Purpose: Antimicrobial resistance is a pressing issue in Ukraine, with healthcare-associated infections caused by multidrug-resistant organisms being a major concern. A recent prospective multicenter study revealed a staggering rate of 48.4% antimicrobial resistance to carbapenems among Enterobacterales causing a healthcare-associated infection. We conducted a systematic survey to investigate the incidence rate and incidence density of carbapenemase-producing Gram-negative bacteria (CPGN) among refugees and war-wounded Ukrainians in connection with the German health system. Methods: From the onset of the war until November 2022, seven Ukrainian patients were admitted to our hospital. Upon admission, screening samples and samples from the focus of suspected infection were taken from all seven patients. The incidence rate and the incidence density of CPGN were calculated as a result of the microbiological findings. We sequenced all CPGN using Illumina technology. Results: The incidence rate of CPGN at our hospital was 0.06 for 2021 and 0.18 for 2022. All seven Ukrainian patients were infected or colonized with at least one CPGN, including K. pneumoniae (14/25), P. aeruginosa (6/25), A. baumannii (1/25), Providencia stutartii (1/25), C. freundii (1/25), and E. coli (2/25). Genomic surveillance revealed that (i) most frequently detected carbapenemases among all sequenced isolates were blaNDM (17/25) and blaOXA-48 (6/25), (ii) most commonly observed plasmid replicons among the K. pneumoniae isolates recovered from Ukrainian patients were Col(pHAD28) (12/14), IncHI1B(pNDM-MAR) (9/14), IncFIB(pNDM-Mar) (12/14), and (iii) clonal relation between the pathogens of the Ukrainian isolates, but not for the isolates from our hospital surveillance system. Conclusion: The rising prevalence of community-acquired colonization and infection with CPGN is having a direct effect on the infection prevention measures, such as higher number of isolations, reprocessing of patient rooms, additional microbiological testing and overall organization within hospitals.
Keywords: Antimicrobial resistance; Carbapenemases; Infection control; cgMLST; Incidence rate; Community-acquired
Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s15010-023-02061-4.
Antimicrobial resistance has changed the ways in which therapeutic approaches to bacterial infections and other ailments must be approached. Responsible and judicious use of antibiotics, increased research into developing new and better treatments, and proper education are all essential to mitigating the potentially dangerous consequences of antimicrobial resistance [[
We conducted a systematic survey to investigate the incidence of multidrug-resistant Gram-negative bacteria among refugees and war-wounded Ukrainians in connection with the German health system, based on whole-genome sequencing analysis. This study aimed to shed light on the incidence of these bacteria among Ukrainians.
At our University Hospital, we have 1411 beds across 26 clinics. Every year, we provide care for around 53,600 inpatients and carry out over 274,000 outpatient consultations. As the only university hospital and facility for basic, standard, and maximum health care in the region of Thuringia (a federal state in Germany), our hospital serves over one million citizens. This study encompasses all clinics and institutes of our hospital. From the onset of the war until November 2022, seven Ukrainian patients were admitted to our hospital. Three of these seven patients were hospitalized due to war wounds, while the remaining four were refugees. On-site surgery was performed to treat the wounded soldiers. Upon admission to our clinic, screening samples and samples from the focus of suspected infection were taken from all seven patients. Patient samples were analyzed in an accredited medical microbiology laboratory using standard operating procedures. The incidence rate and incidence density of CPGN were calculated based on the microbiological findings. The incidence rate was calculated for 2021 and 2022 and given in relation to 100 patient cases. The incidence density was also calculated for 2021 and 2022 and given in relation to 1000 patient days.
We sequence all CPGN at our hospital, regardless of whether colonization or infection had occurred. This enabled us to create a database of over a thousand whole-genome sequenced pathogens.
For microbiological diagnostics, the sample was streaked onto Columbia sheep blood agar (BD, Heidelberg, Germany) and Drigalski lactose agar (Oxoid, ThermoFisher Scientific, Wesel, Germany) for overnight incubation at 37 °C. Colonies were identified by Vitek MS (bioMérieux, Nürtingen, Germany). Antimicrobial susceptibility testing (AST) was performed by determination of minimal inhibitory concentrations (MIC) using Vitek 2 (bioMérieux) and the MICRONAUT-S MDR MRGN Screening microtiter system (Merlin Diagnostika, Bornheim, Germany; distributed by Sifin Diagnostics, Berlin, Germany). Twenty-five isolates from seven Ukrainian patients were sequenced using an Illumina MiniSeq instrument (Illumina, San Diego, CA, USA). DNA was extracted directly from plated microbial cultures using the NexteraTM DNA Flex Microbial Colony Extraction protocol (Illumina, San Diego, CA, USA). The fastq file format was then assembled using Velvet and analyzed with the Ridom SeqSphere + software version 8.4.1. To analyze the species-specific clonal relationship, core genome multilocus sequence typing (cgMLST) was employed. Resistance determinants and plasmids were screened by querying genome assemblies against the ResFinder and PlasmidFinder databases (PlasmidFinder version 2.1.6 (2021-03-27); ResFinder version: 2022-03-17) using the tool ABRicate with minimal coverage and minimal identity settings of 80%, respectively [[
The incidence rate of CPGN at our hospital rose from 0.06 in 2021 to 0.18 in 2022. There was also an increase in incidence density from 2021 (0.08) to 2022 (0.24). This increase was partially attributed to an outbreak of CPGN, which was not associated with Ukrainian patients, resulting in a slight increase in nosocomial cases. However, a significant surge in community-acquired cases was observed in the second and third quarters of 2022 (see Fig. 1), mainly due to the high number of detected CPGN among Ukrainian patients during this period.
