UNASSIGNED: This study was aimed to investigate the multidrug resistance patterns in clinical isolates of Escherichia coli and their correlation with integrons and phylogenetic groupings.
UNASSIGNED: A total of 37 clinical E. coli isolates were evaluated for drug resistance patterns by disk diffusion method. Phylogenetic groupings and the presence of integrons among E. coli were determined by multiplex PCR assays.
UNASSIGNED: Multidrug resistance was identified in 84% of the clinical isolates of E. coli with higher resistance found against cephalosporins (94.6%) and fluoroquinolones (83.8%), while lower resistance was observed against polymyxins (24.3%) and carbapenems (29.7%). Metallo-β-lactamases were found in all carbapenem resistant isolates. The phylogenetic group B2 was the most dominant (40.5%), followed by groups A (35.1%), D (13.5%) and B1 (10.8%). Integrons were detected in 25 (67.6%) isolates and intI1, intI2, and intI3 genes were found in 62.2%, 18.9% and 10.8% of isolates respectively.
UNASSIGNED: Our results show that phylogenetic classification of E. coli is not relevant with antimicrobial resistance. However, there was strong association between the integron classes and resistance against β-lactam and fluoroquinolones antimicrobials. Additionally, this study highlighted that the presence of integrons plays a crucial role in the development of multidrug resistance in clinical isolates of E. coli. Most significantly, this is the first report of detection of three classes of integron among clinical isolates of E. coli in Pakistan.

The rapid emergence of antimicrobial resistance (AMR) in Campylobacter has reinforced its status as a foodborne pathogen of significant public health concern. Resistant Campylobacter is typically transferred to humans via the consumption of contaminated animal products, particularly poultry. The genes associated with antimicrobial resistance in Campylobacter spp. are poorly understood. To address this knowledge gap, we conducted a prevalence survey of AMR Campylobacter across 84 chicken farms in two districts of Bangladesh. Pooled cloacal swabs were collected from chickens and underwent bacteriological testing for Campylobacter spp. with PCR confirmation. Antimicrobial susceptibility was tested against 14 antibiotics by disk diffusion method, and 12 resistance genes were screened in Campylobacter-positive isolates using multiplex PCR. A total of 34 (40.5%) farms were Campylobacter-positive of which 73.5% of isolates were resistant to at least 10 antibiotics. The antimicrobial susceptibility results indicate a high level of resistance against streptomycin (97.1%), clindamycin (97.1%), ampicillin (94.1%), tetracycline (94.1%), erythromycin (91.2%), ciprofloxacin (88.2%), nalidixic acid (85.3%), and imipenem (82.4%), and comparatively a low frequency of resistance to chloramphenicol (47.1%), ceftazidime (44.1%), and colistin (35.3%). Multidrug-resistant (MDR) and extensively drug-resistant Campylobacter were identified in 97.1%, and 50% of isolates, respectively. Ten resistance genes were identified including blaTEM (in 97.1% of isolates), strA-strB (85.9%), tetA (70.6%), tetB (32.4%), qnrS (23.5%), blaCTX-M-1 (20.6%), qnrB (20.6%), blaSHV (8.8%), aadB (5.9%), and qnrA (2.9%). Our findings demonstrate that resistance to ampicillin, tetracycline, and ceftazidime in Campylobacter isolates was significantly (p ≤ 0.05) associated with the presence of blaTEM, tetA, and blaSHV genes, respectively. The high rates of AMR in Campylobacter isolates from our study are not surprising given the liberal use of antimicrobials and incomplete biosecurity provisions on farms. Of particular concern are resistance rates to those classes of antibiotics that should be reserved for human use (azithromycin, ciprofloxacin, and colistin). AMR was more prevalent in chicken farms that used multiple antibiotics, engaged in prophylactic treatment of the birds, and improperly disposed of antibiotic packages. The high prevalence of MDR in chicken-derived Campylobacter isolates from the different regions of our study reinforces the need for more prudent use of antimicrobial compounds in Bangladeshi chicken farms.

