Prevalence, and Microbiological and Clinical Characteristics of Elizabethkingia Isolates from a tertiary hospital in Jiangxi Province, China

doi:10.21203/rs.3.rs-4674119/v1

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Prevalence, and Microbiological and Clinical Characteristics of Elizabethkingia Isolates from a tertiary hospital in Jiangxi Province, China

https://doi.org/10.21203/rs.3.rs-4674119/v1

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Background

Elizabethkingia infections have gradually become life-threatening hospital-acquired infections worldwide with increasing morbidity, multidrug resistance, and poor prognosis. However, information on the epidemiological and clinical characteristics of Elizabethkingia infections in mainland China is limited. The aim of this study was to analyze the molecular and clinical characteristics, and drug susceptibility of clinical Elizabethkingia isolates from a hospital in Jiangxi Province, China.

Results

The mean age of the patients was 61 years (excluding one 13-day-old infant) and 74.8% were male. In total, 85.4% of patients admitted to Intensive Care Unit were infected with Elizabethkingia. COVID-19, respiratory disease, and central venous catheterization rates were significantly different (P <0.05) between the surviving and dying groups. Sequencing of 103 isolates identified 92 strains of Elizabethkingia anopheles, eight strains of Elizabethkingia meningoseptica, two strains of Elizabethkingia bruuniana, and one strain of Elizabethkingia ursingii. The Vitek MS had a correct identification rate of 87% for E. anopheles. More than 90% of the Elizabethkingia isolates were susceptible to minocycline, but resistant to other drugs, including ceftazidime, aztreonam, and imipenem. Resistance genotype analysis showed that blaBlaB and blaCME were highly prevalent in the Elizabethkingia isolates. Molecular typing revealed 29 different PFGE types with clonal transmission between wards.

Conclusions

Multidrug-resistant Elizabethkingiaare beingdetected at increasing rates; a larger database is required for strain identification of this bacterium. This database could be beneficial for the subsequent determination of optimal antimicrobial drugs for the treatment of infections caused by different Elizabethkingia strains. Our PFGE model showed that most isolates had sufficient genetic diversity and clonal transmission; adequate attention should be paid to this pathogen.

The genus Elizabethkingia comprises aerobic, oxidase-positive, glucose-unfermenting, nonautotrophic, gram-negative bacilli that are common in soil, freshwater, saltwater, and hospital environments, but rare in humans (1). Although a rare pathogen, Elizabethkingia meningoseptica, which causes outbreaks of neonatal meningitis, is known to cause life-threatening infections and has been associated with human infections since it was first reported by Elizabeth O. King in 1959 in a neonatal case of meningitis (2, 3). Advances in molecular techniques have revealed that several isolates, previously known as E. meningoseptica, belong to different species with new classifications and nomenclature. To date, at least seven species have been classified into the genus Elizabethkingia including E. meningoseptica, Elizabethkingia anopheles (4), Elizabethkingia miricola (5), Elizabethkingia argenteiflava (6), Elizabethkingia occulta, Elizabethkingia ursingii, and Elizabethkingia bruuniana (7). E. anopheles was isolated from the midgut of a mosquito (Anopheles gambiae) in 2011 (6). The first human infection with E. anophelis was meningitis in a newborn in the Central African Republic in 2013 (8). In 2018, three new species were identified: E. occulta, E. ursingii, and E. bruuniana (9).

Environmental studies have shown that Elizabethkingia can survive in water supply systems and often colonizes sinks, basins, and faucets, creating a potential reservoir of infection within hospitals (10). Elizabethkingia can be introduced to patients through medical equipment contaminated with fluids (e.g., respirators, intubation tubes, fog tents, humidifiers, neonatal incubators, and freezers), and can also be transmitted through wet and dry materials and surfaces, including the hands of hospital staff. Hospital transmission of Elizabethkingia has also been reported in immunocompromised adults in intensive care units (ICUs). Nosocomial outbreaks of Elizabethkingia occur worldwide, especially infections involving ICU patients requiring ventilator support (11, 12). Outbreaks have been mainly related to healthcare and, often, water sources (13, 14). Evidence suggests that most human infections are caused by E. anopheles (15).

