First Report of Extended-Spectrum β-Lactamases Among Clinical Isolates of Klebsiella pneumoniae in Gaza Strip,Palestine

Abstract

Sixteen broad-spectrum cephalosporin-resistant Klebsiella pneumoniae isolates were recovered between April and June 2013 from Palestinian hospitals, Gaza Strip. Genes encoding extended-spectrum beta-lactamases (ESBLs) and other resistance genes were characterized by polymerase chain reaction and sequencing. The following β-lactamase genes were detected: bla_CTX-M-15+ bla_SHV1+ bla_TEM-1 (six isolates), bla_CTX-M-15+ bla_SHV5+ bla_OXA-1 (two isolates), bla_CTX-M-14a (two isolates), bla_CTX-M-15+ bla_SHV33 (one isolate), bla_CTX-M-15+ bla_TEM-1 (one isolate), bla_CTX-M-15+ bla_SHV12+ bla_OXA-1(one isolate), bla_CTX-M-15+ bla_SHV5 (one isolate), bla_CTX-M-15+ bla_SHV1 (one isolate), and bla_CTX-M-3 (one isolate). The ISEcp1 (in four cases truncated by IS26), orf477, or IS903 sequences were found upstream or downstream of bla_CTX-M genes. The aac(6′)-Ib-cr gene was found in seven isolates. The qnrS1 and qnrB1 genes were detected in five isolates and two isolates, respectively. Seven isolates contained class 1 integrons with four gene cassette arrangements: dfrA5 (three isolates), dfrA12-orf-aadA2 (two isolates), dfrA17-aadA5 (one isolate), and aadA1 (one isolate). A high clonal diversity was also observed among studied isolates by pulsed-field gel electrophoresis (12 unrelated profiles). This study demonstrates for the first time the emergence and polyclonal spread of multidrug-resistant ESBL–producing K. pneumoniae isolates among patients in a hospital setting in Gaza Strip, Palestine.

Introduction

Klebsiella pneumoniae is among the most common multiresistant bacteria causing healthcare-associated infections and is mainly responsible for pneumonia, urinary tract infections, neonatal septicemia, and wound infections. Multidrug resistance in K. pneumoniae is most frequently linked with the production of extended-spectrum beta-lactamases (ESBLs).^1,2

ESBLs are plasmid-mediated enzymes derived initially from the classical TEM and SHV-type β-lactamases and hydrolyze oxyimino-beta-lactam agents such as third-generation cephalosporins and aztreonam. Other types have also been identified, in particular, the cefotaximase (CTX-M) ESBLs, which are being reported throughout the world and have largely replaced and outnumbered other types of ESBLs.^3,4

ESBLs were first isolated in Germany in 1983 from K. pneumoniae strain, since then a steady increase in resistance against cephalosporins has been seen among hospitalized patients worldwide.⁵ These ESBLs are most commonly produced by Klebsiella species, but may also occur in other Gram-negative bacteria. ESBL-producing strains of K. pneumoniae have caused major therapeutic problems worldwide since the majority is resistant to various antibiotics commonly used for treatment of most bacterial infections.⁶ Data from the global surveillance system on antimicrobial resistance of microbes (SMART 2008–2010) show that global ESBL-positive rate was 20.1% for K. pneumoniae, with marked geographical differences in the proportion of ESBL production among clinical isolates.⁷ Recently, results from the SENTRY Antimicrobial Surveillance Program found that in 2009–2012, ESBL phenotype rates among Klebsiella spp. were higher in the European Mediterranean regions (35.1%) than in the United States (19.5%).⁸ In the Middle East, several reports highlighted to what extent the spread of ESBL producers has become of serious concern, and this region might indeed be one of the major epicenters of the global ESBL pandemic.^9,10 The clonal dissemination of CTX-M-15–producing K. pneumoniae isolates has been reported in Saudi Arabia, Egypt, Iran, Oman, Lebanon, Gulf Council, Iraq, Kuwait, and Turkey.^9,11–19

No data are available, so far, on ESBL-producing K. pneumoniae in Gaza Strip, Palestine and very scarce data in West Bank, Palestine. There have been reports of bla_CTX-M-15, bla_CTX-M-56, bla_OXA-1, bla_SHV-1, bla_SHV-12, bla_SHV-32, and bla_TEM-1 genes in Gram-negative bacteria isolated from Palestinian patients in West Bank.^20,21

Keeping in view the above facts, we have investigated the molecular characteristics of ESBL-producing K. pneumoniae isolates obtained from Al-Shifa Hospital, Balsam Hospital, and AL-Remal Martyrs Health Center in Gaza Strip, Palestine.