BACKGROUND: Typhoid fever, caused by Salmonella enterica serovar Typhi, is a significant public health concern due to the escalating of antimicrobial resistance (AMR), with limited treatment options for extensively drug-resistant (XDR) S. Typhi strains pose a serious threat to disease management and control. This study aimed to investigate the genomic characteristics, epidemiology and AMR genes of XDR S. Typhi strains from typhoid fever patients in Pakistan.
METHODS: We assessed 200 patients with enteric fever symptoms, confirming 65 S. Typhi cases through culturing and biochemical tests. Subsequent antimicrobial susceptibility testing revealed 40 cases of extensively drug-resistant (XDR) and 25 cases of multi-drug resistance (MDR). Thirteen XDR strains were selected for whole-genome sequencing, to analyze their sequence type, phylogenetics, resistance genes, pathogenicity islands, and plasmid sequences using variety of data analysis resources. Pangenome analysis was conducted for 140 XDR strains, including thirteen in-house and 127 strains reported from other regions of Pakistan, to assess their genetic diversity and functional annotation.
RESULTS: MLST analysis classified all isolates as sequence type 1 (ST-1) with 4.3.1.1. P1 genotype characterization. Prophage and Salmonella Pathogenicity Island (SPI) analysis identified intact prophages and eight SPIs involved in Salmonella\'s invasion and replication within host cells. Genome data analysis revealed numerous AMR genes including dfrA7, sul1, qnrS1, TEM-1, Cat1, and CTX-M-15, and SNPs associated with antibiotics resistance. IncY, IncQ1, pMAC, and pAbTS2 plasmids, conferring antimicrobial resistance, were detected in a few XDR S. Typhi strains. Phylogenetic analysis inferred a close epidemiological linkage among XDR strains from different regions of Pakistan. Pangenome was noted closed among these strains and functional annotation highlighted genes related to metabolism and pathogenesis.
CONCLUSIONS: This study revealed a uniform genotypic background among XDR S. Typhi strains in Pakistan, signifying a persistence transmission of a single, highly antibiotic-resistant clone. The closed pan-genome observed underscores limited genetic diversity and highlights the importance of genomic surveillance for combating drug-resistant typhoid infections.

BACKGROUND: Acinetobacter baumannii international clone II (IC2) is a widespread pandemic clone, however, it is rarely described in South America. The present study reported an outbreak caused by XDR IC2 strains in a clinical setting in Rio de Janeiro in 2022.
METHODS: Molecular epidemiology analysis was conducted with MLST to determine the clonal relationship and to assign a sequence type. The antimicrobial resistance profile of A. baumannii strains was assessed by the disk-diffusion method and MIC determination, and the presence of antibiotic resistance genes was determined by PCR and Sanger sequencing. The whole genome of one representative strain (AB91) was sequenced to prospect its resistome and virulome.
RESULTS: The MLST revealed that all strains belonged to the ST2 (Pasteur scheme) that corresponded to the pandemic IC2 lineage. They presented the XDR phenotype, which was compatible with their resistome composed of several acquired resistance genes and altered housekeeping genes. Additionally, an expressive virulome was revealed in AB91 genome. Genomic comparison with the unique other available IC2 genome from Brazil revealed that outbreaks occurring during (São Paulo - 2020/2021) and after (Rio de Janeiro - 2022) COVID-19 pandemics were caused by the same IC2 lineage.
CONCLUSIONS: This study suggests that the presence of a huge arsenal of resistance and virulence genes may have contributed to the persistence and the successful establishment of IC2 in Brazilian clinical settings during and after the COVID-19 pandemics in response to a series of events, such as the antibiotic overused during that period.

