Β-lactamase Resistance Genes Research Articles

Analysis of the molecular epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae in a hospital in Hunan Province

To examine the molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) and investigate the horizontal transmission of blaKPC and blaNDM genes for the prevention and treatment of CRKP. A total of 49 clinically isolated CRKP strains were retrospectively analyzed from January to December 2022 at The First Hospital of Hunan University of Chinese Medicine. Phenotypic screening was performed using modified carbapenem inactivation assay (mCIM) and EDTA-carbapenem inactivation assay (eCIM). Polymerase chain reaction (PCR) was utilized to identify carbapenem resistance genes, β-lactamase resistance genes, and virulence genes, while multi-locus sequence analysis (MLST) was employed to assess the homology of CRKP strains. Conjugation experiments were conducted to infer the horizontal transmission mechanism of blaKPC and blaNDM genes. The results showed that the study included 49 CRKP strains, with 44 carrying blaKPC and 8 carrying blaNDM, Three strains were identified as blaKPC+blaNDM-CRKP. In this study, 28 out of 49 CRKP strains (57.2%) were found to carry virulence genes. Additionally, one CRKP strain tested positive in the string test and was found to carry both Aerobactin and rmpA virulence genes. MLST results revealed a total of 5 ST types, with ST11 being predominant (41/49, 83.7%). Successful conjugation was observed in all 3 blaKPC-CRKP strains, while only 1 out of 3 blaNDM-CRKP strains showed successful conjugation. The transconjugant exhibited significantly reduced susceptibility to imipenem and cephalosporin antibiotics. In conclusion, the resistance mechanism of CRKP in this study is primarily attributed to the production of KPC enzymes, along with the presence of multiple β-lactamase resistance genes. Additionally, there is a local prevalence of hv-CRKP and blaKPC+blaNDM-CRKP. blaKPC and blaNDM can be horizontally transmitted through plasmids, with varying efficiency among different strains.

Colistin-, cefepime-, and levofloxacin-resistant Salmonella enterica serovars isolated from Egyptian chicken carcasses

ObjectivesThe emergence of multidrug-resistant (MDR) Salmonella strains, especially resistant ones toward critically important antimicrobial classes such as fluoroquinolones and third- and fourth-generation cephalosporins, is a growing public health concern. The current study, therefore, aimed to determine the prevalence, and existence of virulence genes (invA, stn, and spvC genes), antimicrobial resistance profiles, and the presence of β-lactamase resistance genes (blaOXA, blaCTX-M1, blaSHV, and blaTEM) in Salmonella strains isolated from native chicken carcasses in Egypt marketed in Mansoura, Egypt, as well as spotlight the risk of isolated MDR, colistin-, cefepime-, and levofloxacin-resistant Salmonella enterica serovars to public health.MethodsOne hundred fifty freshly dressed native chicken carcasses were collected from different poultry shops in Mansoura City, Egypt between July 2022 and November 2022. Salmonella isolation was performed using standard bacteriological techniques, including pre-enrichment in buffered peptone water (BPW), selective enrichment in Rappaport Vassiliadis broth (RVS), and cultivating on the surface of xylose-lysine-desoxycholate (XLD) agar. All suspected Salmonella colonies were subjected to biochemical tests, serological identification using slide agglutination test, and Polymerase Chain Reaction (PCR) targeting the invasion A gene (invA; Salmonella marker gene). Afterward, all molecularly verified isolates were screened for the presence of virulence genes (stn and spvC). The antimicrobial susceptibility testing for isolated Salmonella strains towards the 16 antimicrobial agents tested was analyzed by Kirby–Bauer disc diffusion method, except for colistin, in which the minimum inhibition concentration (MIC) was determined by broth microdilution technique. Furthermore, 82 cefotaxime-resistant Salmonella isolates were tested using multiplex PCR targeting the β-lactamase resistance genes, including blaOXA, blaCTX-M1, blaSHV, and blaTEM genes.ResultsSalmonella enterica species were molecularly confirmed via the invA Salmonella marker gene in 18% (27/150) of the freshly dressed native chicken carcasses. Twelve Salmonella serotypes were identified among 129 confirmed Salmonella isolates with the most predominant serotypes were S. Kentucky, S. Enteritidis, S. Typhimurium, and S. Molade with an incidence of 19.4% (25/129), 17.1% (22/129), 17.1% (22/129), and 10.9% (14/129), respectively. All the identified Salmonella isolates (n = 129) were positive for both invA and stn genes, while only 31.8% (41/129) of isolates were positive for the spvC gene. One hundred twenty-one (93.8%) of the 129 Salmonella-verified isolates were resistant to at least three antibiotics. Interestingly, 3.9%, 14.7%, and 75.2% of isolates were categorized into pan-drug-resistant, extensively drug-resistant, and multidrug-resistant, respectively. The average MAR index for the 129 isolates tested was 0.505. Exactly, 82.2%, 82.2%, 63.6%, 51.9%, 50.4%, 48.8%, 11.6%, and 10.1% of isolated Salmonella strains were resistant to cefepime, colistin, cefotaxime, ceftazidime/clavulanic acid, levofloxacin, ciprofloxacin, azithromycin, and meropenem, respectively. Thirty-one out (37.8%) of the 82 cefotaxime-resistant Salmonella isolates were β-lactamase producers with the blaTEM as the most predominant β-lactamase resistance gene, followed by blaCTX-M1 and blaOXA genes, which were detected in 21, 16, and 14 isolates respectively).ConclusionThe high prevalence of MDR-, colistin-, cefepime-, and levofloxacin-resistant Salmonella serovars among Salmonella isolates from native chicken is alarming as these antimicrobials are critically important in treating severe salmonellosis cases and boost the urgent need for controlling antibiotic usage in veterinary and human medicine to protect public health.Graphical

