The aim of this paper was to determine in 32 patients from 4 different Mexican hospitals the frequency of opportunistic bacteria in the 2010 to 2011 time period. The patients were divided in 4 groups. Group 1 included 21 HIV positive patients with acute respiratory syndrome. Four HIV positive patients with tuberculosis symptoms were included in Group 2; two patients with tuberculosis symptoms and one asymptomatic person formed Group 3. Reference Group 4 included 4 patients from whom 4 strains of
Immunodeficiency is the main conditioning factor for bacterial infection susceptibility; therefore, immunodeficient patients frequently present several aggregated infections [
It has been established that worldwide 60 million people have active TB and 2-3 million die yearly due to
This bacterium causes infection and is frequently associated to other conventional pathogens [
The objective of this research was to identify the predominant bacterial species in HIV positive and other immunodeficient patients with respiratory symptoms and with or without a TB diagnosis.
The study included 32 patients from four hospitals in the State of Mexico, during the years 2010-2011, and were divided into four groups.
Twenty-one HIV positive patients with acute respiratory syndrome, 16 males and 5 females, aged 31 to 54 years, came from the “Acquired Inmunedeficiency Syndrome (AIDS)” clinic of a main hospital. They presented 41 to 958 CD4 values and <40–110 viral load. They also presented respiratory symptoms, coughing, expectoration, and a 3–20 kg weight loss suggestive of TB. No laboratory tests to confirm the diagnoses were carried out until this study began but the patients received antiretroviral treatment.
Four HIV positive patients with TB syndrome, 3 males and one female 31–37 years old, with 288–576 CD4 values and no detectable viral load were admitted to a specialized AIDS and sexual transmitted disease hospital, with a diagnoses suggestive of TB (cough, expectoration, fever, and a 5–13 kg weight loss). The diagnosis was confirmed by bacilloscopy and the patients received a doTBal shortened strictly supervised tuberculosis treatment (TAES) rifampicin, isoniazid, 3 pyrazinamide, and ethambutol chloride, for six months.
Two patients with a TB syndrome, a 32-years-old male and a 39-year-old female, from an AIDS and sexually transmitted diseases clinic, presented cough, expectoration, fever, and 5 and 10 kg weight loss, respectively, and were diagnosed with TB after positive bacilloscopy. They received a doTBal shortened strictly supervised treatment (TAES) (rifampicin, isoniazid, pyrazinamide, and ethambutol chloride for six months. A sputum sample was obtained from a 30-year-old male companion without symptoms or diagnosis but with laboratory diagnostic tests. He was considered as an asymptomatic high-risk patient.
Four patients were located from a retrospective study (2007–2010) of 186 clinical files of a specialized hospital to detect the number of patients with TB and the most common
In accordance with the guideline for tuberculosis bacteriological diagnoses (MDBT) [
Decontaminated samples were put into in commercial Lowenstein-Jensen (LJ) (BD BBL 0196675) medium, incubated at 37°C for 3-4 weeks, and revised every 7 days. The cultured bacteria were transferred to Petri plates containing Middlebrook 7H10 Agar (Difco, USA) glycerol and OADC enrichment culture medium and incubated at 37°C [
Twenty-eight strains from the patients of Groups 1, 2, and 3 were obtained considering the colony morphology: size, color, surface, and consistency, and 4 strains from the patients in the reference group were acquired from the IMSS Medical Center “Siglo XXl” Tuberculosis Service. The chosen bacteria were smeared, the slides were stained with Ziehl-Neelsen for acid-alcohol resistant bacilli (BAAR) [
For total DNA extraction the Wizard Genomic Purification DNA Kit (Promega A1120) (Isolation of genomic DNA from Gram positive and Gram negative bacteria) was used. For PCR amplifications and sequencing, A 1300–1500 bp fragment of the 16S rRNA gene was amplified with primers 8F (5′-AGA GTT TGA TCA TGG CTC AG-3′) and 1492R (5′-TAC GGT TAC CTT GTT ACG ACT T-3′). After 30 cycles of denaturation at 94°C for 60 s, primer annealing at 55°C for 20 s, and primer extension at 72°C for 60 s, followed by postamplification extension at 72°C for 10 min, amplification was seen in (1%) agarose gel electrophoresis at 90 volts for 30 min. The amplified products were sequenced for Bagdye terminator to Macrogen USA. Sequences corresponding to both regions were assembled and edited using BioEdit software version 7.0.5 [
The identified predominant group of strains were subjected to phylogenetic and biochemical characterization and to antibiotic sensibility studies.
