Prevalence of carbapenemase genes among multidrug-resistant Pseudomonas aeruginosa isolates from tertiary care centers in Southern Thailand

Objectives: To assess the prevalence of carbapenemase genes among multidrug-resistant Pseudomonas aeruginosa (P. aeruginosa) isolates from tertiary care centers in Southern Thailand. Methods: The prevalence of carbapenemase genes in P. aeruginosa isolates collected from patients hospitalized between 2015-2017 in 2 tertiary care hospitals in Songkhla Province, Southern Thailand, was investigated. Standard laboratory procedures were followed and disk diffusion test was used for bacterial identification and susceptibility evaluations. Carbapenemase genes were detected using multiplex polymerase chain reaction (PCR) and genotyping by pulsed field gel electrophoresis. Results: Among the 289 P. aeruginosa isolates, 55% was from sputum, 19.4% was from urine, and 8% was from secretions. The prevalence was 55.7% in carbapenem-resistant multidrug-resistant P. aeruginosa (CR-MDR-PA) and 39.4% in multidrug-resistant P. aeruginosa (MDR-PA). Resistance to imipenem, meropenem, gentamicin, and ceftazidime ranged from 50-60%, and amikacin was the most effective antibiotic (38.4%). The carbapenemase genes bla VIM (27.7%), bla IMP (23.9%), and bla OXA48 (4.8%) were detected; however, bla SPM and bla BIC were not detected in any of the isolates. Pulsed field gel electrophoresis analysis revealed clonal diversity among 17 CR-MDR-PA strains. Conclusion: A high percentage of CR-MDR-PA carries carbapenemase genes in our area; therefore, more emphasis on and application of molecular techniques for infection prevention and control may provide useful insights on disease epidemiology.

Saudi Med J 2022; Vol. 43 (9) https://smj.org.sa M ultidrug-resistant Pseudomonas aeruginosa (MDR-PA) has become a worldwide health problem because it exhibits broad resistance to carbapenems, including "last-line" carbapenems. 1 The prevalence of MDR-PA and extensively drug-resistant P. aeruginosa (XDR-PA) producing carbapenemase has been increasing. One mechanism of this resistance is the degradation of carbapenems by enzyme lactamases such as carbapenemase. Carbapenemases are classified into 3 molecular molecules: class A metallo-β-lactamases (MBLs), which mostly include the enzyme Klebsiella pneumoniae carbapenemase gene (bla KPC ); class B MBLs, such as Verona integron-encoded MBL (bla VIM ), and bla IMP types, which were subsequently mutated to New Delhi MBL 1 (bla NDM-1 ); and class D (oxacillinases or bla OXA ), which is produced by P. aeruginosa. The prevalence of P. aeruginosa infection has increased in the last decade, particularly in healthcare settings, and has been recognized by the Centers of Disease Control and Prevention (CDC). 2 The National Antimicrobial Resistance Surveillance Center reported that the incidence of P. aeruginosa infections in hospitals in Thailand has dramatically increased in the past 15 years. In particular, the Department of Medical Sciences found that the incidence of imipenem (IMP)-resistant P. aeruginosa in the country increased from 14% in 2000 to 47% in 2015, resulting in higher morbidity rates. 3, 4 The transmission of carbapenemase genes among carbapenem-resistant P. aeruginosa (CR-PA) isolates should be carefully considered because many of these genes are carried by plasmids and are easily transferable. Phenotypic techniques, such as the modified Hodge test, for the in vitro identification of carbapenemase production are not very sensitive and specific. 5 Carbapenemase detection may be based on the inhibitory properties of several molecules. Furthermore, although the molecular detection of carbapenemase genes is a viable alternative, it is still seldom used because of its high cost and requirement of data-interpretation expertise. 6 Presently, carbapenem resistance in P. aeruginosa typically results from the formation of class B carbapenemase and has led to a global epidemic of P. aeruginosa infection. 7,8 However, the Clinical Laboratory Standards Institute (CLSI) has not yet established a standardized method for screening and assaying carbapenemase. 9,10 Although several methods are available to diagnose an infection and determine P. aeruginosa resistance, the most accepted is in vitro culture. Susceptibility tests are the gold standard, but they are labor-intensive because only one drug concentration can be tested in each tube. Therefore, understanding the potential resistance mechanisms of P. aeruginosa is imperative to select the effective antimicrobial agents. Thus, this study aimed to examine the prevalence of carbapenemase genes in P. aeruginosa isolates from patients admitted to tertiary care hospitals in Southern Thailand by using the multiplex polymerase chain reaction (mPCR) technique, investigate the antimicrobial susceptibility profile, and then identify any strains that could potentially cause an outbreak by using pulsed field gel electrophoresis (PFGE).
