Abouddihaj Barguigua

First report of a Klebsiella pneumoniae strain coproducing NDM-1, VIM-1 and OXA-48 carbapenemases isolated in Morocco

To the Editor The emergence and dissemination of carbapenem-resistant Enterobacteriaceae represent a significant threat to the management of nosocomial and community-acquired infections (1). Carbapenem-hydrolysing b-lactamases are represented by three molecular classes of b-lactamases: A, B, and D. The best-known carbapenemases are KPC-type (Ambler class A), IMP and VIM types (class B), and OXA48 (class D), mostly identified in Klebsiella pneumoniae as a source of nosocomial outbreaks (2). Recently, a novel metallo-b-lactamase (MBL) named NDM-1 (New Delhi metallo-b-lactamase) has been identified from a K. pneumoniae strain recovered in Sweden from a patient previously hospitalized in India (3). Very recently, NDM-1 producers have been reported in the environment and in communityacquired infections (1, 3). In this study, we report the characterization of a carbapenemase-producing K. pneumoniae strain (Kpp474) isolated from the urine sample recovered from an elderly male (49 years) non-hospitalized patient in Taza (northern Morocco). He had previously received broadspectrum cephalosporins and fluoroquinolones for recurrent urinary tract infections; the treatment was changed to amikacin and the patient recovered. Antibiotic susceptibilities and minimal inhibitory concentrations were determined by disc diffusion and agar dilution methods respectively, in accordance with the recommendations of the Antibiogram Committee of the French Society of Microbiology (http://www.sfm-microbiologie.org). Isolate Kpp474 was resistant to amoxicillin (>256 mg/ L), amoxicillin clavulanate combination (>16/ 8 mg/L), ertapenem (16 mg/L), cefotaxime (>256 mg/L), ceftriaxone (>256 mg/L), cefepime (64 mg/L), ceftazidime (>256 mg/L), aztreonam (64 mg/L) and cefoxitin (>256 mg/L), and had an intermediate susceptibility to imipenem (6 mg/L). In addition, the isolate was also resistant to aminoglycosides (except to amikacin), fluoroquinolones, sulfonamides and tetracycline, but remained susceptible to fosfomycin and colistin. This isolate was positive for modified Hodge test and double-disc synergy test (4, 5), in which only aztreonam showed a positive synergy with clavulanate. We used the previously described PCRs (2, 6) followed by sequencing, to screen and characterize the carbapenem-hydrolysing b-lactamase genes (blaVIM, blaIMP, blaKPC, blaGES, blaNDM and blaOXA-48). In addition, the carriage of plasmid-encoded blaCTX-M, blaTEM, blaOXA-1, blaSHV and blaAmpC b-lactamase genes, the aac(6′)-Ib, aac(3)-II aminoglycoside resistance genes, the quinolone resistance genes qnrA, B, S, the tetracycline resistance genes tetA, and class 1 integron were investigated (7–9). The isolate was positive for blaNDM-1, blaVIM-1 and blaOXA-48. The search for narrow-spectrum b-lactamases, extended spectrum b-lactamase (ESBL) and AmpC genes revealed that Kp474 isolate harboured the narrowspectrum b-lactamase genes blaTEM-1, blaSHV-1 and blaOXA-1, together with the ESBL gene blaCTX-M. However, they did not harbour AmpC-encoding genes. In addition, the plasmid-encoded quinolone resistance genes qnrB and aac(6′)-Ib-cr, aminoglycoside resistance gene aac(3)-II and tetracycline resistance gene tetA were present in the Kp474 isolate. A class 1 integron was detected, with an amplicon of 1.6 kb in length. Plasmid analysis, performed using the alkaline lysis and S1 nuclease-PFGE method (10, 11), revealed that K. pneumoniae Kpp474 harboured five plasmids of ca. 250,130, 60, 52 and 7 kb. The transfer of the b-lactams resistance markers from Kpp 474 to Escherichia coli K12J5 (azide resistant) was performed by mating assays, with selection based on different antibiotics (ertapenem 0.25 mg/L, kanamycin 2 mg/L and ceftazidime 1 mg/L) (12). Two E. coli K12J5 transconjugant isolates (Tc1 and Tc2) were detected with different phenotypes. The Tc1 transconjugant showed an MBL and an ESBL phenotype (4, 5) and was resistant to nalidixic acid, amoxicillin (>256 mg/L), clavulanic acid/amoxicillin (>16/8 mg/L), ceftazidime

Prevalence and genotypic analysis of plasmid-mediated β-lactamases among urinary Klebsiella pneumoniae isolates in Moroccan community

