Jan M Bell, John D Turnidge, Geoffrey W Coombs, Denise A Daley, Thomas Gottlieb, Jenny Robson, Narelle George

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 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 bla_IMP-4, 3 bla_KPC-2, 3 bla_VIM-1, 1 bla_NDM-4, and 1 bla_OXA-181-lke .Commun Dis Intell 2016;40(2):E229–E235.

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

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Introduction

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.

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Methods

Study design

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.

Species identification

Isolates were identified using the routine method for each institution; Vitek®, Phoenix™ Automated Microbiology System, or where available, mass spectrometry (MALDI-TOF).

Susceptibility testing

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 bla_TEM, and bla_SHV 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 (bla_VIM, bla_IMP, and bla_NDM), bla_KPC, and bla_OXA-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

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Results

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).

Table 1: Species tested

Species	Total	%
Escherichia coli	3,493	60.2
Klebsiella pneumoniae	877	15.1
Enterobacter cloacae	343	5.9
Klebsiella oxytoca	226	3.9
Proteus mirabilis	187	3.2
Serratia marcescens	136	2.3
Enterobacter aerogenes	105	1.8
Salmonella species (non Typhi)	94	1.6
Morganella morganii	57	1.0
Citrobacter freundii	53	0.9
Citrobacter koseri	50	0.9
Salmonella Typhi/Paratyphi	26	0.4
Enterobacter asburiae	16	0.3
Raoultella ornithinolytica	15	0.3
Pantoea species	12	0.2
Pantoea agglomerans	12	0.2
Enterobacter species	11	0.2
Providencia stuartii	10	0.2
Other species (n=27)	75	1.3
Total	5,798

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Table 2: Non-susceptibility and resistance rates for the top 6 ranked species tested

		Escherichia coli (%)		Klebsiella pneumoniae (%)		Klebsiella oxytoca (%)		Enterobacter cloacae (%)		Proteus mirabilis (%)		Serratia marcescens (%)
Antimicrobial	Category*	CLSI	EUCAST	CLSI	EUCAST	CLSI	EUCAST	CLSI	EUCAST	CLSI	EUCAST	CLSI	EUCAST
* R = resistant, I = intermediate, NS = non-susceptible (intermediate + resistant), using criteria as published by the Clinical and Laboratory Standards Institute (CLSI) [2014] and European Committee on Antimicrobial Susceptibility Testing (EUCAST) [2014]. † 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.
Ampicillin	I	1.8	–	†	†	†	†	†	†	0.5	–	†	†
	R	50.1	51.9	†	†	†	†	†	†	16.8	17.3	†	†
Amoxycillinclavulanate‡	I	12.7	–	5.1	–	4.4	–	†	†	8.7	–	†	†
	R	8.2	–	5.3	–	8.8	–	†	†	1.6	–	†	†
Ticarcillin-clavulanate	R	9.4	19.3	7.2	11.4	10.4	12.2	24.4	29.7	1.1	1.7	0.0	2.2
Piperacillintazobactam	R	3.2	6.8	4.8	7.2	11.1	11.5	19.0	24.7	1.1	1.6	0.0	0.0
Cefazolin	R	20.5	/	13.0	/	66.0	/	†	†	26.5	/	†	†
Cefoxitin	R	3.8	/	6.2	/	0.9	/	†	†	0.0	/	†	†
Ceftriaxone	NS	9.0	9.0	7.8	7.8	8.0	8.0	27.6	27.6	0.5	0.5	2.9	2.9
Ceftazidime	NS	4.4	8.0	6.1	8.0	0.4	0.4	24.6	27.0	0.0	0.0	2.2	2.2
Cefepime	NS	3.3	6.4	3.7	6.1	0.0	0.0	4.1