β-lactamase and macrolide resistance gene carriage in E. coli isolates among children discharged from inpatient care in western Kenya: a cross-sectional study

Abstract Background Antimicrobial resistance (AMR) is a global threat to infectious disease control, particularly among recently hospitalized children. We sought to determine the prevalence and mitigating factors of resistance in enteric Escherichia coli among children discharged from health facilities in western Kenya. Methods Between June 2016 and November 2019, children aged 1-59 months were enrolled at the point of discharge from hospital. E. coli was isolated by microbiological culture from rectal swabs at baseline. β-lactamases and macrolide resistance-conferring genes were detected by PCR. The modified Poisson regression model was used to assess the predictors mph(A) and CTX-M-type ESBL. Results Of the 238 children whose E. coli isolates were tested, 91 (38.2%) and 109 (45.8%) had detectable CTX-M-type ESBL and mph(A) genes, respectively. Antibiotic treatment during hospitalization (adjusted prevalence ratio [aPR]=2.47; 95%CI:1.12-5.43, p=0.025), length of hospitalization (aPR=1.42; 95%CI:1.00-2.01, p=0.052), and the practice of open defecation (aPR=2.47; 95%CI:1.40-4.36, p=0.002) were independent predictors for CTX-M-type ESBL and for mph(A) genes. Pneumococcal vaccination was associated with a 43% lower likelihood of CTX-M-type ESBL (aPR=0.57; 95%CI:0.38-0.85, p=0.005), whilst measles vaccination was associated with a 32% lower likelihood of mph(A) gene (aPR=0.68; 95%CI:0.49-0.93, p=0.017) in E. coli isolates. Conclusions Among children discharged from hospital, history of vaccination, shorter hospital stay, lack of in-hospital antibiotic exposure and improved sanitation were associated with lower likelihood of AMR genes. AMR control programs should consider strategies beyond antimicrobial stewardship, including improvements in sanitation, increased vaccine coverage and the development of novel vaccines, to mitigate the continued spread of AMR.

Genomic analysis of multidrug-resistant Escherichia coli from Urban Environmental water sources in Accra, Ghana, Provides Insights into public health implications.

Wastewater discharge into the environment in resource-poor countries poses a threat to public health. Studies in this area within these countries are limited, and the use of high-throughput whole-genome sequencing technologies is lacking. Therefore, understanding of environmental impacts is inadequate. The present study investigated the antibiotic resistance profiles and diversity of beta-lactamases in Escherichia coli strains isolated from environmental water sources in Accra, Ghana. Microbiological analyses were conducted on wastewater samples from three hospitals, a sewage and wastewater treatment plant, and water samples from two urban surface water bodies. Confirmed isolates (N = 57) were selected for phenotypic antibiotic resistance profiles. Multi-drug-resistant isolates (n = 25) were genome sequenced using Illumina MiSeq sequencing technology and screened for sequence types, antibiotic resistance, virulence and beta-lactamase genes, and mobile genetic elements. Isolates were frequently resistant to ampicillin (63%), meropenem (47%), azithromycin (46%), and sulfamethoxazole-trimethoprim (42%). Twenty different sequence types (STs) were identified, including clinically relevant ones such as ST167 and ST21. Five isolates were assigned to novel STs: ST14531 (n = 2), ST14536, ST14537, and ST14538. The isolates belonged to phylogroups A (52%), B1 (44%), and B2 (4%) and carried β-lactamase (TEM-1B, TEM-1C, CTX-M-15, and blaDHA-1) and carbapenemase (OXA-1, OXA-181) resistance genes. Dominant plasmid replicons included Col440I (10.2%) and IncFIB (AP001918) (6.8%). Polluted urban environments in Accra are reservoirs for antibiotic-resistant bacteria, posing a substantial public health risk. The findings underscore the need for targeted public health interventions to mitigate the spread of antibiotic-resistant bacteria and protect public health.

