Evolving beta-lactamase epidemiology in Enterobacteriaceae from Italian nationwide surveillance, October 2013: KPC-carbapenemase spreading among outpatients

Extended-spectrum beta-lactamases (ESBLs), AmpC-type beta-lactamases (ACBLs) and carbapenemases are among the most important resistance mechanisms in Enterobacteriaceae . This study investigated the presence of these resistance mechanisms in consecutive non-replicate isolates of Escherichia coli (n = 2,352), Klebsiella pneumoniae (n = 697), and Proteus mirabilis (n = 275) from an Italian nationwide cross-sectional survey carried out in October 2013. Overall, 15.3% of isolates were non-susceptible to extended-spectrum cephalosporins susceptible (ESCR-carbaS), also to carbapenems (ESCR-carbaR). coli CTX-M Acquired ACBLs were less and mostly detected in P. mirabilis . ESCR-carbaR isolates were mostly contributed by pneumoniae (25.1% among K. pneumoniae isolates inpatients and outpatients, respectively), with bla KPC as the most common carbapenemase gene. Results showed an increasing trend for both ESBL and carbapenemase producers in comparison with previous Italian surveys, also among outpatients.


Introduction
Enterobacteriaceae are the most common cause of healthcare associated infections, and beta-lactams are among the most used antibiotics in clinical practice for treatment of these infections [1,2].During the last decades, Enterobacteriaceae with decreased susceptibility to beta-lactams have been increasingly reported worldwide, causing major problems [3][4][5][6][7][8].
The most important resistance mechanism to beta-lactams in Enterobacteriaceae is the production of betalactamases, and the most challenging enzymes of this family are the extended-spectrum beta-lactamases (ESBLs), the AmpC-type beta-lactamases (ACBLs), and the carbapenemases [9][10][11][12].ESBLs are able to hydrolyse a wide range of beta-lactams, including penicillins, narrow-and extended-spectrum cephalosporins and monobactams, but not cephamycins and carbapenems.ESBLs are usually inhibited by conventional beta-lactamase inhibitors.CTX-M-type ESBLs, which emerged in the late 1980s, and demonstrated a high ability to disseminate in clinical settings, rapidly reaching a pandemic diffusion.Nowadays they are the most prevalent plasmid-encoded ESBLs overall [13,14].Acquired ACBLs can confer resistance to penicillins and to most cephalosporins (including cephamycins) but are poorly or not active against monobactams and the zwitterionic oxyimino-cephalosporins, such as cefepime and cefpirome, and the carbapenems.ACBLs are generally less prevalent than ESBLs in Enterobacteriaceae but are nonetheless important for their contribution to beta-lactam resistance, which can be extended also to carbapenems when ACBLs are overproduced in combination with an impermeability defect [10,15,16].Carbapenemase production is the leading resistance mechanism to carbapenems in Enterobacteriaceae.Acquired carbapenemases of the KPC-, VIM-, NDM-and OXA-48-types are the most prevalent, although with a notable geographical variability [6,[17][18][19][20].
In Italy, the most recent data from the European Antimicrobial Resistance Surveillance Network (EARS-Net) reported very high proportions of resistance to extended-spectrum cephalosporins among Escherichia coli (30.1%) and Klebsiella pneumoniae (55.9%), and of resistance to carbapenems among K. pneumoniae (33.5%) [21].However, the EARS-NET data neither cover other enterobacterial species nor describe the resistance mechanisms.This survey, promoted by the Committee for Study of Antibiotics (CoSA) of the Italian Society of Clinical Microbiologists (AMCLI), was carried out to provide an updated picture of the molecular epidemiology of ESBLand carbapenemase-producing Enterobacteriaceae circulating in Italy and to investigate, for the first time, the presence of ACBL producers on a nationwide scale.

