Mutations in the rpsL Gene Are Involved in Streptomycin Resistance in Campylobacter coli

Abstract

To characterize the mechanisms of streptomycin (STR) resistance in Campylobacter coli, we chose 17 isolates that were resistant to STR, erythromycin (ERY), or both, and the putative STR resistance target genes rpsL, rrs, and gidB were analyzed for mutations. The presence of the aadE gene encoding aminoglycoside 6-adenylyltransferase was also evaluated. To reveal putative connection between ERY and STR resistance mechanisms, 13 C. coli isolates initially susceptible to STR and ERY were exposed to STR, and resistant variants were characterized. We also assessed the development of ERY resistance with a similar method. Finally, the effect of the putative CmeABC efflux pump inhibitor phenyl-arginine-β-naphthylamine on STR resistance was tested. Our studies showed an association between mutations in the rpsL gene and STR resistance in C. coli. Further, mutations obtained in vitro were more diverse than those occurring in vivo. However, we observed no resistance associated mutations in the other genes studied, and selection with STR did not result in variants resistant to ERY and vice versa. None of the isolates harbored the aadE gene, and no differences in STR minimum inhibitory concentration levels were detected in the presence or absence of phenyl-arginine-β-naphthylamine. In conclusion, we found that STR resistance was associated with mutations in the rpsL gene, but no obvious association between STR and ERY resistance mechanisms was found in C. coli.

Introduction

Campylobacter species, especially Campylobacter jejuni and Campylobacter coli, are the leading cause of human gastroenteritis worldwide. Most cases are self-limiting and do not require treatment. However, in long-lasting or severe infections and in immunocompromised patients, the use of antimicrobials is warranted.³ The drugs of choice for treating campylobacter infections in humans have traditionally been macrolides and fluoroquinolones.^3,4 In serious campylobacter bacteremia, intravenous aminoglycosides may also be used.² In Finland, the use of aminoglycoside antimicrobials in veterinary medicine has steadily declined over the period of screening, from over 1,000 kg in 1995 to 180 kg in 2008 (of active substance).^7,16 In Denmark, the aminoglycoside usage for production animals has fluctuated between 11 700 kg and 7 100 kg during years 1990–2007.⁶ Many reports exist on the high incidence of streptomycin (STR) resistance in C. coli of animal origin. In Canada, for instance, the rate of resistance has been around 70% and in Italy, it has been close to 90% in C. coli isolates from swine.^14,25,31 In Denmark, C. coli isolates from swine were reported to be more resistant to STR than isolates from broilers or humans.¹ Despite the high prevalence of STR resistance in C. coli isolates from swine, the mechanisms have not been well characterized.

STR belongs to the aminoglycoside group of antimicrobials, and it binds to the 30S subunit of the bacterial ribosome disrupting the elongation process of peptides by making the ribosome more prone to translational errors. The resistance mechanisms for STR have been examined in several bacterial species, including Mycobacterium tuberculosis and Escherichia coli, and resistance-conferring mutations in certain genes have been detected in these species.^10,17,22,24 Since STR affects the protein synthesis of bacteria, genes encoding ribosomal proteins and rRNA, especially the genes rpsL and rrs, have been widely investigated. The rpsL gene encodes ribosomal protein S12, and point mutations in two codons of this gene have been shown to result in STR resistance in, for example, E. coli, M. tuberculosis, and Helicobacter pylori.^10,22,30 A particular mutation in the rpsL gene conferring resistance to STR has also been associated with enhanced synthesis of antimicrobials or proteins in certain Streptomyces species and in E. coli, respectively.^18,28 The rrs gene encodes 16S rRNA, and base substitutions in several positions of the 500 and 900 regions of this gene have been reported to be associated with STR resistance in M. tuberculosis.^17,22

Recently, a new target gene was reported to contribute to STR resistance in M. tuberculosis. The gidB gene encodes a conserved methyl transferase that apparently catalyzes the methylation of base G527 of the rrs gene.²⁴ Mutations in various positions of the gidB gene have been demonstrated to result in low level of STR resistance and in considerable increase of the emergence rate of highly STR resistant (STR^r) bacteria.^24,29 In addition, the products of certain plasmid-mediated genes, such as aadE encoding aminoglycoside 6-adenylyltransferase, have been shown to confer STR resistance in various bacterial species. The gene aadE was first detected by Pinto-Alphandary et al. in Campylobacter spp.²⁶ Two partial and one whole sequence homolog of this gene have been described in two C. jejuni plasmids.^11,23

