Characterization of Neonatal Infections by Gram-Negative Bacilli and Associated Risk Factors, Havana, Cuba.

Infections represent an important problem in neonates because of the high mortality. An increase in neonatal infections has been found in Cuban hospitals in recent years. The aim of this study was to provide evidence on the clinical and microbiological behavior of Gram-negative bacilli that cause neonatal infections in hospitals of Havana, Cuba. It was carried out as a descriptive cross-sectional investigation from September 2017 to July 2018 in The Tropical Medicine Institute "Pedro Kouri" (IPK). Sixty-one Gram-negative bacilli isolated from neonates with infections in six Gyneco-Obstetric and Pediatric Hospitals of Havana were analyzed for their species and antimicrobial susceptibility. Late-onset infections were more common than early-onset ones and included urinary tract infection in the community (87%) and sepsis in hospitals (63.3%). Catheter use (47%) and prolonged stay (38%) were the most frequent risk factors. Species of major pathogens were Escherichia coli (47%) and Klebsiella spp. (26%). The isolated Gram-negative bacilli showed high resistance rates to third-generation cephalosporins, ciprofloxacin and gentamicin, while being more susceptible to carbapenems, fosfomycin, colistin and amikacin. The present study revealed the clinical impact of Gram-negative bacilli in neonatology units in hospitals of Havana. Evaluation of antimicrobial susceptibilities to the isolates from neonates is necessary for selection of appropriate empirical therapy and promotion of the rational antibiotic use.

Comparación de dos métodos comerciales para la determinación de la concentración mínima inhibitoria de meropenem en Klebsiella pneumoniae productora de carbapenemasa tipo KPC / Comparison of two commercial methods for determination of the minimum inhibitory meropenem concentration in KPC carbapenemase-producing Klebsiella pneumoniae

Introducción: El tratamiento de las infecciones por Klebsiella pneumoniae productora de carbapenemasa tipo KPC es complicado debido a las escasas opciones terapéuticas existentes, lo cual obliga a optimizar los esquemas terapéuticos disponibles. Objetivo: Determinar la concordancia de la tarjeta AST-N272 del Sistema Vitek 2 Compact y las tiras M.I.C.ETM Evaluator con la dilución en agar para la determinación de la concentración mínima inhibitoria del meropenem en Klebsiella pneumoniae productora de carbapenemasa tipo KPC. Métodos: Se estudiaron 53 aislados de K. pneumoniae bla KPC positivas no clonales, provenientes de hisopados rectales recolectados en diferentes unidades hospitalarias de Guayaquil, Ecuador, entre enero a junio de 2016. Se determinó la concentración mínima inhibitoria de meropenem por dilución en agar (método de referencia), así como por el sistema Vitek 2 Compact (AST-N272) y las tiras M.I.C.ETM. Se determinó la CMI 50, CMI 90 y la concordancia esencial. Resultados: El rango de la CMI de meropenem de los aislados estudiados fue de 1 a ≥ 32 µg/mL, con una CMI50= 4 µg/mL y una CMI90= ≥ 32 µg/mL. El 86,79 por ciento (n= 46) de los aislados tuvo una CMI≤ 8 µg/mL. Se observó un 94,33 por ciento de concordancia esencial con las tiras M.I.C.ETM, mientras que la tarjeta AST-N272 mostró una concordancia esencial inferior al 50 por ciento. Conclusiones: Los resultados sugieren posibles implicaciones en el tratataminto del paciente, pues reduce opciones terapéuticas en contextos de difícil manejo. Además, resaltan la necesidad de la confirmación de la resistencia a carbapenémicos mediante el método de Kirby Bawer en aquellos laboratorios que tienen métodos automatizados para estudios de susceptibilidad(AU)

