Molecular Characterization of β-Lactam Resistance and Antimicrobial Susceptibility to Possible Therapeutic Options of AmpC-Producing Multidrug-Resistant Proteus mirabilis in a University Hospital of Split,Croatia

Abstract

This study was performed to elucidate genetic relatedness and molecular resistance mechanisms of AmpC-producing multidrug-resistant Proteus mirabilis isolates in University Hospital of Split (UHS), and define efficient antibiotics in vitro. A total of 100 nonrepeated, consecutive, amoxicillin/clavulanate- and cefoxitin-resistant P. mirabilis isolates were collected, mostly from urine (44%) and skin and soft-tissue samples (30%). They were all positive in cefoxitin Hodge test and negative for extended spectrum beta-lactamase production. Pulsed field gel electrophoresis identified four clusters and two singletons, with 79% of isolates in dominant cluster. Molecular characterization and I-CeuI analysis of representatives revealed bla_CMY-16 gene located on chromosome, and insertion element ISEcp1 positioned 110 pb upstream of bla_CMY-16 starting codon. They also harbored bla_TEM-1, except one with bla_TEM-2. They were all resistant to trimethoprim-sulfamethoxazole, all but one to quinolones, and 81% to all aminoglycosides, while 77% were susceptible (S) and 22% intermediate (I) to piperacillin/tazobactam, and 4% were S and 68% I to cefepime. Only 15% were S to ceftolozane/tazobactam. Meropenem, ertapenem, ceftazidime/avibactam, temocillin, and fosfomycin were 100% efficient in vitro. This is the first report of bla_CMY-16 gene in P. mirabilis from hospital samples in Croatia. The findings are in accordance with Italian and Greek reports. The clonal nature of outbreak suggests the high potential of clonal spread. Alternative agents should be considered to spare carbapenem usage.

Introduction

In Proteus mirabilis, likewise in other members of Enterobacterales, acquired AmpC β-lactamases (ABLs) are one of the major clinical concerns worldwide. Their function is characterized by resistance to β-lactam-based β-lactamase inhibitors and even greater ability to hydrolyze cephalosporins and cephamycins than penicillins.¹ Although P. mirabilis originally lacks a chromosomal bla_AmpC gene, it was first reported in France in 1998,² and has been described in many countries since then.^3–12 ABLs most frequently reported in P. mirabilis are members of the CMY/LAT lineage which were most likely introduced to P. mirabilis via horizontal transfer of chromosomal beta-lactamase gene from Citrobacter freundii.¹ Some of European CMY-producing P. mirabilis strains showed significant relatedness, suggesting the possibility of a common origin with acquired and chromosomally inserted bla_CMY gene, followed by clonal spread and further continuous diversification due to mutations.¹⁰ The insertion sequence ISEcp1 and its position upstream and close to bla_CMY genes are associated with transposition and integration of mentioned genes into chromosome.^4,5,7,9,10 It can also be associated with promoter region providing a high-level expression of downstream bla_CMY gene, likewise other bla genes.^13,14 The strains with ABL often produce some other β-lactamase as well, such as TEM-1 or extended spectrum beta-lactamases (ESBLs), which makes detection of ABL phenotype more difficult.

Coproduction of different types of β-lactamases, their overproduction combined with reduction of porin permeability, or mutations that expand hydrolysis spectrum can contribute to a broader spectrum of β-lactam resistance.^15–20 In addition, resistance mechanisms that affect other antimicrobial groups can result in multidrug-resistant (MDR) and extensively drug-resistant strains that are of particular concern.^9,11,21,22 Along with an intrinsic resistance of Proteus species to colistin and tigecycline and intrinsically low activity of imipenem, development of resistance to last-resort antibiotics significantly reduces the treatment choice.

During routine work in University Hospital of Split (UHS), which is located in south Croatia, we noted an increased incidence of MDR P. mirabilis. Compared to previous β-lactam-resistant P. mirabilis isolates in our facility that were susceptible to amoxicillin/clavulanate (AMC) and cefoxitin, and have been proved to produce TEM-52 β-lactamase,^23,24 this new resistance pattern has aroused suspicion that some other underlying mechanism (probably ABL) was acquired.

The aims of this study were to elucidate genetic relatedness of MDR P. mirabilis isolates collected from inpatients at UHS, characterize molecular mechanisms of their β-lactam-resistance, and to define antimicrobial agents that still have good in vitro activity against these strains.

