Phenotypic and genotypic characterization of clinical carbapenem-resistant Enterobacteriaceae isolates from Sokoto, northwest Nigeria.

Emergence and spread of carbapenemase-producing Enterobacteriaceae (CPE) are two of the major problems currently threatening global public health. In Nigeria, interest in CPE is recent. In Sokoto, northwest Nigeria, there are no data on the prevalence and mechanism underlying carbapenem resistance. In this study, we aimed to investigate the presence of clinical carbapenems-resistant Enterobacteriaceae isolates in two leading hospitals in Sokoto, northwest Nigeria. A total of 292 non-duplicate Enterobacteriaceae isolated from clinical specimens processed in the diagnostic laboratories of two hospitals between January and June 2019 were collected. Of these, 129 (44.2 %) and 19 (6.5%) were resistant to third-generation cephalosporin and carbapenems, respectively. RT-PCR revealed that 10 (7.8%), 19 (14.7%) and 46 (35.7%) of the third-generation cephalosporin-resistant isolates harboured bla SHV, bla TEM and bla CTX-M genes, respectively. The modified Carba NP test result showed that only 7 (36.8 %) of the 19 carbapenem-resistant isolates were carbapenemase producing; among them, bla NDM-5 and bla OXA-181 genes were identified in five and two isolates, respectively. However, none of the carbapenemase genes investigated, including bla VIM, bla KPC and bla IMP, was detected in the remaining carbapenem-resistant isolates, suggesting a non-enzymatic mechanism. This study reports for the first time, the emergence of CPE in Sokoto state and the detection of NDM-producing Citrobacter freundii in Nigeria. The observed CPE in this study is a concern in a country where alternative antibiotics are rarely available.

Emergence of plasmid-encoded VIM-2-producing Pseudomonas aeruginosa isolated from clinical samples in Lebanon.

The present study aimed to describe the emergence of carbapenem-resistant Pseudomonas aeruginosa isolated from clinical Lebanese patients. The resistance of these isolates is due to the presence of the plasmid-encoded bla VIM-2 gene. We provide its first description in Lebanon, as well as a description of disruption of the oprD gene by mutations.

Analysis of the prophages carried by human infecting isolates provides new insight into the evolution of Group B Streptococcus species.

OBJECTIVES: Group B Streptococcus (GBS) emerged in the 1970s as a major cause of neonatal infections, and has been increasingly associated with infections in adults since the 1990s. Prophages have been suspected to have driven these epidemiological trends. We have characterized the prophages harboured by 275 human GBS isolates belonging to the major lineages. METHODS: We applied whole genome sequencing (WGS) to 14 isolates representative of the diversity within GBS species, located and identified their prophages. Using prediction tools, we searched for prophage elements potentially involved with the ability of GBS to infect humans. Using the data obtained by WGS, we designed a PCR-based tool and studied the prophage content of 275 isolates. RESULTS: WGS of the 14 isolates revealed 22 prophages (i) distributed into six groups (A-F), (ii) similar to phages and prophages from GBS and non-GBS streptococci recovered from livestock, and (iii) carrying genes encoding factors previously associated with host adaptation and virulence. PCR-based detection of prophages revealed the presence of at least one prophage in 72.4% of the 275 isolates and a significant association between neonatal infecting isolates and prophages C, and between adult infecting isolates and prophages A. CONCLUSIONS: Our results suggest that prophages (possibly animal-associated) have conditioned bacterial adaptation and ability to cause infections in neonates and adults, and support a role of lysogeny with the emergence of GBS as a pathogen in human.

Impact of Exposure of Methicillin-Resistant Staphylococcus aureus to Polyhexanide In Vitro and In Vivo.

