Prevalence and Phenotypic and Molecular Characterization of Carbapenemase-Producing Gram-Negative Bacteria in Gabon

ABSTRACT. Data collection and monitoring of carbapenemase-producing (CP) Gram-negative bacteria (GNB) are often limited. This study determined CP-GNB prevalence in Gabon and the genetic origins of the resistance genes. From January 2016 to March 2018, 869 clinically significant GNB isolates from inpatients and outpatients, and 19 fecal samples (inpatients) were analyzed in the main hospitals of Gabon. Fecal samples were screened using ChromID® CARBA SMART selective chromogenic medium biplates. Species were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry. Antibiotic susceptibility was tested using the disk diffusion method on Müller–Hinton agar, and resistance genes were assessed by multiplex polymerase chain reaction and sequencing. Overall, 1.61% of clinical isolates (14 of 869) and 5.26% of fecal samples (1 of 19) were CP-GNB. The CP-GNB rate was higher among inpatients (2.98%) than outpatients (0.33%), in intensive care units (28.57%, 4 of 14), and in urine samples (35.71%, 5 of 14). The most common CP-GNB were Klebsiella pneumoniae (53.33%) and Acinetobacter baumannii (26.67%). blaOXA-48 was the predominant carbapenemase-encoding gene (40%), followed by blaNDM-5 (33.33%). The A. baumannii multilocus sequence types ST2 and ST78, Enterobacter cloacae ST78, Escherichia coli ST2, and K. pneumonia ST48 and ST147 were found. These data indicate that CP bacteria are present in clinical and carriage samples. Preventive measures are needed to avoid the spread of resistance genes.


INTRODUCTION
The emergence and spread of carbapenemase-producing (CP) Gram-negative bacteria (GNB) represent a serious public health problem because effective therapeutic alternatives (e.g., colistin and cefiderocol) are not widely available in lowincome countries. [1][2][3] Therefore, carbapenems remain often the last available antibiotic class to treat infections caused by multidrug-resistant nonfermenter (Acinetobacter baumannii and Pseudomonas aeruginosa) and fermenter (Enterobacterales) GNB. 4,5 Acquired class A (KPC, IMI, GES), class B (IMP, VIM, NDM), and class D (OXA-48, OXA-181) carbapenemases are the most prevalent determinants of resistance to carbapenems. 6,7 The supporting genes are associated frequently with mobile genetic structures (e.g., plasmids, integrons, transposons) that promote their spread and transmission. CP-GNB spreading is considered an urgent threat because high mortality rates have been reported among infected patients. 8,9 In 2017, the WHO included carbapenem-resistant Enterobacterales, A. baumannii, and P. aeruginosa in the global priority list of antibioticresistant pathogens. 10 CP-GNB have been detected worldwide, 11 but data collection and surveillance of multidrug-resistant GNB are often limited, particularly in middle-and low-income countries, such as many African countries. In Central Africa, only a few studies have reported the presence of isolates that produce carbapenemases, such as NDM-4 in Cameroon, OXA-181 and NDM-1 in Angola, and OXA-181 and NDM-5 in Chad. 4,12 In Gabon, only one study described the presence of NDM-7producing isolates. 13 We investigated the prevalence and genetic characteristics of CP-GNB isolates from clinical and fecal carriage samples from in-and outpatients in Gabon.

MATERIALS AND METHODS
Study area, patients, and specimen collection. Samples were obtained from eight main hospitals and one medical analysis laboratory located in seven of the nine provinces of Gabon (population, 1,811,079 inhabitants among whom more than 93% reside in urban areas) from January 2016 to March 2018 ( Figure 1). The eight hospitals are located in Libreville, the capital city that concentrates almost half of this population (49.5%) 14 and includes the Omar Bongo Ondimba Army Teaching Hospital (HIAOBO), Akanda Army Teaching Hospital, and El Rapha Polyclinic; Lambar en e, which includes the Georges Rawiri Regional Hospital Center; Mouila, which includes the Mouila Regional Hospital Center; Tchibanga, which includes the Benjamin Ngoubou Regional Hospital Center; Koulamoutou, which includes the Paul Moukambi Regional Hospital Center; and Moakokou, which includes the Omar Bongo Ondimba Regional Hospital Center. The Medical Analysis Laboratory of the Interdisciplinary Medical Research Center of Franceville is in Franceville and analyzes samples from two hospitals in Franceville: the Amissa Bongo Regional Hospital Center and the Sino-Gabonese Cooperation Hospital Center. Each hospital has an average of 93 beds (thus, in total, 930 beds).