Materials and Methods

Isolates and susceptibility testing

Sixteen ceftazidime- and/or cefotaxime-resistant K. pneumoniae isolates were recovered from clinical samples obtained in different wards of three major government hospitals in Gaza Strip namely Al-Shifa Hospital, Balsam Hospital, and AL-Remal Martyrs Health Center during April–June 2013, which represent 59.3% of all K. pneumoniae isolated. These isolates were obtained from wound (n = 8), urine (n = 5), and blood (n = 3), and only one isolate per patient was included in the study. Isolates were tested for ESBL production by the double-disk synergy screening method.²² Susceptibility testing to 16 antibiotics was carried out in K. pneumoniae isolates by the disk diffusion method.²² Antibiotics tested were as follows (μg/disk): ampicillin (10), cefoxitin (30), ceftazidime (30), cefotaxime (30), aztreonam (30), gentamicin (10), amikacin (30), tobramycin (10), amoxycillin–clavulanic acid (20/10), nalidixic acid (30), ciprofloxacin (5), imipenem (10), kanamycin (30), trimethoprim–sulfamethoxazole (1.25. 23.75), tetracycline (30), and chloramphenicol (30).

Characterization of β-lactamase genes and genetic environment of bla_CTX-M genes

The genes encoding TEM, SHV, OXA-1, CMY, and CTX-M type β-lactamases and the genetic environment of bla_CTX-M genes were analyzed by polymerase chain reaction (PCR) and sequencing.²³ In addition, plasmidic AmpC β-lactamases were studied using multiplex PCR.²⁴

Nucleotide segments and their deduced amino acid sequences were compared with the sequences included in the GenBank database, as well as with those deposited at the website: (www.lahey.org/Studies/), to describe the particular type of β-lactamase gene. The presence of ISEcp1, IS26, IS903, and orf477 sequences surrounding the bla_CTX-M genes was analyzed by PCR.²³

Pulsed-field gel electrophoresis analysis

The clonal relationship among the K. pneumoniae isolates was determined by pulsed-field gel electrophoresis (PFGE) using XbaI enzyme as previously described.²⁵ Banding patterns were compared by visual analysis and through the use of GelCompar II program (version 6.5 Applied Maths, Ghent, Belgium).

Detection of genes conferring resistance to non-β-lactam antimicrobial agents

The presence of genes associated with resistance to tetracycline [tet(A) and tet(B)], sulfamethoxazole (sul1, sul2, and sul3), gentamicin [aac(3)-I, aac(3)-II, and aac(3)-IV], and quinolones [qnr, qepA, and aac(6′)-Ib-cr] was determined by PCR.^25,26

Detection and characterization of integrons

The presence of intI1 and intI2 genes (encoding classes 1 and 2 integrases, respectively) and the 3′-conserved region (qacEΔ1-sul1 genes) of class 1 integrons were examined by PCR.²⁶ The variable region of classes 1 and 2 integrons was characterized by PCR and sequencing in all intI1- or intI2-positive isolates, respectively, and sequences were compared with those included in GenBank to identify the gene cassettes.

Results

Characterization of β-lactamase genes

The ESBL production was detected in all ceftazidime and/or cefotaxime-resistant K. pneumoniae isolates recovered in the studied hospitals in the analyzed period. β-lactamase genes identified among these isolates were the following ones (Table 1): bla_CTX-M-15 + bla_SHV1+ bla_TEM-1 (six isolates), bla_CTX-M-15 + bla_SHV5 + bla_OXA-1 (two isolates), bla_CTX-M-14a (two isolates), bla_CTX-M-15 + bla_SHV5 (one isolate), bla_CTX-M-3 (one isolate), bla_CTX-M-15 + bla_SHV1(one isolate), bla_CTX-M-15 + bla_TEM-1 (one isolate), bla_CTX-M-15 + bla_SHV12 + bla_OXA-1 (one isolate), and bla_CTX-M-15 + bla_SHV33(one isolate).

Table 1.