The aim of this study is to assess the prevalence and molecular characterization of the extended spectrum β-lactamases (ESBL)-producing Klebsiella pneumoniae isolated from community acquired urinary tract infections and collected in five Moroccan cities during a 2010 survey. In all, 34 (7.5%) of the 453 K. pneumoniae isolates studied were positive for an ESBL phenotype and 91.1% of these isolates were multidrug resistant. The blaCTX-M-15 (n=31) was the most frequent ESBL genes detected, followed equally by blaSHV-28 and blaSHV-12 (n=3), then blaTEM-3, blaSHV-36, blaSHV-110 and blaCTX-M-1 with one isolate for each (n=1). Eight isolates co-expressed more than one ESBL with blaCTX-M-15. The non-ESBL genes detected were blaSHV-1, blaSHV-11, blaSHV-32, blaSHV-26, blaSHV-76, blaTEM-1, blaTEM-1b and blaOXA-1. Plasmid-mediated AmpC β-lactamase genes, blaACT-2, blaDHA-1 and a new β-lacatamase named blaEBC-1464, were detected in 11.7% of isolates. Fourteen (41.1%) isolates harbored qnr genes; qnrA6 (n=1), qnrB1 (n=8), qnrB2 (n=1) and qnrS1 (n=4) types were detected. Twenty-six isolates (76.4%) were positive for aac(6′)-Ib-cr gene. Results of conjugation experiments indicated that blaCTX-M-15, blaTEM-1b, blaOXA-1, aac(6′)-Ib-cr and qnrB1 genes were co-transferred and that these genes were carried by a conjugative plasmid of high molecular weight. With the exception of qnrB1, all the antibiotic resistance genes were clustered in a 12-kb region. The results of this work report the genetic diversity of ESBL genes, with the CTX-M-15 enzyme being most common among ESBL-producing K. pneumoniae in Moroccan community. Furthermore, a major finding is that blaEBC-1464 detection is a first in Morocco.

Epidemiology and virulence of VIM-4 metallo-beta-lactamase-producing Pseudomonas aeruginosa isolated from burn patients in eastern Algeria

In this study, we investigated the prevalence of carbapenem-resistant Pseudomonas aeruginosa (CRPA) in burn patients from eastern Algeria, CRPA virulence factors and the molecular epidemiology of CRPA. The overall prevalence of CRPA was 48.38%. Seven (46.66%) isolates were metallo-β-lactamases (MBL) producers and contained the MBL genes blaVIM-4 (n=6) and blaVIM-2 (n=1). Risk factors for CRPA infection were urinary catheter use and intubation (p=0.008). A high percentage of virulence factors (86.6% of these isolates were able to produce protease; 73.3% of isolates has DNase; and 66.6% were haemolysin positive) was observed in CRPA isolates. Among the seven MBL-producing isolates, four had the same clonal profile. The class 1 integrons, which contained the aadA7 gene cassette, were detected in six isolates. The 16SrRNA methylase gene, rmtB, was detected in one strain. All CRPA isolates were biofilm formers. A study on the kinetics of biofilm production revealed that biofilm production increased when the concentration of imipenem or ciprofloxacin and the incubation time increased. This is the first study to report the presence of VIM-4-producing P. aeruginosa from North Africa and also of the high prevalence of CRPA isolates. Based on our study of burn unit patients, the high percentage of P. aeruginosa with virulence factors and multi-drug resistance is alarming.

Prevalence and characterization of extended spectrum beta-lactamase- producing Enterobacter cloacae strains in Algeria

INTRODUCTION Expended spectrum β-lactamase (ESBL)-producing Enterobacter cloacae is an important nosocomial pathogen. In this study, the prevalence and the molecular epidemiology of ESBL producing E. cloacae strains isolated from various hospitals in Annaba, Algeria were investigated. METHODOLOGY The study involved 63 isolates of E. cloacae obtained during 2009 at the four hospitals in Annaba. The detection of ESBL was performed using the double-disk synergy test and the combined disk test. Minimum inhibitory concentrations (MICs) were determined using the agar dilution method. The presence of bla(CTX-M), bla(SHV), bla(TEM), and bla(DHA) β-lactamase genes was evaluated by PCR, and genomic typing was determined by pulsed-field gel electrophoresis (PFGE) analysis. The clinical and microbiological data were entered into the EpiI Info database. RESULTS Thirty isolates (47.6%) had an ESBL phenotype. Bla(CTX-M) group1 (76%); bla(TEM) (70%) were the most prevalent, followed by bla(DHA) (16.6%) and bla(SHV) (10%). Eighteen strains expressed at least two bla genes. MICs revealed a high level of resistance to cefotaxime, ceftazidime, and cefepime. PFGE revealed an epidemic clonal dissemination of these isolates. Various risk factors associated with the occurrence of ESBL-producing E. cloacae were detected. CONCLUSIONS A higher frequency of ESBL-producing isolates and a diversity of β-lactamases were detected among ESBL-producing E. cloacae; these resulted from an epidemic clonal dissemination and high transference of ESBL genes between bacteria in hospital settings. Strict measures will be required to control the further spread of these pathogens in hospital settings.