During this period, 869 nonduplicate clinical GNB samples were obtained: 569 (65.5%) from patients hospitalized for more than 48 hours and 300 (34.5%) from outpatients. During the same period, fresh stool samples were collected from 19 patients hospitalized for more than 48 hours without digestive pathology at the Omar Bongo Ondimba Army Teaching Hospital and the Mouila Regional Hospital Center to investigate CP-GNB fecal carriage. Briefly, 0.5 g of each fresh stool sample was suspended in 5 mL sterile saline solution (0.9%), and 100-mL aliquots were plated on ChromID V R CARBA SMART selective chromogenic medium biplates (bioM erieux, Marcy-l'Etoile, France), a medium used for the screening of CP Enterobacterales.
DNA was extracted from one single colony for each isolate in a final volume of 100 mL of distilled water by incubation at 95 C for 10 minutes, followed by a centrifugation step. The presence of the bla NDM , bla Oxa48-like , bla GIM , bla PER , bla IMP , bla VIM , bla SPM , bla KPC , bla DIM , bla SIM , bla BIC , bla AIM , bla VEB , bla CTX-M (CTX-M groups 1, 2, 8, 9, and 25), bla TEM , bla SHV , and bla OXA-1-like , the 16S recombinant RNA methylase genes conferring resistance to aminoglycosides (armA, rmtA, rmtB, rmtC, and rmtD), and the plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrB, qnrS, qnrC, qnrD, qepA, aac(69)-Ib-cr, and oqxAB) was assessed by multiplex PCR using a previously published method. 17 Our strategy did not allow discriminating between the aac (69)-Ib gene, conferring resistance to aminoglycosides, and the aac (69)-Ib-cr gene, conferring resistance to aminoglycosides plus quinolones. DNA samples from ESBL-, carbapenemase-, 16S recombinant RNA methylase-and PMQR-positive clinical isolates from the collection of the Montpellier University Hospital bacterial laboratory (Montpellier, France) (previously characterized phenotypically and molecularly), were used as positive controls. PCR products were visualized after electrophoresis on 1.5% agarose gels containing ethidium bromide (Eurobio Scientific, LES ULIS, France) at 100 V for 90 minutes. A 100-bp DNA ladder (Promega, Charbonni eres-les-bains, France) was used as marker size. PCR products were purified using the ExoSAP-IT PCR Product Clean-up Reagent (GE Healthcare, Piscataway, NJ USA), and was sequenced bidirectionally on a  3100 ABI Prism Genetic Analyzer (Applied Biosystems). Nucleotide sequence alignment and analyses were performed online using the BLAST program available at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).
Conjugation experiments. Mating experiments were performed using the azide-resistant Escherichia coli strain J53 as recipient cells. In case of transfer failure, plasmid DNA was extracted with the GeneJET Plasmid Miniprep Kit 18 and transferred into E. coli DH10B cells (Invitrogen, Cergy-Pontoise, France). 19 Plasmids ware characterized by plasmid relaxase gene typing and PCR-based replicon typing. 20 Molecular epidemiology typing. To determine the phylogenetic group of the E. coli CP-GNB isolates, the PCR-based method described by Clermont et al. 21 was used. Multilocus sequence typing (MLST) analysis was performed as described at the Institut Pasteur MLST using whole-genome MLST databases (http://bigsdb.pasteur.fr/) for E. coli, K. pneumoniae, Enterobacter cloacae and A. baumannii.