Controlling the intracellular concentration of antimicrobials is a common mechanism of resistance in bacteria. The CmeABC efflux pump belongs to the resistance-nodulation-cell division type efflux systems and it is the best characterized pump in campylobacter. It has also been associated with multidrug resistance in this species.²⁰ It has been shown to contribute to, for example, macrolide resistance of C. jejuni and C. coli in synergy with other mechanisms.^12,15 However, there is no evidence that this pump has any effect on STR resistance.

We recently found that at a Finnish pig farm wherein tylosin, a macrolide, was used to treat diarrhea in weaned pigs, both STR^r and erythromycin resistant (ERY^r) bacteria emerged. After 13 days of treatment, the most common resistance pattern in the isolates from tylosin-treated pigs was ERY/STR resistance, and the rate of resistance to ERY and STR was significantly higher in the isolates from pigs treated with tylosin as compared with the isolates from untreated pigs (p < 0.01).¹⁹

The aim of our research was to characterize whether any of the STR resistance mechanisms mentioned in the previous paragraphs have a role in C. coli. A further objective was to uncover possible common factors connecting the mechanisms of resistance to STR and ERY. This was accomplished by studying STR^r and/or ERY^r isolates from two pig farms (referred to as primary STR^r and/or ERY^r isolates) and by selecting STR^r and ERY^r variants by cultivating susceptible parent isolates on growth media containing increasing concentrations of STR or ERY (referred to as in vitro acquired STR^r or ERY^r variants, respectively).

Materials and Methods

Growth conditions

All C. coli isolates and variants included in this study were cultivated on Brucella agar (Oxoid, Basingstoke, United Kingdom) supplemented with 5% blood (Labema, Kerava, Finland). Antibimicrobial susceptibility testing and pump inhibitor assays were performed on Müller-Hinton agar (Oxoid) supplemented with 5% blood (Labema). Incubation took place in a microaerobic atmosphere at 37°C for 1–5 days, as considered appropriate. The isolates were confirmed as C. coli by a negative hippurate hydrolysis test and C. coli-specific PCR.²¹ The antibiotics STR (MP Biomedicals, Illkirch Cedex, France) and ERY (Sigma, St. Louis, MO) were included, as considered appropriate (see the next paragraphs). Bacteria were stored in skim milk and glycerol at −70°C.

Primary STR^r and/or ERY^r C. coli isolates

Seventeen C. coli isolates derived from feces of pigs from two pig farms located in different parts of Finland in 2007 were chosen based on their resistance profiles: All of the isolates were resistant to ERY, STR, or both. Additionally, three of the isolates were resistant to ciprofloxacin and nalidixic acid (isolates 22, 36, and 38). At farm I, the sampled pigs were treated with tylosin in association with weaning diarrhea in piglets, whereas at farm II no recent macrolide treatment was reported. Farm I also used penicillin, sulfadiazine-trimethoprim, and amoxicillin for their animals, as needed, and at farm II, sulfadiazine-trimethoprim and penicillin medications were used if required. All isolates except for 17, 20, and 21 were from farm I.

Selection of in vitro acquired Str^r C. coli variants

Thirteen STR susceptible isolates derived in 2007 from five Finnish pig farms in different geographic locations were selected to produce STR^r variants in vitro. These isolates had known resistance profiles for common antimicrobials, STR minimum inhibitory concentration (MIC) level of 2–4 mg/l, and were susceptible to ERY. Two of these parent isolates were resistant to ciprofloxacin and nalidixic acid (parent isolates of variants 5.1, 6.1, and 6.2). The isolates were cultivated successively on agar plates containing increasing concentrations of STR. Three colonies from each concentration were isolated for each parent strain from agar plates containing 8, 32, and 64 mg/l of STR.

Selection of in vitro acquired ERY^r C. coli variants

Two in vitro acquired STR^r variants (MIC STR >1,024 mg/l, susceptible to ERY) and two isolates with STR MIC levels of 3–4 mg/l and susceptible to ERY were used to select ERY^r variants. Selection was carried out, as described earlier, for the in vitro acquired STR^r variants, but with the agar plates containing increasing concentrations of ERY and colonies isolated from plates containing 16 and 32 mg/l of ERY.

