Antimicrobial Resistance and Molecular Characterization of Gene Cassettes From Class 1 Integrons in Pseudomonas Aeruginosa S PDF
Antimicrobial Resistance and Molecular Characterization of Gene Cassettes From Class 1 Integrons in Pseudomonas Aeruginosa S PDF
We investigated the antibiotic-resistance phenotypes and molecularly characterized class 1 integron gene cassettes
from 113 Pseudomonas aeruginosa isolates from patients. Primers specific for the class 1 integron integrase (intI1)
gene were used to screen for these integrons using polymerase chain reactions (PCRs). The variable regions of the
integrons were PCR-amplified and sequenced. Sputum was the most common specimen (69.9%; 79/113) followed
by aseptic sites (21.2%; 24/113). Of the 113 isolates with phenotypic resistance to the tested antimicrobials, the
highest resistances were to ciprofloxacin (CIP) (26.55%), imipenem (IPM) (23.89%), and meropenem (MEM)
(23%). Carbapenem-sensitive P. aeruginosa (CS-PA) isolates displayed 23 patterns, and the predominant multidrug
resistance phenotype was CIP-levofloxacin (7.23%, 6/83). Carbapenem-resistant P. aeruginosa (CR-PA) isolates
displayed 12 patterns, and the predominant multidrug resistance phenotype was IPM-MEM (23.33%, 7/30). Class 1
integrons were detected in 14 (12.4%, 14/113) isolates, 7.22% (6/83) in CS-PA isolates, and 26.67% (8/30) in CR-
PA isolates. Six gene cassette arrays were detected, the most prevalent being aacA4-blaOXA101-aadA5 in five
isolates (4.4%, 5/113). Seventeen gene cassettes were detected. The most prevalent antibiotic-resistance gene
cassettes were aacA4 (6.2%, 7/113), blaOXA-1, and blaOXA-101. Extended-spectrum b-lactamase resistance genes
were detected. Some of the genes carried were similar to those in other species, but some had shared characteristics
among the P. aeruginosa isolates. Long-standing drug resistance genes appeared to be under elimination in
P. aeruginosa, whereas integrons conferring resistance to commonly used clinical drugs such as b-lactamases,
fluoroquinolones, and even carbapenems, as well as some other gene elements, were found to be newly integrated.
670
CLASS 1 INTEGRONS IN PSEUDOMONAS AERUGINOSA 671
starting with 3CS and 5CS. Nucleotide sequences were analyzed Resistance patterns
and compared using BLAST software. The strains were divided into two groups based on their
carbapenem resistance levels (CR-PA, 30 strains; CS-PA, 83
Statistical analysis strains) and the definition rules. The total number of
The data were analyzed using SSPS v17.0 (IBM, Armonk, multidrug-resistant P. aeruginosa (MDR-PA) isolates in the
NY). Comparisons were considered significant if p < 0.05. CS-PA group was 16.87% (14/83), whereas that of CR-PA
was 40% (12/30) (Table 3). The CS-PA isolates displayed
Results 23 patterns, the predominant multidrug resistance pattern of
which was CIP-LEV (7.23%, 6/83) followed by CIP–LEV-
Clinical samples and bacterial isolation IPM-MEM (3.61%, 3/83). ATM and CIP-LEV-IPM patterns
Altogether, 113 P. aeruginosa isolates were collected from were displayed by two strains. Only one strain exhibited
hospitalized patients. Most specimens were sputum (69.9%; another resistance pattern. Twelve patterns were seen for the
79/113), followed by aseptic site specimens (21.2%; 24/113), CR-PA isolates, and the predominant multidrug resistance
blood (5.3%; 6/113), and urine (3.5%; 4/113). CR-PA and pattern was IPM-MEM (23.33%, 7/30), followed by AK-
CS-PA were 30 and 83, respectively. ATM-CAZ-CIP-CSL-FEP-IPM-LEV-MEN-PRL-TOB-TZP
(10%, 3/30). Sixty P. aeruginosa isolates displayed sensi-
Resistance phenotypes tivity to all 13 antimicrobial agents.
