Dissemination of multiresistant Enterobacter cloacae isolates producing OXA-48 and CTX-M-15 in a Spanish hospital

doi:10.1016/j.ijantimicag.2015.07.003

International Journal of Antimicrobial Agents

Volume 46, Issue 4, October 2015, Pages 469-474

https://doi.org/10.1016/j.ijantimicag.2015.07.003 Get rights and content

Highlights

•
Multiresistant Enterobacter cloacae isolates are circulating in a Spanish hospital.
•
The isolates produced OXA-48, CTX-M-15 or both enzymes.
•
The bla_OXA-48 and bla_CTX-M-15 genes were identified in IncL/M and IncF plasmids, respectively.
•
Two main clones caused prolonged outbreaks in separate buildings of the hospital.
•
Intrahospital and interhospital spread of the isolates was observed.

Abstract

Twenty-one multiresistant Enterobacter cloacae isolates producing OXA-48 (n = 10), CTX-M-15 (n = 7) or both (n = 4) β-lactamases were detected in a Spanish hospital during a 1-year period (June 2013 to June 2014). The isolates were also resistant to non-β-lactam antimicrobials, further complicating the therapeutic options. Genotyping of the isolates identified two major clones (ST74 and ST66) that caused prolonged outbreaks in different buildings of the hospital as well as some sporadic isolates (ST78, ST45 and ST295). Isolates belonging to clone 1 (n = 7) were carbapenem-resistant and carried the bla_OXA-48 gene on a conjugative IncL/M plasmid of ca. 65 kb. Clone 2 isolates (n = 11) were resistant to cefepime and harboured the bla_CTX-M-15 gene on an ca. 150-kb, non-conjugative plasmid of the IncF group, co-harbouring the qnrB and aac(6′)-Ib-cr genes encoding quinolone resistance. Four clone 2 isolates were also resistant to carbapenems owing to the co-production of OXA-48. Most of the isolates were recovered from critically ill patients and were admitted to intensive care units; a single patient was transferred from another Spanish hospital. Intrahospital and interhospital dissemination of multiresistant E. cloacae isolates is of major clinical concern as it could lead to endemic nosocomial situations.

Introduction

Enterobacter cloacae is an opportunistic pathogen frequently involved in nosocomial infections [1]. This enterobacterial species is intrinsically resistant to aminopenicillins, amoxicillin/clavulanic acid (AMC) and cephalosporins of early generations owing to a chromosomally encoded AmpC β-lactamase. Isolates overproducing AmpC are also resistant to broad-spectrum cephalosporins. Furthermore, E. cloacae has the ability to acquire additional resistance mechanisms, including plasmid-encoded extended-spectrum β-lactamases (ESBLs) and carbapenemases [1]. Phenotypic detection of ESBL production may be difficult since it can be masked by overexpression of AmpC. Some E. cloacae isolates may also exhibit permeability defects due to the absence of porins or porin mutations, further increasing the minimum inhibitory concentrations (MICs) of β-lactams and carbapenems, particularly ertapenem [1].

In members of the Enterobacteriaceae family, CTX-M-15 and OXA-48 are among the most common enzymes involved in resistance to broad-spectrum cephalosporins and carbapenems, respectively [2], [3]. CTX-M-15 is a class A ESBL encoded by the bla_CTX-M-15 gene, which has been identified in plasmids of varying size (85–200 kb) and structure, and often belonging to the FII incompatibility (Inc) group [4]. OXA-48 is an Ambler class D carbapenemase that confers resistance to penicillins and reduced susceptibility to carbapenems but does not significantly hydrolyse broad-spectrum cephalosporins [3]. The bla_OXA-48 gene has been almost always located on a conjugative plasmid of ca. 62 kb assigned to the IncL/M group and has been identified only in enterobacterial species [5], [6]. Both CTX-M-15 and OXA-48 have been detected in several enterobacterial species, being particularly frequent in Klebsiella pneumoniae, whilst they are relatively uncommon in E. cloacae [2], [3]. Here we report the clinical and genetic features of 21 E. cloacae isolates producing CTX-M-15, OXA-48 or both, which were mainly recovered from critically ill patients, in a Spanish hospital during a 1-year period.

