Jan M Bell, John D Turnidge, Geoffrey W Coombs, Denise A Daley, Thomas Gottlieb, Jenny Robson, Narelle George
The Australian Group on Antimicrobial Resistance performs regular period-prevalence studies to monitor changes in antimicrobial resistance in selected enteric Gram-negative pathogens. The 2014 survey was the second year to focus on blood stream infections. During 2014, 5,798 Enterobacteriaceae species isolates were tested using commercial automated methods (Vitek 2, BioMérieux; Phoenix, BD) and results were analysed using the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (January 2015). Of the key resistances, non-susceptibility to the third-generation cephalosporin, ceftriaxone, was found in 9.0%/9.0% of Escherichia coli (CLSI/EUCAST criteria) and 7.8%/7.8% of Klebsiella pneumoniae, and 8.0%/8.0% K. oxytoca. Non-susceptibility rates to ciprofloxacin were 10.4%/11.6% for E. coli, 5.0%/7.7% for K. pneumoniae, 0.4%/0.4% for K. oxytoca, and 3.5%/6.5% in Enterobacter cloacae. Resistance rates to piperacillin-tazobactam were 3.2%/6.8%, 4.8%/7.2%, 11.1%/11.5%, and 19.0%/24.7% for the same 4 species respectively. Fourteen isolates were shown to harbour a carbapenemase gene, 7 blaIMP-4, 3 blaKPC-2, 3 blaVIM-1, 1 blaNDM-4, and 1 blaOXA-181-lke .Commun Dis Intell 2016;40(2):E229–E235.
Keywords: antibiotic resistance; bacteraemia; gram-negative; Escherichia coli; Enterobacter; Klebsiella
Emerging resistance in common pathogenic members of the Enterobacteriaceae is a world-wide phenomenon, and presents therapeutic problems for practitioners in both the community and in hospital practice. The Australian Group on Antimicrobial Resistance (AGAR) commenced surveillance of the key Gram-negative pathogens, Escherichia coli and Klebsiella species in 1992. Surveys have been conducted biennially until 2008 when annual surveys commenced, alternating between community– and hospital-onset infections.1 In 2004, another genus of Gram-negative pathogens in which resistance can be of clinical importance, Enterobacter species, was added. E. coli is the most common cause of community-onset urinary tract infection, while Klebsiella species are less common but are known to harbour important resistances. Enterobacter species are less common in the community, but of high importance due to intrinsic resistance to first-line antimicrobials in the community. Taken together, the three groups of species surveyed are considered to be valuable sentinels for multi-resistance and emerging resistance in enteric Gram-negative bacilli. In 2013 AGAR commenced the Enterobacteriaceae Sepsis Outcome Programme (EnSOP), which focused on the collection of resistance and some demographic data on all isolates prospectively from patients with bacteraemia. The 2014 survey was the second EnSOP survey.
Resistances of particular interest include resistance to ß-lactams due to ß-lactamases, especially extended-spectrum ß-lactamases, which inactivate the third-generation cephalosporins that are normally considered reserve antimicrobials. Other resistances of interest are to agents important for treatment of these serious infections, such as gentamicin; and resistance to reserve agents such as ciprofloxacin and meropenem.
The objectives of the 2014 surveillance program were to:
- monitor resistance in Enterobacteriaceae isolated from blood;
- examine the extent of co-resistance and multi-resistance; and
- detect emerging resistance to newer last-line agents such as carbapenems.
From 1 January to 31 December 2014, 26 institutions across Australia collected either all or up to 200 isolates from different patient episodes of bacteraemia.
Isolates were identified using the routine method for each institution; Vitek®, Phoenix™ Automated Microbiology System, or where available, mass spectrometry (MALDI-TOF).
Testing was performed by 2 commercial semi-automated methods, Vitek 2 (BioMérieux) or Phoenix (BD), which are calibrated to the ISO reference standard method of broth microdilution. Commercially available Vitek AST-N246, or Phoenix NMIC-203 cards were utilised by all participants throughout the survey period. The Clinical and Laboratory Standards Institute (CLSI) M1002 and European Committee on Antimicrobial Susceptibility Testing (EUCAST) v5.03 breakpoints from January 2015 have been employed in the analysis. For analysis of cefazolin, breakpoints of ≤ 4 for susceptible, and ≥ 8 for resistant were applied due to the restricted minimum inhibitory concentration (MIC) range available on the commercial cards, recognising that the January 2015 breakpoint is actually susceptible ≤ 2 mg/L.
