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Communication

Analysis of Antimicrobial Resistance Genes (ARGs) in Enterobacterales and A. baumannii Clinical Strains Colonizing a Single Italian Patient

by
Alessandra Piccirilli
1,
Elisa Meroni
2,
Carola Mauri
2,
Mariagrazia Perilli
1,*,
Sabrina Cherubini
1,
Arianna Pompilio
3,4,
Francesco Luzzaro
2 and
Luigi Principe
5
1
Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, 67100 L’Aquila, Italy
2
Clinical Microbiology and Virology Unit, “A. Manzoni” Hospital, 23900 Lecco, Italy
3
Department of Medical, Oral and Biotechnological Sciences, “G. d’Annunzio” University of Chieti-Pescara, 66100 Chieti, Italy
4
Center for Advanced Studies and Technology (CAST), “G. d’Annunzio” University of Chieti-Pescara, 66100 Chieti, Italy
5
Clinical Pathology and Microbiology Unit, “S. Giovanni di Dio” Hospital, 88900 Crotone, Italy
*
Author to whom correspondence should be addressed.
Antibiotics 2023, 12(3), 439; https://doi.org/10.3390/antibiotics12030439
Submission received: 10 January 2023 / Revised: 7 February 2023 / Accepted: 20 February 2023 / Published: 23 February 2023
(This article belongs to the Special Issue Antimicrobial Resistance in the Community Setting: The Other Side of the Coin)
Review Reports Versions Notes

Abstract

:
The dramatic increase in infections caused by critically multidrug-resistant bacteria is a global health concern. In this study, we characterized the antimicrobial resistance genes (ARGs) of K. pneumoniae, P. mirabilis, E. cloacae and A. baumannii isolated from both surgical wound and rectal swab of a single Italian patient. Bacterial identification was performed by MALDI-TOF MS and the antimicrobial susceptibility was carried out by Vitek 2 system. The characterization of ARGs was performed using next-generation sequencing (NGS) methodology (MiSeq Illumina apparatus). K. pneumoniae, P. mirabilis and E. cloacae were resistant to most β-lactams and β-lactam/β-lactamases inhibitor combinations. A. baumannii strain was susceptible only to colistin. The presence of plasmids (IncN, IncR, IncFIB, ColRNAI and Col (MGD2)) was detected in all Enterobacterales but not in A. baumannii strain. The IncN plasmid and blaNDM-1 gene were found in K. pneumoniae, P. mirabilis and E. cloacae, suggesting a possible transfer of this gene among the three clinical species. Conjugation experiments were performed using K. pneumoniae (1 isolate), P. mirabilis (2 isolates) and E. cloacae (2 isolates) as donors and E. coli J53 as a recipient. The blaNDM-1 gene was identified by PCR analysis in all transconjugants obtained. The presence of four different bacterial species harboring resistance genes to different classes of antibiotics in a single patient substantially reduced the therapeutic options.

1. Introduction

Antimicrobial resistance represents one of the most serious global public health issues [1]. Several microorganisms known as ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) have emerged as globally critical multidrug-resistant (MDR) pathogens requiring continues monitoring and development of new drugs [2,3]. To date, only few drugs in development are potentially active against ESKAPE pathogens [4]. Among these, the Gram-negative P. aeruginosa, K. pneumoniae and other Enterobacterales are of great concern for their high level of resistance to most antibiotics and, in particular, to carbapenems, often considered as our last line of defense against critical pathogens [5,6,7]. In the last decade, infections caused by carbapenemase-producing Enterobacterales (CPE) have dramatically increased [6,7,8,9,10,11]. In particular, the KPC and NDM variants are the most widespread carbapenemases in clinical strains with a wide range of infections [12,13,14,15].
This study reports a detailed characterization of the antimicrobial resistance genes (ARGs) in P. mirabilis, K. pneumoniae, E. cloacae and A. baumannii, isolated from surgical wound (SW) and rectal swab (RS) of a particular hospitalized patient.

