Detection of Carbapenemase Encoding Gene and Resistance to Cefiderocol in Hospital and Community eXtensive Drug Resistance and Carbapenem-Resistant Pseudomonas aeruginosa Strains in Morocco.

Pseudomonas aeruginosa (Pa) remains among clinically-significant Gram-negative species. The carbapenems are often the last resort for treating infections due to multidrug resistant isolates such as Pa. The carbapenems' efficacy is increasingly compromised by the emergence and the rapid spread of Pa carrying carbapenemases which represent a serious threat to public health. This study aimed to establish the resistance profile and to identify carbapenemase genes in isolates with imipenem resistant phenotypes. Among 134 Pa isolates collected both in the community (46) and hospital (88) from January 2021 to December 2021 in Morocco, 18 (8 were from the community and 10 from the hospital settings) were carbapenem resistant. The identification of these strains has been confirmed using matrix assisted laser desorption ionization-time of flight (MALDI-TOF). The antibiotic susceptibility testing against 16 antibiotics was carried out and interpreted according to the recommendations of the European Committee on Antimicrobial Susceptibility Testing (2021). The worrying antibiotics resistance profiles, which spread to cefiderocol for two isolates, were obtained for all isolates, which were eXtensive Drug Resistance showing highly resistant to all antibiotic categories tested, even to ceftolozane-tazobactam. Colistin (100% susceptible) and cefiderocol (88.88%) were the most active agents against carbapenem-resistant Pa (CRPa). Phenotypic detection by NP-CARBA and NG-CARBA tests of metallo­ß­lactamase (MßL) production was confirmed by PCR amplification and sequencing. Three CRPa isolates coharboring blaVIM-2-blaNDM-1 (two isolates) and blaVIM-2-blaIMP-8 (one isolate) genes were detected. In this study, we describe the coexistence of these MßL genes and the cefiderocol resistance in CRPa strains in Morocco. The alarming antibiotic resistance patterns of all these CRPa isolates and their resistance genes emphasize the importance of antimicrobial susceptibility testing in the choice of antibiotics for treating Pa infections.

New Insight into the Chemical Composition, Antimicrobial and Synergistic Effects of the Moroccan Endemic Thymus atlanticus (Ball) Roussine Essential Oil in Combination with Conventional Antibiotics.

This study reported the volatile profile, the antimicrobial activity and the synergistic potential of essential oil (EO) from the Moroccan endemic Thymus atlanticus (Ball) Roussine, in combination with the antibiotics ciprofloxacin and fluconazole for the first time, to the best of our knowledge. The EO chemical composition was determined by gas chromatography coupled to mass spectrometry (GC-MS) analysis and the antimicrobial activity assessed by the disc diffusion method against three Gram positive (Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus) and three Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli and one clinical isolate, Klebsiella pneumonia). The antifungal activity was evaluated in four pathogenic yeasts (Candida albicans, C. glabrata, C. krusei and C. parapsilosis). The minimum inhibition concentration (MIC) and the synergistic effect with ciprofloxacin and fluconazole were determined by the two-fold dilution technique and checkerboard test, respectively. Twenty-one constituents were identified by GC-MS in the EO, including carvacrol (21.62%) and borneol (21.13%) as the major components. The EO exhibited a significant antimicrobial activity with inhibition zones ranging from 0.7 mm to 22 mm for P. aeruginosa and B. subtilis, respectively, and MIC values varying from 0.56 mg/mL to 4.47 mg/mL. The fractional inhibitory concentration index (FICI) values ranged from 0.25 to 0.50 for bacteria and from 0.25 to 0.28 for yeasts. The maximum synergistic effect was observed for K. pneumonia with a 256-fold gain of antibiotic MIC. Our results have suggested that EO from T. atlanticus may be used alone or in association with antibiotics as a new potential alternative to prevent and control the emergence of resistant microbial strains both in the medical field and in the food industry.

Genotypic characterization of quinolone resistant-Escherichia coli isolates from retail food in Morocco.