The importance of early detection and treatment has been further reinforced by the increasing number of Elizabethkingia infections worldwide in recent years, with high morbidity and mortality rates (16). Due to their Ambler class A serine extended-spectrum β-lactamase (ESBL) gene, blaCME, and the Ambler class B metallo-β-lactamase (MBL) genes, blaBlaB and blaGOB, Elizabethkingia species are intrinsically resistant to a wide variety of β-lactams, contributing to their natural resistance to several commonly used carbapenem antibiotics. Elizabethkingia species are also known to be resistance to quinolones, owing to DNA mutations in their rotamase and/or topoisomerase IV genes (17, 18). Elizabethkingia has the unique ability to acquire multi-drug resistance and survive disinfectants, therefore spread between patients via human/inanimate host material in hospital environments is a concern. Therefore, it is critical to identify the source of infection and establish the kinetics of its spread within hospital environments (2). Elizabethkingia-related infections are complicated by biofilm formation, intracellular invasion, and multidrug resistance of strains; the careful selection of appropriate antimicrobial agents is required.

Three species, E. meningoseptica, E. miricola, and E. anophelis, cannot be distinguished by their phenotypic characteristics, and are often misidentified by biochemical or other commercial systems because of the limited Elizabethkingia database available. Previous studies have misidentified E. anophelis, E. bruniana, E. ursingii, and E. occulta as E. meningoseptica, suggesting an underestimation of the likelihood of infection with these species (18). Most studies investigating Elizabethkingia have used unreliable microbial identification methods; therefore, these studies present the clinical or molecular characteristics of all Elizabethkingia species, not each individual species. Despite their clinical significance, gaps remain in our understanding of the demographics, pathogenicity, and effective treatment options of Elizabethkingia infections.

In this study, the epidemiology, clinical characteristics, and antibiotic susceptibility of Elizabethkingia isolates collected from a hospital affiliated with Nanchang University in 2022 and 2023 were analyzed using 16S rRNA sequencing. We evaluated the susceptibility of Elizabethkingia isolates to 16 antibiotics and compared the results of 16S rRNA sequencing with those of the VITEK MS assay to identify the strains to evaluate the feasibility of this mass spectrometry.

Clinical specimens and identification of Elizabethkingia Isolates

Clinical specimens for bacterial culture were collected from at the First Affiliated Hospital of Nanchang University, a tertiary comprehensive hospital in China with 6,100 beds, between January 2022 and December 2023. A total of 103 specimens were collected from 103 hospitalized patients who were isolated from a variety of sources. The species were initially identified using Vitek MS (bioMérieux). The isolates identified as Elizabethkingia spp. were frozen until use.

Species Identification Using 16S rRNA Gene Sequencing

The 16S rRNA gene was amplified and sequenced using the universal primers 27F: 50- AGAGTTTGATCMTGGCTCAG-30 and 1492R:50-TACGGYTACCTTGTTACGACTT-3'(18). The PCR reaction mixture (50 mL) consisted of 1 uL of each primer, 6 uL of genomic DNA, and 25 uL of 5xPCR Master Mix. PCR was performed with the following conditions: denaturation at 95℃ for 5 minutes; 35 cycles of denaturation at 95℃ for 15 seconds, annealing at 56℃ for 15 seconds, and extension at 72℃ for 15 seconds; and a final extension at 72℃ for 5 minutes. The 1,488 bp product was analyzed via 1% agarose gel electrophoresis and visualized with ethidium bromide staining(34). The assembled 16S rRNA sequences were submitted to the National Center for Biotechnology Information website for comparison with the GenBank sequence database using the Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The similarity of the 16S rRNA sequences of isolates to the type strains in the GenBank sequence databases was examined using the following reference sequences: E. anophelis strain R26, GenBank accession number NR_116021.1; E. meningoseptica type strain 13253, NR_042267.1; E. bruuniana strain SBRL-21-126, NZ_JAMBNJ010000000; and E. ursingii strain G4122,NZ_LNOK01000023)(35). All clinical isolates were identified by Vitek MS and 16sRNA gene sequencing, and with 16sRNA as the gold standard, we compared the accuracy of Vitek MS in identifying Elizabethkingia isolates (21).

Antimicrobial susceptibility testing

In vitro drug susceptibility testing was conducted using the Vitek2-Compact fully automated microbial analysis system. Interpretations of resistance (R), intermediate resistance (I), and sensitivity (S) were performed in accordance with the criteria established by the Clinical Laboratory Standards Institute (M100-S27). PCR amplification was performed to detect the presence of seven resistance genes (blaBlaB, blaGOB, and blaCME) as previously described (19). The amplification primers, systems, and conditions were obtained from the literature.