Methicillin-resistant Staphylococcus aureus (MRSA) resistant to decolonization agents such as mupirocin and chlorhexidine increases the need for development of alternative decolonization molecules. The absence of reported severe adverse reactions and bacterial resistance to polyhexanide makes it an excellent choice as a topical antiseptic. In the present study, we evaluated the in vitro and in vivo capacity to generate strains with reduced polyhexanide susceptibility and cross-resistance with chlorhexidine and/or antibiotics currently used in clinic. Here we report the in vitro emergence of reduced susceptibility to polyhexanide by prolonged stepwise exposure to low concentrations in broth culture. Reduced susceptibility to polyhexanide was associated with genomic changes in the mprF and purR genes and with concomitant decreased susceptibility to daptomycin and other cell wall-active antibiotics. However, the in vitro emergence of reduced susceptibility to polyhexanide did not result in cross-resistance to chlorhexidine. During in vivo polyhexanide clinical decolonization treatment, neither reduced polyhexanide susceptibility nor chlorhexidine cross-resistance was observed. Together, these observations suggest that polyhexanide could be used safely for decolonization of carriers of chlorhexidine-resistant S. aureus strains; they also highlight the need for careful use of polyhexanide at low antiseptic concentrations.

Using MALDI-TOF MS typing method to decipher outbreak: the case of Staphylococcus saprophyticus causing urinary tract infections (UTIs) in Marseille, France.

Staphylococcus saprophyticus is one of the leading causes of urinary tract infections (UTI). In December 2014, our surveillance system identified an abnormal increase in S. saprophyticus causing UTIs in four university hospitals in Marseille, indicating a suspected community S. saprophyticus UTI outbreak. This was detected by our surveillance system BALYSES (Bacterial real-time Laboratory-based Surveillance System). S. saprophyticus/ Escherichia coli UTI ratio increased three-fold from 0.0084 in 2002 to 0.025 in December 2015 in Marseille with an abnormal peak in December 2014, and with an annual estimated ratio trend of 5.10-6 (p-value < 10-3). Matrix-Assisted Laser Desorption Ionisation-Time of Flight Mass Spectrometry (MALDI-TOF MS) spectral analysis of strains was used to analyse strains cluster expansion, comparing strains from Marseille to those from Nice during the same period. MALDI-TOF MS spectral analysis revealed a geographical restricted clonal expansion of the strains clusters in Marseille as compared to Nice. Our finding suggests (i) a geographically restricted expansion of a specific S. saprophyticus strain clusters circulating in Marseille, and (ii) MALDI-TOF MS can be used as a cost-effective tool to investigate an outbreak.

Imipenem heteroresistance in nontypeable Haemophilus influenzae is linked to a combination of altered PBP3, slow drug influx and direct efflux regulation.

OBJECTIVE: To investigate the potential roles of PBPs, efflux pumps and slow drug influx for imipenem heteroresistance in nontypeable Haemophilus influenzae (NTHi). METHODS: Fifty-nine NTHi clinical isolates examined in this study were collected at Geneva University Hospitals between 2009 and 2014. Alterations in PBPs were investigated by gene sequencing. To evaluate the affinities of the PBPs to imipenem, steady-state concentration-response experiments were carried out using imipenem in a competition assay with Bocillin-FL. The effect of the carbonyl cyanide m-chlorophenylhydrazone (CCCP) on imipenem susceptibility was assessed using broth dilution and viable cell counting. Using whole-genome sequencing, we explored the potential roles of outer membrane protein P2 (OmpP2), LytM proteins and the dcw gene cluster in imipenem heteroresistance. RESULTS: All 46 imipenem-heteroresistant isolates (IMIhR) harboured amino acid substitutions in the ftsI gene, which encodes PBP3, corresponding to 25 different mutation patterns that varied from the ftsI gene mutation patterns found in imipenem-susceptible isolates. Among all PBPs, the highest affinity to imipenem was documented for PBP3 (IC50, 0.004 µg/mL). Different amino acid substitutions and insertions were noted in OmpP2, suggesting a relationship with imipenem heteroresistance. The IMIhR isolates were affected by CCCP differently and displayed a higher percentage of killing by imipenem in CCCP-treated cells at concentrations ranging between 0.5 and 8 µg/mL. CONCLUSIONS: The present study provides robust evidence indicating that in combination with the altered PBP3, the slowed drug influx and its enhanced efflux due to the loss of regulation led to the development of imipenem heteroresistance in NTHi.

Comparative genomics of Neisseria meningitidis strains: new targets for molecular diagnostics.