Fecal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae in community setting in Casablanca

Abstract Background: The importance of community-acquired infections due to extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE) has been increasingly recognized in recent years. This study aimed to determine the prevalence of intestinal carriage of ESBL-PE in the community in Casablanca, Morocco. Methods: During 6 months (2013), 93 fecal samples were examined for ESBL-PE. Isolates expressing an ESBL phenotype were investigated for the presence of genes encoding β-lactamases and plasmid-mediated quinolone resistance. Conjugation experiments were done to determine the mobility of ESBL genes. Results: The prevalence of fecal carriage of ESBL-PE was 4.3% (4/93; 95% CI, 0.2–8.4). Klebsiella pneumoniae (n = 2), Enterobacter cloacae (n = 2), Escherichia coli (n = 1), and Serratia odorifera (n = 1) were the ESBL-producing species. Four (66.7%) of these isolates were multidrug-resistant. The blaSHV-12 (n = 5) was the most frequent ESBL gene detected, followed by blaCTX-M-15 (n = 3).The non-ESBL gene detected was blaTEM-1 (n = 5). One isolate harbored the qnrB1 variant. Results of conjugation experiments indicated that blaSHV-12 + blaTEM-1 + qnrB1 and blaCTX-M-15 + blaTEM-1 genes were co-transferred and that these genes were carried by a conjugative plasmid of high molecular weight (125 kb). Conclusion: Our results show the importance of the intestinal tract as a reservoir for ESBL-PE in the community in Morocco.

Rectal carriage of extended-spectrum β-lactamase- and carbapenemase-producing Enterobacteriaceae among hospitalised neonates in a neonatal intensive care unit in Fez, Morocco

OBJECTIVES The aim of this study was to investigate the faecal carriage and molecular epidemiology of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae (ESBLE) isolated from rectal samples of neonates hospitalised in a neonatal intensive care unit (NICU) of a university hospital in Fez, Morocco. METHODS From February-July 2013, all neonates hospitalised in the NICU were screened for ESBLE carriage at discharge. ESBLs were identified by double-disk synergy test, PCR and DNA sequencing analysis. ESBLE were analysed by pulsed-field gel electrophoresis (PFGE), and conjugation was performed by the broth mating method. RESULTS In this study, 169 Enterobacteriaceae were collected from 164 neonates. The prevalence of faecal carriage of ESBLE was 58.0% (98/169), predominantly Klebsiella pneumoniae (65/98; 66.3%). A high rate of multiresistance in ESBLE was noted. blaCTX-M-1 group (78.5%) was the most frequent ESBL gene detected, and all isolates harboured the CTX-M-15 variant. The prevalence of carbapenemase-producing Enterobacteriaceae was 1.8%, and blaOXA-48 was the only gene found in these isolates. Sequencing revealed subgroups corresponding to bla(CTX-M-15,TEM-1,TEM-104,SHV-1,SHV-44,SHV-49andSHV-133) genes. Conjugation experiments showed the transferability of blaCTX-M-15 and blaTEM, but not blaSHV. These genes were carried by a high-molecular-weight conjugative plasmid (ca. 125kb). PFGE profiles demonstrated high clonal dissemination of ESBL-positive strains in the NICU. CONCLUSIONS These results demonstrate the existence of high clonal transmission of ESBLE in a Moroccan NICU. This finding provides useful information to implement a screening policy for resistant Enterobacteriaceae among neonates hospitalised in this ward.

Phenotypic and genotypic characterization of Streptococcus pneumoniae resistant to macrolide in Casablanca, Morocco

