Laboratory surveillance of invasive Haemophilus influenzae disease in Argentina, 2011-2019

Abstract The incorporation of Haemophilus influenzae type b (Hib) vaccine into the Argentine National Immunization Program in 1998 resulted in a dramatic decrease in the incidence of invasive disease due to this serotype. We assessed 1405 H. influenzae (Hi) isolates causing invasive infections referred to the National Reference Laboratory between 2011 and 2019. Non-encapsulated Hi were the most common strains (44.5%), followed by types b (41.1%) and a (10.0%). Significant increase in the proportion of type b was observed, from 31.2% in 2011, to 50% in 2015, correlating with the peak incidence rate, later decreasing to 33.6% by 2019. We compared the genetic relationship between clones circulating during the period of increased Hib incidence (2011-2015) and those of the prevaccination-transition period (1997-1998). Four pulsotypes predominated in both periods, G, M, P and K, G being the most common. Multilocus sequence typing revealed that the 4 pulsotypes belonged to ST6, or one of its simple or double locus variants. Isolates from fully vaccinated individuals did not differ from those of the rest of the population studied. After ruling out aspects associated with emergence of specific clones, we concluded that factors such as low booster coverage rates, delayed vaccination schedules and use of different vaccines may have contributed to the reemergence of Hib infections.

Resumen La introducción de la vacuna contra Haemophilus influenzae tipo b (Hib) en el Programa Nacional de Inmunización de Argentina en 1998 produjo una drástica disminución de la incidencia de enfermedad invasiva causada por este serotipo. En el Laboratorio Nacional de Referencia se estudiaron 1405 aislamientos de H. influenzae causantes de enfermedad invasiva recibidos en el período 2011-2019. H. influenzae no capsulado fue el más frecuente (44,5%), seguido por los tipos b (41,1%) y a (10,0%). Se observó un aumento significativo de la proporción del tipo b, de 31,2% en 2011 a 50% en 2015, que se correlacionó con un pico de incidencia en ese mismo año. Hacia 2019, descendió a 33,6%. Con el objetivo de evaluar los clones circulantes durante el incremento de la proporción de Hib y comparar con el período prevacunal-transición, se determinó la relación genética de una selección de aislamientos de los períodos 1997-1998 y 2011-2015. El análisis por PFGE mostró 4 pulsotipos predominantes en los 2 períodos, G, M, P y K, y el pulsotipo G fue mayoritario en ambos períodos. Por MLST se demostró que los 4 pulsotipos pertenecieron al ST6 o sus variantes (simple o doble locus). Entre los aislamientos de pacientes con vacunación completa no se hallaron clones diferentes respecto del resto de la población. Se postula que las coberturas de vacunación no satisfactorias en las dosis de refuerzo, los esquemas atrasados y el uso de diferentes vacunas pudieron haber contribuido a la reemergencia de Hib.

Laboratory surveillance of invasive Haemophilus influenzae disease in Argentina, 2011-2019.

The incorporation of Haemophilus influenzae type b (Hib) vaccine into the Argentine National Immunization Program in 1998 resulted in a dramatic decrease in the incidence of invasive disease due to this serotype. We assessed 1405 H. influenzae (Hi) isolates causing invasive infections referred to the National Reference Laboratory between 2011 and 2019. Non-encapsulated Hi were the most common strains (44.5%), followed by types b (41.1%) and a (10.0%). Significant increase in the proportion of type b was observed, from 31.2% in 2011, to 50% in 2015, correlating with the peak incidence rate, later decreasing to 33.6% by 2019. We compared the genetic relationship between clones circulating during the period of increased Hib incidence (2011-2015) and those of the prevaccination-transition period (1997-1998). Four pulsotypes predominated in both periods, G, M, P and K, G being the most common. Multi-locus sequence typing revealed that the 4 pulsotypes belonged to ST6, or one of its simple or double locus variants. Isolates from fully vaccinated individuals did not differ from those of the rest of the population studied. After ruling out aspects associated with emergence of specific clones, we concluded that factors such as low booster coverage rates, delayed vaccination schedules and use of different vaccines may have contributed to the reemergence of Hib infections.

High Prevalence of Klebsiella pneumoniae Infections in AnHui Province: Clinical Characteristic and Antimicrobial Resistance.

