Basma Mnif

Emergence of Multidrug-Resistant Klebsiella pneumoniae Isolates Producing VIM-4 Metallo-β-Lactamase, CTX-M-15 Extended-Spectrum β-Lactamase, and CMY-4 AmpC β-Lactamase in a Tunisian University Hospital

ABSTRACT Klebsiella pneumoniae clinical isolates resistant to carbapenems were recovered from 11 patients in the hospital of Sfax, Tunisia. The isolates were closely related as shown by pulsed-field gel electrophoresis, and they produced VIM-4 metallo-enzyme, CTX-M-15 extended-spectrum β-lactamase, and CMY-4 AmpC enzyme. The blaVIM-4 gene is part of a class 1 integron.

Molecular characterization of addiction systems of plasmids encoding extended-spectrum β-lactamases in Escherichia coli

OBJECTIVES Escherichia coli producing CTX-M-type extended-spectrum beta-lactamases (ESBLs) are spreading worldwide. The aim of this work was to investigate the addiction systems carried by the replicons involved in the emergence and spread of ESBLs in relation to ESBL and replicon types. METHODS A collection of 125 TEM, SHV and CTX-M ESBL-producing E. coli isolates and their 125 transconjugants or transformants was analysed. Five plasmid protein antitoxin-regulated systems and three plasmid antisense RNA-regulated systems were sought by PCR. RESULTS Two hundred and ninety-eight plasmid addiction systems were detected in the parental strains (mean 2.38, range 0-6 per strain) and 86 were detected in the recipient strains (mean 0.69, range 0-5 per strain). PemKI, CcdAB, Hok-Sok and VagCD were the most frequently represented systems in both recipient and parental strains. The parental SHV and CTX-M ESBL-producing strains had more addiction systems than the TEM ESBL producers. In the recipient strains, the frequency of addiction systems was significantly higher in IncF plasmids. Among the IncF replicons carrying CTX-M-type enzymes, the frequency of addiction systems was significantly higher in IncF plasmids carrying CTX-M-15 (mean 3.5) or CTX-M-9 (mean 4) than in those carrying CTX-M-14 (mean 0.6). CONCLUSIONS In E. coli producing CTX-M-15 or CTX-M-9 ESBLs, plasmids bearing the bla(CTX-M) gene have multiple addiction systems that could contribute to their maintenance in host strains.

Spread of Klebsiella pneumoniae isolates producing OXA-48 β-lactamase in a Tunisian university hospital

Sir, The emergence and dissemination of Klebsiella pneumoniae isolates harbouring carbapenemases is a serious problem. Since the initial report of the OXA-48 enzyme in a K. pneumoniae isolate from Turkey in 2001, OXA-48 producers have been reported in many countries of the world. – 4 Current reports indicate that OXA-48 producers are widespread, mostly from Mediterranean countries as well as other countries in Europe. – 4 In North Africa, OXA-48 producers have been identified in Morocco and Tunisia. The outbreaks of OXA-48-producing K. pneumoniae isolates have been described in several cities in Turkey, once in the UK and recently in France. In the present report, we describe the spread of OXA-48 associated with CMY-4and CTX-M-14-producing K. pneumoniae clinical isolates in Sfax University Hospital. During a 6 month period (October 2009–March 2010), 153 clinical isolates of K. pneumoniae with reduced susceptibility to extended-spectrum cephalosporins and/or imipenem were recovered in Sfax University Hospital. Among these isolates, 21 (13.7%) produced the blaOXA-48 gene. These isolates were recovered from patients in eight different wards. The antibiogram determined by the disc diffusion method and MICs determined by agar dilution and interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) revealed that all isolates were resistant to ticarcillin (MICs.2048 mg/L) (Table 1). All but one isolate were resistant to extended-spectrum cephalosporins, including cefotaxime (MICs 4–1024 mg/L), ceftazidime (MICs 0.5–256 mg/L) and cefepime (MICs 2–64 mg/L). All isolates but one (Kp11) were susceptible to imipenem (MIC901⁄42 mg/L; MIC range1⁄40.5–8 mg/L). This isolate, Kp11, was intermediate to imipenem but susceptible to extended-spectrum cephalosporins. However, all isolates were resistant to ertapenem (MIC901⁄48 mg/L; MIC range1⁄42–32 mg/L). The b-lactamase genes detected by PCR as described previously and sequencing in the 21 OXA-48-positive K. pneumoniae isolates were as follows: blaOXA-48 (1 isolate); blaOXA-48+blaCMY-4+ blaCTX-M-14 (14 isolates); blaOXA-48+blaCMY-4+blaCTX-M-14+blaOXA-1 (3 isolates); blaOXA-48+blaCMY-4+blaCTX-M-14+blaTEM-1 (1 isolate); blaOXA-48+blaCTX-M-14+blaOXA-1 (1 isolate); and blaOXA-48+blaCMY-4+blaCTX-M-15 (1 isolate). Transferability of the blaOXA-48 gene to Escherichia coli J53 was observed in 20 OXA-48-producing isolates. However, conjugation and electroporation experiments failed for Kp4, suggesting that in this isolate the blaOXA-48 gene might be chromosomally located. However, the blaOXA-48 gene was shown to be mostly plasmid-borne and associated with insertion sequence IS1999 but not integrons. Using a series of PCR primers, two IS1999 insertion sequences were found surrounding the blaOXA-48 gene in all our isolates, as found in the prototype OXA-48-positive K. pneumoniae 11978 isolate from Turkey. Molecular analysis of E. coli transconjugants showed that the blaOXA-48 and blaCMY-4 genes were detected on the same plasmid, explaining the resistance to b-lactams of the K. pneumoniae isolates and their transconjugants. Plasmid analysis showed Research letters