Molecular confirmation of resistances
E. coli and Klebsiella isolates with ceftazidime or ceftriaxone MIC > 1 mg/L, or cefoxitin MIC > 8 mg/L; Enterobacter spp. with cefepime MIC > 1 mg/L; all isolates with ciprofloxacin MIC > 0.25 mg/L; all isolates with meropenem MIC > 0.25 mg/L; and all isolates with amikacin MIC > 32 mg/L were referred to a central laboratory (SA Pathology) for molecular confirmation of resistance.
All referred isolates were screened for the presence of the blaTEM, and blaSHV genes using a real-time polymerase chain reaction (PCR) platform (LC-480) and published primers.4,5 A multiplex real-time TaqMan PCR was used to detect CTX-M-type genes.6 Strains were probed for plasmid-borne AmpC enzymes using the method described by Pérez-Pérez and Hanson,7 and subjected to molecular tests for MBL (blaVIM, blaIMP, and blaNDM), blaKPC, and blaOXA-48-like genes using real-time PCR.8,9 Known plasmid mediated quinolone resistance mechanisms (Qnr, efflux (qepA, oqxAB), and aac(6’)-Ib-cr) were examined by PCR on all referred isolates with ciprofloxacin MIC > 0.25 mg/L using published methods.10,11 All E. coli were examined for presence of the O25b-ST131 clone and its H30- and H30-Rx subclones.12–14
The species isolated, and the numbers of each are listed in Table 1. Three genera, Escherichia spp., Klebsiella spp. and Enterobacter spp. contributed 87.6% of all isolates. Major resistances and non-susceptibilities for the top 6 ranked species are listed in Table 2. Non-susceptibility, (which includes both intermediately resistant and resistant strains), has been included for some agents because these figures provide information about important emerging acquired resistances. Multiple acquired resistances by species are shown in Table 3. Multi-resistance was detected in 13.4% of E. coli isolates, 9.7% of K. pneumoniae, and 12.1% of Ent. cloacae. A more detailed breakdown of resistances and non-susceptibilities by state and territory is provided in the online report from the group (http://www.agargroup.org/surveys).
|Salmonella species (non Typhi)||94||1.6|
|Other species (n=27)||75||1.3|
* R = resistant, I = intermediate, NS = non-susceptible (intermediate + resistant), using criteria as published by the Clinical and Laboratory Standards Institute (CLSI)  and European Committee on Antimicrobial Susceptibility Testing (EUCAST) .
† Considered largely intrinsically resistant due to natural ß-lactamases; – no intermediate category; / no breakpoints defined
‡ For EUCAST interpretation, the clavulanate is fixed at 2 mg/L, rather than a 2:1 ratio used in CLSI guidelines. As all cards used have a 2:1 ratio of clavulanate no EUCAST category has been applied.
|Species||Total||Number of acquired resistances (CLSI breakpoints)|
|0||1||2||3||Cumulative %||4||5||6||7||8||9||10||11||12||13||Cumulative %|
* Antibiotics included: amoxycillin-clavulanate, piperacillin-tazobactam, cefazolin, cefoxitin, ceftriaxone, ceftazidime, cefepime, gentamicin, amikacin, ciprofloxacin, nitrofurantoin, trimethoprim, meropenem;
† Antibiotics included: piperacillin-tazobactam, ceftriaxone, ceftazidime, cefepime, gentamicin, amikacin, ciprofloxacin, nitrofurantoin, trimethoprim, meropenem
|Salmonella spp. (non-Typhi)||71||57||8||4||2|
Moderately high levels of resistance to ampicillin (and therefore amoxycillin) were maintained (50.1%/51.9%, CLSI/EUCAST criteria), with lower rates for amoxycillin-clavulanate (12.7%/- intermediate, 8.2%/20.9% resistant). Non-susceptibility to third-generation cephalosporins was low (ceftriaxone 9.0%/9.0%, ceftazidime 4.4%/8.0%). Moderate levels of resistance were detected to cefazolin (20.5%/-) and trimethoprim (29.2%/29.4%). Ciprofloxacin non-susceptibility was found in 10.4%/11.6% of E. coli isolates. Resistance to ticarcillin-clavulanate (9.4%/19.3%), gentamicin (7.3%/7.5%), piperacillin-tazobactam (3.2%/6.8%), and cefepime (1.6%/2.9%) were low. Nine isolates had elevated meropenem MICs (≥ 0.5 mg/L). For the extended-spectrum ß-lactamase (ESBL)-producing strains, ciprofloxacin and gentamicin resistance was found in 51.6%/51.6% and 33.8%/34.1% respectively.