Statistical analysis. CP-GNB frequencies were compared between men and women, and then between inpatients and outpatients using the x 2 test. Statistical analyses were performed using the R version 4.0.2 (R Foundation for Statistical Computing, Vienna, Austria). Differences were considered significant at the 0.05 confidence level.
Among the 14 clinical CP-GNB isolates, 13 were isolated in hospitals in Libreville, and one in Tchibanga. The only CP-GNB isolate from fecal samples (carriage screening) was isolated in Mouila ( Figure 1, Table 2).
Overall, CP-GNB were detected in 1.61% of clinical samples (14 of 869), with six samples from male patients and eight samples from female patients (P 5 0.76), and 13 samples from inpatients and one sample from an outpatient (P 5 0.03) ( Table 3); and in 5.26% of fecal carriage samples (1 of 19).
Molecular characterization of CP-GNB resistance. Table 1 summarizes the different carbapenemases and associated resistance genes, the phylogroup E. coli, and the plasmids carrying the resistance genes detected in the 15 CP-GNB isolates.
The two E. coli isolates (n 5 1 urinary sample and n 5 1 fecal sample) belonged to phylogroup A, and the resistance genes were carried by the same plasmid (IncFIA). These isolates were from two patients (one inpatient and one outpatient) at hospitals in two different cities (Mouila and Tchibanga), 170 km apart.
The bla NDM-5 gene detected in two K. pneumoniae isolates from blood cultures of two patients hospitalized in the intensive care unit of HIAOBO (Libreville) was on the IncX1 plasmid. Eleven carbapenemase genes (73.33%) were identified on plasmids belonging to three different incompatibility groups (IncX1, IncFIA, and IncFIIK).

DISCUSSION
This study found low rates of CP-GNB in Gabon (1.61% for clinical samples and 5.26% for fecal carriage). The low rate of CP-GNB clinical samples is similar to that reported by a study in Chad (2.5%), 4 but is less than in Cameroon (Central Africa; 11.11%) and Senegal (West Africa; 5.1%). 22,23 Conversely, it is more than that in Burkina Faso (West Africa; 0.9%). 24 This low rate in Gabon could be explained by the high cost of carbapenems, which reduces the risk of excessive use, and therefore promotes the appearance of resistance. The emergence of resistance to carbapenems in Gabon could be the consequence of the implementation in 2008 of a universal health insurance (Caisse Nationale d'Assurance Maladie et de Garantie Sociale) that facilitates the prescription and purchase of these antibiotics. In 2016, Moussounda et al. 13 reported the same prevalence (5.1%) of fecal carriage at the HIAOBO military training hospital in Libreville (samples harboring bla OXA-48 and bla NDM-7 ), whereas Sanou et al. 24 found greater prevalence in Burkina Faso (8.2%). Although the number of fecal samples in our study was very low, our results indicate the presence of CP-GNB carriage in Gabon. This is worrying because it potentially increases the risk of nosocomial and community-acquired CP-GNB infections.
Statistical differences were assessed with the x 2 test. commonly used antibiotics for treating infections caused by A. baumannii, and an increase in carbapenem-resistant A. baumannii strains has been reported worldwide during the past decade. 29 In Africa, carbapenem resistance in A. baumannii has never been detected in Gabon, but only in South Africa, Libya, Egypt, Tunisia, Algeria, and Senegal. [30][31][32] Interestingly, one A. baumannii isolate from urine harbored both the bla OXA-23 and bla OXA-58 genes. Their coexpression is rare and was reported first in France in a strain from a urine sample of a patient without previous hospitalization who had traveled to Indonesia. 26 No patient in our study had a history of travel to Indonesia. Moreover, five CP-GNB isolates carried the bla NDM-5 gene, which belongs to group B of the Ambler classification. To our knowledge, this is the first report of its presence in Gabon. NDM-5 is a variant of the NDM carbapenemase that was identified in a multidrug-resistant E. coli ST648 isolate from the perineum and throat of a patient in the United Kingdom with history of hospitalization in India. 33 Since then, NDM-5 has been detected in other countries, such as Egypt, Algeria, Spain, Japan, Australia, the United States, China, and Chad. 4,34 The presence of metallo-beta-lactamases  in Gabon can be explained by the trade intensification during the past 10 years that facilitates contacts with Asian countries (India and China), and consequently the dissemination of carbapenemase producers. 35 The bla OXA-48 gene is most frequently found in K. pneumoniae (six of eight CP K. pneumoniae isolates in our study), and has often been reported in Turkey, the Middle East, North Africa, and Europe. 36 Its detection is less frequent in Central Africa, and was almost nonexistent in Gabon. Similar to NDM-5, this could be the consequence of trade intensification. On the other hand, the lack of systematic detection of carbapenemases could hide the beginning of local spreading.