All consensus sequences were compiled into a single file (Fasta format) for evolutionary analyses and then aligned with the profile mode of Clustal W 1.8.1 [
For the biochemical characterization each colony was identified using the API 20E (Biomérieux B-20100) and API 50CH profile index (Biomérieux B-50300). The systems were incubated at 35°C and all reactions were read after 20 to 24 h. Results of affirmative or negative tests were recorded and interpreted according to the manufacturer’s directions [
For antibiotic sensitivity the strains were cultured in Mueller-Hinton Agar (MH) (Bioxon, U.S.A); antimicrobial susceptibility was determined with the disk diffusion method accordingly to the National Committee for Clinical Laboratory Standards (NCCLS) [
The demographic characteristics of the patients, the CD4 values, and viral load are shown in Table
Demographic characteristics of the patients, CD4 values and viral load.
Patients | Code | Male/female | Age | Weight loss (kg) | Values of CD4 | Viral load |
---|---|---|---|---|---|---|
Group 1. HIV positive patients with acute respiratory syndrome | ||||||
(1) | E1 | M | 51 | 3 | 47 | 77 |
(2) | NR1b | M | 39 | 20 | 850 | <40 |
(3) | E2 | F | 39 | 6 | 395 | 60 |
(4) | E4 | M | 45 | 6 | 958 | <40 |
(5) | NR5 | F | 44 | 6 | 837 | <40 |
(6) | NR6 | M | 39 | 5 | 377 | <40 |
(7) | NR7 | M | 43 | 6 | ND | ND |
(8) | NR8 | M | 36 | 6 | 329 | <40 |
(9) | NR9 | M | 35 | 10 | 267 | <40 |
(10) | NR10 | M | 40 | 7 | 41 | <40 |
(11) | E6 | M | 31 | 10 | ND | ND |
(12) | NR14 | M | 45 | 8 | 316 | <40 |
(13) | NR15 | M | 31 | 4 | 579 | <40 |
(14) | NR17 | M | 37 | 7 | 814 | 82 |
(15) | NR18 | F | 54 | Without loss | ND | ND |
(16) | NR19 | F | 41 | 15 | 809 | <40 |
(17) | NR21 | M | 30 | 10 | 492 | 111 |
(18) | NR22 | M | 27 | 3 | 474 | 66 |
(19) | NR23 | F | 35 | 16 | 662 | <40 |
(20) | NR24 | M | 32 | 18 | ND | ND |
(21) | NR25 | M | 31 | 3 | 623 | <40 |
Group 2. HIV positive patients with TB syndrome | ||||||
(1) | E9 | M | 35 | 10 | 288 | Indetectable |
(2) | NR41 | M | 31 | 5 | 345 | Indetectable |
(3) | NR42 | F | 26 | Without loss | 576 | Indetectable |
(4) | NR43 | M | 37 | 13 | 438 | Indetectable |
Group 3. Patients with TB syndrome | ||||||
(1) | NR36 | M | 32 | 5 | No | No |
(2) | NR31 | F | 39 | 10 | No | No |
(3) | NR37 | M | 30 | Without loss | No | No |
Group 4. Reference group | ||||||
(1) | NR51 | M | 51 | Without loss | No | No |
(2) | NR52 | F | 51 | Without loss | No | No |
(3) | NR53 | M | 81 | 2 | No | No |
(4) | NR54 | F | 85 | 5 | No | No |
ND: No determined; NO: no CD4 values/no viral load determined.
Of the 28 sputum specimens, 12 were BAAR positive: five strains from Group 1 patients, four strains from Group 2, and three strains from Group 3 patients. All the isolates showed good growth in the two culture media (LJ and Middlebrook 7H10). The acid-fast smear microscopy results (>90%) were given to the health care provider within 24 hours of receipt of all specimens.
The macroscopic and microscopic morphology of the bacteria allowed grouping in four categories. Category A included small size, yellow, round, and creamy like colonies. Ten strains were short bacilli, with round ends, Gram negative, and no BAAR. Category B was small size, dark yellow, round, and wrinkled colonies. In 6 strains the bacilli were elongated, Gram positive, and acid-alcohol resistant. Category C included medium size, cream and yellow color, round, and creamy colonies. Six strains showed straight or slightly curved bacilli, Gram negative, and no BAAR. Category D included a variety of small to medium size, yellow and cream color, smooth, hard, and creamy colonies. Ten strains were of Gram positive diplococci and Gram positive bacilli, no BAAR. The acid-fast smear microscopy results (>90%) were given to the health care provider within 24 hours of receipt of all specimens.
