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JAC Advance Access originally published online on May 11, 2007
Journal of Antimicrobial Chemotherapy 2007 60(1):140-144; doi:10.1093/jac/dkm144
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Community emergence of CTX-M type extended-spectrum ß-lactamases among urinary Escherichia coli from women

P. L. Ho1,*, Winnie W. N. Poon1, S. L. Loke2, Marianne S. T. Leung3, K. H. Chow1, River C. W. Wong1, K. S. Yip1, Eileen L. Lai1, Kenneth W. T. Tsang on behalf of the COMBAT study group4

1 Division of Infectious Diseases, Department of Microbiology and Centre of Infection, The University of Hong Kong, Pokfulam Road, Hong Kong Special Administrative Region, China 2 The Laboratory, St Teresa's Hospital, Prince Edward Road, Hong Kong Special Administrative Region, China 3 Pathlab Medical Laboratories Ltd, Jaffe Road, Hong Kong Special Administrative Region, China 4 Department of Medicine, The University of Hong Kong, Pokfulam Road, Hong Kong Special Administrative Region, China

* Corresponding author. Tel: +852-2855-4897; Fax: +852-2855-1241; E-mail: plho{at}hkucc.hku.hk

Received 21 January 2007; returned 31 January 2007; revised 16 April 2007; accepted 16 April 2007


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Objectives: To conduct a territory-wide study of extended-spectrum ß-lactamases (ESBLs) among community isolates of urinary Escherichia coli from women in Hong Kong.

Methods: Up to 50 consecutive single-patient E. coli isolates, collected from 13 laboratories in 2004, were studied. The ESBLs were characterized by PCR sequencing using specific primers. The epidemiological relationship of the isolates was studied by PFGE and phylogenetic group PCRs.

Results: Forty-two ESBL producers were found among 600 consecutive isolates tested. The ESBL prevalence was 7.3% (15/205) for women aged 18–35 years, 5% (11/219) for women aged 36–50 years, 6.3% (4/63) for women aged 51–64 years and 10.6% (12/113) for women aged ≥65 years (P = 0.3). The ESBL-producing isolates were often multidrug-resistant and CTX-M-14 was found in 37 isolates, CTX-M-15 in 3 isolates and CTX-M-3 in 2 isolates. PFGE revealed no significant clusters among the ESBL producers. Overall, CTX-M-14 producers were significantly more likely to belong to group D than non-ESBL producers [18/37 (48.6%) versus 13/57 (22.8%), P = 0.009]. However, 7 of 13 (53.8%) CTX-M-14 producers from women aged 18–35 years represented phylogenetic group B2, compared with 7 of 24 (29.2%) for women of all other ages (P = 0.1).

Conclusions: The study documented the community emergence of CTX-M as the predominant ESBL type among urinary isolates from women. The spread of CTX-M enzymes among isolates from young women is concerning and deserves close monitoring.

Keywords: antimicrobial resistance epidemiology , urinary tract infections , fluoroquinolones , pulsed-field gel electrophoresis


   Introduction
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A recent issue that concerns the extended-spectrum ß-lactamases (ESBLs) is the global dissemination of the CTX-M ß-lactamases since the mid-1990s. Unlike the TEM and SHV variants, many CTX-M-producing isolates have been reported to be from patients with community-onset infections, especially among urinary Escherichia coli.1 In Asian countries, information pertaining to the CTX-M enzymes relates primarily to isolates obtained from inpatients; little is known about their epidemiology in outpatient isolates. In this study, the occurrence of CTX-M enzyme types and the genetic background of the bacterial host were assessed in a sample of urinary E. coli isolates collected by a surveillance network.


