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Brazilian Journal of Microbiology

Print version ISSN 1517-8382

Braz. J. Microbiol. vol.44 no.2 São Paulo  2013 



Carbapenem and cefoxitin resistance of Klebsiella pneumoniae strains associated with porin OmpK36 loss and DHA-1 β-lactamase production



Weifeng ShiI; Kun LiI; Yun JiI; Qinbo JiangI; Yuyue WangI; Mei ShiI; Zuhuang MiII

IDepartment of Clinical Laboratory, Third Affiliated Hospital of Suzhou University, Changzhou, P.R. China
IIWuxi Clone Gen-Tech Institute, Wuxi, P.R. China

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Clinical isolates of carbapenem-resistant Klebsiella pneumoniae (K. pneumoniae) strains are being increased worldwide. Five pan-resistant K. pneumoniae strains have been isolated from respiratory and ICU wards in a Chinese hospital, and reveal strong resistance to all β-lactams, fluoroquinolones and aminoglycosides. Totally 27 β-lactamase genes and 2 membrane pore protein (porin) genes in 5 K. pneumoniae strains were screened by polymerase chain reaction (PCR). The results indicated that all of 5 K. pneumoniae strains carried blaTEM-1 and blaDHA-1 genes, as well as base deletion and mutation of OmpK35 or OmpK36 genes. Compared with carbapenem-sensitive isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the resistant isolates markedly lacked the protein band of 34-40 kDa, which might be the outer membrane proteins of OmpK36 according to the electrophoresis mobility. In addition, the conjugation test was confirmed that blaDHA-1 mediated by plasmids could be transferred between resistant and sensitive strains. When reserpine (30 µg/mL) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 µg/mL) were added in imipenem and meropenem, the MICs had no change against K. pneumoniae strains. These results suggest that both DHA-1 β-lactamase and loss or deficiency of porin OmpK36 may be the main reason for the cefoxitin and carbapenem resistance in K. pneumoniae strains in our hospital.

Key words: Klebsiella pneumoniae, AmpC β-Lactamase, Porin, molecular biology.




K. pneumoniae is one of the most common pathogens in clinical infections, such as pneumonia, urinary tract infections, sepsis, wound infections, meningitis and other diseases. Multidrug-resistant K. pneumoniae strains are becoming a severe problem worldwide, and it usually carries one or more extended-spectrum β-lactamases (ESBLs) that confers the resistance to expanded-spectrum cephalosporins (Lee et al., 2007; Huang and Hsueh, 2008). In recent years, K. pneumoniae strains have been reported to be resistant to most β-lactamas through mutation genes encoded in chromosome, acquisition of genes from mobile plasmids and integrons (Maltezou et al., 2009; Munoz-Price and Quinn, 2009).

Carbapenem antibiotics are very effective in the treatment of severe bacterial infections caused by ESBL-and AmpC enzymes-producing K. pneumoniae strains so that they have been widely used in clinics. Due to carbapenemases, metallo-β-lactamases and porin loss, carbapenem-resistant K. pneumoniae strains are increasing (Landman et al., 2009; Pournaras et al., 2010). Up to now, it has been reported that the outer membrane proteins of K. pneumoniae strains contain three known porins including OmpK35, OmpK36, and OmpK37 (Hernandez-Alles et al., 1999; Kaczmarek et al., 2006). Furthermore, porins OmpK35 and OmpK36 play critical roles in the penetration of antibiotics into the cells, and the loss of OmpK35 or OmpK36 can resist or reduce susceptibility to cephalosporins and carbapenems, particularly in strains containing Ambler group A, B, C, or D β-lactamase (Doumith et al., 2009; Goldfarb et al., 2009; Kontopoulou et al., 2010).

In 2010, we isolated 5 K. pneumoniae strains resistant to almost all antibiotics from clinical specimens. The minimal inhibitory concentrations (MICs) of piperacillin, piperacillin/tazobactam, amoxicillin/clavulanic acid, cefoperazone/sulbactam, cefoxitin, cefotaxime and aztreonam against 5 K. pneumoniae strains were larger than 128 µg/mL, and the MICs of imipenem and meropenem against 5 K. pneumoniae strains were larger than 32 µg/mL. In addition, these K. pneumoniae strains also revealed the resistance to ciprofloxacin, levofloxacin, gentamicin and amikacin (MIC > 64 µg/mL). The objective of this study was to investigate the cause of high-level carbapenem resistance in K. pneumoniae strains. Except for blaTEM-1 and blaDHA-1 genes, carbapenem-resistant genes were not observed in 5 K. pneumoniae strains; however, the base deletion or mutation of OmpK35 and OmpK36 genes could lead to the alterations of amino acid sequences, protein configuration and pore size of porins, thus impeding the access of antimicrobials. Therefore, high-level resistance to cefoxitin and carbapenem in 5 K. pneumoniae clinical isolates is due to the combinatorial action from the production of blaDHA-1 beta-lactamase and the insertional inactivation or loss of porin OmpK36.