In Morocco, the 13-valent pneumococcal conjugate vaccine (PCV-13) was introduced in the national immunization program (NIP) in October 2010 and replaced by the PCV-10 in July 2012. The present study aimed to determine the prevalence of erythromycin-resistant Streptococcus pneumoniae (ERSP) and to analyze the phenotypic and genotypic characteristics of these isolates in Casablanca, Morocco from January 2007 to December 2014. Isolates were obtained from the Microbiology Laboratory of Ibn Rochd University Hospital Centre of Casablanca. Serogrouping was done using Pneumotest Kit and serotyping by the Quellung capsular swelling. Antibiotic susceptibility pattern was determined by disk diffusion and Etest methods. A total of 655S. pneumoniae isolates were collected from 2007 to 2014 from pediatric and adult patients. Fifty-five percent of these isolates were from invasive pneumococcal diseases. Of the 655 isolates, 92 (14%) were ERSP. Globally, the proportion of ERSP from 2007 to 2010 (before vaccination) and from 2011 to 2014 (after vaccination) were 11.6% and 17.2% (p=0.04), respectively. Of the 92 ERSP, 89%, 4% and 7% displayed constitutive MLSB (resistance to macrolide, lincosamide and streptogramin B), inducible MLSB, and M phenotype (resistance to macrolide only), respectively. ERSP genotypic analysis showed that 90.2% carried the ermB gene, 6.5% the mefE gene, and 3.3% both the genes (ermB+mefE). The most prevalent ERSP serotypes were 6B, 19F and 23F before vaccination and 19F, 6B, 6A and 23F after vaccination. Erythromycin resistance among S. pneumoniae is relatively high in Casablanca. The contribution of PCVs to the reduction in antibiotic use is encouraging but this should be accompanied by a rational use of antibiotic.

Occurrence of plasmid-mediated quinolone resistance and virulence genes in avian Escherichia coli isolates from Algeria

INTRODUCTION The emergence and spread of quinolone-resistant Escherichia coli in poultry products puts consumers at risk of exposure to the strains of E. coli that resist antibiotic treatment. The objective of this study was to define the prevalence and virulence potential of poultry-associated nalidixic acid (NAL)-resistant E. coli in the Annaba city, Algeria. METHODOLOGY In total, 33 samples of retail chicken meat were purchased from various butcher shops and examined for bacterial contamination with NAL-resistant E. coli. These isolates were subjected to antimicrobial susceptibility testing and were also investigated for the presence of plasmid-mediated quinolone resistance (PMQR) genes and virulence genes using conventional polymerase chain reaction (PCR) and DNA sequencing. Phylogenetic grouping of the NAL-resistant E. coli isolates was determined by the conventional multiplex PCR method. RESULTS Twenty-nine (87.8%) products yielded NAL-resistant E. coli. Antibiograms revealed that 96.55% of NAL-resistant E. coli isolates were multidrug resistant (MDR). Resistance was most frequently observed against sulfamethoxazole-trimethoprim (96.6%), tetracycline (96.6%), ciprofloxacin (72%), and amoxicillin (65.5%). Group A was the most prevalent phylogenetic group, followed by groups D, B1, and B2. The PMQR determinants were detected in three isolates with qnrB72 and qnrS1 type identified. Four (13.8%) isolates carried one of the Shiga toxin E. coli-associated genes stx1, stx2, and ehxA alleles. CONCLUSIONS The high prevalence of NAL-resistant E. coli isolated from retail chicken meat with detection of MDR E. coli harboring Shiga toxin genes in this study gives a warning signal for possible occurrence of foodborne infections with failure in antibiotic treatment.

Effect of temperature and plumbing materials on biofilm formation by Legionella pneumophila serogroup 1 and 2-15

Abstract The purpose of our study was to investigate the biofilm formation by Legionella pneumophila serogroup1 and serogroup2-15 on plumbing materials mostly used in building water systems in Morocco. The effect of plumbing materials and temperature were examined. The Atomic Force Microscopy was used to evaluate the roughness and surface topography of the plumbing materials including galvanized steel, stainless steel, copper, Polyvinyl chloride, Polypropylene Random Copolymer and Cross-linked polyethylene. Galvanized steel surfaces supported higher numbers of bacterial cells than that the stainless steel and plastic materials at 37 than 20 and 45 °C. Non-viable counts could be obtained from the copper surfaces. L. pneumophila sg2-15 strains presented a higher ability to biofilm formation than L. pneumophila sg1. The biofilm formation was evaluated by Atomic Force Microscopy.

Adhesion of Legionella pneumophila on glass and plumbing materials commonly used in domestic water systems

ABSTRACT We aimed to investigate the adhesion of Legionella pneumophila serogroup1 and L. pneumophila serogroup2–15 on glass, galvanized steel, stainless steel, copper, Polyvinyl chloride(PVC), Cross-linked polyethylene(PEX-c) and Polypropylene Random Copolymer(PPR). The surface physicochemical properties of both bacterial cells and materials were estimated through contact angle measurements. The roughness and surface topography of the materials were evaluated by Atomic Force Microscopy. The two L. pneumophila serogroups and plumbing materials showed a hydrophobic character, while glass surface was hydrophilic. All strains were adhered to all materials with the exception of copper. The result showed that the adhesion of both L. pneumophila sg1 and sg2–15 was systematically expressed with high intensity on galvanized steel followed by PVC, PEX-c, PPR, stainless steel and the low intensity on glass. The extent of adhesion is in correlation with the surface roughness and acid–bases interactions, while hydrophobicity seems to have no effect in adhesion intensity.