Methods. This retrospective study reviewed the data regarding P. aeruginosa isolates of patients admitted to 2 tertiary care hospitals in Songkhla, Thailand, between August 2015 and March 2017. The study included all consecutive nonduplicate isolates of P. aeruginosa (n=289) resistant to meropenem (MEM) or IMP (based on disk diffusion test findings) from various clinical samples (sputum, blood, urine, secretions [penrose drain, bronchial wash, percutaneous nephrostomy, corneal ulcer, bile, and pleural fluid], pus, tissue, and catheter tip). Bacteria were identified at the hospital's microbiology laboratory using conventional biochemical tests in accordance with the 2015 CLSI guidelines. All P. aeruginosa isolates were stored in 20% glycerol at -80°C until further tests were carried out.
The following primers and conditions for mPCR for carbapenemase gene amplification ( Table 1) were used, as previously described. 13 Pulsed field gel electrophoresis was carried out as previously described by Pfaller et al 15 and modified by Seifer et al. 16 Briefly, an overnight culture of P. aeruginosa with an optical density (OD600) of 0.5 in Luria Bertani broth (Merck KGaA, Darmstadt, Germany) was incubated in 100 mM Tris pH 7.2 buffer containing 100 mM ethylenediaminetetraacetic acid (EDTA; Amresco, Solon, Ohio, USA), 20 mM NaCl (Amresco, Solon, Ohio, USA), and 0.5 mg/ml concentration of proteinase K (Amresco, Solon, Ohio, USA) at 55°C for 10 minutes. Subsequently, an equal volume of 2% UltraPureTM LMP agarose (Invitrogen, Carlsbad, CA, USA) was added, and the solution was placed in a mold to form a solid plug, which was then incubated with cell lysis buffer (50 mM Tris pH 8.0, 100 mM EDTA, 0.1% sodium dodecyl sulfate, 1.0% sarcosine [Amresco, Solon, Ohio, USA], and 0.5 mg/ml concentration of proteinase K) at 55°C for 2 hours. The agarose plug was treated with 10 U of XbaI (Fermentas, USA). 15 The DNA was separated by PFGE using a CHEF-DR III system (Bio-Rad Laboratories, Hercules, CA, USA). Running conditions were 21 hours at 14°C, with an initial switching time of one second and final time of 30 seconds, at 6 V/cm. Band patterns were analyzed using the BioNumerics 7.0 software (Applied Maths, St-Martens-Latem, Belgium) and interpreted according to the Tenover Interpretive Criteria. 17 Statistical analysis. All statistical data were analyzed using the Statistical Package for the Social Sciences, version 23.0 (IBM Corp., Armonk, NY, USA). Categorical variables are reported as numbers and percentages, and each variable was examined by univariate analysis. Multinomial logistic regression was used for calculating odds ratios (ORs), 95% confidence intervals (CIs), and p-values and for further analyzing variables with p<0.05 on univariate analysis. All tests were 2-tailed, and a p-value of <0.05 was considered significant.
The incidence of CR-MDR-PA was the highest, and it was detected mostly from non-wards (46.7%), followed by ICU (9%), medical wards (18.9%), and all other wards (15.4%). However, the infection site had no statistically significant effect on the susceptibility to infection with CR-MDR-PA and MDR-PA.
The dendrogram for genetic similarity was generated using the macrorestriction profile, and data from the 17 most common P. aeruginosa isolates are summarized in Figure 1. All these 17 isolates, which were divided into 13 different genotypes, underwent PFGE. All isolates collected from hospital showed high genetic variation, and 2 were MDR-PA. Other isolates were found from different sites, and they belonged to 5 genotypes. All 17 bla IMP -positive isolates belonged to 11 clusters, but one cluster, which was linked to 2 isolates from ICUobtained sputum samples, had bla NDM , bla oxa48 , bla DIM , and bla GIM .
