Isolation of CRPA from patients and environments in ICU. During the 11 months monitoring period, we collected 54 CRPA isolates in total, 27 of which were isolated from the environments and 27 were isolated from the infected and colonized patients of the 5 different ICUs in a large tertiary hospital, East China. (Fig.1 A) No CRPA was screened from the patients’ stool specimens in this study. Two CRPA strains were screened from sputum samples in two patients, and were judged as colonization. The 25 CRPA strains were isolated from the following samples including sputum (12/25, 48.00%), blood (4/25, 16.00%), urine (3/25, 12.00%), bile (2/25, 8.00%) and so on, and were judged as pathogens of infection. In addition, the 27 CRPA strains were isolated from ICU environmental samples including washbasins (12/27, 44.44%) and ventilator condensate (12/27, 44.44%), the clothes of medical workers (1/27, 3.70%), patient bedding (1/27, 3.70%), and air (1/27, 3.70%). The strains isolated from patients and ventilator condensate were associated with 31 patients, and CRPA strains could also be detected in the sputum of some patients with respiratory infections and in the condensate of used ventilators. (See Supplemental Table 1 for statistical analyses)
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Insert Figure 1 A-B
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Figure 1. Sample distribution and antimicrobial resistance of 54 CRPA isolates. A) Pie chart showing clinical sample distribution. B) Antibiotic resistance profiles. In these figures, asterisks represent significance level (p < 0.05: *, p < 0.01: **, p < 0.001: ***).
MLST,antibiotic resistance genes, virulence factors and antimicrobial susceptibilities of CRPA isolates. A total of 25 STs of CRPA were found in our study. ST463 (13/54, 24.07%) was predominant, followed by ST231 (5/54, 9.26%), ST244 (4/54, 7.41%), ST235 (2/54, 3.70%), ST275 (2/54, 3.70%), and other separated STs accounted for 51.85%. Nine ST463 CRPA strains were isolated from environment and 8 of which were distributed in washbasins from sewage room of each ICUs. Another ST463 strain was isolated from the cuff of a medical worker. The 4 ST463 CRPA strains including 2 strains from sputum and 2 strains from urine were respectively isolated from 4 patients infected with CRPA.
It was noteworthy that all 13 ST463 CRPA strains carried multiple antimicrobial resistance genes, namely, carbapenem resistant (blaKPC-2), aminoglycoside resistance (aph(3')-IIb), cephalosporin resistance (blaOXA-486), and fluoroquinolone resistance genes (crpP). All the 13 ST463 CRPA strains had exoU+/exoS+ virulence genotype, while the coexistence of exoU and exoS genes were not detected in other STs CRPA strain. A total of the 12 CRPA isolates carried the MBLs genes including the blaAFM-1 (7/54, 12.96%), blaVIM-2 (4/54, 7.41%) and blaIMP-1 (1/54, 1.85%). The blaAFM-1-containing CRPA strains respectively belonged to ST463 (3/7), ST275 (2/7), ST712 (2/7). The blaVIM-2-containing CRPA strains belonged to ST 244 (3/4) and ST155 (1/4). The blaIMP-1-containing CRPA strain belonged to ST606. Interestingly, there were 3 ST463 strains harboring both the blaAFM-1 and blaKPC-2 genes, which were isolated from the sewage room’s washbasin and the urine sample of patient in different ICUs.
Antimicrobial susceptibilities were summarized in Figure 1B and Figure 2. All of the 13 ST463 CRPA strains presented as DTR-PA, while the proportion of DTR-PA in the 41 non-ST463 isolates was only 34.15% (13/13, 100% vs, 14/41, 34.15%, p<0.0001). The ceftazidime/avibactam resistance rate of the 54 CRPA strains collected in this study was 42.59% (23/54), with the minimal inhibitory concentrations (MICs) ranges of 16-≥256 µg/mL, 69.57% (16/23) of which showed high levels of drug resistance (MICs ≥ 64µg/mL). (See Supplemental Table 2 for statistical analyses)
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Figure 2. The results of MLST, virulence genes, resistance genes, antimicrobial susceptibilities and biofilm formation abilities among the 54 CRPA isolates. The names of the first line are virulence genes, drug resistance genes and drug sensitivity tests, while the gene categories of the second line represent the functional classification of virulence genes and drug resistance genes respectively. From left to right, the name, sample source, and ST typing of the strain are indicated respectively.