Characteristics of the 16 Broad-Spectrum Cephalosporin-Resistant Klebsiella pneumoniae Strains Detected in Clinical Samples of Unrelated Patients

						Class 1 integron
K. pneumoniae strains	Sample origin	PFGE	Resistance phenotype to non-beta-lactam antibiotics	CTX-M and genetic environment	Resistance genes detected outside integron	IntI1/qacEΔ1+sul1	Integron structure
Gh5	Blood	P1	GM, KAN, AMK, TOB, CHL	^aISEcp1-bla_CTX-M-15- orf477	bla_SHV-5, bla_OXA-1, aac(3)-II	−/−	−
Gh6	Blood	P2	GM, KAN, AMK, TOB, CHL, TET	^aISEcp1-bla_CTX-M-15- orf477	bla_SHV-33, aac(3)-II, tetA	−/−	−
Gh8	Wound	P3	GM, KAN, AMK, TOB, SXT, NAL, CHL, CIP	ISEcp1-bla_CTX-M-14- IS903	aac(3)-II, qnrS1, sul1	+/+	aadA1
Gh9	Blood	P2	GM, KAN, AMK, TOB, SXT, CHL	ISEcp1-bla_CTX-M-15- orf477	bla_SHV-5, bla_OXA1, aac(3)-II, sul1	+/+	dfrA5
Gh13	Urine	P4	GM, TOB, NAL, SXT, CIP	ISEcp1-bla_CTX-M-15- orf477	bla_TEM-1, aac(3)-II, aac(6′)-Ib-cr, sul2, qnrS1	−/−	−
Gh18	Wound	P2	GM, KAN, AMK, TOB, SXT, CHL	^aISEcp1-bla_CTX-M-15- orf477	bla_SHV-12, bla_OXA-1, aac(3)-II, sul1	+/+	dfrA5
Gh19	Wound	P2	GM, KAN, AMK, TOB, SXT, CHL, TET	^aISEcp1-bla_CTX-M-15- orf477	bla_SHV-5, aac(3)-II, sul1, tetA	−/−	−
Gh57	Urine	P5	KAN, AMK, TOB, SXT, NAL,CIP	ISEcp1-bla_CTX-M-15- orf477	bla_SHV-1, bla_TEM-1, aac(6′)-Ib-cr, sul1	+/+	dfrA12-orf-aadA2
Gh66	Urine	P6	GM, KAN, AMK, TOB, SXT, NAL, CIP, TET	ISEcp1-bla_CTX-M-15- orf477	bla_SHV-1, bla_TEM-1, aac(3)-II, aac(6′)-Ib-cr, qnrB1, sul1, sul2, tetA	+/+	dfrA12-orf-aadA2
Gh67	Wound	P7	-	ISEcp1-bla_CTX-M-15- orf477	bla_SHV-1, bla_TEM-1	−/−	−
Gh70	Wound	P8	GM, KAN, TOB, NAL, CIP, TET	ISEcp1-bla_CTX-M-15- orf477	bla_SHV-1, bla_TEM-1, aac(3)-II, aac(6′)-Ib-cr, qnrB1, tetA	−/−	−
Gh94	Wound	P9	GM, KAN, AMK, TOB, SXT, NAL, CHL, CIP, TET	ISEcp1-bla_CTX-M-14- IS903	aac(3)-II, qnrS1, sul1, tetA, tetB	+/+	dfrA17- AadA5
Gh106	Urine	P10	KAN, CHL, CIP, TET	ISEcp1-bla_CTX-M-15- orf477	bla_SHV-1, bla_TEM-1, aac(6′)-Ib-cr, qnrS1, tetB	−/−	−
Gh112	Urine	P11	KAN, AMK, TOB, CHL	ISEcp1-bla_CTX-M-3- orf477	aac(3)-II	+/−	dfrA5
Gh139	Wound	P12	GM, KAN, AMK, NAL, TET	ISEcp1-bla_CTX-M-15- orf477	bla_SHV-1, bla_TEM-1, aac(6′)-Ib-cr, tetA	−/−	−
Gh143	Wound	P13	GM, KAN, AMK, TOB, SXT, NAL, CHL, CIP, TET	ISEcp1-bla_CTX-M-15- orf477	bla_SHV-1, aac(3)-II, aac(6′)-Ib-cr, qnrS1, sul1, tetB	−/−	−

ISEcp1 disrupted by the IS26 element in the opposite orientation.

