John D Turnidge, Thomas Gottlieb, David H Mitchell, Geoffrey W Coombs, Julie C Pearson, Jan M Bell for the Australian Group on Antimicrobial Resistance

Abstract

The Australian Group on Antimicrobial Resistance performs regular period-prevalence studies to monitor changes in antimicrobial resistance in selected enteric Gram-negative pathogens. The 2011 survey focussed on hospital-onset infections, examining isolates from all specimens presumed to be causing disease. In 2011, 1,827 Escherichia coli, 537 Klebsiella species and 269 Enterobacter species were tested using a commercial automated method (Vitek 2, BioMérieux) and results were analysed using Clinical and Laboratory Standards Institute breakpoints from January 2012. Of the key resistances, non-susceptibilty to the third-generation cephalosporin, ceftriaxone, was found in 9.6% of E. coli and 9.5%–12.1% ofKlebsiella spp. Non-susceptibility rates to ciprofloxacin were 10.6% for E. coli, 0.0%–8.3% for Klebsiella spp. and 0.0%–5.0% in Enterobacter spp. Resistance rates to gentamicin were 8.6%, 2.9%–10.9%, and 0.0%–15.6% for the same 3 groups respectively. Eight strains, 5 Klebsiella spp. and 3 Enterobacter spp. were shown to harbour a carbapenemase (IMP-4). Commun Dis Intell 2014;38 (1):E49–E53.

Keywords: antibiotic resistance; hospital onset; gram-negative; Escherichia coli; Enterobacter; Klebsiella

Top of page

Introduction

Emerging resistance in common pathogenic members of the family 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 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 (http://www.agargroup.org/surveys). 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 but prominent in hospital-acquired infections, and of high importance due to intrinsic resistance to first-line antimicrobials. Taken together, the 3 groups surveyed are considered to be valuable sentinels for multi-resistance and emerging resistance in enteric Gram-negative bacilli.

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 include resistance to antibiotics commonly used in the hospital setting such as cefazolin; resistance to agents important for serious infections, such as gentamicin; and resistance to reserve agents such as ciprofloxacin and meropenem.

The objectives of the 2011 surveillance program were to:

• determine the proportion of resistance to the main therapeutic agents in E. coli, Klebsiella species and Enterobacter species in a subset of Australian diagnostic laboratories;
• examine the extent of co-resistance and multi-resistance in these species; and
• detect emerging resistance to extended-spectrum cephalosporins and newer last-line agents such as carbapenems.

Top of page

Methods

Source of isolates

Isolates were collected from patients hospitalised for more than 48 hours. Each institution collected up to 70 E. coli, 20 Klebsiella spp. and 10 Enterobacter spp.

Species identification

Isolates were identified by one of the following methods: Vitek^®; Phoenix™ Automated Microbiology System, Microbact; ATB^®; or agar replication. In addition, some E. coli isolates were identified using chromogenic agar plus spot indole (DMACA).

Susceptibility testing

Testing was performed by a commercial semi-automated method, Vitek^® 2 (BioMérieux), which is calibrated to the ISO reference standard method of broth microdilution. Commercially available Vitek^® AST-N149 cards were utilised by all participants throughout the survey period. The most recent Clinical and Laboratory Standards Institute breakpoints from 2012¹ were employed in the analysis. E. coli ATCC 25922 and E. coli ATCC 35218 were the quality control strains for this survey. For analysis of cefazolin, breakpoints of ≤4 for susceptible and ≥8 for resistant were applied due to the minimum inhibitory concentration (MIC) range available on the Vitek card, recognising that the January 2012 breakpoint is actually susceptible ≤2 mg/L. Ertapenem MICs were performed using Etest™ strips (BioMérieux). 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.

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; and all isolates with ertapenem MIC >0.5 mg/L or meropenem MIC >0.25 mg/L were referred to a central laboratory for molecular confirmation of resistance.

All isolates were screened for the presence of the bla_TEM, and bla_SHV genes using a real-time polymerase chain reaction (PCR) platform (LC-480) and published primers.^2,3 A multiplex real-time TaqMan PCR was used to detect CTX-M-type genes.⁴ Strains were probed for plasmid-borne AmpC enzymes using the method described by Pérez-Pérez and Hanson,^5. and subjected to molecular tests for MBL (bla_VIM, bla_IMP, and bla_NDM), bla_KPC, and bla_OXA-48-like genes using real-time PCR.^6,7

Top of page

Results

In 2011, 2,633 isolates were examined comprising 1,827 E. coli, 537 Klebsiella spp. and 269 Enterobacter spp. (Table 1). The majority of isolates were from urine, while 5.6% of isolates overall were from blood cultures (comprising 4.8% of E. coli isolates, 7.3% of Klebsiella and 8.2% of Enterobacter species). Other sites of isolation reflect the high incidence of these species in nosocomial and pre– and post-operative surgical infections.

