Source Tracking of Carbapenem-Resistant Enterobacteriaceae Outbreaks in Neonatal Patients Revealed Rivers Contaminated with NDM Genes

Source Tracking of Carbapenem-Resistant Enterobacteriaceae Outbreaks in Neonatal Patients Revealed Rivers Contaminated with NDM Genes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Source Tracking of Carbapenem-Resistant Enterobacteriaceae Outbreaks in Neonatal Patients Revealed Rivers Contaminated with NDM Genes Jianshjeng Wang, Changfu Yin, Yuanpeng Lv, Weiwei Yang, Peng Zhao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-395537/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Background: Carbapenem-resistant Enterobacteriaceae (CRE) prevalence in neonatal patients continues to rise in china. However, very few epidemiological data are available to uncover the underlying mechanisms for the increase. This study aimed to elucidate their transmission modes and explore potential sources. Methods: The genotypic features of CRE isolates were analyzed by PCR and sequencing. The elucidation of transmission and sources involved in electronic medical record review, environmental cultures, screening cultures, and prospective case-control analysis. The transferability of carbapenem-resistant genes was performed by Conjugation experiments. Results: A total of 450 CRE isolates recovered from 369 infant patients were genotypically characterised. Carbapenemase genes NDM-1, KPC-2 and IMP-4 accounted for 86.22% (n = 388), 8.89% (n = 40) and 4.44% (n = 20), respectively. After implementation of intensified control measures, CRE rectal prevalence rates in both the 0-3 days and the 4-28 days age group were decreased from 15.53% (100/644) and 27.37% (52/190) to 11.78% (51/433) and 19.59% (19/97) ( P = 0.082 and P = 0.148), respectively. Multivariate analysis showed that gastric lavage (odds ratio [OR]) 3.09, 95% confidence interval [CI] 1.49–6.39) and enema (OR 2.84, 95% CI 1.65–4.91) were independent risk factors for rectal colonisation in the 0-3 days age group, whereas Cephalosporins (OR 2.12, 95% CI 1.14–3.94), gastric lavage (OR 3.44, 95% CI 1.44–8.22) and residence in rural area (OR 2.22, 95% CI 1.19–4.13) were independently associated with rectal colonization in the 4-28 days age group. The transmission tracking of neonatal NDM-1 strains outbreaks revealed rivers contaminated with NDM genes. Most isolates with NDM genes could be successfully transferred. Conclusion: Continued vigilance for importations should be maintained to reduce CRE threat. Infectious Diseases Carbapenem-resistant Outbreak Infection prevention River Figures Figure 1 Figure 2 Figure 3 Background The ever-increasing carbapenem-resistant Enterobacteriaceae (CRE) has become an extremely intractable problem. Recent data from the CHINET surveillance system in China showed that imipenem-resistance rate of Klebsiella pneumoniae (KP) increased from 3.0% in 2005 to 25% in 2018 [ 1 ]. The annual upward trend was also obtained from the China Antimicrobial Resistance Surveillance System ( http://www.carss.cn/ ), which further showed that the annual isolation rate of carbapenem-resistant KP (CRKP) in 2014–2019 was 7.2/8.5/9.3/9.1/10.0/10.1 (%) for pediatric and newborn patients. However, very few detailed epidemiological data are available to uncover the underlying mechanisms for the increase. CRE-associated outbreaks in neonatal patients in China had been increasingly reported [ 2 – 4 ]. Among these CRE isolates, NDM-1 producing KP had attracted much attention because KP with this resistant genotype had been considered to be the leading cause of neonatal carbapenem resistant sepsis in China [ 5 ]. How these strains were introduced into healthcare settings and how they transmitted are still largely unknown to date. In this context, we sought to determine the potential sources and the transmission modes of CRE outbreaks in neonatal patients in a tertiary hospital by performing a series of integrated analysis of clinical, epidemiologic, microbiologic and molecular data. Methods Study design and definition The study was conducted in Hebei General Hospita, a 1830-bed tertiary care hospital located in Hebei, China. The study population was stratified by age as follows: 0–3 days old (DO), 4–28 DO, and 29 DO-1 year old (YO) for infant patients. The demographic, clinical, and epidemiological data were collected via electronic medical record review. A CRE case was defined by the isolation of CRE in any biological sample obtained from the patient. Infection and colonization were classified as either community acquired or healthcare associated, according to the criteria established by the U.S. Centers for Disease Control and Prevention (CDC) [ 6 ]. Patients with known CRE acquisition prior to admission or found to be positive in the first 48 h of admission were regarded as imported cases. Microbiological methods Various clinically indicated samples, screening samples (stool, rectal/perirectal swabs and amniotic fluid), and environmental samples were taken to detect isolates with reduced susceptibility to carbapenems. Bacterial identification and susceptibility testing were routinely performed using the Vitek 2 automated system (bioMe´rieux) and interpreted following the criteria of the latest Clinical Laboratory Standards Institute (CLSI) document. If necessary, MALDI-TOF MS was used for species verification. Meropenem and imipenem susceptibilities were also performed by the Kirby-Bauer disk diffusion method for culture screening purpose. Carbapenem resistance was defined as nonsusceptibility to imipenem or/and meropenem or carbapenemase genes positive. E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as quality controls. Molecular methods Resistance genes were performed by PCR and sequencing, which included genes conferring resistance to class A, B, C and D β-lactam agents [ 7 – 9 ], and gene encoding resistance to colistin (MCR-1) [ 10 ]. Bacterial typing was carried out by multilocus sequence typing (MLST) using primers listed in the online databases ( https://bigsdb.pasteur.fr/klebsiella/klebsiella.html for KP and https://bigsdb.pasteur.fr/ecoli/primers_used.html for E.coli). All alleles and STs were determined according to the MLST database of the Institut Pasteur. Clone relationships were performed by enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) fingerprinting [ 11 ]. Plasmid incompatibility group was determined by a PCR-based replicon typing (PBRT) method [ 12 , 13 ]. Conjugation experiments To determine the transferability of carbapenemase genes, a conjugation experiment was carried out in mixed broth cultures at 35°C [ 14 ]. Carbapenemase positive strains served as donors and azide-resistant E. coli J53 was used as recipient. Briefly, freshly cultured carbapenemase positive donor strains and E. coli J53 recipient strain were separately subcultured into fresh nutrient broth with agitation for approximately 4 h, then mixed at a ratio of 1.5:1 in fresh nutrient broth and incubated overnight without shaking. Transconjugants were selected on eosin methylene blue agar containing azide (200µg/ml) and meropenem (1 mg/L) and inspected by PCRs. Intervention measures No special guidelines regarding CRE prevention in China existed due to the lack of evidence-based epidemiology. Basic infection control measures for CRE employed were based primarily on experience with other multi-drug resistant organisms, which included hand hygiene, contact precautions and/or isolation precautions, healthcare staff education, environmental cleaning and disinfection, aseptic procedures, and antimicrobial stewardship policies. When recognizing outbreaks, an infection control task force was immediately formed, and multidisciplinary meetings were frequently conducted to identify additional effective control measures based on clinical observations, epidemiological surveys and molecular evidence. Monthly environmental surveillance cultures, including air sedimentation cultures, hand swab cultures, and environmental swab cultures which involved high-use medical devices, frequently touched common areas, and areas surrounding the patients, were routinely performed in affected wards, especially ICU, maternity, neonatal, and pediatric wards. Amniotic fluid and rectal swabs or feces from pregnant women were also collected during study period. These screenings were most likely based on the following significant assumptions: (i) Prior to 2015, the neonatal ward and ICU were located on the same floor, and both wards experienced the endemicity of KPC-2 KP strains; (ii) Pregnant women with suspected intrauterine infections might carry these bacteria, which transmitted to the newborns during delivery; (iii) Junior medical staff generally rotated on a regular basis between neonatal and pediatric wards, and their movement may be a contributory factor. To confirm whether infant patients carried CRE in the intestine, we initiated a first prospective rectal screening for the presence of CRE on their admission and during hospitalization during the period from 20th of October 2015 to 30th of April 2017. To observe the efficacy of newly instituted prevention measures on the neonatal colonization, a second prospective rectal surveillance for CRE colonization was performed from September 15, 2017 to April 30, 2018. Sampling sites The Hutuo River in Hebei Province starts from Houciao Village in Pingshan County and flows eastward into the Bohai Sea. The Gangnan Reservoir and Huangbizhuang Reservoir, located on the main stream of Hutuo River, are the only water sources for industry, agriculture and daily life in this region. The Huangbizhuang Reservoir, 28 km downstream of the Gannan Reservoir, also receives water from Yehe River (Fig. 1 ). Starting from the Huangbizhuang Reservoir, the Hutuo River runs through 13 counties, 64 towns and 436 villages. These rivers are closely monitored by the local government and do not directly receive untreated human sewage discharge. Water samples were collected at five sites along Hutuo River (Fig. 1 ) during late July and early August of 2017. At each sampling site, 5000 ml of water samples were collected in sterile bottles and transported directly to the laboratory for processing. Statistical analysis Data were presented as the mean ± SD, number, percentage, or frequency. Student’s t test or Mann-Whitney U test and Chi-squared or Fisher’s exact test were used to compare qualitative and categorical variables, respectively. All variables with a P value ≤ 0.1 in the univariate analysis or that were known risk factors for CRE were included in a backward selection multivariable logistic regression model to identify risk factors independently associated with CRE colonization. Odds ratios (OR) with 95% confidence intervals (CI) were calculated. All tests were two-tailed, with a P value of < 0.05 considered statistically significant. All analyses were performed using SPSS software. Results Genotypic characterization of neonatal and pediatric CRE isolates A total of 163 CRE isolates were recovered from clinical samples of 155 infant patients under the age of 1 year admitted in both neonatal and pediatric wards of our hospital during the period from April 2013 to May 2018. CRE prevalence especially occurred in newborns under three days, accounting for 71.78% (117). KP (95.71%, 156) was the most prevalent organism, followed by E.coli (3.07%, 5) (Table 1 ). The most frequently identified carbapenemase gene was NDM-1 (63.19%, 103), followed by KPC-2 (24.54%, 40), and IMP-4 (12.27%, 20). Table 1 Distribution of carbapenem-resistant Enterobacteriaceae (CRE) isolates in infant patients 0–3 days age group 4–28 days age group 