AMK, amikacin; CHL, chloramphenicol; CIP, ciprofloxacin; GM, gentamicin; KAN, kanamycin; NAL, nalidixic acid; SXT, sulfamethoxazole/trimethoprim; TET, tetracycline; TOB, tobramycin; PFGE, pulsed-field gel electrophoresis.

Genetic environments of bla_CTX-M genes

The ISEcp1 and orf477 sequences were found upstream and downstream of the bla_CTX-M-15 gene, respectively, in all 13 isolates that harbored this gene. The ISEcp1 sequence was truncated in four of these isolates by the IS26 element, located in the opposite position (Table 1). Similarly, the bla_CTX-M-3 gene was found flanked by orf477 and ISEcp1 sequences. The ISEcp1 and IS903 were detected, respectively, from upstream and downstream of the bla_CTX-M-14a gene (Table 1).

Resistance mechanisms to Non-β-lactam antimicrobial agents

Table 1 shows the antibiotic resistance phenotypes and genotypes of the 16 broad-spectrum cephalosporin-resistant K. pneumoniae isolates. Nine of the 16 ESBL isolates harbored sul genes, and in most of the cases contained one sul gene (sul1: 7 isolates; sul2: one isolate; and sul1+sul2: one isolate). Eight of the ESBL isolates harbored tet genes [tet(A): five isolates; tet(B): two isolates; and tet(A) + tet(B): 1 isolate]. Fifteen isolates were also resistant to aminoglycosides, and at least one gene encoding an acetyltransferase [aac(3) or aac(6′)] was found. The aac(3)-II gene was identified in 12 gentamicin-resistant isolates, and the aac(6′)-Ib-cr gene was found in seven isolates. The qnrS1 and qnrB1 genes were detected in five isolates and two isolates, respectively.

Characterization of integrons

Class 1 integrons were present in 7 of the 16 ESBL-positive isolates. The gene cassettes implicated in the resistance to trimethoprim (dfrA5, dfrA12, and dfrA17) and streptomycin/spectinomycin (aadA1, aadA2, and aadA5) were detected into four different genetic arrangements: dfrA5 (three isolates), dfrA12-orf-aadA2 (two isolates), dfrA17-aadA5 (one isolate), and aadA1 (one isolate). The dfrA5 was detected inside a class 1 integron lacking the qacEΔ1 and sul1 genes in one K. pneumoniae isolate (Gh112). No class 2 integron was detected among the studied isolates (Table 1).

Clonal relationship by PFGE

The PFGE analysis demonstrated 12 unrelated pulsotypes among the 16 ESBL-producing isolates. In contrast, the four Gh6, Gh9, Gh18, and Gh19 isolates showed an indistinguishable PFGE pattern (Table 1).

Discussion

Resistance against penicillin and cephalosporins is an increasing problem in both the developed and developing countries. Studies from developing countries, where usage of beta-lactam antibiotics is greatest because of their ready availability and relatively low cost,²⁷ could give more insights and information about this emerging resistance in Gram-negative bacteria. Recently, we reported a fecal carriage of CTX-M-15–producing E. coli isolates as the first report of an ESBL in a Mauritanian hospital, and the dominance of CTX-M-type among ESBL-producing Escherichia coli isolated from both Tunisian and Palestinian hospitals.^28–30 In this study, we characterized ESBL-producing K. pneumoniae recovered from Palestinian hospitals over a 3-month period (April–June 2013). To our knowledge, this is the first study of the genetic background of cefotaxime resistance in clinical K. pneumoniae in Gaza, Palestine and one of the first reports in Palestinian territory. There is only one previous report about the detection of three clinical K. pneumoniae isolates in the West bank harboring non-ESBL SHVs, including SHV-1 and SHV-32, which are unable to confer resistance to broad-spectrum cephalosporins.²⁰ Nonetheless, numerous reports are available from Middle East and North African countries.^9,10

In this report, 14 of the16 K. pneumoniae clinical isolates harbored ESBLs of the CTX-M-1 group, including isolates encoding bla_CTX-M-15 (n = 13) and bla_CTX-M-3 (n = 1) genes. This finding is in agreement with recent reports both in Middle East and other parts of the world, where CTX-M-15 remains the globally predominant ESBL enzyme.^31–34

