Genotypic characterization and clonal relatedness of metallo-β-lactamase-producing non-fermentative gram negative bacteria in the first 5 years of their circulation in Paraguay (2011-2015)

Abstract

Pseudomonas aeruginosa and species of Acinetobacter calcoaceticus-baumanii complex are multiresistant intrahospital opportunistic pathogens, able to acquire carbapenemases and produce outbreaks with high morbidity and mortality. Pseudomonas putida has also emerged with similar characteristics. The aim of this research was to characterize the Metallo-β-lactamases (MBLs) detected by surveillance in Paraguay in the first 5 years of their circulation in hospitals. The coexistence of KPC and OXA-type carbapenemases was also investigated. 70 MBL-producing strains from inpatients were detected from clinical samples and rectal swab from 11 hospitals. The strains were identified by manual, automated, and molecular methods. Antimicrobial susceptibility was studied by Kirby-Bauer and automated methods, while colistin susceptibility was determined by broth macrodilution. MBLs were investigated by synergy with EDTA against carbapenems and PCR, and their variants by sequencing. KPC and OXA-carbapenemases were investigated by PCR. Clonality was studied by pulsed-field gel electrophoresis (PFGE). The results demonstrated the circulation of bla_VIM-2 (60%), bla_NDM-1 (36%), and bla_IMP-18 (4%). The MBL-producing species were P. putida (45.7%), P. aeruginosa (17.2%), A. baumannii (24.3%), A. pittii (5.7%), A. nosocomialis, (4.3%) A. haemolyticus (1.4%), and A. bereziniae (1.4%). PFGE analysis showed one dominant clone for A. baumannii, a predominant clone for half of the strains of P. aeruginosa, and a polyclonal spread for P. putida. In the first 5 years of circulation in Paraguay, MBLs were disseminated as unique variants per genotype, appeared only in Pseudomonas spp. and Acinetobacter spp., probably through horizontal transmission between species and vertical by some successful clones.

Introduction

Gram-negative bacteria resistant to carbapenems, such as Acinetobacter baumannii and Pseudomonas aeruginosa, are emerging causes of health care-associated infections and of global public health concerns [1]. Pseudomonas putida has also emerged as a multiresistant nosocomial opportunistic pathogen [2–4]. The most widespread mechanism of carbapenem resistance is the production of carbapenemases [5]. These enzymes can be classified according to their molecular characteristics into classes A, B, and D. The classes A (ex. KPC) and D (the OXAs) carbapenemases include enzymes that hydrolyze their substrates forming an acyl-enzyme through a serine of the active site, while the class B β-lactamases are metalloenzymes or metallo-β-lactamases (MBLs) that use at least one zinc ion from the active site to facilitate the hydrolysis of beta-lactam. The most important types of MBLs due to epidemiological spread and clinical relevance are IMP, VIM [6], and NDM [7]. SPM is characteristic of P. aeruginosa in Brazil [8].

The surveillance of MBLs dissemination in Paraguay began in 2009, with 21 laboratories members of the survaillance of the Antimicrobial Resistance Network dependent on the Central Laboratory of Public Health—Ministry of Public Health and Social Welfare. The aim of this study was to characterize the MBLs detected among Pseudomonas spp. and A. calcoaceticus-baumannii complex isolates in Paraguay in the first 5 years of their circulation in hospitals. The characterization involved identification of species and clonality of MBL-producing strains, determination of genotype and subtype of MBL, and coexistence of KPC and OXA-type carbapenemases in the strains.

Materials and methods

From November 2009 to December 2015, clinical carbapenem-resistant isolates of Pseudomonas spp. and A. calcoaceticus-baumannii complex collected from hospitalized patients from 11 Paraguayan hospitals were sent to the Central Laboratory of Public Health in Paraguay and submitted to phenotypic detection of MBL by inhibition with EDTA [9]. Only one isolate for each patient was included for further studies, except in one case where two isolates with different resistance phenotypes, recovered from tracheal secretion within 4 months of difference were included.