29–365 days age group Sequence types of KP No. Sequence types of EC No. Other CRE Sequence types of KP No. Sequence types of EC No. Other CRE Sequence types of KP No. Sequence types of EC No. First stool screening ST13 ST20 ST36 ST2068 2 49 19 41 ST1 1 2 a ST13 ST20 ST36 ST2068 ST37 ST45 3 16 14 20 1 1 ST767 ST167 ST524 1 1 1 1 a ST20 ST36 ST2068 ST2267 8 2 5 1 ST196 ST167 ST4542 1 2 1 Second stool screening ST17 ST23 ST20 33 4 3 ST325 20 ST17 ST4 ST2068 14 4 1 ST325 6 ST17 ST23 ST45 ST685 ST36 1 1 1 1 2 ST398 ST469 ST83 1 1 1 Clinical specimens* ST11 ST13 ST20 ST36 ST39 ST307 ST2068 ST17 ST23 ST1786 29 1 33 13 13 15 4 2 2 1 ST471 ST469 ST325 1 1 1 1 b ST11 ST20 ST36 ST2068 ST17 ST1655 ST307 8 10 2 2 3 1 3 ST325 1 1 a ST11 ST20 ST39 ST36 ST719 ST23 ST307 3 4 1 3 1 1 1 ST325 1 *: Clinical specimens including 147 bronchial aspirates or aspirating sputa, 14 blood samples and 2 purulent fluids were detected positive for carbapenemase-producing Enterobacteriaceae. KP, Klebsiella pneumoniae; EC, Escherichia coli; ECL, Enterobacter cloacae. a: NDM-1 ECL; b: IMP-4 Klebsiella oxytoca. ST11, KPC-2-producing KP; ST307, IPM-4-producing KP; Other STs were NDM-1 producers except for 2 KPC-2 and NDM-1 co-producing KP of ST20 strains. Of the 1,650 patients who were prospectively screened for CRE colonization during the two study periods (948 in the first period and 702 in the second period), 257 patients were identified to be colonized by carbapenem-resistant organisms (CROs) in which 244 cases were carried with NDM-1 producing Enterobacteriaceae strains. Among these colonized patients, 30 (12.3%) had developed pulmonary infections with these organisms. Co-carriage of two NDM-1 strains was found in 43 (17.12%) (26 (15.57%) of 167 in the first period vs 17 (22.08%) of 77 in the second period, P = 0.215) of the 244 CRE patients. CRE rectal prevalence rate in the 29 DO-1 YO groups were significantly decreased from 14.29% (15/105) in the first period to 4.07 % (7/172) in the second period ( P = 0.002). The rectal prevalence rates of NDM-1 strains in both the 0–3 DO and the 4–28 DO groups were decreased, but no statistical significances were reached (15.53%, 100/644 vs 11.78%, 51/433, P = 0.082 and 27.37%, 52/190 vs 19.59%, 19/97, P = 0.148, respectively). In the first period, 193 CRE were isolated, including 182 NDM-1 CPKP of 7 STs, with ST20 (40.11%, 73), ST2068 (36.26%, 66), and ST36 (19.23%, 35) being the 1st, 2nd and 3rd most common types, 8 NDM-1 E.coli of 6 STs and 3 NDM-1 Enterobacter cloacae strains. Although transmission events continued to occur during the second period, isolates of ST types or species were completely different from those in the first period, and all isolates were NDM-1-producers consisting of 65 CPKP of 8 STs with a predominance of ST17 (73.85%, 48) and 29 E.coli of 4 STs with ST325 (89.62%, 26) as the major type (Table 1 ). Neonatal outbreak evolution and infection control measures In April 2013, a sporadic epidemic of NDM-1 ST39 KP clone occurred in neonatal ward, involving 13 cases less than 3 days old. The strain was initially isolated in March 2013 from a 5-month-old child with community-acquired pneumonia in the pediatric ward and might subsequently be transmitted to the neonatal ward via rotating medical staff. In July 2014, KPC-2 ST11 KP and IMP-4 ST307 KP clones were introduced into the ward and then spread rapidly, affecting 40 and 17 neonatal cases, respectively. Despite the implementation of basic infection control practices, especially enhanced environmental disinfection and reinforced hand hygiene before and after patient contact, new cases were still emerging. Routine environmental screening demonstrated a heavy environmental contamination with CROs (Supplementary Table 1 and Supplementary Fig. 1). ERIC analysis of patients’ isolates and environmental isolates recovered from both the neonatal ward and adult ICU showed that some Aci.baumannii strains and ST11 KP clones were, respectively, closely related (Supplementary Fig. 1), suggesting an important route of ‘non-patient transfer’ transmission between wards. So a new ward was opened in another building at the end of 2014 to receive new admissions. Those who had been affected remained in the original ward until they were discharged. Extensive environmental screening cultures were performed one month before and after the opening of the new ward, but did not yield any CROs. In November 2015, the new unit was partially closed to external admissions due to NDM-1 ST20 and ST2068 KP clones related outbreaks. To determine the probable sources of the outbreak strains, 342 stool or perianal samples and 286 amniotic fluid samples collected from 342 pregnant women with suspected intrauterine infection in the maternity ward were screened for CROs, but no isolates were identified to be carbapenem-resistant. Therefore, the likely route of CRO through vertical transmission from the mothers at birth was excluded. The fact that there were no CROs circulating in the maternity ward and that no CROs had been isolated from routine obstetric environmental screenings did not support the transmission from maternity to neonatal ward. Next, we speculated that asymptomatic carriers might bring these organisms into the unit, so active rectal screening cultures were initiated for infant patients under one year old. In October 2016, a new NDM-1 ST36 KP clone emerged and spread, affecting 33 cases. In March 2017, the initial results of rectal cultures were fed back to the ward, emphasizing that the frequent community importations of asymptomatically colonised patients and subsequent transmissions should be considered as key elements for intervention. A series of intensified measures were then instituted and implemented, including enhanced environmental disinfection with chlorine-based compound in frequency and extent, especially the carriers’ surroundings, the reduction of the total beds of the room from 14 to 10, increase of staff numbers, and reinforcement of hand hygiene compliance. Afterward, the number of clinical CRE isolates decreased drastically, and the CROs ceased to be detected from the ward environment. In September and October 2017, two new NDM-1 strains of ST325 E.coli and ST17 KP were separately introduced and silently spread, leading to colonization in 23 and 40 cases, respectively. This suggested that these measures were far from sufficient to avoid silent transmissions. We then separately performed prospective matched case-control studies to determine neonatal risk factors associated with rectal colonization of NDM-1-producing Enterobacteriaceae in the 0–3 DO group and the 4–28 DO group. In the multivariate analysis, we found that gastric lavage and enema were independent risk factors for colonization in 0–3 DO group, whereas gastric lavage was a risk factor independently associated with colonization in 4–28 DO group (Table 2 ). These results strongly suggested that improperly sterilized procedures had probably direct causal role in neonatal colonization and transmission. So strict aseptic procedures were emphasized and monitored to insure stringent compliance in caring for high-risk patients. With the addition of these interventions, sporadic new CRE cases were still identified in the following years, but no outbreaks occurred. Table 2 Risk factors associated with rectal colonization of NDM-1-producing Enterobacteriaceae in neonatal patients Variables 4–28 days age group 0–3 days age group Carbapenem-sensitive n = 44 (%) Carbapenem-resistant n = 71 (%) Univariate analysis P value Multivariate analysis OR (95% CI) P value Carbapenem-sensitive n = 150 (%) Carbapenem-resistant n = 151 (%) Univariate analysis P value Multivariate analysis OR (95% CI) P value Age of admission (d) 9.47 ± 7.48 10.18 ± 7.27 0.222 Gender (male) 90 (62.5) 40 (56.34) 0.385 78 (52) 80 (52.98) 0.913 Gestational age (d) 271.67 ± 25.26 270.30 ± 14.39 0.069 267.77 ± 18.65 250.57 ± 23.81 < 0.001 Birth weight (g) 3306.33 ± 568.21 3239.65 ± 526.20 0.592 3080.97 ± 665.74 2637.55 ± 880.633 < 0.001 Prematurity 10 (6.94) 8 (11.27) 0.282 42 (28) 93 (61.59) < 0.001 Cesarean section 54 (37.5) 43 (60.56) 0.001 2.55 (1.37–4.74) 0.003 66 (44) 100 (66.23) < 0.001 2.92 (1.69–5.05) < 0.001 Pulmonary infection 34 (23.61) 20 (28.17) 0.469 18 (12) 23 (15.23) 0.414 Respiratory distress syndrome 12 (8.33) 5 (7.04) 0.741 96 (64) 122 (80.79) 0.001 Acidosis 1 (0.69) 1 (1.41) 0.552 22 (14.67) 53 (35.10) < 0.001 2.25 (1.17–4.33) 0.016 Hyperbilirubinemia 108 (75) 47 (66.20) 0.176 45 (30) 13 (8.61) < 0.001 Respiration Failure 1 (0.69) 2 (2.82) 0.254 7 (4.67) 38 (25.17) 0.999 31 (20.67) 59 (39.07) < 0.001 Anemia 15 (10.42) 10 (14.08) 0.430 11 (7.33) 18 (11.92) 0.177 β-lactam/β-lactamase inhibitor 34 (23.61) 20 (28.17) 0.469 46 (30.67) 52 (34.44) 0.485 Cephalosporins 52 (36.11) 40 (56.34) 0.005 2.12 (1.14–3.94) 0.017 79 (52.67) 94 (62.25) 0.093 Meropenem 10 (6.94) 6 (8.45) 0.692 2 (1.33) 10 (6.62) 0.019 Oxygen inhalation 26 (18·06) 15 (21.13) 0.590 60 (40) 100 (66.23) < 0.001 Sputum suction 62 (43.06) 36 (50.70) 0.290 108 (72) 143 (94.7) < 0.001 Gastric lavage 11 (7.64) 16 (22.54) 0.002 3.44 (1.44–8.22) 0.005 97 (64.67) 138 (91.39) < 0.001 3.09 (1.49–6.39) 0.002 Nasogastric feeding 12 (8.33) 13 (18.31) 0.032 56 (37.33) 85 (59.29) 0.001 Enema 16 (11.11) 13 (18.31) 0.146 40 (26.67) 91 (60.26) < 0.001 2.84 (1.65–4.91) < 0.001 Endotracheal intubation 7 (4.67) 32 (21.19) < 0.001 Residence in rural area 46 (31.94) 37 (52.11) 0.004 2.22 (1.19–4.13) 0.012 72 (48) 72 (47·68) 0.956 Neonatal Outbreak Tracking Revealed Rivers Contaminated With Ndm Genes A detailed analysis of patient admission data combined with cultures and molecular typing revealed that multiple or repeated independent introductions of diverse NDM-1 producers into the neonatal ward direct from the community through colonized/infected infant patients, especially patients older than 3 days, resulted in continued clone transmission events (Fig. 2 ). None of these colonized cases and their family members had any prior history of foreign travel to, or previous exposure to, known areas of high NDM endemicity. Multivariate analysis showed that residence in rural area was an independent risk factor, so we tried to put patients’ home addresses on a regional map to determine the scope of CRE endemicity in the community. We found that most patients lived in the vicinity of Hutuo River or tributaries where people frequently visited in summer (Supplementary Fig. 2). Significantly increased incidences of carriage with community-acquired NDM-1strains were observed during summers (river contact seasons) compared to winters (no river contact seasons) (16 (24.62%) of 65 vs 15 (12%) of 125, P = 0.026). The obvious seasonality in NDM carriage rates might be, in large part, associated with increased chances of exposure to the rivers. To determine the rivers as the likely sources, extensive water samplings were carried out at sampling sites A, B, C, D and E (Fig. 1 ). A total of 83 CROs were recovered and screened for the presence of common carbapenemase genes (Supplementary Table 2 and Table 3 ). NDM producers were only isolated at sampling sites C, D and E (Fig. 1 ). NDM-1 isolates (Aci.baumannii, Aci.lwoffiii, KP of ST37 and ST1306), NDM-5 E.coli of ST167, IMI-3 E.cloacae, OXA-24 Aci.lwoffiii and blaOXA-24, and blaOXA-51 co-producing Aci.baumannii isolates were detected in the rivers. The antimicrobial susceptibility profiles of water NDM isolates were similar to those of clinical isolates, showing high-level resistance to penicillins and cephalosporins but susceptibility to aminoglycosides and quinolones. ERIC analysis showed that water isolates of