Acinetobacter baumannii is one of the most successful pathogens that can cause difficult-to-treat nosocomial infections. Outbreaks and infections caused by multi-drug resistant A. baumannii are prevalent worldwide, with only a few antibiotics are currently available for treatments. Plasmids represent an ideal vehicle for acquiring and transferring resistance genes in A. baumannii. Five extensively drug-resistant A. baumannii clinical isolates from three major Jordanian hospitals were fully sequenced. Whole-Genome Sequences (WGS) were used to study the antimicrobial resistance and virulence genes, sequence types, and phylogenetic relationship of the isolates. Plasmids were characterized In-silico, followed by conjugation, and plasmid curing experiments. Eight plasmids were recovered; resistance plasmids carrying either aminoglycosides or sulfonamide genes were detected. Chromosomal resistance genes included blaOXA-66, blaOXA-91, and blaOXA-23, and the detected virulence factors were involved in biofilm formation, adhesion, and many other mechanisms. Conjugation and plasmid curing experiments resulted in the transfer or loss of several resistance phenotypes. Plasmid profiling along with phylogenetic analyses revealed high similarities between two A. baumannii isolates recovered from two different intensive care units (ICU). The high similarities between the isolates of the study, especially the two ICU isolates, suggest that there is a common A. baumannii strain prevailing in different ICU wards in Jordanian hospitals. Three resistance genes were plasmid-borne, and the transfer of the resistance phenotype emphasizes the role and importance of conjugative plasmids in spreading resistance among A. baumannii clinical strains.

OBJECTIVE: Antimicrobial resistance of nontyphoidal Salmonella (NTS) is a threat to public health worldwide.
METHODS: A study on longitudinally collected NTS isolates from a medical center in Taiwan from 2011 to 2019 was undertaken. The multidrug resistance (MDR) and extensively drug resistance (XDR) phenotypes were determined according to internationally used definitions. Molecular serotyping was performed on the resistant NTS.
RESULTS: Notably 16.1% (870/5412) of the isolates were MDR, while XDR accounted for 2.1% (111/5412). Both MDR and XDR NTS have increased significantly from 2011 to 2019, especially from 2015 to 2017 (MDR from 9.6% in 2015 to 23.1% 2017; XDR from 1.4% in 2016 to 4.7% in 2017). S. Anatum was the commonest NTS serotype expressing MDR and XDR, in 256/559 (45.8%) and 81/111 (73.0%) of the isolates, respectively, followed by S. Typhimurium and S. Goldcoast. Children < 18 years old contributed to 69.0% of all MDR cases and 64.0% of all XDR cases; majority of them aged less than 5 years.
CONCLUSIONS: Increasing MDR and XDR NTS is a threat to public health. MDR and XDR NTS usually caused gastroenteritis in children < 5 years old. Multiple NTS serotypes expressing MDR and XDR indicate multiple food vehicles involved in the transmission. Proper food hygiene practice should never be over-reinforced.

High level carbapenem and extensively drug resistant (XDR) Escherichia coli strain N7, which produces a variant of New Delhi metallo-β-lactamase (NDM-5), was isolated from the influent of the Jungnang wastewater treatment plant located on Han River, Seoul, South Korea. Phenotypic and genotypic resistances to carbapenem were tested using agar and broth dilution methods, and polymerase chain reaction. Whole-genome sequencing was performed to characterize the genetic structure of strain N7. E. coli strain N7, which harbors the bla NDM-5 gene, showed high level of carbapenem resistance at concentrations of doripenem (512 mg/L) and meropenem (256 mg/L), and XDR to 15 antibiotics. Based on the genomic sequence analysis, two plasmids, a hybrid IncHI2/N-type and an IncX3 type, were present. The former contains a cluster (bla NDM-5-ble MBL -trpF-dsbD) bracketed by multi-insertional sequences, IS3000, ISAba125, IS5, and IS26. The latter carries the following resistance genes: bla CTX-14, aac(3)-IV, aadA1, aadA2, aph(3\')-Ia, aph(4)-Ia, sul1, sul2, sul3, dfrA12, fosA3, oqxA, oqxB, mph(A), and floR, and cmlA1. The chromosome, contig3, and contig5 also carry bla CTX-64 and mdf(A), tet(A), and erm(B), tet(M) and aadA22, respectively. Strain N7 also harbors virulence factors such as fimH, flu, ecpABCDE, sfmA, hlyE, and gadA. This study demonstrates the emergence of high level carbapenem resistant XDR E. coli strain N7 containing bla NDM-5 in aquatic environment, Seoul, South Korea. Due to the presence of mobile genetic elements, this strain could horizontally transfer resistance genes, including bla NDM-5 to environmental bacteria. Thus, it is necessary to conduct continuous surveillance for carbapenem resistance in various aquatic environments.

