High Expression of Metallo-β-Lactamase Contributed to the Resistance to Carbapenem in Clinical Isolates of Pseudomonas aeruginosa from Baotou, China

Figures & data

Table 1 Primers Used in This Study

Gene	Primer Sequence (5ʹ→3ʹ)	Amplicon Size (bp)	Annealing Temperature (°C)
SIM	F- TACAAGGGATTCGGCATCG R-TAATGGCCTGTTCCCATGTG	741	60
IMP	F-GAAGGCGTTTATGTTCATAC R-GTATGTTTCAAGAGTGATGC	587	53
GIM	F- CCTGTAGCGTTGCCAGCTTTA R- CAGCCCAAGAGCTAATTGAGG	562	55
VIM	F-GTTTGGTCGCATATCGCAAC R-AATGCGCAGCACCAGGATAG	382	55
SPM	F- CTGCTTGGATTCATGGGCGC R- CCTTTTCCGCGACCTTGATCG	784	55
OprD2	F-CGCCGACAAGAAGAACTAGC R-GTCGATTACAGGATCGACAG	1332	55

Table 2 Distribution of CRPA Isolates in Clinical Specimens

Sources of Specimen	Strains	Ratio (%)
Sputum	52	83.9
Wound	5	8.1
Drainage	3	4.8
Urine	2	3.2
Total	62	100

Table 3 Distribution of CRPA Isolates from Various Clinical Departments

Department	Strains	Ratio (%)
Respiratory	22	35.6
Neurosurgery	13	21.0
Neurology	6	9.7
Orthopaedics	3	4.8
General surgery	3	4.8
Intensive care unit	3	4.8
Health Centers	3	4.8
Urology	2	3.2
Others	7	11.3
Total	62	100

Table 4 Drug Resistance Rates of P. aeruginosa Isolates from 2015 to 2017 (%)

Antimicrobial Agents	2015 (n=143)	2016 (n=105)	2017 (n=95)
Imipenem (IPM) (MIC: 8 µg/mL)	15.4*	26.7	13.8*
Meropenem (MEM) (MIC: 8 µg/mL)	11.5*	23.1	10.3*
Piperacillin (PIP) (MIC: 128 µg/mL)	12.7	20.4	11.5
Ciprofloxacin (CIP) (MIC: 4 µg/mL)	7.1	10.8	5.3
Levofloxacin (LVX) (MIC: 8 µg/mL)	9.9	5.9	5.3
Gentamicin (GEN) (MIC: 16 µg/mL)	9.2	7.8	7.7
Ceftazidime (CAZ) (MIC: 32 µg/mL)	10.2	19.0	14.8
Piperacillin/Tazobactam (TZP) (MIC: 128 µg/mL)	9.9	12.5	8.6
Cefepime (FEP) (MIC: 32 µg/mL)	7.7	15.2	14.9
Tobramycin (TOB) (MIC: 4 µg/mL)	4.6	4.8	3.3
Amikacin (AMK) (MIC: 64 µg/mL)	2.8	5.8	3.3

Notes: Differences between proportions were analyzed using the χ2 test. *p<0.05 compared to resistance rates in 2016.

Figure 1 Drug resistance rates of P. aeruginosa isolates from 2015 to 2017.

Figure 2 IPM-EDTA disk synergy assay for P. aeruginosa standard strain ATCC27853 (left) and P. aeruginosa clinical strain MBL (+) (right). The arrows indicated test papers loaded with IPM or IPM+EDTA, respectively.

Figure 3 Electrophoresis of PCR products of blaIMP, blaVIM, and blaSIM. Left panel: lane 1. CRPA38 isolate without blagGIM PCR product (562 bp); lane 2: CRPA38 isolate without blaSPM PCR product (784 bp); lanes 3 and 4: CRPA38 and CRPA48 isolates with blaVIM PCR product (382 bp); lane 5: CRPA42 isolate with blaIMP PCR product (587 bp). Right panel: lane 1: CRPA30 isolate with blaSIM PCR product (741 bp); lane 2: CRPA35 isolate with blaSIM PCR product (741 bp); lane 3: CRPA36 isolate with blaSIM PCR product (741 bp).

Figure 4 Analysis of partial sequence of OprD2 gene from clinical isolates. (A) Electrophoresis of PCR products for OprD2. Lane 1, 2, 3 and 7: CRPA43, CRPA 52, CRPA 56 and. CRPA 60 isolates with OprD2 PCR products (1332 bp); Lane 4, 5, 6: CRPA01, CRPA02, and CRPA03 isolates without OprD2 PCR products (1332 bp). (B) BLAST of OprD2 PCR products against P. aeruginosa standard strain ATCC27853.

Figure 5 (A) Real-time PCR analysis of mexR mRNA levels from a clinical isolate. # p<0.05 compared to other five strains. (B) Mutations in regulatory genes such as mexR, nalD and nalC may contribute to the overexpression of mexAB-OprM and increased resistance to carbapenem.