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.

Ability of marine-derived fungi isolated from polluted saline environment for enzymatic hydrocarbon remediation.

Marine-derived fungi have attracted much attention due to their ability to present a new biosynthetic diversity. About 50 fungal isolates were obtained from Tunisian Mediterranean seawater and then screened for the presence of lignin-peroxidase (LiP), manganese-dependent peroxidase (MnP), and laccase (Lac) activities. The results obtained from both qualitative and quantitative assays showed that four of marine fungi isolates had a high potential to produce lignin-degrading enzymes. They were characterized taxonomically by a molecular method, based on international spacer (ITS) rDNA sequence analysis, as Chaetomium jodhpurense (MH667651.1), Chaetomium maderasense (MH665977.1), Paraconiothyrium variabile (MH667653.1), and Phoma betae (MH667655.1) which have been reported as producers of ligninolytic enzyme in the literature. The enzymatic activities and culture conditions were optimized using a Fractional Factorial design (2 7- 4). Then, fungal strains were incubated with the addition of 1% of crude oil in 50% of seawater for 25 days to evaluate their abilities to simultaneously degrade hydrocarbon compounds and to produce ligninolytic enzymes. The strain P. variabile exhibited the highest crude oil degradation rate (48.3%). Significant production of ligninolytic enzymes was recorded during the degradation process, which reached 2730 U/L for the MnP, 410 U/L for LiP, and 168.5 U/L for Lac. The FTIR and GC-MS analysis confirmed that the isolates rapidly biodegrade crude oil under ecological and economic conditions.

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.

Isolation of Carbapenem and Colistin Resistant Gram-Negative Bacteria Colonizing Immunocompromised SARS-CoV-2 Patients Admitted to Some Libyan Hospitals.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a devastating effect, globally. We describe, for the first time, the occurrence of carbapenem-resistant bacteria colonizing SARS-CoV-2 patients who developed hospital-associated infections with carbapenemase-producing, Gram-negative bacteria at some isolation centers of SARS-CoV-2 in the eastern part of Libya. In total, at first, 109 samples were collected from 43 patients, with the samples being recovered from oral (n = 35), nasal (n = 45), and rectal (n = 29) cavities. Strain identification was performed via matrix assisted laser desorption ionization-time of flight (MALDI-TOF). Antibiotic susceptibility testing was carried out on Mueller-Hinton agar, using the standard disk diffusion method. MIC determination was confirmed via E-TEST and microdilution standard methods. A molecular study was carried out to characterize the carbapenem and colistin resistance in Gram-negative bacterial strains. All of the positive results were confirmed via sequencing. Klebsiella pneumoniae (n = 32), Citrobacter freundii (n = 21), Escherichia coli (n = 7), and Acinetobacter baumannii (n = 21) were the predominant isolated bacteria. Gram-negative isolates were multidrug-resistant and carried different carbapenem resistance-associated genes, including NDM-1 (56/119; 47.05%), OXA-48 (15/119; 12.60%), OXA-23 (19/119; 15.96%), VIM (10/119; 8.40%), and the colistin resistance mobile gene mcr-1 (4/119; 3.36%). The overuse of antimicrobials, particularly carbapenem antibiotics, during the SARS-CoV-2 pandemic has led to the emergence of multidrug-resistant bacteria, mainly K. pneumoniae, A. baumannii, and colistin-resistant E. coli strains. Increased surveillance as well as the rational use of carbapenem antibiotics and, recently, colistin are required to reduce the propagation of multidrug-resistant strains and to optimally maintain the efficacy of these antibiotics. IMPORTANCE In this work, we describe, for the first time, the occurrence of carbapenem-resistant bacteria colonizing COVID-19 patients who developed hospital-associated infections with carbapenemase-producing, Gram-negative bacteria at some isolation centers of COVID-19 in the eastern part of Libya. Our results confirmed that the overuse of antimicrobials, such as carbapenem antibiotics, during the COVID-19 pandemic has led to the emergence of multidrug-resistant bacteria, mainly K. pneumoniae and A. baumannii, as well as colistin resistance.