Strains and antimicrobial susceptibility testing

In 2010, 5 carbapenem-resistant K. pneumoniae (Kp01, Kp02, Kp03, Kp04 and Kp05) strains were isolated from sputum, urine and deep venous catheter specimens in a Chinese hospital. Among 5 patients, 1 case was from respiratory ward and 4 cases were from ICU ward. The MICs of piperacillin, peracillin/sulbactam, amoxicillin/clavulanic acid, cefoperazone/sulbactam, cefoxitin, cefotaxime, cefepime, aztreonam, imipenem, meropenem, ciprofloxacin, levofloxacin, gentamicin and amikacin were performed by E-test (Oxiod), and E. coli ATCC25922 was used as the control.

Extraction of β-lactamase and three-dimensional test

Briefly, a colony was inoculated into 5 mL of tryptic soy broth overnight and the culture was grown at 35 ºC for 4 h. The cells were concentrated by centrifugation, and crude enzyme extract was prepared by repeated freezethawing cycle for five times. The surface of a Mueller-Hinton agar plate was inoculated with E.coli strains as described by the standard disk diffusion method. A 30 µg cefoxitin disk (Oxoid) was placed on the inoculated agar. With a sterile scalpel blade, 4 slit beginning 5 mm from the edge of the disk was cut in the agar in outward radial direction. By using a pipette, 40 µL of crude enzyme extract was dispensed into each slit, beginning near the disk and moving outward. Slit overfill was avoided. The inoculated media were incubated overnight at 35 ºC. Enhanced growth of the surface organism at the point where the slit intersected the zone of inhibition was considered a positive three-dimensional testing result and was interpreted as the evidence in the presence of AmpC beta-lactamase or ESBL. K. pneumoniae ATCC700603 and Enterobacter cloacae (E. cloacae) 029 M were used as the control strains.

Transconjugation experiments

In order to determine if the resistance was transferable, transconjugation experiments were performed. Five isolates were used as the donors and EC600 (Rif r) was used as the recipient in transconjugation experiments. The organisms were inoculated into 5 mL of Luria-Bertani (LB) broth (Difco) and incubated at 35 ºC for 20 h with shaking. Two organisms were mixed together at a proportion of 1 : 2, and then incubated overnight at 35 ºC with shaking. After centrifugation for 5 min at 4,000 g, the precipitate was mixed with 1 mL of distilled water and inoculated onto

MacConkey agar plates containing 600 µg/mL rifampin and 0.25 µg/mL cefotaxime. Transparent colonies were selected from the agar plates and inoculated into MacConkey agar again for activation. The drug-resistant profile and plasmid electrophoresis were further analyzed.

DNA extraction

Strains were grown overnight on MacConkey agar plates at 37 ºC, and growth from approximately one quarter of a plate was resuspended in 180 µL of distilled water. A total of 200 µL of buffer solution (0.01 mM Tris-HCl, pH 7.8; 0.005 M EDTA; 0.5% sodium dodecyl sulfate) and 20 µL of proteinase K (1 mg/mL) were added. The mixture was incubated at 55 ºC for 2 h, and then 400 µL of phenol-chloroform solution was added, mixed with gentle agitation, and centrifuged at 12,000 g for 5 min. The supernatant was collected and DNA was precipitated after the addition of 0.5 volume of 7.5 M ammonium acetate and 2 volumes of ethanol. DNA was washed with 70% ethanol, dried, and resuspended with 100 µLof Tris-EDTA buffer.

Analysis of β-lactamase and porin genes

The oligonucleotide primers were designed on the basis of the nucleotide sequence in GenBank (Table 1). The 27 β-lactamase and 2 porin genes were screened by PCR. The primer pairs were shown in Table 1. The assay was carried out in 20 µL of reaction mixture containing 0.5 µM each primer, 10 mM KCl, 2 mM MgCl2, 8 mM (NH4)2SO4, 10 mM Tris-HCl (pH 9.0), 200 µM dNTP and1UTaq DNA polymerase. All amplification reactions were performed with an initial denaturation at 93 ºC for 3 min followed by 35 cycles of denaturation at 93 ºC for 1 min, annealing at 55 ºC for 1 min and extension at 72 ºC for 1 min. The final extension was performed at 72 ºC for 7 min. Aliquot (20 µL) of each sample was subjected to electrophoresis by 2% agarose gels. The amplicons were purified with PCR Cleanup kits (Roche Molecular Biochemicals) and sequenced on an ABI PRISM 377 analyzer (Applied Biosystems).

