The co-existence of the colistin resistance (mcr) gene with multiple drug-resistance genes has raised concerns about the possibility of the development of pan-drug-resistant bacteria that will complicate treatment. This study aimed to investigate the antibiotic resistance profiles and co-existence of antibiotic resistance genes among the colistin-resistant Enterobacteriaceae isolates recovered from poultry and poultry meats. The antibiotic susceptibility to various classes of antibiotics was performed using the Kirby-Bauer disk diffusion method and selected antimicrobial resistance genes were detected using PCR in a total of 54 colistin-resistant Enterobacteriaceae isolates including Escherichia coli (E. coli) (n = 32), Salmonella spp. (n = 16) and Klebsiella pneumoniae (K. pneumoniae) (n = 6) isolates. Most of the isolates had multi-drug resistance (MDR), with antibiotic resistance against up to seven classes of antibiotics. All mcr-harbouring, colistin-resistant Enterobacteriaceae isolates showed this MDR (100%) phenotype. The mcr-1 harbouring E. coli isolates were co-harbouring multiple antibiotic resistance genes. The seven most commonly identified resistance genes (^blaTEM, tetA, floR, aac-3-IV, aadA1, fosA, aac(6_)-lb) were detected in an mcr-1-harbouring E. coli isolate recovered from a cloacal swab. The mcr-5 harbouring Salmonella spp. isolate recovered from poultry meats was positive for ^blaTEM, tetA, floR, aac-3-IV, fosA and aac(6_)-lb genes. In conclusion, the colistin-resistant Enterobacteriaceae with mcr genes co-existing multiple clinically important antimicrobial resistance genes in poultry and poultry meats may cause potential future threats to infection treatment choices in humans and animals. Full article

Rare information is available on clinical Enterococcus faecium encountered in Sardinia, Italy. This study investigated the antimicrobial susceptibility profiles and genotypic characteristics of E. faecium isolated at the University Hospital of Sassari, Italy, using the Vitek2 system and PCR, MLST, or WGS. Vitek2 revealed two VanB-type vancomycin-resistant Enterococcus faecium (VREfm) isolates (MICs mg/L = 8 and ≥32) but failed to detect vancomycin resistance in one isolate (MIC mg/L ≤ 1) despite positive genotypic confirmation of vanB gene, which proved to be vancomycin resistant by additional phenotypic methods (MICs mg/L = 8). This vanB isolate was able to increase its vancomycin MIC after exposure to vancomycin, unlike the “classic” occult vanB-carrying E. faecium, becoming detectable by Vitek 2 (MICs mg/L ≥ 32). All three E. faecium had highly mutated vanB₂ operons, as part of a chromosomally integrated Tn1549 transposon, with common missense mutations in VanH and VanB₂ resistance proteins and specific missense mutations in the VanW accessory protein. There were additional missense mutations in VanS, VanH, and VanB proteins in the vanB₂-carrying VREfm isolates compared to Vitek2. The molecular typing revealed a polyclonal hospital-associated E. faecium population from Clade A1, and that vanB₂-VREfm, and nearly half of vancomycin-susceptible E. faecium (VSEfm) analyzed, belonged to ST117. Based on core genome-MLST, ST117 strains had different clonal types (CT), excluding nosocomial transmission of specific CT. Detecting vanB₂-carrying VREfm isolates by Vitek2 may be problematic, and alternative methods are needed to prevent therapeutic failure and spread. Full article

As the prevalence of Staphylococcus aureus infections is of worldwide concern, phenotype and genotype in prevalent MRSA strains require longitudinal investigation. In this study, the antibiotic resistance, virulence gene acquisition, and molecular type were determined on a large scale of nosocomial S. aureus strains in Southern China during 2009–2015. Bacterial identification and antimicrobial susceptibility to 10 antibiotics were tested by Vitek-2. Virulence genes encoding staphylococcal enterotoxins (SEA, SEB, SEC, SED, and SEE), exfoliative toxins (ETA and ETB), Panton–Valentine leukocidin (PVL), and toxic shock syndrome toxin (TSST) were detected by PCR, with SCCmec typing also conducted by multiplex PCR strategy. Genotypes were discriminated by MLST and spaA typing. MLST was performed by amplification of the internal region of seven housekeeping genes. PCR amplification targeting the spa gene was performed for spa typing. No resistance to vancomycin, linezolid, or quinupristin and increase in the resistance to trimethoprim/sulfamethoxazole (55.5%) were identified. A total of nine SCCmec types and subtypes, thirteen STs clustered into thirteen spa types were identified, with ST239-SCCmec III-t037 presenting the predominant methicillin-resistant S. aureus (MRSA) clone. Typically, SCCmec type IX and ST546 were emergent types in China. Isolates positive for both pvl and tsst genes and for both eta and etb genes were also identified. Important findings in this study include: firstly, we have provided comprehensive knowledge on the molecular epidemiology of MRSA in Southern China which fills the gap since 2006 or 2010 from previous studies. Secondly, we have presented the correlation between virulence factors (four major groups) and genotypes (SCCmec, ST and spa types). Thirdly, we have shown evidence for earliest emergence of type I SCCmec from 2012, type VI from 2009 and type XI from 2012 in MRSA from Southern China. Full article