Introduction: The treatment for KPC carbapenemase-producing Klebsiella pneumoniae infections is complicated, due to the scant therapeutic options available, which forces us to optimize the therapies at hand. Objective: Determine the agreement between the AST-N272 card of the Vitek 2 Compact system and the M.I.C.E.TM Evaluator strips, and the agar dilution method for determination of the minimum inhibitory meropenem concentration in KPC carbapenemase-producing Klebsiella pneumoniae. Methods: A study was conducted of 53 positive non-clonal K. pneumoniae bla KPC isolates from rectal swabs collected at several hospitals in Guayaquil, Ecuador, from January to June 2016. Minimum inhibitory meropenem concentration was determined by agar dilution (reference method), the Vitek 2 Compact system (AST-N272) and M.I.C.E.TM strips. Determination was made of MIC 50, MIC 90 and essential agreement. Results: The meropenem MIC range for the isolates studied was 1 to ≥ 32 µg/ml, with MIC50= 4 µg/ml and MIC90= ≥ 32 µg/ml. In 86.79 percent (n= 46) of the isolates MIC was ≤ 8 µg/ml. Essential agreement was 94.33 percent with the M.I.C.E.TM strips and under 50 percent with the AST-N272 card. Conclusions: The results obtained suggest potential implications for the treatment of patients, since therapeutic options are reduced in difficult management contexts. They also highlight the need for confirmation of carbapenem resistance by the Kirby-Bauer procedure in laboratories equipped with automated methods for susceptibility studies(AU)

High Prevalence of CTX-M Type Extended-Spectrum Beta-Lactamase Genes and Detection of NDM-1 Carbapenemase Gene in Extraintestinal Pathogenic Escherichia coli in Cuba.

Increase of extraintestinal pathogenic Escherichia coli (ExPEC) showing resistance to beta-lactams is a major public health concern. This study was conducted as a first molecular epidemiological study on ExPEC in Cuba, regarding prevalence of extended-spectrum beta-lactamases (ESBLs) and carbapenemase genes. A total of 306 ExPEC isolates collected in medical institutions in 16 regions in Cuba (2014-2018) were analyzed for their genotypes and presence of genes encoding ESBL, carbapenemase, plasmid-mediated quinolone resistance (PMQR) determinants by PCR and sequencing. The most common phylogenetic group of ExPEC was B2 (49%), followed by D (23%), A (21%), and B1 (7%). Among ESBL genes detected, blaCTX-M was the most common and detected in 61% of ExPEC, with blaCTX-M-15 being dominant and distributed to all the phylogenetic groups. NDM-1 type carbapenemase gene was identified in two isolates of phylogenetic group B1-ST448. Phylogenetic group B2 ExPEC belonged to mostly ST131 (or its single-locus variant) with O25b allele, harboring blaCTX-M-27, and included an isolate of emerging type ST1193. aac (6')-Ib-cr was the most prevalent PMQR gene (40.5%), being present in 54.5% of CTX-M-positive isolates. These results indicated high prevalence of CTX-M genes and the emergence of NDM-1 gene among recent ExPEC in Cuba, depicting an alarming situation.

High clonal diversity in a non-outbreak situation of clinical ESBL-producing Klebsiella pneumoniae isolates in the first national surveillance program in Cuba.

This work summarized the results obtained in an institutional Klebsiella pneumoniae surveillance program recently implemented in Cuba. Eighteen hospitals from five regions provided a total of 228 K. pneumoniae isolates (164 from admitted patients, four from hospital environmental sources, and 60 isolates from community patients). The genetic relationship was assessed by pulsed-field gel electrophoresis and multilocus sequence typing. Antimicrobial susceptibility was determined by the agar dilution method, and bla(ESBL) genes were sequenced. Fifty four K. pneumoniae isolates were extended-spectrum ß-lactamases (ESBL)-producers (23.6%), mostly due to the CTX-M-15 enzyme (79.6%). ESBL isolates were grouped in 27 different sequence types (STs), being the most prevalent ST15 (15%), ST152 (13%), and both ST48 and ST147 (11%, respectively). Community-acquired criteria could be demonstrated in 60 patients (26%) corresponding to urological (33%), wound (27%), respiratory (27%), and otic (13%) infections. Population structure analysis showed that our isolates corresponded to a highly polyclonal population with 10 nonpreviously described STs, demonstrating the importance of local epidemiological studies. We report the first data of the population structure of ESBL-producing K. pneumoniae isolates obtained in a national multicenter surveillance Cuban program. Results showed that a highly polyclonal ESBL-producer K. pneumoniae population was mainly due to CTX-M-15 carriage, whereas carbapenemases production was not present.

Characterization of Enterococcus faecium with macrolide resistance and reduced susceptibility to quinupristin/dalfopristin in a Japanese hospital: detection of extensive diversity in erm(B)-regulator regions.