Materials and Methods

Study isolates

One hundred consecutive, nonduplicate MDR P. mirabilis isolates were collected from clinically significant samples of patients treated in UHS during a period from April 2013 to February 2014, according to resistance to AMC and cefoxitin that was determined routinely by disk diffusion method and interpreted according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines.²⁵ The identification to species level was performed using conventional biochemical methods.²⁶

Phenotypic characterization of β-lactamases

Double disk synergy test (DDST) was performed using cefotaxime, ceftriaxone, and ceftazidime disks applied next to a disk with amoxicillin and clavulanic acid (Bio-Rad, Marnes la Coquette, France) on Mueller-Hinton (MH) medium (Merck, Darmstadt, Germany) supplemented with 200 μg/mL of cloxacillin (bioMérieux, Marcy L'Etoile, France) for inhibition of AmpC enzymes, and interpreted as recommended.²⁷ Detection of ABL production was performed by cefoxitin Hodge test on MH medium using cefoxitin disk (30 μg; Bio-Rad) and Escherichia coli ATCC 25922, as previously described.²⁸

Molecular epidemiology

Genetic relatedness of all isolates included in this study was assessed by pulsed-field gel electrophoresis (PFGE) of SmaI digested genomic DNA using the CHEF-DR III System (Bio-Rad Laboratories) as previously described.²⁹ PFGE band pattern similarity calculation and clustering were performed by using the Dice Coefficient (1.8% tolerance) and unweighted pair group method with arithmetic mean (BioNumerics v7.6, Belgium). Further molecular characterization and localization of β-lactamase genes, as well as detection of ISEcp1 element, were done on representative isolates.

Molecular characterization of β-lactamase genes

Detection of AmpC beta-lactamase-encoding genes (bla_CMY, bla_MIR, bla_MOX, bla_FOX, bla_LAT, bla_DHA, bla_ACC, and bla_ACT) was performed by PCR.^10,30 The search for other beta-lactamase-encoding genes was also accomplished by PCR, namely: bla_TEM, bla_SHV, bla_CTX-M, bla_PER, bla_VEB, bla_GES, and bla_SME.³¹ All beta-lactamase genes detected by PCR were sequenced by ABI310 using BigDye v1.1 technology (ThermoFisher Scientific).

Localization of bla_AmpC genes and detection of ISEcp1 element

To identify whether bla_AmpC gene location was plasmid or chromosomal, Southern blot analysis of S1-digested and I-CeuI-digested PFGE gels was performed.^5,32 DNA was blotted onto positively charged nylon membrane and hybridized with bla_CMY-specific probe using DIG-High Prime DNA Labeling and Detection Starter Kit I (Roche, Germany). 16s rDNA-specific hybridization probe was also used for Southern blot analysis of I-CeuI-digested PFGE gels.⁵

The detection of ISEcp1 element by PCR and sequencing of the spacer region between ISEcp1 and bla_CMY was performed by using primers ampC5 (5′-CAG CGT TTG CTG CGT G-3′)¹⁰ and ISECP1 (5′-AAA AAT GAT TGA AAG GTG GT-3′).²⁹

Antimicrobial susceptibility testing

Antimicrobial susceptibilities of all isolates were determined by disk diffusion method for all routinely tested antibiotics: amoxicillin alone and combined with clavulanic acid, cefuroxime, cefotaxime, ceftriaxone, ceftazidime, cefoxitin, cefepime, piperacillin/tazobactam, imipenem, meropenem, ertapenem, ciprofloxacin, levofloxacin, trimethoprim-sulfamethoxazole, gentamicin, and amikacin (Bio-Rad). In addition, minimum inhibitory concentrations (MICs) were determined using gradient tests (Liofilchem, Roseto Degli Abruzzi, Italy) for those antimicrobial agents that showed some activity by disk diffusion method: piperacillin/tazobactam, cefepime, imipenem, meropenem, and ertapenem, as well as for not routinely tested antimicrobials that are possible alternatives to carbapenems: temocillin, fosfomycin, ceftazidime/avibactam, and ceftolozane/tazobactam (bioMérieux for the last two mentioned). Interpretative criteria for both disk diffusion method and gradient tests were based on EUCAST breakpoints,²⁵ with the exception of temocillin, which was interpreted according to the British Society for Antimicrobial Chemotherapy (BSAC) standards.³³

