Pattern of cephalosporin and carbapenem-resistant Pseudomonas aeruginosa: a retrospective analysis

Highlights • 1815 clinical isolates were identified• 160 (9%) were resistant to carbapenems and cephalosporins• 93.6% of multidrug-resistant Pseudomonas aeruginosa were negative for carbapenemase genes• The most common resistance was among blood isolates (P-value < 0.00001)


Introduction
Global public health is seriously threatened by antibiotic resistance, which also raises morbidity, mortality, and medical expenses.An important bacterium recognized for its capacity to acquire resistance to several antimicrobial agents is Pseudomonas aeruginosa .Since carbapenems are thought to be the last line of defense against P. aeruginosa , the advent of carbapenem-resistant P. aeruginosa has recently confounded treatment choices [1][2][3] .P. aeruginosa is one of the most virulent organisms and is frequently the leading cause of nosocomial infections including ventilator-associated pneumonia (VAP), catheter-associated urinary according to multiple studies.For instance, P. aeruginosa isolates from diverse clinical specimens in a tertiary care hospital in Riyadh, Saudi Arabia, showed a high rate of carbapenem resistance of 34% in 2004 and 74% in 2009 [7] .An additional study evaluated the susceptibility pattern of inpatient and outpatient isolates of P. aeruginosa in a Saudi hospital over a 6-year period (1998)(1999)(2000)(2001)(2002)(2003).The study showed the following resistance rates of inpatient isolates: piperacillin (3.5% and 16%), ceftazidime (6% and 12.3%), imipenem-cilastatin (1.4% and 11%), and ciprofloxacin (2.3% and 10.7%), in 1998 and 2003, respectively [8] .A recent study from the same hospital showed increasing resistance rates of 8-19% and 18-27% for ceftazidime and imipenem-cilastatin, respectively over the study period 2013-2018 [9] .Management of infectious diseases has become more difficult as a result of the COVID-19 pandemic, including the change in the patterns of antibiotic resistance.Increasing use of antibiotics during the pandemic, both for the treatment of COVID-19 patients initially and as a preumptive therapy in seriously ill patients, may have influenced the development and spread of antibiotic resistant organisms, including P. aeruginosa .Thus, inappropriate antibiotic prescription during the COVID-19 has exacerbated another serious public health catastrophe [10][11][12] .These findings underline how critical it is to keep an eye on and treat antibiotic resistance in P. aeruginosa infections globally, especially during the COVID-19 pandemic.To prevent the spread of multidrug-resistant P. aeruginosa strains, infection control measures and antibiotic stewardship can all be improved by having a thorough understanding of local resistance patterns and trends and the prevalence of carbapenemase genes.This retrospective study aims to evaluate the extent of antimicrobial resistance in P. aeruginosa isolates throughout the COVID-19 pandemic years, particularly to -lactam drugs such as cephalosporins and carbapenems.The study also accounted for the prevalence of carbapenemase genes in P. aeruginosa .

Materials and method
From January 2019 to December 2022, this retrospective study was carried out at the King Fahad Military Medical Complex (KFMMC) microbiology laboratory in Dhahran, Saudi Arabia.As a tertiary care hospital, KFMMC has 335 beds total, including 24 ICU beds.Blood, sputum, tracheal aspirate, urine, and wounds were among the body sites from which P. aeruginosa isolates were examined.Samples were taken from patients in the inpatient wards (intensive care units and geenral hospital units) and outpatient clinics (primary care and specialty clinics).

Pseudomonas identification and susceptibility
Using the BD Phoenix Automated identification and Susceptibility System, P. aeruginosa isolates were identified to the s pecies level.The Clinical and Laboratory Standards Institute (CLSI) breakpoints provided in the BD Phoenix Update Disk Version V7.01A were used to interpret antibacterial susceptibilities.The study concentrated on the susceptibility of P. aeruginosa to carbapenems (imipenem and meropenem) and cephalosporins (cefepime and ceftazidime).Multidrug-resistant (MDR) isolates were those that exhibited non-susceptibility to at least one agent in three antimicrobial categories (aminoglycosides, quinolones, and piperacillin/tazobactam), as well as being resistant to both cephalosporins and carbapenems [13] .

Colistin broth microdilution test
The Compact Antimicrobial Susceptibility Panel (ComASP) broth microdilution test was used to determine the minimum inhibitory concentration (MIC) for Colistin.The test was conducted in accordance with the manufacturer's instructions, and the CLSI guidelines were used to determine the MIC breakpoints.Briefly, a bacterial suspension that is equivalent to standardization of a 0.5 McFarland was prepared and then diluted in saline to reach 1:20 suspension.Subsequently, 400 l of that suspension was added to a Muller Hinton broth, then 100 l was transferred to microtitration plate that contained seven wells of two-fold dilution concentrations (0.25-16 g/ml) of colistin.Finally, the microtitration plate was then incubated overnight at 37°C, and the turbidity was checked visually for the determination of MIC breakpoint according to CLSI guidelines.

