Changes in Multidrug-resistant Escherichia Coli of Neonatal Meningitis During 2001–2019: a Study in Eastern China

Background and objective: Neonatal meningitis (NM) caused by Escherichia coli remains a major health problem in industrialized countries. The purpose of this study was to investigate changes in antimicrobial resistance of E. coli causing neonatal meningitis in a perinatal center in eastern China over the past 19 years. Methods: This survey was investigated during three periods: 2001–2006, 2007–2012, and 2013–2019. NM is diagnosed according to the number of white blood cells in the cerebrospinal uid and the presence of a single potential pathogenic bacterium in culture extracted from blood or cerebrospinal uid of any newborn baby. Changes in the antimicrobial resistance of E. coli were analyzed. Results: A total of 209 cases of NM were identied. E. coli was identied from 69 cases, of which extended-spectrum beta-lactamase (ESBL) production was found in 21 cases. E. coli was the main cause of neonatal meningitis in this study. The overall resistance rate of E. coli to third-generation cephalosporins such as cefotaxime increased from 0% in 2001–2006 to 50% in 2007–2012 and subsequently decreased to 29.0% in 2013–2019. This pattern of change is similar to that of ESBL production. Only 15.0% E. coli found in samples from infants with early-onset meningitis (EOM) produced ESBL, while 40.0% of E. coli from children with late-onset meningitis (LOM) produced ESBL. Conclusion: We concluded that E. coli remains the primary pathogen of NM. Compared to LOM, the percentage of ESBL-producing multi-drug resistant E. coli isolated from EOM is signicantly decreased. Clinicians should consider this trend when choosing appropriate and effective antibiotics as empirical treatments for NM.


Introduction
Bacterial meningitis is related to high mortality and morbidity 1,2 . The mortality rates vary between 10% and 15%, especially in the neonatal period 3,4 . Extraintestinal pathogenic E. coli remains one of the most common bacterial pathogens causing extraintestinal infections, including Neonatal meningitis (NM), septicemia, and urinary tract infections [5][6][7] . Early-onset meningitis (EOM) is de ned as infection occurring within 7 days after birth and is usually acquired by vertical transmission from mother to infant. Lateonset meningitis (LOM) is de ned as infection occurring after 7 days post-birth, generally contracted nosocomially or via community infection. Compared to LOM, infants with conditions of prolonged rupture of membranes (PROM) is more prone to develop EOM with worse outcomes because of chorioamnionitis and amniotic uid contamination 8 . E. coli is the second most common pathogen and was found in 30% of all EOM in developed countries 9 .
Symptoms of NM are generally non-speci c, for which rapid recognition and early initiation of antimicrobial therapy before the availability of blood culture results is crucial. In the 1996 national prospective study of Menin-Gideon's disease in newborns in England and Wales, the mortality rate of neonatal meningitis in the acute stage was 6.6% 4 , while this rate was 22% in a similar study conducted in 1985 10 . Despite the overall improvement of neonatal care from 1985 to 1996, the primary difference between the two studies was an increase in the use of third-generation cephalosporins 11 . A retrospective study by Zhao et al, showed that E. coli is still a prominent pathogen of NM. Antibiotic treatment has always been a routine treatment for this type of infection. However, because of the emergence of drugresistant bacteria, the curative effects of antibiotics have decreased. Presently, E. coli exist with different degrees of resistance to third-generation cephalosporins 12 .
Although studies in developed countries have found that Group B Streptococcus (GBS), E. coli, and Listeria monocytogenes are major organisms in the spread of NM [13][14][15] , the results in developing countries may differ. Data about epidemiology and antimicrobial resistance patterns of NM in developing countries are relatively rare, especially from China where the economy rapidly developed since 21st century. Almost all the reported E. coli isolates from Chinese neonates so far, are susceptible to amikacin, cefoperazone sulbactam and carbapenems 16,17 . As changes in multidrug-resistant E. coli strains occur at an increasing rate globally, the spreading of antimicrobial resistant E. coli is now a public health problem and big concern in China. The aim of this study was to investigate the clinical characteristics, antimicrobial resistance patterns of NM caused by E. coli from 2001 to 2019 in a large tertiary neonatal intensive care unit (NICU) in city Wenzhou, located in Zhejiang province of eastern China. Moreover,we compared the ratio of extended-spectrum beta-lactamase (ESBL)-producing E. coli of NM. We focused on comparing the ratio of ESBL-producing E. coli between EOM and LOM infants. Our study will be helpful in selecting more appropriate antibiotics for empirical treatment in developing countries with similar bacterial spectra and sensitivities.