In our study, many CP-GNB isolates also carried other resistances genes, such as beta-lactamase-encoding genes, PMQR genes, and 16S RNA methylase-encoding genes. In Gabon, as a result of the absence of alternative molecules (e.g., colistin or cefiderocol), these resistant isolates are potentially dangerous because carbapenems are still used as "last-line" treatment of infections caused by resistant GNB, including those producing ESBLs. 37 In addition, one of the CP A. baumannii isolates carried the bla OXA-23 , bla OXA-51 , and armA genes. In 2015, El-Sayed-Ahmed et al. 38 reported the coexistence of bla OXA-23 and armA in A. baumannii isolates in Egypt. This demonstrates that mobile genetic elements (i.e., plasmids) can carry several different genes, thus facilitating the circulation and dissemination of resistance genes among different bacterial species and genera.
The fact that, in two K. pneumoniae isolates from different patients in the same intensive care unit, the bla NDM-5 gene was located on the same IncX1 plasmid supports the hypothesis of a probable nosocomial infection by the nursing staff. 39 It also confirms that plasmids are key genetic elements in the dissemination of antimicrobial drug resistance in bacteria, 40 as suggested by the finding in our study that 73.33% of carbapenemase genes were carried by plasmids.
Among CP-GNB isolates, the sequence type distribution in Enterobacterales shows a majority of ST47 (n 5 6) followed by ST48 (n 5 2) in K. pneumoniae, ST2 in E. coli (n 5 2), and one ST78 in E. cloacae. Among the four A. baumannnii isolates, two belonged to ST2 and two to ST78.
All the OXA-48-producing K. pneumoniae strains belonged to the ST147 clone, and were isolated in the same hospital (the Omar Bongo Ondimba Regional Hospital Center military teaching hospital). The clonal dissemination and outbreak of OXA-48-producing K. pneumoniae were reported in a Chinese hospital in 2016, and K. pneumoniae ST147 was among the two predominant clones associated with the outbreak. 41 In Belgium and Canada, the K. pneumoniae ST147 clone has been associated with ESBL and OXA-48 production. 42 Like E. coli ST410, the K. pneumoniae ST147 clone is considered a high-risk nosocomial clone because of its global distribution, association with various antimicrobial resistance genes, prolonged persistence in the host, efficient transmission among hosts, enhanced fitness and virulence, and capacity to cause severe or recurrent infections. 43 A study in Thailand 44 revealed the presence of ST147 strains in pets. Because this suggests that such strains can be transmitted between humans and companion animals, it is advisable to put in place public health surveillance measures in veterinary hospitals to minimize infection by multidrug-resistant bacteria in pets. Transmission of ST2, ST48, and ST78 clones is responsible of the spread of drug resistance. [45][46][47] A major limitation of our study is the lack of wholegenome sequencing (WGS) data to characterize our isolates. WGS is the best technology for molecular epidemiology studies of antimicrobial resistance and is used to characterize the genetic basis of resistance mechanisms. However, because of its high cost, complexity, and lack of availability in genetic facilities, WGS is not used routinely in low-income countries such as Gabon.

CONCLUSION
Our study highlights the production of carbapenemases by clinical, community, and carriage GNB isolates in Gabon. Good hygiene practice should be encouraged to avoid/reduce the spread of resistance genes within hospitals and to reduce nosocomial infections caused by multiresistant bacteria. Moreover, public authorities should implement a real health policy of prevention.