Study design
Fourteen clinical microbiology laboratories from 13 Italian cities participated in the study (Figure 1).The laboratories were selected so as to provide a countrywide coverage by large laboratories associated with hospitals, representative of most Italian Regions.Twelve of the 14 laboratories had also been involved in the first Italian survey on carbapenemase-producing Enterobacteriaceae, carried out in 2011 [22].The survey period was from 1 to 15 October 2013.
The laboratories consecutively collected all non-replicate (only first isolate from a patient included) clinical isolates of E. coli, K. pneumoniae and Proteus mirabilis, from any site of infection, showing a minimum inhibitory concentration (MIC) > 1 mg/L for extendedspectrum cephalosporins (ESC) (cefotaxime and/or ceftriaxone and/or ceftazidime and/or cefepime), and/or for ertapenem.Participating laboratories determined MICs of ESC and ertapenem by the automated systems routinely used in the respective laboratory: either Vitek 2 (bioMérieux, Marcy l'Etoile, France) or BD Phoenix (Becton, Dickinson and Co., New Jersey, United States (US)).The collected isolates were sent to reference laboratories for confirmation of species identification and characterisation of the resistance mechanisms.
For each isolate, information on the type of clinical specimen and of patient (inpatient or outpatient) were provided.Isolates from patients from nursing homes or other long-term care facilities, and isolates from surveillance specimens, were excluded.Each laboratory also provided information on the total number of nonreplicate clinical isolates of Enterobacteriaceae of the same species observed during the collection period from inpatients and outpatients.

Bacterial identification, antimicrobial susceptibility testing and phenotypic characterisation of resistance mechanisms
At the reference laboratories, identification of collected isolates was confirmed by MALDI-TOF mass spectrometry (Vitek MS, bioMérieux), and susceptibility to ESC (cefotaxime, ceftazidime, cefepime), carbapenems (ertapenem, imipenem, and meropenem), aztreonam, aminoglycosides (amikacin and gentamicin), ciprofloxacin, trimethoprim/sulfamethoxazole was determined by disc diffusion on Mueller-Hinton agar (for P. mirabilis imipenem was not tested) according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology [23].MICs of tigecycline and colistin for carbapenemase producers were determined using reference broth microdilution For each centre the presence of CTX-M extended-spectrum betalactamase and carbapenemase genes is also indicated.
ESBL production was investigated using the double disk method testing the synergistic activity between amoxicillin-clavulanate and cefotaxime, ceftazidime, cefepime, and aztreonam [26].

Molecular characterisation of resistance genes
The presence of carbapenemase genes was investigated by multiplex real time-PCR (mRT-PCR) targeting blaKPC-type,blaVIM-type, blaOXA-48-type and blaNDM-type genes, using primers and conditions reported in Table 1.The presence of blaCTX-M ESBL genes was investigated by a mRT-PCR, able to distinguish among different blaCTX-M-type variants of groups 1, 2, 8/25 and 9, using primers and conditions reported in Table 1.The presence of ACBL genes was investigated by a multiplex PCR targeting genes encoding ACBLs, as described previously [27].For E. coli, specific primers designed to distinguish between acquired and chromosomal ACBL genes were used [27].
The presence of mcr-1-like genes was investigated by a novel real time-PCR (Table 1).

Spectrophotometric assay for carbapenemase activity
In carbapenem non-susceptible isolates that tested negative for known carbapenemase genes, carbapenemase production was further investigated by measuring the imipenem-hydrolysing specific activity in bacterial crude extracts as described previously [28], using a Cary 100 UV-Vis spectrophotometer (Varian, Walnut Creek, California, US).

Statistical analysis
Statistical evaluation of differences between resistance rates observed in this study and previous surveillance studies was carried out with the chi-squared test with Yates correction, using the Stata Statistical Software (release 13, College Station, Texas, US).Since the surveillance studies were overall similar by design but there were a few sampling differences, which could act as confounding factors, the tests were treated rather as exploratory analyses, which may indicate trends.

