Temporal association between antibiotic use and resistance in Gram-negative bacteria

Abstract The β-lactam/lactamase inhibitors (BLBLIs) combination drugs are considered an effective alternative to carbapenems. However, there is a growing concern that the increased use of BLBLIs may lead to increased resistance. This study determined the temporal association between the consumption of BLBLI and the antimicrobial resistance in Gram-negative bacteria. In this retrospective study, electronic data on the Gram-negative bacterial isolates, including A. baumannii, P. aeruginosa, E. coli, and K. pneumoniae from in-patients and susceptibility testing results were retrieved from the medical records of the clinical laboratory. A linear regression and cross-correlation analysis were performed on the acquired data. Increasing trends (p<0.05) in the consumption of BIBLI and carbapenem with a median use of 27.68 and 34.46 DDD/1000 PD per quarter were observed, respectively. A decreased trend (p=0.023) in the consumption of fluoroquinolones with a median use of 29.13 DDD/1000 PD per quarter was observed. The resistance rate of K. pneumoniae was synchronized with the BIBLI and carbapenem consumptions with a correlation coefficient of 0.893 (p=0.012) and 0.951 (p=0.016), respectively. The cross-correlation analysis against the consumption of BIBLI and meropenem resistant K. pneumoniae was peaked at 0-quarter lag (r=951, p=0.016). There was an increasing trend in the consumption of BLBLI and carbapenems. The increasing trend in the rates of resistance to piperacillin/tazobactam, in line with the increasing consumption of BLBLI, suggests that BLBLI has to be used with caution and cannot be directly considered as a long-term alternative to carbapenems.

antibiotic resistance Gram-negative bacteria is still unclear.Our study aimed to determine the association between the consumption of BLBLI, carbapenems, third-generation cephalosporin's, fluoroquinolones, and antibiotic resistance (representative antibiotics of each group) in Gram-negative bacteria, including A. baumannii, E. coli, P. aeruginosa, and K. pneumoniae.It also determined the temporal association between the consumption of BLBLI and the antimicrobial resistance in Gram-negative bacteria for better decision making on the appropriate use of antibiotics.

Methods
This retrospective study was conducted in The Third Xiangya Hospital and Changsha Central Hospital, China between January 2016 and December 2019.Prior to the study, ethics committee approval was obtained from the hospital ethics committee .Electronic data on the Gram-negative bacterial isolates, including A. baumannii, E. coli, P. aeruginosa, and K. pneumoniae from in-patients and susceptibility testing results were retrieved from the medical records of the clinical laboratory.Only the nonrepetitive Gram-negative bacteria data were considered for this analysis.API 20E kits (Biomérieux, France) identified isolates.Antimicrobial susceptibility testing was done by Kirby-Bauer Disk Diffusion method according to guidelines of Clinical and Laboratory Standards Institute published in the respective years of testing.Antibiotic consumption rate was acquired for BIBLI, carbapenem, third-generation cephalosporin and fluoroquinolones class of drugs.Antibiotic consumption was acquired as per WHO 2012 anatomic therapeutic chemical (ATC) classification in DDD/1000 patient-days (PD) (WHOCC, 2019).Since the antibiotic resistance mechanism is similar among the same classes of antibiotics, the ATC classification is widely used to measure the relationship between antibiotic resistance and antibiotic exposure by bacterial isolates (Goossens, 2009).For this study, resistance data for one antibiotic representing each group was used for correlation analyses; piperacillin/tazobactam represented BIBLI, meropenem represented the carbapenems, ceftazidime represented the third-generation cephalosporin's and ciprofloxacin represented the fluoroquinolones.
A linear regression analysis and cross-correlation function analysis were performed.Linear regression analysis was performed to evaluate the trends of antibiotic consumption and antibiotic resistance.A p-value of <0.05 and the R square value of >0.3 was considered as statistically significant.Cross-correlation function analysis performed to assess the association between the quarterly isolation rate of antibiotic susceptibility and a quarterly number of antibiotic usage.The maximum positive correlation coefficient, the lag quarter and its P value were obtained from cross-correlation function analyses.Statistical analyses were performed using MINITAB Ver. 13 (PA, USA) statistical software.

Antibiotic consumption
Increasing trends (p<0.05) in the consumption of BIBLI and carbapenem with a median use of 27.68 and