   Materials and methods
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Study design and patient data

In January 2004, a monitoring group (COMBAT, for COMmunity-Based Antibiotic resistance Trend surveillance) was formed under the coordination of the Centre of Infection at the University of Hong Kong, Hong Kong, China, to conduct a laboratory-based surveillance for emerging antimicrobial resistance in the community.2 The participating microbiology network for surveillance of community-onset ESBL includes six private hospital laboratories and seven stand-alone community laboratories. These laboratories were located in diverse geographic areas and were estimated to provide testing for half of the outpatients in Hong Kong. Between May and December 2004, each laboratory was requested to submit up to 50 consecutive, single-patient urine isolates of E. coli. The inclusion criteria were: (i) female sex, (ii) age ≥18 years, (iii) mid-stream urine specimen, (iv) significant growth at ≥105 cfu/mL and (v) community-onset (from an outpatient or an inpatient within 2 days of hospital admission).

Bacterial identification, antimicrobial susceptibility and ESBL detection

Bacteria were identified to species level by the VITEK GNI system (bioMerieux Vitek Inc., Hazelwood, MO, USA) and their antibiotic susceptibilities were determined by the disc diffusion method according to the CLSI.3 The double-disc synergy test was used to assess for ESBL production in all the isolates.4 Multidrug resistance (MDR) was defined as resistance to three of more of the following non-ß-lactam antibiotics: chloramphenicol, ciprofloxacin, co-trimoxazole, fosfomycin, gentamicin, nitrofurantoin and tetracycline.

Molecular studies

ß-Lactamases related to the TEM, SHV and CTX-M families were sought by PCR and sequencing using primers previously described.5,6 Selected isolates were studied by PFGE of XbaI-digested genomic DNA (Amersham Pharmacia Biotech, Little Chalfont, UK) and patterns were analysed with Gelcompar II software (Applied Maths).5 A multiplex PCR was used to assign the E. coli isolates to one of the four main phylogenetic groups (A, B1, B2 and D).7


   Result
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Patient demographics

A total of 600 isolates was analysed including 519 (86.5%) isolates from outpatients and 81 (13.5%) isolates from inpatients who had been hospitalized for <2 days. The cohort of 600 patients had a mean age of 45.7 years (SD 17.8 and range 18–101 years). The mean ages of patients with ESBL producers and non-producers were similar (49.5 ± 22.4 years for ESBL producers versus 45.4 ± 17.4 years for non-producers, P = 0.2).

Frequency of ESBL production and antimicrobial susceptibility

The double-disc synergy test identified 42 (7%) isolates as ESBL producers and 558 (93%) isolates as non-producers. ESBL producers were found in all 16 laboratories, with numbers ranging from one to eight. The ESBL rate among inpatients with community-onset infection was 9.9% (8/81), compared with 6.5% (34/519) among outpatients (P = 0.3). Prevalence rates of ESBL among community-onset isolates were 7.3% (15/205) for women aged 18–35 years, 5% (11/219) for women aged 36–50 years, 6.3% (4/63) for women aged 51–64 years and 10.6% (12/113) for women aged 65 years and above ({chi}2 test for all groups, P = 0.3).

ESBL-producing isolates were frequently resistant to the non-ß-lactam antibiotics. Isolates with ESBL were more likely than those without to have resistance to ciprofloxacin (ESBL versus non-ESBL, 61.9% versus 22%, P < 0.001), co-trimoxazole (73.8% versus 34.6%, P < 0.01), chloramphenicol (45.2% versus 29.2%, P < 0.001), gentamicin (59.5% versus 20.3%, P < 0.001) and tetracycline (81% versus 60.9%, P < 0.001), and to possess a MDR phenotype (71.4% versus 30.3%, P < 0.001). The great majority of isolates were susceptible to fosfomycin (ESBL versus non-ESBL, 97.6% versus 98.6%) and nitrofurantoin (97.6% versus 89.8%). Susceptibilities to amoxicillin/clavulanic acid and piperacillin/tazobactam were variable. All isolates were susceptible to amikacin and imipenem.