Simultaneous Heavy Metal-Polycyclic Aromatic Hydrocarbon Removal by Native Tunisian Fungal Species.

Multi-contamination by organic pollutants and toxic metals is common in anthropogenic and industrial environments. In this study, the five fungal strains Chaetomium jodhpurense (MH667651.1), Chaetomium maderasense (MH665977.1), Paraconiothyrium variabile (MH667653.1), Emmia lacerata, and Phoma betae (MH667655.1), previously isolated in Tunisia, were investigated for the simultaneous removal and detoxification of phenanthrene (PHE) and benzo[a]anthracene (BAA), as well as heavy metals (HMs) (Cu, Zn, Pb and Ag) in Kirk's media. The removal was analysed using HPLC, ultra-high performance liquid chromatography (UHPLC) coupled to a QToF mass spectrometer, transmission electron microscopy, and toxicology was assessed using phytotoxicity (Lepidium sativum seeds) and Microtox® (Allivibrio fisherii) assays. The PHE and BAA degradation rates, in free HMs cultures, reached 78.8% and 70.7%, respectively. However, the addition of HMs considerably affected the BAA degradation rate. The highest degradation rates were associated with the significant production of manganese-peroxidase, lignin peroxidase, and unspecific peroxygenase. The Zn and Cu removal efficacy was considerably higher with live cells than dead cells. Transmission electron microscopy confirmed the involvement of both bioaccumulation and biosorption processes in fungal HM removal. The environmental toxicological assays proved that simultaneous PAH and HM removal was accompanied by detoxification. The metabolites produced during co-treatment were not toxic for plant tissues, and the acute toxicity was reduced. The obtained results indicate that the tested fungi can be applied in the remediation of sites simultaneously contaminated with PAHs and HMs.

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.

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.

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.

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.

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.

Incidence of OXA-23 and OXA-58 Carbapenemases Coexpressed in Clinical Isolates of Acinetobacter baumannii in Tunisia.

Acinetobacter baumannii is an important opportunistic and multidrug-resistant pathogen responsible for nosocomial infections in health facilities. The aim of this study was to characterize the molecular mechanisms of carbapenem resistance in A. baumannii isolates isolated from Mohamed Kassab Orthopedic Institute in Tunis, Tunisia. Twenty-five imipenem-resistant A. baumannii clinical isolates collected between 2013 and 2016 were identified using API 20NE and were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Carbapenemase activity was detected using microbiological tests and PCR. The epidemiological relatedness of the isolates was studied using multilocus sequence typing (MLST). The isolates were resistant to all antibiotics tested with increased minimum inhibitory concentration values (>32 mg/L). The microbiological tests showed that the 25 A. baumannii were positive for modified Hodge test and for the Carba NP test; however, ß-lactamase activity was not inhibited by EDTA. All the isolates harbored the naturally occurring blaOXA-51-like gene and the blaOXA-23-like carbapenemase gene. Among these isolates, one isolate coexpressed the blaOXA-58 gene. MLST revealed several sequence types (STs) with the predominance of ST2 imipenem-resistant A. baumannii (14/25; 56%). In this study we report the prevalence of ST2 imipenem resistance and for the first time the coexpression of blaOXA-23 and blaOXA-58 in clinical isolates of A. baumannii in a Tunisian hospital.

Prevalence of a New Variant OXA-204 and OXA-48 Carbapenemases Plasmids Encoded in Klebsiella pneumoniae Clinical Isolates in Tunisia.