In line with international trends among community strains of E. coli, most of the strains with ESBL genes harboured genes of the CTX-M type (222/272 = 82%). Over 60% of E. coli with CTX-M group 1 types were found to belong to sequence type 131 (O25b-ST131). ST131 accounted for 68% of E. coli ESBL phenotypes that were ciprofloxacin resistant (MIC > 1 mg/L), and only 6% of ciprofloxacin susceptible ESBL phenotypes. Ninety-one per cent and 41% of O25b-ST131 were associated with the H30 and H30-Rx subclones, respectively, with their reported association with more antibiotic resistances and greater virulence potential.13
K. pneumoniae showed slightly higher levels of resistance to piperacillin-tazobactam and ceftazidime compared with E. coli, but lower rates of resistance to amoxycillin-clavulanate, ticarcillin-clavulanate, cefazolin, ceftriaxone ciprofloxacin, gentamicin, and trimethoprim. Thirteen K. pneumoniae isolates had elevated meropenem MICs. ESBLs were present in 61 of 69 (88%) presumptively ESBL-positive isolates of K. pneumoniae, 47 (77%) of which proved to be of the CTX-M type.
Acquired resistance was common to ticarcillin-clavulanate (24.4%/29.7% and 26.7%/40.6%), piperacillin-tazobactam (19.0%/24.7% and 23.1%/31.7%), ceftriaxone (27.3%/27.3% and 37.5%/37.5%), ceftazidime (24.3%/24.6% and 29.8%/33.7%) and trimethoprim (19.1%/19.1% and 1.9%/1.9%) for Ent. cloacae and Ent. aerogenes, respectively. Cefepime, ciprofloxacin, and gentamicin resistance were all less than 10%. Seventeen of 45 Ent. cloacae tested for ESBL based on a suspicious phenotype, harboured ESBL-encoding genes. Eighteen Ent. cloacae strains had elevated meropenem MICs.
Overall, 14 isolates (14 patients) in 9 institutions from 5 states or territories were found to harbour a carbapenemase gene. BlaIMP-4 was detected in E. cloacae (5) and K. pneumoniae (2); blaKPC-2 was detected in 3 K. pneumoniae isolates from 1 institution; blaVIM-1 was detected in 2 K. pneumoniae; blaNDM-4 in 1 E. coli, and blaOXA-181-like in 1 K. pneumoniae.
AGAR has been tracking resistance in sentinel enteric Gram-negative bacteria since 1992. From 2008, surveillance was segregated into hospital- versus community-onset infections. The last year of hospital-onset only surveillance was 2011.15 In 2013, the first survey of antimicrobial resistance among Enterobacteriaceae isolates from bacteraemic patients throughout Australia was conducted using an approach similar to that conducted by the European EARS-Net program. The 2014 survey was the second survey conducted of antimicrobial resistance among Enterobacteriaceae isolates from bacteraemic patients throughout Australia.
CTX-M-producing E. coli and Klebsiella species and gentamicin- and ciprofloxacin-resistant E. coli continued to be a problem in patients with bacteraemia. Of concern is the high proportion of E. coli that belong to the ST131 H30-Rx subclone, and its reported association with more antibiotic resistance and greater virulence potential.13 Carbapenem resistance attributable to acquired carbapenemases are still uncommon in patients with bacteraemia in Australia, although 5 different types (IMP, KPC, VIM, NDM and OXA-181-like) were detected from 9 of the participating institutions. Compared with many other countries in our region, resistance rates in Australian Gram-negative bacteria are still relatively low,16 but similar to those observed in 2014 in many Western European countries.17
Multi-resistance is being increasingly observed, especially in E. coli and E. cloacae, both of which have multi-resistance rates (as defined by AGAR) above 10%. This is likely to drive more broad-spectrum antibiotic use, and increase the resistance selection pressure for important reserve classes, especially the carbapenemases.