Section snippets

Setting, patients and bacterial isolates

‘Hospital Universitario Central de Asturias’ (HUCA) (Oviedo, Spain) is a 1100-bed university hospital providing care for a population of ca. 342,000 inhabitants in the Asturias region of northern Spain. During the 1-year period from June 2013 to June 2014, all E. cloacae isolates recovered at the hospital (n = 448) were analysed phenotypically for production of carbapenemases and ESBLs. A total of 21 multiresistant isolates, all recovered from different patients, produced a carbapenemase, an ESBL

Antimicrobial susceptibility and genetic basis for resistance to broad-spectrum cephalosporins and carbapenems

As shown in Table 2, 20 of the 21 E. cloacae isolates were resistant to cefotaxime and 11 were also resistant to cefepime. Moreover, 14 isolates displayed intermediate susceptibility or resistance to carbapenems. In addition to β-lactam antibiotics, all E. cloacae isolates were variably resistant to other antimicrobial groups, including quinolones (nalidixic acid and ciprofloxacin), aminoglycosides (gentamicin and tobramycin) and trimethoprim/sulfamethoxazole.

The bla_CTX-M-15 gene was detected

Discussion

Enterobacter cloacae isolates producing OXA-48, CTX-M-15 or both β-lactamases emerged as important pathogens in HUCA, a general university hospital in northern Spain. In fact, they accounted for 4.7% (21/448) of the total E. cloacae isolates recovered over a 1-year period, although they were not previously reported in this region. Together with some sporadic isolates, two major clones (ST74 and ST66, falling in two different clusters according to PFGE analysis) are causing prolonged outbreaks

Conclusions

Intrahospital and interhospital dissemination of multiresistant E. cloacae isolates producing CTX-M-15, OXA-48 or both β-lactamases is a cause of concern due to the limited options remaining for the treatment of affected patients, most with critical underlying conditions. Interestingly, as observed for K. pneumoniae, emergence of both broad-spectrum resistance traits in single E. cloacae isolates corresponds to the transfer of genes having disseminated from community pathogens into nosocomial

References (15)

A. Davin-Regli et al.
Enterobacter aerogenes and Enterobacter cloacae; versatile bacterial pathogens confronting antibiotic treatment
Front Microbiol
(2015)
R. Cantón et al.
Prevalence and spread of extended-spectrum β-lactamase-producing Enterobacteriaceae in Europe
Clin Microbiol Infect
(2008)
A. Potron et al.
Intercontinental spread of OXA-48 β-lactamase-producing Enterobacteriaceae over a 11-year period, 2001 to 2011
Euro Surveill
(2013)
K.L. Hopkins et al.
Replicon typing of plasmids carrying CTX-M or CMY β-lactamases circulating among Salmonella and Escherichia coli isolates
Antimicrob Agents Chemother
(2006)
J. Fernández et al.
Cluster of Escherichia coli isolates producing a plasmid-mediated OXA-48 β-lactamase in a Spanish hospital in 2012
J Clin Microbiol
(2014)
L. Poirel et al.
Genetic features of the widespread plasmid coding for the carbapenemase OXA-48
Antimicrob Agents Chemother
(2012)
Clinical and Laboratory Standards Institute
Performance standards for antimicrobial susceptibility testing; approved standard—twenty-fourth informational supplement. Document M100-S24
(2014)

There are more references available in the full text version of this article.

