Antimicrobial resistance trends among important clinical pathogens reported from the ARINCQ surveillance of bacterial resistance, 2012-2018: multi-center retrospective surveillance study in Chongqing


 Background: Data on the Antimicrobial resistance (AMR) trends of the important clinical isolates in southwestern China is lacking. This study was conducted to monitor the temporal AMR trends of the most common pathogens contributing to the majority of human infections.Methods: A multi-center retrospective surveillance study was conducted from 2012 to 2018. Antimicrobial susceptibility testing was carried out according to unified protocols using the Kirby-Bauer method or automated systems. Results were analyzed according to Clinical and Laboratory Standards Institute (CLSI) 2018 definitions. Data from the China Antimicrobial Resistance Surveillance System (CARSS) in Chongqing (ARINCQ) was analyzed by WHONET 5.6 software. Results: The total number of bacterial isolates was between 49,636 and 128,460 annually. While the isolation rate of Acinetobacter baumannii (A. baumannii) presented decreasing trend, that of Staphylococcus aureus (S. aureus) showed increasing tendency. Escherichia coli (E. coli) showed decreasing susceptibility trends to most antibiotics except carbapenems. Resistance rates of Klebsiella pneumoniae (K. pneumonia) to ceftazidime (CAZ), ceftriaxone (CRO), and cefepime (FEP) are decreasing over time. Pseudomonas aeruginosa (P. aeruginosa) and S. aureus demonstrated obvious declining resistance trends to most of the antibiotics tested. Resistance of Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) to high-level gentamycin are decreasing continuously over time. The resistance rate of A. baumannii to meropenem increased from 48.3% to 57.7%. The isolation rate of carbapenem-resistant K. pneumonia (CRKPN) increased annually from 2.6% in 2012 to 6.1% in 2018. For CRKPN, consistent increasing trends in isolation rates were witnessed in both the children and the elderly groups, with its isolation rate in children being obviously higher than that in the elderly group from 2015 to 2018.Conclusions: The prevalence of CRKPN has been significantly increased in both the children and the elderly groups ever since 2012, which calls for continuous resistance surveillance, colonization screening and clearance, environmental monitoring, and effective antimicrobial strategies. While carbapenems are still active against K. pneumonia and E. coli, vancomycin or linezolid is still effective against S. aureus and Enterococcus spp. Notably, this is the first report of an CRKPN isolate co-harboring triple carbapenemase genes including blaKPC-2, blaNDM-1 and blaIMP-4 worldwide. Keywords: antimicrobial resistance, surveillance, multi-center, carbapenems, Southwestern China, carbapenem-resistant K. pneumonia

harboring triple carbapenemase genes including bla KPC-2 , bla NDM-1 and bla IMP-4 worldwide. To prevent geographical bias, we successively enrolled hospitals in a geographical representative manner, and 59 hospitals have been enrolled in ARINCQ, almost covering all the regions and districts of Chongqing.
Worldwide AMR changing tendencies were reported one after another [1,2,3,4], and the comprehensive and overall data of temporal AMR shifts of important clinical isolates in China was only reported by CHINET, which just included seventeen teaching hospitals [1,5]. We searched pubmed with the terms "antimicrobial resistance of nosocomial pathogenes", and the retrieval results consisted of larger national surveillance studies, reviews, or single case studies, with no results of the studies including comprehensive Chongqing coverage. In addition, the longitudinal province-level AMR surveillance in China was limited, and was mostly about the AMR changing trends of isolates from bloodstream and intra-abdominal infections [6,7,8,9,10]. The overall annual AMR changing trends over long time periods are still lacking in southwestern China. The present study was initiated to provide clear and comprehensive AMR changing trends for the important nosocomial pathogens from 2012 to 2018 in southwestern China, and meanwhile to deeply explore into the cause of the key problems discovered.

Data enrollment criteria
All bacterial data including patients' information, bacterial identification and antimicrobial susceptibility from 59 microbiological laboratories in Chongqing, covering secondary and tertiary hospitals with a ratio of 1.5:1, were acquired from CARSS database in Chongqing (ARINCQ) between 2012 and 2018. Children were defined as patients no older than 14-year-old, adults were defined as patients between 15 and 65 years old, while the elderly were defined as patients older than 65 years old.
Only the first isolate from each individual patient was included in this study.

Bacterial identification and antimicrobial susceptibility test
All the isolates were identified at the species level by semi-or automated systems, such as the VITEK2 compact (bioMérieux, Inc., Durham, NC) system, or the VITEK MS (bioMerieux, Hazelwood, MO, United States) automated system, and so on. Routine antimicrobial susceptibility testings were performed by using either the API system, the BD Phoenix System (Becton Dickinson, America), or the VITEK2 compact (bioMérieux, Inc., Durham, NC) system. According to the breakpoint recommendations by the Clinical and Laboratory Standards Institute, 2018 (CLSI-2018), K.
pneumoniae or E. coli isolates which were resistant to at least one of the carbapenems, with the criteria of minimum inhibitory concentration (MIC) of ≥ 2µg/mL for ertapenem, ≥ 4 µg/mL for imipenem, or ≥ 4µg/mL for meropenem, were defined as carbapenem-resistant Enterobacteriaceae (CRE); while carbapenem-resistant A. baumannii (CRABA) or P. aeruginosa (CRPAE) strains were defined as MIC≥8µg/mL for imipenem or meropenem.