Aci.lwoffiii were unrelated to each other, suggesting horizontal plasmid transmission (Fig. 3 A), while ST37 KP and ST167 E.coli isolates recovered from river were genetically related to those isolated from patients (Fig. 3 B and 3 C). Water ST37 isolate harbored extended-spectrum beta-lactamase genes (ESBLs) (SHV-31/CTX-15) and IncX3 replicon, whereas neonatal ST37 isolate possessed TEM-1/SHV-11/CTX-M-14 and A/C replicon. Both water and clinical isolates of ST167 E.coli had the same ESBLs (TEM-1/CTX-15) and IncFII replicon, but colistin resistance gene MCR-1 was detected only in the latter, suggesting that this strain likely originated from the river and had underwent a complex evolutionary process before transmission to humans. Table 3 Distribution of common carbapenemase genes in river isolates Organisms Sources OXA-24 OXA-24 and OXA-51 NDM IMI-3 Acinetobacter baumannii Gangnan Reservoir 1 Gangnan Reservoir downstream 1 1 Yehe River 1 Huangbizhuang Reservoir 1 3 1 Chahe River 2 Acinetobacter lwoffi Yehe River 5 Huangbizhuang Reservoir 1 1 Chahe River 1 Klebsiella pneumoniae Yehe River 1 Chahe River 1 Escherichia coli Yehe River 1 Enterobacter cloacae Chahe River 1 Transferability Of Isolates With Ndm Genes Via Conjugation A total of 57 infant NDM-1 isolates including 31 KP, 24 E.coli, and 2 E.cloacae were tested for conjugation with E.coli J53, 36 isolates were successfully transferred. The conjugation rates of the NDM plasmids in E.coli and KP were 75% (18/24) and 58.06% (18/31), respectively. Water isolates of ST1306 NDM-1 KP and ST167 NDM-5 E.coli, and one NDM-1Aci.lwoffii isolate were able to transfer, while the ST37 NDM-1 KP strain and one Aci.lwoffii strain that co-produced both NDM-1 and OXA-24 failed to transfer. Discussion In the study, we found that multiple independent introductions of diverse NDM-1 strains from the community and the resultant equipment or environmental contamination potentially contributed to their spread in the neonatal ward. Although basic control measures had been strictly implemented throughout the study period, they were fundamentally inadequate to curb their transmission. Intensified measures developed on the basis of a comprehensive understanding of their transmission had proven to be effective. Neonatal outbreak investigation and transmission tracking revealed that the rivers were contaminated with NDM genes and might serve as large NDM pools for community transmission. The neonatal ward had experienced several outbreaks of NDM-1 strains and finally been terminated through multifaceted intervention measures based on more accurate knowledge of the transmission events. The complete success was attributed to the following three key factors: (i) Active rectal surveillance cultures combined with efficient bacterial typing methods could give more complete transmission information that assisted in allowing for timely targeted control measures to be taken. Previously, CRE were detected only based on clinical samples while the transmission caused by colonization was almost completely ignored, leading to environmental contamination. (ii) Another important component in the success of the intervention was to reduce the workload and improve compliance with control measures. (iii) Risk assessment could reveal equipment- or device-associated transmission and enable precise interventions [ 15 – 18 ], even sometimes without any bacterial evidence [ 17 , 18 ]. Our findings indicated that accurate evaluation of the risk factors that had contributed to neonatal acquisitions could identify potential factors for silent transmission and further improve infection control measures. Walsh et al. reported that NDM organisms largely occurred and disseminated in the community primarily through waterborne and foodborne transmission [ 14 ]. The diffusion of NDM-1 or -5 in China was mainly mediated by X3 plasmid [ 19 – 24 ] and their sources in the community were identified as animals [ 19 , 20 ], migratory birds [ 21 ], fresh vegetables [ 22 ], retail meat [ 23 ], multiple commercial farms [ 24 ], and rivers [ 25 , 26 ]. Some NDM strains also carried MCR-1 [ 20 , 23 , 24 ], which originated in animals and had spread to humans [ 10 ]. Despite the widespread presence of NDM genes in the community, there were few reports to assess the relationships between neonatal patients and the NDM contaminated rivers. Our findings revealed vast, unrecognized NDM contaminated rivers accessible to people. KPC-2 bacteria and OXA-23 Aci.baumannii occurred almost exclusively in healthcare settings, while NDM-1 Aci.lwoffii and OXA-24 Aci.baumannii appeared predominantly in river environment. How these strains occurred and evolved in the rivers remained to be further explored. Most NDM isolates were able to transfer their NDM plasmids via conjugation, and they could serve as potential vectors or reservoirs for transferring themselves and/or NDM genes. This suggested that NDM strains in the rivers could be transferred to families where babies could potentially obtain through daily life contact with their family members who acquired after frequent exposure to or contact with NDM contaminated rivers, or through the use of contaminated washing materials (clothes, bed linen, etc.) washed in the same rivers. Once colonized and adapted to the new human environment, these NDM organisms could further propagate or/and transfer their mobile resistance genes to the microbes that inhabited the human body, which were then introduced into healthcare settings or transmitted to their close contacts in the community. Conclusions CRE would become a persistent problem in the future, largely due to the continuing risk of importation pressures from the community. Hence, continued vigilance and comprehensive epidemiology studies specific for institutions or regions are urgently needed to address the future CRE challenge. Abbreviations CRE Carbapenem-resistant Enterobacteriaceae; CRO:Carbapenem-resistant organism; KP:Klebsiella pneumoniae; CRKP:Carbapenem-resistant Klebsiella pneumoniae; ST:sequence type; DO:Days old; YO:Year old Declarations Acknowledgments We are indebted to clinical staff of the hospital for their extensive support in the investigation of outbreaks; and to Yu-Mei Guo from Shijiazhuang Center for Disease Control and Prevention for technical assistance with ERIC-PCR analysis. Authors’ contributions All authors contributed to this work. JSW and CF Y had made substantial contributions to the conception and design of the study, the acquisition, analysis and interpretation of data. JSW had been responsible for critically drafting and revising the manuscript for all critical intellectual content. YPL, WWY and PZ have made substantial contributions to sample collections, experiments, and data acquisition and interpretation. All authors read and approved the final manuscript. Funding This work was supported by Hebei medical applicable technology tracking project (grant number GL2012049). The funding body played no role in the design of the study, in the collection, analysis, and interpretation of data, and in the writing of the manuscript. Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Protocols for collection of samples as well as the experiment plan and all methods were performed in accordance with the guidelines and regulations of Hebei general hospital and approved by the institutional ethical committees. Written informed consent was obtained from participants or parents prior to their involvement. Consent for publication Not applicable. Competing interests The authors of this study declare no commercial relationships and no conflicts of interests. 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Ding Y, Wang Y, Hsia Y, Sharland M, Heath PT. Systematic review of carbapenem-resistant Enterobacteriaceae causing neonatal sepsis in China. Ann Clin Microbiol Antimicrob. 2019;18(1):36. Horan TC, Andrus M, Dudeck MA. CDC/NHSN surveillance definition of health care-associated infection and criteria for specific types of infections in the acute care setting. Am J Infect Control. 2008;36(5):309–32. Queenan AM, Bush K. Carbapenemases: the Versatile-Lactamases. Clin Microbiol Rev. 2007;20(3):440–58. Dallenne C, Da Costa A, Decré D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae. J Antimicrob Chemother. 2010;65(3):490–5. Sidjabat H, Nimmo GR, Walsh TR, Binotto E, Htin A, Hayashi Y, Li J, Nation RL, George N, Paterson DL. Carbapenem resistance in Klebsiella pneumoniae due to the New Delhi Metallo-β-lactamase. Clin Infect Dis. 2011;52(4):481–4. Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, Doi Y, Tian G, Dong B, Huang X, et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016;16(2):161–8. Cartelle M, del Mar Tomas M, Pertega S, Beceiro A, Dominguez MA, Velasco D, Molina F, Villanueva R, Bou G. Risk factors for colonization and infection in a hospital outbreak caused by a strain of Klebsiella pneumoniae with reduced susceptibility to expanded-spectrum cephalosporins. J Clin Microbiol. 2004;42(9):4242–9. Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods. 2005;63(3):219–28. Compain F, Poisson A, Le Hello S, Branger C, Weill FX, Arlet G, Decré D. Targeting relaxase genes for classifification of the predominant plasmids in Enterobacteriaceae. Int J Med Microbiol. 2014;304(3–4):236–42. Walsh TR, Weeks J, Livermore DM, Toleman MA. Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. Lancet Infect Dis. 2011;11(5):355–62. Kim S, Russell D, Mohamadnejad M, Makker J, Sedarat A, Watson RR, Yang S, Hemarajata P, Humphries R, Rubin Z, et al. Risk Factors Associated with the Transmission of Carbapenem-Resistant Enterobacteriaceae via Contaminated Duodenoscopes. Gastrointest Endosc. 2016;83(6):1121–9. Epstein L, Hunter JC, Arwady MA, Tsai V, Stein L, Gribogiannis M, Frias M, Guh AY, Laufer AS, Black S, et al. New Delhi metallo-β-lactamase-producing carbapenem-resistant Escherichia coli associated with exposure to duodenoscopes. JAMA. 2014;312(14):1447–55. Potron A, Bernabeu S, Cuzon G, Pontiès V, Blanchard H, Seringe E, Naas T, Nordmann P, Dortet L. Analysis of OXA-204 carbapenemase-producing Enterobacteriaceae reveals possible endoscopy-associated transmission, France, 2012 to 2014. Euro Surveill. 2017;22(49):17–48. Humphries RM, Yang S, Kim S, Muthusamy VR, Russell D, Trout AM, Zaroda T, Cheng QJ, Aldrovandi G, Uslan DZ, et al. Duodenoscope-Related Outbreak of a Carbapenem-Resistant Klebsiella pneumoniae Identified Using Advanced Molecular Diagnostics. Clin Infect Dis. 2017;65(7):1159–66. Li J, Bi Z, Ma S, Chen B, Cai C, He J, Schwarz S, Sun C, Zhou Y, Yin J, et al. Inter-host Transmission of Carbapenemase-Producing Escherichia coli among Humans and Backyard Animals. Environ Health Perspect. 2019;127(10):107009. Wang Y, Zhang R, Li J, Wu Z, Yin W, Schwarz S, Tyrrell JM, Zheng Y, Wang S, Shen Z, et al. Comprehensive resistome analysis reveals the prevalence of NDM and MCR-1 in Chinese poultry production. Nat Microbiol. 2017;2:16260. Liao X, Yang RS, Xia J, Chen L, Zhang R, Fang LX, Lei F, Song G, Jia L, Han L, et al. High colonization rate of a novel carbapenem-resistant Klebsiella lineage among migratory birds at Qinghai Lake, China. J Antimicrob Chemother. 2019;74(10):2895–903. Liu BT, Zhang XY, Wan SW, Hao JJ, Jiang RD, Song FJ. Characteristics of Carbapenem-Resistant Enterobacteriaceae in Ready-to-Eat Vegetables in China. Front Microbiol. 2018;9:1147. Zhang Q, Lv L, Huang X, Huang Y, Zhuang Z, Lu J, Liu E, Wan M, Xun H, Zhang Z, et al. Rapid Increase in Carbapenemase-Producing Enterobacteriaceae in Retail Meat Driven by the Spread of the bla NDM-5-Carrying IncX3 Plasmid in China from 2016 to 2018. Antimicrob Agents Chemother. 