Species identification was performed by classical and automated methods (Vitek 2 Compact–Biomerieux, France). For A. calcoaceticus-baumannii complex, in addition to this a multiplex PCR was performed to detect the presence of bla_OXA-51 according to the protocol described in the literature, which allows the detection of bla_OXA-23; bla_OXA-24 and bla_OXA-58 [10]. The bla_OXA-51 negative strains were subjected to species-specific PCR for Acinetobacter pittii and Acinetobacter nosocomialis according to published protocol [11]. Isolates of Acinetobacter spp. that were negative for bla_OXA-51 and species-specific PCR were subjected to rpoβ amplification PCR [12] and sequencing.

Antimicrobial susceptibility tests were performed by automated method (Vitek 2 Compact-Biomerieux, France). Susceptibility to colistin was determined by broth macrodilution [13]. The results were interpreted according to the CLSI manual [13].

On surveillance, routine genotyping of MBLs was performed by PCR for bla_IMP, bla_VIM, and bla_NDM according to the protocol of the regional reference laboratory [14], however, for this work, the genotypes were verified according to multiplex PCR [15]. The bla_SPM—for P. aeruginosa strains—was investigated according to the published protocol [16]. The detection of the Serino-carbapenemase gene bla_KPC was performed for P. aeruginosa isolates [17].

To determine the variant of each MBL genotype, PCR and subsequent sequencing were made according to the published protocol for IMP and VIM [18]. To determine NDM variants, primers designed by the Laboratório de Pesquisa em Infecção Hospitalar (LAPIH) from the Oswaldo Cruz Institute-Fiocruz, Brazil [19] were used (Table 1).

Table 1.

Primers used in this work

	Primer	Gene	Sequence 5′ → 3′	Amplicon size (pb)	Ref
A. pittii	Apit-F	rpoβ	TGGGCAGTTACCAGATTGACCTA	147	[11]
	Apit-R		AACCAGCAGCTTCCATTTGACG
A. nosocomialis	Anos-F	rpoβ	GCCGCTCGTGAACGTGTAATC	394	[11]
	Anos-R		CATCGTGTGGCATATCTTCAAC
Amplification of rpoB	Ac696- F	rpoβ	TAYCGYAAAGAYTTGAAAGAAG	350	[12]
	Ac1093-R		CMACACCYTTGTTMCCRTGA
MBL genotyping	VIM-F	blaVIM	AGTGGTGAGTATCCGACAG	261	[15]
	VIM-R		ATGAAAGTGCGTGGAGAC
	IMP-UF	blaIMP	GGYGTTTWTGTTCATACWTCKTTYGA	404	[15]
	IMP-UR		GGYARCCAAACCACTASGTTATCT
	NDM-F	blaNDM	AGCACACTTCCTATCTCGAC	512	[15]
	NDM-R		GGCGTAGTGCTCAGTGTC
	SPM-F	blaSPM	AGACCGCGATTTCTATTCTT	505	[16]
	SPM-R		AGTTCCTTCGGCTTTATCAT
OXA genotyping	OXA-51 F	blaOXA- 51	TAATGCTTTGATCGGCCTTG	353	[10]
	OXA-51 R		TGGATTGCACTTCATCTTGG
	OXA-23 F	blaOXA- 23	GATCGGATTGGAGAACCAGA	501	[10]
	OXA-23 R		ATTTCTGACCGCATTTCCAT
	OXA-24 F	blaOXA- 24	GGTTAGTTGGCCCCCTTAAA	246	[10]
	OXA-24 R		AGTTGAGCGAAAAGGGGATT
	OXA 58-F	blaOXA- 58	AAGTATTGGGGCTTGTGCTG	599	[10]
	OXA 58-R		CCCCTCTGCGCTCTACATAC
KPC screening	KPC-F	blaKPC	AACAAGGAATATCGTTGATG	916	[17]
	KPC-R		AGATGATTTTCAGAGCCTTA
MBL variants	VIM1-F	blaVIM-1	TGTTAAAAGTTATTAGTAGTTTATTG	801	[18]
	VIM1-R		CTACTCGGCGACTGAGC
	VIM2-F	blaVIM-2	ATGTTCAAACTTTTGAGTAAG	801	[18]
	VIM2-R		CTACTCAACGACTGAGCG
	IMP1-F	blaIMP-1	ATGAGCAAGTTATCTGTATTC	741	[18]
	IMP1-R		TTAGTTGCTTGGTTTTGATGG
	IMP2-F	blaIMP-2	ATGAAGAAATTATTTGTTTTATG	741	[18]
	IMP2-R		TTAGTTACTTGGCTGTGATG
	NDM-F	blaNDM	CGAAGCTGAGCACCGCATTA	764	[19]
	NDM-R		TCAGCGCAGCTTGTCGGC

