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.

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.

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.

The emergence of extensively drug-resistant tuberculosis (XDR-TB) has raised public health concern for global TB control. Although multi drug-resistant tuberculosis (MDR- TB) prevalence and associated genetic mutations in Morocco are well documented, scarce information on XDR TB is available. Hence, the evaluation of pre-XDR and XDR prevalence, as well as the mutation status of gyrA, gyrB, rrs, tlyA genes and eis promoter region, associated with resistance to second line drugs, is of great value for better management of M/XDR TB in Morocco.
To evaluate pre-XDR and XDR prevalence, as well as the mutation status of gyrA, gyrB, rrs, tlyA genes and eis promoter region, associated with resistance to second line drug resistance, in 703 clinical isolates from TB patients recruited in Casablanca, and to assess the usefulness of molecular tools in clinical laboratories for better management of M/XDR TB in Morocco.
Drug susceptibility testing (DST) was performed by the proportional method for first line drugs, and then the selected MDR isolates were tested for second line drugs (Ofloxacin, Kanamycin, Amikacin and Capreomycin). Along with DST, all samples were subjected to rpoB, katG and p-inhA mutation analysis by PCR and DNA sequencing. MDR isolates as well as 30 pan-susceptible strains were subjected to PCR and DNA sequencing of gyrA, gyrB, rrs, tlyA genes and eis promoter, associated with resistance to fluoroquinolones and injectable drugs.
Among the 703 analysed strains, 12.8% were MDR; Ser531Leu and Ser315Thr being the most common recorded mutations within rpoB and katG genes associated with RIF and INH resistance respectively. Drug susceptibility testing for second line drugs showed that among the 90 MDR strains, 22.2% (20/90) were resistant to OFX, 2.22% (2/90) to KAN, 3.33% (3/90) to AMK and 1.11% (1/90) to CAP. Genotypic analysis revealed that 19 MDR strains harbored mutations in the gyrA gene; the most recorded mutation being Asp91Ala accounting for 47.6% (10/21), and 2 isolates harbored mutations in the promoter region of eis gene. No mutation was found in gyrB, rrs and tlyA genes. Moreover, none of the pan-susceptible isolates displayed mutations in targeted genes.
Most of mutations associated with SLD resistance occurred in gyrA gene (codons 90-94) and eis promoter region. These findings highlight the impact of mutations in gyrA on the development of fluroquinolones resistance and provide the first estimates of the proportion of pre-XDR-TB among MDR-TB cases in Morocco.

In Vietnam, a country with high tuberculosis (137/100.000 population) and multidrug-resistant (MDR)-TB burdens (7.8/100.000 population), little is known about the molecular signatures of drug resistance in general and more particularly of second line drug (SLD) resistance. This study is specifically focused on Mycobacterium tuberculosis isolates resistant to four first-line drugs (FLDs) that make TB much more difficult to treat. The aim is to determine the proportion of SLD resistance in these quadruple drug resistant isolates and the genetic determinants linked to drug resistance to better understand the genetic processes leading to quadruple and extremely drug resistance (XDR). 91 quadruple (rifampicin, isoniazid, ethambutol and streptomycin) FLD resistant and 55 susceptible isolates were included. Spoligotyping and 24-locus MIRU-VNTR techniques were performed and 9 genes and promoters linked to FLD and SLD resistance were sequenced. SLD susceptibility testing was carried out on a subsample of isolates. High proportion of quadruple-FLD resistant isolates was resistant to fluoroquinolones (27%) and second-line injectable drugs (30.2%) by drug susceptibility testing. The sequencing revealed high mutation diversity with prevailing mutations at positions katG315, inhA-15, rpoB531, embB306, rrs1401, rpsL43 and gyrA94. The sensitivity and specificity were high for most drug resistances (>86%), but the sensitivity was lower for injectable drug resistances (<69%). The mutation patterns revealed 23.1% of pre-XDR and 7.7% of XDR isolates, mostly belonging to Beijing family. The genotypic diversity and the variety of mutations reflect the existence of various evolutionary paths leading to FLD and SLD resistance. Nevertheless, particular mutation patterns linked to high-level resistance and low fitness costs seem to be favored.

BACKGROUND: Acinetobacter baumannii is an opportunistic pathogen that has become a major cause of concern, since it is a frequent cause of healthcare-associated infections (HAIs). The aim of the study was to describe the occurrence, the management and the control of an outbreak that occurred in an intensive care unit (ICU) of a teaching hospital in Southern Italy caused by multiple strains of extensively drug-resistant A. baumannii (XDRAB).
METHODS: Case-patient was defined as a patient with an healthcare-associated infection caused by an XDRAB isolate identified in a clinically significant culture. Environmental samples were collected from different surfaces. The isolates were identified by typical Gram stain morphology, using the Vitek 2 system (bioMérieux, France) and by MALDI-TOF MS mass spectrometry (bioMèrieux, France). Genotyping was performed through rep-PCR analysis.
RESULTS: A patient presented an XDRAB ventilator-associated pneumonia at admission and was managed with strict isolation precautions until discharge. Five patients had a ventilator-associated pneumonia and two had a central line-associated bloodstream infection. Of the environmental samples, 1 sample obtained from the side of the bed of an infected patient yielded growth of XDRAB. Infection control measures were adopted. Rep-PCR analysis identified four patterns.
CONCLUSIONS: The integration of epidemiological and microbiological data and the application of infection control measures were crucial to bring such an outbreak to a rapid halt. The distinctive characteristic of this study was the complex molecular pattern of the outbreak, which subsided in a short period of time due to adherence to infection-control measures, confirming the fundamental role of molecular typing in the comprehension of outbreaks dynamics and of integrated control interventions for the interruption of epidemic events.

The emergence of drug-resistant tuberculosis (TB) compromises global tuberculosis control. The incidence of multidrug-resistant strains (MDR) defined as resistant to the two main antituberculosis drugs, rifampicin and isoniazid, was raised in the 1990s. Ten percent of these strains have developed additional resistance to the main second-line antituberculosis drugs: fluoroquinolones and aminoglycosides. These strains are defined as extensively drug-resistant (XDR). The prognosis of MDR-TB and XDR-TB is poor due to limited therapeutic resources. However, many new innovations may lead to a radical change in this field. Genotypic testing is now able to detect drug resistance within a few hours. Genotypic diagnosis of rifampicin resistance is now recommended in France for each new case of TB. The currently recommended treatment for MDR-TB is long (18-24 months) and toxic. It is, however, on the verge of being replaced by a 9-month treatment. New antituberculosis drugs such as bedaquiline and delamanid should also improve the prognosis of MDR-TB and XDR-TB.