Serovar and sequence type distribution and phenotypic and genotypic antimicrobial resistance of Salmonella originating from pet animals in Chongqing, China.

A total of 334 Salmonella isolates were recovered from 6,223 pet rectal samples collected at 50 pet clinics, 42 pet shops, 7 residential areas, and 4 plazas. Forty serovars were identified that included all strains except for one isolate that did not cluster via self-agglutination, with Salmonella Typhimurium monophasic variant, Salmonella Kentucky, Salmonella Enteritidis, Salmonella Pomona, and Salmonella Give being the predominant serovars. Fifty-one sequence types were identified among the isolates, and ST198, ST11, ST19, ST451, ST34, and ST155 were the most common. The top four dominant antimicrobials to which isolates were resistant were sulfisoxazole, ampicillin, doxycycline, and tetracycline, and 217 isolates exhibited multidrug resistance. The prevalence of β-lactamase genes in Salmonella isolates was 59.6%, and among these isolates, 185 harbored blaTEM, followed by blaCTX-M (66) and blaOXA (10). Moreover, six PMQR genes, namely, including qnrA (4.8%), qnrB (4.2%), qnrD (0.9%), qnrS (18.9%), aac(6')-Ib-cr (16.5%), and oqxB (1.5%), were detected. QRDR mutations (76.6%) were very common in Salmonella isolates, with the most frequent mutation in parC (T57S) (47.3%). Furthermore, we detected six tetracycline resistance genes in 176 isolates, namely, tet(A) (39.5%), tet(B) (8.1%), tet(M) (7.7%), tet(D) (5.4%), tet(J) (3.3%), and tet(C) (1.8%), and three sulfonamide resistance genes in 303 isolates, namely, sul1 (84.4%), sul2 (31.1%), and sul3 (4.2%). Finally, we found 86 isolates simultaneously harboring four types of resistance genes that cotransferred 2-7 resistance genes to recipient bacteria. The frequent occurrence of antimicrobial resistance, particularly in dogs and cats, suggests that antibiotic misuse may be driving multidrug-resistant Salmonella among pets.IMPORTANCEPet-associated human salmonellosis has been reported for many years, and antimicrobial resistance in pet-associated Salmonella has become a serious public health problem and has attracted increasing attention. There are no reports of Salmonella from pets and their antimicrobial resistance in Chongqing, China. In this study, we investigated the prevalence, serovar diversity, sequence types, and antimicrobial resistance of Salmonella strains isolated from pet fecal samples in Chongqing. In addition, β-lactamase, QRDR, PMQR, tetracycline and sulfonamide resistance genes, and mutations in QRDRs in Salmonella isolates were examined. Our findings demonstrated the diversity of serovars and sequence types of Salmonella isolates. The isolates were widely resistant to antimicrobials, notably with a high proportion of multidrug-resistant strains, which highlights the potential direct or indirect transmission of multidrug-resistant Salmonella from pets to humans. Furthermore, resistance genes were widely prevalent in the isolates, and most of the resistance genes were spread horizontally between strains.

Identification of a novel multi-drug resistant plasmid co-harbouring Extended-Spectrum β-Lactamase resistance genes blaPER-4 and blaOXA-10 in Moellerella wisconsensis of sheep

Identification of a novel multi-drug resistant plasmid co-harbouring Extended-Spectrum β-Lactamase resistance genes blaPER-4 and blaOXA-10 in Moellerella wisconsensis of sheep

Emergence of multidrug-resistant Bacillus spp. derived from animal feed, food and human diarrhea in South-Eastern Bangladesh