Discussion. Pseudomonas aeruginosa is a common cause of nosocomial infections and results in high mortality rates. 18 This study found that MDR-PA isolates were a common cause of infection caused by CR-PA during the study period. This finding may be explained by the heavy use of antibiotics in patients, thereby, increasing the emergence of the XDR phenotype. The incidence of MDR-PA (55.7%) in this study is similar to that in other studies, such as 56% in Egypt. 17 However, in our study, P. aeruginosa infections mostly occurred in the respiratory tract, followed by the urinary tract, and secretions. This result is similar    Carbapenem-resistant MDR-PA infection most commonly occurred in the non-intensive care unit (ICU) wards, with an incidence rate of 46.7%. Bhatt et al 22 reported that the incidence of infections caused by resistant P. aeruginosa was 54.9% in the burn unit in India, consistent with our results. In our study, all resistant isolates were obtained between 2015-2017, demonstrating a 2-fold increase in the incidence of P. aeruginosa infection at Songklanagarind Hospital compared with that in Hat Yai Hospital, Thailand. This result could be explained by the number of available beds, which has an impact on nosocomial infection outbreak. 23 Susceptibility tests showed that 161 CR-MDR-PA isolates tested against 16 antimicrobial agents were highly resistant to AK, GM, CAZ, and TZP. Several isolates were also resistant to LVX, MEM, and IMP. Resistance to quinolones (CIP and LVX) ranged from 30-50% and that to NOR (10.6%) and STFX (2.5%).
As observed, CR-MDR-PA was resistant to various antimicrobial agents, except DA. CR-MDR-PA was previously reported to be susceptible to TZP. 24,25 Furthermore, MBL-producing PA is less sensitive to aztreonam, possibly because of the different resistance mechanisms in P. aeruginosa.
In addition to MDR-PA strains, nearly 90% of the isolates were highly resistant to carbapenem. These isolates were completely resistant to IMP and MEM but were susceptible to AK (86.8%), GM (82.5%), and CIP (71.1%). Therefore, with proper use, AK can still be an effective treatment drug against P. aeruginosa infection.
According to previous studies in Thailand, bla VIM is the most common carbapenemase gene detected in P. aeruginosa and is widely prevalent in the country. 26 In our study, bla VIM (n=80, 22.7%) was the most common, contrary to the result (12.5%) from the study of the clinical CR-PA isolates from Phramongkutklao Hospital, Thailand. 27 Surprisingly, bla DIM was found in our region (18.7%). Likewise, bla DIM was found in 5 out of 200 clinical isolates of P. aeruginosa (2.5%) in India, lower than that observed in our study. 28 The high prevalence of bla DIM may be explained by transgenic gene resistance. These findings clearly demonstrate that other determinants may also be implicated in the prevalence of antibioticresistance genes.
Pulsed field gel electrophoresis analysis revealed that P. aeruginosa harboring bla VIM is the clonally predominant genotype. This major clonality (70%) suggests that cross-transmission is an important mechanism of dissemination, causing high resistance levels among P. aeruginosa isolates. Therefore, continuous surveillance and improved infection control strategies are needed to reduce cross-infection, particularly when majority of the carbapenemase genes are on high mobility.
Study limitations. First, isolates from only 2 hospitals were examined, with most of them being collected from a single institution, thereby limiting the generalization of the results. Second, no DNA sequencing (whole-genome sequencing or at least amplicon sequencing) was carried out to determine the carbapenemase variants that are distributed in our geographical region. Third, the data were retrospectively collected; hence, patient data such as comorbidities and other clinical information could not be collected. Finally, antimicrobial resistance mechanisms and their potential interactions with virulence factor genes were not investigated. More studies will be carried out in the future by multilocus sequencing to elucidate the population genetics of CR-PA isolates from Thailand, and whole-genome sequences will be utilized to explore more epidemiological features of carbapenemaseproducing P. aeruginosa in clinical studies.
In conclusion, the clinical isolates of CR-MDR-PA are highly prevalent in Southern Thailand. Several carbapenemase genes, particularly bla VIM and bla IMP , are present; these genes are associated with genotyping demonstrated in the endemic spread of genetically closely related strains. Our results provide a new possibility for utilizing molecular techniques to control antimicrobial resistance in our region, and prudent antibiotic administration may help limit infection spread. These findings may aid in the improvement of infection control and clinical treatment protocols to lessen the impact of these infections on hospitalized patients.