MIC determination

Preliminary screening of antimicrobial susceptibility of the primary STR^r and/or ERY^r isolates and the parental strains of the in vitro acquired resistant variants was carried out by using VetMIC panels (SVA, Strömsund, Sweden) in cooperation with the Microbiology Unit of the Finnish Food Safety Authority. The STR and ERY MIC values for the primary STR^r and/or ERY^r isolates and in vitro acquired variants were re-determined with E-tests (bioMérieux Suomi Oy, Helsinki, Finland) and/or the agar dilution method.⁵ The C. jejuni American Type Culture Collection (ATCC) strain 33560 was used as a control. The quality control range for ERY MIC (1–8 mg/l) for the control species was taken from the Clinical and Laboratory Standards Institute's guidelines.⁵ Although the quality control range for STR MIC has not been determined for C. jejuni ATCC 33560, we always recorded the STR MIC value to be between 1 and 4 mg/l for this strain with the E-test and agar dilution methods. All the isolates and variants with STR MIC levels of 32–128 mg/l were tested a minimum of twice independently.

Chromosomal and plasmid DNA isolation

Chromosomal DNA was extracted using Pitcher's method²⁷ or Wizard Genomic DNA purification kit (Promega, Madison, WI). Plasmid DNA was isolated with an E.Z.N.A. Plasmid Mini Kit I (Omega Bio-Tek, Norcross, GA).

Molecular analysis of resistance genes

The rrs, rpsL, gidB, 23S rRNA, and aadE gene fragments from the primary STR^r and/or ERY^r isolates and the rpsL and aadE gene fragments from the in vitro acquired STR^r variants and their parent strains were amplified by PCR using primers designed for this study (Table 1). Also, the rrs₅₀₀ fragment was sequenced from five STR^r variants (1.1, 6.1, 10.1, 10.2, and 13.1) and their parent isolates. For the rrs gene, two primer pairs were used to amplify the 500 (rrs₅₀₀) and 900 (rrs₉₀₀) regions of the gene. PCR cycling was as follows: denaturation at 95°C for 30 sec; annealing at 56°C (gidB, rrs₅₀₀), 55°C (rpsL, rrs₉₀₀), or 50°C (aadE, 23S rRNA) for 30 sec; and elongation at 72°C for 60 sec (aadE, rrs₅₀₀, gidB), 35 sec (rrs₉₀₀), or 30 sec (rpsL, 23S rRNA), repeated 30 times. The final extension was carried out at 72°C. Analysis of the amplified products was performed by agarose gel (1.5% w/v) electrophoresis. The C. jejuni plasmid pCG8245 was used as a positive control for the aadE PCR.²³ The PCR products were sequenced by the Institute of Biotechnology, University of Helsinki. The quality of the amplified sequences was analyzed with FinchTV (Geospiza Inc., Seattle, WA) or Staden Package (http://staden.sourceforge.net/) and aligned in ClustalW (EMBL-EBI, Cambridge, United Kingdom).

Table 1.

Primers Used in This Study

Primer	Sequence (5'-3') ^a
rrsF₅₀₀	5′-cgcacgggtgagtaaggtat-3′
rrsR₅₀₀^b	5′-tcgcaatgggtattcttggt-3′
rrsF₉₀₀	5′-ggggagcaaacaggattaga-3′
rrsR₉₀₀^b	5′-aacaatccgaactgggacat-3′
gidBF	5′-gggcatttactttgaatttgga-3′
gidBR^b	5′-cacgcttgtgcctttaagc-3′
rpsLF1^b	5′-ccagcgcttaaaaattgtcc-3′
rpsLFR1^b	5′-tatcaagagcaccacgaacg-3′
aadEF Full	5′-atgagatcagaaaaggaagtttat-3'
aadER Full	5′-ttatttgttatcatcttcaatgcc-3′
ERY23SRNA-F2^b	5′-aattgatggggttagcattagc-3′
ERY23SRNA-R2	5′-caacaatggctcatatacaactgg-3′

Based on the genome sequence of Campylobacter coli RM2228 (accession number NZ_AAFL00000000; gidB: NZ_AAFL01000007, rrs and ERY23SRNA: NZ_AAFL01000009, rpsL: NZ_AAFL01000013) except for aadEF Full and aadER Full, in which the sequences are based on Campylobacter jejuni plasmid pCG8245 (accession number AY701528).

