Pathogens Distribution and Antimicrobial Resistance of Bloodstream Infections at Twenty-Five NICUs in Eastern China, 2016-2019

To describe pathogens distribution and their antimicrobial resistance (AMR) of neonatal bloodstream infections (BSIs) at 25 NICUs in eastern China, 2016-2019. Study We conducted a multicentre retrospective descriptive analysis of all positive blood cultures collected at 25 NICUs of between 2017, and December 31, 2019. From 1 January 2017 to 31 December 2019, a total of 1092 pathogenic bacteria isolates were met inclusion criteria among 66516 specimens. 349 (32%) isolates were responsible for early-onset sepsis (EOS), 702 (64.2%) hospital-acquired late-onset sepsis (HALOS), and 41(3.8%) community-acquired late-onset sepsis (CALOS). The majority isolates causing EOS (198/349, 56.7%) and HALOS (437/702, 62.2%) were gram-negative bacteria; The most frequent pathogens causing EOS were Escherichia coli (95/349, 27.2) and group B streptococcus (GBS; 51/349, 14.6%). E coli were primarily identied among preterm infants (58/95, 61.1%) with GBS primarily among term infants (43/51, 84.3%). GBS and Staphylococcus aureus were responsible for 46.3% (19/36) and 41.5% (17/36) of episodes of CALOS. Isolates causing HALOS were more often resistant than isolates causing EOS and CALOS. K. pneumoniae (196/702, 27.9%) as the most common isolates responsible for HALOS were more likely to be resistant to third-generation cephalosporins (124/196, 63.3%), gentamicin (41/196, 20.9%) and carbapenem (26/196, 13.3%).


Background
Neonatal bloodstream infections (BSIs) is the third most common cause of neonatal morbidity and mortality, and is an ongoing major global public health challenge (1,2). Asia and Africa have the highest burden of BSIs in the world (2). Higher burden of infectious diseases, insu cient surveillance and infection control, abuse of antibiotics contribute to this situation (3,4). As an urgent threat to neonatal health, antimicrobial resistance of pathogens is of increasing importance (4). In 2016, the rst estimate interpret that multi-drug resistant pathogens accounted for approximately 30% of global neonatal sepsis mortality (5). Despite the massive burden, few high-quality data are available and few multicenter related research were reported about pathogen distribution and antimicrobial resistance of neonatal BSIs in China.
Previously, BSIs is divided into early onset sepsis (EOS) and late onset sepsis (LOS) and only few reports distinguished between community-acquired LOS (CALOS) and hospital-acquired LOS (HALOS), further (6). EOS generally re ects vertically transmit from mothers while LOS is due to pathogens acquired after delivery and often from nosocomial infections or community environment (7). Therefore, the spectrums of pathogens causing EOS might be different from CALOS and HALOS. In western counties, GBS were the most frequently isolated pathogens of EOS and the rise of E coli warrants in a recent research (8).
The spread of antimicrobial resistance is now a pressing threat in modern hospitals, and it has been reported in both CALOS and HALOS worldwide. However, in low and middle-income countries, antimicrobial resistance becomes more sever. The risk of emergence and spread of antibiotic resistance in the world health organization (WHO) South East Asia region is thought to be among the highest of all the WHO regions (9,10). So, we conducted a multicenter retrospective study to investigate the regionally representative current data on neonatal pathogens and their antimicrobial resistance patterns in east China.

Study participants
A multicenter retrospective analysis was performed on all positive blood cultures obtained at 25 NICUs of tertiary hospitals from January 1, 2017, and December 31, 2019. Among them, twenty-three tertiary hospitals were located in Shandong province involving 13 major cities, one tertiary hospital in Hebei province and the other one in Inner Mongolia Autonomous Region. The 25 participants included 19 general hospitals and 6 maternal and child health care hospitals, with an average of 55 and 35 beds, respectively. Species identi cation was performed at recruited hospital laboratories on automated detection systems using Bactec FX system (Becton Dickinson, USA) in 15 hospitals and BacT/ALERT 3D system (bioMérieux, France) in 10 hospitals. The pathogens were tested for antibiotics susceptibility following standard established by the Clinical Laboratory Standards Institute (11).

De nitions
Neonatal sepsis was de ned as the growth of a single potentially pathogen (bacterium or fungus) from the blood of an infant who has clinically septic according to de ned criteria and had supportive laboratory evidence of sepsis and antibiotic application time greater than 5 days (12,13). All organisms identi ed in positive blood cultures were classi ed as either a likely contaminant or a potential pathogen.
Organism was a pathogen or a contaminant was made by the local neonatologist according to clinical judgment. EOS was de ned as occurrence of sepsis at or before 72h of life while LOS was de ned as occurrence of sepsis after 72 h of life. Among infants with LOS, infants with sepsis onset ≤ 48 hours after admission were considered as having community-acquired LOS, and those with onset > 48 hours after admission were considered as having hospital-acquired LOS (6). Antimicrobial susceptibilities were categorized as susceptible or resistant (intermediate or resistant) based on microbiology reports. Additional blood cultures obtained considered identical bacterial episodes were not included in the analysis of incidence or antimicrobial resistance unless they identi ed a new pathogen or have different antimicrobial resistance. Preterm was de ned as infants with gestation < 37 weeks.