Molecular Typing

Pulsed-field gel electrophoresis (PFGE) was utilized to appraise the homology of all strains(34). Genomic DNA of Elizabethkingia was fabricated via digestion with the restriction enzyme XhoI for 4 hours at 37°C. The molecular size marker of strain Braenderup H9812 was processed with XbaI(11). Furthermore, the DNA fragments were segregated by employing the CHEF Mapper XA System (Bio-Rad) at 6V/cm for 18h. PFGE band profiles were analyzed with BioNumerics 8.0. Similarity matrices were computed using Dice's coefficients with 1.5% optimization and 1.5% band matching tolerance. Dendrograms were constructed using the unweighted pair group method with arithmetic averages (UPGMA) (36). Isolates were categorized into PFGE subtypes (≥ 95% similarity), PFGE types (85–95% similarity), or different types (< 85% similarity) (21).

Statistical Analysis

The data were analyzed with SPSS 26.0 statistical software. Categorical data were expressed as frequencies and percentages. Chi-squared or Fisher’s exact tests were used to compare categorical variables (sex, underlying diseases, operation, indwelling device, ICU admission, principal disease, fungal infection, and COVID-19). Continuously quantitative data (age, hospitalization duration, temperature, white blood cell count, hemoglobin, neutrophil percentage, platelet count, lymphocyte count, lymphocyte percentage, proealcitonin, C-reactive protine, and serum creatinine) were expressed as the mean ± standard deviation and compared by T test. A P-value of < 0.05 was considered statistically significant.

Identification and Prevalence of Elizabethkingia Isolates

A total of 103 Elizabethkingia isolates, identified by conventional methods, were collected at a university-affiliated hospital in 2022 and 2023. Among the 103 isolates, the species identified using 16S rRNA gene sequencing were 92 isolates (89.3%) of E. anophelis (99.4–100.0% nucleotide identity to E. anophelis type strain R16), eight isolates (7.3%) of E. meningoseptica (99.5–99.9% nucleotide identity to E. meningoseptica type strain ATCC 13253), two isolates (1.9%) of E. bruuniana, and one isolate (1.0%) of E. ursingii. But we found ambiguity in the identification of E. bruuniana and E. ursingii .

A matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) Vitek MS system with an amended database was used and its feasibility for the identification of Elizabethkingia isolates was evaluated. Using VITEK MS, 80.6% of Elizabethkingia isolates (83 of 103) were correctly identified. VITEK MS identified 80 strains (87.0%) of E. anophelis and correctly identified three strains (37.3%) of E. meningoseptica, demonstrating improved accuracy compared with other methods (Table 1). Of these, seven (6.8%) strains of E. anophelis were misidentified as E. miricola, five (4.8%) strains of E. anophelis were misidentified as E. meningoseptica, five (4.8%) strains of E. meningoseptica were misidentified as E. anophelis, and one (1%) strain of E. ursingii was misidentified as E. anophelis. Further, there was one instance each of E. bruuniana being misidentified as E. miricola (1%) and E. bruuniana being misidentified as E. anophelis (1%). These results imply that Vitek MS may be unreliable in identifying E. meningoseptica and E. miricola. Additionally, 16 sputum samples showed concomitant isolates of other bacterial species, such as Acinetobacter baumannii, Acinetobacter SPP, Klebsiella pneumoniae, and Stenotrophomonas maltophilia.

Table 1

Comparison of Vitek MS with 16S rRNA gene sequencing in *Elizabethkingia* isolates identification
16S rRNA sequencing	Vitek MS
	E. anophelis		E. meningoseptica		E. miricola
	Correct discrimination	False discrimination	Correct discrimination	False discrimination	Correct discrimination	False discrimination
E. anophelis(n = 92)	80(87.0%)			5(5.4%)		7(7.6%)
E. meningoseptica(n = 8)		5(62.5%)	3(37.5%)
E. bruuniana(n = 2)		1(50.0%)				1(50.0%)
E. ursingii(n = 1)		1(100.0%)