Cross-resistance to macrolide, lincosamide, and streptogramin B (MLSB) antibiotics is mainly mediated by the erm (erythromycin ribosome methylation) genes that encode 23S rRNA methylases in enterococi, and various mechanisms are involved in the streptogramin B resistance. Prevalence of MLSB resistance and its genetic mechanisms were analyzed for a total of 159 strains of Enterococcus faecium isolated from clinical specimens in a university hospital in Japan from 1997 to 2006. Resistance to erythromycin (EM) and clindamycin was detected in 88.1% and 89.9% of all the strains examined, respectively, and expression of resistance was totally constitutive. Although none of the strain was resistant to quinupristin/dalfopristin (Q/D), 28 strains (17.6%) showed intermediate resistance to Q/D (MIC: 2 µg/ml). The erm(B) gene was detected in 139 strains (87.4%), and msrC was found in all the strains examined, whereas no other known MLSB resistance genes were identified. The erm(B) regulator region (RR) containing a coding region of the leader peptide was classified into 13 genetic variations (L1-L3, M, S1-S7, D, and R genotypes) in 56 strains. However, no relatedness was identified between the erm(B) RR genotype and EM resistance, or reduced susceptibility to Q/D, although most of Q/D-intermediate strains were assigned to the L1, L2, and S1 genotypes. Q/D-intermediate strains were classified into five multiple-locus variable-number tandem-repeat analysis (MLVA) types, including four types of clonal complex (CC)-C1, five sequence types (STs), including four STs of CC-17, and several resistance gene/virulence factor profiles. The present study revealed the occurrence of Q/D-intermediate E. faecium, which are composed of heterogeneous strains in Japan, and more genetic diversity in the erm(B) RRs than those reported previously.

Molecular epidemiologic analysis of Enterococcus faecalis isolates in Cuba by multilocus sequence typing.

We carried out the first study of Enterococcus faecalis clinical isolates in Cuba by multilocus sequence typing linking the molecular typing data with the presence of virulence determinants and the antibiotic resistance genes. A total of 23 E. faecalis isolates recovered from several clinic sources and geographic areas of Cuba during a period between 2000 and 2005 were typed by multilocus sequence typing. Thirteen sequence types (STs) including five novel STs were identified, and the ST 64 (clonal complex [CC] 8), ST 6 (CC2), ST 21(CC21), and ST 16 (CC58) were found in more than one strain. Sixty-seven percent of STs corresponded to STs reported previously in Spain, Poland, and The Netherlands, and other STs (ST115, ST64, ST6, and ST40) were genetically close to those detected in the United States. Prevalence of both antimicrobial resistance genes [aac(6')-aph(2''), aph(3'), ant(6), ant(3'')(9), aph(2'')-Id, aph(2'')-Ic, erm(B), erm(A), erm(C), mef(A), tet(M), and tet(L)] and virulence genes (agg, gelE, cylA, esp, ccf, and efaAfs) were examined by polymerase chain reaction. Aminoglycoside resistance genes aac(6')-Ie-aph(2'')-Ia, aph(3'), ant(6), ant(3'')(9) were more frequently detected in ST6, ST16, ST23, ST64, and ST115. The multidrug resistance was distributed to all STs detected, except for ST117 and singleton ST225. The presence of cyl gene was specifically linked to the ST64 and ST16. Presence of the esp, gel, and agg genes was not specific to any particular ST. This research provided the first insight into the population structure of E. faecalis in Cuba, that is, most Cuban strains were related to European strains, whereas others to U.S. strains. The CC2, CC21, and CC8, three of the biggest CCs in the world, were evidently circulating in Cuba, associated with multidrug resistance and virulence traits.

Genetic diversity of enterococci harboring the high-level gentamicin resistance gene aac(6')-Ie-aph(2'')-Ia or aph(2'')-Ie in a Japanese hospital.