Table 1: Species tested

Group	Species	Total
E. coli	E. coli	1,827
Klebsiella	K. pneumoniae	396
	K. oxytoca	137
	K. pneumoniaesubsp ozaenae	3
	Klebsiella not speciated	1
Total		537
Enterobacter	E. cloacae	180
	E. aerogenes	83
	E. asburiae	3
	E. gergoviae	2
	Enterobacternot speciated	1
Total		269

Top of page

Major resistances and non-susceptibilities are listed in Table 2. Multi-resistance was detected in 12.6% of E. coli isolates, 10.6% of Klebsiella species, and 8.7% of Enterobacter species (Table 3). 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). By way of summary, there were no substantial differences across the states and territories in resistance patterns in contrast to what is seen with resistance patterns in Staphylococcus aureus and Enterococcus spp.

Table 2: Non-susceptibility and resistance rates for the main species tested

Antimicrobial	Category*	E. coli (%)	K. pneumoniae (%)	K. oxytoca (%)	E. cloacae (%)	E. aerogenes (%)
* R = resistant, I = intermediate, NS = non-susceptible (intermediate + resistant). † Considered largely intrinsically resistant due to natural β-lactamases. Testing for resistance to piperacillin-tazobactam was not available for this survey due to a global recall from BioMérieux.
Ampicillin	I	0.9	†	†	†	†
Ampicillin	R	50.5	†	†	†	†
Amoxycillinclavulanate	I	16.1	8.8	4.4	†	†
Amoxycillinclavulanate	R	7.7	6.1	10.2	†	†
Ticarcillin-clavulanate	R	8.0	9.1	11.7	33.9	21.7
Cefazolin	R	22.3	18.4	68.6	†	†
Cefoxitin	R	4.8	4.3	2.2	†	†
Ceftriaxone	NS	9.6	12.1	9.5	43.3	33.7
Ceftazidime	NS	5.8	9.8	3.6	40.6	28.9
Cefepime	NS	1.8	2.3	0.0	4.4	0.0
Meropenem	NS	0.1	0.5	0.0	0.6	0.0
Ertapenem	NS	0.2	1.0	0.0	16.1	4.8
Ciprofloxacin	NS	10.6	8.3	0.0	5.0	0.0
Norfloxacin	NS	10.2	4.8	0.0	4.4	0.0
Gentamicin	NS	8.6	10.9	2.9	15.6	0.0
Trimethoprim	R	23.4	18.7	4.4	27.2	2.4
Nitrofurantoin	NS	5.0	†	†	†	†

Top of page

Table 3: Multiple acquired resistances, by species

		Number of acquired resistances
* Antibiotics included: amoxycillin-clavulanate, cefazolin, cefoxitin, ceftriaxone, ceftazidime, cefepime, gentamicin, amikacin, ciprofloxacin, nitrofurantoin, trimethoprim, meropenem. Antibiotics excluded: ampicillin (intrinsic resistance), ticarcillin-clavulanate, tobramycin, norfloxacin, nalidixic acid, sulfamethoxazole-trimethoprim (high correlation with antibiotics in the included list). † Antibiotics included, ceftriaxone, ceftazidime, cefepime, gentamicin, amikacin, ciprofloxacin, nitrofurantoin, trimethoprim, meropenem. Antibiotics excluded: ampicillin, amoxycillin-clavulanate, cefazolin, and cefoxitin, (all four due to intrinsic resistance); also excluded were ticarcillin-clavulanate, tobramycin, norfloxacin, nalidixic acid, sulfamethoxazole-trimethoprim (high correlation with antibiotics in the included list).
Species	Total	Non-multi-resistant					Multi-resistant
		0	1	2	3	Cumulative %	4	5	6	7	8	9	10	Cumulative %
E. coli	1,827	828	340	278	150		68	48	55	29	26	4	1
%		45.3	18.6	15.2	8.2	87.4	3.7	2.6	3.0	1.6	1.4	0.2	0.1	12.6
Klebsiella spp.*	537	280	158	22	20		20	12	10	11	3	1
%		52.1	29.4	4.1	3.7	89.4	3.7	2.2	1.9	2.0	0.6	0.2		10.6
Enterobacter spp.†	269	107	56	62	18		16	6	3	1
%		39.8	20.8	23.0	6.7	90.3	5.9	2.2	1.1	0.4				9.7

