Abstract
Background
Newborns are particularly susceptible to infection in hospitals, with neonatal sepsis being the most common infection symptom and the third leading cause of neonatal death. Klebsiella pneumoniae is a gram-negative bacterium of Enterobacteriaceae, which is a common pathogen of neonatal septicemia. In this study, we will analyze and evaluate the current status, clinical characteristics, and drug resistance of Klebsiella pneumoniaesepsis infection in Neonatal Intensive Care Unit (NICU), with the aim of providing effective basis for timely and accurate clinical diagnosis and treatment in clinical practice.
Methods
Statistical analysis was performed on 75 cases of Enterobacteriaceae septicemia in infants admitted to NICU in a special obstetrics and gynecology hospital in Shanghai from January 2020 to June 2022. Based on bacterial identification, isolates were divided into the Klebsiella pneumoniae (KP) group (n = 49) and the non-KP Enterobacteriaceae group (n = 26). The infection, clinical characteristics, and bacterial resistance of the two groups of infected patients were compared.
Results
Comparing the clinical characteristics of the two groups, the results showed that most of the subjects in the KP and non-KP groups were premature infants, accounting for 100% and 92.3% of subjects, respectively; late onset was the main disease in both groups, accounting for 93.9% and 80.8% of subjects, respectively. All patients received Peripherally Inserted Central Catheter(PICC). The levels of pro calcitonin and CRP (C-reactive protein) were significantly higher in the KP group compared with those in the non-KP group (p < .05). At the same time, the incidence of thrombocytopenia in the KP group was significantly higher than that in the non-KP group (p < .05). The proportion of antimicrobial drug exposure in the KP group is higher than that in the non-KP group. The drug resistance of the KP group to ceftazidime, ceftriaxone, cefepime, ampicillin/sulbactam, aztreonam, ciprofloxacin and compound sulfamethoxazole was significantly higher than that of the non-KP group, whereas the drug resistance rate to cefotetan, gentamycin and to bramycin was significantly lower than that of the non-KP group, Statistically significant differences (p < .05). 38 cases of Klebsiella pneumoniae producing ESBLs were tested for related resistance genes. The results showed that the main resistance types were SHV and TEM, with detection rates of 60.6% and 28.9%.
Conclusions
This study shows that neonatal sepsis caused by Klebsiella pneumoniae infection has a high incidence and drug resistance in premature and low birth weight infants, and has become a serious public health problem; Clinicians should pay attention to differential diagnosis, Reasonable selection of antibiotics to reduce the generation of drug-resistant bacteria.
1. Introduction
Neonates are especially prone to hospital infection because of the imperfect development of their immune system, combined with factors such as premature delivery, low body weight, vertical transmission between mother and infant, perinatal infection, and unreasonable antibiotic use [Citation1–2]. Neonatal sepsis is of particular concern, which is a systemic disease caused by bacteria, viruses or fungi, related to hemodynamic changes and other clinical manifestations, which can lead to death. Therefore, the 2015 Global Disease burden Study pointed out that neonatal sepsis is the third leading cause of neonatal death [Citation3]. Klebsiella pneumoniais a Gram-negative Enterobacteriaceae that is a common pathogen of neonatal septicemia. Recently, with the widespread use of antibacterial drugs, especially the application of third generation cephalosporins, bacterial drug resistance has been increasing, and several species now contain ultra-broad-spectrum β-lactamase strains (ESBLs) that are resistant to most penicillin and cephalosporin antibiotics. Among them, the treatment of Carbapenem resistant Klebsiella pneumoniae (CRKP) is extremely difficult and attracts scholars’ attention. A study by the World Health Organization in 2017 on multidrug-resistant bacteria showed that CRKP ranks among the top in terms of treatability, mortality rate, medical insurance burden, and trend of drug resistance within 10 years [Citation4]. In this study, 75 children with Enterobacteriaceae bacterial infection septicemia admitted to our Neonatal Intensive Care Unit (NICU) from January 2020 to June 2022 were taken as the research objects. According to the identification results of blood culture bacteria, they were divided into Klebsiella pneumoniae (KP) (n = 49) and non-KP Enterobacteriaceae (n = 26) groups. The clinical characteristics, bacterial resistance, and infection risk factors of the two groups of infected patients were compared to provide an effective basis for timely and accurate clinical diagnosis and treatment.