In 2010, Jaton et al. (False-negative PCR result due to gene polymorphism: the example of Neisseria meningitidis. J Clin Microbiol 2010;48:4590-2) reported an isolate of Neisseria meningitidis serogroup B that was not detected by the ctrA quantitative real-time PCR (qRT-PCR) used in our diagnostic laboratory. Sequence analysis of ctrA revealed several single nucleotide polymorphisms responsible for the negative qRT-PCR. Therefore, we sequenced the genome of this isolate and performed comparative genomics to propose new gene targets for the specific detection of N. meningitidis from clinical specimens. We identified 11 genes as specific to N. meningitidis genomes and common to at least 177 (97%) of the 183 genomes available. Among them, three genes (metA, tauE and shlA) were selected to develop new qRT-PCRs for the detection of N. meningitidis DNA. The three qRT-PCRs were highly sensitive and specific, and they exhibited a good reproducibility when tested on plasmidic positive controls and genomic DNA extracted from strains of N. meningitidis and other relevant bacterial species. The clinical sensitivity and specificity of metA and tauE qRT-PCRs were both 100% based on a testing of cerebrospinal fluid samples positive for N. meningitidis or other clinically relevant bacteria. Despite a 100% specificity, the sensitivity of the shlA qRT-PCR was only 70%. We thus recommend using the metA and/or tauE qRT-PCRs developed here. To prevent PCR failure in the presence of new polymorphic strains, the detection of dual targets by duplex qRT-PCR would be more accurate and suitable for the diagnosis of N. meningitidis from clinical specimens.

Ampicillin-resistant Haemophilus influenzae isolates in Geneva: serotype, antimicrobial susceptibility, and ß-lactam resistance mechanisms.

The purpose of this study was to analyze the molecular mechanisms of ampicillin-resistant Haemophilus influenzae isolated in Geneva, Switzerland. We investigated the association between specific patterns of amino acid substitutions in penicillin-binding protein 3 (with or without ß-lactamase production) and ß-lactam susceptibility. Another main focus for this study was to compare the accuracy of disk diffusion and Etest methods to detect resistance to ampicillin and amoxicillin/clavulanic acid. The antibiotic susceptibility to ß-lactam antibiotics of 124 H. influenzae isolates was determined by disk diffusion and Etest methods, and interpreted by European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) breakpoints. Alterations in PBP3 were investigated by sequencing the ftsI gene. Of the 124 clinical isolates analyzed, ampicillin resistance was found in 36% (45 out of 124). The rate of resistance to amoxicillin/clavulanic acid was 9% and 0.8%, using EUCAST and CLSI breakpoints respectively. For the 78 ß-lactamase negative ampicillin-susceptible (BLNAS) isolates for which the Etest method indicated a high degree of susceptibility (MIC ≤ 1 mg/L), the disk diffusion method revealed resistance to ampicillin and amoxicillin/clavulanic acid in 33 cases (42%). Most common amino acid substitutions were Asn526Lys and Val547Ile, followed by Asp569Ser, Ala502Val, Asp350Asn, Met377Ile, Ile449Val, and Arg517His. The patterns observed were classified into six groups (IIa, IIb, IIc, IId, III-like, and miscellaneous). Continued characterization of both invasive and respiratory H. influenzae isolates is necessary in order to observe changes in the microbiology and epidemiology of this pathogen that could lead to clinical failure when treated by empirical antibiotic therapy.

CTX-M-15-producing Morganella morganii from Hôpital Principal de Dakar, Senegal.

We report the detection and molecular characterization of extended spectrum ß-lactamases in a series of 112 clinical isolates of Enterobacteriaceae from the Hôpital Principal de Dakar, Senegal, including five CTX-M-15-producing Morganella morganii isolates, which are reported for the first time in this country.

Carbapenemase genes and genetic platforms in Gram-negative bacilli: Enterobacteriaceae, Pseudomonas and Acinetobacter species.

The emergence and rapid spread of carbapenemases in Enterobacteriaceae, Pseudomonas and Acinetobacter (EPA) species is becoming a major public health crisis worldwide, and is responsible for large number of hospital-acquired and nosocomial infections. In this article, we review the current knowledge on the classification, phylogeny and genetic platforms of the main carbapenemases already described in Gram-negative bacteria.

[Bacterial genomics and metagenomics: clinical applications and medical relevance]. / Génomique et métagénomique bactériennes: applications cliniques et importance médicale.

