Proteome analysis, genetic characterization, and antibiotic resistance patterns of Klebsiella pneumoniae clinical isolates

Klebsiella pneumoniae (K. pneumoniae) is a member of the ESKAPE group and is responsible for severe community
and healthcare-associated infections. Certain Klebsiella species have very similar phenotypes, which presents a
challenge in identifying K. pneumoniae. Multidrug-resistant K. pneumoniae is also a serious global problem that
needs to be addressed. A total of 190 isolates were isolated from urine (n = 69), respiratory (n = 52), wound (n = 48)
and blood (n = 21) samples collected from various hospitals in the Al-Qassim, Saudi Arabia, between March 2021
and October 2022. Our study aimed to rapidly and accurately detect K. pneumoniae using the Peptide Mass
Fingerprinting (PMF) technique, confirmed by real-time PCR. Additionally, screening for antibiotic susceptibility and
resistance was conducted. The primary methods for identifying K. pneumoniae isolates were culture, Gram staining,
and the Vitek® 2 ID Compact system. An automated MALDI Biotyper (MBT) instrument was used for proteome
identification, which was subsequently confirmed using SYBR green real-time polymerase chain reaction (real-time
PCR) and microfluidic electrophoresis assays. Vitek® 2 AST-GN66 cards were utilized to evaluate the antimicrobial
sensitivity of K. pneumoniae isolates. According to our results, Vitek® 2 Compact accurately identified 178 out of 190
(93.68%) K. pneumoniae isolates, while the PMF technique correctly detected 188 out of 190 (98.95%) isolates with a
score value of 2.00 or higher. Principal component analysis was conducted using MBT Compass software to classify
K. pneumoniae isolates based on their structure. Based on the analysis of the single peak intensities generated by
MBT, the highest peak values were found at 3444, 5022, 5525, 6847, and 7537 m/z. K. pneumoniae gene testing
confirmed the PMF results, with 90.53% detecting entrobactin, 70% detecting 16 S rRNA, and 32.63% detecting
ferric iron uptake. The resistance of the K. pneumoniae isolates to antibiotics was as follows: 64.75% for cefazolin,
62.63% for trimethoprim/sulfamethoxazole, 59.45% for ampicillin, 58.42% for cefoxitin, 57.37% for ceftriaxone,
53.68% for cefepime, 52.11% for ampicillin-sulbactam, 50.53% for ceftazidime, 52.11% for ertapenem, and 49.47%
for imipenem. Based on the results of the double-disk synergy test, 93 out of 190 (48.95%) K. pneumoniae isolates
were extended-spectrum beta-lactamase. In conclusion, PMF is a powerful analytical technique used to identify K. pneumoniae isolates from clinical samples based on their proteomic characteristics. K. pneumoniae isolates have
shown increasing resistance to antibiotics from different classes, including carbapenem, which poses a significant
threat to human health as these infections may become difficult to treat.