Prevalence of carbapenemases among Gram-negative bacteria in Tunisia: first report of KPC-2 producing Acinetobacter baumannii.

INTRODUCTION: The rapid evolution of the antibacterial resistance problem worldwide, including the Mediterranean countries, constitutes a real threat to public health. This study aims to characterize carbapenemase encoding genes among Gram-negative bacteria collected from some Tunisian hospitals. METHODOLOGY: Twenty-two clinical carbapenem-resistant Gram-negative bacteria were recovered, and identified by the matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method. Antibiotic resistance was tested by disk diffusion method on Muller-Hinton Agar. The minimum inhibitory concentration (MIC) for imipenem was revealed by the E-test method. Carbapenemase encoding genes were screened by polymerase chain reaction (PCR). Genetic relatedness was performed by the pulsed field gel electrophoresis (PFGE) method. RESULTS: Our isolates, identified as K. pneumoniae (n = 7), P. mirabilis (n = 1), A. baumannii (n = 13), and P. aeruginosa (n = 1), presented high MIC values for imipenem. Enterobacerales were resistant to carbapenems due to OXA-48 production. Only, four K. pneumoniae harbored the blaNDM-1 gene. VIM-2 production was detected in P. aeruginosa. However, OXA-23 production was observed in A. baumannii isolates, one of which co-produced the KPC-2 enzyme that was identified for the first time in Tunisia in this species. A high genetic diversity was demonstrated by pulsed-field gel electrophoresis in K. pneumoniae and A. baumannii after XbaI and ApaI digestion respectively. CONCLUSIONS: Our findings highlight the spread of various unrelated clones of carbapenemase-producers in some Tunisian hospitals as well as the spread of several carbapenemase types.

Epidemiology, Phenotypic and Genotypic Characterization of Carbapenem-Resistant Gram-Negative Bacteria from a Libyan Hospital.

Antimicrobial resistance, particularly resistance to carbapenems, has become one of the major threats to public health. Seventy-two isolates were collected from patients and hospital environment of Ibn Sina Hospital, Sirte, Libya. Antibiotic susceptibility tests, using the disc diffusion method and E-Test strips, were performed to select carbapenem-resistant strains. The colistin (CT) resistance was also tested by determining the minimum inhibitory concentration (MIC). RT-PCR was conducted to identify the presence of carbapenemase encoding genes and plasmid-mediated mcr CT resistance genes. Standard PCR was performed for positive RT-PCR and the chromosome-mediated CT resistance genes (mgrB, pmrA, pmrB, phoP, phoQ). Gram-negative bacteria showed a low susceptibility to carbapenems. Molecular investigations indicated that the metallo-ß-lactamase New Delhi metallo-beta-lactamases-1 was the most prevalent (n = 13), followed by Verona integron-encoded metallo-beta-lactamase (VIM) enzyme (VIM-2 [n = 6], VIM-1 [n = 1], and VIM-4 [n = 1]) that mainly detected among Pseudomonas spp. The oxacillinase enzyme OXA-23 was detected among six Acinetobacter baumannii, and OXA-48 was detected among one Citrobacter freundii and three Klebsiella pneumoniae, in which one coharbored the Klebsiella pneumoniae carbapenemase enzyme and showed resistance to CT (MIC = 64 µg/mL) by modification in pmrB genes. In this study, we report for the first time the emergence of Pseudomonas aeruginosa carrying the blaNDM-1 gene and belonging to sequence type773 in Libya. Our study reported also for the first time CT resistance by mutation in the pmrB gene among Enterobacteriaceae isolates in Libya.

Detection by Whole-Genome Sequencing of a Novel Metallo-ß-Lactamase Produced by Wautersiella falsenii Causing Urinary Tract Infection in Tunisia.

