Whole-Genome Assessment of Clinical Acinetobacter baumannii Isolates Uncovers Potentially Novel Factors Influencing Carbapenem Resistance

Carbapenems—one of the important last-line antibiotics for the treatment of gram-negative infections—are becoming ineffective for treating Acinetobacter baumannii infections. Studies have identified multiple genes (and mechanisms) responsible for carbapenem resistance. In some A. baumannii strains, the presence/absence of putative resistance genes is not consistent with their resistance phenotype—indicating the genomic factors underlying carbapenem resistance in A. baumannii are not fully understood. Here, we describe a large-scale whole-genome genotype-phenotype association study with 349 A. baumannii isolates that extends beyond the presence/absence of individual antimicrobial resistance genes and includes the genomic positions and pairwise interactions of genes. Ten known resistance genes exhibited statistically significant associations with resistance to imipenem, a type of carbapenem: blaOXA-23, qacEdelta1, sul1, mphE, msrE, ant(3”)-II, aacC1, yafP, aphA6, and xerD. A review of the strains without any of these 10 genes uncovered a clade of isolates with diverse imipenem resistance phenotypes. Finer resolution evaluation of this clade revealed the presence of a 38.6 kbp conserved chromosomal region found exclusively in imipenem-susceptible isolates. This region appears to host several HTH-type DNA binding transcriptional regulators and transporter genes. Imipenem-susceptible isolates from this clade also carried two mutually exclusive plasmids that contain genes previously known to be specific to imipenem-susceptible isolates. Our analysis demonstrates the utility of using whole genomes for genotype-phenotype correlations in the context of antibiotic resistance and provides several new hypotheses for future research.

Imipenem Resistance Mediated by blaOXA-913 Gene in Pseudomonas aeruginosa

Treatment of infectious diseases caused by carbapenem-resistant Pseudomonas aeruginosa is becoming a greater challenge. This study aimed to identify the imipenem resistance mechanism in P. aeruginosa isolated from a dog. Minimum Inhibitory Concentration (MIC) was determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute recommendations. We performed polymerase chain reaction and whole-genome sequencing to detect carbapenem resistance genes. Genomic DNA of P. aeruginosa K19PSE24 was sequenced via the combined analysis of 20-kb PacBio SMRTbell and PacBio RS II. Peptide-Peptide Nucleic Acid conjugates (P-PNAs) targeting the translation initiation region of blaOXA-913 were synthesized. The isolate (K19PSE24) was resistant to imipenem and piperacillin/tazobactam yet was susceptible to most of the tested antimicrobials. Whole-genome sequencing revealed that the K19PSE24 genome comprised a single contig amounting to 6,815,777 base pairs, with 65 tRNA and 12 rRNA genes. K19PSE24 belonged to sequence type 313 and carried the genes aph(3)-IIb, fosA, catB7, crpP, and blaOXA-913 (an allele deposited in GenBank but not described in the literature). K19PSE24 also carried genes encoding for virulence factors (exoenzyme T, exotoxin A, and elastase B) that are associated with adhesion, invasion, and tissue lysis. Nevertheless, we did not detect any of the previously reported carbapenem resistance genes. This is the first report of the blaOXA-913 gene in imipenem-resistant P. aeruginosa in the literature. Notably, no viable colonies were found after co-treatment with imipenem (2 µg/mL) and either of the P-PNAs (12.5 µM or 25 µM). The imipenem resistance in K19PSE24 was primarily due to blaOXA-913 gene carriage.

Characterization of ESBL-Producing Escherichia coli and Klebsiella pneumoniae Isolated from Clinical Samples in a Northern Portuguese Hospital: Predominance of CTX-M-15 and High Genetic Diversity

Background: Enterobacteriaceae are major players in the spread of resistance to β-lactam antibiotics through the action of CTX-M β-lactamases. We aimed to analyze the diversity and genetic characteristics of ESBL-producing Escherichia coli and Klebsiella pneumoniae isolates from patients in a Northern Portuguese hospital. Methods: A total of 62 cefotaxime/ceftazidime-resistant E. coli (n = 38) and K. pneumoniae (n = 24) clinical isolates were studied. Identification was performed by MALDI-TOF MS. Antimicrobial susceptibility testing against 13 antibiotics was performed. Detection of ESBL-encoding genes and other resistance genes, phylogenetic grouping, and molecular typing (for selected isolates) was carried out by PCR/sequencing. Results: ESBL activity was detected in all 62 E. coli and K. pneumoniae isolates. Most of the ESBL-producing E. coli isolates carried a blaCTX-M gene (37/38 isolates), being blaCTX-M-15 predominant (n = 32), although blaCTX-M-27 (n = 1) and blaCTX-M-1 (n = 1) were also detected. Two E. coli isolates carried the blaKPC2/3 gene. The lineages ST131-B2 and ST410-A were detected among the ESBL-producing blood E. coli isolates. Regarding the 24 ESBL-producing K. pneumoniae isolates, 18 carried a blaCTX-M gene (blaCTX-M-15, 16 isolates; blaCTX-M-55, 2 isolates). All K. pneumoniae isolates carried blaSHV genes, including ESBL-variants (blaSHV-12 and blaSHV-27, 14 isolates) or non-ESBL-variants (blaSHV-11 and blaSHV-28, 10 isolates); ten K. pneumoniae isolates also carried the blaKPC2/3 gene and showed imipenem-resistance. ESBL-positive E. coli isolates were ascribed to the B2 phylogenetic group (82%), mostly associated with ST131 lineage and, at a lower rate, to ST410/A. Regarding K. pneumoniae, the three international lineages ST15, ST147, and ST280 were detected among selected isolates. Conclusions: Different ESBL variants of CTX-M (especially CTX-M-15) and SHV-type (specially SHV-12) were detected among CTX/CAZRE. coli and K. pneumoniae isolates, in occasions associated with carbapenemase genes (blaKPC2/3 gene).