Prevalence of Enterobacteriaceae with resistance mechanisms to extended-spectrum cephalosporins and/or carbapenems
During the study period, a total of The ESCR-carbaS phenotypes were contributed by all three species, with higher proportions among P.   mirabilis (23.6%) and E. coli (16.1%) than among K. pneumoniae (9.3%), and with a higher proportion among isolates from inpatients (20.3%) but a remarkable proportion (11.1%) also among those from outpatients (Table 2).
The ESCR-carbaR phenotypes were mostly contributed by K. pneumoniae, with higher proportion among isolates from inpatients (25.1% of K. pneumoniae) but also a notable proportion among those from outpatients (7.7% of K. pneumoniae) (Table 2).
Comparison of these data with results from the earlier Italian nationwide survey on producing ESBL-producing Enterobacteriaceae, carried out in 2003 [29], revealed a notable increase in the proportion of ESBL-producing isolates (from 6.4% to 14.4%; p < 0.001, considering the three target species).Among outpatients, the proportion of ESBL producers increased from 3.4% to 11.0% (p < 0.001), and the increase was especially large among E. coli (from 1.9% to 10.9%; p < 0.001).
The majority of the ESCR-carbaS isolates that were ESBL-negative were positive for an acquired ACBL gene (n = 37, 79%) which, in most cases, belonged to the CMY/LAT/ACT/MIR lineage.Most of the ACBL-positive isolates were P. mirabilis (Figure 2, Table 3).
Comparison of these data with results from the first Italian nationwide survey on carbapenem-resistant Enterobacteriaceae (CRE), carried out in 2011 [22], revealed an increase in the proportion of carbapenemresistant K. pneumoniae isolates (from 11.9% to 18.7%) although the difference was not statistically significant (p = 0.5).KPC-KP remained the major contributors (83.1% of carbapenem-resistant K. pneumoniae in 2013 vs. 87.2% in 2011).Among outpatients, the proportion of KPC-KP increased from 2.2% in 2011 to 4.6% (p < 0.05).
A detailed distribution of the resistance genes in the Italy is reported in Figure 2, and the distribution by species and type of patients is reported in Table 2.

Antimicrobial susceptibility
All ESBL-producing isolates that were carbapenemasenegative remained susceptible to imipenem and meropenem, while ertapenem susceptibility was 98.1% among E. coli and 82.2% among K. pneumoniae (Table 4).
Among the carbapenemase producers, all were nonsusceptible to ertapenem, while most carbapenemase-producing E. coli were susceptible to imipenem and meropenem (80.0% and 60.0%, respectively).Carbapenemase-producing K. pneumoniae were more susceptible to gentamicin than to amikacin (48.7% vs. 16.8%).Broth microdilution assays, carried out with the 113 carbapenemase-producing K. pneumoniae, revealed that 61.7% and 94.4% were susceptible to colistin and tigecycline, respectively, while all five carbapenemase-producing E. coli were susceptible to both drugs (Table 4).These results were overall similar to those reported from the European Survey of Carbapenemase-Producing Enterobacteriaceae (EuSCAPE) survey in 2015 [31], and underscore the remarkable rate of colistin resistance among carbapenemase-producing K. pneumoniae circulating in Italy.No significant differences in susceptibility to these two drugs were detected among inpatients and outpatients (data not shown).

Screening for mcr-1-like genes in colistinresistant isolates
The 43 colistin-resistant carbapenemase-positive K. pneumoniae isolates were screened for the presence of mcr-1-like genes, encoding transferable colistin resistance [32,33].All the tested isolates yielded negative results, revealing that colistin resistance was caused by different mechanisms.