Carbapenemase-producing Klebsiella pneumoniae strains have emerged as a major problem for healthcare systems. The aim of this study was to determine the role and diversity of plasmids harboring carbapenemases encoding genes from a collection of K. pneumoniae isolates recovered between July 2011 and January 2012, with decreased susceptibility to carbapenems. Imipenem (IPM), ertapenem (ETP), meropenem (MEM), and doripenem (DOR) minimum inhibitory concentrations (MICs) were determined by E-test. Carbapenemase production was detected with the modified Hodge test. ß-Lactamases encoding genes were amplified by PCR and sequenced. Plasmid incompatibility groups harbored by carbapenemases producers were investigated using the PCR-based replicon typing method and the clonal relationship of the isolates was investigated by pulse filed electrophoresis. IMP, ertapenem, meropenem, and doripenem MICs ranged between 0.25 and 16 mg/L. Carbapenemase activity was detected in 14 isolates. Two carbapenemases were identified: OXA-48 in 13 isolates and a new variant OXA-204 in 1 isolate, in combination with extended-spectrum ß-lactamases, CTX-M-1, CTX-M-9, CTX-M-14, CTX-M-15, and VEB-8. One isolate produced CMY-2. OXA-48 and the new variant OXA-204 were confirmed as transferable plasmid encoded. The carbapenemase-producing K. pneumoniae harbored plasmids of the A/C, LVPK, and L/M replicon types. Thirteen different pulso types were observed. Three pairs of isolates showed a clonal relatedness. This diversity in ß-lactamases, in pulso types and in plasmid content, shows the ability of OXA-type carbapenemase to disseminate. This is worrying for the control of the increase in antibiotic resistance frequency and necessitates that continuous investigations in the clinical setting remain a high priority to clarify the contribution of antimicrobial use into multiresistance bacterial dissemination.

Co-Occurrence of Plasmid-Mediated AmpC ß-Lactamase Activity Among Klebsiella pneumoniae and Escherichia Coli.

INTRODUCTION: Extended-spectrum ß-lactamases (ESBLs), including the AmpC type, are important mechanisms of resistance among Klebsiella pneumoniae and Escherichia coli isolates. OBJECTIVE: The aim of the study was to investigate the occurrence of AmpC-type ß-lactamase producers isolated from two hospitals in Tripoli, Libya. METHODS: All clinical isolates (76 K. pneumoniae and 75 E. coli) collected over two years (2013-2014) were evaluated for susceptibility to a panel of antimicrobials and were analyzed phenotypically for the ESBL and AmpC phenotype using E-test and ESBL and AmpC screen disc test. Both ESBL and AmpC-positive isolates were then screened for the presence of genes encoding plasmid-mediated AmpC ß-lactamases by polymerase chain reaction (PCR). RESULTS: Of the K. pneumoniae and E. coli tested, 75% and 16% were resistant to gentamicin, 74% and 1.3% to imipenem, 71% and 12% to cefoxitin, 80% and 12% to cefepime, 69% and 22.6% to ciprofloxacin, respectively. None of the E. coli isolates were multidrug resistant compared with K. pneumoniae (65.8%). K. pneumoniae ESBL producers were significantly higher (85.5%) compared with (17.3%) E. coli isolates (P <0.0001, OR=4.93). Plasmid-mediated AmpC genes were detected in 7.9% of K. pneumoniae, and 4% E. coli isolates. There was low agreement between phenotypic and genotypic methods, phenotypic testing underestimated detection of AmpC enzyme and did not correlate well with molecular results. The gene encoding CMY enzyme was the most prevalent (66.6%) of AmpC positive isolates followed by MOX, DHA and EBC. Only one AmpC gene was detected in 5/9 isolates, i.e, blaCMY (n=3), blaMOX (n=1), blaDHA (n=1). However, co-occurrence of AmpC genes were evident in 3/9 isolates with the following distribution: blaCMY and blaEBC (n=1), and blaCMY and blaMOX (n=2). Neither blaFOX nor blaACC was detected in all tested isolates. All AmpC positive strains were resistant to cefoxitin and isolated from patients admitted to intensive care units. CONCLUSION: Further studies are needed for detection of other AmpC variant enzyme production among such isolates. Continued surveillance and judicious antibiotic usage together with the implementation of efficient infection control measures are absolutely required.