Wautersiella falsenii is a rarely non-fermenting Gram-negative bacterium and belongs to the Flavobacteriaceae family. This nosocomial pathogen can cause several human infections, especially among immunocompromised patients. Here, we describe the whole genome sequence of a clinical W. falsenii strain isolated from a urine sample of a 35-year-old woman with a urinary tract infection in Tunisia. We investigated its phenotype and genotype. After bacterial identification by the MALDI-TOF method, the whole-genome sequencing of this strain was performed. This isolate was not susceptible to various antibiotics, including ß-lactams, aminoglycosides, and quinolones. However, it remains susceptible to imipenem (MIC = 0.25 mg/l), ertapenem (MIC = 0.75 mg/l), and meropenem (MIC = 0.19 mg/l). Interestingly, the E-TEST® (MP/MPI) showed a reduced MIC of meropenem +/- EDTA (0.064 µg/ml). Besides, the color change from yellow to red in the ß CARBA test only after 24 hours of incubation can be interpreted in two ways. On the one hand, as a likely low expression of the gene encoding metallo-ß-lactamase. On the other hand, and more likely, it may be a false-positive result because, according to the test manufacturer's recommendations, the test should be read after 30 minutes. Perhaps, therefore, this gene is not expressed in the tested strain. Moreover, the whole-genome sequence analysis demonstrated the presence of a novel chromosomally located subclass B1 metallo-ß-lactamase EBR-like enzyme, sharing 94.92% amino acid identity with a previously described carbapenemase produced by Empedobacter brevis, EBR-1. The results also showed the detection of other antibiotic resistance genes and the absence of plasmids. So far, this study is the first report on the detection of W. falsenii in Tunisia. These findings prove that W. falsenii could be a potential reservoir of antibiotic resistance genes, e.g., ß-lactamases. Collaborative efforts and effective hygiene measures should be established to prevent the emergence of this species in our health care settings.

Polymyxin E-Resistant Gram-Negative Bacteria in Tunisia and Neighboring Countries: Are There Commonalities?

The current global dissemination of polymyxin E resistance constitutes a real public health threat because of the restricted therapeutic options. This review provides a comprehensive assessment of the epidemiology of polymyxin E-resistant bacteria, with special reference to colistin-resistant Gram-negative bacteria in Tunisia and neighboring countries, based on available published data to January 2020. We aimed to determine their prevalence by species and origin, shedding light on the different genes involved and illustrating their genetic support, genetic environment, and geographic distribution. We found that colistin resistance varies considerably among countries. A majority of the research has focused on Algeria (13 of 32), followed by Tunisia (nine of 32), Egypt (nine of 32), and Libya (one of 32). All these reports showed that colistin-resistant Gram-negative bacteria were dramatically disseminated in these countries, as well as in African wildlife. Moreover, high prevalence of these isolates was recorded from various sources (humans, animals, food products, and natural environments). Colistin resistance was mainly reported among Enterobacteriaceae, particularly Klebsiella pneumoniae and Escherichia coli. It was associated with chromosomal mutations and plasmid-mediated genes (mcr). Four mcr variants (mcr1, mcr2, mcr3, and mcr8), mobilized by several plasmid types (IncHI2, IncP, IncFIB, and IncI2), were detected in these countries and were responsible for their rapid spread. Countrywide dissemination of high-risk clones was also observed, including E. coli ST10 and K. pneumoniae ST101 and ST11. Intensified efforts to raise awareness of antibiotic use and legalization thereon are required in order to monitor and minimize the spread of multidrug-resistant bacteria.

Occurrence of NDM-1 and VIM-2 Co-Producing Escherichia coli and OprD Alteration in Pseudomonas aeruginosa Isolated from Hospital Environment Samples in Northwestern Tunisia.