Graph: Fig. 1The incidence density of CPGN at our hospital for 2021 and 2022. The values were divided into community-acquired and nosocomial cases
All seven Ukrainian patients were infected or colonized with at least one CPGN, including carbapenemase-producing P. aeruginosa, A. baumannii, K. pneumoniae, and Providencia stutartii, C. freundii, and E. coli (see Table 1). The minimum inhibitory concentration values for the antibiotics that were tested have been provided in supplementary information (SI 1). Three wounded soldiers, who had already been treated in Ukraine, were found to have wound infections caused by CPGN, as well as colonization with other carbapenemase-producing species. All patients with an infection received a consultation by an Infectious Diseases physician and were treated with antibiotics tailored to the pathogen detection (e.g., cefiderocol).
Table 1 Clinical data of the seven Ukrainian patients were collected. Patients #1–#4 were admitted as refugees, while patients #5–#7 were wounded soldiers
Patient-identifier Admission date Age, sex Cause of admission Sampling Infection/colonization Isolated pathogen Carbapenemase #1 March 76 years, female Transfer from external hospital with known myeloproliferative disease Stool Colonization NDM-3 #2 April 54 years, female Dialysis patient with acute hepatitis b Urine Colonization NDM-1 #3 August 3 years, male Suspected obstructive sleep apnea syndrome with known mucopolysaccharidosis Rectal swab Rectal swab Colonization Colonization OXA-48 NDM-1 #4 September 49 years, female Transfer from external hospital with liver cirrhosis Urine Blood culture Rectal swab Ascites Colonization Infection Colonization Infection NDM-1 NDM-1 NDM-1 NDM #5 April 48 years, male Direct transfer from Ukraine due to war injuries Wounded 04/2022 Multiple bony injuries of the lower extremities on both sides Urologic and abdominal injury from a bullet through the abdomen Wound Blood culture Abdomen Rectal swab Rectal swab Urine Infection Infection Infection Colonization Colonization Colonization IMP-34 NDM-1 OXA-72, -90 NDM-5 OXA-48 NDM-5, OXA-48 #6 June 47 years, male Direct transfer from Ukraine due to war injuries Wounded 05/2022 Open fractures of the upper and lower extremities Skin Skin Colonization Colonization NDM-1 IMP-34 #7 August 35 years, male Direct transfer from Ukraine due to war injuries Wounded 05/2022 Mine injury with fractures of the lower extremities Blood culture Catheter Catheter Rectal swab Rectal swab Rectal swab Deep wound Deep wound Deep wound Infection Colonization Colonization Colonization Colonization Colonization Infection Infection Infection OXA-48 NDM-1 NDM-1 NDM-1, OXA-48 NDM-1, OXA-48 KPC-3 NDM-1 VIM-2 NDM
The most frequently detected carbapenemases among all sequenced isolates were bla
Graph: Fig. 2Illustration of the abundance and diversity of antimicrobial resistance genes within the K. pneumoniae (Kp) in part A and P. aeruginosa (Pa), A. baumannii (Ab), C. freundii (Cf) and E. coli (Ec) species in part B
Most P. aeruginosa isolates were found to carry bla
The most commonly observed plasmid replicons among the K. pneumoniae isolates recovered from Ukrainian patients were Col(pHAD28) (12/14), IncHI1B(pNDM-MAR) (9/14), and IncFIB(pNDM-Mar) (12/14). Plasmid pNDM-MAR, a member of the IncH plasmid family, is a well-known bla
Graph: Fig. 3Mobile genetic element distribution of the Ukrainian isolates (part A). C. freundii isolates were abbreviated within the isolate name with.Cf, K. pneumoniae isolates with.Kp and E. coli with.Ec. B Mobile genetic element distribution of isolates collected in our hospital previously (part B)
Comparative analysis of the antimicrobial resistance genes and the plasmid replicons revealed no within-patient plasmid transfer between the different Gram-negative species, causing colonization or infection of the patients.