Clinical Characteristics of Elizabethkingia Infections

Among the 103 Elizabethkingia isolates, including 36 strains collected in 2022 and 67 strains collected in 2023, the most common site of isolation was the respiratory tract (90.3%), followed by the blood (3.9%). Other sites of isolation included the cerebrospinal fluid (1.9%), urine (1.9%), pleural fluid (1%), and catheter tips (1%) (Fig. 1). Of these patients, 74.8% were male and 25.2% were female; the average age of the patients was 60 ± 18 years (excluding one 13-day-old patient) (Table 2). Prolonged hospital stays (≥ 2 weeks) was observed in 97 patients. Comorbidities were identified in most hospitalized patients, with hypertension being the most prevalent underlying disease (37/103; 35.9%), followed by diabetes mellitus (18/103; 17.5%), and chronic obstructive pulmonary disease (11/103; 10.7%). A large portion of the patients had nervous system disease (59.2%), while 41.4% had cardiovascular disease, and 38.3% had experienced trauma. Furthermore, 88 (85.4%) patients were treated in the ICU, 67 (65.0%) underwent surgery, and 82 (79.6%) received mechanical ventilation. Central venous catheters were placed in 75 patients (72.8%).

A total of 36 deaths occurred, corresponding to a mortality rate of 35.0%. Compared to the survivors, the 36 patients who died were significantly older (67 ± 16 vs 57 ± 18 years; P = 0.006), and had significantly more central venous catheters (86.1 vs. 65.7%, respectively; P = 0.026), and Foley’s catheters (88.9 vs. 71.6%; P =

Table 2

Factors associated with mortality in patients with Elizabethkingia infections
	Total(n = 103)	Survivors (n = 67)	Deaths (n = 36)	p-Value
Age (Years)(mean ± SD)	60 ± 18	57 ± 18	67 ± 16	0.006
Male, n(%)	77(74.8%)	47(70.1%)	30(83.3%)	0.142
Hospitalization duration(days) (mean ± SD)	43 ± 31	45 ± 35	35 ± 17	0.04
Operation, n(%)	67(65.0%)	49(73.1%)	18(50%)	0.019
Indwelling device, n (%)
Mechanical ventilation, n(%)	82(79.6%)	52(77.6%)	30(83.3%)	0.492
Central venous catheter n(%)	75(72.8%)	44(65.7%)	31(86.1%)	0.026
Nasogastric tube n(%)	73(70.9%)	45(67.2%)	28(77.8%)	0.258
Foley’s catheter, n(%)	80(77.7%)	48(71.6%)	32(88.9%)	0.045
Surgical puncture or drain n(%)	47(45.6%)	31(46.3%)	16(44.4)	0.859
ICU admission, n(%)	88(85.4%)	53(79.1%)	35(97.2%)	0.013
COVID-19, n(%)	11(10.7%)	2(3.0%)	9(25%)	0.001
Fungal infection, n(%)	42(40.8%)	23(34.3%)	19(52.8%)	0.069
Underlying diseases, n(%)
Diabetes mellitus, n(%)	18(17.5%)	12(17.9%)	6(16.7%)	0.874
Hypertension, n(%)	37(35.9%)	24(35.8%)	13(36.1%)	0.977
Chronic obstructive pulmonary disease, n(%)	11(10.7%)	3(4.5%)	8(22.2%)	0.005
Principle disease, n(%)
Nervous system, n(%)	61(59.2%)	36(53.7%)	25(69.4%)	0.122
Malignancy, n(%)	5(4.9%)	4(6.0%)	1(2.8%)	0.472
Trauma, n(%)	40(38.3%)	30(44.8%)	11(30.6%)	0.16
Cardiovascular, n(%)	43(41.4%)	28(41.8%)	15(41.7%)	0.99
Digestive, n(%)	21(20.4%)	17(25.4%)	4(11.1%)	0.087
Respiratory, n(%)	35(34.0%)	17(25.4%)	18(50%)	0.012
Temperature(℃) (mean ± SD)	37.7 ± 0.8	37.6 ± 0.7	37.8 ± 0.9	0.338
Laboratory data
White blood cell count (×10^9/L) (mean ± SD)	11.6 ± 7.1	10.9 ± 7.0	13.0 ± 7.1	0.15
Hemoglobin (g/dL) (mean ± SD)	86.7 ± 20.8	87.7 ± 16.0	84.8 ± 27.8	0.496
Platelet count (×10^9/L) (mean ± SD)	226.8 ± 154.0	254.1 ± 146.6	175.8 ± 156.4	0.013
Neutrophil percentage(%)(mean ± SD)	78.4 ± 14.8	77.6 ± 14.4	79.9 ± 15.6	0.455
Lymphocyte count(×10^9/L) (mean ± SD)	1.0 ± 0.6	1.0 ± 0.6	1.1 ± 0.8	0.563
Lymphocyte percentage(%)	11.1 ± 8.5	11.9 ± 9.2	9.4 ± 6.7	0.167
C-reactive protine(mg/L)	66.25 ± 54.3	50.88 ± 38.1	94.4 ± 65.1	0.001
Serum creatinine (mg/dL) (mean ± SD)	106.4 ± 74.2	85.9 ± 55.8	144.5 ± 88.5	0.001
Proealcitonin (ng/mL) (mean ± SD)	2.4 ± 5.1	1.2 ± 3.0	4.4 ± 7.0	0.017