2. Materials and methods
2.1. Research object
From January 2020 to June 2022, the Department of NICU at Shanghai First Maternity and Infant Hospital treated 182 children with sepsis, including 105 cases of Gram-positive cocci infection, 2 cases of Gram-negative pseudomonas bacterial infection, and 75 cases of Gram-negative Enterobacteriaceae bacterial infection. In this study, 75 cases of Enterobacteriaceae bacterial septicemia were selected as the study object.
2.2. Index definition
The delivery methods were divided into Vaginal Delivery and Cesarean Section. According to the onset time, early onset occurs within 3 days of birth, and late onset occurs after 3 days of birth. Based on the gestational week of delivery, the group infants were classified into premature (gestational week: <37 weeks) and full-term infants (gestational week:37–42 weeks). The subjects were also divided into low birth weight (weight < 2.5 kg) or non-low birth weight (weight ≥ 2.5 kg) groups. There were two ways of respiratory support: invasive and noninvasive.
2.3. Sample collection
Venous blood (2–3 ml) from two different regions(left and right groins) was collected before the administration of antibacterial drugs. Blood was collected in BD BACTEC Peds Plus/F Culture Vials(plastic), and then mixed upside down for following culture and testing.
2.4. Judgment of blood test indicators
CRP (C-reactive protein): The clinical significance of CRP is to distinguish between bacterial and viral infections, monitor the severity of the disease, and observe the therapeutic effect. The normal value is CRP < 10mg/L, if CPR is between 10 and 25 mg/L, it indicates viral infection; CRP > 25 mg/L indicates bacterial infection.
Pro calcitonin: It is clinically used to reflect the degree of systemic inflammation, and the normal range is generally less than 0.5 μg/L; 0.5 to 2 μg/L belonging to mild infection; Greater than 2 μg/L is considered a severe infection.
2.5. Detection method for drug resistance phenotype
The blood culture bottle was inserted in the BD BactecTMFX (BD Company, US) for automatic culture. Bacterial isolation, identification, and drug susceptible test were conducted using the VITEK 2 Compact automatic bacteria culture identification instrument with a supporting drug sensitivity card from France BioMerier. The antimicrobial drug sensitivity was evaluated agains CLSI M100-2019.
2.6. Detection method for antibiotic resistance genotypes of ESBLs producing Klebsiella pneumonia
DNA template preparation: Take the KP strain and into an EP tube containing 2 ml of distilled water. Place it in boiling water for 10 min and then in ice for 2 min. Then centrifuge at 12,000 r/min for 2 min. Extract the supernatant and store it at −20 °C for later use. Primer sequence design and amplification conditions from reference [Citation5–6] ().
Table 1. Gene loci and primer sequence.
2.7. Statistical method
This study used WHONET 5.6 and SPSS22.0 software to analyze the data. The count data were expressed as a percentage, and the chi-square or Fisher’s exact test was used for comparison between groups. The difference was statistically significant as p value <.05.
3. Results
3.1. Clinical information
Among 75 infants with Enterobacteriaceae sepsis, 35 (46.7%) were male and 40 (53.3%) were female. The age range of infants at the time of sepsis is 1 d to 3 months, with an average of 5.9 d. Vaginal delivery involved 26 cases (34.7%) while cesarean section was used in 49 cases (65.3%). There were 8 early onset sepsis (10.7%), 67 cases of late onset sepsis (89.3%), 73 cases of premature infants (97.3%), and 2 cases of full-term infants (2.7%); in addition,70 cases (93.3%) had low birth weight.
3.2. Comparison of clinical characteristics between KP and non-KP group
The 75 cases of sepsis caused by Enterobacteriaceae infection were divided into KP (n = 49) and non-KP Enterobacteriaceae group (n = 26) based on the results of bacterial identification. Comparing the clinical characteristics of the two groups, the results showed that most of the subjects in the KP and non-KP groups were premature infants, accounting for 100% and 92.3% of subjects, respectively; late onset was the main disease in both groups, accounting for 93.9% and 80.8% of subjects, respectively. All patients received Peripherally Inserted Central Catheter(PICC). The proportion of antibacterial drug exposure in KP group was higher than that in non KP group, with a statistically significant difference (p < .05). The third generation cephalosporins were mainly exposed, including cefoperazone sodium, cefoperazone sulbactam sodium, ceftazidime, and cefmetazole. In the non-KP group, three patients died in the final outcome ().
Table 2. Comparison of clinical characteristics between KP and non-KP group.
