Mohammed Timinouni

Multidrug Resistance in Salmonella enterica Serotype Typhimurium from Humans in France (1993 to 2003)

ABSTRACT The aim of this study was to determine the distribution of the antimicrobial resistance phenotypes (R types), the phage types and XbaI-pulsed-field gel electrophoresis (PFGE) types, the genes coding for resistance to β-lactams and to quinolones, and the class 1 integrons among a representative sample of Salmonella enterica serotype Typhimurium isolates collected from humans in 2002 through the French National Reference Center for Salmonella (NRC-Salm) network. The trends in the evolution of antimicrobial resistance of serotype Typhimurium were reviewed by using NRC-Salm data from 1993, 1997, 2000, and 2003. In 2002, 3,998 isolates of serotype Typhimurium were registered at the NRC-Salm among 11,775 serotyped S. enterica isolates (34%). The most common multiple antibiotic resistance pattern was resistance to amoxicillin, chloramphenicol, streptomycin and spectinomycin, sulfonamides, and tetracycline (ACSSpSuTe R type), with 156 isolates (48.8%). One isolate resistant to extended-spectrum cephalosporins due to the production of TEM-52 extended-spectrum β-lactamase was detected (0.3%), and one multidrug-resistant isolate was highly resistant to ciprofloxacin (MIC > 32 mg/liter). We found that 57.2% of the isolates tested belonged to the DT104 clone. The main resistance pattern of DT104 isolates was R type ACSSpSuTe (83.2%). However, evolutionary changes have occurred within DT104, involving both loss (variants of Salmonella genomic island 1) and acquisition of genes for drug resistance to trimethoprim or to quinolones. PFGE profile X1 was the most prevalent (74.5%) among DT104 isolates, indicating the need to use a more discriminatory subtyping method for such isolates. Global data from the NRC-Salm suggested that DT104 was the main cause of multidrug resistance in serotype Typhimurium from humans from at least 1997 to 2003, with a roughly stable prevalence during this period.

Influence of humans on evolution and mobilization of environmental antibiotic resistome

The clinical failure of antimicrobial drugs that were previously effective in controlling infectious disease is a tragedy of increasing magnitude that gravely affects human health. This resistance by pathogens is often the endpoint of an evolutionary process that began billions of years ago in non–disease-causing microorganisms. This environmental resistome, its mobilization, and the conditions that facilitate its entry into human pathogens are at the heart of the current public health crisis in antibiotic resistance. Understanding the origins, evolution, and mechanisms of transfer of resistance elements is vital to our ability to adequately address this public health issue.

Ceftriaxone-resistant salmonella enterica serotype Newport, France.

The multidrug-resistant (MDR) Salmonella enterica serotype Newport strain that produces CMY-2 beta-lactamase (Newport MDR-AmpC) was the source of sporadic cases and outbreaks in humans in France during 2000-2005. Because this strain was not detected in food animals, it was most likely introduced into France through imported food products.

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.

Characterization and antibiotic susceptibility of Listeria monocytogenes isolated from poultry and red meat in Morocco.

This study was carried out on 426 samples of raw meats collected from butcheries and supermarkets in Casablanca, Morocco. The samples were examined for the occurrence of Listeria species. Strains of Listeria monocytogenes were characterized by several biochemical tests and confirmed by polymerase chain reaction (PCR). β-hemolytic cultures and nonhemolytic isolates were tested for biochemical properties with the Listeria API test. Among the 43 Listeria species isolates; we identified 10 strains for L. monocytogenes (23.3%), 31 strains for L. innocua (72.1%) and 2 strains for L. welshimeri (4.6%). Strains of L. monocytogenes were separated by multiplex PCR; two serogroups IIb and IVb were thus differentiated. Antibiotic susceptibility of L. monocytogenes to 21 antibiotics was determined by the disk diffusion method. All isolates were susceptible to a wide range of the tested antibiotics with the exception of nalidixic acid, colistine and cephalosporins second and third generation for which they were all resistant.

Ceftazidime-Resistant Salmonella enterica, Morocco

To the Editor: Nontyphoidal salmonellosis (NTS) is a major food-borne illness worldwide. Extended-spectrum cephalosporins (ESCs) are currently preferred drugs for treatment of children with NTS. However, resistance to ESCs has emerged worldwide and has become a serious public health problem. This resistance is caused by production of various class A extended-spectrum β-lactamases (ESBLs) and class C cephalosporinases in Salmonella enterica (1).

Antibiotic resistance determinants and genetic analysis of Salmonella enterica isolated from food in Morocco.

Antimicrobial-resistant non-typhoidal Salmonella (NTS) are an important cause of infection in Africa, but there is a lack of information on their molecular mechanisms of resistance and epidemiology. This study contributes to fill this gap through the characterization by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), plasmid profiling and analysis of antibiotic-resistance determinants of 94 Salmonella enterica strains isolated from food in Morocco. PFGE revealed considerable heterogeneity among the strains, showing 32 pulsotypes. MLST of strains representative of the different serovars evidenced 13 sequence types (STs), three of which were newly identified (ST1694, ST1768 and ST1818) and nine not previously reported in Morocco. Thirty-four strains harbored from one to four plasmids, of IncI1 group in S. Mbandaka, IncFIIA in S. Typhimurium, IncL/M in S. Hadar and S. Blockley. For the first time in Morocco an intact Salmonella Genomic Island 1 (SGI1) carrying the resistance genes aadA2, floR, tetG, blaPSE-1 and sul1 was detected in S. Typhimurium DT104. In serovar Hadar resistance to ampicillin, tetracycline and streptomycin was associated to blaTEM-1, tetA and strA genes respectively, whereas one mutation in gyrA (Asp87Asn) and one in parC (Thr54Ser) genes conferred resistance to nalidixic acid. These findings improve the information on foodborne Salmonella in Morocco, evidencing the presence of MDR strains potentially dangerous to humans, and provide useful data for future studies.

Staphylococcus aureus nasal carriage in a Moroccan dialysis center and isolates characterization

Staphylococcus aureus, which has its ecological niche in the anterior nares, has been shown to cause a variety of infectious diseases mainly for patients in hemodialysis units. We performed this study to evaluate the prevalence of nasal S. aureus carriage among hemodialysis outpatients, to determine the antimicrobial susceptibility of isolates, to characterize the virulence genes, and to identify associated risk factors. Nares swab specimens were obtained from 70 outpatients on hemodialysis between March and June 2010. Samples were plated immediately onto S. aureus specific media and pattern of antibacterial sensitivity was determined using disk diffusion method. Polymerase chain reaction was used to detect nuc, mecA, and genes encoding staphylococcal toxins. Medical record of patients was explored to determine S.aureus carriage risk factors. Nasal screening identified 42.9% S. aureus carriers with only one (3.3%) methicillin‐resistant S. aureus isolate. Among the methicillin‐susceptible S. aureus isolates, high rate of penicillin resistance (81.8%) has been detected. The identified risk factors were male gender and age ≤ 30 years. Research of virulence factors showed a high genetic diversity among the 30 S. aureus isolates. Twenty‐one (70%) of them had at least one virulence gene, of which 3.3% were Panton‐Valentine leukocidin (lukS/F‐PV) genes. S. aureus carriage must be screened for at regular intervals in hemodialysis patients. Setting up a bacterial surveillance system is one of the strategies to understand the epidemiology of methicillin‐resistant S. aureus, to guide local antibiotic policy and prevent spread of antibiotic‐resistant S. aureus.

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.