Comparative Analysis of non-coding small-RNAs in P. aeruginosa Keratitis Strains with Different Antibiotic Susceptibility

Pseudomonas aeruginosa, is a gram-negative bacterium causes opportunistic or nosocomial infections in immunocompromised individuals. In recent years, a steady increase in human corneal infections of P. aeruginosa has been reported with increased multi-drug resistance (MDR) or extensively drug resistance (XDR). Several non-coding sRNAs, has been identified to regulate various physiological processes in P. aeruginosa, including biofilm formation, quorum sensing. However, the regulatory mechanism of sRNAs in MDR/XDR pathways of P. aeruginosa keratitis strains is not yet studied. In this study, we identified bacterial sRNAs in publicly available P. aeruginosa keratitis genomes and investigated their regulatory role in MDR/XDR pathways using bioinformatic analysis. Totally, 46 P. aeruginosa keratitis strains from different geographical regions were included. Of 46, Eight (30%) out of Twenty-seven and Nine (52%) out of Nineteen P. aeruginosa strains from India and Australia were identified as not-MDR. Whereas, 10 (38%) Indian and 9 (47%) Australian strains were identified as MDR. Eight Indian strains were identified as XDR. Out of 46 strains, 23 (50%) carried ExoU, 21(45%) carried ExoS and two (5%) strains carried both ExoU and ExoS, exotoxins for their virulence. The sRNA, SPA0021 was identified in 18 MDR/XDR and 6 not-MDR strains along with UCBPP-PA14. Interestingly, majority of the imipenem resistant P. aeruginosa keratitis strains from the present study was found to be carried SPA0023 sRNA (18 out of 30 strains). The outer membrane porin protein OprD, identified as binding target of SPA0023. Negative regulation or inactivation of OprD, reported in increased imipenem resistance in P. aeruginosa. Mutation analysis revealed that SPA0023 carrying P. aeruginosa keratitis strains contains a lesser number of amino acid changes in OprD protein than other strains. These findings indicate, imipenem resistance in SPA0023 carried strains might arose from the negative regulation or inhibition of OprD by SPA0023. However, functional studies are warranted with large number of P. aeruginosa keratitis strains to confirm the negative regulation of OprD by SPA0023 and imipenem resistance.

Rapid Detection of Imipenem Resistance in Gram-Negative Bacteria Using Tabletop Scanning Electron Microscopy: A Preliminary Evaluation

Background: Enabling faster Antimicrobial Susceptibility Testing (AST) is critical, especially to detect antibiotic resistance, to provide rapid and appropriate therapy and to improve clinical outcomes. Although several standard and automated culture-based methods are available and widely used, these techniques take between 18 and 24 h to provide robust results. Faster techniques are needed to reduce the delay between test and results.Methods: Here we present a high throughput AST method using a new generation of tabletop scanning electron microscope, to evaluate bacterial ultra-structural modifications associated with susceptibilities to imipenem as a proof of concept. A total of 71 reference and clinical strains of Gram-negative bacteria were used to evaluate susceptibility toward imipenem after 30, 60, and 90 min of incubation. The length, width and electron density of bacteria were measured and compared between imipenem susceptible and resistant strains.Results: We correlated the presence of these morphological changes to the bacterial susceptibility and their absence to the bacterial resistance (e.g., Pseudomonas aeruginosa length without [2.24 ± 0.61 μm] and with [2.50 ± 0.68 μm] imipenem after 30 min [p = 3.032E-15]; Escherichia coli width without [0.92 ± 0.07 μm] and with [1.28 ± 0.19 μm] imipenem after 60 min [p = 1.242E-103]). We validated our method by a blind test on a series of 58 clinical isolates where all strains were correctly classified as susceptible or resistant toward imipenem.Conclusion: This method could be a potential tool for rapidly identifying carbapenem-resistance in Enterobacterales in clinical microbiology laboratories in <2 h, allowing the empirical treatment of patients to be rapidly adjusted.