Used for sequencing.

Effect of efflux pump inhibition

The effect of efflux pump inhibition on STR MIC values was tested on the primary STR^r and/or ERY^r isolates with the agar dilution method.⁵ The study was carried out using plates containing 1–1,024 mg/l STR with and without 50 mg/l of the putative pump inhibitor phenyl-arginine-β-naphthylamine (PAβN; Sigma). As a control, the isolates were cultivated on plates containing 0.5–1,024 mg/l ERY with and without 50 mg/l of PAβN and also on plates containing only PAβN. The experiment was repeated twice independently, and the C. jejuni strain ATCC 33560 was used as a control.

Results

Mutations in primary STR^r and/or ERY^r C. coli isolates

The ERY and STR MIC values and mutations in the rpsL and the 23S rRNA genes of the isolates are shown in Table 2. In the rpsL gene, the AAA → AGA (K → R) mutation in codon 43 was detected in all highly STR^r isolates (MIC > 1,024 mg/l). No mutations were found in codon 43 in the isolates 22, 36, and 38 (farm I) having STR MIC of 1 mg/l or in the isolates 17, 20, or 21 (farm II) having STR MIC of 128 mg/l. Also, no mutations were detected in codon 88 in any of the isolates. All of the ERY^r isolates (MIC ≥ 512 mg/l, Table 2) had the A2122G (C. coli_RM2228 numbering)⁸ point mutation in the 23S rRNA gene. No resistance-associated changes were identified in the amplified sequences of the gidB or rrs genes, and the aadE gene was not amplified from the plasmid or chromosomal DNA in any of the 17 primary STR^r and/or ERY^r isolates.

Table 2.

Resistance Patterns and Mutations in the rpsL Gene in the Primary Streptomycin Resistant and/or Erythromycin Resistant Isolates

Isolate	ERY MIC (mg/l) ^a	STR MIC (mg/l) ^a	rpsL Codon 43	rpsL Codon 88	23S rRNA
C. jejuni ATCC 33560	1–4^b	1–4^b	AAA	AAA	—
17	1	128	AAA	AAA	A
18	>1,024	>1,024	AGA	AAA	A2122G
20	1	128	AAA	AAA	A
21	1	128	AAA	AAA	A
22	>1,024	1	AAA	AAA	A2122G
36	>1,024	1	AAA	AAA	A2122G
38	512	1	AAA	AAA	A2122G
39	>1,024	>1,024	AGA	AAA	A2122G
44	>1,024	>1,024	AGA	AAA	A2122G
75^c	>1,024	>1,024	AGA	AAA	A2122G
76^d	>1,024	>1,024	AGA	AAA	A2122G
80	>1,024	>1,024	AGA	AAA	A2122G
81^c	>1,024	>1,024	AGA	AAA	A2122G
82^d	>1,024	>1,024	AGA	AAA	A2122G
83	>1,024	>1,024	AGA	AAA	A2122G
84	>1,024	>1,024	AGA	AAA	A2122G
85	>1,024	>1,024	AGA	AAA	A2122G

Determined with the agar dilution method.

Results in which the MIC value of the C. jejuni American Type Culture Collection (ATCC) 33560 was between these values were accepted.

c,d

Resampling of the same animal.

ERY, erythromycin; MIC, minimum inhibitory concentration; STR, streptomycin.

Mutations in in vitro acquired STR^r C. coli variants

The antimicrobial resistance patterns and mutations in the rpsL gene of the progeny variants are shown in Table 3. No mutations were detected in the parent isolates (data not shown) or in the variants with STR MIC < 1,024 mg/l except for the variant 13.1 having STR MIC of 32 mg/l. All highly STR^r variants (MIC > 1,024 mg/l) had a mutation in either codon 43 or codon 88 of the rpsL gene but never at both sites simultaneously. The AAA → AGA (K → R) mutation in codon 43 was detected most often (nine variants) and it was also the only one detected at this site. More variable mutations were found in codon 88: AAA → AGA (K → R, four variants), AAA → GAA (K → E, two variants), and AAA → CAA (K → Q, one variant) (Table 3). No PCR products for the aadE gene from the plasmid or chromosomal DNA of any of the in vitro acquired variants or their parent isolates were obtained. Also, no mutations were detected in the sequenced fraction of the rrs gene in the isolates 1.1, 6.1, 10.1, 10.2, and 13.1.