BACKGROUND: Klebsiella pneumoniae (K. pneumoniae) causes community-acquired and hospital-acquired pneumonia. The mortality rates of invasive infections caused by hypervirulent K. pneumoniae (HvKP) are extremely high. However, the microbiological characteristics and clinical manifestations of K. pneumoniae in AnHui province still remain unclear. PURPOSE: To show the high prevalence of HvKP infections regarding clinical characteristics and antimicrobial resistance in Anhui province. PATIENTS AND METHODS: A retrospective analysis was conducted to study the clinical data of 115 strains of K. pneumoniae from July 2019 to March 2020 in The First Affiliated Hospital of AnHui Medical University. The virulence genes, capsular types, carbapenemase genes, and molecular subtypes of these hypervirulent isolates were detected. RESULTS: Overall, 59.1% (68/115) cases were HvKP infections, mainly from the department of intensive care unit (ICU, n=14, 20.6%) and the department of respiratory and critical care (n=13, 19.1%). K2 was the most prevalent capsular serotype (n=26), followed by K1 (n=21). The results of MLST identification of 68 strains showed that ST23 (n=15, 22.1%) was the most common type of ST, followed by ST11 and ST65 (n=12, 17.6%), ST86 (n=9, 13.2%), and ST412 (n=6, 8.8%). Among 68 hvKP strains, 12 isolates were carbapenem resistant, and all except two harboured KPC. CONCLUSION: The high incidence of carbapenemase producing HvKP in the Anhui province, especially the higher mortality of HvKP, should be paid more attention. Meanwhile, epidemiological surveillance and clinical treatment strategies should be continuously determined and implemented.

Capsular type diversity of Mannheimia haemolytica determined by multiplex real-time PCR and indirect hemagglutination in clinical isolates from cattle, sheep, and goats in Spain.

This study compares the utility of a commercially available multiplex q-PCR assay for serotyping A1, A2, and A6 M. haemolytica serotypes with indirect hemagglutination, for determining the relative distribution of M. haemolytica capsular types associated with respiratory disorders in cattle, sheep, and goats. For the 129 isolates analyzed, both q-PCR and IHA assays exhibited nearly complete agreement for capsular types A1 (k = 0.965) and A2 (k = 0.888) and substantial agreement for A6 (k = 0.801). Despite the overall good performance of the commercial q-PCR, its effectiveness differed between the host origin of the isolates. The serotype was identified by q-PCR in 83.3 % of cattle, 77.8 % of goat, and 53.8 % of sheep isolates. Combining the results of both methods, A1 was the most prevalent in cattle and sheep (55.6 % and 22.25 %, respectively) but was not detected in goats, A2 was the most prevalent in goats (61.1 %) and the second most prevalent in cattle (16.7 %) and sheep (20.5 %). The prevalence of A6 was 7.4 %, 5.1 %, and 16.7 % in cattle, sheep, and goats, respectively. Other capsular types determined exclusively by IHA were A16 in cattle, A9 in goats, and A7, A8, A9, and A13 in sheep. Capsular type diversity was greater in sheep (H = 0.601) than in cattle (H = 0.408) and goat (H = 0.330) isolates. The commercial multiplex q-PCR is a valuable tool, alternative to IHA, for identifying isolates of capsular types A1, A2, and A6, the most frequent serotypes of M. haemolytica associated with respiratory disease in ruminants. However, when testing sheep isolates it should be complemented with immunological assays due to the wider range of serotypes implicated.

Fourier transform infrared spectroscopy: unlocking fundamentals and prospects for bacterial strain typing.

The need to identify highly related bacterial strains is ancient in clinical, industrial, or environmental microbiology. Strategies based on different phenotypic and genotypic principles have been used since the early 1930s with variable outcomes and performances, accompanying the evolution of bacterial features' knowledge as well as technologies, instruments, and data analysis tools. Today, more than ever, the implementation of bacterial typing methods that combine a high reliability and accuracy with a rapid, low-cost, and user-friendly performance is highly desirable, especially for clinical microbiology. FT-IR developments for bacterial discrimination at the infra-species level settled on the identification of bacterial groups previously defined by phenotypic or genotypic typing methods. Therefore, this review provides a brief historical overview of main bacterial strain typing methods, and a comprehensive analysis of the fundamentals and applications of Fourier transform infrared spectroscopy, a phenotypic-based method with potential for routine strain typing. The different studies on FT-IR-based strain typing of diverse Gram-negative and Gram-positive bacterial species are discussed in light of genotypic, phenotypic, and biochemical aspects, in order to definitively give this methodology credit to be widely accepted by microbiologists. Importantly, the discriminatory biochemical fingerprints observed on FT-IR spectra have been consistently correlated with sugar-based coating structures that besides reflecting strain variation are also of high relevance for the specificity in pathogen-host interactions. Thus, FT-IR-based bacterial typing might not only be useful for quick and reliable strain typing but also to help understanding the diversity, evolution, and host adaptation factors of key bacterial pathogens or subpopulations.