2. Results

2.1. Antimicrobial Susceptibility

Overall, six clinical isolates obtained from SW and RS were analyzed against a large panel of antibiotics. As given in Table 1, the E. cloacae and K. pneumoniae isolates showed resistance to carbapenems (ertapenem, imipenem and meropenem), cephalosporins (cefotaxime, ceftazidime and cefepime), amoxicillin–clavulanic acid, piperacillin–tazobactam, ceftolozane–tazobactam and ceftazidime–avibactam. However, E. cloacae and K. pneumoniae isolates were susceptible to amikacin, gentamycin and colistin. A similar susceptibility profile was found for P. mirabilis isolates. The A. baumannii strain was susceptible only to colistin.

2.2. Multi-Locus Sequence Typing (MLST) and Plasmid Multi-Locus Sequence Typing (pMLST)

The draft genome of the six isolates recovered for this study was obtained using MiSeq Illumina platform. The sequence analysis revealed that the total number of sequenced nucleotides was 4.6–4.8 Mb for E. cloacae isolates, 3.7–4.3 Mb for P. mirabilis isolates, 5.7 Mb for K. pneumoniae and 3.7 Mb for A. baumannii (Table 2). The MLST of K. pneumoniae, E. cloacae and A. baumannii indicates that these strains belonged to the lineages ST4587, ST45 and ST2, respectively. To date, molecular characteristics and genetic relationships among Proteus spp. have not been elucidated, and for this reason, the MLST analysis of P. mirabilis was not launched.

2.3. ARGs and Mobile Genetic Elements

With the exception of A. baumannii SW, plasmid replicons have been found in all strains (Table 2). Of note, P. mirabilis isolates carried IncN and IncQ1 incompatibility plasmids. Both E. cloacae RS and E. cloacae SW isolates harbored IncN, IncFIB (pECLA) and IncFII (pECLA) plasmids, but E. cloacae RS had, in addition, ColRNAI. K. pneumoniae RS carried the IncN, IncR, IncFIB (K), Col (MGD2) and ColRNAI plasmids. Overall, E. cloacae RS, E. cloacae SW, P. mirabilis RS, P. mirabilis SW and K. pneumoniae RS shared the same IncN plasmid belonging to the ST7 pMLST scheme. Different insertion sequences (ISs) were identified in the six isolates analyzed. Both E. cloacae RS and E. cloacae SW carried transposon Tn2, IS6100 (IS6 family), ISKpn8 (IS3 family) and ISSen3 (IS21 family). Nevertheless, in E. cloacae RS, ISSen4 (IS3 family), IS26 (IS6 family) and ISKpn19 (ISKra4) were also detected. ISKpn19 and IS6100 were identified in P. mirabilis, E. cloacae and K. pneumoniae isolates (Table 2). In A. baumannii, ISAba24 (IS66 family), ISAba26 (IS256 family) and IS26 sequence insertions were found. Table 2 displayed a detailed analysis of the resistome of the six clinical strains. Both P. mirabilis SW and RS isolates showed the same resistance genes: (a) blaNDM-1 and blaTEM-1B, β-lactams resistance genes; (b) qnrS1, a plasmid mediating resistance to fluoroquinolones; (c) aadA1 that confers resistance to aminoglycosides (streptomycin and spectinomycin); (d) strA-strB chromosomal gene cluster conferring resistance to streptomycin; (e) sul2, tet (J) and catA1, conferring resistance to sulphonamides, tetracycline and chloramphenicol, respectively. The E. cloacae SW and RS isolates harbored strB, strA, sul2, dfrA14, qnrS1, catA2, blaNDM-1, blaTEM-1B and blaACT-15, whereas K. pneumoniae RS had qnrS1, oqxB, dfrA14, fosA, blaNDM-1 and blaLEN-22. A. baumannii harbored blaOXA-23, blaADC-25, blaOXA-66, armA, strA, strB, mph (E), msr (E), sul2 and tetB genes.