2019;63(8):e00573-19. Xiang R, Zhang AY, Ye XL, Kang ZZ, Lei CW, Wang HN. Various sequence types of Enterobacteriaceae carrying blaNDM-5 gene from commercial chicken farms in China. Antimicrob Agents Chemother. 2018;62(10):e00779-18. Zhao Q, Berglund B, Zou H, Zhou Z, Xia H, Zhao L, Nilsson LE, Li X. Dissemination of bla(NDM-5) via IncX3 plasmids in carbapenem-resistant Enterobacteriaceae among humans and in the environment in an intensive vegetable cultivation area in eastern China. Environ Pollut. 2021;273:116370. Khan H, Miao X, Liu M, Ahmad S, Bai X. Behavior of last resort antibiotic resistance genes (mcr-1 and bla(NDM-1)) in a drinking water supply system and their possible acquisition by the mouse gut flora. Environ Pollut. 2020;259:113818. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-395537","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":38978582,"identity":"6ef24807-5bc7-40c5-9f84-376f5c41906d","order_by":0,"name":"Jianshjeng Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIie2RMQrCQBBFZ1mIzUjaWcwhIoIgBNejCIKVh1hI6wEU75HKYoLFNkHbQJpUqZMbGHKCSSe4r5riPz6fAQgEfhIGIMgwXrh5yjkxV55X9crS+iDMpqXvaPf8INSg+uEiUbjapNQ1qB5Om3shULZcqZa4QZ1wpJcyxbdM/MaIjmKF12MLI4oVO23hExKWuWyLufluRby31udlP0gUIAZN06WcJD8Su/EfwmwgEAj8KV+LQDj9YWqeGQAAAABJRU5ErkJggg==","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jianshjeng","middleName":"","lastName":"Wang","suffix":""},{"id":38978583,"identity":"180a5bc9-37d8-4b30-8b32-603da5c7472a","order_by":1,"name":"Changfu Yin","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Changfu","middleName":"","lastName":"Yin","suffix":""},{"id":38978584,"identity":"5676a94b-1fd2-4387-8001-de228ddb2cd5","order_by":2,"name":"Yuanpeng Lv","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuanpeng","middleName":"","lastName":"Lv","suffix":""},{"id":38978585,"identity":"4700714f-d682-4060-90da-3460a6e473b6","order_by":3,"name":"Weiwei Yang","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weiwei","middleName":"","lastName":"Yang","suffix":""},{"id":38978586,"identity":"7a8b43e8-e8f5-45ea-9561-9b3a17c81009","order_by":4,"name":"Peng Zhao","email":"","orcid":"","institution":"Hebei General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2021-04-05 14:49:38","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-395537/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-395537/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":11412902,"identity":"2471ee0a-3f55-4a1c-97e7-856bf31f4696","added_by":"auto","created_at":"2021-07-13 14:16:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":111622,"visible":true,"origin":"","legend":"Location of sampling sites and home address-based distribution of infant patients with carbapenemase-producing Enterobacteriaceae (CPE). To explore the scope of CPE transmission in the community, neonatal patients’ home addresses were indicated by solid circles and pentacles on the map, which represented hospital-acquired and conmmunity-acquired, respectively. Arrows indicated sample collection points.","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-395537/v2/7136da4a27553d14c2133890.png"},{"id":11412903,"identity":"fbe27728-4b5c-48f7-b6ea-c93b2e34092e","added_by":"auto","created_at":"2021-07-13 14:16:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":269851,"visible":true,"origin":"","legend":"Distribution of infant patients with some represent NDM-1 Klebsiella pneumoniae strains of different STs according to months\n\n","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-395537/v2/fb2aa39a70a874983cb2ea33.png"},{"id":11412900,"identity":"498cfa01-d50f-4dd1-baac-e049841af9d2","added_by":"auto","created_at":"2021-07-13 14:16:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":572977,"visible":true,"origin":"","legend":"Genotypic relationships of carbapenem-resistant organisms from rivers and patients. To observe the genetic relationships between clinical and water isolates, adult CPE isolates were also used for genetic analysis. Dendrogram depicting the genetic relatedness among the isolates was obtained by cluster analysis of ERIC profiles according to Dice’s similarity coefficient and the unweighted pair-group method with arithmetic mean UPGMA (the position tolerance and optimization were set at 1.0 and 1.5%, respectively). Panel A shows NDM-1 producing Acinetobacter spp.. The fecal isolate from a neonatal patient displayed a banding profile distinct from those river isolates. Panel B shows K.pneumoniae isolates of ST37. The river and neonatal patient isolates showed similar banding patterns. Panel C shows NDM-5 producing E.coli isolates. The river and adult patient isolates of ST167 exhibited identical banding patterns.","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-395537/v2/f658bbbec69163b63525c190.png"},{"id":13703619,"identity":"d5e8119a-b701-40ca-bc71-e380c7f322d3","added_by":"auto","created_at":"2021-09-17 13:42:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1122567,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-395537/v2/de9ab1b1-6bb3-489e-b41f-7251c7f4ac24.pdf"},{"id":11413185,"identity":"71bdfaf8-a2a9-41d0-9ee7-60dc7f5d6e96","added_by":"auto","created_at":"2021-07-13 14:19:53","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":1952859,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-395537/v2/eb3ee0b1113993ddc3c7a864.docx"}],"financialInterests":"","formattedTitle":"Source Tracking of Carbapenem-Resistant Enterobacteriaceae Outbreaks in Neonatal Patients Revealed Rivers Contaminated with NDM Genes","fulltext":[{"header":"Background","content":" \u003cp\u003eThe ever-increasing carbapenem-resistant Enterobacteriaceae (CRE) has become an extremely intractable problem. Recent data from the CHINET surveillance system in China showed that imipenem-resistance rate of Klebsiella pneumoniae (KP) increased from 3.0% in 2005 to 25% in 2018 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The annual upward trend was also obtained from the China Antimicrobial Resistance Surveillance System (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.carss.cn/\u003c/span\u003e\u003c/span\u003e), which further showed that the annual isolation rate of carbapenem-resistant KP (CRKP) in 2014\u0026ndash;2019 was 7.2/8.5/9.3/9.1/10.0/10.1 (%) for pediatric and newborn patients. However, very few detailed epidemiological data are available to uncover the underlying mechanisms for the increase.\u003c/p\u003e \u003cp\u003eCRE-associated outbreaks in neonatal patients in China had been increasingly reported [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among these CRE isolates, NDM-1 producing KP had attracted much attention because KP with this resistant genotype had been considered to be the leading cause of neonatal carbapenem resistant sepsis in China [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. How these strains were introduced into healthcare settings and how they transmitted are still largely unknown to date.\u003c/p\u003e \u003cp\u003eIn this context, we sought to determine the potential sources and the transmission modes of CRE outbreaks in neonatal patients in a tertiary hospital by performing a series of integrated analysis of clinical, epidemiologic, microbiologic and molecular data.\u003c/p\u003e "},{"header":"Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and definition\u003c/h2\u003e \u003cp\u003e The study was conducted in Hebei General Hospita, a 1830-bed tertiary care hospital located in Hebei, China. The study population was stratified by age as follows: 0\u0026ndash;3 days old (DO), 4\u0026ndash;28 DO, and 29 DO-1 year old (YO) for infant patients. The demographic, clinical, and epidemiological data were collected via electronic medical record review. A CRE case was defined by the isolation of CRE in any biological sample obtained from the patient. Infection and colonization were classified as either community acquired or healthcare associated, according to the criteria established by the U.S. Centers for Disease Control and Prevention (CDC) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Patients with known CRE acquisition prior to admission or found to be positive in the first 48 h of admission were regarded as imported cases.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMicrobiological methods\u003c/h2\u003e \u003cp\u003eVarious clinically indicated samples, screening samples (stool, rectal/perirectal swabs and amniotic fluid), and environmental samples were taken to detect isolates with reduced susceptibility to carbapenems. Bacterial identification and susceptibility testing were routinely performed using the Vitek 2 automated system (bioMe\u0026acute;rieux) and interpreted following the criteria of the latest Clinical Laboratory Standards Institute (CLSI) document. If necessary, MALDI-TOF MS was used for species verification. Meropenem and imipenem susceptibilities were also performed by the Kirby-Bauer disk diffusion method for culture screening purpose. Carbapenem resistance was defined as nonsusceptibility to imipenem or/and meropenem or carbapenemase genes positive. E. coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as quality controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMolecular methods\u003c/h2\u003e \u003cp\u003eResistance genes were performed by PCR and sequencing, which included genes conferring resistance to class A, B, C and D β-lactam agents [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and gene encoding resistance to colistin (MCR-1) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Bacterial typing was carried out by multilocus sequence typing (MLST) using primers listed in the online databases ( \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bigsdb.pasteur.fr/klebsiella/klebsiella.html\u003c/span\u003e\u003c/span\u003e for KP and \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bigsdb.pasteur.fr/ecoli/primers_used.html\u003c/span\u003e\u003c/span\u003e for E.coli). All alleles and STs were determined according to the MLST database of the Institut Pasteur. Clone relationships were performed by enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) fingerprinting [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Plasmid incompatibility group was determined by a PCR-based replicon typing (PBRT) method [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eConjugation experiments\u003c/h2\u003e \u003cp\u003eTo determine the transferability of carbapenemase genes, a conjugation experiment was carried out in mixed broth cultures at 35\u0026deg;C [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Carbapenemase positive strains served as donors and azide-resistant E. coli J53 was used as recipient. Briefly, freshly cultured carbapenemase positive donor strains and E. coli J53 recipient strain were separately subcultured into fresh nutrient broth with agitation for approximately 4 h, then mixed at a ratio of 1.5:1 in fresh nutrient broth and incubated overnight without shaking. Transconjugants were selected on eosin methylene blue agar containing azide (200\u0026micro;g/ml) and meropenem (1 mg/L) and inspected by PCRs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIntervention measures\u003c/h2\u003e \u003cp\u003eNo special guidelines regarding CRE prevention in China existed due to the lack of evidence-based epidemiology. Basic infection control measures for CRE employed were based primarily on experience with other multi-drug resistant organisms, which included hand hygiene, contact precautions and/or isolation precautions, healthcare staff education, environmental cleaning and disinfection, aseptic procedures, and antimicrobial stewardship policies.