This study was conducted to assess the retail food as a possible vehicle for antimicrobial resistant, particularly quinolones resistant and pathogenic Escherichia coli. We determined the prevalence and characteristics of nalidixic acid (Nal) resistant E. coli isolates from diverse retail food samples. In all, 70 (28%) of 250 E. coli isolates studied were Nal-resistant E. coli and 91% of these were multi-drug resistant. Plasmid mediated quinolone resistance genes were identified in 32 isolates, including aac(6')-Ib-cr (n = 16), qnrS1 (n = 11) and qnrB19 (n = 7). Mutations in gyr A and par C genes were detected among 80% of the isolates, and the isolates showed substitution Ser83-Leu and Asp87-Asn in gyrA and Ser80-Ile in parC. In addition, three different gene cassettes were identified (aadA1, aadA7, aac(3)-Id) in 18%. Virulence-associated genes stx1, eae, sfa, hlyA and stx2 were found in six (8%), three (4%), two (3%), three (4%) and three (4%) isolates, respectively. E. coli isolates of phylogenetic group A were dominant (64%, 45/70). Pulsed field gel electrophoresis revealed none epidemiological relationship between these isolates. The results of this work report the higher frequency of Nal-resistant E. coli isolates from Moroccan retail food samples including MDR and pathogenic isolates.

Prevalence and types of extended spectrum ß-lactamases among urinary Escherichia coli isolates in Moroccan community.

This study was designed to characterize extended-spectrum-ß-lactamases (ESBL) produced by Escherichia coli isolates causing community urinary tract infections over a 2-year period (2010 and 2011) in a Moroccan large geographical region. Molecular characterization was done by using PCR and sequencing of the ß-lactamases genes and plasmid-mediated quinolone resistance determinants. Among 1174 isolates, 49 (4.1%) were ESBL producers. The blaCTx-M-15 (n = 31) was the most frequent ESBL gene detected, followed by blaCTx-M-1 (n = 5), blaSHV-12 (n = 6), blaPER-2 (n = 3), then blaTEM-3, blaTEM-20, blaTEM-158, blaSHV-27, blaSHV-28, blaSHV-36, blaSHV-125, blaCTx-M-14 and blaCTx-M-27 with one isolate for each. The non-ESBL genes detected were blaTEM-70 (n = 1), blaTEM-176 (n = 1), blaTEM-104 (n = 6), blaTEM-1 (n = 15) and blaOxA-1 (n = 12). Plasmid mediated AmpC ß-lactamases genes; blaACT-5 (n = 1), blaDHA-1(n = 2) and blaCMY-2 (n = 4) were detected in seven isolates (14.2%). The blaOxA-48 (n = 1) and blaIMP-1 (n = 1) carbapenemases genes were detected among five carbapenem-resistant E. coli. Five isolates (10.2%) harboured qnr genes, qnrB1 (n = 3), qnrB2 (n = 1) and qnrS1 (n = 1) type were detected. Thirty isolates (61.2%) were positive for aac(6')-Ib-cr gene. The class 1 integron was detected in twenty two (44.8%) isolates. Phylogenetic grouping revealed that 22 (44.8%) isolates belonged to group A, while 15 (30.6%), 11 (22.4%) and 1 (2%) belonged to B2, D and B1. Results of conjugation experiments indicated that blaCTx-M-15, blaTEM-1, 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. The results of this work reports the genetic diversity of ESBL genes, with the CTX-M-15 enzyme being the most common among ESBL-producing E. coli in Moroccan community.

Occurrence of OXA-48 and NDM-1 carbapenemase-producing Klebsiella pneumoniae in a Moroccan university hospital in Casablanca, Morocco.

The purpose of this investigation was to determine the prevalence and the characteristics of carbapenemase-producing Klebsiellapneumoniae isolates recovered from various clinical specimens in the university hospital of Casablanca, in Morocco. We conducted a prospective study on a total of 166 K. pneumoniae isolates collected from June to August 2011. The strains suspected to carry carbapenemase showed reduced susceptibility to imipenem or ertapenem. The PCR and a sequencing strategy were used to identify carbapenemases, expended spectrum ß-lactamases (ESBL), plasmid-mediated AmpC ß-lactamases, plasmid mediated quinolone resistance and aminoglycoside resistance determinants. The clonal relationships between isolates were analyzed by pulsed field gel electrophoresis (PFGE). Among the 166 K. pneumoniae isolates studied, 11 (6%) were carbapenemases producers, 9 of which harbored blaOXA-48 and 2 were positive for blaNDM-1. All carbapenemase-producing K. pneumoniae were also ESBL producers and the blaCTX-M-15 was the most frequent ESBL gene detected (n=9), blaCTX-M-28 and blaSHV-28 were also encountered in one isolate each. The K.pneumoniae isolates carried also non-ESBL genes blaTEM-1 (n=9), blaSHV-1 (n=8) and blaOXA-1 (n=3). Five isolates harbored qnr genes, qnrS1 (n=3) and qnrB1 (n=2) variants. Six isolates were positive for aac(6')-Ib-cr gene and two for aac(3)-II gene. The class 1 integron was detected in five isolates. PFGE has revealed the presence of a clonal dissemination in our hospital. The results of conjugation experiments indicated that blaOXA-48+blaCTX-M-15, blaOXA-48+blaCTX-M-28, blaNDM-1+blaCTX-M-15+blaTEM-1+blaOXA-1+qnrS1+aac(6')-Ib-cr and blaNDM-1+blaCTX-M-15+blaTEM-1+qnrB1+aac(6')-Ib-cr genes were co-transferred and that these genes were carried by a conjugative plasmid of high molecular weight.