An extensively-drug resistant (XDR) Escherichia coli W60 was isolated from the urine sample of a patient. The genetic basis for its XDR phenotype was investigated, particularly the basis for its resistance toward β-lactam/BLI (β-Lactamase Inhibitor) combinations. Following determination of the XDR phenotype, third generation genomic sequencing was performed to identify genetic structures in E. coli W60. Further cloning analysis was performed to identify determinants of β-lactam/BLI combination resistance. It was found that E. coli W60 is resistant to nearly all of the tested antibiotics including all commonly used β-lactam/BLI combinations. Analysis of the genomic structures in E. coli W60 showed two novel transferable plasmids are responsible for the resistance phenotypes. Further genetic analysis showed bla NDM-5 leads to high resistance to β-lactam/BLI combinations, which was enhanced by co-expressing ble MBL. pECW602 harbors a truncated bla TEM that is not functional due to the loss of the N-terminal signal peptide coding region. Research performed in this work leads to several significant conclusions: the XDR phenotype of E. coli W60 can be attributed to the presence of transferable multidrug resistance plasmids; NDM-5 confers high resistance to β-lactam/BLI combinations; co-expression of ble MBL enhances resistance caused by NDM-5; the signal peptides of TEM type β-lactamases are essential for their secretion and function. Findings of this work show the danger of transferable multidrug resistance plasmids and metallo-β-lactamases, both of which should be given more attention in the analysis and treatment of multidrug resistant pathogens.

A major challenge in the treatment of infections has been the rise of extensively drug resistance (XDR) and multidrug resistance (MDR) in Acinetobacter baumannii. The goals of this study were to determine the pattern of antimicrobial susceptibility, blaOXA and carO genes among burn-isolated A. baumannii strains. In this study, 100 A. baumannii strains were isolated from burn patients and their susceptibilities to different antibiotics were determined using disc diffusion testing and broth microdilution. Presence of carO gene and OXA-type carbapenemase genes was tested by PCR and sequencing. SDS-PAGE was done to survey CarO porin and the expression level of carO gene was evaluated by Real-Time PCR. A high rate of resistance to meropenem (98%), imipenem (98%) and doripenem (98%) was detected. All tested A. baumannii strains were susceptible to colistin. The results indicated that 84.9% were XDR and 97.9% of strains were MDR. In addition, all strains bore blaOXA-51 like and blaOXA-23 like and carO genes. Nonetheless, blaOXA-58 like and blaOXA-24 like genes were harbored by 0 percent and 76 percent of strains, respectively. The relative expression levels of the carO gene ranged from 0.06 to 35.01 fold lower than that of carbapenem-susceptible A. baumannii ATCC19606 and SDS - PAGE analysis of the outer membrane protein showed that all 100 isolates produced CarO. The results of current study revealed prevalence of blaOXA genes and changes in carO gene expression in carbapenem resistant A.baumannii.

Multi-, extensively-, and pan-drug resistant bacteria are a threat to our health today, because their wide resistance spectra make their infections difficult to cure. In this work, we isolated an extensively drug resistant (XDR) Klebsiella pneumoniae 2-1 strain from the stool sample of a patient diagnosed of colorectal cancer. K. pneumoniae 2-1 was found to be resistant to all the antibiotics tested except for cefepime, tigecycline, and ceftazidime-avibactam. By sequencing the complete genome of K. pneumoniae 2-1, we found it contains a chromosome of 5.23 Mb and two circular plasmids with the size of 246 and 90 kb. The larger plasmid, pKP21HI1 was found to be a new conjugation-defective plasmid belonging to incompatibility group HI1B and a new sequence type. Further comparative genomics analysis and antimicrobial resistance gene analysis showed that although a great deal of changes took place on the chromosome of K. pneumoniae 2-1 in comparison with the reference genome, the extensively drug resistance phenotype of K. pneumoniae 2-1 is primarily due to the two multidrug resistant plasmids it contains. This work explains the genetic and mechanistic basis of the extensive drug resistance of K. pneumoniae 2-1, and found that plasmids play key roles in the strong antibiotic resistance of bacteria.