbapenemase-producing K. pneumoniae isolates are a public health concern. This study investigated the existence of some ESBL and carbapenemase genes among clinical isolates of K. pneumoniae in Southwest Nigeria and additionally determined their circulating clones. Materials and Methods: Various clinical samples from 420 patients from seven tertiary hospitals within Southwestern Nigeria were processed between February 2018 and July 2019. These samples were cultured on blood agar and MacConkey agar, and the isolated bacteria were identified by Microbact GNB 12E. All K. pneumoniae were confirmed by polymerase chain reaction (PCR) using the 16s rRNA gene. Antibiotic susceptibility testing (AST) was done on these isolates, and the PCR was used to evaluate the common ESBL-encoding genes and carbapenem resistance genes. Genotyping was performed using multi-locus sequencing typing (MLST). Results: The overall prevalence of K. pneumoniae in Southwestern Nigeria was 30.5%. The AST revealed high resistance rates to tetracyclines (67.2%), oxacillin (61.7%), ampicillin (60.2%), ciprofloxacin (58.6%), chloramphenicol (56.3%), and lowest resistance to meropenem (43.0%). All isolates were susceptible to polymyxin B. The most prevalent ESBL gene was the TEM gene (47.7%), followed by CTX-M (43.8%), SHV (39.8%), OXA (27.3%), CTX-M-15 (19.5%), CTX-M-2 (11.1%), and CTX-M-9 (10.9%). Among the carbapenemase genes studied, the VIM gene (43.0%) was most detected, followed by OXA-48 (28.9%), IMP (22.7%), NDM (17.2%), KPC (13.3%), CMY (11.7%), and FOX (9.4%). GIM and SPM genes were not detected. MLST identified six different sequence types (STs) in this study. The most dominant ST was ST307 (50%, 5/10), while ST258, ST11, ST147, ST15, and ST321 had (10%, 1/10) each. Conclusion: High antimicrobial resistance in K. pneumoniae is a clear and present danger for managing infections in Nigeria. Additionally, the dominance of a successful international ST307 clone highlights the importance of ensuring that genomic surveillance remains a priority in the hospital environment in Nigeria.

Characterization of antibiotic resistance genes and mobile elements in extended-spectrum β-lactamase-producing Escherichia coli strains isolated from hospitalized patients in Guangdong, China.

This study aimed to investigate the high-resolution phenotypic and genotypic characterization of extended-spectrum β-lactamase (ESBL)-producing E. coli strains isolated from hospitalized patients to explore the resistance genes and mobile genetic elements (MGEs) involved in horizontal dissemination. Between May and September 2021, a total of 216 ESBL-producing E. coli isolates were recovered from multiple departments. The identification of strains was performed using MALDI-TOF mass spectrometry and PCR, while antibiotic susceptibility testing was carried out using the Vitek 2 COMPACT system to determine resistance patterns, while PCR was used to detect different resistance genes and MGEs. In addition, a conjugation assay was performed to investigate the horizontal gene transfer of resistance genes. Selected isolates underwent whole-genome sequencing using the Illumina MiSeq platform. A total of 216 out of 409 E. coli isolates recovered from a tertiary hospital were observed to be ESBL-producing, giving a carriage rate of 52.8%, as determined by phenotypic screening. The most frequent sources of ESBL-producing E. coli isolates were urine (129/216, 59.72%,) and blood (50/216, 23.14%). The most prevalent ESBL genes identified were blaCTX-M (60.18%), blaTEM (40.27%), and blaSHV (18.05%). Three E. coli isolates were found to carry the genes blaNDM, mcr-1, and fosA3 genes. The most prevalent MGEs were IS26 (95.37%), Int (87.03%), and IncFIB (76.85%). Whole-genome sequencing analysis of eight MDR E. coli strains revealed that these isolates belonged to eight different sequence types (STs) and serotypes and were found to harbor multiple plasmid replicons and virulence factors. This study highlights a high incidence of antibiotic resistance genes and MGEs associated with the dissemination of ESBLs and other resistance genes.

Genetic Profiling of Pathogens Associated with Pyroxia of Unknown Origin Treated for Plasmodiasis in Bayelsa State, Nigeria

Antimicrobial resistance of Salmonella is an increasing problem and has become a public health issue worldwide. Moreover the evidences on hazard of therapeutic failure due to the increasing incidence of antimicrobial resistance among typhoidal antibodies and malaria are on the increase. This study was aimed at the genetic profiling of pathogens associated with Pyroxia of unknown origin treated for Plasmodiasis in Bayelsa State, Nigeria. A total of 1200 samples (200 Stool and 1000 blood) were collected from consented subjects in Federal Medical Center and Niger Delta University Teaching Hospital Bayelsa State, Nigeria. Blood samples were used for detection of Malaria parasites and Typhoidal antibodies while the stool samples were used for culturing on Salmonella-Shigella Agar. Antibiotic sensitivity of the pure isolates were carried out using sensitivity disc, biochemical analysis using API 20E was carried out to actually differentiate bacterial from each isolates and finally by genetic profiling of the resistant genes of the pure isolates using polymerase chain reaction. Antibiogram of the isolates showed high resistance to Oxacillin (73.6 %), Ampicillin (66.6%), Tetracycline (60.0%), and Erythromycin (53.3%) while Gentamycin, 22(73.3%), Chloramphenicol 18(60%) and Azithromycin 18(60%) had the highest sensitivity. The distribution of resistant genes from the genetic profiling showed that out of 20 amplified isolates, NDM resistant genes were 17(85%) followed by Bla TEM genes 14(70%) followed by CTXM and SHV genes as 11(55%) and 10(50%) respectively. The bacterial isolates showed high AMR and MDR to the commonly used antimicrobial drugs and the distribution of ESBL genes revealed a high level of resistant genes in Bayelsa State.