Hospital environments constitute the main reservoir of multidrug-resistant bacteria. In this study we aimed to investigate the presence of Gram-negative bacteria in one Northwestern Tunisian hospital environment, and characterize the genes involved in bacterial resistance. A total of 152 environmental isolates were collected from various surfaces and isolated using MacConkey medium supplemented with cefotaxime or imipenem, with 81 fermenter bacteria (27 Escherichia coli, and 54 Enterobacter spp., including 46 Enterobacter cloacae), and 71 non-fermenting bacteria (69 Pseudomonas spp., including 54 Pseudomonas aeruginosa, and 2 Stenotrophomonas maltophilia) being identified by the MALDI-TOF-MS method. Antibiotic susceptibility testing was performed by disk diffusion method and E-Test was used to determine MICs for imipenem. Several genes implicated in beta-lactams resistance were characterized by PCR and sequencing. Carbapenem resistance was detected among 12 isolates; nine E. coli (blaNDM-1 (n = 8); blaNDM-1 + blaVIM-2 (n = 1)) and three P. aeruginosa were carbapenem-resistant by loss of OprD porin. The whole-genome sequencing of P. aeruginosa 97H was determined using Illumina MiSeq sequencer, typed ST285, and harbored blaOXA-494. Other genes were also detected, notably blaTEM (n = 23), blaCTX-M-1 (n = 10) and blaCTX-M-9 (n = 6). These new epidemiological data imposed new surveillance strategies and strict hygiene rules to decrease the spread of multidrug-resistant bacteria in this area.

First Description of Various Bacteria Resistant to Heavy Metals and Antibiotics Isolated from Polluted Sites in Tunisia.

Environmental bacteria belonging to various families were isolated from polluted water collected from ten different sites in Tunisia. Sites were chosen near industrial and urban areas known for their high degree of pollution. The aim of this study was to investigate cross-resistance between heavy metals (HM), i.e., silver, mercury and copper (Ag, Hg, and Cu), and antibiotics. In an initial screening, 80 isolates were selected on ampicillin, and 39 isolates, retained for further analysis, could grow on a Tris-buffered mineral medium with gluconate as carbon source. Isolates were identified based on their 16S rRNA gene sequence. Results showed the prevalence of antibiotic resistance genes, especially all isolates harbored the bla TEM gene. Some of them (15.38%) harbored bla SHV. Moreover, several were even ESBLs and MBLs-producers, which can threaten the human health. On the other hand, 92.30%, 56.41%, and 51.28% of the isolates harbored the heavy metals resistance genes silE, cusA, and merA, respectively. These genes confer resistance to silver, copper, and mercury. A cross-resistance between antibiotics and heavy metals was detected in 97.43% of our isolates.

High Carbapenem Resistance Caused by VIM and NDM Enzymes and OprD Alteration in Nonfermenter Bacteria Isolated from a Libyan Hospital.

Acinetobacter baumannii and Pseudomonas aeruginosa are among the most prevalent pathogens causing a wide range of serious infections in hospitalized patients and contaminating intensive care units and inanimate surfaces. The purpose of this study was to investigate the mechanism of carbapenem resistance in clinical and hospital environmental isolates of A. baumannii and P. aeruginosa recovered from a Libyan hospital. From a total of 82 Gram-negative bacteria, 8 isolates of A. baumannii and 3 isolates of P. aeruginosa exhibited resistance to imipenem with minimum inhibitory concentrations ranging from 16 to >32 µg/mL. Five isolates of A. baumannii harbored blaOXA-23 gene, from which three isolates were collected from patients and two from hospital environment. Only one isolate harbored blaNDM-1 gene, which was responsible for carbapenem resistance in A. baumannii. The OprD gene seems to be disturbed by an insertion sequence (IS) in two isolates and affected by polymorphism in one isolate. Pulsed-field gel electrophoresis results showed high genetic diversity among carbapenemase producing A. baumannii. This study highlights the dissemination of blaOXA-23 and blaNDM-1 genes in a Libyan setting. Therefore, infection prevention and control practices, antimicrobial stewardship initiatives, and antimicrobial resistance surveillance systems should be implemented to prevent the wide spread of antimicrobial resistance.

Characterization of O25b-ST131 Escherichia coli Clone Producing CTX-M-15, DHA-4, and CMY-42 in Urinary Tract Infections in a Tunisian Island.