Comparison of PFGE and multilocus sequence typing for analysis of Klebsiella pneumoniae isolates.

Klebsiella pneumoniae represents an important nosocomial pathogen causing urinary, respiratory and blood infections (Brisse et al., 2006; Podschun & Ullmann, 1998). Hospital outbreaks due to K. pneumoniae are frequent and especially feared when caused by multidrug-resistant strains, such as extended-spectrum blactamase producers (Paterson & Bonomo, 2005). DNA-based strain typing methods are used to distinguish K. pneumoniae clinical isolates in order to understand transmission patterns and to help management of hospital infections. Molecular serotyping, based on PCR– RFLP of the cps operon responsible for capsular polysaccharide expression, has a higher discriminatory ability than traditional K typing (Brisse et al., 2004), and ribotyping is also applicable to K. pneumoniae (Brisse & Verhoef, 2001). Nevertheless, the most commonly used method is PFGE analysis of macrorestriction fragments (Arlet et al., 1994). The main advantage of PFGE lies in its high discriminatory power (Hansen et al., 2002), but PFGE is technically demanding and requires a high level of coordination (e.g. http://www.cdc.gov/ pulsenet) to achieve inter-laboratory reproducibility. In contrast, multilocus sequence typing (MLST) provides unambiguous data that are suitable for global epidemiology and evolutionary studies (Maiden et al., 1998). A MLST method was previously developed for K. pneumoniae, and analysis of nosocomial isolates showed that MLST can discriminate among epidemiologically unrelated isolates (Diancourt et al., 2005). However, the discriminatory power of MLST was not compared to that of PFGE. In our previous study (Diancourt et al., 2005), 28 isolates belonged to 11 groups that were not distinguished by MLST nor by ribotyping. Among these 11 groups, 5 comprised isolates from distinct countries or separated by large sampling times. For these apparently unrelated cases the isolates could be suspected as being genotypically undistinguishable due to an insufficient discriminatory power of MLST and ribotyping, rather than due to an undocumented epidemiological link. We report here on the comparison of the discriminatory power of PFGE with previously reported methods.

Molecular epidemiology of extended-spectrum beta-lactamase-producing Escherichia coli in Tunisia and characterization of their virulence factors and plasmid addiction systems