Prevalence of high-level gentamicin resistance genes aac(6')-Ie-aph(2'')-Ia and aph(2'')-Ie, which encode distinct aminoglycoside-modifying enzymes, was analyzed for a total of 1128 clinical isolates of enterococci obtained in a Japanese hospital during a period between 1997 and 2007. The aac(6')-Ie-aph(2'')-Ia was detected in 40.1%, 12.9%, and 3.6% of Enterococcus faecalis, E. faecium, and other enterococcal species, respectively, and aph(2'')-Ie was detected in 3.3% of E. faecium. During the study period, detection rate of aac(6')-Ie-aph(2'')-Ia in E. faecium increased from 4% (1997-1998) to 28% (2006-2007), whereas generally constant in E. faecalis. By the analysis of IS256-flanking patterns of aac(6')-Ie-aph(2'')-Ia, truncated forms of Tn5281 lacking IS256 at the 3'-end, 5'-end, and both ends of aac(6')-Ie-aph(2'')-Ia were identified in 4.6%, 32.4%, and 34.2% of E. faecalis strains, respectively, while the composite Tn5281-like element with IS256 at both sides was detected in 28.7% of the strains. A truncated form of Tn5281 lacking IS256 at the 5'-end was predominant in other enterococcal species. Among 14 E. faecalis and 10 E. faecium strains harboring aac(6')-Ie-aph(2'')-Ia, 8 and 6 different sequence types (STs) were identified by multilocus sequence typing, respectively. Some E. faecalis STs (ST4, ST16, ST64, and ST223) were found in more than one strain, and ST4 and ST64 were associated with different IS256-flanking patterns. STs of five among six E. faecium strains with aac(6')-Ie-aph(2'')-Ia (ST78, ST203, and ST418) belonged to the clonal complex (CC)17, which is known as globally emerging lineage of vancomycin- or ampicillin-resistant E. faecium clones. E. faecium strains with aph(2'')-Ie were classified into newly assigned STs, ST426, and its single locus variant ST427, which also belonged to CC17. Therefore, it was suggested that E. faecium of CC17 is prone to acquire high-level gentamicin resistance genes, and aph(2'')-Ie is distributed to specific E. faecium clones that are distinct from those having aac(6')-Ie-aph(2'')-Ia.

Genetic diversity of the low-level vancomycin resistance gene vanC-2/vanC-3 and identification of a novel vanC subtype (vanC-4) in Enterococcus casseliflavus.

An intrinsic low-level vancomycin resistance (VanC phenotype) in Enterococcus casseliflavus is conferred by either of two subtypes of vanC genes, that is, vanC-2 or vanC-3, which are genetically closely related. To know genetic diversity of vanC-2/C-3 genes among E. casseliflavus, nucleotide sequences of vanC-2/C-3 and other genetic components in vanC gene cluster (vanXYc, vanTc, vanRc, and vanSc) were analyzed for nine clinical isolates and four standard strains that showed low-level vancomycin resistance. While the vanC-2/C-3 gene sequences showed 93-100% identities among the strains examined, two genetic groups were discriminated by phylogenetic analysis: one closely related to the previously reported vanC-2 or vanC-3 genes (vanC-2/C-3 genotype) with 98-100% identity, and the other distinct from the vanC-2/C-3 genotype (93-95% identity). The latter group found in three clinical isolates was considered as a new subtype of vanC and tentatively designated as vanC-4. Between strains with the vanC-2/C-3 genotype and those with vanC-4, vanXYc genes were also genetically discriminated with 92-93% identity. Similar sequence diversity was observed for vanTc, vanRc, and vanSc (88-93% identity). Clonal relatedness among the E. casseliflavus strains was investigated by phylogenetic analysis of atpA gene. While among E. casseliflavus strains with vanC-2/C-3 genotype, extremely high sequence identities of atpA were found (98.7% or higher), these strains showed slightly lower identity to those with vanC-4 (94-96%). These two groups of E. casseliflavus strains were also discriminated by genotyping with arbitrarily primed PCR. These findings indicated that among E. casseliflavus there are at least two genetic lineages with the distinct vanC genes, that is, a single subtype including previously known vanC-2/C-3, and a novel subtype vanC-4.

Enzyme-linked immunosorbent assay for quantitative determination of capsular polysaccharide production in Streptococcus pneumoniae clinical isolates.

A simple, specific, sensitive and reproducible ELISA has been developed to quantify the level of CPS (capsular polysaccharide) production in supernatants of Streptococcus pneumoniae cell cultures. CPSs from Strep. pneumoniae have been widely used as vaccine antigens. The quantification method is based on two type-23F serotype-specific polyclonal antibodies: IgG, purified from sera of mice immunized with a pneumococcal type-23F CPS conjugate, used in the coating step, and a serotype-specific rabbit serum as the second antibody. Solutions of purified type-23F CPS were used as standards. The relationship between A(492) and type-23F CPS concentration was linear over the range 1-310 ng/ml (r=0.989), with 1 ng/ml as the lower limit of sensitivity. The specificity of ELISA was assessed because purified type-19F CPS and cell-wall polysaccharide samples were not detected after their evaluation by the ELISA described in the present study. Repeatability and intermediate precision of the assay were good, the coefficients of variation being 3 and 10% respectively. This ELISA allowed selection of an appropriate vaccine strain, for a natural polysaccharide vaccine, among several 23F pneumococcal clinical isolates and constituted a valuable analytical tool for Strep. pneumoniae fermentation and CPS purification follow-up.