2.4. Conjugation Experiments and PCR Assays

Conjugation experiments were performed using E. coli J53 strain as a recipient and P. mirabilis (RS and SW), E. cloacae (RS and SW) and K. pneumoniae RS as donors. Conjugational transfer of meropenem resistance was ascertained in all three systems, and the presence of blaNDM-1 was confirmed by PCR in all transconjugants obtained.

3. Discussion

Here, we described the molecular characterization of ARGs of K. pneumoniae, E. cloacae, P. mirabilis and A. baumannii clinical strains isolated from different sample sites of a single hospitalized patient. The case was not epidemiologically related to other hospitalized patients, and no information was available about the stay of the patient in LTCF. The characterization of ARGs was performed by NGS analyzing the resistome of all strains. On the basis of the draft genome analysis, we have noted that the IncN plasmid was found in all Enterobacterales analyzed. The IncN belongs to a broad-host-range plasmids with a size of 30 to 70 Kb that, often, carry a great variety of resistance genes, including blaCTX-M, blaVIM and blaNDM [16,17,18,19]. In this study, IncN was simultaneously present with IncQ1 in P. mirabilis, with IncFIB and ColRNAI in E. cloacae and with IncR in K. pneumoniae. The IncQ1 belongs to the MOBQ group with a medium–small size (8–14 Kb) and, often, they carry sul-strA-strB gene cluster [18]. The IncR (40–160 Kb) is a mobilizable plasmid frequently cointegrated with IncN plasmid [19], the same cluster found in P. mirabilis and E. cloacae (this study). The β-lactams resistance genes found in K. pneumoniae, P. mirabilis and E. cloacae were blaNDM-1, blaACT-15, blaLEN-22 and blaTEM-1B. However, only blaNDM-1 was found in all Enterobacterales, and for this reason, we have supposed that IncN plasmid harbored blaNDM-1 gene. On the basis of conjugation experiments, we have presumed the circulation of blaNDM-1 gene via IncN plasmid among K. pneumoniae, P. mirabilis and E. cloacae isolated from the single patient. The wide distribution of blaNDM-1 and its natural variants among clinical and community-acquired Enterobacterales is related to the fact that they can be carried by different plasmid types (IncA/C, IncF, IncL/M or untypable) that are readily self-transmissible by conjugation [20]. The blaNDM promiscuity is related to its high mobilization capacity into plasmids or chromosomes [21]. Patients simultaneously infected and/or colonized with multiple species of CPE are more frequently observed [22,23,24]. Several cases of interspecies exchange of identical blaKPC-, blaOXA-48− and blaNDM-1-carrying plasmids have been described [22,23,24,25,26]. In particular, those involving the blaNDM-1 were mainly related to the horizontal spread of broad-host-range IncC plasmids (formerly, IncA/C2) [26]. Invasive infections by MBL-producing Enterobacterales are associated with high mortality rates (>30%), especially, in the hospital setting when critically ill patients are involved [27,28]. The spread of CPE is significantly increasing in healthcare settings and, also, in long-term care facilities (LTCFs) [15,16,17]. The draft genome analysis of A. baumannii strain, analyzed in this study, exhibited the presence of blaOXA-23, blaOXA-66 and blaADC-25 genes. It is very common to find the simultaneous presence of blaOXA-23 and blaOXA-66 in the genome of carbapenem-resistant A. baumannii strains [29,30]. The OXA-66 is an OXA-51-like enzyme, intrinsically overexpressed in A. baumannii strains, which is able to confer high resistance to carbapenems [31]. The blaADC genes are also chromosomally encoded in A. baumannii strains [32]. The presence of ISAba24 and ISAba26 upstream the blaOXA genes indicates the plausible presence of a strong promoter that drives expression of the downstream genes and facilitates the transferability of resistance determinants [33,34]. In particular, the LTCFs represent an important ARGs’ reservoir in older resident people who are more vulnerable to bacterial infections due to multiple chronic illnesses.