BackgroundAntimicrobial resistance poses a huge risk to human health worldwide, while Bangladesh is confronting the most severe challenge between the food supply and the huge consumption of antibiotics annually. More importantly, probiotics containing Bacillus spp. are claimed to be an alternative to antimicrobial stewardship programs. However, their antibiotic resistance remains elusive. Thus, we employed the antimicrobial susceptibility test and PCR to assess the prevalence of resistance, including multidrug resistance (MDR) and resito-genotyping of isolated Bacillus spp.ResultsThe phenotypic profile showed that Bacillus spp. were 100% sensitive to gentamicin (2 µg/mL), whereas lowered sensitivity to levofloxacin (67.8%, 0.5–1 µg/mL), ciprofloxacin (62.3%, 0.5–1 µg/mL), clindamycin (52.2%, 0.25–0.5 µg/mL), amoxicillin-clavulanic acid (37.6%, 0.06 µg/mL), azithromycin (33.4%, 1–2 µg/mL), tetracycline (25.6%, 2–4 µg/mL), nitrofurantoin (21.1%, 16–32 µg/mL), co-trimoxazole (19.2%, 2 µg/mL), and erythromycin (18.8%, 0.25–0.5 µg/mL). The strains were completely resistant to penicillin, amoxicillin-clavulanic acid, cefixime, ceftriaxone, vancomycin, and co-trimoxazole, and a species-specific trend was seen in both phenotypic and genotypic resistance patterns. Genotypic resistance indicated prevalence of the bla1 (71.5%), tetA (33%), erm1 (27%), blaTEM (13.1%), blaCTX-M-1/blaCTX-M-2 /sul1 (10.1%), blaSHV (9.6%), and qnrS (4.1%) genes. The β-lactamase resistance gene bla1 was found in all penicillin-resistant (MIC ≥ 32 µg/mL) Bacillus spp. One hundred ninety-one isolates (89.6%) were MDR, with 100% from diarrhea, 90.3% from food, and 88.7% from animal feed.ConclusionBased on the MIC value and profile analysis of antibiotic resistance genes, this is the first study that Bacillus spp. antimicrobial susceptibilities have been identified in Bangladesh, and our study will shed light on the adverse effects of feed-borne Bacillus spp. emerging from animal feed to the food chain. A comprehensive investigation is urgently needed by policymakers on tolerance limits and harmful effects in the animal industry.

Antimicrobial resistance profiles of Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae strains isolated from broiler chickens

Globally, the spread of multidrug-resistant Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae from food to humans poses a severe threat to public health. The aim of this study was to assess the co-occurrence of colistin and β-lactamase resistance genes in E. coli, K. pneumoniae, and P. aeruginosa strains isolated from faeces of abattoir broiler chickens. The E. coli, P. aeruginosa and K. pneumoniae isolates were successfully detected from faecal samples by polymerase chain reaction (PCR) at infection rates of 60.7%, 22.5% and 16.7% respectively. The isolates displayed the highest levels of antibiotic resistance (AR) against ampicillin (82.3%) and amoxicillin-clavulanic acid (74.2%) for E. coli, followed by cefoxitin (70.6%) for K. pneumoniae, whilst P. aeruginosa displayed 26.1% antibiotic resistance (AR) against both ampicillin and colistin sulphate. The colistin mcr-1 gene was harboured by 46.8%, 47.1% and 21.7%, E. coli, K. pneumonia and P. aeruginosa isolates respectively. Ten out of 62 (16.1%), 6/17 (35.3%), 4/23 (17.4%) isolates were phenotypically classified as ESBL E. coli, K. pneumoniae, and P. aeruginosa respectively. The ESBL-E. coli isolates respectively possessed blaCTX-M (60%), blaTEM (20%) and blaCTX-M-9 (10%) genes. The ESBL-K. pneumoniae harboured, blaCTX-M (50%), blaOXA (33%), blaCARB (17%), and blaCTX-M-9 (17%) genes respectively, whilst, P. aeruginosa isolates respectively carried blaTEM (75%), blaCTX-M (50%), blaOXA (25%) and blaCARB (25%) genes. Molecular analysis identified the blaCTX-Mβ-lactamase-encoding genes collectively from E. coli, P. aeruginosa, K. pneumoniae isolates. Colistin and β-lactamase genes were present in only 16.7%, 6.9%, and 2.9% of E. coli, K. pneumoniae, and P. aeruginosa isolates, respectively. A total of 17, 7 and 3 isolates for E. coli, K. pneumoniae and P. aeruginosa respectively carried both colistin and β-lactamase antibiotics resistant genes. This is a public health threat that points to a challenge in the treatment of infections caused by these zoonotic bacteria. Data generated from this study will contribute to formulation of new strategies for combating spread of E. coli, K. pneumoniae, and P. aeruginosa isolates as well as prevention of their AR development.

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.