0.045) placed. Furthermore, the distribution of primary diseases showed a significant difference, with a higher percentage of respiratory diseases in patients who died compared to survivors (50.0 vs. 25.4%; P = 0.012); COVID-19 was the most significant risk factor associated with mortality (25 vs. 3%; P = 0.001). Laboratory data showed that C-reactive protein, serum creatinine, and procalcitonin levels were significantly different between the survival and death groups ( P < 0.05); however, white blood cell count, hemoglobin level, neutrophil percentage, lymphocyte count, and lymphocyte percentage showed no significant differences.

Table 3

Antimicrobial Susceptibilities of 103 Elizabethkingia Isolates Determined by the Vitek2-Compact fully automated microbial analysis system
No. of isolates with result/total no. of isolates tested (%)
	E.Anophelis			E.Meningoseptic			E.Bruuniana			E.Ursingii			Total isolates
antimicrobial agents	S	I	R	S	I	R	S	I	R	S	I	R	S	I	R
Piperacillin-tazobactam	26/90(28.9)	3/90(3.3)	61/90(67.8)	6/8(75)	0	2/8(25)	0	1/2(50)	1/2(50)	0	0	1/1(100)	32/1(31.7)	4/101 (4.0)	65/101 (63.3)
Ticarcillin-clavulanic acid	2/55(3.6)	6/55(10.9)	47/55(85.5)	0	0	2/2(100)	0	0	1/1(100)	0	0	1/1(100)	2/59(3.4)	6/59(10.2)	51/59(86.4)
Ceftazidime	0	1/68(1.5)	67/68(98.5)	0	0	2/2(100)	0	0	2/2(100)	0	0	1/1(100)	0	1/73(1.4)	72/73(98.6)
Cefepime	0	5/91(5.5)	86/91(94.5)	0	0	8/8(100)	0	0	2/2(100)	0	0	1/1(100)	0	5/102(4.9)	97/102(95.1)
Cefoperazone-sulbactam	2 (12.5)	2/16 (12.5)	12/16 (75)	0	0	1/1(100)	0	0	1/1(100)	0	0	0	2/18(11.1)	2/18(11.1)	14/18(77.8)
Aztreonam	0	0	91/91 (100)	0	0	8/8(100)	0	0	2/2(100)	0	0	1/1(100)	0	0	102/102(100)
Imipenem	2/92 (2.2)	0	90/92 (97.8)	0	0	8/8(100)	0	0	2/2(100)	0	0	1/1(100)	2/103(1.9)	0	101/103(98.1)
Meropenem	0	2/62 (3.2)	60/62 (96.8)	0	0	2/2(100)	0	0	1/1(100)	0	0	1/1(100)	0	2/66(3.0)	64/66(97.0)
Amikacin	3/92 (3.3)	1/92 (1.1)	88/92 (95.6)	0	0	8/8(100)	1/2	0	1/2 (50)	0	0	1/1(100)	4/103 (3.9)	1/103 (1.0)	98/103 (95.1)
Ciprofloxacin	21/92 (22.8)	3/92 (3.3)	68/92 (73.9)	1/8 (12.5)	0	7/8 (87.5)	2/2(100)	0	0	0	0	1/1(100)	24/103 (23.3)	3/103 (2.9)	76/103 (73.8)
Levofloxacin	30/92 (32.6)	0	62/92 (67.4)	1/8 (12.5)	0	7/8 (87.5)	1/2 (50)	0	1/2 (50)	0	0	1/1(100)	32/103 (31.1)	0	71/103 (68.9)
Trimethoprim-sulfamethoxazole	71/89 (79.8)	0	18/89 (20.2)	8/8(100)	0	0	1/2 (50)	0	1/2 (50)	1/1(100)	0	0	81/100 (81)	0	19/100 (19)
Doxycycline	54/61 (88.5)	1/61 (1.6)	6/61 (9.9)	2/2(100)	0	0	1/1(100)	0	0	1/1(100)	0	0	58/65 (89.3)	1/65 (1.5)	6/65 (9.2)
Minocycline	59/61 (96.7)	0	2/61 (3.3)	2/2(100)	0	0	1/1(100)	0	0	1/1(100)	0	0	63/65 (96.9)	0	2/65 (3.1)
Gentamicin	1/31 (3.2)	4/31 (12.9)	26/31 (83.9)	0	0	6/6(100)	0	0	1/1(100)	0	0	0	1/38 (2.6)	4/38 (10.5)	33/38 (86.9)
Tobramycin	3/92 (3.3)	0	89/92 (96.7)	0	0	8/8(100)	1/2 (50)	0	1/2 (50)	0	0	1/1(100)	4/103 (3.9)	0	99/103 (96.1)