Cited by (42)

High-risk international clones ST66, ST171 and ST78 of Enterobacter cloacae complex causing blood stream infections in Spain and carrying bla<inf>OXA-48</inf> with or without mcr-9
2023, Journal of Infection and Public Health
Citation Excerpt :
Clinical isolates identified as E. cloacae, assigned to these STs and resistant to critically important antibiotics have been reported in different countries. For instance ST66 and ST78 were associated with blaOXA-48 and/or blaCTX-M-15 (our hospital in 2013-2014; [4]); ST78 with blaVIM-1 and mcr-9 (the Netherlands; [41]); blaKPC-2 (Israel and China), blaCTX-M-15 (France), blaCTX-M-9 (Latvia), blaNDM-1 and blaCTX-M-3 (China), blaNDM-7 (Spain), blaVIM-1 (Greece, Italy and Spain) and blaOXA-48 (Turkey) [42–45]; and ST171 with blaKPC-3 (widely spread in the United States), blaNDM-1 (China, Guatemala, Brazil and South Africa), blaKPC-2 (Colombia) and blaKPC-4 (United Kingdom) [42,43]. When analyzing the SNP-based tree it is clear that two clusters are formed: cluster A (comprising ST78 isolates) and cluster B (englobing ST171 and ST66 isolates).
In the last years, Enterobacter cloacae complex has become an important threat associated with nosocomial infections (including bacteraemia). These bacteria have the ability to acquire mobile genetic elements with antimicrobial resistance genes, reducing the number of therapies available for treatment of the infections they cause. Multidrug resistant isolates of the E. cloacae complex have been causing blood stream infections in a hospital in northern Spain. The aim of this study was to report the spread of E. cloacae complex isolates carrying bla_OXA-48 with or without mcr-9 which were involved in blood stream infections, in a Spanish hospital.
All Enterobacter spp. isolates recovered from blood cultures of patients admitted to a tertiary Spanish hospital, over a five-year period were recovered. Of those, OXA-48-producing isolates were selected for further analysis (19 E. xiangfangensis isolates and a single E. hoffmannii). Bacterial identification, antimicrobial susceptibility, DNA sequencing, molecular typing, resistome analysis and plasmid characterization was performed.
20 isolates were positive for bla_OXA-48, harbored by IncL/M plasmids. They belonged to the international high-risk clones ST66, ST171 and ST78. They produced the extended-spectrum β-lactamases CTX-M-15 and/or CTX-M-9 and 40 % of them (n = 8) also carried the mcr-9 gene, located on IncHI2 plasmids. However, they were susceptible to colistin.
The presence of bla_OXA-48, together with at least one bla_CTX-M gene in our multidrug resistant high-risk E. cloacae complex clones is worrisome. Also, the additional presence of mcr-9 in some of them is of concern as it could potentially be transferred into other hosts or acquire mutations that might led to emerging colistin resistance. Surveillance systems are essential to detect these difficult-to-treat bacteria which, apart from causing live-threatening infections, can spread important resistance threats.
Enterobacter cloacae: A probable etiological agent associated with slow growth in the giant freshwater prawn Macrobrachium rosenbergii
2021, Aquaculture
Macrobrachium rosenbergii, the giant freshwater prawn, is an economically crucial freshwater shrimp that has been severely affected by slowing growth in recent years. In Gaoyou County, Jiangsu Province, China, Enterobacter cloacae infection frequently occurs in M. rosenbergii hatcheries, and E. cloacae has been detected with high prevalence in slow-growing prawns. In this study, the growth of prawns was found to be significantly reduced after E. cloacae infection. In addition, under co-culture of healthy and slow-growing prawns, slow-growing prawns were infectious and able to cause the slow growth of healthy prawns. The expression of growth-related genes after E. cloacae infection was analyzed, and differential expression of growth-related genes between slow-growing and healthy prawns was investigated. The results showed that the expression of CHIT3, Cat, JHEH, EcR, and RXR was downregulated after E. cloacae infection and that of EcR was upregulated after E. cloacae infection. Similarly, the expression of CHIT3, Cat, JHEH, EcR, and RXR was downregulated in slow-growing prawns compared to that in healthy prawns, and that of EcR was upregulated in slow-growing prawns compared to that in healthy prawns. The present results reveal a high prevalence of E. cloacae in slow-growing M. rosenbergii and suggest that this pathogen is the probable cause of the slow growth in M. rosenbergii.