Results

Study isolates

Based on the resistance pattern mentioned earlier, a total of 100 nonrepeated P. mirabilis isolates were collected. They were isolated from various clinically significant specimens, obtained from patients hospitalized in 12 different wards. Isolates were most frequently obtained from urine samples (44%) and samples from skin and soft tissue infections (30%). The remaining isolates were obtained from the lower respiratory tract (12%), bloodstream (5%), and other sterile sites (9%). With regard to the hospital wards, isolates were most frequently detected in different internal medicine wards (n = 36), different surgery wards (n = 28), and intensive care units (n = 18). The rest of the isolates were collected from the patients at infectious diseases ward (n = 8), two medical wards (n = 8), and psychiatry ward (n = 2). Although these isolates were collected over a 10-month period, they were part of an outbreak at UHS that started before the collection period and lasted over a year before the incidence was reduced to sporadic cases.

Characteristics of the isolates in phenotypic β-lactamase detection

All isolates were negative for ESBL production as judged by the DDST performed on MH medium supplemented with cloxacillin. All of them were positive for AmpC production by cefoxitin Hodge test.

Clonal relationship of the isolates

Analyzed collection of isolates exhibited polyclonal genetic background, comprised four clusters and two single isolates (≥85% genetic linkage level³⁴). The most dominant cluster GC1 included 79% of isolates. They were collected at all the above mentioned wards, which underline the high potential of clonal dissemination of MDR P. mirabilis among patients hospitalized at different hospital units.

The remaining three clusters cover 19% of the isolates and came from ICU, surgery, and two medical wards. The singletons are from the internal medicine ward and the plastic surgery ward.

Molecular characterization and location of β-lactamase genes and ISEcp1

Gene bla_CMY-16 was detected in all 21 representative isolates included in further analysis. Coproduction of TEM beta-lactamase was observed in all isolates, of which TEM-1 was detected in 20 and TEM-2 in one representative isolate (Table 1). No large plasmids (>50 kb) were detected by S1-nuclease-PFGE and plasmid position of bla_CMY gene was not successfully determined using hybridization with bla_CMY-specific probe. Southern blot of I-CeuI-digested PFGE gels revealed that blaCMY-16 and 16s rDNA probes hybridized to the same DNA bands, indicating chromosomal localization of bla_CMY-16 gene in all representative isolates. PCR and sequencing confirmed the presence of ISEcp1 element in all of them as well, with an insertion located 110 pb upstream of bla_CMY-16 starting codon.

Table 1.

Epidemiological Data and Genotypic Characteristics of 21 Multidrug-Resistant AmpC-Producing Proteus mirabilis Representative Isolates Collected from Clinical Samples in University Hospital Split

Isolate	Date	PFGE profile	Specimen	Ward	ESBL	AmpC	CMYseq	TEMseq	ISEcp1
I/4	Apr 13	3	BA	PULM	−	+	CMY-16	TEM-1	+
I/7	Apr 13	4	WS	INT	−	+	CMY-16	TEM-1	+
I/10	Apr 13	1	WS	ICU	−	+	CMY-16	TEM-1	+
I/12	Apr 13	1	WS	INT	−	+	CMY-16	TEM-1	+
I/22	Jun 13	4	U	SUR (N)	−	+	CMY-16	TEM-1	+
I/23	Jun 13	2	AbdA	SUR (A)	−	+	CMY-16	TEM-1	+
I/27	Jun 13	3	U	NEU	−	+	CMY-16	TEM-1	+
I/29	Jun 13	1	S	PULM	−	+	CMY-16	TEM-1	+
I/34	Jun 13	Single	WS	SUR (P)	−	+	CMY-16	TEM-1	+
I/49	Jul 13	2	U	SUR (U)	−	+	CMY-16	TEM-1	+
I/55	Aug 13	3	AbsA	SUR (G)	−	+	CMY-16	TEM-1	+
II/25	Nov 13	3	WS	INF	−	+	CMY-16	TEM-1	+
II/39	Nov 13	3	U	PSY	−	+	CMY-16	TEM-1	+
II/7	Dec 13	3	U	INT	−	+	CMY-16	TEM-1	+
II/21	Dec 13	3	AbsA	SUR	−	+	CMY-16	TEM-1	+
II/35	Dec 13	3	U	SUR (G)	−	+	CMY-16	TEM-1	+
II/43	Dec 13	2	U	INT	−	+	CMY-16	TEM-1	+
II/50	Dec 13	3	U	ICU	−	+	CMY-16	TEM-1	+
II/16	Feb 14	4	B	ICU	−	+	CMY-16	TEM-1	+
II/48	Feb 14	Single	U	INT	−	+	CMY-16	TEM-1	+
II/49	Feb 14	4	U	SUR (U)	−	+	CMY-16	TEM-2	+