Outer membrane protein analysis

K. pneumoniae was inoculated overnight in 5 mL of LB medium at 35 ºC with shaking, the culture was added in 200 mL of LB liquid medium at 35 ºC with shaking for 5 h. After centrifugation (4 ºC) for 5 min at 4,000 g, the supernatant was discarded. The precipitate was washed three times with 50 mM (pH 7.0) phosphate buffer solution (PBS), and suspended in 10 mM Tris-HCl (pH 7.4). The ultrasonic fragmentation of bacteria was performed in ice bath conditions. In order to remove unbroken bacteria, centrifugation was performed at 4,000 g for 30 min, and then the supernatant was centrifuged at 25,000 g for another 30 min. The precipitation was resuspended with buffer, and phenylmethylsulfonyl was added. The outer membrane protein was denatured at 100 ºC for 5 min before electrophoresis and then separated by SDS-PAGE with Mini-sub cell horizontal electrophoresis tank (Biorad Co.). The gel concentration was 12.0%, and stained with Coomassie brilliant blue R250 for 30 min.

Inhibitory effects of reserpine and CCCP against efflux

Susceptibility test was carried out using agar dilution method. MIC changes were observed in the absence or presence of reserpine (J & K Chemical) and CCCP (Sigma) at concentrations of 30 µg/mL and 50 µg/mL, respectively. An inoculum of each isolate at the amount of 5 x 104 cfu/mL was inoculated onto MH medium containing serial dilutions of imipenem and meropenem. A phenotype for positive efflux was detectable after at least 4-fold dilutions of MIC for imipenem or meropenem in the absence or presence of reserpine and CCCP.



Antimicrobial susceptibility test

In vitro tests showed that the MICs of piperacillin, piperacillin/tazobactam, amoxicillin/clavulanic acid, cefoperazone/sulbactam, cefoxitin, cefotaxime and aztreonam against 5 K. pneumoniae strains were larger than 128 µg/mL, and the MICs of imipenem or meropenem against 5 K. pneumoniae strains were larger than 32 µg/mL. In addition, 5 K. pneumoniae strains also revealed strong resistance to ciprofloxacin, levofloxacin, gentamicin and amikacin (Table 2).



Three dimension test

All 5 isolates exhibited positive three-dimensional test results, and their extracts promoted the growth of one surface organism of E.coli ATCC25922 on MH agar. E. cloacae 029 M could produce AmpC enzyme to exhibit an interference with the growth of ATCC25922. ATCC700603 was used as the negative control (Figure 1).



Sequence analysis of β-lactamase, OmpK35 and OmpK36 genes

The PCR and sequence analysis showed that Kp01, Kp02, Kp03, Kp04 and Kp05 contained TEM and DHA β-lactamase genes. Compared with blaTEM-1 (Accession No. FJ668746) and blaDHA-1 (Accession No. HM193083) in GenBank, these strains shared 100% identity. Similarly, OmpK35 and OmpK36 genes in 5 multidrug-resistant K. pneumoniae strains were detected by PCR amplification and sequence analysis. Among 5 K. pneumoniae strains, the base deletions of OmpK35 and OmpK36 genes were observed in Kp01 and Kp03 strains, and the base deletion of OmpK35 gene was also observed in Kp02 and Kp05. In OmpK36 gene, insertion and deletion as well as the mutations of short DNA fragment (1-5 bp) could result in the alteration of open reading frame and early termination of translation. In addition, mutations of OmpK35 gene occurred in Kp04 (Table 3). Compared with GU945384 in GenBank, OmpK35 gene mutations of G C at base 465 and T C at base 466 in Kp04 could also result in the substitution from Gln to His at position 155 and substitution from Tyr to His at position 156. A new subtype has been registered in GenBank (Accession No. HQ259027).



Transconjugation test of K. pneumoniae

EC600 was used as the recipient in the conjugation studies. In addition, blaDHA-1 resistant genes from Kp01, Kp02, Kp03 and Kp05 were transferred into the recipient EC600 and confirmed by PCR. EC600 resulted in drug resistance from Kp01, Kp02, Kp03 and Kp05 to a certain degree, and the MICs of piperacillin, piperacillin/sulbactam, amoxicillin/clavulanic acid and cefotaxime exhibited 2-16 fold increase (Table 4).



SDS-PAGE of outer membrane protein

The outer membrane proteins of 4 strains were detected by SDS-PAGE. Compared with the sensitive strains, the lack of outer membrane proteins in 3 carbepenem-resistant K. pneumoniae strains (Kp01, Kp02 and Kp04) were detected by SDS-PAGE, and molecular weights of these outer membrane proteins were 34-40 kDa, which suggested that only porin loss of OmpK36 in Kp01, Kp02 and Kp04 strains when compared with K. pneumoniae ATCC700603 and sensitive strains. In contrast, the porin of OmpK35 and OmpK36 was remained in Kp03 (Figure 2).