BackgroundExtended-spectrum β-lactamases (ESBLs), particularly CTX-M- type ESBLs, are among the most important resistance determinants spreading worldwide in Enterobacteriaceae. The aim of this study was to characterize a collection of 163 ESBL-producing Escherichia coli collected in Tunisia, their ESBL-encoding plasmids and plasmid associated addiction systems.ResultsThe collection comprised 163 ESBL producers collected from two university hospitals of Sfax between 1989 and 2009. 118 isolates harbored blaCTX-M gene (101 blaCTX-M-15 gene and 17 blaCTX-M-14 gene). 49 isolates carried blaSHV-12 gene, 9 blaSHV-2a gene and only 3 blaTEM-26 gene. 16 isolates produced both CTX-M and SHV-12. The 101 CTX-M-15-producing isolates were significantly associated to phylogroup B2 and exhibiting a high number of virulence factors. 24 (23.7%) of the group B2 isolates belonged to clonal complex ST131. Pulsed-field gel electrophoresis (PFGE) typing revealed a genetic diversity of the isolates. 144 ESBL determinants were transferable mostly by conjugation. The majority of plasmid carrying blaCTX-M-15 genes (72/88) were assigned to various single replicon or multireplicon IncF types and had significantly a higher frequency of addiction systems, notably the VagCD module.ConclusionThis study demonstrates that the dissemination of CTX-M-15 producing E. coli in our setting was due to the spread of various IncF-type plasmids harboring multiple addiction systems, into related clones with high frequency of virulence determinants.

Endocarditis Due to Kytococcus schroeteri: Case Report and Review of the Literature

ABSTRACT We report the third case of endocarditis caused by the newly described micrococcal species Kytococcus schroeteri. A 49-year-old woman was admitted to the hospital with suspected prosthetic valve endocarditis. Five blood cultures and prosthetic valve cultures grew the same type of organism, initially identified as Micrococcus sp. Assignment to the genus Kytococcus was suggested by the arginine dihydrolase activity and resistance to oxacillin. After sequencing of the 16S rRNA genes, the isolate was recognized as K. schroeteri. The patient was treated first with vancomycin combined with gentamicin and later with pristinamycin and rifampin. Three cases of K. schroeteri endocarditis described within a short period of time might indicate a specific pathogenicity of this new species. The isolation of kytococci from normally sterile sites should not be overlooked.

Diversity of β-Lactamases in Pseudomonas aeruginosa Isolates Producing Metallo-β-Lactamase in Two Tunisian Hospitals

This study was conducted to identify the β-lactamase content of 30 metallo-β-lactamase-producing Pseudomonas aeruginosa isolated in 2007 from two Tunisian hospitals and to investigate their genetic relatedness. All these isolates produced VIM-2. bla(PER-1), bla(PSE-1), bla(OXA-2), and bla(OXA-10) were identified in 17, 5, 21, and 1 isolates, respectively. These enzymes were often associated in the same isolate: 26 isolates had at least two β-lactamases. The predominant serotype was O12. Pulsed-field gel electrophoresis revealed genetic diversity among the metallo-β-lactamase-producing P. aeruginosa isolates. This is the first report on the existence of bla(PER-1), bla(PSE-1), bla(OXA-2), and bla(OXA-10) in Tunisia.

Analysis of Borderline Oxacillin-Resistant Staphylococcus aureus (BORSA) Strains Isolated in Tunisia

ABSTRACT Twenty-three strains of Staphylococcus aureus with borderline resistance to oxacillin were studied. These strains were not detected by the cefoxitin test, tests for penicillin-binding protein 2a (PBP2a), mecA, and mecA LGA251 were negative, and the strains were genetically unrelated. To detect all strains resistant to oxacillin, laboratories should routinely test for both cefoxitin and oxacillin.

Colistin-tigecycline versus colistin-imipenem-cilastatin combinations for the treatment of Acinetobacter baumannii ventilator-acquired pneumonia: a prognosis study.