\u003c/p\u003e \u003cp\u003eWhen recognizing outbreaks, an infection control task force was immediately formed, and multidisciplinary meetings were frequently conducted to identify additional effective control measures based on clinical observations, epidemiological surveys and molecular evidence.\u003c/p\u003e \u003cp\u003eMonthly environmental surveillance cultures, including air sedimentation cultures, hand swab cultures, and environmental swab cultures which involved high-use medical devices, frequently touched common areas, and areas surrounding the patients, were routinely performed in affected wards, especially ICU, maternity, neonatal, and pediatric wards. Amniotic fluid and rectal swabs or feces from pregnant women were also collected during study period. These screenings were most likely based on the following significant assumptions: (i) Prior to 2015, the neonatal ward and ICU were located on the same floor, and both wards experienced the endemicity of KPC-2 KP strains; (ii) Pregnant women with suspected intrauterine infections might carry these bacteria, which transmitted to the newborns during delivery; (iii) Junior medical staff generally rotated on a regular basis between neonatal and pediatric wards, and their movement may be a contributory factor.\u003c/p\u003e \u003cp\u003eTo confirm whether infant patients carried CRE in the intestine, we initiated a first prospective rectal screening for the presence of CRE on their admission and during hospitalization during the period from 20th of October 2015 to 30th of April 2017. To observe the efficacy of newly instituted prevention measures on the neonatal colonization, a second prospective rectal surveillance for CRE colonization was performed from September 15, 2017 to April 30, 2018.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSampling sites\u003c/h2\u003e \u003cp\u003eThe Hutuo River in Hebei Province starts from Houciao Village in Pingshan County and flows eastward into the Bohai Sea. The Gangnan Reservoir and Huangbizhuang Reservoir, located on the main stream of Hutuo River, are the only water sources for industry, agriculture and daily life in this region. The Huangbizhuang Reservoir, 28 km downstream of the Gannan Reservoir, also receives water from Yehe River (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Starting from the Huangbizhuang Reservoir, the Hutuo River runs through 13 counties, 64 towns and 436 villages. These rivers are closely monitored by the local government and do not directly receive untreated human sewage discharge.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWater samples were collected at five sites along Hutuo River (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) during late July and early August of 2017. At each sampling site, 5000 ml of water samples were collected in sterile bottles and transported directly to the laboratory for processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, number, percentage, or frequency. Student\u0026rsquo;s t test or Mann-Whitney U test and Chi-squared or Fisher\u0026rsquo;s exact test were used to compare qualitative and categorical variables, respectively. All variables with a \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026le;\u0026thinsp;0.1 in the univariate analysis or that were known risk factors for CRE were included in a backward selection multivariable logistic regression model to identify risk factors independently associated with CRE colonization. Odds ratios (OR) with 95% confidence intervals (CI) were calculated. All tests were two-tailed, with a \u003cem\u003eP\u003c/em\u003e value of \u0026lt;\u0026thinsp;0.05 considered statistically significant. All analyses were performed using SPSS software.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGenotypic characterization of neonatal and pediatric CRE isolates\u003c/h2\u003e \u003cp\u003eA total of 163 CRE isolates were recovered from clinical samples of 155 infant patients under the age of 1 year admitted in both neonatal and pediatric wards of our hospital during the period from April 2013 to May 2018. CRE prevalence especially occurred in newborns under three days, accounting for 71.78% (117). KP (95.71%, 156) was the most prevalent organism, followed by E.coli (3.07%, 5) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The most frequently identified carbapenemase gene was NDM-1 (63.19%, 103), followed by KPC-2 (24.54%, 40), and IMP-4 (12.27%, 20).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution of carbapenem-resistant Enterobacteriaceae (CRE) isolates in infant patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"17\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003e0\u0026ndash;3 days age group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c12\" namest=\"c8\"\u003e \u003cp\u003e4\u0026ndash;28 days age group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c17\" namest=\"c14\"\u003e \u003cp\u003e29\u0026ndash;365 days age group\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence types of KP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSequence types of EC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOther CRE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSequence types of KP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSequence types of EC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eOther CRE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e \u003cp\u003eSequence types of KP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c16\"\u003e \u003cp\u003eSequence types of EC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c17\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFirst stool screening\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eST13\u003c/p\u003e \u003cp\u003eST20\u003c/p\u003e \u003cp\u003eST36\u003c/p\u003e \u003cp\u003eST2068\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e49\u003c/p\u003e \u003cp\u003e19\u003c/p\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eST1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eST13\u003c/p\u003e \u003cp\u003eST20\u003c/p\u003e \u003cp\u003eST36\u003c/p\u003e \u003cp\u003eST2068\u003c/p\u003e \u003cp\u003eST37\u003c/p\u003e \u003cp\u003eST45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e3\u003c/p\u003e \u003cp\u003e16\u003c/p\u003e \u003cp\u003e14\u003c/p\u003e \u003cp\u003e20\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eST767\u003c/p\u003e \u003cp\u003eST167\u003c/p\u003e \u003cp\u003eST524\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eST20\u003c/p\u003e \u003cp\u003eST36\u003c/p\u003e \u003cp\u003eST2068\u003c/p\u003e \u003cp\u003eST2267\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e8\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e5\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003eST196\u003c/p\u003e \u003cp\u003eST167\u003c/p\u003e \u003cp\u003eST4542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSecond stool screening\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eST17\u003c/p\u003e \u003cp\u003eST23\u003c/p\u003e \u003cp\u003eST20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eST325\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eST17\u003c/p\u003e \u003cp\u003eST4\u003c/p\u003e \u003cp\u003eST2068\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e14\u003c/p\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eST325\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eST17\u003c/p\u003e \u003cp\u003eST23\u003c/p\u003e \u003cp\u003eST45\u003c/p\u003e \u003cp\u003eST685\u003c/p\u003e \u003cp\u003eST36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003eST398\u003c/p\u003e \u003cp\u003eST469\u003c/p\u003e \u003cp\u003eST83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical specimens*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eST11\u003c/p\u003e \u003cp\u003eST13\u003c/p\u003e \u003cp\u003eST20\u003c/p\u003e \u003cp\u003eST36\u003c/p\u003e \u003cp\u003eST39\u003c/p\u003e \u003cp\u003eST307\u003c/p\u003e \u003cp\u003eST2068\u003c/p\u003e \u003cp\u003eST17\u003c/p\u003e \u003cp\u003eST23\u003c/p\u003e \u003cp\u003eST1786\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e29\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e33\u003c/p\u003e \u003cp\u003e13\u003c/p\u003e \u003cp\u003e13\u003c/p\u003e \u003cp\u003e15\u003c/p\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eST471\u003c/p\u003e \u003cp\u003eST469\u003c/p\u003e \u003cp\u003eST325\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eST11\u003c/p\u003e \u003cp\u003eST20\u003c/p\u003e \u003cp\u003eST36\u003c/p\u003e \u003cp\u003eST2068\u003c/p\u003e \u003cp\u003eST17\u003c/p\u003e \u003cp\u003eST1655\u003c/p\u003e \u003cp\u003eST307\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e8\u003c/p\u003e \u003cp\u003e10\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003cp\u003e3\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eST325\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eST11\u003c/p\u003e \u003cp\u003eST20\u003c/p\u003e \u003cp\u003eST39\u003c/p\u003e \u003cp\u003eST36\u003c/p\u003e \u003cp\u003eST719\u003c/p\u003e \u003cp\u003eST23\u003c/p\u003e \u003cp\u003eST307\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e3\u003c/p\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e3\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003eST325\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"17\"\u003e*: Clinical specimens including 147 bronchial aspirates or aspirating sputa, 14 blood samples and 2 purulent fluids were detected positive for carbapenemase-producing Enterobacteriaceae. KP, Klebsiella pneumoniae; EC, Escherichia coli; ECL, Enterobacter cloacae. a: NDM-1 ECL; b: IMP-4 Klebsiella oxytoca. ST11, KPC-2-producing KP; ST307, IPM-4-producing KP; Other STs were NDM-1 producers except for 2 KPC-2 and NDM-1 co-producing KP of ST20 strains.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOf the 1,650 patients who were prospectively screened for CRE colonization during the two study periods (948 in the first period and 702 in the second period), 257 patients were identified to be colonized by carbapenem-resistant organisms (CROs) in which 244 cases were carried with NDM-1 producing Enterobacteriaceae strains. Among these colonized patients, 30 (12.3%) had developed pulmonary infections with these organisms. Co-carriage of two NDM-1 strains was found in 43 (17.12%) (26 (15.57%) of 167 in the first period vs 17 (22.08%) of 77 in the second period, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.215) of the 244 CRE patients. CRE rectal prevalence rate in the 29 DO-1 YO groups were significantly decreased from 14.29% (15/105) in the first period to 4.07 % (7/172) in the second period (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002). The rectal prevalence rates of NDM-1 strains in both the 0\u0026ndash;3 DO and the 4\u0026ndash;28 DO groups were decreased, but no statistical significances were reached (15.53%, 100/644 vs 11.78%, 51/433, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.082 and 27.37%, 52/190 vs 19.59%, 19/97, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.148, respectively).