Characteristics of the ESBL-producing isolates

All 42 isolates with an ESBL phenotype tested positive for blaCTX-M using the consensus primers. Subsequent PCR and sequencing with group-specific primers showed the presence of CTX-M-14 in 37 isolates, CTX-M-15 in 3 isolates and CTX-M-3 in 2 isolates. The 15 CTX-M producers from women aged 18–35 years included 13 CTX-M-14 and 2 CTX-M-15 isolates. Both CTX-M-3 producers were found in elderly women aged over 65 years. As demonstrated by the inhibition zone diameters, all 42 isolates with an ESBL phenotype exhibited a higher level of resistance to ceftriaxone than ceftazidime (mean diameters ± SD; 12.9 ± 3.7 mm versus 25.7 ± 4.4 mm, P < 0.001). Ceftazidime inhibition zone diameters for CTX-M-15 isolates were significantly smaller than for CTX-M-14 isolates (mean diameters ± SD; 13.7 ± 1.5 mm versus 26.9 ± 2.9 mm, P < 0.001). In addition, TEM-1a was found in 2 isolates and TEM-1b in 24 isolates. One isolate produced an SHV-type enzyme.

Epidemiological typing of strains

A subset of 99 isolates including the 42 ESBL-positive and 57 ESBL-negative isolates was analysed further by PFGE and phylogenetic group PCRs. The 57 ESBL-negative isolates were chosen to represent the most frequent antibiotic patterns (represented by five or more isolates). PFGE analysis showed that the isolates had highly diverse banding patterns (Figure 1). Most ESBL-producing isolates were not clonally related but some CTX-M-14 producers shared clonality with non-producers. Of the 42 ESBL-producing E. coli isolates, 19 (45.2%) were derived from phylogenetic group D, 15 (35.7%) from group B2, 2 (4.8%) from group B1 and 6 (14.3%) from group A (Table 1). Half of the 37 CTX-M-14 producers were from group D. Therefore, CTX-M-14 producers were significantly more likely to belong to group D than non-ESBL producers [18/37 (48.6%) versus 13/57 (22.8%), P = 0.009]. However, 7 of 13 (53.8%) CTX-M-14 producers from women aged 18–35 years were phylogenetic group B2, compared with 7 of 24 (29.2%) for women of all other ages (P = 0.1). Isolates producing CTX-M group 1 enzymes (CTX-M-15 and CTX-M-3) were from diverse phylogenetic groups. As for the non-ESBL producers, the predominant phylogenetic group was B2 (57.9%).


Figure 1
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  		Figure 1.. Dendrogram showing the genetic relatedness of 99 E. coli isolates. Each gradation on the scale represents a 5% difference in similarity index. The boxes indicate clusters with both ESBL- and non-ESBL-producers at ≥85% similarity (UPMAG, Dice, black vertical line). CTX-M-14 producers are shaded in black, CTX-M-3 producers are shaded in dark grey and CTX-M-15 producers are shaded in light grey.

 


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  		Table 1.. Distribution of E. coli isolates according to phylogenetic group

 

   Discussion
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 Abstract
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 Materials and methods
 Result
 Discussion
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The findings from this study indicated that the CTX-M family is the dominant enzyme type among urinary E. coli with an ESBL phenotype in Hong Kong. It is striking that 7.3% of the isolates from women aged 18–35 years were ESBL producers, although the ESBL rate among older women was higher. Our further analysis showed that this occurred mainly because of the emergence of the group 9 enzyme, CTX-M-14. In East Asia and Spain,1 group 9 enzymes including CTX-M-14 were found to predominate in recent studies.

When compared with control isolates without ESBL, our findings clearly showed that isolates harbouring CTX-M-14 belonged largely to phylogenetic group D. This is in line with recent observations,8 although non-ESBL-producing control isolates were not included in these previous studies. The finding that one-third of our CTX-M-14 producers belonged to phylogenetic group B2 deserves further investigation. Many previous studies have highlighted that group B2 isolates often carry multiple virulence factors that were absent in the non-B2 groups.9 Therefore, transmission of CTX-M-14 to more virulent B2 host strains could underscore the emergence of this enzyme in urinary E. coli from younger women.