The dissemination of extended-spectrum ß-lactamases encoding genes in Escherichia coli, especially in the uropathogenic O25b-ST131 E. coli clone, constitutes a real concern. We aimed to identify the molecular mechanisms of resistance to cephalosporins among E. coli clinical isolates and to estimate the prevalence of the uropathogenic O25b-ST131 clone in our study. Forty-two cephalosporin-resistant E. coli implicated in urinary tract infections were collected from the Regional Hospital of a southeastern Tunisian Island from April 2015 to August 2016. Molecular screening of ß-lactamases encoding genes by PCR and sequencing showed that the majority of our isolates harbored blaCTX-M gene (blaCTX-M-15 [n = 36], blaCTX-M-14 [n = 2]). Nevertheless, the blaSHV, blaTEM, and blaOXA-1 genes were not detected. Various class C ß-lactamases encoding genes were observed in association or not with blaCTX-M genes and were as follows: blaampC (n = 14), blaCMY-42 (n = 7), blaCMY-2 (n = 1), and blaDHA-4 (n = 1). The research of O25b-ST131 clone was carried out by duplex PCR (pabB and trpA genes) and revealed that most of our isolates (n = 30) belonged to this clone. We also noted that the majority of our isolates belonged to the B2 phylogenetic group (n = 32), five isolates to the B1 phylogenetic group, three isolates to the D phylogenetic group, and only two isolates belonged to the A phylogenetic group. Our study provides new epidemiological information about E. coli clinical isolates in this area. Indeed, this is the first report of CTX-M-14 producing O25b-ST131 E. coli in our country and the first report of DHA-4 and CMY-42 producing E. coli in Tunisia.

Carbapenemase Producing Gram-Negative Bacteria in Tunisia: History of Thirteen Years of Challenge.

The wide spread of multidrug-resistant bacteria, particularly carbapenem-resistant Gram-negative bacteria (CR-GNB), constitutes a major public health threat worldwide, owing to the limited therapeutic options. This review will describe and uncover the Tunisian experience in the challenge against carbapenem resistance. Indeed, we illuminate on the dissemination of CR-GNB in different hospitals, animals, and other natural environments in this country. We resumed the different carbapenemase variants detected from various bacterial species and mapped their regional distribution, basing on Tunisian published data during a period extended from 2006, the date of its first description in Tunisia, to February 2019. We also resumed the different mobile genetic elements implicated in their dissemination. This review shows that the majority of the research reports focused in the north and the coastal cities in spite of the fact that KPC and IMP carbapenemases were uncommonly detected in our country. However, VIM, NDM-1, and OXA-48 enzymes were usually reported with the predominance of OXA-48 among Enterobacteriaceae. Furthermore, OXA-23, OXA-51, and OXA-58 carbapenemases constituted the main mechanism conferring carbapenem resistance among Acinetobacter baumannii in Tunisia. Collaborative efforts and raising awareness of the threat of antibiotic resistance are required in order to minimize the spread of multidrug-resistant bacteria.

First Report of SHV-148-Type ESBL and CMY-42-Type AmpC ß-Lactamase in Klebsiella pneumoniae Clinical Isolates in Tunisia.

Extended-spectrum beta-lactamase producing Enterobacteriaceae present a real problem worldwide. We aimed to investigate the molecular mechanisms of resistance to antibiotics among Klebsiella pneumoniae clinical isolates collected from a Hospital in the southeast of Tunisia. Eighteen cephalosporin-resistant K. pneumoniae were recovered between April 2015 and August 2016. Molecular characterization of antimicrobial resistance encoding genes was performed by PCR and sequencing. Results revealed several types of Ambler class A ß-lactamase encoding genes among our isolates: [blaCTXM-15 (15), blaSHV-28 (6), blaSHV-1 (2), blaSHV-148 (1), blaSHV-61 (1), blaSHV-76 (1), blaSHV-186 (1), blaTEM-1 (8)]. The association of blaOXA-1 was observed in nine isolates. However, the class C ß-lactamase encoding genes were detected in four isolates [blaCMY-4 (2), blaCMY-42 (1), blaACT-35 (1)]. Molecular typing of K. pneumoniae isolates by pulsed-field gel electrophoresis showed 16 unrelated pulsotypes proving a high diversity among our isolates. Our study provides new epidemiological information showing a huge diversity of ß-lactamase encoding genes among our isolates. In fact, this is the first report of SHV-76, SHV-148, and SHV-186 in Tunisia. This is also the first report of CMY-42 and ACT-35 producing K. pneumoniae in our country.

