Microbial Drug Resistance

Since the start of the COVID-19 pandemic, our reliance on disinfectants and sanitizers and the use thereof has grown. While this may protect human health, it may be selecting for antimicrobial-resistant microorganisms, including those that are not only capable of growth in the presence of disinfectants but also thrive using this as an energy source. Furthermore, there is a growing concern in emerging nosocomial pathogens, which have shown resistance to antibiotics and disinfectants. This rise in resistance has led to the investigation of various mechanisms behind resistance, such as biofilms, efflux pumps, and mobile genetic elements. Although many resistance mechanisms have been identified, it was discovered that some potentially pathogenic microbes could metabolize these compounds, which remains an avenue for further investigation. Investigating alternative metabolic pathways in microorganisms capable of growth using disinfectants as their sole carbon and energy source may provide insight into the metabolism of quaternary ammonium compound (QAC)-based antimicrobials. Many of the metabolic reactions proposed include hydroxylation, N-dealkylation, N-demethylation, and β-oxidation of QACs. If clear metabolic pathways and reactions are elucidated, possible alternative approaches to QACs may be advised. Alternatively, this may provide opportunities for biodegradation of the compounds that adversely affect the environment.

Pseudomonas aeruginosa is an opportunist pathogen usually associated with life threatening infections and exhibits a set of intrinsic and acquired antimicrobial mechanisms. Although resistance to penicillins-like compounds is commonly associated with the chromosomal Pseudomonas-derived cephalosporinases β-lactamase, the real contribution of OXA-50, a second chromosomally encoded β-lactamase, remains unclear. In this study, we characterized the biochemical properties of OXA-50, OXA-488, and OXA-494. Both oxacilinases differ from OXA-50 in two amino acids each. The bla_OXA-50, bla_OXA-488, and bla_OXA-494 were cloned into pET26b⁺ that was transformed into Escherichia coli DH5α strain, expressed in E. coli BL21 strain, and then purified for obtaining the hydrolytic parameters. Benzylpenicillin was the preferential substrate instead of oxacillin. Besides, OXA-488 showed a threefold increase in catalytic efficiency for benzylpenicillin, and it was twofold more efficient in hydrolyzing imipenem, compared with OXA-50, although such carbapenemase activity was considered weak. In addition, OXA-488 and OXA-494 showed an increased affinity for penicillins, which contributed to the increased catalytic efficiency against ampicillin, especially OXA-488. Chromosomally encoded resistance mechanisms are usually overshadowed by acquired mechanisms. However, understanding their real contribution is essential to comprehend the versatile profiles verified in P. aeruginosa isolates. Such information can help to choose the best therapy in a scenario of limited options.

Reports on multidrug-resistant hypervirulent Klebsiella pneumoniae (MDR-hvKP) in recent years indicate the wide-spreading trend of MDR-hvKP. The co-occurrence of hypervirulence and drug resistance poses a great challenge to clinical treatment. In this study, molecular characteristics of an MDR strain hvKP247 and 30 clinically isolated hvKP strains were characterized. The whole genome of hvKP247 belonging to sequence type (ST) 5214 and capsule serotype K1 was sequenced and analyzed. Conjugation experiments were performed to evaluate transferability of the plasmids in hvKP247. We found two new STs among our isolates, ST5570 and ST5571. The ST5214 hvKP247 contained two transferable plasmids: a hybrid virulence plasmid (pHvKP247-vir) carrying transfer-related modules that had self-transferable ability, and a drug-resistant plasmid (pHvKP247-MDR) that could be indirectly transferred with the help of pHvKP247-vir. The virulence-related genes were located on the pHvKP247-vir and chromosomal ICEKp1 mobile genetic element. In conclusion, the hybrid virulence plasmid and the drug-resistant plasmid are co-transferred, which emphasizes the importance of raising public awareness of the simultaneous spread of virulence and resistance genes of MDR-hvKP strains.

Staphylococcus epidermidis, a major skin bacterium, can cause opportunistic infections. Use of antimicrobial agents against Cutibacterium acnes for acne treatment becomes a risk factor for emergence of antimicrobial-resistant skin bacteria. In this study, the impact of antimicrobial treatment of acne vulgaris on S. epidermidis antimicrobial resistance was assessed. A total of 344 S. epidermidis strains isolated from patients with acne vulgaris who visited hospital (165 strains) and dermatological clinics (179 strains), respectively, were analyzed. Except for doxycycline, the resistance rates were higher in strains isolated from patients who had used antimicrobials for acne treatment than in those isolated from patients who had not used antimicrobials. The prevalence rates of strains with erm(C) from patients who used macrolides and clindamycin (hospital, 78.0%; clinics, 61.3%) and those of strains with tet(M) from patients who used tetracyclines (hospital, 27.5%; clinics, 42.4%) were significantly higher than those of strains from patients who did not use antimicrobials (p < 0.05). All strains with erm(A) (8/8) and 91.7% strains with erm(C) (156/170) showed high-level resistance to macrolides and clindamycin (MIC ≥256 μg/mL). Furthermore, almost all strains with tet(M) showed resistance to minocycline. Our results showed that the use of antimicrobials for acne treatment may lead to an increased prevalence of antimicrobial-resistant S. epidermidis. In particular, the emergence of minocycline-resistant strains with tet(M) owing to the use of tetracyclines (doxycycline and minocycline) is a critical issue. Appropriate antimicrobial use for acne treatment may be an important strategy to prevent the emergence of antimicrobial-resistant skin bacteria.