Table 2 shows the antimicrobial resistance phenotypes of
Prevalence of class 1 integrons and amplification
the isolates toward 13 antimicrobial agents. Antimicrobial
products from the variable regions
susceptibility testing revealed that of the 113 isolates ex-
pressing phenotypic resistance to the tested antimicrobials, Class 1 integrons were detected in 14 (12.4%, 14/113)
the most common resistance was to CIP (26.55%) followed isolates (Table 3), 7.22% (6/83) in the CS-PA isolates and
by IPM (23.89%) and MEM (23%); all other antimicrobials 26.67% (8/30) in the CR-PA isolates. The variable regions
had values of <20% (Table 2). P. aeruginosa multidrug- of the class 1 integrons were amplified and analyzed suc-
resistant isolates (resistant to at least three antimicrobial cessfully for 10 intI1-positive isolates, but these regions
classes) were recovered from the two sets of isolates inves- failed to amplify in four intI1-positive isolates. In the re-
tigated in our study. Isolates with resistance to all the anti- maining 10 intI1-positive isolates, only partial variable re-
microbials tested occurred only in the CR-PA isolates. The gion sequences were amplified and sequenced.
highest prevalence of resistance in the CR-PA isolates was to Six gene cassette arrays were detected (Table 3), the most
IPM (93.33%) followed by MEM (86.67%), LEV (56.67%), prevalent of which were aacA4-blaOXA101-aadA5 (detected
and CIP (53.33%); all other antimicrobials had values of in five strains), whereas qnrvc-gcu165-arr2-dfrA22e, aacA4-
<40%. In the CS-PA isolates, the most common resistance gcu35-blaOXA-1-catB3, aadB-aac(6¢)-1I-pse-1, dhfr2-aacA4-
was to LEV (16.87%), followed by CIP (15.66%); all other aadA1-tna, and AadA22 were detected in 2, 2, 1, 1, and 1
antimicrobials had values of <20%. Comparison of drug re- isolates, respectively. aacA4-blaOXA101-aadA5, aacA4-
sistance in the CS-PA and CR-PA groups revealed that, with gcu35-blaOXA-1-catB3, and qnrvc-gcu165-arr2-dfrA22e gene
the exception of GEN and ATM, the other 11 antimicrobial cassette arrays were detected in CR-PA at 5/30, 2/30, and 2/30,
agents differed, and the differences were significant. respectively, whereas aadB-aac(6¢)-1I-pse-1, dhfr2-aacA4-
aadA1-tna, and AadA22 were detected in CS-PA in 1, 1, and 1
Table 2. Resistance Phenotypes and Comparison isolates, respectively. Strain numbers 39 and 70 both contained
of Carbapenem-Resistant Pseudomonas aeruginosa two gene cassette arrays (qnrvc-gcu165-arr2-dfrA22e and
with Carbapenem-Sensitive P. aeruginosa aacA4-gcu35-blaOXA-1-catB3).
CR-PA CS-PA Total (%) p Seventeen gene cassettes were detected, including
blaOXA-1/101, pse-1, aacA4, aac6-II, aadA1/A5//B,
AK 23.33 0 7.07 0.000 AadA22, arr2, catB3, dfrA22e, dhfr2, tna, qnrvc, and gcu35/
ATM 26.67 14.46 17.70 0.222 165 (Table 3). The most prevalent antibiotic resistance gene
CAZ 36.67 3.61 12.39 0.000 cassettes were aacA4 (7/113), which confers resistance to
CIP 53.33 15.66 26.55 0.000 aminoglycosides, aadA5 (5/113), which confers resistance
CSL 36.67 6.02 15.04 0.000 to streptomycin and spectinomycin, and blaOXA-101 (5/
FEP 36.67 0 9.73 0.000 113), which confers resistance to b-lactamases. The struc-
GEN 16.67 6.02 9.73. 0.079 tural characteristics and translation directions of the variable
IPM 93.33 0 23.89 —
LEV 56.67 16.87 27.4 0.000 region gene cassettes are shown in Fig. 1.