Data Collection
Neonatal cases were de ned as infants 28 days or less of age. All newborns diagnosed with purulent meningitis in the NICU of the Second A liated Hospital of Wenzhou Medical University and Yuying Children's Hospital during the study periods were included in this retrospective cohort study. Sensitivity and speci city test results and ESBL statuses were reported by our clinical laboratory, which conducts routine microbiological examinations according to the standards formulated by the American Clinical and Laboratory Standards Association. Because the present study covered an extended time period, bacterial species were identi ed by either traditional biochemical techniques or automated methods via VITEK system (Vitek 2 compact, BioMerieux, France). Initially, the manual Kirby-Bauer disk diffusion method or the recent gram-negative drug sensitivity card (BioMerieux, France) was used to determine the antibiotic sensitivity of bacterial isolates. This study covered three time periods: [2001][2002][2003][2004][2005][2006][2007][2008][2009][2010][2011][2012], and 2013-2019. All cases were identi ed by registration and hospital diagnosis records and were con rmed by detailed chart reviews.
Relevant clinical data were extracted from the patients' medical records. We collected each infant's gestational age, sex, birth weight, mode of delivery, and instance of fever (> 38℃). To calculate the incidence of NM, data on the number of total live births in the hospital during these three periods were also collected. This research study was approved by the institutional ethics committee of the Second A liated Hospital of Wenzhou Medical College and Yuying Children's Hospital. The patients' parents consent to review their medical records which was allowed by the Institutional Ethics Committee. The treatment of con dentiality of patient data strictly follows the rules formulated by the institution and conforms to the Helsinki Declaration.

Statistical Analyses
SPSS software (version 23.0) was used for statistical analyses. The patients' basic clinical features and blood culture results and the antimicrobial susceptibilities of their relative E. coli strains were analyzed. The Kolmogorov-Smirnov test was used to test the normality of continuous variables. The data of normal distribution are described as means ± standard deviations and were analyzed by Student's t-test of variance. The data with non-normal distribution are described as medians and ranges and were analyzed using the Wilcoxon signed rank test or the Mann-Whitney U-test. Classi cation data were analyzed using the chi-square test or Fisher's exact test. A p-value of < 0.05 of the predicted variable was considered signi cant.

Bacterial pathogens in Neonatal Meningitis
The proportions of different bacterial pathogens causing NM were compared in the three periods as shown in Table 1 thus GBS became the most frequently isolated gram-positive bacteria from 2013 to 2019. Over the past two decades, the proportion of E. coli as the pathogen causing NM remained relatively stable, above 30% in the three periods and was still the primary bacterial pathogen conferring neonatal meningitis. The proportion of enterococcus causing NM decreased, although the differences were not statistically signi cant, similar to Staphylococcus aureus. Besides, the proportion of Klebsiella also remained relatively stable, about 3% in every period.