3.3. Comparison of blood test indexes between KP and non-KP Enterobacteriaceae groups
The test results of the KP and non-KP groups showed that there was no significant difference in the white blood cell count between the two groups. However, levels of pro calcitonin and CRP were significantly higher in the KP group compared with those in the non-KP group (p < .05). At the same time, the incidence of thrombocytopenia in the KP group was significantly higher than that in the non-KP group (p < .05) ().
Table 3. Comparison of test indexes between KP and non-KP enterobacteriaceae groups.
3.4. Distribution of bacteria in non-KP Enterobacteriaceae group
Most bacteria identified in the 26 non-KP patients were Escherichia coli (13 cases). Serratia marcescens, Enterobacter aerogenes, and Enterobacter cloacae were also detected in 6, 4, and 3 cases, respectively.
3.5. Analysis of drug resistance in two groups
The drug resistance of bacteria in the KP group to ceftazidime, ceftriaxone, cefepime, ampicillin/sulbactam, aztreonam, ciprofloxacin, and sulfamethoxazole was significantly higher than that of the non-KP group, where as the drug resistance rate to cefotetan, gentamycin, and tobramycin was significantly lower than that of the non-KP group ().
Table 4. Analysis of bacterial resistance in two groups.
3.6. Resistance genotypes of ESBLs-producing Klebsiella pneumoniae
PCR amplification technology was used to detect the 38 cases of ESBLs-producing Klebsiella pneumoniae β- Lactam related resistance genes. Overall, the genes of SHV, TEM, CTX-M-1, CTX-M-2, and CTX-M-9, were detected in 60.6%, 28.9%, 2.6%, 2.6%, and 5.3% of the 38 cases, respectively ().
Table 5. Resistance genotypes of ESBLs -producing Klebsiella pneumoniae.
4. Discussion
Klebsiell apneumonia is widely distributed in nature and occurs in the respiratory tract and intestinal tract of healthy people. K. pneumonia is not only a conditional pathogen but also an important no socomial pathogen. In this study, the number of KP isolates detected in NICU blood culture from January 2020 to June 2022 in our hospital accounted for 26.9% of the total number of positive blood cultures in the same period, from mainly late onset premature infants (93.9%). These patients experienced an extended hospital stay with an average of 47.8 d, consistent with domestic and foreign reports [Citation7–10].
The incidence of thrombocytopenia and procalciton in and CRP elevation in patients with K. pneumoniae sepsis was significantly higher than that in the patients in the non-KP groups. Infection is an important factor leading to thrombocytopenia. In this study, K. pneumoniae, can adsorb onto the surface of platelets as an antigen, activate complement, and thus destroy platelets; Secondly, the enterotoxin produced by K. pneumoniae can directly damage vascular endothelial cells and platelets, increasing platelet destruction, resulting in significantly higher thrombocytopenia in the KP group compared to the non KP group. Both procalcitonin and CRP were inflammatory markers that increase during the inflammatory response. The main difference between the two was that procalcitonin specifically increases in systemic bacterial infections and sepsis, and the degree of increase was positively correlated with the severity of the disease, while CRP was only related to the inflammatory response, and the degree of increase was not significantly related to the severity of the disease. In this study, the enterotoxin produced by Klebsiella pneumoniae caused sepsis. Therefore, the levels of pro calicetin and CRP were significantly higher in the KP group compared with those in the non KP group [Citation11–14]. Therefore, when patients with suspected infection appear in the NICU, it is necessary to promptly monitor relevant infection indicators and thereby enable timely judgments to be made based on clinical manifestations, for active treatment and to be alert to any adverse outcomes caused by sepsis.
The wide application of β-lactam antibiotics, especially the third generation cephalosporins, has enabled Gram-negative Klebsiellato become the most common ESBL-producing bacteria [Citation15]. Bacterial DNA encoding the ESBL enzymes can be transferred between the same or different bacterial species through genetic combination, transformation, and transduction, resulting in drug resistance in previously drug susceptible bacteria. Due to different policies for the use of antibiotics and the selective pressure of bacteria on different drugs, the prevalence categories of ESBLs vary in different countries and regions [Citation16]. There are literature reports [Citation17–19] that TEM and SHV types are mainly prevalent in North America and Western Europe, while CTM-M types are prevalent in Eastern Europe, Asia, and South America. In China [Citation20], the CTX-M type is mainly present in South China like Guangdong province, while TEM type is more common in Northern China including Shanxi province. In this study, 38 cases of ESBLs-producing Klebsiella pneumoniae were tested for related resistance genes, and the results showed that the resistance types were mainly SHV and TEM, with detection rates of 60.6% and 28.9%, respectively.