This study is the first to identify CTX-M-3, a variant of CTX-M-15 in K. pneumoniae in Palestine. CTX-M-15 differs from CTX-M-3 in only one amino acid at position 240 (Asp→Gly), which confers increased catalytic activity against ceftazidime.³⁵ According to previous reports, the variant CTX-M-3 is very unusual in K. pneumoniae in North Africa and the Middle East. Most reports of bla_CTX-M-3 gene have been from Turkey and Algeria.^36,37 CTX-M-3 was recently described in clinical isolates of K. pneumoniae in Saudi Arabia.¹²

This study is also the first to identify CTX-M-14 in Palestine. CTX-M-14 is among the most successful family member of the CTX-M-ESBLs. The bla_CTX-M-14 gene has previously been reported in K. pneumoniae and E.coli isolates at low frequency in Jordan, Lebanon, Saudi Arabia, Kuwait, and Egypt.^11,38–40

The bla_CTX-M gene has been previously found to be associated to the ISEcp1 sequence. This IS element contains typical −35 and −10 putative promoter regions and may enhance the expression of β-lactamase genes.^41,42 In our study, we found the presence of ISEcp1 upstream of bla_CTX-M-15, bla_CTX-M-3, and bla_CTX-M-14 in all isolates containing those genes. Interestingly, this IS was found to be disrupted by the IS26 element in the opposite orientation in four of these isolates. A similar organization was reported in Tunisia and Algeria.^26,37 It is interesting to note that the presence of IS26 flanking ISEcp1 may affect the impact of this insertion sequence on the mobilization and expression of the bla_CTX-M-15 gene.²⁸

In the present study, all CTX-M-15–producing isolates harbor more than one β-lactamase gene, and nine isolates harbor three kinds, namely bla_TEM, bla_SHV, and bla_CTX-M. K. pneumoniae isolates carrying multiple β-lactamase genes have been previously reported in other studies.^13,43–45

Four isolates coexpressed bla_CTX-M-15 along with other ESBLs. Three isolates harbored both SHV-5 and CTX-M-15, and only one isolate coproduced both SHV-12 and CTX-M-15.

Similarly, coexistence of CTX-M-15 with SHV12 or SHV-5 has been recently described in K. pneumoniae isolates from Iran, Saudi Arabia, and Egypt.^{12, 45–46}

The coexistence of multiple ESBL genes within individual isolates is of concern because it has been reported that such isolates had a greater resistance to various classes of antibiotics mediated through acquisition of genes located on mobile genetic elements.⁴⁷

In our study, all ESBL-producing isolates showed a low level of susceptibility to most antimicrobial agents tested and this could be explained by the fact that the ESBL producers usually carry a multiresistant plasmid, and these also carry genes conferring resistance to β-lactam and non-β-lactam antibiotics.⁴⁸

A significant correlation between resistance to broad-spectrum cephalosporins and resistance to ciprofloxacin was already reported.⁴⁹ Previous studies emphasized that plasmid-mediated quinolone resistance (PMQR) genes are often linked with ESBL production as they can sometimes be found on the same mobile genetic elements.^31,50

The screening for PMQR determinants among the 16 ESBL-producing isolates showed the presence of qnrS1 genes in 2 CTX-M-14–producing isolates, aac-(6′)-Ib-cr in 2 CTX-M-15–producing isolates, qnrS1+ aac-(6′)-Ib-cr and qnrB1 + aac-(6′)-Ib-cr in 3 and 2 CTX-M-15–producing K. pneumoniae, respectively. These results support previous findings from different countries suggesting a larger dissemination of aac(6′)-Ib-cr with qnr determinants, especially in ESBL-producing K. pneumoniae carrying the bla_CTX-M-15 gene.^37,51–57 The current study may be the first Palestinian study, which reports the detection of qnrB2, qnrS, and aac(6′)-Ib-cr genes in ESBL-positive K. pneumoniae from clinical specimens. As a matter of fact, there is only one previous reference from Palestine that reports the detection of aac(6′)-Ib-cr and qnrB1 genes among ESBL-producing Enterobacteriaceae.²⁰