Characterization of Pasteurella multocida involved in rabbit infections.

In rabbit, P. multocida is considered a predominant pathogenic agent; despite this, few data on the molecular epidemiology are available so far. The aim of this work was to characterize P. multocida isolates from rabbit affected by various diseases in Italy. Comparison was made to reference strains from other countries. Thirty-nine isolates were tested using PCRs to detect the genes coding capsular antigens, virulence factors and lipopolysaccharide structures (LPS). Multilocus sequence typing (MLST) was performed and 19 STs registered that belonged to 9 clonal complexes. Italian isolates were all related to P. multocida subsp. P. multocida. Three sequence types dominated (ST9, ST50 and ST74). The isolates were assigned to capsular types A (20/39), D (9/39) and F (10/39), to virulence genes pfhA (13/39), hgbB (21/39) and pfhA+hgbB (4/39) (one without virulence factors) and the isolates either belonged to the LPS genotypes 3 (22/39) or 6 (17/39). The clonal relationships of the Italian strains from rabbit had similarity to previously reported rabbit isolates that belonged to ST9, ST74, ST204 and ST206, however, they differed from other rabbit references strains that belonged to six other STs. In particular, ST9 with capsular type F has been previously reported from diseased rabbit in Czech Republic and ST74 has been observed for older rabbit isolates. ST50 has probably been reported from Spain. ST9 and ST50 have previously also been reported from birds and pig, respectively, whereas ST74 has exclusively been reported from pig. It remains to be investigated if the isolates obtained from diseased rabbit in Italy represent introductions from other host or they are primarily of rabbit origin.

A capsule/lipopolysaccharide/MLST genotype D/L6/ST11 of Pasteurella multocida is likely to be strongly associated with swine respiratory disease in China.

Pasteurella multocida is a leading cause of respiratory disease in pigs worldwide. In this study, we determined the genetic characteristics of 115 P. multocida isolates from the lungs of pigs with respiratory disease in China in 2015 using capsular typing, lipopolysaccharide (LPS) genotyping, and virulence genotyping based on the detection of virulence-associated genes. The results showed that the isolates belonged to three capsular types: A (49.6%), D (46.1%), and nontypable (4.3%); and two LPS genotypes: L3 (22.6%) and L6 (77.4%). When combining the capsular types with the LPS genotypes, a genotype group D: L6 (46.1%) was the most prevalent among the strains. Among the 23 virulence-associated genes detected in this study, a small number of them displayed a certain level of "genotype-preference". We found that pfhA, hgbA, and hgbB had a close association with P. multocida LPS genotypes, while tadD was more associated with P. multocida capsular types. In addition, multilocus sequence typing (MLST) on 40 P. multocida isolates identified four sequence types: ST3, ST10, ST11, and ST16, and the distribution of ST11 was significantly higher than the other MLST genotypes. Interestingly, all of the ST11 isolates detected in this study were genotype D: L6 strains and they were 100% positive for hgbB. Our data suggest that a capsule/LPS/MLST genotype D/L6/ST11 is likely to be strongly associated with respiratory clinical manifestation of the disease in pigs.

Capsular Polysaccharide Types and Virulence-Related Traits of Epidemic KPC-Producing Klebsiella pneumoniae Isolates in a Chinese University Hospital.

Klebsiella pneumoniae is an important human pathogen associated with a variety of diseases and the prevalence of blaKPC carrying K. pneumoniae (KPC-Kp) is rapidly increasing. Capsule is an important virulence factor in K. pneumoniae. In this study, we determined to first systematically characterize capsular polysaccharide (CPS) and virulence traits in KPC-Kp strains. A total of 56 KPC-Kp isolates were recovered from clinical samples in a Chinese hospital, which were assigned to clonal lineages by multilocus sequence typing (MLST). Capsule typing (wzi sequencing and wzc polymerase chain reaction [PCR]) and virulence genes were characterized by molecular approaches. The virulence of these strains was determined by biofilm formation, serum killing resistance, phagocytosis, and infection models. Six different STs were found among 56 KPC-Kp isolates: 76.8% (43 of 56 isolates) belonged to ST11, 6 isolates belonged to ST147, 4 isolates belonged to ST15, 1 isolate belonged to ST1456, 1 isolate belonged to ST65, and 1 isolate was ST23. Based on the wzi gene DNA sequences and wzc PCR, these 56 strains were classified as capsular type wzi47-K47 (n = 37), wzi64-K64 (n = 8), wzi8-K8 (n = 4), wzi37-K37 (n = 4), wzi53-K53 (n = 1), wzi125-K2 (n = 1), and wzi1-K1 (n = 1). Heterogeneity was detected in biofilm formation and phagocytosis among different CPS types. ST11 strains were less virulent than other ST strains. KPC-Kp strains exhibit variability of virulence-associated traits. Differences were associated with the ST types and CPS.