Metallo-ß-lactamases and class D carbapenemases in south-east Tunisia: Implication of mobile genetic elements in their dissemination.

Carbapenem resistance in Gram-negative bacteria constitutes a major clinical problem. We characterized molecular features among carbapenem-resistant Gram-negative clinical isolates collected from Southeastern Tunisian Island Hospital. Eighteen carbapenem-resistant clinical isolates (13 Klebsiella pneumoniae, 1 Proteus mirabilis, 1 Enterobacter cloacae, 3 Acinetobacter baumannii) were recovered during April 2015-August 2016. Molecular characterization of antimicrobial resistance was performed using polymerase chain reaction (PCR) and sequencing. Molecular typing of carbapenemase-producing K. pneumoniae was performed by pulsed-field gel electrophoresis (PFGE) after XbaI digestion and multilocus sequence typing (MLST). Conjugation experiments were conducted and type/number/size of plasmids were characterized by PCR-Based-Replicon-Typing and PFGE after S1 digestion. Carbapenemase genes were detected in K. pneumoniae [blaNDM-1(8), blaNDM-1+blaOXA-48(1), blaOXA-48(4)], P. mirabilis [blaOXA-48(1)], E. cloacae [blaVIM-2(1)] and A. baumannii [blaOXA-23(3)]. K. pneumoniae isolates were typed as ST15, ST1412 and ST147 and showed seven different pulsotypes. The genetic structure surrounding blaNDM-1 was composed of ISAba125 and ble. The blaVIM-2 carried by E. cloacae was located within the variable region of a class1 integron and blaOXA-48 gene was inserted into Tn1999.2. IncA/C and IncFIIA replicons were implicated in dissemination of blaNDM-1 and a non-typeable 48.5 kb plasmid in the propagation of blaOXA-48. The emergence of carbapenemase-producing Gram-negative species in a Tunisian hospital shows the need for preventive strategies and hygiene measures to minimize their spread. Although conjugative plasmids play an important role in rapid carbapenemase genes dissemination, other mobile genetic elements, such as insertion sequences, transposons and integrons, are involved in acquisition of these resistances.

Association of HLA-G +3142 C>G polymorphism and breast cancer in Tunisian population.

HLA-G is highly expressed in cancer. Also, it is associated to its progression. Here, we explored the relationship between two HLA-G polymorphisms with breast cancer (BC) and tried to make a correlation with sHLA-G levels. We genotyped 104 patients with BC and 83 controls (CTRL) for HLA-G 14-bp insertion/deletion (Ins/Del) and HLA-G +3142 C>G polymorphisms. The mutations were identified with PCR and PCR-RFLP. The sHLA-G dosage was performed on plasma samples by a specific ELISA. A significant association with BC was found concerning the G allele in the +3142 C>G polymorphism (p = 0.0004). The G/G genotype is the protective genotype (1 % in BC patients vs. 13.1 % in CTRL, OR 0.065, 95 % CI 0.008-0.523). No statistically significant differences were observed for the 14-bp Ins/Del polymorphism between BC patients and controls frequencies. The protection by G/G genotype of +3142 C>G polymorphism is maintained in young patients (<50 years, p = 0.0006) and in early-diagnosed BC patients (<50 years, p = 0.0033). In addition, an association was found between the haplotypes inferred by both HLA-G polymorphisms and BC susceptibility. Indeed, the (DelG) haplotype is found as the protective haplotype against BC (OR 0.269, 95 % CI 0.081-0.895, p = 0.023). The ELISA dosage of sHLA-G revealed increased levels in BC compared to CTRL (p < 0.0001). We demonstrated also that sHLA-G is closely associated with advanced stages of BC without significance. sHLA-G is increased in TNM IV and SBR III subgroups. It is also enhanced in patients with a tumor size over 20 mm and in triple-negative patients. Taken together, our findings demonstrate, for the first time, the association of HLA-G +3142 C>G polymorphism with BC susceptibility in Tunisian population. Our results revealed also a potential implication of sHLA-G in advanced stages of BC.