With the comparative analysis of the obtained sequences deposited in Gen Bank, the principal species were identified: ten strains were identified as
Sequenced identified bacteria and the similarity percentage calculated with the BLASTN program.
Patients | Code | BLASTN program | Morphology group | Similarity percentage |
---|---|---|---|---|
Group 1. HIV positive patients with acute respiratory syndrome | ||||
(1) | E1 | Category D | 99 | |
(2) | NR1b | Category A | 100 | |
(3) | E2 | Category D | 100 | |
(4) | E4 | Category A | 100 | |
(5) | NR5 | Category A | 99 | |
(6) | NR6 | Category B | 99 | |
(7) | NR7 | Category D | 99 | |
(8) | NR8 | Category D | 99 | |
(9) | NR9 | Category A | 99 | |
(10) | NR10 | Category D | 99 | |
(11) | E6 | Category D | 97 | |
(12) | NR14 | Category D | 99 | |
(13) | NR15 | Category A | 99 | |
(14) | NR17 | Category A | 100 | |
(15) | NR18 | Category D | 97 | |
(16) | NR19 | Category C | 99 | |
(17) | NR21 | Category C | 99 | |
(18) | NR22 | Category C | 99 | |
(19) | NR23 | Category C | 99 | |
(20) | NR24 | Category A | 99 | |
(21) | NR25 | Category E | 99 | |
Group 2. HIV positive patients with TB syndrome | ||||
(1) | E9 | Category C | 99 | |
(2) | NR41 | Category C | 99 | |
(3) | NR42 | Category A | 99 | |
(4) | NR43 | Category A | 100 | |
Group 3. Patients with a TB syndrome | ||||
(1) | NR36 | Category B | 100 | |
(2) | NR31 | Category A | 99 | |
(3) | NR37 | Category B | 100 | |
Group 4. Reference group | ||||
(1) | NR51 | Category B | 97 | |
(2) | NR52 | Category B | 99 | |
(3) | NR53 | Category B | 99 | |
(4) | NR54 | Category B | 99 |
The analysis of the sequencing of fragment 1367 bp without gaps from 16S rRNA gene was carried out by comparison with five species of the genus
Phylogenetic tree based on 1367 bp without gaps from 16S rRNA gene.
Since
Most of the
Antibiogram results for microbial.
Antibiotic | NR5 | NR9 | N17 | NR24 | NR43 | NR31 |
---|---|---|---|---|---|---|
Amikacin (AK) | R | I | R | S | S | R |
Ampicillin (AM) | S | R | R | R | R | R |
Carbenicillin (CB) | R | R | R | R | R | R |
Cefotaxima (CTX) | S | R | R | I | R | R |
Ceftriaxone (CRO) | R | R | R | I | R | R |
Choramphenicol (CL) | R | I | I | R | R | I |
Gentamycin (GE) | R | I | I | R | S | R |
Netilmicin (NET) | R | R | R | R | R | R |
Nitrofurantoin (NF) | I | R | R | R | R | R |
Pefloxacin (PEF) | R | R | R | R | R | R |
R: Resistant; I: Intermediate; S: Susceptible.
The symptoms of the included patients are similar to those produced by
García et al. (2003) reported that 100% of the patients admitted in a hospital present with pulmonary diseases mostly due to opportunistic infections (98.7%), and in HIV positive patients, the 3 principal causes were, TB, bacterial pneumonia and other no specified infectious neumonia [
The results of this investigation indicate that not all respiratory syndrome in immunodeficient patients is due to TB. The patients studied presented with productive cough but the identified organisms were
The mostly isolated bacterium was
On the other hand, Domínguez et al. in 2008 reported that the most frequently isolated Gram negative bacillus is
In general,
In Group 2 we expected to isolate
As to the patients in Group 3, it is interesting that their sputum was negative for Ziehl-Neelsen but culture positive. This result implies an epidemiologic factor. The related persons in contact with the TB patient are acid-fast bacteria negative and are sent home without diagnosis or treatment but they are a focus of infection for other persons.
In the present study, we confirmed bacterial identification by means of the amplified sequence of 16S rRNA gene. Molecular biology techniques are a useful tool for the confirmation of the isolated organism, as is the case of
By means of the API 20E and 50CH biochemical assays practiced on
From the 32 included patients, the following strains were identified: ten strains of
Even though, initially the aim our study was the search and identification of
Our research confirms the presence of opportunistic bacteria such as
The authors declare that they do not have any conflict of interests.
This work is product derived from the thesis of degree of the Doctorate in Sciences of the Health, registered in the PNPC-CONACYT, and of the economic contribution of project SIEA-UAEM2742/2008U.