Community-onset Klebsiella pneumoniae pneumonia in Taiwan: clinical features of the disease and associated microbiological characteristics of isolates from pneumonia and nasopharynx.

Klebsiella pneumoniae is an important cause of community-onset pneumonia in Asian countries and South Africa. We investigated the clinical characteristics of K. pneumoniae causing community-onset pneumonia, and the associated microbiological features between K. pneumoniae isolates from pneumonia and those from the nasopharynx in Taiwan. This study was conducted at the Taipei Veterans General Hospital during July, 2012 to February, 2014. The clinical characteristics in patients with community-onset K. pneumoniae pneumonia were analyzed. K. pneumoniae isolates from the nasopharynx of adults attending otorhinolaryngology outpatient clinics were collected to compare their microbiological features with those from pneumonia. Capsular genotypes, antimicrobial susceptibility, and multilocus sequence type (MLST) were determined among these strains. Ninety-one patients with community-onset K. pneumoniae pneumonia were enrolled. We found a high mortality (29.7%) among these patients. Capsular types K1, K2, K5, K20, K54, and K57 accounted for ∼70% of the K. pneumoniae isolates causing pneumonia, and ∼70% of all the K. pneumoniae strains isolated from the nasopharynx of patients in outpatient clinics. The MLST profiles further demonstrated the genetic relatedness between most pneumonia isolates and those from the nasopharynx. In conclusion, our results show that community-onset pneumonia caused by K. pneumoniae was associated with high mortality and could have a reservoir in the nasopharynx. To tackle this high-mortality disease, the distribution of capsular types in the nasopharynx might have implications for future vaccine development.

Caracterización de los tipos capsulares de Pasteurella multocida en exudado faríngeo de bovinos productores de carne clínicamente sanos en el estado de Querétaro / Characterization of capsular types of Pasteurella multocida isolated from clinically healthy beef cattle pharyngeal exudate in the state of Querétaro

El objetivo del presente estudio fue identificar y caracterizar los tipos capsulares de P. multocida en exudado faríngeo en bovinos destinados a la producción de carne en el estado de Querétaro. Se obtuvieron, mediante hisopo, 227 muestras de exudado faríngeo de animales clínicamente sanos en una planta de sacrificio ubicada en el municipio de Ezequiel Montes, Querétaro. Las muestras se sembraron en agar sangre y se incubaron a 37°C por 24 h en aerobiosis. Las cepas aisladas fueron identificadas mediante características morfológicas, pruebas bioquímicas convencionales y el microsistema comercial API 20NE. La tipificación de los grupos capsulares A y D se realizó por medio de una PCR múltiple para la amplificación de los genes hyaD-hyaC y dcbF, respectivamente. De acuerdo con los valores establecidos por el software API WEB, se logró la identificación de 14.09% (32/227) de cepas de P. multocida, que mostraron 96% de identidad y una tipicidad de 1 a P. multocida. Por medio de la PCR múltiple se logró la amplificación de los genes hyaD-hyaC correspondientes al grupo capsular A en el 100% (32/32) de las cepas identificadas previamente como P. multocida. No existen datos similares en México sobre la identificación y caracterización de P. multocida en bovinos destinados a la producción de carne. Con los resultados obtenidos se corrobora que, de manera similar a otros países de Europa y América, en México el grupo capsular predominante de P. multocida es el A.