Macrogen Inc.-Korea performed sequencing. Using the Genbank database through the BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) the sequences were analyzed, and further studied by multiple alignments with BioEdit software comparing them with reference sequences available from NCBI.

To perform PFGE, the PulseNet protocol was used [20]. Restriction enzymes (Invitrogen by Thermo Fisher Scientific) and PFGE running conditions for P. aeruginosa, P. putida and Acinetobacter spp. (protocols not included in PulseNet Network) were taken from the scientific literature [21, 22] with adjusted running times, 18.8 h for Acinetobacter spp. and 18.7 h for Pseudomonas spp. Salmonella serovar Braenderup H9812 (restricted with Xba I) was used as the standard strain. The images of gels were analyzed (Gel Doc EZ Imager-BioRad) using Gel Compare II Software (Applied Maths) to obtain the relationship between patterns. We determine clusters using the Unweighted Pair Group Mean (UPGMA) method, with 90% Dice’s similarity coefficients among patterns to define each clonal group.

Results

Out of 1289 clinical carbapenem-resistant isolates of Pseudomonas spp. and A. calcoaceticus-baumannii complex studied between 2009 and 2015, 70 strains were MBL positive (5.4%). The first MBL-positive strain appeared in 2011 and the prevalence was 5.8% in 2011, 2.4% in 2012, 3.2% in 2013, 5.9% in 2014, and 12.8% in 2015 (see Fig. 1).

Fig. 1.

Number of MBL-positive isolates out of the total strains studied per year

The bla_VIM (n = 60%) was the most frequently MBL gene detected, followed by bla_NDM (n = 36%) and bla_IMP (n = 4%). No bla_SPM or bla_KPC genes were detected. Only the bla_OXA-51 was detected in 65% of Acinetobacter spp. isolates (17 strains), since in A. pittii (4 strains), A. nosocomialis (3 strains), A. bereziniae (1 strain), and A. haemolyticus (1 strain) OXA-type carbapenemases were not detected.

Regarding geographical origin, all the positive strains came from the Capital and Central Department of Paraguay. Concerning the source, 67% came from several clinical samples, and 33% from rectal swabs. bla_NDM was detected mainly in rectal swabs (64%), bla_VIM was mainly associated with clinical samples (83.3%) and the only three bla_IMP were isolated from blood culture, cerebrospinal fluid, and pleural fluid, respectively.

About the association between MBL genotype and bacterial species, bla_VIM was detected in P. aeruginosa (n = 12) and P. putida (n = 30). bla_IMP was detected in two P. putida strains and one A. pittii strain, while bla_NDM appeared only in species of Acinetobacter spp.: A. baumannii (n = 17), A. nosocomialis (n = 3), A. pittii (n = 3), A. haemolyticus (n = 1) and A. bereziniae (n = 1). Figure 2 shows the proportion of detection of each genotype with respect to the carrier species and the time. bla_VIM was the most prevalent genotype until 2014, mainly associated with P. putida.

Fig. 2.