Antimicrobial susceptibilities and Genotype of Elizabethkingia Isolates

The drug susceptibilities of the 103 Elizabethkingia isolates were determined using the Vitek2-Compact fully automated microbial analysis system (Table 3). All isolates were resistant to aztreonam. All 103 isolates were susceptible to at least one antimicrobial agent tested. Sixty-three of 65 isolates (96.9%) tested were susceptible to minocycline, 58/65 (89.3%) were susceptible to

doxycycline, and 81/100 (81.0%) were susceptible to trimethoprim-sulfamethoxazole. Over 95% of the tested isolates were resistant to ceftazidime, imipenem, meropenem, amikacin and tobramycin. In addition, one E. anophelis isolate was resistant to all antibiotics tested; however, most

Elizabethkingia isolates were only sensitive to two or three antibiotics tested (Table 4).

A total of 69 Elizabethkingia strains carried β-lactamase genes. Of these, 68 Elizabethkingia isolates carried blaBlaB and seven carried blaCME; none carried blaGOB. Six Elizabethkingia isolates harbored both blaBlaB and blaCME genes (Fig. 2). Accordingly, strains carrying these resistance genes were found to be resistant to ceftazidime, cefepime, meropenem, and imipenem, with resistance rates > 90%; this effect was particularly pronounced in E. anophelis and E. meningoseptica. Strains with GryA, GyrB, ParC, and ParE genes were resistant to fluoroquinolones, with approximately 30% susceptibility to ciprofloxacin, which was higher in strains carrying the RND gene for the efflux pump.

Table 4

The number of susceptible antibiotics of 103 isolates of *Elizabethkingia* isolates
Number of Susceptible Antibiotics	All Isolates (n = 103)		Number of Episodes (%)
Number of Susceptible Antibiotics	All Isolates (n = 103)	E.Anophelis(92)	E.Meningoseptic(8)	E.Bruuniana(2)	E.Ursingii(1)
0	1(1%)	1(1.1%)	0	0	0
1	5(4.8%)	5(5.4%)	0	0	0
2	29(28.2%)	24(26.1%)	5(62.5%)	0	0
3	42(40.8%)	38(41.3%)	2(25%)	1(50%)	1(100%)
4	17(16.5%)	15(16.3%)	1(12.5%)	1(50%)	0
5	6(5.8%)	6(6.5%)	0	0	0
6	2(1.9%)	2(2.2%)	0	0	0
8	1(1%)	1(1.1%)	0	0	0

Molecular typing of Elizabethkingia Isolates

Nineteen Elizabethkingia isolates were found to be resistant to XhoI digestion. The remaining 84 isolates clustered into 29 different pulsed-field gel electrophoresis (PFGE) types (Fig. 2). In particular, 74 E. anophelis isolates were divided into 22 clusters designated A–V, seven E. meningoseptica isolates were divided into five clusters designated A–E, and two E. bruuniana and one E. ursingii isolate were divided into two clusters designated A and B. PFGE typing was most common for the J-type with 42 strains, 14 of which belonged to the same subtype. Of these 14 strains, all were from patients admitted to the ICU of the same department within a three month span from which clonal strains of the same subtype were collected (most from sputum) indicating the presence of clonal transmission in the ICU. Most patients with the same subtype experienced cerebrovascular accidents, and received mechanical ventilation and indwelling tubes during hospitalization. Similar antimicrobial susceptibility patterns were observed for different subtypes of the same clustered strains.