Top of page

Escherichia coli

Moderately high levels of resistance to ampicillin (and therefore amoxycillin) were observed (50.5%), with lower rates for amoxycillin-clavulanate (16.1% intermediate, and 7.7% resistant) (Table 2). Non-susceptibility to third-generation cephalosporins has increased slowly compared with the 2009 survey (ceftriaxone 9.6%, ceftazidime 5.8%, compared with 7.2% and 4.2% respectively in 2009). Most of the strains with extended-spectrum ß-lactamase (ESBL) genes harboured genes of the CTX-M type (68%, 128/189). Moderate levels of resistance were detected to cefazolin (22.3%) and trimethoprim (23.4%). Ciprofloxacin non-susceptibility was found in 10.6% of E. coli isolates. Ciprofloxacin resistance was found in 51.1% and gentamicin resistance was found in 42.6% of ESBL-producing strains. Resistance to ticarcillin-clavulanate, cefepime, and gentamicin were below 5%. Two isolates had elevated meropenem MICs (≥0.5 mg/L) but 73 strains (4.0%) had ertapenem MICs above wild-type (>0.06 mg/L), 89% of which contained CTX-M or plasmid-borne AmpC genes. None harboured a known carbapenemase.

Klebsiella species

These showed slightly higher levels of resistance to cefazolin and ceftriaxone compared with E. coli, but lower rates of resistance or non-susceptibility to ticarcillin-clavulanate, cefazolin, ceftriaxone, ceftazidime, and gentamicin (Table 2). ESBLs were present in 48 of 53 presumptively ESBL-positive isolates of K. pneumoniae, 35 of which proved to be of the CTX-M type. Five of 7 Klebsiella species (5 K. pneumoniae and 1 K. oxytoca) with elevated meropenem MICs (≥ 0.5 mg/L) harboured bla_IMP-4, while 30 additional strains had elevated ertapenem MICs (>0.06 mg/L), but none of these harboured a known carbapenemase.

Enterobacter species

Acquired resistance was common to ticarcillin-clavulanate (29.7%), ceftriaxone (40.1%), ceftazidime (36.4%) and trimethoprim (19.3%) (Table 2). Rates of resistance to cefepime, ciprofloxacin, and gentamicin were all less than 11%. Twenty-seven of 88 strains tested for ESBL based on a suspicious phenotype, harboured ESBL-encoding genes. Five strains had elevated meropenem MICs (≥ 0.5 mg/L) three of which harboured bla_IMP-4, while 39% of strains had ertapenem MICs above wild type (>0.125 mg/L), related to the presence of stably-derepressed chromosomal AmpC ß-lactamase.

Top of page

Discussion

Comparing these results with those from the first hospital-onset survey in 2009, there is a small but noticeable increase in resistance or non-susceptibility rates to some reserve antibiotics. For example, rates of resistance in E. coli for ceftriaxone rose from 7.2% to 9.6% and for non-susceptibility to ciprofloxacin rose from 8.1% to 10.6%. Such rises were not observed in Klebsiella or Enterobacter species. Although originally thought to be primarily community-associated, the great bulk of extended-spectrum ß-lactamases detected were of the CTX-M type, suggesting that this group has become the dominant form in hospital infections as well. Plasmid-borne AmpC ß-lactamases also appear to be increasing substantially, up from 31 strains with genes detected encoding one of these enzymes in 2009, to 51 strains in 2011.

The greatest concern is the emergence of carbapenemases which affect the ‘last-line’ ß-lactams such as meropenem. In 2009, we detected 5 strains of Klebsiella with a carbapenemase, all of which were bla_IMP-4. In this 2011 survey, we found 8 strains, 5 Klebsiella spp. and 3 Enterobacter sp., all of which were also bla_IMP-4. This carbapenemase appears to have become endemic in Australia, albeit at a very low level presently. So far our surveys have not detected other carbapenemases, such as KPC-2 and NDM-1, which are known to be prevalent in other countries. However, there are published reports of the detection on these carbapenemases in Australia, all so far imported by overseas visitors or Australian returning from overseas.^10,11 Surveys such as those conducted by AGAR are critical to determining whether such unwelcome resistances might become established in Australia.

