ABSTRACT

To analyse the genotypes of clinical isolates of Extended-Spectrum-β-Lactamase-Producing (ESBLproducing) Proteus mirabilis (P. mirabilis) and the mechanisms of antimicrobial resistance, to guide reasonable use of antibiotics and to avoid nosocomial outbreak infections by ESBL-producing P. mirabilis. 125 clinical isolates of P. mirabilis were collected from the Drug-Resistant Bacteria Surveillance Center of Anhui Province (from Jan 2009 to May 2010). Searching for the genotypes of ESBLs was perfomed by PCR amplification and DNA sequencing, and performed conjugation test simultaneously. Among ESBL-producing strains, CTX-M was the major genotype (3 CTX-M-13 and 1 CTX-M-3). TEM-1b spectrum β-lactamase was also prevalence in P. mirabilis. The diversity of β-lactamases in P. mirabilis and the emergency of multi-drug-resistance clinical strains will present serious threat to clinical therapy and even will lead to outbreak of nosocomial infections. Our study emphasizes the need for enhanced supervision of ESBL-producing P. mirabilis. Timely and reasonable drug-resistance data are indispensable to clinical therapy.

Keywords:Genotype; Extended-Spectrum-β-Lactamase; Antimicrobial Resistance; Proteus Mirabilis

1. Introduction

Proteus mirabilis is one of the most common gram-negative pathogens encountered in clinical specimens and can cause a variety of communityor hospital-acquired illnesses, including urinary tract, wound, and blood stream infections. This organism is intrinsically resistant to nitrofurantoin and tetracycline, but it is naturally susceptible to ß-lactams, aminoglycosides, fluoroquinolones, and trimethoprim sulfamethoxazole [1] .

Extended-spectrum ß-lactamases (ESBLs) have become increasingly common worldwide and have emerged as a major source of antimicrobial resistance in gram-negative pathogens. Except for Escherichia coli and Klebsiella pneumoniae, Proteus mirabilis is another common ESBL-producing gram-negative pathogen. It has been found that most ESBLs were derivatives of TEM-1 type, TEM-2 type and SHV-1 type of β-lactamase, which is composed of one or several point code gene mutation. In recent years, in addition to the TEM-type ESBLs, there also were increasing reports of CTX-M-type ESBLs procuced by Proteus Mirabilis [2] -[8] .

ESBLs confer resistance to penicillins, cephalosporins, aztreonam, and also associated with resistance to other classes of nonpenicillin antibiotics, including fluoroquinolones, aminoglycosides, trimethoprimsulfamethoxazole, and ß-lactam/ß-lactamase inhibitor combinations. Thus, ESBL-producing organisms often possess a multidrug resistance phenotype. Detection and susceptibility results of ESBL-producing Proteus mirabilis play an essential role in the treatment of infections caused by this pathogen and also in controlling the spread of ESBLs.

The aim of the present study was to evaluate the prevalence and the molecular distribution of ESBL-producing Proteus mirabilis in local area. We examined 125 clinical isolates of Proteus mirabilis, detected the existence of ESBLs and the antimicrobial resistance of the ESBL-producing strains.

2. Materials and Methods

2.1. Stains

125 stored isolates were from the Drug-Resistant Bacteria Surveillance Center of Anhui Province (from Jan 2009 to May 2010).

Organism identification was performed by Microscan GN combo card (Dade Behring, West Sacramento, CA, USA.). Pathogens identified as ESBL producers were subjected to PCR amplication and gene sequencing tests. E. coli ATCC 25922 was used as negative control for ESBL production. Standard ESBL-producing strains (TEM-1, TEM-26, SHV-5, CTX-M-3, CTX-M-24, TOHO-1, OXA-1, OXA-2, OXA-10) were used as positive control. All tests were performed according to CLSI guidelines. Minimum inhibitory concentrations (MIC) of 13 antimicrobials(ampicillin, ampicilin/sulbactam, piperacillin, piperacillin/tazobactam, cefoperazone, cefoperazone/sulbactam, ceftazidime, cefotaxime, gentamicin, amikacin, aztreonam, imipenem and ciprofloxacin) were determined by the micro-broth dilution method.

2.2. Polymerase Chain Reaction (PCR)

ESBL genes (blaTEM, blaSHV, blaCTX-M, blaTOHO and blaOXA) were amplified by PCR with the following sets of primers (Table 1).

2.3. Agar Gel Electrophoresis Test

Agar gel electrophoresis test (DYY-10C, Beijing Six-one Instrument Company) was used to compare PCR products with the standard ESBL-producing strains.

2.4. Gene Sequencing Test

Sequencing of PCR amplicons were performed twice on both strands with an ABI Prism 3730 DNA Squencer (Perkin-Elmer, Applied Biosystems Division), by the dideoxy chain termination method of Sanger method. The nucleotide sequences were analysed with the BLAST.

3. Results

3.1. PCR and Agar Gel Electrophoresis Test Results

125 stains of Proteus mirabilis were amplified by PCR with the former primers. There were 9 positive strains, including 5 blaTEM, 3 blaCTX-M-13 and 1 blaCTX-M-3. No positive strain was detected in the types of SHV, OXA-1, OXA-2, OXA-10, and TOHO-1.