MEM 86.67 0 23.0 —
PIP 36.67 13.25 19.46 0.006 Comparison between integron-positive
TOB 30.00 4.81 12.39 0.001 and integron-negative isolates
TZP 30.00 6.02 12.39 0.001
Of the 14 integron-positive isolates, >70% were resistant
AK, amikacin; ATM, aztreonam; CAZ, ceftazidime; CIP, cipro- to CIP, LEV, and TOB, and >50% were resistant to the other
floxacin; CR-PA, carbapenem-resistant P. aeruginosa; CSL, cefoper- drugs. In the integron-negative isolates, only resistance to
azone/sulbactam; CS-PA, carbapenem-sensitive P. aeruginosa; FEP,
cefepime; GEN, gentamicin; IPM, imipenem; LEV, levofloxacin; IPM and MEM was close to 20%, whereas resistance to the
MEM, meropenem; PRL, piperacillin; TOB, tobramycin; TZP, other drugs was <20% or close to 0. Comparison of the drug
piperacillin/tazobactam. sensitivity of the two groups revealed that the drug
Table 3. Antimicrobial Resistance Phenotypes and Molecular Characterization
of Gene Cassettes in Class 1 Integrons
GenBank
Sample no. Resistance pattern Int Length Gene cassettes accession no.
1, 2, 4, 8, 9, 15, 17, 20, 22, 26, Sensitive to all drugs - - -
27, 29, 30, 31, 34, 38, 38, 44,
45, 47, 49, 50, 52, 53, 55, 56,
57, 60, 62, 63, 64, 65, 66, 67,
68, 69, 73, 74, 75, 76, 77, 78,
79, 80, 82, 84, 89, 93, 95, 96,
98, 102, 106, 107, 109, 110,
112, 114, 115, 116
CS-PA
3, 83 ATM - - -
117 PRL - - -
25, 51, 92, 94, 99, 105 CIP, LEV - - -
85 ATM, PRL - - -
13 LEV, PRL - - -
41 ATM, CIP, LEV - - -
100 ATM, CIP, LEV + - -
43 CAZ, PRL, TZP - - -
5, 88 CIP, LEV, IPM - - -
61 CIP, LEV, PRL - - -
104 CIP, LEV, GEN + - -
35 ATM, CSL, PRL + 1.1k AadA22 CP041394.1
99.48%
23 GEN, PRL, TOB, TZP + 2.1k aadB-aac(6¢)- DQ266447.1
1I-pse-1 97.6
32 ATM, CSL, PRL, TZP - - -
6, 11, 19 CIP, LEV, IPM, MEM - -
48 ATM, CIP, LEV, MEM - - -
16 CSL, LEV, IPM, MEM - - -
87 ATM, CIP, GEN, LEV, TOB - - -
108 ATM, CAZ, CSL, PRL, TOB - - -
18 CAZ, CIP, FEP, GEN, LEV, TOB + - -
111 AK, ATM, CIP, CSL, GEN, TOB + 3.2k dhfr2-aacA4-
aadA1-tna
97 ATM, CIP, CSL, GEN, LEV, PRL, - -
TOB
42 ATM, CAZ, CIP, CSL, GEN, LEV, - - -
PRL, TOB, TZP
CR-PA
21 IPM, MEM, LEV - - -
14 IPM, MEM, PRL - - -
7, 10, 12, 40, 59, 81, 113 IPM, MEM - - -
72 CAZ, IPM, MEM, PRL - - -
103 CIP, FEP, LEV, IPM, MEM - - -
28 ATM, CAZ, CSL, FEP, IPM, MEM, - - -
PRL
33 ATM, CAZ, CSL, IPM, MEM, PRL, - - -
TZP
54 CIP, CSL, FEP, GEN, IPM, LEV, + - -
MEN, PRL, TOB, TZP
71 AK, ATM, CAZ, CIP, CSL, FEP, + 2.1k aacA4-blaOXA101-
IPM, MEN, PRL, TOB, TZP aadA5
36, 46, 101 AK, ATM, CAZ, CIP, CSL, FEP, + 2.1k aacA4-blaOXA101-
IPM, LEV, MEN, PRL, TOB, TZP aadA5
39, 70 AK, CAZ, CIP, CSL, FEP, GEN, + 2.2k+2.8k qnrvc-gcu165- KU984332
IPM, LEV, MEN, PRL, TOB, TZP arr2-dfrA22e KU984333
aacA4-gcu35-
blaOXA-1-catB3
24 AK, ATM, CAZ, CIP, CSL, FEP, + 2.1k aacA4-blaOXA101- KM111259.1
GEN, IPM, LEV, MEN, PRL, aadA5 99.35%
TOB, TZP
+, presence of the PCR product; -, absence of the PCR product.