Distribution of E. coli Neonatal Meningitis From 2001 to 2019
The varies in antibiotic susceptibility of all E. coli strains isolated from infants with NM in the three periods are presented in Fig. 1. As shown in Fig. 1 3.6 Term and premature infants of E. coli Neonatal Meningitis Figure 3 shows the results of the antibiotic susceptibility testing on meningitis-causing E. coli comparing data from term and premature infants. Compared to data from term infants, the composition ratio of E. coli encephalitis in premature infants was signi cantly lower (75.4-24.6%, respectively; p < 0.01). The resistance rate of premature infants with E. coli meningitis to ampicillin was 85.7%, while that of term infants was 63.8%, with no statistical difference. In isolates from premature infants, the resistance rate to levo oxacin was slightly higher than that in term infants (not signi cant, 33.3% vs 20.4%, p = 0.305). Of the E. coli isolates from premature infants, 40.0% were resistant to third-generation cephalosporins (such as cefotaxime), also slightly higher rate than that from term infants (31.3%, p = 0.530). This is similar to ESBL-producing E. coli which represented 40.0% of all E. coli isolated from preterm infants as compared to 30.0% of E. coli in term infants (p = 0.639). In general, the majority of E. coli strains isolated from term and premature infants with meningitis in the studied hospital were sensitive to amoxicillin-clavulanic acid, amikacin, cefoxitin, sulposhen, imipenem and meropenem. Figure 4 shows the results of the antibiotic susceptibility testing on E. coli causing meningitis grouped by EOM vs LOM among neonates. The resistance rate of the late-onset group to second-generation cephalosporins such as cefuroxime was higher than those isolated from early-onset group ( Large cohort studies, NM remains a substantial cause of sepsis-related morbidity and mortality in the term and near-term infant 23 . We described how meningitis-related pathogens have changed from 2001 to 2019 in a city located in China. Bacterial resistance to commonly used antibiotics has become a global problem 24 , and regional differences exist 13,25 . When selecting empirical antibiotics, clinicians should consider local epidemiology (if known), early vs. late disease onset, antimicrobial resistance patterns, and availability within resource constraints 26 .There are relatively rare data about NM from developing countries, and the bacterial pro le may be quite different. We investigated the current research in a large third-class hospital in eastern China over the last two decades. The ndings shows the ratio of meningitis caused by CoNS has signi cantly decreased in the NICU of studied hospital, and the ratio of GBS meningitis has signi cantly increased, although E. coli persists as the mian etiologic agent of NM in the hospital. The most common pathogens(E. coli and GBS) we reported here are similar to those reported by Wiswell et al 27 . A multi-center survey of neonatal purulent meningitis from 13 hospitals in northern China showed that E. coli was the most common pathogen of NM with a rate of 21.1% among the infants of NM 28 , which is lower than that in our study (33%,69/209). We found out that GBS become the rst common pathogen in our region, possibly resulted from the dramatic socioeconomic changes happened in China due to industrialization. Due to industrialization, people are more accustomed to drink milk and thus more maternal colonization in vagina and breast of pregnant women. This usually result in neonatal infection.