Nasopharyngeal colonization by Moraxella catarrhalis and study of antimicrobial susceptibility in healthy children from Cuban day-care centers.

The prevalence of nasopharyngeal carriage of Moraxella catarrhalis was determined for the first time in Cuba. One-hundred fifty healthy children attending three day-care centers in the municipality of Marianao, Havana City were studied. The percentage of recovering bacteria in nasal and pharyngeal swabs was compared. Antimicrobial susceptibilities to ampicillin, trimethoprim-sulfamethoxazole, tetracycline, cefotaxime, ceftriaxone, chloramphenicol, erythromycin, azithromycin, amoxicillin/clavulanate, and norfloxacin were determined by the disk diffusion method according to recommendations of the National Committee for Clinical Laboratory Standards. Sixty-five percent of the children studied carried Moraxella catarrhalis. The nasal cavity was the main isolation site for this organism (81% of positive cultures). Most strains were highly susceptible to the antimicrobial agents tested, except to ampicillin (53.6% resistance). This study provides evidence of the need for continued surveillance of antimicrobial susceptibility of Moraxella catarrhali, in order to determine optimal empiric therapy for community-acquired respiratory tract infections produced by this pathogen.

Enterococci spp. isolated from Cuba: species frequency of occurrence and antimicrobial susceptibility profile.

This study investigated the species distribution and antimicrobial resistance among 99 enterococci isolated from hospitalized patients in 12 hospitals in Cuba from October 2000 to September 2001. Species identification was performed by WIDER Automatic System (Francisco Soria Melguizo, Madrid, Spain), and the susceptibility testing was performed by disk diffusion, agar dilution, and E-test methods. Enterococcus faecalis was the most prevalent (85%) species, followed by E. faecium (10%), E. gallinarum (2%), E. casseliflavus (2%), and E. durans-hirae (1%). A higher percentage of resistance to ampicillin (50%), fosfomycin (40%), ciprofloxacin (30%), norfloxacin (20%), and tetracycline (90%) was detected in E. faecium isolates, whereas E. faecalis strains showed higher rates of resistance to erythromycin (52.4%), chloramphenicol (34.5%), rifampicin (62.5%), moxifloxacin (3%), and nitrofurantoin (2.4%). Resistance to glycopeptide was detected in E. faecalis (1.2%) and E. faecium (10%). Thirty-one E. faecalis (37%) and 3 E. faecium (30%) showed a high-level resistance to gentamicin. The results of this work will be very helpful to guide empirical antimicrobial therapy and the implementation of infection control measures in Cuban hospitals.

Portadores nasales de Staphylococcus aureus resistente a la meticilina entre niños cubanos que asisten a círculos infantiles / Nasal carriers of methicillin-resistant Staphylococcus aureus among cuban children attending day-care centers

FUNDAMENTO. Recientes pero escasos informes de cepas de Staphylococcuss aureus resistentes a la meticilina (SARM) entre niños no expuestos a los factores de riesgos asociados con su adquisición, nos indujeron a investigar su circulación en la comunidad. PACIENTES Y MÉTODOS. Durante los meses de septiembre y octubre de 1997 se tomaron exudados nasales y faríngeos a 358 niños menores de 5 años, atendidos en tres círculos infantiles del municipio Marianao de la Ciudad de La Habana, Cuba. Las cepas de S. aureus aisladas fueron caracterizadas por susceptibilidad a antimicrobianos utilizando el método de Kirby-Bauer. Se confirmó la resistencia a la meticilina a través del método " Oxacillin Salt-Agar Screening-Plate" recomendado por el Comité Nacional de Procedimientos Estándar del Laboratorio Clínico. RESULTADOS. El 18,7 por ciento de los niños portaban cepas de S. aureus en el tracto respiratorio superior. En el 2,2 por ciento eran cepas de SARM. Los índices más altos de resistencia se revelaron para la eritromicina (50,7 por ciento) y tetraciclina (29,9 por ciento). Todas las cepas fueron susceptibles a la ciprofloxacina. CONCLUSIONES. Nuestros resultados proporcionan evidencias sobre el aislamiento de cepas de SARM entre niños sanos atendidos en círculos infantiles, e indican la posibilidad de que éstas se establezcan y diseminen de forma acelerada en la comunidad (AU)