In this study, K. pneumonia isolates cultured from infants in the KP group were resistant to most cephalosporins and penicillins, and the resistance rate to cefazolin, cefuroxime, ceftazidime, ceftriaxone, cefotaxime, cefepime, and ampicillin was >70% or more, which was related to the high proportion of K. pneumonia strains producing ESBLs. We found that 77.8% of the KP group produced ESBLs. This is consistent with both national and international research [Citation21–22]. Studies have reported that the resistance rates to amikacin and ciprofloxacin of ESBL-producing bacteria isolated from adults were 69.6% and 73.3%, respectively [Citation23–24], where as the resistance rates to quinolones drugs were 12.3%–18.1% [Citation25]. The drug resistance test results for the KP group in this study showed that the drug resistance rate to amikacin was zero, whereas the drug resistance rate to ciprofloxacin was 86.4%, which may be related to clinical drug use. This phenomenon proves that the high frequency of broad-spectrum antibiotics used to treat infection will exert selective pressure on pathogenic bacteria and lead to their increased drug resistance.
In summary, the increasing drug resistance of K. pneumoniae has become a serious public health problem, although the onset of K. pneumoniae septicemia may be clinically overlooked. Clinicians should pay attention to differential diagnosis, avoid missed diagnosis, and review relevant laboratory indicators in a timely fashion. In general, for children, antibiotics should be used reasonably in combination with clinical characteristics, and the indications for venous catheterization should be strictly controlled. Furthermore, the neonatal ward should maintain a policy of strictly disinfection and isolation. The medical staff should strengthen their practice in aseptic operations, minimize invasive operations, shorten the catheter retention time, attach importance to monitoring the detection of pathogenic bacteria, reduce infection opportunities, and actively and effectively prevent infections. Hospitals and clinics should ensure rational selection of antibiotics and strict restrictions on preventive drug use. An understanding and strict grasp of the application of third generation cephalosporins could help reduce the production of drug-resistant bacteria.
There were some shortcomings in this study. This study was a single center retrospective study with a small sample size. Only cases of neonatal bloodstream infection with Klebsiella pneumoniae in our hospital were analyzed, and no other infection samples were added. There was a certain degree of information and selection bias, and the representativeness was not strong. If it was necessary to prove the infection status of Klebsiella pneumoniae sepsis in newborns in the local area, large sample multi center data support was needed.
Authors contributions
HM wrote the first draft of the manuscript. JWX contributed to patient informed consent and information collection. YHZ contributed to patient informed consent and data acquisition. RYZ contributed to the data analysis. JW contributed to the study design and finaldraft of the manuscript. All authors read and approved the final manuscript.
Ethical approval
This research has been performed in accordance with the ethical standards as laid down in the 1964\Declaration of Helsinki and its later amendments or comparable ethical standards. This study was approved by the Ethics Committee of the Shanghai First Maternity and Infant Hospital affiliated to Tongji University (Reference number: KS18175; Date: 21/11/2018). Written informed consent was obtained from the parents/legal guardian of the eligible infants before recruitment.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Data availability statement
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.
Additional information
Funding
References
- Wenwen C, Shihe G, Kai Y, et al. Analysis of high risk factors of nosocomial infection in neonatal intensive care unit. Anhui Med J. 2015;192(2):1–7.
- Xiujuan M, Anhua W. How to cope with the severe challenge of hospital infection caused by multi drug resistant bacteria. Chin J Infect Control. 2019;18(3):185–192.
- Wang H, Naghavi M, Allen C, et al. Global, regional, and national life expectancy, all-cause mortality, and cause- specific mortality for 249 causes of death, 1980-2015: a systematic analysis for the global burden of disease study 2015. Lancet. 2016;388(10053):1459–1544. doi: 10.1016/S0140-6736(16)31012-1.
- Tacconelli E, Carrara E, Savoldi A, et al. WHO pathogens priority list working group. Discovery, research, and development of new antibiotics: the who priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis. 2018;18(3):318–327. doi: 10.1016/S1473-3099(17)30753-3.
- Wang Q, Li G, Zheng B. Antimicrobial susceptibility and resistance genes of carbapenem-resistant Klebsiella pneumoniae in China from 2017 to 2018. Chin J Infect Control. 2021;20(05):437–442.