Table 3.

Resistance Patterns and Mutations in the rpsL Gene in the In Vitro Acquired Streptomycin Resistant Variants

Variant	ERY MIC (mg/l) ^a	STR MIC (mg/l) ^a	rpsL Codon 43	rpsL Codon 88
1.1^b	0.25	32	AAA	AAA
1.2^c	0.25	>1,024	AAA	AGA
2.1^b	<0.063	>1,024^d	AAA	AGA
2.2^e	<0.063	>1,024^d	AAA	AGA
3.1^c	<0.063	>1,024	AAA	AGA
4.1^e	0.25	>1,024^d	AGA	AAA
5.1^e	0.125	>1,024^d	AAA	GAA
6.1^c	0.25	64	AAA	AAA
6.2^e	0.25	>1,024	AAA	CAA
7.1^e	0.25	>1,024	AGA	AAA
8.1^b	0.5	>1,024^d	AGA	AAA
8.2^b	2	>1,024^d	AGA	AAA
8.3^c	1	>1,024	AGA	AAA
9.1^c	4	>1,024^d	AGA	AAA
9.2^e	4	>1,024^d	AGA	AAA
10.1^b	1	4	AAA	AAA
10.2^c	4	128	AAA	AAA
11.1^c	4	>1,024	AGA	AAA
12.1^e	0.5	>1,024	AGA	AAA
13.1^c	0.5	32	AAA	GAA

Determined with the agar dilution method unless stated otherwise.

Isolated from STR concentration of 8 mg/l.

Isolated from STR concentration of 32 mg/l.

Determined with E-tests.

Isolated from STR concentration of 64 mg/l.

MIC values of in vitro acquired ERY^r C. coli variants

Despite repeated attempts, one of the STR susceptible isolates failed to grow on plates containing 1 or 2 mg/l (1–2 × MIC) of ERY. The STR MIC of the progeny variants from the other isolate initially susceptible to STR did not change with exposure to increasing ERY concentrations even when they were resistant to ERY (MIC > 256 mg/l, data not shown).

Effect of efflux pump inhibition

No differences in the STR MIC values of the primary STR^r and/or ERY^r isolates in the presence or absence of the efflux pump inhibitor PAβN were detected, but the ERY MIC values decreased on average fourfold in the presence of PAβN (data not shown).

Discussion

The STR resistance-conferring mutations in codons 43 and 88 of the rpsL gene resulting in an amino acid shift of K → R at the corresponding sites in ribosomal protein S12 have been well documented in several bacterial species, including M. tuberculosis and E. coli.^9,10,22 However, the association of these mutations with STR resistance in campylobacters has not been previously described. Our results show that each of the C. coli isolates and variants with a high level of STR resistance (MIC > 1,024 mg/l) had a mutation in codon 43 or codon 88 of the rpsL gene. This is consistent with a previous study conducted on a related species, H. pylori, in which all the STR^r mutants had a K → R amino acid shift mutations in codons cause aa shifts, not vice versa causing change in codon 43 or 88.³⁰

The AAA → AGA (K → R) mutation in codon 43 was the most frequently detected both in primary STR^r isolates and in vitro acquired STR^r variants. Interestingly, the mutations leading to amino acid changes K → R, K → E, and K → Q in codon 88 were observed only in the in vitro acquired STR^r variants. Also other authors describe diverse mutations in the corresponding codon in M. tuberculosis and E. coli.^9,13,29 In our in vitro acquired STR^r isolates, all the rpsL mutants were highly resistant to STR (MIC > 1,024 mg/l), except for one variant that had the K88 → E amino acid change and STR MIC of 32 mg/l. However, another variant with a similar change in codon 88 had STR MIC > 1,024. This is consistent with an earlier study on M. tuberculosis, in which a K88 → R mutation led to STR MIC values of 16, 256, and 1,024 mg/l.⁹ There are also other studies reporting more heterogeneous MIC levels in association with mutations in codon 88, including when the mutation was K → E.^13,22

In addition, there is evidence of increased protein or antibiotic synthesis activity in bacteria with mutation in codon 88/87: In Streptomyces lividans, Streptomyces coelicolor, and E. coli, this has been shown to be the consequence of the K88/87 → E mutations.^18,28 Whether this mutation affects the protein synthesis rate of campylobacters remains to be elucidated.