Elizabethkingia isolates cause serious nosocomial infections and outbreaks worldwide, but have received relatively little attention. In this study, we used 16S rRNA gene sequencing as a reference method for the species identification of Elizabethkingia spp. collected over two years and analyzed the characteristics of Elizabethkingia spp. obtained from clinical samples. We found that E. anophelis, but not E. meningoseptica, accounted for the majority of human infections with the genus Elizabethkingia, and that the isolation rate of Elizabethkingia almost doubled from 36 strains collected in 2022 to 67 strains in 2023, with increasing detection of E. anophelis. In fact, evidence suggests that E. anopheles, rather than E. meningoseptica, dominates Elizabethkingia in clinical settings (19). Therefore, we must focus on nosocomial Elizabethkingia infections.

Advances in microbial identification techniques have made it possible to identify several emerging unusual bacteria that cause disease, primarily in immunocompromised patients. Traditional identification systems are poor at identifying rare species and can easily lead to misidentification, misdiagnosis, treatment failure, and underestimation of the incidence of infection (20). MALDI-TOF, Vitek mass spectrometry (VMS), and molecular identification techniques (16S rRNA, rpoB gene sequencing, and whole genome sequencing) have become useful tools for accurate identification of microorganisms (17, 21). These tools have an excellent discrimination ability, especially for rare opportunistic bacteria. In this study, we found ambiguity in the identification of Ebr97, Ebr131, and Eur74 by 16S rRNA gene sequencing, which may have been due to the presence of multiple copies of the different sequences, as well as the fact these are highly variable regions of 16S rRNA (16). PFGE mapping showed better resolution of clonal relationships, indicating that Ebr131 is more closely related to Ebr97, with a similarity of 85.7%. The rpoB gene is a single-copy gene with a higher phylogenetic evolutionary resolution than the 16S rRNA genes, thus allows for accurate differentiation of Elizabethkingia at the species level (9, 17).

Studies have shown that most patients with Elizabethkingia infections have underlying chronic diseases such as diabetes, cardiovascular diseases, and pulmonary diseases (22, 23). Our study yielded similar results. Previous studies have shown that E. meningoseptica is commonly isolated from ICUs in India (24, 25) and Taiwan (26). In the present study, the emergency ICU, trauma ICU, and general ICU were ranked in the top three Elizabethkingia sources. These data consistently suggest that Elizabethkingia favors infection in immunocompromised patients. In patients infected with Elizabethkingia, the mortality rate ranges from 20 to 40% (27). The major risk factors for patients with Elizabethkingia infection include ICU admission, surgery, and the use of an indwelling device. Other risk factors include COVID-19, prolonged hospitalization, and underlying diseases (28). In this study, COVID-19, respiratory illness, mechanical ventilation, and central venous cannulation were risk factors for mortality in patients with Elizabethkingia infection. Elizabethkingia can form biofilms in wet environments or on water-related equipment, facilitating its spread in hospital environments (28, 29). Elizabethkingia was identified in the ward during environmental surveillance sampling; a retrospective review of clinical data revealed that Elizabethkingia had also been identified in the sputum of patients admitted to the ward in the previous ten days, leading to the hypothesis that transmission of this bacterium may occur between caregivers and patients, and that environmental surfaces and shared medical equipment may also place patients at risk of Elizabethkingia infection (30).

Elizabethkingia infections are challenging because they tend to exhibit inherent resistance to antimicrobial agents (including beta-lactams and inhibitors, aminoglycosides, macrolides, tetracycline, vancomycin, and carbapenems) (16). Genomic and proteomic analyses have confirmed the presence of multidrug resistance genes and drug efflux systems in Elizabethkingia (31, 32). These strains showed differential susceptibilities to doxycycline, ceftazidime, imipenem, meropenem, amikacin, and tobramycin. The high prevalence of blaBlaB and blaCME genes in the present study is consistent with broad-spectrum resistance to beta-lactams, including carbapenems. Several genes associated with drug resistance have been identified in Elizabethkingia. MBL genes are of global concern as they can confer resistance to carbapenems and almost all β-lactams (33). Elizabethkingia is the only organism known to carry two distinct MBL genes (blaBlaB and blaGOB) and the blaCME gene, which can confer resistance to cephalosporins (21). Resistance genes, including gyrA, gyrB, parC, and parE, and efflux pump genes, including RND, MFS, MATE, and ABC, were detected in Elizabethkingia isolates. The presence of multiple drug resistance genes in Elizabethkingia increases the difficulty of clinical treatment (27).