Comparative analyses of the faecal resistome against β-lactam and quinolone antibiotics in humans and livestock using metagenomic sequencing

To assess the prevalence and abundance of antibiotic resistance genes in human and livestock gut microbiomes, 87 humans (healthy individuals and patients with Clostridioides difficile infection (CDI)) and 108 livestock (swine, cattle, and chickens) were enrolled. Gut microbiomes and fluoroquinolone-resistant Escherichia coli isolates were sequenced, and mobile genetic elements adjacent to the β-lactamase (bla) and transferable quinolone resistance (qnr) genes were compared using metagenomic contigs. Each group of humans and livestock exhibited distinctive microbiota and resistome compositions in the gut. Concerning the resistome of bla and qnr, the prevalence rates between chickens and patients with CDI were the most similar (R2 = 0.46); blaTEM, blaOXA, blaCTX-M, and qnrS were highly prevalent in both groups. According to genomic and phylogenetic analyses, blaCTX-M and blaOXA expressed lineage specificity to either humans or livestock, while qnrS and blaTEM displayed a shared lineage between humans and livestock. A qnrS1 mobilome comprising five genes, including two recombinases, a transposase, and a plasmid gene, is commonly found in human and chicken gut microbiomes. Humans and chickens showed the most similar gut resistomes to β-lactams and quinolones. QnrS and blaTEM displayed especially strong co-occurrence between the guts of humans and livestock.

Genomic profiling of extended-spectrum β-lactamase-producing Escherichia coli from Pets in the United Arab Emirates: Unveiling colistin resistance mediated by mcr-1.1 and its probable transmission from chicken meat – A One Health perspective

BackgroundThe United Arab Emirates (UAE) has witnessed rapid urbanization and a surge in pet ownership, sparking concerns about the possible transfer of antimicrobial resistance (AMR) from pets to humans and the environment. This study delves into the whole-genome sequencing analysis of ESBL-producing E. coli strains from healthy cats and dogs in the UAE, which exhibit multidrug resistance (MDR). Additionally, it provides a genomic exploration of the mobile colistin resistance gene mcr-1.1, marking the first instance of its detection in Middle Eastern pets. MethodsWe investigate 17 ESBL-producing E. coli strains from healthy UAE pets using WGS and bioinformatics analysis to identify genes encoding virulence factors, assign diverse typing schemes to the isolates, and scrutinize the presence of AMR genes. Furthermore, we characterized plasmid contigs housing the mcr-1.1 gene and conducted phylogenomic analysis to evaluate their relatedness to previously identified UAE isolates. ResultsOur study unveiled a variety of virulence factor-encoding genes within the isolates, with fimH emerging as the most prevalent. Regarding β-lactamase resistance genes, the blaCTX group 1 gene family predominated, with CTX-M-15 found in 52.9% (9/17) of the isolates, followed by CTX-M-55 in 29.4% (5/17). These isolates were categorized into multiple sequence types (STs), with the epidemic ST131 being the most frequent. The presence of the mcr-1.1 gene, linked to colistin resistance, was confirmed in two isolates. These isolates belonged to ST1011 and displayed distinct profiles of β-lactamase resistance genes. Phylogenomic analysis revealed close connections between the isolates and those from chicken meat in the UAE. ConclusionOur study underscores the presence of MDR ESBL-producing E. coli in UAE pets. The identification of mcr-1.1-carrying isolates warrants the urgency of comprehensive AMR surveillance and highlights the role of companion animals in AMR epidemiology. These findings underscore the significance of adopting a One Health approach to mitigate AMR transmission risks effectively.

Ultrafast detection of β-lactamase resistance in Klebsiella pneumoniae from blood culture by nanopore sequencing.

Aim: This study aimed to assess the ultra-fast method using MinION™ sequencing for rapid identification of β-lactamase-producing Klebsiella pneumoniae clinical isolates from positive blood cultures. Methods: Spiked-blood positive blood cultures were extracted using the ultra-fast method and automated DNA extraction for MinION sequencing. Raw reads were analyzed for β-lactamase resistance genes. Multilocus sequence typing and β-lactamase variant characterization were performed after assembly. Results: The ultra-fast method identified clinically relevant β-lactamase resistance genes in less than 1h. Multilocus sequence typing and β-lactamase variant characterization required 3-6 h. Sequencing quality showed no direct correlation with pore number or DNA concentration. Conclusion: Nanopore sequencing, specifically the ultra-fast method, is promising for the rapid diagnosis of bloodstream infections, facilitating timely identification of multidrug-resistant bacteria in clinical samples.

In Silico Analysis of Antibiotics Resistance Genes in Lactobacillus plantarum Kita-3 isolated from Halloumi Cheese

Previous studies found that antibiotics resistance genes on plasmids in Lactobacillus make them unsafe for food purposes due to the capability of genes to transfer to pathogenic microorganisms. In contrast, Lactobacillus is widely used as a probiotic. This study assessed the antibiotics susceptibility of Lactobacillus plantarum Kita-3 isolated from Halloumi cheese using eight antibiotics. Genome sequencing was performed using the NovaSeq 6000 sequencing platform to detect the presence of antibiotics resistance genes on chromosomes and plasmids. L. plantarum Kita-3 was resistant to clindamycin, streptomycin, chloramphenicol and susceptible to tetracycline, ampicillin, kanamycin, erythromycin, ciprofloxacin. Genome sequencing of the L. plantarum Kita-3 verified the presence of the tetracycline, fluoroquinolones, β-lactamase resistance genes, and multidrug resistance efflux. Kita-3 did not have transposable elements, gene transfer agents, and plasmid-related functions. Overall, this study resulted in the antibiotics resistance profile of L. plantarumKita-3 to assess the risk of antibiotics resistance genes transfer to other bacteria. This study can provide essential data on the safe use of Lactobacillus plantarum Kita-3 as probiotics.