Among 100 isolates analyzed by PFGE, 21 representatives have been selected for further molecular analysis by random choice from every cluster and every different ward, and with an inclusion of both singletons.

A, abdominal surgery; AbdA, abdominal aspirate; AbsA, abscess aspirate; B, blood; BA, bronchial aspirate; ESBL, extended spectrum beta-lactamase; G, general surgery; ICU, intensive care unit; INF, infectious diseases; INT, internal medicine; N, neurosurgery; NEU, neurology; P, plastic surgery; PFGE, pulsed-field gel electrophoresis; PSY, psychiatry; PULM, pulmonology; S, sputum; SUR, surgery; U, urine; U, urology; WS, wound swab.

Antimicrobial susceptibility testing

All 100 isolates had MDR phenotype and were resistant to amoxicillin alone and combined with clavulanic acid, cefuroxime, cefotaxime, ceftriaxone, ceftazidime, cefoxitin, and trimethoprim-sulfamethoxazole. All but one were resistant to quinolones, and 81% were resistant to all aminoglycosides. They remained susceptible to carbapenems, along with piperacillin/tazobactam and cefepime, but with varying levels of susceptibility for the last two. Gradient tests performed for remaining susceptible antibiotics and new potential carbapenem alternatives showed that 77% of the isolates were susceptible (S) and 22% had intermediate susceptibility (I) to piperacillin/tazobactam, while 4% were S, and 68% I to cefepime (Table 2). Only 15% of isolates were S to ceftolozane/tazobactam. All isolates were fully susceptible to meropenem and ertapenem, while imipenem showed slightly higher MICs as expected,³⁴ thus 89.0% of isolates were S, and 11.0% I to it. Ceftazidime/avibactam, temocillin, and fosfomycin also showed 100% of efficacy in vitro.

Table 2.

In Vitro Susceptibility of Antimicrobial Agents That Remained Fully or Partially Active Against Multidrug-Resistant AmpC-producing Proteus mirabilis Isolates Collected from Clinical Samples in University Hospital Split (n = 100)

Antimicrobial agent	MIC (μg/mL)			MIC interpretation ^a (%)
Antimicrobial agent	50%	90%	Range	Susceptible	Intermediate	Resistant
Piperacillin/tazobactam	4	12	1–32	77.0	22.0	1.0
Ceftolozane/tazobactam	3	12	0.125–24	15.0	0	85.0
Ceftazidime/avibactam	0.094	0.25	<0.026–0.5	100	0	0
Cefepime	3	8	0.75–32	4.0	68.0	28.0
Imipenem	0.38	3	0.094–6	89.0	11.0	0
Meropenem	0.047	0.094	0.016–0.38	100	0	0
Ertapenem	0.016	0.032	0.008–0.19	100	0	0
Temocillin	2	3	1–6	100	0	0
Fosfomycin	0.75	2	0.19–8	100	0	0

MICs were determined using gradient tests, and interpretative criteria were based on EUCAST breakpoints,²⁵ except for temocillin where MICs were interpreted using breakpoints for Enterobacterales recommended by BSAC.³³

BSAC, British Society for Antimicrobial Chemotherapy; EUCAST, European Committee on Antimicrobial Susceptibility Testing; MIC, minimum inhibitory concentration.

Discussion

This study highlights the emergence of acquired ABL in P. mirabilis clinical isolates in UHS, that cause constitutively expressed cephalosporin resistance, and can be combined with other resistance mechanisms significantly reducing the choice of antimicrobial treatment options. It also highlights the high potential of clonal spread of such strains in clinical environment.