We investigated the in vitro susceptibility of ceftazidime-avibactam (CZA) resistant Stenotrophomonas maltophilia to the associations aztreonam/amoxicillin-clavulanate (ATM-AMC) and ATM-CZA. Forty clinical isolates of S. maltophilia recovered from sputum samples of 40 cystic fibrosis people were selected from the collection of the Nantes University Hospital, based on their resistance to CZA. Minimum inhibitory concentrations (MICs) of ATM-CZA and ATM-AMC were determined for each isolate by an Etest strip superposition method, and by Etest for each individual antibiotic. MICs of CZA, ATM, and AMC ranged from 12 to ≥256, ≥256, and 16 to ≥256 mg/L, respectively. Synergistic effects were observed with the ATM-CZA combination for all isolates (fractional inhibitory concentration index range of 0.01 to 0.27), with combination MICs ranging from 0.75 to 16 mg/L (MIC_50/90 = 3/12 mg/L), corresponding to a decrease of at least 16-folds in the MIC of ATM. In 23 (57.5%) S. maltophilia isolates, the association of AMC to ATM was also synergistic and combination MICs were ≤16 mg/L (EUCAST breakpoint for ATM resistance in Pseudomonas aeruginosa). Our results show that ATM-CZA or ATM-AMC could be alternative therapeutic options against some highly resistant S. maltophilia. This encourages further experimental studies, in particular time-kill analyses, and clinical trials to delineate conditions required for use of these combinations in practice.

The study describes the first isolation of multidrug-resistant (MDR) Klebsiella pneumoniae ST16, Escherichia coli ST131 (Esc), and Enterobacter hormaechei subsp. steigerwaltii ST93 (Enterobacter cloacae complex [ECC]) in Sri Lanka. Eight MDR strains of uropathogenic Enterobacterales isolated from hospital acquired urinary tract infections (UTIs) were analyzed using genomic sequencing and comparative genomics. Isolates carried multiple carbapenemase, AmpC, and ESBL (extended-spectrum β-lactamase) genes. ECC manifested both bla_NDM-4 and bla_OXA-181. The K. pneumoniae strains harbored fimbrial genes that facilitate pathogenesis of UTI. Several extraintestinal pathogenic E. coli associated virulence genes were identified in Esc. The efflux pump gene, acrA, and the T6SS gene cluster were detected in ECC. Many antimicrobial resistance (AMR) and virulence genes were identified associated with mobile genetic elements. ISEcp1 flanked upstream of bla_CTX-M-15. The carbapenemase genes were carried on ColKP3 plasmids and were associated with ISEcp1. In Esc, the AMR gene bla_TEM-1B and virulence gene traT were found on an IncF plasmid replicon. In K. pneumoniae the AMR genes sul1 and tetB present on IncR plasmid replicons and were associated with the insertion sequence IS6100. In Kp5, bla_LAP-2 and qnrS1 coexisted and were flanked by ISEcl. AMR gene clusters, conferring resistance to multiple antimicrobial classes, flanked by mobile elements were identified in seven isolates.

Aeromonas hydrophila and other closely related Aeromonas species cause motile aeromonad septicemia, a common fish disease. The disease affects many aquaculture sectors potentially requiring antimicrobial treatments. Therefore, researchers and laboratory diagnosticians need criteria called epidemiological cutoff values (ECVs) to determine whether a bacterial isolate has developed decreased susceptibility to an antimicrobial. To generate ECVs for this bacterium, we assembled a diverse collection of 245 isolates previously identified as A. hydrophila from fish. Using rpoD sequencing, we confirmed that 97 of the 245 isolates were A. hydrophila. We allocated the isolates among three laboratories and tested their susceptibility against eight antimicrobials using standard Clinical and Laboratory Standards Institute (CLSI) disk diffusion and broth microdilution methods. The resulting frequency distributions were statistically analyzed to determine wild-type cutoff estimates, which, along with scatterplots, were used to estimate potential ECVs. In collaboration with the CLSI, aquaculture working group, we proposed ECVs for six of the eight antimicrobials tested. Subsequently, the CLSI Subcommittee on Veterinary Antimicrobial Susceptibility Testing reviewed our data and approved the ECVs to be added to the 2020 edition of the VET04 performance standards for antimicrobial susceptibility testing of aquatic bacteria.