Inhibitory effects of reserpine and CCCP on the efflux of K. pneumoniae

All strains with a concentration of5x104 cfu/mL can grow in the presence of 256 µg/mL reserpine and 128 µg/mL CCCP. When reserpine (30 µg/mL) and CCCP (50 µg/mL) were added in imipenem and meropenem, no change of MICs was observed in the presence of reserpine and CCCP.



In the present study, we screened 27 β-lactamase genes including Ambler class A, B, C and D by using PCR technique. The metallo-β-lactamases, OXA-type and KPC-type β-lactamases were not detected by PCR in 5 K. pneumoniae strains. However, all of 5 strains carried blaTEM-1 and blaDHA-1 β-lactamase genes. DHA-1, a plasmid-mediated AmpC type β-lactamase belonging to Ambler class C, can confer the resistance to oxyiminocephalosporins (cefotaxime and ceftazidime) and cephamycins (cefoxitin and moxalactam), and transfer of the resistance has been confirmed by transconjugation (Lee, 2007; Ding et al., 2008; Yamada et al., 2009). In the present study, blaDHA-1 resistant genes from Kp01, Kp02, Kp03 and Kp05 may be transferred into the recipient EC600, leading to 2-16 fold MIC increase of piperacillin, piperacillin/sulbactam, amoxicillin/clavulanic acid and cefotaxime. These findings indicate that blaDHA-1 β-lactamase gene can spread rapidly between the same and different bacteria.

The base deletion of OmpK35 and OmpK36 genes in Kp01 and Kp03 was simultaneously observed. Similarly, the base deletion of OmpK35 also existed in Kp02 and Kp05. In addition, there were still genetic insertion of OmpK36 in Kp02, Kp04 and Kp05. SDS-PAGE analysis of the OMPs revealed the only loss of OmpK36 in Kp01, Kp02 and Kp04 strains when compared with K. pneumoniae ATCC700603 and a sensitive strains. However, the loss of porin was not observed in Kp03, which suggested that mutations or base deletions of porin-coding genes can lead to the alterations of open reading frame, amino acid sequences and protein configuration. Thus, the pore size of porin OmpK35 or OmpK36 can be affected and the accessibility of drugs can be impeded. Moreover, the porin OmpK36 plays an important role in the resistance or reduced susceptibility to carbapenems in K. pneumoniae strains (Hernandez-Alles, 1999; Wang et al., 2009). In this study, high-level carbapenem resistance in 5 K. pneumoniae strains was associated with the deletion or mutation of porin genes. The lack or deficiency of outer membrane protein can result in the change of permeability and reduction of cell accessibility for antibiotics or other drugs, which plays an important role in the main channels (Martinez-Martinez, 2008; Endimiani et al., 2009; Jiang et al., 2009; Landman, 2009). Therefore, blaDHA-1 β-lactamases combined with the absence of outer membrane proteins may confer to the carbapenems and cefoxitin resistance in K. pneumoniae strains (Su et al., 2008; Jiang et al., 2010).

Efflux systems that contribute to antibiotic resistance have been described for a number of clinically important bacteria (Guglierame et al., 2006; Coyne et al., 2010; Husain and Nikaido, 2010). The overexpression of multidrug efflux pumps can lead to low-level multi-drug resistance (Guglierame, 2006; Piddock, 2006). It has been reported that reserpine and CCCP were used as the pumps inhibitors (Shi et al., 2005). Reserpine is a well-established inhibitor of efflux pumps among Gram-positive microorganisms and non-fermenting Gram-negative microorganisms. CCCP can destroy the proton gradient of bacterial transcytoplasm membrane, causing transport proteins to lose energy supply and eventually leading to the increasing accumulation of drug concentration (Zhang et al., 2010). In this study, when reserpine and CCCP were added, MIC changes of imipenem and meropenem were not observed. The results indicated that the resistance of K. pneumoniae strains to carbapenems seems to be irrelevant to efflux.

Taken all together, the expression of porin OmpK36 coupled with blaDHA-1 β-lactamase genes plays an important role in conferring resistance of K. pneumoniae strains to carbapenems and cefoxitin in our hospital. Our findings highlight the urgent need to develop the strategies for the prevention and control of infections. Limited application of antimicrobials, especially for fluoroquinolones and cephalosporins, may be the effective strategies.



We really thank J Zhang and YH. Feng for their help of electrophoresis analysis.



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Send correspondence to:
W. Shi
Department of Clinical Laboratory, Third Affiliated Hospital of Suzhou University
No.185 Juqian Road
213003 Changzhou, P.R. China

Submitted: April 03, 2011
Approved: September 10, 2012



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