This is the first report of CMY-16 type of ABL in P. mirabilis hospital isolates in Croatia. The same type was found in Italy and Greece; therefore, we can assume that CMY-16 is a dominant type of acquired ABL in P. mirabilis in northern parts of the Mediterranean region.^5,7,9,10 The location of bla_CMY-16 gene on chromosome and presence of ISEcp1, as well as its position 110 pb upstream of bla_CMY-16 starting codon, are also in accordance with Italian and Greek reports.^5,7,9,10

Cephalosporin resistance in studied isolates was constitutively expressed, showing complete growth-to-disk resistance to all cephalosporins except cefepime (which showed varying levels of susceptibility). This kind of resistance profile could be an outcome of high-level expression, which in some enterobacteria could be a result of failed acquisition of AmpR, and/or consequence of acquired ISEcp1 seeing that this sequence can be inserted in the AmpR-binding site upstream of bla_CMY gene and provide promoter region to drive high expression of bla_CMY, as well as some other bla genes.^13,14,35 The findings of ISEcp1 in this study go in favor of the latter mentioned hypothesis.

Cefepime resistance is more often the result of ESBL production,^15,16 but since these were not detected in our study isolates, it may indicate the selection of mutants with extended-spectrum AmpC cephalosporinases (ESACs), enzymes with structural modification in vicinity of active site leading to broadened hydrolysis spectrum.^19,20

The TEM-1 and TEM-2 β-lactamases found here do not have influential role in resistance profile in ABL-producing isolates.^17,18,36

The constitutive expression and overexpression of AmpC enzyme significantly reduce treatment options, especially when this is accompanied with other resistance mechanisms. The investigation of antimicrobial resistome in similar MDR P. mirabilis isolate from northern Italy (IT NO-051/03) that carried bla_CMY-16 and bla_TEM-1b genes, also revealed the resistance genes to aminoglycosides, quinolones, chloramphenicol, tetracyclines, trimethoprim, and sulfonamides,²² which was out of the scope of our study, but phenotypic testing revealed a similar profile in most of our isolates.

One of possible treatment options for MDR P. mirabilis-related infections is piperacillin/tazobactam, to which 77% of our isolates were susceptible, and further 22% were susceptible to increased exposure (Table 2). Tazobactam is a weak inducer and a weak inhibitor of ABLs, but can still be sometimes effective in combinations such as with piperacillin.^1,37 Although it has been shown not to be inferior to carbapenems for treatment of infections caused by Enterobacterales with chromosomal inducible ABLs,^38,39 there are still no sufficient studies that show its clinical efficacy on strains with constitutive high-level production. Thus, this treatment option should be considered with caution until more studies are done.^7,40

Ceftolozane/tazobactam is a novel antipseudomonal β-lactam/β-lactamase inhibitor combination that shows variable in vitro activity against AmpC-producing enterobacteria.⁴¹ Only 15% of isolates were susceptible in this study, which supports a strong recommendation against the use of this antibiotic in infections due to AmpC-producing Enterobacterales.⁴²

Oppositely, ceftazidime/avibactam is broadly active against Enterobacterales with ESBL and/or AmpC enzymes,⁴³ and is recommended as an alternative to carbapenems for infections caused with such strains.⁴² Avibactam is a novel β-lactamase inhibitor structurally different than the other inhibitors clinically used so far, and in combination with ceftazidime, it has demonstrated absolute in vitro efficacy in this study (100%).

In regard to the aforementioned cefepime, no strong evidence was found to suggest that it was inferior to carbapenems in treatment of bloodstream infections due to chromosomal AmpC Enterobacterales,³⁸ but it seems that its efficiency can be taken for certain only for strains with MIC ≤2mg/L, since cefepime treatment of higher MIC values have shown to be inferior.⁴⁴ Furthermore, many AmpC-producing organisms are susceptible to cefepime when they are tested in conventional inoculum, but MICs dramatically increase when 100-fold-inoculum is used.⁴⁵ Accordingly, British Society for Antimicrobial Chemotherapy/Healthcare Infection Society/British Infection Association (BSAC/HIS/BIA) Joint Working Party, likewise EUCAST, recommends cefepime for treatment of infections caused by ESBL- or AmpC-producing bacteria with MIC ≤1 mg/L, however, it does not recommend cefepime even at increased dose for isolates with MIC >1 mg/L or those producing both ESBL and AmpC.^27,40 In this study, MIC_50% for cefepime was 3 μg/mL, which indicates that higher MIC values can be expected in such strains.