Discussion
This study provides an updated picture of the prevalence, distribution, beta-lactamase profiles and susceptibilities of ESBL-, carbapenemase-and ACBLproducing E. coli, K. pneumoniae and P. mirabilis circulating in Italy.
Compared with the earlier Italian nationwide survey on ESBL-producing Enterobacteriaceae, carried out in 2003 [29], the proportion of ESBL-producing isolates (considering the three target species) showed a notable increase, especially among outpatients and among E. coli.At the level of resistance mechanisms, this epidemiological evolution was associated with a remarkable increase in the prevalence of CTX-M-type enzymes, which, in 2013, represented by far the most common type of ESBL in E. coli and in K. pneumoniae.Interestingly, despite their ability to spread, the genes encoding these resistance mechanisms have not disseminated in P. mirabilis, where the ESCR phenotype was found to be relatively common (23.6% of isolates) but due to other mechanisms, i. e. ESBLs other than CTX-M-type or ACBLs, mostly of the CMY/LAT/ ACT/MIR lineage.A similar epidemiological evolution was observed in other European Countries including Belgium (with 77% vs 46% of CTX-M-positive isolates, among ESBL-producing E. coli, in 2008 vs 2006) [34], Spain (with 72% vs 52% of CTX-M-positive isolates among ESBL-producing E. coli in 2006 vs 2000) [35].
Concerning carbapenem resistance, a notable increase was observed among K. pneumoniae in comparison with data from the first Italian nationwide survey on CRE, carried out in 2011 [22], with KPC-KP remaining the major contributors of CRE endemicity in Italy and other types of carbapenemases remaining uncommon.These findings underscore the ability of carbapenemase-producing Enterobacteriaceae (CPE) to rapidly disseminate and establish conditions of high-level endemicity, and the notion that CPE are associated with a heightened epidemiological risk and deserve special attention, as readily acknowledged by the European Centre for Disease Prevention and Control (ECDC) [36].Indeed, the data from the EARS-NET surveillance system indicate that there is an overall increasing spread of CRE in some European countries [21], and results from the EuSCAPE project have shown that some regions are experiencing a worrisome prevalence of CPE per 10,000 hospital admissions, ranging from four to six for Italy, Greece and Spain [37].
Interestingly, KPC-KP isolates were also found from outpatients, at a rate that was approximately double compared with that found in the 2011 survey (4.6% vs 2.2%) [22].Since a significant proportion of these outpatients could have been recently hospitalised or otherwise have had contact with healthcare services, this figure may not reflect accurately the prevalence of KPC-KP in the community, in Italy.However, this finding points to an increasing dissemination of CPE also outside the hospital setting, likely reflecting the ability of KPC-KP to establish persistent intestinal colonisation even after hospital discharge [38,39].It might herald a possible dissemination in the community like the one previously witnessed with CTX-M-producing E. coli [40].Indeed, recent reports from the US and Spain have described the emergence of CPE infections also in the community, underscoring this possibility [41,42].Such a scenario should be avoided, and suitable infection control measures should be enforced to address this phenomenon in settings of high CRE endemicity like Italy and some other European countries [37].
In this grim scenario, the good news was that (i) overall carbapenem susceptibility was retained by P. mirabilis, with no carbapenemase-positive isolates detected in this species despite the high endemicity of CPE; and that (ii) no mcr-1-like transferable colistin resistance genes were detected among the colistin-resistant CPE isolates, suggesting that this worrisome resistance mechanism remains uncommon among colistin-resistant CPE circulating in Italy.Updated molecular surveillance data on resistance genes to beta-lactams are of increasing importance due to the introduction on the market of new antibiotics for resistant Gram-negatives that are based on new beta-lactamase inhibitors (e. g. avibactam), which will be useful for treatment of infections caused by ESBL and carbapenemase producers [43].The new inhibitors, however, are not active against all beta-lactamases e. g. avibactam is able to inhibit class A but not class B beta-lactamases.Thus the possibility of using these new drugs will depend on the specific mechanism of resistance, and rapid testing for these resistance mechanisms will become an essential step in antibiotic stewardship programs.In this perspective, this survey underscores the major role of beta-lactamase genes as resistance determinants in Enterobacteriaceae in the Italian epidemiological setting, and provides relevant information for the selection of the most suitable diagnostic strategies based on molecular detection of beta-lactam resistance mechanisms.A limitation of this study is that, due to the study design, laboratorybased surveillance, information about risk factors for developing antibiotic resistance was not available.Future studies are warranted to investigate these aspects, which are relevant to understand the reasons for the epidemiological differences observed in different European countries and to contextualise infection control policies.

Table 1
Sequence of primers and probes used in nationwide surveillance survey of the molecular epidemiology of ESBL-and carbapenemase-producing Enterobacteriaceae, Italy, October 2013

Table 4
Antimicrobial susceptibility testing results for the investigated isolates, nationwide surveillance survey, Italy, October 2013 (n=652 isolates) COL and TIG were tested only for carbapenemase producers. a