Emergence of Escherichia coli isolates producing NDM-1 carbapenemase from waterfowls in Hainan island, China
2020, Acta Tropica
Citation Excerpt :
Above all, the results identified in this study indicate that ST10 belonging to phylogroup E is an important reservoir of blaNDM-1 in waterfowls of this tropical region. Our study identified a variety of carbapenem-resistant E. coli and similarity in their clonal relatedness, which is consistent with the studies of others (Fernandez et al., 2015; Hargreaves et al., 2015). The present results reminded most common clonal complexes of E. coli carrying NDM-1-carbapenemase in waterfowls should be taken as serious concern.
Carbapenems are traditionally recognized to be the last resort drugs to treat infections due to MDR organisms such as E. coli. As such, the emergence of New Delhi metallo-β-lactamase-producing E. coli strains have become a challenging threat to the public health. In this regard, we examined the molecular characteristics of carbapenem-resistant E. coli (CRE) isolated from waterfowls in China's tropical island, Hainan. A total of 311 single E. coli strains were obtained from 20 various farms of healthy ducks and geese in 2 districts of Hainan island. The CRE strains were initially identified via phenotypic resistance and modified Hodge test. PCR assay and subsequent nucleotide sequencing were used to detect different types of carbapenemase encoding genes (bla_NDM, bla_VIM, bla_IMP, bla_OXA and bla_KPC). In addition, MLST and PFGE analyses were also performed. Among the 311 E. coli strains, 8 strains were detected to produce a single type of carbapenemase i.e. NDM-1 (2.6%). A total of 5 sequence types (STs) were observed, of which ST10 was the most prevalent accounting for 37.5% (3/8). Moreover, these 8 isolates yielded 6 different PFGE clusters but showed approximately related PFGE types, suggesting the propagation of similar clone between the farms. This is the first report on the identification of NDM-1-producing E. coli from waterfowls in Hainan island, China. Our results emphasize the need for better efforts to control the further spread of NDM-1-producing E. coli strains in this tropical island.
Colistin-Resistant mcr-Positive Enterobacteriaceae in Fresh Vegetables, an Increasing Infectious Threat in China
2019, International Journal of Antimicrobial Agents
Citation Excerpt :
Besides E. coli reported in vegetables [4,6,7], one mcr-1-positive E. cloacae isolate from green pepper was also identified in this study. E. cloacae is a prevalent clinical pathogen around the world [14,15], and mcr-1 has been reported in clinical E. cloacae isolates in China [16] and France [17]. Recently, an E. cloacae isolate from Jinghang Grand Canal, China, was found to carry mcr-1 [18].
The presence of mobilized colistin resistance (mcr) genes is a global concern. However, data concerning mcr in fresh vegetables, a reservoir for antibiotic resistance genes, are still rare. In this study, mcr genes were analysed in 528 vegetable samples from 53 supermarkets or farmer's markets in 23 cities of 9 provinces in China, and the mcr-positive Enterobacteriaceae were characterized. Nineteen (3.6%) samples carried one or more mcr-positive isolates, and the highest three detection rates were found in carrot, pak choi and green pepper. Twenty-four mcr-1-positive isolates (23 Escherichia coli and one Enterobacter cloacae) were obtained, and E. coli isolates showed high genetic diversity. Different multilocus sequence type (MLST) isolates were also observed within the same sample. All 24 isolates showed multidrug resistance, and 14 carried bla_CTX-M genes. Most isolates harbored similarly conjugative IncX4-type (∼33 kb) or IncI2-type (∼60 kb) mcr-1-bearing plasmids. The sequenced prevalent IncX4 plasmid and IncI2 plasmid from tomato were similar to the relevant plasmids from animals and clinical isolates in various countries. mcr-1-bearing IncHI2/ST3 plasmid highly similar to that carrying 14 resistance genes from E. coli of chicken was also observed. In conclusion, a high prevalence of mcr-1 in fresh vegetables was found in China, and the dissemination of mcr-1 was mediated by similar IncX4 or IncI2 plasmids. The plasmids from vegetables showed high similarity to plasmids from clinical isolates, indicating MCR-1-producers in ready-to-eat vegetables may pose a huge threat to public health and measures need to be taken to ensure food safety.