Carbapenems are usually considered as the drug of choice in case of MDR AmpC-producing Enterobacterales.^1,38 EUCAST does not advise imipenem for treatment of serious infections caused by P. mirabilis due to higher MIC values than those of other carbapenems.⁴⁶ The consequence of selective pressure due to carbapenem use is appearance of carbapenem-resistant strains, thus ABL and carbapenemase coproducing P. mirabilis strains have been reported.^9,11 The other possible mechanisms of acquired carbapenem resistance are high-level production of ABL in combination with porin loss^47,48 or selection of ESACs.^19,20

As possible carbapenem alternatives, we included temocillin and fosfomycin in antimicrobial susceptibility testing and both drugs showed 100% in vitro activity. Interpretation for temocillin was made according to BSAC, which is the only organization that has determined clinical breakpoints. Temocillin is a 6-α-methoxy derivate of ticarcillin, clinically and microbiologically effective against ESBL and/or derepressed AmpC-producing Enterobacterales and could be used as an alternative and a more narrowed therapeutic option than carbapenems.^49–51 In Europe, it is available in Belgium, United Kingdom, and Luxembourg only, and currently approved for treatment of septicemia and urinary and lower respiratory tract infections due to Enterobacterales. Although tested in vitro in this study, these excellent results should raise awareness of its possible use as an effective narrow spectrum option in other European countries as well.

Fosfomycin, like temocillin, is an old antibiotic which has been recovered because of possible activity against MDR isolates, including ESBL- and AmpC-producing Enterobacterales.⁵² It is approved for peroral use for uncomplicated urinary tract infections (UTI), and available as intravenous option for combination treatment of many different site infections, due to its favorable pharmacokinetic characteristics and excellent distribution to many tissues and organs.⁵³ In this study, it showed great results with 100% in vitro activity against MDR P. mirabilis strains. However, with an exception of a randomized controlled trial (RCT) for complicated UTI, including pyelonephritis and ongoing RCT for bacteremic UTI, all other sites of infection require more clinical evidence for recommendation of intravenous fosfomycin.^54,55

In conclusion, a suitable carbapenem replacement is a desirable option to limit selective pressure of carbapenems, and more clinical studies are needed to compare carbapenem alternatives for treatment of infections caused with MDR AmpC-producing P. mirabilis strains and other similar enterobacteria. It is worth mentioning that Proteus spp. have an intrinsic resistance to tigecycline and colistin, antibiotics that are usually left as the last resort for the treatment of infections caused by MDR gram-negative organisms. In fact, due to high incidence of infections caused by MDR Acinetobacter baumannii in our facility and subsequent therapy with colistin, it is possible that a selective pressure of colistin led to outbreak with MDR P. mirabilis. Possible further acquisition of carbapenem resistance could turn this pathogen to be one of the major concerns in hospital care-associated infections. The clonal nature of the outbreak suggests the high epidemic potential of such strains, so their spread should be monitored in a clinical environment and genetic background should be identified to control the spread of resistance mechanisms. Suitable infection control measures have to be the cornerstone in the fight against the dissemination of these strains.

Footnotes

Disclosure Statement

All authors certify that they have no actual or potential commercial associations that might create conflict of interest in connection with this article.

Funding Information

The authors received no financial support for the research, authorship, and/or publication of this article.

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Molecular Characterization of β-Lactam Resistance and Antimicrobial Susceptibility to Possible Therapeutic Options of AmpC-Producing Multidrug-Resistant Proteus mirabilis in a University Hospital of Split,Croatia

Abstract

Introduction

Materials and Methods

Study isolates

Phenotypic characterization of β-lactamases

Molecular epidemiology

Molecular characterization of β-lactamase genes

Localization of blaAmpC genes and detection of ISEcp1 element

Antimicrobial susceptibility testing

Results

Study isolates

Characteristics of the isolates in phenotypic β-lactamase detection

Clonal relationship of the isolates

Molecular characterization and location of β-lactamase genes and ISEcp1

Antimicrobial susceptibility testing

Discussion

Footnotes

Disclosure Statement

Funding Information

References

Localization of bla_AmpC genes and detection of ISEcp1 element