PFGE typing reveals genetic diversity and clonal transmission. Although E. anophelis is genotypically highly diverse, clonal transmission has been observed in several pairs of patients from the same or different departments. From April to August in 2022, E. meningoseptica isolates were genetically homogeneous (2/7 strains were type A and 2/7 strains were type B) in the hematology and emergency ICU wards, suggesting recent clonal amplification and persistence between the wards. Previous reports have found that the acquisition of Elizabethkingia may be associated with water sources or water-related equipment, such as sinks and hand hygiene sink aerators in the hospital environment (11, 29). Clonal transmission may be mediated by the hands of hospital staff or patients; therefore, there is a need for better hand hygiene and environmental cleanliness if an isolate is detected in hospitals (15).

It should be noted that this study has several limitations. (i) This is a single-center study with some bias in the data, and follow-up studies with larger and more extensive multicenter are needed. (ii) We have not made any further distinction between Elizabethkingia isolates by sequencing the 32rpoB gene and by sequencing the whole genome. (iii) No further exploration of biofilm formation was performed .

Elizabethkingia infection has become an important public health concern; therefore, it is crucial to understand its clinical, molecular, and genetic characteristics. In the present study, 16S rRNA gene sequencing was performed on 103 Elizabethkingia isolates. Microbiological characterization of the identified Elizabethkingia isolates revealed the resistance patterns and genetic diversity of the clinical isolates at this site. As much research and clinical practice continue to rely on automated bacteriological identification systems to characterize Elizabethkingia, upgrading MALDI-TOF mass spectrometry with expanded reference databases, or the use of molecular techniques, is necessary to accurately characterize these microorganisms. Elizabethkingia exhibits variable susceptibility to various antibiotics; therefore, if antimicrobial susceptibility testing is used as a guide, treatment will be more reliable. Our results show that minocycline has the potential to become the drug of choice for patients with Elizabethkingia infections; however, clinical trials are required. Further research is required to determine the optimal antimicrobial agents for these life-threatening infections, either alone or in combination.

Data availability

All data generated or analyzed during this study are included in this published article and raw data will be available upon request. The sequences of detected genes were submitted to the GenBank database under accession numbers PQ057235-PQ057337

Acknowledgements

The authors thank all the members in the laboratory.

Funding

This research was funded by the National Natural Science Foundation of China (grant number 82102411, 82260403,32370195); the Clinical Research Nurture Project of the First Affiliated Hospital of Nanchang University (grant number YFYLCYJPY202201); Science and Technology Program Project of the Health and Wellness Commission of Jiangxi Province(grant number 202130167).

Author information

Xiuhua Kang and Huaming Guo contributed equally.

Authors and Affiliations

^a Jiangxi Medical Center for Critical Public Health Events, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang 330052, China.

^b Infection Control Division, the First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P. R. China.

^c.First Clinical Medical College, Nanchang University, Nanchang, China.

^dDepartment of Clinical Laboratory, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, 17 Yongwai Zhengjie, Nanchang 330006, China

^e China-Japan Friendship Jiang Xi Hospital, National Regional Center for Respiratory Medicine, Nanchang 330006, Jiangxi, China

Contributions

The study was designed by DDW and YL, while HMG, STZ , PL, YFM and WZZ conducted the experiments. YL, HMG, LZ and XHK carried out the analysis, and HMG , XHK and DDW drafted the manuscript. All authors contributed to and approved the final version of the article .

Corresponding author

Correspondence to Yang Liu or Dandan Wei

Ethics declarations

Ethics approval and consent to participate

Based on the rules of the Ethical Committee of our institute, this study did not require informed consent statement, because all isolates were recovered from clinical specimens during routine diagnostic procedures and these isolates were not specific to this study. In addition, the patients were not available to us. Based on the points mentioned above, the Ethics Committee of the First Affiliated Hospital of Nanchang University approved our project and allowed this study to be conducted without informed consent statement.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interest.

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Prevalence, and Microbiological and Clinical Characteristics of Elizabethkingia Isolates from a tertiary hospital in Jiangxi Province, China

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Background

Methods

Species Identification Using 16S rRNA Gene Sequencing

Antimicrobial susceptibility testing

Molecular Typing

Statistical Analysis

Results

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