Interspecies plasmid transfer appears rare in sequential infections with extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae
2019, Diagnostic Microbiology and Infectious Disease
Citation Excerpt :
Horizontal transfer of extended-spectrum β-lactamase (ESBL) genes via mobile genetic elements, i.e., ESBL-carrying plasmids, and the emergence of successful virulent bacterial clones are key reasons for the rapid spread of antibiotic multiresistance among Enterobacteriaceae (Brolund and Sandegren, 2016; Mathers et al., 2015). The role of interspecies plasmid spread of resistance genes in polyclonal outbreaks of ESBL- or carbapenemase-producing Enterobacteriaceae is well documented (Bocanegra-Ibarias et al., 2017; Dautzenberg et al., 2014; Fernandez et al., 2015; Muller et al., 2016a; Stillwell et al., 2015). However, the extent of a change of ESBL-carrying bacterial species and possible transfer of ESBL-carrying plasmids between consecutive isolates in patients with repetitive infections with ESBL-producing Enterobacteriaceae (EPE) is, to our knowledge, largely unexplored.
From a cohort of 1836 Swedish patients infected with ESBL-producing Enterobacteriaceae (EPE) during 2004–2014, 513 patients with recurrent EPE infection were identified. Only in 14 of the 513 patients was a change of species (ESBL-E. coli to ESBL-K. pneumoniae or vice versa) found between the index and subsequent infection. Eleven sequential urine isolates from 5 of the 14 patients were available for further analysis of possible transfer of ESBL-carrying plasmids. The plasmid content was studied using optical DNA mapping (ODM), PCR-based replicon typing, and ESBL gene sequencing. ODM allowed us to directly compare whole plasmids between isolates and found similar ESBL-carrying plasmids in 3 out of the 5 patients. The ODM results and the rarity in shift of species between ESBL-E. coli and ESBL-K. pneumoniae imply that in recurrent EPE infections interspecies plasmid transfer is uncommon.
Enterobacter cloacae associated with mass mortality in zoea of giant freshwater prawns Macrobrachium rosenbergii and control with specific chicken egg yolk immunoglobulins (IgY)
2019, Aquaculture
Outbreaks of mass mortality among cultured zoea of M. rosenbergii occurred in different hatcheries of Gaoyou country of China in April 2017. Of the isolates obtained from the diseased zoea, XL3–1 was pathogenic to the zoea and postlarva of M. rosenbergii in the virulence test, with a LD50 value 3.2 × 10⁶ CFU/mL and 3.5 × 10⁶ CFU/mL, respectively. XL3–1 was identified as Enterobacter cloacae by biochemical characteristics and molecular tools. Detection of virulence-associated genes by PCR indicated that XL3–1 was positive for irp2, fhuA, sodB, sltA, flaD and OmpX. According to the antibiotic resistant assay, the isolate was sensitive to quinolones, while they were resistant to cephalosporins and penicillins. This study also investigated the protection of specific chicken egg yolk antibodies (IgY) in E. cloacae-infected M. rosenbergii. The highest titer against E. cloacae of the obtained specific IgY by ELISA was 1: 2¹³. M. rosenbergii treated with specific egg yolk powder could achieve the survival rate of 67% after E. cloacae infection, while M. rosenbergii treated with non-specific egg yolk powder or no egg yolk powder could achieve survival rates of 20% and 17%, respectively. As well, the bacterial burden in hepatopancreas was significantly lower in M. rosenbergii treated with specific egg yolk powder than those treated with non-specific egg yolk powder. This study clarified the virulence characteristics and antibiotic resistance of E. cloacae, and provided a strategy to control the disease by using specific IgY.