We investigated the epidemiological connection between Ukrainian isolates and the clinical isolates in our database. We observed a clonal relationship between the pathogens for the Ukrainian isolates, but not for the isolates from our clinical surveillance database. No nosocomial transmissions were detected within our hospital. However, due to the limited medical history and language barrier, it is not possible to evaluate whether there is a possible connection within the home country. For K. pneumoniae, we mostly detected isolates belonging to sequence type 395 (7/13) among the Ukrainian isolates. In our surveillance database, we have identified several MLST sequence types (STs) of bacterial isolates. Among these, ST307 (7% of all K. pneumoniae isolates in our database), ST147 (6%), and ST45 (6%) are the most prevalent in K. pneumoniae. Additionally, we have observed ST395 (6%) to be the most frequently detected ST in our database for this species.
For E. coli, we have sequenced MLST ST131 (E. coli MLST Warwick, 29%) most frequently in our database. Conversely, ST167 and ST46 of the Ukrainian isolates are only sporadically present or not at all in our database. In P. aeruginosa, we have identified MLST ST235 (31%) as the most commonly detected ST in our database. The STs isolated from Ukrainian patients (ST654, ST1047, ST773) have yet to appear in our database. Notably, we isolated pathogens from the same species without high genomic similarity and different antimicrobial resistance profiles within one focus, e.g., wounds (see Table 1, patients #3, #5, #7). This phenomenon is not usually observed for our daily, routinely infectious cases, but from the wounded soldiers.
We determined an increase in the incidence rate and incidence density for carbapenemase-producing bacteria from 2021 to 2022 at our hospital. This rise is mainly due to the high number of carbapenem-resistant isolates that we detected in Ukrainian patients. All Ukraine patients, who were admitted at our hospital until November 2022 were colonized or infected with at least one CPGN, which are insensitive to reserve antibiotics. High prevalence of these pathogens causes extensive infection prevention measures. In addition, the risk of nosocomial transmission increases and poses the danger of life-threatening infections that can no longer be treated with antibiotics. Furthermore infections are a major contributor to the rising cost of healthcare [[
The increased incidence rate for carbapenemase-producing bacteria within our hospital point up the importance of stringent screening protocols and pre-emptive isolation measures in clinical settings, particularly for war-wounded patients. This proactive approach is critical to ensure the safety of the patients most at risk. Another way to protect vulnerable patient groups from colonization and infection with carbapenemase-producing bacteria is to utilize biocides such as chlorhexidine for bathing patients [[
Our report substantiates the recommendations of the ECDC [[
We were able to calculate the incidence rate of carbapenem-resistant strains due to our systematic survey of all patients in our hospital. However, this study is limited to the patients of one hospital. One limitation of the present survey is the small number of patients we could include in our observations. Furthermore, long-read sequencing is the more suitable method to characterize plasmid replicons. For economic reasons, the isolates were not additionally sequenced using a long-read technique.
The incidence rate and incidence density of CPGN have been on the rise, resulting in a greater number of patients being isolated. This has had a significant impact on the organization of examinations and inpatient processes within hospitals, leading to an increase in the number of comprehensive final disinfections. Our clinic has been successful in preventing nosocomial transmission, which validates the efficacy of the hygiene measures and barriers we have implemented. However, the increasing incidence rate of carbapenemase-producing pathogens also highlights the importance of active screening and pre-emptive isolation.
The increasing prevalence of community-acquired colonization and infection with CPGN is increasing epidemiologic pressure on inpatient care. As a result, drastic and costly infection prevention measures are needed to avoid nosocomial transmission and life-threatening infections.
Seamless microbiological surveillance and whole-genome sequencing can create an effective early warning system to combat this issue.
We are grateful to Monika Cristofolini for her assistance in gathering epidemiological data to determine the incidence rate and incidence density. Her invaluable contribution has been instrumental in helping us to gain a better understanding of the situation.
All authors contributed to the study conception and design. The first draft of the manuscript was written by CS and MZ. Material preparation, data collection and analysis were performed by CS, RS and MZ. All authors read and approved the final manuscript.
Open Access funding enabled and organized by Projekt DEAL. No funding received for this study.
Whole genome sequencing (WGS) data are available under study ID PRJEB58365 (ERP143427) at the European Nucleotide Archive (ENA).
The authors have no relevant financial or non-financial interests to disclose. The authors have no competing interests to declare that are relevant to the content of this article. The authors have no financial or proprietary interests in any material discussed in this article.
This study was approved by the Ethics committee of the Jena University Hospital (Reg-Nr.: 2023-2918-Daten) conducted in accordance with the relevant ethical guidelines and regulations. Informed consent was waived by the Ethics committee considering the study design and adherence to the relevant guidelines. In particular, study data were deidentified to protect privacy and preserve the confidentiality of the study participants.
Below is the link to the electronic supplementary material.
Graph: Supplementary file1 (DOCX 19 KB)
• ST
- Sequence type
• Cf
- C. freundii
• Kp
- K. pneumoniae
• Ec
- E. coli
• cgMLST
- Core genome multilocus sequence typing
• CPGN
- Carbapenemase-producing Gram-negative bacteria
• MIC
- Minimal inhibitory conentration
• ECDC
- European Centre for Disease Prevention and Control
By Claudia Stein; Maria Zechel; Riccardo Spott; Mathias W. Pletz and Frank Kipp
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