Introduction
Gram-negative bacteria, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae are associated with nosocomial infections, including urinary tract infections, bloodstream infections, catheter-associated infections and ventilator-associated pneumonia (Peleg and Hooper, 2010).The increasing drug resistance towards various antibiotics leads to severe life-threatening conditions and poses a challenge in the treatment of Gram-negative bacterial infections.In addition, the emergence of multi-drug resistant (MDR) Gram-negative bacteria adds to the burden of treatment challenges and increased morbidity and mortality (Gurjar et al., 2013;Righi et al., 2017;Shah and Shah, 2015;Yang et al., 2021).By 2050, in the Asia-Pacific region, an estimated 4.73 million deaths will occur annually due to global antimicrobial-resistant and will lead to a comprehensive economic loss of up to $100 trillion (Shah and Shah, 2015;Teillant et al., 2015).Beta-lactam antibiotics were the drug of choice for the treatment of Gram-negative bacterial infections.However, the emergence of extended-spectrum β-lactamase (ESBL) producing microorganisms raised a global alarm.Subsequently, carbapenems were introduced and effectively used against ESBL producing and MDR Gram-negative bacteria (Livermore and Woodford, 2006;Mansy et al., 2020).Worldwide, MDR organisms, including A. baumannii, P. aeruginosa, ESBL-or carbapenemaseproducing Enterobacteriaceae have been increasingly reported (Peleg and Hooper, 2010).Reports on carbapenem resistance have raised serious concern and indicated the limited therapeutic options available for the treatment (Lee et al., 2005).To overcome carbapenem resistance, β-lactam/lactamase inhibitors (BLBLIs) combination drugs such as piperacillin/tazobactam and ampicillin/sulbactam were introduced to increase the efficacy of β-lactam antibiotics (Drawz and Bonomo, 2010).However, there is a growing concern that the increased use of BLBLIs may lead to increased resistance.
Studies evaluating the association of antibiotic consumption and resistance are as old as the antibiotics.They are affected by several parameters, including the extent of prior antibiotic exposure, control group selection, measurements of resistance outcomes and inadequate adjustment for important confounding variables (Schechner et al., 2013).Thus, several indicators were used to measure antibiotic use; however, it is still debatable on which is the best.Three different metrics viz., (i) defined daily dose (DDD), (ii) days of therapy (DOT), (iii) length of therapy (LOT) are available for the measurement of antibiotic use.However, the World Health Organization (WHO) proposed DDD as the commonly used metric; it is expressed as DDDs per 100,000 population (for outpatient use) and DDDs per 1,000 patient-days (for in-patient use) (WHOCC, 2013).The DDD metrics method is easy to collect, no patientlevel data is required, it allows standardized comparisons between drugs and between settings irrespective of route.The other two metrics require patient-level data to be collected (Schechner et al., 2013).Hence, in our study, we have adapted the DDD metrics to measure antibiotic use.There is a general agreement that the increased use of antibiotics will lead to increased resistance (Hsueh et al., 2005).The association between BLBLI consumption and Brazilian Journal of Biology, 2023, vol.83, e239323 3/6 Association of Gram-negative bacterial resistance and antibiotic use 34.46 DDD/1000 PD per quarter were observed, respectively.A decreased trend (p=0.023) in the consumption of fluoroquinolones with a median use of 29.13 DDD/1000 PD per quarter was observed.The consumption of thirdgeneration cephalosporin was stable over the study period (Figure 1).
The resistance rate of K. pneumoniae showed an increased, steadily increased, and decreased trend towards piperacillin/tazobactam, meropenem and ceftazidime over the study period, respectively.The resistance rate of A. baumannii showed an increased and a marginal decrease trend towards ceftazidime and piperacillin/tazobactam, respectively.The resistance rate of P. aeruginosa showed an increased trend towards ceftazidime and piperacillin/ tazobactam.The resistance rate of E. coli showed a decreased trend towards piperacillin/tazobactam while it maintained almost a stable trend against all the other antibiotics included in this study.Ciprofloxacin resistance was almost stable in all microorganisms included in this study (Figure 2).

Correlation and cross-correlation analysis
The resistance rate of K. pneumoniae was synchronized with the BIBLI and carbapenem consumptions with a correlation coefficient of 0.893 (p=0.012) and 0.951 (p=0.016),respectively.E. coli resistance rate showed a negative correlation with the consumption of BIBLI with a correlation coefficient of 0.428 (p=0.047),respectively.E. coli resistance remained stable against ceftazidime and did correlate well with the stable use of third-generation cephalosporin's (r=0.714,p=0.027).The resistance rate of A. baumannii was on an increasing trend against ceftazidime and did not correlate with the stable use of third-generation cephalosporin's (r=0.262,p=0.141).There was a weak correlation between the P. aeruginosa resistance and the consumption of piperacillin/tazobactam (r=0.305,p=0.045) and ceftazidime (r=0.301,p=0.039).The cross-correlation analysis against the consumption of BIBLI and meropenem resistant K. pneumoniae was peaked at 0-quarter lag (r=951, p=0.016) (Table 1).