View all citing articles on Scopus

View full text

Article preview

International Journal of Antimicrobial Agents

Highlights

Abstract

Introduction

Section snippets

Setting, patients and bacterial isolates

Antimicrobial susceptibility and genetic basis for resistance to broad-spectrum cephalosporins and carbapenems

Discussion

Conclusions

References (15)

Enterobacter aerogenes and Enterobacter cloacae; versatile bacterial pathogens confronting antibiotic treatment

Front Microbiol

Prevalence and spread of extended-spectrum β-lactamase-producing Enterobacteriaceae in Europe

Clin Microbiol Infect

Intercontinental spread of OXA-48 β-lactamase-producing Enterobacteriaceae over a 11-year period, 2001 to 2011

Euro Surveill

Replicon typing of plasmids carrying CTX-M or CMY β-lactamases circulating among Salmonella and Escherichia coli isolates

Antimicrob Agents Chemother

Cluster of Escherichia coli isolates producing a plasmid-mediated OXA-48 β-lactamase in a Spanish hospital in 2012

J Clin Microbiol

Genetic features of the widespread plasmid coding for the carbapenemase OXA-48

Antimicrob Agents Chemother

Performance standards for antimicrobial susceptibility testing; approved standard—twenty-fourth informational supplement. Document M100-S24

Cited by (42)

High-risk international clones ST66, ST171 and ST78 of Enterobacter cloacae complex causing blood stream infections in Spain and carrying bla<inf>OXA-48</inf> with or without mcr-9

Enterobacter cloacae: A probable etiological agent associated with slow growth in the giant freshwater prawn Macrobrachium rosenbergii

Emergence of Escherichia coli isolates producing NDM-1 carbapenemase from waterfowls in Hainan island, China

Colistin-Resistant mcr-Positive Enterobacteriaceae in Fresh Vegetables, an Increasing Infectious Threat in China

Interspecies plasmid transfer appears rare in sequential infections with extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae

Enterobacter cloacae associated with mass mortality in zoea of giant freshwater prawns Macrobrachium rosenbergii and control with specific chicken egg yolk immunoglobulins (IgY)

Short CommunicationDissemination of multiresistant Enterobacter cloacae isolates producing OXA-48 and CTX-M-15 in a Spanish hospital

Highlights

Abstract

Introduction

Section snippets

Setting, patients and bacterial isolates

Antimicrobial susceptibility and genetic basis for resistance to broad-spectrum cephalosporins and carbapenems

Discussion

Conclusions

Enterobacter aerogenes and Enterobacter cloacae; versatile bacterial pathogens confronting antibiotic treatment

Front Microbiol

Prevalence and spread of extended-spectrum β-lactamase-producing Enterobacteriaceae in Europe

Clin Microbiol Infect

Intercontinental spread of OXA-48 β-lactamase-producing Enterobacteriaceae over a 11-year period, 2001 to 2011

Euro Surveill

Replicon typing of plasmids carrying CTX-M or CMY β-lactamases circulating among Salmonella and Escherichia coli isolates

Antimicrob Agents Chemother

Cluster of Escherichia coli isolates producing a plasmid-mediated OXA-48 β-lactamase in a Spanish hospital in 2012

J Clin Microbiol

Genetic features of the widespread plasmid coding for the carbapenemase OXA-48

Antimicrob Agents Chemother

Performance standards for antimicrobial susceptibility testing; approved standard—twenty-fourth informational supplement. Document M100-S24

Short Communication
Dissemination of multiresistant Enterobacter cloacae isolates producing OXA-48 and CTX-M-15 in a Spanish hospital