4. Materials and Methods

4.1. Clinical Case Description

In September 2020, an 88-year-old woman was admitted to the Emergency Department of the A. Manzoni Hospital (Lecco, Italy) following an accidental fall. The X-ray revealed a displaced fracture of the left femur involving the lesser trochanter. The patient’s medical history revealed previous fractures of the same femur, presumed autoimmune liver disease, diabetes, obesity and lower limb polyneuropathy. No recent hospitalization was reported. Four days after admission, the patient underwent surgery after washing the fracture site. One week after surgery, the patient was discharged from the Orthopedics unit to an LTCF; but a few days later, she was newly admitted to the General Medicine of the A. Manzoni Hospital, showing hypotension and diffuse icterus. Based on the hospital protocol for patients coming from LTCF, a rectal swab for CPE screening was performed, whereas an antimicrobial treatment was empirically initiated with amoxicillin/clavulanic acid (0.625 g, tid). Laboratory data showed increased values of lipases, bilirubin, AST and ALT enzymes, thus suggesting hepatic dysfunction. Based on appropriate imaging, acute cholecystitis with gallbladder hydrops was diagnosed and empiric therapy was then changed to piperacillin/tazobactam (4.5 g, tid) and gentamicin (240 mg, once a daily). Bacterial isolates were recovered from MacConckey agar (bioMérieux, Marcy l’Etoile, France), after an 18–22 h incubation period in aerobic conditions (37 °C) in the context of a laboratory clinical routine. In particular for rectal swabs, bacterial isolates resistant to carbapenems were also recovered from chromogenic Brilliance CRE agar (Thermo Fisher Scientific, Waltham, MA, USA). Cultures from SW were positive for E. cloacae complex SW and P. mirabilis SW, both producing an NDM-type carbapenemase. The RS performed at the same time showed positive for E. cloacae complex RS, P. mirabilis RS and K. pneumoniae isolates, all of them producing an NDM-type enzyme. Subsequently, a carbapenem-resistant A. baumannii SW was also isolated from the wound. The patient was discharged after 45 days in good health conditions, and a home care regimen was assessed.

4.2. Strains Identification and Antibiotic Susceptibility Testing

The bacterial identification was performed by MALDI-TOF mass spectrometry (Vitek MS, bioMérieux, Marcy l’Étoile, France). The antimicrobial susceptibility was determined using both the Vitek 2 system (bioMérieux, Marcy l’Étoile, France) and the Sensititre™ Gram Negative Panel (ThermoFisher, Waltham, MA, USA). Susceptibility results were interpreted according to current EUCAST criteria. Carbapenemase production was first assessed using phenotypic methods, including an immunochromatographic assay (RESIST-4 O.K.N.V., Coris Bio-Concept, Gembloux, Belgium) and specific inhibitor disks (KPC+MBL Confirm ID Kit, Rosco Diagnostica), Figure S1. The clinical strains analyzed in this study were from the surgical wound (E. cloacae SW, P. mirabilis SW, A. baumannii SW) and rectal swab (E. cloacae RS, P. mirabilis RS, K. pneumoniae RS) samples.

4.3. Resistome Analysis

Total DNA of P. mirabilis (2 isolates), E. cloacae (2 isolates), K. pneumoniae (1 isolate) and A. baumannii (1 isolate) was extracted using a modified protocol, as previously reported [16,17]. Libraries were sequenced using the Illumina MiSeq system by 2 × 300 paired-end approach (Illumina, San Diego, CA, USA) [16,17]. Paired-end reads were assembled using Velvet (v.1.2.10) [35]. The resistome and plasmidome were analyzed using ResFinder 4.1 (available online: https://cge.cbs.dtu.dk/services/ResFinder/ (accessed on 14 January 2022)) and PlasmidFinder 2.1 (available online: https://cge.cbs.dtu.dk/services/PlasmidFinder/ (accessed on 16 January 2022)), respectively [36,37]. MobileElementFinder was used to identify mobile genetic elements and their relation to ARGs [38]. The Pasteur multi-locus sequence typing (MLST) scheme was used to assign the ST (available online: https://bigsdb.pasteur.fr/index.html (accessed on 16 January 2022)) [39].