Dear Editor, Ventilator-acquired pneumonia (VAP) still remains a major issue in intensive care units (ICUs). Several studies highlighted that Pseudomonas aeruginosa or Acinetobacter baumannii (Ab) are two leading causes of mortality in this setting [1]. Multidrug resistance is one of the hallmarks of Ab infections. Hence, a renewed interest in colistin as a salvage therapy has emerged in the last few decades. More recently, experimental studies testing the susceptibility of Ab to tigecycline gave promising results [2]. However, a phase 3, multicenter, randomized, double-blind study has shown that tigecycline use was associated with higher mortality in the subgroup of patients with VAP when compared to imipenem/cilastatin [3]. The aim of our study was to compare two regimens for the treatment of patients with Ab VAP: colistin– tigecycline versus colistin– imipenem/cilastatin. Between 1 January 2009 and 30 September 2014, 79 adult patients admitted in the ICU of Habib Bourguiba University Hospital developed Ab VAP and were treated either with colistin–tigecycline [Tige(?) group; n = 19] or colistin–imipenem/cilastatin [Tige(-) group; n = 60]. Colistin–tigecycline regimen was used only when Ab isolated strains were resistant to imipenem. Colistin was given at 2 MIU (million international units) every 8 h without a loading dose. Tigecycline was given at 100 mg twice a day during the first 48 h and then at 50 mg twice a day. Imipenem was given at 500 mg every 6 h. Median [interquartile range (IQR)] age was 42 [27–60] years. Median [IQR] SOFA score was 7 [5–10]. Median [IQR] from the initiation of mechanical ventilation to VAP onset was 7 [5–10] days. Subsequent Ab bloodstream infections were recorded in 10 patients and were significantly more frequent in the Tige(?) group (26.3 versus 8.3 %; p = 0.04). Median [IQR] MIC of tigecycline in the Tige(?) group was 1 [0.4–1.8] mg/l. Crude ICU mortality was significantly lower in the Tige(?) group (26.3 versus 53.3 %; p = 0.04). The Kaplan–Meier estimates of the probability of day 28 survival showed no significant differences between the two groups [HR = 0.52; 95 % CI 0.20–1.34; p = 0.166] (Fig. 1). The inverse probability of treatment weighted (IPTW) Cox proportional hazards model adjusted for variables found to be significantly associated with death 28 days after VAP diagnosis showed no significant difference between Tige(?) and Tige(-) groups [HR = 0.76, 95 % CI 0.44–1.33; p = 0.34]. Unlike Freire et al.’s study [3], our findings show the colistin–tigecycline regimen to be as effective as colistin– imipenem/cilastatin in patients with Ab VAP. This difference can be related to two major factors: first, tigecycline and imipenem were both used in combination with colistin; second, our patients received highdose tigecycline regimen during the first 48 h. In fact, Burkhardt et al. [4] reported that the concentration of tigecycline in the intracellular epithelial lining fluid was particularly high, whereas it was significantly lower in the extracellular fluid. Hence, using tigecycline with a high dosage may improve its efficacy in VAP patients. Moreover, these 48 h represent the time needed to achieve an effective concentration of colistin at the infection site [5]. The major limitations of our study are its

Carbapenemase-producing Salmonella enterica serotype Kentucky ST198, North Africa

in the coding sequence. These mutations have previously been shown to result in increased azithromycin resistance due to induced expression of the MtrCDE efflux pump. 8 The genes erm(A), erm(B), erm(C) and erm(F), encoding rRNA methylases, and mef(A), encoding an efflux pump, were not present in these isolates. Molecular epidemiology analysis of the isolates was performed using the N. gonorrhoeae multiantigen sequence typing (NG-MAST) method, 9 which assigns an ST based on a combination of the highly variable por and tbpB alleles. The isolates were assigned ST3102 and ST1866, which are almost identical and differ only by one SNP in por. Thus far, the high-level azithromycin-resistant N. gonorrhoeae strains that have been isolated in various countries belong to 16 different STs, of which ST649 isolates appear to be most widespread. 5 – 7 The por and tbpB alleles of these STs differ by a total of 33–92 SNPs from the por and tbpB alleles of the strains described in this study, except for the Australian ST10133 isolate, 7 which contains an identical tbpB allele (33) and a por allele (2573) that only differs by 16 and 17 SNPs from the por alleles of ST3102 and ST1866, respectively. Interestingly, it was reported that this isolate was most likely contracted in China or Hong Kong, which might explain its closer genetic relationship. How widespread high-level azithromycin-resistant strains are in China remains to be determined. Several years earlier, in 2009, two ST1866 strains were isolated from patients in the city of Nanjing, 10 which is located in a neighbouring province. These strains were reported to have an azithromycin MIC of .64 mg/L. It is not known whether these strains actually are directly related to the ST1866 isolate described in this study, but if they are related it would indicate that this high-level azithromycin-resistant ST might be spreading in China. Azithromycin susceptibility testing is currently not the standard in China. However, the identification of high-level azithromycin-resistant N. gonorrhoeae isolates stresses the need for a more thorough overview of azithromycin susceptibility in China, particularly when ceftriaxone and azithromycin dual antimicrobial therapy is considered as a future first-line therapy. These high-level azithromycin-resistant isolates might have a major impact on the efficacy of this dual antimicrobial therapy.