\u003c/p\u003e \u003cp\u003eIn the first period, 193 CRE were isolated, including 182 NDM-1 CPKP of 7 STs, with ST20 (40.11%, 73), ST2068 (36.26%, 66), and ST36 (19.23%, 35) being the 1st, 2nd and 3rd most common types, 8 NDM-1 E.coli of 6 STs and 3 NDM-1 Enterobacter cloacae strains. Although transmission events continued to occur during the second period, isolates of ST types or species were completely different from those in the first period, and all isolates were NDM-1-producers consisting of 65 CPKP of 8 STs with a predominance of ST17 (73.85%, 48) and 29 E.coli of 4 STs with ST325 (89.62%, 26) as the major type (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eNeonatal outbreak evolution and infection control measures\u003c/h2\u003e \u003cp\u003eIn April 2013, a sporadic epidemic of NDM-1 ST39 KP clone occurred in neonatal ward, involving 13 cases less than 3 days old. The strain was initially isolated in March 2013 from a 5-month-old child with community-acquired pneumonia in the pediatric ward and might subsequently be transmitted to the neonatal ward via rotating medical staff. In July 2014, KPC-2 ST11 KP and IMP-4 ST307 KP clones were introduced into the ward and then spread rapidly, affecting 40 and 17 neonatal cases, respectively. Despite the implementation of basic infection control practices, especially enhanced environmental disinfection and reinforced hand hygiene before and after patient contact, new cases were still emerging. Routine environmental screening demonstrated a heavy environmental contamination with CROs (Supplementary Table\u0026nbsp;1 and Supplementary Fig.\u0026nbsp;1). ERIC analysis of patients\u0026rsquo; isolates and environmental isolates recovered from both the neonatal ward and adult ICU showed that some Aci.baumannii strains and ST11 KP clones were, respectively, closely related (Supplementary Fig.\u0026nbsp;1), suggesting an important route of \u0026lsquo;non-patient transfer\u0026rsquo; transmission between wards. So a new ward was opened in another building at the end of 2014 to receive new admissions. Those who had been affected remained in the original ward until they were discharged. Extensive environmental screening cultures were performed one month before and after the opening of the new ward, but did not yield any CROs.\u003c/p\u003e \u003cp\u003eIn November 2015, the new unit was partially closed to external admissions due to NDM-1 ST20 and ST2068 KP clones related outbreaks. To determine the probable sources of the outbreak strains, 342 stool or perianal samples and 286 amniotic fluid samples collected from 342 pregnant women with suspected intrauterine infection in the maternity ward were screened for CROs, but no isolates were identified to be carbapenem-resistant. Therefore, the likely route of CRO through vertical transmission from the mothers at birth was excluded. The fact that there were no CROs circulating in the maternity ward and that no CROs had been isolated from routine obstetric environmental screenings did not support the transmission from maternity to neonatal ward. Next, we speculated that asymptomatic carriers might bring these organisms into the unit, so active rectal screening cultures were initiated for infant patients under one year old.\u003c/p\u003e \u003cp\u003eIn October 2016, a new NDM-1 ST36 KP clone emerged and spread, affecting 33 cases. In March 2017, the initial results of rectal cultures were fed back to the ward, emphasizing that the frequent community importations of asymptomatically colonised patients and subsequent transmissions should be considered as key elements for intervention. A series of intensified measures were then instituted and implemented, including enhanced environmental disinfection with chlorine-based compound in frequency and extent, especially the carriers\u0026rsquo; surroundings, the reduction of the total beds of the room from 14 to 10, increase of staff numbers, and reinforcement of hand hygiene compliance. Afterward, the number of clinical CRE isolates decreased drastically, and the CROs ceased to be detected from the ward environment.\u003c/p\u003e \u003cp\u003eIn September and October 2017, two new NDM-1 strains of ST325 E.coli and ST17 KP were separately introduced and silently spread, leading to colonization in 23 and 40 cases, respectively. This suggested that these measures were far from sufficient to avoid silent transmissions. We then separately performed prospective matched case-control studies to determine neonatal risk factors associated with rectal colonization of NDM-1-producing Enterobacteriaceae in the 0\u0026ndash;3 DO group and the 4\u0026ndash;28 DO group. In the multivariate analysis, we found that gastric lavage and enema were independent risk factors for colonization in 0\u0026ndash;3 DO group, whereas gastric lavage was a risk factor independently associated with colonization in 4\u0026ndash;28 DO group (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results strongly suggested that improperly sterilized procedures had probably direct causal role in neonatal colonization and transmission. So strict aseptic procedures were emphasized and monitored to insure stringent compliance in caring for high-risk patients. With the addition of these interventions, sporadic new CRE cases were still identified in the following years, but no outbreaks occurred.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRisk factors associated with rectal colonization of NDM-1-producing Enterobacteriaceae in neonatal patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003e4\u0026ndash;28 days age group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c12\" namest=\"c8\"\u003e \u003cp\u003e0\u0026ndash;3 days age group\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarbapenem-sensitive\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;44 (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCarbapenem-resistant\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;71 (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnivariate analysis\u003c/p\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eMultivariate analysis\u003c/p\u003e \u003cp\u003eOR (95% CI) \u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCarbapenem-sensitive\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;150 (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCarbapenem-resistant\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;151 (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eUnivariate analysis\u003c/p\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003eMultivariate analysis\u003c/p\u003e \u003cp\u003eOR (95% CI) \u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge of admission (d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.47\u0026thinsp;\u0026plusmn;\u0026thinsp;7.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.18\u0026thinsp;\u0026plusmn;\u0026thinsp;7.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender (male)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90 (62.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40 (56.34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.385\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e78 (52)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e80 (52.98)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.913\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGestational age (d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e271.67\u0026thinsp;\u0026plusmn;\u0026thinsp;25.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e270.30\u0026thinsp;\u0026plusmn;\u0026thinsp;14.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.069\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e267.77\u0026thinsp;\u0026plusmn;\u0026thinsp;18.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e250.57\u0026thinsp;\u0026plusmn;\u0026thinsp;23.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBirth weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3306.33\u0026thinsp;\u0026plusmn;\u0026thinsp;568.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3239.65\u0026thinsp;\u0026plusmn;\u0026thinsp;526.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.592\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3080.97\u0026thinsp;\u0026plusmn;\u0026thinsp;665.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2637.55\u0026thinsp;\u0026plusmn;\u0026thinsp;880.633\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrematurity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (6.94)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (11.27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.282\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e42 (28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e93 (61.59)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCesarean section\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54 (37.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43 (60.56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.55\u003c/p\u003e \u003cp\u003e(1.37\u0026ndash;4.74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e66 (44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100 (66.23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e2.92 (1.69\u0026ndash;5.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePulmonary infection\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34 (23.61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20 (28.17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.469\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e18 (12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e23 (15.23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRespiratory distress syndrome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12 (8.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (7.04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.741\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e96 (64)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e122 (80.79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcidosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (0.69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (1.41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.552\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e22 (14.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e53 (35.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e2.25\u003c/p\u003e \u003cp\u003e(1.17\u0026ndash;4.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHyperbilirubinemia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e108 (75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47 (66.20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.176\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e45 (30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e13 (8.61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRespiration\u0026nbsp;Failure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (0.69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (2.82)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.254\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7 (4.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e38 (25.17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3.46\u003c/p\u003e \u003cp\u003e(1.41\u0026ndash;8.45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcleistocardia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e67 (46.53)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44 (61.97)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.033\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e96 (64)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e83 (54.97)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypocalcemia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (4.17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (4.23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e31 (20.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e59 (39.07)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnemia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15 (10.42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (14.08)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.430\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11 (7.