Roles of intrinsic and acquired resistance determinants in multidrug-resistant clinical Pseudomonas aeruginosa in Bangladesh

Introduction: Pseudomonas aeruginosa is an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter spp.) pathogen and one of the leading etiologies in multiple nosocomial infections. Treatment of P. aeruginosa is becoming increasingly difficult due to its ever-increasing antibiotic resistance trends. This study investigated clinical multidrug resistance (MDR) P. aeruginosa (MDR-PA), their intrinsic resistance determinants, including the presence of chromosomal AmpC β-lactamase (Ampicillinase), decreased expression of outer membrane porin protein OprD and selected acquired β-lactamase resistance genes.
 Methods: Out of 238 clinical specimens, including urines from urinary tract-infected patients, wound swabs, burn swabs, and catheter aspirates, were collected from two major hospitals in Savar, Dhaka, Bangladesh. Samples were inoculated with Cetrimide agar to isolate presumptive P. aeruginosa. Bacteria were identified by cultural, biochemical characterization, 16S rDNA sequencing, and phylogenetic analysis. Virulence-associated genes of P. aeruginosa, namely, toxA, lasB, and plcH, were identified by polymerase chain reaction (PCR). Antibiotic susceptibilities of the isolates were investigated against ten antibiotics belonging to seven groups by disc-diffusion method followed by a selected minimum inhibitory concentration (MIC) assay. Phenotypic expression of Metallo-β-lactamases (MBLs) production was checked by the double disc synergistic test selectively among the imipenem-resistant isolates. Acquisition of β-lactam resistance trait was examined by PCR detection of bla-genes variants. Mutational loss of the OprD was analyzed by PCR to investigate intrinsic resistance determinants. Phenotypic overexpression of chromosomal AmpC was assayed with the identification of the AmpC gene by PCR. The expression level of OprD was assessed by real-time quantitative PCR (RT-qPCR).
 Results: Fifty-three P. aeruginosa was identified, with an overall isolation of 22.3% (53/238), where urine remains the most prevalent source. Virulence genes toxA, lasB, and plcH were identified in the isolates of 92.4%, 96.2%, and 94.3%. The highest phenotypic antimicrobial resistance was observed against ampicillin and ceftriaxone (100%), followed by cefotaxime (96%), tetracycline (89%), azithromycin (72%), imipenem (31%), ciprofloxacin (29%), levofloxacin (29%), gentamycin (27%) and ceftazidime (14%). The antibiogram pattern revealed 85% of isolates as multidrug-resistant, while 12% were considered extensively drug-resistant (XDR)-P. aeruginosa. The carriage of β-lactamase genes blaTEM, blaSHV, and blaOXA was detected in 4%, 2%, and 2% cephalosporin-resistant isolates, respectively. Double disc synergistic test revealed 87% of imipenem-resistant isolates expressing MBL-mediated resistance phenomenon. All seven ceftazidime-resistant isolates showed the presence of the AmpC gene with phenotypic overproduction of the AmpC enzyme, indicating AmpC-mediated ceftazidime resistance. Mutational loss of OprD was observed in 12% of phenotypically multidrug-resistant isolates, and RT-qPCR analysis revealed reduced expression of OprD porin protein at various levels in the outer membrane of multidrug-resistant isolates.
 Conclusions: This study depicts the high prevalence of MDR-PA in clinical specimens in Bangladesh. The identified intrinsic and acquired antimicrobial resistance determinants play synergistic roles in emerging MDR-PA.
 Bangladesh Journal of Medical Science Vol. 22 No. 03 July’23 Page : 489-507

Distribution and transmission of β-lactamase resistance genes in meal-to-milk chain on dairy farm