In this study, the detection of the qnr determinants (43.75%) among ESBL-positive isolates was higher than some other reports from Turkey (8.8%), Saudi Arabia (22.5%), Kuwait (15.6%), and Iran (32.5%), but lower than reports from India (82.6%). In contrast, the prevalence of aac(6′)-Ib-cr (43.75%) was also higher than Turkey (23.5%) and Kuwait (15%) and lower than previous reports from Saudi Arabia (55.7%), Iran (80%), and India (56.5%).^{50,55,56,58,59}

Resistance to gentamicin is also common in ESBL-producing K. pneumoniae isolates, as reported in several parts of the world, including north Africa^60,37 and the Middle East.^12,61,62 In this study, a marked resistance was observed to aminoglycosides (amikacin: 87.5%; gentamicin: 75%; and kanamycin: 75%). This finding concurs with recent studies of ESBL-producing K. pneumoniae isolates in Saudi Arabia, which indicated that more than 80% of the isolates were nonsusceptible to gentamicin.¹²

Several studies have documented the relationship between gentamicin resistance and the presence of aac(3)-II gene encoding the gentamicin modifying enzyme AAC(3)-II.^63,64 In agreement with these reports, aac(3)-II gene was detected in most gentamicin resistance isolates in this study.

ESBL-producing K. pneumoniae have been reported to harbor higher rates of class 1 integrons compared to non-ESBL isolates.^65–68 In the current study, class 1 integrons were commonly identified, and most of the genes included in the variable region encoded resistance to trimethoprim (dfrA5, dfrA12, and dfrA17) and streptomycin/spectinomycin (aadA1, aadA2, and aadA5). Similar to our study, class 1 integrons, including dfr and aadA gene cassettes, have been the most prevalent type of integrons in E. coli isolates in Palestine.²⁰ The current study is the first to characterize class 1 integrons in K. pneumoniae isolate in Palestinian territory.

The 3′-conserved segments of the class 1 integrons usually contain the genetic determinants qacEΔ1 and sul1.⁶⁹ It is important to underline that one of the 7 integron-positive isolates did not contain the conserved qacEΔ1-sul1 region. Defective integrons lacking the qacE1-sul1 region have been previously reported in Europe and North Africa, but have never been found in the Middle East.^23,70,71

ESBL-producing isolates were genetically heterogeneous, and 12 unique PFGE patterns were identified. This finding suggested that the high prevalence of CTX-M K. pneumoniae in our hospitals was mainly due to horizontal dissemination of gene transfer between unrelated isolates compared with the transmission of a single clone.

However, a certain degree of intrahospital spread of these resistant isolates was discovered. Four isolates showed an indistinguishable PFGE pattern and all belonged to the same hospital. These results indicate that clonal spread may play a minor role in the dissemination of bla ESBL genes in our hospital.

Conclusion

In conclusion, our study provides baseline information on antibiotic resistance patterns mediated by ESBL-positive K. pneumoniae isolates in Gaza strip, Palestine.

To our knowledge, this is the first report of ESBL genes and class 1 integron among K. pneumoniae isolates from Gaza Strip hospitals. Several ESBL genes were detected in clinical samples in the studied hospitals, but the bla_CTX-M-15 was the predominant one. The data presented herein illustrate the high genetic diversity among ESBL-positive clinical K. pneumoniae isolates from Gaza Strip, indicating the potential horizontal transfer of ESBL genes in different K. pneumoniae clones. The results should encourage further studies allowing us to gain knowledge of the genetic lineages of ESBL-positive clinical K. pneumoniae circulating in Palestinian hospitals, as well as in the evolution of ESBL types and their frequencies.

Footnotes

Disclosure Statement

No competing financial interests exist.

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First Report of Extended-Spectrum β-Lactamases Among Clinical Isolates of Klebsiella pneumoniae in Gaza Strip,Palestine

Abstract

Introduction

Materials and Methods

Isolates and susceptibility testing

Characterization of β-lactamase genes and genetic environment of blaCTX-M genes

Pulsed-field gel electrophoresis analysis

Detection of genes conferring resistance to non-β-lactam antimicrobial agents

Detection and characterization of integrons

Results

Characterization of β-lactamase genes

Genetic environments of blaCTX-M genes

Resistance mechanisms to Non-β-lactam antimicrobial agents

Characterization of integrons

Clonal relationship by PFGE

Discussion

Conclusion

Footnotes

Disclosure Statement

References

Characterization of β-lactamase genes and genetic environment of bla_CTX-M genes

Genetic environments of bla_CTX-M genes