Australian Capital Territory
Peter Collignon and Susan Bradbury, The Canberra Hospital
New South Wales
Thomas Gottlieb and Graham Robertson, Concord Hospital
John Ferguson and Jack (Ian) Winney, John Hunter Hospital
James Branley and Donna Barbaro, Nepean Hospital
George Kotsiou and Peter Huntington, Royal North Shore Hospital
David Mitchell and Lee Thomas, Westmead Hospital
Rob Baird and Jann Hennessy, Royal Darwin Hospital
Enzo Binotto and Bronwyn Thomsett, Pathology Queensland Cairns Base Hospital
Graeme Nimmo and Narelle George, Pathology Queensland Central Laboratory
Petra Derrington and Sharon Dal-Cin, Pathology Queensland Gold Coast Hospital
Robert Horvath, Pathology Queensland Prince Charles Hospital
Naomi Runnegar and Joel Douglas, Pathology Queensland Princess Alexandra Hospital
Jenny Robson and Georgia Peachey, Sullivan Nicolaides Pathology
Kelly Papanoum and Nicholas Wells, SA Pathology (Flinders Medical Centre)
Morgyn Warner and Kija Smith, SA Pathology (Royal Adelaide Hospital)
John Turnidge and Jan Bell, SA Pathology (Women’s and Children’s Hospital)
Louise Cooley and Rob Peterson, Royal Hobart Hospital
Denis Spelman, Amanda Dennison and Christopher Lee, Alfred Hospital
Benjamin Howden and Peter Ward, Austin Hospital
Tony Korman and Despina Kotsanas, Southern Health, Monash Medical Centre
Andrew Daley and Gena Gonis, Royal Women’s Hospital
Mary Jo Waters and Linda Joyce, St Vincent’s Hospital
David McGechie and Rebecca Wake, PathWest Laboratory Medicine - WA, Fremantle Hospital
Ronan Murray and Barbara Henderson, PathWest Laboratory Medicine – WA, Queen Elizabeth II Hospital
Owen Robinson, Denise Daley and Geoffrey Coombs, PathWest Laboratory Medicine – WA, Royal Perth Hospital
Sudha Pottumarthy-Boddu and Fay Kappler, St John of God Pathology
Prof John D Turnidge1
Ms Jan M Bell2
A/Prof Geoffrey W Coombs3,4
Ms Denise A Daley5
A /Prof Thomas Gottlieb6
Dr Jenny Robson7
Ms Narelle George8
- Departments of Pathology, Paediatrics and Molecular and Biomedical Sciences, University of Adelaide, Adelaide, South Australia
- Microbiology and Infectious Diseases Directorate, SA Pathology, Adelaide, South Australia
- Australian Collaborating Centre for Enterococcus and Staphylococcus Species (ACCESS) Typing and Research, School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western Australia
- Department of Microbiology and Infectious Diseases, PathWest Laboratory Medicine-WA, Fiona Stanley Hospital, Murdoch, Western Australia
- Australian Group on Antimicrobial Resistance, Fiona Stanley Hospital, Murdoch, Western Australia
- Concord Hospital, Concord, New South Wales
- Sullivan Nicolaides Pathology, Queensland
- Microbiology, Pathology Queensland, Royal Brisbane and Women’s Hospital, Herston, Queensland
Corresponding author: Prof John D Turnidge, Departments of Pathology, Paediatrics and Molecular and Biomedical Sciences, University of Adelaide, ADELAIDE SA 8000. Telephone: +61 417 811 552. Email: email@example.com
- Australian Group on Antimicrobial Resistance. Survey reports. [online]. Available from: http://www.agargroup.org/surveys
- Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Twenty-Fifth Informational Supplement M100–S25. Villanova, PA, USA 2015.