Antibiotics have been widely used in animal husbandry, which leads to high risk of food-borne transfer of antibiotic resistance genes (ARGs). The present study investigated the distribution of β-lactamase resistance genes (β-RGs) on dairy farm in the Songnen Plain of western Heilongjiang Province, China, to provide mechanistic insights into food-borne transmission of β-RGs through “meal-to-milk” chain under practically relevant circumstances. The results demonstrated that the abundance of β-RGs (91%) was much higher than that of other ARGs in the livestock farms. The blaTEM exhibited the content as high as 94.55% among all ARGs, and higher than 98% blaTEM was detected in meal, water and milk sample. The metagenomic taxonomy analysis indicated that the blaTEM should be carried by tnpA-04 (7.04%) and tnpA-03 (1.48%) hosted in Pseudomonas genus (15.36%) and Pantoea (29.02%) genus. Both tnpA-04 and tnpA-03 in the milk sample were identified to be the key mobile genetic elements (MGEs) responsible for transferring blaTEM along the “meal-manure-soil-surface water-milk” chain. The ARGs transfer across ecological boundaries underscored the need to evaluate potential dissemination of high-risk Proteobacteria and Bacteroidetes carried by humans and animals. They were capable of producing expanded-spectrum β-lactamases (ESBLs) and destroying commonly used antibiotics, leading to possible risk of food-borne horizontal transmission of ARGs. This study not only has important environmental implications for identifying the pathway for ARGs transfer, but also highlights the demand for appropriate policy toward safe regulation of dairy farm and husbandry products.

Characteristics of antibiotic resistance mechanisms and genes of Klebsiella pneumoniae.

Klebsiella pneumoniae is an important multidrug-resistant (MDR) pathogen that can cause a range of infections in hospitalized patients. With the growing use of antibiotics, MDR K. pneumoniae is more prevalent, posing additional difficulties and obstacles in clinical therapy. To provide a valuable reference to deeply understand K. pneumoniae, and also to provide the theoretical basis for clinical prevention of such bacteria infections, the antibiotic resistance and mechanism of K. pneumoniae are discussed in this article. We conducted a literature review on antibiotic resistance of K. pneumoniae. We ran a thorough literature search of PubMed, Web of Science, and Scopus, among other databases. We also thoroughly searched the literature listed in the papers. We searched all antibiotic resistance mechanisms and genes of seven important antibiotics used to treat K. pneumoniae infections. Antibiotics such as β-lactams, aminoglycosides, and quinolones are used in the treatment of K. pneumoniae infection. With both chromosomal and plasmid-encoded ARGs, this pathogen has diverse resistance genes. Carbapenem resistance genes, enlarged-spectrum β-lactamase genes, and AmpC genes are the most often β-lactamase resistance genes. K. pneumoniae is a major contributor to antibiotic resistance worldwide. Understanding K. pneumoniae antibiotic resistance mechanisms and molecular characteristics will be important for the design of targeted prevention and novel control strategies against this pathogen.

Serotype distribution, trend of multidrug resistance and prevalence of β-lactamase resistance genes in human Salmonella isolates from clinical specimens in Guizhou, China.

Salmonella, one of the major causes of foodborne infections, can cause bacterial foodborne illness. We investigated the serotype distribution, multidrug resistance (MDR), and β-lactamase resistance genes of human Salmonella isolates collected from clinical specimens in Guizhou, China, between 2013 and 2018. A total of 363 Salmonella isolates were collected from clinical specimens at 17 surveillance hospitals. Twenty-four serotypes were identified by sliding agglutination test. S. Enteritidis (33.9%), Salmonella 4,[5],12:i:- (24.0%), S. Typhimurium (16.3%), S. London (6.3%), and S. Derby (3.9%) were the top five serotypes. In 2018, the most common serotype changed from S. Enteritidis to S. Typhimurium. Among the 363 Salmonella isolates, 97.5% of isolates were resistant to at least one class of antimicrobial agents. For cephalosporins, ceftriaxone had the highest resistance rate of 10.5%, and cefepime and cefoxitin were 8.0% and 2.2%, respectively. Three hundred and one (82.9%) Salmonella isolates showed MDR. Salmonella 4,[5],12:i:- had the highest MDR rate with 94.2%, followed by S. London (91.3%) and S. Typhimurium (88.1%). Multidrug resistance rates of Salmonella isolates in Guizhou from 2013 to 2017 increased from 75.8% to 86.7%. Sixteen isolates (4.4%) showed extensive drug resistance. One hundred thirty-four antimicrobial resistance patterns were found. Two hundred and forty-one (66.4%) isolates carried at least one β-lactamase resistance gene. The blaTEM gene (61.2%) was the most prevalent resistant gene in all Salmonella isolates, followed by the blaCTX-M gene (6.1%) and blaOXA-1 gene (4.1%). Our findings showed that the MDR rate of Salmonella isolates from Guizhou province increased year by year. Therefore, systematic and long-term surveillance on MDR Salmonella isolates from clinical patients should be further strengthened.

Prevalence of antimicrobial resistance and virulence genes in Klebsiella pneumoniae and Congenetic Raoultella Isolates from captive giant pandas.