4.4. Conjugation Assays

Conjugation experiments were performed using Escherichia coli J53 (rifampicin-resistant strain) as a recipient and P. mirabilis (RS and SW), E. cloacae (RS and SW) and K. pneumoniae RS strains as donors. Transconjugants were selected on Luria–Bertani (LB) agar plates supplemented with 300 mg/L rifampicin and 2 mg/L meropenem or 100 mg/L ampicillin. The detection sensitivity of the assay was ≥5 × 10–7 transconjugants per recipient.

4.5. PCR Experiments

One colony of each transconjugant was picked, dissolved in 100 µL of sterile H2O and boiled at 100 °C. The mixture was harvested at 14,000× g, and 2 µL of supernatant was used for PCR experiments with specific NDM-1 primers. Primers and PCR conditions were from our previous study [40].

5. Conclusions

In this study, we have characterized the ARGs from four different bacterial species isolated from a single patient, who was admitted to the Orthopedics unit of the A. Manzoni Hospital. The patient was then discharged from the Orthopedics to an LTCF. After a few days, the patient was newly admitted to the General Medicine of the same hospital for different problems. This represents a clear example of bacterial pathogens spreading from community to hospital settings and vice versa. Unfortunately, we have no information about the epidemiological situation of the LTCF that accommodated the patient. Results obtained in this study showed the persistence of blaNDM-1 in three different Enterobacterales species isolated from a single patient. NDM producers are not commonly related to the Italian epidemiologic context, but they are emerging and increasingly reported [41,42]. Moreover, infections caused by pathogens harboring ARGs that confer resistance to different classes of antibiotics substantially reduced the therapeutic options, especially, when bacteria harbored MBLs.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antibiotics12030439/s1. Figure S1: Immunochromatographic assay and specific inhibitor disks to detect carbapenemases.