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e18 (11.92)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-lactam/β-lactamase inhibitor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34 (23.61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20 (28.17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.469\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e46 (30.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e52 (34.44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.485\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCephalosporins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52 (36.11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40 (56.34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.12\u003c/p\u003e \u003cp\u003e(1.14\u0026ndash;3.94)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e79 (52.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e94 (62.25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMeropenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 (6.94)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (8.45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.692\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2 (1.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e10 (6.62)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxygen inhalation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26 (18\u0026middot;06)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (21.13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.590\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e60 (40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100 (66.23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSputum suction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62 (43.06)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36 (50.70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e108 (72)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e143 (94.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGastric lavage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11 (7.64)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (22.54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.44\u003c/p\u003e \u003cp\u003e(1.44\u0026ndash;8.22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e97 (64.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e138 (91.39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3.09 (1.49\u0026ndash;6.39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNasogastric feeding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12 (8.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13 (18.31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e56 (37.33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e85 (59.29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnema\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16 (11.11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13 (18.31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e40 (26.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e91 (60.26)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e2.84 (1.65\u0026ndash;4.91)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndotracheal intubation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7 (4.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e32 (21.19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResidence in rural area\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46 (31.94)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37 (52.11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.22 (1.19\u0026ndash;4.13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e72 (48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e72 (47\u0026middot;68)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.956\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \n\u003ch2\u003eNeonatal Outbreak Tracking Revealed Rivers Contaminated With Ndm Genes\u003c/h2\u003e\n \u003cp\u003eA detailed analysis of patient admission data combined with cultures and molecular typing revealed that multiple or repeated independent introductions of diverse NDM-1 producers into the neonatal ward direct from the community through colonized/infected infant patients, especially patients older than 3 days, resulted in continued clone transmission events (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). None of these colonized cases and their family members had any prior history of foreign travel to, or previous exposure to, known areas of high NDM endemicity. Multivariate analysis showed that residence in rural area was an independent risk factor, so we tried to put patients\u0026rsquo; home addresses on a regional map to determine the scope of CRE endemicity in the community. We found that most patients lived in the vicinity of Hutuo River or tributaries where people frequently visited in summer (Supplementary Fig.\u0026nbsp;2). Significantly increased incidences of carriage with community-acquired NDM-1strains were observed during summers (river contact seasons) compared to winters (no river contact seasons) (16 (24.62%) of 65 vs 15 (12%) of 125, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.026). The obvious seasonality in NDM carriage rates might be, in large part, associated with increased chances of exposure to the rivers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the rivers as the likely sources, extensive water samplings were carried out at sampling sites A, B, C, D and E (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A total of 83 CROs were recovered and screened for the presence of common carbapenemase genes (Supplementary Table\u0026nbsp;2 and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). NDM producers were only isolated at sampling sites C, D and E (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). NDM-1 isolates (Aci.baumannii, Aci.lwoffiii, KP of ST37 and ST1306), NDM-5 E.coli of ST167, IMI-3 E.cloacae, OXA-24 Aci.lwoffiii and blaOXA-24, and blaOXA-51 co-producing Aci.baumannii isolates were detected in the rivers. The antimicrobial susceptibility profiles of water NDM isolates were similar to those of clinical isolates, showing high-level resistance to penicillins and cephalosporins but susceptibility to aminoglycosides and quinolones. ERIC analysis showed that water isolates of Aci.lwoffiii were unrelated to each other, suggesting horizontal plasmid transmission (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), while ST37 KP and ST167 E.coli isolates recovered from river were genetically related to those isolated from patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Water ST37 isolate harbored extended-spectrum beta-lactamase genes (ESBLs) (SHV-31/CTX-15) and IncX3 replicon, whereas neonatal ST37 isolate possessed TEM-1/SHV-11/CTX-M-14 and A/C replicon. Both water and clinical isolates of ST167 E.coli had the same ESBLs (TEM-1/CTX-15) and IncFII replicon, but colistin resistance gene MCR-1 was detected only in the latter, suggesting that this strain likely originated from the river and had underwent a complex evolutionary process before transmission to humans.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution of common carbapenemase genes in river isolates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrganisms\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSources\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOXA-24\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOXA-24 and OXA-51\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNDM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIMI-3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eAcinetobacter baumannii\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGangnan Reservoir\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGangnan Reservoir downstream\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYehe River\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuangbizhuang Reservoir\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChahe River\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAcinetobacter lwoffi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYehe River\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuangbizhuang Reservoir\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChahe River\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eKlebsiella pneumoniae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYehe River\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChahe River\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEscherichia coli\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYehe River\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnterobacter cloacae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChahe River\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \n\u003ch2\u003eTransferability Of Isolates With Ndm Genes Via Conjugation\u003c/h2\u003e\n \u003cp\u003eA total of 57 infant NDM-1 isolates including 31 KP, 24 E.coli, and 2 E.cloacae were tested for conjugation with E.coli J53, 36 isolates were successfully transferred. The conjugation rates of the NDM plasmids in E.coli and KP were 75% (18/24) and 58.06% (18/31), respectively. Water isolates of ST1306 NDM-1 KP and ST167 NDM-5 E.coli, and one NDM-1Aci.lwoffii isolate were able to transfer, while the ST37 NDM-1 KP strain and one Aci.lwoffii strain that co-produced both NDM-1 and OXA-24 failed to transfer.\u003c/p\u003e "},{"header":"Discussion","content":" \u003cp\u003eIn the study, we found that multiple independent introductions of diverse NDM-1 strains from the community and the resultant equipment or environmental contamination potentially contributed to their spread in the neonatal ward. Although basic control measures had been strictly implemented throughout the study period, they were fundamentally inadequate to curb their transmission. Intensified measures developed on the basis of a comprehensive understanding of their transmission had proven to be effective. Neonatal outbreak investigation and transmission tracking revealed that the rivers were contaminated with NDM genes and might serve as large NDM pools for community transmission.\u003c/p\u003e \u003cp\u003eThe neonatal ward had experienced several outbreaks of NDM-1 strains and finally been terminated through multifaceted intervention measures based on more accurate knowledge of the transmission events. The complete success was attributed to the following three key factors: (i) Active rectal surveillance cultures combined with efficient bacterial typing methods could give more complete transmission information that assisted in allowing for timely targeted control measures to be taken. Previously, CRE were detected only based on clinical samples while the transmission caused by colonization was almost completely ignored, leading to environmental contamination. (ii) Another important component in the success of the intervention was to reduce the workload and improve compliance with control measures. (iii) Risk assessment could reveal equipment- or device-associated transmission and enable precise interventions [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], even sometimes without any bacterial evidence [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our findings indicated that accurate evaluation of the risk factors that had contributed to neonatal acquisitions could identify potential factors for silent transmission and further improve infection control measures.