(1) Background: During the COVID-19 outbreak, several studies showed an increased prevalence of extended-spectrum β-lactamase producing Enterobacterales (ESBL-PE) carriage in intensive care units (ICUs). Our objective was to assess the impact of antibiotic prescriptions on the acquisition of ESBL-PE in ICUs during the COVID-19 crisis. (2) Methods: We conducted an observational study between 1 April 2020, and 31 December 2021, in the medical-surgical ICU of the Cayenne General Hospital. We defined two periods: Period 1 with routine, empirical antibiotic use, and Period 2 with no systematic empiric antibiotic prescription. (3) Results: ICU-acquired ESBL-PE carriage was 22.8% during Period 1 and 9.4% during Period 2 (p = 0.005). The main isolated ESBL-PE was Klebsiella pneumoniae (84.6% in Period 1 and 58.3% in Period 2). When using a generalized linear model with a Poisson family, exposure to cefotaxime was the only factor independently associated with ESBL-PE acquisition in ICU (p = 0.002, IRR 2.59 (95% IC 1.42–4.75)). The propensity scores matching estimated the increased risk for cefotaxime use to acquire ESBL-PE carriage at 0.096 (95% CI = 0.02–0.17), p = 0.01. (4) Conclusions: Exposure to cefotaxime in patients with severe COVID-19 is strongly associated with the emergence of ESBL-PE in the context of maximal infection control measures. Full article

(1) Background: Linezolid plays an important role in the treatment of invasive infections caused by vancomycin-resistant enterococci after its introduction to clinical practice. However, a detailed examination of linezolid-nonsusceptible enterococci (LNSE) is required. In this study, we attempted to analyze the mechanisms of LNSE strains isolated from a tertiary care hospital. (2) Methods: From 2019 to 2020, 18 Enterococcus faecalis, 14 E. faecium, and 2 E. gallinarum clinical isolates were collected at Severance Hospital. Agar dilution was performed to evaluate precise linezolid minimum inhibitory concentrations (MICs). Short-read whole-genome sequencing (WGS) was used to analyze resistance determinants. (3) Results: The presence of the optrA gene was likely the primary resistance mechanism in these three species, typically demonstrating a MIC value of 8 μg/mL. The co-existence of the cfr(D) and poxtA2 gene was the second major mechanism, primarily predicting a phenotype showing intermediate susceptibility to linezolid. G2576U mutation on 23S rRNA was only found in E. faecium; it mediated the most significant increase in linezolid MIC. (4) Conclusion: This is the first report examining poxtA2–cfr(D) co-harboring clinical enterococcal isolates in Korea and demonstrating the poxtA EF9F6-harboring clinical E. gallinarum strain worldwide. The comparison with resistance-gene-containing fragments in the isolates obtained from different countries and different sources demonstrated the spread of linezolid-resistance genes and suggested the possibility of foodborne transmission. Full article

Background: Whole-genome sequencing is one of the best ways to investigate resistance mechanisms of clinical isolates as well as to detect and identify circulating multi-drug-resistant (MDR) clones or sub-clones in a given hospital setting. Methods: Here, we sequenced 37 isolates of Acinetobacter baumannii, 10 Klebsiella pneumoniae, and 5 Pseudomonas aeruginosa collected from the biobank of the hospital setting of the King Fahad Medical City. Complete phenotypic analyses were performed, including MALDI-TOF identification and antibiotic susceptibility testing. After the genome assembly of raw data, exhaustive genomic analysis was conducted including full resistome determination, genomic SNP (gSNP) analysis, and comparative genomics. Results: Almost all isolates were highly resistant to all tested antibiotics, including carbapenems and colistin. Resistome analysis revealed many antibiotic resistance genes, including those with resistance to β-lactams, aminoglycosides, macrolides, tetracyclines, sulfamids, quinolones, and phenicols. In A. baumannii isolates, the endemic carbapenemase bla_OXA-23 gene was detected in 36 of the 37 isolates. Non-synonymous mutations in pmrB were detected in almost all of the isolates and likely mediated colistin resistance. Interestingly, while classical analyses, such as MLST, revealed the predominance of an ST2 clone in A. baumannii isolates, the genomic analysis revealed the presence of five circulating sub-clones and identified several isolate transmissions between patients. In the 10 K. pneumoniae isolates, several resistance genes were identified, and the observed carbapenem resistance was likely mediated by overexpression of the detected extended-spectrum-β-lactamase (ESBL) genes associated with low membrane permeability as few carbapenemase genes were detected with just bla_OXA-48 in three isolates. Colistin resistance was mediated either by non-synonymous mutations in the MgrB regulator, PmrA, PmrB, and PhoQ proteins or the presence of the MCR-1 protein. Here, gSNP analysis also revealed the existence of bacterial clones and cases of isolate transmissions between patients. The five analyzed P. aeruginosa isolates were highly resistant to all tested antibiotics, including carbapenems mediated by loss or truncated OprD porin, and colistin resistance was associated with mutations in the genes encoding the PmrA, PmrB, or PhoQ proteins. Conclusion: We demonstrate here the usefulness of whole-genome sequencing to exhaustively investigate the dissemination of MDR isolates at the sub-clone level. Thus, we suggest implementing such an approach to monitor the emergence and spread of new clones or sub-clones, which classical molecular analyses cannot detect. Moreover, we recommend increasing the surveillance of the endemic and problematic colistin resistance mcr-1 gene to avoid extensive dissemination. Full article