Number of MBL-positive isolates per year of each bacterial species according to MBL genotype

Regarding the MBL variant genes, all bla_NDM sequences aligned 100% with the reference sequence bla_NDM-1 (GenBank FN396876.1), bla_VIM, 38/42 strains aligned 99 to 100% with the reference sequence bla_VIM-2 (GenBank FN396876.1) (it was not possible to determine the bla_VIM allelic variant from four isolates, because they were not viable at the time of sequencing). Concerning to bla_IMP, the sequence of one P. putida isolate and A. pittii corresponded to bla_IMP-18. The bla_IMP allelic variant of one P. putida was not sequenced due to lack of viability too.

Concerning resistance profile (antibiotype), it was variable, and Table 2 shows the summary. The definition of resistance classification was according to Magiorakos et al. [23]. Only colistin maintained 100% susceptibility in all isolates (MIC range ≤ 0.5 to 1 μg/mL, MIC50 and MIC90 = 0.5 μg/mL) (Fig. 3). Regarding P. aeruginosa, less than 50% were also susceptible to amikacin (5 strains), aztreonam (4 strains), and/or ciprofloxacin (3 strains). Concerning to P. putida, most of the isolates remained susceptible to amikacin (97%). All Acinetobacter spp. remained susceptible to tigecycline; some strains were also susceptible to amikacin (46%), gentamicin (42%), ciprofloxacin (38.5%), trimethoprim-sulfamethoxazole (35%), and ampicillin-sulbactam (11.6%). P. putida has a higher proportion of XDR (78%) and VIM antibiotypes II and III coincide with some of the IMP. Several of the isolates share the same pattern. The antibiotype mentioned in Table 2 is the same as the dendrograms that appear later (Figs. 4, 5, 6, 7, and 8).

Table 2.

Antimicrobial resistance characteristics of the MBL-positive strains

Species	Genotype	Nr. of strains	Resistance pattern (not-sensitive)	Sensitive pattern	AT	RP
P. aeruginosa	VIM
		7	CAZ, IPM, MPM, PIT, CPM, ATM, GEN, CIP,AMI	COL	I	XDR
		2	CAZ, IPM, MPM, PIT, CPM, GEN, CIP	ATM,AMI,COL	II	MDR
		1	CAZ, IPM, MPM, PIT, CPM, ATM, GEN	CIP,AMI,COL	III	MDR
		2	CAZ, IPM, MPM, PIT, CPM, GEN	ATM,CIP,AMI,COL	IV	MDR
P. putida	VIM	1	CAZ, IPM, MPM, PIT, CPM, ATM, GEN, CIP,AMI	COL	I	XDR
	VIM IMP	13 1	CAZ, IPM, MPM, PIT, CPM, ATM, GEN, CIP CAZ, IPM, MPM, PIT, CPM, ATM, GEN, CIP	AMI,COL AMI,COL	II II	XDR XDR
	VIM IMP	9 1	CAZ, IPM, MPM, PIT, CPM, ATM, CIP CAZ, IPM, MPM, PIT, CPM, ATM, CIP	GEN,AMI,COL GEN,AMI,COL	III III	XDR XDR
	VIM	4	CAZ, IPM, MPM, PIT, CPM, ATM, GEN	CIP,AMI,COL	IV	MDR
	VIM	2	CAZ, IPM, MPM, PIT, CPM, GEN, CIP	ATM, AMI,COL	V	MDR
	VIM	1	CAZ, IPM, MPM, PIT, CPM, GEN	ATM,CIP,AMI,COL	VI	MDR
Acinetobacter spp.	NDM	13 Ab	CAZ,IPM, MPM, PIT, CPM, GEN, AMI, CIP, SUT,AMS	COL,TGC	I	XDR
		2 Ab	CAZ,IPM, MPM, PIT, CPM, GEN, CIP, SUT, AMS	COL,AMI,TGC	II	MDR
		1 An	CAZ,IPM, MPM, PIT, CPM, SUT, AMS	COL,GEN,AMI,CIP, TGC	III	MDR
		1 Ab	CAZ,IPM, MPM, PIT, CPM, CIP, SUT	COL,GEN,AMI,AMS,TGC	IV	MDR
		7 Ap, An, Ab, Abe *	CAZ,IPM, MPM, PIT, CPM	COL,GEN,AMI,CIP, SUT,TGC	V	MDR
		1 Ah	CAZ,IPM, MPM, PIT, CPM, AMI	COL,GEN,CIP,SUT, AMS,TGC	VI	MDR
	IMP	1 Ap	CAZ,IPM, PIT, CPM, AMS	MPM,COL,GEN,AMI,CIP,SUT, AMS,TGC	VII	MDR