ATM, aztreonam (30 mg); IPM, imipenem (10 mg).
673
674 LIU ET AL.
resistance rates for the 13 antimicrobial agents in the Altogether, 113 P. aeruginosa isolates were collected,
integron-positive isolates were higher than those of the primarily from sputum (69.9%; 79/113), aseptic site speci-
integron-negative isolates, and the difference was significant mens (21.2%; 24/113), blood (5.3%; 6/113), and urine
(Table 4). (3.5%; 4/113).
The isolates from our study were CR-PA and CS-PA
types, comprising 30 and 83, respectively. Excluding IPM
Discussion and MEM, of the 11 other drugs, only GEN did not show a
In recent years, the emergence and spread of MDR bac- significant difference in resistance. Resistance in the CR-PA
teria have brought great challenges to the clinical treatment group was greater than that of the CS-PA group, and the
of bacterial infections. P. aeruginosa, a clinically important difference was significant, a finding also shown by Gu B21
Pseudomonas species, causes nosocomial infections world- and colleagues. This finding suggests that new drug-resistant
wide. During transmission of bacterial drug resistance, the genes are captured continuously by P. aeruginosa under the
movement of genes and other DNA sequences (e.g., drug selection pressure of an antibacterial agent, which enables
resistance plasmids, transposons, insertion sequences, and P. aeruginosa to survive if various medicines are adminis-
integrons, among others) plays an important role. Increased tered. In addition, based on the integron carrier, the col-
antibiotic resistance in P. aeruginosa isolates can be at- lected strains were divided into integron-positive and
tributed to their ability to obtain resistance by the movement integron-negative groups, from which drug resistance was
of genetic elements. Because of the role played by class 1 compared. The results showed more integron-positive
integrons in the rapid dissemination of multiple genes en- groups than integron-negative groups in all 13 commonly
coding antibiotic resistance,6 we analyzed the prevalence of used clinical drugs, and the difference was significant. This
class 1 integrons in P. aeruginosa. result is similar to that of other scholars.5,21–23
Class 1 integrons were detected in 14 (12.4%, 14/113)
isolates, a similar finding to other research from China.5,21–23
Table 4. Comparison Between Integron-Positive However, this result is quite different from those of studies
and Integron-Negative Isolates in which the determinants of drug resistance related to in-
tegrons were found in the isolates from several P. aeruginosa
int+ (%) int- (%) p outbreaks.6,24,25 Studies have shown that almost half of all
AK 50.00 0.00 0.000 drug-resistant P. aeruginosa strains carry integrons.6,26 It
ATM 57.14 12.12 0.000 has been suggested that the homozygous carriage rate for
CAZ 57.14 6.06 0.000 P. aeruginosa in China is lower than that in other coun-
CIP 78.57 18.18 0.000 tries, and the results of our study are in accordance with
CSL 64.29 7.07 0.000 data from China.5,21
FEP 64.29 2.02 0.000 We detected six gene clusters containing 17 gene cas-
GEN 50.00 3.03 0.000 settes in the variable regions of the isolates. These gene
IPM 57.14 20.20 0.003 cassettes contain not only aminoglycoside and trimethoprim
LEV 71.43 21.21 0.000 drug-resistant genes (the antibiotics for which were used
MEM 57.14 18.18 0.001 commonly in China in the early to mid-20th century) but
PIP 64.29 13.13 0.000
TOB 71.43 3.03 0.000 also genes encoding resistance to b-lactamases, fluor-
TZP 64.29 5.05 0.000 oquinolones, and even carbapenem (the antibiotics for
which are used widely at present). Previously, these genes
int+: integron-positive isolates; int-: integron negative isolates. were underdetected or detected mainly in other species,
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