Correlation between staphylococcal biofilm formation in vitro and potential for catheter-related infections.

INTRODUCTION: The present study evaluated biofilm-forming capacity and the presence of both icaA and icaD genes among staphylococcal strains isolated from catheter-related infections and blood culture. METHODOLOGY: Ninety staphylococcal isolates, which included 45 strains of catheter infection origin and 45 strains of blood culture origin, were tested for their ability to produce biofilm using microtiter test plates and a catheter test. The presence of icaA and icaD genes was determined by polymerase chain reaction (PCR). RESULTS: Of the 45 strains of catheter infection origin, 22 (48.88%) formed biofilm. In comparison, only 10 (22.22%) of the 45 strains of blood culture origin formed biofilms. Similar results were obtained from both the microplate test and catheter test. In the 32 strains that were able to form biofilm, 30 were positive for icaA and icaD genes, and the remaining 2 strains were negative for both genes. Fifteen staphylococcal strains of all origins presented only the icaA locus and did not form biofilm. In 88 of 90 tested strains (97.77%), there was a positive correlation between biofilm production and presence of icaA and icaD genes, and between no biofilm production and absence of both or only one of the tested genes. CONCLUSIONS: The ability of staphylococcal isolates to form biofilm in vitro appears to be an indication of a virulence trait that enhances the ability of isolates to cause catheter-related infections. In addition, our results indicate an important role of ica genes and phenotypic variability of biofilm production as virulence factors in staphylococcal infections.

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

BACKGROUND: The importance of community-acquired infections due to extended-spectrum ß-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 ß-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.

Prevalence and genotypic analysis of plasmid-mediated ß-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 ß-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 bla(CTX-M-15) (n=31) was the most frequent ESBL genes detected, followed equally by bla(SHV-28) and bla(SHV-12) (n=3), then bla(TEM-3), bla(SHV-36), bla(SHV-110) and bla(CTX-M-1) with one isolate for each (n=1). Eight isolates co-expressed more than one ESBL with bla(CTX-M-15). The non-ESBL genes detected were bla(SHV-1), bla(SHV-11), bla(SHV-32), bla(SHV-26), bla(SHV-76), bla(TEM-1), bla(TEM-1b) and bla(OXA-1). Plasmid-mediated AmpC ß-lactamase genes, bla(ACT-2), bla(DHA-1) and a new ß-lacatamase named bla(EBC-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 bla(CTX-M-15), bla(TEM-1b), bla(OXA-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 bla(EBC-1464) detection is a first in Morocco.

Characterization of extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates from the community in Morocco.

Of 803 community Escherichia coli (n = 767) and Klebsiella pneumoniae (n = 36) isolates collected from patients with urinary tract infections in three Moroccan cities, 10 E. coli (1.3%) and 2 K. pneumoniae (5.6 %) isolates were shown to produce extended-spectrum ß-lactamases (ESBLs). PFGE revealed that the E. coli isolates comprised seven distinct genotypes. The presence of plasmids in the 12 isolates was revealed by conjugation experiments of plasmids from these Enterobacteriaceae strains with E. coli K(12)J(5), with further isolation of the plasmids in the transconjugants. Subsequent nucleotide sequencing indicated that the plasmids encoded the bla(CTX-M), bla(OXA), bla(TEM) and bla(SHV) genes, including genes for CTX-M-15 (n = 11), OXA-1 (n = 11), TEM-1b (n = 4), SHV-5 (n = 1) and SHV-1 (n = 2). Identification of plasmid-mediated quinolone-resistance genes was performed by PCR. The aac(6')Ib-cr variant was detected in all strains, and two strains co-expressed qnrS1, bla(CTX-M-15) and bla(OXA-1) genes. The presence of ESBLs in the Enterobacteriaceae strains studied was probably due to the dissemination of resistance plasmids with the predominant genotype of bla(CTX-M-15.).