- Woodford N, Fagan EJ, Ellington MJ. Multiplex PCR for rapid detection of genes encoding CTX-M extended-spectrum β-lactamases. J Antimicrob Chemother. 2006;57(1):154–155. doi: 10.1093/jac/dki412.
- Lin C, Falin X, Ming N, et al. Pathogen and clinical characteristics of premature infants with septicemia . Chin J Contemp Pediatr. 2019;21(9):881–885.
- Mwananyanda L, Pierre C, Mwansa J, et al. Preventing bloodstreaminfections and death in Zambian neonates: impact of a low-cost infection control bundle. Clin Infect Dis. 2019;69(8):1360–1367. doi: 10.1093/cid/ciy1114.
- Chen Y, Brook TC, Soe CZ, et al. Preterm infants harbour diverse Klebsiella populations, including atypical species that encode and produce an array of antimicrobial resistance- and virulence-associated factors. Microb Genom. 2020;6(6):e000377.
- Bingjie W, Fen P, Hong Z, et al. Distribution characteristics and drug resistance analysis of carbapenem resistant enterobacteriaceae bacteria in children from. Chin J Microbiol Immunol. 2016;2019(8):583–590.
- Xing C, Chi Z, Tao X. The application of procalcitonin, C-reactive protein, and blood cell parameters in the differential diagnosis of sepsis. Lab Med. 2020;35(12):1284–1287.
- Ying X. Changes in serum procalcitonin and C-reactive protein levels and bacterial distribution in children with bloodstream infection. J Clin Ration Drug Use. 2018;11(30):124–125.
- Shijie H. The clinical significance of applying quantitative detection of serum procalcitonin in the diagnosis of bacterial infections. Chin Foreign Med Res. 2019;17(9):58–59.
- Chenjing H, Huafeng X. Wang yin the clinical significance of quantitative detection of serum procalcitonin in the diagnosis of bacterial infections. Lab Med. 2015;30(10):980–982.
- Qingde Z, Jingzhi M, Xiuzhen Z. Study on molecular epidemiology of hospital infection of Klebsiella pneumoniae producing ESBL. Chin J Hosp Infect. 2000;10(1):10–12.
- Song JE, Jeong H, Lim YS, et al. An outbreak of KPC producing Klebsiella pneumoniae linked with an index case of community-acquired KPC-producing isolate: epidemiological investigation and whole genome sequencing analysis. Microb Drug Resist. 2019;12(45):1457–1464.
- Harb L, Boeckman J, Newkirk H, et al. Complete genome sequence of the novel Klebsiella pneumonia phage Marfa. Microbiol Resour Announc. 2019;8(29):935–947.
- Zhang R, Hu YY, Zhou HW, et al. Emergence of mcr-1 and the tet(A) variant in a Klebsiella pneumoniae isolate from the faeces of a healthy person. J Med Microbiol. 2019;22(85):341–452.
- Zhao M, Lepak AJ, Marchillo K, et al. In vivo pharmacodynamic target determination for delafloxacin against Klebsiella pneumonia and Pseudomonas aerugionsa in the neutropenic murine pneumonia model. Antimicrob Agents Chemother. 2019;63(10):2125–2138. doi: 10.1128/AAC.01131-19.
- Xiaokui W. The effect of different Chinese and Western antibacterial drugs on the inhibition of endotoxin release by Klebsiella pneumoniae. Anti Infective Pharm. 2019;16(4):574–576.
- Pokhrel B, Koirala T, Shah G, et al. Bacteriological profile and antibiotic susceptibility of neonatal sepsis in neonatal intensive care unit of a tertiary hospital in Nepal. BMC Pediatr. 2018;18(1):208. doi: 10.1186/s12887-018-1176-x.
- Jiang Y, Kuang L, Wang H, et al. The clinical characteristics of neonatal sepsis infection in southwest China . Intern Med. 2016;55(6):597–603. doi: 10.2169/internalmedicine.55.3930.
- Qing’an L, Wendong L, Qingyu X, et al. Extended spectrum entero bacteriaceae bacteria production in some areas of shanghai β-detection of lactamase and drug sensitivity. Chin J Tuberculosis Respiratory Heterosis. 2000;23(7):420–423.
- Qing’an L, Wendong L. Qingyu X, et al. Super broad spectrum β- detection of drug resistance of Klebsiella pneumoniae with lactamase. Chin J Lab Med. 2001;24(4):203.
- Jiatai L, Allan JW, Min Y. Monitoring of bacterial resistance in China library. Chin Med J. 200l;8l(1):8–16.