New sequencing technologies provide in a short time and at low cost high amount of genomic sequences useful for applications such as: a) development of diagnostic PCRs and/or serological tests; b) detection of virulence factors (virulome) or genes/SNPs associated with resistance to antibiotics (resistome) and c) investigation of transmission and dissemination of bacterial pathogens. Thus, bacterial genomics of medical importance is useful to clinical microbiologists, to infectious diseases specialists as well as to epidemiologists. Determining the microbial composition of a sample by metagenomics is another application of new sequencing technologies, useful to understand the impact of bacteria on various non-infectious diseases such as obesity, asthma, or diabetes. Genomics and metagenomics will likely become a specialized diagnostic analysis.

Nutrition advocacy and national development: the PROFILES programme and its application.

Investment in nutritional programmes can contribute to economic growth and is cost-effective in improving child survival and development. In order to communicate this to decision-makers, the PROFILES nutrition advocacy and policy development programme was applied in certain developing countries. Effective advocacy is necessary to generate financial and political support for scaling up from small pilot projects and maintaining successful national programmes. The programme uses scientific knowledge to estimate development indicators such as mortality, morbidity, fertility, school performance and labour productivity from the size and nutritional condition of populations. Changes in nutritional condition are estimated from the costs, coverage and effectiveness of proposed programmes. In Bangladesh this approach helped to gain approval and funding for a major nutrition programme. PROFILES helped to promote the nutrition component of an early childhood development programme in the Philippines, and to make nutrition a top priority in Ghana's new national child survival strategy. The application of PROFILES in these and other countries has been supported by the United States Agency for International Development, the United Nations Children's Fund, the World Bank, the Asian Development Bank, the Micronutrient Initiative and other bodies.

PIP: This paper describes a database approach to nutrition policy development and advocacy. The PROFILES nutrition advocacy and policy development program was applied in such developing countries as Bangladesh, Philippines, and Ghana. It has been used to increase awareness among decision-makers of the need for greater investment in nutrition, to facilitate the design and selection of programs, and to promote particular interventions that were already being designed. The program uses scientific knowledge to estimate development indicators such as mortality, morbidity, fertility, school performance, and labor productivity from the size and nutritional condition of populations. Nutritional condition changes were estimated from the costs, coverage and effectiveness of proposed programs. This nutritional program contributes to economic growth and is cost-effective in improving child survival and development. Thus, this approach has been successful: in Bangladesh, it played a vital role in bringing the government to accept the young child nutrition program and obtaining approval for assisted funding; in the Philippines, PROFILES also helped in developing and advocating an early childhood development program that included a large nutrition component in combination with health, family planning and educational components; in Ghana, it helped make nutrition the government's top priority, as evidenced by the Ministry of Health's new child survival strategy.

[Our experience with laminectomy in the treatment of spinal cord disease caused by cervical arthrosis: apropos of 42 cases]. / Notre expérience de la laminectomie dans le traitement de la myélopathie par arthrose cervicale: àpropos de 42 cas.

Cervical spondylotic arthRosis may lead to a myelopathy and the question which can rise is: posterior surgical decompression is adequate regarding the others surgical technics discussed? In this paper the authors describe the postoperative results of 42 patients who have been operated on by laminectomy from 1971 to 1995 at Fann Hospital in Dakar. The median age was 42 years and the median delay between the onset of neurological disorders and operation was 5.9 months. All patients showed symptoms of spinal cord and root disturbances. Neuro-imaging studies with plan X-rays and myelography reveal arthrosis and the impact of this arthrosis on nervous system. With a follow up of 6 months we found a mortality rate of 4.7% (two cases of death), 66.8% of good results and 28.5% of fail. Eighteen months post surgery results were respectively 52.3% and 45.7% of good and poor results. The complications of the laminectomy were peri operative hemorrhage (19%); sepsis (21.4%); spine cord traumatic injuries (9.5%) spondylolisthesis (4.7%). Laminectomy has been found to improve "spine syndrome"; paresthesia; fasciculations, and sensory deficits. Also three level laminectomy and surgery which can take place before six months are good prognosis factors. The authors stressed on laminectomy because of our low medical care situations particularly that no other surgical procedures through the modern literature leads to better results.