Author Contributions

Conceptualization, M.P. and A.P. (Alessandra Piccirilli); methodology, S.C., E.M. and C.M.; investigation, S.C. and A.P. (Alessandra Piccirilli); data curation, A.P. (Alessandra Piccirilli), S.C. and M.P.; writing—original draft preparation, A.P. (Alessandra Piccirilli) and M.P.; writing—review and editing, M.P., A.P. (Alessandra Piccirilli), L.P., F.L. and A.P. (Arianna Pompilio); visualization, A.P. (Alessandra Piccirilli), A.P. (Arianna Pompilio), F.L. and M.P.; supervision, A.P. (Alessandra Piccirilli), L.P. and M.P.; project administration, M.P. and A.P. (Alessandra Piccirilli); funding acquisition, A.P. (Alessandra Piccirilli) All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the University of L’Aquila, grant number 07_PROGETTO_RICERCA_ATENEO (Alessandra Piccirilli).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We wish to thank Anna Toso (Toronto Catholic District School Board, Toronto, Canada) for the language revision of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Table
Table 1. Susceptibility profile of isolates obtained from surgical wound (SW) and rectal swab (RS).
Table 1. Susceptibility profile of isolates obtained from surgical wound (SW) and rectal swab (RS).
E. cloacae SW
E. cloacae RS		P. mirabilis SW
P. mirabilis RS		A. baumanniiK. pneumoniae RS
Antimicrobial AgentMIC (mg/L)Interpretation MIC (mg/L)Interpretation MIC (mg/L)Interpretation MIC (mg/L)Interpretation
Amoxicillin/clavulanic acid>16R>16R>16R>16R
Piperacillin/tazobactam>64R>32R>64R>64R
Cefepime>16R4I16R8R
Cefotaxime>32R16R>32R16R
Ceftazidime>32R>32R>32R>32R
Ceftazidime/avibactam>16R>16R>16R>16R
Ceftolozane/tazobactam>32R>32R>32R>32R
Ciprofloxacin>2R0.5I>2R>2R
Ertapenem>4R>4R>4R>4R
Imipenem>8R>8R>8R>8R
Meropenem>8R>8R>8R>8R
Amikacin≤1S4S>32R≤1S
Gentamycin≤1S≤1S>8R≤1S
Colistin0.5S<4R≤0.5S≤0.5S
Minimum Inhibitory Concentration (MIC) interpretation: R, resistant; I, intermediate; S, susceptible.
Table
Table 2. Resistome of the six clinical strains isolated from surgical wound (SW) and rectal swab (RS) of a single patient.
Table 2. Resistome of the six clinical strains isolated from surgical wound (SW) and rectal swab (RS) of a single patient.
StrainsGenome Size (bp)MLST
(Pasteur)		Plasmid Replicons/pMLSTMobile Genetic ElementsΒ-lactams Resistant GenesOther ARGs
Proteus mirabilis RS4.342.694noneIncN, IncQ1/
IncN: ST7		ISKpn19, IS6100, ISVsa5 (IS10R), IS26blaNDM-1,
blaTEM-1B		aadA1, strB, strA, sul2, dfrA1, dfrA14, qnrS1, tet(J), catA1
Proteus mirabilis SW3.796.792noneIncN, IncQ1/
IncN: ST7		ISKpn19, IS26, IS6100blaNDM-1,
blaTEM-1B		aadA1, strB, strA, sul2, dfrA1, dfrA14, qnrS1, tet(J), catA1
Enterobacter cloacae RS4.617.198ST45IncN, IncFIB(pECLA), IncFII(pECLA), ColRNAI/
IncN: ST7		Tn2, ISKpn19, IS26, IS6100, ISSen4 (IS3, Group IS407), ISSen3(Family IS21), ISKpn8 (Family IS3)blaNDM-1,
blaTEM-1B,
blaACT-15		strB, strA, sul2, dfrA14, qnrS1,
catA2
Enterobacter cloacae SW4.781.639ST45IncN, IncFIB(pECLA), IncFII(pECLA)/
IncN: ST7		Tn2
ISSen3 (Family IS21)
ISKpn8 (Family IS3)
IS6100		blaNDM-1,
blaTEM-1B,
blaACT-15		strB, strA, sul2, dfrA14, qnrS1, catA2
Klebsiella pneumoniae RS5.757.187ST4587IncN, IncR, Col(MGD2), IncFIB(K), ColRNAI/IncN: ST7-likeISKpn19, ISKpn21, IS6100, IS5075 (Family IS110)blaNDM-1,
blaLEN-22		qnrS1, oqxB, dfrA14, fosA
Acinetobacter baumannii SW3.737.728ST2noneISAba24 (Family IS66)
ISAba26 (Family IS256)
IS26		blaOXA-23, blaADC-25, blaOXA-66armA, strA, strB, mph(E), msr(E), sul2, tetB
MLST, Multi-Locus Sequence Typing; pMLST, plasmid Multi-Locus Sequence Typing; ARGs, Antimicrobial Resistance Genes.
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Piccirilli, A.; Meroni, E.; Mauri, C.; Perilli, M.; Cherubini, S.; Pompilio, A.; Luzzaro, F.; Principe, L. Analysis of Antimicrobial Resistance Genes (ARGs) in Enterobacterales and A. baumannii Clinical Strains Colonizing a Single Italian Patient. Antibiotics 2023, 12, 439. https://doi.org/10.3390/antibiotics12030439

AMA Style

Piccirilli A, Meroni E, Mauri C, Perilli M, Cherubini S, Pompilio A, Luzzaro F, Principe L. Analysis of Antimicrobial Resistance Genes (ARGs) in Enterobacterales and A. baumannii Clinical Strains Colonizing a Single Italian Patient. Antibiotics. 2023; 12(3):439. https://doi.org/10.3390/antibiotics12030439

Chicago/Turabian Style

Piccirilli, Alessandra, Elisa Meroni, Carola Mauri, Mariagrazia Perilli, Sabrina Cherubini, Arianna Pompilio, Francesco Luzzaro, and Luigi Principe. 2023. "Analysis of Antimicrobial Resistance Genes (ARGs) in Enterobacterales and A. baumannii Clinical Strains Colonizing a Single Italian Patient" Antibiotics 12, no. 3: 439. https://doi.org/10.3390/antibiotics12030439

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