Top of page

Agar participants

Australian Capital Territory

Peter Collignon and Susan Bradbury, The Canberra Hospital

New South Wales

Thomas Gottlieb and Glenn Funnell, Concord Hospital

Miriam Paul and Richard Jones, Douglass Hanly Moir Pathology

James Branley and Donna Barbaro, Nepean Hospital

George Kotsiou and Peter Huntington, Royal North Shore Hospital

Colin MacLeod and Bradley Watson, Royal Prince Alfred Hospital

Iain Gosbell and Annabelle LeCordier, South West Area Pathology Service

David Mitchell and Lee Thomas, Westmead Hospital

Top of page

Northern Territory

Rob Baird and Jann Hennessy, Royal Darwin Hospital

Queensland

Enzo Binotto and Bronwyn Thomsett, Pathology Queensland Cairns Base Hospital

Graeme Nimmo and Narelle George, Pathology Queensland Central Laboratory

Petra Derrington and Dale Thorley, Pathology Queensland Gold Coast Hospital

Chris Coulter and Sonali Coulter, Pathology Queensland Prince Charles Hospital

Joan Faoagali and Gweneth Lye, Pathology Queensland Princess Alexandra Hospital

Jenny Robson and Georgia Peachey, Sullivan Nicolaides Pathology

South Australia

Kelly Papanoum and Hendik Pruul, SA Pathology, Flinders Medical Centre

Morgyn Warner and Fleur Manno, SA Pathology, Royal Adelaide Hospital

John Turnidge and Jan Bell, SA Pathology, Women’s and Children’s Hospital

Top of page

Tasmania

Mhisti Rele and Kathy Wilcox, Launceston General Hospital

Alistair McGregor and Rob Peterson, Royal Hobart Hospital

Victoria

Denis Spelman and Michael Huysmans, Alfred Hospital

Ben Howden and Peter Ward, Austin Hospital

Tony Korman and Despina Kotsanas, Southern Health, Monash Medical Centre

Sue Garland and Gena Gonis, Royal Women’s Hospital

Mary Jo Waters and Linda Joyce, St Vincent’s Hospital

Western Australia

David McGechie and Rebecca Wake, PathWest Laboratory Medicine, WA Fremantle Hospital

Ronan Murray and Barbara Henderson, PathWest Laboratory Medicine, WA Queen Elizabeth II Hospital

Keryn Christiansen and Geoffrey Coombs, PathWest Laboratory Medicine, WA Royal Perth Hospital

Victoria D’Abrera and Sindy Budalich, St John of God Pathology

Author details

John D Turnidge^1,2

Thomas Gottlieb³

David H Mitchell⁴

Geoffrey W Coombs^5,6

Julie C Pearson⁶

Jan M Bell¹

1. Microbiology and Infectious Diseases, SA Pathology, Women’s and Children’s Hospital, North Adelaide, South Australia
2. Departments of Pathology, Paediatrics and Molecular Biosciences, University of Adelaide, South Australia
3. Department of Microbiology and Infectious Diseases, Concord, Concord, New South Wales
4. Centre for Infectious Diseases and Microbiology, Westmead Hospital, Westmead, New South Wales
5. Australian Collaborating Centre for Enterococcus and Staphylococcus Species (ACCESS) Typing and Research, School of Biomedical Sciences, Curtin University, Perth, Western Australia
6. Department of Microbiology and Infectious Diseases, PathWest Laboratory Medicine, WA, Royal Perth Hospital, Perth, Western Australia

Corresponding author: Professor John Turnidge, Microbiology and Infectious Diseases, SA Pathology, Women’s and Children’s Hospital, 72 King William Road, NORTH ADELAIDE SA. Telephone: +61 8 8161 6873 Email: john.turnidge AT health.sa.gov.au

Top of page

References

1. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Twenty-Second Informational Supplement M100–S22. Villanova, PA, USA 2012.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. Turnidge J, Gottlieb T, Mitchell D, Pearson J for the Australian Group for Antimicrobial Resistance. Gram-negative Survey, 2008 Antimicrobial Susceptibility Report. 2011. Adelaide: Australian Group for Antimicrobial Resistance. Available from: http://www.agargroup.org/files/AGAR%20GNB08%20Report%20FINAL.pdf
9. 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.
10. Sidjabat H, Nimmo GR, Walsh TR, Binotto E, Htin A, Hayashi Y, Li J, Nation RL, George N, Paterson DL. Carbapenem resistance in Klebsiella pneumoniae due to the New Delhi metallo-ß-lactamase. Clin Infect Dis 2011;52(4):481–484.
11. Coatsworth NR, Huntington PG, Hardiman RP, Hudson BJ, Fernandes CJ. A case of carbapenemase-producing Klebsiella pneumoniae in Australia. Pathology 2012;44(1):42–44.

Top of page