Statistical analysis
In order to do the statistical analysis, Microsoft Office Excel was used.Charts were used as appropriate to illustrate the data, and variables were reported as numbers and percentages.Comparisons between different groups was done using X-square and a P -value of < 0.05 was considered significant.

Results
During the study period, a total of 1815 clinical isolates of P. aeruginosa were identified during the study period.And of those 160 (9%) were cephalosporins and carbapenems resistant organisms.Of those 94 (58.7%) were MDR ( Table 1 ).Of the 597 P. aeruginosa in 2019, 40 (6.7%) were resistant to carbapenems and cephalosporins and 13 (32.5%) of these were MDR.There were 393 P. aeruginosa in 2020, 44 (11.2%) exhibited cephalosporin and carbapenem resistance and 38 (86.3%) of the resistant isolates were MDR.Of the 369 isolates of P. aeruginosa in 2021, 26 (7%) were resistant to cephalosporins and carbapenems and 15 (57.7%) of these were MDR.Of the 456 P. aeruginosa in 2022, 50 (11%) exhibited cephalosporin and carbapenem resistance, and 28 (56%) of those were MDR.There was a significant difference in the percentage between inpatient and intensive care isolates compared to outpatient isolates ( P -value is < 0.00001) ( Figure 1 ).The most common phynotypes were: cefepime sensitive, ceftazidim sensitive, imipenem resistant, and meropenem sensitive followed by cefepime sensitive, ceftazidim sensitive, imipenem sensitive and meropenem resis- tant ( Figure 2 ).Only one (1%) of the MDR P. aeruginosa were colistin resistant.
Of the 42 P. aeruginosa isolates from blood samples, eight (19%) were resistant to cephalosporins and carbapenems compared to 26 (3.5%) of the 750 the urine isolates.In 588 P. aeruginosa isolates of respiratory samples, 73 (12.4%) showed resistance to cephalosporins and carbapenems compared to 39 (15.3%) of 255 of the isolates from wound samples ( Table 2 ).The most common resistance was among blood (19%) compared to other sites ( P -value was < 0.00001) ( Table 2 ).

Discussion
This study shows that cephalosporin and carbapenem resistance is a prevalent occurence among P. aeruginosa isolates.The rates of resistance are consistent with earlier studies done in healthcare settings, which also found considerable levels of resistance and is consistent with observations from other regions [14] .In our study, the prevalence of P. aeruginosa resistance to cephalosporins and carbapenems fluctuated between 6.7% and 11.2% over the course of the study.Recent studies have  [15] .However, a different study found that antibiotic resistance pf P. aeruginosa increased during the pandemic period compared to the prepandemic period [16] .It is important to note that 6.4% of the MDR P. aeruginosa had detectable carbapenemase genes using GenXpert CARPA-R kit.The NDM, IMP, and VIM carbapenemase genes, respectively, were present in three (3.2%),two (2.1%), and one (1%) of the isolates.These findings are in line with prior studies of carbapenemase-producing P. aeruginosa [17] .The absence of carbapenemase genes does not rule out the possibility of other resistance mechanisms, and it is crucial to note given that Pseudomonas can acquire resistance through a variety of genetic mechanisms, including mutations in target sites and activation of efflux pumps [18] .Only one (1%) of the MDR P. aeruginosa were colistin resistant.This finding is close to 3.1% resistance reported in one study from Saudi Arabia [19] .Although, the majority of MDR P. aeruginosa lacked detectable carbapenemase genes, a small proportion tested positive for NDM, IMP, or VIM carbapenemase genes.This highlights the significance of meticulous surveillance and infection control techniques as a means of preventing the emergence of resistant strains by illuminating the complex nature of Pseudomonas' resistance mechanisms.However, the fact that only one hospital was included in the study is a limitation of the study, and the findings might not be applicable to other institutions.Another limitation is the retrospective nature and not including the demographics and outcome of the included patients.Thus, furhter surviellance from different centers and different regions is required to further charcaterize the evolution of the resistance pattern on P. aeruginosa.

Figure 1 .
Figure 1. of cephalosporins and carbapenems Pseudomonas aeruginosa cases per location.ICU, care unit.The bar represents the number of isolates (Left Y-axis) and the line represents the percentage of resistance (left Y-axis).

Table 1
Distribution of cephalosporins and carbapenems resistant Pseudomonas aeruginosa over the study period.

Table 2
Distribution of cephalosporins and carbapenems resistant Pseudomonas aeruginosa cases by body sites.

Table 3
The prevalence of carbapenemase enzymes (sing Genexpert CARBA-R kit test) and colistin resistance among MDR Pseudomonas aeruginosa isolates.