Multidrug-Resistant Phenotypes of Escherichia coli Isolates in Wild Canarian Egyptian Vultures (Neophron percnopterus majorensis)

The presence of multidrug-resistant (MDR) Escherichia coli in cloacal samples from Canarian Egyptian vultures was investigated. Samples were obtained from chicks (n = 65) and from adults and immature birds (n = 38). Antimicrobial susceptibility to 16 antibiotics included in 12 different categories was determined for 103 E. coli isolates. MDR was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories. Forty-seven different resistance phenotypes were detected: 31 MDR (41 isolates) and 16 non-MDR (62 isolates). One isolate was resistant to all 12 antimicrobial categories and 2 phenotypes included resistance to 9 antimicrobial categories. Imipenem resistance was included in five MDR phenotypes, corresponding to five different isolates. Statistically significant differences in prevalence of MDR-phenotypes were found between chicks in nests and the rest of the animals, probably due to the shorter exposure time of chicks to antimicrobials. The main risk derived from MDR bacteria in scavengers is that it threatens the treatment of wild animals in rescue centres and could be transferred to other animals in the facilities. In addition to this, it could pose a health risk to veterinarians or other staff involved in wildlife protection programmes.

Rapid ResaImipenem/Acinetobacter NP test for detection of carbapenem susceptibility/resistance in Acinetobacter baumannii

The Rapid ResaImipenem/Acinetobacter NP test was developed for the identification of carbapenem resistance among Acinetobacter baumannii isolates. The principle of this test is based on the reduction of resazurin (a viability colorant) by metabolically active bacterial cells, hence detecting bacterial growth, in the presence of a defined concentration of imipenem chosen to be slightly above that defining imipenem resistance (6 μg/ml). The bacterial growth is visually detected by a color change from blue (resazurin) to purple or pink (resorufin product). A total of 110 A. baumannii isolates, among which 61 were imipenem-resistant, were used to evaluate the test performance. The sensitivity and specificity of the test were found to be 100%, by comparison with broth microdilution taken as the reference standard method. The Rapid ResaImipenem/Acinetobacter NP test is highly specific and sensitive, and easy to implement in routine microbiology laboratories and results are obtained within 2 h 30. It does not require any specific equipment.

Genetic and phenotypic diversity of methicillin-resistant Staphylococcus aureus among Japanese inpatients in the early 1980s

To trace the linkage between Japanese healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) strains in the early 1980s and the 2000s onward, we performed molecular characterizations using mainly whole-genome sequencing. Among the 194 S. aureus strains isolated, 20 mecA-positive MRSA (10.3%), 8 mecA-negative MRSA (4.1%) and 3 mecA-positive methicillin-susceptible S. aureus (MSSA) (1.5%) strains were identified. The most frequent sequence type (ST) was ST30 (n = 11), followed by ST5 (n = 8), ST81 (n = 4), and ST247 (n = 3). Rates of staphylococcal cassette chromosome mec (SCCmec) types I, II, and IV composed 65.2%, 13.0%, and 17.4% of isolates, respectively. Notably, 73.3% of SCCmec type I strains were susceptible to imipenem unlike SCCmec type II strains (0%). ST30-SCCmec I (n = 7) and ST5-SCCmec I (n = 5) predominated, whereas only two strains exhibited imipenem-resistance and were tst-positive ST5-SCCmec II, which is the current Japanese HA-MRSA genotype. All ST30 strains shared the common ancestor strain 55/2053, which caused the global pandemic of Panton-Valentine leukocidin-positive MSSA in Europe and the United States in the 1950s. Conspicuously more heterogeneous, the population of HA-MRSA clones observed in the 1980s, including the ST30-SCCmec I clone, has shifted to the current homogeneous population of imipenem-resistant ST5-SCCmec II clones, probably due to the introduction of new antimicrobials.

Imipenem Resistance Gram-Negative Bacilli Isolated from Patients of a Tertiary Care Hospital in Dhaka, Bangladesh

Introduction: The emergence of antibiotics resistance bacteria is a persistent global problem affecting public health. The occurrence and widespread resistance to Imipenem among hospital bacterial isolates can constitute a significant threat to chemotherapy. Objectives: This cross-sectional study aimed to investigate the imipenem resistant pattern among gram-negative bacilli isolated from different samples in Dhaka medical college hospital. Methods: A total of 300 samples (wound swab, urine, endotracheal aspirate, blood, and sputum) were collected from July 2015 to June 2016. Two hundred four gram-negative bacilli were isolated and tested for resistance to imipenem by the disc diffusion method. Results: Among 204-gram negative bacilli, 39.21% imipenem resistance was detected by the Disc Diffusion method. 14.7% imipenem resistance gram-negative bacilli were from wound swab. 83.33% of Acinetobacter baumannii were Imipenem resistant. Conclusions: Imipenem resistance is widespread among gram-negative bacilli isolated from human infections. Imipenem resistance (39.21%) found in the study is quite worrisome. J Shaheed Suhrawardy Med Coll, December 2020, Vol.12(1); 3-5