Discussion
The overuse and misuse of antibiotics is the major cause of dissemination of antibiotic resistance (Zhang et al., 2019).There was an increasing trend (p<0.05) in the consumption of BIBLI with a median use of 27.68 and 34.46 DDD/1000 PD per quarter was observed in our study.Similar results were published in a study from Korea on K. pneumoniae (Ryu et al., 2018).In addition, we reported that there was an increasing trend in the use of carbapenems, which is similar to that reported in China (Hao et al., 2020;Zhang et al., 2019).We reported a median increased trend (p<0.05) in the consumption of carbapenem with a median use of 34.46 DDD/1000 PD per quarter.A study result from China exactly corroborates our findings; the study reported an increasing trend of carbapenem consumption with an average of 32.68 DDDs/1,000 pd (Hao et al., 2020).According to a report from Europe, the average consumption of carbapenem was only 0.05 DDDs/1,000 pd, which is much lesser than that reported in our study (ECDC, 2016).China is considered as the largest antibiotic consumer in the world (Heddini et al., 2009), explains the largest BLBLI and carbapenem consumption compared to European data.Our hospital is a tertiary care centre; handling a large number of critical care patients with multiple diseases could also be another reason for the highest consumption of both BLBLI and carbapenem drugs.
The increasing rate of ESBL producing organisms, including A. baumannii, P. aeruginosa, K. pneumoniae has raised serious concern and limits the clinical therapeutic options (Lee et al., 2005;Paterson and Bonomo, 2005;Peleg and Hooper, 2010).Although carbapenems are considered effective against ESBL producing Gram-negative bacteria, the overconsumption of carbapenems led to ineffective treatment.The BLBLI is considered as an effective alternative to carbapenems (Tamma and Rodriguez-Bano, 2017).However, the increased use of these drugs led to increased resistance, which undermines the efficacy of these drugs  Association of Gram-negative bacterial resistance and antibiotic use (Tamma and Rodriguez-Bano, 2017).Establishing the temporal association between the use of antibiotics and the resistance rates will help in developing appropriate protocol for BLBLI.Several studies have focused on the temporal association of antibiotic use and antibiotic resistance (Goossens, 2009;Hao et al., 2020;Mansy et al., 2020;Ryu et al., 2018).In our study, there was a positive temporal correlation observed between BLBLI utilization and piperacillin/ tazobactam resistance in K. pneumoniae.Our result is in complete agreement with previous studies (Mansy et al., 2020;Ryu et al., 2018).Similarly, our findings are consistent with the results of another study, which reported a significant correlation between the use of piperacillin/ tazobactam and its resistance in Enterobacteriaceae (Lai et al., 2011).Another study reported that Klebsiella sp.showed a significant positive correlation with BLBLI, cephalosporin, and fluoroquinolones, while in our study, there was a positive correlation with only BLBLI and a negative correlation with third-generation cephalosporin use (Wang et al., 2004).Similar to piperacillin/tazobactam resistance, in our study, there was a positive temporal correlation between the carbapenem utilization and meropenem resistance in K. pneumoniae.However, there was no significant association between the use of fluoroquinolones and ciprofloxacin-resistant K. pneumoniae.In our study, there was a significant correlation between the use of third-generation cephalosporin and ceftazidime resistant E. coli.The consumption rate of third-generation cephalosporin was stable; consistent with its consumption, the ceftazidime resistance in E. coli was also found to establish a stable trend.While ceftazidime resistance in K. pneumoniae showed a negative correlation to the use of third-generation cephalosporin.The low level of fluctuation and low magnitude of the utilization of fluoroquinolones could be the possible reason for our results (Peterson, 2005).In contrast, a previous study reported a positive correlation between ceftazidime resistance in K. pneumoniae and the use of third-generation cephalosporin (Mansy et al., 2020).The fluoroquinolone use was in a declining trend, although not significantly correlated the ciprofloxacin resistance in A. baumannii, E. coli, P. aeruginosa and K. pneumoniae were on a slightly increasing trend.These could possibly be due to selection pressure of antibiotic usage on resistant isolates (Mansy et al., 2020;Ryu et al., 2018).The cross-correlation analysis against the consumption of BIBLI and meropenem resistant K. pneumoniae was peaked at a 0-quarter lag.The first study, which determined the temporal association of BIBLI consumption resistance to K. pneumoniae, reported a significant temporal correlation between BLBLI use and piperacillin/tazobactam resistance in K. pneumoniae (Ryu et al., 2018).The strong correlation on the resistant Gram-negative bacteria with the increasing trend in the consumption of BLBLI suggests that BLBLI should be used with caution and cannot be considered as an ideal long-term alternate to carbapenems.As the consumption of BLBLI is on the rise with an increasing trend in the antibiotic resistance, we suggest that BLBLI may be used as along with carbapenems or as carbapenem sparing-option (Peterson, 2005).
The following are the limitations of our study: first, the study was not conducted at the individual level; hence should be considered as an ecological level and may not reflect the biological association at the individual level.Second, this is a bi-centric study; hence the result could probably be based on the local patient population and infection control measures adopted in our hospitals, and one should be cautious in applying these results for the general population of China.Thus, a larger multicentre study involving larger patient population and wide drug classes is required to evaluate the antibiotic stewardship program to balance the use and resistance of Gram-negative microorganisms against different classes of antibiotics.

Conclusion
There was an increasing trend in the consumption of BLBLI and carbapenems.The increasing trend in the rates of resistance to piperacillin/tazobactam, in line with the increasing consumption of BLBLI, suggests that BLBLI should be used with caution and cannot be directly considered as a long-term alternative to carbapenems.

Table 1 .
Cross-correlation analysis between the consumption of BLBLI and antibiotic resistance of Gram-negative bacteria.