^AbA. baumannii, ^AnA. nosocomialis, ^AbeA. bereziniae, ^ApA. pittii, ^AhA. haemolyticus, *7: Ap n = 3, An n = 2, Ab n = 1, Abe n = 1. AT antibiotype, RP resistance profile, CAZ ceftazidime, CPM cefepime, IPM imipenem, MPM meropenem, PIT piperacillin/tazobactam, ATM aztreonam, GEN gentamicin, AMI amikacin, CIP ciprofloxacin, COL colistin, SUT trimethoprim/sulphametoxazole, AMS ampicillin/sulbactam, TGC tigecycline

Fig. 3.

Minimal inhibitory concentrations of colistin. Abbreviations: Ahae: A. haemolyticus, Abe: A. bereziniae, Apit: Acinetobacter pittii, Anos: A. nosocomialis, Aba: A. baumannii, Pae: P. aeruginosa, Pput: P. putida

Fig. 4.

Dendrogram of molecular typing of P. aeruginosa isolates. Abbreviations: tr: traqueal, re: rectal swab, ur: urine, ab: abdominal fluid, ca: catheter, Lab: origin (hospital laboratory) GT: genotype, PT: pulsotype, AT: antibiotype

Fig. 5.

Dendrogram of molecular typing of P. putida strains. Abbreviations: ur: urine, sp: sputum, bl: blood, pl: pleural fluid, re: rectal swab, tr: traqueal, cf: cerebrospinal fluid, ca: catheter, ab: abdominal fluid, GT: genotype, PT: pulsotype, AT: antibiotype

Fig. 6.

Dendrogram of molecular typing of A. baumannii isolates. Abbreviations: ur: urine, re: rectal swab, ps: ps: secretion, tr: traqueal, bl: blood, GT: genotype, PT: pulsotype, AT: antibiotype

Fig. 7.

Dendrogram of molecular typing of A. pittii strains. Abbreviations: bl: blood, re: rectal swab, cf: cerebrospinal fluid, GT: genotype, PT: pulsotype, AT: antibiotype. Obs: The strains A. pittii 1 and A. pittii 2 were studied in a previous publication [17]

Fig. 8.

Dendrogram of molecular typing of A. nosocomialis isolates. Abreviations: re: rectal swab, GT: genotype, PT: pulsotype, AT: antibiotype

About molecular typing with PFGE, VIM-2-producing P. aeruginosa (Fig. 4) revealed six different patterns or pulsotypes and four antibiotypes. Six isolates (50%) belong to dominant pulsotype C, including four subtypes. These strains were isolated in 2013 and 2015, came from four different hospitals and had the same antibiotype. The strains were isolated from both clinical samples and rectal swabs. Despite presenting different antibiotypes, the two isolates from the same patient (Pae 62 and Pae 69 isolates) belonged to the same pulsotype (pattern A). Figure 4 shows the dendrogram and complementary data of the strains studied.

PFGE analysis for P. putida (Fig. 5) showed 25 different pulsotypes in the VIM-producing strains with only six antibiotypes, and no large clonal group was observed. However, there are isolates from different hospitals belonging to the same pulsotype. The IMP-producing strains belonged to different clones than VIM-producing isolates but share some of the antibiotypes. The strains came from both clinical samples and rectal swabs.

The dendrogram for NDM-producing A. baumannii isolates (Fig. 6) revealed five different pulsotypes (A-E) and three antibiotypes (I, II, and V). Pulsotype A including seven subtypes (A1-A7) was found in 13 strains, 12 of them from the same hospital (L1), and all except one with the same antibiotype. The first NDM-producing A. baumannii isolate detected in 2014 recovered from a different hospital (L6) also belonged to the pulsotype A. The four remaining strains presented different patterns. In this group of strains, 12/17 came from rectal swabs.