- European Committee on Antimicrobial Susceptibility Testing (2014). Breakpoint tables for interpretation of MICs and zone diameters. Version 5.0, January 2015. Accessed on 1 January 2015. Available from: http://www.eucast.org/clinical_breakpoints/
- Hanson ND, Thomson KS, Moland ES, Sanders CC, Berthold G, Penn RG. Molecular characterization of a multiply resistant Klebsiella pneumoniae encoding ESBLs and a plasmid-mediated AmpC. J Antimicrob Chemother 1999;44(3):377–380.
- Chia JH, Chu C, Su LH, Chiu CH, Kuo AJ, Sun CF, et al. Development of a multiplex PCR and SHV melting-curve mutation detection system for detection of some SHV and CTX-M b-lactamases of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae in Taiwan. J Clin Microbiol 2005;43(9):4486–4491.
- Birkett CI, Ludlam HA, Woodford N, Brown DFJ, Brown NM, Roberts MTM, et al. Real-time TaqMan PCR for rapid detection and typing of genes encoding CTX-M extended-spectrum ß-lactamases. J Med Microbiol 2007;56(Pt 1):52–55.
- Perez-Perez FJ, Hanson ND. Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 2002;40(6):2153–2162.
- Poirel L, Héritier C, Tolün V, Nordmann P. Emergence of oxacillinase-mediated resistance to imipenem in Klebsiella pneumoniae. Antimicrob Agents Chemother 2004;48(1):15–22.
- Mendes RE, Kiyota KA, Monteiro J, Castanheira M, Andrade SS, Gales AC, et al. Rapid detection and identification of metallo-ß-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis. J Clin Microbiol 2007;45(2):544–547.
- Cattoir V, Poirel L, Rotimi V, Soussy C-J, Nordmann P. Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBL-producing enterobacterial isolates. J Antimicrob Chemother 2007;60(2):394–397.
- Ciesielczuk H, Hornsey M, Choi V, Woodford N, Wareham DW. Development and evaluation of a multiplex PCR for eight plasmid-mediated quinolone-resistance determinants. J Med Microbiol 2013;62(Pt 12):1823–1827.
- Dhanjii H, Doumith M, Clermont O, Denamur E, Hope R, Livermore DM, et al. Real-time PCR for detection of the O25b-ST131 clone of Escherichia coli and its CTX-M-15-like extended-spectrum ß-lactamases. J Antimicrob Agents 2010;36(4):355–358.
- Banerjee R, Robicsek A, Kuskowski MA, Porter S, Johnston BD, Sokurenko E, et al. Molecular epidemiology of Escherichia coli sequence type 131 and its H30 and H30-Rx subclones among extended-spectrum-ß-lactamase-positive and -negative E. coli clinical isolates from the Chicago region, 2007 to 2010. Antimicrob Agents Chemother 2013;57(12):6385–6388.
- Colpan A, Johnston B, Porter S, Clabots C, Anway R, Thao L, et al. Escherichia coli sequence type 131 (ST131) subclone H30 as an emergent multidrug-resistant pathogen among US veterans. Clin Infect Dis 2013;57(9):1256–1265.
- Turnidge J, Gottlieb T, Mitchell D, Pearson J, Bell J, for the Australian Group for Antimicrobial Resistance. Gram-negative survey 2011 antimicrobial susceptibility report. 2011 Adelaide. Available from: http://www.agargroup.org/files/AGAR%20GNB08%20Report%20FINAL.pdf
- Sheng WH, Badal RE, Hsueh PR; SMART Program. Distribution of extended-spectrum ß-lactamases, AmpC ß-lactamases, and carbapenemases among Enterobacteriaceae isolates causing intra-abdominal infections in the Asia–Pacific region: results of the study for Monitoring Antimicrobial Resistance Trends (SMART). Antimicrob Agents Chemother 2013;57(7):2981–2988.
- European Centre for Disease Prevention and Control. Annual epidemiological report antimicrobial resistance and healthcare-associated infections 2014. Available from: http://ecdc.europa.eu/en/publications/_layouts/forms/Publication_DispForm.aspx?List=4f55ad51-4aed-4d32-b960-af70113dbb90&ID=1292