Distribution and genetic relatedenss of ST463 strains in ICU. Figure 3. described the isolation time and space of the 54 CRPA strains and the spatial distribution of the 5 different ICUs (including ICU1, ICU2, ICU3, ICU4, and ICU5) located in 3 different buildings in the tertiary hospital. At different time periods, ST463 strains were detected in washbasins of sewage room in each ICUs. In addition, one ST463 strain (PA-F6) was isolated from the cuff of a health care worker in ICU1. It was found that CRPA strains with other ST types colonized in different washbasins near beds in ICU5. The CRPA strains were isolated from the sputum and ventilator condensate of patient P6, all of which exhibited the ST231 phenotype. Patient P4 was found to have long-term colonization of CRPA strains in the ventilator condensate, but no CRPA colonization/infection was found in patient’s respiratory samples during hospitalization. However, we isolated carbapenem-sensitive P. aeruginosa from sputum of the patient.
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Insert Figure 3 A-B
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Figure 3. A) Spatial location characteristics of the 5 ICU wards in 3 different buildings. B) Schematic diagram of CRPA strains collected from environments and patients in the ICU during the 11-month surveillance. The beds in a black box represent that they are arranged in the same room. Blue, green and red shade are used to represent CRPA strains isolated from ventilator condensate, ward environment and patients respectively. Strains belonging to the same patient are marked in the red dashed boxes and red arrows represent the transfer routes of patients. ST463-CRPA-positive sites are marked with black bolded boxes.
The SNP difference between the PA-A3 and PA-A6 isolated from the same washbasin with 1-month interval in ICU4 was 41 SNPs. However, PA-B4 differed from PA-B5 by only 22 SNPs differences, which were also isolated from the same washbasin in ICU2 with 1-month interval. Furthermore, it was found that PA-D3 isolated from sewage room’s washbasin in ICU5 had a SNP difference of 33-36 SNPs compared to the PA-A3 and PA-A6 isolated from the ICU4 located only one floor away from ICU5. Interestingly, the 2 ST463 strains (PA-H1, PA-D3) with 6-month interval colonized in different washbasins from different buildings revealed a close genetic relationship, which are only 8 SNP differences. In addition, the SNP difference between JYPA156 isolated from the urine sample of an infected patient and environmental strains PA-H1 and PA-D3 ranged from 22 to 30 SNPs. The isolation time of patient strains is between the two environmental strains, and all the three strains were isolated from different ICUs. Notably, the SNP numbers between Alcaligenes faecalis metallo-β-lactamase (AFM)-producing ST463-CRPA strains (JYPA156, PA-A3, PA-A6) mentioned above ranged within 41-59. (Fig. 3B and Fig. 4)
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Insert Figure 4 A-B
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Figure 4. Genome phylogenetic tree of ST463 isolates generated by panaroo and iTOL. A)The colored stripes represent the sample source. B) SNP matrix diagram of ST463 strains.
Biofilm Forming Ability. The proportion of environmental CRPA strains with stronger biofilm forming ability was significantly higher than that of CRPA strains related to patients (17/27, 62.96% vs 9/27, 33.33%, P<0.05). Furthermore, the proportion of ST463 isolates showed stronger biofilm forming ability was significantly higher compared with the non-ST463 isolates (10/13, 76.92% vs 16/41, 39.02%, P < 0.05).
Virulence Assessment. The mortality of infected larvae by the two CRPA strains (JYPA121, ST1743 and JYPA526, ST1026) isolated from colonized patients had no difference from that by the low-virulence control ATCC9027 (P > 0.05). Then, we compared the mortality of infected larvae between CRPA strains from environmental samples and infected patients’ samples. The mortalities of infected larvae by the two groups were similar to that by the high-virulence control group PA14, with no significant difference (P > 0.05) (Fig. 5A). It also showed that the mortality of infected larvae of environmental CRPA strains was similar to those of strains from the different sources of infected patients (Fig. 5B). While, the mortality of the ST463 strains infected larvae group was significantly higher than in the non-ST463 strains infected (P < 0.05) (Fig. 5C).
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Figure 5. Virulence potential in a G. mellonella infection model.