In this study, no substantial clusters of CTX-M producers were noted in the PFGE analysis. The finding is consistent with findings from some areas in which there was little or no clonality among isolates with CTX-M enzymes.10 In the UK, Canada and Spain, however, clonally related CTX-M-producing E. coli was responsible for some community outbreak urinary tract infections.10

In Hong Kong, ~80% of the total outpatient service is provided by doctors in private practice. In an attempt to ensure a good and fair representation of isolates from all regions of Hong Kong, each laboratory was requested to contribute up to 50 isolates. The advantage of this approach is that the final sample will not be dominated by one or two large laboratories. Since all isolates in this study were obtained from private laboratories, the results from this study may not reflect the situation among outpatients in the public sector. Since ESBL-producing E. coli may also emerge in other patient populations, further studies assessing the epidemiology among inpatients, males and children should be performed.

In conclusion, this study found significant rates of ESBL among urinary E. coli from adult women of all ages in the Hong Kong community, largely due to the spread of CTX-M-14 ß-lactamase. The emergence of CTX-M-14 among community strains from young women is concerning and deserves close monitoring.


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None to declare.


   Acknowledgements
 
Part of the work was submitted by W. W. N. P. to the University of Hong Kong in partial fulfillment of the requirement for Master in Medical Sciences. The work is supported by research grants from RGC (HKU 7513/06M) and the Research Fund for the Control of Infectious Diseases (RFCID) of the Health, Welfare and Food Bureau of the Hong Kong SAR Government. Members of the COMBAT study group: K. W. Chan, K. H. Chow, L. C. Chu, M. Y. Fong, P. L. Ho, Y. C. Kong, C. H. Lam, T. H. Lee, M. S. T. Leung, K. H. Li, M. Lincoln, S. L. Loke, J. Ng, W. C. Li, K. W. T. Tsang, S. W. Tsang and M. Wah.


   References
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1 Bonnet R. Growing group of extended-spectrum ß-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother (2004) 48:1–14.[Free Full Text]

2 Ho PL, Cheung C, Mak GC, et al. Molecular epidemiology and household transmission of community-associated methicillin-resistant Staphylococcus aureus in Hong Kong. Diagn Microbiol Infect Dis (2007) 57:145–51.[CrossRef][ISI][Medline]

3 Clinical and Laboratory Standard Institute. Performance Standards for Antimicrobial Susceptibility Testing: Fifteen Informational Supplement M100-S15 (2005) Wayne, PA, USA: CLSI.

4 Ho PL, Chow KH, Yuen KY, et al. Comparison of a novel, inhibitor-potentiated disc-diffusion test with other methods for the detection of extended-spectrum ß-lactamases in Escherichia coli and Klebsiella pneumoniae. J Antimicrob Chemother (1998) 42:49–54.[Abstract/Free Full Text]

5 Ho PL, Shek RH, Chow KH, et al. Detection and characterization of extended-spectrum ß-lactamases among bloodstream isolates of Enterobacter spp. in Hong Kong, 2000–2002. J Antimicrob Chemother (2005) 55:326–32.[Abstract/Free Full Text]

6 Saladin M, Cao VT, Lambert T, et al. Diversity of CTX-M ß-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. FEMS Microbiol Lett (2002) 209:161–8.[ISI][Medline]

7 Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol (2000) 66:4555–8.[Abstract/Free Full Text]

8 Branger C, Zamfir O, Geoffroy S, et al. Genetic background of Escherichia coli and extended-spectrum ß-lactamase type. Emerg Infect Dis (2005) 11:54–61.[ISI][Medline]

9 Johnson JR, Stell AL. Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J Infect Dis (2000) 181:261–72.[CrossRef][ISI][Medline]

10 Livermore DM, Canton R, Gniadkowski M, et al. CTX-M: changing the face of ESBLs in Europe. J Antimicrob Chemother (2007) 59:165–74.[Abstract/Free Full Text]


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