To study antimicrobial resistance and virulence genes of Klebsiella pneumoniae and Raoultella strains isolated from captive giant pandas. Non-duplicate fecal samples were collected from 128 giant pandas during 2017-2019. All isolated microbial strains were tested for antimicrobial drug susceptibility using BD verification panels. Four extended-spectrum β-lactamase resistance genes, nine virulence genes and six capsular serotype genes were detected using PCR. 42 K. pneumoniae and nine Raoultella strains were isolated from different giant pandas. Antibiotic resistance rates were 1.9%-23.5%, except for ampicillin, and 7.8% of the isolates were multidrug-resistant to 7-10 antibiotic classes. This is the first time that a multidrug-resistant R. ornithinolytica strain has been isolated from captive giant pandas. The blaTEM, blaCTX-M, blaSHV and blaDHA genes were detected in four MDR ESBL- K. pneumoniae strains. The rmpA, iutA, ybtS, iroN and iroB genes were positively detected in 11.7% of the isolates. Capsular serotype (K2, K5, K54 and K57) genes were all detected in four K. pneumoniae strains, and one was identified as hypervirulent. This study showed that MDR ESBL- K. pneumoniae, hypervirulent K. pneumoniae, MDR R. ornithinolytica and the colistin-resistant strain may pose risks to captive giant pandas and their keepers, and that the diversity of antibiotic resistance and virulence genes in Klebsiella and Raoultella should be monitored regularly.

Detection of Virulence and β-lactamase resistance genes of non-typhoidal Salmonella isolates from human and animal origin in Egypt "one health concern"

BackgroundNon-typhoidal Salmonella (NTS) is a major foodborne zoonotic pathogen worldwide. In the current study, Various NTS strains were isolated from (cows, milk and dairy products in addition to humans) in New Valley and Assiut Governorate, Egypt. NTS were firstly serotyped and tested by antibiotic sensitivity test. Secondly, some virulence genes and Antibiotic resistance genes have been identified by using PCR. Finally, Phylogenesis was performed depending on the invA gene, for two S. typhimurium isolates (one of animal origin and the other of human origin for evaluating zoonotic potential).ResultsOut of 800 examined samples, the total number of isolates was 87 (10.88%), which were classified into 13 serotypes, with the most prevalent being S. Typhimurium and S. enteritidis. Both bovine and human isolates showed the highest resistance to clindamycin and streptomycin, with 90.80% of the tested isolates exhibiting MDR. The occurrence of the invA gene was 100%, while 72.22%, 30.56%, and 94.44% of the examined strains were positive for stn, spvC, and hilA genes, respectively. Additionally, blaOXA-2 was detected in 16.67% (6/ 36) of the tested isolates, while blaCMY-1 was detected in 30.56% (11of 36) of the tested isolates. Phylogenesis revealed a high degree of similarity between the two isolates.ConclusionsThe high occurrence of MDR strains of NTS in both human and animal samples with high degree of genetic similarity, shows that cows, milk and milk product may be a valuable source of human infection with NTS and interfere with treatment procedures.

Antimicrobial susceptibility profile and detection of extended spectrum β-lactamase resistance genes in pseudomonas aeruginosa isolated from post-operative wounds

The spread of Pseudomonas aeruginosa is difficult to control with disinfectants and antibiotics due to the resistance found in P. aeruginosa isolates. The study investigated the susceptibility profile and detect extended spectrum β-lactamase (ESBL) resistance genes in the Pseudomonas aeruginosa isolated from post-operative wound infection. Wound swabs were cultured on MacConkey and sub-cultured on Cetrimide agar. The isolates were identified base on their macroscopic and microscopic characteristics as well as 16SrRNA. Disc diffusion technique was used to check the susceptibility profile of the isolates and the extended spectrum beta-lactam (ESBL) producers were determined by growing the isolates on Mueller Hinton agar plate in the presence of amoxyclav. 40 positive isolates were recovered in the study with 21 isolates been multi-drug resistance (MDR). Before plasmid curing, Streptomycin, ceftazidime, Augmentin, and Gentamicin has the highest resistance rate of 80% (n=32), 60% (n=24), 60% (n=24) and 60% (n=24) respectively. However, after plasmid curing, the result showed reduction in resistance rates. Streptomycin and ceftazidime showed high reduction in resistance from 80% to 25% and from 60 to 12.7% respectively indicating that resistance was plasmid-mediated. Pseudomonas aeruginosa F23 and Pseudomonas aeruginosa S15 isolates shown to possess both the plcH and bla PER genes with bands amplicons size of 307bp and 520bp respectively. Antibiotic susceptibility pattern after plasmid curing showed that 45% of the isolates produces beta-lactamase enzymes i.e., 18 out of 40 isolates were inactive to all beta-lactam group of antibiotics.