The emergence of carbapenem-resistant Acinetobacter calcoaceticus-baumannii complex (CRACB) in clinical environments is a significant global concern. These critical pathogens have shown resistance to a broad spectrum of antibacterial drugs, including carbapenems, mostly due to the acquisition of various β-lactamase genes. Clinical samples (n = 1985) were collected aseptically from multiple sources and grown on blood and MacConkey agar. Isolates and antimicrobial susceptibility were confirmed with the VITEK-2 system. The modified Hodge test confirmed the CRACB phenotype, and specific PCR primers were used for the molecular identification of bla_OXA and bla_NDM genes. Of the 1985 samples, 1250 (62.9%) were culture-positive and 200 (43.9%) were CRACB isolates. Of these isolates, 35.4% were recovered from pus samples and 23.5% from tracheal secretions obtained from patients in intensive care units (49.3%) and medical wards (20.2%). An antibiogram indicated that 100% of the CRACB isolates were resistant to β-lactam antibiotics and β-lactam inhibitors, 86.5% to ciprofloxacin, and 83.5% to amikacin, while the most effective antibiotics were tigecycline and colistin. The CRACB isolates displayed resistance to eight different AWaRe classes of antibiotics. All isolates exhibited the bla_OXA-51 gene, while bla_OXA-23 was present in 94.5%, bla_VIM in 37%, and bla_NDM in 14% of the isolates. The bla_OXA-51, bla_OXA-23, and bla_OXA-24 genes co-existed in 13 (6.5%) isolates. CRACB isolates with co-existing bla_OXA-23, bla_OXA-24, bla_NDM, bla_OXA-51 and bla_VIM genes were highly prevalent in clinical samples from Pakistan. CRACB strains were highly critical pathogens and presented resistance to virtually all antibacterial drugs, except tigecycline and colistin. Full article

Aminoglycosides are widely used to treat infections of Pseudomonas aeruginosa. Genes encoding aminoglycoside-modifying enzymes (AMEs), acquired by horizontal gene transfer, are commonly associated with aminoglycoside resistance, but their effects have not been quantified. The aim of this research was to determine the extent to which AMEs increase the antibiotic tolerance of P. aeruginosa. Bioinformatics analysis identified AME-encoding genes in 48 out of 619 clinical isolates of P. aeruginosa, with ant(2′)-Ia and aac(6′)-Ib3, which are associated with tobramcyin and gentamicin resistance, being the most common. These genes and aph(3′)-VIa (amikacin resistance) were deleted from antibiotic-resistant strains. Antibiotic minimum inhibitory concentrations (MICs) were reduced by up to 64-fold, making the mutated bacteria antibiotic-sensitive in several cases. Introduction of the same genes into four antibiotic-susceptible P. aeruginosa strains increased the MIC by up to 128-fold, making the bacteria antibiotic-resistant in all cases. The cloned genes also increased the MIC in mutants lacking the MexXY-OprM efflux pump, which is an important contributor to aminoglycoside resistance, demonstrating that AMEs and this efflux pump act independently in determining levels of aminoglycoside tolerance. Quantification of the effects of AMEs on antibiotic susceptibility demonstrates the large effect that these enzymes have on antibiotic resistance. Full article

Tebipenem-pivoxil hydrobromide, an orally bioavailable carbapenem, is currently in clinical development for the treatment of extended-spectrum β-lactamase- and AmpC-producing Enterobacterales. Previously, tebipenem was found to possess antimicrobial activity against the biothreat pathogens, Burkholderia pseudomallei and Burkholderia mallei. Thus, herein, tebipenem was evaluated against a panel of 150 curated strains of Burkholderia cepacia complex (Bcc) and Burkholderia gladioli, pathogens that infect people who are immunocompromised or have cystic fibrosis. Using the provisional susceptibility breakpoint of 0.12 mg/L for tebipenem, 100% of the Bcc and B. gladioli tested as being provisionally resistant to tebipenem. Bcc and B. gladioli possess two inducible chromosomal β-lactamases, PenA and AmpC. Using purified PenA1 and AmpC1, model β-lactamases expressed in Burkholderia multivorans ATCC 17616, PenA1 was found to slowly hydrolyze tebipenem, while AmpC1 was inhibited by tebipenem with a k₂/K value of 1.9 ± 0.1 × 10³ M⁻¹s⁻¹. In addition, tebipenem was found to be a weak inducer of bla_PenA1 expression. The combination of the slow hydrolysis by PenA1 and weak induction of bla_PenA1 likely compromises the potency of tebipenem against Bcc and B. gladioli. Full article