Data Collection and Statistical Analyses
The medical record of each infant with positive blood culture was reviewed by a local neonatologist and data were documented onto a uni ed standardized worksheet. These included the medical institution, medical record number, gestational age, birth weight, gender, date of birth, date of blood cultures obtained, organisms identi ed, clinical signi cance of organisms and antimicrobial susceptibility. Other clinical data were also collected including body temperature, heart rate, white blood cell count (WBC), procalcitonin (PCT) and C-reactive protein (CRP) in 72h before and after blood cultures collected. The worksheet of each infant with positive blood cultures from 25 NICUs were reexamined critically by a neonatologist served as an investigator and a microbiologist.
The ethics committees of all 25 participating hospitals approved the study and allowed data sharing. Procedures were in accordance with the Helsinki Declaration of the World Medical Association.
Frequencies, proportion and summary statistics were used to describe relevant variables.
Pathogen distribution Table II shows the pathogenic bacteria distribution that caused neonatal EOS, community-acquired and hospital-acquired late-onset BSIs. The proportion of pathogens responsible for early-onset BSI was half of hospital-acquired late-onset BSI (32.0% vs 64.3%). GNB accounted for 56.7% of pathogens causing early-onset BSI and 62.2% of hospital-acquired late-onset BSI. E coli and GBS were the most common pathogenic bacteria of EOS, accounting for 27.2% (95 of 349) and 14.6% (51 of 349). 84.3 % of GBS (43 of 51) was identi ed in term infants and 15.7% (8 of 51) in preterm. In contrast, E coli was responsible for 61.1% (58 of 95) of pathogens in preterm, and 38.9% (37 of 95) in term infants.
K pneumonia, the leading pathogen of hospital-acquired late-onset BSI, was involved in 27.9% of pathogens (196 of 702), followed by E coli (15.7%, 110 of 702) and Fungi (12.8%, 90 of 702). 97.8% (90/92) fungi were identi ed responsible for hospital-acquired late-onset BSI. Compared with neonates with early-onset BSI, infants with hospital-acquired late-onset BSI were born at a lower gestational age. K pneumonia primarily collected among preterm infants (135 of 196 [68.9%]). GBS and staphylococcus aureus were responsible for 46.3% (19 of 41) and 41.5 % (17 of 41) of pathogens of community-acquired late-onset BSI. There was a strong gestational age predominance, with all pathogens identi ed from term infant.

Antimicrobial resistance
Overall AMR rates were high, especially among gram negative organisms responsible for hospitalacquired late-onset BSI (Table II)