Molecular typing with PFGE for A. pittii (Fig. 7) revealed three different pulsotypes (A–C) for two antibiotypes, one of them also present in A. baumannii (V, see Fig. 6). The first two NDM-producing A. pittii isolates, recovered from the same hospital, belonged to different pulsotypes (pulsotype A and C), already investigated in a previous publication [24]. Pulsotype A has with two closely related strains from the same hospital, isolated in different years, and the B and C pulsotypes are observed in one strain each. All NDM-positive strains showed the same antibiotypes, and they came from the same hospital. The IMP positive strain shows its own pulsotypes and antibiotype.

A. nosocomialis strains revealed two different pulsotypes (Fig. 8) and two antibiotypes, one of them (V) also present in A. baumannii and A. pittii. Two strains were grouped as A1 and A2 subtypes, while the third strain have a different pulsotype. All strains were isolated from the rectal swab.

Two strains were not included in the previous dendrograms, since they were typified as A. haemolyticus (antibiotype VI present in this single strain) and A. bereziniae (antibiotype V also present in A. baumannii and A. pittii).

Discussion

There are few studies published about intrahospital carbapenemases in Paraguayan non-fermenting gram-negative rods using molecular methods. One of them is from Pasteran et al. [24] which includes our strains A. piitii 1 and A. pitti 2 (see Fig. 7). Pasteran et al. identified those two strains as A. pittii by MALDI-TOF and were not clonally related by PFGE. These findings are consistent with our results. The study published by Rodriguez et al. [25] including A. baumannii paraguayan strains among others, found only OXA-23 carbapenemase and no MBL. None of these strains is part of the present work. In 2021, Melgarejo et al. [26] studying fermenting and non-fermenting gram-negative bacilli in Paraguay, found strains of A. baumannii with OXA 51, OXA 23, NDM, and NDM + OXA 58, and P. aeruginosa with NDM, but all those strains were isolated in 2021. Our work shows the beginning and the first years of MBLs dissemination in non-fermenting gram-negative bacilli and their behavior over time. Since then, MBL-producing strains have not ceased to be detected and as expected, the isolates increased gradually. It is possible to affirm that in Paraguay the MBL began to appear in BGNNF to spread years later in fermenting gram-negative bacilli also since the first publication in an Enterobacterales is from the year 2016 [27].

Regarding the MBL-producing species, P. putida, P. aeruginosa and A. baumannii, were the most prevalent in this investigation, as in another studies [8]. As expected, A. baumannii turned out to be the most prevalent of its genus (65%), followed by A. pittii and A. nosocomialis. This last two species, as well as A. haemolyticus and A. bereziniae, less prevalent, have been sporadically described as species involved in hospital infections [28, 29]. The most frequent association between bacterial species and genotype was observed with P. putida and VIM (43%), followed by A. baumannii associated with NDM (24%). It is interesting to note that NDM was detected in Acinetobacter spp. from 2011 to 2014 in “non-baumannii” species (A. pittii, A. haemolyticus, A. nosocomialis, A. bereziniae). However, appeared in A. baumannii in 2015 with practically absolute predominance.

Although IMP, VIM, and NDM genotypes are the most frequent MBLs in the world, [7, 30], the frequency is variable, characteristic of each country, region, or hospital, consequently, studies published, show a variable prevalence [31, 32]. Before the description of NDM, the most frequently detected MBLs in the world were IMP and VIM type, being VIM the most predominant [5, 30]. In our study, the distribution percentage found was 60% VIM, 36% NDM, and 4% IMP, but the prevalence varied over to the years. In fact, the VIM-producing, the most frequently isolated until 2014, was surpassed in 2015 by NDM associated to A. baumannii evidencing the great dissemination capacity of the mechanism in this genomespecies. In 2015 by the way, NDM was considered to becoming the most commonly and distributed carbapenemase worldwide [30].