Detection of Carbapenemase genes
Polymerase chain reaction was used to detect the potential presence of carbapenemase genes including bla KPC , bla NDM , bla VIM , bla IMP , bla GES , bla OXA-48-like , and bla OXA-181-like , and all the variants of these carbapenemase genes were confirmed by sequencing. Moreover, the Carba NP test and sCIM were performed on all isolates to determine whether any bacteria produced carbapenemases by phenotypic methods but were negative by genotypic methods, or vice versa [11].

Statistical analyses
Raw data was firstly processed by WHONET 5.6 software (version 5.6, http://www.whonet.org/ software.html) and then analyzed on SPSS v.23.0 (SPSS, Chicago, IL, USA) software. The changing trends in AMR of each species and in the isolation rates of the main resistance phenotypes over the study period were determined using the linear trend analysis method. A statistically significant trend was established if the P value was <0.05. The P value was provided whenever there was a significant trend in the resistance. Moreover, the regression coefficient was calculated and indicated the change in the percentage of resistant isolates (on the y-axis) over time (on the x-axis) [12,13]. A negative slope (−) indicated a decrease in resistance over time, whereas a positive slope (+) presented an increase in resistance over time. The difference in AMR between children and non-children was further assessed by Pearson Chi-square test. Statistical significance was confirmed if a two-tailed P value was no more than 0.05.

Changing trends of the isolation percentages of the main clinical pathogen species
Between 2012 and 2018, the number of non-repetitive bacterial isolates ranged between 49,636 and 128,460 annually. The ratio of specimen types during the study period did not change. The percentages of the seven investigated species (E. coli, K. pneumoniae, P. aeruginosa, A. baumannii, S. aureus, E. faecium, and E. faecalis) among total isolates were shown in Table 1. While the annual total percentage of the seven pathogen species among the total isolates didn't change much, accounting from 60.2 to 63.4%, the percentage of A. baumannii showed a slowly decreasing trend, whereas the ratio of S. aureus presented a slowly increasing tendency (Table 1).

P. aeruginosa and baumannii complex
The resistance rates of P. aeruginosa to all the eleven investigated antibiotics were declining with statistical significance. The AMR rates of P. aeruginosa to all the antibiotics tested were less than 26% during the study period and all were below 19% in 2018 (Table 4).   (Table 6).

E. faecalis and E. faecium
The resistance rates of E. faecalis to VAN

Isolation rates of CRKPN and CRECO according to different age groups
As for CRKPN isolation rates, all the three groups witnessed statistically annual increasing trends over  and 5% (4/79), respectively. bla VIM, bla GES, and bla OXA were not detected in this study. 11.4% (9/79) of the strains harbored two or three types of carbapenemase genes (bla KPC-2 and bla NDM-1 , n=7;

Discussion
While a nationwide multi-center study in Korea showed increased AMRs to 3rd CEPs both for ECO and KPN from 2004 to 2012 [14], our study demonstrated gradual decreasing resistance tendencies for most antibiotics including third and fourth-generation cephalosporins (3 rd and 4 th CEPs), which was congruent with a previous report from CHINET in China which declared decreasing resistance trends to 3 rd and 4 th CEPs for ECO and KPN during 2012-2017 [1].
The emergence of CRE raises a global health-care threat. CRE infections are related to high mortality because therapeutic options are very limited [15,16,17,18]. As to the isolation rates of carbapenemresistant Enterobateriaceae, that of CRKPN was much higher than that of CRECO according to Figure   2. Although the isolation rate of CRKPN in western China was lower compared with that from CHINET [1], the increasing CRKPN isolation rates are still noteworthy, especially more attention should be paid to the CRE isolated from children and the elders.
To further clarify the epidemiology of CRKPN in different patient age cohorts, the annual changing trends of isolation rates of CRKPN with a comparison with those of CRECO stratified by age were shown in Figure 3 and 4. It could be concluded that the isolation rates of CRKPN in both the children and the elders increased consistently over time, which contributed significantly to continuous increasing annual CRKPN isolation rates. Notably, more attention should be paid to CRKPN in children its isolation rates in children were approximately twice higher than those in adults from 2015 to 2018.
Carbapenemase-producing Enterobacteriaceae (CPE) is the most pervasive antibiotic resistance threat to health services worldwide. The most globally widespread carbapenemase genes are typically carried on mobile genetic elements which can be freely exchanged between bacterial strains and  [19].
As to the geographical distribution of the carbapenemase genes, bla NDM was predominant in the middle of Chongqing. The differential compositions of carbapenemase gene types in different areas in Chongqing might due to nosocomial spread or un-rational uses of anibiotics in different regions.
To our relief, the AMR of P. aeruginosa in Chongqing all showed decreasing tendencies, which was consistent with the results from CHINET [1]. Though significant decreases in resistance trends were also seen in P. aeruginosa to amikacin and gentamicin in a 10-year study from 2005 to 2014 in the third largest tertiary healthcare university hospital in Serbia, significant increasing trends were witnessed in resistance rates to imipenem and meropenem [20]. In order to comprehensively understand the antibiotic resistance situation in P. aeruginosa, more detailed hierarchical researches of P. aeruginosa should be executed.
While important temporal decreases in susceptibility rates among A. baumannii complex isolates were observed for all the antimicrobial agents tested from medical centers enrolled in the SENTRY Program [21], our study demonstrated an approximate 10 percent increase of the annual resistance rates of A.
baumannii complex to meropenem, which was similar to that of a multi-center study report by K.  Declarations the version to be published, and agree to be accountable for all aspects of the work. SS, SQN and SFH designed this study, participated in statistic calculations and interpretation of data, and drafted this manuscript. LY,CMZ and JDS collected the raw data of ARINCQ by Whonet 5.6.

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