\u003c/p\u003e \u003cp\u003eWalsh \u003cem\u003eet al.\u003c/em\u003e reported that NDM organisms largely occurred and disseminated in the community primarily through waterborne and foodborne transmission [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The diffusion of NDM-1 or -5 in China was mainly mediated by X3 plasmid [\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and their sources in the community were identified as animals [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], migratory birds [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], fresh vegetables [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], retail meat [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], multiple commercial farms [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and rivers [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Some NDM strains also carried MCR-1 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], which originated in animals and had spread to humans [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Despite the widespread presence of NDM genes in the community, there were few reports to assess the relationships between neonatal patients and the NDM contaminated rivers. Our findings revealed vast, unrecognized NDM contaminated rivers accessible to people. KPC-2 bacteria and OXA-23 Aci.baumannii occurred almost exclusively in healthcare settings, while NDM-1 Aci.lwoffii and OXA-24 Aci.baumannii appeared predominantly in river environment. How these strains occurred and evolved in the rivers remained to be further explored.\u003c/p\u003e \u003cp\u003eMost NDM isolates were able to transfer their NDM plasmids via conjugation, and they could serve as potential vectors or reservoirs for transferring themselves and/or NDM genes. This suggested that NDM strains in the rivers could be transferred to families where babies could potentially obtain through daily life contact with their family members who acquired after frequent exposure to or contact with NDM contaminated rivers, or through the use of contaminated washing materials (clothes, bed linen, etc.) washed in the same rivers. Once colonized and adapted to the new human environment, these NDM organisms could further propagate or/and transfer their mobile resistance genes to the microbes that inhabited the human body, which were then introduced into healthcare settings or transmitted to their close contacts in the community.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eCRE would become a persistent problem in the future, largely due to the continuing risk of importation pressures from the community. Hence, continued vigilance and comprehensive epidemiology studies specific for institutions or regions are urgently needed to address the future CRE challenge.\u003c/p\u003e "},{"header":"Abbreviations","content":" \u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCRE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCarbapenem-resistant Enterobacteriaceae; CRO:Carbapenem-resistant organism; KP:Klebsiella pneumoniae; CRKP:Carbapenem-resistant Klebsiella pneumoniae; ST:sequence type; DO:Days old; YO:Year old\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are indebted to clinical staff of the hospital for their extensive support in the investigation of outbreaks; and to Yu-Mei Guo from Shijiazhuang Center for Disease Control and Prevention for technical assistance with ERIC-PCR analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to this work. JSW\u0026nbsp;and\u0026nbsp;CF Y\u0026nbsp;had made substantial contributions to the conception and design of the study, the acquisition, analysis and interpretation of data.\u0026nbsp;JSW\u0026nbsp;had been responsible for critically drafting and revising the manuscript for all critical intellectual content.\u0026nbsp;YPL, WWY and PZ have made substantial contributions to sample collections, experiments, and data acquisition and interpretation.\u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Hebei medical applicable technology tracking project (grant number GL2012049). The funding body played no role in the design of the study, in the collection, analysis, and interpretation of data, and in the writing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtocols for collection of samples as well as the experiment plan and all methods were performed in accordance with the guidelines and regulations of Hebei general hospital and approved by the institutional ethical committees. Written informed consent was obtained from participants or parents prior to their involvement.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors of this study declare no commercial relationships and no conflicts of interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHu F, Guo Y, Yang Y, Zheng Y, Wu S, Jiang X, Zhu D, Wang F. China Antimicrobial Surveillance Network (CHINET) Study Group. Resistance reported from China antimicrobial surveillance network (CHINET) in 2018. Eur J Clin Microbiol Infect Dis. 2019;38(12):2275\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Li X, Wang M, Yue H, Li P, Liu Y, Cao W, Yao D, Liu L, Zhou X, et al. 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JAMA. 2014;312(14):1447\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePotron A, Bernabeu S, Cuzon G, Ponti\u0026egrave;s V, Blanchard H, Seringe E, Naas T, Nordmann P, Dortet L. Analysis of OXA-204 carbapenemase-producing Enterobacteriaceae reveals possible endoscopy-associated transmission, France, 2012 to 2014. Euro Surveill. 2017;22(49):17\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHumphries RM, Yang S, Kim S, Muthusamy VR, Russell D, Trout AM, Zaroda T, Cheng QJ, Aldrovandi G, Uslan DZ, et al. Duodenoscope-Related Outbreak of a Carbapenem-Resistant Klebsiella pneumoniae Identified Using Advanced Molecular Diagnostics. Clin Infect Dis. 2017;65(7):1159\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Bi Z, Ma S, Chen B, Cai C, He J, Schwarz S, Sun C, Zhou Y, Yin J, et al. Inter-host Transmission of Carbapenemase-Producing Escherichia coli among Humans and Backyard Animals. Environ Health Perspect. 2019;127(10):107009.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Zhang R, Li J, Wu Z, Yin W, Schwarz S, Tyrrell JM, Zheng Y, Wang S, Shen Z, et al. Comprehensive resistome analysis reveals the prevalence of NDM and MCR-1 in Chinese poultry production. Nat Microbiol. 2017;2:16260.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiao X, Yang RS, Xia J, Chen L, Zhang R, Fang LX, Lei F, Song G, Jia L, Han L, et al. High colonization rate of a novel carbapenem-resistant Klebsiella lineage among migratory birds at Qinghai Lake, China. J Antimicrob Chemother. 2019;74(10):2895\u0026ndash;903.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu BT, Zhang XY, Wan SW, Hao JJ, Jiang RD, Song FJ. Characteristics of Carbapenem-Resistant Enterobacteriaceae in Ready-to-Eat Vegetables in China. Front Microbiol. 2018;9:1147.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Q, Lv L, Huang X, Huang Y, Zhuang Z, Lu J, Liu E, Wan M, Xun H, Zhang Z, et al. Rapid Increase in Carbapenemase-Producing Enterobacteriaceae in Retail Meat Driven by the Spread of the bla NDM-5-Carrying IncX3 Plasmid in China from 2016 to 2018. Antimicrob Agents Chemother. 2019;63(8):e00573-19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiang R, Zhang AY, Ye XL, Kang ZZ, Lei CW, Wang HN. Various sequence types of Enterobacteriaceae carrying blaNDM-5 gene from commercial chicken farms in China. Antimicrob Agents Chemother. 2018;62(10):e00779-18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Q, Berglund B, Zou H, Zhou Z, Xia H, Zhao L, Nilsson LE, Li X. Dissemination of bla(NDM-5) via IncX3 plasmids in carbapenem-resistant Enterobacteriaceae among humans and in the environment in an intensive vegetable cultivation area in eastern China. Environ Pollut. 2021;273:116370.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan H, Miao X, Liu M, Ahmad S, Bai X. Behavior of last resort antibiotic resistance genes (mcr-1 and bla(NDM-1)) in a drinking water supply system and their possible acquisition by the mouse gut flora. Environ Pollut. 2020;259:113818.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Carbapenem-resistant, Outbreak, Infection prevention, River ","lastPublishedDoi":"10.21203/rs.3.rs-395537/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-395537/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003e\u0026nbsp;Carbapenem-resistant Enterobacteriaceae (CRE) prevalence in neonatal patients continues to rise in china. However, very few epidemiological data are available to uncover the underlying mechanisms for the increase. This study aimed to elucidate their transmission modes and explore potential sources. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003eThe genotypic features of CRE isolates were analyzed by PCR and sequencing. The elucidation of transmission and sources involved in electronic medical record review, environmental cultures, screening cultures, and prospective case-control analysis.\u003cstrong\u003e \u003c/strong\u003eThe transferability of carbapenem-resistant genes was performed by Conjugation experiments.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u0026nbsp;\u003c/strong\u003eA total of\u003cstrong\u003e \u003c/strong\u003e450 CRE isolates recovered from 369 infant patients were genotypically characterised. Carbapenemase genes NDM-1, KPC-2 and IMP-4 accounted for 86.22% (n = 388), 8.89% (n = 40) and 4.44% (n = 20), respectively. After implementation of intensified control measures, CRE rectal prevalence rates in both the 0-3 days and the 4-28 days age group were decreased from 15.53% (100/644) and 27.37% (52/190) to 11.78% (51/433) and 19.59% (19/97) (\u003cem\u003eP \u003c/em\u003e= 0.082 and \u003cem\u003eP \u003c/em\u003e= 0.148), respectively. Multivariate analysis showed that gastric lavage (odds ratio [OR]) 3.09, 95% confidence interval [CI] 1.49–6.39) and enema (OR 2.84, 95% CI 1.65–4.91) were independent risk factors for rectal colonisation in the 0-3 days age group, whereas Cephalosporins (OR 2.12, 95% CI 1.14–3.94), gastric lavage (OR 3.44, 95% CI 1.44–8.22) and residence in rural area (OR 2.22, 95% CI 1.19–4.13) were independently associated with rectal colonization in the 4-28 days age group. The transmission tracking of neonatal NDM-1 strains outbreaks revealed rivers contaminated with NDM genes. Most isolates with NDM genes could be successfully transferred.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u0026nbsp;\u003c/strong\u003eContinued vigilance for importations should be maintained to reduce CRE threat. \u003c/p\u003e","manuscriptTitle":"Source Tracking of Carbapenem-Resistant Enterobacteriaceae Outbreaks in Neonatal Patients Revealed Rivers Contaminated with NDM Genes","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2021-07-13 14:16:51","doi":"10.21203/rs.3.rs-395537/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2021-04-06 01:13:21","doi":"10.21203/rs.3.rs-395537/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aac13476-0160-429c-8b66-250f97e85cfa","owner":[],"postedDate":"July 13th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":5662111,"name":"Infectious Diseases"}],"tags":[],"updatedAt":"2021-07-13T14:16:51+00:00","versionOfRecord":[],"versionCreatedAt":"2021-07-13 14:16:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-395537","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-395537","identity":"rs-395537","version":["v2"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}