Discussion
In this multicenter retrospective describing study, we highlights the pathogen distribution and AMR of neonatal early-onset BSI, hospital-acquired late-onset BSI and community-acquired late-onset BSI in China, a lower-middle income country with the paucity of high-quality data. This is one of the few multicenter large samples studies from Asian countries having regional representation and the rst to identi ed key differences between three types of BSI. In our study 32% isolates were responsible for earlyonset BSI, 64.2% hospital-acquired late-onset BSI, and 3.8%community-acquired late-onset BSI. Earlyonset BSI, hospital-acquired late-onset BSI, and community-acquired late-onset BSI have distinct primary pathogens and AMR. High AMR in K. pneumoniae were the main isolates in hospital-acquired late-onset BSI.
In our study, GNB predominated in both early-onset BSI and hospital-acquired late-onset BSI. These data are markedly different from the data from most high-income counties, including the United States, where GBS continues to be the most common cause of early-onset BSI and CONS is responsible for the greatest proportion of late-onset sepsis cases in hospitalized neonates (14)(15)(16). E coli was an important GNP in low-income settings and was the second most common early-onset BSI pathogen in a large US prospective cohort study (17). Similar to recent reports from Chinese and other Asian counties, GNP predominated in the pathogens causing EOS in our study, with the top one E. coli responsible for 27.2% (17,22,23). GBS was second common pathogen of EOS accounting for 14.6%. Previously, GBS was reported to be a rare cause of EOS and was documented in only few reports from Chinese and other Asian countries (6,18,19). A recent worldwide systematic review had the same report that the incidence for infant GBS disease was the lowest in Asia (20). However, GBS infection emerged as the leading cause of EOS in developed counties since 1970s (21). A previous prospective surveillance of a national neonatal research network study in US showed that although GBS remains the most common pathogen of EOS, there has been a shift from GBS to E. coli as the predominant pathogen associated with EOS (22). Similarly, a latest surveillance from the same national neonatal network demonstrated the rising proportion of E. coli (36.6%) and decreasing of the GBS (30.2%) (8). According to this pattern, it can be found that E. coli might have played an increasingly important role in EOS.
We found that K pneumonia was the most frequently pathogen with hospital-acquired LOS, followed by E. coli, which is parallel with the results of a latest study in south China (23). Meanwhile, CoNS was the third common pathogen causing LOS accounting for 11.2%, with majority of identi ed from VLBW. Some previous Chinese studies showed that 54.7% to 67.1% of LOS was caused by CoNS (19,24). It is necessary to eliminate the pollution factors of culture contaminate before making a correct judgment. As shown in our multicenter cohort study, eighty percent of CoNS was identi ed as contaminants. Differences in pathogen distribution in our motherland between different studies may be partially explained by the de nition of CoNS infection. By contrast, CoNS was signi cantly predominant in western countries, 24.2% in Australia, 28.3% in the North America, 31.5% in Greek and 36.5% in Switzerland, most often no more than 40% infections (25). In addition to contaminating factors, the explanations for the difference to these studies is likely to be multifactorial and may include different gestational age strati cation as well as whether central venous catheterization was used in the enrolled infants.
Community-acquired LOS was the most common presentation in term newborns, accounting for 3.8% of all pathogens. E coli and GBS were the leading pathogens. Compared with early-onset BSI and hospitalacquired late-onset BSI, community-acquired late-onset BSI has received little attention, and often is reported in combination with data from infants up to 3 months of age or just being ignored (6).
Antimicrobial resistance pathogens causing neonatal late-onset BSI is a rapidly emerging, potentially disastrous problem (26). For GPB, meticillin resistance was identi ed in 77.8% of CoNS and 80% of S aureus. Simlar to report from Asia, more than half of the Staphylococcus aureus were resistant to methicillin. Research found that the most frequent mechanism of MDR in GNB was due to ESBL production. Similar to that previously reports from Asian NICUs (27,28), approximately half of GNPs were resistant to third-generation cephalosporins. Of particular concern, about 63.3% of K pneumonia was resistant to third-generation cephalosporins and they continue to be the recommended second line treatment for infants with neonatal sepsis in Asian countries. Although this was similar to that previously reported (29), it is worrying as K pneumonia was the most common pathogen and caused most nosocomial infection.
Alternative antibiotics for treatment of neonatal sepsis in the face of such resistance include piperacillin tazobactam, amikacin and carbapenems. However, resistance to these antibiotics of K pneumonia is emerging (30). Resistance to alternative antibiotics is even emerging, seriously limiting our antibiotic options. Accurate diagnosis and e cient therapy are necessary to prevent Antimicrobial resistance. Therefore, knowledge of common bacteriological agent and their resistance pattern for prescription of empiric treatment are necessary. The great difference and overuse of antibiotic in neonates is a worldwide concern, while in China more than two-thirds of inpatient newborns were used antibiotic products in a two year study (31).
Our study has several limitations. First, this is a multi-center retrospective study and no uni ed standard for differentiating contaminants from pathogen exists. The proportion of contaminants may be overstated or also underestimate mainly judged by local neonatologists based on the experiences. Second, our study was not population-based and, although 25 NICUs throughout the east China were included, rendering ndings regional representative, we were unable to calculate the incidence rates. Third, we are not on the basis of two positive blood cultures because empirical antibiotics are usually started after only one set of blood culture is obtained in most centres in China, and may underestimate the proportion of true pathogenic bacteria.

Conclusion
Escherichia coli, K. pneumonia and GBS were the most common pathogens in EOS, hospital-acquired and community-acquired late-onset BSI, respectively. High AMR in K. pneumoniae causing hospital-acquired late-onset BSI warrants continued study and devise measures to prevent it in low-income and middleincome countries.
Abbreviations CONS: Coagulase negative staphylococcus aureus; AMR: Antimicrobial resistance; BSI: Bloodstream infections; GBS Group B streptococcus; EOS: early-onset sepsis; HALOS: hospital-acquired late-onset sepsis; CALOS: community-acquired late-onset sepsis; WHO: World health organization Declarations JL, played a role in the analysis and interpretation of the data and in preparing and drafting the manuscript. The co-rst authors, participated in the design of the study, the collection and interpretation of the data and writing the manuscript. All authors listed on the manuscript approved the submission of this version of the manuscript and take full responsibility for the manuscript.

Availability of data and materials
The data that support the ndings of this study are available from the corresponding authors upon reasonable request.

Ethics approval and consent to participate
The Institutional Review Board of Shandong Provincial Hospital A liated with Shandong University approved this project. All authors have signed written informed consent and approved the submission of this version of the manuscript and take full responsibility for the manuscript. The legal guardian of all participants signed an informed consent form that their data could be used for various clinical studies.