Since we had three primary STR^r isolates and three in vitro acquired STR^r variants with MIC values of 32–128 mg/l and lacking mutations in the rpsL gene, we wanted to explore other genes associated with STR resistance. However, no resistance-related mutations were found in the sequenced fractions of the rrs gene in the primary STR^r isolates or in the in vitro acquired STR^r variants studied, suggesting that it does not have a role in STR resistance in C. coli. In M. tuberculosis, mutations in the rrs gene have been shown to result in STR resistance.²² However, this species has only one copy of the 16S rRNA-encoding gene, whereas C. coli has three. Therefore, it can be expected that mutations in this gene are less likely to occur in C. coli under STR exposure. Moreover, no changes were found in the gidB gene of the primary STR^r and/or ERY^r isolates, and none of the isolates or the in vitro acquired STR^r variants seemed to harbor the aadE gene as detected in plasmid pCG8245 isolated from C. jejuni.²³

Over the past 4 years, no aminoglycoside treatment had been reported from either of the pig farms from which our primary STR^r and/or ERY^r isolates originated. Our porcine C. coli isolates from several Finnish pig farms have not had typically high STR MIC values (2–8 mg/l, data not shown). Nevertheless, STR resistance seems to be common among porcine C. coli isolates in many countries.^1,14,25 The treatment of pigs with tylosin at farm I was followed by increased isolation of bacteria with high MIC values for both ERY and STR, raising the question of whether the use of macrolides exerted selection pressure also for the development of STR resistance.¹⁹ However, we failed to find a common factor, for example, the CmeABC efflux pump, linking the resistance mechanisms of these antimicrobials. Also, in other studies, resistance to STR and ERY seem not to be unambiguously linked, because strains highly resistant to both are often also resistant to antimicrobials from other antimicrobial groups, for example, tetracyclines or fluoroquinolones.^14,25,31 It can be hypothesized that STR^r strains have persisted a long time at the farms from which our primary STR^r and/or ERY^r isolates originated, even though aminoglycoside usage in veterinary medicine has declined in Finland.

To our knowledge, this is the first study to reveal a connection between STR resistance in C. coli and mutations in the rpsL gene. No mutations in the other potential target genes were observed in the isolates studied, and no obvious association between STR and ERY resistance was detected. It also seems evident that there are other unknown factors contributing to the lower level of STR resistance in C. coli, and mutations occurring in vivo differ from those obtained in vitro.

Footnotes

Acknowledgments

This study was funded by the Academy of Finland (FCoE MiFoSa grant no. 118602) and the Ministry of Agriculture and Forestry (grant no. 4491/502/2006). The C. jejuni plasmid pCG8245 was kindly provided to us by Patricia Guerry from the Naval Medical Center, Silver Spring, Maryland. Technician Anna-Kaisa Keskinen is acknowledged for excellent technical assistance.

Disclosure Statement

No competing financial interests exist.

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Mutations in the rpsL Gene Are Involved in Streptomycin Resistance in Campylobacter coli

Abstract

Introduction

Materials and Methods

Growth conditions

Primary STRr and/or ERYr C. coli isolates

Selection of in vitro acquired Strr C. coli variants

Selection of in vitro acquired ERYr C. coli variants

MIC determination

Chromosomal and plasmid DNA isolation

Molecular analysis of resistance genes

Effect of efflux pump inhibition

Results

Mutations in primary STRr and/or ERYr C. coli isolates

Mutations in in vitro acquired STRr C. coli variants

MIC values of in vitro acquired ERYr C. coli variants

Effect of efflux pump inhibition

Discussion

Footnotes

Acknowledgments

Disclosure Statement

References

Primary STR^r and/or ERY^r C. coli isolates

Selection of in vitro acquired Str^r C. coli variants

Selection of in vitro acquired ERY^r C. coli variants

Mutations in primary STR^r and/or ERY^r C. coli isolates

Mutations in in vitro acquired STR^r C. coli variants

MIC values of in vitro acquired ERY^r C. coli variants