3.2. Gene Sequencing Test

Sequencing analysis identified that 5 stains harboured blaTEM-1b, 3 harboured blaCTX-M-14(1 together with blaTEM-1b), and 1 harboured blaCTX-M-3 (Table 2).

3.3. Antimicrobial Susceptibility Tests

All the PCR positive strains expressed high resistance to ampicillin, piperacillin and cefoperazon, which MICs were >256, >256 and >64 respectively. But all the strains were sensitive to Imipenem, and the MICs ranged from 1 to 4. As showed in Table 3, the 4 CTX-M-positive P. mirabilis were all resistant to cefotaxime and ceftazidime, and, at the same time showed high resistance to the other antimicrobials listed in the table. On the contrary, the 5 TEM-1b-positive strains were all sensitive to cefotaxime and ceftazidime, which are one group of

Note: Abbreviations for antimicrobial agents follow (CLSI2012 breakpoints for susceptibility [S] and resistance [R] in micrograms per milliliter] are given in parentheses): AMP, ampicillin (S ≤ 8, R ≥ 32); SAM, ampicillin plus sulbactam (S ≤ 8/4, R ≥ 32/16); PIP, piperacillin (S ≤ 16, R ≥ 128); TZP, piperacillin plus tazobactam (S ≤ 16/4, R ≥ 128/4); CPZ, cefoperazone (S ≤ 16,R ≥ 64); CPS, cefoperazone plus sulbactam (S ≤ 16/8, R ≥ 64/32,t); CTX, cefotaxime (S ≤ 8, R ≥ 64); CAZ, ceftazidime (S ≤ 8, R ≥ 32); ATM, aztreonam (S ≤ 8, R ≥ 32); IPM, imipenem (S ≤ 4, R ≥ 16); AMK, amikacin (S ≤ 16, R ≥ 32); GEN, gentamicin (S ≤ 4, R ≥ 8); CIP, ciprofloxacin (S ≤ 1, R ≥ 4).

β-lac-temases and not belong to the family of ESBL. Also they were widely susceptible to ampicillin-sulbactam, pi-peracillin-tazobactam, cefoperazone-sulbactam, aztreonam, amikacin, gentamicin and ciprofloxacin.

4. Discussion

The present study revealed that the prevalent genetypes in ESBL-producing Proteus mirabilis in local area was CTX-M type (3 were CTX-M-14 type, 1 was CTX-M-13 type, and 1 was CTX-M-3 type). Besides, TEM-type β-lactemases were ubiquitous in clinical original Proteus mirabilis.

ESBLs have emerged as a major source of antimicrobial resistance in gram-negative pathogens and generally encoded by plasmid-borne genes. ESBL-producing organisms often possess a multidrug resistance phenotype. In spite of the worldwide use of β-lactam antimicrobial agents, which is considered as the main reason for the emergence of ESBLs [9] , the distributions of ESBLs are far from uniform. Some literatures reported that TEM- type ESBLs was prevalent in Proteus mirabilis [7] [10] -[13] . In 2000, TEM-72 ESBL was identified in Proteus mirabilis in Italy, who had 4 substituents in amino acid sequence: Q39K, M182T, G238S and E240K compared to TEM-1 type [14] . TEM-92 ESBL was isolated from Proteus mirabilis by French researchers in 2001 [15] . In a study of Enterobacteria, 76 stains were detected to produce ESBLs in 106 stains of Proteus mirabilis, and their genotypes were TEM-20, TEM-26, TEM-47, TEM-52 and TEM-87 [16] . In the current study, we only detected the existent of TEM-1 β-lactamase in P. mirabilis.

But the emergence of more and more CTX-M type ESBLs in Proteus mirabilis had aroused wide concern [17] [18] . 2003, Japanese researchers detected 19 stains of CTX-M-2 ESBL-producing Proteus mirabilis in a outbreak of nosocomial infection, which are all presented as multi-drug resistant phenotype [19] . Researchers in Hong Kong area detected CTX-M-13 and CTX-M-14 types of ESBLs from P. mirabilis. 13 genotype positive strains were analyzed. There were 8 CTX-M-14 ESBLs, which all simultaneous with TEM-2 gene. And 3 strains were CTX-M-13 type ESBLs and one of them was together with TEM-2 gene. There still were one TEM-11 and one TEM-1 strains [20] .

In the century, CTX-M-type ESBLs develop quickly and they have higher potentiality to hydrolyze cefotaxime than ceftazidime. Even they has the lower prevalence ratio than TEM-type ESBLs in Proteus mirabilis, they should receive more attention to their wide spectrum of substrate specificity. ESBL production was associated with severe adverse outcomes, including higher overall and infection-related mortality, increased length of stay, delay in appropriate therapy, discharge to chronic care, and higher costs [21] .

The diversity of distribution of ESBLs in P. mirabilis remind us it’s important to detect the genotype and the antimicrobial susceptibility pattern which are critical in the therapy of infections caused by ESBL-positive bacteria. Our results showed that except for imipenem, amikacin and piperacillin/tazobactam might be effective drugs in vitro too.

Acknowledgments

We thank all the members from the division of Clinical Microbiology (Dept. of Clinical Laboratory, the First Affiliated Hospital of Anhui Medical University) for their collecting of the bacterial stains and technical contributions to this work.

Funding: This work was supported by a grant (Code: 81171606) from the National Natural Science Foundation of China

References

NOTES

^*Corresponding author.