Distribution of E. coli causing EOM and LOM
In the past 10 years, the annual deliveries number in our hospital has increased obviously,with coli usually colonizes the maternal reproductive tract and can cause early neonatal infection 29,30 . However, the multidrug-resistant ESBL-producing bacteria observed in a large-scale sub-Saharan Africa study are obtained from polluted hospital environments, which increase the risk of death 18 . Our results are consistent with these ndings. All ve children born at home during our study periods were term infants and were infected with ESBL-negative E. coli. The proportion of ESBL-producing E. coli isolated from infants with EOM was signi cantly lower than that of infants with LOM (15.0% and 40.0%, respectively). In a similar way as presented in the French national survey, EOM from E. coli was statistically less frequent than LOM from E. coli (18% and 33%, respectively, P < 0.01) 31 .Notably, the antimicrobial resistance rate of E. coli isolated from infants with EOM and from infants with LOM increased with regard to third-generation cephalosporins for example cefotaxime (15% and 41.9%, respectively), and to second-generation cephalosporins such as cefuroxime (15% and 43.9%, respectively), both the difference were considered signi cant. This may due to ESBL-producing E. coli infection spreads in the community and contaminated hospital environment with the mobile genetic elements of E. coli strains, such as plasmids. A lasted research revealed that ESBL-producing E. coli Loading [MathJax]/jax/output/CommonHTML/jax.js spreaded frequently in households with babies and improving community health was helpful to prevent the spread of ESBL-producing E. coli 32 .
The incidence of NM was between 0.12‰-1‰ in infants carried to term and 3‰ in premature infants 11,33,34 . A regional retrospective study conducted in Sweden from 1987-1996 estimated at 0.3 per 1000 live births 35 . This is in accordance with the UK and Ireland where reported an incidence of 0.38 per 1000 live births 36 . The incidence of NM much higher at 0.8-6.1 per 1000 live births, with a mortality of 40-58% in developing countries 13 .The incidence of culture-con rmed NM in our perinatal center increased from 0. Neonatal E. coli causing meningitis is related to high mortality and morbidity 37,38 . In a French national survey about neonatal bacterial meningitis 444 Cases from 2001 to 2007, reported the neonatal mortality rate of bacterial meningitis was 13%,while the mortality rate was twice as high in preterm (26%) than in term infants (10%),and neonatal mortality rate with E. coli meningitis was 12% (15/123) 31 . In 2015, it was reported that in Britain, the most common cause of neonatal meningitis caused by gram-negative bacteria was E. coli K1, with a mortality rate of 10-15% 39 . As reported from1997-2017 in Sweden, the pathogens with the highest NM mortality rate are gram-negative bacteria, Klebsiella pneumoniae (33%; 2/6), and E. coli (11%; 2/18) 40  Which is similar to the estimated 10% mortality rate of neonatal meningitis in developed countries 13 .
For infected infants < 60 days of age, the World Health Organization recommends using penicillins (such as ampicillin or penicillin) and aminoglycosides such as gentamicin or third-generation cephalosporins (such as ceftriaxone or cefotaxime) 13  Additionally, the proportion of premature infants with E. coli meningitis who were birth weight < 1500 gm in our NICU increased from 4.2-11.4%, which may be the result of the comprehensive actions of many factors. With the general progress of neonatal nursing and the improvement of NICU doctors' clinical skills, an increasing number of extremely premature infants have been successfully treated. In addition, this should related to most maternal immunoglobulins do not cross the placenta before 32 weeks gestation, the extremely preterm infants are at signi cantly higher risk for infections. 44 Furthermore,early initiation of breastfeeding may be protective against infections which due to transfer of immunoglobulin A, however, term infants usually with breastfeeding at home 45 .
Interestingly, E. coli is one of the most common microorganisms producing L-asparaginase, and Lglutamine releases ammonia through enzymatic reaction, which makes E. coli obtain acid resistance. In a recent study, which revealed the divalent cations except magnesium almost completely inhibited L-

Conclusion
In conclusion, we studied the changes in the pattern of drug resistance in neonatal-meningitis-causing E. coli in a large perinatal medical center in Wenzhou located at eastern China. E. coli remains the major cause of neonatal bacterial meningitis despite GBS incidence has signi cantly increased. While the proportion of ESBL-producing multi-drug resistant E. coli in NM isolates has signi cantly decreased in recent 7 years bene t from the compliance and strict use of antibiotics. Therefore, the third-generation cephalosporins on the whole are still effective to E. coli NM. This study covered neonate meningitis cases across 19 years in a single hospital. The study has its limitation, such as the incidence of NM in term and premature infants were not calculated. Another limitation is that only con rmed cases before hospital discharge were calculated, further studies are needed to collect the follow-up as long-term neurologic morbidity and physical disability. The third-generation cephalosporins and penicillin would be a rst option for NM when occured in developing countries each anti infection of E. coli and GBS. Continuous monitoring of antibiotic susceptibility in NM isolates is necessary to ensure the effectiveness of the standard empirical treatment.