Regarding the allelic variants described, VIM-1 and VIM-2 are the most frequent, reported in Europe [7, 33] and Asia [7]. In Latin American countries VIM-2 has been mostly detected [7] in Chile and Venezuela [34], Argentina [35], Brazil [36], Uruguay [37], and other countries, especially in P. aeruginosa followed by P. fluorescens. In this study, we demonstrate the circulation of VIM-2, associated with P. putida and P. aeruginosa. The allelic variant NDM-1, found in the present study, has been reported around the world, including Latin American countries such as Colombia, where it was detected in A. baumannii and A. nosocomialis [38], NDM-1 was also confirmed in A. baumannii [39] and in A. pittii [40] in Brazil. The IMP has been described with a lower prevalence than others. IMP-1 was the first variant described, followed later by IMP-2 [41], subsequently more variants were described. In this study, the IMP-18 subtype was detected in a lower prevalence. This variant has been detected in P. aeruginosa in Brazil [42].

In this work, only OXA-51, intrinsic to A. baumannii, was detected. None other OXA were found associated with MBL. In fact, the coexistence of OXA-type carbapenemase with NDM is unusual but has been documented [43].

Multiple clonalities of P. putida has been reported [44]. Some studies reported several P. putida clones with the same VIM-2 transposon in plasmids, together with a high proportion of MBL-producing strains compared to P. aeruginosa, suggesting that P. putida is a reservoir of these elements MDR transferable [44]. This situation could have been observed in this study, where in 2014 P. aeruginosa was not isolated and 43% of the VIM-2-producing strains were P. putida versus 17% of P. aeruginosa.

European reports suggest intrahospital transmission of VIM associated with P. aeruginosa [45]. Publications from Argentina [35], Spain [45], Colombia [46], and Brazil [47] show polyclonal propagation as well as spread by successful clones. In our study, we observed intra and out-off-hospital spread of the predominant clonal group. An outbreak would have started in one hospital, lasted 2 years and spread at two other hospitals.

Clonal diversity as well as dissemination of successful clonal groups of A. baumannii has been demonstrated in several studies [48, 49]. All this has been verified in the present work. NDM-producing A. baumannii presented a group of strains with a common clonal origin (13/17 isolates), all epidemiologically related, since 12 were from the same hospital (L1) and from the same year (2015). This could describe an outbreak in a hospital (L1) that would have originated in another hospital (L6), which had only one closely related isolate (95% similarity).

With this study, we ascertain the intra and interhospital dissemination of MBL-type carbapenemases, through polyclonal and successful clones capable of generating outbreaks, evidenced with the pulsotype A of A. baumannii, pulsotypes C of P. aeruginosa and R of P. putida. We also observed evidence of dissemination by mobile genetic elements due the presence of the same MBL variant in different species like VIM-2 in both P. aeruginosa and P. putida, IMP-18 in both P. putida and A. pitti, and NDM-1 in different Acinetobacter species. The presence of the same genotype variant and the same antibiotype in clonally unrelated strains suggests the spread of resistance by mobile genetic elements. It will be very interesting to compare this work with the subsequent evolution of carbapenemases in non-fermenting gram-negative rods in Paraguay, and the study of mobile genetic elements in more recent times.

Obviously, it is necessary to maintain and strengthen the surveillance of MBLs, as well as in-hospital containment measures to avoid the dissemination of these resistance mechanisms and others that could be successfully transferred through mobile genetic elements sometimes associated with successful clones, both within the same hospital, as between hospitals in Paraguay. The transfer of patients from one hospital to another or the fact that in Paraguay health personnel work at the same time in different hospitals, could be part of the problem of the spread of colonizing bacteria of intrahospital origin.

Funding

This work was supported by grants from the IEBAS-FOCEM Project “Research, Education and Biotechnologies applied to Health” (agreement COF 03/11).

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethical approval

Ethical approval was received from the Institutional Ethics Committee (IEC) of the Central Laboratory of Public Health, (approval no. CEI / LCSP 41/030314).

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Conflict of interest

The authors declare no competing interests.