The objective of the present study was to identify and characterize capsular types of P. multocida isolated from beef cattle pharyngeal exudate in the state of Querétaro. Two hundred and twenty seven pharyngeal exudate swab samples from clinically healthy animals in a slaughterhouse in the municipality of Ezequiel Montes, Querétaro were obtained. Samples were seeded in blood agar and incubated at 37°C for 24 h under aerobiosis. Strains were identified through morphological characteristics, conventional biochemical tests and commercial API 20NE Micro-System. Capsular typing of groups A and D was performed by a multiplex PCR for amplification of genes hyaD-hyaC and dcbF, respectively. According to the values established by API WEB software, it was possible to identify 14.09% (32/227) of P. multocida strains, which showed an identification percentage of 96% and a typicality of 1 to P. multocida. By multiplex PCR, the amplification of genes hyaD-hyaC, correspondent to capsular group A in 100% (32/32) of the strains previously identified as P. multocida, was achieved. There are no similar data in Mexico on the identification and characterization of P. multocida in beef cattle. With the results obtained it is confirmed that, in a similar way with other countries of Europe and America, capsular type A of P. multocida is predominant in Mexico.

Caracterización fenotípica y molecular de cepas de Pasteurella multocida aisladas de exudado nasal de bovinos, en dos cuencas lecheras de México / Phenotypic and molecular strain characterization of Pasteurella multocida isolated from cattle nasal exudate from two dairy complexes in Mexico

Two hundred and fifty strains of P. multocida isolated from nasal exudate were obtained, 182 clinically healthy bovine strains and 68 clinically ill with pneumonia bovine strains, from two dairy complexes, one in the Tizayuca region of Hidalgo state (n = 81), and another in the Region Lagunera of the states of Coahuila and Durango (n = 169), Mexico. Strains were identifed by conventional biochemical tests and API 20NE commercial system. Capsular typing was performed by testing hyauloronidase and acrifavine, as well as by a multiplex PCR for amplification of genes hyaC-hyaD and dcbF. The overall results of hyaluronidase by the test showed that 90.4% (226/250) of the strains were capsular type A and through the acrifavine test 9.6% (24/250) was capsular type D. Using the multiplex PCR, 92% (230/250) was capsular type A and 8% (20/250) was capsular type D. The comparison of results between biochemical tests and PCR are consistent in identifying strains of capsular type A but not with the capsular type D. It was possible to confrm that capsular type A of P. multocida is predominat in Mexico.

Se obtuvieron 250 cepas de P. multocida aisladas de exudado nasal, 182 cepas de bovinos clínicamente sanos y 68 cepas de bovinos clínicamente enfermos de neumonía, de dos complejos lecheros, uno en la región de Tizayuca estado de Hidalgo (n = 81), y otro en la Región Lagunera de los estados de Coahuila y Durango (n = 169), México. Las cepas fueron identificadas mediante pruebas bioquímicas convencionales y el sistema comercial API 20NE. La tipificación capsular se realizó por medio de las pruebas de hiauloronidasa y acrifavina, así como por medio de una PCR múltiple para la amplificación de los genes hyaD-hyaC y dcbF. Los resultados globales mediante la prueba de hialuronidasa mostraron que 90.4% (226/250) de las cepas fueron del tipo capsular A y por medio de la prueba de acrifavina, 9.6% (24/250) fue del tipo capsular D. Por medio de la PCR múltiple, 92% (230/250) fue tipo capsular A y 8% (20/250) fue tipo capsular D. La comparación de los resultados entre las pruebas bioquímicas y la técnica de PCR concuerdan en la identificación de las cepas del tipo capsular A, pero no así con las del tipo capsular D. Se corrobora que en México el tipo capsular predominante de P. multocida es el A.

Distributions of pathogenic capsular types and in vitro antimicrobial susceptibility of different serotypes of Streptococcus suis isolated from clinically healthy sows from 10 provinces in China / 中华流行病学杂志

Objective To determine the distributions of major pathogenic capsular types and in vitro antimicrobial susceptibility of different serotypes of Streptococcus suis isolated from clinically healthy sows in China. Methods Tonsil specimens of clinically healthy sows from 10 different provinces in China were collected, a total of 421 S.suis were isolated. Capsular types of S.suis were decided using the sera agglutination reaction. Antimicrobial susceptibility testing was performed using a broth microdilution method and the differences between serotypes were decided statistically. Results The prevalent capsular types of S.suis isolated from clinically healthy sows were 9(26.6% ), 3 (23.5%) and 7(15.7% ) types, respectively. 7.4% of isolates were confirmed to be S.suis type 2. Overall, differences in antimicrobial susceptibility among serotypes of S. suis were found. By comparison, lower resistance was observed for S.suis type 2 from clinically healthy sows. Conclusion The prevalence of pathogenic S.suis serotypes from clinically healthy sows again indicates S.suis is a conditional pathogenic bacterium. Differential prevention and treatment regimes should be considered according to antimicrobial susceptibility of different serotypes of S.suis.